Biomedical subjects

L E Heasley

Publications and source records attributed to L E Heasley.

At least 19 recordsLinked to original sources

PubMed2001

Isoforms of Jun kinase are differentially expressed and activated in human monocyte/macrophage (THP-1) cells.

Ten isoforms of c-jun N-terminal kinase (JNK) have been described that arise by differential mRNA splicing of three genes. In that the relative expression and function of these different JNK proteins in human monocytic cells is not known, we have examined the JNK isoforms in THP-1 monocyte/macrophage cells. Differentiation of THP-1 cells by exposure to 10(-8) M PMA for 42-48 h enhances cellular responses to LPS, including enhanced activation of total JNK activity and increased phosphorylation of p54 JNK as well as p46 JNK. Examination of JNK proteins on Western blots reveals a predominance of p46 JNK1 and p54 JNK2 proteins. Clearing of lysates by immunoprecipitation of JNK1(99% effective) removes 46% of the JNK enzymatic activity (p < 0.01), whereas clearing of JNK1 plus JNK2 (70% effective) depletes the sample of 72% of the JNK activity (p < 0.01). Further analysis, undertaken with real-time RT-PCR, revealed that 98% of the JNK messages code for three isoforms: JNK1beta1, JNK2alpha1, and JNK2alpha2. The p54 JNK that is phosphorylated in LPS-stimulated, PMA-differentiated THP-1 cells is most likely JNK2alpha2 because 97% of the p54 JNK-encoding messages code for JNK2alpha2. By analogous reasoning, the p46 JNKs that are not heavily phosphorylated, but account for approximately half of the N-terminal c-jun kinase enzymatic activity, are most likely either JNK1beta1 or JNK2alpha1 because they account for 98% of the messages that can code for 46kDa JNKS:

Blotting, Western↗

PubMed2001

Autocrine and paracrine signaling through neuropeptide receptors in human cancer.

Autocrine and paracrine signaling leading to stimulation of tumor cell growth is a common theme in human cancers. In addition to polypeptide growth factors such as EGF family members which signal through receptor tyrosine kinases, accumulating evidence supports the autocrine and paracrine involvement of specific neuropeptides with defined physiologic actions as neurotransmitters and gut hormones in lung, gastric, colorectal, pancreatic and prostatic cancers. These neuropeptides, including gastrin-releasing peptide, neuromedin B, neurotensin, gastrin, cholecystokinin and arginine vasopressin bind seven transmembrane-spanning receptors that couple to heterotrimeric G proteins. Studies with human small cell lung cancer (SCLC) cells support a requirement for balanced signaling through G(q) and G(12/13) proteins leading to intracellular Ca2+ mobilization, PKC activation and regulation of the ERK and JNK MAP kinase pathways. While specific neuropeptide antagonists offer promise for interrupting the single neuropeptide autocrine systems operating in pancreatic and prostatic cancers, SCLC is exemplified by multiple, redundant neuropeptide autocrine systems such that tumor growth cannot be inhibited with a single specific antagonist. However, a novel class of neuropeptide derivatives based on the substance P sequence have been defined that exhibit broad specificity for neuropeptide receptors and induce apoptosis in SCLC by functioning as biased agonists that stimulate discordant signal transduction. Thus, interruption of autocrine and paracrine neuropeptide signaling with specific antagonists or broad-spectrum biased agonists offer promising new therapeutic approaches to the treatment of human cancers.

Antineoplastic Agents↗

PubMed2001

Induction of cytosolic phospholipase A2 by oncogenic Ras is mediated through the JNK and ERK pathways in rat epithelial cells.

