Tet3-mediated hydroxymethylation of epigenetically silenced genes contributes to bone morphogenic protein 7-induced reversal of kidney fibrosis - PubMed (original) (raw)

Tet3-mediated hydroxymethylation of epigenetically silenced genes contributes to bone morphogenic protein 7-induced reversal of kidney fibrosis

Björn Tampe et al. J Am Soc Nephrol. 2014 May.

Abstract

Methylation of CpG island promoters is an epigenetic event that can effectively silence transcription over multiple cell generations. Hypermethylation of the Rasal1 promoter contributes to activation of fibroblasts and progression of kidney fibrosis. Here, we explored whether such causative hypermethylation could be reversed through endogenous mechanisms and whether such reversal of hypermethylation is a constituent of the antifibrotic activity of bone morphogenic protein 7 (BMP7). We show that successful inhibition of experimental kidney fibrosis through administration of BMP7 associates with normalization of Rasal1 promoter hypermethylation. Furthermore, this reversal of pathologic hypermethylation was achieved specifically through Tet3-mediated hydroxymethylation. Collectively, our findings reveal a new mechanism that may be exploited to facilitate therapeutic DNA demethylation to reverse kidney fibrosis.

Copyright © 2014 by the American Society of Nephrology.

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Figures

Figure 1.

Figure 1.

Ameliorated experimental renal fibrosis upon BMP7 treatment is associated with reversal of aberrant Rasal1 promoter methylation. (A) Histology of UUO-challenged kidneys. The panels display representative photomicrographs of Masson’s trichrome-stained (MTS) kidney sections from control and UUO kidneys treated with either vehicle or BMP7. Original magnification, ×10; scale bar, 200 _µ_m. (B) Rasal1 methylation in UUO kidneys. We performed methylated DNA immunoprecipitation (MeDIP) to assess the effect of antifibrotic BMP7 treatment on Rasal1 methylation in mouse kidneys that were challenged with UUO. In this assay, fragmented total kidney DNA (input DNA) was exposed to antibodies that specifically capture 5mC DNA. The captured DNA is eluted and analyzed by primers specific to the Rasal1 promoter. Electrophoresis of PCR products was performed on a Bioanalyzer. The upper panel displays a virtual gel of Rasal1 PCR products of captured (methylated; 5mC) DNA; the lower panel displays Rasal1 PCR products of input DNA (to control for equal loading in immunoprecipitation). Rasal1 was hypermethylated after 7 days of ureteral ligation (_n_=4), and Rasal1 hypermethylation was ameliorated in BMP7-treated mice (_n_=4). (C) Rasal1 mRNA expression in UUO kidneys. Rasal1 expression was analyzed by quantitative RT-PCR in RNA isolated from total kidneys. For statistical analysis, UUO mice treated with vehicle or BMP7 were compared with controls arbitrarily set to one. Rasal1 expression was suppressed in kidneys of mice that had been challenged with UUO and received vehicle buffer (_n_=4), but it was increased in kidneys of BMP7-treated mice (_n_=4). Experiments were done in triplicate, and data are presented as means±SDs. **P<0.01; ****P<0.0001. (D) Histology of kidneys from diabetic mice (DN kidneys). Representative photomicrographs of MTS kidney sections from control mice, mice that had been made diabetic with DN, and diabetic mice treated with BMP7 are displayed. Original magnification, ×10; scale bar, 200 _µ_m. (E) Rasal1 methylation in DN kidneys. E displays a virtual gel of MeDIP analysis. PCR products of control input DNA and immunoprecipitated DNA (5mC DNA) of nonfibrotic control kidneys, fibrotic DN kidneys (6 months post-DN injection), and DN kidneys of BMP7-treated mice (DN month 6 + BMP7) are displayed. Rasal1 is hypermethylated in fibrotic kidneys of diabetic mice (_n_=5). Rasal1 methylation is ameliorated in kidneys of mice that had been treated with antifibrotic BMP7 (_n_=4). (F) Rasal1 expression in DN kidneys. Rasal1 mRNA expression was assessed in whole-kidney lysates. The graph summarizes Rasal1 expression levels in relation to control kidneys (no diabetes). Compared with fibrotic DN kidneys (_n_=5), Rasal1 expression was increased on BMP7 treatment (_n_=4). Experiments were done in triplicate, and data are presented as means±SDs. **P<0.01; ***P<0.001. (G) Rasal1 hydroxymethylation (5hmC) in UUO kidneys. We immunoprecipitated hydroxymethylated DNA using antibodies to 5hmC (hydroxy-MeDIP) and performed PCR using primers specific to Rasal1 promoter. The upper panel shows a virtual gel image of Rasal1 PCR products of hydroxymethylated DNA (5hmC DNA), and the lower panel shows PCR products of input DNA as controls for equal loading. Hydroxymethylation was not detected in kidneys from control mice (_n_=4) or fibrotic kidneys of mice that had been challenged with UUO (_n_=4), but it was present in UUO kidneys of mice that had been treated with BMP7 (_n_=4). (H) Rasal1 hydroxymethylation (5hmC) in DN kidneys. Hydroxymethylation was not detected in kidneys from control mice (_n_=3) or fibrotic kidneys of diabetic mice (_n_=5); antifibrotic BMP7 therapy caused de novo Rasal1 hydroxymethylation (_n_=4). (I–K) Tet mRNA expression levels in UUO and DN kidneys. We isolated mRNA from whole-kidney lysates and analyzed for Tet expression levels by quantitative RT-PCR in fibrotic kidneys of mice that had been challenged with UUO and DN and treated with antifibrotic BMP7. The bar graphs summarize relative expression in each group. Expression of Tet1 and Tet2 were not significantly altered in fibrotic UUO (_n_=4), DN kidneys (_n_=5) or response to BMP7 (_n_=4 in each group). Tet3 expression was suppressed in fibrotic kidneys challenged with UUO (_n_=4) and DN (_n_=5), and ameliorated fibrosis on BMP7 treatment was associated with increased Tet3 expression (_n_=4 in each group). Experiments were done in triplicate, and data are presented as means±SDs. ****P<0.0001.

