Morphological transformation induced by activation of the mitogen-activated protein kinase pathway requires suppression of the T-type Ca2+ channel.

Transformation of fibroblasts by various oncogenes, including ras, mos, and src accompanies with characteristic morphological changes from flat to round (or spindle) shapes. Such morphological change is believed to play an important role in establishing malignant characteristics of cancer cells. Activation of the mitogen-activated protein kinase (MAPK) pathway is a converging downstream event of transforming activities of many oncogene products commonly found in human cancers. Intracellular calcium is known to regulate cellular morphology. In fibroblasts, Ca2+ influx is primarily controlled by two types of Ca2+ channels (T- and L-types). Here, we report that the T-type current was specifically inhibited in cells expressing oncogenically activated Ras as well as gain-of-function mutant MEK (MAPK/extracellular signal-regulated kinase (ERK) kinase, a direct activator of MAPK), whereas treatment of ras-transformed cells with a MEK-specific inhibitor restored T-type Ca2+ channel activity. Using a T-type Ca2+ channel antagonist, we further found that suppression of the T-type Ca2+ channel by the activated MAPK pathway is a prerequisite event for the induction and/or maintenance of transformation-associated morphological changes.     

Disruption of the murine protein kinase Cbeta gene promotes gallstone formation and alters biliary lipid and hepatic cholesterol metabolism

The protein kinase C (PKC) family of Ca(2+) and/or lipid-activated serine-threonine protein kinases is implicated in the pathogenesis of obesity and insulin resistance. We recently reported that protein kinase Cβ (PKCβ), a calcium-, diacylglycerol-, and phospholipid-dependent kinase, is critical for maintaining whole body triglyceride homeostasis. We now report that PKCβ deficiency has profound effects on murine hepatic cholesterol metabolism, including hypersensitivity to diet-induced gallstone formation. The incidence of gallstones increased from 9% in control mice to 95% in PKCβ(-/-) mice. Gallstone formation in the mutant mice was accompanied by hyposecretion of bile acids with no alteration in fecal bile acid excretion, increased biliary cholesterol saturation and hydrophobicity indices, as well as hepatic p42/44(MAPK) activation, all of which enhance susceptibility to gallstone formation. Lithogenic diet-fed PKCβ(-/-) mice also displayed decreased expression of hepatic cholesterol-7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CYP8b1). Finally, feeding a modified lithogenic diet supplemented with milk fat, instead of cocoa butter, both increased the severity of and shortened the interval for gallstone formation in PKCβ(-/-) mice and was associated with dramatic increases in cholesterol saturation and hydrophobicity indices. Taken together, the findings reveal a hitherto unrecognized role of PKCβ in fine tuning diet-induced cholesterol and bile acid homeostasis, thus identifying PKCβ as a major physiological regulator of both triglyceride and cholesterol homeostasis.     

Gonadotropin-releasing hormone induction of extracellular-signal regulated kinase is blocked by inhibition of calmodulin

Our previous studies demonstrate that GnRH-induced ERK activation required influx of extracellular Ca2+ in alphaT3-1 and rat pituitary cells. In the present studies, we examined the hypothesis that calmodulin (Cam) plays a fundamental role in mediating the effects of Ca2+ on ERK activation. Cam inhibition using W7 was sufficient to block GnRH-induced reporter gene activity for the c-Fos, murine glycoprotein hormone alpha-subunit, and MAPK phosphatase (MKP)-2 promoters, all shown to require ERK activation. Inhibition of Cam (using a dominant negative) was sufficient to block GnRH-induced ERK but not c-Jun N-terminal kinase activity activation. The Cam-dependent protein kinase (CamK) II inhibitor KN62 did not recapitulate these findings. GnRH-induced phosphorylation of MAPK/ERK kinase 1 and c-Raf kinase was blocked by Cam inhibition, whereas activity of phospholipase C was unaffected, suggesting that Ca2+/Cam modulation of the ERK cascade potentially at the level of c-Raf kinase. Enrichment of Cam-interacting proteins using a Cam agarose column revealed that c-Raf kinase forms a complex with Cam. Reconstitution studies reveal that recombinant c-Raf kinase can associate directly with Cam in a Ca2+-dependent manner and this interaction is reduced in vitro by addition of W7. Cam was localized in lipid rafts consistent with the formation of a Ca2+-sensitive signaling platform including the GnRH receptor and c-Raf kinase. These data support the conclusion that Cam may have a critical role as a Ca2+ sensor in specifically linking Ca2+ flux with ERK activation within the GnRH signaling pathway.     

