TGFβ signaling pathways regulate a number of keratinocyte functions during epidermal carcinogenesis and wound healing, including proliferation, survival, and migration. TGFβ can induce expression of the matrix metalloproteinase MMP-9, which has critical roles in promoting extracellular matrix remodeling and angiogenesis during tumorigenesis and tissue repair. Integrin α3β1 is a cell adhesion receptor for laminin-332/laminin-5 with important roles in the survival and motility of epidermal keratinocytes. We previously reported that α3β1 induces the expression of MMP-9 in immortalized keratinocytes. In the current study, we show that endogenous TGFβ is required for maximal MMP-9 expression, and that α3β1 is required for full induction of MMP-9 protein and mRNA in response to TGFβ. This regulation was not observed in non-immortalized, primary keratinocytes, indicating that coordinate regulation of MMP-9 by α3β1 and TGFβ is a property of immortalized cells. α3β1 did not regulate endogenous TGFβ gene expression, TGFβ bioavailability, or TGFβ-Smad signaling. However, the combined inductive effects of TGFβ and α3β1 on MMP-9 were suppressed by a Src family kinase (SFK) inhibitor, indicating involvement of an SFK pathway. These findings may reflect a novel role for α3β1 in augmenting TGFβ-mediated induction of MMP-9 in immortalized or transformed keratinocytes during skin carcinogenesis.
TGFβ; α3β1 integrin; MMP-9; keratinocyte
Matrix expansion is an early change in age-related maculopathy. The aim of this study was to determine whether connective tissue growth factor (CTGF) regulates the production of extracellular matrix components by retinal pigmented epithelial (RPE) cells.
ARPE-19 cells were treated with CTGF and analyzed for fibronectin, laminin, and MMP-2 by RT-qPCR, Western blot analysis, or zymography. Cells were also pretreated with an MEK-1/2 inhibitor (PD98059) or a p38 inhibitor (SB203580) and an anti-CTGF antibody to analyze the signaling contributing to fibronectin, laminin, and MMP-2 production. Human maculas were analyzed for mRNA using laser capture microdissected RPE cells and by immunohistochemistry for the topographic distribution of CTGF.
CTGF induced fibronectin mRNA (P = 0.006) and protein (P = 0.006), and laminin mRNA (P = 0.006) and protein (P = 0.02) by ARPE-19 cells. CTGF also induced MMP-2 mRNA (P = 0.002) and protein secretion (P = 0.04). Using zymography, CTGF increased the latent and active forms of MMP-2 compared to controls (P = 0.02). An anti-CTGF antibody inhibited fibronectin, laminin, and MMP-2 after CTGF stimulation. CTGF increased the phosphorylation of p38 and ERK1/2. Fibronectin and MMP-2 mRNA and protein were suppressed by a MEK-1/2 inhibitor, but not with a p38 inhibitor. Laminin expression was suppressed by both inhibitors. RT-qPCR analysis showed that macular RPE cells from human donors express CTGF. Immunohistochemistry of human maculas showed strong labeling of CTGF in Bruch membrane, including basal deposits and drusen.
CTGF is increased in basal deposits and drusen of AMD specimens, and it induces matrix protein production in ARPE-19 cells through the ERK (p42/p44mapk) and p38mapk signaling pathways.
Cartilage damage in osteoarthritis (OA) is considered an imbalance between catabolic and anabolic factors, favoring the catabolic side. We assessed whether adenoviral overexpression of transforming growth factor-β (TGFβ) enhanced cartilage repair and whether TGFβ-induced fibrosis was blocked by local expression of the intracellular TGFβ inhibitor Smad7. We inflicted cartilage damage by injection of interleukin-1 (IL-1) into murine knee joints. After 2 days, we injected an adenovirus encoding TGFβ. On day 4, we measured proteoglycan (PG) synthesis and content. To examine whether we could block TGFβ-induced fibrosis and stimulate cartilage repair simultaneously, we injected Ad-TGFβ and Ad-Smad7. This was performed both after IL-1-induced damage and in a model of primary OA. In addition to PG in cartilage, synovial fibrosis was measured by determining the synovial width and the number of procollagen I-expressing cells. Adenoviral overexpression of TGFβ restored the IL-1-induced reduction in PG content and increased PG synthesis. TGFβ-induced an elevation in PG content in cartilage of the OA model. TGFβ-induced synovial fibrosis was strongly diminished by simultaneous synovial overexpression of Smad7 in the synovial lining. Of great interest, overexpression of Smad7 did not reduce the repair-stimulating effect of TGFβ on cartilage. Adenoviral overexpression of TGFβ stimulated repair of IL-1- and OA-damaged cartilage. TGFβ-induced synovial fibrosis was blocked by locally inhibiting TGFβ signaling in the synovial lining by simultaneously transfecting it with an adenovirus overexpressing Smad7.
