Indian hedgehog is an essential component of mechanotransduction complex to stimulate chondrocyte proliferation

Indian hedgehog (Ihh), a member of the vertebrate hedgehog morphogen family, is a key signaling molecule that controls chondrocyte proliferation and differentiation. In this study, we show a novel function of Ihh. Namely, it acts as an essential mediator of mechanotransduction in cartilage. Cyclic mechanical stress greatly induces the expression of Ihh by chondrocytes. This induction is abolished by gadolinium, an inhibitor of stretch-activated channels. This suggests that the IHH gene is mechanoresponsive. The mechano-induction of Ihh is essential for stimulating chondrocyte proliferation by mechanical loading. The presence of an Ihh functional blocking antibody during loading completely abolishes the stimulatory effect of mechanical load on proliferation. Furthermore, Ihh mediates the mechanotransduction process in a bone morphogenic protein (BMP)-dependent and parathyroid hormone-related peptide-independent manner. BMP 2/4 are up-regulated by mechanical stress through the induction of Ihh, and BMP antagonist noggin inhibits mechanical stimulation of chondrocyte proliferation. This suggests BMP lies downstream of Ihh in mechanotransduction pathway. Our data suggest that Ihh may transduce mechanical signals during cartilage growth and repair processes.     

Indian hedgehog in the late-phase differentiation in mouse chondrogenic EC cells, ATDC5: upregulation of type X collagen and osteoprotegerin ligand mRNAs

Endochondral bone formation includes a cascade of cellular events such as proliferation, maturation, hypertrophic conversion and calcification of chondrocytes and the cartilage replacement by bone. During these processes, hypertrophic conversion and calcification of chondrocytes (the late-phase differentiation) is a crucial process of chondrogenic differentiation. Indian hedgehog (Ihh), a secreted protein expressed in early hypertrophic chondrocytes, is thought to be involved in regulation of hypertrophic conversion via a feedback loop through the perichondrium. In the present study, we showed by Northern analysis and in situ hybridization that Smoothened (Smo), a key component in hedgehog signal transduction, was expressed in chondrocytes in both adult mice and mouse embryos at 16 days post-coitum in vivo, suggesting that Ihh directly acts on chondrocytes. We previously reported that Ihh, Patched and Smo were all expressed in differentiated ATDC5 cells. Exogenously administered mouse recombinant N-terminal protein of Ihh (mrIhh-N) upregulated the gene expression of type X collagen, a phenotypic marker of hypertrophic chondrocytes, as well as osteoprotegerin ligand (OPGL), a potent stimulator of osteoclastogenesis and osteoclast activity, while it did not modulate the expression of Ihh itself, bone morphogenetic protein (BMP)-4, BMP-6, transforming growth factor (TGF)-beta1 and TGF-beta2 in differentiated ATDC5 cells. Moreover, when added to the osteoclast cultures, mrIhh-N markedly stimulated the formation of resorption pits on dentine slices. Our data support the hypothesis that Ihh stimulated the late-phase chondrogenic differentiation in differentiated ATDC5 cells and upregulated the gene expression of OPGL in these cells.     

Indian hedgehog: its roles and regulation in endochondral bone development

Normal endochondral bone development requires the coordination of chondrocyte proliferation and differentiation. Indian hedgehog (Ihh) is a morphogen produced by chondrocytes in the early stage of terminal differentiation and plays several key roles in this process. Ihh regulates growth of adjacent proliferative chondrocytes and can also regulate the rate of differentiation of chondrocytes indirectly through its stimulation of parathyroid hormone-related protein (PTHrP). In this review, we focus on recent studies that have identified new functions of Ihh and how Ihh itself is being regulated.     

BMP and Ihh/PTHrP signaling interact to coordinate chondrocyte proliferation and differentiation.

During endochondral ossification, two secreted signals, Indian hedgehog (Ihh) and parathyroid hormone-related protein (PTHrP), have been shown to form a negative feedback loop regulating the onset of hypertrophic differentiation of chondrocytes. Bone morphogenetic proteins (BMPs), another family of secreted factors regulating bone formation, have been implicated as potential interactors of the Ihh/PTHrP feedback loop. To analyze the relationship between the two signaling pathways, we used an organ culture system for limb explants of mouse and chick embryos. We manipulated chondrocyte differentiation by supplementing these cultures either with BMP2, PTHrP and Sonic hedgehog as activators or with Noggin and cyclopamine as inhibitors of the BMP and Ihh/PTHrP signaling systems. Overexpression of Ihh in the cartilage elements of transgenic mice results in an upregulation of PTHrP expression and a delayed onset of hypertrophic differentiation. Noggin treatment of limbs from these mice did not antagonize the effects of Ihh overexpression. Conversely, the promotion of chondrocyte maturation induced by cyclopamine, which blocks Ihh signaling, could not be rescued with BMP2. Thus BMP signaling does not act as a secondary signal of Ihh to induce PTHrP expression or to delay the onset of hypertrophic differentiation. Similar results were obtained using cultures of chick limbs. We further investigated the role of BMP signaling in regulating proliferation and hypertrophic differentiation of chondrocytes and identified three functions of BMP signaling in this process. First we found that maintaining a normal proliferation rate requires BMP and Ihh signaling acting in parallel. We further identified a role for BMP signaling in modulating the expression of IHH: Finally, the application of Noggin to mouse limb explants resulted in advanced differentiation of terminally hypertrophic cells, implicating BMP signaling in delaying the process of hypertrophic differentiation itself. This role of BMP signaling is independent of the Ihh/PTHrP pathway.     

