Academic literature on the topic 'HHIPL1'

DNA methylation is one of the several epigenetic modifications that together with genetic aberrations are hallmarks of tumorigenesis including those emanating from the pituitary gland. In this study, we examined DNA methylation across 27 578 CpG sites spanning more than 14 000 genes in the major pituitary adenoma subtypes. Genome-wide changes were first determined in a discovery cohort comprising non-functioning (NF), growth hormone (GH), prolactin (PRL)-secreting and corticotroph (CT) adenoma relative to post-mortem pituitaries. Using stringent cut-off criteria, we validated increased methylation by pyrosequencing in 12 of 16 (75%) genes. Overall, these criteria identified 40 genes in NF, 21 in GH, six in PRL and two in CT that were differentially methylated relative to controls. In a larger independent cohort of adenomas, for genes in which hypermethylation had been validated, different frequencies of hypermethylation were apparent, where the KIAA1822 (HHIPL1) and TFAP2E genes were hypermethylated in 12 of 13 NF adenomas whereas the COL1A2 gene showed an increase in two of 13 adenomas. For genes showing differential methylation across and between adenoma subtypes, pyrosequencing confirmed these findings. In three of 12 genes investigated, an inverse relationship between methylation and transcript expression was observed where increased methylation of EML2, RHOD and HOXB1 is associated with significantly reduced transcript expression. This study provides the first genome-wide survey of adenoma, subtype-specific epigenomic changes and will prove useful for identification of biomarkers that perhaps predict or characterise growth patterns. The functional characterisation of identified genes will also provide insight of tumour aetiology and identification of new therapeutic targets.

Vertebrate Hedgehog (HH) signaling is controlled by several ligand-binding antagonists including Patched-1 (PTCH1), PTCH2, and HH-interacting protein 1 (HHIP1), whose collective action is essential for proper HH pathway activity. However, the molecular mechanisms used by these inhibitors remain poorly understood. In this paper, we investigated the mechanisms underlying HHIP1 antagonism of HH signaling. Strikingly, we found evidence that HHIP1 non–cell-autonomously inhibits HH-dependent neural progenitor patterning and proliferation. Furthermore, this non–cell-autonomous antagonism of HH signaling results from the secretion of HHIP1 that is modulated by cell type–specific interactions with heparan sulfate (HS). These interactions are mediated by an HS-binding motif in the cysteine-rich domain of HHIP1 that is required for its localization to the neuroepithelial basement membrane (BM) to effectively antagonize HH pathway function. Our data also suggest that endogenous, secreted HHIP1 localization to HS-containing BMs regulates HH ligand distribution. Overall, the secreted activity of HHIP1 represents a novel mechanism to regulate HH ligand localization and function during embryogenesis.

Genetic variants in Hedgehog interacting protein (HHIP) have consistently been associated with the susceptibility to develop chronic obstructive pulmonary disease and pulmonary function levels, including the forced expiratory volume in 1 s (FEV1), in general population samples by genome-wide association studies. However, in vivo evidence connecting Hhip to age-related FEV1 decline and emphysema development is lacking. Herein, using Hhip heterozygous mice (Hhip+/−), we observed increased lung compliance and spontaneous emphysema in Hhip+/− mice starting at 10 mo of age. This increase was preceded by increases in oxidative stress levels in the lungs of Hhip+/− vs. Hhip+/+ mice. To our knowledge, these results provide the first line of evidence that HHIP is involved in maintaining normal lung function and alveolar structures. Interestingly, antioxidant N-acetyl cysteine treatment in mice starting at age of 5 mo improved lung function and prevented emphysema development in Hhip+/− mice, suggesting that N-acetyl cysteine treatment limits the progression of age-related emphysema in Hhip+/− mice. Therefore, reduced lung function and age-related spontaneous emphysema development in Hhip+/− mice may be caused by increased oxidative stress levels in murine lungs as a result of haploinsufficiency of Hhip.

