c-Abl Kinase Is Required for Satellite Cell Function Through Pax7 Regulation.

Satellite cells (SCs) are tissue-specific stem cells responsible for adult skeletal muscle regeneration and maintenance. SCs function is critically dependent on two families of transcription factors: the paired box (Pax) involved in specification and maintenance and the Muscle Regulatory Factors (MRFs), which orchestrate myogenic commitment and differentiation. In turn, signaling events triggered by extrinsic and intrinsic stimuli control their function via post-translational modifications, including ubiquitination and phosphorylation. In this context, the Abelson non-receptor tyrosine kinase (c-Abl) mediates the activation of the p38 α/ß MAPK pathway, promoting myogenesis. c-Abl also regulates the activity of the transcription factor MyoD during DNA-damage stress response, pausing differentiation. However, it is not clear if c-Abl modulates other key transcription factors controlling SC function. This work aims to determine the role of c-Abl in SCs myogenic capacity via loss of function approaches in vitro and in vivo. Here we show that c-Abl inhibition or deletion results in a down-regulation of Pax7 mRNA and protein levels, accompanied by decreased Pax7 transcriptional activity, without a significant effect on MRF expression. Additionally, we provide data indicating that Pax7 is directly phosphorylated by c-Abl. Finally, SC-specific c-Abl ablation impairs muscle regeneration upon acute injury. Our results indicate that c-Abl regulates myogenic progression in activated SCs by controlling Pax7 function and expression.

USP7-dependent control of myogenin stability is required for terminal differentiation in skeletal muscle progenitors.

Satellite cells (SCs) are myogenic progenitors responsible for skeletal muscle regeneration and maintenance. Upon activation, SCs enter a phase of robust proliferation followed by terminal differentiation. Underlying this myogenic progression, the sequential expression of muscle regulatory transcription factors (MRFs) and the downregulation of transcription factor paired box gene 7 (Pax7) are key steps regulating SC fate. In addition to transcriptional regulation, post-translational control of Pax7 and the MRFs provides another layer of spatiotemporal control to the myogenic process. In this context, previous work showed that Pax7 is ubiquitinated by the E3 ligase neural precursor cell-expressed developmentally downregulated protein 4 and interacts with several proteins related to the ubiquitin-proteasome system, including the deubiquitinase ubiquitin-specific protease 7 (USP7). Although USP7 functions in diverse cellular contexts, its role(s) during myogenesis remains poorly explored. Here, we show that USP7 is transiently expressed in adult muscle progenitors, correlating with the onset of myogenin expression, while it is downregulated in newly formed myotubes/myofibers. Acute inhibition of USP7 activity upon muscle injury results in persistent expression of early regeneration markers and a significant reduction in the diameter of regenerating myofibers. At the molecular level, USP7 downregulation or pharmacological inhibition impairs muscle differentiation by affecting myogenin stability. Co-immunoprecipitation and in vitro activity assays indicate that myogenin is a novel USP7 target for deubiquitination. These results suggest that USP7 regulates SC myogenic progression by enhancing myogenin stability.

Galectin-8 as an immunosuppressor in experimental autoimmune encephalomyelitis and a target of human early prognostic antibodies in multiple sclerosis.

Galectin-8 (Gal-8) is a member of a glycan-binding protein family that regulates the immune system, among other functions, and is a target of antibodies in autoimmune disorders. However, its role in multiple sclerosis (MS), an autoimmune inflammatory disease of the central nervous system (CNS), remains unknown. We study the consequences of Gal-8 silencing on lymphocyte subpopulations and the development of experimental autoimmune encephalitis (EAE), to then assess the presence and clinical meaning of anti-Gal-8 antibodies in MS patients. Lgals8/Lac-Z knock-in mice lacking Gal-8 expression have higher polarization toward Th17 cells accompanied with decreased CCR6+ and higher CXCR3+ regulatory T cells (Tregs) frequency. These conditions result in exacerbated MOG35-55 peptide-induced EAE. Gal-8 eliminates activated Th17 but not Th1 cells by apoptosis and ameliorates EAE in C57BL/6 wild-type mice. ß-gal histochemistry reflecting the activity of the Gal-8 promoter revealed Gal-8 expression in a wide range of CNS regions, including high expression in the choroid-plexus. Accordingly, we detected Gal-8 in human cerebrospinal fluid, suggesting a role in the CNS immune-surveillance circuit. In addition, we show that MS patients generate function-blocking anti-Gal-8 antibodies with pathogenic potential. Such antibodies block cell adhesion and Gal-8-induced Th17 apoptosis. Furthermore, circulating anti-Gal-8 antibodies associate with relapsing-remitting MS (RRMS), and not with progressive MS phenotypes, predicting clinical disability at diagnosis within the first year of follow-up. Our results reveal that Gal-8 has an immunosuppressive protective role against autoimmune CNS inflammation, modulating the balance of Th17 and Th1 polarization and their respective Tregs. Such a role can be counteracted during RRMS by anti-Gal-8 antibodies, worsening disease prognosis. Even though anti-Gal-8 antibodies are not specific for MS, our results suggest that they could be a potential early severity biomarker in RRMS.

Epidermal growth factor receptor endocytic traffic perturbation by phosphatidate phosphohydrolase inhibition: new strategy against cancer.

Epidermal growth factor receptor (EGFR) exaggerated (oncogenic) function is currently targeted in cancer treatment with drugs that block receptor ligand binding or tyrosine kinase activity. Because endocytic trafficking is a crucial regulator of EGFR function, its pharmacological perturbation might provide a new anti-tumoral strategy. Inhibition of phosphatidic acid (PA) phosphohydrolase (PAP) activity has been shown to trigger PA signaling towards type 4 phosphodiesterase (PDE4) activation and protein kinase A inhibition, leading to internalization of empty/inactive EGFR. Here, we used propranolol, its l- and d- isomers and desipramine as PAP inhibitors to further explore the effects of PAP inhibition on EGFR endocytic trafficking and its consequences on EGFR-dependent cancer cell line models. PAP inhibition not only made EGFR inaccessible to stimuli but also prolonged the signaling lifetime of ligand-activated EGFR in recycling endosomes. Strikingly, such endocytic perturbations applied in acute/intermittent PAP inhibitor treatments selectively impaired cell proliferation/viability sustained by an exaggerated EGFR function. Phospholipase D inhibition with FIPI (5-fluoro-2-indolyl des-chlorohalopemide) and PDE4 inhibition with rolipram abrogated both the anti-tumoral and endocytic effects of PAP inhibition. Prolonged treatments with a low concentration of PAP inhibitors, although without detectable endocytic effects, still counteracted cell proliferation, induced apoptosis and decreased anchorage-independent growth of cells bearing EGFR oncogenic influences. Overall, our results show that PAP inhibitors can counteract EGFR oncogenic traits, including receptor overexpression or activating mutations resistant to current tyrosine kinase inhibitors, perturbing EGFR endocytic trafficking and perhaps other as yet unknown processes, depending on treatment conditions. This puts PAP activity forward as a new suitable target against EGFR-driven malignancy.