Stimulatory effects of insulin-like growth factor-I on growth plate chondrogenesis are mediated by nuclear factor-kappaB p65

Insulin-like growth factor-I (IGF-I) is an important regulator of endochondral ossification. However, little is known about the signaling pathways activated by IGF-I in growth plate chondrocytes. We have previously shown that NF-kappaB-p65 facilitates growth plate chondrogenesis. In this study, we first cultured rat metatarsal bones with IGF-I and/or pyrrolidine dithiocarbamate (PDTC), a known NF-kappaB inhibitor. The IGF-I-mediated stimulation of metatarsal growth and growth plate chondrogenesis was neutralized by PDTC. In rat growth plate chondrocytes, IGF-I induced NF-kappaB-p65 nuclear translocation. The inhibition of NF-kappaB-p65 expression and activity (by p65 short interfering RNA and PDTC, respectively) in chondrocytes reversed the IGF-I-mediated induction of cell proliferation and differentiation and the IGF-I-mediated prevention of cell apoptosis. Moreover, the inhibition of the phosphatidylinositol 3-kinase and Akt abolished the effects of IGF-I on NF-kappaB activation. In conclusion, our findings indicate that IGF-I stimulates growth plate chondrogenesis by activating NF-kappaB-p65 in chondrocytes.     

Proepithelin stimulates growth plate chondrogenesis via nuclear factor-kappaB-p65-dependent mechanisms

Proepithelin, a previously unrecognized growth factor in cartilage, has recently emerged as an important regulator for cartilage formation and function. In the present study, we provide several lines of evidences in proepithelin-mediated induction of cell proliferation, differentiation, and apoptosis in the metatarsal growth plate. Proepithelin-mediated stimulation of metatarsal growth and growth plate chondrogenesis was neutralized by pyrrolidine dithiocarbamate, a known NF-κB inhibitor. In rat growth plate chondrocytes, proepithelin induced NF-κB-p65 nuclear translocation, and nuclear NF-κB-p65 initiated its target gene cyclin D1 to regulate chondrocyte functions. The inhibition of NF-κB-p65 expression and activity (by p65 short interfering RNA (siRNA) and pyrrolidine dithiocarbamate, respectively) in chondrocytes reversed the proepithelin-mediated induction of cell proliferation and differentiation and the proepithelin-mediated prevention of cell apoptosis. Moreover, the inhibition of the phosphatidylinositol 3-kinase and Akt abolished the effects of proepithelin on NF-κB activation. Finally, using siRNA and antisense strategies, we demonstrated that endogenously produced proepithelin by chondrocytes is important for chondrocyte growth in serum-deprived conditions. These results support the hypothesis that the induction of NF-κB activity of in growth plate chondrocytes is critical in proepithelin-mediated growth plate chondrogenesis and longitudinal bone growth.     

Stanniocalcin 1 acts as a paracrine regulator of growth plate chondrogenesis

During embryogenesis, the expression of mammalian stanniocalcin (STC1) in the appendicular skeleton suggests its involvement in the regulation of longitudinal bone growth. Such a role is further supported by the presence of dwarfism in mice overexpressing STC1. Yet, the STC 1 inhibitory effect on growth may be related to both postnatal metabolic abnormalities and prenatal defective bone formation. In our study, we used an organ culture system to evaluate the effects of STC on growth plate chondrogenesis, which is the primary determinant of longitudinal bone growth. Fetal rat metatarsal bones were cultured in the presence of recombinant human STC (rhSTC). After 3 days, rhSTC suppressed metatarsal growth, growth plate chondrocyte proliferation and hypertrophy/differentiation, and extracellular matrix synthesis. In addition, rhSTC increased the number of apoptotic chondrocytes in the growth plate. In cultured chondrocytes, rhSTC increased phosphate uptake, reduced chondrocyte proliferation and matrix synthesis, and induced apoptosis. All these effects were reversed by culturing chondrocytes with rhSTC and phosphonoformic acid, an inhibitor of phosphate transport. The rhSTC-mediated inhibition of metatarsal growth and growth plate chondrocyte proliferation and hypertrophy/differentiation was abolished by culturing metatarsals with rhSTC and phosphonoformic acid. Taken together, our findings indicate that STC1 inhibits longitudinal bone growth directly at the growth plate. Such growth inhibition, likely mediated by an increased chondrocyte phosphate uptake, results from suppressed chondrocyte proliferation, hypertrophy/differentiation, and matrix synthesis and by increased apoptosis. Last, the expression of both STC1 and its binding site in the growth plate would support an autocrine/paracrine role for this growth factor in the regulation of growth plate chondrogenesis.     

