Suberoylanilide hydroxamic acid (SAHA) inhibits transforming growth factor-beta 2-induced increases in aqueous humor outflow resistance

Transforming growth factor-beta 2 (TGF-β2) is highly concentrated in the aqueous humor of primary open-angle glaucoma patients. TGF-β2 causes fibrosis of outflow tissues, such as the trabecular meshwork (TM), and increases intraocular pressure by increasing resistance to aqueous humor outflow. Recently, histone deacetylase (HDAC) activity was investigated in fibrosis in various tissues, revealing that HDAC inhibitors suppress tissue fibrosis. However, the effect of HDAC inhibitors on fibrosis in the eye was not determined. Here, we investigated the effect of suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor, on TGF-β2-induced increased resistance to aqueous humor outflow. We found that SAHA suppressed TGF-β2-induced outflow resistance in perfused porcine eyes. Moreover, SAHA cotreatment suppressed TGF-β2-induced ocular hypertension in rabbits. The permeability of monkey TM (MTM) and Schlemm’s canal (MSC) cell monolayers was decreased by TGF-β2 treatment. SAHA inhibited the effects of TGF-β2 on the permeability of these cells. TGF-β2 also increased the expression of extracellular matrix proteins (fibronectin and collagen type I or IV) in MTM, MSC, and human TM (HTM) cells, while SAHA inhibited TGF-β2-induced extracellular matrix protein expression in these cells. SAHA also inhibited TGF-β2-induced phosphorylation of Akt and ERK, but did not inhibit Smad2/3 phosphorylation, the canonical pathway of TGF-β signaling. Moreover, SAHA induced the expression of phosphatase and tensin homolog, a PI3K/Akt signaling factor, as well as bone morphogenetic protein 7, an endogenous antagonist of TGF-β. These results imply that SAHA prevents TGF-β2-induced increases in outflow resistance and regulates the non-Smad pathway of TGF-β signaling in TM and MSC cells.     

TGF-beta and fibrosis

Transforming growth factor-beta (TGF-beta) isoforms are multifunctional cytokines that play a central role in wound healing and in tissue repair. TGF-beta is found in all tissues, but is particularly abundant in bone, lung, kidney and placental tissue. TGF-beta is produced by many but not all parenchymal cell types, and is also produced or released by infiltrating cells such as lymphocytes, monocytes/macrophages, and platelets. Following wounding or inflammation, all these cells are potential sources of TGF-beta. In general, the release and activation of TGF-beta stimulates the production of various extracellular matrix proteins and inhibits the degradation of these matrix proteins, although exceptions to these principles abound. These actions of TGF-beta contribute to tissue repair, which under ideal circumstances leads to the restoration of normal tissue architecture and may involve a component of tissue fibrosis. In many diseases, excessive TGF-beta contributes to a pathologic excess of tissue fibrosis that compromises normal organ function, a topic that has been the subject of numerous reviews [1-3]. In the following chapter, we will discuss the role of TGF-beta in tissue fibrosis, with particular emphasis on renal fibrosis.     

Serine Threonine Receptor-Associated Protein (STRAP) plays a role in the maintenance of mesenchymal morphology

The stromal tissue, made of extracellular matrix and mesenchymal cells, is vital for the functional design of all complex tissues. Fibroblasts are key components of stromal tissue and play a crucial role during organ development, wound repair, angiogenesis and fibrosis. We have previously reported the identification of a novel WD-domain protein, STRAP¹ that inhibits transforming growth factor-β (TGF-β) signaling and enhances tumorigenicity via TGF-β-dependent and TGF-β-independent mechanisms. Here, we report, for the first time, that deletion of STRAP from Mouse Embryonic Fibroblasts (MEFs) results in a loss of mesenchymal morphology. These cells lose their spindle shape and exhibit features of an epithelial morphology. Gene expression profiling has confirmed that deletion of STRAP affects expression of sets of genes important for diverse functions including cell–cell adhesion and cell polarization, and upregulates E-cadherin expression leading to the formation of adherens junctions, subsequent localization of β-catenin to the cell membrane and downregulation of the mesenchymal markers like LEF1 (lymphoid enhancer-binding factor 1). Upregulation of WT1 (Wilms tumor homolog 1) in STRAP null MEFs plays a role in E-cadherin induction. Finally, stable expression of STRAP in these cells results in a loss of WT1 and E-cadherin expressions, and a reversal from epithelial to the mesenchymal morphology. Thus, these results provide a novel TGF-β-independent function of STRAP and describe a mechanism for the role of STRAP in the maintenance of mesenchymal morphology.     

