The Diagnostic Value of the Interstitial Biomarkers KL-6 and SP-D for the Degree of Fibrosis in Combined Pulmonary Fibrosis and Emphysema

The combined pulmonary fibrosis and emphysema (CPFE) was reported first in 1990, but it has been comparatively underestimated until recently. Although the diagnostic findings of both emphysematous and fibrotic regions are detectable by high-resolution computed tomography (HRCT) of the chest, the degree of progressive fibrosis, which increases with emphysematous lesions, is difficult to evaluate. In this study, we hypothesized that the biomarkers for pulmonary fibrosis, surfactant protein D (SP-D), and KL-6 would serve as good indicators of fibrotic lesions in CPFE. We recruited 46 patients who had been diagnosed in our hospital with both emphysema and fibrosis by their CT scan image from April 2003 to March 2008. The correlation among their pulmonary function tests, composite physiologic index (CPI), and the serum levels of SP-D and KL-6 was evaluated. We found a correlation between KL-6 and %VC, %TLC, or CPI and between SP-D and %VC or CPI. Interestingly, the combined product of KL-6 and SP-D (KL-6xSP-D) was found to highly correlate with %VC and %TLC or CPI. These results show that both KL-6 and SP-D, and especially the product of SP-D and KL-6, are good indicators of the presence of fibrotic lesions in the lungs of CPFE patients

A novel function of RING finger protein 10 in transcriptional regulation of the myelin-associated glycoprotein gene and myelin formation in Schwann cells.

Myelin-associated glycoprotein (MAG) has been detected in Schwann cells prior to the onset of myelination, suggesting its functions in the initiation of myelination. However, transcriptional regulatory mechanisms of MAG remain to be elucidated. Here, we analyzed the promoter of the MAG gene by using luciferase reporter systems in the primary rat Schwann cells. We identified a novel cis-acting element located 160 bp upstream from the MAG transcription initiation site. Using the identified cis-element as a bait, we performed yeast one-hybrid screening and isolated a cDNA encoding a RNF10 as a putative trans-acting protein. When overexpressed in Schwann cells, RNF10 enhanced the activity of the MAG promoter. When RNF10 expression in Schwann cells was knocked down by siRNA, endogenous MAG mRNA and protein expression decreased. Furthermore, we evaluated myelin synthesis using Schwann cell-DRG neuron cocultures. When Schwann cells were infected with retrovirus expressing RNF10 siRNA, myelin formation was inhibited. These data suggest that RNF10 regulates MAG expression and is required for myelin formation

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

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

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

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