Search

Background & Aims: The function of β-catenin as a transcriptional coactivator of T-cell factor-4 (TCF-4) is crucial for colorectal carcinogenesis. However, β-catenin has no nuclear localization signal, and the mechanisms by which β-catenin is imported into the nucleus and forms a complex with the TCF-4 nuclear protein are poorly understood. Methods: Proteins of 2 colorectal cancer cell lines, HCT-116 and DLD1, were immunoprecipitated with anti-TCF-4 antibody and analyzed directly by nanoflow liquid chromatography and mass spectrometry. The functional significance of nuclear pore complex (NPC) proteins in Wnt signaling was evaluated by in vitro and in vivo sumoylation, luciferase reporter, and colony formation assays. Results: TCF-4 interacted with a large variety of NPC proteins including ras-related nuclear protein (Ran), Ran binding protein-2 (RanBP2), and Ran GTPase-activating protein-1 (RanGAP1). The NPC protein RanBP2 functioned as the small ubiquitin-related modifier (SUMO) E3 ligase of TCF-4, and sumoylation of TCF-4 enhanced the interaction between TCF-4 and β-catenin. The overexpression of NPC proteins increased the nuclear import of the TCF-4 and β-catenin proteins and enhanced the transcriptional activity. NPC proteins increased the growth of colorectal cancer cells, whereas sentrin-specific protease-2 inhibited it. Conclusions: Through a comprehensive proteomics approach, we revealed that NPC functions as a novel regulator of Wnt signaling and is possibly involved in colorectal carcinogenesis. A new drug targeting the interactions of TCF-4 with NPC proteins as well as their sumoylation activity might be effective for suppressing aberrant Wnt signaling and the proliferation of colorectal cancer cells.

Graft-versus-host disease (GVHD) mediated by alloreactive donor T cells is the most dreaded complication after allogeneic bone marrow transplantation (BMT). Conditioning therapy in the context of BMT creates a proinflammatory milieu, which is thought to be central to the development of GVHD. Interfering with the conditioning-induced inflammatory response could be an approach to prevent GVHD without compromising the graft-versus-malignancy reaction. Histone deacetylase (HDAC) inhibitors belong to a new family of anti-cancer drugs with potent anti-inflammatory properties and have recently been shown to reduce the development of GVHD. The aim of this study was understand the mechanisms underlying the downregulation of GVHD after treatment with the HDAC inhibitor suberonylanilide hydroxamic acid (SAHA). Using the fully MHC-mismatched strain combination B6 to BALB/c, treatment with SAHA resulted in a significantly reduced GVHD mortality. Thus, at days +10 or +37 post-BMT survival for vehicle-treated or SAHA-treated mice was 33 % versus 86 % and 8 % versus 57 % respectively (Chi2 test, p = 0,027 and p = 0,02, respectively). This was associated with a significant reduction in IFN-g and IL-5 serum levels of SAHA-treated animals. As we could not detect any effect of SAHA treatment on T cell activation or T cell expansion in vitro and in vivo, we hypothesized that the inhibitory effect of SAHA treatment on the development of GVHD might be primarily due to an interference in the early events of the inflammatory cascade occurring after conditioning and initial alloactivation. Therefore, we performed gene expression profiling studies in classical GVHD target organs of animals treated with SAHA or vehicle to further understand the mechanisms underlying this effect. SAHA treated animals revealed a significant upregulation of the mRNA expression of the Protein inhibitor of activated stat 1 (PIAS1) gene in the liver compared to vehicle-treated animals. To further strengthen the hypothesis that SAHA might exert its action by interfering with inflammatory reaction and subsequent signaling through the JAK/STAT pathway, we analyzed the effects of SAHA on STAT-1, 3, and 5 activation and expression of SOCS-1 and SOCS-3 in vitro and in vivo. Thus, BALB/c responder splenocytes were incubated with or without irradiated B6 stimulators in the presence or absence of LPS in order to allow for the separate analysis of LPS and alloactivation-induced JAK/STAT activation. Treatment for 24 hours with SAHA completely inhibited phosphorylation of STAT-1 and STAT-3 in response to LPS and alloactivation using western blot analysis. Furthermore, analysis of liver tissue from GVHD animals showed a sustained expression of SOCS-1 protein in SAHA treated animals whereas SOCS-1 was downregulated in the absence of SAHA. In conclusion our data suggest that the inhibitory effect of SAHA on the development of GVHD is associated with an inhibition of the JAK/STAT signaling pathway. Further studies are warranted to understand the precise mechanisms how SAHA interferes with JAK/STAT signaling and how this leads to inhibition of GVHD. However, it is conceivable that interfering with inflammatory signaling pathways using pharmacological inhibitors of the JAK/STAT pathway might provide a highly attractive treatment strategy for the prevention of GVHD.

