RNF10 negatively regulates Schwann cell proliferation.

<p>(A)The time course of cell number was assessed for Schwann cells infected with retrovirus expressing RNF10 siRNA, or control EGFP siRNA by using the MTT assay. Compared to control cells, the <i>RNF10</i>-knockdown Schwann cells exhibited a 40% greater increase. (B) BrdU incorporation was significantly increased (80% greater) in stable RNF10 knockdown cells compared with controls. *p<0.01; Student's t test. Error bars show mean±SD.</p

Luciferase activity of Schwann cells transiently transfected with a <i>MAG</i> promoter construct.

<p>(A) Transient cotransfections of Schwann cells and ROS cells using various rat <i>MAG</i> reporter constructs. The largest <i>MAG</i>-luciferase reporter plasmid had a 2.7-kb promoter. Numerous sequences with 5′ deletion were constructed, including reporter plasmids containing 283-, 162-, 153-, and 77-bp segments of the MAG promoter. A sequence with internal deletion of a segment from −162 to −153 was also constructed. The observed firefly luciferase activity is normalized with the Renilla luciferase activity and the results are expressed as fold induction compared with empty vector in Schwann cells. Deletion of a region between −162 and −153 greatly reduced the luciferase activity. In contrast, the luciferase activity showed subtle changes in the ROS cell. (B, C) To examine the positive effects of the sequence between −162 and −153 on the promoter activity, we generated and analyzed various lengths of tandem repeats downstream from −162 (B) and upstream from −143 (C). The normalized luciferase activity is expressed as fold induction compared with the value of either p162 in (B) or p77 in (C) respectively. Only the reporter constructs bearing 20-bp tandem repeats (−162 to −143) could increase the luciferase activity in a repeat number-dependent manner. Therefore, we believe that this 20-bp sequence (5′-ACAAGGGCCCCTTTGTGCCC-3′) is required and sufficient for the activation of the <i>MAG</i> promoter and that it is a <i>cis</i>-acting element.</p

Maximum systolic and diastolic blood pressure pressure-lowering values at 6 h after administration of FBPs to SHRs (10 mg/kg BW, n = 6).

<p>* <i>p</i><0.05,</p><p>** <i>p</i><0.01, versus the negative control group as evaluated by Student's <i>t</i>-test.</p><p>Maximum systolic and diastolic blood pressure pressure-lowering values at 6 h after administration of FBPs to SHRs (10 mg/kg BW, n = 6).</p

<i>In vitro</i> ACE-inhibitory activities (IC₅₀) of FBPs.

<p>Data represent the mean ± S.E. (n = 3). Different letters indicate significant differences between each FBP (<i>p</i><0.05, one-way ANOVA followed by Tukey's test).</p><p><i>In vitro</i> ACE-inhibitory activities (IC₅₀) of FBPs.</p

Vascular tension changes after incubation with angiotensin I and DVWY, FDART, FQ, WTFR, and captopril.

<p>Tension changes were measured using SHR thoracic aorta rings incubated in 0.2 µM angiotensin I with DVWY, FDART, FQ, WTFR, and captopril (positive control). Data represent the mean ± S.E. (n = 3). *<i>p</i><0.05, **<i>p</i><0.01 versus the negative control group (Krebs solution treatment) by Student's <i>t</i>-test.</p

Comparison of the promoter region of the MAG gene.

<p>Sequence alignments of the MAG promoter region. Identities are indicated by dots and gaps by dashes. Bold characters show the 20 bp MAG promoter cis-element (−162/−143), designated “SSE”. SSE sequence is completely conserved between Rat, Mouse and Human.</p

Angiotensin II production after incubation with angiotensin I and DVWY, FDART, FQ, WTFR, and captopril.

<p>Angiotensin II production was measured in SHR aorta rings incubated in 0.2 µM angiotensin I with DVWY, FDART, FQ, WTFR, and captopril (positive control). Data represent the mean ± S.E. (n = 3). *<i>p</i><0.05, **<i>p</i><0.01 versus the negative control group (Krebs solution treatment), Student's <i>t</i>-test.</p

FBP concentration-vasorelaxation curves of phenylephrine-preconstricted thoracic aorta rings from SHRs.

