J. Biol. Chem.-2016-Rozycki-227-43

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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 291, NO. 1, pp. 227–243, January 1, 2016
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Myocardin-related Transcription Factor Regulates Nox4

Protein Expression
LINKING CYTOSKELETAL ORGANIZATION TO REDOX STATE *
Received for publication, June 24, 2015, and in revised form, October 15, 2015 Published, JBC Papers in Press, November 10, 2015, DOI 10.1074/jbc.M115.674606
Matthew Rozycki‡, Janne Folke Bialik‡§, Pam Speight‡, Qinghong Dan‡, Teresa E. T. Knudsen§, Stephen G. Szeto‡,
Darren A. Yuen‡, Katalin Szászi‡¶, Stine F. Pedersen§, and András Kapus‡¶储1
From the ‡Keenan Research Centre for Biomedical Science of the St. Michael’s Hospital, Departments of ¶Surgery and
储
Biochemistry, University of Toronto, Toronto, Ontario M5B 1T8, Canada and the §Department of Biology, Section for Cell Biology
and Physiology, University of Copenhagen, Copenhagen DK-2100, Denmark
Background: Generation of myofibroblasts, the culprit of fibrosis, requires cytoskeleton remodeling and Nox4 (NADPH
oxidase) expression. The link between these events is unknown.
Results: Down-regulation/inhibition of the cytoskeleton-controlled transcriptional coactivators, myocardin-related transcrip-
tion factor (MRTF), and TAZ/YAP abrogates Nox4 expression.
Conclusion: MRTF and TAZ/YAP are essential for Nox4 expression.
Significance: We show new mechanisms whereby the cytoskeleton regulates cellular redox state and fibrogenesis.
Downloaded from http://www.jbc.org/ by guest on January 18, 2018

TGF␤-induced expression of the NADPH oxidase Nox4 is cient; Nox4 expression also requires TGF␤-activated Smad3
essential for fibroblast-myofibroblast transition. Rho has been and TAZ/YAP, two contact- and cytoskeleton-regulated
implicated in Nox4 regulation, but the underlying mechanisms Smad3-interacting coactivators. Down-regulation/inhibition of
are largely unknown. Myocardin-related transcription factor TAZ/YAP mitigates injury-induced epithelial Nox4 expression
(MRTF), a Rho/actin polymerization-controlled coactivator of in vitro and in vivo. These findings uncover new MRTF- and
serum response factor, drives myofibroblast transition from TAZ/YAP-dependent mechanisms, which link cytoskeleton
various precursors. We have shown that TGF␤ is necessary but remodeling and redox state and impact epithelial plasticity and
insufficient for epithelial-myofibroblast transition in intact epi- myofibroblast transition.
thelia; the other prerequisite is the uncoupling of intercellular
contacts, which induces Rho-dependent nuclear translocation
of MRTF. Because the Nox4 promoter harbors a serum response Organ fibrosis is a dysregulated form of tissue repair in
factor/MRTF cis-element (CC(A/T)6GG box), we asked if MRTF response to chronic injury, characterized by excessive extracel-
(and thus cytoskeleton organization) could regulate Nox4 lular matrix deposition and the consequent loss of tissue archi-
expression. We show that Nox4 protein is robustly induced in tecture, which ultimately leads to organ failure (1, 2). Fibropro-
kidney tubular cells exclusively by combined application of con- liferative diseases, including renal, hepatic, pulmonary, and
tact uncoupling and TGF␤. Nox4 knockdown abrogates epithe- cardiovascular fibrosis, and systemic sclerosis are thought to
lial-myofibroblast transition-associated reactive oxygen species contribute to 45% of all deaths in the Western world (2). The
production. Laser capture microdissection reveals increased major pathogenic culprit of organ fibrosis is the myofibroblast,
Nox4 expression in the tubular epithelium also during obstruc- a contractile, extracellular matrix-producing mesenchymal
tive nephropathy. MRTF down-regulation/inhibition sup- cell, hallmarked by the expression of ␣-smooth muscle actin
presses TGF␤/contact disruption-provoked Nox4 protein and (SMA)2 (3). Although myofibroblasts are scarce in normal
mRNA expression, Nox4 promoter activation, and reactive oxy- parenchymal organs, they are abundantly formed during fibro-
gen species production. Mutation of the CC(A/T)6GG box elim- genesis from various precursors via phenotypic reprogram-
inates the synergistic activation of the Nox4 promoter. Jas- ming. Therefore, both their cellular origins and the molecular
plakinolide-induced actin polymerization synergizes with mechanisms leading to their formation and activation have
TGF␤ to facilitate MRTF-dependent Nox4 mRNA expression/ become central questions in fibrosis research. Although the
promoter activation. Moreover, MRTF inhibition prevents identity of myofibroblast progenitors in various fibrotic dis-
Nox4 expression during TGF␤-induced fibroblast-myofibro- eases is a contentious issue, potential sources include resident
blast transition as well. Although necessary, MRTF is insuffi- fibroblasts, the epithelium, the endothelium, pericytes, and
* This work was supported by Canadian Institute of Health Research Grants

2
MOP-86535, MOP-106625, and MOP-130463 (to A. K.), the Kidney Founda- The abbreviations used are: SMA, smooth muscle actin; CArG, CC(A/T)6GG;
tion of Canada (to A. K. and K. S.), and by Natural Sciences and Engineering DCF-DA, dichlorofluorescein diacetate; DKD, diabetic kidney disease;
Research Council of Canada Grant RGPIN227-908-13 (to A. K.) and Grant EMyT, epithelial-myofibroblast transition; JK, jasplakinolide; LCM, low cal-
RGPIN 327407 (to K. S.). The authors declare that they have no conflicts of cium medium; LCMi, laser caption microdissection; MRTF, myocardin-re-
interest with the contents of this article. lated transcription factor; NR, non-related; Nox, NADPH Oxidase; qPCR, real
1
To whom correspondence should be addressed: Keenan Research Centre time quantitative PCR; ROS, reactive oxygen species; SBE, Smad-binding
for Biomedical Science, Rm. 621, 209 Victoria St., Toronto, Ontario M5B 1T8, element; SRF, serum response factor; UUO, unilateral ureteric obstruction;
Canada. Tel.: 416-847-1751; E-mail: kapusa@smh.ca. VP, verteporfin; PFA, paraformaldehyde; VP, verteporfin.
JANUARY 1, 2016 • VOLUME 291 • NUMBER 1 JOURNAL OF BIOLOGICAL CHEMISTRY 227

MRTF Regulates Nox4 Expression
bone marrow-derived fibrocytes (4 – 6). Irrespective of the par- sought to determine whether MRTF and the state of the actin
ticular source, the pleiotropic cytokine TGF␤ plays a central skeleton might regulate Nox4 expression and Nox4-dependent
role in the phenotype shift and the ensuing activation of a myo- ROS production. We investigated this problem in the context
genic program (SMA expression) in all precursors (6). of myofibroblast transition, particularly in EMyT, wherein
Previously, our group has explored the mechanisms through Smad3- and Rho-dependent processes can be effectively dis-
which kidney tubular cells transform to myofibroblasts, a pro- sected. We show that MRTF signaling and thus the state of the
cess termed epithelial-myofibroblast transition (EMyT). We actin skeleton are important permissive inputs for Nox4
have shown that TGF␤, although necessary, is insufficient for expression, which in turn is critical for myofibroblast transi-
EMyT, implying that an intact epithelium is largely resistant to tion. These studies reveal a new mechanistic link between cyto-
the transforming effect of this cytokine; the other pre-requisite skeleton organization and cellular redox state (ROS genera-
is the disruption or absence of intercellular contacts (7, 8). We tion). Moreover, we also provide evidence that TAZ and YAP,
denoted this mechanism the two-hit scheme. Contact disrup- two cell contact-dependent and Smad3-interacting (33) and
tion (e.g. during wounding) facilitates activation of the Rho fam- Hippo pathway-regulated transcriptional co-activators, are
ily small GTPases Rac1 and RhoA, promoting F-actin polymer- also required for Nox4 expression.
ization, which in turn induces the nuclear translocation of the
transcriptional coactivator, myocardin-related transcription Experimental Procedures
factor (MRTF) (9 –11). In resting cells MRTF is sequestered in Reagents—NADPH oxidase inhibitor VAS2870, dichloro-
the cytosol through association with G-actin, which masks its fluorescein diacetate (DCF-DA), jasplakinolide (JK), MRTF/
nuclear localization sequence. Once in the nucleus, MRTF SRF inhibitor CCG-1423, and TAZ/YAP inhibitor verteporfin
were purchased from Sigma. TGF␤ was purchased from R&D

