Journal articles on the topic 'Keratinocyte'
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1
Hildesheim, J., U. Kuhn, C. L. Yee, R. A. Foster, K. B. Yancey, and J. C. Vogel. "The hSkn-1a POU transcription factor enhances epidermal stratification by promoting keratinocyte proliferation." Journal of Cell Science 114, no. 10 (May 15, 2001): 1913–23. http://dx.doi.org/10.1242/jcs.114.10.1913.
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Skn-1a is a POU transcription factor that is primarily expressed in the epidermis and is known to modulate the expression of several genes associated with keratinocyte differentiation. However, the formation of a stratified epidermis requires a carefully controlled balance between keratinocyte proliferation and differentiation, and a role for Skn-1a in this process has not been previously demonstrated. Here, our results show, surprisingly, that human Skn-1a contributes to epidermal stratification by primarily promoting keratinocyte proliferation and secondarily by enhancing the subsequent keratinocyte differentiation. In organotypic raft cultures of both primary human keratinocytes and immortalized HaCaT keratinocytes, human Skn-1a expression is associated with increased keratinocyte proliferation and re-epithelialization of the dermal substrates, resulting in increased numbers of keratinocytes available for the differentiation process. In these same raft cultures, human Skn-1a expression enhances the phenotypic changes of keratinocyte differentiation and the upregulated expression of keratinocyte differentiation genes. Conversely, expression of a dominant negative human Skn-1a transcription factor lacking the C-terminal transactivation domain blocks keratinocytes from proliferating and stratifying. Keratinocyte stratification is dependent on a precise balance between keratinocyte proliferation and differentiation, and our results suggest that human Skn-1a has an important role in maintaining epidermal homeostasis by promoting keratinocyte proliferation.
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Burks, Hope E., Christopher Arnette, Jennifer Koetsier, Joshua Broussard, Quinn Roth-Carter, Pedram Gerami, Jodi Johnson, and Kathleen Green. "Abstract 3186: Keratinocyte desmosomal cadherin Desmoglein 1 as a mediator and target of paracrine signaling in the melanoma niche." Cancer Research 82, no. 12_Supplement (June 15, 2022): 3186. http://dx.doi.org/10.1158/1538-7445.am2022-3186.
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Abstract Melanoma is an aggressive cancer arising from the transformation of melanocytes residing in the basal layer of the epidermis, where they are in direct contact with surrounding keratinocytes. The role of keratinocytes in shaping the melanoma tumor microenvironment, however, remains understudied. We previously showed that the keratinocyte-specific desmosomal cadherin, desmoglein 1 (Dsg1) is reduced in response to acute UV exposure. Here, we show that Dsg1 protein is reduced in keratinocytes surrounding melanoma lesions but is unchanged in keratinocytes adjacent to benign nevi. As Dsg1 reduction by UV exposure is transient, we hypothesized that the persistent loss of Dsg1 observed in the melanoma tumor niche is due to melanoma-keratinocyte paracrine crosstalk. To address this idea, keratinocytes were cultured in conditioned media from melanocytes or melanoma cells. Melanoma conditioned media reduced keratinocyte Dsg1 mRNA and protein levels, suggesting that Dsg1 downregulation in keratinocytes is maintained by melanoma cells post-transformation. Keratinocytes in melanoma conditioned media exhibited reduced Grhl1 (Grainyhead like 1), a transcriptional activator of Dsg1, and increased Snai2, a repressor of Grhl1 expression and epidermal differentiation. These data support the idea that increased Snai2 stimulated by factors secreted by melanoma cells reduces keratinocyte Dsg1 mRNA expression by repressing Grhl1. To determine the impact of keratinocyte Dsg1 loss on melanoma cell behavior, melanoma cells were grown in conditioned media from control or Dsg1-deficient keratinocytes. Melanoma cell migration increased in a trans-well assay when melanoma cells were grown in conditioned media from Dsg1-deficient keratinocytes compared to from control keratinocytes. Supporting the in vivo importance of this finding, we found a significant, negative correlation between keratinocyte Dsg1 expression and melanoma cell movement within the melanoma tumor niche in patient samples. Downregulation of keratinocyte Dsg1 by melanoma cell conditioned media increased Erk1/2 signaling upstream of pro-migratory CXCL1 production, while pharmacological inhibition of keratinocyte Erk1/2 abrogated this effect. Furthermore, inhibition of the CXCL1 receptor, CXCR2 on melanoma cells decreased migration in melanoma cells grown in Dsg1-depleted keratinocyte conditioned media. Together, these data support the idea that paracrine crosstalk between melanoma cells and keratinocytes resulting in chronic keratinocyte Dsg1 reduction is important for melanoma progression and melanoma cell movement in the early stages of metastasis. Citation Format: Hope E. Burks, Christopher Arnette, Jennifer Koetsier, Joshua Broussard, Quinn Roth-Carter, Pedram Gerami, Jodi Johnson, Kathleen Green. Keratinocyte desmosomal cadherin Desmoglein 1 as a mediator and target of paracrine signaling in the melanoma niche [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3186.
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Yan, Feng-Juan, Yong-Jian Wang, Song-En Wang, and Hai-Ting Hong. "PKCα/ERK/C7ORF41 axis regulates epidermal keratinocyte differentiation through the IKKα nuclear translocation." Biochemical Journal 478, no. 4 (February 24, 2021): 839–54. http://dx.doi.org/10.1042/bcj20200879.
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Aberrant differentiation of keratinocytes disrupts the skin barrier and causes a series of skin diseases. However, the molecular basis of keratinocyte differentiation is still poorly understood. In the present study, we examined the expression of C7ORF41 using tissue microarrays by immunohistochemistry and found that C7ORF41 is specifically expressed in the basal layers of skin epithelium and its expression is gradually decreased during keratinocytes differentiation. Importantly, we corroborated the pivotal role of C7ORF41 during keratinocyte differentiation by C7ORF41 knockdown or overexpression in TPA-induced Hacat keratinocytes. Mechanismly, we first demonstrated that C7ORF41 inhibited keratinocyte differentiation mainly through formatting a complex with IKKα in the cytoplasm, which thus blocked the nuclear translocation of IKKα. Furthermore, we also demonstrated that inhibiting the PKCα/ERK signaling pathway reversed the reduction in C7ORF41 in TPA-induced keratinocytes, indicating that C7ORF41 expression could be regulated by upstream PKCα/ERK signaling pathway during keratinocyte differentiation. Collectively, our study uncovers a novel regulatory network PKCα/ERK/C7ORF41/IKKα during keratinocyte differentiation, which provides potential therapeutic targets for skin diseases.
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Toda, K., T. L. Tuan, P. J. Brown, and F. Grinnell. "Fibronectin receptors of human keratinocytes and their expression during cell culture." Journal of Cell Biology 105, no. 6 (December 1, 1987): 3097–104. http://dx.doi.org/10.1083/jcb.105.6.3097.
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Keratinocyte attachment to fibronectin (FN) substrata was inhibited by the peptide Gly-Arg-Gly-Asp-Ser-Pro-Cys, but not by the variant peptide Gly-Arg-Gly-Glu-Ser-Pro. The RGDS-containing peptide did not inhibit keratinocyte adhesion to collagen. Keratinocyte adhesion to FN substrata also was inhibited by polyclonal anti-FN receptor antibodies originally prepared against the 140-kD FN receptors of Chinese hamster ovary (CHO) cells. Anti-CHO FN receptor antibodies did not, however, inhibit keratinocyte adhesion to collagen substrata. A monoclonal antibody designated VM-1 that was prepared against human basal keratinocytes inhibited keratinocyte adhesion to collagen but not to FN. Based on these results, we conclude that keratinocytes have distinct FN and collagen receptors. Experiments were performed to compare the expression of FN receptors on keratinocytes freshly isolated from skin and keratinocytes harvested from cell cultures. Cells harvested from keratinocyte cultures were able to neutralize the inhibitory activity of anti-CHO FN receptor antibodies and were able to attach and spread on anti-CHO FN receptor-coated substrata. Cells freshly harvested from skin, however, did not neutralize the antibodies, nor did they attach and spread on antibody-coated substrata. To learn more about the biochemical nature of the keratinocyte FN receptors, we performed immunoaffinity chromatography and immunoprecipitation experiments using the anti-CHO FN receptor antibodies. Extracts from metabolically radiolabeled, 10-d cultured keratinocytes contained FN receptors that had a 135-kD component under reducing conditions and 115- and 155-kD components under nonreducing conditions. Similar components were observed in extracts from surface-radiolabeled cells indicating that the FN receptors were expressed on keratinocyte cell surfaces. On the other hand, extracts from metabolically radiolabeled, 1-d cultured keratinocytes lacked intact FN receptors but contained a component that migrated at 48 kD under reducing conditions and 50 kD under nonreducing conditions. Because this fragment was not detected in surface-radiolabeled keratinocytes that were freshly isolated from skin, it seems likely that the fragment was located inside the cells rather than on the cell surface. A 50-kD FN receptor fragment also was observed in extracts from 10-d cultured keratinocytes if leupeptin and pepstatin were omitted from the extraction buffer. The results suggested that human keratinocytes cultured for 10 d express the 140-kD class of FN receptors, but that these receptors are not expressed on the surfaces of keratinocytes freshly isolated from skin.(ABSTRACT TRUNCATED AT 400 WORDS)
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Darmstadt, Gary L., Laurel Mentele, Philip Fleckman, and Craig E. Rubens. "Role of Keratinocyte Injury in Adherence ofStreptococcus pyogenes." Infection and Immunity 67, no. 12 (December 1, 1999): 6707–9. http://dx.doi.org/10.1128/iai.67.12.6707-6709.1999.
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ABSTRACT Keratinocytes injured acutely by UVB light or lipopolysaccharide were used to test the hypothesis that keratinocyte injury promotes bacterial adherence and the development of group A streptococcal skin infections. Injury did not affect adherence to undifferentiated and differentiated keratinocytes, but keratinocyte differentiation promoted adherence four- to fivefold.
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HUANG, YI-CHAU, TZU-WEI WANG, JUI-SHENG SUN, and FENG-HUEI LIN. "EFFECT OF CALCIUM ION CONCENTRATION ON KERATINOCYTE BEHAVIORS IN THE DEFINED MEDIA." Biomedical Engineering: Applications, Basis and Communications 18, no. 01 (February 25, 2006): 37–41. http://dx.doi.org/10.4015/s1016237206000087.
