Smad3 Couples Pak1 With the Antihypertrophic Pathway Through the E3 Ubiquitin Ligase, Fbxo32

Abstract

Pathological cardiac hypertrophy is regarded as a critical intermediate step toward the development of heart failure. Many signal transduction cascades are demonstrated to dictate the induction and progression of pathological hypertrophy; however, our understanding in regulatory mechanisms responsible for the suppression of hypertrophy remains limited. In this study, we showed that exacerbated hypertrophy induced by pressure overload in cardiac-deleted Pak1 mice was attributable to a failure to upregulate the antihypertrophic E3 ligase, Fbxo32, responsible for targeting proteins for the ubiquitin-degradation pathway. Under pressure overload, cardiac overexpression of constitutively active Pak1 mice manifested strong resilience against pathological hypertrophic remodeling. Mechanistic studies demonstrated that subsequent to Pak1 activation, the binding of Smad3 on a critical singular AGAC -286 -binding site on the FBXO32 promoter was crucial for its transcriptional regulation. Pharmacological upregulation of Fbxo32 by Berberine ameliorated hypertrophic remodeling and improved cardiac performance in cardiac-deficient Pak1 mice under pressure overload. Our findings discover Smad3 and Fbxo32 as novel downstream components of the Pak1-dependent signaling pathway for the suppression of hypertrophy. This discovery opens a new venue for opportunities to identify novel targets for the management of cardiac hypertrophy.

The p21‐activated kinase 1 (Pak1) signalling pathway in cardiac disease: from mechanistic study to therapeutic exploration

“…Previous studies have demonstrated that Smad3 can be phosphorylated by JNK in lung epithelial cells, p38 in breast carcinoma cells or Pak1 in mesangial cells (Chen et al , ; Velden et al , ; Kamaraju and Roberts, ). This is interesting as a lack of Smad3 phosphorylation was also demonstrated in Pak1‐deficient hearts under hypertrophic conditions (Tsui et al , ). This study further confirmed that knockdown of MKK7 or JNK abolished the phenylephrine‐induced phosphorylation of Smad3 despite the presence of Pak1, indicating that Pak1 activation of Smad3 is likely to occur through the MKK7/JNK pathway (Tsui et al , ).…”

“…This is interesting as a lack of Smad3 phosphorylation was also demonstrated in Pak1‐deficient hearts under hypertrophic conditions (Tsui et al , ). This study further confirmed that knockdown of MKK7 or JNK abolished the phenylephrine‐induced phosphorylation of Smad3 despite the presence of Pak1, indicating that Pak1 activation of Smad3 is likely to occur through the MKK7/JNK pathway (Tsui et al , ). These results highlight the therapeutic potential of Fbxo32 itself and a pharmacological activator of Fbxo32, berberine, has been reported to improve cardiac function (Huang et al , ; Lau et al , ; Zhang et al , ).…”

“…Consistently, the cardioprotective effects of Pak1 were further confirmed by subjecting mice with cardiac‐specific overexpression of Ad‐CaPak1 (Pak1 cTG ) to TAC stress. The detrimental effects of TAC stress were significantly reduced in the Pak1 cTG mice; the TAC‐induced increases in fibrosis, LV mass, LV end‐diastolic diameter, LV end‐systolic diameter and reduced fraction shortening were diminished in these mice (Tsui et al , ).…”

“…A novel anti‐hypertrophic pathway of Pak1 was identified through small mother against decapentaplegic 3 (Smad3) transcriptional regulation of F‐box protein 32 (Fbxo32) (Tsui et al , ). In the absence of Pak1 in the heart TAC stress led to exacerbated increased protein levels of calcineurin as the up‐regulation of Fbxo32 was abolished.…”

“…In control NRCMs PE stress induces an up‐regulation of Fbxo32 at the mRNA and protein levels, which was eliminated in Pak1 knockdown NRCMs, resulting in the accumulation of calcineurin. Ad‐CaPak1‐infected NRCMs showed elevated protein expression levels of Fbxo32 and subsequent down‐regulation of calcineurin (Tsui et al , ). Thus, Pak1 negatively regulates calcineurin, through Fbxo32, in response to hypertrophic stress.…”