Up-regulation of nPKC contributes to proliferation of mice pulmonary artery smooth muscle cells in hypoxia-induced pulmonary hypertension.

This study is designed to investigate the role of novel protein kinases C (nPKC) in mediating pulmonary artery smooth muscle cells (PASMCs) proliferation in pulmonary hypertension (PH) and the underlying mechanisms. Mouse PASMCs was isolated using magnetic separation technology. The PASMCs were divided into 24 h group, 48 h group and 72 h group according to different hypoxia treatment time, then detected cell proliferation rate and nPKC expression level in each group. We treated PASMCs with agonists or inhibitors of PKCdelta (PKCδ) and PKCepsilon (PKCε) and exposed them to hypoxia or normoxia for 72 h, then measured the proliferation of PASMCs. We also constructed a lentiviral vector containing siRNA fragments for inhibiting PKCδ and PKCε to transfected PASMCs, then examined their proliferation. PASMCs isolated successfully by magnetic separation method and were in good condition. Hypoxia promoted the proliferation of PASMCs, and the treatment for 72 h had the most significant effect. Hypoxia upregulated the expression of PKCδ and PKCε in mouse PASMCs, leading to PASMCs proliferation. Moreover, Our study demonstrated that hypoxia induced upregulation of PKCδ and PKCε expression resulting to the proliferation of PASMCs via up-regulating the phosphorylation of AKT and ERK. Our study provides clear evidence that increased nPKC expression contributes to PASMCs proliferation and uncovers the correlation between AKT and ERK pathways and nPKC-mediated proliferation of PASMCs. These findings may provide novel targets for molecular therapy of pulmonary hypertension.

Tectoridin inhibits the progression of colon cancer through downregulating PKC/p38 MAPK pathway.

Colon cancer is one of the most familiar malignancies worldwide, with high morbidity and high mortality. This study intended to explore the role and mechanism of tectoridin (TEC) in regulating the progression of colon cancer. First, colon cancer cell lines (HCT116 and SW480 cells) were treated with different doses of TEC (0-200 µM). Then, CCK8 and clone formation experiments were performed to detect cell proliferation. Flow cytometry and western blot were conducted to examine apoptosis. Subsequently, Transwell assay and wound-healing test was employed to determine the effect of TEC on colon cancer cell invasion and migration. Next, western blot was performed to monitor the PKC/p38 MAPK pathway activation. In addition, a tumor model was established in nude mice to explore the effect of TEC on tumor growth in vivo. TEC dose-dependently dampened the proliferation, migration and invasion of colon cancer cells and facilitated their apoptosis. In addition, TEC abated the tumor cell growth in vivo. Besides, TEC dose-dependently suppressed the expression of PKC and p38 MAPK. Moreover, inhibiting the PKC pathway almost cancel out the anti-tumor effects induced by TEC. TEC attenuates the colon cancer progression by inhibiting the PKC/p38 MAPK pathway.

Regulation of mGluR1 on the Expression of PKC and NMDAR in Aluminum-Exposed PC12 Cells.

Aluminum demonstrates clear neurotoxicity and can cause Alzheimer's disease (AD)-like symptoms, including cognitive impairment. One toxic effect of aluminum is a decrease in synaptic plasticity, but the specific mechanism remains unclear. In this study, PC12 cells were treated with Al(mal)3 to construct a toxic cell model. (S)-3,5-Dihydroxyphenylglycine (DHPG), α-methyl-4-carboxyphenylglycine (MCPG), and mGluR1-siRNA were used to interfere with the expression of metabotropic glutamate receptor subtype 1 (mGluR1). Polymerase chain reaction and western blotting were used to investigate the expression of mGluR1, protein kinase C (PKC), and N-methyl-D-aspartate receptor (NMDAR) subunits. ELISA was used to detect PKC enzyme activity. In PC12 cells, mRNA and protein expressions of PKC and NMDAR subunits were inhibited by Al(mal)3. Aluminum may further regulate the expression of NMDAR1 and NMDAR2B through mGluR1 to regulate PKC enzyme activity, thereby affecting learning and memory functions. Furthermore, the results implied that the mGluR1-PKC-NMDAR signaling pathway may predominately involve positive regulation. These findings provide new targets for studying the neurotoxic mechanism of aluminum.

