Role In Crosstalk Research Articles

Dual Genetic Encoding of Acetyl-lysine and Non-deacetylatable Thioacetyl-lysine Mediated by Flexizyme.

Acetylation of lysine residues is an important post-translational protein modification. Lysine acetylation in histones and its crosstalk with other post-translational modifications in histone and non-histone proteins are crucial to DNA replication, DNA repair, and transcriptional regulation. We incorporated acetyl-lysine (AcK) and the non-hydrolyzable thioacetyl-lysine (ThioAcK) into full-length proteins in vitro, mediated by flexizyme. ThioAcK and AcK were site-specifically incorporated at different lysine positions into human histone H3, either individually or in pairs. We demonstrate that the thioacetyl group in histone H3 could not be removed by the histone deacetylase sirtuin type 1. This method provides a powerful tool to study protein acetylation and its role in crosstalk between post-translational modifications.

Autoimmune Liver Disease Post-Liver Transplantation: A Summary and Proposed Areas for Future Research.

Autoimmune liver diseases (AILD) are rare diseases with a reported prevalence of less than 50 per 100 000 population. As the research landscape and our understanding of AILDs and liver transplantation evolves, there remain areas of unmet needs. One of these areas of unmet needs is prevention of disease recurrence after liver transplantation. Disease recurrence is not an insignificant event because allograft loss with the need for retransplantation can occur. Patients transplanted for AILD are more likely to experience acute rejection compared to those transplanted for non-AILD, and the reason(s) behind this observation is unclear. Tasks for the future include a better understanding of the pathogenesis of AILD, definition of the precise pathogenetic mechanisms of recurrent AILD, and development of strategies that can identify recipients at risk for disease recurrence. Importantly, the role of crosstalk between alloimmune responses and autoimmune responses in AILD is an important area that needs further study.This article reviews the relevant literature of de novo autoimmune hepatitis, recurrent autoimmune hepatitis, recurrent primary sclerosing cholangitis, and recurrent primary biliary cirrhosis in terms of the clinical entity, the scientific advancements, and future scientific goals to enhance our understanding of these diseases.

Eicosopentaneoic Acid and Other Free Fatty Acid Receptor Agonists Inhibit Lysophosphatidic Acid- and Epidermal Growth Factor-Induced Proliferation of Human Breast Cancer Cells.

Many key actions of ω-3 (n-3) fatty acids have recently been shown to be mediated by two G protein-coupled receptors (GPCRs) in the free fatty acid receptor (FFAR) family, FFA1 (GPR40) and FFA4 (GPR120). n-3 Fatty acids inhibit proliferation of human breast cancer cells in culture and in animals. In the current study, the roles of FFA1 and FFA4 were investigated. In addition, the role of cross-talk between GPCRs activated by lysophosphatidic acid (LPA), and the tyrosine kinase receptor activated by epidermal growth factor (EGF), was examined. In MCF-7 and MDA-MB-231 human breast cancer cell lines, both LPA and EGF stimulated proliferation, Erk activation, Akt activation, and CCN1 induction. LPA antagonists blocked effects of LPA and EGF on proliferation in MCF-7 and MDA-MB-231, and on cell migration in MCF-7. The n-3 fatty acid eicosopentaneoic acid inhibited LPA- and EGF-induced proliferation in both cell lines. Two synthetic FFAR agonists, GW9508 and TUG-891, likewise inhibited LPA- and EGF-induced proliferation. The data suggest a major role for FFA1, which was expressed by both cell lines. The results indicate that n-3 fatty acids inhibit breast cancer cell proliferation via FFARs, and suggest a mechanism involving negative cross-talk between FFARS, LPA receptors, and EGF receptor.

Role of Crosstalk between Epithelial and Immune Cells, the Epimmunome, in Allergic Rhinitis Pathogenesis.

