Colon cancer develops over a period of 10 to 15 years, providing a window of opportunity for chemoprevention and early intervention. However, few molecular targets for effective colon cancer chemoprevention have been characterized and validated. Protein kinase CβII (PKCβII) plays a requisite role in the initiation of colon carcinogenesis in a preclinical mouse model by promoting proliferation and increased β-catenin accumulation. In this study, we test the hypothesis that PKCβII is an effective target for colon cancer chemoprevention using enzastaurin (LY317615), a PKCβ-selective inhibitor, in a mouse model of colon carcinogenesis. We find that enzastaurin potently reduces azoxymethane-induced colon tumor initiation and progression by inhibiting PKCβII-mediated tumor cell proliferation and β-catenin accumulation. Biochemically, enzastaurin reduces expression of the PKCβII- and β-catenin/T-cell factor–regulated genes PKCβII, cyclooxygenase II, and vascular endothelial growth factor, three genes implicated in colon carcinogenesis. Our results show that enzastaurin is an effective chemopreventive agent in a mouse model of sporadic colon cancer that significantly reduces both tumor initiation and progression by inhibiting expression of proproliferative genes. Thus, PKCβII is an important target for colon cancer chemoprevention and the PKCβ-selective inhibitor enzastaurin may represent an effective chemopreventive agent in patients at high risk for colon cancer.
We previously demonstrated that elevated expression of either protein kinase CβII (PKCβII) or PKCι/λ enhances colon carcinogenesis in mice. Here we use novel bi-transgenic mice to determine the relative importance of PKCβII and PKCι/λ in colon carcinogenesis in two complimentary models of colon cancer in vivo. Bi-transgenic mice over-expressing PKCβII and constitutively active PKCι (PKCβII/caPKCι) or kinase-deficient, dominant negative PKCι (PKCβII/kdPKCι) in the colon exhibit a similar increase in colon tumor incidence, tumor size and tumor burden in response to azoxymethane (AOM) when compared to non-transgenic littermates. However, PKCβII/kdPKCι mice develop predominantly benign colonic adenomas whereas PKCβII/caPKCι mice develop malignant carcinomas. In contrast, PKCβ deficient (PKCβ−/−) mice fail to develop tumors even in the presence of caPKCι. Our previous data indicated that PKCβII drives tumorigenesis and proliferation by activating β-catenin/Apc signaling. Consistent with this conclusion, genetic deletion of PKCβ has no effect on spontaneous tumorigenesis in APCmin/+ mice. In contrast, tissue-specific knock out of PKCλ significantly suppresses intestinal tumor formation in APCmin/+ mice. Our data demonstrate that PKCβII and PKCι/λ serve distinct, non-overlapping functions in colon carcinogenesis. PKCβII is required for AOM-induced tumorigenesis, but is dispensable for tumor formation in ApcMin/+ mice. PKCι/λ promotes tumor progression in both AOM- and APCmin/+-induced tumorigenesis. Thus PKCβII and PKCι, whose expression is elevated in both rodent and human colon tumors, collaborate to drive colon tumor formation and progression, respectively.
colon carcinogenesis; transgenic mice; β-catenin; proliferation; Adenomatous polyposis coli (Apc); intestinal tumorigenesis
Myocardial remodeling and heart failure (HF) are common sequelae of many forms of cardiovascular disease and a leading cause of mortality worldwide. Accumulation of damaged cardiac proteins in heart failure has been described. However, how protein quality control (PQC) is regulated and its contribution to HF development are not known. Here, we describe a novel role for activated protein kinase C isoform βII (PKCβII) in disrupting PQC. We show that active PKCβII directly phosphorylated the proteasome and inhibited proteasomal activity in vitro and in cultured neonatal cardiomyocytes. Importantly, inhibition of PKCβII, using a selective PKCβII peptide inhibitor (βIIV5-3), improved proteasomal activity and conferred protection in cultured neonatal cardiomyocytes. We also show that sustained inhibition of PKCβII increased proteasomal activity, decreased accumulation of damaged and misfolded proteins and increased animal survival in two rat models of HF. Interestingly, βIIV5-3-mediated protection was blunted by sustained proteasomal inhibition in HF. Finally, increased cardiac PKCβII activity and accumulation of misfolded proteins associated with decreased proteasomal function were found also in remodeled and failing human hearts, indicating a potential clinical relevance of our findings. Together, our data highlights PKCβII as a novel inhibitor of proteasomal function. PQC disruption by increased PKCβII activity in vivo appears to contribute to the pathophysiology of heart failure, suggesting that PKCβII inhibition may benefit patients with heart failure. (218 words)
Încreasing evidence demonstrates that protein kinase C βII (PKCβII) promotes colon carcinogenesis. We previously reported that colonic PKCβII is induced during colon carcinogenesis in rodents and humans, and that elevated expression of PKCβII in the colon of transgenic mice enhances colon carcinogenesis. Here, we demonstrate that PKCβII represses transforming growth factor β receptor type II (TGFβRII) expression and reduces sensitivity to TGF-β–mediated growth inhibition in intestinal epithelial cells. Transgenic PKCβII mice exhibit hyperproliferation, enhanced colon carcinogenesis, and marked repression of TGFβRII expression. Chemopreventive dietary ω-3 fatty acids inhibit colonic PKCβII activity in vivo and block PKCβII-mediated hyperproliferation, enhanced carcinogenesis, and repression of TGFβRII expression in the colonic epithelium of transgenic PKCβII mice. These data indicate that dietary ω-3 fatty acids prevent colon cancer, at least in part, through inhibition of colonic PKCβII signaling and restoration of TGF-β responsiveness.
