Role of protein kinase C in the translational regulation of lipoprotein lipase in adipocytes

The hypertriglyceridemia of diabetes is accompanied by decreased lipoprotein lipase (LPL) activity in adipocytes. Although the mechanism for decreased LPL is not known, elevated glucose is known to increase diacylglycerol, which activates protein kinase C (PKC). To determine whether PKC is involved in the regulation of LPL, we studied the effect of 12-O-tetradecanoyl phorbol 13-acetate (TPA) on adipocytes. LPL activity was inhibited when TPA was added to cultures of 3T3-F442A and rat primary adipocytes. The inhibitory effect of TPA on LPL activity was observed after 6 h of treatment, and was observed at a concentration of 6 nM. 100 nM TPA yielded maximal (80%) inhibition of LPL. No stimulation of LPL occurred after short term addition of TPA to cultures. To determine whether TPA treatment of adipocytes decreased LPL synthesis, cells were labeled with [35S]methionine and LPL protein was immunoprecipitated. LPL synthetic rate decreased after 6 h of TPA treatment. Western blot analysis of cell lysates indicated a decrease in LPL mass after TPA treatment. Despite this decrease in LPL synthesis, there was no change in LPL mRNA in the TPA-treated cells. Long term treatment of cells with TPA is known to down-regulate PKC. To assess the involvement of the different PKC isoforms, Western blotting was performed. TPA treatment of 3T3-F442A adipocytes decreased PKC alpha, beta, delta, and epsilon isoforms, whereas PKC lambda, theta, zeta, micro, iota, and gamma remained unchanged or decreased minimally. To directly assess the effect of PKC inhibition, PKC inhibitors (calphostin C and staurosporine) were added to cultures. The PKC inhibitors inhibited LPL activity rapidly (within 60 min). Thus, activation of PKC did not increase LPL, but inhibition of PKC resulted in decreased LPL synthesis by inhibition of translation, indicating a constitutive role of PKC in LPL gene expression.     

Activation of ERK during DNA damage-induced apoptosis involves protein kinase Cdelta

We have previously shown that protein kinase C (PKC) acts upstream of caspases to regulate cisplatin-induced apoptosis. Since extracellular signal-regulated kinases (ERKs) have also been implicated in DNA damage-induced apoptosis, we have examined if ERK signaling pathway acts downstream of PKC in the regulation of cisplatin-induced apoptosis. PKC activator PDBu induced ERK1/2 phosphorylation which was inhibited by general PKC inhibitor bisindolylmaleimide and G? 6983 as well as the MEK inhibitor U0126 but not by the PKCdelta inhibitor rottlerin. Cisplatin caused a concentration-dependent activation of ERK1/2 in HeLa cells. The level of ERK2 was decreased in HeLa cells that acquired resistance to cisplatin (HeLa/CP). The MEK inhibitor U0126 inhibited cisplatin-induced ERK activation and attenuated cisplatin-induced cell death. Inhibition of PKCdelta by rottlerin or depletion of PKCdelta by siRNA inhibited cisplatin-induced ERK activation. These results suggest that cisplatin-induced DNA damage results in activation of ERK1/2 via PKCdelta.     