Mutations in ras genes have been detected with high frequency in nonsmall cell lung cancer cells (NSCLC) and contribute to transformed growth of these cells. It has previously been shown that expression of oncogenic forms of Ras in these cells is associated with elevated expression of cytosolic phospholipase A(2) (cPLA(2)) and cyclooxygenase-2 (COX-2), resulting in high constitutive levels of prostaglandin production. To determine whether expression of constitutively active Ras is sufficient to induce expression of these enzymes in nontransformed cells, normal lung epithelial cells were transfected with H-Ras. Stable expression of H-Ras increased expression of cPLA(2) and COX-2 protein. Transient transfection with H-Ras increased promoter activity for both enzymes. H-Ras expression also activated all three families of MAP kinase: ERKs, JNKs, and p38 MAP kinase. Expression of constitutively active Raf did not increase either cPLA(2) or COX-2 promoter activity, but inhibition of the ERK pathway with pharmacological agents or expression of dominant negative ERK partially blocked the H-Ras-mediated induction of cPLA(2) promoter activity. Expression of dominant negative JNK kinases decreased cPLA(2) promoter activity in NSCLC cell lines and inhibited H-Ras-mediated induction in normal epithelial cells, whereas expression of constructs encoding constitutively active JNKs increased promoter activity. Inhibition of p38 MAP kinase or NF-kappaB had no effect on cPLA(2) expression. Truncational analysis revealed that the region of the cPLA(2) promoter from -58 to +12 contained sufficient elements to mediate H-Ras induction. We conclude that expression of oncogenic forms of Ras directly increases cPLA(2) expression in normal epithelial cells through activation of the JNK and ERK pathways.

Animals↗

PubMed2000

Akt negatively regulates the cJun N-terminal kinase pathway in PC12 cells.

The protein serine/threonine kinase Akt is a target of phosphatidylinositol 3-kinase that mediates many of the trophic actions of growth factors on cells. In PC12 cells, complete removal of serum leads to rapid stimulation of the cJun N-terminal kinase (JNK) pathway. Inclusion of insulin-like growth factor-1, a stimulator of Akt in PC12 cells, inhibits JNK activation in this setting, whereas addition of wortmannin to PC12 cells in the presence of serum stimulates JNK activity, suggesting that growth factor-mediated signaling through the phosphatidylinositol 3-kinase/Akt pathway chronically inhibits the JNK pathway in PC12 cells. To explore the possible role of Akt as a negative regulator of JNK activity in PC12 cells, a myristoylated, gain-of-function Akt polypeptide (Myr-Akt) was expressed by retrovirus-mediated gene transfer. Stimulation of JNK activity by serum withdrawal or UV irradiation in PC12 cell clones stably expressing Myr-Akt was inhibited approximately 95% or 50%, respectively, relative to control transfected PC12 cells. Phosphorylation of both JNKs and a proximal activator, MAP kinase kinase 4 (MKK4), in response to UV irradiation was inhibited in Myr-Akt-expressing PC12 cells. Furthermore, transient expression of Myr-Akt strongly inhibited cJun transactivation mediated by MEKK1 or MKK7-JNK3, a gain-of-function MKK7-JNK fusion protein. Interestingly, inhibited JNK activation in the Myr-Akt-expressing PC12 cells is associated with marked induction of JNK-interacting protein-1 (JIP-1). We propose that negative regulation of the JNK pathway through Akt-dependent induction of specfic JIP proteins contributes to the antiapoptotic actions of Akt in neuronal cell types.

Adaptor Proteins, Signal Transducing↗

PubMed2000

MAPK mediation of hypertonicity-stimulated cyclooxygenase-2 expression in renal medullary collecting duct cells.

We have previously shown that hypertonicity stimulates cyclooxygenase-2 (COX-2) expression in cultured medullary epithelial cells. The aims of the present study were (i) to examine the role of cytoplasmic signaling through MAPK pathways in tonicity regulation of COX-2 expression in collecting duct cells and (ii) to assess the possible contribution of COX-2 to the survival of inner medullary collecting duct (IMCD) cells under hypertonic conditions. In mIMCD-K2 cells, a cell line derived from mouse IMCDs, hypertonicity induced a marked increase in COX-2 protein expression. The stimulation was reduced significantly by inhibition of MEK1 (PD-98059, 5-50 microm) and p38 (SB-203580, 5-100 microm) and was almost abolished by the combination of the two compounds. To study the role of JNK in tonicity-stimulated COX-2 expression, IMCD-3 cell lines stably transfected with dominant-negative mutants of three JNKs (JNK-1, -2, and -3) were used. Hypertonicity-stimulated COX-2 protein expression was significantly reduced in dominant-negative JNK-2-expressing cells and was unchanged in dominant-negative JNK-1- and JNK-3-expressing cells compared with controls. The reduction of COX-2 expression was associated with greatly reduced viability of dominant-negative JNK-2-expressing cells during hypertonicity treatment. 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) (2-8 microm), an inhibitor of Src kinases, reduced the tonicity-stimulated COX-2 expression in a dose-dependent manner, whereas PP3, an inactive analog of PP2, had no effect. Inhibition of COX-2 activity by NS-398 (30-90 microm) and SC-58236 (10-20 microm) significantly reduced viability of mIMCD-K2 cells subjected to prolonged hypertonic treatment. We conclude that 1) all three members of the MAPK family (ERK, JNK-2, and p38) as well as Src kinases are required for tonicity-stimulated COX-2 expression in mouse collecting duct cells and that 2) COX-2 may play a role in cell survival of medullary cells under hypertonic conditions.