associates with serum response factor (SRF), and the complex
binds to CC(A/T)6GG elements (CArG box) present in the pro- Systems (Minneapolis, MN). Commercially available antibod-
moter of a cohort of target genes, including those encoding ies were from the following sources: TAZ/YAP and phospho-
cytoskeletal proteins (e.g. SMA) (12, 13). Thus, cytoskeleton myosin light chain II (Thr-18/Ser-19), Cell Signaling Technol-
remodeling is not only an early feature of the phenotypic shift ogies (Danvers, MA); tubulin and SMA, Sigma; GAPDH, Santa
but also a key driver of the ensuing transcriptional reprogram- Cruz Biotechnology (Santa Cruz, CA); and Nox4, Novus Bio-
ming, thereby linking cell structure to gene expression via the logicals (Littleton CO). The rabbit polyclonal MRTF antibody
MRTF/SRF pathway (14). Accordingly, we and others have (BSAC) has been described previously (34). Given the concerns
shown that genetic or pharmacological inhibition of MRTF regarding the specificity of commercially available Nox4 anti-
prevents myofibroblast transition (8, 10) and lessens organ bodies and the variability of batches (35), experiments were
fibrosis (15–17). completed only with antibodies that recognized a protein of the
Recently, redox signaling has emerged as another TGF␤-in- proper molecular weight and of which immunoreactivity was
duced mechanism that plays a central role in myofibroblast lost upon Nox4 siRNA treatment.
transition. Specifically the NADPH oxidase (Nox) isoform Cell Culture—LLC-PK1 (Cl 4) cells, a porcine proximal tubu-
Nox4 (18), a downstream target of TGF␤ effectors Smad2/3, lar epithelial cell line (a kind gift from R. C. Harris, Vanderbilt
has been implicated as a chief mediator of fibroblast-myofibro- University School of Medicine, Nashville, TN), and C3H-
blast transition (19 –23). As opposed to other NADPH oxidase 10T1/2 cells, a mouse embryonic mesenchymal cell line (Amer-
variants whose activity is controlled by stimulus-induced ican Type Culture Collection, Manassas, VA), were cultured in
assembly with regulatory subunits (p47, p67, and Rac1), Nox4 is low-glucose DMEM (Invitrogen) supplemented with 10% fetal
thought to be constitutively active and is regulated primarily at bovine serum and 1% streptomycin/penicillin solution (Invitro-
the level of its expression (24, 25). Nox4-mediated reactive oxygen). NRK-F (rat kidney fibroblasts, American Type Culture
gen species (ROS) production was found to be necessary for the Collection) were cultured in high-glucose DMEM (Invitrogen)
induction of key myofibroblast features, including contractility, with similar supplements. To induce contact disruption, cells
matrix production (extra domain A fibronectin, collagen), and were washed three times with phosphate-buffered saline (PBS;
SMA expression (19, 22, 26). Accordingly, interventions that Invitrogen) and then cultured in nominally calcium-free
suppress ROS signaling or directly target Nox4 have been DMEM (LCM; Invitrogen), as in our earlier studies (8, 30).
shown to attenuate fibrosis in mouse models of chronic kidney Where indicated, cells were treated with 5 ng/ml TGF␤. For
disease (27–29). Moreover, we found that EMyT is also associ- siRNA transfections, LLC-PK1 cells were cultured in antibiot-
ated with Smad3-dependent Nox4 expression (30). ic-free medium.
Cognizant of the key role of cytoskeleton remodeling and ROS Detection—Nox4 activity and ROS production were mea-
Nox4 expression in myofibroblast transition, we asked whether sured using a DCF-DA assay, as described previously (36 –38).
these processes might be causally linked. Such a hypothesis is Confluent monolayers were washed 1 time with PBS and 1 time
supported by the recent finding that RhoA acts upstream of with serum-free medium or LCM and then incubated at 37 °C
Nox4-mediated ROS generation (31). Moreover, we noted that with 50 ␮M DCF-DA for 30 min. Following the incubation
the Nox4 promoter contains a CArG box. Intriguingly, period, cells were washed 2 times with PBS and then one time
increased actin polymerization has been proposed to correlate with serum-free medium or LCM. Plates were immediately
with enhanced ROS production in the context of cellular aging read using SpectraMax 5Me fluorescent plate reader (493 nm
and apoptosis; however, a causal link and the potential mecha- excitation, 520 nm emission, Molecular Devices, Sunnyvale,
nisms remained enigmatic (32). In light of this scenario, we CA) or Fluoroskan Ascent FL plate reader (485 nm excitation,
228 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291 • NUMBER 1 • JANUARY 1, 2016

526 nm emission, Lab Systems, Mumbai, India). Two subse- TTA GCC GAA-3⬘), and GAPDH (sense, 5⬘-GCA AAG TGG
quent readings were then made at 30-min intervals followed by ACA TGG TCG CCA TCA-3⬘, and antisense, 5⬘-AGC TTC
cellular lysis. The results were normalized by dividing the CCA TTC TCA GCC TTG ACT-3⬘) were used. qPCR analysis
obtained fluorescence values by the absorbance from a BCA was performed in a Bio-Rad iCycler Thermal Cycler with iQ5
protein assay (absorbance 562 nm) to generate fluorescence Multicolor real time PCR detection system (95 °C 3 min; 48 ⫻
units. 96 °C 30 s, 55 °C 1 min) with GAPDH as reference gene. Data
Plasmids and Transfection—The Nox4 luciferase construct were analyzed using iQ5 software. Melting curves always con-
was a gift from Dr. Thannickal (University of Alabama, Bir- firmed the presence of only one amplicon. Calculation of the
mingham) and has been described previously (39). The HA- relative expression ratio was done using ⌬⌬Ct.
MRTF B construct has been described previously (40). The Western Blotting—Following the indicated treatments, cells
SMA luciferase construct was a gift from S. H. Phan (University were first washed two times with PBS and then lysed with Tri-
of Michigan Medical School, Ann Arbor) and contains the ton lysis buffer (30 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM
proximal 765 bp of the rat SMA promoter cloned into a pGL3- EGTA, 20 mM sodium fluoride, and 1% Triton X-100) supple-
basic vector. The thymidine kinase promoter-driven Renilla was mented with 1 mM sodium vanadate, 1 mM phenylmethylsulfo-
purchasedfromPromega(Madison,WI).Transfectionsofthecon- nyl fluoride, and Complete Mini Protease Inhibitor (Roche
structs were completed using JetPRIME (Polyplus Transfection Applied Science, Mississauga, Ontario, Canada). In experi-
SA, New York) in accordance with the manufacturer’s guidelines. ments that measured relative phosphomyosin II levels, a RIPA
The inactivation of the CArG box in human Nox4 promoter con- lysis buffer was used (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5%
structs was achieved by standard PCR-based mutagenesis using sodium deoxycholate, 1% Triton X-100, pH 7.4) that was sup-

