Jessica M. Overstreet

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.

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.

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.

Loss of tumour suppressor PTEN expression in renal injury initiates SMAD3- and p53-dependent fibrotic responses

Deregulation of the tumour suppressor PTEN occurs in lung and skin fibrosis and diabetic and ischaemic renal injury. However, the potential role of PTEN and associated mechanisms in the progression of kidney fibrosis is unknown. Tubular and interstitial PTEN expression was dramatically decreased in several models of renal injury, including aristolochic acid nephropathy (AAN), streptozotocin (STZ)‐mediated injury and ureteral unilateral obstruction (UUO), correlating with Akt, p53 and SMAD3 activation and fibrosis. Stable silencing of PTEN in HK‐2 human tubular epithelial cells induced dedifferentiation and CTGF, PAI‐1, vimentin, α‐SMA and fibronectin expression, compared to HK‐2 cells expressing control shRNA. Furthermore, PTEN knockdown stimulated Akt, SMAD3 and p53Ser15 phosphorylation, with an accompanying decrease in population density and an increase in epithelial G1 cell cycle arrest. SMAD3 or p53 gene silencing or pharmacological blockade partially suppressed fibrotic gene expression and relieved growth inhibition orchestrated by deficiency or inhibition of PTEN. Similarly, shRNA suppression of PAI‐1 rescued the PTEN loss‐associated epithelial proliferative arrest. Moreover, TGFβ1‐initiated fibrotic gene expression is further enhanced by PTEN depletion. Combined TGFβ1 treatment and PTEN silencing potentiated epithelial cell death via p53‐dependent pathways. Thus, PTEN loss initiates tubular dysfunction via SMAD3‐ and p53‐mediated fibrotic gene induction, with accompanying PAI‐1‐dependent proliferative arrest, and cooperates with TGFβ1 to induce the expression of profibrotic genes and tubular apoptosis. Copyright

Tumor suppressor ataxia telangiectasia mutated functions downstream of TGF-β1 in orchestrating profibrotic responses

Effective therapy to prevent organ fibrosis, which is associated with more than half of all mortalities, remains elusive. Involvement of tumor suppressor ataxia telangiectasia mutated (ATM) in the TGF‐β1 pathway related to renal fibrosis is largely unknown. ATM activation (pATMSer1981) increased 4‐fold in the tubulointerstitial region of the unilateral ureteral obstruction‐injured kidney in mice correlating with SMAD3 and p53Ser15 phosphorylation and elevated levels of p22phox subunit of the NADPH oxidases (NOXs), and fibrotic markers, plasminogen activator inhibitor‐1 (PAI‐1), and fibronectin, when compared to contralateral (contra) or sham controls. In fact, ATM is rapidly phosphorylated at Ser1981 by TGF‐b1 stimulation. Stable silencing and pharmacologic inhibition of ATM ablated TGF‐β1‐induced p53 activation (>95%) and subsequent PAI‐1, fibronectin, connective tissue growth factor, and p21 expression in human kidney 2 (HK‐2) tubular epithelial cells and normal rat kidney‐49 fibroblasts (NRK‐49F). ATM or p53 depletion in HK‐2 cells, moreover, bypassed TGF‐β1‐mediated cytostasis evident in control short hairpin RNA‐expressing HK‐2 cells. Interestingly, stable silencing of NOX subunits, p22phox and p47phox, in HK‐2 cells blocked TGF‐β1‐induced pATMSer1981 (>90%) and target gene induction via p53‐dependent mechanisms. Furthermore, NRK‐49F fibroblast proliferation triggered by conditioned media from TGF‐β1‐stimulated, control vector‐transfected HK‐2 cells decreased (~50%) when exposed to conditioned media from ATM‐deficient, TGF‐β1‐treated HK‐2 cells. Thus, TGF‐β1 promotes NOX‐dependent ATM activation leading to p53‐mediated fibrotic gene reprogramming and growth arrest in HK‐2 cells. Furthermore, TGF‐β1/ATM‐initiated paracrine factor secretion by dysfunctional renal epithelium promotes interstitial fibroblast growth, suggesting a role of tubular ATM in mediating epithelial‐mesenchymal cross‐talk highlighting the translational benefit of targeting the NOX/ATM/p53 axis in renal fibrosis.—Overstreet, J. M., Samarakoon, R., Cardona‐Grau, D., Goldschmeding, R., Higgins, P. J. Tumor suppressor ataxia telangiectasia mutated functions downstream of TGF‐β1 in orchestrating profibrotic responses. FASEB J. 29, 1258‐1268 (2015). www.fasebj.org

Loss of expression of protein phosphatase magnesium-dependent 1A during kidney injury promotes fibrotic maladaptive repair.

