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Regulation of TGF-β receptor hetero-oligomerization and signaling by endoglin.

Pomeraniec L, Hector-Greene M, Ehrlich M, Blobe GC, Henis YI - Mol. Biol. Cell (2015)

Bottom Line: However, such studies, especially in live cells, are missing for the endothelial cell coreceptor endoglin and for the ALK1 type I receptor, which enables endothelial cells to respond to TGF-β by activation of both Smad2/3 and Smad1/5/8.ALK1 and ALK5 bind to endoglin with differential dependence on TβRII, which plays a major role in recruiting ALK5 to the complex.Signaling data indicate a role for the quaternary receptor complex in regulating the balance between TGF-β signaling to Smad1/5/8 and to Smad2/3.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.

No MeSH data available.


Related in: MedlinePlus

Endoglin expression enhances phosphorylation of Smad1/5/8 but not of Smad2/3. (A) Biochemical analysis of Smad activation in response to TGF-β1 was performed in mouse embryonic endothelial cells derived from endoglin wild-type (+/+) or endoglin  (−/−) mice. MEEC+/+ and MEEC−/− cells were serum starved (6 h) and stimulated (30 min) with TGF-β1 at the indicated concentrations. Lysates were resolved by SDS–PAGE and subjected to Western blot analysis with antibodies to pSmad1/5/8, pSmad2, tSmad1, tSmad2, and β-actin. Data are representative of at least three independent experiments. (B) Densitometric analysis of band intensities of pSmad1/5/8 relative to β-actin; similar results were obtained for calibration relative to tSmad1. (C) Densitometric analysis of band intensities of pSmad2 relative to β-actin; similar results were obtained relative to tSmad2. Bars represent normalized mean ± SEM of three independent experiments. In B and C, results were calibrated relative to the value in MEEC+/+ cells stimulated with 100 pM TGF-β1, taken as 100%. Asterisks indicate significant differences between the values obtained in MEEC+/+ and MEEC−/− cells stimulated with the same TGF-β1 concentration (*p < 0.05).
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Figure 9: Endoglin expression enhances phosphorylation of Smad1/5/8 but not of Smad2/3. (A) Biochemical analysis of Smad activation in response to TGF-β1 was performed in mouse embryonic endothelial cells derived from endoglin wild-type (+/+) or endoglin (−/−) mice. MEEC+/+ and MEEC−/− cells were serum starved (6 h) and stimulated (30 min) with TGF-β1 at the indicated concentrations. Lysates were resolved by SDS–PAGE and subjected to Western blot analysis with antibodies to pSmad1/5/8, pSmad2, tSmad1, tSmad2, and β-actin. Data are representative of at least three independent experiments. (B) Densitometric analysis of band intensities of pSmad1/5/8 relative to β-actin; similar results were obtained for calibration relative to tSmad1. (C) Densitometric analysis of band intensities of pSmad2 relative to β-actin; similar results were obtained relative to tSmad2. Bars represent normalized mean ± SEM of three independent experiments. In B and C, results were calibrated relative to the value in MEEC+/+ cells stimulated with 100 pM TGF-β1, taken as 100%. Asterisks indicate significant differences between the values obtained in MEEC+/+ and MEEC−/− cells stimulated with the same TGF-β1 concentration (*p < 0.05).

Mentions: Because endoglin appears to form complexes with TβRII, ALK5, and ALK1, we examined its ability to modulate TGF-β signaling via ALK5 (phopho-Smad2 [pSmad2] formation) and/or ALK1 (pSmad1/5/8 formation). To this end, we used endoglin- murine embryonic endothelial cells derived from endoglin−/− mice (MEEC−/−) and their wild-type control (MEEC+/+). The cells were stimulated by increasing concentrations of TGF-β1, and the resulting phosphorylation of Smad2 and Smad1/5/8 was measured by immunoblotting (Figure 9). A low but measurable level of pSmad1/5/8 was observed in both cell lines already before addition of TGF-β1. Stimulation by TGF-β1 increased the pSmad1/5/8 levels in both cell types but was significantly higher in MEEC+/+ cells at TGF-β1 concentrations ≥10 pM. A markedly different scenario was observed for Smad2 phosphorylation, for which no significant differences between MEEC+/+ and MEEC−/− cells were detected without or with TGF-β1 stimulation (Figure 9, A and C). In light of earlier findings that demonstrated that TGF-β–mediated phosphorylation of Smad1/5/8 proceeds via ALK1 in human umbilical vein endothelial cells and mouse embryonic endothelial cells (Oh et al., 2000; Goumans et al., 2002), we conclude that endoglin expression alters the balance between the activation of ALK1-dependent (Smad1/5/8) and ALK5-dependent (Smad2/3) TGF-β signaling.


