Abstract
This study was performed to define the roles of actin-binding proteins in the regulation of actin filament assembly associated with cellular signal transduction pathways in stromal cell proliferation. Genistein, a tyrosine protein kinase inhibitor, decreased the intracellular Ca(2+) and attenuated cell proliferation and DNA synthesis through the beta-catenin and cyclin D1 pathway in human umbilical CD105-positive cells. Immunoprecipitation studies using anti-beta-actin antibody revealed that several actin-binding proteins implicated in cells include formin-2 (FMN-2), caldesmon (CaD), tropomyosin (Tm), and profilin. Protein levels of these proteins in whole cell lysates were not significantly changed by genistein. Three Tm isoforms, Tm-1, Tm-2, and Tm-4, were found to be present in cells. Genistein caused a reduction in levels of mRNAs coding for Tm-1 and Tm-4, but had no significant effect on Tm-2 mRNA levels. Immunofluorescence confocal scanning microscopy indicated that changes in the subcellular distribution of Tm and CaD, in which the diffuse cytosolic staining was shifted to show colocalization with actin stress fibers. In contrast, genistein-induced accumulation of FMN-2 and profilin in the peri-nuclear area. Silencing of FMN-2 by small interfering RNA resulted in increases of intracellular Ca(2+) and rendered genistein resistance in decreasing intracellular Ca(2+) in cells. These results provide the novel findings that genistein acts by modulating the cellular distribution of actin-binding proteins in association with alterations of cellular signal transduction pathways in human stromal cell proliferation.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Antigens, CD / analysis
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Antigens, CD / metabolism
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Biomarkers / analysis
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Biomarkers / metabolism
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Calcium Signaling / drug effects
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Calcium Signaling / physiology
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Calmodulin-Binding Proteins / drug effects
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Calmodulin-Binding Proteins / metabolism
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Cell Proliferation / drug effects
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Cells, Cultured
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Cyclin D1 / drug effects
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Cyclin D1 / metabolism
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Cytoplasm / drug effects
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Cytoplasm / metabolism
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Endoglin
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Genistein / pharmacology*
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Humans
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Infant, Newborn
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Mesenchymal Stem Cells / cytology
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Mesenchymal Stem Cells / drug effects
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Mesenchymal Stem Cells / metabolism
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Microfilament Proteins / drug effects*
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Microfilament Proteins / metabolism
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Nerve Tissue Proteins / drug effects
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Nerve Tissue Proteins / genetics
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Nerve Tissue Proteins / metabolism
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Profilins / drug effects
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Profilins / metabolism
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Protein Isoforms / drug effects
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Protein Isoforms / genetics
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Protein Isoforms / metabolism
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Protein Kinase Inhibitors / pharmacology
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RNA Interference / drug effects
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RNA, Messenger / drug effects
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RNA, Messenger / metabolism
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Receptors, Cell Surface / analysis
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Receptors, Cell Surface / metabolism
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Signal Transduction / drug effects
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Signal Transduction / physiology
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Stromal Cells / cytology
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Stromal Cells / drug effects*
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Stromal Cells / metabolism*
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Tropomyosin / drug effects
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Tropomyosin / genetics
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Tropomyosin / metabolism
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Umbilical Cord
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beta Catenin / drug effects*
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beta Catenin / metabolism
Substances
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Antigens, CD
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Biomarkers
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Calmodulin-Binding Proteins
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ENG protein, human
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Endoglin
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Microfilament Proteins
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Nerve Tissue Proteins
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Profilins
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Protein Isoforms
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Protein Kinase Inhibitors
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RNA, Messenger
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Receptors, Cell Surface
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Tropomyosin
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beta Catenin
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Cyclin D1
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Genistein