Nandrolone, an anabolic steroid, stabilizes Numb protein through inhibition of mdm2 in C2C12 myoblasts.
Nandrolone, an anabolic steroid, slows denervation atrophy of rat muscle,  prevents denervation-induced nuclear accumulation of intracellular domain of  the Notch receptor, and elevates expression of Numb. Numb acts as an inhibitor  of Notch signaling and promotes myogenic differentiation of satellite cells.  Turnover of Numb is regulated by mdm2, an E3 ubiquitin ligase. With these  considerations in mind, we investigated the effects of nandrolone on the  expression of Numb and mdm2 proteins and determined the effect of mdm2 on  nandrolone-induced alterations in Numb protein in C2C12 myoblasts. When C2C12  cells were cultured in a medium favoring differentiation (Dulbecco modified  Eagle medium containing 2% horse serum), nandrolone up-regulated Numb protein  levels in a time-dependent manner and prolonged Numb protein half-life from 10  to 18 hours. In contrast, nandrolone reduced the expression of mdm2 protein.  To determine whether the decreased mdm2 expression induced by nandrolone was  responsible for the increased levels and prolonged half-life of Numb protein  in this cell line, mdm2-small interfering RNA (siRNA) was employed to  inhibit mdm2 expression. Compared to cells transfected with scrambled siRNA  (negative control), transfection with mdm2-siRNA increased basal Numb protein  expression but abolished the further increase in Numb protein levels by  nandrolone. In addition, transfection of mdm2-siRNA mimicked the effect of  nandrolone to prolong the half-life of Numb protein. Moreover, when C2C12  cells were forced to overexpress mdm2, there was a significant decline in the  expression of both basal and inducible Numb protein. Our data suggest that  nandrolone, by a novel mechanism for this agent in a muscle cell type,  increases Numb protein levels in C2C12 myoblasts by stabilizing Numb protein  against degradation, at least in part, via suppression of mdm2 expression.
National Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J Peter VA Medical Center, Bronx, New York 10468, USA. chris.cardozo@mssm.edu
Xin-Hua
XH
Liu
Shen
S
Yao
Alice C
AC
Levine
Alexander
A
Kirschenbaum
Jiangping
J
Pan
Yong
Y
Wu
Weiping
W
Qin
Lauren
L
Collier
William A
WA
Bauman
Christopher P
CP
Cardozo
D000818:Animals; D002460:Cell Line; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D032446:Myoblasts / Q000378:metabolism; D009277:Nandrolone / Q000494:pharmacology*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2 / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D015854:Up-Regulation
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C106293:Numb protein, mouse; D009277:Nandrolone; C497207:Mdm2 protein, mouse; D051736:Proto-Oncogene Proteins c-mdm2