Hydrogen peroxide-induced c-fos expression is mediated by arachidonic acid release: role of protein kinase C

We found previously that stimulation of c-fos and c-myc mRNA expression are early events in hydrogen peroxide-induced growth in rat aortic smooth muscle (RASM) cells. In the present study, we investigated the role of phospholipase A2 (PLA2) and protein kinase C (PKC) in mediating hydrogen peroxide-induced c-fos mRNA expression in RASM cells. Mepacrine and p-bromophenacylbromide, potent inhibitors of PLA2 activity, blocked hydrogen peroxide-induced c-fos mRNA expression. Arachidonic acid, a product of PLA2 activity, stimulated the expression of c-fos mRNA with a time course similar to that of hydrogen peroxide. PKC down-regulation attenuated both hydrogen peroxide and arachidonic acid-induced c-fos mRNA expression by 50%. Nordihydroguaiaretic acid (a lipoxygenase-cytochrome P450 monooxygenase inhibitor) significantly inhibited both hydrogen peroxide and arachidonic acid-induced c-fos mRNA expression, whereas indomethacin (a cyclooxygenase inhibitor) had no effect. Together, these findings indicate that 1) hydrogen peroxide-induced c-fos mRNA expression is mediated by PLA2-dependent arachidonic acid release, 2) both PKC-dependent and independent mechanisms are involved in hydrogen peroxide-induced expression of c-fos mRNA and 3) arachidonic acid metabolism via the lipoxygenase-cytochrome P450 monooxygenase pathway appears to be required for hydrogen peroxide-induced expression of c-fos mRNA.     

Intracellular signaling events associated with the induction of DNA synthesis in human B lymphocytes. II. Different pathways triggered by IL-2 and IL-4

In attempts to detect associations between early signaling events triggered by interleukins and the induction of DNA synthesis, inhibitors of various second messenger pathways were tested for their effects on IL-2- and IL-4-elicited mitogenesis in preactivated human B lymphocytes. Inhibitors of phosphoinositidase C and of InsP3-induced calcium release suppressed IL-4- but not IL-2-mediated proliferation. The response to both lymphokines was also impaired by an inhibitor of the calcium/calmodulin complex and was modulated by variations of the [Ca2+]i. PKC inhibitors and PK-C depletion did not significantly alter the response to IL-2 and IL-4. The response to IL-2, but not to IL-4, was inhibited by cAMP analogues or by agents that raise cAMP. In contrast, IL-4, but not IL-2, stimulated cAMP accumulation in activated B cells. Taken together, these observations indicate that IL-2 and IL-4 use different signaling pathways to induce the G1-->S transition in these cells and suggest that the IL-4 inhibition of the B cell response to IL-2 may result from its effect on cAMP generation.     

Mechanical stretch induces hypertrophic responses in cardiac myocytes of angiotensin II type 1a receptor knockout mice

Many lines of evidence have suggested that angiotensin II (AngII) plays an important role in the development of cardiac hypertrophy through AngII type 1 receptor (AT1). To determine whether AngII is indispensable for the development of mechanical stress-induced cardiac hypertrophy, we examined the activity of mitogen-activated protein kinase (MAPK) family and the expression of the c-fos gene as hypertrophic responses after stretching cultured cardiac myocytes of AT1a knockout (KO) mice. When cardiac myocytes were stretched by 20% for 10 min, extracellular signal-regulated protein kinases (ERKs) were strongly activated in KO cardiomyocytes as well as wild type (WT) myocytes. Both basal and stimulated levels of ERKs were higher in cardiomyocytes of KO mice than in those of WT mice. Activation of another member of the MAPK family, p38(MAPK), and expression of the c-fos gene were also induced by stretching cardiac myocytes of both types of mice. An AT1 antagonist attenuated stretch-induced activation of ERKs in WT cardiomyocytes but not in KO cardiomyocytes. Down-regulation of protein kinase C inhibited stretch-induced ERK activation in WT cardiomyocytes, whereas a broad spectrum tyrosine kinase inhibitor (genistein) and selective inhibitors of epidermal growth factor receptor (tyrphostin, AG1478, and B42) suppressed stretch-induced activation of ERKs in KO cardiac myocytes. Epidermal growth factor receptor was phosphorylated at tyrosine residues by stretching cardiac myocytes of KO mice. These results suggest that mechanical stretch could evoke hypertrophic responses in cardiac myocytes that lack the AT1 signaling pathway possibly through tyrosine kinase activation.     

