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.     

Cell cycle genes in chondrocyte proliferation and differentiation.

Coordinated proliferation and differentiation of growth plate chondrocytes controls longitudinal growth of endochondral bones. While many extracellular factors regulating these processes have been identified, much less is known about the intracellular mechanisms transducing and integrating these extracellular signals. Recent evidence suggests that cell cycle proteins play an important role in the coordination of chondrocyte proliferation and differentiation. Our current knowledge of the function and regulation of cell cycle proteins in endochondral ossification is summarized.     

The PI3K pathway regulates endochondral bone growth through control of hypertrophic chondrocyte differentiation

Background  

The majority of our bones develop through the process of endochondral ossification that involves chondrocyte proliferation and hypertrophic differentiation in the cartilage growth plate. A large number of growth factors and hormones have been implicated in the regulation of growth plate biology, however, less is known about the intracellular signaling pathways involved. PI3K/Akt has been identified as a major regulator of cellular proliferation, differentiation and death in multiple cell types.     

Reduced chondrocyte proliferation and chondrodysplasia in mice lacking the integrin-linked kinase in chondrocytes

Chondrocyte proliferation and differentiation requires their attachment to the collagen type II-rich matrix of developing bone. This interaction is mediated by integrins and their cytoplasmic effectors, such as the integrin-linked kinase (ILK). To elucidate the molecular mechanisms whereby integrins control these processes, we have specifically inactivated the ILK gene in growth plate chondrocytes using the Cre-lox methodology. Mice carrying an ILK allele flanked by loxP sites (ILK-fl) were crossed to transgenic mice expressing the Cre recombinase under the control of the collagen type II promoter. Inactivation of both copies of the ILK-fl allele lead to a chondrodysplasia characterized by a disorganized growth plate and to dwarfism. Expression of chondrocyte differentiation markers such as collagen type II, collagen type X, Indian hedgehog and the PTH-PTHrP receptor was normal in ILK-deficient growth plates. In contrast, chondrocyte proliferation, assessed by BrdU or proliferating cell nuclear antigen labeling, was markedly reduced in the mutant growth plates. Cell-based assays showed that integrin-mediated adhesion of primary cultures of chondrocytes from mutant animals to collagen type II was impaired. ILK inactivation in chondrocytes resulted in reduced cyclin D1 expression, and this most likely explains the defect in chondrocyte proliferation observed when ILK is inactivated in growth plate cells.     

Low mitochondrial DNA variation among American alligators and a novel non-coding region in crocodilians

We analyzed 1317-1823 base pairs (bp) of mitochondrial DNA sequence beginning in the 5' end of cytochrome b (cyt b) and ending in the central domain of the control region for 25 American alligators (Alligator mississippiensis) and compared these to a homologous sequence from a Chinese alligator (A. sinensis). Both species share a non-coding spacer between cyt b and tRNA(Thr). Chinese alligator cyt b differs from that of the American alligator by 17.5% at the nucleotide level and 13.8% for inferred amino acids, which is consistent with their presumed ancient divergence. Only two cyt b haplotypes were detected among the 25 American alligators (693-1199 bp surveyed), with one haplotype shared among 24 individuals. One alligator from Mississippi differed from all other alligators by a single silent substitution. The control region contained only slightly more variation among the 25 American alligators, with two variable positions (624 bp surveyed), yielding three haplotypes with 22, two, and one individuals in each of these groups. Previous genetic studies examining allozymes and the proportion of variable microsatellite DNA loci also found low levels of genetic diversity in American alligators. However, in contrast with allozymes, microsatellites, and morphology, the mtDNA data shows no evidence of differentiation among populations from the extremes of the species range. These results suggest that American alligators underwent a severe population bottleneck in the late Pleistocene, resulting in nearly homogenous mtDNA among all American alligators today.     

Endocrine regulation of the growth plate

Longitudinal bone growth occurs at the growth plate by endochondral ossification. Within the growth plate, chondrocyte proliferation, hypertrophy, and cartilage matrix secretion result in chondrogenesis. The newly formed cartilage is invaded by blood vessels and bone cells that remodel the newly formed cartilage into bone tissue. This process of longitudinal bone growth is governed by a complex network of endocrine signals, including growth hormone, insulin-like growth factor I, glucocorticoid, thyroid hormone, estrogen, androgen, vitamin D, and leptin. Many of these signals regulate growth plate function, both by acting locally on growth plate chondrocytes and also indirectly by modulating other endocrine signals in the network. Some of the local effects of hormones are mediated by changes in paracrine factors that control chondrocyte proliferation and differentiation. Many human skeletal growth disorders are caused by abnormalities in the endocrine regulation of the growth plate. This review provides an overview of the endocrine signals that regulate longitudinal bone growth, their interactions, and the mechanisms by which they affect growth plate chondrogenesis.     

