1alpha,25-dihydroxyvitamin D(3) and 24R,25-dihydroxyvitamin D(3) modulate growth plate chondrocyte physiology via protein kinase C-dependent phosphorylation of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase.
Membrane-mediated increases in protein kinase C (PKC) activity and PKC-dependent physiological responses of growth plate chondrocytes to vitamin D metabolites depend on the state of endochondral maturation; 1alpha,25-dihydroxyvitamin D(3) [1alpha,25-(OH)(2)D(3)] regulates growth zone (GC) cells, whereas 24R,25-(OH)(2)D(3) regulates resting zone (RC) cells. Different mechanisms, including protein kinase A signaling, mediate the effects of 1alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3) on PKC, suggesting that different mechanisms may also regulate any MAPK involvement in the physiological responses. This study used confluent cultures of rat costochondral chondrocytes as a model. 1alpha,25-(OH)(2)D(3) stimulated MAPK specific activity in GC in a time- and dose-dependent manner, evident within 9 min. 24R,25-(OH)(2)D(3) stimulated MAPK in RC; increases were dose dependent, occurred after 9 min, and were greatest at 90 min. In both cells the effect was due to ERK1/2 activation (p42 > p44 in GC; p42 = p44 in RC). MAPK activation was dependent on PKC, but not protein kinase A. The effect of 1alpha,25-(OH)(2)D(3) required phospholipase C, and the effect of 24R,25-(OH)(2)D(3) required phospholipase D. Inhibition of cyclooxygenase activity reduced the effect of 1alpha,25-(OH)(2)D(3) on MAPK in GC and enhanced the effect of 24R,25-(OH)(2)D(3) in RC. Based on MAPK inhibition with PD98059, ERK1/2 MAPK mediated the effect of 24R,25-(OH)(2)D(3) on [(3)H]thymidine incorporation and [(35)S]sulfate incorporation by RC, but only partially mediated the effect of 1alpha,25-(OH)(2)D(3) on GC. ERK1/2 was not involved in the regulation of alkaline phosphatase specific activity by either metabolite. This paper supports the hypothesis that 1alpha,25-(OH)(2)D(3) regulates the physiology of GC via rapid membrane-mediated signaling pathways, and some, but not all, of the response to 1alpha,25-(OH)(2)D(3) is via the ERK family of MAPKs. In contrast, 24R,25-(OH)(2)D(3) exerts its effects on RC via PKC-dependent MAPK. Whereas 1alpha,25-(OH)(2)D(3) increases MAPK activity via phospholipase C and increased prostaglandin production, 24R,25-(OH)(2)D(3) increases MAPK via phospholipase D and decreased prostaglandin production. The cell specificity, metabolite stereospecificity, and the dependence on PKC argue for the participation of membrane receptors for 1alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3) in the regulation of ERK1/2 in the growth plate.
Schwartz
Z
Z
Department of Orthopedics, University of Texas Health Science Center, San Antonio 78229, USA.
Ehland
H
H
Sylvia
V L
VL
Larsson
D
D
Hardin
R R
RR
Bingham
V
V
Lopez
D
D
Dean
D D
DD
Boyan
B D
BD
D015650:24,25-Dihydroxyvitamin D 3 / Q000494:pharmacology*; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002117:Calcitriol / Q000494:pharmacology*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000502:physiology*; D004789:Enzyme Activation / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006132:Growth Plate / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D007202:Indicators and Reagents; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D010740:Phospholipases / Q000378:metabolism; D010766:Phosphorylation; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D011509:Proteoglycans / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects*
D007202:Indicators and Reagents; D011509:Proteoglycans; D012333:RNA, Messenger; D015650:24,25-Dihydroxyvitamin D 3; D011451:Prostaglandin-Endoperoxide Synthases; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D010740:Phospholipases; D000469:Alkaline Phosphatase; D002117:Calcitriol