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. 2010 Mar;176(3):1139-47.
doi: 10.2353/ajpath.2010.090459. Epub 2010 Jan 21.

Bone morphogenetic protein-7 inhibits proximal tubular epithelial cell Smad3 signaling via increased SnoN expression

Dong Dong Luo  1 , Aled Phillips, Donald Fraser
Affiliations

Bone morphogenetic protein-7 inhibits proximal tubular epithelial cell Smad3 signaling via increased SnoN expression

Dong Dong Luo et al. Am J Pathol. 2010 Mar.

Abstract

Bone morphogenetic protein-7 (BMP-7) improves outcome in animal models of fibrotic renal disease by opposing transforming growth factor beta1 (TGF-beta)-dependent fibrosis. However, the underlying mechanisms remain obscure. Here, we studied the effect of BMP-7 on response to TGF-beta in the proximal tubular cell line HK-2 (PTC). BMP-7 specifically limited Smad3 but not Smad2 signaling. BMP-7 did not inhibit Smad3 phosphorylation or nuclear accumulation, nor did BMP-7 alter phosphorylated Smad3 dephosphorylation or degradation. However, BMP-7 treatment reduced Smad3 DNA binding to a consensus Smad binding element probe, and chromatin immunoprecipitation showed reduced Smad3 binding to the plasminogen activator inhibitor-1 promoter in PTCs treated with BMP-7 and TGF-beta compared with TGF-beta alone. Degradation of the transcriptional repressor SnoN has recently been shown to be necessary for Smad3 (but not Smad2) signaling. SnoN expression was transiently lost in PTCs after TGF-beta stimulation, but BMP-7 prevented this. Furthermore, BMP-7 had no effect on Smad3 signaling after siRNA-mediated SnoN knockdown, whereas prevention of SnoN degradation with the proteasome inhibitor MG132 reproduced the inhibitory action of BMP-7 on Smad3 signaling. We conclude that BMP-7 prevents TGF-beta-mediated loss of the transcriptional repressor SnoN and hence specifically limits Smad3 DNA binding, altering the balance of transcriptional responses to TGF-beta in PTCs. These results provide an important mechanistic insight into a key regulator of TGF-beta signaling.

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Figures

Figure 1
Figure 1
Regulation of profibrotic TGF-β transcriptional _targets by BMP-7. A–C: qRT-PCR analysis shows expression of profibrotic TGF-β-inducible genes in PTCs after 24 hours with or without1 ng/ml TGF-β incubation with or without 50 ng/ml BMP-7. A: TGF-β. B: Connective tissue growth factor (CTGF). C: PAI-1. D: PAI-1 expression after 8 days of culture with or without 1 ng/ml TGF-β with or without 50 ng/ml BMP-7. n = 3, mean of duplicate determinations. Error bars indicate +SEM. Representative data from one of three experiments giving similar results. *P < 0.01 versus control. **P < 0.05 versus TGF-β.
Figure 2
Figure 2
Regulation of Smad signaling response to TGF-β by BMP-7. HK-2 cells were transfected with Smad3-responsive (A and B) or Smad2-responsive (C) plasmids and then were incubated with BMP-7 for various times or doses, before incubation with 1 ng/ml TGF-β (black bar, control medium followed by TGF-β; gray bars, BMP-7 followed by TGF-β) or control medium (white bars) for 6 hours. A: Effect on Smad3 signaling of time course of 50 ng/ml BMP-7 incubation. B: Effect on Smad3 signaling of 24 hours preincubation with a BMP-7 dose range of 0 to 2000 ng/ml. C: Effect on Smad2 signaling of a 24-hour preincubation with BMP-7 at a dose range of 0 to 2000 ng/ml. *P < 0.05 versus TGF-β. RLU, relative light units.
Figure 3
Figure 3
Time course of Smad phosphorylation and dephosphorylation/degradation in response to TGF-β and BMP-7. A: HK-2 cells were incubated with 50 ng/ml BMP-7 for 0 to 24 hours before incubation with 1 ng/ml TGF-β for 1 hour. Whole-cell lysates were immunoblotted with antibodies against Phospho-Smads (PSmad) 1, 2, 3, and total Smads 1 and 3. Stripping and reprobing for glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used to confirm approximately equal loading. B–D: Time course of Smad1/3 dephosphorylation/degradation. HK-2 cells were incubated with control medium (B and C) or 50 ng/ml BMP-7 (D) for 24 hours before incubation with 1 ng/ml TGF-β for 30 minutes. Cells were washed extensively and incubated in cytokine-free control medium (B) or medium containing the Alk5 kinase inhibitor SB431542 (C and D) for time points up to 5 hours. Residual Phospho-Smad3 activity was detected by immunoblot before stripping and reprobing for total Smad3.
Figure 4
Figure 4
Nuclear accumulation of Smad3. HK-2 cells were incubated with 50 ng/ml BMP-7 or control medium for 24 hours before incubation with 1 ng/ml TGF-β or control medium for 1 hour. A: Immunoblotting of nuclear extracts for Phospho-Smad (PSmad) 1/3 and subsequent reprobing for c-Jun to confirm approximately equal loading. B: Immunofluorescent localization of Smad3. HK-2 cells were incubated with 50 ng/ml BMP-7 or control medium for 24 hours in eight-well chamber slides before incubation with 1 ng/ml TGF-β or control medium for 1 hour and detection of Smad3 by immunofluorescence microscopy.
Figure 5
Figure 5
BMP-7 inhibits Smad3 DNA binding. A and B: Electrophoretic mobility shift assay with a consensus Smad binding element probe. HK-2 cells were incubated with BMP-7 or control (Ctrl) medium for 24 hours before incubation with 1 ng/ml TGF-β or control medium for 1 hour. Bold arrow, retarded probe; arrow, supershifted probe. A: Electrophoretic mobility shift assay performed with nuclear protein extract and consensus Smad binding element (SBE) probe. B: Supershift assay performed with antibodies to Smad3, 4, and 5. Sm, Smad. C and D: ChIP: Smad3 binding to the PAI-1 promoter. After chromatin immunoprecipitation with Smad3 antibody or pre-immune globulin, PAI-1 promoter Smad binding elements (SBE) were detected by qRT-PCR. Data are presented as Smad3-precipitated signal/pre-immune globulin-precipitated signal, normalized to control. C: HK-2 cells were incubated with TGF-β for time points to 24 hours before ChIP. D: HK-2 cells were incubated with BMP-7 and TGF-β as indicated for 6 hours before ChIP. RE, Relative Expression.
Figure 6
Figure 6
BMP-7 prevents TGF-β-mediated down-regulation of the repressor of Smad3 transcription, SnoN. A–C: HK-2 cells were incubated with 1 ng/ml TGF-β, 50 ng/ml BMP-7, or both, for time points to 24 hours before immunoblotting of whole-cell extracts. Subsequently, blots were reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to confirm approximately equal loading. A: SnoN and Ski expression after TGF-β. B: SnoN expression after BMP-7. C: SnoN expression after TGF-β and BMP-7. D and E: HK-2 cells were incubated with 50 ng/ml BMP-7 or control medium for 24 hours before incubation with 1 ng/ml TGF-β or control medium for 30 minutes and immunoblotting of whole-cell extracts for SnoN. Subsequently, blots were reprobed for GAPDH to confirm approximately equal loading. D: Representative immunoblot of four experiments giving similar results. E: Densitometry of four independent experiments. F: HK-2 cells were transfected with SnoN siRNA together with Smad3 responsive (Scr) (CAGA luciferase) and control (Renilla) vectors for 48 hours. Subsequently, cells were incubated with BMP-7 or control medium for 24 hours and then incubated with TGF-β for 6 hours. Data are presented as firefly luciferase/Renilla luciferase, normalized to the TGF-β control. G: HK-2 cells were transfected with Ski siRNA together with Smad3-responsive (Scr) (CAGA luciferase) and control (Renilla) vectors for 48 hours. Subsequently, cells were incubated with BMP-7 or control medium for 24 hours and then incubated with TGF-β for 6 hours. Data are presented as firefly luciferase/Renilla luciferase, normalized to TGF-β control. RLU, relative light units.
Figure 7
Figure 7
Effect of proteasome inhibitor MG132 on SnoN expression, Smad3 signaling, and TGF-β-dependent PAI-1 expression. A and B: HK-2 cells were incubated with control medium or 1 ng/ml TGF-β for 30 minutes with or without 10 μmol/L MG132, and immunoblots of whole-cell extracts for SnoN are shown. Subsequently, blots were reprobed for GAPDH to confirm approximately equal loading. A: Representative immunoblot of four experiments giving similar results. B: Densitometry of four independent experiments. C: HK-2 cells were transfected with Smad3 and Renilla control reporter constructs for 48 hours, before incubation with control medium or 1 ng/ml TGF-β for 6 hours with or without 10 μmol/L MG132. Data are presented as firefly/Renilla luciferase activity, normalized to control. D: HK-2 cells were incubated with control medium or 1 ng/ml TGF-β for 24 hours with or without 10 μmol/L MG132 before detection of PAI-1 mRNA by qRT-PCR. *P < 0.001 versus control; **P < 0.01 versus TGF-β. RLU, relative light units.
Figure 8
Figure 8
Proposed mechanism of regulation of Smad3 signaling by BMP7. A: In the absence of TGF-β, Smads shuttle into and out of the nucleus. SnoN binds to Smad binding elements and prevents R-Smad binding. B: After ligand binding, active TGF-β receptor complex leads to R-Smad phosphorylation. R-Smad-Arkadia-SnoN complexes lead to SnoN degradation. R-Smad-Smad4 complexes accumulate in the nucleus and bind to DNA. C: BMP7 prevents loss of SnoN expression. R-Smad-Smad4 complexes accumulate in the nucleus, but DNA binding to consensus Smad binding elements is prevented by SnoN. Arkadia-dependent SnoN degradation appears necessary for Smad3 (or Smad2Δ-exon3)-dependent responses, but not Smad1/Smad4-dependent responses or responses driven by Smad2-Smad4-FoxH1 complexes (see Levy et al26).
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