A novel transgenic mouse strain expressing PKCßII demonstrates expansion of B1 and marginal zone B cell populations.

Protein kinase Cß (PKCß) expressed in mammalian cells as two splice variants, PKCßI and PKCßII, functions in the B cell receptor (BCR) signaling pathway and contributes to B cell development. We investigated the relative role of PKCßII in B cells by generating transgenic mice where expression of the transgene is directed to these cells using the Eµ promoter (Eµ-PKCßIItg). Our findings demonstrate that homozygous Eµ-PKCßIItg mice displayed a shift from IgD+IgMdim toward IgDdimIgM+ B cell populations in spleen, peritoneum and peripheral blood. Closer examination of these tissues revealed respective expansion of marginal zone (MZ)-like B cells (IgD+IgM+CD43negCD21+CD24+), increased populations of B-1 cells (B220+IgDdimIgM+CD43+CD24+CD5+), and higher numbers of immature B cells (IgDdimIgMdimCD21neg) at the expense of mature B cells (IgD+IgM+CD21+). Therefore, the overexpression of PKCßII, which is a phenotypic feature of chronic lymphocytic leukaemia cells, can skew B cell development in mice, most likely as a result of a regulatory influence on BCR signaling.

High Glucose Stimulates Mineralocorticoid Receptor Transcriptional Activity Through the Protein Kinase C ß Signaling.

Activation of mineralocorticoid receptor (MR) is shown in resistant hypertension including diabetes mellitus. Although protein kinase C (PKC) signaling is involved in the pathogenesis of diabetic complications, an association between PKC and MR is not known. Activation of PKCα and PKCß by TPA (12-O-Tetradecanoylphorbol 13-acetate) increased MR proteins and its transcriptional activities in HEK293-MR cells. In contrast, a high glucose condition resulted in PKCß but not PKCα activation, which is associated with elevation of MR protein levels and MR transcriptional activities. Reduction of endogenous PKCß by siRNA decreased those levels. Interestingly, high glucose did not affect MR mRNA levels, but rather decreased ubiquitination of MR proteins. In db/db mice kidneys, levels of phosphorylated PKCß2, MR and Sgk-1 proteins were elevated, and the administration of PKC inhibitor reversed these changes compared to db/+ mice. These data suggest that high glucose stimulates PKCß signaling, which leads to MR stabilization and its transcriptional activities.

Topography of Protein Kinase C ßII in Benign and Malignant Melanocytic Lesions.

BACKGROUND: Protein kinase C ßII promotes melanogenesis and affects proliferation of melanocytic cells but is frequently absent or decreased in melanoma cells in vitro. OBJECTIVE: To investigate PKC-ßII expression and spatial distribution within a lesion in various benign and malignant melanocytic proliferations. METHODS: Expression of PKC-ßII was semiquantitatively assessed in the various existing compartments (intraepidermal [not nested], junctional [nested], and dermal) of benign (n = 43) and malignant (n = 28) melanocytic lesions by immunohistochemistry. RESULTS: Melanocytes in the basal layer of normal skin or in lentigo simplex stained strongly for PKC-ßII. Common nevi lacked completely PKC-ßII. All other lesions expressed variably PKC-ßII, with cutaneous melanoma metastases displaying the lowest rate of positivity (14%). In the topographical analysis within a lesion, PKC-ßII expression was largely retained in the intraepidermal and junctional part of all other lesions (dysplastic nevus, lentigo maligna, and melanoma). Reduced expression of PKC-ßII was found in the dermal component of benign and malignant lesions ( P = .041 vs intraepidermal). PKC-ßII expression in the various compartments did not differ significantly between benign and malignant lesions. CONCLUSIONS: The current study revealed a significant correlation between PKC-ßII expression and spatial localization of melanocytes, with the lowest expression found in the dermal compartment and the highest in the epidermal compartment.

Hyperosmolarity-Induced Down-Regulation of Claudin-2 Mediated by Decrease in PKCß-Dependent GATA-2 in MDCK Cells.