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Calcium ion concentration is proposed to be involved in the regulation of the proliferative capacity of keratinocytes, based on its significant actions in the skin. These actions are mediated by Ca2+ influx and inhibition of cell proliferation. To define Ca2+ action in the keratinocyte we investigated its effects on the proliferation and differentiation using the primary keratinocytes model. Primary keratinocytes were incubated in DMEM (containing 1.2mM calcium ion concentration) or DK11 medium (containing 0.4 mM calcium ion concentration) or K medium (containing 0.03mM calcium ion concentration). Cell viability was assessed with the MTT assay. Crystal violet assay was evaluated the proliferation rate and colony formation size of keratinocyte. Real-time PCR used to determine the terminal differentiated keratinocyte which expressed Caspase-14. Proliferation assays and real°Vtime PCR were correlated with either proliferation or differentiation in cultured human skin epidermal keratinocytes. High Ca2+ concentration was inhibited the cell viability and proliferation rate of keratinocyte. Ca2+ also increased caspases-14 expression, and inhibited cell viability, and cell colony forming efficiency. These results are consistent with Ca2+ induction of the keratinocyte differentiation. Thus, the overall Ca2+ actions connote protective functions for the epidermis that appear to include the triggering or acceleration of the differentiation.
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Hatterschide, Joshua, Amelia E. Bohidar, Miranda Grace, Tara J. Nulton, Hee Won Kim, Brad Windle, Iain M. Morgan, Karl Munger, and Elizabeth A. White. "PTPN14 degradation by high-risk human papillomavirus E7 limits keratinocyte differentiation and contributes to HPV-mediated oncogenesis." Proceedings of the National Academy of Sciences 116, no. 14 (March 20, 2019): 7033–42. http://dx.doi.org/10.1073/pnas.1819534116.
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High-risk human papillomavirus (HPV) E7 proteins enable oncogenic transformation of HPV-infected cells by inactivating host cellular proteins. High-risk but not low-risk HPV E7 target PTPN14 for proteolytic degradation, suggesting that PTPN14 degradation may be related to their oncogenic activity. HPV infects human keratinocytes but the role of PTPN14 in keratinocytes and the consequences of PTPN14 degradation are unknown. Using an HPV16 E7 variant that can inactivate retinoblastoma tumor suppressor (RB1) but cannot degrade PTPN14, we found that high-risk HPV E7-mediated PTPN14 degradation impairs keratinocyte differentiation. Deletion ofPTPN14from primary human keratinocytes decreased keratinocyte differentiation gene expression. Related to oncogenic transformation, both HPV16 E7-mediated PTPN14 degradation andPTPN14deletion promoted keratinocyte survival following detachment from a substrate. PTPN14 degradation contributed to high-risk HPV E6/E7-mediated immortalization of primary keratinocytes and HPV+but not HPV−cancers exhibit a gene-expression signature consistent with PTPN14 inactivation. We find that PTPN14 degradation impairs keratinocyte differentiation and propose that this contributes to high-risk HPV E7-mediated oncogenic activity independent of RB1 inactivation.
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Ademi, Hyrije, Katarzyna Michalak-Micka, Ueli Moehrlen, Thomas Biedermann, and Agnes S. Klar. "Effects of an Adipose Mesenchymal Stem Cell-Derived Conditioned medium and TGF-β1 on Human Keratinocytes In Vitro." International Journal of Molecular Sciences 24, no. 19 (September 29, 2023): 14726. http://dx.doi.org/10.3390/ijms241914726.
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Human keratinocytes play a crucial role during skin wound healing and in skin replacement therapies. The secretome of adipose-derived stem cells (ASCs) has been shown to secrete pro-healing factors, among which include TGF-β1, which is essential for keratinocyte migration and the re-epithelialization of cutaneous wounds during skin wound healing. The benefits of an ASC conditioned medium (ASC-CM) are primarily orchestrated by trophic factors that mediate autocrine and paracrine effects in keratinocytes. Here, we evaluated the composition and the innate characteristics of the ASC secretome and its biological effects on keratinocyte maturation and wound healing in vitro. In particular, we detected high levels of different growth factors, such as HGF, FGFb, and VEGF, and other factors, such as TIMP1 and 4, IL8, PAI-1, uPA, and IGFBP-3, in the ASC-CM. Further, we investigated, using immunofluorescence and flow cytometry, the distinct effects of a human ASC-CM and/or synthetic TGF-β1 on human keratinocyte proliferation, migration, and cell apoptosis suppression. We demonstrated that the ASC-CM increased keratinocyte proliferation as compared to TGF-β1 treatment. Further, we found that the ASC-CM exerted cell cycle progression in keratinocytes via regulating the phases G1, S, and G2/M. In particular, cells subjected to the ASC-CM demonstrated increased DNA synthesis (S phase) compared to the TGF-β1-treated KCs, which showed a pronounced G0/G1 phase. Furthermore, both the ASC-CM and TGF-β1 conditions resulted in a decreased expression of the late differentiation marker CK10 in human keratinocytes in vitro, whereas both treatments enhanced transglutaminase 3 and loricrin expression. Interestingly, the ASC-CM promoted significantly increased numbers of keratinocytes expressing epidermal basal keratinocyte markers, such DLL1 and Jagged2 Notch ligands, whereas those ligands were significantly decreased in TGF-β1-treated keratinocytes. In conclusion, our findings suggest that the ASC-CM is a potent stimulator of human keratinocyte proliferation in vitro, particularly supporting basal keratinocytes, which are crucial for a successful skin coverage after transplantation. In contrast, TGF-β1 treatment decreased keratinocyte proliferation and specifically increased the expression of differentiation markers in vitro.
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Moon, Jadie Yoonjoo, Sonya Wolf, Christopher Audu, William Melvin, Amrita Joshi, Kevin Mangum, Emily Barrett, et al. "IL-17A-mediated JMJD3 regulation of integrin alpha 3 gene expression alters migration of diabetic keratinocytes and impairs wound repair." Journal of Immunology 210, no. 1_Supplement (May 1, 2023): 63.01. http://dx.doi.org/10.4049/jimmunol.210.supp.63.01.
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Abstract Keratinocytes are key structural cells in cell migration during wound repair. Keratinocyte migration is dysfunctional in diabetes and results in non-healing, however, the reason for this is unclear. Using bulk and single cell sequencing, we identified that Th17 CD4+ T-cells predominate as does an IL-17A signature in human diabetic wound tissue compared to non-diabetic controls. The effects of increased IL-17A in diabetic skin on keratinocyte function is unknown. To examine this, we subjected isolated keratinocytes to scratch injury and a 24hr rIL-17A (20ng) stimulation. We observed a significant decrease in migration rate in IL-17A stimulated keratinocytes compared to their controls. Next, we examined migration related genes in keratinocytes from mice fed with a normal (ND) or a high fat (HFD) diet. Integrin alpha 3 subunit, Itga3, (a protein known to delay keratinocyte migration during wound re-epithelialization) was higher in diabetic (HFD) keratinocytes compared to ND controls at baseline and with rIL-17A. We analyzed epigenetic enzymes known to increase gene expression and identified that JMJD3, a histone demethylase that relaxes chromatin, was increased both in diabetic keratinocytes from our human scRNA-seq and in murine diabetic keratinocytes with rIL17A. Further, treatment with a JMJD3-specific inhibitor (GSK-J4) decreased Itga3 in diabetic keratinocytes, suggesting that JMJD3 may be relevant to IL-17A-mediated regulation of integrin alpha 3 and other keratinocyte migration genes. Continued investigation into upstream IL-17A signaling in normal and diabetic keratinocytes that alters downstream migration is crucial for our understanding of impaired keratinocyte functions associated with diabetic wound repair. NIH T32 AI 007413, Research Training in Experimental Immunology Rackham Pre-Candidate Graduate Student Research Grant, University of Michigan
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Norris, D. A., S. B. Ryan, R. M. Kissinger, K. A. Fritz, and S. T. Boyce. "Systematic comparison of antibody-mediated mechanisms of keratinocyte lysis in vitro." Journal of Immunology 135, no. 2 (August 1, 1985): 1073–79. http://dx.doi.org/10.4049/jimmunol.135.2.1073.
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Abstract We have conducted a systematic comparison of lysis of TNP-coated keratinocyte targets by TNP-specific antibody, by antibody plus complement, by antibody-dependent cellular cytotoxicity (ADCC), and by natural killing with the use of monocyte, lymphocyte, and neutrophil effectors. With chromium-release assays, human keratinocytes, HEp-2 cells (transformed human keratinocytes), PAM 212 cells (transformed mouse keratinocytes), and RSC (transformed rabbit keratinocytes) were all susceptible to monocyte- and lymphocyte-mediated ADCC (p less than 0.01 to p less than 0.02). All trypsinized keratinocyte targets were also susceptible to natural killing by monocyte or lymphocyte effectors (p = 0.05 to p less than 0.001). Antibody and antibody plus complement were poor mediators of keratinocyte lysis. If protein and complex lipid synthesis of keratinocytes were inhibited by 16-hr cycloheximide preincubation, then keratinocytes were susceptible to complement-mediated lysis, implying that the resistance of these cells to complement may be due to repair of transmembrane pores. Comparison of chromium-release assays with fluorescein diacetate dye uptake viability assays showed that human keratinocytes were still susceptible to monocyte and lymphocyte ADCC but not to antibody, antibody plus complement, or natural killing. The reproducible and uniform susceptibility of normal and transformed keratinocyte targets from three different species to monocyte and lymphocyte ADCC supports the hypothesis that this mechanism of cellular lysis may be important in antibody-associated diseases of epidermal cytotoxicity.
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Hancock, K., and I. M. Leigh. "Cultured keratinocytes and keratinocyte grafts." BMJ 299, no. 6709 (November 11, 1989): 1179–80. http://dx.doi.org/10.1136/bmj.299.6709.1179.
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Zhu, Guoqin, Jie Liu, Yuan Wang, Naixin Jia, Weiyi Wang, Junhui Zhang, Yi Liang, Hao Tian, and Jiaping Zhang. "ADAM17 Mediates Hypoxia-Induced Keratinocyte Migration via the p38/MAPK Pathway." BioMed Research International 2021 (October 28, 2021): 1–11. http://dx.doi.org/10.1155/2021/8328216.
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Although hypoxia has been shown to promote keratinocyte migration and reepithelialization, the underlying molecular mechanisms remain largely unknown. ADAM17, a member of the metalloproteinase superfamily, has been implicated in a variety of cellular behaviors such as proliferation, adhesion, and migration. ADAM17 is known to promote cancer cell migration under hypoxia, and whether or how ADAM17 plays a role in hypoxia-induced keratinocyte migration has not been identified. Here, we found that ADAM17 expression and activity were significantly promoted in keratinocytes under hypoxic condition, inhibition of ADAM17 by TAPI-2, or silencing of ADAM17 using small interfering RNA which suppressed the hypoxia-induced migration of keratinocytes significantly, indicating a pivotal role for ADAM17 in keratinocyte migration. Further, we showed that p38/MAPK was activated by hypoxia. SB203580, an inhibitor of p38/MAPK, significantly attenuated the upregulation of ADAM17 as well as the migration of keratinocytes induced by hypoxia. Activation of p38/MAPK by MKK6 (Glu) overexpression, however, had adverse effects. Taken together, our study demonstrated that hypoxia-induced keratinocyte migration requires the p38/MAPK-ADAM17 signal axis, which sheds new light on the regulatory mechanisms of keratinocyte migration. Our study might also help in developing therapeutic strategies to facilitate wound healing in vivo, where cells are migrated in a hypoxic microenvironment.
