1.Effects of overexpression of PEMT2 on expression and translocation of different PKC isoforms in rat hepatoma cells.
Ya-li LI ; Ke-li MA ; Wei ZOU ; Quan XIA ; Zhao-chun CUI
Chinese Journal of Hepatology 2005;13(9):678-681
OBJECTIVETo explore the mechanism of cell proliferation inhibition by transfecting phosphatidylethanolamine N-methyltransferase 2 gene (PEMT2).
METHODSThe expression and translocation of different isoforms of protein kinase C (PKC) in cells were observed with immunocytochemistry and Western blot techniques. The content of diacylglycerol (DAG) was analyzed with high performance thin layer chromatography (HPTLC) technique.
RESULTSTransfection of PEMT2 can inhibit the expression of cPKC alpha, but obviously promotes the expression and translocation from cytosol to plasma membrane of cPKC beta2. At the same time, the content of DAG was decreased in the transfected cells. Expression and translocation of other PKC isoforms were not changed by PEMT2 transfection.
CONCLUSIONEffects of overexpression of PEMT2 on the expression and translocation of different PKC isoforms might be related to the mechanism of cell proliferation inhibition and apoptosis induced by transfecting PEMT2.
Animals ; Liver Neoplasms, Experimental ; enzymology ; pathology ; Phosphatidylethanolamine N-Methyltransferase ; biosynthesis ; genetics ; Protein Isoforms ; Protein Kinase C ; biosynthesis ; genetics ; Rats ; Transfection
2.A Study on Altered Expression of Serine Palmitoyltransferase and Ceramidase in Psoriatic Skin Lesion.
Kyung Kook HONG ; Hee Ryung CHO ; Won Chul JU ; Yunhi CHO ; Nack In KIM
Journal of Korean Medical Science 2007;22(5):862-867
Ceramides are the main lipid component maintaining the lamellae structure of stratum corneum, as well as lipid second messengers for the regulation of cellular proliferation and/or apoptosis. In our previous study, psoriatic skin lesions showed marked decreased levels of ceramides and signaling molecules, specially protein kinase C-alpha (PKC-alpha) and c-jun N-terminal kinase (JNK) in proportion to the psoriasis area and severity index (PASI) scores, which suggested that the depletion of ceramide is responsible for epidermal hyperproliferation of psoriasis via downregulation of proapoptotic signal cascade such as PKC-alpha and JNK. In this study, we investigated the protein expression of serine palmitoyltransferase (SPT) and ceramidase, two major ceramide metabolizing enzymes, in both psoriatic epidermis and non-lesional epidermis. The expression of SPT, the ceramide generating enzyme in the de novo synthesis in psoriatic epidermis, was significantly less than that of the non-lesional epidermis, which was inversely correlated with PASI score. However, the expression of ceramidase, the degradative enzyme of ceramides, showed no significant difference between the lesional epidermis and the non-lesional epidermis of psoriatic patients. This might suggest that decreased expression of SPT protein is one of the important causative factors for decreased ceramide levels in psoriasis.
Adolescent
;
Adult
;
Amidohydrolases/*biosynthesis/metabolism
;
Apoptosis
;
Cell Proliferation
;
Ceramidases
;
Ceramides/chemistry
;
Child
;
Epidermis/metabolism
;
Female
;
Humans
;
JNK Mitogen-Activated Protein Kinases/metabolism
;
Male
;
Models, Biological
;
Protein Kinase C-alpha/metabolism
;
Psoriasis/*blood/diagnosis
;
Serine C-Palmitoyltransferase/*biosynthesis
3.Aldosterone stimulating PDGF-B expression in HSC via activation of EGR-1.
Xu LI ; Ying MENG ; Xi-shan YANG ; Ping-sheng WU ; Zhen-shu ZHANG
Chinese Journal of Hepatology 2005;13(8):567-570
OBJECTIVEIt is known that intrahepatic renin-angiotensin-aldosterone system (RAAS) plays a key role in liver fibrogenesis. Aldosterone (Aldo), the principal effector molecule of the RAAS, exerts local effects on cell growth and fibrogenesis. However, the signal transduction mechanisms underlying the effects of Aldo on hepatic fibrogenesis remain to be fully elucidated. The present study aims to investigate the signal transduction mechanism underlying the effects of Aldo on extracellular signal-regulated kinase 1/2 (ERK1/2), early growth response-1 (EGR-1) and on the platelet-derived growth factor-B (PDGF-B).
