Specific Inhibitor Of Protein Kinase Research Articles

CXCL13–CXCR5 co-expression regulates epithelial to mesenchymal transition of breast cancer cells during lymph node metastasis

We investigated the expression of -CXC chemokine ligand 13 (CXCL13) and its receptor -CXC chemokine receptor 5 (CXCR5) in 98 breast cancer (BC) patients with infiltrating duct carcinoma, out of which 56 were found lymph node metastasis (LNM) positive. Interestingly, co-expression of CXCL13 and CXCR5 showed a significant correlation with LNM. Since, epithelial to mesenchymal transition (EMT) is highly associated with metastasis we investigated EMT-inducing potential of CXCL13 in BC cell lines. In CXCL13-stimulated BC cells, expression of various mesenchymal markers (Vimentin, N-cadherin), EMT regulators (Snail, Slug), and matrix metalloproteinase-9 (MMP9) was increased, whereas the expression of epithelial marker E-cadherin was found to be decreased. In addition, expression of receptor activator of nuclear factor kappa-B ligand(RANKL), which is known to regulate MMP9 expression via Src activation, was also significantly increased after CXCL13 stimulation. Using specific protein kinase inhibitors, we confirmed that CXCL13 stimulated EMT and MMP9 expression via RANKL-Src axis in BC cell lines. To further validate this observation, we examined gene expression patterns in primary breast tumors and detected significantly higher expression of various mesenchymal markers and regulators in CXCL13-CXCR5 co-expressing patients. Therefore, this study showed the EMT-inducing potential of CXCL13 as well as demonstrated the prognostic value of CXCL13-CXCR5 co-expression in primary BC. Moreover, CXCL13-CXCR5-RANKL-Src axis may present a therapeutic target in LNM positive BC patients.

Characterization of WZ4003 and HTH-01-015 as selective inhibitors of the LKB1-tumour-suppressor-activated NUAK kinases

The related NUAK1 and NUAK2 are members of the AMPK (AMP-activated protein kinase) family of protein kinases that are activated by the LKB1 (liver kinase B1) tumour suppressor kinase. Recent work suggests they play important roles in regulating key biological processes including Myc-driven tumorigenesis, senescence, cell adhesion and neuronal polarity. In the present paper we describe the first highly specific protein kinase inhibitors of NUAK kinases namely WZ4003 and HTH-01-015. WZ4003 inhibits both NUAK isoforms (IC50 for NUAK1 is 20 nM and for NUAK2 is 100 nM), whereas HTH-01-015 inhibits only NUAK1 (IC50 is 100 nM). These compounds display extreme selectivity and do not significantly inhibit the activity of 139 other kinases that were tested including ten AMPK family members. In all cell lines tested, WZ4003 and HTH-01-015 inhibit the phosphorylation of the only well-characterized substrate, MYPT1 (myosin phosphate-targeting subunit 1) that is phosphorylated by NUAK1 at Ser445. We also identify a mutation (A195T) that does not affect basal NUAK1 activity, but renders it ~50-fold resistant to both WZ4003 and HTH-01-015. Consistent with NUAK1 mediating the phosphorylation of MYPT1 we find that in cells overexpressing drug-resistant NUAK1[A195T], but not wild-type NUAK1, phosphorylation of MYPT1 at Ser445 is no longer suppressed by WZ4003 or HTH-01-015. We also demonstrate that administration of WZ4003 and HTH-01-015 to MEFs (mouse embryonic fibroblasts) significantly inhibits migration in a wound-healing assay to a similar extent as NUAK1-knockout. WZ4003 and HTH-01-015 also inhibit proliferation of MEFs to the same extent as NUAK1 knockout and U2OS cells to the same extent as NUAK1 shRNA knockdown. We find that WZ4003 and HTH-01-015 impaired the invasive potential of U2OS cells in a 3D cell invasion assay to the same extent as NUAK1 knockdown. The results of the present study indicate that WZ4003 and HTH-01-015 will serve as useful chemical probes to delineate the biological roles of the NUAK kinases.

