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Peripheral nerve injury repair requires a certain degree of cooperation between axon regeneration and Wallerian degeneration. Therefore, investigating how axon regeneration and degeneration work together to repair peripheral nerve injury may uncover the molecular mechanisms and signal cascades underlying peripheral nerve repair and provide potential strategies for improving the low axon regeneration capacity of the central nervous system. In this study, we applied weighted gene co-expression network analysis to identify differentially expressed genes in proximal and distal sciatic nerve segments from rats with sciatic nerve injury. We identified 31 and 15 co-expression modules from the proximal and distal sciatic nerve segments, respectively. Functional enrichment analysis revealed that the differentially expressed genes in proximal modules promoted regeneration, while the differentially expressed genes in distal modules promoted neurodegeneration. Next, we constructed hub gene networks for selected modules and identified a key hub gene, Kif22, which was up-regulated in both nerve segments. In vitro experiments confirmed that Kif22 knockdown inhibited proliferation and migration of Schwann cells by modulating the activity of the extracellular signal-regulated kinase signaling pathway. Collectively, our findings provide a comparative framework of gene modules that are co-expressed in injured proximal and distal sciatic nerve segments, and identify Kif22 as a potential therapeutic target for promoting peripheral nerve injury repair via Schwann cell proliferation and migration. All animal experiments were approved by the Institutional Animal Ethics Committee of Nantong University, China (approval No. S20210322-008) on March 22, 2021.

Cdc42 is a key factor in directed cell migration and accumulates at the leading edge of migrating cells. However, what kind of proteins control Cdc42 and when is unclear. After mechanical wounding, protein kinase C α (PKCα), a conventional PKC isozyme, begins to accumulate at the edges of cells adjacent to the wounded cells (WCs). In this study, we hypothesized that PKCα may be implicated in directed cell migration at an early stage before Cdc42 controls the migration. We focused on the spatiotemporal distribution of PKCα, Cdc42, and Rac1 before cell migration. After wounding, at the edges of cells adjacent to the WCs, PKCα accumulation, Cdc42 accumulation, Rac1 accumulation, and filopodia formation occurred in that order. The PKCα inhibitor suppressed Cdc42 accumulation at the cell edges. These results suggest that inhibition of PKCα activity inhibits cell migration. In addition, it is not Cdc42 but PKCα that may decide the direction of cell migration. • PKCα accumulates at edge of cells faster than Cdc42 after mechanical wounding. • PKCα controls Cdc42 and directed cell migration. • PKCα, not Cdc42, is a trigger for directed cell migration after mechanical wounding. [ABSTRACT FROM AUTHOR]

The metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), inhibits pro-oncogenic signaling in pancreatic cancer (PC). This investigation dissected a novel mechanism induced by NDRG1 on WNT/β-catenin signaling in multiple PC cell types. NDRG1 overexpression decreased β-catenin and downregulated glycogen synthase kinase-3β (GSK-3β) protein levels and its activation. However, β-catenin phosphorylation at Ser33, Ser37, and Thr41 are classically induced by GSK-3β was significantly increased after NDRG1 overexpression, suggesting a GSK-3β-independent mechanism. Intriguingly, NDRG1 overexpression upregulated protein kinase Cα (PKCα), with PKCα silencing preventing β-catenin phosphorylation at Ser33, Ser37, and Thr41, and decreasing β-catenin expression. Further, NDRG1 and PKCα were demonstrated to associate, with PKCα stabilization occurring after NDRG1 overexpression. PKCα half-life increased from 1.5 ± 0.8 h (3) in control cells to 11.0 ± 2.5 h (3) after NDRG1 overexpression. Thus, NDRG1 overexpression leads to the association of NDRG1 with PKCα and PKCα stabilization, resulting in β-catenin phosphorylation at Ser33, Ser37, and Thr41. The association between PKCα, NDRG1, and β-catenin was identified, with the formation of a potential metabolon that promotes the latter β-catenin phosphorylation. This anti-oncogenic activity of NDRG1 was multi-modal, with the above mechanism accompanied by the downregulation of the nucleo-cytoplasmic shuttling protein, p21-activated kinase 4 (PAK4), which is involved in β-catenin nuclear translocation, inhibition of AKT phosphorylation (Ser473), and decreased β-catenin phosphorylation at Ser552 that suppresses its transcriptional activity. These mechanisms of NDRG1 activity are important to dissect to understand the marked anti-cancer efficacy of NDRG1-inducing thiosemicarbazones that upregulate PKCα and inhibit WNT signaling., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Glucose fluctuations may contribute to large conductance calcium activated potassium (BK) channel dysfunction. However, the underlying mechanisms remain elusive. The aim of this study was to investigate the molecular mechanisms involved in BK channel dysfunction as a result of glucose fluctuations. A rat diabetic model was established through the injection of streptozotocin. Glucose fluctuations in diabetic rats were induced via consumption and starvation. Rat coronary arteries were isolated and coronary vascular tensions were measured after three weeks. Rat coronary artery smooth muscle cells were isolated and whole-cell BK channel currents were recorded using a patch clamp technique. Human coronary artery smooth muscle cells in vitro were used to explore the underlying mechanisms. After incubation with iberiotoxin (IBTX), the Δ tensions (% Max) of rat coronary arteries in the controlled diabetes mellitus (C-DM), the uncontrolled DM (U-DM) and the DM with glucose fluctuation (GF-DM) groups were found to be 84.46 ± 5.75, 61.89 ± 10.20 and 14.77 ± 5.90, respectively (P <.05), while the current densities of the BK channels in the three groups were 43.09 ± 4.35 pA/pF, 34.23 ± 6.07 pA/pF and 17.87 ± 4.33 pA/pF, respectively (P <.05). The Δ tensions (% Max) of rat coronary arteries after applying IBTX in the GF-DM rats injected with 0.9% sodium chloride (NaCl) (GF-DM + NaCl) and the GF-DM rats injected with N-acetyl-L-cysteine (NAC) (GF-DM + NAC) groups were found to be 8.86 ± 1.09 and 48.90 ± 10.85, respectively (P <.05). Excessive oxidative stress and the activation of protein kinase C (PKC) α and nuclear factor (NF)-κB induced by glucose fluctuations promoted the decrease of BK-β1 expression, while the inhibition of reactive oxygen species (ROS), PKCα, NF-κB and muscle ring finger protein 1 (MuRF1) reversed this effect. Glucose fluctuations aggravate BK channel dysfunction via the ROS overproduction and the PKCα/NF-κB/MuRF1 signaling pathway. Illustration showing the role of PKCα/NF-κB/MuRF1 signaling in the regulation of BK channel in response to glucose fluctuation. Under conditions of glucose fluctuations, there is an activation of PKC, which in turn promotes NF-κB/p65 nuclear translocation, facilitates MuRF1-dependent BK-β1 protein degradation. Unlabelled Image • Glucose fluctuations aggravate coronary vasodilation dysfunction. • BK channels are impaired by BK-β1 degradation due to glucose fluctuations. • ROS overproduction is enhanced by glucose fluctuations. • Glucose fluctuations activate PKCα/NF-κB/MuRF1 signaling pathway. [ABSTRACT FROM AUTHOR]