a high fidelity proofreading polymerase. The primer pair and plemented as described above. Protein concentration was
subsequent mutations (in parentheses) were as follows: 5⬘- determined using a BCA assay (Thermo Scientific). SDS-PAGE
TATTGGTTTAACAAAAATTTATTAAATATCTATTTT- and Western blotting was performed as described previously
GCAT-3⬘ and 5⬘-ATGCAAAATAGATATTTAATAAATTT- (8). Briefly, blots were blocked in Tris-buffered saline contain-
TTGTTAAACCAATA-3⬘ (C⫺860/A; C⫺859/A; G⫺852/A and ing 3% BSA and incubated with the primary antibody overnight.
G⫺851/A). All new constructs were verified by sequencing. Antibody binding was visualized with the corresponding per-
Luciferase Reporter Assays—Luciferase assays were com- oxidase-conjugated secondary antibodies and the enhanced
pleted in accordance with our previously published data (8, 30, chemiluminescence method (GE Healthcare, Mississauga,
40). Briefly, cells were transfected at 60% confluence with lucif- Ontario, Canada). Where indicated, blots were stripped and
erase reporter constructs (0.25 ␮g/ml) and the thymidine reprobed to demonstrate equal loading or to detect levels of
kinase promoter-driven Renilla (0.025 ␮g/ml). Following the down-regulated proteins. Densitometry was performed using a
indicated treatments, cells were lysed using 1⫻ passive lysis GS800 densitometer and Quantity One software (Bio-Rad).
buffer (Promega). Subsequently, luciferase activity was mea- Immunofluorescence Microscopy—Cells were seeded onto
sured using a dual assay luciferase kit (Promega). glass coverslips, grown to confluence, and then treated as indi-
RNA Interference—RNA interference was used to target the cated. The coverslips were fixed using 4% paraformaldehyde
following porcine-specific sequences: MRTF A, 5⬘-CCAAGG- (PFA; Canemco & Marivac, Lakefield, Quebec, Canada) for 20
AGCUGAAGCCAAAUU-3⬘; MRTF B, 5⬘-CGACAAACACC- min. Next, PFA was quenched with 100 mM glycine. Cells were
GUAGCAAAUU-3⬘; Yap, 5⬘-UCAAAGCGCUCCAGUGAA- then permeabilized with 0.1% Triton X-100 in PBS and then
AUU-3⬘; Taz, 5⬘-GGAAGAAGAUCCUGCCUGAUU-3⬘; and blocked with 3% bovine serum albumin for 1 h. Samples were
Nox4, 5⬘-CCGCAGACCUGGAUUUUU-3⬘. siRNA synthesis incubated with the indicated primary antibodies for 1 h, washed
was completed by Applied Biosystems (Burlington, Ontario, with PBS, and then incubated with 4⬘,6-diamidino-2-phenylin-
Canada) or by Thermo Scientific (Waltham, MA). Nonrelated dole (DAPI; Lonza, Basel, Switzerland) and fluorescently
(NR) control siRNA was purchased from Applied Biosystems labeled secondary antibody (Jackson ImmunoResearch, West
(Silencer Negative Control siRNA number 2). siRNA transfec- Grove, PA) for 1 h. Dako (Burlington, Ontario, Canada) mount-
tions were completed using either JetPRIME or Lipofectamine ing medium was used to mount coverslips onto slides. Images
RNAiMAX (Invitrogen) in accordance with the manufacturer’s were collected using a WaveFX spinning-disk microscopy sys-
guidelines. tem (Quorum Technologies, Guelph, Canada) equipped with
Real Time Quantitative PCR (qPCR)—After treatment, an ORCA-Flash4.0 digital camera. Z sections were collapsed
mRNA was extracted using the RNeasy mini kit (Qiagen into a maximum intensity projection using MetaMorph Pre-
Venlo, Netherlands) according to the manufacturer’s instruc- miere software (Molecular Devices) to generate the presented
tions as in Ref. 41. mRNA was reverse-transcribed using images.
iScriptTM reverse transcription (Bio-Rad). iQTM SYBR威 Green Unilateral Ureteric Obstruction—All animal studies were
qPCR was performed in triplicate on each cDNA sample using carried out according to a protocol approved by the animal care
1 ␮l of cDNA and 10 ␮l of 2⫻ iQTM SYBR威 Green Supermix committee of the St. Michael’s Hospital. Male C57BL/6 mice
(Bio-Rad), 2 ␮M forward and reverse primers. Specific primers (6 – 8 weeks old) were purchased from The Jackson Laboratory
targeting Nox4 (sense, 5⬘-GGA ACG CAC TAC CAG GAT (Bar Harbor, ME) and underwent either left-sided unilateral
GT-3⬘, and antisense, 5⬘-CAG GTC TGC GGA AAG TTA ureteral obstruction (UUO) or sham surgery. Briefly, a left-
GC-3⬘), MRTF (sense, 5⬘-CAG ATG GCA CCA CCC ATA sided flank incision was made in anesthetized mice, and the left
TCC TTA-3⬘, and antisense, 5⬘-AGC GGA GCG ATT TCA kidney and ureter were identified. Subsequently, the left ureter

was obstructed via two 4-0 silk suture knots, just distal to the scribed using Sens iScript Reverse Transcription kit (Qiagen)
renal pelvis. One or 2 weeks post-surgery, the mice were sacri- using oligo(dT) primer. The PCR was performed using iProof
ficed, and the left kidneys were harvested. Half of the kidney High Fidelity PCR kit (Bio-Rad). The following primers were
was snap-frozen in liquid nitrogen, and the other half was fixed used to evaluate cell-specific markers: Tamm-Horsfall protein
and embedded in paraffin or frozen fresh in Optimum Cutting (sense, 5⬘-TCA GCC TGA AGA CCT CCC TA-3⬘, and anti-
Temperature (OCT) medium. The samples were stored at sense, 5⬘-TGT GGC ATA GCA GTT GGT CA-3⬘); basic amino
⫺80 °C until processed. For studies using verteporfin, male acid transporter (sense, 5⬘-ACG TCT TCC TCG TGG TTC
mice (6 – 8 weeks old) underwent left-sided UUO or sham sur- TG-3⬘, and antisense, 5⬘-GCC ATC TCT TAG GGA GCT
gery. UUO mice were randomized to receive intraperitoneal T-3⬘); Nephrin (sense, 5⬘-GTG TTT TTC TTC GGG TGT
injection of either verteporfin (100 mg/kg) every other day, dis- CA-3⬘, and antisense, 5⬘-CCA CTT TCG TCA GGG GAG
solved in 10% DMSO/PBS, or 10% DMSO/PBS vehicle control. TA-3⬘); collagen I (sense, 5⬘-CCT GG TAA AGA TGG TGC
Treatment began immediately post-surgery and continued C-3⬘, and antisense, 5⬘-CAC CAG GTT CAC CTT TCG CAC
until day 7. C-3⬘); and GAPDH (sense, 5⬘-ACC ACA GTC CAT GCC ATC
Immunofluorescent Staining of Kidney Sections—Snap-fro- AC-3⬘, and antisense, 5⬘-CAC CAC CCT GTT GCT GTA
zen kidney sections from sham or UUO animals were embed- GCC-3⬘).
ded on glass coverslips and fixed using 4% PFA for 20 min. PFA Statistical Analysis—Data are presented as representative
was subsequently quenched using 100 mM glycine and then blots or images from at least three similar experiments or as
permeabilized with 0.1% Triton X-100 in PBS. Samples were means ⫾ S.E. of the mean (S.E.) for the number of experiments
blocked using 5% donkey serum for 1 h and then incubated with indicated. Statistical significance was determined by two-tailed

primary antibody overnight at 4 °C. The following day, samples Student’s t test or one-way analysis of variance (Tukey or Dunn
were incubated with DAPI and fluorescently labeled secondary post hoc testing for parametric and nonparametric analysis of
antibody for 1 h. Samples were then immersed in Dako mount- variance, as appropriate), using Prism and Instat software. p ⬍
ing medium and mounted on coverslips. Analysis of fixed sam- 0.05 was accepted as significant. *, p ⬍ 0.05; **, p ⬍ 0.01.
ples was completed using an Olympus IX81 microscope as
above. Results
Whole Kidney Lysates—Kidney tissues from sham and UUO Fibrogenic Transition of the Epithelium Is Associated with
(7 and 14 days) mice were cut into very small pieces with a razor Increased Nox4 Expression, Which Contributes to the Ensuing
blade. The samples were homogenized with a rotor-stator ROS Production—Although fibroblasts require only TGF␤ for
homogenizer in ice-cold RIPA buffer containing 50 mM Tris- myofibroblast transition, the epithelium becomes susceptible
HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxy- to such TGF␤-provoked phenotypic shift only when the integ-
cholate, 0.1% SDS, and 1 mM EDTA supplemented with fresh 1 rity of intercellular contacts is compromised (8, 10). To assess
mM PMSF, 1 mM sodium vanadate, 1⫻ protease inhibitor mix- whether such synergy between TGF␤ and cell contact-depen-
ture. The samples were centrifuged at 12,000 rpm for 20 min at dent signaling is also required for enhanced ROS production
4 °C. Protein content of the supernatants was determined, and and Nox4 expression, we investigated these processes in the
samples were analyzed using SDS-PAGE and Western blotting context of our previously characterized two-hit scheme (7, 8,
as above. 10). To this end, LLC-PK1 tubular cells were exposed to TGF␤
Laser Capture Microdissection (LCMi)—Mouse kidneys from or LCM (which uncouples the intercellular contacts) or the
sham and unilateral UUO (7 and 14 days)-treated animals were combination of these stimuli, which provokes EMyT. To mon-
dissected, placed in cryomolds, embedded in Tissue-Tek OCT itor the ensuing intracellular ROS production, cells that had
medium, immediately frozen in liquid nitrogen, and stored at been treated with these stimuli were briefly loaded with DCF-
⫺80 °C. The tissues were sectioned at 2–5 ␮m in a cryostat and DA, and the extent and rate of change in DCF fluorescence were
mounted on glass slides. Rapid H&E staining was performed as followed for an hour (Fig. 1, A and B). Untreated cells exhibited
reported previously (42) with minor modification. Briefly, fro- slow ROS production, which was not measurably affected by
zen sections were fixed with 70% ethanol for 1 min and then TGF␤. Note that our previous studies have verified that conflu-
washed with RNase-free water for 10 s. Sections were stained ent epithelial cells do not show general unresponsiveness to
rapidly with Mayer’s hematoxylin solution (Sigma) for 30 s, TGF␤, and the TGF␤ receptor remains accessible and func-
washed with RNase-free water for 10 s, dehydrated with an tional, as evidenced by rapid Smad3 phosphorylation and other
ethanol gradient, and counterstained with alcoholic eosin Y downstream events (7). Yet, this response is not sufficient to
solution (Sigma) for 30 s. Sections were washed three times induce lasting ROS production (above) or SMA expression (8).
with 100% ethanol (10 s each) and then two times with xylenes LCM alone caused a very modest albeit significant increase in
(5 min each). After air-drying for 10 min, LCMi was performed the rate of ROS production (Fig. 1B). Importantly, TGF␤ ⫹
using the PixCell II laser capture microdissection system and LCM induced a substantial rise in the extent and rate (2.5-fold)
CapSure LCMi Caps (Arcturus Mountain View, CA). The fol- of ROS generation (Fig. 1B). These results imply that markedly
lowing parameters were set for dissection: spot size,7.5 ␮m; enhanced ROS generation occurred only under conditions that
power, 5 milliwatts; duration, 650 ␮s. A total of 100 –200 target promote myofibroblast transition. Next, we investigated
tubules and interstitial areas were captured for each sample. whether Nox4 expression followed a similar pattern. In
Total RNA from LCMi-captured samples was isolated using the untreated epithelial cells basal Nox4 expression was marginal.
RNeasy micro RNA isolation kit (Qiagen) and reverse-tran- Again, TGF␤ failed to elicit a detectable effect, whereas LCM


