Wimolpak Sriwai

Gi-coupled receptors mediate phosphorylation of CPI-17 and MLC20 via preferential activation of the PI3K/ILK pathway

Sustained smooth-muscle contraction or its experimental counterpart, Ca2+ sensitization, by G(q/13)-coupled receptor agonists is mediated via RhoA-dependent inhibition of MLC (myosin light chain) phosphatase and MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation by a Ca2+-independent MLCK (MLC kinase). The present study identified the corresponding pathways initiated by G(i)-coupled receptors. Somatostatin acting via G(i)1-coupled sstr3 receptor, DPDPE ([D-Pen2,D-Pen5]enkephalin; where Pen is penicillamine) acting via G(i)2-coupled delta-opioid receptors, and cyclopentyl adenosine acting via G(i)3-coupled adenosine A1 receptors preferentially activated PI3K (phosphoinositide 3-kinase) and ILK (integrin-linked kinase), whereas ACh (acetylcholine) acting via G(i)3-coupled M2 receptors preferentially activated PI3K, Cdc42 (cell division cycle 42)/Rac1, PAK1 (p21-activated kinase 1) and p38 MAPK (mitogen-activated protein kinase). Only agonists that activated ILK induced sustained CPI-17 (protein kinase C potentiated inhibitor 17 kDa protein) phosphorylation at Thr38, MLC20 phosphorylation at Ser19, and contraction, consistent with recent evidence that ILK can act as a Ca2+-independent MLCK capable of phosphorylating the MLC phosphatase inhibitor, CPI-17, at Thr38. ILK activity, and CPI-17 and MLC20 phosphorylation were inhibited by LY294002 and in muscle cells expressing ILK(R211A) or treated with siRNA (small interfering RNA) for ILK. ACh acting via M2 receptors activated ILK, and induced CPI-17 and MLC20 phosphorylation and muscle contraction, but only after inhibition of p38 MAPK; all these responses were inhibited in cells expressing ILK(R211A). Conversely, ACh activated PAK1, a step upstream of p38 MAPK, whereas the three other agonists did so only in cells transfected with ILK(R211A) or siRNA for ILK. The results demonstrate reciprocal inhibition between two pathways downstream of PI3K, with ILK inhibiting PAK1, and p38 MAPK inhibiting ILK. Sustained contraction via G(i)-coupled receptors is dependent on CPI-17 and MLC20 phosphorylation by ILK.

Gq-dependent signalling by the lysophosphatidic acid receptor LPA3 in gastric smooth muscle: reciprocal regulation of MYPT1 phosphorylation by Rho kinase and cAMP-independent PKA

The present study characterized the signalling pathways initiated by the bioactive lipid, LPA (lysophosphatidic acid) in smooth muscle. Expression of LPA(3) receptors, but not LPA(1) and LPA(2), receptors was demonstrated by Western blot analysis. LPA stimulated phosphoinositide hydrolysis, PKC (protein kinase C) and Rho kinase (Rho-associated kinase) activities: stimulation of all three enzymes was inhibited by expression of the G(alphaq), but not the G(alphai), minigene. Initial contraction and MLC(20) (20 kDa regulatory light chain of myosin II) phosphorylation induced by LPA were abolished by inhibitors of PLC (phospholipase C)-beta (U73122) or MLCK (myosin light-chain kinase; ML-9), but were not affected by inhibitors of PKC (bisindolylmaleimide) or Rho kinase (Y27632). In contrast, sustained contraction, and phosphorylation of MLC(20) and CPI-17 (PKC-potentiated inhibitor 17 kDa protein) induced by LPA were abolished selectively by bisindolylmaleimide. LPA-induced activation of IKK2 {IkappaB [inhibitor of NF-kappaB (nuclear factor kappaB)] kinase 2} and PKA (protein kinase A; cAMP-dependent protein kinase), and degradation of IkappaBalpha were blocked by the RhoA inhibitor (C3 exoenzyme) and in cells expressing dominant-negative mutants of IKK2(K44A) or RhoA(N19RhoA). Phosphorylation by Rho kinase of MYPT1 (myosin phosphatase targeting subunit 1) at Thr(696) was masked by phosphorylation of MYPT1 at Ser(695) by PKA derived from IkappaB degradation via RhoA, but unmasked in the presence of PKI (PKA inhibitor) or C3 exoenzyme and in cells expressing IKK2(K44A). We conclude that LPA induces initial contraction which involves activation of PLC-beta and MLCK and phosphorylation of MLC(20), and sustained contraction which involves activation of PKC and phosphorylation of CPI-17 and MLC(20). Although Rho kinase was activated, phosphorylation of MYPT1 at Thr(696) by Rho kinase was masked by phosphorylation of MYPT1 at Ser(695) via cAMP-independent PKA derived from the NF-kappaB pathway.

