Abstract
Fibro/Adipogenic Progenitors (FAPs) define a stem cell population playing a pro-regenerative role after muscle damage. When removed from their natural niche, FAPs readily differentiate into adipocytes or fibroblasts. This digressive differentiation potential, which is kept under tight control in the healthy muscle niche, contributes to fat and scar infiltrations in degenerative myopathies, such as in Duchenne Muscular Dystrophy (DMD). Controlling FAP differentiation by means of small molecules may contribute to delay the adverse consequences of the progressive pathological degeneration while offering, at the same time, a wider temporal window for gene therapy and cell-based strategies. In a high content phenotypic screening, we identified the immunosuppressant, azathioprine (AZA) as a negative modulator of FAP adipogenesis. We show here that AZA negatively affects the adipogenic propensity of FAPs purified from wild type and mdx mice by impairing the expression of the master adipogenic regulator, peroxisome proliferator-activated receptor γ (PPARγ). We show that this inhibition correlates with a decline in the activation of the AKT-mTOR axis, the main pathway that transduces the pro-adipogenic stimulus triggered by insulin. In addition, AZA exerts a cytostatic effect that has a negative impact on the mitotic clonal process that is required for the terminal differentiation of the preadipocyte-committed cells.
Highlights
Muscle regeneration is governed by a complex cellular crosstalk that is activated after damage[1]
Each differentiation phenotype was assessed by specific staining, demonstrating that the preparation of muscle mononuclear cells had the potential to differentiate into alkaline phosphatase (ALP)-positive osteoblast precursors, α-smooth muscle actin (α-SMA)-myofibroblasts, Oil Red O (ORO)-positive adipocytes or myosin heavy chain (MyHC)-positive myotubes (Supplementary Fig. S1A–I)
Among all the tested molecules, AZA reduced the percentage of the ORO-positive cells, revealing a significant negative perturbation of the intrinsic adipogenic potential of some cell sub-population(s) within the muscle mononuclear cell preparation (Fig. 1C–E)
Summary
Muscle regeneration is governed by a complex cellular crosstalk that is activated after damage[1]. The heterogeneous muscle mononuclear cell populations can be separated from the fibres and cultivated in vitro where differentiation can be monitored in conditions in which the crosstalk between the different mononuclear populations is allowed to proceed[14]. This experimental system partially recapitulates the in vivo cellular www.nature.com/scientificreports/. Context and can be used for screening strategies aimed at selecting molecules affecting differentiation We used such a complex, albeit robust, in vitro system to identify new drugs limiting adipogenesis. AZA treatment impairs FAP adipogenesis by downregulating the transcription factor peroxisome proliferator-activated receptor γ (PPARγ) as a consequence of an attenuation of AKT-mTOR signaling and of a mitotic delay
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