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Wilkinson, J. Erby, C. A. Smith, Maja M. Suter, and Robert M. Lewis. "Antigen expression in cultured oral keratinocytes from dogs." American Journal of Veterinary Research 52, no. 3 (March 1, 1991): 445–48. http://dx.doi.org/10.2460/ajvr.1991.52.03.445.
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SUMMARY Oral keratinocytes from dogs were cultured on either collagen gels or artificial matrices at the air-liquid interface, and the expression of keratinocyte antigens and basement membrane components was determined, using various monoclonal and polyclonal antibodies. Keratinocytes grown on collagen gels expressed pemphigus vulgaris, pemphigus foliaceous, and bullous pemphigoid antigens. Diffuse, suprabasal, and superficial keratinocyte membrane differentiation antigens identified by various monoclonal antibodies also were expressed in a pattern identical to that observed in the native tissue. Laminin and type-IV collagen were deposited at the keratinocyte-collagen interface in a patchy distribution. When synthetic matrices were used, the oral keratinocytes differentiated, but to a lesser extent than cells grown on collagen gels. Antigen expression for cells grown on synthetic matrices was similar to that for cells on collagen, except for failure of the keratinocytes on synthetic membranes to express superficial cell antigens and pemphigus foliaceous antigens.
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Elliott, G., D. van de Meent, J. van Dijk, and M. Mol. "Human keratinocyte sensitivity towards inflammatory cytokines varies with culture time." Mediators of Inflammation 1, no. 6 (1992): 385–90. http://dx.doi.org/10.1155/s0962935192000589.
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Proliferating keratinocyte cultures have been reported to synthesize higher concentrations of prostaglandin (PG) E than confluent ones. As interleukin-1 (IL-1) stimulates keratinocyte PGE synthesis we investigated whether the degree of confluency of the keratinocyte culture modified the response of the cells to IL-1. It was found that IL-1α (100 U/ml) stimulated PGE2synthesis by proliferating (7 days in culture) but not differentiating (14 days in culture) keratinocytes. Similar effects were observed using tumour necrosis factor-α. Both arachidonic acid (AA) and the calcium ionophore A23187 stimulated PGE2synthesis by 7 and 14 day cultures although the increase was greatest when 7 day cultures were used. Our data indicate that there is a specific down-regulation of the mechanism(s) by which some inflammatory cytokines stimulate keratinocyte eicosanoid synthesis as cultured keratinocytes begin to differentiate.
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Prince, Tessa, Andrew J. McBain, and Catherine A. O'Neill. "Lactobacillus reuteri Protects Epidermal Keratinocytes from Staphylococcus aureus-Induced Cell Death by Competitive Exclusion." Applied and Environmental Microbiology 78, no. 15 (May 11, 2012): 5119–26. http://dx.doi.org/10.1128/aem.00595-12.
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ABSTRACTRecent studies have suggested that the topical application of probiotic bacteria can improve skin health or combat disease. We have utilized a primary human keratinocyte culture model to investigate whether probiotic bacteria can inhibitStaphylococcus aureusinfection. Evaluation of the candidate probioticsLactobacillus reuteriATCC 55730,Lactobacillus rhamnosusAC413, andLactobacillus salivariusUCC118 demonstrated that bothL. reuteriandL. rhamnosus, but notL. salivarius, reducedS. aureus-induced keratinocyte cell death in both undifferentiated and differentiated keratinocytes. Keratinocyte survival was significantly higher if the probiotic was applied prior to (P< 0.01) or simultaneously with (P< 0.01) infection withS. aureusbut not when added after infection had commenced (P> 0.05). The protective effect ofL. reuteriwas not dependent on the elaboration of inhibitory substances such as lactic acid.L. reuteriinhibited adherence ofS. aureusto keratinocytes by competitive exclusion (P= 0.026).L. salivariusUCC118, however, did not inhibitS. aureusfrom adhering to keratinocytes (P> 0.05) and did not protect keratinocyte viability.S. aureusutilizes the α5β1 integrin to adhere to keratinocytes, and blocking of this integrin resulted in a protective effect similar to that observed with probiotics (P= 0.03). This suggests that the protective mechanism forL. reuteri-mediated protection of keratinocytes was by competitive exclusion of the pathogen from its binding sites on the cells. Our results suggest that use of a topical probiotic prophylactically could inhibit the colonization of skin byS. aureusand thus aid in the prevention of infection.
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Tsukui, Kei, Takuya Kakiuchi, Hidetomo Sakurai, and Yoshihiro Tokudome. "Shotokuseki Extract Promotes Keratinocyte Differentiation Even at a Low Calcium Concentration." Applied Sciences 12, no. 5 (February 22, 2022): 2270. http://dx.doi.org/10.3390/app12052270.
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The switch between keratinocyte proliferation and differentiation is regulated by extracellular calcium levels, requiring high concentrations (>1 mol/L) of extracellular calcium to induce differentiation. The Shotokuseki extract (SE) contains various ions such as calcium, but its effect on keratinocytes is unknown. This study focused on calcium-induced differentiation of keratinocytes and investigated the effects of simultaneous application of calcium and other ions on keratinocyte differentiation. The expression of differentiation markers increased when SE was added to a keratinocyte culture but not when only calcium was added at the same concentration present in SE. The calcium concentration in SE was found to be too low (0.01 mol/L) to induce differentiation of keratinocytes. In addition, the application of SE increased intracellular calcium concentration compared with calcium solution alone. Therefore, the induction of keratinocyte differentiation by SE is not calcium-dependent, or SE may alter the calcium sensitivity of keratinocytes. In our study, we found that simultaneous application of multiple ions and/or the application of trace ions may alter calcium sensitivity and the epidermal cell response. The function of ion transporters associated with these ions and the response of cells to ions depends largely on the balance among various ions and the function of trace ions.
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Jung, Joon Min, Tai Kyung Noh, Soo Youn Jo, Su Yeon Kim, Youngsup Song, Young-Hoon Kim, and Sung Eun Chang. "Guanine Deaminase in Human Epidermal Keratinocytes Contributes to Skin Pigmentation." Molecules 25, no. 11 (June 5, 2020): 2637. http://dx.doi.org/10.3390/molecules25112637.
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Epidermal keratinocytes are considered as the most important neighboring cells that modify melanogenesis. Our previous study used microarray to show that guanine deaminase (GDA) gene expression is highly increased in melasma lesions. Hence, we investigated the role of GDA in skin pigmentation. We examined GDA expression in post-inflammatory hyperpigmentation (PIH) lesions, diagnosed as Riehl’s melanosis. We further investigated the possible role of keratinocyte-derived GDA in melanogenesis by quantitative PCR, immunofluorescence staining, small interfering RNA-based GDA knockdown, and adenovirus-mediated GDA overexpression. We found higher GDA positivity in the hyperpigmentary lesional epidermis than in the perilesional epidermis. Both UVB irradiation and stem cell factor (SCF) plus endothelin-1 (ET-1) were used, which are well-known melanogenic stimuli upregulating GDA expression in both keratinocyte culture alone and keratinocyte and melanocyte coculture. GDA knockdown downregulated melanin content, while GDA overexpression promoted melanogenesis in the coculture. When melanocytes were treated with UVB-exposed keratinocyte-conditioned media, the melanin content was increased. Also, GDA knockdown lowered SCF and ET-1 expression levels in keratinocytes. GDA in epidermal keratinocytes may promote melanogenesis by upregulating SCF and ET-1, suggesting its role in skin hyperpigmentary disorders.
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Barker, J. N., M. L. Jones, C. L. Swenson, V. Sarma, R. S. Mitra, P. A. Ward, K. J. Johnson, J. C. Fantone, V. M. Dixit, and B. J. Nickoloff. "Monocyte chemotaxis and activating factor production by keratinocytes in response to IFN-gamma." Journal of Immunology 146, no. 4 (February 15, 1991): 1192–97. http://dx.doi.org/10.4049/jimmunol.146.4.1192.
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Abstract Monocytes accumulate in the epidermis and along the dermo-epidermal junction in several different inflammatory skin diseases. To determine whether human epidermal keratinocytes elaborate a specific chemotaxin responsible for the accumulation of monocytes at these anatomic sites, monocyte chemotactic activity in conditioned 16-h cultured keratinocyte supernatants were assayed using human peripheral blood monocytes as the target cell. Dilutional analysis revealed directed monocyte migration in IFN-gamma-treated (100 U/ml) keratinocyte supernatants (80% maximal FMLP response) which was 10-fold more than IFN-gamma itself or untreated keratinocyte activity alone. Gel filtration chromatography revealed that this activity eluted just ahead of a 12.5-kDa molecular mass marker. Blocking studies demonstrated that a rabbit polyclonal antibody to monocyte chemotaxis and activating factor (MCAF) inhibited all monocyte chemotaxis by greater than 80%. Keratinocytes were metabolically labeled with 35S-cysteine/methionine, and after 16 h incubation the supernatants immunoprecipitated with the same anti-MCAF antibody. MCAF was detected as a protein doublet of 12 and 9 kDa only in IFN-gamma-treated (100 U/ml) keratinocyte supernatants. Incubation with IFN-gamma and TNF-alpha (250 U/ml) in combination resulted in increased production of MCAF protein. By Northern blot analysis, MCAF mRNA was constitutively expressed in keratinocytes and upregulated only in the presence of IFN-gamma. TNF-alpha, IL-1 beta, transforming growth factor-beta and phorbol esters had no positive or negative influence on MCAF mRNA. These studies demonstrate that biologically active MCAF is elaborated by human epidermal keratinocytes upon activation by IFN-gamma, a cytokine also required for the induction of adherence between monocytes and keratinocytes. Keratinocyte-derived MCAF is likely to be important in the regulation of cutaneous monocyte trafficking and may also be responsible for the recruitment of Langerhans cells and dermal dendrocytes, which share many phenotypic features with monocytes/macrophages, to their anatomic locations in skin.
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Zuniga, Kameel, Neda Ghousifam, John Sansalone, Kris Senecal, Mark Van Dyke, and Marissa Nichole Rylander. "Keratin Promotes Differentiation of Keratinocytes Seeded on Collagen/Keratin Hydrogels." Bioengineering 9, no. 10 (October 15, 2022): 559. http://dx.doi.org/10.3390/bioengineering9100559.
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Keratinocytes undergo a complex process of differentiation to form the stratified stratum corneum layer of the skin. In most biomimetic skin models, a 3D hydrogel fabricated out of collagen type I is used to mimic human skin. However, native skin also contains keratin, which makes up 90% of the epidermis and is produced by the keratinocytes present. We hypothesized that the addition of keratin (KTN) in our collagen hydrogel may aid in the process of keratinocyte differentiation compared to a pure collagen hydrogel. Keratinocytes were seeded on top of a 100% collagen or 50/50 C/KTN hydrogel cultured in either calcium-free (Ca-free) or calcium+ (Ca+) media. Our study demonstrates that the addition of keratin and calcium in the media increased lysosomal activity by measuring the glucocerebrosidase (GBA) activity and lysosomal distribution length, an indication of greater keratinocyte differentiation. We also found that the presence of KTN in the hydrogel also increased the expression of involucrin, a differentiation marker, compared to a pure collagen hydrogel. We demonstrate that a combination (i.e., containing both collagen and kerateine or “C/KTN”) hydrogel was able to increase keratinocyte differentiation compared to a pure collagen hydrogel, and the addition of calcium further increased the differentiation of keratinocytes. This multi-protein hydrogel shows promise in future models or treatments to increase keratinocyte differentiation into the stratum corneum.
