Protein kinase A-dependent inhibition of alkaline phosphatase release by SaOS-2 human osteoblastic cells: studies in new mutant cell lines that express a cyclic AMP-resistant phenotype.
We have established mutant SaOS-2 cell lines that express a cyclic AMP (cAMP)-resistant phenotype to investigate the regulation and functional importance of orthophosphoric-monoester phosphohydrolase alkaline optimum (ALPase) in the action of parathyroid hormone (PTH). Cells were stably transfected with a plasmid that directs the synthesis of a mutant form of the type I regulatory subunit of protein kinase A (PKA) under the control of the metallothionein promotor. There was no significant difference between parental SaOS-2 cells and the mutant lines in the affinity or number of receptors for 125I-Nle8,18Tyr34bPTH1-34NH2, either in the absence or presence of Zn2+. When cAMP-dependent gene transcription was examined using transient transfection with a somatostatin promoter-chloramphenicol acetyl transferase (CAT) reporter plasmid, CAT activity stimulated by human PTH and dibutyryl cAMP (DBcAMP) was inhibited by greater than 90% in the presence of Zn2+ in the mutant cell lines. In contrast, activation by a phorbol ester of a pentameric collagenase promoter/CAT construct containing five tandem copies of the AP-1 response element (5x-TRE-CAT) was unaffected in Zn(2+)-treated mutant cells. The inhibitory actions of PTH and DBcAMP on ALPase release were blunted by up to 80-90% in the mutant cell lines in the presence of Zn2+; there were no significant differences in the magnitude of inhibitory effects between these agonists. We conclude that the inhibitory action of PTH on ALPase release in SaOS-2 cells is mediated via activation of PKA. These cAMP-resistant cell lines will be especially useful in elucidating signal transduction mechanism(s) for PTH in human osteoblastic cells.
Fukayama
S
S
Endocrine Unit, Massachusetts General Hospital, Boston.
Kearns
A K
AK
Skurat
R M
RM
Tashjian
A H
AH
Bringhurst
F R
FR
D000469:Alkaline Phosphatase / Q000378:metabolism*; D002460:Cell Line; D000242:Cyclic AMP / Q000502:physiology; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D006801:Humans; D066298:In Vitro Techniques; D015759:Ionomycin / Q000494:pharmacology; D009154:Mutation; D010006:Osteoblasts / Q000201:enzymology*; D010281:Parathyroid Hormone / Q000502:physiology*; D011493:Protein Kinase C / Q000502:physiology; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011953:Receptors, Cyclic AMP / Q000235:genetics; D015398:Signal Transduction; D014162:Transfection; D014660:Vasoactive Intestinal Peptide / Q000502:physiology
D010281:Parathyroid Hormone; D011953:Receptors, Cyclic AMP; D014660:Vasoactive Intestinal Peptide; D015759:Ionomycin; D000242:Cyclic AMP; D011494:Protein Kinases; D011493:Protein Kinase C; D000469:Alkaline Phosphatase