Figure 2.

Figure 2.

BMP7 normalizes TGF-_β_1–induced methylation of Rasal1 through hydoxymethylation in primary renal fibroblasts. (A) Rasal1 methylation (5mC). Rasal1 methylation in primary mouse kidney fibroblasts in response to TGF-_β_1 and/or BMP7 for 1, 2, 5, and 10 days was analyzed by MeDIP. After 2 days of TGF-_β_1 stimulation, Rasal1 methylation was detected. After removal of TGF-_β_1 (after 5 days when Rasal1 was robustly methylated), Rasal1 remained hypermethylated and transcriptionally silenced (assessed at day 10 of tissue culture when cells had been exposed to 5 days of TGF-_β_1 and 5 additional days of growth factor-free media). When TGF-_β_1 incubation was followed by exposure to BMP7 for 5 days, Rasal1 was no longer found to be methylated. (B) Rasal1 expression in response to TGF-_β_1 and BMP7. Primary mouse kidney fibroblasts were subjected to TGF-_β_1 for 1, 2, 5, and 10 days. Exposure to TGF-_β_1 caused rapid suppression of Rasal1 mRNA within 1 day without Rasal1 methylation. Prolonged exposure to TGF-_β_1 (starting after 2 days) causes Rasal1 methylation, and Rasal1 mRNA expression levels remain suppressed, even when the TGF-_β_1 was removed after 5 days. Exposure to BMP7 normalized Rasal1 mRNA expression levels. Experiments were done in triplicate, and data are presented as means. ***P<0.001. P values were calculated respective to 5 days TGF-_β_1+5 days untreated. (C) Proliferation of primary mouse kidney fibroblasts. Primary mouse fibroblasts were seeded into six-well plates at a density of 60,000 cells per well, trypsinized, and counted after 1, 2, 5, and 10 days. Compared with fibroblasts cultured in growth factor-free media, TGF-_β_1 increased fibroblast proliferation, even when the TGF-_β_1 was replaced with growth factor-free media after 5 days. Treatment with BMP7 decreased TGF-_β_1–induced fibroblast proliferation. Experiments were replicated four times, and data are presented as means. **P<0.01. P values were calculated respective to 5 days TGF-_β_1+5 days untreated. (D) Rasal1 hydroxymethylation (5hmC) in response to TGF-_β_1 and BMP7. The upper panel shows a virtual gel image of Rasal1 PCR products of immunoprecipitated 5hmC DNA, and the lower panel shows PCR products of input DNA as controls for equal loading in hydroxy-MeDIP. BMP7 induced Rasal1 hydroxymethylation. (E–G) Tet mRNA expression on treatment with TGF-_β_1 and BMP7. Tet expression levels were analyzed by quantitative RT-PCR. Whereas Tet1 and Tet2 were not significantly regulated on exposure to TGF-_β_1 and/or BMP7, treatment with TGF-_β_1 suppressed Tet3, whereas BMP7 normalized mRNA expression levels. Experiments were done in triplicate, and data are presented as means±SDs. *P<0.05; ***P<0.001. (H) Tet3 protein levels on TGF-_β_1 and/or BMP7 treatment. On exposure to TGF-_β_1 and/or BMP7 for 2 days, Tet3 analyzed by Western blot was suppressed after treatment with TGF-_β_1, whereas BMP7 restored altered protein levels. (I) Quantification of Tet3 protein levels on treatment with TGF-_β_1 and/or BMP7. Tet3 band density was quantified relative to _α_-tubulin as control for equal loading. Measurements were done in triplicate, and data are presented as means±SD. ***P<0.001; ****P<0.0001. n.s., not significant.