protein kinase C; colon carcinogenesis; ω-3 fatty acids; transforming growth factor β; hyperproliferation
A major mechanism by which cancers escape control by the immune system is by blocking the differentiation of myeloid cells into dendritic cells (DCs), immunostimulatory cells that activate anti-tumor T cells. Tumor-dependent activation of signal transducer and activator of transcription 3 (STAT3) signaling in myeloid progenitor cells is thought to cause this block in their differentiation. In addition, a signaling pathway through protein kinase C βII (PKCβII) is essential for the differentiation of myeloid cells into DCs. Here, we found in humans and mice that breast cancer cells substantially decreased the abundance of PKCβII in myeloid progenitor cells through a mechanism involving the enhanced activation of STAT3 signaling by soluble, tumor-derived factors (TDFs). STAT3 bound to previously undescribed negative regulatory elements within the promoter of PRKCB, which encodes PKCβII. We also found a previously undescribed counter-regulatory mechanism through which the activity of PKCβII inhibited tumor-dependent STAT3 signaling by decreasing the abundance of cell-surface receptors, such as cytokine and growth factor receptors, that are activated by TDFs. Together, these data suggest that a previously unrecognized crosstalk mechanism between the STAT3 and PKCβII signaling pathways provides the molecular basis for the tumor-induced blockade in the differentiation of myeloid cells, and suggest that enhancing PKCβII activity may be a therapeutic strategy to alleviate cancer-mediated suppression of the immune system.
The ubiquitous enzyme Protein Kinase C (PKC) has been linked to the pathogenesis of vascular injury, but the cell-specific and discrete functions of the βII isoform have yet to be discovered in this setting. Our previous findings demonstrated significantly increased PKCβII in the membrane fraction of injured femoral arteries in wild type (WT) mice and revealed reduction of neointimal expansion in PKCβ-/- mice after acute vascular injury. As PKCβ-/- mice are globally devoid of PKCβ, we established novel transgenic (Tg) mice to test the hypothesis that the action of PKCβII specifically in smooth muscle cells (SMCs) mediates the formation of neointimal lesions in response to arterial injury.
Tg mice expressing SM22α promoter-targeted mouse carboxyl-terminal deletion mutant PKCβII were produced using standard techniques, subjected to femoral artery injury and compared with littermate controls. Smooth muscle cells (SMC) were isolated from wild-type (WT) and Tg mice and exposed to a prototypic stimulus, tumor necrosis factor (TNF)-α. Multiple strategies were employed in vivo and in vitro to examine the molecular mechanisms underlying the specific effects of SMC PKCβII in neointimal expansion.
In vivo and in vitro analyses demonstrated that PKCβII activity in SMCs was critical for neointimal expansion in response to arterial injury, at least in part via regulation of ERK1/2, Egr-1 and induction of MMP-9.
These data identify the SMC-specific regulatory role of PKCβII in neointimal expansion in response to acute arterial injury, and suggest that targeted inactivation of PKCβII may be beneficial in limiting restenosis via suppression of the neointima-mediating effects of Egr-1 and MMP-9.
arterial injury; transgenic mouse; mutant PKCβII; signal transduction; SMC
Stanniocalcin-1(STC-1) is up-regulated in several cancers including gastric cancer. Evidences suggest that STC-1 is associated with carcinogenesis and angiogenic process. However, it is unclear on the exact role for STC-1 in inducing angiogenesis and tumorigeneisis.
BGC/STC cells (high-expression of STC-1) and BGC/shSTC cells (low- expression of STC-1) were constructed to investigate the effect of STC-1 on the xenograft tumor growth and angiogenesis in vitro and in vivo. ELISA assay was used to detect the expression of vascular endothelial growth factor (VEGF) in the supernatants. Neutralizing antibody was used to inhibit VEGF expression in supernatants. The expression of phosphorylated -PKCβII, phosphorylated -ERK1/2 and phosphorylated -P38 in the BGC treated with STC-1protein was detected by western blot.
