RSPO3: From Wnt Signaling Enhancer to Dual-Pathway Mechanism of Angiogenesis Regulation

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This article systematically elucidates the dual mechanisms by which RSPO3, as a core member of the R-spondin family, regulates angiogenesis through both the canonical Wnt/β-catenin pathway and the non-canonical Gαi1/3-Akt-mTOR pathway, while analyzing its pivotal role in vascular development and pathological angiogenesis.

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RSPO3: From Wnt Signaling Enhancer to Dual-Pathway Mechanism of Angiogenesis Regulation

Summary
This article systematically elaborates on the molecular characteristics and biological functions of RSPO3 as a core member of the R-spondin family, highlighting its dual regulatory mechanisms in angiogenesis through both the canonical Wnt/β-catenin pathway and the non-canonical Gαi1/3-Akt-mTOR pathway, while analyzing its critical role in vascular development and pathological angiogenesis.

I. Molecular Characteristics and Family Position of RSPO3

The R-spondin family consists of four members (RSPO1 to RSPO4), encoding four evolutionarily highly conserved small secreted glycoproteins with 40% to 60% sequence and domain similarity among members. RSPO3 is one of the most functionally diverse members of this family, featuring four main functional domains: an N-terminal signal peptide for protein secretion; two adjacent furin-like domains (FU1 and FU2) responsible for binding to LGR4/5/6 receptors and RNF43/ZNRF3; a TSP1 domain involved in protein-protein interactions; and a C-terminal region rich in basic amino acids, potentially mediating binding to cell surface heparan sulfate proteoglycans. The human RSPO3 gene is located on chromosome 6q22.33, encoding a protein of approximately 272 amino acid residues with a molecular weight of about 30 to 35 kDa.

II. RSPO3 Regulates Angiogenesis via the Canonical Wnt/β-catenin Pathway

The most classical molecular function of RSPO3 lies in its enhancement of canonical Wnt signaling through LRP6 mediation. In the canonical Wnt pathway, RSPO3 binds to LGR4/5/6 receptors and promotes the clearance of E3 ubiquitin ligases RNF43 and ZNRF3, thereby relieving their inhibitory ubiquitination of Wnt receptor Frizzled and amplifying Wnt/β-catenin signaling. Activated β-catenin translocates to the nucleus and binds to TCF/LEF transcription factors, driving the expression of downstream target genes, including vascular endothelial growth factor (VEGF). As a core regulator of angiogenesis, VEGF promotes endothelial cell proliferation, migration, and lumen formation, ultimately driving neovascularization. Additionally, RSPO3 maintains cellular lineage balance during vascular development by inhibiting hematopoietic cell differentiation. Thus, RSPO3 is considered a key signaling protein mediating angiogenesis and vascular development.

III. RSPO3 Regulates Angiogenesis via the Non-canonical Gαi1/3-Akt-mTOR Pathway

Although the Wnt/β-catenin pathway is an important route for RSPO3-mediated angiogenesis regulation, recent studies have revealed a novel molecular mechanism independent of canonical Wnt signaling. The latest research has identified Gαi1/3 as key signaling proteins in RSPO3-induced Akt-mTOR activation and angiogenesis both in vitro and in vivo.

Gαi proteins belong to the α-subunit family of heterotrimeric G proteins, traditionally thought to bind only to G protein-coupled receptors. Studies have shown that in cultured human umbilical vein endothelial cells and human brain microvascular endothelial cells, RSPO3 induces the formation of the LGR4-Gαi1/3-Gab1 signaling complex, mediating downstream Akt-mTOR activation. This process is entirely independent of Wnt/β-catenin signaling—Gαi1/3 silencing does not affect active β-catenin accumulation, and β-catenin knockdown does not alter Gαi1/3 expression or RSPO3-induced Akt-mTOR activation.

Functional validation demonstrated that Gαi1/3 silencing significantly inhibited RSPO3-induced endothelial cell migration, invasion, proliferation, and in vitro vessel formation, while Gαi1/3 overexpression enhanced these responses. In vivo experiments showed that endothelial cell-specific knockdown of Gαi1/3 in mice markedly suppressed RSPO3 overexpression-induced Akt-mTOR activation and retinal angiogenesis, whereas endothelial cell-specific overexpression of Gαi1/3 promoted retinal neovascularization. This study is the first to reveal a novel molecular mechanism by which RSPO3 promotes vascular development and angiogenesis distinct from the traditional Wnt/β-catenin pathway, further enriching our understanding of Gαi proteins' critical role in regulating abnormal retinal vascular development and pathological angiogenesis.

IV. Synergistic Significance and Translational Value of RSPO3's Dual Signaling Pathways

RSPO3's coordinated regulation of angiogenesis through both Wnt/β-catenin-VEGF and Gαi1/3-Akt-mTOR pathways holds significant biological and translational implications. The Wnt/β-catenin pathway primarily provides long-feedback regulation by transcriptionally controlling VEGF expression, supplying critical growth factor signals for angiogenesis. In contrast, the Gαi1/3-Akt-mTOR pathway directly drives angiogenesis execution through rapid activation of endothelial cell survival, proliferation, and migration effectors, representing a rapid-response mechanism. The temporal and spatial coordination of these two pathways enables RSPO3 to precisely regulate different stages of vascular development. This discovery also offers new intervention targets for treating angiogenesis-related disorders—selectively targeting the Gαi1/3 downstream pathway may allow precise inhibition of pathological angiogenesis without affecting Wnt/β-catenin-mediated tissue homeostasis.

V. Conclusion

As a core member of the R-spondin family, RSPO3 plays an irreplaceable role in vascular development and pathological angiogenesis through its dual regulatory capacity via both canonical Wnt/β-catenin and non-canonical Gαi1/3-Akt-mTOR pathways. Recombinant human RSPO3 protein, as an essential tool for basic research and drug development, will continue to provide critical support for unraveling angiogenesis mechanisms and exploring therapeutic strategies for related diseases.

In RSPO3-related basic research and drug screening, high-quality recombinant human RSPO3 protein serves as a core tool for receptor binding analysis, signaling pathway investigation, and functional validation. To meet these research needs, Uni offers RSPO3 Protein, Human, suitable for applications including RSPO3-LGR4/5/6 receptor binding analysis, exploration of Wnt/β-catenin and Gαi1/3-Akt-mTOR signaling mechanisms, and in vitro activity evaluation of anti-RSPO3 antibody drugs.

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This article is reviewed and published by the technical expert team of UA

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