FIGURE 1. ROS production, Nox4 protein, and mRNA expression increase upon double hit-induced EMyT. A, LLC-PK1 cells were treated as indicated for
72 h and then loaded with 50 ␮M DCF-DA as described under “Experimental Procedures.” DCF fluorescence was determined after the indicated times and
normalized to the total cellular protein. Lines represent mean fluorescence units (n ⫽ 3). B, values from A were used to express the rate of fluorescence increase.
Bars represent mean fold relative to untreated cells (n ⫽ 3). C, LLC-PK1 cells were treated for 48 h with one or both stimuli of the two-hit scheme, as indicated
(control, serum-free; TGF␤, 5 ng/ml; LCM, low calcium medium). Lysates were then subjected to Western blotting for Nox4 and GAPDH. D, LLC-PK1 cells were
treated as indicated for either 24 or 72 h. mRNA was extracted, and qPCR was performed to determine relative amounts of Nox4 mRNA. Bars represent mean
fold increase in Nox4 mRNA levels (n ⫽ 3). Note that the y axis is logarithmic, and data are normalized to 24 h LCM ⫹ TGF␤ condition to show fold changes,
because this was always readily detectable although the basal levels were often near the threshold of detectability.
induced weak Nox4 protein expression. However, simultane- to UUO for 1, 3, or 7 days (Fig. 2D). A 3-fold significant increase
ous application of these stimuli led to robust Nox4 expression, was seen as early as day 3. Thus, although the exact role of Nox4
indicating strong synergy between these inputs (Fig. 1C). in the injured tubular epithelium as well as the role of the epi-
Accordingly, 24 h of combined treatment significantly in- thelium in fibrogenesis remain a matter of debate (see “Discus-
creased Nox4 mRNA (⬎10-fold), which continued to grow fur- sion”), these observations indicate that fibrosis is associated
ther by 72 h (Fig. 1D). Thus, in kidney epithelial cells, the two- with enhanced tubular Nox4 expression and validate the two-
hit condition induces Nox4 expression at the transcriptional hit model as a relevant in vitro approach to address the under-
level, the onset of which precedes EMyT. lying mechanisms.
Next, we wished to examine whether epithelial Nox4 overex- Having established robust Nox4 expression under two-hit
pression occurs in an animal model of kidney fibrosis (Fig. 2). conditions, we asked whether Nox4 is a significant contributor
UUO appeared particularly relevant, as both TGF␤-induced to ROS production in this setting. To test this, cells were trans-
Smad3 activation (43) and heightened Rho signaling (44) fected with NR or Nox4-specific siRNA, the latter of which
(partly due to ensuing mechanical stretch) have been impli- potently (⬎70%) inhibited Nox4 protein expression (Figs. 3A
cated as key pathogenic mechanisms in this fibrosis model. As and also 8C). Subsequently, cells were treated according to the
expected, UUO induced strong expression of fibrotic markers two-hit scheme followed by DCF fluorescence measurements
(e.g. fibroblast specific protein-1 (FSP-1) and SMA), which was (Fig. 3B). Nox4 siRNA-transfected and LCM ⫹ TGF␤-treated
accompanied by a remarkable rise in total Nox4 protein levels cells showed significantly suppressed accumulation of DCF fluo-
(Fig. 2A). Importantly, immunofluorescence staining of kidney rescence compared with their NR siRNA-transfected counter-
sections revealed that the most prominent increase in Nox4 parts (Fig 3B, panels i–iii). Nox4 silencing reduced the rate of
expression occurred in the tubular epithelium (Fig. 2B). To sub- ROS generation by ⬇50% (Fig. 3C), which nonetheless re-
stantiate tubular Nox4 changes by an independent method, we mained higher than that of the non-stimulated control. Taken
performed laser capture microdissection. Using a variety of together, these results show that ROS production is heightened
tubular (Tamm Horsfall protein and basic amino acid trans- during EMyT, and a substantial portion of this effect is due to
porter), glomerular (nephrin), and interstitial (collagen I) mark- the ensuing Nox4 expression.
ers, we verified the successful isolation of the tubular compart- Nox4 Expression Is Regulated by Myocardin-related Tran-
ment (Fig. 2C). Nox4 mRNA was then determined by qPCR in scription Factor—TGF␤ has been described to stimulate Nox4
tubular samples obtained from sham animals or mice exposed expression in mesenchymal cells, presumably through SBE in


FIGURE 2. Nox4 protein levels and mRNA are up-regulated in the tubular compartment during renal fibrogenesis. A, lysates were prepared from the left
kidney of sham animals or mice challenged with UUO for the indicated times. Western blotting was used to probe for Nox4, SMA, FSP-1, as well as tubulin as
a loading control. Sham controls are 14 days post-intervention. B, kidneys were collected from mice subject to the indicated intervention. Sections were
mounted and stained for Nox4 and DAPI. T denotes the tubular lumen. Scale bars, 10 ␮m. C, verification of successful separation of the tubular and interstitial
compartments. Approximately 100 laser spots were collected for each sample from Sham (S) or 7-day UUO (U) mice; tub, tubular samples; int, interstitial
samples; wk, whole kidney. Samples were analyzed by end-point RT-PCR (35 cycles) for various markers. Tubular markers: Tamm Horsfall protein (THP) and basic
amino acid transporter (BAT); glomerular marker, nephrin; interstitial marker, collagen I. Control (Cont), no cDNA was added to the reaction; M, size markers.
Note that interstitial areas from UUO mice contain no tubular or glomerular markers. The slight tubular signal in sham animals is due to the fact that there is very
little interstitium among the tubules in healthy mice. D, mice were subject to indicated treatments, and subsequently tubular remnants were isolated using
LCMi as described under “Experimental Procedures.” Nox4 mRNA levels were then measured using qPCR analysis. Bars represent mean fold changes in
expression (n ⫽ 3).
the Nox4 promoter (39, 45). However, as shown above, TGF␤ effect. We compared the activation of the Nox4 promoter in
alone was insufficient to elicit well detected Nox4 protein cells transfected with NR or MRTF siRNA (Fig. 4C). Elimina-
expression in tubular cells, indicating that Smad3 signaling is tion of MRTF significantly mitigated the two-hit-induced acti-
qualitatively or quantitatively insufficient for this response in vation of the promoter, nearly abolishing the potentiating effect
the intact epithelium. Searching for a mediator, MRTF ap- of LCM. Moreover, an inactivating mutation of the CArG box
peared as an attractive candidate because of the following: 1) we had a similar effect, i.e. it significantly suppressed the synergy
have shown that MRTF, downstream to RhoA/Rac1 signaling, between TGF␤ and LCM (Fig. 4D). Taken together MRTF, act-
is indispensable for EMyT (8, 10, 40) and TGF␤ prolongs and ing through the identified CArG box, is a significant contribu-
augments the contact disruption-induced nuclear accumula- tor to Nox4 up-regulation.
tion of MRTF (8); 2) Rho has been recently implicated as an We argued that if MRTF is indeed critical for Nox4 expres-
important input for fibroblast-myofibroblast transition (46); sion, and Nox4 expression is a significant mechanism for the
and 3) we noted a CArG box in the Nox4 promoter (Fig. 4C, red ensuing rise in ROS production, then MRTF down-regulation
letters). Based on this rationale, we tested whether MRTF might should reduce ROS generation. To test this assumption,
contribute to Nox4 expression in the epithelium. We efficiently changes in DCF fluorescence were monitored in cells trans-
down-regulated both isoforms of MRTF (Fig. 4A) and com- fected with NR or MRTF-specific siRNA and exposed to the
pared stimulus-induced changes in Nox4 expression in NR or individual and combined stimuli of the two-hit scheme (Fig. 4,
MRTF siRNA-transfected cells. MRTF silencing abolished both E, panels i–iii, and F). In agreement with our hypothesis, MRTF
LCM-induced and LCM ⫹ TGF␤-induced Nox4 protein ex- down-regulation significantly inhibited the extent (Fig. 4E,
pression (Fig. 4A). To assess whether MRTF down-regulation panels i–iii) and rate (Fig. 4F) of ROS production, which was
affected Nox4 mRNA, we performed qPCR measurements. most prominent upon combined stimulation. Remarkably, the
MRTF siRNA caused ⬎60% reduction in the two-hit provoked extent of the inhibition obtained by MRTF silencing was essen-
rise in Nox4 mRNA (Fig. 4B). Next, we sought to assess whether tially the same as observed upon Nox4 down-regulation.
MRTF indeed affects the activity of the Nox4 proximal pro- Together, these results suggest that MRTF is required for
moter and whether the putative CArG box is involved in this enhanced ROS generation in the transitioning epithelium.


