Activin E is a transforming growth factor β ligand that signals specifically through activin receptor-like kinase 7

Abstract

Activins are one of the three distinct subclasses within the greater Transforming growth factor β (TGFβ) superfamily. First discovered for their critical roles in reproductive biology, activins have since been shown to alter cellular differentiation and proliferation. At present, members of the activin subclass include activin A (ActA), ActB, ActC, ActE, and the more distant members myostatin and GDF11. While the biological roles and signaling mechanisms of most activins class members have been well-studied, the signaling potential of ActE has remained largely unknown. Here, we characterized the signaling capacity of homodimeric ActE. Molecular modeling of the ligand:receptor complexes showed that ActC and ActE shared high similarity in both the type I and type II receptor binding epitopes. ActE signaled specifically through ALK7, utilized the canonical activin type II receptors, ActRIIA and ActRIIB, and was resistant to the extracellular antagonists follistatin and WFIKKN. In mature murine adipocytes, ActE invoked a SMAD2/3 response via ALK7, like ActC. Collectively, our results establish ActE as a specific signaling ligand which activates the type I receptor, ALK7.

Keywords: adipocytes; molecular mechanisms; receptor tyrosine kinases; transforming growth factors.

Figure 1.. Modeling of the Activin E signaling complex.

(A) Schematic representation of a generic dimeric TGFβ dimeric ligand. (B) Schematic diagram of the signaling complex consisting of the dimeric ligand (blue), type I (yellow), and type II (orange) receptors. (C) Overview of the ActE:RIIB:ALK7 ternary complex generated by using AlphaFold 2.3.0. (D) Sequence alignment of the activin subclass annotated based on models of activin ternary complexes (PDB: 7OLY [ActA:RIIB:ALK4], ActB:RIIB:ALK7, ActC:RIIB:ALK7, and ActE:ALK7). Residues are colored if the BSA ≥ 15 Ų — yellow (type I) and orange (type II) within 7OLY. Residues that are divergent between the ligands are marked with asterisks. The knuckle region of the ligand central to binding the type II receptor is boxed. (E) and (F) Identity matrix comparing residues across between ligands with (E) comparing the type I and type II receptor and (F) dividing the type I receptor epitope into chain A (fingers, red) and chain B (prehelix/wrist, green).

Figure 2.. Construct design and expression of Activin E.

(A) Schematic of the full-length ActE constructs (Construct I and Construct II) used for mammalian cell expression in ExpiCHO-S cells. Asterisks denote the known N-linked glycosylation site on the prodomain. (B) Schematic of the different processing states of ActE and corresponding sizes (without glycosylation). (C) Western blot analysis (α-Flag) showing expression of Constructs I and II along with empty vector (EV) in ExpiCHO-S media under both non-reducing (left) and reducing (right) conditions. Flag + control is Flag-BAP fusion protein run under non-reducing conditions (left).

Figure 3.. Transient expression of Activin E activates signaling through ALK7.

(A) CAGA-luciferase reporter assay in HEK293T293 cells in response to transfection of ActA (10 ng), ActC (10 ng), or ActE Construct I (5 or 50 ng), and ActE Construct II (5 or 50 ng). (B–D) Similar assay to (A) modified by transfection of SB-431542 resistant type I receptors; ALK4-ST (B), ALK5-ST (C), and ALK7-ST (D) type I receptors. All cells were transfected with 100 ng total of DNA containing either empty vector (EV) and ligand or EV, ligand, and type I receptor. Data normalized to cells not transfected with ligand DNA constructs (EV alone or EV and type I receptor). Each point represents a technical replicate within triplicate experiments with bars displaying the mean ± SD. In (B–D), cells were treated with 10 μM SB-431542 to inhibit signaling activity of endogenous receptors.

Figure 4.. Activin E conditioned media signals though ALK7.

