MAPK mediates PKC-dependent contraction of cat esophageal and lower esophageal sphincter circular smooth muscle

Esophageal (ESO) circular muscle contraction and lower esophageal sphincter (LES) tone are PKC dependent. Because MAPKs may be involved in PKC-dependent contraction, we examined ERK1/ERK2 and p38 MAPKs in ESO and LES. In permeabilized LES muscle cells, ERK1/2 antibodies reduced 1,2-dioctanoylglycerol (DG)- and threshold ACh-induced contraction, which are PKC dependent, but not maximal ACh, which is calmodulin dependent. LES tone was reduced by the ERK1/2 kinase inhibitor PD-98059 and by the p38 MAPK inhibitor SB-203580. In permeable ESO cells, ACh contraction was reduced by ERK1/ERK2 and p38 MAPK antibodies and by PD-98059 and SB-203580. ACh increased MAPK activity and phosphorylation of MAPK and of p38 MAPK. The 27-kDa heat shock protein (HSP27) antibodies reduced ACh contraction. HSP27 and p38 MAPK antibodies together caused no greater inhibition than either one alone. p38 MAPK and HSP27 coprecipitated after ACh stimulation, suggesting that HSP27 is linked to p38 MAPK. These data suggest that PKC-dependent contraction in ESO and LES is mediated by the following two distinct MAPK pathways: ERK1/2 and HSP27-linked p38 MAPK.     

Tamoxifen elicits its anti-estrogen effects in growth plate chondrocytes by inhibiting protein kinase C

17 beta-Estradiol (E(2)) regulates growth plate cartilage cells via classical nuclear receptor mechanisms, as well as by direct effects on the chondrocyte membrane. These direct effects are stereospecific, causing a rapid increase in protein kinase C (PKC) specific activity, are only found in cells from female rats and are mimicked by E(2)-bovine serum albumin (BSA), which cannot penetrate the cell membrane. E(2) and E(2)-BSA stimulate alkaline phosphatase specific activity and proteoglycan sulfation in female rat costochondral cartilage cell cultures, but traditional nuclear receptors do not appear to be involved. This study examined the effect of the anti-estrogen tamoxifen on these markers of chondrocyte differentiation; the gender-specificity of tamoxifen's effect on PKC, if tamoxifen has an effect on vitamin D metabolite-stimulated PKC, which is mediated via specific membrane receptors (1,25-mVDR; 24,25-mVDR) and whether the effect of tamoxifen is mediated by nuclear estrogen receptors. Tamoxifen dose-dependently inhibited the effect of E(2)-BSA on PKC, alkaline phosphatase and proteoglycan sulfation in confluent cultures of female resting zone (RC) cells and growth zone (GC) (prehypertrophic/upper hypertrophic zones) cells, suggesting that its action is at the membrane and not cell maturation-dependent. Neither the estrogen receptor (ER) antagonist ICI 182780 nor the ER agonist diethylstilbesterol affected E(2) or E(2)-BSA-stimulated PKC in female chondrocytes. Tamoxifen also inhibited the increase in PKC activity due to 1 alpha,25-(OH)(2)D(3) or 24R,25-(OH)(2)D(3) in growth plate cells derived from either female or male rats. Inhibition of PKC by tamoxifen may be a general property of membrane receptors involved in rapid responses to hormones.     

