Nemo-Like Kinase (NLK) Is a Pathological Signaling Effector in the Mouse Heart.

Nemo-like kinase (NLK) is an evolutionary conserved serine/threonine protein kinase implicated in development, proliferation and apoptosis regulation. Here we identified NLK as a gene product induced in the hearts of mice subjected to pressure overload or myocardial infarction injury, suggesting a potential regulatory role with pathological stimulation to this organ. To examine the potential functional consequences of increased NLK levels, cardiac-specific transgenic mice with inducible expression of this gene product were generated, as well as cardiac-specific Nlk gene-deleted mice. NLK transgenic mice demonstrated baseline cardiac hypertrophy, dilation, interstitial fibrosis, apoptosis and progression towards heart failure in response to two surgery-induced cardiac disease models. In contrast, cardiac-specific deletion of Nlk from the heart, achieved by crossing a Nlk-loxP allele containing mouse with either a mouse containing a ß-myosin heavy chain promoter driven Cre transgene or a tamoxifen inducible α-myosin heavy chain promoter containing transgene driving a MerCreMer cDNA, protected the mice from cardiac dysfunction following pathological stimuli. Mechanistically, NLK interacted with multiple proteins including the transcription factor Stat1, which was significantly increased in the hearts of NLK transgenic mice. These results indicate that NLK is a pathological effector in the heart.

Mnk1 (Mitogen-Activated Protein Kinase-Interacting Kinase 1) Deficiency Aggravates Cardiac Remodeling in Mice.

Identifying the key factor involved in cardiac remodeling is critically important for developing novel strategies to protect against heart failure. Here, the role of Mnk1 (mitogen-activated protein kinase-interacting kinase 1) in cardiac remodeling was clarified. Cardiac remodeling was induced by transverse aortic constriction in Mnk1-knockout mice and their wild-type control mice. After 4 weeks of transverse aortic constriction, Mnk1-knockout mice developed exaggerated cardiac hypertrophy, fibrosis, dysfunction, and cardiomyocyte apoptosis and showed increased ERK1/2 (extracellular signal-regulated kinase 1/2) activation along with reduced sprouty2 expression. In line with the in vivo studies, Mnk1 knockdown by Mnk1 siRNA transfection induced exaggerated angiotensin II-induced cardiomyocyte hypertrophy in neonatal rat ventricular myocytes (NRVMs). Moreover, adenovirus-mediated overexpression of Mnk1 in NRVMs protected cardiomyocytes from angiotensin II-induced hypertrophy. In addition, overexpression of sprouty2 rescued NRVMs with Mnk1 knockdown from angiotensin II-induced hypertrophy. In accordance with the in vivo studies, as compared with the control group, Mnk1 knockdown led to hyperphosphorylation of ERK1/2 and suppression of the sprouty2 expression in angiotensin II-treated NRVMs; furthermore, Mnk1 overexpression led to hypophosphorylation of ERK1/2 in angiotensin II-treated NRVMs. In addition, sprouty2 overexpression suppressed the activation of ERK1/2 in angiotensin II-treated NRVMs with Mnk1 knockdown. Impressively, MnK1-knockout mice with overexpression of sprouty2 exhibited signs of a blunted cardiac hypertrophic response. Mnk1 likely carries out a suppressive function in cardiac hypertrophy via regulating the sprouty2/ERK1/2 pathway. It implicates Mnk1 in the development of cardiac remodeling.

Virus-induced gene silencing in plant MAPK research.

Virus-induced gene silencing (VIGS) technology has become more and more widely used in various plant species for rapid screening of gene functions. VIGS does not require time-consuming tissue culture steps that are needed for stable transformation in most plant species and it can be used for studying gene function even in plants that are very difficult to stably transform. Furthermore, VIGS technology provides high gene silencing efficiency (up to 95 %) and specificity. Here, we describe a VIGS protocol that can be used for studying the functions of MAPKs and other genes in a wild tobacco species, Nicotiana attenuata. This method is also suitable for other Nicotiana species and tomato with minor modifications.

