Dissertations / Theses on the topic 'Zc3h10'

Mitochondria play a crucial role in energy metabolism. Mitochondria have their own genome (mtDNA), whose replication and transcription are mainly regulated by the mitochondrial transcription factor A (Tfam). Recent researches demonstrate how mitochondria participate to a large number of cellular processes like cell cycle and differentiation. Our goal is to identify new mitochondrial regulators to light up the molecular mechanisms underlying mitochondrial function biology. We used a high throughput screening in 293 cells in order to identify positive mitochondrial regulators. By these means, we identified Zinc Finger CCCH-type containing 10 (Zc3h10) as the best hit. Following experiments demonstrated that Zc3h10 knockdown decreased mitochondrial function and differentiation in myotubes. RNA immunoprecipitation assay indicates that Zc3h10 is able to bind 410 transcripts. Several target genes are involved in energy metabolism and iron balance. Notably, Zc3h10 downregulation in C2C12 leads to iron overload while its overexpression restores ferric ion content to control levels. Collectively, our findings annotate Zc3h10 as a new mitochondrial regulator in skeletal muscle.

Mitochondria play a crucial role in many cellular processes, and they are essential organelles for the cell’s health. Beside their contribution to energy production, they are key regulators of tissue development and cell differentiation. In this context, a new mitochondrial regulator, zinc finger CCCH-type containing 10 (Zc3h10) protein has been recently discovered, and we validated its role during white adipocytes differentiation. The commitment of mesenchymal stem cells to pre-adipocytes is stimulated by hormonal induction. During adipocytes differentiation, pre-adipocytes undergo blunted protein synthesis, cytoskeleton remodeling and increased mitochondrial function to support anabolic pathways. All these molecular changes enable differentiation into mature adipocytes. We found that Zc3h10 is a critical regulator of the early stages of adipogenesis. Indeed, Zc3h10 depletion in pre-adipocytes resulted in increased protein translation and impaired filamentous (F)-actin remodeling, thus resulting in mitochondrial and metabolic dysfunction, incorrect mitotic clonal expansion (MCE), impaired lipid accumulation and terminal differentiation. In contrast, Zc3h10 overexpression yielded mature adipocytes with remarkably increased lipid droplet size. Overall, our results establish Zc3h10 as a fundamental pro-adipogenic transcription factor that represses protein synthesis and promotes F-actin/mitochondria dynamics to ensure proper energy metabolism, MCE and favor lipid accumulation to allow final adipocytes differentiation.

Le CBC intervient dans de nombreuses étapes du métabolisme des ARN, telle que l’épissage, la maturation de l’extrémité 3’, la dégradation, l’export et la traduction. Ainsi, le CBC constitue un complexe majeur qui peut orchestrer les différentes étapes de maturation des ARN. Récemment, nous avons identifié le complexe CBCAP, composé de CBC, ARS2 et PHAX. Nous avons montré que la protéine ARS2 stimule la formation des extrémités 3’ de plusieurs familles d’ARN dont les snARN. De plus, ARS2 stimule le recrutement de PHAX sur le CBC. Ainsi, nous proposons un modèle où CBC-ARS2 stimule la formation de l’extrémité 3’ des pré-snARN et recrute PHAX pour favoriser leur export. Une autre étude a identifié un autre complexe le CBCN, constitué de CBC, Ars2, et de ZC3H18-NEXT au lieu de PHAX. CBCN recrute l’exosome et stimule la dégradation de certains ARN, comme les PROMPTS et les transcrits «read-through » des snARN et des ARNm d’histone. Ainsi, PHAX et ZC3H18 destinent leur ARN cibles vers l’export ou la dégradation. Il a été montré que PHAX reconnait et lie spécialement les ARN de petite taille. D’une manière remarquable, nos données de CLIP-Seq et de RIP suivie par des analyses avec des puces « All genes » montrent que PHAX lie aussi d’autres familles d’ARN. En effet, PHAX lie les ARNm ainsi que des ARN non-codant avec une