PKCα Protein Research Articles

Abstract 189: Protein kinase C alpha suppresses AKT activation in endometrial cancer

Abstract Endometrial cancer (EC) is the most common gynecological malignancy and the fourth leading cancer in women in the United States. Hyperactivation of the PI3K/AKT pathway is observed in ∼90% of EC cases and this pathway is the most prominent driver of endometrial carcinogenesis. Mutations in multiple PI3K/AKT pathway components (including PTEN, PIK3CA, PIK3R1, AKT) often coexist in EC, indicating that EC development requires full, unopposed activation of PI3K/AKT signaling. In this study, we identified PKCα as a negative regulator of AKT activation that suppresses endometrial carcinogenesis. Our analysis revealed that PKCα protein and mRNA are reduced or lost in 50% of EC cell lines and ∼60% of human ECs. PKCα deficiency was also observed in hyperplastic endometrial lesions in murine models with allelic knock-in of mutant Pten (ptenΔ4-5/+, ptenC124R/+ and ptenC129E/+) or endometrial-specific Pten deletion (ptenpr-/-, ptenltf-/-), indicating that loss of PKCα is an early event in endometrial carcinogenesis. Mechanistic analysis has further revealed that loss of PKCα is independent of changes in PI3K/AKT signaling; however, the enzyme acts as a potent negative regulator of the PI3K/AKT pathway in EC cells. PKC agonists reduced AKT phosphorylation/activity in EC cells that retain PKCα, via a PP2A-dependent mechanism. Conversely, PKCα knockdown led to increased AKT phosphorylation. Notably, while PKC agonists did not affect AKT in EC cells with loss of PKCα, exogenous expression of the enzyme in these cells restored the inhibitory effect of the agonists on PI3K/AKT signaling. Collectively these data indicate that PKCα has a restraining effect on PI3K/AKT signaling in EC cells, providing a basis for its loss during endometrial tumor progression. Physiological relevance of these findings was established using cell lines, patient samples and mouse models. PKCα restoration in human EC cells decreased expression of oncogenes such as cyclin D1 and Id1 and abolished their anchorage-independent growth, supporting a tumor suppressive role for the isozyme in this tissue. Analysis of PKCα expression in patient specimens determined that loss of PKCα expression correlates with high grade disease. Finally, in mice heterozygous for mutant Pten, PKCα knockout led to a 3-fold increase in endometrial tumor burden at three months, confirming a role of PKCα as a tumor suppressor in the endometrium. Taken together, our results provide evidence that PKCα 1) negatively regulates AKT signaling and the expression of important oncogenes in EC cells; and 2) functions as tumor suppressor in the endometrium whose loss is associated with disease progression. Thus, PKCα signaling represents a promising biomarker for risk stratification in early stage disease and may provide insight into therapeutic strategies for late stage disease. Supported by NIH grants CA036727, CA016056, and DK60632. Citation Format: Alice H. Hsu, Kathryn J. Curry, Kang-Sup Shim, Peter Frederick, Carl D. Morrison, Baojiang Chen, Subodh M. Lele, Takiko Daikoku, Sudhansu K. Dey, Gustavo Leone, Adrian R. Black, Jennifer D. Black. Protein kinase C alpha suppresses AKT activation in endometrial cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 189.

Low expression of PKCα and high expression of KRAS predict poor prognosis in patients with colorectal cancer.

The current study aimed to determine the association between protein kinase Cα (PKCα) and Kirsten rat sarcoma viral oncogene homolog (KRAS) expression and the response to folinic acid, 5-fluorouracil and oxaliplatin (FOLFOX regimen) in patients with colorectal cancer (CRC). The protein levels of PKCα and KRAS were analyzed by immunohistochemistry in tissue samples from patients with CRC and in non-cancerous tissues, including 152 cases of colorectal adenocarcinoma, 30 cases of colorectal adenoma and 20 normal colonic mucosa samples. The association between PKCα and KRAS expression and clinicopathological features was analyzed. The rates of positive PKCα protein expression in patients with poorly, moderately and well-differentiated adenocarcinoma were 16.7% (6/36), 40.0% (24/60), and 57.1% (32/56), respectively (P<0.013). The rate of positive KRAS expression in CRC patients was significantly higher than in patients with colon adenoma and normal colon mucosa (P<0.001). Expression levels of KRAS were associated with the degree of differentiation of CRC (P<0.001). Expression of PKCα was negatively correlated with KRAS expression in CRC tissues. The mean progression-free survival (PFS) times in patients with high and low expression of PKCα were 43.9 and 38.8 months, respectively (P<0.001). The mean PFS times were 38.5 and 45.5 months in patients with high and low expression of KRAS, respectively (P=0.001). In conclusion, low PKCα and high KRAS expression predicted relatively poor prognosis in patients with CRC.

Expression of cell cycle proteins in cortical neurons-Correlation with glutamate-induced neurotoxicity.

