Ihh Signaling Research Articles

Tougu Xiaotong formula induces chondrogenic differentiation in association with transforming growth factor-β1 and promotes proliferation in bone marrow stromal cells.

Indian hedgehog (Ihh), one of the hedgehog gene families, is indicated in the regulation of chondrocyte differentiation. Tougu Xiaotong formula (TXF), a traditional Chinese medicinal compound, has been used for the treatment of bone and joint disease. However, the underlying molecular mechanisms of TXF on the function of bone marrow stromal cells (BMSCs) remain unclear. In the present study, the affect of TXF on proliferation and chondrogenic differentiation was investigated in primary BMSCs from four‑week‑old Sprague Dawley rats. The cell viability in BMSCs treated with TXF was higher compared to the untreated cells. Additionally, the percentage of G(0)/G(1) phase cells was significantly decreased, whereas that of the S phase cells was significantly increased. Furthermore, following TXF treatment, cyclin D1, cyclin‑dependent kinase 4 (CDK4) and CDK6 expression in BMSCs was significantly enhanced. The results showed that TXF had no cytotoxicity to BMSCs. To explore the effect of TXF on the differentiation in BMSCs, whether TXF induced chondrogenic differentiation of BMSCs by the regulation of Ihh signaling pathway was investigated. The protein expression of Ihh, Patched and Smoothened in the induction group were significantly increased when compared to those in the control group, and the highest protein level of Ihh was in the induction group that was treated with the combination of TXF and transforming growth factor‑β1 (TGF‑β1). In addition, TXF combined with TGF‑β1 significantly induced the protein expression of cartilage oligomeric matrix protein and collagen II compared to the TGF‑β1 group. Taken together, these results indicate that TXF promotes the proliferation via accelerating the G(1)/S transition, and induces chondrogenic differentiation in BMSCs by activation of the Ihh signaling pathway in association with TGF‑β1.

Mesenchymal stem cells overexpressing Ihh promote bone repair.

BackgroundIndian hedgehog (Ihh) signaling pathway is known to play key roles in various aspects of normal endochondral bone development. This study tested the potential roles of high Ihh signaling in the context of injury-induced bone regeneration.MethodsA rabbit tibia defect model was established to test the effects of the implant of Ihh/mesenchymal stem cells (MSCs)/scaffold complex. Computed tomography (CT), gross observation, and standard histological and immunohistological techniques were used to evaluate the effectiveness of the treatment. In vitro studies with MSCs and C3H10T1/2 cells were also employed to further understand the cellular and molecular mechanisms.ResultsWe found that the implanted Ihh/MSCs/scaffold complex promoted bone repair. Consistently, in vitro study found that Ihh induced the upregulation of chondrocytic, osteogenic, and vascular cell markers, both in C3H10T1/2 cells and MSCs.ConclusionsOur study has demonstrated that high Ihh signaling in a complex with MSCs enhanced bone regeneration effectively in a clinically relevant acute injury model. Even though the exact underlying mechanisms are still far from clear, our primary data suggested that enhanced chondrogenesis, osteogenesis, and angiogenesis of MSCs at least partially contribute to the process. This study not only has implications for basic research of MSCs and Ihh signaling pathway but also points to the possibility of direct application of this specific paradigm to clinical bone repair.Electronic supplementary materialThe online version of this article (doi:10.1186/s13018-014-0102-7) contains supplementary material, which is available to authorized users.

Overexpression of Shox2 leads to congenital dysplasia of the temporomandibular joint in mice.

Our previous study reported that inactivation of Shox2 led to dysplasia and ankylosis of the temporomandibular joint (TMJ), and that replacing Shox2 with human Shox partially rescued the phenotype with a prematurely worn out articular disc. However, the mechanisms of Shox2 activity in TMJ development remain to be elucidated. In this study, we investigated the molecular and cellular basis for the congenital dysplasia of TMJ in Wnt1-Cre; pMes-stop Shox2 mice. We found that condyle and glenoid fossa dysplasia occurs primarily in the second week after the birth. The dysplastic TMJ of Wnt1-Cre; pMes-stop Shox2 mice exhibits a loss of Collagen type I, Collagen type II, Ihh and Gli2. In situ zymography and immunohistochemistry further demonstrate an up-regulation of matrix metalloproteinases (MMPs), MMP9 and MMP13, accompanied by a significantly increased cell apoptosis. In addition, the cell proliferation and expressions of Sox9, Runx2 and Ihh are no different in the embryonic TMJ between the wild type and mutant mice. Our results show that overexpression of Shox2 leads to the loss of extracellular matrix and the increase of cell apoptosis in TMJ dysplasia by up-regulating MMPs and down-regulating the Ihh signaling pathway.