Glucagon like peptide-1 increases visfatin expression in 3T3-L1 adipocytes via PKA pathway

Objective To test whether glucagon like peptide-1 (GLP-1) would regulate the expression of visfatin in adipocytes,and to explore the mechanism of this effect.Methods Fully differentiated 3T3-L1 adipocytes were treated with GLP-1.Total RNA was extracted for analyzing the level of visfatin mRNA by quantitative RT-PCR.The media were collected for measuring the level of visfatin protein by enzyme linked immuno-assay (ELISA).In order to test the involvement of PKA pathway,the adipocytes were pretreated with a specific pharmacological PKA inhibitor H89 for 30 min before GLP-1 was added.Results GLP-1 increased visfatin expression in a time-and dose-dependent manner.The level of visfatin significantly increased at the concentration of 10 10 mol/L GLP-1 (P＜0.05),and reached the peak at 10-9 mol/L (P＜0.01).After incubation for 18 hours,GLP-1 dominantly increased the level of visfatin (P＜0.05).Inhibition of PKA pathway by H89 partially blocked the effect of GLP-1 on visfatin expression.Conclusions GLP-1 may enhance the expression of visfatin in 3T3-L1 adipocyte via the PKA pathway,which might contribute to the improvement in glucose homeostasis. Key words: Glucagon like peptide-1; Visfatin; Adipocyte; Diabetes

Angiotensin-(1–7) Modulates Renal Vascular Resistance Through Inhibition of p38 Mitogen-Activated Protein Kinase in Apolipoprotein E–Deficient Mice

Apolipoprotein E-deficient (apoE(-/-)) mice fed on Western diet are characterized by increased vascular resistance and atherosclerosis. Previously, we have shown that chronic angiotensin (Ang)-(1-7) treatment ameliorates endothelial dysfunction in apoE(-/-) mice. However, the mechanism of Ang-(1-7) on vasoconstrictor response to Ang II is unknown. To examine Ang-(1-7) function, we used apoE(-/-) and wild-type mice fed on Western diet that were treated via osmotic minipumps either with Ang-(1-7) (82 μg/kg per hour) or saline for 6 weeks. We show that Ang II-induced renal pressor response was significantly increased in apoE(-/-) compared with wild-type mice. This apoE(-/-)-specific response is attributed to reactive oxygen species-mediated p38 mitogen-activated protein kinase activation and subsequent phosphorylation of myosin light chain (MLC(20)), causing renal vasoconstriction. Here, we provide evidence that chronic Ang-(1-7) treatment attenuated the renal pressor response to Ang II in apoE(-/-) mice to wild-type levels. Ang-(1-7) treatment significantly decreased renal inducible nicotinamide adenine dinucleotide phosphate subunit p47phox levels and, thus, reactive oxygen species production that in turn causes decreased p38 mitogen-activated protein kinase activity. The latter has been confirmed by administration of a specific p38 mitogen-activated protein kinase inhibitor SB203580 (5 μmol/L), causing a reduced renal pressor response to Ang II in apoE(-/-) but not in apoE(-/-) mice treated with Ang-(1-7). Moreover, Ang-(1-7) treatment had no effect in Mas(-/-)/apoE(-/-) double-knockout mice confirming the specificity of Ang-(1-7) action through the Mas-receptor. In summary, Ang-(1-7) modulates vascular function via Mas-receptor activation that attenuates pressor response to Ang II in apoE(-/-) mice by reducing reactive oxygen species-mediated p38 mitogen-activated protein kinase activity.

CD137 stimulation and p38 MAPK inhibition improve reactivity in an in vitro model of glioblastoma immunotherapy.