Previously, we have shown that active protein kinase Cα (PKCα) promotes recovery of mitochondrial function after injury in vitro [Nowak G & Bakajsova D (2012) Am J Physiol Renal Physiol 303, F515‐F526]. This study examined whether PKCα regulates recovery of mitochondrial and kidney functions after ischemia‐induced acute injury (AKI) in vivo. Markers of kidney injury were increased after bilateral ischemia and returned to normal levels in wild‐type (WT) mice. Maximum mitochondrial respiration and activities of respiratory complexes and FoF1‐ATPase decreased after ischemia and recovered in WT mice. Reperfusion after ischemia was accompanied by translocation of active PKCα to mitochondria. PKCα deletion reduced mitochondrial respiration and activities of respiratory complex I and FoF1‐ATPase in noninjured kidneys, indicating that PKCα is essential in developing fully functional renal mitochondria. These changes in PKCα‐deficient mice were accompanied by lower levels of complex I subunits (NDUFA9 and NDUFS3) and the γ‐subunit of FoF1‐ATPase. Also, lack of PKCα exacerbated ischemia‐induced decreases in respiration, complex I and FoF1‐ATPase activities, and blocked their recovery after injury, indicating a crucial role of PKCα in promoting mitochondrial recovery after AKI. Further, PKCα deletion exacerbated acetylation and succinylation of key mitochondrial proteins of energy metabolism after ischemia due to decreases in deacetylase and desuccinylase (sirtuin3 and sirtuin5) levels in renal mitochondria. Thus, our data show a novel role for PKCα in regulating levels of mitochondrial sirtuins and acetylation and succinylation of key mitochondrial proteins. We conclude that PKCα deletion: (a) affects renal physiology by decreasing mitochondrial capacity for maximum respiration; (b) blocks recovery of mitochondrial functions, renal morphology, and functions after AKI; and (c) decreases survival after AKI. Enzymes: Protein kinase C: EC 2.7.11.13; NADH : ubiquinone reductase (H+‐translocating; complex I): EC 7.1.1.2; FoF1‐ATPase (H+‐transporting two‐sector ATPase): EC 7.1.2.2; Succinate : ubiquinone oxidoreductase (complex II): EC 1.3.5.1; Ubiquinol : cytochrome‐c reductase (complex III): EC 7.1.1.8; Cytochrome c oxidase (complex IV): EC 1.9.3.1; NAD‐dependent protein deacetylase sirtuin‐3, mitochondrial: EC 2.3.1.286; NAD‐dependent protein deacetylase sirtuin‐5, mitochondrial: EC 3.5.1.‐; Proteinase K (peptidase K): EC 3.4.21.64. [ABSTRACT FROM AUTHOR]

• PKCα is upregulated in Schwann cells of distal nerve stumps as long as 28 days in rat sciatic nerve transection model. • PKCα promotes Schwann cell proliferation and migration in SCs. • PKCα promotes proliferation and migration via ERK pathway. Wallerian degeneration (WD) and axon regeneration generally take place following peripheral nerve injury (PNI). Schwann cells (SCs) and macrophages play major role in WD. SCs, acting as repair cells and primary signal mediators, dedifferentiate and proliferate to remove the debris, form Büngner's bands and secrete trophic factors during these processes. However, the underlying mechanisms remain poorly understood. Here, we found that protein kinase Cα (PKCα), a serine/threonine kinase, expressed in SCs was significantly up-regulated after PNI. Activating PKCα with phorbol 12-myristate 13-acetate (PMA), a phorbol ester binds and activates PKCα) promoted SCs proliferation and migration. While, silence of PKCα by siRNAs inhibited these processes. PD184352, an inhibitor of MEK1, reversed the effect induced by PMA on SCs. Mechanism studies revealed that PKCα functioned through activating the ERK signaling pathway. Furthermore, PKCα also exhibited a neuroprotective role by upregulating the expression of neurotrophic factors in SCs. To sum up, this study offers novel insights for clarifying our understanding of the involvement of PKCα in the mechanism of peripheral nerve degeneration as well as regeneration. [ABSTRACT FROM AUTHOR]

Mechanical wounding of an endothelial monolayer induces an immediate Ca2+ wave. Several hours later, the denuded area is covered by endothelial cells (ECs) that migrate to the wound. This migration process is closely related to protein kinase Cα (PKCα), a Ca2+-dependent protein that translocates from the cytosol to the cell membrane. Because the cells adjacent to the wounded area are the first to migrate into the wound, we investigated whether a mechanical wound immediately induces PKCα translocation in adjacent cells. We monitored Ca2+ dynamics and PKCα translocation simultaneously using fluorescent microscopy. For this simultaneous observation, we used Fura-2–acetoxymethyl ester to visualize Ca2+ and constructed a green fluorescent protein-tagged fusion protein to visualize PKCα. Mechanical wounding of the endothelial monolayer induced an immediate Ca2+ wave in cells adjacent to the wounded cells before their migration. Almost concurrently, PKCα in the neighboring cells translocated to the cell membrane, then accumulated at the periphery near the wounded cell. This report is the first description of this biphasic and directed translocation of PKCα in cells before cell migration. Our results may provide new insights into the directed migration of ECs.

Lung adenocarcinoma is the most common histologic subtype of lung cancer. The aim of the present study was to assess the expression of hepatoma-derived growth factor (HDGF) and protein kinase Cα (PRKCA) in lung adenocarcinoma (LADC), and to determine the association between the combined expression of these two proteins and clinicopathological characteristics of patients with LADC. The expression of HDGF and PRKCA mRNA was assessed by GEO database analysis, and HDGF and PRKCA protein levels were examined by immunohistochemistry using a tissue microarray. High HDGF and PRKCA expression was observed in LADC tissue compared to normal samples, and increased HDGF and PRKCA expression was associated with AJCC clinical stage, tumor classification, node classification, and lymph node metastasis. GEO database analysis revealed no significant differences between HDGF mRNA and PRKCA mRNA in LADC tissue. However, high PRKCA protein expression was associated with high HDGF protein expression, and patients with high HDGF and PRKCA expression exhibited poorer overall survival rates than patients with low expression levels of the two proteins. The results of the present study suggest that upregulation of both HDGF and PRKCA may be an unfavourable factor for lung adenocarcinoma progression. [ABSTRACT FROM AUTHOR]

Lipin-1 is an Mg2+-dependent phosphatidate phosphatase that facilitates the dephosphorylation of phosphatidic acid to generate diacylglycerol. Little is known about the expression and function of lipin-1 in normal human epidermal keratinocytes (NHEKs). Here, we demonstrate that lipin-1 is present in basal and spinous layers of the normal human epidermis, and lipin-1 expression is gradually downregulated during NHEK differentiation. Interestingly, lipin-1 knockdown (KD) inhibited keratinocyte differentiation and caused G1 arrest by upregulating p21 expression. Cell cycle arrest by p21 is required for commitment of keratinocytes to differentiation, but must be downregulated for the progress of keratinocyte differentiation. Therefore, reduced keratinocyte differentiation results from sustained upregulation of p21 by lipin-1 KD. Lipin-1 KD also decreased the phosphorylation/activation of protein kinase C (PKC)α, whereas lipin-1 overexpression increased PKCα phosphorylation. Treatment with PKCα inhibitors, like lipin-1 KD, stimulated p21 expression, while lipin-1 overexpression reduced p21 expression, implicating PKCα in lipin-1-induced regulation of p21 expression. Taken together, these results suggest that lipin-1-mediated downregulation of p21 is critical for the progress of keratinocyte differentiation after the initial commitment of keratinocytes to differentiation induced by p21, and that PKCα is involved in p21 expression regulation by lipin-1.