Molecular regulation of NADPH oxidase 5 via the MAPK pathway

The mechanisms controlling the activity of NADPH oxidase 5 (Nox5) are unique in that they are independent of the protein: protein interactions that coordinate the activation of other Nox isoforms. Instead, the primary driving force for Nox5 activity is calcium. However, in a previous study we reported that the protein kinase C (PKC)-agonist PMA could induce a sustained activation of Nox5 that was independent of calcium changes. This apparent calcium-independent activation was found to be mediated by the PKC-dependent phosphorylation of specific serine and threonine residues on Nox5 which increased the calcium sensitivity of the enzyme and enabled activation at resting levels of calcium. However, the specific kinase(s) mediating the phosphorylation and activation of Nox5 are not known. As PKC can activate the MEK/ERK1/2 signaling pathway, we hypothesized that Nox5 is activated by the coordinated phosphorylation of both MAPK and PKC pathways. The inhibition of MEK1 using PD-98059 and U-0126 significantly reduced the phosphorylation and activity of Nox5 in response to PMA but not to the calcium-mobilizing stimulus ionomycin. Dominant negative MEK1 and knockdown of endogenous MEK1/2 using a specific small interfering RNA also inhibited Nox5 activity in response to PMA. The mutation of S498 to a nonphosphorylatable residue and to a lesser degree T494 blocked the ability of ERK to stimulate Nox5 activity. However, a constitutively active form of MEK1 failed to increase Nox5 activity in the absence of PMA stimulation. These results suggest that the MEK/ERK1/2 pathway is necessary but not sufficient to regulate the PMA-dependent activation of Nox5.     

Prevention of TNF-alpha-induced apoptosis in polyamine-depleted IEC-6 cells is mediated through the activation of ERK1/2

It has been documented that polyamines play a critical role in the regulation of apoptosis in intestinal epithelial cells. We have recently reported that protection from TNF-alpha/cycloheximide (CHX)-induced apoptosis in epithelial cells depleted of polyamines is mediated through the inactivation of a proapoptotic mediator, JNK. In this study, we addressed the involvement of the MAPK pathway in the regulation of apoptosis after polyamine depletion of IEC-6 cells. Polyamine depletion by alpha-difluromethylornithine (DFMO) resulted in the sustained activation of ERK in response to TNF-alpha/CHX treatment. Pretreatment of polyamine-depleted IEC-6 cells with a cell membrane-permeable MEK1/2 inhibitor, U-0126, significantly inhibited TNF-alpha/CHX-induced ERK phosphorylation and significantly increased DNA fragmentation, JNK activity, and caspase-3 activity in response to TNF-alpha/CHX. Moreover, the dose dependency of U-0126-mediated inhibition of TNF-alpha/ CHX-induced ERK phosphorylation correlated with the reversal of the antiapoptotic effect of DFMO. IEC-6 cells expressing constitutively active MEK1 had decreased TNF-alpha/CHX-induced JNK phosphorylation and were significantly protected from apoptosis. Conversely, a dominant-negative MEK1 resulted in high basal activation of JNK, cytochrome c release, and spontaneous apoptosis. Polyamine depletion of the dominant-negative MEK1 cells did not prevent JNK activation or cytochrome c release and failed to confer protection from both TNF-alpha/CHX and camptothecin-induced apoptosis. Finally, expression of a dominant-negative mutant of JNK significantly protected IEC-6 cells from TNF-alpha/CHX-induced apoptosis. These data indicate that polyamine depletion results in the activation of ERK, which inhibits JNK activation and protects cells from apoptosis.     

PKC epsilon -mediated ERK1/2 activation involved in radiation-induced cell death in NIH3T3 cells

Protein kinase C (PKC) isoforms play distinct roles in cellular functions. We have previously shown that ionizing radiation activates PKC isoforms (alpha, delta, epsilon, and zeta), however, isoform-specific sensitivities to radiation and its exact mechanisms in radiation mediated signal transduction are not fully understood. In this study, we showed that overexpression of PKC isoforms (alpha, delta, epsilon, and zeta) increased radiation-induced cell death in NIH3T3 cells and PKC epsilon overexpression was predominantly responsible. In addition, PKC epsilon overexpression increased ERK1/2 activation without altering other MAP-kinases such as p38 MAPK or JNK. Co-transfection of dominant negative PKC epsilon (PKC epsilon -KR) blocked both PKC epsilon -mediated ERK1/2 activation and radiation-induced cell death, while catalytically active PKC epsilon construction augmented these phenomena. When the PKC epsilon overexpressed cells were pretreated with PD98059, MEK inhibitor, radiation-induced cell death was inhibited. Co-transfection of the cells with a mutant of ERK1 or -2 (ERK1-KR or ERK2-KR) also blocked these phenomena, and co-transfection with dominant negative Ras or Raf cDNA revealed that PKC epsilon -mediated ERK1/2 activation was Ras-Raf-dependent. In conclusion, PKC epsilon -mediated ERK1/2 activation was responsible for the radiation-induced cell death.     

