Protein kinase C (PKC) family plays a critical role in many developmental events, including oocyte activation, completion of the second meiosis and initiation of the first mitosis, compaction, and blastocysts formation as well. But little is known of its many isozymes. Studies have shown that 10 isozymes of PKC and its anchor protein, RACK, are expressed in the course from 2 cell stage through blastocyst stage in mouse. We reviewed here the recent studies on the location pattern and expression levels of different PKC isozymes. Those studies indicated that the isozymes were very important for every stage of preimplantation embryonic development, especially at the early 4-cell stage. Some are increased temporarily in nucleus, which indicated that they might control and regulate the remolding of embryonic nucleus. We also analyzed the possible functions of PKCs in the somatic nuclear transferred embryos.
Animals ; Embryonic Development ; physiology ; Fertilization ; physiology ; Isoenzymes ; metabolism ; physiology ; Oocytes ; physiology ; Protein Kinase C ; metabolism ; physiology ; Receptors for Activated C Kinase ; Receptors, Cell Surface ; metabolism

Protein-protein interactions between viruses and hosts are common during viral infection and replication. In this study, a cDNA library from larvae of Plutella xylostella was constructed and used for screening of genes encoding proteins interacting with Plutella xylostella granulovirus (PlxyGV) proteins. Two cDNA clones containing genes encoding proteins interacting with PlxyGV PP31 were identified by yeast two-hybrid assays. Sequence analysis showed that the genes encoded homologues of receptor for activated protein C kinase (RACK) and methionine aminopeptidase 2 (MetAP2), respectively. The P. xylostella rack gene and the PlxyGV pp31 was expressed in an E. coli strain to produce proteins fused with a 6-His or a GST tag. It was shown that the rack was expressed as a 38kD peptide as prospected. The 38kD His-tagged peptide was co-purified with GST-PP31 by GST-bind resin in GST-pulldown assays, confirming interaction between the PlxyGV PP31 and the RACK protein of P. xylostella.
Aminopeptidases ; genetics ; physiology ; Animals ; Gene Library ; Granulovirus ; physiology ; Metalloendopeptidases ; genetics ; physiology ; Moths ; virology ; Receptors for Activated C Kinase ; Receptors, Cell Surface ; genetics ; physiology

Adenosine is a naturally occurring breakdown product of adenosine triphosphate and plays an important role in different physiological and pathological conditions. Adenosine also serves as an important trigger in ischemic and remote preconditioning and its release may impart cardioprotection. Exogenous administration of adenosine in the form of adenosine preconditioning may also protect heart from ischemia-reperfusion injury. Endogenous release of adenosine during ischemic/remote preconditioning or exogenous adenosine during pharmacological preconditioning activates adenosine receptors to activate plethora of mechanisms, which either independently or in association with one another may confer cardioprotection during ischemia-reperfusion injury. These mechanisms include activation of K(ATP) channels, an increase in the levels of antioxidant enzymes, functional interaction with opioid receptors; increase in nitric oxide production; decrease in inflammation; activation of transient receptor potential vanilloid (TRPV) channels; activation of kinases such as protein kinase B (Akt), protein kinase C, tyrosine kinase, mitogen activated protein (MAP) kinases such as ERK 1/2, p38 MAP kinases and MAP kinase kinase (MEK 1) MMP. The present review discusses the role and mechanisms involved in adenosine preconditioning-induced cardioprotection.
Adenosine Triphosphate ; Adenosine* ; Heart ; Inflammation ; Mitogen-Activated Protein Kinase Kinases ; Nitric Oxide ; Phosphotransferases ; Protein Kinase C ; Protein-Tyrosine Kinases ; Proto-Oncogene Proteins c-akt ; Receptors, Opioid ; Receptors, Purinergic P1 ; Reperfusion Injury

