Ubiquitin-like protein FAT10 promotes renal fibrosis by stabilizing USP7 to prolong CHK1-mediated G2/M arrest in renal tubular epithelial cells

Abstract

Renal fibrosis is the pathological hallmark of chronic kidney disease that is influenced by numerous factors. Arrest of renal tubular epithelial cells (RTECs) in G2/M phase is closely correlated with the progression of renal fibrosis; however, the mechanisms mediating these responses remain poorly defined. In this study, we observed that human leukocyte antigen-F adjacent transcript 10 (FAT10) deficiency abolished hypoxia-induced upregulation of checkpoint kinase 1 (CHK1) expression in RTECs derived from FAT10^+/+ and FAT10^-/- mice. Further investigations revealed that FAT10 contributes to CHK1-mediated G2/M arrest and production of pro-fibrotic cytokines in RTECs exposed to hypoxia. Mechanistically, FAT10 directly interacted with and stabilized the deubiquitylating enzyme ubiquitin specific protease 7 (USP7) to mediate CHK1 upregulation, thereby promoting CHK1-mediated G2/M arrest in RTECs. In animal model, FAT10 expression was upregulated in the obstructed kidneys of mice induced by unilateral ureteric obstruction injury, and FAT10^-/- mice exhibited reduced unilateral ureteric obstruction injury induced-renal fibrosis compared with FAT10^+/+ mice. Furthermore, in a cohort of patients with calculi-related chronic kidney disease, upregulated FAT10 expression was positively correlated with renal fibrosis and the USP7/CHK1 axis. These novel findings indicate that FAT10 prolongs CHK1-mediated G2/M arrest via USP7 to promote renal fibrosis, and inhibition of the FAT10/USP7/CHK1 axis might be a plausible therapeutic approach to alleviate renal fibrosis in chronic kidney disease.

Keywords: FAT10; cell cycle (G2/M) arrest; checkpoint kinase 1; renal fibrosis; ubiquitin specific protease 7.

Figure 1

Hypoxia-induced CHK1 upregulation in RTECs is depend on FAT10. (A) Upon hypoxia treatment for 24 h, mass spectroscopic analysis was performed to detect protein expression in RTECs from FAT10^+/+ mice (n = 3) and FAT10^−/− mice (n = 3). (B) Determination (left) and quantification (right) of the FAT10 and CHK1 protein levels in FAT10^+/+ RTECs or FAT10^−/− RTECs after hypoxia injury. Tubulin was used as a loading control. ^*P < 0.05, ^**P < 0.01. (C) Determination (left) and quantification (right) of the FAT10 and CHK1 protein levels in HK-2 cells following treatment with hypoxia or without hypoxia. ^*P < 0.05, ^**P < 0.01. (D) The protein and mRNA levels of FAT10 and CHK1 in HK-2 cells following treatment with hypoxia or without hypoxia. ^**P < 0.01. (E) Determination (left) and quantification (right) of the FAT10 and CHK1 protein levels in HK-2 cells transfected with shFAT10 following hypoxic injury. ^**P < 0.01. (F) Upon hypoxia treatment for 24 h, the mRNA levels of FAT10 and CHK1 in HK-2 cells transfected with shFAT10. ^**P < 0.01.

Figure 2

FAT10 is required for CHK-1-mediated G2/M arrest in RTECs under hypoxia treatment. (A) Western blotting showing the protein expression of CHK1, CDKN1A, TGF-β and CTGF in CHK1-silencing HK-2 cells following hypoxia injury. Tubulin was used as a loading control. (B) TGF-β and CTGF in the culture supernatants was measured in culture supernatants by ELISA assay. ^**P < 0.01. (C) Detection for cell cycle of CHK1-silencing HK-2 cells following hypoxia injury. Results are expressed as peak diagram (left) and calculated distribution for cells in G0/G1, S and G2/M phases (right). ^*P < 0.05. (D) Upon hypoxia treatment, western blotting of FAT10, CHK1, CDKN1A, TGF-β and CTGF in HK-2 cells stably transfected with shFAT10 in the presence or absence of Flag-CHK1. (E) Detection for cell cycle of FAT10-silencing HK-2 cells in the presence or absence of Flag-CHK1 following hypoxia injury. Results are expressed as peak diagram (left) and calculated distribution for cells in G0/G1, S and G2/M phases (right). ^*P < 0.05. (F) Western blotting showing the protein expression of FAT10, CHK1, CDKN1A, TGF-β and CTGF in FAT10^+/+ RTECs and FAT10^−/− RTECs following treatment with hypoxia or without hypoxia. (G) Detection for cell cycle of FAT10^+/+ RTECs and FAT10^−/− RTECs following treatment with hypoxia or without hypoxia. Results are expressed as peak diagram (left) and calculated distribution for cells in G0/G1, S and G2/M phases (right). ^*P < 0.05.

