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MEK inhibition augments Raf activity, but has variable effects on mitogenesis, in vascular smooth muscle cells
R H Weiss1, E A Maga, A Ramirez
1Division of Nephrology, Department of Internal Medicine, University of California, Davis 95616, USA.
Abstract:
Proteins comprising the mitogen-activated protein (MAP) kinase signaling cascade are activated by a variety of growth factors, but the precise role of this series of kinase reactions, especially Raf kinase and MAP kinase kinase (MEK), in vascular smooth muscle (VSM) cell mitogenesis 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 and Raf-1 activity in VSM cells stimulated with serum as well as with growth 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-derived growth factor (PDGF)-BB, or alpha-thrombin, preincubation of the cells with 50 microM PD-98059 for 1 h inhibits stimulation by PDGF and thrombin, but not by serum. There is a dose-dependent inhibition of the mitogenic effect by PD-98059 in all cases; these results are not affected when PD-98059 is added at times ranging from 4 h before to 2 h after growth factor addition (times at which PD-98059 exerts its inhibitory effect). In the presence of PD-98059, stimulated MAP kinase activity is attenuated when growth factors are added between 10 min and 4 h, times which correspond to both early and sustained phases of MAP kinase activity. In addition, Raf-1 activity is markedly increased by incubation of the cells with PD-98059,despite attenuation of hyperphosphorylation of this kinase. Thus growth factors coupled to both tyrosine kinase and G protein receptors require components of the MAP kinase cascade (MEK and/or MAP kinase) for VSM cell mitogenesis, whereas serum is capable of stimulatory effects in the absence of active MEK and MAP kinase. Furthermore, there exists a functional feedback stimulatory effect of inhibited MEK on its upstream activator Raf-1 in the case of serum as well as growth factors coupled to tyrosine kinase and G protein receptors.
Insights
MAP kinase kinase (MEK) and MAP kinase are crucial for vascular smooth muscle cell growth stimulated by growth factors, but not by serum. Inhibiting MEK paradoxically increases Raf-1 activity, suggesting a feedback loop.
Area of Science:
- Molecular Biology
- Cell Signaling
- Vascular Biology
Background:
- The mitogen-activated protein (MAP) kinase pathway regulates cell growth.
- The specific role of MAP kinase kinase (MEK) and Raf kinase in vascular smooth muscle (VSM) cell proliferation is not fully understood.
- Growth factors activate MAP kinase signaling, but their precise downstream effects in VSM cells require further investigation.
Purpose of the Study:
- To investigate the role of MEK in VSM cell DNA synthesis and proliferation.
- To determine the involvement of MEK and MAP kinase in growth factor-induced VSM cell mitogenesis.
- To examine the effect of MEK inhibition on Raf-1 activity in VSM cells.
Main Methods:
- Utilized PD-98059, a selective MEK inhibitor, to study VSM cell responses.
- Stimulated VSM cells with serum, platelet-derived growth factor (PDGF)-BB, and alpha-thrombin.
- Assessed DNA synthesis, MAP kinase activity, and Raf-1 activity under various treatment conditions.
Main Results:
- PD-98059 inhibited PDGF-BB and alpha-thrombin-induced DNA synthesis but not serum-induced DNA synthesis.
- MEK inhibition attenuated MAP kinase activity in response to growth factors.
- Incubation with PD-98059 increased Raf-1 activity, despite reduced hyperphosphorylation.
Conclusions:
- MEK and MAP kinase are essential for growth factor-mediated VSM cell proliferation.
- Serum-induced VSM cell proliferation can occur independently of active MEK and MAP kinase.
- MEK inhibition triggers a feedback mechanism that stimulates upstream Raf-1 activity.