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.     

Regulation of TGF-beta1/MAPK-mediated PAI-1 gene expression by the actin cytoskeleton in human mesangial cells

The importance of transforming growth factor-beta1 (TGF-beta1) in plasminogen activator inhibitor-1 (PAI-1) gene expression has been established, but the precise intracellular mechanisms are not fully understood. Our hypothesis is that the actin cytoskeleton is involved in TGF-beta1/MAPK-mediated PAI-1 expression in human mesangial cells. Examination of the distributions of actin filaments (F-actin), alpha-actinin, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) by immunofluorescence and immunoprecipitation revealed that ERK and JNK associate with alpha-actinin along F-actin and that TGF-beta1 stimulation promote the dissociation of ERK and JNK with F-actin. Disassembly of the actin cytoskeleton inhibited phosphorylation of ERK and JNK and modulated PAI-1 expression and promoter activity under both basal and TGF-beta1-stimulated conditions. Stabilizing actin prevented dephosphorylation of ERK and JNK. ERK and JNK inhibitors and overexpressed dominant negative mutants antagonized the ability of TGF-beta1 to increase PAI-1 expression and promoter activity. Disassembly of F-actin also inhibited AP-1 DNA binding activity as determined by electrophoretic mobility shift assay using AP-1 consensus oligonucleotides derived from human PAI-1 promoter. F-actin stabilization prevented loss of AP-1 DNA binding activity. Therefore, changes in actin cytoskeleton modulate the ability of TGF-beta1 to stimulate PAI-1 expression through a mechanism dependent on the activation of MAPK/AP-1 pathways.     

Transforming growth factor-beta 1-induced activation of the ERK pathway/activator protein-1 in human lung fibroblasts requires the autocrine induction of basic fibroblast growth factor

Transforming growth factor-beta (TGF-beta) is involved in multiple processes including cell growth and differentiation. In particular, TGF-beta has been implicated in the pathogenesis of fibrotic lung diseases. In this study, we examined regulation of the mitogen-activated protein kinase pathway by TGF-beta1 in primary human lung fibroblasts. TGF-beta1 treatment resulted in extracellular signal-regulated kinase (ERK) pathway activation in a delayed manner, with maximal activity at 16 h. ERK activation occurred concomitantly with the induction of activator protein-1 (AP-1) binding, a nuclear factor required for activation of multiple genes involved in fibrosis. AP-1 binding was dependent on ERK activation, since the MEK-1 (mitogen-activated protein kinase kinase) inhibitor PD98059 inhibited TGF-beta1-induced binding. Induction of the receptor tyrosine kinase-linked growth factor, basic fibroblast growth factor (bFGF) protein expression temporally paralleled the activation of ERK/AP-1. Induction of AP-1 by TGF-beta1-conditioned medium was observed at 2 h, similar to AP-1 induction in response to exogenous bFGF. Dependence of ERK/AP-1 activation on bFGF induction was demonstrated by inhibition of TGF-beta1-induced ERK/AP-1 activation when conditioned medium from TGF-beta1-treated cells was incubated with bFGF-neutralizing antibody. Together, these results demonstrate that TGF-beta1 regulates the autocrine induction of bFGF, resulting in activation of the ERK mitogen-activated protein kinase pathway and induction of AP-1 binding.     

Involvement of p38 mitogen-activated protein kinase in basic fibroblast growth factor-induced interleukin-6 synthesis in osteoblasts.

We previously showed that basic fibroblast growth factor (bFGF)-induced activation of protein kinase C (PKC) via phosphatidylinositol-hydrolyzing phospholipase C and phosphatidylcholine-hydrolyzing phospholipase D suppresses interleukin-6 (IL-6) synthesis by bFGF itself in osteoblast-like MC3T3-E1 cells. In the present study, we further investigated the mechanism underlying the bFGF-induced IL-6 synthesis in MC3T3-E1 cells. bFGF time-dependently induced the phosphorylation of p38 mitogen-activated protein (MAP) kinase. SB203580, a specific inhibitor of p38 MAP kinase, suppressed the bFGF-induced IL-6 synthesis dose-dependently. The phosphorylation of p38 MAP kinase by bFGF was suppressed by TMB-8, an inhibitor of intracellular Ca(2+) mobilization, or the depletion of extracellular Ca(2+) with EGTA. A23187, a Ca-ionophore, stimulated the phosphorylation of p38 MAP kinase. SB203580 inhibited the A23187-induced synthesis of IL-6. 1-Oleoyl-2-acetyl-sn-glycerol, a synthetic diacylglycerol activating PKC, reduced the bFGF-induced IL-6 synthesis. 12-O-Tetradecanoylphorbol-13-acetate, an activator of PKC, attenuated the phosphorylation of p38 MAP kinase by bFGF, but did not affect the A23187-induced phosphorylation. These results strongly suggest that bFGF-induced IL-6 synthesis is mediated via p38 MAP kinase activation in osteoblasts, and that PKC acts at a point upstream from p38 MAP kinase.     

