Distinct roles of Smad2-, Smad3-, and ERK-dependent pathways in transforming growth factor-beta1 regulation of pancreatic stellate cellular functions

Pancreatic stellate cells (PSCs) play a major role in promoting pancreatic fibrosis. Transforming growth factor-beta(1) (TGF-beta(1)) regulates PSC activation and proliferation in an autocrine manner. The intracellular signaling pathways of the regulation were examined in this study. Immunoprecipitation and immunocytochemistry revealed that Smad2, Smad3, and Smad4 were functionally expressed in PSCs. Adenovirus-mediated expression of Smad2, Smad3, or dominant-negative Smad2/3 did not alter TGF-beta(1) mRNA expression level or the amount of autocrine TGF-beta(1) peptide. However, expression of dominant-negative Smad2/3 inhibited PSC activation and enhanced their proliferation. Co-expression of Smad2 with dominant-negative Smad2/3 restored PSC activation inhibited by dominant-negative Smad2/3 expression without changing their proliferation. By contrast, co-expression of Smad3 with dominant-negative Smad2/3 attenuated PSC proliferation enhanced by dominant-negative Smad2/3 expression without altering their activation. Exogenous TGF-beta(1) increased TGFbeta(1) mRNA expression in PSCs. However, PD98059, a specific inhibitor of mitogen-activated protein kinase kinase (MEK1), inhibited ERK activation by TGF-beta(1), and consequently attenuated TGF-beta(1) enhancement of its own mRNA expression in PSCs. We propose that TGF-beta(1) differentially regulates PSC activation, proliferation, and TGF-beta(1) mRNA expression through Smad2-, Smad3-, and ERK-dependent pathways, respectively.     

Angiotensin II promotes the proliferation of activated pancreatic stellate cells by Smad7 induction through a protein kinase C pathway

Activated pancreatic stellate cells (PSCs) play major roles in promoting pancreatic fibrosis. We previously reported that angiotensin II (Ang II) enhances activated PSC proliferation through EGF receptor transactivation. In the present study, we elucidated a novel intracellular mechanism by which Ang II stimulates cellular proliferation. TGF-beta1 inhibits activated PSC proliferation via a Smad3 and Smad4-dependent pathway in an autocrine manner. We demonstrated that Ang II inhibited TGF-beta1-induced nuclear accumulation of Smad3 and Smad4. Furthermore, Ang II rapidly induced inhibitory Smad7 mRNA expression. Adenovirus-mediated Smad7 overexpression inhibited TGF-beta1-induced nuclear accumulation of Smad3 and Smad4, and potentiated activated PSC proliferation. PKC inhibitor Go6983 blocked the induction of Smad7 mRNA expression by Ang II. In addition, 12-O-tetradecanoyl-phorbol 13-acetate, a PKC activator, increased Smad7 mRNA expression. These results suggest that Ang II enhances activated PSC proliferation by blocking autocrine TGF-beta1-mediated growth inhibition by inducing Smad7 expression via a PKC-dependent pathway.     

TGF-beta signaling preserves RECK expression in activated pancreatic stellate cells

Activated pancreatic stellate cells (PSCs) play a pivotal role in the pathogenesis of pancreatic fibrosis, but the detailed mechanism for dysregulated accumulation of extracellular matrix (ECM) remains unclear. Cultured rat PSCs become activated by profibrogenic mediators, but these mediators failed to alter the expression levels of matrix metalloproteinases (MMPs) to the endogenous tissue inhibitors of metalloproteinases (TIMPs). Here, we examined the expression of RECK, a novel membrane-anchored MMP inhibitor, in PSCs. Although RECK mRNA levels were largely unchanged, RECK protein expression was barely detected at 2, 5 days after plating PSCs, but appeared following continued in vitro culture and cell passage which result in PSC activation. When PSCs at 5 days after plating (PSCs-5d) were treated with pepstatin A, an aspartic protease inhibitor, or TGF-beta1, a profibrogenic mediator, RECK protein was detected in whole cell lysates. Conversely, Smad7 overexpression or suppression of Smad3 expression in PSCs after passage 2 (PSCs-P2) led to the loss of RECK protein expression. These findings suggest that RECK is post-translationally processed in pre-activated PSCs but protected from proteolytic degradation by TGF-beta signaling. Furthermore, collagenolytic activity of PSCs-5d was greatly reduced by TGF-beta1, whereas that of PSCs-P2 was increased by anti-RECK antibody. Increased RECK levels were also observed in cerulein-induced acute pancreatitis. Therefore, our results suggest for the first time proteolytic processing of RECK as a mechanism regulating RECK activity, and demonstrate that TGF-beta signaling in activated PSCs may promote ECM accumulation via a mechanism that preserves the protease inhibitory activity of RECK.     

