Notch and NOXA-related pathways in melanoma cells

Notch receptor-mediated intracellular events represent an ancient cell signaling system, and alterations in Notch expression are associated with various malignancies in which Notch may function as an oncogene or less commonly as a tumor suppressor. Notch signaling regulates cell fate decisions in the epidermis, including influencing stem cell dynamics and growth/differentiation control of cells in skin. Because of increasing evidence that the Notch signaling network is deregulated in human malignancies, Notch receptors have become attractive targets for selective killing of malignant cells. Compared with proliferating normal human melanocytes, melanoma cell lines are characterized by markedly enhanced levels of activated Notch-1 receptor. By using a small molecule gamma-secretase inhibitor (GSI) consisting of a tripeptide aldehyde, N-benzyloxycarbonyl-Leu-Leu-Nle-CHO, which can block processing and activation of all four different Notch receptors, we identified a specific apoptotic vulnerability in melanoma cells. GSI triggers apoptosis in melanoma cells, but only G2/M growth arrest in melanocytes without subsequent cell death. Moreover, GSI treatment induced a pro-apoptotic BH3-only protein, NOXA, in melanoma cells but not in normal melanocytes. The use of GSI to induce NOXA induction overcomes the apoptotic resistance of melanoma cells, which commonly express numerous cell survival proteins such as Mcl-1, Bcl-2, and survivin. Taken together, these results highlight the concept of synthetic lethality in which exposure to GSI, in combination with melanoma cells overexpressing activated Notch receptors, has lethal consequences, producing selective killing of melanoma cells, while sparing normal melanocytes. By identifying signaling pathways that contribute to the transformation of melanoma cells (e.g. Notch signaling), and anti-cancer agents that achieve tumor selectivity (e.g., GSI-induced NOXA), this experimental approach provides a useful framework for future therapeutic strategies in cutaneous oncology.

p53 prevents progression of nevi to melanoma predominantly through cell cycle regulation

p53 is the central member of a critical tumor suppressor pathway in virtually all tumor types, where it is silenced mainly by missense mutations. In melanoma, p53 predominantly remains wild type, thus its role has been neglected. To study the effect of p53 on melanocyte function and melanomagenesis, we crossed the 'high-p53'Mdm4+/- mouse to the well-established TP-ras0/+ murine melanoma progression model. After treatment with the carcinogen dimethylbenzanthracene (DMBA), TP-ras0/+ mice on the Mdm4+/- background developed fewer tumors with a delay in the age of onset of melanomas compared to TP-ras0/+ mice. Furthermore, we observed a dramatic decrease in tumor growth, lack of metastasis with increased survival of TP-ras0/+: Mdm4+/- mice. Thus, p53 effectively prevented the conversion of small benign tumors to malignant and metastatic melanoma. p53 activation in cultured primary melanocyte and melanoma cell lines using Nutlin-3, a specific Mdm2 antagonist, supported these findings. Moreover, global gene expression and network analysis of Nutlin-3-treated primary human melanocytes indicated that cell cycle regulation through the p21WAF1/CIP1 signaling network may be the key anti-melanomagenic activity of p53.

Synergism between Wnt3a and heparin enhances osteogenesis via a phosphoinositide 3-kinase/Akt/RUNX2 pathway

A new strategy has emerged to improve healing of bone defects using exogenous glycosaminoglycans by increasing the effectiveness of bone-anabolic growth factors. Wnt ligands play an important role in bone formation. However, their functional interactions with heparan sulfate/heparin have only been investigated in non-osseous tissues. Our study now shows that the osteogenic activity of Wnt3a is cooperatively stimulated through physical interactions with exogenous heparin. N-Sulfation and to a lesser extent O-sulfation of heparin contribute to the physical binding and optimal co-stimulation of Wnt3a. Wnt3a-heparin signaling synergistically increases osteoblast differentiation with minimal effects on cell proliferation. Thus, heparin selectively reduces the effective dose of Wnt3a needed to elicit osteogenic, but not mitogenic responses. Mechanistically, Wnt3a-heparin signaling strongly activates the phosphoinositide 3-kinase/Akt pathway and requires the bone-related transcription factor RUNX2 to stimulate alkaline phosphatase activity, which parallels canonical beta-catenin signaling. Collectively, our findings establish the osteo-inductive potential of a heparin-mediated Wnt3a-phosphoinositide 3-kinase/Akt-RUNX2 signaling network and suggest that heparan sulfate supplementation may selectively reduce the therapeutic doses of peptide factors required to promote bone formation.

