Hyaluronan facilitates transforming growth factor-β1-dependent proliferation via CD44 and epidermal growth factor receptor interaction

Fibroblast proliferation is an early feature of progressive tissue fibrosis and is largely regulated by the cytokine transforming growth factor-β1 (TGF-β1). In the oral mucosa, fibroblasts have a unique phenotype and demonstrate healing with no fibrosis/scarring. Our previous studies show that whereas dermal fibroblasts proliferate in response to TGF-β1, oral fibroblasts have an antiproliferative response to this cytokine. Hyaluronan (HA) was directly linked to this TGF-β1-dependent response. The aim of this study was to understand the underlying mechanism through which HA regulates TGF-β-dependent responses. Using patient-matched oral and dermal fibroblasts, we show that TGF-β1-dependent proliferation is mediated through the HA receptor CD44, whereas the TGF-β1-mediated antiproliferative response is CD44-independent. Furthermore, overexpression of HAS2 (HA synthase-2) in oral cells modifies their response, and they subsequently demonstrate a proliferative, CD44-dependent response to TGF-β1. We also show that epidermal growth factor (EGF) and its receptor (EGFR) are essential for TGF-β1/HA/CD44-dependent proliferation. Increased HA levels promote EGFR and CD44 coupling, potentiating signal transduction through the MAPK/ERK pathway. Thus, in a HA-rich environment, late ERK1/2 activation results from EGFR/CD44 coupling and leads to a proliferative response to TGF-β1. In comparison, in a non-HA-rich environment, only early ERK1/2 activation occurs, and this is associated with an antiproliferative response to TGF-β1. In summary, HA facilitates TGF-β1-dependent fibroblast proliferation through promoting interaction between CD44 and EGFR, which then promotes specific MAPK/ERK activation, inducing cellular proliferation.     

Accelerated re-epithelialization in beta3-integrin-deficient- mice is associated with enhanced TGF-beta1 signaling

The upregulation of TGF-beta1 and integrin expression during wound healing has implicated these molecules in this process, but their precise regulation and roles remain unclear. Here we report that, notably, mice lacking beta(3)-integrins show enhanced wound healing with re-epithelialization complete several days earlier than in wild-type mice. We show that this effect is the result of an increase in TGF-beta1 and enhanced dermal fibroblast infiltration into wounds of beta(3)-null mice. Specifically, beta(3)-integrin deficiency is associated with elevated TGF-beta receptor I and receptor II expression, reduced Smad3 levels, sustained Smad2 and Smad4 nuclear localization and enhanced TGF-beta1-mediated dermal fibroblast migration. These data indicate that alpha(v)beta(3)-integrin can suppress TGF-beta1-mediated signaling, thereby controlling the rate of wound healing, and highlight a new mechanism for TGF-beta1 regulation by beta(3)-integrins.     

CCL18-stimulated upregulation of collagen production in lung fibroblasts requires Sp1 signaling and basal Smad3 activity

A CC chemokine CCL18 stimulates collagen production in pulmonary fibroblasts through an unknown signaling mechanism. In this study, involvement of Sp1 and Smad3 in CCL18 signaling in primary human pulmonary fibroblast cultures was investigated. Phosphorylation of Sp1, DNA-binding by Sp1, and the activity of an Sp1-dependent reporter were all increased in response to CCL18 stimulation. CCL18 did not stimulate a detectable increase in Smad3 phosphorylation or Smad3/4 DNA-binding activity, although some basal phosphorylation and DNA binding by Smad3/4 were noted. Transient overexpression of dominant negative mutants of Sp1 and Smad3 abrogated CCL18-dependent upregulation as well as basal production of collagen. These observations suggested that CCL18 activates collagen production in pulmonary fibroblasts through an Sp1-dependent pathway that also requires basal Smad3 activity. Possible involvement of autocrine TGF-beta in CCL18 signaling was considered. CCL18 stimulated increases in collagen mRNA and protein production without detectable changes in TGF-beta1, -beta2, and -beta3 mRNA or protein levels. Neutralizing anti-TGF-beta antibodies, latency-associated peptide, ALK5-specific inhibitor SD431542, and an inhibitor of the protease-dependent TGF-beta activation aprotinin, each failed to block CCL18-stimulated collagen production. These observations suggest that both CCL18 signaling in pulmonary fibroblasts and basal Smad3 activity are independent of autocrine TGF-beta.     

