Novel members of the mitogen-activated protein kinase activator family in Xenopus laevis.

Mitogen-activated protein (MAP) kinases comprise an evolutionarily conserved family of proteins that includes at least three vertebrate protein kinases (p42, p44, and p55 MAPK) and five yeast protein kinases (SPK1, MPK1, HOG1, FUS3, and KSS1). Members of this family are activated by a variety of extracellular agents that influence cellular proliferation and differentiation. In Saccharomyces cerevisiae, there are multiple physiologically distinct MAP kinase activation pathways composed of structurally related kinases. The recently cloned vertebrate MAP kinase activators are structurally related to MAP kinase activators in these yeast pathways. These similarities suggest that homologous kinase cascades are utilized for signal transduction in many, if not all, eukaryotes. We have identified additional members of the MAP kinase activator family in Xenopus laevis by a polymerase chain reaction-based analysis of embryonic cDNAs. One of the clones identified (XMEK2) encodes a unique predicted protein kinase that is similar to the previously reported activator (MAPKK) in X. laevis. XMEK2, a highly expressed maternal mRNA, is developmentally regulated during embryogenesis and expressed in brain and muscle. Expression of XMEK2 in yeast cells suppressed the growth defect associated with loss of the yeast MAP kinase activator homologs, MKK1 and MKK2. Partial sequence of a second cDNA clone (XMEK3) identified yet another potential MAP kinase activator. The pattern of expression of XMEK3 is distinct from that of p42 MAPK and XMEK2. The high degree of amino acid sequence similarity of XMEK2, XMEK3, and MAPKK suggests that these three are related members of an amphibian family of protein kinases involved in the activation of MAP kinase. Discovery of this family suggests that multiple MAP kinase activation pathways similar to those in yeast cells exist in vertebrates.     

mRNA distribution within dendrites: Relationship to afferent innervation

The majority of neuronal mRNAs are confined to cell bodies, but a few mRNAs are present at high levels in dendrites. Here we report an initial analysis of the relationship between afferent innervation and the distribution of mRNA within dendritic fields. In situ hybridization techniques were used to compare the subcellular distribution of dendritic mRNAs in principal neurons of the hippocampal formation in vivo. The mRNA encoding the α subunit of calcium/calmodulin dependent protein kinase II (CAMII kinase) was present at high levels throughout the layers that contain the dendrites of hippocampal pyramidal cells and dentate granule cells. In contrast, the mRNA encoding the high molecular weight microtubule-associated protein MAP2 had a more limited distribution. In the dentate gyrus, labeling for MAP2 was present in a discrete band in the lamina containing proximal dendrites and decreased to low levels in laminae containing distal dendrites. This laminar pattern resembles the distinct terminations of the commissural/associational projection (high MAP2 labeling) and the entorhinal projection (lower MAP2 labeling) upon dendrites of granule cells. To determine if the differential distribution of dendritic mRNAs was regulated by either the presence or activity of afferents, we evaluated mRNA distribution in the dentate molecular layer following (1) removal of the entorhinal input by lesions of the entorhinal cortex or (2) prolonged delivery of potentiating stimulation to entorhinal afferents. Denervation led to modest decreases in the levels of mRNAs for both CAMII and MAP2 but did not lead to detectable alterations in mRNA distribution. Also, prolonged stimulation did not lead to detectable alterations in MAP2 or CAMII mRNA distribution, although such stimulation clearly elevated the expression of mRNA for glial fibrillary acidic protein (GFAP). © 1995 John Wiley & Sons, Inc.     

