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. 2010 Jan;21(1):93-102.
doi: 10.1681/ASN.2009020146. Epub 2009 Nov 19.

NAD(P)H oxidase mediates TGF-beta1-induced activation of kidney myofibroblasts

Corry D Bondi  1 , Nagaraj Manickam, Duck Yoon Lee, Karen Block, Yves Gorin, Hanna E Abboud, Jeffrey L Barnes
Affiliations

NAD(P)H oxidase mediates TGF-beta1-induced activation of kidney myofibroblasts

Corry D Bondi et al. J Am Soc Nephrol. 2010 Jan.

Abstract

TGF-beta1 expression closely associates with activation and conversion of fibroblasts to a myofibroblast phenotype and synthesis of an alternatively spliced cellular fibronectin variant, Fn-ED-A. Reactive oxygen species (ROS), such as superoxide, which is a product of NAD(P)H oxidase, also promote the transition of fibroblasts to myofibroblasts, but whether these two pathways are interrelated is unknown. Here, we examined a role for NAD(P)H oxidase-derived ROS in TGF-beta1-induced activation of rat kidney fibroblasts and expression of alpha-smooth muscle actin (alpha-SMA) and Fn-ED-A. In vitro, TGF-beta1 stimulated formation of abundant stress fibers and increased expression of both alpha-SMA and Fn-ED-A. In addition, TGF-beta1 increased both the activity of NADPH oxidase and expression of Nox2 and Nox4, homologs of the NAD(P)H oxidase family, indicating that this growth factor induces production of ROS. Small interfering RNA targeted against Nox4 markedly inhibited TGF-beta1-induced stimulation of NADPH oxidase activity and reduced alpha-SMA and Fn-ED-A expression. Inhibition of TGF-beta1 receptor 1 blocked Smad3 phosphorylation; reduced TGF-beta1-enhanced NADPH oxidase activity; and decreased expression of Nox4, alpha-SMA, and Fn-ED-A. Diphenyleneiodonium, an inhibitor of flavin-containing enzymes such as the Nox oxidases, had no effect on TGF-beta1-induced Smad3 but reduced both alpha-SMA and Fn-ED-A protein expression. The Smad3 inhibitor SIS3 reduced NADPH oxidase activity, Nox4 expression, and blocked alpha-SMA and Fn-ED-A, indicating that stimulation of myofibroblast activation by ROS is downstream of Smad3. In addition, TGF-beta1 stimulated phosphorylation of extracellular signal-regulated kinase (ERK1/2), and this was inhibited by blocking TGF-beta1 receptor 1, Smad3, or the Nox oxidases; ERK1/2 activation increased alpha-SMA and Fn-ED-A. Taken together, these results suggest that TGF-beta1-induced conversion of fibroblasts to a myofibroblast phenotype involves a signaling cascade through Smad3, NAD(P)H oxidase, and ERK1/2.