Indian hedgehog signaling regulates transcription and expression of collagen type X via Runx2/Smads interactions.

Indian hedgehog (Ihh) is essential for chondrocyte differentiation and endochondral ossification and acts with parathyroid hormone-related peptide in a negative feedback loop to regulate early chondrocyte differentiation and entry to hypertrophic differentiation. Independent of this function, we and others recently reported independent Ihh functions to promote chondrocyte hypertrophy and matrix mineralization in vivo and in vitro. However, the molecular mechanisms for these actions and their functional significance are still unknown. We recently discovered that Ihh overexpression in chondrocytes stimulated the expression of late chondrocyte differentiation markers and induced matrix mineralization. Focusing on collagen type X (Col10α1) expression and transcription, we observed that hedgehog downstream transcription factors GLI-Krüppel family members (Gli) 1/2 increased COL10A1 promoter activity and identified a novel Gli1/2 response element in the 250-bp basic promoter. In addition, we found that Ihh induced Runx2 expression in chondrocytes without up-regulating other modulators of chondrocyte maturation such as Mef2c, Foxa2, and Foxa3. Runx2 promoted Col10α1 expression in cooperation with Ihh. Further analyses using promoter assays, immunofluorescence, and binding assays showed the interaction of Gli1/2 in a complex with Runx2/Smads induces chondrocyte differentiation. Finally, we could demonstrate that Ihh promotes in vitro matrix mineralization using similar molecular mechanisms. Our data provide an in vitro mechanism for Ihh signaling to positively regulate Col10α1 transcription. Thus, Ihh signaling could be an important player for not only early chondrocyte differentiation but maturation and calcification of chondrocytes.

Fetal jaw movement affects Ihh signaling in mandibular condylar cartilage development: The possible role of Ihh as mechanotransduction mediator

ObjectiveJaw movement is an important mechanical factor for prenatal development of the condylar cartilage of mandible. Fetal jaw movement restriction has been shown to cause deformity of the mandibular condyle. We hypothesized that this treatment affects the expression of mechanosensitive molecules, namely Indian hedgehog (Ihh) and Parathyroid hormone related protein (PTHrP) in the condyle. Experimental methodsWe restrained jaw movement by suturing the jaw of E15.5 mouse embryos and allowed them to develop until E18.5 using exo utero system, and analyzed them by immunohistochemistry and in situ hybridization methods. ResultsMorphological, histomorphometric and immunohistochemical study showed that the mandibular condylar cartilage was reduced and deformed, the volume and total cell numbers in the condylar cartilage were also reduced, and number and/or distribution of 5-bromo-2′-deoxyuridine-positive cells, Ihh-positive cells in the mesenchymal and pre-hypertrophic zones were significantly and correspondingly decreased in the sutured group. Using in situ hybridization, reduced expression of Ihh, PTHrP and their related receptors were observed in condylar cartilage of the sutured embryos. ConclusionsOur results revealed that the altered mechanical stress induced by prenatal jaw movement restriction decreased proliferating cells, the amount of cartilage, and altered expression of the Ihh and PTHrP, suggesting that Ihh act as mechanotransduction mediators in the development of mandibular condylar cartilage.

Wnt7b can replace Ihh to induce hypertrophic cartilage vascularization but not osteoblast differentiation during endochondral bone development.

Indian hedgehog (Ihh) is an essential signal that regulates endochondral bone development. We have previously shown that Wnt7b promotes osteoblast differentiation during mouse embryogenesis, and that its expression in the perichondrium is dependent on Ihh signaling. To test the hypothesis that Wnt7b may mediate some aspects of Ihh function during endochondral bone development, we activated Wnt7b expression from the R26-Wnt7b allele with Col2-Cre in the Ihh−/− mouse. Artificial expression of Wnt7b rescued vascularization of the hypertrophic cartilage in the Ihh−/− mouse, but failed to restore orthotopic osteoblast differentiation in the perichondrium. Similarly, Wnt7b did not recover Ihh-dependent perichondral bone formation in the Ihh−/−; Gli3−/− embryo. Interestingly, Wnt7b induced bone formation at the diaphyseal region of long bones in the absence of Ihh, possibly due to increased vascularization in the area. Thus, Ihh-dependent expression of Wnt7b in the perichondrium may contribute to vascularization of the hypertrophic cartilage during endochondral bone development.