The migration of dendritic cells (DCs) to secondary lymphoid organs depends on chemoattraction through the interaction of the chemokine receptors with chemokines. However, the mechanism of how lymphoid chemokines attract DCs to lymphoid organs remains unclear. Here, we demonstrate the mechanism of DC migration in response to the lymphoid chemokine CCL21. CCL21-mediated DC migration is controlled by the regulation of sarcoplasmic reticulum Ca²⁺ ATPase 2 (SERCA2) expression rather than through the activation of mitogen-activated protein kinases CCL21-exposed mature DCs (mDCs) exhibited decreased SERCA2 expression but not decreased phospholamban (PLB) or Hax-1 expression, which are known to be SERCA2-interacting proteins. In addition, CCL21 did not affect the mRNA levels of SERCA2 or its interacting protein Hax-1. Interestingly, SERCA2 expression was inversely related to DC migration in response to chemokine stimulation. The migratory capacity of CCL21-treated mDCs was decreased by the phospholipase C inhibitor U73122 and by the protein kinase C inhibitor BAPTA-AM. The migratory capacities of mDCs were increased in response to SERCA2 siRNA expression but were decreased by SERCA2 overexpression. In addition, DCs treated with a SERCA2-specific inhibitor (cyclopiazonic acid) had significantly increased migratory capacities as mDCs regardless of SERCA2 expression. Moreover, SERCA2 expression was dependent on DC maturation induced by cytokines or Toll-like receptor agonists. Therefore, the migratory capacities differed in differentially matured DCs. Taken together, these results suggest that SERCA2 contributes to the migration of CCL21-activated DCs as an important feature of the adaptive immune response and provide novel insights regarding the role of SERCA2 in DC functions.
Adaptive Immunity ; Adenosine Triphosphatases* ; Chemokine CCL21 ; Chemokines ; Cytokines ; Dendritic Cells* ; Mitogen-Activated Protein Kinases ; Protein Kinase C ; Receptors, Chemokine ; RNA, Messenger ; RNA, Small Interfering ; Sarcoplasmic Reticulum* ; Toll-Like Receptors ; Type C Phospholipases

To understand the roles of purinergic receptors and cellular molecules below the receptors in the vascular inflammatory response, we determined if extracellular nucleotides up-regulated chemokine expression in vascular smooth muscle cells (VSMCs). Human aortic smooth muscle cells (AoSMCs) abundantly express P2Y1, P2Y6, and P2Y11 receptors, which all respond to extracellular nucleotides. Exposure of human AoSMCs to NAD+ , an agonist of the human P2Y11 receptor, and NADP+ as well as ATP, an agonist for P2Y1 and P2Y11 receptors, caused increase in chemokine (C-C motif) ligand 2 gene (CCL2) transcript and CCL2 release; however, UPT did not affect CCL2 expression. CCL2 release by NAD+ and NADP+ was inhibited by a concentration dependent manner by suramin, an antagonist of P2-purinergic receptors. NAD+ and NADP+ activated protein kinase C and enhanced phosphorylation of mitogen-activated protein kinases and Akt. NAD(+)- and NADP(+)-mediated CCL2 release was significantly attenuated by SP6001250, U0126, LY294002, Akt inhibitor IV, RO318220, GF109203X, and diphenyleneiodium chloride. These results indicate that extracellular nucleotides can promote the proinflammatory VSMC phenotype by up-regulating CCL2 expression, and that multiple cellular elements, including phosphatidylinositol 3-kinase, Akt, protein kinase C, and mitogen-activated protein kinases, are involved in that process.
Adenosine Triphosphate ; Butadienes ; Chromones ; Humans ; Indoles ; Maleimides ; Mitogen-Activated Protein Kinases ; Morpholines ; Muscle, Smooth, Vascular ; Myocytes, Smooth Muscle ; Nitriles ; Nucleotides ; Onium Compounds ; Phenotype ; Phosphatidylinositol 3-Kinase ; Phosphorylation ; Protein Kinase C ; Receptors, Purinergic ; Suramin

OBJECTIVETo observe the expression and clinical implication of receptor for activated C kinase 1 (RACK1) in mononuclear cells and coronary atherosclerotic plaques from patients with coronary artery disease.

METHODSmRNA and protein expressions of RACK1 were detected in mononuclear cells from 29 patients with stable angina pectoris (SAP), 41 patients with acute coronary syndrome (ACS) and 30 healthy volunteers. RACK1 protein expression was also detected by immunohistochemistry in 17 coronary atherosclerotic plaques and 6 normal autopsy coronary samples.

