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Sample records for factor receptor coexpression

  1. The coexpression and prognostic significance of c-MET, fibroblast growth factor receptor 2, and human epidermal growth factor receptor 2 in resected gastric cancer: a retrospective study

    PubMed Central

    Jia, Yong-Xu; Li, Teng-Fei; Zhang, Dan-Dan; Fan, Zong-Min; Fan, Hui-Jie; Yan, Jie; Chen, Li-Juan; Tang, Hong; Qin, Yan-Ru; Li, Xing-Ya

    2016-01-01

    Molecular-targeted therapy against tyrosine kinase receptors (RTKs) plays an important role in gastric cancer (GC) treatment. Understanding the correlation between RTK coexpression could better guide clinical drug use. In the present study, the coexpression status of c-MET, fibroblast growth factor receptor 2 (FGFR2), and human epidermal growth factor receptor 2 (HER2) in human GC and their clinical significance in clinical therapy were explored. Immunohistochemical (IHC) staining, quantitative real-time polymerase chain reaction and fluorescent in situ hybridization were performed in 143 cases of GC who had undergone gastrectomy without preoperative chemoradiotherapy. Their association with clinicopathological features and clinical prognosis was analyzed. The frequencies of c-MET, FGFR2, and HER2 overexpression were 47.6% (68/143), 34.3% (49/143), and 10.5% (15/143), respectively. In the RTK coexpression study, 30.1% of patients (43/143) were positive for only one RTK, 25.8% (37/143) were positive for two RTKs, 3.5% (5/143) had triple-positive status, and 40.6% (58/143) had triple-negative status. In survival analysis, the overexpression of c-MET, FGFR2, and HER2 were significantly associated with overall survival (OS) (P=0.018, 0.004, and 0.049, respectively). In coexpression analysis, patients with triple-positive GC had the poorest OS (P=0.013). In conclusion, RTK coexpression is significantly associated with poor clinical outcome in GC. PMID:27729801

  2. Coexpression of transforming growth factor-alpha and épidermal growth factor receptor in capillary hemangioblastomas of the central nervous system.

    PubMed Central

    Reifenberger, G.; Reifenberger, J.; Bilzer, T.; Wechsler, W.; Collins, V. P.

    1995-01-01

    The expression of epidermal growth factor receptor (EGFR) and the pre-pro form of one of its ligands, transforming growth factor-alpha (TGF-alpha), was studied by Northern blotting in a series of 14 capillary hemangioblastomas of the central nervous system. A constant coexpression of EGFR and pre-pro-TGF-alpha mRNAs was found. Immunocytochemical investigation of an extended series of 51 capillary hemangioblastomas revealed that the stromal cells in these tumors showed immunoreactivity with monoclonal antibodies to EGFR and TGF-alpha. Analysis of gene dosage by Southern blotting in 20 tumors indicated a normal gene copy number of EGFR and TGF alpha in all cases. Our findings suggest that autocrine and/or juxtacrine growth stimulation via the EGFR may contribute to tumor growth in capillary hemangioblastomas. Images Figure 2 Figure 1 Figure 3 PMID:7639324

  3. Immunohistochemical co-expression status of cytokeratin 5/6, androgen receptor, and p53 as prognostic factors of adjuvant chemotherapy for triple negative breast cancer.

    PubMed

    Maeda, Tetsuyo; Nakanishi, Yoko; Hirotani, Yukari; Fuchinoue, Fumi; Enomoto, Katsuhisa; Sakurai, Kenichi; Amano, Sadao; Nemoto, Norimichi

    2016-03-01

    Triple negative breast cancer (TNBC) is immunohistochemically characterised by the lack of expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor type 2 (HER2). TNBC is known for its poor prognosis and high recurrence probability. There is no effective targeted treatment for TNBC, but only adjuvant chemotherapies. There are two TNBC subtypes, basal-like and non-basal-like, which are defined based on positive cytokeratin (CK) 5/6 and/or epidermal growth factor receptor (EGFR) expression. In particular, CK5/6 expression is reported to correlate with TNBC recurrence. TNBC lacks ER-α expression, but some TNBCs are known to express the androgen receptor (AR). Moreover, although p53 accumulation is detected in various malignant tumors, its influence on adjuvant chemotherapy for patients with TNBC remains unclear. The aim of this study was to assess the combined immunohistochemical expression of CK 5/6, AR, and p53 as a potential prognostic marker of adjuvant chemotherapy for patients with TNBC. The expression of CK5/6, AR, and p53 in formalin-fixed and paraffin-embedded (FFPE) surgical sections from 52 patients with TNBC was analysed by immunohistochemistry (IHC) and the co-expression patterns in individual cells were investigated by immunofluorescent (IF) staining. Low AR expression was correlated with high clinical stage (P < 0.05) and low nuclear grade (P < 0.05). The expression of CK5/6 and p53 did not correlate with clinicopathological features. Patients who needed adjuvant chemotherapy presented the worst prognosis. In particular, when the IHC expression pattern was CK5/6 (-), AR (-), and p53 (+), the disease free survival (DFS) and overall survival (OS) were the worst. On the other hand, patients with AR (+) and p53 (-) TNBC presented a good prognosis. The analysis of the co-expression status of these three markers showed that no cells presented both AR and CK5/6 expression. Furthermore, TP53 m

  4. Coexpression of platelet-derived growth factor (PDGF) and PDGF-receptor genes by primary human astrocytomas may contribute to their development and maintenance.

    PubMed Central

    Maxwell, M; Naber, S P; Wolfe, H J; Galanopoulos, T; Hedley-Whyte, E T; Black, P M; Antoniades, H N

    1990-01-01

    The present studies investigated the expression of the two PDGF genes (c-sis/PDGF-2 and PDGF-1) and the PDGF-receptor b gene (PDGF-R) in 34 primary human astrocytomas. Northern blot analysis demonstrated the coexpression of the c-sis/PDGF-2 protooncogene and the PDGF-R gene in all astrocytomas examined. The majority of the tumors also expressed the PDGF-1 gene. There was no correlation between the expression of the two PDGF genes. Nonmalignant human brain tissue expressed the PDGF-R and PDGF-1 genes but not the c-sis/PDGF-2 protooncogene. In situ hybridization of astrocytoma tissue localized the expression of the c-sis and PDGF-R mRNA's in tumor cells. Capillary endothelial cells also expressed c-sis mRNA. In contrast, nonmalignant human brain tissue expressed only PDGF-R mRNA but not c-sis/PDGF-2 mRNA. The coexpression of a potent mitogenic growth factor protooncogene (c-sis) and its receptor gene in astrocytoma tumor cells suggests the presence of an autocrine mechanism that may contribute to the development and maintenance of astrocytomas. The expression of c-sis mRNA in tumor cells but not in nonmalignant brain cells may serve as an additional diagnostic criterion for the detection of astrocytomas in small tissue specimen using in situ hybridization for the detection of c-sis mRNA and/or immunostaining for the recognition of its protein product. Images PMID:2164040

  5. Co-expression of the androgen receptor and the transcription factor ZNF652 is related to prostate cancer outcome.

    PubMed

    Callen, David F; Ricciardelli, Carmela; Butler, Miriam; Stapleton, Alan; Stahl, Jurgen; Kench, James G; Horsfall, David J; Tilley, Wayne D; Schulz, Renee; Nesland, Jahn M; Neilsen, Paul M; Kumar, Raman; Holm, Ruth

    2010-04-01

    ZNF652, a DNA binding transcription factor, was previously suggested to be differentially expressed in prostate cancer. This study investigated if the expressions of ZNF652 and androgen receptor (AR) in prostate cancer are associated with prostate specific antigen (PSA) defined relapse. ZNF652 and AR immunoreactivity were evaluated in prostate tissues from a cohort of 121 patients with prostate cancer and associations with disease outcome determined. To assess if ZNF652 can influence AR expression, or vice versa, levels of expression of ZNF652, AR and PSA were determined in the prostate cell line LNCaP following induction of AR activity by 5alpha-dihydrotestosterone, or knockdown of ZNF652 expression. Two thirds of prostate tumors retained high levels of ZNF652 (71/109 cases) and 50% of tumors high levels of AR (57/113). There was a significant decrease (p=0.005) in relapse-free survival of patients with high expression levels of both ZNF652 and AR and this was independent of preoperative PSA and seminal vesicle involvement. Modulation of either AR or ZNF652 expression levels in LNCaP cells was not associated with any corresponding changes to the levels of either ZNF652 or AR, respectively. High levels of expression of both AR and ZNF652 in clinically organ-defined prostate cancer are associated with a statistically increased risk of relapse. The ZNF652 and AR transcription factors are acting independently and it is proposed that the continued maintenance of expression of ZNF652 in AR positive cells results in a gene expression pattern that contributes to the relapse.

  6. Localization and distribution of neurons that co-express xeroderma pigmentosum-A and epidermal growth factor receptor within Rosenthal's canal.

    PubMed

    Guthrie, O'neil W

    2015-10-01

    Xeroderma pigmentosum-A (XPA) is a C4-type zinc-finger scaffolding protein that regulates the removal of bulky-helix distorting DNA damage products from the genome. Phosphorylation of serine residues within the XPA protein is associated with improved protection of genomic DNA and cell death resistance. Therefore, kinase signaling is one important mechanism for regulating the protective function of XPA. Previous experiments have shown that spiral ganglion neurons (SGNs) may mobilize XPA as a general stress response to chemical and physical ototoxicants. Therapeutic optimization of XPA via kinase signaling could serve as a means to improve DNA repair capacity within neurons following injury. The kinase signaling activity of the epidermal growth factor receptor (EGFR) has been shown in tumor cell lines to increase the repair of DNA damage products that are primarily repaired by XPA. Such observations suggest that EGFR may regulate the protective function of XPA. However, it is not known whether SGNs in particular or neurons in general could co-express XPA and EGFR. In the current study gene and protein expression of XPA and EGFR were determined from cochlear homogenates. Immunofluorescence assays were then employed to localize neurons expressing both EGFR and XPA within the ganglion. This work was then confirmed with double-immunohistochemistry. Rosenthal's canal served as the reference space in these experiments and design-based stereology was employed in first-order stereology quantification of immunoreactive neurons. The results confirmed that a population of SGNs that constitutively express XPA may also express the EGFR. These results provide the basis for future experiments designed to therapeutically manipulate the EGFR in order to regulate XPA activity and restore gene function in neurons following DNA damage.

  7. Impact of estrogen receptor (ER) and human epidermal growth factor receptor-2 (HER2) co-expression on breast cancer disease characteristics: implications for tumor biology and research.

    PubMed

    Alqaisi, Abeer; Chen, Li; Romond, Edward; Chambers, Mara; Stevens, Mark; Pasley, Grace; Awasthi, Mukta; Massarweh, Suleiman

    2014-11-01

    ER and HER2 are critical drivers of breast cancer biology and can interact when co-expressed, but less data describe the impact of ER/HER2 co-expression on clinical disease characteristics. We studied the impact of ER and HER2 (co)-expression in a cohort of 1,187 patients with invasive breast cancer and compared disease characteristics among different groups according to ER and HER2 status. Age, tumor size, grade, nodal status, TNM stage, and metastatic sites were compared and significance determined using the appropriate t tests. All p values were two-tailed. Compared to ER-negative/HER2-negative disease as the control group, ER expression was associated with older age, smaller tumors, lower grade, earlier TNM stage, and increased bone involvement in de novo metastasis, while HER2 had no significant impact on these characteristics. ER and HER2 co-expression was associated with lower grade and higher bone involvement in de novo metastasis, reflecting a retained impact for ER. HER2 impact on ER-positive disease was reflected by younger age, higher grade and TNM stage, and increased frequency of visceral involvement in de novo metastasis. Within the ER-positive/HER2-positive group, triple positive breast cancer (ER+/PgR+/HER2+) was associated with younger age compared to ER+/PgR-/HER2+ disease (mean age of 50.8 vs. 56 years, p = 0.0226). PgR was also associated with younger age in ER+/HER2- disease with a mean age of 57.6 years in ER+/PgR+/HER2- disease vs. 63.4 years in ER+/PgR-/HER2- disease (p < 0.0001). In conclusion, ER has a profound impact on breast cancer characteristics, including a retained impact when co-expressed with HER2. Similarly, HER2 dramatically modulates ER-positive breast cancer making it more aggressive. PgR association with young age may be related to hormonal levels of the premenopausal state, with HER2 providing an earlier growth advantage in triple positive disease, suggesting a specific dependence for this subset on high estrogen

  8. Co-expression of Dsb proteins enables soluble expression of a single-chain variable fragment (scFv) against human type 1 insulin-like growth factor receptor (IGF-1R) in E. coli.

    PubMed

    Sun, Xue-Wen; Wang, Xiao-Hua; Yao, Yan-Bing

    2014-12-01

    Type 1 insulin-like growth factor receptor (IGF-1R) is a promising therapeutic target for cancer treatment. A single-chain variable fragment (scFv) against human IGF-1R forms inclusion body when expressed in periplasmic space of E. coli routinely. Here, we described that co-expression of appropriate disulfide bonds (Dsb) proteins known to catalyze the formation and isomerization of Dsb can markedly recover the soluble expression of target scFv in E. coli. A 50 % recovery in solubility of the scFv was observed upon co-expression of DsbC alone, and a maximum solubility (80 %) was obtained when DsbA and DsbC were co-expressed in combination. Furthermore, the soluble scFv present full antigen-binding activity with IGF-1R, suggesting its correct folding. This study also suggested that the selection of Dsb proteins should be tested case-by-case if the approach of co-expression of Dsb system is adopted to address the problem of insoluble expression of proteins carrying Dsb.

  9. Low co-expression of epidermal growth factor receptor and its chaperone heat shock protein 90 is associated with worse prognosis in primary glioblastoma, IDH-wild-type.

    PubMed

    Sartori, Elsa; Langer, Rupert; Vassella, Erik; Hewer, Ekkehard; Schucht, Philippe; Zlobec, Inti; Berezowska, Sabina

    2017-10-01

    Epidermal growth factor receptor (EGFR) is a major oncogenic driver in glioblastoma (GBM) without mutations in the isocitrate dehydrogenase gene (IDH-wildtype). Heat shock protein 90 (HSP90) is a regulator of the stability of oncogenic proteins including EGFR, thereby acting as a molecular chaperone. We investigated the expression of EGFR and its chaperone HSP90 in GBM, IDH-wildtype. Tissue availability permitted analysis of 237/449 consecutive GBM cases, among them 214 IDH-wildtype (90.3%). The expression of EGFR and HSP90 was analysed by immunohistochemistry on a tissue microarray containing various tumour regions. The expression intensity (EI), and an expression score (ES) combining the percentage of stained cells with EI were determined for both markers. Overall, there was a positive correlation between EGFR and HSP90 expression (EI; r=0.275, P<0.001; ES, r=0.333, P<0.001). The expression of EGFR and HSP90 was significantly higher in the tumour centre, compared to the infiltration front (EI, P=0.002; ES, P<0.001). Survival data were available in 96 IDH-wildtype cases, and high expression of EGFR (ES only) was in trend associated with better outcome, but failed to meet statyistical significance (P=0.061). A combination of EGFR and HSP90, however, discriminated between different prognostic groups, with EGFRlow/HSP90low tumours showing the worst prognosis in univariate analysis (P=0.001), and in multivariate analysis including the other relevant prognostic factors age, MGMT status and postoperative treatment [n=76; hazard ratio (HR)=0.571; 95% confidence interval (CI) 0.328-0.996; P=0.048]. EGFR expression stratified most pronounced among HSP90low tumours, where the EGFRhigh phenotype was associated with longer survival. Our results reveal a variable reliance on the signalling pathway by EGFR in GBM, IDH-wildtype. Low co-expression was associated with worse prognosis.

  10. Coexpression of the platelet-derived growth factor (PDGF) B chain and the PDGF. beta. receptor in isolated pancreatic islet cells stimulates DNA synthesis

    SciTech Connect

    Welsh, M.; Hallberg, A.; Welsh, N.; Arkhammar, P.; Nilsson, T.; Berggren, P.O. ); Claesson-Welsh, L.; Heldin, C.H. ); Betsholtz, C. ); Berggren, P.O. )

    1990-08-01

    Suspensions rich in pancreatic {beta} cells were transfected by means of electroporation or by using the liposome technique with DNA constructs coding for the {beta} chain of platelet-derived growth factor (PDGF) and the PDGF {alpha} and {beta} receptors to induce a mitotic response in this slowly replicating cell type. Transfection with the B-chain construct induced synthesis of the PDGF B-chain homodimer (PDGF-BB) as assessed by the presence of {sup 125}I-labeled PDGF-BB competing activity in the conditioned medium of the transfected islet cells. Moreover, islet cells transfected with the PDGF {beta}-receptor construct exhibited increased immunofluorescence staining with a PDGF {beta}-receptor antibody. These cells also displayed increased {sup 125}I-labeled PDGF-BB binding compared with control transfected cells. The {beta} cells exhibited elevated levels of ({sup 3}H)inositol trisphosphate after transfection with the B-chain and {beta}-receptor constructs, indicating activation of phospholipase C. Islet cells transfected with the different receptor constructs exhibited different patterns of tyrosine phosphorylation upon ligand activation. The results demonstrate that pancreatic islet cells can be stimulated to increase DNA synthesis by transfection with the PDGF {beta}-receptor gene, whereas cotransfection with the {alpha}-receptor gene may attenuate the growth response.

  11. Modeling the Effects of HER/ErbB1-3 Coexpression on Receptor Dimerization and Biological Response

    SciTech Connect

    Shankaran, Harish; Wiley, H. S.; Resat, Haluk

    2006-06-01

    The human epidermal growth factor receptor (HER/ErbB) system comprises the epidermal growth factor receptor (EGFR/HER1) and three other homologues viz. HERs2-4. This receptor system plays a critical role in cell proliferation and differentiation. Over-expression of these receptors can be associated with poor prognosis in cancers of the epithelium. It is believed that the dimerization pattern among members of the HER family may play a key role in controlling downstream signaling and the eventual biological response. Here, we examine the effect of co-expressing varying levels of HERs1-3 on the receptor dimerization patterns using mathematical modeling. The model integrates biochemical reactions such as ligand binding, receptor dimerization and phosphorylation with biophysical trafficking reactions to predict the concentrations of activated receptors in various cellular compartments. Our results indicate that co-expression of EGFR with HER2 or HER3 biases signaling to the cell surface and retards signal down-regulation. In addition, simultaneous co-expression of HERs1-3 leads to preferential formation of HER2-HER3 heterodimers, which are known to be potent inducers of cell growth and transformation. We further examined the effect of receptor dimerization patterns on cell phenotype using a simple phenomenological model. Results indicate that co-expression of HER2 and HER3 at low to moderate levels may enable cells to match the phenotype of a high HER2 expresser.

  12. Co-expression of vascular endothelial growth factor (VEGF) and its receptors (flk-1 and flt-1) in hormone-induced mammary cancer in the Noble rat

    PubMed Central

    Xie, B; Tam, N N C; Tsao, S W; Wong, Y C

    1999-01-01

    Vascular endothelial growth factor (VEGF) is recognized to play a predominant role in breast cancer prognosis. The action of VEGF is mediated by two high-affinity receptors with ligand-stimulated tyrosine kinase activity: VEGFR-1/flt-1 and VEGFR-2/flk-1, which are expressed mainly in vascular endothelial cells. To the best of our knowledge, no previous studies on the expression of these receptors in breast cancer cells has been made. We have established a new animal model for breast cancer, using a combination of 17β-oestradiol and testosterone as ‘carcinogens’. Taking advantage of the animal model, we have demonstrated that mammary cancer cells expressed not only high levels of VEGF but also, surprisingly, its receptors (flt-1 and flk-1) in mammary cancer cells. Intense reactivities to VEGF, flt-1 and flk-1 were observed in mammary cancer cells, especially in invasive mammary carcinoma. Western blot analysis confirmed the increase in flk-1 and flt-1 proteins in induced mammary cancers. Based on these observations, we hypothesize that in mammary cancer, VEGF regulates, in addition to endothelial proliferation and angiogenesis, also growth of cancer cells by an autocrine mechanism mediated through its receptors. To further verify this hypothesis, we investigated the correlation between cellular proliferation and the expression of VEGF, flt-1 and flk-1. Using double-labelling immunocytochemistry, we have shown a correlation between high VEGF activity and Ki-67 expression. The Ki-67 indices in the areas of strong and weak VEGF reactivities were 58.3% and 3.7% respectively. Similarly, there was also a correlation of strong flk-1 and Ki-67 reactivity. The Ki-67 indices for areas of strong and weak flk-1 reactivities were 53.9% and 3.1% respectively. On the other hand, there was a reverse correlation between flt-1 and Ki-67 activities. These results indicate that overexpression of VEGF and flk-1 is correlated with high Ki-67 index. The data, therefore, suggest that

  13. Genome-wide coexpression of steroid receptors in the mouse brain: Identifying signaling pathways and functionally coordinated regions.

    PubMed

    Mahfouz, Ahmed; Lelieveldt, Boudewijn P F; Grefhorst, Aldo; van Weert, Lisa T C M; Mol, Isabel M; Sips, Hetty C M; van den Heuvel, José K; Datson, Nicole A; Visser, Jenny A; Reinders, Marcel J T; Meijer, Onno C

    2016-03-08

    Steroid receptors are pleiotropic transcription factors that coordinate adaptation to different physiological states. An important target organ is the brain, but even though their effects are well studied in specific regions, brain-wide steroid receptor targets and mediators remain largely unknown due to the complexity of the brain. Here, we tested the idea that novel aspects of steroid action can be identified through spatial correlation of steroid receptors with genome-wide mRNA expression across different regions in the mouse brain. First, we observed significant coexpression of six nuclear receptors (NRs) [androgen receptor (Ar), estrogen receptor alpha (Esr1), estrogen receptor beta (Esr2), glucocorticoid receptor (Gr), mineralocorticoid receptor (Mr), and progesterone receptor (Pgr)] with sets of steroid target genes that were identified in single brain regions. These coexpression relationships were also present in distinct other brain regions, suggestive of as yet unidentified coordinate regulation of brain regions by, for example, glucocorticoids and estrogens. Second, coexpression of a set of 62 known NR coregulators and the six steroid receptors in 12 nonoverlapping mouse brain regions revealed selective downstream pathways, such as Pak6 as a mediator for the effects of Ar and Gr on dopaminergic transmission. Third, Magel2 and Irs4 were identified and validated as strongly responsive targets to the estrogen diethylstilbestrol in the mouse hypothalamus. The brain- and genome-wide correlations of mRNA expression levels of six steroid receptors that we provide constitute a rich resource for further predictions and understanding of brain modulation by steroid hormones.

  14. Modeling the effects of HER/ErbB1-3 co-expression on receptor dimerization and biological response

    SciTech Connect

    Shankaran, Harish; Wiley, H. S.; Resat, Haluk

    2006-06-01

    The human epidermal growth factor receptor (HER/ErbB) system comprises the epidermal growth factor receptor (EGFR/HER1) and three other homologues viz. HER2-4. This receptor system plays a critical role in cell proliferation and differentiation and receptor over-expression can be associated with poor prognosis in cancers of the epithelium. Here, we examine the effect of co-expressing varying levels of HER1-3 on the receptor dimerization patterns using a detailed kinetic model for ErbB heterodimerization and trafficking. Our results indicate that co-expression of EGFR with HER2 or HER3 biases signaling to the cell surface and retards signal down-regulation. In addition, simultaneous co-expression of HER1-3 leads to preferential formation of HER2-HER3 heterodimers, which are known to be potent inducers of cell growth and transformation. Analysis of the parameter dependencies in the model reveals that measurements of HER3 phosphorylation and HER2 internalization ratio may prove to be especially useful for the estimation of critical model parameters. Further, we examined the effect of receptor dimerization patterns on cell phenotype using a simple phenomenological model. Results indicate that co-expression of EGFR with HER2 and HER3 at low to moderate levels may enable cells to match the phenotype of a high HER2 expresser.

  15. Coexpression of striatal dopamine receptor subtypes and excitatory amino acid subunits.

    PubMed

    Ariano, M A; Larson, E R; Noblett, K L; Sibley, D R; Levine, M S

    1997-08-01

    The striatal cellular coexpression patterns for the D(1A) and D2 dopamine (DA) receptor subtypes and the ionotropic excitatory amino acid (EAA) subunits of the N-methyl-D-aspartate (NMDA-R1) and the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) (GluR1 and GluR2/3) receptor subunits were examined morphologically. Their coincidence was assessed by visualization of mRNA transcripts, localization of encoded receptor proteins, and binding analysis using concurrently paired methods of fluorescence detection. The findings indicated that 1) mRNA transcripts for both receptor systems were detected in the medium-sized neuron population, and the distribution of receptor message closely reflected protein and binding patterns, with the exception of the GluR1 subunit; 2) both DA receptor mRNA transcripts were coexpressed with each ionotropic EAA receptor subunit examined and with each other, and NMDA and AMPA receptor subunits also showed coincident expression; 3) D(1A) DA receptor protein was detected in neurons which coexpressed EAA subunit proteins; and 4) GluR2/3 and NMDA-R1 subunit proteins were coexpressed in medium-sized neurons which also demonstrated D2 DA receptor binding sites. These findings suggest morphological receptor "promiscuity" since the coexpression patterns between DA and EAA receptors were found in all permutations. The results provide a spatial framework for physiological findings describing functional interactions between the two DA receptor types and between specific DA and EAA receptors in the striatum.

  16. Genome-wide coexpression of steroid receptors in the mouse brain: Identifying signaling pathways and functionally coordinated regions

    PubMed Central

    Lelieveldt, Boudewijn P. F.; Grefhorst, Aldo; van Weert, Lisa T. C. M.; Mol, Isabel M.; Sips, Hetty C. M.; van den Heuvel, José K.; Datson, Nicole A.; Visser, Jenny A.; Meijer, Onno C.

    2016-01-01

    Steroid receptors are pleiotropic transcription factors that coordinate adaptation to different physiological states. An important target organ is the brain, but even though their effects are well studied in specific regions, brain-wide steroid receptor targets and mediators remain largely unknown due to the complexity of the brain. Here, we tested the idea that novel aspects of steroid action can be identified through spatial correlation of steroid receptors with genome-wide mRNA expression across different regions in the mouse brain. First, we observed significant coexpression of six nuclear receptors (NRs) [androgen receptor (Ar), estrogen receptor alpha (Esr1), estrogen receptor beta (Esr2), glucocorticoid receptor (Gr), mineralocorticoid receptor (Mr), and progesterone receptor (Pgr)] with sets of steroid target genes that were identified in single brain regions. These coexpression relationships were also present in distinct other brain regions, suggestive of as yet unidentified coordinate regulation of brain regions by, for example, glucocorticoids and estrogens. Second, coexpression of a set of 62 known NR coregulators and the six steroid receptors in 12 nonoverlapping mouse brain regions revealed selective downstream pathways, such as Pak6 as a mediator for the effects of Ar and Gr on dopaminergic transmission. Third, Magel2 and Irs4 were identified and validated as strongly responsive targets to the estrogen diethylstilbestrol in the mouse hypothalamus. The brain- and genome-wide correlations of mRNA expression levels of six steroid receptors that we provide constitute a rich resource for further predictions and understanding of brain modulation by steroid hormones. PMID:26811448

  17. Co-Expression of GRK2 Reveals a Novel Conformational State of the µ-Opioid Receptor

    PubMed Central

    Nickolls, Sarah A.; Humphreys, Sian; Clark, Mellissa; McMurray, Gordon

    2013-01-01

    Agonists at the µ-opioid receptor are known to produce potent analgesic responses in the clinical setting, therefore, an increased understanding of the molecular interactions of ligands at this receptor could lead to improved analgesics. As historically morphine has been shown to be a poor recruiter of β-arrestin in recombinant cell systems and this can be overcome by the co-expression of GRK2, we investigated the effects of GRK2 co-expression, in a recombinant µ-opioid receptor cell line, on ligand affinity and intrinsic activity in both β-arrestin recruitment and [35S]GTPγS binding assays. We also investigated the effect of receptor depletion in the β-arrestin assay. GRK2 co-expression increased both agonist Emax and potency in the β-arrestin assay. The increase in agonist potency could not be reversed using receptor depletion, supporting that the effects were due to a novel receptor conformation not system amplification. We also observed a small but significant effect on agonist KL values. Potency values in the [35S]GTPγS assay were unchanged; however, inverse agonist activity became evident with GRK2 co-expression. We conclude that this is direct evidence that the µ-opioid receptor is an allosteric protein and the co-expression of signalling molecules elicits changes in its conformation and thus ligand affinity. This has implications when describing how ligands interact with the receptor and how efficacy is determined. PMID:24376730

  18. Attenuation of Eph Receptor Kinase Activation in Cancer Cells by Coexpressed Ephrin Ligands

    PubMed Central

    Falivelli, Giulia; Lisabeth, Erika Mathes; de la Torre, Elena Rubio; Perez-Tenorio, Gizeh; Tosato, Giovanna; Salvucci, Ombretta; Pasquale, Elena B.

    2013-01-01

    The Eph receptor tyrosine kinases mediate juxtacrine signals by interacting “in trans” with ligands anchored to the surface of neighboring cells via a GPI-anchor (ephrin-As) or a transmembrane segment (ephrin-Bs), which leads to receptor clustering and increased kinase activity. Additionally, soluble forms of the ephrin-A ligands released from the cell surface by matrix metalloproteases can also activate EphA receptor signaling. Besides these trans interactions, recent studies have revealed that Eph receptors and ephrins coexpressed in neurons can also engage in lateral “cis” associations that attenuate receptor activation by ephrins in trans with critical functional consequences. Despite the importance of the Eph/ephrin system in tumorigenesis, Eph receptor-ephrin cis interactions have not been previously investigated in cancer cells. Here we show that in cancer cells, coexpressed ephrin-A3 can inhibit the ability of EphA2 and EphA3 to bind ephrins in trans and become activated, while ephrin-B2 can inhibit not only EphB4 but also EphA3. The cis inhibition of EphA3 by ephrin-B2 implies that in some cases ephrins that cannot activate a particular Eph receptor in trans can nevertheless inhibit its signaling ability through cis association. We also found that an EphA3 mutation identified in lung cancer enhances cis interaction with ephrin-A3. These results suggest a novel mechanism that may contribute to cancer pathogenesis by attenuating the tumor suppressing effects of Eph receptor signaling pathways activated by ephrins in trans. PMID:24348920

  19. Evolution of Spatially Coexpressed Families of Type-2 Vomeronasal Receptors in Rodents

    PubMed Central

    Francia, Simona; Silvotti, Lucia; Ghirardi, Filippo; Catzeflis, François; Percudani, Riccardo; Tirindelli, Roberto

    2015-01-01

    The vomeronasal organ (VNO) is an olfactory structure for the detection of pheromones. VNO neurons express three groups of unrelated G-protein-coupled receptors. Type-2 vomeronasal receptors (V2Rs) are specifically localized in the basal neurons of the VNO and are believed to sense protein pheromones eliciting specific reproductive behaviors. In murine species, V2Rs are organized into four families. Family-ABD V2Rs are expressed monogenically and coexpress with family-C V2Rs of either subfamily C1 (V2RC1) or subfamily C2 (V2RC2), according to a coordinate temporal diagram. Neurons expressing the phylogenetically ancient V2RC1 coexpress family-BD V2Rs or a specific group of subfamily-A V2Rs (V2RA8-10), whereas a second neuronal subset (V2RC2-positive) coexpresses a recently expanded group of five subfamily-A V2Rs (V2RA1-5) along with vomeronasal-specific Major Histocompatibility Complex molecules (H2-Mv). Through database mining and Sanger sequencing, we have analyzed the onset, diversification, and expansion of the V2R-families throughout the phylogeny of Rodentia. Our results suggest that the separation of V2RC1 and V2RC2 occurred in a Cricetidae ancestor in coincidence with the evolution of the H2-Mv genes; this phylogenetic event did not correspond with the origin of the coexpressing V2RA1-5 genes, which dates back to an ancestral myomorphan lineage. Interestingly, the evolution of receptors within the V2RA1-5 group may be implicated in the origin and diversification of some of the V2R putative cognate ligands, the exocrine secreting peptides. The establishment of V2RC2, which probably reflects the complex expansion and diversification of family-A V2Rs, generated receptors that have probably acquired a more subtle functional specificity. PMID:25539725

  20. Chemokine receptor co-expression reveals aberrantly distributed TH effector memory cells in GPA patients.

    PubMed

    Lintermans, Lucas L; Rutgers, Abraham; Stegeman, Coen A; Heeringa, Peter; Abdulahad, Wayel H

    2017-06-14

    Persistent expansion of circulating CD4(+) effector memory T cells (TEM) in patients with granulomatosis with polyangiitis (GPA) suggests their fundamental role in disease pathogenesis. Recent studies have shown that distinct functional CD4(+) TEM cell subsets can be identified based on expression patterns of chemokine receptors. The current study aimed to determine different CD4(+) TEM cell subsets based on chemokine receptor expression in peripheral blood of GPA patients. Identification of particular circulating CD4(+) TEM cells subsets may reveal distinct contributions of specific CD4(+) TEM subsets to the disease pathogenesis in GPA. Peripheral blood of 63 GPA patients in remission and 42 age- and sex-matched healthy controls was stained immediately after blood withdrawal with fluorochrome-conjugated antibodies for cell surface markers (CD3, CD4, CD45RO) and chemokine receptors (CCR4, CCR6, CCR7, CRTh2, CXCR3) followed by flow cytometry analysis. CD4(+) TEM memory cells (CD3(+)CD4(+)CD45RO(+)CCR7(-)) were gated, and the expression patterns of chemokine receptors CXCR3(+)CCR4(-)CCR6(-)CRTh2(-), CXCR3(-)CCR4(+)CCR6(-)CRTh2(+), CXCR3(-)CCR4(+)CCR6(+)CRTh2(-), and CXCR3(+)CCR4(-)CCR6(+)CRTh2(-) were used to distinguish TEM1, TEM2, TEM17, and TEM17.1 cells, respectively. The percentage of CD4(+) TEM cells was significantly increased in GPA patients in remission compared to HCs. Chemokine receptor co-expression analysis within the CD4(+) TEM cell population demonstrated a significant increase in the proportion of TEM17 cells with a concomitant significant decrease in the TEM1 cells in GPA patients compared to HC. The percentage of TEM17 cells correlated negatively with TEM1 cells in GPA patients. Moreover, the circulating proportion of TEM17 cells showed a positive correlation with the number of organs involved and an association with the tendency to relapse in GPA patients. Interestingly, the aberrant distribution of TEM1 and TEM17 cells is modulated in CMV

  1. Significance of Co-expression of Epidermal Growth Factor Receptor and Ki67 on Clinical Outcome in Patients With Anal Cancer Treated With Chemoradiotherapy: An Analysis of NRG Oncology RTOG 9811.

    PubMed

    Doll, Corinne M; Moughan, Jennifer; Klimowicz, Alexander; Ho, Clement K; Kornaga, Elizabeth N; Lees-Miller, Susan P; Ajani, Jaffer A; Crane, Christopher H; Kachnic, Lisa A; Okawara, Gordon S; Berk, Lawrence B; Roof, Kevin S; Becker, Mark J; Grisell, David L; Ellis, Robert J; Sperduto, Paul W; Marsa, Gerald W; Guha, Chandan; Magliocco, Anthony M

    2017-03-01

    To measure co-expression of EGFR and Ki67 proteins in pretreatment tumor biopsies of anal cancer patients enrolled on NRG Oncology RTOG 9811, a phase III trial comparing 5-fluorouracil/mitomycin-C/radiation therapy (Arm A) versus 5-fluorouracil/cisplatin/radiation therapy (Arm B), and to correlate expression with clinical outcome. EGFR and Ki67 co-expression was measured after constructing a tissue microarray using fluorescence immunohistochemistry and automated quantitative image analysis. The Ki67 score within EGFR high versus low areas (Ki67ratio in EGFRhigh:low) in each tumor core was analyzed at the median, quartiles, and as a continuous variable. Associations between the tumor markers and clinical endpoints (overall and disease-free survival, locoregional and colostomy failure, and distant metastases) were explored. A total of 282 pretreatment tumors were analyzed from NRG Oncology RTOG 9811. Of evaluated specimens, 183 (65%, n=89, Arm A; n=94, Arm B) were eligible and analyzable. There were no significant differences in baseline characteristics or outcomes between analyzable and unanalyzable patient cases. Median follow-up was 6.0 years. On multivariate analysis, after adjusting for gender, patients with Ki67ratio in EGFRhigh:low ≥median had worse overall survival (hazard ratio 2.41, 95% confidence interval 1.38-4.19, P=.0019). After adjusting for N stage and largest tumor dimension, patients with Ki67ratio in EGFRhigh:low ≥ median had a higher risk of a disease-free failure (hazard ratio 1.85, 95% confidence interval 1.18-2.92, P=.0078). Technical validation with an independent anal cancer patient cohort was performed and shows a very similar biomarker score distribution. High Ki67ratio in EGFRhigh:low is associated with worse clinical outcome in this subset of patients with anal cancer treated with chemoradiation on NRG Oncology RTOG 9811. Evaluation within a clinical trial will be required to determine whether patients with these tumor

  2. Erythropoietin and erythropoietin receptor coexpression is associated with poor survival in stage I non-small cell lung cancer.

    PubMed

    Saintigny, Pierre; Besse, Benjamin; Callard, Patrice; Vergnaud, Anne-Claire; Czernichow, Sébastien; Colombat, Magali; Girard, Philippe; Validire, Pierre; Breau, Jean-Luc; Bernaudin, Jean-François; Soria, Jean-Charles

    2007-08-15

    This study was designed to evaluate the prognostic effect of erythropoietin (EPO) and EPO receptor (EPO-R) expression in stage I non-small cell lung cancer (NSCLC) patients. EPO and EPO-R expression in 158 tumor samples from resected stage I NSCLC was evaluated using immunohistochemistry and tissue array technology. EPO-R and EPO were highly expressed in 20.9% and 35.4% of tumors, respectively. High EPO-R expression compared with negative or low-level expression was associated with a poor 5-year disease-specific survival (60.6% versus 80.8%; P = 0.01, log-rank test). High EPO expression compared with negative and low-level expression was associated with a trend toward a poor 5-year disease-specific survival (69.6% versus 80.4%; P = 0.13, log-rank test). A high level of EPO-R and EPO coexpression was associated with a poor 5-year disease-specific survival compared with other groups of patients (50.0% versus 80.0% survival at the end of follow-up; P = 0.005, log-rank test). In multivariate analysis for disease-specific survival, high-level EPO-R and EPO coexpression was an independent prognostic factor for disease-specific survival (hazard ratio, 2.214; 95% confidence interval, 1.012-4.848; P = 0.046). These results establish the pejorative prognostic value of EPO and EPO-R expression in early-stage resected NSCLC and suggest a potential paracrine and/or autocrine role of endogenous EPO in NSCLC aggressiveness.

  3. Human brain endothelium: coexpression and function of vanilloid and endocannabinoid receptors.

    PubMed

    Golech, Susanne Andrea; McCarron, Richard M; Chen, Ye; Bembry, Joliet; Lenz, Frederick; Mechoulam, Raphael; Shohami, Esther; Spatz, Maria

    2004-12-06

    The arachidonic acid derivative, 2-arachidonoyl-glycerol (2-AG), was initially isolated from gut and brain; it is also produced and released from blood and vascular cells. Many of the 2-AG-induced cellular responses (i.e., neuromodulation, cytoprotection and vasodilation) are mediated by cannabinoid receptors CB1 and CB2. The findings presented here demonstrate the expression of CB1, CB2 and TRPV1 receptors on cerebromicrovascular endothelial cells (HBEC). The expression of TRPV1, CB1 and CB2 receptor mRNA and proteins were demonstrated by RT-PCR and polyclonal antibodies, respectively. The endocannabinoid 2-AG, and other related compounds [anandamide (ANA), methanandamide (m-ANA), N-(4-hydroxyphenyl-arachidonyl-ethanolamide) (AM404) and capsaicin] dose-dependently stimulated Ca2+ influx in HBEC. The selective TRPV1 receptor antagonist (capsazepine), CB1 receptor antagonist (SR141716A) and CB2 receptor antagonist (SR144528) inhibited these responses. The effects of capsaicin, a specific agonist for TRPV1 receptors, were inhibited by capsazepine, but only weakly by CB1 or CB2 receptor antagonists. 2-AG also induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP); this response was mediated by VR1 receptors. These studies clearly indicate that 2-AG and other related compounds may function as agonists on VR1 receptors, as well as CB1 and CB2 receptors, and implicated these factors in various HBEC functions.

  4. Glutamate transporter type 3 attenuates the activation of N-methyl-D-aspartate receptors co-expressed in Xenopus oocytes.

    PubMed

    Zuo, Zhiyi; Fang, Hongyu

    2005-06-01

    We studied the regulation of N-methy-D-aspartate receptor (NMDAR) current/activation by glutamate transporter type 3 (EAAT3), a neuronal EAAT in vivo, in the restricted extracellular space of a biological model. This model involved co-expressing EAAT3 and NMDAR (composed of NMDAR1-1a and NMDAR2A) in Xenopus oocytes. The NMDAR current was reduced in the co-expression oocytes but not in oocytes expressing NMDAR only when the flow of glutamate-containing superfusate was stopped. The degree of this current reduction was glutamate concentration-dependent. No reduction of NMDAR current was observed in Na+-free solution or when NMDA, a non-substrate for EAATs, was used as the agonist for NMDAR. In the continuous flow experiments, the dose-response curve of glutamate-induced current was shifted to the right-hand side in co-expression oocytes compared with oocytes expressing NMDAR alone. The degree of this shift depended on the abundance of EAAT3 in the co-expression oocytes. Thus, the glutamate concentrations sensed by NMDAR locally were lower than those in the superfusates. These results suggest that EAAT3 regulates the amplitude of NMDAR currents at pre-saturated concentrations of glutamate to EAAT3. Thus, EAATs, by rapidly regulating glutamate concentrations near NMDAR, modulate NMDAR current/activation.

  5. Co-expression of neuropeptide Y Y1 and Y5 receptors results in heterodimerization and altered functional properties.

    PubMed

    Gehlert, Donald R; Schober, Douglas A; Morin, Michelle; Berglund, Magnus M

    2007-12-03

    Centrally administered neuropeptide Y (NPY) produces anxiolytic and orexigenic effects by interacting with Y1 and Y5 receptors that are colocalized in many brain regions. Therefore, we tested the hypothesis that co-expression of Y1 and Y5 receptors results in heterodimerization, altered pharmacological properties and altered desensitization. To accomplish this, the carboxyl-termini of Y1 and Y5 receptors were fused with Renilla luciferase and green fluorescent protein and the proximity of the tagged receptors assessed using bioluminescent resonance energy transfer. Under basal conditions, cotransfection of tagged Y1 receptor and Y5 produced a substantial dimerization signal that was unaffected by the endogenous, nonselective agonists, NPY and peptide YY (PYY). Selective Y5 agonists produced an increase in the dimerization signal while Y5 antagonists also produced a slight but significant increase. In the absence of agonists, selective antagonists decreased dimerization. In functional studies, Y5 agonists produced a greater inhibition of adenylyl cyclase activity in Y1/Y5 cells than cells expressing Y5 alone while NPY and PYY exhibited no difference. With PYY stimulation, the Y1 antagonist became inactive and the Y5 antagonist exhibited uncompetitive kinetics in the Y1/Y5 cell line. In confocal microscopy studies, Y1/Y5 co-expression resulted in increased Y5 signaling following PYY stimulation. Addition of both Y1 and Y5 receptor antagonists was required to significantly decrease PYY-induced internalization. Therefore, Y1/Y5 co-expression results in heterodimerization, altered agonist and antagonist responses and reduced internalization rate. These results may account for the complex pharmacology observed when assessing the responses to NPY and analogs in vivo.

  6. Co-expression of the Follicle Stimulating Hormone Receptor and Stem Cell Markers: A Novel Approach to Target Ovarian Cancer Stem Cells

    DTIC Science & Technology

    2012-09-01

    Award Number: 11 1 0623 TITLE: Co-expression of the Follicle Stimulating Hormone Receptor and Stem...Annual 3. DATES COVERED 1 Sep 2011 – 31 Aug 2012 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Coexpression of the Follicle Stimulating Hormone ...Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT The purpose of this project is to determine whether the Follicle-stimulating Hormone Receptor (FSHR) is co

  7. Progression of Lung Cancer Is Associated with Increased Dysfunction of T Cells Defined by Coexpression of Multiple Inhibitory Receptors.

    PubMed

    Thommen, Daniela S; Schreiner, Jens; Müller, Philipp; Herzig, Petra; Roller, Andreas; Belousov, Anton; Umana, Pablo; Pisa, Pavel; Klein, Christian; Bacac, Marina; Fischer, Ozana S; Moersig, Wolfgang; Savic Prince, Spasenija; Levitsky, Victor; Karanikas, Vaios; Lardinois, Didier; Zippelius, Alfred

    2015-12-01

    Dysfunctional T cells present in malignant lesions are characterized by a sustained and highly diverse expression of inhibitory receptors, also referred to as immune checkpoints. Yet, their relative functional significance in different cancer types remains incompletely understood. In this study, we provide a comprehensive characterization of the diversity and expression patterns of inhibitory receptors on tumor-infiltrating T cells from patients with non-small cell lung cancer. In spite of the large heterogeneity observed in the amount of PD-1, Tim-3, CTLA-4, LAG-3, and BTLA expressed on intratumoral CD8(+) T cells from 32 patients, a clear correlation was established between increased expression of these inhibitory coreceptors and progression of the disease. Notably, the latter was accompanied by a progressively impaired capacity of T cells to respond to polyclonal activation. Coexpression of several inhibitory receptors was gradually acquired, with early PD-1 and late LAG-3/BTLA expression. PD-1 blockade was able to restore T-cell function only in a subset of patients. A high percentage of PD-1(hi) T cells was correlated with poor restoration of T-cell function upon PD-1 blockade. Of note, PD-1(hi) expression marked a particularly dysfunctional T-cell subset characterized by coexpression of multiple inhibitory receptors and thus may assist in identifying patients likely to respond to inhibitory receptor-specific antibodies. Overall, these data may provide a framework for future personalized T-cell-based therapies aiming at restoration of tumor-infiltrating lymphocyte effector functions.

  8. Tissue and cell-type co-expression networks of transcription factors and wood component genes in Populus trichocarpa.

    PubMed

    Shi, Rui; Wang, Jack P; Lin, Ying-Chung; Li, Quanzi; Sun, Ying-Hsuan; Chen, Hao; Sederoff, Ronald R; Chiang, Vincent L

    2017-05-01

    Co-expression networks based on transcriptomes of Populus trichocarpa major tissues and specific cell types suggest redundant control of cell wall component biosynthetic genes by transcription factors in wood formation. We analyzed the transcriptomes of five tissues (xylem, phloem, shoot, leaf, and root) and two wood forming cell types (fiber and vessel) of Populus trichocarpa to assemble gene co-expression subnetworks associated with wood formation. We identified 165 transcription factors (TFs) that showed xylem-, fiber-, and vessel-specific expression. Of these 165 TFs, 101 co-expressed (correlation coefficient, r > 0.7) with the 45 secondary cell wall cellulose, hemicellulose, and lignin biosynthetic genes. Each cell wall component gene co-expressed on average with 34 TFs, suggesting redundant control of the cell wall component gene expression. Co-expression analysis showed that the 101 TFs and the 45 cell wall component genes each has two distinct groups (groups 1 and 2), based on their co-expression patterns. The group 1 TFs (44 members) are predominantly xylem and fiber specific, and are all highly positively co-expressed with the group 1 cell wall component genes (30 members), suggesting their roles as major wood formation regulators. Group 1 TFs include a lateral organ boundary domain gene (LBD) that has the highest number of positively correlated cell wall component genes (36) and TFs (47). The group 2 TFs have 57 members, including 14 vessel-specific TFs, and are generally less correlated with the cell wall component genes. An exception is a vessel-specific basic helix-loop-helix (bHLH) gene that negatively correlates with 20 cell wall component genes, and may function as a key transcriptional suppressor. The co-expression networks revealed here suggest a well-structured transcriptional homeostasis for cell wall component biosynthesis during wood formation.

  9. Coexpression of Nuclear Receptors and Histone Methylation Modifying Genes in the Testis: Implications for Endocrine Disruptor Modes of Action

    PubMed Central

    Anderson, Alison M.; Carter, Kim W.; Anderson, Denise; Wise, Michael J.

    2012-01-01

    Background Endocrine disruptor chemicals elicit adverse health effects by perturbing nuclear receptor signalling systems. It has been speculated that these compounds may also perturb epigenetic mechanisms and thus contribute to the early origin of adult onset disease. We hypothesised that histone methylation may be a component of the epigenome that is susceptible to perturbation. We used coexpression analysis of publicly available data to investigate the combinatorial actions of nuclear receptors and genes involved in histone methylation in normal testis and when faced with endocrine disruptor compounds. Methodology/Principal Findings The expression patterns of a set of genes were profiled across testis tissue in human, rat and mouse, plus control and exposed samples from four toxicity experiments in the rat. Our results indicate that histone methylation events are a more general component of nuclear receptor mediated transcriptional regulation in the testis than previously appreciated. Coexpression patterns support the role of a gatekeeper mechanism involving the histone methylation modifiers Kdm1, Prdm2, and Ehmt1 and indicate that this mechanism is a common determinant of transcriptional integrity for genes critical to diverse physiological endpoints relevant to endocrine disruption. Coexpression patterns following exposure to vinclozolin and dibutyl phthalate suggest that coactivity of the demethylase Kdm1 in particular warrants further investigation in relation to endocrine disruptor mode of action. Conclusions/Significance This study provides proof of concept that a bioinformatics approach that profiles genes related to a specific hypothesis across multiple biological settings can provide powerful insight into coregulatory activity that would be difficult to discern at an individual experiment level or by traditional differential expression analysis methods. PMID:22496781

  10. Serotonin-2C and -2A Receptor Co-expression on Cells in the Rat Medial Prefrontal Cortex

    PubMed Central

    Nocjar, Christine; Alex, Katherine D; Sonneborn, Alex; Abbas, Atheir I; Roth, Bryan L; Pehek, Elizabeth A

    2015-01-01

    Neural function within the medial prefrontal cortex (mPFC) regulates normal cognition, attention and impulse control, implicating neuroregulatory abnormalities within this region in mental dysfunction related to schizophrenia, depression and drug abuse. Both serotonin -2A (5-HT2A) and -2C (5-HT2C) receptors are known to be important in neuropsychiatric drug action and are distributed throughout the mPFC. However, their interactive role in serotonergic cortical regulation is poorly understood. While the main signal transduction mechanism for both receptors is stimulation of phosphoinositide production, they can have opposite effects downstream. 5-HT2A versus 5-HT2C receptor activation oppositely regulates behavior and can oppositely affect neurochemical release within the mPFC. These distinct receptor effects could be caused by their differential cellular distribution within the cortex and/or other areas. It is known that both receptors are located on GABAergic and pyramidal cells within the mPFC, but it is not clear whether they are expressed on the same or different cells. The present work employed immunofluorescence with confocal microscopy to examine this in layers V-VI of the prelimbic mPFC. The majority of GABA cells in the deep prelimbic mPFC expressed 5-HT2C receptor immunoreactivity. Furthermore, most cells expressing 5-HT2C receptor immunoreactivity notably co-expressed 5-HT2A receptors. However, 27% of 5-HT2C receptor immunoreactive cells were not GABAergic, indicating that a population of prelimbic pyramidal projection cells could express the 5-HT2C receptor. Indeed, some cells with 5-HT2C and 5-HT2A receptor co-labeling had a pyramidal shape and were expressed in the typical layered fashion of pyramidal cells. This indirectly demonstrates that 5-HT2C and 5-HT2A receptors may be commonly co-expressed on GABAergic cells within the deep layers of the prelimbic mPFC and perhaps co-localized on a small population of local pyramidal projection cells. Thus a

  11. Co-Expression of Wild-Type P2X7R with Gln460Arg Variant Alters Receptor Function

    PubMed Central

    Aprile-Garcia, Fernando; Metzger, Michael W.; Paez-Pereda, Marcelo; Stadler, Herbert; Acuña, Matías; Liberman, Ana C.; Senin, Sergio A.; Gerez, Juan; Hoijman, Esteban; Refojo, Damian; Mitkovski, Mišo; Panhuysen, Markus; Stühmer, Walter; Holsboer, Florian; Deussing, Jan M.; Arzt, Eduardo

    2016-01-01

    The P2X7 receptor is a member of the P2X family of ligand-gated ion channels. A single-nucleotide polymorphism leading to a glutamine (Gln) by arginine (Arg) substitution at codon 460 of the purinergic P2X7 receptor (P2X7R) has been associated with mood disorders. No change in function (loss or gain) has been described for this SNP so far. Here we show that although the P2X7R-Gln460Arg variant per se is not compromised in its function, co-expression of wild-type P2X7R with P2X7R-Gln460Arg impairs receptor function with respect to calcium influx, channel currents and intracellular signaling in vitro. Moreover, co-immunoprecipitation and FRET studies show that the P2X7R-Gln460Arg variant physically interacts with P2X7R-WT. Specific silencing of either the normal or polymorphic variant rescues the heterozygous loss of function phenotype and restores normal function. The described loss of function due to co-expression, unique for mutations in the P2RX7 gene so far, explains the mechanism by which the P2X7R-Gln460Arg variant affects the normal function of the channel and may represent a mechanism of action for other mutations. PMID:26986975

  12. Co-expression of the human cannabinoid receptor coding region splice variants (hCB₁) affects the function of hCB₁ receptor complexes.

