Rohan Samarakoon

TGF-β signaling in tissue fibrosis: redox controls, target genes and therapeutic opportunities.

During development of TGF-β1-initiated fibroproliferative disorders, NADPH oxidases (NOX family members) generate reactive oxygen species (ROS) resulting in downstream transcription of a subset genes encoding matrix structural elements and profibrotic factors. Prominent among the repertoire of disease-implicated genes is the TGF-β1 target gene encoding the potent profibrotic matricellular protein plasminogen activator inhibitor-1 (PAI-1 or SERPINE1). PAI-1 is the major physiologic inhibitor of the plasmin-based pericellular cascade and a causative factor in the development of vascular thrombotic and fibroproliferative disorders. ROS generation in response to TGF-β1 stimulation is rapid and precedes PAI-1 induction; engagement of non-SMAD (e.g., EGFR, Src kinase, MAP kinases, p53) and SMAD2/3 pathways are both required for PAI-1 expression and are ROS-dependent. Recent findings suggest a novel role for p53 in TGF-β1-induced PAI-1 transcription that involves ROS generation and p53/SMAD interactions. Targeting ROS and ROS-activated cellular events is likely to have therapeutic implications in the management of fibrotic disorders, particularly in the context of prolonged TGF-β1 signaling.

PAI-1: An Integrator of Cell Signaling and Migration

Cellular migration, over simple surfaces or through complex stromal barriers, requires coordination between detachment/re-adhesion cycles, involving structural components of the extracellular matrix and their surface-binding elements (integrins), and the precise regulation of the pericellular proteolytic microenvironment. It is now apparent that several proteases and protease inhibitors, most notably urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1), also interact with several cell surface receptors transducing intracellular signals that significantly affect both motile and proliferative programs. These events appear distinct from the original function of uPA/PAI-1 as modulators of the plasmin-based proteolytic cascade. The multifaceted interactions of PAI-1 with specific matrix components (i.e., vitronectin), the low-density lipoprotein receptor-related protein-1 (LRP1), and the uPA/uPA receptor complex have dramatic consequences on the migratory phenotype and may underlie the pathophysiologic sequalae of PAI-1 deficiency and overexpression. This paper focuses on the increasingly intricate role of PAI-1 as a major mechanistic determinant of the cellular migratory phenotype.

TGF-β1 → SMAD/p53/USF2 → PAI-1 transcriptional axis in ureteral obstruction-induced renal fibrosis

Chronic kidney disease constitutes an increasing medical burden affecting 26 million people in the United States alone. Diabetes, hypertension, ischemia, acute injury, and urological obstruction contribute to renal fibrosis, a common pathological hallmark of chronic kidney disease. Regardless of etiology, elevated TGF-β1 levels are causatively linked to the activation of profibrotic signaling pathways initiated by angiotensin, glucose, and oxidative stress. Unilateral ureteral obstruction (UUO) is a useful and accessible model to identify mechanisms underlying the progression of renal fibrosis. Plasminogen activator inhibitor-1 (PAI-1), a major effector and downstream target of TGF-β1 in the progression of several clinically important fibrotic disorders, is highly up-regulated in UUO and causatively linked to disease severity. SMAD and non-SMAD pathways (pp60c-src, epidermal growth factor receptor [EGFR], mitogen-activated protein kinase, p53) are required for PAI-1 induction by TGF-β1. SMAD2/3, pp60c-src, EGFR, and p53 activation are each increased in the obstructed kidney. This review summarizes the molecular basis and translational significance of TGF-β1-stimulated PAI-1 expression in the progression of kidney disease induced by ureteral obstruction. Mechanisms discussed here appear to be operative in other renal fibrotic disorders and are relevant to the global issue of tissue fibrosis, regardless of organ site.

