Akt1 Mediates α-Smooth Muscle Actin Expression and Myofibroblast Differentiation via Myocardin and Serum Response Factor

Maha Abdalla,Anna Goc,Lakshman Segar,Payaningal R Somanath

doi:10.1074/jbc.m113.504290

Maha Abdalla, Anna Goc + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m113.504290

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Abstract

Myofibroblast (MF) differentiation, marked by the de novo expression of smooth muscle α-actin (αSMA) stress fibers, plays a central role in wound healing and its persistence is a hallmark of fibrotic diseases. We have previously shown that Akt1 is necessary for wound healing through matrix regulation. However, the role of Akt1 in regulating MF differentiation with implications in fibrosis remains poorly defined. Here, we show that sustained activation of Akt1 was associated with a 6-fold increase in αSMA expression and assembly; an effect that is blunted in cells expressing inactive Akt1 despite TGFβ stimulation. Mechanistically, Akt1 mediated TGFβ-induced αSMA synthesis through the contractile gene transcription factors myocardin and serum response factor (SRF), independent of mammalian target of rapamycin in mouse embryonic fibroblasts and fibroblasts overexpressing active Akt1. Akt1 deficiency was associated with decreased myocardin, SRF, and αSMA expressions in vivo. Furthermore, sustained Akt1-induced αSMA synthesis markedly decreased upon RNA silencing of SRF and myocardin. In addition to its integral role in αSMA synthesis, we also show that Akt1 mediates fibronectin splice variant expression, which is required for MF differentiation, as well as total fibronectin, which generates the contractile force that promotes MF differentiation. In summary, our results constitute evidence that sustained Akt1 activation is crucial for TGFβ-induced MF formation and persistent differentiation. These findings highlight Akt1 as a novel potential therapeutic target for fibrotic diseases.

Highlights

Significance of Akt1 in myofibroblast differentiation is unknown
Akt1 Inactivation Abolishes TGF␤-induced Myofibroblast Differentiation—We first determined that 72 h is the optimal time for TGF␤-induced MF differentiation in NIH 3T3 fibroblasts, as measured by a 5-fold increase in ␣SMA expression)
To examine whether Akt1 activation is required for ␣SMA expression, the marker for MF differentiation, fibroblasts transfected with constitutively active Akt1 and inactive dominant-negative-Akt1 (DN-Akt1; Akt1 K179M), respectively, were serum starved and treated with control PBS or TGF␤ (100 pM) for 72 h

Summary

Background

Results: Akt mediates myofibroblast differentiation via serum response factor (SRF) and myocardin signaling leading to ␣-smooth muscle synthesis. Akt mediated TGF␤-induced ␣SMA synthesis through the contractile gene transcription factors myocardin and serum response factor (SRF), independent of mammalian target of rapamycin in mouse embryonic fibroblasts and fibroblasts overexpressing active Akt. Our results constitute evidence that sustained Akt activation is crucial for TGF␤-induced MF formation and persistent differentiation These findings highlight Akt as a novel potential therapeutic target for fibrotic diseases. Our studies constitute evidence of a novel signaling cascade that links Akt to ␣SMA synthesis through enhanced expression of myocardin and SRF, a previously uncharacterized link in MFs. We demonstrate the dual role of Akt in mediating ␣SMA synthesis and ED-A FN splice variant along with total fibronectin. Our studies suggest that Akt is a potential therapeutic target in fibrotic diseases

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION