Tumor suppressor ataxia telangiectasia mutated ... - The FASEB Journal

The FASEB Journal article fj.14-262527. Published online December 5, 2014.

The FASEB Journal • Research Communication

Tumor suppressor ataxia telangiectasia mutated functions downstream of TGF-b1 in orchestrating profibrotic responses Jessica M. Overstreet,* Rohan Samarakoon,*,1 Diana Cardona-Grau,† Roel Goldschmeding,‡ and Paul J. Higgins*,1 *Center for Cell Biology and Cancer Research and †Division of Urology, Albany Medical College, Albany, New York, USA; and ‡Department of Pathology, University Medical Center, Utrecht, The Netherlands 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-b1 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 TGFb1 stimulation. Stable silencing and pharmacologic inhibition of ATM ablated TGF-b1-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-b1-mediated cytostasis evident in control short hairpin RNAexpressing HK-2 cells. Interestingly, stable silencing of NOX subunits, p22phox and p47phox, in HK-2 cells blocked TGF-b1-induced pATMSer1981 (>90%) and target gene induction via p53-dependent mechanisms. Furthermore, NRK-49F fibroblast proliferation triggered by conditioned media from TGF-b1-stimulated, control vector-transfected HK-2 cells decreased (∼50%) when exposed to conditioned media from ATM-deficient, TGF-b1-treated HK-2 cells. Thus, TGF-b1 promotes NOX-dependent ATM activation leading to p53-mediated fibrotic gene reprogramming ABSTRACT

Abbreviations: AKI, acute kidney injury; ALK5, TGF-b type I receptor kinase; a-SMA, a-smooth muscle actin; ATM, ataxia telangiectasia mutated; CKD, chronic kidney disease; con shRNA, control short hairpin RNA; contra, contralateral; COX2, cyclooxygenase-2; CTGF, connective tissue growth factor; ECM, extracellular matrix; EGFR, epidermal growth factor receptor; FBS, fetal bovine serum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; H2O2, hydrogen peroxide; HK-2, human kidney 2; NOX, NADPH oxidase; NRK-49F, normal rat kidney-49fibroblast; PAI-1, plasminogen activator inhibitor-1; PCNA, proliferating cell nuclear antigen; pSMAD3, phosphoSMAD3; ROS, reactive oxygen species; shRNA, short hairpin RNA; UUO, unilateral ureteral obstruction

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and growth arrest in HK-2 cells. Furthermore, TGFb1/ATM-initiated paracrine factor secretion by dysfunctional renal epithelium promotes interstitial fibroblast growth, suggesting a role of tubular ATM in mediating epithelial-mesenchymal crosstalk 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-b1 in orchestrating profibrotic responses. FASEB J. 29, 000–000 (2015). www.fasebj.org Key Words: kidney fibrosis • p53 • ROS • NOX FIBROTIC DISORDERS OF the renal, pulmonary, cardiac, and hepatic systems are associated with significant morbidity and mortality (1). Effective therapy to prevent or attenuate the progression to organ failure, however, remains a major clinical problem (2). Regardless of the involved tissue and primary causative insult (e.g., ischemia, toxicants, and diabetes), persistent elevation of TGF-b1 is a major contributor to disease progression (1, 3–6). TGF-b1 is the principal driver of renal fibrosis, which is characterized by tubular cell injury/apoptosis, infiltration of inflammatory cells, interstitial fibroblast proliferation, and excess extracellular matrix (ECM) synthesis, leading to the loss of organ function and evolution to chronic kidney disease (CKD) (3, 7). TGF-b1 activates the TGF-b type I receptor kinase (ALK-5) receptor canonical SMAD2/SMAD3 pathway as well as noncanonical signaling [e.g., via epidermal growth factor receptor (EGFR) and p53], collectively driving fibrotic gene transcription (7–9). Such signal integration may have pathophysiologic consequences. Indeed, TGF-b1 stimulates the activation and assembly of p53-SMAD3 transcriptional complexes required for expression of the renal fibrotic genes, plasminogen activator inhibitor-1 (PAI-1), connective tissue growth factor (CTGF), and TGF-b1 (10). These findings are consistent with simultaneous activation of SMAD3 and p53 and their downstream 1 Correspondence: Center for Cell Biology & Cancer Research, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208, USA. E-mail: [email protected] (R.S.); [email protected] (P.J.H.) doi: 10.1096/fj.14-262527