Integration of non-SMAD and SMAD signaling in TGF-β1-induced plasminogen activator inhibitor type-1 gene expression in vascular smooth muscle cells

Overexpression of plasminogen activator inhibitor-1 (SERPINE1, PAI-1), the major physiological inhibitor of pericellular plasmin generation, is a significant causative factor in the progression of vascular disorders (e.g. arteriosclerosis, thrombosis, perivascular fibrosis) as well as a biomarker and a predictor of cardiovascular-disease associated mortality. PAI-1 is a temporal/spatial regulator of pericellular proteolysis and ECM accumulation impacting, thereby, vascular remodeling, smooth muscle cell migration, proliferation and apoptosis. Within the specific context of TGF-beta1-initiated vascular fibrosis and neointima formation, PAI-1 is a member of the most prominently expressed subset of TGF-beta1-induced transcripts. Recent findings implicate EGFR/pp60c-src-->MEK/ERK1/2 and Rho/ROCK-->SMAD2/3 signaling in TGF-beta1-stimulated PAI-1 expression in vascular smooth muscle cells. The EGFR is a direct upstream regulator of MEK/ERK1/2 while Rho/ROCK modulate both the duration of SMAD2/3 phosphorylation and nuclear accumulation. E-box motifs (CACGTG) in the PE1/PE2 promoter regions of the human PAI-1 gene, moreover, are platforms for a MAP kinase-directed USF subtype switch (USF-1-->USF-2) in response to growth factor addition suggesting that the EGFR-->MEK/ERK axis impacts PAI-1 expression, at least partly, through USF-dependent transcriptional controls. This paper reviews recent data suggesting the essential cooperativity among the EGFR-->MAP kinase cascade, the Rho/ROCK pathway and SMADs in TGF-beta1-initiated PAI-1 expression. The continued clarification of mechanistic controls on PAI-1 transcription may lead to new targeted therapies and clinically-relevant options for the treatment of vascular diseases in which PAI-1 dysregulation is a major underlying pathogenic feature.

Induction of renal fibrotic genes by TGF-β1 requires EGFR activation, p53 and reactive oxygen species

While transforming growth factor-β (TGF-β1)-induced SMAD2/3 signaling is a critical event in the progression of chronic kidney disease, the role of non-SMAD mechanisms in the orchestration of fibrotic gene changes remains largely unexplored. TGF-β1/SMAD3 pathway activation in renal fibrosis (induced by ureteral ligation) correlated with epidermal growth factor receptor(Y845) (EGFR(Y845)) and p53(Ser15) phosphorylation and induction of disease causative target genes plasminogen activator inhibitor-1 (PAI-1) and connective tissue growth factor (CTGF) prompting an investigation of the mechanistic involvement of EGFR and tumor suppressor p53 in profibrotic signaling. TGF-β1, PAI-1, CTGF, p53 and EGFR were co-expressed in the obstructed kidney localizing predominantly to the tubular and interstitial compartments. Indeed, TGF-β1 activated EGFR and p53 as well as SMAD2/3. Genetic deficiency of either EGFR or p53 or functional blockade with AG1478 or Pifithrin-α, respectively, effectively inhibited PAI-1and CTGF induction and morphological transformation of renal fibroblasts as did SMAD3 knockdown or pretreatment with the SMAD3 inhibitor SIS3. Reactive oxygen species (ROS)-dependent mechanisms initiated by TGF-β1 were critical for EGFR(Y845) and p53(Ser15) phosphorylation and target gene expression. The p22(Phox) subunit of NADPH oxidase was also elevated in the fibrotic kidney with an expression pattern similar to p53 and EGFR. EGF stimulation alone initiated, albeit delayed, c-terminal SMAD3 phosphorylation (that required the TGF-β1 receptor) and rapid ERK2 activation both of which are necessary for PAI-1 and CTGF induction in renal fibroblasts. These data highlight the extensive cross-talk among SMAD2/3, EGFR and p53 pathways essential for expression of TGF-β1-induced fibrotic target genes.

