Chapter2: Krϋppel-like Factor 15 Suppresses Renal Glomerular Mesangial Cell Proliferation Via Enhancing P53 SUMO1 Conjugation
May 12, 2022
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3 RESULTS
3.1|Screening of KLF15-binding genes through ChIP-Seq in primary renal glomerular MCs
To identify the direct binding partner genes of KLF15, we performed ChlP-Seq analysis and ultimately screened 2478 genes. GO analysis of these genes through the tool at the website www.uniprot.org (Figure 1A, B)revealed molecular function terms associated with 1941 genes, cellular component terms related to 1662 genes and biological process terms related to 1766 genes. Since our aim was to find out how KLF15 affects MCs, we focused on cell process-related genes. Among the 1315 cell process-related genes,74 genes were found to participate in cell cycle processes; specifically, these genes participated in the mitotic cell cycle, the cell cycle phase transition and other processes(Figure 1C, D). Furthermore, we analysed the genes involved in growth and found that they were related to developmental growth, cell growth and other types of growth(Figure 1E).
3.2|Screening of differentially regulated genes in KLF15-overexpressing renal glomerular MCs using SILAC and LC/MS
SILAC and LC/MS analysis of HRMCs overexpressing KLF15 compared to parental cells led to the identification of 1357 proteins. We used the DAVID and IPA to acquire the GO domains and en-riched pathways of the quantified proteins identified by SILAC. Interestingly, many proteins' biological function-related terms were among the top 30 significantly enriched GO terms, including the regulation of the cellular amino acid metabolic process, proteasome complex, protein binding, and transcription and translation terms, all of which are closely related to ubiquitination(Figure 2A). Figure 2B shows the top 30 significantly enriched pathway terms. Several biological process terms, including the proteasome and neurodegenerative disease terms, were also related to ubiquitination.
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3.3 | Bioinformatics analysis of KLF15-binding genes that are potentially associated with renal glomerular MC proliferation
To explore the KLF15-binding genes that are potentially associated with renal glomerular MC proliferation, we performed a bioinformatics analysis of the ChIP-Seq data and the SILAC-LC/MS data. Fifty-two genes were screened (Table 2 and Figure 2C), and five of these genes, including SUMO1, macrophage migration inhibitory factor (MIF), eukaryotic initiation factor (EIF), insulin-like growth factor(IGF) and reticulocalbin (RCN), were closely related to cell proliferation. Since the GO and pathway analyses of the differentially expressed proteins suggested that the screened genes were related mainly to the ubiquitination process, we concluded that SUMO1, a member of the ubiquitin-like (UBL)protein family, participates in post-translational modification similar to ubiquitination as the target gene of KLF15.
Increasing amounts of evidence have indicated that HG is one of the major factors inducing the development of diabetic nephropathy, and it promotes MC proliferation and increased matrix synthesis in vitro.25A previous study has shown that PDGF-BB is essential for MC proliferation preceding the development of glomerulosclerosis in experimental glomerulonephritis.26 After confirming that MCs were stimulated by HG or PDGF-BB in vitro, we detected changes in the expression of KLF15 and SUMO1 at both the RNA and protein
levels and found that these molecules had similar trends (Figure 2D-l). Finally, we determined SUMO1 to be the target gene of KLF15.
3.4|KLF15 up-regulates SUMO-1 gene expression by binding to a 16 bp CACCCA-SUMO-1 promoter region and a C+A-rich motif
We used the MEME suite(http://meme-suite.org/tools/meme)to characterize the KLF15-binding motifs of the 52 screened genes from the KLF15 ChlP-Seq data and identified the KLF-binding motif (Figure 3A).In addition, we further analysed more than one thousand bases upstream of the SUMO1 promoter and found that KLF15 could recognize and bind to a 16 bp sequence (nucleotides -205~-199)including -CACCCA-(Figure 3B).ChIP-PCR confirmed that KLF15 is bound to the SUMO1 promoter, and the results showed significantly higher expression of SUMO1 in the anti-KLF15 antibody group than in the background control group (Figure 3C).