C2-phytoceramide perturbs lipid rafts and cell integrity in Saccharomyces cerevisiae in a sterol-dependent manner.

Specific ceramides are key regulators of cell fate, and extensive studies aimed to develop therapies based on ceramide-induced cell death. However, the mechanisms regulating ceramide cytotoxicity are not yet fully elucidated. Since ceramides also regulate growth and stress responses in yeast, we studied how different exogenous ceramides affect yeast cells. C2-phytoceramide, a soluble form of phytoceramides, the yeast counterparts of mammalian ceramides, greatly reduced clonogenic survival, particularly in the G2/M phase, but did not induce autophagy nor increase apoptotic markers. Rather, the loss of clonogenic survival was associated with PI positive staining, disorganization of lipid rafts and cell wall weakening. Sensitivity to C2-phytoceramide was exacerbated in mutants lacking Hog1p, the MAP kinase homolog of human p38 kinase. Decreasing sterol membrane content reduced sensitivity to C2-phytoceramide, suggesting sterols are the targets of this compound. This study identified a new function of C2-phytoceramide through disorganization of lipid rafts and induction of a necrotic cell death under hypo-osmotic conditions. Since lipid rafts are important in mammalian cell signaling and adhesion, our findings further support pursuing the exploitation of yeast to understand the basis of synthetic ceramides' cytotoxicity to provide novel strategies for therapeutic intervention in cancer and other diseases.

S100A9 interaction with TLR4 promotes tumor growth.

By breeding TRAMP mice with S100A9 knock-out (S100A9(-/-)) animals and scoring the appearance of palpable tumors we observed a delayed tumor growth in animals devoid of S100A9 expression. CD11b(+) S100A9 expressing cells were not observed in normal prostate tissue from control C57BL/6 mice but were readily detected in TRAMP prostate tumors. Also, S100A9 expression was observed in association with CD68(+) macrophages in biopsies from human prostate tumors. Delayed growth of TRAMP tumors was also observed in mice lacking the S100A9 ligand TLR4. In the EL-4 lymphoma model tumor growth inhibition was observed in S100A9(-/-) and TLR4(-/-), but not in RAGE(-/-) animals lacking an alternative S100A9 receptor. When expression of immune-regulating genes was analyzed using RT-PCR the only common change observed in mice lacking S100A9 and TLR4 was a down-regulation of TGFß expression in splenic CD11b(+) cells. Lastly, treatment of mice with a small molecule (ABR-215050) that inhibits S100A9 binding to TLR4 inhibited EL4 tumor growth. Thus, S100A9 and TLR4 appear to be involved in promoting tumor growth in two different tumor models and pharmacological inhibition of S100A9-TLR4 interactions is a novel and promising target for anti-tumor therapies.

RAGE and ICAM-1 cooperate in mediating leukocyte recruitment during acute inflammation in vivo.

The receptor for advanced glycation end products (RAGE) contributes to the inflammatory response in many acute and chronic diseases. In this context, RAGE has been identified as a ligand for the beta(2)-integrin Mac-1 under static in vitro conditions. Because intercellular adhesion molecule (ICAM)-1 also binds beta(2)-integrins, we studied RAGE(-/-), Icam1(-/-), and RAGE(-/-) Icam1(-/-) mice to define the relative contribution of each ligand for leukocyte adhesion in vivo. We show that trauma-induced leukocyte adhesion in cremaster muscle venules is strongly dependent on RAGE and ICAM-1 acting together in an overlapping fashion. Additional in vivo experiments in chimeric mice lacking endothelium-expressed RAGE and ICAM-1 located the adhesion defect to the endothelial compartment. Using microflow chambers coated with P-selectin, CXCL1, and soluble RAGE (sRAGE) demonstrated that sRAGE supports leukocyte adhesion under flow conditions in a Mac-1- but not LFA-1-dependent fashion. A static adhesion assay revealed that wild-type and RAGE(-/-) neutrophil adhesion and spreading were similar on immobilized sRAGE or fibrinogen. These observations indicate a crucial role of endothelium-expressed RAGE as Mac-1 ligand and uncover RAGE and ICAM-1 as a new set of functionally linked adhesion molecules, which closely cooperate in mediating leukocyte adhesion during the acute trauma-induced inflammatory response in vivo.