Growth factors that contribute to the pathogenesis of many types of cancers also induce activation of the JAK/STAT signaling pathway. Several STAT family members, most notably Stat3 and Stat5, have been found constitutively activated in various hematopoietic malignancies. Recent findings in multiple myeloma suggest that activation of Stat3 contributes to tumor cell proliferation and confers resistance to apoptosis. In the present study, blast cells from patients with various stages of myeloid leukemia and myelodysplastic syndrome (MDS) were analyzed for STAT activation. Our findings indicate that isoforms of STATs 1, 3, and 5 are found constitutively activated in the absence of exogenous growth factors. Similar patterns of STAT activation were also found in several factor-independent myeloid leukemia cell lines, including KG1a and HEL 92.1.7. Blocking tyrosine kinase signaling differentially affects STAT DNA-binding activity, cell proliferation, and apoptosis in the various cell lines, suggesting that divergent signaling pathways lead to constitutive STAT activation in myeloid malignancies. We conclude that constitutive activation of STAT proteins in some of the critical pathways involved in cellular transformation, and that interfering with STAT signaling results in apoptosis of tumor cells. These results provide evidence of a role for STAT signaling in the pathogenesis of myeloid leukemia, and thus identify STAT proteins as potential molecular targets for therapeutic intervention.

NF-kappa B is an important regulator of immunity and inflammation, and its activation pathway has been studied extensively. The mechanisms that downregulate the activity of NF-kappa B have also received a lot of attention, particularly since its activity needs to be terminated to prevent chronic inflammation and subsequent tissue damage. The COMMD family has been identified as a new group of proteins involved in NF-kappa B termination. All ten COMMD members share the structurally conserved carboxy-terminal motif, the COMM domain, and are ubiquitously expressed. They seem to play distinct and non-redundant roles in various physiological processes, including NF-kappa B signaling. In this review, we describe the mechanisms and proteins involved in the termination of canonical NF-kappa B signaling, with a specific focus on the role of the COMMD family in the down-modulation of NF-kappa B. (C) 2013 Elsevier B.V. All rights reserved.

DJ-1 is a protein involved in multiple physiological processes, including cancer, Parkinson’s disease, and male fertility. It is unknown how DJ-1 functions in the apparently different systems. The crystal structure of DJ-1 at 1.6 Å resolution shows that DJ-1 is a helix-strand-helix sandwich and forms a dimer. The DJ-1 structure is similar to the members of the intracellular protease PfpI family. However, the catalytic triad of Cys–His–Glu is not strictly conserved in DJ-1, implying that DJ-1 has a different catalytic mechanism if it acts as a protease or DJ-1 serves as a regulatory protein in the physiological processes. The structure shows that Leu166 positions in the middle of a helix and thus predicts that the L166P mutation will bend the helix and impact the dimerization of DJ-1. As a result, the conformational changes may diminish the DJ-1 binding with its partner, leading to the familial Parkinson’s disease caused by the single L166P mutation.

Summary The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway is involved in many cellular processes, including cell growth and differentiation, immune functions and cancer. It is activated by various cytokines, growth factors, and protein tyrosine kinases (PTKs) and regulates the transcription of many genes. Of the four JAK isoforms and seven STAT isoforms known, JAK2 and STAT3 are highly expressed in the brain where they are present in the postsynaptic density (PSD). Here, we demonstrate a new neuronal function for the JAK/STAT pathway. Using a variety of complementary approaches, we show that the JAK/STAT pathway plays an essential role in the induction of NMDA-receptor dependent long-term depression (NMDAR-LTD) in the hippocampus. Therefore, in addition to established roles in cytokine signaling, the JAK/STAT pathway is involved in synaptic plasticity in the brain., Highlights ► Identification of a specific role of the JAK/STAT pathway in synaptic plasticity ► Activation of JAK2 and STAT3 is required for NMDAR-LTD ► Activation of STAT3 in dendrites and nucleus during NMDAR-LTD ► Nuclear translocation of STAT3 is not required for NMDAR-LTD, The authors demonstrate a new neuronal function for the JAK/STAT pathway in the induction of NMDA-receptor-dependent long-term depression (NMDAR-LTD) in the hippocampus.