Cell-type-specific activation of PAK2 by transforming growth factor beta independent of Smad2 and Smad3

Transforming growth factor beta (TGF-beta) causes growth arrest in epithelial cells and proliferation and morphological transformation in fibroblasts. Despite the ability of TGF-beta to induce various cellular phenotypes, few discernible differences in TGF-beta signaling between cell types have been reported, with the only well-characterized pathway (the Smad cascade) seemingly under identical control. We determined that TGF-beta receptor signaling activates the STE20 homolog PAK2 in mammalian cells. PAK2 activation occurs in fibroblast but not epithelial cell cultures and is independent of Smad2 and/or Smad3. Furthermore, we show that TGF-beta-stimulated PAK2 activity is regulated by Rac1 and Cdc42 and dominant negative PAK2 or morpholino antisense oligonucleotides to PAK2 prevent the morphological alteration observed following TGF-beta addition. Thus, PAK2 represents a novel Smad-independent pathway that differentiates TGF-beta signaling in fibroblast (growth-stimulated) and epithelial cell (growth-inhibited) cultures.     

Polarity of response to transforming growth factor-beta1 in proximal tubular epithelial cells is regulated by beta-catenin

Transforming growth factor-beta1 (TGF-beta1)-mediated loss of proximal tubular epithelial cell-cell interaction is regulated in a polarized fashion. The aim of this study was to further explore the polarity of the TGF-beta1 response and to determine the significance of R-Smad-beta-catenin association previously demonstrated to accompany adherens junction disassembly. Smad3 signaling response to TGF-beta1 was assessed by activity of the Smad3-responsive reporter gene construct (SBE)(4)-Lux and by immunoblotting for phospho-Smad proteins. Similar results were obtained with both methods. Apical application of TGF-beta1 led to increased Smad3 signaling compared with basolateral stimulation. Association of Smad proteins with beta-catenin was greater following basolateral TGFbeta-1 stimulation, as was the expression of cytoplasmic Triton-soluble beta-catenin. Inhibition of beta-catenin expression by small interfering RNA augmented Smad3 signaling. Lithium chloride, a GSK-3 inhibitor, increased expression of beta-catenin and attenuated TGF-beta1-dependent Smad3 signaling. Lithium chloride did not influence degradation of Smad3 but resulted in decreased nuclear translocation. Smad2 activation as assessed by Western blot analysis and activity of the Smad2-responsive reporter constructs ARE/MF1 was also greater following apical as compared with basolateral TGFbeta-1 stimulation, suggesting that this is a generally applicable mechanism for the regulation of TGF-beta1-dependent R-Smads. Caco-2 cells are a colonic carcinoma cell line, with known resistance to the anti-proliferative effects of TGF-beta1 and increased expression of beta-catenin. We used this cell line to address the general applicability of our observations. Inhibition of beta-catenin in this cell line by small interfering RNA resulted in increased TGF-beta1-dependent Smad3 phosphorylation and restoration of TGF-beta1 anti-proliferative effects.     

A tale of two proteins: differential roles and regulation of Smad2 and Smad3 in TGF-beta signaling

Transforming growth factor-beta (TGF-beta) is an important growth inhibitor of epithelial cells, and insensitivity to this cytokine results in uncontrolled cell proliferation and can contribute to tumorigenesis. Smad2 and Smad3 are direct mediators of TGF-beta signaling, however little is known about the selective activation of Smad2 versus Smad3. The Smad2 and Smad3 knockout mouse phenotypes and studies comparing Smad2 and Smad3 activation of TGF-beta target genes, suggest that Smad2 and Smad3 have distinct roles in TGF-beta signaling. The observation that TGF-beta inhibits proliferation of Smad3-null mammary gland epithelial cells, whereas Smad3 deficient fibroblasts are only partially growth inhibited, suggests that Smad3 has a different role in epithelial cells and fibroblasts. Herein, the current understanding of Smad2 and Smad3-mediated TGF-beta signaling and their relative roles are discussed, in addition to potential mechanisms for the selective activation of Smad2 versus Smad3. Since alterations in the TGF-beta signaling pathway play an important role in promoting tumorigenesis and cancer progression, methods for therapeutic targeting of the TGF-beta signaling pathway are being pursued. Determining how Smad2 or Smad3 differentially regulate the TGF-beta response may translate into developing more effective strategies for cancer therapy.     

