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.

Abstract

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 12rl-Nle818Tyr31bPTH,_3.NH2, 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 >90%/o 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 Zn2+-treated mutant cells. The inhibitory actions of PTH and DBcAMP on ALPase release were blunted by § Corresponding author. 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.

HMGB1 expression and release by bone cells

“…PTH, via the activation of the protein kinase A (PKA) signaling pathway, attenuates the release of HMGB1 from primary osteoblasts and MC3T3‐E1 cells, yet enhances release from UMR 106‐01 cells. PTH‐induced activation of PKA also attenuates osteocalcin and alkaline phosphatase release in osteoblast‐like cells (Fukayama et al, 1991; Lajeunesse et al, 1991) while increasing alkaline phosphatase activity (present work and Nakatani et al, 1984). The PTH receptor is functionally coupled to both the PKA and protein kinase C (PKC) response limbs via the activation of adenylyl cyclase and phospholipase Cb, respectively, and in bone cells PTH regulates most of its response proteins through the PKA pathway (Swarthout et al, 2002).…”

Intermittent administration of PTH(1-34) regulates the osteoblastic differentiation of human periodontal ligament cells via protein kinase C- and protein kinase A-dependent pathways in vitro

“…In contrast to this view, Fukayama et al. (26) reported on a PKA‐dependent inhibition of ALP release by SaOS‐2 human osteoblastic cells. These apparent discrepancies stress the need for a refined view when interpreting data relative to previously published material that was obtained in different cell culture systems or following different experimental protocols.…”

“…These results strengthen the findings of previous reports on the predominant role of the cAMP/PKA‐dependent pathway in PTH signaling for the regulation of osteoblastic differentiation parameters, including ALP specific activity and bone sialoprotein (23–25). Conflicting evidence regarding the role of PKA as an activator or inhibitor of PTH‐induced target gene transcription (10,24,26,27) exists with the majority of the data obtained in osteoblast studies, supporting the concept that multiple mechanisms are involved in the PTH response by demonstrating the involvement of both cAMP and PKC in PTH signal transduction with a predominance of PKA as an activator of ALP activity (3,10,24). In contrast to this view, Fukayama et al.…”

A New Action of Parathyroid Hormone

“…SaOS-2 Ca#4A cells are a subclone of SaOS-2 cells in which enhancement of cAMP formation does not activate PKA due to the overexpression of a mutant PKA regulatory subunit (36). hPTH-(1-34) (100 nM) stimulated an acute increase in ECAR in these cells, while 1 mM 8-Br-cAMP did not produce any increase in ECAR, demonstrating that the cAMP-resistant phenotype was being expressed (data not shown 34 ]bPTH-(7-34)NH 2 (E) for 25 min and then sequentially exposed to increasing concentrations of hPTH-(1-34) (0.01-500 nM) for 2-min intervals, and ECAR was monitored.…”