Abstract
PDZRN3 is a PDZ domain-containing RING-finger family protein that functions in various developmental processes. We previously showed that expression of PDZRN3 is induced together with that of MyoD during the early phase of skeletal muscle regeneration in vivo. We here show that PDZRN3 suppresses apoptosis and promotes proliferation in myoblasts in a manner dependent on cyclin A2. Depletion of PDZRN3 in mouse C2C12 myoblasts by RNA interference reduced the proportion of Ki-67-positive cells and the level of Akt phosphorylation, implicating PDZRN3 in regulation of both cell proliferation and apoptosis. Exposure of C2C12 cells as well as of C3H10T1/2 mesenchymal stem cells and NIH-3T3 fibroblasts to various inducers of apoptosis including serum deprivation resulted in a greater increase in the amount of cleaved caspase-3 in PDZRN3-depleted cells than in control cells. The abundance of cyclin A2 was reduced in PDZRN3-depleted C2C12 myoblasts, as was that of Mre11, which contributes to the repair of DNA damage. Overexpression of cyclin A2 restored the expression of Mre11 and Ki-67 as well as attenuated caspase-3 cleavage in PDZRN3-depleted cells deprived of serum. These results indicate that PDZRN3 suppresses apoptosis and promotes proliferation in myoblasts and other cell types by maintaining cyclin A2 expression.
Highlights
PDZ domain-containing RING-finger protein 3 (PDZRN3 or LNX3) was originally identified in silico as a homolog of LNX1 (PDZRN2), whose function as E3 ubiquitin ligase for NUMB is thought to contribute to cell fate determination at the level of inhibition of Notch signaling[1]
We found that RNA interference (RNAi)-mediated depletion of PDZRN3 by infection with an adenovirus encoding a short hairpin RNA specific for PDZRN3 mRNA (KD) in C2C12 myoblasts maintained in growth medium (GM) resulted in a significant decline in cell number compared with control cells infected with an adenovirus encoding a scrambled version of the PDZRN3 shRNA sequence (Scramb) (Fig. 1a,b)
Serum deprivation resulted in a time-dependent increase in H2AX phosphorylation in C2C12 myoblasts (Fig. 5d,e), consistent with previous observations[22], and this effect was significantly augmented in PDZRN3-depleted cells compared with control cells (Fig. 5d,e)
Summary
PDZ domain-containing RING-finger protein 3 (PDZRN3 or LNX3) was originally identified in silico as a homolog of LNX1 (PDZRN2), whose function as E3 ubiquitin ligase for NUMB is thought to contribute to cell fate determination at the level of inhibition of Notch signaling[1]. Regeneration of skeletal muscle is initiated by the activation of satellite cells (myogenic stem cells) in response to injury These cells give rise to myoblasts that express the transcription factor MyoD, proliferate (early phase of differentiation), and subsequently differentiate into myocytes that express the transcription factor myogenin and fuse to form myotubes (late phase of differentiation). We previously showed that depletion of PDZRN3 inhibited the late phase of differentiation of myocytes into myotubes[2,16], and that the expression of PDZRN3 is induced together with that of MyoD during regeneration of injured skeletal muscle in vivo[2]. We found that PDZRN3 regulates apoptosis and proliferation through cyclin A2 in these cells, and that such regulation appears common to other cell types that express PDZRN3
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