Regulation of peroxisome proliferator-activated receptor-gamma-mediated gene expression. A new mechanism of action for high density lipoprotein

Cellular cholesterol content reflects a balance of lipid influx by lipoprotein receptors and endogenous synthesis and efflux to cholesterol acceptor particles. The beneficial effect of high density lipoprotein (HDL) in protecting against the development of cardiovascular disease is thought to be mediated predominately through its induction of cellular cholesterol efflux and "reverse cholesterol transport" from peripheral tissues to the liver. We tested the hypothesis that HDL could inhibit cellular lipid accumulation by modulating expression of peroxisome proliferator-activated receptor-gamma (PPARgamma)-responsive genes. To this end, we evaluated expression of two PPARgamma-responsive genes, CD36, a receptor for oxidized low density lipoprotein, and aP2, a fatty acid-binding protein. HDL decreased expression of macrophage CD36 and aP2 in a dose-dependent manner. HDL also decreased aP2 expression in fibroblasts, reduced accumulation of lipid, and slowed differentiation of fibroblasts into adipocytes. HDL stimulated mitogen-activated protein (MAP) kinase activity, and inhibition of CD36 expression was blocked by co-incubation with a MAP kinase inhibitor. HDL increased expression of PPARgamma mRNA and protein, induced translocation of PPARgamma from the cytoplasm to the nucleus, and increased PPARgamma phosphorylation. Our data demonstrate that despite induction and translocation of PPARgamma in response to HDL, MAP kinase-mediated phosphorylation of PPARgamma inhibited expression of PPARgamma-responsive genes and suggest mechanisms by which HDL may inhibit cellular lipid accumulation.     

Coupling of Serotonin 5-HT1B Receptors to Activation of Mitogen-Activated Protein Kinase (ERK-2) and p70 S6 Kinase Signaling Systems

Abstract: Little is known about the coupling of serotonin 5-HT_1B receptors to cellular signals other than cyclic AMP. In the present studies, the activation by 5-HT_1B receptors of p70 S6 kinase and the mitogen-activated protein kinase (MAP kinase) ERK-2 was investigated. Studies were performed by using both nontransfected Chinese hamster ovary (CHO) cells, which express endogenous receptors at a very low density, and a stable transfected CHO cell line expressing 5-HT_1B receptors at 230 fmol/mg of membrane protein, a density similar to that expressed in cortex. In nontransfected cells, 5-HT was found to stimulate a greater than twofold increase in MAP kinase activity with an EC₅₀ of 20 n M . Reflecting increased density of receptors, 5-HT caused a greater than eightfold activation of ERK-2 in transfected cells with an EC₅₀ of 2 n M . 5-HT was found to also stimulate p70 S6 kinase in both nontransfected and transfected cells. The stimulation was sixfold in both types of cells, but the EC₅₀ for 5-HT was fourfold lower in transfected cells. The coupling of 5-HT_1B receptors to ERK-2 and to p70 S6 kinase was inhibited by pertussis toxin, inhibitors of phosphatidylinositol 3-kinase, and by the inhibitor of MAP kinase kinase PD098059. Activation of p70 S6 kinase, but not ERK-2, was also inhibited by rapamycin. These findings demonstrate that 5-HT_1B receptors couple to ERK-2 and p70 S6 kinase through overlapping, but nonidentical, pathways.     

Regulation of corticotropin-releasing factor receptor type 2beta mRNA by mitogen-activated protein kinases in aortic smooth muscle cells

The actions of the corticotropin-releasing factor (CRF) family of peptides are mediated by the seven transmembrane-domain G-protein-coupled receptors, the CRF receptors. CRF receptor type 2beta (CRFR2beta) messenger RNA (mRNA) is expressed primarily in the cardiovascular system, where its levels are decreased by urocortin 1 (Ucn1), a novel peptide in the CRF family. In a previous study, we reported that CRFR2beta mRNA levels were partially down-regulated via the cAMP-protein kinase A pathway. This study focused on the involvement of the intracellular mitogen-activated protein (MAP) kinase pathway in the modulation of CRFR2beta mRNA levels. Ribonuclease protection assays showed that decreases in CRFR2beta mRNA levels induced by Ucn1 and cAMP were attenuated by the p38 MAP kinase inhibitor SB202190 or SB203580. This finding suggested that the p38 MAP kinase pathway was involved in this regulation. Anisomycin, a classic p38 kinase activator, increased CRFR2beta mRNA levels in A7r5 cells. This effect of anisomycin was completely reversed by H7, a serine/threonine kinase inhibitor, while both p38 kinase and MAP kinase kinase inhibitors failed to block the increase in CRFR2beta mRNA levels caused by anisomycin. As anisomycin can activate Jun amino terminal kinases, as well as p38 MAP kinase, it is possible that other MAP kinases, such as Jun amino terminal kinases, also contribute to the increase in gene levels. Alternatively, anisomycin may increase CRFR2beta mRNA levels indirectly as a consequence of blocking protein synthesis.     

