Transcriptional regulation of the murine urokinase-type plasminogen activator gene in skeletal myoblasts

We have previously shown that urokinase-type plasminogen activator (uPA) is highly expressed in murine C2C12 myoblasts and that antibodies against uPA are able to block both myoblast fusion and differentiation. Here we show t he characterization of cis-acting elements in the mouse uPA promoter in vit ro which are involved in uPA gene expression in C2C12 myoblast cells. DNase I hypersensitive (HS) site analysis revealed the presence of three HS site s in myoblasts. Deletion analysis of stably transfected uPA-promoter constr ucts revealed that at least two of the three HS sites accounted for the hig h transcriptional expression in C2C12 cells. One was located at -2.4 kb and corresponded to a known PEA3/AP1(A) element and the other one was located at -4.9 kb and contained a CArG box and a CRE element. So far, no regulator y function had been assigned to this CRE/CArG element. Both HS sites alone were able to activate transcription of a heterologous promoter and showed a cooperative effect when placed together. Electrophoretic mobility-shift as says using myoblast nuclear extracts and specific antibodies demonstrated t hat cJun, JunD and ATF2 bound to the PEA3/AP1(A) element, whereas the CRE/C ArG element bound SRF. Altogether, these results suggest that high uPA expr ession in myoblasts is dependent on the cooperation of two regulatory sites in the uPA promoter.