The mechanisms which regulate cyclin H/cdk7 phosphorylation of RARα have not been elucidated. To determine if RA itself could affect the phosphorylation status of its receptor, we treated SCC4 and SCC25 lines with 1 μM all trans RA for 16 hours. As shown in Fig. 1A, RA treatment decreased expression of both cyclin H and cdk7 in SCC4 and SCC25 cells by 3 fold. To determine if reduced cyclin H and cdk7 expression correlated with decreased RARα phosphorylation, we immunoprecipitated the receptor from vehicle and RA treated SCC4 and SCC25 cells. As shown in Fig. 1B, relative phosphorylation levels of RARα decreased by 3 fold, similar to the reduction in cyclin H and cdk7 expression. Previous studies have shown that the amino terminus of the estrogen receptor is phosphorylated by MAPK proteins [12]. To determine the participation of MAPKs in RARα phosphorylation, we performed RARα immunoprecipitation on SCC25 cells treated with the MEK/ERK inhibitor PD98059 or p38 inhibitory drug SB203580. Treatment with these drugs did not change relative RARα phosphorylation levels (Fig. 1B). We concluded that RA repressed cyclin H/cdk7 expression which correlated with reduced RARα phosphorylation levels.
RA is a potent inhibitor of cellular proliferation [13]. To determine the effects of hypophosphorylated RARα on the growth of human cancer cells, we expressed an S77A RARα expression construct in SCC25 cells. The serine residue at position 77 was previously shown to be the target of cyclin H/cdk7 phosphorylation [7]. As shown in Fig. 2, expression of the S77A mutant resulted in decreased relative phosphorylation of immunoprecipitated RARα in stable clones compared to G418 resistant control cells. To confirm that this effect was due to expression of the hypophosphorylated RARα mutant, we also created stable clones expressing either HA tagged RARα or the S77A mutant. As shown in Fig. 3, only clones expressing HA-RARα and not the HA-S77A mutant showed phosphorylation on serine residues. These experiments indicate that the relative amount of phosphorylated RARα in SCC25 cells was decreased by the S77A mutant. SCC25 cells expressing the S77A mutant RARα proliferated at markedly reduced rates compared to control clones (Fig. 4). The S77A RARα mutants grew at only 30–50% of the rate of G418 resistant controls in culture. By comparison, proliferation of clones expressing HA tagged RARα was 90% of G418 resistant control cells. We concluded that decreased RARα phosphorylation produces inhibition of proliferation in SCC25 cells.
To determine if decreased proliferation of the mutant RARα clones correlated with inhibition of cell cycle progression, we performed BrdU incorporation analysis. As shown in Fig. 5, less than half the percentage of S77A mutant cells incorporated BrdU compared to the G418 resistant control clones. By comparison, there were no significant differences in BrdU incorporation between HA tagged RARα clones and G418 resistant control cells. These results indicate that the G1 to S phase transition of the cell cycle is inhibited in S77A mutant clones. To corroborate this conclusion, we examined expression of the G1 phase cyclin dependent kinase inhibitors p21WAF1/Cip1 and p27Kip1 in G418 resistant and S77A mutant clones. We also determined the expression of the S phase marker cyclin A in these cells. As shown in Fig. 6, expression of the G1 phase markers p21WAF1/Cip1 and p27Kip1 in the mutant RARα clones was up to 3 fold higher compared to control cells. In contrast, expression of the S phase marker cyclin A was decreased by 3 fold in two clones (RAR1 and 2) and was undetectable in 2 others (RAR3 and 4) when compared to G418 resistant control cells. These results indicate that the decreased proliferation observed in S77A RARα mutant clones is due to inhibition of cell cycle progression at the G1 to S phase transition.
The antiproliferative effects of RA have been attributed to its inhibition of AP-1 transcriptional activity [13]. While the S77A RARα mutant has been shown to inhibit transactivation of RA response elements [7], its effects on AP-1 activity have not been characterized. We first addressed this issue by transiently transfecting a heterologous promoter containing a consensus AP-1 site fused to the luciferase reporter gene [11] along with wild type RARα or S77A mutant expression vectors. In the absence of ligand, the S77A RARα mutant inhibited AP-1 activity from this construct by 50%, similar to treatment with 1 μM all trans RA (Fig. 7). The wild type receptor possessed slight anti-AP-1 activity in the absence of ligand (20% reduction compared to control cells transfected with blank expression vector). These results indicate that hypophosphorylated RARα can mimic the effects of RA by inhibiting AP-1 activity.
To determine if reductions in AP-1 protein levels could account for the observed transcriptional inhibition by the S77A mutant RARα, we compared expression of fos and jun proteins in G418 resistant control clones and S77A RARα expressing cells. As shown in Fig. 8, Fra-1 expression was undetectable in 3 of 4 RARα mutant clones. Expression of c-jun was reduced by 3 to 4 fold in the S77A RARα cells compared to G418 resistant control clones. JunB expression was also decreased by up to 20 fold in mutant RARα expressing cells. Expression of c-fos, Fra-2, FosB, and JunD was not detected under normal growth conditions in SCC25 cells. These results indicate that decreased AP-1 protein expression correlates with AP-1 transcriptional inhibition by hypophosphorylated RARα.
Upstream of AP-1 transcription factors, RA has been shown to inhibit epidermal growth factor receptor (EGFR) expression [14]. To determine if hypophosphorylated RARα could inhibit EGFR expression, we examined expression of the growth factor receptor by western blot in control and S77A mutant cells. As shown in Fig. 9, EGFR expression was undetectable in 3 of 4 mutant RARα clones and was reduced by 50% in the remaining clone (RAR1). To determine if this dramatically decreased EGFR expression could inhibit downstream components of EGF signaling, we also examined activation of ERK1 (a terminal effector in the mitogen activated protein kinase pathway). Activated ERK1 in S77A mutant cells was expressed at less than 10% of the levels found in G418 resistant control clones. Overall ERK1 expression was not affected by hypophosphorylated RARα. These results indicate that components of the EGFR signaling pathway, upstream of AP-1 transcription factors, are inhibited by hypophosphorylated RARα.
Activation of growth factor receptors has been shown to induce expression of AP-1 proteins [15]. To determine if EGF signaling correlated with AP-1 activity in SCC25 cells, we first incubated cultures in 1% depleted serum for 16 hours to inhibit AP-1 protein expression. We then stimulated these cultures with 10 ng/ml EGF for up to 4 hours. As shown in Fig. 10, EGF produced dramatic induction of AP-1 protein expression within 15 minutes after its addition. c-fos expression peaked at 100 fold over unstimulated levels at 2 hours following EGF addition. Following a similar time course, c-jun expression increased by 25 fold following stimulation by EGF. Induction of Fra-1 and JunB expression was also noted, although the increase was of less magnitude than that observed for c-fos and c-jun. The effects of EGF on AP-1 protein expression were completely blocked by the EGFR kinase inhibitor tyrphostin AG1478 (data not shown), indicating that growth factor receptor signaling directly correlates with AP-1 protein expression. These data suggest that in the unliganded state, hypophosphorylated RARα can mimic the effects of RA on EGFR expression, ERK1 activity, and AP-1 protein levels.