Requirement for Ras/Rac1-mediated p38 and c-Jun N-terminal kinase signaling in Stat3 transcriptional activity induced by the Src oncoprotein.

Signal transducers and activators of transcription (STATs) are transcription factors that mediate normal biologic responses to cytokines and growth factors. However, abnormal activation of certain STAT family members, including Stat3, is increasingly associated with oncogenesis. In fibroblasts expressing the Src oncoprotein, activation of Stat3 induces specific gene expression and is required for cell transformation. Although the Src tyrosine kinase induces constitutive Stat3 phosphorylation on tyrosine, activation of Stat3-mediated gene regulation requires both tyrosine and serine phosphorylation of Stat3. We investigated the signaling pathways underlying the constitutive Stat3 activation in Src oncogenesis. Expression of Ras or Rac1 dominant negative protein blocks Stat3-mediated gene regulation induced by Src in a manner consistent with dependence on p38 and c-Jun N-terminal kinase (JNK). Both of these serine/threonine kinases and Stat3 serine phosphorylation are constitutively induced in Src-transformed fibroblasts. Furthermore, inhibition of p38 and JNK activities suppresses constitutive Stat3 serine phosphorylation and Stat3-mediated gene regulation. In vitro kinase assays with purified full-length Stat3 as the substrate show that both JNK and p38 can phosphorylate Stat3 on serine. Moreover, inhibition of p38 activity and thus of Stat3 serine phosphorylation results in suppression of transformation by v-Src but not v-Ras, consistent with a requirement for Stat3 serine phosphorylation in Src transformation. Our results demonstrate that Ras- and Rac1-mediated p38 and JNK signals are required for Stat3 transcriptional activity induced by the Src oncoprotein. These findings delineate a network of tyrosine and serine/threonine kinase signaling pathways that converge on Stat3 in the context of oncogenesis.

The effect of nitrogen dioxide exposure on the release of surfactant isolated from neonatal rabbit type II pneumocytes in culture.

Nitrogen dioxide (NO2) is a well-known environmental air toxin, produced from a variety of sources, including cigarette smoke. Because of the growing knowledge of the harmful effects of passive smoking on children, we decided to study the effect of NO2 exposure on the release of surfactant from isolated neonatal type II pulmonary epithelial cells. After isolation from 1 to 4 day old rabbits, type II epithelial cells were allowed to adhere for 18 hours, washed, media changed, and were exposed to either 5% CO2 in room air or NO2, 5 ppm, for 2 hours (all results mean +/- sd; comparisons, paired t-test). There was no difference in cell number or viability prior to exposure. Cells exposed to NO2 had an increase in LDH release [LDH activity in media/(LDH in media+cells) x 100], air 12.6 +/- 2.2%, NO2 21.7 +/- 3.7%, (p < 0.05). NO2-exposed cells also had an increase in total phospholipid (microgram/cell culture dish) in media compared to air exposed, air 170.13 +/- 7.54, NO2 195.15 +/- 11.2, (p < 0.05). 3H-choline incorporation as a precursor to disaturated phosphatidylcholine (DSPC) was also conducted during exposure to either air or NO2. Incorporation of 3H-choline into surfactant lipid was increased in media from cells after NO2 exposure compared to air, 58.23 +/- 15.16 air, 76.81 +/- 19.86 NO2 (cpm/microgram protein; p < 0.05). These results show that 2 hours of 5 ppm NO2 exposure is associated with an increase in release of surfactant from neonatal type II cells in culture.

Vitamin E distribution and modulation of the physical state and function of pulmonary endothelial cell membranes.

