Acid Sphingomyelinase Deficiency Prevents AdipoRon-induced Transcription Factor EB activation and Differentiation in Arterial Smooth Muscle Cells

AdipoRon is a selective agonist of adiponectin receptor and has been reported to protect against vascular remodeling by preventing smooth muscle cell (SMC) dedifferentiation. Our recent studies have demonstrated that activation of transcription factor EB (TFEB) and its downstream autophagy signaling contribute to adipoRon-induced SMC dedifferentiation. This study aims to examine whether acid sphingomyelinase is involved in mediating adipoRon-induced TFEB activation in SMCs. In cultured arterial SMCs isolated from wild-type (Smpd1+/+) mice, adipoRon induced expression of acid sphingomyelinase (ASM; gene symbol Smpd1) and ceramide production, which is accompanied by TFEB nuclear translocation and upregulated transcription of genes involved in autophagy pathway and enhanced autophagic flux pathways. However, such adipoRon-induced ceramide, TFEB activation, and autophagic signaling were suppressed in SMCs from acid sphingomyelinase gene knockout (Smpd1-/-) mice. Consistently, adipoRon suppressed serum-induced cell proliferation and inhibited SMC migration in Smpd1+/+ SMCs as characterized by wound-healing retardation, F-actin reorganization, and matrix metalloproteinase-9 downregulation; whereas these inhibitory effects of adipoRon on proliferation and migration were attenuated in Smpd1-/- SMCs. Mechanistically, adipoRon increased the expression of protein phosphatase 2A (PP2A) and calcineurin in Smpd1+/+ SMCs but not Smpd1-/- SMCs. Pharmacological inhibition of PP2A by okadaic acid (OA) blocked adipoRon-induced TFEB activation and gene expression inSmpd1+/+ SMCs. Furthermore, activation of calcineurin by lysosomal TRPML1 channel agonist ML-SA1 could similarly activate TFEB and downstream autophagic signaling in Smpd1+/+ and Smpd1-/- SMCs. Inhibiting calcineurin by FK506 and cyclosporin A (CsA) prevented AdipoRon-induced TFEB and autophagy signaling activation. Together, these data suggest that adipoRon-induced TFEB signaling in SMCs is dependent on the ASM-mediated activation of phosphatase PP2A and calcineurin. This study provides novel mechanistic insights into understanding the therapeutic effects of adipoRon on TFEB signaling and pathological vascular remodeling.