Effects of marine noise pollution on Mediterranean fishes and invertebrates: A review.

Marine noise pollution (MNP) can cause a multitude of impacts on many organisms, but information is often scattered and general outcomes difficult to assess. We have reviewed the literature on MNP impacts on Mediterranean fish and invertebrates. Both chronic and acute MNP produced by various human activities - e.g. maritime traffic, pile driving, air guns - were found to cause detectable effects on intra-specific communication, vital processes, physiology, behavioral patterns, health status and survival. These effects on individuals can extend to inducing population- and ecosystem-wide alterations, especially when MNP impacts functionally important species, such as keystone predators and habitat forming species. Curbing the threats of MNP in the Mediterranean Sea is a challenging task, but a variety of measures could be adopted to mitigate MNP impacts. Successful measures will require more accurate information on impacts and that effective management of MNP really becomes a priority in the policy makers' agenda.

The topoisomerase 1-interacting protein BTBD1 is essential for muscle cell differentiation.

DNA topoisomerase I (Topo1) contributes to vital biological functions, but its regulation is not clearly understood. The BTBD1 protein was recently cloned on the basis of its interaction with the core domain of Topo1 and is expressed particularly in skeletal muscle. To determine BTBD1 functions in this tissue, the in vitro model used was the C2C12 mouse muscle cell line, which expresses BTBD1 mainly after myotube differentiation. We studied the effects of a stably overexpressed BTBD1 protein truncated of the 108 N-terminal amino-acid residues and harbouring a C-terminal FLAG tag (Delta-BTBD1). The proliferation speed of Delta-BTBD1 C2C12 cells was significantly decreased and no myogenic differentiation was observed, although these cells maintained their capacity to enter adipocyte differentiation. These alterations could be related to Topo1 deregulation. This hypothesis is further supported by the decrease in nuclear Topo1 content in Delta-BTBTD1 proliferative C2C12 cells and the switch from the main peripheral nuclear localization of Topo1 to a mainly nuclear diffuse localization in Delta-BTBTD1 C2C12 cells. Finally, this study demonstrated that BTBD1 is essential for myogenic differentiation.

Increased MAPK signaling during in vitro muscle wounding.

Regeneration of skeletal muscle upon injury is a complex process, involving activation of satellite cells, followed by migration, fusion, and regeneration of damaged myofibers. Previous work concerning the role of the mitogen activated protein (MAP) kinase signaling pathways in muscle injury comes primarily from studies using chemically induced wounding. The purpose of this study was to test the hypothesis that physical injury to skeletal muscle cells in vitro activates the MAP kinase signaling pathways. We demonstrate that extracellular signal regulated kinases (ERKs) 1, 2, and p38 are rapidly and transiently activated in response to injury in C2C12 cells, and are primarily localized to cells adjacent to the wound bed. Culture medium from wounded cells is able to stimulate activation of p38 but not ERK in unwounded cells. These results suggest that both ERK and p38 are involved in the response of muscle cells to physical injury in culture, and reflect what is seen in whole tissues in vivo.

Inhibition of myogenesis enables adipogenic trans-differentiation in the C2C12 myogenic cell line.

C2C12 cells are a well-established model system for studying myogenesis. We examined whether inhibiting the process of myogenesis via expression of dominant negative (DN) mitogen-activated protein kinase kinase-3 (MKK3) facilitated the trans-differentiation of these cells into adipocytes. Cells expressing DN MKK3 respond to rosiglitazone, resulting in adipocyte formation. The effects of rosiglitazone appear to be potentiated through peroxisome proliferator activating receptor-gamma. This trans-differentiation is inhibited by the use of the phosphoinositide-3 (PI3) kinase inhibitor, LY294002. These results indicate that preventing myogenesis through expression of DN MKK3 facilitates adipocytic trans-differentiation, and involves PI3 kinase signalling.

