Fibro/Adipogenic Progenitors (FAPs) are muscle-interstitial progenitors mediating pro-myogenic signals that are critical for muscle homeostasis and regeneration. In myopathies, the autocrine/paracrine constraints controlling FAP adipogenesis are released causing fat infiltrates. Here, by combining pharmacological screening, high-dimensional mass cytometry and in silico network modeling with the integration of single-cell/bulk RNA sequencing data, we highlighted the canonical WNT/ GSK/β-catenin signaling as a crucial pathway modulating FAP adipogenesis triggered by insulin signaling. Consistently, pharmacological blockade of GSK3, by the LY2090314 inhibitor, stabilizes β-catenin and represses PPARγ expression abrogating FAP adipogenesis ex vivo while limiting fatty degeneration in vivo. Furthermore, GSK3 inhibition improves the FAP pro-myogenic role by efficiently stimulating, via follistatin secretion, muscle satellite cell (MuSC) differentiation into mature myotubes. Combining, publicly available single-cell RNAseq datasets, we characterize FAPs as the main source of WNT ligands inferring their potential in mediating autocrine/paracrine responses in the muscle niche. Lastly, we identify WNT5a, whose expression is impaired in dystrophic FAPs, as a crucial WNT ligand able to restrain the detrimental adipogenic differentiation drift of these cells through the positive modulation of the β-catenin signaling.

Fibro-adipogenic progenitors (FAPs) promote satellite cell differentiation in adult skeletal muscle regeneration. However, in pathological conditions, FAPs are responsible for fibrosis and fatty infiltrations. Here we show that the NOTCH pathway negatively modulates FAP differentiation both in vitro and in vivo. However, FAPs isolated from young dystrophin-deficient mdx mice are insensitive to this control mechanism. An unbiased mass spectrometry–based proteomic analysis of FAPs from muscles of wild-type and mdx mice suggested that the synergistic cooperation between NOTCH and inflammatory signals controls FAP differentiation. Remarkably, we demonstrated that factors released by hematopoietic cells restore the sensitivity to NOTCH adipogenic inhibition in mdx FAPs. These results offer a basis for rationalizing pathological ectopic fat infiltrations in skeletal muscle and may suggest new therapeutic strategies to mitigate the detrimental effects of fat depositions in muscles of dystrophic patients.

Degradation of the endoplasmic reticulum (ER) via selective autophagy (ER‐phagy) is vital for cellular homeostasis. We identify FAM134A/RETREG2 and FAM134C/RETREG3 as ER‐phagy receptors, which predominantly exist in an inactive state under basal conditions. Upon autophagy induction and ER stress signal, they can induce significant ER fragmentation and subsequent lysosomal degradation. FAM134A, FAM134B/RETREG1, and FAM134C are essential for maintaining ER morphology in a LC3‐interacting region (LIR)‐dependent manner. Overexpression of any FAM134 paralogue has the capacity to significantly augment the general ER‐phagy flux upon starvation or ER‐stress. Global proteomic analysis of FAM134 overexpressing and knockout cell lines reveals several protein clusters that are distinctly regulated by each of the FAM134 paralogues as well as a cluster of commonly regulated ER‐resident proteins. Utilizing pro‐Collagen I, as a shared ER‐phagy substrate, we observe that FAM134A acts in a LIR‐independent manner and compensates for the loss of FAM134B and FAM134C, respectively. FAM134C instead is unable to compensate for the loss of its paralogues. Taken together, our data show that FAM134 paralogues contribute to common and unique ER‐phagy pathways.

In Duchenne muscular dystrophy (DMD), the absence of the dystrophin protein causes a variety of poorly understood secondary effects. Notably, muscle fibers of dystrophic individuals are characterized by mitochondrial dysfunctions, as revealed by a reduced ATP production rate and by defective oxidative phosphorylation. Here, we show that in a mouse model of DMD (mdx), fibro/adipogenic progenitors (FAPs) are characterized by a dysfunctional mitochondrial metabolism which correlates with increased adipogenic potential. Using high-sensitivity mass spectrometry–based proteomics, we report that a short-term high-fat diet (HFD) reprograms dystrophic FAP metabolism in vivo. By combining our proteomic dataset with a literature-derived signaling network, we revealed that HFD modulates the β-catenin–follistatin axis. These changes are accompanied by significant amelioration of the histological phenotype in dystrophic mice. Transplantation of purified FAPs from HFD-fed mice into the muscles of dystrophic recipients demonstrates that modulation of FAP metabolism can be functional to ameliorate the dystrophic phenotype. Our study supports metabolic reprogramming of muscle interstitial progenitor cells as a novel approach to alleviate some of the adverse outcomes of DMD.