    PubMed

    Bagher, Amina M; Laprairie, Robert B; Kelly, Melanie E M; Denovan-Wright, Eileen M

    2013-12-05

    The human type 1 cannabinoid (hCB1) receptor is expressed at high levels in the central nervous system. mRNA variants of the coding region of this receptor, human cannabinoid hCB1a and hCB1b receptors, have been identified, their biological function remains unclear. The present study demonstrated that the three human cannabinoid hCB1 coding region variants are expressed in the human and monkey (Macaca fascicularis) brain. Western blot analyses of homogenates from different regions of the monkey brain demonstrated that proteins with the expected molecular weights of the cannabinoid CB1, CB1a and CB1b receptors were co-expressed throughout the brain. Given the co-localization of these receptors, we hypothesized that physical interactions between the three splice variants may affect cannabinoid pharmacology. The human cannabinoid hCB1, hCB1a, and hCB1b receptors formed homodimers and heterodimers, as determined by BRET in transiently transfected HEK 293A cells. We found that the co-expression of the human cannabinoid hCB1 and each of the splice variants increased cell surface expression of the human cannabinoid hCB1 receptor and increased Gi/o-dependent ERK phosphorylation in response to cannabinoid agonists. Therefore, the human cannabinoid hCB1 coding region splice variants play an important physiological role in the activity of the endocannabinoid system.

  13. Coexpression of stemness factors Oct4 and Nanog predict liver resection.

    PubMed

    Yin, Xin; Li, Yi-Wei; Zhang, Bo-Heng; Ren, Zheng-Gang; Qiu, Shuang-Jian; Yi, Yong; Fan, Jia

    2012-09-01

    Oct4 and Nanog are two major transcription factors related to the stem cell self-renewal and differentiation. The aim of this study was to evaluate the correlation between these two stemness markers with recurrence, metastasis, and prognosis of hepatocellular carcinoma (HCC). Expression of Oct4 and Nanog was evaluated by immunohistochemistry in a random cohort of 228 HCC patients (cohort A), predominantly hepatitis B related, and validated in another independent cohort of 95 patients (cohort B). Survival analysis was performed by univariate and multivariate analyses. Oct4 and Nanog expression levels in 5 HCC cell lines with different metastatic potential were detected by Western blot assay and quantitative real-time PCR assay. In tissue microarrays, coexpression of Oct4 and Nanog was dramatically associated with big tumor size (P = .001) and vascular invasion (P = .02) and was an independent predictor of postoperative recurrence (hazard ratio [HR] = 1.57, 95 % confidence interval [95 % CI] 1.21-2.04, P = .01) and poor prognosis (HR = 2.20, 95 % CI 1.71-2.88, P < .001). This association was further validated in patients in cohort B. Importantly, this correlation remained significant in patients with early-stage HCC or alpha-fetoprotein (AFP) negative HCC. In addition, expression of Oct4 and Nanog increased in a concordant manner with the increase of metastatic potential in human HCC cell lines. Coexpression of stemness markers Oct4 and Nanog in HCC indicated the aggressive tumor behaviors and predicted a worse clinical outcome, which may be a useful biomarker to identify patients at high risk of postoperative recurrence.

  14. Androgen Receptor Variant AR-V9 Is Coexpressed with AR-V7 in Prostate Cancer Metastases and Predicts Abiraterone Resistance.

    PubMed

    Kohli, Manish; Ho, Yeung; Hillman, David W; Van Etten, Jamie L; Henzler, Christine; Yang, Rendong; Sperger, Jamie M; Li, Yingming; Tseng, Elizabeth; Hon, Ting; Clark, Tyson; Tan, Winston; Carlson, Rachel E; Wang, Liguo; Sicotte, Hugues; Thai, Ho; Jimenez, Rafael; Huang, Haojie; Vedell, Peter T; Eckloff, Bruce W; Quevedo, Jorge F; Pitot, Henry C; Costello, Brian A; Jen, Jin; Wieben, Eric D; Silverstein, Kevin A T; Lang, Joshua M; Wang, Liewei; Dehm, Scott M

    2017-08-15

    Purpose: Androgen receptor (AR) variant AR-V7 is a ligand-independent transcription factor that promotes prostate cancer resistance to AR-targeted therapies. Accordingly, efforts are under way to develop strategies for monitoring and inhibiting AR-V7 in castration-resistant prostate cancer (CRPC). The purpose of this study was to understand whether other AR variants may be coexpressed with AR-V7 and promote resistance to AR-targeted therapies.Experimental Design: We utilized complementary short- and long-read sequencing of intact AR mRNA isoforms to characterize AR expression in CRPC models. Coexpression of AR-V7 and AR-V9 mRNA in CRPC metastases and circulating tumor cells was assessed by RNA-seq and RT-PCR, respectively. Expression of AR-V9 protein in CRPC models was evaluated with polyclonal antisera. Multivariate analysis was performed to test whether AR variant mRNA expression in metastatic tissues was associated with a 12-week progression-free survival endpoint in a prospective clinical trial of 78 CRPC-stage patients initiating therapy with the androgen synthesis inhibitor, abiraterone acetate.Results: AR-V9 was frequently coexpressed with AR-V7. Both AR variant species were found to share a common 3' terminal cryptic exon, which rendered AR-V9 susceptible to experimental manipulations that were previously thought to target AR-V7 uniquely. AR-V9 promoted ligand-independent growth of prostate cancer cells. High AR-V9 mRNA expression in CRPC metastases was predictive of primary resistance to abiraterone acetate (HR = 4.0; 95% confidence interval, 1.31-12.2; P = 0.02).Conclusions: AR-V9 may be an important component of therapeutic resistance in CRPC. Clin Cancer Res; 23(16); 4704-15. ©2017 AACR. ©2017 American Association for Cancer Research.

  15. Co-Expression of Two Subtypes of Melatonin Receptor on Rat M1-Type Intrinsically Photosensitive Retinal Ganglion Cells

    PubMed Central

    Sheng, Wen-Long; Chen, Wei-Yi; Yang, Xiong-Li; Zhong, Yong-Mei; Weng, Shi-Jun

    2015-01-01

    Intrinsically photosensitive retinal ganglion cells (ipRGCs) are involved in circadian and other non-image forming visual responses. An open question is whether the activity of these neurons may also be under the regulation mediated by the neurohormone melatonin. In the present work, by double-staining immunohistochemical technique, we studied the expression of MT1 and MT2, two known subtypes of mammalian melatonin receptors, in rat ipRGCs. A single subset of retinal ganglion cells labeled by the specific antibody against melanopsin exhibited the morphology typical of M1-type ipRGCs. Immunoreactivity for both MT1 and MT2 receptors was clearly seen in the cytoplasm of all labeled ipRGCs, indicating that these two receptors were co-expressed in each of these neurons. Furthermore, labeling for both the receptors were found in neonatal M1 cells as early as the day of birth. It is therefore highly plausible that retinal melatonin may directly modulate the activity of ipRGCs, thus regulating non-image forming visual functions. PMID:25714375

  16. In vivo neuronal co-expression of mu and delta opioid receptors uncovers new therapeutic perspectives

    PubMed Central

    Erbs, Eric; Faget, Lauren; Veinante, Pierre; Kieffer, Brigitte L; Massotte, Dominique

    2015-01-01

    Opioid receptors belong to the G protein coupled receptor family. They modulate brain function at all levels of neural integration and therefore impact on autonomous, sensory, emotional and cognitive processing. In vivo functional interaction between mu and delta opioid receptors are known to take place though it is still debated whether interactions occur at circuitry, cellular or molecular level. Also, the notion of receptor crosstalk via mu-delta heteromers is well documented in vitro but in vivo evidence remains scarce. To identify neurons in which receptor interactions could take place, we designed a unique double mutant knock-in mouse line that expresses functional red-fluorescent mu receptors and green-fluorescent delta receptors. We mapped mu and delta receptor distribution and co-localization throughout the nervous system and created the first interactive brain atlas with concomitant mu-delta visualization at subcellular resolution (http://mordor.ics-mci.fr/). Mu and delta receptors co-localize in neurons from subcortical networks but are mainly detected in separate neurons in the forebrain. Also, co-immunoprecipitation experiments indicated physical proximity in the hippocampus, a prerequisite to mu-delta heteromerization. Altogether, data suggest that mu-delta functional interactions take place at systems level for high-order emotional and cognitive processing whereas mu-delta may interact at cellular level in brain networks essential for survival, which has potential implications for innovative drug design in pain control, drug addiction and eating disorders. PMID:25938125

  17. In vivo neuronal co-expression of mu and delta opioid receptors uncovers new therapeutic perspectives.

    PubMed

    Erbs, Eric; Faget, Lauren; Veinante, Pierre; Kieffer, Brigitte L; Massotte, Dominique

    2014-09-01

    Opioid receptors belong to the G protein coupled receptor family. They modulate brain function at all levels of neural integration and therefore impact on autonomous, sensory, emotional and cognitive processing. In vivo functional interaction between mu and delta opioid receptors are known to take place though it is still debated whether interactions occur at circuitry, cellular or molecular level. Also, the notion of receptor crosstalk via mu-delta heteromers is well documented in vitro but in vivo evidence remains scarce. To identify neurons in which receptor interactions could take place, we designed a unique double mutant knock-in mouse line that expresses functional red-fluorescent mu receptors and green-fluorescent delta receptors. We mapped mu and delta receptor distribution and co-localization throughout the nervous system and created the first interactive brain atlas with concomitant mu-delta visualization at subcellular resolution (http://mordor.ics-mci.fr/). Mu and delta receptors co-localize in neurons from subcortical networks but are mainly detected in separate neurons in the forebrain. Also, co-immunoprecipitation experiments indicated physical proximity in the hippocampus, a prerequisite to mu-delta heteromerization. Altogether, data suggest that mu-delta functional interactions take place at systems level for high-order emotional and cognitive processing whereas mu-delta may interact at cellular level in brain networks essential for survival, which has potential implications for innovative drug design in pain control, drug addiction and eating disorders.

  18. Co-expression of a heat shock transcription factor to improve conformational quality of recombinant protein in Escherichia coli.

    PubMed

    Hsu, Shao-Yen; Lin, Yu-Sheng; Li, Shu-Jyuan; Lee, Wen-Chien

    2014-09-01

    A co-expression system was established in Escherichia coli for enhancing the cellular expression of heat shock transcription factor, sigma 32 (σ(32)). A Shine-Dalgarno sequence and the rpoH gene of E. coli, which encodes σ(32), were cloned into a bacterial plasmid containing a gene fusion encoding a doubly tagged N-acetyl-d-neuraminic acid aldolase (GST-Neu5Ac aldolase-5R). After the IPTG induction, a substantially higher level of sigma 32 was observed up to 3 h in the co-expression cells, but an enhancement in the solubility of target protein was manifest only in the first hour. Nevertheless, the co-expression of sigma 32 led to higher level of Neu5Ac aldolase enzymatic activity in both the soluble and insoluble (inclusion body) fractions. The Neu5Ac aldolase activity of the supernatant from the lysate of cells co-expressing GST-Neu5Ac aldolase-5R and recombinant σ(32) was 3.4-fold higher at 3 h postinduction than that in cells overexpressing GST-Neu5Ac aldolase-5R in the absence of recombinantly expressed σ(32). The results of acrylamide quenching indicated that the conformational quality of the fusion protein was improved by the co-expression of recombinant σ(32). Thus, the increased level of intracellular σ(32) might have created favorable conditions for the proper folding of recombinant proteins through the cooperative effects of chaperones/heat shock proteins expressed by the E. coli host, which resulted in smaller inclusion bodies, improved conformational quality and a higher specific activity of the overexpressed GST-Neu5Ac aldolase-5R protein. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  19. Unique Responses are Observed in Transient Receptor Potential Ankyrin 1 and Vanilloid 1 (TRPA1 and TRPV1) Co-Expressing Cells.

    PubMed

    Sadofsky, Laura R; Sreekrishna, Koti T; Lin, Yakang; Schinaman, Renee; Gorka, Kate; Mantri, Yogita; Haught, John Christian; Huggins, Thomas G; Isfort, Robert J; Bascom, Charles C; Morice, Alyn H

    2014-06-11

    Transient receptor potential (TRP) ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) receptors are implicated in modulation of cough and nociception. In vivo, TRPA1 and TRPV1 are often co-expressed in neurons and TRPA1V1 hetero-tetramer formation is noted in cells co-transfected with the respective expression plasmids. In order to understand the impact of TRP receptor interaction on activity, we created stable cell lines expressing the TRPA1, TRPV1 and co-expressing the TRPA1 and TRPV1 (TRPA1V1) receptors. Among the 600 compounds screened against these receptors, we observed a number of compounds that activated the TRPA1, TRPV1 and TRPA1V1 receptors; compounds that activated TRPA1 and TRPA1V1; compounds that activated TRPV1 and TRPA1V1; compounds in which TRPA1V1 response was modulated by either TRPA1 or TRPV1; and compounds that activated only TRPV1 or TRPA1 or TRPA1V1; and one compound that activated TRPA1 and TRPV1, but not TRPA1V1. These results suggest that co-expression of TRPA1 and TRPV1 receptors imparts unique activation profiles different from that of cells expressing only TRPA1 or TRPV1.

  20. Co-expression network analysis reveals transcription factors associated to cell wall biosynthesis in sugarcane.

    PubMed

    Ferreira, Savio Siqueira; Hotta, Carlos Takeshi; Poelking, Viviane Guzzo de Carli; Leite, Debora Chaves Coelho; Buckeridge, Marcos Silveira; Loureiro, Marcelo Ehlers; Barbosa, Marcio Henrique Pereira; Carneiro, Monalisa Sampaio; Souza, Glaucia Mendes

    2016-05-01

    Sugarcane is a hybrid of Saccharum officinarum and Saccharum spontaneum, with minor contributions from other species in Saccharum and other genera. Understanding the molecular basis of cell wall metabolism in sugarcane may allow for rational changes in fiber quality and content when designing new energy crops. This work describes a comparative expression profiling of sugarcane ancestral genotypes: S. officinarum, S. spontaneum and S. robustum and a commercial hybrid: RB867515, linking gene expression to phenotypes to identify genes for sugarcane improvement. Oligoarray experiments of leaves, immature and intermediate internodes, detected 12,621 sense and 995 antisense transcripts. Amino acid metabolism was particularly evident among pathways showing natural antisense transcripts expression. For all tissues sampled, expression analysis revealed 831, 674 and 648 differentially expressed genes in S. officinarum, S. robustum and S. spontaneum, respectively, using RB867515 as reference. Expression of sugar transporters might explain sucrose differences among genotypes, but an unexpected differential expression of histones were also identified between high and low Brix° genotypes. Lignin biosynthetic genes and bioenergetics-related genes were up-regulated in the high lignin genotype, suggesting that these genes are important for S. spontaneum to allocate carbon to lignin, while S. officinarum allocates it to sucrose storage. Co-expression network analysis identified 18 transcription factors possibly related to cell wall biosynthesis while in silico analysis detected cis-elements involved in cell wall biosynthesis in their promoters. Our results provide information to elucidate regulatory networks underlying traits of interest that will allow the improvement of sugarcane for biofuel and chemicals production.

  1. Co-expression of sialic acid receptors compatible with avian and human influenza virus binding in emus (Dromaius novaehollandiae).

    PubMed

    Gujjar, Naveen; Chothe, Shubhada K; Gawai, Shashikant; Nissly, Ruth; Bhushan, Gitanjali; Kanagaraj, Vijayarani; Jayarao, Bhushan M; Kathaperumal, Kumanan; Subbiah, Madhuri; Kuchipudi, Suresh V

    2017-01-01

    Influenza A viruses (IAVs) continue to threaten animal and human health with constant emergence of novel variants. While aquatic birds are a major reservoir of most IAVs, the role of other terrestrial birds in the evolution of IAVs is becoming increasingly evident. Since 2006, several reports of IAV isolations from emus have surfaced and avian influenza infection of emus can lead to the selection of mammalian like PB2-E627K and PB2-D701N mutants. However, the potential of emus to be co-infected with avian and mammalian IAVs is not yet understood. As a first step, we investigated sialic acid (SA) receptor distribution across major organs and body systems of emu and found a widespread co-expression of both SAα2,3Gal and SAα2,6Gal receptors in various tissues that are compatible with avian and human IAV binding. Our results suggest that emus could allow genetic recombination and hence play an important role in the evolution of IAVs.

  2. Coexpression of human somatostatin receptor-2 (SSTR2) and SSTR3 modulates antiproliferative signaling and apoptosis

    PubMed Central

    2012-01-01

    Background Somatostatin (SST) via five Gi coupled receptors namely SSTR1-5 is known to inhibit cell proliferation by cytostatic and cytotoxic mechanisms. Heterodimerization plays a crucial role in modulating the signal transduction pathways of SSTR subtypes. In the present study, we investigated human SSTR2/SSTR3 heterodimerization, internalization, MAPK signaling, cell proliferation and apoptosis in HEK-293 cells in response to SST and specific agonists for SSTR2 and SSTR3. Results Although in basal conditions, SSTR2 and SSTR3 colocalize at the plasma membrane and exhibit heterodimerization, the cell surface distribution of both receptors decreased upon agonist activation and was accompanied by a parallel increase in intracellular colocalization. Receptors activation by SST and specific agonists significantly decreased cAMP levels in cotransfected cells in comparison to control. Agonist-mediated modulation of pERK1/2 was time and concentration-dependent, and pronounced in serum-deprived conditions. pERK1/2 was inhibited in response to SST; conversely receptor-specific agonist treatment caused inhibition at lower concentration and activation at higher concentration. Strikingly, ERK1/2 phosphorylation was sustained upon prolonged treatment with SST but not with receptor-specific agonists. On the other hand, SST and receptor-specific agonists modulated p38 phosphorylation time-dependently. The receptor activation in cotransfected cells exhibits Gi-dependent inhibition of cell proliferation attributed to increased PARP-1 expression and TUNEL staining, whereas induction of p21 and p27Kip1 suggests a cytostatic effect. Conclusion Our study provides new insights in SSTR2/SSTR3 mediated signaling which might help in better understanding of the molecular interactions involving SSTRs in tumor biology. PMID:22651821

  3. Evidence for limited D1 and D2 receptor coexpression and colocalization within the dorsal striatum of the neonatal mouse.

    PubMed

    Biezonski, Dominik K; Trifilieff, Pierre; Meszaros, Jozsef; Javitch, Jonathan A; Kellendonk, Christoph

    2015-06-01

    The striatum is the major input nucleus of the basal ganglia involved in reward processing, goal-directed behaviors, habit learning, and motor control. The striatum projects to the basal ganglia output nuclei via the "direct" and "indirect" pathways, which can be distinguished by their projection fields and their opposing effects on behavior. In adult animals, the functional opposition is modulated by the differential actions of D1 and D2 dopamine receptors (D1R, D2R), the expression of which is largely separated between these pathways. To determine whether a similar degree of separation exists earlier in development, we used dual-label immunohistochemistry to map dorsal-striatal D1R and D2R expression at the promoter level in postnatal day 0 (PD0) Drd1a-tdTomato/Drd2-GFP BAC transgenic mice, and at the receptor level by costaining for native D1R and D2R in wildtype (WT) PD0 animals. To assess for potential molecular interactions between D1R and D2R we also employed a recently developed proximity-ligation assay (PLA). Limited coexpression and colocalization of the D1R and D2R proteins was found in clusters of neurons endemic to the "patch" compartment as identified by costaining with tyrosine hydroxylase, but not outside these clusters. Moreover, in contrast to our recent findings where we failed to detect a D1R-D2R PLA signal in the adult striatum, in PD0 striatum we did identify a clear PLA signal for this pair of receptors. This colocalization at close proximity points to a possible role for D1R/D2R-mediated crosstalk in early striatal ontogeny.

  4. Coexpression of heparanase activity, cathepsin L, tissue factor, tissue factor pathway inhibitor, and MMP-9 in proliferative diabetic retinopathy

    PubMed Central

    Siddiquei, Mohammad Mairaj; Nawaz, Mohd Imtiaz; De Hertogh, Gert; Mohammad, Ghulam; Alam, Kaiser; Mousa, Ahmed; Opdenakker, Ghislain

    2016-01-01

    Purpose Heparanase cleaves heparan sulfate side chains of heparan sulfate proteoglycans, activity that is implicated in angiogenesis. Proteolytic cleavage of proheparanase by cathepsin L leads to the formation of catalytically active heparanase. We investigated the expression levels of heparanase enzymatic activity and correlated these with the levels of cathepsin L, the angiogenic factors tissue factor (TF) and matrix metalloproteinase-9 (MMP-9), and the angiostatic factor tissue factor pathway inhibitor (TFPI) in proliferative diabetic retinopathy (PDR). Methods Vitreous samples from 25 patients with PDR and 20 nondiabetic patients and epiretinal membranes from 12 patients with PDR were studied with enzyme-linked immunosorbent assay, western blot analysis, and immunohistochemistry. Results We observed a significant increase in the expression of heparanase activity in vitreous samples from patients with PDR compared to the nondiabetic controls (p=0.027). Significant positive correlations were found between the levels of heparanase activity and the levels of cathepsin L (r=0.51; p=0.001), TF (r=0.6; p<0.0001), and TFPI (r=0.49; p=0.001). The expression levels of cathepsin L (p=0.019), TF (p<0.0001), TFPI (p<0.0001), and MMP-9 (p=0.029) were significantly higher in the vitreous samples with detected heparanase activity compared to the vitreous samples with undetected heparanase activity. Western blot analysis demonstrated proteolytic cleavage of TFPI in the vitreous samples from patients with PDR. In the epiretinal membranes, cathepsin L, TF, and TFPI were expressed in vascular endothelial cells and CD45-expressing leukocytes. Significant positive correlations were detected between the number of blood vessels that expressed CD31 and the number of blood vessels that expressed TF (r=0.9; p<0.0001) and TFPI (r=0.81; p=0.001). Conclusions The coexpression of these angiogenesis regulatory factors suggests cross-talk between these factors and pathogenesis of PDR

  5. Selective coexpression of VEGF receptor 2 in EGFRvIII-positive glioblastoma cells prevents cellular senescence and contributes to their aggressive nature.

    PubMed

    Jones, Karra A; Gilder, Andrew S; Lam, Michael S; Du, Na; Banki, Michael A; Merati, Aran; Pizzo, Donald P; VandenBerg, Scott R; Gonias, Steven L

    2016-05-01

    In glioblastoma (GBM), the gene for epidermal growth factor receptor (EGFR) is frequently amplified. EGFR mutations also are common, including a truncation mutation that yields a constitutively active variant called EGFR variant (v)III. EGFRvIII-positive GBM progresses rapidly; however, the reason for this is not clear because the activity of EGFRvIII is attenuated compared with EGF-ligated wild-type EGFR. We hypothesized that EGFRvIII-expressing GBM cells selectively express other oncogenic receptors that support tumor progression. Mining of The Cancer Genome Atlas prompted us to test whether GBM cells in culture, which express EGFRvIII, selectively express vascular endothelial growth factor receptor (VEGFR)2. We also studied human GBM propagated as xenografts. We then applied multiple approaches to test the effects of VEGFR2 on GBM cell growth, apoptosis, and cellular senescence. In human GBM, EGFR overexpression and EGFRvIII positivity were associated with increased VEGFR2 expression. In GBM cells in culture, EGFRvIII-initiated cell signaling increased expression of VEGFR2, which prevented cellular senescence and promoted cell cycle progression. The VEGFR-selective tyrosine kinase inhibitor cediranib decreased tumor DNA synthesis, increased staining for senescence-associated β-galactosidase, reduced retinoblastoma phosphorylation, and increased p27(Kip1), all markers of cellular senescence. Similar results were obtained when VEGFR2 was silenced. VEGFR2 expression by GBM cells supports cell cycle progression and prevents cellular senescence. Coexpression of VEGFR2 by GBM cells in which EGFR signaling is activated may contribute to the aggressive nature of these cells. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  6. Sulfation of the FLAG epitope is affected by co-expression of G protein-coupled receptors in a mammalian cell model

    PubMed Central

    Hunter, Morag Rose; Grimsey, Natasha Lillia; Glass, Michelle

    2016-01-01

    G protein-coupled receptors (GPCRs) are important therapeutic targets and therefore extensively studied. Like most transmembrane proteins, there has been considerable difficulty in developing reliable specific antibodies for them. To overcome this, epitope tags are often used to facilitate antibody recognition in studies on fundamental receptor signalling and trafficking. In our study of cannabinoid CB1/dopamine D2 interactions we sought to generate HEK293 cells expressing FLAG-tagged D2 for use in antibody-based assays of GPCR localisation and trafficking activity, however observed that stable FLAG-hD2 expression was particularly challenging to maintain. In contrast, when expressed in cell lines expressing hCB1 robust and stable FLAG-hD2 expression was observed. We hypothesised that co-expression of CB1 might stabilise surface FLAG-hD2 expression, and therefore investigated this further. Here, we describe the observation that co-expression of either cannabinoid CB1 or CB2 receptors in HEK293 decreases the sulfation of a FLAG epitope appended at the N-terminus of the dopamine D2 receptor. Sulfation alters epitope recognition by some anti-FLAG antibodies, leading to the detection of fewer receptors, even though expression is maintained. This demonstrates that cannabinoid receptor expression modifies posttranslational processing of the FLAG-hD2 receptor, and importantly, has wider implications for the utilisation and interpretation of receptor studies involving epitope tags. PMID:27273047

  7. Identification of Estrogen Receptor Dimer Selective Ligands Reveals Growth-Inhibitory Effects on Cells That Co-Express ERα and ERβ

    PubMed Central

    Powell, Emily; Shanle, Erin; Brinkman, Ashley; Li, Jun; Keles, Sunduz; Wisinski, Kari B.; Huang, Wei; Xu, Wei

    2012-01-01

    Estrogens play essential roles in the progression of mammary and prostatic diseases. The transcriptional effects of estrogens are transduced by two estrogen receptors, ERα and ERβ, which elicit opposing roles in regulating proliferation: ERα is proliferative while ERβ is anti-proliferative. Exogenous expression of ERβ in ERα-positive cancer cell lines inhibits cell proliferation in response to estrogen and reduces xenografted tumor growth in vivo, suggesting that ERβ might oppose ERα's proliferative effects via formation of ERα/β heterodimers. Despite biochemical and cellular evidence of ERα/β heterodimer formation in cells co-expressing both receptors, the biological roles of the ERα/β heterodimer remain to be elucidated. Here we report the identification of two phytoestrogens that selectively activate ERα/β heterodimers at specific concentrations using a cell-based, two-step high throughput small molecule screen for ER transcriptional activity and ER dimer selectivity. Using ERα/β heterodimer-selective ligands at defined concentrations, we demonstrate that ERα/β heterodimers are growth inhibitory in breast and prostate cells which co-express the two ER isoforms. Furthermore, using Automated Quantitative Analysis (AQUA) to examine nuclear expression of ERα and ERβ in human breast tissue microarrays, we demonstrate that ERα and ERβ are co-expressed in the same cells in breast tumors. The co-expression of ERα and ERβ in the same cells supports the possibility of ERα/β heterodimer formation at physio- and pathological conditions, further suggesting that targeting ERα/β heterodimers might be a novel therapeutic approach to the treatment of cancers which co-express ERα and ERβ. PMID:22347418

  8. SeqEnrich: A tool to predict transcription factor networks from co-expressed Arabidopsis and Brassica napus gene sets.

    PubMed

    Becker, Michael G; Walker, Philip L; Pulgar-Vidal, Nadège C; Belmonte, Mark F

    2017-01-01

    Transcription factors and their associated DNA binding sites are key regulatory elements of cellular differentiation, development, and environmental response. New tools that predict transcriptional regulation of biological processes are valuable to researchers studying both model and emerging-model plant systems. SeqEnrich predicts transcription factor networks from co-expressed Arabidopsis or Brassica napus gene sets. The networks produced by SeqEnrich are supported by existing literature and predicted transcription factor-DNA interactions that can be functionally validated at the laboratory bench. The program functions with gene sets of varying sizes and derived from diverse tissues and environmental treatments. SeqEnrich presents as a powerful predictive framework for the analysis of Arabidopsis and Brassica napus co-expression data, and is designed so that researchers at all levels can easily access and interpret predicted transcriptional circuits. The program outperformed its ancestral program ChipEnrich, and produced detailed transcription factor networks from Arabidopsis and Brassica napus gene expression data. The SeqEnrich program is ideal for generating new hypotheses and distilling biological information from large-scale expression data.

  9. CO-EXPRESSION OF ALPHA-2A-ADRENERGIC AND DELTA-OPIOID RECEPTORS IN SUBSTANCE P TERMINALS IN RAT DORSAL HORN

    PubMed Central

    Riedl, Maureen S.; Schnell, Stephen A.; Overland, Aaron C.; Chabot-Doré, Anne-Julie; Taylor, Anna M.; Ribeiro-Da-Silva, Alfredo; Elde, Robert P.; Wilcox, George L.; Stone, Laura S.

    2009-01-01

    Agonists acting at α2-adrenergic and opioid receptors (α2ARs and ORs, respectively) inhibit pain transmission in the spinal cord. When co-administered, agonists activating these receptors interact in a synergistic manner. Although the existence of α2AR/OR synergy has been well characterized, its mechanism remains poorly understood. The formation of hetero-oligomers has been proposed as a molecular basis for interactions between neuronal G-protein-coupled receptors. The relevance of hetero-oligomer formation to spinal analgesic synergy requires demonstration of the expression of both receptors within the same neuron as well as the localization of both receptors in the same neuronal compartment. We used immunohistochemistry to investigate the spatial relationship between α2ARs and ORs in the rat spinal cord to determine if co-expression could be demonstrated between these receptors. We observed extensive co-localization between α2A-adrenergic and delta-opioid receptors (DOP) on substance P (SP)-immunoreactive (ir) varicosities in the superficial dorsal horn of the spinal cord and in peripheral nerve terminals in the skin. α2AAR- and DOP-ir elements were co-localized in subcellular structures of 0.5 μm or less in diameter in isolated nerve terminals. Furthermore, co-incubation of isolated synaptosomes with α2AR and DOP agonists resulted in a greater-than-additive increase in the inhibition of K+-stimulated neuropeptide release. These findings suggest that co-expression of the synergistic receptor pair α2AAR-DOP on primary afferent nociceptive fibers may represent an anatomical substrate for analgesic synergy, perhaps due to protein-protein interactions such as hetero-oligomerization. PMID:19180644

  10. A dominant negative mutation suppresses the function of normal epidermal growth factor receptors by heterodimerization.

    PubMed Central

    Kashles, O; Yarden, Y; Fischer, R; Ullrich, A; Schlessinger, J

    1991-01-01

    Recent studies provide evidence that defective receptors can function as a dominant negative mutation suppressing the action of wild-type receptors. This causes various diminished responses in cell culture and developmental disorders in murine embryogenesis. Here, we describe a model system and a potential mechanism underlying the dominant suppressing response caused by defective epidermal growth factor (EGF) receptors. We used cultured 3T3 cells coexpressing human wild-type receptors and an inactive deletion mutant lacking most of the cytoplasmic domain. When expressed alone, EGF was able to stimulate the dimerization of either wild-type or mutant receptors in living cells as revealed by chemical covalent cross-linking experiments. In response to EGF, heterodimers and homodimers of wild-type and mutant receptors were observed in cells coexpressing both receptor species. However, only homodimers of wild-type EGF receptors underwent EGF-induced tyrosine autophosphorylation in living cells. These results indicate that the integrity of both receptor moieties within receptor dimers is essential for kinase activation and autophosphorylation. Moreover, the presence of mutant receptors in cells expressing wild-type receptors diminished the number of high-affinity binding sites for EGF, reduced the rate of receptor endocytosis and degradation, and diminished biological signalling via EGF receptors. We propose that heterodimerization with defective EGF receptors functions as a dominant negative mutation suppressing the activation and response of normal receptors by formation of unproductive heterodimers. Images PMID:1705006

  11. GABAB1 and GABAB2 receptor subunits co-expressed in cultured human RPE cells regulate intracellular Ca2+ via Gi/o-protein and phospholipase C pathways.

    PubMed

    Cheng, Z-Y; Wang, X-P; Schmid, K L; Han, X-G

    2014-11-07

    GABAB receptors associate with Gi/o-proteins that regulate voltage-gated Ca(2+) channels and thus the intracellular Ca(2+) concentration ([Ca(2+)]i), there is also reported cross-regulation of phospholipase C. These associations have been studied extensively in the brain and also shown to occur in non-neural cells (e.g. human airway smooth muscle). More recently GABAB receptors have been observed in chick retinal pigment epithelium (RPE). The aims were to investigate whether the GABAB receptor subunits, GABAB1 and GABAB2, are co-expressed in cultured human RPE cells, and then determine if the GABAB receptor similarly regulates the [Ca(2+)]i of RPE cells and if phospholipase C is involved. Human RPE cells were cultured from five donor eye cups. Evidence for GABAB1 and GABAB2 mRNAs and proteins in the RPE cell cultures was investigated using real time polymerase chain reaction, western blots and immunofluorescence. The effects of the GABAB receptor agonist baclofen, antagonist CGP46381, a Gi/o-protein inhibitor pertussis toxin, and the phospholipase C inhibitor U73122 on [Ca(2+)]i in cultured human RPE were demonstrated using Fluo-3. Both GABAB1 and GABAB2 mRNA and protein were identified in cell cultures of human RPE; antibody staining was co-localized to the cell membrane and cytoplasm. One-hundred micromolars of baclofen caused a transient increase in the [Ca(2+)]i of RPE cells regardless of whether Ca(2+) was added to the buffer. Baclofen-induced increases in the [Ca(2+)]i were attenuated by pre-treatment with CGP46381, pertussis toxin, and U73122. GABAB1 and GABAB2 are co-expressed in cell cultures of human RPE. GABAB receptors in RPE regulate the [Ca(2+)]i via a Gi/o-protein and phospholipase C pathway. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

  12. C-terminal truncated cannabinoid receptor 1 coexpressed with G protein trimer in Sf9 cells exists in a precoupled state and shows constitutive activity.

    PubMed

    Chillakuri, Chandramouli Reddy; Reinhart, Christoph; Michel, Hartmut

    2007-12-01

    We have investigated the existence of a precoupled form of the distal C-terminal truncated cannabinoid receptor 1 (CB1-417) and heterotrimeric G proteins in a heterologous insect cell expression system. CB1-417 showed higher production levels than the full-length receptor. The production levels obtained in our expression system were double the values reported in the literature. We also observed that at least the distal C-terminus of the receptor was not involved in receptor dimerization, as was predicted in the literature. Using fluorescence resonance energy transfer, we found that CB1-417 and Galpha(i1)beta(1)gamma(2) proteins were colocalized in the cells. GTPgammaS binding assays with the Sf9 cell membranes containing CB1-417 and the G protein trimer showed that the receptor could constitutively activate the Galpha(i1) protein in the absence of agonists. A CB1-specific antagonist (SR 141716A) inhibited this constitutive activity of the truncated receptor. We found that the CB1-417/Galpha(i1)beta(1)gamma(2) complex could be solubilized from Sf9 cell membranes and coimmunoprecipitated. In this study, we have proven that the receptor and G proteins can be coexpressed in higher yields using Sf9 cells, and that the protein complex is stable in detergent solution. Thus, our system can be used to produce sufficient quantities of the protein complex to start structural studies.

  13. Effect of transmembrane pressure on Factor VIII yield in ATF perfusion culture for the production of recombinant human Factor VIII co-expressed with von Willebrand factor.

    PubMed

    Kim, Seung-Chul; An, Sora; Kim, Hyun-Ki; Park, Beom-Soo; Na, Kyu-Heum; Kim, Byung-Gee

    2016-10-01

    In this study, we evaluated three cell retention devices, an alternating tangential flow (ATF) system, a spin-filter, and a Centritech Lab III centrifuge, for the production of recombinant human Factor VIII co-expressed with von Willebrand factor. From the results, it was found that the FVIII activity in bioreactor was significantly higher in the ATF perfusion culture than two other perfusion cultures. Moreover, the FVIII activity yield was unexpectedly low in the ATF perfusion culture. We have, therefore, studied the reasons for this low FVIII activity yield. It was revealed that the inactivation and the surface adsorption of FVIII onto the harvest bag were not the main reasons for the low yield in the ATF perfusion culture. The FVIII activity yield was not increased by the use of a hollow fiber filter with 0.5 μm pore size instead of 0.2 μm pore size. Additionally, the retention of FVIII molecules by the hollow fiber filter was a dominant factor in the low FVIII activity yield in the ATF perfusion culture. We demonstrated that FVIII yield was significantly improved by controlling transmembrane pressure (TMP) across the hollow fiber filter membrane. Taken together, these results suggest that TMP control could be an efficient method for the enhancement of FVIII yield in an ATF perfusion culture.

  14. Coexpressed Catalase Protects Chimeric Antigen Receptor-Redirected T Cells as well as Bystander Cells from Oxidative Stress-Induced Loss of Antitumor Activity.

    PubMed

    Ligtenberg, Maarten A; Mougiakakos, Dimitrios; Mukhopadhyay, Madhura; Witt, Kristina; Lladser, Alvaro; Chmielewski, Markus; Riet, Tobias; Abken, Hinrich; Kiessling, Rolf

    2016-01-15

    Treatment of cancer patients by adoptive T cell therapy has yielded promising results. In solid tumors, however, T cells encounter a hostile environment, in particular with increased inflammatory activity as a hallmark of the tumor milieu that goes along with abundant reactive oxygen species (ROS) that substantially impair antitumor activity. We present a strategy to render antitumor T cells more resilient toward ROS by coexpressing catalase along with a tumor specific chimeric Ag receptor (CAR) to increase their antioxidative capacity by metabolizing H2O2. In fact, T cells engineered with a bicistronic vector that concurrently expresses catalase, along with the CAR coexpressing catalase (CAR-CAT), performed superior over CAR T cells as they showed increased levels of intracellular catalase and had a reduced oxidative state with less ROS accumulation in both the basal state and upon activation while maintaining their antitumor activity despite high H2O2 levels. Moreover, CAR-CAT T cells exerted a substantial bystander protection of nontransfected immune effector cells as measured by CD3ζ chain expression in bystander T cells even in the presence of high H2O2 concentrations. Bystander NK cells, otherwise ROS sensitive, efficiently eliminate their K562 target cells under H2O2-induced oxidative stress when admixed with CAR-CAT T cells. This approach represents a novel means for protecting tumor-infiltrating cells from tumor-associated oxidative stress-mediated repression.

  15. Hippocampal GABAergic interneurons coexpressing alpha7-nicotinic receptors and connexin-36 are able to improve neuronal viability under oxygen-glucose deprivation.

    PubMed

    Voytenko, L P; Lushnikova, I V; Savotchenko, A V; Isaeva, E V; Skok, M V; Lykhmus, O Yu; Patseva, M A; Skibo, G G

    2015-08-07

    The hippocampal interneurons are very diverse by chemical profiles and rather inconsistent by sensitivity to CI. Some hippocampal GABAergic interneurons survive certain time after ischemia while ischemia-sensitive interneurons and pyramidal neurons are damaged. GABAergic signaling, nicotinic receptors expressing α7-subunit (α7nAChRs(+)) and connexin-36 (Cx36(+), electrotonic gapjunctions protein) contradictory modulate post-ischemic environment. We hypothesized that hippocampal ischemia-resistant GABAergic interneurons coexpressing glutamate decarboxylase-67 isoform (GAD67(+)), α7nAChRs(+), Cx36(+) are able to enhance neuronal viability. To check this hypothesis the histochemical and electrophysiological investigations have been performed using rat hippocampal organotypic culture in the condition of 30-min oxygen-glucose deprivation (OGD). Post-OGD reoxygenation (4h) revealed in CA1 pyramidal layer numerous damaged cells, decreased population spike amplitude and increased pair-pulse depression. In these conditions GAD67(+) interneurons displayed the OGD-resistance and significant increase of GABA synthesis/metabolism (GAD67-immunofluorescence, mitochondrial activity). The α7nAChRs(+) and Cx36(+) co-localizations were revealed in resistant GAD67(+) interneurons. Under OGD: GABAA-receptors (GABAARs) blockade increased cell damage and exacerbated the pair-pulse depression in CA1 pyramidal layer; α7nAChRs and Cx36-channels separate blockades sufficiently decreased cell damage while interneuronal GAD67-immunofluorescence and mitochondrial activity were similar to the control. Thus, hippocampal GABAergic interneurons co-expressing α7nAChRs and Cx36 remained resistant certain time after OGD and were able to modulate CA1 neuron survival through GABAARs, α7nAChRs and Cx36-channels activity. The enhancements of the neuronal viability together with GABA synthesis/metabolism normalization suggest cooperative neuroprotective mechanism that could be used for increase in

  16. Co-expression patterns of the neuropeptides vasoactive intestinal peptide and cholecystokinin with the transduction molecules alpha-gustducin and T1R2 in rat taste receptor cells.

    PubMed

    Shen, T; Kaya, N; Zhao, F-L; Lu, S-G; Cao, Y; Herness, S

    2005-01-01

    Taste receptor cells are primary sensory receptors utilized by the nervous system to detect the presence of gustatory stimuli in the oral cavity. These cells are particularly heterogeneous and may be divided into various subtypes based on morphological, histochemical, or physiological criteria. One example is the heterogeneous expression of neuropeptides, such as cholecystokinin and vasoactive intestinal polypeptide. These peptides are hypothesized to participate in the transduction processes. To pursue examination of this hypothesis, this study explored the relationship of peptide expression with two important and mostly non-overlapping transductive elements--the taste-specific G protein gustducin, involved in bitter and sweet transduction cascades, and the seven transmembrane taste receptor T1R2, hypothesized to respond to sweet compounds. Double labeling experiments were performed on taste buds of the posterior rat tongue combining immunocytochemistry for peptide expression and in situ hybridization experiments for either gustducin or T1R2 expression. Additionally, vasoactive intestinal peptide (VIP)-expression in posterior taste receptor cells was confirmed using the technique of RT-PCR. More than half (56%) of the CCK-expressing taste receptor cells co-expressed alpha-gustducin mRNA whereas far fewer (15%) co-expressed T1R2 mRNA. A majority of VIP-expressing taste receptor cells co-expressed alpha-gustducin mRNA (60%) whereas only 19% of these cells co-expressed T1R2 mRNA. More remarkable was the observation that these two peptides displayed almost identical expression patterns with these signal transduction molecules, suggesting that peptides are not randomly expressed with relation to signal transduction molecules. This observation supports the hypothesis that peptides may play roles in transduction. Further physiological exploration will be required to elucidate the nature of these roles.

  17. Interaction of glucocorticosteroid receptor and wild-type or mutated 90-kDa heat shock protein coexpressed in baculovirus-infected Sf9 cells.

    PubMed

    Cadepond, F; Binart, N; Chambraud, B; Jibard, N; Schweizer-Groyer, G; Segard-Maurel, I; Baulieu, E E

    1993-11-15

    Coexpression of the human glucocorticosteroid receptor (hGR) and chicken 90-kDa heat shock protein alpha (chsp90) in recombinant baculovirus-infected Sf9 cells is a system that provides a large quantity of wild-type chsp90-hGR complexes able to bind hormone ([3H]triamcinolone acetonide; TA), sedimenting at 8 S, and displaceable to 11 S by BF4 and D7 alpha anti-chsp90 monoclonal antibodies. Thus, we were able to examine the effects of selective chsp90 mutations on hetero-oligomeric complex formation. Two deletions involved hydrophilic regions, A between amino acids 221 and 290 and B between amino acids 530 and 581, and the third, Z, removed a central leucine heptad repeat region (amino acids 392-419). When these chsp90 mutants were expressed, the lack of displacement of [3H]TA receptor complexes on sucrose gradient by specific chsp90 antibodies was consistent with the formation of [3H]TA receptor complexes containing only endogenous insect hsp90. By using an immunoadsorption method and sedimentation analysis, we found that the deletion of region A precluded the interaction of chsp90 with the hGR, while B and Z deletions led to formation of abnormal complexes with the hGR, which displayed large forms (> 10 S), were unable to bind hormone, and apparently formed only small amounts of tightly bound nuclei hGR upon in vivo hormone treatment. As a whole, the data are consistent with distinct roles of hsp90 regions in hGR function.

  18. Impact of fusion to Gα(i2) and co-expression with RGS proteins on pharmacological properties of human cannabinoid receptors CB₁R and CB₂R.

    PubMed

    Sutor, Sarah; Heilmann, Jörg; Seifert, Roland

    2011-08-01

    G protein coupled receptor (GPCR)-Gα fusion proteins are often employed to investigate receptor/G protein interaction. In this study, the impact of Gα fusion proteins on pharmacology of CBRs, both mediating signals through Gα(i) proteins, were investigated. Gα(i2) was fused to the C-terminus of the CBRs or co-expressed with non-fused Gα(i2) in Sf9 cells, always together with Gβ₁γ₂. Furthermore, the impact of RGS proteins on CBR signaling in combination with the CBR fusion approach was examined, using RGS4 and RGS19 as paradigms. CBR ligands were characterized in the steady-state GTPase assay and pharmacological properties of ligands in the different test systems were correlated. Fusion of CBRs to Gα(i2) enhanced the maximal stimulatory effects of ligands compared to the co-expression system, especially for CB₂R. RGS4, but not RGS19, behaved as a GTPase-activating protein at CBRs in the Gα(i2) co-expression and fusion system. Fusion of GPCR, most prominently CB₂R, to Gα(i2) , and co-expression with RGS4 altered the pharmacological properties of ligands. Our data suggest that fusion of CB₂R to Gα(i2) and co-expression with RGS4 impedes with conformational changes. Moreover, our results support the concept of ligand-specific receptor conformations. Finally, this paper describes the most sensitive CBR test system currently available. © 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.

  19. Receptor-ligand requirements for increased NK cell polyfunctional potential in slow progressors infected with HIV-1 coexpressing KIR3DL1*h/*y and HLA-B*57.

    PubMed

    Kamya, Philomena; Boulet, Salix; Tsoukas, Christos M; Routy, Jean-Pierre; Thomas, Réjean; Côté, Pierre; Boulassel, Mohamed-Rachid; Baril, Jean-Guy; Kovacs, Colin; Migueles, Stephen A; Connors, Mark; Suscovich, Todd J; Brander, Christian; Tremblay, Cecile L; Bernard, Nicole

    2011-06-01

    Carriage of the natural killer (NK) receptor genotype KIR3DL1*h/*y with its HLA-B*57 ligand (*h/*y+B*57) is associated with slow time to AIDS and low viral load (VL). To provide a functional basis for these epidemiological observations, we assessed whether HIV-1-infected slow progressors (SP) carrying the *h/*y+B*57 compound genotype would have increased NK cell polyfunctional potential in comparison to SP with other killer immunoglobulin-like receptor (KIR)/HLA compound genotypes and whether this enhanced polyfunctionality was dependent upon the coexpression of both KIR3DL1*h/*y and HLA-B*57. The functional potential of NK cells was investigated by stimulating peripheral blood mononuclear cells with HLA-devoid targets or single HLA transfectants. Multiparametric flow cytometry was used to detect NK cells with seven functional profiles representing all permutations of CD107a expression and gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) secretion. NK cells from individuals carrying KIR3DL1 receptor-HLA-Bw4 ligand pairs had greater trifunctional responses than those from KIR3DL1 homozygotes (hmz), who were Bw6 homozygotes. NK cells from subjects carrying the *h/*y+B*57 genotypes exhibited the highest trifunctional potential, and this was dependent on cocarriage of the NK receptor and its ligand. Trifunctional cells secreted more of each function tested on a per-cell basis than each corresponding monofunctional NK subset. Although VL influenced NK functionality, individuals with defined KIR/HLA genotypes exhibited differences in NK cell polyfunctionality that could not be accounted for by VL alone. The protective effect of HLA-B*57 on slow progression to AIDS and low VL may be mediated through its interaction with KIR3DL1 alleles to educate NK cells for potent activity upon stimulation.

  20. Coexpression of adrenomedullin and its receptor component proteins in the reproductive system of the rat during gestation

    PubMed Central

    2010-01-01

    Background Adrenomedullin (ADM), a novel vasorelaxant peptide, was found in human/rat ovaries and uteri. Plasma ADM level increases in pregnant women and pregnant rats. Methods The gene expression levels of Adm and its receptor components - Crlr, Ramp1, Ramp2 and Ramp3, the ADM peptide concentration and localization in the rat female reproductive system during gestation were studied by real-time RT-PCR, EIA and immunohistochemical techniques. Results The mRNAs of Adm and its receptor component and ADM were differentially distributed between implantation sites and inter-implantation sites of the pregnant uterus. The day on which vaginal sperm were found was taken to be pregnancy day 1. The Adm mRNA levels in the implantation sites of the uteri in mid- (day 12) and late pregnancy (day 17) were more than 10-fold higher than those in nonpregnancy, pre-implantation (day 3) or early (day 7) pregnancy. ADM was localized in the endometrial stroma with increased immunoreactivity from nonpregnancy to pregnancy. The ADM level and the mRNA levels of Adm, Crlr, Ramp2 and Ramp3 in the corpus luteum all increased in late pregnancy compared with early pregnancy. The gene expression of Adm and it receptor components and intense immunostaining of ADM were also found in the oviduct during pregnancy. Conclusions The gene expressions levels of Adm and its receptor components - Crlr, Ramp1, Ramp2 and Ramp3, and ADM peptide concentration exhibited a spatio-temporal pattern in the rat female reproductive system during gestation and this suggests that ADM may play important roles in gestation. PMID:21034462

  1. Co-expression of CD133, CD44v6 and human tissue factor is associated with metastasis and poor prognosis in pancreatic carcinoma.

    PubMed

    Chen, Kai; Li, Zhonghu; Jiang, Peng; Zhang, Xi; Zhang, Yujun; Jiang, Yan; He, Yu; Li, Xiaowu

    2014-08-01

    The metastasis-related molecules CD133, CD44v6 and human tissue factor (TF) have been shown to be associated with tumor invasion and metastasis. This study aimed to determine whether co-expression of these three molecules was associated with metastasis and overall prognosis in pancreatic carcinoma. We analyzed the expression profiles of these three molecules by immunohistochemistry and evaluated the relationship of their expression profiles with metastasis and prognosis in 109 pancreatic carcinomas. The results showed that the expression levels of CD133, CD44v6 and TF were increased in pancreatic carcinoma. Co-expression of CD133, CD44v6 and TF (tri-expression) was also detected in pancreatic carcinoma. Clinical analysis showed that individual expression of CD133, CD44v6 or TF was associated with vessel invasion, lymph node metastasis and liver metastasis, while tri-expression was associated with lymph node metastasis. Survival analysis showed that patients with co-expression of CD133 and TF or tri-expression had lower and the lowest overall survival rates, respectively. Univariate analysis showed that T-factor, lymph node metastasis, TNM stage, and individual levels or tri-expression of CD133, CD44v6 and TF were survival risk factors. Multivariate analysis showed that tri-expression of CD133, CD44v6 and TF was an independent predictor of survival. These results suggest that overexpression of CD133, CD44v6 and TF is associated with pancreatic carcinoma metastasis. Tri-expression of these three molecules may be a useful predictor for pancreatic carcinoma prognosis.