Genome-wide association studies have identified chromosome 14q32 as a locus for coronary artery disease in humans. The disease associated variants fall in a gene called Hedgehog interacting protein-like 1 (HHIPL1), which encodes an uncharacterised sequence homologue of an antagonist of Hedgehog signalling. Here, is presented the investigation of HHIPL1 function and its role in atherosclerosis. In vitro analysis was undertaken in order to determine the molecular and cellular function of the protein. Epitope tagged HHIPL1 protein was present in the media of transfected cells and immunoprecipitated with GFP tagged Sonic Hedgehog (SHH) protein, demonstrating that HHIPL1 is a secreted interactor of SHH. HHIPL1 gene expression was also measured in cardiovascular cell types and found that it is expressed in aortic smooth muscle cells (HASMC), but not in other disease relevant cell types. During atherogenesis smooth muscle cells migrate and proliferate into the tunica intima. Therefore, the effect of HHIPL1 on HASMC phenotype was examined following HHIPL1 knockdown. Down regulation of HHIPL1 through siRNA resulted in a significant reduction in both HASMC proliferation and migration, suggesting a regulatory role for HHIPL1 in smooth muscle cell phenotype. Next, the role of HHIPL1 in atherosclerosis in vivo was examined. In atherosclerotic mouse aortas (Apoe-/-) Hhipl1 expression increased with disease progression. In order to further investigate the effect of Hhipl1 on atherosclerosis Hhipl1 knockout mice, which are phenotypically normal, were crossed onto two hyperlipidemic atherosclerosis prone backgrounds. Double knockout mice (Hhipl1-/-; Apoe-/-, Hhipl1-/-; Ldlr-/-) displayed a sunstantial reduction of over 60% in atherosclerotic lesion size compared with controls. Moreover, Hhipl1-/- lesions were characterised by reduced smooth muscle cell content, but unaltered lipid and macrophage profile. These data represent the first experimental investigation of HHIPL1 and demonstrate that HHIPL1 is a proatherogenic protein that regulates smooth muscle cell proliferation and migration, presumably through its involvement in Hedgehog signalling.

The thymus is a primary lymphoid organ that supports the development of functional T cells through its unique stromal architecture. The most important and abundant component of this microenvironment are thymic epithelial cells, or TECs. TEC development, function and maintenance are critically dependent on the expression of the master transcription factor Foxn1. The Hedgehog-Interacting Protein (Hhip) was identified as a novel candidate target of Foxn1-mediated gene expression. Hhip is an inhibitor of Hedgehog (Hh) signalling, an embryonic developmental pathway which is also vital for normal T cell development. I first provided evidence that Foxn1 indirectly modulated the Hh signalling pathway via hhip. To identify a role for Hhip in thymus biology, I investigated the consequences of a constitutive deficiency for hhip expression in embryonic and neonatal mice, and demonstrated that loss of hhip resulted in an upregulation of Hh signalling via the coreceptors Gas1 and Boc, affecting the relative frequencies of cortical and medullary TECs in a dose-dependent manner and favouring medullary TEC development. I also generated a novel transgenic mouse model with a targeted loss of hhip in Foxn1-expressing cells, to specifically delineate the role of Hhip in TECs. This approach additionally circumvented the limitation of neonatal lethality in constitutive hhip-deficient mice. TEC-targeted loss of hhip expression not only impacted TEC sublineage decisions, but also affected the ability of TECs to perform positive thymocyte selection, resulting in the generation of T cells with reduced TCR signal strength which were less responsive to mitogenic stimuli. In aggregate, the experimental data here presents the first evidence that Hhip plays an important role in regulating TEC differentiation and function.

