Activins and inhibins both belong to the TGF-β superfamily and are formed by different combinations of inhibin subunits, but they exert opposing effects on the regulation of pituitary follicle-stimulating hormone (FSH). Activins promote FSH synthesis and secretion, whereas inhibins restrict activin-associated signaling by competing for activin receptors. In addition to reproductive endocrine regulation, the activin system participates in embryonic development, hematopoiesis, immune regulation, tissue repair, fibrosis, muscle and bone metabolism, and tumor progression. Activin ligands, receptors, and endogenous regulatory proteins have become important targets in disease-mechanism research, biomarker detection, and targeted drug development.

Keywords: activins; inhibins; Activin A; INHBA; ACVR1B; ACVR2A; ACVR2B; SMAD2/3; reproductive regulation; fibrosis; targeted therapy

1 Molecular Composition of Activins and Inhibins

1.1 Inhibin Subunits and Dimeric Forms

(1) Composition of β Subunits

INHBA, INHBB, INHBC, and INHBE encode the inhibin βA, βB, βC, and βE subunits, respectively. Different β subunits combine as homodimers or heterodimers to form different types of activins.

Activin A consists of two βA subunits, Activin B consists of two βB subunits, and Activin AB consists of one βA and one βB subunit. Different dimers differ in tissue distribution, receptor binding, and biological functions.

(2) The α Subunit and Inhibin Formation

INHA encodes the inhibin α subunit. The α subunit forms heterodimers with either the βA or βB subunit to generate Inhibin A and Inhibin B, respectively.

Inhibins are not simply inactive forms of activins but are endocrine and paracrine factors with independent structures and receptor-regulatory mechanisms.

Table 1 Major Molecular Composition of Activins and Inhibins

1.2 Molecular Characteristics of Activin A

Activin A is the most extensively studied activin and consists of two inhibin βA subunits. INHBA encodes the βA subunit rather than the complete Activin A dimer.

Activin A is widely expressed in the gonads, pituitary gland, placenta, immune tissues, blood vessels, and connective tissues. Its effects are strongly dependent on cell type, concentration, and treatment duration. In some cells, Activin A inhibits proliferation and promotes differentiation; under conditions of persistent inflammation or tissue injury, excessive Activin A signaling may also promote fibroblast activation, extracellular matrix deposition, and tissue fibrosis.

1.3 Functional Relationship Between Activins and Inhibins

(1) FSH Regulation

Activins promote pituitary FSH synthesis and secretion, whereas inhibins are produced by the gonads and provide negative feedback to suppress FSH release. Together, they maintain the dynamic balance of the pituitary-gonadal axis.

(2) Competition at the Receptor Level

Inhibins generally do not act by directly binding Activin A. Instead, with the assistance of the coreceptor TGFBR3, also known as betaglycan, they bind ACVR2A or ACVR2B and thereby reduce the opportunity for activins to interact with type II receptors.

(3) Ligand Neutralization

Follistatin directly binds ligands such as Activin A and Activin B and prevents them from binding type II receptors, making it an important extracellular neutralizing protein in the activin system.

2 Activin Receptors and Signal Transduction

2.1 Formation of Receptor Complexes

(1) Type II Receptor Binding

Activin A first binds the type II receptors ACVR2A or ACVR2B on the plasma membrane. Both receptors contain serine/threonine kinase domains and can recruit and phosphorylate type I receptors.

(2) Recruitment of Type I Receptors

The major type I receptor in canonical Activin A signaling is ACVR1B, also known as ALK4. After ligand binding to the type II receptor, ACVR1B is recruited into the complex and activated through phosphorylation by the type II receptor.

A complete signaling complex generally consists of one activin dimer, two type II receptors, and two type I receptors.

2.2 Canonical SMAD2/3 Signaling Pathway

(1) SMAD2/3 Phosphorylation

Activated ACVR1B phosphorylates the receptor-regulated SMAD proteins SMAD2 and SMAD3. Phosphorylated SMAD2/3 forms a complex with SMAD4 and enters the nucleus.

(2) Target-Gene Regulation

After entering the nucleus, the SMAD complex cooperates with transcription factors, coactivators, or corepressors to regulate target-gene expression. Related genes are involved in the cell cycle, differentiation, extracellular matrix production, inflammatory responses, and tissue repair.