Recently, the prevalence of allergic rhinitis has been dramatically increasing worldwide. As conventional therapies for allergic rhinitis, such as antihistamines, leukotriene receptor antagonists, nasal sprays and allergen immunotherapy, have limitations, the development of new drugs is required. Recent studies have revealed that epithelial cell-derived cytokines, including thymic stromal lymphopoietin, interleukin (IL)-25 and IL-33, are able to control immune cells, such as dendritic cells and T cells, thereby acting as 'master switches' in allergic disease. In addition, new roles have been identified for follicular helper T cells and regulatory B cells in allergic disease, and they are considered to be promising targets for new therapies. Thus, crosstalk between epithelial and immune cells, the epimmunome, underlies the pathogenesis of allergic rhinitis. Greater understanding of the epimmunome may lead to breakthroughs in the development of new treatments for allergic rhinitis and will help us cure many patients suffering from its severe symptoms in the future.

New insights into the bioactivity of peptides from probiotics.

Probiotics are unique bacteria that offer several therapeutic benefits to human beings when administered in optimum amounts. Probiotics are able to produce antimicrobial substances, which stimulate the body's immune responses. Here, we review in detail the anti-infective peptides derived from probiotics and their potential immunomodulatory and anti-inflammatory activities, including a major role in cross-talk between probiotics and gut microbiota under adverse conditions. Insights from the engineered cell surface of probiotics may provide novel anti-infective therapy by heterologous expression of receptor peptides of bacterial toxins. It may be possible to use antigenic peptides from viral pathogens as live vaccines. Another possibility is to generate antiviral peptides that bind directly to virus particles, while some peptides exert anti-inflammatory and anticancer effects. Some extracellular polymeric substances might serve as anti-infective peptides. These avenues of treatment have remained largely unexplored to date, despite their potential in generating powerful anti-inflammatory and anti-infective products.

Identification of Major Signaling Pathways in Prion Disease Progression Using Network Analysis.

Prion diseases are transmissible neurodegenerative diseases that arise due to conformational change of normal, cellular prion protein (PrPC) to protease-resistant isofrom (rPrPSc). Deposition of misfolded PrpSc proteins leads to an alteration of many signaling pathways that includes immunological and apoptotic pathways. As a result, this culminates in the dysfunction and death of neuronal cells. Earlier works on transcriptomic studies have revealed some affected pathways, but it is not clear which is (are) the prime network pathway(s) that change during the disease progression and how these pathways are involved in crosstalks with each other from the time of incubation to clinical death. We perform network analysis on large-scale transcriptomic data of differentially expressed genes obtained from whole brain in six different mouse strain-prion strain combination models to determine the pathways involved in prion diseases, and to understand the role of crosstalks in disease propagation. We employ a notion of differential network centrality measures on protein interaction networks to identify the potential biological pathways involved. We also propose a crosstalk ranking method based on dynamic protein interaction networks to identify the core network elements involved in crosstalk with different pathways. We identify 148 DEGs (differentially expressed genes) potentially related to the prion disease progression. Functional association of the identified genes implicates a strong involvement of immunological pathways. We extract a bow-tie structure that is potentially dysregulated in prion disease. We also propose an ODE model for the bow-tie network. Predictions related to diseased condition suggests the downregulation of the core signaling elements (PI3Ks and AKTs) of the bow-tie network. In this work, we show using transcriptomic data that the neuronal dysfunction in prion disease is strongly related to the immunological pathways. We conclude that these immunological pathways occupy influential positions in the PFNs (protein functional networks) that are related to prion disease. Importantly, this functional network involvement is prevalent in all the five different mouse strain-prion strain combinations that we studied. We also conclude that the dysregulation of the core elements of the bow-tie structure, which belongs to PI3K-Akt signaling pathway, leads to dysregulation of the downstream components corresponding to other biological pathways.

