Neutral sphingomyelinase 3 modulates myotube density and is regulated by microRNA‐133

The neutral sphingomyelinase (nSMase) family is a group of enzymes that catalyzes the conversion of sphingomyelin to ceramide in a pH dependent manner. Sphingomyelinase activity and sphingolipid signaling play a role in the regulation of cellular processes. Our current data suggest that a novel, muscle‐specific neutral sphingomyelinase, nSMase3, may regulate muscle cell size. Overexpression of nSMase3 in C2C12 myotubes causes a decrease in myotube density, while treatment with an nSMase3 siRNA causes an increase in myotube density. MicroRNA‐133 (miR‐133) is a muscle‐specific small RNA molecule that has roles in proliferation and differentiation of myoblasts. A sequence within the 3′ untranslated region (UTR) of nSMase3 contains a predicted miR‐133 binding site. We hypothesize that miR‐133 inhibits nSMase3 translation and therefore plays a role in its regulation of cell size. In support of our hypothesis, expression of miR‐133 progressively increased while nSMase3 protein levels decreased during myotube differentiation. To test our hypothesis, a luciferase reporter gene containing the 3′ UTR of nSMase3 was cloned and used in a transient transfection assay. Co‐transfection of the reporter gene and miR‐133 decreased luciferase activity. These data suggest that miR‐133 is capable of regulating the expression of nSMase3 and, thus, may have a role in the regulation of muscle size.