A novel regulatory mechanism of the bone morphogenetic protein (BMP) signaling pathway involving the carboxyl-terminal tail domain of BMP type II receptor

Abstract

Bone morphogenetic protein (BMP) signaling regulates many different biological processes, including cell growth, differentiation, and embryogenesis. BMPs bind to heterogeneous complexes of transmembrane serine/threonine (Ser/Thr) kinase receptors known as the BMP type I and II receptors (BMPRI and BMPRII). BMPRII phosphorylates and activates the BMPRI kinase, which in turn activates the Smad proteins. The cytoplasmic region of BMPRII contains a "tail" domain (BMPRII-TD) with no enzymatic activity or known regulatory function. The discovery of mutations associated with idiopathic pulmonary artery hypertension mapping to BMPRII-TD underscores its importance. Here, we report that Tribbles-like protein 3 (Trb3) is a novel BMPRII-TD-interacting protein. Upon BMP stimulation, Trb3 dissociates from BMPRII-TD and triggers degradation of Smad ubiquitin regulatory factor 1 (Smurf1), which results in the stabilization of BMP receptor-regulated Smads and potentiation of the Smad pathway. Downregulation of Trb3 inhibits BMP-mediated cellular responses, including osteoblast differentiation of C2C12 cells and maintenance of the smooth muscle phenotype of pulmonary artery smooth muscle cells. Thus, Trb3 is a critical component of a novel mechanism for regulation of the BMP pathway by BMPRII.

FIG. 1.

Trb3 and BMPRII form a complex in mammalian cells. (A) Schematic diagram of Trb3 and yeast two-hybrid clones. Yeast two-hybrid clones are fusions between Gal4 AD and mouse Trb3 cDNAs. (B) Cos7 cells were transiently transfected with the indicated Flag-tagged BMPRII constructs with or without HA-tagged Trb3 expression plasmid. Twenty-four hours after transfection, cells were lysed, immunoprecipitated (IP) with anti-Flag antibody, and immunoblotted (IB) with anti-HA antibodies. Total cell lysates were immunoblotted with anti-Flag or anti-HA antibody to visualize the expression levels of BMPRII and Trb3. The result is summarized in the panel below. (C) Untransfected PASMCs (top) or PASMCs transfected with a Flag-tagged BMPRII (WT or ΔT) expression construct were subjected to immunofluorescence staining with anti-human Trb3 antibodies conjugated with FITC (green) or anti-Flag antibody conjugated with Cy3 (red), respectively. Nuclei were visualized by DNA staining with DAPI. (D) Interaction between Trb3 and BMPRII-TD was tested by yeast two-hybrid analysis. Trb3 deletion mutant cDNAs fused to Gal4 AD were transformed into yeast expressing the bait construct containing a fusion of human BMPRII-TD and Gal4 DB. Interaction was scored by growth on a histidine-depleted plate. (E) Cos7 cells were transfected with a vector encoding Flag-tagged Trb3 (WT) or the ΔK mutant with or without BMPRII expression vector as indicated. Total cell lysates were immunoprecipitated with anti-BMPRII antibodies, followed by immunoblot analysis with anti-Flag antibody. Total cell lysates were subjected to immunoblot analysis with anti-BMPRII, anti-Flag (for Trb3), and anti-p38MAPK (loading control) antibodies.

FIG. 2.

Trb3 dissociates from BMPRII upon BMP4 stimulation. (A) Cos7 cells were transfected with Flag-BMPRII (WT, Δ860, or K1) and HA-Trb3, followed by treatment with 3 nM BMP4 for 2 h. The amount of BMPRII-Trb3 complex was examined by immunoprecipitation (IP) with anti-Flag antibody, followed by immunoblot analysis (IB) with anti-Trb3 antibody. Total cell lysates were subjected to immunoblot analysis with anti-Trb3 and anti-Flag (for BMPRII) antibodies. (B) PASMCs transfected with the Flag-BMPRII (WT) expression construct were treated with (+BMP4) or without (none) 3 nM BMP4 for 2 h and subjected to immunofluorescence staining with anti-human Trb3 antibodies conjugated with Cy3 (red) or anti-Flag antibody FITC (green), respectively. Colocalization of Trb3 and BMPRII was reduced from 66% ± 2.5% to 32% ± 0.5% upon BMP4 treatment (means ± standard errors for three independent experiments; n = 50).

FIG. 3.