Mathematical modeling of the cancer cell’s control circuitry : paving the way to individualized therapeutic strategies

Cancer is a disease of signal transduction in which the dysregulation of the network of intracellular and extracellular signaling cascades is sufficient to thwart the cells finely-tuned biochemical control mechanisms. A keen interest in the mathematical modeling of cell signaling networks and the regulation of signal transduction has emerged in recent years, and has produced a glimmer of insight into the sophisticated feedback control and network regulation operating within cells. In this review, we present an overview of published theoretical studies on the control aspects of signal transduction, emphasizing the role and importance of mechanisms such as ‘ultrasensitivity’ and feedback loops. We emphasize that these exquisite and often subtle control strategies represent the key to orchestrating ‘simple’ signaling behaviors within the complex intracellular network, while regulating the trade-off between sensitivity and robustness to internal and external perturbations. Through a consideration of these apparent paradoxes, we explore how the basic homeostasis of the intracellular signaling network, in the face of carcinogenesis, can lead to neoplastic progression rather than cell death. A simple mathematical model is presented, furnishing a vivid illustration of how ‘control-oriented’ models of the deranged signaling networks in cancer cells may enucleate improved treatment strategies, including patient-tailored combination therapies, with the potential for reduced toxicity and more robust and potent antitumor activity.

ATM, RAD50 and p53 in breast cancer

Breast cancer is the most commonly occurring cancer among women, and its incidence is increasing worldwide. Positive family history is a well established risk factor for breast cancer, and it is suggested that the proportion of breast cancer that can be attributed to genetic factors may be as high as 30%. However, all the currently known breast cancer susceptibility genes are estimated to account for 20-30% of familial breast cancer, and only 5% of the total breast cancer incidence. It is thus likely that there are still other breast cancer susceptibility genes to be found. Cellular responses to DNA damage are crucial for maintaining genomic integrity and preventing the development of cancer. The genes operating in DNA damage response signaling network are thus good candidates for breast cancer susceptibility genes. The aim of this study was to evaluate the role of three DNA damage response associated genes, ATM, RAD50, and p53, in breast cancer. ATM, a gene causative for ataxia telangiectasia (A-T), has long been a strong candidate for a breast cancer susceptibility gene because of its function as a key DNA damage signal transducer. We analyzed the prevalence of known Finnish A-T related ATM mutations in large series of familial and unselected breast cancer cases from different geographical regions in Finland. Of the seven A-T related mutations, two were observed in the studied familial breast cancer patients. Additionally, a third mutation previously associated with breast cancer susceptibility was also detected. These founder mutations may be responsible for excess familial breast cancer regionally in Northern and Central Finland, but in Southern Finland our results suggest only a minor effect, if any, of any ATM genetic variants on familial breast cancer. We also screened the entire coding region of the ATM gene in 47 familial breast cancer patients from Southern Finland, and evaluated the identified variants in additional cases and controls. All the identified variants were too rare to significantly contribute to breast cancer susceptibility. However, the role of ATM in cancer development and progression was supported by the results of the immunohistochemical studies of ATM expression, as reduced ATM expression in breast carcinomas was found to correlate with tumor differentiation and hormone receptor status. Aberrant ATM expression was also a feature shared by the BRCA1/2 and the difficult-to-treat ER/PR/ERBB2-triple-negative breast carcinomas. From the clinical point of view, identification of phenotypic and genetic similarities between the BRCA1/2 and the triple-negative breast tumors could have an implication in designing novel targeted therapies to which both of these classes of breast cancer might be exceptionally sensitive. Mutations of another plausible breast cancer susceptibility gene, RAD50, were found to be very rare, and RAD50 can only be making a minor contribution to familial breast cancer predisposition in UK and Southern Finland. The Finnish founder mutation RAD50 687delT seems to be a null allele and may carry a small increased risk of breast cancer. RAD50 is not acting as a classical tumor suppressor gene, but it is possible that RAD50 haploinsufficiency is contributing to cancer. In addition to relatively rare breast cancer susceptibility alleles, common polymorphisms may also be associated with increased breast cancer risk. Furthermore, these polymorphisms may have an impact on the progression and outcome of the disease. Our results suggest no effect of the common p53 R72P polymorphism on familial breast cancer risk or breast cancer risk in the population, but R72P seems to be associated with histopathologic features of the tumors and survival of the patients; 72P homozygous genotype was an independent prognostic factor among the unselected breast cancer patients, with a two-fold increased risk of death. These results present important novel findings also with clinical significance, as codon 72 genotype could be a useful additional prognostic marker in breast cancer, especially among the subgroup of patients with wild-type p53 in their tumors.