Under physiological conditions, upon differentiation neurons become irreversibly post-mitotic by down-regulating cell cycle progression. However, recent studies have provided evidence that aberrant expression of cell cycle related proteins; especially cyclins, cyclin-dependent kinases, and their inhibitors are accompanied by programmed cell death in neurons. This abnormal phenotype has been postulated to contribute to the pathophysiology of different neurodegenerative diseases. Glutamate is the most abundant and major excitatory neurotransmitter in the central nervous system but high concentrations are reported to be involved in the pathology of many neurodegenerative diseases. The mechanisms of glutamate neurotoxicity have been intensively investigated over the past decades but still remain not fully understood. In this study, we hypothesized that aberrant regulation of cell cycle proteins may be involved in glutamate-induced neurotoxicity in primary cultures of rat cortical neurons. The results have shown that, glutamate treatment caused apoptosis by inducing active caspase-3 and p53 expression. Together with this, an increase in cyclin D1 and Cdk4 protein levels, localization of cyclin D1 to nucleus, and a decrease in the cell cycle inhibitor p27 were observed. After glutamate treatment we also detected up-regulation of protein kinase C-α (PKC-α) protein expression. Altogether, the data reported in this study show for the first time that glutamate in cortical neurons changes simultaneously the expression levels of a number of key cell cycle proteins and cell homeostasis regulators. © 2016 BioFactors, 42(4):358-367, 2016.

Inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ signaling mediates delayed myogenesis in Duchenne muscular dystrophy fetal muscle

Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disorder characterized by muscle wasting and premature death. The defective gene is dystrophin, a structural protein, absence of which causes membrane fragility and myofiber necrosis. Several lines of evidence showed that in adult DMD patients dystrophin is involved in signaling pathways that regulate calcium homeostasis and differentiation programs. However, secondary aspects of the disease, such as inflammation and fibrosis development, might represent a bias in the analysis. Because fetal muscle is not influenced by gravity and does not suffer from mechanical load and/or inflammation, we investigated 12-week-old fetal DMD skeletal muscles, highlighting for the first time early alterations in signaling pathways mediated by the absence of dystrophin itself. We found that PLC/IP3/IP3R/Ryr1/Ca(2+) signaling is widely active in fetal DMD skeletal muscles and, through the calcium-dependent PKCα protein, exerts a fundamental regulatory role in delaying myogenesis and in myofiber commitment. These data provide new insights into the origin of DMD pathology during muscle development.

Inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ signaling mediates delayed myogenesis in Duchenne muscular dystrophy fetal muscle.

Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disorder characterized by muscle wasting and premature death. The defective gene is dystrophin, a structural protein, absence of which causes membrane fragility and myofiber necrosis. Several lines of evidence showed that in adult DMD patients dystrophin is involved in signaling pathways that regulate calcium homeostasis and differentiation programs. However, secondary aspects of the disease, such as inflammation and fibrosis development, might represent a bias in the analysis. Because fetal muscle is not influenced by gravity and does not suffer from mechanical load and/or inflammation, we investigated 12-week-old fetal DMD skeletal muscles, highlighting for the first time early alterations in signaling pathways mediated by the absence of dystrophin itself. We found that PLC/IP3/IP3R/Ryr1/Ca(2+) signaling is widely active in fetal DMD skeletal muscles and, through the calcium-dependent PKCα protein, exerts a fundamental regulatory role in delaying myogenesis and in myofiber commitment. These data provide new insights into the origin of DMD pathology during muscle development.

Expression of Protein Kinase C Isoforms in Pancreatic Islets and Liver of Male Goto-Kakizaki Rats, a Model of Type 2 Diabetes.

Protein kinase C (PKC) is a family of protein kinases controlling protein phosphorylation and playing important roles in the regulation of metabolism. We have investigated expression levels of PKC isoforms in pancreatic islets and liver of diabetic Goto-Kakizaki (GK) rats with and without insulin treatment to evaluate their association with glucose homeostasis. mRNA and protein expression levels of PKC isoforms were assessed in pancreatic islets and liver of Wistar rats and GK rats with or without insulin treatment. PKCα and PKCζ mRNA expressions were down-regulated in islets of GK compared with Wistar rats. PKCα and phosphorylated PKCα (p-PKCα) protein expressions were decreased in islets of GK compared with insulin-treated GK and Wistar rats. PKCζ protein expression in islets was reduced in GK and insulin-treated GK compared with Wistar rats, but p-PKCζ was decreased only in GK rats. Islet PKCε mRNA and protein expressions were lower in GK compared with insulin-treated GK and Wistar rats. In liver, PKCδ and PKCζ mRNA expressions were decreased in both GK and insulin-treated GK compared with Wistar rats. Hepatic PKCζ protein expression was diminished in both GK rats with and without insulin treatment compared with Wistar rats. Hepatic PKCε mRNA expression was down-regulated in insulin-treated GK compared with GK and Wistar rats. PKCα, PKCε, and p-PKCζ expressions were secondary to hyperglycaemia in GK rat islets. Hepatic PKCδ and PKCζ mRNA expressions were primarily linked to hyperglycaemia. Additionally, hepatic PKCε mRNA expression could be under control of insulin.

