TGF-β1 promotes expression of fibrosis-related genes through the induction of histone variant H3.3 and histone chaperone HIRA

Abstract

Renal fibrosis is a histological manifestation that occurs in almost every type of chronic kidney disease. Histone variant H3.3 and its chaperone, histone cell cycle regulation defective homolog A (HIRA), serve as epigenetic marks that regulate transcriptional activity. In this study, we assessed the roles of histone H3.3 and HIRA in unilateral ureteral-obstruction (UUO) mice. In UUO mice, the levels of histone H3.3 and HIRA were significantly upregulated in the kidneys. These upregulated levels were decreased by a TGF-β1 neutralizing antibody. TGF-β1 induced histone H3.3 and HIRA expression in vitro via a Smad3-dependent pathway in normal rat kidney (NRK)-52E cells. Additionally, knockdown of HIRA expression decreased histone H3.3 expression and fibrogenesis in NRK-52E cells after TGF-β1 stimulation. Chromatin immunoprecipitation analysis revealed that promoters of fibrosis-related genes were immunoprecipitated with both histone H3.3 and HIRA in NRK-52E cells. Lastly, in human kidney biopsies from patients diagnosed with IgA nephropathy, histone H3.3 and HIRA immunostaining correlated positively with areas of fibrosis and estimated glomerular filtration rate. In conclusion, TGF-β1 induces expression of histone H3.3 and HIRA, which regulates expression of fibrosis-related genes.

Figure 1

Histone H3.3 and HIRA are up-regulated in the kidney after obstructive injury. (A) Histone H3.3 (H3f3a and H3f3b) and (B) Hira mRNA levels were determined by quantitative real-time polymerase chain reaction (qRT-PCR) 7 days after sham or UUO surgery. UUO-induced (C) histone H3.3 and (D) HIRA protein levels were confirmed by western blotting. Histone H3 for histone H3.3 and β-Actin for HIRA were used as internal controls. Data are means ± S.D. *P < 0.05, (t test; n = 5 mice per group).

Figure 2

TGF-β1 induces histone H3.3 and HIRA expression and injection of neutralizing TGF-β1 antibody reduces histone H3.3 and HIRA expression after UUO. UUO mice were treated with mouse immunoglobulin G1 (IgG1) or neutralizing TGF-β1 antibody (TGF-β1-Ab). (A) Images of HE and Masson’s trichrome staining show histological change in UUO mice with control IgG1 or TGF-β1-Ab. (B) mRNA levels of histone H3.3 (H3f3a and H3f3b) and (C) Hira were determined in kidneys by qRT-PCR. (D) Representative western blot analysis of histone H3.3 and HIRA is shown. Histone H3 for histone H3.3 and β-Actin for HIRA were used as internal controls. Relative levels of expression are shown in the lower panel. (E) Images of histone H3.3 and HIRA staining demonstrating the levels of histone H3.3 and HIRA after treatment with control IgG1 or TGF-β1-Ab. Bar = 100 μm. Data are means ± S.D. *P < 0.05, (one-way ANOVA followed by the post hoc t test with Bonferroni correction; n = 5 mice per group).

Figure 3

TGF-β1 induces histone H3.3 and HIRA expression in renal epithelial and fibroblast cells. Representative western blotting analysis shows the levels of histone H3.3 and HIRA proteins in TGF-β1-stimulated NRK-52E and NRK-49F cells at various doses (time; 24 hours) for (A) histone H3.3 and (B) HIRA, and time points (TGF-β1; 1.0 ng/mL) for (C) histone H3.3 and (D) HIRA. Histone H3 for histone H3.3 and β-Actin for HIRA were used as internal controls. Expression levels were compared with vehicle-treated control. Relative levels of expression are shown in the lower panel. Data are means ± S.D. *P < 0.05, **P < 0.01, (one-way ANOVA followed by Dunnett’s post hoc test based on vehicle-treated controls; n = 5 samples per group).