The circadian oscillator gene GIGANTEA mediates a long-term response of the Arabidopsis thaliana circadian clock to sucrose.

Circadian clocks are 24-h timing devices that phase cellular responses; coordinate growth, physiology, and metabolism; and anticipate the day-night cycle. Here we report sensitivity of the Arabidopsis thaliana circadian oscillator to sucrose, providing evidence that plant metabolism can regulate circadian function. We found that the Arabidopsis circadian system is particularly sensitive to sucrose in the dark. These data suggest that there is a feedback between the molecular components that comprise the circadian oscillator and plant metabolism, with the circadian clock both regulating and being regulated by metabolism. We used also simulations within a three-loop mathematical model of the Arabidopsis circadian oscillator to identify components of the circadian clock sensitive to sucrose. The mathematical studies identified GIGANTEA (GI) as being associated with sucrose sensing. Experimental validation of this prediction demonstrated that GI is required for the full response of the circadian clock to sucrose. We demonstrate that GI acts as part of the sucrose-signaling network and propose this role permits metabolic input into circadian timing in Arabidopsis.

Predicting Cell Cycle Regulated Genes by Causal Interactions

The fundamental difference between classic and modern biology is that technological innovations allow to generate high-throughput data to get insights into molecular interactions on a genomic scale. These high-throughput data can be used to infer gene networks, e. g., the transcriptional regulatory or signaling network, representing a blue print of the current dynamical state of the cellular system. However, gene networks do not provide direct answers to biological questions, instead, they need to be analyzed to reveal functional information of molecular working mechanisms. In this paper we propose a new approach to analyze the transcriptional regulatory network of yeast to predict cell cycle regulated genes. The novelty of our approach is that, in contrast to all other approaches aiming to predict cell cycle regulated genes, we do not use time series data but base our analysis on the prior information of causal interactions among genes. The major purpose of the present paper is to predict cell cycle regulated genes in S. cerevisiae. Our analysis is based on the transcriptional regulatory network, representing causal interactions between genes, and a list of known periodic genes. No further data are used. Our approach utilizes the causal membership of genes and the hierarchical organization of the transcriptional regulatory network leading to two groups of periodic genes with a well defined direction of information flow. We predict genes as periodic if they appear on unique shortest paths connecting two periodic genes from different hierarchy levels. Our results demonstrate that a classical problem as the prediction of cell cycle regulated genes can be seen in a new light if the concept of a causal membership of a gene is applied consequently. This also shows that there is a wealth of information buried in the transcriptional regulatory network whose unraveling may require more elaborate concepts than it might seem at first.

