Transforming growth factor-beta induces collagenase-3 expression by human gingival fibroblasts via p38 mitogen-activated protein kinase

Human collagenase-3 (matrix metalloproteinase 13 (MMP-13)) is characterized by exceptionally wide substrate specificity and restricted tissue specific expression. Human skin fibroblasts in culture express MMP-13 only when they are in three-dimensional collagen (Ravanti, L., Heino, J., López-Otín, C., and K?h?ri. V.-M. (1999) J. Biol. Chem. 274, 2446-2455). Here we show that MMP-13 is expressed by fibroblasts during normal human gingival wound repair. Expression of MMP-13 by human gingival fibroblasts cultured in monolayer or in collagen gel was induced by transforming growth factor-beta1 (TGF-beta1). Treatment of gingival fibroblasts with TGF-beta1 activated two distinct mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinase 1/2 (ERK1/2) in 15 min and p38 MAPK in 1 and 2 h. Induction of MMP-13 expression by TGF-beta1 was blocked by SB203580, a specific inhibitor of p38 MAPK, but not by PD98059, a selective inhibitor of ERK1/2 activation. Adenovirus-mediated expression of dominant negative p38alpha and c-Jun potently inhibited induction of MMP-13 expression in gingival fibroblasts by TGF-beta1. Infection of gingival fibroblasts with adenovirus for constitutively active MEK1 resulted in activation of ERK1/2 and JNK1 and up-regulation of collagenase-1 (MMP-1) and stromelysin-1 (MMP-3) production but did not induce MMP-13 expression. In addition, activation of p38 MAPK by constitutively active MKK6b or MKK3b was not sufficient to induce MMP-13 expression. These results show that TGF-beta-elicited induction of MMP-13 expression by gingival fibroblasts is dependent on the activity of p38 MAPK and the presence of functional AP-1 dimers. These observations demonstrate a fundamental difference in the regulation of collagenolytic capacity between gingival and dermal fibroblasts and suggest a role for MMP-13 in rapid turnover of collagenous matrix during repair of gingival wounds, which heal with minimal scarring.     

Transforming growth factor-beta1 stimulates vascular endothelial growth factor 164 via mitogen-activated protein kinase kinase 3-p38alpha and p38delta mitogen-activated protein kinase-dependent pathway in murine mesangial cells

Transforming growth factor-beta1 (TGF-beta1) is a potent inducer of extracellular matrix synthesis leading to progressive glomerular fibrosis. The intracellular signaling mechanisms involved in this process remain incompletely understood. The p38 mitogen-activated protein kinase (MAPK) is a major stress signal transducing pathway that is rapidly activated by TGF-beta1 in mesangial cells. We have previously demonstrated MKK3 as the immediate upstream MAPK kinase required for selective activation of p38 MAPK isoforms, p38alpha and p38delta, and stimulation of pro-alpha1(I) collagen by TGF-beta1 in murine mesangial cells. In this study, we further sought to determine MAPK kinase 3 (MKK3)-dependent TGF-beta1 responses by gene expression profiling analysis utilizing mesangial cells isolated from Mkk3-/- mice compared with Mkk3+/+ controls. Interestingly, vascular endothelial growth factor (VEGF) was identified as a TGF-beta1-induced gene affected by deletion of Mkk3. VEGF is a well known endothelial mitogen, whose actions in nonendothelial cell types are still not well understood. We confirmed that TGF-beta1 increased VEGF mRNA and protein synthesis of VEGF164 and VEGF188 isoforms in wild-type mesangial cells. However, in the Mkk3-/- mesangial cells, both TGF-beta1-induced VEGF mRNA and VEGF164 protein expression were inhibited, whereas TGF-beta1-induced VEGF188 protein expression was unaffected. Furthermore, transfection of dominant negative mutants of p38alpha and p38delta resulted in marked inhibition of TGF-beta1-induced VEGF164 expression but not VEGF188, and treatment with recombinant mouse VEGF164 increased collagen and fibronectin mRNA expression in mesangial cells. Taken together, our findings suggest a critical role for the MKK3-p38alpha and p38delta MAPK pathway in mediating VEGF164 isoform-specific stimulation by TGF-beta1 in mesangial cells. Further, VEGF164 stimulates collagen and fibronectin expression in mesangial cells and thus in turn enhances TGF-beta1-induced extracellular matrix and may play an important role in progressive glomerular fibrosis.     