Abstract B05: An immune modulatory role for the Fes protein tyrosine kinase

Abstract The Fes protein tyrosine kinase is abundantly expressed in phagocytic immune cells, including tumor associated macrophages. In addition to the C-terminal kinase and central SH2 domains, Fes contains an N-terminal F-BAR domain that confers unique membrane binding and bending properties which implicate it in the regulation of dynamic membrane-cytoskeletal remodeling processes associated with phagocytosis, receptor endocytosis, vesicular trafficking and cross-talk between receptor systems. In addition to roles in signaling downstream from a variety of cytokine receptors, including those for IL-3, -4, -5, and GM-CSF, Fes has also been implicated in regulating signaling downstream from the TLR-4 receptor for LPS. Fes knockout mice display enhanced sensitivity to LPS, and this was shown to be associated with increased NF-κB signaling and TNFα production from Fes-/- macrophages. Recent studies have also implicated Fes in cross-talk between MHC-I and the NF-κB and IRF-3 pathways in macrophages. Activation of these pro-inflammatory NF-κB and IRF-3 pathways so called Signal 3 - in antigen presenting cells (APCs) is associated with more robust activation of T cells. Interestingly, tumor onset in the MMTV-Neu transgenic mouse model of breast cancer is significantly delayed in Fes-/-, and this was associated with increased frequency of T and B lymphocytes in the premalignant mammary glands. These observations suggest that Fes contributes to the regulation of signaling downstream from the MHC-I and TLR receptors such that Fes disruption in TLR-engaged APCs would lead to enhanced activation of CD8+ T cells through increased Signal 3 from the T-cell receptor-engaged APCs. Using a lymphocytic choriomeningitis virus (LCMV) model of immune activation we show that LPS stimulated Fes-/- macrophages promote more robust activation of CD8+ T cells than Fes+/+ macrophages. Furthermore, LPS stimulated Fes-/- macrophages show increased phosphorylation of TBK-1 and IRF-3. We also observed that Fes co-localizes with MHC-I in dynamic vesicular structures within macrophages. These observations are consistent with a model where Fes regulates Signal 3 in APCs through roles in cross-talk between MHC-I and the NF-kB and IRF-3 signaling pathways. This suggests that Fes plays an immune checkpoint role at the level of Signal 3, and that Fes inhibition could promote tumor immunity through increased Signal 3 driven T cell activation. Citation Format: Faizah Mesfer Alotaibi, Connie Zhang, Sam Basta, Peter A. Greer. An immune modulatory role for the Fes protein tyrosine kinase. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr B05.

Abstract 12518: MicroRNA 139-5p Coordinates APLNR-CXCR4 Crosstalk During Vascular Maturation

Rationale: Sprouting angiogenesis is governed by the concept of tip/stalk cells that guides our understanding of the transition from vascular sprouting to maturation and ultimately quiescence. The VEGF and Notch signaling pathways have been extensively described in regulating the discrimination between these two cell populations. However, several additional tip and stalk cell specific genes have been identified. To date, unresolved questions remain, and our understanding of the mechanisms by which these signaling processes are integrated is incomplete. Objective: We set out to investigate novel mechanisms by which signaling pathways involving two G protein coupled receptors (GPCRs), expressed in a mutually exclusive fashion in the tip/stalk cell populations, are intricately linked in vascular development. Methods/Results: Using a combination of in vivo and in vitro techniques, we demonstrate the critical role of crosstalk between APLNR and CXCR4 in vascular maturation. We show robust flow induced expression of the stalk cell specific APLNR, that leads to marked suppression of CXCR4 expression, a mechanism to achieve tip cell restricted expression of the latter. Retinas from Apln (ligand), Aplnr (receptor) and endothelial specific Aplnr deleted mice show retarded vascular expansion, reduced vascularized area and fewer vascular branch points. These phenotypes are in part due to increased expression of Cxcr4 in Apln-/- and Aplnr-/- retinal vessels as Cxcr4 inhibition through a selective inhibitor can ameliorate the Aplnr phenotype. The crosstalk between the two GPCRs was found to involve a key shear responsive microRNA, miR-139-5p, which is upregulated by APLN/APLNR signaling and directly targets CXCR4 in endothelial cells. In accordance, Apln-/- and Aplnr-/- retinal endothelial cells showed depleted levels of miR-139-5p. Lastly, we demonstrate that atorvastatin, an HMG-CoA reductase inhibitor shown to enhance APLNR signaling, can induce miR-139-5p expression and rescue the vascular phenotypes associated with APLN/APLNR deficiency. Conclusions: These findings provide key mechanistic insights into a critical microRNA based crosstalk between two GPCR signaling cascades, which regulates important steps in vascular maturation.