Smad4 is required for the normal organization of the cartilage growth plate

Smad4 is the central intracellular mediator of transforming growth factor-beta (TGF-beta) signals. To study the role of Smad4 in skeletal development, we introduced a conditional mutation of the gene in chondrocytes using Cre--loxP system. We showed that Smad4 was expressed strongly in prehypertrophic and hypertrophic chondrocytes. The abrogation of Smad4 in chondrocytes resulted in dwarfism with a severely disorganized growth plate characterized by expanded resting zone of chondrocytes, reduced chondrocyte proliferation, accelerated hypertrophic differentiation, increased apoptosis and ectopic bone collars in perichondrium. Meanwhile, Smad4 mutant mice exhibited decreased expression of molecules in Indian hedgehog/parathyroid hormone-related protein (Ihh/PTHrP) signaling. The cultured mutant metatarsal bones failed to response to TGF-beta1, while the hypertrophic differentiation was largely inhibited by Sonic hedgehog (Shh). This indicated that Ihh/PTHrP inhibited the hypertrophic differentiation of chondrocytes independent of the Smad4-mediated TGF-beta signals. All these data provided the first genetic evidence demonstrating that Smad4-mediated TGF-beta signals inhibit the chondrocyte hypertrophic differentiation, and are required for maintaining the normal organization of chondrocytes in the growth plate.     

Phosphate regulates embryonic endochondral bone development

Phosphate is required for terminal differentiation of hypertrophic chondrocytes during postnatal growth plate maturation. In vitro models of chondrocyte differentiation demonstrate that 7 mM phosphate, a concentration analogous to that of the late gestational fetus, activates the mitochondrial apoptotic pathway in hypertrophic chondrocytes. This raises the question as to whether extracellular phosphate modulates chondrocyte differentiation and apoptosis during embryonic endochondral bone formation. To address this question, we performed investigations in the mouse metatarsal culture model that recapitulates in vivo bone development. Metatarsals were cultured for 4, 8, and 12 days with 1.25 and 7 mM phosphate. Metatarsals cultured with 7 mM phosphate showed a decrease in proliferation compared to those cultured in 1.25 mM phosphate. This decrease in proliferation was accompanied by an early enhancement in hypertrophic chondrocyte differentiation, associated with an increase in FGF18 expression. By 8 days in culture, an increase caspase‐9 activation and apoptosis of hypertrophic chondrocytes was observed in the metatarsals cultured in 7 mM phosphate. Immunohistochemical analyses of embryonic bones demonstrated activation of caspase‐9 in hypertrophic chondrocytes, associated with vascular invasion. Thus, these investigations demonstrate that phosphate promotes chondrocyte differentiation during embryonic development and implicate a physiological role for phosphate activation of the mitochondrial apoptotic pathway during embryonic endochondral bone formation. J. Cell. Biochem. 108: 668–674, 2009. © 2009 Wiley‐Liss, Inc.     

Glycogen synthase kinase 3 controls endochondral bone development: contribution of fibroblast growth factor 18

Glycogen synthase kinase 3 (GSK3) inhibits signaling pathways that are essential for bone development. To study the requirement for GSK activity during endochondral bone development, we inhibited GSK3 in cultured metatarsal bones with pharmacological antagonists. Interestingly, we find that inhibition of GSK3 strongly repressed chondrocyte and perichondrial osteoblast differentiation. Moreover, chondrocyte proliferation was inhibited, whereas perichondrial cell proliferation was stimulated. These results mirror the effects of fibroblast growth factor signaling (FGF), suggesting the FGF expression is induced. Indeed, we showed that (1) FGF18 expression is stimulated following inhibition of GSK3 and (2) GSK3 regulates FGF18 expression through the control of beta-catenin levels. Stimulation of cultured metatarsal with FGF18 had similar effects on the differentiation and proliferation of chondrocytes and perichondrial cells as GSK3 repression. This suggests that the regulation of FGF18 expression is a major function of GSK3 during endochondral bone development. Consistent with this, we showed that the effect of GSK3 inhibition on chondrocyte proliferation is repressed in tissues lacking a receptor for FGF18, FGF receptor 3.     