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Pfeifer, João P. H., Vitor H. Santos, Gustavo Rosa, Jaqueline B. Souza, Marcos Jun Watanabe, Carlos E. Fonseca-Alves, Elenice Deffune, and Ana L. G. Alves. "Isolation, cultivation and immunofluorescence characterization of lamellar keratinocytes from equine hoof by using explants." Pesquisa Veterinária Brasileira 39, no. 4 (April 2019): 292–98. http://dx.doi.org/10.1590/1678-5150-pvb-5747.
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ABSTRACT: The importance of the hoof to the horse health is clear, and the current knowledge regarding the cellular aspects of hoof keratinocytes is poor. Studies on equine keratinocyte culture are scarce. Developing keratinocyte cultures in vitro is a condition for studies on molecular biology, cell growth and differentiation. Some methods have already been established, such as those for skin keratinocyte culture. However, few methodologies are found for lamellar keratinocytes. The objective of this study was to standardize the equine hoof keratinocyte isolation and cultivation, and then characterize the cell immunophenotype. For this, the primary culture method used was through explants obtained from three regions of the equine hoof (medial dorsal, dorsal, and lateral dorsal). After the cell isolation and cultivation, the cell culture and its explants were stained with anti-pan cytokeratin (pan-CK) (AE1/AE3), vimentin (V9), p63 (4A4), and Ki-67 (MIB-1) antibodies. Cells were grown to third passage, were positive for pan-CK, p63 and Ki-67, and few cells had vimentin positive expression. As for the explants, the epidermal laminae were not stained for vimentin or Ki-67. However, some cells presented positive pan-CK and p63 expression. This study demonstrated the viability of lamellar explants of equine hooves as a form of isolating keratinocytes in primary cultures, as well as characterized the proliferation ability of such keratinocytes in monolayers.
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Zarkoob, Hoda, Sathivel Chinnathambi, John C. Selby, and Edward A. Sander. "Substrate deformations induce directed keratinocyte migration." Journal of The Royal Society Interface 15, no. 143 (June 2018): 20180133. http://dx.doi.org/10.1098/rsif.2018.0133.
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Cell migration is an essential part of many (patho)physiological processes, including keratinocyte re-epithelialization of healing wounds. Physical forces and mechanical cues from the wound bed (in addition to biochemical signals) may also play an important role in the healing process. Previously, we explored this possibility and found that polyacrylamide (PA) gel stiffness affected human keratinocyte behaviour and that mechanical deformations in soft (approx. 1.2 kPa) PA gels produced by neighbouring cells appeared to influence the process of de novo epithelial sheet formation. To clearly demonstrate that keratinocytes do respond to such deformations, we conducted a series of experiments where we observed the response of single keratinocytes to a prescribed local substrate deformation that mimicked a neighbouring cell or evolving multicellular aggregate via a servo-controlled microneedle. We also examined the effect of adding either Y27632 or blebbistatin on cell response. Our results indicate that keratinocytes do sense and respond to mechanical signals comparable to those that originate from substrate deformations imposed by neighbouring cells, a finding that could have important implications for the process of keratinocyte re-epithelialization that takes place during wound healing. Furthermore, the Rho/ROCK pathway and the engagement of NM II are both essential to substrate deformation-directed keratinocyte migration.
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Zhang, Chen, ZhenLai Zhu, JiXin Gao, LuTing Yang, ErLe Dang, Hui Fang, Shuai Shao, et al. "Plasma exosomal miR-375-3p regulates mitochondria-dependent keratinocyte apoptosis by targeting XIAP in severe drug-induced skin reactions." Science Translational Medicine 12, no. 574 (December 16, 2020): eaaw6142. http://dx.doi.org/10.1126/scitranslmed.aaw6142.
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Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are severe drug-induced cutaneous reactions characterized by keratinocyte apoptosis. Exosomes are nanometer-sized membranous vesicles in body fluids. They contain functional proteins, mRNAs, and miRNAs, which induce immune dysfunction and influence disease progression. However, their roles and mechanisms in SJS/TEN remain unknown. Our results demonstrate that exosomes isolated from the plasma of patients with SJS/TEN were 30 to 200 nm in diameter and expressed CD9, CD63, CD81, and TSG101 exosome marker proteins. miR-375-3p was markedly up-regulated in 35 patients with SJS/TEN and correlated with clinical severity. Plasma exosomes were internalized by human primary keratinocytes and promoted keratinocyte apoptosis in vitro. Furthermore, miR-375-3p overexpression promoted intrinsic (mitochondria-dependent) apoptosis of human primary keratinocytes via down-regulation of the X-linked inhibitor of apoptosis protein (XIAP), a key apoptosis regulator in primary human keratinocytes. In sum, our study indicates that the circulating exosomal miR-375-3p enters keratinocytes, down-regulates XIAP, and induces keratinocyte apoptosis in patients with SJS/TEN.
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Saito, Nao, Hongjiang Qiao, Teruki Yanagi, Satoru Shinkuma, Keiko Nishimura, Asuka Suto, Yasuyuki Fujita, et al. "An annexin A1–FPR1 interaction contributes to necroptosis of keratinocytes in severe cutaneous adverse drug reactions." Science Translational Medicine 6, no. 245 (July 16, 2014): 245ra95. http://dx.doi.org/10.1126/scitranslmed.3008227.
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Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are life-threatening, cutaneous adverse drug reactions that are accompanied by keratinocyte cell death. Dead keratinocytes from SJS/TEN lesions exhibited necrosis, by morphological criteria. Supernatant from peripheral blood mononuclear cells (PBMCs) that had been exposed to the causative drug from patients with SJS/TEN induced the death of SJS/TEN keratinocytes, whereas supernatant from PBMCs of patients with ordinary drug skin reactions (ODSRs) exposed to the same drug did not. Keratinocytes from ODSR patients or from healthy controls were unaffected by supernatant from SJS/TEN or ODSR PBMCs. Mass spectrometric analysis identified annexin A1 as a key mediator of keratinocyte death; depletion of annexin A1 by a specific antibody diminished supernatant cytotoxicity. The necroptosis-mediating complex of RIP1 and RIP3 was indispensable for SJS/TEN supernatant–induced keratinocyte death, and SJS/TEN keratinocytes expressed abundant formyl peptide receptor 1 (FPR1), the receptor for annexin A1, whereas control keratinocytes did not. Inhibition of necroptosis completely prevented SJS/TEN-like responses in a mouse model of SJS/TEN. Our results demonstrate that a necroptosis pathway, likely mediated by annexin 1 acting through the FPR1 receptor, contributes to SJS/TEN.
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Di Fusco, Davide, Carmine Stolfi, Antonio Di Grazia, Vincenzo Dinallo, Federica Laudisi, Irene Marafini, Alfredo Colantoni, Ivan Monteleone, and Giovanni Monteleone. "Albendazole negatively regulates keratinocyte proliferation." Clinical Science 134, no. 7 (April 2020): 907–20. http://dx.doi.org/10.1042/cs20191215.
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Abstract Background: Increased keratinocyte proliferation occurs in the skin of psoriatic patients and is supposed to play a role in the pathogenesis of this disorder. Compounds interfering with keratinocyte proliferation could be useful in the management of psoriatic patients. Aim: To investigate whether albendazole, an anti-helmintic drug that regulates epithelial cell function in various systems, inhibits keratinocyte proliferation in models of psoriasis. Methods: Aldara-treated mice received daily topical application of albendazole. Keratinocyte proliferation and keratin (K) 6 and K16 expression were evaluated by immunohistochemistry and Western blotting and inflammatory cells/mediators were analysed by immunohistochemistry and real-time PCR. In human keratinocytes (HEKa and HaCaT) treated with albendazole, cell cycle and proliferation, keratins and cell cycle-associated factors were evaluated by flow cytometry, colorimetric assay and Western blotting respectively. Results: Aldara-treated mice given albendazole exhibited reduced epidermal thickness, decreased number of proliferating keratinocytes and K6/K16 expression. Reduction of CD3- and Ly6G-positive cells in the skin of albendazole-treated mice associated with inhibition of IL-6, TNF-α, IL-1β, IL-17A, IL-36, CCL17, CXCL1, CXCL2 and CXCL5 expression. Treatment of keratinocytes with albendazole reduced K6/K16 expression and reversibly inhibited cell growth by promoting accumulation of cells in S-phase. This phenomenon was accompanied by down-regulation of CDC25A, a phosphatase regulating progression of cell cycle through S-phase, and PKR-dependent hyper-phosphorylation of eIF2α, an inhibitor of CDC25 translation. In Aldara-treated mice, albendazole activated PKR, enhanced eIF2α phosphorylation and reduced CDC25A expression. Conclusions: Data show that albendazole inhibits keratinocyte proliferation and exerts therapeutic effect in a murine model of psoriasis.
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Raife, TJ, EM Demetroulis, and SR Lentz. "Regulation of thrombomodulin expression by all-trans retinoic acid and tumor necrosis factor-alpha: differential responses in keratinocytes and endothelial cells." Blood 88, no. 6 (September 15, 1996): 2043–49. http://dx.doi.org/10.1182/blood.v88.6.2043.bloodjournal8862043.
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Thrombomodulin is a cell-surface anticoagulant glycoprotein expressed by vascular endothelial cells and epidermal keratinocytes. Thrombomodulin expression in endothelial cells is regulated by retinoic acid and tumor necrosis factor-alpha (TNF), agents that also modulate epidermal differentiation. We examined thrombomodulin function and regulation of thrombomodulin expression by all-trans retinoic acid (ATRA) and TNF in human keratinocytes and endothelial cells. Untreated keratinocytes and endothelial cells expressed thrombomodulin of comparable activity and apparent thrombin affinity. Incubation of keratinocytes with 10 mumol/L ATRA for 24 hours increased thrombomodulin activity 5.4 +/- 0.9-fold (mean +/- SE), with equivalent increases observed in thrombomodulin protein (5.5 +/- 2.1-fold) and mRNA (4.2 +/- 1.2-fold). Incubation of keratinocytes with 1.0 nmol/L TNF markedly increased expression of keratinocyte transglutaminase, but had no effect on thrombomodulin activity, protein, or mRNA. In endothelial cells, ATRA produced a small increase in thrombomodulin activity (1.9 +/- 0.1-fold), and incubation with TNF for 24 hours decreased thrombomodulin activity 83% +/- 7%. The activity profile of keratinocyte thrombomodulin exhibited a distinct maximum near 1.0 mmol/L Ca2+. These results demonstrate that keratinocyte thrombomodulin is regulated by retinoids and Ca2+, but not by TNF, and that regulation of thrombomodulin expression differs in keratinocytes and endothelial cells.