The primary feature of osteoarthritis is cartilage loss. In addition, osteophytes can frequently be observed. Transforming growth factor‐β (TGFβ) has been suggested to be associated with protection against cartilage damage and new cartilage formation as seen in osteophytes.
To study TGFβ and TGFβ signalling in experimental osteoarthritis to gain insight into the role of TGFβ in cartilage degradation and osteophyte formation during osteoarthritis progression.
Histological sections of murine knee joints were stained immunohistochemically for TGFβ3 and phosphorylated SMAD‐2 (SMAD‐2P). Expression patterns were studied in two murine osteoarthritis models, representing spontaneous (STR/ort model) and instability‐associated osteoarthritis (collagenase‐induced instability model).
TGFβ3 and SMAD‐2P staining was increasingly reduced in cartilage during osteoarthritis progression in both models. Severely damaged cartilage was negative for TGFβ3. In contrast, bone morphogenetic protein‐2 (BMP‐2) expression was increased. In chondrocyte clusters, preceding osteophyte formation, TGFβ3 and SMAD‐2P were strongly expressed. In early osteophytes, TGFβ3 was found in the outer fibrous layer, in the peripheral chondroblasts and in the core. Late osteophytes expressed TGFβ3 only in the fibrous layer. SMAD‐2P was found throughout the osteophyte at all stages. In the late‐stage osteophytes, BMP‐2 was strongly expressed.
Data show that lack of TGFβ3 is associated with cartilage damage, suggesting loss of the protective effect of TGFβ3 during osteoarthritis progression. Additionally, our results indicate that TGFβ3 is involved in early osteophyte development, whereas BMP might be involved in late osteophyte development.
Cell migration during wound healing is a complex process that involves the expression of a number of growth factors and cytokines. One of these factors, transforming growth factor-beta (TGFβ) controls many aspects of normal and pathological cell behavior. It induces migration of keratinocytes in wounded skin and of epithelial cells in damaged cornea. Furthermore, this TGFβ-induced cell migration is correlated with the production of components of the extracellular matrix (ECM) proteins and expression of integrins and matrix metalloproteinases (MMPs). MMP digests ECMs and integrins during cell migration, but the mechanisms regulating their expression and the consequences of their induction remain unclear. It has been suggested that MMP-14 activates cellular signaling processes involved in the expression of MMPs and other molecules associated with cell migration. Because of the manifold effects of MMP-14, it is important to understand the roles of MMP-14 not only the cleavage of ECM but also in the activation of signaling pathways.
wound healing; migration; matrix metalloproteinase; transforming growth factor; skin; cornea
Extracellular matrix (ECM) molecules modify gene expression through attachment-dependent (i.e., focal adhesion related) integrin receptor signalling. It was previously unknown whether the same molecules acting as soluble peptides could generate signal cascades without the associated mechanical anchoring, a condition that may be encountered during matrix remodelling, degradation and relevant to invasion and metastatic processes. In the current study the role of ECM ligand regulated gene expression through this attachment independent process was examined.
It was observed that fibronectin, laminin, collagens type I and II induce Smad2 activation in MCF-10A and MCF-7 cells. This activation is not caused by TGFβ ligand contamination or autocrine TGF involvement and is 3–5 fold less robust than the TGFβ1 ligand. The resulting nuclear translocation of Smad4 in response to ECM ligand indicates downstream transcriptional responses occurring. Co-immunoprecipitation experiments determined that type II collagen and laminin act through interaction with integrin α2β1 receptor complex. The ECM ligand induced Smad activation (termed signalling crosstalk) resulted cell type and ligand specific transcriptional changes which are distinct from the TGFβ ligand induced responses.
These findings demonstrate that cell-matrix communication is more complex than previously thought. Soluble ECM peptides drive transcriptional regulation through corresponding adhesion and non-attachment related processes. The resultant gene expressional patterns correlate with pathway activity and not by the extent of Smad activation. These results extend the complexity and the existing paradigms of ECM-cell communication to ECM ligand regulation without the necessity of mechanical coupling.
Extracellular matrix; TGFβ signalling; Breast Cancer; Gene expression
While transforming growth factor β (TGFβ) signaling plays a critical role in chondrocyte metabolism, the TGFβ signaling pathways and target genes involved in cartilage homeostasis and the development of osteoarthritis (OA) remain unclear. Using an in vitro cell culture method and an in vivo mouse genetic approach, we undertook this study to investigate TGFβ signaling in chondrocytes and to determine whether Mmp13 and Adamts5 are critical downstream target genes of TGFβ signaling.