Dendritic cell vaccination has become an interesting option for cancer immunotherapy. Tumor-lysate-pulsed dendritic cells (DC) can prime naïve T cells and induce the regression of established tumors including gliomas as shown in various animal models. Despite hopeful results even in clinical studies, the outcome for many patients is still unsatisfying. In the present study, we tested the combination of tumor-lysate-pulsed dendritic cells (TPDC) with a monoclonal antibody against CD137, a monoclonal antibody against CD25 (daclizumab) and a specific p38 mitogen-activated protein kinase (p38 MAPK) inhibitor (SB203580) for improving immunostimulation in an in vitro model of immunotherapy for human gliomas. We observed a higher secretion of interferon gamma by TPDC-primed peripheral blood mononuclear cells (PBMC) that were incubated with an antibody against CD137 or the p38 MAPK inhibitor. In addition, we observed higher specific lysis of tumor cells after incubation of PBMC with the p38 MAPK inhibitor or the anti-CD137 antibody. In contrast, incubation of TPDC-primed PBMC with the anti-CD25 antibody did enhance neither interferon gamma secretion nor cellular cytotoxicity. Cell depletion experiments demonstrated that the immune reaction induced by TPDC is strongly dependent on CD4-positive and CD8-positive cells. Incubation of DC during maturation and antigen loading with the anti-CD137 antibody did not enhance cytotoxicity and interferon gamma secretion in comparison with application of the anti-CD137 antibody during priming. In conclusion, our data suggest that p38 MAPK inhibition and anti-CD137 antibodies can enhance the immune response against glioblastoma cells.

Radio-sensitization of human leukaemic MOLT-4 cells by DNA-dependent protein kinase inhibitor, NU7441

We studied the effect of pre-incubation with NU7441, a specific inhibitor of DNA-dependent protein kinase (DNA-PK), on molecular mechanisms triggered by ionizing radiation (IR). The experimental design involved four groups of human T-lymphocyte leukaemic MOLT-4 cells: control, NU7441-treated (1 μM), IR-treated (1 Gy), and combination of NU7441 and IR. We used flow cytometry for apoptosis assessment, Western blotting and ELISA for detection of proteins involved in DNA repair signalling and epifluorescence microscopy for detection of IR-induced phosphorylation of histone H2A.X. We did not observe any major changes in the amount of DNA-PK subunits Ku70/80 caused by the combination of NU7441 and radiation. Their combination led to an increased phosphorylation of H2A.X, a hallmark of DNA damage. However, it did not prevent up-regulation of neither p53 (and its phosphorylation at Ser 15 and 392) nor p21. We observed a decrease in the levels of anti-apoptotic Mcl-1, cdc25A phosphatase, cleavage of PARP and a significant increase in apoptosis in the group treated with combination. In conclusion, the combination of NU7441 with IR caused increased phosphorylation of H2A.X early after irradiation and subsequent induction of apoptosis. It was efficient in MOLT-4 cells in 10× lower concentration than the inhibitor NU7026. NU7441 proved as a potent radio-sensitizing agent, and it might provide a platform for development of new radio-sensitizers in radiotherapy.

Regulation of MSK1‐Mediated NF‐κB Activation Upon UVB Irradiation

Nuclear Factor Kappa-B (NF-κB) is a transcription factor that controls expression of genes involved in the immune and inflammatory responses as well as being a key component in the onset of cancers. In this study, we provided evidence that mitogen- and stress-activated protein kinase (MSK1) is responsible for a noncanonical late-phase activation of NF-κB upon UVB irradiation. Our data demonstrated that following UVB irradiation, MSK1 is activated via phosphorylation at the 24h time point coinciding with translocation of NF-κB into the nucleus. Investigations into the signaling pathways upstream of MSK1 through the use of specific inhibitors for mitogen-activated protein kinase and p38 revealed that both kinases are required for full phosphorylation during the late phase (24h), while p38 is paramount for phosphorylation during the early phase (6h). Electromobility shift assays (EMSA) showed that inhibition of MSK1 resulted in a marked reduction in NF-κB binding affinity without altering the nuclear translocation of NF-κB. Supershift EMSA implicate that the p65, but not p50, isoform of NF-κB is involved in late-phase activation in response to UVB irradiation. Together, the results of these studies shed light onto a novel pathway of MSK1-mediated late-phase activation of NF-κB in response to UVB irradiation.