Cholesterol induces pancreatic β cell apoptosis through oxidative stress pathway

Type 2 diabetes is often associated with high blood cholesterol. Here, we investigated the effect of cholesterol loading on MIN6 cells derived from pancreatic β cells. Exposure of MIN6 cells to cholesterol-induced apoptosis in time- and dose-dependent manner. Treatment with methyl-β-cyclodextrin that removes cholesterol from plasma membrane prevented the cells from cholesterol-induced apoptosis. Western blot analysis revealed that the levels of phosphorylated-p38 mitogen-activated protein kinase (P-p38 MAPK) and c-Jun N-terminal kinases (P-JNK) were significantly increased after the cholesterol loading, suggesting that the stress-activated protein kinase signaling was stimulated. A specific p38 inhibitor rescued MIN6 cells from cholesterol-induced apoptosis, while JNK inhibitor failed, suggesting the importance of activation of p38 MAPK signaling in response to cholesterol. The expression of Bip and CHOP, the endoplasmic reticulum (ER) stress markers, remained unaffected, indicating that the ER stress may not be involved in the cytotoxicity of cholesterol on the ΜΙΝ6 cells. The intracellular concentration of reactive oxygen species measured by use of 2′,7′-dichlorofluorescin diacetate was significantly increased after cholesterol loading, demonstrating the induced apoptosis was mediated through oxidative stress. Addition of reduced form of glutathione in the medium rescued MIN6 cells from apoptosis induced by cholesterol loading. Taken together, these results demonstrate that the free cholesterol loading can induce apoptosis of MIN6 cells mediated by oxidative stress and the activation of p38 MAPK signaling.     

Cholesterol Perturbation in Mice Results in p53 Degradation and Axonal Pathology through p38 MAPK and Mdm2 Activation

Perturbation of lipid metabolism, especially of cholesterol homeostasis, can be catastrophic to mammalian brain, as it has the highest level of cholesterol in the body. This notion is best illustrated by the severe progressive neurodegeneration in Niemann-Pick Type C (NPC) disease, one of the lysosomal storage diseases, caused by mutations in the NPC1 or NPC2 gene. In this study, we found that growth cone collapse induced by genetic or pharmacological disruption of cholesterol egress from late endosomes/lysosomes was directly related to a decrease in axonal and growth cone levels of the phosphorylated form of the tumor suppressor factor p53. Cholesterol perturbation-induced growth cone collapse and decrease in phosphorylated p53 were reduced by inhibition of p38 mitogen-activated protein kinase (MAPK) and murine double minute (Mdm2) E3 ligase. Growth cone collapse induced by genetic (npc1−/−) or pharmacological modification of cholesterol metabolism was Rho kinase (ROCK)-dependent and associated with increased RhoA protein synthesis; both processes were significantly reduced by P38 MAPK or Mdm2 inhibition. Finally, in vivo ROCK inhibition significantly increased phosphorylated p53 levels and neurofilaments in axons, and axonal bundle size in npc1−/− mice. These results indicate that NPC-related and cholesterol perturbation-induced axonal pathology is associated with an abnormal signaling pathway consisting in p38 MAPK activation leading to Mdm2-mediated p53 degradation, followed by ROCK activation. These results also suggest new targets for pharmacological treatment of NPC disease and other diseases associated with disruption of cholesterol metabolism.     

Cholesterol is the major component of native lipoproteins activating the p38 mitogen-activated protein kinases

Elevated low-density lipoprotein (LDL) levels induce activation of the p38 mitogen-activated protein kinase (MAPK), a stress-activated protein kinase potentially participating in the development of atherosclerosis. The nature of the lipoprotein components inducing p38 MAPK activation has remained unclear however. We show here that both LDLs and high-density lipoproteins (HDLs) have the ability to stimulate the p38 MAPKs with potencies that correlate with their cholesterol content. Cholesterol solubilized in methyl-beta-cyclodextrin was sufficient to activate the p38 MAPK pathway. Liposomes made of phosphatidylcholine (PC) or sphingomyelin, the two main phospholipids found in lipoproteins, were unable to stimulate the p38 MAPKs. In contrast, PC liposomes loaded with cholesterol potently activated this pathway. Reducing the cholesterol content of LDL particles lowered their ability to activate the p38 MAPKs. Cell lines representative of the three main cell types found in blood vessels (endothelial cells, smooth muscle cells and fibroblasts) all activated their p38 MAPK pathway in response to LDLs or cholesterol-loaded PC liposomes. These results indicate that elevated cholesterol content in lipoproteins, as seen in hypercholesterolemia, favors the activation of the stress-activated p38 MAPK pathway in cells from the vessel wall, an event that might contribute to the development of atherosclerosis.     