Trb3 is essential for the BMP-mediated signaling pathway. (A) The Xvent2-luc reporter construct was cotransfected with increasing amounts of Trb3 expression plasmid (250, 500, or 1,000 ng) into P19 cells. Transcriptional activity in the presence (+BMP4) or absence (none) of 3 nM BMP4 treatment was monitored by a luciferase assay. Data are plotted as means ± standard errors for three independent experiments. *, P < 0.05; **, P < 0.001 (for presence versus absence of BMP4 treatment). (B) PA-1 cells were infected with adenovirus (Ad) carrying Flag-tagged mouse Trb3 (WT), the Trb3 (ΔK) mutant, or β-galactosidase (LacZ) as a control. Twenty-four hours after virus infection, PA-1 cells were stimulated with or without 3 nM BMP4 for 20 h and were subjected to a luciferase assay. Total cell lysates of PA-1 cells were subjected to Western blot analysis by anti-Flag antibody to examine expression of Trb3 (WT or ΔK). The average levels of induction of reporter activity observed upon BMP4 stimulation are plotted on the ordinate axis with standard errors. The increase in induction observed upon Trb3 (WT) virus infection is statistically significant compared to the value for uninfected or LacZ virus-infected cells (*, P < 0.001). IB, immunoblot analysis. (C) PA-1 cells were transfected with low (60 nM) or high (120 nM) concentrations of nontargeting siRNA (control) or siRNA directed against mouse Trb3 for 48 h. Total RNAs were isolated, and endogenous Trb3 mRNA expression was analyzed by RT-PCR. The result was normalized to the hypoxanthine-guanine phosphoribosyl transferase expression level. The difference in normalized Trb3 mRNA expression between control siRNA-transfected and Trb3 siRNA-transfected cells (low and high) is statistically significant (*, P < 0.001). (D) PA-1 cells transfected with 120 nM siRNA (control or mTrb3) were coinfected with adenovirus carrying Trb3 or LacZ (control) cDNA. Forty-eight hours after treatment with or without 3 nM BMP4, cells were subjected to a luciferase assay. The data represent the means and standard errors for induction (n-fold) of reporter activity observed upon BMP4 stimulation in three independent experiments. Values labeled with the same letters do not differ significantly from one another (P > 0.05). (E) C2C12 cells were transfected with low (60 nM) or high (120 nM) concentrations of control siRNA or Trb3 siRNA, followed by 3 nM BMP4 stimulation for 72 h. Histochemical analysis of ALP activity was performed by phase-contrast microscopy. Following histochemical ALP analysis, the ALP staining was quantified using the image documentation system Imagestore 7500 (Packard). The graph shows averages for three independent ALP assays. The ALP activities for low and high doses of control or Trb3 siRNA-transfected cells treated with BMP4 are shown as relative activities by normalizing the ALP activity of mock-transfected cells treated with BMP4 to 100%. The difference in ALP activity between control siRNA-transfected cells and cells transfected with Trb3 siRNA (low and high concentrations) is statistically significant (*, P < 0.001).

FIG. 4.

Trb3 is essential for the BMP-mediated signal in PASMCs. (A) PASMCs were transiently transfected with 250 nM of siRNA directed against human Trb3 (hTrb3) or nontargeting siRNA (control) for 48 h, followed by stimulation with (+BMP4) or without (none) BMP4 (3 nM) for 48 h. Cells were stained with FITC-conjugated antibodies against SMC markers, SMA (αSM-Actin), or calponin (αCalponin; green) and DAPI (blue). Downregulation of endogenous Trb3 in PASMCs by siRNA was confirmed by FITC-anti-Trb3 (αTrb3) antibody staining. (B) PASMCs were transfected with 250 nM siRNA directed against human Trb3 (hTrb3) or nontargeting siRNA (control) for 48 h, followed by extraction of total RNA and RT-PCR analysis. SMA and Trb3 mRNA expression relative to GAPDH mRNA expression was measured by real-time PCR. Data are plotted as means ± standard errors for three independent experiments. *, P < 0.001 (for presence versus absence of BMP4 treatment); **, P < 0.05 (for Trb3 treatment versus control); αSMA, anti-SMA. (C) PASMCs were transiently transfected with 250 nM siRNA directed against human Trb3 or nontargeting siRNA (control) for 48 h, followed by adenovirus infection with siRNA-resistant mouse Trb3 (WT or ΔK) and stimulation with or without BMP4 (3 nM) for 48 h. Cells were subjected to staining with FITC-conjugated anti-SMA or anti-Flag antibody (αFlag; green) and DAPI (blue).

FIG. 5.