TLR9 ligation in pancreatic stellate cells promotes tumorigenesis.

Modulation of Toll-like receptor (TLR) signaling can have protective or protumorigenic effects on oncogenesis depending on the cancer subtype and on specific inflammatory elements within the tumor milieu. We found that TLR9 is widely expressed early during the course of pancreatic transformation and that TLR9 ligands are ubiquitous within the tumor microenvironment. TLR9 ligation markedly accelerates oncogenesis, whereas TLR9 deletion is protective. We show that TLR9 activation has distinct effects on the epithelial, inflammatory, and fibrogenic cellular subsets in pancreatic carcinoma and plays a central role in cross talk between these compartments. Specifically, TLR9 activation can induce proinflammatory signaling in transformed epithelial cells, but does not elicit oncogene expression or cancer cell proliferation. Conversely, TLR9 ligation induces pancreatic stellate cells (PSCs) to become fibrogenic and secrete chemokines that promote epithelial cell proliferation. TLR9-activated PSCs mediate their protumorigenic effects on the epithelial compartment via CCL11. Additionally, TLR9 has immune-suppressive effects in the tumor microenvironment (TME) via induction of regulatory T cell recruitment and myeloid-derived suppressor cell proliferation. Collectively, our work shows that TLR9 has protumorigenic effects in pancreatic carcinoma which are distinct from its influence in extrapancreatic malignancies and from the mechanistic effects of other TLRs on pancreatic oncogenesis.

Abstract 4319: Clinical significance and possible role of GPNMB in patients with breast cancer

Abstract Glycoprotein non-metastatic B (GPNMB) is a type I transmembrane protein, which is isolated from differential expression assay using metastatic melanoma cells. The physiological function is very little known but may be supposed to be associated with cell invasion and motility particularly in breast cancer cells. Here we investigated the role of GPNMB in breast cancer. First we checked expression of GPNMB by RT-PCR and western blot in several cancer cell lines including breast, gastric, and colon cancer followed by establishment of GPNMB measurement by ELISA because it's reported that GPNMB is shed at extracellular domain by sheddase such as ADAM10. GPMNB expressed in breast (5/6:83%), gastric (3/6:50%), colon (1/7:14.3%) cancer cell lines. Of breast cancer cell lines, GPNMB was highly expressed in SK-BR3 (HER2 positive), BT474 (HER2/ER positive), MDA-MB-157 (Triple negative) cells. Shed GPNMB in culture medium was measurable and correlated with expression of each cell line. Next we evaluated serum GPNMB in patients with breast (n = 164; primary 119, metastatic 43), gastric (n = 38), and colorectal (n = 50) cancer in our institute from 2011.9-2014.2.) This study was approved by the central ethics committee of Gifu University. Serum GPNMBs were 9.403, 5.751, 6.550 ng/ml, respectively. GPNMB for breast cancer patients was statistically higher than those by colorectal cancer patients (p = 0.018). Of breast cancer patients, GPNMB for HER2-type patients was higher than those for Luminal type and DCIS patients (p = 0.0386, p = 0.0195, respectively). Those for triple negative patients was also higher than those for DCIS patients (p = 0.0459). Interestingly, serum GPNMB was dramatically reduced in accordance with chemotherapy in some patients. Based on these clinical observations, we further investigated relationship between GPNMP and HER2 in vitro. Blockage of GPNMB induced not only HER2 but also EGFR expression. On the other hand, inhibition of HER2 by trastuzumab increased expression of GPNMB. Depletion of GPNMB increased sensitivity of trastuzumab, suggesting that GPNMB may play an important role in crosstalk of signal transduction for breast cancer. These notions may suggest a novel therapeutic strategy to overcome HER2 positive breast cancer. Citation Format: Manabu Futamura, Masako Kanematsu, Atsuko Yamada, Kasumi Morimitsu, Akemi Morikawa, Ryutaro Mori, Kazuhiro Yoshida. Clinical significance and possible role of GPNMB in patients with breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4319. doi:10.1158/1538-7445.AM2015-4319