RESULTS(1) mRNA expression of RACK1 was significantly upregulated in mononuclear cells from patients with ACS compared with those from patients with SAP (18.71 ± 5.45 vs. 12.18 ± 4.14, P < 0.05), and the latter was also significantly higher than in healthy controls (12.18 ± 4.14 vs. 3.65 ± 1.57, P < 0.05). (2) Similar changes were observed for protein expression of RACK1 for the three groups. (3) Increased expression of RACK1 was found in atherosclerotic plaques, especially in unstable plaques, positive RACK1 stain was evidenced in foam cells, inflammatory cells, smooth muscle cells and endothelial cells.

CONCLUSIONSThe expression of RACK1 is significantly upregulated in mononuclear cells from patients with coronary artery disease, especially in patients with ACS, and in coronary atherosclerotic plaques, especially in unstable plaques. Our results thus suggest that RACK1 might play an important role in the development and progression of coronary artery disease.

Adult ; Aged ; Case-Control Studies ; Coronary Artery Disease ; blood ; genetics ; pathology ; Female ; Gene Expression ; Humans ; Leukocytes ; metabolism ; Male ; Middle Aged ; Receptors for Activated C Kinase ; Receptors, Cell Surface ; genetics ; metabolism

To explore the intracellular signal pathways for beta-VLDL induced very low density lipoprotein receptor (VLDL-R) transcription up-regulation and their effects on lipid accumulation in macrophages, Western Blot was used to examine phosphorylated ERK1/2 protein and regulated effects by different singal kinase inhibitants. It was found that beta-VLDL induced an increase in ERK1/2 activity in a protein kinase C (PKC)-dependent manner in murine RAW264.7 macrophages. By using different protein kinases inhibitors or activators, it was observed that the effect of beta-VLDL induced VLDL receptor transcription, which was monitored by RT-PCR analysis of VLDL receptor mRNA, was not affected by the inhibitor of p38 kinase and cAMP analog, but extremely abolished by pretreating cells with PD98059, an inhibitor of ERK and GF 109203X, an inhibitor of PKC. These results demonstrated that the PKC-ERK1/2 cascade is the essential signaling pathway by which beta-VLDL activated VLDL-R mRNA expression. Inhibition of the ERK1/2 signaling cascade resulted in suppression of the cellular lipid accumulation induced by beta-VLDL in macrophages.
Cells, Cultured ; Lipoproteins, VLDL ; metabolism ; Macrophages ; cytology ; metabolism ; Mitogen-Activated Protein Kinase 1 ; metabolism ; physiology ; Mitogen-Activated Protein Kinase 3 ; metabolism ; physiology ; Protein Kinase C ; antagonists & inhibitors ; metabolism ; Receptors, LDL ; biosynthesis ; genetics ; Signal Transduction ; Transcription Factors ; metabolism ; Transcription, Genetic ; Up-Regulation

TLR6 forms a heterodimer with TLR2 and TLR4. While proinflammatory roles of TLR2 and TLR4 are well documented, the role of TLR6 in inflammation is poorly understood. In order to understand mechanisms of action of TLR6 in inflammatory responses, we investigated the effects of FSL-1, the TLR6 ligand, on expression of chemokine CCL2 and cytokine IL-1beta and determined cellular factors involved in FSL-1-mediated expression of CCL2 and IL-1beta in mononuclear cells. Exposure of human monocytic leukemia THP-1 cells to FSL-1 resulted not only in enhanced secretion of CCL2 and IL-1beta, but also profound induction of their gene transcripts. Expression of CCL2 was abrogated by treatment with OxPAPC, a TLR-2/4 inhibitor, while treatment with OxPAPC resulted in partially inhibited expression of IL-1beta. Treatment with FSL-1 resulted in enhanced phosphorylation of Akt and mitogen-activated protein kinases and activation of protein kinase C. Treatment with pharmacological inhibitors, including SB202190, SP6001250, U0126, Akt inhibitor IV, LY294002, GF109203X, and RO318220 resulted in significantly attenuated FSL-1-mediated upregulation of CCL2 and IL-1beta. Our results indicate that activation of TLR6 will trigger inflammatory responses by upregulating expression of CCL2 and IL-1beta via TLR-2/4, protein kinase C, PI3K-Akt, and mitogen-activated protein kinases.
Butadienes ; Chemokine CCL2 ; Chromones ; Humans ; Imidazoles ; Indoles ; Inflammation ; Leukemia ; Macrophages ; Maleimides ; Mitogen-Activated Protein Kinases ; Morpholines ; Nitriles ; Phosphatidylcholines ; Phosphorylation ; Protein Kinase C ; Pyridines ; Toll-Like Receptor 6 ; Toll-Like Receptors ; Up-Regulation

