The PKC Activator 12-O-tetradecanoylphorbol 13-acetate Research Articles

Protein kinase C phosphorylates the EphA2 receptor on serine 892 in the regulatory linker connecting the kinase and SAM domains

The EphA2 receptor tyrosine kinase signals through two distinct mechanisms, one regulated by tyrosine phosphorylation and the other by serine/threonine phosphorylation. Serine 892 (S892) is one of the major serine/threonine phosphorylation sites in EphA2, but little is known about its regulation and function. S892 is located in the linker connecting the EphA2 kinase and SAM domains, and is part of a cluster of five phosphorylated residues that includes the well characterized S897. EphA2 can be phosphorylated on S897 by the RSK, AKT and PKA kinases to promote a non-canonical form of signaling that plays an important role in cancer malignancy. Here we show that the Protein Kinase C (PKC) family phosphorylates the EphA2 S892 motif in vitro and in cells. By using a newly developed phosphospecific antibody, we detected EphA2 S892 phosphorylation in a variety of cell lines. As expected for a PKC target site, the PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) increases S892 phosphorylation whereas the broad-spectrum PKC inhibitor Go 6983 inhibits both basal and TPA-induced S892 phosphorylation. Besides phosphorylating S892, PKC can also increase EphA2 phosphorylation on S897 through the MEK kinase, which regulates the ERK-RSK signaling axis. We also found that S892 and S897 phosphorylation induced by PKC activation can be downregulated by ephrin ligand-induced EphA2 canonical signaling. Our data reveal that the PKC family contributes to the phosphorylation cluster in the EphA2 kinase-SAM linker, which regulates EphA2 non-canonical signaling and cancer malignancy.

DGKγ Knock-Out Mice Show Impairments in Cerebellar Motor Coordination, LTD, and the Dendritic Development of Purkinje Cells through the Activation of PKCγ.

Diacylglycerol kinase γ (DGKγ) regulates protein kinase C (PKC) activity by converting DG to phosphatidic acid (PA). DGKγ directly interacts with PKCγ and is phosphorylated by PKCγ, resulting in the upregulation of lipid kinase activity. PKC dysfunction impairs motor coordination, indicating that the regulation of PKC activity is important for motor coordination. DGKγ and PKC are abundantly expressed in cerebellar Purkinje cells. However, the physiological role of DGKγ has not been elucidated. Therefore, we developed DGKγ knock-out (KO) mice and tested their cerebellar motor coordination. In DGKγ KO mice, cerebellar motor coordination and long-term depression (LTD) were impaired, and the dendrites of Purkinje cells from DGKγ KO mice were significantly retracted. Interestingly, treatment with the cPKC inhibitor Gö6976 (Gö) rescued the dendritic retraction of primary cultured Purkinje cells from DGKγ KO mice. In contrast, treatment with the PKC activator 12-o-tetradecanoylphorbol 13-acetate (TPA) reduced morphologic alterations in the dendrites of Purkinje cells from wild-type (WT) mice. In addition, we confirmed the upregulation of PKCγ activity in the cerebellum of DGKγ KO mice and rescued impaired LTD in DGKγ KO mice with a PKCγ-specific inhibitor. Furthermore, impairment of motor coordination observed in DGKγ KO mice was rescued in tm1c mice with DGKγ reexpression induced by the FLP-flippase recognition target (FRT) recombination system. These results indicate that DGKγ is involved in cerebellar LTD and the dendritic development of Purkinje cells through the regulation of PKCγ activity, and thus contributes to cerebellar motor coordination.

