Bioinformatic analysis identifies epidermal development genes that contribute to melanoma progression.

Several diagnostic and prognostic markers for melanoma have been identified in last few years. However, their actual contribution to melanoma progression have not been investigated in detail. This study was aimed to identify genes, biological processes, and signaling pathways implicated in melanoma progression by applying bioinformatics analysis. We identified nine differentially expressed genes (DEGs) (IL36RN, KRT6A, KRT6B, KRT16, S100A7, SPRR1A, SPRR1B, SPRR2B, and KLK7) that were upregulated in primary melanoma compared with metastatic melanoma in all five datasets analyzed. All these genes except IL36RN, both form a protein-protein interaction network and have cellular functions associated with constitutive processes of keratinocytes. Thus, they were generically termed Epidermal Development and Cornification (EDC) genes. The differential expression of these genes in primary and metastatic melanoma was confirmed in the TCGA-SKCM cohort. High expression of the EDC genes correlated with reduced tumor thickness in primary melanoma and shorter survival in metastatic melanoma. Analysis of DEGs from primary melanoma patients displaying high or low expression of all eight EDC revealed that the upregulated genes are enriched in biological process related to cell migration, extracellular matrix organization, invasion, and Epithelial-Mesenchymal Transition. Further analysis of enriched curated oncogenic genesets together with RPPA data of phosphorylated proteins revealed the activation of MEK, ATF2, and EGFR pathways in tumors displaying high expression of EDC genes. Thus, EDC genes may contribute to melanoma progression by promoting the activation of MEK, ATF2, and EGFR pathways together with biological processes associated with tumor aggressiveness.

The yeast two-component SLN1 branch of the HOG pathway and the scaffolding activity of Pbs2 modulate the response to endoplasmic reticulum stress induced by tunicamycin.

In addition to the UPR pathway, yeast cells require components of the HOG pathway to respond to ER stress. In this work, we found that unphosphorylated Sln1 and Ssk1 are required to mount an appropriate response to Tn. We also found that the MAPKKKs Ssk2 participates in the Tn response, but its osmo-redundant protein Ssk22 does not. We also found that the Pbs2 docking sites for Ssk2 (RDS-I and KD) are partially dispensable when mutated separately; however, the prevention of Ssk2 binding to Pbs2, by the simultaneous mutation of RDS-I and KD, caused strong sensitivity to Tn. In agreement with the lack of Hog1 phosphorylation during Tn treatment, a moderate resistance to Tn is obtained when a Pbs2 version lacking its kinase activity is expressed; however, the presence of mutual Pbs2-Hog1 docking sites is essential for the Tn response. Finally, we detected that Tn induced a transcriptional activation of some components of the SLN1 branch. These results indicate that the Tn response requires a complex formed by the MAPK module and components of the SLN1 branch but not their canonical osmoregulatory activities.

c-Jun integrates signals from both MEK/ERK and MKK/JNK pathways upon vaccinia virus infection.

Usurpation of the host's signalling pathways is a common strategy employed by viruses to promote their successful replication. Here we show that infection with the orthopoxvirus vaccinia virus (VACV) leads to sustained stimulation of c-Jun activity during the entire infective cycle. This stimulation is temporally regulated through MEK/ERK or MKK/JNK pathways, i.e. during the early/mid phase (1 to 6 hpi) and in the late phase (9 to 24 hpi) of the infective cycle, respectively. As a transcriptional regulator, upon infection with VACV, c-Jun is translocated from the cytoplasm to the nucleus, where it binds to the AP-1 DNA sequence found at the promoter region of its target genes. To investigate the role played by c-Jun during VACV replication cycle, we generated cell lines that stably express a c-Jun-dominant negative (DNc-Jun) mutation. Our data revealed that c-Jun is required during early infection to assist with viral DNA replication, as demonstrated by the decreased amount of viral DNA found in the DNc-Jun cells. We also demonstrated that c-Jun regulates the expression of the early growth response gene (egr-1), a gene previously shown to affect VACV replication mediated by MEK/ERK signalling. VACV-induced stimulation of the MKK/JNK/JUN pathway impacts viral dissemination, as we observed a significant reduction in both viral yield, during late stages of infection, and virus plaque size. Collectively, our data suggest that, by modulating the host's signalling pathways through a common target such as c-Jun, VACV temporally regulates its infective cycle in order to successfully replicate and subsequently spread.