Resistin-like molecule ß acts as a mitogenic factor in hypoxic pulmonary hypertension via the Ca2+-dependent PI3K/Akt/mTOR and PKC/MAPK signaling pathways.

BACKGROUND: Pulmonary arterial smooth muscle cell (PASMC) proliferation plays a crucial role in hypoxia-induced pulmonary hypertension (HPH). Previous studies have found that resistin-like molecule ß (RELM-ß) is upregulated de novo in response to hypoxia in cultured human PASMCs (hPASMCs). RELM-ß has been reported to promote hPASMC proliferation and is involved in pulmonary vascular remodeling in patients with PAH. However, the expression pattern, effects, and mechanisms of action of RELM-ß in HPH remain unclear. METHODS: We assessed the expression pattern, mitogenetic effect, and mechanism of action of RELM-ß in a rat HPH model and in hPASMCs. RESULTS: Overexpression of RELM-ß caused hemodynamic changes in a rat model of HPH similar to those induced by chronic hypoxia, including increased mean right ventricular systolic pressure (mRVSP), right ventricular hypertrophy index (RVHI) and thickening of small pulmonary arterioles. Knockdown of RELM-ß partially blocked the increases in mRVSP, RVHI, and vascular remodeling induced by hypoxia. The phosphorylation levels of the PI3K, Akt, mTOR, PKC, and MAPK proteins were significantly up- or downregulated by RELM-ß gene overexpression or silencing, respectively. Recombinant RELM-ß protein increased the intracellular Ca2+ concentration in primary cultured hPASMCs and promoted hPASMC proliferation. The mitogenic effects of RELM-ß on hPASMCs and the phosphorylation of PI3K, Akt, mTOR, PKC, and MAPK were suppressed by a Ca2+ inhibitor. CONCLUSIONS: Our findings suggest that RELM-ß acts as a cytokine-like growth factor in the development of HPH and that the effects of RELM-ß are likely to be mediated by the Ca2+-dependent PI3K/Akt/mTOR and PKC/MAPK pathways.

Antidiabetic effect of a flavonoid-rich extract from Sophora alopecuroides L. in HFD- and STZ- induced diabetic mice through PKC/GLUT4 pathway and regulating PPARα and PPARγ expression.

HEADINGS ETHNOPHARMACOLOGICAL RELEVANCE: Sophora alopecuroides L. is a traditional ethnopharmacological plant, which is widely used in traditional Chinese medicine and Mongolian and Uighur medicine to ameliorate "thirst disease". AIM OF THE STUDY: This study aimed to investigate the antidiabetic activities and mechanisms of a flavonoid-rich extract from Sophora alopecuroides L. (SA-FRE) both in vivo and vitro. MATERIALS AND METHODS: The main six chemical constituents of SA-FRE were elucidated based on an off-line semi-preparative liquid chromatography nuclear magnetic resonance (LC-NMR) protocol. Myc-GLUT4-mOrange-L6 cell models and mouse model with diabetes induced by high-fat diet combined with STZ injection were respectively adopted to investigate the antidiabetic effects of SA-FRE both in vitro and vivo. RESULTS: In vivo, 4-week treatment of SA-FRE ameliorated hyperglycemia, dyslipidemia, and insulin resistance in diabetic mice. Mechanically, SA-FRE regulated PPARα and PPARγ expression in white adipose tissue (WAT) and liver, thereby ameliorating dyslipidemia. Moreover, SA-FRE increased the phosphorylation of PKC and further stimulated the GLUT4 expression in WAT and skeletal muscle, thus increasing the glucose utilization in vivo. In vitro, 50 µg/mL SA-FRE increased GLUT4 translocation to about 1.91-fold and glucose uptake to 1.82-fold in L6-myotubes. SA-FRE treatment increased the GLUT4 expression at both gene and protein levels. Furthermore, only Gö6983, a PKC inhibitor, reversed the SA-FRE-induced GLUT4 translocation and expression at the gene and protein levels. CONCLUSIONS: Generally, SA-FRE ameliorated hyperglycemia, dyslipidemia, and insulin resistance partly through activating PKC/GLUT4 pathway and regulating PPARα and PPARγ expression.

