PKCα Overexpression Research Articles

PKCα suppresses osteoblastic differentiation

Protein kinase C (PKC) plays an essential role in cellular signal transduction for mediating a variety of biological functions. There are 11 PKC isoforms and these isoforms are believed to play distinct roles in cells. Although the role of individual isoforms of PKC has been investigated in many fields, little is known about the role of PKC in osteoblastic differentiation. Here, we investigated which isoforms of PKC are involved in osteoblastic differentiation of the mouse preosteoblastic cell line MC3T3-E1. Treatment with Gö6976, an inhibitor of PKCα and PKCβI, increased alkaline phosphatase (ALP) activity as well as gene expression of ALP and Osteocalcin (OCN), and enhanced calcification of the extracellular matrix. Concurrently, osteoblastic cell proliferation decreased at a concentration of 1.0 μM. In contrast, a PKCβ inhibitor, which inhibits PKCβI and PKCβII, did not significantly affect osteoblastic differentiation or cell proliferation. Knockdown of PKCα using MC3T3-E1 cells transfected with siRNA also induced an increase in ALP activity and in gene expression of ALP and OCN. In contrast, overexpression of wild-type PKCα decreased ALP activity and attenuated osteoblastic differentiation markers including ALP and OCN, but promoted cell proliferation. Taken together, our results indicate that PKCα suppresses osteoblastic differentiation, but promotes osteoblastic cell proliferation. These results imply that PKCα may have a pivotal role in cell signaling that modulates the differentiation and proliferation of osteoblasts.

Intracellular Calcium Release and Protein Kinase C Activation Stimulate Sonic Hedgehog Gene Expression During Gastric Acid Secretion

Hypochlorhydria during Helicobacter pylori infection inhibits gastric Sonic Hedgehog (Shh) expression. We investigated whether acid-secretory mechanisms regulate Shh gene expression through intracellular calcium (Ca2(+)(i))-dependent protein kinase C (PKC) or cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) activation. We blocked Hedgehog signaling by transgenically overexpressing a secreted form of the Hedgehog interacting protein-1, a natural inhibitor of hedgehog ligands, which induced hypochlorhydria. Gadolinium, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) + 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), PKC-overexpressing adenoviruses, and PKC inhibitors were used to modulate Ca(2+)(i)-release, PKC activity, and Shh gene expression in primary gastric cell, organ, and AGS cell line cultures. PKA hyperactivity was induced in the H(+)/K(+)-β-cholera-toxin-overexpressing mice. Mice that expressed secreted hedgehog-interacting protein-1 had lower levels of gastric acid (hypochlorhydria), reduced production of somatostatin, and increased gastrin gene expression. Hypochlorhydria in these mice repressed Shh gene expression, similar to the levels obtained with omeprazole treatment of wild-type mice. However, Shh expression also was repressed in the hyperchlorhydric H(+)/K(+)-β-cholera-toxin model with increased cAMP, suggesting that the regulation of Shh was not solely acid-dependent, but pertained to specific acid-stimulatory signaling pathways. Based on previous reports that Ca(2+)(i) release also stimulates acid secretion in parietal cells, we showed that gadolinium-, thapsigargin-, and carbachol-mediated release of Ca(2+)(i) induced Shh expression. Ca(2+)-chelation with BAPTA + EGTA reduced Shh expression. Overexpression of PKC-α, -β, and -δ (but not PKC-ϵ) induced an Shh gene expression. In addition, phorbol esters induced a Shh-regulated reporter gene. Secretagogues that stimulate gastric acid secretion induce Shh gene expression through increased Ca(2+)(i)-release and PKC activation. Shh might be the ligand transducing changes in gastric acidity to the regulation of G-cell secretion of gastrin.

PKC-ALPHA MEDIATES NEPHRIN ENDOCYTOSIS: PP.9.354

Background: Mikroalbuminuria is an early lesion during the development of diabetic nephropathy. The loss of high molecular weight proteins in the urine is usually associated with decreased expression of slit diaphragm proteins. Nephrin, is the major component of the glomerular slit diaphragm and loss of nephrin has been well described in rodent models of experimental diabetes as well as in human diabetic nephropathy. Methodology/Principal Findings: In this manuscript we analyzed the role of PKCalpha (PKCα) on endocytosis of nephrin in podocytes. We found that treatment of diabetic mice with a PKCα-inhibitor (GÖ6976) leads to preserved nephrin expression and reduced proteinuria. In addition, we found that PKCα deficient mice are completely protected from LPS induced transient proteinuria. In vitro, we found that high glucose but also LPS stimulation would induce PKCα protein expression. We can demonstrate that PKCα mediates nephrin endocytosis in podocytes and that overexpression of PKCα leads to an augmented endocytosis response. After PKCactivation, we demonstrate an inducible association of PKCα, PICK1 and nephrin in podocytes. Moreover we can demonstrate a strong induction of PKCα in podocytes of patients with diabetic nephropathy. Conclusions/Significance: We therefore conclude that activation of PKCα is a pathomechanistic key event during the development of diabetic nephropathy. PKCα is involved in reduction of nephrin surface expression and therefore PKCα inhibition might be a novel target olecule for anti-proteinuric therapy.