Regulation of the JNK pathway by TGF-beta activated kinase 1 in rheumatoid arthritis synoviocytes

c-Jun N-terminal kinase (JNK) contributes to metalloproteinase (MMP) gene expression and joint destruction in inflammatory arthritis. It is phosphorylated by at least two upstream kinases, the mitogen-activated protein kinase kinases (MEK) MKK4 and MKK7, which are, in turn, phosphorylated by MEK kinases (MEKKs). However, the MEKKs that are most relevant to JNK activation in synoviocytes have not been determined. These studies were designed to assess the hierarchy of upstream MEKKs, MEKK1, MEKK2, MEKK3, and transforming growth factor-β activated kinase (TAK)1, in rheumatoid arthritis (RA). Using either small interfering RNA (siRNA) knockdown or knockout fibroblast-like synoviocytes (FLSs), MEKK1, MEKK2, or MEKK3 deficiency (either alone or in combination) had no effect on IL-1β-stimulated phospho-JNK (P-JNK) induction or MMP expression. However, TAK1 deficiency significantly decreased P-JNK, P-MKK4 and P-MKK7 induction compared with scrambled control. TAK1 knockdown did not affect p38 activation. Kinase assays showed that TAK1 siRNA significantly suppressed JNK kinase function. In addition, MKK4 and MKK7 kinase activity were significantly decreased in TAK1 deficient FLSs. Electrophoretic mobility shift assays demonstrated a significant decrease in IL-1β induced AP-1 activation due to TAK1 knockdown. Quantitative PCR showed that TAK1 deficiency significantly decreased IL-1β-induced MMP3 gene expression and IL-6 protein expression. These results show that TAK1 is a critical pathway for IL-1β-induced activation of JNK and JNK-regulated gene expression in FLSs. In contrast to other cell lineages, MEKK1, MEKK2, and MEKK3 did not contribute to JNK phosphorylation in FLSs. The data identify TAK1 as a pivotal upstream kinase and potential therapeutic target to modulate synoviocyte activation in RA.     

Diverse signaling pathways regulate fibroblast differentiation and transformation through Rho kinase activation

This study examined the role of agonist-induced Rho kinase (ROCK) involvement in the morphological outcome of pulmonary-derived fibroblasts. Normal human lung fibroblasts (NHLF) spontaneously differentiate into network-like structures in a two-dimensional growth factor reduced Matrigel matrix-based assay. Sphingosine 1-phosphate (SPP), a bioactive phospholipid that regulates angiogenesis, inhibited fibroblast morphogenesis in a dose-dependent manner, virtually eliminating the presence of multi-cellular structures at 500 nM. Pretreatment with the Rho kinase-specific inhibitor, H1152, eradicated the high dose SPP-induced inhibition. Similarly, NHLFs transfected with Rho kinase siRNA prevented SPP-induced inhibition of the fibroblast morphogenesis. Alternatively, transforming growth factor-beta1 (TGF-beta1), a cytokine recognized as a key mediator of wound healing, terminally differentiates NHLF into myofibroblasts as evidenced by the expression of the smooth muscle cell isoform of alpha-actin (alpha-SMA). H1152 suppressed TGF-beta1-induced alpha-SMA expression in a dose-dependent manner. Similarly, treatment with Rho kinase siRNA reduced alpha-SMA expression by greater than 50%. SPP treatment had no effect on TGF-beta1-induced transformation into myofibroblasts, and TGF-beta1 treatment did not alter fibroblast morphogenesis. This study suggests a dual regulatory role for Rho kinase in cellular regulation of fibroblasts in which SPP-induced Rho kinase activation via a G-protein coupled receptor suppresses fibroblast morphogenesis while TGF-beta1-induced Rho kinase activation through a serine/threonine kinase receptor culminates in transformation into myofibroblasts.     

