The switch in question involves R-spondins, a family of four secreted glycoproteins that act as potent amplifiers of canonical WNT signaling. RSPOs are best known for their role in stem cell biology, where they bind LGR4/5/6 receptors and the E3 ubiquitin ligases ZNRF3 and RNF43, thereby stabilizing WNT receptors at the plasma membrane and preventing their internalization. What has remained murky is whether RSPO-mediated signaling plays any role in the muscle stromal compartment. Using single-cell RNA sequencing of whole hindlimb muscle from C57BL/6J mice, the researchers found that Rspo gene expression was concentrated almost exclusively in the FAP population, marked by Pdgfra, Thy1 and Ly6a, with Rspo3 showing the highest expression among all four family members. Droplet digital PCR on freshly isolated FAPs confirmed that Rspo3 was the most abundant, with Rspo2 running a close second, while muscle stem cells expressed both genes only weakly.

That expression pattern raised an obvious question: if FAPs are making their own R-spondins, what are those proteins doing to FAP fate? The answer, it turns out, is that they are restraining the cells from becoming fat. When the researchers cultured FAPs in adipogenic induction medium supplemented with recombinant RSPO2 or RSPO3 alone, lipid accumulation, measured by Oil Red O staining at days 7 and 10 of differentiation, was barely affected. But when either RSPO was combined with WNT3A, the canonical WNT ligand, the anti-adipogenic effect was dramatically enhanced. Western blotting revealed that the combination treatment strongly suppressed PPARγ and C/EBPα, the two master transcription factors that drive adipogenesis, as well as FABP4, a mature adipocyte marker, during the early phase of differentiation.

To test whether endogenous R-spondins perform a similar function, the team used pools of four siRNAs each to knock down Rspo2, Rspo3, or both genes simultaneously in cultured FAPs. Individual knockdowns produced only modest increases in lipid accumulation, but the dual knockdown, which achieved over 60 percent reduction of both transcripts, markedly enhanced adipogenic differentiation, with elevated expression of PPARγ, C/EBPα and FABP4 detectable as early as day 3. The redundancy between the two ligands, the authors note, is consistent with overlapping activities described among RSPO family members in other tissues, and suggests that basal RSPO signaling helps maintain FAPs in a non-adipogenic state under normal conditions, likely through an autocrine mechanism in which the progenitors regulate their own fate.

Mechanistically, the researchers traced the anti-adipogenic effect to β-catenin, the central transcriptional co-activator of canonical WNT signaling. When they knocked down Ctnnb1, the gene encoding β-catenin, using siRNA, the inhibitory effect of RSPO3 and WNT3A on lipid accumulation and adipogenic marker expression was partially reversed. Conversely, when they used FAPs derived from mice carrying a β-catenin gain-of-function allele, in which Cre-mediated excision of exon 3 removes the phosphorylation sites that normally target β-catenin for degradation, adipogenic differentiation was strongly suppressed. Activation was confirmed by roughly 60-fold and 30-fold increases in Axin2, a known downstream WNT target, at days 3 and 10 of differentiation respectively. Together, these experiments establish canonical WNT/β-catenin signaling as the central mediator of RSPO function in FAPs.

The in vivo validation came from a glycerol-induced muscle injury model, in which 50 percent glycerol injected into the tibialis anterior muscle promotes ectopic adipogenesis. When the researchers delivered recombinant RSPO3 and WNT3A proteins directly into the injured muscle over two consecutive days, FABP4 immunofluorescence intensity was significantly reduced compared to vehicle controls, with the highest-intensity FABP4-positive cells, the top 10 percent, particularly depleted. PLIN1 immunofluorescence showed a similar trend toward lower adipocyte density, and RT-qPCR confirmed decreased expression of adipogenic genes in the treated muscle. A retrospective power analysis based on mouse-level corrected total cell fluorescence values calculated a statistical power of approximately 99.97 percent with four mice per group, underscoring the robustness of the effect.