Shear-induced tyrosine phosphorylation in endothelial cells requires Rac1-dependent production of ROS

The shear-induced intracellular signal transduction pathway invascular endothelial cells involves tyrosine phosphorylation andactivation of mitogen-activated protein (MAP) kinase, which may beresponsible for the sustained release of nitric oxide. MAP kinase isknown to be activated by reactive oxygen species (ROS), such asH₂O₂,in several cell types. ROS production in ligand-stimulatednonphagocytic cells appears to require the participation of aRas-related small GTP-binding protein, Rac1. We hypothesized that Rac1might serve as a mediator for the effect of shear stress on MAP kinaseactivation. Exposure of bovine aortic endothelial cells to laminarshear stress of 20 dyn/cm² for5-30 min stimulated total cellular and cytosolic tyrosine phosphorylation as well as tyrosine phosphorylation of MAP kinase. Treating endothelial cells with the antioxidantsN-acetylcysteine and pyrrolidinedithiocarbamate inhibited in a dose-dependent manner theshear-stimulated increase in total cytosolic and, specifically, MAPkinase tyrosine phosphorylation. Hence, the onset of shear stresscaused an enhanced generation of intracellular ROS, as evidenced by anoxidized protein detection kit, which were required for theshear-induced total cellular and MAP kinase tyrosine phosphorylation. Total cellular and MAP kinase tyrosine phosphorylation was completely blocked in sheared bovine aortic endothelial cells expressing adominant negative Rac1 gene product (N17rac1). We concluded that theGTPase Rac1 mediates the shear-induced tyrosine phosphorylation of MAPkinase via regulation of the flow-dependent redox changes inendothelial cells in physiological and pathological circumstances.     

Cellular stress induces the tyrosine phosphorylation of caveolin-1 (Tyr(14)) via activation of p38 mitogen-activated protein kinase and c-Src kinase. Evidence for caveolae, the actin cytoskeleton, and focal adhesions as mechanical sensors of osmotic stress

Environmental stressors have been recently shown to activate intracellular mitogen-activated protein (MAP) kinases, such as p38 MAP kinase, leading to changes in cellular functioning. However, little is known about the downstream elements in these signaling cascades. In this study, we show that caveolin-1 is phosphorylated on tyrosine 14 in NIH 3T3 cells after stimulation with a variety of cellular stressors (i.e. high osmolarity, H2O2, and UV light). To detect this phosphorylation event, we employed a phosphospecific monoclonal antibody probe that recognizes only tyrosine 14-phosphorylated caveolin-1. Since p38 MAP kinase and c-Src have been previously implicated in the stress response, we next assessed their role in the tyrosine phosphorylation of caveolin-1. Interestingly, we show that the p38 inhibitor (SB203580) and a dominant-negative mutant of c-Src (SRC-RF) both block the stress-induced tyrosine phosphorylation of caveolin-1 (Tyr(P)(14)). In contrast, inhibition of the p42/44 MAP kinase cascade did not affect the tyrosine phosphorylation of caveolin-1. These results indicate that extracellular stressors can induce caveolin-1 tyrosine phosphorylation through the activation of well established upstream elements, such as p38 MAP kinase and c-Src kinase. However, heat shock did not promote the tyrosine phosphorylation of caveolin-1 and did not activate p38 MAP kinase. Finally, we show that after hyperosmotic shock, tyrosine-phosphorylated caveolin-1 is localized near focal adhesions, the major sites of tyrosine kinase signaling. In accordance with this localization, disruption of the actin cytoskeleton dramatically potentiates the tyrosine phosphorylation of caveolin-1. Taken together, our results clearly define a novel signaling pathway, involving p38 MAP kinase activation and caveolin-1 (Tyr(P)(14)). Thus, tyrosine phosphorylation of caveolin-1 may represent an important downstream element in the signal transduction cascades activated by cellular stress.     