Figure 3.

Figure 3.

BMP7 normalizes TGF-_β_1–induced methylation of Rasal1 through Tet3-dependent hydroxymethylation. Fibroblasts were maintained in serum-free control media (Rasal1 unmethylated) or media supplemented with TGF-_β_1 (Rasal1 hypermethylated) for 5 or 10 days. For analysis of possible Rasal1 demethylation, TGF-_β_1–containing media were removed after 5 days and replaced with either serum-free control media or media containing BMP7 for additional 5 days. For analysis of possible involvement of Tet proteins in Rasal1 demethylation and Rasal1 hydroxymethylation, cells were transfected with small interfering RNA (siRNA) oligonucleotides specifically targeting Tet1, Tet2, or Tet3 or scrambled control nucleotides. (A) Impact of Tet knockdown on Rasal1 methylation (5mC). The upper panel shows virtual gel images of Rasal1 PCR products of immunoprecipitated 5mC DNA, and the lower panel shows PCR products of input DNA as controls for equal loading. Panels display representative analysis of cells that had been transfected with scrambled siRNA or siRNAs targeting Tet1, Tet2, or Tet3. Tet3 knockdown but not Tet1 or Tet2 knockdown prevents Rasal1 demethylation in response to BMP7. (B) Impact of Tet3 knockdown on Rasal1 hydroxymethylation (5hmC). The upper panel shows a virtual gel image of Rasal1 PCR products of immunoprecipitated 5hmC DNA, and the lower panel shows PCR products of input DNA as controls for equal loading in hydroxy-MeDIP. BMP7-induced hydroxymethylation is only observed when Tet3 is present. (C) Impact of Tet3 knockdown on Rasal1 mRNA expression. Rasal1 mRNA expression levels were analyzed by quantitative RT-PCR. Bar graphs summarize relative Rasal1 mRNA expression in control cells (transfected with scrambled siRNA) on Tet1 and Tet2 knockdown as well as Tet3 knockdown. Rasal1 mRNA expression was only restored by BMP7 when Tet3 was present. Experiments were done in triplicate, and data are presented as means. **P<0.01. P values were calculated respective to 5 days TGF-_β_1+5 days untreated. (D) Impact of Tet3 knockdown on fibroblast proliferation. The bar graphs summarize relative cell counts after 5 and 10 days. Cells were transfected with either scrambled or siRNA-specific for Tet1, Tet2, or Tet3. BMP7 failed to blunt TGF-_β_1–induced proliferation when Tet3 was knocked down. Experiments were replicated four times, and data are presented as means. *P<0.05; **P<0.01; ***P<0.001. P values were calculated respective to 5 days TGF-_β_1+5 days untreated.

Figure 4.

Figure 4.

Tet3-dependent hydroxymethylation reverses profibrotic promoter methylation. (Top panel) Under physiologic conditions, cytosin bases within the Rasal1 CpG island promoter are unmethylated (referred to as naked cytosine), and Rasal1 is open for transcription. (Middle panel) Prolonged exposure to TGF-_β_1 causes methylation of cytosine residues within the Rasal1 CpG island promoter (referred to as 5mC) through a process involving the enzyme Dnmt1, causing transcriptional silencing of Rasal1. (Bottom panel) BMP7 induces Tet3 to convert 5mC into 5hmC. Such hydroxymethylation ultimately allows DNA repair mechanisms to replace hydroxymethylated cytosine with naked cytosine. Rasal1 is open for transcription.

Comment in

References

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