Regulation of TGF-β receptor hetero-oligomerization and signaling by endoglin.

Pomeraniec L, Hector-Greene M, Ehrlich M, Blobe GC, Henis YI - Mol. Biol. Cell (2015)

Endoglin expression enhances phosphorylation of Smad1/5/8 but not of Smad2/3. (A) Biochemical analysis of Smad activation in response to TGF-β1 was performed in mouse embryonic endothelial cells derived from endoglin wild-type (+/+) or endoglin  (−/−) mice. MEEC+/+ and MEEC−/− cells were serum starved (6 h) and stimulated (30 min) with TGF-β1 at the indicated concentrations. Lysates were resolved by SDS–PAGE and subjected to Western blot analysis with antibodies to pSmad1/5/8, pSmad2, tSmad1, tSmad2, and β-actin. Data are representative of at least three independent experiments. (B) Densitometric analysis of band intensities of pSmad1/5/8 relative to β-actin; similar results were obtained for calibration relative to tSmad1. (C) Densitometric analysis of band intensities of pSmad2 relative to β-actin; similar results were obtained relative to tSmad2. Bars represent normalized mean ± SEM of three independent experiments. In B and C, results were calibrated relative to the value in MEEC+/+ cells stimulated with 100 pM TGF-β1, taken as 100%. Asterisks indicate significant differences between the values obtained in MEEC+/+ and MEEC−/− cells stimulated with the same TGF-β1 concentration (*p < 0.05).
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Figure 9: Endoglin expression enhances phosphorylation of Smad1/5/8 but not of Smad2/3. (A) Biochemical analysis of Smad activation in response to TGF-β1 was performed in mouse embryonic endothelial cells derived from endoglin wild-type (+/+) or endoglin (−/−) mice. MEEC+/+ and MEEC−/− cells were serum starved (6 h) and stimulated (30 min) with TGF-β1 at the indicated concentrations. Lysates were resolved by SDS–PAGE and subjected to Western blot analysis with antibodies to pSmad1/5/8, pSmad2, tSmad1, tSmad2, and β-actin. Data are representative of at least three independent experiments. (B) Densitometric analysis of band intensities of pSmad1/5/8 relative to β-actin; similar results were obtained for calibration relative to tSmad1. (C) Densitometric analysis of band intensities of pSmad2 relative to β-actin; similar results were obtained relative to tSmad2. Bars represent normalized mean ± SEM of three independent experiments. In B and C, results were calibrated relative to the value in MEEC+/+ cells stimulated with 100 pM TGF-β1, taken as 100%. Asterisks indicate significant differences between the values obtained in MEEC+/+ and MEEC−/− cells stimulated with the same TGF-β1 concentration (*p < 0.05).
Mentions: Because endoglin appears to form complexes with TβRII, ALK5, and ALK1, we examined its ability to modulate TGF-β signaling via ALK5 (phopho-Smad2 [pSmad2] formation) and/or ALK1 (pSmad1/5/8 formation). To this end, we used endoglin- murine embryonic endothelial cells derived from endoglin−/− mice (MEEC−/−) and their wild-type control (MEEC+/+). The cells were stimulated by increasing concentrations of TGF-β1, and the resulting phosphorylation of Smad2 and Smad1/5/8 was measured by immunoblotting (Figure 9). A low but measurable level of pSmad1/5/8 was observed in both cell lines already before addition of TGF-β1. Stimulation by TGF-β1 increased the pSmad1/5/8 levels in both cell types but was significantly higher in MEEC+/+ cells at TGF-β1 concentrations ≥10 pM. A markedly different scenario was observed for Smad2 phosphorylation, for which no significant differences between MEEC+/+ and MEEC−/− cells were detected without or with TGF-β1 stimulation (Figure 9, A and C). In light of earlier findings that demonstrated that TGF-β–mediated phosphorylation of Smad1/5/8 proceeds via ALK1 in human umbilical vein endothelial cells and mouse embryonic endothelial cells (Oh et al., 2000; Goumans et al., 2002), we conclude that endoglin expression alters the balance between the activation of ALK1-dependent (Smad1/5/8) and ALK5-dependent (Smad2/3) TGF-β signaling.

Bottom Line: However, such studies, especially in live cells, are missing for the endothelial cell coreceptor endoglin and for the ALK1 type I receptor, which enables endothelial cells to respond to TGF-β by activation of both Smad2/3 and Smad1/5/8.ALK1 and ALK5 bind to endoglin with differential dependence on TβRII, which plays a major role in recruiting ALK5 to the complex.Signaling data indicate a role for the quaternary receptor complex in regulating the balance between TGF-β signaling to Smad1/5/8 and to Smad2/3.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.

No MeSH data available.


Related in: MedlinePlus