Abstract 4608: Overexpression of PKCα alters gene expression pattern in T47D breast cancer cells associated with hormone-independent growth and tamoxifen resistance

Abstract Background: Tamoxifen (TAM) resistant breast cancer is a major obstacle in the clinical management of breast cancer and the mechanisms are not fully understood. Our lab has previously shown that overexpression of protein kinase C α (PKCα) in T47D breast cancer cells leads to a hormone-independent and TAM-resistant phenotype (Chisamore et. al., 2001, Clin. Cancer Res.7:3156-3165). Furthermore, we found that PKCα overexpression correlates with TAM-resistance and disease recurrence in patients (Tonetti et. al., 2003, Br. J. Cancer 88:1400-1402). Taken together, these findings suggest overexpression of PKCα is important in the progression of breast cancer to a hormone-independent and TAM-resistant phenotype. Methods: To determine the mechanism whereby overexpression of PKCα can mediate this progression, gene expression profiles of T47D: A18/neo and T47D: A18/PKCα cells following treatment with 0.1% EtOH or 10 nM 17β-estradiol (E2) for 24 hours were compared utilizing the Affymetrix Human Gene ST 1.0 Array. Total RNA was harvested for cRNA labeling and hybridization to Affymetrix ST 1.0 GeneChips. Data was analyzed using Partek Genomics statistical package (Partek, Inc). ANOVA test was used to calculate significance of the differential expression. Gene Ontology (GO) term enrichment was carried out using the Functional Annotation Clustering tool in DAVID Bioinformatics Database. Results: The basal expression of more than one thousand genes are differently expressed in T47D cells just by overexpressing PKCα, including genes related to cell cycle progression, gene expression regulation, protein transport and localization and metabolic processes. Interestingly, many of the E2-regulated genes in T47D: A18/neo cells have similar expression patterns in T47D: A18/PKCα cells in the absence of E2 treatment. Thirty six genes are up regulated by E2 in T47D: A18/neo cells and they have higher basal expression levels in T47D: A18/PKCα cells compared with T47D: A18/neo cells, including TFF1, CXCL12, STC2 and MYC. On the other hand, seventy six genes are down regulated by E2 in T47D: A18/neo cells that also have lower basal expression levels in the PKCα-overexpressing cells compared to T47D: A18/neo, including ERBB2, TGFB2 and TGFB3. Furthermore, E2 does not have a great influence on the gene expression levels in T47D: A18/PKCα cells. Conclusion: These results suggest that PKCα overexpression may lead to estrogen-independent activation of ER target genes and result in a pattern of gene expression similar to that in T47D cells treated with E2. This altered activation of ER by PKCα may lead to a gene expression pattern that mediates the progression to the estrogen-independent growth and TAM resistance. Pharmacological inhibition of this progression by inhibiting PKCα may offer treatment options for TAM-resistant breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4608.

Estradiol Induced Regression of T47D:A18/PKCα Tumor Involves Translocation of Estrogen Receptor alpha from the Nucleus to the Cytoplasm.

Abstract Background: Resistance to tamoxifen (TAM) is one of the most challenging problems in the treatment of breast cancer. In our laboratory we have developed a PKCα overexpressing T47D:A18 cell line which is TAMresistant and 17β estradiol (E2) independent compared to the E2 dependent T47D:A18/neo cell line (Chisamore et al, 2001). PKCα overexpression is known to be associated with TAM treatment failure in breast cancer (Tonetti et al, 2003). This T47D:A18/PKCα model exhibits E2-induced tumor regression in in vivo or when grown in matrigel (3D matrix) but not on 2D plastic. Recently we have demonstrated that tumor regression involves participation of estrogen receptor alpha (ERα), Fas/FasL and the extracellular matrix (Zhang et al, 2009). Interestingly membrane impermeable E2-BSA conjugate mediates growth inhibition of T47D:A18/PKCα colonies in matrigel similar to E2 suggesting a potential role of extranuclear ERα. On the basis of our preliminary data we investigated whether extranuclear ERα may play a critical role in E2-induced tumor regression.Materials and methods: T47D:A18/PKCα cells were injected into mammary fat pads of 20 ovariectomized athymic mice. Mice were left untreated and tumors were allowed to grow until the mean tumor size reached 0.5 cm2, then 10 mice were implanted with a 1.0 cm E2 capsule and the other 10 continued with no treatment (control). T47D:A18/neo tumors were also established in 10 athymic mice in the presence of E2. Four micrometer thick sections were prepared from the paraffin embedded T47D:A18/PKCα tumor tissues of control and E2-treated mice and T47D:A18/neo tumors. For immunofluorescence staining, alexa fluor 488 and Cy3 fluorescence tagged secondary antibodies were used against ERα and β-actin primary antibodies respectively.Results: Immunofluorescence images of T47D:A18/PKCα tumor sections from untreated control mice showed that the ERα was mainly localized in the nucleus. Interestingly, treatment with E2 resulted in significant translocation of ERα from the nucleus to the cytoplasm and the plasma membrane of tumor cells. Immunofluorescence analysis of tumor sections obtained from E2 treated mice indicated that approximately 5-10% of cells contained ERα localized to the plasma membrane whereas the majority of ERα was localized to the cytosol. However in T47D:A18/neo tumors the majority of ERα was found in the nucleus suggesting the involvement of the ERE mediated classical pathway in tumor growth.Discussion: This is the first time we are reporting that E2-induced T47D:A18/PKCα tumor regression is accompanied by translocation of ERα to extranuclear sites. E2-induced tumor regression observed in our pre-clinical model suggests E2 or an E2-like compound may be a potential treatment option for patients harboring PKCα overexpressing tumors. Furthermore the extranuclear ERα signaling pathway may be an attractive therapeutic target to treat PKCα overexpressing tumors that are refractory to current endocrine therapies. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4140.