Figure 4

TGF-β1-induced histone H3.3 and HIRA expression is suppressed by knockdown of Smad3 in NRK-52E cells. NRK-52E cells were treated with Smad3 siRNA (si-Smad3) or negative control (si-Neg) oligonucleotides. (A) histone H3.3 (H3f3b) and Hira mRNA levels were determined by qRT-PCR in stimulated NRK-52E cells with or without TGF-β1 (1.0 ng/mL, 24 hours). Representative western blot analysis for (B) histone H3.3, (C) HIRA, (D) Smad3, and (E) phosphorylated Smad3 (p-Smad3) in stimulated NRK-52E cells with or without TGF-β1 (1.0 ng/mL, 30 minutes or 24 hours). Because p-Smad3 reaches a peak 30 minutes after TGF-β1 stimulation, this time point was only used in p-Smad3 experiments. Histone H3 for histone H3.3, and β-Actin for HIRA, Smad3, and p-Smad were used as internal controls. Relative levels of expression are shown in the lower panel. Data are means ± S.D. *P < 0.05, **P < 0.01, (one-way ANOVA followed by the post hoc t test with Bonferroni correction; n = 5 samples per group).

Figure 5

Knockdown of HIRA in NRK-52E cells inhibits TGF-β1-induced fibrogenesis by suppressing histone H3.3 expression. NRK-52E cells were treated with Hira siRNA (si-HIRA) or negative control (si-Neg) oligonucleotides. (A) Hira, (B) histone H3.3 (H3f3b), (C) α-SMA (Acta2), and collagen1 (Colla1) mRNA levels were determined by qRT-PCR in stimulated NRK-52E cells with or without TGF-β1 (1.0 ng/mL, 24 hours). Representative western blot analysis for (A) HIRA, (B) histone H3.3 and (C) α-SMA. Histone H3 for histone H3.3 and β-Actin for HIRA and α-SMA were used as internal controls. Relative levels of expression are in the lower panel. Data are means ± S.D. *P < 0.05, **P < 0.01, (one-way ANOVA followed by the post hoc t test with Bonferroni correction; n = 5 samples per group).

Figure 6

HIRA co-localizes with histone H3.3 at activated promoters of fibrotic genes in NRK-52E cells. Treatment with neutralizing TGF-β1 antibody (2.0 μg/mL) was performed at the same time as TGF-β1 (1.0 ng/mL) stimulation. Chromatin immunoprecipitation (ChIP) analysis of the binding of (A) histone H3.3 and (B) HIRA proteins to Col1a1, Ctgf, Pai1 and Acta2 promoters in NRK-52E cells is shown. Immunoprecipitated DNA and input DNA were subjected to qPCR. Results were normalized to input DNA and rabbit immunoglobulin G (IgG) was used as a negative control. Data are means ± S.D. *P < 0.05, **P < 0.01, (one-way ANOVA followed by the post hoc t test with Bonferroni correction; n = 5 samples per group).

Figure 7

HIRA expression correlates with the expression of histone H3.3, the degree of fibrosis and estimated glomerular filtration rate (eGFR) in human kidney biopsy specimens (n = 28). (A) Representative images of histone H3.3, HIRA, Masson’s trichrome staining and α-SMA demonstrate that levels of histone H3.3, HIRA, and the size of fibrotic areas increase with the decline of eGFR in patients with IgA nephropathy (IgAN). Columns show images from the same patient. (B) HIRA expression correlated positively with histone H3.3 expression (ρ = 0.46, P = 0.015), Masson’s trichrome-positive areas (ρ = 0.39, P = 0.038), α-SMA levels (ρ = 0.42, P = 0.026) and eGFR (ρ = −0.53, P = 0.014). Histone H3.3 expression was positively correlated with Masson’s trichrome-positive areas (ρ = 0.52, P = 0.004), α-SMA levels (ρ = 0.42, P = 0.027) and eGFR (ρ = −0.68, P = 0.008). Spearman’s correlation coefficient test was used. Bar = 50 μm. The Japanese GFR equation based on serum creatinine was used to determine eGFR. eGFR (mL/minute/1.73 m²) = 194 × Scr^−1.094 × Age^−0.287 × 0.739 (if female).