légère préférence pour les snARN (en comparaison avec ZC3H18). Afin de mieux comprendre le rôle de PHAX et ZC3H18, j’ai tout d’abord démontré si les deux protéines se lient simultanément au CBC. Pour ce faire, J’ai réalisé des tests de compétitions entre PHAX et ZC3H18, in vivo, et j’ai montré que la surexpression de ZC3H18 déplace PHAX du CBC et vice versa. Puis en utilisant la technique de « Tethering Assays » j’ai pu montrer que PHAX et ZC3H18 ont des effets opposés sur la biogénèse des ARNm. De plus PHAX semble avoir un effet positif sur la maturation des ARNm et ce, en empêchant ZC3H18 et l’exosome d’être recruter. Nous avons aussi montré que la déplétion de PHAX et ZC3H18 a des conséquences fonctionnelles sur le taux des formes matures des snARN. Dans le but de caractériser la protéine ZC3H18, j’ai réalisé un crible double-hybride et j’ai montré que ZC3H18 interagit avec plusieurs facteurs d’épissage. J’ai aussi identifié les domaines de ZC3H18 impliqués dans ses différentes interactions. D’une manière intéressante, l’interaction de ZC3H18 avec certains facteurs d’épissage peut être exclusive à son interaction avec NEXT. De plus, des expériences de protéomique réalisés sur un des facteurs d’épissage trouvé dans le crible, montrent qu’il co-purifie au sein d’un complexe qui pourrait faire le lien entre la coiffe et la machinerie d’épissage. En accord avec ces résultats, nos données de RNA-seq montrent que la déplétion de ZC3H18 engendre un défaut d’épissage pour des introns qui sont proches de la coiffe et ceci pour un nombre restreint de gènes. Ainsi, notre travail décode davantage le rôle de la coiffe dans les différentes étapes de maturation des ARN et suggère un modèle où la séquence des transcrits naissant stimule la formation d’un complexe spécifique à cet ARN parmi plusieurs autres
The cap binding complex (CBC) plays a key role in a number of gene expression pathways and has been proposed to participate in the discrimination of RNA families. It also enhances many RNA processing steps, including transcription, splicing, 3’end formation, degradation, export and translation.Recently, we identified the CBCAP complex, composed of CBC, Ars2 and PHAX. We showed that Ars2 stimulates snRNA 3'-end processing as well as PHAX binding to the CBC, hence coupling snRNA maturation with their export. Other studies showed that the CBC and ARS2 can form another complex that contains ZC3H18-NEXT instead of PHAX. This complex, named CBCN, is a cofactor of the RNA exosome and is involved in the degradation of cryptic RNAs such as PROMPTs and read-through transcripts at histone and snRNA genes. Thus, PHAX and ZC3H18 target specific families of capped RNA toward either export or degradation. Previous studies proposed that PHAX binds specifically to small RNAs and discriminates them over other RNA species. Surprisingly, our CLIP-Seq and RIP-microarrays data showed that in contrast to expectations, PHAX was not specific for snRNAs. It also binds mRNAs as well as other non-coding RNAs and has a weak preference for snRNAs comparing to ZC3H18. To better understand the role of PHAX and ZC3H18, Ifirst determined whether PHAX and ZC3H18 can bind simultaneously to the CBC. Competitive LUMIER IPs indicated that binding of these proteins is mutually exclusive. I then used tethering assays and could show that PHAX and ZC3H18 have opposite effect on mRNA biogenesis. These data go against a model where binding of PHAX or ZC3H18 discriminate RNA families, and instead suggest promiscuous binding for these proteins. In addition, PHAX may exert a positive effect on mRNA processing by preventing binding of ZC3H18 and recruitment of the RNA exosome. Last but not least, our RT-QPCR data show that PHAX and ZC3H18 depletions have functional consequences on the level of mature snRNA, and this is due to a competition between both proteins which occur on those snRNA read-through transcripts.To further explore the role of ZC3H18, I performed a two-hybrid screen and identified several splicing factors. I could validate these interactions, identify the domains involved and show that binding of some of these factors is exclusive with that of NEXT. Importantly, proteomic experiments with one of these factors identified a complex that makes the link between the cap and the splicing machinery. In agreement, RNA-Seq analysis of ZC3H18 knock-down cells showed alterations in splicing of cap-proximal introns, for a small set of genes.Altogether, this work reveals how the multiple roles of the RNA cap are achieved at the biochemical level, and suggests that the nascent RNA sequence triggers formation of one among several mutually exclusive complexes

