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Inhibitors of tyrosine phosphatases and apoptosis reprogram lineage-marked differentiated muscle to myogenic progenitor cells.

ABSTRACT: Muscle regeneration declines with aging and myopathies, and reprogramming of differentiated muscle cells to their progenitors can serve as a robust source of therapeutic cells. Here, we used the Cre-Lox method to specifically label postmitotic primary multinucleated myotubes and then utilized small molecule inhibitors of tyrosine phosphatases and apoptosis to dedifferentiate these myotubes into proliferating myogenic cells, without gene overexpression. The reprogrammed, fusion competent, muscle precursor cells contributed to muscle regeneration in vitro and in vivo and were unequivocally distinguished from reactivated reserve cells because of the lineage marking method. The small molecule inhibitors downregulated cell cycle inhibitors and chromatin remodeling factors known to promote and maintain the cell fate of myotubes, facilitating cell fate reversal. Our findings enhance understanding of cell-fate determination and create novel therapeutic approaches for improved muscle repair.

SUBMITTER: Paliwal P

PROVIDER: S-EPMC3183467 | biostudies-other | 2011 Sep

REPOSITORIES: biostudies-other

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Inhibitors of tyrosine phosphatases and apoptosis reprogram lineage-marked differentiated muscle to myogenic progenitor cells.

Paliwal Preeti P Conboy Irina M IM

Chemistry & biology 20110901 9

Muscle regeneration declines with aging and myopathies, and reprogramming of differentiated muscle cells to their progenitors can serve as a robust source of therapeutic cells. Here, we used the Cre-Lox method to specifically label postmitotic primary multinucleated myotubes and then utilized small molecule inhibitors of tyrosine phosphatases and apoptosis to dedifferentiate these myotubes into proliferating myogenic cells, without gene overexpression. The reprogrammed, fusion competent, muscle ...[more]

PMID: 21944754

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Project description:In all, 15 aryl-containing phosphonates have been synthesized and tested for their effect on protein-tyrosine phosphatase (PTPase) activity. Two compounds, (naphth-2-yl) difluoromethylphosphonic acid (12) and (napthy-1-yl) difluoromethylphosphonic acid (13) have been found to inhibit dephosphorylation of [32P]insulin receptors by PTP-1B, a protein tyrosine phosphatase (PTPase), with IC50 values of 40-50 microM. Compound 12 competitively inhibited insulin-receptor dephosphorylation by PTP-1B. Compound 12 also inhibited PTP-1B-catalysed dephosphorylation of a synthetic tyrosine phosphorylated substrate poly(Glu80-Tyr20) at the same potency, indicating that 12 acted via interaction with the PTPase. Additionally, 12 inhibited insulin-receptor PTPase(s) and epridermal-growth-factor-receptor PTPase(s) present in solubilized membranes from CHO (Chinese-hamster ovary)/HIRc and A431 cells respectively. IC50 values of 40-50 microM were obtained in all cases with compound 12. Of note is the fact that these compounds did not have any effect on insulin-receptor autophosphorylation. Nine out of the 15 compounds potently inhibited serine/threonine phosphatase PP-2A activity without any effect on serine/threonine phosphatase PP-1 when tested at a concentration as high as 675 microM. The most potent compounds acting toward PP-2A had IC50 values of 45-50 microM. These PP-2A inhibitors could be useful tools for studying serine/threonine-phosphatase-mediated signal transduction. Two compounds, 12 and 13, inhibited both tyrosine phosphatase PTP-1B and serine/threonine phosphatase PP-2A with similar potency; IC50 values being 40-50 microM in both cases. Details of the synthesis of compounds 10, 11 and 13 are given in Supplementary Publication SUP 50177 (6 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1995) 305, 9.

Project description:BACKGROUND:Degeneration of smooth muscles in sphincters can cause debilitating diseases such as fecal incontinence. Skeletal muscle-derived cells have been effectively used in clinics for the regeneration of the skeletal muscle sphincters, such as the external anal or urinary sphincter. However, little is known about the in vitro smooth muscle differentiation potential and in vivo regenerative potential of skeletal muscle-derived cells. METHODS:Myogenic progenitor cells (MPC) were isolated from the skeletal muscle and analyzed by flow cytometry and in vitro differentiation assays. The differentiation of MPC to smooth muscle cells (MPC-SMC) was evaluated by immunofluorescence, flow cytometry, patch-clamp, collagen contraction, and microarray gene expression analysis. In vivo engraftment of MPC-SMC was monitored by transplanting reporter protein-expressing cells into the pyloric sphincter of immunodeficient mice. RESULTS:MPC derived from human skeletal muscle expressed mesenchymal surface markers and exhibit skeletal myogenic differentiation potential in vitro. In contrast, they lack hematopoietic surface marker, as well as adipogenic, osteogenic, and chondrogenic differentiation potential in vitro. Cultivation of MPC in smooth muscle differentiation medium significantly increases the fraction of alpha smooth muscle actin (aSMA) and smoothelin-positive cells, while leaving the number of desmin-positive cells unchanged. Smooth muscle-differentiated MPC (MPC-SMC) exhibit increased expression of smooth muscle-related genes, significantly enhanced numbers of CD146- and CD49a-positive cells, and in vitro contractility and express functional Cav and Kv channels. MPC to MPC-SMC differentiation was also accompanied by a reduction in their skeletal muscle differentiation potential. Upon removal of the smooth muscle differentiation medium, a major fraction of MPC-SMC remained positive for aSMA, suggesting the definitive acquisition of their phenotype. Transplantation of murine MPC-SMC into the mouse pyloric sphincter revealed engraftment of MPC-SMC based on aSMA protein expression within the host smooth muscle tissue. CONCLUSIONS:Our work confirms the ability of MPC to give rise to smooth muscle cells (MPC-SMC) with a well-defined and stable phenotype. Moreover, the engraftment of in vitro-differentiated murine MPC-SMC into the pyloric sphincter in vivo underscores the potential of this cell population as a novel cell therapeutic treatment for smooth muscle regeneration of sphincters.

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Inhibitors of tyrosine phosphatases and apoptosis reprogram lineage-marked differentiated muscle to myogenic progenitor cells.

Publications

Inhibitors of tyrosine phosphatases and apoptosis reprogram lineage-marked differentiated muscle to myogenic progenitor cells.

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