A-MYB IS EXPRESSED IN BOVINE VASCULAR SMOOTH-MUSCLE CELLS DURING THE LATE G(1)-TO-S-PHASE-TRANSITION AND COOPERATES WITH C-MYC TO MEDIATE PROGRESSION TO S-PHASE

The Myb family of transcription factors is defined by homology within the DNA binding domain and includes c-Myb, A-Myb, and B-Myb. The prote in products of the myb genes all bind the Myb-binding site (RIBS) [YG( A/G)C(A/C/G)GTT(G/A)]. A-myb has been found to display a limited patte rn of expression. Here we report that bovine aortic smooth muscle cell s (SMCs) express A-myb. Sequence analysis of isolated bovine A-myb cDN A clones spanning the entire coding region indicated extensive homolog y with the human gene, including the putative transactivation domain. Expression of A-myb was cell cycle dependent; levels of A-myb RNA incr eased in the late G(1)-to-S phase transition following serum stimulati on of serum-deprived quiescent SMC cultures and peaked in S phase. Nuc lear run-on analysis revealed that an increased rate of transcription can account for most of the increase in A-myb RNA levels. Treatment of SMC cultures with 5,6-dichlorobenzimidazole riboside, a selective inh ibitor of RNA polymerase II, indicated an approximate 4-h half-life fo r A-myb mRNA during the S phase of the cell cycle. Expression of A-myb by SMCs was stimulated by basic fibroblast growth factor, in a cell d ensity-dependent fashion. Cotransfection of a human A-myb expression v ector activated a multimerized RIBS element-driven reporter construct approximately 30-fold in SMCs. The activity of c-myb and c-myc promote rs, which both contain multiple RIBS elements, were similarly transact ivated, approximately 30- and 50-fold, respectively, upon cotransfecti on with human A-myb. Lastly, A-myb RNA levels could be increased by a combination of phorbol ester plus insulin-like growth factor 1. To tes t the role of myb family members in progression through the cell cycle , we comicroinjected c-myc and myb expression vectors into serum-depri ved quiescent SMCs. The combination of c-myc and either A-myb or c-myb but not B-myb synergistically led to entry into S phase, whereas micr oinjection of any vector alone had little effect on S phase entry. Thu s, these results suggest that A-myb is a potent transactivator in bovi ne SMCs and that its expression induces progression into S phase of th e cell cycle.