Claudin 1 Is Highly Upregulated by PKC in MCF7 Human Breast Cancer Cells and Correlates Positively with PKCε in Patient Biopsies

Recent studies provide compelling evidence to suggest that the tight junction protein claudin 1, aberrantly expressed in several cancer types, plays an important role in cancer progression. Dysregulation of claudin 1 has been shown to induce epithelial mesenchymal transition (EMT). Furthermore, activation of the ERK signaling pathway by protein kinase C (PKC) was shown to be necessary for EMT induction. Whether PKC is involved in regulating breast cancer progression has not been addressed. The PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) was used to investigate the effect of PKC activity on claudin 1 transcription and protein levels, subcellular distribution, and alterations in EMT markers in human breast cancer (HBC) cell lines. As well, tissue microarray analysis (TMA) of a large cohort of invasive HBC biopsies was conducted to investigate correlations between claudin 1 and PKC isomers. TPA upregulated claudin 1 levels in all HBC cell lines analyzed. In particular, a high induction of claudin 1 protein was observed in the MCF7 cell line. TPA treatment also led to an accumulation of claudin 1 in the cytoplasm. Additionally, we demonstrated that the upregulation of claudin 1 was through the ERK signaling pathway. In patient biopsies, we identified a significant positive correlation between claudin 1, PKCα, and PKCε in ER+ tumors. A similar correlation between claudin 1 and PKCε was identified in ER− tumors, and high PKCε was associated with shorter disease-free survival. Collectively, these studies demonstrate that claudin 1 and the ERK signaling pathway are important players in HBC progression.

Protein kinase Cα inhibitor enhances the sensitivity of human pancreatic cancer HPAC cells to Clostridium perfringens enterotoxin via claudin-4

Protein kinase C (PKC) is overexpressed in cancer, including pancreatic cancer, compared with normal tissue. Moreover, PKCα is considered one of the biomarkers for the diagnosis of cancers. In several human cancers, the claudin tight junction molecules are abnormally regulated and are thus promising molecular targets for diagnosis and therapy with Clostridium perfringens enterotoxin (CPE). In order to investigate the changes of tight junction functions of claudins via PKCα activation in pancreatic cancer cells, the well-differentiated human pancreatic cancer cell line HPAC, with its highly expressed tight junction molecules and well-developed barrier function, was treated with the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA). Treatment with TPA modified the activity of phosphoPKCα and caused an increase of the Snail family members Snail, Slug and Smad-interacting protein 1 and a decrease of E-cadherin. In HPAC cells treated with TPA, downregulation of claudin-1 and mislocalization of claudin-4 and occludin around the nuclei were observed, together with a decrease in the numbers of tight junction strands and an increase in phosphorylation of claudin-4. The barrier function and the cytotoxicity of CPE were significantly decreased on TPA treatment. All such changes after TPA treatment were prevented by inhibitors of panPKC and PKCα. These findings suggest that, in human pancreatic cancer cells, PKCα activation downregulates tight junction functions as a barrier and as a receptor of CPE via the modification of claudin-1 and -4 during epithelial to mesenchymal transition-like changes. PKCα inhibitors might represent potential therapeutic agents against human pancreatic cancer cells by use of CPE cytotoxicity via claudin-4.

Factors related to degradation of articular cartilagein osteoarthritis: a review

Objectives: Osteoarthritis (OA) is a common joint deterioration initiated by multiple factors. To better understand related factors in the development of this disease, we focused on the mechanical stress loaded on articular cartilage. Materials and Methods: The anterior cruciate ligaments of rabbit knee joints were transected, and expression of protein kinase C (PKC) examined immunohistochemically. The PKC activator 12-o-tetradecanoyl-phorbol-13-acetate (TPA) was then administered intraarticularly. To determine the involvement of gas mediators, a cartilage defect was made on the medial femoral condyle of rabbit knee joints. Hydrostatic pressure was loaded on the cartilage taken from the surrounding defects, and levels of superoxide anion and nitric oxide (NO) were measured. Bovine chondrocytes were subjected to cyclic mechanical stretch using a Flexercell Strain Instrument. Proteoglycan synthesis and PKC activity were measured. Expression of matrix metal loproteinase (MMP)-3 and tissue inhibitors of metalloproteinase (TIMP)-1 in articular cartilages obtained from OA patients were examined using Northern blots. Results: Chondrocytes from experimentally induced OA were stained positively with anti-α-PKC antibody. Intraarticular administration of TPA prevented the development of OA changes. Cyclic tensile stretch loaded on chondrocytes decreased proteoglycan synthesis and PKC activity. Thus, PKC is involved in the stress-mediated degradation of articular cartilage. Cartilage defects led to degradation of surrounding cartilage and to enhanced superoxide anion and NO synthesis. We also noted increased and decreased expressions of MMP-3 and TIMP-1 mRNA in human OA cartilage, respectively. Conclusion: PKC, gas mediators (superoxide anion, NO), and proteinases are all involved in OA.