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Gwak, So-Jung, Sang-Soo Kim, Kyungeun Sung, Joungho Han, Cha Yong Choi, and Byung-Soo Kim. "Synergistic Effect of Keratinocyte Transplantation and Epidermal Growth Factor Delivery on Epidermal Regeneration." Cell Transplantation 14, no. 10 (November 2005): 809–17. http://dx.doi.org/10.3727/000000005783982521.
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Both keratinocyte transplantation and epidermal growth factor (EGF) delivery stimulate epidermal regeneration. In this study, we hypothesized that the combined therapy of keratinocyte transplantation and EGF delivery accelerates epidermal regeneration compared to the single therapy of either keratinocyte transplantation or EGF delivery. To test this hypothesis, we utilized fibrin matrix as a keratinocyte/EGF delivery vehicle for epidermal regeneration. Full-thickness wounds were created on the dorsum of athymic mice, and human keratinocytes and EGF in fibrin matrix were sprayed onto the wounds to regenerate epidermal layers (group 1). As controls, human keratinocytes in fibrin matrix (group 2), EGF in fibrin matrix (group 3), or fibrin matrix alone (group 4) was sprayed onto the wounds. Spraying keratinocytes suspended in fibrin matrix did not affect the keratinocyte viability, as the cell viabilities before and after spraying were not different. EGF was released from fibrin matrix for 3 days. The wounds were analyzed with histology and immunohistochemistry at 1 and 3 weeks after treatments. Compared with the control groups, initial wound closure rate was highest in group 1. Histological analyses indicated that group 1 exhibited faster and better epidermal regeneration than the other groups. Immunohistochemical analyses showed that regenerated epithelium in groups 1 and 2 stained positively for human involucrin at 3 weeks, whereas the tissue sections of the groups 3 and 4 stained negatively. Human laminin was detected at the dermal–epidermal junction of the regenerated tissues in groups 1 and 2 at 3 weeks and was not detected in groups 3 and 4. The epidermal thickness of the regenerated tissues in group 1 was significantly thicker than that of the other groups at all time points. These results suggest that the combined therapy of keratinocyte transplantation and EGF delivery is more efficacious for epidermal regeneration than each separate therapy alone.
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Shirai, Yasuhito, Shoko Morioka, Megumi Sakuma, Ken-ichi Yoshino, Chihiro Otsuji, Norio Sakai, Kaori Kashiwagi, et al. "Direct binding of RalA to PKCη and its crucial role in morphological change during keratinocyte differentiation." Molecular Biology of the Cell 22, no. 8 (April 15, 2011): 1340–52. http://dx.doi.org/10.1091/mbc.e10-09-0754.
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During differentiation, keratinocytes undergo a dramatic shape change from small and round to large and flat, in addition to production of proteins necessary for the formation of epidermis. It has been shown that protein kinase C (PKC) η is crucial for keratinocyte differentiation. However, its role in this process has yet to be fully elucidated. Here, we show that catalytic activity is not necessary for enlarged and flattened morphology of human keratinocytes induced by overexpression of PKCη, although it is important for gene expression of the marker proteins. In addition, we identify the small G protein RalA as a binding partner of PKCη, which binds to the C1 domain, an indispensable region for the morphological change. The binding led activation of RalA and actin depolymerization associated with keratinocyte differentiation. siRNA techniques proved that RalA is involved in not only the keratinocyte differentiation induced by PKCη overexpression but also normal keratinocyte differentiation induced by calcium and cholesterol sulfate. These results provide a new insight into the molecular mechanism of cytoskeletal regulation leading to drastic change of cell shape.
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Tolg, Cornelia, Muhan Liu, Katelyn Cousteils, Patrick Telmer, Khandakar Alam, Jenny Ma, Leslie Mendina, James B. McCarthy, Vincent L. Morris, and Eva A. Turley. "Cell-specific expression of the transcriptional regulator RHAMM provides a timing mechanism that controls appropriate wound re-epithelialization." Journal of Biological Chemistry 295, no. 16 (March 12, 2020): 5427–48. http://dx.doi.org/10.1074/jbc.ra119.010002.
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Prevention of aberrant cutaneous wound repair and appropriate regeneration of an intact and functional integument require the coordinated timing of fibroblast and keratinocyte migration. Here, we identified a mechanism whereby opposing cell-specific motogenic functions of a multifunctional intracellular and extracellular protein, the receptor for hyaluronan-mediated motility (RHAMM), coordinates fibroblast and keratinocyte migration speed and ensures appropriate timing of excisional wound closure. We found that, unlike in WT mice, in Rhamm-null mice, keratinocyte migration initiates prematurely in the excisional wounds, resulting in wounds that have re-surfaced before the formation of normal granulation tissue, leading to a defective epidermal architecture. We also noted aberrant keratinocyte and fibroblast migration in the Rhamm-null mice, indicating that RHAMM suppresses keratinocyte motility but increases fibroblast motility. This cell context–dependent effect resulted from cell-specific regulation of extracellular signal-regulated kinase 1/2 (ERK1/2) activation and expression of a RHAMM target gene encoding matrix metalloprotease 9 (MMP-9). In fibroblasts, RHAMM promoted ERK1/2 activation and MMP-9 expression, whereas in keratinocytes, RHAMM suppressed these activities. In keratinocytes, loss of RHAMM function or expression promoted epidermal growth factor receptor–regulated MMP-9 expression via ERK1/2, which resulted in cleavage of the ectodomain of the RHAMM partner protein CD44 and thereby increased keratinocyte motility. These results identify RHAMM as a key factor that integrates the timing of wound repair by controlling cell migration.
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Fang, Wei-Cheng, and Cheng-Che E. Lan. "The Epidermal Keratinocyte as a Therapeutic Target for Management of Diabetic Wounds." International Journal of Molecular Sciences 24, no. 5 (February 21, 2023): 4290. http://dx.doi.org/10.3390/ijms24054290.
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Diabetes mellitus (DM) is an important cause of chronic wounds and non-traumatic amputation. The prevalence and number of cases of diabetic mellitus are increasing worldwide. Keratinocytes, the outermost layer of the epidermis, play an important role in wound healing. A high glucose environment may disrupt the physiologic functions of keratinocytes, resulting in prolonged inflammation, impaired proliferation, and the migration of keratinocytes and impaired angiogenesis. This review provides an overview of keratinocyte dysfunctions in a high glucose environment. Effective and safe therapeutic approaches for promoting diabetic wound healing can be developed if molecular mechanisms responsible for keratinocyte dysfunction in high glucose environments are elucidated.
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Scheau, Cristian, Constantin Caruntu, and Ana Caruntu. "Current Approach and Future Directions in the Diagnosis and Prognosis of Keratinocyte Carcinomas." Journal of Clinical Medicine 12, no. 12 (June 11, 2023): 3974. http://dx.doi.org/10.3390/jcm12123974.
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Rodeck, U., M. Jost, C. Kari, D. T. Shih, R. M. Lavker, D. L. Ewert, and P. J. Jensen. "EGF-R dependent regulation of keratinocyte survival." Journal of Cell Science 110, no. 2 (January 15, 1997): 113–21. http://dx.doi.org/10.1242/jcs.110.2.113.
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Tissue organization and maintenance within multicellular organisms is in part dependent on the ability of cells to undergo programmed cell death or apoptosis. Conversely, disruption of cell death pathways often is associated with tumor development. At present, the molecular control of apoptosis in epithelial cells is poorly understood. Here we describe evidence linking epidermal growth factor-receptor (EGF-R) activation to survival of normal human keratinocytes in culture. Inhibition of EGF-R activation by an anti-EGF-R antagonistic monoclonal antibody (mAb 425), followed by detachment of keratinocytes from the substratum, induced extensive death with several features of apoptosis in keratinocyte cultures. Other, non-epithelial normal human cells including melanocytes and fibroblasts, did not show this effect. Similar to EGF-R blockade by mAb 425, inhibition of the EGF-R tyrosine kinase activity using tyrphostin AG1478 resulted in lack of attachment and extensive cell death upon passaging. Attachment to keratinocyte-derived ECM partially resuced mAb 425-treated keratinocytes from cell death, indicating that adhesion-dependent and EGF-R-dependent signal transduction pathways serve partially overlapping but not redundant roles in supporting keratinocyte survival.
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Serezani, Ana Paula, Gunseli Bozdogan, Daniel Walsh, Purna Krishnamurthy, Sarita Sehra, Shreevrat Goenka, Dan Spandau, and Mark Kaplan. "Limited dependence of IL-4-regulated keratinocyte differentiation and gene expression on STAT6 (HYP6P.261)." Journal of Immunology 192, no. 1_Supplement (May 1, 2014): 118.6. http://dx.doi.org/10.4049/jimmunol.192.supp.118.6.
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Abstract Epidermal Differentiation Complex (EDC) genes are critical for barrier function in the skin, and Th2 cytokines that promote allergic inflammation inhibit EDC gene expression in keratinocytes. To further define the effects of the atopic immune response on keratinocyte differentiation, we investigated IL-4-mediated changes in gene expression in human keratinocytes via RNA-seq. Human keratinocytes were differentiated for 2 days with calcium chloride and then stimulated with IL-4 for 24 hours, or keratinocytes were differentiated with calcium chloride and chronically stimulated with IL-4 for 5 days. At day 2, IL-4 increased expression of genes associated with cell cycle, and decreased expression of genes involved with cell adhesion. At day 5 of differentiation, IL-4-stimulated keratinocytes showed increased expression of genes involved with cell projection and adhesion, and decreased expression of genes that participate in wound repair, epidermis development and host defense. STAT6 inhibition by siRNA significantly altered the expression of less than 20% of genes induced by IL-4. Thus, although IL-4 dramatically alters keratinocyte gene expression and differentiation, only a subset of this response is dependent upon STAT6, and eliminating STAT6 expression has IL-4-independent changes in gene expression. This suggests that keratinocyte gene regulation by IL-4 requires additional molecular pathways.
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Jameson, Julie, Anne Costanzo, and Kristen Taylor. "Obesity compromises the ability of skin γδ T cells to regulate skin homeostasis and barrier function (166.6)." Journal of Immunology 186, no. 1_Supplement (April 1, 2011): 166.6. http://dx.doi.org/10.4049/jimmunol.186.supp.166.6.
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Abstract Within the epidermis of the skin resides a unique population of γδ T lymphocytes that play key roles in skin homeostasis and wound repair. During obesity and related metabolic disease the skin-resident γδ T lymphocytes become dysfunctional due the chronic inflammatory environment. This dysfunction contributes to the non-healing wounds associated with disease. We are now investigating how skin γδ T cell dysfunction impacts normal keratinocyte homeostasis. Interestingly, in lean mice we have determined that the number of keratinocytes in the epidermis is directly proportional to the number of closely neighboring γδ T cells. Once obesity and related metabolic disease ensues, the keratinocyte numbers resemble areas that are devoid of γδ T cells in lean mice and keratinocytes are unable to maintain normal homeostatic numbers. Furthermore, keratinocytes in obese mice exhibit diminished proliferation and accelerated differentiation. This results in a dysregulated epidermis in which fewer basal keratinocytes express the proliferation marker keratin 5 and more exhibit early expression of the differentiation marker keratin 1. Additionally, skin γδ T cells downregulate CD103 in obese mice, while keratinocytes alter expression of its ligand E-cadherin. Together these studies demonstrate that dysfunctional γδ T cells in the epidermis of obese mice contribute to impaired keratinocyte homeostasis ultimately resulting in compromised skin barrier function.