The recent advances in molecular and computational biology have made possible the study of complicated transcriptional regulatory networks that control a wide range of biological processes and phenotypic traits. In this thesis, several approaches were combined including next generation sequencing, gene expression profiling, chromatin and RNA immunoprecipitation, bioinformatics and genome editing methods in order to characterize the biological significance of the ZBED6 and ZC3H11A genes. A mutation in the binding site of ZBED6, located in an intron of IGF2, disrupts the binding and leads to 3-fold upregulation of IGF2 mRNA in pig muscle tissues. The first part of the thesis presents a detailed functional characterization of ZBED6. Transient silencing of ZBED6 expression in mouse myoblasts led to increased Igf2 expression (~2-fold). ChIP-seq analysis of ZBED6 and histone modifications showed that ZBED6 preferentially binds active promoters and modulates their transcriptional activities (paper I). In the follow-up studies using CRISPR/Cas9 we showed that either the deletion of ZBED6 or its binding site in Igf2 (Igf2ΔGGCT) led to more than 30-fold up-regulation of Igf2 expression in myoblasts. Differentiation of these genetically engineered cells resulted in hypertrophic myotubes. Transcriptome analysis revealed ~30% overlap between the differentially expressed genes in Zbed6-/- and Igf2ΔGGCT myotubes, with significant enrichment of muscle-specific genes. ZBED6-overexpression in myoblasts led to cell cycle arrest, reduced cell viability, reduced mitochondrial activities and impaired the differentiation of myoblasts (paper II). Further studies on cancer cells showed that ZBED6 influences the growth of colorectal cancer cells with dramatic changes in the transcription of hundreds of cancer-related genes (paper III). The phenotypic characterization of Zbed6-/- and Igf2pA/mG mouse models showed that the ZBED6-Igf2 axis has a major effect on regulating muscle growth and the growth of internal organs. Transcriptome analysis demonstrated a massive up-regulation of Igf2 expression (~30-fold) in adult tissues, but not in fetal tissues, of transgenic mice (paper IV). In the second part of the thesis we investigated the cellular function of Zc3h11a, the gene harboring ZBED6 in one of its first introns. The function of the ZC3H11A protein is so far poorly characterized. We show that ZC3H11A is a novel stress-induced protein that is required for efficient mRNA export from the nucleus. The inactivation of ZC3H11A inhibited the growth of multiple viruses including HIV, influenza, HSV and adenoviruses (paper V).

Cystic fibrosis (CF) is the most common fatal genetic disease affecting Canadian Caucasians. The majority of CF patients suffered from perpetual inflammation caused by Pseudomonas aeruginosa (P. aeruginosa). Previous, we found cells lacking functionalCFTR exhibit IL6 hypersecretion phenotype and a concomitant hyperactivity of p38 MAPK in response to P. aeruginosa. Therefore, I dedicated my master's thesis to investigate the link between increased IL6 production and p38 MAPK. ZC3H12A, an IL6-specific ribonuclease, was found to be a novel substrate of p38 MAPK and negatively regulated IL6 production from P. aeruginosa-stimulated airway epithelial cells. Together, these discoveries demonstrated a direct link between a specific signal transduction pathway and am RNA stability modulator, both of which contribute to the regulation of IL6 gene expression at a post-transcriptional level.
