A role for p21ras/MAP kinase in TCR-mediated activation of LFA-1

LFA-1 is a beta2 integrin that plays well-characterized roles in adhesion of T lymphocytes to APC, T cell-mediated cytolysis, and leukocyte-endothelial cell interactions. Although it is clear that LFA-1 must undergo affinity or avidity changes to bind its cellular ligand ICAM-1, the intracellular signaling pathways involved are not well characterized. Here, we show that the Ras-mitogen-activated protein kinase (MAPK) signaling pathway is also involved in TCR-activated LFA-1 adhesion. Expression of a dominant negative form of p21ras in a thymocyte cell line inhibits, while constitutively active p21ras both enhances and sustains, subsequent TCR-triggered adhesion to isolated ICAM-1. However, the Ras/MAPK pathway alone is not sufficient for activating T cell LFA-1, as inhibition of both downstream MAPK/extracellular regulated kinase kinase (MEK) activity and phosphatidylinositol 3-kinase activity is required for complete inhibition of adhesion.     

Two different signal transduction pathways can be activated by transforming growth factor beta 1 in epithelial cells

Signal transduction initiated by transforming growth factor beta 1 (TGF beta 1) was studied in two sublines of the same colon carcinoma cell line, which respond in opposite ways to TGF beta 1, by proliferation or by growth inhibition. TGF beta 1 activates ras proteins within 5 min of addition when it acts to inhibit growth but not when it acts as a mitogen. In both cases TGF beta 1 also rapidly modulates the activities of three protein kinases, detected by their in gel kinase activity on the mitogen-activated protein kinase (MAP kinase) substrate, myelin basic protein (MBP). When TGF beta 1 acts as a mitogen for U9 cells, it increases the activity of MBP kinases of 57, 105, and 130 kDa within 10 min of the addition without detectably activating ras proteins. When TGF beta 1 inhibits the growth of HD3 cells, it activates ras proteins and the 57-kDa MBP kinase within 5 min but inhibits the activity of the 105- and 130-kDa MBP kinases. In HD3 cells ras activation occurred in two signal transduction pathways, one from TGF beta 1 leading to growth inhibition and one from epidermal growth factor (EGF) leading to proliferation. In addition to ras proteins, EGF activates a different set of MBP kinases in HD3 cells than does TGF beta 1, MBP kinases of 85, 57, and 44 kDa. The latter is likely to be the 44-kDa MAP kinase extracellular signal-regulated kinase (erk) 1, because EGF treatment of HD3 cells activates erk1 by increasing its phosphotyrosine level. Therefore, in two closely related epithelial cell lines TGF beta 1 activates two different signal transduction pathways, one ras-dependent and one ras-independent, and modulates the activities of a set of MBP kinases.     

p21 ras and phosphatidylinositol-3 kinase are required for survival of wild-type and NF1 mutant sensory neurons

Nerve growth factor (NGF) is a required differentiation and survival factor for sympathetic and a majority of neural crest-derived sensory neurons in the developing vertebrate peripheral nervous system. Although much is known about the function of NGF, the intracellular signaling cascade that it uses continues to be a subject of intense study. p21 ras signaling is considered necessary for sensory neuron survival. How additional intermediates downstream or in parallel may function has not been fully understood yet. Two intracellular signaling cascades, extra cellular regulated kinase (erk) and phosphatidylinositol-3 (PI 3) kinase, transduce NGF signaling in the pheochromocytoma cell line PC12. To elucidate the role these cascades play in survival and differentiation, we used a combination of recombinant adenoviruses and chemical inhibitors to perturb these pathways in sensory neurons from wild-type mice and mice deficient for neurofibromin in which the survival and differentiation pathway is constitutively active. We demonstrate that ras activity is both necessary and sufficient for the survival of embryonic sensory neurons. Downstream of ras, however, the erk cascade is neither required nor sufficient for neuron survival or overall differentiation. Instead, the activity of PI 3 kinase is necessary for the survival of the wild-type and neurofibromin-deficient neurons. Therefore, we conclude that in sensory neurons, NGF acts via a signaling pathway, which includes both ras and PI 3 kinase.     