The kinases MSK1 and MSK2 act as negative regulators of Toll-like receptor signaling.

The kinases MSK1 and MSK2 are activated 'downstream' of the p38 and Erk1/2 mitogen-activated protein kinases. Here we found that MSK1 and MSK2 were needed to limit the production of proinflammatory cytokines in response to stimulation of primary macrophages with lipopolysaccharide. By inducing transcription of the mitogen-activated protein kinase phosphatase DUSP1 and the anti-inflammatory cytokine interleukin 10, MSK1 and MSK2 exerted many negative feedback mechanisms. Deficiency in MSK1 and MSK2 prevented the binding of phosphorylated transcription factors CREB and ATF1 to the promoters of the genes encoding interleukin 10 and DUSP1. Mice doubly deficient in MSK1 and MSK2 were hypersensitive to lipopolysaccharide-induced endotoxic shock and showed prolonged inflammation in a model of toxic contact eczema induced by phorbol 12-myristate 13-acetate. Our results establish MSK1 and MSK2 as key components of negative feedback mechanisms needed to limit Toll-like receptor-driven inflammation.

The kinase Mirk/Dyrk1B mediates cell survival in pancreatic ductal adenocarcinoma.

Ductal adenocarcinoma of the pancreas is almost uniformly lethal as this cancer is invariably detected at an advanced stage and is resistant to treatment. The serine/threonine kinase Mirk/Dyrk1B has been shown to be antiapoptotic in rhabdomyosarcomas. We have now investigated whether Mirk might mediate survival in another cancer in which Mirk is widely expressed, pancreatic ductal adenocarcinoma. Mirk was an active kinase in each pancreatic cancer cell line where it was detected. Mirk knockdown by RNA interference (RNAi) reduced the clonogenicity of Panc1 pancreatic cancer cells 4-fold and decreased tumor cell number, showing that Mirk mediates survival in these cells. Mirk knockdown by synthetic duplex RNAis in Panc1, AsPc1, and SU86.86 pancreatic cancer cells induced apoptosis and enhanced the apoptosis induced by gemcitibine. Mirk knockdown did not increase the abundance or activation of Akt. However, four of five pancreatic carcinoma cell lines exhibited either elevated Mirk activity or elevated Akt activity, suggesting that pancreatic cancer cells primarily rely on Mirk or Akt for survival signaling. Mirk protein was detected by immunohistochemistry in 25 of 28 cases (89%) of pancreatic ductal adenocarcinoma, with elevated expression in 11 cases (39%). Increased expression of Mirk was seen in pancreatic carcinomas compared with primary cultures of normal ductal epithelium by serial analysis of gene expression and by immunohistochemistry. Thus, Mirk is a survival factor for pancreatic ductal adenocarcinoma. Because knockout of Mirk does not cause embryonic lethality, Mirk is not essential for normal cell growth and may represent a novel therapeutic target.

A novel UBA and UBX domain protein that binds polyubiquitin and VCP and is a substrate for SAPKs.

A widely expressed protein containing UBA (ubiquitin-associated) and UBX (ubiquitin-like) domains was identified as a substrate of SAPKs (stress-activated protein kinases). Termed SAKS1 (SAPK substrate-1), it was phosphorylated efficiently at Ser200 in vitro by SAPK3/p38gamma, SAPK4/p38delta and JNK (c-Jun N-terminal kinase), but weakly by SAPK2a/p38alpha, SAPK2b/p38beta2 or ERK (extracellular-signal-regulated kinase) 2. Ser200, situated immediately N-terminal to the UBX domain, became phosphorylated in HEK-293 (human embryonic kidney) cells in response to stressors. Phosphorylation was not prevented by SB 203580 (an inhibitor of SAPK2a/p38alpha and SAPK2b/p38beta2) and/or PD 184352 (which inhibits the activation of ERK1 and ERK2), and was similar in fibroblasts lacking both SAPK3/p38gamma and SAPK4/p38delta or JNK1 and JNK2. SAKS1 bound ubiquitin tetramers and VCP (valosin-containing protein) in vitro via the UBA and UBX domains respectively. The amount of VCP in cell extracts that bound to immobilized GST (glutathione S-transferase)-SAKS1 was enhanced by elevating the level of polyubiquitinated proteins, while SAKS1 and VCP in extracts were coimmunoprecipitated with an antibody raised against S5a, a component of the 19 S proteasomal subunit that binds polyubiquitinated proteins. PNGase (peptide N-glycanase) formed a 1:1 complex with VCP and, for this reason, also bound to immobilized GST-SAKS1. We suggest that SAKS1 may be an adaptor that directs VCP to polyubiquitinated proteins, and PNGase to misfolded glycoproteins, facilitating their destruction by the proteasome.