TGFβ receptor type II (TGFβRII)–conditional knockout (KO) (TGFβRIICol2ER) mice were generated by breeding TGFβRIIflox/flox mice with Col2-CreER–transgenic mice. Histologic, histomorphometric, and gene expression analyses were performed. In vitro TGFβ signaling studies were performed using chondro-genic rat chondrosarcoma cells. To determine whether Mmp13 and Adamts5 are critical downstream target genes of TGFβ signaling, TGFβRII/matrix metalloproteinase 13 (MMP-13)– and TGFβRII/ADAMTS-5–double-KO mice were generated and analyzed.
Inhibition of TGFβ signaling (deletion of the Tgfbr2 gene in chondrocytes) resulted in up-regulation of Runx2, Mmp13, and Adamts5 expression in articular cartilage tissue and progressive OA development in TGFβRIICol2ER mice. Deletion of the Mmp13 or Adamts5 gene significantly ameliorated the OA-like phenotype induced by the loss of TGFβ signaling. Treatment of TGFβRIICol2ER mice with an MMP-13 inhibitor also slowed OA progression.
Mmp13 and Adamts5 are critical downstream target genes involved in the TGFβ signaling pathway during the development of OA.
Fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients share many similarities with transformed cancer cells, including spontaneous production of matrix metalloproteinases (MMPs). Altered or chronic activation of proto-oncogenic Ras family GTPases is thought to contribute to inflammation and joint destruction in RA, and abrogation of Ras family signaling is therapeutic in animal models of RA. Recently, expression and post-translational modification of Ras guanine nucleotide releasing factor 1 (RasGRF1) was found to contribute to spontaneous MMP production in melanoma cancer cells. Here, we examine the potential relationship between RasGRF1 expression and MMP production in RA, reactive arthritis, and inflammatory osteoarthritis synovial tissue and FLS.
Expression of RasGRF1, MMP-1, MMP-3, and IL-6 was detected in synovial tissue by immunohistochemistry and stained sections were evaluated by digital image analysis. Expression of RasGRF1 in FLS and synovial tissue was also assessed by immunoblotting. Double staining was performed to detect proteins in specific cell populations, and cells producing MMP-1 and MMP-3. RasGRF1 expression was manipulated in RA FLS by cDNA transfection and gene silencing, and effects on MMP-1, TIMP-1, MMP-3, IL-6, and IL-8 production measured by ELISA.
Expression of RasGRF1 was significantly enhanced in RA synovial tissue, and detected in FLS and synovial macrophages in situ. In cultured FLS and synovial biopsies, RasGRF1 was detected by immunoblotting as a truncated fragment lacking its negative regulatory domain. Production of MMP-1 and MMP-3 in RA but not non-RA synovial tissue positively correlated with expression of RasGRF1 and co-localized in cells expressing RasGRF1. RasGRF1 overexpression in FLS induced production of MMP-3, and RasGRF1 silencing inhibited spontaneous MMP-3 production.
Enhanced expression and post-translational modification of RasGRF1 contributes to MMP-3 production in RA synovial tissue and the semi-transformed phenotype of RA FLS.
Transforming growth factor beta (TGFβ) plays a central role in morphogenesis, growth, and cell differentiation. This cytokine is particularly important in cartilage where it regulates cell proliferation and extracellular matrix synthesis. While the action of TGFβ on chondrocyte metabolism has been extensively catalogued, the modulation of specific genes that function as mediators of TGFβ signalling is poorly defined. In the current study, elements of the Smad component of the TGFβ intracellular signalling system and TGFβ receptors were characterised in human chondrocytes upon TGFβ1 treatment.
Human articular chondrocytes were incubated with TGFβ1. Then, mRNA and protein levels of TGFβ receptors and Smads were analysed by RT-PCR and western blot analysis. The role of specific protein 1 (Sp1) was investigated by gain and loss of function (inhibitor, siRNA, expression vector).
We showed that TGFβ1 regulates mRNA levels of its own receptors, and of Smad3 and Smad7. It modulates TGFβ receptors post-transcriptionally by affecting their mRNA stability, but does not change the Smad-3 and Smad-7 mRNA half-life span, suggesting a potential transcriptional effect on these genes. Moreover, the transcriptional factor Sp1, which is downregulated by TGFβ1, is involved in the repression of both TGFβ receptors but not in the modulation of Smad3 and Smad7. Interestingly, Sp1 ectopic expression permitted also to maintain a similar expression pattern to early response to TGFβ at 24 hours of treatment. It restored the induction of Sox9 and COL2A1 and blocked the late response (repression of aggrecan, induction of COL1A1 and COL10A1).