Figure 3

FAT10 interacting with USP7 and stabilising USP7 expression. (A) A partial list of FAT10-associated proteins were indicated by immunoprecipitation-mass spectrometry. (B) Protein levels of FAT10 and USP7 in FAT10-overexpressing or FAT10-silenced HK-2 cells were detected by western blotting. Tubulin was used as a loading control. (C) Determination (left) and quantification (right) of FAT10 and USP7 protein levels in HK-2 cells or FAT10-silenced HK-2 cells following treatment with hypoxia or without hypoxia. ^*P < 0.05, ^**P < 0.01. (D) Western blot showing USP7 protein levels in HK-2 cells following treatment with 10 μM MG132 at different times. (E) HK-2 cells transduced with shFAT10 or Flag-FAT10 were treated with MG132. Cells were collected at 6 h and immunoblotted with the antibodies indicated. (F and G) Representative (F) and quantitative (G) results of USP7 protein level in FAT10-overexpression or FAT10-silencing cells. The cells were treated with cycloheximide (CHX, 100 μg/ml) for indicated time points were subjected to western blot analysis. The degradation rate of USP7 protein was calculated according to the ratio of USP7/tubulin. The quantification data represent mean ± SD from three independent experiments and were statistically analyzed with Student’s t-test, ^*P < 0.05, ^**P < 0.01. (H and I) Competitive binding of USP7 was analyzed in a GST-pull down experiment. HEK-293 T cells were transfected with the indicated constructs and lysed for IP using anti-His beads to detect GST binding. (J) Knockdown or exogenous expression of FAT10 in HK-2 cells altered the ubiquitination of USP7. The cells in each group were treated with MG132. (K) Western blotting showing the protein expression of FAT10 and USP7 in FAT10^+/+ RTECs and FAT10^−/− RTECs following treatment with hypoxia or without hypoxia. (L) Ubiquitinated USP7 in in FAT10^+/+ RTECs and FAT10^−/− RTECs following treatment with hypoxia or without hypoxia. The cells in each group were treated with MG132.

Figure 4

FAT10 regulates CHK1 expression through USP7. (A and B) Western blotting and qRT-PCR analyses of CHK1 expression levels in HK-2 cells stably transfected with shNC or shUSP7. (C and D) Western blotting and qRT-PCR analyses of CHK1 expression levels in HK-2 cells stably transfected with control vector or Flag-USP7. ^**P < 0.01. Tubulin was used as a loading control. (E) Knockdown or exogenous expression of USP7 altered the ubiquitination of CHK1 in HK-2 cells. The cells in each group were treated with MG132. (F) Western blotting of FAT10, CHK1 and USP7 in HK-2 cells stably transfected with Flag-FAT10 in the presence or absence of shUSP7. (G) Ubiquitinated CHK1 in HK-2 cells stably transfected with Flag-FAT10 in the presence or absence of shUSP7. The cells in each group were treated with MG132. (H and I) Upon hypoxia treatment, western blotting of FAT10, CHK1, CDKN1A, TGF-β and CTGF in FAT10-silencing HK-2 transfected with Flag-USP7 (H) or in FAT10-overexpression HK-2 transfected with shUSP7 (I). (J and K) Detection for cell cycle of FAT10-silencing HK-2 transfected with Flag-USP7 (J) or FAT10-overexpression HK-2 transfected with shUSP7 (K) following hypoxia injury. Results are expressed as peak diagram (left) and calculated distribution for cells in G2/M phases (right). ^*P < 0.05.

Figure 5

UUO-induced kidney fibrosis was suppressed in FAT10-deficient mice. (A and B) Representative HP and FAT10 staining in the kidneys from FAT10^+/+ (A) and FAT10^−/− (B) mice subjected to either UUO or sham operation; scale bar = 50 μm. (C and D) Representative Masson’s trichrome (C) and picrosirius red staining (D) of kidney sections from FAT10^+/+ and FAT10^−/− mice with or without UUO for 7 days. (E and F) Immunohistochemistry of protein expression of α-SMA (E) and Collagen IV (F) in obstructed kidneys from FAT10^+/+ and FAT10^−/− mice subjected to either UUO or sham operation. (G and H) Bar graph (right) shows quantification of fibrotic areas in histological sections; ^**P < 0.01 versus FAT10^+/+ mice at the same time point; n = 6. (I and J) Bar graph shows quantification of areas of positive cells; ^**P < 0.01 versus FAT10^+/+ mice at the same time point; n = 6.

Figure 6

FAT10 was up-regulated and positively correlated with renal fibrosis in patients with calculi related chronic kidney disease. (A) Representative photos of renal sections from normal kidney and biopsy samples from patients with calculi related chronic kidney disease. IHC, immunohistochemistry; scale bar = 50 μm. (B) The expression of renal FAT10 at initial biopsy positively correlated with the tubular interstitial fibrosis index in a partial correlation analysis. (C–F) Scatter plots show a positive correlation between FAT10 and USP7, CHK1, CDKN1A, TGF-β, respectively. (G–I) The statistical analysis data show that the expression level of USP7 was positively correlated with the expression level of CHK1, CDKN1A and TGF-β. ^*P < 0.05, ^**P < 0.01.

Figure 7

Model summarizing the role of FAT10 in renal fibrosis response to UUO injury. Upon UUO injury, FAT10 up-regulation stabilizes USP7 expression, thereby leading to CHK1-mediated G2/M arrest in RTECs, which further drives fibrogenic responses.