Transforming growth factor-beta-Smad signaling pathway negatively regulates nontypeable Haemophilus influenzae-induced MUC5AC mucin transcription via mitogen-activated protein kinase (MAPK) phosphatase-1-dependent inhibition of p38 MAPK

In contrast to the extensive studies on the role of transforming growth factor-beta (TGF-beta) in regulating cell proliferation, differentiation, and apoptosis over the past decade, relatively little is known about the exact role of TGF-beta signaling in regulating host response in infectious diseases. Most of the recent studies have suggested that TGF-beta inhibits macrophage activation during infections with pathogens such as Trypanosoma cruzi and Leishmania, thereby favoring virulence. In certain situations, however, there is also evidence that TGF-beta has been correlated with enhanced resistance to microbes such as Candida albicans, thus benefiting the host. Despite these distinct observations that mainly focused on macrophages, little is known about how TGF-beta regulates host primary innate defensive responses, such as up-regulation of mucin, in the airway epithelial cells. Moreover, how the TGF-beta-Smad signaling pathway negatively regulates p38 mitogen-activated protein kinase (MAPK), a key pathway mediating host response to bacteria, still remains largely unknown. Here we show that nontypeable Haemophilus influenzae, a major human bacterial pathogen of otitis media and chronic obstructive pulmonary diseases, strongly induces up-regulation of MUC5AC mucin via activation of the Toll-like receptor 2-MyD88-dependent p38 path-way. Activation of TGF-beta-Smad signaling, however, leads to down-regulation of p38 by inducing MAPK phophatase-1, thereby acting as a negative regulator for MUC5AC induction. These studies may bring new insights into the novel role of TGF-beta signaling in attenuating host primary innate defensive responses and enhance our understanding of the signaling mechanism underlying the cross-talk between TGF-beta-Smad signaling pathway and the p38 MAPK pathway.     

Transforming growth factor beta1 rescues serum deprivation-induced apoptosis via the mitogen-activated protein kinase (MAPK) pathway in macrophages

Cell death and cell survival are central components of normal development and pathologic states. Transforming growth factor beta1 (TGF-beta1) is a pleiotropic cytokine that regulates both cell growth and cell death. To better understand the molecular mechanisms that control cell death or survival, we investigated the role of TGF-beta1 in the apoptotic process by dominant-negative inhibition of both TGF-beta1 and mitogen-activated protein kinase (MAPK) signaling pathways. Murine macrophages (RAW 264.7) undergo apoptosis following serum deprivation, as determined by DNA laddering assay. However, apoptosis is prevented in serum-deprived macrophages by the presence of exogenous TGF-beta1. Using stably transfected RAW 264.7 cells with the kinase-deleted dominant-negative mutant of TbetaR-II (TbetaR-IIM) cDNA, we demonstrate that this protective effect by TGF-beta1 is completely abrogated. To determine the downstream signaling pathways, we examined TGF-beta1 effects on the MAPK pathway. We show that TGF-beta1 induces the extracellular signal-regulated kinase (ERK) activity in a time-dependent manner up to 4 h after stimulation. Furthermore, TGF-beta1 does not rescue serum deprivation-induced apoptosis in RAW 264.7 cells transfected with a dominant-negative mutant MAPK (ERK2) cDNA or in wild type RAW 264.7 cells in the presence of the MAPK kinase (MEK1) inhibitor. Taken together, our data demonstrate for the first time that TGF-beta1 is an inhibitor of apoptosis in cultured macrophages and may serve as a cell survival factor via TbetaR-II-mediated signaling and downstream intracellular MAPK signaling pathway.     

