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.     

Growth factor regulation of human growth plate chondrocyte proliferation in vitro

Linear growth occurs as the result of growth plate chondrocytes undergoing proliferative and hypertrophic phases. Paracrine feedback loops that regulate the entry of chondrocytes into the hypertrophic phase have been shown and similar pathways likely exist for the proliferative phase. Human long-bone growth plate chondrocytes were cultured in vitro. The proliferative effects of a variety of factors were determined by [3H]thymidine uptake and the gene expression profile of these cells was determined by DNA microarray analysis. Serum, insulin-like growth factor (IGF)-I and -II, transforming growth factor-beta (TGF-beta, fibroblast growth factor (FGF)-1, -2, and -18, and platelet-derived growth factor (PDGF)-BB were potent stimulators of proliferation. FGF-10, testosterone, and bone morphogenetic proteins (BMP)-2, -4, and -6 inhibited proliferation. Microarray analysis showed that the genes for multiple members of the IGF-I, TGF-beta, FGF, and BMP pathways were expressed, suggesting the presence of autocrine/paracrine pathways that regulate the proliferative phase of growth plate-mediated growth.     

PTHrP and Indian hedgehog control differentiation of growth plate chondrocytes at multiple steps

In developing murine growth plates, chondrocytes near the articular surface (periarticular chondrocytes) proliferate, differentiate into flat column-forming proliferating cells (columnar chondrocytes), stop dividing and finally differentiate into hypertrophic cells. Indian hedgehog (Ihh), which is predominantly expressed in prehypertrophic cells, stimulates expression of parathyroid hormone (PTH)-related peptide (PTHrP) which negatively regulates terminal chondrocyte differentiation through the PTH/PTHrP receptor (PPR). However, the roles of PTHrP and Ihh in regulating earlier steps in chondrocyte differentiation are unclear. We present novel mouse models with PPR abnormalities that help clarify these roles. In mice with chondrocyte-specific PPR ablation and mice with reduced PPR expression, chondrocyte differentiation was accelerated not only at the terminal step but also at an earlier step: periarticular to columnar differentiation. In these models, upregulation of Ihh action in the periarticular region was also observed. In the third model in which the PPR was disrupted in about 30% of columnar chondrocytes, Ihh action in the periarticular chondrocytes was upregulated because of ectopically differentiated hypertrophic chondrocytes that had lost PPR. Acceleration of periarticular to columnar differentiation was also noted in this mouse, while most of periarticular chondrocytes retained PPR signaling. These data suggest that Ihh positively controls differentiation of periarticular chondrocytes independently of PTHrP. Thus, chondrocyte differentiation is controlled at multiple steps by PTHrP and Ihh through the mutual regulation of their activities.     

Localization of Indian hedgehog and PTH/PTHrP receptor expression in relation to chondrocyte proliferation during mouse bone development

We have developed a useful approach to examine the pattern of gene expression in comparison to cell proliferation, using double in situ hybridization and immunofluorescence. Using this system, we examined the expression of Indian hedgehog (Ihh) and PTH/PTHrP receptor (PPR) mRNA in relation to chondrocyte proliferation during embryonic mouse bone development. Both genes are expressed strongly in prehypertrophic and early hypertrophic chondrocytes, and there is a strong correlation between upregulation of both Ihh and PPR expression and chondrocyte cell cycle arrest. At embryonic day (E14.5), PPR mRNA upregulation begins in the columnar chondrocytes just prior to cell cycle exit, but at later time points expression is only observed in the postproliferative region. In contrast, Ihh mRNA expression overlaps slightly with the region of columnar proliferating chondrocytes at all stages. This study provides further evidence that in the developing growth plate, cell cycle exit and upregulation of Ihh and PPR mRNA expression are coupled.     

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.     

FGF18 is required for early chondrocyte proliferation, hypertrophy and vascular invasion of the growth plate

Fibroblast growth factor 18 (FGF18) has been shown to regulate chondrocyte proliferation and differentiation by signaling through FGF receptor 3 (FGFR3) and to regulate osteogenesis by signaling through other FGFRs. Fgf18(-/-) mice have an apparent delay in skeletal mineralization that is not seen in Fgfr3(-/-) mice. However, this delay in mineralization could not be simply explained by FGF18 signaling to osteoblasts. Here we show that delayed mineralization in Fgf18(-/-) mice was closely associated with delayed initiation of chondrocyte hypertrophy, decreased proliferation at early stages of chondrogenesis, delayed skeletal vascularization and delayed osteoclast and osteoblast recruitment to the growth plate. We further show that FGF18 is necessary for Vegf expression in hypertrophic chondrocytes and the perichondrium and is sufficient to induce Vegf expression in skeletal explants. These findings support a model in which FGF18 regulates skeletal vascularization and subsequent recruitment of osteoblasts/osteoclasts through regulation of early stages of chondrogenesis and VEGF expression. FGF18 thus coordinates neovascularization of the growth plate with chondrocyte and osteoblast growth and differentiation.     