GABA(B) receptors couple to Gαq to mediate increases in voltage-dependent calcium current during development.

Metabotropic GABA(B) receptors are known to modulate the activity of voltage-dependent calcium channels. Previously, we have shown that GABA(B) receptors couple to a non-Gi/o G-protein to enhance calcium influx through L-type calcium channels by activating protein kinase C in neonatal rat hippocampal neurons. In this study, the components of this signaling pathway were investigated further. Gαq was knocked down using morpholino oligonucleotides prior to examining GABA(B) -mediated enhancement of calcium influx. When Gαq G-proteins were eliminated using morpholino-mediated knockdown, the enhancing effects of the GABA(B) receptor agonist baclofen (10 μM) on calcium current or entry were eliminated. These data suggest that GABA(B) receptors couple to Gαq to regulate calcium influx. Confocal imaging analysis illustrating colocalization of GABA(B) receptors with Gαq supports this hypothesis. Furthermore, baclofen treatment caused translocation of PKCα (protein kinase C α) but not PKCβ or PKCε, suggesting that it is the α isoform of PKC that mediates calcium current enhancement. Inhibition of calcium/calmodulin-dependent kinase II did not affect the baclofen-mediated enhancement of calcium levels. In summary, activation of GABA(B) receptors during development leads to increased calcium in a subset of neurons through Gαq signaling and PKCα activation without the involvement of calcium/calmodulin-dependent kinase II. Activation of GABA(B) receptors in the neonatal rat hippocampus enhances voltage-dependent calcium currents independently of Gi/o . In this study, knockdown of Gαq with morpholino oligonucleotides abolished enhancement of calcium influx and protein kinase Cα was activated by GABA(B) receptors. Therefore, we hypothesize that GABA(B) receptors couple to Gq to activate PKCα leading to enhancement of L-type calcium current.

Jatropha curcas Protein Concentrate Stimulates Insulin Signaling, Lipogenesis, Protein Synthesis and the PKCα Pathway in Rat Liver.

Jatropha curcas is an oil seed plant that belongs to the Euphorbiaceae family. Nontoxic genotypes have been reported in Mexico. The purpose of the present work was to evaluate the effect of a Mexican variety of J. curcas protein concentrate (JCP) on weight gain, biochemical parameters, and the expression of genes and proteins involved in insulin signaling, lipogenesis, cholesterol and protein synthesis in rats. The results demonstrated that short-term consumption of JCP increased serum glucose, insulin, triglycerides and cholesterol levels as well as the expression of transcription factors involved in lipogenesis and cholesterol synthesis (SREBP-1 and LXRα). Moreover, there was an increase in insulin signaling mediated by Akt phosphorylation and mTOR. JCP also increased PKCα protein abundance and the activation of downstream signaling pathway targets such as the AP1 and NF-κB transcription factors typically activated by phorbol esters. These results suggested that phorbol esters are present in JCP, and that they could be involved in the activation of PKC which may be responsible for the high insulin secretion and consequently the activation of insulin-dependent pathways. Our data suggest that this Mexican Jatropha variety contains toxic compounds that produce negative metabolic effects which require caution when using in the applications of Jatropha-based products in medicine and nutrition.

Abstract 4095: Protein kinase C alpha (PKCα) is a novel regulator of FOXC2 and p120-catenin in triple negative and endocrine resistant breast cancer

Abstract Loss of E-cadherin, a core member of the epithelial adherens junction (AJ), leads to the induction of the epithelial-mesenchymal transition (EMT), a phenomenon where epithelial cells de-differentiate towards a more motile and invasive mesenchymal cell type. Because of its stabilizing function in the AJ, p120-catenin has caught attention in the context of tumor development and progression. We previously reported that expression of protein kinase C alpha (PKCα) imparts a hormone-independent, and tamoxifen-resistant phenotype in T47D:A18/PKCα breast cancer cells. Our preliminary data strongly indicate a positive relationship between PKCα and FOXC2, a forkhead transcription factor shown to be a central mediator of the EMT program and tumor metastasis. Data mining from Oncomine database revealed a strong correlation between PRKCA and FOXC2 in basal A triple negative breast cancer (TNBC) cell lines. In this study, two representative cell lines, HCC1937 and HCC1143, were chosen to investigate the relationship between PKCα, FOXC2, and p120-catenin, and how their interplay alters migration and invasion of TNBC and endocrine resistant breast cancer. Cell lines (T47D:A18/PKCα, HCC1143, HCC1937) were maintained as previously described and according to ATCC guidelines. Expression of PKCα and FOXC2 were manipulated using siRNA targeting PKCα and FOXC2 respectively. Transcripts level of FOXC2 (FOXC2) and CTNND1(p120-catenin) were measured using quantitative PCR. PKCα, FOXC2, and p120-catenin protein expression was measured by Western blot using PKCα (GE-Healthcare, 1:200), FOXC2 (Abcam, 1:500), and p120-catenin (Cell Signaling, 1:500) antibodies respectively. All three cell lines were found to co-express high level of PKCα and FOXC2. Knockdown of PKCα resulted in a down-regulation of FOXC2 transcripts and protein. However, knockdown of FOXC2 did not alter expression of PKCα, indicating PKCα is an upstream regulator of FOXC2, and not vice versa. Downregulation of either PKCα or FOXC2 led to a rescue of p120-catenin, and not E-cadherin, transcripts. Re-expression of p120-catenin transcripts also resulted in re-expression of the protein. Knockdown of FOXC2 in T47D:A18/ PKCα cells led to a significant reduction in migratory and invasive potential of these cells, to the same extent as knockdown of PKCα For the first time, we are reporting PKCα as a novel regulator of FOXC2 in TNBC and endocrine-resistant breast cancer. We also identified p120-catenin as a potential target of PKCα and FOXC2. Down-regulation of p120-catenin by either PKCα or FOXC2 may be a critical event in the dissolution of the AJ, which facilitates migration of breast cancer cells. Citation Format: Thao ND Pham, Bethany Perez-White, Debra A. Tonetti. Protein kinase C alpha (PKCα) is a novel regulator of FOXC2 and p120-catenin in triple negative and endocrine resistant breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4095. doi:10.1158/1538-7445.AM2015-4095