Staurosporine induces dopaminergic neurite outgrowth through AMP-activated protein kinase/mammalian target of rapamycin signaling pathway

Axonal degeneration of dopaminergic neurons is one of the pathological features in the early stages of Parkinson disease. Promotion of axonal outgrowth of the remaining dopaminergic neurons leads to the recovery of the nigrostriatal pathway. Staurosporine (STS), a wide-spectrum kinase inhibitor, induces neurite outgrowth in various cell types, although its mechanism of action remains elusive. In this study, we analyzed which protein kinase is involved in STS-induced neurite outgrowth. We have previously established the method to measure the length of dopaminergic neurites that extend from a mesencephalic cell region, which is formed on a coverslip by an isolation wall. By means of this method, we clarified that STS treatment causes dopaminergic axonal outgrowth in mesencephalic primary cultures. Among the specific protein kinase inhibitors we tested, compound C (C.C), an AMP-activated protein kinase (AMPK) inhibitor, promoted dopaminergic neurite outgrowth. STS as well as C.C elevated the phosphorylation level of 70-kDa ribosomal protein S6 kinase, a downstream target of mammalian target of rapamycin (mTOR) signaling pathway. The STS- and C.C-induced dopaminergic neurite outgrowth was suppressed by rapamycin, an mTOR inhibitor. Furthermore, the application of C.C rescued 1-methyl-4-phenylpyridinium ion (MPP+)-induced dopaminergic neurite degeneration. These results suggest that STS induces dopaminergic axonal outgrowth through mTOR signaling pathway activation as a consequence of AMPK inhibition.

Differential 12-O-Tetradecanoylphorbol-13-acetate-induced activation of rat mammary carcinoma susceptibility Fbxo10 variant promoters via a PKC-AP1 pathway.

Rat mammary carcinoma susceptibility 5a1 (Mcs5a1), which is concordant to human MCS5A1 breast cancer risk locus, mediates susceptibility by a non-mammary cell-autonomous mechanism associated with T cell differential expression of F-box protein 10 (Fbxo10). Human FBXO10, an evolutionarily conserved ubiquitin ligase gene, was shown to have a potential role in regulating cell death by controlling the degradation of Bcl-2, a key protein involved in apoptosis. Breast cancer susceptibility is controlled by interactions between environmental and genetic factors; therefore, we sought to determine if breast cancer risk-associated environmental chemicals interact with Mcs5a1 variants using luciferase reporter constructs containing 4.2 kb Fbxo10 promoters based on alleles of mammary cancer susceptible Wistar Furth (WF) and resistant Wistar Kyoto (WKY) rat strains. 12-O-Tetradecanoylphorbol-13-acetate (TPA) induced activation of a 4.2 kb WF Fbxo10 promoter region, but lower levels of activation of the homologous WKY Fbxo10 promoter region. Using general and specific protein kinase inhibitors, we identified a protein kinase C (PKC) pathway that mediated TPA activation. We narrowed the possible PKCs to a member of the atypical PKC isoforms, namely PKCµ. We also determined that activator protein 1 (AP1) family member c-Fos mediated TPA activation of the 4.2 kb WF Fbxo10 promoter. TPA was shown to induce endogenous FBXO10 mRNA and FBXO10 protein in Jurkat cells, a human T cell line, with a maximal level of expression from 1.5 to 2.5 h after exposure. These results indicate that FBXO10/Fbxo10 expression is regulated by a PKC-dependent pathway acting through c-Fos, which binds AP1-specific DNA elements in Mcs5a1.

