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

Abstract

Fibro‐adipogenic progenitors (FAPs) are muscle‐resident stromal cells that support skeletal muscle regeneration and homeostasis but can drive pathological adipogenesis. In this study, we investigated the role of R‐spondins (RSPOs), a family of WNT signaling modulators, in FAP adipogenic differentiation. FAPs were differentiated in adipogenic induction medium (AIM) supplemented with RSPO2 or RSPO3 in combination with WNT3A, followed by assessment of adipogenic differentiation using western blot and Oil Red O (ORO) analyses. siRNA‐mediated knockdown of Rspo 2, Rspo 3, and Ctnnb1 , as well as constitutive activation of β-catenin, was performed to investigate their roles in FAP adipogenesis. For in vivo validation, a mixture of recombinant RSPO3 and WNT3A was injected into glycerol‐injured tibialis anterior muscles, followed by assessment of adipogenesis and transcriptomic analysis. Here, we identify Rspo 2 and Rspo 3 as key autocrine regulators of FAP fate through modulation of WNT/β‐catenin signaling. Their siRNA‐mediated knockdown markedly enhances FAP adipogenic differentiation. Conversely, RSPO2 and RSPO3 synergize with WNT3A to suppress FAP adipogenesis, an effect dependent on β‐catenin as demonstrated by attenuation following Ctnnb1 knockdown. Constitutive β‐catenin activation similarly restrains adipogenic differentiation. In vivo , intramuscular delivery of recombinant RSPO3 and WNT3A proteins significantly reduces ectopic adipogenesis induced in glycerol‐injured tibialis anterior muscle, accompanied by downregulation of adipogenic markers and transcriptional programs. Collectively, these findings establish RSPO2 and RSPO3 as previously unrecognized suppressors of FAP adipogenesis acting through canonical WNT/β‐catenin signaling.