These data help to better understand the negative feedback loop in the TGFβ signalling system, and enlighten an interesting role of Sp1 to regulate TGFβ response.
Signaling pathways enabling transforming growth factor-beta (TGFβ)’s conversion from a tumor suppressor to a tumor promoter are not well characterized. TGFβ utilizes intracellular SMADs to mediate growth suppression; however, TGFβ-induced proliferative pathways may become more apparent when SMAD signaling is abrogated. Here, we determined regulation of the tumor suppressor PTEN by TGFβ utilizing SMAD4-null colon cancer cells. TGFβ downregulated PTEN mRNA and simultaneously induced growth proliferation. TGFβ also induced both SMAD2 and SMAD3 nuclear translocation, but only triggered SMAD2-specific transcriptional activity in the absence of SMAD4. Interference of SMAD2 with DN-SMAD2 enhanced TGFβ-induced cell proliferation, but downregulation of PTEN expression by TGFβ was unaffected. TGFβ increased PI3K tyrosine phosphorylation, and inhibition of PI3K pharmacologically or by DN-p85 transfection reversed both TGFβ-induced PTEN suppression and TGFβ-induced cell proliferation. Thus, TGFβ activates PI3K to downregulate PTEN for enhancement of cell proliferation that is independent of SMAD proteins.
transforming growth factor; PTEN; PI3K; colonic cancer; SMAD
Functional inactivation of the tumor suppressor Smad4 in colorectal and pancreatic carcinogenesis occurs coincident with the transition to invasive growth. Breaking the basement membrane (BM) barrier, a prerequisite for invasive growth, can be due to tumor induced proteolytic tissue remodeling or to reduced synthesis of BM molecules by incipient tumor cells. Laminin-332 (laminin-5), a heterotrimeric BM component composed of α3-, β3- and γ2-chains, has recently been identified as a target structure of Smad4 and represents the first example for expression control of an essential BM component by a tumor and invasion suppressor. Biochemically Smad4 is a transmitter of signals of the TGFβ superfamily of cytokines. We have reported previously, that Smad4 functions as a positive transcriptional regulator of constitutive and of TGFβ-induced transcription of all three genes encoding Laminin-332, LAMA3, LAMB3 and LAMC2.
Promoter-reporter constructs harboring 4 kb upstream regions, each of the three genes encoding Laminin-322 as well as deletion and mutations constructs were established. Promoter activities and TGFβ induction were assayed through transient transfections in Smad4-negative human cancer cells and their stable Smad4-positive derivatives. Functionally relevant binding sites were subsequently confirmed through chromatin immunoprecipitation.
Herein, we report that Smad4 mediates transcriptional regulation through three different mechanisms, namely through Smad4 binding to a functional SBE site exclusively in the LAMA3 promoter, Smad4 binding to AP1 (and Sp1) sites presumably via interaction with AP1 family components and lastly a Smad4 impact on transcription of AP1 factors. Whereas Smad4 is essential for positive regulation of all three genes, the molecular mechanisms are significantly divergent between the LAMA3 promoter as compared to the LAMB3 and LAMC2 promoters.
We hypothesize that this divergence in modular regulation of the three promoters may lay the ground for uncoupled regulation of Laminin-332 in Smad4-deficient tumor cells in response to stromally expressed cytokines acting on budding tumor cells.
Transforming growth factor beta (TGFβ) is a growth factor with many faces. In our osteoarthritis (OA) research we have found that TGFβ can be protective as well as deleterious for articular cartilage. We postulate that the dual effects of TGFβ on chondrocytes can be explained by the fact that TGFβ can signal via different receptors and related Smad signaling routes. On chondrocytes, TGFβ not only signals via the canonical type I receptor ALK5 but also via the ALK1 receptor. Notably, signaling via ALK5 (Smad2/3 route) results in markedly different chondrocyte responses than ALK1 signaling (Smad1/5/8), and we postulate that the balance between ALK5 and ALK1 expression on chondrocytes will determine the overall effect of TGFβ on these cells. Importantly, signaling via ALK1, but not ALK5, stimulates MMP-13 expression by chondrocytes. In cartilage of ageing mice and in experimental OA models we have found that the ALK1/ALK5 ratio is significantly increased, favoring TGFβ signaling via the Smad1/5/8 route, changes in chondrocyte differentiation and MMP-13 expression. Moreover, human OA cartilage showed a significant correlation between ALK1 and MMP-13 expression. In this paper we summarize concepts in OA, its link with ageing and disturbed growth factor responses, and a potential role of TGFβ signaling in OA development.