The signalling profile of recombinant human orexin-2 receptor

Orexin-A and orexin-B orchestrate their diverse central and peripheral effects via two G-protein coupled receptors, OX1R and OX2R, which activate multiple G-proteins. In many tissues, orexins activate extracellular signal-regulated kinase (ERK(1/2)) and p38 mitogen-activated protein kinase (MAPK); however, the mechanism by which OX2R alone mediates MAPK activation is not understood. This study describes the intracellular signalling pathways involved in OX2R-mediated ERK(1/2) and p38 MAPK activation. In HEK-293 cells stably over-expressing recombinant human OX2R, orexin-A/B resulted in a rapid, dose and time dependent increase in activation of ERK(1/2) and p38 MAPK, with maximal activation at 10 min for ERK(1/2) and 30 min for p38 MAPK. Using dominant-negative G-proteins and selective inhibitors of intracellular signalling cascades, we determined that orexin-A and orexin-B induced ERK(1/2) and p38 MAPK activation through multiple G-proteins and different intracellular signalling pathways. ERK(1/2) activation involves Gq/phospholipase C (PLC)/protein kinase C (PKC), Gs/adenylyl cyclase (AC)/cAMP/protein kinase A (PKA) and Gi cascades; however, the Gq/PLC/PKC pathway, as well as PKA is not required for OX2R-mediated p38 MAPK activation. Interestingly, orexin-B-induced ERK(1/2) activation is predominantly mediated through the Gq/PLC/PKC pathway. In conclusion, this is the first comprehensive signalling study of the human OX2R recombinant receptor, showing ERK(1/2) and p38 MAPK activation are regulated by differential signalling pathways in HEK-293 cells, and that the ERK(1/2) activation is severely affected by naturally occurring mutants associated with narcolepsy. Moreover, it is evident that the human OX2R has ligand specific effects, with orexin-B being more potent in this transfected system and this distinct modulation of the MAPKs through OX2R, may translate to the regulation of diverse biological actions of orexins.     

Selective induction of glial glutamate transporter GLT-1 by hypertonic stress in C6 glioma cells.

Glial glutamate transporter GLT-1 mRNA was selectively induced in C6 glioma cells exposed to hypertonic stress (HS), while the expression of two other subtypes, GLAST and EAAC1, was suppressed. HS increased phosphorylation of the MAPK family, ERK, p38 MAPK, and JNK. Treatment with a PKC inhibitor showed that phosphorylation of both p38 MAPK and JNK is PKC-dependent but ERK phosphorylation is independent. Inhibition of either ERK or p38 MAPK did not abolish GLT-1 mRNA induction. Inhibition of PKC also had no effect. These findings indicate that the induction of GLT-1 mRNA by HS is independent of the MAPK pathways. This is the first report that the expression of glial glutamate transporters is osmotically regulated.     

Icaritin induces growth inhibition and apoptosis of human prostatic smooth muscle cells in an estrogen receptor-independent manner

Icaritin has selective estrogen receptor (ER) modulating activity. ERs are expressed in the prostate stroma, and estrogens have an important role in the pathology of benign prostatic hyperplasia (BPH). However, the impact of icaritin on BPH was not studied. Human prostatic smooth muscle cells (PSMCs) were treated with 0–100 μM icaritin, also using 10 μM ICI182780 as a specific ER antagonist. The effects on cell growth and apoptosis were determined by cell counting and sandwich-enzyme-immunoassay. Western blotting was employed to illustrate the possible mechanisms. Cell growth was strongly inhibited by icaritin, and this was accompanied by an augmented apoptosis. Few changes in icaritin-induced growth inhibition and apoptosis were observed after pretreatment in the presence of ICI182780. Consistent with growth inhibition and apoptosis induction, icaritin decreased cyclin D1 and CDK4 expression and increased Bax/Bcl-2 ratio in human PSMCs. Furthermore, icaritin induced sustained phosphorylation of extracellular signal-regulated kinase (ERK) in human PSMCs. PD98059, a specific ERK inhibitor, blocked the activation of ERK by icaritin and abolished the icaritin-induced growth inhibition and apoptosis. The results indicate that icaritin reduces growth and induces apoptosis in human PSMCs via ERK signaling pathway without involvement of ERs.     