METHODSIn vitro, hepatic stellate cell (HSC)-T6 cell line was treated with Aldo for 10 min, 0.5 h, 1 h, 2 h and 3 h. Protein expression of phospho-p42/44 was detected by Western blot. In addition, HSC-T6 were preincubated for 1 h or not at all with U0126 (an inhibitor of the MAPK/ERK kinase), and antioxidant-N-acetylcysteine (NAC) prior to exposure to Aldo for the indicated times. Protein expressions of phospho-p42/44 and PDGF-B were measured by Western blot. DNA biding activity of EGR-1 was analyzed by electrophoretic gel mobility shift assay (EMSA). By means of immunohistochemistry, expression of PDGF-B was detected.
RESULTSAldo induced phospho-p42/44 expression could be abrogated by U0126; NAC did not inhibit phospho-p42/44 expression. Gel shift study showed that stimulation of HSC by Aldo markedly increased the EGR-1 DNA binding activity, which was abrogated by U0126, reaching a maximum at 60 minutes, and then declined progressively. NAC did not reduce the EGR-1 activity. Aldo increased the PDGF-B protein level in HSC, which was not attenuated by NAC and U0126.
CONCLUSIONSStimulation of HSC by Aldo results in activation of EGR-1 via ERK1/2 pathway, leading to up-regulation of PDGF-B expression.
Aldosterone ; pharmacology ; Cell Line ; Early Growth Response Protein 1 ; biosynthesis ; genetics ; Hepatocytes ; cytology ; metabolism ; Humans ; Mitogen-Activated Protein Kinase 3 ; biosynthesis ; genetics ; Proto-Oncogene Proteins c-sis ; biosynthesis ; genetics ; Signal Transduction
4.Cell type-specific upregulation of myristoylated alanine-rich C kinase substrate and protein kinase C-alpha, -beta I, -beta II, and -delta in microglia following kainic acid-induced seizures.
Su Yong EUN ; Eun Hae KIM ; Kee Seok KANG ; Hwa Jung KIM ; Sangmee Ahn JO ; Soon Jong KIM ; Su Hyun JO ; Sang Jeong KIM ; Perry J BLACKSHEAR ; Jun KIM
Experimental & Molecular Medicine 2006;38(3):310-319
Myristoylated alanine-rich C kinase substrate (MARCKS) is a widely distributed protein kinase C (PKC) substrate and has been implicated in actin cytoskeletal rearrangement in response to extracellular stimuli. Although MARCKS was extensively examined in various cell culture systems, the physiological function of MARCKS in the central nervous system has not been clearly understood. We investigated alterations of cellular distribution and phosphorylation of MARCKS in the hippocampus following kainic acid (KA)-induced seizures. KA (25 mg/kg, i.p.) was administered to eight to nine week-old C57BL/6 mice. Behavioral seizure activity was observed for 2 h after the onset of seizures and was terminated with diazepam (8 mg/kg, i.p.). The animals were sacrificed and analyzed at various points in time after the initiation of seizure activity. Using double-labeling immunofluorescence analysis, we demonstrated that the expression and phosphorylation of MARCKS was dramatically upregulated specifically in microglial cells after KA-induced seizures, but not in other types of glial cells. PKC alpha, beta I, beta II and delta, from various PKC isoforms examined, also were markedly upregulated, specifically in microglial cells. Moreover, immunoreactivities of phosphorylated MARCKS were co-localized in the activated microglia with those of the above isoforms of PKC. Taken together, our in vivo data suggest that MARCKS is closely linked to microglial activation processes, which are important in pathological conditions, such as neuroinflammation and neurodegeneration.
Up-Regulation/drug effects
;
Time Factors
;
Seizures/chemically induced/*metabolism
;
Protein Kinase C-delta/analysis
;
Protein Kinase C-alpha/analysis
;
Protein Kinase C/*analysis
;
Protein Biosynthesis/drug effects
;
Phosphorylation/drug effects
;
Microscopy, Confocal
;
Microglia/cytology/drug effects/*metabolism
;
Mice, Inbred C57BL
;
Mice
;
Membrane Proteins/*analysis/metabolism
;
Kainic Acid/*toxicity
;
Isoenzymes/analysis
;
Intracellular Signaling Peptides and Proteins/*analysis/metabolism
;
Immunohistochemistry
;
Animals
5.Effect of bufalin-inducing apoptosis on Bcl-2 and PKC in HL-60 cells.