Improving the Post-Stroke Therapeutic Potency of Mesenchymal Multipotent Stromal Cells by Cocultivation With Cortical Neurons: The Role of Crosstalk Between Cells.

The goal of the present study was to maximally alleviate the negative impact of stroke by increasing the therapeutic potency of injected mesenchymal multipotent stromal cells (MMSCs). To pursue this goal, the intercellular communications of MMSCs and neuronal cells were studied in vitro. As a result of cocultivation of MMSCs and rat cortical neurons, we proved the existence of intercellular contacts providing transfer of cellular contents from one cell to another. We present evidence of intercellular exchange with fluorescent probes specifically occupied by cytosol with preferential transfer from neurons toward MMSCs. In contrast, we observed a reversed transfer of mitochondria (from MMSCs to neural cells). Intravenous injection of MMSCs in a postischemic period alleviated the pathological indexes of a stroke, expressed as a lower infarct volume in the brain and partial restoration of neurological status. Also, MMSCs after cocultivation with neurons demonstrated more profound neuroprotective effects than did unprimed MMSCs. The production of the brain-derived neurotrophic factor was slightly increased in MMSCs, and the factor itself was redistributed in these cells after cocultivation. The level of Miro1 responsible for intercellular traffic of mitochondria was increased in MMSCs after cocultivation. We conclude that the exchange by cellular compartments between neural and stem cells improves MMSCs' protective abilities for better rehabilitation after stroke. This could be used as an approach to enhance the therapeutic benefits of stem cell therapy to the damaged brain. The idea of priming stem cells before practical use for clinical purposes was applied. Thus, cells were preconditioned by coculturing them with the targeted cells (i.e., neurons for the treatment of brain pathological features) before the transfusion of stem cells to the organism. Such priming improved the capacity of stem cells to treat stroke. Some additional minimal study will be required to develop a detailed protocol for coculturing followed by cell separation.

Role of Gravin in Compartmentalization of cAMP Signaling

A‐kinase anchoring proteins (AKAPs) play a critical role in signaling by targeting cAMP dependent signaling complexes to specific cellular sites. Gravin, a multivalent AKAP that targets PKA and possibly PDE4 to the cell membrane, undergoes redistribution to the cytosol in response to elevated [Ca2+]i suggesting it may play a role in cross‐talk between Ca2+ and cAMP dependent pathways. We have shown previously that gravin redistribution alters PKA activity in cytosolic and membrane regions, but the effect of gravin redistribution on cAMP levels in these compartments is unknown. To begin assessing this, we used a FRET based cAMP biosensor (lck‐EPAC2‐camps) targeted to the membrane to determine the effect of gravin expression on cAMP levels. Treatment of cells expressing the biosensor and gravin with IBMX showed a greater increase in cAMP levels compared to cells lacking gravin. In addition, cells expressing gravin and the biosensor, showed a significantly higher increase in cAMP when treated with rolipram (PDE4)/forskolin compared to cilostamide (PDE3)/forskolin. These experiments indicate that gravin expression affects cAMP levels at the membrane following PDE inhibition and sets the stage for determining the role of gravin redistribution in regulating cytosolic and membrane cAMP levels. EPAC2‐camps was a gift from Drs. V. Nikolaev and M. Lohse. Supported by NIH grants 1P30GM103329 and 1S100D016250‐01.