Transcriptomic profiling of the RSPO3/WNT3A-treated muscles provided a systems-level view of what the treatment was doing. RNA sequencing identified 1,122 upregulated and 1,270 downregulated genes at a fold change threshold of 1.5 and an FDR cutoff of 0.05. Pathway enrichment analysis revealed that downregulated genes were strongly enriched in adipogenesis, PPAR signaling, and oxidative phosphorylation, consistent with the metabolic reprogramming that accompanies adipocyte differentiation. Transcription factor enrichment using the TRRUST database identified SREBF1, PPARG and CEBPA as central nodes, and ten downstream adipogenic targets, including Adipoq, Retn, Fabp4 and Slc2a4, were coordinately suppressed. Interestingly, MyoD and MEF2C, two major myogenic regulators, were also identified among the affected transcription factors, suggesting that RSPO3/WNT3A may influence myogenesis during muscle regeneration as well.

On the upregulated side, the picture was more complicated. Genes enriched in focal adhesion, cell adhesion, and PI3K-Akt-mTOR signaling pathways were elevated, with several extracellular matrix genes, including Col1a1, Col1a2, Col5a1 and Fn1, showing increased expression, potentially regulated by SP1, STAT3 and SMAD3. Preliminary results showed that both RSPO2 and RSPO3, in combination with WNT3A, increased COL1A1 and CTGF protein expression in cultured FAPs, raising the possibility that suppression of adipogenesis is accompanied by a reciprocal shift toward ECM production and fibrogenic processes. Notably, despite the expected activation of canonical WNT signaling, pathway enrichment analyses did not prominently identify WNT-related terms, which the authors attribute to the transient nature of WNT target gene activation by negative feedback and to the timing of muscle harvest after RSPO/WNT administration.

The in vivo loss-of-function experiments, however, left the endogenous role of R-spondins somewhat inconclusive. The researchers used conditional Rspo3 knockout mice, achieving over 90 percent ablation efficiency, combined with electroporation of Rspo2 siRNA into the injured tibialis anterior muscle, which achieved roughly 30 to 50 percent knockdown. Across four groups, control, Rspo2 knockdown, Rspo3 conditional knockout, and combined knockdown/knockout, no significant differences in adipogenesis were observed by FABP4 corrected total cell fluorescence or adipogenic marker gene expression. The authors suggest that the suboptimal Rspo2 knockdown, compensatory mechanisms from other RSPO family members, or the complexity of the in vivo niche, where multiple signaling pathways converge to regulate FAP fate, may explain the discrepancy with the in vitro results. A genetic Rspo2/Rspo3 double knockout model, they conclude, will be necessary to fully resolve the contribution of endogenous RSPO signaling in vivo.

Nevertheless, the study identifies RSPO2 and RSPO3 as previously unrecognized suppressors of FAP adipogenesis acting through canonical WNT/β-catenin signaling, and suggests a potential therapeutic strategy to modulate FAP behavior, preserving their native functions in muscle homeostasis and regeneration while limiting pathological adipogenesis. The implications extend to conditions characterized by pathological intramuscular fat accumulation, including Duchenne muscular dystrophy, aging-related sarcopenia, and metabolic disorders, where FAP-derived fat infiltration degrades muscle function. The authors caution, however, that further investigation into the balance between adipogenic and fibrogenic outcomes will be essential before such findings can be translated into clinical applications, given the preliminary evidence that RSPO/WNT signaling may also promote a fibrogenic phenotype in FAPs. Whether delivering RSPO proteins, or small molecules that mimic their WNT-potentiating activity, can safely tip the fate of muscle-resident progenitors away from fat and toward repair remains an open question, but the molecular switch they have uncovered now offers a concrete target for intervention.

Subject of Research: R-spondins suppress adipogenic differentiation in fibro-adipogenic progenitors via autocrine WNT/β-catenin signaling

Article Title: R-spondins suppress adipogenic differentiation in fibro-adipogenic progenitors via autocrine WNT/β-catenin signaling

Article References: Fransisca, J., Oh, Y., Sarmito, B., Alymkulova, N., Hwang, Y., & Yoon, J. K. (2026). R-spondins suppress adipogenic differentiation in fibro-adipogenic progenitors via autocrine WNT/β-catenin signaling. Frontiers in Cell and Developmental Biology, 14, Article 1942626. https://doi.org/10.3389/fcell.2026.1942626