(1) Background: It is known that sickle cells contain a higher amount of Ca2+ compared to healthy red blood cells (RBCs). The increased Ca2+ is associated with the most severe symptom of sickle cell disease (SCD), the vaso-occlusive crisis (VOC). The Ca2+ entry pathway received the name of Psickle but its molecular identity remains only partly resolved. We aimed to map the involved Ca2+ signaling to provide putative pharmacological targets for treatment. (2) Methods: The main technique applied was Ca2+ imaging of RBCs from healthy donors, SCD patients and a number of transgenic mouse models in comparison to wild-type mice. Life-cell Ca2+ imaging was applied to monitor responses to pharmacological targeting of the elements of signaling cascades. Infection as a trigger of VOC was imitated by stimulation of RBCs with lysophosphatidic acid (LPA). These measurements were complemented with biochemical assays. (3) Results: Ca2+ entry into SCD RBCs in response to LPA stimulation exceeded that of healthy donors. LPA receptor 4 levels were increased in SCD RBCs. Their activation was followed by the activation of Gi protein, which in turn triggered opening of TRPC6 and CaV2.1 channels via a protein kinase Cα and a MAP kinase pathway, respectively. (4) Conclusions: We found a new Ca2+ signaling cascade that is increased in SCD patients and identified new pharmacological targets that might be promising in addressing the most severe symptom of SCD, the VOC.

Both decreased autophagy positive regulator AMP activated protein kinase (AMPK) level and promoted mitophagy are observed in oxygen‐glucose deprivation (OGD) cardiomyocytes treated with pigment epithelium‐derived factor (PEDF). This contradictory phenomenon and its underlying mechanisms have not been thoroughly elucidated. Our previous study reveals that PEDF increases the protein kinase Cα (PKCα) and phospho‐PKCα (p‐PKCα) contents to promote mitophagy. Thus, we investigated the association between PKCα and mitophagy. Here we identify an interaction between PKCα and Unc‐51‐like kinase 1 (ULK1), essential component of mitophagy. Further analyses show this is a direct interaction within a domain of ULK1 that termed the serine/threonine‐rich domain (S/T domain). Notably, a deletion mutant ULK1 that lacks the binding domain is defective in mediating PEDF‐induced mitophagy. Furthermore, we demonstrate that ULK1 is phosphorylated at Ser317/555/777 and Raptor is also phosphorylated by phospho‐PKCα. Phospho‐ULK1 (p‐ULK1) at these sites are all essential for PEDF‐induced mitophagy and reduce the release of mitochondrial ROS and DNA. This study therefore identifies a previously uncharacterized interaction between the ULK1 and PKCα that can replace the AMPK‐dependent mitophagy processes. [ABSTRACT FROM AUTHOR]

Abnormal blood-brain barrier (BBB) is a common pathological feature in brain damage. In the present study, a brain microvascular endothelial cell (BMEC) model was established to determine the role of the toll-like receptor 4 (TLR4)/protein kinase Cα (PKCα)/occludin signaling pathway in BBB dysfunction. Three small interfering (si)RNAs directed against PKCα were designed to investigate the molecular mechanisms of PKCα underlying BBB damage. BMECs were divided into 4 groups: Control group, TAK-242 (a TLR4 inhibitor) group, PKCα-siRNA group and TAK-242+PKCα-siRNA group. The results indicated that siRNA-3 was the most effective at silencing PKCα gene expression. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis indicated no significant difference of TLR4 mRNA expression levels between three different treated groups and the Control group. However, PKCα mRNA expression in the PKCα-siRNA and TAK-242+PKCα-siRNA groups were significantly decreased compared with that in Control and TAK-242 groups. In addition, occludin mRNA expression in PKCα-siRNA and TAK-242+PKCα-siRNA groups were significantly higher compared with the Control group. Meanwhile, occluding expressions in three treated groups were also significantly higher compared with the Control group. Furthermore, TAK-242 treatment, PKCα-siRNA treatment, and TAK-242+PKCα-siRNA treatment could promote occludin junctional labeling compared with the Control group. The permeability of PKCα-siRNA and TAK-242+PKCα-siRNA groups was significantly promoted compared with the control group. The TLR4/PKCα/occludin signaling pathway was closely related to BBB damage. The present study will lead to an improved molecular understanding of BBB damage in the future. [ABSTRACT FROM AUTHOR]

Hypoxic pulmonary hypertension (HPH) may contribute to vascular remodeling, and pulmonary artery smooth muscle cell (PASMC) proliferation has an important role in this process. However, no relevant information concerning the role and mechanism of protein kinase C (PKC)α in hypoxia‑induced rat PASMC proliferation has been elucidated. The present study aimed to further investigate this by comparison of rat PASMC proliferation among normoxia for 72 h (21% O2), hypoxia for 72 h (3% O2), hypoxia + promoter 12‑myristate 13‑acetate control, hypoxia + safingol control, hypoxia + PD98059 control and hypoxia + U0126 control groups. The present study demonstrated that protein expression levels of PKCα in rat PASMCs were elevated. In conclusion, through activating the extracellular signal‑regulated 1/2 signaling pathway, PKCα is involved in and initiates PASMC proliferation, thus bringing about pulmonary artery hypertension. These results add to the understanding of the mechanism PKCα in PH formation and lays a theoretical basis for prevention as well as treatment of HPH. [ABSTRACT FROM AUTHOR]

Brain microvascular endothelial cells (BMECs) are the primary component of the blood-brain barrier (BBB). Tight junction (TJ) proteins, including claudin, occludin and zonula occludens (ZO)-1, ZO-2 and ZO-3, maintain the structural integrity of BMECs. Ethanol activates the assembly and disassembly of TJs, which is a process that is regulated by protein kinase C (PKC). In addition, ethanol treatment leads to the loss of structural integrity, which damages the permeability of the BBB and subsequently affects central nervous system homeostasis, thus allowing additional substances to enter the brain. However, the mechanisms underlying ethanol-induced loss of BBB structure remain unknown. It has been hypothesized that long-term exposure to ethanol reduces the expression of claudin-5, occludin and ZO-1 via the PKC signaling pathway, thereby affecting BBB structural integrity. In the current study, the human cerebral microvascular endothelial cell line, HCMEC/D3, was treated with 50, 100, 200 and 400 mM ethanol for 24, 48 and 72 h. Cell viability was determined using an MTS assay. The expression of claudin-5, occludin and ZO-1 protein and mRNA was measured using western blot analysis and reverse transcription-quantitative polymerase chain reaction, respectively. Following the pretreatment of HCMEC/D3 cells with the PKCα-specific inhibitor, safingol (10 μmol/l), the expression of claudin-5, occludin, ZO-1 and phosphorylated (p)-PKCα was measured using western blot analysis, and PKCα localization was determined by immunofluorescence. With increasing concentrations of ethanol, the expression of claudin-5, occludin and ZO-1 protein decreased, while the expression of claudin-5, occludin and ZO-1 mRNA increased. Exposure to ethanol significantly increased the expression of p-PKCα, whereas no significant effect on the expression of PKCα was observed. Following 48 h treatment with 200 mM ethanol, the expression of claudin-5, occludin and ZO-1 protein was significantly decreased when compared with the control. By contrast, the expression of p-PKCα was increased, and increased translocation of PKCα from the cytoplasm to the nuclear membrane and nucleus was observed. In addition, the results demonstrated that safingol significantly reversed these effects of ethanol. In conclusion, long-term exposure to ethanol downregulates the expression of claudin-5, occludin and ZO-1 protein in HCMEC/D3 s, and this effect may be mediated via activation of PKCα. [ABSTRACT FROM AUTHOR]

Kidney cancer is among the most important causes of cancer-associated mortality worldwide. The present study aimed to evaluate protein kinase C α (PKCα) expression in kidney cancer tissues and cell lines, and its significance in apoptosis and migration. Expression of PKCα was analyzed using quantitative polymerase chain reaction and western blotting. In addition, the inhibitor of PKCα (calphostin C and GO6976) was used to treat kidney cancer cells. The ACHN cell line was generated with PKCα-small-interfering RNA (siRNA) and a stable expression of PKCα, in order to facilitate the analysis of apoptosis and migration of PKCα during knockdown and inactivation. Flow cytometry was used to determine the rates of apoptosis. Immunohistochemical staining was used to identify the localization of PKCα in renal clear cell carcinoma and normal sections. PKCα expression in normal tissues was found to be greater than in cancerous tissues. Furthermore, apoptosis was not promoted with PKCα inhibitors or PKCα-siRNA treatment, and a decrease of the migration ability was observed following transfection with PKCα-dominant negative. The results indicated that inhibition of PKCα might not contribute to apoptosis progression in kidney carcinoma. [ABSTRACT FROM AUTHOR]

