Caveolin-1 knockout mice have increased bone size and stiffness.

UNLABELLED: The skeletal phenotype of the cav-1(-/-) mouse, which lacks caveolae, was examined. muCT and histology showed increased trabecular and cortical bone caused by the gene deletion. Structural changes were accompanied by increased mechanical properties. Cell studies showed that cav-1 deficiency leads to increased osteoblast differentiation. These results suggest that cav-1 helps to maintain osteoblast progenitors in a less differentiated state. INTRODUCTION: The absence of caveolin-1 in cellular membranes causes dysregulated signaling. To understand the role of the caveolar microdomain in bone homeostasis, we examined the skeletal phenotype of 5- and 8-wk-old cav-1(-/-) mice. MATERIALS AND METHODS: High-resolution microCT imaging showed a region-specific effect of cav-1 deficiency on the skeleton. At 5 wk, cav-1(-/-) mice had increased epiphyseal bone volume (+58.4%, p = 0.05); at 8 wk, metaphyseal bone volume was increased by 77.4% (p = 0.008). Cortical bone at the femoral mid-diaphysis showed that the periosteal area of cav-1(-/-) mice significantly exceeded that of cav-1(+/+) mice by 23.9% and 16.3% at 5 and 8 wk, respectively, resulting in increased mechanical properties (I(max): +38.2%, p = 0.003 and I(mi): +23.7%, p = 0.03). RESULTS: Histomorphometry complemented microCT results showing increased bone formation rate (BFR) at trabecular and cortical sites at 5 wk, which supported findings of increased bone at 8 wk in cav-1(-/-) mice. Formal mechanical testing of the femoral diaphysis confirmed increased bone structure: stiffness increased 33% and postyield deflection decreased 33%. Stromal cells from cav-1(-/-) marrow showed a 23% increase in von Kossa-positive nodules; osteoclastogenesis was also modestly increased in cav-1-deficient marrow. Knockdown of cav-1 with siRNA in wildtype stromal cells increased alkaline phosphatase protein and expression of osterix and Runx2, consistent with osteoblast differentiation. CONCLUSIONS: These data suggest that cav-1 helps to maintain a less differentiated state of osteoblast progenitor cells, and the absence of cav-1 causes bone to mature more rapidly. Caveolin-1 may thus be a target for altering skeletal homeostasis.

1alpha,25(OH)2D3 is an autocrine regulator of extracellular matrix turnover and growth factor release via ERp60 activated matrix vesicle metalloproteinases.

Growth plate chondrocytes produce proteoglycan-rich type II collagen extracellular matrix (ECM). During cell maturation and hypertrophy, ECM is reorganized via a process regulated by 1alpha,25(OH)(2)D(3) and involving matrix metalloproteinases (MMPs), including MMP-3 and MMP-2. 1alpha,25(OH)(2)D(3) regulates MMP incorporation into matrix vesicles (MVs), where they are stored until released. Like plasma membranes (PM), MVs contain the 1alpha,25(OH)(2)D(3)-binding protein ERp60, phospholipase A(2) (PLA(2)), and caveolin-1, but appear to lack nuclear Vitamin D receptors (VDRs). Chondrocytes produce 1alpha,25(OH)(2)D(3) (10(-8)M), which binds ERp60, activating PLA(2), and resulting lysophospholipids lead to MV membrane disorganization, releasing active MMPs. MV MMP-3 activates TGF-beta1 stored in the ECM as large latent TGF-beta1 complexes, consisting of latent TGF-beta1 binding protein, latency associated peptide, and latent TGF-beta1. Others have shown that MMP-2 specifically activates TGF-beta2. TGF-beta1 regulates 1alpha,25(OH)(2)D(3)-production, providing a mechanism for local control of growth factor activation. 1alpha,25(OH)(2)D(3) activates PKCalpha in the PM via ERp60-signaling through PLA(2), lysophospholipid production, and PLCbeta. It also regulates distribution of phospholipids and PKC isoforms between MVs and PMs, enriching the MVs in PKCzeta. Direct activation of MMP-3 in MVs requires ERp60. However, when MVs are treated with 1alpha,25(OH)(2)D(3), PKCzeta activity is decreased and PKCalpha is unaffected, suggesting a more complex feedback mechanism, potentially involving MV lipid signaling.

Regulation of growth plate chondrocytes by 1,25-dihydroxyvitamin D3 requires caveolae and caveolin-1.

