Sublytic C5b‐9 induces proliferation of glomerular mesangial cells via ERK5/MZF1/RGC‐32 axis activated by FBXO28‐TRAF6 complex

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  Figure 1. The expression and roles of RGC‐32 in GMC proliferation and ECM secretion exposed to sublytic C5b‐9. A and B, RGC‐32 protein in the renal tissues of Thy‐1N rats (A) and in the GMCs exposed to sublytic C5b‐9 (B) for different time‐points was examined using IB assay. **P < 0.01 vs 0 hour time‐point. C, RGC‐32 protein in the renal tissues of Thy‐1N and NRS rats at 10 hours was examined using IB analysis. **P < 0.01 vs NRS group. D, Rat GMCs were divided into different groups. At 10 hours after treatment, RGC‐32 protein was determined using IB experiments. **P < 0.01 vs other groups. E and F, Rat GMCs were transfected with pIRES2‐EGFP/RGC‐32 or pIRES2‐EGFP for 48 hours and 72 hours and then the protein levels of RGC‐32, cyclin D2, PCNA, FN and collagen IV in the GMCs were detected using IB assay (E, 48 hours) and cellular proliferation was also determined with CCK‐8 assay (F, 72 hours). **P < 0.01 vs pIRES2‐EGFP group. G and H, Rat GMCs were transfected with shRGC‐32 or shCTR for 48 hours followed by sublytic C5b‐9 treatment for different time‐points. The expression of RGC‐32 at 10 hours as well as cyclin D2, PCNA, FN and collagen IV at 18 hours were detected using IB assay (G).…

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  Figure 2. Analysis of RGC‐32 gene promoter and detection of potential transcriptional factors. A, Luciferase reporter plasmids of RGC‐32 gene promoter (FL or different deletion fragments) and pRL‐SV40 were co‐transfected into rat GMCs for 48 hours followed by sublytic C5b‐9 stimulation for 10 hours and then the luciferase activity was detected. **P < 0.01 vs FL group. B, Transcriptional factor binding elements in the −194 ~ +8 nt region of rat RGC‐32 gene promoter were predicted using TFsearch software. C and D, The protein levels of MZF1 and SP‐1 were detected using IB experiments both in the renal tissues of Thy‐1N rats (C) and in the GMCs induced by sublytic C5b‐9 (D). **P < 0.01 vs 0 hour time‐point. E and F, MZF1 protein in the renal tissues of Thy‐1N and NRS rats at 10 hours was examined using IB analysis (E) and IHC staining (F, Magnification, ×400). **P < 0.01 vs NRS group. G, Rat GMCs were divided into different groups. At 10 hours after treatment, MZF1 protein was determined using IB assay. **P < 0.01 vs other groups. Results from one representative experiment out of three are shown. Data are represented as means ± SD (n = 6 in vivo, n = 3 in vitro in each group or each time‐point)

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  Figure 3. The effects of MZF1 on RGC‐32 gene activation as well as GMC proliferation and ECM production induced by sublytic C5b‐9. A, The plasmids of pIRES2‐EGFP/MZF1 and pGL3/RGC‐32 (FL or different deletion fragments) were co‐transfected into the GMCs and then the luciferase activity was detected at 48 hours after transfection. **P < 0.01 vs FL group. B, The plasmids of pIRES2‐EGFP/MZF1‐Flag and pIRES2‐EGFP were transfected into rat GMCs respectively and then the anti‐Flag antibody or IgG control was used to pull down DNA‐protein complex from the GMCs at 48 hours after transfection. Subsequently, immunoprecipitated DNA was amplified using PCR using a pair of primers for the −220 ~ −50 nt region of RGC‐32 gene promoter. C, The plasmids of pGL3/RGC‐32 (FL), pRL‐SV40 and shMZF1 or shCTR were co‐transfected into rat GMCs for 48 hours followed by sublytic C5b‐9 treatment for 10 hours and then the luciferase activity was detected. **P < 0.01 vs shCTR + sublytic C5b‐9 group. D and E, Rat GMCs were transfected with pIRES2‐EGFP/MZF1 or pIRES2‐EGFP for 48 hours and 72 hours and then the expression levels of MZF1, RGC‐32, cyclin D2, PCNA, FN and collagen IV were determined using IB analysis (D, 48…