  2. Confirmation of warfarin resistance of naturally occurring VKORC1 variants by coexpression with coagulation factor IX and in silico protein modelling

    PubMed Central

    2014-01-01

    Background VKORC1 has been identified some years ago as the gene encoding vitamin K epoxide reductase (VKOR) – the target protein for coumarin derivates like warfarin or phenprocoumon. Resistance against warfarin and other coumarin-type anticoagulants has been frequently reported over the last 50 years in rodents due to problems in pest control as well as in thrombophilic patients showing variable response to anticoagulant treatment. Many different mutations have already been detected in the VKORC1 gene leading to warfarin resistance in rats, mice and in humans. Since the conventional in vitro dithiothreitol (DTT)-driven VKOR enzymatic assay often did not reflect the in vivo status concerning warfarin resistance, we recently developed a cell culture-based method for coexpression of VKORC1 with coagulation factor IX and subsequent measurement of secreted FIX in order to test warfarin inhibition in wild-type and mutated VKORC1. Results In the present study, we coexpressed wild-type factor IX with 12 different VKORC1 variants which were previously detected in warfarin resistant rats and mice. The results show that amino acid substitutions in VKORC1 maintain VKOR activity and are associated with warfarin resistance. When we projected in silico the amino acid substitutions onto the published three-dimensional model of the bacterial VKOR enzyme, the predicted effects matched well the catalytic mechanism proposed for the bacterial enzyme. Conclusions The established cell-based system for coexpression of VKORC1 and factor IX uses FIX activity as an indicator of carboxylation efficiency. This system reflects the warfarin resistance status of VKORC1 mutations from anticoagulant resistant rodents more closely than the traditional DTT-driven enzyme assay. All mutations studied were also predicted to be involved in the reaction mechanism. PMID:24491178

  3. Co-expression modules of NF1, PTEN and sprouty enable distinction of adult diffuse gliomas according to pathway activities of receptor tyrosine kinases

    PubMed Central

    Xue, Yang; Wu, Chenxing; Yao, Kun; Zhang, Chuanbao; Jin, Qiang; Huang, Rong; Li, Jiuyi; Sun, Yingyu; Su, Xiaodong; Jiang, Tao; Fan, Xiaolong

    2016-01-01

    Inter-individual variability causing elevated signaling of receptor tyrosine kinases (RTK) may have hampered the efficacy of targeted therapies. We developed a molecular signature for clustering adult diffuse gliomas based on the extent of RTK pathway activities. Glioma gene modules co-expressed with NF1 (NF1-M), Sprouty (SPRY-M) and PTEN (PTEN-M) were identified, their signatures enabled robust clustering of adult diffuse gliomas of WHO grades II-IV from five independent data sets into two subtypes with distinct activities of RAS-RAF-MEK-MAPK cascade and PI3K-AKT pathway (named RMPAhigh and RMPAlow subtypes) in a morphology-independent manner. The RMPAhigh gliomas were associated with poor prognosis compared to the RMPAlow gliomas. The RMPAhigh and RMPAlow glioma subtypes harbored unique sets of genomic alterations in the RTK signaling-related genes. The RMPAhigh gliomas were enriched in immature vessel cells and tumor associated macrophages, and both cell types expressed high levels of pro-angiogenic RTKs including MET, VEGFR1, KDR, EPHB4 and NRP1. In gliomas with major genomic lesions unrelated to RTK pathway, high RMPA signature was associated with short survival. Thus, the RMPA signatures capture RTK activities in both glioma cells and glioma microenvironment, and RTK signaling in the glioma microenvironment contributes to glioma progression. PMID:27385209

  4. Co-expression of alpha7 and beta2 nicotinic acetylcholine receptor subunit mRNAs within rat brain cholinergic neurons.

    PubMed

    Azam, L; Winzer-Serhan, U; Leslie, F M

    2003-01-01

    Nicotine enhances cognitive and attentional processes through stimulation of the basal forebrain cholinergic system. Although muscarinic cholinergic autoreceptors have been well characterized, pharmacological characterization of nicotinic autoreceptors has proven more difficult. The present study used double-labeling in situ hybridization to determine expression of nicotinic acetylcholine receptor (nAChR) subunit mRNAs within basal forebrain cholinergic neurons in order to gain information about possible nAChR autoreceptor properties. Cholinergic cells of the mesopontine tegmentum and striatal interneurons were also examined, as were septohippocampal GABAergic neurons that interact with cholinergic neurons to regulate hippocampal activity. alpha7 and beta2 nAChR mRNAs were found to be co-expressed in almost all cholinergic cells and in the majority of GABAergic neurons examined. alpha4 nAChR mRNA expression was restricted to cholinergic cells of the nucleus basalis magnocellularis, and to non-cholinergic cells of the medial septum and mesopontine tegmentum. These data suggest possible regional differences in the pharmacological properties of nicotinic autoreceptors on cholinergic cells. Whereas most cholinergic cells express rapidly desensitizing alpha7 homomers or alpha7beta2 heteromers, cortical projection neurons may also express a pharmacologically distinct alpha4beta2 nAChR subtype. There may also be differential nAChR regulation of cholinergic and non-cholinergic cells within the mesopontine tegmentum that are implicated in acquisition of nicotine self-administration.

  5. Coexpression Analysis Identifies Rice Starch Regulator1, a Rice AP2/EREBP Family Transcription Factor, as a Novel Rice Starch Biosynthesis Regulator1[W][OA

    PubMed Central

    Fu, Fang-Fang; Xue, Hong-Wei

    2010-01-01

    Starch biosynthesis is important for plant development and is a critical factor in crop quality and nutrition. As a complex metabolic pathway, the regulation of starch biosynthesis is still poorly understood. We here present the identification of candidate regulators for starch biosynthesis by gene coexpression analysis in rice (Oryza sativa). Starch synthesis genes can be grouped into type I (in seeds; sink tissues) and type II (in vegetative tissues; source tissues), and 307 and 621 coexpressed genes are putatively involved in the regulation of starch biosynthesis in rice seeds and vegetative tissues, respectively. Among these genes, Rice Starch Regulator1 (RSR1), an APETALA2/ethylene-responsive element binding protein family transcription factor, was found to negatively regulate the expression of type I starch synthesis genes, and RSR1 deficiency results in the enhanced expression of starch synthesis genes in seeds. Seeds of the knockout mutant rsr1 consistently show the increased amylose content and altered fine structure of amylopectin and consequently form the round and loosely packed starch granules, resulting in decreased gelatinization temperature. In addition, rsr1 mutants have a larger seed size and increased seed mass and yield. In contrast, RSR1 overexpression suppresses the expression of starch synthesis genes, resulting in altered amylopectin structure and increased gelatinization temperature. Interestingly, a decreased proportion of A chains in rsr1 results in abnormal starch granules but reduced gelatinization temperature, whereas an increased proportion of A chains in RSR1-overexpressing plants leads to higher gelatinization temperatures, which is novel and different from previous reports, further indicating the complicated regulation of starch synthesis and determination of the physicochemical properties of starch. These results demonstrate the potential of coexpression analysis for studying rice starch biosynthesis and the regulation of a

  6. Matrix factorization reveals aging-specific co-expression gene modules in the fat and muscle tissues in nonhuman primates

    PubMed Central

    Wang, Yongcui; Zhao, Weiling; Zhou, Xiaobo

    2016-01-01

    Accurate identification of coherent transcriptional modules (subnetworks) in adipose and muscle tissues is important for revealing the related mechanisms and co-regulated pathways involved in the development of aging-related diseases. Here, we proposed a systematically computational approach, called ICEGM, to Identify the Co-Expression Gene Modules through a novel mathematical framework of Higher-Order Generalized Singular Value Decomposition (HO-GSVD). ICEGM was applied on the adipose, and heart and skeletal muscle tissues in old and young female African green vervet monkeys. The genes associated with the development of inflammation, cardiovascular and skeletal disorder diseases, and cancer were revealed by the ICEGM. Meanwhile, genes in the ICEGM modules were also enriched in the adipocytes, smooth muscle cells, cardiac myocytes, and immune cells. Comprehensive disease annotation and canonical pathway analysis indicated that immune cells, adipocytes, cardiomyocytes, and smooth muscle cells played a synergistic role in cardiac and physical functions in the aged monkeys by regulation of the biological processes associated with metabolism, inflammation, and atherosclerosis. In conclusion, the ICEGM provides an efficiently systematic framework for decoding the co-expression gene modules in multiple tissues. Analysis of genes in the ICEGM module yielded important insights on the cooperative role of multiple tissues in the development of diseases. PMID:27703186

  7. Matrix factorization reveals aging-specific co-expression gene modules in the fat and muscle tissues in nonhuman primates

    NASA Astrophysics Data System (ADS)

    Wang, Yongcui; Zhao, Weiling; Zhou, Xiaobo

    2016-10-01

    Accurate identification of coherent transcriptional modules (subnetworks) in adipose and muscle tissues is important for revealing the related mechanisms and co-regulated pathways involved in the development of aging-related diseases. Here, we proposed a systematically computational approach, called ICEGM, to Identify the Co-Expression Gene Modules through a novel mathematical framework of Higher-Order Generalized Singular Value Decomposition (HO-GSVD). ICEGM was applied on the adipose, and heart and skeletal muscle tissues in old and young female African green vervet monkeys. The genes associated with the development of inflammation, cardiovascular and skeletal disorder diseases, and cancer were revealed by the ICEGM. Meanwhile, genes in the ICEGM modules were also enriched in the adipocytes, smooth muscle cells, cardiac myocytes, and immune cells. Comprehensive disease annotation and canonical pathway analysis indicated that immune cells, adipocytes, cardiomyocytes, and smooth muscle cells played a synergistic role in cardiac and physical functions in the aged monkeys by regulation of the biological processes associated with metabolism, inflammation, and atherosclerosis. In conclusion, the ICEGM provides an efficiently systematic framework for decoding the co-expression gene modules in multiple tissues. Analysis of genes in the ICEGM module yielded important insights on the cooperative role of multiple tissues in the development of diseases.

  8. Receptor-Ligand Requirements for Increased NK Cell Polyfunctional Potential in Slow Progressors Infected with HIV-1 Coexpressing KIR3DL1*h/*y and HLA-B*57▿†

    PubMed Central

    Kamya, Philomena; Boulet, Salix; Tsoukas, Christos M.; Routy, Jean-Pierre; Thomas, Réjean; Côté, Pierre; Boulassel, Mohamed-Rachid; Baril, Jean-Guy; Kovacs, Colin; Migueles, Stephen A.; Connors, Mark; Suscovich, Todd J.; Brander, Christian; Tremblay, Cecile L.; Bernard, Nicole

    2011-01-01

    Carriage of the natural killer (NK) receptor genotype KIR3DL1*h/*y with its HLA-B*57 ligand (*h/*y+B*57) is associated with slow time to AIDS and low viral load (VL). To provide a functional basis for these epidemiological observations, we assessed whether HIV-1-infected slow progressors (SP) carrying the *h/*y+B*57 compound genotype would have increased NK cell polyfunctional potential in comparison to SP with other killer immunoglobulin-like receptor (KIR)/HLA compound genotypes and whether this enhanced polyfunctionality was dependent upon the coexpression of both KIR3DL1*h/*y and HLA-B*57. The functional potential of NK cells was investigated by stimulating peripheral blood mononuclear cells with HLA-devoid targets or single HLA transfectants. Multiparametric flow cytometry was used to detect NK cells with seven functional profiles representing all permutations of CD107a expression and gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) secretion. NK cells from individuals carrying KIR3DL1 receptor–HLA-Bw4 ligand pairs had greater trifunctional responses than those from KIR3DL1 homozygotes (hmz), who were Bw6 homozygotes. NK cells from subjects carrying the *h/*y+B*57 genotypes exhibited the highest trifunctional potential, and this was dependent on cocarriage of the NK receptor and its ligand. Trifunctional cells secreted more of each function tested on a per-cell basis than each corresponding monofunctional NK subset. Although VL influenced NK functionality, individuals with defined KIR/HLA genotypes exhibited differences in NK cell polyfunctionality that could not be accounted for by VL alone. The protective effect of HLA-B*57 on slow progression to AIDS and low VL may be mediated through its interaction with KIR3DL1 alleles to educate NK cells for potent activity upon stimulation. PMID:21471235

  9. Upregulation of epidermal growth factor receptor 4 in oral leukoplakia

    PubMed Central

    Kobayashi, Hiroshi; Kumagai, Kenichi; Gotoh, Akito; Eguchi, Takanori; Yamada, Hiroyuki; Hamada, Yoshiki; Suzuki, Satsuki; Suzuki, Ryuji

    2013-01-01

    In the present study, we investigate the expression profile of the epidermal growth factor receptor family, which comprises EGFR/ErbB1, HER2/ErbB2, HER3/ErbB3 and HER4/ErbB4 in oral leukoplakia (LP). The expression of four epidermal growth factor receptor (EGFR) family genes and their ligands were measured in LP tissues from 14 patients and compared with levels in 10 patients with oral lichen planus (OLP) and normal oral mucosa (NOM) from 14 healthy donors by real-time polymerase chain reaction (PCR) and immunohistochemistry. Synchronous mRNA coexpression of ErbB1, ErbB2, ErbB3 and ErbB4 was detected in LP lesions. Out of the receptors, only ErbB4 mRNA and protein was more highly expressed in LP compared with NOM tissues. These were strongly expressed by epithelial keratinocytes in LP lesions, as shown by immunohistochemistry. Regarding the ligands, the mRNA of Neuregulin2 and 4 were more highly expressed in OLP compared with NOM tissues. Therefore, enhanced ErbB4 on the keratinocytes and synchronous modulation of EGFR family genes may contribute to the pathogenesis and carcinogenesis of LP. PMID:23492901

  10. TF-Cluster: A pipeline for identifying functionally coordinated transcription factors via network decomposition of the shared coexpression connectivity matrix (SCCM)

    PubMed Central

    2011-01-01

    Background Identifying the key transcription factors (TFs) controlling a biological process is the first step toward a better understanding of underpinning regulatory mechanisms. However, due to the involvement of a large number of genes and complex interactions in gene regulatory networks, identifying TFs involved in a biological process remains particularly difficult. The challenges include: (1) Most eukaryotic genomes encode thousands of TFs, which are organized in gene families of various sizes and in many cases with poor sequence conservation, making it difficult to recognize TFs for a biological process; (2) Transcription usually involves several hundred genes that generate a combination of intrinsic noise from upstream signaling networks and lead to fluctuations in transcription; (3) A TF can function in different cell types or developmental stages. Currently, the methods available for identifying TFs involved in biological processes are still very scarce, and the development of novel, more powerful methods is desperately needed. Results We developed a computational pipeline called TF-Cluster for identifying functionally coordinated TFs in two steps: (1) Construction of a shared coexpression connectivity matrix (SCCM), in which each entry represents the number of shared coexpressed genes between two TFs. This sparse and symmetric matrix embodies a new concept of coexpression networks in which genes are associated in the context of other shared coexpressed genes; (2) Decomposition of the SCCM using a novel heuristic algorithm termed "Triple-Link", which searches the highest connectivity in the SCCM, and then uses two connected TF as a primer for growing a TF cluster with a number of linking criteria. We applied TF-Cluster to microarray data from human stem cells and Arabidopsis roots, and then demonstrated that many of the resulting TF clusters contain functionally coordinated TFs that, based on existing literature, accurately represent a biological process

  11. Clinical benefit of lapatinib-based therapy in patients with HER2-positive breast tumors co-expressing the truncated p95HER2 receptor

    PubMed Central

    Scaltriti, Maurizio; Chandarlapaty, Sarat; Prudkin, Ludmila; Aura, Claudia; Jimenez, José; Angelini, Pier Davide; Sánchez, Gertrudis; Guzman, Marta; Parra, Josep Lluis; Ellis, Catherine; Gagnon, Robert; Koehler, Maria; Gomez, Henry; Geyer, Charles; Cameron, David; Arribas, Joaquin; Rosen, Neal; Baselga, José

    2011-01-01

    Purpose A subgroup of HER2 overexpressing breast tumors co-expresses p95HER2, a truncated HER2 receptor that retains a highly functional HER2 kinase domain but lacks the extracellular domain and results in intrinsic trastuzumab resistance. We hypothesized that lapatinib, a HER2 tyrosine kinase inhibitor, would be active in these tumors. We have studied the correlation between p95HER2 expression and response to lapatinib, both in preclinical models and in the clinical setting Experimental design Two different p95HER2 animal models were used for preclinical studies. Expression of p95HER2 was analyzed in HER2 overexpressing breast primary tumors from a first line lapatinib monotherapy study (EGF20009) and a second line lapatinib in combination with capecitabine study (EGF100151). p95HER2 expression was correlated with overall response rate (complete + partial response), clinical benefit rate (complete response + partial response + stable disease ≥ 24 weeks) and progression-free survival using logistic regression and Cox-proportional hazard models. Results Lapatinib inhibited tumor growth and HER2 downstream signaling of p95HER2 expressing tumors. A total of 68 and 156 tumors from studies EGF20009 and EGF100151 were evaluable, respectively, for p95HER2 detection. The percentage of p95HER2 positive patients was 20.5% in the EGF20009 study and 28.5% in the EGF100151 study. In both studies there was no statistically significant difference in progression-free survival, clinical benefit rate and overall response rate between p95HER2-positive and p95HER2-negative tumors. Conclusions Lapatinib as a monotherapy or in combination with capecitabine appears to be equally effective in patients with p95HER2-positive and p95HER2-negative HER2-positive breast tumors. PMID:20406840

  12. Connections between EM2-containing terminals and GABA/μ-opioid receptor co-expressing neurons in the rat spinal trigeminal caudal nucleus

    PubMed Central

    Li, Meng-Ying; Wu, Zhen-Yu; Lu, Ya-Cheng; Yin, Jun-Bin; Wang, Jian; Zhang, Ting; Dong, Yu-Lin; Wang, Feng

    2014-01-01

    Endomorphin-2 (EM2) demonstrates a potent antinociceptive effect via the μ-opioid receptor (MOR). To provide morphological evidence for the pain control effect of EM2, the synaptic connections between EM2-immunoreactive (IR) axonal terminals and γ-amino butyric acid (GABA)/MOR co-expressing neurons in lamina II of the spinal trigeminal caudal nucleus (Vc) were investigated in the rat. Dense EM2-, MOR- and GABA-IR fibers and terminals were mainly observed in lamina II of the Vc. Within lamina II, GABA- and MOR-neuronal cell bodies were also encountered. The results of immunofluorescent histochemical triple-staining showed that approximately 14.2 or 18.9% of GABA-IR or MOR-IR neurons also showed MOR- or GABA-immunopositive staining in lamina II; approximately 45.2 and 36.1% of the GABA-IR and MOR-IR neurons, respectively, expressed FOS protein in their nuclei induced by injecting formalin into the left lower lip of the mouth. Most of the GABA/MOR, GABA/FOS, and MOR/FOS double-labeled neurons made close contacts with EM2-IR fibers and terminals. Immuno-electron microscopy confirmed that the EM2-IR terminals formed synapses with GABA-IR or MOR-IR dendritic processes and neuronal cell bodies in lamina II of the Vc. These results suggest that EM2 might participate in pain transmission and modulation by binding to MOR-IR and GABAergic inhibitory interneuron in lamina II of the Vc to exert inhibitory effect on the excitatory interneuron in lamina II and projection neurons in laminae I and III. PMID:25386121

  13. Adverse effect on syngeneic islet transplantation by transgenic coexpression of decoy receptor 3 and heme oxygenase-1 in the islet of NOD mice.

    PubMed

    Huang, S-H; Lin, G-J; Chien, M-W; Chu, C-H; Yu, J-C; Chen, T-W; Hueng, D-Y; Liu, Y-L; Sytwu, H-K

    2013-03-01

    Decoy receptor 3 (DcR3) blocks both Fas ligand- and LIGHT-induced pancreatic β-cell damage in autoimmune diabetes. Heme oxygenase 1 (HO-1) possesses antiapoptotic, anti-inflammatory, and antioxidative effects that protect cells against various forms of attack by the immune system. Previously, we have demonstrated that transgenic islets overexpressing DcR3 or murine HO-1 (mHO-1) exhibit longer survival times than nontransgenic islets in syngeneic islet transplantation. In this study, we evaluated whether DcR3 and mHO-1 double-transgenic islets of NOD mice could provide better protective effects and achieve longer islet graft survival than DcR3 or mHO-1 single-transgenic islets after islet transplantation. We generated DcR3 and mHO-1 double-transgenic NOD mice that specifically overexpress DcR3 and HO-1 in islets. Seven hundred islets isolated from double-transgenic, single-transgenic, or nontransgenic NOD mice were syngeneically transplanted into the kidney capsules of newly diabetic female recipients. Unexpectedly, there was no significant difference in the survival time between double-transgenic or nontransgenic NOD islet grafts, and the survival times of double-transgenic NOD islet grafts were even shorter than those of DcR3 or mHO-1 single-transgenic islets. Our data indicate that transplantation of double-transgenic islets that coexpress HO-1 and DcR3 did not result in a better outcome. On the contrary, this strategy even caused an adverse effect in syngeneic islet transplantation.

  14. Evidence for epidermal growth factor (EGF)-induced intermolecular autophosphorylation of the EGF receptors in living cells.

    PubMed Central

    Honegger, A M; Schmidt, A; Ullrich, A; Schlessinger, J

    1990-01-01

    In response to epidermal growth factor (EGF) stimulation, the intrinsic protein tyrosine kinase of EGF receptor is activated, leading to tyrosine phosphorylation of several cellular substrate proteins, including the EGF receptor molecule itself. To test the mechanism of EGF receptor autophosphorylation in living cells, we established transfected cell lines coexpressing a kinase-negative point mutant of EGF receptor (K721A) with an active EGF receptor mutant lacking 63 amino acids from its carboxy terminus. The addition of EGF to these cells caused tyrosine phosphorylation of the kinase-negative mutant by the active receptor molecule, demonstrating EGF receptor cross-phosphorylation in living cells. After internalization the kinase-negative mutant and CD63 have separate trafficking pathways. This limits their association and the extent of cross-phosphorylation of K721A by CD63. The coexpression of the kinase-negative mutant together with active EGF receptors in the same cells suppressed the mitogenic response toward EGF as compared with that in cells that express active receptors alone. The presence of the kinase-negative mutant functions as a negative dominant mutation suppressing the response of active EGF receptors, probably by interfering with EGF-induced signal transduction. It appears, therefore, that crucial events of signal transduction occur before K721A and active EGF receptors are separated by their different endocytic itineraries. Images PMID:2164634

  15. Altered metabolite accumulation in tomato fruits by coexpressing a feedback-insensitive AroG and the PhODO1 MYB-type transcription factor.

    PubMed

    Xie, Qingjun; Liu, Zhongyuan; Meir, Sagit; Rogachev, Ilana; Aharoni, Asaph; Klee, Harry J; Galili, Gad

    2016-12-01

    Targeted manipulation of phenylalanine (Phe) synthesis is a potentially powerful strategy to boost biologically and economically important metabolites, including phenylpropanoids, aromatic volatiles and other specialized plant metabolites. Here, we use two transgenes to significantly increase the levels of aromatic amino acids, tomato flavour-associated volatiles and antioxidant phenylpropanoids. Overexpression of the petunia MYB transcript factor, ODORANT1 (ODO1), combined with expression of a feedback-insensitive E. coli 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (AroG), altered the levels of multiple primary and secondary metabolites in tomato fruit, boosting levels of multiple secondary metabolites. Our results indicate that coexpression of AroG and ODO1 has a dual effect on Phe and related biosynthetic pathways: (i) positively impacting tyrosine (Tyr) and antioxidant related metabolites, including ones derived from coumaric acid and ferulic acid; (ii) negatively impacting other downstream secondary metabolites of the Phe pathway, including kaempferol-, naringenin- and quercetin-derived metabolites, as well as aromatic volatiles. The metabolite profiles were distinct from those obtained with either single transgene. In addition to providing fruits that are increased in flavour and nutritional chemicals, coexpression of the two genes provides insights into regulation of branches of phenylpropanoid metabolic pathways.

  16. Diversification of Neu differentiation factor and epidermal growth factor signaling by combinatorial receptor interactions.

    PubMed Central

    Pinkas-Kramarski, R; Soussan, L; Waterman, H; Levkowitz, G; Alroy, I; Klapper, L; Lavi, S; Seger, R; Ratzkin, B J; Sela, M; Yarden, Y

    1996-01-01

    The ErbB family includes two receptors, ErbB-1 and ErbB-3, that respectively bind to epidermal growth factor and Neu differentiation factor, and an orphan receptor, ErbB-2. Unlike ErbB-1 and ErbB-2, the intrinsic tyrosine kinase of ErbB-3 is catalytically impaired. By using interleukin-3-dependent cells that ectopically express the three ErbB proteins or their combinations, we found that ErbB-3 is devoid of any biological activity but both ErbB-1 and ErbB-2 can reconstitute its extremely potent mitogenic activity. Transactivation of ErbB-3 correlates with heterodimer formation and is reflected in receptor phosphorylation and the transregulation of ligand affinity. Inter-receptor interactions enable graded proliferative and survival signals: heterodimers are more potent than homodimers, and ErbB-3-containing complexes, especially the ErbB-2/ErbB-3 heterodimer, are more active than ErbB-1 complexes. Nevertheless, ErbB-1 signaling displays dominance over ErbB-3 when the two receptors are coexpressed. Although all receptor combinations activate the mitogen-activated protein kinases ERK and c-Jun kinase, they differ in their rate of endocytosis and in coupling to intervening signaling proteins. It is conceivable that combinatorial receptor interactions diversify signal transduction and confer double regulation, in cis and in trans, of the superior mitogenic activity of the kinase-defective ErbB-3. Images PMID:8665853

  17. Co-expression of ovine LPS receptor CD14 with Mannheimia haemolytica leukotoxin receptor LFA-1 or Mac-1 does not enhance leukotoxin-induced cytotoxicity

    USDA-ARS?s Scientific Manuscript database

    Leukotoxin (Lkt) and LPS are the major virulence determinants of Mannheimia haemolytica that contribute to the pathogenesis of bovine and ovine pneumonic pasteurellosis. We have previously identified bovine and ovine CD18 as the functional receptor for Lkt. LPS complexes with Lkt resulting in incre...

  18. Differential Coexpression Analysis Reveals Extensive Rewiring of Arabidopsis Gene Coexpression in Response to Pseudomonas syringae Infection

    PubMed Central

    Jiang, Zhenhong; Dong, Xiaobao; Li, Zhi-Gang; He, Fei; Zhang, Ziding

    2016-01-01

    Plant defense responses to pathogens involve massive transcriptional reprogramming. Recently, differential coexpression analysis has been developed to study the rewiring of gene networks through microarray data, which is becoming an important complement to traditional differential expression analysis. Using time-series microarray data of Arabidopsis thaliana infected with Pseudomonas syringae, we analyzed Arabidopsis defense responses to P. syringae through differential coexpression analysis. Overall, we found that differential coexpression was a common phenomenon of plant immunity. Genes that were frequently involved in differential coexpression tend to be related to plant immune responses. Importantly, many of those genes have similar average expression levels between normal plant growth and pathogen infection but have different coexpression partners. By integrating the Arabidopsis regulatory network into our analysis, we identified several transcription factors that may be regulators of differential coexpression during plant immune responses. We also observed extensive differential coexpression between genes within the same metabolic pathways. Several metabolic pathways, such as photosynthesis light reactions, exhibited significant changes in expression correlation between normal growth and pathogen infection. Taken together, differential coexpression analysis provides a new strategy for analyzing transcriptional data related to plant defense responses and new insights into the understanding of plant-pathogen interactions. PMID:27721457

  19. Differential Coexpression Analysis Reveals Extensive Rewiring of Arabidopsis Gene Coexpression in Response to Pseudomonas syringae Infection.

    PubMed

    Jiang, Zhenhong; Dong, Xiaobao; Li, Zhi-Gang; He, Fei; Zhang, Ziding

    2016-10-10

    Plant defense responses to pathogens involve massive transcriptional reprogramming. Recently, differential coexpression analysis has been developed to study the rewiring of gene networks through microarray data, which is becoming an important complement to traditional differential expression analysis. Using time-series microarray data of Arabidopsis thaliana infected with Pseudomonas syringae, we analyzed Arabidopsis defense responses to P. syringae through differential coexpression analysis. Overall, we found that differential coexpression was a common phenomenon of plant immunity. Genes that were frequently involved in differential coexpression tend to be related to plant immune responses. Importantly, many of those genes have similar average expression levels between normal plant growth and pathogen infection but have different coexpression partners. By integrating the Arabidopsis regulatory network into our analysis, we identified several transcription factors that may be regulators of differential coexpression during plant immune responses. We also observed extensive differential coexpression between genes within the same metabolic pathways. Several metabolic pathways, such as photosynthesis light reactions, exhibited significant changes in expression correlation between normal growth and pathogen infection. Taken together, differential coexpression analysis provides a new strategy for analyzing transcriptional data related to plant defense responses and new insights into the understanding of plant-pathogen interactions.

  20. Co-expression of the protease furin in Nicotiana benthamiana leads to efficient processing of latent transforming growth factor-β1 into a biologically active protein.

    PubMed

    Wilbers, Ruud H P; Westerhof, Lotte B; van Raaij, Debbie R; van Adrichem, Marloes; Prakasa, Andreas D; Lozano-Torres, Jose L; Bakker, Jaap; Smant, Geert; Schots, Arjen

    2016-08-01

    Transforming growth factor beta (TGF-β) is a signalling molecule that plays a key role in developmental and immunological processes in mammals. Three TGF-β isoforms exist in humans, and each isoform has unique therapeutic potential. Plants offer a platform for the production of recombinant proteins, which is cheap and easy to scale up and has a low risk of contamination with human pathogens. TGF-β3 has been produced in plants before using a chloroplast expression system. However, this strategy requires chemical refolding to obtain a biologically active protein. In this study, we investigated the possibility to transiently express active human TGF-β1 in Nicotiana benthamiana plants. We successfully expressed mature TGF-β1 in the absence of the latency-associated peptide (LAP) using different strategies, but the obtained proteins were inactive. Upon expression of LAP-TGF-β1, we were able to show that processing of the latent complex by a furin-like protease does not occur in planta. The use of a chitinase signal peptide enhanced the expression and secretion of LAP-TGF-β1, and co-expression of human furin enabled the proteolytic processing of latent TGF-β1. Engineering the plant post-translational machinery by co-expressing human furin also enhanced the accumulation of biologically active TGF-β1. This engineering step is quite remarkable, as furin requires multiple processing steps and correct localization within the secretory pathway to become active. Our data demonstrate that plants can be a suitable platform for the production of complex proteins that rely on specific proteolytic processing. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  1. Intestinal trefoil factor (TFF 3) and pS2 (TFF 1), but not spasmolytic polypeptide (TFF 2) mRNAs are co-expressed in normal, hyperplastic, and neoplastic human breast epithelium.

    PubMed

    Poulsom, R; Hanby, A M; Lalani, E N; Hauser, F; Hoffmann, W; Stamp, G W

    1997-09-01

    pS2-TFF 1 is expressed in breast cancers and has been investigated as a potential prognostic factor reflecting oestrogen dependence. The relationship to the expression of other trefoil peptides, human spasmolytic polypeptide (hSP-TFF 2) and intestinal trefoil factor (hITF/hPI.B-TFF 3) is documented here. Fifty-seven breast specimens were selected from surgical pathology archives and included five normal breasts (two lactating), seven benign proliferative lesions, 11 ductal carcinomas in situ (DCIS), three lobular carcinomas in situ (LCIS), 24 invasive ductal carcinomas (IDC), and seven invasive lobular carcinomas (ILC). The comparative distribution of trefoil mRNAs was assessed by in situ hybridization using 35S-labelled riboprobes and immunohistochemical staining for pS2-TFF 1 and hSP-TFF 2. pS2-TFF 1 and hITF/hPI.B-TFF 3 mRNA were focally present at low signal intensity in normal and benign breast. Both pS2-TFF 1 and hITF/hPI.B-TFF 3 were expressed in all DCIS, LCIS and ILC, and 21/24 IDC. Overall, expression patterns of pS2-TFF 1 and hITF/hPI.B-TFF 3 coincided, but hITF/hPI.B-TFF 3 mRNA was usually found in a greater proportion of cells. Expression of hSP-TFF 2 peptide or mRNA was not detected in any of these cases. MCF 7 breast carcinoma cells also expressed hITF/hPI.B-TFF 3 and pS2-TFF 1 mRNAs but not hSP-TFF 2. hITF/hPI.B-TFF 3 co-expression with pS2-TFF 1 may act as a prognostic factor, but also raises questions about the regulatory pathway for pS2-TFF 1 hITF/hPI.B-TFF 3. Trefoil factors have effects on cell motility and spreading in vitro, and co-expression of hITF/hPI.B-TFF 3 with pS2-TFF 1 could be functionally significant if they form a heterodimer or compete for receptor binding. Absence of hSP-TFF 2 expression may be of equal relevance to tumour cell biology.

  2. Interaction specificity and coexpression of rice NPR1 homologs 1 and 3 (NH1 and NH3), TGA transcription factors and Negative Regulator of Resistance (NRR) proteins.

    PubMed

    Chern, Mawsheng; Bai, Wei; Ruan, Deling; Oh, Taeyun; Chen, Xuewei; Ronald, Pamela C

    2014-06-11

    The nonexpressor of pathogenesis-related genes 1, NPR1 (also known as NIM1 and SAI1), is a key regulator of SA-mediated systemic acquired resistance (SAR) in Arabidopsis. In rice, the NPR1 homolog 1 (NH1) interacts with TGA transcriptional regulators and the Negative Regulator of Resistance (NRR) protein to modulate the SAR response. Though five NPR1 homologs (NHs) have been identified in rice, only NH1 and NH3 enhance immunity when overexpressed. To understand why NH1 and NH3, but not NH2, NH4, or NH5, contribute to the rice immune response, we screened TGA transcription factors and NRR-like proteins for interactions specific to NH1 and NH3. We also examined their co-expression patterns using publicly available microarray data. We tested five NHs, four NRR homologs (RHs), and 13 rice TGA proteins for pair-wise protein interactions using yeast two-hybrid (Y2H) and split YFP assays. A survey of 331 inter-family interactions revealed a broad, complex protein interaction network. To investigate preferred interaction partners when all three families of proteins were present, we performed a bridged split YFP assay employing YFPN-fused TGA, YFPC-fused RH, and NH proteins without YFP fusions. We found 64 tertiary interactions mediated by NH family members among the 120 sets we examined. In the yeast two-hybrid assay, each NH protein was capable of interacting with most TGA and RH proteins. In the split YFP assay, NH1 was the most prevalent interactor of TGA and RH proteins, NH3 ranked the second, and NH4 ranked the third. Based on their interaction with TGA proteins, NH proteins can be divided into two subfamilies: NH1, NH2, and NH3 in one family and NH4 and NH5 in the other.In addition to evidence of overlap in interaction partners, co-expression analyses of microarray data suggest a correlation between NH1 and NH3 expression patterns, supporting their common role in rice immunity. However, NH3 is very tightly co-expressed with RH1 and RH2, while NH1 is strongly

  3. Neurotrophic Factor Receptor Expression and in vitro Nerve Growth of Geniculate Ganglion Neurons That Supply Divergent Nerves

    PubMed Central

    Yamout, Adam; Spec, Andrej; Cosmano, Jason; Kashyap, Manoj; Rochlin, M. William

    2016-01-01

    We investigated which neurotrophic factors may contribute to the divergence of two peripheral nerves emanating from the geniculate ganglion. We compared receptor mRNA profiles of the neurons that supply the nerves, and also the growth of their neurites in response to neurotrophic factors in culture. Three mRNAs, Gfra2, TrkA, and TrkC, were differentially expressed. Only one ligand, Neurturin, promoted substantially different nerve regrowth from the nerves, and therefore may contribute to nerve divergence. Three receptor mRNAs were expressed in 100% of the neurons: TrkB, TrkB.T2 (kinaselacking isoform), and NCAM-140. Ligands for these Trks and FRα-1 promoted more outgrowth than ligands for the other receptors. NT-3 and BDNF synergistically promoted outgrowth. Finally, receptors are coexpressed at random rates, arguing against the existence of neuronal subtypes defined by a combinatorial code of these receptors. PMID:16137986

  4. Galanin is Co-Expressed with Substance P, Calbindin and Corticotropin-Releasing Factor (CRF) in The Enteric Nervous System of the Wild Boar (Sus scrofa) Small Intestine.

    PubMed

    Czujkowska, A; Arciszewski, M B

    2016-04-01

    Galanin is a neuropeptide widely present in the enteric nervous system of numerous animal species and exhibiting neurotransmittery/neuromodulatory roles. Colocalization patterns of galanin with substance P (SP), corticotropin-releasing factor (CRF) and calbindin were studied in the small intestine of the wild boar using immunofluorescence technique. We demonstrated the presence of SP in substantial populations of galanin-immunoreactive (IR) submucous neurons. Additionally, different amounts of nerve fibres exhibiting simultaneous presence of galanin and SP were noted in the small intestinal smooth musculature, submucous ganglia, lamina muscularis mucosae and mucosa. In the wild boar duodenum, jejunum and ileum, the co-expression of galanin and calbindin was limited to minor populations of submucous neurons only. Single galanin-/CRF-IR nerve fibres were exclusively present in the duodenal and jejunal (but not ileal) mucosa. These results strongly suggest that galanin participates in neuronal control of the wild boar small intestine also by functional co-operation with other biologically active neuropeptides.

  5. Analysis of transient receptor potential ankyrin 1 (TRPA1) in frogs and lizards illuminates both nociceptive heat and chemical sensitivities and coexpression with TRP vanilloid 1 (TRPV1) in ancestral vertebrates.

    PubMed

    Saito, Shigeru; Nakatsuka, Kazumasa; Takahashi, Kenji; Fukuta, Naomi; Imagawa, Toshiaki; Ohta, Toshio; Tominaga, Makoto

    2012-08-31

    Transient receptor potential ankyrin 1 (TRPA1) and TRP vanilloid 1 (V1) perceive noxious temperatures and chemical stimuli and are involved in pain sensation in mammals. Thus, these two channels provide a model for understanding how different genes with similar biological roles may influence the function of one another during the course of evolution. However, the temperature sensitivity of TRPA1 in ancestral vertebrates and its evolutionary path are unknown as its temperature sensitivities vary among different vertebrate species. To elucidate the functional evolution of TRPA1, TRPA1s of the western clawed (WC) frogs and green anole lizards were characterized. WC frog TRPA1 was activated by heat and noxious chemicals that activate mammalian TRPA1. These stimuli also activated native sensory neurons and elicited nocifensive behaviors in WC frogs. Similar to mammals, TRPA1 was functionally co-expressed with TRPV1, another heat- and chemical-sensitive nociceptive receptor, in native sensory neurons of the WC frog. Green anole TRPA1 was also activated by heat and noxious chemical stimulation. These results suggest that TRPA1 was likely a noxious heat and chemical receptor and co-expressed with TRPV1 in the nociceptive sensory neurons of ancestral vertebrates. Conservation of TRPV1 heat sensitivity throughout vertebrate evolution could have changed functional constraints on TRPA1 and influenced the functional evolution of TRPA1 regarding temperature sensitivity, whereas conserving its noxious chemical sensitivity. In addition, our results also demonstrated that two mammalian TRPA1 inhibitors elicited different effect on the TRPA1s of WC frogs and green anoles, which can be utilized to clarify the structural bases for inhibition of TRPA1.

  6. Analysis of Transient Receptor Potential Ankyrin 1 (TRPA1) in Frogs and Lizards Illuminates Both Nociceptive Heat and Chemical Sensitivities and Coexpression with TRP Vanilloid 1 (TRPV1) in Ancestral Vertebrates*

    PubMed Central

    Saito, Shigeru; Nakatsuka, Kazumasa; Takahashi, Kenji; Fukuta, Naomi; Imagawa, Toshiaki; Ohta, Toshio; Tominaga, Makoto

    2012-01-01

    Transient receptor potential ankyrin 1 (TRPA1) and TRP vanilloid 1 (V1) perceive noxious temperatures and chemical stimuli and are involved in pain sensation in mammals. Thus, these two channels provide a model for understanding how different genes with similar biological roles may influence the function of one another during the course of evolution. However, the temperature sensitivity of TRPA1 in ancestral vertebrates and its evolutionary path are unknown as its temperature sensitivities vary among different vertebrate species. To elucidate the functional evolution of TRPA1, TRPA1s of the western clawed (WC) frogs and green anole lizards were characterized. WC frog TRPA1 was activated by heat and noxious chemicals that activate mammalian TRPA1. These stimuli also activated native sensory neurons and elicited nocifensive behaviors in WC frogs. Similar to mammals, TRPA1 was functionally co-expressed with TRPV1, another heat- and chemical-sensitive nociceptive receptor, in native sensory neurons of the WC frog. Green anole TRPA1 was also activated by heat and noxious chemical stimulation. These results suggest that TRPA1 was likely a noxious heat and chemical receptor and co-expressed with TRPV1 in the nociceptive sensory neurons of ancestral vertebrates. Conservation of TRPV1 heat sensitivity throughout vertebrate evolution could have changed functional constraints on TRPA1 and influenced the functional evolution of TRPA1 regarding temperature sensitivity, whereas conserving its noxious chemical sensitivity. In addition, our results also demonstrated that two mammalian TRPA1 inhibitors elicited different effect on the TRPA1s of WC frogs and green anoles, which can be utilized to clarify the structural bases for inhibition of TRPA1. PMID:22791718

  7. Nerve growth factor regulates the expression of bradykinin binding sites on adult sensory neurons via the neurotrophin receptor p75.

    PubMed

    Petersen, M; Segond von Banchet, G; Heppelmann, B; Koltzenburg, M

    1998-03-01

    Neurotrophins mediate specific effects on sensory neurons through tyrosine kinase receptors. Most of these neurons also co-express the neurotrophin receptor p75 (p75NTR), but its function has remained obscure. We now show that nerve growth factor but not brain-derived neurotrophic factor or neurotrophin-3 selectively increases the expression of bradykinin binding sites on cultured dorsal root ganglion neurons from adult mouse via p75NTR. This up-regulation of bradykinin binding sites did not occur in neurons from mice lacking p75NTR or in neurons from wild-type mice treated with p75NTR-blocking antibody, indicating that tyrosine kinase receptors alone are not sufficient to trigger this physiological neuronal response. Thus, the interaction of nerve growth factor with p75NTR is an important factor contributing to chronic pain conditions.

  8. PDGF-alpha receptor and myelin basic protein mRNAs are not coexpressed by oligodendrocytes in vivo: a double in situ hybridization study in the anterior medullary velum of the neonatal rat.

    PubMed

    Butt, A M; Hornby, M F; Ibrahim, M; Kirvell, S; Graham, A; Berry, M

    1997-01-01

    Platelet-derived growth factor (PDGF) is a growth-regulatory dimer with A and B subunits. PDGF-AA, acting via PDGF receptors of the alpha-unit subtype (PDGF-alphaR), is implicated in the differentiation of oligodendrocyte precursors and in the survival of newly formed oligodendrocytes, which gradually lose expression of PDGF-alphaR. However, it is unclear whether terminally differentiated oligodendrocytes express PDGF-alphaR in vivo. To address this question, and to help clarify the role of PDGF-AA in late oligodendrocyte differentiation, we have used double in situ hybridization with digoxigenin- and fluorescein-labeled riboprobes to relate PDGF-alphaR mRNA and myelin basic protein (MBP) mRNA expression in the isolated intact anterior medullary velum (AMV) of rats ages Postnatal Day (P) 10-12 and P30-32. In parallel experiments, AMV were immunolabeled with the oligodendrocyte-specific monoclonal antibody Rip to provide information on oligodendrocyte development and the extent of myelination. At P10, the AMV contained tracts in which axons ranged from unmyelinated to fully myelinated, whereas myelination was complete in P30-32 AMV. The first oligodendrocytes to express MBP mRNA or Rip were promyelinating oligodendrocytes, which had a "star-burst" morphology and had not yet begun to form myelin sheaths. As myelination proceeded, MBP mRNA became dispersed throughout oligodendrocyte units, comprising cell somata, processes, and internodal myelin sheaths. By P30-32, MBP mRNA had been redistributed to the myelin sheaths only, reflecting a change in the site of protein synthesis in mature myelinated axon tracts. At no stage of oligodendrocyte differentiation did we observe cellular coexpression of mRNA for PDGFalphaR and MBP. Our results indicated that oligodendrocytes lost the expression of PDGFalphaR prior to gaining that of myelin gene products, and preclude an action of PDGF-AA on Rip+/MBP+ star-burst promyelinating oligodendrocytes. The spatial and temporal

  9. Tissue factor- and factor X-dependent activation of protease-activated receptor 2 by factor VIIa

    PubMed Central

    Camerer, Eric; Huang, Wei; Coughlin, Shaun R.

    2000-01-01

    Protease-activated receptor 2 (PAR2) is expressed by vascular endothelial cells and other cells in which its function and physiological activator(s) are unknown. Unlike PAR1, PAR3, and PAR4, PAR2 is not activatable by thrombin. Coagulation factors VIIa (FVIIa) and Xa (FXa) are proteases that act upstream of thrombin in the coagulation cascade and require cofactors to interact with their substrates. These proteases elicit cellular responses, but their receptor(s) have not been identified. We asked whether FVIIa and FXa might activate PARs if presented by their cofactors. Co-expression of tissue factor (TF), the cellular cofactor for FVIIa, together with PAR1, PAR2, PAR3, or PAR4 conferred TF-dependent FVIIa activation of PAR2 and, to lesser degree, PAR1. Responses to FXa were also observed but were independent of exogenous cofactor. The TF/FVIIa complex converts the inactive zymogen Factor X (FX) to FXa. Strikingly, when FX was present, low picomolar concentrations of FVIIa caused robust signaling in cells expressing TF and PAR2. Responses in keratinocytes and cytokine-treated endothelial cells suggested that PAR2 may be activated directly by TF/FVIIa and indirectly by TF/FVIIa-generated FXa at naturally occurring expression levels of TF and PAR2. These results suggest that PAR2, although not activatable by thrombin, may nonetheless function as a sensor for coagulation proteases and contribute to endothelial activation in the setting of injury and inflammation. More generally, these findings highlight the potential importance of cofactors in regulating PAR function and specificity. PMID:10805786

  10. “Related to ABA-Insensitive3(ABI3)/Viviparous1 and AtABI5 transcription factor co-expression in cotton enhances drought stress adaptation”

    PubMed Central

    Mittal, Amandeep; Gampala, Srinivas S. L.; Ritchie, Glen L.; Payton, Paxton; Burke, John J.; Rock, Christopher D.

    2014-01-01

    Drought tolerance is an important trait being pursued by the agbiotech industry. Abscisic acid (ABA) is a stress hormone that mediates a multitude of processes in growth and development, water use efficiency (WUE), and gene expression during seed development and in response to environmental stresses. Arabidopsis B3-domain transcription factor Related to ABA-Insensitive3 (ABI3)/Viviparous1 (namely, AtRAV2) and basic leucine zipper (bZIPs) AtABI5 or AtABF3 transactivated ABA- inducible promoter: GUS reporter expression in a maize mesophyll protoplast transient assay and showed synergies in reporter transactivation when co-expressed. Transgenic cotton (Gossypium hirsutum) expressing AtRAV1/2 and/or AtABI5 showed resistance to imposed drought stress under field and greenhouse conditions and exhibited improved photosynthetic and WUEs associated with absorption through larger root system and greater leaf area. We observed synergy for root biomass accumulation in the greenhouse, intrinsic WUE in the field, and drought tolerance in stacked AtRAV and AtABI5 double-transgenic cotton. We assessed AtABI5 and AtRAV1/2 involvement in drought stress adaptations though reactive oxygen species scavenging and osmotic adjustment by marker gene expression in cotton. Deficit irrigation-grown AtRAV1/2 and AtABI5 transgenics had “less stressed” molecular and physiological phenotypes under drought, likely due to improved photoassimilation and root and shoot sink strengths and enhanced expression of endogenous GhRAV and genes for antioxidant and osmolyte biosynthesis. Over-expression of bZIP and RAV TFs could impact sustainable cotton agriculture and potentially other crops under limited irrigation conditions. PMID:24483851

  11. Differentially Coexpressed Disease Gene Identification Based on Gene Coexpression Network.

    PubMed

    Jiang, Xue; Zhang, Han; Quan, Xiongwen

    2016-01-01

    Screening disease-related genes by analyzing gene expression data has become a popular theme. Traditional disease-related gene selection methods always focus on identifying differentially expressed gene between case samples and a control group. These traditional methods may not fully consider the changes of interactions between genes at different cell states and the dynamic processes of gene expression levels during the disease progression. However, in order to understand the mechanism of disease, it is important to explore the dynamic changes of interactions between genes in biological networks at different cell states. In this study, we designed a novel framework to identify disease-related genes and developed a differentially coexpressed disease-related gene identification method based on gene coexpression network (DCGN) to screen differentially coexpressed genes. We firstly constructed phase-specific gene coexpression network using time-series gene expression data and defined the conception of differential coexpression of genes in coexpression network. Then, we designed two metrics to measure the value of gene differential coexpression according to the change of local topological structures between different phase-specific networks. Finally, we conducted meta-analysis of gene differential coexpression based on the rank-product method. Experimental results demonstrated the feasibility and effectiveness of DCGN and the superior performance of DCGN over other popular disease-related gene selection methods through real-world gene expression data sets.

  12. Differentially Coexpressed Disease Gene Identification Based on Gene Coexpression Network

    PubMed Central

    Quan, Xiongwen

    2016-01-01

    Screening disease-related genes by analyzing gene expression data has become a popular theme. Traditional disease-related gene selection methods always focus on identifying differentially expressed gene between case samples and a control group. These traditional methods may not fully consider the changes of interactions between genes at different cell states and the dynamic processes of gene expression levels during the disease progression. However, in order to understand the mechanism of disease, it is important to explore the dynamic changes of interactions between genes in biological networks at different cell states. In this study, we designed a novel framework to identify disease-related genes and developed a differentially coexpressed disease-related gene identification method based on gene coexpression network (DCGN) to screen differentially coexpressed genes. We firstly constructed phase-specific gene coexpression network using time-series gene expression data and defined the conception of differential coexpression of genes in coexpression network. Then, we designed two metrics to measure the value of gene differential coexpression according to the change of local topological structures between different phase-specific networks. Finally, we conducted meta-analysis of gene differential coexpression based on the rank-product method. Experimental results demonstrated the feasibility and effectiveness of DCGN and the superior performance of DCGN over other popular disease-related gene selection methods through real-world gene expression data sets. PMID:28042568

  13. Overexpression of the Rho-guanine nucleotide exchange factor ECT2 inhibits nuclear translocation of nuclear receptor CAR in the mouse liver.

    PubMed

    Hosseinpour, Fardin; Timsit, Yoav; Koike, Chika; Matsui, Kenji; Yamamoto, Yukio; Moore, Rick; Negishi, Masahiko

    2007-10-16

    Various drugs such as phenobarbital (PB) trigger translocation of constitutive active/adrostane receptor (CAR) from the cytoplasm into the nucleus of mouse liver cells without directly binding to the receptor. We have now characterized the guanine nucleotide exchange factor epithelial cell-transforming gene 2 (ECT2) as a PB-inducible factor as well as a cellular signal that represses PB-triggered nuclear translocation of CAR. When CFP-tagged ECT2 was co-expressed with YFP-tagged CAR in the liver of Car(-/-) mice, ECT2 repressed CAR nuclear translocation. Coexpression of various deletion mutants delineated this repressive activity to the tandem Dbl homology/pleckstrin homology domains of ECT2 and to their cytosolic expression. CAR directly bound to the PH domain. Thus, ECT2 may comprise a part of the PB response signal regulating the intracellular trafficking of CAR.

  14. A novel bispecific diabody targeting both vascular endothelial growth factor receptor 2 and epidermal growth factor receptor for enhanced antitumor activity.

    PubMed

    Xu, Menghuai; Jin, Haizhen; Chen, Zhiguo; Xie, Wei; Wang, Youfu; Wang, Yang; Wang, Min; Zhang, Juan; Acheampong, Desmond Omane

    2016-03-01

    Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2) are receptor tyrosine kinases known to play critical roles in the development and progression of tumors. Based on the cross-talk between EGFR and VEGFR2 signal pathways, we designed and produced a bispecific diabody (bDAb) targeting both EGFR and VEGFR2 simultaneously. The bispecific molecule (EK-02) demonstrated that it could bind to HUVEC (VEGFR2 high-expressing) and A431 (EGFR overexpressing) cells. Additionally, similar to the parental antibodies, it was able to inhibit proliferation and migration, and induced apoptosis in these cells (HUVECs and A431), demonstrating that it had retained the functional properties of its parental antibodies. Furthermore, the efficacy of EK-02 was evaluated using the human colon adenocarcinoma cell line HT29 (VEGFR2 and EGFR coexpressing). In vitro assay showed that EK-02 could bind to HT29 cells, restrain cell growth and migration, and induce apoptosis with enhanced efficacy compared to both parental antibodies. Further, it inhibited the neovascularization and tumor formation on an HT29 cell bearing chicken chorioallantoic membrane (CAM) tumor model in vivo. In conclusion, these data suggest that the novel bDAb (EK-02) has antiangiogenesis and antitumor capacity both in vitro and in vivo, and can possibly be used as cotargeted therapy for the treatment of EGFR and VEGFR2 overexpressing tumors. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:294-302, 2016. © 2016 American Institute of Chemical Engineers.

  15. Phenotyping of human melanoma cells reveals a unique composition of receptor targets and a subpopulation co-expressing ErbB4, EPO-R and NGF-R.