Cyclooxygenase-2 is required for activated pancreatic stellate cells to respond to proinflammatory cytokines

Cyclooxygenase-2 (COX-2) mediates various inflammatory responses and is expressed in pancreatic tissue from patients with chronic pancreatitis. To examine the role of COX-2 in chronic pancreatitis, we investigated its participation in regulating functions of pancreatic stellate cells (PSCs), using isolated rat PSCs. COX-2 was expressed in culture-activated PSCs but not in freshly isolated quiescent PSCs. TGF-1, IL-1, and IL-6 enhanced COX-2 expression in activated PSCs, concomitantly increasing the expression of -smooth muscle actin (-SMA), a parameter of PSC activation. The COX-2 inhibitor NS-398 blocked culture activation of freshly isolated quiescent PSCs. NS-398 also inhibited the enhancement of -SMA expression by TGF-1, IL-1, and IL-6 in activated PSCs. These data indicate that COX-2 is required for the initiation and promotion of PSC activation. We further investigated the mechanism by which cytokines enhance COX-2 expression in PSCs. Adenovirus-mediated expression of dominant negative Smad2/3 inhibited the increase in expression of COX-2, -SMA, and collagen-1 mediated by TGF-1 in activated PSCs. Moreover, dominant negative Smad2/3 expression attenuated the expression of COX-2 and -SMA enhanced by IL-1 and IL-6. Anti-TGF- neutralizing antibody also attenuated the increase in COX-2 and -SMA expression caused by IL-1 and IL-6. IL-6 as well as IL-1 enhanced TGF-1 secretion from PSCs. These data indicate that Smad2/3-dependent pathway plays a central role in COX-2 induction by TGF-1, IL-1, and IL-6. Furthermore, IL-1 and IL-6 promote PSC activation by enhancing COX-2 expression indirectly through Smad2/3-dependent pathway by increasing TGF-1 secretion from PSCs. transforming growth factor-; interleukin; Smad; autocrine; pancreatic fibrosis     

Autocrine loop between TGF-beta1 and IL-1beta through Smad3- and ERK-dependent pathways in rat pancreatic stellate cells

Pancreatic stellate cells (PSCs) are activated during pancreatitis and promote pancreatic fibrosis by producing and secreting ECMs such as collagen and fibronectin. IL-1 has been assumed to participate in pancreatic fibrosis by activating PSCs. Activated PSCs secrete various cytokines that regulate PSC function. In this study, we have examined IL-1 secretion from culture-activated PSCs as well as its regulatory mechanism. RT-PCR and ELISA have demonstrated that PSCs express IL-1 mRNA and secrete IL-1 peptide. Inhibition of TGF-₁ activity secreted from PSCs by TGF-₁-neutralizing antibody attenuated IL-1 secretion from PSCs. Exogenous TGF-₁ increased IL-1 expression and secretion by PSCs in a dose-dependent manner. Adenovirus-mediated expression of dominant-negative (dn)Smad2/3 expression reduced both basal and TGF-₁-stimulated IL-1 expression and secretion by PSCs. Coexpression of Smad3 with dnSmad2/3 restored IL-1 expression and secretion by PSCs, which were attenuated by dnSmad2/3 expression. In contrast, coexpression of Smad2 with dnSmad2/3 did not alter them. Furthermore, inhibition of IL-1 activity secreted from PSCs by IL-1-neutralizing antibody attenuated TGF-₁ secretion from PSCs. Exogenous IL-1 enhanced TGF-₁ expression and secretion by PSCs. IL-1 activated ERK, and PD-98059, a MEK1 inhibitor, blocked IL-1 enhancement of TGF-₁ expression and secretion by PSCs. We propose that an autocrine loop exists between TGF-₁ and IL-1 in activated PSCs through Smad3- and ERK-dependent pathways. fibrosis; cytokine; chronic pancreatitis     