PKC downregulation upon rapamycin treatment attenuates mitochondrial disease.

Leigh syndrome is a fatal neurometabolic disorder caused by defects in mitochondrial function. Mechanistic target of rapamycin (mTOR) inhibition with rapamycin attenuates disease progression in a mouse model of Leigh syndrome (Ndufs4 knock-out (KO) mouse); however, the mechanism of rescue is unknown. Here we identify protein kinase C (PKC) downregulation as a key event mediating the beneficial effects of rapamycin treatment of Ndufs4 KO mice. Assessing the impact of rapamycin on the brain proteome and phosphoproteome of Ndufs4 KO mice, we find that rapamycin restores mitochondrial protein levels, inhibits signalling through both mTOR complexes and reduces the abundance and activity of multiple PKC isoforms. Administration of PKC inhibitors increases survival, delays neurological deficits, prevents hair loss and decreases inflammation in Ndufs4 KO mice. Thus, PKC may be a viable therapeutic target for treating severe mitochondrial disease.

Expression of syndecan-1, PKC and VEGF in rats with acute kidney injury and correlation between syndecan-1 and renal function.

OBJECTIVE: This study was designed to investigate the expression of syndecan-1 (Sdc-1), protein kinase C (PKC) and vascular endothelial growth factor (VEGF) in rats with acute kidney injury, as well as the association between Sdc-1 and indicators [such as serum creatinine (Scr) and blood urea nitrogen (BUN)] related to renal function. MATERIALS AND METHODS: A total of 120 clean grade 2-week-old SD rats were selected and randomized into experimental group and control group (n=60). At 12 h (T1), 24 h (T2), 36 h (T3), 48 h (T4) after the model was established, 3 mL blood from abdominal aorta was taken, and Sdc-1, PKC, VEGF, serum creatinine (Scr), urea nitrogen (BUN) and other indicators were detected by Enzyme-Linked Immunosorbent Assay (ELISA). RESULTS: The expression levels of Sdc-1, PKC and VEGF in the experimental group were increasing from T1 to T4, with statistically significant difference between every two time points (p<0.05); the expression levels of Scr and BUN in the experimental group was increasing from T1 to T4, with statistically significant difference between every two time points (p<0.05). The level of Sdc-1 in the serum of rats in the experimental group was positively correlated with Scr (r=0.668, p<0.001), negatively correlated with BUN (r=0.722, p<0.001), and positively correlated with BUN (r=0.722, p<0.001); PKC level was positively correlated with Scr (r=0.589, p<0.001), BUN (r=0.788, p<0.001), and VEGF level was positively correlated with Scr (r=0.666, p<0.001), BUN (r=0.784, p<0.001). CONCLUSIONS: As the concentration of syndecan-1 increases gradually, renal dysfunction aggravates accordingly, so syndecan-1 can be used as a marker of acute kidney injury and can be used to judge the degree of kidney injury at an early stage.

A dose-dependent response to MEK inhibition determines hypoblast fate in bovine embryos.

BACKGROUND: The segregation of the hypoblast and the emergence of the pluripotent epiblast mark the final stages of blastocyst formation in mammalian embryos. In bovine embryos the formation of the hypoblast has been partially studied, and evidence shows that MEK signalling plays a limited role in the segregation of this lineage. Here we explored the role of different signalling pathways during lineage segregation in the bovine embryo using immunofluorescence analysis of NANOG and SOX17 as readouts of epiblast and hypoblast, respectively. RESULTS: We show that SOX17 starts to be expressed in 16-32-cell stage embryos, whereas NANOG is first detected from 8-cell stage. SOX17 is first co-expressed with NANOG, but these markers become mutually exclusive by the late blastocyst stage. By assessing the expression kinetics of NANOG/SOX17 we show that inhibition of MEK signalling can eliminate SOX17 expression in bovine blastocysts, without altering NANOG expression. Modulation of WNT, PKC and LIF did not affect NANOG expression in the epiblast when used in combination with the ERK inhibitor. CONCLUSIONS: This study shows that SOX17 can be used as a reliable early marker of hypoblast in the bovine, and based on its expression profile we show that the hypoblast segregates in day 7 blastocysts. Furthermore, SOX17 expression is abolished using 1 µM of PD0325901, without affecting the NANOG population in the epiblast. Modulation of WNT, PKC and LIF are not sufficient to support enhanced NANOG expression in the epiblast when combined with ERK inhibitor, indicating that additional signalling pathways should be examined to determine their potential roles in epiblast expansion.