Protein kinase C-mediated inhibition of transmembrane signalling through CCK(A) and CCK(B) receptors.

1. The rat CCK(A) and CCK(B) receptors were stably expressed in Chinese hamster ovary (CHO-09) cells in order to compare modes of signal transduction and effects of protein kinase C (PKC) thereupon. 2. Spectrofluorophotometry of Fura-2-loaded cells revealed that both receptors retained their pharmacological characteristics following expression in CHO cells. Sulphated cholecystokinin-(26-33)-peptide amide (CCK-8-S) increased the cytosolic Ca2+ concentration ([Ca2+]i) in CCK(A) cells, measured as an increase in Fura-2 fluorescence emission ratio, 1000 fold more potently than its non-sulphated form (CCK-8-NS) (EC50 values of 0.19 nM and 0.18 microM, respectively). By contrast, CCK-8-S and CCK-8-NS were equally potent in CCK(B) cells (EC50 values of 0.86 nM and 1.18 nM, respectively). The CCK(A) receptor agonist JMV-180 increased [Ca2+]i only in CCK(A) cells. Likewise, pentagastrin increased [Ca2+]i only in CCK(B) cells. Finally, CCK-8-S-induced Ca2+ signalling through the CCK(A) receptor was most potently inhibited by the CCK(A) receptor antagonist L364,718, whereas the CCK(B) receptor antagonist L365,260 was more potent in CCK(B) cells. 3. Receptor-mediated activation of adenylyl cyclase was measured in the presence of the inhibitor of cyclic nucleotide phosphodiesterase activity, 3-isobutyl-1-methylxanthine. CCK-8-S and, to a lesser extent, CCK-8-NS, but not JMV-180 or pentagastrin, stimulated the accumulation of cyclicAMP in CCK(A) cells. By contrast, none of these agonists increased cyclicAMP in CCK(B) cells. 4. Short-term (3 min) pretreatment with the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) evoked a rightward shift of the dose-response curve for the Ca2+ mobilizing effect of CCK-8-S in both cell lines. In addition, short-term TPA pretreatment markedly reduced CCK-8-S-induced cyclicAMP accumulation in CCK(A) cells. In both cases, the inhibitory effect of TPA was abolished by the PKC inhibitors, GF-109203X and staurosporine, whereas no inhibition was observed with the inactive phorbol ester, 4-alpha-phorbol 12-myristate 13-acetate. 5. During prolonged TPA treatment, the cells gradually recovered from phorbol ester inhibition and in the case of CCK-8-S-induced Ca2+ mobilization complete recovery was achieved after 24 h of TPA treatment. Western blot analysis revealed that this recovery was paralleled by down-regulation of PKC-alpha, suggesting the involvement of this PKC isotype in the inhibitory action of TPA. 6. This study demonstrates that following expression in CHO cells (i) both CCK(A) and CCK(B) receptors are coupled to Ca2+ mobilization, (ii) only CCK(A) receptors are coupled to cyclicAMP formation and (iii) with both receptors signalling is inhibited by PKC.

Contribution of Protein Kinase C to p53-dependent WAF1 Induction Pathway after Heat Treatment in Human Glioblastoma Cell Lines

To examine whether protein kinase C (PKC) contributes to p53-dependent WAF1 induction after heat treatment, the effects of calphostin C (CAL), a specific inhibitor of PKC, on WAF1 induction were analyzed by PKC activity and gel mobility-shift assays and Western blot analysis in human glioblastoma cell lines. Heat-induced accumulation of WAF1 in A-172 cells carrying wild-typep53(wtp53) was suppressed by CAL in a dose-dependent manner. In T98G cells carrying mutantp53(mp53), no significant accumulation of WAF1 was observed after heat treatment and CAL exerted no significant effects on this response of T98G cells. In accordance with the accumulation of WAF1, heat-induced activation of the binding ability of p53 to p53 consensus sequence (p53 CON) was suppressed by CAL in A-172 cells but no DNA-binding activity was observed in the mp53 in T98G cells. PKC in A-172 cells was activated rapidly (within 5 min) after heat treatment in the membrane fraction but not in the cytosolic fraction. When the cell lines were treated with the PKC activator, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), WAF1 was accumulated in A-172 cells in a dose-dependent manner but not in T98G cells. In addition, the cellular contents of WAF1 after heating did not increase in A-172 cells transformed with mp53.These results suggest that PKC contributes to heat-induced signal transduction leading to p53-dependent WAF1 induction in a way that PKC is involved in the specific DNA-binding activation of p53.

