Growth hormone regulation of the growth hormone receptor mRNA in cultured rat epiphyseal chondrocytes.
This study was designed to investigate whether growth hormone (GH) influences the expression of its own receptor in chondrocytes. To investigate this possibility GH-receptor mRNA was measured in cultured rat epiphyseal chondrocytes in the absence or presence of GH under various experimental conditions. Chondrocytes were isolated enzymatically from epiphyseal growth plates of the proximal tibia of 20-day-old male rats and cultured in monolayer in Ham's F-12 medium supplemented with 10% calf serum and 1% of a serum substitute. The cells were seeded at various densities (100,000-1,000,000 cells per flask) and cultured for 14 days. Subsequently, the calf serum-containing medium and the cells cultured for various periods of time (0-24 h) before total nucleic acid preparation. GH-receptor mRNA was measured with a solution hybridization technique using [35S]UTP-labeled RNA growth hormone receptor cloned from rat liver cDNA. Human GH (hGH; 50 ng/ml) increased GH-receptor mRNA after 3 h and maximal levels were seen 12 h after GH addition. This effect of hGH was time and dose dependent with a significant effect of hGH at a concentration of 0.5 ng/ml and a maximal effect at 50 ng/ml. The hGH-stimulated increase of GH-receptor mRNA was completely blocked by actinomycin-C1 (1.0-0.1 micrograms/ml), while cycloheximide (10 micrograms/ml) only slightly counteracted the hGH effect. Rat and human GH were equally potent, and ovine prolactin was effective at 500 ng/ml but not 5 and 50 ng/ml. A high dose of insulin-like growth factor-I (IGF-I; 1 microgram/ml) caused a small stimulatory effect and addition of 10% calf serum caused a marked increase in GH-receptor mRNA. The level of GH receptor mRNA after 14 days of culture was inversely proportional to the cell density at the start of culture. These results show that GH specifically regulates mRNA levels for its own receptor in rat epiphyseal chondrocytes by interacting with somatogenic binding sites. These findings also suggest a transcription-dependent regulatory system between the GH-receptor and the GH-receptor gene.
Department of Physiology, University of Göteborg, Sweden.
A
A
Nilsson
B
B
Carlsson
L
L
Mathews
O G
OG
Isaksson
D000818:Animals; D015152:Blotting, Northern; D002478:Cells, Cultured; D003470:Culture Media; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000031:analogs & derivatives / Q000494:pharmacology; D005786:Gene Expression Regulation; D013006:Growth Hormone / Q000378:metabolism / Q000494:pharmacology*; D006132:Growth Plate / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008297:Male; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011986:Receptors, Somatotropin / Q000235:genetics*
D003470:Culture Media; D012333:RNA, Messenger; D011986:Receptors, Somatotropin; C030013:cactinomycin; D003609:Dactinomycin; D013006:Growth Hormone; D003513:Cycloheximide