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target genes, PAI-1 and CTGF, in the tubulointerstitial region of the fibrotic kidney in response to unilateral ureteral obstruction (UUO) in rats (10). Furthermore, tubularspecific ablation of p53 in mice, or Pifithrin-a-mediated inactivation of p53, prevented tubular cell cycle arrest, fibrotic factor secretion, and the transition from acute kidney injury (AKI) to CKD, further suggestive of the profibrotic role of p53 in disease progression (11, 12). However, upstream control of p53 activation by TGF-b1 has not been identified. One potential regulator of p53 function in the context of tissue injury is tumor suppressor ataxia telangiectasia mutated (ATM). ATM mutation, originally described in patients with ataxia telangiectasia, leads to genomic instability causing a predisposition to cancer (13). ATM is a Ser/ Thr kinase that mediates the DNA damage repair response (14). Emerging evidence highlights ATM involvement in noncancer pathologies, including AKI (11) and bleomycin-induced lung damage (15). In fact, ATM is activated (phosphorylation at Ser1981) by stress signals such as hydrogen peroxide (H2O2) and bleomycin (16). Furthermore, in ischemia reperfusion renal injury and aristolochic acid nephropathy, ATM activation mediates tubular epithelial G2/M cell cycle arrest and subsequent induction of fibrotic factors (e.g., TGF-b and CTGF) by the injured epithelium to promote fibroblast growth and disease progression (11). The involvement of ATM in TGF-b signaling in the context of renal fibrotic injury has not been evaluated. This study describes concurrent ATM, p53, and TGFb1/SMAD3 activation in the obstructed kidney. TGF-b1 also promotes ATMSer1981 phosphorylation in human kidney 2 (HK-2) human renal tubular epithelial cells. Stable gene silencing and pharmacologic inhibition of ATM eliminated p53-dependent expression of profibrotic proteins and tubular growth inhibition in response to TGF-b1. NADPH oxidases (NOXs) are required for ATM activation downstream of TGF-b1, highlighting the translational relevance of the NOX/ATM/p53 axis in suppressing aberrant tissue repair evident in the fibrotic kidney. MATERIALS AND METHODS Cell culture HK-2 human proximal tubular epithelial cells and normal rat kidney-49fibroblast (NRK-49F) rat renal fibroblasts were grown in DMEM supplemented with 10% fetal bovine serum (FBS). Cells with stable lentiviral infection were cultured in DMEM/10% FBS/5 mg/ml puromycin. HK-2 and NRK-49F cells were serum deprived for 1 d prior to 2 ng/ml human recombinant TGF-b1 (R&D Systems, Minneapolis, MN, USA) treatment. The ATM pharmacologic inhibitor KU-55933 (Tocris Bioscience, Bristol, United Kingdom) was pretreated for 1 h, whereas pretreatment with Nutlin-3a (Cayman Chemicals, Ann Arbor, MI, USA) occurred 3 d prior to TGF-b1 addition. Stable genetic silencing using lentivirus infection Semiconfluent (50%) HK-2 or NRK-49F cells plated in 6-well plates were incubated with Polybrene at a final concentration of 5 mg/ml prior to infection with control short hairpin RNA (con shRNA) or ATM, p53, p47phox, or p22phox shRNA lentiviral particles (2–5 ml; all from Santa Cruz Biotechnology, Santa Cruz, CA,