Epidermal growth factor up-regulates transforming growth factor-beta receptor type II in human dermal fibroblasts via p38 mitogen-activated protein kinase pathway

TGF-beta receptors (TbetaRs) are serine/threonine kinase receptors that bind to TGF-beta and propagate intracellular signaling through Smad proteins. TbetaRs are repressed in some human cancers and expressed at high levels in several fibrotic diseases. We demonstrated that epidermal growth factor (EGF) up-regulates type II TGF-beta receptor (TbetaRII) expression in human dermal fibroblasts. EGF-mediated induction of TbetaRII expression was inhibited by the treatment of fibroblasts with a specific p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, whereas MEK inhibitor PD98059 did not block the up-regulation of TbetaRII by EGF. EGF induced the TbetaRII promoter activity, and this induction was significantly blocked by SB203580, but not by PD98059. The overexpression of the dominant negative form of p38alpha or p38beta significantly reduced the induction of TbetaRII promoter activity by EGF. These results indicate that the EGF-mediated induction of TbetaRII expression involves the p38 MAPK signaling pathway. The EGF-mediated induction of TbetaRII expression may participate in a synergistic interplay between EGF and TGF-beta signaling pathway.     

p38 mitogen-activated protein kinase (MAPK) promotes cholesterol ester accumulation in macrophages through inhibition of macroautophagy

p38 MAPK has been strongly implicated in the development of atherosclerosis, but its role in cholesterol ester accumulation in macrophages and formation of foam cells, an early step in the development of atherosclerosis, has not been investigated. We addressed this issue and made some brand new observations. First, elevated intracellular cholesterol level induced by the exposure to LDL-activated p38 MAPK and activation of p38 MAPK with anisomycin increased the ratio of cholesterol esters over free cholesterol, whereas inhibition of p38 MAPK with SB203580 or siRNA reduced the LDL loading-induced intracellular accumulation of free cholesterol and cholesterol esters in macrophages. Second, exposure to LDL cholesterol inhibited autophagy in macrophages, and inhibition of autophagy with 3-methyladenine increased intracellular accumulation of cholesterol (free cholesterol and cholesterol esters), whereas activation of autophagy with rapamycin decreased intracellular accumulation of free cholesterol and cholesterol esters induced by the exposure to LDL cholesterol. Third, LDL cholesterol loading-induced inhibition of autophagy was prevented by blockade of p38 MAPK with SB203580 or siRNA. Neutral cholesterol ester hydrolase was co-localized with autophagosomes. Finally, LDL cholesterol loading and p38 activation suppressed expression of the key autophagy gene, ulk1, in macrophages. Together, our results provide brand new insight about cholesterol ester accumulation in macrophages and foam cell formation.     

Endothelin-1 induces vascular endothelial growth factor synthesis in osteoblasts: involvement of p38 mitogen-activated protein kinase

We previously reported that endothelin-1 (ET-1) stimulates p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of ET-1 on the synthesis of vascular endothelial growth factor (VEGF) in these cells. ET-1 significantly stimulated VEGF secretion time-dependently 18 hours after the stimulation. The stimulatory effect was dose-dependent in the range between 0.1 nM and 0.1 micro;M. BQ123, an antagonist of endothelin(A) (ET(A)) receptor, inhibited the ET-1-induced VEGF secretion. The ET-1-induced VEGF secretion was suppressed by SB203580 and PD169316, inhibitors of p38 MAP kinase, but not PD98059, an inhibitor of the upstream kinase that activates p44/p42 MAP kinase. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester, stimulated VEGF secretion. Calphostin C, a specific PKC inhibitor, suppressed the VEGF secretion by ET-1. TPA-induced VEGF secretion was suppressed by SB203580. Taken together, our results strongly suggest that ET-1 stimulates VEGF synthesis via ET(A) receptor in osteoblasts and that p38 MAP kinase is involved at a point downstream from PKC in the VEGF synthesis.     

Transforming growth factor-beta1 (TGF-beta)-induced apoptosis of prostate cancer cells involves Smad7-dependent activation of p38 by TGF-beta-activated kinase 1 and mitogen-activated protein kinase kinase 3