La fibrose kystique (FK) est la maladie génétique mortelle la plus fréquente parmi les canadiens d'origine caucasienne. La majorité des personnes atteintes souffrent d'une inflammation chronique causée par la bactérie Pseudomonas aeruginosa (P. aeruginosa). Précédemment, nous avons découvert que des cellules dépourvues de protéine CFTR fonctionnelle présentent un phénotype d'hypersécrétion d'interleukin-6 et d'hyperactivité concomitante de la MAPK p38 en réponse à la bactérie. J'ai donc consacré ma maîtrise à étudier le lien potentiel entre la MAPK p38 et la production élevée d'IL6. La ribonuclease ZC3H12A, spécifique envers IL6, fut découverte comme un nouveau substrat de la MAPK p38. Ces découvertes ont démontré un lien direct entre une voie spécifique de transduction du signal et un modulateur de stabilité de l'ARNm, les deux contribuant à la régulation post-transcriptionnelle de l'expression d'IL6.

Coronary artery disease (CAD), characterized by the narrowing of coronary arteries through the complex manifestation and development of atherosclerosis, is a complex disease and one of the leading causes of death worldwide. Both genetic and environmental factors are believed to contribute equally to the risk of CAD. Recently, a study identified a non-synonymous coding variant, rs11556924, (MAF, 0.38) in ZC3HC1 associated with protection against CAD (p= 9.8x10-18; OR= 0.90). NIPA, encoded by ZC3HC1, is a characterized F-Box protein and regulator of cell cycle. Since the amino acid change (Arg363His) is in a conserved region of NIPA and is predicted to have functional effects (Polyphen-2), this study aimed at understanding the functional implications of this amino acid change on NIPA and cell cycle regulation. Here we are able to effectively show a) allele specific differences in mRNA expression in whole blood, b) a slight structural difference between NIPA363Arg and NIPA363His variants, c) proliferation rates of NIPA363Arg expressing cells were significantly increased, and d) phosphorylation of a critical serine residue in close proximity to aa.363 is not statistically different between the two variants. These results suggest that rs11556924 plays a direct role in development of CAD through its disruption of cell cycle regulation and NIPA mRNA availability. This study is the first to identify a molecular basis for the association of rs11556924 to CAD development.

Primary immunodeficiencies (PIDs) are a heterogeneous group of disorders causing immune dysfunction that manifest with increased susceptibility to infection. Some PID patients may also have autoimmune and autoinflammatory manifestations. In many cases, PIDs are monogenic disorders that follow Mendelian inheritance and mutations in more than 250 genes have been shown to cause PIDs. However, in the majority of PID patients the causative mutations remain unknown. Here I report a study of a patient from a consanguineous family who presented in infancy with colitis, autoimmune hepatitis, autoimmune anemia and thrombocytopenia. The patient also suffered recurrent respiratory infections leading to bronchiectasis and had several episodes of severe varicella zoster virus (VZV) infections, including pneumonia and meningitis. Immunologically, the patient had increased IgM and IgG levels, absent IgA, low specific antibodies and multiple auto-antibodies, including anti-Interferon- antibodies. Whole blood stimulation assays identified an increased production of the pro-inflammatory cytokine IL-6. Throughout his life the patient received immunosuppressive therapy. Whole exome sequencing of the patient discovered a homozygous frameshift mutation in the ZC3H12A gene that encodes the Regnase-1 protein also known as MCPIP1. Regnase-1 is a regulatory RNase that directly degrades mRNAs of several pro-inflammatory genes, e.g. mRNA of cytokine IL-6, thus curbing the immune activation. The presentation of the patient resembled the phenotype of the Regnase-1-knockout mice that developed spontaneous systemic inflammation, disorganisation of lymphoid organs, severe anaemia and hyperimmunoglobulinemia, with the increased production of IL-6. I studied expression of the mutant Regnase-1 protein using commercial antibodies; also a new custom-made antibody that detects the truncated mutant Regnase-1 protein was developed. Analysis of the patient-derived cells demonstrated absence of the full-length Regnase-1 protein. Cloning and forced expression of the truncated mutant protein showed that it is mislocalized inside the cells and is functionally impaired. Studies of the iPSC-derived macrophages, EBV-transformed B cells and primary fibroblasts of the patient demonstrated increased levels of the IL-6 mRNA in the resting cells. They also showed impaired regulation of the truncated mutant Regnase-1 protein and IL-6 mRNA levels after cell stimulation. Mutations in Regnase-1 have never been associated with human diseases previously. Therefore, this study describes a novel PID caused by the Regnase-1 deficiency.