T47D/PKCα Cells Are Nonmigratory and Noninvasive In Vitro Despite Tamoxifen Resistance In Vivo.

Abstract Background: We have previously reported that overexpression of PKCα in T47D (T47D/PKCα) breast cancer cells results in hormone independence, growth inhibition by estradiol, and tamoxifen resistance (Chisamore, 2001). This has clinical relevance because we have shown that PKCα overexpression may predict disease recurrence and tamoxifen treatment failure suggesting consideration of other treatments (Tonetti, 2003). Further characterization reveals that T47D/PKCα cells are noninvasive in vitro compared to T47D/neo control cells in Matrigel invasion assays and that knockdown of PKCα reverses the phenotype (Perez White, AACR 2009). We have further characterized the in vitro phenotype of T47D/PKCα cells using migration assay and profiling of proteins associated with epithelial-mesenchymal transition (EMT). Adherens junctions are made of a complex of proteins including E-cadherin whose dissolution is thought to play a role in EMT. E-cadherin can be transcriptionally repressed by the embryonic transcription factor Snail1. Generally, cells that lose expression of E-cadherin gain expression of N-cadherin and/or vimentin which is correlated to increased migration, invasion, and metastasis.Methods: To determine migration, cells were plated in serum-free media into Boyden chambers for 24 h with media containing 10% fetal bovine serum as chemoattractant. Protein expression levels were determined by western blot of whole cell extracts and transcript levels by RT-qPCR. Statistical analyses were performed by t-test with GraphPad Prism 5.Results: Migration assay results show that T47D/PKCα cells are nonmigratory compared to T47D/neo cells (P < 0.0001). Western blots show that there is significantly lower expression of E-cadherin in T47D/PKCα cells compared to T47D/neo cells (P < 0.005). However, transcripts of E-cadherin are expressed at 3-fold higher levels in T47D/PKCα cells compared to T47D/neo cells. Neither cell line expresses vimentin, N-cadherin, or Snail1 proteins.Conclusions: T47D/PKCα cells have reduced invasive and migratory potential even though they have decreased expression of E-cadherin. Results indicate that regulation of E-cadherin may be due to translational repression or increased degradation due to increased transcripts and absence of a transcriptional repressor. In summary, despite the fact that PKCα overexpression correlates with tamoxifen resistance, these tumors may have a better prognosis due to the less invasive and migratory nature of these cells. Future mechanistic and clinical studies are needed to determine the full prognostic impact of PKCα tumor overexpression in patients. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6173.

2-(4-Hydroxyphenyl)-Benzo[b]thiophen-6-ol, an Estrogen-Like Compound, Induces Apoptosis in T47D/PKCα Breast Cancer Cells.

