ABSTRACT
Little progress has been made toward the use of embryonic stem (ES) cells to study and isolate skeletal muscle progenitors. This is due to the paucity of paraxial mesoderm formation during embryoid body (EB) in vitro differentiation and to the lack of reliable identification and isolation criteria for skeletal muscle precursors. Here we show that expression of the transcription factor Pax3 during embryoid body differentiation enhances both paraxial mesoderm formation and the myogenic potential of the cells within this population. Transplantation of Pax3-induced cells results in teratomas, however, indicating the presence of residual undifferentiated cells. By sorting for the PDGF-alpha receptor, a marker of paraxial mesoderm, and for the absence of Flk-1, a marker of lateral plate mesoderm, we derive a cell population from differentiating ES cell cultures that has substantial muscle regeneration potential. Intramuscular and systemic transplantation of these cells into dystrophic mice results in extensive engraftment of adult myofibers with enhanced contractile function without the formation of teratomas. These data demonstrate the therapeutic potential of ES cells in muscular dystrophy.
Subject(s)
Cell Differentiation , Embryonic Stem Cells/cytology , Muscle, Skeletal/physiology , Regeneration , Animals , Cardiotoxins , Cell Transplantation , DNA-Binding Proteins/metabolism , Dystrophin/metabolism , Embryonic Stem Cells/transplantation , Mesoderm/embryology , Mice , Mice, Inbred mdx , Muscle Contraction , Muscle Development , Muscular Dystrophy, Animal , PAX3 Transcription Factor , Paired Box Transcription Factors/isolation & purification , Paired Box Transcription Factors/metabolism , Receptor, Platelet-Derived Growth Factor alpha/metabolism , Teratoma/pathology , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
Little is known about factors that stimulate transcription of the p53 tumor suppressor gene. Here, we report that the human pituitary homeobox 1 (hPitx1) transcription factor increases the expression of p53 at the mRNA and protein levels in human mammary carcinoma (MCF-7) cells. Increased p53 mRNA expression was due to activation of the p53 promoter by hPitx1. hPitx1 bound directly to the p53 promoter and functionally utilized two hPitx1 consensus elements. The predominant consensus element utilized by hPitx1 to stimulate p53 transcription was located within the first exon of the p53 gene. A hPitx1 mutant (hPitx1-R141P) acting as a dominant inhibitor repressed p53 transcription. Forced expression of hPitx1 resulted in cell-cycle arrest and p53-dependent apoptosis in p53-replete MCF-7 cells. Furthermore, hPitx1 stimulated the transcription of p53 target genes involved in cell-cycle arrest and apoptosis (p21 and PTGF-beta), again in a p53-dependent manner. Depletion of endogenous hPitx1 by small interfering RNA (siRNA) in MCF-7 cells resulted in decreased basal expression of p53 and consequently of p21 and placental transforming growth factor beta (PTGF-beta). Depletion of p53 by siRNA dramatically attenuated hPitx1-induced apoptosis in MCF-7 cells. Thus, p53 is a direct transcriptional target gene of hPitx1. This observation is concordant with the recent identification of hPitx1 as a tumor suppressor gene.
Subject(s)
Apoptosis , Genes, p53 , Paired Box Transcription Factors/genetics , Paired Box Transcription Factors/metabolism , Transcriptional Activation , Tumor Suppressor Protein p53/metabolism , Cell Cycle , Cell Division , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cytokines/genetics , DNA/metabolism , Growth Differentiation Factor 15 , Humans , Mutation , Paired Box Transcription Factors/isolation & purification , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering , Transcription, Genetic , Transfection , Tumor Cells, Cultured , Tumor Suppressor Protein p53/isolation & purification , Up-RegulationABSTRACT
Pax genes encode transcription factors that are critical regulators of key developmental processes in evolutionarily diverse animal phyla. Here we report the first isolation of a Pax gene from a crustacean: a Pax group III gene we have termed CdpaxIII that contains highly conserved DNA-binding domains, the paired domain and homeodomain. CdpaxIII is expressed in the embryo, in adult limb muscle during both quiescence and regeneration, and during the distinct process of epimorphic limb regeneration. Interestingly, CdpaxIII is expressed as two distinct alternate transcripts, one of which is novel in lacking a large portion of its paired domain.