Myofibroblast Differentiation and Enhanced Tgf-B Signaling in Cystic Fibrosis Lung Disease

Rationale

TGF-β, a mediator of pulmonary fibrosis, is a genetic modifier of CF respiratory deterioration. The mechanistic relationship between TGF-β signaling and CF lung disease has not been determined.

Objective

To investigate myofibroblast differentiation in CF lung tissue as a novel pathway by which TGF-β signaling may contribute to pulmonary decline, airway remodeling and tissue fibrosis.

Methods

Lung samples from CF and non-CF subjects were analyzed morphometrically for total TGF-β₁, TGF-β signaling (Smad2 phosphorylation), myofibroblast differentiation (α-smooth muscle actin), and collagen deposition (Masson trichrome stain).

Results

TGF-β signaling and fibrosis are markedly increased in CF (p<0.01), and the presence of myofibroblasts is four-fold higher in CF vs. normal lung tissue (p<0.005). In lung tissue with prominent TGF-β signaling, both myofibroblast differentiation and tissue fibrosis are significantly augmented (p<0.005).

Conclusions

These studies establish for the first time that a pathogenic mechanism described previously in pulmonary fibrosis is also prominent in cystic fibrosis lung disease. The presence of TGF-β dependent signaling in areas of prominent myofibroblast proliferation and fibrosis in CF suggests that strategies under development for other pro-fibrotic lung conditions may also be evaluated for use in CF.     

Molecular Mechanism Responsible for Fibronectin-controlled Alterations in Matrix Stiffness in Advanced Chronic Liver Fibrogenesis

Fibrosis is characterized by extracellular matrix (ECM) remodeling and stiffening. However, the functional contribution of tissue stiffening to noncancer pathogenesis remains largely unknown. Fibronectin (Fn) is an ECM glycoprotein substantially expressed during tissue repair. Here we show in advanced chronic liver fibrogenesis using a mouse model lacking Fn that, unexpectedly, Fn-null livers lead to more extensive liver cirrhosis, which is accompanied by increased liver matrix stiffness and deteriorated hepatic functions. Furthermore, Fn-null livers exhibit more myofibroblast phenotypes and accumulate highly disorganized/diffuse collagenous ECM networks composed of thinner and significantly increased number of collagen fibrils during advanced chronic liver damage. Mechanistically, mutant livers show elevated local TGF-β activity and lysyl oxidase expressions. A significant amount of active lysyl oxidase is released in Fn-null hepatic stellate cells in response to TGF-β1 through canonical and noncanonical Smad such as PI3 kinase-mediated pathways. TGF-β1-induced collagen fibril stiffness in Fn-null hepatic stellate cells is significantly higher compared with wild-type cells. Inhibition of lysyl oxidase significantly reduces collagen fibril stiffness, and treatment of Fn recovers collagen fibril stiffness to wild-type levels. Thus, our findings indicate an indispensable role for Fn in chronic liver fibrosis/cirrhosis in negatively regulating TGF-β bioavailability, which in turn modulates ECM remodeling and stiffening and consequently preserves adult organ functions. Furthermore, this regulatory mechanism by Fn could be translated for a potential therapeutic target in a broader variety of chronic fibrotic diseases.     

TGF-β-Elicited Induction of Tissue Inhibitor of Metalloproteinases (TIMP)-3 Expression in Fibroblasts Involves Complex Interplay between Smad3, p38α, and ERK1/2

Transforming growth factor-β (TGF-β) promotes extracellular matrix deposition by down-regulating the expression of matrix degrading proteinases and upregulating their inhibitors. Tissue inhibitor of metalloproteinases (TIMP)-3 is an ECM-associated specific inhibitor of matrix degrading metalloproteinases. Here, we have characterized the signaling pathways mediating TGF-β-induced expression of TIMP-3. Basal and TGF-β-induced TIMP-3 mRNA expression was abolished in Smad4-deficient mouse embryonic fibroblasts and restoring Smad4 expression rescued the response. Inhibition of Smad signaling by expression of Smad7 and dominant negative Smad3 completely abolished TGF-β-elicited expression of TIMP-3 in human fibroblasts, whereas overexpression of Smad3 enhanced it. Inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) activation with PD98059 and p38 mitogen-activated protein kinase activity by SB203580 resulted in suppression of TGF-β-induced TIMP-3 expression, indicating that ERK1/2 and p38 MAPK mediate the effect of TGF-β on TIMP-3 expression. Specific activation of p38α and ERK1/2 by constitutively active mutants of MKK3b or MEK1, respectively, and simultaneous co-expression of Smad3 resulted in induction of TIMP-3 expression in the absence of TGF-β indicating that Smad3 co-operates with p38 and ERK1/2 in the induction of TIMP-3 expression. These results demonstrate the complex interplay between Smad3, p38α, and ERK1/2 signaling in the regulation of TIMP-3 gene expression in fibroblasts, which may play a role in inflammation, tissue repair, and fibrosis.     

