Sphingosine-1-phosphate stimulates human Caco-2 intestinal epithelial proliferation via p38 activation and activates ERK by an independent mechanism

Sphingosine-1-phosphate (S-1-P) has been identified as an extracellular mediator and an intracellular second messenger that may modulate cell motility, adhesion, proliferation, and differentiation and cancer cell invasion. Widely distributed, S-1-P is most abundant in the intestine. Although S-1-P is likely to modulate various intracellular pathways, activation of the mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase 1 (ERK1), ERK2, and p38 is among the best-characterized S-1-P effects. Because the MAPKs regulate proliferation, we hypothesized that S-1-P might stimulate intestinal epithelial cell proliferation by MAPK activation. Human Caco-2 intestinal epithelial cells were cultured on a fibronectin matrix because fibronectin is an important constituent of the gut mucosal basement membrane. We assessed ERK1, ERK2, and p38 activation by Western blotting with antibodies specific for their active forms and proliferation by Coulter counting at 24 h. Specific MAP kinase kinase (MEK) and p38 inhibitors PD98059 (20 microM) and SB202190 and SB203580 (10 and 20 microM) were used to probe the role of ERK and p38 in S-1-P-mediated proliferation. Three or more similar studies were pooled for the analysis. S-1-P stimulated Caco-2 proliferation and dose-responsively activated ERK1, ERK2, and p38. Proliferation peaked at 5 microM, yielding a cell number 166.3 +/- 2.7% of the vehicle control (n = 6, P < 0.05). S-1-P also maximally stimulated ERK1, ERK2, and p38 at 5 microM, to 164.4 +/- 19.9%, 232.2 +/- 38.5%, and 169.2 +/- 20.5% of the control, respectively. Although MEK inhibition prevented S-1-P activation of ERK1 and ERK2 and slightly but significantly inhibited basal Caco-2 proliferation, MEK inhibition did not block the S-1-P mitogenic effect. However, pretreatment with 10 microM SB202190 or SB203580 (putative p38 inhibitors) attenuated the stimulation of proliferation by S-1-P. Twenty micromolars of SB202190 or SB203580 completely blocked the mitogenic effect of S-1-P. Ten to twenty micromolars of SB202190 and SB203580 also dose-dependently ablated the effects of 5 microM S-1-P on heat shock protein 27 accumulation, a downstream consequence of p38 MAPK activation. Consistent with the reports in some other cell types, S-1-P appears to activate ERK1, ERK2, and p38 and to stimulate proliferation. However, in contrast to the mediation of the S-1-P effects in some other cell types, S-1-P appears to stimulate human intestinal epithelial proliferation by activating p38. ERK activation by S-1-P is not required for its mitogenic effect.     

The inhibitory effect of dexamethasone on platelet-derived growth factor-induced vascular smooth muscle cell migration through up-regulating PGC-1α expression

Dexamethasone has been shown to inhibit vascular smooth muscle cell (VSMC) migration, which is required for preventing restenosis. However, the mechanism underlying effect of dexamethasone remains unknown. We have previously demonstrated that peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1 alpha (PGC-1α) can inhibit VSMC migration and proliferation. Here, we investigated the role of PGC-1α in dexamethasone-reduced VSMC migration and explored the possible mechanism. We first examined PGC-1α expression in cultured rat aortic VSMCs. The results revealed that incubation of VSMCs with dexamethasone could significantly elevate PGC-1α mRNA expression. In contrast, platelet-derived growth factor (PDGF) decreased PGC-1α expression while stimulating VSMC migration. Mechanistic study showed that suppression of PGC-1α by small interfering RNA strongly abrogated the inhibitory effect of dexamethasone on VSMC migration, whereas overexpression of PGC-1α had the opposite effect. Furthermore, an analysis of MAPK signal pathways showed that dexamethasone inhibited ERK and p38 MAPK phosphorylation in VSMCs. Overexpression of PGC-1α decreased both basal and PDGF-induced p38 MAPK phosphorylation, but it had no effect on ERK phosphorylation. Finally, inhibition of PPARγ activation by a PPARγ antagonist GW9662 abolished the suppressive effects of PGC-1α on p38 MAPK phosphorylation and VSMC migration. These effects of PGC-1α were enhanced by a PPARγ agonist troglitazone. Collectively, our data indicated for the first time that one of the anti-migrated mechanisms of dexamethasone is due to the induction of PGC-1α expression. PGC-1α suppresses PDGF-induced VSMC migration through PPARγ coactivation and, consequently, p38 MAPK inhibition.     

