ABSTRACT
[This corrects the article on p. 395 in vol. 39.].
ABSTRACT
Sox11 deletion mice are known to exhibit developmental defects of craniofacial skeletal malformations, asplenia, and hypoplasia of the lung, stomach, and pancreas. Despite the importance of Sox11 in the developing skeleton, the role of Sox11 in osteogenesis has not been studied yet. In this study, we identified that Sox11 is an important transcription factor for regulating the proliferation and survival of osteoblast precursor cells as well as the self-renewal potency of mesenchymal progenitor cells via up-regulation of Tead2. Furthermore, Sox11 also plays an important role in the segregation of functional osteoblast lineage progenitors from osteochondrogenic progenitors. Facilitation of osteoblast differentiation from mesenchymal cells was achieved by enhanced expression of the osteoblast lineage specific transcription factors Runx2 and Osterix. Morpholino-targeted disruption of Sox11 in zebrafish impaired organogenesis, including the bones, which were under mineralized. These results indicated that Sox11 plays a crucial role in the proliferation and survival of mesenchymal and osteoblast precursors by Tead2, and osteogenic differentiation by regulating Runx2 and Osterix.
Subject(s)
Cell Differentiation/physiology , Cell Proliferation , Mesenchymal Stem Cells/metabolism , Osteoblasts/metabolism , SOXC Transcription Factors/metabolism , Animals , Cell Line , Cell Survival/physiology , Core Binding Factor Alpha 1 Subunit/genetics , Core Binding Factor Alpha 1 Subunit/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Humans , Mesenchymal Stem Cells/cytology , Mice , Osteoblasts/cytology , SOXC Transcription Factors/genetics , Sp7 Transcription Factor , TEA Domain Transcription Factors , Transcription Factors/genetics , Transcription Factors/metabolism , Zebrafish/embryology , Zebrafish/geneticsABSTRACT
Hoxc8 is a homeobox gene family member, which is essential for growth and differentiation. Mgl1, a mouse homologue of the Drosophila tumor suppressor gene lgl, was previously identified as a possible target of Hoxc8. However, the biological effects and underlying molecular mechanism of Hoxc8 regulation on Mgl1 has not been fully established. The endogenous expression patterns of Hoxc8 were inversely correlated with those of Mgl1 in different types of cells and tissues. Here we showed that Hoxc8 overexpression downregulated the Mgl1 mRNA expression. Characterization of the ~2 kb Mgl1 promoter region revealed that the upstream sequence contains several putative Hox core binding sites and chromatin immunoprecipitation assay confirmed that Hoxc8 directly binds to the 5' upstream region of Mgl1. The promoter activity of this region was diminished by Hoxc8 expression but resumed by knockdown of Hoxc8 using siRNA against Hoxc8. Functional study of Mgl1 in C3H10T1/2 cells revealed a significant reduction in cell adhesion upon expression of Hoxc8. Taken together, our data suggest that Hoxc8 downregulates Mgl1 expression via direct binding to the promoter region, which in turn reduces cell adhesion and concomitant cell migration.
Subject(s)
Asialoglycoproteins , Fibroblasts/metabolism , Gene Expression Regulation, Developmental , Homeodomain Proteins/metabolism , Lectins, C-Type , Membrane Proteins , Animals , Asialoglycoproteins/chemistry , Asialoglycoproteins/genetics , Asialoglycoproteins/metabolism , Base Sequence , Cell Adhesion/drug effects , Cell Adhesion/genetics , Cell Movement/drug effects , Cell Movement/genetics , Cells, Cultured , Chromatin Immunoprecipitation , Female , Fibroblasts/cytology , Gene Silencing/drug effects , Homeodomain Proteins/antagonists & inhibitors , Homeodomain Proteins/genetics , Lectins, C-Type/chemistry , Lectins, C-Type/genetics , Lectins, C-Type/metabolism , Male , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Inbred ICR , Molecular Sequence Data , Promoter Regions, Genetic , Protein Binding , RNA, Small Interfering/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Transcription, GeneticABSTRACT
Protein transduction domains (PTDs) have been shown to cross the biological cell membranes efficiently through a receptor and energy independent mechanism. Because of its ease in membrane transducing ability, PTDs could be used as a gene delivery vector. Since we already have shown that purified Hoxc8 homeoprotein has the ability to cross the cellular membrane, we analyzed the possibility of the third helix of the Hoxc8 homeodomain as a useful gene delivery vector. For that purpose, a 16-aa long synthetic oligopeptide Hoxc8 Protein Transduction Domain (HPTD) was chemically synthesized and then tested to see whether the HPTD could form a complex with DNA or not. Gel retardation analysis revealed that the HPTD interacts with plasmid DNA efficiently but failed to transfer the DNA into the cells. However, HPTD can enhance the efficiency of gene transfer in combination with Lipofectamine which doubled the gene transfer rate into COS-7 cells compared with the DNA/Lipofectamine control. An MTT assay indicated that the amount of HPTD used in the complex for the transfection did not show any cytotoxicty in COS-7 cells. The TEM studies showed compact particle formation in the presence of HPTD. These results indicate that the HPTD could be a good candidate adjuvant molecule to enhance the gene transfer efficiency of Lipofectamine in eukaryotic cells.