Fibroblast growth factor-2 has opposite effects on human breast cancer MCF-7 cell growth depending on the activation level of the mitogen-activated protein kinase pathway

In human breast cancer MCF-7 and MCF-7ras cells, we demonstrated that whereas insulin had a mitogenic effect on both cell lines, fibroblast growth factor-2 (FGF-2) had opposite effects, stimulating MCF-7 and inhibiting MCF-7ras cell proliferation. The inhibitory signal induced by FGF-2 was related to sustained mitogen-activated protein kinase (MAPK) activation in MCF-7ras cells, while transient MAPK activation was associated with MCF-7 cell proliferation. FGF-2 was further used in combination with insulin or cAMP. In MCF-7 cells, insulin and cAMP reversed the mitogenic effect of FGF-2. In MCF-7ras cells, insulin did not modify the inhibitory effect of FGF-2, but cAMP markedly enhanced it. These effects were also associated with an increased level and duration of MAPK activation. PD98056 abolished the effect of FGF-2 on DNA synthesis in both cell lines, demonstrating that the dual effect of FGF-2 on cell proliferation is dependent on the activity of the extracellular-signal-regulated kinase 1 and 2 (ERK1/2) signalling pathway.     

Ultrastructural studies of the female breast: I. 9 + 0 cilia in myoepithelial cells

1 Phenylethanolamine-N-methyltransferase (PNMT) activity in adrenal gland was lower than the control values at day 24 of rabbit pregnancy and fell throughout the gestational period. 2 Catechol-O-methyltransferase (COMT) activity during pregnancy and parturition followed a similar pattern to PNMT. 3 Adrenaline content of the adrenal gland declined significantly during the last few days of pregnancy but at parturition showed a tendency to return towards normal. 4 The biochemical mechanisms which result in a decline in PNMT and COMT activities and adrenaline concentration appear to be the consequence of modifications in endocrine gland secretion during pregnancy.     

Randomized, double-blind, placebo-controlled study of ascorbate on the preventive effect of nitrate tolerance in patients with congestive heart failure

Mitogen-activated protein kinase (MAPK) is a serine-threonine kinase that is activated by various extracellular stimuli. Extracellular signal-regulated kinases (ERK1 and ERK2), an MAPK subfamily, are activated by many oncogenes, such as ras and raf, and they induce cell proliferation. myc is also an oncogene and one of the targets of ERKs. Mutations of ras and overexpression of myc were found in various human cancers, and ERKs were also reported to play a role in carcinogenesis. In this study, we examined 39 biopsy specimens of oral squamous cell carcinoma (OSCC) and 5 of normal gingival mucosa for the expression of ERK protein and the proliferation marker, MIB-1 (Ki-67 antibody). Thirteen OSCC specimens and five normal gingival biopsies were also examined for the expression of ERKs mRNA by in situ hybridization. Double staining for ERKs and MIB-1 was also performed. Histologically, 18 patients (46%) were diagnosed with well-differentiated SCC, 17 (44%) with moderately differentiated SCC, and 4 (10%) with poorly differentiated SCC. The histologic grade correlated with the MIB-1 index. The localization of ERK1 was similar to that of ERK2. Positive signals for ERK proteins were localized in superficial keratinocytes in normal gingival mucosa, whereas these mRNAs were weakly positive in the basal and spinous layer. Basal and suprabasal cells were positive for MIB-1. In well-differentiated and moderately differentiated OSCC, positive signals for ERK mRNA and proteins were found at higher levels than in normal gingival mucosa in keratotic cells around cancer pearls. Some cells showed positive signals for ERKs and MIB-1. Furthermore, most cancer cells in poorly differentiated SCC were positive for both ERK and MIB-1. The histologic grade was statistically related to the percentage of cells positive for both ERK and MIB-1. This suggested that ERKs might be related to proliferation in OSCC.     

The different forms of the prolactin receptor in the rat corpus luteum: developmental expression and hormonal regulation in pregnancy

The corpora lutea of pregnancy in the rat are highly dependent on the action of PRL and PRL-like hormones to hypertrophy and to produce progesterone needed for the maintenance of gestation. Two forms of the PRL receptor (PRL-R), designated as long (PRL-RL) and short (PRL-RS), have been described in rat tissues. To determine whether both forms are present in the corpus luteum during pregnancy and to examine the developmental and hormonal regulation of their expression, total RNA isolated from corpora lutea at different stages of pregnancy and from highly luteinized granulosa cells subjected to different hormonal treatments were analyzed by semiquantitative RT-PCR. Immunoblotting of luteal proteins from early and late pregnancy was also performed to determine if the pattern of PRL-R proteins follows that of PRL-R messenger RNA (mRNA) expression. In addition, the correlation between the well characterized PRL-regulated gene, 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), and PRL-R gene expression was investigated during the time of luteolysis. Both PRL-RL and PRL-RS mRNA and protein were expressed in corpora lutea of pregnancy, with the long form being the most dominant at all stages. Whereas no changes in mRNA level of either PRL-RL or PRL-RS were found until day 20 of gestation, a profound decline in PRL-R mRNA and protein for both receptor types occurred at the end of pregnancy. This drop in PRL-R expression was accompanied by a sharp and abrupt expression of 20alpha-HSD mRNA. Studies performed in vivo and in luteinized cells in culture indicate that PRL can up-regulate the expression of the PRL-RL mRNA, an effect prevented by the tyrosine kinase inhibitor, genistein. PRL-RL mRNA was also selectively increased by cAMP. In summary, the results of this investigation have established that: 1) the corpus luteum of pregnancy expresses both the short and long forms of the PRL-R with the long form being more abundant; 2) the mRNA for both forms of the PRL-R remains at constant levels throughout pregnancy but drops before parturition; 3) the decline in PRL-R mRNA at the end of pregnancy is accompanied by a dramatic rise in 20alpha-HSD; 4) PRL is able to increase the expression of PRL-R mRNA; and that 5) both A kinase and tyrosine kinase mediated pathways appear to participate in the up-regulatory mechanism involved in PRL-R mRNA expression.     

Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts

NIH 3T3 fibroblasts stably transformed with a constitutively active isoform of p21(Ras), H-RasV12 (v-H-Ras or EJ-Ras), produced large amounts of the reactive oxygen species superoxide (.O2-). .O2- production was suppressed by the expression of dominant negative isoforms of Ras or Rac1, as well as by treatment with a farnesyltransferase inhibitor or with diphenylene iodonium, a flavoprotein inhibitor. The mitogenic activity of cells expressing H-RasV12 was inhibited by treatment with the chemical antioxidant N-acetyl-L-cysteine. Mitogen-activated protein kinase (MAPK) activity was decreased and c-Jun N-terminal kinase (JNK) was not activated in H-RasV12-transformed cells. Thus, H-RasV12-induced transformation can lead to the production of .O2- through one or more pathways involving a flavoprotein and Rac1. The implication of a reactive oxygen species, probably .O2-, as a mediator of Ras-induced cell cycle progression independent of MAPK and JNK suggests a possible mechanism for the effects of antioxidants against Ras-induced cellular transformation.     

The A and the extended V N-terminal regions of streptococcal protein I/IIf mediate the production of tumour necrosis factor alpha in the monocyte cell line THP-1

Signal transduction is ubiquitously involved in the initiation of physiological signals that lead to growth and proliferation of cells. The signaling cascade mediated by the mitogen-activated protein kinase (MAPK) is considered essential for T cell growth and function. Therefore, it was of interest to determine the influence of age on the induction of MAPK in mitogen-activated T cells. T cells from young (4-6 months) and old (24-26 months) rats responded to concanavalin A (Con A) stimulation by increasing MAPK, c-jun amino terminal kinase (JNK), and p21ras activities. The time course of induction of MAPK/JNK and p21ras activities was similar in T cells isolated from young and old rats. The induction of JNK activity did not change significantly with age; however, the induction of MAPK and p21ras activities was significantly less (50 to 65%) in T cells from old rats than in T cells from young rats. Although the relative protein levels of p42 and p44 MAPK did not change with age, the proportion of the phosphorylated p44 MAPK decreased with age. In addition, it was found that the in vitro kinase activities of the T cell receptor-associated protein tyrosine kinase Lck (p56Lck) and ZAP-70 but not Fyn (p59Fyn) were lower in T cells from old rats than in T cells from young rats. The decline in activities of these signaling molecules with age was not associated with changes in their corresponding protein levels. Thus, our results demonstrate that aging alters the activation of the signal transduction cascade that leads to T cell activation.     

Angiotensin II type 2 receptor is upregulated in human heart with interstitial fibrosis, and cardiac fibroblasts are the major cell type for its expression