Abrogation of transforming growth factor-beta signaling by SMAD7 inhibits collagen gel contraction of human dermal fibroblasts

Human fibroproliferative disorders like hypertrophic scarring of the skin are characterized by increased contractility and excess extracellular matrix synthesis. A beneficial role of transforming growth factor (TGF)-beta in wound healing was proposed; however, chronic stimulation by this cytokine leads to fibrosis. In the present report, the intracellular TGF-beta signaling in fibroblasts derived from hypertrophic scars and normal skin was examined. In an attempt to intervene in profibrogenic TGF-beta functions, ectopic expression of Smad7 or dominant negative Smads3/4 completely inhibited contractility of scar-derived and normal fibroblasts after suspension in collagen gels. Both cell types displayed constitutive Smad2/3 phosphorylation and (CAGA)9-MLP-Luc activity with expression and phosphorylation of Smad3 being predominant in hypertrophic scar-derived fibroblasts. Down-regulation of intrinsic signaling with various TGF-beta antagonists, e.g. soluble TGF-beta receptor, latency-associated peptide, and anti-TGF-beta1 antibodies, confirms autocrine TGF-beta stimulation of both cell populations. Further, Smad7 expression inhibited alpha1 (I) collagen and alpha-smooth muscle actin expression. In summary, our data indicate that autocrine TGF-beta/Smad signaling is involved in contractility and matrix gene expression of fibroblasts from normal and hypertrophic scars. Smad7 inhibits these processes and may exert beneficial effects on excessive scar formation.     

Hyaluronan regulates transforming growth factor-beta1 receptor compartmentalization

Transforming growth factor-beta1 (TGF-beta1) is a key cytokine involved in the pathogenesis of fibrosis in many organs. We previously demonstrated in renal proximal tubular cells that the engagement of the extracellular polysaccharide hyaluronan with its receptor CD44 attenuated TGF-beta1 signaling. In the current study we examined the potential mechanism by which the interaction between hyaluronan (HA) and CD44 regulates TGF-beta receptor function. Affinity labeling of TGF-beta receptors demonstrated that in the unstimulated cells the majority of the receptor partitioned into EEA-1-associated non-lipid raft-associated membrane pools. In the presence of exogenous HA, the majority of the receptors partitioned into caveolin-1 lipid raft-associated pools. TGF-beta1 increased the association of activated/phosphorylated Smad proteins with EEA-1, consistent with activation of TGF-beta1 signaling following endosomal internalization. Following addition of HA, caveolin-1 associated with the inhibitory Smad protein Smad7, consistent with the raft pools mediating receptor turnover, which was facilitated by HA. Antagonism of TGF-beta1-dependent Smad signaling and the effect of HA on TGF-beta receptor associations were inhibited by depletion of membrane cholesterol using nystatin and augmented by inhibition of endocytosis. The effect of HA on TGF-beta receptor trafficking was inhibited by inhibition of HA-CD44 interactions, using blocking antibody to CD44 or inhibition of MAP kinase activation. In conclusion, we have proposed a model by which HA engagement of CD44 leads to MAP kinase-dependent increased trafficking of TGF-beta receptors to lipid raft-associated pools, which facilitates increased receptor turnover and attenuation of TGF-beta1-dependent alteration in proximal tubular cell function.     

Antagonistic effects of TNF-alpha on TGF-beta signaling through down-regulation of TGF-beta receptor type II in human dermal fibroblasts

