ECT2-PLXNB2 Signaling Inhibits Ribosome Biogenesis Leading to Kidney Fibrosis

Background: During kidney fibrosis, the G2/M arrest and senescence of Tubular Epithelial Cells (TECs) are key pathogenic events. Ribosome Biogenesis (RiBi) refers to the assembling of ribosome complex, and RNA Pol1 catalyzing rDNA transcription is essential in RiBi. When RiBi is inhibited, ribosomal proteins RPL5 and RPL11 combine with HDM2 to form an Impaired Ribosome Biogenesis Checkpoint (IRBC) complex, which subsequently results in p53 upregulation. Formerly, it is reported RiBi partakes in senescence, however, whether RiBi is involved in kidney fibrosis is uncovered. It is reported that ECT2 plays important roles in G2/M arrest, aging and fibrosis, however whether ECT2 could regulate RiBi is unknown. Currently, we aim to investigate whether and how ECT2 regulate RiBi during renal fibrosis. Methods: The UUO and in vitro TGF-β stimuli were utilized to setup fibrosis models. CX5461 was administrated to inhibit rDNA transcription. EU staining was used to detect the synthesis of nascent RNA. Cell proliferation and senescence were observed by CCK8 and SA-β-Gal staining respectively. Besides, RT-qPCR, WB, Masson, IF and multiple IF, Flow cytometry, Co-IP and transcriptome sequencing were used in this study. Results: In vitro and in vivo, multiple IF and EU staining revealed a significant reduction of nascent RNA during fibrosis, and which was reconfirmed by the decline of 47S rRNA level tested via RT-qPCR. Above findings indicated that RiBi was depressed in fibrotic status. Moreover, using CX5461 successfully disrupted RiBi along with kidney fibrosis, G2/M arrest and cell senescence. Interestingly, CX5461 treatment increased the binding of HDM2 to RPL5 and RPL11. Next, we found ECT2 induced RiBi failure, contrarily, blocking ECT2 mitigated the inhibition of RiBi and kidney fibrosis caused by CX5461. Furthermore, transcriptome sequencing indicated that PLXNB2 was one of the down-regulated genes of ECT2. Knocking down PLXNB2 inhibited RiBi and promoted fibrosis, however, this effect could be partially abolished by co-transfection with ECT2 siRNA. These data implied that the elevated ECT2 depressed PLXNB2, and which consequently led to the failure of RiBi and kidney fibrosis. Conclusion: Collectively, our findings indicated that blocking RiBi initiated G2/M arrest and senescence of TECs as well as kidney fibrosis. ECT2 mediated PLXNB2 decline was a potential mechanism accounting for the impaired RiBi in fibrosis.