STC-1 could promote angiogenesis in vitro and in vivo, and the angiogenesis was consistent with VEGF expression in vitro. Inhibition of VEGF expression in supernatants with neutralizing antibody markedly abolished angiogenesis induced by STC-1 in vitro. The process of STC-1-regulated VEGF expression was mediated via PKCβII and ERK1/2.
STC-1 promotes the expression of VEGF depended on the activation of PKCβII and ERK1/2 pathways. VEGF subsequently enhances tumor angiogenesis which in turn promotes the gastric tumor growth.
STC-1; angiogenesis; VEGF; PKCβII; ERK1/2
Protein kinase C βII (PKCβII) levels increase in the myocardium of patients with end-stage heart failure (HF). Also targeted over-expression of PKCβII in the myocardium of mice leads to dilated cardiomyopathy associated with inflammation, fibrosis and myocardial dysfunction. These reports suggest a deleterious role of PKCβII in HF development. Using a post-myocardial infarction (MI) model of heart failure in rats, we determined the benefit of chronic inhibition of PKCβII on the progression of heart failure over a period of 6 weeks after the onset of symptoms and the cellular basis for these effects. Four weeks after MI, rats with HF signs that were treated for 6 weeks with the PKCβII selective inhibitor (βIIV5-3 conjugated to TAT47-57 alone) (3mg/kg/day) showed improved fractional shortening (from 21% to 35%) compared to control (TAT47-57 alone). Formalin-fixed mid-ventricle tissue sections stained with picrosirius red, hematoxylin-eosin and toluidine blue dyes exhibited a 150% decrease in collagen deposition, a two-fold decrease in inflammation and a 30% reduction in mast cell degranulation, respectively, in rat hearts treated with the selective PKCβII inhibitor. Further, a 90% decrease in active TGFβ1 and a significant reduction in SMAD2/3 phosphorylation indicated that the selective inhibition of PKCβII attenuates cardiac remodeling mediated by the TGF-SMAD signaling pathway. Therefore, sustained selective inhibition of PKCβII in a post-MI HF rat model improves cardiac function and is associated with inhibition of pathological myocardial remodeling.
Protein kinase; PKCβII inhibitor peptide; cardiac remodeling; heart failure; myocardial infarction; mast cells, myocardial fibrosis; inflammation
Protein kinase C βII (PKCβII) levels increase in the myocardium of patients with end-stage heart failure (HF). Also targeted overexpression of PKCβII in the myocardium of mice leads to dilated cardiomyopathy associated with inflammation, fibrosis and myocardial dysfunction. These reports suggest a deleterious role of PKCβII in HF development. Using a post-myocardial infarction (MI) model of HF in rats, we determined the benefit of chronic inhibition of PKCβII on the progression of HF over a period of 6 weeks after the onset of symptoms and the cellular basis for these effects. Four weeks after MI, rats with HF signs that were treated for 6 weeks with the PKCβII selective inhibitor (βIIV5-3 conjugated to TAT47–57 carrier peptide) (3 mg/kg/day) showed improved fractional shortening (from 21% to 35%) compared to control (TAT47–57 carrier peptide alone). Formalin-fixed mid-ventricle tissue sections stained with picrosirius red, haematoxylin and eosin and toluidine blue dyes exhibited a 150% decrease in collagen deposition, a two-fold decrease in inflammation and a 30% reduction in mast cell degranulation, respectively, in rat hearts treated with the selective PKCβII inhibitor. Further, a 90% decrease in active TGFβ1 and a significant reduction in SMAD2/3 phosphorylation indicated that the selective inhibition of PKCβII attenuates cardiac remodelling mediated by the TGF-SMAD signalling pathway. Therefore, sustained selective inhibition of PKCβII in a post-MI HF rat model improves cardiac function and is associated with inhibition of pathological myocardial remodelling.
protein kinase; PKCβII inhibitor peptide; cardiac remodelling; heart failure; myocardial infarction; mast cells, myocardial fibrosis; inflammation
The aim of this study was to examine the endothelial distribution and activity of selected PKC isoforms in coronary vessels with respect to their functional impact on endothelial permeability under the experimental conditions relevant to diabetes.
Methods and Results
En face immunohistochemistry demonstrated a significant increase of PKCβII and decrease of PKCδ expression in coronary arterial endothelium of Zucker diabetic rats. To test whether changes in PKC expression alter endothelial barrier properties, we measured the transcellular electric resistance in human coronary microvascular endothelial monolayers and found that either PKCβII overexpression or PKCδ inhibition disrupted the cell–cell adhesive barrier. Three-dimensional fluorescence microscopy revealed that hyperpermeability was caused by altered PKC activity in association with distinct translocation of PKCβII to the cell–cell junction and PKCδ localization to the cytosol. Further analyses in fractionated endothelial lysates confirmed the differential redistribution of these isozymes. Additionally, FRET analysis of PKC subcellular dynamics demonstrated a high PKCβII activity at the cell surface and junction, whereas PKCδ activity is concentrated in intracellular membrane organelles.