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USA) in Polybrene for 24 h. Cells were recovered in fresh 10% FBS/DMEM for 24 h after which stably expressing clones were selected using complete media supplemented with puromycin dihydrochloride (5 mg/ml) for 3 d. Immunoblotting of ATM, p53, p47phox, or p22phox confirmed knockdown efficiency. Immunoblotting Cells were lysed in Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) containing 5% 2-ME. Extracted protein (;30 mg) was separated on a 10% SDS-4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid gel and transferred to nitrocellulose. Primary antibodies included rabbit anti-PAI-1 [1:3000 (10)]; mouse antiphospho-ATMSer1981 (1:1000; Abcam Incorporated, Cambridge, MA, USA); rabbit anti-phospho-SMAD3 (pSMAD3) (1:1000; Abcam); rabbit anti-a-smooth muscle actin (a-SMA) (1:3000; Sigma-Aldrich, St. Louis, MO, USA); goat anti-CTGF (1:1000; Santa Cruz Biotechnology); rabbit anti-GAPDH (glyceraldehyde 3phosphate dehydrogenase) (1:5000; Santa Cruz Biotechnology); rabbit anti-b-actin (1:5000; Santa Cruz Biotechnology); mouse anti-fibronectin (1:10,000; BD Biosciences, Franklin Lakes, NJ, USA); rabbit anti-p21 (1:1000; Santa Cruz Biotechnology); rabbit anti-phospho-p53Ser15 (1:1000; Santa Cruz Biotechnology); rabbit anti-p53 (1:1000; Santa Cruz Biotechnology); rabbit antip22phox (1:1000; Santa Cruz Biotechnology); rabbit antip47phox (1:1000; Abcam); rabbit anti-SMAD3 (1:1000; Abcam); mouse anti-ATM (1:1000; Santa Cruz Biotechnology); and rabbit anti-PCNA (proliferating cell nuclear antigen) (1:1000; Santa Cruz Biotechnology). Blots were quantified using densitometry. Growth arrest studies in HK-2 cells with stable ATM depletion Sparsely confluent (30%) HK-2 cells with con shRNA or ATM shRNA expression were serum starved for 1 d and treated with TGF-b1 for 24 h prior to the addition of 1% FBS/DMEM for 3 or 7 d to stimulate proliferation. Phase-contrast images as well as automated cell count analysis using the Sceptor 2.0 Handheld Automated Cell Counter (EMD Millipore, Billerica, MA, USA; according to the manufacturer’s recommendations) evaluated total cell number. Western blot analysis of the extracted lysates determined gene expression patterns. Similar experiments were performed in p53 shRNA and con shRNA stably expressing HK-2 cells. For evaluation of the effect of Nutlin-3A (a p53 activator) in ATM-depleted HK-2 cell proliferation, subconfluent con shRNA and ATM shRNA-expressing HK-2 cells were serum starved for 1 d, pretreated with Nutlin-3A, or remained untreated for 3 d prior to adding TGF-b1 and 1% serum to stimulate growth for 3 d. Assessments on the role of tubular ATM in epithelial communication with fibroblasts Identically seeded and serum-starved (1 d) HK-2 cells with con or ATM shRNA expression were stimulated with TGF-b1 for 24 h prior to adding 1% serum for 3 d. HK-2 cells were washed with PBS prior to adding fresh 0.5% FBS/DMEM and maintained at 37°C for an additional 48 h. Conditioned media isolated from HK-2 epithelial cells were directly added to similarly confluent NRK-49F fibroblasts for 2 d. Relative NRK-49F fibroblast proliferation was measured using the Sceptor 2.0 Handheld Automated Cell Counter (according to the manufacturer’s recommendations) as described above. UUO Mice were fed a standard pellet diet with continuous access to water and housed in standard cages on a 12 h light-dark cycle

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OVERSTREET ET AL.

Figure 1. Increased activation of ATM in the fibrotic kidney following UUO in mice. A) Obstructed (UUO) and contra control kidneys were removed from mice following 14 d of experimental UUO. Paraffin kidney sections were stained with phosphoATMSer1981 (pATMSer1981). Sham kidneys derived from mice receiving a surgical incision without kidney manipulation served as an additional control. Scale bars, 60 mm. B) Histogram depicts the expression level of pATMSer1981 quantified from 56 adjacent areas (see red inset; 7 3 8 grid, 100 mm per square) of the contra and UUO mouse kidneys. Statistical significance was determined using the Student’s t test; **P , 0.01. C) Western blot analysis for pATMSer1981, total ATM, pSMAD3, total SMAD3, p-p53Ser15, p22phox expression, and profibrotic markers (a-SMA and PAI-1) in 3 individual kidney lysates from Contra and UUOinjured mice following 14 d of ureteral ligation. GAPDH is a loading control. D–I) Western blot analysis (C) quantified in graph by calculating the mean 6 SD for protein levels in obstructed compared to Contra kidney in 3 individual mice (n = 3). J) Immunoblotting of pATMSer1981, p-p53Ser15, and p22phox expression in kidney lysates from Contra and obstructed (UUO) mouse kidneys after 7 d of ureteral ligation. K–M) Graphs depict the mean 6 SD of protein expression patterns from ( J). *P , 0.05; **P , 0.01; ***P , 0.001. with a constant environmental temperature. Surgery was performed in accordance with the guidelines of the Experimental Animal Ethics Committee of the University of Utrecht. In brief, mice anesthetized using isoflurane-oxygen inhalation under aseptic conditions received a small incision in the flank allowing the permanent ligation of the left ureter prior to suturing the wound. On d 7 or 14 postsurgery following anesthetization by intraperitoneal injection of ketamine-xylazine-atropine, the obstructed (UUO) and contra kidneys were isolated for

ATM MEDIATES PROFIBROTIC TGF-b1 SIGNALING

experiments. Sham kidneys were obtained from animals undergoing the surgical procedure but without ureteral ligation. Immunohistochemistry Kidney sections were deparaffinized in xylene and rehydrated in ethanol followed by antigen retrieval by heating in sodium citrate buffer. Sections were incubated in BLOXALL (Vector