Parathyroid hormone activation of map kinase in rat duodenal cells is mediated by 3',5'-cyclic AMP and Ca(2+)

In a previous study, we demonstrated that parathyroid hormone (PTH) stimulates in rat duodenal cells (enterocytes) the phosphorylation and activity of extracellular signal-regulated mitogen-activated protein kinase (MAPK) isoforms ERK1 and ERK2. As PTH activates adenylyl cyclase (AC) and phospholipase C and increases intracellular Ca(2+) in these cells, in the present study we evaluated the involvement of cAMP, Ca(2+) and protein kinase C (PKC) on PTH-induced MAPK activation. We found that MAPK phosphorylation by the hormone did not depend on PKC activation. PTH response could, however, be mimicked by addition of forskolin (5-15 microM), an AC activator, or Sp-cAMP (50-100 microM), a cAMP agonist, and suppressed to a great extent by the AC inhibitor, compound Sq-22536 (0.2-0.4 mM) and the cAMP antagonist Rp-cAMP (0.2 mM). Removal of external Ca(2+) (EGTA 0.5 mM), chelation of intracellular Ca(2+) with BAPTA (5 microM), or blockade of L-type Ca(2+)-channels with verapamil (10 microM) significantly decreased PTH-activation of MAPK. Furthermore, a similar degree of phosphorylation of MAPK was elicited by the Ca(2+) mobilizing agent thapsigargin, the Ca(2+) ionophore A23187, ionomycin and membrane depolarization with high K(+). Inclusion of the calmodulin inhibitor fluphenazine (50 microM) did not prevent hormone effects on MAPK. Taken together, these results indicate that cAMP and Ca(2+) play a role upstream in the signaling mechanism leading to MAPK activation by PTH in rat enterocytes. As Ca(2+) and cAMP antagonists did not block totally PTH-induced MAPK phosphorylation, it is possible that linking of the hormone signal to the MAPK pathway may additionally involve Src, which has been previously shown to be rapidly activated by PTH. Of physiological significance, in agreement with the mitogenic role of the MAPK cascade, PTH increased enterocyte DNA synthesis, and this effect was blocked by the specific inhibitor of MAPK kinase (MEK) PD098059, indicating that hormone modulation of MAPK through these messenger systems stimulates duodenal cell proliferation.     

Basic fibroblast growth factor evokes a rapid glutamate release through activation of the MAPK pathway in cultured cortical neurons

We examined the possibility that basic fibroblast growth factor (bFGF) is involved in synaptic transmissions. We found that bFGF rapidly induced the release of glutamate and an increase in the intracellular Ca2+ concentration through voltage-dependent Ca2+ channels in cultured cerebral cortical neurons. bFGF also evoked a significant influx of Na+. Tetanustoxin inhibited the bFGF-induced glutamate release, revealing that bFGF triggered exocytosis. The mitogen-activated protein kinase (MAPK) pathway was required for these acute effects of bFGF. We also found that pretreatment with bFGF significantly enhanced high K+-elicited glutamate release also in a MAPK activation-dependent manner. Therefore, we propose that bFGF exerts promoting effects on excitatory neuronal transmission via activation of the MAPK pathway.     

RhoA and p38 MAPK mediate apoptosis induced by cellular cholesterol depletion

Cholesterol is essential for cell viability, and homeostasis of cellular cholesterol is crucial to various cell functions. Here we examined the effect of cholesterol depletion on apoptosis and the mechanisms underlying this effect in NIH3T3 cells. We show that chronic cholesterol depletion achieved with lipoprotein-deficient serum (LPDS) and 25-hydroxycholesterol (25-HC) treatment resulted in a significant increase in cellular apoptosis and caspase-3 activation. This effect is not due to a deficiency of nonsterol isoprenoids, intermediate metabolites of the cholesterol biosynthetic pathway, but rather to low cholesterol levels, since addition of cholesterol together with LPDS and 25-HC nearly abolished apoptosis, whereas addition of farnesyl pyrophosphate or geranylgeranyl-pyrophosphate did not reverse the cell viability loss induced by LPDS plus 25-HC treatment. These effects were accompanied by an increase in ERK, JNK and p38 MAPK activity. However, only the inhibition of p38 MAPK with the specific inhibitor SB203580 or the overexpression of a kinase defective MKK6 resulted in a significant decrease in apoptosis and caspase-3 cleavage induced by cholesterol depletion. Furthermore, LPDS plus 25-HC increased RhoA activity, and this effect was reversed by addition of exogenous cholesterol. Finally, overexpression of the dominant negative N19RhoA inhibited p38 MAPK phosphorylation and apoptosis induced by low cholesterol levels. Together, our results demonstrate that cholesterol depletion induces apoptosis through a RhoA- and p38 MAPK-dependent mechanism.     