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Figures

Figure 1.
Figure 1.
TGF-β1 induces a ROS-dependent kidney fibroblast conversion to a myofibroblast phenotype. (A and B) TGF-β1 induces a myofibroblast phenotype as indicated by an increase in α-SMA and Fn-ED-A protein expression by immunofluorescence microscopy (A) and Western blotting (B). DPI inhibits TGF-β1–enhanced expression of both proteins, implicating a role for ROS in kidney myofibroblast activation. Data are means ± SEM from three independent experiments. *P < 0.05 versus control; #P < 0.05 versus TGF-β1; §P < 0.05 versus TGF-β1+inhibitor.
Figure 2.
Figure 2.
Effect of TGF-β1 on production of ROS in kidney fibroblasts is shown. Fibroblasts were serum-starved and treated with 1 ng/ml TGF-β1 for the indicated periods. 2′,7′-dichlorodihydrofluorescein (DCF) fluorescence reflecting the relative levels of ROS was imaged with a confocal laser scanning fluorescence microscope. (A through C) Representative photomicrographs show DCF fluorescence in kidney fibroblasts under basal conditions (A), 15 min after addition of TGF-β1 (B), and in cells pretreated with DPI (10 μM) before TGF-β1 (C). (D) TGF-β1 induced a sustained increase in the rate of NADPH oxidase activity (relative luminescent units per minute per milligram of protein) in these cells, peaking at 30 min after exposure as illustrated in a representative graph of three separate experiments. DPI inhibits both ROS detection by DCF (C) and NADPH oxidase activity (D). (E) Western analysis indicates that kidney fibroblasts express Nox2 and Nox4 as early as 5 min after TGF-β1 treatment, which progressively increases over time. Kidney fibroblasts do not express Nox1 or Nox3 homologs. *Nox4 seems to be the predominant homolog expressed by kidney fibroblasts given the amount of lysate loaded onto gels was one fourth that for all other homologs. (HEK: HEK293 cell lysates were used as a positive control for Nox1 and Nox3). (F and G) NADPH oxidase activation was verified in primary kidney fibroblast (A2F11) showing TGF-β1 induction of NADPH oxidase activity in a representative graph (F) and inhibition of growth factor–stimulated α-SMA and Fn-ED-A protein expression by DPI (G).
Figure 3.
Figure 3.
Nox4 mediates kidney myofibroblast activation in response to TGF-β1. siRNA complementary to Nox4 (siNox4) and scrambled siRNA (Scr) were examined in TGF-β1–induced myofibroblast activation. (A) The effect of siRNA in kidney fibroblasts was confirmed by the observation that siNox4 decreased basal Nox4 protein expression relative to Scr siRNA. (B through E) siNox4 also reduced TGF-β1–induced increments in NADPH oxidase activity (B) as well as α-SMA (C and D) and Fn-ED-A (C and E) protein expression, indicating a role for superoxide generation by this homolog in kidney myofibroblast activation. Data are means ± SEM from three independent experiments. *P < 0.05 versus control; #P < 0.05 versus TGF-β1; §P < 0.05 versus TGF-β1+Scr.
Figure 4.
Figure 4.
TGF-β1 stimulates kidney myofibroblast α-SMA and Fn-ED-A expression, NADPH oxidase activity, and Nox4 via TGFBR. To determine a role for NAD(P)H oxidase–derived ROS in TGF-β1 signaling of kidney myofibroblast activation, we used experiments inhibiting TGFR1 with SB and determining expression levels of α-SMA and Fn-ED-A, NADPH oxidase activity, and Nox4 protein. (A through C) SB inhibited TGF-β1–induced increases in α-SMA and Fn-ED-A expression (A) and substantially reduced NADPH oxidase activity shown in a representative graph (B) as well as Nox4 protein expression (C). Data are means ± SEM from three independent experiments. *P < 0.05 versus control; #P < 0.05 versus TGF-β1; §P < 0.05 versus TGF-β1+inhibitor.
Figure 5.
Figure 5.
TGF-β1–induced myofibroblast activation involves Smad3 regulation of NAD(P)H oxidase. To assess the NAD(P)H oxidase–derived ROS relative to p-Smad3, we treated kidney fibroblasts with SB, DPI, or an inhibitor of Smad3 (SIS3) before stimulation with TGF-β1. Smad3 phosphorylation and expression levels of α-SMA, Fn-ED-A, Nox4, and NADPH oxidase activity were examined. (A) TGF-β1 activates pSmad3 by 5 min progressively increasing over 1 h. (B and C) TGF-β1 activation of p-Smad3 is inhibited by SB (B); however, DPI had no effect on TGF-β1–induced Smad3 phosphorylation (C). (D and E) Inhibition of p-Smad3 by SIS3 blocked TGF-β1–induced stimulation of NADPH oxidase activity (D) and Nox4 (E), indicating that NAD(P)H oxidase is downstream of Smad3. (F and G) Similarly, SIS3 inhibited TGF-β1–induced stimulation of α-SMA (F) and Fn-ED-A (G) expression. Data are means ± SEM from three independent experiments. *P < 0.05 versus control; #P < 0.05 versus TGF-β1.
Figure 6.
Figure 6.
ERK1/2 has a central role in Smad3 and NAD(P)H oxidase signaling of TGF-β1–induced myofibroblast activation. (A) TGF-β1 induced a rapid expression of p-ERK1/2 at 5 min, disappearing thereafter, and was absent up to 24 h after stimulation by growth factor; therefore, all subsequent studies were performed at 5 min after stimulation with TGF-β1. (B and C) UO126, an inhibitor of MEK, completely eliminates TGF-β1–induced ERK1/2 phosphorylation (B) and substantially reduces α-SMA and Fn-ED-A protein expression (C). (D) In addition, inhibition of TGFBR with SB blocks p-ERK expression, indicating that TGF-β1–induced myofibroblast activation is partially regulated through this signal transduction molecule. (E) Similarly, DPI inhibits p-ERK, indicating that this signal protein is downstream of TGFBR and ROS. (F) SIS3 inhibition of Smad3 also inhibited p-ERK1/2 expression, supporting a TGFBR/Smad3/ROS/ERK1/2 signaling cascade in TGF-β1 stimulation of kidney myofibroblast activation and matrix synthesis (see Figure 7). Data are means ± SEM from three independent experiments. *P < 0.05 versus control; #P < 0.05 versus TGF-β1; §P < 0.05 versus TGF-β1+inhibitor.
Figure 7.
Figure 7.
Proposed signaling cascade involving Smad3, ROS, and ERK1/2 in TGF-β1–induced kidney myofibroblast activation and matrix synthesis is shown.
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