Activin A inhibits BMP-signaling by binding ACVR2A and ACVR2B

Abstract

Background: Activins are members of the TGF-β family of ligands that have multiple biological functions in embryonic stem cells as well as in differentiated tissue. Serum levels of activin A were found to be elevated in pathological conditions such as cachexia, osteoporosis and cancer. Signaling by activin A through canonical ALK4-ACVR2 receptor complexes activates the transcription factors SMAD2 and SMAD3. Activin A has a strong affinity to type 2 receptors, a feature that they share with some of the bone morphogenetic proteins (BMPs). Activin A is also elevated in myeloma patients with advanced disease and is involved in myeloma bone disease.

Results: In this study we investigated effects of activin A binding to receptors that are shared with BMPs using myeloma cell lines with well-characterized BMP-receptor expression and responses. Activin A antagonized BMP-6 and BMP-9, but not BMP-2 and BMP-4. Activin A was able to counteract BMPs that signal through the type 2 receptors ACVR2A and ACVR2B in combination with ALK2, but not BMPs that signal through BMPR2 in combination with ALK3 and ALK6.

Conclusions: We propose that one important way that activin A regulates cell behavior is by antagonizing BMP-ACVR2A/ACVR2B/ALK2 signaling.

Figure 1

Activin A inhibited BMP-9-induced apoptosis. (A) The expression of the type I receptors ALK4 and ALK7 in eight human myeloma cell lines was determined using QRT-PCR. The delta delta Ct method using GAPDH as housekeeping gene was used to determine the relative levels of mRNA compared to the expression of ALK7 in cell line JJN-3 (Ct-value = 33) which was set to 1. (B) Phosphorylation of SMAD2 was determined using immunoblotting in INA-6 and IH-1 cells treated with activin A (10 ng/mL) or TGF-β (5 ng/mL) for 4 hours. GAPDH was used as loading control. INA-6 (C) and IH-1 cells (D) were treated for three days with activin A (10 ng/mL) and the indicated concentrations of BMP-9 before cell viability was determined by flow cytometry using annexin V/PI labeling. Cells that were negative for both annexin V and PI were considered viable. Phosphorylation of SMAD1/5/8 or SMAD2 was determined using immunoblotting in INA-6 (E) and IH-1 cells (F) treated with activin A (10 ng/mL) and/or BMP-9 (0.5 ng/mL) for one, six and 24 hours. GAPDH was used as loading control.

Figure 2

Activin A inhibited cell death induced by BMP-6 and BMP-9, but not by BMP-2 and BMP-4. IH-1 (A) and INA-6 cells (B) were treated with the indicated concentrations of BMP-9, activin A (10 ng/mL) and the inhibitor SB431542 (5 μM) for three days before cell growth was determined using the CellTiter Glo assay. Relative luciferase units (RLU) reflected the amount of ATP in each well, represented as mean ± SEM from three independent experiments. P-values were determined by T-test (*P < 0.05, **P < 0.01). (C) IH-1 cells were treated for three days with activin A (10 ng/mL), BMP-2 (5 ng/mL), BMP-4 (2.5 ng/mL), BMP-6 (25 ng/mL), BMP-9 (0.5 ng/mL) and follistatin (625 ng/mL) before cell growth was determined as in (A).

Figure 3

Activin A competes with BMP-6 and BMP-9 in binding to the type II receptors ACVR2A and ACVR2B. (A) Expression of the type II receptors ACVR2A and ACVR2B in INA-6 and IH-1 cells were determined using QRT-PCR. The delta delta Ct method using GAPDH as housekeeping gene was used to determine the relative levels of mRNA compared to the expression of ACVR2A in INA-6 (Ct-value = 33) which was set to 1. (B) INA-6 cells were treated with BMP-6 (50 ng/mL) or BMP-9 (0.25 ng/mL) for three days in the presence of the soluble Fc-receptors ACVR1/ALK2, BMPR2, ACVR2A or ACVR2B (5 μg/mL) and cell growth was determined using the CellTiter Glo assay. Relative luciferase units (RLU) reflected the amount of ATP in each well, represented as mean ± SEM from three independent experiments. P-values were determined by T-test (*P < 0.05, **P < 0.01). (C) INA-6 cells were treated with BMP-6 (25 ng/mL) with or without activin A (10 ng/mL) and soluble Fc-receptors (5 μg/mL) where indicated and cell growth was determined as in (B). (D) Soluble Fc-receptor ACVR2A and ACVR2B (1 μg/mL) was coated in wells and treated with BMP-9 (15 ng/mL) and different concentrations of activin A (1.5-50 ng/mL). Bound BMP-9 was expressed as absorbance (OD) using BMP-9 DuoSet detection reagents. Shown are mean ± SEM values from three independent experiments. P-values were determined by T-test (*P < 0.05, **P < 0.01). (E) Soluble receptors were coated into plates as in (D) and treated with BMP-9 (15 ng/mL), activin A (15 ng/mL) and different concentrations of follistatin (300–1000 ng/mL). Binding of BMP-9 was determined as in (D).