    PubMed

    Mirkina, Irina; Hadzijusufovic, Emir; Krepler, Clemens; Mikula, Mario; Mechtcheriakova, Diana; Strommer, Sabine; Stella, Alexander; Jensen-Jarolim, Erika; Höller, Christoph; Wacheck, Volker; Pehamberger, Hubert; Valent, Peter

    2014-01-01

    Malignant melanoma is a life-threatening skin cancer increasingly diagnosed in the western world. In advanced disease the prognosis is grave. Growth and metastasis formation in melanomas are regulated by a network of cytokines, cytokine-receptors, and adhesion molecules. However, little is known about surface antigens and target expression profiles in human melanomas. We examined the cell surface antigen profile of human skin melanoma cells by multicolor flow cytometry, and compared their phenotype with 4 melanoma cell lines (A375, 607B, Mel-Juso, SK-Mel28). Melanoma cells were defined as CD45-/CD31- cells co-expressing one or more melanoma-related antigens (CD63, CD146, CD166). In most patients, melanoma cells exhibited ErbB3/Her3, CD44/Pgp-1, ICAM-1/CD54 and IGF-1-R/CD221, but did not express CD20, ErbB2/Her2, KIT/CD117, AC133/CD133 or MDR-1/CD243. Melanoma cell lines were found to display a similar phenotype. In most patients, a distinct subpopulation of melanoma cells (4-40%) expressed the erythropoietin receptor (EPO-R) and ErbB4 together with PD-1 and NGF-R/CD271. Both the EPO-R+ and EPO-R- subpopulations produced melanoma lesions in NOD/SCID IL-2Rgamma(null) (NSG) mice in first and secondary recipients. Normal skin melanocytes did not express ErbB4 or EPO-R, but expressed a functional KIT receptor (CD117) as well as NGF-R, ErbB3/Her3, IGF-1-R and CD44. In conclusion, melanoma cells display a unique composition of surface target antigens and cytokine receptors. Malignant transformation of melanomas is accompanied by loss of KIT and acquisition of EPO-R and ErbB4, both of which are co-expressed with NGF-R and PD-1 in distinct subfractions of melanoma cells. However, expression of EPO-R/ErbB4/PD-1 is not indicative of a selective melanoma-initiating potential.

  16. Phenotyping of Human Melanoma Cells Reveals a Unique Composition of Receptor Targets and a Subpopulation Co-Expressing ErbB4, EPO-R and NGF-R

    PubMed Central

    Krepler, Clemens; Mikula, Mario; Mechtcheriakova, Diana; Strommer, Sabine; Stella, Alexander; Jensen-Jarolim, Erika; Höller, Christoph; Wacheck, Volker; Pehamberger, Hubert; Valent, Peter

    2014-01-01

    Malignant melanoma is a life-threatening skin cancer increasingly diagnosed in the western world. In advanced disease the prognosis is grave. Growth and metastasis formation in melanomas are regulated by a network of cytokines, cytokine-receptors, and adhesion molecules. However, little is known about surface antigens and target expression profiles in human melanomas. We examined the cell surface antigen profile of human skin melanoma cells by multicolor flow cytometry, and compared their phenotype with 4 melanoma cell lines (A375, 607B, Mel-Juso, SK-Mel28). Melanoma cells were defined as CD45−/CD31− cells co-expressing one or more melanoma-related antigens (CD63, CD146, CD166). In most patients, melanoma cells exhibited ErbB3/Her3, CD44/Pgp-1, ICAM-1/CD54 and IGF-1-R/CD221, but did not express CD20, ErbB2/Her2, KIT/CD117, AC133/CD133 or MDR-1/CD243. Melanoma cell lines were found to display a similar phenotype. In most patients, a distinct subpopulation of melanoma cells (4–40%) expressed the erythropoietin receptor (EPO-R) and ErbB4 together with PD-1 and NGF-R/CD271. Both the EPO-R+ and EPO-R− subpopulations produced melanoma lesions in NOD/SCID IL-2Rgammanull (NSG) mice in first and secondary recipients. Normal skin melanocytes did not express ErbB4 or EPO-R, but expressed a functional KIT receptor (CD117) as well as NGF-R, ErbB3/Her3, IGF-1-R and CD44. In conclusion, melanoma cells display a unique composition of surface target antigens and cytokine receptors. Malignant transformation of melanomas is accompanied by loss of KIT and acquisition of EPO-R and ErbB4, both of which are co-expressed with NGF-R and PD-1 in distinct subfractions of melanoma cells. However, expression of EPO-R/ErbB4/PD-1 is not indicative of a selective melanoma-initiating potential. PMID:24489649

  17. Endoglin is an accessory protein that interacts with the signaling receptor complex of multiple members of the transforming growth factor-beta superfamily.

    PubMed

    Barbara, N P; Wrana, J L; Letarte, M

    1999-01-08

    Endoglin (CD105) is a transmembrane glycoprotein that binds transforming growth factor (TGF)-beta1 and -beta3, and coprecipitates with the Ser/Thr kinase signaling receptor complex by affinity labeling of endothelial and leukemic cells. The present study shows that in addition to TGF-beta1 and -beta3, endoglin interacts with activin-A, bone morphogenetic protein (BMP)-7, and BMP-2 but requires coexpression of the respective ligand binding kinase receptor for this association. Endoglin cannot bind ligands on its own and does not alter binding to the kinase receptors. It binds TGF-beta1 and -beta3 by associating with the TGF-beta type II receptor and interacts with activin-A and BMP-7 via activin type II receptors, ActRII and ActRIIB, regardless of which type I receptor partner is coexpressed. However, endoglin binds BMP-2 by interacting with the ligand binding type I receptors, ALK3 and ALK6. The formation of heteromeric signaling complexes was not altered by the presence of endoglin, although it was coprecipitated with these complexes. Endoglin did not interact with BMP-7 through complexes containing the BMP type II receptor, demonstrating specificity of its action. Our data suggest that endoglin is an accessory protein of multiple kinase receptor complexes of the TGF-beta superfamily.

  18. Coagulation factor VII is regulated by androgen receptor in breast cancer

    PubMed Central

    Naderi, Ali

    2014-01-01

    Androgen receptor (AR) is widely expressed in breast cancer; however, there is limited information on the key molecular functions and gene targets of AR in this disease. In this study, gene expression data from a cohort of 52 breast cancer cell lines was analyzed to identify a network of AR co-expressed genes. A total of three hundred genes, which were significantly enriched for cell cycle and metabolic functions, showed absolute correlation coefficients (| CC |) of more than 0.5 with AR expression across the dataset. In this network, a subset of 35 “AR-signature” genes were highly co-expressed with AR (| CC| > 0.6) that included transcriptional regulators PATZ1, NFATC4, and SPDEF. Furthermore, gene encoding coagulation factor VII (F7) demonstrated the closest expression pattern with AR (CC= 0.716) in the dataset and factor VII protein expression was significantly associated to that of AR in a cohort of 209 breast tumors. Moreover, functional studies demonstrated that AR activation results in the induction of factor VII expression at both transcript and protein levels and AR directly binds to a proximal region of F7 promoter in breast cancer cells. Importantly, AR activation in breast cancer cells induced endogenous factor VII activity to convert factor X to Xa in conjunction with tissue factor. In summary, F7 is a novel AR target gene and AR activation regulates the ectopic expression and activity of factor VII in breast cancer cells. These findings have functional implications in the pathobiology of thromboembolic events and regulation of factor VII/tissue factor signaling in breast cancer. PMID:25447311

  19. Expression of transforming growth factor alpha, epidermal growth factor receptor and epidermal growth factor in precursor lesions to gastric carcinoma.

    PubMed Central

    Filipe, M. I.; Osborn, M.; Linehan, J.; Sanidas, E.; Brito, M. J.; Jankowski, J.

    1995-01-01

    Epidermal growth factor (EGF), its related peptide transforming growth factor (TGF-alpha) and their common receptor (EGFR) have been implicated in the control of cell proliferation and differentiation in the gastrointestinal epithelium and may play an important role in gastric carcinogenesis. We compared the immunohistochemical expression and topographic distribution of these peptides using Western blot analysis in gastric carcinoma precursor lesions and in non-cancer tissue. We observed: (i) increased and extended expression of TGF-alpha in normal mucosa and hyperplasia in carcinoma fields compared with non-cancer controls; (ii) increased expression of EGFR in intestinal metaplasia (IM) from carcinoma fields compared with controls; (iii) EGF expression was not detected in normal mucosa and only weakly in IM; (iv) coexpression of TGF-alpha/EGFR and EGF/EGFR was higher in intestinal metaplasia in carcinoma fields than in non-cancer controls. We conclude that altered expression of TGF-alpha/EGFR is associated with morphological changes during gastric carcinogenesis. In this regard increased expression of TGF-alpha is a very early event which is subsequently followed by up-regulation of EGFR and this has important biological and clinical implications. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:7819044

  20. Expression of type 1 corticotropin-releasing factor receptor in the guinea pig enteric nervous system.

    PubMed

    Liu, Sumei; Gao, Xiang; Gao, Na; Wang, Xiyu; Fang, Xiucai; Hu, Hong-Zhen; Wang, Guo-Du; Xia, Yun; Wood, Jackie D

    2005-01-17

    Reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, electrophysiological recording, and intraneuronal injection of the neuronal tracer biocytin were integrated in a study of the functional expression of corticotropin-releasing factor (CRF) receptors in the guinea pig enteric nervous system. RT-PCR revealed expression of CRF1 receptor mRNA, but not CRF2, in both myenteric and submucosal plexuses. Immunoreactivity for the CRF1 receptor was distributed widely in the myenteric plexus of the stomach and small and large intestine and in the submucosal plexus of the small and large intestine. CRF1 receptor immunoreactivity was coexpressed with calbindin, choline acetyltransferase, and substance P in the myenteric plexus. In the submucosal plexus, CRF1 receptor immunoreactivity was found in neurons that expressed calbindin, substance P, choline acetyltransferase, or neuropeptide Y. Application of CRF evoked slowly activating depolarizing responses associated with elevated excitability in both myenteric and submucosal neurons. Histological analysis of biocytin-filled neurons revealed that both uniaxonal neurons with S-type electrophysiological behavior and neurons with AH-type electrophysiological behavior and Dogiel II morphology responded to CRF. The CRF-evoked depolarizing responses were suppressed by the CRF1/CRF2 receptor antagonist astressin and the selective CRF1 receptor antagonist NBI27914 and were unaffected by the selective CRF2 receptor antagonist antisauvagine-30. The findings support the hypothesis that the CRF1 receptor mediates the excitatory actions of CRF on neurons in the enteric nervous system. Actions on enteric neurons might underlie the neural mechanisms by which stress-related release of CRF in the periphery alters intestinal propulsive motor function, mucosal secretion, and barrier functions.

  1. Human epidermal growth factor receptor 3 in head and neck squamous cell carcinomas.

    PubMed

    Rysman, Bénédicte; Mouawad, François; Gros, Abigaëlle; Lansiaux, Amélie; Chevalier, Dominique; Meignan, Samuel

    2016-04-01

    Human epidermal growth factor receptor 3 (HER3) is a member of the human epidermal growth factor receptor (HER) family. The main characteristic of HER3 is that it does not possess tyrosine kinase activity, unlike other HERs. The role of HER3 in tumorigenesis has now been recognized, particularly in head and neck squamous cell carcinomas (HNSCCs). Despite conflicting studies, HER3 was found to be overexpressed in HNSCC samples, and correlates with disease progression and poor survival, especially when it is coexpressed with other HERs. HER3 is a significant factor in HNSCC treatment resistance. Indeed, HER3 is a major mechanism described for cetuximab resistance because of modification of epidermal growth factor receptor (EGFR) internalization and by phosphotidylinositol-3-kinase (PI3K)/AKT signaling pathway activation. HER3 also affects resistance to tyrosine kinase inhibitors (TKIs) and thereby promotes treatment escape and radiotherapy resistance by activation of the survival signaling pathway. To counteract this, pharmacologic inhibitors of HER3 are currently in development and could significantly improve HNSCC treatment. © 2016 Wiley Periodicals, Inc. Head Neck 38: E2412-E2418, 2016.

  2. kappa-Opioid receptor activates an inwardly rectifying K+ channel by a G protein-linked mechanism: coexpression in Xenopus oocytes.

    PubMed

    Ma, G H; Miller, R J; Kuznetsov, A; Philipson, L H

    1995-05-01

    cRNAs encoding the kappa-opioid receptor and an inwardly rectifying, G protein-coupled, K+ channel were coinjected into Xenopus oocytes. The effects of kappa-opioid receptor agonists and antagonists on the membrane currents in these oocytes were studied using the two-electrode voltage-clamp technique. The kappa-opioid receptor agonists U69593 and dynorphin A induced a concentration-dependent inward current (EC50 of approximately 0.3 microM and approximately 30 nM, respectively) after coinjection of both cRNAs, whereas the mu-opioid receptor agonist [D-Ala2,N-MePhe4,Gly5-ol]enkephalin (10 microM) and the delta-opioid receptor agonist [D-Pen2,5]enkephalin (1 microM) had no effect. The agonist-induced inward current was reversible upon washing out of the agonists and was inhibited in the presence of the K+ channel blocker Ba2+ (0.1 mM). The specific kappa-opioid receptor antagonist norbinaltorphimine (0.1 microM) and the nonspecific opioid receptor antagonist naloxone (1 microM) abolished the agonist-induced currents. Furthermore, the agonist-induced currents exhibited rapid desensitization in the continuous presence of the agonists or after repeated application. Preincubation of the coinjected oocytes with pertussis toxin (400 ng/ml for 3 days of 1.5 microgram/ml for 24 hr) abolished most of the agonist-induced activation of the inwardly rectifying K+ current. We therefore conclude that specific stimulation of the kappa-opioid receptor can activate the inwardly rectifying K+ channel through a pertussis toxin-sensitive G protein.

  3. Vicia villosa agglutinin labels a subset of neurons coexpressing both the mu opioid receptor and parvalbumin in the developing rat subiculum.

    PubMed

    Bausch, S B; Chavkin, C

    1996-12-23

    Vicia villosa agglutinin (VVA), anti-parvalbumin antiserum and an affinity-purified anti-mu opioid receptor antibody were used to triple-label neurons in the postnatal rat subiculum. VVA labeled a subset of mu opioid receptor-positive neurons that were also immunoreactive for parvalbumin. The morphology of the triple-labeled neurons was heterogeneous, and included multipolar, ovoid and pyramidal-shaped neurons. Neurons single-labeled for the mu opioid receptor, VVA or parvalbumin were also morphologically heterogeneous. The postnatal development of mu opioid receptor immunoreactivity (IR), parvalbumin-IR and VVA binding was investigated using triple-labeling immunocytochemistry. Mu opioid receptor-IR appeared first and was present at postnatal day 1 (P1). Parvalbumin-IR was first observed in somata at P10, followed by proximal and distal dendrites at P15 and P20 respectively. Faint VVA labeling was seen first at P10 and surrounded a limited number of neurons. The intensity of labeling and the number of neurons labeled with VVA increased between P10 and P20; however, both measures remained below adult levels at P20. This study further illustrates the neurochemical heterogeneity of interneurons in the hippocampal formation and shows the developmentally early appearance of mu opioid receptor-IR compared to the late appearance of VVA binding and parvalbumin-IR.

  4. Squamous cell carcinomas escape immune surveillance via inducing chronic activation and exhaustion of CD8+ T Cells co-expressing PD-1 and LAG-3 inhibitory receptors

    PubMed Central

    Mishra, Ameet K.; Kadoishi, Tanya; Wang, Xiaoguang; Driver, Emily; Chen, Zhangguo; Wang, Xiao-Jing; Wang, Jing H.

    2016-01-01

    Squamous cell carcinoma (SCC) is the second commonest type of skin cancer. Moreover, about 90% of head and neck cancers are SCCs. SCCs develop at a significantly higher rate under chronic immunosuppressive conditions, implicating a role of immune surveillance in controlling SCCs. It remains largely unknown how SCCs evade immune recognition. Here, we established a mouse model by injecting tumor cells derived from primary SCCs harboring KrasG12D mutation and Smad4 deletion into wild-type (wt) or CD8−/− recipients. We found comparable tumor growth between wt and CD8−/− recipients, indicating a complete escape of CD8+ T cell-mediated anti-tumor responses by these SCCs. Mechanistically, CD8+ T cells apparently were not defective in infiltrating tumors given their relatively increased percentage among tumor infiltrating lymphocytes (TILs). CD8+ TILs exhibited phenotypes of chronic activation and exhaustion, including overexpression of activation markers, co-expression of programmed cell death 1 (PD-1) and lymphocyte activation gene-3 (LAG-3), as well as TCRβ downregulation. Among CD4+ TILs, T regulatory cells (Tregs) were preferentially expanded. Contradictory to prior findings in melanoma, Treg expansion was independent of CD8+ T cells in our SCC model. Unexpectedly, CD8+ T cells were required for promoting NK cell infiltration within SCCs. Furthermore, we uncovered AKT-dependent lymphocyte-induced PD-L1 upregulation on SCCs, which was contributed greatly by combinatorial effects of CD8+ T and NK cells. Lastly, dual blockade of PD-1 and LAG-3 inhibited the tumor growth of SCCs. Thus, our findings identify novel immune evasion mechanisms of SCCs and suggest that immunosuppressive mechanisms operate in a cancer-type specific and context-dependent manner. PMID:27835902

  5. Squamous cell carcinomas escape immune surveillance via inducing chronic activation and exhaustion of CD8+ T Cells co-expressing PD-1 and LAG-3 inhibitory receptors.

    PubMed

    Mishra, Ameet K; Kadoishi, Tanya; Wang, Xiaoguang; Driver, Emily; Chen, Zhangguo; Wang, Xiao-Jing; Wang, Jing H

    2016-12-06

    Squamous cell carcinoma (SCC) is the second commonest type of skin cancer. Moreover, about 90% of head and neck cancers are SCCs. SCCs develop at a significantly higher rate under chronic immunosuppressive conditions, implicating a role of immune surveillance in controlling SCCs. It remains largely unknown how SCCs evade immune recognition. Here, we established a mouse model by injecting tumor cells derived from primary SCCs harboring KrasG12D mutation and Smad4 deletion into wild-type (wt) or CD8-/- recipients. We found comparable tumor growth between wt and CD8-/- recipients, indicating a complete escape of CD8+ T cell-mediated anti-tumor responses by these SCCs. Mechanistically, CD8+ T cells apparently were not defective in infiltrating tumors given their relatively increased percentage among tumor infiltrating lymphocytes (TILs). CD8+ TILs exhibited phenotypes of chronic activation and exhaustion, including overexpression of activation markers, co-expression of programmed cell death 1 (PD-1) and lymphocyte activation gene-3 (LAG-3), as well as TCRβ downregulation. Among CD4+ TILs, T regulatory cells (Tregs) were preferentially expanded. Contradictory to prior findings in melanoma, Treg expansion was independent of CD8+ T cells in our SCC model. Unexpectedly, CD8+ T cells were required for promoting NK cell infiltration within SCCs. Furthermore, we uncovered AKT-dependent lymphocyte-induced PD-L1 upregulation on SCCs, which was contributed greatly by combinatorial effects of CD8+ T and NK cells. Lastly, dual blockade of PD-1 and LAG-3 inhibited the tumor growth of SCCs. Thus, our findings identify novel immune evasion mechanisms of SCCs and suggest that immunosuppressive mechanisms operate in a cancer-type specific and context-dependent manner.

  6. Epidermal growth factor receptor signaling in tissue

    SciTech Connect

    Shvartsman, Stanislav; Wiley, H. S.; Lauffenburger, Douglas A.

    2004-08-01

    Abstract: A peptide purified from the salivary gland of a mouse was shown few years ago to accelerate incisor eruption and eyelid opening in newborn mice, and was named epidermal growth factor (EGF). The members of this family of peptide growth factors had been identified in numerous physiological and pathological contexts. EGF binds to a cell surface EGF receptor, which induces a biochemical modification (phosphorylation) of the receptor's cytoplasmic tail. There is a growing consensus in the research community that, in addition to cellular and molecular studies, the dynamics of the EGFR network and its operation must be examined in tissues. A key challenge is to integrate the existing molecular and cellular information into a system-level description of the EGFR network at the tissue and organism level. In this paper, the two examples of EGFR signaling in tissues are described, and the recent efforts to model EGFR autocrine loops, which is a predominant mode of EGFR activation in vivo, are summarized.

  7. Craniopharyngiomas express embryonic stem cell markers (SOX2, OCT4, KLF4, and SOX9) as pituitary stem cells but do not coexpress RET/GFRA3 receptors.

    PubMed

    Garcia-Lavandeira, Montserrat; Saez, Carmen; Diaz-Rodriguez, Esther; Perez-Romero, Sihara; Senra, Ana; Dieguez, Carlos; Japon, Miguel A; Alvarez, Clara V

    2012-01-01

    Adult stem cells maintain some markers expressed by embryonic stem cells and express other specific markers depending on the organ where they reside. Recently, stem/progenitor cells in the rodent and human pituitary have been characterized as expressing GFRA2/RET, PROP1, and stem cell markers such as SOX2 and OCT4 (GPS cells). Our objective was to detect other specific markers of the pituitary stem cells and to investigate whether craniopharyngiomas (CRF), a tumor potentially derived from Rathke's pouch remnants, express similar markers as normal pituitary stem cells. We conducted mRNA and Western blot studies in pituitary extracts, and immunohistochemistry and immunofluorescence on sections from normal rat and human pituitaries and 20 CRF (18 adamantinomatous and two papillary). Normal pituitary GPS stem cells localized in the marginal zone (MZ) express three key embryonic stem cell markers, SOX2, OCT4, and KLF4, in addition to SOX9 and PROP1 and β-catenin overexpression. They express the RET receptor and its GFRA2 coreceptor but also express the coreceptor GFRA3 that could be detected in the MZ of paraffin pituitary sections. CRF maintain the expression of SOX2, OCT4, KLF4, SOX9, and β-catenin. However, RET and GFRA3 expression was altered in CRF. In 25% (five of 20), both RET and GFRA3 were detected but not colocalized in the same cells. The other 75% (15 of 20) lose the expression of RET, GFRA3, or both proteins simultaneously. Human pituitary adult stem/progenitor cells (GPS) located in the MZ are characterized by expression of embryonic stem cell markers SOX2, OCT4, and KLF4 plus the specific pituitary embryonic factor PROP1 and the RET system. Redundancy in RET coreceptor expression (GFRA2 and GFRA3) suggest an important systematic function in their physiological behavior. CRF share the stem cell markers suggesting a common origin with GPS. However, the lack of expression of the RET/GFRA system could be related to the cell mislocation and deregulated

  8. Functional Analysis and Characterization of Differential Coexpression Networks

    PubMed Central

    Hsu, Chia-Lang; Juan, Hsueh-Fen; Huang, Hsuan-Cheng

    2015-01-01

    Differential coexpression analysis is emerging as a complement to conventional differential gene expression analysis. The identified differential coexpression links can be assembled into a differential coexpression network (DCEN) in response to environmental stresses or genetic changes. Differential coexpression analyses have been successfully used to identify condition-specific modules; however, the structural properties and biological significance of general DCENs have not been well investigated. Here, we analyzed two independent Saccharomyces cerevisiae DCENs constructed from large-scale time-course gene expression profiles in response to different situations. Topological analyses show that DCENs are tree-like networks possessing scale-free characteristics, but not small-world. Functional analyses indicate that differentially coexpressed gene pairs in DCEN tend to link different biological processes, achieving complementary or synergistic effects. Furthermore, the gene pairs lacking common transcription factors are sensitive to perturbation and hence lead to differential coexpression. Based on these observations, we integrated transcriptional regulatory information into DCEN and identified transcription factors that might cause differential coexpression by gain or loss of activation in response to different situations. Collectively, our results not only uncover the unique structural characteristics of DCEN but also provide new insights into interpretation of DCEN to reveal its biological significance and infer the underlying gene regulatory dynamics. PMID:26282208

  9. Functional Analysis and Characterization of Differential Coexpression Networks.

    PubMed

    Hsu, Chia-Lang; Juan, Hsueh-Fen; Huang, Hsuan-Cheng

    2015-08-18

    Differential coexpression analysis is emerging as a complement to conventional differential gene expression analysis. The identified differential coexpression links can be assembled into a differential coexpression network (DCEN) in response to environmental stresses or genetic changes. Differential coexpression analyses have been successfully used to identify condition-specific modules; however, the structural properties and biological significance of general DCENs have not been well investigated. Here, we analyzed two independent Saccharomyces cerevisiae DCENs constructed from large-scale time-course gene expression profiles in response to different situations. Topological analyses show that DCENs are tree-like networks possessing scale-free characteristics, but not small-world. Functional analyses indicate that differentially coexpressed gene pairs in DCEN tend to link different biological processes, achieving complementary or synergistic effects. Furthermore, the gene pairs lacking common transcription factors are sensitive to perturbation and hence lead to differential coexpression. Based on these observations, we integrated transcriptional regulatory information into DCEN and identified transcription factors that might cause differential coexpression by gain or loss of activation in response to different situations. Collectively, our results not only uncover the unique structural characteristics of DCEN but also provide new insights into interpretation of DCEN to reveal its biological significance and infer the underlying gene regulatory dynamics.

  10. Prognostic values of ETS-1, MMP-2 and MMP-9 expression and co-expression in breast cancer patients.

    PubMed

    Puzovic, V; Brcic, I; Ranogajec, I; Jakic-Razumovic, J

    2014-01-01

    The aim of this study was to analyse expression of ETS-1 protein and two gelatinases (MMP-2 and MMP-9) and their possible prognostic value in breast carcinoma patients, as well as correlation of their expression with other known prognostic factors such as tumor size, grade, vascular invasion, steroid receptor values, HER2 values and proliferative index. The expression of MMP-2, MMP-9 and ETS-1 was immunohistochemicaly analysed in 121 consecutive primary breast carcinoma patients who underwent surgery at the Clinical Hospital Centre Zagreb during 2002. Three representative areas from each tumor paraffin blocks were taken and arranged on a recipient paraffin block with predefined coordinates for simultaneous analyses of multiple tissue samples (TMA). ETS-1, MMP-2 and MMP-9 expression and co-expression were correlated with other clinico-pathological parameters and based on the available clinical follow up data survival analysis was performed. The ETS-1 protein is found to be expressed in tumor cell nuclei and cytoplasm as well as in stromal lymphocytes, fibroblasts and endothelial cells. MMP-2 and MMP-9 were found to be expressed in cytoplasm of both, tumor and stromal cells. For our analysis only tumor cell expression was used for statistical analysis. We found 56,2% ETS-1 positive tumors, 77,7% were MMP-2 positive, and MMP-9 was expressed in 90% of primary breast carcinomas. There were no significant correlations between MMP-s expression and other patohistological prognostic factors, but expression of ETS-1 was significantly correlated with higher tumor size and grade, as well as with negative steroid receptors. Co-expression of MMP-2, MMP-9 and ETS-1 was found in 40,5 % of tumors, and more commonly was found in tumors larger than 2 cm, high grade tumors, and steroid receptor negative tumors. In univariate analysis, statistically significant negative impact on overall survival (OS) had tumor size, nuclear and tumor grade, ETS-1 expression in tumor cells, co-expression

  11. Prostate-specific antigen and gross cystic disease fluid protein-15 are co-expressed in androgen receptor-positive breast tumours.

    PubMed Central

    Hall, R. E.; Clements, J. A.; Birrell, S. N.; Tilley, W. D.

    1998-01-01

    Androgens regulate breast cancer cell proliferation via androgen receptor (AR)-mediated mechanisms. To investigate further the androgen-responsiveness of human breast tumours, we examined the immunohistochemical expression of the AR and two androgen-regulated proteins, prostate-specific antigen (PSA) and gross cystic disease fluid protein-15 (GCDFP-15), in 72 primary breast tumours. AR immunoreactivity was present in the nuclei of breast tumour cells and was correlated with oestrogen receptor (ER; P < 0.05) and progesterone receptor (PR; P < 0.01) status. PSA and GCDFP-15 immunoreactivity was present in the cytoplasm of tumour cells but not the adjacent stromal cells. AR-positive cells were present in 85% (61/72) of breast tumours, and 98% (43/44) of PSA-positive and 92% (44/48) of GCDFP-15-positive tumours were also positive for AR. Positive immunoreactivity for both PSA and GCDFP-15 in breast tumours was highly dependent on AR status (odds ratios of 24.0 and 4.5 respectively), but unrelated to age, ER and PR status and axillary lymph node involvement. PSA immunoreactivity was more frequently observed in moderate and well-differentiated tumours and was significantly (P < 0.001) associated with GCDFP-15 immunoreactivity. In conclusion, PSA and GCDFP-15 immunoreactivity was dependent on the presence of AR, but not ER or PR in primary breast tumours. Images Figure 1 PMID:9703283

  12. Transforming growth factor-beta receptor requirements for the induction of the endothelin-1 gene.

    PubMed

    Castañares, Cristina; Redondo-Horcajo, Mariano; Magan-Marchal, Noemi; Lamas, Santiago; Rodriguez-Pascual, Fernando

    2006-06-01

    Expression of the endothelin (ET)-1 gene is subject to complex regulation by numerous factors, among which the cytokine transforming growth factor-beta (TGF-beta) is one of the most important. TGF-beta action is based on the activation of the Smad signaling pathway. Smad proteins activate transcription of the gene by cooperation with activator protein-1 (AP-1) at specific sites on the ET-1 promoter. Smad signaling pathway is initiated by binding of the cytokine to a heteromeric complex of type I and type II receptors. Signal is then propagated to the nucleus by specific members of the Smad family. Most cell types contain a type I receptor known as ALK5. However, endothelial cells are unique because they coexpress an additional type I receptor named ALK1. These forms do not constitute redundant receptors with the same function, but they actually activate different Smad-mediated expression programs that lead to specific endothelial phenotypes. TGF-beta/ALK5/Smad3 pathway is associated to a mature endothelium because it leads to inhibition of cell migration/proliferation. Conversely, TGF-beta/ALK1/Smad5 activates both processes and is more related to the angiogenic state. We have analyzed the TGF-beta receptor subtype requirements for the activation of the ET-1 gene. For that purpose, we have overexpressed type I receptor and Smad isoforms in endothelial cells and analyzed the effect on ET-1 expression. Our experiments indicate that TGF-beta induces ET-1 expression preferentially through the activation of the ALK5/Smad3 pathway and, therefore, the expression of the vaso-constrictor may be associated to a quiescent and mature endothelial phenotype.

  13. Clinical significance of coexpression of L-type amino acid transporter 1 (LAT1) and ASC amino acid transporter 2 (ASCT2) in lung adenocarcinoma

    PubMed Central

    Yazawa, Tomohiro; Shimizu, Kimihiro; Kaira, Kyoichi; Nagashima, Toshiteru; Ohtaki, Yoichi; Atsumi, Jun; Obayashi, Kai; Nagamori, Shushi; Kanai, Yoshikatsu; Oyama, Tetsunari; Takeyoshi, Izumi

    2015-01-01

    Background: L-type amino acid transporter 1 (LAT1) and ASC amino acid transporter 2 (ASCT2) have been associated with tumor growth and progression. However, the clinical significance of LAT1 and ASCT2 coexpression in the prognosis of patients with lung adenocarcinoma remains unclear. Methods: In total, 222 patients with surgically resected lung adenocarcinoma were investigated retrospectively. Tumor sections were stained immunohistochemically for LAT1, ASCT2, CD98, phosphorylated mammalian target-of-rapamycin (p-mTOR), and Ki-67, and microvessel density (MVD) was determined by staining for CD34. Epidermal growth factor receptor (EGFR) mutation status was also examined. Results: LAT1 and ASCT2 were positively expressed in 22% and 40% of cases, respectively. Coexpression of LAT1 and ASCT2 was observed in 12% of cases and was associated significantly with disease stage, lymphatic permeation, vascular invasion, CD98, Ki-67, and p-mTOR. Only LAT1 and ASCT2 coexpression indicated a poor prognosis for lung adenocarcinoma. Furthermore, this characteristic was recognized in early-stage patients, especially those who had wild-type, rather than mutated, EGFR. Multivariate analysis confirmed that the coexpression of LAT1 and ASCT2 was an independent factor for predicting poor outcome. Conclusions: LAT1 and ASCT2 coexpression is an independent prognostic factor for patients with lung adenocarcinoma, especially during the early stages, expressing wild-type EGFR. PMID:26279756

  14. Co-expression of Arabidopsis transcription factor, AtMYB12, and soybean isoflavone synthase, GmIFS1, genes in tobacco leads to enhanced biosynthesis of isoflavones and flavonols resulting in osteoprotective activity.

    PubMed

    Pandey, Ashutosh; Misra, Prashant; Khan, Mohd P; Swarnkar, Gaurav; Tewari, Mahesh C; Bhambhani, Sweta; Trivedi, Ritu; Chattopadhyay, Naibedya; Trivedi, Prabodh K

    2014-01-01

    Isoflavones, a group of flavonoids, restricted almost exclusively to family Leguminosae are known to exhibit anticancerous and anti-osteoporotic activities in animal systems and have been a target for metabolic engineering in commonly consumed food crops. Earlier efforts based on the expression of legume isoflavone synthase (IFS) genes in nonlegume plant species led to the limited success in terms of isoflavone content in transgenic tissue due to the limitation of substrate for IFS enzyme. In this work to overcome this limitation, the activation of multiple genes of flavonoid pathway using Arabidopsis transcription factor AtMYB12 has been carried out. We developed transgenic tobacco lines constitutively co-expressing AtMYB12 and GmIFS1 (soybean IFS) genes or independently and carried out their phytochemical and molecular analyses. The leaves of co-expressing transgenic lines were found to have elevated flavonol content along with the accumulation of substantial amount of genistein glycoconjugates being at the highest levels that could be engineered in tobacco leaves till date. Oestrogen-deficient (ovariectomized, Ovx) mice fed with leaf extract from transgenic plant co-expressing AtMYB12 and GmIFS1 but not wild-type extract exhibited significant conservation of trabecular microarchitecture, reduced osteoclast number and expression of osteoclastogenic genes, higher total serum antioxidant levels and increased uterine oestrogenicity compared with Ovx mice treated with vehicle (control). The skeletal effect of the transgenic extract was comparable to oestrogen-treated Ovx mice. Together, our results establish an efficient strategy for successful pathway engineering of isoflavones and other flavonoids in crop plants and provide a direct evidence of improved osteoprotective effect of transgenic plant extract.

  15. Autonomous Stimulation of Cancer Cell Plasticity by the Human NKG2D Lymphocyte Receptor Coexpressed with Its Ligands on Cancer Cells

    PubMed Central

    Cai, Xin; Dai, Zhenpeng; Reeves, Rebecca S.; Caballero-Benitez, Andrea; Duran, Kate L.; Delrow, Jeffrey J.; Porter, Peggy L.

    2014-01-01

    The stimulatory NKG2D receptor on lymphocytes promotes tumor immune surveillance by targeting ligands selectively induced on cancer cells. Progressing tumors counteract by employing tactics to disable lymphocyte NKG2D. This negative dynamic is escalated as some human cancer cells co-opt expression of NKG2D, thereby complementing the presence of its ligands for autonomous stimulation of oncogenic signaling. Clinical association data imply relationships between cancer cell NKG2D and metastatic disease. Here we show that NKG2D promotes cancer cell plasticity by induction of phenotypic, molecular, and functional signatures diagnostic of the epithelial–mesenchymal transition, and of stem-like traits via induction of Sox9, a key transcriptional regulator of breast stem cell maintenance. These findings obtained with model breast tumor lines and xenotransplants were recapitulated by ex vivo cancer cells from primary invasive breast carcinomas. Thus, NKG2D may have the capacity to drive high malignancy traits underlying metastatic disease. PMID:25291178

  16. Coexpression of two functionally independent p58 inhibitory receptors in human natural killer cell clones results in the inability to kill all normal allogeneic target cells.

    PubMed Central

    Vitale, M; Sivori, S; Pende, D; Moretta, L; Moretta, A

    1995-01-01

    In the present study, we define a group of natural killer (NK) clones (group 0) that fails to lyse all of the normal allogeneic target cells analyzed. Their specificity for HLA class I molecules was suggested by their ability to lyse class I-negative target cells and by the fact that they could lyse resistant target cells in the presence of selected anti-class I monoclonal antibodies. The use of appropriate target cells represented by either HLA-homozygous cell lines or cell transfectants revealed that these clones recognized all the HLA-C alleles. By the use of monoclonal antibodies directed to either GL183 or EB6 molecules, we showed that the EB6 molecules were responsible for the recognition of Cw4 and related alleles, while the GL183 molecules recognized Cw3 (and related C alleles). These data suggest that the GL183 and the EB6 molecules can function, in individual NK clones, as independent receptors for two different groups of HLA-C alleles, (which include all known alleles for locus C), thus resulting in their inability to lyse all normal HLA-C+ target cells. Indirect immunofluorescence and fluorescence-activated cell sorting analysis revealed that the presently defined GL183+EB6+ group 0 NK clones brightly express EB6 molecules (EB6bright) while the GL183+EB6+ group 2 clones (unable to recognize Cw4) express an EB6dull phenotype. These data also imply that the density of EB6 receptors may be critical for the generation of an optimal negative signal upon interaction with appropriate HLA-C alleles. PMID:7724594

  17. The transcription factor RUNX2 regulates receptor tyrosine kinase expression in melanoma

    PubMed Central

    Boregowda, Rajeev K.; Medina, Daniel J.; Markert, Elke; Bryan, Michael A.; Chen, Wenjin; Chen, Suzie; Rabkin, Anna; Vido, Michael J.; Gunderson, Samuel I.; Chekmareva, Marina; Foran, David J.; Lasfar, Ahmed; Goydos, James S.; Cohen-Solal, Karine A.

    2016-01-01

    Receptor tyrosine kinases-based autocrine loops largely contribute to activate the MAPK and PI3K/AKT pathways in melanoma. However, the molecular mechanisms involved in generating these autocrine loops are still largely unknown. In the present study, we examine the role of the transcription factor RUNX2 in the regulation of receptor tyrosine kinase (RTK) expression in melanoma. We have demonstrated that RUNX2-deficient melanoma cells display a significant decrease in three receptor tyrosine kinases, EGFR, IGF-1R and PDGFRβ. In addition, we found co-expression of RUNX2 and another RTK, AXL, in both melanoma cells and melanoma patient samples. We observed a decrease in phosphoAKT2 (S474) and phosphoAKT (T308) levels when RUNX2 knock down resulted in significant RTK down regulation. Finally, we showed a dramatic up regulation of RUNX2 expression with concomitant up-regulation of EGFR, IGF-1R and AXL in melanoma cells resistant to the BRAF V600E inhibitor PLX4720. Taken together, our results strongly suggest that RUNX2 might be a key player in RTK-based autocrine loops and a mediator of resistance to BRAF V600E inhibitors involving RTK up regulation in melanoma. PMID:27102439

  18. The transcription factor RUNX2 regulates receptor tyrosine kinase expression in melanoma.

    PubMed

    Boregowda, Rajeev K; Medina, Daniel J; Markert, Elke; Bryan, Michael A; Chen, Wenjin; Chen, Suzie; Rabkin, Anna; Vido, Michael J; Gunderson, Samuel I; Chekmareva, Marina; Foran, David J; Lasfar, Ahmed; Goydos, James S; Cohen-Solal, Karine A

    2016-05-17

    Receptor tyrosine kinases-based autocrine loops largely contribute to activate the MAPK and PI3K/AKT pathways in melanoma. However, the molecular mechanisms involved in generating these autocrine loops are still largely unknown. In the present study, we examine the role of the transcription factor RUNX2 in the regulation of receptor tyrosine kinase (RTK) expression in melanoma. We have demonstrated that RUNX2-deficient melanoma cells display a significant decrease in three receptor tyrosine kinases, EGFR, IGF-1R and PDGFRβ. In addition, we found co-expression of RUNX2 and another RTK, AXL, in both melanoma cells and melanoma patient samples. We observed a decrease in phosphoAKT2 (S474) and phosphoAKT (T308) levels when RUNX2 knock down resulted in significant RTK down regulation. Finally, we showed a dramatic up regulation of RUNX2 expression with concomitant up-regulation of EGFR, IGF-1R and AXL in melanoma cells resistant to the BRAF V600E inhibitor PLX4720. Taken together, our results strongly suggest that RUNX2 might be a key player in RTK-based autocrine loops and a mediator of resistance to BRAF V600E inhibitors involving RTK up regulation in melanoma.

  19. Subspace differential coexpression analysis: problem definition and a general approach.

    PubMed

    Fang, Gang; Kuang, Rui; Pandey, Gaurav; Steinbach, Michael; Myers, Chad L; Kumar, Vipin

    2010-01-01

    In this paper, we study methods to identify differential coexpression patterns in case-control gene expression data. A differential coexpression pattern consists of a set of genes that have substantially different levels of coherence of their expression profiles across the two sample-classes, i.e., highly coherent in one class, but not in the other. Biologically, a differential coexpression patterns may indicate the disruption of a regulatory mechanism possibly caused by disregulation of pathways or mutations of transcription factors. A common feature of all the existing approaches for differential coexpression analysis is that the coexpression of a set of genes is measured on all the samples in each of the two classes, i.e., over the full-space of samples. Hence, these approaches may miss patterns that only cover a subset of samples in each class, i.e., subspace patterns, due to the heterogeneity of the subject population and disease causes. In this paper, we extend differential coexpression analysis by defining a subspace differential coexpression pattern, i.e., a set of genes that are coexpressed in a relatively large percent of samples in one class, but in a much smaller percent of samples in the other class. We propose a general approach based upon association analysis framework that allows exhaustive yet efficient discovery of subspace differential coexpression patterns. This approach can be used to adapt a family of biclustering algorithms to obtain their corresponding differential versions that can directly discover differential coexpression patterns. Using a recently developed biclustering algorithm as illustration, we perform experiments on cancer datasets which demonstrates the existence of subspace differential coexpression patterns. Permutation tests demonstrate the statistical significance for a large number of discovered subspace patterns, many of which can not be discovered if they are measured over all the samples in each of the classes

  20. Frequent co-expression of EGFR and NeuGcGM3 ganglioside in cancer: it's potential therapeutic implications.

    PubMed

    Palomo, Addys González; Santana, Rancés Blanco; Pérez, Xiomara Escobar; Santana, Damián Blanco; Gabri, Mariano Rolando; Monzon, Kalet León; Pérez, Adriana Carr

    2016-10-01

    Interaction between epidermal growth factor receptor (EGFR) signaling with GM3 ganglioside expression has been previously described. However, little is known about EGFR and NeuGcGM3 co-expression in cancer patients and their therapeutic implications. In this paper, we evaluate the co-expression of EGFR and NeuGcGM3 ganglioside in tumors from 92 patients and in two spontaneous lung metastasis models of mice (Lewis lung carcinoma (3LL-D122) in C57BL/6 and mammary carcinoma (4T1) in BALB/c). As results, co-expression of EGFR and NeuGcGM3 ganglioside was frequently observed in 63 of 92 patients (68 %), independently of histological subtype. Moreover, EGFR is co-expressed with NeuGcGM3 ganglioside in the metastasis of 3LL-D122 and 4T1 murine models. Such dual expression appears to be therapeutically relevant, since combined therapy with mAbs against these two molecules synergistically increase the survival of mice treated. Overall, our results suggest that NeuGcGM3 and EGFR may coordinately contribute to the tumor cell biology and that therapeutic combinations against these two targets might be a valid strategy to explore.

  1. Analysis of functional and pathway association of differential co-expressed genes: a case study in drug addiction.

    PubMed

    Li, Zi-hui; Liu, Yu-feng; Li, Ke-ning; Duanmu, Hui-zi; Chang, Zhi-qiang; Li, Zhen-qi; Zhang, Shan-zhen; Xu, Yan

    2012-02-01

    Drug addiction has been considered as a kind of chronic relapsing brain disease influenced by both genetic and environmental factors. At present, many causative genes and pathways related to diverse kinds of drug addiction have been discovered, while less attention has been paid to common mechanisms shared by different drugs underlying addiction. By applying a co-expression meta-analysis method to mRNA expression profiles of alcohol, cocaine, heroin addicted and normal samples, we identified significant gene co-expression pairs. As co-expression networks of drug group and control group constructed, associated function term pairs and pathway pairs reflected by co-expression pattern changes were discovered by integrating functional and pathway information respectively. The results indicated that respiratory electron transport chain, synaptic transmission, mitochondrial electron transport, signal transduction, locomotory behavior, response to amphetamine, negative regulation of cell migration, glucose regulation of insulin secretion, signaling by NGF, diabetes pathways, integration of energy metabolism, dopamine receptors may play an important role in drug addiction. In addition, the results can provide theory support for studies of addiction mechanisms.

  2. EGFR and PDGFRA co-expression and heterodimerization in glioblastoma tumor sphere lines.

    PubMed

    Chakravarty, Debyani; Pedraza, Alicia M; Cotari, Jesse; Liu, Angela H; Punko, Diana; Kokroo, Aushim; Huse, Jason T; Altan-Bonnet, Gregoire; Brennan, Cameron W

    2017-08-22

    Concurrent amplifications of EGFR and PDGFRA have been reported in up to 5% of glioblastoma (GBM) and it remains unclear why such independent amplification events, and associated receptor overexpression, would be adaptive during glioma evolution. Here, we document that EGFR and PDGFRA protein co-expression occurs in 37% of GBM. There is wide cell-to-cell variation in the expressions of these receptor tyrosine kinases (RTKs) in stable tumor sphere lines, frequently defining tumor cell subpopulations with distinct sensitivities to growth factors and RTK inhibitors. We also find evidence for functional transactivation of PDGFRA by EGFR and EGF-induced receptor heterodimerization, both of which are abolished by EGFR inhibitors. These results indicate that GBM growth responses to targeted therapies previously tested in clinical trials are strongly influenced by the balance of EGFR and PDGFRA activation in individual cells, which is heterogeneous at baseline.

  3. Anti-oncogenic activity of signalling-defective epidermal growth factor receptor mutants.

    PubMed Central

    Redemann, N; Holzmann, B; von Rüden, T; Wagner, E F; Schlessinger, J; Ullrich, A

    1992-01-01

    Overexpression and autocrine activation of the epidermal growth factor receptor (EGF-R) cause transformation of cultured cells and correlate with tumor progression in cancer patients. Dimerization and transphosphorylation are crucial events in the process by which receptors with tyrosine kinase activity generate normal and transforming cellular signals. Interruption of this process by inactive receptor mutants offers the potential to inhibit ligand-induced cellular responses. Using recombinant retroviruses, we have examined the effects of signalling-incompetent EGF-R mutants on the growth-promoting and transforming potential of ligand-activated, overexpressed wild-type EGF-R and the v-erbB oncogene product. Expression of a soluble extracellular EGF-R domain had little if any effect on the growth and transformation of NIH 3T3 cells by either tyrosine kinase. However, both a kinase-negative EGF-R point mutant (HERK721A) and an EGF-R lacking 533 C-terminal amino acids efficiently inhibited wild-type EGF-R-mediated, de novo DNA synthesis and cell transformation in a dose-dependent manner. Furthermore, coexpression with the v-erbBES4 oncogene product in NIH 3T3 cells resulted in transphosphorylation of the HERK721A mutant receptor and reduced soft-agar colony growth but had no effect in a focus formation assay. These results demonstrate that signalling-defective receptor tyrosine kinase mutants differentially interfere with oncogenic signals generated by either overexpressed EGF-R or the retroviral v-erbBES4 oncogene product. Images PMID:1346334

  4. Co-expression of pregnane X receptor and ATP-binding cassette sub-family B member 1 in peripheral blood: A prospective indicator for drug resistance prediction in non-small cell lung cancer

    PubMed Central

    KONG, QINGNUAN; HAN, ZENGLEI; ZUO, XIAOLI; WEI, HONGJUN; HUANG, WEIQING

    2016-01-01

    The aim of the present study was to investigate the protein expression profiling of pregnane X receptor (PXR) and ATP-binding cassette sub-family B member 1 (ABCB1; also known as MDR1 or P-gp), present in the peripheral blood mononuclear cells (PBMCs) and cancerous tissues of cases of non-small cell lung cancer (NSCLC). Furthermore, the study aimed to assess the feasibility of predicting drug resistance through the medium of PBMCs. Of the subjects included in the study, 37 were histopathologically diagnosed with NSCLC and 17 were control patients without cancer. ThinPrep liquid-based smears with cytosine were applied in the examination of the PBMCs and proved quite effective in preserving the morphology and surface antigens of the lymphocytes. Measurements of expression levels in the PBMCs and cancerous tissues were obtained by immunohistochemical means. The results showed that, with the exception of the selective PXR expression in the normal lung tissues, the two types of proteins existed extensively throughout the PBMCs, normal tissues and tumors. Among the cancer patients, prior to chemotherapy, a significant rise in ABCB1 expression could be observed in the PBMCs, together with a similar rise in ABCB1 and PXR expression in the tumor specimens. Marked upregulation of the two proteins was detected in the PBMCs following 1 cycle of first-line chemotherapy. ABCB1 expression, correlated with PXR, persisted mostly in the PBMCs and tissue samples. When bound to and activated by ligands, PXR translocates from the cytoplasm to the nucleus of the cells. PXR subsequently binds to its DNA response elements as a heterodimer with the retinoid X receptor. A PXR translocation of moderate or low differentiation was identified in 3 cases of adenocarcinoma, which were co-expressing the two genes in the PBMCs prior to chemotherapy. During follow-up visits, tumor recurrence was observed within 3 months in 5 cases, which were characterized by PXR translocation. These findings

  5. Co-expression of pregnane X receptor and ATP-binding cassette sub-family B member 1 in peripheral blood: A prospective indicator for drug resistance prediction in non-small cell lung cancer.

    PubMed

    Kong, Qingnuan; Han, Zenglei; Zuo, Xiaoli; Wei, Hongjun; Huang, Weiqing

    2016-05-01

    The aim of the present study was to investigate the protein expression profiling of pregnane X receptor (PXR) and ATP-binding cassette sub-family B member 1 (ABCB1; also known as MDR1 or P-gp), present in the peripheral blood mononuclear cells (PBMCs) and cancerous tissues of cases of non-small cell lung cancer (NSCLC). Furthermore, the study aimed to assess the feasibility of predicting drug resistance through the medium of PBMCs. Of the subjects included in the study, 37 were histopathologically diagnosed with NSCLC and 17 were control patients without cancer. ThinPrep liquid-based smears with cytosine were applied in the examination of the PBMCs and proved quite effective in preserving the morphology and surface antigens of the lymphocytes. Measurements of expression levels in the PBMCs and cancerous tissues were obtained by immunohistochemical means. The results showed that, with the exception of the selective PXR expression in the normal lung tissues, the two types of proteins existed extensively throughout the PBMCs, normal tissues and tumors. Among the cancer patients, prior to chemotherapy, a significant rise in ABCB1 expression could be observed in the PBMCs, together with a similar rise in ABCB1 and PXR expression in the tumor specimens. Marked upregulation of the two proteins was detected in the PBMCs following 1 cycle of first-line chemotherapy. ABCB1 expression, correlated with PXR, persisted mostly in the PBMCs and tissue samples. When bound to and activated by ligands, PXR translocates from the cytoplasm to the nucleus of the cells. PXR subsequently binds to its DNA response elements as a heterodimer with the retinoid X receptor. A PXR translocation of moderate or low differentiation was identified in 3 cases of adenocarcinoma, which were co-expressing the two genes in the PBMCs prior to chemotherapy. During follow-up visits, tumor recurrence was observed within 3 months in 5 cases, which were characterized by PXR translocation. These findings

  6. Co-expression of the transcription factors CEH-14 and TTX-1 regulates AFD neuron-specific genes gcy-8 and gcy-18 in C. elegans.

    PubMed

    Kagoshima, Hiroshi; Kohara, Yuji

    2015-03-15

    A wide variety of cells are generated by the expression of characteristic sets of genes, primarily those regulated by cell-specific transcription. To elucidate the mechanism regulating cell-specific gene expression in a highly specialized cell, AFD thermosensory neuron in Caenorhabditis elegans, we analyzed the promoter sequences of guanylyl cyclase genes, gcy-8 and gcy-18, exclusively expressed in AFD. In this study, we showed that AFD-specific expression of gcy-8 and gcy-18 requires the co-expression of homeodomain proteins, CEH-14/LHX3 and TTX-1/OTX1. We observed that mutation of ttx-1 or ceh-14 caused a reduction in the expression of gcy-8 and gcy-18 and that the expression was completely lost in double mutants. This synergy effect was also observed with other AFD marker genes, such as ntc-1, nlp-21and cng-3. Electrophoretic mobility shift assays revealed direct interaction of CEH-14 and TTX-1 proteins with gcy-8 and gcy-18 promoters in vitro. The binding sites of CEH-14 and TTX-1 proteins were confirmed to be essential for AFD-specific expression of gcy-8 and gcy-18 in vivo. We also demonstrated that forced expression of CEH-14 and TTX-1 in AWB chemosensory neurons induced ectopic expression of gcy-8 and gcy-18 reporters in this neuron. Finally, we showed that the regulation of gcy-8 and gcy-18 expression by ceh-14 and ttx-1 is evolutionally conserved in five Caenorhabditis species. Taken together, ceh-14 and ttx-1 expression determines the fate of AFD as terminal selector genes at the final step of cell specification.

  7. Fibroblast growth factor receptor 4 (FGFR4) and fibroblast growth factor 19 (FGF19) autocrine enhance breast cancer cells survival.