Attenuation of colon inflammation through activators of the retinoid X receptor (RXR)/peroxisome proliferator-activated receptor gamma (PPARgamma) heterodimer. A basis for new therapeutic strategies.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is highly expressed in the colon mucosa and its activation has been reported to protect against colitis. We studied the involvement of PPARgamma and its heterodimeric partner, the retinoid X receptor (RXR) in intestinal inflammatory responses. PPARgamma(1/)- and RXRalpha(1/)- mice both displayed a significantly enhanced susceptibility to 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis compared with their wild-type littermates. A role for the RXR/PPARgamma heterodimer in the protection against colon inflammation was explored by the use of selective RXR and PPARgamma agonists. TNBS-induced colitis was significantly reduced by the administration of both PPARgamma and RXR agonists. This beneficial effect was reflected by increased survival rates, an improvement of macroscopic and histologic scores, a decrease in tumor necrosis factor alpha and interleukin 1beta mRNA levels, a diminished myeloperoxidase concentration, and reduction of nuclear factor kappaB DNA binding activity, c-Jun NH(2)-terminal kinase, and p38 activities in the colon. When coadministered, a significant synergistic effect of PPARgamma and RXR ligands was observed. In combination, these data demonstrate that activation of the RXR/PPARgamma heterodimer protects against colon inflammation and suggest that combination therapy with both RXR and PPARgamma ligands might hold promise in the clinic due to their synergistic effects.

Tumor necrosis factor alpha-mediated inhibition of melanogenesis is dependent on nuclear factor kappa B activation.

Melanogenesis is a physiological process resulting in the synthesis of melanin pigments which play a crucial protective role against skin photocarcinogenesis. In vivo, solar ultraviolet light triggers the secretion of numerous keratinocyte-derived factors that are implicated in the regulation of melanogenesis. Among these, tumor necrosis factor alpha (TNFalpha), a cytokine implicated in the pro-inflammatory response, down-regulates pigment synthesis in vitro. In this report, we aimed to determine the molecular mechanisms by which this cytokine inhibits melanogenesis in B16 melanoma cells. First, we show that TNFalpha inhibits the activity and protein expression of tyrosinase which is the key enzyme of melanogenesis. Further, we demonstrate that this effect is subsequent to a down-regulation of the tyrosinase promoter activity in both basal and cAMP-induced melanogenesis. Finally, we present evidence indicating that the inhibitory effect of TNFalpha on melanogenesis is dependent on nuclear factor kappa B (NFkappaB) activation. Indeed, overexpression of this transcription factor in B16 cells is sufficient to inhibit tyrosinase promoter activity. Furthermore, a mutant of inhibitory kappa B (IkappaB), that prevents NFkappaB activation, is able to revert the effect of TNFalpha on the tyrosinase promoter activity. Taken together, our results clarify the mechanisms by which TNFalpha inhibits pigmentation and point out the key role of NFkappaB in the regulation of melanogenesis.

The JNK pathway regulates the In vivo deletion of immature CD4(+)CD8(+) thymocytes.

The extracellular signal-regulated kinase (ERK), the c-Jun NH2-terminal kinase (JNK), and p38 MAP kinase pathways are triggered upon ligation of the antigen-specific T cell receptor (TCR). During the development of T cells in the thymus, the ERK pathway is required for differentiation of CD4(-)CD8(-) into CD4(+)CD8(+) double positive (DP) thymocytes, positive selection of DP cells, and their maturation into CD4(+) cells. However, the ERK pathway is not required for negative selection. Here, we show that JNK is activated in DP thymocytes in vivo in response to signals that initiate negative selection. The activation of JNK in these cells appears to be mediated by the MAP kinase kinase MKK7 since high levels of MKK7 and low levels of Sek-1/MKK4 gene expression were detected in thymocytes. Using dominant negative JNK transgenic mice, we show that inhibition of the JNK pathway reduces the in vivo deletion of DP thymocytes. In addition, the increased resistance of DP thymocytes to cell death in these mice produces an accelerated reconstitution of normal thymic populations upon in vivo DP elimination. Together, these data indicate that the JNK pathway contributes to the deletion of DP thymocytes by apoptosis in response to TCR-derived and other thymic environment- mediated signals.

The neuronal death induced by endotoxic shock but not that induced by excitatory amino acids requires TNF-alpha.