Human plasminogen kringle 1–5 inhibits angiogenesis and induces thrombomodulin degradation in a protein kinase A-dependent manner

Kringle 1–5 (K1–5), an endogenous proteolytic fragment of human plasminogen (Plg), is an angiostatin-related protein that inhibits angiogenesis. Many angiostatin-related proteins have been identified, but the detailed molecular mechanisms underlying their antiangiogenic effects remain unclear. Thrombomodulin (TM) is a transmembrane glycoprotein that plays a major role in the anticoagulation process in endothelial cells. Previously, we demonstrated that recombinant TM could interact with Plg to enhance Plg activation. In the present study, we investigated the interaction between TM and K1–5, and their functions in endothelial cells. We found that K1–5 colocalized with TM and directly interacted with TM through the TM lectin-like domain. After K1–5 interacted with TM, it induced TM internalization and degradation. In addition, the K1–5-induced TM internalization and degradation in proteasomes after ubiquitin modification were dependent on protein kinase A (PKA). Moreover, a PKA-specific inhibitor reversed the effects of K1–5 on cell migration and tube formation. Consistent with these findings, TM overexpression resulted in increased cell migration; moreover, K1–5 inhibited the increase of TM-mediated cell migration in a PKA-dependent manner. We determined that TM acts as a K1–5 receptor and that K1–5 induces TM internalization, ubiquitination, and degradation through the PKA pathway, by which K1–5 may inhibit endothelial cell migration and tube formation.

Discrete Control of TRPV4 Channel Function in the Distal Nephron by Protein Kinases A and C

We have recently documented that the Ca(2+)-permeable TRPV4 channel, which is abundantly expressed in distal nephron cells, mediates cellular Ca(2+) responses to elevated luminal flow. In this study, we combined Fura-2-based [Ca(2+)]i imaging with immunofluorescence microscopy in isolated split-opened distal nephrons of C57BL/6 mice to probe the molecular determinants of TRPV4 activity and subcellular distribution. We found that activation of the PKC pathway with phorbol 12-myristate 13-acetate significantly increased [Ca(2+)]i responses to flow without affecting the subcellular distribution of TRPV4. Inhibition of PKC with bisindolylmaleimide I diminished cellular responses to elevated flow. In contrast, activation of the PKA pathway with forskolin did not affect TRPV4-mediated [Ca(2+)]i responses to flow but markedly shifted the subcellular distribution of the channel toward the apical membrane. These actions were blocked with the specific PKA inhibitor H-89. Concomitant activation of the PKA and PKC cascades additively enhanced the amplitude of flow-induced [Ca(2+)]i responses and greatly increased basal [Ca(2+)]i levels, indicating constitutive TRPV4 activation. This effect was precluded by the selective TRPV4 antagonist HC-067047. Therefore, the functional status of the TRPV4 channel in the distal nephron is regulated by two distinct signaling pathways. Although the PKA-dependent cascade promotes TRPV4 trafficking and translocation to the apical membrane, the PKC-dependent pathway increases the activity of the channel on the plasma membrane.

Role of Tyrosine Phosphorylation in the Muscarinic Activation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride (Cl(-)) channel, which plays an important role in physiological anion and fluid secretion, and is defective in several diseases. Although its activation by PKA and PKC has been studied extensively, its regulation by receptors is less well understood. To study signaling involved in CFTR activation, we measured whole-cell Cl(-) currents in BHK cells cotransfected with GPCRs and CFTR. In cells expressing the M3 muscarinic acetylcholine receptor, the agonist carbachol (Cch) caused strong activation of CFTR through two pathways; the canonical PKA-dependent mechanism and a second mechanism that involves tyrosine phosphorylation. The role of PKA was suggested by partial inhibition of cholinergic stimulation by the specific PKA inhibitor Rp-cAMPS. The role of tyrosine kinases was suggested by Cch stimulation of 15SA-CFTR and 9CA-CFTR, mutants that lack 15 PKA or 9 PKC consensus sequences and are unresponsive to PKA or PKC stimulation, respectively. Moreover the residual Cch response was sensitive to inhibitors of the Pyk2 and Src tyrosine kinase family. Our results suggest that tyrosine phosphorylation acts on CFTR directly and through inhibition of the phosphatase PP2A. Results suggest that PKA and tyrosine kinases contribute to CFTR regulation by GPCRs that are expressed at the apical membrane of intestinal and airway epithelia.

