OBJECTIVETo study the role of protein kinase C-delta (PKC-delta) in hyperthermia-induced apoptosis in human tongue squamous cell carcinoma Tca8113 cells.

METHODSTca8113 cells were treated at 43 degrees C in a heating water bath for 0, 40, 80, 120 min after pretreatment with Rottlerin, a specific inhibitor of PKC-delta, and equal volume dimethyl sulfoxide (DMSO) for 30 min, respectively. The cells were stained by propidium iodide (PI) and Rhodamine 123 to analysis apoptotic rate and the changes of mitochondrial transmembrane potential by flow cytometry (FCM). The total proteins were extracted for Western blotting analysis of activation and proteolysis of PKC-delta, and for colorimetric assay of relative activity of Caspase-3.

RESULTSHyperthermia could induce proteolysis and activation of PKC-delta, and this was attenuated by Rottlerin. Apoptotic rate, decreasing of mitochondrial transmembrane potential and activity of Caspase-3 which being induced by hyperthermia in Tca8113 cells were inhibited by PKC-delta specific inhibitor Rottlerin. There were significantly statistical differences in apoptosis rates, mitochondrial transmembrane potential and activity of Caspase-3 between Rottlerin- and non-Rottlerin-pretreated cells after hyperthermia for 40, 80, 120 min (P < 0.01).

CONCLUSIONActivated PKC-delta may facilitate hyperthermia-induced apoptosis in Tca8113 cells, and may be one of the mechanisms of apoptosis induced by hyperthermia.

Acetophenones ; Apoptosis ; Benzopyrans ; Carcinoma, Squamous Cell ; Humans ; Protein Kinase C ; Protein Kinase C-delta

Our previous study demonstrated that a bacterial siderophore, deferoxamine (DFO), could trigger inflammatory signals in human intestinal epithelial cells as a single stimulus, leading to IL-8 production via ERK1/2 and p38 phosphorylation and NF-kappa B-independent mechanism. In the present study, we proved that a novel protein kinase C (PKC)isoform, PKCdelta, is necessary for DFO-induced IL-8 production. Pretreatment of HT-29 cells with rottlerin showed remarkable inhibition of DFO-induced IL-8 production. In contrast, a conventional PKC inhibitor Go6976 did not show significant inhibition of DFO-induced IL-8 production. DFO induced strong phosphorylation of PKCdelta in the epithelial cells. Overexpression of PKCdelta resulted in enhanced PKCdelta phosphorylation, while transfection with dominant-negative PKCdelta vector failed DFO-induced phosphorylation. In addition, transfection of HT-29 cells with siRNA targeting endogenous PKCdelta, which suppressed PKCdelta expression, attenuated DFO-induced IL-8 production. These results demonstrate that PKCdelta plays an important role in regulating iron chelator-induced IL-8 production in human intestinal epithelial cells.
Deferoxamine ; Epithelial Cells* ; HT29 Cells ; Humans* ; Interleukin-8* ; Iron ; Phosphorylation ; Protein Kinase C* ; Protein Kinase C-delta* ; Protein Kinases* ; RNA, Small Interfering ; Transfection

PURPOSE: Protein kinase C (PKC) has been implicated in a wide variety of cellular processes. Although PKC-delta is implicated in cell growth inhibition, as well as in cell differentiation, apoptosis, and tumor suppression, its role in atherosclerosis remains unclear. This study aimed to identify the mechanism of PKC-delta in the development of atherosclerosis. METHODS: To induce atherosclerosis, we performed allograft transplantations on aortas in mice. At 2, 4, and 6 weeks after transplantation, grafted aortas were obtained to compare the degree of atherosclerosis between wild type and PKC-delta (-/-) aorta. Alloantibody levels in the recipient mice's blood were measured. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to quantitatively measure chemokine and cytokine mRNA expression of the inflammation from the harvested aorta. RESULTS: Atherosclerosis was more severe in the PKC-delta (-/-) aorta than in the wild type aorta. Alloantibody levels were higher in the mice grafted with aorta from the PKC-delta (-/-) mice than in the mice grafted with aorta from the wild type mice. RT-PCR revealed higher expressions of MRP-2, MCP-1, MIP-1alpha, and IL-2 in the mice grafted with aorta from the PKC-delta (-/-) mice than the wild type mice. CONCLUSION: Aorta allograft transplantation is a useful modality for inducing atherosclerosis. PKC-delta may be a negative regulator of atherosclerosis.
Animals ; Aorta ; Apoptosis ; Atherosclerosis ; Cell Differentiation ; Chemokine CCL3 ; Inflammation ; Interleukin-2 ; Mice ; Protein Kinase C ; Protein Kinase C-delta ; Protein Kinases ; RNA, Messenger ; Transplantation, Homologous ; Transplants