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Figure 2. Activation and requirement of ATM in TGF-b-induced gene expression in renal tubular epithelial cells and fibroblasts. A) Immunoblotting for pATMSer1981, phospho-p53Ser15 (p-p53Ser15), and pSMAD3 provided an assessment of activation kinetics following a time course of TGF-b1 stimulation (0–120 min). Histograms in (B) and (C) measure pATMSer1981 and p-p53Ser15, respectively, determined by the mean 6 SD. *P , 0.05, **P , 0.01, and ***P , 0.001 vs. untreated controls. D–H) Con shRNAexpressing or ATM shRNA-expressing HK-2 renal epithelial cells remained untreated or treated with TGF-b1 for 6 (E) or 24 h (G) and immunoblotted for PAI-1. Knockdown efficiency of ATM was confirmed in HK-2 cells (D). F and H) Plotted data of (E) and (G) represent the mean 6 SD for TGF-b1-induced PAI-1 expression arbitrarily setting levels in untreated cells with con shRNA as 1. **P , 0.01; ***P , 0.001. I–L) Western blot analysis of TGF-b1-dependent profibrotic gene expression in ATM-depleted cells compared to mock controls. TGF-b1-mediated SMAD3 activation is evident in both con shRNA and ATM shRNA-expressing cells. Graphs depict the mean 6 SD of fibronectin (I, J), p21 (I, K), and COX-2 (I, L) in ATM knockdown cells compared to their control counterparts where untreated, con shRNA was arbitrarily set as 1. *P , 0.05; **P , 0.01. M, N) Expression of PAI-1 protein following stable generation of NRK-49F rat renal fibroblasts expressing control or ATM shRNA in response to TGF-b1 treatment for 6 or 24 h. Efficiency of ATM knockdown in NRK-49Fs was measured by blotting for ATM (M). GAPDH (A, D, E, G, M, N) and actin (I) provided loading controls.

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Figure 3. Pharmacologic inhibition of ATM blocks TGF-b-stimulated PAI-1 induction in renal tubular epithelial cells and fibroblasts. A–H) HK-2 cells were pretreated with KU-55933 at the indicated doses followed by TGF-b1 exposure for 6 (F) or 24 h (A). Western blotting assessed PAI-1, fibronectin, and p21 expression. Inhibitor function was confirmed using pATMSer1981 as a readout. B–E, G, H) Histograms depict the expression of PAI-1 (C, G), fibronectin (D), and p21 (E, H) as the mean 6 SD in triplicate experiments. *P , 0.05; **P , 0.01; ***P , 0.001. I, J) Effect of TGF-b1-mediated PAI-1 and fibronectin protein levels following pretreatment with KU-55933 in NRK-49F fibroblasts at 6 (I) and 24 h (J). GAPDH (F) and actin (A, I, J) provided the loading controls. In all histograms, untreated HK-2 or NRK-49F controls were arbitrarily set as 1. Laboratories, Burlingame, CA, USA) to quench endogenous peroxidase activity prior to blocking in 10% normal goat serum. Mouse anti-phospho-ATMSer1981 (1:500; Abcam) antibody in 1% bovine serum albumin was added to sections for 30 min followed by incubation with appropriate secondary biotinylated antibody for another 30 min. VECTASTAIN Elite ABC was added for 30 min, reactions were developed with ImmPACT 3,39diaminobenzidine peroxidase substrate, and sections were counterstained with Hematoxylin QS (H-3404; all from Vector Laboratories).


Protein expression and localization in the tissue samples were measured using the morphometric analysis as previously described by our laboratory (17). Statistical analysis Histograms were plotted as the mean 6 SD. The statistical comparisons between experimental groups were performed using the

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Figure 4. ATM is a mediator of TGF-b-induced growth arrest in renal tubular epithelial cells. A) Schematic of experimental plan for growth arrest studies in HK-2 cells. Serum-starved (1 d) subconfluent (30%) HK-2 cells were treated with TGF-b for 1 d followed by 1% serum for 3 or 7 d to stimulate proliferation. B) Phase-contrast images of con shRNA- and ATM shRNA-expressing HK-2 cells stimulated with or without TGF-b1 followed by 3 d of growth in 1% serum. C, E) Relative cell count of epithelial cells stimulated with TGF-b for 3 (C) or 7 d (E) was depicted in histograms (mean 6 SD) setting relative cell count in untreated con shRNA as 1 at both time points. *P , 0.05; **P , 0.01; ***P , 0.001. D) Immunoblotting of p21 and PCNA expression in HK-2 cells following TGF-b stimulation for 3 d with or without ATM depletion. GAPDH is a loading control. N.S., not significant. 2-tailed Student’s t test. P values ,0.05 were considered statistically significant, and P , 0.05, P , 0.01, and P , 0.001 were represented as “*,” **,” and “***” in histograms, respectively.