Highlights d Pathogenic signaling pathways are rewired in type 2 diabetic islets d The GSK3-PDX1 axis is compromised in type 2 diabetic islets d The GSK3 kinase is a crucial regulator of GLUT2 and insulin expression d GSK3 inhibition restores the ability of diabetic islets to secrete insulin

Activin receptor type-1C (ALK-7) Adiponectin (ADIPOQ) Alkaline phosphatase, tissue-nonspecific isozyme (ALPL) Alpha smooth muscle actin (a-SMA, ACTA2) Amyotrophic lateral sclerosis (ALS) Angiopoietin-1 receptor (TEK/TIE2) Anterior cruciate ligament (ACL) AT-rich interactive domain-containing protein 5B (ARID5B) Beta-sarcoglycan (SGCB) Bone morphogenetic protein 1 (BMP-1) Bone morphogenetic protein 4 (BMP-4) Bone morphogenic protein 2 (BMP-2) Bone morphogenic protein 3 (BMP-3) Cancer-associated cachexia (CAC) Cardiotoxin (CTX) Catenin beta-1 (CTNNB1, b-catenin) C-C chemokine receptor type 2 (CCR2) C-C motif chemokine 2 (MCP-1) C-C motif chemokine 7 (CCL7) CCAAT/enhancer-binding protein alpha (C/EBPa) CCN family member 4/WNT1-inducible-signaling pathway protein 1 (CCN4/WISP-1) Chronic inflammatory myopathy (CIM) Chronic kidney disease (CKD) Collagen alpha-1(I) chain (COL1A1) Collagen alpha-1(III) chain (COL3A1) Collagen alpha-1(VIII) chain (COL8A1) Collagen alpha-1(XII) chain (COL12A1) Collagen alpha-2(I) chain (COL1A2) C-X-C motif chemokine 14 (CXCL14) C-X-C motif chemokine 5 (CXCL5) Delta-like protein 1/3/4 (DELTA1, DELTA3, DELTA4) Wingless-related integration site (WNT)

Muscle resident fibro-adipogenic progenitors (FAPs), support muscle regeneration by releasing cytokines that stimulate the differentiation of myogenic stem cells. However, in non-physiological contexts (myopathies, atrophy, aging) FAPs cause fibrotic and fat infiltrations that impair muscle function. We set out to perform a fluorescence microscopy-based screening to identify compounds that perturb the differentiation trajectories of these multipotent stem cells. From a primary screen of 1,120 FDA/EMA approved drugs, we identified 34 compounds as potential inhibitors of adipogenic differentiation of FAPs isolated from the murine model (mdx) of Duchenne muscular dystrophy (DMD). The hit list from this screen was surprisingly enriched with compounds from the glucocorticoid (GCs) chemical class, drugs that are known to promote adipogenesis in vitro and in vivo. To shed light on these data, three GCs identified in our screening efforts were characterized by different approaches. We found that like dexamethasone, budesonide inhibits adipogenesis induced by insulin in sub-confluent FAPs. However, both drugs have a pro-adipogenic impact when the adipogenic mix contains factors that increase the concentration of cAMP. Gene expression analysis demonstrated that treatment with glucocorticoids induces the transcription of Gilz/Tsc22d3, an inhibitor of the adipogenic master regulator PPARγ, only in anti-adipogenic conditions. Additionally, alongside their anti-adipogenic effect, GCs are shown to promote terminal differentiation of satellite cells. Both the anti-adipogenic and pro-myogenic effects are mediated by the glucocorticoid receptor and are not observed in the presence of receptor inhibitors. Steroid administration currently represents the standard treatment for DMD patients, the rationale being based on their anti-inflammatory effects. The findings presented here offer new insights on additional glucocorticoid effects on muscle stem cells that may affect muscle homeostasis and physiology.In muscular dystrophies the degeneration of muscle tissue is initially compensated by efficient regeneration that neutralize muscle loss 1 . However, over time, the regenerative potential in muscles of patients affected by myopathies is impaired and myofibers repair is curbed by the formation of fibrotic scars and fat infiltrations, ultimately leading to decreased muscle function 2 . Muscle fibro-adipogenic progenitors play an important role in these processes. FAPs are muscle mesenchymal stem cells residing in the muscle fibers interstitial space. FAPs express the SCA1, CD34 and PDGFRα (CD140a) antigens while they are negative for the hematopoietic and endothelial markers, CD45 and CD31, and for the satellite marker α7 integrin (ITGA7) 2-4 . FAPs contribute to muscle regeneration by secreting IGF-1 and IL-6 3 , by facilitating the clearance of necrotic debris 5 and by promoting the formation of extra-cellular matrix 6 . In addition to this pro-regenerative roles, FAPs are responsible for the formation of ectopic tissue infiltrations ...

In the tumor microenvironment, cancer cells experience hypoxia resulting in the accumulation of misfolded/unfolded proteins largely in the endoplasmic reticulum (ER). Consequently, ER proteotoxicity elicits unfolded protein response (UPR) as an adaptive mechanism to resolve ER stress. In addition to canonical UPR, proteotoxicity also stimulates the selective, autophagy-dependent, removal of discrete ER domains loaded with misfolded proteins to further alleviate ER stress. These mechanisms can favor cancer cell growth, metastasis, and long-term survival. Our investigations reveal that during hypoxia-induced ER stress, the ER-phagy receptor FAM134B targets damaged portions of ER into autophagosomes to restore ER homeostasis in cancer cells. Loss of FAM134B in breast cancer cells results in increased ER stress and reduced cell proliferation. Mechanistically, upon sensing hypoxia-induced proteotoxic stress, the ER chaperone BiP forms a complex with FAM134B and promotes ER-phagy. To prove the translational implication of our mechanistic findings, we identified vitexin as a pharmacological agent that disrupts FAM134B-BiP complex, inhibits ER-phagy, and potently suppresses breast cancer progression in vivo.