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


BACKGROUND
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.


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.
Boyan
Barbara D
BD
Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332-0363, USA. barbara.boyan@bme.gatech.edu
Wong
Kevin L
KL
Wang
Liping
L
Yao
Hai
H
Guldberg
Robert E
RE
Drab
Marek
M
Jo
Hanjoong
H
Schwartz
Zvi
Z
D000818:Animals; D021941:Caveolae / Q000378:metabolism*; D051242:Caveolin 1 / Q000378:metabolism*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D006132:Growth Plate / Q000166:cytology* / Q000648:ultrastructure; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D018345:Mice, Knockout; D046529:Microscopy, Electron, Transmission; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000494:pharmacology
D051242:Caveolin 1; C118756:dihydroxy-vitamin D3; D014807:Vitamin D; D011493:Protein Kinase C