Nicotine stimulates adhesion molecular expression via calcium influx and mitogen-activated protein kinases in human endothelial cells

To evaluate the effect of nicotine on endothelium dysfunction and development of vascular diseases, we investigated the influence on adhesion molecular expression mediated by nicotine and the mechanism of this effect in human umbilical vein endothelial cells (HUVECs). The result showed that nicotine could induce surface/soluble vascular cell adhesion molecule (VCAM-1) and endothelial selectin (E-selectin) expression in a time-response decline manner and the peak appeared at 15 min. This action could be mediated by mitogen-activated protein kinase/extracellular signal regulated kinase 1/2 (MAPK/ERK1/2) and MAPK/p38 because their activation could be distinctly blocked by MAPK inhibitors, PD098059 or SB203580. Mecamylamine (non-selective nicotinic receptor inhibitor), alpha-bungarotoxin (alpha7 nicotinic receptor inhibitor) could block Ca2+ accumulation, and then, prevented the phosphorylation on ERK1/2 and p38. They also inhibited the surface/soluble VCAM-1, E-selectin production of HUVECs modulated by nicotine. Therefore, we concluded that: (i) nicotine obviously up-regulates VCAM-1 and E-selectin expression at 15 min in HUVECs, (ii) nicotine activates HUVECs triggered by the ERK1/2 and p38 phosphorylation with an involvement of intracellular calcium mobilization chiefly mediated by alpha7 nicotinic receptor, (iii) intracellular Ca2+ activates a sequential pathway from alpha7 nicotinic receptor to the phosphorylation of ERK1/2, p38. These elucidate that nicotine activates HUVECs through fast signal transduction pathway and arguments their capacity of adhesion molecular production. Further more nicotine may contribute its influence to the progression of vascular disease such as atherosclerotic lesion.     

Ras/mitogen-activated protein kinase (MAPK) signaling modulates protein stability and cell surface expression of scavenger receptor SR-BI

The mitogen-activated protein kinase (MAPK) Erk1/2 has been implicated to modulate the activity of nuclear receptors, including peroxisome proliferator activator receptors (PPARs) and liver X receptor, to alter the ability of cells to export cholesterol. Here, we investigated if the Ras-Raf-Mek-Erk1/2 signaling cascade could affect reverse cholesterol transport via modulation of scavenger receptor class BI (SR-BI) levels. We demonstrate that in Chinese hamster ovary (CHO) and human embryonic kidney (HEK293) cells, Mek1/2 inhibition reduces PPARα-inducible SR-BI protein expression and activity, as judged by reduced efflux onto high density lipoprotein (HDL). Ectopic expression of constitutively active H-Ras and Mek1 increases SR-BI protein levels, which correlates with elevated PPARα Ser-21 phosphorylation and increased cholesterol efflux. In contrast, SR-BI levels are insensitive to Mek1/2 inhibitors in PPARα-depleted cells. Most strikingly, Mek1/2 inhibition promotes SR-BI degradation in SR-BI-overexpressing CHO cells and human HuH7 hepatocytes, which is associated with reduced uptake of radiolabeled and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyane-labeled HDL. Loss of Mek1/2 kinase activity reduces SR-BI expression in the presence of bafilomycin, an inhibitor of lysosomal degradation, indicating down-regulation of SR-BI via proteasomal pathways. In conclusion, Mek1/2 inhibition enhances the PPARα-dependent degradation of SR-BI in hepatocytes.     

Epidermal growth factor potentiates cholecystokinin/gastrin receptor-mediated Ca2+ release by activation of mitogen-activated protein kinases