Signalling pathways involved in multisite phosphorylation of the transcription factor ATF-2.

The multisite phosphorylation of the transcription factor ATF-2 was investigated using transformed embryonic fibroblasts from wild-type mice and mice deficient in c-Jun N-terminal kinases (JNK)1 and 2, and in the presence and absence of inhibitors of p38 mitogen-activated protein kinase (p38 MAPK) and the classical MAP kinase cascade. In wild-type cells, p38 MAPK and extracellular signal-regulated protein kinase (ERK)1/2 were not rate limiting for the phosphorylation of Thr69, Thr71 or Ser90. In JNK-deficient cells, p38 MAPK substituted for JNK partially in the phosphorylation of Thr69 and p38 MAPK or ERK1/2 in the phosphorylation of Thr71. JNK was the only MAP kinase that phosphorylated Ser90 under the conditions examined.

A Toll-like receptor 2 ligand stimulates Th2 responses in vivo, via induction of extracellular signal-regulated kinase mitogen-activated protein kinase and c-Fos in dendritic cells.

The adaptive immune system can generate distinct classes of responses, but the mechanisms that determine this are poorly understood. In this study, we demonstrate that different Toll-like receptor (TLR) ligands induce distinct dendritic cell (DC) activation and immune responses in vivo. Thus, Escherichia coli LPS (TLR-4 stimulus), activates DCs to produce abundant IL-12(p70), but little IL-10, and stimulates Th1 and Tc1 responses. In contrast, Pam-3-cys (TLR-2 stimulus) elicits less IL-12(p70), but abundant IL-10, and favors Th2 and T cytotoxic 2 (Tc2) responses. These distinct responses likely occur via differences in extracellular signal-regulated kinase signaling in DCs. Thus, Pam-3-cys induces enhanced extracellular signal-regulated kinase signaling, compared with LPS, resulting in suppressed IL-12(p70) and enhanced IL-10 production, as well as enhanced induction of the transcription factor, c-Fos. Interestingly, DCs from c-fos(-/-) mice produce more IL-12(p70), but less IL-10, compared with control DCs. Therefore, different TLR ligands induce distinct cytokines and signaling in DCs, and differentially bias Th responses in vivo.

A p38 inhibitor allows to dissociate differentiation and apoptotic processes triggered upon LIF withdrawal in mouse embryonic stem cells.

Mouse embryonic stem cells remain pluripotent when maintained in the presence of leukemia inhibitory factor (LIF). Upon LIF withdrawal, most cells differentiate into various lineages, while some die by apoptosis within 3 days. We have analyzed the activation pattern of the mitogen-activated protein kinase (MAPK) families and characterized the expression profile of selected genes modulated during differentiation or apoptosis. We show that p38 MAPKs are activated first, during the apoptotic crisis, while extracellular-regulated kinases and c-Jun N-terminal kinases are induced after the apoptotic crisis in differentiated cells. However, by using both p38 kinase inhibitors (PD169316 and SB203580) and a p38alpha(-/-) cell line, we demonstrate that p38alpha activation is rather a consequence than a cause of apoptosis. We thus reveal novel properties of PD169316, which induces cell survival without impairing cell differentiation, and identify PD169316-sensitive targets like the fibroblast growth factor-5, Brachyury and bcl-2 genes. Finally, we demonstrate that overexpression of the PD169316 - regulated bcl-2 gene prevents LIF withdrawal - induced cell death.