Transforming growth factor β (TGFβ) plays a critical role in connective tissue remodeling by fibroblasts during development, tissue repair, and fibrosis. We investigated the molecular pathways in the transmission of TGFβ signals that lead to features of connective tissue remodeling, namely formation of an α-smooth muscle actin (α-SMA) cytoskeleton, matrix contraction, and expression of profibrotic genes. TGFβ causes the activation of focal adhesion kinase (FAK), leading to JNK phosphorylation. TGFβ induces JNK-dependent actin stress fiber formation, matrix contraction, and expression of profibrotic genes in fak+/+, but not fak−/−, fibroblasts. Overexpression of MEKK1, a kinase acting upstream of JNK, rescues TGFβ responsiveness of JNK-dependent transcripts and actin stress fiber formation in FAK-deficient fibroblasts. Thus we propose a FAK-MEKK1-JNK pathway in the transmission of TGFβ signals leading to the control of α-SMA cytoskeleton reorganization, matrix contraction, and profibrotic gene expression and hence to the physiological and pathological effects of TGFβ on connective tissue remodeling by fibroblasts.
Increased blood pressure, leading to mechanical stress on vascular smooth muscle cells (VSMC), is a known risk factor for vascular remodeling via increased activity of matrix metalloproteinase (MMP) within the vascular wall. This study aimed to identify cell surface mechanoreceptors and intracellular signaling pathways that influence VSMC to produce MMP in response to mechanical stretch (MS). When VSMC was stimulated with MS (0–10% strain, 60 cycles/min), both production and gelatinolytic activity of MMP-2, but not MMP-9, were increased in a force-dependent manner. MS-enhanced MMP-2 expression and activity were inhibited by molecular inhibition of Akt using Akt siRNA as well as by PI3K/Akt inhibitors, LY293002 and AI, but not by MAPK inhibitors such as PD98059, SP600125 and SB203580. MS also increased Akt phosphorylation in VSMC, which was attenuated by AG1295, a PDGF receptor (PDGFR) inhibitor, but not by inhibitors for other receptor tyrosine kinase including EGF, IGF, and FGF receptors. Although MS activated PDGFR-α as well as PDGFR-β in VSMC, MS-induced Akt phosphorylation was inhibited by molecular deletion of PDGFR-β using siRNA, but not by inhibition of PDGFR-α. Collectively, our data indicate that MS induces MMP-2 production in VSMC via activation of Akt pathway, that is mediated by activation of PDGFR-β signaling pathways.
The purpose of this study was to investigate the mechanism of expression of matrix metalloproteinase-13 (MMP-13) induced by nitric oxide (NO). Human chondrocytes (HCs) were stimulated with a NO donor (MAHMA-NONOate), then mitogen-activated protein kinases’ (MAPKs) and nuclear factor κB’ (NF-κB) activations and MMP-13′ expression were assayed by Western blot analysis. Additionally, the intracellular signalling of NO was investigated using the inhibitors of MAPKs and NF-κB. NO-induced MMP-13 expression was not suppressed by extracellular signal-regulated kinase (ERK) inhibitor (PD98059) or inhibitors of p38 kinase (SB203580), but was inhibited by a c-jun terminal kinase (JNK) inhibitor (SP600125) and inhibitors of NF-κB (SN-50). Additionally, SP600125 treatment reduced NF-κB activation, but SN-50 treatment did not significantly affect JNK activation. These results suggest that NO induces MMP-13 expression by JNK and NF-κB activation in HCs.
The metastasis‐associated protein S100A4 promotes the progression of cancer by regulating the remodelling of the extracellular matrix. The expression of S100A4 in vivo is shown and the functional role of S100A4 in the pathogenesis of osteoarthritis and rheumatoid arthritisis is explored. The expression of S100A4 in rheumatoid arthritis, osteoarthritis and normal synovial tissues was determined by immunohistochemistry. The expression of matrix metalloproteinase (MMP) mRNA was measured in rheumatoid arthritis and osteoarthritis synovial fibroblasts treated and untreated with S100A4 oligomer by real‐time polymerase chain reaction. Levels of released MMPs were confirmed by ELISA in cell culture supernatants. S100A4 protein was expressed in rheumatoid arthritis and osteoarthritis synovial tissues, in contrast with normal synovium. S100A4 up regulated MMP‐3 mRNA in rheumatoid arthritis synovial fluid, with a peak after 6 h. This resulted in release of MMP‐3 protein. MMP‐1, MMP‐9 and MMP‐13 mRNA were also up regulated in synovial fluid, but with different kinetics. MMP‐14 mRNA showed no change. Thus, S100A4 protein is expressed in synovial tissues of patients with rheumatoid arthritis and osteoarthritis in contrast with healthy people. It induces the expression and release of MMP‐3 and other MMPs from synovial fluid. The data suggest that S100A4‐producing cells could be involved in the pathogenesis of osteoarthritis and rheumatoid arthritis, including pannus formation and joint destruction.
