Modulation of c-myc, max, and mad gene expression during neural differentiation of embryonic stem cells by all-trans-retinoic acid

Abstract

c-Myc regulates cellular proliferation, differentiation, and apoptosis. Temporal expression of c-Myc during all-trans-retinoic acid (RA)-mediated neural differentiation in murine embryonic stem cell (ES) was investigated. Correlation to the modulation of dimerizing partners Max and Mad that may influence c-Myc signaling and transcription regulation was elucidated for the first time in these cells. In RA-treated cells, increase in c-myc mRNA was detected by reverse transcriptase polymerase chain reaction on days 11 and 14 of differentiation compared with the vehicle-treated controls. The results were further corroborated by ribonuclease protection assay (RPA). Western blots revealed an increase in c-Myc protein only on day 14 of differentiation in RA-treated cells. Increases in max and mad gene transcription detected by RPA at times of elevated c-Myc in RA-treated ES cells suggest that a transient increase in c-Myc protein expression may influence differential dimerization of Myc partners needed for signaling in the neural differentiation of ES cells.

Figure 1

Increased c-myc mRNA expression in spontaneously dividing ES cells at 12 and 24 h as detected by RT-PCR. The mRNA expression was normalized to house keeping gene β-actin at 0 = undifferentiated, 12, 24, 36, and 48 h after initiation of differentiation. The products were amplified at 33 cycles in the exponential phase of PCR reaction and the gel representative of exponential amplification of c-myc is shown (inset). The results are expressed as mean ± SEM (n = 3). The photograph of representative gel shows β-actin and c-myc mRNA at respective times. *Significantly different from undifferentiated group at p ≤ 0.05.

Figure 2

Differential expression of c-myc, max, and mad mRNA expression by RNase protection assay (RPA). (A) Representative gel of RPA showing only c-myc, max, and mad (indicated on the left) mRNA expression at 0 = undifferentiated, 24 and 48 h. L32 represents the housekeeping gene. A nonhybridized probe set was run as a size marker. (B) The relative mRNA expression normalized to housekeeping gene L32 at 0 = undifferentiated, 24 and 48 h after differentiation. The results are expressed as mean ± SEM (n = 2). *Significantly different from undifferentiated group at p ≤ 0.05.

Figure 3

Modulation of c-myc mRNA expression by RT-PCR during RA-mediated ES neural differentiation. The cells were treated with RA on days 8, 9, and 10 of differentiation. (A) The photographs of representative gels show β-actin and c-myc mRNA on days 11, 14, 17, and 21 of differentiation. (B) The relative c-myc gene expression was normalized to β-actin. The results are expressed as mean ± SEM (n = 3). *Significantly different from concurrent control p ≤ 0.05.

Figure 4

Alteration in c-myc expression during RA-mediated ES differentiation. (A) Representative gel of RNase protection assay. A nonhybridized probe was run as a size marker. The names of the genes are listed on the left. L32 represents the housekeeping gene. Treatment groups are indicated as control: (lane 1) 11-day vehicle-treated, (lane 3) 14-day vehicle-treated, (lane 5) 17-day vehicle-treated, and (lane 7) 21-day vehicle-treated. RA-exposed: (lane 2) 11-day RA-treated, (lane 4) 14-day RA-treated, (lane 6) 17-day RA-treated, and (lane 8) 21-day RA-treated. (B) The relative mRNA expression is indicated against L32. Pooled RNA from three independent wells was used for the assay, and the results are expressed as mean ± SEM (n = 2). *Significantly different from concurrent control at p ≤ 0.05.

Figure 5

Alteration in c-Myc protein expression during differentiation by Western blot. (A) Fifteen micrograms of cytosolic cell lysate was electrophoretically separated and transferred onto nitrocellulose membranes. After incubation with primary and secondary antibody, the proteins were visualized autoradiographically with chemiluminiscent reagents and quantified as pixels. Results expressed are mean ± SEM (n = 3). Representative gel shows c-Myc expression at 0 = undifferentiated, 12, 24, 36, and 48 h after initiation of differentiation. *Significantly different from undifferentiated group at p ≤ 0.05. (B) Alteration in c-Myc protein expression during RA-mediated ES differentiation on days 11, 14, 17, and 21 of differentiation. The results are expressed as mean ± SEM (n = 3). *Significantly different from concurrent control at p ≤ 0.05. Representative photograph of gel is shown. Treatment groups are indicated as control: (lane 1) 11-day vehicle-treated, (lane 3) 14-day vehicle-treated, (lane 5) 17-day vehicle-treated, and (lane 7) 21-day vehicle-treated. RA-exposed: (lane 2) 11-day RA-treated, (lane 4) 14-day RA-treated, (lane 6) 17-day RA-treated, and (lane 8) 21-day RA-treated.

Figure 6

Alterations in max expression during RA-mediated ES differentiation. The relative mRNA expression is indicated against L32. Pooled RNA from three independent wells was used for the assay, and the results of a representative experiment are expressed as mean ± SE (n = 2). *Significantly different from concurrent control at p ≤ 0.05.

Figure 7

Alterations in mad expression during RA-mediated ES differentiation. The relative mRNA expression is indicated against L32. Pooled RNA from three independent wells was used for the assay, and the results are expressed as mean ± SE (n = 2). *Significantly different from concurrent control at p ≤ 0.05.