Pulmonary edema is a major cause of mortality due to acute lung injury (ALI). The involvement of protein kinase C-delta (PKC-delta) in ALI has been a controversial topic. Here we investigated PKC-delta function in ALI using PKC-delta knockout (KO) mice and PKC inhibitors. Our results indicated that although the ability to produce proinflammatory mediators in response to LPS injury in PKC-delta KO mice was similar to that of control mice, they showed enhanced recruitment of neutrophils to the lung and more severe pulmonary edema. PKC-delta inhibition promoted barrier dysfunction in an endothelial cell layer in vitro, and administration of a PKC-delta-specific inhibitor significantly increased steady state vascular permeability. A neutrophil transmigration assay indicated that the PKC-delta inhibition increased neutrophil transmigration through an endothelial monolayer. This suggests that PKC-delta inhibition induces structural changes in endothelial cells, allowing extravasation of proteins and neutrophils.
Acute Lung Injury* ; Animals ; Capillary Permeability* ; Endothelial Cells ; Lung ; Mice ; Mortality ; Neutrophils ; Protein Kinase C-delta* ; Protein Kinases* ; Pulmonary Edema

OBJECTIVETo investigate the expressions of protein kinase C (PKC) isoforms in X-ray-exposed HepG2 cells and identify the PKC isoforms that induce radioresistance in HepG2 cells.

METHODSCultured HepG2 cells were divided into control group and 6 Gy radiation group for corresponding treatments. The fluorescence intensity (FI) and the percentage of positive cells were determined using flow cytometry.

RESULTSThe FI of PKCalpha and PKCdelta were 2.28 and 5.05 in the radiation group, respectively, significantly higher than those in the control group (P<0.05). The percentages of PKCalpha- and PKCdelta -positive cells were significantly higher in the radiation group than in the control group (P<0.05). The FI and the percentages of PKC zeta, gamma, epsilon, zeta positive cells were rather low and showed no significant differences between the two groups (P>0.05); PKCbeta expression was not detected in the two groups of cells. The apoptosis rates of the control and radiation groups were 1.73% and 20.90%, respectively.

CONCLUSIONPKCalpha and PKCdelta may be involved in protecting HepG2 cells from radiation-induced apoptosis.

Apoptosis ; physiology ; radiation effects ; Hep G2 Cells ; Humans ; Isoenzymes ; classification ; metabolism ; Protein Kinase C-alpha ; metabolism ; Protein Kinase C-delta ; metabolism ; Radiation Tolerance ; Signal Transduction ; drug effects ; physiology

OBJECTIVETo study the function of 5 single nucleotide polymorphisms (SNPs) of the PRKCZ gene, a susceptibility gene for type 2 diabetes in Han population of North China, in the pathogenesis of the disease.

METHODSBioinformatic methods and reporter gene activity determination were used to analyze the function of the 5 SNPs.

RESULTSThe reporter gene activities of different alleles of 2 SNPs, rs427811 and rs809912, were obviously different, which implies that these 2 SNPs might be susceptibility loci of the disease.

CONCLUSIONThe PRKCZ gene is further confirmed to be a susceptibility gene for type 2 diabetes in Han population of North China. Two SNPs in the gene play a role in the pathogenesis of the disease by affecting the expression level of PRKCZ gene.

Alleles ; Asian Continental Ancestry Group ; Diabetes Mellitus, Type 2 ; genetics ; Ethnic Groups ; Genetic Predisposition to Disease ; Humans ; Polymorphism, Single Nucleotide ; Protein Kinase C ; genetics ; Protein Kinase C-delta

AIMTo investigate the effects of sulfated polymannuroguluronate (SPMG), a novel candidate anti-AIDS drug in Phase II clinical trial, on Tat-induced release of proinflammatory cytokines (i.e., TNFalpha, IL-1beta and IL-6) and its related mechanism.

METHODSThe effects of SPMG on Tat induced TNFalpha (4 h), IL-1beta and IL-6 (6 h) secretion in THP-1 cells were measured by ELISA. Western blotting analysis was used to study the effects of SPMG on Tat induced PKCzeta, PKCtheta and PKCsigma phosphorylation.