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  Figure 4. The activation and roles of ERK5 in MZF1 and RGC‐32 expression as well as GMC proliferation and ECM secretion in response to sublytic C5b‐9. A and B, ERK5 phosphorylation in the renal tissues of Thy‐1N rats (A) and in the GMCs exposed to sublytic C5b‐9 (B) was examined using IB assay. *P < 0.05, **P < 0.01 vs 0 hour time‐point. C, ERK5 phosphorylation in the renal tissues of Thy‐1N and NRS rats at 5 hours was examined by IB analysis. **P < 0.01 vs NRS groups. D, Rat GMCs were treated in different groups for 5 hours and then ERK5 phosphorylation was detected using IB. **P < 0.01 vs other groups. E and F, Rat GMCs were transfected with pEGFP‐N1/ERK5 or pEGFP‐N1 for 48 hours and 72 hours and then the expression levels of ERK5, MZF1, RGC‐32, cyclin D2, PCNA, FN and collagen IV were detected using IB (E, 48 hours). Meantime, cellular proliferation was also determined with CCK‐8 assay (F, 72 hours). *P < 0.05, **P < 0.01 vs pEGFP‐N1 group. G and H, Rat GMCs were transfected with shERK5 or shCTR for 48 hours followed by sublytic C5b‐9 treatment for different time‐points. The expression levels of p‐ERK5, t‐ERK5 at 5 hours, MZF‐1 and RGC‐32 at 10 hours, as well as cyclin D2, PCNA, FN and…

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  Figure 5. The expression of FBXO28 and TRAF6 in the renal tissues of Thy‐1N rats and in the GMCs stimulated with sublytic C5b‐9. A and B, The protein levels of FBXO28 and TRAF6 in the renal tissues of Thy‐1N rats (A) and in the GMCs exposed to sublytic C5b‐9 (B) for different time‐points were examined using IB assay. *P < 0.05, **P < 0.01 vs 0 hour time‐point. C‐E, The protein levels of FBXO28 and TRAF6 in the renal tissues of Thy‐1N and NRS rats at 5 hours were examined using IB analysis (C) and IHC staining (D and E, Magnification, ×400) respectively. **P < 0.01 vs NRS groups. F, Rat GMCs were divided into different groups. At 5 hours after treatment, the protein levels of FBXO28 and TRAF6 were determined using IB experiments. **P < 0.01 vs other groups. Results from one representative experiment out of three are shown. Data are represented as means ± SD (n = 6 in vivo, n = 3 in vitro in each group or each time‐point)

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  Figure 6. FBXO28‐TRAF6 complex‐mediated ERK5 K63‐ubiquitination is necessary for ERK5/MZF1/RGC‐32 activation as well as GMC proliferative changes in response to sublytic C5b‐9. A and B, Rat GMCs were transfected with shFBXO28 (A) or shTRAF6 (B) for 48 hours followed by sublytic C5b‐9 treatment for different time‐points. The expression levels of FBXO28, TRAF6 and ERk5 at 5 hours, MZF‐1 and RGC‐32 at 10 hours, as well as cyclin D2, PCNA, FN and collagen IV at 18 hours were detected using IB. C and D, Co‐IP experiment was done to pull down ERK5 protein with anti‐ERK5 from FBXO28‐dificient GMCs (C) or TRAF6‐dificient GMCs (D) exposed to sublytic C5b‐9 for 5 hours, the then IB assay was used to detect K63‐ubiquitin, ERK5, FBXO28 and TRAF6 in the co‐IP‐complex as well as FBXO28, TRAF6, ERK5, p‐ERK5, MZF1 and RGC‐32 in WCE. E, TRAF6‐dificient GMCs was transfected with pcDNA3.1/HA‐TRAF6WT or pcDNA3.1/HA‐TRAF6C70A for 48 hours and then the levels of K63‐ubiquitin, ERK5 and HA‐TRAF6 in the co‐IP‐complex and HA‐TRAF6, ERK5 and p‐ERK5 in WCE were detected using IB analysis. F, 293T cells were transfected with pcDNA3.1/FBXO28‐His, pcDNA3.1/FBXO28∆1‐His or pcDNA3.1/FBXO28∆2‐His and pEGFP‐N1/ERK5‐Flag…

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  Figure 7. Knockdown of renal FBXO28, TRAF6, ERK5, MZF1 and RGC‐32 gene abolished GMC proliferation and ECM secretion in Thy‐1N rats. LV‐shFBXO28, LV‐shTRAF6, LV‐shERK5, LV‐shMZF1 and LV‐shRGC‐32 were used to silence target genes, and then Thy‐1N was induced 4 days later. A, The protein levels of FBXO28, TRAF6, p‐ERK5, t‐ERK5, MZF1, RGC‐32, cyclin D2, PCNA, FN and collagen IV in the renal tissues of rats were detected using IB assay (at 5 hours for FBXO28, TRAF6, p‐ERK5 and t‐ERK5, at 10 hours for MZF1 and RGC‐32, on 7 d for cyclin D2, PCNA, FN and collagen IV). B and C, The numbers of total glomerular cells in different groups of rats on 7 d after Thy‐1N induction was observed using Haematoxylin and eosin under LM (Magnification, ×400). D, GMC proliferation and ECM accumulation in different groups of rats on 7 d after Thy‐1N induction were evaluated by EM. E, The content of urinary protein (mg/24 h) of rats on day 7 after Thy‐1N induction was detected. F, A schematic drawing for the pathway we revealed. **P < 0.01 vs Thy‐1N group and LV‐shCTR + Thy‐1N group. Results from one representative experiment out of three are shown. Data are represented as means ± SD (n = 6 in each group)