Role of PKCα in electroacupuncture-induced reduction of acute kidney injury induced by endotoxic shock in rabbits: the relationship with Nrf2/HO-1 pathway

Objective To evaluate the role of protein kinase Ca (PKCα) in electroacupuncture (EA)-induced reduction of acute kidney injury (AKI) induced by endotoxic shock, and the relationship with nuclear factor E2-related factor 2/heme oxygenase-1 Nrf2/HO-1 pathway in rabbits. Methods Eighty healthy male New Zealand white rabbits, aged 2 months, weighing 1.5-2.0 kg, were randomly divided into 8 groups (n= 10 each) using a random number table: sham operation group (group S), group AKI, specific PKCα inhibitor chelerythrine + AKI group (group CHA), chelerythrine group (group Che), dimethyl sulfoxide (DMSO) group (group D), EA at acupoints + AKI group (group EA), EA at non-acupoints + AKI group (group SEA), and EA at acupoints + chelerythrine + AKI group (group CEA). Bilateral 30 min EA (disperse-dense wave, wave length 0.2-0.6 ms, frequency 2/15 Hz, intensity 1-2 mA) stimulation of Zusanli and Shenshu acupoints was performed once a day for 4 days before establishment of the model and during the process of establishment of the model in EA and CEA groups.In group SEA, EA was performed at the points 0.5 cm lateral to the acupoints of Zusanli and Shenshu with the same parameters.The animals were anesthetized with iv 20% urethane 5 ml/kg, tracheostomized and kept spontaneous breathing.Lipopolysaccharide 5 mg/kg (in 2 ml of normal saline) was injected via the auricular vein to establish the model of endotoxic shock-induced AKI in AKI, CHA, EA , SEA and CEA groups, while the equal volume of normal saline was given in S, Che and D groups.At 30 min before establishment of the model, chelerythrine 5 mg/kg (in 0.5 ml of 1% DMSO) was injected intravenously in CHA and CEA groups, the equal volume of chelerythrine was given in Che group, while the equal volume of DMSO was given in group D. At 6 h after lipopolysaccharide or normal saline injection, blood samples were taken from the internal carotid artery for determination of serum urea nitrogen (BUN) and creatinine (Cr) concentrations.The rabbits were then sacrificed by exsanguinations.The kidney specimens were removed for microscopic examination of pathologic changes which were scored and for determination of superoxide dismutase (SOD) activities, malondialdehyde (MDA) contents, and expression of PKCα protein and HO-1 protein, and expression of Nrf2 in nucleoprotein and total protein. Results Compared with group S, the serum BUN and Cr concentrations were significantly increased, MDA contents were increased, the activities of SOD were decreased, the kidney injury scores were increased, and the expression of PKCα protein, HO-1 protein, and Nrf2 in nucleoprotein and total protein was up-regulated in AKI, CHA, EA, SEA and CEA groups.Compared with group AKI, the serum BUN and Cr concentrations were significantly decreased, MDA contents were decreased, the activities of SOD were increased, the kidney injury scores were decreased, and the expression of PKCα protein, HO-1 protein, and Nrf2 in nucleoprotein and total protein was up-regulated in group EA, and the serum BUN and Cr concentrations were significantly increased, MDA contents were increased, the activities of SOD were decreased, the kidney injury scores were increased, and the expression of PKCα protein, HO-1 protein, and Nrf2 in nucleoprotein and total protein was down-regulated in CHA and CEA groups.The serum BUN and Cr concentrations were significantly higher, MDA contents were higher, the activities of SOD were lower, the kidney injury scores were higher, and the expression of PKCα protein, HO-1 protein, and Nrf2 in nucleoprotein and total protein was lower in group CEA than in group EA, and in CHA group than in CEA group. Conclusion PKCα mediates reduction of endotoxic shock-induced AKI by EA of Zusanli and Shenshu acupoints in rabbits, and the mechanism may be related to activation of Nrf2/HO-1. Key words: Electric stimulation therapy; Protein kinase C-alpha; NF-E2-related factor2; Heme oxygenase-1; Shock, septic; Acute kidney injury