Differential Activation of Protein Kinase C δ and ε Gene Expression by Gonadotropin-releasing Hormone in αT3-1 Cells: AUTOREGULATION BY PROTEIN KINASE C

The effect of gonadotropin-releasing hormone (GnRH) upon protein kinase C (PKC) delta and PKCepsilon gene expression was investigated in the gonadotroph-derived alphaT3-1 cell line. Stimulation of the cells with a stable analog [D-Trp6]GnRH (GnRH-A) resulted in a rapid elevation of PKCepsilon mRNA levels (1 h), while PKCdelta mRNA levels were elevated only after 24 h of incubation. The rapid elevation of PKCepsilon mRNA by GnRH-A was blocked by pretreatment with a GnRH antagonist or actinomycin D. The PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA), but not the Ca2+ ionophore ionomycin, mimicked the rapid effect of GnRH-A upon PKCepsilon mRNA elevation. Additionally, the rapid stimulatory effect of GnRH-A was blocked by the selective PKC inhibitor GF109203X, by TPA-mediated down-regulation of endogenous PKC, or by Ca2+ removal. Interestingly, serum-starvation (24 h) advanced the stimulation of PKCdelta mRNA levels by GnRH-A and the effect could be detected at 1 h of incubation. The rapid effect of GnRH-A upon PKCdelta mRNA levels in serum-starved cells was mimicked by TPA, but not by ionomycin, and was abolished by down-regulation of PKC or by Ca2+ removal. Preactivation of alphaT3-1 cells with GnRH-A for 1 h followed by removal of ligand and serum resulted in elevation of PKCdelta mRNA levels after 24 h of incubation. Western blot analysis revealed that GnRH-A and TPA stimulated (within 5 min) the activation and some degradation of PKCdelta and PKCepsilon. We conclude that Ca2+ and PKC are involved in GnRH-A elevation of PKCdelta and PKCepsilon mRNA levels, with Ca2+ being necessary but not sufficient, while PKC is both necessary and sufficient to mediate the GnRH-A response. A serum factor masks PKCdelta but not PKCepsilon mRNA elevation by GnRH-A, and its removal exposes preactivation of PKCdelta mRNA by GnRH-A which can be memorized for 24 h. PKCdelta and PKCepsilon gene expression evoked by GnRH-A is autoregulated by PKC, and both isotypes might participate in the neurohormone action.

Effect of the protein phosphatase inhibitor okadaic acid on FSH-induced granulosa cell steroidogenesis.

To address a possible role of type 1 and 2A serine/threonine protein phosphatases (PP1 and PP2A) in regulating granulosa cell hormonal responses, we investigated the effects of okadaic acid (OA) on FSH- and cAMP-induced steroidogenesis in these cells. When added alone (0.01-1 nmol/l), the cell-permeant phosphatase inhibitor did not affect progesterone and 3 beta-hydroxysteroid dehydrogenase/delta 5-4 isomerase (3 beta-HSD) enzyme activity, whereas when added with FSH it dose-dependently augmented (minimal effective dose, 0.1 nmol/l) gonadotropin-stimulated steroidogenesis in cultured granulosa cells. A similar stimulatory effect of the toxin was observed in cells cultured for 48 h with the cell-permeant analogue dibutyryl cAMP (1 mmol/l), or when granulosa cells were stimulated with the cAMP-inducing agents cholera toxin (1 microgram/ml), forskolin (15 mumol/l) or 1-methyl-3-isobutyl-xanthine (0.1 mmol/l). The observed effect of OA on FSH-supported granulosa cell steroidogenesis was not a consequence of increased cAMP generation, and time course experiments also revealed that a minimal time period of 12 h was necessary for OA (0.1 and 1 nmol/l) to significantly enhance FSH-induced progesterone and 3 beta-HSD enzyme activity. Since OA also inhibits the dephosphorylation of protein kinase C (PKC) substrates, we also compared the effect of OA and the PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) on FSH-induced granulosa cell steroidogenic activity. While activation of the PKC pathway with the tumor promoter TPA (10 nmol/l) inhibited progesterone and cAMP accumulation in FSH-stimulated granulosa cells, treatment with OA augmented steroidogenesis and did not affect gonadotropin-induced cAMP generation. Collectively these results suggest that PP1 and PP2A may be important in regulating the phosphorylation state of proteins implicated in the cAMP-protein kinase A-stimulated steroidogenic activity of these cells.