Indian Hedgehog Mediates Gastrin-Induced Proliferation in Stomach of Adult Mice

Loss of expression of Sonic Hedgehog (Shh) from parietal cells results in hypergastrinemia in mice, accompanied by increased expression of Indian Hedgehog (Ihh) and hyperproliferation of surface mucous cells. We investigated whether hypergastrinemia induces gastric epithelial proliferation by activating Ihh signaling in mice. We studied mice with parietal cell-specific deletion of Shh (PC-Shh(KO)) and hypergastrinemia, crossed with gastrin-deficient (GKO) mice (PC-Shh(KO)/GKO). When mice were 3-4 months old, gastric tissues were collected and analyzed by histology, for incorporation of bromodeoxyuridine, and for expression of the surface mucous cell marker Ulex europaeus. PC-Shh(KO)/GKO mice were given gastrin infusions for 7 days; gastric surface epithelium was collected and expression of Ihh was quantified by laser capture microdissection followed by quantitative reverse transcriptase polymerase chain reaction. Mouse stomach-derived organoids were incubated with or without inhibitors of WNT (DKK1) or Smoothened (vismodegib) and then cocultured with immortalized stomach mesenchymal cells, to assess proliferative responses to gastrin. Gastric tissues from PC-Shh(KO)/GKO mice with hypergastrinemia had an expanded surface pit epithelium, indicated by a significant increase in numbers of bromodeoxyuridine- and Ulex europaeus-positive cells, but there was no evidence for hyperproliferation. Gastrin infusion of PC PC-Shh(KO)/GKO mice increased expression of Ihh and proliferation within the surface epithelium compared with mice given infusions of saline. In gastric organoids cocultured with immortalized stomach mesenchymal cells, antagonists of WNT and Smoothened inhibited gastrin-induced proliferation and WNT activity. Activity of WNT in media collected from immortalized stomach mesenchymal cells correlated with increased expression of glioma-associated oncogene homolog 1, and was inhibited by DKK1 or vismodegib. Ihh signaling mediates gastrin-induced proliferation of epithelial cells in stomachs of adult mice.

Tu1651 Indian Hedgehog Is Required for Intestinal Adenoma Formation

Introduction: Activating mutations in the Hedgehog pathway are found in basal cell carcinomas and medulloblastomas. The role of Hedgehog signaling in intestinal tumorigenesis has not yet been clarified. Hedgehog is expressed by differentiated enterocytes and signals in a paracrine manner from the epithelium to the mesenchyme. Hedgehog controls mesenchymal factors such as Bone Morphogenetic Proteins and Activins which negatively regulate the proliferation of precursor cells. Since Indian Hedgehog (Ihh) is the major Hedgehog expressed in the intestinal epithelium we decided to study the potential role of Ihh signaling in intestinal adenoma formation.Methods: In order to study the effect of loss of Ihh signaling in sporadic tumorigenesis we conditionally deleted Ihh in adult mice in the intestinal epithelium using the Cyp1a1Cre promoter. These compound mice were crossed with conditional mutant Apc mice to generate Cyp1a1Cre-Ihh-Apc mice. At 4-7 weeks of age mice were intraperitoneally injected with 80 mg/kg β-naphthoflavone for 5 days. Cyp1a1Cre-IhhApc littermates served as controls. Four months after recombination, we sacrificed and analyzed the mice (n=15). Results: Ihh expression was upregulated in polyps of Cyp1a1CreIhh-Apc mice as assessed by in situ hybridization. Deletion of Ihh resulted in a marked reduction of 90.3% in the number of polyps (17.2 ± 13.65 polyps per mouse vs. 1.7 ± 1.5, P < 0.01). The average polyp size did not differ between groups. Neither did the number of proliferating or apoptotic cells in the polyps, as assessed by immunostaining for BrdU and cleaved caspase 3. Hh responsive cells in the adenomawere restricted to themesenchyme. In those adenomas that did develop in Ihh mutant mice, we observed a remarkable loss of α-sma/desmin double positive smooth muscle cells. COX2 is expressed early in adenoma to carcinoma transition and believed to play an important role in adenoma formation. Stromal cells are known to be a source of COX2. In accordance with this, examination of Cox2 expression by Q-PCR and immunostaining showed up regulation in polyps ofCyp1a1Cre-Ihh -Apc mice in compared to normal intestinal epithelium (P < 0.01, resp. P < 0.05). In contrast, Cox2 expression in the polyps of Cyp1a1Cre-Ihh-Apc mice was not significantly up regulated. Conclusion: Surprisingly, in contrast to its role as an anti-proliferative signal in the normal epithelium, Ihh acts as a tumor promoter. Our data show that Ihh signaling is increased during intestinal adenoma formation and as in the normal intestine Ihh signaling is exclusively from the epithelium to the mesenchyme. Loss of Ihh signaling is associated with major changes in the composition of the adenoma mesenchyme and decreased Cox2 expression, which significantly impairs adenoma formation.