The inhibitory Smad7, a direct target gene for transforming growth factor-beta (TGF-beta), mediates TGF-beta1-induced apoptosis in several cell types. Herein, we report that apoptosis of human prostate cancer PC-3U cells induced by TGF-beta1 or Smad7 overexpression is caused by a specific activation of the p38 mitogen-activated protein kinase pathway in a TGF-beta-activated kinase 1 (TAK1)- and mitogen-activated protein kinase kinase 3 (MKK3)-dependent manner. Expression of dominant negative p38, dominant negative MKK3, or incubation with the p38 selective inhibitor [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole], prevented TGF-beta1-induced apoptosis. The expression of Smad7 was required for TGF-beta-induced activation of MKK3 and p38 kinases, and endogenous Smad7 was found to interact with phosphorylated p38 in a ligand-dependent manner. Ectopic expression of wild-type TAK1 promoted TGF-beta1-induced phosphorylation of p38 and apoptosis, whereas dominant negative TAK1 reduced TGF-beta1-induced phosphorylation of p38 and apoptosis. Endogenous Smad7 was found to interact with TAK1, and TAK1, MKK3, and p38 were coimmunoprecipitated with Smad7 in transiently transfected COS1 cells. Moreover, ectopically expressed Smad7 enhanced the coimmunoprecipitation of HA-MKK3 and Flag-p38, supporting the notion that Smad7 may act as a scaffolding protein and facilitate TAK1- and MKK3-mediated activation of p38.     

Down-regulation of cytokine-induced interleukin-8 requires inhibition of p38 mitogen-activated protein kinase (MAPK) via MAPK phosphatase 1-dependent and -independent mechanisms

Down-regulation of overabundant interleukin (IL)-8 present in cystic fibrosis (CF) airways could ease excessive neutrophil burden and its deleterious consequences for the lung. IL-8 production in airway epithelial cells, stimulated with e.g. inflammatory cytokines IL-1β and tumor necrosis factor (TNF)-α, is regulated by several signaling pathways including nuclear factor (NF)-κB and p38 mitogen-activated protein kinase (MAPK). We previously demonstrated that the anti-inflammatory drugs dexamethasone and ibuprofen suppress NF-κB; however, only dexamethasone down-regulates cytokine-induced IL-8, highlighting the importance of non-NF-κB mechanisms. Here, we tested the hypothesis that down-regulation of cytokine-induced IL-8 requires modulation of the MAPK phosphatase (MKP)-1/p38 MAPK/mRNA stability pathway. The effects of dexamethasone (5 nm) and ibuprofen (480 μm) on this pathway and IL-8 were studied in CF (CFTE29o-, CFBE41o-) and non-CF (1HAEo-) airway epithelial cells. We observed that dexamethasone, but not ibuprofen, destabilizes IL-8 mRNA and up-regulates MKP-1 mRNA. Further, siRNA silencing of MKP-1, via p38 MAPK, leads to IL-8 overproduction and diminishes the anti-IL-8 potential of dexamethasone. However, MKP-1 overexpression does not significantly alter IL-8 production. By contrast, direct inhibition of p38 MAPK (inhibitor SB203580) efficiently suppresses IL-8 with potency comparable with dexamethasone. Similar to dexamethasone, SB203580 decreases IL-8 mRNA stability. Dexamethasone does not affect p38 MAPK activation, which excludes its effects upstream of p38 MAPK. In conclusion, normal levels of MKP-1 are necessary for a full anti-IL-8 potential of pharmacological agents; however, efficient pharmacological down-regulation of cytokine-induced IL-8 also requires direct effects on p38 MAPK and mRNA stability independently of MKP-1.     

Dexamethasone causes sustained expression of mitogen-activated protein kinase (MAPK) phosphatase 1 and phosphatase-mediated inhibition of MAPK p38

The stress-activated protein kinase p38 stabilizes a number of mRNAs encoding inflammatory mediators, such as cyclooxygenase 2 (Cox-2). In HeLa cells the anti-inflammatory glucocorticoid dexamethasone destabilizes Cox-2 mRNA by inhibiting p38 function. Here we demonstrate that this effect is phosphatase dependent. Furthermore, in HeLa cells dexamethasone induced the sustained expression of mitogen-activated protein kinase phosphatase 1 (MKP-1), a potent inhibitor of p38 function. The inhibition of p38 and the induction of MKP-1 by dexamethasone occurred with similar dose dependence and kinetics. No other known p38 phosphatases were induced by dexamethasone, and other cell types which failed to express MKP-1 also failed to inhibit p38 in response to dexamethasone. The proinflammatory cytokine interleukin 1 (IL-1) induced MKP-1 expression in a p38-dependent manner and acted synergistically with dexamethasone to induce MKP-1 expression. In HeLa cells treated with IL-1 or IL-1 and dexamethasone, the dynamics of p38 activation mirrored the expression of MKP-1. These observations suggest that MKP-1 participates in a negative-feedback loop which regulates p38 function and that dexamethasone may inhibit proinflammatory gene expression in part by inducing MKP-1 expression.     

