Diabetic retinopathy and signaling mechanism for activation of matrix metalloproteinase-9

In the pathogenesis of diabetic retinopathy, H-Ras (a small molecular weight G-protein) and matrix metalloproteinase-9 (MMP9) act as pro-apoptotic, accelerating the apoptosis of retinal capillary cells, a phenomenon that predicts its development and the activation of MMP9 is under the control of H-Ras. The goal of this study is to elucidate the cellular mechanism by which H-Ras activates MMP9 culminating in the development of diabetic retinopathy. Using isolated retinal endothelial cells, the effect of regulation of H-Ras downstream signaling cascade, Raf-1, MEK, and ERK, was investigated on glucose-induced activation of MMP9. In vitro results were confirmed in the retina obtained from diabetic mice manipulated for MMP9 gene, and also in the retinal microvasculature obtained from human donors with diabetic retinopathy. Regulation of Raf-1/MEK/ERK by their specific siRNAs and pharmacologic inhibitors prevented glucose-induced activation of MMP9 in retinal endothelial cells. In MMP9-KO mice, diabetes had no effect on retinal MMP9 activation, and H-Ras/Raf-1/MEK signaling cascade remained normal. Similarly, donors with diabetic retinopathy had increased MMP9 activity in their retinal microvessels, the site of histopathology associated with diabetic retinopathy, and this was accompanied by activated H-Ras signaling pathway (Raf-1/ERK). Collectively, these results suggest that Ras/Raf-1/MEK/ERK cascade has an important role in the activation of retinal MMP9 resulting in the apoptosis of its capillary cells. Understanding the upstream mechanism responsible for the activation of MMP9 should help identify novel molecular targets for future pharmacological interventions to inhibit the development/progression of diabetic retinopathy.     

Small molecular weight G-protein, H-Ras, and retinal endothelial cell apoptosis in diabetes

We have demonstrated that the expressions of small molecular weight G-protein, H-Ras, and its effector protein, Raf-1, are increased in the retina in diabetes, and the specific inhibitors of Ras function inhibit glucose-induced apoptosis of retinal capillary cells. This study is to examine the contributory roles for H-Ras in glucose-induced apoptosis of retinal endothelial cells by genetic manipulation of functionally active H-Ras levels. Bovine retinal endothelial cells were transfected with the plasmids of either wild type (WT), constitutively active (V12) or dominant-negative (N17) H-Ras. Glucose-induced increase in apoptosis, nitric oxide (NO) levels and activation of NF-κB and caspase-3 were determined in these genetically manipulated cells. Exposure of bovine retinal endothelial cells to 20 mM glucose significantly increased H-Ras activation as determined by Raf-1 binding assay. Overexpression of V12 in the endothelial cells further increased their glucose-induced apoptosis by 40%, NO levels by about 50%, and activated NF-κB and caspase-3 by about 30–40% compared to the untransfected cells incubated in 20 mM glucose. In contrast, overexpression of the inactive mutant, N17, inhibited glucose-mediated increases in apoptotic cell death, NO levels and NF-κB and caspase-3 activation; the values were significantly different (p < 0.02) compared to those obtained from the untransfected cells incubated under similar conditions. Our findings demonstrate that H-Ras activation is important in the activation of the specific signaling events leading to the accelerated retinal capillary cell apoptosis in hyperglycemic conditions, suggesting the possible use of H-Ras inhibitors to inhibit the pathogenesis of diabetic retinopathy.     

Oncogenic Ras leads to Rho activation by activating the mitogen-activated protein kinase pathway and decreasing Rho-GTPase-activating protein activity