Programmed cell senescence: role of IL-6 in the pituitary.

IL-6 is a pleiotropic cytokine with multiple pathophysiological functions. As a key factor of the senescence secretome, it can not only promote tumorigenesis and cell proliferation but also exert tumor suppressive functions, depending on the cellular context. IL-6, as do other cytokines, plays important roles in the function, growth and neuroendocrine responses of the anterior pituitary gland. The multiple actions of IL-6 on normal and adenomatous pituitary function, cell proliferation, angiogenesis and extracellular matrix remodeling indicate its importance in the regulation of the anterior pituitary. Pituitary tumors are mostly benign adenomas with low mitotic index and rarely became malignant. Premature senescence occurs in slow-growing benign tumors, like pituitary adenomas. The dual role of IL-6 in senescence and tumorigenesis is well represented in pituitary tumor development, as it has been demonstrated that effects of paracrine IL-6 may allow initial pituitary cell growth, whereas autocrine IL-6 in the same tumor triggers senescence and restrains aggressive growth and malignant transformation. IL-6 is instrumental in promotion and maintenance of the senescence program in pituitary adenomas.

MAPKs' status at early stages of renal carcinogenesis and tumors induced by ferric nitrilotriacetate.

Renal cell carcinoma (RCC) is asymptomatic at early stages, and thus, initial diagnosis frequently occurs at advanced or even metastatic stages, leading to a high rate of mortality. Ferric nitrilotriacetate (FeNTA)-induced RCC model is a useful tool to analyze molecular events at different stages of the carcinogenesis process in vivo. MAPKs' alterations seem to play an important role in the development and maintenance of human RCC tumors. Based on the above, p38α/ß/γ, JNK1/2, and ERK1/2 statuses were studied at early stages of FeNTA-induced renal carcinogenesis (1 and 2 months of carcinogen treatment) as well as in tumor tissue. MAPKs showed distinct response along carcinogenesis process, either as total proteins and/or as their phosphorylated forms. While the increase in total and phospho-p38α/ß levels became lower as carcinogenesis progressed, p38γ overexpression grew. Instead, total JNK2 diminished, but JNK1 was elevated at all studied times, and p-JNK1 levels increased at early stages, but not in tumors. In contrast, p-JNK2 rose at 2 months of treatment and in tumor tissue. Increased levels of p-ERK1/2 were observed at all stages analyzed. Very interestingly, at 1 and 2 months of FeNTA treatment, no alterations in MAPKs were found in liver or lung, where no primary tumors are induced with the scheme of FeNTA administration followed here. In conclusion, MAPKs' behavior evolved differentially as renal carcinogenesis advanced, even among isoforms of the same family, but it did not change in other tissues. All this strongly suggests a role of these kinases in FeNTA-induced RCC tumor development and maintenance.

Understanding intellectual disability through RASopathies.

Intellectual disability, commonly known as mental retardation in the International Classification of Disease from World Health Organization, is the term that describes an intellectual and adaptive cognitive disability that begins in early life during the developmental period. Currently the term intellectual disability is the preferred one. Although our understanding of the physiological basis of learning and learning disability is poor, a general idea is that such condition is quite permanent. However, investigations in animal models suggest that learning disability can be functional in nature and as such reversible through pharmacology or appropriate learning paradigms. A fraction of the cases of intellectual disability is caused by point mutations or deletions in genes that encode for proteins of the RAS/MAP kinase signaling pathway known as RASopathies. Here we examined the current understanding of the molecular mechanisms involved in this group of genetic disorders focusing in studies which provide evidence that intellectual disability is potentially treatable and curable. The evidence presented supports the idea that with the appropriate understanding of the molecular mechanisms involved, intellectual disability could be treated pharmacologically and perhaps through specific mechanistic-based teaching strategies.

Analysis of the BRAF V600E mutation in primary cutaneous melanoma.