A Critical Role for the mTORC2 Pathway in Lung Fibrosis

A characteristic of dysregulated wound healing in IPF is fibroblastic-mediated damage to lung epithelial cells within fibroblastic foci. In these foci, TGF-β and other growth factors activate fibroblasts that secrete growth factors and matrix regulatory proteins, which activate a fibrotic cascade. Our studies and those of others have revealed that Akt is activated in IPF fibroblasts and it mediates the activation by TGF-β of pro-fibrotic pathways. Recent studies show that mTORC2, a component of the mTOR pathway, mediates the activation of Akt. In this study we set out to determine if blocking mTORC2 with MLN0128, an active site dual mTOR inhibitor, which blocks both mTORC1 and mTORC2, inhibits lung fibrosis. We examined the effect of MLN0128 on TGF-β-mediated induction of stromal proteins in IPF lung fibroblasts; also, we looked at its effect on TGF-β-mediated epithelial injury using a Transwell co-culture system. Additionally, we assessed MLN0128 in the murine bleomycin lung model. We found that TGF-β induces the Rictor component of mTORC2 in IPF lung fibroblasts, which led to Akt activation, and that MLN0128 exhibited potent anti-fibrotic activity in vitro and in vivo. Also, we observed that Rictor induction is Akt-mediated. MLN0128 displays multiple anti-fibrotic and lung epithelial-protective activities; it (1) inhibited the expression of pro-fibrotic matrix-regulatory proteins in TGF-β-stimulated IPF fibroblasts; (2) inhibited fibrosis in a murine bleomycin lung model; and (3) protected lung epithelial cells from injury caused by TGF-β-stimulated IPF fibroblasts. Our findings support a role for mTORC2 in the pathogenesis of lung fibrosis and for the potential of active site mTOR inhibitors in the treatment of IPF and other fibrotic lung diseases.     

Dimethylfumarate Attenuates Renal Fibrosis via NF-E2-Related Factor 2-Mediated Inhibition of Transforming Growth Factor-β/Smad Signaling

TGF-β plays a key role in the development of renal fibrosis. Suppressing the TGF-β signaling pathway is a possible therapeutic approach for preventing this disease, and reports have suggested that Nrf2 protects against renal fibrosis by inhibiting TGF-β signaling. This study examines whether dimethylfumarate (DMF), which stimulates Nrf2, prevents renal fibrosis via the Nrf2-mediated suppression of TGF-β signaling. Results showed that DMF increased nuclear levels of Nrf2, and both DMF and adenovirus-mediated overexpression of Nrf2 (Ad-Nrf2) decreased PAI-1, alpha-smooth muscle actin (α-SMA), fibronectin and type 1 collagen expression in TGF-β-treated rat mesangial cells (RMCs) and renal fibroblast cells (NRK-49F). Additionally, DMF and Ad-Nrf2 repressed TGF-β-stimulated Smad3 activity by inhibiting Smad3 phosphorylation, which was restored by siRNA-mediated knockdown of Nrf2 expression. However, downregulation of the antioxidant response element (ARE)-driven Nrf2 target genes such as NQO1, HO-1 and glutathione S-transferase (GST) did not reverse the inhibitory effect of DMF on TGF-β-induced upregulation of profibrotic genes or extracellular matrix proteins, suggesting an ARE-independent anti-fibrotic activity of DMF. Finally, DMF suppressed unilateral ureteral obstruction (UUO)-induced renal fibrosis and α-SMA, fibronectin and type 1 collagen expression in the obstructed kidneys from UUO mice, along with increased and decreased expression of Nrf2 and phospho-Smad3, respectively. In summary, DMF attenuated renal fibrosis via the Nrf2-mediated inhibition of TGF-β/Smad3 signaling in an ARE-independent manner, suggesting that DMF could be used to treat renal fibrosis.     