Shifts in temperature within the physiologic range modify strand-specific expression of select human microRNAs

Previous studies have revealed that clinically relevant changes in temperature modify clinically relevant gene expression profiles through transcriptional regulation. Temperature dependence of post-transcriptional regulation, specifically, through expression of miRNAs has been less studied. We comprehensively analyzed the effect of 24 h exposure to 32°C or 39.5°C on miRNA expression profile in primary cultured human small airway epithelial cells (hSAECs) and its impact on expression of a targeted protein, protein kinase C α (PKCα). Using microarray, and solution hybridization-based nCounter assays, with confirmation by quantitative RT-PCR, we found significant temperature-dependent changes in expression level of only five mature human miRNAs, representing only 1% of detected miRNAs. Four of these five miRNAs are the less abundant passenger (star) strands. They exhibited a similar pattern of increased expression at 32°C and reduced expression at 39.5°C relative to 37°C. As PKCα mRNA has multiple potential binding sites for three of these miRNAs, we analyzed PKCα protein expression in HEK 293T cells and hSAECs. PKCα protein levels were lowest at 32°C and highest at 39.5°C and specific miRNA inhibitors reduced these effects. Finally, we analyzed cell-cycle progression in hSAECs and found 32°C cells exhibited the greatest G1 to S transition, a process known to be inhibited by PKCα, and the effect was mitigated by specific miRNA inhibitors. These results demonstrate that exposure to clinically relevant hypothermia or hyperthermia modifies expression of a narrow subset of miRNAs and impacts expression of at least one signaling protein involved in multiple important cellular processes.

Platelet derived growth factor receptor α-phospholipase Cγ1-protein kinase Cα signal transductionin in human lung cancer cell GLC-82 for combination of the drugs tumor necrosis factor-related apoptosis-inducing ligand and proteasome inhibitor MG132

Objective To tumor necrosis factor- related apoptosis- inducing ligand(TRAIL)combined with a protease inhibitor MG132 observed in human lung adenocarcinoma cell line GLC- 82 inhibition of proliferation and platelet derived growth factor receptor α(PDGFRα)- phospholipase Cγ1(PLCγ1)-protein kinase Cα(PKCα)signal transduction pathway. Methods Methyl thiazol tetrazolium(MTT)assay inhibition of TRAIL combined with a protease inhibitor MG132 on GLC- 82 cell growth detection; using flow cytometry to detect the effects of TRAIL combined with a protease inhibitor MG132 on GLC- 82 cell apoptosis, using SP immunohistochemical detection of protease inhibitors MG132 joint expression of TRAIL- associated protein PDGFRα, PLCγ1,PKCα's, Western blotting to determine the effect of protease inhibitors MG132 TRAIL combined for GLC- 82 cells PDGFRα, PLCγ1, PKCαprotein expression. Results MTT assay inhibition of TRAIL combined with a protease inhibitor MG132 on GLC- 82 cell growth detection. The inhibitory rates were(34.92±3.95)%,(33.17±1.78)% and(76.96±1.28)%, respectively; using flow cytometry to detect the effects of TRAIL combined with a protease inhibitor MG132 on GLC- 82 cell apoptosis. Apoptosis rates of the four group were 6.78%, 38.72%, 45.60% and 91.74%, respectively. Using SPimmunohistochemical detection of protease inhibitors MG132 joint expression of TRAIL- associated protein PDGFRα, PLCγ1, PKCα's, Western blotting to determine the effect of protease inhibitors MG132 TRAIL combined for GLC- 82 cells PDGFRα, PLCγ1, PKCα protein expression. Conclusion TRAIL combined protease inhibitor MG132 on human lung adenocarcinoma GLC- 82 cells significantly inhibited the growth, can reduce the level of GLC- 82 cells PDGFRα- PLCγ1- PKCα each protein signaling pathway, the incidence of lung cancer and PDGFRα- PLCγ1- PKCα enhanced signal transduction pathways are closely related. Key words: Tumor necrosis factor-related apoptosis-inducing ligand; MG132; Lung adenocarcinoma; Signal transduction

Protein kinase C isoforms α, δ and ε are differentially expressed in mouse ovaries at different stages of postnatal development.

