Objective: Hex (hematopoietically expressed homeobox), a member of homeobox family of transcription factors, has been implicated in the vascular development because of its expression in hemangioblast, a hypothetical stem cell that gives rise to both angioblasts and hematopoietic lineages. In the present study, we examined the role of Hex in the differentiation of vascular smooth muscle cells.
Methods and results: We constructed adenovirus expressing Hex, to which we refer to as AxCA/Hex, and transduced murine embryonic fibroblasts, 10T1/2 cells. Northern blot analyses showed that Hex increased the mRNA levels of smooth muscle alpha-actin and SM22alpha but not of calponin and smooth muscle myosin heavy chain. Transient transfection assays showed that Hex activates the transcription from the SM22alpha promoter in a CArG box-dependent manner. Electrophoretic mobility shift assays demonstrate that Hex is not able to bind to CArG box, but binding of serum responsive factor (SRF) to CArG box is enhanced in AxCA/Hex-transduced cells. Recombinant Hex protein produced by in vitro translation system augmented the binding activity of SRF to CArG box. Immunoprecipitation experiments revealed the physical association between Hex and SRF.
Conclusions: Hex induces transcription of the SM22alpha gene by facilitating the interaction between SRF and its cognate binding site in pluripotent embryonic fibroblasts. This study demonstrates that Hex, a hematopoietically expressed homeobox protein, induces transcription of the SM22alpha gene by facilitating the interaction between SRF and its cognate binding site in embryonic fibroblasts. These findings will provide the clue for understanding the mechanisms by which bone marrow-derived SMC precursor cells undergo differentiation.