Macrophage recruitment in obese adipose tissue.

Obesity is characterized as a chronic state of low-grade inflammation with progressive immune cell infiltration into adipose tissues. Adipose tissue macrophages play critical roles in the establishment of the chronic inflammatory state and metabolic dysfunctions. The novel discovery that pro-inflammatory macrophages are recruited to obese adipose tissue prompted an increased interest in the interplay between immune cells and metabolism. Since this discovery, many works have been published investigating the factors that lead to macrophage recruitment, the phenotypic change of adipose tissue macrophages, and metabolic dysfunctions. Adipokines and chemokines are key mediators that play crucial roles in crosstalk between adipocytes and macrophages and in regulating the adipose tissue inflammation. In the present review, we discuss the obesity-mediated adipose tissue remodelling, and particularly, the role of adipokines/chemokines in macrophage recruitment to obese adipose tissue. This review provides new insights into the physiological role of these factors and identifies a potential therapeutic target for obesity and associated disorders.

The Potential Role of Th9 Cell Related Cytokine and Transcription Factors in Patients with Hepatic Alveolar Echinococcosis.

Human alveolar echinococcosis (AE) is a lethal parasitic infectious disease which may lead to liver failure if left untreated. It is caused by the larval stage of the fox tapeworm Echinococcus multilocularis and usually develops a substantial infiltrative occupation in solid organs. During the infection, T helper subsets are known to play crucial role in crosstalk between the parasite and human host. Th9 cells, a new member of CD4+ T cell family which is characterized by its specific cytokine IL-9 and transcription factors PU.1 and IRF-4, have been known recently to have a critical role in allergic diseases, and cancers as well as the parasitic infection. To assess the potential role of Th9 cells during the infection, the mRNA levels of IL-9, PU.1, and IRF-4 both in peripheral blood mononuclear cells and in liver tissues were, respectively, detected by using real-time PCR. The plasma concentration levels of IL-9 were detected by using enzyme linked immunosorbent assay (ELISA). Th9 related cytokine IL-9 and transcription factors PU.1 and IRF-4 mRNA levels elevated both in PBMCs, and in hepatic lesion and paralesion tissues in AE patients. This may facilitate the infiltrative growth of the parasite and its persistence in human host.

Dual Face of Immune Microenvironment in Myeloma

An important property of human multiple myeloma (MM) is the growth of tumor cells and their precursors in the bone marrow microenvironment. This specialized microenvironment includes a distinct component of immune cells that includes cells of innate as well as adaptive immune system. Growing body of evidence points to a central role for cross-talk between this immune microenvironment and tumor cells in regulating the growth of tumor cells in myeloma. These interactions play a dual role-with some interactions promoting tumor growth while others suppress growth of tumor cells. Cross talk between T and B cells has been implicated in normal B/plasma cell differentiation and may also play a role in pathogenesis of myeloma, explaining increased risk of plasma cell malignancies in certain settings such as Gaucher disease. The bone marrow of MM patients is often infiltrated with innate cells such as dendritic cells (DCs) and cross-talk between myeloid/plasmacytoid DCs or macrophages may promote growth and survival of MM cells. This cross-talk may also have fundamental implications for inducing genomic instability in tumor cells, such as via the induction of cytidine deaminases in tumor cells. Genomic instability is an early feature of human MM and many of the mutations found in MM tumor cells are also present in the precursor states. The immune system has the capacity to recognize these precursor lesions and even in the setting of clinical MM, the tumor bed contains T cells that can be recruited to kill tumor cells. Specific targets of tumor immunity may differ between preclinical and clinical malignancy. Immunity to certain stem cell-associated antigens are in particular associated with reduced risk of progression to clinical MM. With advanced disease, tumor infiltrating T cells encounter several immune inhibitory checkpoints which leads to suppression of function of tumor-infiltrating T cells. In addition to T cells, cells of the innate immune system such as natural killer and natural killer T cells may also be attractive targets of protective tumor immunity in MM. Immunologic approaches may also be combined with other strategies such as other immunomodulatory drugs (IMiDs) already in clinical use in MM. Recent advances in mouse modeling provide new opportunities to better understand how to harness the immune system against human MM. Such approaches have the advantage of the potential for durable control of tumor cells via immunologic memory. Such approaches are now entering the clinic as attractive targets to restore anti-tumor immunity, both in the context of therapy or prevention of MM. Disclosures Dhodapkar: Celgene: Research Funding. Off Label Use: checkpoint blockade drugs, imids.

