MEK INHIBITION AUGMENTS RAF ACTIVITY, BUT HAS VARIABLE EFFECTS ON MITOGENESIS, IN VASCULAR SMOOTH-MUSCLE CELLS

Proteins comprising the mitogen-activated protein (MAP) kinase signali ng cascade are activated by a variety of growth factors, but the preci se role of this series of kinase reactions, especially Raf kinase and MAP kinase kinase (MEK), in vascular smooth muscle (VSM) cell mitogene sis is not known. In this study, a specific and selective inhibitor of MEK, PD-98059, was used to examine the role of MEK in DNA synthesis a nd Raf-l activity in VSM cells stimulated with serum as well as with g rowth factors encompassing both tyrosine kinase and G protein-coupled receptor classes. Although significant increases in DNA synthesis are seen after stimulation of VSM cells with either 10% serum, platelet-de rived growth factor (PDGF)-BB, or alpha-thrombin, preincubation of the cells with 50 mu M PD-98059 for 1 h inhibits stimulation by PDGF and thrombin, but not by serum. There is a dose-dependent inhibition of th e mitogenic effect by PD-98059 in all cases; these results are not aff ected when PD-98059 is added at times ranging from 4 h before to 2 h a fter growth factor addition (times at which PD-98059 exerts its inhibi tory effect). In the presence of PD-98059, stimulated MAP kinase activ ity is attenuated when growth factors are added between 10 min and 4 h , times which correspond to both early and sustained phases of MAP kin ase activity. In addition, Raf-l activity is markedly increased by inc ubation of the cells with PD-98059, despite attenuation of hyperphosph orylation of this kinase. Thus growth factors coupled to both tyrosine kinase and G protein receptors require components of the MAP kinase c ascade (MEK and/or MAP kinase) for VSM cell mitogenesis, whereas serum is capable of stimulatory effects in the absence of active MEK and MA P kinase. Furthermore, there exists a functional feedback stimulatory effect of inhibited MEK on its upstream activator Raf-l in the case of serum as well as growth factors coupled to tyrosine kinase and G prot ein receptors.