Animals↗

PubMed2000

TRH regulates Kv1.5 gene expression through a Galphaq-mediated PLC-independent pathway.

Thyrotropin-releasing hormone (TRH) decreases transcription of the Kv1.5 K(+) channel gene in GH(3) pituitary cells. Here, we examine whether TRH utilizes Gq activated phospholipase C, Gs or Gi to produce this response. We report that expression of constitutively active Galphaq mimicked and occluded the TRH effect. In contrast, expression of activated Galpha(S) or Galpha(i2) had no effect on Kv1. 5 mRNA expression. Furthermore, pertussis and cholera toxins failed to block the TRH-induced decrease in channel mRNA. Surprisingly, despite the role of Gq, the phospholipase C inhibitor U73122 did not alter down-regulation of channel mRNA by TRH, although it abolished the TRH-induced increase in intracellular [Ca(2+)] and up-regulation of c-fos mRNA. Furthermore, depletion of an intracellular Ca(2+) pool or inhibition of protein kinase C did not block the TRH-induced decrease in Kv1.5 mRNA. These results indicate that TRH-induced down-regulation of Kv1.5 gene expression is mediated by Galphaq proteins, but does not require PLC activation.

Animals↗

PubMed2000

Akt/protein kinase B up-regulates Bcl-2 expression through cAMP-response element-binding protein.

In our previous study we showed that insulin-like growth factor-I induces a cAMP-response element (CRE) site-containing Bcl-2 promoter through a novel signaling pathway involving mitogen-activated protein kinase kinase 6/p38beta mitogen-activated protein kinase/MAP kinase-activated protein kinase-3/cAMP-response element-binding protein (CREB) (Pugazhenthi, S., Miller, E., Sable, C., Young, P., Heidenreich, K. A., Boxer, L. M., and Reusch, J. E.-B. (1999) J. Biol. Chem. 274, 27529-27535). In the present investigation, we define a second pathway contributing to CREB-dependent up-regulation of Bcl-2 expression as a novel anti-apoptotic function of Akt signaling. To examine the role of Akt on Bcl-2 expression, a series of transient transfections using a luciferase reporter gene driven by the promoter region of Bcl-2 containing a CRE were carried out. Pharmacological inhibition of phosphatidylinositol (PI) 3-kinase, the upstream kinase of Akt, with LY294002 led to a 45% decrease in Bcl-2 promoter activity. The reporter activity was enhanced 2.3-fold by overexpression of active p110 subunit of PI 3-kinase and inhibited 44% by the dominant negative p85 subunit of PI 3-kinase. Cotransfection with 3-phosphoinositide-dependent kinase (PDK1), which is required for the full activation of Akt, resulted in enhanced luciferase activity. Insulin-like growth factor-I-mediated induction of Bcl-2 promoter activity was decreased significantly (p < 0.01) by the dominant negative forms of p85 subunit of PI 3-kinase, PDK1, and Akt. These data indicate that regulation of Bcl-2 expression by IGF-I involves a signaling cascade mediated by PI 3-kinase/PDK1/Akt/CREB. Furthermore, we measured the Bcl-2 mRNA in PC12 cells overexpressing Akt by real-time quantitative reverse transcription-polymerase chain reaction using the TaqMan(TM) fluorogenic probe system. We observed a 2.1-fold increase in Bcl-2 mRNA levels in the Akt cell line compared with control PC12 cells, supporting the observation that enhanced CREB activity by Akt signaling leads to increased Bcl-2 promoter activity and cell survival.

