Abstract
Parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHRP) regulate Na+/H+ exchanger activity in osteoblastic cells, although the signaling components involved are not precisely defined. Since these peptide hormones can stimulate production of diverse second messengers (i.e. cAMP and diacylglycerol) that activate protein kinase A (PKA) and protein kinase C (PKC) in target cells, it is conceivable that either one or both of these pathways can participate in modulating exchanger activity. To discriminate among these possibilities, a series of synthetic PTH and PTHRP fragments were used that stimulate adenylate cyclase and/or PKC. In the osteoblastic cell line UMR-106, human PTH(1-34) and PTHRP(1-34) augmented adenylate cyclase activity, whereas PTH(3-34), PTH(28-42), and PTH(28-48) had no effect. Nevertheless, all these peptide fragments were found to enhance PKC translocation from the cytosol to the membrane in a dose-dependent (10(-11) to 10(-7) M) manner. PTHRP(1-16), a biologically inert fragment, was incapable of influencing either the PKA or PKC pathway. PTH(1-34) and PTHRP(1-34), but not PTH(3-34), PTH(28-42), PTH(28-48), or PTHRP(1-16), elevated Na+/H+ exchanger activity, implicating cAMP as the transducing signal. In accordance with this observation, forskolin (10 microM), which directly stimulates adenylate cyclase, also activated Na+/H+ exchanger activity. The involvement of PKA was verified when the highly specific PKA inhibitor, H-89, completely abolished the stimulatory effect of PTH(1-34) and forskolin on Na+/H+ exchange. In addition, Northern blot analysis revealed the presence of only the NHE-1 isoform of the Na+/H+ exchanger in UMR-106 cells. In summary, these results indicated that PTH and PTHRP activate the Na+/H+ exchanger NHE-1 isoform in osteoblastic UMR-106 cells exclusively via a cAMP-dependent pathway.
Highlights
¶ Recipient of a studentship award from the MRC. ʈ Recipient of a Chercheur-Boursier (Scholarship) from the Fond de la Recherche en Santedu Quebec
To verify the linkage of the Parathyroid hormone (PTH) receptor to these signaling pathways in our UMR-106 cell line, experiments were conducted using a series of synthetic fragments of human PTH and parathyroid hormone-related peptide (PTHRP) that should allow one to distinguish between activation of the protein kinase A (PKA) and protein kinase C (PKC) pathways
This study has identified that the Naϩ/Hϩ exchanger isoform present in UMR-106 cells is NHE-1, which is the most broadly distributed isoform in mammalian tissues
Summary
¶ Recipient of a studentship award from the MRC. ʈ Recipient of a Chercheur-Boursier (Scholarship) from the Fond de la Recherche en Santedu Quebec. Numerous studies have shown that Naϩ/Hϩ exchanger activity is acutely regulated by a wide variety of stimuli that activate diverse signal transduction systems (i.e. PKA, PKC, Ca2ϩ/calmodulindependent protein kinase II, tyrosine kinases), in many cases the precise mechanisms have yet to be fully elucidated (reviewed in Ref. 10) This extensive functional and regulatory diversity appears to be generated by tissue-specific expression of multiple isoforms of the Na/H exchanger as well as by variations in the signaling repertoire of individual cell types (reviewed in Ref. 11). Using UMR106 cells as a model system, we wished to examine in greater detail the reported linkage of PTH/PTHRP to the PKA and PKC signaling pathways, and possibly to another pathway involving G␣13, in regulating Naϩ/Hϩ exchanger activity. Our results demonstrated that only the NHE-1 isoform is expressed in UMR-106 cells and that PTH and PTHRP selectively activate NHE-1 via a pathway involving PKA
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