Transformation by oncogenic Ras requires signaling through Rho family proteins including RhoA, but the mechanism(s) whereby oncogenic Ras regulates the activity of RhoA is (are) unknown. We examined the effect of Ras on RhoA activity in NIH 3T3 cells either stably transfected with H-Ras(V12) under control of an inducible promoter or transiently expressing the activated H-Ras. Using a novel method to quantitate enzymatically the GTP bound to Rho, we found that expression of the oncogenic Ras increased Rho activity approximately 2-fold. Increased Rho activity was associated with increased plasma membrane binding of RhoA and decreased activity of the Rho/Ras-regulated p21(WAF1/CIP1) promoter. RhoA activation by oncogenic Ras could be explained by a decrease in cytosolic p190 Rho-GAP activity and translocation of p190 Rho-GAP from the cytosol to a detergent-insoluble cytoskeletal fraction. Pharmacologic inhibition of the Ras/Raf/MEK/ERK pathway prevented Ras-induced activation of RhoA and translocation of p190 Rho-GAP; expression of constitutively active Raf-1 kinase or MEK was sufficient to induce p190 Rho-GAP translocation. We conclude that in NIH 3T3 cells oncogenic Ras activates RhoA through the Raf/MEK/ERK pathway by decreasing the cytosolic activity and changing the subcellular localization of p190 Rho-GAP.     

H-Ras signaling and K-Ras signaling are differentially dependent on endocytosis

Endocytosis is required for efficient mitogen-activated protein kinase (MAPK) activation by activated growth factor receptors. We examined if H-Ras and K-Ras proteins, which are distributed across different plasma membrane microdomains, have equal access to the endocytic compartment and whether this access is necessary for downstream signaling. Inhibition of endocytosis by dominant interfering dynamin-K44A blocked H-Ras but not K-Ras-mediated PC12 cell differentiation and selectively inhibited H-Ras- but not K-Ras-mediated Raf-1 activation in BHK cells. H-Ras- but not K-Ras-mediated Raf-1 activation was also selectively dependent on phosphoinositide 3-kinase activity. Stimulation of endocytosis and endocytic recycling by wild-type Rab5 potentiated H-Ras-mediated Raf-1 activation. In contrast, Rab5-Q79L, which stimulates endocytosis but not endocytic recycling, redistributed activated H-Ras from the plasma membrane into enlarged endosomes and inhibited H-Ras-mediated Raf-1 activation. Rab5-Q79L expression did not cause the accumulation of wild-type H-Ras in enlarged endosomes. Expression of wild-type Rab5 or Rab5-Q79L increased the specific activity of K-Ras-activated Raf-1 but did not result in any redistribution of K-Ras from the plasma membrane to endosomes. These results show that H-Ras but not K-Ras signaling though the Raf/MEK/MAPK cascade requires endocytosis and endocytic recycling. The data also suggest a mechanism for returning Raf-1 to the cytosol after plasma membrane recruitment.     

Activation of the Raf/MAP kinase cascade by the Ras-related protein TC21 is required for the TC21-mediated transformation of NIH 3T3 cells

TC21 is a member of the Ras superfamily of small GTP-binding proteins and, like Ras, has been implicated in the regulation of growth-stimulating pathways. Point mutations introduced into TC21 based on equivalent H-Ras oncogenic mutations are transforming in cultured cells, and oncogenic mutations in TC21 have been isolated from several human tumours. The mechanism of TC21 signalling in transformation is poorly understood. While activation of the serine/threonine kinases Raf-1 and B-Raf has been implicated in signalling pathways leading to transformation by H-Ras, it has been argued that TC21 does not activate Raf-1 or B-Raf. Since the Raf-signalling pathway is important in transformation by other Ras proteins, we assessed whether the Raf pathway is important to transformation by TC21. Raf-1 and B-Raf are constitutively active in TC21-transformed cells and the ERK/MAPK cascade is required for the maintenance of the transformed state. We demonstrate that oncogenic V23 TC21, like Ras, interacts with Raf-1 and B-Raf (but not with A-Raf), resulting in the translocation of the Raf proteins to the plasma membrane and in their activation. Furthermore, using point mutations in the effector loop of TC21, we show that the interaction of TC21 with Raf-1 is crucial for transformation.     

Diabetes-induced activation of caspase-3 in retina: effect of antioxidant therapy