Abstract Background: Tamoxifen (TAM) treatment failure is a major obstacle encountered in the clinical setting. Our lab has previously shown that constitutive overexpression of PKCα imparts a TAM resistant/ hormone independent phenotype in the T47D:A18 breast cancer cell line. Furthermore 17β-estradiol (E2) inhibits tumor growth in vivo as well as inhibits colony formation when cells are grown in 3D Matrigel (Zhang, 2009). Before TAM treatment was introduced, breast cancer patients received high-dose E2 and diesthystilbesterol (DES) treatment. DES treated patients may have a survival advantage over TAM treated patients in the long term (Peethambaram,1999). Others have shown that E2 can have inhibitory effects on MCF-7 cells both in vitro and in vivo (Lewis-Wambi, 2009). Taken together, this suggests an estrogenic compound may be efficacious for TAM-resistant breast cancer. The present study was designed to evaluate the effect of an estrogenic compound, 2-(4-hydroxyphenyl)-benzo[b]thiophen-6-ol (BTC), related to the SERMs raloxifene and arzoxifene, on the T47D:A18/PKCα breast cancer cell line.Methods: To determine if BTC can activate estrogen regulated genes we preformed an ERE-luciferase reporter assay 24 hours after BTC treatment. 3D Matrigel colony formation assay was performed to determine if BTC could inhibit colony formation. Colonies were stained and counted on day 10. To determine if apoptosis was occurring, cells were grown in Matrigel and the TUNEL Assay was performed on Day 6.Results: T47D:A18/PKCα cells showed a 31-fold induction in ERE-luciferase reporter activity when treated with BTC (10-7M) compared to 7-fold induction when treated with E2. In the T47D:A18/Neo parental cell line, BTC induced a 40-fold induction whereas estradiol induced a 96-fold induction. In the Matrigel colony formation assay T47D:A18/PKCα cells formed significantly fewer colonies when treated with BTC. Conversely, in the T47D:A18/Neo parental cell line BTC enhanced colony formation similar to E2 treatment. Our results indicate that BTC was able to induce significant apoptosis on day 6 (P= 0.038) as determined by the TUNEL Assay. Therefore BTC acts similarly to estradiol by inhibiting T47D:A18/PKCα colony formation and inducing apoptosis when cells are grown in Matrigel.Conclusion: These findings suggest that BTC is a potential lead compound in the treatment of PKCα overexpressing breast cancer. Furthermore, BTC was shown to be a potent inducer of the cytoprotective enzyme NQO1 in the Hepa 1c1c7 cell system as well as a strong activator of antioxidant responsive elements (Yu, 2007) indicating it may have the added benefit of chemoprotective effects which are absent in estradiol treatment. Further understanding of the mechanism in which BTC induces apoptosis in the T47D:A18/PKCα cell line may result in a clinical advantage to estradiol treatment including fewer side effects. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5140.

Phosphorylation of α6-Tubulin by Protein Kinase Cα Activates Motility of Human Breast Cells

Engineered overexpression of protein kinase Calpha (PKCalpha) was previously shown to endow nonmotile MCF-10A human breast cells with aggressive motility. A traceable mutant of PKCalpha (Abeyweera, T. P., and Rotenberg, S. A. (2007) Biochemistry 46, 2364-2370) revealed that alpha6-tubulin is phosphorylated in cells expressing traceable PKCalpha and in vitro by wild type PKCalpha. Gain-of-function, single site mutations (Ser-->Asp) were constructed at each PKC consensus site in alpha6-tubulin (Ser158, Ser165, Ser241, and Thr337) to simulate phosphorylation. Following expression of each construct in MCF-10A cells, motility assays identified Ser165 as the only site in alpha6-tubulin whose pseudophosphorylation reproduced the motile behavior engendered by PKCalpha. Expression of a phosphorylation-resistant mutant (S165N-alpha6-tubulin) resulted in suppression of MCF-10A cell motility stimulated either by expression of PKCalpha or by treatment with PKCalpha-selective activator diacylglycerol-lactone. MCF-10A cells treated with diacylglycerol-lactone showed strong phosphorylation of endogenous alpha-tubulin that could be blocked when S165N-alpha6-tubulin was expressed. The S165N mutant also inhibited intrinsically motile human breast tumor cells that express high endogenous PKCalpha levels (MDA-MB-231 cells) or lack PKCalpha and other conventional isoforms (MDA-MB-468 cells). Comparison of Myc-tagged wild type alpha6-tubulin and S165N-alpha6-tubulin expressed in MDA-MB-468 cells demonstrated that Ser165 is also a major site of phosphorylation for endogenously active, nonconventional PKC isoforms. PKC-stimulated motility of MCF-10A cells was nocodazole-sensitive, thereby implicating microtubule elongation in the mechanism. These findings support a model in which PKC phosphorylates alpha-tubulin at Ser165, leading to microtubule elongation and motility.

Deletion of SM-B, the high ATPase isoform of myosin, upregulates the PKC-mediated signal transduction pathway in murine urinary bladder smooth muscle

Detrusor smooth muscle (DSM) hypertrophy induced by partial bladder outlet obstruction (PBOO) is associated with changes in the NH2-terminal myosin heavy chain isoform from predominantly SM-B to SM-A, alteration in the Ca2+ sensitization pathway, and the contractile characteristics from phasic to tonic in rabbits. We utilized the SM-B knockout (KO) mouse to determine whether a shift from SM-B to SM-A without PBOO is associated with changes in the signal transduction pathway mediated via PKC and CPI-17, which keeps the myosin phosphorylation (MLC20) level high by inhibiting the myosin phosphatase. DSM strips from SM-B KO mice generated more force in response to electrical field stimulation, KCl, carbachol, and phorbol 12,13-dibutyrate than that of age-matched wild-type mice. There was no difference in the ED50 for carbachol but the maximum response was greater for the SM-B KO mice. DSM from SM-B KO mice revealed increased mass and hypertrophy. The KO mice also showed an overexpression of PKC-alpha, increased levels of phospho-CPI-17, and an elevated level of IP3 and DAG upon stimulation with carbachol. Two-dimensional gel electrophoresis revealed an increased level of MLC20 phosphorylation in response to carbachol. Together, these changes may be responsible for the higher level of force generation and maintenance by the DSM from the SM-B KO bladders. In conclusion, our data show that ablation of SM-B is associated with alteration of PKC-mediated signal transduction and CPI-17-mediated Ca2+ sensitization pathway that regulate smooth muscle contraction. Interestingly, similar changes are also present in PBOO-induced DSM compensatory response in the rabbit model in which SM-B is downregulated.

