Peroxisome proliferator-activated receptor gamma ligands inhibit transforming growth factor-beta-induced, hyaluronan-dependent, T cell adhesion to orbital fibroblasts

Thyroid eye disease is characterized by the infiltration of leukocytes and accumulation of hyaluronan (HA) in orbital tissue. Inflamed orbital tissue expands in size due to excessive HA and to the formation of scar tissue (fibrosis) and/or adipose accumulation. Transforming growth factor β (TGF-β) acts as a key inducer of fibrosis by enhancing extracellular matrix production. Treatment of primary human orbital fibroblasts with TGF-β led to significant increases in both HA synthesis and secretion. TGF-β also strongly induced hyaluronan synthase 1 (HAS1) and HAS2 mRNA levels, which increased 50- and 6-fold, respectively. Remarkably, the addition of the peroxisome proliferator-activated receptor (PPARγ) ligands pioglitazone (Pio) or rosiglitazone (Rosi) to TGF-β-treated orbital fibroblasts attenuated HA synthesis and reduced HAS1 and HAS2 mRNA levels. The attenuation of TGF-β function by Pio and Rosi was independent of PPARγ activity. Furthermore, Pio and Rosi treatment inhibited TGF-β-induced T cell adhesion to orbital fibroblasts. Our findings demonstrate that TGF-β plays an important role in HA synthesis and in the inflammatory response by enhancing or facilitating inflammatory cell infiltration and adhesion to orbital tissue. Pio and Rosi exhibit anti-fibrotic and anti-inflammatory activity and may be useful in treating thyroid eye disease.     

A rapid transient increase in hyaluronan synthase-2 mRNA initiates secretion of hyaluronan by corneal keratocytes in response to transforming growth factor beta

Keratocytes of the corneal stroma produce transparent extracellular matrix devoid of hyaluronan (HA); however, in corneal pathologies and wounds, HA is abundant. We previously showed primary keratocytes cultured under serum-free conditions to secrete matrix similar to that of normal stroma, but serum and transforming growth factor beta (TGFbeta) induced secretion of fibrotic matrix components, including HA. This study found HA secretion by primary bovine keratocytes to increase rapidly in response to TGFbeta, reaching a maximum in 12 h and then decreasing to <5% of the maximum by 48 h. Cell-free biosynthesis of HA by cell extracts also exhibited a transient peak at 12 h after TGFbeta treatment. mRNA for hyaluronan synthase enzymes HAS1 and HAS2 increased >10- and >50-fold, respectively, in 4-6 h, decreasing to near original levels after 24-48 h. Small interfering RNA against HAS2 inhibited the transient increase of HAS2 mRNA and completely blocked HA induction, but small interfering RNA to HAS1 had no effect on HA secretion. HAS2 mRNA was induced by a variety of mitogens, and TGFbeta acted synergistically to induce HAS2 by as much as 150-fold. In addition to HA synthesis, treatment with TGFbeta induced degradation of fluorescein-HA added to culture medium. These results show HA secretion by keratocytes to be initiated by a rapid transient increase in the HAS2 mRNA pool. The very rapid induction of HA expression in keratocytes suggests a functional role of this molecule in the fibrotic response of keratocytes to wound healing.     

Differential effect of growth factors on hyaluronan synthase gene expression in fibroblasts exposed to oxidative stress

The aim of this study was to evaluate how growth factors (PDGF-BB, EGF, and TGF-1beta) modulate hyaluronan synthase (HAS) activities in normal or stressed cultured human skin fibroblasts. The effects of concomitant treatment with cytokines and FeSO4 plus ascorbate on HAS mRNA expression, protein synthesis, and hyaluronic acid (HA) concentrations were also studied. Treatment of fibroblasts with growth factors up-regulated HAS gene expression and increased HAS enzymes and HA production. PDGF-BB induced HAS mRNA expression, protein synthesis, and HA production more efficiently than EGF and TGF-1beta. EGF was less effective than TGF-1beta. In addition, TGF-1beta reduced the expression and synthesis of HAS3, while PDGF-BB and EGF had the opposite effect. Concomitant treatment with growth factors and the oxidant was able to further increase HAS mRNA expression, once again with the exception of HAS3 with TGF-1beta. HAS protein synthesis was reduced, while HA levels were unaffected in comparison to those obtained from exposure to FeSO4 plus ascorbate alone. In conclusion, although growth factors plus the oxidant synergistically induced HAS mRNA expression in part, enzyme production was not correlated with this increase. Moreover, the increase in HAS mRNA levels was not translated into a consequent rise in HA concentration.     