Apoptosis of retinal endothelial cells and pericytes is postulated to contribute to the development of retinopathy in diabetes. The goal of this study is to investigate diabetes-induced activation of retinal caspase-3, an apoptosis executer enzyme, in retina, and examine the effects of antioxidants on the activation. Caspase-3 activation was determined in the retina of alloxan diabetic rats (2-14 months duration) and in the isolated retinal capillary cells (endothelial cells and pericytes) by measuring cleavage of caspase-3 specific fluorescent substrate, and cleavage of caspase-3 holoenzyme and poly (ADP ribosyl) polymerase. Effect of antioxidants on the activation of caspase-3 was determined by feeding a group of diabetic rats diet supplemented with a comprehensive mixture of antioxidants, including Trolox, alpha-tocopherol, N-acetyl cysteine, ascorbic acid, beta-carotene and selenium for 2-14 months, and also under in vitro conditions by incubating isolated retinal capillary cells with antioxidants with wide range of actions. Caspase-3 was activated in the rat retina at 14 months of diabetes (P < 0.05 vs. normal), but not at 2 months of diabetes, and administration of antioxidants for the entire duration inhibited this activation. In the isolated retinal capillary cells incubated in 25 mM glucose medium, caspase-3 activity was increased by 50% compared to the cells incubated in 5 mM glucose (P < 0.02), and antioxidants or caspase-3 inhibitor inhibited this increase. Our results suggest that increased oxidative stress in diabetes is involved in the activation of retinal caspase-3 and apoptosis of endothelial cells and pericytes. Antioxidants might be inhibiting the development of diabetic retinopathy by inhibiting microvascular apoptosis.     

Stretch-induced Raf-1 activation in mesangial cells requires actin cytoskeletal integrity

Glomerular capillary hypertension is a determinant of glomerulosclerosis and is modelled in vitro by exposure of mesangial cells to cyclic mechanical strain. In response to strain, Erk is activated and mediates extracellular matrix accumulation and mesangial cell proliferation. Erk activation is dependent on an intact cytoskeleton. Since Raf-1 lies upstream of Erk in response to numerous stimuli, and since its activation is dependent on membrane recruitment, we postulated that the cytoskeleton was essential for Raf-1 membrane recruitment and Erk activation. Primary rat mesangial cells (passages 8-20) were stretched at 1 Hz and 27 kPa. Raf-1 was both phosphorylated on serine-338 (S338) and activated within 2 min of strain. The Raf-1 inhibitor, GW5074, dose-dependently blocked strain-induced Erk activation and Raf-1 phosphorylation. Although phosphatidylinositol-3-kinase (PI3-K) may mediate Raf-1 activation, PI3-K inhibition with wortmannin or LY294002 had no effect on stretch-induced Raf-1 activation. Cytoskeletal disruption with cytochalasin D and the Rho-kinase inhibitor, Y-27632, however, blocked both Raf-1 phosphorylation and activation. Furthermore, membrane localization of Raf-1 was increased by strain and prevented by cytoskeletal disruption. Thus, strain leads to rapid membrane localization, S338 phosphorylation, and activation of Raf-1. These events are independent of PI3-K, but require Rho-kinase activation and an intact actin cytoskeleton.     

Cyclic AMP Inhibits Activation of Mitogen-Activated Protein Kinase and Cell Proliferation in Response to Growth Factors in Cultured Rat Cortical Astrocytes

Abstract: The cyclic AMP (cAMP)-induced inhibitory effect on cell proliferation was examined through inhibition of mitogen-activated protein kinase (MAP kinase) activation in cultured rat cortical astrocytes. Basic fibroblast growth factor (bFGF) at 10 ng/ml maximally stimulated MAP kinase activity, which peaks during 10 min and prolonged for 24 h. Likewise, DNA synthesis was maximally potentiated with 10 ng/ml bFGF and correlated with MAP kinase activity in a dose-dependent manner. Dibutyryl cAMP (dbcAMP) at 1 m M and isoproterenol at 10 µ M inhibited MAP kinase activation and DNA synthesis potentiation with bFGF and platelet-derived growth factor to the control level in cultured astrocytes and C6 glioma cells. The stimulation with bFGF caused a prominent translocation of MAP kinase from the cytosol to the nucleus after 1 h in astrocytes. Treatment of the cells with dbcAMP and isoproterenol completely prevented the translocation of MAP kinase. In experiments with ³²P-labeled cultured astrocytes, phosphorylation of Raf-1 was apparently stimulated with bFGF. Treatment with dbcAMP or isoproterenol had a greatly inhibitory effect on the stimulation of Raf-1 phosphorylation with bFGF. Consistent with the effect on Raf-1 phosphorylation, dbcAMP and isoproterenol completely prevented bFGF-induced phosphorylation of MAP kinase kinases, target proteins of Raf-1. Our observations suggest that cAMP-induced suppression of cell growth in astrocytes is due to the inhibitory effect on activation of MAP kinase and its translocation to the nucleus and that the site of the cAMP action is located at Raf-1 or the upstream site of Raf-1.     