Relationship between protein kinase C and adenylyl cyclase activity in the regulation of growth hormone secretion by human pituitary somatotrophinomas.

To determine the potential role of protein kinase C (PKC) and its relationship to adenylyl cyclase activity in controlling growth hormone (GH) secretion by human pituitary somatotrophinomas. Twenty-eight somatotrophinomas were placed into cell culture, and the in vitro effects of the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) and the PKC inhibitor staurosporine on basal and GH-releasing hormone (GHRH)-stimulated GH secretion were examined. In addition, the influence of chronic exposure of cultured somatotrophinoma cells to TPA on the rate of inositol 1,4,5-trisphosphate production was determined. Each tumor was assessed for the presence of gsp oncogenes, and thus constitutive adenylyl cyclase activity, by direct sequence analysis of polymerase chain reaction-generated deoxyribonucleic acid. GH secretory responses of tumors with and without these oncogenes were compared. TPA consistently stimulated GH secretion by cultured somatotrophinoma cells. There was no difference in response between somatotrophinomas with and without gsp oncogenes, and the effects did not correlate with the variable stimulation exerted by GHRH. Tumors in which GHRH had no significant effect nevertheless responded to TPA. In combination, TPA and GHRH exerted additive stimulation. TPA treatment of cultured somatotrophinoma cells eventually resulted in suppression of inositol 1,4,5-trisphosphate production, probably reflecting down-regulation of membrane phosphatidylinositol hydrolysis, a second messenger system that also generates the endogenous PKC activator diacylglycerol. GHRH had no effect on phosphatidylinositol hydrolysis. In contrast to the effects of TPA, the PKC inhibitor staurosporine tended to reduce GH secretion, although this effect was not observed in all tumors examined. As with TPA, the effects of staurosporine did not correlate with presence or absence of gsp oncogenes. Furthermore, staurosporine did not reduce the stimulatory effects exerted by GHRH on GH secretion. These results demonstrate a role for the phosphatidylinositol-PKC second messenger cascade in controlling GH secretion by human pituitary somatotrophinomas. The results also show that the system operates relatively independent of intracellular adenylyl cyclase and, thus, protein kinase A.

Protein kinase C activator inhibits progression of osteoarthritis induced in rabbit knee joints

To study the role of protein kinase C (PKC) in cartilage tissue in osteoarthritis, experimental osteoarthritis was induced in the knee joints of rabbits by resection of the anterior cruciate ligament (ACL). At 4 weeks after the operation, osteoarthritic changes varying from surface irregularities and cleft formation to loss of the tangential layer were observed, and cloning or hypocellularity of the chondrocytes was observed mainly in the transitional and radial layers. The PKC activator 12-o-tetradecanoyl-phorbol-13-acetate (TPA) or non-PKC-activating phorbol ester 4 α-phorbol-12,13-didecanate (PDD) was administered intraarticularly once a week from the day of the operation for 3 weeks. Histologic evaluation with a rating scale was carried out. In the TPA-administered group, cartilage structures were preserved almost completely, and score of the cartilage lesion was significantly less than that in animals administered PDD or in nonadministered controls. A chondroprotective role of PKC under mechanical stress was suggested.