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Chodakewitz, J. A., J. Lacy, S. E. Edwards, N. Birchall, and D. L. Coleman. "Macrophage colony-stimulating factor production by murine and human keratinocytes. Enhancement by bacterial lipopolysaccharide." Journal of Immunology 144, no. 6 (March 15, 1990): 2190–96. http://dx.doi.org/10.4049/jimmunol.144.6.2190.
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Abstract CSF have a broad range of effects on differentiated cells outside the bone marrow. Site-specific elaboration of these factors may influence local immune reactions. Keratinocytes have been demonstrated to produce a number of immunoactive cytokines, including factors capable of modifying macrophage function. We have previously identified at least two products of keratinocytes that induce DNA synthesis by elicited peritoneal macrophages; one factor has been identified as granulocyte-macrophage CSF. In the present study, the second keratinocyte product has been characterized and identified as macrophage-CSF (M-CSF). Conditioned media from cultures of normal human keratinocytes and the transformed murine keratinocyte cell line PAM 212 induce formation of macrophage colonies in soft agar as well as dose-dependent proliferation of the M-CSF-dependent cell line BAC1.2F5. The bioactivity in both assays is blocked by neutralizing anti-M-CSF antibody. Western blot analysis of cell lysates from both PAM 212 and normal human keratinocytes demonstrates multiple molecular mass forms of M-CSF (45 to 98 kDa). Northern blot analysis (PAM 212 cells) and in situ hybridization (normal keratinocytes) demonstrate expression of M-CSF mRNA. Stimulation of keratinocytes with LPS increases M-CSF synthesis as measured both by bioactivity and level of mRNA expression. Thus, both murine and human keratinocytes produce M-CSF in vitro. Furthermore, production of keratinocyte-derived M-CSF is increased by bacterial LPS. CSF production by keratinocytes may play an important role in regulating the cutaneous immune response.
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Kuhn, Ulrich, Atsushi Terunuma, Wolfgang Pfutzner, Ruth Ann Foster, and Jonathan C. Vogel. "In Vivo Assessment of Gene Delivery to Keratinocytes by Lentiviral Vectors." Journal of Virology 76, no. 3 (February 1, 2002): 1496–504. http://dx.doi.org/10.1128/jvi.76.3.1496-1504.2002.
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ABSTRACT For skin gene therapy, introduction of a desired gene into keratinocyte progenitor or stem cells could overcome the problem of achieving persistent gene expression in a significant percentage of keratinocytes. Although keratinocyte stem cells have not yet been completely characterized and purified for gene targeting purposes, lentiviral vectors may be superior to retroviral vectors at gene introduction into these stem cells, which are believed to divide and cycle slowly. Our initial in vitro studies demonstrate that lentiviral vectors are able to efficiently transduce nondividing keratinocytes, unlike retroviral vectors, and do not require the lentiviral accessory genes for keratinocyte transduction. When lentiviral vectors expressing green fluorescent protein (GFP) were directly injected into the dermis of human skin grafted onto immunocompromised mice, transduction of dividing basal and nondividing suprabasal keratinocytes could be demonstrated, which was not the case when control retroviral vectors were used. However, flow cytometry analysis demonstrated low transduction efficiency, and histological analysis at later time points provided no evidence for progenitor cell targeting. In an alternative in vivo method, human keratinocytes were transduced in tissue culture (ex vivo) with either lentiviral or retroviral vectors and grafted as skin equivalents onto immunocompromised mice. GFP expression was analyzed in these human skin grafts after several cycles of epidermal turnover, and both the lentiviral and retroviral vector-transduced grafts had similar percentages of GFP-expressing keratinocytes. This ex vivo grafting study provides a good in vivo assessment of gene introduction into progenitor cells and suggests that lentiviral vectors are not necessarily superior to retroviral vectors at introducing genes into keratinocyte progenitor cells during in vitro culture.
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Maarof, Manira, Shiplu Roy Chowdhury, Aminuddin Saim, Ruszymah Bt Hj Idrus, and Yogeswaran Lokanathan. "Concentration Dependent Effect of Human Dermal Fibroblast Conditioned Medium (DFCM) from Three Various Origins on Keratinocytes Wound Healing." International Journal of Molecular Sciences 21, no. 8 (April 22, 2020): 2929. http://dx.doi.org/10.3390/ijms21082929.
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Fibroblasts secrete many essential factors that can be collected from fibroblast culture medium, which is termed dermal fibroblast conditioned medium (DFCM). Fibroblasts isolated from human skin samples were cultured in vitro using the serum-free keratinocyte-specific medium (Epilife (KM1), or define keratinocytes serum-free medium, DKSFM (KM2) and serum-free fibroblast-specific medium (FM) to collect DFCM-KM1, DFCM-KM2, and DFCM-FM, respectively). We characterised and evaluated the effects of 100–1600 µg/mL DFCM on keratinocytes based on attachment, proliferation, migration and gene expression. Supplementation with 200–400 µg/mL keratinocyte-specific DFCM-KM1 and DFCM-KM2 enhanced the attachment, proliferation and migration of sub-confluent keratinocytes, whereas 200–1600 µg/mL DFCM-FM significantly increased the healing rate in the wound healing assay, and 400–800 µg/mL DFCM-FM was suitable to enhance keratinocyte attachment and proliferation. A real-time (RT2) profiler polymerase chain reaction (PCR) array showed that 42 genes in the DFCM groups had similar fold regulation compared to the control group and most of the genes were directly involved in wound healing. In conclusion, in vitro keratinocyte re-epithelialisation is supported by the fibroblast-secreted proteins in 200–400 µg/mL DFCM-KM1 and DFCM-KM2, and 400–800 µg/mL DFCM-FM, which could be useful for treating skin injuries.
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Sharlow, E. R., C. S. Paine, L. Babiarz, M. Eisinger, S. Shapiro, and M. Seiberg. "The protease-activated receptor-2 upregulates keratinocyte phagocytosis." Journal of Cell Science 113, no. 17 (September 1, 2000): 3093–101. http://dx.doi.org/10.1242/jcs.113.17.3093.
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The protease-activated receptor-2 (PAR-2) belongs to the family of seven transmembrane domain receptors, which are activated by the specific enzymatic cleavage of their extracellular amino termini. Synthetic peptides corresponding to the tethered ligand domain (SLIGRL in mouse, SLIGKV in human) can activate PAR-2 without the need for receptor cleavage. PAR-2 activation is involved in cell growth, differentiation and inflammatory processes, and was shown to affect melanin and melanosome ingestion by human keratinocytes. Data presented here suggest that PAR-2 activation may regulate human keratinocyte phagocytosis. PAR-2 activation by trypsin, SLIGRL or SLIGKV increased the ability of keratinocytes to ingest fluorescently labeled microspheres or E. coli K-12 bioparticles. This PAR-2 mediated increase in keratinocyte phagocytic capability correlated with an increase in actin polymerization and *-actinin reorganization, cell surface morphological changes and increased soluble protease activity. Moreover, addition of serine protease inhibitors downmodulated both the constitutive and the PAR-2 mediated increases in phagocytosis, suggesting that serine proteases mediate this functional activity in keratinocytes. PAR-2 involvement in keratinocyte phagocytosis is a novel function for this receptor.
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Schmidt, Morna F., Maria Feoktistova, Diana Panayotova-Dimitrova, Ramona A. Eichkorn, and Amir S. Yazdi. "Pitfalls in the Application of Dispase-Based Keratinocyte Dissociation Assay for In Vitro Analysis of Pemphigus Vulgaris." Vaccines 10, no. 2 (January 28, 2022): 208. http://dx.doi.org/10.3390/vaccines10020208.
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Pemphigus vulgaris (PV) is a chronic, life-altering autoimmune disease due to the production of anti-desmoglein antibodies causing the loss of cell–cell adhesion in keratinocytes (acantholysis) and blister formation in both skin and mucous membranes. The dispase-based keratinocyte dissociation assay (DDA) is the method of choice to examine the pathogenic effect of antibodies and additional co-stimuli on cell adhesion in vitro. Despite its widespread use, there is a high variability of experimental conditions, leading to inconsistent results. In this paper, we identify and discuss pitfalls in the application of DDA, including generation of a monolayer with optimized density, appropriate culturing conditions to obtain said monolayer, application of mechanical stress in a standardized manner, and performing consistent data processing. Importantly, we describe a detailed protocol for a successful and reliable DDA and the respective ideal conditions for three different types of human keratinocytes: (1) primary keratinocytes, (2) the HaCaT spontaneously immortalized keratinocyte cell line, and (3) the recently characterized HaSKpw spontaneously immortalized keratinocyte cell line. Our study provides detailed protocols which guarantee intra- and inter-experimental comparability of DDA.
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de Jong, Sarah Jill, Amandine Créquer, Irina Matos, David Hum, Vignesh Gunasekharan, Lazaro Lorenzo, Fabienne Jabot-Hanin, et al. "The human CIB1–EVER1–EVER2 complex governs keratinocyte-intrinsic immunity to β-papillomaviruses." Journal of Experimental Medicine 215, no. 9 (August 1, 2018): 2289–310. http://dx.doi.org/10.1084/jem.20170308.
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Patients with epidermodysplasia verruciformis (EV) and biallelic null mutations of TMC6 (encoding EVER1) or TMC8 (EVER2) are selectively prone to disseminated skin lesions due to keratinocyte-tropic human β-papillomaviruses (β-HPVs), which lack E5 and E8. We describe EV patients homozygous for null mutations of the CIB1 gene encoding calcium- and integrin-binding protein-1 (CIB1). CIB1 is strongly expressed in the skin and cultured keratinocytes of controls but not in those of patients. CIB1 forms a complex with EVER1 and EVER2, and CIB1 proteins are not expressed in EVER1- or EVER2-deficient cells. The known functions of EVER1 and EVER2 in human keratinocytes are not dependent on CIB1, and CIB1 deficiency does not impair keratinocyte adhesion or migration. In keratinocytes, the CIB1 protein interacts with the HPV E5 and E8 proteins encoded by α-HPV16 and γ-HPV4, respectively, suggesting that this protein acts as a restriction factor against HPVs. Collectively, these findings suggest that the disruption of CIB1–EVER1–EVER2-dependent keratinocyte-intrinsic immunity underlies the selective susceptibility to β-HPVs of EV patients.