JNK-dependent phosphorylation of c-Jun on serine 63 mediates nitric oxide-induced apoptosis of neuroblastoma cells.

c-Jun NH2-terminal kinases (JNKs) potentiate transcriptional activity of c-Jun by phosphorylating serines 63 and 73. Moreover, JNK and c-Jun can modulate apoptosis. However, an involvement of nitric oxide (NO)-induced phosphorylation of c-Jun on Ser-63 and Ser-73 in apoptosis has not been explored. We report that in SH-Sy5y neuroblastoma cells, NO induced apoptosis following JNK activation and phosphorylation of c-Jun almost exclusively on Ser-63. Importantly, NO-induced apoptosis and caspase-3 activity were inhibited in cells stably transformed with dominant-negative c-Jun in which Ser-63 is mutated to alanine (S63A), but not in cells transformed with dominant-negative c-Jun (S73A). Ser-63 of c-Jun (but not Ser-73) was required for NO-induced, c-Jun-dependent transcriptional activity. NO-induced apoptosis, Ser-63 phosphorylation of c-Jun, and caspase-3 activity were all inhibited in SH-Sy5y cells transformed with dominant-negative jnk. A caspase-3 inhibitor prevented apoptosis but not c-Jun phosphorylation. In a different neuroblastoma cell line, NO-induced Ser-63 phosphorylation of c-Jun and apoptosis were blocked by a specific JNK inhibitor. We conclude that NO-inducible apoptosis is mediated by JNK-dependent Ser-63 phosphorylation of c-Jun upstream of caspase-3 activation in neuroblastoma cells.

5-fluorouracil blocks transforming growth factor-beta-induced alpha 2 type I collagen gene (COL1A2) expression in human fibroblasts via c-Jun NH2-terminal kinase/activator protein-1 activation.

5-Fluorouracil (5-FU), a pyrimidine analog widely used in cancer chemotherapy and in glaucoma surgery, has recently shown some efficacy in the treatment of keloids, scars that overgrow the boundaries of original wounds. Given the physiopathological importance of transforming growth factor-beta (TGF-beta) in keloid and scar formation, we have examined whether the clinical benefits from 5-FU treatment may result from its capacity to interfere with TGF-beta signaling and resulting activation of type I collagen gene expression. Using various molecular approaches to study the mechanisms underlying 5-FU effects, we have demonstrated that 5-FU antagonizes TGF-beta-driven COL1A2 transcription and associated type I collagen production by dermal fibroblasts. In addition, 5-FU inhibits both SMAD3/4-specific transcription and formation of SMAD/DNA complexes induced by TGF-beta. 5-FU induces c-Jun phosphorylation and activates both AP-1-specific transcription and DNA binding. Overexpression of an antisense c-jun expression vector, or that of a dominant-negative form of MKK4 that interferes with c-Jun N-terminal kinase (JNK) activation, blocks the inhibitory activity of 5-FU on TGF-beta-induced COL1A2 transcription. Furthermore, in a cellular context devoid of JNK activity (i.e., JNK-/- fibroblasts), 5-FU inhibits neither formation of SMAD/DNA complexes nor SMAD-driven COL1A2 transcription in response to TGF-beta. Together, these results identify 5-FU as a potent inhibitor of TGF-beta/SMAD signaling, capable of blocking TGF-beta-induced, SMAD-driven up-regulation of COL1A2 gene expression in a JNK-dependent manner. We thus provide a molecular explanation to the observed clinical benefits of 5-FU in the treatment of keloids and hypertrophic scars.