Transforming growth factor-beta stimulates the production of the extracellular matrix, whereas TNF-alpha has antifibrotic activity. Understanding the molecular mechanism underlying the antagonistic activities of TNF-alpha against TGF-beta is critical in the context of tissue repair and maintenance of tissue homeostasis. In the present study, we demonstrated a novel mechanism by which TNF-alpha blocks TGF-beta-induced gene and signaling pathways in human dermal fibroblasts. We showed that TNF-alpha prevents TGF-beta-induced gene trans activation, such as alpha2(I) collagen or tissue inhibitor of metalloproteinases 1, and TGF-beta signaling pathways, such as Smad3, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinases, without inducing levels of inhibitory Smad7 in human dermal fibroblasts. TNF-alpha down-regulates the expression of type II TGF-beta receptor (TbetaRII) proteins, but not type I TGF-beta receptor (TbetaRI), in human dermal fibroblasts. However, neither TbetaRII mRNA nor TbetaRII promoter activity was decreased by TNF-alpha. TNF-alpha-mediated decrease of TbetaRII protein expression was not inhibited by the treatment of fibroblasts with either a selective inhibitor of I-kappaB-alpha phosphorylation, BAY 11-7082, or a mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor, PD98059. Calpain inhibitor I (ALLN), a protease inhibitor, inhibits TNF-alpha-mediated down-regulation of TbetaRII. We found that TNF-alpha triggered down-regulation of TbetaRII, leading to desensitization of human dermal fibroblasts toward TGF-beta. Furthermore, these events seemed to cause a dramatic down-regulation of alpha2(I) collagen and tissue inhibitor of metalloproteinases 1 in systemic sclerosis fibroblasts. These results indicated that TNF-alpha impaired the response of the cells to TGF-beta by regulating the turnover of TbetaRII.     

Adipocytes recognize and degrade oxidized low density lipoprotein through CD36

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine involved in controlling critical cellular activities including proliferation, differentiation, extracellular matrix production, and apoptosis. TGF-beta signals are mediated by a family of Smad proteins, of which Smad2 and Smad3 are downstream intracellular targets of serine/threonine kinase receptors of TGF-beta. Although Smad2 and Smad3 are crucial for TGF-beta signaling, little is known about the regulation of their expression. In this study, we investigated the expression of Smad2 and Smad3 in an in vivo animal model of lung fibrosis induced by bleomycin. We found that the expression of Smad3 was regulated in lungs during bleomycin-induced pulmonary fibrosis. The decline of Smad3 mRNA was evident at day three of post-bleomycin instillation and the expression of Smad3 continually decreased during the reparative phase of lung injury (days 8 and 12), whereas the expression of Smad2 showed little change after bleomycin administration. We further investigated whether the expression of Smad3 was regulated by TGF-beta in an in vitro lung fibroblast culture system. Our results show an immediate translocation of Smad3 protein from the cytoplasm to the nucleus and a delayed down-regulation of Smad3 mRNA by TGF-beta in lung fibroblasts. These studies provide direct evidence for a differential regulation of Smad3 expression that is distinct from that of Smad2 during bleomycin-induced pulmonary fibrosis and suggest a ligand-induced negative feedback loop that modulates cellular TGF-beta signaling.     

Phosphatidylinositol 3-kinase is involved in alpha2(I) collagen gene expression in normal and scleroderma fibroblasts

TGF-beta is implicated in the pathogenesis of fibrotic disorders. It has been shown that Smad3 promotes the human alpha2(I) collagen (COL1A2) gene expression by TGF-beta1 in human dermal fibroblasts. Here, we investigated the role of phosphatidylinositol 3-kinase (PI3K) in the COL1A2 gene expression in normal and scleroderma fibroblasts. In normal fibroblasts, the PI3K inhibitor, LY294002, significantly decreased the basal and the TGF-beta1-induced increased stability of COL1A2 mRNA. The TGF-beta1-induced COL1A2 promoter activity, but not the basal activity, was significantly attenuated by LY294002 or the dominant negative mutant of p85 subunit of PI3K, while the constitutive active mutant of p110 subunit of PI3K did not affect the basal or the TGF-beta1-induced COL1A2 promoter activity. LY294002 significantly decreased the phosphorylation of Smad3 induced by TGF-beta1. Furthermore, the transient overexpression of 2xFYVE, which induces the mislocalization of FYVE domain proteins, decreased the TGF-beta1-induced Smad3 phosphorylation to a similar extent to LY294002. In scleroderma fibroblasts, the blockade of PI3K significantly decreased the mRNA stability and the promoter activity of the COL1A2 gene. Furthermore, LY294002 and the transient overexpression of 2xFYVE completely diminished the constitutive phosphorylation of Smad3. These results indicate that 1) the basal activity of PI3K is necessary for the COL1A2 mRNA stabilization in normal and scleroderma fibroblasts, 2) there is an unidentified FYVE domain protein specifically interacting with Smad3, and 3) the basal activity of PI3K and the FYVE domain protein are indispensable for the efficient TGF-beta/Smad3 signaling in normal fibroblasts and for the establishment of the constitutive activation of TGF-beta/Smad3 signaling in scleroderma fibroblasts.