Effect of x-irradiation on growth and the expression of parathyroid hormone-related peptide and Indian hedgehog in the tibial growth plate of the rat

AIM: To study the effect of irradiation on the longitudinal growth and the expression of parathyroid hormone-related peptide (PTHrP) and Indian hedgehog (IHh) in tibial growth plates of rats. METHODS: At 3 weeks of age, 30 male rats received a single fraction of irradiation (8 Gy) to their right hind limb, and small groups of animals were sacrificed 1, 2, 3, 5, 7, 10, 15, and 26 weeks after irradiation. Weight and length of both irradiated and nonirradiated tibiae were measured, and sections of the tibiae were stained with HE. PTHrP and IHh were visualized using immunohistochemical techniques. RESULTS: Radiation resulted in persistent growth delay of the irradiated tibiae, with a difference in length of more than 10% between the irradiated and the nonirradiated tibiae 15 weeks or more after irradiation. The growth plate architecture was disturbed, and the expression of both PTHrP and IHh was decreased in the irradiated tibiae. CONCLUSION: As PTHrP and IHh are key regulators of both the pace and the synchronization of the differentiation of growth plate chondrocytes, the reduced expression of PTHrP and IHh may contribute to the changes found after irradiation.     

Ovariectomy causes cell proliferation and matrix synthesis in the growth plate cartilage of the adult rat

The in vivo effects of ovariectomy in rats have been studied on cell proliferation and matrix synthesis in the growth plate cartilage by assessing immunohistochemically the levels of proliferating cell nuclear antigen and chondroitin sulphate proteoglycan(s). The serum levels of insulin-like growth factor-I and growth hormone were also measured by radioimmunoassay procedures. At 5 weeks after ovariectomy, the serum levels of the growth factor were significantly higher than those in sham-operated rats. In contrast, the level of growth hormone was lower. The nuclear staining of proliferating cell nuclear antigen was generally seen in the zone of proliferative chondrocytes from both groups of rats. Whereas almost all chondrocytes in the proliferative zone of ovariectomized rats expressed proliferating cell nuclear antigen immunoreactivity, fewer did so in that of the sham rats. Quantitative image analysis by ACAS 570 laser cytometry demonstrated that the n uclear antigen-positive sites in ovariectomized rats had significantly higher integrated values (staining intensity), areas and perimeters than those in sham rats. In addition, the number of chondroitin sulphate proteoglycan-immunoreactive cells in the proliferative chondrocytes was also higher in ovariectomized rats than in sham ones. These results suggest that ovariectomy significantly stimulates the cell proliferation and matrix synthesis in the growth plate cartilage, probably through the higher serum level of insulin-like growth factor-I.     

PIM-2 is an independent regulator of chondrocyte survival and autophagy in the epiphyseal growth plate

The overall goal of the investigation was to examine the activity and role of the PIM serine/threonine protein kinases in the growth plate. We showed for the first time that PIM-2 was highly expressed in epiphyseal chondrocytes and that the kinase was required for critical activities linked to cell survival. These activities were independent of those mediated by Akt-1. It was noted that PIM-2 protected chondrocytes from rapamycin sensitized (TOR inhibited) cell death. Since inhibition of mTOR caused autophagy, we examined the autophagic response of PIM-2 silenced cells. We showed that PIM-2 promoted expression and organization of autophagic proteins LC3, and Beclin-1 and enhanced lysosomal acidification. At the same time, PIM-2 modulated the activity of a key regulator of apoptosis, BAD. Since BAD inhibition and Beclin-1 expression activated autophagy, it is likely that induction of the autophagic pathway would serve to inhibit apoptosis and preserve the life of the terminally differentiated chondrocyte. We conclude that PIM-2 regulates a new intermediate stage in the differentiation pathway, the induction of autophagy.     