Transforming growth factor-β and atherosclerosis: interwoven atherogenic and atheroprotective aspects

Age-related progression of cardiovascular disease is by far the largest health problem in the US and involves vascular damage, progressive vascular fibrosis and the accumulation of lipid-rich atherosclerotic lesions. Advanced lesions can restrict flow to key organs and can trigger occlusive thrombosis resulting in a stroke or myocardial infarction. Transforming growth factor-beta (TGF-β) is a major orchestrator of the fibroproliferative response to tissue damage. In the early stages of repair, TGF-β is released from platelets and activated from matrix reservoirs; it then stimulates the chemotaxis of repair cells, modulates immunity and inflammation and induces matrix production. At later stages, it negatively regulates fibrosis through its strong antiproliferative and apoptotic effects on fibrotic cells. In advanced lesions, TGF-β might be important in arterial calcification, commonly referred to as “hardening of the arteries”. Because TGF-β can signal through multiple pathways, namely the SMADs, a MAPK pathway and the Rho/ROCK pathways, selective defects in TGF-β signaling can disrupt otherwise coordinated pathways of tissue regeneration. TGF-β is known to control cell proliferation, cell migration, matrix synthesis, wound contraction, calcification and the immune response, all being major components of the atherosclerotic process. However, many of the effects of TGF-β are essential to normal tissue repair and thus, TGF-β is often thought to be “atheroprotective”. The present review attempts to parse systematically the known effects of TGF-β on both the major risk factors for atherosclerosis and to isolate the role of TGF-β in the many component pathways involved in atherogenesis.     

Targeting of Proteoglycan Synthesis Pathway: A New Strategy to Counteract Excessive Matrix Proteoglycan Deposition and Transforming Growth Factor-β1-Induced Fibrotic Phenotype in Lung Fibroblasts

Stimulation of proteoglycan (PG) synthesis and deposition plays an important role in the pathophysiology of fibrosis and is an early and dominant feature of pulmonary fibrosis. Transforming growth factor-β1 (TGF-β1) is a major cytokine associated with fibrosis that induces excessive synthesis of matrix proteins, particularly PGs. Owing to the importance of PGs in matrix assembly and in mediating cytokine and growth factor signaling, a strategy based on the inhibition of PG synthesis may prevent excessive matrix PG deposition and attenuates profibrotic effects of TGF-β1 in lung fibroblasts. Here, we showed that 4-MU4-deoxy-β-D-xylopyranoside, a competitive inhibitor of β4-galactosyltransferase7, inhibited PG synthesis and secretion in a dose-dependent manner by decreasing the level of both chondroitin/dermatan- and heparin-sulfate PG in primary lung fibroblasts. Importantly, 4-MU4-deoxy-xyloside was able to counteract TGF-β1-induced synthesis of PGs, activation of fibroblast proliferation and fibroblast-myofibroblast differentiation. Mechanistically, 4-MU4-deoxy-xyloside treatment inhibited TGF-β1-induced activation of canonical Smads2/3 signaling pathway in lung primary fibroblasts. The knockdown of β4-galactosyltransferase7 mimicked 4-MU4-deoxy-xyloside effects, indicating selective inhibition of β4-galactosyltransferase7 by this compound. Collectively, this study reveals the anti-fibrotic activity of 4-MU4-deoxy-xyloside and indicates that inhibition of PG synthesis represents a novel strategy for the treatment of lung fibrosis.     

Secreted Phosphoprotein 24 kD (Spp24) and Spp14 Affect TGF-β Induced Bone Formation Differently

Transforming growth factor-β (TGF-β) and bone morphogenetic proteins (BMPs) have opposing but complementary functions in directing bone growth, repair, and turnover. Both are found in the bone matrix. Proteins that bind to and affect the activity of these growth factors will determine the relative abundance of the growth factors and, therefore, regulate bone formation. Secreted phosphoprotein 24 kD (Spp24) is a bone matrix protein that has been demonstrated to bind to and affect the activity of BMPs. The arginine-rich carboxy terminus of Spp24 is proteolytically processed to produce three other predictable truncation products (Spp18.1, Spp16.0, and Spp14.5). In this work, we report that kinetic data obtained by surface plasmon resonance demonstrate that Spp24 and the three C-terminal truncation products all bind to TGF-β1 and TGF-β2 with a similar but somewhat less affinity than they bind BMP-2; that, as in the case of BMP-2, the full-length (FL) form of Spp24 binds TGF-β with greater affinity than do the truncation products; that FL-Spp24 inhibits TGF-β2 induced bone formation in vivo, but Spp14.5 does not; and that co-administration of FL-Spp24 or Spp14.5 with TGF-β2 in vivo is associated with a reduction in the amount of cartilage, relative to new bone, present at the site of injection. This finding is consistent with the observation that low-dose TGF-β administration in vivo is associated with greater bone formation than high-dose TGF-β administration, and suggests that one function of Spp24 and its truncation products is to down-regulate local TGF-β activity or availability during bone growth and development. The similarities and differences of the interactions between Spp24 proteins and TGF-β compared to the interaction of the Spp24 proteins and BMPs have significant implications with respect to the regulation of bone metabolism and with respect to engineering therapeutic proteins for skeletal disorders.     