Protein kinase C-zeta and phosphoinositide-dependent protein kinase-1 are required for insulin-induced activation of ERK in rat adipocytes.

The mechanisms used by insulin to activate the multifunctional intracellular effectors, extracellular signal-regulated kinases 1 and 2 (ERK1/2), are only partly understood and appear to vary in different cell types. Presently, in rat adipocytes, we found that insulin-induced activation of ERK was blocked (a) by chemical inhibitors of both phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC)-zeta, and, moreover, (b) by transient expression of both dominant-negative Deltap85 PI3K subunit and kinase-inactive PKC-zeta. Further, insulin effects on ERK were inhibited by kinase-inactive 3-phosphoinositide-dependent protein kinase-1 (PDK-1), and by mutation of Thr-410 in the activation loop of PKC-zeta, which is the target of PDK-1 and is essential for PI3K/PDK-1-dependent activation of PKC-zeta. In addition to requirements for PI3K, PDK-1, and PKC-zeta, we found that a tyrosine kinase (presumably the insulin receptor), the SH2 domain of GRB2, SOS, RAS, RAF, and MEK1 were required for insulin effects on ERK in the rat adipocyte. Our findings therefore suggested that PDK-1 and PKC-zeta serve as a downstream effectors of PI3K, and act in conjunction with GRB2, SOS, RAS, and RAF, to activate MEK and ERK during insulin action in rat adipocytes.     

Knockout of PKC alpha enhances insulin signaling through PI3K

Insulin stimulates glucose transport and certain other metabolic processes by activating atypical PKC isoforms (lambda, zeta, iota) and protein kinase B (PKB) through increases in D3-polyphosphoinositides derived from the action of PI3K. The role of diacylglycerol-sensitive PKC isoforms is less clear as they have been suggested to be both activated by insulin and yet inhibit insulin signaling to PI3K. Presently, we found that insulin signaling to insulin receptor substrate 1-dependent PI3K, PKB, and PKC lambda, and downstream processes, glucose transport and activation of ERK, were enhanced in skeletal muscles and adipocytes of mice in which the ubiquitous conventional diacylglycerol-sensitive PKC isoform, PKC alpha, was knocked out by homologous recombination. On the other hand, insulin provoked wortmannin-insensitive increases in immunoprecipitable PKC alpha activity in adipocytes and skeletal muscles of wild-type mice and rats. We conclude that 1) PKC alpha is not required for insulin-stimulated glucose transport, and 2) PKC alpha is activated by insulin at least partly independently of PI3K, and largely serves as a physiological feedback inhibitor of insulin signaling to the insulin receptor substrate 1/PI3K/PKB/PKC lambda/zeta/iota complex and dependent metabolic processes.     

Role of RAF/MEK/ERK pathway,p‐STAT‐3 and Mcl‐1 in sorafenib activity in human pancreatic cancer cell lines