Ca2+ transport by chondrocyte mitochondria of the epiphyseal growth plate

Summary In a study of the Ca²⁺ kinetics of mitochondria of chick epiphyseal chondrocytes, the rate of Ca²⁺ uptake was linear up to a medium Ca²⁺ concentration of 30 m. The half maximal transport rate occurred at 34 m Ca²⁺. The Ca²⁺ uptake rate, expressed as a function of time, was 35 nmoles/mg protein/min; the presence of Mg²⁺ had little effect on Ca²⁺ accumulation. While these kinetic parameters did not differ significantly from mitochondria of cells of nonmineralizing tissues, the respiratory characteristics of the chondrocyte organelles exhibited functional differences. Thus, up to 350 nmoles Ca²⁺/mg protein, chondrocyte mitochondria performed coupled oxidative phosphorylation. Calcium uptake was energy supported, while Ca²⁺ binding was low. Addition of respiratory inhibitors and uncouplers to these mitochondria resulted in a rapid loss of more than 80% of the total Ca²⁺. The Ca/Pi ratio of the extrudate was very similar to the ratio of these ions in cartilage septum fluid. In the most mineralized zones of the epiphyseal plate, there was little change in the state 4 respiratory rate, but nonspecific Ca²⁺ binding was elevated and a high percentage of the total Ca²⁺ was in a nonextrudable form. The results indicate that in cells preparing for mineralization, much of the total mitochondrial Ca²⁺ is in a form that can be transported to the calcification front. In cells close to the calcification front, nonextrudable Ca²⁺ may form calcium phosphate granules described by other investigators.     

A greenprint for growth: signalling the pattern of proliferation

The shoot and root apical meristems (SAM and RAM, respectively) of plants serve both as sites of cell division and as stem cell niches. The SAM is also responsible for the initiation of new leaves, whereas the analogous process of lateral root initiation occurs in the pericycle, a specialized layer of cells that retains organogenic potential within an otherwise non-dividing region of the root. A picture is emerging of how cell division, growth, and differentiation are coordinated in the meristems and lateral organ primordia of plants. This is starting to reveal striking parallels between the control of stem cell maintenance in both shoots and roots, and to provide information on how signalling from developmental processes and the environment impact on cell behaviour within meristems.     

An integrated agent-mathematical model of the effect of intercellular signalling via the epidermal growth factor receptor on cell proliferation

We have previously developed Epitheliome, a software agent representation of the growth and repair characteristics of epithelial cell populations, where cell behaviour is governed by a number of simple rules. In this paper, we describe how this model has been extended to incorporate an example of a molecular 'mechanism' behind a rule-in this case, how signalling by both endogenous and exogenous ligands of the epidermal growth factor receptor (EGFR) can impact on the proliferation of cell agents. We have developed a mathematical model representing release of endogenous ligand by cells, three-dimensional diffusion of the secreted molecules through a volume of cell culture medium, ligand-receptor binding, and bound receptor internalization and trafficking. Information relating to quantities of molecular species associated with each cell agent is frequently exchanged between the agent and signalling models, and the ratio of bound to free receptors determines cell cycle progression and hence the proliferative behaviour of the cell agents. We have applied this integrated model to examine the effect of plating density on tissue growth via autocrine/paracrine signalling. This predicts that cell growth is dependent on the concentration of exogenous ligand, but where this is limited, then growth becomes dependent on cell density and the availability of endogenous ligand. We have further modified the calcium concentration of the medium to modulate the formation of intercellular bonds between cells and shown that the increased propensity for cells to form colonies in physiological calcium does not result in significantly different patterns of receptor occupancy. In conclusion, our approach demonstrates that by combining agent-based and mathematical modelling paradigms, it is possible to probe the complex feedback relationship between the behaviour of individual cells and their interaction with one another and their environment.     

From fruitflies to mammals: mechanisms of signalling via the Sonic hedgehog pathway in lung development

Chorioamnionitis is frequently associated with preterm deliveries before 30 weeks gestation. Chorioamnionitis correlates both with an increased risk of bronchopulmonary dysplasia and with a decreased risk of respiratory distress syndrome. Both interleukin-1α and endotoxin can induce inflammation in the fetal lungs and lung maturation after preterm birth when given by intra-amniotic injection. Inflammation can also result in an arrest of alveolarization, and this lung developmental abnormality is prominent in the lungs of preterm infants that die of bronchopulmonary dysplasia. The mechanisms by which infection/inflammation can have both beneficial and injurious effects on the preterm lung remain to be characterized.