Connective tissue growth factor/CCN2 stimulates actin disassembly through Akt/protein kinase B-mediated phosphorylation and cytoplasmic translocation of p27(Kip-1)

Connective tissue growth factor (CTGF/CCN2) is a 38-kDa secreted protein, a prototypic member of the CCN family, which is up-regulated in many diseases, including atherosclerosis, pulmonary fibrosis, and diabetic nephropathy. We previously showed that CTGF can cause actin disassembly with concurrent down-regulation of the small GTPase Rho A and proposed an integrated signaling network connecting focal adhesion dissolution and actin disassembly with cell polarization and migration. Here, we further delineate the role of CTGF in cell migration and actin disassembly in human mesangial cells, a primary target in the development of renal glomerulosclerosis. The functional response of mesangial cells to treatment with CTGF was associated with the phosphorylation of Akt/protein kinase B (PKB) and resultant phosphorylation of a number of Akt/PKB substrates. Two of these substrates were identified as FKHR and p27(Kip-1). CTGF stimulated the phosphorylation and cytoplasmic translocation of p27(Kip-1) on serine 10. Addition of the PI-3 kinase inhibitor LY294002 abrogated this response; moreover, addition of the Akt/PKB inhibitor interleukin (IL)-6-hydroxymethyl-chiro-inositol-2(R)-2-methyl-3-O-octadecylcarbonate prevented p27(Kip-1) phosphorylation in response to CTGF. Immunocytochemistry revealed that serine 10 phosphorylated p27(Kip-1) colocalized with the ends of actin filaments in cells treated with CTGF. Further investigation of other Akt/PKB sites on p27(Kip-1), revealed that phosphorylation on threonine 157 was necessary for CTGF mediated p27(Kip-1) cytoplasmic localization; mutation of the threonine 157 site prevented cytoplasmic localization, protected against actin disassembly and inhibited cell migration. CTGF also stimulated an increased association between Rho A and p27(Kip-1). Interestingly, this resulted in an increase in phosphorylation of LIM kinase and subsequent phosphorylation of cofilin, suggesting that CTGF mediated p27(Kip-1) activation results in uncoupling of the Rho A/LIM kinase/cofilin pathway. Confirming the central role of Akt/PKB, CTGF-stimulated actin depolymerization only in wild-type mouse embryonic fibroblasts (MEFs) compared to Akt-1/3 (PKB alpha/gamma) knockout MEFs. These data reveal important mechanistic insights into how CTGF may contribute to mesangial cell dysfunction in the diabetic milieu and sheds new light on the proposed role of p27(Kip-1) as a mediator of actin rearrangement.

Molecular mechanisms for a switch-like mating decision in Saccharomyces cerevisiae