2008 Thomas W. Smith Memorial Lecture—Protein Kinase C α as a Novel Therapeutic Target for Treating Heart Failure

We have shown that protein kinase C (PKC) α functions as a proximal regulator of Ca 2+ handling in cardiac myocytes (Braz et al, Nat. Med. 10:248, 2004). Deletion of PKC α in the mouse resulted in augmented sarcoplasmic reticulum Ca 2+ loading, enhanced Ca 2+ transients, and augmented contractility, whereas overexpression of PKCα in the heart blunted contractility. Mechanistically, PKCα regulates Ca 2+ handling by altering inhibitor-1 phosphorylation, which suppresses protein phosphatase-1 activity, thus modulating phospholamban activity and sarcoplasmic reticulum Ca 2+ AT-Pase 2 (SERCA2). Acute inhibition of PKCα with the pharmacologic agents Ro-32-0432 or Ro-31-8220 significantly augmented cardiac contractility in vivo or in an isolated work performing heart preparation in wild-type mice, but not in PKC α-deficient mice. Ro-32-0432 also acutely increased cardiac contractility in two different models of heart failure in vivo. Moreover, acute or chronic treatment with Ro-32-8220 in a mouse model of heart failure, due to deletion of the muscle lim protein (MLP) gene, significantly augmented cardiac contractility and restored normal pump function. Adenoviral-mediated gene therapy with a dominant negative PKCα cDNA rescued heart failure in a chronic rat model of postinfarction cardiomyopathy. Moreover, expression of dominant-negative PKCα in cardiac myocytes using a cardiac-specific transgenic system (tetracycline-regulated) also enhanced cardiac contractility and antagonized heart failure due to myocardial infarction injury. Finally, another PKCα/β inhibitor, ruboxistaurin (LY333531), antagonized heart failure after long-term pressure overload in mice. PKCα is the dominant conventional PKC isoform expressed in the adult human heart, providing potential relevance of these findings to human pathophysiology. Indeed, pharmacological inhibition of PKCα may serve as a novel therapeutic strategy for either enhancing cardiac contractility in the setting of severe functional deterioration or as a long-term treatment option to prevent worsening of heart failure in earlier stages.

UV irradiation increases ROS production via PKCδ signaling in primary murine fibroblasts

Ultraviolet (UV) irradiation is a major environmental factor responsible for a high incidence of premature skin aging, referred to as photoaging, as well as skin cancer and melanoma. UVA irradiation represents 90% of the solar UV light reaching the earth's surface, and yet the mechanisms by which it exerts its biological effects are not clear. UVA penetrates into the skin tissue, reaching the basal layers of the active dividing cells and, therefore, the contribution of UVA to skin damage may be significant. The majority of UVA energy is absorbed by unidentified photosensitizers in the cells which are postulated to generate reactive oxygen species (ROS). It has been believed that both chronological aging and photoaging share the same molecular features and, as such, it is very common to utilize UV irradiation for induction of skin aging. To determine the involvement of protein kinase isoforms in chronological aging and photoaging, we utilized in vitro aging model systems of primary murine fibroblasts and primary fibroblasts isolated from PKC null mice. We show for the first time distinct involvement of PKC isoforms PKCdelta and PKCalpha in photoaging versus cellular senescence. While chronological aging is accompanied by overexpression and activation of PKCalpha, UV irradiation and ROS production are associated with photoaging accompanied by PKCdelta downregulation and nuclear translocation.

Coordinated phosphorylation of insulin receptor substrate-1 by glycogen synthase kinase-3 and protein kinase CβII in the diabetic fat tissue