Sphingosylphosphorylcholine stimulates expression of fibronectin through TGF-beta1-Smad-dependent mechanism in human mesenchymal stem cells

Sphingosylphosphorylcholine (SPC) has been reported to stimulate the expression of fibronectin (FN), which plays a key role in cell recruitment and adhesion during wound healing. In a previous study, we reported that SPC induces differentiation of human adipose tissue-derived mesenchymal stem cells (hATSCs) to smooth muscle-like cell types through ERK-dependent autocrine secretion of TGF-beta1 and delayed activation of the TGF-beta1-Smad pathway. In the present study, we demonstrated that SPC dose- and time-dependently increased the expression of FN in hATSCs. Pretreatment of the cells with U0126, an MEK inhibitor, markedly attenuated the SPC-induced expression of FN and delayed phosphorylation of Smad2, suggesting that ERK is involved in the SPC induction of FN expression through activation of Smad2. In addition, the SPC-induced expression of FN and delayed activation of Smad2 were abrogated by SB-431542, a TGF-beta type I receptor kinase inhibitor, or anti-TGF-beta1 neutralizing antibody. Furthermore, the SPC-induced expression of FN was abrogated by adenoviral expression of Smad7, an inhibitory Smad, or short interference RNA (siRNA)-mediated depletion of endogenous Smad2 expression, suggesting that SPC induces the expression of FN through ERK-dependent activation of the TGF-beta1-Smad2 crosstalk pathway. Adhesion of U937 monocytic cells to hATSCs was enhanced by pretreatment of hATSCs with SPC or TGF-beta1 for 4 days, and the peptide GRGDSP (an antagonist of fibronectin receptors) blocked the adhesion of U937 cells to the hATSCs. These results led us to suggest that SPC-induced FN expression plays a pivotal role in the wound healing by stimulating adhesion and recruitment of leukocytes.     

IL-6 and tumor necrosis factor-alpha. Autocrine and paracrine cytokines involved in B cell function

IL-6 and TNF-alpha are synthesized and secreted by normal tonsillar B cells after stimulation with the polyclonal B cell activator Staphylococcus aureus Cowan strain 1 (SAC) and IL-2 as well as spontaneously by in vivo activated B cells from patients with hypergammaglobulinemia. Using specific neutralizing antibodies, both factors were shown to be involved in autocrine and/or paracrine regulation of B cell differentiation. IgG induced by SAC/IL-2 stimulation was reduced 73% with an anti-IL-6 antibody and 40% with an anti-TNF-alpha antibody. Similar effects of these antibodies were observed on the spontaneous in vitro IgG production by lymphoblastic B cells from six patients with hypergammaglobulinemia. Kinetic studies with SAC/IL-2-activated B cells revealed that the anti-TNF-alpha antibody must be present at the beginning of the culture to exert an effect on Ig production, whereas the anti-IL-6 antibody reduced Ig production even if added as late as day 3. This sequential action of TNF-alpha and IL-6 on B cell differentiation was reflected by different kinetics of release of these two cytokines into the supernatant of SAC/IL-2 activated B cells; TNF-alpha peaked at 24 h and IL-6 at 96 h after stimulation. In addition, it was shown that IL-6 production by in vitro-activated B cells was partially blocked by an anti-TNF-alpha antibody suggesting that TNF-alpha regulates IL-6 production in normal B cells via an autocrine pathway. We also investigated the effects of TGF-beta on TNF-alpha and IL-6 production by normal B cells. Although TGF-beta inhibited Ig production by in vitro-activated and in vivo-activated B cells, it did not inhibit the release of these cytokines from normal B cells. Furthermore, TGF-beta did not inhibit the induction of nuclear factor-IL-6 nor the expression of IL-6R on activated B cells. Thus, although the biologic effects of anti-IL-6 and TGF-beta on B cell Ig production are similar, their mechanisms of actions appear to be distinct.     