BackgroundThe protein kinase C (PKC) is a family of serine/threonine kinases that consists of 12 different isoforms. Since PKC isoform expressions are known to be specific for different cell types and postnatal developmental stages, we aimed to determine immunolocalizations and protein expression levels of different PKC isoforms in pre-pubertal, pubertal and adult mouse ovaries.MethodsOvaries were obtained from postnatal day 1 (PND1) and PND7 of pre-pubertal, PND21 of pubertal and PND60 of adult mice. Immunolocalizations of PKCα, PKCδ and PKCε isoforms were determined and immunostainings in different cellular components of all follicular stages were evaluated by H-Score. PKCα, PKCδ and PKCε protein expression levels were determined by Western blot. The bands were quantified via ImageJ software. The data obtained from H-Score and ImageJ evaluations were analyzed by ANOVA statistical test.ResultsPKCα immunostainings were more intense in oocytes when compared to granulosa and theca cells at different follicular stages of all groups. The Western blot analysis revealed that PKCα expression was significantly higher in PND60 adult ovaries. Conversely, PKCδ immunostainings were more intense in granulosa cells. According to the Western blot analysis, PKCδ protein expression was also higher in PND60 and significantly lower in PND1 ovaries. PKCε immunostaining was more apparent in oocytes. PKCε protein expression was significantly higher in adult PND60 and pubertal PND21 ovaries when compared to pre-pubertal PND7 and PND1 ovaries. Interestingly, PKCε immunostaining was significantly higher in primordial follicles, though PKCα and PKCδ immunostainings were more apparent in larger follicles. PKCα immunostainings of corpora lutea (CL) were significantly higher when compared to follicles in PND60 ovaries.ConclusionsThis study demonstrates that PKCα, PKCδ and PKCε isoforms are differentially expressed in particular cellular components of pre-pubertal, pubertal and adult mouse ovarian follicles. Therefore, we suggest that each PKC isoform has unique functions that are controlled by gonadotropin dependent mechanisms during follicular growth, oocyte maturation, ovulation and luteinization.

PKC-α contributes to high NaCl-induced activation of NFAT5 (TonEBP/OREBP) through MAPK ERK1/2.

High NaCl in the renal medullary interstitial fluid powers the concentration of urine but can damage cells. The transcription factor nuclear factor of activated T cells 5 (NFAT5) activates the expression of osmoprotective genes. We studied whether PKC-α contributes to the activation of NFAT5. PKC-α protein abundance was greater in the renal medulla than in the cortex. Knockout of PKC-α reduced NFAT5 protein abundance and expression of its target genes in the inner medulla. In human embryonic kidney (HEK)-293 cells, high NaCl increased PKC-α activity, and small interfering RNA-mediated knockdown of PKC-α attenuated high NaCl-induced NFAT5 transcriptional activity. Expression of ERK1/2 protein and phosphorylation of ERK1/2 were higher in the renal inner medulla than in the cortex. Knockout of PKC-α decreased ERK1/2 phosphorylation in the inner medulla, as did knockdown of PKC-α in HEK-293 cells. Also, knockdown of ERK2 reduced high NaCl-dependent NFAT5 transcriptional activity in HEK-293 cells. Combined knockdown of PKC-α and ERK2 had no greater effect than knockdown of either alone. Knockdown of either PKC-α or ERK2 reduced the high NaCl-induced increase of NFAT5 transactivating activity. We have previously found that the high NaCl-induced increase of phosphorylation of Ser(591) on Src homology 2 domain-containing phosphatase 1 (SHP-1-S591-P) contributes to the activation of NFAT5 in cell culture, and here we found high levels of SHP-1-S591-P in the inner medulla. PKC-α has been previously shown to increase SHP-1-S591-P, which raised the possibility that PKC-α might be acting through SHP-1. However, we did not find that knockout of PKC-α in the renal medulla or knockdown in HEK-293 cells affected SHP-1-S591-P. We conclude that PKC-α contributes to high NaCl-dependent activation of NFAT5 through ERK1/2 but not through SHP-1-S591.