Diabetes-induced Activation of Caspase-3 in Retina: Effect of Antioxidant Therapy

Apoptosis of retinal endothelial cells and pericytes is postulated to contribute to the development of retinopathy in diabetes. The goal of this study is to investigate diabetes-induced activation of retinal caspase-3, an apoptosis executer enzyme, in retina, and examine the effects of antioxidants on the activation. Caspase-3 activation was determined in the retina of alloxan diabetic rats (2-14 months duration) and in the isolated retinal capillary cells (endothelial cells and pericytes) by measuring cleavage of caspase-3 specific fluorescent substrate, and cleavage of caspase-3 holoenzyme and poly (ADP ribosyl) polymerase. Effect of antioxidants on the activation of caspase-3 was determined by feeding a group of diabetic rats diet supplemented with a comprehensive mixture of antioxidants, including Trolox, &#102 -tocopherol, N -acetyl cysteine, ascorbic acid, &#103 -carotene and selenium for 2-14 months, and also under in vitro conditions by incubating isolated retinal capillary cells with antioxidants with wide range of actions. Caspase-3 was activated in the rat retina at 14 months of diabetes ( P <0.05 vs. normal), but not at 2 months of diabetes, and administration of antioxidants for the entire duration inhibited this activation. In the isolated retinal capillary cells incubated in 25 mM glucose medium, caspase-3 activity was increased by 50% compared to the cells incubated in 5 mM glucose ( P <0.02), and antioxidants or caspase-3 inhibitor inhibited this increase. Our results suggest that increased oxidative stress in diabetes is involved in the activation of retinal caspase-3 and apoptosis of endothelial cells and pericytes. Antioxidants might be inhibiting the development of diabetic retinopathy by inhibiting microvascular apoptosis.     

Calmodulin modulates H-Ras mediated Raf-1 activation

We have previously demonstrated that, in COS-1 cells, inhibition of calmodulin increases Ras-GTP levels although it decreases Raf-1 activity and consequently MAPK. The present study analyzes the role of calmodulin in the regulation of Raf-1. First we show, using FRET microscopy, that inhibition of Raf-1 was not a consequence of a decreased interaction between H-Ras and Raf-1. Besides, the analysis of the phosphorylation state of Raf-1 showed that calmodulin, through downstream PI3K, is essential to ensure the Ser338-Raf-1 phosphorylation, critical for Raf-1 activation. We also show that the expression of a dominant negative mutant of PI3K impairs the calmodulin-mediated Raf-1 activation; in addition, both calmodulin and PI3K inhibitors decrease phospho-Ser338 and Raf-1 activity from upstream active H-Ras (H-RasG12V) and this effect is dependent on endocytosis. Importantly, in H-Ras depleted COS-1 cells, calmodulin does not modulate MAPK activation. Altogether, the results suggest that calmodulin regulation of MAPK in COS-1 cells relies upon H-Ras control of Raf-1 activity and involves PI3K.     

Transmembrane signaling pathway mediates oxidized low-density lipoprotein-induced expression of plasminogen activator inhibitor-1 in vascular endothelial cells