Taken together, these data suggest that PKCβII and PKCδ counter-regulate coronary endothelial barrier properties by targeting distinctive subcellular sites. Imbalanced PKCβII/PKCδ expression and activity may contribute to endothelial hyperpermeability and coronary dysfunction in diabetes.
diabetes; inflammation; permeability; protein kinase; FRET
Serine/threonine protein kinase C βII isoform (PKCβII) or the pain receptor transient receptor potential vanilloid 1 (TRPV1) have been separately implicated in mediating heat hyperalgesia during inflammation or diabetic neuropathy. However, detailed information on the role of PKC βII in nociceptive signaling mediated by TRPV1 is lacking. This study presents evidence for activation and translocation of the PKC βII isoform as a signaling event in nociception mediated by activation of TRPV1 by capsaicin. We show that capsaicin induces translocation of cytosolic PKCβII isoform fused with enhanced green fluorescence protein (PKCβII-EGFP) in dorsal root ganglion (DRG) neurons. We also show capsaicin-induced translocation in Chinese Hamster Ovarian (CHO) cells co-transfected with TRPV1 and PKCβII-EGFP, but not in CHO cells expressing PKCβII-EGFP alone. By contrast, the PKC activator phorbol-12-myristate-13-acetate (PMA) induced translocation of PKCβII-EGFP which was sustained and independent of calcium or TRPV1. In addition PMA-induced sensitization of TRPV1 to capsaicin response in DRG neurons was attenuated by PKCβII blocker CGP 53353. Capsaicin response via TRPV1 in the DRG neurons was confirmed by TRPV1 antagonist AMG 9810. These results suggested a novel and potential signaling link between PKCβII and TRPV1. These cell culture models provide a platform for investigating mechanisms of painful neuropathies mediated by nociceptors expressing the pain sensing gene TRPV1, and its regulation by the PKC isoform PKCβII.
pain; protein kinase C; transient receptor potential vanilloid-1; real-time translocation; dorsal root ganglion neurons; nociceptive signaling
PURPOSE: Angiogenesis plays an important role in pancreas cancer pathobiology. Pancreatic tumor cells secrete vascular endothelial growth factor (VEGF), activating endothelial cell protein kinase C beta (PKCβ) that phosphorylates GSK3β to suppress apoptosis and promote endothelial cell proliferation and microvessel formation. We used Enzastaurin (Enz) to test the hypothesis that inhibition of PKCβ results in radiosensitization of endothelial cells in culture and in vivo. MATERIALS/METHODS: We measured PKCβ phosphorylation, VEGF pathway signaling, colony formation, and capillary sprout formation in primary human dermal microvessel endothelial cells (HDMECs) after Enz or radiation (RT) treatment. Microvessel density and tumor volume of human pancreatic cancer xenografts in nude mice were measured after treatment with Enz, RT, or both. RESULTS: Enz inhibited PKCβ and radiosensitized HDMEC with an enhancement ratio of 1.31 ± 0.05. Enz combined with RT reduced HDMEC capillary sprouting to a greater extent than either agent alone. Enz prevented radiation-induced GSK3β phosphorylation of serine 9 while having no direct effect on VEGFR phosphorylation. Treatment of xenografts with Enz and radiation produced greater reductions in microvessel density than either treatment alone. The reduction in microvessel density corresponded with increased tumor growth delay. CONCLUSIONS: Enz-induced PKCβ inhibition radiosensitizes human endothelial cells and enhances the antiangiogenic effects of RT. The combination of Enz and RT reduced microvessel density and resulted in increased growth delay in pancreatic cancer xenografts, without increase in toxicity. These results provide the rationale for combining PKCβ inhibition with radiation and further investigating such regimens in pancreatic cancer.
Phosphorylation of the adaptor protein p66shc is essential for p66shc-mediated oxidative stress. We investigated the role of the reducing protein/DNA repair enzyme apurinic/apyrimidinic endonuclease1 (APE1) in modulating protein kinase CβII (PKCβII)-mediated p66shc phosphorylation in cultured endothelial cells and PKC-mediated vasoconstriction of arteries.