Involvement of Estrogen in Rapid Pain Modulation in the Rat Spinal Cord

The pivotal role of estrogens in the pain sensitivity has been investigated in many ways. Traditionally, it is ascribed to the slow genomic changes mediated by classical nuclear estrogen receptors (ER), ER?? and ER??, depending on peripheral estrogens. Recently, it has become clear that estrogens can also signal through membrane ERs (mERs), such as G-protein-coupled ER1 (GPER1), mediating the non-genomic effects. However, the spinal specific role played by ERs and the underlying cellular mechanisms remain elusive. The present study investigated the rapid estrogenic regulation of nociception at the spinal level. Spinal administration of 17??-estradiol (E2), the most potent natural estrogen, acutely produced a remarkable mechanical allodynia and thermal hyperalgesia without significant differences among male, female and ovariectomized (Ovx) rats. E2-induced the pro-nociceptive effects were partially abrogated by ICI 182,780 (ERs antagonist), and mimicked by E2-BSA (a mER agonist). Inhibition of local E2 synthesis by 1,4,6-Androstatrien-3,17-dione (ATD, a potent irreversible aromatase inhibitor), or blockade of ERs by ICI 182,780 produced an inhibitory effect on the late phase of formalin nociceptive responses. Notably, lumbar puncture injection of G15 (a selective GPER1 antagonist) resulted in similar but more efficient inhibition of formalin nociceptive responses as compared with ICI 182,780. At the cellular level, the amplitude and decay time of spontaneous inhibitory postsynaptic currents were attenuated by short E2 or E2-BSA treatment in spinal slices. These results indicate that estrogen acutely facilitates nociceptive transmission in the spinal cord via activation of membrane-bound estrogen receptors.     

Sphingosine kinase 1 is involved in dibutyryl cyclic AMP-induced granulocytic differentiation through the upregulation of extracellular signal-regulated kinase, but not p38 MAP kinase, in HL60 cells

The role of sphingosine kinase (SPHK) in the dibutyryl cyclic AMP (dbcAMP)-induced granulocytic differentiation of HL60 cells was investigated. During differentiation, SPHK activity was increased, as were mRNA and protein levels of SPHK1, but not of SPHK2. Pretreatment of HL60 cells with N,N-dimethylsphingosine (DMS), a potent SPHK inhibitor, completely blocked dbcAMP-induced differentiation. The phosphorylation of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 MAPK was also increased during dbcAMP-induced differentiation. Pretreatment of HL60 cells with the MEK inhibitor, U0126, but not the p38 MAPK inhibitor, SB203580, completely suppressed dbcAMP-induced ERK1/2 activation and granulocytic differentiation, but did not affect the increase in SPHK activity. DMS inhibited dbcAMP-induced ERK1/2 activation, but had little effect on p38 MAPK activation. DMS had no effect on the dbcAMP-induced membrane translocation of protein kinase C (PKC) isozymes, and PKC inhibitors had no significant effect on ERK activation. The overexpression of wild-type SPHK1, but not dominant negative SPHK1, resulted in high basal levels of ERK1/2 phosphorylation and stimulated granulocytic differentiation in HL60 cells. These data show that SPHK1 participates in the dbcAMP-induced differentiation of HL60 cells by activating the MEK/ERK pathway.     

Roles of mitogen-activated protein kinase pathways during <Emphasis Type="Italic">Escherichia coli-</Emphasis>induced apoptosis in U937 cells

Escherichia coli (E. coli) infections play an important and growing role in the clinic. In the present study, we investigated the involvement of members of the mitogen-activated protein kinase (MAPK) superfamily, including extracellular signal-regulated kinase (ERK), c-Jun NH₂-terminal kinase (JNK) and p38 MAPK, and caspase-3 and 9 activity in E. coli-induced apoptosis in human U937 cells. We found that E. coli induces apoptosis in U937 cell lines in a dose- and time-dependent manner, p38 MAPK and JNK were activated after 10 min of infection with E. coli. In contrast, ERK1/2 was down-regulated in a time-dependent manner. The levels of total (phosphorylation state-independent) p38 MAPK, JNK and ERK1/2 did not change in E. coli-infected U937 cells at all times examined. Moreover, exposure of U937 cells to E. coli led to caspase-3 and 9 activity. For the evaluation of the role of MAPKs, PD98059, SB203580 and SP600125 were used as MAPKs inhibitors for ERK1/2, p38 MAPK and JNK. Inhibition of ERK1/2 with PD98059 caused further enhancement in apoptosis and caspase-3 and 9 activity, while a selective p38 MAPK inhibitor, SB203580 and JNK inhibitor, SP600125 significantly inhibited E. coli-induced apoptosis and caspase-3 and 9 activity in U937 cells. The results were further confirmed by the observation that the caspase inhibitors Z-DEVD-FMK and Z-LEHD-FMK blocked E. coli-induced U937 apoptosis. Taken together, we have shown that E. coli increase p38 MAPK and JNK and decrease ERK1/2 phosphorylation and increase caspase-3 and 9 activity in U937 cells.     

