The analysis of genome evolution in the MHC class III region and function of extracellular matrix tenascin-X

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To identify proteins that interact with extracellular matrix tenascin-X (TNX) in the extracellular environment, we searched for TNX-binding proteins using a yeast two-hybrid system. We used mouse TNX-specific fibronectin type III repeats (mTNX/FNIIIィイD213-25ィエD2) as a bait. We found that vascular endothelial growth factor B (VEGF-B) binds to mTNX/FNIIIィイD213-25ィエD2. This interaction was confirmed by pull-down assays and coimmunoprecipitation assays. mTNX/FNIIIィイD213 -25ィエD2 specifically interacted with both altenative splice isoforms, VEGF-BィイD2186ィエD2 and VEGF-BィイD2167ィエD2, but not with other family members. Likewise, the full-length TNX could also bind to both VEGF-B isoforms. The minimal region of TNX that interacts with VEGF-B was mapped to the FNIII repeats (mTNX/FNIIIィイD213-25ィエD2) that were used for a bait but not to other characteristic domains of TNX. The TNX-binding site of VECJF-B was located in the N-terminal 115-amino-acids region. mTNX/FNIIIィイD213-25ィエD2 did not prevent t … More he interaction of VEGF-B with VEGFR-1 (VEGF receptor 1), and VEGF-B could simultaneously bind to both mTNX/FNIIIィイD213-25ィエD2 and VEGFR-1. A conditioned medium from transfected 293T cells coexpressing full-length TNX and VEGF-B can enhance DNA synthesis in ECV304 cells. These findings suggest that TNX is responsible for proliferation of the cells.To elucidate the molecular basis of the TNX gene expression, the promoter region of the mouse TNX gene (mTnx) has been characterized. The two adjacent transcription initiation sites were identified at 68 and 67 bp upstream of the previously known 5'- untranslated exon. Transient transfection of L and 293T cells with 5'-deletion constructs of the promoter region linked to the luciferase reporter revealed that the region (-141 to -136) containing a transcription factor Sp1-binding element contributes to the expression of mTnx. Site-directed mutagenesis of the Sp1-binding region confirmed this result. Electrophoretic mobility shift analysis using nuclear extracts obtained from the cells demonstrated that a distinct Sp1-DNA complex is formed at the element. Our results show that Sp1 plays a critical role in the gene expression of mTnx. Less