PKA negatively regulates PP2Cβ to activate NF-κB-mediated inflammatory signaling

Protein phosphatase 2Cβ (PP2Cβ) was found to act as a negative regulator of NF-κB-mediated inflammatory signaling; however, its regulatory mechanism has not been examined. Here, we show that protein kinase A (PKA) phosphorylates the PP2Cβ, which was inhibited by PKA-specific inhibitor, H89. Mutation analysis of serine residues in PP2Cβ revealed that Ser-195 in PP2Cβ is phosphorylated by PKA. Importantly, PKA inhibition by H89 abrogated the Forskolin-induced destabilization of PP2Cβ against ubiquitin-dependent proteosomal degradation pathway. Furthermore, H89 treatment efficiently reversed the negative effect of Forskolin on the anti-inflammatory function of PP2Cβ. Collectively, these data suggest that PKA destabilizes PP2Cβ upon inflammatory stimuli via phosphorylation of Ser-195 in PP2Cβ.

Novel Role of KT5720 on Regulating Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel Activity and Dorsal Root Ganglion Neuron Excitability

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed in dorsal root ganglion (DRG) neurons, which are involved in diverse mechanisms that regulate DRG functions. Protein kinase A (PKA) is an essential kinase that plays a key role in almost all types of cells; it regulates the ion channel activity, the intracellular Ca(2+) concentration, as well as modulates cellular signals transduction. Nevertheless, the effect of PKA inhibition on the HCN channel activity in DRG neuron remains to be elucidated. Here we investigated the impact of PKA inhibition on the HCN channel activity and DRG neurons excitability. Our patch-clamp experiments both under whole-cell and single-channel conditions demonstrated that PKA inhibition with KT5720, a cell membrane permeable PKA-specific inhibitor, significantly attenuated HCN channel currents. Current clamp recording on freshly isolated DRG neurons showed KT5720 reduced overshoot amplitude and enhanced the threshold of the action potential. Moreover, our live-cell Ca(2+) imaging experiments illustrated KT5720 markedly reduced the intracellular Ca(2+) level. Collectively, this is the first report that addresses KT5720 attenuates the HCN channel activity and intracellular Ca(2+), thus reducing DRG neurons excitability. Therefore, our data strongly suggest that PKA is a potential target for curing HCN and DRG neuron relevant diseases.

Focal Adhesion Kinase Regulates Collagen I–Induced Airway Smooth Muscle Phenotype Switching

Increased extracellular matrix (ECM) deposition and airway smooth muscle (ASM) mass are major contributors to airway remodeling in asthma. Recently, we demonstrated that the ECM protein collagen I, which is increased surrounding asthmatic ASM, induces a proliferative, hypocontractile ASM phenotype. Little is known, however, about the signaling pathways involved. Using bovine tracheal smooth muscle, we investigated the role of focal adhesion kinase (FAK) and downstream signaling pathways in collagen I-induced ASM phenotype modulation. Phosphorylation of FAK was increased during adhesion to both uncoated and collagen I-coated culture dishes, without differences between these matrices. Nor were any differences found in cellular adhesion. Inhibition of FAK activity by overexpression of the FAK deletion mutants FAT (focal adhesion targeting domain) and FRNK (FAK-related nonkinase) attenuated adhesion. After attachment, FAK phosphorylation increased in a time-dependent manner in cells cultured on collagen I, whereas no activation was found on an uncoated plastic matrix. In addition, collagen I increased in a time- and concentration-dependent manner the cell proliferation, which was fully inhibited by FAT and FRNK. Similarly, the specific pharmacologic FAK inhibitor PF-573228 [6-((4-((3-(methanesulfonyl)benzyl)amino)-5-trifluoromethylpyrimidin-2-yl) amino)-3,4-dihydro-1H-quinolin-2-one] as well as specific inhibitors of p38 mitogen-activated protein kinase (MAPK) and Src also fully inhibited collagen I-induced proliferation, whereas partial inhibition was observed by inhibition of phosphatidylinositol-3-kinase (PI3-kinase) and mitogen-activated protein kinase kinase (MEK). The inhibition of cell proliferation by these inhibitors was associated with attenuation of the collagen I-induced hypocontractility. Collectively, the results indicate that induction of a proliferative, hypocontractile ASM phenotype by collagen I is mediated by FAK and downstream signaling pathways.