Xin TIAN ; Ping-Ping WANG ; Yun-Peng LIU ; Ke-Zuo HOU ; Bo JIN ; Ying LUO ; Xiu-Juan QU
Journal of Experimental Hematology 2007;15(1):67-71
Previous study revealed that bufalin can inhibit proliferation, and induce apoptosis in some human cancer cell lines. However, the mechanism of its anticancer effect has not been fully understood. The present study was designed to investigate the effects of bufalin-induced apoptosis on Bcl-2 and PKC in human leukemic HL-60 cells. The cell viability was determined by trypan blue dye exclusion. The apoptosis was detected by morphology, flow cytometry and DNA agarose gel electrophoresis. The expressions of Bcl-2 and PKC were analyzed by Western blot, and activity of PKC was assayed by [gamma-(32)P] isotope incorporation method. The results showed as follows: (1) proliferation of HL-60 cells was inhibited by bufalin and the IC(50) at 24, 48, 72 hours were (25.8 +/- 2.1), (8.0 +/- 1.2) and (2.3 +/- 0.3) nmol/L, respectively. (2) apoptosis of HL-60 cells was induced when the cells were treated with bufalin at concentration of 50 nmol/L for 24 hours. (3) compared with control, treatment with bufalin at concentration of 50 nmol/L for 6 - 24 hours resulted in downregulation of protein expression, decrease of phosphorylation, and cleavage of Bcl-2, simultaneously. (4) the activity of total PKC was unchanged when HL-60 cells were exposed to 1 - 100 nmol/L bufalin for 30 minutes, but PKCbetaII underwent translocation from cytosol to membrane. It is concluded that apoptosis induced by bufalin is associated with downregulation of protein expression, dephosphorylation, and cleavage of Bcl-2 in HL-60 cells.
Apoptosis
;
drug effects
;
Bufanolides
;
pharmacology
;
Cell Proliferation
;
drug effects
;
Drugs, Chinese Herbal
;
pharmacology
;
HL-60 Cells
;
Humans
;
Materia Medica
;
pharmacology
;
Phosphorylation
;
Protein Kinase C
;
biosynthesis
;
genetics
;
Proto-Oncogene Proteins c-bcl-2
;
biosynthesis
;
genetics
6.Roles of PKCα on the biological functions of T cells.
Li-Fen YANG ; Hui-Min KONG ; Xiao-Qing ZHANG ; Fei YIN
Chinese Journal of Contemporary Pediatrics 2015;17(12):1354-1359
OBJECTIVETo study the roles of PKCα on the proliferation, apoptosis, differentiation, cytokine production and inducible regulatory T cell (iTreg) induction of T cells.
METHODST cells from WT (PKCα⁺/⁺) or PKCα knockout (PKCα⁻/⁻) mice were isolated and cultured in vitro. T cell proliferation and apoptosis were determined using ³H thymidine incorporation and CSFE/Annexin V staining. Cytokines production (IL-2, IL-4, IFN-γ and IL-17) was detected using ELISA. CD4⁺T cells were isolated and cultured in vitro via Th17 or iTreg biased condition. Flow cytometry was used to detect the cell differentiation.
RESULTSThe production of IL-2 upon TCR stimulation increased, while the contents of IL-4 and IL-17 decreased in the PKCα⁻/⁻ group compared with the PKCα⁺/⁺ group. The differentiation rate of Th17 cells decreased, while the iTreg production increased in the PKCα⁻/⁻ group compared with the PKCα⁺/⁺ group.
CONCLUSIONSPKC-α is proinflammatory.
Animals ; Cell Differentiation ; Cytokines ; biosynthesis ; Lymphocyte Activation ; Mice ; Protein Kinase C-alpha ; physiology ; Receptors, Antigen, T-Cell ; physiology ; T-Lymphocytes ; physiology ; Th17 Cells ; immunology
7.Expression of PKC in rat hepatic fibrosis and the effect of Dan-shao-hua-xian Capsule on its expression pattern.