We aimed to investigate the correlation between a disintegrin and metalloprotease with thrombospondin motif 2 (ADAMTS-2) and transforming growth factor-β1 (TGF-β1) in clinical human cirrhotic tissues.
The liver tissues of 24 patients (16 cases with cirrhotic portal hypertension as the cirrhosis group and eight cases with healthy livers as the normal group) were collected. Immunohistochemistry and Western blots were performed to evaluate the protein expression levels of ADAMTS-2 and TGF-β1. Western blots for other key mediators of cirrhotic progression, including SMAD2, SMAD3, TGF-β receptor II (TGFβRII), matrix metalloproteinases 2 (MMP2), and tissue inhibitor of matrix metalloproteinases 2 (TIMP2), were also performed.
Cirrhotic tissues showed higher percentages of collagen. The protein expression levels of ADAMTS-2 and TGF-β1 were significantly higher in the cirrhotic group as compared to the matched normal group (p<0.05), and there was a positive correlation between these two proteins (r=0.862, p<0.01). The protein expressions of MMP2, TIMP2, and TGFβRII, as well as the phosphorylated forms of SMAD2 and SMAD3, were significant higher in the cirrhotic group (p<0.01 or p<0.05).
These findings suggested that ADAMTS-2 and TGF-β1 may play important roles in the pathogenesis of human cirrhosis; specifically, TGF-β1 may induce the expression of ADAMTS-2 through the TGFβ/SMAD pathway.
A disintegrin and metalloprotease with thrombospondin motif-2; Transforming growth factor beta 1; Cirrhosis; Humans
To examine the relative importance of tumour necrosis factor‐receptor 1 (TNF‐R1) and TNF‐R2 and their signalling pathways for pro‐inflammatory and pro‐destructive features of early‐passage synovial fibroblasts (SFB) from rheumatoid arthritis (RA) and osteoarthritis (OA).
Cells were stimulated with tumour necrosis factor (TNF)α or agonistic anti‐TNF‐R1/TNF‐R2 monoclonal antibodies. Phosphorylation of p38, ERK and JNK kinases was assessed by western blot; proliferation by bromodesoxyuridine incorporation; interleukin (IL)6, IL8, prostaglandin E2 (PGE2) and matrix metalloproteinase (MMP)‐1 secretion by ELISA; and MMP‐3 secretion by western blot. Functional assays were performed with or without inhibition of p38 (SB203580), ERK (U0126) or JNK (SP600125).
In RA‐ and OA‐SFB, TNFα‐induced phosphorylation of p38, ERK or JNK was exclusively mediated by TNF‐R1. Reduction of proliferation and induction of IL6, IL8 and MMP‐1 were solely mediated by TNF‐R1, whereas PGE2 and MMP‐3 secretion was mediated by both TNF‐Rs. In general, inhibition of ERK or JNK did not significantly alter the TNFα influence on these effector molecules. In contrast, inhibition of p38 reversed TNFα effects on proliferation and IL6/PGE2 secretion (but not on IL8 and MMP‐3 secretion). The above effects were comparable in RA‐ and OA‐SFB, except that TNFα‐induced MMP‐1 secretion was reversed by p38 inhibition only in OA‐SFB.
In early‐passage RA/OA‐SFB, activation of MAPK cascades and pro‐inflammatory/pro‐destructive features by TNFα is predominantly mediated by TNF‐R1 and, for proliferation and IL6/PGE2 secretion, exclusively regulated by p38. Strikingly, RA‐SFB are insensitive to p38 inhibition of MMP‐1 secretion. This indicates a resistance of RA‐SFB to the inhibition of pro‐destructive functions and suggests underlying structural/functional alterations of the p38 pathway, which may contribute to the pathogenesis or therapeutic sensitivity of RA, or both.