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Maas-Szabowski, N., A. Shimotoyodome, and N. E. Fusenig. "Keratinocyte growth regulation in fibroblast cocultures via a double paracrine mechanism." Journal of Cell Science 112, no. 12 (June 15, 1999): 1843–53. http://dx.doi.org/10.1242/jcs.112.12.1843.
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Epithelial-mesenchymal interactions play an important role in regulating tissue homeostasis and repair. For skin, the regulatory mechanisms of epidermal-dermal interactions were studied in cocultures of normal human epidermal keratinocytes (NEK) and dermal fibroblasts (HDF) rendered postmitotic by alpha-irradiation (HDFi). The expression kinetics of different cytokines and their receptors with presumed signalling function in skin were determined at the RNA and protein level in mono- and cocultured NEK and HDFi. In cocultured HDFi, mRNA and protein synthesis of keratinocyte growth factor (KGF) (FGF-7) was strongly enhanced, whereas in cocultured keratinocytes interleukin (IL)-1alpha and -1beta mRNA expression increased compared to monocultures. Thus we postulated that IL-1, which had no effect on keratinocyte proliferation, induced in fibroblasts the expression of factors stimulating keratinocyte proliferation, such as KGF. The functional significance of this reciprocal modulation was substantiated by blocking experiments. Both IL-1alpha and -1beta-neutralizing antibodies and IL-1 receptor antagonist significantly reduced keratinocyte proliferation supposedly through abrogation of KGF production, because IL-1 antibodies blocked the induced KGF production. These data indicate a regulation of keratinocyte growth by a double paracrine mechanism through release of IL-1 which induces KGF in cocultured fibroblasts. Thus IL-1, in addition to its proinflammatory function in skin, may play an essential role in regulating tissue homeostasis.
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Bailey, Lakiea J., Vivek Choudhary, and Wendy B. Bollag. "Possible Role of Phosphatidylglycerol-Activated Protein Kinase C-βII in Keratinocyte Differentiation." Open Dermatology Journal 11, no. 1 (October 24, 2017): 59–71. http://dx.doi.org/10.2174/1874372201711010059.
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Background: The epidermis is a continuously regenerating tissue maintained by a balance between proliferation and differentiation, with imbalances resulting in skin disease. We have previously found that in mouse keratinocytes, the lipid-metabolizing enzyme phospholipase D2 (PLD2) is associated with the aquaglyceroporin, aquaporin 3 (AQP3), an efficient transporter of glycerol. Our results also show that the functional interaction of AQP3 and PLD2 results in increased levels of phosphatidylglycerol (PG) in response to an elevated extracellular calcium level, which triggers keratinocyte differentiation. Indeed, we showed that directly applying PG can promote keratinocyte differentiation. Objective: We hypothesized that the differentiative effects of this PLD2/AQP3/PG signaling cascade, in which AQP3 mediates the transport of glycerol into keratinocytes followed by its PLD2-catalyzed conversion to PG, are mediated by protein kinase CβII (PKCβII), which contains a PG-binding domain in its carboxy-terminus. Method: To test this hypothesis we used quantitative RT-PCR, western blotting and immunocytochemistry. Results: We first verified the presence of PKCβII mRNA and protein in mouse keratinocytes. Next, we found that autophosphorylated (activated) PKCβII was redistributed upon treatment of keratinocytes with PG. In the unstimulated state phosphoPKCβII was found in the cytosol and perinuclear area; treatment with PG resulted in enhanced phosphoPKCβII localization in the perinuclear area. PG also induced translocation of phosphoPKCβII to the plasma membrane. In addition, we observed that overexpression of PKCβII enhanced calcium- and PG-induced keratinocyte differentiation without affecting calcium-inhibited keratinocyte proliferation. Conclusion: These results suggest that the PG produced by the PLD2/AQP3 signaling module may function by activating PKCβII.
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Liu, Jianwei, Fanghui Geng, Hongying Sun, Xiaxia Wang, Hui Zhang, Qiaozhen Yang, and Jie Zhang. "Candida albicans induces TLR2/MyD88/NF-κB signaling and inflammation in oral lichen planus-derived keratinocytes." Journal of Infection in Developing Countries 12, no. 09 (September 30, 2018): 780–86. http://dx.doi.org/10.3855/jidc.8062.
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Introduction: The risk of oral lichen planus (OLP), a chronic inflammatory oral mucosal disease, becoming malignant increases by 21-fold in patients with fungal infection. This study examined the impact of Candida albicans exposure on Toll-like receptor (TLR) signaling in primary keratinocyte cultures obtained from OLP patients.
Methodology: Following co-culture of primary OLP keratinocyte cultures with C. albicans for 24 hours, inflammatory cytokine concentrations were determined by ELISA. TLR2, MyD88, and NF-κBp65 mRNA and protein expression were assessed using quantitative RT-PCR and Western blot analyses, respectively. Keratinocyte apoptosis was also determined by flow cytometry.
Results: IL-10, IL-8, IL-2, and TNF-ɑ levels were significantly higher following co-culture with C. albicans (all p ≤ 0.034). MyD88, NF-κB p65, and TLR2 mRNA (all p < 0.001) and protein (all p ≤ 0.004) expression levels were significantly higher in OLP keratinocytes following C. albicans exposure. Finally, the apoptosis rates of OLP keratinocytes were 21.2%, 29.4%, and 25.4% for the control cells and 3.9%, 5.6%, and 4.4% for those exposed to C. albicans, suggesting that co-culture with C. albicans inhibits the apoptosis of OLP keratinocytes.
Conclusions: C. albicans activates the TLR2/MyD88/NF-κB signaling pathway in OLP keratinocytes, resulting in increased cytokine expression and decreased keratinocyte apoptosis. Two key events in the pathogenesis of OLP and its progression to malignancy, namely increased inflammation and decreased apoptosis, were induced by exposure to C. albicans. Thus, targeting this signaling pathway may represent a novel therapeutic strategy to prevent OLP malignant transformation.
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Wolf, Sonya, Christopher Audu, Jadie Y. Moon, Amrita D. Joshi, William J. Melvin, Emily C. Barrett, Kevin D. Mangum, and Katherine A. Gallagher. "Diabetic wound keratinocytes induce macrophage JMJD3-mediated Nlrp3expression via IL1R signaling." Journal of Immunology 210, no. 1_Supplement (May 1, 2023): 143.05. http://dx.doi.org/10.4049/jimmunol.210.supp.143.05.
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Abstract Type 2 diabetic (T2D) wound Macrophages (Mϕs) display a delayed chronic inflammatory state that prevents the resolution of wound inflammation. Increased NLRP3 inflammasome gene expression and activation in Mϕs contributes to impaired wound healing in T2D and is induced by diabetic wound environment factors, yet the molecular mechanisms regulating this are unclear. Our lab has shown that keratinocytes regulate Mϕ phenotype during wound healing by producing proinflammatory cytokines. Thus, this project explores the molecular mechanisms by which keratinocytes drive increased Nlrp3 expression in diabetic wound Mϕs.Here, we found that Nlrp3 expression is increased in diabetic wound Mϕs late post-injury, and this can be induced by stimulation of Mϕs with diabetic wound keratinocyte supernatant. Further, we found that stimulation of Mϕs with diabetic wound keratinocyte supernatant decreased H3K27me3 deposition at the Nlrp3 promoter. Using genetically engineered mice (Jmjd3 fl/fl lyz2cre), we demonstrate that JMJD3, a demethylase specific for H3K27, controls Mϕ-mediated Nlrp3 expression and inflammasome activation during wound healing. Importantly, we show that diabetic wound keratinocytes induce Jmjd3 expression in Mϕs via IL1R/MyD88 upstream signaling. Interestingly, we found that IL1α is increased in human and murine wound diabetic keratinocytes compared to non-diabetic controls and stimulation of Mϕs with IL1α increases Nlrp3 expression via a JMJD3-mediated mechanism. Thus, our data suggest a role for keratinocyte IL1α in driving increased Nlrp3 expression in diabetic wound Mϕs via JMJD3. Continued investigation into the keratinocyte-Mϕ axis is key to developing novel treatments for non-healing T2D wounds. P30 DK089503, 1K99DK133828-01, 5K12 GM111725
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Oldak, Monika, Hans Smola, Monique Aumailley, Francisco Rivero, Herbert Pfister, and Sigrun Smola-Hess. "The Human Papillomavirus Type 8 E2 Protein Suppresses β4-Integrin Expression in Primary Human Keratinocytes." Journal of Virology 78, no. 19 (October 1, 2004): 10738–46. http://dx.doi.org/10.1128/jvi.78.19.10738-10746.2004.
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ABSTRACT Human papillomaviruses (HPVs) infect keratinocytes of skin and mucosa. Homeostasis of these constantly renewing, stratified epithelia is maintained by balanced keratinocyte proliferation and terminal differentiation. Instructions from the extracellular matrix engaging integrins strongly regulate these keratinocyte functions. The papillomavirus life cycle parallels the differentiation program of stratified epithelia, and viral progeny is produced only in terminally differentiating keratinocytes. Whereas papillomavirus oncoproteins can inhibit keratinocyte differentiation, the viral transcription factor E2 seems to counterbalance the impact of oncoproteins. In this study we show that high expression of HPV type 8 (HPV8) E2 in cultured primary keratinocytes leads to strong down-regulation of β4-integrin expression levels, partial reduction of β1-integrin, and detachment of transfected keratinocytes from underlying structures. Unlike HPV18 E2-expressing keratinocytes, HPV8 E2 transfectants did not primarily undergo apoptosis. HPV8 E2 partially suppressed β4-integrin promoter activity by binding to a specific E2 binding site leading to displacement of at least one cellular DNA binding factor. To our knowledge, we show for the first time that specific E2 binding contributes to regulation of a cellular promoter. In vivo, decreased β4-integrin expression is associated with detachment of keratinocytes from the underlying basement membrane and their egress from the basal to suprabasal layers. In papillomavirus disease, β4-integrin down-regulation in keratinocytes with higher E2 expression may push virally infected cells into the transit-amplifying compartment and ensure their commitment to the differentiation process required for virus replication.
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Burks, Hope E., Christopher R. Arnette, Jennifer L. Koetsier, Joshua A. Broussard, Valarie Ogwo, Quinn R. Roth-Carter, Pedram Gerami, Jodi L. Johnson, and Kathleen J. Green. "Abstract PR07: The role of keratinocyte desmoglein 1 in shaping the melanoma tumor niche." Cancer Research 80, no. 19_Supplement (October 1, 2020): PR07. http://dx.doi.org/10.1158/1538-7445.mel2019-pr07.
