The roles of phosphatidylinositol 3-kinase and protein kinase Czeta for thrombopoietin-induced mitogen-activated protein kinase activation in primary murine megakaryocytes.

Thrombopoietin (TPO) stimulates a network of intracellular signaling pathways that displays extensive cross-talk. We have demonstrated previously that the ERK/mitogen-activated protein kinase pathway is important for TPO-induced endomitosis in primary megakaryocytes (MKs). One known pathway by which TPO induces ERK activation is through the association of Shc with the penultimate phosphotyrosine within the TPO receptor, Mpl. However, several investigators found that the membrane-proximal half of the cytoplasmic domain of Mpl is sufficient to activate ERK in vitro and support base-line megakaryopoiesis in vivo. Using BaF3 cells expressing a truncated Mpl (T69Mpl) as a tool to identify non-Shc/Ras-dependent signaling pathways, we describe here novel mechanisms of TPO-induced ERK activation mediated, in part, by phosphoinositide 3-kinase (PI3K). Similar to cells expressing full-length receptor, PI3K was activated by its incorporation into a complex with IRS2 or Gab2. Furthermore, the MEK-phosphorylating activity of protein kinase Czeta (PKCzeta) was also enhanced after TPO stimulation of T69Mpl, contributing to ERK activity. PKCzeta and PI3K also contribute to TPO-induced ERK activation in MKs, confirming their physiological relevance. Like in BaF3 cells, a TPO-induced signaling complex containing p85PI3K is detectable in MKs expressing T61Mpl and is probably responsible for PI3K activation. These data demonstrate a novel role of PI3K and PKCzeta in steady-state megakaryopoiesis.

Arsenite inhibits Ras-dependent activation of ERK but activates ERK in the presence of oncogenic Ras in baboon vascular smooth muscle cells.

Exposure to arsenical compounds enhances the risk of atherosclerosis. The reason is unknown but it might be because an effect of arsenite (As3+) on plaque smooth muscle cells (SMCs) activation of extracellular signal-regulated kinase (ERK), a crucial mediator of SMC function. We found that arsenite inhibits the activation of ERK by platelet-derived growth factor-BB (PDGF-BB). This inhibitory effect depends on the time of arsenite exposure, is reversible, and is attenuated by preincubation of SMCs with the antioxidant N-acetyl-cysteine. These observations are consistent with the assumption that oxidative stress is involved. The blockade of ERK by arsenite may be mediated by an inhibition of Ras as arsenite prevents GTP-loading of Ras in response to PDGF-BB. Moreover, the Ras blockade by arsenite is not specific for PDGF-BB because it was also observed following stimulation of SMCs with EGF. To address the role of Ras, we expressed constitutively active, GTP-bound Ha-Ras (V12Ras). Unexpectedly, in V12Ras expressing-SMCs, arsenite stimulates ERK, but still decreases ERK activity in the presence of PDGF-BB. Our data suggest that arsenite inhibits the Ras/ERK pathway in SMCs, and that arsenite may activate ERK in Ras-transformed cells by mechanisms different from those employed by growth factors.

G-protein expression and intimal hyperplasia after arterial injury: a role for Galpha(i) proteins.

PURPOSE: Guanine nucleotide binding protein (G-protein) coupled receptors are involved in smooth muscle cell proliferation, but the role of G-proteins in arterial intimal hyperplasia has not been defined. This study examines the expression of G-proteins in the developing intimal hyperplasia after balloon injury of the rat carotid artery and specifically tests the hypothesis that the pertussis toxin sensitive G(i) G-protein subunit plays a role in the initiation of intimal hyperplasia. METHODS: In vitro responses to serum stimulation (10% fetal bovine serum) were examined in the presence and absence of pertussis toxin (PTx). After a standard balloon injury in male Sprague-Dawley rats, the expression of G-protein subunits (alpha(o), alpha(i), alpha(q), alpha(s), and betagamma) was determined by means of Western blotting in the first 28 days. Thereafter, a second set of animals was allocated to control and PTx-treated (a Galpha(i) inhibitor; 500 ng/mL in an externally applied 30% pluronic gel) groups. Smooth muscle cell proliferation was estimated by means of thymidine analogue 5-bromo-2' deoxyuridine incorporation 2 days after injury, and vessel dimensions were determined by means of videomorphometry 14 days after injury. RESULTS: There was inhibition of DNA synthesis and smooth muscle cell proliferation in response to serum with an IC(50) of 100 ng/mL. Three days after balloon injury, there was an increase in Galpha(i3) expression, which decreased at days 7, 14, and 28, compared with the uninjured carotid. Galpha(q) expression increased in a time-dependent manner. There was a marked time-dependent increase in Gbetagamma in the 28 days. Galpha(i2) and Galpha(s) isoforms (45 and 52 kDa) did not change significantly with time. There was no major change in Galpha(i1) and Galpha(o) in the study period. At 14 days, PTx treatment reduced intimal hyperplasia by 52% (63 +/- 4 microm vs. 30 +/- 5 microm, control vs. PTx; P <.001). Medial smooth muscle cell proliferation at day 2 was decreased in the PTx group, compared with that in the gel-coated group (15% +/- 2% and 26% +/- 3%; P = .02). CONCLUSION: After balloon injury, there is a time-dependent increase in G-protein expression, which is subunit specific. Activation of PTx sensitive G-proteins (Galpha(i)) is involved during the initiation of intimal hyperplasia after arterial injury, and their inhibition results in a decrease in early medial cell proliferation. This acute interruption of G(i) signaling produces a long-term decrease in intimal hyperplasia.

Raf-1 is activated by the p38 mitogen-activated protein kinase inhibitor, SB203580.

SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imi dazole) is widely used as a specific inhibitor of p38 mitogen-activated protein kinase (MAPK). Here, we report that SB203580 activates the serine/threonine kinase Raf-1 in quiescent smooth muscle cells in a dose-dependent fashion. The concentrations of SB203580 required lie above those necessary to inhibit p38 MAPK and we were unable to detect basal levels of active p38 MAPK. SB203580 does not directly activate Raf-1 in vitro, and fails to activate Ras, MEK, and ERK in intact cells. In vitro, however, SB203580-stimulated Raf-1 activates MEK1 in a coupled assay. We conclude that activation of Raf-1 by SB203580 is not mediated by an inhibition of p38 MAPK, is Ras-independent, and is uncoupled from MEK/ERK signaling.