We studied, using organotypic hippocampal slices in culture, the role of pro-inflammatory cytokines, oxygen radicals and nitric oxide in neuronal death induced either by endotoxic insult [interferon (IFN) gamma, 24 h followed by lipopolysaccharide, 24 h] or by glutamate receptor-mediated excitotoxic insult. We demonstrated that neuronal death induced by endotoxic insult was absolutely dependent on the synthesis of tumour necrosis factor alpha (TNF-alpha). Indeed, TNF-alpha antibodies and SB203580, an inhibitor of p38 stress kinase known to block TNF-alpha and other cytokine synthesis, completely protected neurons from the endotoxic insult. Inhibiting oxygen radical and nitric oxide productions also reduced the endotoxic shock. We also showed that after priming the cultures with IFN-gamma, TNF-alpha was unable to induce neuronal death unless oxygen-free radicals were exogenously provided. In contrast, although glutamate receptor-induced excitotoxicity was associated with a low TNF-alpha synthesis and a modest activation of p38 stress kinase, neither TNF-alpha antibodies nor SB203580 were able to decrease excitotoxic neuronal insult. We did not reduce glutamate receptor-induced neuronal death with superoxide dismutase plus catalase. In conclusion, although inflammation follows glutamate receptor-mediated neurotoxicity, the mechanisms by which an endotoxic insult triggers neuronal death are different from those involved in excitotoxicity.

Solar ultraviolet light activates extracellular signal-regulated kinases and the ternary complex factor in human normal keratinocytes.

Exposure to ultraviolet radiation of solar light is responsible for inflammation, premature skin aging and is the main cause of human skin carcinogenesis. While the noxious consequences of U.V. exposure are known, the molecular events triggered by this radiation are poorly understood. We observed that U.V.-A and U.V.-B irradiation of human keratinocytes induces the activation of tyrosine kinase pathways leading to the tyrosine phosphorylation of several cellular proteins. We also observed a stimulation of the Stress Activated Protein kinases (SAPKs), p38 and JNK, and an activation of the transcription factors AP-1 in response to U.V.-A and U.V.-B radiation. Furthermore, we clearly demonstrated that physiological U.V. doses are able to activate the Extracellular signal-Regulated Kinases, ERK1 and ERK2, which could explain the activation of the Ternary Complex Factor. Thus, in human keratinocytes, solar U.V. light activates multiple signalling pathways that could be involved in skin inflammation following U.V.-induced skin injury or in U.V.-induced skin carcinogenesis.

Translation initiation factor IF2 of the myxobacterium Stigmatella aurantiaca: presence of a single species with an unusual N-terminal sequence.

The structural gene for translation initiation factor IF2 (infB) was isolated from the myxobacterium Stigmatella aurantiaca on a 5.18-kb BamHI genomic restriction fragment. The infB gene (ca. 3.16 kb) encodes a 1,054-residue polypeptide with extensive homology within its G domain and C terminus with the equivalent regions of IF2s from Escherichia coli, Bacillus subtilis, Bacillus stearothermophilus, and Streptococcus faecium. The N-terminal region does not display any significant homology to other known proteins. The S. aurantiaca infB gene encodes a single protein which cross-reacted with antiserum to E. coli IF2 and was able to complement an E. coli infB mutant. The S. aurantiaca IF2 is distinguished from all other IF2s by a sequence of 160 residues near the N terminus that has an unusual composition, made up essentially of alanine, proline, valine, and glutamic acid. Within this sequence, the pattern PXXXAP is repeated nine times. Complete deletion of this sequence did not affect the factor's function in initiation of translation and even increased its capacity to complement the E. coli infB mutant.

Reprogramming the signalling requirement for AP-1 (activator protein-1) activation during differentiation of precursor CD4+ T-cells into effector Th1 and Th2 cells.

Upon antigenic stimulation, precursor CD4+ helper T-cells differentiate into two subsets of effector cells, Th1 and Th2. These two subpopulations are defined by the pattern of cytokine expression that distinguishes these differentiated cells from their precursors. We have used reporter transgenic mice here to show that, during differentiation of precursor T-cells into effector Th1 or Th2 cells, high levels of preformed activator protein (AP)-1 complexes are accumulated. However, upon stimulation, the preformed AP-1 complexes in effector Th2 cells, but not in Th1 cells, are able to induce high levels of AP-1 transcriptional activity. Furthermore, in contrast to precursor T-cells, the induction of AP-1 transcriptional activity is independent of calcium and co-stimulatory signals in effector Th2 cells. This AP-1 transcriptional activity appears to correlate with the presence of JunB complexes, which accumulate differentially in effector Th2 cells, but not in precursor CD4+ T-cells or effector Th1 cells. Unlike precursor cells, the activation of AP-1 does not appear to be mediated by c-Jun N-terminal kinase (JNK) in effector Th2 cells. These results indicate that during differentiation of T-cells, and probably other cell types, the signal requirements for the AP-1 transcription machinery are reprogrammed to enable the differentiated cells to perform their specialized functions.