Animals↗

PubMed2000

Prolactin stimulates activation of c-jun N-terminal kinase (JNK).

In recent years the mitogen-activated protein (MAP) kinase family has expanded to include both c-jun N-terminal kinases (JNKs), and the p38/HOG1 family in addition to the extracellular regulated kinase (ERK) family. These kinases are activated by a variety of growth factors, as well as extra- and intracellular insults such as osmotic stress, UV light, and chemotherapeutic agents. Stimulation of the PRL-dependent Nb2 cell line with PRL results in the rapid activation of JNK as determined by the glutathione-S-transferase (GST)-jun kinase assay. Activation was maximal 30 min after stimulation with 50 nM rat PRL (rPRL) and decreased after that time. Dose response studies indicated that concentrations as low as 10 nM rPRL resulted in maximal activation. The interleukin-3 (IL-3)-dependent myeloid progenitor cell line 32Dcl3 was transfected with the long, Nb2, and short forms of the rat PRL receptor (rPRLR), as well as the long form of the human PRLR (hPRLR). The long and Nb2 forms of the PRLR were able to stimulate activation of JNK; however, the short form of the rPRLR was not. This corresponds with the inability of the short form of the rPRLR to stimulate proliferation of 32Dcl3 cells. Activation of JNK in 32Dcl3 cells expressing the long form of the hPRLR was maximal at 30 min after stimulation with 100 nM ovine PRL (oPRL) and declined after that time. Dose response studies indicated that activation of JNK was maximal after 30 min at a concentration of 10 nM, and the amount of activated JNK declined at the highest concentration of oPRL, 100 nM. Immunoblot analysis with an antibody that recognizes the activated (phosphorylated) forms of JNK1 and JNK2 indicated that both JNK1 and JNK2 isoforms were activated in 32D/hPRLR cells stimulated with oPRL. A recombinant human adenovirus expressing a kinase-inactive mutant of JNK1 (APF mutant) was used to determine the biological effect of blocking JNK activity in Nb2 cells. Expression of the JNK1-APF mutant inhibited cellular proliferation and induced DNA fragmentation typical of cells undergoing apoptosis. These data suggest that activation of JNKs may be important in mitogenic signaling and/or suppression of apoptosis in Nb2 cells.

Animals↗

PubMed1999

Synergistic effects of new chemopreventive agents and conventional cytotoxic agents against human lung cancer cell lines.

Non-small cell lung cancer (NSCLC) cells have constitutively high expression of cytosolic phospholipase A2 (cPLA2) and cyclooxygenase (COX) 2. These NSCLC cells also have increased prostaglandin expression (PGE2). Many lung cancers also express 12-lipoxygenase RNA and 12-lipoxygenase protein and biosynthesize 12(S)-hydroxyeicosatetraenoic acid, which correlates with their metastatic potential. Several studies have demonstrated that COX-1 and COX-2 inhibitors could inhibit the in vitro growth of human lung cancer cell lines. In this report, we evaluated the growth-inhibitory effects of sulindac sulfide, a COX-1 and COX-2 inhibitor; exisulind (sulindac sulfone), a novel proapoptotic agent that does not inhibit COX enzymes; and nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor on human lung cancer cell lines. We compared these effects with those of 13-cis-retinoic acid, a chemoprevention agent, and with the cytotoxic chemotherapeutic agents paclitaxel and cisplatin, alone or in combination. Our goal was to develop new chemoprevention and treatment strategies. Each of the six agents tested inhibited the in vitro growth of three NSCLC and three SCLC cell lines at the highest concentration. Paclitaxel was the most potent agent (IC50 = 0.003-0.150 microM); sulindac sulfide, NDGA, and 13-cis-retinoic acid had intermediate potency (IC50 = 4-80 microM), and cisplatin and exisulind were the least potent (IC50 = 150-500 microM). Combination studies showed synergistic interactions for sulindac sulfide, exisulind, and NDGA with paclitaxel, cisplatin, and 13-cis-retinoic acid, regardless of drug-resistance phenotype. At high concentrations, the combination of 13-cis-retinoic acid and each of the five other drugs resulted in a strong synergistic effect. These studies provide a rationale for chemoprevention (exisulind +/- retinoic acid +/- NDGA) and therapeutic (exisulind +/- paclitaxel +/- cisplatin) studies in patients at risk for, or with, lung cancer.