Atherosclerotic cardiovascular disease is the number one cause of death for adults in Western society. Plasminogen activator inhibitor-1 (PAI-1), the major physiological inhibitor of plasminogen activators, has been implicated in both thrombogenesis and atherogenesis. Previous studies demonstrated that copper-oxidized low-density lipoprotein (C-oLDL) stimulated production of PAI-1 in vascular endothelial cells (EC). The present study examined the involvement of lectin-like oxidized LDL receptor-1 (LOX-1) and Ras/Raf-1/ERK1/2 pathway in the upregulation of PAI-1 in cultured EC induced by oxidized LDLs. The results demonstrated that C-oLDL or FeSO(4)-oxidized LDL (F-oLDL) increased the expression of PAI-1 or LOX-1 in human umbilical vein EC (HUVEC) or coronary artery EC (HCAEC). Treatment with C-oLDL significantly increased the levels of H-Ras mRNA, protein, and the translocation of H-Ras to membrane fraction in EC. LOX-1 blocking antibody, Ras farnesylation inhibitor (FTI-277), or small interference RNA against H-Ras significantly reduced C-oLDL or LDL-induced expression of H-Ras and PAI-1 in EC. Incubation with C-oLDL or F-oLDL increased the phosphorylation of Raf-1 and ERK1/2 in EC compared with LDL or vehicle. Treatment with Raf-1 inhibitor blocked Raf-1 phosphorylation and the elevation of PAI-1 mRNA level in EC induced by C-oLDL or LDL. Treatment with PD-98059, an ERK1/2 inhibitor, blocked C-oLDL or LDL-induced ERK1/2 phosphorylation or PAI-1 expression in EC. The results suggest that LOX-1, H-Ras, and Raf-1/ERK1/2 are implicated in PAI-1 expression induced by oxidized LDLs or LDL in cultured EC.     

Neuronal nitric oxide synthase-induced S-nitrosylation of H-Ras inhibits calcium ionophore-mediated extracellular-signal-regulated kinase activity

nNOS (neuronal nitric oxide synthase) is a constitutively expressed enzyme responsible for the production of NO* from L-arginine and O2. NO* acts as both an intra- and an inter-cellular messenger that mediates a variety of signalling pathways. Previous studies from our laboratory have demonstrated that nNOS production of NO* blocks Ca2+-ionophore-induced activation of ERK1/2 (extracellular-signal-regulated kinase 1/2) of the mitogen-activated protein kinases through a mechanism involving Ras G-proteins and Raf-1 kinase. Herein we describe a mechanism by which NO* blocks Ca2+-mediated ERK1/2 activity through direct modification of H-Ras. Ca2+-mediated ERK1/2 activation in NO*-producing cells could be restored by exogenous expression of constitutively active mitogen-activated protein kinase kinase 1. In contrast, exogenous expression of constitutively active mutants of Raf-1 and H-Ras only partially restored ERK1/2 activity, by 50% and 10% respectively. On the basis of these findings, we focused on NO*-mediated mechanisms of H-Ras inhibition. Assays for GTP loading and H-Ras interactions with the Ras-binding domain on Raf-1 demonstrated a decrease in H-Ras activity in the presence of NO*. We demonstrate that S-nitrosylation of H-Ras occurs in nNOS-expressing cells activated with Ca2+ ionophore. Mutation of a putative nitrosylation site at Cys118 inhibited S-nitrosylation and restored ERK1/2 activity by constitutively active H-Ras even in the presence of NO*. These findings indicate that intracellular generation of NO* by nNOS leads to S-nitrosylation of H-Ras, which interferes with Raf-1 activation and propagation of signalling through ERK1/2.     

Bcl-2 inhibits Bax translocation from cytosol to mitochondria during drug-induced apoptosis of human tumor cells

The pro-apoptotic protein, Bax, has been reported to translocate from cytosol to mitochondria following exposure of cells to apoptotic stresses including cytokine withdrawal and treatment with glucocorticoids and cytotoxic drugs. These observations, coupled with reports showing that Bax causes the release of mitochondrial cytochrome c, implicate Bax as a central mediator of the apoptotic process. In this report we demonstrate by subcellular fractionation a significant shift in Bax localization from cytosol to cellular membranes in two human tumor cell lines exposed to staurosporine or etoposide. Immunofluorescence studies confirmed that Bax specifically relocalized to the mitochondria. This redistribution of Bax occurred in concert with, or just prior to, proteolytic processing of procaspase-3, activation of DEVD-specific cleavage activity and degradation of poly(ADP-ribose) polymerase. However, Bax membrane translocation was independent of caspase activity as determined using the broad-range caspase inhibitor z-VAD-fmk. High level overexpression of the anti-apoptotic protein Bcl-2 prevented Bax redistribution to the mitochondria, caspase activation and apoptosis following exposure to staurosporine or etoposide. These data confirm the role of Bax in mitochondrial cytochrome c release, and indicate that prevention of Bax translocation to the mitochondrial membrane represents a novel mechanism by which Bcl-2 inhibits drug-induced apoptosis.     