Promoter Characterization and Role of cAMP/PKA/CREB in the Basal Transcription of the Mouse ORMDL3 Gene

Orosomucoid 1-like 3 (ORMDL3) gene was strongly linked with the development of asthma in genetic association studies, and its expression could be significantly induced by allergen in airway epithelial cells of mice. However, the expression mechanism of ORMDL3 was still unclear. Here we have identified and characterized the mouse ORMDL3 gene promoter. Deletion constructs of the 5′ flanking region were fused to a luciferase reporter gene. After transient transfection in mouse fibroblast cell line NIH3T3, a CRE (−27/−20) binding CREB was identified in the core promoter region. Deletion or mutation of the CRE consensus sequence resulted in a significant loss of the promoter activity. EMSA and ChIP assays demonstrated the binding of CREB to the core promoter. Knocking down endogenous CREB led to a reduction in ORMDL3 expression. Conversely, overexpression of CREB up-regulated ORMDL3 expression. Moreover, forskolin, a PKA activator, could facilitate the phosphorylation of CREB, which in turn heightens ORMDL3 expression. H-89, a PKA-specific inhibitor, could significantly inhibit ORMDL3 expression. This study delineates the characterization of mouse ORMDL3 gene promoter and shows signaling pathway cAMP/PKA/CREB plays an important role in regulating ORMDL3 expression, which will be helpful for future animal model studies regarding the regulation or function of ORMDL3 gene.

Separate control of TRPV4 activity and trafficking in the distal nephron

We have recently documented that Ca2+‐permeable TRPV4 channel, which is abundantly expressed in the distal nephron cells, mediates cellular Ca2+ responses to elevated luminal flow. The current research combined Fura‐2 Ca2+ imaging with immunofluorescent microscopy in split‐opened distal nephrons of C57BL/6 mice to probe molecular determinants regulating subcellular distribution and activity of TRPV4. We found that activation of PKA pathway with forskolin does not affect TRPV4‐ mediated Ca2+ responses to flow, while markedly shifting subcellular distribution of the channel towards the apical membrane. These actions are blocked with a specific PKA inhibitor H‐89. Activation of PKC pathway with PMA significantly increases Ca2+ responses to flow without affecting the subcellular distribution of TRPV4. In contrast, inhibition of PKC with BIM‐1 diminishes cellular responses to elevated flow. Concomitant activation of PKA and PKC cascades greatly increases basal Ca2+ levels indicating constitutive TRPV4 activation and this effect is precluded by a selective TRPV4 antagonist, HC‐067047. Therefore, functional status of TRPV4 channel in the distal nephron is regulated by two distinct signaling pathways. While PKA cascade appears to be responsible for TRPV4 trafficking and translocation to the apical membrane, PKC‐dependent pathway increases the activity of the channel on the plasma membrane.

Mycoplasma fermentans MALP-2 induces heme oxygenase-1 expression via mitogen-activated protein kinases and Nrf2 pathways to modulate cyclooxygenase 2 expression in human monocytes.

Heme oxygenase-1 (HO-1) is a stress-inducible rate-limiting enzyme in heme degradation that confers cytoprotection against oxidative injury and performs a vital function in the maintenance of cell hemostasis. Increasing numbers of reports have indicated that mycoplasma-derived membrane lipoproteins/lipopeptides, such as macrophage-activating lipopeptide-2 (MALP-2), function as agents that stimulate the immune system by producing various inflammatory mediators, such as cytokines and cyclooxygenase 2 (COX-2), which play roles in the pathogenesis of inflammatory responses during mycoplasma infection. Here, we report that MALP-2 induced HO-1 mRNA and protein expression and upregulated HO-1 enzyme activity in THP-1 cells. Specific inhibitors of mitogen-activated protein kinases (MAPKs), SB203580, PD98059, and SP600125, significantly abolished HO-1 expression. In addition, MALP-2 also induced NF-E2-related factor 2 (Nrf2) translocation, and the silencing of Nrf2 expression in THP-1 cells decreased the levels of MALP-2-mediated HO-1 expression. Furthermore, COX-2 protein expression levels were upregulated in THP-1 cells in response to MALP-2, and transfection with small interfering RNAs of HO-1 significantly increased COX-2 accumulation. These results demonstrate that MALP-2 induces HO-1 expression via MAPKs and Nrf2 pathways and, furthermore, that MALP-2-induced COX-2 expression was modulated by HO-1 in THP-1 cells.