Antineoplastic Agents↗

PubMed1999

Stress- and cell type-dependent regulation of transfected c-Jun N-terminal kinase and mitogen-activated protein kinase kinase isoforms.

The cJun N-terminal kinases (JNKs) are encoded by three genes generating ten protein kinase polypeptides and are activated in settings of cell stress, mitogenesis, differentiation and morphogenesis. The specific role of the JNK family members in these diverse cell programmes is largely undefined. In this study, we tested the hypothesis that individual JNK isoforms would exhibit distinct patterns of regulation within cells. The cDNAs encoding five haemagglutinin (HA)-tagged JNK isoforms (p46JNK1alpha, p54JNK2alpha, p54JNK2beta, p46JNK3 and p54JNK3) were expressed in cultured rat PC12 phaeochromocytoma cells and human small-cell lung cancer (SCLC) cells by retrovirus-mediated gene transfer. In addition, HA-tagged forms of the dual-specificity mitogen-activated protein kinase kinases (MKKs), MKK4 and MKK7, which are specific activators of the JNK enzymes, were similarly expressed. Reverse transcription and PCR revealed that JNK3 is endogenously expressed in SCLC cells, but not in either chromaffin or neuronally differentiated PC12 cells. MKK4 and MKK7 were endogenously expressed in both PC12 cells and SHP77 cells. Immunoprecipitation and analysis of the JNKs expressed in SCLC cells revealed strong stimulation of all five JNK isoforms by UV radiation. Hypertonic stress, elicited by mannitol, also significantly stimulated these same JNKs, although the JNK3 isoforms were most strongly activated. In PC12 cell transfectants, however, selective and equal activation of p54JNK2alpha and p54JNK3 by UV and osmotic stress was observed, with little or no activation of JNK1alpha or JNK2beta. In contrast with the broad activation of the JNK enzymes by UV in SCLC cells, only HA-MKK4 was stimulated by UV exposure in these cells, whereas osmotic stress stimulated both HA-MKK4 and HA-MKK7. These findings indicate selective activation of JNK and MKK isoforms in a manner that is dependent upon the specific cell stress and the cell type.

Animals↗

PubMed1998

In vivo regulation of MAP kinases in Ratus norvegicus renal papilla by water loading and restriction.

In cultured renal cells, hypertonicity activates multiple mitogen-activated protein kinases (MAPKs) and enhances the expression of heat shock proteins (HSPs). In rats, 24 h water restriction increased mean urinary osmolality (Uosm) from 2, 179+/-153 mOsm/kg to 2,944+/-294 mOsm/kg (P < 0.001) and was associated with significant (P < 0.05) increases in the papillary activity of c-Jun NH2-terminal protein kinase (JNK) by 22%, extracellular signal-regulated protein kinase (ERK) by 49%, and p38 MAPK by 15%. Conversely, 24 h of water-loading (Uosm 473+/-33 mOsm/kg) caused suppression of JNK activity by 43% (P < 0.001), ERK by 39% (P < 0.05), and p38 MAPK by 26% (P < 0.05). No such modulation was observed in the isotonic cortex. c-Jun phosphorylation was decreased in papilla from water-loaded rats by 45% versus controls. Expression of Hsp 110, inducible Hsp 70, and Hsp 25 was greater in the hyperosmotic papilla than the isosmotic cortex but was not affected by the animal's hydration state. In cultured inner medullary collecting duct cells, HSP expression was maximal at 500 mOsm/kg, while activation of JNK continued to increase. We conclude that under basal conditions of hydration, these HSPs are maximally expressed in the hypertonic inner medulla, while the activation of all three members of the MAPK family approaches but is not maximal.

Animals↗

PubMed1998

Expression of catalytically inactive phospholipase Cbeta disrupts phospholipase Cbeta and mitogen-activated protein kinase signaling and inhibits small cell lung cancer growth.