Bortezomib Is Cytotoxic to the Human Growth Plate and Permanently Impairs Bone Growth in Young Mice

Bortezomib, a novel proteasome inhibitor approved for the treatment of cancer in adults, has recently been introduced in pediatric clinical trials. Any tissue-specific side effects on bone development have to our knowledge not yet been explored. To address this, we experimentally studied the effects of bortezomib in vivo in young mice and in vitro in organ cultures of rat metatarsal bones and human growth plate cartilage, as well as in a rat chondrocytic cell line. We found that bortezomib while efficiently blocking the ubiquitin/proteasome system (UPS) caused significant growth impairment in mice, by increasing resting/stem-like chondrocyte apoptosis. Our data support a local action of bortezomib, directly targeting growth plate chondrocytes leading to decreased bone growth since no suppression of serum levels of insulin-like growth factor-I (IGF-I) was observed. A local effect of bortezomib was confirmed in cultured rat metatarsal bones where bortezomib efficiently caused growth retardation in a dose dependent and irreversible manner, an effect linked to increased chondrocyte apoptosis, mainly of resting/stem-like chondrocytes. The cytotoxicity of bortezomib was also evaluated in a unique model of cultured human growth plate cartilage, which was found to be highly sensitive to bortezomib. Mechanistic studies of apoptotic pathways indicated that bortezomib induced activation of p53 and Bax, as well as cleavage of caspases and poly-ADP-ribose polymerase (PARP) in exposed chondrocytes. Our observations, confirmed in vivo and in vitro, suggest that bone growth could potentially be suppressed in children treated with bortezomib. We therefore propose that longitudinal bone growth should be closely monitored in ongoing clinical pediatric trials of this promising anti-cancer drug.     

TGF-beta/Smad3 signals repress chondrocyte hypertrophic differentiation and are required for maintaining articular cartilage

Endochondral ossification begins from the condensation and differentiation of mesenchymal cells into cartilage. The cartilage then goes through a program of cell proliferation, hypertrophic differentiation, calcification, apoptosis, and eventually is replaced by bone. Unlike most cartilage, articular cartilage is arrested before terminal hypertrophic differentiation. In this study, we showed that TGF-beta/Smad3 signals inhibit terminal hypertrophic differentiation of chondrocyte and are essential for maintaining articular cartilage. Mutant mice homozygous for a targeted disruption of Smad3 exon 8 (Smad3(ex8/ex8)) developed degenerative joint disease resembling human osteoarthritis, as characterized by progressive loss of articular cartilage, formation of large osteophytes, decreased production of proteoglycans, and abnormally increased number of type X collagen-expressing chondrocytes in synovial joints. Enhanced terminal differentiation of epiphyseal growth plate chondrocytes was also observed in mutant mice shortly after weaning. In an in vitro embryonic metatarsal rudiment culture system, we found that TGF-beta1 significantly inhibits chondrocyte differentiation of wild-type metatarsal rudiments. However, this inhibition is diminished in metatarsal bones isolated from Smad3(ex8/ex8) mice. These data suggest that TGF-beta/Smad3 signals are essential for repressing articular chondrocyte differentiation. Without these inhibition signals, chondrocytes break quiescent state and undergo abnormal terminal differentiation, ultimately leading to osteoarthritis.     