(3) Dependence on Cellular Context

The same Activin A stimulus may produce different outcomes in different cells. These differences arise from variations in receptor abundance, SMAD coregulators, chromatin state, and the activity of other signaling pathways.

Figure 1. Canonical and non-canonical Activin A signaling pathways

2.3 Noncanonical Signaling Pathways

(1) MAPK-Associated Signaling

Activin A can regulate ERK1/2, p38 MAPK, and JNK signaling in certain cells, thereby affecting cell proliferation, stress responses, migration, and inflammatory-factor expression.

(2) PI3K/AKT Signaling

Activin A can also crosstalk with the PI3K/AKT pathway and affect cell survival, metabolism, and protein synthesis.

(3) Wnt-Associated Signaling

During embryonic development, stem-cell differentiation, and tissue remodeling, activin signaling can cooperate with the Wnt pathway to regulate cell fate. The strength and direction of noncanonical signaling depend on cell type and experimental conditions.

2.4 Negative Regulation of Activin Signaling

(1) Follistatin directly binds activin ligands and prevents them from binding ACVR2A or ACVR2B.

(2) Inhibins compete for type II receptors with the assistance of TGFBR3, thereby reducing formation of activin receptor complexes.

(3) Inhibitory SMAD proteins, receptor internalization, and protein degradation can limit signaling duration.

(4) The relative expression levels of activins, inhibins, Follistatin, and receptors jointly determine the final signaling strength.

3 Physiological Functions of Activins and Inhibins

3.1 Reproductive Endocrine Regulation

(1) Pituitary FSH Regulation

Activins promote FSH-associated gene expression and secretion, whereas inhibins provide negative-feedback signals from the gonads to prevent persistent excessive elevation of FSH.

(2) Female Reproductive Function

Activins and inhibins participate in follicular development, dominant-follicle selection, ovulation, menstrual-cycle regulation, embryo implantation, and pregnancy maintenance. Changes in the expression of ligands, receptors, and Follistatin at different reproductive stages jointly maintain local homeostasis.

(3) Male Reproductive Function

The activin system can influence testicular development, Sertoli-cell function, spermatogenesis, and androgen-associated regulation. Inhibin B is an important feedback regulator in the male reproductive axis.

3.2 Hematopoiesis and Cell Differentiation

Activin-associated pathways participate in hematopoietic progenitor-cell differentiation and erythrocyte maturation. Activin A can also be combined with factors such as BMP, VEGF, and FGF to induce differentiation of pluripotent stem cells toward mesodermal or hematopoietic lineages.

Ligand-trap proteins constructed from the extracellular domains of ACVR2A or ACVR2B can alter the availability of multiple TGF-β superfamily ligands and thereby regulate late-stage erythroid maturation. Their effects are generally not restricted to a single activin ligand.

3.3 Tissue Development and Injury Repair

(1) Tissue Formation

Activin signaling participates in embryonic development, organ formation, and cell-fate determination. Signaling strength during different temporal windows can influence whether cells continue proliferating, enter differentiation, or undergo tissue reorganization.

(2) Wound Repair

After tissue injury, Activin A can regulate the responses of inflammatory cells, epithelial cells, and fibroblasts. Moderate and transient signaling supports tissue repair, whereas persistent or excessive signaling may promote abnormal scarring and fibrosis.

3.4 Muscle, Bone, and Metabolic Regulation

(1) Muscle and Bone Tissues

ACVR2A and ACVR2B recognize multiple TGF-β superfamily ligands associated with muscle mass and bone metabolism. Modulating the ligand-binding capacity of these receptors can affect muscle growth, bone remodeling, and tissue regeneration.

(2) Adipose Tissue and Energy Metabolism

Activin E and INHBE-associated signaling have mainly attracted attention in liver and adipose-metabolism research. Changes in INHBE expression may be associated with lipid metabolism, obesity, and altered insulin sensitivity.

3.5 Immune and Inflammatory Regulation

Activin A can be produced by macrophages, dendritic cells, vascular cells, and tissue stromal cells and can affect immune-cell differentiation, cytokine expression, and tissue infiltration.

Activin A is not inherently a fixed pro-inflammatory or anti-inflammatory factor. Its effects are influenced by cellular source, local concentration, stimulation duration, and disease stage and therefore need to be evaluated in specific models.