ZC3H11A (ZC3) protein has been reported to be part of the TREX (TRanscription-EXport) nuclear export system for mammalian cells. According to our previous publication, ZC3 not only plays an unelucidated role in the TREX complex, but also supports the growth of several human nucleus replicating virus, such as influenza virus, adenovirus (HAdV), herpes simplex virus and HIV. We thought to further elucidate the role of ZC3 in immunological stress based on previous observations that ZC3 was upregulated in stress condition. Our previous experiment tested the effect of knocking out ZC3 in HeLa cell then stimulating the cells with IL-1β to induce immunological stress. It showed that IL-1β stimulated ZC3 knockout Hela cells produce more than double fold IL6 compared to IL-1β stimulated HeLa Cas 9 wild type. Since IL-6 is downstream of NFkB signalling pathway, we aimed to explore a possible role of ZC3 protein in mammalian cell’s NFkB pathway. Our primary results showed that NFkB pathway might be more upregulated in ZC3 KO cells than in wild type HeLa Cas9 cells. This up-regulation was found to be correlated to defective IkBα inhibitory mRNA biogenesis in knockout cells. Our results indicate that ZC3 might play a role in IkBα inhibitory mRNA biogenesis, process, and/or export. Further work is needed to describe the exact role of ZC3 in IKBα mRNA biogenesis.

Mouse epiblast stem cells (EpiSC) are pluripotent cells derived of the epiblast of post-implantation blastocysts that can self-renew indefinitely in culture, display lineage-restricted differentiation, and appear to closely resemble human embryonic stem cells (ESC). Despite significant advances in the last decade, the precise molecular mechanisms and many master regulator (MR) genes underlying stem cell self-renewal, pluripotency, interactions with surrounding cells, and lineage-specific differentiation still remain elusive. The goal of this thesis is to address these gaps of knowledge using a systematic approach to identify novel MR genes and functionally validate them using genetically modified mouse models.In order to elucidate MR genes that control understudied biological processes, previous work in the Shen lab have computationally reconstructed the regulatory network of EpiSC and interrogated the EpiSC interactome with pluripotency signatures of EpiSC lines. One MR gene of interest from the previous analysis is ZC3H13, which encodes a protein that has been previously shown to be a crucial for N6-methyladenosine modification in RNA (m⁶A). This suggests a novel connection between m⁶A epitranscriptional modifications and primed state pluripotency. In my thesis research, I have shown that Zc3h13 is essential for proper trophoblast lineage differentiation and the importance of m6A modifications in early embryonic development. Using two Zc3h13 knockout mouse lines, I have found that Zc3h13 null embryos are embryonic lethal at the peri-implantation stage due to a failure to implant into the uterus. In vitro outgrowth analysis revealed a lack of trophoblast giant cells in Zc3h13 null outgrowths, and the lack of enlarged nuclei in the Zc3h13 null outgrowth suggests a failure in endoreduplication. Immunofluorescence analysis of Zc3h13 null blastocysts showed that the trophectoderm cells of Zc3h13 null blastocyst expressed trophectoderm specific factors at abnormal levels, indicating a severe dysregulation of the trophectoderm regulatory network. To elucidate the effects of Zc3h13 knockout on pluripotency, I also performed a detailed immunofluorescence analysis of Zc3h13 null inner cell mass (ICM), which expressed pluripotency factors at normal levels. However, Zc3h13 null blastocysts were less efficient at generating ESC lines and the Zc3h13 KO ESC generated were morphologically abnormal. Dot blot and mass spectrometry analysis showed that Zc3h13 KO ESC had a dramatically lower level of m⁶A modification, suggesting a connection between m6A epitranscriptional modification and endoreduplication. Interestingly, chimera and teratoma analysis showed that while Zc3h13 KO ESC can contribute to derivatives of the three primary lineages, Zc3h13 KO ESC has a bias towards neuroectoderm differentiation. In this thesis, I have shown the importance of m6A transcriptional regulation in trophoblast giant cell differentiation. Taken together, my studies can help further the understanding of the biological functions of m⁶A modifications as well as the relationship between transcriptional regulation and cell fate transition. My work highlights another level of gene regulation through epitranscriptional modification and the importance of the epitranscriptomic landscape in cell fate transition and development.