Fibroblast growth factor 21 (FGF21) inhibits chondrocyte function and growth hormone action directly at the growth plate

Fibroblast growth factor 21 (FGF21) modulates glucose and lipid metabolism during fasting. In addition, previous evidence indicates that increased expression of FGF21 during chronic food restriction is associated with reduced bone growth and growth hormone (GH) insensitivity. In light of the inhibitory effects on growth plate chondrogenesis mediated by other FGFs, we hypothesized that FGF21 causes growth inhibition by acting directly at the long bones' growth plate. We first demonstrated the expression of FGF21, FGFR1 and FGFR3 (two receptors known to be activated by FGF21) and β-klotho (a co-receptor required for the FGF21-mediated receptor binding and activation) in fetal and 3-week-old mouse growth plate chondrocytes. We then cultured mouse growth plate chondrocytes in the presence of graded concentrations of rhFGF21 (0.01-10 μg/ml). Higher concentrations of FGF21 (5 and 10 μg/ml) inhibited chondrocyte thymidine incorporation and collagen X mRNA expression. 10 ng/ml GH stimulated chondrocyte thymidine incorporation and collagen X mRNA expression, with both effects prevented by the addition in the culture medium of FGF21 in a concentration-dependent manner. In addition, FGF21 reduced GH binding in cultured chondrocytes. In cells transfected with FGFR1 siRNA or ERK 1 siRNA, the antagonistic effects of FGF21 on GH action were all prevented, supporting a specific effect of this growth factor in chondrocytes. Our findings suggest that increased expression of FGF21 during food restriction causes growth attenuation by antagonizing the GH stimulatory effects on chondrogenesis directly at the growth plate. In addition, high concentrations of FGF21 may directly suppress growth plate chondrocyte proliferation and differentiation.     

L-type calcium channels in growth plate chondrocytes participate in endochondral ossification

Longitudinal bone growth occurs by a process called endochondral ossification that includes chondrocyte proliferation, differentiation, and apoptosis. Recent studies have suggested a regulatory role for intracellular Ca(2+) (Ca(i) (2+)) in this process. Indirect studies, using Ca(2+) channel blockers and measurement of Ca(i) (2+), have provided evidence for the existence of Ca(2+) channels in growth plate chondrocytes. Furthermore, voltage-gated Ca(2+) channels (VGCC), and specifically L- and T-type VGCCs, have been recently described in murine embryonic growth plates. Our aim was to assess the effect of L-type Ca(2+) channel blockers on endochondral ossification in an organ culture. We used cultures of fetal rat metatarsal rudiments at 20 days post gestational age, with the addition of the L-type Ca(2+) channel blockers verapamil (10-100 microM) or diltiazem (10-200 microM) to the culture medium. Longitudinal bone growth, chondrocyte differentiation (number of hypertrophic chondrocytes), and cell proliferation (incorporation of tritiated thymidine) were measured. Verapamil dose-dependently decreased growth, the number of hypertrophic chondrocytes, and cell proliferation, at concentrations of 10-100 microM. Growth and the number of hypertrophic chondrocytes decreased significantly with diltiazem at 50-100 microM, and proliferation decreased significantly at concentrations of 10-200 microM. Additionally, there was no increase in apoptosis over physiological levels with either drug. We confirmed the presence of L-type VGCCs in rat rudiments using immunohistochemistry, and showed that the antagonists did not alter the pattern of VGCC expression. In conclusion, our data suggest that L-type Ca(2+) channel activity in growth plate chondrocytes is necessary for normal longitudinal growth, participating in chondrocyte proliferation and differentiation.     

BMP-5 expression increases during chondrocyte differentiation in vivo and in vitro and promotes proliferation and cartilage matrix synthesis in primary chondrocyte cultures

Bone morphogenetic proteins (BMPs) play pivotal roles in bone and cartilage growth and repair. Through phenotypes of short-ear (se) mice, which have BMP-5 mutations, a role for BMP-5 in some specific aspects of skeletogenesis and cartilage growth is known. This report examines BMP-5 expression in the growth plate and in differentiating cultures of primary chondrocytes, and the effects of addition of BMP-5 or its inhibition by anti-BMP-5 antibody in chondrocyte cultures. By laser capture microdissection and immunohistochemistry, we found that BMP-5 is expressed in proliferating zone (PZ) chondrocytes and that the expression increases sharply with hypertrophic differentiation. A similar pattern was observed in differentiating cultures of primary chondrocytes, with BMP-5 expression increasing as cells differentiated, in contrast to other BMPs. BMP-5 added to cultures increased cell proliferation early in the culture period and also stimulated cartilage matrix synthesis. Also, BMP-5 addition to the cultures activated phosphorylation of Smad 1/5/8 and p38 MAP kinase and caused increased nuclear accumulation of phospho-Smads. Anti-BMP-5 antibody inhibited the endogenous BMP-5, reducing cell proliferation and phospho-Smad nuclear accumulation. Together, the results demonstrate that BMP-5 is normally an important regulator of chondrocyte proliferation and differentiation. Whether other BMPs may compensate in BMP-5 loss-of-function mutations is discussed.     