Selective interaction of JNK protein kinase isoforms with transcription factors.

The JNK protein kinase is a member of the MAP kinase group that is activated in response to dual phosphorylation on threonine and tyrosine. Ten JNK isoforms were identified in human brain by molecular cloning. These protein kinases correspond to alternatively spliced isoforms derived from the JNK1, JNK2 and JNK3 genes. The protein kinase activity of these JNK isoforms was measured using the transcription factors ATF2, Elk-1 and members of the Jun family as substrates. Treatment of cells with interleukin-1 (IL-1) caused activation of the JNK isoforms. This activation was blocked by expression of the MAP kinase phosphatase MKP-1. Comparison of the binding activity of the JNK isoforms demonstrated that the JNK proteins differ in their interaction with ATF2, Elk-1 and Jun transcription factors. Individual members of the JNK group may therefore selectively target specific transcription factors in vivo.

MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway.

The p38 mitogen-activated protein (MAP) kinase signal transduction pathway is activated by proinflammatory cytokines and environmental stress. The detection of p38 MAP kinase in the nucleus of activated cells suggests that p38 MAP kinase can mediate signaling to the nucleus. To test this hypothesis, we constructed expression vectors for activated MKK3 and MKK6, two MAP kinase kinases that phosphorylate and activate p38 MAP kinase. Expression of activated MKK3 and MKK6 in cultured cells caused a selective increase in p38 MAP kinase activity. Cotransfection experiments demonstrated that p38 MAP kinase activation causes increased reporter gene expression mediated by the transcription factors ATF2 and Elk-1. These data demonstrate that the nucleus is one target of the p38 MAP kinase signal transduction pathway.

Cdc42 and PAK-mediated signaling leads to Jun kinase and p38 mitogen-activated protein kinase activation.

The PAK family of protein kinases has been suggested as a potential target of the Cdc42 and Rac GTPases based on studies in vitro. We show that PAK-3 is activated by Cdc42 in vivo. Both, activated (GTPase-defective) Cdc42 and a constitutively active PAK-3 mutant stimulated the activity of Jun kinase 1 (JNK1) in transfected cells. Activated Cdc42 also stimulated the activity of the related p38 mitogen-activated protein kinase but was a less effective activator of ERK2. The effect of Cdc42 on JNK activity was similar to that of the potent inflammatory cytokine interleukin-1 (IL-1). The observation that a dominant-negative Cdc42 mutant inhibited IL-1 activation of JNK1 indicates a role for Cdc42 in IL-1 signaling. These results suggest that Cdc42 and PAK may mediate the effects of cytokines on transcriptional regulation.

Independent human MAP-kinase signal transduction pathways defined by MEK and MKK isoforms.

Mammalian mitogen-activated protein (MAP) kinases include extracellular signal-regulated protein kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38 subgroups. These MAP kinase isoforms are activated by dual phosphorylation on threonine and tyrosine. Two human MAP kinase kinases (MKK3 and MKK4) were cloned that phosphorylate and activate p38 MAP kinase. These MKK isoforms did not activate the ERK subgroup of MAP kinases, but MKK4 did activate JNK. These data demonstrate that the activators of p38 (MKK3 and MKK4), JNK (MKK4), and ERK (MEK1 and MEK2) define independent MAP kinase signal transduction pathways.

Transcription factor ATF2 regulation by the JNK signal transduction pathway.

Treatment of cells with pro-inflammatory cytokines or ultraviolet radiation causes activation of the c-Jun NH2-terminal protein kinase (JNK). Activating transcription factor-2 (ATF2) was found to be a target of the JNK signal transduction pathway. ATF2 was phosphorylated by JNK on two closely spaced threonine residues within the NH2-terminal activation domain. The replacement of these phosphorylation sites with alanine inhibited the transcriptional activity of ATF2. These mutations also inhibited ATF2-stimulated gene expression mediated by the retinoblastoma (Rb) tumor suppressor and the adenovirus early region 1A (E1A) oncoprotein. Furthermore, expression of dominant-negative JNK inhibited ATF2 transcriptional activity. Together, these data demonstrate a role for the JNK signal transduction pathway in transcriptional responses mediated by ATF2.

JNK2 contains a specificity-determining region responsible for efficient c-Jun binding and phosphorylation.