白细胞介素-6在急性胰腺炎中的作用及其机制研究

目的:明确白细胞介素-6(IL-6)在小鼠急性胰腺炎中的作用及其机制研究。方法:通过胰胆管结扎的方法诱导小鼠急性胰腺炎;分离小鼠胰腺腺泡细胞。采用ELISA方法检测胰腺组织或腺泡细胞裂解物中的细胞因子;通过western blot分析检测组织或细胞中IL-6或ERK表达。结果:IL-6浓度在胰腺组织和腺泡细胞中显著增加(P0.05)。在离体原代小鼠腺泡细胞,TNF-α刺激增加IL-6释放(P0.05);与此同时,IL-6刺激可增加其它促炎性细胞因子的释放,两者都涉及ERK MAP激酶通路。黄酮类化合物木犀草素抑制IL-6刺激引起白细胞介素-6(IL-6)和人巨嗜细胞激活蛋白-1(CCL2/MCP-1)释放。最后进一步证实,IL-6激活人胰腺组织中的ERK。结论:IL-6在急性胰腺炎中增加,激活炎症通路并加重急性胰腺炎。     

Regulation of TGF-beta 1-induced connective tissue growth factor expression in airway smooth muscle cells

Transforming growth factor (TGF)-beta may play an important role in airway remodeling, and the fibrogenic effect of TGF-beta may be mediated through connective tissue growth factor (CTGF) release. We investigated the role of MAPKs and phosphatidylinositol 3-kinase (PI3K) and the effects of inflammatory cytokines on TGF-beta-induced CTGF expression in human airway smooth muscle cells (ASMC). We examined whether Smad signal was involved in the regulatory mechanisms. TGF-beta 1 induced a time- and concentration-dependent expression of CTGF gene and protein as analyzed by real-time RT-PCR and Western blot. Inhibition of ERK and c-jun NH(2)-terminal kinase (JNK), but not of p38 MAPK and PI3K, blocked the effect of TGF-beta 1 on CTGF mRNA and protein expression and on Smad2/3 phosphorylation. T helper lymphocyte 2-derived cytokines, IL-4 and IL-13, attenuated TGF-beta 1-stimulated mRNA and protein expression of CTGF and inhibited TGF-beta 1-stimulated ERK1/2 and Smad2/3 activation in ASMC. The proinflammatory cytokines tumor necrosis factor-alpha and IL-1 beta reduced TGF-beta 1-stimulated mRNA expression of CTGF but did not inhibit TGF-beta-induced Smad2/3 phosphorylation. TGF-beta 1-stimulated CTGF expression is mediated by mechanisms involving ERK and JNK pathways and is downregulated by IL-4 and IL-13 through modulation of Smad and ERK signals.     

Pressure activates rat pancreatic stellate cells

Pancreatic stellate cells (PSCs) play a central role in development of pancreatic fibrosis. In chronic pancreatitis, pancreatic tissue pressure is higher than that of the normal pancreas. We here evaluate the effects of pressure on the activation of rat PSCs. PSCs were isolated from the pancreas of Wistar rat using collagenase digestion and centrifugation with Nycodenz gradient. Pressure was applied to cultured rat PSCs by adding compressed helium gas into the pressure-loading apparatus to raise the internal pressure. Cell proliferation rate was assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation. MAPK protein levels and alpha-smooth muscle actin (alpha-SMA) expression were evaluated by Western blot analysis. Concentration of activated transforming growth factor-beta1 (TGF-beta1) secreted from PSCs into culture medium was determined by ELISA. Collagen type I mRNA expression and collagen secretion were assessed by quantitative PCR and Sirius red dye binding assay, respectively. Application of pressure significantly increased BrdU incorporation and alpha-SMA expression. In addition, pressure rapidly increased the phosphorylation of p44/42 and p38 MAPK. Treatment of PSCs with an MEK inhibitor and p38 MAPK inhibitor suppressed pressure-induced cell proliferation and alpha-SMA expression, respectively. Moreover, pressure significantly promoted activated TGF-beta1 secretion, collagen type I mRNA expression, and collagen secretion. Our results demonstrate that pressure itself activates rat PSCs and suggest that increased pancreatic tissue pressure may accelerate the development of pancreatic fibrosis in chronic pancreatitis.     