Cross-regulation of VPAC2 receptor internalization by m2 receptors via c-Src-mediated phosphorylation of GRK2

The aim of the study was to examine the mechanisms by which ACh, acting via m2 receptors, regulates GRK2-mediated VPAC(2) receptor desensitization in gastric smooth muscle cells. VIP induced VPAC(2) receptor phosphorylation and internalization in freshly dispersed smooth muscle cells. Co-stimulation with acetylcholine (ACh), in the presence of m3 receptor antagonist, 4-DAMP, augmented VPAC(2) receptor phosphorylation and internalization. The m2 receptor antagonist methoctramine or the c-Src inhibitor PP2 blocked the effect of ACh, suggesting that the augmentation was mediated by c-Src, derived from m2 receptor activation. ACh induced activation of c-Src and phosphorylation of GRK2 and the effects of ACh were blocked by methoctramine, PP2, or by uncoupling of m2 receptors from G(i3) with pertussis toxin. In conclusion, we identified a novel mechanism of cross-regulation of GRK2-mediated phosphorylation and internalization of G(s)-coupled VPAC(2) receptors by G(i)-coupled m2 receptors via tyrosine phosphorylation of GRK2 and stimulation of GRK2 activity.

Diabetes-induced oxidative stress mediates upregulation of RhoA/Rho kinase pathway and hypercontractility of gastric smooth muscle

The pathogenesis of diabetes-associated motility disorders are multifactorial and attributed to abnormalities in extrinsic and intrinsic innervation, and a decrease in the number of interstitial cells of Cajal, and nNOS expression and activity. Here we studied the effect of hyperglycemia on smooth muscle function. Using smooth muscles from the fundus of ob/ob mice and of wild type (WT) mice treated with 30 mM glucose (HG), we identified the molecular mechanism by which hyperglycemia upregulates RhoA/Rho kinase pathway and muscle contraction. RhoA expression, Rho kinase activity and muscle contraction were increased, while miR-133a expression was decreased in smooth muscle of ob/ob mice and in smooth muscle treated with HG. Intraperitoneal injections of pre-miR-133a decreased RhoA expression in WT mice and reversed the increase in RhoA expression in ob/ob mice. Intraperitoneal injections of antagomiR-133a increased RhoA expression in WT mice and augmented the increase in RhoA expression in ob/ob mice. The effect of pre-miR-133a or antagomiR-133a in vitro in smooth muscle treated with HG was similar to that obtained in vivo, suggesting that the expression of RhoA is negatively regulated by miR-133a and a decrease in miR-133a expression in diabetes causes an increase in RhoA expression. Oxidative stress (levels of reactive oxygen species and hydrogen peroxide, and expression of superoxide dismutase 1 and NADPH oxidase 4) was increased in smooth muscle of ob/ob mice and in HG-treated smooth muscle. Treatment of ob/ob mice with N-acetylcysteine (NAC) in vivo or addition of NAC in vitro to HG-treated smooth muscle reversed the effect of glucose on the expression of miR-133a and RhoA, Rho kinase activity and muscle contraction. NAC treatment also reversed the decrease in gastric emptying in ob/ob mice. We conclude that oxidative stress in diabetes causes a decrease in miR-133a expression leading to an increase in RhoA/Rho kinase pathway and muscle contraction.