Estradiol-17beta protects against hypoxia-induced hepatocyte injury through ER-mediated upregulation of Bcl-2 as well as ER-independent antioxidant effects

Although many previous studies have suggested that estrogen functions as a cytoprotective agent under oxidative stress conditions, the underlying mechanism by which this effect is exerted remains to be elucidated. This study assessed the effects of estradiol-17β （E2） （10^-8s M） on hypoxia-induced cell injury and its related signaling in primary cultured chicken hepatocytes. Hypoxic conditions were found to augment the level of DNA damage and to reduce cell viability and the level of [^3H]-thymidine incorporation, and these phenomena were prevented through treatment with E2. Hypoxia also increased caspase-3 expression, but showed no evidence of an influence on the expression of Bcl-2. However, E2 induced an increase in the level of Bcl-2 expression under hypoxic conditions and reduced the level of caspase-3 expression. The effects of E2 on Bcl-2 and caspase expression were blocked by ICI 182780 （E2 receptor （ER） antagonist, 10＂7 M）. In addition, hypoxia resulted in an increase in the intracellular reactive oxygen species （ROS） generated. These effects were blocked by E2, but not by E2-BSA and ICI 182780. Hypoxia also activated p38 mitogen-activated protein kinase （MAPK）, c-JUN N-terminal kinase/stress-activated protein kinase （JNK/SAPK） and nuclear factor-kB （NF-kB）. These effects were blocked by E2, but not by ICI 182780. The inhibition of p38 MAPK and JNK/SAPK blocked NF-kB activation. In conclusion, E2 was found to protect against hypoxia-induced cell injury in chicken hepatocytes through ER-mediated upregulation of Bcl-2 expression and through reducing the activity of ROS-dependent p38 MAPK, JNK/ SAPK and NF-kB.     

17 beta-estradiol-BSA conjugates and 17 beta-estradiol regulate growth plate chondrocytes by common membrane associated mechanisms involving PKC dependent and independent signal transduction

Nuclear receptors for 17 beta-estradiol (E(2)) are present in growth plate chondrocytes from both male and female rats and regulation of chondrocytes through these receptors has been studied for many years; however, recent studies indicate that an alternative pathway involving a membrane receptor may also be involved in the cell response. E(2) was found to directly affect the fluidity of chondrocyte membranes derived from female, but not male, rats. In addition, E(2) activates protein kinase C (PKC) in a nongenomic manner in female cells, and chelerythrine, a specific inhibitor of PKC, inhibits E(2)-dependent alkaline phosphatase activity and proteoglycan sulfation in these cells, indicating PKC is involved in the signal transduction mechanism. The aims of the present study were: (1) to examine the effect of a cell membrane-impermeable 17 beta-estradiol-bovine serum albumin conjugate (E(2)-BSA) on chondrocyte proliferation, differentiation, and matrix synthesis; (2) to determine the pathway that mediates the membrane effect of E(2)-BSA on PKC; and (3) to compare the action of E(2)-BSA to that of E(2). Confluent, fourth passage resting zone (RC) and growth zone (GC) chondrocytes from female rat costochondral cartilage were treated with 10(-9) to 10(-7) M E(2) or E(2)-BSA and changes in alkaline phosphatase specific activity, proteoglycan sulfation, and [(3)H]-thymidine incorporation measured. To examine the pathway of PKC activation, chondrocyte cultures were treated with E(2)-BSA in the presence or absence of GDP beta S (inhibitor of G-proteins), GTP gamma S (activator of G-proteins), U73122 or D609 (inhibitors of phospholipase C [PLC]), wortmannin (inhibitor of phospholipase D [PLD]) or LY294002 (inhibitor of phosphatidylinositol 3-kinase). E(2)-BSA mimicked the effects of E(2) on alkaline phosphatase specific activity and proteoglycan sulfation, causing dose-dependent increases in both RC and GC cell cultures. Both forms of estradiol inhibited [(3)H]-thymidine incorporation, and the effect was dose-dependent. E(2)-BSA caused time-dependent increases in PKC in RC and GC cells; effects were observed within three minutes in RC cells and within one minute in GC cells. Response to E(2) was more robust in RC cells, whereas in GC cells, E(2) and E(2)-BSA caused a comparable increase in PKC. GDP beta S inhibited the activation of PKC in E(2)-BSA-stimulated RC and GC cells. GTP gamma S increased PKC in E(2)-BSA-stimulated GC cells, but had no effect in E(2)-BSA-stimulated RC cells. The phosphatidylinositol-specific PLC inhibitor U73122 blocked E(2)-BSA-stimulated PKC activity in both RC and GC cells, whereas the phosphatidylcholine-specific PLC inhibitor D609 had no effect. Neither the PLD inhibitor wortmannin nor the phosphatidylinositol 3-kinase inhibitor LY294022 had any effect on E(2)-BSA-stimulated PKC activity in either RC or GC cells. The classical estrogen receptor antagonist ICI 182780 was unable to block the stimulatory effect of E(2)-BSA on PKC. Moreover, the classical receptor agonist diethylstilbestrol (DES) had no effect on PKC, nor did it alter the stimulatory effect of E(2)-BSA. The specificity of the membrane response to E(2) was also demonstrated by showing that the membrane receptor for 1 alpha,25-(OH)(2)D(3) was not involved. These data indicate that the rapid nongenomic effect of E(2)-BSA on PKC activity in RC and GC cells is dependent on G-protein-coupled PLC and support the hypothesis that many of the effects of E(2) involve membrane-associated mechanisms independent of classical estrogen receptors. (c) 2001 Wiley-Liss, Inc.     