RESULTS ATM activation in the fibrotic kidney correlates with p53 and SMAD3 activation and profibrotic markers To investigate the potential role of ATM in renal fibrosis, the expression pattern of ATM was assessed in the UUO mouse model of renal injury. Dysmorphic tubules with expansion of the interstitial space were evident in the obstructed kidney within 14 d of UUO (Fig. 1A). Immunohistochemical staining (;4-fold; **P , 0.01) and Western blot analysis (;5-fold; **P , 0.01) revealed a significant increase of injury-associated ATM phosphorylation (pATMSer1981) in both the tubular epithelium and interstitium compared to the contra or sham control kidneys (Fig. 1A–D). Up-regulation of 2 profibrotic genes, PAI-1 (Fig. 1C, E; **P , 0.01) and a-SMA (Fig. 1C, F; ***P , 0.001), was similarly evident in the UUO-injured kidney 6

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relative to the contra control. Activation of ATM correlated with an increase in p53Ser15 phosphorylation (p-p53Ser15; Fig. 1C, H; *P , 0.05) and elevated expression of the NOX subunit, p22phox (Fig. 1C, I, *P , 0.05) at 14 d following obstruction. Similarly, lysates derived from the ligated kidney at d 7 [UUO (d 7)] revealed activation of ATM (Fig. 1J, K; *P , 0.05) and p53 (Fig. 1J, L; *P , 0.05), which correlated with increased p22phox levels (Fig. 1J, M, *P , 0.05) compared to the respective contra controls. Consistent with UUO as a TGF-b1-driven model (7, 18), there was a dramatic increase in pSMAD3 levels (Fig. 1C, G; ***P , 0.001), which correlated with the time course of pATMSer1981 expression. Such findings promoted the investigation of ATM kinase as a target of TGF-b1-dependent signaling. Critical role of ATM kinase in TGF-b1-mediated gene reprogramming TGF-b1 rapidly promoted ATM phosphorylation (Fig. 2A, B; *P , 0.05 at 15 min), which preceded the phosphorylation of a known TGF-b1 target, p53 (Fig. 2A, C;


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Figure 5. ATM is required for p53 phosphorylation downstream of TGF-b in renal epithelial cells. A–F) ATM knockdown in HK-2 cells eliminated p53Ser15 phosphorylation in response to TGF-b stimulation at 0.5, 1 (A), 2 (C), or 24 h (E) compared to con shRNA cultures. Graphs (mean 6 SD) in (B), (D), and (F) represent the quantification of (A), (C), and (E), respectively, arbitrarily setting p-p53Ser15 levels in untreated con shRNA cells as 1 for both experiments (n = 3). G, H) The effect of KU-55933 pretreatment on TGF-b1-induced p53Ser15 phosphorylation. H) Histogram reflects the dose-dependent reduction in p-p53Ser15 by TGF-b quantified by the mean 6 SD of 3 independent experiments setting p-p53Ser15 levels in untreated controls as 1. I) Phasecontrast images of con shRNA and p53 shRNA-expressing HK-2 cells treated with or without TGF-b followed by a 3 d stimulation of 1% serum to promote growth. Western blot analysis confirmed the knockdown efficiency of p53. J) Graphs depict the relative cell count of above cultures (mean 6 SD) setting relative cell count in untreated con shRNA as 1. K) Schematic of experimental plan involving the p53 activator Nutlin-3A. Sparsely confluent (30%) HK-2 cells were serum deprived for 1 d, pretreated with Nutlin-3a for 3 d, followed by TGF-b and 1% serum treatment for 3 d. L) Plotted cell count is the mean 6 SD of 3 experiments arbitrarily setting cell count in unstimulated, con shRNA-expressing HK-2 cells as 1. GAPDH (A, C, E, I) and actin (G) confirmed equal loading. *P , 0.05; **P , 0.01; ***P , 0.001. N.S., not significant.


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Figure 6. NOX subunits, p47phox and p22phox, are necessary for TGF-b1-dependent profibrotic gene induction. A) Knockdown efficiency of the p22phox and p47phox subunits was confirmed by Western blot analysis. B) TGF-b1 treatment for 6 or 24 h promoted the expression of PAI-1 in the control cells, but not p22phox-depleted renal epithelial cells, whereas HK-2 cells with p47phox silencing have reduced PAI-1 expression compared to con shRNA in response to TGF-b1. C) PAI-1 expression is shown in the graph (mean 6 SD) setting protein levels in untreated, con shRNA as 1. *P , 0.05; **P , 0.01; ***P , 0.001. D) TGF-b1-dependent p21 and fibronectin were ablated in p47phox and p22phox knockdown HK-2 cells. GAPDH (A), actin (B), and ERK2 (D) acted as loading controls.