Transforming Growth Factor-β (TGF-β) Expression Is Increased in the Subsynovial Connective Tissue in a Rabbit Model of Carpal Tunnel Syndrome

Carpal tunnel syndrome (CTS) is an idiopathic disease that results from increased fibrosis of the subsynovial connective tissue (SSCT). A recent study found overexpression of both transforming growth factor-β (TGF-β) and connective tissue growth factor (CTGF) in the SSCT of CTS patients. This study investigated TGF-β and CTGF expression in a rabbit model of CTS, in which SSCT fibrosis is induced by a surgical injury. Levels of TGF-β1 and CTGF at 6, 12, 24 weeks after injury were determined by immunohistochemistry A significant increase in TGF-β1 and a concomitant significant increase in CTGF were found at 6 weeks, in addition to higher cell density compared to normal (all p<0.05), Interestingly, CTGF expression was reduced at 12 and 24 weeks, suggesting that an initial insult results in a time limited response. We conclude that this rabbit model mimics the fibrosis found in human CTS, and may be useful to study pathogenetic mechanisms of CTS in vivo.     

The AMPK Agonist AICAR Inhibits TGF-β1 Induced Activation of Kidney Myofibroblasts

Activation of interstitial myofibroblasts and excessive production of extracellular matrix proteins are common pathways that contribute to chronic kidney disease. In a number of tissues, AMP-activated kinase (AMPK) activation has been shown to inhibit fibrosis. Here, we examined the inhibitory effect of the AMPK activator, 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR), on renal fibrosis in vivo and TGF-β1-induced renal fibroblasts activation in vitro. A unilateral ureteral obstruction (UUO) model was induced in male BALB/c mice. Mice with UUO were administered AICAR (500 mg/Kg/day) or saline intraperitoneally 1 day before UUO surgery and daily thereafter. Both kidneys were harvested 7 days after surgery for further analysis. For the in vitro studies, NRK-49F rat fibroblasts were pre-incubated with AICAR before TGF-β1 stimulation. The inhibitory effects of AICAR on signaling pathways down-stream of TGF-β1 were analyzed. In UUO model mice, administration of AICAR attenuated extracellular matrix protein deposition and the expression of α-smooth muscle actin (α-SMA), type I collagen and fibronectin. Pre-incubation of NRK-49F cells with AICAR inhibited TGF-β1-induced myofibroblast activation. Silencing of AMPKα1 by siRNA or by blocking AMPK activation with Compound C diminished the inhibitory effect of AICAR. Moreover, the inhibitory effects of AICAR on TGF-β1-mediated myofibroblast activation were associated with down-regulation of ERK 1/2 and STAT3. Our results suggest that AICAR reduces tubulointerstitial fibrosis in UUO mice and inhibits TGF-β1-induced kidney myofibroblast activation. AMPK activation by AICAR may have therapeutic potential for the treatment of renal tubulointerstitial fibrosis.     

Stimulation of chondrogenesis in limb bud mesoderm cells by recombinant human bone morphogenetic protein 2B (BMP-2B) and modulation by transforming growth factor β1 and β2

Bone morphogenetic protein 2B (BMP-2B) also called BMP-4 is one of a family of cartilage and bone-inductive proteins derived from bone matrix and belongs to the transforming growth factor β (TGF-β) superfamily. These bone-inductive proteins isolated from adult bone may be involved in bone repair. However, they may also play a role in cartilage and bone formation during embryonic development. To test whether BMP-2B influences cartilage formation by embryonic cells, recombinant human BMP-2B was applied to cultured limb bud mesoderm plated at three different densities. BMP-2B stimulated cartilage formation as assessed by Alcian blue staining and incorporation of radioactive sulfate into sulfated proteoglycans. Cells cultured at all three densities in the presence of 10 ng/ml BMP-2B formed a nearly continuous sheet of cartilage with abundant extracellular matrix and type II collagen. In addition, when cells were cultured in 0.5% serum in the presence of 10 ng/ml of BMP-2B for 5 days there was an increase in alkaline phosphatase as detected by histochemical and biochemical methods. Transforming growth factor β isoforms (TGF-β₁ and TGF-β₂) inhibited sulfate incorporation into proteoglycans in a dose-dependent manner. This inhibition by TGFβ was overcome by recombinant BMP-2B. This study demonstrates that recombinant BMP-2B stimulates cartilage formation by chick limb bud mesoderm in vitro and is further modulated by TGF-β isoforms.