Figure 1.
RXR-γ expression is increased in DRGN/SC cocultures.
(A) The mRNA levels of all six RARs and RXRs were analyzed by Q-RT-PCR. The results are shown as the mean value ± SD relative to the levels found in whole adult rat brain (dashed line). (B) The mRNA levels of RAR-β and RXR-γ were analyzed by Q-RT-PCR in isolated SCs or myelination-competent DRGN/SC cocultures and compared to isolated DRG neurons and total adult rat brain. The results are shown as the mean value ± SEM relative to the levels found in isolated SCs.
Figure 2.
RXR-γ levels in SCs are induced by axonal contact mimickers.
(A–D) Isolated SCs were adapted for 24 hours to the absence of the axonal contact mimickers BPE and forskolin and subsequently treated for 48 hours with 2 µM forskolin (Fk), 60 µg/ml BPE or the combination of both, and the levels of RXR-γ (A), RAR-β (B), MAG (C) and Egr2 (D) were analyzed by Q-RT-PCR. In all instances, the levels reached after the acute combined treatment were compared to SCs continuously grown in the presence of BPE and Fk (BPE+Fk always). (E–F) SCs were adapted for 24 hours to the absence the axonal contact mimickers and subsequently treated for 48 hours with 2 µM forskolin (Fk), 5 µM cyclopamine (CPM) or the combination of both, and the levels of RXR-γ (E) and RAR-β (F) were analyzed by Q-RT-PCR. All values are shown as the mean ± SD relative to their respective controls (cells grown in the presence of vehicle). Significance was assessed by unpaired Student's t-test. n.s.: non-significant; *: p<0.05; **: p<0.01; ***: p<0.005.
Figure 3.
Protein synthesis inhibition abrogates RXR-γ, while increasing RAR-β mRNA steady state mRNA levels.
SCs were treated with the axonal mimickers forskolin and BPE for 24 hours in the presence or absence of 10 µg/ml of the protein synthesis inhibitor cycloheximide (CHX), and RXR-γ (A) and RAR-β (B) mRNA levels were determined by Q-RT-PCR. All values are shown as the mean ± SEM relative to their respective controls in the absence of cycloheximide.
Figure 4.
RA upregulates RAR-β and downregulates RXR-γ in myelinating cocultures.
(A) DRGN/SC cocultures were allowed to myelinate for 7 days in the presence or absence of 1 µM RA, and the relative levels of RAR-α, -β and -γ, as well as RXR-α, -β and -γ were determined by Q-RT-PCR. All values are shown as the mean ± SD relative to their respective controls (sister cultures treated only with vehicle). (B) Myelinating cocultures were treated with 1 µM RA from the time of ascorbic acid induction and the levels of all six retinoid receptors were determined by Q-RTR-PCR at different times of treatment. All values are shown as the mean ± SD relative to their respective controls (myelinating cocultures treated only with vehicle for the same length of time).
Figure 5.
RA upregulates RAR-β mRNA and downregulates RXR-γ mRNA in isolated SCs.
SCs were treated with 1 µM RA for the times indicated and the relative levels of all six retinoid receptors were determined by Q-RT-PCR. All values are shown as the mean ± SD relative to their respective controls.
Figure 6.
Influence of axonal contact mimickers on RXR-γ and RAR-β regulation by RA.
RXR-γ downregulation is higher in the presence of axonal contact mimickers, while RAR-β upregulation was similar irrespective of the growth conditions. Isolated SCs were adapted for 24 hours to the absence of the axonal contact mimickers BPE and forskolin and subsequently treated for 48 hours with 1 µM RA in the presence or absence of 2 µM forskolin, 60 µg/ml BPE or the combination of both, and the mRNA levels of RAR-β (A) and RXR-γ (B) were analyzed by Q-RT-PCR. In all instances, the levels reached after the acute combined treatment were compared to SCs continuously grown in the presence of BPE and Fk (BPE+Fk always). All values are shown as the mean ± SEM relative to their respective controls.
Figure 7.
RAR-β and RXR-γ regulation is mediated by RAR.
(A) Myelinating cocultures were treated for 7 days with 1 µM of the natural retinoids all-trans-retinoic acid (RA) and 9-cis-retinoic acid (9-cis), or the synthetic compound TTNPB, and the relative mRNA levels of RAR-β and RXR-γ were determined by RT-Q-PCR. (B) Isolated SCs were treated for 48 hours with different doses of all-trans-retinoic acid (RA), TTNPB or 9-cis-retinoic acid (9-cis), and the relative levels of RAR-β and RXR-γ mRNAs were determined by RT-Q-PCR. All values are shown as the mean ± SD relative to their respective controls.
Figure 8.
Transcriptional regulation of RAR-β and RXR-γ by RA.
(A) The levels of newly synthesized hnRNA for RAR-β and RXR-γ were determined by RT-Q-PCR in myelinating cocultures treated with 1 µM RA for the indicated times. All values are shown as the mean ± SD relative to their respective controls. (B) Newly synthesized RAR-β and RXR-γ hnRNA was determined in isolated SCs treated or not with 1 µM RA for 48 hours. All values are shown as the mean ± SD relative to their respective controls. (C) SCs were treated with 1 µM RA in combination or not with the axonal mimickers forskolin and BPE for 24 hours in the presence or absence of 10 µg/ml of the protein synthesis inhibitor cycloheximide (CHX), and RAR-β and RXR-γ mRNA levels were determined by Q-RT-PCR. All values are shown as the mean ± SEM relative to their respective controls in the absence of cycloheximide and retinoic acid. (D) Isolated SCs were pretreated or not with 1 µM RA for 24 hours and the stability of RAR-β and RXR-γ mRNA was determined by incubating the cells with 10 µg/ml actinomycin D for varying times in the continuous presence or not of the retinoid. All values are shown as the mean ± SD relative to their respective time zero. RA-treated cells presented RAR-β mRNA levels 59 times higher than control cells, while RXR-γ levels were only 40% of those from control cultures.
Figure 9.
Retinoic acid and axonal contact mimickers regulate RXR-γ protein levels.
Isolated SCs were adapted for 24 hours to the absence of the axonal contact mimickers BPE and forskolin and subsequently treated for 48 hours with 1 µM RA in the presence or absence of 2 µM forskolin and 60 µg/ml BPE, and the protein levels of RXR-γ were analyzed by western blot. The upper panel depicts the quantitation of the blots. The data are presented as the mean value ± SEM referred as the percentage over the controls. Significance was assessed by unpaired Student's t-test. *: p<0.05; ***: p<0.005.