High glucose increases extracellular matrix production in pancreatic stellate cells by activating the renin-angiotensin system

Pancreatic stellate cells (PSCs) are involved in pancreatic inflammation and fibrosis. Recent studies have shown that blocking the renin-angiotensin system (RAS) attenuates pancreatic inflammation and fibrosis. However, there are few data about the direct effects of high glucose on extracellular matrix (ECM) protein synthesis and angiotensin II (Ang II) induction in PSCs. PSCs were isolated from male Sprague-Dawley rats and cultured in medium containing 5.5 mM (LG group) or 27 mM D-glucose (HG group). Levels of Ang II and transforming growth factor-beta (TGF-beta) in culture media were measured and Ang II-positive cells were counted. We used real-time polymerase chain reaction (PCR) to detect Ang II receptor expression and Western blot analysis for the expression of ECM proteins such as connective-tissue growth factor (CTGF) and collagen type IV. Cells were also treated with an Ang II-receptor antagonist (candesartan, 10 microM) or angiotensin-converting enzyme (ACE) inhibitor (ramiprilat, 100 nM). Thymidine uptake by PSCs increased fourfold with high glucose treatment. Ang II levels and the proportion of Ang II-positive PSCs were significantly increased after 6 h under high-glucose conditions. TGF-beta concentrations also increased significantly with high glucose. After 72 h, the expression of CTGF and collagen type IV proteins in high-glucose cultures increased significantly and this increase was effectively attenuated by the candesartan or the ramiprilat. All together, high glucose induced PSCs proliferation and ECM protein synthesis, and these effects were attenuated by an Ang II-receptor antagonist. The data suggest that pancreatic inflammation and fibrosis aggravated by hyperglycemia, and Ang II play an important role in this pathogenesis.     

Curcumin blocks activation of pancreatic stellate cells

Activated pancreatic stellate cells (PSCs) play a pivotal role in the pathogenesis of pancreatic fibrosis and inflammation. Inhibition of activation and cell functions of PSCs is a potential target for the treatment of pancreatic fibrosis and inflammation. The polyphenol compound curcumin is the yellow pigment in curry, and has anti-inflammatory and anti-fibrotic properties. We here evaluated the effects of curcumin on the activation and cell functions of PSCs. PSCs were isolated from rat pancreas tissue and used in their culture-activated, myofibroblast-like phenotype unless otherwise stated. The effects of curcumin on proliferation, alpha-smooth muscle actin gene expression, monocyte chemoattractant protein (MCP)-1 production, and collagen expression were examined. The effect of curcumin on the activation of freshly isolated cells in culture was also assessed. Curcumin inhibited platelet-derived growth factor (PDGF)-induced proliferation, alpha-smooth muscle actin gene expression, interleukin-1beta- and tumor necrosis factor (TNF)-alpha-induced MCP-1 production, type I collagen production, and expression of type I and type III collagen genes. Curcumin inhibited PDGF-BB-induced cyclin D1 expression and activation of extracellular signal-regulated kinase (ERK). Curcumin inhibited interleukin-1beta- and TNF-alpha-induced activation of activator protein-1 (AP-1) and mitogen-activated protein (MAP) kinases (ERK, c-Jun N-terminal kinase (JNK), and p38 MAP kinase), but not of nuclear factor-kappaB (NF-kappaB). In addition, curcumin inhibited transformation of freshly isolated cells to myofibroblast-like phenotype. In conclusion, curcumin inhibited key cell functions and activation of PSCs.     

