Role of protein kinase-A in homologous down-regulation of parathyroid hormone (PTH)/PTH-related peptide receptor messenger ribonucleic acid in human osteoblast-like SaOS-2 cells.
Homologous down-regulation of PTH/PTH-related peptide (PTHrP) receptor expression occurs in several PTH-responsive osteoblastic cell lines, but the mechanisms responsible are not well understood. We have used wild-type SaOS-2 human osteoblastic cells, in which homologous PTH/PTHrP receptor down-regulation occurs within 4 h, and a mutant cAMP-resistant subclone (Ca4A strain), to investigate the mechanisms by which PTH/PTHrP receptor mRNA is regulated. SaOS-2 cells expressed a single 2.2- to 2.5-kilobase transcript of PTH/PTHrP receptor mRNA, as assessed by Northern blot analysis of total RNA with a cDNA probe encoding the human PTH/PTHrP receptor. Homologous down-regulation of this PTH/PTHrP receptor mRNA first became significant when SaOS-2 cells had been treated with human (h) PTH-(1-34) (10(-7) M) for 8-12 h. By 24 h, steady state levels of PTH/PTHrP receptor mRNA were reduced by about 50%. This effect was mimicked by both (Bu)2cAMP (DBcAMP; 0.5 mM) and forskolin (Fsk; 10(-5) M). In contrast, down-regulation of PTH/PTHrP receptor mRNA by hPTH-(1-34), DBcAMP or Fsk was almost completely blocked in cAMP-resistant Ca4A cells. Short term (4-6 h) treatment with hPTH-(1-34), DBcAMP, or Fsk did not reduce steady state levels of PTH/PTHrP receptor mRNA in either SaOS-2 or Ca4A cells, although down-regulation was induced by 4-6 h of treatment with active phorbol esters such as 12-O-tetradecanoyl phorbol-13-acetate (200 nM) or phorbol-12,13-didecanoate (200 nM). Neither thapsigargin (1 microM) nor ionomycin (200 nM), both of which stimulate calcium transients in these cells, altered PTH/PTHrP receptor mRNA expression. Treatment with hPTH-(39-84) and hPTH-(53-84), which do not activate either cAMP-dependent protein kinase or protein kinase-C, but do stimulate 45Ca2+ uptake in these cells, did not alter PTH/PTHrP receptor mRNA expression. In the presence of actinomycin-D (1 microgram/ml), down-regulation of PTH/PTHrP receptor mRNA by hPTH-(1-34) was not observed. Cycloheximide (10 micrograms/ml) did not block down-regulation of PTH/PTHrP receptor mRNA induced by hPTH-(1-34). We conclude that homologous down-regulation of PTH/PTHrP receptor mRNA in SaOS-2 cells occurs later than the decline in functional surface receptors via a mechanism that does not involve enhanced mRNA degradation or new protein synthesis, but is dependent upon cAMP/cAMP-dependent protein kinase.
Fukayama
S
S
Endocrine Unit, Massachusetts General Hospital, Boston 02114.
Schipani
E
E
Jüppner
H
H
Lanske
B
B
Kronenberg
H M
HM
Abou-Samra
A B
AB
Bringhurst
F R
FR
D015152:Blotting, Northern; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D015536:Down-Regulation; D006801:Humans; D010006:Osteoblasts / Q000378:metabolism*; D010281:Parathyroid Hormone / Q000494:pharmacology; D011493:Protein Kinase C / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism*; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone / Q000235:genetics*
D010281:Parathyroid Hormone; D012333:RNA, Messenger; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone; D003609:Dactinomycin; D003513:Cycloheximide; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C