Downregulation of FUSE-binding protein and c-myc by tRNA synthetase cofactor p38 is required for lung cell differentiation.

p38 is associated with a macromolecular tRNA synthetase complex. It has an essential role as a scaffold for the complex, and genetic disruption of p38 in mice causes neonatal lethality. Here we investigated the molecular mechanisms underlying lethality of p38-mutant mice. p38-deficient mice showed defects in lung differentiation and respiratory distress syndrome. p38 was found to interact with FUSE-binding protein (FBP), a transcriptional activator of c-myc. Binding of p38 stimulated ubiquitination and degradation of FBP, leading to downregulation of c-myc, which is required for differentiation of functional alveolar type II cells. Transforming growth factor-beta (TGF-beta) induced p38 expression and promoted its translocation to nuclei for the regulation of FBP and c-myc. Thus, this work identified a new activity of p38 as a mediator of TGF-beta signaling and its functional importance in the control of c-myc during lung differentiation.

p38 Mitogen-activated protein kinase regulation of JB6 Cl41 cell transformation promoted by epidermal growth factor.

The relationship between cell transformation and p38 MAP kinase, a major mitogen-activated protein (MAP) kinase pathway converting signals of various extracellular stimuli into expression of specific target genes through activation of transcription factors, still remains unclear. The aim of the present study was to investigate the role of the p38 MAP kinase pathway in epidermal growth factor (EGF)-induced cell transformation in JB6 cells. Our data show that a dominant negative mutant of p38 MAP (DN-p38) kinase inhibits EGF-promoted JB6 Cl41 cell transformation and that SB202190, an inhibitor of p38 MAP kinase, also inhibits JB6 Cl41 cell transformation in a dose-dependent manner. Moreover, our results show that DN-p38 MAP kinase inhibits the phosphorylation of EGF-stimulated activating transcription factor-2 (ATF-2) and signal transducer and activator of transcription 1 (STAT1). Additionally, DN-p38 MAP kinase inhibits EGF-induced phosphorylation of c-Myc (Thr58/Ser62). Gel shift assays indicate that DN-p38 MAP kinase inhibits EGF-induced activator protein-1 (AP-1) DNA binding in a dose-dependent manner. These results show that p38 MAP kinase plays a key role in the regulation of EGF-induced cell transformation in JB6 cells through regulation of phosphorylation of p38 MAP kinase and activation of its target genes in phosphorylation, c-Myc cell transformation-related genes, and AP-1 binding ability.

JNK1 is required for maintenance of neuronal microtubules and controls phosphorylation of microtubule-associated proteins.

Microtubules (MTs) play an important role in elaboration and maintenance of axonal and dendritic processes. MT dynamics are modulated by MT-associated proteins (MAPs), whose activities are regulated by protein phosphorylation. We found that a member of the c-Jun NH(2)-terminal protein kinase (JNK) subgroup of MAP kinases, JNK1, is involved in regulation of MT dynamics in neuronal cells. Jnk1(-/-) mice exhibit disrupted anterior commissure tract formation and a progressive loss of MTs within axons and dendrites. MAP2 and MAP1B polypeptides are hypophosphorylated in Jnk1(-/-) brains, resulting in compromised ability to bind MTs and promote their assembly. These results suggest that JNK1 is required for maintaining the cytoskeletal integrity of neuronal cells and is a critical regulator of MAP activity and MT assembly.

c-Jun NH(2)-terminal kinase is essential for the regulation of AP-1 by tumor necrosis factor.

The c-Jun NH(2)-terminal kinase (JNK) is activated by the cytokine tumor necrosis factor (TNF). This pathway is implicated in the regulation of AP-1-dependent gene expression by TNF. To examine the role of the JNK signaling pathway, we compared the effects of TNF on wild-type and Jnk1(-/-) Jnk2(-/-) murine embryo fibroblasts. We show that JNK is required for the normal regulation of AP-1 by TNF. The JNK-deficient cells exhibited decreased expression of c-Jun, JunD, c-Fos, Fra1, and Fra2; decreased phosphorylation of c-Jun and JunD; and decreased AP-1 DNA binding activity. The JNK-deficient cells also exhibited defects in the regulation of the AP-1-related transcription factor ATF2. These changes were associated with marked defects in TNF-regulated gene expression. The JNK signal transduction pathway is therefore essential for AP-1 transcription factor regulation in cells exposed to TNF.