Tu1653 Farnesoid X Receptor (FXR) Represses Matrix Metalloproteinase 7 (MMP7) Expression in Intestinal Epithelial Cells

Introduction: Activating mutations in the Hedgehog pathway are found in basal cell carcinomas and medulloblastomas. The role of Hedgehog signaling in intestinal tumorigenesis has not yet been clarified. Hedgehog is expressed by differentiated enterocytes and signals in a paracrine manner from the epithelium to the mesenchyme. Hedgehog controls mesenchymal factors such as Bone Morphogenetic Proteins and Activins which negatively regulate the proliferation of precursor cells. Since Indian Hedgehog (Ihh) is the major Hedgehog expressed in the intestinal epithelium we decided to study the potential role of Ihh signaling in intestinal adenoma formation.Methods: In order to study the effect of loss of Ihh signaling in sporadic tumorigenesis we conditionally deleted Ihh in adult mice in the intestinal epithelium using the Cyp1a1Cre promoter. These compound mice were crossed with conditional mutant Apc mice to generate Cyp1a1Cre-Ihh-Apc mice. At 4-7 weeks of age mice were intraperitoneally injected with 80 mg/kg β-naphthoflavone for 5 days. Cyp1a1Cre-IhhApc littermates served as controls. Four months after recombination, we sacrificed and analyzed the mice (n=15). Results: Ihh expression was upregulated in polyps of Cyp1a1CreIhh-Apc mice as assessed by in situ hybridization. Deletion of Ihh resulted in a marked reduction of 90.3% in the number of polyps (17.2 ± 13.65 polyps per mouse vs. 1.7 ± 1.5, P < 0.01). The average polyp size did not differ between groups. Neither did the number of proliferating or apoptotic cells in the polyps, as assessed by immunostaining for BrdU and cleaved caspase 3. Hh responsive cells in the adenomawere restricted to themesenchyme. In those adenomas that did develop in Ihh mutant mice, we observed a remarkable loss of α-sma/desmin double positive smooth muscle cells. COX2 is expressed early in adenoma to carcinoma transition and believed to play an important role in adenoma formation. Stromal cells are known to be a source of COX2. In accordance with this, examination of Cox2 expression by Q-PCR and immunostaining showed up regulation in polyps ofCyp1a1Cre-Ihh -Apc mice in compared to normal intestinal epithelium (P < 0.01, resp. P < 0.05). In contrast, Cox2 expression in the polyps of Cyp1a1Cre-Ihh-Apc mice was not significantly up regulated. Conclusion: Surprisingly, in contrast to its role as an anti-proliferative signal in the normal epithelium, Ihh acts as a tumor promoter. Our data show that Ihh signaling is increased during intestinal adenoma formation and as in the normal intestine Ihh signaling is exclusively from the epithelium to the mesenchyme. Loss of Ihh signaling is associated with major changes in the composition of the adenoma mesenchyme and decreased Cox2 expression, which significantly impairs adenoma formation.