GnRH activates ERK1/2 leading to the induction of c-fos and LHbeta protein expression in LbetaT2 cells

GnRH acts on pituitary gonadotropes to stimulate the synthesis and release of LH and FSH. However, the signaling pathways downstream of the GnRH receptor that mediate these effects are not fully understood. In this paper, we demonstrate that GnRH activates ERK, c-Jun N-terminal kinase, and p38MAPK in the LbetaT2 gonadotrope cell line. Phosphorylation of both ERK and p38MAPK are stimulated rapidly, 30- to 50-fold in 5 min, but activation of c-Jun N-terminal kinase has slower kinetics, reaching only 10-fold after 30 min. Activation of ERK by GnRH is blocked by inhibition of MAPK kinase (MEK) and partially blocked by inhibition of PKC and calcium, but not PI3K or p38MAPK signaling. We demonstrate that phosphorylated ERK accumulates in the nucleus in a PKC-dependent manner. We also show that GnRH induces c-fos and LHbeta subunit protein expression in LbetaT2 cells via MEK. Experiments with EGTA or calcium channel antagonists indicated that calcium influx is important for the induction of both genes by GnRH. In conclusion, these results show that GnRH activates all three MAPK subfamilies in LbetaT2 cells and induces c-fos and LHbeta protein expression through calcium and MEK-dependent mechanisms. These results also demonstrate that the nuclear translocation of ERK by GnRH requires PKC signaling.     

Differential activation of MAP kinase family members triggered by CD99 engagement

The molecular basis for the modulatory properties of CD99 is not well understood. Treatment of human Jurkat T lymphocytes with anti-CD99 antibody led to activation of three mitogen-activated protein kinase (MAPK) members, ERK, JNK, and p38 MAPK, along with homotypic aggregation. While phosphorylation of ERK and JNK was inhibited by the pretreatment of a PKC inhibitor, bisindolylmaleimide I, activation of p38 MAPK was upregulated by the same pretreatment. The signaling pathways to MAPKs by CD99 engagement were independent of PI-3 kinase, distinguishing from those by CD3 engagement. Among MAPKs, ERK pathway was essential for homotypic aggregation together with intracytoplasmic Ca(2+).     