Role of JNK, p38, and ERK in platelet-derived growth factor-induced vascular proliferation, migration, and gene expression.

OBJECTIVE: We investigated the comparative roles of mitogen-activated protein (MAP) kinases, including c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38, in vascular smooth muscle cell (VSMC) proliferation, migration, and gene expression. METHODS AND RESULTS: VSMCs were infected with recombinant adenovirus containing dominant-negative mutants of ERK, p38, and JNK (Ad-DN-ERK, Ad-DN-p38, and Ad-DN-JNK, respectively) to specifically inhibit the respective MAP kinases and then stimulated with platelet-derived growth factor (PDGF)-BB. Ad-DN-ERK attenuated PDGF-BB-induced VSMC proliferation more potently than Ad-DN-p38 or Ad-DN-JNK, indicating the dominant role of ERK in VSMC proliferation. Ad-DN-ERK, Ad-DN-p38, and Ad-DN-JNK similarly inhibited PDGF-induced VSMC migration. Ad-DN-ERK and Ad-DN-JNK suppressed PDGF-BB-induced downregulation of cyclin-dependent kinase inhibitor p27Kip1, whereas Ad-DN-p38 decreased PDGF-BB-induced upregulation of p21Cip1. Ad-DN-ERK inhibited PDGF-BB-induced plasminogen activator inhibitor type-1 (PAI-1), monocyte chemoattractant protein-1, and transforming growth factor-beta1 expressions, Ad-DN-p38 blocked monocyte chemoattractant protein-1 and transforming growth factor-beta1 expression but not PAI-1, whereas Ad-DN-JNK suppressed only PAI-1 expression. Moreover, in vivo gene transfer of Ad-DN-p38 to rat carotid artery caused the inhibition of intimal hyperplasia by balloon injury, indicating the involvement of p38 in vascular remodeling in vivo. CONCLUSIONS: We propose that these 3 MAP kinases participate in vascular diseases via differential molecular mechanisms and are new therapeutic targets for treatment of vascular diseases.

The role of the Ah receptor and p38 in benzo[a]pyrene-7,8-dihydrodiol and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide-induced apoptosis.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants in the environment. Benzo[a]pyrene (B[a]P), a prototypical member of this class of chemicals, affects cellular signal transduction pathways and induces apoptosis. In this study, the proximate carcinogen of B[a]P metabolism, trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-dihydrodiol) and the ultimate carcinogen, B[a]P-r-7,t-8-dihydrodiol-t-9,10-epoxide(+/-) (BPDE-2) were found to induce apoptosis in human HepG2 cells. Apoptosis initiated by B[a]P-7,8-dihydrodiol was linked to activation of the Ah receptor and induction of CYP1A1, an event that can lead to the formation of BPDE-2. With both B[a]P-7,8-dihydrodiol and BPDE-2 treatment, changes in anti- and pro-apoptotic events in the Bcl-2 family of proteins correlated with the release of mitochondrial cytochrome c and caspase activation. The onset of apoptosis as monitored by caspase activation was linked to mitogen-activated protein (MAP) kinases. Utilizing mouse hepa1c1c7 cells and the Arnt-deficient BPRc1 cells, activation of MAP kinase p38 by B[a]P-7,8-dihydrodiol was shown to be Ah receptor-dependent, indicating that metabolic activation by CYP1A1 was required. This was in contrast to p38 activation by BPDE-2, an event that was independent of Ah receptor function. Confirmation that MAP kinases play a critical role in BPDE-2-induced apoptosis was shown by inhibiting caspase activation of poly(ADP-ribose)polymerase 1 (PARP-1) by chemical inhibitors of p38 and ERK1/2. Furthermore, mouse embryo p38-/- fibroblasts were shown to be resistant to the actions of BPDE-2-induced apoptosis as determined by annexin V analysis, cytochrome c release, and cleavage of PARP-1. These results confirm that the Ah receptor plays a critical role in B[a]P-7,8-dihydrodiol-induced apoptosis while p38 MAP kinase links the actions of an electrophilic metabolite like BPDE-2 to the regulation of programmed cell death.