NANDROLONE-INDUCED NUCLEAR ACCUMULATION OF MYOD PROTEIN IS MEDIATED BY NUMB, A NOTCH INHIBITOR, IN C2C12 MYOBLASTS

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SAT 338-357-Steroid Hormone Actions
Basic/Translational
Saturday, June 15, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SAT-351
Xin-Hua Liu*1, Yong Wu2, Lauren Collier1, William A Bauman2 and Christopher Pratt Cardozo3
1J.J. Peter VA Medical Center, Bronx, NY, 2James J. Peters VA Medical Center, Bronx, NY, 3James J. Peters Veteran Affairs Medical Center, Bronx, NY
Androgen signaling via the androgen receptor (AR) is a key pathway that contributes to myogenic progenitor differentiation. In addition, accumulated evidence has demonstrated that the myogenic differentiation factor D (MyoD) and Numb, a Notch inhibitor, play key roles in regulating myogenic differentiation. Nandrolone, an anabolic steroid, has been shown to upregulate both MyoD and Numb expression in myogenic cells. However, the molecular mechanisms by which MyoD is upregulated by nandrolone are unclear. In addition, the crosstalk between nandrolone, MyoD and Numb are not well delineated. With these considerations in mind, we examined the effects of nandrolone on the expression of MyoD mRNA and protein, and determined the interactive effect of MyoD and Numb in the presence or absence of nandrolone in differentiating C2C12 myoblasts. Real-time PCR revealed a moderated, but significant, increase in MyoD mRNA levels (65%) within 2 days after nandrolone treatment. To evaluate the effects of nandrolone on MyoD protein expression and intracellular distribution, we conducted time course assays of MyoD induction by nandrolone in cytosolic and the nuclear fractions of the cells. Western blot analysis demonstrated that nandrolone-induced increases in MyoD protein levels occurred in the nuclei of the cells, which started by 16h with a peak effect (4.6-fold) at 3d. The levels remained constant for at least 5d after administration of the steroid. By contrast, cytoplasmic MyoD levels were upregulated later, beginning at day 2, with a lower amplitude (2.5-fold) and a short duration of peak expression, after which the MyoD levels decreased to its basal level. To determine whether elevated Numb expression is necessary for the induction of MyoD by nandrolone, Numb-siRNA was employed to inhibit the expression of Numb. Cells transfected with Numb-siRNA demonstrated significant reductions in Numb mRNA and protein. Gene knockdown with Numb-siRNA did not alter basal levels of MyoD protein but prevented nandrolone-induced upregulation of nuclear accumulation of MyoD protein compared to the cells transfected with non-silencing random siRNA (negative control). In contrast, transfection with Numb-siRNA had no effect on the levels of nandrolone-induced MyoD mRNA. These data suggest that nandrolone upregulated MyoD expression and activity, mainly, through a posttranslational mechanism that, at least in part, promotes MyoD nuclear localization. In addition, the data imply that nandrolone-induced Numb expression is a critical step in the process of nandrolone-promoted MyoD activation.

Nothing to Disclose: XHL, YW, LC, WAB, CPC

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: This work was supported by the Veterans Health Administration, Rehabilitation Research and Development Service (Grants B4162C, F7756R and F6997R).