Abstract 4211: Protein kinase C alpha (PKCα) signaling in endometrial cancer

Abstract Endometrial cancer is the most common gynecological malignancy in the US and the fourth most common cancer in women. Despite its high incidence, there is limited knowledge of the molecular etiology of this disease. With obesity being a major risk factor, the incidence of endometrial cancer has been on a steady incline and is expected to become an increasing cause of cancer mortality in future years. Thus, improved understanding of endometrial tumorigenesis is of critical importance. Our analysis of human endometrial tumors identified loss of the signaling molecule PKCα in approximately 60% of endometrioid (Type I) tumors. Loss of PKCα trends with increasing tumor grade and is associated with aggressive disease features such as lymphovascular involvement and myometrial invasion, pointing to an important role for this molecule in regulating endometrial cancer progression. Inactivation of the tumor suppressor PTEN and deregulation of the PI3K/AKT pathway are key drivers of Type I endometrial cancer. Thus, mice with allele-specific knock-in of cancer-related Pten mutants (i.e., PtenΔ4-5, PtenC124R, and PtenG120E) offer unique models for the disease. Precancerous endometrial hyperplasias arising in these mice show loss of PTEN and increased AKT activity. While PKCα is expressed in the normal murine endometrial epithelium at all phases of the estrus cycle, the enzyme is uniformly lost in these precancerous lesions. Thus, disruption of PKCα signaling can occur early in endometrial tumorigenesis. In human endometrial cancer cell lines, low PKCα levels also correlate with PTEN loss; however, shRNA-mediated knockdown of PTEN in PTEN expressing cells did not affect PKCα expression indicating that downregulation of the kinase is not the direct result of changes in PTEN activity. Notably, PKC agonists suppress AKT activity in cells expressing high levels PKCα (HEC-1-A, HEC-50) but not in PKCα-low cells (Ishikawa, RL95-2). Collectively, these data suggest that, in addition to PTEN inactivation, loss of PKCα is required for full activation of the PI3K/AKT signaling pathway during endometrial carcinogenesis. Analysis of the molecular basis for loss of PKCα expression in endometrial tumors identified multiple regulatory mechanisms. PKCα protein expression in tumors generally parallels that of its mRNA and promoter activity assays point to transcriptional mechanisms for PKCα downregulation. Differential mRNA and protein stability were identified as additional levels of regulation of PKCα expression in endometrial cancer. Taken together, our findings indicate that 1) PKCα signaling regulates the PI3K/AKT pathway in the endometrial epithelium; 2) concomitant loss of PKCα and PTEN may act in a co-operative manner in regulation of endometrial turmorigenesis; and 3) loss of PKCα can occur by multiple mechanisms, as often seen with tumor suppressive molecules during neoplastic progression. Supported by NIH grants CA036727, CA016056, and DK60632. Citation Format: Alice H. Hsu, Kathryn J. Curry, Kang-Sup Shim, Peter Frederick, Carl D. Morrison, Baojing Chen, Subodh M. Lele, Gustavo Leone, Adrian R. Black, Jennifer D. Black. Protein kinase C alpha (PKCα) signaling in endometrial cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4211. doi:10.1158/1538-7445.AM2014-4211

Phospholipase D1 increases Bcl-2 expression during neuronal differentiation of rat neural stem cells.

We studied the possible role of phospholipase D1 (PLD1) in the neuronal differentiation, including neurite formation of neural stem cells. PLD1 protein and PLD activity increased during neuronal differentiation. Bcl-2 also increased. Downregulation of PLD1 by transfection with PLD1 siRNA or a dominant-negative form of PLD1 (DN-PLD1) inhibited both neurite outgrowth and Bcl-2 expression. PLD activity was dramatically reduced by a PLCγ (phospholipase Cγ) inhibitor (U73122), a Ca(2+)chelator (BAPTA-AM), and a PKCα (protein kinase Cα) inhibitor (RO320432). Furthermore, treatment with arachidonic acid (AA) which is generated by the action of PLA2 (phospholipase A2) on phosphatidic acid (a PLD1 product), increased the phosphorylation of p38 MAPK and CREB, as well as Bcl-2 expression, indicating that PLA2 is involved in the differentiation process resulting from PLD1 activation. PGE2 (prostaglandin E2), a cyclooxygenase product of AA, also increased during neuronal differentiation. Moreover, treatment with PGE2 increased the phosphorylation of p38 MAPK and CREB, as well as Bcl-2 expression, and this effect was inhibited by a PKA inhibitor (Rp-cAMP). As expected, inhibition of p38 MAPK resulted in loss of CREB activity, and when CREB activity was blocked with CREB siRNA, Bcl-2 production also decreased. We also showed that the EP4 receptor was required for the PKA/p38MAPK/CREB/Bcl-2 pathway. Taken together, these observations indicate that PLD1 is activated by PLCγ/PKCα signaling and stimulate Bcl-2 expression through PLA2/Cox2/EP4/PKA/p38MAPK/CREB during neuronal differentiation of rat neural stem cells.

MicroRNA-200b targets protein kinase Cα and suppresses triple-negative breast cancer metastasis

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with poor prognosis and lacks effective targeted therapies. The microRNA-200 (miR-200) family is found to inhibit or promote breast cancer metastasis; however, the underlying mechanism is not well understood. This study was performed to investigate the effect and mechanism of miR-200b on TNBC metastasis and identify targets for developing more efficient treatment for TNBC. We found that miR-200 family expression levels are significantly lower in highly migratory TNBC cells and metastatic TNBC tumors than other types of breast cancer cells and tumors. Ectopically expressing a single member (miR-200b) of the miR-200 family drastically reduces TNBC cell migration and inhibits tumor metastasis in an orthotopic mouse mammary xenograft tumor model. We identified protein kinase Cα (PKCα) as a new direct target of miR-200b and found that PKCα protein levels are inversely correlated with miR-200b levels in 12 kinds of breast cancer cells. Inhibiting PKCα activity or knocking down PKCα levels significantly reduces TNBC cell migration. In contrast, forced expression of PKCα impairs the inhibitory effect of miR-200b on cell migration and tumor metastasis. Further mechanistic studies revealed that PKCα downregulation by miR-200b results in a significant decrease of Rac1 activation in TNBC cells. These results show that loss of miR-200b expression plays a crucial role in TNBC aggressiveness and that miR-200b suppresses TNBC cell migration and tumor metastasis by targeting PKCα. Our findings suggest that miR-200b and PKCα may serve as promising therapeutic targets for metastatic TNBC.