**P , 0.01 at 30 min), as well as the activation of the canonical SMAD3 pathway (Fig. 2A, at 15 min). To determine the potential role of ATM in the TGF-b1 signaling pathway, introduction of ATM shRNA lentiviral particles into HK-2 human renal epithelial cells was used to efficiently reduce ATM protein levels (Fig. 2D; .95%). ATM knockdown completely abolished PAI-1 expression (Fig. 2E–H; .95%, **P , 0.01 for T6 and ***P , 0.001 for T24) in response to TGF-b1 treatment, seen in the con shRNAexpressing HK-2 cells. ATM depletion, moreover, decreased fibronectin (Fig. 2I, J, **P , 0.01 at T24), p21 (Fig. 2I, K, **P , 0.01 at T24), cyclooxygenase-2 (COX-2; Fig. 2I, L, *P , 0.05 at T24), and CTGF (Fig. 2I) in TGF-b1stimulated renal epithelial cells. TGF-b1-induced SMAD3 activation, however, was intact in ATM-depleted HK-2 cells (Fig. 2I). Similarly, ATM silencing in rat renal fibroblasts (Fig. 2M; .95%) inhibited TGF-b1-dependent PAI-1 expression compared to con shRNA-expressing NRK-49F cells (Fig. 2N; .95%). ATM activation blockade (Fig. 3A, B), by pretreatment with the ATM inhibitor KU-55933 prior to TGF-b1 stimulation, dose dependently decreased expression of TGF-b1 target genes, PAI-1 (Fig. 3A, C, F, G; .90% at 10 mM; **P , 0.01), fibronectin (Fig. 3A, D; **P , 0.01), and p21 (Fig. 3A, E, F, H; ***P , 0.001). This response to KU-55933 was not restricted to renal epithelial cells because pharmacologic inhibition of ATM attenuated the increase in PAI-1 (.95% at 10 mM) and fibronectin in response to TGF-b1 in NRK49F renal fibroblasts (Fig. 3I, J). Collectively, genetic silencing and pharmacologic blockade approaches demonstrate a requirement for ATM in profibrotic gene reprogramming driven by TGF-b1 in both renal epithelial cells and fibroblasts. ATM mediates the cytostatic response in renal epithelial cells Because the expression of the growth arrest gene p21 was ATM dependent in HK-2 cells (Fig. 2I, K), it was necessary to evaluate the putative role of ATM in TGF-b1-mediated cytostasis. Serum-deprived (1 d) subconfluent (30%) HK-2 cells were treated with TGF-b1 prior to 1% serum stimulation for 3 d to promote proliferation (schematic in Fig. 4A). Exposure of HK-2 cells to TGF-b1 for 3 (Fig. 4B, C) or 7 (Fig. 4E) d resulted in a marked decrease (;40%; *P , 0.05 at 8

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T3d) in cell number compared to the control cultures. In contrast, ATM depletion in HK-2 cells enhanced proliferation (2-fold; ***P , 0.001) and completely bypassed growth arrest in response to TGF-b1 (Fig. 4B, C, E). This phenotypic response correlated with an increase in PCNA, and loss of p21 expression (Fig. 4D), respectively. Collectively, these data suggest that TGF-b1-induced cytostasis requires ATM. ATM-regulated gene expression and cytostatic response downstream of TGF-b1 require p53-dependent mechanisms p53 is a tumor suppressor that regulates growth arrest, senescence, and apoptosis (19). It was previously established that TGF-b1 promoted p53 activation (p-p53Ser15) necessary for p53 and SMAD cooperation in the transcriptional control of profibrotic genes in HK-2 renal epithelial cells (10), although the upstream kinase of p53 activation is unclear. Stable genetic silencing of ATM (Fig. 5A–F; .95%, ***P , 0.001 at T1, T2, and T24) or ATM blockade by the pharmacologic inhibitor, KU-55933 (Fig. 5G, H; .95%, **P , 0.01 at 10 mM), ablated TGF-b1-induced p53 phosphorylation (p-p53Ser15) in renal epithelial cells. Similar to the observations in the ATM-depleted HK-2 cells, TGF-b1-induced growth arrest evident in the con shRNA-expressing cultures was eliminated in p53 shRNAexpressing HK-2 cells (Fig. 5I, J; ***P , 0.001, con vs. p53 at T3d). To investigate the causal involvement of p53 downstream of ATM, HK-2 cells were pretreated with Nutlin-3a (which inhibits p53-MDM2 interactions promoting an increase in p53 expression), followed by TGF-b1 and 1% serum stimulation for 3 d (schematic in Fig. 5K). Nutlin-3a pretreatment reduced the growth of ATM shRNAexpressing cells to levels comparable to con shRNAexpressing cells (Fig. 5L). Furthermore, bypass of growth arrest, evident in TGF-b1-treated ATM-depleted HK-2 cells, was restored with Nutlin-3a pretreatment (Fig. 5L; **P , 0.01), suggesting that p53 is a downstream target of ATM in TGF-b1-mediated proliferative arrest. NOXs promote the activation of ATM and p53 necessary for TGF-b1 target genes Elevated reactive oxygen species (ROS) is a major cause of the progression and maintenance of fibrotic disease


OVERSTREET ET AL.