Protease-Activated Receptor-2 Reduces Cyclohexamide-Induced Apoptosis in K562 Myeloid Leukemia Cells

Myelodysplastic syndrome (MDS) is clonal disease of hematopoietic precursor cells that is characterized by pancytopenia. Patients with MDS have an increase in the risk of developing acute myeloid leukemia (AML), characterized by unlimited growth and accumulation of blast cells in hematopoietic tissues. Blast cells derived from a group of patients with AML produce and secrete tryptase. These patients exhibit increased expression of bone marrow tryptase and serum tryptase levels. There is evidence that PAR-2, cleaved and activated by tryptase, contributes to tumor progression and proliferation in various cancer cell types. However, the role of PAR-2 in the context of myeloid leukemia remains unknown. Aims. The aim of the present study was to determine the effect of PAR-2 activation on leukemic cell survival and proliferation. Methods. Protein expression of PAR-2 was examined in K562 cells, a human myeloid leukemia cell line, by RT-PCR. K562 cells were treated with the PAR-2 activating peptide SLIGRL-NH2 (1, 10 and 100 μM) for up to 72 hours in serumfree media. Media containing serum and serum free media were used as positive and negative controls, respectively. Cell proliferation in response to agonist peptide was evaluated by the MTT assay after exposure to serum-free media, SLIGRL-NH2 or serum-containing media. Caspase-3 and cleaved caspase-3 protein levels were determined by Western blots in the combined presence of PAR-2 activating peptide and cyclohexamide (50 μg/ml). Results. PAR-2 was detected in K562 cells. These cells showed a significant increase in cell growth as determined by MTT assay after treatment with serum-containing media. However, PAR2 activating peptide did not significantly alter cell growth as determined absorbance by MTT assay when compared to controls at any of the time points examined. Treatment with cyclohexamide alone significantly increased cleaved caspase-3. The PAR-2 activating peptide alone did not alter K562 cell apoptosis, as indicated by measurement of cleaved caspase-3. However, the effect of cyclohexamide on caspase-3 cleavage was significantly decreased in the present of PAR-2 activating peptide, suggesting anti-apoptotic activity. Conclusions. Our studies show that PAR-2 activation does not stimulate cellular proliferation in myeloid leukemia cells. Instead, PAR-2 activating peptide reduces leukemic cell response to cyclohexamide-induced apoptosis. The results imply that enhanced tryptase levels during MDS or AML may, via activating PAR-2, offer protection of malignant cells against chemotherapeutic agents thereby result in a survival advantage of the malignant clone.

S1624 Inhibition of Sustained Smooth Muscle Contraction By PKA and PKG Is Mediated By Phosphorylation of Gα13 and Inhibition of Gα13-Dependent Activation of RHOA

A, valproic acid, apicidin and MS275). NTR1 mRNA and protein expression was assessed by Northern and Western blots, respectively. NTR1 promoter activity was assessed by luciferase assay. (ii) The role of GSK-3β and ERK signaling pathways was determined using the selective inhibitors SB-216763 (for GSK-3β) and U0126 or PD98059 (for MEK/ERK) as well as siRNA to GSK-3β. (iii) To assess the functional effects of HDACI regulation of NTR, the effect of NaBT on NT-mediated induction of COX-2, c-myc and IL-8 expression was assessed by Northern blot and RNase protection assay. RESULTS. (i) Treatment with all HDACIs resulted in the potent down-regulation of endogenous NTR1 mRNA, protein and promoter activity. Northern blot analyses showed that HDACIs down-regulate NTR1 mRNA in a time-and dose-dependent fashion in all five NTR1-positive cell lines. Notably, NTR1mRNAwas dramatically decreased after 4 h of NaBT treatment in HT29 cells suggesting that NTR1 may be a direct target gene of HDACIs. (ii) Overexpression of GSK-3β decreased NTR1 promoter activity (> 30%); inhibition of GSK-3β increased NTR1 expression in HT29 and SW480 cells, indicating that GSK-3β is a negative regulator of ERK and NTR1. Consistent with our previous findings, HDACIs significantly decreased phosphorylated ERK while increasing GSK-3β. Selective MEK/ERK inhibitors suppressed NTR1 mRNA expression in a timeand dose-dependent fashion, and reduced NTR1 promoter activity by ~70%. (iii) Pretreatment with NaBT prevented NT-mediated COX-2 and c-myc expression and attenuated NT-induced IL-8 expression. CONCLUSIONS. HDACIs suppress endogenous NTR1 expression and function in CRC cell lines; this effect may involve both a direct and/or an indirect mechanism through the GSK-3β/ERK pathway. The down-regulation of NTR1 in CRCs may represent an important mechanism for the anti-cancer effects of HDACIs.