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Abstract Melanoma is an aggressive cancer arising from transformation of melanocytes residing in the basal layer of the epidermis, where they are in direct contact with surrounding keratinocytes. Keratinocytes and melanocytes communicate through both contact and paracrine signaling, a niche called the pigmentary unit. Ultraviolet (UV) radiation-induced melanocyte mutations drive melanoma formation and progression, but the role of keratinocytes and an altered microenvironment in melanomagenesis is understudied. We showed that the keratinocyte-specific desmosomal cadherin, desmoglein 1 (Dsg1), is reduced in response to acute UV exposure. Here, we show that Dsg1 is progressively reduced in regions surrounding human dysplastic nevi, melanoma in situ, and stage I/II melanomas, preceding loss of E-cadherin that occurs in later stages. Since we previously showed that Dsg1 suppresses EGFR/Erk1/2 signaling and regulates keratinocyte cytokine profiles, we hypothesized that altered keratinocyte Dsg1 expression governs melanocyte behavior and transformation through paracrine signaling. Dsg1 knockdown via shRNA elevated phospho-EGFR, phospho-Stat3, phospho-ERK and nuclear NFκB, accompanied by increases in IL-1α, IL-1β, IL-6, and IL-8, and CXCL1 proinflammatory cytokine mRNAs and secretion of IL6, IL8, and CXCL1. Melanocytes grown in conditioned media from Dsg1-deficient keratinocytes increased MITF transcription, while AXL transcript levels remained unchanged, a signature associated with early-stage melanoma. Consistent with increased MITF, melanocytes cultured in conditioned media from Dsg1-depleted keratinocytes increased pigment secretion, reversible by inhibiting the melanocortin 1 receptor. Melanocytes incorporated into Dsg1-deficient 3D human skin equivalents mis-localized to suprabasal layers, similar to pagetoid behavior in early melanoma. This, combined with the loss of keratinocyte Dsg1 surrounding human melanoma lesions, led us to examine mechanisms of sustained Dsg1 loss in the tumor niche. Keratinocytes cultured in melanoma cell-conditioned media reduced Dsg1 expression, suggesting that Dsg1 downregulation is maintained by melanoma cells post-transformation. We postulate that keratinocyte Dsg1 downregulation may initially be part of the skin’s protective response against UV exposure through its role in increasing pigmentation. However, chronic Dsg1 loss may contribute to melanoma initiation and be important for the maintenance of the promelanoma tumor niche. This abstract is also being presented as Poster A02. Note: This abstract was not presented at the conference. Citation Format: Hope E. Burks, Christopher R. Arnette, Jennifer L. Koetsier, Joshua A. Broussard, Valarie Ogwo, Quinn R. Roth-Carter, Pedram Gerami, Jodi L. Johnson, Kathleen J. Green. The role of keratinocyte desmoglein 1 in shaping the melanoma tumor niche [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR07.
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Moltrasio, Chiara, Maurizio Romagnuolo, and Angelo Valerio Marzano. "Epigenetic Mechanisms of Epidermal Differentiation." International Journal of Molecular Sciences 23, no. 9 (April 28, 2022): 4874. http://dx.doi.org/10.3390/ijms23094874.
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Keratinocyte differentiation is an essential process for epidermal stratification and stratum corneum formation. Keratinocytes proliferate in the basal layer of the epidermis and start their differentiation by changing their functional or phenotypical type; this process is regulated via induction or repression of epidermal differentiation complex (EDC) genes that play a pivotal role in epidermal development. Epidermal development and the keratinocyte differentiation program are orchestrated by several transcription factors, signaling pathways, and epigenetic regulators. The latter exhibits both activating and repressive effects on chromatin in keratinocytes via the ATP-dependent chromatin remodelers, histone demethylases, and genome organizers that promote terminal keratinocyte differentiation, and the DNA methyltransferases, histone deacetylases, and Polycomb components that stimulate proliferation of progenitor cells and inhibit premature activation of terminal differentiation-associated genes. In addition, microRNAs are involved in different processes between proliferation and differentiation during the program of epidermal development. Here, we bring together current knowledge of the mechanisms controlling gene expression during keratinocyte differentiation. An awareness of epigenetic mechanisms and their alterations in health and disease will help to bridge the gap between our current knowledge and potential applications for epigenetic regulators in clinical practice to pave the way for promising target therapies.
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Nanba, Daisuke, Fujio Toki, Sota Tate, Matome Imai, Natsuki Matsushita, Ken Shiraishi, Koji Sayama, Hiroshi Toki, Shigeki Higashiyama, and Yann Barrandon. "Cell motion predicts human epidermal stemness." Journal of Cell Biology 209, no. 2 (April 20, 2015): 305–15. http://dx.doi.org/10.1083/jcb.201409024.
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Image-based identification of cultured stem cells and noninvasive evaluation of their proliferative capacity advance cell therapy and stem cell research. Here we demonstrate that human keratinocyte stem cells can be identified in situ by analyzing cell motion during their cultivation. Modeling experiments suggested that the clonal type of cultured human clonogenic keratinocytes can be efficiently determined by analysis of early cell movement. Image analysis experiments demonstrated that keratinocyte stem cells indeed display a unique rotational movement that can be identified as early as the two-cell stage colony. We also demonstrate that α6 integrin is required for both rotational and collective cell motion. Our experiments provide, for the first time, strong evidence that cell motion and epidermal stemness are linked. We conclude that early identification of human keratinocyte stem cells by image analysis of cell movement is a valid parameter for quality control of cultured keratinocytes for transplantation.
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Ogawa, Eisaku, Tomohiro Edamitsu, Hidetaka Ohmori, Kazuyoshi Kohu, Mineo Kurokawa, Hiroshi Kiyonari, Masanobu Satake, and Ryuhei Okuyama. "Transcription Factors Runx1 and Runx3 Suppress Keratin Expression in Undifferentiated Keratinocytes." International Journal of Molecular Sciences 23, no. 17 (September 2, 2022): 10039. http://dx.doi.org/10.3390/ijms231710039.
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The Runt-related transcription factor (Runx) family has been suggested to play roles in stem cell regulation, tissue development, and oncogenesis in various tissues/organs. In this study, we investigated the possible functions of Runx1 and Runx3 in keratinocyte differentiation. Both Runx1 and Runx3 proteins were detected in primary cultures of mouse keratinocytes. Proteins were localized in the nuclei of undifferentiated keratinocytes but translocated to the cytoplasm of differentiated cells. The siRNA-mediated inhibition of Runx1 and Runx3 expression increased expression of keratin 1 and keratin 10, which are early differentiation markers of keratinocytes. In contrast, overexpression of Runx1 and Runx3 suppressed keratin 1 and keratin 10 expression. Endogenous Runx1 and Runx3 proteins were associated with the promoter sequences of keratin 1 and keratin 10 genes in undifferentiated but not differentiated keratinocytes. In mouse skin, the inhibition of Runx1 and Runx3 expression by keratinocyte-specific gene targeting increased the ratios of keratin 1- and keratin 10-positive cells in the basal layer of the epidermis. On the other hand, inhibition of Runx1 and Runx3 expression did not alter the proliferation capacity of cultured or epidermal keratinocytes. These results suggest that Runx1 and Runx3 likely function to directly inhibit differentiation-induced expression of keratin 1 and keratin 10 genes but are not involved in the regulation of keratinocyte proliferation.
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Dickson, Mark A., William C. Hahn, Yasushi Ino, Vincent Ronfard, Jenny Y. Wu, Robert A. Weinberg, David N. Louis, Frederick P. Li, and James G. Rheinwald. "Human Keratinocytes That Express hTERT and Also Bypass a p16INK4a-Enforced Mechanism That Limits Life Span Become Immortal yet Retain Normal Growth and Differentiation Characteristics." Molecular and Cellular Biology 20, no. 4 (February 15, 2000): 1436–47. http://dx.doi.org/10.1128/mcb.20.4.1436-1447.2000.
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ABSTRACT Normal human cells exhibit a limited replicative life span in culture, eventually arresting growth by a process termed senescence. Progressive telomere shortening appears to trigger senescence in normal human fibroblasts and retinal pigment epithelial cells, as ectopic expression of the telomerase catalytic subunit, hTERT, immortalizes these cell types directly. Telomerase expression alone is insufficient to enable certain other cell types to evade senescence, however. Such cells, including keratinocytes and mammary epithelial cells, appear to require loss of the pRB/p16INK4a cell cycle control mechanism in addition to hTERT expression to achieve immortality. To investigate the relationships among telomerase activity, cell cycle control, senescence, and differentiation, we expressed hTERT in two epithelial cell types, keratinocytes and mesothelial cells, and determined the effect on proliferation potential and on the function of cell-type-specific growth control and differentiation systems. Ectopic hTERT expression immortalized normal mesothelial cells and a premalignant, p16INK4a-negative keratinocyte line. In contrast, when four keratinocyte strains cultured from normal tissue were transduced to express hTERT, they were incompletely rescued from senescence. After reaching the population doubling limit of their parent cell strains, hTERT+ keratinocytes entered a slow growth phase of indefinite length, from which rare, rapidly dividing immortal cells emerged. These immortal cell lines frequently had sustained deletions of the CDK2NA/INK4A locus or otherwise were deficient in p16INK4a expression. They nevertheless typically retained other keratinocyte growth controls and differentiated normally in culture and in xenografts. Thus, keratinocyte replicative potential is limited by a p16INK4a-dependent mechanism, the activation of which can occur independent of telomere length. Abrogation of this mechanism together with telomerase expression immortalizes keratinocytes without affecting other major growth control or differentiation systems.
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Liarte, Sergio, Ángel Bernabé-García, Mónica Rodríguez-Valiente, José M. Moraleda, Gregorio Castellanos, and Francisco J. Nicolás. "Amniotic Membrane Restores Chronic Wound Features to Normal in a Keratinocyte TGF-β-Chronified Cell Model." International Journal of Molecular Sciences 24, no. 7 (March 25, 2023): 6210. http://dx.doi.org/10.3390/ijms24076210.
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Unsuccessful wound closure in chronic wounds can be linked to altered keratinocyte activation and their inability to re-epithelize. Suggested mechanisms driving this impairment involve unbalanced cytokine signaling. However, the molecular events leading to these aberrant responses are poorly understood. Among cytokines affecting keratinocyte responses, Transforming Growth Factor-β (TFG-β) is thought to have a great impact. In this study, we have used a previously characterized skin epidermal in vitro model, HaCaT cells continuously exposed to TGF-β1, to study the wound recovery capabilities of chronified/senescent keratinocytes. In this setting, chronified keratinocytes show decreased migration and reduced activation in response to injury. Amniotic membrane (AM) has been used successfully to manage unresponsive complicated wounds. In our in vitro setting, AM treatment of chronified keratinocytes re-enabled migration in the early stages of wound healing, also promoting proliferation at later stages. Interestingly, when checking the gene expression of markers known to be altered in TGF-β chronified cells and involved in cell cycle regulation, early migratory responses, senescence, and chronic inflammation, we discovered that AM treatment seemed to reset back to keratinocyte status. The analysis of the evolution of both the levels of keratinocyte activation marker cytokeratin 17 and the spatial-temporal expression pattern of the proliferation marker Ki-67 in human in vivo biopsy samples suggests that responses to AM recorded in TGF-β chronified HaCaT cells would be homologous to those of resident keratinocytes in chronic wounds. All these results provide further evidence that sustained TGF-β might play a key role in wound chronification and postulate the validity of our TGF-β chronified HaCaT in vitro model for the study of chronic wound physiology.