Role of c-Jun N-terminal kinase in the PDGF-induced proliferation and migration of human adipose tissue-derived mesenchymal stem cells

Platelet-derived growth factor (PDGF) is a critical regulator of proliferation and migration for mesenchymal type cells. In this study, we examined the role of mitogen-activated protein (MAP) kinases in the PDGF-BB-induced proliferation and migration of human adipose tissue-derived mesenchymal stem cells (hATSCs). The PDGF-induced proliferation was prevented by a pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor, SP600125. However, it was not prevented by a pretreatment with a p38 MAP kinase inhibitor, SB202190, and a specific inhibitor of the upstream kinase of extracellular signal-regulated kinase (ERK1/2), U0126. Treatment with PDGF induced the activation of JNK and ERK in hATSCs, and pretreatment with SP600125 specifically inhibited the PDGF-induced activation of JNK. Treatment with PDGF induced the cell cycle transition from the G0/G1 phase to the S phase, the elevated expression of cyclin D1, and the phosphorylation of Rb, which were prevented by a pretreatment with SP600125. In addition, the PDGF-induced migration of hATSCs was completely blocked by a pretreatment with SP600125, but not with U0126 and SB202190. These results suggest that JNK protein kinase plays a key role in the PDGF-induced proliferation and migration of mesenchymal stem cells.     

Integrin and FAK-mediated MAPK activation is required for cyclic strain mitogenic effects in Caco-2 cells

Rhythmic strain stimulates Caco-2 proliferation. We asked whether mitogen-activated protein kinase (MAPK) activation mediates strain mitogenicity and characterized upstream signals regulating MAPK. Caco-2 cells were subjected to strain on collagen I-precoated membranes or antibodies to integrin subunits. Twenty-four hours of cyclic strain increased cell numbers compared with static conditions. MAPK-extracellular signal-regulated kinase (ERK) kinase inhibition (20 microM PD-98059) blocked strain mitogenicity. p38 Inhibition (10 microM SB-202190) did not. Strain rapidly and time-dependently activated focal adhesion kinase (FAK), paxillin, ERK1 and 2, and p38 on collagen. c-Jun NH(2)-terminal kinase (JNK)1 and 2 exhibited delayed activation. Similar activation occurred when Caco-2 cells were subjected to strain on a substrate of functional antibody to the alpha2-, alpha3-, alpha6-, or beta1-integrin subunits but not on a substrate of functional antibody to the alpha5-subunit. FAK inhibition by FAK397 transfection blocked ERK2 and JNK1 activation by in vitro kinase assays, but pharmacological protein kinase C inhibition did not block ERK1 or 2 activation by strain. Strain-induced ERK signals mediate strain's mitogenic effects and may require integrins and FAK activation.     

p38 MAP kinase negatively regulates angiotensin II-mediated effects on cell cycle molecules in human coronary smooth muscle cells

Many of the signaling events in VSMC stimulated by angiotensin II (AngII) are mediated by members of the mitogen-activated protein kinase (MAPK) family, including p38 MAPK. The role of p38 MAPK in AngII-mediated cell cycle regulation is poorly understood. Therefore, we examined the involvement of p38 MAPK signaling in AngII-stimulated DNA synthesis, phosphorylation of the retinoblastoma protein (Rb), and expression of the G1-phase cyclin D1 in human coronary artery smooth muscle cells (CASMC). AngII (1 microM) stimulated p38 MAPK and ERK1/2 activation. Pretreatment with the p38 MAPK inhibitors SB203580 (10 microM) (SB) or SKF-86002 (10 microM) (SKF) potently inhibited AngII-induced p38 MAPK activation, but enhanced AngII-mediated ERK1/2 activation. AngII-induced-phosphorylation of Rb (Ser 795 and Ser 807/811), -cyclin D1 expression, and -DNA synthesis was also markedly enhanced by pharmacological inhibition of the p38 MAPK pathway. The present study demonstrates that p38 MAPK negatively regulates AngII-induced ERK1/2 activity, Rb phosphorylation, cyclin D1 expression, and DNA-synthesis in human CASMC. These findings support an important role for p38 MAPK in modulating AngII-mediated VSMC hyperplasia.     