Previous studies have demonstrated the important role of angiotension II (AngII) in promoting proliferation of myofibroblasts (myoFbs) and myocardial fibrosis. However, the underlying mechanisms and the role of oxygen free radicals in the proliferation of myofibroblasts induced by AngII are unclear. The present study was designed to shed light on this issue through exploration of AngII signaling pathways via in vitro experiments. Primary cultures of neonatal rat myoFbs were divided into five groups which were treated with AngII (10−8 to 10−6 M), AngII with the antioxidant N-acetyl-L-cysteine (NAC), or normal culture medium. We observed the proliferation of myoFbs as induced by AngII at different concentrations with MTT. Reactive oxygen species (ROS) levels in myoFbs were detected by monitoring the fluorescence of 2′,7′-dichlorofluorescein. The contents and levels of oxygen free radicals (OH·) in the three groups were detected by spectrophotometer, immunocytochemical staining, and confocal fluorescence. Western blot and image analysis were used to measure membrane translocation and expression of phospho-protein kinase Cα. MyoFbs incubated with AngII (10−8 to 10−6 M) for 24 h increased their rate of proliferation, the content of OH·, and expression of ROS (P

The susceptibility to drug abuse may be associated with the structural and/or functional changes in the reward-related brain regions induced by drug exposure during sensitive periods of embryonic development. Previously, we have found that prenatal morphine exposure during embryonic days 17–20 may be crucial for developing the susceptibility to morphine reward after hatching. However, the underlying structure and cellular mechanisms need further investigation. In the present study, the chicks of a few days old, which were prenatally exposed to morphine during E17-20, obviously showed higher preference for the morphine-paired chamber and hyperactivity during the expression of morphine conditioned place preference (CPP), and the reduction in membrane-bound of PKCα of the bilateral intermediate medial mesopallium (IMM) assayed immunologically. These results indicate that the decreased expression of PKCα in IMM may participate in the development of the susceptibility to the rewarding effects of morphine in chicks prenatally exposed to morphine, and provide further support for the cross-species evolutionary concordance among amniotes. [ABSTRACT FROM AUTHOR]

ABSTRACT Background and objective Ventilator-induced lung injury ( VILI) is commonly associated with respiratory barrier dysfunction; however, the mechanisms have not been fully elucidated. This study aimed to determine the order and components of the signalling pathway that mediates the degradation of adherin junction of p120-catenin in VILI. Methods For the in vivo study, C57BL/6 mice were pre-treated with inhibitors for 60 min prior to 4 h of mechanical ventilation. For the in vitro study, mouse lung epithelial 12 ( MLE-12) cells were pre-treated with inhibitors for 60 min or small interfering RNA ( siRNA) for 48 h prior to cyclic stretch at 20% for 4 h. The protein levels of protein kinase Cα ( PKCα), activated c-Src and p120-catenin were determined via western blot analysis. Lung injury was determined via HE staining, immunofluorescence, wet/dry ratio and lung injury scores. Results High tidal volume mechanical ventilation and 20% cyclic stretch resulted in the degradation of p120-catenin. Inhibitors of PKCα blocked c-Src kinase activation and p120-catenin degradation in VILI. Inhibitors of c-Src kinase or PP2 or siRNA blocked p120-catenin degradation but not PKCα activation. Conclusion The current findings demonstrates that PKCα and c-Src kinase participate in VILI. PKCα activation phosphorylates c-Src kinase and further decreases p120-catenin in VILI. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Ventricular arrhythmias and sudden cardiac deaths are among the leading causes of mortality in patients with heart failure, and the underlying mechanisms remain incompletely understood. Chronic elevation of angiotensin II (ANGII) is known to be one of the main contributors to heart failure.Objective: We tested whether ANGII can alter ventricular conduction and Na(+) current using transgenic mice with cardiomyocyte-restricted overexpression of ANGII type 1 receptor (AT1R).Methods: We used surface electrocardiograms along with current- and voltage-clamp techniques to characterize the electrophysiological properties of AT1R mice while the underlying regulatory mechanisms were explored using reverse transcription/quantitative polymerase chain reaction, Western blots, and immunofluorescence techniques.Results: Electrophysiological data indicated that chronic AT1R activation in ventricular myocytes caused a 60% reduction in Na(+) current density that slowed the maximal velocity of the action potential upstroke, leading to a prolongation of the QRS complex. These changes occur independently of cardiac hypertrophy, suggesting a direct role for ANGII/AT1R in slowing ventricular conduction. Western blots demonstrated a selective increase in sarcolemmal protein kinase Cα (PKCα) in AT1R mice, indicating PKCα activation. Furthermore, immunofluorescence analysis showed reorganization of PKCα expression to sarcolemma and colocalization with NaV1.5 in AT1R myocytes. The involvement of PKCα in regulating Na(+) current was subsequently demonstrated in human-induced pluripotent stem cell-derived cardiomyocytes where ANGII treatment reduced Na(+) current density. Concomitant treatment with αV5-3, a PKCα translocation inhibitor peptide, blocked the ANGII effect.Conclusion: Overall, this study suggests that in mouse and human cardiomyocytes, PKCα is an important mediator of the ANGII-induced reduction in Na(+) current and may contribute to ventricular arrhythmias. [ABSTRACT FROM AUTHOR]

Tumor cell motility exhibits a crucial role in tumor development. Therefore, the present study aimed to investigate whether thymol could reduce C6 glioma cell migration. Cell viability was determined using the EZ-Cytox Cell Viability kit. The scratch wound healing and Boyden chamber assays were performed to test C6 glioma cell migration in the presence of fetal bovine serum (FBS). Additionally, the study investigated whether signaling proteins relevant to C6 glioma cell migration, i.e., extracellular signal-regulated kinases (ERK)1/2, protein kinase Cα (PKCα), matrix metallopeptidase (MMP)9 and MMP2, were affected by thymol treatment. Up to 30 µM, thymol did not alter cell viability, whereas 100 µM thymol induced the death of ~20% of the cells. Furthermore, thymol (30 µM) significantly reduced FBS-induced migration. In the FBS-stimulated C6 glioma cells, thymol (30 µM) suppressed PKCα and ERK1/2 phosphorylation. MMP9 and MMP2 production was also significantly reduced by treatment with 30 µM thymol in the C6 glioma cells. Taken together, these results indicate that thymol attenuates C6 glioma cell migration. Additionally, the study suggests that the effect of thymol on the FBS-induced migration of C6 glioma cells affects PKCα and ERK1/2 signaling, and suppresses MMP9 and MMP2 production. [ABSTRACT FROM AUTHOR]

Malignant gliomas are refractory to conventional therapies, including surgery, radiotherupy and chemotherapy. Thus, a variety of therapies such as the inhibition of angiogenesis and signal transcluction pathways have been attempted. In the present study, we have evaluated the combined effect of enclostatin, an inhibitor of angiogenes, amd a DNA enzyme targeting the protein kinase Cα(PKCα)gene expression. Inhibition of PKCα by a nuclease- esistant DNA enzyme eliminated PKCα gene expression and induced apoptosis in most glioma cells. To assess the efficacy of endostatin and the PKCα DNA enzyme in vivo, rats are bearing the intracranial tumor BT4C were given a combined treatment of endostatin and the PKCα enzyme. Survival was significantly longer than those treated with the inactive form (P