Transformed growth of human small cell lung cancer (SCLC) is mediated by autocrine signaling through multiple G protein-coupled neuropeptide receptors. To define the role of Gq and its effector, phospholipase Cbeta (PLCbeta), in SCLC growth, we expressed a COOH-terminal fragment of PLCbeta1 (PLCbetaCT) that is catalytically inactive and is predicted to behave as a competitive inhibitor of Gq signaling. Using endogenous muscarinic receptors as indicators of Gq-coupled receptor signaling status, we observed that stable expression of PLCbetaCT in NCI-H345 SCLC cells significantly inhibited muscarinic receptor-mediated phospholipase C activation and intracellular Ca2+ mobilization. In addition, PLCbetaCT expression reduced the basal activity of protein kinase C as well as the receptor-stimulated activity of the extracellular signal-regulated kinases, consistent with the sequential requirement for Gq, PLCbeta, and protein kinase C in the regulation of the extracellular signal-regulated kinases by neuropeptide and muscarinic receptors in SCLC. By contrast, muscarinic agonist stimulation of the c-Jun NH2-terminal kinases was not inhibited in SCLC cells expressing PLCbetaCT, indicating that other G proteins such as the G12,13 family members mediate c-Jun NH2-terminal kinase activation by neuropeptides and muscarinic agonists. Finally, soft agar colony formation by the SCLC cells expressing PLCbetaCT, but not growth in suspension culture, was markedly reduced, indicating that signaling through Gq and PLCbeta by autocrine-signaling neuropeptide receptors is a dominant pathway involved in the transformed growth of SCLC.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1998

Dominant-negative c-Jun NH2-terminal kinase 2 sensitizes renal inner medullary collecting duct cells to hypertonicity-induced lethality independent of organic osmolyte transport.

The c-Jun NH2-terminal protein kinases (JNKs), as well as the extracellular signal-regulated protein kinases (ERKs) and p38 mitogen-activated protein kinase, are activated in renal cells in response to extracellular hypertonicity. To determine whether activation of JNKs by hypertonicity is isoform-specific, renal inner medullary collecting duct cells were stably transfected with cDNA's encoding hemagglutinin (HA)-tagged JNK1 and JNK2 isoforms, and the expressed kinases were immunoprecipitated with an anti-HA antibody. Whereas both recombinant kinases were equivalently expressed, only immunoprecipitates from the HA-JNK2 cells displayed hypertonicity-inducible JNK activity. Furthermore, expression of dominant-negative JNK2 (HA-JNK2-APF) in stable clones inhibited hypertonicity-induced JNK activation by 40-70%, whereas expression of dominant-negative JNK1 (HA-JNK1-APF) had no significant inhibitory effect. Independent HA-JNK2-APF (but not HA-JNK1-APF) clones displayed greatly reduced viability relative to neomycin controls after 16 h of exposure to 600 mosM/kg hypertonic medium with percent survival of 20.5 +/- 2.7 and 31.5 +/- 7.3 for two independent HA-JNK2-APF clones compared with 80.1 +/- 1.0 for neomycin controls (p < 0.001, n = 5, mean +/- S.E.). However, neither JNK mutant blocked either regulatory volume increase or hypertonicity-induced enhancement of uptake of inositol, an organic osmolyte putatively involved in long term adaptation to hypertonicity. These results define JNK2 as the primary hypertonicity-activated JNK isoform in IMCD-3 cells and demonstrate its central importance in cellular survival in a hypertonic environment by a mechanism independent of acute regulatory volume increase as well as regulation of organic osmolyte uptake.

Adaptation, Physiological↗

PubMed1997

Suppression of smooth-muscle alpha-actin expression by platelet-derived growth factor in vascular smooth-muscle cells involves Ras and cytosolic phospholipase A2.