Trb3 mediates downregulation of Smurf1. (A) Cos7 cells were transfected with Flag-tagged Trb3 (WT) or the ΔK mutant with or without the Myc-Smurf1 (C710A) mutant. Total cell lysates were subjected to immunoprecipitation (IP) with anti-Flag antibody, followed by immunoblot analysis (IB) with anti-Smurf1 antibody to examine the amount of the Trb3-Smurf1 complex. Total cell lysates were subjected to immunoblot analysis with anti-Smurf1 and anti-Flag antibodies (for Trb3). (B) Interaction between Trb3 and Smurf1 was examined by yeast two-hybrid analysis. Trb3 deletion mutant cDNAs fused to Gal4 AD were transformed into yeast expressing the bait construct, a fusion of Smurf1 (WT) and Gal4 DB. Interaction was scored by growth on a histidine-depleted plate. (C) Cos7 cells were transfected with Myc-tagged Smurf1 with increasing amounts of the Flag-tagged Trb3 (WT or ΔK) construct. The amounts of Smurf1, Trb3, and p38MAPK (loading control) were examined by immunoblot analysis with anti-Myc antibody (for Smurf1), anti-Flag antibody (for Trb3), and anti-p38MAPK antibodies, respectively. (D) Cos7 cells were transfected with Myc-tagged Smurf1 (WT) or a catalytically inactive Smurf1 mutant (Cys710Ala) mutated in the HECT domain with or without Flag-tagged Trb3. The amounts of Smurf1 (WT or CA), Trb3, and p38MAPK (loading control) were examined by immunoblot analysis. (E) Cos7 cells overexpressing the Myc-Smurf1 expression construct and increasing amounts of the Flag-Trb3 construct were treated with or without 5 μM lactacystin for 24 h. Total cell lysates were subjected to immunoblot analysis with anti-Myc (for Smurf1), anti-Flag (for Trb3), and anti-p38MAPK (loading control) antibodies. (F) Cos7 cells were transfected with polyhistidine-tagged ubiquitin (Ub), Myc-Smurf1 (C710A), or the Flag-Trb3 construct as indicated. Total cell lysates were subjected to purification with Ni-nitrilotriacetic acid bead affinity column chromatography. The eluates from the column were subjected to immunoblot analysis with anti-Smurf1 antibody.

FIG. 6.

Trb3 alters Smad1 level via regulation of Smurf1. (A) PASMCs were transfected with siRNA (200 nM) against Trb3 or nontargeting siRNA (control). Levels of expression of endogenous Smurf1, Smad1, Trb3, and p38MAPK (loading control) were examined by immunoblot analysis (IB). Lanes 1 and 2 represent duplicate control siRNA transfections, and lanes 3 and 4 represent duplicate Trb3 siRNA transfections. (B) Cos7 cells were transfected with Flag-tagged RhoA, Flag-Trb3, and Myc-Smurf1. The amounts of RhoA, Smurf1, and Trb3 were examined by immunoblot analysis with anti-Myc antibody (for Smurf1) and anti-Flag antibody (for RhoA and Trb3). (C) The Xvent2-luc reporter was transfected with increasing amounts of Trb3 (WT) or (ΔK) and Smurf1 in P19 cells. The transcriptional activation in the presence and absence of BMP4 was monitored by a luciferase assay. The results are shown as induction levels observed upon BMP4 treatment. The difference in induction between untransfected cells and cells transfected with Smurf1 alone or Smurf1 with a high dose of Trb3 (WT) is statistically significant (*, P < 0.001; **, P < 0.001). (D) The TGFβ-specific reporter construct 3TP-lux was transfected into Mv1Lu cells with increasing amounts of Trb3 constructs (0.25, 0.5, and 1 μg). The transcriptional activation was measured by a luciferase assay upon stimulation with 100 pM TGFβ1 (+TGFβ) for 20 h. Data are plotted as means ± standard errors for three independent experiments. *, P < 0.001 (for presence versus absence of TGFβ treatment).

FIG. 7.

BMPRII-TD plays a role in regulation of Smurf1 by Trb3. (A) Cos7 cells were transfected with Myc-Smurf1, Flag-Trb3, and increasing amounts of Flag-BMPRII (WT), Flag-BMPRII (ΔT), Flag-BMPRII-TD (Tail), or Flag-BMPRII (Δ860) expression plasmid. Cell lysates were subjected to immunoblot analysis (IB) with anti-Myc (for Smurf1), anti-Trb3, anti-Flag (for BMPRII), and anti-p38MAPK (loading control) antibodies. Groups of images from different parts of the same gel are presented in the top three panels. (B) The Xvent2-luc reporter construct was transfected with or without the Trb3 expression construct and increasing amounts of BMPRII-TD (Tail) expression plasmid (0, 500, or 750 ng) into P19 cells. Transcriptional activity in the presence or absence of 3 nM BMP4 treatment was monitored by a luciferase assay. (C) Cos7 cells were transfected with Flag-Trb3, Flag-BMPRII, and increasing amounts of the catalytically inactive Myc-Smurf1 (C710A) mutant. The amount of the BMPRII-Trb3 complex was examined by immunoprecipitation with anti-BMPRII antibodies, followed by immunoblot analysis with anti-Flag antibody. Total cell lysates were subjected to immunoblot analysis with anti-Flag (for BMPRII and Trb3) and anti-Myc (for Smurf1) antibodies. (D) PASMCs were infected with adenovirus carrying Flag-Trb3 or GFP (control), followed by stimulation with 3 nM BMP4 for 24 h. Total cell lysates were prepared and subjected to immunoblot analysis with anti-Smurf1, anti-Flag (for Trb3), and anti-GAPDH (loading control). (E) Schematic diagram of the mechanism for regulation of the BMP signaling pathway by Trb3. Upon BMP4 treatment, Trb3 is released from BMPRII-TD and forms a complex with Smurf1 that leads to its degradation, which in turn stabilizes targets of Smurf1, such as Smad1 and RhoA, and activates the BMP pathway.