Protein kinase Cα (PKCα) phosphorylates the Ser33/37/Thr41 residues of β-catenin, which lacks a typical PKCα canonical sequence, but little is known about its underlying mechanism. Here we showed that Ser33/Ser37/Thr41 of β-catenin fragments encompassing the armadillo repeats 1–5 (β-catenin 1–781 , β-catenin 1–682 , and β-catenin 1–422 ) are phosphorylated by PKCα whereas β-catenin 1–138 lacking these repeats is not phosphorylated. Binding-site analysis revealed that PKCα directly interacts with β-catenin through the sites on the armadillo repeats 1–5. In addition, axin fragments (365–500), which interacts with β-catenin through armadillo repeats 3–5, disrupted PKCα/β-catenin association and inhibited β-catenin phosphorylation by PKCα. In HEK293 cells, the levels of β-catenin 1–781 and β-catenin 1–422 were decreased whereas the amount of β-catenin 1–138 was unchanged by pharmacological stimulation of PKCα. Our results suggest that the association of PKCα with the armadillo repeats of β-catenin placed the Ser33/37/Thr41 residues of β-catenin in close proximity to PKCα, thereby facilitating PKCα-mediated β-catenin phosphorylation. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) can differentiate toward various lineages, including the osteogenic lineage, and thus hold great potential for clinic purposes. By using pharmacological inhibitors, protein kinase C (PKC) signaling has been shown to either negatively or positively regulate differentiation of bone, however, due to the low transfection efficiency in MSCs, the role of individual PKC isoforms is still not fully understood. In this study, we established a TAT peptide-mediated transduction system that efficiently delivered PKCα proteins into MSCs in a non-invasive fashion. The increased PKCα protein levels significantly promoted osteogenic differentiation in the murine mesenchymal C3H10T1/2 cells and in primary MSCs from both human and mouse, as demonstrated by the enhanced activity of the osteoblast marker, alkaline phosphatase, and the enhanced expression of the key transcription factor runx2. Mineralization is an important functional indication for bone differentiation. Our results further showed that PKCα promoted expression of the important osteocalcin gene and the accumulation of calcium minerals. Taken together, this study provides direct evidence showing that PKCα positively regulates osteogenic differentiation and demonstrates that the TAT peptide-mediated method enables functional study of specific PKC isoforms in MSCs without using viral infection. This may promote the application of PKCs in therapeutic treatment. [ABSTRACT FROM AUTHOR]

Background Selenoprotein S (SelS) is an important endoplasmic reticulum and plasma membrane-located selenoprotein implicated in inflammatory responses and insulin resistance. However, the effects of SelS on endothelial cells (ECs) have not been reported. In the present study, the role of SelS in oxidative stress and the underlying mechanism were investigated in human ECs. Methods A SelS over-expression plasmid (pc-SelS) and a SelS-siRNA plasmid were transfected into human umbilical vein endothelial cells (American Type Culture Collection, USA). The cells were divided into four groups: control, SelS over-expression (transfected with pc-SelS), vector control, and SelS knockdown (transfected with siRNA-SelS). After treating the cells with H2O2, the effects of oxidative stress and the expression of caveolin-1 (Cav-1) and protein kinase Cα (PKCα) were investigated. Results Following treatment with H2O2, over-expression of SelS significantly increased cell viability and superoxide dismutase (SOD) activity, and decreased malondialdehyde (MDA) production and Cav-1 gene and protein expression. However, no effects on PKCα were observed. In contrast, knockdown of SelS significantly decreased cell viability, SOD activity, and PKCα gene and protein expression, and increased MDA production and Cav-1 gene and protein expression. Conclusions SelS protects ECs from oxidative stress by inhibiting the expression of Cav-1 and PKCα. [ABSTRACT FROM AUTHOR]

ABSTRACT Sunifiram is a novel pyrrolidone nootropic drug structurally related to piracetam, which was developed for neurodegenerative disorder like Alzheimer's disease. Sunifiram is known to enhance cognitive function in some behavioral experiments such as Morris water maze task. To address question whether sunifiram affects N-methyl- D-aspartate receptor (NMDAR)-dependent synaptic function in the hippocampal CA1 region, we assessed the effects of sunifiram on NMDAR-dependent long-term potentiation (LTP) by electrophysiology and on phosphorylation of synaptic proteins by immunoblotting analysis. In mouse hippocampal slices, sunifiram at 10-100 nM significantly enhanced LTP in a bell-shaped dose-response relationship which peaked at 10 nM. The enhancement of LTP by sunifiram treatment was inhibited by 7-chloro-kynurenic acid (7-ClKN), an antagonist for glycine-binding site of NMDAR, but not by ifenprodil, an inhibitor for polyamine site of NMDAR. The enhancement of LTP by sunifilam was associated with an increase in phosphorylation of α-amino-3-hydroxy-5-methylisozazole-4-propionate receptor (AMPAR) through activation of calcium/calmodulin-dependent protein kinase II (CaMKII) and an increase in phosphorylation of NMDAR through activation of protein kinase Cα (PKCα). Sunifiram treatments at 1-1000 nM increased the slope of field excitatory postsynaptic potentials (fEPSPs) in a dose-dependent manner. The enhancement was associated with an increase in phosphorylation of AMPAR receptor through activation of CaMKII. Interestingly, under the basal condition, sunifiram treatments increased PKCα (Ser-657) and Src family (Tyr-416) activities with the same bell-shaped dose-response curve as that of LTP peaking at 10 nM. The increase in phosphorylation of PKCα (Ser-657) and Src (Tyr-416) induced by sunifiram was inhibited by 7-ClKN treatment. The LTP enhancement by sunifiram was significantly inhibited by PP2, a Src family inhibitor. Finally, when pretreated with a high concentration of glycine (300 μM), sunifiram treatments failed to potentiate LTP in the CA1 region. Taken together, sunifiram stimulates the glycine-binding site of NMDAR with concomitant PKCα activation through Src kinase. Enhancement of PKCα activity triggers to potentiate hippocampal LTP through CaMKII activation. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Abstract: Previous studies have demonstrated the important role of angiotension II (AngII) in promoting proliferation of myofibroblasts (myoFbs) and myocardial fibrosis. However, the underlying mechanisms and the role of oxygen free radicals in the proliferation of myofibroblasts induced by AngII are unclear. The present study was designed to shed light on this issue through exploration of AngII signaling pathways via in vitro experiments. Primary cultures of neonatal rat myoFbs were divided into five groups which were treated with AngII (10−8 to 10−6 M), AngII with the antioxidant N-acetyl-L-cysteine (NAC), or normal culture medium. We observed the proliferation of myoFbs as induced by AngII at different concentrations with MTT. Reactive oxygen species (ROS) levels in myoFbs were detected by monitoring the fluorescence of 2′,7′-dichlorofluorescein. The contents and levels of oxygen free radicals (OH ) in the three groups were detected by spectrophotometer, immunocytochemical staining, and confocal fluorescence. Western blot and image analysis were used to measure membrane translocation and expression of phospho-protein kinase Cα. MyoFbs incubated with AngII (10−8 to 10−6 M) for 24h increased their rate of proliferation, the content of OH , and expression of ROS (P<0.01 vs. control group), whereas these parameters decreased in the presence of NAC. Immunocytochemistry, confocal fluorescence staining and image analysis showed that AngII could promote the translocation and expression of p-PKCα in membrane, and the antioxidant NAC blocked this increase (P<0.01). Western blot results also showed that NAC could inhibit the expression of p-PKCα. [Copyright &y& Elsevier]