BRAF V600E is the most common mutation in cutaneous melanomas, and has been described in 30-72% of such cases. This mutation results in the substitution of valine for glutamic acid at position 600 of the BRAF protein, which consequently becomes constitutively activated. The present study investigated the BRAF V600E mutation frequency and its clinical implications in a group of 77 primary cutaneous melanoma patients treated in a cancer reference center in Brazil. Mutation analysis was accomplished by polymerase chain reaction, restriction fragment length polymorphism, and automated DNA sequencing. The chi-squared and Fischer exact tests were used for comparative analyses. The BRAF V600E mutation was detected in 54/77 (70.1%) melanoma subjects. However, no statistically significant association was found between the presence of the mutation and clinical or prognostic parameters. Our results demonstrated that the BRAF V600E mutation is a common event in melanomas, representing an important molecular target for novel therapeutic approaches in such tumors.

In vitro manganese exposure disrupts MAPK signaling pathways in striatal and hippocampal slices from immature rats.

The molecular mechanisms mediating manganese (Mn)-induced neurotoxicity, particularly in the immature central nervous system, have yet to be completely understood. In this study, we investigated whether mitogen-activated protein kinases (MAPKs) and tyrosine hydroxylase (TH) could represent potential targets of Mn in striatal and hippocampal slices obtained from immature rats (14 days old). The aim of this study was to evaluate if the MAPK pathways are modulated after subtoxic Mn exposure, which do not significantly affect cell viability. The concentrations of manganese chloride (MnCl2; 10-1,000 µM) caused no change in cell viability in slices exposed for 3 or 6 hours. However, Mn exposure significantly increased extracellular signal-regulated kinase (ERK) 1/2, as well as c-Jun N-terminal kinase (JNK) 1/2/3 phosphorylation at both 3 and 6 hours incubations, in both brain structures. Furthermore, Mn exposure did not change the total content or phosphorylation of TH at the serine 40 site in striatal slices. Thus, Mn at concentrations that do not disrupt cell viability causes activation of MAPKs (ERK1/2 and JNK1/2/3) in immature hippocampal and striatal slices. These findings suggest that altered intracellular MAPKs signaling pathways may represent an early event concerning the effects of Mn in the immature brain.

Ubiquitin proteasome system and the atypical kinase PfPK7 are involved in melatonin signaling in Plasmodium falciparum.

We previously reported that melatonin modulates the Plasmodium falciparum erythrocytic cycle by increasing schizont stage population as well as diminishing ring stage population. In addition, the importance of calcium and cAMP in melatonin signaling pathway in P. falciparum was also demonstrated. Nevertheless, the molecular effectors of the indoleamine signaling pathway remain elusive. We now demonstrate by real-time PCR that melatonin treatment up-regulates genes related to ubiquitin/proteasome system (UPS) components and that luzindole, a melatonin receptor antagonist, inhibits UPS transcription modulation. We also show that protein kinase PfPK7, a P. falciparum orphan kinase, plays a crucial role in the melatonin transduction pathway, since following melatonin treatment of P. falciparum parasites where pfpk7 gene is disrupted (pfpk7(-) parasites) (i) the ratio of asexual stages remain unchanged, (ii) the increase in cytoplasmatic calcium in response to melatonin was strongly diminished and (iii) up-regulation of UPS genes did not occur. The wild-type melatonin-induced alterations in cell cycle features, calcium rise and UPS gene transcription were restored by re-introduction of a functional copy of the pfpk7 gene in the pfpk7(-) parasites.

Periodic mechanical stress activates MEK1/2-ERK1/2 mitogenic signals in rat chondrocytes through Src and PLCγ1