Crosstalk between Signaling Pathways Provided by Single and Multiple Protein Phosphorylation Sites

Cellular fate depends on the spatiotemporal separation and integration of signaling processes that can be provided by phosphorylation events. In this study, we identify the crucial points in signaling crosstalk that can be triggered by discrete phosphorylation events on a single target protein. We integrated the data on individual human phosphosites with the evidence on their corresponding kinases, the functional consequences of phosphorylation on activity of the target protein and corresponding pathways. Our results show that there is a substantial fraction of phosphosites that can play critical roles in crosstalk between alternative and redundant pathways and regulatory outcome of phosphorylation can be linked to a type of phosphorylated residue. These regulatory phosphosites can serve as hubs in the signal flow and their functional roles are directly connected to their specific properties. Namely, phosphosites with similar regulatory functions are phosphorylated by the same kinases and participate in regulation of similar biochemical pathways. Such sites are more likely to cluster in sequence and space unlike sites with antagonistic outcomes of their phosphorylation on a target protein. In addition, we found that in silico phosphorylation of sites with similar functional consequences has comparable outcomes on a target protein stability. An important role of phosphorylation sites in biological crosstalk is evident from the analysis of their evolutionary conservation.

Leptin Inhibits Preproinsulin mRNA Expression Induced by Suppression of Cytokine Signalling 3 in Beta-Cells

Aim: To study the role of crosstalk between SOCS3 and leptin on insulin expression in rat insulinoma (RIN-5AH) cells that inducibly express SOCS3 mRNA. Materials and Methods: SOCS3 and preproinsulin mRNA expression induced by 5 μM ponasterone A, and the effects of leptin on SOCS3 and preproinsulin mRNA levels were detected by RT-PCR and quantitative PCR, respectively. The effects of SOCS3 on STAT3 phosphorylation were investigated by Western blot analysis. Results: We discovered that SOCS3 regulates preproinsulin mRNA levels in a dose-dependent and timedependent manner. The insulin-suppressing effect of leptin appears to be mediated through reducing the suppressive effects of SOCS3 on STAT3 phosphorylation. Conclusion: Our findings suggest that leptin inhibits preproinsulin mRNA expression induced by SOCS3 in RIN-5AH beta-cells.

Crosstalk between IGF-1R and other Tumor Promoting Pathways

Insulin-like growth factor 1 receptor (IGF-1R) is important in cancer pathogenesis and progression. While its signaling pathway is an interesting therapeutic target, recent clinical trials have exhibited limited effects; however, significant crosstalks between IGF- 1R and other signaling pathways have garnered increasing attention. These complex networks include interactions between IGF-1R and receptor tyrosine kinases (RTKs), including insulin receptor (IR), epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), mesenchymal-epithelial transition factor (MET), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor (FGFR). Furthermore, IGF-1R also is related to steroid hormones, including estrogen receptors alpha and beta (ER! and ER"), androgen receptor (AR), and progesterone receptor (PR). Cumulatively, actions of crosstalk between IGF-1R, and RTKs/steroid hormones promote tumorigenesis, as demonstrated by the effectiveness of recently proposed therapeutic strategies. These therapeutic strategies, primarily pertaining to crosstalk-cotargeting, exhibited notable advantages in overcoming resistance to conventional chemotherapy and conventional endocrine therapy. Furthermore, these techniques offer benefits beyond the limited effects of single- agent targeting previously reported. Thus, the role of crosstalk between IGF-1R and RTKs/steroid hormones, including strategies to block these pathways in combination with recent development in this field, were reviewed and the potential future cancer therapeutics suggested by this rationale were considered.