High Cell Density Upregulates Calcium Oscillation by Increasing Calcium Store Content via Basal Mitogen-Activated Protein Kinase Activity

Calcium releases of non-excitable cells are generally a combination of oscillatory and non-oscillatory patterns, and factors affecting the calcium dynamics are still to be determined. Here we report the influence of cell density on calcium increase patterns of clonal cell lines. The majority of HeLa cells seeded at 1.5 x 10⁴/cm² showed calcium oscillations in response to histamine and ATP, whereas cells seeded at 0.5 x 10⁴/cm² largely showed transient and sustained calcium increases. Cell density also affected the response of HEK293 cells to ATP in a similar manner. High cell density increased the basal activity of the mitogen-activated protein (MAP) kinase and calcium store content, and both calcium oscillation and calcium store content were down-regulated by a MAP kinase inhibitor, U0126. Thus, MAP kinase-mediated regulation of calcium store likely underlie the effect of cell density on calcium oscillation. Calcium increase patterns of HeLa cells were conserved at any histamine concentrations tested, whereas the overexpression of histamine H1 receptor, which robustly increased histamine-induced inositol phospholipid hydrolysis, converted calcium oscillations to sustained calcium increases only at high histamine concentrations. Thus, the consequence of modulating inositol phospholipid metabolism was distinct from that of changing cell density, suggesting the effect of cell density is not attributed to inositol phospholipid metabolism. Collectively, our results propose that calcium increase patterns of non-excitable cells reflect calcium store, which is regulated by the basal MAP kinase activity under the influence of cell density.     

Mitogen-activated protein kinase-activated protein (MAPKAP) kinase 2 deficiency protects brain from ischemic injury in mice

Mitogen-activated protein (MAP) kinase-activated protein kinase 2 (MK2) is one of several kinases directly regulated by p38 MAP kinase. A role of p38 MAP kinase in ischemic brain injury has been previously suggested by pharmacological means. In the present study, we provide evidence for a role of MK2 in cerebral ischemic injury using MK2-deficient (MK2(-/-)) mice. MK2(-/-) mice subjected to focal ischemia markedly reduced infarct size by 64 and 76% after transient and permanent ischemia, respectively, compared with wild-type mice. Furthermore, MK2(-/-) mice had significant reduction in neurological deficits. Real-time PCR analysis identified a significantly lower expression in interleukin-1beta mRNA (53% reduction) but not in tumor necrosis factor-alpha mRNA in MK2(-/-) mice over wild-type animals after ischemic injury. The significant reduction in interleukin-1beta was also confirmed in MK2(-/-) mice by enzyme-linked immunosorbent assay. The marked neuroprotection from ischemic brain injury in MK2(-/-) mice was not associated with the alteration of hemodynamic or systemic variables, activation of caspase-3, or apoptosis. Our data provide new evidence for the involvement of MAP kinase pathway in focal ischemic brain injury and suggest that this effect might be associated with the expression of interleukin-1beta in the ischemic brain tissue.     