Hyperosmolarity decreases claudin-2 expression in renal tubular epithelial cells, but the molecular mechanism remains undefined. Here, we found that the hyperosmolarity-induced decrease in claudin-2 expression is inhibited by Go6983, a non-selective protein kinase C (PKC) inhibitor, and PKCß specific inhibitor in Madin-Darby canine kidney II cells. Hyperosmolarity increased intracellular free Ca(2+) concentration and phosphorylated PKCß level, which were inhibited by RN-1734, an antagonist of transient receptor potential vanilloid 4 channel. Phorbol 12-myristate 13-acetate, a PKC activator, decreased claudin-2 expression. These results indicate hyperosmolarity decreases claudin-2 expression mediated by the activation of RN-1734-sensitive channel and PKCß. Hyperosmolarity decreased promoter activity of claudin-2, which was inhibited by Go6983 and PKCß inhibitor similar to those in real-time PCR and Western blotting. The effect of hyperosmolarity on promoter activity was not observed in the construct of -469/-6, a deletion mutant. Claudin-2 has hyperosmolarity-sensitive region in its promoter, which includes GATA binding site. Hyperosmolarity decreased the nuclear level of GATA-2, which was inhibited by Go6983 and PKCß inhibitor. Mutation of GATA binding site decreased the basal promoter activity and inhibited the effect of hyperosmolarity. In contrast, the hyperosmolarity-induced decrease in reporter activity and claudin-2 expression were rescued by over-expression of wild type GATA-2. Chromatin immunoprecipitation assay showed that GATA-2 bound to promoter region of claudin-2. These results suggest that hyperosmolarity decreases the expression level of claudin-2 via a decrease in PKCß-dependent GATA-2 transcriptional activity in renal tubular epithelial cells.

Propofol protects against angiotensin II-induced mouse hippocampal HT22 cells apoptosis via inhibition of p66Shc mitochondrial translocation.

Hippocampal neuronal oxidative stress and apoptosis have been reported to be involved in cognitive impairment, and angiotensin II could induce hippocampal oxidative stress and apoptosis. Propofol is a widely used intravenous anesthetic agent in clinical practice, and it demonstrates significant neuroprotective activities. In this study, we investigated the mechanism how propofol protected mouse hippocampal HT22 cells against angiotensin II-induced oxidative stress and apoptosis. Cell viability was evaluated with CCK8 kit. Protein expressions of active caspase 3, cytochrome c, p66(Shc), p-p66(shc)-Ser(36), protein kinase C ßII (PKCßII), Pin-1 and phosphatase A2 (PP2A) were measured by Western blot. Superoxide anion (O2(.-)) accumulation was measured with the reduction of ferricytochrome c. Compared with the control group, angiotensin II up-regulated expression of PKCßII, Pin-1 and PP2A, induced p66(Shc)-Ser(36) phosphorylation, and facilitated p66(Shc) mitochondrial translocation, resulting in O2(.-) accumulation, mitochondrial cytochrome c release, caspase 3 activation, and the inhibition of cell viability. Importantly, we found propofol inhibited angiotensin II-induced PKCßII and PP2A expression and improved p66(Shc) mitochondrial translocation, O2(.-) accumulation, mitochondrial cytochrome c release, caspase 3 activation, inhibition of cell viability. On the other hand, propofol had no effects on angiotensin II-induced Pin-1 expression and p66(Shc)-Ser(36) phosphorylation. Moreover, the protective effects of propofol on angiotensin II-induced HT22 apoptosis were similar with calyculin A, an inhibitor of PP2A and CGP53353, an inhibitor of PKCßII. However, the protective effect of propofol could be reversed by FTY720, an activator of PP2A, rather than PMA, an activator of PKCßII. Our data indicated that propofol down-regulated PP2A expression, inhibiting dephosphorylation of p66(Shc)-Ser(36) and p66(Shc) mitochondrial translocation, decreasing O2(.-) accumulation, reducing mitochondrial cytochrome c release, inhibiting caspase 3 activation. By these mechanisms, it protects mouse hippocampal HT22 cells against angiotensin II-induced apoptosis.

Somatostatin receptor-2 negatively regulates ß-adrenergic receptor mediated Ca(2+) dependent signaling pathways in H9c2 cells.

In the present study, we report that somatostatin receptor 2 (SSTR2) plays a crucial role in modulation of ß1AR and ß2AR mediated signaling pathways that are associated with increased intracellular Ca(2+) and cardiac complications. In H9c2 cells, SSTR2 colocalizes with ß1AR or ß2AR in receptor specific manner. SSTR2 selective agonist inhibits isoproterenol and formoterol stimulated cAMP formation and PKA phosphorylation in concentration dependent manner. In the presence of SSTR2 agonist, the expression of PKCα and PKCß was comparable to the basal condition, however SSTR2 agonist inhibits isoproterenol or formoterol induced PKCα and PKCß expression, respectively. Furthermore, the activation of SSTR2 not only inhibits calcineurin expression and its activity, but also blocks NFAT dephosphorylation and its nuclear translocation. SSTR2 selective agonist abrogates isoproterenol mediated increase in cell size and protein content (an index of hypertrophy). Taken together, the results described here provide direct evidence in support of cardiac protective role of SSTR2 via modulation of Ca(2+) associated signaling pathways attributed to cardiac hypertrophy.

A new PKCα/ß/TBX3/E-cadherin pathway is involved in PLCε-regulated invasion and migration in human bladder cancer cells.