TGF-β1-induced plasminogen activator inhibitor-1 expression in vascular smooth muscle cells requires pp60c-src/EGFRY845 and Rho/ROCK signaling

TGF-beta1 and its target gene encoding plasminogen activator inhibitor-1 (PAI-1) are major causative factors in the pathology of tissue fibrosis and vascular disease. The increasing complexity of TGF-beta1 action in the cardiovascular system requires analysis of specific TGF-beta1-initiated signaling events that impact PAI-1 transcriptional regulation in a physiologically-relevant cell system. TGF-beta1-induced PAI-1 expression in both primary cultures and in an established line (R22) of vascular smooth muscle cells (VSMC) was completely blocked by inhibition of epidermal growth factor receptor (EGFR) activity or adenoviral delivery of a kinase-dead EGFR(K721A) construct. TGF-beta1-stimulated PAI-1 expression, moreover, was preceded by EGFR phosphorylation on Y845 (a src kinase target residue) and required pp60(c-src) activity. Infection of VSMC with an adenovirus encoding the EGFR(Y845F) mutant or transfection with a dominant-negative pp60(c-src) (DN-Src) expression vector effectively decreased TGF-beta1-stimulated, but not PDGF-induced, PAI-1 expression implicating the pp60(c-src) phosphorylation site EGFR(Y845) in the inductive response. Consistent with these findings, TGF-beta1 failed to induce PAI-1 synthesis in src kinase-deficient (SYF(-/-/-)) fibroblasts and reexpression of a wild-type pp60(c-src) construct in SYF(-/-/-) cells rescued the PAI-1 response to TGF-beta1. TGF-beta1-induced EGFR activation, but not SMAD2 activation, moreover, was virtually undetectable in SYK(-/-/-) fibroblasts in comparison to wild type (SYK(+/+/+)) counterparts, confirming an upstream signaling role of src family kinases in EGFR(Y845) phosphorylation. Genetic EGFR deficiency or infection of VSMCs with EGFR(K721A) virtually ablated TGF-beta1-stimulated ERK1/2 activation as well as PAI-1 expression but not SMAD2 phosphorylation. Transient transfection of a dominant-negative RhoA (DN-RhoA) expression construct or pretreatment of VSMC with C3 transferase (a Rho inhibitor) or Y-27632 (an inhibitor of p160ROCK, a downstream effector of Rho) also dramatically attenuated the TGF-beta1-initiated PAI-1 inductive response. In contrast to EGFR pathway blockade, interference with Rho/ROCK signaling effectively inhibited TGF-betaR-mediated SMAD2 phosphorylation and nuclear accumulation. TGF-beta1-stimulated SMAD2 activation, moreover, was not sufficient to induce PAI-1 expression in the absence of EGFR signaling both in VSMC and mouse embryonic fibroblasts. Thus, two distinct pathways involving the EGFR/pp60(c-src)/MEK-ERK pathway and Rho/ROCK-dependent SMAD2 activation are required for TGF-beta1-induced PAI-1 expression in VSMC. The identification of such novel interactions between two TGF-beta1-activated signaling networks that specifically impact PAI-1 transcription in VSMC may provide therapeutically-relevant targets to manage the pathophysiology of PAI-1-associated cardiovascular/fibrotic diseases.

Plasminogen activator inhibitor type-1 gene expression and induced migration in TGF-β1-stimulated smooth muscle cells is pp60c-src/MEK-dependent