The transcriptional activity of c-Jun is augmented through phosphorylation at two sites by a c-Jun amino-terminal kinase (JNK). All cells express two distinct JNK activities, 46 and 55 kD in size. It is not clear which of them is the more important c-Jun kinase and how they specifically recognize c-Jun. The 46-kD form of JNK was identified as a new member of the MAP kinase group of signal-transducing enzymes, JNK1. Here, we report the molecular cloning of the 55-kD form of JNK, JNK2, which exhibits 83% identity and similar regulation to JNK1. Despite this close similarity, the two JNKs differ greatly in their ability to interact with c-Jun. JNK2 binds c-Jun approximately 25 times more efficiently than JNK1, and as a result has a lower Km toward c-Jun than JNK1. The structural basis for this difference was investigated and traced to a small beta-strand-like region near the catalytic pocket of the enzyme. Modeling suggests that this region is solvent exposed and therefore is likely to serve as a docking site that increases the effective concentration of c-Jun near JNK2. These results explain how two closely related MAP kinases can differ in their ability to recognize specific substrates and thereby elicit different biological responses.

Differential activation of ERK and JNK mitogen-activated protein kinases by Raf-1 and MEKK.

Growth factors activate mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases (ERKs) and Jun kinases (JNKs). Although the signaling cascade from growth factor receptors to ERKs is relatively well understood, the pathway leading to JNK activation is more obscure. Activation of JNK by epidermal growth factor (EGF) or nerve growth factor (NGF) was dependent on H-Ras activation, whereas JNK activation by tumor necrosis factor alpha (TNF-alpha) was Ras-independent. Ras activates two protein kinases, Raf-1 and MEK (MAPK, or ERK, kinase) kinase (MEKK). Raf-1 contributes directly to ERK activation but not to JNK activation, whereas MEKK participated in JNK activation but caused ERK activation only after overexpression. These results demonstrate the existence of two distinct Ras-dependent MAPK cascades--one initiated by Raf-1 leading to ERK activation, and the other initiated by MEKK leading to JNK activation.

Signal transduction by tumor necrosis factor mediated by JNK protein kinases.

JNK protein kinases are distantly related to mitogen-activated protein kinases (ERKs) and are activated by dual phosphorylation on Tyr and Thr. The JNK protein kinase group includes the 46-kDa isoform JNK1. Here we describe the molecular cloning of a second member of the JNK group, the 55-kDa protein kinase JNK2. The activities of both JNK isoforms are markedly increased by exposure of cells to UV radiation. Furthermore, JNK protein kinase activation is observed in cells treated with tumor necrosis factor. Although both JNK isoforms phosphorylate the NH2-terminal activation domain of the transcription factor c-Jun, the activity of JNK2 was approximately 10-fold greater than that of JNK1. This difference in c-Jun phosphorylation correlates with increased binding of c-Jun to JNK2 compared with JNK1. The distinct in vitro biochemical properties of these JNK isoforms suggest that they may have different functions in vivo. Evidence in favor of this hypothesis was obtained from the observation that JNK1, but not JNK2, complements a defect in the expression of the mitogen-activated protein kinase HOG1 in the yeast Saccharomyces cerevisiae. Together, these data indicate a role for the JNK group of protein kinases in the signal transduction pathway initiated by proinflammatory cytokines and UV radiation.

c-Jun N-terminal phosphorylation correlates with activation of the JNK subgroup but not the ERK subgroup of mitogen-activated protein kinases.

c-Jun transcriptional activity is stimulated by phosphorylation at two N-terminal sites: Ser-63 and -73. Phosphorylation of these sites is enhanced in response to a variety of extracellular stimuli, including growth factors, cytokines, and UV irradiation. New members of the mitogen-activated protein (MAP) kinase group of signal-transducing enzymes, termed JNKs, bind to the activation domain of c-Jun and specifically phosphorylate these sites. However, the N-terminal sites of c-Jun were also suggested to be phosphorylated by two other MAP kinases, ERK1 and ERK2. Despite these reports, we find that unlike the JNKs, ERK1 and ERK2 do not phosphorylate the N-terminal sites of c-Jun in vitro; instead they phosphorylate an inhibitory C-terminal site. Furthermore, the phosphorylation of c-Jun in vivo at the N-terminal sites correlates with activation of the JNKs but not the ERKs. The ERKs are probably involved in the induction of c-fos expression and thereby contribute to the stimulation of AP-1 activity. Our study suggests that two different branches of the MAP kinase group are involved in the stimulation of AP-1 activity through two different mechanisms.