Vitamin E, a dietary antioxidant, is thought to incorporate into the lipid bilayer of biological membranes. We evaluated the lipid composition and distribution of [3H]-vitamin E in various membranes of pulmonary endothelial cells and determined whether vitamin E incorporation caused alterations in membrane structure and function in these cells. Following 6-, 12-, 18-, 24-, and 48-h incubation periods, vitamin E incorporation values were 3.0, 5.7, 6.9, 7.2, and 6.8 nmol/mg protein or 3.8, 7.3, 8.8, 9.2, and 8.7 nmol/mg phospholipid in mitochondrial membranes and 2.0, 4.4, 5.2, 5.3, and 5.0 nmol/mg protein or 3.5, 7.7, 9.1, 9.3, and 8.8 nmol/mg phospholipid in microsomal membranes, respectively. Vitamin E incorporation into the plasma membranes was greater than in mitochondrial and microsomal membranes after 12-, 24-, and 48-h incubations (18.9, 20.8, and 19.6 nmol/mg protein, respectively [P less than .001] versus mitochondria and microsomes or 12.2, 13.4, and 12.6 nmol/mg phospholipid, respectively [P less than .05] versus mitochondria and microsomes). The total phospholipid content, as well as the unsaturation index of the fatty acid content of these membranes, were in the same order, (i.e., plasma membrane greater than mitochondrial membranes and microsomal membranes). The physical state of the intact plasma membrane and the mitochondrial and microsomal membranes were measured by monitoring fluorescence anisotropies (rs) of the molecular probes, diphenylhexatriene (DPH) and trimethylamino-DPH (TMA-DPH). Vitamin E incorporation caused significant increases in rs for DPH (P less than .01) and TMA-DPH (P less than .01) in all three membranes compared to controls. Similar increases in rs values for DPH and TMA-DPH were observed in lipid vesicles prepared from these membranes. Following vitamin E incorporation, 5-hydroxytryptamine (5-HT) transport was measured as an index of plasma membrane function. Vitamin E incorporation resulted in an 18% reduction (P less than .05) in 5-HT uptake. These results indicate that vitamin E was distributed nonuniformly in endothelial cell membranes but resulted in comparable decreases in fluidity in all three membranes. In addition to its role as an antioxidant, vitamin E may alter the membrane physical state and modulate a variety of endothelial cell functions, including 5-HT transport.

Involvement of lipoxygenase in lysophosphatidic acid-stimulated hydrogen peroxide release in human HaCaT keratinocytes.

Although it is now recognized that low levels of reactive oxygen species (ROS) are required for the mitogenic response, mitogen-induced signalling pathways that regulate ROS generation in non-phagocytic cells remain largely uncharacterized. Using a real-time assay for measuring hydrogen peroxide (H(2)O(2)) formation, we analysed H(2)O(2) release in human HaCaT keratinocytes in response to lysophosphatidic acid (LPA), a mitogen for keratinocytes. LPA rapidly increased H(2)O(2) release in HaCaT cells. Unlike LPA-induced mitogen-activated protein (MAP) kinase activation, LPA-stimulated H(2)O(2) release was independent of the tyrosine kinase activity of the epidermal growth factor (EGF) receptor. Calcium chelators, phospholipase A(2) inhibitors, and lipoxygenase inhibitors effectively blocked LPA-stimulated H(2)O(2) release, whereas cyclooxygenase inhibitors were without effect. Addition of 5-lipoxygenase products 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and leukotriene B(4), but not 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene C(4), restored LPA-stimulated H(2)O(2) release in cells treated with the lipoxygenase inhibitors nordihydroguaiaretic acid and Zileuton. These results suggest that the lipoxygenase products 5-HPETE and leukotriene B(4) are required for LPA-stimulated H(2)O(2) release in HaCaT cells.

Suppression of fibroblast cell cycle progression in G1 phase by N-acetylcysteine.

The antioxidant N-acetyl-L-cysteine (NAC) has been increasingly used as an experimental tool to assess involvement of reactive oxygen species in cell signaling and is being evaluated as a preventive and therapeutic agent for cancer and pulmonary diseases related to inflammation and oxidative stress. However, a detailed characterization of the effect of NAC on cell cycle progression has not been reported. In the present study, modulation of cell cycle progression by NAC was analyzed in mouse fibroblast NIH3T3 cells grown in 10% fetal bovine serum. Complete inhibition of NIH3T3 cell proliferation was obtained with 20 mM NAC. Inhibition of cell proliferation by NAC (at or below 20 mM) was not due to cell death, and the antiproliferative effect of NAC was reversible. Flow cytometric analysis of cell cycle phase distribution indicated that NAC blocked the cell cycle in the G1 phase. Consistent with this observation, NAC inhibited DNA synthesis. After releasing the G1-block by NAC, S phase re-entry occurred between 8 and 12 h, suggesting that NAC blocked the cell cycle in early to mid-G1 phase. NAC prevented activation of mitogen-activated protein (MAP) kinases p42MAPK and p44MAPK and inhibited expression of cyclin D1, but had no effect on the levels of proliferating cell nuclear antigen. Incubation of cells with PD98059, a specific inhibitor of MAP kinase kinase 1, partially arrested the cell cycle in the G1 phase. These results indicate that the antiproliferative effect of NAC is linked in part to inhibition of the MAP kinase pathway.