Figure 7. TGF-b1-mediated ATM activation (pATMSer1981) and p53 phosphorylation (p-p53Ser15) require NOX1/NOX2/NOX4. A) Western blot analysis of TGF-b1-mediated p53Ser15 phosphorylation following genetic silencing of p47phox and p22phox in renal epithelial cells. GAPDH (A) confirmed equal loading. B, C) Histograms (mean 6 SD) illustrate relative p-p53Ser15 and pATMSer1981 levels, respectively, in p47phox or p22phox-depleted HK-2 cells following TGF-b1 exposure. *P , 0.05; **P , 0.01; ***P , 0.001.

(20–22). Rapid generation of ROS by TGF-b1 is necessary for p53-regulated gene expression (10); the specific underlying mechanism, however, needs further clarification. To evaluate NOX as a candidate of ROS generation, gene knockdown of the regulatory subunits, p22phox (a common subunit in NOX1/NOX2/NOX4) and p47phox (a common subunit in NOX1/NOX2), was utilized as an experimental approach. Stable knockdown of p22phox and p47phox (Fig. 6A) attenuated the expression of PAI-1 following TGF-b1 exposure compared to con shRNA-expressing cells (Fig. 6B, C; **P , 0.01 for p22phox at T24 and **P , 0.01 for p47phox at T24). p21 and fibronectin expression in response to TGFb1 was similarly inhibited (Fig. 6D). Furthermore, TGF-b1-dependent p53 activation was completely blocked in p22phox-depleted (.95%) HK-2 cells and partially inhibited in the p47phox-depleted cells compared to the con shRNA-expressing cells (Fig. 7A, B). ATM implication in redox sensing (16) prompted an investigation as to whether NOX proteins were upstream regulators of ATM activation. TGF-b1-stimulated pATMSer1981 was inhibited by p22phox and p47phox knockdown in renal

epithelial cells (Fig. 7C; .90% at T1 and T2; **P , 0.01), establishing the role of NOX in the TGF-b1 pathway leading to ATM/p53 activation. Role of tubular ATM-induced paracrine factors in promoting renal fibroblast proliferation Injury-induced (e.g., aristolochic acid) growth-arrested epithelial cells in the kidney secrete fibrotic factors CTGF and TGF-b1, promoting proliferation of fibroblasts (11). Given the evidence that ATM/p53-driven mechanisms mediate epithelial growth inhibition (Figs. 4C and 5J ) and profibrotic factor secretion (e.g., CTGF, PAI-1, fibronectin, and TGF-b1) in HK-2 cells [Fig. 2; (10)], it is important to examine if tubular ATM-derived fibrotic factors, acting in a paracrine manner, promote interstitial renal fibroblast growth, a hallmark of fibrosis (7). Con shRNA and ATM shRNA-expressing epithelial cells were subjected to a 1 d TGF-b1 + 3 d 1% serum-stimulation to induce a growth arrest state, washed with PBS to minimize any existing

Figure 8. Increased renal fibroblast proliferation by conditioned media derived from TGF-b1-stimulated con shRNA-, but not ATM shRNA-, expressing HK-2 cells. A) Schematic of epithelial-fibroblast crosstalk. Con shRNA or ATM-depleted HK-2 cells were treated with TGF-b1 for 1 d followed by additon of 1% serum for 3 d to promote growth arrest, washed with PBS to remove any residual TGF-b1 prior to addition of fresh 0.5% FBS/DMEM for 48 h. Conditioned media isolated from epithelial cells were directly added to semiconfluent NRK-49F fibroblasts at similar cell density for 2 d. B) Plots (mean 6 SD) represent relative NRK49F cell counts of over 100 (2 3 2 mm) fields for each experimental condition. ***P , 0.001.


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DISCUSSION

Figure 9. Proposed model of ATM activation downstream of TGFb signaling in the context of renal biology. TGF-b ligand binding initiates ALK5-dependent signal transduction resulting in the activation of the SMAD2/SMAD3/SMAD4. In a collateral pathway, TGF-b utilizes the p47phox and p22phox-dependent NOXs to generate ROS necessary for ATM activation as well as its downstream substrate p53. p53 and SMAD, in turn, form a complex that binds to TGF-b target gene promoters (e.g., PAI-1) stimulating optimal gene expression. Other ROS-dependent non-SMAD elements, including c-src/EGFR/MAPK, function to recruit additional transcriptional elements as part of a highly interactive canonical and noncanonical TGF-b transcriptional complex.