Skóra stanowi zewnętrzną powłokę ciała. Składa się z naskórka, skóry właściwej oraz tkanki podskórnej. Naskórek zbudowany jest z kilku warstw, które odpowiedzialne są za prawidłowe funkcjonowanie tej tkanki. Najliczniejszy typ komórek obecny w naskórku to keratynocyty, jednakże poszczególne warstwy naskórka są również skolonizowane przez komórki układu odpornościowego. Skóra stanowi aktywną barierę immunologiczną, która chroni przed przedostawaniem się potencjalnie szkodliwych substancji środowiskowych i mikroorganizmów. Utrzymanie homeostazy immunologicznej w skórze zależy od precyzyjnie wyregulowanej komunikacji między poszczególnymi komponentami komórkowymi skóry, a środowiskiem mikrobiologicznym. Rozregulowanie któregokolwiek z czynników biorących udział w odpowiedzi zapalnej oraz zachwianie równowagi fizjologicznej skóry jest główną przyczyną rozwoju chorób skóry o podłożu zapalnym. Do najczęstszych chorób skóry, u podstaw których leży stan zapalny należy łuszczyca, która jest przewlekłą, nawracającą chorobą zapalna skóry występującą u 2 do 3% ludzkiej populacji. Etiopatogeneza choroby nie jest w pełni poznana. Bierze się pod uwagę udział czynników genetycznych i immunologicznych, w tym zwiększoną proliferację keratynocytów pod wpływem aktywowanych limfocytów T. Leczenie chorób skóry nie zawsze daje trwałe i pozytywne rezultaty, nawet pomimo stosowania terapii farmakologicznych jak i nowoczesnych zabiegów chirurgicznych. Ponadto, często powracające zmiany skórne wywołane czynnikami zewnętrznymi, stresem czy uwarunkowaniami genetycznymi utrudniają dobranie prawidłowej terapii leczenia. Wiele modulatorów odpowiedzi zapalnej odgrywa istotną rolą w utrzymaniu prawidłowej homeostazy naskórka. Białko MCPIP1, kodowane przez gen ZC3H12A, jest negatywnym regulatorem stanu zapalnego w wyniku aktywności RNazowej tego białka, która pozwala na degradację mRNA cytokin prozapalnych, takich jak IL-1b czy IL-6. Dodatkowo, białko MCPIP1 jest negatywnym regulatorem czynników transkrypcyjnych NF-kB oraz AP-1, przez co może uczestniczyć w kontroli szeregu procesów komórkowych, takich jak proliferacja, apoptoza czy różnicowanie. Celem badań prowadzonych w ramach pracy doktorskiej było określenie roli białka MCPIP1 w procesach fizjologicznych oraz patofizjologicznych naskórka, ze szczególnym uwzględnieniem patogenezy łuszczycy. W poniższej pracy wykazano, iż poziom białka MCPIP1 wzrasta w trakcie różnicowania pierwotnych ludzkich keratynocytów, podczas gdy poziom mRNA MCPIP1 spada. Potwierdzono również, że białko MCPIP1 lokalizuje się w tych warstwach naskórka, gdzie komórki intensywnie różnicują. Stosując system nadekspresji MCPIP1 zaobserwowano, iż zwiększenie poziomu białka MCPIP1 w keratynocytach powoduje zahamowanie procesu proliferacji z jednoczesną aktywacją procesu ich różnicowania. Mechanizm tej regulacji oparty jest na aktywności RNazowej MCPIP1. Przy użyciu mysiego modelu naskórka pozbawionego genu kodującego białko Mcpip1 w keratynoctach warstwy podstawnej wykazano, iż Mcpip1 jest kluczowym regulatorem homeostazy naskórka. Udowodniono, że usunięcie tego białka w keratynocytach wpływa na profil mRNA pierwotnych mysich keratynocytów, ich aktywność proliferacyjną oraz na zwiększenie grubości naskórka nowonarodzonych myszy. Brak białka Mcpip1 w naskórku powoduje rozwój lokalnego stanu zapalnego skóry wraz z wiekiem myszy, który objawia się rozwojem dysplazji naskórka, silnym naciekiem komórek układu immunologicznego w skórze właściwej oraz aktywacją cytokin prozapalnych. Ponadto, progresywnemu