Sea urchin deciliation induces thermoresistance and activates the p38 mitogen-activated protein kinase pathway

In this study, we demonstrate by a variety of approaches (ie, morphological analysis, Western blots, immunolocalization, and the use of specific antibodies) that hyperosmotic deciliation stress of sea urchin embryos induces a thermotolerant response. Deciliation is also able to activate a phosphorylation signaling cascade the effector of which might be the p38 stress-activated protein kinase because we found that the administration of the p38 inhibitor SB203580 to sea urchin deciliated gastrula embryos makes the hyperosmotic deciliation stress lethal.     

Interleukin (IL)-17 enhances tumor necrosis factor-alpha-stimulated IL-6 synthesis via p38 mitogen-activated protein kinase in osteoblasts

Inflammatory cytokines are well known to play crucial roles in the pathogenesis of rheumatoid arthritis. Among them, interleukin (IL)-17 is a cytokine that is mainly synthesized by activated T cells and its receptors are present in osteoblasts. The synthesis of IL-6, known to stimulate osteoclastic bone resorption, is reportedly responded to bone resorptive agents such as tumor necrosis factor-alpha (TNF-alpha) in osteoblasts. It has been reported that IL-17 enhances TNF-alpha-stimulated IL-6 synthesis in osteoblast-like MC3T3-E1 cells. We previously showed that sphingosine 1-phosphate (S1-P) mediates TNF-alpha-stimulated IL-6 synthesis in these cells. In the present study, we investigated the mechanism of IL-17 underlying enhancement of IL-6 synthesis in MC3T3-E1 cells. IL-17 induced phosphorylation of p38 mitogen-activated protein (MAP) kinase. SB203580 and PD169316, specific inhibitors of p38 MAP kinase, significantly reduced the enhancement by IL-17 of TNF-alpha-stimulated IL-6 synthesis. IL-17 also amplified S1-P-stimulated IL-6 synthesis, and the amplification by IL-17 was suppressed by SB203580. Anisomycin, an activator of p38 MAP kinase, which alone had no effect on IL-6 level, enhanced the IL-6 synthesis stimulated by TNF-alpha. SB203580 and PD169316 inhibited the amplification by anisomycin of the TNF-alpha-induced IL-6 synthesis. Taken together, our results strongly suggest that IL-17 enhances TNF-alpha-stimulated IL-6 synthesis via p38 MAP kinase activation in osteoblasts.     

Transforming growth factor-beta stimulates parathyroid hormone-related protein and osteolytic metastases via Smad and mitogen-activated protein kinase signaling pathways

Transforming growth factor (TGF)-beta promotes breast cancer metastasis to bone. To determine whether the osteolytic factor parathyroid hormone-related protein (PTHrP) is the primary mediator of the tumor response to TGF-beta, mice were inoculated with MDA-MB-231 breast cancer cells expressing a constitutively active TGF-beta type I receptor. Treatment of the mice with a PTHrP-neutralizing antibody greatly decreased osteolytic bone metastases. There were fewer osteoclasts and significantly decreased tumor area in the antibody-treated mice. TGF-beta can signal through both Smad and mitogen-activated protein (MAP) kinase pathways. Stable transfection of wild-type Smad2, Smad3, or Smad4 increased TGF-beta-stimulated PTHrP secretion, whereas dominant-negative Smad2, Smad3, or Smad4 only partially reduced TGF-beta-stimulated PTHrP secretion. When the cells were treated with a variety of protein kinases inhibitors, only specific inhibitors of the p38 MAP kinase pathway significantly reduced both basal and TGF-beta-stimulated PTHrP production. The combination of Smad dominant-negative blockade and p38 MAP kinase inhibition resulted in complete inhibition of TGF-beta-stimulated PTHrP production. Furthermore, TGF-beta treatment of MDA-MB-231 cells resulted in a rapid phosphorylation of p38 MAP kinase. Thus, the p38 MAP kinase pathway appears to be a major component of Smad-independent signaling by TGF-beta and may provide a new molecular target for anti-osteolytic therapy.     

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.