Adiponectin attenuates angiotensin II-induced oxidative stress in renal tubular cells through AMPK and cAMP-Epac signal transduction pathways

Obesity is a risk factor for chronic kidney disease (CKD) progression. Circulating levels of adiponectin, an adipokine, decrease with obesity and play a protective role in the cardiovascular system. We hypothesized that adiponectin might also protect the kidney. Because activation of the renin-angiotensin system (RAS) is a contributor to CKD progression, we tested our hypothesis by studying the interactions between adiponectin and angiotensin II (ANG II) in renal tubular cells. Primary human renal proximal tubule cells expressed both adiponectin receptor 1 and 2 (adipoR1 and R2). ANG II-induced NADPH oxidase activation and oxidative stress were attenuated by adiponectin and dependent on adipoR1. Activation of AMPK with 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) mimicked, while inhibition of AMPK with compound C abrogated, the effect of adiponectin on ANG II-induced activation of NADPH oxidase. Similarly, the effect of adiponectin was recapitulated by the stable cAMP analogs 4-chlorophenylthio (pCPT)-cAMP and dibutyryl (db)-cAMP and blocked by the adenylate cyclase inhibitor SQ22536. Adiponectin did not activate PKA in renal tubular cells, and the specific PKA inhibitor myristoylated PKI (14-22) amide failed to block the inhibitory effect of adiponectin on ANG II-induced NADPH oxidase activation. In contrast, the specific Epac activator 8-(4-chlorophenylthio)-2'-O-methyl (8-CPT-2-O-Me)-cAMP blocked ANG II-induced activation of NADPH oxidase, an effect that was reversed by coincubation with the AMPK inhibitor compound C. Finally, adiponectin attenuated ANG II-induced NF-κB activation and fibronectin protein expression. These in vitro findings support the hypothesis that adiponectin may attenuate the deleterious effects of ANG II in the kidney and play a protective role in CKD.

Drosophila Bestrophin-1 Currents Are Regulated by Phosphorylation via a CaMKII Dependent Mechanism

Cell swelling induced by hypo-osmotic stress results in activation of volume-regulated anion channels (VRAC) that drive a compensatory regulatory volume decrease. We have previously shown that the Best1 gene in Drosophila encodes a VRAC that is also activated by increases in intracellular Ca2+. The role of Best1 as a VRAC has recently been independently confirmed by the Clapham lab in an unbiased RNAi screen. Although dBest1 is clearly a volume-regulated channel, its mechanisms of regulation remain unknown. Here we investigate Drosophila Best1 (dBest1) regulation using the Drosophila S2 cell model system. Because dBest1 activates slowly after establishing whole-cell recording, we tested the hypothesis that the channel is activated by phosphorylation. Two experiments indicate that phosphorylation is required for dBest1 activation: nonspecific protein kinase inhibitors or intracellular perfusion with the non-hydrolyzable ATP analog AMP-PNP dramatically reduce the amplitude of dBest1 currents. Furthermore, intracellular perfusion with ATP-γ-S augments channel activation. The kinase responsible for dBest1 activation is likely Ca2+/calmodulin dependent kinase II (CaMKII), because specific inhibitors of this kinase dramatically inhibit dBest1 current activation. Neither specific PKA inhibitors nor inactive control inhibitors have effects on dBest1currents. Our results demonstrate that dBest1 currents are regulated by phosphorylation via a CaMKII dependent mechanism.