Furthermore, we performed a dual-luciferase reporter assay to confirm the targeting relationship between SUMO1 and KLF15. The wild-type SUMO1 promoter group showed higher luciferase activity than the pGL3 vector and mutant groups in HRMCs, and the activity was significantly increased by overexpression of KLF15. The enhancements in luciferase activity were reversed by transfection with a plasmid expressing the mutant promoter region(Figure 3D). Taken together, these data suggest that SUMO1 is a direct transcriptional target of KLF15.
3.5 |Screening of P53 as the SUMOylation substrate of SUMO1
SUMO modifications are widely expressed post-translational modifications in eukaryotes. Reversible conjugation of these modifications to substrate proteins is also known as SUMOylation, which plays important role in regulating the functions of the target proteins and further participates in various biological processes, such as nucleocytoplasmic transport, transcriptional regulation, apoptosis, protein stabilization, stress responses and cell cycle progression.27 To explore the downstream signalling molecules directly conjugated by SUMO1, we performed a network analysis of SUMO1 using the IPA database, and the data showed that SUMO1 could directly con-jugate to P53, APP, JUN and AKT, among other proteins(Figure 4A-C). We initially selected P53 as a downstream molecule of SUMO1 because it is a well-known protein that is closely related to cell proliferation.28.29 Furthermore, we used SUMOsp software to predict the possible SUMOylation sequences of P53invariousspecies and found that P53 had the SUMOylation sequence（Figure 4D）. To determine whether P53 is indeed modified by SUMOylation, we transiently transfected HRMCs with a SUMO1 overexpression plasmid or SUMO1 siRNA.Both the IP and Western blot results revealed trends in expression changes of SUMO1-P53and P53 that were consistent with the changes in SUMO1 (Figure 4E-J). These data indicate that P53 is a SUMOylation substrate of SUMO1.
3.6 |KLF15 inhibits MC proliferation by promoting SUMO1 expression and P53 SUMOylation
To establish the regulation of P53 SUMOylation and MC proliferation by KLF15 and SUMO1, we intervened with KLF15 or SUMO1 expression in HRMCs and treated HRMCs with PDGF-BB. Among PDGF-BB-treated HRMCs, cells transfected with the KLF15 overexpression plasmid exhibited higher expression of SUMO1-P53, P53, KLF15 and SUMO1 than cells transfected with the KLF15 control plasmid. The same changes in SUMO1-P53, P53 and SUMO1 were found in the SUMO1 overexpression plasmid-transfected cells, while KLF15 expression was unchanged in these cells (Figure 5A-F). PDGF-BB is one of the most effective growth factors of MCs described thus far, and proliferative response of HRMCs to PDGF-BB was observed. As shown in Figure 5G, H, the percentage of EdU-positive cells was significantly increased by the administration of recombinant PDGF-BB. The EdU staining results indicated that both KLF15 and SUMO1 overexpression inhibited PDGF-BB-induced cell proliferation (Figure 5G, H).
To gain further insight into the roles of KLF15 and SUMO1 in proliferating HRMCs, we transfected cells with only SUMO1 siRNA or cotransfected them with SUMO1 siRNA and a KLF15 overexpression plasmid. Down-regulation of SUMO1 did not affect the expression of KLF15, but the expression levels of SUMO1-P53 and P53 were obviously decreased after SUMO1 siRNA transfection(Figure6A-C). In addition, when the expression of SUMO1 was inhibited, SUMO1-P53 and P53 expression levels were also suppressed even in cells overexpressing KLF15 (Figure 6D-F). Then, MC proliferation was evaluated using an EdU staining assay. SUMO1 RNAi enhanced the proliferative effect of PDGF-BBon HRMCs, and the group cotransfected with the KLF15 overexpression plasmid and SUMO1 siRNA had more EdU-positive cells than the group cotransfected with the overexpression plasmid and the hybrid sequence control siRNA(Figure 6G, H). We, therefore, conclude that KLF15 suppresses MC proliferation by enhancing P53 SUMOylation.