FIGURE 3. Nox4 expression is a major contributor to ROS production during EMyT. A, LLC-PK1 cells were transfected with 50 nM NR or Nox4-specific siRNA at 60%
confluence. Forty eight hours post-transfection, lysates were collected and Western blotting was performed for the indicated proteins. Bars represent mean fold Nox4
levels relative to 48 h LCM ⫹ TGF␤ (n ⫽ 4). B, LLC-PK1 cells were transfected with 50 nM NR (panel i) or Nox4 siRNA (panel ii) at 60% confluence followed by the indicated
treatment for 24 h. Cells were subsequently incubated with 50 ␮M DCF-DA, and fluorescence was monitored as in Fig. 1. Data are expressed as mean fluorescence units
(n ⫽ 3) for each condition. The functions representing the 24-h LCM ⫹ TGF␤ condition from panels i and ii were plotted on the same graph (panel iii). C, values from (B,
panel iii) were used to calculate the rate of fluorescence increase. Bars represent mean fold relative to control (NR siRNA)-transfected cells (n ⫽ 3).
Given that MRTF is involved in Nox4 expression and ROS conducted on fibroblasts emphasize the key importance of
production, we surmised that these processes should be poten- Smad3-dependent signaling through an SBE in the Nox4 pro-
tiated by enhanced F-actin polymerization, a key regulator of moter (39), and we found that Smad3 is also required for this
MRTF localization. An intriguing aspect of this possibility is process in the transitioning epithelium (30). To address the
that actin polymerization and augmented oxidative stress have above question, initially we confirmed the need for the MRTF/
been proposed to be coupled (32); however, the underlying SRF axis for the regulation of the Nox4 promoter using yet
mechanisms remained elusive. To test this assumption, we another (pharmacological) approach. CCG-1423 is a potent
treated the cells with the actin polymerizing drug, JK, and inhibitor of MRTF or the MRTF/SRF interaction (47). Pretreat-
TGF␤ alone or in combination. Although these stimuli had ment with this drug strongly reduced the activity of the Nox4
modest effect in themselves, their combination resulted in a promoter induced by the double hit scheme compared with
significant activation of the Nox4 promoter (Fig. 5A) and a large vehicle control (Fig. 6A). Thus, in agreement with the siRNA
increase in Nox4 mRNA level (Fig. 5B). Importantly, siRNA- data, the full-length promoter is sensitive to the pharmacolog-
mediated down-regulation of MRTF suppressed the JK ⫹ ical inhibition of MRTF. Next, we compared the double hit-
TGF␤-induced rise in Nox4 mRNA by 70% (Fig. 5C). To mon- induced activation of various truncation constructs of the Nox4
itor the functional consequences of JK treatment, DCF fluores- promoter. The shortest construct tested (⫺741), which lacks
cence was followed in the absence and presence of this drug and both the SBE and the CArG box, showed much less (only
TGF␤. JK itself promoted ROS production, which was further marginal) activation (Fig. 6B). Interestingly, the construct
enhanced by TGF␤ (which alone had no effect) (Fig. 5D). Sim- that contains the CArG box but not the SBE (⫺1434) exhib-
ilar results were obtained in cells transfected with NR siRNA ited a similarly weak response (Fig. 6B). Furthermore, over-
(Fig. 5E). In contrast, Nox4-specific siRNA reduced the effect of expression of MRTF (which can strongly induce the SMA
JK and completely prevented any further stimulation by TGF␤ promoter) failed to activate the full-length Nox4 promoter
(Fig. 5F). Accordingly, ROS production was significantly higher (Fig. 6C). Together, these results show that SRF/MRTF,
in NR siRNA-transfected than in Nox4 siRNA-transfected cells although essential, are not sufficient for Nox4 promoter acti-
(Fig. 5G). Taken together, JK (and thus actin polymerization) vation, implying that these factors play an indispensable,
and TGF␤ synergistically provoke Nox4 expression, and JK permissive role in this process.
augments ROS production partially through Nox4. TAZ and YAP Contribute to the Regulation of Epithelial Nox4
Although these results imply that MRTF is necessary for Expression—The fact that TGF␤ (and Smad3 signaling) is nec-
Nox4 expression, it remained an open question whether MRTF essary for Nox4 expression together with our finding that epi-
activation is sufficient for this process. In fact, previous studies thelial Nox4 expression also requires contact injury prompted

FIGURE 4. MRTF is necessary for Nox4 expression and ROS production during EMyT. A, LLC-PK1 cells were transfected with 50 nM NR or MRTF A ⫹ B siRNA
at 60% confluence. Upon reaching confluence, cells were treated as indicated for 48 h followed by cell lysis and Western blotting for the indicated proteins. Bars
represent fold change in Nox4 levels relative to those obtained after 48 h of LCM ⫹ TGF␤ treatment (n ⫽ 3). B, LLC-PK1 cells were transfected with 50 nM NR or
MRTF A ⫹ B siRNA at 60% confluence and then treated as indicated for 72 h followed by mRNA extraction. qPCR was used to determine Nox4 mRNA levels. Data
were normalized to mRNA levels obtained after LCM ⫹TGF␤ treatment. Bars represent mean fold (n ⫽ 3). C, LLC-PK1 cells were transfected with the Nox4
luciferase reporter (Nox4-Luc) and the indicated siRNA at 60% confluence. Upon reaching confluence, cells were treated according to the two-hit scheme as
indicated for 24 h, followed by the determination of luciferase activity. Bars represent mean fold change relative to control (n ⫽ 6). The CArG box is shown above
the graph. SBE designates the previously described Smad-binding element. D, LLC-PK1 cells were transfected with the wild type (WT) or CArG mutant Nox4
promoter (as shown above the graphs) at 60% confluence and upon reaching confluence were treated as indicated for 24 h followed by measurement of
luciferase activity. Bars represent mean fold change in luciferase activity relative to control (n ⫽ 9). E, LLC-PK1 cells were transfected with 50 nM NR siRNA (panel
i) or 50 nM MRTF A⫹B siRNA (panel ii) as above. Cells were subsequently incubated with 50 ␮M DCF-DA, and fluorescence was measured (n ⫽ 3) for each
condition. For better comparability, values obtained in the LCM ⫹ TGF␤-treated NR-transfected (panel i) and MRTF A ⫹ B siRNA-transfected (panel ii) samples
were plotted on the same graph (panel iii) (n ⫽ 3). F, rate of fluorescence increase was determined from E, panel iii (n ⫽ 3).