Class II of metabotropic glutamate receptors modulate the evoked release of adenosine and glutamate from rat hippocampal slices

In rat hippocampal slices, the novel metabotropic glutamate receptor (mGluR) ligand, (1 S,1′ R,2′ R,3′ R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) enhanced the stimulation of polyphosphoinositide (PPI) hydrolysis elicited by quisqualate or by submaximal concentrations of ibotenate or (1 S,3 R)-1aminocyclopentane-1,3-dicarboxylic acid (1 S,3 R-ACPD). The enhancing effect of DCG-IV was (i) specific for mGluR agonists, (ii) restricted to hippocampal slice preparation, (iii) reversible, and (iv) not subject to homologous desensitization. In addition, DGC-IV did not interact with l-2-amino-4-phosphonobutanoate (AP4), a noncompetitive antagonist of mGluRs coupled to PPI hydrolysis in brain slices [32]. The action of DCG-IV on quisqualate-stimulated PPI hydrolysis was insensitive to antagonists of ionotropic glutamate receptors and did not appear to be a consequence of a reduction in the intracellular levels of cAMP [14]. When the stimulation of PPI hydrolysis was measured as a function of the incubation time, DCG-IV potentiated quisqualate-stimulated PPI hydrolysis after 60 min of incubation, when quisqualate had already reached its maximal effect. Knowing that activation of protein kinase C (PKC) limits the extent of mGluR agonist-stimulated PPI hydrolysis over time, we have studied the enhancing effect of DCG-IV in the presence of the PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA). As expected [9], TPA reduced quisqualate-stimulated PPI hydrolysis in control slices, but was inactive in slices incubated in the presence of DCG-IV. Taken collectively, these results suggest that DCG-IV positively modulates the activity of mGluRs coupled to PPI hydrolysis through a mechanism, which involves PKC-mediated phosphorylation processes.

(2 S,1′ R,2′ R,3′ R)-2-(2,3-dicarboxycyclopropyl) glycine positively modulates metabotropic glutamate receptors coupled to polyphosphoinositide hydrolysis in rat hippocampal slices

In rat hippocampal slices, the novel metabotropic glutamate receptor (mGluR) ligand, (1 S,1′ R,2′ R,3′ R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) enhanced the stimulation of polyphosphoinositide (PPI) hydrolysis elicited by quisqualate or by submaximal concentrations of ibotenate or (1 S,3 R)-1aminocyclopentane-1,3-dicarboxylic acid (1 S,3 R-ACPD). The enhancing effect of DCG-IV was (i) specific for mGluR agonists, (ii) restricted to hippocampal slice preparation, (iii) reversible, and (iv) not subject to homologous desensitization. In addition, DGC-IV did not interact with l-2-amino-4-phosphonobutanoate (AP4), a noncompetitive antagonist of mGluRs coupled to PPI hydrolysis in brain slices [32]. The action of DCG-IV on quisqualate-stimulated PPI hydrolysis was insensitive to antagonists of ionotropic glutamate receptors and did not appear to be a consequence of a reduction in the intracellular levels of cAMP [14]. When the stimulation of PPI hydrolysis was measured as a function of the incubation time, DCG-IV potentiated quisqualate-stimulated PPI hydrolysis after 60 min of incubation, when quisqualate had already reached its maximal effect. Knowing that activation of protein kinase C (PKC) limits the extent of mGluR agonist-stimulated PPI hydrolysis over time, we have studied the enhancing effect of DCG-IV in the presence of the PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA). As expected [9], TPA reduced quisqualate-stimulated PPI hydrolysis in control slices, but was inactive in slices incubated in the presence of DCG-IV. Taken collectively, these results suggest that DCG-IV positively modulates the activity of mGluRs coupled to PPI hydrolysis through a mechanism, which involves PKC-mediated phosphorylation processes.

1,25(OH)2 vitamin D3 activates PKC-alpha in Caco-2 cells: a mechanism to limit secosteroid-induced rise in [Ca2+]i.

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.

Regulation of glycoprotein hormone free alpha-subunit secretion and intracellular alpha-subunit content in primary pituitary cells.