4 Dysregulation of the Activin System and Disease

4.1 Reproductive-System Disorders

Imbalances among activins, inhibins, and Follistatin may disrupt follicular development, ovulation, androgen synthesis, and pregnancy maintenance and are associated with polycystic ovary syndrome, gonadal dysfunction, and pregnancy-related complications.

Inhibin A and Inhibin B can also be used to assess certain aspects of gonadal function, but the results need to be interpreted together with age, sex, reproductive cycle, and other hormonal indicators.

4.2 Inflammation and Fibrosis

Persistently enhanced Activin A-ACVR2A/ACVR2B-ACVR1B-SMAD2/3 signaling can promote fibroblast activation, collagen production, and extracellular matrix deposition.

This process can participate in fibrosis of the lung, liver, kidney, and other tissues. Activin A also affects inflammatory-factor expression, allowing chronic inflammation and fibrosis to form a mutually reinforcing regulatory network.

4.3 Tumor Initiation and Progression

(1) Growth-Inhibitory Effects

In certain cells or during early stages of tumor development, SMAD2/3 signaling can inhibit cell proliferation and promote differentiation.

(2) Tumor-Promoting Effects

In advanced tumors or specific microenvironments, activin signaling may also promote cell migration, invasion, matrix remodeling, and immunosuppression.

(3) Biomarker Research

Abnormal expression of INHBA, INHBB, ACVR2A, and ACVR2B can be studied in ovarian cancer, prostate cancer, breast cancer, lung cancer, and other tumors. Increased expression of a single ligand alone is insufficient to determine whether it has tumor-promoting or tumor-suppressive effects, and receptor expression and SMAD-signaling status should also be evaluated.

4.4 Neurological and Musculoskeletal Disorders

Activin receptor signaling can affect neuronal stress, muscle mass, bone formation, and tissue regeneration. ACVR2B-targeted strategies have therefore been investigated in muscle atrophy, cachexia, and certain neuromuscular diseases.

Because ACVR2A and ACVR2B can bind multiple ligands, broad receptor blockade may simultaneously affect hematopoietic, vascular, metabolic, and reproductive systems, requiring careful control of targeting range and dose.

5 Activin-Targeted Therapeutic Strategies

5.1 Ligand-Trapping Receptor Fusion Proteins

ACVR2A-Fc and ACVR2B-Fc are formed by fusing the receptor extracellular domain with an Fc fragment and can act as soluble ligand-trapping molecules that compete for ligands in the circulation or tissues.

ACVR2A-Fc molecules can be used in research on hematologic diseases and related receptor pharmacology, whereas ACVR2B-Fc molecules are commonly used in studies of muscle mass, bone metabolism, and cachexia.

These fusion proteins can bind multiple TGF-β superfamily ligands simultaneously, and their effects cannot be simply interpreted as specific inhibition of Activin A.

5.2 Ligand- and Receptor-Targeting Antibodies

(1) Ligand-Neutralizing Antibodies

Antibodies against Activin A or related inhibin subunits can be used for detection, ligand neutralization, and disease-mechanism research. The specificity of ligand antibodies needs to distinguish among complete dimers, free subunits, and precursor proteins.

(2) ACVR2A and ACVR2B Antibodies

Antibodies against ACVR2A or ACVR2B can be used for receptor-expression detection, ligand-binding analysis, and receptor-functional intervention. Different antibodies may exhibit binding, blocking, or receptor-modulating activities, and their pharmacological properties cannot be inferred solely from the target name.

(3) ACVR1B/ALK4 Antibodies

ACVR1B antibodies can be used to detect expression and localization of the canonical type I receptor for Activin A and to assist in verifying whether SMAD2/3 signaling depends on ALK4.

5.3 Major Targets and Research Directions

5.4 Key Limitations of Targeted Therapy

(1) Activin ligands and receptors have many-to-many relationships, and intervention against a single receptor may simultaneously affect multiple ligands.

(2) The same activin may produce different effects in different tissues and disease stages.

(3) Systemic inhibition may affect reproductive, hematopoietic, vascular, muscular, and bone metabolism.

(4) Fc fusion-induced dimerization and multivalent binding can enhance ligand trapping, and the pharmacological properties of these proteins are not completely identical to those of natural membrane receptors.

(5) Targeted therapy requires selection of suitable models or populations based on ligand levels, receptor expression, and SMAD-signaling activity.