Constitutive E2F1 overexpression delays endochondral bone formation by inhibiting chondrocyte differentiation

Longitudinal bone growth results from endochondral ossification, a process that requires proliferation and differentiation of chondrocytes. It has been shown that proper endochondral bone formation is critically dependent on the retinoblastoma family members p107 and p130. However, the precise functional roles played by individual E2F proteins remain poorly understood. Using both constitutive and conditional E2F1 transgenic mice, we show that ubiquitous transgene-driven expression of E2F1 during embryonic development results in a dwarf phenotype and significantly reduced postnatal viability. Overexpression of E2F1 disturbs chondrocyte maturation, resulting in delayed endochondral ossification, which is characterized by reduced hypertrophic zones and disorganized growth plates. Employing the chondrogenic cell line ATDC5, we investigated the effects of enforced E2F expression on the different phases of chondrocyte maturation that are normally required for endochondral ossification. Ectopic E2F1 expression strongly inhibits early- and late-phase differentiation of ATDC5 cells, accompanied by diminished cartilage nodule formation as well as decreased type II collagen, type X collagen, and aggrecan gene expression. In contrast, overexpression of E2F2 or E2F3a results in only a marginal delay of chondrocyte maturation, and increased E2F4 levels have no effect. These data are consistent with the notion that E2F1 is a regulator of chondrocyte differentiation.     

A network of transcriptional and signaling events is activated by FGF to induce chondrocyte growth arrest and differentiation

Activating mutations in FGF receptor 3 (FGFR3) cause several human dwarfism syndromes by affecting both chondrocyte proliferation and differentiation. Using microarray and biochemical analyses of FGF-treated rat chondrosarcoma chondrocytes, we show that FGF inhibits chondrocyte proliferation by initiating multiple pathways that result in the induction of antiproliferative functions and the down-regulation of growth-promoting molecules. The initiation of growth arrest is characterized by the rapid dephosphorylation of the retinoblastoma protein (pRb) p107 and repression of a subset of E2F target genes by a mechanism that is independent of cyclin E-Cdk inhibition. In contrast, hypophosphorylation of pRb and p130 occur after growth arrest is first detected, and may contribute to its maintenance. Importantly, we also find a number of gene expression changes indicating that FGF promotes many aspects of hypertrophic differentiation, a notion supported by in situ analysis of developing growth plates from mice expressing an activated form of FGFR3. Thus, FGF may coordinate the onset of differentiation with chondrocyte growth arrest in the developing growth plate.     

Cartilage formation in growth plate and arteries: from physiology to pathology

Vascular calcifications are the consequence of several pathological conditions such as atherosclerosis, diabetes, hypercholesterolemia and chronic renal insufficiency. They are associated with risks of amputation, ischemic heart disease, stroke and increased mortality. A growing body of evidence indicates that vascular smooth muscle cells (VSMCs) undergo chondrogenic commitment eventually leading to vascular calcification, by mechanisms similar to those governing ossification in the cartilage growth plate. Our knowledge of the formation of cartilage growth plate can therefore help us to understand why and how arteries calcify and, consequently, develop new therapeutic strategies. Reciprocally, thorough consideration of the events leading to ectopic chondrocyte differentiation appears crucial to further increase our understanding of growth plate formation. In this context, we will review the effects of known or suspected factors that promote chondrogenic differentiation in growth plate and arteries.     

Interaction of TGFβ and BMP signaling pathways during chondrogenesis

TGFβ and BMP signaling pathways exhibit antagonistic activities during the development of many tissues. Although the crosstalk between BMP and TGFβ signaling pathways is well established in bone development, the relationship between these two pathways is less well defined during cartilage development and postnatal homeostasis. We generated hypomorphic mouse models of cartilage-specific loss of BMP and TGFβ signaling to assess the interaction of these pathways in postnatal growth plate homeostasis. We further used the chondrogenic ATDC5 cell line to test effects of BMP and TGFβ signaling on each other's downstream targets. We found that conditional deletion of Smad1 in chondrocytes resulted in a shortening of the growth plate. The addition of Smad5 haploinsufficiency led to a more severe phenotype with shorter prehypertrophic and hypertrophic zones and decreased chondrocyte proliferation. The opposite growth plate phenotype was observed in a transgenic mouse model of decreased chondrocytic TGFβ signaling that was generated by expressing a dominant negative form of the TGFβ receptor I (ΔTβRI) in cartilage. Histological analysis demonstrated elongated growth plates with enhanced Ihh expression, as well as an increased proliferation rate with altered production of extracellular matrix components. In contrast, in chondrogenic ATDC5 cells, TGFβ was able to enhance BMP signaling, while BMP2 significantly reduces levels of TGF signaling. In summary, our data demonstrate that during endochondral ossification, BMP and TGFβ signaling can have antagonistic effects on chondrocyte proliferation and differentiation in vivo. We also found evidence of direct interaction between the two signaling pathways in a cell model of chondrogenesis in vitro.     

VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation.

Hypertrophic chondrocytes in the epiphyseal growth plate express the angiogenic protein vascular endothelial growth factor (VEGF). To determine the role of VEGF in endochondral bone formation, we inactivated this factor through the systemic administration of a soluble receptor chimeric protein (Flt-(1-3)-IgG) to 24-day-old mice. Blood vessel invasion was almost completely suppressed, concomitant with impaired trabecular bone formation and expansion of hypertrophic chondrocyte zone. Recruitment and/or differentiation of chondroclasts, which express gelatinase B/matrix metalloproteinase-9, and resorption of terminal chondrocytes decreased. Although proliferation, differentiation and maturation of chondrocytes were apparently normal, resorption was inhibited. Cessation of the anti-VEGF treatment was followed by capillary invasion, restoration of bone growth, resorption of the hypertrophic cartilage and normalization of the growth plate architecture. These findings indicate that VEGF-mediated capillary invasion is an essential signal that regulates growth plate morphogenesis and triggers cartilage remodeling. Thus, VEGF is an essential coordinator of chondrocyte death, chondroclast function, extracellular matrix remodeling, angiogenesis and bone formation in the growth plate.