Parathyroid hormone activation of MAP kinase in rat duodenal cells is mediated by 3´,5´-cyclic AMP and Ca2+

GENTILI CLAUDIA; MORELLI SUSANA; BOLAND, RICARDO; RUSSO DE BOLAND ANA

BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS

Elseiver Science Inc

Lugar: Holanda; Año: 2001 vol. 1540 p. 201 - 212

0304-4165

In a previous study, we demonstrated that parathyroid hormone (PTH) stimulates in rat duodenal cells (enterocytes) the phosphorylation and activity of extracellular signal-regulated mitogen-activated protein kinase (MAPK) isoforms ERK1 and ERK2. As PTH activates adenylyl cyclase (AC) and phospholipase C and increases intracellular Ca2. in these cells, in the present study we evaluated the involvement of cAMP, Ca,  and protein kinase C (PKC) on PTH-induced MAPK activation. We found that MAPK phosphorylation by the hormone did not depend on PKC activation. PTH response could, however, be mimicked by addition of forskolin (5-15 uM), an AC activator, or Sp-cAMP (50-100 uM), a cAMP agonist, and suppressed to a great extent by the AC inhibitor, compound Sq-22536 (0.2-0.4 mM) and the cAMP antagonist Rp-cAMP (0.2 mM). Removal of external Ca2. (EGTA 0.5 mM), chelation of intracellular Ca2. with BAPTA (5 uM), or blockade of L-type Ca2.-channels with verapamil (10 uM) significantly decreased PTH-activation of MAPK. Furthermore, a similar degree of phosphorylation of MAPK was elicited by the Ca2. mobilizing agent thapsigargin, the Ca ionophore A23187, ionomycin and membrane depolarization with high K.. Inclusion of the calmodulin inhibitor fluphenazine (50 uM) did not prevent hormone effects on MAPK. Taken together, these results indicate that cAMP and Ca2. play a role upstream in the signaling mechanism leading to MAPK activation by PTH in rat enterocytes. As Ca and cAMP antagonists did not block totally PTH-induced MAPK phosphorylation, it is possible that linking of the hormone signal to the MAPK pathway may additionally involve Src, which has been previously shown to be rapidly activated by PTH. Of physiological significance, in agreement with the mitogenic role of the MAPK cascade, PTH increased enterocyte DNA synthesis, and this effect was blocked by the specific inhibitor of MAPK kinase (MEK) PD098059, indicating that hormone modulation of MAPK through these messenger systems stimulates duodenal cell proliferation.2. in these cells, in the present study we evaluated the involvement of cAMP, Ca,  and protein kinase C (PKC) on PTH-induced MAPK activation. We found that MAPK phosphorylation by the hormone did not depend on PKC activation. PTH response could, however, be mimicked by addition of forskolin (5-15 uM), an AC activator, or Sp-cAMP (50-100 uM), a cAMP agonist, and suppressed to a great extent by the AC inhibitor, compound Sq-22536 (0.2-0.4 mM) and the cAMP antagonist Rp-cAMP (0.2 mM). Removal of external Ca2. (EGTA 0.5 mM), chelation of intracellular Ca2. with BAPTA (5 uM), or blockade of L-type Ca2.-channels with verapamil (10 uM) significantly decreased PTH-activation of MAPK. Furthermore, a similar degree of phosphorylation of MAPK was elicited by the Ca2. mobilizing agent thapsigargin, the Ca ionophore A23187, ionomycin and membrane depolarization with high K.. Inclusion of the calmodulin inhibitor fluphenazine (50 uM) did not prevent hormone effects on MAPK. Taken together, these results indicate that cAMP and Ca2. play a role upstream in the signaling mechanism leading to MAPK activation by PTH in rat enterocytes. As Ca and cAMP antagonists did not block totally PTH-induced MAPK phosphorylation, it is possible that linking of the hormone signal to the MAPK pathway may additionally involve Src, which has been previously shown to be rapidly activated by PTH. Of physiological significance, in agreement with the mitogenic role of the MAPK cascade, PTH increased enterocyte DNA synthesis, and this effect was blocked by the specific inhibitor of MAPK kinase (MEK) PD098059, indicating that hormone modulation of MAPK through these messenger systems stimulates duodenal cell proliferation. and protein kinase C (PKC) on PTH-induced MAPK activation. We found that MAPK phosphorylation by the hormone did not depend on PKC activation. PTH response could, however, be mimicked by addition of forskolin (5-15 uM), an AC activator, or Sp-cAMP (50-100 uM), a cAMP agonist, and suppressed to a great extent by the AC inhibitor, compound Sq-22536 (0.2-0.4 mM) and the cAMP antagonist Rp-cAMP (0.2 mM). Removal of external Ca2. (EGTA 0.5 mM), chelation of intracellular Ca2. with BAPTA (5 uM), or blockade of L-type Ca2.-channels with verapamil (10 uM) significantly decreased PTH-activation of MAPK. Furthermore, a similar degree of phosphorylation of MAPK was elicited by the Ca2. mobilizing agent thapsigargin, the Ca ionophore A23187, ionomycin and membrane depolarization with high K.. Inclusion of the calmodulin inhibitor fluphenazine (50 uM) did not prevent hormone effects on MAPK. Taken together, these results indicate that cAMP and Ca2. play a role upstream in the signaling mechanism leading to MAPK activation by PTH in rat enterocytes. As Ca and cAMP antagonists did not block totally PTH-induced MAPK phosphorylation, it is possible that linking of the hormone signal to the MAPK pathway may additionally involve Src, which has been previously shown to be rapidly activated by PTH. Of physiological significance, in agreement with the mitogenic role of the MAPK cascade, PTH increased enterocyte DNA synthesis, and this effect was blocked by the specific inhibitor of MAPK kinase (MEK) PD098059, indicating that hormone modulation of MAPK through these messenger systems stimulates duodenal cell proliferation.), an AC activator, or Sp-cAMP (50-100 uM), a cAMP agonist, and suppressed to a great extent by the AC inhibitor, compound Sq-22536 (0.2-0.4 mM) and the cAMP antagonist Rp-cAMP (0.2 mM). Removal of external Ca2. (EGTA 0.5 mM), chelation of intracellular Ca2. with BAPTA (5 uM), or blockade of L-type Ca2.-channels with verapamil (10 uM) significantly decreased PTH-activation of MAPK. Furthermore, a similar degree of phosphorylation of MAPK was elicited by the Ca2. mobilizing agent thapsigargin, the Ca ionophore A23187, ionomycin and membrane depolarization with high K.. Inclusion of the calmodulin inhibitor fluphenazine (50 uM) did not prevent hormone effects on MAPK. Taken together, these results indicate that cAMP and Ca2. play a role upstream in the signaling mechanism leading to MAPK activation by PTH in rat enterocytes. As Ca and cAMP antagonists did not block totally PTH-induced MAPK phosphorylation, it is possible that linking of the hormone signal to the MAPK pathway may additionally involve Src, which has been previously shown to be rapidly activated by PTH. Of physiological significance, in agreement with the mitogenic role of the MAPK cascade, PTH increased enterocyte DNA synthesis, and this effect was blocked by the specific inhibitor of MAPK kinase (MEK) PD098059, indicating that hormone modulation of MAPK through these messenger systems stimulates duodenal cell proliferation.2. (EGTA 0.5 mM), chelation of intracellular Ca2. with BAPTA (5 uM), or blockade of L-type Ca2.-channels with verapamil (10 uM) significantly decreased PTH-activation of MAPK. Furthermore, a similar degree of phosphorylation of MAPK was elicited by the Ca2. mobilizing agent thapsigargin, the Ca ionophore A23187, ionomycin and membrane depolarization with high K.. Inclusion of the calmodulin inhibitor fluphenazine (50 uM) did not prevent hormone effects on MAPK. Taken together, these results indicate that cAMP and Ca2. play a role upstream in the signaling mechanism leading to MAPK activation by PTH in rat enterocytes. As Ca and cAMP antagonists did not block totally PTH-induced MAPK phosphorylation, it is possible that linking of the hormone signal to the MAPK pathway may additionally involve Src, which has been previously shown to be rapidly activated by PTH. Of physiological significance, in agreement with the mitogenic role of the MAPK cascade, PTH increased enterocyte DNA synthesis, and this effect was blocked by the specific inhibitor of MAPK kinase (MEK) PD098059, indicating that hormone modulation of MAPK through these messenger systems stimulates duodenal cell proliferation.2.-channels with verapamil (10 uM) significantly decreased PTH-activation of MAPK. Furthermore, a similar degree of phosphorylation of MAPK was elicited by the Ca2. mobilizing agent thapsigargin, the Ca ionophore A23187, ionomycin and membrane depolarization with high K.. Inclusion of the calmodulin inhibitor fluphenazine (50 uM) did not prevent hormone effects on MAPK. Taken together, these results indicate that cAMP and Ca2. play a role upstream in the signaling mechanism leading to MAPK activation by PTH in rat enterocytes. As Ca and cAMP antagonists did not block totally PTH-induced MAPK phosphorylation, it is possible that linking of the hormone signal to the MAPK pathway may additionally involve Src, which has been previously shown to be rapidly activated by PTH. Of physiological significance, in agreement with the mitogenic role of the MAPK cascade, PTH increased enterocyte DNA synthesis, and this effect was blocked by the specific inhibitor of MAPK kinase (MEK) PD098059, indicating that hormone modulation of MAPK through these messenger systems stimulates duodenal cell proliferation.2. mobilizing agent thapsigargin, the Ca ionophore A23187, ionomycin and membrane depolarization with high K.. Inclusion of the calmodulin inhibitor fluphenazine (50 uM) did not prevent hormone effects on MAPK. Taken together, these results indicate that cAMP and Ca2. play a role upstream in the signaling mechanism leading to MAPK activation by PTH in rat enterocytes. As Ca and cAMP antagonists did not block totally PTH-induced MAPK phosphorylation, it is possible that linking of the hormone signal to the MAPK pathway may additionally involve Src, which has been previously shown to be rapidly activated by PTH. Of physiological significance, in agreement with the mitogenic role of the MAPK cascade, PTH increased enterocyte DNA synthesis, and this effect was blocked by the specific inhibitor of MAPK kinase (MEK) PD098059, indicating that hormone modulation of MAPK through these messenger systems stimulates duodenal cell proliferation... Inclusion of the calmodulin inhibitor fluphenazine (50 uM) did not prevent hormone effects on MAPK. Taken together, these results indicate that cAMP and Ca2. play a role upstream in the signaling mechanism leading to MAPK activation by PTH in rat enterocytes. As Ca and cAMP antagonists did not block totally PTH-induced MAPK phosphorylation, it is possible that linking of the hormone signal to the MAPK pathway may additionally involve Src, which has been previously shown to be rapidly activated by PTH. Of physiological significance, in agreement with the mitogenic role of the MAPK cascade, PTH increased enterocyte DNA synthesis, and this effect was blocked by the specific inhibitor of MAPK kinase (MEK) PD098059, indicating that hormone modulation of MAPK through these messenger systems stimulates duodenal cell proliferation.2. play a role upstream in the signaling mechanism leading to MAPK activation by PTH in rat enterocytes. As Ca and cAMP antagonists did not block totally PTH-induced MAPK phosphorylation, it is possible that linking of the hormone signal to the MAPK pathway may additionally involve Src, which has been previously shown to be rapidly activated by PTH. Of physiological significance, in agreement with the mitogenic role of the MAPK cascade, PTH increased enterocyte DNA synthesis, and this effect was blocked by the specific inhibitor of MAPK kinase (MEK) PD098059, indicating that hormone modulation of MAPK through these messenger systems stimulates duodenal cell proliferation. and cAMP antagonists did not block totally PTH-induced MAPK phosphorylation, it is possible that linking of the hormone signal to the MAPK pathway may additionally involve Src, which has been previously shown to be rapidly activated by PTH. Of physiological significance, in agreement with the mitogenic role of the MAPK cascade, PTH increased enterocyte DNA synthesis, and this effect was blocked by the specific inhibitor of MAPK kinase (MEK) PD098059, indicating that hormone modulation of MAPK through these messenger systems stimulates duodenal cell proliferation.