![Figure 4. GFs and ATRA activate RARα through MAP kinases. MAP kinases can mimic the action of GFs on ATRA-induced RARα activity. U-937 cells were transfected with pGAL4(UAS)5-luc, pMB1-CMV-lacZ and pGAL4(DBD)-RARα1 (A), or pGAL4(DBD)-RARα2 (B). Data are presented as mean ± SD of 3 (A) or 2 (B) independent experiments done in triplicates. Induction of exogenous RARα1 (A) and RARα2 (B) transcriptional activity by ATRA and G/GM-CSF was measured after 18 hours. MEK-1 and-2 inhibitor (U0126) and p38 inhibitor (SB203580) were added 10 minutes prior to differentiating agents in SFM. The inhibitors had no effect on the basal luciferase activity, and β-galactosidase level remained unchanged. (C) This experiment is as in panel B but measures the efficiency of the MAP kinase-inhibitors on the activities of the endogenous RARα receptors. U-937 cells have been transiently transfected with the ATRA-responsive reporter PREP4-(DR5G)3-tk-luc and the pMB1-CMV-lacZ control plasmid. Data are shown from 3 independent experiments done in duplicates, mean ± SD. (D) ATRA-induced activity of a reporter (PREP4-(DR5G)3-tk-luc) was measured 18 hours after cotransfection with either constitutively activated (active) MEK (pBABE-MEK-1 E217/E221) or equal amount of empty vector (inactive) control (pBABE-puro). In a separate experiment, expression of p38α-2 (pEXV-CSBP-2 [active] versus equal amount of empty pEXV vector control [inactive], as indicated) also stimulated ATRA-induced reporter activity. Data are shown from one representative experiment each, which has been repeated with similar results. Values are presented as mean ± SD from quadruplicates. (E) The additional increase of ATRA-induced GAL4-driven luciferase activity by G/GM-CSF has been measured in U-937 cells comparing the pGAL4(DBD)-RARα2 with the pGAL4(DBD)-RARα2 (A74/77) mutant. Amino acid numbers 74 and 77 are relative to the RARα1 sequence. The numbering for the identical amino acid residues in the common B Region of RARα2 is 69 and 72, respectively. Data are shown from 3 independent experiments done in quadruplicates, mean ± SD. (F) Western blot analysis for MAPKAPK-2, the substrate of p38 kinase, and ERK-1/-2, substrate of MEK-1/-2, indicating induction of phosphorylation of both kinases by ATRA and GFs, as well as by FCS, compared with serum-free control C. Inhibition by SB203580 and U0126 occurs at low concentrations of 1 μM and 10 μM, respectively. For panels A-E, numbers above bars indicate mean values. The statistical significance of differences between control and treated samples is indicated by asterisks, and between treated samples by plus symbols. ***/+++P < .001, **/++P < .01, */+P < .05.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/105/1/10.1182_blood-2004-03-1074/6/zh80010571740004.jpeg?Expires=1735583778&Signature=ekKrys7XDWTifZ~xeNTH3oDgkGLTQ6gbASflifwUQ4QJaSOHbm9ZrjZ61AK1bOIo3ehBp8xsrSv0gzGlhxuEfrWwFdRq8~zvkI8CmrLO47naxdgYWYlJbYSB2s0KTeSN8sg80dDe-udwkGE-TNGq75VPprmmX~vlDD5vkc4CTyUiDFnAOEoILgR5QMzj8FZMFUvi98Hip2AM1ogwAgieUcP-qOWoiG7t3GEesU3jHNEfpZbrgpE5fx6-Bos15zviNCJiIXnU~CbuhErkcyRkMiDyxO8WNMQcqs8k9EmO3sxz3mf~iOM-ZVdKaG--0Rv~E7iyCwxHBhIe81uMjWKmKQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
GFs and ATRA activate RARα through MAP kinases. MAP kinases can mimic the action of GFs on ATRA-induced RARα activity. U-937 cells were transfected with pGAL4(UAS)5-luc, pMB1-CMV-lacZ and pGAL4(DBD)-RARα1 (A), or pGAL4(DBD)-RARα2 (B). Data are presented as mean ± SD of 3 (A) or 2 (B) independent experiments done in triplicates. Induction of exogenous RARα1 (A) and RARα2 (B) transcriptional activity by ATRA and G/GM-CSF was measured after 18 hours. MEK-1 and-2 inhibitor (U0126) and p38 inhibitor (SB203580) were added 10 minutes prior to differentiating agents in SFM. The inhibitors had no effect on the basal luciferase activity, and β-galactosidase level remained unchanged. (C) This experiment is as in panel B but measures the efficiency of the MAP kinase-inhibitors on the activities of the endogenous RARα receptors. U-937 cells have been transiently transfected with the ATRA-responsive reporter PREP4-(DR5G)3-tk-luc and the pMB1-CMV-lacZ control plasmid. Data are shown from 3 independent experiments done in duplicates, mean ± SD. (D) ATRA-induced activity of a reporter (PREP4-(DR5G)3-tk-luc) was measured 18 hours after cotransfection with either constitutively activated (active) MEK (pBABE-MEK-1 E217/E221) or equal amount of empty vector (inactive) control (pBABE-puro). In a separate experiment, expression of p38α-2 (pEXV-CSBP-2 [active] versus equal amount of empty pEXV vector control [inactive], as indicated) also stimulated ATRA-induced reporter activity. Data are shown from one representative experiment each, which has been repeated with similar results. Values are presented as mean ± SD from quadruplicates. (E) The additional increase of ATRA-induced GAL4-driven luciferase activity by G/GM-CSF has been measured in U-937 cells comparing the pGAL4(DBD)-RARα2 with the pGAL4(DBD)-RARα2 (A74/77) mutant. Amino acid numbers 74 and 77 are relative to the RARα1 sequence. The numbering for the identical amino acid residues in the common B Region of RARα2 is 69 and 72, respectively. Data are shown from 3 independent experiments done in quadruplicates, mean ± SD. (F) Western blot analysis for MAPKAPK-2, the substrate of p38 kinase, and ERK-1/-2, substrate of MEK-1/-2, indicating induction of phosphorylation of both kinases by ATRA and GFs, as well as by FCS, compared with serum-free control C. Inhibition by SB203580 and U0126 occurs at low concentrations of 1 μM and 10 μM, respectively. For panels A-E, numbers above bars indicate mean values. The statistical significance of differences between control and treated samples is indicated by asterisks, and between treated samples by plus symbols. ***/+++P < .001, **/++P < .01, */+P < .05.
