thapsigargin and phorbolol-myristate-acetate

Fluid and exocrine secretion of mucins by salivary mucous glands is regulated predominantly by parasympathetic activation of muscarinic receptors. A direct role for subsequent putative signaling steps, phospholipase C (PLC), increased intracellular calcium ([Ca(2+)](i)), and isoforms of protein kinase C (PKC) in mediating muscarinic exocrine secretion has not been elucidated, and these are potential therapeutic targets to enhance mucin secretion in hyposalivary patients. We found that muscarinic-induced mucin secretion by rat sublingual tubulo-acini was dependent upon PLC activation and the subsequent increase in [Ca(2+)](i), and further identified a transient PKC-independent component of secretion dependent upon Ca(2+) release from intracellular stores, whereas sustained secretion required entry of extracellular Ca(2+). Interactions among carbachol, PKC inhibitors, phorbol 12-myristate 13-acetate, and thapsigargin to modulate [Ca(2+)](i) implicated conventional PKC isoforms in mediating sustained secretion. With increasing times during carbachol perfusion of glands, in situ, PKC-α redistributed across glandular membrane compartments and underwent a rapid and persistent accumulation near the luminal borders of mucous cells. PKC-β1 displayed transient localization near luminal borders, whereas the novel PKCs, PKC-δ or PKC-ε, displayed little or no redistribution in mucous cells. Collective results implicate synergistic interactions between diacylglycerol (DAG) and increasing [Ca(2+)](i) levels to activate cPKCs in mediating sustained muscarinic-induced secretion.

Topics: Acinar Cells; Animals; Calcium; Carbachol; Diglycerides; Drug Synergism; Enzyme Activation; Isoenzymes; Mucins; Muscarinic Agonists; Protease Inhibitors; Protein Kinase C; Rats; Rats, Wistar; Receptors, Muscarinic; Signal Transduction; Sublingual Gland; Tetradecanoylphorbol Acetate; Thapsigargin; Type C Phospholipases

Osteoprotegerin (OPG), a secreted member of the tumor necrosis receptor superfamily, is a potent inhibitor of osteoclast formation and bone resorption. Parathyroid hormone (PTH), a potent inducer of osteoclast formation, suppresses OPG mRNA expression in vitro and in vivo. To determine the molecular basis of this inhibition, we analyzed the effects of PTH on the human OPG promoter (-5917 to +19) fused with beta-galactosidase reporter gene in stable and transient transfections into rat osteoblast-like UMR106 cells. The effect of PTH on OPG promoter expression was biphasic and concentration-dependent. PTH (1-100 nM) induced the transcriptional activity of the OPG promoter (1.7-fold) at 8 h followed by a gradual decrease with maximal inhibition (6.6-fold) at 24-48 h. To ascertain the signal transduction pathways mediating PTH (1-38) effects on OPG gene expression, we compared the effects of PTH with PTH analogs, parathyroid hormone-related protein 1-34 (PTHrP 1-34), forskolin, 3-isobutyl-1-methylxanthine (IBMX), dibutyryl cAMP, phorbol-12-myristate-13-acetate (PMA), thapsigargin and calcium ionophore A23187. PTH 1-31 and PTHrP 1-34, which stimulate the cAMP/PKA pathway, and other activators of cAMP/PKA, forskolin, IBMX, N(6), O(2')-dibityryl adenosine 3',5'-cyclic monophosphate (dibutyryl cAMP), all elicited a similar biphasic response on OPG promoter expression. PTH analogs PTH 3-34 and PTH 7-34, that do not stimulate cAMP production, had no effect on OPG expression. In contrast, phorbol-12-myristate-13-acetate (PMA), an activator of PKC, stimulated OPG promoter expression, while thapsigargin and calcium ionophore A23187, which increase intracellular Ca(2+), showed a dose-dependent inhibition of OPG promoter expression. To delineate the promoter sequences that mediate the inhibitory effects of PTH on OPG transcription, we analyzed systematic deletions of the OPG promoter for responsiveness in transient transfection assays. The major inhibitory effects of PTH were localized to 391 bp (-372 to +19) of the proximal promoter. Deletions of the promoter region led to a complete loss of responsiveness. Taken together, these results demonstrate that the inhibitory effects of PTH on OPG are mediated at the transcriptional level through cis elements in the proximal promoter. The similar biphasic response of OPG to PTH, PTH 1-31, PTHrP 1-34, forskolin, IBMX and dibutyryl cAMP suggests that PTH regulates OPG transcription via activation of the cAMP/PKA signal transduct

Topics: 1-Methyl-3-isobutylxanthine; Animals; Bucladesine; Calcimycin; Colforsin; Gene Expression; Glycoproteins; Osteoblasts; Osteoprotegerin; Parathyroid Hormone; Parathyroid Hormone-Related Protein; Peptide Fragments; Promoter Regions, Genetic; Proteins; Rats; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Signal Transduction; Tetradecanoylphorbol Acetate; Thapsigargin