    PubMed

    Tiong, Kai Hung; Tan, Boon Shing; Choo, Heng Lungh; Chung, Felicia Fei-Lei; Hii, Ling-Wei; Tan, Si Hoey; Khor, Nelson Tze Woei; Wong, Shew Fung; See, Sze-Jia; Tan, Yuen-Fen; Rosli, Rozita; Cheong, Soon-Keng; Leong, Chee-Onn

    2016-09-06

    Basal-like breast cancer is an aggressive tumor subtype with poor prognosis. The discovery of underlying mechanisms mediating tumor cell survival, and the development of novel agents to target these pathways, is a priority for patients with basal-like breast cancer. From a functional screen to identify key drivers of basal-like breast cancer cell growth, we identified fibroblast growth factor receptor 4 (FGFR4) as a potential mediator of cell survival. We found that FGFR4 mediates cancer cell survival predominantly via activation of PI3K/AKT. Importantly, a subset of basal-like breast cancer cells also secrete fibroblast growth factor 19 (FGF19), a canonical ligand specific for FGFR4. siRNA-mediated silencing of FGF19 or neutralization of extracellular FGF19 by anti-FGF19 antibody (1A6) decreases AKT phosphorylation, suppresses cancer cell growth and enhances doxorubicin sensitivity only in the FGFR4+/FGF19+ breast cancer cells. Consistently, FGFR4/FGF19 co-expression was also observed in 82 out of 287 (28.6%) primary breast tumors, and their expression is strongly associated with AKT phosphorylation, Ki-67 staining, higher tumor stage and basal-like phenotype. In summary, our results demonstrated the presence of an FGFR4/FGF19 autocrine signaling that mediates the survival of a subset of basal-like breast cancer cells and suggest that inactivation of this autocrine loop may potentially serve as a novel therapeutic intervention for future treatment of breast cancers.

  8. Tissue Factor, Protease Activated Receptors and Pathologic Heart Remodeling

    PubMed Central

    Antoniak, Silvio; Sparkenbaugh, Erica; Pawlinski, Rafal

    2015-01-01

    Tissue factor is the primary initiator of coagulation cascade and plays an essential role in hemostasis and thrombosis. In addition, tissue factor and coagulation proteases contribute to the many cellular responses via activation of protease activated receptors. Heart is the organ demonstrating high levels of constitutive tissue factor expression. This review focuses on the role of tissue factor, coagulation proteases and protease activated receptors in heart hemostasis and the pathological heart remodeling associated with myocardial infarction, viral myocarditis and hypertension. PMID:25104210

  9. Epidermal Growth Factor Receptor Transactivation by the Cannabinoid Receptor (CB1) and Transient Receptor Potential Vanilloid 1 (TRPV1) Induces Differential Responses in Corneal Epithelial Cells

    DTIC Science & Technology

    2010-01-01

    Epidermal growth factor receptor transactivation by the cannabinoid receptor (CB1) and transient receptor potential vanilloid 1 ( TRPV1 ) induces...Available online 7 July 2010 Keywords: cannabinoid receptor 1 (CB1) transient receptor potential vanilloid 1 ( TRPV1 ) epidermal growth factor receptor (EGFR...release of endogenous metabolites that are cannabinoid receptor 1 (CB1) and transient receptor potential vanilloid 1 ( TRPV1 ) agonists. We determined

  10. Gene Coexpression Network Topology of Cardiac Development, Hypertrophy, and Failure

    PubMed Central

    Dewey, Frederick E.; Perez, Marco V.; Wheeler, Matthew T.; Watt, Clifton; Spin, Joshua; Langfelder, Peter; Horvath, Stephen; Hannenhalli, Sridhar; Cappola, Thomas P.; Ashley, Euan A.

    2011-01-01

    Background Network analysis techniques allow a more accurate reflection of underlying systems biology to be realized than traditional unidimensional molecular biology approaches. Here, using gene coexpression network analysis, we define the gene expression network topology of cardiac hypertrophy and failure and the extent of recapitulation of fetal gene expression programs in failing and hypertrophied adult myocardium. Methods and Results We assembled all myocardial transcript data in the Gene Expression Omnibus (n = 1617). Since hierarchical analysis revealed species had primacy over disease clustering, we focused this analysis on the most complete (murine) dataset (n = 478). Using gene coexpression network analysis, we derived functional modules, regulatory mediators and higher order topological relationships between genes and identified 50 gene co-expression modules in developing myocardium that were not present in normal adult tissue. We found that known gene expression markers of myocardial adaptation were members of upregulated modules but not hub genes. We identified ZIC2 as a novel transcription factor associated with coexpression modules common to developing and failing myocardium. Of 50 fetal gene co-expression modules, three (6%) were reproduced in hypertrophied myocardium and seven (14%) were reproduced in failing myocardium. One fetal module was common to both failing and hypertrophied myocardium. Conclusions Network modeling allows systems analysis of cardiovascular development and disease. While we did not find evidence for a global coordinated program of fetal gene expression in adult myocardial adaptation, our analysis revealed specific gene expression modules active during both development and disease and specific candidates for their regulation. PMID:21127201

  11. Expression and autoregulation of transforming growth factor beta receptor mRNA in small-cell lung cancer cell lines.

    PubMed Central

    Nørgaard, P.; Spang-Thomsen, M.; Poulsen, H. S.

    1996-01-01

    In small-cell lung cancer cell lines resistance to growth inhibition by transforming growth factor (TGF)-beta 1, was previously shown to correlate with lack of TGF-beta receptor I (RI) and II (RII) proteins. To further investigate the role of these receptors, the expression of mRNA for RI, RII and beta-glycan (RIII) was examined. The results showed that loss of RII mRNA correlated with TGF-beta 1 resistance. In contrast, RI-and beta-glycan mRNA was expressed by all cell lines, including those lacking expression of these proteins. According to Southern blot analysis, the loss of type II mRNA was not due to gross structural changes in the gene. The effect of TGF-beta 1 on expression of TGF-beta receptor mRNA (receptor autoregulation) was examined by quantitative Northern blotting in four cell lines with different expression of TGF-beta receptor proteins. In two cell lines expressing all three TGF-beta receptor proteins beta-glycan mRNA was rapidly down-regulated and this effect was sustained throughout the 24 h observation period. RI and RII mRNAs were slightly increased 24 h after treatment. In one cell line sensitive to growth inhibition by TGF-beta, 1 but lacking beta-glycan expression, and one cell line expressing only beta-glycan and thus TGF-beta 1 -resistant, no autoregulation of mRNA of either TGF-beta receptor was demonstrated. The results suggest that TGF-beta 1 regulates the expression of its receptors, in particular beta-glycan, and that this effect is dependent on co-expression of beta-glycan, RI and RII. Images Figure 1 Figure 2 Figure 4 PMID:8624260

  12. Analyzing the effects of co-expression of chick (Gallus gallus) melanocortin receptors with either chick MRAP1 or MRAP2 in CHO cells on sensitivity to ACTH(1-24) or ACTH(1-13)NH2: Implications for the avian HPA axis and avian melanocortin circuits in the hypothalamus.

    PubMed

    Thomas, Alexa L; Maekawa, Fumihiko; Kawashima, Takaharu; Sakamoto, Hirotaka; Sakamoto, Tatsuya; Davis, Perry; Dores, Robert M

    2017-09-06

    In order to better understand the roles that melanocortin receptors (cMCRs) and melanocortin-2 receptor accessory proteins (cMRAP1 and cMRAP2) play in the HPA axis and hypothalamus, adrenal gland and hypothalamus mRNA from 1day-old white leghorn chicks (Gallus gallus), were analyzed by real-time PCR. mRNA was also made for kidney, ovary, and liver. Mrap1 mRNA could be detected in adrenal tissue, but not in any of the other tissues, and mrap2 mRNA was also detected in the adrenal gland. Finally, all five melanocortin receptors mRNAs could be detected in the adrenal gland; mc2r and mc5r mRNAs were the most abundant. To evaluate any potential interactions between MRAP1 and the MCRs that may occur in adrenal cells, individual chick mcr cDNA constructs were transiently expressed in CHO cells either in the presence or absence of a chick mrap1 cDNA, and the transfected cells were stimulated with hACTH(1-24) at concentrations ranging from 10(-13)M to 10(-6)M. As expected, MC2R required co-expression with MRAP1 for functional expression; whereas, co-expression of cMC3R with cMRAP1 had no statistically significant effect on sensitivity to hACTH(1-24). However, co-expression of MC4R and MC5R with MRAP1, increased sensitivity for ACTH(1-24) by approximately 35 fold and 365 fold, respectively. However, co-expressing of cMRAP2 with these melanocortin receptors had no effect on sensitivity to hACTH(1-24). Since the real-time PCR analysis detected mrap2 mRNA and mc4r mRNA in the hypothalamus, the interaction between cMC4R and cMRAP2 with respect to sensitivity to ACTH(1-13)NH2 stimulation was also evaluated. However, no effect, either positive or negative, was observed. Finally, the highest levels of mc5r mRNA were detected in liver cells. This observation raises the possibility that in one-day old chicks, activation of the HPA axis may also involve a physiological response from liver cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Induction of nerve growth factor receptors on cultured human melanocytes

    SciTech Connect

    Peacocke, M.; Yaar, M.; Mansur, C.P.; Chao, M.V.; Gilchrest, B.A. )

    1988-07-01

    Normal differentiation and malignant transformation of human melanocytes involve a complex series of interactions during which both genetic and environmental factors play roles. At present, the regulation of these processes is poorly understood. The authors have induced the expression of nerve growth factor (NGF) receptors on cultured human melanocytes with phorbol 12-tetradecanoate 13-acetate and have correlated this event with the appearance of a more differentiated, dendritic morphology. Criteria for NGF receptor expression included protein accumulation and cell-surface immunofluorescent staining with a monoclonal antibody directed against the human receptor and induction of the messenger RNA species as determined by blot-hybridization studies. The presence of the receptor could also be induced by UV irradiation or growth factor deprivation. The NGF receptor is inducible in cultured human melanocytes, and they suggest that NGF may modulate the behavior of this neural crest-derived cell in the skin.

  14. CD95 death receptor and epidermal growth factor receptor (EGFR) in liver cell apoptosis and regeneration.

    PubMed

    Reinehr, Roland; Häussinger, Dieter

    2012-02-01

    Recent evidence suggests that signaling pathways towards cell proliferation and cell death are much more interconnected than previously thought. Whereas not only death receptors such as CD95 (Fas, APO-1) can couple to both, cell death and proliferation, also growth factor receptors such as the epidermal growth factor receptor (EGFR) are involved in these opposing kinds of cell fate. EGFR is briefly discussed as a growth factor receptor involved in liver cell proliferation during liver regeneration. Then the role of EGFR in activating CD95 death receptor in liver parenchymal cells (PC) and hepatic stellate cells (HSC), which represent a liver stem/progenitor cell compartment, is described summarizing different ways of CD95- and EGFR-dependent signaling in the liver. Here, depending on the hepatic cell type (PC vs. HSC) and the respective signaling context (sustained vs. transient JNK activation) CD95-/EGFR-mediated signaling ends up in either liver cell apoptosis or cell proliferation.

  15. Activation of 5-HT7 receptors increases neuronal platelet-derived growth factor β receptor expression.

    PubMed

    Vasefi, Maryam S; Kruk, Jeff S; Liu, Hui; Heikkila, John J; Beazely, Michael A

    2012-03-09

    Several antipsychotics have a high affinity for 5-HT7 receptors yet despite intense interest in the 5-HT7 receptor as a potential drug target to treat psychosis, the function and signaling properties of 5-HT7 receptors in neurons remain largely uncharacterized. In primary mouse hippocampal and cortical neurons, as well as in the SH-SY5Y cell line, incubation with 5-HT, 5-carboxamidotryptamine (5-CT), or 5-HT7 receptor-selective agonists increases the expression of platelet-derived growth factor (PDGF)β receptors. The increased PDGFβ receptor expression is cyclic AMP-dependent protein kinase (PKA)-dependent, suggesting that 5-HT7 receptors couple to Gα(s) in primary neurons. Interestingly, up-regulated PDGFβ receptors display an increased basal phosphorylation state at the phospholipase Cγ-activating tyrosine 1021. This novel linkage between the 5-HT7 receptor and the PDGF system may be an important GPCR-neurotrophic factor signaling pathway in neurons.

  16. PLANEX: the plant co-expression database.

    PubMed

    Yim, Won Cheol; Yu, Yongbin; Song, Kitae; Jang, Cheol Seong; Lee, Byung-Moo

    2013-05-20

    The PLAnt co-EXpression database (PLANEX) is a new internet-based database for plant gene analysis. PLANEX (http://planex.plantbioinformatics.org) contains publicly available GeneChip data obtained from the Gene Expression Omnibus (GEO) of the National Center for Biotechnology Information (NCBI). PLANEX is a genome-wide co-expression database, which allows for the functional identification of genes from a wide variety of experimental designs. It can be used for the characterization of genes for functional identification and analysis of a gene's dependency among other genes. Gene co-expression databases have been developed for other species, but gene co-expression information for plants is currently limited. We constructed PLANEX as a list of co-expressed genes and functional annotations for Arabidopsis thaliana, Glycine max, Hordeum vulgare, Oryza sativa, Solanum lycopersicum, Triticum aestivum, Vitis vinifera and Zea mays. PLANEX reports Pearson's correlation coefficients (PCCs; r-values) that distribute from a gene of interest for a given microarray platform set corresponding to a particular organism. To support PCCs, PLANEX performs an enrichment test of Gene Ontology terms and Cohen's Kappa value to compare functional similarity for all genes in the co-expression database. PLANEX draws a cluster network with co-expressed genes, which is estimated using the k-mean method. To construct PLANEX, a variety of datasets were interpreted by the IBM supercomputer Advanced Interactive eXecutive (AIX) in a supercomputing center. PLANEX provides a correlation database, a cluster network and an interpretation of enrichment test results for eight plant species. A typical co-expressed gene generates lists of co-expression data that contain hundreds of genes of interest for enrichment analysis. Also, co-expressed genes can be identified and cataloged in terms of comparative genomics by using the 'Co-expression gene compare' feature. This type of analysis will help interpret

  17. Co-expression of Japanese encephalitis virus prM-E-NS1 antigen with granulocyte-macrophage colony-stimulating factor enhances humoral and anti-virus immunity after DNA vaccination.

    PubMed

    Gao, Na; Chen, Wei; Zheng, Qun; Fan, Dong-ying; Zhang, Jun-lei; Chen, Hui; Gao, George F; Zhou, De-shan; An, Jing

    2010-03-10

    Japanese encephalitis virus (JEV) is an agent of Japanese encephalitis, and granulocyte-macrophage colony-stimulating factor (GM-CSF) is an attractive DNA vaccine adjuvant for its antigen presentation. In the present study, we have constructed DNA vaccines that carried JEV prM-E-NS1 genes with or without the GM-CSF gene. Immunization with the bicistronic plasmid pCAG-JEGM that co-expresses GM-CSF and viral prM-E-NS1, resulted in the highest IgG response and sufficient protection against virus-challenged BALB/c mice. However, much to our surprise, co-inoculation of the GM-CSF plasmid with the pCAG-JE plasmid expressing viral prM-E-NS1 lead to a low antibody titer and a relatively low survival rate. Moreover, anamnestic antibody-mediated protection played a dominant role in the mice JEV challenge model, according to the enhancement of post-challenge neutralizing antibody titers and further adoptive transfer experiments. Taken together, this study should encourage further development of JEV DNA vaccine strategies and caution against the use of cytokines as an adjuvant.

  18. Novel Drosophila receptor that binds multiple growth factors

    SciTech Connect

    Rosner, M.R.; Thompson, K.L.; Garcia, V.; Decker, S.J.

    1986-05-01

    The authors have recently reported the identification of a novel growth factor receptor from Drosophila cell cultures that has dual binding specificity for both insulin and epidermal growth factor (EGF). This 100 kDa protein is also antigenically related to the cytoplasmic region of the mammalian EGF receptor-tyrosine kinase. They now report that this protein binds to mammalian nerve growth factor and human transforming growth factor alpha as well as insulin and EGF with apparent dissociation constants ranging from 10/sup -6/ to 10/sup -8/ M. The 100 kDa protein can be affinity-labeled with these /sup 125/I-labeled growth factors after immunoprecipitation with anti-EGF receptor antiserum. These four growth factors appear to share a common binding site, as evidenced by their ability to block affinity labelling by /sup 125/I-insulin. No significant binding to the 100 kDa protein was observed with platelet-derived growth factor, transforming growth factor beta, or glucagon. The 100 kDa Drosophila protein has a unique ligand-binding spectrum with no direct counterpart in mammalian cells and may represent an evolutionary precursor of the mammalian receptors for these growth factors.

  19. ATTED-II in 2016: A Plant Coexpression Database Towards Lineage-Specific Coexpression.

    PubMed

    Aoki, Yuichi; Okamura, Yasunobu; Tadaka, Shu; Kinoshita, Kengo; Obayashi, Takeshi

    2016-01-01

    ATTED-II (http://atted.jp) is a coexpression database for plant species with parallel views of multiple coexpression data sets and network analysis tools. The user can efficiently find functional gene relationships and design experiments to identify gene functions by reverse genetics and general molecular biology techniques. Here, we report updates to ATTED-II (version 8.0), including new and updated coexpression data and analysis tools. ATTED-II now includes eight microarray- and six RNA sequencing-based coexpression data sets for seven dicot species (Arabidopsis, field mustard, soybean, barrel medick, poplar, tomato and grape) and two monocot species (rice and maize). Stand-alone coexpression analyses tend to have low reliability. Therefore, examining evolutionarily conserved coexpression is a more effective approach from the viewpoints of reliability and evolutionary importance. In contrast, the reliability of species-specific coexpression data remains poor. Our assessment scores for individual coexpression data sets indicated that the quality of the new coexpression data sets in ATTED-II is higher than for any previous coexpression data set. In addition, five species (Arabidopsis, soybean, tomato, rice and maize) in ATTED-II are now supported by both microarray- and RNA sequencing-based coexpression data, which has increased the reliability. Consequently, ATTED-II can now provide lineage-specific coexpression information. As an example of the use of ATTED-II to explore lineage-specific coexpression, we demonstrate monocot- and dicot-specific coexpression of cell wall genes. With the expanded coexpression data for multilevel evaluation, ATTED-II provides new opportunities to investigate lineage-specific evolution in plants. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

  20. Roles of hepatocyte growth factor/scatter factor and the met receptor in the early development of the metanephros

    PubMed Central

    1995-01-01

    Several lines of evidence suggest that hepatocyte growth factor/scatter factor (HGF/SF), a soluble protein secreted by embryo fibroblasts and several fibroblast lines, may elicit morphogenesis in adjacent epithelial cells. We investigated the role of HGF/SF and its membrane receptor, the product of the c-met protooncogene, in the early development of the metanephric kidney. At the inception of the mouse metanephros at embryonic day 11, HGF/SF was expressed in the mesenchyme, while met was expressed in both the ureteric bud and the mesenchyme, as assessed by reverse transcription PCR, in situ hybridization, and immunohistochemistry. To further investigate the expression of met in renal mesenchyme, we isolated 13 conditionally immortal clonal cell lines from transgenic mice expressing a temperature-sensitive mutant of the SV-40 large T antigen. Five had the HGF/SF+/met+ phenotype and eight had the HGF/SF-/met+ phenotype. None had the HGF/SF+/met- nor the HGF/SF-/met- phenotypes. Thus the renal mesenchyme contains cells that express HGF/SF and met or met alone. When metanephric rudiments were grown in serum-free organ culture, anti- HGF/SF antibodies (a) inhibited the differentiation of metanephric mesenchymal cells into the epithelial precursors of the nephron; (b) increased cell death within the renal mesenchyme; and (c) perturbed branching morphogenesis of the ureteric bud. These data provide the first demonstration for coexpression of the HGF/SF and met genes in mesenchymal cells during embryonic development and also imply an autocrine and/or paracrine role for HGF/SF and met in the survival of the renal mesenchyme and in the mesenchymal-epithelial transition that occurs during nephrogenesis. They also confirm the postulated paracrine role of HGF/SF in the branching of the ureteric bud. PMID:7822413

  1. Dynamic co-expression network analysis of lncRNAs and mRNAs associated with venous congestion

    PubMed Central

    Li, Jinshun; Xu, Yuqin; Xu, Jia; Wang, Jinhua; Wu, Liying

    2016-01-01

    Venous congestion and volume overload are important in cardiorenal syndromes, in which multiple regulated factors are involved, including long non-coding RNAs (lncRNAs). To investigate the underlying role of lncRNAs in regulating the development of venous congestion, an Affymetrix microarray associated with peripheral venous congestion was annotated, then a bipartite dynamic lncRNA-mRNA co-expression network was constructed in which nodes indicated lncRNAs or mRNAs. The nodes were connected when the lncRNAs or mRNAs were dynamically co-expressed. Following functional analysis of this network, several dynamic alternative pathways were identified, including the calcium signaling pathway during venous congestion development. Additionally, certain lncRNAs (LINC00523, LINC01210 and RP11-435O5.5) were identified that may potentially dynamically regulate certain proteins, including plasma membrane calcium ATPase (PMCA) and G protein-coupled receptor (GPCR), in the calcium signaling pathway. Particularly, the dynamically regulated switch of LINC00523 from co-expression with PMCA to GPCR may be involved in damage to steady state intracellular calcium. In brief, the current study demonstrated a potential novel mechanism of lncRNA function during venous congestion. PMID:27431002

  2. Investigating the Combinatory Effects of Biological Networks on Gene Co-expression

    PubMed Central

    Zhang, Cheng; Lee, Sunjae; Mardinoglu, Adil; Hua, Qiang

    2016-01-01

    Co-expressed genes often share similar functions, and gene co-expression networks have been widely used in studying the functionality of gene modules. Previous analysis indicated that genes are more likely to be co-expressed if they are either regulated by the same transcription factors, forming protein complexes or sharing similar topological properties in protein-protein interaction networks. Here, we reconstructed transcriptional regulatory and protein-protein networks for Saccharomyces cerevisiae using well-established databases, and we evaluated their co-expression activities using publically available gene expression data. Based on our network-dependent analysis, we found that genes that were co-regulated in the transcription regulatory networks and shared similar neighbors in the protein-protein networks were more likely to be co-expressed. Moreover, their biological functions were closely related. PMID:27445830

  3. A novel computational approach for the prediction of networked transcription factors of aryl hydrocarbon-receptor-regulated genes.

    PubMed

    Kel, Alexander; Reymann, Susanne; Matys, Volker; Nettesheim, Paul; Wingender, Edgar; Borlak, Jürgen

    2004-12-01

    A novel computational method based on a genetic algorithm was developed to study composite structure of promoters of coexpressed genes. Our method enabled an identification of combinations of multiple transcription factor binding sites regulating the concerted expression of genes. In this article, we study genes whose expression is regulated by a ligand-activated transcription factor, aryl hydrocarbon receptor (AhR), that mediates responses to a variety of toxins. AhR-mediated change in expression of AhR target genes was measured by oligonucleotide microarrays and by reverse transcription-polymerase chain reaction in human and rat hepatocytes. Promoters and long-distance regulatory regions (>10 kb) of AhR-responsive genes were analyzed by the genetic algorithm and a variety of other computational methods. Rules were established on the local oligonucleotide context in the flanks of the AhR binding sites, on the occurrence of clusters of AhR recognition elements, and on the presence in the promoters of specific combinations of multiple binding sites for the transcription factors cooperating in the AhR regulatory network. Our rules were applied to search for yet unknown Ah-receptor target genes. Experimental evidence is presented to demonstrate high fidelity of this novel in silico approach.

  4. Topical administration of adrenergic receptor pharmaceutics and nerve growth factor

    PubMed Central

    Steinle, Jena J

    2010-01-01

    Topical application of nerve growth factor (NGF) and adrenergic receptor pharmaceutics are currently in use for corneal ulcers and glaucoma. A recent interest in the neuroprotective abilities of NGF has led to a renewed interest in NGF as a therapeutic for retinal and choroidal diseases. NGF can promote cell proliferation through actions of the TrkA receptor or promote apoptosis through receptor p75NTR. This understanding has led to novel interest in the role of NGF for diseases of the posterior eye. The role of β-adrenergic receptor agonists and antagonists for treatments of glaucoma, diabetic retinopathy, and their potential mechanisms of action, are still under investigation. This review discusses the current knowledge and applications of topical NGF and adrenergic receptor drugs for ocular disease. PMID:20668722

  5. The epidermal growth factor receptor family: Biology driving targeted therapeutics

    PubMed Central

    Wieduwilt, M. J.; Moasser, M. M.

    2011-01-01

    The epidermal growth factor family of receptor tyrosine kinases (ErbBs) plays essential roles in regulating cell proliferation, survival, differentiation and migration. The ErbB receptors carry out both redundant and restricted functions in mammalian development and in the maintenance of tissues in the adult mammal. Loss of regulation of the ErbB receptors underlies many human diseases, most notably cancer. Our understanding of the function and complex regulation of these receptors has fueled the development of targeted therapeutic agents for human malignancies in the last 15 years. Here we review the biology of ErbB receptors, including their structure, signaling, regulation, and roles in development and disease, then briefly touch on their increasing roles as targets for cancer therapy. PMID:18259690

  6. Understanding Cytokine and Growth Factor Receptor Activation Mechanisms

    PubMed Central

    Atanasova, Mariya; Whitty, Adrian

    2012-01-01

    Our understanding of the detailed mechanism of action of cytokine and growth factor receptors – and particularly our quantitative understanding of the link between structure, mechanism and function – lags significantly behind our knowledge of comparable functional protein classes such as enzymes, G protein-coupled receptors, and ion channels. In particular, it remains controversial whether such receptors are activated by a mechanism of ligand-induced oligomerization, versus a mechanism in which the ligand binds to a pre-associated receptor dimer or oligomer that becomes activated through subsequent conformational rearrangement. A major limitation to progress has been the relative paucity of methods for performing quantitative mechanistic experiments on unmodified receptors expressed at endogenous levels on live cells. In this article we review the current state of knowledge on the activation mechanisms of cytokine and growth factor receptors, critically evaluate the evidence for and against the different proposed mechanisms, and highlight other key questions that remain unanswered. New approaches and techniques have led to rapid recent progress in this area, and the field is poised for major advances in the coming years, which promises to revolutionize our understanding of this large and biologically and medically important class of receptors. PMID:23046381

  7. Astrocyte Mitogen Inhibitor Related to Epidermal Growth Factor Receptor

    NASA Astrophysics Data System (ADS)

    Nieto-Sampedro, Manuel

    1988-06-01

    Epidermal growth factor (EGF) is a well-characterized polypeptide hormone with diverse biological activities, including stimulation of astrocyte division. A soluble astrocyte mitogen inhibitor, immunologically related to the EGF receptor, is present in rat brain. Injury to the brain causes a time-dependent reduction in the levels of this inhibitor and the concomitant appearance of EGF receptor on the astrocyte surface. Intracerebral injection of antibody capable of binding the inhibitor caused the appearance of numerous reactive astrocytes. EGF receptor-related inhibitors may play a key role in the control of glial cell division in both normal and injured brain.

  8. Voltage-gated ion channel Kv4.3 is associated with Rap guanine nucleotide exchange factors and regulates angiotensin receptor type 1 signaling to small G-protein Rap.

    PubMed

    Potapova, Irina A; Cohen, Ira S; Doronin, Sergey V

    2007-09-01

    The voltage-gated potassium channel Kv4.3 was coexpressed with its beta-subunit Kv channel-interacting protein 2 and the angiotensin type 1 receptor in HEK-293 cells. Proteomic analysis of proteins coimmunoprecipitated with Kv4.3 revealed that Kv4.3 is associated with Rap guanine nucleotide exchange factors MR-GEF and EPAC-1. Previously, we demonstrated that Kv4.3 interacts with the angiotensin type 1 receptor in HE293 cells and cardiac myocytes. On the basis of this, we investigated the angiotensin type 1 receptor signaling to small G-proteins Ras and Rap-1 in the presence and absence of the Kv4.3-Kv channel-interacting protein 2 macromolecular complex. Ras activation was not significantly affected by coexpression of Kv4.3 and Kv channel-interacting protein 2. Ras exhibited a rapid activation-inactivation pattern with maximum activity at 2.5 min after addition of angiotensin II. In contrast, activation of Rap-1 was affected dramatically by coexpression of Kv4.3 and Kv channel-interacting protein 2 with the angiotensin type 1 receptor. In the absence of Kv4.3 and Kv channel-interacting protein 2, stimulation of the angiotensin type 1 receptor resulted in steady activation of Rap-1 that reached a plateau 25 min after addition of angiotensin II. In the presence of Kv4.3 and Kv channel-interacting protein 2, Rap-1 reaches a maximum activity 2.5 min after addition of angiotensin II and then deactivates rapidly, demonstrating a pattern of activation similar to that of Ras. Our findings show that Kv4.3 regulates angiotensin type 1 receptor signaling to the small G-protein Rap-1.

  9. Linking Hematopoietic Differentiation to Co-Expressed Sets of Pluripotency-Associated and Imprinted Genes and to Regulatory microRNA-Transcription Factor Motifs

    PubMed Central

    Hamed, Mohamed; Trumm, Johannes; Spaniol, Christian; Sethi, Riccha; Irhimeh, Mohammad R.; Fuellen, Georg; Paulsen, Martina

    2017-01-01

    Maintenance of cell pluripotency, differentiation, and reprogramming are regulated by complex gene regulatory networks (GRNs) including monoallelically-expressed imprinted genes. Besides transcriptional control, epigenetic modifications and microRNAs contribute to cellular differentiation. As a model system for studying the capacity of cells to preserve their pluripotency state and the onset of differentiation and subsequent specialization, murine hematopoiesis was used and compared to embryonic stem cells (ESCs) as a control. Using published microarray data, the expression profiles of two sets of genes, pluripotent and imprinted, were compared to a third set of known hematopoietic genes. We found that more than half of the pluripotent and imprinted genes are clearly upregulated in ESCs but subsequently repressed during hematopoiesis. The remaining genes were either upregulated in hematopoietic progenitors or in differentiated blood cells. The three gene sets each consist of three similarly behaving gene groups with similar expression profiles in various lineages of the hematopoietic system as well as in ESCs. To explain this co-regulation behavior, we explored the transcriptional and post-transcriptional mechanisms of pluripotent and imprinted genes and their regulator/target miRNAs in six different hematopoietic lineages. Therewith, lineage-specific transcription factor (TF)-miRNA regulatory networks were generated and their topologies and functional impacts during hematopoiesis were analyzed. This led to the identification of TF-miRNA co-regulatory motifs, for which we validated the contribution to the cellular development of the corresponding lineage in terms of statistical significance and relevance to biological evidence. This analysis also identified key miRNAs and TFs/genes that might play important roles in the derived lineage networks. These molecular associations suggest new aspects of the cellular regulation of the onset of cellular differentiation and

  10. Modulation of the NMDA Receptor Through Secreted Soluble Factors.

    PubMed

    Cerpa, Waldo; Ramos-Fernández, Eva; Inestrosa, Nibaldo C

    2016-01-01

    Synaptic activity is a critical determinant in the formation and development of excitatory synapses in the central nervous system (CNS). The excitatory current is produced and regulated by several ionotropic receptors, including those that respond to glutamate. These channels are in turn regulated through several secreted factors that function as synaptic organizers. Specifically, Wnt, brain-derived neurotrophic factor (BDNF), fibroblast growth factor (FGF), and transforming growth factor (TGF) particularly regulate the N-methyl-D-aspartate receptor (NMDAR) glutamatergic channel. These factors likely regulate early embryonic development and directly control key proteins in the function of important glutamatergic channels. Here, we review the secreted molecules that participate in synaptic organization and discuss the cell signaling behind of this fine regulation. Additionally, we discuss how these factors are dysregulated in some neuropathologies associated with glutamatergic synaptic transmission in the CNS.

  11. A glioma classification scheme based on coexpression modules of EGFR and PDGFRA.

    PubMed

    Sun, Yingyu; Zhang, Wei; Chen, Dongfeng; Lv, Yuhong; Zheng, Junxiong; Lilljebjörn, Henrik; Ran, Liang; Bao, Zhaoshi; Soneson, Charlotte; Sjögren, Hans Olov; Salford, Leif G; Ji, Jianguang; French, Pim J; Fioretos, Thoas; Jiang, Tao; Fan, Xiaolong

    2014-03-04

    We hypothesized that key signaling pathways of glioma genesis might enable the molecular classification of gliomas. Gene coexpression modules around epidermal growth factor receptor (EGFR) (EM, 29 genes) or platelet derived growth factor receptor A (PDGFRA) (PM, 40 genes) in gliomas were identified. Based on EM and PM expression signatures, nonnegative matrix factorization reproducibly clustered 1,369 adult diffuse gliomas WHO grades II-IV from four independent databases generated in three continents, into the subtypes (EM, PM and EM(low)PM(low) gliomas) in a morphology-independent manner. Besides their distinct patterns of genomic alterations, EM gliomas were associated with higher age at diagnosis, poorer prognosis, and stronger expression of neural stem cell and astrogenesis genes. Both PM and EM(low)PM(low) gliomas were associated with younger age at diagnosis and better prognosis. PM gliomas were enriched in the expression of oligodendrogenesis genes, whereas EM(low)PM(low) gliomas were enriched in the signatures of mature neurons and oligodendrocytes. The EM/PM-based molecular classification scheme is applicable to adult low-grade and high-grade diffuse gliomas, and outperforms existing classification schemes in assigning diffuse gliomas to subtypes with distinct transcriptomic and genomic profiles. The majority of the EM/PM classifiers, including regulators of glial fate decisions, have not been extensively studied in glioma biology. Subsets of these classifiers were coexpressed in mouse glial precursor cells, and frequently amplified or lost in an EM/PM glioma subtype-specific manner, resulting in somatic copy number alteration-dependent gene expression that contributes to EM/PM signatures in glioma samples. EM/PM-based molecular classification provides a molecular diagnostic framework to expedite the search for new glioma therapeutic targets.

  12. CXCL13-CXCR5 co-expression regulates epithelial to mesenchymal transition of breast cancer cells during lymph node metastasis.

    PubMed

    Biswas, Subir; Sengupta, Suman; Roy Chowdhury, Sougata; Jana, Samir; Mandal, Gunjan; Mandal, Palash Kumar; Saha, Nipun; Malhotra, Vivek; Gupta, Arnab; Kuprash, Dmitry V; Bhattacharyya, Arindam

    2014-01-01

    We investigated the expression of -CXC chemokine ligand 13 (CXCL13) and its receptor -CXC chemokine receptor 5 (CXCR5) in 98 breast cancer (BC) patients with infiltrating duct carcinoma, out of which 56 were found lymph node metastasis (LNM) positive. Interestingly, co-expression of CXCL13 and CXCR5 showed a significant correlation with LNM. Since, epithelial to mesenchymal transition (EMT) is highly associated with metastasis we investigated EMT-inducing potential of CXCL13 in BC cell lines. In CXCL13-stimulated BC cells, expression of various mesenchymal markers (Vimentin, N-cadherin), EMT regulators (Snail, Slug), and matrix metalloproteinase-9 (MMP9) was increased, whereas the expression of epithelial marker E-cadherin was found to be decreased. In addition, expression of receptor activator of nuclear factor kappa-B ligand (RANKL), which is known to regulate MMP9 expression via Src activation, was also significantly increased after CXCL13 stimulation. Using specific protein kinase inhibitors, we confirmed that CXCL13 stimulated EMT and MMP9 expression via RANKL-Src axis in BC cell lines. To further validate this observation, we examined gene expression patterns in primary breast tumors and detected significantly higher expression of various mesenchymal markers and regulators in CXCL13-CXCR5 co-expressing patients. Therefore, this study showed the EMT-inducing potential of CXCL13 as well as demonstrated the prognostic value of CXCL13-CXCR5 co-expression in primary BC. Moreover, CXCL13-CXCR5-RANKL-Src axis may present a therapeutic target in LNM positive BC patients.

  13. Platelet-activating factor: receptors and signal transduction.

    PubMed

    Chao, W; Olson, M S

    1993-06-15

    During the past two decades, studies describing the chemistry and biology of PAF have been extensive. This potent phosphoacylglycerol exhibits a wide variety of physiological and pathophysiological effects in various cells and tissues. PAF acts, through specific receptors and a variety of signal transduction systems, to elicit diverse biochemical responses. Several important future directions can be enumerated for the characterization of PAF receptors and their attendant signalling mechanisms. The recent cloning and sequence analysis of the gene for the PAF receptor will allow a number of important experimental approaches for characterizing the structure and analysing the function of the various domains of the receptor. Using molecular genetic and immunological technologies, questions relating to whether there is receptor heterogeneity, the precise mechanism(s) for the regulation of the PAF receptor, and the molecular details of the signalling mechanisms in which the PAF receptor is involved can be explored. Another area of major significance is the examination of the relationship between the signalling response(s) evoked by PAF binding to its receptor and signalling mechanisms activated by a myriad of other mediators, cytokines and growth factors. A very exciting recent development in which PAF receptors undoubtedly play a role is in the regulation of the function of various cellular adhesion molecules. Finally, there remain many incompletely characterized physiological and pathophysiological situations in which PAF and its receptor play a crucial signalling role. Our laboratory has been active in the elucidation of several tissue responses in which PAF exhibits major autocoid signalling responses, e.g. hepatic injury and inflammation, acute and chronic pancreatitis, and cerebral stimulation and/or trauma. As new experimental strategies are developed for characterizing the fine structure of the molecular mechanisms involved in tissue injury and inflammation, the

  14. ALCOdb: Gene Coexpression Database for Microalgae.

    PubMed

    Aoki, Yuichi; Okamura, Yasunobu; Ohta, Hiroyuki; Kinoshita, Kengo; Obayashi, Takeshi

    2016-01-01

    In the era of energy and food shortage, microalgae have gained much attention as promising sources of biofuels and food ingredients. However, only a small fraction of microalgal genes have been functionally characterized. Here, we have developed the Algae Gene Coexpression database (ALCOdb; http://alcodb.jp), which provides gene coexpression information to survey gene modules for a function of interest. ALCOdb currently supports two model algae: the green alga Chlamydomonas reinhardtii and the red alga Cyanidioschyzon merolae. Users can retrieve coexpression information for genes of interest through three unique data pages: (i) Coexpressed Gene List; (ii) Gene Information; and (iii) Coexpressed Gene Network. In addition to the basal coexpression information, ALCOdb also provides several advanced functionalities such as an expression profile viewer and a differentially expressed gene search tool. Using these user interfaces, we demonstrated that our gene coexpression data have the potential to detect functionally related genes and are useful in extrapolating the biological roles of uncharacterized genes. ALCOdb will facilitate molecular and biochemical studies of microalgal biological phenomena, such as lipid metabolism and organelle development, and promote the evolutionary understanding of plant cellular systems.

  15. ALCOdb: Gene Coexpression Database for Microalgae

    PubMed Central

    Aoki, Yuichi; Okamura, Yasunobu; Ohta, Hiroyuki; Kinoshita, Kengo; Obayashi, Takeshi

    2016-01-01

    In the era of energy and food shortage, microalgae have gained much attention as promising sources of biofuels and food ingredients. However, only a small fraction of microalgal genes have been functionally characterized. Here, we have developed the Algae Gene Coexpression database (ALCOdb; http://alcodb.jp), which provides gene coexpression information to survey gene modules for a function of interest. ALCOdb currently supports two model algae: the green alga Chlamydomonas reinhardtii and the red alga Cyanidioschyzon merolae. Users can retrieve coexpression information for genes of interest through three unique data pages: (i) Coexpressed Gene List; (ii) Gene Information; and (iii) Coexpressed Gene Network. In addition to the basal coexpression information, ALCOdb also provides several advanced functionalities such as an expression profile viewer and a differentially expressed gene search tool. Using these user interfaces, we demonstrated that our gene coexpression data have the potential to detect functionally related genes and are useful in extrapolating the biological roles of uncharacterized genes. ALCOdb will facilitate molecular and biochemical studies of microalgal biological phenomena, such as lipid metabolism and organelle development, and promote the evolutionary understanding of plant cellular systems. PMID:26644461

  16. Expression of leukemia inhibitory factor and leukemia inhibitory factor receptor in the canine pituitary gland and corticotrope adenomas.

    PubMed

    Hanson, J M; Mol, J A; Meij, B P

    2010-05-01

    Leukemia inhibitory factor (LIF) is a pleiotropic cytokine of the IL-6 family that activates the hypothalamic-pituitary-adrenal axis and promotes corticotrope cell differentiation during development. The aim of this study was to investigate the expression of LIF and its receptor (LIFR) in the canine pituitary gland and in corticotrope adenomas, and to perform a mutation analysis of LIFR. Using immunohistochemistry, immunofluorescence, and quantitative expression analysis, LIF and LIFR expression were studied in pituitary glands of control dogs and in specimens of corticotrope adenoma tissue collected through hypophysectomy in dogs with pituitary-dependent hypercortisolism (PDH, Cushing's disease). Using sequence analysis, cDNA was screened for mutations in the LIFR. In the control pituitary tissues and corticotrope adenomas, there was a low magnitude of LIF expression. The LIFR, however, was highly expressed and co-localized with ACTH(1-24) expression. Cytoplasmatic immunoreactivity of LIFR was preserved in corticotrope adenomas and adjacent nontumorous cells of pars intermedia. No mutation was found on mutation analysis of the complete LIFR cDNA. Surprisingly, nuclear to perinuclear immunoreactivity for LIFR was present in nontumorous pituitary cells of the pars distalis in 10 of 12 tissue specimens from PDH dogs. These data show that LIFR is highly co-expressed with adrenocorticotropic hormone (ACTH) and alpha-melanocyte-stimulating hormone (alpha-MSH) in the canine pituitary gland and in corticotrope adenomas. Nuclear immunoreactivity for LIFR in nontumorous cells of the pars distalis may indicate the presence of a corticotrope adenoma. Copyright (c) 2009 Elsevier Inc. All rights reserved.

  17. Placental growth factor and vascular endothelial growth factor receptor-2 in human lung development.

    PubMed

    Janér, Joakim; Andersson, Sture; Haglund, Caj; Karikoski, Riitta; Lassus, Patrik

    2008-08-01

    We examined the pulmonary expression of 2 proangiogenic factors, namely, placental growth factor and vascular endothelial growth factor receptor-2, during lung development and acute and chronic lung injury in newborn infants. Six groups were included in an immunohistochemical study of placental growth factor and vascular endothelial growth factor receptor-2, that is, 9 fetuses, 4 preterm and 8 term infants without lung injury who died soon after birth, 5 preterm infants with respiratory distress syndrome of <2 days and 7 with respiratory distress syndrome of >10 days, and 6 with bronchopulmonary dysplasia. Placental growth factor concentrations in tracheal aspirate fluid were measured in 70 samples from 20 preterm infants during the first postnatal week. In immunohistochemical analyses, placental growth factor staining was seen in bronchial epithelium and macrophages in all groups. Distal airway epithelium positivity was observed mostly in fetuses and in preterm infants who died soon after birth. Vascular endothelial growth factor receptor-2 staining was seen in vascular endothelium in all groups and also in lymphatic endothelium in fetuses. Vascular endothelial growth factor receptor-2 staining in arterial endothelium was associated with higher and staining in venous endothelium with lower gestational age. In capillaries, less vascular endothelial growth factor receptor-2 staining was seen in bronchopulmonary dysplasia. The mean placental growth factor protein concentration in tracheal aspirate fluid during the first postnatal week was 0.64 +/- 0.42 pg/mL per IgA-secretory component unit. Concentrations during the first postnatal week were stable. Lower placental growth factor concentrations correlated with chorioamnionitis and lactosyl ceramide positivity. The vascular endothelial growth factor receptor-2 staining pattern seems to reflect ongoing differentiation and activity of different endothelia. Lower vascular endothelial growth factor receptor-2 expression

  18. Cell and molecular biology of epidermal growth factor receptor.

    PubMed

    Ceresa, Brian P; Peterson, Joanne L

    2014-01-01

    The epidermal growth factor receptor (EGFR) has been one of the most intensely studied cell surface receptors due to its well-established roles in developmental biology, tissue homeostasis, and cancer biology. The EGFR has been critical for creating paradigms for numerous aspects of cell biology, such as ligand binding, signal transduction, and membrane trafficking. Despite this history of discovery, there is a continual stream of evidence that only the surface has been scratched. New ways of receptor regulation continue to be identified, each of which is a potential molecular target for manipulating EGFR signaling and the resultant changes in cell and tissue biology. This chapter is an update on EGFR-mediated signaling, and describes some recent developments in the regulation of receptor biology.

  19. Functional interaction between co-expressed MAGE-A proteins

    PubMed Central

    Laiseca, Julieta E.; Ladelfa, María F.; Cotignola, Javier; Peche, Leticia Y.; Pascucci, Franco A.; Castaño, Bryan A.; Galigniana, Mario D.; Schneider, Claudio

    2017-01-01

    MAGE-A (Melanoma Antigen Genes-A) are tumor-associated proteins with expression in a broad spectrum of human tumors and normal germ cells. MAGE-A gene expression and function are being increasingly investigated to better understand the mechanisms by which MAGE proteins collaborate in tumorigenesis and whether their detection could be useful for disease prognosis purposes. Alterations in epigenetic mechanisms involved in MAGE gene silencing cause their frequent co-expression in tumor cells. Here, we have analyzed the effect of MAGE-A gene co-expression and our results suggest that MageA6 can potentiate the androgen receptor (AR) co-activation function of MageA11. Database search confirmed that MageA11 and MageA6 are co-expressed in human prostate cancer samples. We demonstrate that MageA6 and MageA11 form a protein complex resulting in the stabilization of MageA11 and consequently the enhancement of AR activity. The mechanism involves association of the Mage A6-MHD domain to MageA11, prevention of MageA11 ubiquitinylation on lysines 240 and 245 and decreased proteasome-dependent degradation. We experimentally demonstrate here for the first time that two MAGE-A proteins can act together in a non-redundant way to potentiate a specific oncogenic function. Overall, our results highlight the complexity of the MAGE gene networking in regulating cancer cell behavior. PMID:28542476

  20. Functional interaction between co-expressed MAGE-A proteins.

    PubMed

    Laiseca, Julieta E; Ladelfa, María F; Cotignola, Javier; Peche, Leticia Y; Pascucci, Franco A; Castaño, Bryan A; Galigniana, Mario D; Schneider, Claudio; Monte, Martin

    2017-01-01

    MAGE-A (Melanoma Antigen Genes-A) are tumor-associated proteins with expression in a broad spectrum of human tumors and normal germ cells. MAGE-A gene expression and function are being increasingly investigated to better understand the mechanisms by which MAGE proteins collaborate in tumorigenesis and whether their detection could be useful for disease prognosis purposes. Alterations in epigenetic mechanisms involved in MAGE gene silencing cause their frequent co-expression in tumor cells. Here, we have analyzed the effect of MAGE-A gene co-expression and our results suggest that MageA6 can potentiate the androgen receptor (AR) co-activation function of MageA11. Database search confirmed that MageA11 and MageA6 are co-expressed in human prostate cancer samples. We demonstrate that MageA6 and MageA11 form a protein complex resulting in the stabilization of MageA11 and consequently the enhancement of AR activity. The mechanism involves association of the Mage A6-MHD domain to MageA11, prevention of MageA11 ubiquitinylation on lysines 240 and 245 and decreased proteasome-dependent degradation. We experimentally demonstrate here for the first time that two MAGE-A proteins can act together in a non-redundant way to potentiate a specific oncogenic function. Overall, our results highlight the complexity of the MAGE gene networking in regulating cancer cell behavior.

  1. Vascular growth factors and receptors in capillary hemangioblastomas and hemangiopericytomas.

    PubMed Central

    Hatva, E.; Böhling, T.; Jääskeläinen, J.; Persico, M. G.; Haltia, M.; Alitalo, K.

    1996-01-01

    Capillary hemangioblastomas and hemangiopericytomas are highly vascular central nervous system tumors of controversial origin. Of interest in their pathogenesis are mechanisms regulating endothelial cell growth. The endothelial cell mitogen vascular endothelial growth factor (VEGF) stimulates angiogenesis, and together with its two receptor tyrosine kinases VEGFR-1(FLT1) and VEGFR-2(KDR), is up-regulated during the malignant progression of gliomas. We have analyzed the expression of VEGF and its receptors, the related placental growth factor (PlGF) and the endothelial receptors FLT4 and Tie by in situ hybridization in capillary hemangioblastomas and hemangiopericytomas. VEGF mRNA was up-regulated in all of the hemangiopericytomas studied and highly expressed in the stromal cells of hemangioblastomas. In addition, some hemangioblastoma tumor cells expressed high levels of PlGF. Significantly elevated levels of Tie mRNA, Tie protein, VEGFR-1, and VEGFR-2 but not FLT4 mRNAs were observed in the endothelia of both tumor types. In hemangioblastomas, however, the receptors were also highly expressed by a subpopulation of stromal cells. Consistent results were obtained for a human hemangioblastoma cell line in culture. Up-regulation of the endothelial growth factors and receptors may result in autocrine or paracrine stimulation of endothelial cells and their precursors involved in the genesis of these two vascular tumors. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8774132

  2. Growth factor control of epidermal growth factor receptor kinase activity via an intramolecular mechanism.

    PubMed

    Koland, J G; Cerione, R A

    1988-02-15

    The mechanism by which the protein kinase activity of the epidermal growth factor (EGF) receptor is activated by binding of growth factor was investigated. Detergent-solubilized receptor in monomeric form was isolated by sucrose density gradient centrifugation and both its kinase and autophosphorylation activities monitored. In a low ionic strength medium and with MnCl2 as an activator, the activity of the monomeric receptor was EGF-independent. However, with 0.25 M ammonium sulfate present, the MnCl2-stimulated kinase activity was strikingly EGF-dependent. In contrast, the kinase activity expressed in the presence of MgCl2 showed growth factor control in the absence of added salt. Under the conditions of these experiments there was apparently little tendency for growth factor to induce aggregation of the receptor, indicating that the allosteric activation of the receptor kinase by EGF occurred via an intramolecular mechanism. Whereas detergent-solubilized receptor was the subject of these studies, the kinase activity of cell surface receptors might also be controlled by an intramolecular mechanism. These results indicate that an individual receptor molecule has the potential to function as a transmembrane signal transducer.

  3. The ontogeny of epidermal growth factor receptors during mouse development

    SciTech Connect

    Adamson, E.D.; Meek, J.

    1984-05-01

    In an attempt to understand the role(s) of epidermal growth factor (EGF) in vivo during murine development, we have examined the /sup 125/I-EGF binding characteristics of EGF-receptors in membrane preparations of tissues from the 12th day of gestation to parturition. Using autoradiography, the earliest time that we could detect EGF-receptors was on trophoblast cells cultured for 3 days as blastocyst outgrowths. Trophoblast eventually forms a large portion of the placenta, where EGF-receptors have long been recognized. We measured the number and affinity of EGF-receptors on tissues dissected from conceptuses from the 12th day of gestation in order to identify a stage when tissues may be most sensitive to EGF. Whereas the number of EGF receptors increases during gestation for all tissues examined, the affinity of the receptors declines for carcass and placenta and remains relatively unchanged for brain and liver. This suggests that EGF may function differently throughout development. Our hypothesis is that EGF (or its embryonic equivalent) initially stimulates proliferation in embryonic cells and then stimulates differentiation as the tissues mature. In the adult, its main role could be to stimulate tissue repair after damage.

  4. Macrophage Migration Inhibitory Factor-CXCR4 Receptor Interactions

    PubMed Central

    Rajasekaran, Deepa; Gröning, Sabine; Schmitz, Corinna; Zierow, Swen; Drucker, Natalie; Bakou, Maria; Kohl, Kristian; Mertens, André; Lue, Hongqi; Weber, Christian; Xiao, Annie; Luker, Gary; Kapurniotu, Aphrodite; Lolis, Elias; Bernhagen, Jürgen

    2016-01-01

    An emerging number of non-chemokine mediators are found to bind to classical chemokine receptors and to elicit critical biological responses. Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine that exhibits chemokine-like activities through non-cognate interactions with the chemokine receptors CXCR2 and CXCR4, in addition to activating the type II receptor CD74. Activation of the MIF-CXCR2 and -CXCR4 axes promotes leukocyte recruitment, mediating the exacerbating role of MIF in atherosclerosis and contributing to the wealth of other MIF biological activities. Although the structural basis of the MIF-CXCR2 interaction has been well studied and was found to engage a pseudo-ELR and an N-like loop motif, nothing is known about the regions of CXCR4 and MIF that are involved in binding to each other. Using a genetic strain of Saccharomyces cerevisiae that expresses a functional CXCR4 receptor, site-specific mutagenesis, hybrid CXCR3/CXCR4 receptors, pharmacological reagents, peptide array analysis, chemotaxis, fluorescence spectroscopy, and circular dichroism, we provide novel molecular information about the structural elements that govern the interaction between MIF and CXCR4. The data identify similarities with classical chemokine-receptor interactions but also provide evidence for a partial allosteric agonist compared with CXCL12 that is possible due to the two binding sites of CXCR4. PMID:27226569

  5. Biochemical and biological properties of the nerve growth factor receptor

    SciTech Connect

    Taniuchi, M.