The role of smoothened for the osteophyte formation in mouse osteoarthritis

Objective: Osteoarthritis (OA) shows a progressive degeneration of articular cartilage and osteophyte formation. Endochondral ossification is considered an important pathophysiological process for the osteophyte formation. Chondrocyte hypertrophy is also an essential process required for endochondral bone formation. It is reported that the expression of Indian hedgehog (Ihh) in pre- and early hypertrophic chondrocytes of growth plate and osteophytes, and a PTHrP-independent role of Ihh signaling in regulating chondrocyte hypertrophy. Therefore, in this study, we investigated whether the deletion of smoothened (Smo), an essential transducer of Hedgehog signaling, affected osteophyte formation in mouse osteoarthritis. Methods: To investigate the role of Smo in osteophyte formation, we crossed Smo/flox mice, which express a null allele of Smo upon exposure to Cre recombinase, with RosaCreER(T) mice to generate Smo conditional knockout (CKO) mice upon administration of Tamoxifen. CKO mice and Cre-negative control mice were administrated Tamoxifen on 3, 5 weeks after birth and 3days, 1day before surgery, resulting in downregulated Hedgehog signaling. OA was surgically induced in 8-week-old CKO mice and Cre-negative control mice using the anterior cruciate ligament transection model. 1week after surgery, mice were caged in climbing cage to accelerate degenerative change. Mice were sacrificed at 4, 6, 8 weeks after surgery. Histomorphometric and immunohistochemical (IHC) assessment was performed. Result: The immature osteophyte-like lesion composed of chondrocytes and cartilage was observed at the posterior margins of tibia in both CKO mice and Cre-negative control mice in 4 weeks post-surgery. However, the maturity of osteophytes was suppressed in CKO mice compared to Cre-negative control mice at 6 and 8 weeks post-surgery. In contrast, Cre-negative control mice showed mature ossifying lesion and hypertrophic chondrocytes in osteophytes, and had a larger and more mature osteophytes at 6 and 8 weeks post-surgery by histomorphometric analysis. Furthermore, it is revealed that lesser expression of type X collagen in osteophytes of CKO mice. These data suggests that the deletion of Smo delayed the formation of osteophytes and the suppression of the endochondral ossification process with blocking the hypertrophy of chondrocytes and type X collagen expression. Conclusions: Smo plays a crucial role of osteophyte formation in mouse osteoarthritis, especially, the maturity of osteophytes through endochondral ossification process.

Stem cells and their niche in homeostasis/regeneration and disease

The Minisymposium ”Stem Cells and Their Niche in Homeostasis/Regeneration and Disease” was aimed at highlighting the importance of cell biology aspects in understanding stem cell biology. Tudorita (Doina) Tumbar (Cornell University) started the session by summarizing two models of stem cell behavior during normal homeostasis. Stem cells can divide asymmetrically to generate a stem cell and a more differentiated cell. Conversely, she showed that hair follicle stem cells behave as a population: some differentiate and eventually die, while others self-renew by symmetric divisions. During hair follicle stem cell G0 quiescence, the transcription factor Runx1 drives a reversible step of differentiation to progenitors. The latter succumb irreversibly to rapid proliferation and terminal differentiation upon activation of tissue signals. Pantelis Rompolas (Valentica Greco's Lab, Yale University) reported evidence for a direct link between a specific niche location and cell fate, using in vivo imaging to trace single hair-follicle stem cells long term in live mice. Furthermore, by utilizing laser ablation, he showed that hair follicle stem cells may be dispensable and that other epithelial populations can be reprogrammed to support hair regeneration by entering the niche. Gage Crump (University of Southern California) presented evidence for a transfating of chondrocytes to the osteoblasts that rebuild bone during jawbone regeneration in zebrafish. Such transfating is not observed during development of the same bone, challenging the notion that adult regeneration of an organ simply recapitulates its development. He also presented evidence for a novel role of Ihh signaling in this process and for a similar process in mammalian bone regeneration. Heike Kroeger (Jonathan Lin's Lab, University of California, San Diego) presented a study that links unfolded protein response signaling to hESC differentiation and maturation via the function of ATF6. hESC activates the ATF6 signaling during hESC differentiation through the FGF2 signaling cascade. ATF6 signaling leads to dramatic expansion of the endoplasmic reticulum (ER) and enhances hESC differentiation. These findings provide a new mechanism underlying hESC differentiation that involves ATF6’s induction and activation of ER functions. Daniela Malide (National Institutes of Health) reported the use of combinatorial 5 fluorescent proteins to mark hematopoietic stem and progenitor cells that allowed in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allowed noninvasive fate mapping of multicolored hematopoietic stem/progenitor cell–derived cells in intact nonhematopoietic tissues for extensive periods of time following transplantation, demonstrating clearly delineated clones progressing from multicolored to monochromatic over time. Abby Gerhold (Paul Maddox's Lab, University of North Carolina, Chapel Hill) reported in vivo live imaging of Caenorhabditis elegans germ-line stem cells to investigate how mitotic progression is influenced by the physiological context. She provided evidence that 1) the duration of chromosome congression is reduced during developmental expansion of the stem cell pool, and 2) dietary restriction induces a spindle assembly checkpoint–dependent delay in anaphase onset, suggesting the basic mitotic process is affected by physiological factors. Yukiko Yamashita (University of Michigan, Ann Arbor) discussed her group's recent identification of Klp10A kinesin as the first stem cell–specific centrosomal component in the Drosophila male germ line. Klp10A seems to specifically regulate the length of stem cells’ mother centrosomes. She presented additional data suggesting Klp10A regulates stem cell behavior through the regulation of nanos mRNA.