(A–C) Luciferase reporter assay response to recombinant mature ActA, ActC, or GDF11 (5 nM) along with conditioned media of ActE Construct I, Construct II, or empty vector (EV) in (CAGA₁₂)-luciferase HEK293T293 cells transfected with SB-431542 resistant ALK4-ST (A), ALK5-ST (B), or ALK7-ST (C) type I receptors. Media was concentrated 20× and diluted two-fold for the titration (2.5×, 5×, 10×, 20×). Data normalized to transfected cells with no ligand treatment. (D) Effects of the ALK7 neutralizing antibody (nAb) on recombinant ActA or ActE conditioned media samples on (CAGA₁₂)-luciferase promoter activity in cells expressing the designated SB-431542 resistant type I receptor. (ns = not significant) (****P < 0.0001) (****P < 0.0001) (E). Western blot analysis under reducing and non-reducing conditions of elution samples from the purification of flag-tagged mature ActE from construct 2 using flag affinity resin. (F) Luciferase reporter activity of pooled fractions from (E) in (CAGA₁₂)-luciferase HEK293T cells transfected with the ALK7-ST. Data normalized to untreated control cells. In (A–D, and F) each point represents a technical replicate within triplicate experiments with bars displaying the mean ± SD. In (A–D, and F), cells were treated with 10 μM SB-431542 to inhibit signaling activity of endogenous receptors.

Figure 5.. Activin E signals via the activin type II receptors.

(A) Schematic diagram of activin type II receptor Fc-fusion proteins used as decoy receptors. (B and C) (CAGA₁₂)-luciferase HEK293T293 cells transfected with ALK4-ST or ALK7-ST receptor constructs and treated with ActA (0.62 nM), ActE conditioned media (20×), ActC (0.62 nM), and 10 μM SB-431542 in the presence of increasing amounts (6.25, 12.5, 25 nM) of either ActRIIA-Fc (****P < 0.0001) (****P < 0.0001) (****P = 0.0002) (B) or ActRIIB-Fc (****P < 0.0001) (P = 0.3132) (P = 0.0549) (ns = not significant) (C). (D) Schematic representation of neutralizing antibodies targeting the extracellular domains (ECDs) of the activin type II receptors. (E) (CAGA₁₂)-luciferase HEK293T293 cells either untransfected or transfected with the ALK7-ST type I receptor and treated with ActA (0.62 nM), ActE conditioned media (20×), ActC (0.62 nM), and 10 μM SB-431542 in the presence or absence of neutralizing antibodies targeting ActRIIA, ActRIIB, or both (2.5 µg/ml) (****P < 0.0001) (****P < 0.0001) (****P < 0.0001) (ns = not significant). In (B, C, and E) each point represents a technical replicate within triplicate experiments with bars displaying the mean ± SD. Data are represented as percentages of uninhibited signal (B, C, and E).

Figure 6.. Activin E is resistant to antagonism by Follistatin and WFIKKN.

(A) (CAGA₁₂)-luciferase HEK293T cells transfected with ALK4-ST or ALK7-ST receptor constructs and treated with recombinant GDF11 (0.62 nM), ActE conditioned media (20×), EV conditioned media (20×), and ActC (0.62 nM) in the presence or absence of increasing amounts of FS288 (12.5 and 25 nM) (****P < 0.0001) (*P = 0.0105) (***P = 0.0004) (P = 0.2746) (P = 0.8552) (ns = not significant). (B) (CAGA₁₂)-luciferase HEK293T cells transfected with ALK4-ST or ALK7-ST receptor constructs and treated with recombinant GDF11 (0.62 nM), ActE conditioned media (20×), EV conditioned media (20×), and ActC (0.62 nM) in the presence or absence of increasing amounts of WFIKKN2 (12.5 and 25 nM) (****P < 0.0001) (P = 0.3687) (P = 0.1983) (ns = not significant). Each point represents a technical replicate within triplicate experiments with bars displaying the mean ± SD. Data are normalized to untreated control cells. In (A and B), cells were treated with 10 μM SB-431542 to inhibit signaling activity of endogenous receptors.

Figure 7.. Activin E activates phosphorylation of SMAD2 via ALK7 in mature adipocytes.

(A) Western blot analysis showing phosphorylated SMAD2 (pSMAD2) and total SMAD2/3 in SVF-derived adipocytes following treatment with EV conditioned media, ActE conditioned media (20×), or ActC in the presence or absence of an ALK7 neutralizing antibody for 1 h. (B) Western blot analysis showing phosphorylated SMAD2 (pSMAD2) and total SMAD2/3 in SVF-derived adipocytes following treatment with EV conditioned media, ActE conditioned media (20×), or ActC in the presence or absence of FS288 for 1 h. Data shown are representative of two independent experiments.