Differential regulation of type-II and type-X collagen synthesis by parathyroid hormone-related protein in chick growth-plate chondrocytes.
Parathyroid hormone-related protein is a critical autocrine regulator of endochondral ossification in the growth plate, as demonstrated by the severe disruption of growth-plate structure and function in parathyroid hormone-related protein-deficient transgenic mice. In the present study, the effects of parathyroid hormone-related protein on the synthesis of collagen mRNA and protein were studied in short-term cultures of isolated chick growth-plate chondrocytes. Parathyroid hormone-related protein selectively inhibits type-X collagen protein synthesis with no significant effect on type-II collagen protein synthesis. These effects were present in all maturationally distinct populations of chondrocytes separated by countercurrent centrifugal elutriation. In cultures of resting chondrocytes, the onset of type-X collagen expression was inhibited, while the synthesis of type-X collagen was decreased in cultures of hypertrophic chondrocytes. Synthesis of type-II and type-X collagen mRNA was examined by nonradioactive in situ hybridization with synthetic oligonucleotide cDNA probes, and the level of expression was quantified using digital image analysis. Dose-dependent suppression of type-X collagen gene expression by parathyroid hormone-related protein was observed, with no significant effect on type-II collagen mRNA detected. The results were confirmed by analysis of Northern blots of total chondrocyte mRNA. These experiments demonstrated differential transcriptional regulation of type-II and type-X collagen, with selective suppression of type-X collagen expression, by parathyroid hormone-related protein in growth-plate chondrocytes. In addition, excellent agreement was found between traditional protein and mRNA analyses and microscopic digital image analysis techniques, supporting the use of this convenient and sensitive assay method. Parathyroid hormone-related protein inhibits chondrocyte maturation and is known to stimulate proliferation, suggesting that this autocrine factor may function to regulate premature hypertrophy in the growth plate.
O'Keefe
R J
RJ
Department of Orthopaedics, University of Rochester, NY 14642, USA.
Loveys
L S
LS
Hicks
D G
DG
Reynolds
P R
PR
Crabb
I D
ID
Puzas
J E
JE
Rosier
R N
RN
D000818:Animals; D015152:Blotting, Northern; D002356:Cartilage / Q000166:cytology / Q000378:metabolism*; D002478:Cells, Cultured; D002645:Chickens; D003094:Collagen / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006132:Growth Plate / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism
C470121:PTHLH protein, human; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins; D012333:RNA, Messenger; D003094:Collagen