Cross talk between glucocorticoid and estrogen receptors occurs at a subset of proinflammatory genes

Glucocorticoids exert potent anti-inflammatory effects by repressing proinflammatory genes. We previously demonstrated that estrogens repress numerous proinflammatory genes in U2OS cells. The objective of this study was to determine if cross talk occurs between the glucocorticoid receptor (GR) and estrogen receptor (ER)α. The effects of dexamethasone (Dex) and estradiol on 23 proinflammatory genes were examined in human U2OS cells stably transfected with ERα or GR. Three classes of genes were regulated by ERα and/or GR. Thirteen genes were repressed by both estradiol and Dex (ER/GR-repressed genes). Five genes were repressed by ER (ER-only repressed genes), and another five genes were repressed by GR (GR-only repressed genes). To examine if cross talk occurs between ER and GR at ER/GR-repressed genes, U2OS-GR cells were infected with an adenovirus that expresses ERα. The ER antagonist, ICI 182780 (ICI), blocked Dex repression of ER/GR-repressed genes. ICI did not have any effect on the GR-only repressed genes or genes activated by Dex. These results demonstrate that ICI acts on subset of proinflammatory genes in the presence of ERα but not on GR-activated genes. ICI recruited ERα to the IL-8 promoter but did not prevent Dex recruitment of GR. ICI antagonized Dex repression of the TNF response element by blocking the recruitment of nuclear coactivator 2. These findings indicate that the ICI-ERα complex blocks Dex-mediated repression by interfering with nuclear coactivator 2 recruitment to GR. Our results suggest that it might be possible to exploit ER and GR cross talk for glucocorticoid therapies using drugs that interact with ERs.     

Heterogeneity of the signal transduction pathways for VEGF-induced MAPKs activation in human vascular endothelial cells.

Vascular endothelial growth factor (VEGF) activates ERK and p38 MAPK in endothelial cells (ECs). The present study was aimed to compare its intracellular signal transduction pathways between three primary cultures of human ECs including human aortic ECs (HAECs), human umbilical vein ECs (HUVECs), and human microvascular ECs (HMVECs). VEGF activated ERK and p38 MAPK in all of three ECs. Isoforms of p38 MAPK that were activated by VEGF in HUVECs were p38-alpha and p38-delta. GF109203X, a specific inhibitor of PKC, markedly inhibited VEGF-induced activation of ERK and p38 MAPK in HAECs and HUVECs, whereas it exhibited little effect in HMVECs. In contrast, dominant negative mutant of Ha-Ras almost completely abrogated VEGF-induced activation of ERK and p38 MAPK in HMVECs. Although dominant negative mutant of Ha-Ras substantially inhibited the basal activities of ERK and p38 MAPK, it exhibited marginal effect on VEGF-induced activation of ERK and p38 MAPK in HUVECs and HAECs. The activation of Ras by VEGF appeared to be most prominent in HMVECs. These results indicate that intracellular signal transduction pathways for VEGF-induced activation of MAPKs are heterogeneous and vary depending on the origin of ECs.Copyright 2001 Wiley-Liss, Inc.     

PKC-dependent delayed metabolic preconditioning is independent of transient MAPK activation

In this study we used an in vitro model of delayed preconditioning to investigate activation of mitogen-activated protein kinases (MAPKs) and their potential role in protection. Neonatal rat cardiomyocytes were preconditioned using a buffer containing glycolytic inhibitors and low pH (minimal metabolic preconditioning; MMPC) consisting of modified Krebs buffer, 10 mM 2-deoxyglucose, and 20 mM lactate, pH 6.8, for 2 h followed by 24 h of simulated reperfusion before lethal simulated ischemia (LSI). MAPK activation during the MMPC protocol was determined using phospho-specific antisera and the effect on protection determined following LSI. Rapid, transient phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 MAPK was observed during each of the MMPC, reperfusion, and LSI phases; an effect blocked by MAPK inhibitors PD-98059 and SB-203580, respectively, but not by the protein kinase C (PKC) inhibitor Ro31-8220. However, although MMPC was blocked by Ro31-8220, treatment with the MAPK inhibitors during the preconditioning protocol did not block delayed protection conferred by MMPC. Thus the data suggest that, in this model of delayed preconditioning, protection appears to be PKC dependent but independent of ERK1/2 or p38 MAPK activation.