TGF-b1 influence, and maintained in fresh media with low serum (0.5%) for an additional 2 d (schematic in Fig. 8A). Conditioned media derived from HK-2 cells from each experimental condition were directly added to NRK-49F fibroblasts (maintained at a similar cell density) for 2 d prior to measuring total cell counts (schematic in Fig. 8A). Renal fibroblasts incubated with conditioned medium from con shRNA-expressing HK-2 cells treated with TGF-b1 (TGF-b1+Con shRNA) underwent a 2.5-fold increase (Fig. 8B; ***P , 0.001) in cell growth relative to NRK-49F fibroblasts receiving con shRNA-expressing, untreated HK-2-derived conditioned media (con shRNA). Interestingly, increased fibroblast proliferation triggered by conditioned media from (TGF-b1 plus Con shRNA) HK-2 cells was decreased over 50% (***P , 0.001) in NRK-49F cells identically treated with conditioned media from (TGF-b1 plus ATM shRNA) HK-2 cells, suggesting that tubular ATM activation by TGF-b1 drives paracrine factor secretion leading to epithelialmesenchymal crosstalk in the progression of renal fibrosis. 10

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CKD, which affects more than 13% of the U.S. population, is a major public health burden due to the increased prevalence of diabetes and hypertension; there is insufficient availability of renal dialysis and transplants to meet patient needs (3). Identification of novel elements and molecular pathways associated with the initiation and progression of fibrosis is of high clinical significance given that fibrotic disorders are refractory to current therapy (1, 3). This study identifies ATM as a critical downstream target of TGF-b1, which not only regulates the expression of fibrotic factors (e.g., PAI-1 and fibronectin) in both renal epithelial cells and fibroblasts but also mediates the epithelial cytostatic response. These findings are in agreement with simultaneous activation of TGF-b1/SMAD3 and ATM with profibrotic genes, such as PAI-1, in the tubular and interstitial compartments of the fibrotic kidney. Elevated PAI-1, a causative factor in renal fibrosis, mediates proliferative arrest in response to TGF-b1 in human keratinocytes (23). However, involvement of this Ser protease in TGF-b1-induced growth arrest in renal epithelial cells remains to be determined. Stably silencing p22phox and p47phox subunits, furthermore, conclusively establish the upstream involvement of NOX1/NOX2/NOX4 in ATM phosphorylation in TGFb1-stimulated HK-2 cells. Tubular ATM-dependent secretion of paracrine factors in response to TGF-b1 promotes renal fibroblast growth suggestive of the role of this tumor suppressor in epithelial-mesenchymal crosstalk in fibrosis. Indeed, p53 is a profibrotic effector, which orchestrates TGF-b1-initiated p53/SMAD3-dependent transcription of fibrotic genes (e.g., TGF-b1, CTGF, and PAI-1) in renal HK2 and NRK-49F cells (10, 17). This study also identifies, for the first time to our knowledge, ATM as the upstream kinase that mediates TGF-b1-dependent p53 activation via the NOX1/NOX2/NOX4 pathway (Fig. 9). Ureteral ligation, ischemia reperfusion, and toxininduced renal injury are accompanied by ATMSer1981 phosphorylation (Fig. 1A) (11, 24). Although oxidative stress has been implicated in the progression of UUO injured-, ischemic-, and diabetic-induced renal disease or tissue fibrosis in general (22) and free radicals such as H2O2 are known to activate ATM (16), the precise regulators of ATM activation in the diseased kidney are not clear. This study identifies TGF-b1 as a potent and rapid inducer of ATMSer1981 phosphorylation via redox-sensitive mechanisms leading to maladaptive renal fibrotic responses (25), including expression of PAI-1, CTGF, and fibronectin as well as epithelial growth inhibition and fibroblast proliferation. Such findings may have widespread pathologic significance in renal disease because TGF-b1 is a major transducer of renal injury/aberrant repair regardless of etiology (e.g., ischemic, obstructive, diabetic, or hypertensive) (1, 3, 7). Elevated TGF-b1 signaling is causatively linked to fibrosis (7, 26). In fact, urinary TGF-b1 levels have prognostic implications in children with obstructed uropathy/nephropathy (27) and patients exhibiting fibrosis in a failing kidney allograft (28). Components of the non-SMAD pathway (e.g., NOX, p53, and EGFR) of TGFb1 signaling are viable targets in antifibrotic therapies. Inhibition of p53 suppressed the transition of acute injury to chronic renal failure (11), and the NOX1/NOX4


OVERSTREET ET AL.

inhibitor GKT137831 is currently in phase 2 clinical trials for the treatment of diabetic nephropathy (29). Identification of a NOX/ATM/p53 axis in transducing fibrotic signaling is a previously unexplored therapeutic opportunity that complements direct targeting of TGF-b1 currently in clinical trials for various fibrotic disorders. This work was supported by U.S. National Institutes of Health National Institute of General Medical Sciences Grant GM057242 (to P.J.H.). J.M.O., R.S., and P.J.H. conceived and designed the experiments. J.M.O., R.S., and D.C.-G. performed the experiments. J.M.O. and R.S. analyzed the data. R.G. contributed key reagents/materials. J.M.O., R.S., and P.J.H. wrote the paper. The authors declare no conflicts of interest.

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