Brain-specific expression of MAP2 detected using a cloned cDNA probe

We describe the isolation of a set of overlapping cDNAs encoding mouse microtubule associated protein 2 (MAP2), using an anti-MAP antiserum to screen a mouse brain cDNA expression library cloned in bacteriophage lambda gt11. The authenticity of these clones was established by the following criteria: (a) three non-identical clones each expressing a MAP2 immunoreactive fusion protein were independently isolated from the expression library; each of these clones cross-hybridized at the nucleic acid level; (b) anti-MAP antiserum was affinity purified using nitrocellulose-bound fusion protein; these antibodies detected only MAP2 in an immunoblot experiment of whole brain microtubule protein; (c) a series of cDNA "walking" experiments was done so as to obtain a non-overlapping cloned fragment corresponding to a different part of the same mRNA molecule. Upon subcloning this non-overlapping fragment into plasmid expression vectors, a fusion protein was synthesized that was immunoreactive with an anti-MAP2 specific antiserum. Thus, a single contiguous cloned mRNA molecule encodes at least two MAP2-specific epitopes; (d) the cloned cDNA probes detect an mRNA species in mouse brain that is of a size (approximately 9 kb) consistent with the coding capacity required by a 250,000-D protein. The MAP2-specific cloned cDNA probes were used in RNA blot transfer experiments to assay for the presence of MAP2 mRNA in a variety of mouse tissues. Though brain contained abundant quantities of MAP2 mRNA, no corresponding sequences were detectable in RNA prepared from liver, kidney, spleen, stomach, or thymus. We conclude that the expression of MAP2 is brain-specific. Use of the MAP2 specific cDNA probes in genomic Southern blot transfer experiments showed the presence of a single gene encoding MAP2 in mouse. The microheterogeneity of MAP2 is therefore ascribable either to alternative splicing within a single gene, or to posttranslational modification(s), or both. Under conditions of low stringency, the mouse MAP2 cDNA probe cross-hybridizes with genomic sequences from rat, human, and (weakly) chicken, but not with sequences in frog, Drosophila, or sea urchin DNA. Thus, there is significant interspecies divergence of MAP2 sequences. The implications of the above observations are discussed in relationship to the potential biological function of MAP2.     

Fibroblast growth factor (FGF)-2 directly stimulates mature osteoclast function through activation of FGF receptor 1 and p42/p44 MAP kinase

We previously reported that fibroblast growth factor-2 (FGF-2) acts not only on osteoblasts to stimulate osteoclastic bone resorption indirectly but also on mature osteoclasts directly. In this study, we investigated the mechanism of this direct action of FGF-2 on mature osteoclasts using mouse and rabbit osteoclast culture systems. FGF-2 stimulated pit formation resorbed by isolated rabbit osteoclasts moderately from low concentrations (>/=10(-12) m), whereas at high concentrations (>/=10(-9) m) it showed stimulation on pit formation resorbed by unfractionated bone cells very potently. FGF-2 (>/=10(-12) m) also increased cathepsin K and MMP-9 mRNA levels in mouse and rabbit osteoclasts. Among FGF receptors (FGFR1 to 4) only FGFR1 was detected on isolated mouse osteoclasts, whereas all FGFRs were identified on mouse osteoblasts. FGF-2 (>/=10(-12) m) up-regulated the phosphorylation of cellular proteins, including p42/p44 mitogen-activated protein (MAP) kinase, and increased the kinase activity of immunoprecipitated FGFR1 in mouse osteoclasts. The stimulation of FGF-2 on mouse and rabbit osteoclast functions was abrogated by PD-98059, a specific inhibitor of p42/p44 MAP kinase. These results strongly suggest that FGF-2 acts directly on mature osteoclasts through activation of FGFR1 and p42/p44 MAP kinase, causing the stimulation of bone resorption at physiological or pathological concentrations.     

Loss of p27 Expression Through RAS->BRAF->MAP Kinase-Dependent Pathway in Human Thyroid Carcinomas

In the present study, we report that the RAS->BRAF->MAP kinase cascade plays a crucial role in the regulation of the Skp2/p27 pathway in thyroid cancer cells and that this is critical for cell proliferation. In vitro studies with cellular models of human thyroid carcinoma cells demonstrated that the adoptive expression of oncogenic RET/PTC1, Ha-RASV12 or BRAFV600E enhances Skp2 and reduces p27 protein expression in a MAP kinase-dependent manner; that RAS->BRAF->MAP kinase–dependent control of p27 expression in thyroid cancer cells occurs by regulating the stability of Skp2 and p27 protein; and that antisense oligonucleotides to p27 suppress growth arrest induced by MEK inhibitors. Finally, analysis of human thyroid carcinomas indicated that MAP kinase-positive thyroid tumours—as detected by immunostaining for p-ERK—presented high p27 degradative activity and low levels of p27 protein (N=30; P