Les changements évolutifs nous instruisent sur les nombreuses innovations permettant à chaque organisme de maximiser ses aptitudes en choisissant le partenaire approprié, telles que les caractéristiques sexuelles secondaires, les patrons comportementaux, les attractifs chimiques et les mécanismes sensoriels y répondant. L'haploïde de la levure Saccharomyces cerevisiae distingue son partenaire en interprétant le gradient de la concentration d'une phéromone sécrétée par les partenaires potentiels grâce à un réseau de protéines signalétiques de type kinase activées par la mitose (MAPK). La décision de la liaison sexuelle chez la levure est un événement en "tout–ourien", à la manière d'un interrupteur. Les cellules haploïdes choisissent leur partenaire sexuel en fonction de la concentration de phéromones qu’il produit. Seul le partenaire à proximité sécrétant des concentrations de phéromones égales ou supérieures à une concentration critique est retenu. Les faibles signaux de phéromones sont attribués à des partenaires pouvant mener à des accouplements infructueux. Notre compréhension du mécanisme moléculaire contrôlant cet interrupteur de la décision d'accouplement reste encore mince. Dans le cadre de la présente thèse, je démontre que le mécanisme de décision de la liaison sexuelle provient de la compétition pour le contrôle de l'état de phosphorylation de quatre sites sur la protéine d'échafaudage Ste5, entre la MAPK, Fus3, et la phosphatase,Ptc1. Cette compétition résulte en la dissociation de type « intérupteur » entre Fus3 et Ste5, nécessaire à la prise de décision d'accouplement en "tout-ou-rien". Ainsi, la décision de la liaison sexuelle s'effectue à une étape précoce de la voie de réponse aux phéromones et se produit rapidement, peut-être dans le but de prévenir la perte d’un partenaire potentiel. Nous argumentons que l'architecture du circuit Fus3-Ste5-Ptc1 génère un mécanisme inédit d'ultrasensibilité, ressemblant à "l'ultrasensibilité d'ordre zéro", qui résiste aux variations de concentration de ces protéines. Cette robustesse assure que l'accouplement puisse se produire en dépit de la stochasticité cellulaire ou de variations génétiques entre individus.Je démontre, par la suite, qu'un évènement précoce en réponse aux signaux extracellulaires recrutant Ste5 à la membrane plasmique est également ultrasensible à l'augmentation de la concentration de phéromones et que cette ultrasensibilité est engendrée par la déphosphorylation de huit phosphosites en N-terminal sur Ste5 par la phosphatase Ptc1 lorsqu'elle est associée à Ste5 via la protéine polarisante, Bem1. L'interférence dans ce mécanisme provoque une perte de l'ultrasensibilité et réduit, du même coup, l'amplitude et la fidélité de la voie de réponse aux phéromones à la stimulation. Ces changements se reflètent en une réduction de la fidélité et de la précision de la morphologie attribuable à la réponse d'accouplement. La polarisation dans l'assemblage du complexe protéique à la surface de la membrane plasmique est un thème général persistant dans tous les organismes, de la bactérie à l'humain. Un tel complexe est en mesure d'accroître l'efficacité, la fidélité et la spécificité de la transmission du signal. L'ensemble de nos découvertes démontre que l'ultrasensibilité, la précision et la robustesse de la réponse aux phéromones découlent de la régulation de la phosphorylation stoichiométrique de deux groupes de phosphosites sur Ste5, par la phosphatase Ptc1, un groupe effectuant le recrutement ultrasensible de Ste5 à la membrane et un autre incitant la dissociation et l'activation ultrasensible de la MAPK terminal Fus3. Le rôle modulateur de Ste5 dans la décision de la destinée cellulaire étend le répertoire fonctionnel des protéines d'échafaudage bien au-delà de l'accessoire dans la spécificité et l'efficacité des traitements de l'information. La régulation de la dynamique des caractères signal-réponse à travers une telle régulation modulaire des groupes de phosphosites sur des protéines d'échafaudage combinées à l'assemblage à la membrane peut être un moyen général par lequel la polarisation du destin cellulaire est obtenue. Des mécanismes similaires peuvent contrôler les décisions cellulaires dans les organismes complexes et peuvent être compromis dans des dérèglements cellulaires, tel que le cancer. Finalement, sur un thème relié, je présente la découverte d'un nouveau mécanisme où le seuil de la concentration de phéromones est contrôlé par une voie sensorielle de nutriments, ajustant, de cette manière, le point prédéterminé dans lequel la quantité et la qualité des nutriments accessibles dans l'environnement déterminent le seuil à partir duquel la levure s'accouple. La sous-unité régulatrice de la kinase à protéine A (PKA),Bcy1, une composante clé du réseau signalétique du senseur aux nutriments, interagit directement avec la sous-unité α des petites protéines G, Gpa1, le premier effecteur dans le réseau de réponse aux phéromones. L'interaction Bcy1-Gpa1 est accrue lorsque la cellule croit en présence d'un sucre idéal, le glucose, diminuant la concentration seuil auquel la décision d'accouplement est activée. Compromettre l'interaction Bcy1-Gpa1 ou inactiver Bcy1 accroît la concentration seuil nécessaire à une réponse aux phéromones. Nous argumentons qu'en ajustant leur sensibilité, les levures peuvent intégrer le stimulus provenant des phéromones au niveau du glucose extracellulaire, priorisant la décision de survie dans un milieu pauvre ou continuer leur cycle sexuel en choisissant un accouplement.