    1988-01-01

    We have utilized a monoclonal antibody (192-IgG) to study the rat nerve growth factor receptor. After intraocular injection, {sup 125}I-192-IgG was retrogradely transported in sympathetic neuronal axons to the superior cervical ganglion. When the sciatic nerve was ligated to induce the accumulation of axonally transported materials, 192-IgG immunostaining was observed on both sides of the ligature, indicating that NGF receptors are transported in both orthograde and retrograde directions. By using {sup 125}I-NGF crosslinking and 192-IgG immunoprecipitation, we detected receptor molecules throughout the rat brain, thereby supporting the hypothesis that NGF is active in the central nervous system. We also discovered that sciatic nerve transection leads to a dramatic increase in the amount of NGF receptor found in the distal portion of the nerve. Immunostaining revealed that all Schwann cells in the distal axotomized nerve were expressing NGF receptors. We examined phosphorylation of NGF receptor in cultured sympathetic neurons and PC12 cells. We also examined pharmacological effects of 192-IgG. Systemic injection of 192-IgG into neonatal rats caused a permanent partial sympathectomy in a dose-dependent manner; a maximum of 50% of the cells were killed.

  6. Systems Biological Analysis of Epidermal Growth Factor Receptor Internalization Dynamics for Altered Receptor Levels*

    PubMed Central

    Schmidt-Glenewinkel, Hannah; Reinz, Eileen; Eils, Roland; Brady, Nathan R.

    2009-01-01

    Epidermal growth factor (EGF) receptor (EGFR) overexpression is a hallmark of many cancers. EGFR endocytosis is a critical step in signal attenuation, raising the question of how receptor expression levels affect the internalization process. Here we combined quantitative experimental and mathematical modeling approaches to investigate the role of the EGFR expression level on the rate of receptor internalization. Using tetramethylrhodamine-labeled EGF, we established assays for quantifying EGF-triggered EGFR internalization by both high resolution confocal microscopy and flow cytometry. We determined that the flow cytometry approach was more sensitive for examining large populations of cells. Mathematical modeling was used to investigate the relationship between EGF internalization kinetics, EGFR expression, and internalization machinery. We predicted that the standard parameter used to assess internalization kinetics, the temporal evolution r(t) of the ratio of internalized versus surface-located ligand·receptor complexes, does not describe a straight line, as proposed previously. Instead, a convex or concave curve occurs depending on whether initial receptor numbers or internalization adaptors are limiting the uptake reaction, respectively. To test model predictions, we measured EGF-EGFR binding and internalization in cells expressing different levels of green fluorescent protein-EGFR. As expected, surface binding of rhodamine-labeled EGF increased with green fluorescent protein-EGFR expression level. Unexpectedly, internalization of ligand· receptor complexes increased linearly with increasing receptor expression level, suggesting that receptors and not internalization adaptors were limiting the uptake in our experimental model. Finally, determining the ratio of internalized versus surface-located ligand·receptor complexes for this cell line confirmed that it follows a convex curve, supporting our model predictions. PMID:19297331

  7. Therapeutic Targeting of Fibroblast Growth Factor Receptors in Gastric Cancer

    PubMed Central

    Fujimori, Yoshitaka; Otsuki, Sho; Sato, Yuya; Nakagawa, Masatoshi

    2015-01-01

    Chemotherapy has become the global standard treatment for patients with metastatic or unresectable gastric cancer (GC), although outcomes remain unfavorable. Many molecular-targeted therapies inhibiting signaling pathways of various tyrosine kinase receptors have been developed, and monoclonal antibodies targeting human epidermal growth factor receptor 2 (HER2) have become standard therapy for HER2-positive GC. An inhibitor of vascular endothelial growth factor receptor 2 or MET has also produced promising results in patients with GC. Fibroblast growth factor receptors (FGFR) play key roles in tumor growth via activated signaling pathways in GC. Genomic amplification of FGFR2 leads to the aberrant activation found in GC tumors and is related to survival in patients with GC. This review discusses the clinical relevance of FGFR in GC and examines FGFR as a potential therapeutic target in patients with GC. Preclinical studies in animal models suggest that multitargeted tyrosine kinase inhibitors (TKIs), including FGFR inhibitor, suppress tumor cell proliferation and delay tumor progression. Several TKIs are now being evaluated in clinical trials as treatment for metastatic or unresectable GC harboring FGFR2 amplification. PMID:26000013

  8. Platelet-activating factor (PAF) receptor and genetically engineered PAF receptor mutant mice.

    PubMed

    Ishii, S; Shimizu, T

    2000-01-01

    Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a biologically active phospholipid mediator. Although PAF was initially recognized for its potential to induce platelet aggregation and secretion, intense investigations have elucidated potent biological actions of PAF in a broad range of cell types and tissues, many of which also produce the molecule. PAF acts by binding to a unique G-protein-coupled seven transmembrane receptor. PAF receptor is linked to intracellular signal transduction pathways, including turnover of phosphatidylinositol, elevation in intracellular calcium concentration, and activation of kinases, resulting in versatile bioactions. On the basis of numerous pharmacological reports, PAF is thought to have many pathophysiological and physiological functions. Recently advanced molecular technics enable us not only to clone PAF receptor cDNAs and genes, but also generate PAF receptor mutant animals, i.e., PAF receptor-overexpressing mouse and PAF receptor-deficient mouse. These mutant mice gave us a novel and specific approach for identifying the pathophysiological and physiological functions of PAF. This review also describes the phenotypes of these mutant mice and discusses them by referring to previously reported pharmacological and genetical data.

  9. Identification of the epidermal growth factor receptor as the receptor for Salmonella Rck-dependent invasion.

    PubMed

    Wiedemann, Agnès; Mijouin, Lily; Ayoub, Mohammed Akli; Barilleau, Emilie; Canepa, Sylvie; Teixeira-Gomes, Ana Paula; Le Vern, Yves; Rosselin, Manon; Reiter, Eric; Velge, Philippe

    2016-12-01

    The Salmonella Rck outer membrane protein binds to the cell surface, which leads to bacterial internalization via a Zipper mechanism. This invasion process requires induction of cellular signals, including phosphorylation of tyrosine proteins, and activation of c-Src and PI3K, which arises as a result of an interaction with a host cell surface receptor. In this study, epidermal growth factor receptor (EGFR) was identified as the cell signaling receptor required for Rck-mediated adhesion and internalization. First, Rck-mediated adhesion and internalization were shown to be altered when EGFR expression and activity were modulated. Then, immunoprecipitations were performed to demonstrate the Rck-EGFR interaction. Furthermore, surface plasmon resonance biosensor and homogeneous time-resolved fluorescence technologies were used to demonstrate the direct interaction of Rck with the extracellular domain of human EGFR. Finally, our study strongly suggests a noncompetitive binding of Rck and EGF to EGFR. Overall, these results demonstrate that Rck is able to bind to EGFR and thereby establish a tight adherence to provide a signaling cascade, which leads to internalization of Rck-expressing bacteria.-Wiedemann, A., Mijouin, L., Ayoub, M. A., Barilleau, E., Canepa, S., Teixeira-Gomes, A. P., Le Vern, Y., Rosselin, M., Reiter, E., Velge, P. Identification of the epidermal growth factor receptor as the receptor for Salmonella Rck-dependent invasion.

  10. Analysis of differentially co-expressed genes based on microarray data of hepatocellular carcinoma.

    PubMed

    Wang, Y; Jiang, T; Li, Z; Lu, L; Zhang, R; Zhang, D; Wang, X; Tan, J

    2017-01-01

    Hepatocellular carcinoma (HCC) is the third leading cause of cancer related death worldwide. Although great progress in diagnosis and management of HCC have been made, the exact molecular mechanisms remain poorly understood. The study aims to identify potential biomarkers for HCC progression, mainly at transcription level. In this study, chip data GSE 29721 was utilized, which contains 10 HCC samples and 10 normal adjacent tissue samples. Differentially expressed genes (DEGs) between two sample types were selected by t-test method. Following, the differentially co-expressed genes (DCGs) and differentially co-expressed Links (DCLs) were identified by DCGL package in R with the threshold of q < 0.25. Afterwards, pathway enrichment analysis of the DCGs was carried out by DAVID. Then, DCLs were mapped to TRANSFAC database to reveal associations between relevant transcriptional factors (TFs) and their target genes. Quantitative real-time RT-PCR was performed for TFs or genes of interest. As a result, a total of 388 DCGs and 35,771 DCLs were obtained. The predominant pathways enriched by these genes were Cytokine-cytokine receptor interaction, ECM-receptor interaction and TGF-β signaling pathway. Three TF-target interactions, LEF1-NCAM1, EGR1-FN1 and FOS-MT2A were predicted. Compared with control, expressions of the TF genes EGR1, FOS and ETS2 were all up-regulated in the HCC cell line, HepG2; while LEF1 was down-regulated. Except NCAM1, all the target genes were up-regulated in HepG2. Our findings suggest these TFs and genes might play important roles in the pathogenesis of HCC and may be used as therapeutic targets for HCC management.

  11. Role of fibroblast growth factor receptor signaling in kidney development.

    PubMed

    Bates, Carlton M

    2007-03-01

    Fibroblast growth factor receptors (Fgfrs) are expressed in the ureteric bud and metanephric mesenchyme of the developing kidney. Furthermore, in vitro and in vivo studies have shown that exogenous fibroblast growth factors (Fgfs) increase growth and maturation of the metanephric mesenchyme and ureteric bud. Deletion of fgf7, fgf10, and fgfr2IIIb (the receptor isoform that binds Fgf7 and Fgf10) in mice lead to smaller kidneys with fewer collecting ducts and nephrons. Overexpression of a dominant negative receptor isoform in transgenic mice has revealed more striking defects including renal aplasia or severe dysplasia. Moreover, deletion of many fgf ligands and receptors in mice results in early embryonic lethality, making it difficult to determine their roles in kidney development. Recently, conditional targeting approaches revealed that deletion of fgf8 from the metanephric mesenchyme interrupts nephron formation. Furthermore, deletion of fgfr2 from the ureteric bud resulted in both ureteric bud branching and stromal mesenchymal patterning defects. Deletion of both fgfr1 and fgfr2 in the metanephric mesenchyme resulted in renal aplasia, characterized by defects in metanephric mesenchyme formation and initial ureteric bud elongation and branching. Thus, Fgfr signaling is critical for growth and patterning of all renal lineages at early and later stages of kidney development.

  12. Expression of the estrogen receptor α, progesterone receptor and epidermal growth factor receptor in papillary thyroid carcinoma tissues

    PubMed Central

    CHEN, DAN; QI, WENJING; ZHANG, PENGXIN; GUAN, HONGWEI; WANG, LIFEN

    2015-01-01

    The present study aimed to determine the protein expression, in addition to the clinical value of the expression, of estrogen receptor α (ERα), progesterone receptor (PR) and epidermal growth factor receptor (EGFR) in papillary thyroid carcinoma (PTC). The expression of ERα, PR and EGFR was examined immunohistochemically on paraffin-embedded thyroid tissues obtained from 64 patients with PTC and 14 patients with nodular thyroid goiter (NTG). The expression level of ERα, PR and EGFR was found to be significantly elevated in the PTC tissues compared with the NTG tissues. In addition, the expression of ERα was found to be correlated with the size of PTC tumors. However, there was no significant difference between the expression levels of ERα, PR and EGFR in males and females with PTC. Thus, immunohistochemical evaluation of ERα, PR and EGFR expression in patients with PTC may aid in the prediction of the prognosis of patients with PTC. PMID:26171022

  13. Expression of androgen receptor in breast cancer & its correlation with other steroid receptors & growth factors

    PubMed Central

    Mishra, Ashwani K.; Agrawal, Usha; Negi, Shivani; Bansal, Anju; Mohil, R.; Chintamani, Chintamani; Bhatnagar, Amar; Bhatnagar, Dinesh; Saxena, Sunita

    2012-01-01

    Background & objectives: Breast cancer is the second most common malignancy in Indian women. Among the members of the steroid receptor superfamily the role of estrogen and progesterone receptors (ER and PR) is well established in breast cancer in predicting the prognosis and management of therapy, however, little is known about the clinical significance of androgen receptor (AR) in breast carcinogenesis. The present study was aimed to evaluate the expression of AR in breast cancer and to elucidate its clinical significance by correlating it with clinicopathological parameters, other steroid receptors (ER and PR) and growth factors receptors (EGFR and CD105). Methods: Expression of AR, ER, PR, epidermal growth factor receptor (EGFR) and endoglin (CD105) was studied in 100 cases of breast cancer by immunohistochemistry (IHC). Risk ratio (RR) along with 95% confidence interval (CI) was estimated to assess the strength of association between the markers and clinicopathological characteristics. Categorical principal component analysis (CATPCA) was applied to obtain new sets of linearly combined expression, for their further evaluation with clinicopathological characteristics (n=100). Results: In 31 cases presenting with locally advanced breast cancer (LABC), the expression of AR, ER, PR, EGFR and CD105 was associated with response to neoadjuvant chemotherapy (NACT). The results indicated the association of AR+ (P=0.001) and AR+/EGFR- (P=0.001) with the therapeutic response to NACT in LABC patients. The AR expression exhibited maximum sensitivity, specificity and likelihood ratio of positive and negative test. The present results showed the benefit of adding AR, EGFR and CD105 to the existing panel of markers to be able to predict response to therapy. Interpretation & conclusions: More studies on the expression profiles of AR+, AR+/CD105+ and AR+/EGFR- in larger set of breast cancer patients may possibly help in confirming their predictive role for therapeutic response

  14. Nuclear transportation of exogenous epidermal growth factor receptor and androgen receptor via extracellular vesicles.

    PubMed

    Read, Jolene; Ingram, Alistair; Al Saleh, Hassan A; Platko, Khrystyna; Gabriel, Kathleen; Kapoor, Anil; Pinthus, Jehonathan; Majeed, Fadwa; Qureshi, Talha; Al-Nedawi, Khalid

    2017-01-01

    Epidermal growth factor receptor (EGFR) plays a central role in the progression of several human malignancies. Although EGFR is a membrane receptor, it undergoes nuclear translocation, where it has a distinct signalling pathway. Herein, we report a novel mechanism by which cancer cells can directly transport EGFR to the nucleus of other cells via extracellular vesicles (EVs). The transported receptor is active and stimulates the nuclear EGFR pathways. Interestingly, the translocation of EGFR via EVs occurs independently of the nuclear localisation sequence that is required for nuclear translocation of endogenous EGFR. Also, we found that the mutant receptor EGFRvIII could be transported to the nucleus of other cells via EVs. To assess the role of EVs in the regulation of an actual nuclear receptor, we studied the regulation of androgen receptor (AR). We found that full-length AR and mutant variant ARv7 are secreted in EVs derived from prostate cancer cell lines and could be transported to the nucleus of AR-null cells. The EV-derived AR was able to bind the androgen-responsive promoter region of prostate specific antigen, and recruit RNA Pol II, an indication of active transcription. The nuclear-translocated AR via EVs enhanced the proliferation of acceptor cells in the absence of androgen. Finally, we provide evidence that nuclear localisation of AR could occur in vivo via orthotopically-injected EVs in male SCID mice prostate glands. To our knowledge, this is the first study showing the nuclear translocation of nuclear receptors via EVs, which significantly extends the role of EVs as paracrine transcriptional regulators. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. UV activates growth factor receptors via reactive oxygen intermediates

    PubMed Central

    1996-01-01

    Exposure of mammalian cells to UV irradiation induces rapid and transient expression of early growth response-1 gene (Egr-1) encoding a transcription factor that plays a role in cell survival. These signals from the irradiated cell surface are likely to involve more than one pathway, and we show here that an essential pathway involves activation of several growth factor receptors by reactive oxygen intermediates (ROI). UVC irradiation causes the tyrosine phosphorylation of EGF receptor (EGFR) in mouse NIH 3T3 fibroblasts and HC11 mouse mammary cells. EGFR activation by irradiation of cells is abrogated by suramin, by antioxidants, and by the presence of a dominant negative EGFR. UV induces the formation of complexes between activated EGFR and SOS, Grb2, PLC gamma, and SHC that can be precipitated with antibodies to EGFR. The activation of EGFR by UV is mimicked by H2O2, suggesting that ROI may function upstream of EGFR activation. Our observations support the hypothesis that ROI and growth factor receptors operate in the early steps of the UV signal that lead to the enhanced expression and activity of Egr-1. PMID:8601609

  16. Fibroblast growth factor receptor levels decrease during chick embryogenesis

    PubMed Central

    1990-01-01

    Two putative receptors for fibroblast growth factor (FGF) of approximately 150 and 200 kD were identified in membrane preparations from chick embryos. Specific binding (femtomoles/milligram) of 125I- aFGF to whole chick embryonic membranes was relatively constant from day 2 to 7, then decreased fivefold between days 7 and 13. Day-19 chick embryos retained 125I-aFGF binding at low levels to brain, eye, and liver tissues but not to skeletal muscle or cardiac tissues. The 200-kD FGF receptor began to decline between day 4.5 and 7 and was barely detectable by day 9, whereas the 150-kD FGF receptor began to decline by day 7 but was still detectable in day-9 embryonic membranes. It is not known whether the two FGF-binding proteins represent altered forms of one polypeptide, but it is clear that their levels undergo differential changes during development. Because endogenous chick FGF may remain bound to FGF receptor in membrane preparations, membranes were treated with acidic (pH 4.0) buffers to release bound FGF; such treatment did not affect 125I-aFGF binding and moderately increased the number of binding sites in day-7 and -19 embryos. Consequently, the observed loss of high affinity 125I-aFGF binding sites and FGF-binding polypeptides most likely represents a loss of FGF receptor protein. These experiments provide in vivo evidence to support the hypothesis that regulation of FGF receptor levels may function as a mechanism for controlling FGF-dependent processes during embryonic development. PMID:2153684

  17. Identification of ciliary neurotrophic factor (CNTF) residues essential for leukemia inhibitory factor receptor binding and generation of CNTF receptor antagonists.

    PubMed Central

    Di Marco, A; Gloaguen, I; Graziani, R; Paonessa, G; Saggio, I; Hudson, K R; Laufer, R

    1996-01-01

    Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containing the ligand-specific alpha-receptor subunit (CNTFR alpha) and the signal transducers gp130 and leukemia inhibitory factor receptor-beta (LIFR). The D1 structural motif, located at the beginning of the D-helix of human CNTF, contains two amino acid residues, F152 and K155, which are conserved among all cytokines that signal through LIFR. The functional importance of these residues was assessed by alanine mutagenesis. Substitution of either F152 or K155 with alanine was found to specifically inhibit cytokine interaction with LIFR without affecting binding to CNTFR alpha or gp130. The resulting variants behaved as partial agonists with varying degrees of residual bioactivity in different cell-based assays. Simultaneous alanine substitution of both F152 and K155 totally abolished biological activity. Combining these mutations with amino acid substitutions in the D-helix, which enhance binding affinity for the CNTFR alpha, gave rise to a potent competitive CNTF receptor antagonist. This protein constitutes a new tool for studies of CNTF function in normal physiology and disease. Images Fig. 1 Fig. 6 PMID:8799186

  18. Methods for studying the platelet-derived growth factor receptor

    SciTech Connect

    Bowen-Pope, D.F.; Ross, R.

    1985-01-01

    Platelet-derived growth factor (PDGF) is a basic 30,000-dalton protein circulating in normal blood sequestered within the platelet alpha granule. Radioiodinated PDGF shows saturable (e.g., 60,000-120,000 receptors per diploid human fibroblast) high affinity binding to culture PDGF-responsive cells. The apparent dissociation constant reported for this binding interaction has varied widely. This paper focuses on factors which affect (/sup 125/I)PGDF binding and on the development of a radioreceptor assay for PDGF.

  19. Conformational thermostabilisation of corticotropin releasing factor receptor 1

    PubMed Central

    Kean, James; Bortolato, Andrea; Hollenstein, Kaspar; Marshall, Fiona H.; Jazayeri, Ali

    2015-01-01

    Recent technical advances have greatly facilitated G-protein coupled receptors crystallography as evidenced by the number of successful x-ray structures that have been reported recently. These technical advances include novel detergents, specialised crystallography techniques as well as protein engineering solutions such as fusions and conformational thermostabilisation. Using conformational thermostabilisation, it is possible to generate variants of GPCRs that exhibit significantly increased stability in detergent micelles whilst preferentially occupying a single conformation. In this paper we describe for the first time the application of this technique to a member of a class B GPCR, the corticotropin releasing factor receptor 1 (CRF1R). Mutational screening in the presence of the inverse agonist, CP-376395, resulted in the identification of a construct with twelve point mutations that exhibited significantly increased thermal stability in a range of detergents. We further describe the subsequent construct engineering steps that eventually yielded a crystallisation-ready construct which recently led to the solution of the first x-ray structure of a class B receptor. Finally, we have used molecular dynamic simulation to provide structural insight into CRF1R instability as well as the stabilising effects of the mutants, which may be extended to other class B receptors considering the high degree of structural conservation. PMID:26159865

  20. Vascular Endothelial Growth Factor Receptor -2 in Breast Cancer

    PubMed Central

    Guo, Shanchun; Colbert, Laronna S.; Fuller, Miles; Zhang, Yuanyuan; Gonzalez-Perez, Ruben R.

    2010-01-01

    Investigations over the last decade have established the essential role of growth factors and their receptors during angiogenesis and carcinogenesis. The vascular endothelial growth factor receptor (VEGFR) family in mammals contains three members, VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1) and VEGFR-3 (Flt-4), which are transmembrane tyrosine kinase receptors that regulate the formation of blood and lymphatic vessels. In the early 1990s, the above VEGFR were structurally characterized by cDNA cloning. Among these three receptors, VEGFR-2 is generally recognized to have a principal role in mediating VEGF-induced responses. VEGFR-2 is considered as the earliest marker for endothelial cell development. Importantly, VEGFR-2 directly regulates tumor angiogenesis. Therefore, several inhibitors of VEGFR-2 have been developed and many of them are now in clinical trials. In addition to targeting endothelial cells, the VEGF/VEGFR-2 system works as an essential autocrine/paracrine process for cancer cell proliferation and survival. Recent studies mark the continuous and increased interest in this related, but distinct, function of VEGF/VEGFR-2 in cancer cells: the autocrine/paracrine loop. Several mechanisms regulate VEGFR-2 levels and modulate its role in tumor angiogenesis and physiologic functions, i.e.: cellular localization/trafficking, regulation of cis-elements of promoter, epigenetic regulation and signaling from Notch, cytokines/growth factors and estrogen, etc. In this review, we will focus on updated information regarding VEGFR-2 research with respect to the molecular mechanisms of VEGFR-2 regulation in human breast cancer. Investigations in the activation, function, and regulation of VEGFR-2 in breast cancer will allow the development of new pharmacological strategies aimed at directly targeting cancer cell proliferation and survival. PMID:20462514

  1. Role of fibroblast growth factor receptor signaling in kidney development.

    PubMed

    Bates, Carlton M

    2011-09-01

    Fibroblast growth factor receptors (Fgfrs) are expressed throughout the developing kidney. Several early studies have shown that exogenous fibroblast growth factors (Fgfs) affect growth and maturation of the metanephric mesenchyme (MM) and ureteric bud (UB). Transgenic mice that over-express a dominant negative receptor isoform develop renal aplasia/severe dysplasia, confirming the importance of Fgfrs in renal development. Furthermore, global deletion of Fgf7, Fgf10, and Fgfr2IIIb (isoform that binds Fgf7 and Fgf10) in mice leads to small kidneys with fewer collecting ducts and nephrons. Deletion of Fgfrl1, a receptor lacking intracellular signaling domains, causes severe renal dysgenesis. Conditional targeting of Fgf8 from the MM interrupts nephron formation. Deletion of Fgfr2 from the UB results in severe ureteric branching and stromal mesenchymal defects, although loss of Frs2α (major signaling adapter for Fgfrs) in the UB causes only mild renal hypoplasia. Deletion of both Fgfr1 and Fgfr2 in the MM results in renal aplasia with defects in MM formation and initial UB elongation and branching. Loss of Fgfr2 in the MM leads to many renal and urinary tract anomalies as well as vesicoureteral reflux. Thus, Fgfr signaling is critical for patterning of virtually all renal lineages at early and later stages of development.

  2. Topography of human placental receptors for epidermal growth factor.

    PubMed

    Rao, C V; Ramani, N; Chegini, N; Stadig, B K; Carman, F R; Woost, P G; Schultz, G S; Cook, C L

    1985-02-10

    These studies were undertaken to determine whether term human placental microvillus plasma membranes, which are exposed to maternal blood, and basolateral plasma membranes, which are in close proximity to fetal blood capillaries, contain receptors for epidermal growth factor (EGF). These two highly purified membranes bound 125I-EGF with similar affinity (apparent dissociation constants, 0.07-0.12 nM, but the total number of available receptors was greater in microvillus (8.2 pmol/mg protein) compared to basolateral (4.9 pmol/mg protein) plasma membranes. Detailed characterization of 125I-EGF binding to these membranes revealed numerous similarities as well as differences. The two membranes contained two major (155 and 140 kDa) and at least three minor (115, 175, and 210 kDa) specific 125I-EGF binding proteins. The 115-kDa protein was only found in basolateral plasma membranes. The 155-kDa protein was predominantly labeled in microvillus, whereas the 140-kDa protein was labeled predominantly in basolateral plasma membranes. The addition of protease inhibitors did not alter the multiple 125I-EGF binding proteins pattern found in these membranes. EGF stimulated phosphorylation of 140- and 155-kDa proteins in both microvillus and basolateral plasma membranes. However, the 155-kDa protein was phosphorylated to a greater extent in microvillus, whereas both 140- and 155-kDa proteins were phosphorylated equally in basolateral plasma membranes. Light and electron microscope autoradiographic studies revealed that 125I-EGF preferentially associated with microvillus plasma membranes. The data demonstrates the presence of EGF receptors in outer cell membranes of syncytiotrophoblasts and suggests that maternal EGF may influence syncytiotrophoblast function by binding to receptors in microvillus plasma membranes, while fetal EGF may also influence syncytiotrophoblast function but via receptors in basolateral plasma membranes.

  3. Inhibiting the Epidermal Growth Factor Receptor | Center for Cancer Research

    Cancer.gov

    The Epidermal Growth Factor Receptor (EGFR) is a widely distributed cell surface receptor that responds to several extracellular signaling molecules through an intracellular tyrosine kinase, which phosphorylates target enzymes to trigger a downstream molecular cascade. Since the discovery that EGFR mutations and amplifications are critical in a number of cancers, efforts have been under way to develop and use targeted EGFR inhibitors. These efforts have met with some spectacular successes, but many patients have not responded as expected, have subsequently developed drug-resistant tumors, or have suffered serious side effects from the therapies to date. CCR Investigators are studying EGFR from multiple vantage points with the goal of developing even better strategies to defeat EGFR-related cancers.

  4. Signal Transduction by Vascular Endothelial Growth Factor Receptors

    PubMed Central

    Koch, Sina; Claesson-Welsh, Lena

    2012-01-01

    Vascular endothelial growth factors (VEGFs) are master regulators of vascular development and of blood and lymphatic vessel function during health and disease in the adult. It is therefore important to understand the mechanism of action of this family of five mammalian ligands, which act through three receptor tyrosine kinases (RTKs). In addition, coreceptors like neuropilins (NRPs) and integrins associate with the ligand/receptor signaling complex and modulate the output. Therapeutics to block several of the VEGF signaling components have been developed with the aim to halt blood vessel formation, angiogenesis, in diseases that involve tissue growth and inflammation, such as cancer. In this review, we outline the current information on VEGF signal transduction in relation to blood and lymphatic vessel biology. PMID:22762016

  5. Brassinosteroid signal transduction from receptor kinases to transcription factors.

    PubMed

    Kim, Tae-Wuk; Wang, Zhi-Yong

    2010-01-01

    Brassinosteroids (BRs) are growth-promoting steroid hormones in plants. Genetic studies in Arabidopsis illustrated the essential roles of BRs in a wide range of developmental processes and helped identify many genes involved in BR biosynthesis and signal transduction. Recently, proteomic studies identified missing links. Together, these approaches established the BR signal transduction cascade, which includes BR perception by the BRI1 receptor kinase at the cell surface, activation of BRI1/BAK1 kinase complex by transphosphorylation, subsequent phosphorylation of the BSK kinases, activation of the BSU1 phosphatase, dephosphorylation and inactivation of the BIN2 kinase, and accumulation of unphosphorylated BZR transcription factors in the nucleus. Mass spectrometric analyses are providing detailed information on the phosphorylation events involved in each step of signal relay. Thus, the BR signaling pathway provides a paradigm for understanding receptor kinase-mediated signal transduction as well as tools for the genetic improvement of the productivity of crop plants.

  6. Phosphorylation of insulin-like growth factor I receptor by insulin receptor tyrosine kinase in intact cultured skeletal muscle cells

    SciTech Connect

    Beguinot, F.; Smith, R.J.; Kahn, C.R.; Maron, R.; Moses, A.C.; White, M.F.

    1988-05-03

    The interaction between insulin and insulin-like growth factor I (IGF I) receptors was examined by determining the ability of each receptor type to phosphorylate tyrosine residues on the other receptor in intact L6 skeletal muscle cells. This was made possible through a sequential immunoprecipitation method with two different antibodies that effectively separated the phosphorylated insulin and IGF I receptors. After incubation of intact L6 cells with various concentrations of insulin or IGF I in the presence of (/sup 32/P)-orthophosphate, insulin receptors were precipitated with one of two human polyclonal anti-insulin receptor antibodies (B2 or B9). Phosphorylated IGF I receptors remained in solution and were subsequently precipitated by anti-phosphotyrosine antibodies. The identifies of the insulin and IGF I receptor ..beta..-subunits in the two immunoprecipitates were confirmed by binding affinity, by phosphopeptide mapping after trypsin digestion, and by the distinct patterns of expression of the two receptors during differentiation. Stimulated phosphorylation of the ..beta..-subunit of the insulin receptor correlated with the occupancy of the ..beta..-subunit of the insulin receptor by either insulin or IGF I as determined by affinity cross-linking. Similarly, stimulation of phosphorylation of the ..beta..-subunit of the IGF I receptor by IGF I correlated with IGF I receptor occupancy. In contrast, insulin stimulated phosphorylation of the ..beta..-subunit of the IGF I receptor at hormone concentrations that were associated with significant occupancy of the insulin receptor but negligible IGF I receptor occupancy. These findings indicate that the IGF I receptor can be a substrate for the hormone-activated insulin receptor tyrosine kinase activity in intact L6 skeletal muscle cells.

  7. Spectral tuning by opsin coexpression in retinal regions that view different parts of the visual field.

    PubMed

    Dalton, Brian E; Loew, Ellis R; Cronin, Thomas W; Carleton, Karen L

    2014-12-22

    Vision frequently mediates critical behaviours, and photoreceptors must respond to the light available to accomplish these tasks. Most photoreceptors are thought to contain a single visual pigment, an opsin protein bound to a chromophore, which together determine spectral sensitivity. Mechanisms of spectral tuning include altering the opsin, changing the chromophore and incorporating pre-receptor filtering. A few exceptions to the use of a single visual pigment have been documented in which a single mature photoreceptor coexpresses opsins that form spectrally distinct visual pigments, and in these exceptions the functional significance of coexpression is unclear. Here we document for the first time photoreceptors coexpressing spectrally distinct opsin genes in a manner that tunes sensitivity to the light environment. Photoreceptors of the cichlid fish, Metriaclima zebra, mix different pairs of opsins in retinal regions that view distinct backgrounds. The mixing of visual pigments increases absorbance of the corresponding background, potentially aiding the detection of dark objects. Thus, opsin coexpression may be a novel mechanism of spectral tuning that could be useful for detecting prey, predators and mates. However, our calculations show that coexpression of some opsins can hinder colour discrimination, creating a trade-off between visual functions. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

  8. Spectral tuning by opsin coexpression in retinal regions that view different parts of the visual field

    PubMed Central

    Dalton, Brian E.; Loew, Ellis R.; Cronin, Thomas W.; Carleton, Karen L.

    2014-01-01

    Vision frequently mediates critical behaviours, and photoreceptors must respond to the light available to accomplish these tasks. Most photoreceptors are thought to contain a single visual pigment, an opsin protein bound to a chromophore, which together determine spectral sensitivity. Mechanisms of spectral tuning include altering the opsin, changing the chromophore and incorporating pre-receptor filtering. A few exceptions to the use of a single visual pigment have been documented in which a single mature photoreceptor coexpresses opsins that form spectrally distinct visual pigments, and in these exceptions the functional significance of coexpression is unclear. Here we document for the first time photoreceptors coexpressing spectrally distinct opsin genes in a manner that tunes sensitivity to the light environment. Photoreceptors of the cichlid fish, Metriaclima zebra, mix different pairs of opsins in retinal regions that view distinct backgrounds. The mixing of visual pigments increases absorbance of the corresponding background, potentially aiding the detection of dark objects. Thus, opsin coexpression may be a novel mechanism of spectral tuning that could be useful for detecting prey, predators and mates. However, our calculations show that coexpression of some opsins can hinder colour discrimination, creating a trade-off between visual functions. PMID:25377457

  9. Epidermal growth factor receptors in the canine antrum

    SciTech Connect

    Zimmerman, R.P.; Gates, T.S.; Boehmer, C.G.; Mantyh, P.W.

    1988-11-01

    In this study we localized receptor binding sites for /sup 125/I-human epidermal growth factor (hEGF) in the antrum of the adult canine stomach. High levels of specific /sup 125/I-hEGF binding sites were observed over the mucosa and muscularis mucosa, whereas specific binding sites were not detectable over the submucosa, external circular and longitudinal muscle or myenteric neurons. These results are in agreement with previous studies which indicated that EGF stimulates the proliferation of cultured epithelial cells and inhibits gastric acid secretion. This suggests that EGF may be a useful therapeutic agent in the healing of gastric ulcers.

  10. Epidermal growth factor and its receptors in human pancreatic carcinoma

    SciTech Connect

    Chen, Y.F.; Pan, G.Z.; Hou, X.; Liu, T.H.; Chen, J.; Yanaihara, C.; Yanaihara, N. )

    1990-05-01

    The role of epidermal growth factor (EGF) in oncogenesis and progression of malignant tumors is a subject of vast interest. In this study, radioimmunoassay and radioreceptor assay of EGF were established. EGF contents in malignant and benign pancreatic tumors, in normal pancreas tissue, and in culture media of a human pancreatic carcinoma cell line were determined. EGF receptor binding studies were performed. It was shown that EGF contents in pancreatic carcinomas were significantly higher than those in normal pancreas or benign pancreatic tumors. EGF was also detected in the culture medium of a pancreatic carcinoma cell line. The binding of 125I-EGF to the pancreatic carcinoma cells was time and temperature dependent, reversible, competitive, and specific. Scatchard analysis showed that the dissociation constant of EGF receptor was 2.1 X 10(-9) M, number of binding sites was 1.3 X 10(5) cell. These results indicate that there is an over-expression of EGF/EGF receptors in pancreatic carcinomas, and that an autocrine regulatory mechanism may exist in the growth-promoting effect of EGF on tumor cells.

  11. Identification of Transcriptional Modules and Key Genes in Chickens Infected with Salmonella enterica Serovar Pullorum Using Integrated Coexpression Analyses

    PubMed Central

    2017-01-01

    Salmonella enterica Pullorum is one of the leading causes of mortality in poultry. Understanding the molecular response in chickens in response to the infection by S. enterica is important in revealing the mechanisms of pathogenesis and disease progress. There have been studies on identifying genes associated with Salmonella infection by differential expression analysis, but the relationships among regulated genes have not been investigated. In this study, we employed weighted gene coexpression network analysis (WGCNA) and differential coexpression analysis (DCEA) to identify coexpression modules by exploring microarray data derived from chicken splenic tissues in response to the S. enterica infection. A total of 19 modules from 13,538 genes were associated with the Jak-STAT signaling pathway, the extracellular matrix, cytoskeleton organization, the regulation of the actin cytoskeleton, G-protein coupled receptor activity, Toll-like receptor signaling pathways, and immune system processes; among them, 14 differentially coexpressed modules (DCMs) and 2,856 differentially coexpressed genes (DCGs) were identified. The global expression of module genes between infected and uninfected chickens showed slight differences but considerable changes for global coexpression. Furthermore, DCGs were consistently linked to the hubs of the modules. These results will help prioritize candidate genes for future studies of Salmonella infection. PMID:28529955

  12. Identification of Transcriptional Modules and Key Genes in Chickens Infected with Salmonella enterica Serovar Pullorum Using Integrated Coexpression Analyses.

    PubMed

    Liu, Bao-Hong; Cai, Jian-Ping

    2017-01-01

    Salmonella enterica Pullorum is one of the leading causes of mortality in poultry. Understanding the molecular response in chickens in response to the infection by S. enterica is important in revealing the mechanisms of pathogenesis and disease progress. There have been studies on identifying genes associated with Salmonella infection by differential expression analysis, but the relationships among regulated genes have not been investigated. In this study, we employed weighted gene coexpression network analysis (WGCNA) and differential coexpression analysis (DCEA) to identify coexpression modules by exploring microarray data derived from chicken splenic tissues in response to the S. enterica infection. A total of 19 modules from 13,538 genes were associated with the Jak-STAT signaling pathway, the extracellular matrix, cytoskeleton organization, the regulation of the actin cytoskeleton, G-protein coupled receptor activity, Toll-like receptor signaling pathways, and immune system processes; among them, 14 differentially coexpressed modules (DCMs) and 2,856 differentially coexpressed genes (DCGs) were identified. The global expression of module genes between infected and uninfected chickens showed slight differences but considerable changes for global coexpression. Furthermore, DCGs were consistently linked to the hubs of the modules. These results will help prioritize candidate genes for future studies of Salmonella infection.

  13. Generation of monoclonal antibody targeting fibroblast growth factor receptor 3.

    PubMed

    Gorbenko, Olena; Ovcharenko, Galyna; Klymenko, Tetyana; Zhyvoloup, Olexandr; Gaman, Nadia; Volkova, Darija; Gout, Ivan; Filonenko, Valeriy

    2009-08-01

    Fibroblast growth factor receptor 3 (FGFR3) is a member of the FGFR family of receptor tyrosine kinases, whose function has been implicated in diverse biological processes, including cell proliferation, differentiation, survival, and tumorigenesis. Deregulation of FGFR3 signaling has been implicated with human pathologies, including cancer. Activating mutations in FGFR3 gene are frequently detected in bladder cancer, multiple myeloma, and noninvasive papillary urothelial cell carcinomas, while the overexpression of the receptor is observed in thyroid lymphoma and bladder cancer. The main aim of this study was to generate hybridoma clones producing antibody that could specifically recognize FGFR3/S249C mutant, but not the wild-type FGFR. To achieve this, we used for immunization bacterially expressed fragment of FGFR3 corresponding to loops II-III of the extracellular domain (GST-His/FGFR3/S249C-LII-III), which possesses oncogenic mutation at Ser249 detected in at least 50% of bladder cancers. Primary ELISA screening allowed us to isolate several hybridoma clones that showed specificity towards FGFR3/S249C, but not FGFR3wt protein. Unfortunately, these clones were not stable during single-cell cloning and expansion and lost the ability to recognize specifically FGFR3/S249C. However, this study allowed us to generate several monoclonal antibodies specific towards both FGFR3wt and FGFR3/S249C recombinant proteins. Produced hybridomas secreted MAbs that were specific in Western blotting towards bacterially expressed FGFR3wt and FGFR3/S249C, as well as the full-length receptors ectopically expressed in Sf21 and HEK293 cells. Moreover, transiently expressed wild-type and oncogenic forms of FGFR were efficiently immunoprecipitated with selected antibodies from the lysates of infected Sf21 and transiently transfected HEK293. In summary, generated antibodies should be useful as tools for examining the expression pattern and biological functions of FGFR3 in normal and

  14. Down-Modulation of Lung Immune Responses by Interleukin-10 and Transforming Growth Factor β (TGF-β) and Analysis of TGF-β Receptors I and II in Active Tuberculosis

    PubMed Central

    Bonecini-Almeida, M. Glória; Ho, John L.; Boéchat, Neio; Huard, Richard C.; Chitale, Sadhana; Doo, Howard; Geng, Jiayuan; Rego, Lorena; Lazzarini, Luiz Claudio Oliveira; Kritski, AfrÂnio L.; Johnson, Warren D.; McCaffrey, Timothy A.; Silva, José R. Lapa e

    2004-01-01

    Immune factors influencing progression to active tuberculosis (TB) remain poorly defined. In this study, we investigated the expression of immunoregulatory cytokines and receptors by using lung bronchoalveolar lavage cells obtained from patients with pulmonary TB, patients with other lung diseases (OLD patients), and healthy volunteers (VOL) by using reverse transcriptase PCR, a transforming growth factor β (TGF-β) bioactivity assay, and an enzyme immunoassay. TB patients were significantly more likely than OLD patients to coexpress TGF-β receptor I (RI) and RII mRNA, as well as interleukin-10 (IL-10) mRNA (thereby indicating the state of active gene transcription in the alveolar cells at harvest). In contrast, gamma interferon (IFN-γ) and IL-2 mRNA was seen in both TB and OLD patients. Likewise, significantly elevated pulmonary steady-state protein levels of IL-10, IFN-γ, and bioactive TGF-β were found in TB patients versus those in OLD patients and VOL. These data suggest that the combined production of the immunosuppressants IL-10 and TGF-β, as well as coexpression of TGF-β RI and RII (required for cellular response to TGF-β), may act to down-modulate host anti-Mycobacterium tuberculosis immunity and thereby allow uncontrolled bacterial replication and overt disease. Delineating the underlying mechanisms of M. tuberculosis-triggered expression of these immune elements may provide a molecular-level understanding of TB immunopathogenesis. PMID:15102771

  15. Coregulation of Epidermal Growth Factor Receptor/Human Epidermal Growth Factor Receptor 2 (HER2) Levels and Locations: Quantitative Analysis of HER2 Overexpression Effects

    SciTech Connect

    Hendriks, Bart S.; Opresko, Lee; Wiley, H. S.; Lauffenburger, Douglas A.

    2003-03-01

    Elevated expression of human epidermal growth factor receptor 2 (HER2) is know to alter cell signalilng and behavioral responses implicated in tumor progression. However, multiple diverse mechanisms may be involved in these overall effects, including signaling by HER2 itself, modulation of signalilng by epidermal growth factor receptor (EGFR) and modification of trafficking dynamics for both EGFR and HER2. Continued....

  16. Regulation of growth factor receptor degradation by ADP-ribosylation factor domain protein (ARD) 1.

    PubMed

    Meza-Carmen, Victor; Pacheco-Rodriguez, Gustavo; Kang, Gi Soo; Kato, Jiro; Donati, Chiara; Zhang, Chun-Yi; Vichi, Alessandro; Payne, D Michael; El-Chemaly, Souheil; Stylianou, Mario; Moss, Joel; Vaughan, Martha

    2011-06-28

    ADP-ribosylation factor domain protein 1 (ARD1) is a 64-kDa protein containing a functional ADP-ribosylation factor (GTP hydrolase, GTPase), GTPase-activating protein, and E3 ubiquitin ligase domains. ARD1 activation by the guanine nucleotide-exchange factor cytohesin-1 was known. GTPase and E3 ligase activities of ARD1 suggest roles in protein transport and turnover. To explore this hypothesis, we used mouse embryo fibroblasts (MEFs) from ARD1-/- mice stably transfected with plasmids for inducible expression of wild-type ARD1 protein (KO-WT), or ARD1 protein with inactivating mutations in E3 ligase domain (KO-E3), or containing persistently active GTP-bound (KO-GTP), or inactive GDP-bound (KO-GDP) GTPase domains. Inhibition of proteasomal proteases in mifepristone-induced KO-WT, KO-GDP, or KO-GTP MEFs resulted in accumulation of these ARD1 proteins, whereas KO-E3 accumulated without inhibitors. All data were consistent with the conclusion that ARD1 regulates its own steady-state levels in cells by autoubiquitination. Based on reported growth factor receptor-cytohesin interactions, EGF receptor (EGFR) was investigated in induced MEFs. Amounts of cell-surface and total EGFR were higher in KO-GDP and lower in KO-GTP than in KO-WT MEFs, with levels in both mutants greater (p = 0.001) after proteasomal inhibition. Significant differences among MEF lines in content of TGF-β receptor III were similar to those in EGFR, albeit not as large. Differences in amounts of insulin receptor mirrored those in EGFR, but did not reach statistical significance. Overall, the capacity of ARD1 GTPase to cycle between active and inactive forms and its autoubiquitination both appear to be necessary for the appropriate turnover of EGFR and perhaps additional growth factor receptors.

  17. Distribution of brain-derived neurotrophic factor and TrkB receptor proteins in the fetal and postnatal hippocampus and cerebellum of the guinea pig.

    PubMed

    Dieni, Sandra; Rees, Sandra

    2002-12-16

    This study investigates the distribution of brain-derived neurotrophic factor protein (BDNF) and its receptor, TrkB, during the development of hippocampus and cerebellum in a long-gestation species, the guinea pig. In the granule cell populations of both structures, BDNF immunoreactivity (-IR) was exclusive to postmigratory, mature neurons. In dentate granule cells, TrkB-IR was coexpressed with BDNF-IR, suggesting that the ligand-receptor interaction could occur by means of an autocrine/paracrine mechanism. In cerebellar granule cells, TrkB-IR was detected in both pre- and postmigratory cells, indicating that immature neurons are also BDNF-responsive. With advancing gestational age an increase in the intensity of BDNF-IR in granule cells was accompanied by concomitant increases in the staining and areal growth of the associated mossy fiber layer in the hippocampus, and the molecular layer in the cerebellum. The developmental increase in BDNF- and TrkB-IR in the neuropil of both structures coincided with periods of significant growth in all strata, indicating a role for BDNF and TrkB in process outgrowth. In the hippocampus, CA2, CA3, and hilar, neurons demonstrated both BDNF- and TrkB-IR during development and maturation, whereas CA1 neurons showed TrkB-IR throughout this period but only transient BDNF-IR in early gestation. In the fetal cerebellum, Purkinje cell bodies coexpressed BDNF-IR and TrkB-IR. In the postnatal period, BDNF-IR was down-regulated but TrkB-IR persisted, indicating that mature Purkinje cells might retain their responsiveness to BDNF. Thus, we have demonstrated in both the hippocampus and cerebellum that the spatiotemporal distribution of BDNF-IR and TrkB-IR coincides with the maturation of granule cells prenatally and with significant periods of neuropil growth, both prenatally and in the immediate postnatal period.

  18. Proteinase-activated receptor-2 transactivation of epidermal growth factor receptor and transforming growth factorreceptor signaling pathways contributes to renal fibrosis.

    PubMed

    Chung, Hyunjae; Ramachandran, Rithwik; Hollenberg, Morley D; Muruve, Daniel A

    2013-12-27

    Chronic kidney diseases cause significant morbidity and mortality in the population. During renal injury, kidney-localized proteinases can signal by cleaving and activating proteinase-activated receptor-2 (PAR2), a G-protein-coupled receptor involved in inflammation and fibrosis that is highly expressed in renal tubular cells. Following unilateral ureteric obstruction, PAR2-deficient mice displayed reduced renal tubular injury, fibrosis, collagen synthesis, connective tissue growth factor (CTGF), and α-smooth muscle actin gene expression at 7 days, compared with wild-type controls. In human proximal tubular epithelial cells in vitro, PAR2 stimulation with PAR2-activating peptide (PAR2-AP) alone significantly up-regulated the expression of CTGF, a potent profibrotic cytokine. The induction of CTGF by PAR2-AP was synergistically increased when combined with transforming growth factor-β (TGF-β). Consistent with these findings, treating human proximal tubular epithelial cells with PAR2-AP induced Smad2/3 phosphorylation in the canonical TGF-β signaling pathway. The Smad2 phosphorylation and CTGF induction required signaling via both the TGFβ-receptor and EGF receptor suggesting that PAR2 utilizes transactivation mechanisms to initiate fibrogenic signaling. Taken together, our data support the hypothesis that PAR2 synergizes with the TGFβ signaling pathway to contribute to renal injury and fibrosis.

  19. Vascular endothelial growth factor receptor 3 directly regulates murine neurogenesis

    PubMed Central

    Calvo, Charles-Félix; Fontaine, Romain H.; Soueid, Jihane; Tammela, Tuomas; Makinen, Taija; Alfaro-Cervello, Clara; Bonnaud, Fabien; Miguez, Andres; Benhaim, Lucile; Xu, Yunling; Barallobre, Maria-José; Moutkine, Imane; Lyytikkä, Johannes; Tatlisumak, Turgut; Pytowski, Bronislaw; Zalc, Bernard; Richardson, William; Kessaris, Nicoletta; Garcia-Verdugo, Jose Manuel; Alitalo, Kari; Eichmann, Anne; Thomas, Jean-Léon

    2011-01-01

    Neural stem cells (NSCs) are slowly dividing astrocytes that are intimately associated with capillary endothelial cells in the subventricular zone (SVZ) of the brain. Functionally, members of the vascular endothelial growth factor (VEGF) family can stimulate neurogenesis as well as angiogenesis, but it has been unclear whether they act directly via VEGF receptors (VEGFRs) expressed by neural cells, or indirectly via the release of growth factors from angiogenic capillaries. Here, we show that VEGFR-3, a receptor required for lymphangiogenesis, is expressed by NSCs and is directly required for neurogenesis. Vegfr3:YFP reporter mice show VEGFR-3 expression in multipotent NSCs, which are capable of self-renewal and are activated by the VEGFR-3 ligand VEGF-C in vitro. Overexpression of VEGF-C stimulates VEGFR-3-expressing NSCs and neurogenesis in the SVZ without affecting angiogenesis. Conversely, conditional deletion of Vegfr3 in neural cells, inducible deletion in subventricular astrocytes, and blocking of VEGFR-3 signaling with antibodies reduce SVZ neurogenesis. Therefore, VEGF-C/VEGFR-3 signaling acts directly on NSCs and regulates adult neurogenesis, opening potential approaches for treatment of neurodegenerative diseases. PMID:21498572

  20. Role of fibroblast growth factor receptor signaling in kidney development.

    PubMed

    Bates, Carlton M

    2011-08-01

    Fibroblast growth factor receptors (Fgfrs) consist of four signaling family members and one nonsignaling "decoy" receptor, Fgfr-like 1 (Fgfrl1), all of which are expressed in the developing kidney. Several studies have shown that exogenous fibroblast growth factors (Fgfs) affect growth and maturation of the metanephric mesenchyme (MM) and ureteric bud (UB) in cultured tissues. Transgenic and conditional knockout approaches in whole animals have shown that Fgfr1 and Fgfr2 (predominantly the IIIc isoform) in kidney mesenchyme are critical for early MM and UB formation. Conditional deletion of the ligand, Fgf8, in nephron precursors or global deletion of Fgfrl1 interrupts nephron formation. Fgfr2 (likely the IIIb isoform signaling downstream of Fgf7 and Fgf10) is critical for ureteric morphogenesis. Moreover, Fgfr2 appears to act independently of Frs2α (the major signaling adapter for Fgfrs) in regulating UB branching. Loss of Fgfr2 in the MM leads to many kidney and urinary tract anomalies, including vesicoureteral reflux. Thus Fgfr signaling is critical for patterning of virtually all renal lineages at early and later stages of development.

  1. Polychlorinated Biphenyls Disrupt Hepatic Epidermal Growth Factor Receptor Signaling.

    PubMed

    Hardesty, Josiah E; Wahlang, Banrida; Falkner, K Cameron; Clair, Heather B; Clark, Barbara J; Ceresa, Brian P; Prough, Russell A; Cave, Matthew C

    2016-07-26

    1. Polychlorinated biphenyls (PCBs) are persistent environmental pollutants that disrupt hepatic xenobiotic and intermediary metabolism, leading to metabolic syndrome and nonalcoholic steatohepatitis (NASH). 2. Since phenobarbital indirectly activates Constitutive Androstane Receptor (CAR) by antagonizing growth factor binding to the epidermal growth factor receptor (EGFR), we hypothesised that PCBs may also diminish EGFR signaling. 3. The effects of the PCB mixture Aroclor 1260 on the protein phosphorylation cascade triggered by EGFR activation were determined in murine (in vitro and in vivo) and human models (in vitro). EGFR tyrosine residue phosphorylation was decreased by PCBs in all models tested. 4. The IC50 values for Aroclor 1260 concentrations that decreased Y1173 phosphorylation of EGFR were similar in murine AML-12 and human HepG2 cells (∼2-4 μg/mL). Both dioxin and non-dioxin-like PCB congeners decreased EGFR phosphorylation in cell culture. 5. PCB treatment reduced phosphorylation of downstream EGFR effectors including Akt and mTOR, as well as other phosphoprotein targets including STAT3 and c-RAF in vivo. 6. PCBs diminish EGFR signaling in human and murine hepatocyte models and may dysregulate critical phosphoprotein regulators of energy metabolism and nutrition, providing a new mechanism of action in environmental diseases.

  2. Pregnane X receptor, constitutive androstane receptor and hepatocyte nuclear factors as emerging players in cancer precision medicine.