Serine/threonine phosphorylation of insulin receptor substrate-1 (IRS-1) is an important negative modulator of insulin signaling. Previously, we showed that glycogen synthase kinase-3 (GSK-3) phosphorylates IRS-1 at Ser(332). However, the fact that GSK-3 requires prephosphorylation of its substrates suggested that Ser(336) on IRS-1 was the "priming" site phosphorylated by an as yet unknown protein kinase. Here, we sought to identify this "priming kinase" and to examine the phosphorylation of IRS-1 at Ser(336) and Ser(332) in physiologically relevant animal models. Of several stimulators, only the PKC activator phorbol ester PMA enhanced IRS-1 phosphorylation at Ser(336). Treatment with selective PKC inhibitors prevented this PMA effect and suggested that a conventional PKC was the priming kinase. Overexpression of PKCalpha or PKCbetaII isoforms in cells enhanced IRS-1 phosphorylation at Ser(336) and Ser(332), and in vitro kinase assays verified that these two kinases directly phosphorylated IRS-1 at Ser(336). The expression level and activation state of PKCbetaII, but not PKCalpha, were remarkably elevated in the fat tissues of diabetic ob/ob mice and in high-fat diet-fed mice compared with that from lean animals. Elevated levels of PKCbetaII were also associated with enhanced phosphorylation of IRS-1 at Ser(336/332) and elevated activity of GSK-3beta. Finally, adenoviral mediated expression of PKCbetaII in adipocytes enhancedphosphorylation of IRS-1 at Ser(336). Taken together, our results suggest that IRS-1 is sequentially phosphorylated by PKCbetaII and GSK-3 at Ser(336) and Ser(332). Furthermore, these data provide evidence for the physiological relevance of these phosphorylation events in the pathogenesis of insulin resistance in fat tissue.

Protein Kinase Cα and ζ Differentially Regulate Death-Inducing Signaling Complex Formation in Cigarette Smoke Extract-Induced Apoptosis

Cigarette smoke, a major risk factor in emphysema, causes cell death by incompletely understood mechanisms. Death-inducing signaling complex (DISC) formation is an initial event in Fas-mediated apoptosis. We demonstrate that cigarette smoke extract (CSE) induces DISC formation in human lung fibroblasts (MRC-5) and promotes DISC trafficking from the Golgi complex to membrane lipid rafts. We demonstrate a novel role of protein kinase C (PKC) in the regulation of DISC formation and trafficking. The PKC isoforms, PKCalpha, zeta, epsilon, and eta, were activated by CSE exposure. Overexpression of wild-type PKCalpha inhibited, while PKCzeta promoted, CSE-induced cell death. Dominant-negative (dn)PKCzeta protected against CSE-induced cell death by suppressing DISC formation and caspase-3 activation, while dnPKCalpha enhanced cell death by promoting these events. DISC formation was augmented by wortmannin, an inhibitor of PI3K. CSE-induced Akt phosphorylation was reduced by dnPKCalpha, but it was increased by dnPKCzeta. Expression of PKCalpha in vivo inhibited DISC formation, caspase-3/8 activation, lung injury, and cell death after prolonged cigarette smoke exposure, whereas expression of PKCzeta promoted caspase-3 activation. In conclusion, CSE-induced DISC formation is differentially regulated by PKCalpha and PKCzeta via the PI3K/Akt pathway. These results suggest that modulation of PKC may have therapeutic potential in the prevention of smoke-related lung injury.

Activation of Bone Morphogenetic Protein Signaling by a Gemini Vitamin D3 Analogue Is Mediated by Ras/Protein Kinase Cα

Bone morphogenetic proteins (BMP) are members of the transforming growth factor-beta superfamily, and they play an important role for embryonic development, for bone and cartilage formation, and during carcinogenesis. We have previously shown that the novel Gemini vitamin D(3) analogue, Ro-438-3582 [Ro3582; 1 alpha,25-dihydroxy-20S,21(3-hydroxy-3-methylbutyl)-23-yne-26,27-hexafluorocholecalciferol], inhibited cell proliferation and activated the BMP/Smad signaling pathway in MCF10AT1 breast epithelial cells. In this report, we investigated the upstream signaling pathways responsible for the activation of BMP/Smad signaling by Ro3582. Among seven different serine/threonine kinase inhibitors that we tested, protein kinase C (PKC) inhibitors blocked the effects of Ro3582 on the phosphorylation of Smad1/5, mRNA synthesis for BMP-2 and BMP-6, and cell growth in MCF10AT1 cells. Overexpression of PKC alpha, but not PKC epsilon, PKC delta or PKC zeta isoforms, increased Ro3582-induced phosphorylation of Smad1/5, suggesting that PKC alpha mediates the activation of Smad signaling and inhibition of cell proliferation. Interestingly, the activation of Smad signaling by Ro3582 was shown in Ha-ras-transfected MCF10AT1 cells, but not in the parent cell line (MCF10A without Ras). Inhibiting Ras activity blocked the translocation of PKC alpha to the plasma membrane and the phosphorylation of Smad1/5 induced by Ro3582, indicating that Ras is necessary for the activation of PKC alpha and Smad signaling. In conclusion, Ro3582 inhibits cell proliferation and activates BMP/Smad signaling via a Ras and PKC alpha pathway in breast epithelial cells.