Maintenance and differentiation of neural stem cells

The adult mammalian brain contains self-renewable, multipotent neural stem cells (NSCs) that are responsible for neurogenesis and plasticity in specific regions of the adult brain. Extracellular matrix, vasculature, glial cells, and other neurons are components of the niche where NSCs are located. This surrounding environment is the source of extrinsic signals that instruct NSCs to either self-renew or differentiate. Additionally, factors such as the intracellular epigenetics state and retrotransposition events can influence the decision of NSC`s fate into neurons or glia. Extrinsic and intrinsic factors form an intricate signaling network, which is not completely understood. These factors altogether reflect a few of the key players characterized so far in the new field of NSC research and are covered in this review. (C) 2010 John Wiley & Sons, Inc. WIREs Syst Biol Med 2011 3 107-114 DOI:10.1002/wsbm:100

TGF-beta 1 modulates the homeostasis between MMPs and MMP inhibitors through p38 MAPK and ERK1/2 in highly invasive breast cancer cells

Background: Metastasis is the main factor responsible for death in breast cancer patients. Matrix metalloproteinases (MMPs) and their inhibitors, known as tissue inhibitors of MMPs (TIMPs), and the membrane-associated MMP inhibitor (RECK), are essential for the metastatic process. We have previously shown a positive correlation between MMPs and their inhibitors expression during breast cancer progression; however, the molecular mechanisms underlying this coordinate regulation remain unknown. In this report, we investigated whether TGF-beta 1 could be a common regulator for MMPs, TIMPs and RECK in human breast cancer cell models. Methods: The mRNA expression levels of TGF-beta isoforms and their receptors were analyzed by qRT-PCR in a panel of five human breast cancer cell lines displaying different degrees of invasiveness and metastatic potential. The highly invasive MDA-MB-231 cell line was treated with different concentrations of recombinant TGF-beta 1 and also with pharmacological inhibitors of p38 MAPK and ERK1/2. The migratory and invasive potential of these treated cells were examined in vitro by transwell assays. Results: In general, TGF-beta 2, T beta RI and T beta RII are over-expressed in more aggressive cells, except for T beta RI, which was also highly expressed in ZR-75-1 cells. In addition, TGF-beta 1-treated MDA-MB-231 cells presented significantly increased mRNA expression of MMP-2, MMP-9, MMP-14, TIMP-2 and RECK. TGF-beta 1 also increased TIMP-2, MMP-2 and MMP-9 protein levels but downregulated RECK expression. Furthermore, we analyzed the involvement of p38 MAPK and ERK1/2, representing two well established Smad-independent pathways, in the proposed mechanism. Inhibition of p38MAPK blocked TGF-beta 1-increased mRNA expression of all MMPs and MMP inhibitors analyzed, and prevented TGF-beta 1 upregulation of TIMP-2 and MMP-2 proteins. Moreover, ERK1/2 inhibition increased RECK and prevented the TGF-beta 1 induction of pro-MMP-9 and TIMP-2 proteins. TGF-beta 1-enhanced migration and invasion capacities were blocked by p38MAPK, ERK1/2 and MMP inhibitors. Conclusion: Altogether, our results support that TGF-beta 1 modulates the mRNA and protein levels of MMPs (MMP-2 and MMP-9) as much as their inhibitors (TIMP-2 and RECK). Therefore, this cytokine plays a crucial role in breast cancer progression by modulating key elements of ECM homeostasis control. Thus, although the complexity of this signaling network, TGF-beta 1 still remains a promising target for breast cancer treatment.