    PubMed

    De Mattia, Elena; Cecchin, Erika; Roncato, Rossana; Toffoli, Giuseppe

    2016-09-01

    Great research effort has been focused on elucidating the contribution of host genetic variability on pharmacological outcomes in cancer. Nuclear receptors have emerged as mediators between environmental stimuli and drug pharmacokinetics and pharmacodynamics. The pregnane X receptor, constitutive androstane receptor and hepatocyte nuclear factors have been reported to regulate transcription of genes that encode drug metabolizing enzymes and transporters. Altered nuclear receptor expression has been shown to affect the metabolism and pharmacological profile of traditional chemotherapeutics and targeted agents. Accordingly, polymorphic variants in these genes have been studied as pharmacogenetic markers of outcome variability. This review summarizes the state of knowledge about the roles played by pregnane X receptor, constitutive androstane receptor and hepatocyte nuclear factor expression and genetics as predictive markers of anticancer drug toxicity and efficacy, which can improve cancer precision medicine.

  3. Tumor necrosis factor receptor-associated factor 5 is an essential mediator of ischemic brain infarction.

    PubMed

    Wang, Lang; Lu, Yanyun; Guan, Hongjing; Jiang, Dingsheng; Guan, Yu; Zhang, Xin; Nakano, Hiroyasu; Zhou, Yan; Zhang, Yan; Yang, Li; Li, Hongliang

    2013-08-01

    Tumor necrosis factor receptor-associated factor 5 (TRAF5) is an adaptor protein of the tumor necrosis factor (TNF) receptor superfamily and the interleukin-1 receptor/Toll-like receptor superfamily and plays important roles in regulating multiple signaling pathways. This study was conducted to investigate the role of TRAF5 in the context of brain ischemia/reperfusion (I/R) injury. Transient occlusion of the middle cerebral artery was performed on TRAF5 knockout mice (KO), neuron-specific TRAF5 transgene (TG), and the appropriate controls. Compared with the WT mice, the TRAF5 KO mice showed lower infarct volumes and better outcomes in the neurological tests. A low neuronal apoptosis level, an attenuated blood-brain barrier (BBB) disruption and an inhibited inflammatory response were exhibited in TRAF5 KO mice. TRAF5 TG mice exhibited an opposite phenotype. Moreover, the Akt/FoxO1 signaling pathway was enhanced in the ischemic brains of the TRAF5 KO mice. These results provide the first demonstration that TRAF5 is a critical mediator of I/R injury in an experimental stroke model. The Akt /FoxO1 signaling pathway probably plays an important role in the biological function of TRAF5 in this model. © 2013 International Society for Neurochemistry.

  4. Results With Accelerated Partial Breast Irradiation in Terms of Estrogen Receptor, Progesterone Receptor, and Human Growth Factor Receptor 2 Status

    SciTech Connect

    Wilder, Richard B.; Curcio, Lisa D.; Khanijou, Rajesh K.; Eisner, Martin E.; Kakkis, Jane L.; Chittenden, Lucy; Agustin, Jeffrey; Lizarde, Jessica; Mesa, Albert V.; Macedo, Jorge C.; Ravera, John; Tokita, Kenneth M.

    2010-11-01

    Purpose: To report our results with accelerated partial breast irradiation (APBI) in terms of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2/neu) status. Methods and Materials: Between February 2003 and June 2009, 209 women with early-stage breast carcinomas were treated with APBI using multicatheter, MammoSite, or Contura brachytherapy to 34 Gy in 10 fractions twice daily over 5-7 days. Three patient groups were defined by receptor status: Group 1: ER or PR (+) and HER-2/neu (-) (n = 180), Group 2: ER and PR (-) and HER-2/neu (+) (n = 10), and Group 3: ER, PR, and HER-2/neu (-) (triple negative breast cancer, n = 19). Median follow-up was 22 months. Results: Group 3 patients had significantly higher Scarff-Bloom-Richardson scores (p < 0.001). The 3-year ipsilateral breast tumor control rates for Groups 1, 2, and 3 were 99%, 100%, and 100%, respectively (p = 0.15). Group 3 patients tended to experience relapse in distant sites earlier than did non-Group 3 patients. The 3-year relapse-free survival rates for Groups 1, 2, and 3 were 100%, 100%, and 81%, respectively (p = 0.046). The 3-year cause-specific and overall survival rates for Groups 1, 2, and 3 were 100%, 100%, and 89%, respectively (p = 0.002). Conclusions: Triple negative breast cancer patients typically have high-grade tumors with significantly worse relapse-free, cause-specific, and overall survival. Longer follow-up will help to determine whether these patients also have a higher risk of ipsilateral breast tumor relapse.

  5. Coexpression of Normally Incompatible Developmental Pathways in Retinoblastoma Genesis

    PubMed Central

    McEvoy, Justina; Flores-Otero, Jacqueline; Zhang, Jiakun; Nemeth, Katie; Brennan, Rachel; Bradley, Cori; Krafcik, Fred; Rodriguez-Galindo, Carlos; Wilson, Matthew; Xiong, Shunbin; Lozano, Guillermina; Sage, Julien; Fu, Ligia; Louhibi, Lotfi; Trimarchi, Jeff; Pani, Amar; Smeyne, Richard; Johnson, Dianna; Dyer, Michael A.

    2012-01-01

    SUMMARY It is widely believed that the molecular and cellular features of a tumor reflect its cell of origin and can thus provide clues about treatment targets. The retinoblastoma cell of origin has been debated for over a century. Here, we report that human and mouse retinoblastomas have molecular, cellular, and neurochemical features of multiple cell classes, principally amacrine/horizontal interneurons, retinal progenitor cells, and photoreceptors. Importantly, single-cell gene expression array analysis showed that these multiple cell type-specific developmental programs are coexpressed in individual retinoblastoma cells, which creates a progenitor/neuronal hybrid cell. Furthermore, neurotransmitter receptors, transporters, and biosynthetic enzymes are expressed in human retinoblastoma, and targeted disruption of these pathways reduces retinoblastoma growth in vivo and in vitro. PMID:21840489

  6. Epidermal growth factor receptor degradation: an alternative view of oncogenic pathways.

    PubMed

    Kirisits, Andreas; Pils, Dietmar; Krainer, Michael

    2007-01-01

    Positive regulation of epidermal growth factor receptor signalling is related to many human malignancies. Besides overexpression and gain of function mutations, the escape from negative regulation through an increase in epidermal growth factor receptor stability has evolved as yet another key factor contributing to enhanced receptor activity. Intensive research over the past years has provided considerable evidence concerning the molecular mechanisms which provide epidermal growth factor receptor degradation. c-Cbl mediated ubiquitination, endocytosis via clathrin-coated pits, endosomal sorting and lysosomal degradation have become well-investigated cornerstones. Recent findings on the interdependency of the endosomal sorting complexes required for transport in multivesicular body sorting, stress the topicality of receptor tyrosine kinase downregulation. Here, we review the degradation pathway of the epidermal growth factor receptor, following the receptor from ligand binding to the lysosome and illustrating different modes of oncogenic deregulation.

  7. Mammary tumorigenesis induced by fibroblast growth factor receptor 1 requires activation of the epidermal growth factor receptor.

    PubMed

    Bade, Lindsey K; Goldberg, Jodi E; Dehut, Hazel A; Hall, Majken K; Schwertfeger, Kathryn L

    2011-09-15

    Fibroblast growth factor receptor 1 (FGFR1) is an oncoprotein with known involvement in mammary tumorigenesis. To understand how FGFR1 signaling promotes mammary tumorigenesis, an inducible FGFR1 (iFGFR1) system was created previously. Previous studies have demonstrated that upon iFGFR1 activation in vivo, the epidermal growth factor (EGF) ligands amphiregulin (AREG) and epiregulin (EREG) are upregulated. Both AREG and EREG interact with the EGF receptor (EGFR). Here, we investigated whether the FGFR1-induced increase in AREG and EREG expression might coordinately increase EGFR signaling to promote mammary tumorigenesis. Treatment of mouse mammary epithelial cells with either AREG or EREG conferred a greater migratory potential, increased cellular proliferation and increased extracellular regulated kinase 1/2 (ERK1/2) activation. These effects could be blocked with the EGFR-specific inhibitor erlotinib, suggesting that they are EGFR-dependent. In transgenic mice with iFGFR1 under the control of the mouse mammary tumor virus (MMTV) promoter, iFGFR1 activation also led to increased mammary epithelial cell proliferation that was inhibited with erlotinib. Taken together, these data suggest that AREG and EREG mediate tumorigenic phenotypes by activating EGFR signaling, and that the oncogenic potential of FGFR1 requires EGFR activation to promote mammary tumorigenesis.

  8. Recombinant pigment epithelium-derived factor PEDF binds vascular endothelial growth factor receptors 1 and 2.

    PubMed

    Johnston, Erin K; Francis, Mary K; Knepper, Janice E

    2015-08-01

    Angiogenesis, or the formation of new blood vessels, is stimulated by angiogenic factors such as vascular endothelial growth factor (VEGF). Pigment epithelium-derived factor (PEDF) is a potent inhibitor of angiogenesis. To explore the mechanism by which PEDF acts, recombinant PEDF was expressed with a 6x-His tag (for purification) and a green fluorescent protein (GFP) tag. The PEDF fusion protein was confirmed to be active in inhibition of endothelial cell proliferation and migration. Direct binding of PEDF to both vascular endothelial growth factor receptor-1 (VEGFR-1) and VEGFR-2 was demonstrated in an in vitro assay similar to an enzyme-linked immunosorbent assay (ELISA). PEDF was shown by immune-confocal microscopy to be localized within treated endothelial cells. When VEGF-stimulated endothelial cells were incubated with PEDF the VEGF receptors showed intracellular localization. These data suggest that the interaction between PEDF and VEGFR-1 or VEGFR-2 may be a possible mechanism for inhibiting angiogenesis. PEDF may be binding to the VEGF receptors to promote their internalization and/or degradation to limit VEGF responses in treated cells.

  9. Nuclear receptors, nuclear-receptor factors, and nuclear-receptor-like orphans form a large paralog cluster in Homo sapiens.

    PubMed

    Garcia-Vallvé, S; Palau, J

    1998-06-01

    We studied a human protein paralog cluster formed by 38 nonredundant sequences taken from the Swiss-Prot database and its supplement, TrEMBL. These sequences include nuclear receptors, nuclear-receptor factors and nuclear-receptor-like orphans. Working separately with both the central cysteine-rich DNA-binding domain and the carboxy-terminal ligand-binding domain, we performed multialignment analyses that included drawings of paralog trees. Our results show that the cluster is highly multibranched, with considerable differences in the amino acid sequence in the ligand-binding domain (LBD), and 17 proximal subbranches which are identifiable and fully coincident when independent trees from both domains are compared. We identified the six recently proposed subfamilies as groups of neighboring clusters in the LBD paralog tree. We found similarities of 80%-100% for the N-terminal transactivation domain among mammalian ortholog receptors, as well as some paralog resemblances within diverse subbranches. Our studies suggest that during the evolutionary process, the three domains were assembled in a modular fashion with a nonshuffled modular fusion of the LBD. We used the EMBL server PredictProtein to make secondary-structure predictions for all 38 LBD subsequences. Amino acid residues in the multialigned homologous domains--taking the beginning of helix H3 of the human retinoic acid receptor-gamma as the initial point of reference--were substituted with H or E, which identify residues predicted to be helical or extended, respectively. The result was a secondary structure multialignment with the surprising feature that the prediction follows a canonical pattern of alignable alpha-helices with some short extended elements in between, despite the fact that a number of subsequences resemble each other by less than 25% in terms of the similarity index. We also identified the presence of a binary patterning in all of the predicted helices that were conserved throughout the 38

  10. Changing the insulin receptor to possess insulin-like growth factor I ligand specificity

    SciTech Connect

    Andersen, A.S.; Kjeldsen, T.; Wiberg, F.C.; Christensen, P.M.; Rasmussen, J.S.; Norris, K.; Moeller, K.B.; Moeller, N.P.H. )

    1990-08-14

    To examine the role of the N-terminal part of the insulin-like growth factor I (IGF-I) receptor and insulin receptor in determining ligand specificity, the authors prepared an expression vector encoding a hybrid receptor where exon 1 (encoding the signal peptide and seven amino acids of the {alpha}-subunit), exon 2, and exon 3 of the insulin receptor were replaced with the corresponding IGF-I receptor cDNA (938 nucleotides). To allow direct quantitative comparison of the binding capabilities of this hybrid receptor with those of the human IGF-I receptor and the insulin receptor, all three receptors were expressed in baby hamster kidney (BHK) cells as soluble molecules and partially purified before characterization. The hybrid IGF-I/insulin receptor bound IGF-I with an affinity comparable to that of the wild-type IGF-I receptor. In contrast, the hybrid receptor no longer displayed high-affinity binding of insulin. These results directly demonstrate that it is possible to change the specificity of the insulin receptor to that of the IGF-I receptor and, furthermore, that the binding specificity for IGF-I is encoded within the nucleotide sequence from 135 to 938 of the IGF-I receptor cDNA. Since the hybrid receptor only bound insulin with low affinity, the insulin binding region is likely to be located within exons 2 and 3 of the insulin receptor.

  11. Glucocorticoids and atrial natriuretic factor receptors on vascular smooth muscle.

    PubMed

    Yasunari, K; Kohno, M; Murakawa, K; Yokokawa, K; Takeda, T

    1990-11-01

    The effect of glucocorticoids on the atrial natriuretic factor (ANF)-mediated formation of cyclic guanosine monophosphate (cGMP) by intact vascular smooth muscle cells (VSMC) was studied in rats. Cultured VSMC were obtained from the renal arteries of 14-week-old Wistar rats by the explant method. Micromolar concentrations of dexamethasone, given as pretreatment for 48 hours, suppressed the ANF-mediated response. The dexamethasone-induced suppression was detectable at 6 hours and reached a maximum 24 hours after administration in a dose-dependent manner. Inhibitors of protein synthesis blocked this effect of the glucocorticoid. The basal activity of guanylate cyclase in the dexamethasone-treated cells was lower than in the control cells. Other steroids having glucocorticoid action mimicked this suppression of the ANF-mediated response. This suppression was blocked by a glucocorticoid receptor antagonist. The results suggest that glucocorticoids suppress ANF-mediated cGMP formation by VSMC through glucocorticoid type II receptors and the induction of protein synthesis. Suppression of the ANF-mediated response may play a role in glucocorticoid-induced hypertension.

  12. Epidermal Growth Factor Receptor Cell Survival Signaling Requires Phosphatidylcholine Biosynthesis

    PubMed Central

    Crook, Matt; Upadhyay, Awani; Ido, Liyana J.; Hanna-Rose, Wendy

    2016-01-01

    Identification of pro-cell survival signaling pathways has implications for cancer, cardiovascular, and neurodegenerative disease. We show that the Caenorhabditis elegans epidermal growth factor receptor LET-23 (LET-23 EGFR) has a prosurvival function in counteracting excitotoxicity, and we identify novel molecular players required for this prosurvival signaling. uv1 sensory cells in the C. elegans uterus undergo excitotoxic death in response to activation of the OSM-9/OCR-4 TRPV channel by the endogenous agonist nicotinamide. Activation of LET-23 EGFR can effectively prevent this excitotoxic death. We investigate the roles of signaling pathways known to act downstream of LET-23 EGFR in C. elegans and find that the LET-60 Ras/MAPK pathway, but not the IP3 receptor pathway, is required for efficient LET-23 EGFR activity in its prosurvival function. However, activation of LET-60 Ras/MAPK pathway does not appear to be sufficient to fully mimic LET-23 EGFR activity. We screen for genes that are required for EGFR prosurvival function and uncover a role for phosphatidylcholine biosynthetic enzymes in EGFR prosurvival function. Finally, we show that exogenous application of phosphatidylcholine is sufficient to prevent some deaths in this excitotoxicity model. Our work implicates regulation of lipid synthesis downstream of EGFR in cell survival and death decisions. PMID:27605519

  13. Argos inhibits epidermal growth factor receptor signalling by ligand sequestration.

    PubMed

    Klein, Daryl E; Nappi, Valerie M; Reeves, Gregory T; Shvartsman, Stanislav Y; Lemmon, Mark A

    2004-08-26

    The epidermal growth factor receptor (EGFR) has critical functions in development and in many human cancers. During development, the spatial extent of EGFR signalling is regulated by feedback loops comprising both well-understood activators and less well-characterized inhibitors. In Drosophila melanogaster the secreted protein Argos functions as the only known extracellular inhibitor of EGFR, with clearly identified roles in multiple stages of development. Argos is only expressed when the Drosophila EGFR (DER) is activated at high levels, and downregulates further DER signalling. Although there is ample genetic evidence that Argos inhibits DER activation, the biochemical mechanism has not been established. Here we show that Argos inhibits DER signalling without interacting directly with the receptor, but instead by sequestering the DER-activating ligand Spitz. Argos binds tightly to the EGF motif of Spitz and forms a 1:1 (Spitz:Argos) complex that does not bind DER in vitro or at the cell surface. Our results provide an insight into the mechanism of Argos function, and suggest new strategies for EGFR inhibitor design.

  14. Epidermal Growth Factor Receptor in Prostate Cancer Derived Exosomes

    PubMed Central

    Kharmate, Geetanjali; Hosseini-Beheshti, Elham; Caradec, Josselin; Chin, Mei Yieng; Tomlinson Guns, Emma S.

    2016-01-01

    Exosomes proteins and microRNAs have gained much attention as diagnostic tools and biomarker potential in various malignancies including prostate cancer (PCa). However, the role of exosomes and membrane-associated receptors, particularly epidermal growth factor receptor (EGFR) as mediators of cell proliferation and invasion in PCa progression remains unexplored. EGFR is frequently overexpressed and has been associated with aggressive forms of PCa. While PCa cells and tissues express EGFR, it is unknown whether exosomes derived from PCa cells or PCa patient serum contains EGFR. The aim of this study was to detect and characterize EGFR in exosomes derived from PCa cells, LNCaP xenograft and PCa patient serum. Exosomes were isolated from conditioned media of different PCa cell lines; LNCaP xenograft serum as well as patient plasma/serum by differential centrifugation and ultracentrifugation on a sucrose density gradient. Exosomes were confirmed by electron microscopy, expression of exosomal markers and NanoSight™ analysis. EGFR expression was determined by western blot analysis and ELISA. This study demonstrates that exosomes may easily be derived from PCa cell lines, serum obtained from PCa xenograft bearing mice and clinical samples derived from PCa patients. Presence of exosomal EGFR in PCa patient exosomes may present a novel approach for measuring of the disease state. Our work will allow to build on this finding for future understanding of PCa exosomes and their potential role in PCa progression and as minimal invasive biomarkers for PCa. PMID:27152724

  15. Epidermal growth factor receptor activation in developing rat kidney.

    PubMed

    Cybulsky, A V; Goodyer, P R; McTavish, A J

    1994-09-01

    Epidermal growth factor (EGF) binding increases in late-gestational rat kidney and then falls toward basal adult levels postnatally during the 1st wk. We report that the increase in EGF binding is accompanied by an increase in EGF receptor (EGFR) protein and activation of EGFR tyrosine kinase. Multiple proteins were endogenously tyrosine phosphorylated in kidney membranes from fetal rats, and the phosphorylation pattern was similar in rats ranging from 16 to 21 days of gestation. Tyrosine phosphorylation was, however, almost undetectable in 12-wk adult rat kidneys (controls). Among the phosphoproteins in fetal kidney, a prominent 170-kDa protein was identified as EGFR. Endogenous tyrosine phosphorylation of EGFR (reflecting receptor activation) was 30-fold higher in fetal kidney membranes than in adult (3- to 7-fold higher when adjusted for differences in EGF binding or EGFR protein content). The EGFR substrate, phospholipase C-gamma 1, was tyrosine phosphorylated in fetal kidneys but not adult, and a greater proportion was membrane-associated in fetal kidneys, consistent with activation of phospholipase C-gamma 1. Thus EGFR tyrosine kinase activity is increased in late-gestational rat kidney. Induction and activation of EGFR may mediate perinatal renal cell growth and development.

  16. Epidermal Growth Factor Receptor Cell Proliferation Signaling Pathways

    PubMed Central

    Wee, Ping; Wang, Zhixiang

    2017-01-01

    The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is commonly upregulated in cancers such as in non-small-cell lung cancer, metastatic colorectal cancer, glioblastoma, head and neck cancer, pancreatic cancer, and breast cancer. Various mechanisms mediate the upregulation of EGFR activity, including common mutations and truncations to its extracellular domain, such as in the EGFRvIII truncations, as well as to its kinase domain, such as the L858R and T790M mutations, or the exon 19 truncation. These EGFR aberrations over-activate downstream pro-oncogenic signaling pathways, including the RAS-RAF-MEK-ERK MAPK and AKT-PI3K-mTOR pathways. These pathways then activate many biological outputs that are beneficial to cancer cell proliferation, including their chronic initiation and progression through the cell cycle. Here, we review the molecular mechanisms that regulate EGFR signal transduction, including the EGFR structure and its mutations, ligand binding and EGFR dimerization, as well as the signaling pathways that lead to G1 cell cycle progression. We focus on the induction of CYCLIN D expression, CDK4/6 activation, and the repression of cyclin-dependent kinase inhibitor proteins (CDKi) by EGFR signaling pathways. We also discuss the successes and challenges of EGFR-targeted therapies, and the potential for their use in combination with CDK4/6 inhibitors. PMID:28513565

  17. Functional role of hepatocyte growth factor receptor during sperm maturation.

    PubMed

    Catizone, A; Ricci, G; Galdieri, M

    2002-01-01

    Mammalian spermatozoa acquire motility and fertilizing capacity during their transit through the epididymis. Hepatocyte growth factor (HGF) is a pleiotropic cytokine with potent motogenic capacities that has been identified in different organs, including the mammalian male genital tract. In mice, HGF is present in the testis and, in large amounts, in the distal part of the epididymis. In prepuberal rats, we have demonstrated that HGF is synthesized by the peritubular myoid cells and in men, HGF is present in significant quantities in seminal plasma. It has been suggested that in mice, HGF has a role in initiating sperm motility, whereas in men, no significant correlations between HGF concentration and sperm motility have been found. In the present paper we report that in rats, HGF receptor, c-met, is expressed in testicular and epididymal spermatozoa. Through immunocytochemistry, we have found that c-met is exclusively localized on the head in testicular sperm. A different localization of c-met has been found in sperm isolated from caput and cauda epididymidis. Cells isolated from epididymal caput show a c-met localization exclusively restricted to the head in most cells. In a minority of caput epididymis spermatozoa the receptor is localized both in the cell head and along the flagellum. Spermatozoa isolated from the epididymal cauda were quite homogeneous, showing the receptor localized along the entire cell surface. We also report that HGF is synthesized and secreted by the rat epididymis as indicated by the scatter effect of epididymal cell homogenate and culture medium on MDCK cells. To clarify whether HGF is involved in the acquisition of sperm motility in the epididymis, its maintenance, or both, spermatozoa isolated from caput epididymidis have been cultured in medium alone or supplemented with HGF. The results obtained indicated that HGF has a positive effect on the maintenance of sperm motility which, in the absence of HGF, significantly decreases during

  18. Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor–Resistant Disease

    PubMed Central

    Ohashi, Kadoaki; Maruvka, Yosef E.; Michor, Franziska; Pao, William

    2013-01-01

    Purpose EGFR-mutant lung cancer was first described as a new clinical entity in 2004. Here, we present an update on new controversies and conclusions regarding the disease. Methods This article reviews the clinical implications of EGFR mutations in lung cancer with a focus on epidermal growth factor receptor tyrosine kinase inhibitor resistance. Results The discovery of EGFR mutations has altered the ways in which we consider and treat non–small-cell lung cancer (NSCLC). Patients whose metastatic tumors harbor EGFR mutations are expected to live longer than 2 years, more than double the previous survival rates for lung cancer. Conclusion The information presented in this review can guide practitioners and help them inform their patients about EGFR mutations and their impact on the treatment of NSCLC. Efforts should now concentrate on making EGFR-mutant lung cancer a chronic rather than fatal disease. PMID:23401451

  19. Epidermal growth factor receptor-targeted therapy for pancreatic cancer.

    PubMed

    Xiong, Henry Q; Abbruzzese, James L

    2002-10-01

    Epidermal growth factor receptor (EGFR) plays an important role in tumor development and maintenance. It is a cell surface molecule that mediates signal transduction from the cell surface to cytoplasm. Elevated expression of EGFR or its ligand correlates with worse prognosis in a variety of human cancers. Therefore, blockade of EGFR activity would provide a novel strategy for the treatment of cancer. Two classes of EGFR inhibitors, monoclonal antibodies and tyrosine kinase inhibitors, have been described. The preclinical activity of these EGFR inhibitors and phase I clinical data are summarized in this article. A phase II trial of the EGFR inhibitor IMC-C225 in combination with gemcitabine for patients with advanced pancreatic cancer is discussed. Copyright 2002, Elsevier Science (USA). All rights reserved.

  20. A comprehensive pathway map of epidermal growth factor receptor signaling

    PubMed Central

    Oda, Kanae; Matsuoka, Yukiko; Funahashi, Akira; Kitano, Hiroaki

    2005-01-01

    The epidermal growth factor receptor (EGFR) signaling pathway is one of the most important pathways that regulate growth, survival, proliferation, and differentiation in mammalian cells. Reflecting this importance, it is one of the best-investigated signaling systems, both experimentally and computationally, and several computational models have been developed for dynamic analysis. A map of molecular interactions of the EGFR signaling system is a valuable resource for research in this area. In this paper, we present a comprehensive pathway map of EGFR signaling and other related pathways. The map reveals that the overall architecture of the pathway is a bow-tie (or hourglass) structure with several feedback loops. The map is created using CellDesigner software that enables us to graphically represent interactions using a well-defined and consistent graphical notation, and to store it in Systems Biology Markup Language (SBML). PMID:16729045

  1. Targeting fibroblast growth factor receptor signaling in hepatocellular carcinoma.

    PubMed

    Cheng, Ann-Lii; Shen, Ying-Chun; Zhu, Andrew X

    2011-01-01

    Hepatocellular carcinoma (HCC) is the primary type of liver cancer, and both the age-adjusted incidence and mortality of HCC have steadily increased in recent years. Advanced HCC is associated with a very poor survival rate. Despite accumulating data regarding the risk factors for HCC, the mechanisms that contribute to HCC tumorigenesis remain poorly understood. Signaling through the fibroblast growth factor (FGF) family is involved in fibrosis and its progression to cirrhosis of the liver, which is a risk factor for the development of HCC. Furthermore, several alterations in FGF/FGF receptor (FGFR) signaling correlate with the outcomes of HCC patients, suggesting that signaling through this family of proteins contributes to the development or progression of HCC tumors. Currently, there are no established systemic treatments for patients with advanced HCC in whom sorafenib treatment has failed or who were unable to tolerate it. Recently, several multikinase inhibitors that target FGFRs have demonstrated some early evidence of antitumor activity in phase I/II trials. Therefore, this review discusses the molecular implications of FGFR-mediated signaling in HCC and summarizes the clinical evidence for novel FGFR-targeted therapies for HCC currently being studied in clinical trials.

  2. Targeting Extracellular Domains D4 and D7 of Vascular Endothelial Growth Factor Receptor 2 Reveals Allosteric Receptor Regulatory Sites

    PubMed Central

    Hyde, Caroline A. C.; Giese, Alexandra; Stuttfeld, Edward; Abram Saliba, Johan; Villemagne, Denis; Schleier, Thomas; Binz, H. Kaspar

    2012-01-01

    Vascular endothelial growth factors (VEGFs) activate three receptor tyrosine kinases, VEGFR-1, -2, and -3, which regulate angiogenic and lymphangiogenic signaling. VEGFR-2 is the most prominent receptor in angiogenic signaling by VEGF ligands. The extracellular part of VEGF receptors consists of seven immunoglobulin homology domains (Ig domains). Earlier studies showed that domains 2 and 3 (D23) mediate ligand binding, while structural analysis of dimeric ligand/receptor complexes by electron microscopy and small-angle solution scattering revealed additional homotypic contacts in membrane-proximal Ig domains D4 and D7. Here we show that D4 and D7 are indispensable for receptor signaling. To confirm the essential role of these domains in signaling, we isolated VEGFR-2-inhibitory “designed ankyrin repeat proteins” (DARPins) that interact with D23, D4, or D7. DARPins that interact with D23 inhibited ligand binding, receptor dimerization, and receptor kinase activation, while DARPins specific for D4 or D7 did not prevent ligand binding or receptor dimerization but effectively blocked receptor signaling and functional output. These data show that D4 and D7 allosterically regulate VEGFR-2 activity. We propose that these extracellular-domain-specific DARPins represent a novel generation of receptor-inhibitory drugs for in vivo applications such as targeting of VEGFRs in medical diagnostics and for treating vascular pathologies. PMID:22801374

  3. Targeting extracellular domains D4 and D7 of vascular endothelial growth factor receptor 2 reveals allosteric receptor regulatory sites.

    PubMed

    Hyde, Caroline A C; Giese, Alexandra; Stuttfeld, Edward; Abram Saliba, Johan; Villemagne, Denis; Schleier, Thomas; Binz, H Kaspar; Ballmer-Hofer, Kurt

    2012-10-01

    Vascular endothelial growth factors (VEGFs) activate three receptor tyrosine kinases, VEGFR-1, -2, and -3, which regulate angiogenic and lymphangiogenic signaling. VEGFR-2 is the most prominent receptor in angiogenic signaling by VEGF ligands. The extracellular part of VEGF receptors consists of seven immunoglobulin homology domains (Ig domains). Earlier studies showed that domains 2 and 3 (D23) mediate ligand binding, while structural analysis of dimeric ligand/receptor complexes by electron microscopy and small-angle solution scattering revealed additional homotypic contacts in membrane-proximal Ig domains D4 and D7. Here we show that D4 and D7 are indispensable for receptor signaling. To confirm the essential role of these domains in signaling, we isolated VEGFR-2-inhibitory "designed ankyrin repeat proteins" (DARPins) that interact with D23, D4, or D7. DARPins that interact with D23 inhibited ligand binding, receptor dimerization, and receptor kinase activation, while DARPins specific for D4 or D7 did not prevent ligand binding or receptor dimerization but effectively blocked receptor signaling and functional output. These data show that D4 and D7 allosterically regulate VEGFR-2 activity. We propose that these extracellular-domain-specific DARPins represent a novel generation of receptor-inhibitory drugs for in vivo applications such as targeting of VEGFRs in medical diagnostics and for treating vascular pathologies.

  4. The RON and MET oncogenes are co-expressed in human ovarian carcinomas and cooperate in activating invasiveness.

    PubMed

    Maggiora, Piera; Lorenzato, Annalisa; Fracchioli, Stefano; Costa, Barbara; Castagnaro, Massimo; Arisio, Riccardo; Katsaros, Dionyssios; Massobrio, Marco; Comoglio, Paolo M; Flavia Di Renzo, Maria

    2003-08-15

    RON is a member of the receptor tyrosine kinase gene family that includes the MET oncogene, whose germline mutations have been causally related to human tumorigenesis. In vitro, RON and MET receptors cross-talk, synergize in intracellular signaling, and cooperate in inducing morphogenic responses. Here we show that the RON and MET oncogenes were expressed in 55% and 56% of human ovarian carcinomas, respectively, and were significantly coexpressed in 42% (P < 0.001). In ovarian carcinoma samples and cell lines we did not find mutations in RON and MET gene kinase domain, nor coexpression of RON and MET receptor ligands (MSP and HGF, respectively). We show that motility and invasiveness of ovarian cancer cells coexpressing MET and RON receptors were elicited by HGF and, to a lesser extent, by MSP. More interestingly, invasion of both reconstituted basement membrane and collagen gel was greatly enhanced by the simultaneous addition of the two ligands. These data suggest that coexpression of the MET and RON receptors confer a selective advantage to ovarian cancer cells and might promote ovarian cancer progression.

  5. Autophosphorylation is essential for the in vivo function of the Lotus japonicus Nod factor receptor 1 and receptor-mediated signalling in cooperation with Nod factor receptor 5.

    PubMed

    Madsen, Esben B; Antolín-Llovera, Meritxell; Grossmann, Christina; Ye, Juanying; Vieweg, Syndi; Broghammer, Angelique; Krusell, Lene; Radutoiu, Simona; Jensen, Ole N; Stougaard, Jens; Parniske, Martin

    2011-02-01

    Soil-living rhizobia secrete lipochitin oligosaccharides known as Nod factors, which in Lotus japonicus are perceived by at least two Nod-factor receptors, NFR1 and NFR5. Despite progress in identifying molecular components critical for initial legume host recognition of the microsymbiont and cloning of downstream components, little is known about the activation and signalling mechanisms of the Nod-factor receptors themselves. Here we show that both receptor proteins localize to the plasma membrane, and present evidence for heterocomplex formation initiating downstream signalling. Expression of NFR1 and NFR5 in Nicotiana benthamiana and Allium ampeloprasum (leek) cells caused a rapid cell-death response. The signalling leading to cell death was abrogated using a kinase-inactive variant of NFR1. In these surviving cells, a clear interaction between NFR1 and NFR5 was detected in vivo through bimolecular fluorescence complementation (BiFC). To analyse the inter- and intramolecular phosphorylation events of the kinase complex, the cytoplasmic part of NFR1 was assayed for in vitro kinase activity, and autophosphorylation on 24 amino acid residues, including three tyrosine residues, was found by mass spectrometry. Substitution of the phosphorylated amino acids of NFR1 identified a single phosphorylation site to be essential for NFR1 Nod-factor signalling in vivo and kinase activity in vitro. In contrast to NFR1, no in vitro kinase activity of the cytoplasmic domain of NFR5 was detected. This is further supported by the fact that a mutagenized NFR5 construct, substituting an amino acid essential for ATP binding, restored nodulation of nfr5 mutant roots. © 2010 The Authors. The Plant Journal © 2010 Blackwell Publishing Ltd.

  6. Arctigenin induced gallbladder cancer senescence through modulating epidermal growth factor receptor pathway.

    PubMed

    Zhang, Mingdi; Cai, Shizhong; Zuo, Bin; Gong, Wei; Tang, Zhaohui; Zhou, Di; Weng, Mingzhe; Qin, Yiyu; Wang, Shouhua; Liu, Jun; Ma, Fei; Quan, Zhiwei

    2017-05-01

    Gallbladder cancer has poor prognosis and limited therapeutic options. Arctigenin, a representative dibenzylbutyrolactone lignan, occurs in a variety of plants. However, the molecular mechanisms involved in the antitumor effect of arctigenin on gallbladder cancer have not been fully elucidated. The expression levels of epidermal growth factor receptor were examined in 100 matched pairs of gallbladder cancer tissues. A positive correlation between high epidermal growth factor receptor expression levels and poor prognosis was observed in gallbladder cancer tissues. Pharmacological inhibition or inhibition via RNA interference of epidermal growth factor receptor induced cellular senescence in gallbladder cancer cells. The antitumor effect of arctigenin on gallbladder cancer cells was primarily achieved by inducing cellular senescence. In gallbladder cancer cells treated with arctigenin, the expression level of epidermal growth factor receptor significantly decreased. The analysis of the activity of the kinases downstream of epidermal growth factor receptor revealed that the RAF-MEK-ERK signaling pathway was significantly inhibited. Furthermore, the cellular senescence induced by arctigenin could be reverted by pcDNA-epidermal growth factor receptor. Arctigenin also potently inhibited the growth of tumor xenografts, which was accompanied by the downregulation of epidermal growth factor receptor and induction of senescence. This study demonstrates arctigenin could induce cellular senescence in gallbladder cancer through the modulation of epidermal growth factor receptor pathway. These data identify epidermal growth factor receptor as a key regulator in arctigenin-induced gallbladder cancer senescence.

  7. Expression of nerve growth factor and its receptor, tyrosine kinase receptor A, in rooster testes.

    PubMed

    Ma, Wei; Wang, Chunqiang; Su, Yuhong; Tian, Yumin; Zhu, Hongyan

    2015-10-01

    Nerve growth factor (NGF), which is required for the survival and differentiation of the nervous system, is also thought to play an important role in the development of mammalian reproductive tissues. To explore the function of NGF in the male reproductive system of non-mammalian animals, we determined the presence of NGF and its receptor, tyrosine kinase receptor A (TrkA), in rooster testes and investigated the regulation of NGF and TrkA expression by follicle-stimulating hormone (FSH). The mRNA and protein levels of NGF and TrkA in 6-week-old rooster testes were lower than those in 12-, 16- or 20-week age groups; levels were highest in the 16-week group. Immunohistochemistry showed that NGF and TrkA were both detected in spermatogonia, spermatocytes and spermatids. NGF immunoreactivity was observed in Leydig cells and strong TrkA signals were present in Sertoli cells. Meanwhile, FSH increased TrkA transcript levels in rooster testes in a dose-dependent manner. We present novel evidence for the developmental and FSH-regulated expression of the NGF/TrkA system, and our findings suggest that the NGF/TrkA system may play a prominent role in chicken spermatogenesis. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Identification of a novel immunoreceptor tyrosine-based activation motif-containing molecule, STAM2, by mass spectrometry and its involvement in growth factor and cytokine receptor signaling pathways.

    PubMed

    Pandey, A; Fernandez, M M; Steen, H; Blagoev, B; Nielsen, M M; Roche, S; Mann, M; Lodish, H F

    2000-12-08

    In an effort to clone novel tyrosine-phosphorylated substrates of the epidermal growth factor receptor, we have initiated an approach coupling affinity purification using anti-phosphotyrosine antibodies to mass spectrometry-based identification. Here, we report the identification of a signaling molecule containing a Src homology 3 domain as well as an immunoreceptor tyrosine-based activation motif (ITAM). This molecule is 55% identical to a previously isolated molecule designated signal transducing adaptor molecule (STAM) that was identified as an interleukin (IL)-2-induced phosphoprotein and is therefore designated STAM2. Tyrosine phosphorylation of STAM2 is induced by growth factors such as epidermal growth factor and platelet-derived growth factor as well as by cytokines like IL-3. Several of the deletion mutants tested except the one containing only the amino-terminal region underwent tyrosine phosphorylation upon growth factor stimulation, implying that STAM2 is phosphorylated on several tyrosine residues. STAM2 is downstream of the Jak family of kinases since coexpression of STAM2 with Jak1 or Jak2 but not an unrelated Tec family kinase, Etk, resulted in its tyrosine phosphorylation. In contrast to epidermal growth factor receptor-induced phosphorylation, this required the ITAM domain since mutants lacking this region did not undergo tyrosine phosphorylation. Finally, overexpression of wild type STAM2 led to an increase in IL-2-mediated induction of c-Myc promoter activation indicating that it potentiates cytokine receptor signaling.

  9. Conversion of epidermal growth factor receptor 2 and hormone receptor expression in breast cancer metastases to the brain

    PubMed Central

    2012-01-01

    Introduction We investigated the status of estrogen receptor alpha (ERα), progesterone receptor (PR), and epidermal growth factor receptor 2 (HER2) in primary tumor and in the corresponding brain metastases in a consecutive series of breast cancer patients. Additionally, we studied factors potentially influencing conversion and evaluated its association with survival. Methods The study group included 120 breast cancer patients. ERα, PR, and HER2 status in primary tumors and in matched brain metastases was determined centrally by immunohistochemistry and/or fluorescence in situ hybridization. Results Using the Allred score of ≥ 3 as a threshold, conversion of ERα and PR in brain metastases occurred in 29% of cases for both receptors, mostly from positive to negative. Conversion of HER2 occurred in 14% of patients and was more balanced either way. Time to brain relapse and the use of chemotherapy or trastuzumab did not influence conversion, whereas endocrine therapy induced conversion of ERα (P = 0.021) and PR (P = 0.001), mainly towards their loss. Receptor conversion had no significant impact on survival. Conclusions Receptor conversion, particularly loss of hormone receptors, is a common event in brain metastases from breast cancer, and endocrine therapy may increase its incidence. Receptor conversion does not significantly affect survival. PMID:22898337

  10. Activity of ixabepilone in oestrogen receptor-negative and oestrogen receptor-progesterone receptor-human epidermal growth factor receptor 2-negative metastatic breast cancer.

    PubMed

    Pivot, Xavier B; Li, Rubi K; Thomas, Eva S; Chung, Hyun-Cheol; Fein, Luis E; Chan, Valorie F; Jassem, Jacek; de Mendoza, Fernando Hurtado; Mukhopadyay, Pralay; Roché, Henri H

    2009-11-01

    Oestrogen receptor (ER)-negative breast cancer, including oestrogen receptor-, progesterone receptor- and human epidermal growth factor receptor 2-negative (ER/PR/HER2-negative) breast cancer, is more aggressive than ER-positive disease. A major limitation in the treatment of ER-negative disease subtypes is the inherent insensitivity to hormonal agents (tamoxifen, aromatase inhibitors) that are widely used in the treatment of breast cancer. Thus, therapeutic options for poor prognosis patients with ER-negative breast cancer are limited to a handful of chemotherapeutic agents, and new agents are needed to improve the treatment of this disease. Ixabepilone, a novel epothilone B analogue with low susceptibility to cellular mechanisms that confer resistance to taxanes and other chemotherapeutic agents, has demonstrated potent preclinical antitumour activity in multiple models, including those with primary or acquired drug resistance. This review summarises the results of a prospective subset analysis from a phase III clinical trial evaluating ixabepilone for the treatment of metastatic breast cancer (MBC), in which efficacy and safety were evaluated in patients with ER-negative and ER/PR/HER2-negative disease.

  11. Stem cell growth factor receptor in canine vs. feline osteosarcomas

    PubMed Central

    Wolfesberger, Birgitt; Fuchs-Baumgartinger, Andrea; Hlavaty, Juraj; Meyer, Florian R.; Hofer, Martin; Steinborn, Ralf; Gebhard, Christiane; Walter, Ingrid

    2016-01-01

    Osteosarcoma is considered the most common bone cancer in cats and dogs, with cats having a much better prognosis than dogs, since the great majority of dogs with osteosarcoma develop distant metastases. In search of a factor possibly contributing to this disparity, the stem cell growth factor receptor KIT was targeted, and the messenger (m)RNA and protein expression levels of KIT were compared in canine vs. feline osteosarcomas, as well as in normal bone. The mRNA expression of KIT was quantified by reverse transcription-quantitative polymerase chain reaction, and was observed to be significantly higher in canine (n=14) than in feline (n=5) osteosarcoma samples (P<0.001). KIT protein expression was evaluated by immunohistochemistry, which revealed that 21% of canine osteosarcoma samples did not exhibit KIT staining in their neoplastic cells, while in 14% of samples, a score of 1 (<10% positive tumour cells) was observed, and in 50% and 14% of samples, a score of 2 (10–50% positivity) and 3 (>50% positivity), respectively, was observed. By contrast, the cancer cells of all the feline bone tumour samples analysed were entirely negative for KIT. Notably, canine and feline osteocytes of healthy bone tissue lacked any KIT expression. These results could be the first evidence that KIT may be involved in the higher aggressiveness of canine osteosarcoma compared with feline osteosarcoma. PMID:27698817

  12. Epidermal growth factor receptor tyrosine kinase inhibitors as anticancer agents.

    PubMed

    Ciardiello, F

    2000-01-01

    The epidermal growth factor receptor (EGFR)-driven autocrine growth pathway has been implicated in the development and progression of the majority of the most common human epithelial cancers, making the blockade of this growth pathway a promising anticancer therapeutic strategy. Different approaches have been developed to block EGFR activation and/or function in cancer cells. In the past 15 years, various anti-EGFR blocking monoclonal antibodies (MAb), recombinant proteins containing transforming growth factor-alpha (TGFalpha) or EGF fused to toxins, and tyrosine kinase inhibitors (TKIs) have been generated and their biological and potentially therapeutic properties characterised. One of these agents, MAb IMC-C225, a chimeric human-mouse IgG1 MAb, is the first anti-EGFR agent to enter phase II to III clinical trials in patients with cancer. Several small compounds that block the ligand-induced activation of the EGFR tyrosine kinase have been developed. Among these EGFR-TKIs, various quinazoline-derived agents have been synthesised and have shown promising activity as anticancer agents in preclinical models. ZD1839 ('Iressa'), an anilinoquinazoline, is an orally active, selective EGFR-TKI which is currently under clinical evaluation in phase II to III clinical trials in patients with cancer. Preclinical data for ZD1839 strongly support the possibility of potentiating the antitumour activity of conventional chemotherapy with agents that selectively block the EGFR.

  13. The Orphan Nuclear Receptor SHP Inhibits Hepatocyte Nuclear Factor 4 and Retinoid X Receptor Transactivation: Two Mechanisms for Repression

    PubMed Central

    Lee, Yoon-Kwang; Dell, Helen; Dowhan, Dennis H.; Hadzopoulou-Cladaras, Margarita; Moore, David D.

    2000-01-01

    The orphan nuclear hormone receptor SHP interacts with a number of other nuclear hormone receptors and inhibits their transcriptional activity. Several mechanisms have been suggested to account for this inhibition. Here we show that SHP inhibits transactivation by the orphan receptor hepatocyte nuclear factor 4 (HNF-4) and the retinoid X receptor (RXR) by at least two mechanisms. SHP interacts with the same HNF-4 surface recognized by transcriptional coactivators and competes with them for binding in vivo. The minimal SHP sequences previously found to be required for interaction with other receptors are sufficient for interaction with HNF-4, although deletion results indicate that additional C-terminal sequences are necessary for full binding and coactivator competition. These additional sequences include those associated with direct transcriptional repressor activity of SHP. SHP also competes with coactivators for binding to ligand-activated RXR, and based on the ligand-dependent interaction with other nuclear receptors, it is likely that coactivator competition is a general feature of SHP-mediated repression. The minimal receptor interaction domain of SHP is sufficient for full interaction with RXR, as previously described. This domain is also sufficient for full coactivator competition. Functionally, however, full inhibition of RXR transactivation requires the presence of the C-terminal repressor domain, with only weak inhibition associated with this receptor interaction domain. Overall, these results suggest that SHP represses nuclear hormone receptor-mediated transactivation via two separate steps: first by competition with coactivators and then by direct effects of its transcriptional repressor function. PMID:10594021

  14. Development of the epidermal growth factor receptor inhibitor OSI-774.

    PubMed

    Grünwald, Viktor; Hidalgo, Manuel

    2003-06-01

    The epidermal growth factor receptor (EGFR) is a transmembrane receptor involved in the regulation of a complex array of essential biological processes such as cell proliferation and survival. Dysregulation of the EGFR signaling network has been frequently reported in multiple human cancers and has been associated with the processes of tumor development, growth, proliferation, metastasis, and angiogenesis. Inhibition of the EGFR was associated with antitumor effects in preclinical models. On the basis of these data, therapeutics targeting the EGFR were explored in clinical trials. OSI-774 is a small-molecule selective inhibitor of the EGFR tyrosine kinase. In preclinical studies, OSI-774 inhibited the phosphorylation of the EGFR in a dose-dependent and concentration-dependent manner resulting in cell cycle arrest and induction of apoptosis. In in vivo studies, this agent caused tumor growth inhibition and showed synergistic effects when combined with conventional chemotherapy. Subsequent single-agent phase I studies and phase I studies in combination with chemotherapy showed that the agent has a good safety profile and induced tumor growth inhibition in a substantial number of patients with a variety of different solid tumors. Preliminary reports from phase II studies confirmed the excellent tolerability of OSI-774 and showed encouraging preliminary activity. Phase III studies have either been completed or are ongoing in several tumor types such as lung cancer and pancreatic cancer. In summary, OSI-774 is a novel inhibitor of the EGFR tyrosine kinase that has shown promising activity in initial studies and is currently undergoing full development as an anticancer drug.

  15. Developmental regulation of human truncated nerve growth factor receptor

    SciTech Connect

    DiStefano, P.S.; Clagett-Dame, M.; Chelsea, D.M.; Loy, R. )

    1991-01-01

    Monoclonal antibodies (designated XIF1 and IIIG5) recognizing distinct epitopes of the human truncated nerve growth factor receptor (NGF-Rt) were used in a two-site radiometric immunosorbent assay to monitor levels of NGF-Rt in human urine as a function of age. Urine samples were collected from 70 neurologically normal subjects ranging in age from 1 month to 68 years. By using this sensitive two-site radiometric immunosorbent assay, NGF-Rt levels were found to be highest in urine from 1-month old subjects. By 2.5 months, NGF-Rt values were half of those seen at 1 month and decreased more gradually between 0.5 and 15 years. Between 15 and 68 years, urine NGF-Rt levels were relatively constant at 5% of 1-month values. No evidence for diurnal variation of adult NGF-Rt was apparent. Pregnant women in their third trimester showed significantly elevated urine NGF-Rt values compared with age-matched normals. Affinity labeling of NGF-Rt with 125I-NGF followed by immunoprecipitation with ME20.4-IgG and gel autoradiography indicated that neonatal urine contained high amounts of truncated receptor (Mr = 50 kd); decreasingly lower amounts of NGF-Rt were observed on gel autoradiograms with development, indicating that the two-site radiometric immunosorbent assay correlated well with the affinity labeling technique for measuring NGF-Rt. NGF-Rt in urines from 1-month-old and 36-year-old subjects showed no differences in affinities for NGF or for the monoclonal antibody IIIG5. These data show that NGF-Rt is developmentally regulated in human urine, and are discussed in relation to the development and maturation of the peripheral nervous system.

  16. Constitutive and inducible co-expression systems for non-viral osteoinductive gene therapy.

    PubMed

    Feichtinger, G A; Hacobian, A; Hofmann, A T; Wassermann, K; Zimmermann, A; van Griensven, M; Redl, H

    2014-02-19

    Tissue regenerative gene therapy requires expression strategies that deliver therapeutic effective amounts of transgenes. As physiological expression patterns are more complex than high-level expression of a singular therapeutic gene, we aimed at constitutive or inducible co-expression of 2 transgenes simultaneously. Co-expression of human bone morphogenetic protein 2 and 7 (BMP2/7) from constitutively expressing and doxycycline inducible plasmids was evaluated in vitro in C2C12 cells with osteocalcin reporter gene assays and standard assays for osteogenic differentiation. The constitutive systems were additionally tested in an in vivo pilot for ectopic bone formation after repeated naked DNA injection to murine muscle tissue. Inductor controlled differentiation was demonstrated in vitro for inducible co-expression. Both co-expression systems, inducible and constitutive, achieved significantly better osteogenic differentiation than single factor expression. The potency of the constitutive co-expression systems was dependent on relative expression cassette topology. In vivo, ectopic bone formation was demonstrated in 6/13 animals (46% bone formation efficacy) at days 14 and 28 in hind limb muscles as proven by in vivo µCT and histological evaluation. In vitro findings demonstrated that the devised single vector BMP2/7 co-expression strategy mediates superior osteoinduction, can be applied in an inductor controlled fashion and that its efficiency is dependent on expression cassette topology. In vivo results indicatethatco-expression of BMP2/7 applied by non-viral naked DNA gene transfer effectively mediates bone formation without the application of biomaterials, cells or recombinant growth factors, offering a promising alternative to current treatment strategies with potential for clinical translation in the future.

  17. The F-BAR Protein PACSIN2 Regulates Epidermal Growth Factor Receptor Internalization

    PubMed Central

    de Kreuk, Bart-Jan; Anthony, Eloise C.; Geerts, Dirk; Hordijk, Peter L.

    2012-01-01

    Signaling via growth factor receptors, including the epidermal growth factor (EGF) receptor, is key to various cellular processes, such as proliferation, cell survival, and cell migration. In a variety of human diseases such as cancer, aberrant expression and activation of growth factor receptors can lead to disturbed signaling. Intracellular trafficking is crucial for proper signaling of growth factor receptors. As a result, the level of cell surface expression of growth factor receptors is an important determinant for the outcome of downstream signaling. BAR domain-containing proteins represent an important family of proteins that regulate membrane dynamics. In this study, we identify a novel role for the F-BAR protein PACSIN2 in the regulation of EGF receptor signaling. We show that internalized EGF as well as the (activated) EGF receptor translocated to PACSIN2-positive endosomes. Furthermore, loss of PACSIN2 increased plasma membrane expression of the EGF receptor in resting cells and increased EGF-induced phosphorylation of the EGF receptor. As a consequence, EGF-induced activation of Erk and Akt as well as cell proliferation were enhanced in PACSIN2-depleted cells. In conclusion, this study identifies a novel role for the F-BAR-domain protein PACSIN2 in regulating EGF receptor surface levels and EGF-induced downstream signaling. PMID:23129763

  18. AXL and GAS6 co-expression in lung adenocarcinoma as a prognostic classifier.

    PubMed

    Seike, Masahiro; Kim, Cheol-Hong; Zou, Fenfei; Noro, Rintaro; Chiba, Mika; Ishikawa, Arimi; Κunugi, Shinobu; Kubota, Kaoru; Gemma, Akihiko

    2017-06-01

    AXL, a receptor tyrosine kinase implicated in cell survival, proliferation, and migration, is also associated with acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor therapy. However, its prognostic significance in lung adenocarcinoma (AD) remains unclear. We therefore evaluated the prognostic significance of the expression of AXL and/or its ligand, growth arrest-specific 6 (GAS6), in completely resected lung AD. We evaluated the relationship between AXL, GAS6, and vimentin expression, as determined by immunohistochemistry (IHC) analysis, with overall survival and disease-free survival in 113 patients with stages I-III lung AD. Protein expression was also assayed using western blot analysis in 10 lung AD cell lines. AXL-positive (AXL+), GAS6-positive (GAS6+), or AXL+/GAS6+ staining was significantly associated with vimentin-positive (vimentin+) expression. AXL+/GAS6+ and vimentin+ showed a negative tendency toward an association with EGFR mutation. AXL+, GAS6+, or AXL+/GAS6+ status significantly correlated with poor overall survival. In stage I cases, AXL+/GAS6+ status significantly correlated with poor overall survival and disease-free survival, especially in cases with wild-type EGFR. In multivariate analysis, AXL/GAS6 classifications in stage I as well as in stages I-III lung AD were found to be independent factors for poor patient outcomes. Unlike lung AD cell lines with mutant EGFR, almost all cells with wild-type EGFR showed AXL and vimentin co-expression as determined by western blotting. AXL+ and GAS6+ expression is relevant to a poor prognosis in resected lung AD patients at stage I. AXL/GAS6 might serve as crucial predictive and prognostic biomarkers and targets to identify individuals at high risk of post-operative death.