Sorafenib is a multikinase inhibitor that has shown promising therapeutic results in different tumor histotypes, both as a single agent or in combination with other treatments. We analyzed the in vitro activity of sorafenib in pancreatic cancer, one of the most lethal and chemo‐radio‐resistant tumors, using four human pancreatic cancer cell lines (t3m4, Capan 1, Capan 2, and MiaPaca 2), characterized by different K‐ras gene status and RAF/MEK/ERK profile. Sorafenib exerted a strong anti‐proliferative effect independently of RAS/RAF/MEK/ERK and induced various degrees of apoptosis in the cell lines. The mechanisms involved were explored in detail in t3m4 and Capan 1, in which sorafenib induced the highest and lowest levels of apoptosis, respectively. In t3m4, the RAF/AKT/STAT‐3 rather than the RAF/MEK/ERK pathway was involved, whereas in Capan 1 cells there was a strong decrease in pMEK and pERK which was not accompanied by an important reduction in RAF, AKT, and STAT‐3 proteins or in their phosphorylation. Moreover, U0126‐induced MEK inhibition did not induce apoptosis in any cell line, reinforcing the hypothesis of a MEK/ERK‐independent mechanism of sorafenib activity. Mcl‐1 appears to play a crucial role in sorafenib‐induced apoptosis. In fact, both protein and mRNA were downregulated in t3m4 and upregulated in Capan 1, in which siRNA‐induced silencing resulted in the same level of apoptosis as observed in t3m4. Our results show that sorafenib exerts anti‐proliferative and pro‐apoptotic activity in pancreatic cancer cells. Used singly or in combination with other drugs, it could therefore represent valid treatment for pancreatic cancer. J. Cell. Physiol. 220: 214–221, 2009. © 2009 Wiley‐Liss, Inc.     

The Raf/MEK/extracellular signal-regulated kinase 1/2 pathway can mediate growth inhibitory and differentiation signaling via androgen receptor downregulation in prostate cancer cells

Upregulated ERK1/2 activity is correlated with androgen receptor (AR) downregulation in certain prostate cancer (PCa) that exhibits androgen deprivation-induced neuroendocrine differentiation, but its functional relevance requires elucidation. We found that sustained ERK1/2 activation using active Raf or MEK1/2 mutants is sufficient to induce AR downregulation at mRNA and protein levels in LNCaP. Downregulation of AR protein, but not mRNA, was blocked by proteasome inhibitors, MG132 and bortezomib, indicating that the pathway regulation is mediated at multiple points. Ectopic expression of a constitutively active AR inhibited Raf/MEK/ERK-mediated regulation of the differentiation markers, neuron-specific enolase and neutral endopeptidase, and the cyclin-dependent kinase inhibitors, p16^INK4A and p21^CIP1, but not Rb phosphorylation and E2F1 expression, indicating that AR has a specific role in the pathway-mediated differentiation and growth inhibitory signaling. However, despite the sufficient role of Raf/MEK/ERK, its inhibition using U0126 or ERK1/2 knockdown could not block androgen deprivation-induced AR downregulation in an LNCaP neuroendocrine differentiation model, suggesting that additional signaling pathways are involved in the regulation. We additionally report that sustained Raf/MEK/ERK activity can downregulate full length as well as hormone binding domain-deficient AR isoforms in androgen-refractory C4-2 and CWR22Rv1, but not in LAPC4 and MDA-PCa-2b. Our study demonstrates a novel role of the Raf/MEK/ERK pathway in regulating AR expression in certain PCa types and provides an insight into PCa responses to its aberrant activation.     

PKC and ERK1/2 regulate amylase promoter activity during differentiation of a salivary gland cell line

The addition of transforming growth factor alpha (TGFalpha) to a human submandibular gland cell line (HSG) cultured on basement membrane extract Matrigel, synergistically activates the acinar cell-specific salivary amylase promoter. Signaling through beta1 integrins and increased phosphorylation of ERK1/2 are involved in the increased promoter activity. Phorbol-12-myristate-13-acetate (PMA) and thapsigargin increase amylase promoter activity, suggesting that phorbol ester and calcium-dependent protein kinase C (PKC) pathways are also involved. The combination of specific inhibitors of PKC and MEK1 inhibits the amylase promoter. Inhibitors of the calcium-dependent PKC isoforms alpha, beta, and gamma decrease the promoter activity; however, PKCbeta is not detectable in HSG cells. TGFalpha alters the cellular localization of PKCalpha but not -gamma, suggesting PKCalpha is involved in TGFalpha upregulation of the amylase promoter. Furthermore, rottlerin, a PKCdelta-specific inhibitor, increases the promoter activity, suggesting PKC isoforms differentially regulate the amylase promoter. In conclusion, beta1-integrin and TGFalpha signaling pathways regulate the amylase promoter activity in HSG cells. In response to Matrigel and TGFalpha, the activation of both PKCalpha and phosphorylation of ERK1/2 results in synergistic activation of the amylase promoter. Published 2000 Wiley-Liss, Inc.     