Differential coupling of alpha1-, alpha2-, and beta-adrenergic receptors to mitogen-activated protein kinase pathways and differentiation in transfected PC12 cells

Three adrenergic receptor families that selectively activate three different G proteins (alpha1/Gq/11, alpha2/Gi, and beta/Gs) were used to study mitogen-activated protein kinase (MAPK) activation and differentiation in PC12 cells. PC12 cells were stably transfected with alpha1A-, alpha2A-, or beta1-adrenergic receptors (ARs) in an inducible expression vector, and subclones were characterized. Norepinephrine stimulated inositol phosphate formation in alpha1A-transfected cells, inhibited cyclic adenosine 3'5'-monophosphate (cAMP) formation in alpha2A-transfected cells, and stimulated cAMP formation in beta1-transfected cells. Nerve growth factor activated extracellular signal-regulated kinases (ERKs) in all cell lines; however, norepinephrine activated ERKs only in alpha1A- and beta1-transfected cells but not in alpha2A-transfected cells. Norepinephrine also activated c-Jun NH2-terminal kinase and p38 MAPK in alpha1A-transfected cells but not in beta1- or alpha2A-transfected cells. Norepinephrine caused differentiation of PC12 cells expressing alpha1A-ARs but not those expressing beta1- or alpha2A-ARs. However, norepinephrine acted synergistically with nerve growth factor in promoting differentiation of cells expressing beta1-ARs. Whereas ERKs are activated by Gi- but not Gs-linked receptors in many fibroblastic cell lines, we observed the opposite in PC12 cells. The results show that activation of the different G protein signaling pathways has different effects on MAPKs and differentiation in PC12 cells, with Gq signaling pathways activating all three major MAPK pathways.     

Peptide hormone and growth factor regulation of nuclear proto-oncogenes and specific functions in adrenal cells

Among the large number of immediate early genes, nuclear proto-oncogenes of the Fos and Jun families, have been postulated to be involved in the long-term effects of several growth factors on cell differentiation and/or multiplication. Since adrenal cell differentiated functions appear to be regulated by specific hormones and growth factors, the effects of these factors on proto-oncogene mRNA levels were analysed in bovine adrenal fasciculata cells (BAC) in culture. Corticotropin (ACTH) and insulin-like growth factor I increased c-fos and jun-B mRNA, but had no effect on c-jun mRNA and these early changes were associated with a later increase in BAC specific function [ACTH receptors, cytochrome P450 17 alpha) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD)] and an enhanced steroidogenic responsiveness to both ACTH and angiotensin-II (A-II). On the other hand, A-II increased the three proto-oncogene (c-fos, c-jun and jun-B) mRNAs, induced a decrease of P450 17 alpha and 3 beta-HSD and caused a marked homologous and heterologous (ACTH) densitization. Transforming growth factor beta 1 which only increased jun-B mRNA, markedly reduced BAC differentiated functions and the steroidogenic responsiveness to both ACTH and A-II. Thus, it is postulated that the proto-oncoproteins encoded by the immediate early genes may play a role in the long-term effects of peptide hormones and growth factors on BAC differentiated functions.     

Cell-free degradation of p27(kip1), a G1 cyclin-dependent kinase inhibitor, is dependent on CDK2 activity and the proteasome

Entry into S phase is dependent on the coordinated activation of CDK4,6 and CDK2 kinases. Once a cell commits to S phase, there must be a mechanism to ensure the irreversibility of this decision. The activity of these kinases is inhibited by their association with p27. In many cells, p27 plays a major role in the withdrawal from the cell cycle in response to environmental cues. Thus, it is likely that p27 is a target of the machinery required to ensure the irreversibility of S-phase entry. We have been interested in understanding the mechanisms regulating p27 at the G1/S transition. In this report, we define a cell-free degradation system which faithfully recapitulates the cell cycle phase-specific degradation of p27. We show that this reaction is dependent on active CDK2 activity, suggesting that CDK2 activity is directly required for p27 degradation. In addition to CDK2, other S-phase-specific factors are required for p27 degradation. At least some of these factors are ubiquitin and proteasome dependent. We discuss the relationships between CDK2 activity, ubiquitin-dependent, and possibly ubiquitin-independent proteasomal activities in S-phase extracts as related to p27.     