Methods and results
Oxidized low-density lipoprotein (oxLDL)induced p66shc phosphorylation at serine 36 residue and PKCβII phosphorylation in mouse endothelial cells. Adenoviral overexpression of APE1 resulted in reduction of oxLDL-induced p66shc and PKCβII phosphorylation. Phorbol 12-myristate 13-acetate (PMA), which stimulates PKCs, induced p66shc phosphorylation and this was inhibited by a selective PKCβII inhibitor. Adenoviral overexpression of PKCβII also increased p66shc phosphorylation. Overexpression of APE1 suppressed PMA-induced p66shc phosphorylation. Moreover, PMA-induced p66shc phosphorylation was augmented in cells in which APE1 was knocked down. PMA increased cytoplasmic APE1 expression, compared with the basal condition, suggesting the role of cytoplasmic APE1 against p66shc phosphorylation. Finally, vasoconstriction induced by phorbol-12,13, dibutylrate, another PKC agonist, was partially inhibited by transduction of Tat-APE1 into arteries.
APE1 suppresses oxLDL-induced p66shc activation in endothelial cells by inhibiting PKCβII-mediated serine phosphorylation of p66shc, and mitigates vasoconstriction induced by activation of PKC.
p66shc; Apurinic/apyrimidinic endonuclease1; Oxidized LDL; Protein kinase C; Endothelial cells
Coordinated translation initiation is coupled with cell cycle progression and cell growth, whereas excessive ribosome biogenesis and translation initiation often lead to tumor transformation and survival. Hepatocellular carcinoma (HCC) is among the most common and aggressive cancers worldwide and generally displays inherently high resistance to chemotherapeutic drugs. We found that RACK1, the receptor for activated C-kinase 1, was highly expressed in normal liver and frequently upregulated in HCC. Aberrant expression of RACK1 contributed to in vitro chemoresistance as well as in vivo tumor growth of HCC. These effects depended on ribosome localization of RACK1. Ribosomal RACK1 coupled with PKCβII to promote the phosphorylation of eukaryotic initiation factor 4E (eIF4E), which led to preferential translation of the potent factors involved in growth and survival. Inhibition of PKCβII or depletion of eIF4E abolished RACK1-mediated chemotherapy resistance of HCC in vitro. Our results imply that RACK1 may function as an internal factor involved in the growth and survival of HCC and suggest that targeting RACK1 may be an efficacious strategy for HCC treatment.
Accelerated atherosclerosis is the leading cause of morbidity and mortality in diabetic patients. Hyperglycemia is a recognized independent risk factor for heightened atherogenesis in diabetes mellitus (DM). However, our understanding of the mechanisms underlying glucose damage to the vasculature remains incomplete.
High glucose and hyperglycemia reduced upregulation of the NF-κB inhibitory and atheroprotective protein A20 in human coronary endothelial (EC) and smooth muscle cell (SMC) cultures challenged with Tumor Necrosis Factor alpha (TNF), aortae of diabetic mice following Lipopolysaccharide (LPS) injection used as an inflammatory insult and in failed vein-grafts of diabetic patients. Decreased vascular expression of A20 did not relate to defective transcription, as A20 mRNA levels were similar or even higher in EC/SMC cultured in high glucose, in vessels of diabetic C57BL/6 and FBV/N mice, and in failed vein grafts of diabetic patients, when compared to controls. Rather, decreased A20 expression correlated with post-translational O-Glucosamine-N-Acetylation (O-GlcNAcylation) and ubiquitination of A20, targeting it for proteasomal degradation. Restoring A20 levels by inhibiting O-GlcNAcylation, blocking proteasome activity, or overexpressing A20, blocked upregulation of the receptor for advanced glycation end-products (RAGE) and phosphorylation of PKCβII, two prime atherogenic signals triggered by high glucose in EC/SMC. A20 gene transfer to the aortic arch of diabetic ApoE null mice that develop accelerated atherosclerosis, attenuated vascular expression of RAGE and phospho-PKCβII, significantly reducing atherosclerosis.
High glucose/hyperglycemia regulate vascular A20 expression via O-GlcNAcylation-dependent ubiquitination and proteasomal degradation. This could be key to the pathogenesis of accelerated atherosclerosis in diabetes.
mTORC2 has been shown to be involved in cytoskeletal regulation, but the mechanisms by which this takes place are poorly understood. This study shows that PKCβII is specifically required for mTORC2-dependent activation of adenylyl cyclase 9 and back retraction during neutrophil chemotaxis to chemoattractants.
Chemotaxis is a process by which cells polarize and move up a chemical gradient through the spatiotemporal regulation of actin assembly and actomyosin contractility, which ultimately control front protrusions and back retractions. We previously demonstrated that in neutrophils, mammalian target of rapamycin complex 2 (mTORC2) is required for chemoattractant-mediated activation of adenylyl cyclase 9 (AC9), which converts ATP into cAMP and regulates back contraction through MyoII phosphorylation. Here we study the mechanism by which mTORC2 regulates neutrophil chemotaxis and AC9 activity. We show that inhibition of protein kinase CβII (PKCβII) by CPG53353 or short hairpin RNA knockdown severely inhibits chemoattractant-induced cAMP synthesis and chemotaxis in neutrophils. Remarkably, PKCβII-inhibited cells exhibit specific and severe tail retraction defects. In response to chemoattractant stimulation, phosphorylated PKCβII, but not PKCα, is transiently translocated to the plasma membrane, where it phosphorylates and activates AC9. mTORC2-mediated PKCβII phosphorylation on its turn motif, but not its hydrophobic motif, is required for membrane translocation of PKCβII. Inhibition of mTORC2 activity by Rictor knockdown not only dramatically decreases PKCβII activity, but it also strongly inhibits membrane translocation of PKCβII. Together our findings show that PKCβII is specifically required for mTORC2-dependent AC9 activation and back retraction during neutrophil chemotaxis.