The mitogenic effects of periodic mechanical stress on chondrocytes have been studied extensively but the mechanisms whereby chondrocytes sense and respond to periodic mechanical stress remain a matter of debate. We explored the signal transduction pathways of chondrocyte proliferation and matrix synthesis under periodic mechanical stress. In particular, we sought to identify the role of the MEK1/2-ERK1/2 signaling pathway in chondrocyte proliferation and matrix synthesis following cyclic physiologic mechanical compression. Under periodic mechanical stress, both rat chondrocyte proliferation and matrix synthesis were significantly increased (P < 0.05) and were associated with increases in the phosphorylation of Src, PLCγ1, MEK1/2, and ERK1/2 (P < 0.05). Pretreatment with the MEK1/2-ERK1/2 selective inhibitor, PD98059, and shRNA targeted to ERK1/2 reduced periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis (P < 0.05), while the phosphorylation levels of Src-Tyr418 and PLCγ1-Tyr783 were not inhibited. Proliferation, matrix synthesis and phosphorylation of MEK1/2-Ser217/221 and ERK1/2-Thr202/Tyr204 were inhibited after pretreatment with the PLCγ1 inhibitor U73122 in chondrocytes in response to periodic mechanical stress (P < 0.05), while the phosphorylation site of Src-Tyr418 was not affected. Inhibition of Src activity with PP2 and shRNA targeted to Src abrogated chondrocyte proliferation and matrix synthesis (P < 0.05) and attenuated PLCγ1, MEK1/2 and ERK1/2 activation in chondrocytes subjected to periodic mechanical stress (P < 0.05). These findings suggest that periodic mechanical stress promotes chondrocyte proliferation and matrix synthesis in part through the Src-PLCγ1-MEK1/2-ERK1/2 signaling pathway, which links these three important signaling molecules into a mitogenic cascade.

Periodic mechanical stress activates MEK1/2-ERK1/2 mitogenic signals in rat chondrocytes through Src and PLCγ1.

The mitogenic effects of periodic mechanical stress on chondrocytes have been studied extensively but the mechanisms whereby chondrocytes sense and respond to periodic mechanical stress remain a matter of debate. We explored the signal transduction pathways of chondrocyte proliferation and matrix synthesis under periodic mechanical stress. In particular, we sought to identify the role of the MEK1/2-ERK1/2 signaling pathway in chondrocyte proliferation and matrix synthesis following cyclic physiologic mechanical compression. Under periodic mechanical stress, both rat chondrocyte proliferation and matrix synthesis were significantly increased (P < 0.05) and were associated with increases in the phosphorylation of Src, PLCγ1, MEK1/2, and ERK1/2 (P < 0.05). Pretreatment with the MEK1/2-ERK1/2 selective inhibitor, PD98059, and shRNA targeted to ERK1/2 reduced periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis (P < 0.05), while the phosphorylation levels of Src-Tyr418 and PLCγ1-Tyr783 were not inhibited. Proliferation, matrix synthesis and phosphorylation of MEK1/2-Ser217/221 and ERK1/2-Thr202/Tyr204 were inhibited after pretreatment with the PLCγ1 inhibitor U73122 in chondrocytes in response to periodic mechanical stress (P < 0.05), while the phosphorylation site of Src-Tyr418 was not affected. Inhibition of Src activity with PP2 and shRNA targeted to Src abrogated chondrocyte proliferation and matrix synthesis (P < 0.05) and attenuated PLCγ1, MEK1/2 and ERK1/2 activation in chondrocytes subjected to periodic mechanical stress (P < 0.05). These findings suggest that periodic mechanical stress promotes chondrocyte proliferation and matrix synthesis in part through the Src-PLCγ1-MEK1/2-ERK1/2 signaling pathway, which links these three important signaling molecules into a mitogenic cascade.

A role for the MAPK/ERK pathway in oligodendroglial differentiation in vitro: stage specific effects on cell branching.

The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway is important for both long-term survival and timing of the progression of oligodendrocyte differentiation. Oligodendroglial cells treated with MEK inhibitor were distinguished by using stage specific markers: NG2 proteoglycan, A2B5, 2'3'nucleotide-cyclic 3'phosphodiesterase (CNPase) and myelin basic protein (MBP), and classified according to their morphology into different developmental stages. Treatment significantly increased the number of cells with more immature morphologies and decreased the number of mature cells. Furthermore, it increased the number of rounded cells that could not be classified into any of the oligodendroglial developmental stages. The strongest effects were usually observed shortly after treatment. Rounded cells were CNPase/MBP positive and they were not stained by anti-NG2 or A2B5, indicating that they were mature cells unable either to extend and/or to maintain their processes. These data showed an effect of the MAPK/ERK pathway on oligodendroglial branching, with possible consequences for the formation of the myelin sheath.

Detection of a mitochondrial kinase complex that mediates PKA-MEK-ERK-dependent phosphorylation of mitochondrial proteins involved in the regulation of steroid biosynthesis.