Androgen receptor (AR) belongs to the super-family of nuclear hormone receptors that employ complex molecular mechanisms to guide the development and physiological functions of their target tissues. Our recent work has led to the identification of four novel proteins that recognize AR zinc-finger region (ZFR) both in vivo and in vitro. One is a small nuclear RING-finger protein that possesses separate interaction interfaces for AR and for other transcription activators such as Spl. The second is a nuclear serine/threonine protein kinase (androgen-receptor-interacting nuclear protein kinase; ANPK); however, the receptor itself does not seem to be a substrate for this kinase. The third one is dubbed androgen-receptor-interacting protein 3 (ARIP3) and is a novel member of the PIAS (protein inhibitor of activated STAT) protein family. The fourth protein, termed ARIP4, is a nuclear ATPase that belongs to the SNF2-like family of chromatin remodelling proteins. All four proteins exhibit a punctate nuclear pattern when expressed in cultured cells. Each protein modulates AR-dependent transactivation in co-transfection experiments; their activating functions are not restricted to AR. Current work is aimed at elucidating the biochemical and functional properties of these AR-interacting proteins and at finding the partner proteins that form complexes with them in vivo.

Hypoxic niches help maintain mesenchymal stromal cell properties, and their amplification under hypoxia sustains their immature state. However, how MSCs maintain their genomic integrity in this context remains elusive, since hypoxia may prevent proper DNA repair by downregulating expression of BRCA1 and RAD51. Here, we find that the ING1b tumor suppressor accumulates in adipose-derived stromal cells (ADSCs) upon genotoxic stress, owing to SUMOylation on K193 that is mediated by the E3 small ubiquitin-like modifier (SUMO) ligase protein inhibitor of activated STAT protein γ (PIAS4). We demonstrate that ING1b finely regulates the hypoxic response by triggering HIF1α proteasomal degradation. On the contrary, when mutated on its SUMOylation site, ING1b failed to efficiently decrease HIF1α levels. Consistently, we observed that the adipocyte differentiation, generally described to be downregulated by hypoxia, was highly dependent on ING1b expression, during the early days of this process. Accordingly, contrary to what was observed with HIF1α, the absence of ING1b impeded the adipogenic induction under hypoxic conditions. These data indicate that ING1b contributes to adipogenic induction in adipose-derived stromal cells, and thus hinders the phenotype maintenance of ADSCs.

It is becoming increasingly apparent that the chronic gut inflammation observed in the idiopathic inflammatory bowel diseases (e.g. ulcerative colitis, Crohn's disease) is associated with enhanced production of leukocyte-derived oxidants. Oxidants such as hydrogen peroxide are known to activate certain transcription factors such as nuclear transcription factor kappa beta. Nuclear transcription factor kB (NF-kappa B) is a ubiquitous transcription factor and pleiotropic regulator of numerous genes involved in the immune and inflammatory responses. This transcription factor is activated via the selective phosphorylation, ubiquination and degradation of its inhibitor protein I-kB thereby allowing translocation of NF-kappa B into the nucleus where it upregulates the transcription of a variety of adhesion molecules (e.g. ICAM-1, VCAM-1), cytokines (TNF, IL-1, IL-6) and enzymes (iNOS). The proteolytic degradation of the post-translationally modified I-kappa B is known to be mediated by the 26S proteasome complex. Based upon work from our laboratory, we propose that inhibition of NF-kappa B activation produces significant anti inflammatory activity which may be mediated by the inhibition of transcription of certain pro-inflammatory mediators and adhesion molecules.

The Arabidopsis thaliana genes PROTEIN INHIBITOR OF ACTIVATED STAT LIKE1 (PIAL1) and PIAL2 encode proteins with SP-RING domains, which occur in many ligases of the small ubiquitin-related modifier (SUMO) conjugation pathway. We show that PIAL1 and PIAL2 function as SUMO ligases capable of SUMO chain formation and require the SUMO-modified SUMO-conjugating enzyme SCE1 for optimal activity. Mutant analysis indicates a role for PIAL1 and 2 in salt stress and osmotic stress responses, whereas under standard conditions, the mutants show close to normal growth. Mutations in PIAL1 and 2 also lead to altered sulfur metabolism. We propose that, together with SUMO chain binding ubiquitin ligases, these enzymes establish a pathway for proteolytic removal of sumoylation substrates.