h-89 and Bone-Neoplasms

It has been shown that spinal PKA/CREB signaling pathway is involved in neuropathic and inflammatory pain, but its effects on bone cancer pain have not previously been investigated. The aim of this study was to examine the potential role of the spinal PKA/CREB signaling pathway in the development of bone cancer pain.. A bone cancer pain model was made by inoculation of Walker 256 cells into the intramedullary space of rat tibia. Western blot analysis examined the expression of PKAca (PKA catalytic subunit) and phospho-CREB (p-CREB) protein levels. The authors further investigated effects of intrathecal treatment with H-89 (a PKA inhibitor, 8 nmol) or forskolin (a PKA agonist, 10 nmol) on nociceptive behavior and the expression of PKAca and p-CREB.. On days 6, 9, and 15 after inoculation, the expression of PKAca and p-CREB protein levels were higher in the bone cancer pain rats compared to the sham rats. On day 9, intrathecal administration of H-89 significantly attenuated bone cancer-induced mechanical allodynia as well as upregulation of PKAca and p-CREB protein levels. These effects were completely abolished by intrathecal pretreatment with the PKA agonist forskolin.. The results suggest that the spinal PKA/CREB signaling pathway may participate in the development of bone cancer pain. The findings of this study may provide an evidence for developing novel analgesics to block bone cancer pain.

Topics: Animals; Bone Neoplasms; Colforsin; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Female; Isoquinolines; Pain; Pain Measurement; Posterior Horn Cells; Rats; Rats, Sprague-Dawley; Signal Transduction; Sulfonamides; Up-Regulation

Bone cancer pain is difficult to treat and has a strong impact on the quality of life of patients. Few therapies have emerged because the molecular mechanisms underlying bone cancer pain are poorly understood. Recently, T-cell death-associated gene 8 (TDAG8) has been shown to participate in complete Freund's adjuvant-induced chronic inflammatory pain. In this study, we aimed to examine whether TDAG8 and its downstream protein kinase A (PKA) pathway are involved in bone cancer pain. A bone cancer pain model was made by inoculation of Walker 256 cells into the intramedullary space of rat tibia. Spinal TDAG8 expression was increased after inoculation with tumor cells. Intrathecal TDAG8 siRNA attenuated bone cancer pain behaviors during the initiation and maintenance phases; there were also concomitant decreases in TDAG8 mRNA and protein levels in spinal cord. Moreover, we found spinal PKA and phosphorylated cAMP response element-binding (pCREB) protein levels were up-regulated in the rat model of bone cancer pain. Knockdown of TDAG8 resulted in reduced bone cancer pain-induced spinal PKA and pCREB protein expression in two procedures. Furthermore, intrathecal H-89 (a PKA inhibitor) significantly attenuated bone cancer pain behaviors in rats. Our results suggest a causal relationship between TDAG8 expression and the initiation and maintenance of bone cancer pain. Activation of spinal TDAG8 contributes to bone cancer pain through the PKA signaling pathway in rats. These findings may lead to novel strategies for the treatment of bone cancer pain.

Topics: Animals; Bone Neoplasms; Carcinoma 256, Walker; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Female; Isoquinolines; Neoplasm Transplantation; Pain; Posterior Horn Cells; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Receptors, G-Protein-Coupled; RNA, Small Interfering; Sulfonamides; Up-Regulation

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