Academic literature on the topic 'Hedgehog interacting protein'

Basal cell carcinomas (BCCs) are driven by abnormal hedgehog signaling and highly overexpress several hedgehog target genes. We report here our use of one of these target genes, hedgehog-interacting protein (Hip1), as a tumor-associated antigen for immunoprevention of BCCs in Ptch1+/− mice treated with ionizing radiation. Hip1 mRNA is expressed in adult mouse tissues at levels considerably lower than those in BCCs. Immunization with either of two large recombinant Hip1 polypeptides was well tolerated in Ptch1+/− mice, induced B and T cell responses detectable by enzyme-linked immunosorbent assay, Western blot, delayed type hypersensitivity, and enzyme-linked immunospot assay, and reduced the number of BCCs by 42% (P < 0.001) and 32% (P < 0.01), respectively. We conclude that immunization with proteins specifically up-regulated by hedgehog signaling may hold promise as a preventive option for patients such as those with the basal cell nevus syndrome who are destined to develop large numbers of BCCs.

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.

The Hedgehog (Hh) pathway is an embryonic development pathway, driven by peptide ligands called hedgehogs. During progression of prostate cancer (PCa), Hh signaling is increased, with especially high activation in castration resistant disease (CRPC). Evidence is lacking for canonical Hh signaling in PCa, indicating the likelihood that non-canonical pathways are involved. Our work shows that transcriptionally active androgen receptor (AR) binding to Gli proteins drives non-canonical Hh signaling in PCa. Androgen-sensitive LNCaP and androgen-independent LNCaP-AI and LN95 cells were transfected with a Gli-promoter driven luciferase reporter and treated with R1881, enzalutamide, or both, and luciferase activity was measured. Androgen treatment (R1881) induced Gli transcriptional activity while enzalutamide reversed this effect. Similarly, siRNA knockdown of full-length AR (AR-FL) suppressed R1881-induced Gli transcription. Western blot and qPCR confirmed increased expression of endogenous Gli target genes Gli1 and Ptch1 with androgen treatment. Androgen treatment stabilized expression of full-length active Gli3 in a dose-dependent manner but this was reversed by AR knockdown using siRNA. AR binds to Gli3 at the protein processing domain (PPD), which, given the above data, suggests that AR binding to Gli stabilizes full-length Gli3 by preventing phosphorylation and ubiquitination of the PPD, thereby stopping proteolytic cleavage and proteosomal degradation from occurring. Finally, we found that an AR-binding decoy peptide derived from the Gli2 C-terminus can compete with Gli3 for binding to AR, suppressing Gli transcriptional activity in PCa cells. Our data supports the idea that transcriptionally active AR binding to Gli proteins provides a means for Hh signaling to occur. Not only does AR co-activate Gli transcriptional activity, but it also alters the proteolytic processing of Gli proteins by preventing phosphorylation and ubiquitination of the Gli PPD by competing with β-TrCP for binding to Gli. Collectively, our findings show the importance of AR-Gli interaction in PCa progression.
Medicine, Faculty of
Graduate

La régulation post-transcriptionnelle de l'expression génique joue un rôle essentiel dans divers processus cellulaires pendant le développement. Les protéines de liaison à l'ARN (RBP) sont des médiateurs fondamentaux des régulations post-transcriptionnelles qui contrôlent l'expression de l'ARNm en reconnaissant des séquences spécifiques dans les transcrits cibles. Smaug est une protéine de liaison à l'ARN conservée de la levure jusqu’à l’homme qui est essentielle pendant l'embryogenèse précoce de la drosophile. Smaug reconnaît et lie des éléments de reconnaissance de Smaug (SRE) dans ses ARNm cibles et recrute des facteurs supplémentaires, via des interactions protéine-protéine, qui régulent l'ARNm lié. Un concept qui émerge est celui des voies de signalisation pouvant moduler l'activité des RBP par des modifications post-traductionnelles, en ajoutant ainsi une couche supplémentaire dans le contrôle de l'expression des gènes.Au cours de mon travail de thèse, j'ai cherché à mettre en évidence que la voie de signalisation Hedgehog régule Smaug en favorisant sa phosphorylation. Mon travail montre que la signalisation HH diminue les niveaux de protéines Smaug affectant sa capacité à réprimer la traduction de l'ARNm. Cet effet négatif semble dépendre de l'interaction entre Smaug et le transducteur de signal HH, Smoothened. De plus, Smaug est constitutivement phosphorylée dans son domaine de liaison à l'ARN, ce qui semble être nécessaire pour la formation des foci cytoplasmiques de Smaug
Post-transcriptional regulation of gene expression plays a critical role in a variety of cellular processes during development. RNA binding proteins are fundamental mediators of post-transcriptional regulations that control mRNA expression by recognizing specific cis acting elements within the target transcripts. Smaug is a highly conserved sequence specific RNA-binding protein that is essential during Drosophila early embryogenesis. Smaug binds Smaug Recognition Elements (SRE) in the target mRNA and recruits additional factors, via protein-protein interactions, that regulate the bound mRNA. An emergent concept that signaling pathways can modulate RBP activity by post-translation modifications adds a new layer in the control of gene expression. During my thesis work, I sought to understand how the Hedgehog pathway regulates Smaug by promoting its phosphorylation. My work shows that HH signaling downregulates Smaug protein levels affecting its ability to repress mRNA translation. This negative effect seems to be dependent on the interaction between Smaug and the HH signal transducer Smoothened. Moreover, Smaug is constitutively phosphorylated in its RNA binding domain, which appears to be necessary for cytoplasmic Smaug foci formation

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.

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.

While hyaluronic acid (HA) hydrogels provide a stable 3D environment that is conducive to the chondrogenesis of mesenchymal stem cells (MSCs) in the presence of growth factors [1], the neocartilage that is formed remains inferior to native tissue, even after long culture durations. Additionally, MSCs eventually transit into a hypertrophic phenotype after chondrogenic induction, resulting in the calcification of the ECM after ectopic transplantation [2]. From a material design perspective, variation in the HA hydrogel scaffold crosslinking density via changes in the HA macromer concentration can influence chondrogenesis of MSCs and neocartilage formation [3]. Recent studies have also demonstrated that dynamic compression enhances the expression of chondrogenic markers and cartilage matrix synthesis by MSCs encapsulated in various hydrogels, including agarose [4], alginate [5] and fibrin [6]. Furthermore, mechanical signals also regulate growth plate and articular cartilage chondrocyte hypertrophy via the IHH-PTHrP (India hedgehog, Parathyroid hormone-related protein) pathway [7]. In contrast to biologically inert scaffold materials, HA is capable of interacting with cells (including MSCs) via cell surface receptors (CD44, CD54, and CD168) [8; 9]. Therefore the objectives of this study were to (i) evaluate the effects of both hydrogel crosslinking and dynamic compressive loading on (i) chondrogenesis and cartilage matrix production/distribution of human MSCs encapsulated in HA gels and (ii) hypertrophic differentiation of human MSCs using an in vitro MSC hypertrophy model [10].