3.7|Global SUMO1 and P53 expression in glomerular MCs is negatively correlated with MC proliferation in rat Thy-1 nephritis
Anti-Thy1 nephritis is a classical model of self-limited mesangial proliferative glomerulonephritis with a proliferative phase and a recovery phase. We injected a Thy1 antibody into Wistar rats to create this model. Both serum urea nitrogen and creatinine have no significant change between control rats and the model rats (Figure S1). Marked mesangial proliferation and ECM accumulation were observed during the proliferative phase (days 5and 7)in the model rats, and the number of MCs decreased during the recovery phase on day 10 (Figure 7A). We also detected the expression changes in the cell proliferation marker PCNA by IHC (Figure 7A, B).PCNA levels were increased on day 5, peaked on day 7 and decreased on day 10 (Figure 7F). Western blot analysis showed that the protein expression of P53, SUMO1 and KLF15 in isolated glomeruli was lower in the model groups than in the control group (Figure 7C, D), consistent with the immunohistochemical results (Figure 7E, F). These results indicate that the abnormal proliferation of MCs in anti-Thy1 model rats is related to the low-level expression of KLF15 and SUMO1. Interfering with the expression of these molecules is expected to alleviate the pathological phenotype in rats.
4 DISCUSSION
The glomeruli are the filtration units of the kidney. Disruption of glomerular function can be caused by primary glomerular pathology or can be secondary to systemic diseases. Mesangial changes were seen following glomerular injury including hyperproliferation of MCs followed by excessive production of ECM (mesangial expansion) and production of chemoattractant for inflammatory cells. Therefore, modulation of MC responses, especially abnormal proliferation, is a novel therapeutic approach. KLF15 is a protein that plays a regulatory role as a transcription factor by binding to specific DNA sequences. Many studies have reported that KLF15 is involved in the regula-tion of cell proliferation. For example, it has been found that KLF15 participates in the inhibition of MC proliferation-related signalling pathways,15,30but its direct target gene has not been reported in the literature. Therefore, this study involved mainly joint screening of transcription and translation levels to identify the direct target gene of KLF15.
KLF15 regulates different genes in different species and in different tissues and organs. In the process of regulating the proliferation of MCs, KLF15 affects the expression of multiple genes and participates in multiple pathways.131,32 To explore the direct target genes of KLF15, we used ChIP-Seg.Klein RH and his colleagues used ChlP-seq technology to study the regulation of KLF7 on the differentiation of corneal epithelial cells.33 Ying M et al used this technique to study the inhibitory effect of KLF9on the pluripotency of glioblastoma.34Compared with ChlP-chip, ChIP-Seq enables true whole-genome analysis with higher resolution, higher detection sensitivity and lower sample size demand.35 We used ChP to obtain the DNA fragments directly bound by KLF15, and after comparison and analysis with GenBank, we screened 2478 possible target genes. Through GO and pathway analyses, we identified many target genes involved in the cell cycle and proliferation processes.
ChlP-Seq experiments require PCR for amplification of the detection signal, and some degree of bias during the amplification process is inevitable. In addition, ChIP-Seq obtains only the genes that KLF15 can bind and does not indicate the changes that occur in the expression of these genes when the expression of KLF15 changes. We used SILAC-LC/MS proteomics analysis to compensate for these shortcomings. This technique provides information on all the proteins whose expression changes upon regulation by KLF15, including the proteins directly regulated by KLF15 and the proteins that are indirectly regulated or post-translationally modified. HRMCs were cultured using SILAC, and KLF15 was overexpressed in the cells through plasmid transfection. The proteins were collected for LC/MS detection, and 1357 differentially expressed proteins were obtained. GO and pathway analyses revealed that some of the differentially expressed proteins were involved in ubiquitination. The gene and protein data were intersected. The genes not related to the research purpose and genes not directly regulated by KLF15 were removed; ultimately,52 genes were screened. Among these, the five genes with the most obvious expression differences that were the most closely related to proliferation were selected. Given the combined results of pathway analysis, SUMO1 and KLF15 expression analysis in proliferating HRMCs, ChlP-PCR and dual-luciferase reporter assays, the protein SUMO1 was selected as the target gene of KLF15.