Role of cholesterol in the regulation of growth plate chondrogenesis and longitudinal bone growth

Inborn errors of cholesterol synthesis are associated with multiple systemic abnormalities, including skeletal malformations. The regulatory role of cholesterol during embryogenesis appears to be mediated by Shh, a signaling molecule in which activity depends on molecular events involving cholesterol. Based on this evidence, we hypothesized that cholesterol, by modifying the activity of Ihh (another of the Hedgehog family proteins) in the growth plate, regulates longitudinal bone growth. To test this hypothesis, we treated rats with AY 9944, an inhibitor of the final reaction of cholesterol synthesis. After 3 weeks, AY 9944 reduced the cumulative growth, tibial growth, and the tibial growth plate height of the rats. To determine whether cholesterol deficiency affects bone growth directly at the growth plate, we then cultured fetal rat metatarsal bones in the presence of AY 9944. After 4 days, AY 9944 suppressed metatarsal growth and growth plate chondrocyte proliferation and hypertrophy. The inhibitory effect on chondrocyte hypertrophy was confirmed by the AY 9944-mediated decreased expression of collagen X. Lastly, AY 9944 decreased the expression of Ihh in the metatarsal growth plate. We conclude that reduced cholesterol synthesis in the growth plate, possibly by altering the normal activity of Ihh, results in suppressed longitudinal bone growth and growth plate chondrogenesis.     

Parathyroid hormone-related peptide (PTHrP)-dependent and -independent effects of transforming growth factor beta (TGF-beta) on endochondral bone formation.

Previously, we showed that expression of a dominant-negative form of the transforming growth factor beta (TGF-beta) type II receptor in skeletal tissue resulted in increased hypertrophic differentiation in growth plate and articular chondrocytes, suggesting a role for TGF-beta in limiting terminal differentiation in vivo. Parathyroid hormone-related peptide (PTHrP) has also been demonstrated to regulate chondrocyte differentiation in vivo. Mice with targeted deletion of the PTHrP gene demonstrate increased endochondral bone formation, and misexpression of PTHrP in cartilage results in delayed bone formation due to slowed conversion of proliferative chondrocytes into hypertrophic chondrocytes. Since the development of skeletal elements requires the coordination of signals from several sources, this report tests the hypothesis that TGF-beta and PTHrP act in a common signal cascade to regulate endochondral bone formation. Mouse embryonic metatarsal bone rudiments grown in organ culture were used to demonstrate that TGF-beta inhibits several stages of endochondral bone formation, including chondrocyte proliferation, hypertrophic differentiation, and matrix mineralization. Treatment with TGF-beta1 also stimulated the expression of PTHrP mRNA. PTHrP added to cultures inhibited hypertrophic differentiation and matrix mineralization but did not affect cell proliferation. Furthermore, terminal differentiation was not inhibited by TGF-beta in metatarsal rudiments from PTHrP-null embryos; however, growth and matrix mineralization were still inhibited. The data support the model that TGF-beta acts upstream of PTHrP to regulate the rate of hypertrophic differentiation and suggest that TGF-beta has both PTHrP-dependent and PTHrP-independent effects on endochondral bone formation.     