rozwojowi lokalnego stanu zapalnego towarzyszy rozwój ogólnoustrojowego stanu zapalnego, który manifestuje się utratą wagi, patologicznym powiększeniem śledziony oraz węzłów chłonnych myszy. Analizując proces patofizjologiczny naskórka przy użyciu mysiego modelu łuszczycy wykazano, iż białko Mcpip1 jest kluczowym inicjatorem rozwoju łuszczycy. Jego brak w keratynocytach prowadzi do zwiększenia dysplazji naskórka, aktywacji ścieżki sygnalizacyjnej Il-17A/Il-23A, Il-36 oraz aktywacji czynnika transkrypcyjnego Stat3. Wykorzystując mysi model gojenia ran dowiedziono, iż Mcpip1 może być ważnym czynnikiem regulującym migrację keratynocytów w trakcie gojenia się ran. Podsumowywując, w niniejszej pracy doktorskiej udowodniono, że białko MCPIP1 może być kluczowym regulatorem biologii keratynocytów oraz homeostazy skóry, poprzez kontrolę procesu proliferacji, różnicowania keratynocytów oraz inicjacji i progresji stanu zapalnego skóry.
The skin provides an external barrier of the body. It consists of the epidermis, dermis and subcutaneous tissue. The epidermis is made of several layers that are responsible for the proper functioning of this tissue. The most numerous cell types present in the epidermis are keratinocytes, however, individual layers of the epidermis are also colonized by immune cells. The skin provides an active immune barrier that protects against the penetration of potentially harmful environmental substances and microorganisms. The maintenance of immunological homeostasis in the skin depends on the precisely regulated communication between individual skin cell components and the microbiological environment. Deregulation of any of the factors involved in the inflammatory response and disturbance of skin physiology are the main cause of the development of inflammatory skin diseases. Psoriasis is one of the most common skin conditions with inflammatory backqround. Psoriasis is a chronic, recurrent inflammatory skin disease that occurs in 2 to 3% of the human population. The etiopathogenesis of the disease is not fully understood. The contribution of genetic and immunological factors is taken into account, including increased proliferation of keratinocytes under the influence of activated T lymphocytes. Treatment of skin diseases does not always give lasting and positive results, even despite the use of pharmacological therapies and modern surgical procedures. In addition, often recurring skin changes due to external factors, stress or genetic conditions make difficulties in choosing the right therapy for treatment. Many inflammatory response modulators play an important role in maintaining normal epidermal homeostasis. The MCPIP1 protein, encoded by the ZC3H12A gene, is a negative regulator of inflammation due to its RNase activity. This activity allows degradation of mRNA coding for proinflammatory cytokines, such as IL-1b and IL-6. In addition, the MCPIP1 protein is a negative regulator of the transcription factors NF-kB and AP-1, and thus may participate in the control of several cellular processes, such as proliferation, apoptosis and differentiation. The aim of the research carried out as a part of the doctoral dissertation was to determine the role of the MCPIP1 protein in physiological and pathophysiological processes of the epidermis, particulary in pathogenesis of psoriasis. The following work demonstrates that protein levels of MCPIP1 increase during differentiation of primary human keratinocytes, while MCPIP1 mRNA levels decrease. It was also confirmed that the MCPIP1 