h-89 and Osteosarcoma

Parathyroid hormone (PTH)-related peptide (PTHrP) can modulate the proliferation and differentiation of a number of cell types including osteoblasts. PTHrP can activate a G protein-coupled PTH/PTHrP receptor, which can interface with several second-messenger systems. In the current study, we have examined the signaling pathways involved in stimulated type I collagen and alkaline phosphatase expression in the human osteoblast-derived osteosarcoma cells, MG-63. By use of Northern blotting and histochemical analysis, maximum induction of these two markers of osteoblast differentiation occurred after 8 h of treatment with 100 nM PTHrP-(1-34). Chemical inhibitors of adenylate cyclase (H-89) or of protein kinase C (chelerythrine chloride) each diminished PTHrP-mediated type I collagen and alkaline phosphatase stimulation in a dose-dependent manner. These effects of PTHrP could also be blocked by inhibiting the Ras-mitogen-activated protein kinase (MAPK) pathway with a Ras farnesylation inhibitor, B1086, or with a MAPK inhibitor, PD-98059. Transient transfection of MG-63 cells with a mutant form of Galpha, which can sequester betagamma-subunits, showed significant downregulation of PTHrP-stimulated type I collagen expression, as did inhibition of phosphatidylinositol 3-kinase (PI 3-kinase) by wortmannin. Consequently, the betagamma-PI 3-kinase pathway may be involved in PTHrP stimulation of Ras. Collectively, these results demonstrate that, acting via its G protein-coupled receptor, PTHrP can induce indexes of osteoblast differentiation by utilizing multiple, perhaps parallel, signaling pathways.

Topics: Alkaloids; Benzophenanthridines; Blotting, Northern; Cell Differentiation; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; GTP-Binding Protein alpha Subunits, Gs; Heterotrimeric GTP-Binding Proteins; Humans; Isoquinolines; Mitogen-Activated Protein Kinases; Mutation; Osteoblasts; Osteosarcoma; Parathyroid Hormone-Related Protein; Phenanthridines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase C; Proteins; ras Proteins; Signal Transduction; Sulfonamides; Transfection; Tumor Cells, Cultured

In previous studies, we have shown that calcitonin gene-related peptide (CGRP) acutely inhibits 45Ca2+ uptake in osteoblastic UMR106 cells, and we have proposed that ATP-sensitive potassium (K(ATP)) channels are involved in mediating this action of CGRP. To directly test this proposed mechanism, we have now examined the effects of CGRP on both membrane potential (Em) and K+ mobilization in UMR106 cells, using specific fluorometric dye assays. CGRP (0.01-100 nM) induced membrane hyperpolarization in a dose-dependent manner, with a half maximal effect (ED50) at approximately 0.2 nM and a maximal effect at 100 nM. Both pinacidil (Pina; a K(ATP) channel opener) and forskolin (FSK) induced similar membrane hyperpolarization, but the actions of these three agents could be easily distinguished: both CGRP and Pina actions were well antagonized by glibenclamide (Glib; a selective K(ATP) channel blocker), whereas FSK action was strongly attenuated only by tetraethylammonium (a K(Ca) channel blocker) or compound H-89 (an inhibitor of cAMP-dependent protein kinases). Cells pretreated with Pina no longer responded to CGRP, but they could still respond to FSK; furthermore, pretreatment with FSK failed to block successive treatment with either CGRP or Pina. In parallel with observed changes in Em, CGRP (0.01-100 nM) decreased intracellular K+ concentrations ([K+]i) in a dose-dependent manner, with an ED50 identical to that obtained for alterations in Em. This action of CGRP was sensitive to Glib and had only slight sensitivity to tetraethylammonium; this CGRP effect was mimicked by Pina but not by FSK. Interestingly, CGRP significantly elicited changes in cell shape by a Glib-sensitive mechanism that included notable decreases in cross-sectional cytoplasmic area. These observations strongly support our proposal that CGRP primarily stimulates K+ efflux via activation of K(ATP) channels and thereby induces membrane hyperpolarization in UMR106 cells. Furthermore, our data also suggest that this cascade of initial cellular events may result in rapid changes in cell morphology and decreases in cellular area of the type that are thought to act as triggers for proliferation and/or differentiation in many cellular phenotypes.