Dexamethasone (DEX), one of the corticosteroid hormones, is one of the most common therapeutic strategies in ophthalmological treatment. Despite its widespread use and clinical efficiency, little is known regarding the specific effects of DEX on cell growth, differentiation and cell death in human trabecular meshwork cells. The presence of the glucocorticoid receptor (GR, dexamethasone receptor) in TM-5 cell line, which was derived from the primary human trabecular meshwork cells, was verified by RT-PCR and western blot analysis. The effects of DEX on the cellular proliferation of TM5 cells were measured by a BrdU incorporation assay. Western blot analysis were used to examine the effects of DEX on the Ras/MEK/ERK signaling pathway. The total Ras, MEK1/2 and ERK1/2 protein levels as well as the levels of activated (phosphorylated) form were both significantly increased by the DEX treatment for 5 days. Both MEK1/2 and ERK1/2 were significantly activated by phosphorylation after 10 minutes. The dependence of this increased cell proliferation on GR activation by DEX and the sustained activation of ERK was examined using RU486 (a GR inhibitor) and U0126 (a MEK inhibitor). Both RU486 and U0126 prevented the induction of cell proliferation by the DEX treatment in the TM5 cells. In conclusion this study demonstrated that GR is expressed in TM5 cells. Secondly, DEX treatment for 5 days stimulates cell proliferation in TM5 cells, and that this increased proliferation effect is mediated by the Ras/MEK/ERK pathway.
Cell Division/*drug effects ; Cells, Cultured ; Dexamethasone/*pharmacology ; Human ; Mitogen-Activated Protein Kinase Kinases/metabolism ; Mitogen-Activated Protein Kinases/metabolism ; Protein-Serine-Threonine Kinases/metabolism ; Protein-Tyrosine Kinase/metabolism ; Proto-Oncogene Proteins c-raf/metabolism ; Receptors, Glucocorticoid/physiology ; Trabecular Meshwork/cytology/*drug effects

Neural stem cells and neural progenitor cells (NPCs) exist throughout life and are mobilized to replace neurons, astrocytes and oligodendrocytes after injury. Stromal cell-derived factor 1 (SDF-1, now named CXCL12) and its receptor CXCR4, an α-chemokine receptor, are critical for NPC migration into damaged areas of the brain. Our previous studies demonstrated that immune activated and/or HIV-1-infected human monocyte-derived-macrophages (MDMs) induced a substantial increase of SDF-1 production by human astrocytes. However, matrix metalloproteinase (MMP)-2, a protein up-regulated in HIV-1-infected macrophages, is able to cleave four amino acids from the N-terminus of SDF-1, resulting in a truncated SDF-1(5-67). In this study, we investigate the diverse signaling and function induced by SDF-1α and SDF-1(5-67) in human cortical NPCs. SDF-1(5-67) was generated by incubating human recombinant SDF-1α with MMP-2 followed by protein determination via mass spectrometry, Western blotting and ELISA. SDF-1α induced time-dependent phosphorylation of extracellular signal-regulated kinases (ERK) 1/2, Akt-1, and diminished cyclic adenosine monophosphate (cAMP). In contrast, SDF-1(5-67) failed to induce these signaling. SDF-1α activation of CXCR4 induced migration of NPCs, an effect that is dependent on ERK1/2 and Akt-1 pathways; whereas SDF-1(5-67) failed to induce NPC migration. This observation provides evidence that MMP-2 may affect NPC migration through post-translational processing of SDF-1α.
Cell Movement ; Cells, Cultured ; Chemokine CXCL12 ; metabolism ; Cyclic AMP ; metabolism ; Humans ; Matrix Metalloproteinase 2 ; metabolism ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Neural Stem Cells ; cytology ; metabolism ; Phosphorylation ; Proteolysis ; Proto-Oncogene Proteins c-akt ; metabolism ; Receptors, CXCR4 ; metabolism ; Signal Transduction