TLR3 activation evokes IL-6 secretion, autocrine regulation of Stat3 signaling and TLR2 expression in human bronchial epithelial cells

Human bronchial epithelial cells exposed to synthetic double-stranded RNA (poly I:C) exhibited increased IL-6 and RANTES secretion and TLR2 expression that was inhibited following TLR3 silencing. Increased NF-κB and Stat3 phosphorylation were detected after poly I:C exposure and pretreatment with neutralizing antibody targeting IL-6 receptor α (IL-6Rα -nAb) or blocking Jak2 and Stat3 activity inhibited Stat3 phosphorylation. TLR2 up-regulation by poly I:C was also reduced by IL-6Rα-nAb and inhibitors of Jak2, Stat3 and NF-κB phosphorylation, whereas RANTES secretion was unaffected, but abolished following NF-κB inhibition. Treatment with exogenous IL-6 failed to increase TLR2. These findings demonstrate that TLR3 activation differentially regulates TLR expression through autocrine signaling involving IL-6 secretion, IL-6Rα activation and subsequent phosphorylation of Stat3. The results also indicate that NF-κB and Stat3 are required for TLR3-dependent up-regulation of TLR2 and that its delayed expression was due to a requirement for IL-6-dependent Stat3 activation.     

Transforming growth factor Beta1 induction of tissue inhibitor of metalloproteinases 3 in articular chondrocytes is mediated by reactive oxygen species

Transforming growth factor beta1 (TGF-beta1) stimulates cartilage extracellular matrix synthesis but, in excess, evokes synovial inflammation, hyperplasia, and osteophyte formation in arthritic joints. TGF-beta1 induces tissue inhibitor of metalloproteinases 3 (TIMP-3), an inhibitor of cartilage-damaging matrix metalloproteianases and aggrecanases. We investigated the role of reactive oxygen species (ROS) in TIMP-3 induction by TGF-beta1. In primary human and bovine chondrocytes, ROS scavenger and antioxidant N-acetylcysteine (NAC) inhibited TGF-beta1-induced TIMP-3 mRNA and protein increases. Ebselen and ascorbate also reduced this induction. TGF-beta1 time-dependently induced ROS production that was suppressed by NAC. Hydrogen peroxide, a ROS, induced TIMP-3 RNA. The TIMP-3 increase induced by TGF-beta1 was partly Smad2-dependent. TGF-beta1-stimulated Smad2 phosphorylation was inhibited by NAC. Reduced glutathione and L-cysteine also blocked Smad2 and TIMP-3 induction by TGF-beta1, whereas a nonthiol, N-acetylalanine, did not. Smad2 was not activated by H2O2. Smad2 phosphorylation was independent, and TIMP-3 expression was dependent, on new protein synthesis. TGF-beta-stimulated ERK and JNK phosphorylation was also inhibited by NAC. However, inhibitory actions of NAC were not mediated by ERK activation. Thus, ROS mediate TGF-beta1-induced TIMP-3 gene expression. Blocking TGF-beta1-induced gene expression by modulating cellular redox status with thiols can be potentially beneficial for treating arthritic and other disorders caused by excessive TGF-beta1.     

Pancreatic stellate cell-potentiated insulin secretion from Min6 cells is independent of interleukin 6-mediated pathway

Pancreatic stellate cells (PSCs) secrete various factors, which can influence the β-cell function. The identification of stellate cell infiltration into the islets in pancreatic diseases suggests possible existence of cross-talk between these cells. To elucidate the influence of PSCs on β-cell function, mouse PSCs were cocultured with Min6 cells using the Transwell inserts. Glucose-stimulated insulin secretion from Min6 cells in response to PSCs was quantified by enzyme-linked immunosorbent assay and insulin gene expression was measured by quantitative polymerase chain reaction. Upon cytometric identification of IL6 in PSC culture supernatants, Min6 cells were cultured with IL6 to assess its influence on the insulin secretion and gene expression. PLC-IP₃ pathway inhibitors were added in the cocultures, to determine the influence of PSC-secreted IL6 on Glucose-stimulated insulin secretion from Min6 cells. Increased insulin secretion with a concomitant decrease in total insulin content was noticed in PSC-cocultured Min6 cells. Although increased GSIS was noted from IL6-treated Min6 cells, no change in the total insulin content was noted. Coculture of Min6 cells with PSCs or their exposure to IL6 did not alter either the expression of β-cell-specific genes or that of miRNA-375. PSC-cocultured Min6 cells, in the presence of PLC-IP₃ pathway inhibitors (U73122, Neomycin, and Xestospongin C), did not revoke the observed increase in GSIS. In conclusion, the obtained results indicate that augmented insulin secretion from Min6 cells in response to PSC secretions is independent of IL6-mediated PLC-IP₃ pathway.     