Protein kinase C zeta plays a central role in activation of the p42/44 mitogen-activated protein kinase by endotoxin in alveolar macrophages

Human alveolar macrophages respond to endotoxin (LPS) by activation of a number of mitogen-activated protein kinase pathways, including the p42/44 (extracellular signal-related kinase (ERK)) kinase pathway. In this study, we evaluated the role of the atypical protein kinase C (PKC) isoform, PKC zeta, in LPS-induced activation of the ERK kinase pathway. Kinase activity assays showed that LPS activates PKC zeta, mitogen-activated protein/ERK kinase (MEK, the upstream activator of ERK), and ERK. LPS did not activate Raf-1, the classic activator of MEK. Pseudosubstrate-specific peptides with attached myristic acid are cell permeable and can be used to block the activity of specific PKC isoforms in vivo. We found that a peptide specific for PKC zeta partially blocked activation of both MEK and ERK by LPS. We also found that this peptide blocked in vivo phosphorylation of MEK after LPS treatment. In addition, we found that LPS caused PKC zeta to bind to MEK in vivo. These observations suggest that MEK is an LPS-directed target of PKC zeta. PKC zeta has been shown in other systems to be phosphorylated by phosphatidylinositol (PI) 3-kinase-dependent kinase. We found that LPS activates PI 3-kinase and causes the formation of a PKC zeta/PI 3-kinase-dependent kinase complex. These data implicate the PI 3-kinase pathway as an integral part of the LPS-induced PKC zeta activation. Taken as a whole, these studies suggest that LPS activates ERK kinase, in part, through activation of an atypical PKC isoform, PKC zeta.     

Involvement of p21(Waf1/Cip1) in protein kinase C alpha-induced cell cycle progression

Protein kinase C (PKC) plays an important role in the regulation of glioma growth; however, the identity of the specific isoform and mechanism by which PKC fulfills this function remain unknown. In this study, we demonstrate that PKC activation in glioma cells increased their progression through the cell cycle. Of the six PKC isoforms that were present in glioma cells, PKC alpha was both necessary and sufficient to promote cell cycle progression when stimulated with phorbol 12-myristate 13-acetate. Also, decreased PKC alpha expression resulted in a marked decrease in cell proliferation. The only cell cycle-regulatory molecule whose expression was rapidly altered and increased by PKC alpha activity was the cyclin-cyclin-dependent kinase (CDK) inhibitor p21(Waf1/Cip1). Coimmunoprecipitation studies revealed that p21(Waf1/Cip1) upregulation was accompanied by an incorporation of p21(Waf1/Cip1) into various cyclin-CDK complexes and that the kinase activity of these complexes was increased, thus resulting in cell cycle progression. Furthermore, depletion of p21(Waf1/Cip1) by antisense strategy attenuated the PKC-induced cell cycle progression. These results suggest that PKC alpha activity controls glioma cell cycle progression through the upregulation of p21(Waf1/Cip1), which facilitates active cyclin-CDK complex formation.     