碩士
南台科技大學
生物科技系
93
The Hz-1 virus is a member of insect baculovirus, which can establish both persistent and productive infection in several lepidopteran cell lines. One results revealed that both heat and UV treatments of Hz-1 virus reduced the enhancement of hhi1 promoter activity indicating that the protein(s) on the nucleocapsid was candidate of activator to enhance the expression of hhi1. We further cloned Hz1V011 and Hz1V046, the two ORFs showing homologies to two baculovirus structure proteins: p74 and vp91-capsid associated protein, and performed cotransfection of pKS/hsp-V011 and pKS/hsp-V046 with a plasmid which expressed luciferase gene by hhi1 promoter (-688/+277) respectively. The results showed that both Hz1V011 and Hz1V046 could activate hhi1 promoter (-688/+277). Previous studies of temporal hhi1 promoter activity revealed that hhi1 promoter activity decreased from (-688/+277) to (-403/+277). These results strongly suggested that activator(s) bind to hhi1 promoter at region between –688 and -403. The computer similarity analysis of region between –688 and -403 with database of HGP in Japan showed 100% identity of five motifs: HSF (-592/-588, and –645/-641), CdxA (-480/-485), AML-1a (-655/-660, -578/-584), within –688/-403. We subjected gel mobility shift assay with fragment (–688/-557) labeled with Cy-3, however, failed to find any protein binding to this region. In order to study the profile of HHI1 during viral infection, we prepared an antibody generated by injecting New Zealand white rabbit with antigen covering amino acid number 200 to 400 of HHI1. From the results of Western blot, a 40-kd subunit was detected by 12 hours after Hz-1 viral infection and decreased 24 hours after infection. This data suggested that HHI1 protein could be cleavaged during viral infection.

博士
國立成功大學
生物科技研究所碩博士班
96
Previously we found that the upstream region of polh gene of baculovirus can induce a high level background expression of the CMVm promoter, therefore impossible to establish a tetracycline regulatory expression system (TRES) in baculovirus. In this study, we disrupted this upstream region of the polh gene, and for the first time successfully constructed a TRES in the genome of the baculovirus AcMNPV. We have used this system to regulate the expression of one of the early gene of baculovirus, lef2. LEF2 is one of the 18 late expression factors found in AcMNPV. Its cellular function is not yet fully understood, however, it is known that all lefs genes plays important roles during late and very late gene expression stages in baculovirus life cycle. We have over-expressed lef2 gene using the TRES, and discovered a ten-fold up-regulation of the polh promoter. The inducible TRES is therefore a useful tool in examining the cellular function of a particular baculovirus gene, as well as for stimulating the late or very late baculovirus promoters (p10 or polh) for protein expression. Another focus of this study is finding the early gene regulating mechanism of the HzNV-1 virus. HzNV-1 is a type viral species which can produce both latent and productive infections in insect cells. Previously, our laboratory have identified two highly-expressed transcripts, HzNV-1 Hind III-1 (hhi1) and persistency-associated gene 1 (pag1), from the HzNV-1 infected Trichoplusia ni TN-368 cells. In Sf-21 cells, the hhi1 gene can be activated by either AcMNPV or HzNV-1 virus infection. By co-transfection with AcMNPV viral DNA, we identified the active promoter region to be -62~+227 of the hhi1 gene. By progressive deletion of viral genomic DNA, we found two AcMNPV genes, ie1 and p35, together played an important role in hhi1 gene activation. Microarray study showed that two AcMNPV early genes, orf21 and orf25, also regulated by the co-operation between IE1 and P35, suggesting this co-operative gene regulation mechanism using ie1 and p35 gene products also occurred in the regulation of AcMNPV genome. We therefore discovered a new gene regulation mechanism within the AcMNPV genome. The co-operation between ie1 and p35 is not only up-regulates hhi1 gene expression, but also regulates early gene expression in the genome of AcMNPV. We also demonstrated that the expression of hhi1 gene in Sf-21 cells was sufficient to induce apoptotic cell death, mainly via the caspase 3 dependent pathway. Currently, two genes, the Hz-iap2 and PAT1 from HzNV-1, can inhibit hhi1-induced apoptosis.

碩士
國立臺灣師範大學
生物學系
84
Hz-1 virus is an insect virus. It can induce both productive and persistent viral infections in the Lepidopteran culture cells. In order to understand the molecular switches of productive and persistent viral infections, gene expression and regulation specifically associated to these two phases should be compared. During persistent viral infection, there is only one gene, the persistence-associated gene 1(pag1), is expressed. Since pag1 is also expressed as an immediate early (IE) gene during productive viral infection, it is necessary to compare pag1 and other productive specific IE genes to illucidate the controlling mechanisms as why all the other productive-specific IE genes are turned off during persistent viral infection but not the pag1 gene. Most of the immediate early genes of Hz-1 virus were found to locate in Hind III A, D, and I fragments previously. In this study, a sequence of 7704 base pair region has been determined in Hind III-I fragment. Computer analysis revealed the presence of two Open Reading Frames(ORF) of 1851 bp and 3426 bp onthis region. Between two ORFs, a 431 bp gap region was found. The 3''-RACE study showed the 3'' end of hhi1 gene is located at nucleotide 7478.TestCode analysis of hhi1 gene and the result of RT-PCR showed that the gap region between these two ORFs is not removed by splicing. Forthermore, in vitro translation assay also revealed that a 226 KD protein could be generated by hhi1 gene suggesting that it is resulted by a single ORF of 5670 base long.