TMJ Degeneration in SAMP8 Mice is Accompanied by Deranged Ihh Signaling

The temporomandibular joint (TMJ) functions as a load-bearing diarthrodial joint during mastication, and its continuous use and stress can lead to degeneration over age. Using senescence-accelerated (SAMP8) mice that develop early osteoarthritis-like changes in synovial joints at high frequency, we analyzed possible molecular mechanisms of TMJ degeneration and tested whether and how malocclusion may accelerate it. Condylar articular cartilage in young SAMP8 mice displayed early-onset osteoarthritic changes that included reductions in superficial/chondroprogenitor cell number, proteoglycan/collagen content, and Indian hedgehog (Ihh)-expressing chondrocytes. Following malocclusion induced by tooth milling, the SAMP8 condyles became morphologically defective, displayed even lower proteoglycan levels, and underwent abnormal chondrocyte maturation compared with malocclusion-treated condyles in wild-type mice. Malocclusion also induced faster progression of pathologic changes with increasing age in SAMP8 condyles as indicated by decreased PCNA-positive proliferating chondroprogenitors and increased TUNEL-positive apoptotic cells. These changes were accompanied by steeper reductions in Ihh signaling and by expression of matrix metalloproteinase 13 at the chondro-osseous junction in SAMP8 articular cartilage. In sum, we show for the first time that precocious TMJ degeneration in SAMP8 mice is accompanied by—and possibly attributable to—altered Ihh signaling and that occlusal dysfunction accelerates progression toward degenerative TMJ disease in this model.

Disrupting the Indian hedgehog signaling pathway in vivo attenuates surgically induced osteoarthritis progression in Col2a1-CreER T2 ; Ihhfl/fl mice

IntroductionPrevious observations implicate Indian hedgehog (Ihh) signaling in osteoarthritis (OA) development because it regulates chondrocyte hypertrophy and matrix metallopeptidase 13 (MMP-13) expression. However, there is no direct genetic evidence for the role of Ihh in OA, because mice with cartilage or other tissue-specific deletion of the Ihh gene die shortly after birth. We evaluated the role of Ihh in vivo via a Cre-loxP-mediated approach to circumvent the early death caused by Ihh deficiency.MethodsTo evaluate the role of Ihh in OA development, Ihh was specifically deleted in murine cartilage using an Ihh conditional deletion construct (Col2a1-CreERT2; Ihhfl/fl). The extent of cartilage degradation and OA progression after Ihh deletion was assessed by histological analysis, immunohistochemistry, real-time PCR and in vivo fluorescence molecular tomography (FMT) 2 months after OA was induced by partial medial meniscectomy. The effect of Ihh signaling on cartilage was compared between Ihh-deleted mice and their control littermates.ResultsOnly mild OA changes were observed in Ihh-deleted mice, while control mice displayed significantly more cartilage damage. Typical OA markers such as type X collagen and MMP-13 were decreased in Ihh-deleted mice. In vivo FMT demonstrated decreased cathepsins and MMP activity in knee joints of animals with deletion of Ihh.ConclusionsThese findings support the protective role of Ihh deletion in surgically induced OA. Thus, our findings suggest the potential to develop new therapeutic strategies that can prevent and treat OA by inhibiting Ihh signaling in chondrocytes.

Sirt6 regulates postnatal growth plate differentiation and proliferation via Ihh signaling

Sirtuin 6 (Sirt6) is a mammalian homologue of NAD+-dependent histone deacetylase Sir2. Although Sirt6−/− mice exhibit growth retardation, the role of Sirt6 in cartilage metabolism is unclear. The aim of this study was to investigate the Sirt6 signaling pathway in cartilage metabolism. Immunohistological evaluation of the tibial growth plate in Sirt6−/− mice exhibited impaired proliferation and differentiation of chondrocytes, reduced expression of Indian hedgehog (Ihh), and a senescent phenotype. When Sirt6 was knocked down in chondrocytes in vitro, expression of Ihh and its downstream genes were reduced. Impaired differentiation by Sirt6 silencing was completely rescued by administration of a Hh signal agonist. When sirtuins were activated, chondrocyte differentiation was enhanced together with activation of Ihh signal, and these effects were abrogated by Sirt6 silencing. ChIP assay revealed the affinity of ATF4 to the Ihh promoter was markedly decreased by Sirt6 knockdown. These data indicate Sirt6 directly controls proliferation and differentiation of chondrocytes.