Global transcriptional response of Caulobacter crescentus to iron availability

Abstract Background In the alpha subclass of proteobacteria iron homeostasis is controlled by diverse iron responsive regulators. Caulobacter crescentus, an important freshwater α-proteobacterium, uses the ferric uptake repressor (Fur) for such purpose. However, the impact of the iron availability on the C. crescentus transcriptome and an overall perspective of the regulatory networks involved remain unknown. Results In this work we report the identification of iron-responsive and Fur-regulated genes in C. crescentus using microarray-based global transcriptional analyses. We identified 42 genes that were strongly upregulated both by mutation of fur and by iron limitation condition. Among them, there are genes involved in iron uptake (four TonB-dependent receptor gene clusters, and feoAB), riboflavin biosynthesis and genes encoding hypothetical proteins. Most of these genes are associated with predicted Fur binding sites, implicating them as direct targets of Fur-mediated repression. These data were validated by β-galactosidase and EMSA assays for two operons encoding putative transporters. The role of Fur as a positive regulator is also evident, given that 27 genes were downregulated both by mutation of fur and under low-iron condition. As expected, this group includes many genes involved in energy metabolism, mostly iron-using enzymes. Surprisingly, included in this group are also TonB-dependent receptors genes and the genes fixK, fixT and ftrB encoding an oxygen signaling network required for growth during hypoxia. Bioinformatics analyses suggest that positive regulation by Fur is mainly indirect. In addition to the Fur modulon, iron limitation altered expression of 113 more genes, including induction of genes involved in Fe-S cluster assembly, oxidative stress and heat shock response, as well as repression of genes implicated in amino acid metabolism, chemotaxis and motility. Conclusions Using a global transcriptional approach, we determined the C. crescentus iron stimulon. Many but not all of iron responsive genes were directly or indirectly controlled by Fur. The iron limitation stimulon overlaps with other regulatory systems, such as the RpoH and FixK regulons. Altogether, our results showed that adaptation of C. crescentus to iron limitation not only involves increasing the transcription of iron-acquisition systems and decreasing the production of iron-using proteins, but also includes novel genes and regulatory mechanisms.

TGF-β1 modulates the homeostasis between MMPs and MMP inhibitors through p38 MAPK and ERK1/2 in highly invasive breast cancer cells

Abstract Background Metastasis is the main factor responsible for death in breast cancer patients. Matrix metalloproteinases (MMPs) and their inhibitors, known as tissue inhibitors of MMPs (TIMPs), and the membrane-associated MMP inhibitor (RECK), are essential for the metastatic process. We have previously shown a positive correlation between MMPs and their inhibitors expression during breast cancer progression; however, the molecular mechanisms underlying this coordinate regulation remain unknown. In this report, we investigated whether TGF-β1 could be a common regulator for MMPs, TIMPs and RECK in human breast cancer cell models. Methods The mRNA expression levels of TGF-β isoforms and their receptors were analyzed by qRT-PCR in a panel of five human breast cancer cell lines displaying different degrees of invasiveness and metastatic potential. The highly invasive MDA-MB-231 cell line was treated with different concentrations of recombinant TGF-β1 and also with pharmacological inhibitors of p38 MAPK and ERK1/2. The migratory and invasive potential of these treated cells were examined in vitro by transwell assays. Results In general, TGF-β2, TβRI and TβRII are over-expressed in more aggressive cells, except for TβRI, which was also highly expressed in ZR-75-1 cells. In addition, TGF-β1-treated MDA-MB-231 cells presented significantly increased mRNA expression of MMP-2, MMP-9, MMP-14, TIMP-2 and RECK. TGF-β1 also increased TIMP-2, MMP-2 and MMP-9 protein levels but downregulated RECK expression. Furthermore, we analyzed the involvement of p38 MAPK and ERK1/2, representing two well established Smad-independent pathways, in the proposed mechanism. Inhibition of p38MAPK blocked TGF-β1-increased mRNA expression of all MMPs and MMP inhibitors analyzed, and prevented TGF-β1 upregulation of TIMP-2 and MMP-2 proteins. Moreover, ERK1/2 inhibition increased RECK and prevented the TGF-β1 induction of pro-MMP-9 and TIMP-2 proteins. TGF-β1-enhanced migration and invasion capacities were blocked by p38MAPK, ERK1/2 and MMP inhibitors. Conclusion Altogether, our results support that TGF-β1 modulates the mRNA and protein levels of MMPs (MMP-2 and MMP-9) as much as their inhibitors (TIMP-2 and RECK). Therefore, this cytokine plays a crucial role in breast cancer progression by modulating key elements of ECM homeostasis control. Thus, although the complexity of this signaling network, TGF-β1 still remains a promising target for breast cancer treatment.