Protein phosphatase magnesium‐dependent‐1A (PPM1A) dephosphorylates SMAD2/3, which suppresses TGF‐β signaling in keratinocytes and during Xenopus development; however, potential involvement of PPM1A in chronic kidney disease is unknown. PPM1A expression was dramatically decreased in the tubulointerstitium in obstructive and aristolochic acid nephropathy, which correlates with progression of fibrotic disease. Stable silencing of PPM1A in human kidney‐2 human renal epithelial cells increased SMAD3 phosphorylation, stimulated expression of fibrotic genes, induced dedifferentiation, and orchestrated epithelial cell‐cycle arrest via SMAD3‐mediated connective tissue growth factor and plasminogen activator inhibitor‐1 up‐regulation. PPM1A stable suppression in normal rat kidney‐49 renal fibroblasts, in contrast, promoted a SMAD3dependent connective tissue growth factor and plasminogen activator inhibitor‐1–induced proliferative response. Paracrine factors secreted by PPM1A‐depleted epithelial cells augmented fibroblast proliferation (>50%) compared with controls. PPM1A suppression in renal cells further enhanced TGF‐b1–induced SMAD3 phosphorylation and fibrotic gene expression, whereas PPM1A overexpression inhibited both responses. Moreover, phosphate tensin homolog on chromosome 10 depletion in human kidney‐2 cells resulted in loss of expression and decreased nuclear levels of PPM1A, which enhanced SMAD3‐mediated fibrotic gene induction and growth arrest that were reversed by ectopic PPM1A expression. Thus, phosphate tensin homolog on chromosome 10 is an upstream regulator of renal PPM1A deregulation. These findings establish PPM1A as a novel repressor of the SMAD3 pathway in renal fibrosis and as a new therapeutic target in patients with chronic kidney disease.—Samarakoon, R., Rehfuss, A., Khakoo, N.S., Falke, L. L., Dobberfuhl, A.D., Helo, S., Overstreet, J.M., Goldschmeding, R., Higgins, P. J. Loss of expression of protein phosphatase magnesium‐dependent 1A during kidney injury promotes fibrotic maladaptive repair. FASEBJ. 30, 3308–3320 (2016). www.fasebj.org

Deregulation of Hippo–TAZ pathway during renal injury confers a fibrotic maladaptive phenotype

Although yes‐associated protein (YAP) and transcriptional coactivator with PDZ‐binding motif (TAZ), nuclear transducers of the Hippo pathway, are mostly silent in adult organs, aberrant activation of YAP/TAZ promotes tumorigenesis and abnormal tissue repair. The extent of involvement of TAZ in chronic kidney disease (CKD) is unknown. In our study, increased TAZ nuclear accumulation and expression in the tubulointerstitium was readily evident in 3 models of renal injury including obstructive, aristolochic acid (AA), and diabetic nephropathy, correlating with fibrosis progression. Stable TAZ overexpression in human kidney (HK)‐2 epithelial cells promoted connective tissue growth factor (CTGF), fibronectin, vimentin, and p21 expression, epithelial dedifferentiation, and growth inhibition, in part, via Sma mothers against decapentaplegic homologue (SMAD)‐3‐dependent CTGF induction. CTGF secretion by TAZ‐overexpressing epithelium also triggered proliferative defects in nonengineered HK‐2 cells confirming a nonautonomous role of TAZ (via a paracrine mechanism) in orchestrating kidney epithelial cell‐cell communication. Renal tubular‐specific induction of TGF‐β1 in mice and TGF‐β1 stimulation of HK‐2 cells resulted in TAZ protein up‐regulation. TAZ stable silencing in HK‐2 cells abrogated TGF‐β1–induced expression of target genes without affecting SMAD3 phosphorylation, which is also crucial for fibrotic reprogramming. Thus, TAZ was activated in fibrosis through TGF‐β1–dependent mechanisms and sustained TAZ signaling promotes epithelial maladaptive repair. TAZ is also a novel non‐SMAD downstream effector of renal TGF‐β1 signaling, establishing TAZ as a new antifibrosis target for treatment of CKD.—Anorga, S., Overstreet, J. M., Falke, L. L., Tang, J., Goldschmeding, R. G., Higgins, P. J., Samarakoon, R. Deregulation of Hippo‐TAZ pathway during renal injury confers a fibrotic maladaptive phenotype. FASEB J. 32, 2644–2657 (2018). www.fasebj.org

Fatty acid receptor modulator PBI-4050 inhibits kidney fibrosis and improves glycemic control

Extensive kidney fibrosis occurs in several types of chronic kidney diseases. PBI-4050, a potentially novel first-in-class orally active low-molecular weight compound, has antifibrotic and antiinflammatory properties. We examined whether PBI-4050 affected the progression of diabetic nephropathy (DN) in a mouse model of accelerated type 2 diabetes and in a model of selective tubulointerstitial fibrosis. eNOS-/- db/db mice were treated with PBI-4050 from 8-20 weeks of age (early treatment) or from 16-24 weeks of age (late treatment). PBI-4050 treatment ameliorated the fasting hyperglycemia and abnormal glucose tolerance tests seen in vehicle-treated mice. In addition, PBI-4050 preserved (early treatment) or restored (late treatment) blood insulin levels and increased autophagy in islets. PBI-4050 treatment led to significant improvements in lifespan in the diabetic mice. Both early and late PBI-4050 treatment protected against progression of DN, as indicated by reduced histological glomerular injury and albuminuria, slow decline of glomerular filtration rate, and loss of podocytes. PBI-4050 inhibited kidney macrophage infiltration, oxidative stress, and TGF-β-mediated fibrotic signaling pathways, and it also protected against the development of tubulointerstitial fibrosis. To confirm a direct antiinflammatory/antifibrotic effect in the kidney, further studies with a nondiabetic model of EGFR-mediated proximal tubule activation confirmed that PBI-4050 dramatically decreased the development of the associated tubulointerstitial injury and macrophage infiltration. These studies suggest that PBI-4050 attenuates development of DN in type 2 diabetes through improvement of glycemic control and inhibition of renal TGF-β-mediated fibrotic pathways, in association with decreases in macrophage infiltration and oxidative stress.