Indian Hedgehog Signaling Promotes Chondrocyte Differentiation in Enchondral Ossification in Human Cervical Ossification of the Posterior Longitudinal Ligament

Histological, immunohistochemical, and immunoblot analyses of the expression of Indian hedgehog (Ihh) signaling in human cervical ossification of the posterior longitudinal ligament (OPLL). To examine the hypothesis that Ihh signaling in correlation with Sox9 and parathyroid-related peptide hormone (PTHrP) facilitates chondrocyte differentiation in enchondral ossification process in human cervical OPLL. In enchondral ossification, certain transcriptional factors regulate cell differentiation. OPLL is characterized by overexpression of these factors and disturbance of the normal cell differentiation process. Ihh signaling is essential for enchondral ossification, especially in chondrocyte hypertrophy. Samples of ossified ligaments were harvested from 45 patients who underwent anterior cervical decompressive surgery for symptomatic OPLL, and 6 control samples from patients with cervical spondylotic myelopathy/radiculopathy without OPLL. The harvested sections were stained with hematoxylin-eosin and toluidine blue, examined by transmission electron microscopy, and immunohistochemically stained for Ihh, PTHrP, Sox9, type X, XI collagen, and alkaline phosphatase. Immunoblot analysis was performed in cultured cells derived from the posterior longitudinal ligaments in the vicinity of the ossified plaque and examined for the expression of these factors. The ossification front in OPLL contained chondrocytes at various differentiation stages, including proliferating chondrocytes in fibrocartilaginous area, hypertrophic chondrocytes around the calcification front, and apoptotic chondrocytes near the ossified area. Immunoreactivity for Ihh and Sox9 was evident in proliferating chondrocytes and was strongly positive for PTHrP in hypertrophic chondrocytes. Mesenchymal cells with blood vessel formation were positive for Ihh, PTHrP, and Sox9. Cultured cells from OPLL tissues expressed significantly higher levels of Ihh, PTHrP, and Sox9 than those in non-OPLL cells. Our results indicated that overexpression of Ihh signaling promotes abnormal chondrocyte differentiation in enchondral ossification and enhances bone formation in OPLL.

The Ihh signal is essential for regulating proliferation and hypertrophy of cultured chicken chondrocytes

The Indian hedgehog (Ihh) signal plays a vital role in regulating proliferation and hypertrophy of chondrocytes. To investigate its function in postnatal chicken (Gallus gallus) chondrocytes, cyclopamine was used to inhibit Ihh signaling. The MTT and ALP assays revealed the downgrade-proliferation and upgrade-differentiation of chondrocytes. To further elucidate the mechanism, the mRNA expression levels of Ihh, parathyroid hormone related protein (PTHrP), Gli-2, Bcl-2, Bone Morphogenetic Protein 6 (BMP-6), type X collagen (Col X) and type II collagen (Col II) were detected by quantitative real-time RT-PCR analysis, and the protein expressions of Ihh, Col X, and Col II were determined using Western blot analysis. After the Ihh signal was blocked, chondrocytes demonstrated high expression levels of PTHrP and Col X and low levels of Gli-2, BMP-6, Bcl-2 and Col II although Ihh expression was increased. Based on these results, the Ihh signal is essential for balancing chicken chondrocyte proliferation and hypertrophy, and the regulatory function of PTHrP acts in an Ihh-dependent manner. Furthermore, BMP-6 and Bcl-2 played roles in maintaining the development of chondrocytes and may be downstream regulatory factors of Ihh signaling.

Indian Hedgehog Controls Proliferation and Differentiation in Skin Tumorigenesis and Protects against Malignant Progression

Mutations in the hedgehog pathway drive the formation of tumors in many different organs, including the development of basal cell carcinoma in the skin. However, little is known about the role of epidermal Indian hedgehog (Ihh) in skin physiology. Using mouse genetics, we identified overlapping and distinct functions of Ihh in different models of epidermal tumorigenesis. Epidermal deletion of Ihh resulted in increased formation of benign squamous papilloma. Strikingly, Ihh-deficient mice showed an increase in malignant squamous cell carcinoma and developed lung and lymph node metastases. In a sebaceous gland tumor model, Ihh deficiency inhibited tumor cell differentiation. More mechanistically, IHH stimulated cell proliferation by activating the transcription factor GLI2 in human keratinocytes and human tumors. Thus, our results uncover important functions for Ihh signaling in controlling proliferation, differentiation, malignant progression, and metastasis of epithelial cancer, establishing Ihh as a gatekeeper for controlling the grade of tumor malignancy.