The role of p70S6K in hepatic stellate cell collagen gene expression and cell proliferation

During fibrosis the hepatic stellate cell (HSC) undergoes a complex activation process characterized by increased proliferation and extracellular matrix deposition. The 70-kDa ribosomal S6 kinase (p70S6K) is activated by mitogens, growth factors, and hormones in a phosphatidylinositol 3-kinase-dependent manner. p70S6K regulates protein synthesis, proliferation, and cell cycle control. Because these processes are involved in HSC activation, we investigated the role of p70S6K in HSC proliferation, cell cycle control, and type I collagen expression. Platelet-derived growth factor (PDGF) stimulated p70S6K phosphorylation, which was blocked by LY294002, an inhibitor of phosphatidylinositol 3-kinase. Rapamycin blocked phosphorylation of p70S6K but had no affect on PDGF-induced Akt phosphorylation, positioning p70S6K downstream of Akt. Transforming growth factor-beta, which inhibits HSC proliferation, did not affect PDGF-induced p70S6K phosphorylation. Rapamycin treatment did not affect alpha1(I) collagen mRNA but reduced type I collagen protein secretion. Expression of smooth muscle alpha-actin was not affected by rapamycin treatment, indicating that HSC activation was not altered. Rapamycin inhibited serum-induced DNA synthesis approximately 2-fold. Moreover, rapamycin decreased expression of cyclins D1, D3, and E but not cyclin D2, Rb-Ser780, and Rb-Ser795. Together, p70S6K plays a crucial role in HSC proliferation, collagen expression, and cell cycle control, thus representing a potential therapeutic target for liver fibrosis.     

Requirement of mitogen-activated protein kinase kinase 3 (MKK3) for activation of p38alpha and p38delta MAPK isoforms by TGF-beta 1 in murine mesangial cells

Transforming growth factor-beta1 (TGF-beta1) is a potent inducer of extracellular matrix (ECM) synthesis that leads to renal fibrosis. Intracellular signaling mechanisms involved in this process remain incompletely understood. Mitogen-activated protein kinase (MAPK) is a major stress signal-transducing pathway, and we have previously reported activation of p38 MAPK by TGF-beta1 in rat mesangial cells and its role in the stimulation of pro-alpha1(I) collagen. In this study, we further investigated the mechanism of p38 MAPK activation by TGF-beta1 and the role of MKK3, an upstream MAPK kinase of p38 MAPK, by examining the effect of targeted disruption of the Mkk3 gene. We first isolated glomerular mesangial cells from MKK3-null (Mkk3-/-) and wild-type (Mkk3+/+) control mice. Treatment with TGF-beta1 induced rapid phosphorylation of MKK3 as well as p38 MAPK within 15 min in cultured wild-type (Mkk3+/+) mouse mesangial cells. In contrast, TGF-beta1 failed to induce phosphorylation of either MKK3 or p38 MAPK in MKK3-deficient (Mkk3-/-) mouse mesangial cells, indicating that MKK3 is required for TGF-beta1-induced p38 MAPK activation. TGF-beta1 selectively activated the p38 MAPK isoforms p38alpha and p38delta in wild-type (Mkk3+/+) mesangial cells, but not in MKK3-deficient (Mkk3-/-) mesangial cells. Thus, activation of p38alpha and p38delta is dependent on the activation of upstream MKK3 by TGF-beta1. Furthermore, MKK3 deficiency resulted in a selective disruption of TGF-beta1-stimulated up-regulation of pro-alpha1(I) collagen expression but not TGF-beta1 induction of fibronectin and PAI-1. These data demonstrate that the MKK3 is a critical component of the TGF-beta1 signaling pathway, and its activation is required for subsequent p38alpha and p38delta MAPK activation and collagen stimulation by TGF-beta1.     