Background: Different studies have established that cholinergic neurodegeneration could be a major pathological feature of Alzheimer's disease (AD). Thus, enhancement of the central cholinergic neurotransmission has been regarded as one of the most promising strategies for the symptomatic treatment of AD, mainly by means of reversible acetylcholinesterase inhibitors (AChEIs). The cognitive-enhancing properties of both huprine X, a new AChEI, and the structurally related huperzine A, as well as their effects on the regulation of several neurochemical processes related to AD have been studied in triple transgenic mice (3xTg-AD). Methods: Seven-month-old homozygous 3xTg-AD male mice, which received chronic intraperitoneal treatment with either saline, huprine X (0.12 μmol·kg-1) or huperzine A (0.8 μmol·kg-1) were subjected to a battery of behavioural tests after 3 weeks of treatment and thereafter the brains were dissected to study the neurochemical effects induced by the two AChEIs. Results: Treatments with huprine X and huperzine A improved learning and memory in the Morris water maze and some indicators of emotionality without inducing important adverse effects. Moreover, huprine X and huperzine A activate protein kinase C/mitogen-activated protein kinase pathway signalling, α-secretases (ADAM 10 and TACE) and increase the fraction of phospho-glycogen synthase kinase 3-β. Conclusion: Results obtained herein using a sample of 3xTg-AD animals strongly suggest that the treatment with the two AChEIs not only improves the cognitive performance of the animals but also induces some neurochemical changes that could contribute to the beneficial effects observed. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: It has been supposed that green tea polyphenols (GTPs) have neuroprotective effects on brain damage after brain ischemia in animal experiments. Little is known regarding GTPs’ protective effects against the blood-brain barrier (BBB) disruption after ischemic stroke. We investigated the effects of GTPs on the expression of claudin-5, occludin, and ZO-1, and the corresponding cellular mechanisms involved in the early stage of cerebral ischemia. Methods: Male Wistar rats were subjected to a middle cerebral artery occlusion (MCAO) for 0, 30, 60, and 120 min. GTPs (400 mg/kg/day) or vehicle was administered by intragastric gavage twice a day for 30 days prior to MCAO. At different time points, the expression of claudin-5, occludin, ZO-1, and PKCα signaling pathway in microvessel fragments of cerebral ischemic tissue were evaluated. Results: GTPs reduced BBB permeability at 60 min and 120 min after ischemia as compared with the vehicle group. Transmission electron microscopy also revealed that GTPs could reverse the opening of tight junction (TJ) barrier at 60 min and 120 min after MACO. The decreased mRNA and protein expression levels of claudin-5, occludin, and ZO-1 in microvessel fragments of cerebral ischemic tissue were significantly prevented by treatment with GTPs at the same time points after ischemia in rats. Furthermore, GTPs could attenuate the increase in the expression levels of PKCα mRNA and protein caused by cerebral ischemia. Conclusions: These results demonstrate that GTPs may act as a potential neuroprotective agent against BBB damage at the early stage of focal cerebral ischemia through the regulation of TJ and PKCα signaling. [ABSTRACT FROM AUTHOR]

Abstract: Activation of peroxisome proliferator-activated receptor (PPAR) isoforms (α, β/δ, and γ) is known to inhibit platelet aggregation. In the present study, we examined whether PPARs-mediated pathways contribute to the antiplatelet activity of magnolol, a compound purified from Magnolia officinalis. Magnolol (20–60μM) dose-dependently enhanced the activity and intracellular level of PPAR-β/γ in platelets. In the presence of selective PPAR-β antagonist (GSK0660) or PPAR-γ antagonist (GW9662), the inhibition of magnolol on collagen-induced platelet aggregation and intracellular Ca2+ mobilization was significantly reversed. Moreover, magnolol-mediated up-regulation of NO/cyclic GMP/PKG pathway and Akt phosphorylation leading to increase of eNOS activity were markedly abolished by blocking PPAR-β/γ activity. Additionally, magnolol significantly inhibited collagen-induced PKCα activation through a PPAR-β/γ and PKCα interaction manner. The arachidonic acid (AA) or collagen-induced thromboxane B2 formation and elevation of COX-1 activity caused by AA were also markedly attenuated by magnolol. However, these above effects of magnolol on platelet responses were strongly reduced by simultaneous addition of GSK0660 or GW9662, suggesting that PPAR-β/γ-mediated processes may account for magnolol-regulated antiplatelet mechanisms. Similarly, administration of PPAR-β/γ antagonists remarkably abolished the actions of magnolol in preventing platelet plug formation and prolonging bleeding time in mice. Taken together, we demonstrate for the first time that the antiplatelet and anti-thrombotic activities of magnolol are modulated by up-regulation of PPAR-β/γ-dependent pathways. [Copyright &y& Elsevier]

Abstract: The aim of this study was to investigate the inhibitory mechanism of pure curcumin on WT1 expression in leukemic K562 cells. Pure curcumin suppressed WT1 expression, independent of effects on protein degradation or WT1 mRNA stability. Chromatin immunoprecipitation and reporter gene assays indicate that pure curcumin treatment attenuates WT1 auto-regulation. Interestingly, PKCα inhibition mimicks the repressive effects of pure curcumin in K562 cells. Conversely, myristoylated PKCα over-expression increased WT1 expression and reversed the inhibitory effect of pure curcumin. Our study indicates that pure curcumin attenuates WT1 auto-regulatory function through inhibition of PKCα signaling in K562 cells. [Copyright &y& Elsevier]

Abstract: Novel types of PKCα activators based on isobenzofuranone bearing a myo-inositol moiety were designed and synthesized. The derivatives with bulky substituents on the myo-inositol moiety significantly activated PKCα, but their binding sites were not the same as that of phorbol ester. [Copyright &y& Elsevier]

Abstract: Protein interacting with C kinase 1 (PICK1) is a PDZ-containing protein that binds to AMPA receptor (AMPAR) GluR2 subunit and protein kinase Cα (PKCα) in the central neurons. It functions as a targeting and transport protein, presents the activated form of PKCα to synaptic GluR2, and participates in synaptic AMPAR trafficking in the nervous system. Thus, PICK1 might be involved in many physiological and pathological processes triggered via the activation of AMPARs. We report herein that PICK1 knockout mice display impaired mechanical and thermal pain hypersensitivities during complete Freund’s adjuvant (CFA)-induced inflammatory pain maintenance. Acute transient knockdown of spinal cord PICK1 through intrathecal injection of PICK1 antisense oligodeoxynucleotide had a similar effect. In contrast, knockout and knockdown of spinal cord PICK1 did not affect incision-induced guarding pain behaviors or mechanical or thermal pain hypersensitivities. We also found that PICK1 is highly expressed in dorsal horn, where it interacts with GluR2 and PKCα. Injection of CFA into a hind paw, but not a hind paw incision, increased PKCα-mediated GluR2 phosphorylation at Ser880 and GluR2 internalization in dorsal horn. These increases were absent when spinal cord PICK1 was deficient. Given that dorsal horn PKCα-mediated GluR2 phosphorylation at Ser880 and GluR2 internalization contribute to the maintenance of CFA-induced inflammatory pain, our findings suggest that spinal PICK1 may participate in the maintenance of persistent inflammatory pain, but not in incision-induced post-operative pain, through promoting PKCα-mediated GluR2 phosphorylation and internalization in dorsal horn neurons. [Copyright &y& Elsevier]