Image Credits: AI Generated

DOI: 10.3389/fcell.2026.1942626

Keywords: R-spondins, suppress, adipogenic, differentiation, fibro-adipogenic, progenitors, autocrine, catenin, signaling, scientific research

The WNT/β-catenin pathway is one of the most intensively studied signaling networks in liver cancer biology. Under basal conditions, cytoplasmic β-catenin is held in check by a destruction complex containing APC, Axin, and GSK-3β, which tags the protein for phosphorylation and degradation. When WNT ligands engage their receptors, this process is inhibited, allowing β-catenin to accumulate and translocate to the nucleus, where it partners with TCF/LEF transcription factors to drive the expression of target genes controlling proliferation, differentiation, migration, and survival. Dysregulation of this pathway is a well-established molecular feature of hepatocellular carcinoma, and because it integrates signals governing cell fate, alterations in its activity may represent an important molecular interface between metabolic stress and cancer-associated phenotypes in the liver.

Non-alcoholic fatty liver disease, recently redefined as metabolic dysfunction-associated steatotic liver disease, is the most prevalent chronic liver disease worldwide and carries an increased risk of hepatocellular carcinoma. Disease progression is accompanied by excessive lipid accumulation, oxidative stress, chronic inflammation, mitochondrial dysfunction, and metabolic disturbances that can impair hepatic homeostasis. Lipid overload promotes the production of pro-inflammatory cytokines and reactive oxygen species, contributing to lipid peroxidation that can damage cellular macromolecules and membrane integrity. Persistent oxidative and inflammatory stress may also contribute to DNA damage and genomic instability, providing a mechanistic bridge between metabolic injury and altered cellular signaling. Defining how specific fatty acids alter cancer-relevant signaling pathways may therefore illuminate the molecular connection between metabolic liver disease and hepatocellular carcinoma.

To establish a reproducible lipid-loading model, the researchers first used the MTT assay to determine suitable exposure conditions. Palmitate reduced HepG2 cell viability in a concentration-dependent manner, with estimated IC50 values of approximately 0.8 millimolar after 24 hours and 0.7 millimolar after 48 hours. Oleate was somewhat better tolerated, with IC50 values of approximately 1.0 and 0.9 millimolar at the same time points. Because no significant difference emerged between the 24- and 48-hour values, the shorter exposure was selected for all subsequent experiments. The IC50 concentrations were treated as standardized, experimentally defined exposure levels associated with a measurable cellular response rather than as representations of physiological fatty acid concentrations in vivo.

Combining the two fatty acids proved more challenging than combining their individual tolerances would suggest. When the IC50 concentrations of palmitate and oleate were simply added together, metabolic viability collapsed to roughly 20 percent of control levels, leaving too few viable cells for molecular analysis. The team therefore evaluated lower combinations and settled on 0.4 millimolar palmitate plus 0.8 millimolar oleate, which maintained approximately 50 percent viability while still delivering a robust cellular response. Oil Red O staining confirmed that all treatments significantly increased intracellular lipid droplet accumulation, with the combined treatment producing the highest lipid content of all, indicating a stronger steatotic effect than either fatty acid alone. Lipid droplet accumulation rose progressively with increasing combined concentrations, and an inverse relationship was observed between cell viability and intracellular lipid deposition.

The transcriptional analysis revealed a strikingly asymmetric molecular landscape. Palmitate produced a coordinated activation of canonical WNT/β-catenin signaling components, significantly upregulating WNT expression by 1.53-fold, β-catenin transcript by 3.60-fold, TCF by 1.63-fold, and Axin2, a canonical downstream target gene, by 3.12-fold. In contrast, oleate produced a qualitatively different response, strongly inducing LEF expression by 5.79-fold, the highest transcriptional response among all examined genes, and elevating GSK-3β transcript by 2.27-fold, without significantly altering WNT or TCF expression. Both individual fatty acids suppressed Axin1 transcript levels by approximately 89 percent relative to control, a theoretically significant finding because Axin1 serves as an important scaffold of the β-catenin destruction complex.