TNF‐receptor; synovial fibroblast; p38 MAP kinase; interleukin; matrix metalloproteinase
PPARγ agonists inhibit liver fibrosis, but the mechanisms involved are uncertain. We hypothesized that PPARγ agonists inhibit transforming growth factor (TGF)β1-activation of TGFβ receptor (TGFβR)-1 signaling in quiescent stellate cells, thereby abrogating Smad3-dependent induction of extracellular matrix (ECM) genes, such as PAI-1 and collagen-1αI. To test this, human HSC were cultured to induce a quiescent phenotype, characterized by lipid accumulation and PPARγ expression and transcriptional activity. These adipocytic HSC were then treated with TGFβ1 ± a TGFβR-1 kinase inhibitor (SB431542) or a PPARγ agonist (GW7845). TGFβ1 caused dose- and time-dependent increases in Smad3 phosphorylation, followed by induction of collagen and PAI-1 expression. Like the TGFβR-1 kinase inhibitor, the PPARγ agonist caused dose-dependent inhibition of all of these responses without effecting HSC proliferation or viability. Thus, the anti-fibrotic actions of PPARγ agonists reflect their ability to inhibit TGFβ1-TGFβR1 signaling that initiates ECM gene expression in quiescent HSC.
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by destruction of bone and cartilage, which is mediated, in part, by synovial fibroblasts. Matrix metalloproteinases (MMPs) are a large family of proteolytic enzymes responsible for matrix degradation. Macrophage migration inhibitory factor (MIF) is a cytokine that induces the production of a large number of proinflammatory molecules and has an important role in the pathogenesis of RA by promoting inflammation and angiogenesis.
In the present study, we determined the role of MIF in RA synovial fibroblast MMP production and the underlying signaling mechanisms. We found that MIF induces RA synovial fibroblast MMP-2 expression in a time-dependent and concentration-dependent manner. To elucidate the role of MIF in MMP-2 production, we produced zymosan-induced arthritis (ZIA) in MIF gene-deficient and wild-type mice. We found that MMP-2 protein levels were significantly decreased in MIF gene-deficient compared with wild-type mice joint homogenates. The expression of MMP-2 in ZIA was evaluated by immunohistochemistry (IHC). IHC revealed that MMP-2 is highly expressed in wild-type compared with MIF gene-deficient mice ZIA joints. Interestingly, synovial lining cells, endothelial cells, and sublining nonlymphoid mononuclear cells expressed MMP-2 in the ZIA synovium. Consistent with these results, in methylated BSA (mBSA) antigen-induced arthritis (AIA), a model of RA, enhanced MMP-2 expression was also observed in wild-type compared with MIF gene-deficient mice joints. To elucidate the signaling mechanisms in MIF-induced MMP-2 upregulation, RA synovial fibroblasts were stimulated with MIF in the presence of signaling inhibitors. We found that MIF-induced RA synovial fibroblast MMP-2 upregulation required the protein kinase C (PKC), c-jun N-terminal kinase (JNK), and Src signaling pathways. We studied the expression of MMP-2 in the presence of PKC isoform-specific inhibitors and found that the PKCδ inhibitor rottlerin inhibits MIF-induced RA synovial fibroblast MMP-2 production. Consistent with these results, MIF induced phosphorylation of JNK, PKCδ, and c-jun. These results indicate a potential novel role for MIF in tissue destruction in RA.
Transforming growth factor β2 (TGFβ2) is highly expressed in a variety of different cancer cell lines. Using Z12 cells, a mutant of 293 cells with overexpression of TGFβ2, we found that the cyclic adenosine monophosphate (cAMP)-responsive element (CRE) sequence in the promoter of the TGFβ2 gene is crucial for its increased expression. Further, constitutive phosphorylation of CRE-binding protein (CREB) is increased in these cells. Treating Z12 cells with either the PI3 kinase inhibitor LY294002 or the p38 MAPK inhibitor SB203580 significantly inhibited both the phosphorylation of CREB and expression of TGFβ2. In addition, treating Z12 or cancer cell lines with either of these 2 inhibitors significantly decreased their secretion of TGFβ2. These data suggest that activated PI3 kinase and p38 MAPK play important roles in high expression of TGFβ2 in cancer cells by stimulating the phosphorylation of CREB, which activates the CRE in the promoter of the TGFβ2 gene. We have identified an important link between PI3 kinase, p38 MAPK, and TGFβ2, providing an additional rationale for using inhibitors of these kinases as therapeutic drugs in cancer.
Transforming growth factor β, (TGFβ) plays a central role in extracellular matrix remodeling. We hypothesized that TGFβ signaling is involved in cervical remodeling. This study evaluates patterns within this signaling pathway.