Protein kinase C alpha is a target for the meprin B metalloproteinase (690.14)

Meprin metalloproteinases have been shown to play a role in the progression of diabetic nephropathy (DN) in both mice and humans. However, the mechanisms involved are not fully understood. The objective of this study was to determine whether PKC, a serine/threonine kinase known to play a role in DN, is a meprin substrate. Activated forms of purified recombinant meprin A and B were incubated for 0‐4 h with cytosolic‐enriched proteins from meprin αβ double knockout mouse kidneys (which lack endogenous meprins) and protein lysates extracted from Mardin‐Darby canine kidney (MDCK) cells (which do not express meprins). The levels of PKCα were determined by Western blot analysis using anti‐PKCα specific antibodies coupled with optic densitometry. Incubation with meprin B significantly reduced the levels of PKCα present in the kidney proteins and MDCK lysates. This decrease was not observed in proteins incubated with meprin A or control reactions without meprins, suggesting isoform‐specific degradation of PKCα. To confirm the degradation, human PKCα proteins were expressed in yeast, purified by GST‐affinity chromatography, and incubated with the activated meprins. These data suggest that meprin B may impact kidney function via proteolysis of PKCα. Consistently, PKC isoforms are upregulated in DN and PKCα has been shown to play a role in the development of albuminuria in mice with streptozotocin‐induced type 1 diabetes.Grant Funding Source: Supported by NIGMS (SC3GM102049)

Significance of expression of PKCα, Annexin A2 and S100A10 proteins in gastric cancer

目的：观察蛋白激酶Cα（protein kinase Cα，PKCα）、AnnexinA2和S100A10蛋白在胃癌组织中的表达及意义，为获取胃癌诊断相关的蛋白分子提供实验依据．方法：Westernblot分析PKCα、AnnexinA2和S100AIO蛋白在正常胃黏膜与胃癌组织中的表达情况；免疫组织化学染色观察组织芯片中三者的表达情况，并分析其临床病理学意义．结果：（1）Westernblot检测结果显示，PKCα、AnnexinA2和S100A10蛋白在胃癌组织中表达较正常胃黏膜组织中高（P〈0．01）；（21免疫组织化学染色结果显示，PKC仪蛋白在正常胃黏膜及胃癌组织中的阳性表达率分别为8．82％（3／34）和76．54％（62／811：AnnexinA2蛋白在正常胃黏膜及胃癌组织中的阳性表达率分别为5．88％（2／34）和79．01％（64／811：S100A10蛋白在正常胃黏膜及胃癌组织中的阳性表达率分别为2．94％（1／34）、59．26％（48／81）；（3）PKCα、AnnexinA2和S100A10蛋白在胃癌组织中的阳性表达率较正常胃黏膜组织高（P〈0．01）．结论：PKCα、AnnexinA2 和S100A10蛋白在胃癌组织中表达较正常胃黏膜组织中表达高，其表达与胃癌的发生及分化程度有关．

Protein kinase C isoforms ¿, ¿ and ¿ are differentially expressed in mouse ovaries at different stages of postnatal development

BackgroundThe protein kinase C (PKC) is a family of serine/threonine kinases that consists of 12 different isoforms. Since PKC isoform expressions are known to be specific for different cell types and postnatal developmental stages, we aimed to determine immunolocalizations and protein expression levels of different PKC isoforms in pre-pubertal, pubertal and adult mouse ovaries.MethodsOvaries were obtained from postnatal day 1 (PND1) and PND7 of pre-pubertal, PND21 of pubertal and PND60 of adult mice. Immunolocalizations of PKCα, PKCδ and PKCε isoforms were determined and immunostainings in different cellular components of all follicular stages were evaluated by H-Score. PKCα, PKCδ and PKCε protein expression levels were determined by Western blot. The bands were quantified via ImageJ software. The data obtained from H-Score and ImageJ evaluations were analyzed by ANOVA statistical test.ResultsPKCα immunostainings were more intense in oocytes when compared to granulosa and theca cells at different follicular stages of all groups. The Western blot analysis revealed that PKCα expression was significantly higher in PND60 adult ovaries. Conversely, PKCδ immunostainings were more intense in granulosa cells. According to the Western blot analysis, PKCδ protein expression was also higher in PND60 and significantly lower in PND1 ovaries. PKCε immunostaining was more apparent in oocytes. PKCε protein expression was significantly higher in adult PND60 and pubertal PND21 ovaries when compared to pre-pubertal PND7 and PND1 ovaries. Interestingly, PKCε immunostaining was significantly higher in primordial follicles, though PKCα and PKCδ immunostainings were more apparent in larger follicles. PKCα immunostainings of corpora lutea (CL) were significantly higher when compared to follicles in PND60 ovaries.ConclusionsThis study demonstrates that PKCα, PKCδ and PKCε isoforms are differentially expressed in particular cellular components of pre-pubertal, pubertal and adult mouse ovarian follicles. Therefore, we suggest that each PKC isoform has unique functions that are controlled by gonadotropin dependent mechanisms during follicular growth, oocyte maturation, ovulation and luteinization.