Diabetic Hemodialysis: Vitamin D Supplementation and its Related Signaling Pathways Involved in Insulin and Lipid Metabolism.

BACKGROUND: This study was conducted to determine the effects of vitamin D supplementation on some of the gene expressions related to insulin and lipid metabolism in diabetic hemodialysis (HD) patients. METHODS: A double-blind, randomized, placebo-controlled clinical trial was carried out in 55 patients with diabetic HD. The current project used two groups in which each subject received vitamin D supplements (50,000 IU, n=28) or placebo (50,000 IU, n=27) every 2 weeks for 12 weeks. Gene expression analyses (RT-PCR) were included to obtain the rate of gene expression of the related insulin and lipid metabolism genes in peripheral blood mononuclear cells (PBMCs) of patients with diabetic HD. RESULTS: Our data revealed that consumption of vitamin D supplementation enables to overexpress the peroxisome proliferation-activated receptor gamma (PPAR-γ) (P=0.001), AKT (P=0.04), PI3K (P=0.02), insulin receptor substrate-1 (IRS1) (P0.008) and glucose transporter type 4 (GLUT-4) (P=0.01) and downregulate the expression of protein kinase C (PKC) (P=0.001) in patients with diabetic HD than control group following the 12-week intervention. In addition, vitamin D supplementation downregulated low-density lipoprotein receptor (LDLR) (P=0.03) expression in the subjects with diabetic HD than the control group. Vitamin D supplementation did not show any effects on the expression of pyruvate dehydrogenase kinase 1 (PDK1) (P=0.37), IRS2 (P=0.90) and lipoprotein (a) [Lp(a)] (P=0.05). CONCLUSION: Our findings confirmed that diabetic HD subjects who received the vitamin D supplementation (for 12 weeks), showed a significant overexpression in the PPAR-γ, AKT, PI3K, IRS1 and GLUT4 genes, and also showed a significant downregulation in the PKC and LDLR genes. Moreover, no effects on PDK1, IRS2 and Lp(a) expression were observed.

PKM-ζ Expression Is Important in Consolidation of Memory in Prelimbic Cortex Formed by the Process of Behavioral Tagging.

Memories are acquired and stored in two forms, short-term memory (STM) and long-term memory (LTM). For the consolidation of LTM, de novo protein synthesis is required, which are also known as plasticity related proteins. Long-term potentiation is a form of synaptic plasticity and it is considered as a cellular model of learning and memory. One of the Long-term potentiation specific plasticity related proteins, PKM-ζ, is required for the formation of LTM as well as for the maintenance of Long-term potentiation. In our study, we have shown that for the consolidation of LTM, in addition to Long-term potentiation -specific plasticity related proteins, synaptic tags are required to interact with each other. In the present study, we investigated the involvement of Long-term potentiation -specific PKM-ζ and learning tags within a critical time window, which are required for the formation of LTM without affecting STM. Behavioral tagging is an established model for the assessment of some forms of learning and memory. Despite being studied for LTM formation for many years, no studies so far have investigated the role of PKM-ζ in Behavioral tagging model. Hence, by using these two different memories based tasks (i.e., Inhibitory avoidance and Novel object recognition tasks), we observed how PKM-ζ activated by exposing a novel arena after a weak training and led to the consolidation of memory. These findings thus show how the process of behavioral tagging activates Long-term potentiation -specific PKM-ζ for the formation of LTM.

Long non-coding RNA CCAT1/miR-148a/PKCζ prevents cell migration of prostate cancer by altering macrophage polarization.