The effects and interactions of GnRH, TRH, a cAMP analog, a protein kinase-C (PKC) activator, a calcium ionophore, and a calcium channel blocker on pituitary glycoprotein hormone free alpha-subunit secretion and intracellular free alpha-subunit content were investigated. Treatment of dispersed rat pituitary cells with GnRH (100 nM) effected a time-dependent release of alpha-subunit, reaching a 4.5-fold increase (P < 0.05) at 24 h. Smaller effects were observed with TRH (10 nM). A rapid and progressive fall in intracellular alpha-subunit content was observed for 8 h after stimulation with GnRH (61% decrease; P < 0.05) or TRH (55% decrease; P < 0.05), which then remained constant at 24 h. The cAMP analogue 8-bromo-cAMP augmented a late release of alpha-subunit (4.5-fold increase at 24 h; P < 0.05) without affecting levels of alpha-subunit within the cells. Co-addition of 8-bromo-cAMP with GnRH or TRH arrested the marked fall in intracellular alpha-subunit seen with GnRH or TRH alone. These results suggest that although cAMP is capable of stimulating alpha-subunit secretion and maintaining cell content in the face of GnRH- and TRH-stimulated secretion, it does not mediate their effects on alpha-subunit. Like GnRH, the PKC activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) rapidly stimulated alpha-subunit secretion (1.7-fold increase at 4 h; P < 0.05) and progressively lowered cell content over 24h (73% decrease; P < 0.01). This similarity of action and the lack of demonstration of additive effects of TPA with GnRH or TRH imply a role for PKC as a mediator of GnRH and TRH action on alpha-subunit. Using verapamil (50 microM) to block L-type calcium channels had no effect on either basal or GnRH-stimulated alpha-secretion over 24 h. The calcium ionophore A23187 (3 microM) blocked the stimulatory effects of GnRH on alpha-subunit release and alone inhibited free alpha-subunit secretion (28% decrease at 24 h; P < 0.05). Our results suggest that neither cAMP nor an influx of extracellular calcium mediates the effects of GnRH or TRH on free alpha-subunit secretion. Accordingly, we postulate that PKC is involved in the actions of GnRH and TRH on alpha-subunit in rat pituitary cells, although further studies are required in PKC-depleted cells to confirm this hypothesis.

Regulation of glycoprotein hormone free alpha-subunit secretion and intracellular alpha-subunit content in primary pituitary cells

The effects and interactions of GnRH, TRH, a cAMP analog, a protein kinase-C (PKC) activator, a calcium ionophore, and a calcium channel blocker on pituitary glycoprotein hormone free alpha-subunit secretion and intracellular free alpha-subunit content were investigated. Treatment of dispersed rat pituitary cells with GnRH (100 nM) effected a time-dependent release of alpha-subunit, reaching a 4.5-fold increase (P < 0.05) at 24 h. Smaller effects were observed with TRH (10 nM). A rapid and progressive fall in intracellular alpha-subunit content was observed for 8 h after stimulation with GnRH (61% decrease; P < 0.05) or TRH (55% decrease; P < 0.05), which then remained constant at 24 h. The cAMP analogue 8-bromo-cAMP augmented a late release of alpha-subunit (4.5-fold increase at 24 h; P < 0.05) without affecting levels of alpha-subunit within the cells. Co-addition of 8-bromo-cAMP with GnRH or TRH arrested the marked fall in intracellular alpha-subunit seen with GnRH or TRH alone. These results suggest that although cAMP is capable of stimulating alpha-subunit secretion and maintaining cell content in the face of GnRH- and TRH-stimulated secretion, it does not mediate their effects on alpha-subunit. Like GnRH, the PKC activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) rapidly stimulated alpha-subunit secretion (1.7-fold increase at 4 h; P < 0.05) and progressively lowered cell content over 24h (73% decrease; P < 0.01). This similarity of action and the lack of demonstration of additive effects of TPA with GnRH or TRH imply a role for PKC as a mediator of GnRH and TRH action on alpha-subunit. Using verapamil (50 microM) to block L-type calcium channels had no effect on either basal or GnRH-stimulated alpha-secretion over 24 h. The calcium ionophore A23187 (3 microM) blocked the stimulatory effects of GnRH on alpha-subunit release and alone inhibited free alpha-subunit secretion (28% decrease at 24 h; P < 0.05). Our results suggest that neither cAMP nor an influx of extracellular calcium mediates the effects of GnRH or TRH on free alpha-subunit secretion. Accordingly, we postulate that PKC is involved in the actions of GnRH and TRH on alpha-subunit in rat pituitary cells, although further studies are required in PKC-depleted cells to confirm this hypothesis.