Oxidative stress and the development of diabetic retinopathy: Contributory role of matrix metalloproteinase-2

Matrix metalloproteinases (MMPs) degrade extracellular matrix and regulate many functions including cell signaling. Oxidative stress is implicated in the development of diabetic retinopathy, and MMP-2, the most ubiquitous member of the MMP family, is sensitive to oxidative stress. This study aimed to determine the regulation of MMP-2 by oxidative stress in the development of diabetic retinopathy and the role of MMP-2 in the apoptosis of retinal capillary cells. The effects of mitochondrial superoxide scavenger on glucose-induced alterations in MMP-2, and its proenzyme activator MT1-MMP and physiological inhibitor TIMP-2, were determined in retinal endothelial cells, and the regulation of their glucose-induced accelerated apoptosis by the inhibitors of MMP-2 was accessed. To confirm in vitro results, the effects of antioxidant supplementation on MMP-2, MT1-MMP, and TIMP-2 were investigated in the retina of streptozotocin-induced diabetic rats. Glucose-induced activation of retinal capillary cell MMP-2 and MT1-MMP and decrease in TIMP-2 were inhibited by superoxide scavengers, and their accelerated apoptosis was prevented by the inhibitors of MMP-2. Antioxidant therapies, which have been shown to inhibit oxidative stress, capillary cell apoptosis, and retinopathy in diabetic rats, ameliorated alterations in retinal MMP-2 and its regulators. Thus, MMP-2 has a proapoptotic role in the loss of retinal capillary cells in diabetes, and the activation of MMP-2 is under the control of superoxide. This suggests a possible use of MMP-2-targeted therapy to inhibit the development of diabetic retinopathy.     

Activation of protein kinase C inhibits TRAIL-induced caspases activation, mitochondrial events and apoptosis in a human leukemic T cell line

TRAIL causes apoptosis in numerous types of tumor cells. However, the mechanisms regulating TRAIL-induced apoptosis remain to be elucidated. We have investigated the role of PKC in regulating TRAIL-induced mitochondrial events and apoptosis in the Jurkat T cell line. We found a caspase-dependent decline in mitochondrial membrane potential and translocation of cytochrome c from mitochondria into the cytosol in response to TRAIL. Both these events were prevented by PKC activation. Moreover, PKC activation considerably reduced the activation of caspases, PARP cleavage and apoptosis when induced upon TRAIL treatment. MAPK activation was involved in the mechanism of PKC-mediated inhibition of TRAIL-induced cytochrome c release from mitochondria. Furthermore, inhibition of the MAPK pathway partially reversed the PKC-mediated inhibition of TRAIL-induced apoptosis. Besides, PKC activation may also inhibit the TRAIL-induced apoptosis through a MAPK-independent mechanism. Altogether, these results indicate a negative role of PKC in the regulation of apoptotic signals generated upon TRAIL receptor activation.     

RKIP downregulation induces the HBx-mediated Raf-1 mitochondrial translocation

The Raf-1 kinase inhibitory protein (RKIP) can regulate multiple key signaling pathways. Specifically, RKIP binds to Raf-1 kinase and inhibits the Ras-Raf-1-MEK1/2- ERK1/2 pathway. Additionally, Raf-1 has been shown to translocate to mitochondria and thereby protect cells from stress-mediated apoptosis. Recently, HBx was found to stimulate the mitochondrial translocation of Raf-1, contributing to the anti-apoptotic effect. We found that RKIP was downregulated during HBx-mediated hepatocarcinogenesis. In this study, we show that RKIP bound to Raf-1 and consequently inhibited the translocation of Raf-1 into mitochondria. This promoted the apoptosis of cells treated with apoptotic stimulus. Thus, the downregulation of RKIP increased the level of free Raf-1 and thereby elevated the mitochondrial translocation of Raf-1 during HBx-mediated hepatocarcinogenesis. The elevated Raf-1 mitochondrial translocation induced the increased anti-apoptotic effect and subsequently promoted HBx-mediated hepatocarcinogenesis.     