Protein kinase C alpha and epsilon differentially modulate hepatocyte growth factor-induced epithelial proliferation and migration

Protein kinase C (PKC) isoenzymes require membrane translocation for physiological activation. We have recently shown that the growth factors such as epidermal growth factor and hepatocyte growth factor (HGF), but not keratinocyte growth factor (KGF), regulate PKCα activation to promote epithelial wound healing [Sharma, G.D., Ottino, P., Bazan, H.E.P., 2005. Epidermal and hepatocyte growth factors, but not keratinocyte growth factor, modulate protein kinase C alpha translocation to the plasma membrane through 15( S)-hydroxyeicosatetraenoic acid synthesis. J. Biol. Chem. 280, 7917—7924]. Protein kinase C alpha (PKCα) and protein kinase C epsilon (PKCɛ) are two differentially regulated isoenzymes. While PKCα requires Ca 2+ for its activation, PKEɛ is Ca 2+ independent. However, growth factor-induced activation of these enzymes and their specific regulation of epithelial migration and proliferation have not been explored. In the present study, we overexpressed PKCɛ fused to green fluorescent protein to examine its translocation in real-time to the plasma membrane in living human corneal epithelial cells. Stimulation with HGF and KGF demonstrated translocation of PKCɛ to the plasma membrane. Because HGF activates both PKCs, this growth factor was used to stimulate wound healing. PKCα or PKCɛ-genes were knocked down individually without affecting the basal expression of the other PKC isoforms. Gene knockdown of PKCα significantly inhibited HGF-stimulated proliferation of human corneal epithelial cells. In contrast, PKCɛ-gene-silencing severely impaired the HGF-stimulated migratory ability of human corneal epithelial cells. When migrating epithelial cells in the cornea wound bed after injury were transfected with specific PKCα- or PKCɛ-siRNA, there was a significant delay in wound healing. Corneal wound healing stimulated with HGF in similar conditions was also inhibited. On the other hand, overexpression of PKCα or PKCɛ-genes fused with green fluorescent protein in migrating corneal epithelium accelerated repair of the epithelial defect. Our findings demonstrate that PKCα and PKCɛ modulate different stages of wound healing stimulated by HGF and contribute to epithelial repair by playing selective regulatory roles in epithelial proliferation and migration, both crucial to corneal wound healing.

Downregulation of GLP-1 and GIP Receptor Expression by Hyperglycemia

Stimulation of insulin secretion by the incretin hormones glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) has been found to be diminished in type 2 diabetes. We hypothesized that this impairment is due to a defect at the receptor level induced by the diabetic state, particularly hyperglycemia. Gene expression of incretin receptors, GLP-1R and GIPR, were significantly decreased in islets of 90% pancreatectomized (Px) hyperglycemic rats, with recovery when glucose levels were normalized by phlorizin. Perifused islets isolated from hyperglycemic Px rats showed reduced insulin responses to GLP-1 and GIP. To examine the acute effect of hyperglycemia on incretin receptor expression, a hyperglycemic clamp study was performed for 96 h with reduction of GLP-1 receptor expression but increase in GIP receptor expression. Similar findings were found when islets were cultured at high glucose concentrations for 48 h. The reduction of GLP-1 receptor expression by high glucose was prevented by dominant-negative protein kinase C (PKC)alpha overexpression, whereas GLP-1 receptor expression was reduced with wild-type PKCalpha overexpression. Taken together, GLP-1 and GIP receptor expression is decreased with chronic hyperglycemia, and this decrease likely contributes to the impaired incretin effects found in diabetes.

Sphingosylphosphorylcholine-induced interleukin-6 production is mediated by protein kinase C and p42/44 extracellular signal-regulated kinase in human dermal fibroblasts

Sphingosylphosphorylcholine (SPC) has been reported as a novel lipid mediator that exerts various actions on wound healing process. The aim of this study is to evaluate the involvement of interleukin-6 (IL-6) in SPC-induced wound healing acceleration. We performed immunohistochemical analysis to demonstrate the IL-6 induction by SPC. To analyze the signaling events, skin fibroblasts were treated with SPC, and then RT-PCR, ELISA and Western blot analyses were carried out. SPC markedly induced interleukin-6 (IL-6) expression in rabbit ear wound. SPC also induced IL-6 expression at both the mRNA and protein levels in human dermal fibroblasts cultured in vitro. SPC rapidly phosphorylated p42/44 extracellular signal-regulated kinase (ERK). Pretreatment with PD 98059, a specific MAPK kinase 1/2 inhibitor, markedly suppressed SPC-induced IL-6 expression in a dose-dependent manner. Protein kinase C (PKC) activation by phorbol myristate acetate (PMA) potentiated IL-6 mRNA expression, whereas PKC inhibition by bisindolylmaleimide blocked SPC-induced p42/44 ERK phosphorylation and IL-6 expression. Over-expression of PKCalpha markedly induced the IL-6 expression and p42/44 ERK activation. These results suggest that SPC-induced IL-6 production is mediated by PKC-dependent p42/44 ERK activation in human dermal fibroblasts cultured in vitro.