The cervix of non pregnant and timed pregnant rats were obtained. mRNA expression of TGFβ1, TGFβ-Receptor 1 (TβR1), TβR2, and TβR3 was evaluated. Four animals were sacrificed for each time point. Western blotting was performed for protein expression. Smad2 and 3 phosphorylation were assessed to evaluate TGFβ activation.
TGFβ1 mRNA increased through day 21 and declined on day 22 (ANOVA p=0.001). TβR1 expression was unchanged. TβR2 and TβR3 mRNA expression was similar to TGFβ1. TβR3 protein expression was similar to mRNA. Smad2 phosphorylation paralleled changes in TβR3.
Components of the TGFβ signaling pathway increase during pregnancy along with Smad2 activation. The decline on day 22 correlates with a transition to the ripening phase supporting a role in cervical remodeling.
Cervix; Cervical remodeling; Extracellular Matrix; TGFβ
We sought to define the relationship between cytokine stimulated release of matrix metalloproteinases (MMPs) and cell migration using adult rat cardiac fibroblasts. Interleukin-1β (IL-1β) increased release of MMP-2, 3, and 9, and TIMP-1, by 3–6-fold, measured by immunoblotting and gel zymography. Tumor necrosis factor-α (TNFα) augmented IL-1 stimulated release of MMP-9, but not MMP-2 or -3. Transforming growth factor-β1 (TGFβ1) attenuated all the responses to IL-1β. IL-1β was also the most robust stimulus of adult rat cardiac fibroblast migration, measured in Boyden chamber assays. The combination of IL-1β plus TNFα substantially enhanced migration, whereas TGFβ1 strongly inhibited the migratory response to IL-1β. The pan-selective MMP inhibitor GM 6001 effectively blocked IL-1β stimulated migration. Pharmacologic inhibitors selective for ERK, JNK, and p38 MAP kinase pathways inhibited the IL-1β regulation of individual MMPs. Increased MMP activity associated with migration of cardiac fibroblasts may be important determinants of cytokine-directed remodeling of injured myocardium.
Cytokines; fibroblasts; MAP kinases; matrix metalloproteinases; migration
We have discovered that fibrillin-1, which forms extracellular microfibrils, can regulate the bioavailability of transforming growth factor (TGF) β1, a powerful cytokine that modulates cell survival and phenotype. Altered TGFβ signaling is a major contributor to the pathology of Marfan syndrome (MFS) and related diseases. In the presence of cell layer extracellular matrix, a fibrillin-1 sequence encoded by exons 44–49 releases endogenous TGFβ1, thereby stimulating TGFβ receptor–mediated Smad2 signaling. This altered TGFβ1 bioavailability does not require intact cells, proteolysis, or the altered expression of TGFβ1 or its receptors. Mass spectrometry revealed that a fibrillin-1 fragment containing the TGFβ1-releasing sequence specifically associates with full-length fibrillin-1 in cell layers. Solid-phase and BIAcore binding studies showed that this fragment interacts strongly and specifically with N-terminal fibrillin-1, thereby inhibiting the association of C-terminal latent TGFβ-binding protein 1 (a component of the large latent complex [LLC]) with N-terminal fibrillin-1. By releasing LLC from microfibrils, the fibrillin-1 sequence encoded by exons 44–49 can contribute to MFS and related diseases.
The mechanism underlying the ability of fibroblasts to contract a collagen gel matrix is largely unknown. Fibroblasts from scarred (lesional) areas of patients with the fibrotic disease scleroderma show enhanced ability to contract collagen relative to healthy fibroblasts. Thrombospondin 1 (TSP1), an activator of latent transforming growth factor (TGF)β, is overexpressed by scleroderma fibroblasts. In this report we investigate whether activation of latent TGFβ by TSP1 plays a key role in matrix contraction by normal and scleroderma fibroblasts.
We use the fibroblast populated collagen lattices (FPCL) model of matrix contraction to show that interfering with TSP1/TGFβ binding and knockdown of TSP1 expression suppressed the contractile ability of normal and scleroderma fibroblasts basally and in response to TGFβ. Previously, we have shown that ras/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) mediates matrix contraction basally and in response to TGFβ.
During mechanical stimulation in the FPCL system, using a multistation tensioning-culture force monitor (mst-CFM), TSP1 expression and p-ERK activation in fibroblasts are enhanced. Inhibiting TSP1 activity reduced the elevated activation of MEK/ERK and expression of key fibrogenic proteins. TSP1 also blocked platelet-derived growth factor (PDGF)-induced contractile activity and MEK/ERK activation.
TSP1 is a key mediator of matrix contraction of normal and systemic sclerosis fibroblasts, via MEK/ERK.