BACKGROUND: Macrophage polarization plays an important role in tumor microenvironment, which regulated the prognosis of prostate cancer. However, the potential role of it is still need further identification. METHODS: The M1 Macrophages were inducted using 100 ng/mL LPS and 100 ng/mL IFN-γ, the M1 Macrophages were inducted using 20 ng/mL IL-4. TAMs were obtained by culturing monocytes for 7 days in RPMI 1640 10% FBS with 50% of conditioned medium from PC-3 cells real-time PCR was performed to determine the expression of miR-148a, CCAT1, and PKCζ. Western blot was used to measure the level of PKCζ. The cytokine IL-10 was determined using ELISA. Transwell chamber was carried out to determine cell migration. Luciferase reporter assay was used to determine the relationship between miR-148a and PKCζ. RESULTS: The expression of miR-148a was highest in TAMs, while CCAT1 and PKCζ were highest in M1 Macrophages. Overexpressed miR-148a promoted the level of IL-10 and cell migration. Down-regulated CCAT1 promoted the level of IL-10 and cell migration, while this effects were abolished by co-transfection of si-CCAT1 and miR-148a inhibitor. PKCζ is the target gene of miR-148a. The effects of overexpressed miR-148a on the level of IL-10, genes expression, and cell migration were abolished by miR-148a mimic and pcDNA-PKCζ. In vivo experiments verified the effects of CCAT1 and miR-148a on tumor growth. CONCLUSION: CCAT1 knockdown promoted M2 macrophages polarization by up-regulating miR-148a, while miR-148a up-regulation promoted M2 macrophages polarization by down-regulating the expression of PKCζ.

5-HT2A Receptor-Induced Morphological Reorganization of PKCγ-Expressing Interneurons Gates Inflammatory Mechanical Allodynia in Rat.

Mechanical allodynia, a widespread pain symptom that still lacks effective therapy, is associated with the activation of a dorsally directed polysynaptic circuit within the spinal dorsal horn (SDH) or medullary dorsal horn (MDH), whereby tactile inputs into deep SDH/MDH can gain access to superficial SDH/MDH, eliciting pain. Inner lamina II (IIi) interneurons expressing the γ isoform of protein kinase C (PKCγ+) are key elements for allodynia circuits, but how they operate is still unclear. Combining behavioral, ex vivo electrophysiological, and morphological approaches in an adult rat model of facial inflammatory pain (complete Freund's adjuvant, CFA), we show that the mechanical allodynia observed 1 h after CFA injection is associated with the following (1) sensitization (using ERK1/2 phosphorylation as a marker) and (2) reduced dendritic arborizations and enhanced spine density in exclusively PKCγ+ interneurons, but (3) depolarized resting membrane potential (RMP) in all lamina IIi PKCγ+/PKCγ- interneurons. Blocking MDH 5HT2A receptors (5-HT2AR) prevents facial mechanical allodynia and associated changes in the morphology of PKCγ+ interneurons, but not depolarized RMP in lamina IIi interneurons. Finally, activation of MDH 5-HT2AR in naive animals is enough to reproduce the behavioral allodynia and morphological changes in PKCγ+ interneurons, but not the electrophysiological changes in lamina IIi interneurons, induced by facial inflammation. This suggests that inflammation-induced mechanical allodynia involves strong morphological reorganization of PKCγ+ interneurons via 5-HT2AR activation that contributes to open the gate for transmission of innocuous mechanical inputs to superficial SDH/MDH pain circuitry. Preventing 5-HT2AR-induced structural plasticity in PKCγ+ interneurons might represent new avenues for the specific treatment of inflammation-induced mechanical hypersensitivity.SIGNIFICANCE STATEMENT Inflammatory or neuropathic pain syndromes are characterized by pain hypersensitivity such as mechanical allodynia (pain induced by innocuous mechanical stimuli). It is generally assumed that mechanisms underlying mechanical allodynia, because they are rapid, must operate at only the level of functional reorganization of spinal or medullary dorsal horn (MDH) circuits. We discovered that facial inflammation-induced mechanical allodynia is associated with rapid and strong structural remodeling of specifically interneurons expressing the γ isoform of protein kinase C (PKCγ) within MDH inner lamina II. Moreover, we elucidated a 5-HT2A receptor to PKCγ/ERK1/2 pathway leading to the behavioral allodynia and correlated morphological changes in PKCγ interneurons. Therefore, descending 5-HT sensitize PKCγ interneurons, a putative "gate" in allodynia circuits, via 5-HT2A receptor-induced structural reorganization.