6 Experimental Design for Activin Research

6.1 Ligand-Receptor Binding Assays

Recombinant Activin A, ACVR2A, and ACVR2B proteins can be used in ELISA, surface plasmon resonance, biolayer interferometry, and cell-binding assays to analyze ligand-receptor affinity, receptor selectivity, and competitive inhibition.

His-tagged receptor proteins can be used for immobilization and affinity measurements, whereas Fc-fusion proteins with independent ligand-binding capacity, such as ACVR2A-Fc and ACVR2B-Fc, can be used in ligand-trapping experiments. Fc-mediated dimerization may increase apparent binding strength, whereas the extracellular domains of type I receptors such as ALK4 generally require type II receptors for ligand-complex assembly; therefore, ligand-trapping capacity cannot be inferred solely from the presence of an Fc tag.

6.2 Cellular Signaling Assays

(1) Canonical Signaling

After Activin A treatment, SMAD2/3 phosphorylation, nuclear translocation of SMAD complexes, and downstream gene expression can be measured. Short-term treatment is suitable for analyzing receptor activation and SMAD phosphorylation, whereas long-term treatment is suitable for observing transcriptional and cellular phenotypic changes.

(2) Noncanonical Signaling

Phosphorylation of ERK1/2, p38 MAPK, JNK, and AKT can be used to analyze Activin A-associated noncanonical signaling. Receptor blockade or SMAD-intervention controls should be included to distinguish parallel signaling from secondary effects.

6.3 Cell Differentiation and Functional Research

(1) Stem-Cell Differentiation

Activin A can be combined with BMP, VEGF, FGF, and other factors to induce differentiation of pluripotent stem cells toward mesodermal and hematopoietic lineages. Differentiation outcomes can be evaluated through gene-expression and cell-surface-marker analysis.

(2) Cell Proliferation

Specific cells exhibit proliferation-inhibitory responses to Activin A and can be used to evaluate the biological activity of recombinant proteins. Protein purity alone cannot substitute for a functional activity assay.

(3) Migration and Invasion

Exogenous Activin A or Activin B stimulation and intervention in INHBA or INHBB expression can be used to investigate migration, invasion, and extracellular matrix changes in tumor cells and stromal cells.

6.4 Protein Quantification and Biomarker Research

Activin A ELISA kits can be used to measure protein levels in serum, plasma, and cell-culture supernatants. During sample analysis, free ligand, Follistatin-bound ligand, and receptor-bound complexes may exhibit different antibody-recognition efficiencies.

Biomarker research should also integrate receptor expression, p-SMAD2/3 levels, and relevant clinical or histological indicators to avoid making mechanistic conclusions solely from Activin A concentrations.

6.5 Key Experimental Design Considerations

(1) Species Matching

Human, mouse, and rat activin systems are highly conserved, but cross-species activity should still be confirmed through binding or cellular functional assays.

(2) Protein Form

Mature activin dimers, individual inhibin subunits, precursor proteins, and receptor-Fc fusion proteins should be distinguished. Different forms cannot be used as equivalent reagents.

(3) Effects of Tags

His and Fc tags may affect protein conformation, dimerization, and apparent affinity. When comparing different proteins, identical tags or corresponding tag controls should be used.

(4) Purity and Aggregation State

In addition to SDS-PAGE purity, the monomeric proportion of ligand and receptor-fusion proteins should be evaluated by SEC-HPLC. Aggregated proteins may enhance nonspecific binding or abnormal receptor crosslinking.

(5) Concentration and Time

Activin A signaling is strongly concentration- and time-dependent. Experiments should include concentration and time gradients and should avoid relying on a single high-concentration treatment that may produce nonphysiological results.

7 Products

7.1 Activin, Inhibin, and Receptor Proteins

7.2 Antibodies for Detecting Activins, Inhibins, and Their Receptors

7.3 ACVR2A- and ACVR2B-Targeting Antibodies

7.4 Activin A Detection Kits

The activin system consists of multiple dimeric ligands, shared receptors, and extracellular regulatory proteins, and its effects cannot be fully explained by a single ligand or receptor. Combining recombinant-protein stimulation, ligand-receptor binding, SMAD2/3-signaling detection, antibody intervention, and protein quantification allows more accurate analysis of the roles of activins in reproductive regulation, inflammatory fibrosis, and targeted therapy.

References

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