UNLABELLED: We examined the role of caveolae and caveolin-1 in the mechanism of 1alpha,25(OH)(2)D(3) action in growth plate chondrocytes. We found that caveolae are required for rapid 1alpha,25(OH)(2)D(3)-dependent PKC signaling, and caveolin-1 must be present based on studies using chondrocytes from Cav-1(-/-) mice. INTRODUCTION: 1,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] regulates endochondral ossification in part through membrane-associated mechanisms, including protein kinase C (PKC) signaling activated by a membrane-associated 1alpha,25(OH)(2)D(3)-binding protein, ERp60. We tested the hypothesis that caveolae are required for 1alpha,25(OH)(2)D(3) action and play an important role in regulating chondrocyte biology and growth plate physiology. MATERIALS AND METHODS: Rat costochondral chondrocytes were examined for caveolae by transmission electron microscopy of cultured cells and of cells in situ. Western blots and confocal microscopy were used to detect caveolae proteins including caveolin-1 (Cav-1) and 1alpha,25(OH)(2)D(3) receptors. Caveolae cholesterol was depleted with beta-cyclodextrin (CD) and effects of 1alpha,25(OH)(2)D(3) on PKC, DNA synthesis, alkaline phosphatase, and proteoglycan production determined. Chondrocytes from Cav-1(-/-) and C57BL/6 wildtype mice were also treated with 1alpha,25(OH)(2)D(3). Epiphyses and costochondral junctions of 8-week-old male Cav-1(-/-) and wildtype mice (N = 8) were compared by histomorphometry and microCT. Data were analyzed by ANOVA and Bonferroni for posthoc comparisons. RESULTS: Growth zone chondrocytes had caveolae and Cav-1, -2, and -3. Resting zone chondrocytes, which do not exhibit a rapid 1alpha,25(OH)(2)D(3)-dependent increase in PKC activity, also had these caveolins, but caveolae were larger and fewer in number. ERp60 but not VDR co-localized with Cav-1 in plasma membranes and in lipid rafts. CD-treatment blocked 1alpha,25(OH)(2)D(3) effects on all parameters tested. The Cav-1(-/-) cells did not respond to 1alpha,25(OH)(2)D(3), although 1alpha,25(OH)(2)D(3) increased PKC, alkaline phosphatase, and [(35)S]-sulfate incorporation in wildtype C57BL/6 cells. Histology and microCT showed that Cav-1(-/-) growth plates were longer and had more hypertrophic cells in each column. Growth plate changes were reflected in the metaphysis. CONCLUSIONS: The membrane-mediated effects of 1alpha,25(OH)(2)D(3) require caveolae and Cav-1, and Cav-1 deficiency results in altered growth plate physiology.

Plasma membrane requirements for 1alpha,25(OH)2D3 dependent PKC signaling in chondrocytes and osteoblasts.

1,25-Dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] acts on chondrocytes and osteoblasts through traditional nuclear Vitamin D receptor (VDR) mechanisms as well as through rapid actions on plasma membranes that initiate intracellular signaling pathways. We have investigated the mechanisms involved in activation of protein kinase C (PKC) and downstream biological responses that depend on the latter pathway. These studies show that PKC activation depends on presence of a membrane receptor ERp60 and rapid increases in phospholipase A(2) (PLA(2)) activity. Cells that are responsive to 1alpha,25(OH)(2)D(3) express PLA(2) activating protein (PLAA), suggesting a link between ERp60 and PLA(2). Increased PLA(2) results in increased arachidonic acid release and formation of lysophospholipid, which then activates phospholipase C beta (PLCbeta), leading to rapid formation of inositol-trisphosphate (IP3) and diacylglycerol (DAG). PLA(2), PLC, and DAG are all associated with lipid rafts including caveolae in many cells, suggesting that the caveolar environment may be an important mediator of PKC activation by 1alpha,25(OH)(2)D(3). Here, we use the VDR(-/-) mouse costochondral cartilage growth plate to examine the expression of ERp60 and PLAA in vivo in 1alpha,25(OH)(2)D(3)-responsive hypertrophic chondrocytes (growth zone cells) and in resting zone cells that do not respond to this Vitamin D metabolite in vitro. In addition, we determined if intact lipid rafts are required for the response of rat costochondral cartilage growth zone cells to 1alpha,25(OH)(2)D(3). The results show that ERp60 and PLAA are localized to 1alpha,25(OH)(2)D(3)-responsive growth zone cells and metaphyseal osteoblasts, even in VDR(-/-) mice. Disruption of lipid rafts using beta-cyclodextrin blocks the activation of PKC by 1alpha,25(OH)(2)D(3) and reduces the ability of 1alpha,25(OH)(2)D(3) to regulate [(35)S]-sulfate incorporation.