Hhip (Hedgehog interacting protein), un antagoniste de la voie de signalisation Hegehog (Hh) a était devouverte comme un antagoniste des 3 ligands Hh, soit Sonic (Shh), Indian (Ihh) et Desert (Dhh). La protéines Hhip régularise la fonction cellulaire autant par voie (Hh) canonique que non-canonique. Elle est formée de 700 acides aminés et est fortement exprimée dans les tissus riches en cellules endothéliales, comme les reins et le pancréas. Toutefois, son rôle dans le fonctionnement des cellules bêta matures soit en condition de bonne santé ou de maladie comme dans des conditions d’obésité provoquée par une diète riche en gras ainsi que son role dans les maladies chronique du rein et la dysfonction rénale. Les souris en déficience de Hhip (Hhip-/-) ont une malformation des ilots pancréatiques (une diminution de 45% des ilots et de 40% de la prolifération des cellules beta) et un problème pulmonaire qui cause la mort post-natale. L’objectif de notre étude initiale était de démontrer le role de Hhip dans le pancréas, en utilisant un KO corporel entier en réponse à une diète riche en gras (HFD) et la dysfonction des cellules beta in vivo et ex vivo sur des souris hétérozygotes pour Hhip (Hhip+/-) et des souris contrôles (Hhip +/+) Suite à une HFD, toutefois, les souris mâles et femelles HFD-Hhip+/+ ont développé une intolérance sévère au glucose (IPGTT) et cette intolérance a été améliorée chez les souris HFD-Hhip+/-. Associé a cette intolérance, les males HFD-Hhip+/- démontraient une hyperinsulinémie et leur taux d’insuline plasmatique (phase 1 et 2), contrairement aux souris males HFD-Hhip+/+, augmentait de façon significative. Dans les îlots de souris Hhip+/+, l’augmentation de Hhip induite par une HFD a été observée principalement dans les cellules bêta mais aucunement dans les cellules alpha. Sans varier le nombre total d’îlots et la quantité de cellules bêta, les souris mâles HFD-Hhip+/+ avaient un nombre supérieur de gros îlots dans lesquels le taux d’insuline était diminué. La structure de ces îlots était désorganisée, démontrant une évidente invasion des cellules alpha au coeur des îlots bêta, le stress oxidatif (8-OHdG et NADPH oxidase 2 (Nox 2)) est aussi augmentée. En revanche, chez les souris mâles HFD-Hhip+/-, il a été possible d’observer une augmentation du nombre de petits îlots, de la prolifération des cellules bêta, et aussi de la sécrétion d’insuline stimulée par le glucose (GSIS), une amélioration du stress oxidatif et un maintien de l’intégrité des îlots ont été démontré. In vitro, la protéine recombinante Hhip (rHhip) a accentué le stress oxidatif (Nox2 et l’activité de NADPH oxidase 2) et a causé une diminution du nombre de cellules bêta ; par contre, le siRNA-Hhip augmente le GSIS et abolit la stimulation de l’expression du gène Nox2 induite par le palmitate de sodium (PA)-BSA. Grace a ces observations, il est démontré que les genes Hhip pancréatiques inhibe la sécrétion d’insuline en altérant la structure des ilots et en favorisant l’expression du gene Nox2 dans les ilots en réponse à la dysfonction des cellules beta suite a une diète riche en gras HFD. Le diabète engendre des risques élevés de complication tel que des problèmes chroniques des reins caractérisés par une perte graduelle des fonctions rénales. Cette situation a été récemment reliée au taux élevé d’obésité. On a aussi démontré dans notre modèle de diabète gestationnel que l’augmentation de Hhip causait des irrégularités durant la néphrogénèse des rejetons [127]. Ensuite, nos données récentes démontrent que, chez les souris adultes, l’hyperglycémie a provoqué une forte expression du gene Hhip rénales causant ainsi l’apoptose des cellules épithéliales des glomérules et la transition endothéliale à mésenchymateuse (EndoMT) - liée à fibrose rénale [128]. Dans l’étude présente, on a établi que la surexpression de Hhip dans les cellules des tubules proximaux rénaux contribuait au développement initial des problèmes chroniques des reins suite a une HFD de 14 semaines. Un gain de poids significatif a été observé chez les souris du groupe HFD