Functional adipocyte glucose disposal is a key component of global glucose homeostasis. PKCβII is involved in rat skeletal muscle cell ISGT. Western blot analysis and Real-Time PCR revealed 3T3-L1 cells developmentally regulated PKCβ splicing such that PKCβI was downregulated and PKCβII was upregulated during the course of differentiation. An initial glucose uptake screen using PKC inhibitor LY379196 pointed to a PKC isozyme other than PKCζ mediating 3T3-L1 adipocyte ISGT. Subsequent use of PKCβII inhibitor CGP53353 pointed to a role for PKCβII in ISGT. Western blot analysis showed that CGP53353 specifically inhibited phosphorylation of PKCβII Serine 660. Subcellular fractionation and immunofluorescence demonstrated that PKCβII regulates GLUT4 translocation. Further western blot, immunofluorescence and co-immunoprecipitation analysis reveal that PKCβII inhibition does not affect mTORC2 activity yet abrogates phosphorylation of Akt Serine 473. PKCβII regulates GLUT4 translocation by regulating Akt phosphorylation and thus activity.
PKCβII; GLUT4; Akt; mTORC2
The centrosome is important for microtubule organization and cell cycle progression in animal cells. Recently, mutations in the centrosomal protein, pericentrin, have been linked to human microcephalic osteodysplastic primordial dwarfism (MOPD II), a rare genetic disease characterized by severe growth retardation and early onset of type 2 diabetes among other clinical manifestations. While the link between centrosomal and cell cycle defects may account for growth deficiencies, the mechanism linking pericentrin mutations with dysregulated glucose homeostasis and pre-pubertal onset of diabetes is unknown. In this report we observed abundant expression of pericentrin in quiescent pancreatic β-cells of normal animals which led us to hypothesize that pericentrin may have a critical function in β-cells distinct from its known role in regulating cell cycle progression. In addition to the typical centrosome localization, pericentrin was also enriched with secretory vesicles in the cytoplasm. Pericentrin overexpression in β-cells resulted in aggregation of insulin-containing secretory vesicles with cytoplasmic, but not centrosomal, pericentriolar material and an increase in total levels of intracellular insulin. RNAi- mediated silencing of pericentrin in secretory β-cells caused dysregulated secretory vesicle hypersecretion of insulin into the media. Together, these data suggest that pericentrin may regulate the intracellular distribution and secretion of insulin. Mice transplanted with pericentrin-depleted islets exhibited abnormal fasting hypoglycemia and inability to regulate blood glucose normally during a glucose challenge, which is consistent with our in vitro data. This previously unrecognized function for a centrosomal protein to mediate vesicle docking in secretory endocrine cells emphasizes the adaptability of these scaffolding proteins to regulate diverse cellular processes and identifies a novel target for modulating regulated protein secretion in disorders such as diabetes.
Protein kinase C beta (PKCβ) expression in breast cancer is associated with a more aggressive tumor phenotype, yet the mechanism for how PKCβ is pro-tumorigenic in this disease is still unclear. Interestingly, while it is known that PKCβ mediates angiogenesis, immunity, fibroblast function and adipogenesis, all components of the mammary tumor microenvironment (TME), no study to date has investigated whether stromal PKCβ is functionally relevant in breast cancer. Herein, we evaluate mouse mammary tumor virus–polyoma middle T-antigen (MMTV–PyMT) induced mammary tumorigenesis in the presence and absence of PKCβ. We utilize two model systems: one where PKCβ is deleted in both the epithelial and stromal compartments to test the global requirement for PKCβ on tumor formation, and second, where PKCβ is deleted only in the stromal compartment to test its role in the TME. MMTV–PyMT mice globally lacking PKCβ live longer and develop smaller tumors with decreased proliferation and decreased macrophage infiltration. Similarly, when PKCβ is null exclusively in the stroma, PyMT-driven B6 cells form smaller tumors with diminished collagen deposition. These experiments reveal for the first time a tumor promoting role for stromal PKCβ in MMTV–PyMT tumorigenesis. In corroboration with these results, PKCβ mRNA (Prkcb) is increased in fibroblasts isolated from MMTV–PyMT tumors. These data were confirmed in a breast cancer patient cohort. Combined these data suggest the continued investigation of PKCβ in the mammary TME is necessary to elucidate how to effectively target this signaling pathway in breast cancer.