Repression of p27kip1 synthesis by platelet-derived growth factor in BALB/c 3T3 cells

We have investigated the regulation of p27kip1, a cyclin-dependent kinase inhibitor, in BALB/c 3T3 cells during growth factor-stimulated transition from quiescence (G0) to a proliferative (G1) state. The level of p27kip1 protein falls dramatically after mitogenic stimulation and is accompanied by a decrease in cyclin E associated p27kip1, as well as a transient increase in cyclin D1-associated p27kip1 that later declines concomitantly with the loss of total p27kip1. Analysis of metabolically labelled cells revealed that cyclin D2, cyclin D3, and cdk4 were also partnered with p27kip1 in quiescent BALB/c 3T3 cells and that this association decreased after platelet-derived growth factor (PDGF) treatment. Furthermore, the decline in p27kip1 and reduced association with cyclin D3, initiated by the addition of PDGF but not plasma-derived factors, suggested that these changes are involved in competence, the first step in the exit from G0. Synthesis of p27kip1 as determined by incorporation of [35S]methionine was repressed upon mitogenic stimulation, and PDGF was sufficient to elicit this repression within 2 to 3 h. Pulse-chase experiments demonstrated the reduced rate of synthesis was not the result of an increased rate of degradation. Full repression of p27kip1 synthesis required the continued presence of PDGF and failed to occur in the presence of the RNA polymerase inhibitor 5,6-dichlorobenzimidazole riboside. These characteristics demonstrate that repression was a late effect of PDGF and was consistent with our finding that conditional expression of activated H-ras did not affect synthesis of p27kip1. Northern (RNA) analysis of p27kip1 mRNA revealed that the repression was not accompanied by a corresponding decrease in p27kip1 mRNA, suggesting that the PDGF-regulated decrease in p27kip1 expression occurred through a translational mechanism.     

Dietary gamma-linolenic acid enhances mouse macrophage-derived prostaglandin E1 which inhibits vascular smooth muscle cell proliferation

We previously demonstrated that macrophages isolated from mice fed gamma-linolenic acid (GLA)-enriched diets reduce vascular smooth muscle cell (SMC) proliferation in a cyclooxygenase-dependent fashion and may therefore favorably modulate the atherogenic process. The present study was conducted to elucidate the mechanism(s) by which dietary GLA influences the ability of macrophages to modulate SMC growth programs. Resident peritoneal macrophages were isolated from C57BL/6 female mice fed diets containing variable GLA compositions at 10% (wt/wt), treated with various antibodies and co-cultured with cycling naive vascular SMC isolated from nonpurified diet-fed mice. Smooth muscle cell proliferation and intracellular cAMP levels were measured after co-culture. In parallel experiments, cycling naive vascular SMC isolated from nonpurified diet-fed mice were dosed with exogenous prostaglandin E1 (PGE1 ) for various periods and challenged with cycloheximide for 4 h (8-12 h after PGE1 addition), and intracellular cAMP levels were measured at various time points. Macrophages isolated from mice fed GLA-enriched dietary oils significantly reduced SMC proliferation in co-culture compared with controls (macrophages from mice fed a corn oil diet containing no GLA). Anti-PGE1 antiserum treatment (1:50 or 1:100) blocked the ability of GLA-enriched macrophages to down-regulate SMC proliferation, a response reversed by exogenous PGE1 treatment. Macrophages isolated from mice fed GLA-enriched dietary oils elevated SMC intracellular cAMP levels in a biphasic fashion. In addition, exogenous PGE1 (1 nmol/L to 10 micromol/L) exerted a similar biphasic cAMP response in SMC, and the second phase of cAMP elevation was antagonized by cycloheximide. In conclusion, dietary GLA enhances mouse macrophage-derived prostaglandin E1, which inhibits vascular SMC proliferation.     