Abstract: Impaired glutamatergic activity and synaptic dysfunction contributing to excitotoxicity and neuronal degeneration has been observed in the diabetic retina. Here we analyzed the expression changes and trafficking abnormalities of the AMPA glutamate receptor 2 subunit (GluR2) and its regulators protein kinase Cα (PKCα) and PKC-interacting protein 1 (PICK1) in the rat retina during the early phases of streptozotocin-(STZ-) induced diabetes. Diabetes was induced in Long Evans rats by injection of STZ. Two and six weeks after induction of diabetes, immunohistochemistry and in situ hybridization were performed on retinal paraffin sections to investigate the expression and localization of GluR2 and its regulators PKCα and PICK1. The cellular distribution and trafficking of these proteins in retinae were also investigated by subcellular fractionation and western blotting. While no significant changes were observed for GluR2 transcripts, we observed a strong increase in GluR2 immunoreactivity, predominantly in the ganglion cell layer (GCL) and the inner plexiform layer (IPL), as early as two weeks of diabetes. GluR2/3 immunoreactivity was further increased from the GCL to OPL after 6 weeks of diabetes. Increased expression of a phosphorylated non-synaptic population of GluR2 was detected in the GCL, the IPL and in distinct photoreceptor cells within the outer nuclear layer (ONL) of diabetic animals. Further, the PICK1 retinal distribution was unchanged two and six weeks after onset of diabetes and in both control and diabetic rat retinae the PKCα immunoreactivity remained the same. However, phosphorylated PKCα immunoreactivity was increased in diabetic retina as compared to control and peaked after 6 weeks of diabetes. Activated PKCα was almost completely lost in all membrane fractions and primarily recovered in the cytosolic fraction. These results are consistent with PKCα being re-localized in the diabetic retina. The observations indicate a diabetes-dependent increase in the activation of PKCα and a disturbed GluR2 regulation by altered internalization and recycling. [Copyright &y& Elsevier]

Abstract: High levels of specific prolactin-releasing peptide (PrRP) binding sites have been found in the myocardium; however, the functional importance of PrRP in the regulation of cardiac function is unknown. In isolated perfused rat hearts, infusion of PrRP (1–100 nM) induced a dose-dependent positive inotropic effect. Inhibition of cAMP catabolism by IBMX, a phosphodiesterase inhibitor, failed to augment the contractile effect of PrRP. The protein phosphatase (PP1/PP2A) inhibitor calyculin A increased the inotropic response to PrRP, whereas the PP2A inhibitor okadaic acid had no effect. Ro32-0432, a protein kinase Cα (PKCα) inhibitor, significantly enhanced the inotropic effect of PrRP as well as the phosphorylation of phospholamban at Ser-16. In conclusion, the present data define a hitherto unrecognized role for PrRP in the regulation of cardiovascular system by showing that PrRP exerts a direct positive inotropic effect. Moreover, our results suggest that the cAMP-independent inotropic response to PrRP is suppressed by concurrent activation of PKCα and PP1. [Copyright &y& Elsevier]

Background: Increasing evidence has suggested that differentiation of adventitial fibroblasts (AFs) to myofibroblasts plays an important role in arterial remodeling. The molecular mechanisms by which myofibroblast formation is regulated still remain largely unknown. This study aimed to evaluate the role of cyclic nucleotide phosphodiesterase 1A (PDE1A) in the formation of adventitial myofibroblasts induced by transforming growth factor (TGF)-β1. Methods and Results: AFs were cultured by the explant method. Western blot and immunocytochemistry were applied for α-smooth muscle actin (SMA) or protein kinase C (PKC) α protein analysis. Results showed that TGF-β1 upregulated PDE1A protein expression in rat aortic AFs and pharmacological inhibition of PDE1A blocked TGF-β1-induced α-SMA expression, a marker of myofibroblast formation, suggesting that the upregulation of PDE1A may mediate TGF-β1-induced AF transformation. Moreover, calphostin C (a PKC inhibitor) inhibited TGF-β1-induced α-SMA expression, whereas phorbol-12-myristate-13-acetate (a PKC activator) induced it. Finally, the upregulation of PKCα expression by TGF-β1 was also inhibited by PDE1A inhibition. Conclusions: Taken together, our data suggest that TGFβ1 induces α-SMA expression and myofibroblast formation via a PDE1A-PKCα-dependent mechanism. Our study thus unveils a novel signaling mechanism underlying TGF-β1-induced adventitial myofibroblast formation. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: We recently proposed a novel gene regulation system responding to specifically and abnormally activated intracellular enzymes in diseased cells. In the present study, we focused on protein kinase C (PKC)α, which is hyper-activated in most tumor cells, as a trigger for transgene regulation. We prepared cationic copolymers comprising hydrophilic and neutral polymers in main chains and cationic peptide substrates with different contents in side chains. Our copolymer with high peptide content (>3 mol%) condensed with pDNA more weakly than with poly(l-lysine) (pLL) having a similar molecular weight, but gene suppression was nearly identical to that of pLL, probably due to the steric hindrance of the main chains in our copolymer. Steric hindrance of the main chains barely affected the phosphorylation reaction of the pendant peptide. In cell and mouse experiments, higher gene expression was observed in complexes of pDNA with copolymers pended PKCα-specific substrate peptide than that in complexes with negative copolymers pended peptide substituted phosphorylation site of serine residues with alanine. These results indicate that our system can recognize intracellular PKCα as a trigger to regulate transgene expression, and may be useful for tumor gene therapy. [Copyright &y& Elsevier]

Abstract: Efficient enantio-selective synthesis of conformationally constrained diacylglycerol analogues, 7-substituted isobenzofuranone derivatives, originally developed by us as PKCα ligands, was achieved by asymmetric dihydroxylation and γ-lactone formation via ortho-lithiation and carboxylation. A series of derivatives having straight and/or branched side chains were synthesized and evaluated, and low-nanomolar-concentration affinity ligands and highly potent PKCα activators were found among them. These potent ligands induced phenotypic change of K562 cells, which is characteristic of PKC activators. [Copyright &y& Elsevier]

Abstract: Vasodilator responses induced by okadaic acid were investigated in canine basilar artery precontracted with 80 mM KCl. Okadaic acid (1 µM) relaxed the artery and this relaxant effect was partially inhibited by Gö6976, a conventional protein kinase C inhibitor, and calphostin C, an inhibitor of conventional and novel PKCs. Rottlerin, a specific inhibitor of PKCδ, did not influence okadaic acid''s effect. KCl increased phosphorylation of 20,000-Dalton myosin light chain (MLC20) at Ser-19. Okadaic acid additionally increased MLC20 phosphorylation at Thr-18 and Thr-9, resulting in triphosphorylation of MLC20. This phosphorylation was inhibited by Gö6976. Okadaic acid stimulated phosphorylation of PKCα and 17,000-Dalton PKC-potentiated inhibitory phosphoprotein (CPI-17), and Gö6976 inhibited these phosphorylations. These results suggest that okadaic acid''s relaxant effect involves MLC20 triphosphorylation through a direct phosphorylation by PKCα and an indirect phosphorylation by inhibition of myosin light chain phosphatase through PKCα-mediated CPI-17 phosphorylation. [Copyright &y& Elsevier]

The hypoxia-inducible factors (HIFs) play a central role in oxygen homeostasis. HIF prolyl hydroxylases (PHDs) modify HIFα subunits and thereby target them for proteasomal degradation. Mammalian PHDs comprise three isozymes, PHD1, PHD2 and PHD3, and belong to the iron(II)-2-oxoglutarate-dependent dioxygenase family. We have expressed full-length human PHD1 in Escherichia coli, and purified it to apparent homogeneity by immobilized Ni-affinity chromatography, cation-exchange HPLC followed by gel filtration. Fe2+ was found to have EC50 value of 0.64 μM and the purified enzyme showed maximal activity at 10 μM Fe2+. The IC50 values for transition metal ions, Co2+, Ni2+ and Cu2+, were 58, 35 and 220 μM, respectively, in the presence of 100 μM Fe2+. Mn2+ did not affect the activity <1 mM. Many transcription-related proteins are regulated by phosphorylation. Thus, recombinant PHD1 was examined for in vitro phosphorylation using protein kinase A, protein kinase Cα, casein kinase I and II and Erk2. The protein was most strongly phosphorylated by protein kinase Cα, and the phosphorylation sites were found to be Ser-132, Ser-226 and Ser-234. Mutation of Ser-132 or Ser-234 to Asp or Glu diminished the enzymatic activity to 25–60%, while mutation of Ser-226 scarcely influenced the activity. [ABSTRACT FROM PUBLISHER]

