Bisperoxovanadium, a phospho‐tyrosine phosphatase inhibitor, reprograms myogenic cells to acquire a pluripotent, circulating phenotype

Abstract

Satellite cells are the main source of myogenic progenitors in postnatal skeletal muscle, but their use in cell therapy for muscle disorders is limited because these cells cannot be delivered through circulation and they are rapidly exhausted in severe myopathies. The search for alternative donor cells is ongoing, but none of the candidates so far show all the features required for successful colonization and repair of diseased muscle. In this study, we show that bisperoxovanadium, a phospho-tyrosine phosphatase inhibitor, induces myogenic cells to acquire a gene expression profile and a differentiation potential consistent with the phenotype of a circulating precursors, while maintaining their myogenic potential. These effects are mediated, at least in part, by NF-B activation through the Tyr42-IB-␣ phosphorylation, as shown by the expression of the dominant negative mutant form of the p50 NF-B subunit. Moreover, when bisperoxovanadium-treated cells are injected into the femoral artery of ␣-sarcoglican null dystrophic mice, they are able to circulate and to reach muscle tissue; importantly, they contribute to muscle regeneration, as shown by the expression of ␣-sarcoglican in some fibers. Our observations indicate that bisperoxovanadium, or similar compounds, may prove very valuable to obtain and to expand, from committed cells, multipotent cell populations suitable for gene-cell therapy applications and may help to understand the molecular basis of genome reprogramming and "stem-ness."-Castaldi, L., Serra, C., Moretti, F., Messina, G., Paoletti, R., Sampaolesi, M., Torgovnick, A., Baiocchi, M., Padula, F., Pisaniello A., Molinaro, M., Cossu, G., Levrero, M., Bouché, M. Bisperoxovanadium, a phospho-tyrosine phosphatase inhibitor, reprograms myogenic cells to acquire a pluripotent, circulating phenotype. FASEB J. 21, 3573-3583 (2007)

Inhibitors of Tyrosine Phosphatases and Apoptosis Reprogram Lineage-Marked Differentiated Muscle to Myogenic Progenitor Cells

“…Hence we reasoned that global transient inactivation of tyrosine phosphatases would reset signaling in myotubes, making them receptive to mitogens present in growth medium conditions and propelling them into cell cycle as well as towards less differentiated state. Earlier BpV (a tyrosine phosphatase inhibitor) was reported to delay differentiation of dividing C2C12 into myotubes (Castaldi et al, 2007) and other studies also indicated that a small percentage of myotubes that enter S-phase upon over-expression of genes fail to proliferate and succumb to apoptosis (Endo and Nadal-Ginard, 1998; Latella et al, 2000). Therefore, we reasoned that if BpV was able to trigger the process of myotube de-differentiation and that the addition of an apoptosis inhibitor (Q-VD) in our studies may help in survival of those myotubes that might undergo massive restructuring of cell cytoskeleton simultaneously with the breakage of post-mitotic arrest.…”

PKCθ signaling is required for myoblast fusion by regulating the expression of caveolin-3 and β1D integrin upstream focal adhesion kinase

“…Primary cultures were prepared from total limb muscles of WT, PKCθ −/− , or mPKC-K/R mice, as previously described (Castaldi et al , 2007). Muscle-derived cells were grown on collagen-coated dishes, in GM (DMEM containing 20% horse serum, HS, 3% chick embryo extract, EE, all from Invitrogen, Carlsbad, CA) in a humidified 5% CO 2 atmosphere at 37°C.…”

“…The muscle fiber mean CSA was determined by measuring the CSA of all fibers in the entire section, using Scion Image 4.0.3.2 software (NIH, Bethesda, MD). Immunofluorescence analysis of cells, cryosections, or isolated myofibers was performed as previously described (Castaldi et al , 2007; Aulino et al , 2010). Nuclei were counterstained with Hoechst 33342 (Fluka) or with TO-PRO-3 (Invitrogen), and the samples were analyzed under an epifluorescence Zeiss Axioskop 2 Plus microscope (Carl Zeiss, Oberkochen, Germany) or a Leica Leitz DMRB microscope fitted with a DFC300FX camera (Leica, Wetzlar, Germany).…”

Protein kinase Cθ is required for cardiomyocyte survival and cardiac remodeling

“…The following primary antibodies were used: the antimyosin heavy-chain MF20, 35 the anti-ANF (Bachem, Bubendorf, Switzerland); the anti-caveolin-3, the anti-PKC- α , - δ , - ɛ , - η , - θ and the anti-phospho Thr538 PKC θ (BD Bioscience, Franklin Lake, NJ, USA); the anti-Akt, the anti-phosphoAkt, the anti-phosphoGSK3 β and the anti phospho-p38 (Cell Signalling Inc., Danvers, MA, USA); the anti-caspase-9 (Assay Designs, Ann Arbor, MI, USA); the anti-caspase-8 (Santa Cruz Biotec, Santa Cruz, CA, USA); the anti- α -tubulin (Sigma-Aldrich, St. Louis, MO, USA).…”

“…For immunofluorescence analysis, cryosections were fixed in 4% paraformaldehyde, as described above, whereas cultured cells were fixed in ethanol/acetone (1 : 1 ratio) at −20°C for 20 min, and processed as described previously. 35 Samples were examined under a Zeiss Axioskop 2 Plus fluorescence microscope (Zeiss, Gottingen, Germany), as mentioned above. The mean CMC CSA in the heart tissue, as well as the mean cell area in cultured CMCs, were measured using the Scion Image 4.0.3.2 software (NIH, Bethesda, MD, USA).…”