Establishment of a growth hormone responsive chondrogenic cell line from fetal rat tibia.
Reproducible effects of growth hormone (GH) on primary isolated cells in monolayer are highly dependent on the culture conditions and/or the fraction of GH responsive cells. To study the effect of GH at the cellular level, a homogenous cell line with both GH responsiveness and chondrogenic properties was established. Primary isolated cells from 18-day-old fetal rat tibia were subcultured using a strict protocol for passages (every third day and a seeding density of 15,000/cm2). Of six established cell lines, one fetal tibia cell line No. 5 (FTC 5) expressed adipogenic and chondrogenic properties at a low frequency. Cells from FTC 5 were subcultured in soft agar suspension with the addition of bovine GH (100 ng/ml). After 14 days in culture eight monoclonal cell lines were established from individual large colonies. Two subclones, FTC 5:3 and FTC 5:6, expressed a chondrogenic phenotype as demonstrated by chondrocyte foci, alcian blue staining and production of type II collagen. Further characterization of FTC 5:3 revealed specific binding of bovine GH with an affinity of 1.7 x 10(9) M-1, and approximately 7300 receptors/cell. Northern blot analysis of FTC 5:3 with a 32P-labeled RNA probe complementary to an extracellular part of the rat GH receptor, revealed two major labeled bands (4.0 and 1.2 kilobases). Both GH and insulin-like growth factor-I (IGF-I) stimulated 3H-thymidine uptake in FTC 5:3 (194 +/- 28% and 405 +/- 127% over control, respectively), while proteoglycan synthesis, as measured by [35S]sulphate uptake, was stimulated by IGF-I only (101 +/- 18% over control).(ABSTRACT TRUNCATED AT 250 WORDS)
Ohlsson
C
C
Department of Physiology, University of Göteborg, Sweden.
Nilsson
A
A
Swolin
D
D
Isaksson
O G
OG
Lindahl
A
A
D000818:Animals; D015152:Blotting, Northern; D002356:Cartilage / Q000166:cytology / Q000187:drug effects* / Q000196:embryology*; D002455:Cell Division / Q000187:drug effects; D002999:Clone Cells; D003094:Collagen / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D013006:Growth Hormone / Q000378:metabolism / Q000494:pharmacology*; D007150:Immunohistochemistry; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D007621:Karyotyping; D010641:Phenotype; D012313:RNA / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D011986:Receptors, Somatotropin / Q000235:genetics / Q000378:metabolism; D013977:Tibia / Q000196:embryology
D011986:Receptors, Somatotropin; D012313:RNA; D007334:Insulin-Like Growth Factor I; D013006:Growth Hormone; D003094:Collagen; D004247:DNA