Immunohistochemical Localization of Bone Morphogenetic Proteins (BMPs) and their Receptors in Solitary and Multiple Human Osteochondromas

The expression of bone morphogenetic proteins (BMPs) and their cognate receptors (BMPRs) in osteochondromas has not been investigated. We determined the immunohistochemical localization and distribution of BMP-2/4, -6 and -7; BMP receptors BMPR-1A, BMPR-1B and BMPR-2; signal transducing proteins phosphorylated Smad1/5/8; and BMP antagonist noggin in the cartilaginous cap of solitary (SO) and multiple (MO) human osteochondromas and compared these with bovine growth plate and articular cartilage. The distribution and localization patterns for BMP-6, BMP-7, BMPR-1A and BMPR-2 were similar between the cartilaginous cap and the growth plate. BMP-2/4 and BMPR-1B were present throughout the growth plate. However, BMP-2/4 and phosphorylated Smad1/5/8 were mainly detected in proliferating chondrocytes of the cartilaginous cap. Also, BMPR-1B was found in hypertrophic chondrocytes of SO and proliferating chondrocytes of MO. Noggin was observed in resting chondrocytes and, to a lesser extent, in clustered proliferating chondrocytes in SO. On the other hand, noggin in MO was observed in proliferating chondrocytes. Since BMPs can stimulate proliferation and hypertrophic differentiation of chondrocytes, these findings suggest that there is an imbalance of BMP-2/4 and noggin interactions that may lead to abnormal regulation of chondrocyte proliferation and differentiation in the cartilaginous cap of human osteochondromas.     

Chondrocyte apoptosis enhanced at the growth plate: a physeal response to a diaphyseal fracture

Post-traumatic overgrowth of growing long bones is a common clinical phenomenon in paediatric traumatology and is the result of an enhanced stimulation of the nearby growth plate after fracture. To date, the exact post-fractural reactions of the growth plate are poorly understood. The aim of this study has been to determine the impact of fracture on the frequency of chondrocyte apoptosis of the growth plate. Rats sustained a mid-diaphyseal closed fracture of the left tibia or were left untreated. All animals were killed 3, 10, 14 or 29 days after trauma. The left and right tibiae were harvested and apoptotic chondrocytes of the proximal tibial growth plate were detected by TUNEL staining. The apoptosis percentage of physeal chondrocytes was statistically compared among fractured bones, intact contra-lateral bones and control bones. The physeal apoptosis rate of the fractured bone was significantly higher than that of the contra-lateral intact bone (valid for all evaluated days) and the control bone (valid from day 10 onwards). Contra-lateral intact tibiae never showed significantly higher apoptosis rates compared with control tibiae. Thus, mid-diaphyseal fracture influences the nearby growth plate by stimulating chondrocyte programmed cell death, which is associated with cartilage resorption and bone replacement. The lack of a significant difference between the intact contra-lateral and the intact control bone suggests that fracture only has a local effect that contributes to the greater apoptosis rate of the adjacent physis.     

FGF signaling targets the pRb-related p107 and p130 proteins to induce chondrocyte growth arrest

Unregulated FGF signaling affects endochondral ossification and long bone growth, causing several genetic forms of human dwarfism. One major mechanism by which FGFs regulate endochondral bone growth is through their inhibitory effect on chondrocyte proliferation. Because mice with targeted mutations of the retinoblastoma (Rb)-related proteins p107 and p130 present severe endochondral bone defects with excessive chondrocyte proliferation, we have investigated the role of the Rb family of cell cycle regulators in the FGF response. Using a chondrocyte cell line, we found that FGF induced a rapid dephosphorylation of all three proteins of the Rb family (pRb, p107, and p130) and a blockade of the cells in the G1 phase of the cell cycle. This cell cycle block was reversed by inactivation of Rb proteins with viral oncoproteins such as polyoma large T (PyLT) antigen and Adenovirus E1A. Expression of a PyLT mutant that efficiently binds pRb, but not p107 and p130, allowed the cells to be growth inhibited by FGF, suggesting that pRb itself is not involved in the FGF response. To investigate more precisely the role of the individual Rb family proteins in FGF-mediated growth inhibition, we used chondrocyte micromass culture of limb bud cells isolated from mice lacking Rb proteins individually or in combination. Although wild-type as well as Rb-/- chondrocytes were similarly growth inhibited by FGF, chondrocytes null for p107 and p130 did not respond to FGF. Furthermore, FGF treatment of metatarsal bone rudiments obtained from p107-/-;p130-/- embryos failed to inhibit proliferation of growth plate chondrocytes, whereas rudiments from p107-null or p130-null embryos showed only a slight inhibition of growth. Our findings indicate that p107 and p130, but not pRb, are critical effectors of FGF-mediated growth inhibition in chondrocytes.