comparativement aux groupes ND. Les souris du groupe HFD ont développé une intolérance au glucose mais sans changement apparent à la sensibilité à l’insuline ni à l’hypertension (pression arterielle) même si ces souris mâles avaient des légers dépôts du gras périrénal. Les fonctions rénales telle que mesurées par le taux de filtration glomérulaire restaient normales dans tous les groupes révélant ainsi que ces deux facteurs (HFD et surexpression de Hhip) n’avaient aucune influence sur l’hyperfiltration rénale. Néanmoins, la morphologie rénale a révélé que les souris du groupe HFD présentaient une lésion infraclinique et des signes de vacuolisation tubulaire et des lésions par rapport aux souris ND. Cette pathologie de lésion tubulaire et de vacuolisation était plus prononcée chez les souris transgéniques (Hhip-Tg) que chez les souris non-Tg, ce qui favorisait l'apoptose des cellules tubulaires bénignes et un stress oxydatif accru. En conclusion, l'obésité provoquée par l'HFD a eu des effets néfastes sur la tolérance au glucose et de légères modifications morphologiques des reins, caractérisées par la présence d'une néphrose osmotique, une augmentation du stress oxydatif rénal et une apoptose pouvant être induites par une augmentation de la FABP4 rénale. Cela a été exacerbé par la surexpression de Hhip dans les tubules rénaux proximaux.
Hedgehog interacting protein (Hhip), a signaling molecule in the Hedgehog Hh pathway, was originally discovered as a putative antagonist of all 3 secreted Hh ligands, i.e., Sonic (Shh), Indian (Ihh), and Desert (Dhh). Hhip regulates cell function via either canonical- or non-canonical Hh pathway. Hhip encodes a protein of 700 amino acids, and is abundantly expressed in vascular endothelial cell-rich tissues, including the pancreas, and kidneys. To date, less is known about Hhip’s expression pattern in mature islet cells, and its function under normal and/or disease conditions, such as diet induced-obesity, as well as its role in chronic kidney disease, and kidney dysfunction. Hhip null mice (Hhip-/-) display markedly impaired pancreatic islet formation (45% reduction of islet mass with a decrease of beta cell proliferation by 40%), however Hhip-/- mice die shortly after birth mainly due to lung defects. In our first study, we systemically studied the role of pancreatic Hhip expression by using a whole body knock out in response to 8 weeks high fat diet (HFD) insult, and HFD-mediated beta cell dysfunction in vivo, ex vivo and in vitro using heterozygous (Hhip+/-) vs. wild type (Hhip+/+) mice. Both HFD-fed Hhip+/+ male and female mice developed severe glucose intolerance (IPGTT), which was ameliorated in male and female HFD-Hhip+/- mice. Associated with this glucose intolerance, was hyperinsulinemia, which was observed only in HFD-fed male Hhip+/- mice. HFD-fed Hhip+/- mice had high levels of circulating plasma insulin in both insulin secretion phases compared to HFD fed Hhip+/+ mice. In the pancreas, Hhip expression was increased in the islets of HFD-Hhip+/+ mice, mainly co-localized in beta cells and none in alpha cells. While maintaining the total islet number, and beta cell mass, male HFD-Hhip+/+ mice had a higher number of larger islets, in which insulin content was reduced; islet architecture was disoriented, with evident invasion of alpha cells into the central core of beta cells; and an evident increase in oxidative stress markers (8-OHdG and NADPH oxidase 2 (Nox 2)). In contrast, male HFD-Hhip+/- mice had a higher number of smaller islets, with increased beta cell proliferation, pronounced glucose stimulated insulin secretion (GSIS), ameliorated oxidative stress and preserved islet integrity. In vitro, recombinant Hhip (rHhip) dose-dependently increased oxidative stress (Nox2 and NADPH activity), and decreased the number of insulin-positive beta cells, while siRNA-Hhip enhanced GSIS, and abolished the stimulation of sodium palmitate (PA)-BSA on Nox2 gene expression. We believe our data highlights a novel finding as to how pancreatic Hhip gene inhibits insulin secretion, by altering