<p>Dose dependent response to cumulatively increasing concentration of DVWY (A), FDART (B), VVG (C), FQ (D), VAE (E), and WTFR (F). Vasorelaxant rate is expressed as the percentage reduction of evoked contraction. Each data point and bar represent the mean ± S.E. (n = 3). *<i>p</i><0.05, **<i>p</i><0.01, versus pre-treatment tension, Student's <i>t</i>-test.</p

RNF10 activates <i>MAG</i> promoter activity in Schwann cells.

<p>(A) Schwann cells were cotransfected with an expression construct for RNF10 (pcDNA3.1-RNF10) and <i>MAG</i>-promoter-LUC with (p77C, p162C) or without (p77, p153) the tandem SSE region. Overexpression of RNF10 increased the <i>MAG</i> promoter activity in Schwann cells by 100% when compared with the activity in the control cells only when the reporter constructs contained the SSE region. Thus, RNF10 increases the promoter activity in a <i>cis</i>-element-dependent manner. (B) Cotransfection of an expression construct for RNF10 (pcDNA3.1-RNF10) and <i>MAG</i>-promoter-LUC containing the tandem SSE into ROS cells. The <i>MAG</i> promoter activity in the ROS cells increased by 60% when compared with the activity in the control cells. However, the degree of activation in ROS cells was not as high as that in the Schwann cells. (C) There was no difference in endogenous RNF10 mRNA expression between SC and ROS cells. (D) Cotransfection of an expression construct for the RING finger domain-deletion mutant of RNF10 (ΔRFD) and <i>MAG</i>-promoter-LUC containing the tandem SSE into Schwann cells. The RING finger domain-deletion construct did not activate the <i>MAG</i> promoter. (E) Indirect immunofluorescence of Schwann cells indicated the nuclear localization of RNF10. Schwann cells stably expressed Myc epitope-tagged RNF10 by retrovirus-mediated gene transfer. Cells were immunostained with an anti-Myc antibody, and the nucleus was stained with Hoechst 33342. The arrow heads show that tagged RNF10 was almost exclusively located in the nucleus in dot-like structures. The normalized luciferase activity is expressed as fold induction compared with the value of p77/mock or p153/mock in (A), p77/mock in (B) and p77C/mock in (D). *p<0.01; Student's t test. Error bars show mean±SD.</p

Specific silencing of the <i>RNF10</i> gene with RNF10 siRNA in Schwann cells.

<p>(A) Schwann cells were transfected with either RNF10 siRNA or control EGFP siRNA expression vector. At 48 h after transfection, quantitative RT-PCR analysis showed that the relative <i>RNF10</i> mRNA levels in the RNF10 siRNA-treated cells had reduced to 10% of that in the control EGFP siRNA-treated cells (left). Retroviral siRNA showed approximately the same results (right). (B) Downregulation of <i>MAG</i> promoter activity by RNF10 siRNA. Schwann cells were cotransfected with a <i>MAG</i>-promoter-LUC containing tandem SSE and RNF10 vector or control EGFP siRNA expression vector. Luciferase activity was measured 48 h posttransfection. RNF10 siRNA suppressed the promoter activity to 40% of that in the control cells. (C) EMSA using Retrovirus-mediated RNF10 siRNA Schwann cells. Schwann cells were stably transfected with a retrovirus-based RNF10 siRNA and control EGFP siRNA. Nuclear extracts were used for the EMSA with a WT probe. DNA-protein complex was observed with the control nuclear extract (arrow), while the nuclear proteins of RNF10 siRNA Schwann cells could not form a DNA-protein complex. (D) Schwann cells were infected with a retrovirus expressing RNF10 siRNA or control EGFP siRNA, selected in puromycin, and analyzed for the expression of MAG by quantitative RT-PCR analysis. Retrovirus-mediated RNF10 siRNA specifically reduced the MAG mRNA expression levels to 25% of that in the control. No difference was observed in the mRNA levels of MPZ and MBP between the RNF10 siRNA-treated and control cells. (E) Western blotting using an anti-MAG or anti-actin antibody of Schwann cells infected with either control EGFP siRNA or RNF10 siRNA-expressing retrovirus. RNF10 siRNA markedly reduced the MAG protein expression in Schwann cells. *p<0.01; Student's t test. Error bars show mean±SD.</p