Role of the ERK pathway in the activation of store-mediated calcium entry in human platelets

Extracellular signal-regulated kinases (ERKs), are common participants in a broad variety of signal transduction pathways. Several studies have demonstrated the presence of ERKs in human platelets and their activation by the physiological agonist thrombin. Here we report the involvement of the ERK cascade in store-mediated Ca(2+) entry in human platelets. Treatment of dimethyl-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid-loaded platelets with thapsigargin to deplete the intracellular Ca(2+) stores resulted in a time- and concentration-dependent activation of ERK1 and ERK2. Incubation with either U0126 or PD 184352, specific inhibitors of mitogen-activated protein kinase kinase (MEK), prevented thapsigargin-induced ERK activation. Furthermore, U0126 and PD 184352 reduced Ca(2+) entry stimulated by thapsigargin or thrombin, in a concentration-dependent manner. The role of ERK in store-mediated Ca(2+) entry was found to be independent of phosphatidylinositol 3- and 4-kinases, the tyrosine kinase pathway, and actin polymerization but sensitive to treatment with inhibitors of Ras, suggesting that the ERK pathway might be a downstream effector of Ras in mediating store-mediated Ca(2+) entry in human platelets. In addition, we have found that store depletion stimulated ERK activation does not require PKC activity. This study demonstrates for the first time a novel mechanism for regulation of store-mediated Ca(2+) entry in human platelets involving the ERK cascade.     

Protein kinase Cdelta overexpression enhances radiation sensitivity via extracellular regulated protein kinase 1/2 activation, abolishing the radiation-induced G(2)-M arrest.

Protein kinase C (PKC) has been widely implicated in regulation ofcell growth/cell cycle progression and apoptosis. However,the role of PKCdelta in radiosensitivity and cell cycle regulation remains unclear. Overexpression of PKCdelta increased Ca2+-independent PKC activity without altering other PKC isoforms (PKCalpha, -beta1, -epsilon, and -zeta), and extracellular regulated protein kinase (ERK) 1/2 activity was also increased in PKCdelta-specific manner. A clonogenic survival assay showed that PKCdelta-overexpressed cells had more radiosensitivity and pronounced induction of apoptosis than control cells. Flow cytometric analysis revealed that PKCdelta made the cells escape from radiation-induced G(2)-M arrest. Moreover, p53 and p21(Waf) induction by radiation were higher in PKCdelta-overexpressed cells than control cells, and PKCdelta-mediated apoptosis was reduced, when radiation-induced ERK1/2 activity was inhibited by PD98059. Furthermore, PKCdelta antisense and rottlerin, PKC inhibitor-abrogated PKCdelta-mediated radiosensitivity and reduced ERK1/2 activity to the control vector level. These results demonstrated that PKCdelta overexpression enhanced radiation-induced apoptosis and radiosensitivity via ERK1/2 activation, thereby abolishing the radiation-induced G(2)-M arrest and finally apoptosis.     

Differential regulation of Cl- transport proteins by PKC in Calu-3 cells

Cl- transport proteins expressed in a Calu-3 airway epithelial cell line were differentiated by function and regulation by protein kinase C (PKC) isotypes. mRNA expression of Cl- transporters was semiquantitated by RT-PCR after transfection with a sense or antisense oligonucleotide to the PKC isotypes that modulate the activity of the cystic fibrosis transmembrane conductance regulator [CFTR (PKC-epsilon)] or of the Na/K/2Cl (NKCC1) cotransporter (PKC-delta). Expression of NKCC1 and CFTR mRNAs and proteins was independent of antisense oligonucleotide treatment. Transport function was measured in cell monolayers grown on a plastic surface or on filter inserts. With both culture methods, the antisense oligonucleotide to PKC-epsilon decreased the amount of PKC-epsilon and reduced cAMP-dependent activation of CFTR but not alpha(1)-adrenergic activation of NKCC1. The antisense oligonucleotide to PKC-delta did not affect CFTR function but did block alpha(1)-adrenergic activation of NKCC1 and reduce PKC-delta mass. These results provide the first evidence for mRNA and protein expression of NKCC1 in Calu-3 cells and establish the differential regulation of CFTR and NKCC1 function by specific PKC isotypes at a site distal to mRNA expression and translation in airway epithelial cells.