Ihh and Runx2/Runx3 Signaling Interact to Coordinate Early Chondrogenesis: A Mouse Model

Endochondral bone formation begins with the development of a cartilage intermediate that is subsequently replaced by calcified bone. The mechanisms occurring during early chondrogenesis that control both mesenchymal cell differentiation into chondrocytes and cell proliferation are not clearly understood in vertebrates. Indian hedgehog (Ihh), one of the hedgehog signaling molecules, is known to control both the hypertrophy of chondrocytes and bone replacement; these processes are particularly important in postnatal endochondral bone formation rather than in early chondrogenesis. In this study, we utilized the maternal transfer of 5E1 to E12.5 in mouse embryos, a process that leads to an attenuation of Ihh activity. As a result, mouse limb bud chondrogenesis was inhibited, and an exogenous recombinant IHH protein enhanced the proliferation and differentiation of mesenchymal cells. Analysis of the genetic relationships in the limb buds suggested a more extensive role for Ihh and Runx genes in early chondrogenesis. The transfer of 5E1 decreased the expression of Runx2 and Runx3, whereas an exogenous recombinant IHH protein increased Runx2 and Runx3 expression. Moreover, a transcription factor Gli1 in hedgehog pathway enhances the direct induction of both Runx2 and Runx3 transcription. These findings suggested that Ihh signaling plays an important role in chondrocyte proliferation and differentiation via interactions with Runx2 and Runx3.

Constitutive Activation of Gli2 Impairs Bone Formation in Postnatal Growing Mice

Indian hedgehog (Ihh) signaling is indispensable for osteoblast differentiation during endochondral bone development in the mouse embryo. We have previously shown that the Gli2 transcription activator critically mediates Ihh function in osteoblastogenesis. To explore the possibility that activation of Hedgehog (Hh) signaling may enhance bone formation, we generated mice that expressed a constitutively active form of Gli2 in the Osx-lineage cells. Unexpectedly, these mice exhibited severe osteopenia due to a marked decrease in osteoblast number and function, although bone resorption was not affected. Quantitative analyses of the molecular markers indicated that osteoblast differentiation was impaired in the mutant mouse. However, the osteoblast-lineage cells isolated from these mice exhibited more robust osteoblast differentiation than normal in vitro. Similarly, pharmacological stimulation of Hh signaling enhanced osteoblast differentiation from Osx-expressing cells isolated from the wild-type mouse. Thus, even though Hh signaling directly promotes osteoblast differentiation in vitro, constitutive activation of this pathway impairs bone formation in vivo, perhaps through an indirect mechanism.

Stimulation of superficial zone protein accumulation by hedgehog and Wnt signaling in surface zone bovine articular chondrocytes

To determine the roles of the hedgehog and Wnt signaling pathways in accumulation of superficial zone protein (SZP) in surface zone articular chondrocytes. Explant cultures of disks of surface zone cartilage or isolated chondrocytes from the surface zone of articular cartilage of bovine stifle joints were cultured in serum-free chemically defined medium. Accumulation of SZP in the culture medium, in response to hedgehog proteins (sonic hedgehog [SHH] and Indian hedgehog [IHH]), Wnt proteins (Wnt-3a, Wnt-5a, and Wnt-11), agonists of the Wnt/β-catenin pathway (glycogen synthase kinase 3β [GSK-3β] inhibitors), and antagonists of the Wnt/β-catenin pathway, was investigated. The interaction between transforming growth factor β1 (TGFβ1) and hedgehog proteins or antagonists of the Wnt/β-catenin pathway was also investigated. Hedgehog proteins stimulated SZP accumulation. Activation of the Wnt/β-catenin pathway by Wnt-3a and GSK-3β inhibitors led to inhibition of SZP accumulation; however, Wnt-5a and Wnt-11 had no influence on SZP accumulation. Conversely, antagonists of the Wnt/β-catenin pathway stimulated SZP accumulation. In addition, there were combinatorial effects of TGFβ1 and hedgehog proteins or antagonists of the Wnt/β-catenin pathway on SZP accumulation. SHH and IHH signaling has a stimulatory effect on SZP accumulation in surface zone cartilage and isolated articular chondrocytes. These findings provide insight into the regulatory mechanisms of articular cartilage homeostasis and maintenance by morphogens.