Topics: Adenosine Triphosphate; Animals; Calcitonin Gene-Related Peptide; Colforsin; Enzyme Inhibitors; Glyburide; Guanidines; Isoquinolines; Membrane Potentials; Osteoblasts; Osteosarcoma; Pinacidil; Potassium; Potassium Channel Blockers; Potassium Channels; Protein Kinase Inhibitors; Rats; Sulfonamides; Tetraethylammonium; Tumor Cells, Cultured

Osteocalcin (OC) is a bone-specific extracellular matrix protein expressed by mature osteoblasts during late stages of differentiation. Previous studies have shown that forskolin, an activator of adenylate cyclase, stimulated OC production. Because PTH has been shown to activate several intracellular signal transduction pathways including cAMP, inositol phosphate and intracellular calcium mobilization, we investigated whether PTH action on cAMP accumulation leads to OC promoter activation. The rat OC promoter (1095 bp) was cloned into the promoterless luciferase gene reporter vector. The transcriptional activity of the rat OC promoter was evaluated after transfection of SaOS-2, an osteosarcoma cell line, with the OC promoter followed by treatment with PTH. Maximal OC promoter activity was observed within 4-8 h after the addition of 10(-8) M PTH, whereas very little induction was seen after 24 and 48 h of treatment. The induction of OC promoter activity by PTH was concentration dependent. PTH analogs (PTH 1-84, PTH 1-34, and PTH 1-31) that stimulate intracellular cAMP accumulation, induced OC promoter activity, whereas other PTH analogs (PTH 3-34, PTH 7-34, PTH 13-34, and PTH 53-84) that do not stimulate cAMP production had no effect on OC promoter activation. Furthermore, PTH activation of the OC promoter was significantly enhanced in the presence of 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor. Inactivation of cAMP-dependent protein kinase A activity by either a selective protein kinase A inhibitor, H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5 isoquinolinesulfonamide), or antisense oligonucleotide directed against the regulatory subunit of cAMP-dependent protein kinase A, led to a corresponding loss of OC promoter activation by PTH. 5' deletion analysis of the OC promoter demonstrated that the promoter (1095 bp) exhibited the greatest response to PTH, whereas the -198 bp construct of the OC promoter, containing only one cAMP response element and OC box, was no longer responsive. The constructs with further deletions (-120, -92, and -74) retained PTH responsiveness, but to a lesser extent. In summary, our results indicate that PTH activation of the OC promoter is a rapid event and mediated by the cAMP-dependent protein kinase A pathway. Although the novel cAMP response region overlapping the OC box is required for activation, full activation may require several cis-acting cAMP response elements or other response elements.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Base Sequence; Bone Neoplasms; Cyclic AMP; Dose-Response Relationship, Drug; Drug Synergism; Gene Deletion; Gene Expression Regulation; Genes, Reporter; Humans; Isoquinolines; Luciferases; Molecular Sequence Data; Osteocalcin; Osteosarcoma; Parathyroid Hormone; Phosphodiesterase Inhibitors; Promoter Regions, Genetic; Protein Kinase Inhibitors; Rats; Sulfonamides; Teriparatide; Time Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured

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.

Topics: Animals; Bone Neoplasms; Cell Line; Cell Membrane; CHO Cells; Colforsin; Cricetinae; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytosol; Enzyme Inhibitors; Humans; Isoquinolines; Kinetics; Osteosarcoma; Parathyroid Hormone; Parathyroid Hormone-Related Protein; Protein Kinase C; Proteins; Rats; Recombinant Proteins; Second Messenger Systems; Sodium-Hydrogen Exchangers; Sulfonamides; Tetradecanoylphorbol Acetate; Transfection; Tumor Cells, Cultured

1. Bone resorptive factors, prostaglandin E2 and parathyroid hormone are shown to suppress alkaline phosphatase activity in a rat osteoblastic cell line. 2. Phorbol myristate acetate, but not dibutyryl cAMP or calcium ionophore can suppress alkaline phosphatase activity. 3. The protein kinase C inhibitors (H89, staurosporine) are able to block the suppression of alkaline phosphatase activity induced by prostaglandin E2 and parathyroid hormone. 4. These data suggest that protein kinase C is involved in the inhibition of alkaline phosphatase activity induced by prostaglandin E2 and parathyroid hormone.

Topics: Alkaline Phosphatase; Alkaloids; Animals; Bucladesine; Calcimycin; Cyclic AMP; Cycloheximide; Dinoprostone; Isoquinolines; Osteoblasts; Osteosarcoma; Parathyroid Hormone; Protein Kinase C; Rats; Staurosporine; Sulfonamides; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured