1,25(OH)2 vitamin D3 activates PKC-alpha in Caco-2 cells: a mechanism to limit secosteroid-induced rise in [Ca2+]i
Marc BissonnetteXiao-Ying TienSharon M. NiedzielaSusanne C. HartmannBrendan P. FrawleyHemant K. RoyMichael D. SitrinRobert L. PerlmanThomas A. Brasitus
59
Citation
0
Reference
10
Related Paper
Citation Trend
Abstract:
Our laboratory recently reported that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] rapidly increases the breakdown of membrane phosphoinositides, raises intracellular calcium concentration ([Ca2+]i), and translocates protein kinase C (PKC) from the cytosolic to the particulate fraction of Caco-2 cells. In the present experiments, we found that Caco-2 cells contained predominantly the alpha- and zeta-isoforms of PKC, with minimally detectable amounts of PKC-beta and -epsilon by Western blotting. 1,25(OH)2D3 and the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) each caused time-dependent translocations of PKC-alpha, but not PKC-zeta. TPA treatment of these cells for 24 h induced a significant concentration-dependent downregulation of PKC-alpha, but not PKC-zeta. Since PKC inhibits phospholipase C-induced mobilization of Ca2+ in other cells, we examined the effects of staurosporine and H-7, PKC inhibitors, and TPA on 1,25(OH)2D3-stimulated increase in [Ca2+]i. As previously demonstrated by our laboratory, 1,25(OH)2D3 caused a biphasic increase in [Ca2+]i, with an initial elevation (transient phase) followed by a sustained increase (plateau phase). We previously demonstrated that the transient phase is mediated, at least in part, by an increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] stimulated by the secosteroid. Acute pretreatment with staurosporine or H-7 caused a significant stimulation, whereas acute TPA pretreatment caused a significant inhibition of the 1,25(OH)2D3-induced increase in the transient phase of [Ca2+]i. Preincubation of Caco-2 cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxy-methyl ester (BAPTA-AM) abolished both the rise in [Ca2+]i and the increase in particulate-associated PKC-alpha stimulated by 1,25(OH)2D3. Moreover, downregulation of PKC-alpha by chronic TPA treatment significantly augmented the transient phase of the 1,25(OH)2D3-stimulated rise in [Ca2+]i but had no effect on the 1,25(OH)2D3-induced change in Ins(1,4,5)P3 concentration. Furthermore, in these PKC-alpha downregulated cells staurosporine no longer increased the secosteroid-stimulated transient rise in [Ca2+]i. These results indicate that 1,25(OH)2D3, which increases [Ca2+]i and diacylglycerol, activates PKC-alpha, but not PKC-zeta. The alpha-isoform, in turn, limits the secosteroid-stimulated rise in [Ca2+]i, at a step distal to Ins(1,4,5)P3 accumulation in Caco-2 cells.Keywords:
Staurosporine
PKC alpha
Inositol trisphosphate
Staurosporine
Phorbol
Cite
Citations (15)
Considerable evidence suggests that protein kinase C activation participates in the regulation of vascular smooth muscle tone. The objective of the current study was to examine the relations between inhibition of protein kinase C (PKC) and myosin light-chain kinase (MLCK) and vasorelaxation and blood pressure regulation in spontaneously hypertensive rats (SHR). Putative PKC inhibitors from two chemical classes, staurosporinelike (staurosporine and K252A) and isoquinolinesulfonamides (H7 and HA1004), were tested for their ability to 1) inhibit PKC and MLCK from SHR aorta, 2) relax isolated SHR aorta, and 3) lower blood pressure in conscious SHR. A rank order of potency for the inhibition of PKC and MLCK was established, with the staurosporinelike compounds (staurosporine PKC IC50 = 54 nM) clearly more potent than the isoquinolinesulfonamides (H7 PKC IC50 = 128 microM). The rank order of potency for inhibition of PKC was retained for inhibition of MLCK for all compounds. Staurosporine (EC50 = 75 nM) and H7 (EC50 = 2 microM) caused concentration-dependent relaxation of SHR aorta, but only staurosporine produced vasorelaxation at concentrations consistent with the inhibition of PKC or MLCK. Dose-dependent reductions in arterial pressure of SHR were demonstrated after intravenous injection of staurosporine and HA1004. A single intravenous injection of staurosporine (0.3 mg/kg) lowered blood pressure for more than 10 hours. Staurosporine also lowered blood pressure after oral administration. The depressor response to staurosporine was unaffected by sympathetic beta-adrenergic blockade. In conclusion, the vasorelaxant and antihypertensive actions of staurosporine in SHR are consistent with the inhibition of PKC but could also be equally related to inhibition of MLCK. Not all PKC inhibitors produce vasorelaxation and lower blood pressure. Moreover, the lack of correlation between in vitro vasodilation and PKC or MLCK inhibition for the isoquinolinesulfonamide protein kinase inhibitors H7 and HA1004 suggests that these agents do not cause vasorelaxation in SHR by inhibition of these enzymes.
Staurosporine
Phorbol
Cite
Citations (34)
The protein kinase C (PKC) family of isoenzymes is believed to mediate a wide range of signal-transduction pathways in many different cell types. A series of bisindolylmaleimides have been evaluated as inhibitors of members of the conventional PKC family (PKCs-alpha, -beta, -gamma) and of a representative of the new, Ca(2+)-independent, PKC family, PKC-epsilon. In contrast with the indolocarbazole staurosporine, all the bisindolylmaleimides investigated showed slight selectivity for PKC-alpha over the other isoenzymes examined. In addition, bisindolylmaleimides bearing a conformationally restricted side-chain were less active as inhibitors of PKC-epsilon. Most noticeable of these was Ro 32-0432, which showed a 10-fold selectivity for PKC-alpha and a 4-fold selectivity for PKC-beta I over PKC-epsilon.
Staurosporine
PKC alpha
Alpha (finance)
Cite
Citations (524)
Staurosporine
Protein kinase inhibitor
Cite
Citations (14)
In the human T-cell lymphoma line, HuT 78, proliferation and phorbol ester-induced growth arrest and differentiation were inhibited by the protein kinase C (PKC) inhibitor, staurosporine. By contrast, an alternative PKC inhibitor, H-7, inhibited proliferation but not phorbol ester-induced growth arrest. The cell line was found to contain both alpha and beta isoforms of PKC by Western blot techniques. A cell line, K-4, was cloned from HuT 78 in the presence of H-7 and this clone was found to be positive for PKC-alpha only. PKC-beta did not return on cultivation in the absence of H-7. Proliferation of K-4 was insensitive to inhibition with both H-7 and staurosporine. However, phorbol ester-induced growth arrest remained staurosporine sensitive. Phorbol-stimulated IL-2 secretion was minimal in the PKC-beta-deficient cell line. These data suggest that PKC-beta may be a regulatory enzyme for proliferation and stimulated interleukin-2 secretion in HuT 78 cells. Heterogeneity of responses to PKC activation may reflect the use of different isozymes in different intracellular pathways. The K-4 cell line should provide a useful tool in the dissection of involvement of PKC isozymes in cellular function.
Staurosporine
Rottlerin
Phorbol
clone (Java method)
PKC alpha
Cite
Citations (28)
Two main forms of protein kinase C (PKC 1 and PKC 2) are detected in homogenates of rat hepatocytes using DEAE-cellulose column chromatography. The activity of these forms paradoxically, is rapidly decreased by treatment in vivo with phorbol myristate acetate (PMA). The dose-response curves to PMA for decreasing the activities of PKC 1 and 2 were shifted to the right by the potent and selective PKC inhibitors, staurosporine and calphostin-C. The decreases induced by 100 nM PMA were dose-dependently blocked by these inhibitors. It is concluded that activation of PKC is required and precedes such decreases in activity induced by the active phorbol ester.
Staurosporine
Calphostin C
Calphostin
Phorbol
Tetradecanoylphorbol Acetate
Cite
Citations (2)
Staurosporine
Phorbol
Cite
Citations (77)
The potent inhibitors of protein kinase C (PKC), H7, staurosporine, and staurosporine derivatives, were examined for their inhibitory effects on novel PKC (nPKC) isozymes δ and ε. H7 and staurosporine, usually used as selective inhibitors of PKC, showed similar inhibitory effects on cPKC (a mixture of cPKCα,β, and γ) and nPKCδ and ε. The inhibitory effects of K252a, a non‐selective protein kinase inhibitor, on cPKC was 3.2‐ and 22‐fold higher than those on nPKCε and δ, respectively. The staurosporine derivatives UCN‐01 and UCN‐01‐Me also showed selective inhibition of cPKC.
Staurosporine
Alpha (finance)
Cite
Citations (49)
The aim of the present study was to investigate the effects of staurosporine on phorbol-myristate acetate (PMA)-induced activation of protein kinase C (PKC) and the desensitization of leukotriene D4 (LTD4)-stimulated Ca2+ mobilization in rat basophilic leukemia (RBL-1) cells. Staurosporine, one of the most potent PKC inhibitors known to date, markedly inhibited partially purified PKC from RBL-1 cells with an IC50 of 3 nM. Exposure of RBL-1 cells to PMA resulted in inhibition of LTD4-stimulated Ca2+ mobilization. However, prior treatment of the cells with staurosporine completely prevented PMA-induced desensitization of LTD4-stimulated Ca2+ mobilization. This reversal of Ca2+ desensitization by staurosporine was dose dependent with an IC50 of 0.1 microM. Treatment of RBL-1 cells with PMA resulted in translocation and activation of PKC from the cytosol to the membrane fraction. Pretreatment of RBL-1 cells with staurosporine inhibited the PMA-induced activation of PKC in the membrane fraction. The inhibition of PKC activity by staurosporine was time and dose dependent with an IC50 of 0.9 microM. These results show that PMA-induced heterologous desensitization is mediated by PKC and staurosporine prevented this process by directly inhibiting PKC in intact RBL-1 cells.
Staurosporine
Phorbol
Cite
Citations (64)
Staurosporine
Phorbol
Chelerythrine
Bisindolylmaleimide
Cite
Citations (20)