Smad proteins differentially regulate transforming growth factor-β-mediated induction of chondroitin sulfate proteoglycans

Traumatic injury to the CNS results in increased expression and deposition of chondroitin sulfate proteoglycans (CSPGs) that are inhibitory to axonal regeneration. Transforming growth factor-β (TGF-β) has been implicated as a major mediator of these changes, but the mechanisms through which TGF-β regulates CSPG expression are not known. Using lentiviral expressed Smad-specific ShRNA we show that TGF-β induction of CSPG expression in astrocytes is Smad-dependent. However, we find a differential dependence of the synthetic machinery on Smad2 and/or Smad3. TGF-β induction of neurocan and xylosyl transferase 1 required both Smad2 and Smad3, whereas induction of phosphacan and chondroitin synthase 1 required Smad2 but not Smad3. Smad3 knockdown selectively reduced induction of chondroitin-4-sulfotransferase 1 and the amount of 4-sulfated CSPGs secreted by astrocytes. Additionally, Smad3 knockdown in astrocytes was more efficacious in promoting neurite outgrowth of neurons cultured on the TGF-β-treated astrocytes. Our data implicate TGF-β Smad3-mediated induction of 4-sulfation as a critical determinant of the permissiveness of astrocyte secreted CSPGs for axonal growth.     

Smad3-mediated myocardin silencing: a novel mechanism governing the initiation of smooth muscle differentiation

Both TGF-β and myocardin (MYOCD) are important for smooth muscle cell (SMC) differentiation, but their precise role in regulating the initiation of SMC development is less clear. In TGF-β-induced SMC differentiation of pluripotent C3H10T1/2 progenitors, we found that TGF-β did not significantly induce Myocd mRNA expression until 18 h of stimulation. On the other hand, early SMC markers such as SM α-actin, SM22α, and SM calponin were detectable beginning 2 or 4 h after TGF-β treatment. These results suggest that Myocd expression is blocked during the initiation of TGF-β-induced SMC differentiation. Consistent with its endogenous expression, Myocd promoter activity was not elevated until 18 h following TGF-β stimulation. Surprisingly, Smad signaling was inhibitory to Myocd expression because blockade of Smad signaling enhanced Myocd promoter activity. Overexpression of Smad3, but not Smad2, inhibited Myocd promoter activity. Conversely, shRNA knockdown of Smad3 allowed TGF-β to activate the Myocd promoter in the initial phase of induction. Myocd was activated by PI3 kinase signaling and its downstream target Nkx2.5. Interestingly, Smad3 did not affect PI3 kinase activity. However, Smad3 physically interacted with Nkx2.5. This interaction blocked Nkx2.5 binding to the Myocd promoter in the early stage of TGF-β induction, leading to inhibition of Myocd mRNA expression. Moreover, Smad3 inhibited Nkx2.5-activated Myocd promoter activity in a dose-dependent manner. Taken together, our results reveal a novel mechanism for Smad3-mediated inhibition of Myocd in the initiation phase of SMC differentiation.     

Macrophage-stimulated cardiac fibroblast production of IL-6 is essential for TGF β/Smad activation and cardiac fibrosis induced by angiotensin II

Interleukin-6 (IL-6) is an important cytokine participating in multiple biologic activities in immune regulation and inflammation. IL-6 has been associated with cardiovascular remodeling. However, the mechanism of IL-6 in hypertensive cardiac fibrosis is still unclear. Angiotensin II (Ang II) infusion in mice increased IL-6 expression in the heart. IL-6 knockout (IL-6-/-) reduced Ang II-induced cardiac fibrosis: 1) Masson trichrome staining showed that Ang II infusion significantly increased fibrotic areas of the wild-type mouse heart, which was greatly suppressed in IL-6-/- mice and 2) immunohistochemistry staining showed decreased expression of α-smooth muscle actin (α-SMA), transforming growth factor β1 (TGF-β1) and collagen I in IL-6-/- mouse heart. The baseline mRNA expression of IL-6 in cardiac fibroblasts was low and was absent in cardiomyocytes or macrophages; however, co-culture of cardiac fibroblasts with macrophages significantly increased IL-6 production and expression of α-SMA and collagen I in fibroblasts. Moreover, TGF-β1 expression and phosphorylation of TGF-β downstream signal Smad3 was stimulated by co-culture of macrophages with cardiac fibroblasts, while IL-6 neutralizing antibody decreased TGF-β1 expression and Smad3 phosphorylation in co-culture of macrophage and fibroblast. Taken together, our results indicate that macrophages stimulate cardiac fibroblasts to produce IL-6, which leads to TGF-β1 production and Smad3 phosphorylation in cardiac fibroblasts and thus stimulates cardiac fibrosis.     