islet integrity, and promoting Nox2 gene expression in beta cells in response to HFD-mediated beta cell dysfunction. Diabetes presents high risk factors associated with complications such as chronic kidney disease (CKD) characterized by a gradual loss in kidney function. The increased incidence of diabetic related kidney complications has been recently correlated with increase rate of obesity. We recently established that impaired nephrogenesis in kidneys of offsprings of our murine model of maternal diabetes was associated with upregulation of Hhip gene expression [127]. Subsequently, our recent data also shows that hyperglycemia induced increased renal Hhip gene expression in adult murine kidneys leading to apoptosis of glomerular epithelial cells and endothelial to mesenchymal transition (Endo-MT) - related renal fibrosis [128]. In this current study, we demonstrated how Hhip overexpression in renal proximal tubular cells, contributes to early development of chronic kidney disease after 14 weeks of HFD. Mice in HFD-fed groups showed significantly greater weight gain as compared to mice in ND fed groups. IPGTT revealed that HFD fed mice also developed glucose intolerance, with no apparent changes in insulin sensitivity. HFD did not impact hypertension, even though we had a modest trend of increase in perirenal fat deposit in the HFD fed subgroups. Renal function as measured by the glomerular filtration rate was normal in all four subgroups, indicating that neither HFD, nor Hhip overexpression promoted renal hyperfiltration. Nonetheless, renal morphology revealed HFD kidneys had subclinical injury, presented signs of tubular vacuolization and damage compared to ND fed mice. This pathology of tubular damage and vacuolization was more pronounced in HFD-fed transgenic (Hhip-Tg) mice compared to non-Tg mice, and this promoted mild tubular cell apoptosis and enhanced oxidative stress. In conclusion, HFD feeding-induced obesity led to detrimental effects on glucose toleranc,e and mild morphological changes in kidneys, characterized by the presence of osmotic nephrosis, increased renal oxidative stress, and apoptosis which might be mediated by an increase in renal FABP4. This was exacerbated by the over-expression of Hhip in the renal proximal tubules.

碩士
國立陽明大學
生化暨分子生物研究所
102
The cross-talk between oncogenic pathways and stemness pathways play important roles in tumor initiation and progression. Recent years, the aberrant activations of stemness signaling such as Hedgehog (HH), hypoxia-inducible factor, and Wnt pathways, and the stemness factors like Oct-4 and Sox-2 have been reported in lung cancers. However, most researches to date focused on the impact of positive regulators of stemness pathways in oncogenesis, but less on the importance of negative regulators. Hedgehog interaction protein (HHIP) is a membrane protein that binds to HH ligands with an affinity comparable to Ptch-1 (the native HH ligand receptor), and HHIP overexpression attenuates HH signaling by capturing HH ligands. HHIP has been found to be down-regulated in several types of cancers through promoter hyper-methylation. In lung cancer, however, its role and importance has not been identified. Here, we show that HHIP was significantly repressed in lung cancer cell lines and human lung tumor samples through epigenetic silencing. Overexpression of HHIP in lung cancer cells blocked the auto-loop induction of endogenous HH pathway, and inhibited the invasiveness of cancer cells. We also found that in starvation state, HH pathway was autonomously induced which then mediated the expression of HGF and cMET phosphorylation, while HHIP overexpression blocked such inductions and significantly repressed cell proliferation rate. Furthermore, HHIP reduced the size of spheroids formed by lung cancer cells in serum-free 3D matrix. In summary, our results indicate that HHIP is a key regulator of HH signaling that was silenced in lung cancer and thus potentiates cancer cells to activate HH stemness pathway in adverse conditions to acquire survival and metastasis abilities.