Activation of protein kinase C inhibits 45Ca-accumulation in cultures of osteoblast-like cells: possible involvement of insulin-like growth factor-I

In a previous report, we have demonstrated that exogenous insulin-like growth factor-I (IGF-I) stimulates 45Ca-accumulation into extracellular matrix in long-term cultures of osteoblast-like MC3T3-E1 cells and that 45Ca-accumulation occurs even in the cultures without exogenous IGF-I. In this study, effects of protein kinase C (PKC) on IGF-I secretion and 45Ca-accumulation into extracellular matrix were examined in 6-week cultured MC3T3-E1 cells. The MC3T3-E1 cells secreted IGF-I spontaneously. The PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA) suppressed IGF-I secretion in a dose-dependent manner. 4 alpha-Phorbol 12,13-didecanoate (4 alpha-PDD), which is inactive for PKC, had little effect on the secretion. 1-Oleoyl-2-acetylglycerol, a specific activator for PKC, also suppressed the IGF-I secretion dose dependently. H-7, a PKC inhibitor, recovered the inhibitory effect of TPA. On the other hand, TPA inhibited the 45Ca-accumulation into extracellular matrix in cultures of these cells dose dependently, whereas 4 alpha-PDD was ineffective in this capacity. The TPA-induced inhibition of 45Ca-accumulation was recovered almost to the control level by H-7. Exogenous IGF-I recovered the inhibitory effect of TPA on 45Ca-accumulation. In spite of the inhibitory effects of TPA as above, TPA had little effect on DNA synthesis in these cells. These results suggest that the activation of PKC inhibits calcification via suppression of IGF-I secretion in osteoblast-like cells.

Selective potentiation of N-methyl-D-aspartate-induced current by protein kinase C in Xenopus oocytes injected with rat brain RNA.

Glutamate receptors and protein kinase C (PKC) may play significant roles in long-term potentiation in hippocampus. To clarify the regulatory involvement of PKC in the functions of glutamate receptors, we examined the effects of PKC activation on current response induced by the activation of each subtype of glutamate receptor in Xenopus oocytes injected with rat brain RNA. Treatment with the PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), potentiated N-methyl-D-aspartate (NMDA)-induced current by about 2.5-fold, although it did not affect kainate-induced current at all. Quisqualate-mediated oscillatory current was almost abolished by this treatment. The TPA-induced potentiation of NMDA current was suppressed by staurosporine, an inhibitor of protein kinases. Pretreatment with 4-O-methyl-TPA, an inactive phorbol ester, had no effect on NMDA current. Current response mediated by NMDA receptors would thus appear to be modulated by PKC.

Effects of H-7 (protein kinase inhibitor) and phorbol ester on aortic strips from spontaneously hypertensive rats

We investigated the vascular responsiveness to vasoactive agents and the inhibition by H-7 (1-(5-isoquinoline-sulfonyl)-2-methylpiperazine), which inhibits cyclic nucleotide-dependent protein kinases and protein kinase C(PKC) equally potently in helically cut strips of thoracic aortas from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). The susceptibility of norepinephrine (NE)- and angiotensin II (Ang II)-induced contractions to H-7 was significantly higher in the aortas from SHR than in those from WKY. H-7 decreased the contractile responses to KCl to a similar extent in both strains without affecting the high K +-stimulated Ca 2+ influx. H-7 produced a shift to the right of the dose-response curve for the PKC activator, 12-o-tetradecanoylphorbol-13-acetate (TPA) in the case of SHR aortas, while no such shift was noted in tissues from WKY. Functional alterations in the PKC branch of the Ca 2 messenger system in vascular smooth muscle may play an important role in SHR during the sustained contraction.