RESULTSSPMG (50 to 100 microg x mL(-1)) markedly suppressed TNFalpha, IL-1beta and IL-6 secretion in Tat activated THP-1 cells. In THP-1 cells the phosphorylation levels of PKCzeta, PKCtheta and PKCsigma significantly increased following Tat stimulation, and only PKCsigma phosphorylation levels was inhibited by SPMG (50 to 100 microg x mL(-1)).

CONCLUSIONSPMG suppresses the secretion of proinflammatory cytokines in THP-1 cells may be by inhibiting PKCsigma activation.

Cell Line, Tumor ; Gene Products, tat ; pharmacology ; Humans ; Interleukin-1beta ; secretion ; Interleukin-6 ; secretion ; Isoenzymes ; metabolism ; Phosphorylation ; Polysaccharides ; pharmacology ; Protein Kinase C ; metabolism ; Protein Kinase C-delta ; metabolism ; Protein Kinase C-theta ; Tumor Necrosis Factor-alpha ; secretion

Animals ; Mice ; Myocardium/metabolism* ; Oxidative Stress ; Physical Conditioning, Animal ; Protein Kinase C-delta/metabolism* ; Src Homology 2 Domain-Containing, Transforming Protein 1/metabolism* ; Swimming

OBJECTIVETo investigate the expression of ASK1 and PRKCD in the process of monocyte differentiation, and explore their role in functional changes of hypersplenism spleen macrophages (Mφ) in portal hypertension (PH).

METHODSU937 cells were stimulated to differentiate into monocyte/macrophage-like cells by cultivation in PMA and the mRNA expressions of ASK1 and PRKCD were detected by q-PCR and the changes of protein expression were identified by western blot analysis. The secretion of phagocytose related cytokines such as IL-10 and TNF-α were tested by ELISA, and the function of the macrophage-like cells were studied by chicken red blood cell phagocytose test.

RESULTSThe expressions of PRKCD and ASK1 mRNA were gradually decreased along with the cell differentiation, while the secretion of TNF-α was increased, IL-10 secretion reached a maximum at 24 h after PAM stimulation, and then gradually fell. The expression of ASK1 and p-ASK1 were rapidly increased compared with the non-stimulated U937 cells, while the expression of PRKCD and p-PRKCD were sightly declined. The phagocytose test show that U937 cells induced with PMA were able to swallow the chicken red blood cell.

CONCLUSIONUp-regulated protein expression of ASK1 and p-ASK1 and down-regulated protein expression of PRKCD and p-PRKCD in the process of PMA induced monocyte differentiation, are consist with the expression changes of splenic macrophage phagocytosis in hypersplenism, which leads to increased activity of Mφ.

Cell Differentiation ; Down-Regulation ; Humans ; Hypersplenism ; Hypertension, Portal ; Interleukin-10 ; secretion ; MAP Kinase Kinase Kinase 5 ; physiology ; Macrophages ; cytology ; Phagocytosis ; Protein Kinase C-delta ; physiology ; RNA, Messenger ; Tumor Necrosis Factor-alpha ; secretion ; U937 Cells

OBJECTIVETo search for the disease-associated haplotype in the PRKCZ gene, a susceptibility gene for type 2 diabetes in Han population of North China, by case-control study and linkage disequilibrium (LD) analysis using single nucleotide polymorphisms (SNPs).

METHODSSNPs located in the PRKCZ gene were chosen from public SNP domain by bioinformatic methods and single base extension (SBE) method was used to genotype the loci in 173 sporadic type 2 diabetes patients and 152 normal individuals to perform case-control study and LD analysis. Haplotype block were constructed in these populations.

RESULTSSeveral SNPs in the PRKCZ gene were found to be associated with the disease. The SNPs formed different haplotype block pattern in case and control groups. The frequencies of the haplotypes formed by 5 SNPs were statistically different between the two groups.

CONCLUSIONThe haplotype formed by 5 SNPs in the PRKCZ gene may be associated with type 2 diabetes in Han population of China, which is confirmed from statistics to be a susceptibility gene for the disease.

Alleles ; Asian Continental Ancestry Group ; Case-Control Studies ; Diabetes Mellitus, Type 2 ; genetics ; Ethnic Groups ; Genetic Predisposition to Disease ; Haplotypes ; Humans ; Linkage Disequilibrium ; Polymorphism, Single Nucleotide ; Protein Kinase C ; genetics ; Protein Kinase C-delta