Influence of PKC-α overexpression on HSP70 and cardioprotection

Recent research has indicated that the protein kinase C (PKC) isoforms and the heat shock proteins (HSPs) are involved in cardioprotection. We have investigated the possible interaction between these two protein families. We have found that adenoviral-mediated expression of PKC-alpha in neonatal rat ventricular myocytes (NRVM) not only increases the expression of HSP70 but also protects against simulated ischemia-reperfusion. In addition, Western blots of PKC-alpha-infected NRVM indicated that other HSPs are not induced in the same manner as HSP70. In an effort to determine the mechanism of induction of HSP70 by PKC-alpha, we tested a chimeric construct that linked the luciferase reporter gene to the 5'-promoter region of HSP70 in myogenic H9c2 cells. When PKC-alpha was expressed, the 5'-promoter region of the HSP70 responded robustly, indicating that PKC-alpha induction of HSP70 expression is through transcription activation. Electrophoretic mobility shift assay determined that overexpression of PKC-alpha, PKC-delta, or PKC-epsilon did not induce activation of heat shock factor-1 (HSF-1). Therefore, induction of HSP70 by PKC-alpha is independent of heat shock factor-1 activation. We also measured cellular injury by assessing creatine kinase (CK) release from NRVM after simulated ischemia to determine cardioprotection. NRVM infected with the wild-type adenoviral construct AdwtPKC-alpha released 54% less CK than control NRVM. Experiments using small interfering RNA against HSP70 indicate that loss of PKC-alpha-induced HSP70 expression results in increased CK release or a loss of protection. Our results show that there is a close interaction between PKC-alpha and HSP70, independent of heat shock factor-1 activation, and that the protection conferred by PKC-alpha overexpression is mediated by the transcriptionally induced expression of HSP70.

Pharmacological- and Gene Therapy-Based Inhibition of Protein Kinase Cα/β Enhances Cardiac Contractility and Attenuates Heart Failure

The conventional protein kinase C (PKC) isoform alpha functions as a proximal regulator of Ca2+ handling in cardiac myocytes. Deletion of PKCalpha in the mouse results in augmented sarcoplasmic reticulum Ca2+ loading, enhanced Ca2+ transients, and augmented contractility, whereas overexpression of PKCalpha in the heart blunts contractility. Mechanistically, PKCalpha directly regulates Ca2+ handling by altering the phosphorylation status of inhibitor-1, which in turn suppresses protein phosphatase-1 activity, thus modulating phospholamban activity and secondarily, the sarcoplasmic reticulum Ca2+ ATPase. In the present study, we show that short-term inhibition of the conventional PKC isoforms with Ro-32-0432 or Ro-31-8220 significantly augmented cardiac contractility in vivo or in an isolated work-performing heart preparation in wild-type mice but not in PKCalpha-deficient mice. Ro-32-0432 also increased cardiac contractility in 2 different models of heart failure in vivo. Short-term or long-term treatment with Ro-31-8220 in a mouse model of heart failure due to deletion of the muscle lim protein gene significantly augmented cardiac contractility and restored pump function. Moreover, adenovirus-mediated gene therapy with a dominant-negative PKCalpha cDNA rescued heart failure in a rat model of postinfarction cardiomyopathy. PKCalpha was also determined to be the dominant conventional PKC isoform expressed in the adult human heart, providing potential relevance of these findings to human pathophysiology. Pharmacological inhibition of PKCalpha, or the conventional isoforms in general, may serve as a novel therapeutic strategy for enhancing cardiac contractility in certain stages of heart failure.

Nicotine-induced upregulation of human neuronal nicotinic α7-receptors is potentiated by modulation of cAMP and PKC in SH-EP1-hα7 cells

Chronic nicotine exposure induces upregulation of nicotinic receptors, but the mechanisms underlying this phenomenon are not well understood. The aim of this study was to examine the role of different second messenger systems in the nicotine-induced upregulation of α7-nicotinic receptors in SH-EP1-hα7 human epithelial cells. We show here that chronic exposure to nicotine results in accumulation of cAMP. Furthermore, an enhanced cAMP signalling potentiates nicotine-induced upregulation of α7-nicotinic receptors measured by [ 3H]methyllycaconitine ([ 3H]MLA) binding suggesting that cAMP is involved in the α7-nicotinic receptor upregulation. Down-regulation of protein kinase C (PKC) with a phorbol ester abolishes the nicotine-induced upregulation of α7-nicotinic receptors. Furthermore, overexpression of PKCα in SH-EP1-hα7 cells results in potentiation of nicotine-evoked upregulation indicating that PKC has a role in regulation of α7-nicotinic receptor number. The Ca 2+-calmodulin kinase II (CaMKII) and extracellular signal regulated kinase 1/2 (ERK1/2) appear not to participate in α7-nicotinic receptor upregulation since the specific inhibitors of these kinases did not have an effect on the nicotine-induced upregulation. Taken together this study provides evidence that nicotine induces accumulation of cAMP and that the upregulation mechanisms of α7-nicotinic receptors are potentiated both by cAMP and PKC. As nicotine-evoked upregulation of heteromeric nicotinic receptors in SH-SY5Y cells was unaffected by the treatment with drugs affecting cAMP signalling or PKC activity, our results suggest that the upregulation mechanisms of homomeric α7-nicotinic receptors and heteromeric nicotinic receptors differ from each other.