Developmental transitions in amygdala PKC isoforms and AMPA receptor expression associated with threat memory in infant rats.

Although infants learn and remember, they rapidly forget, a phenomenon known as infantile amnesia. While myriad mechanisms impact this rapid forgetting, the molecular events supporting memory maintenance have yet to be explored. To explore memory mechanisms across development, we used amygdala-dependent odor-shock conditioning and focused on mechanisms important in adult memory, the AMPA receptor subunits GluA1/2 and upstream protein kinases important for trafficking AMPAR, protein kinase M zeta (PKMζ) and iota/lambda (PKCι/λ). We use odor-shock conditioning in infant rats because it is late-developing (postnatal day, PN10) and can be modulated by corticosterone during a sensitive period in early life. Our results show that memory-related molecules did not change in pups too young to learn threat (PN8) but were activated in pups old enough to learn (PN12), with increased PKMζ-PKCι/λ and GluA2 similar to that observed in adult memory, but with an uncharacteristic decrease in GluA1. This molecular signature and behavioral avoidance of the conditioned odor was recapitulated in PN8 pups injected with CORT before conditioning to precociously induce learning. Blocking learning via CORT inhibition in older pups (PN12) blocked the expression of these molecules. PN16 pups showed a more adult-like molecular cascade of increased PKMζ-PKCι/λ and GluA1-2. Finally, at all ages, zeta inhibitory peptide (ZIP) infusions into the amygdala 24 hr after conditioning blocked memory. Together, these results identify unique features of memory processes across early development: AMPAR subunits GluA1/2 and PKC isoform expression are differentially used, which may contribute to mechanisms of early life forgetting.

Copper-induced activation of TRP channels promotes extracellular calcium entry and activation of CaMK, PKA, PKC, PKG and CBLPK leading to increased expression of antioxidant enzymes in Ectocarpus siliculosus.

The existence of functional Transient Receptor Potential (TRP) channels was analyzed in Ectocarpus siliculosus using agonists of human TRPs and specific antagonists of TRPA1, TRPC5, TRPM8 and TRPV; intracellular calcium was detected for 60 min. Increases in intracellular calcium were observed at 13, 29, 39 and 50-52 min, which appeared to be mediated by the activation of TRPM8/V1 at 13 min, TRPV1 at 29 min, TRPA1/V1 at 39 min and TRPA1/C5 at 50-52 min. In addition, intracellular calcium increases appear to be due to extracellular calcium entry, not requiring protein kinase activation. On the other hand, 2.5 µM copper exposure induced increased intracellular calcium at 13, 29, 39 and 51 min, likely due to the activation of a TRPA1/V1 at 13 min, TRPA1/C5/M8 at 29 min, TRPC5/M8 at 39 min, and a TRPC5/V1 at 51 min. The increases in intracellular calcium induced by copper were due to extracellular calcium entry and required protein kinase activation. Furthermore, from 3 to 24 h, copper exposure induced an increase in the level of transcripts encoding antioxidant enzymes such as superoxide dismutase, ascorbate peroxidase, glutathione reductase and peroxiredoxin. The described upregulation decreased with inhibitors of CaMK, PKA, PKC, PKG and CBLPK, as well as with a mixture of TRP inhibitors. Thus, copper induces the activation of TRP channels allowing extracellular calcium entry as well as the activation of CaMK, PKA, PKC, PKG and CBLPK leading to increased expression of genes encoding antioxidant enzymes in E. siliculosus.

Peripheral Synthesis of an Atypical Protein Kinase C Mediates the Enhancement of Excitability and the Development of Mechanical Hyperalgesia Produced by Nerve Growth Factor.

