PDZRN3 regulates differentiation of myoblasts into myotubes through transcriptional and posttranslational control of Id2

Abstract

PDZRN3 (also known as LNX3) is a member of the PDZ domain-containing RING finger protein family. We previously showed that PDZRN3 is essential for differentiation of myoblasts into myotubes and that depletion of PDZRN3 inhibits such differentiation downstream of the upregulation of myogenin, a basic helix-loop-helix (bHLH) transcription factor required for completion of the differentiation process. However, the mechanism by which PDZRN3 controls this process has remained unclear. Myogenin is rendered active during the late stage of myogenic differentiation by the downregulation of Id2, a negative regulator of bHLH transcription factors. We now show that depletion of PDZRN3 inhibits the differentiation of C2C12 cells by inducing the upregulation of Id2 and thereby delaying its downregulation. Knockdown of Id2 by RNA interference restores the differentiation of PDZRN3-depleted cells. Luciferase reporter assays revealed that a putative binding site for STAT5b in the Id2 gene promoter is required for the upregulation of Id2 expression by PDZRN3 depletion. In addition, the amount of phosphorylated Id2 was reduced and that of the nonphosphorylated protein concomitantly increased in PDZRN3-depleted cells, with the inhibitory effect of Id2 on bHLH transcription factors having previously been shown to be attenuated by phosphorylation of Id2 catalyzed by the complex of Cdk2 with cyclin A2 or E1. Indeed, the expression of cyclin A2, but not that of cyclin E1, was reduced in PDZRN3-depleted cells. Our results thus indicate that PDZRN3 plays a key role in the differentiation of myoblasts into myotubes by regulating Id2 at both transcriptional and posttranslational levels.

PDZRN3 protects against apoptosis in myoblasts by maintaining cyclin A2 expression

“…We previously showed that the expression of cyclin A2 was significantly reduced at both protein and mRNA levels, whereas that of other cyclins such as cyclin E1 and cyclin D1 was unaffected, in PDZRN3-depleted C2C12 myoblasts 16 . This previous analysis was performed with confluent cells.…”

“…Although apoptosis-related signaling and DNA damage are required for differentiation from myoblasts to myotubes 22,[28][29][30][31] , such differentiation is inhibited by depletion of PDZRN3 2,16 . This apparent inconsistency might be explained in part by our previous observation that the inhibition of such differentiation by PDZRN3 depletion is mediated by the up-regulation of Id2 16 . DNA damage before the induction of myoblasts to myotubes differentiation has also been shown to inhibit the differentiation process 32,33 .…”

“…BrdU was obtained from BD Pharmingen (San Diego, CA). Polyclonal antibodies to PDZRN3 were generated and purified as described previously 16 . Rabbit polyclonal antibodies to cyclin A2 (GTX103042) and to Mre11 (GTX118741) were obtained from GeneTex (Irvine, CA), those to cytochrome c (#21680) were from Signalway Antibody (College Park, MD), those to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 2275-PC) were from Trevigen (Gaithersburg, MD), and those to cleaved caspase-3 (#9661), to Ser 15 -phosphorylated p53 (#9284), to Ser 473 -phosphorylated Akt C2C12 cells infected with adenoviruses encoding KD or Scramb shRNAs.…”

“…RNAi. Recombinant adenoviruses encoding shRNAs specific for mouse PDZRN3 mRNA (KD and KD*) or an shRNA corresponding to a scrambled version of KD (Scramb) were constructed as described previously 16 . Unless www.nature.com/scientificreports www.nature.com/scientificreports/ indicated otherwise, C2C12 cells were seeded at a density of 2 × 10 4 cells per well in a 48-well plate, cultured in GM for 3 h, and infected with adenoviruses at a multiplicity of infection of 100 plaque-forming units per cell for 24 h, after which the medium was replaced with fresh GM and the cells were cultured for 24 h to ~90% confluence before analysis.…”

“…We previously showed that PDZRN3 is essential for differentiation from C2C12 myoblasts to myotubes 2,16 . Long-term serum deprivation of C2C12 myoblasts induces not only apoptosis but also entry into G 0 phase of the cell cycle followed by myogenic differentiation [17][18][19] .…”