Autocrine extracellular signal-regulated kinase (ERK) activation in normal human keratinocytes: metalloproteinase-mediated release of amphiregulin triggers signaling from ErbB1 to ERK

ErbB signaling through extracellular signal-regulated kinase (ERK) has been implicated in regulating the expression of ErbB ligands in hyperproliferative skin disorders and wound healing. Here, we characterize the process of autocrine ERK activation in cultured normal human keratinocytes (NHKs) subjected to growth factor (GF) deprivation. Basal ERK phosphorylation was lower after 48 h than after 24 h of GF deprivation, and lowest at 30-60 min after an additional medium change. ERK phosphorylation was markedly increased by low concentrations of epidermal growth factor (EGF) (0.2-1 ng/ml) that provoked only a limited increase in ErbB1 tyrosine phosphorylation and internalization. Basal ErbB tyrosine phosphorylation and ERK phosphorylation were inhibited by two different ErbB receptor tyrosine kinase inhibitors, by the ErbB1-specific neutralizing monoclonal antibody 225 IgG, by two different metalloproteinase inhibitors, and by neutralizing antibodies against amphiregulin (AR). In contrast, these responses were unaffected by neutralizing antibodies against other ErbB1 ligands or the ErbB2 inhibitors geldanamycin and AG825. The time course of autocrine ERK phosphorylation correlated with the appearance of soluble AR, and two different metalloproteinase inhibitors blocked AR release. These results define an amphiregulin- and ErbB1-dependent mechanism by which autocrine ERK activation is maintained in NHKs, even when ErbB1 autophosphorylation and internalization are limited.     

Thrombin induced secretion of macrophage migration inhibitory factor (MIF) and its effect on nuclear signaling in endothelium

The procoagulant thrombin stimulates endothelial cells (EC) to undergo rapid cytoskeleton changes via signaling pathways that induce multiple phenotypic changes, including alterations in permeability, vasomotor tone, adhesion molecule synthesis, and leukocyte trafficking. We studied a novel role of thrombin's action on the endothelium that results in MIF secretion, which is linked to myosin light chain (MLC) and extracellular signal-regulated kinase (ERK(1/2))-dependent nuclear signaling. In bovine pulmonary artery EC (BPAEC), thrombin treatment induced intracellular MLC phosphorylation within 15 min, followed by a significant increase in MIF secretion within 30 min. Thrombin treatment induced biphasic ERK(1/2) phosphorylation with an early phase occurring at 15 min and a later phase at 120 min. To understand the role of MIF secretion in thrombin-induced biphasic activation of ERK(1/2), BPAE cells were treated with (i) recombinant MIF, and (ii) the medium collected from thrombin-treated BPAE cells. These studies demonstrated a sustained monophasic ERK(1/2) phosphorylation. Inhibition of MIF secretion by MIF siRNA or antisense-MIF treatment, along with a neutralizing antibody, attenuated the thrombin-induced second phase ERK phosphorylation, suggesting a direct involvement of MIF in the second phase of ERK(1/2) activation. Pretreatment of BPAE cells with an ERK kinase inhibitor and with antisense-MIF significantly inhibited thrombin-induced nuclear factor kappa (NF-kappaB) activation. These results indicate that MIF secretion and ERK phosphorylation both play a necessary role in thrombin induced NF-kappaB activation.