protein kinase C beta; breast cancer; mammary neoplasms (experimental); tumor microenvironment; stroma; fibroblasts
Insulin stimulates phosphorylation cascades, including phosphatidylinositol-3-kinase (PI3K), phosphatidylinositol-dependent kinase (PDK1), Akt, and protein kinase C (PKC). Myristoylated alanine-rich C-kinase substrate (MARCKS), a PKCβII substrate, could link the effects of insulin to insulin-stimulated glucose transport (ISGT) via phosphorylation of its effector domain since MARCKS has a role in cytoskeletal rearrangements.
We examined phosphoPKCβII after insulin treatment of L6 myocytes, and cytosolic and membrane phosphoMARCKS, MARCKS and phospholipase D1 in cells pretreated with LY294002 (PI3K inhibitor), CG53353 (PKCβII inhibitor) or W13 (calmodulin inhibitor), PI3K, PKCβII and calmodulin inhibitors, respectively, before insulin treatment, using western blots. ISGT was examined after cells had been treated with inhibitors, small inhibitory RNA (siRNA) for MARCKS, or transfection with MARCKS mutated at a PKC site. MARCKS, PKCβII, GLUT4 and insulin receptor were immunoblotted in subcellular fractions with F-actin antibody immunoprecipitates to demonstrate changes following insulin treatment. GLUT4 membrane insertion was followed after insulin with or without CG53353.
Insulin increased phosphoPKCβII(Ser660 and Thr641); LY294002 blocked this, indicating its activation by PI3K. Insulin treatment increased cytosolic phosphoMARCKS, decreased membrane MARCKS and increased membrane phospholipase D1 (PLD1), a protein regulating glucose transporter vesicle fusion resulted. PhosphoMARCKS was attenuated by CG53353 or MARCKS siRNA. MARCKS siRNA blocked ISGT. Association of PKCβII and GLUT4 with membrane F-actin was enhanced by insulin, as was that of cytosolic and membrane MARCKS. ISGT was attenuated in myocytes transfected with mutated MARCKS (Ser152Ala), whereas overproduction of wild-type MARCKS enhanced ISGT. CG53353 blocked insertion of GLUT4 into membranes of insulin treated cells.
The results suggest that PKCβII is involved in mediating downstream steps of ISGT through MARCKS phosphorylation and cytoskeletal remodelling.
F-actin; Glucose transporter 4; Insulin-stimulated glucose uptake; L6 myocytes; MARCKS; Phospholipase D1; PKCβ
The transcription factor RelB is required for proper development and function of dendritic cells (DCs), and its expression is upregulated early during differentiation from a variety of progenitors. We explored this mechanism of upregulation in the KG1 cell line model of a DC progenitor and in the differentiation-resistant KG1a subline. RelB expression is relatively higher in untreated KG1a cells but is upregulated only during differentiation of KG1 by an early enhancement of transcriptional elongation, followed by an increase in transcription initiation. Restoration of protein kinase CβII (PKCβII) expression in KG1a cells allows them to differentiate into DCs. We show that PKCβII also downregulated constitutive expression of NF-κB in KG1a-transfected cells and restores the upregulation of RelB during differentiation by increased transcriptional initiation and elongation. The two mechanisms are independent and sensitive to PKC signaling levels. Conversely, RelB upregulation was inhibited in primary human monocytes where PKCβII expression was knocked down by small interfering RNA targeting. Altogether, the data show that RelB expression during DC differentiation is controlled by PKCβII-mediated regulation of transcriptional initiation and elongation.
Rapid repair of epithelial wounds is essential for intestinal homeostasis, and involves cell proliferation and migration, which in turn are mediated by multiple cellular signaling events including PKC activation. PKC isoforms have been implicated in regulating cell proliferation and migration, however, the role of PKCs in intestinal epithelial cell (IEC) wound healing is still not completely understood. In the current work we used phorbol 12-myristate 13-acetate (PMA), a well recognized agonist of classical and non-conventional PKC subfamilies to investigate the effect of PKC activation on IEC wound healing. We found that PMA treatment of wounded IEC monolayers resulted in 5.8±0.7-fold increase in wound closure after 24 hours. The PMA effect was specifically mediated by PKCβII, as its inhibition significantly diminished the PMA-induced increase in wound closure. Furthermore, we show that the PKCβII-mediated increase in IEC wound closure after PMA stimulation was mediated by increased cell spreading/cell migration but not proliferation. Cell migration was mediated by PKCβII dependent actin cytoskeleton reorganization, enhanced formation of lamellipodial extrusions at the leading edge and increased activation of the focal adhesion protein, paxillin. These findings support a role for PKCβII in IEC wound repair and further demonstrate the ability of epithelial cells to migrate as a sheet thereby efficiently covering denuded surfaces to recover the intestinal epithelial barrier.