Abstract: We examined the adhesion and proliferation of immortalized endothelial cells GP8.39 (ECs) onto polyethyleneterephtalate (PET) and polyhydroxymethylsiloxane (PHMS) thin films, functionalized by UV-O3 treatment and/or protein immobilization. The modified surface topography showed partial oxidation for both polymers, a slight increase in wettability and monopolar basic character for PET, and a hydrophilic bipolar acid–base behaviour for PHMS. UV-O3 treatment did not induce significant roughness changes (under 1 nm) as shown by atomic force spectroscopy measurements (AFM). The EC adhesion and spreading onto untreated and modified surfaces were investigated both before and after immobilization of collagen (CA) and fibronectin (FN) adlayers. AFM analyses showed an open-weave protein layer on both untreated polymers which became a tight-woven net after UV-O3 irradiation of underlying films. On day 5 after seeding, cell count analyses on irradiated PET surfaces, CA/FN-coated or not, showed EC adhesion and proliferation significantly greater than those on untreated polymers, indicating that UV-O3 irradiation promoted fast endothelialization. A less pronounced EC spreading behaviour on treated PHMS was observed. In ECs grown on irradiated and CA- or FN-coated PET, the levels of phospho-protein kinase Cα (p-PKCα, phospho-ERK1/2, and phospho-cytosolic phospholipase A2 (p-cPLA2), all enzymes taken as signaling markers of cell adhesion and proliferation, decreased in comparison to those in CA- or FN-coated untreated PET. In contrast, in ECs grown on UV-O3-treated PHMS, Western blot analyses showed increased levels of p-PKCα, p-ERK1/2 and p-cPLA2 in comparison with cells grown onto untreated polymer. The growth response of ECs to the substrates was related to the changes of polarity properties of UV-O3-treated polymer films, from hydrophobic/neutral towards hydrophilic/charged layers, and the signaling pathway remodelling to the cell proliferation degree. [Copyright &y& Elsevier]

Abstract: Abstract: Members of the protein kinase C (PKC) isoenzyme family play a central role in the tumorigenesis of several tissues. However, little is known about subtype specific intracellular expression of PKC in human hepatocellular carcinomas. Methods: We investigated PKC isoforms mRNA expression in 42 HCC specimens using reverse transcription polymerase chain reaction analysis, and the correlation between PKC isoforms expression and clinicopathologic parameters. Results: We found that PKCα, PKCδ and PKCι mRNA were significantly increased in HCCs as compared to the corresponding non-cancerous liver tissues. PKCα expression also significantly correlated with tumor size (P <0.05) and TNM stage (P <0.05), but PKCδ and PKCι did not. The log-rank analysis revealed that patients with higher PKCα mRNA expression in the HCC tissues had significantly shorter survival rate than patients with lower PKCα mRNA expression (P <0.01). Conclusions: Our results suggested that the PKCα may be a prognostic factor for the survival of patients with HCC. [Copyright &y& Elsevier]

The presence of serotonin 1A receptor (5-HT1A-R) in the hippocampus, amygdala, and most regions of the frontal cortex is essential between postnatal day-5–21 (P5–21) for the expression of normal anxiety levels in adult mice. Thus, the 5-HT1A-R plays a crucial role in this time window of brain development. We show that the 5-HT1A-R-mediated stimulation of extracellular signal-regulated kinases 1 and 2 (Erk1/2) in the hippocampus undergoes a transition between P6 and P15. At P6, a protein kinase C (PKC) isozyme is required for the 5-HT1A-R ⇒Erk1/2 cascade, which causes increased cell division in the dentate gyrus. By contrast, at P15, PKCα participates downstream of Erk1/2 to augment synaptic transmission through the Schaffer Collateral pathway but does not cause increased cell division. Our data demonstrate that the 5-HT1A-R ⇒Erk1/2 cascade uses PKC isozymes differentially, first boosting the cell division to form new hippocampal neurons at P6 and then undergoing a plastic change in mechanism to strengthen synaptic connections in the hippocampus at P15. [ABSTRACT FROM AUTHOR]

Both protein kinase Cα-dependent Na+/Ca2+ exchanger1 (NCX1) phosphorylation and calcineurin activity are required for the depression of NCX activity observed in chronically phenylephrine (PE)-treated hypertrophic neonatal rat cardiomyocytes. In this study, we explored the possibility that the same changes occur in vivo hypertrophy. In the hypertrophic hearts of thoracic aortic-banded (TAB) mice, NCX1 phosphorylation increased significantly compared with control hearts. Furthermore, the TAB-induced cardiac hypertrophy was much less prominent in transgenic mice overexpressing an NCX1 mutant having defective phosphorylation sites. These data suggest that the phosphorylation status of NCX1 may play an important role in the pathogenesis of load-induced cardiac hypertrophy. [ABSTRACT FROM AUTHOR]

Na+/Ca2+ exchanger (NCX) activity is markedly inhibited in hypertrophic neonatal rat cardiomyocytes subjected to chronic phenylephrine treatment. This inhibition is reversed partially and independently by acute inhibition of calcineurin and protein kinase C (PKC) activities. Similar NCX inhibition occurs in CCL39 cells expressing cloned wild-type NCX1, when they are infected with adenoviral vectors carrying activated calcineurin A and then treated acutely with phorbol myristoyl acetate or protein phosphatase-1 inhibitors. The data obtained with these cells suggest that calcineurin activity, PKCα-mediated NCX1 phosphorylation, and the central loop of NCX1 (possibly its β1 repeat) are required for the observed NCX inhibition. We observe partial inhibition of NCX activity independent of NCX1 phosphorylation when CCL39 cells are infected with activated calcineurin A but not further treated with phorbol myristoyl acetate or phosphatase inhibitors. Calcineurin thus appears to downregulate NCX activity via two independent mechanisms, one involving NCX1 phosphorylation and the other not involving NCX1 phosphorylation. These data indicate the existence of a novel regulatory mechanism for NCX1 involving calcineurin and PKC, which may be important in cardiac pathology. [ABSTRACT FROM AUTHOR]

Abstract: In the biogenesis of HDL by exogenous apolipoprotein (apo) A-I in rat astrocytes, apoA-I induced translocation of phospholipase Cγ (PLCγ) and PKCα to cytosolic lipid protein particle (CLPP) [Ito et al., 2004. J. Lipid Res. 45, 2269] and caused tyrosine-phosphorylation of PLCγ in CLPP in the initial 5min. It also induced translocation of caveolin-1 and newly synthesized cholesterol and phospholipid to CLPP, and increased cholesterol biosynthesis prior to the HDL biogenesis [Ito et al., 2002a. J. Biol. Chem. 277, 7929]. Cyclosporin A (CsA), an indirect inhibitor of protein phosphatase 2B (PP2B) and a potential inhibitor of ABC transporter A1 (ABCA1), suppressed all of these apoA-I-induced cellular events. CsA, however, did not affect the basal lipid release by the production of HDL with endogenous apoE, except for moderate decrease of its cholesterol content. Direct inhibitors of PP2B, inhibited only the release of lipids by apoA-I and had no effect on other apoA-I-induced events. CsA thus interferes with cellular cholesterol homeostasis independently of PP2B inhibition, perhaps by direct inhibition of ABCA1 reactivity to exogenous apoA-I, although PP2B may be involved in the lipid release step. CsA could therefore cause some neurological side effects by interfering with cellular cholesterol homeostasis in the brain. [Copyright &y& Elsevier]