FIGURE 5. Pharmacologically provoked actin polymerization synergizes with TGF␤ to induce Nox4 promoter activation, mRNA expression, and ROS
production in an MRTF-dependent manner. A, LLC-PK1 cells were transfected with the Nox4-Luc promoter and upon reaching confluence were exposed to
either vehicle (DMSO) or the actin polymerization-inducing drug jasplakinolide (1 ␮M) in the absence or presence of TGF␤ for 24 h. Normalized Nox4-luciferase
activities were then determined under each condition. Data are expressed as fold change relative to control, mean (n ⫽ 3). B, LLC-PK1 cells were treated as in
A followed by qPCR analysis of Nox4 mRNA levels. Data are expressed as fold change in mRNA relative to the level obtained upon TGF␤ exposure (n ⫽ 3). C,
LLC-PK1 cells were transfected with 50 nM NR or MRTF A⫹B siRNA and then treated as indicated for 24 h. Subsequently, Nox4 mRNA was determined by qPCR
analysis. Note that MRTF down-regulation effectively suppressed Nox4 mRNA expression induced by JK ⫹ TGF␤, mean (n ⫽ 3). D–G, Nox4 expression
contributes to JK-promoted ROS generation. D, to assess the impact of actin polymerization on ROS production, LLC-PK1 cells were treated for 24 h with DMSO
or 1 ␮M JK in the absence or presence of TGF␤. After DCF-DA loading, fluorescence was followed in the indicated time range. Data are expressed as mean (n ⫽
3). E and F, cells were transfected with either 50 nM NR (E) or Nox4 siRNA (F) and treated and processed as in D. G, LLC-PK1 cells were treated with TGF␤ and 1
␮M JK for 24 h with the addition of either NR or Nox4 siRNA as in E and F. Following treatment, cells were loaded with DCF-DA as above, and fluorescence was
measured for the indicated time range. Date are expressed as mean (n ⫽ 6). Note significant inhibition in ROS production in Nox4 down-regulated cells in which
the synergy between JK and TGF␤ was lost.
us to ask whether the TGF␤-dependent inputs might also be increase in TAZ and (to a smaller extent) in YAP protein level,
contact-sensitive. The Hippo pathway effector TAZ and YAP which was further stimulated by contact disruption (Fig. 7A).
are good candidates to link contact disruption to enhanced The mechanism of this effect is subject to ongoing studies and
Smad3 signaling, because these factors translocate to the will not be addressed here (see under “Discussion”). Impor-
nucleus upon the uncoupling of intercellular junctions (41, 48) tantly, transfection with TAZ and YAP siRNA efficiently sup-
and can act as nuclear Smad3 retention factors (48). Further- pressed both the basal level and the stimulus-induced increase
more, crosstalk between TGF␤ and TAZ/YAP signaling has in the expression of these proteins and concomitantly pre-
been described, and depending on the target, these pathways vented the double hit-provoked induction of Nox4 expression
may be synergistic or antagonistic (49). To test whether TAZ (Fig. 7A). To substantiate this finding, we measured the activity
and YAP might be involved in the up-regulation of Nox4 in the of the Nox4 promoter in cells transfected with NR or TAZ and
transitioning epithelium, we examined Nox4 expression upon YAP siRNA. Knockdown of TAZ and YAP (TAZ/YAP) expres-
down-regulation of both TAZ and YAP (Fig. 7). In cells trans- sion led to a significant drop in the basal promoter activity,
fected with NR siRNA, TGF␤ itself caused a substantial abolished the TGF␤-induced rise, and accordingly strongly


FIGURE 6. MRTF is necessary but not sufficient to drive the Nox4 promoter. A, LLC-PK1 cells were grown to 60% confluence and transfected with the Nox4
Luc construct. Upon reaching confluence, cells were treated as indicated for 24 h in the presence of DMSO or 4 ␮M CCG-1432. Subsequently, lysates were
collected, and luciferase activity was measured. Bars represent mean fold luciferase activity relative to control (DMSO) normalized to Renilla (n ⫽ 3). B, LLC-PK1
cells were grown to confluence and transfected with the indicated promoter constructs (WT, ⫺741 and ⫺1434 Nox4-Luc). Upon reaching confluence, cells
were treated as indicated for 24 h, and lysates were collected for measurement of luciferase activity. Bars represent mean fold change in luciferase activity
relative to control (Cont) normalized to Renilla (n ⫽ 3). C, LLC-PK1 cells were grown to 60% confluence and transfected with pCMV5 or HA-MRTF in addition to
the Nox4 Luc construct. Cells were subsequently serum-starved for 24 h, and lysates were collected for luciferase activity measurement. Bars represent mean
fold luciferase activity relative to control (pCMV5) normalized to Renilla (n ⫽ 3).
suppressed the level achieved by the two-hit stimulation (Fig. (51). As expected, LCM ⫹ TGF␤ triggered SMA expression in
7B). Given that suppression of TAZ/YAP was sufficient for vehicle-treated control cells. This effect was entirely blocked by
abrogation of Nox4 expression in vitro, we next determined VAS2870 (Fig. 8A). Subsequent experiments using the SMA
whether this was also the case in a mouse model of obstructive promoter luciferase reporter revealed that NADPH oxidases
nephropathy using the UUO model. Mice were treated with the are critical for the activation of the SMA promoter, as VAS2870
TAZ/YAP inhibitor verteporfin (VP) (50). Under control con- strongly suppressed both the LCM-induced response and the
ditions, UUO mice demonstrated up-regulation of tubular synergistic action of LCM ⫹ TGF␤ (Fig. 8B). To assess whether
Nox4 as assessed by immunofluorescence analysis (Fig. 7C) and Nox4 is a significant contributor to the transition, similar
also had significantly increased levels of tubular Nox4 mRNA experiments were performed using NR or Nox4-specific
relative to sham mice (Fig. 7D). In comparison, treatment with siRNA. We followed two critical features, myosin phosphory-
VP led to substantial reduction in both of these parameters (Fig. lation and SMA protein expression, both of which were
7, C and D). Taken together, these results demonstrate that strongly induced by LCM ⫹ TGF␤ and nearly abolished by
TAZ and YAP are necessary for efficient Nox4 expression in Nox4 down-regulation (Fig. 8C). In accordance with these find-
tubular cells challenged with fibrogenic stimuli both in vitro ings, silencing of Nox4 significantly suppressed the activation of
and in vivo. the SMA promoter in the epithelium (Fig. 8D). Taken together,
NADPH Oxidase Activity and Nox4 Expression Are Necessary Nox4 is a chief mediator of EMyT.
for the Myogenic Shift of the Epithelium—In non-epithelial MRTF Plays a Critical Role in Nox4 Expression in Mesenchy-
progenitors, in which TGF␤ is sufficient for myofibroblast tran- mal Myofibroblast Progenitors—Recent research suggests that
sition, ROS production was shown to be necessary for the the major source of myofibroblasts during experimental fibro-
induction of key myofibroblast features, including increased genesis is not the epithelium but various mesenchymal progen-
contractility and SMA expression (19, 21, 22). To determine itors, such as interstitial fibroblasts and possibly pericytes
whether ROS are also essential for double hit-provoked epithe- (52–54). Nox4 is induced in response to TGF␤ in these mesen-
lial-myofibroblast transition, we initially tested the effect of the chymal cell types and is necessary for acquisition of key myofi-
general NADPH oxidase inhibitor VAS2870 (Fig. 8, A and B) broblast features (23). We therefore sought to assess whether