Conditional Ablation of the Heparan Sulfate-synthesizing Enzyme Ext1 Leads to Dysregulation of Bone Morphogenic Protein Signaling and Severe Skeletal Defects

Increasing evidence indicates that heparan sulfate (HS) is an integral component of many morphogen signaling pathways. However, its mechanisms of action appear to be diverse, depending on the type of morphogen and the developmental contexts. To define the function of HS in skeletal development, we conditionally ablated Ext1, which encodes an essential glycosyltransferase for HS synthesis, in limb bud mesenchyme using the Prx1-Cre transgene. These conditional Ext1 mutant mice display severe limb skeletal defects, including shortened and malformed limb bones, oligodactyly, and fusion of joints. In developing limb buds of mutant mice, chondrogenic differentiation of mesenchymal condensations is delayed and impaired, whereas the area of differentiation is diffusely expanded. Correspondingly, the distribution of both bone morphogenic protein (BMP) signaling domains and BMP2 immunoreactivity in the mutant limb mesenchyme is broadened and diffuse. In micromass cultures, chondrogenic differentiation of mutant chondrocytes is delayed, and the responsiveness to exogenous BMPs is attenuated. Moreover, the segregation of the pSmad1/5/8-expressing chondrocytes and fibronectin-expressing perichondrium-like cells surrounding chondrocyte nodules is disrupted in mutant micromass cultures. Together, our results show that HS is essential for patterning of limb skeletal elements and that BMP signaling is one of the major targets for the regulatory role of HS in this developmental context.     

Discovery of sonic hedgehog expression in postnatal growth plate chondrocytes: differential regulation of sonic and Indian hedgehog by retinoic acid

Sonic hedgehog (Shh) is a key signal protein in early embryological patterning of limb bud development. Its analog, Indian hedgehog (Ihh), primarily expressed during early cartilage development in prehypertrophic chondrocytes, regulates proliferation and suppresses terminal differentiation of postnatal growth plate (GP) chondrocytes. We report here for the first time that both Shh and Ihh mRNA are expressed in the GP of rapidly growing 6-week-old broiler-strain chickens. They are also expressed in other tissues such as articular chondrocytes, kidney, and bone. In situ hybridization and RT-PCR analyses reveal Shh in all zones of the GP, with peak expression in late hypertrophy. Using primary cultures of GP chondrocytes in serum-containing medium, we followed the patterns of Shh and Ihh mRNA expression as the cultures matured and mineralized. We find a cyclical expression of both hedgehog genes during the early period of culture development between day 10 and 14; when one is elevated, the other tended to be suppressed, suggesting that the two hedgehogs may play complementary roles during GP development. Retinoic acid (RA), a powerful modulator of gene expression in cell differentiation, stimulates GP chondrocytes toward terminal differentiation, enhancing mineral formation. We find that RA strongly suppresses Ihh, but enhances expression of Shh in this system. While Ihh suppresses maturation of GP chondrocytes to hypertrophy, we hypothesize that Shh acts to push these cells toward hypertrophy.     

Ihh signaling is directly required for the osteoblast lineage in the endochondral skeleton

Indian hedgehog (Ihh) is indispensable for development of the osteoblast lineage in the endochondral skeleton. In order to determine whether Ihh is directly required for osteoblast differentiation, we have genetically manipulated smoothened (Smo), which encodes a transmembrane protein that is essential for transducing all Hedgehog (Hh) signals. Removal of Smo from perichondrial cells by the Cre-LoxP approach prevents formation of a normal bone collar and also abolishes development of the primary spongiosa. Analysis of chimeric embryos composed of wild-type and Smo(n/n) cells indicates that Smo(n/n) cells fail to contribute to osteoblasts in either the bone collar or the primary spongiosa but generate ectopic chondrocytes. In order to assess whether Ihh is sufficient to induce bone formation in vivo, we have analyzed the bone collar in the long bones of embryos in which Ihh was artificially expressed in all chondrocytes by the UAS-GAL4 bigenic system. Although ectopic Ihh does not induce overt ossification along the entire cartilage anlage, it promotes progression of the bone collar toward the epiphysis, suggesting a synergistic effect between ectopic Ihh and endogenous factors such as the bone morphogenetic proteins (BMPs). In keeping with this model, Hh signaling is further found to be required in BMP-induced osteogenesis in cultures of a limb-bud cell line. Taken together, these results demonstrate that Ihh signaling is directly required for the osteoblast lineage in the developing long bones and that Ihh functions in conjunction with other factors such as BMPs to induce osteoblast differentiation. We suggest that Ihh acts in vivo on a potential progenitor cell to promote osteoblast and prevent chondrocyte differentiation.     