Transforming growth factor‐β1 (TGF‐β1) stimulates expression of plasminogen activator inhibitor type‐1 (PAI‐1), a serine protease inhibitor (SERPIN) important in the control of stromal barrier proteolysis and cell‐to‐matrix adhesion. Pharmacologic agents that target MEK (PD98059, U0126) or src family (PP1) kinases attenuated TGF‐β1‐dependent PAI‐1 transcription in R22 aortic smooth muscle cells. Pretreatment with PP1 at concentrations that inhibited TGF‐β1‐dependent PAI‐1 expression also blocked ERK1/2 activation/nuclear accumulation suggesting that the required src kinase activity is upstream of ERK1/2 in the TGF‐β1‐initiated signaling cascade. The IC50 of the PP1‐sensitive kinase, furthermore, specifically implied involvement of pp60c‐src in PAI‐1 induction. Indeed, addition of TGF‐β1 to quiescent R22 cells resulted in a 3‐fold increase in pp60c‐src autophosphorylation and kinase activity. Transfection of a dominant‐negative pp60c‐src construct, moreover, reduced TGF‐β1‐induced PAI‐1 expression levels to that of unstimulated controls or PP1‐pretreated cells. A ≥170 kDa protein that co‐immunoprecipitated with TGF‐β1‐activated pp60c‐src was also phosphorylated transiently in response to TGF‐β1. TGF‐β1 is known to transactivate the 170 kDa EGF receptor (EGFR) by autocrine HB‐EGF or TGF‐α mechanisms suggesting involvement of EGFR activation in certain TGF‐β1‐initiated responses. Incubation of quiescent R22 cells with the EGFR‐specific inhibitor AG1478 prior to growth factor (EGF or TGF‐β1) addition effectively blocked EGFR activation as determined by direct visualization of receptor internalization. AG1478 suppressed (in a dose‐dependent fashion) EGF‐induced PAI‐1 protein levels and, at a final concentration of 2.5 μM, virtually eliminated EGF‐dependent PAI‐1 synthesis. More importantly, AG1478 similarly repressed inducible PAI‐1 levels in TGF‐β1‐stimulated R22 cultures. PP1, PD98059, and U0126 also inhibited TGF‐β1‐dependent cell motility at concentrations that significantly attenuated PAI‐1 expression. Consistent with the AG1478‐associated reductions in EGF‐ and TGF‐β1‐stimulated PAI‐1 expression, pretreatment of R22 cell cultures with AG1478 effectively suppressed growth factor‐stimulated cell motility. These data indicate that two major phenotypic characteristics of TGF‐β1‐exposure (i.e., transcription of specific target genes [e.g., PAI‐1], increased cell motility) are linked in the R22 vascular smooth muscle cell system, require pp60c‐src kinase activity and MEK signaling and involve activation of an AG1478‐sensitive (likely EGFR‐dependent) pathway.

Differential requirement for MEK/ERK and SMAD signaling in PAI-1 and CTGF expression in response to microtubule disruption

Colchicine and nocodazole, both established microtubule disruptors, are useful tools to investigate cytoskeletal-dependent signaling cascades and the associated downstream transcriptional targets. Since cytoskeletal events impact pathophysiologic consequences in the vascular system, the signaling requirements underlying colchicine-stimulated expression of PAI-1 and CTGF, two prominent cell deformation-sensitive fibrosis-initiating proteins, were evaluated in vascular smooth muscle cells. Microtubule disruption rapidly induced EGFR transactivation (at the src kinase-sensitive EGFR(Y845) site) in a ROS-dependent manner. Genetic deficiency of EGFR, inhibition of EGFR signaling with AG1478 or introduction of a kinase-deficient EGFR construct effectively blocked colchicine-stimulated PAI-1 and CTGF expression. MEK/ERK involvement downstream of ROS generation was critical for PAI-1, but not CTGF, expression following cytoskeletal perturbation suggesting bifurcation of signaling pathways downstream of EGFR activation. Colchicine also stimulated SMAD2/3 phosphorylation by a Rho/ROCK-dependent mechanism independent of TGF-beta1 release or receptor activity. Rho/ROCK signaling initiated by tubulin network collapse was required for both CTGF and PAI-1 induction. Colchicine-initiated SMAD3 phosphorylation, however, was essential for PAI-1, but not CTGF, expression further highlighting divergence of signaling events downstream of Rho/ROCK that mediate microtubule deformation-associated changes in profibrotic gene transcription.

Redox-induced Src kinase and caveolin-1 signaling in TGF-β1-initiated SMAD2/3 activation and PAI-1 expression.