TGF-β1 induces EMT reprogramming of porcine bladder urothelial cells into collagen producing fibroblasts-like cells in a Smad2/Smad3-dependent manner

Activation of fibroblasts and their differentiation into myofibroblasts, excessive collagen production and fibrosis occurs in a number of bladder diseases. Similarly, conversion of epithelial cells into mesenchymal cells (EMT) has been shown to increase fibroblasts like cells. TGF-β1 can induce the EMT and the role of TGF-β1-induced EMT during bladder injury leading to fibrosis and possible organ failure is gaining increasing interest. Here we show that EMT and fibrosis in porcine bladder urothelial (UC) cells are Smad dependent. Fresh normal porcine bladder urothelial cells were grown in culture with or without TGF-β1 and EMT markers were assessed. TGF-β1 treatment induced changes in cellular morphology as depicted by a significant decrease in the expression of E-cadherin and corresponding increase in N-cadherin and α-SMA. We knocked down Smad2 and Smad3 by Smad specific siRNA. Downregulation of E-cadherin expression by TGF-β1 was Smad3-dependent, whereas N-cadherin and α-SMA were dependent on both Smad2 and Smad3. Connective tissue growth factor (CTGF/CCN2), matrix metalloproteinase-2 and -9 (MMP-2, MMP-9) has been shown to play important roles in the pathogenesis of fibrosis. Induction of these genes by TGF-β1 was found to be time dependent. Upregulation of CTGF/CCN2 by TGF-β1 was Smad3 dependent; whereas MMP-2 was Smad2 dependent. Smad2 and Smad3 both participated in MMP-9 expression. TGF-β1 reprogrammed mesenchymal fibroblast like cells robustly expressed collagen I and III and these was inhibited by SB-431542, a TGF-β receptor inhibitor. Our results indicate that EMT of porcine bladder UC cells is TGF-β1 dependent and is mediated through Smad2 and Smad3. TGF-β1 may be an important factor in the development of bladder fibrosis via an EMT mechanism. This identifies a potential amenable therapeutic target.     

Hyaluronan Controls the Deposition of Fibronectin and Collagen and Modulates TGF-β1 Induction of Lung Myofibroblasts

The contribution of hyaluronan-dependent pericellular matrix to TGF-β1-driven induction and maintenance of myofibroblasts is not understood. Hyaluronan is an extracellular matrix (ECM) glycosaminoglycan important in cell adhesion, proliferation and migration, and is implicated in myofibroblast formation and maintenance. Reduced turnover of hyaluronan has been linked to differentiation of myofibroblasts and potentiation of lung fibrosis. Fibronectin is a fibril forming adhesive glycoprotein that is also upregulated following induction with TGF-β1. Although they are known to bind each other, the interplay between hyaluronan and fibronectin in the pericellular matrix during myofibroblast induction and matrix assembly is not clear. This study addresses the role of hyaluronan and its interaction with fibrillar matrix components during myofibroblast formation. Hyaluronan and fibronectin were increased and co-localized in the ECM following myofibroblast induction by TGF-β1. Inhibition of hyaluronan synthesis in TGF-β1-induced lung myofibroblasts over a 4 day period with 4-methyl umbelliferone (4-MU) further enhanced myofibroblast morphology, caused increased deposition of fibronectin and type I collagen in the ECM, and increased expression of alpha-smooth muscle actin and hyaluronan synthase 2 (HAS2) mRNA. Hyaluronan oligosaccharides or hyaluronidase treatment, which more effectively disrupted the pericellular matrix, had similar effects. CD44 and β1 integrins co-localized in the cell membrane and along some stress fibers. However, CD44 and hyaluronan were specifically excluded from focal adhesions, and associated primarily with cortical actin. Time-lapse imaging of the immediate effects of hyaluronidase digestion showed that hyaluronan matrix primarily mediates attachment of membrane and cortical actin between focal contacts, suggesting that surface adhesion through hyaluronan and CD44 is distinct from focal adhesion through β1 integrins and fibronectin. Fluorescein-labeled hyaluronan bound regularly along fibronectin fibers and co-localized more with β1 integrin and less with CD44. Therefore, the hyaluronan matrix can interfere with the assembly of fibrillar ECM components, and this interplay regulates the degree of myofibroblast formation. These data also suggest that adhesion through hyaluronan matrix impacts cytoskeletal organization, and is potentially part of a clutch mechanism that regulates stick and slip of myofibroblasts by affecting the adhesion to and organization of fibronectin and collagen.