Indian hedgehog signals independently of PTHrP to promote chondrocyte hypertrophy

Chondrocyte hypertrophy is an essential process required for endochondral bone formation. Proper regulation of chondrocyte hypertrophy is also required in postnatal cartilage homeostasis. Indian hedgehog (Ihh) and PTHrP signaling play crucial roles in regulating the onset of chondrocyte hypertrophy by forming a negative feedback loop, in which Ihh signaling regulates chondrocyte hypertrophy by controlling PTHrP expression. To understand whether there is a PTHrP-independent role of Ihh signaling in regulating chondrocyte hypertrophy, we have both activated and inactivated Ihh signaling in the absence of PTHrP during endochondral skeletal development. We found that upregulating Ihh signaling in the developing cartilage by treating PTHrP(-/-) limb explants with sonic hedgehog (Shh) protein in vitro, or overexpressing Ihh in the cartilage of PTHrP(-/-) embryos or inactivating patched 1 (Ptch1), a negative regulator of hedgehog (Hh) signaling, accelerated chondrocyte hypertrophy in the PTHrP(-/-) embryos. Conversely, when Hh signaling was blocked by cyclopamine or by removing Smoothened (Smo), a positive regulator of Hh signaling, chondrocyte hypertrophy was delayed in the PTHrP(-/-) embryo. Furthermore, we show that upregulated Hh signaling in the postnatal cartilage led to accelerated chondrocyte hypertrophy during secondary ossification, which in turn caused reduction of joint cartilage. Our results revealed a novel role of Ihh signaling in promoting chondrocyte hypertrophy independently of PTHrP, which is particularly important in postnatal cartilage development and homeostasis. In addition, we found that bone morphogenetic protein (Bmp) and Wnt/beta-catenin signaling in the cartilage may both mediate the effect of upregulated Ihh signaling in promoting chondrocyte hypertrophy.     

Involvement of the MAP kinase pathways in induction of GADD45 following UV radiation

Indian hedgehog (Ihh) is highly expressed in prehypertrophic chondrocytes in vivo and has been proposed to regulate the proliferation and maturation of chondrocytes and bone collar formation in the growth plate. In high-density cultures of rabbit growth-plate chondrocytes, Ihh mRNA was also expressed at the highest level in the prehypertrophic stage. To explore endogenous factors that regulate Ihh expression in chondrocytes, we examined the effects of various growth factors on Ihh mRNA expression in this system. Retinoic acid (RA) and bone morphogenetic protein-2 enhanced Ihh mRNA expression, whereas PTH/PTH-related peptide (PTHrP) markedly suppressed Ihh expression. RA at more than 10(-8) M induced the expression of Ihh and Patched 1 (Ptc1) within 3 h, before it increased the type X collagen mRNA level at 6-24 h. Cycloheximide blocked the up-regulation of Ihh by RA, indicating the requirement of de novo protein synthesis for this stimulation. These findings suggest that RA is involved in the up-regulation of Ihh during endochondral bone formation. In contrast to RA, PTH (1-84) at 10(-7) M abolished the mRNA expression of Ihh and Ptc1 within 2-4 h, before it suppressed the expression of type X collagen at 12-24 h. The inhibition of Ihh expression by PTH (1-84) did not require de novo protein synthesis. PTH (1-34), PTHrP (1-34), and (Bu)(2)cAMP also suppressed Ihh expression. On the other hand, Ihh has been reported to induce PTHrP synthesis in the perichondrium. Consequently, the direct inhibitory action of PTH/PTHrP on Ihh appears to be a negative feedback mechanism that prevents excess PTHrP accumulation in cartilage.     

Wnt9a signaling is required for joint integrity and regulation of Ihh during chondrogenesis

Joints, which separate skeleton elements, serve as important signaling centers that regulate the growth of adjacent cartilage elements by controlling proliferation and maturation of chondrocytes. Accurate chondrocyte maturation is crucial for endochondral ossification and for the ultimate size of skeletal elements, as premature or delayed maturation results predominantly in shortened elements. Wnt9a has previously been implicated as being a player in joint induction, based on gain-of function experiments in chicken and mouse. We show that loss of Wnt9a does not affect joint induction, but results to synovial chondroid metaplasia in some joints. This phenotype can be enhanced by removal of an additional Wnt gene, Wnt4, suggesting that Wnts are playing a crucial role in directing bi-potential chondro-synovioprogenitors to become synovial connective tissue, by actively suppressing their chondrogenic potential. Furthermore, we show that Wnt9a is a temporal and spatial regulator of Indian hedgehog (Ihh), a central player of skeletogenesis. Loss of Wnt9a activity results in transient downregulation of Ihh and reduced Ihh-signaling activity at E12.5-E13.5. The canonical Wnt/beta-catenin pathway probably mediates regulation of Ihh expression in prehypertrophic chondrocytes by Wnt9a, because embryos double-heterozygous for Wnt9a and beta-catenin show reduced Ihh expression, and in vivo chromatin immunoprecipitation demonstrates a direct interaction between the beta-catenin/Lef1 complex and the Ihh promoter.