Although PLCε has been verified to enhance bladder cancer cell invasion, the signaling pathways responsible for this remain elusive. Protein kinase C (PKCα/ß), which is involved in cancer development and progression, has been demonstrated to be activated by PLCε. However, the roles of PKCα/ß in PLCε-mediated bladder carcinoma cell invasion and migration have not been clearly identified. In this study, to determine what role PKCα/ß plays in PLCε-mediated bladder cancer cell invasion and migration, we silenced PLCε gene by adenovirus-shPLCε in T24 and BIU-87 cells and then revealed that it significantly inhibited cell migration and invasion. Further research indicated that cell bio-function of PLCε-regulated was related with PKCα/ß activity. These in vitro findings were supported by data from bladder carcinoma patient samples. In 35 case bladder cancer tumor samples, PLCε-overexpressing tumors showed significantly higher positive rates of PKCα/ß membrane immunohistochemistry staining than PLCε-low-expressing tumors. Mechanistically, study further showed that PLCε knockdown gene induced E-cadherin expression and decreased TBX3 expression, both of which were dependent on PKCα/ß activity. In addition, we demonstrated that treatment cells with TBX3-specific shorting hairpin RNA (shRNA) up-regulated E-cadherin expression and inhibited cell invasion/migration. Moreover, in in vivo experiment, immunohistochemistry analysis of Ad-shPLCε-infected tumor tissue showed low expression levels of phospho-PKCα/ß and TBX3 and high expression levels of E-cadherin compared with those of the control group. In summary, our findings uncover that PKCα/ß is critical for PLCε-mediated cancer cell invasion and migration and provide valuable insights for current and future Ad-shPLCε and PKCα/ß clinical trials.

Effects of Trigonella foenum-graecum (L.) on retinal oxidative stress, and proinflammatory and angiogenic molecular biomarkers in streptozotocin-induced diabetic rats.

The aim of the present study was to investigate the protective effects of Trigonella foenum-graecum Linn. (fenugreek) in Streptozotocin-induced diabetic rat retina. Fenugreek (100 and 200 mg/kg body weights) treatment was carried out for 24 weeks and evaluated for inflammatory [tumor necrosis factor (TNF)-α and interleukin (IL)-1ß] and angiogenic [vascular endothelial growth factor (VEGF) and protein kinase C (PKC)-ß] molecular biomarkers. Retinal oxidative stress was evaluated by estimating antioxidant (Glutathione, Superoxide dismutase, and Catalase) parameters. Fluorescein angiography was performed to detect retinal vascular leakage. Electron microscopy was performed to determine basement membrane thickness. In the present study, significant rises in the expressions of retinal inflammatory (TNF-α and IL-1ß) and angiogenic (VEGF and PKC-ß) molecular biomarkers were observed in diabetic retinae compared with normal retinae. However, fenugreek-treated retinae showed marked inhibition in the expression of inflammatory and angiogenic molecular biomarkers. Moreover, results from the present study showed positive modulatory effects of fenugreek on retinal oxidative stress. Fluorescein angiograms and fundus photographs obtained from diabetic retinae showed retinal vascular leakage. On the other hand, fenugreek-treated retinae did not show vascular leakage. Further, thickened BM was recorded in diabetic retina compared with normal retinae. However, fenugreek-treated retinae showed relatively lesser thickening of capillary BM. In conclusion, it may be postulated that fenugreek has great potential in preventing diabetes-induced retinal degeneration in humans after regular consumption in the specified dosage.

Modulation of Rack-1/PKCßII signalling by soluble AßPPα in SH-SY5Y cells.

The soluble amyloid ß precursor protein α (sAßPPα) released after α-secretase cleavage of the amyloid ß precursor protein (AßPP) has several functions including modulation of neuronal excitability and synaptic plasticity; it has been suggested that some of these effects are mediated by activation of NF-κB via induction of PI3K/Akt signaling pathway. We have recently described the presence of several consensus binding sites of c-Rel transcription factor in the promoter region of the GNB2L1 gene, coding for the Receptor for Activated C Kinase -1 (RACK-1). We investigated whether sAßPPα could influence the expression of RACK-1 through NF-κB involvement. Our data demonstrate that sAßPPα regulates RACK-1 gene expression through PI3K/Akt-dependent pathway, inducing c-Rel nuclear translocation and NF-κB activation. Since RACK-1 is the scaffold of protein kinase C ßII (PKCßII), we turned our attention to this kinase in order to evaluate whether sAßPPα could also influence PKCßII signalling demonstrating that sAßPPα induces PKCßII translocation and interaction with its scaffold with consequent RACK-1/PKCßII complex increase in membrane. Altogether these results suggest the existence of an interesting loop between the functions of the metabolic products of AßPP and the role of PKC and that the impact of a dysregulated AßPP metabolism occurring in several conditions (from physiological aging to injury response) may have consequences on the potential protective functions of the non amyloidogenic sAßPPα.