Role of phosphatidic acid in the coupling of the ERK cascade

The production of phosphatidic acid plays a crucial role in the activation of the ERK cascade. This role was linked to the binding of phosphatidate to a specific polybasic site within the kinase domain of Raf-1. Here we show that phosphatidate promotes ERK phosphorylation in intact cells but does not activate Raf in vitro. The kinase suppressor of Ras (KSR) contains a sequence homologous to the phosphatidate binding site of Raf-1. Direct binding of phosphatidate to synthetic peptides derived from the sequences of the binding domains of Raf-1 and KSR was demonstrated by spectroscopic techniques. The specificity of these interactions was confirmed using synthetic lipids and mutated peptides in which the core of the phosphatidic acid binding domain was disrupted. Insulin and exogenous dioleoyl phosphatidate induced a rapid translocation of a mouse KSR1-EGFP construct to the plasma membrane of HIRcB cells. Mutation of two arginines located in the core of the putative phosphatidate binding site abolished dioleoyl phosphatidate- and insulin-induced translocation of KSR1. Overexpression of the mutant KSR1 in HIRcB cells inhibited insulin-dependent MEK and ERK phosphorylation. The addition of dioleoyl phosphatidate or insulin increased the co-localization of KSR1 and H-Ras and promoted the formation of plasma membrane patches enriched in both proteins and phosphatidic acid. These results, in conjunction with our previous work, suggest the formation of phosphatidate-enriched membrane microdomains that contain all components of the ERK cascade. We propose that these domains act as molecular scaffolds in the coupling of signaling events.     

Stabilization and translocation of p53 to mitochondria is linked to Bax translocation to mitochondria in simvastatin-induced apoptosis

Statins are cholesterol-lowing drugs with pleiotropic effects including cytotoxicity to cancer cells. In this study, we investigated the signaling pathways leading to apoptosis by simvastatin. Simvastatin induced cardinal features of apoptosis including increased DNA fragmentation, disruption of mitochondrial membrane potential (MMP), and increased caspase-3 activity by depleting isoprenoids in MethA fibrosarcoma cells. Interestingly, the simvastatin-induced apoptosis was accompanied by p53 stabilization involving Mdm2 degradation. The apoptosis was ameliorated in p53 knockdown clones of MethA cells as well as p53^−/− HCT116 cells. The stabilized p53 protein translocated to mitochondria with Bax, and cytochrome c was released into cytosol. Moreover, knockdown or deficiency of p53 expression reduced both Bax translocation to mitochondria and MMP disruption in simvastatin-induced apoptosis. Taken together, these all indicate that stabilization and translocation of p53 to mitochondria is involved in Bax translocation to mitochondria in simvastatin-induced apoptosis.     

H-Ras/mitogen-activated protein kinase pathway inhibits integrin-mediated adhesion and induces apoptosis in osteoblasts

We have studied the relevance of H-Ras and its downstream effectors to osteoblast functions. 1) Purified human osteoblasts highly expressed integrins beta1, alpha4, alpha5, alpha6 and the activation epitope of beta1. However, these molecules were markedly down-regulated on osteoblasts transfected with expression vector encoding fully activated H-Ras(V12), H-Ras(V12)T35S, activating Raf-1/mitogen-activated protein kinase (MAPK), or an active Raf-1 but not on cells having H-Ras(V12)Y40C, a phosphoinositide 3-kinase (PI3K)-binding mutant. 2) Although osteoblasts spontaneously adhered to fibronectin and laminin in beta1-dependent manner, the expression of H-Ras(V12) or H-Ras(V12)T35S, but not H-Ras(V12)Y40C, in osteoblasts reduced their adhesion. 3) Osteoblasts bearing H-Ras(V12), H-Ras(V12)T35S, or Raf-1 failed to proliferate, whereas those with H-Ras(V12)Y40C proliferated well. (4) The up-regulation of Fas and down-regulation of Bcl-2 were observed in osteoblasts expressing H-Ras(V12), H-Ras(V12)T35S, or Raf-1. (5) Most of the cells having H-Ras(V12), H-Ras(V12)T35S, or Raf-1 became annexin-V(high)/propidium iodide (PI)(high or low) and terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling (TUNEL)(high)/PI(low) after 24 and 72 h incubation, respectively. Thus, we propose that H-Ras signals followed by Raf-1/MAPK pathway but not PI3K not only reduces beta(1)-mediated adhesion of osteoblasts to matrix proteins but induces apoptosis presumably via the Fas up-regulation and Bcl-2 down-regulation.