Small differences in amplitude, duration, and temporal patterns of change in the concentration of free intracellular Ca2+ ([Ca2+](i)) can profoundly affect cell physiology, altering programs of gene expression, cell proliferation, secretory activity, and cell survival. We report a novel mechanism for amplitude modulation of [Ca2+](i) that involves mitogen-activated protein kinase (MAPK). We show that epidermal growth factor (EGF) potentiates gastrin-(1-17) (G17)-stimulated Ca2+ release from intracellular Ca2+ stores through a MAPK-dependent pathway. G17 activation of the cholecystokinin/gastrin receptor (CCK(2)R), a G protein-coupled receptor, stimulates release of Ca2+ from inositol 1,4,5-triphosphate-sensitive Ca2+ stores. Pretreating rat intestinal epithelial cells expressing CCK(2)R with EGF increased the level of G17-stimulated Ca2+ release from intracellular stores. The stimulatory effect of EGF on CCK(2)R-mediated Ca2+ release requires activation of the MAPK kinase (MEK)1,2/extracellular signal-regulated kinase (ERK)1,2 pathway. Inhibition of the MEK1,2/ERK1,2 pathway by either serum starvation or treatment with selective MEK1,2 inhibitors PD98059 and U0126 or expression of a dominant-negative mutant form of MEK1 decreased the amplitude of the G17-stimulated Ca2+ release response. Activation of the MEK1,2/ERK1,2 pathway either by pretreating cells with EGF or by expression of constitutively active K-ras (K-rasV12G) or MEK1 (MEK1*) increased the amplitude of G17-stimulated Ca2+ release. Although EGF, MEK1*, and K-rasV12G activated the MEK1,2/ERK1,2 pathway, they did not increase [Ca2+](i) in the absence of G17. These data demonstrate that the activation state of the MEK1,2/ERK1,2 pathway can modulate the amplitude of the CCK(2)R-mediated Ca2+ release response and identify a novel mechanism for cross-talk between EGF receptor- and CCK(2)R-regulated signaling pathways.     

Involvement of intermediary metabolites in the pathway of extracellular Ca2+-induced mitogen-activated protein kinase activation in human fibroblasts.

Human fibroblasts in culture will grow in serum-free medium containing serum replacement factors, but without protein growth factors, as long as the Ca2+ level is 1.0-2.0 mM. When the Ca2+ is reduced to 0.1 mM, the cells stop cycling, but they can be reinduced to cycle by raising the Ca2+ level to 1.0 mM Ca2+ or to higher concentrations that result in activation of mitogen-activated protein kinase (MAPK). We now report that exposure of human fibroblasts to extracellular Ca2+ increased the level of inositol (1,4,5)-trisphosphate in the cytoplasm and caused a transient rise in the concentration of intracellular free Ca2+. Ca2+-induced MAPK activation was partly abolished by treatment of the cells with pertussis toxin. It was also decreased by treatment of cells with thapsigargin, which depletes intracellular Ca2+ stores; with phorbol 12-myristyl 13-acetate (PMA), which down-regulates protein kinase C (PKC); with the calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide HCl (W-7), and calmidazolium (24571); as well as with lanthanum, a Ca2+ channel inhibitor. Ca2+ stimulation did not result in phosphorylation of the c-raf-1 protein. Our results suggest that extracellular Ca2+ stimulates MAPK activation through a pathway(s) involving a pertussis toxin-sensitive G protein, phospholipase C, intracellular free Ca2+, calmodulin, and PKC.     

Dietary taurine enhances cholesterol degradation and reduces serum and liver cholesterol concentrations in rats fed a high-cholesterol diet

Summary.  The effect of taurine on hypercholesterolemia induced by feeding a high-cholesterol (HC) diet (10 g/kg) to rats was examined. When taurine was supplemented to HC for 2 wk, serum total cholesterol significantly decreased and serum HDL-cholesterol increased compared with the HC diet group. In the hypercholesterolemic rats fed the HC diet, the excretion of fecal bile acids and hepatic cholesterol 7α-hydroxylase (CYP7A1) activity and its mRNA level increased significantly, and the supplementation of taurine further enhanced these indexes, indicating an increase in cholesterol degradation. Agarose gel electrophoresis revealed that, in hypercholesterolemic rats fed the HC diet, the serum level of the heavier VLDL increased significantly, but taurine repressed this increase and normalized this pattern. Significant correlations were observed between the time-dependent increase of CYP7A1 gene expression and the decrease of blood cholesterol concentration in rats fed the HC diet supplemented with taurine. These results suggest that the hypocholesterolemic effects of taurine observed in the hypocholesterolemic rats fed the HC diet were mainly due to the enhancement of cholesterol degradation and the excretion of bile acid. Received December 4, 2001 Accepted January 2, 2002 Published online September 10, 2002 Acknowledgment This work was supported by a grant of Taisho Pharmaceutical Co., Ltd (Japan). We thank J. I. Gordon for their generous gifts of cDNAs. Authors' address: Dr. Hidehiko Yokogoshi, School of Food and Nutritional Sciences, The University of Shizuoka, Shizuoka 4228526, Japan, E-mail: yokogosi@u-shizuoka-ken.ac.jp