ABSTRACT
The malignant potential of smooth muscle tumors correlates strongly with the disappearance of gamma-smooth muscle isoactin, a lineage-specific marker of smooth muscle development. In this paper, we identify a 36-base pair regulatory motif containing an AT-rich domain, CArG box, and a non-canonical NK-2 homeodomain-binding site that has the capacity to regulate smooth muscle-specific gene expression in cultured intestinal smooth muscle cells. Serum-response factor associates with an NK-2 transcription factor via protein-protein interactions and binds to the core CArG box element. Our studies suggest that the NK-2 transcription factor that associates with serum-response factor during smooth muscle differentiation is Nkx2-3. Myocyte-specific enhancer factor 2 binding to this regulatory complex was also observed but limited to uterine smooth muscle tissues. Smooth muscle neoplasms displayed altered transcription factor binding when compared with normal myometrium. Differential nuclear accessibility of serum-response factor protein during smooth muscle differentiation and neoplastic transformation was also observed. Thus, we have identified a unique regulatory complex whose differential binding properties and nuclear accessibility are associated with modulating gamma-smooth muscle isoactin-specific gene expression in both normal and neoplastic tissues.
Subject(s)
Cell Differentiation , DNA-Binding Proteins/metabolism , Homeodomain Proteins/metabolism , Muscle, Smooth/cytology , Neoplasms/metabolism , Nuclear Proteins/metabolism , Transcription Factors/metabolism , 3T3 Cells , Animals , Animals, Newborn , Base Sequence , Cell Nucleus/metabolism , Drosophila Proteins , MEF2 Transcription Factors , Mice , Molecular Sequence Data , Muscle, Smooth/metabolism , Myogenic Regulatory Factors , Promoter Regions, Genetic , Rats , Serum Response FactorABSTRACT
BACKGROUND & AIMS: Cultured gastrointestinal smooth muscle cells have been shown to dedifferentiate and reinitiate their myogenic program in vitro. The aim of this study was to determine whether the cellular phenotypes observed in vitro were similar to those previously characterized in vivo. METHODS: Differential isoactin expression was examined in primary cultures of intestinal smooth muscle cells (ISMCs) by Northern blot and immunohistochemical analysis. Cellular phenotype was determined for cultured ISMCs grown at high density, at low density, in the presence and absence of serum supplementation, and on several distinct substrates including collagen type IV, laminin, fibronectin, and plastic. RESULTS: The unique patterns of isoactin protein and gene expression observed in cultured ISMCs indicate that distinct cellular phenotypes were present in vitro. The production and maintenance of these distinct smooth muscle cell phenotypes was dependent on cell density, serum supplementation, and substrate used. CONCLUSIONS: Cultured ISMCs appear to recapitulate a portion of their in vivo myogenic program in vitro, providing a unique opportunity for the molecular mechanisms controlling gastrointestinal smooth muscle myogenesis and pathogenesis to begin to be identified.
Subject(s)
Actins/biosynthesis , Intestine, Small/cytology , Muscle, Smooth/cytology , Actins/genetics , Animals , Animals, Newborn , Cell Culture Techniques/methods , Cell Division , Cells, Cultured , Collagen , Culture Media, Serum-Free , Embryonic and Fetal Development , Fibronectins , Intestine, Small/embryology , Intestine, Small/metabolism , Kinetics , Laminin , Muscle, Smooth/embryology , Muscle, Smooth/metabolism , Phenotype , Plastics , Protein Biosynthesis , Rats , Rats, Sprague-Dawley , Transcription, GeneticABSTRACT
Gastrointestinal smooth muscle development proceeds by the linear differentiation of distinct smooth muscle cell phenotypes. In an effort to identify specific gene products associated with distinct smooth muscle cell phenotypes, we performed differential display on smooth muscle myoblasts versus immature smooth muscle myocytes. This analysis identified a novel short-chain alcohol dehydrogenase-like isozyme which was preferentially expressed in smooth muscle myoblasts over immature and mature smooth muscle myocytes. We postulate that this novel short-chain alcohol dehydrogenase-like isozyme may play a role in potentiating the dedifferentiation of smooth muscle cells in vitro.