Platelet-derived growth factor (PDGF), which is a potent mitogen for vascular smooth-muscle cells (VSMC), also inhibits the expression of specific smooth-muscle proteins, including smooth-muscle alpha-actin (SM-alpha-actin), in these cells. The goal of this study was to identify signalling pathways mediating these distinct effects. In rat aortic VSMC, PDGF caused a rapid activation of Ras and Raf, leading to the activation of mitogen-activated protein kinases (ERKs). Cells stably transfected with constitutively active Ras (H-Ras) expressed low levels of SM-alpha-actin protein. Arginine vasopressin, which stimulated SM-alpha-actin promoter activity in wild-type cells or controls (Neo; transfected with a plasmid lacking an insert), failed to do so in cells transiently expressing H-Ras. The effects of Ras on suppression of SM-alpha-actin expression were not mediated by the Raf/ERK pathway, since cells stably expressing constitutively active Raf (BxB-Raf) had normal levels of SM-alpha-actin protein, and stimulation of SM-alpha-actin promoter activity by vasopressin was unaffected in cells transiently expressing BxB-Raf. Furthermore a specific inhibitor of ERK activation had no effect on SM-alpha-actin expression. Exposure of wild-type VSMC to PDGF, or stable expression of Ras but not Raf, also resulted in constitutive increases in prostaglandin E2 production and cytosolic phospholipase A2 (cPLA2) activity, which was mediated by an increased expression of cPLA2 protein. Transient expression of cPLA2 in wild-type VSMC inhibited the stimulation of SM-alpha-actin promoter activity by vasopressin. These results suggest that PDGF-induced inhibition of SM-alpha-actin expression is mediated through a Ras-dependent/Raf independent pathway involving the induction of cPLA2 and eicosanoid production.

Actins↗

PubMed1997

Induction of cytosolic phospholipase A2 by oncogenic Ras in human non-small cell lung cancer.

Mutations in Ras family members that confer oncogenic potential are frequently observed in specific human cancers. We report that human non-small cell lung cancer (NSCLC) lines that harbor oncogenic mutations in Ki-Ras (H460, A549, H2122) synthesized high levels of prostaglandin E2 (PGE2) compared with NSCLC lacking Ras mutations or non-transformed lung epithelial cells (BEAS-2B). This increased PGE2 production was mediated by constitutively high expression of 85-kDa cytosolic phospholipase A2 (cPLA2) and cyclooxygenase 2 (COX-2). The increased expression of cPLA2 protein was mediated through elevated mRNA levels and activation of the cPLA2 promoter. Induction of cPLA2 promoter activity was blocked by expression of dominant-negative forms of Ras. Inhibition of Ras by the farnesyltransferase inhibitor BZA-5B inhibited prostaglandin synthesis in H2122 cells by decreasing expression of both cPLA2 and COX-2. Finally, inhibitors of eicosanoid synthesis blocked anchorage-independent growth of NSCLC lines exhibiting Ki-Ras mutations. These results identify cPLA2 as a novel Ras-inducible regulator of eicosanoid synthesis that participates in cellular transformation.

Carcinoma, Non-Small-Cell Lung↗

PubMed1997

c-Jun NH2-terminal kinase regulation of the apoptotic response of small cell lung cancer cells to ultraviolet radiation.

Exposure of cultured small cell lung cancer (SCLC) cells to UV radiation induces apoptosis. We observed that the UV sensitivity of a panel of SCLC lines and the activation of c-Jun NH2-terminal kinases (JNKs) by UV in the individual SCLC lines, assessed by binding and phosphorylation of glutathione S-transferase (GST)-c-Jun fusion proteins, ranged widely. In fact, increased JNK activity in this assay was closely correlated with decreased sensitivity to apoptosis following UV irradiation. Increased JNK activity was also detected in anti-JNK1 immune complexes collected from UV-irradiated SCLC cells, although the level of activity was similar among the various SCLC lines and correlated poorly with UV sensitivity. Immunoblot analysis of JNK polypeptides that bound to GST-c-Jun revealed at least two JNK polypeptides, one of which appeared only in extracts from UV-irradiated SCLC. To test the role of JNKs in UV-induced apoptosis, nonphosphorylatable mutants of JNK1 or JNK2 in which the phosphorylation site Thr-Pro-Tyr is changed to Ala-Pro-Phe (JNK-APF) and are predicted to behave as competitive inhibitors were stably expressed in SCLC. Expression of JNK1-APF or JNK2-APF significantly reduced UV-stimulated JNK activity. However, JNK1-APF markedly increased the resistance of the cells to UV-induced apoptosis, while JNK2-APF did not influence SCLC sensitivity to UV. The findings suggest that UV-stimulated JNK1 activation promotes UV-induced SCLC apoptosis, while a JNK isoform that is variably activated among the SCLC lines may signal a UV-protective response. We hypothesize that integration of distinct JNK activities dictates the relative responsiveness of SCLC to UV and ionizing radiation.