FIGURE 7. TAZ and YAP are necessary for Nox4 expression. A, LLC-PK1 cells were grown to 60% confluence and transfected with either 100 nM NR or TAZ ⫹
YAP siRNA. Upon reaching confluence, cells were treated according to the individual or combined stimulation of the two-hit scheme for 48 h. Lysates were
collected and analyzed by Western blotting for YAP, TAZ, Nox4, and tubulin. B, LLC-PK1 cells at 60% confluence were transfected with 100 nM NR or TAZ ⫹ YAP
siRNA along with the Nox4-Luc construct. Upon reaching confluence, cells were treated as indicated for 24 h, and luciferase activity was measured. Data are
expressed as fold change in normalized luciferase activity compared with the untreated control (n ⫽ 3). C, kidneys were collected from Sham, UUO, and UUO
⫹ VP mice 7 days post-surgery. Sections were mounted and stained for Nox4 and DAPI. T denotes the tubular lumen. Scale bars, 10 ␮m. D, mice were subject
to the indicated treatments, and subsequently tubular remnants were isolated using LCMi as described under “Experimental Procedures.” Nox4 mRNA levels
were then measured using qPCR analysis. Bars represent mean fold changes in expression (n ⫽ 3).
MRTF signaling was also an important regulator of Nox4 in that the Hippo pathway effectors TAZ/YAP are required for
mesenchymal cells. To this end, we first used 10T1/2 fibroblast/ epithelial up-regulation of Nox4 both in vitro and in vivo; and 3)
pericyte-like cells, in which TGF␤ induced substantial up-reg- that in an epithelial context TGF␤ is insufficient to provoke
ulation of the Nox4 protein (Fig. 9A). Importantly, pretreat- Nox4 expression, and a second input is also required (two hit
ment of the cells with the MRTF/SRF inhibitor CCG-1423 model) that may emanate from the uncoupling of the intercel-
completely abrogated the TGF␤-provoked increase in Nox4 lular contacts or other mechanical stimuli, which can activate
protein expression (Fig. 9B) and eliminated the activation of the both MRTF and TAZ/YAP signaling.
Nox4 promoter (Fig. 9C). To verify that these effects reflect ROS in general and Nox4 in particular have been shown to
promoter activation in a CArG box-specific manner, we com- play important roles in a variety of physiological and patholog-
pared the impact of TGF␤ on the WT and CArG box-mutated ical processes (55). Because Nox4 activity is primarily regulated
Nox4 promoter (Fig. 9D). The mutation rendered the promoter
via Nox4 expression (56, 57), it is imperative to understand the
completely insensitive to TGF␤. Similar results were obtained
inputs and mechanisms that control this process. Several tran-
in normal rat kidney fibroblasts (NRK-F cells) (Fig. 9E). Taken
scription factors and the corresponding cis-elements have been
together, these results imply that, similar to the findings in
implicated in transcriptional regulation of Nox4, implying the
tubular epithelial cells, MRTF and MRTF/SRF-mediated CArG
multifactorial nature of this process. These include Smad3
box-dependent Nox4 expression plays an important role also in
myofibroblast transition from mesenchymal progenitors. (39, 45), which through AP1/Smad-binding elements (39)
couples Nox4 to TGF␤ signaling and thereby fibrogenesis;
Discussion Hif1␣ (58), Nrf2 (59) and Oct-1 (60), which link Nox4
This study allows new mechanistic insight into the regulation expression to ROS generation per se under conditions of
of Nox4, particularly in an epithelial setting. The key new find- hypoxia, hyperoxia, and shear stress; NF␬B (61) and STAT
ings include the following: 1) that MRTF is a major permissive proteins (62), mediators of inflammatory gene expression;
regulator of Nox4 expression, a mechanism that unravels a new and E2F (63), a regulator of proliferation. This study enriches
link between actin polymerization and the regulation of cellular this picture by two important findings indicating that MRTF
redox state, present both in epithelial cells and fibroblasts; 2) and TAZ/YAP, two main cytoskeleton-regulated transcrip-


FIGURE 8. EMyT is dependent upon Nox4 expression and ROS production. A, medium of LLC-PK1 cells was supplemented with vehicle (DMSO) or the
general NADPH inhibitor VAS2870 (10 ␮M), and cells were then treated as indicated for 72 h. Lysates were collected, and Western blotting was performed for
the indicated proteins. B, LLC-PK1 cells were grown to 60% confluence and transfected with the SMA-Luc promoter construct. Upon reaching confluence, cells
were treated as indicated for 24 h, and the medium was supplemented with either DMSO or 10 ␮M VAS870. Luciferase activity was measured, normalized to
Renilla, and expressed as fold change relative to control (n ⫽ 3). C, LLC-PK1 cells were transfected with 50 nM NR or Nox4 siRNA at 60% confluence. Upon
reaching confluence, cells were treated for 72 h as indicated. Lysates were then collected, and Western blotting was performed on indicated proteins. D,
LLC-PK1 cells were co-transfected with either NR or Nox4 siRNA along with the SMA-Luc reporter system. Upon reaching confluence, cells were treated as
indicated for 48 h, and luciferase activity was measured as above (n ⫽ 3).
tional coactivators, are important novel regulators/modula- It is important to note that changes in actin polymerization may
tors of Nox4 expression. have acute and direct effects on ROS production differing from
The role of the MRTF/SRF pathway is evidenced by the facts the transcriptional effects described herein. For example, actin
that the Nox4 promoter harbors a perfect CArG box and that depolymerization was shown to promote p47phox-dependent,
down-regulation or pharmacological inhibition of MRTF or the Nox2-mediated superoxide production in neutrophils (65) and
MRTF/SRF interaction or mutation of the CArG box abrogates endothelial cells (66). The underlying mechanism is likely
the activation of the Nox4 promoter, suppresses Nox4 mRNA 2-fold as follows: actin depolymerization enhances receptor-
and protein expression, and reduces the ensuing ROS produc- induced signaling pathways leading to the assembly of Nox2
tion in response to various fibrogenic stimuli both in epithelial from cytosolic and membrane-bound components, and it may
and mesenchymal cells. This recognition is a significant step also remove physical constraints from the association of these.
forward because it directly links the state of the cytoskeleton to Indeed, our earlier studies indicated that F-actin depolymeriza-
the redox state of the cell. Previous studies have revealed that tion triggers several tyrosine kinase pathways (67), which may
enhanced actin polymerization (reduced actin dynamics) leads participate in the regulation of NADPH oxidase enzymes. In
to augmented ROS production, which in turn promotes cellular contrast to Nox2, Nox4 activity does not require the association
aging and apoptosis (32). Although the underlying mechanisms of the oxidase with cytosolic partners. Thus, cytoskeleton orga-
remained largely undefined, actin polymerization was pro- nization may exert differential effects both in terms of timing
posed to alter mitochondrial ROS production in yeast (64). (acute regulation versus transcription) and in terms of isoform
Here, we show that actin polymerization, directly triggered by specificity.
JK, potentiates TGF␤-induced Nox4 mRNA expression and Our findings also explain, at least in part, the regulation of
ROS production in the epithelium in an MRTF-dependent Nox4 by Rho GTPase. A recent elegant paper reported that
manner. We therefore propose that MRTF, whose nucleocyto- TGF␤-induced Rho/Rho kinase activation is an essential
plasmic traffic is regulated by actin polymerization (14), serves upstream mechanism for Nox4 expression and activity in renal
as a key mediator through which cytoskeletal changes impact fibroblasts and their transition to myofibroblasts (31). Mecha-
the redox state. This represents a novel long term (transcrip- nistically, the authors have implicated polymerase (DNA-di-
tional) mechanism of communication between these systems. rected) ␦-interacting protein 2 (Poldip2), a recently described