Sulfur dioxide inhibits vascular smooth muscle cell proliferation via suppressing the Erk/MAP kinase pathway mediated by cAMP/PKA signaling.

The present study was designed to investigate the role of endogenous sulfur dioxide (SO2) in vascular smooth muscle cell (VSMC) proliferation, and explore the possible role of cross-talk between cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) and extracellular signal-regulated kinase (Erk)/mitogen-activated protein kinase (MAPK) pathways in this action. By cell counting, growth curve depict, flow cytometry and bromodeoxyuridine (BrdU) labeling assays, we found that SO2 inhibited VSMC proliferation by preventing cell cycle progression from G1 to S phase and by reducing DNA synthesis. SO2 synthase aspartate aminotransferase (AAT1 and AAT2) overexpression significantly inhibited serum-induced proliferating cell nuclear antigen (PCNA) protein expression in VSMCs, demonstrated by western blot analysis. Moreover, overexpression of AAT1 or AAT2 markedly reduced incorporation of BrdU in serum-treated VSMCs. By contrast, either AAT1 or AAT2 knockdown significantly exacerbated serum-stimulated VSMC proliferation. Thus, both exogenous- and endogenous-derived SO2 suppressed serum-induced VSMC proliferation. However, annexin V-propidium iodide (PI) staining and cell cycle analysis demonstrated that SO2 did not influence VSMC apoptosis in the serum-induced proliferation model. In a platelet-derived growth factor (PDGF)-BB-stimulated VSMC proliferation model, SO2 dephosphorylated the active sites of Erk1/2, MAPK kinase 1/2 and RAF proto-oncogene serine/threonine-protein kinase (c-Raf) induced by PDGF-BB. However, the inactivation of the three kinases of the Erk/MAPK pathway was not due to the separate interferences on them by SO2 simultaneously, but a consequence of the influence on the upstream activity of the c-Raf molecule. Hence, we examined the cAMP/PKA pathway, which could inhibit Erk/MAPK transduction in VSMCs. The results showed that SO2 could stimulate the cAMP/PKA pathway to block c-Raf activation, whereas the Ser259 site on c-Raf had an important role in SO2-induced suppression of Erk/MAPK pathway. The present study firstly demonstrated that SO2 exerted a negative regulation of VSMC proliferation via suppressing the Erk/MAPK pathway mediated by cAMP/PKA signaling.

Prostatic Disease Associated withTrichomonas vaginalis

Trichomonas vaginalis is an extracellular protozoan parasite that binds to the epithelium of the human urogenital tract during infection, and causes contact-dependent cytotoxicity. Neutrophils, macrophages and mast cells known to be involved in innate immunity produced proinflammatory cytokines by stimulation with T. vaginalis. Crosstalk between vaginal epithelial cells (VEC) infected with T. vaginalis and mast cells showed increased inflammatory response compared with that by VECs only. In addition, we confirmed that T. vaginalis caused prostatitis in rat by injection via urethra, and prostate epithelial and stromal cells reacted with trichomonads produced cytokines, including interleukin-1β, CXCL8, and CCL2, resulting in increased migration of neutrophils and monocytes. In further study, we will investigate the role of crosstalk between prostate cells infected with T. vaginalis and inflammatory cells on prostatic cell proliferation or prostatic cancerous change. However, it has not yet been determined whether prostate cancer is associated with T. vaginalis infection. In order to determine their association, a serologic test showing high sensitivity and specificity is necessary. In addition, a molecular diagnostic test with improved sensitivity should be developed for early detection and treatment of trichomoniasis.