Apoptosis↗

PubMed1997

Analysis of mutant platelet-derived growth factor receptors expressed in PC12 cells identifies signals governing sodium channel induction during neuronal differentiation.

The mechanisms governing neuronal differentiation, including the signals underlying the induction of voltage-dependent sodium (Na+) channel expression by neurotrophic factors, which occurs independent of Ras activity, are not well understood. Therefore, Na+ channel induction was analyzed in sublines of PC12 cells stably expressing platelet-derived growth factor (PDGF) beta receptors with mutations that eliminate activation of specific signalling molecules. Mutations eliminating activation of phosphatidylinositol 3-kinase (PI3K), phospholipase C gamma (PLC gamma), the GTPase-activating protein (GAP), and Syp phosphatase failed to diminish the induction of type II Na+ channel alpha-subunit mRNA and functional Na+ channel expression by PDGF, as determined by RNase protection assays and whole-cell patch clamp recording. However, mutation of juxtamembrane tyrosines that bind members of the Src family of kinases upon receptor activation inhibited the induction of functional Na+ channels while leaving the induction of type II alpha-subunit mRNA intact. Mutation of juxtamembrane tyrosines in combination with mutations eliminating activation of PI3K, PLC gamma, GAP, and Syp abolished the induction of type II alpha-subunit mRNA, suggesting that at least partially redundant signaling mechanisms mediate this induction. The differential effects of the receptor mutations on Na+ channel expression did not reflect global changes in receptor signaling capabilities, as in all of the mutant receptors analyzed, the induction of c-fos and transin mRNAs still occurred. The results reveal an important role for the Src family in the induction of Na+ channel expression and highlight the multiplicity and combinatorial nature of the signaling mechanisms governing neuronal differentiation.

Animals↗

PubMed1997

Multiple mitogen-activated protein kinases are regulated by hyperosmolality in mouse IMCD cells.

Inner medullary collecting duct (IMCD) cells adapt to a hypertonic environment by synthesizing transporters that allow for accumulation of organic osmolytes. To examine for activation of additional mitogen-activated protein (MAP) kinases, extracts of IMCD-3 cells subjected to a hypertonic medium (600 mosmol/kgH2O) for 15 min were fractionated by Mono Q fast-performance liquid chromatography and assayed with the epidermal growth factor receptor [EGFR-(662-681)] peptide as substrate. Three peaks of activity were identified. Western blotting revealed that these peaks coincided with Jun NH2-terminal kinase (JNK), extracellular signal-regulated protein kinases, ERK1 and ERK2, and p38 MAP kinase. To assess the functional significance of ERK2 activation in IMCD-3 cells, the effect of PD-098059, an inhibitor of the upstream regulatory protein kinase MAP/ERK kinase (MEK) was assessed. PD-098059 inhibited ERK activation by hypertonicity. Yet, the stimulation of inositol uptake, a marker of adaptation, after 16 h was unaltered. Direct measurements of JNK activity [phosphorylation of GST-cJun-(1-79)] revealed a marked (20- to 40-fold) increase in activity as medium osmolality was increased from 300 to 900 mosmol/kgH2O with either NaCl or mannitol. Urea induced a more modest increase in activity. The response is prompt and detected as early as 2 min after exposure, reaching a maximum activation at 10-15 min. Downregulation of cellular protein kinase C (PKC) by chronic exposure to phorbol esters only minimally attenuated the JNK response to hyperosmolality, indicating a lack of involvement of PKC. We conclude that, in IMCD-3 cells, inhibition of ERK activation by hyperosmolality does not prevent osmoregulatory increase in inositol transport. This is not consistent with a role for ERKs in the response. The roles for JNK and p38 have not been ruled out, and these pathways may represent the initiating event in the subsequent transcription of organic osmolyte transporter genes and adaptation to extracellular hypertonicity.

Animals↗