Eicosapentaenoic acid inhibits PDGF-induced mitogenesis and cyclin D1 expression via TGF-beta in mesangial cells

Eicosapentaenoic acid (EPA), an omega-3 polyunsaturated fatty acid derived from fish oil, is efficacious in glomerular diseases where mesangial proliferation is a key event. We examined the mechanisms of action of EPA on platelet-derived growth factor (PDGF)-stimulated rat mesangial cell mitogenesis. EPA dose-dependently inhibited PDGF-stimulated [(3)H]-thymidine incorporation. PDGF-induced PDGF receptor autophosphorylation, an initial event for PDGF signaling, was not affected by 2 micro g/ml EPA. Similarly, PDGF-stimulated activation of extracellular signal-regulated kinase (ERK) was not altered. On the other hand, EPA inhibited cyclin-dependent kinase 4 (CDK4) activation and cyclin D1 protein induction, a critical step for G1/S progression. TGF-beta secretion assessed by ELISA and bioassay was increased by EPA at 18 h. Coincubation with anti-TGF-beta antibody inhibited the EPA-induced suppression of [(3)H]-thymidine incorporation and cyclin D1 expression. SB203580, an inhibitor of p38, a downstream kinase of TGF-beta, did not affect EPA's growth inhibitory effect. These results demonstrate that EPA inhibits PDGF-stimulated mesangial cell mitogenesis and cyclin D1 expression via TGF-beta.     

ECM‐dependent mRNA expression profiles and phosphorylation patterns of p130Cas,FAK, ERK and p38 MAPK of osteoblast‐like cells

Osteoblast cells synthesize collagen‐rich ECM (extracellular matrix) in response to various environmental cues, but little is known about ECM‐dependent variations in phosphorylation patterns. Using MC3T3 E1 osteoblast‐like cells and mouse whole‐genome microarrays, we investigated molecular signalling affected by collagen‐based ECMs. A genome‐wide expression analysis revealed that cells grown in the 3D collagen matrix partially suppressed the genes associated with cell adhesion and cell cycling. Western analysis demonstrated that the expression of the active (phosphorylated) form of p130Cas, FAK (focal adhesion kinase) and ERK1/2 (extracellular‐signal‐regulated protein kinase 1/2) was reduced in cells grown in the 3D matrix. Conversely, phosphorylation of p38 MAPK (p38 mitogen‐activated protein kinase) was elevated in the 3D matrix, and its up‐regulation was linked to an increase in mRNA levels of dentin matrix protein 1 and bone sialoprotein. Although multiple characteristics such as surface topography, chemical composition and mechanical properties differ in the preparations of our collagen‐rich milieu, our observations support the notion that geometrical alterations in ECM environments can alter the phosphorylation pattern of p130Cas, FAK, ERK1/2 and p38 MAPK and lead to a differential developmental fate.     

The Interaction of Endothelin-1 and TGF-β1 Mediates Vascular Cell Remodeling

Background

Pulmonary arterial hypertension is characterized by increased thickness of pulmonary vessel walls due to both increased proliferation of pulmonary arterial smooth muscle cell (PASMC) and deposition of extracellular matrix. In patients suffering from pulmonary arterial hypertension, endothelin-1 (ET-1) synthesis is up-regulated and may increase PASMC activity and vessel wall remodeling through transforming growth factor beta-1 (TGF-β1) and connective tissue growth factor.

Objective

To assess the signaling pathway leading to ET-1 induced proliferation and extracellular matrix deposition by human PASMC.

Methods

PASMC were serum starved for 24 hours before stimulation with either ET-1 and/or TGF-β1. ET-1 was inhibited by Bosentan, ERK1/2 mitogen activated protein kinase (MAPK) was inhibited by U0126 and p38 MAPK was inhibited by SB203580.

Results

ET-1 increased PASMC proliferation when combined with serum. This effect involved the mitogen activated protein kinases (MAPK) ERK1/2 MAPK and was abrogated by Bosentan which caused a G1- arrest through activation of p27^(Kip). Regarding the contribution of extracellular matrix deposition in vessel wall remodeling, TGF-β1 increased the deposition of collagen type-I and fibronectin, which was further increased when ET-1 was added mainly through ERK1/2 MAPK. In contrast, collagen type-IV was not affected by ET-1. Bosentan dose-dependently reduced the stimulatory effect of ET-1 on collagen type-I and fibronectin, but had no effect on TGF-β1.

Conclusion and Clinical Relevance

ET-1 alone does not induce PASMC proliferation and extracellular matrix deposition. However, ET-1 significantly up-regulates serum induced proliferation and TGF-β1 induced extracellular matrix deposition, specifically of collagen type-I and fibronectin. The synergistic effects of ET-1 on serum and TGF-β1 involve ERK1/2 MAPK and may thus present a novel mode of action in the pathogenesis of pulmonary arterial hypertension.     

Vascular endothelial growth factor-induced secretion of fibronectin is ERK dependent

In severe asthma, cytokines and growth factors contribute to the proliferation of smooth muscle cells and blood vessels, and to the increased extracellular matrix deposition that constitutes the process of airway remodeling. Vascular endothelial growth factor (VEGF), which regulates vascular permeability and angiogenesis, also modulates the function of nonendothelial cell types. In this study, we demonstrate that VEGF induces fibronectin secretion by human airway smooth muscle (ASM) cells. In addition, stimulation of ASM with VEGF activates ERK, but not p38MAPK, and fibronectin secretion is ERK dependent. Both ERK activation and fibronectin secretion appear to be mediated through the VEGF receptor flt-1, as evidenced by the effects of the flt-1-specific ligand placenta growth factor. Finally, we demonstrate that ASM cells constitutively secrete VEGF, which is increased in response to PDGF, transforming growth factor-beta, IL-1beta, and PGE(2). We conclude that ASM-derived VEGF, through modulation of the extracellular matrix, may play an important role in airway remodeling seen in asthma.     

Endoglin modulation of TGF-beta1-induced collagen synthesis is dependent on ERK1/2 MAPK activation.

BACKGROUND/AIMS: Transforming growth factor-beta1 (TGF-beta1) plays a pivotal role in the extracellular matrix accumulation observed in fibrotic diseases. Endoglin is an important component of the TGF-beta receptor complex highly expressed in tissues undergoing fibrotic processes. Endoglin expression regulates the effect of TGF-beta on extracellular matrix synthesis. The purpose of our study has been to understand the molecular mechanism by which endoglin exerts its effects on fibrosis and the possible role of MAP kinases in these effects. METHODS: We have assessed in mock and in endoglin-transfected L6E9 myoblasts the effect of TGF-beta1 on collagen mRNA by Northern blot and effect of TGF-beta1 on collagen content in the cultured medium by [(3)H]-Proline incorporation into collagen proteins. Total and activated MAPK and their role on collagen synthesis were assessed by Western blot. RESULTS: TGF-beta1 induced an increase on alpha(2) (I) collagen mRNA expression and collagen accumulation in mock-transfected myoblasts, whereas the response was much lower in endoglintransfected cells. TGF-beta1 activated the ERK1/2 and p38 MAPK pathways but not the JNK pathway in L6E9 myoblasts. TGF-beta1-induced alpha(2) (I) collagen mRNA expression and collagen accumulation were completely inhibited by SB203580, in either mock or endoglintransfected myoblasts. PD98059 increased TGF-beta1 induced-collagen synthesis and accumulation in endoglin-transfected myoblasts but not in mock cells. CONCLUSION: Our studies demonstrate that TGF-beta1- induced collagen synthesis is mediated by p38 MAPK activation in L6E9 myoblasts. Furthermore, endoglin expression reduces basal and TGF-beta1 induced collagen synthesis when ERK1/2 pathway is operating.     

MAP kinases in lung endothelial permeability induced by microtubule disassembly

Lung endothelial barrier function is regulated by multiple signaling pathways, including mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinases (ERK) 1/2 and p38. We have recently shown involvement of microtubule (MT) disassembly in endothelial cell (EC) barrier failure. In this study, we examined potential involvement of ERK1/2 and p38 MAPK in lung EC barrier dysfunction associated with MT disassembly. MT inhibitors nocodazole (0.2 microM) and vinblastine (0.1 microM) induced sustained activation of Ras-Raf-MEK1/2-ERK1/2 and MKK3/6-p38-MAPKAPK2 MAPK cascades in human and bovine pulmonary EC, as detected by phosphospecific antibodies and in MAPK activation assays. These effects were linked to increased permeability assessed by measurements of transendothelial electrical resistance and cytoskeletal remodeling analyzed by morphometric analysis of EC monolayers. MT stabilization by taxol (5 microM, 1 h) attenuated nocodazole-induced ERK1/2 and p38 MAPK activation and phosphorylation of p38 MAPK substrate 27-kDa heat shock protein and regulatory myosin light chains, the proteins involved in actin polymerization and actomyosin contraction. Importantly, only pharmacological inhibition of p38 MAPK by SB-203580 (20 microM, 1 h) attenuated nocodazole-induced MT depolymerization, actin remodeling, and EC barrier dysfunction, whereas the MEK/ERK1/2 inhibitor U0126 (5 microM, 1 h) exhibited no effect. These data suggest a direct link between p38 MAPK activation, remodeling of MT network, and EC barrier regulation.     