Protein-level measurements added crucial nuance to the transcriptional picture. Palmitate significantly increased WNT-1 protein expression by 1.30-fold and β-catenin protein by 1.19-fold, consistent with the corresponding mRNA changes. Oleate and the combined treatment also significantly elevated β-catenin transcript levels, by 2.87-fold and 2.88-fold respectively, but neither produced a corresponding increase in β-catenin protein abundance, which remained comparable to or slightly lower than control levels. Palmitate also significantly elevated Glypican-3 protein expression by 1.35-fold, whereas oleate reduced GPC3 protein levels, although not significantly. These findings demonstrate that palmitate promotes WNT and β-catenin expression at both transcriptional and protein levels, whereas oleate partially attenuates this response despite producing a common phenotype of intracellular lipid accumulation.

The authors interpret these results as evidence that palmitate generates a molecular environment more compatible with enhanced canonical WNT/β-catenin signaling, whereas oleate does not produce the same coordinated pattern. Mechanistically, the simultaneous suppression of Axin1 and induction of Axin2 in palmitate-treated cells is compatible with a shift toward enhanced pathway signaling, because reduced Axin1 may favor β-catenin stabilization while increased Axin2 may reflect activation of a downstream transcriptional response and an accompanying negative-feedback mechanism. The oleate-associated increase in β-catenin transcript without corresponding protein accumulation suggests that transcriptional regulation alone is insufficient to infer sustained canonical WNT activity, and that posttranscriptional and posttranslational mechanisms may contribute to the dissociation between mRNA and protein abundance. The pronounced induction of LEF by oleate should not be interpreted as direct evidence of canonical WNT activation, because TCF and LEF can participate in overlapping as well as context-dependent transcriptional programs.

The combined palmitate/oleate treatment generally produced intermediate molecular responses, attenuating several of the effects induced by each fatty acid alone while maintaining significant activation of selected pathway components. However, the authors caution that because the palmitate concentration differed between the palmitate-alone and combined-treatment conditions, these differences cannot be interpreted as direct evidence of an oleate-mediated protective or antagonistic effect. Rather, they indicate that combined fatty acid exposure produced a molecular response distinct from that observed with palmitate alone. This distinction may be particularly relevant in metabolic liver disease, where hepatocytes are exposed to mixtures of fatty acids rather than to individual lipid species, and where the balance between pathway-promoting and pathway-restraining signals may depend on fatty acid composition.

Several limitations frame the conclusions. The study was performed exclusively in HepG2 cells, a hepatocellular carcinoma-derived model with a transformed phenotype and potentially altered WNT/β-catenin regulation, so the findings cannot be directly extrapolated to normal hepatocytes or to the early stages of metabolic dysfunction-associated steatotic liver disease. Functional WNT/β-catenin activation was not directly assessed through β-catenin nuclear localization, active non-phosphorylated β-catenin measurement, or β-catenin-dependent transcriptional assays, and GSK-3β phosphorylation status and enzymatic activity were not evaluated. Lipidomic and detailed metabolic analyses were not performed, so specific intracellular lipid species, oxidative stress responses, endoplasmic reticulum stress, mitochondrial function, and inflammatory mediators remain unexplored. The MTT assay provides an indirect assessment of metabolic activity and cannot independently distinguish changes in metabolic activity from alterations in cell number or specific cell death mechanisms. Future studies incorporating functional assays, primary or non-transformed hepatocyte models, and appropriate in vivo approaches will be required to validate these findings and clarify the specific contribution and interaction of these fatty acids.

Subject of Research: Fatty acid composition and WNT/β-catenin signaling in lipid-loaded HepG2 cells

Article Title: Differential Modulation of WNT/β‐Catenin Pathway‐Associated Molecular Responses by Palmitate and Oleate in Lipid‐Loaded HepG2 Cells

Article References: Al‐Haeri, M., Mohammadi, Y., Pashaei‐Asl, R., & Bahreini, E. (2026). Differential Modulation of WNT /β‐Catenin Pathway‐Associated Molecular Responses by Palmitate and Oleate in Lipid‐Loaded HepG2 Cells. Cancer Reports, 9(10), Article e70703. https://doi.org/10.1002/cnr2.70703

Image Credits: AI Generated

DOI: 10.1002/cnr2.70703

Keywords: Differential, Modulation, Catenin, Pathway, Associated, Molecular, Responses, Palmitate, Oleate, Lipid, Loaded, HepG2