Nerve growth factor (NGF) plays a key role in the initiation as well as the prolonged heightened pain sensitivity of the inflammatory response. Previously, we showed that NGF rapidly augmented both the excitability of isolated rat sensory neurons and the mechanical sensitivity of the rat's hind paw. The increase in excitability and sensitivity was blocked by the myristoylated pseudosubstrate inhibitor of atypical PKCs (mPSI), suggesting that an atypical PKC may play a key regulatory role in generating this heightened sensitivity. Our findings raised the question as to whether NGF directs changes in translational control, as suggested for long-lasting long-term potentiation (LTP), or whether NGF leads to the activation of an atypical PKC by other mechanisms. The current studies demonstrate that enhanced action potential (AP) firing produced by NGF was blocked by inhibitors of translation, but not transcription. In parallel, in vitro studies showed that NGF elevated the protein levels of PKMζ, which was also prevented by inhibitors of translation. Intraplantar injection of NGF in the rat hind paw produced a rapid and maintained increase in mechanical sensitivity whose onset was delayed by translation inhibitors. Established NGF-induced hypersensitivity could be transiently reversed by injection of rapamycin or mPSI. These results suggest that NGF produces a rapid increase in the synthesis of PKMζ protein in the paw that augments neuronal sensitivity and that the ongoing translational expression of PKMζ plays a critical role in generating as well as maintaining the heightened sensitivity produced by NGF.

Regulatory connection between the expression level of classical protein kinase C and pruning of climbing fibers from cerebellar Purkinje cells.

Cerebellar Purkinje cells (PCs) express two members of the classical protein kinase C (cPKC) subfamily, namely, PKCα and PKCγ. Previous studies on PKCγ knockout (KO) mice have revealed a critical role of PKCγ in the pruning of climbing fibers (CFs) from PCs during development. The question remains as to why only PKCγ and not PKCα is involved in CF synapse elimination from PCs. To address this question, we assessed the expression levels of PKCγ and PKCα in wild-type (WT) and PKCγ KO PCs using PC-specific quantitative real-time reverse transcription-polymerase chain reaction, western blotting, and immunohistochemical analysis. The results revealed that the vast majority of cPKCs in PCs were PKCγ, whereas PKCα accounted for the remaining minimal fraction. The amount of PKCα was not up-regulated in PKCγ KO PCs. Lentiviral expression of PKCα in PKCγ KO PCs resulted in a 10-times increase in the amount of PKCα mRNA in the PKCγ KO PCs, compared to that in WT PCs. Our quantification showed that the expression levels of cPKC mRNA in PKCγ KO PCs increased roughly from 1% to 22% of that in WT PCs solely through PKCα expression. The up-regulation of PKCα in PKCγ KO PCs significantly rescued the impaired CF synapse elimination. Although both PKCα and PKCγ are capable of pruning supernumerary CF synapses from developing PCs, these results suggest that the expression levels of cPKCs in PKCγ KO PCs are too low for CF pruning.

Expression of Plant Receptor Kinases in Yeast.

The budding yeast Saccharomyces cerevisiae is a useful system to express recombinant proteins and analyze protein-protein interaction. Membrane-spanning proteins like plant receptor kinases find their way to the plasma membrane when expressed in yeast and seem to retain their structure and function. Here, we describe a general yeast DNA transformation procedure based on lithium acetate, salmon sperm DNA, and polyethylene glycol used to express recombinant proteins. Yeast cells expressing plant receptor kinases can be used for in vivo and in vitro studies of receptor function.

PKCζ in prostate cancer cells represses the recruitment and M2 polarization of macrophages in the prostate cancer microenvironment.