Background Plasminogen activator inhibitor-1 (PAI-1), a major regulator of the plasmin-based pericellular proteolytic cascade, is significantly increased in human arterial plaques contributing to vessel fibrosis, arteriosclerosis and thrombosis, particularly in the context of elevated tissue TGF-β1. Identification of molecular events underlying to PAI-1 induction in response to TGF-β1 may yield novel targets for the therapy of cardiovascular disease. Principal Findings Reactive oxygen species are generated within 5 minutes after addition of TGF-β1 to quiescent vascular smooth muscle cells (VSMCs) resulting in pp60c-src activation and PAI-1 expression. TGF-β1-stimulated Src kinase signaling sustained the duration (but not the initiation) of SMAD3 phosphorylation in VSMC by reducing the levels of PPM1A, a recently identified C-terminal SMAD2/3 phosphatase, thereby maintaining SMAD2/3 in an active state with retention of PAI-1 transcription. The markedly increased PPM1A levels in triple Src kinase (c-Src, Yes, Fyn)-null fibroblasts are consistent with reductions in both SMAD3 phosphorylation and PAI-1 expression in response to TGF-β1 compared to wild-type cells. Activation of the Rho-ROCK pathway was mediated by Src kinases and required for PAI-1 induction in TGF-β1-stimulated VSMCs. Inhibition of Rho-ROCK signaling blocked the TGF-β1-mediated decrease in nuclear PPM1A content and effectively attenuated PAI-1 expression. TGF-β1-induced PAI-1 expression was undetectable in caveolin-1-null cells, correlating with the reduced Rho-GTP loading and SMAD2/3 phosphorylation evident in TGF-β1-treated caveolin-1-deficient cells relative to their wild-type counterparts. Src kinases, moreover, were critical upstream effectors of caveolin-1Y14 phosphoryation and initiation of downstream signaling. Conclusions TGF-β1-initiated Src-dependent caveolin-1Y14 phosphorylation is a critical event in Rho-ROCK-mediated suppression of nuclear PPM1A levels maintaining, thereby, SMAD2/3-dependent transcription of the PAI-1 gene.

Redox control of p53 in the transcriptional regulation of TGF-β1 target genes through SMAD cooperativity

Transforming growth factor-β1 (TGF-β1) regulates the tissue response to injury and is the principal driver of excessive scarring leading to fibrosis and eventual organ failure. The TGF-β1 effectors SMAD3 and p53 are major contributors to disease progression. While SMAD3 is an established pro-fibrotic factor, the role of p53 in the TGF-β1-induced fibrotic program is not clear. p53 gene silencing, genetic ablation/subsequent rescue, and pharmacological inhibition confirmed that p53 was required for expression of plasminogen activator inhibitor-1 (PAI-1), a major TGF-β1 target gene and a key causative element in fibrotic disorders. TGF-β1 regulated p53 activity by stimulating p53(Ser15 and 9) phosphorylation and acetylation, promoting interactions with activated SMADs and subsequent binding of p53/SMAD3 to the PAI-1 promoter in HK-2 human renal tubular epithelial cells and HaCaT human keratinocytes. Immunohistochemistry revealed prominent co-induction of SMAD3, p53 and PAI-1 in the tubular epithelium of the obstructed kidney consistent with a potential in vivo role for p53 and SMADs in TGF-β1-driven renal fibrosis. TGF-β1-initiated phosphorylation of p53(Ser15) and up-regulation of expression of several pro-fibrotic genes, moreover, was dependent on the rapid generation of reactive oxygen species (ROS). shRNA silencing of the p22(Phox) subunit of NADP(H) oxidases in HK-2 cells partially attenuated (over 50%) p53(Ser15) phosphorylation and PAI-1 induction. These studies highlight the role of free radicals in p53 activation and subsequent pro-fibrotic reprogramming by TGF-β1 via the SMAD3-p53 transcriptional axis. Present findings provide a rationale for therapeutic targeting of SMAD3-p53 in aberrant TGF-β1 signaling associated with renal fibrosis.