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

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

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

Schwann cell nuclear extract and synthesized RNF10 can bind to the 20-bp <i>MAG</i> promoter <i>cis</i>-element (−162/−143) designated as “SSE.”

<p>(A) EMSA for the binding of nuclear factors. The binding of Schwann cell nuclear extracts (10 µg) with the DIG-labeled <i>MAG</i> promoter fragment (−167/−138) was tested in the absence of a specific competitor (lane 1) and in the presence of a 100-fold molar excess of the unlabeled fragment (lane 2). The arrow indicates DNA-protein complexes. (B) RNF10 was in vitro translated from RNF10-pCITE4 plasmid. DNA binding activity of RNF10 was analyzed using DIG-labeled <i>MAG</i> promoter oligonucleotide as probe. Translation reaction products with empty vector pCITE4 were used as control. 100-fold molar excess of the unlabeled fragment was used as competitor. Arrows denote the positions of the DNA–RNF10 complex. RNF10 is able to form specific DNA-protein complex with <i>MAG</i> promoter oligonucleotide containing “SSE” sequence. (C) Serial 20-bp mutant oligonucleotides of the −162/−153 region of the <i>MAG</i> promoter <i>cis</i>-element were generated. The flanking sequences are indicated in small typeface, and mutated bases are indicated in italics and bold. (D) EMSA using DIG-labeled mutant oligonucleotides and in vitro-translated RNF10. Any mutation abrogated DNA-protein complex formation in varying degrees. The arrow indicates the DNA-RNF10 complexes. (E) EMSA-supershift analysis of Myc-RNF10. Myc epitope-tagged RNF10 was expressed in Schwann cells by retrovirus-mediated gene transfer. Nuclear extracts were used for the EMSA with a WT probe. DNA-protein complex was observed (arrow). Anti-Myc antibody was added for supershift analysis. The presence of the anti-Myc antibody created the supershift band (arrowhead). (F) Myc-RNF10 was expressed in Schwann cells by retroviral transduction. ChIP assay followed by PCR analyses was performed on Myc-tagged RNF10. A portion of the rat Gap<i>dh</i> promoter region that does not contain “SSE”-like sequence was amplified to control for specificity. Input DNA was obtained from formaldehyde-crosslinked sonicated chromatin without immunoprecipitation. IgG was used as negative control.</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

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

STarT Back Tool.

<p>Response options for items 1–8 are “disagree” (0 points) or “agree” (1 point). Responses to item 9 are on a scale of 1–5: “not at all,” “slightly,” “moderately,” “very much,” or “extremely.” The first three options (“not at all,” “slightly,” and “moderately”) are scored as 0, and the remaining two options (“very much” and “extremely”) are scored as 1. Items 1–4 constitute the physical subscale. Items 5–9 constitute the psychosocial subscale.</p

The associations between magnetic resonance imaging findings and low back pain: A 10-year longitudinal analysis

<div><p>Purpose</p><p>To conduct a 10-year longitudinal analysis of the relationship between magnetic resonance imaging (MRI) findings and low back pain (LBP).</p><p>Materials and methods</p><p>Ninety-one volunteers with a history of LBP, but without current LBP were recruited between 2005 and 2006. Participants’ baseline demographics and MRI findings were recorded. All volunteers were invited for a follow-up MRI in 2016; of these, 49 volunteers (53.8%) participated in the follow-up. We enquired whether they had LBP history during the 10 years between the baseline and follow-up examinations. Sagittal T1 and T2-weighted MRI were used to assess the intervertebral space from T12/L1 to L5/S1. We evaluated the presence of disc degeneration by Pfirrmann’s grading system, disc bulging, high intensity zone (HIZ), spondylolisthesis, and any type of Modic changes in the follow-up MRIs. We compared the follow-up MRI findings with the baseline findings; the progress of each finding over the 10 years were also compared between the groups with (n = 36) and without (n = 13) LBP.</p><p>Results</p><p>Average age of the study participants at follow-up was 44.8 years; 25 were female and 24 were male. Average age, sex, body mass index, and smoking habits of those who did and did not participate in the follow-up study, as well as the demographic characteristics of those who did and did not have LBP history during the 10 years, were not significantly different. Compared with the group without LBP history, the group that had LBP history during the 10 years did not have a significantly increased prevalence of disc degeneration, disc bulging, and HIZ in the follow-up and baseline MRIs. Spondylolisthesis and any type of Modic changes were also not associated with LBP history during the 10 years.</p><p>Conclusions</p><p>Follow-up MRI findings consistent with Pfirrmann grading ≥4, disc bulging, HIZ, spondylolisthesis, and any type of Modic changes were not associated with LBP history during the 10 years between the baseline and follow-up study. The progresses of these findings were also not associated with the LBP history. In addition, baseline MRI findings were not associated with LBP history during the 10 years; therefore, our data suggest that baseline MRI findings cannot predict future LBP.</p></div

Mean number of absences for the three STarT-J risk groups.

<p>The linear trend was tested using the Jonckheere-Terpstra test (p < 0.0001). STarT-J: The Japanese version of the STarT Back Tool.</p

Spearman’s correlation coefficients for the STarT-J and related measures.

<p>Spearman’s correlation coefficients for the STarT-J and related measures.</p