Transforming growth factor-beta in calf articular cartilage organ cultures: synthesis and distribution.
Transforming growth factor beta 1 (TGF-beta 1) has been shown to play a prominent role in controlling proteoglycan synthesis and breakdown as measured following addition to organ cultures of calf articular cartilage (Morales, T. I., and Roberts, A. B., J. Biol. Chem., 263, 12,828-12,831, 1988). In this study, we compare two closely related TGF-beta isoforms, TGF-beta 1 and TGF-beta 2, both by assessing the effects of exogenous peptide as well as by analyzing the biosynthesis and total amount of these two isoforms in cartilage explants. Added exogenously, TGF-beta 1 and TGF-beta 2 induce a comparable increase in proteoglycan synthesis over basal controls with saturation at approximately 5 ng/ml. Synthesis of TGF-beta by basal calf cartilage cultures is demonstrated by (i) immunolocalization of intracellular TGF-beta, (ii) Northern blot analysis of steady-state mRNA levels, and (iii) immunoprecipitation of metabolically labeled TGF-beta from tissue extracts and conditioned culture medium. The net amount of extractable TGF-beta 1 and TGF-beta 2 in the basal cartilage cultures was assessed by a functional assay involving inhibition of proliferation of CCL-64 mink lung epithelial cells and by sandwich enzyme-linked immunosorbent assay. The predominant isoform was TGF-beta 1 (60-85%) and the total TGF-beta 1 + TGF-beta 2 was in excess of the amount required for maximal activation of proteoglycan synthesis. The level of both isoforms was maintained relatively constant between Days 2 and 7 of culture despite a sharp (approximately two to fourfold) drop in proteoglycan synthesis. This suggests that cartilage contains a large pool of TGF-beta which is not readily accessible to the chondrocyte. We propose that much of the polypeptide is sequestered in the matrix awaiting release upon demand.
Morales
T I
TI
Bone Research Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892.
Joyce
M E
ME
Sobel
M E
ME
Danielpour
D
D
Roberts
A B
AB
D000818:Animals; D015152:Blotting, Northern; D002358:Cartilage, Articular / Q000187:drug effects / Q000378:metabolism*; D002417:Cattle; D003545:Cysteine / Q000378:metabolism; D006025:Glycosaminoglycans / Q000096:biosynthesis; D007700:Kinetics; D009924:Organ Culture Techniques; D011509:Proteoglycans / Q000096:biosynthesis; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology
D006025:Glycosaminoglycans; D011509:Proteoglycans; D016212:Transforming Growth Factor beta; D012313:RNA; D003545:Cysteine