MAPKs and signal transduction in the control of gastrointestinal epithelial cell proliferation and differentiation

Mitogen-activated protein kinase (MAPK) pathways are activated by several stimuli and transduce the signal inside cells, generating diverse responses including cell proliferation, differentiation, migration and apoptosis. Each MAPK cascade comprises a series of molecules, and regulation takes place at different levels. They communicate with each other and with additional pathways, creating a signaling network that is important for cell fate determination. In this review, we focus on ERK, JNK, p38 and ERK5, the major MAPKs, and their interactions with PI3K-Akt, TGFβ/Smad and Wnt/β-catenin pathways. More importantly, we describe how MAPKs regulate cell proliferation and differentiation in the rapidly renewing epithelia that lines the gastrointestinal tract and, finally, we highlight the recent findings on nutritional aspects that affect MAPK transduction cascades.

Global transcriptional response of Caulobacter crescentus to iron availability

BACKGROUND: In the alpha subclass of proteobacteria iron homeostasis is controlled by diverse iron responsive regulators. Caulobacter crescentus, an important freshwater α-proteobacterium, uses the ferric uptake repressor (Fur) for such purpose. However, the impact of the iron availability on the C. crescentus transcriptome and an overall perspective of the regulatory networks involved remain unknown. RESULTS: In this work we report the identification of iron-responsive and Fur-regulated genes in C. crescentus using microarray-based global transcriptional analyses. We identified 42 genes that were strongly upregulated both by mutation of fur and by iron limitation condition. Among them, there are genes involved in iron uptake (four TonB-dependent receptor gene clusters, and feoAB), riboflavin biosynthesis and genes encoding hypothetical proteins. Most of these genes are associated with predicted Fur binding sites, implicating them as direct targets of Fur-mediated repression. These data were validated by β-galactosidase and EMSA assays for two operons encoding putative transporters. The role of Fur as a positive regulator is also evident, given that 27 genes were downregulated both by mutation of fur and under low-iron condition. As expected, this group includes many genes involved in energy metabolism, mostly iron-using enzymes. Surprisingly, included in this group are also TonB-dependent receptors genes and the genes fixK, fixT and ftrB encoding an oxygen signaling network required for growth during hypoxia. Bioinformatics analyses suggest that positive regulation by Fur is mainly indirect. In addition to the Fur modulon, iron limitation altered expression of 113 more genes, including induction of genes involved in Fe-S cluster assembly, oxidative stress and heat shock response, as well as repression of genes implicated in amino acid metabolism, chemotaxis and motility. CONCLUSIONS: Using a global transcriptional approach, we determined the C. crescentus iron stimulon. Many but not all of iron responsive genes were directly or indirectly controlled by Fur. The iron limitation stimulon overlaps with other regulatory systems, such as the RpoH and FixK regulons. Altogether, our results showed that adaptation of C. crescentus to iron limitation not only involves increasing the transcription of iron-acquisition systems and decreasing the production of iron-using proteins, but also includes novel genes and regulatory mechanisms