Growth factor dependence of progression through G1 and S phases of adult rat hepatocytes in vitro. Evidence of a mitogen restriction point in mid-late G1

Several hepatocyte mitogens have been identified, but the signals triggering the G0/G1 transition and cell cycle progression of hepatocytes remain unknown. Using hepatocyte primary cultures, we investigated the role of epidermal growth factor/pyruvate during the entry into and progression through the G1 phase and analyzed the expression of cell cycle markers. We show that the G0/G1 transition occurs during hepatocyte isolation as evidenced by the expression of early genes such as c-fos, c-jun, and c-myc. In culture, hepatocytes progress through G1 regardless of growth factor stimulation until a restriction point (R point) in mid-late G1 beyond which they cannot complete the cell cycle without mitogenic stimulation. Changes in cell cycle gene expression were associated with progression in G1; the cyclin E mRNA level is low early in G1 but increases at the G1/S boundary, while the protein is constantly detected during cell cycle but undergoes a change of electrophoretic mobility in mid-late G1 after the R point. In addition, a drastic induction of cyclin D1 mRNA and protein, and to a lesser extent of cyclin D2 mRNA, takes place in mitogen-stimulated cells after the R point. In contrast, cyclin D3 mRNA appears early in G1, remains constant in stimulated cells, but accumulates in unstimulated arrested cells, paralleling the cyclin-dependent kinase 4 mRNA expression. These results characterize the different steps of G1 phase in hepatocytes.     

Somatostatin receptor-mediated signaling in smooth muscle. Activation of phospholipase C-beta3 by Gbetagamma and inhibition of adenylyl cyclase by Galphai1 and Galphao

In COS-7 cells, all five cloned somatostatin receptors are coupled via inhibitory G proteins to activation of an unidentified phospholipase C-beta (PLC-beta) isozyme and inhibition of adenylyl cyclase. In the present study, intestinal smooth muscle cells (SMC) that express only one receptor type, sstr3, and possess a full complement of G proteins and PLC-beta isozymes were used to identify the PLC-beta isozyme and the G proteins coupled to it and to adenylyl cyclase. Somatostatin-14 bound with high affinity to intestinal SMC; stimulated D-myo-inositol-1,4,5-trisphosphate (IP3) formation, Ca2+ release, and contraction; and inhibited forskolin-stimulated cAMP formation in a pertussis toxin-sensitive fashion. Somatostatin also stimulated phosphoinositide hydrolysis in plasma membranes. Only those somatostatin analogs that shared a high affinity for sstr3 receptors elicited muscle contraction. IP3 formation, Ca2+ release, and contraction in permeabilized SMC and phosphoinositide hydrolysis in plasma membranes were inhibited (approximately 80%) by pretreatment with antibodies to PLC-beta3 but not other PLC-beta isozymes, and by antibodies to Gbeta but not Galpha. Inhibition of cAMP formation was partially blocked by antibody to Galphai1 or Galphao and additively blocked by a combination of both antibodies. Somatostatin-stimulated [35S]GTPgammaS-Galpha complexes in plasma membranes were bound selectively by Galphai1 and Galphao antibodies. We conclude that in smooth muscle sstr3 is coupled to Gi1 and Go; the alpha subunits of both G proteins mediate inhibition of adenylyl cyclase, while the betagamma subunits mediate activation of PLC-beta3.     

Is the Beck Depression Inventory reliable over time? An evaluation of multiple test-retest reliability in a nonclinical college student sample

The staurosporine-induced G1 cell cycle arrest was analyzed in a variety of cell lines which includes human tumor cell lines and oncogene-transformed NIH3T3 cell lines. All the cell lines which were sensitive to staurosporine-induced G1 arrest contained a functional retinoblastoma protein (pRB). However, when pRB-lacking fibroblast cells derived from pRB knockout mice were tested they were also sensitive to G1 arrest by staurosporine, indicating that the inactivation of pRB alone is not sufficient for the abrogation of staurosporine-induced G1 arrest. In searching for a common event caused by staurosporine, the cyclin-dependent kinase (CDK) inhibitor protein p27kip1 but not p21cip1 was found to accumulate after staurosporine treatment in all the cell lines examined. This accumulation occurred regardless of the induction of the G1 arrest. The result indicates that the accumulation of p27kip1 is the cell's primary response to staurosporine and that the capability of staurosporine to induce G1 arrest depends on the integrity of cell cycle regulatory components which are downstream of p27kip1.