TGF-ß1-PML SUMOylation-peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) form a positive feedback loop to regulate cardiac fibrosis.

Transforming growth factor-ß (TGF-ß) signaling pathway is involved in fibrosis in most, if not all forms of cardiac diseases. Here, we evaluate a positive feedback signaling the loop of TGF-ß1/promyelocytic leukemia (PML) SUMOylation/Pin1 promoting the cardiac fibrosis. To test this hypothesis, the mice underwent transverse aortic constriction (3 weeks) were developed and the morphological evidence showed obvious interstitial fibrosis with TGF-ß1, Pin1 upregulation, and increase in PML SUMOylation. In neonatal mouse cardiac fibroblasts (NMCFs), we found that exogenous TGF-ß1 induced the upregulation of TGF-ß1 itself in a time- and dose-dependent manner, and also triggered the PML SUMOylation and the formation of PML nuclear bodies (PML-NBs), and consequently recruited Pin1 into nuclear to colocalize with PML. Pharmacological inhibition of TGF-ß signal or Pin1 with LY364947 (3 µM) or Juglone (3 µM), the TGF-ß1-induced PML SUMOylation was reduced significantly with downregulation of the messenger RNA and protein for TGF-ß1 and Pin1. To verify the cellular function of PML by means of gain- or loss-of-function, the positive feedback signaling loop was enhanced or declined, meanwhile, TGF-ß-Smad signaling pathway was activated or weakened, respectively. In summary, we uncovered a novel reciprocal loop of TGF-ß1/PML SUMOylation/Pin1 leading to myocardial fibrosis.

Manipulating PML SUMOylation via Silencing UBC9 and RNF4 Regulates Cardiac Fibrosis.

The promyelocytic leukemia protein (PML) is essential in the assembly of dynamic subnuclear structures called PML nuclear bodies (PML-NBs), which are involved in regulating diverse cellular functions. However, the possibility of PML being involved in cardiac disease has not been examined. In mice undergoing transverse aortic constriction (TAC) and arsenic trioxide (ATO) injection, transforming growth factor ß1 (TGF-ß1) was upregulated along with dynamic alteration of PML SUMOylation. In cultured neonatal mouse cardiac fibroblasts (NMCFs), ATO, angiotensin II (Ang II), and fetal bovine serum (FBS) significantly triggered PML SUMOylation and the assembly of PML-NBs. Inhibition of SUMOylated PML by silencing UBC9, the unique SUMO E2-conjugating enzyme, reduced the development of cardiac fibrosis and partially improved cardiac function in TAC mice. In contrast, enhancing SUMOylated PML accumulation, by silencing RNF4, a poly-SUMO-specific E3 ubiquitin ligase, accelerated the induction of cardiac fibrosis and promoted cardiac function injury. PML colocalized with Pin1 (a positive regulator for TGF-ß1 mRNA expression in PML-NBs) and increased TGF-ß1 activity. These findings suggest that the UBC9/PML/RNF4 axis plays a critical role as an important SUMO pathway in cardiac fibrosis. Modulating the protein levels of the pathway provides an attractive therapeutic target for the treatment of cardiac fibrosis and heart failure.