Regulation of IL-11 expression in intestinal myofibroblasts: role of c-Jun AP-1- and MAPK-dependent pathways

IL-11 inhibits the activation of NF-kappaB and induces the Th2 polarization of CD4+ T cells. The clinical utility of IL-11 is being investigated in Crohn's disease. However, physiological secretion of IL-11 in the intestine remains unclear. In this study, we investigated IL-11 secretion in human intestinal subepithelial myofibroblasts (SEMFs). Intestinal SEMFs were isolated from the human colonic mucosa. IL-11 secretion and mRNA expression were determined by ELISA and Northern blot analysis. The activating protein (AP)-1-DNA binding activity was evaluated by EMSA. IL-11 secretion was induced by IL-1beta and transforming growth factor (TGF)-beta1. These were also observed at the mRNA level. The EMSAs demonstrated that both IL-1beta and TGF-beta1 induced AP-1 activation within 2 h after stimulation, and a blockade of AP-1 activation by the recombinant adenovirus containing a dominant negative c-Jun markedly reduced the IL-1beta- and TGF-beta1-induced IL-11 mRNA expression. IL-1beta and TGF-beta1 induced an activation of ERK p42/44 and p38 MAP kinases, and the MAP kinase inhibitors (SB-202190, PD-98059, and U-0216) significantly reduced the IL-1beta- and TGF-beta1-induced IL-11 secretion. The upregulation of IL-11 mRNA by IL-1beta- and TGF-beta1 was also mediated by a p38 MAP kinase-mediated mRNA stabilization. The combination of IL-1beta and TGF-beta1 additively enhanced IL-11 secretion. Intestinal SEMFs secreted IL-11 in response to IL-1beta- and TGF-beta1. Mucosal IL-11 secretion might be important as an anti-inflammatory response in the pathogenesis of intestinal inflammation.     

Reversible inhibition of the protein phosphatase 1 by hydrogen peroxide. Potential regulation of eIF2 alpha phosphorylation in differentiated PC12 cells

Oxidative inactivation of protein tyrosine phosphatases and calcineurin is a well established mechanism; however, little information with regard to the effect of oxidants on PP1 and PP2A activity is available. Herein, we show that PP1 activity is inhibited by H(2)O(2) treatment in differentiated PC12 cells both in vitro and in vivo experiments. Thiol-antioxidant N-acetyl-cysteine (NAC) and reduced glutathione (GSH), when added in vitro to lysates from H(2)O(2)-treated cells, reversed PP1 inhibition. H(2)O(2) treatment increased eIF2 alpha phosphorylated levels (eIF2 alpha P) in a time- and dose-dependent fashion and promoted protein synthesis inhibition. Interestingly, NAC pretreatment protected cells from H(2)O(2)-induced PP1 inactivation and, consequently, it abolished increased H(2)O(2)-induced eIF2 alpha phosphorylation and protein synthesis inhibition. In addition, PP1 inhibitor tautomycin prevented both NAC-induced PP1 reactivation and eIF2 alpha P dephosphorylation in H(2)O(2)-treated cells. Taken together, our findings support a role for PP1 in eIF2 alpha phosphorylation and oxidative stress-triggered translation down regulation.     

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.     

DLPC decreases TGF-beta1-induced collagen mRNA by inhibiting p38 MAPK in hepatic stellate cells

Dilinoleoylphosphatidylcholine (DLPC), the active component of polyenylphosphatidylcholine extracted from soybeans, decreases collagen accumulation induced by TGF-beta1 in cultured hepatic stellate cells (HSCs). Because DLPC exerts antioxidant effects and TGF-beta1 generates oxidative stress, we evaluated whether the antifibrogenic effect of DLPC is linked to its antioxidant action. In passage 1 culture of rat HSCs, TGF-beta1 induced a concentration-dependent increase in procollagen-alpha(1)(I) mRNA levels and enhanced intracellular H(2)O(2) and superoxide anion formation and lipid peroxidation but decreased GSH levels. These changes were prevented by DLPC. Upregulation of collagen mRNA by TGF-beta1 was likewise inhibited by catalase and p38 MAPK inhibitor SB-203580, suggesting involvement of H(2)O(2) and p38 MAPK signaling in this process. TGF-beta1 or addition of H(2)O(2) to HSCs activated p38 MAPK with a rise in procollagen mRNA level; these changes were blocked by catalase and SB-203580 and likewise by DLPC. alpha-Smooth muscle actin abundance in HSCs was not altered by TGF-beta1 treatment (with or without DLPC), indicating that downregulation of procollagen mRNA by DLPC was not due to alteration in HSC activation. These results demonstrate that DLPC prevents TGF-beta1-induced increase in collagen mRNA by inhibiting generation of oxidative stress and associated H(2)O(2)-dependent p38 MAPK activation, which explains its antifibrogenic effect. DLPC, an innocuous phospholipid, may be considered for prevention and treatment of liver fibrosis.