In addition to ubiquitin, increasing numbers of UBLproteins,3637 including SUMO,38 neural precursor cell-expressed, developmentally down-regulated 8(NEDD8)3940 and interferon-stimulated gene 15(ISG15),41 are being identified. These modifying proteins powerfully regulate a variety of biological processes. The covalent addition of SUMO to substrates, termed SUMOylation, is a post-translational modification involved in a series of cellular processes, including nuclear-cytosolic transport, transcriptional regulation, apoptosis, protein stability control, stress responses and cell cycle progression.27SUMO is typically attached to acceptor lysine residues of protein substrates harbouring a consensus sequence and contributes to the regulation of protein-protein interactions as well as to subcellular compartmentalization and protein stability.2 sUMO1, as a member of the SUMOS family, can affect cell proliferation by modifying substrate and stabilizing cell cycle regulatory proteins.43 Therefore, combined with the literature report and the comprehensive screening and analysis results, we focused on how KLF15 regulates the effect of SUMO1 on mesangial cell proliferation.
To identify the substrates of SUMO1, we performed a network analysis of SUMO1 using the IPA database and SUMOsp software. Combined with published research on P53, our initial screening data suggested P53 as a downstream molecule of SUMO1 with the SUMOylation sequence Y-K-x-D/E. Previous studies have shown that P53 was a substrate of SUMOylation,45 and enhanced P53 SUMO1 conjugation promoted the stability and activity of P53 and induced se-nescence.46 In our study, interference with the expression of SUMO1 in HRMCs produced the same change trends in SUMO1-P53 and P53, indicating that P53was a SUMOylation substrate of SUMO1.
Emerging evidence has indicated that SUMOylation and de-SUMOylation play roles in numerous nephropathic diseases, such as renal dysgenesis, renal carcinoma, glomerular disease, podocyte apoptosis, and renal medulla hypertonicity, acute kidney injury and nephrolithiasis.7-51 To clarify the relationship among KLF15, SUMO1, P53 SUMOylation and MC proliferation, we performed a series of transfection treatments on cells that had undergone PDGF-BB-induced proliferation. Overexpression of KLF15 up-regulated the expression of SUMO1, while overexpression of SUMO1 did not affect the expression of KLF15. Either KLF15 or SUMO1 overexpression increased the SUMOylation of P53 and antagonized the cell proliferation induced by PDGF-BB. When the expression of SUMO1 was inhibited, the SUMOylation of P53 was also inhibited, and KLF15 lost its antagonistic effect on cell proliferation. In vivo. the proliferation of MCs gradually increased with aggravation of mesangial proliferative nephritis, while the expression of KLF15, SUMO1 and P53 decreased significantly. Considering the integrated observations in cells and tissues, we preliminarily conclude that KLF15 regulates the SUMOylation of P53 through SUMO1, thereby inhibiting MC proliferation.
5 CONCLUSIONS
Some studies have indicated that KLF15 plays an important role in regulating the proliferation of MCs. In this work, we explored the mechanisms of MC proliferation suppression mediated by this transcription factor. We identified SUMO1 as the primary target of KLF15 in MCs via bioinformatics analyses involving ChIP-Seq and SILAC-LC/MS. Furthermore, with the aid of the IPA database and SUMOsp software, we determined that P53 was a direct substrate of SUMO1. In vitro and in vivo experiments confirmed that KLF15 could promote the expression of SUMO1 and the SUMOylation of P53 then inhibit the proliferation of MCs(Figure S2). These results contribute to the understanding of the regulatory role of KLF15 in MC proliferation and provide a theoretical basis for finding new treatments for MC proliferation-related kidney diseases.