Many viruses take advantage of receptor-mediated endocytosis in order to enter target cells. We have utilized influenza virus and Semliki Forest virus (SFV) to define a role for protein kinase C βII (PKCβII) in endocytic trafficking. We show that specific PKC inhibitors prevent influenza virus infection, suggesting a role for classical isoforms of PKC. We also examined virus entry in cells overexpressing dominant-negative forms of PKCα and -β. Cells expressing a phosphorylation-deficient form of PKCβII (T500V), but not an equivalent mutant form of PKCα, inhibited successful influenza virus entry—with the virus accumulating in late endosomes. SFV, however, believed to enter cells from the early endosome, was unaffected by PKCβII T500V expression. We also examined the trafficking of two cellular ligands, transferrin and epidermal growth factor (EGF). PKCβII T500V expression specifically blocked EGF receptor trafficking and degradation, without affecting transferrin receptor recycling. As with influenza virus, in PKCβII kinase-dead cells, EGF receptor was trapped in a late endosome compartment. Our findings suggest that PKCβII is an important regulator of a late endosomal sorting event needed for influenza virus entry and infection.
Significant up-regulation of the protein kinase CβII (PKCβII) develops during heart failure and yet divergent functional outcomes are reported in animal models. The goal here is to investigate PKCβII modulation of contractile function and gain insights into downstream targets in adult cardiac myocytes. Increased PKCβII protein expression and phosphorylation developed after gene transfer into adult myocytes while expression remained undetectable in controls. The PKCβII was distributed in a perinuclear pattern and this expression resulted in diminished rates and amplitude of shortening and re-lengthening compared to controls and myocytes expressing dominant negative PKCβII (PKCβDN). Similar decreases were observed in the Ca2+ transient and the Ca2+ decay rate slowed in response to caffeine in PKCβII-expressing myocytes. Parallel phosphorylation studies indicated PKCβII targets phosphatase activity to reduce phospholamban (PLB) phosphorylation at residue Thr17 (pThr17-PLB). The PKCβ inhibitor, LY379196 (LY) restored pThr17-PLB to control levels. In contrast, myofilament protein phosphorylation was enhanced by PKCβII expression, and individually, LY and the phosphatase inhibitor, calyculin A each failed to block this response. Further work showed PKCβII increased Ca2+- activated, calmodulin-dependent kinase IIδ (CaMKIIδ) expression and enhanced both CaMKIIδ and protein kinase D (PKD) phosphorylation. Phosphorylation of both signaling targets also was resistant to acute inhibition by LY. These later results provide evidence PKCβII modulates contractile function via intermediate downstream pathway(s) in cardiac myocytes.
Protein kinase C; cardiac myocyte; contractile function; gene transfer
Background: Diabetes is associated with an increased risk of cardiac microvascular disease. The mechanisms by which this damage occurs are unknown. However, research suggests that signaling through the sphingosine-1-phosphates receptor 1 and 3 (S1P1/3) by FTY720, a sphiongolipid drug that is structually similar to SIP, may play a role in the treatment on cardiac microvascular dysfunction in diabetes. We hypothesized that FTY720 might exert the cardioprotective effects of S1P1 and S1P3 viaprotein kinase C-beta (PKCβ II) signaling pathway.
Methodology/Principal Findings: Transthoracic echocardiography was performed to detect the change of cardiac function. Scanning and transmission electron microscope with lanthanum tracer were used to determine microvascular ultrastructure and permeability in vivo. Apoptosis was detected by TUNEL and CD31 dual labeling in paraffin-embedded sections. Laser capture miscrodissection was used to assess cardiac micovascular endothelial cells (CMECs) in vivo. RT-PCR and Western blot analysis were used to determine the mRNA levels and protein expression of S1P1, S1P3, and PKCβ II. In the diabetic rats vs. controls, cardiac capillaries showed significantly higher density; CD31 positive endothelial cells were significantly reduced; the apoptosis index of cardiac endothlial cells was significantly higher. And FTY720 could increase the expressional level of S1P1 and boost S1P3 trasnslocation from membrane to nuclear, then ameliorate cardiac microvascular barrier impairment and pathologic angiogenesis induced by diabetes. In addition, overexpression of PKCβ II significantly decreased the protective effect of FTY720.
Conclusions: Our study represents that the deregulation of S1P1 and S1P3 is an important signalresponsible for cardiac microvascular dysfunction in diabetes. FTY720 might be competent to serve as a potential therapeutic approach for diabetic heart disease through ameliorating cardiac microvascular barrier impairment and pathologic angiogenesis, which might be partly dependent on PKCβII-mediated signaling pathway.