Qin YANG ; Ru-jia XIE ; Xin-hua LUO ; Bing HAN ; Ting YANG ; Li FANG ; Ming-liang CHENG
Chinese Journal of Hepatology 2005;13(9):707-708
Animals
;
Capsules
;
Drugs, Chinese Herbal
;
therapeutic use
;
Liver Cirrhosis
;
drug therapy
;
enzymology
;
Male
;
Phytotherapy
;
Protein Kinase C
;
biosynthesis
;
genetics
;
Rats
;
Rats, Wistar
9.Effect of overexpression of Smad7 gene on cell proliferation.
Yan-ying HUO ; Kai-tai ZHANG ; Bang-yin LI ; Qin-zhi XU ; Rui-feng DUAN ; Ying-chun HU ; Xiao-qiong XIANG ; Gang LI ; De-chang WU
Chinese Journal of Oncology 2004;26(9):521-524
OBJECTIVETo study the effect of overexpression of Smad7 gene on cell proliferation in human bronchial epithelial cell lines.
METHODSHuman bronchial epithelial cell lines, BEP2D and BERP35T2 cells, were cotransfected with the mammalian expression vectors PCISmad7.neo and pMyc-SEAP, the latter was ac-myc cis-acting enhancer element fused with alkaline phosphatase (SEAP) reporter gene. Expression of c-myc, p15 and p21 mRNA was detected by RT-PCR before and after stable transfection of Smad7 into BEP2D and BERP35T2 cells in order to study the regulation of TGF-beta-mediated growth inhibition.
RESULTSAfter BEP2D and BERP35T2 cells transfected with Smad7, the transcriptional activity of c-myc was significantly increased. Smad7 overexpressing cells showed upregulation of c-myc expression and downregulation of p15 and p21 expression, which contributed to the loss of TGF-beta responses in these cells.
CONCLUSIONOverexpression of Smad7 may facilitate cell proliferation by antagonizing TGF-beta-mediated antiproliferative gene responses.
Bronchi ; cytology ; Cell Proliferation ; Cell Transformation, Neoplastic ; Cells, Cultured ; Cyclin-Dependent Kinase Inhibitor p15 ; biosynthesis ; genetics ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; genetics ; Epithelial Cells ; cytology ; Humans ; Proto-Oncogene Proteins c-myc ; biosynthesis ; genetics ; Signal Transduction ; Smad7 Protein ; biosynthesis ; genetics ; Transfection ; Transforming Growth Factor beta ; biosynthesis ; genetics
10.Cloning, expression, purification of protein kinase Cdelta and its preliminary application in drug lead compounds screening.
Chinese Journal of Biotechnology 2005;21(2):300-304
Protein kinase Cdelta (PKCdelta) is a member of protein kinase C family, which possess phospholipid-dependent serine and threonine kinase activity. PKCdelta is a potential drug target of diabetes and some cancers. The abnormal activation of PKCdelta can arouse diabetes and some cancers. Therefore the specific inhibitors of PKCdelta can be applied in the research and development of the drug candidate of these diseases. The present aim is to obtain active recombinant PKCdelta from COS1 cells. For cloning of mouse PKCdelta a pair of specific primers were designed based on the published sequence of this gene. The cDNA of full coding region was obtained by RT-PCR. The amplified cDNA was subsequently cloned into FLAG-tagged pcDNA3.0 and its sequence was confirmed by DNA sequencing analysis. FLAG-tagged pcDNA3.0-PKCdelta was transfected into COS1 cells. A cell strain which can stably express PKCdelta was obtained by G418 screening. FLAG-tagged PKCdelta in the supernant of COS1 cells extracts was absorbed by anti-FLAG resin and eluted by FLAG peptide. The purified protein appeared as a single band on both SDS-PAGE and western blotting, indicating that it was chemical and antigenic pure. By kinase assay, the recombinant PKCdelta was active. Positive inhibitor, staurosporine, was used to prove the enzyme could be greatly inhibited. Several compounds have been found to inhibit the enzyme, which indicates the preliminary application in drug lead compounds screening.
Animals
;
COS Cells
;
Cercopithecus aethiops
;
Cloning, Molecular
;
Drug Delivery Systems
;
Drug Evaluation, Preclinical
;
Humans
;
Mice
;
Protein Kinase C-delta
;
antagonists & inhibitors
;
biosynthesis
;
genetics
;
isolation & purification
;
Recombinant Proteins
;
antagonists & inhibitors
;
biosynthesis
;
genetics
;
isolation & purification
;
Staurosporine
;
pharmacology