protein localizes in the layers of the epidermis, where cells intensively differentiate. Using the MCPIP1 overexpression system, it was observed that increased level of MCPIP1 in human keratinocytes inhibits the proliferation process with the simultaneous activation of the keratinocyte differentiation. The mechanism of this regulation is based on MCPIP1 RNase activity. Using a mouse model of the epidermis lacking the gene encoding the Mcpip1 in keratinocytes of the basal layer, it was shown that Mcpip1 is a key regulator of epidermal homeostasis. It was proved that the removal of this protein in keratinocytes affects the mRNA profile of primary mouse keratinocytes, their proliferative activity and the increase in the thickness of the epidermis of newborn mice. Lack of Mcpip1 protein in the epidermis causes the development of local inflammation of the skin with the aging of the mice, which is manifested by the development of epidermal dysplasia, a strong infiltration of immune cells in the dermis and the activation of proinflammatory cytokines. In addition, the progressive development of local inflammation is accompanied by the development of systemic inflammation, which is manifested by weight loss, pathological enlargement of the spleen and lymph nodes. Analyzing the pathophysiological process of the epidermis using a mouse model of psoriasis, it was shown that the Mcpip1 is a key initiator of psoriasis development through increased activation of epidermal dysplasia, activation the Il-17A/Il-23A, Il-36 signaling pathways and the Stat3 transcription factor. Using the mouse model of wound healing, it was proved that Mcpip1 can be an important factor regulating re-epithelization and migration of keratinocytes during wound healing. Summing up, in this dissertation it was proved that the MCPIP1 protein can be a key regulator of keratinocyte biology and skin homeostasis, through the active participation of this protein in the process of proliferation, differentiation of keratinocytes and the control of initiation and progression of skin inflammation.

碩士
臺北醫學大學
醫學科學研究所
102
Monocyte chemotactic protein induced protein 3 (MCPIP3), also name as ZC3H12C (Zine finger CCCH domain-containing protein 12C), belongs to CCCH-zinc finger protein family member. MCPIP3 protein contains a highly conserved of the CCCH-zinc finger domain and Nedd4-BP1, YacP Nuclease (NYN) domain. Previous studies were shown that MCPIP3 inhibits the expression of pro-inflammatory genes, vascular cell adhesion molecule-1 (VCAM-1) and E-cadherin expression in endothelial cells, but the role and function of MCPIP3 in cancer cells are unknown. In human colorectal tissues. In previous experiments was found that most of cancer tissues are increased at the mRNA level of MCPIP3 compared to normal tissues. To understand the function of MCPIP3, our data investigated HA-MCPIP3/T-REx-293 cells which can be induced the MCPIP3 expression by doxycycline. Overexpression of MCPIP3 changed the epithelial-mesenchymal transition (EMT)-related marker genes expression correlated with reduced E-cadherin and increased Vimentin. To further investigate the functions of MCPIP3 in human colorectal cancer cells, overexpression of MCPIP3 by transduction of lentiviral in SW620 and HCT116 cell lines Overexpression of MCPIP3 significantly decreased cell migration and increased E-cadherin expression in both SW620 and HCT116 cells. These results suggest that MCPIP3 may play a negative role in cell proliferation and migration of human colorectal cancer cells.