FIGURE 9. Nox4 expression also requires MRTF activity in mesenchymal myofibroblast progenitors. A, 10T1/2 cells were treated as indicated for 48 h.
Subsequently, Western blotting was performed for Nox4 and tubulin. B, 10T1/2 cells were subjected to indicated treatments for 48 h in the presence of either
DMSO or 4 ␮M CCG-1432. Following treatment, lysates were collected, and Western blotting was performed for Nox4 and ␤-actin. C, 10T1/2 cells were
transfected with the Nox4-luc promoter at 60% confluence. One day later, cells were treated as indicated for 24 h in presence of DMSO or 4 ␮M CCG-1432.
Subsequently luciferase activity was measured (n ⫽ 3). D and E, 10T1/2 cells (D) were transfected with the WT or CArG mutant Nox4 promoter at 60%
confluence. One day later cells were treated as indicated for 24 h, and luciferase activity was determined (n ⫽ 6). E, NRK-F cells were transfected with the WT or
CArG mutant Nox4-luc construct at 60% confluence. One day later cells were treated as indicated for 24 h and luciferase activity was measured (n ⫽ 3).
Nox4 enhancer (68), which was also induced by Rho. However, stream target and an upstream activator of a particular path-
Poldip2 expression does not, a priori, explain the increase in way (71). Thus, whereas TGF␤ is a prime inducer of Nox4
Nox4 protein levels. Because Rho is a major upstream regulator expression, Nox4-derived ROS facilitated TGF␤ signaling both
of MRTF and, as our previous studies show, nuclear accumula- by promoting the liberation of active TGF␤ from the latent
tion of MRTF is abolished by Rho kinase inhibition (10), our complex (72) and by enhancing TGF␤-induced Smad2/3 phos-
data provide a straightforward mechanism linking Rho and phorylation (19). Similarly, Nox4 can be driven by and activate
Nox4. Moreover, in silico analysis suggests the presence of a NF␬B (73) as well as Hif1␣ and -2␣ (74, 75). Our findings
putative CArG box in the promoter of Poldip2 as well. Indeed, together with earlier reports extend this notion for the Rho/
although the presence of and requirement for an intact CArG Nox4 relationship as well. Nox4-derived ROS has been sug-
box in the Nox4 promoter is highly suggestive of direct action of gested to promote Rho activation (68), whereas active Rho can
SRF and presumably MRTF at this locus, MRTF/SRF may also induce MRTF translocation, which in turn stimulates Nox4
contribute indirectly by controlling other Nox4 regulators. expression.
Future studies should validate this intriguing possibility. Our Interestingly, the vast majority of the literature has examined
data also imply that MRTF, although necessary, is not sufficient the regulation of Nox4 expression in vascular smooth muscle,
to drive the Nox4 promoter; TGF␤-dependent signaling endothelium, and fibroblasts, although information in the epi-
remains essential. Thus, MRTF is a permissive and potentiating thelium is scarce, despite the fact that the tubules were reported
but not directly inductive factor for this promoter. There is to possess 80% of all Nox4 activity in the kidney (35). Two
precedence for such permissive role of MRTF with regards to additional reasons lend importance to the epithelial context,
VE-cadherin (69) and CCN1 (70) expression. Furthermore, SRF namely, the epithelium differs from the other tissues both in
overexpression alone also failed to drive the promoter (data not terms of the stimuli required for Nox4 induction and in terms of
shown), excluding that the Nox4 promoter is SRF- but not the ensuing (patho)physiological consequences (see below).
MRTF-sensitive. Future work should clarify how TGF␤-acti- Considering the first point, our data show that, similar to the
vated transcription factors synergize with SRF/MRTF to induce induction of EMyT, epithelial Nox4 expression requires two-
Nox4 expression. hit conditions; although TGF␤ is sufficient in other tissues, it
A characteristic feature of Nox4-related signaling is the pres- does not provoke a major increase in Nox4 in the epithelium.
ence of positive feedback loops, wherein Nox4 is both a down- The other prerequisite is the injury of the intercellular contacts,

which translates into enhanced contractility (30). This finding (81). According to this, limited early Nox4-mediated ROS pro-
is concordant with the fact that TGF␤ is a weak and transient duction may be protective because, presumably through Nrf2
Rho activator in the intact epithelium and that prolonged and Hif1␣, it may induce antioxidant genes (80). In addition,
nuclear MRTF accumulation requires both stimuli (8, 10). At Nox4 is a mediator of cellular senescence (82) and autophagy
the same time, MRTF overexpression (or Rho activation, data (83), both of which were proposed to lessen fibrosis (84, 85).
not shown) is also insufficient, and this implies that TGF␤ is However, sustained activation of Nox4 likely contributes to tis-
also indispensable. This is in agreement with our earlier data sue damage and fibrosis. In addition, whether epithelial Nox4
showing that Smad3 down-regulation abrogates Nox4 expres- (at least initially) serves more protective roles, while Nox4
sion under double hit conditions (30). The dependence of Nox4 expression in fibroblasts is pathogenic, remains an open ques-
expression both on TGF␤ and the loss of cell contacts tion, which can be addressed by tissue-specific knockouts.
prompted us to investigate the potential involvement of the Although most studies found increased renal Nox4 expres-
Hippo pathway effectors TAZ and YAP, which, similar to sion during chronic injury (27, 86, 87), progressive loss was also
MRTF, are also regulated by these inputs (41, 48). Indeed, reported (35). We found that epithelial Nox4 increases during
knockdown of TAZ/YAP suppressed Nox4 induction. Similar UUO as detected by immunofluorescence as well as by qPCR
results were obtained in vivo, using the TAZ/YAP inhibitor performed on tubular samples obtained by laser capture micro-
verteporfin. This is likely due to the fact that TAZ/YAP are dissection. In the UUO model, the primary insult is a pressure-
Smad3 nuclear retention factors, which can facilitate TGF␤- induced stretch of the tubular compartment, which then can
induced gene expression (49, 76). Thus, the mechanism under- lead to local cytoskeleton remodeling and thus MRTF activa-
lying the synergy between contact disruption and TGF␤ is tion. This in turn can potentiate tubular Nox4 expression. It is
2-fold: TGF␤ prolongs MRTF nuclear accumulation induced

conceivable that initially this mechanism is adaptive and reno-
by contact disassembly-provoked Rho activation (8), and con- protective, although upon sustained overwhelming injury it
tact injury translocates TAZ/YAP to the nucleus, which in turn becomes fibrogenic. It is noteworthy that the epithelium is only
prolongs TGF␤-induced Smad3 accumulation and gene a minor source of myofibroblasts via EMyT in vivo (52). This
expression. In addition, we noted that TGF␤ (in itself) strongly view assigns a key but indirect role to the epithelium in fibro-
elevates TAZ/YAP levels. Our ongoing studies address the genesis; upon injury it assumes a secretory phenotype and
mechanism of this intriguing phenomenon. In any case, this primes the underlying mesenchyme in a paracrine fashion (53,
finding further confirms that the intact epithelium remains 88 –90). We propose that MRTF is a key mediator of both the
responsive to TGF␤ (although EMyT does not develop) and partial (secretory) and the full-blown (EMyT) phenotype shift.
that TGF␤ may prime the cells for injury-induced Nox4 expres- In fact, our ongoing studies suggest that a subtle interplay
sion through this indirect pathway, too. Importantly, TAZ/YAP between the MRTF and TAZ/YAP pathways may be responsi-
are regulated by many fibrogenic conditions, including matrix ble for preventing the shift to go “all the way” to a myogenic
stiffness (77, 78) and hyperglycemia (79). Clearly, the involve- (SMA-expressing) stage. In any case, the recognition of the role
ment of TAZ and YAP in the regulation of the redox state of the of the cytoskeleton-MRTF/SRF axis in the regulation of Nox4
cell warrants further mechanistic studies. expression both in epithelial and mesenchymal cells should
Finally, the pathophysiological role of Nox4 should be con- facilitate our understanding of the biology of this intriguing
sidered. There is strong evidence that Nox4 is up-regulated dur- enzyme, which seems to be involved both in preserving tissue
ing and is required for fibroblast-myofibroblast transition in integrity and promoting fibrosis.
vitro (19, 21–23). Moreover, Nox4 down-regulation by siRNA
or antisense oligonucleotides proved to be protective in a lung Author Contributions—M. R. designed, performed, and analyzed
fibrosis model (22) and reduced matrix deposition in diabetic experiments shown in Figs. 1 and 3–9, wrote part of the manuscript,
kidney disease (DKD) (27), whereas new Nox1/4-selective and prepared the figures; J. F. B. designed, conducted, and analyzed
pharmacological inhibitors exerted potent kidney-protective qPCR experiments shown in Figs. 1, 4, and 5 with the participation of
and antifibrotic effects in DKD models (28, 29). All these find- T. E. T. K. P. S. generated construct by mutagenesis an provided
ings suggest that Nox4 protein levels rise under fibrogenic (pre- experimental advice; Q. D. performed the laser capture microdissec-
tion studies; S. G. S. and D. A. Y. were involved in the in vivo exper-
dominantly diabetic) conditions, and this contributes to the
iments and data analysis; K. S. and S. F. P. contributed to the design
ensuing nephropathy and fibrosis. Consistent with this sce-
of studies, critical interpretation of the data, and the final assembly of
nario and our new findings, a recent paper reports that MRTF- the manuscript; A. K. conceived and conceptualized the work and
deficient mice are largely protected against DKD (16). How- wrote the majority of the manuscript. All authors reviewed the
ever, results obtained with whole body Nox4 knock-out mice results and approved the final version of the manuscript.
are controversial or more complex. One study found that Nox4
deletion indeed mitigated the progression of DKD on the Acknowledgment—We are indebted to Dr. A. Manea for valuable
apoE⫺/⫺ background (29). However, Babelova et al. (35) using discussions.
four chronic kidney injury models detected no protective effect
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Myocardin-related Transcription Factor Regulates Nox4 Protein Expression:
LINKING CYTOSKELETAL ORGANIZATION TO REDOX STATE
Matthew Rozycki, Janne Folke Bialik, Pam Speight, Qinghong Dan, Teresa E. T.
Knudsen, Stephen G. Szeto, Darren A. Yuen, Katalin Szászi, Stine F. Pedersen and
András Kapus
J. Biol. Chem. 2016, 291:227-243.
doi: 10.1074/jbc.M115.674606 originally published online November 10, 2015
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