The expression pattern of angiotensin (Ang) II type 2 receptor (AT2-R) in the remodeling process of human left ventricles (LVs) remains poorly defined. We analyzed its expression at protein, mRNA, and cellular levels using autopsy, biopsy, or operation LV samples from patients with failing hearts caused by acute (AMI) or old (OMI) myocardial infarction and idiopathic dilated cardiomyopathy (DCM) and also examined functional biochemical responses of failing hearts to Ang II. In autopsy samples from the nonfailing heart group, the ratio of AT1-R and AT2-R was 59% and 41%, respectively. The expression of AT2-R was markedly increased in DCM hearts at protein (3.5-fold) and mRNA (3.1-fold) levels compared with AMI or OMI. AT1-R protein and mRNA levels in AMI hearts showed 1.5- and 2.1-fold increases, respectively, whereas in OMI and DCM hearts, AT1-R expression was significantly downregulated. AT1-R-mediated response in inositol phosphate production was significantly attenuated in LV homogenate from failing hearts compared with nonfailing hearts. AT2-R sites were highly localized in the interstitial region in either nonfailing or failing heart, whereas AT1-R was evenly distributed over myocardium at lower densities. Mitogen-activated protein kinase (MAPK) activation by Ang II was significantly decreased in fibroblast compartment from the failing hearts, and pretreatment with AT2-R antagonist caused an additional significant increase in Ang II-induced MAPK activity (36%). Cardiac hypertrophy suggested by atrial and brain natriuretic peptide levels was comparably increased in OMI and DCM, whereas accumulation of matrix proteins such as collagen type 1 and fibronectin was much more prominent in DCM than in OMI. These findings demonstrate that (1) AT2-R expression is upregulated in failing hearts, and fibroblasts present in the interstitial regions are the major cell type responsible for its expression, (2) AT2-R present in the fibroblasts exerts an inhibitory effect on Ang II-induced mitogen signals, and (3) AT1-R in atrial and LV tissues was downregulated during chronic heart failure, and AT1-R-mediated functional biochemical responsiveness was decreased in the failing hearts. Thus, the expression level of AT2-R is likely determined by the extent of interstitial fibrosis associated with heart failure, and the expression and function of AT1-R and AT2-R are differentially regulated in failing human hearts.     

p38 Mitogen-activated protein kinase is a critical component of the redox-sensitive signaling pathways activated by angiotensin II. Role in vascular smooth muscle cell hypertrophy

Angiotensin II induces an oxidant stress-dependent hypertrophy in cultured vascular smooth muscle cells. To investigate the growth-related molecular targets of H2O2, we examined the redox sensitivity of agonist-stimulated activation of the mitogen-activated protein kinase (MAPK) family. We show here that angiotensin II elicits a rapid increase in intracellular H2O2 and a rapid and robust phosphorylation of both p42/44MAPK (16-fold) and p38MAPK (15-fold). However, exogenous H2O2 activates only p38MAPK (14-fold), and diphenylene iodonium, an NADH/NADPH oxidase inhibitor, attenuates angiotensin II-stimulated phosphorylation of p38MAPK, but not p42/44MAPK. Furthermore, in cells stably transfected with human catalase, angiotensin II-induced intracellular H2O2 generation is almost completely blocked, resulting in inhibition of phosphorylation of p38MAPK, but not p42/44MAPK, and a subsequent partial decrease in angiotensin II-induced hypertrophy. Specific inhibition of either the p38MAPK pathway with SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H- imidaz ole) or the p42/44MAPK pathway with PD98059 (2-(2'-amino-3'-methoxyphenyl)oxanaphthalen-4-one) also partially, but significantly, attenuates angiotensin II-induced hypertrophy; however, simultaneous blockade of both pathways has an additive inhibitory effect, indicating that the hypertrophic response to angiotensin II requires parallel, independent activation of both MAPK pathways. These results provide the first evidence that p38MAPK is a critical component of the oxidant stress (H2O2)-sensitive signaling pathways activated by angiotensin II in vascular smooth muscle cells and indicate that it plays a crucial role in vascular hypertrophy.     

G(o)-protein alpha-subunits activate mitogen-activated protein kinase via a novel protein kinase C-dependent mechanism

Mitogen-activated protein kinase (MAPK) is activated in response to both receptor tyrosine kinases and G-protein-coupled receptors. Recently, Gi-coupled receptors, such as the alpha 2A adrenergic receptor, were shown to mediate Ras-dependent MAPK activation via a pathway requiring G-protein beta gamma subunits (G beta gamma) and many of the same intermediates involved in receptor tyrosine kinase signaling. In contrast, Gq-coupled receptors, such as the M1 muscarinic acetylcholine receptor (M1AChR), activate MAPK via a pathway that is Ras-independent but requires the activity of protein kinase C (PKC). Here we show that, in Chinese hamster ovary cells, the M1AChR and platelet-activating factor receptor (PAFR) mediate MAPK activation via the alpha-subunit of the G(o) protein. G(o)-mediated MAPK activation was sensitive to treatment with pertussis toxin but insensitive to inhibition by a G beta gamma-sequestering peptide (beta ARK1ct). M1AChR and PAFR catalyzed G(o) alpha-subunit GTP exchange, and MAPK activation could be partially rescued by a pertussis toxin-insensitive mutant of G(o) alpha but not by similar mutants of Gi. G(o)-mediated MAPK activation was insensitive to inhibition by a dominant negative mutant of Ras (N17Ras) but was completely blocked by cellular depletion of PKC. Thus, M1AChR and PAFR, which have previously been shown to couple to Gq, are also coupled to G(o) to activate a novel PKC-dependent mitogenic signaling pathway.