NAD(P)H oxidase mediates TGF-β1-induced activation of kidney myofibroblasts

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

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Abstract

TGF-β1 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-β1-induced activation of rat kidney fibroblasts and expression of α-smooth muscle actin (α-SMA) and Fn-ED-A. In vitro, TGF-β1 stimulated formation of abundant stress fibers and increased expression of both α-SMA and Fn-ED-A. In addition, TGF-β1 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-β1-induced stimulation of NADPH oxidase activity and reduced α-SMA and Fn-ED-A expression. Inhibition of TGF-β1 receptor 1 blocked Smad3 phosphorylation; reduced TGF-β1-enhanced NADPH oxidase activity; and decreased expression of Nox4, α-SMA, and Fn-ED-A. Diphenyleneiodonium, an inhibitor of flavin-containing enzymes such as the Nox oxidases, had no effect on TGF-β1-induced Smad3 but reduced both α-SMA and Fn-ED-A protein expression. The Smad3 inhibitor SIS3 reduced NADPH oxidase activity, Nox4 expression, and blocked α-SMA and Fn-ED-A, indicating that stimulation of myofibroblast activation by ROS is downstream of Smad3. In addition, TGF-β1 stimulated phosphorylation of extracellular signal-regulated kinase (ERK1/2), and this was inhibited by blocking TGF-β1 receptor 1, Smad3, or the Nox oxidases; ERK1/2 activation increased α-SMA and Fn-ED-A. Taken together, these results suggest that TGF-β1-induced conversion of fibroblasts to a myofibroblast phenotype involves a signaling cascade through Smad3, NAD(P)H oxidase, and ERK1/2.

Original languageEnglish (US)
Pages (from-to)93-102
Number of pages10
JournalJournal of the American Society of Nephrology
Volume21
Issue number1
DOIs
StatePublished - Jan 2010

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Myofibroblasts
NADPH Oxidase
Kidney
Reactive Oxygen Species
Fibroblasts
Oxidoreductases
Phosphorylation
Phenotype
Stress Fibers
Mitogen-Activated Protein Kinase 1
Fibronectins
Superoxides
Small Interfering RNA
Smooth Muscle
Actins
Intercellular Signaling Peptides and Proteins
Enzymes

ASJC Scopus subject areas

  • Nephrology

Cite this

Bondi, C. D., Manickam, N., Lee, D. Y., Block, K., Gorin, Y., Abboud, H. E., & Barnes, J. L. (2010). NAD(P)H oxidase mediates TGF-β1-induced activation of kidney myofibroblasts. Journal of the American Society of Nephrology, 21(1), 93-102. https://doi.org/10.1681/ASN.2009020146

NAD(P)H oxidase mediates TGF-β1-induced activation of kidney myofibroblasts. / Bondi, Corry D.; Manickam, Nagaraj; Lee, Duck Yoon; Block, Karen; Gorin, Yves; Abboud, Hanna E.; Barnes, Jeffrey L.

In: Journal of the American Society of Nephrology, Vol. 21, No. 1, 01.2010, p. 93-102.

Research output: Contribution to journalArticle

Bondi, CD, Manickam, N, Lee, DY, Block, K, Gorin, Y, Abboud, HE & Barnes, JL 2010, 'NAD(P)H oxidase mediates TGF-β1-induced activation of kidney myofibroblasts', Journal of the American Society of Nephrology, vol. 21, no. 1, pp. 93-102. https://doi.org/10.1681/ASN.2009020146
Bondi, Corry D. ; Manickam, Nagaraj ; Lee, Duck Yoon ; Block, Karen ; Gorin, Yves ; Abboud, Hanna E. ; Barnes, Jeffrey L. / NAD(P)H oxidase mediates TGF-β1-induced activation of kidney myofibroblasts. In: Journal of the American Society of Nephrology. 2010 ; Vol. 21, No. 1. pp. 93-102.
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