Salidroside inhibits platelet-derived growth factor–induced proliferation and migration of airway smooth muscle cells

Abnormal proliferation and migration of airway smooth muscle cells (ASMCs) have been found to be important for the airway remodeling during the pathogenesis of asthma. Salidroside a bioactive glucoside that exerts antitumor activity via inhibiting the cell proliferation and migration of cancer cells. The aim of the current study was to evaluate the effects of salidroside on the proliferation and migration of ASMCs. Our results showed that salidroside inhibited the proliferation and migration of ASMCs in response to platelet-derived growth factor (PDGF) stimulation. Salidroside markedly attenuated the PDGF-induced production of matrix metalloproteinase 2 (MMP-2) and MMP-9 in ASMCs. The levels of contractile phenotype markers including smooth muscle α-actin and calponin were reduced in response to PDGF stimulation, which was attenuated by salidroside pretreatment. Salidroside diminished the increase in the expression levels of type I collagen and fibronectin in PDGF-stimulated ASMCs. Furthermore, salidroside blocked the PDGF-induced activation of the nuclear factor-κB (NF-κB) pathway in ASMCs. The results suggested that salidroside functionally regulated the proliferation, migration, phenotype plasticity, and extracellular matrix deposition in PDGF-induced ASMCs and the NF-κB pathway might be implicated in the effects of salidroside on ASMCs induced by PDGF.     

Alpha2beta1 integrin promotes T cell survival and migration through the concomitant activation of ERK/Mcl-1 and p38 MAPK pathways

Integrin-mediated attachment to extracellular matrix (ECM) is crucial for cancer progression. Malignant T cells such as acute lymphoblastic leukemia (T-ALL) express β1 integrins, which mediate their interactions with ECM. However, the role of these interactions in T-ALL malignancy is still poorly explored. In the present study, we investigated the effect of collagen; an abundant ECM, on T-ALL survival and migration. We found that collagen through α2β1 integrin promotes the survival of T-ALL cell lines in the absence of growth factors. T-ALL cell survival by collagen is associated with reduced caspase activation and maintenance of Mcl-1 levels. Collagen activated both ERK and p38 MAPKs but only MAPK/ERK was required for collagen-induced T-ALL survival. However, we found that α2β1 integrin promoted T-ALL migration via both ERK and p38. Together these data indicate that α2β1 integrin signaling can represent an important signaling pathway in T-ALL pathogenesis and suggest that its blockade could be beneficial in T-ALL treatment.     

Catalase potentiates interleukin-1beta-induced expression of nitric oxide synthase in rat vascular smooth muscle cells

The role of reactive oxygen species (ROS) in regulating the expression of the inducible nitric oxide synthase (iNOS) was studied in rat aortic vascular smooth muscle cells (VSMC). We hypothesized that ROS regulate iNOS expression through the mitogen-activated protein kinases ERK and p38(MAPK). We found that interleukin-1beta (IL-1beta) stimulated the production of hydrogen peroxide (H2O2) which could be inhibited by loading the cells with the H2O2-scavenging enzyme catalase. Inhibition of the upstream ERK1,2 activator MEK1,2 with U0126 prevented IL-1beta-stimulated iNOS expression, while the p38MAPK inhibitor SB03580 potentiated iNOS expression. Loading the cells with catalase enhanced ERK activation and iNOS expression but had no effect on p38MAPK activation or PDGF-induced ERK activation. These data indicated that H2O2 negatively regulates iNOS expression through ERK inhibition independently of p38MAPK. The present results outline a novel role for H2O2 in suppressing signaling pathways leading to gene expression such as iNOS in VSMC in response to cytokines.