Tumor-associated macrophages are key regulators of the complex interplay between tumor and tumor microenvironment. M2 Macrophages, one type of tumor-associated macrophages, are involved in prostate cancer growth and progression. Protein kinase C zeta has been shown to suppress prostate cancer cell growth, invasion, and metastasis as a tumor suppressor; however, its role in chemotaxis and activation of tumor-associated macrophages remains unclear. Here, we investigated the role of protein kinase C zeta of prostate cancer cells in regulation of macrophage chemotaxis and M2 phenotype activation. Immunohistochemistry was performed to analyze the expression of protein kinase C zeta and the number of CD206+ M2 macrophages in human prostate tissue. Macrophage chemotaxis and polarization were examined using Transwell migration assays and a co-culture system. Quantitative real-time polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay were used to detect M2 markers, protein kinase C zeta, interleukin-4, and interleukin-10 expression. We found the expression of protein kinase C zeta increased in prostate cancer tissues, especially in the early stage, and was negatively associated with tumor grade and the number of CD206+ macrophages. Inhibition of protein kinase C zeta expression in prostate cancer cells promoted chemotaxis of peripheral macrophages and acquisition of M2 phenotypic features. These results were further supported by the finding that silencing of endogenous protein kinase C zeta promoted the expression of prostate cancer cell-derived interleukin-4 and interleukin-10. These results suggest that protein kinase C zeta plays an important role in reducing infiltration of tumor-associated macrophages and activation of a pro-tumor M2 phenotype, which may constitute an important mechanism by which protein kinase C zeta represses cancer progression.

Higher PKD3 expression in hepatocellular carcinoma (HCC) tissues predicts poorer prognosis for HCC patients.

AIM: Protein kinase D (PKD) acts as a key mediator in several cancer development signaling pathways. The aim of this study was to investigate the clinical significance and prognostic value of PKD3 expression in hepatocellular carcinoma (HCC) patients after hepatectomy. METHODS: PKD3 mRNA and protein expression levels in tumor and matched non-tumoral (NT) tissues, HCC cell lines were evaluated by quantitative PCR (qRT-PCR), western blotting and immunohistochemical staining (IHC). Additionally, PKD3 mRNA expression in HCC tissues correlated with clinicopathological characteristics and survival. RESULTS: PKD3 mRNA and protein expression was elevated in HCC tissues and HCC cell lines. Our data also showed that in HCC patients after resection, a high-expression of PKD3 mRNA and protein significantly correlated with multiple tumor nodules (P=0.009, P=0.020, respectively), poor tumor differentiation (P=0.001, P=0.004, respectively), high serum AFP level (P=0.005, P=0.002, respectively), vascular invasion (P=0.006, P=0.009, respectively) and advanced AJCC stage (P=0.001, P=0.022, respectively). A Kaplan-Meier analysis indicated that an elevated PKD3 mRNA expression correlated with shorter overall survival (OS) (P<0.001) and disease-free survival (DFS) (P=0.008). Moreover, multivariate analysis showed that a high-expression of PKD3 was an independent prognostic factor for three-year overall survival rate. CONCLUSIONS: Our findings suggest that abnormal PKD3 expression might contribute to HCC progression. Furthermore, high PKD3 expression predicts a poor prognosis in HCC patients after hepatectomy.

The effect of cortisol in rat steatotic and non-steatotic liver transplantation from brain-dead donors.

In the present study, we examined the effects of cortisol on steatotic and non-steatotic liver grafts from brain-dead donors and characterized the underlying mechanisms involved. Non-steatotic liver grafts showed reduced cortisol and increased cortisone levels in association with up-regulation of enzymes that inactivate cortisol. Conversely, steatotic liver grafts exhibited increased cortisol and reduced cortisone levels. The enzymes involved in cortisol generation were overexpressed, and those involved in cortisol inactivation or clearance were down-regulated in steatotic liver grafts. Exogenous administration of cortisol negatively affected hepatic damage and survival rate in non-steatotic liver transplantation (LT); however, cortisol treatment up-regulated the phosphoinositide 3-kinase (PI3K)-protein kinase C (PKC) pathway, resulting in protection against the deleterious effects of brain-dead donors on damage and inflammatory response in steatotic LT as well as in increased survival of recipients. The present study highlights the differences in the role of cortisol and hepatic mechanisms that regulate cortisol levels based on the type of liver. Our findings suggest that cortisol treatment is a feasible and highly protective strategy to reduce the adverse effects of brain-dead donor livers in order to ultimately improve liver graft quality in the presence of steatosis, whereas cortisol treatment would not be recommended for non-steatotic liver grafts.