In order to achieve the goal of this article, as an example we will describe the strategies followed to analyze the presence of the multi-kinase complex at the mitochondria and the posttranslational modification of two key mitochondrial proteins, which participate in the regulation of cholesterol transport across the mitochondrial membranes and in the regulation of steroid biosynthesis. Hormones, ions or growth factors modulate steroid biosynthesis by the posttranslational phosphorylation of proteins. The question still remains on how phosphorylation events transmit a specific signal to its mitochondrial site of action. Cholesterol transport requires specific interactions in mitochondria between several proteins including a multi-kinase complex. The presence of this multi-kinase complex at the mitochondria reveals the importance of the posttranslational modification of mitochondrial proteins for its activity and functions. The activation of PKA triggers the posttranslational modification of the mitochondrial acyl-CoA thioesterase (Acot2), which releases arachidonic acid (AA) in the mitochondria, and the activation of a kinase cascade that leads to the phoshorylation of the steroidogenic acute regulatory (StAR) protein. The function of StAR is to facilitate the access of cholesterol to the first enzyme of the biosynthesis process and its induction is dependent on Acot2 and intramitochondrial AA release. Truncation of the StAR protein is associated with the steroid deficiency disease, congenital lipoid adrenal hyperplasia.

Xanthomonas campestris overcomes Arabidopsis stomatal innate immunity through a DSF cell-to-cell signal-regulated virulence factor.

Pathogen-induced stomatal closure is part of the plant innate immune response. Phytopathogens using stomata as a way of entry into the leaf must avoid the stomatal response of the host. In this article, we describe a factor secreted by the bacterial phytopathogen Xanthomonas campestris pv campestris (Xcc) capable of interfering with stomatal closure induced by bacteria or abscisic acid (ABA). We found that living Xcc, as well as ethyl acetate extracts from Xcc culture supernatants, are capable of reverting stomatal closure induced by bacteria, lipopolysaccharide, or ABA. Xcc ethyl acetate extracts also complemented the infectivity of Pseudomonas syringae pv tomato (Pst) mutants deficient in the production of the coronatine toxin, which is required to overcome stomatal defense. By contrast, the rpfF and rpfC mutant strains of Xcc, which are unable to respectively synthesize or perceive a diffusible molecule involved in bacterial cell-to-cell signaling, were incapable of reverting stomatal closure, indicating that suppression of stomatal response by Xcc requires an intact rpf/diffusible signal factor system. In addition, we found that guard cell-specific Arabidopsis (Arabidopsis thaliana) Mitogen-Activated Protein Kinase3 (MPK3) antisense mutants were unresponsive to bacteria or lipopolysaccharide in promotion of stomatal closure, and also more sensitive to Pst coronatine-deficient mutants, showing that MPK3 is required for stomatal immune response. Additionally, we found that, unlike in wild-type Arabidopsis, ABA-induced stomatal closure in MPK3 antisense mutants is not affected by Xcc or by extracts from Xcc culture supernatants, suggesting that the Xcc factor might target some signaling component in the same pathway as MPK3.

LmxPK4, a mitogen-activated protein kinase kinase homologue of Leishmania mexicana with a potential role in parasite differentiation.

Members of the mitogen-activated protein (MAP) kinase cascade are important for the establishment of a Leishmania mexicana infection and are involved in flagellar length control, although the underlying molecular mechanisms remain to be elucidated. This study reports the cloning and characterization of LmxPK4, a MAP kinase kinase homologue of L. mexicana displaying putative plant-like regulatory phosphorylation sites. The recombinant protein has autophosphorylating activity and phosphorylates myelin basic protein. An LmxPK4 gene deletion mutant showed a proliferation defect after infection of macrophages and no or delayed lesion development in mice. Irrespective of the onset of lesion development parasites showed an early and homogeneous lesion development in re-infection experiments. This is indicative for a compensation of the null mutant phenotype. Additionally, this phenotype could be reverted by reintroduction of the wild-type gene into the deletion background. Mutants expressing loss-of-function or N-terminally truncated versions of LmxPK4 retained the null mutant phenotype. LmxPK4 is stage-specifically expressed in promastigotes and during differentiation to amastigotes, but is not detectable in amastigotes isolated from the mammalian host. Moreover, its in vitro kinase activity increases with temperature rise up to 40 degrees C. Our results suggest that LmxPK4 is involved in the differentiation process and affects virulence of Leishmania mexicana.