  19. Epidermal Growth Factor Receptor Overexpression as a Target for Auger Electron Radiotherapy of Breast Cancer

    DTIC Science & Technology

    1999-08-01

    proportion of estrogen receptor-negative and hormone-resistant breast cancers. Our objective is to construct a human epidermal growth factor (hEGF...61 5 INTRODUCTION Overexpression of the epidermal growth factor receptor (EGFR) occurs in a high proportion of estrogen receptor-negative and...Lac Iq promotor induced by isopropyl-b- D -thiogalactopyranoside (IPTG). The DNA sequence of the final hEGF-CH1 construct was confirmed (FUi. 2). BamHJ

  20. A transcription factor active on the epidermal growth factor receptor gene.

    PubMed Central

    Kageyama, R; Merlino, G T; Pastan, I

    1988-01-01

    We have developed an in vitro transcription system for the epidermal growth factor receptor (EGFR) oncogene by using nuclear extracts of A431 human epidermoid carcinoma cells, which overproduce EGFR. We found that a nuclear factor, termed EGFR-specific transcription factor (ETF), specifically stimulated EGFR transcription by 5- to 10-fold. In this report, ETF, purified by using sequence-specific oligonucleotide affinity chromatography, is shown by renaturing material eluted from a NaDodSO4/polyacrylamide gel to be a protein with a molecular mass of 120 kDa. ETF binds to the promoter region, as measured by DNase I "footprinting" and gel-mobility-shift assays, and specifically stimulates the transcription of the EGFR gene in a reconstituted in vitro transcription system. These results suggest that ETF could play a role in the overexpression of the cellular oncogene EGFR. Images PMID:3393529

  1. Exploring timing activation of functional pathway based on differential co-expression analysis in preimplantation embryogenesis

    PubMed Central

    Wang, Shanshan; Yang, Lei; Liao, Mingzhi; Wei, Zhuying; Bai, Chunling; Li, Guangpeng

    2016-01-01

    Recent genome-wide omics studies have confirmed the early embryogenesis strictly dependent on the rigorous spatiotemporal activation and multilevel regulation. However, the full effect of functional pathway was not considered. To obtain complete understanding of the gene activation during early development, we performed systematic comparisons based on differential co-expression analysis for bovine preimplantation embryo development (PED). The results confirmed that the functional pathways actively transcribes as early as the 2-cell and 4-cell waves, which Basal transcription factor, Endocytosis and Spliceosome pathway can represent first signs of embryonic activity. Endocytosis act as one of master activators for uncovering a series of successive waves of maternal pioneer signal regulator with the help of Spliceosome complex. Furthermore, the results showed that pattern recognition receptors began to perform its essential function at 4-cell stage, which might be needed to coordinate the later major activation. And finally, our work presented a probable dynamic landscape of key functional pathways for embryogenesis. A clearer understanding of early embryo development will be helpful for Assisted Reproductive Technology (ART) and Regenerative Medicine (RM). PMID:27705919

  2. A Null Model for Pearson Coexpression Networks

    PubMed Central

    Gobbi, Andrea; Jurman, Giuseppe

    2015-01-01

    Gene coexpression networks inferred by correlation from high-throughput profiling such as microarray data represent simple but effective structures for discovering and interpreting linear gene relationships. In recent years, several approaches have been proposed to tackle the problem of deciding when the resulting correlation values are statistically significant. This is most crucial when the number of samples is small, yielding a non-negligible chance that even high correlation values are due to random effects. Here we introduce a novel hard thresholding solution based on the assumption that a coexpression network inferred by randomly generated data is expected to be empty. The threshold is theoretically derived by means of an analytic approach and, as a deterministic independent null model, it depends only on the dimensions of the starting data matrix, with assumptions on the skewness of the data distribution compatible with the structure of gene expression levels data. We show, on synthetic and array datasets, that the proposed threshold is effective in eliminating all false positive links, with an offsetting cost in terms of false negative detected edges. PMID:26030917

  3. A null model for Pearson coexpression networks.

    PubMed

    Gobbi, Andrea; Jurman, Giuseppe

    2015-01-01

    Gene coexpression networks inferred by correlation from high-throughput profiling such as microarray data represent simple but effective structures for discovering and interpreting linear gene relationships. In recent years, several approaches have been proposed to tackle the problem of deciding when the resulting correlation values are statistically significant. This is most crucial when the number of samples is small, yielding a non-negligible chance that even high correlation values are due to random effects. Here we introduce a novel hard thresholding solution based on the assumption that a coexpression network inferred by randomly generated data is expected to be empty. The threshold is theoretically derived by means of an analytic approach and, as a deterministic independent null model, it depends only on the dimensions of the starting data matrix, with assumptions on the skewness of the data distribution compatible with the structure of gene expression levels data. We show, on synthetic and array datasets, that the proposed threshold is effective in eliminating all false positive links, with an offsetting cost in terms of false negative detected edges.

  4. Modeling the epidermal growth factor -- epidermal growth factor receptor l2 domain interaction: implications for the ligand binding process.

    PubMed

    Jorissen, Robert N; Treutlein, Herbert R; Epa, V Chandana; Burgess, Antony W

    2002-06-01

    Signaling from the epidermal growth factor (EGF) receptor is triggered by the binding of ligands such as EGF or transforming growth factor alpha (TGF-alpha) and subsequent receptor dimerization. An understanding of these processes has been hindered by the lack of structural information about the ligand-bound, dimerized EGF receptor. Using an NMR-derived structure of EGF and a homology model of the major ligand binding domain of the EGF receptor and experimental data, we modeled the binding of EGF to this EGF receptor fragment. In this low resolution model of the complex, EGF sits across the second face of the EGF receptor L2 domain and EGF residues 10-16, 36-37, 40-47 bind to this face. The structural model is largely consistent with previously published NMR data describing the residues of TGF-alpha which interact strongly with the EGF receptor. Other EGF residues implicated in receptor binding are accounted by our proposal that the ligand binding is a two-step process with the EGF binding to at least one other site of the receptor. This three-dimensional model is expected to be useful in the design of ligand-based antagonists of the receptor.

  5. Anti-Epidermal Growth Factor Receptor Gene Therapy for Glioblastoma

    PubMed Central

    Hicks, Martin J.; Chiuchiolo, Maria J.; Ballon, Douglas; Dyke, Jonathan P.; Aronowitz, Eric; Funato, Kosuke; Tabar, Viviane; Havlicek, David; Fan, Fan; Sondhi, Dolan; Kaminsky, Stephen M.; Crystal, Ronald G.

    2016-01-01

    Glioblastoma multiforme (GBM) is the most common and aggressive primary intracranial brain tumor in adults with a mean survival of 14 to 15 months. Aberrant activation of the epidermal growth factor receptor (EGFR) plays a significant role in GBM progression, with amplification or overexpression of EGFR in 60% of GBM tumors. To target EGFR expressed by GBM, we have developed a strategy to deliver the coding sequence for cetuximab, an anti-EGFR antibody, directly to the CNS using an adeno-associated virus serotype rh.10 gene transfer vector. The data demonstrates that single, local delivery of an anti-EGFR antibody by an AAVrh.10 vector coding for cetuximab (AAVrh.10Cetmab) reduces GBM tumor growth and increases survival in xenograft mouse models of a human GBM EGFR-expressing cell line and patient-derived GBM. AAVrh10.CetMab-treated mice displayed a reduction in cachexia, a significant decrease in tumor volume and a prolonged survival following therapy. Adeno-associated-directed delivery of a gene encoding a therapeutic anti-EGFR monoclonal antibody may be an effective strategy to treat GBM. PMID:27711187

  6. Molecular Docking and Interactions of Pueraria Tuberosa with Vascular Endothelial Growth Factor Receptors

    PubMed Central

    Asthana, S.; Agarwal, T.; Singothu, S.; Samal, A.; Banerjee, I.; Pal, K.; Pramanik, K.; Ray, S. S.

    2015-01-01

    Pueraria tuberosa is known for its therapeutic potentials in cardiovascular disorders, but its effect in angiogenesis has not been studied so far. In this study, a computational approach has been applied to elucidate the role of the phytochemicals in inhibition of angiogenesis through modulation of vascular endothelial growth factor receptors: Vascular endothelial growth factor receptor-1 and vascular endothelial growth factor receptor-2, major factors responsible for angiogenesis. Metabolite structures retrieved from PubChem and KNApSAcK – 3D databases, were docked using AutoDock4.2 tool. Hydrogen bond and molecular docking, absorption, distribution, metabolism and excretion and toxicity predictions were carried out using UCSF Chimera, LigPlot+ and PreADMET server, respectively. From the docking analysis, it was observed that puerarone and tuberostan had significant binding affinity for the intracellular kinase domain of vascular endothelial growth factor receptors-1 and vascular endothelial growth factor receptor-2 respectively. It is important to mention that both the phytochemicals shared similar interaction profile as that of standard inhibitors of vascular endothelial growth factor receptors. Also, both puerarone and tuberostan interacted with Lys861/Lys868 (adenosine 5’-triphosphate binding site of vascular endothelial growth factor receptors-1/vascular endothelial growth factor receptors-2), thus providing a clue that they may enforce their inhibitory effect by blocking the adenosine 5’-triphosphate binding domain of vascular endothelial growth factor receptors. Moreover, these molecules exhibited good drug-likeness, absorption, distribution, metabolism and excretion properties without any carcinogenic and toxic effects. The interaction pattern of the puerarone and tuberostan may provide a hint for a novel drug design for vascular endothelial growth factor tyrosine kinase receptors with better specificity to treat angiogenic disorders. PMID:26664060

  7. Indoxyl Sulfate Downregulates Mas Receptor via Aryl Hydrocarbon Receptor/Nuclear Factor-kappa B, and Induces Cell Proliferation and Tissue Factor Expression in Vascular Smooth Muscle Cells.

    PubMed

    Ng, Hwee-Yeong; Bolati, Wulaer; Lee, Chien-Te; Chien, Yu-Shu; Yisireyili, Maimaiti; Saito, Shinichi; Pei, Sung-Nan; Nishijima, Fuyuhiko; Niwa, Toshimitsu

    2016-01-01

    Angiotensin converting enzyme-related carboxypeptidase 2/angiotensin (Ang)-(1-7)/Mas receptor axis is protective in the development of chronic kidney disease and cardiovascular disease. This study is aimed at investigating whether indoxyl sulfate (IS) affects Mas receptor expression, cell proliferation and tissue factor expression in vascular smooth muscle cells, and if Ang-(1-7), an activator of Mas receptor, counteracts the IS-induced effects. IS was administered to normotensive and hypertensive rats. Human aortic smooth muscle cells (HASMCs) were cultured with IS. IS reduced the expression of Mas receptor in the aorta of normotensive and hypertensive rats. IS downregulated the Mas receptor expression in a time- and dose-dependent manner in HASMCs. Knockdown of aryl hydrocarbon receptor (AhR) and nuclear factor-kappa B (NF-x03BA;B) inhibited IS-induced downregulation of Mas receptor. Further, IS stimulated cell proliferation and tissue factor expression in HASMCs. Ang-(1-7) attenuated IS-induced cell proliferation and tissue factor expression in HASMCs. Ang-(1-7) suppressed phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and NF-x03BA;B in HASMCs. IS downregulated the expression of Mas receptor via AhR/NF-x03BA;B, and induced cell proliferation and tissue factor expression in HASMCs. Ang-(1-7) inhibited IS-induced cell proliferation and tissue factor expression by suppressing the phosphorylation of ERK1/2 and NF-x03BA;B p65. © 2016 S. Karger AG, Basel.

  8. Studying the complex expression dependences between sets of coexpressed genes.

    PubMed

    Huerta, Mario; Casanova, Oriol; Barchino, Roberto; Flores, Jose; Querol, Enrique; Cedano, Juan

    2014-01-01

    Organisms simplify the orchestration of gene expression by coregulating genes whose products function together in the cell. The use of clustering methods to obtain sets of coexpressed genes from expression arrays is very common; nevertheless there are no appropriate tools to study the expression networks among these sets of coexpressed genes. The aim of the developed tools is to allow studying the complex expression dependences that exist between sets of coexpressed genes. For this purpose, we start detecting the nonlinear expression relationships between pairs of genes, plus the coexpressed genes. Next, we form networks among sets of coexpressed genes that maintain nonlinear expression dependences between all of them. The expression relationship between the sets of coexpressed genes is defined by the expression relationship between the skeletons of these sets, where this skeleton represents the coexpressed genes with a well-defined nonlinear expression relationship with the skeleton of the other sets. As a result, we can study the nonlinear expression relationships between a target gene and other sets of coexpressed genes, or start the study from the skeleton of the sets, to study the complex relationships of activation and deactivation between the sets of coexpressed genes that carry out the different cellular processes present in the expression experiments.

  9. COXPRESdb: a database of coexpressed gene networks in mammals.

    PubMed

    Obayashi, Takeshi; Hayashi, Shinpei; Shibaoka, Masayuki; Saeki, Motoshi; Ohta, Hiroyuki; Kinoshita, Kengo

    2008-01-01

    A database of coexpressed gene sets can provide valuable information for a wide variety of experimental designs, such as targeting of genes for functional identification, gene regulation and/or protein-protein interactions. Coexpressed gene databases derived from publicly available GeneChip data are widely used in Arabidopsis research, but platforms that examine coexpression for higher mammals are rather limited. Therefore, we have constructed a new database, COXPRESdb (coexpressed gene database) (http://coxpresdb.hgc.jp), for coexpressed gene lists and networks in human and mouse. Coexpression data could be calculated for 19 777 and 21 036 genes in human and mouse, respectively, by using the GeneChip data in NCBI GEO. COXPRESdb enables analysis of the four types of coexpression networks: (i) highly coexpressed genes for every gene, (ii) genes with the same GO annotation, (iii) genes expressed in the same tissue and (iv) user-defined gene sets. When the networks became too big for the static picture on the web in GO networks or in tissue networks, we used Google Maps API to visualize them interactively. COXPRESdb also provides a view to compare the human and mouse coexpression patterns to estimate the conservation between the two species.

  10. Signal transduction induced in endothelial cells by growth factor receptors involved in angiogenesis

    PubMed Central

    Hofer, Erhard; Schweighofer, Bernhard

    2010-01-01

    Summary New vessel formation during development and in the adult is triggered by concerted signals of largely endothelial-specific receptors for ligands of the VEGF, angiopoietin and ephrin families. The signals and genes induced by these receptors operate in the context of additional signals transduced by non-endothelial specific growth factor receptors, inflammatory cytokine receptors as well as adhesion molecules. We summarize here available data on characteristic signaling of the VEGF receptor-2 and the current state of knowledge regarding the additional different receptor tyrosine kinases of the VEGF, Tie and Ephrin receptor families. Furthermore, the potential cross-talk with signals induced by other growth factors and inflammatory cytokines as well as the modulation by VE-cadherin is discussed. PMID:17334501

  11. Coexpression of PD-1, 2B4, CD160 and KLRG1 on exhausted HCV-specific CD8+ T cells is linked to antigen recognition and T cell differentiation.

    PubMed

    Bengsch, Bertram; Seigel, Bianca; Ruhl, Marianne; Timm, Jörg; Kuntz, Martin; Blum, Hubert E; Pircher, Hanspeter; Thimme, Robert

    2010-06-10

    Exhausted CD8+ T cell responses during chronic viral infections are defined by a complex expression pattern of inhibitory receptors. However, very little information is currently available about the coexpression patterns of these receptors on human virus-specific CD8+ T cells and their correlation with antiviral functions, T cell differentiation and antigen recognition. We addressed these important aspects in a cohort of 38 chronically HCV infected patients and found a coexpression of inhibitory receptors such as 2B4, CD160 and KLRG1 in association with PD-1 in about half of the HCV-specific CD8+ T cell responses. Importantly, this exhaustive phenotype was associated with low and intermediate levels of CD127 expression, an impaired proliferative capacity, an intermediate T cell differentiation stage and absence of sequence variations within the corresponding epitopes, indicating ongoing antigen triggering. In contrast, a low expression of inhibitory receptors by the remaining HCV-specific CD8+ T cells occurred in concert with a CD127hi phenotype, an early T cell differentiation stage and presence of viral sequence variations within the corresponding epitopes. In sum, these results suggest that T cell exhaustion contributes to the failure of about half of HCV-specific CD8+ T cell responses and that it is determined by a complex interplay of immunological (e.g. T cell differentiation) and virological (e.g. ongoing antigen triggering) factors.

  12. Teaching resources. Growth factor and receptor tyrosine kinases.

    PubMed

    Aaronson, Stuart

    2005-02-22

    This Teaching Resource provides lecture notes and slides for a graduate-level class on ligand regulation of signaling by receptor tyrosine kinases and receptors involved in the Wnt canonical pathway. It is part of a series of lectures that constitute the Cell Signaling Systems course. A description of the lecture, along with a set of slides used to present this information, is provided.

  13. Dynamic tracing for epidermal growth factor receptor mutations in urinary circulating DNA in gastric cancer patients.

    PubMed

    Shi, Xiu-Qin; Xue, Wen-Hua; Zhao, Song-Feng; Zhang, Xiao-Jian; Sun, Wukong

    2017-02-01

    The mutations of epidermal growth factor receptor are detected in gastric cancer, indicating its suitability as a target for receptor tyrosine kinase inhibitors, as well as a marker for clinical outcome of chemotherapeutic treatments. However, extraction of quality tumor tissue for molecular processes remains challenging. Here, we aimed to examine the clinical relevance of urinary cell-free DNA as an alternative tumor material source used specifically for monitoring epidermal growth factor receptor mutations. Therefore, 120 gastric cancer patients with epidermal growth factor receptor mutations and 100 healthy controls were recruited for the study. The gastric patients also received epidermal growth factor receptor inhibitor treatment for a serial monitoring study. Paired primary tumor specimens were obtained with blood and urine samples, which were taken at a 1-month interval for a duration of 12 months. We found that urinary cell-free DNA yielded a close agreement of 92% on epidermal growth factor receptor mutation status when compared to primary tissue at baseline, and of 99% epidermal growth factor receptor mutation status when compared to plasma samples at different time points. Thus, our data suggest that urinary cell-free DNA may be a reliable source for screening and monitoring epidermal growth factor receptor mutations in the primary gastric cancer.

  14. Causal co-expression method with module analysis to screen drugs with specific target.

    PubMed

    Yu, Shuhao; Zheng, Lulu; Li, Yixue; Li, Chunyan; Ma, Chenchen; Yu, Yang; Li, Xuan; Hao, Pei

    2013-04-10

    The considerable increase of investment in research and development by the pharmaceutical industry over the past three decades has not added the number of approved new drugs. An important issue ignored by drug discovery practice is the multi-dimensional interaction network between drugs and their targets. Thus, it is essential to view drug actions through the lens of network biology. In the current study, based on the co-expression network of transcription factors and their downstream genes, we proposed a novel approach, called causal co-expression method with module analysis, to screen drugs with specific target and fewer side effects. We presented a causal co-expression method with module analysis and it could be used in analyzing the microarray data of different drug candidates. At first, the differential wiring value (DW) was calculated to find some causal transcription factors (TFs) by combining with differential expression genes in the regulated networks. After the discovery of the causal TFs, co-expression module analysis method was applied to mine molecular pharmacology pathways around these causal TFs at molecular level. We applied our methods to two drug candidates, Argyrin A and Bortezomib, both with anti-cancer activities. We first obtained some differentially expressed transcription factors of cells treated with Argyrin A or Bortezomib. Nearly all these transcription factors are associated with the tumor suppressor protein p27kip1. Furthermore, module analysis showed that Bortezomib inhibited tumor growth not specifically by cell cycle and cell proliferation pathway, but through many basic metabolic processes which result in cell toxicity. In contrast, Argyrin A had influence on cell cycle, and was involved in DNA damage repair at the same time, showing that Argyrin A was a more suitable drug for anti-cancer treatment. Our study revealed that the causal co-expression method with module analysis was effective and can be used as a tool to evaluate drug

  15. Oncogenic fingerprint of epidermal growth factor receptor pathway and emerging epidermal growth factor receptor blockade resistance in colorectal cancer

    PubMed Central

    Sobani, Zain A; Sawant, Ashwin; Jafri, Mikram; Correa, Amit Keith; Sahin, Ibrahim Halil

    2016-01-01

    Epidermal growth factor receptor (EGFR) has been an attractive target for treatment of epithelial cancers, including colorectal cancer (CRC). Evidence from clinical trials indicates that cetuximab and panitumumab (anti-EGFR monoclonal antibodies) have clinical activity in patients with metastatic CRC. The discovery of intrinsic EGFR blockade resistance in Kirsten RAS (KRAS)-mutant patients led to the restriction of anti-EGFR antibodies to KRAS wild-type patients by Food and Drug Administration and European Medicine Agency. Studies have since focused on the evaluation of biomarkers to identify appropriate patient populations that may benefit from EGFR blockade. Accumulating evidence suggests that patients with mutations in EGFR downstream signaling pathways including KRAS, BRAF, PIK3CA and PTEN could be intrinsically resistant to EGFR blockade. Recent whole genome studies also suggest that dynamic alterations in signaling pathways downstream of EGFR leads to distinct oncogenic signatures and subclones which might have some impact on emerging resistance in KRAS wild-type patients. While anti-EGFR monoclonal antibodies have a clear potential in the management of a subset of patients with metastatic CRC, further studies are warranted to uncover exact mechanisms related to acquired resistance to EGFR blockade. PMID:27777877

  16. Coexpression of GMP-140 and PAF by endothelium stimulated by histamine or thrombin: a juxtacrine system for adhesion and activation of neutrophils

    PubMed Central

    1991-01-01

    The adhesion of polymorphonuclear leukocytes (PMNs) to vascular endothelial cells (EC) is an early and fundamental event in acute inflammation. This process requires the regulated expression of molecules on both the EC and PMN. EC stimulated with histamine or thrombin coexpress two proadhesive molecules within minutes: granule membrane protein 140 (GMP-140), a member of the selectin family, and platelet-activating factor (PAF), a biologically active phospholipid. Coexpression of GMP-140 and PAF is required for maximal PMN adhesion and the two molecules act in a cooperative fashion. The component of adhesion mediated by EC-associated PAF requires activation of CD11/CD18 integrins on the PMN and binding of these heterodimers to counterreceptors on the EC. GMP-140 also binds to a receptor on the PMN; however, it tethers the PMN to the EC without requiring activation of CD11/CD18 integrins. This component of the adhesive interaction is blocked by antibodies to GMP-140 or by GMP-140 in the fluid phase. Experiments with purified GMP-140 indicate that binding to its receptor on the PMN does not directly induce PMN adhesiveness but that it potentiates the CD11/CD18-dependent adhesive response to PAF by a mechanism that involves events distal to the PAF receptor. Tethering of the PMN to the EC by GMP-140 may also be required for efficient interaction of PAF with its receptor on the PMN. These observations define a complex cell recognition system in which tethering of PMNs by a selectin, GMP-140, facilitates juxtacrine activation of the leukocytes by a signaling molecule, PAF. The latter event recruits the third component of the adhesive interaction, the CD11/CD18 integrins. PMID:1717478

  17. The Pseudo Signal Peptide of the Corticotropin-releasing Factor Receptor Type 2A Prevents Receptor Oligomerization*

    PubMed Central

    Teichmann, Anke; Rutz, Claudia; Kreuchwig, Annika; Krause, Gerd; Wiesner, Burkhard; Schülein, Ralf

    2012-01-01

    N-terminal signal peptides mediate the interaction of native proteins with the translocon complex of the endoplasmic reticulum membrane and are cleaved off during early protein biogenesis. The corticotropin-releasing factor receptor type 2a (CRF2(a)R) possesses an N-terminal pseudo signal peptide, which represents a so far unique domain within the large protein family of G protein-coupled receptors (GPCRs). In contrast to a conventional signal peptide, the pseudo signal peptide remains uncleaved and consequently forms a hydrophobic extension at the N terminus of the receptor. The functional consequence of the presence of the pseudo signal peptide is not understood. Here, we have analyzed the significance of this domain for receptor dimerization/oligomerization in detail. To this end, we took the CRF2(a)R and the homologous corticotropin-releasing factor receptor type 1 (CRF1R) possessing a conventional cleaved signal peptide and conducted signal peptide exchange experiments. Using single cell and single molecule imaging methods (fluorescence resonance energy transfer and fluorescence cross-correlation spectroscopy, respectively) as well as biochemical experiments, we obtained two novel findings; we could show that (i) the CRF2(a)R is expressed exclusively as a monomer, and (ii) the presence of the pseudo signal peptide prevents its oligomerization. Thus, we have identified a novel functional domain within the GPCR protein family, which plays a role in receptor oligomerization and which may be useful to study the functional significance of this process in general. PMID:22689579

  18. Isolation of an additional member of the fibroblast growth factor receptor family, FGFR-3

    SciTech Connect

    Keegan, K.; Hayman, M.J. ); Johnson, D.E.; Williams, L.T. )

    1991-02-15

    The fibroblast growth factors are a family of polypeptide growth factors involved in a variety of activities including mitogenesis, angiogenesis, and wound healing. Fibroblast growth factor receptors (FGFRs) have previously been identified in chicken, mouse, and human and have been shown to contain an extracellular domain with either two or three immunoglobulin-like domains, a transmembrane domain, and a cytoplasmic tyrosine kinase domain. The authors have isolated a human cDNA for another tyrosine kinase receptor that is highly homologous to the previously described FGFR. Expression of this receptor cDNA in COS cells directs the expression of a 125-kDa glycoprotein. They demonstrate that this cDNA encodes a biologically active receptor by showing that human acidic and basic fibroblast growth factors activate this receptor as measured by {sup 45}Ca{sup 2+} efflux assays. These data establish the existence of an additional member of the FGFR family that they have named FGFR-3.

  19. Sperm Epidermal Growth Factor Receptor (EGFR) Mediates α7 Acetylcholine Receptor (AChR) Activation to Promote Fertilization

    PubMed Central

    Jaldety, Yael; Glick, Yair; Orr-Urtreger, Avi; Ickowicz, Debby; Gerber, Doron; Breitbart, Haim

    2012-01-01

    To attain fertilization the spermatozoon binds to the egg zona pellucida (ZP) via sperm receptor(s) and undergoes an acrosome reaction (AR). Several sperm receptors have been described in the literature; however, the identity of this receptor is not yet certain. In this study, we suggest that the α7 nicotinic acetylcholine receptor (α7nAChR) might be a sperm receptor activated by ZP to induce epidermal growth factor receptor (EGFR)-mediated AR. We found that isolated ZP or α7 agonists induced the AR in sperm from WT but not α7-null spermatozoa, and the induced AR was inhibited by α7 or EGFR antagonists. Moreover, α7-null sperm showed very little binding to the egg, and microfluidic affinity in vitro assay clearly showed that α7nAChR, as well as EGFR, interacted with ZP3. Induction of EGFR activation and the AR by an α7 agonist was inhibited by a Src family kinase (SFK) inhibitor. In conclusion we suggest that activation of α7 by ZP leads to SFK-dependent EGFR activation, Ca2+ influx, and the acrosome reaction. PMID:22577141

  20. Characterization of a comparative model of the extracellular domain of the epidermal growth factor receptor.

    PubMed

    Jorissen, R N; Epa, V C; Treutlein, H R; Garrett, T P; Ward, C W; Burgess, A W

    2000-02-01

    The Epidermal Growth Factor (EGF) receptor is a tyrosine kinase that mediates the biological effects of ligands such as EGF and transforming growth factor alpha. An understanding of the molecular basis of its action has been hindered by a lack of structural and mutational data on the receptor. We have constructed comparative models of the four extracellular domains of the EGF receptor that are based on the structure of the first three domains of the insulin-like growth factor-1 (IGF-1) receptor. The first and third domains of the EGF receptor, L1 and L2, are right-handed beta helices. The second and fourth domains of the EGF receptor, S1 and S2, consist of the modules held together by disulfide bonds, which, except for the first module of the S1 domain, form rod-like structures. The arrangement of the L1 and S1 domains of the model are similar to that of the first two domains of the IGF-1 receptor, whereas that of the L2 and S2 domains appear to be significantly different. Using the EGF receptor model and limited information from the literature, we have proposed a number of regions that may be involved in the functioning of the receptor. In particular, the faces containing the large beta sheets in the L1 and L2 domains have been suggested to be involved with ligand binding of EGF to its receptor.

  1. Characterization of a comparative model of the extracellular domain of the epidermal growth factor receptor.

    PubMed Central

    Jorissen, R. N.; Epa, V. C.; Treutlein, H. R.; Garrett, T. P.; Ward, C. W.; Burgess, A. W.

    2000-01-01

    The Epidermal Growth Factor (EGF) receptor is a tyrosine kinase that mediates the biological effects of ligands such as EGF and transforming growth factor alpha. An understanding of the molecular basis of its action has been hindered by a lack of structural and mutational data on the receptor. We have constructed comparative models of the four extracellular domains of the EGF receptor that are based on the structure of the first three domains of the insulin-like growth factor-1 (IGF-1) receptor. The first and third domains of the EGF receptor, L1 and L2, are right-handed beta helices. The second and fourth domains of the EGF receptor, S1 and S2, consist of the modules held together by disulfide bonds, which, except for the first module of the S1 domain, form rod-like structures. The arrangement of the L1 and S1 domains of the model are similar to that of the first two domains of the IGF-1 receptor, whereas that of the L2 and S2 domains appear to be significantly different. Using the EGF receptor model and limited information from the literature, we have proposed a number of regions that may be involved in the functioning of the receptor. In particular, the faces containing the large beta sheets in the L1 and L2 domains have been suggested to be involved with ligand binding of EGF to its receptor. PMID:10716183

  2. Human corpus luteum: presence of epidermal growth factor receptors and binding characteristics

    SciTech Connect

    Ayyagari, R.R.; Khan-Dawood, F.S.

    1987-04-01

    Epidermal growth factor receptors are present in many reproductive tissues but have not been demonstrated in the human corpus luteum. To determine the presence of epidermal growth factor receptors and its binding characteristics, we carried out studies on the plasma cell membrane fraction of seven human corpora lutea (days 16 to 25) of the menstrual cycle. Specific epidermal growth factor receptors were present in human corpus luteum. Insulin, nerve growth factor, and human chorionic gonadotropin did not competitively displace epidermal growth factor binding. The optimal conditions for corpus luteum-epidermal growth factor receptor binding were found to be incubation for 2 hours at 4 degrees C with 500 micrograms plasma membrane protein and 140 femtomol /sup 125/I-epidermal growth factor per incubate. The number (mean +/- SEM) of epidermal growth factor binding sites was 12.34 +/- 2.99 X 10(-19) mol/micrograms protein; the dissociation constant was 2.26 +/- 0.56 X 10(-9) mol/L; the association constant was 0.59 +/- 0.12 X 10(9) L/mol. In two regressing corpora lutea obtained on days 2 and 3 of the menstrual cycle, there was no detectable specific epidermal growth factor receptor binding activity. Similarly no epidermal growth factor receptor binding activity could be detected in ovarian stromal tissue. Our findings demonstrate that specific receptors for epidermal growth factor are present in the human corpus luteum. The physiologic significance of epidermal growth factor receptors in human corpus luteum is unknown, but epidermal growth factor may be involved in intragonadal regulation of luteal function.

  3. Expression of the forkhead transcription factor FOXP1 is associated both with hypoxia inducible factors (HIFs) and the androgen receptor in prostate cancer but is not directly regulated by androgens or hypoxia.

    PubMed

    Banham, Alison H; Boddy, Jane; Launchbury, Rosalind; Han, Cheng; Turley, Helen; Malone, Peter R; Harris, Adrian L; Fox, Stephen B

    2007-07-01

    FOXP1 is a member of the winged helix or forkhead transcription factors. Recent studies have indicated possible roles for FOXP1 as a candidate tumor suppressor gene and a potential estrogen receptor (ER) co-regulator in the development of breast cancer. This study investigated whether FOXP1 has a similar relationship to the androgen receptor (AR) in prostate cancer and how these factors relate to the presence of hypoxia. FOXP1, the AR and various hypoxia-regulated proteins (HIF-1alpha, HIF-2alpha, and VEGF) were measured with immunohistochemistry using a tissue microarray constructed from 167 archival radical prostatectomies. Statistical analyses compared the co-expression of these factors both with each other and conventional parameters including patient age, pre-operative prostate specific antigen (PSA), post-operative Gleason score, capsular invasion, surgical margin status, tumor volume, and PSA recurrence. The influence of hypoxia, dihydrotestosterone, and the AR blocker Casodex was investigated in prostate cell lines VCaP and LNCaP in vitro. Expression of nuclear FOXP1 was significantly positively correlated with AR (P = 0.0001), hypoxia inducible factor 1alpha (HIF-1alpha) (P = 0.01), HIF-2alpha (P = 0.0001), and vascular endothelial growth factor (VEGF) (P = 0.007) expression. A positive significant relationship was also identified with the post-operative Gleason score (P = 0.03) but not with the other variables, including PSA recurrence (P > 0.05). There was no significant change in expression in FOXP1 protein levels under conditions of hypoxia (0.1%), dihydrotestosterone stimulation (10 or 100 nM), or androgen blockade with Casodex (1, 10, or 50 microM). These findings suggest that there may be a hormonal and hypoxia independent regulatory mechanism coordinating the expression of HIFs, the AR, and FOXP1 in prostate tumors.

  4. MICAL-like1 mediates epidermal growth factor receptor endocytosis

    PubMed Central

    Abou-Zeid, Nancy; Pandjaitan, Rudy; Sengmanivong, Lucie; David, Violaine; Le Pavec, Gwenaelle; Salamero, Jean; Zahraoui, Ahmed

    2011-01-01

    Small GTPase Rabs are required for membrane protein sorting/delivery to precise membrane domains. Rab13 regulates epithelial tight junction assembly and polarized membrane transport. Here we report that Molecule Interacting with CasL (MICAL)-like1 (MICAL-L1) interacts with GTP-Rab13 and shares a similar domain organization with MICAL. MICAL-L1 has a calponin homology (CH), LIM, proline rich and coiled-coil domains. It is associated with late endosomes. Time-lapse video microscopy shows that green fluorescent protein–Rab7 and mcherry-MICAL-L1 are present within vesicles that move rapidly in the cytoplasm. Depletion of MICAL-L1 by short hairpin RNA does not alter the distribution of a late endosome/lysosome-associated protein but affects the trafficking of epidermal growth factor receptor (EGFR). Overexpression of MICAL-L1 leads to the accumulation of EGFR in the late endosomal compartment. In contrast, knocking down MICAL-L1 results in the distribution of internalized EGFR in vesicles spread throughout the cytoplasm and promotes its degradation. Our data suggest that the N-terminal CH domain associates with the C-terminal Rab13 binding domain (RBD) of MICAL-L1. The binding of Rab13 to RBD disrupts the CH/RBD interaction, and may induce a conformational change in MICAL-L1, promoting its activation. Our results provide novel insights into the MICAL-L1/Rab protein complex that can regulate EGFR trafficking at late endocytic pathways. PMID:21795389

  5. A third distinct tumor necrosis factor receptor of orthopoxviruses

    PubMed Central

    Loparev, Vladimir N.; Parsons, Joseph M.; Knight, Janice C.; Panus, Joanne Fanelli; Ray, Caroline A.; Buller, R. Mark L.; Pickup, David J.; Esposito, Joseph J.

    1998-01-01

    Cowpox virus Brighton red strain (CPV) contains a gene, crmD, which encodes a 320-aa tumor necrosis factor receptor (TNFR) of 44% and 22% identity, respectively, to the CPV TNFR-like proteins, cytokine response modifiers (crm) CrmB and CrmC. The crmD gene was interrupted in three other cowpox strains examined and absent in various other orthopoxviruses; however, four strains of ectromelia virus (ECT) examined contained an intact crmD (97% identity to CPV crmD) and lacked cognates of crmB and crmC. The protein, CrmD, contains a transport signal; a 151-aa cysteine-rich region with 21 cysteines that align with human TNFRII ligand-binding region cysteines; and C-terminal region sequences that are highly diverged from cellular TNFR C-terminal region sequences involved in signal transduction. Bacterial maltose-binding proteins containing the CPV or ECT CrmD cysteine-rich region bound TNF and lymphotoxin-α (LTα) and blocked their in vitro cytolytic activity. Secreted viral CrmD bound TNF and LTα and was detectable after the early stage of replication, using nonreducing conditions, as 60- to 70-kDa predominant and 90- to 250-kDa minor disulfide-linked complexes that were able to be reduced to a 46-kDa form and deglycosylated to a 38-kDa protein. Cells infected with CPV produced extremely low amounts of CrmD compared with ECT. Possessing up to three TNFRs, including CrmD, which is secreted as disulfide-linked complexes in varied amounts by CPV and ECT, likely enhances the dynamics of the immune modulating mechanisms of orthopoxviruses. PMID:9520445

  6. Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling

    PubMed Central

    Mutoh, Shingo; Sobhany, Mack; Moore, Rick; Perera, Lalith; Pedersen, Lee; Sueyoshi, Tatsuya; Negishi, Masahiko

    2017-01-01

    Phenobarbital is a central nervous system depressant that also indirectly activates nuclear receptor constitutive active androstane receptor (CAR), which promotes drug and energy metabolism, as well as cell growth (and death), in the liver. We found that phenobarbital activated CAR by inhibiting epidermal growth factor receptor (EGFR) signaling. Phenobarbital bound to EGFR and potently inhibited the binding of EGF, which prevented the activation of EGFR. This abrogation of EGFR signaling induced the dephosphorylation of receptor for activated C kinase 1 (RACK1) at Tyr52, which then promoted the dephosphorylation of CAR at Thr38 by the catalytic core subunit of protein phosphatase 2A. The findings demonstrated that the phenobarbital-induced mechanism of CAR dephosphorylation and activation is mediated through its direct interaction with and inhibition of EGFR. PMID:23652203

  7. Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling.

    PubMed

    Mutoh, Shingo; Sobhany, Mack; Moore, Rick; Perera, Lalith; Pedersen, Lee; Sueyoshi, Tatsuya; Negishi, Masahiko

    2013-05-07

    Phenobarbital is a central nervous system depressant that also indirectly activates nuclear receptor constitutive active androstane receptor (CAR), which promotes drug and energy metabolism, as well as cell growth (and death), in the liver. We found that phenobarbital activated CAR by inhibiting epidermal growth factor receptor (EGFR) signaling. Phenobarbital bound to EGFR and potently inhibited the binding of EGF, which prevented the activation of EGFR. This abrogation of EGFR signaling induced the dephosphorylation of receptor for activated C kinase 1 (RACK1) at Tyr(52), which then promoted the dephosphorylation of CAR at Thr(38) by the catalytic core subunit of protein phosphatase 2A. The findings demonstrated that the phenobarbital-induced mechanism of CAR dephosphorylation and activation is mediated through its direct interaction with and inhibition of EGFR.

  8. Noncontiguous domains of the alpha-factor receptor of yeasts confer ligand specificity.

    PubMed

    Sen, M; Marsh, L

    1994-01-14

    The Saccharomyces cerevisiae alpha-factor receptor has a 3400-fold higher affinity for the S. cerevisiae alpha-factor peptide (c-alpha-f) than for the Saccharomyces kluyveri alpha-factor peptide (k-alpha-f) as determined by competition for [3H] c-alpha-f binding. The S. kluyveri alpha-factor receptor has an approximately 2-fold higher affinity for k-alpha-f than for c-alpha-f. The S. kluyveri receptor gene (k-STE2) is incompletely regulated by S. cerevisiae mating type and poorly expressed on the surface of an S. cerevisiae mating type a strain. A chimeric receptor (c/k1) with amino acid residues 1-45 derived from S. cerevisiae and amino acid residues 46-427 from S. kluyveri exhibits the binding specificity of the S. kluyveri receptor. However, chimeric receptors containing residues 1-168 (c/k2) or 1-250 (c/k3) from S. cerevisiae and the remainder from the S. kluyveri receptor exhibit specificities similar to one another, but intermediate between the parent S. cerevisiae and S. kluyveri receptors. The relative ability of c-alpha-f and k-alpha-f to induce growth arrest in strains expressing chimeric receptors parallels relative affinity. Thus, two noncontiguous domains that include putative extracellular loops 1 and 3 and associated transmembrane segments, but exclude the extracellular NH2 terminus and loop 2, appear to contribute to alpha-factor receptor ligand specificity. COOH-terminal regions of the S. kluyveri receptor appear to confer a desensitization defect when expressed in S. cerevisiae. The S. cerevisiae receptor truncated at residue 296 retains ligand specificity for growth arrest.

  9. Novel structural co-expression analysis linking the NPM1-associated ribosomal biogenesis network to chronic myelogenous leukemia

    PubMed Central

    Chan, Lawrence WC; Lin, Xihong; Yung, Godwin; Lui, Thomas; Chiu, Ya Ming; Wang, Fengfeng; Tsui, Nancy BY; Cho, William CS; Yip, SP; Siu, Parco M.; Wong, SC Cesar; Yung, Benjamin YM

    2015-01-01

    Co-expression analysis reveals useful dysregulation patterns of gene cooperativeness for understanding cancer biology and identifying new targets for treatment. We developed a structural strategy to identify co-expressed gene networks that are important for chronic myelogenous leukemia (CML). This strategy compared the distributions of expressional correlations between CML and normal states, and it identified a data-driven threshold to classify strongly co-expressed networks that had the best coherence with CML. Using this strategy, we found a transcriptome-wide reduction of co-expression connectivity in CML, reflecting potentially loosened molecular regulation. Conversely, when we focused on nucleophosmin 1 (NPM1) associated networks, NPM1 established more co-expression linkages with BCR-ABL pathways and ribosomal protein networks in CML than normal. This finding implicates a new role of NPM1 in conveying tumorigenic signals from the BCR-ABL oncoprotein to ribosome biogenesis, affecting cellular growth. Transcription factors may be regulators of the differential co-expression patterns between CML and normal. PMID:26205693

  10. DEPENDENCE OF PPAR LIGAND-INDUCED MAPK SIGNALING ON EPIDERMAL GROWTH FACTOR RECEPTOR TRANSACTIVATION HEPARIN-BINDING EGF CLEAVAGE MEDIATES ZINC-INDUCED EGF RECEPTOR PHOSPHORYLATION

    EPA Science Inventory

    Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that function as ligand-activated transcription factors regulating lipid metabolism and homeostasis. In addition to their ability to regulate PPAR-mediated gene transcription, PPARalpha and gamma li...

  11. DEPENDENCE OF PPAR LIGAND-INDUCED MAPK SIGNALING ON EPIDERMAL GROWTH FACTOR RECEPTOR TRANSACTIVATION HEPARIN-BINDING EGF CLEAVAGE MEDIATES ZINC-INDUCED EGF RECEPTOR PHOSPHORYLATION

    EPA Science Inventory

    Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that function as ligand-activated transcription factors regulating lipid metabolism and homeostasis. In addition to their ability to regulate PPAR-mediated gene transcription, PPARalpha and gamma li...

  12. Heregulin-Induced Growth Factor Receptor Signaling and Breast Carcinogenesis

    DTIC Science & Technology

    1995-07-17

    driven receptor heterodimers and receptor cross-phosphorylation [Goldman, et al., 1990; Wada, et al., 1990; Qian, et al., 1992; Spivak -Kroizman, et al...in a 1:1 stoichiometry [ Spivak -Kroizman, et al., 1994], yet FGFs are monomeric, it has been proposed that HSPG binding potentiates FGF stimulation of...E. coli. They are also supplying us with human recombinant amphiregulin (AR). We have also established collaborations with Dr. Michael Klagsbrun

  13. Epidermal growth factor receptor transactivation by intracellular prostaglandin E2-activated prostaglandin E2 receptors. Role in retinoic acid receptor-β up-regulation.

    PubMed

    Fernández-Martínez, Ana B; Lucio Cazaña, Francisco J

    2013-09-01

    The pharmacological modulation of renoprotective factor vascular endothelial growth factor-A (VEGF-A) in the proximal tubule has therapeutic interest. In human proximal tubular HK-2 cells, treatment with all-trans retinoic acid or prostaglandin E2 (PGE2) triggers the production of VEGF-A. The pathway involves an initial increase in intracellular PGE2, followed by activation of EP receptors (PGE2 receptors, most likely an intracellular subset) and increase in retinoic acid receptor-β (RARβ) expression. RARβ then up-regulates transcription factor hypoxia-inducible factor-1α (HIF-1α), which increases the transcription and production of VEGF-A. Here we studied the role in this pathway of epidermal growth factor receptor (EGFR) transactivation by EP receptors. We found that EGFR inhibitor AG1478 prevented the increase in VEGF-A production induced by PGE2- and all-trans retinoic acid. This effect was due to the inhibition of the transcriptional up-regulation of RARβ, which resulted in loss of the RARβ-dependent transcriptional up-regulation of HIF-1α. PGE2 and all-trans retinoic acid also increased EGFR phosphorylation and this effect was sensitive to antagonists of EP receptors. The role of intracellular PGE2 was indicated by two facts; i) PGE2-induced EGFR phosphorylation was substantially prevented by inhibitor of prostaglandin uptake transporter bromocresol green and ii) all-trans retinoic acid treatment, which enhanced intracellular but not extracellular PGE2, had lower effect on EGFR phosphorylation upon pre-treatment with cyclooxygenase inhibitor diclofenac. Thus, EGFR transactivation by intracellular PGE2-activated EP receptors results in the sequential activation of RARβ and HIF-1α leading to increased production of VEGF-A and it may be a target for the therapeutic modulation of HIF-1α/VEGF-A. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. The role of EGF receptor transmodulation in embryonal carcinoma-derived growth factor-induced mitogenesis.

    PubMed Central

    Heath, J K; Mahadevan, L; Foulkes, J G

    1986-01-01

    Exposure of quiescent 10T1/2 fibroblast cells to embryonal carcinoma-derived growth factor (ECDGF) results in a rapid temperature and ECDGF concentration-dependent inhibition of [125I]EGF binding to the epidermal growth factor (EGF) receptor (transmodulation). ECDGF predominantly inhibits the association of [125I]EGF with a high affinity subclass of EGF receptors, and induces increased phosphorylation of the EGF receptor on serine and threonine residues. No mitogenic effect of EGF can be detected in the presence of ECDGF concentrations which induce maximal EGF receptor transmodulation. ECDGF-induced EGF receptor transmodulation is sensitive to phorbol ester-induced desensitization whereas ECDGF-induced DNA synthesis is unaffected by prolonged pre-treatment with biologically active phorbol ester. These findings suggest that EGF receptor transmodulation is not essential for ECDGF mitogenicity but may inhibit EGF-induced DNA synthesis. Images Fig. 5. PMID:3489616

  15. Photoreceptors of Nrl -/- mice coexpress functional S- and M-cone opsins having distinct inactivation mechanisms.

    PubMed

    Nikonov, Sergei S; Daniele, Lauren L; Zhu, Xuemei; Craft, Cheryl M; Swaroop, Anand; Pugh, Edward N

    2005-03-01

    The retinas of mice null for the neural retina leucine zipper transcription factor (Nrl-/-) contain no rods but are populated instead with photoreceptors that on ultrastructural, histochemical, and molecular criteria appear cone like. To characterize these photoreceptors functionally, responses of single photoreceptors of Nrl-/- mice were recorded with suction pipettes at 35-37 degrees C and compared with the responses of rods of WT mice. Recordings were made either in the conventional manner, with the outer segment (OS) drawn into the pipette ("OS in"), or in a novel configuration with a portion of the inner segment drawn in ("OS out"). Nrl-/- photoreceptor responses recorded in the OS-out configuration were much faster than those of WT rods: for dim-flash responses tpeak = 91 ms vs. 215 ms; for saturating flashes, dominant recovery time constants, tau(D) = 110 ms vs. 240 ms, respectively. Nrl-/- photoreceptors in the OS-in configuration had reduced amplification, sensitivity, and slowed recovery kinetics, but the recording configuration had no effect on rod response properties, suggesting Nrl-/- outer segments to be more susceptible to damage. Functional coexpression of two cone pigments in a single mammalian photoreceptor was established for the first time; the responses of every Nrl-/- cell were driven by both the short-wave (S, lambda(max) approximately 360 nm) and the mid-wave (M, lambda(max) approximately 510 nm) mouse cone pigment; the apparent ratio of coexpressed M-pigment varied from 1:1 to 1:3,000 in a manner reflecting a dorso-ventral retinal position gradient. The role of the G-protein receptor kinase Grk1 in cone pigment inactivation was investigated in recordings from Nrl-/-/Grk1-/- photoreceptors. Dim-flash responses of cells driven by either the S- or the M-cone pigment were slowed 2.8-fold and 7.5-fold, respectively, in the absence of Grk1; the inactivation of the M-pigment response was much more seriously retarded. Thus, Grk1 is essential to

  16. Uncovering co-expression gene network modules regulating fruit acidity in diverse apples.

    PubMed

    Bai, Yang; Dougherty, Laura; Cheng, Lailiang; Zhong, Gan-Yuan; Xu, Kenong

    2015-08-16

    Acidity is a major contributor to fruit quality. Several organic acids are present in apple fruit, but malic acid is predominant and determines fruit acidity. The trait is largely controlled by the Malic acid (Ma) locus, underpinning which Ma1 that putatively encodes a vacuolar aluminum-activated malate transporter1 (ALMT1)-like protein is a strong candidate gene. We hypothesize that fruit acidity is governed by a gene network in which Ma1 is key member. The goal of this study is to identify the gene network and the potential mechanisms through which the network operates. Guided by Ma1, we analyzed the transcriptomes of mature fruit of contrasting acidity from six apple accessions of genotype Ma_ (MaMa or Mama) and four of mama using RNA-seq and identified 1301 fruit acidity associated genes, among which 18 were most significant acidity genes (MSAGs). Network inferring using weighted gene co-expression network analysis (WGCNA) revealed five co-expression gene network modules of significant (P < 0.001) correlation with malate. Of these, the Ma1 containing module (Turquoise) of 336 genes showed the highest correlation (0.79). We also identified 12 intramodular hub genes from each of the five modules and 18 enriched gene ontology (GO) terms and MapMan sub-bines, including two GO terms (GO:0015979 and GO:0009765) and two MapMap sub-bins (1.3.4 and 1.1.1.1) related to photosynthesis in module Turquoise. Using Lemon-Tree algorithms, we identified 12 regulator genes of probabilistic scores 35.5-81.0, including MDP0000525602 (a LLR receptor kinase), MDP0000319170 (an IQD2-like CaM binding protein) and MDP0000190273 (an EIN3-like transcription factor) of greater interest for being one of the 18 MSAGs or one of the 12 intramodular hub genes in Turquoise, and/or a regulator to the cluster containing Ma1. The most relevant finding of this study is the identification of the MSAGs, intramodular hub genes, enriched photosynthesis related processes, and regulator genes in a

  17. Expression of growth factors and growth factor receptors in human cleft-affected tissue.

    PubMed

    Krivicka, Benita; Pilmane, Mara; Akota, Ilze

    2013-01-01

    OBJECTIVE. To investigate cleft disordered tissue in children with cleft palate and cleft lip with or without alveolar clefting for detection of local tissue growth factors and growth factor receptors and compare findings. Design. Morphological analysis of human tissue. Patients. Three groups were studied: 14 patients with cleft palate at the age from eight months to 18 years and two months, 12 patients with cleft lip with or without alveolar clefting in the age from four months to 15 years and four months and 11 control patients. RESULTS. In general, cleft palate disordered tissue showed more prominent expression of BMP2/4 (z=3.574; p=0.0004) and TGFβ (z=2.127; p=0.033), while expression of TGFBR3 significantly higher was only in connective tissue (z=3.822; p=0.0001). Cleft lip affected tissue showed significantly pronounced expression of FGFR1 in general as well as separately in epithelium. CONCLUSIONS. The marked and statistically significant expression of BMP 2/4 in cleft palate disordered soft tissue probably is delayed, but still proliferation and differentiation as well as tissue, especially, bone remodeling contributing signal. Cleft palate affected tissue show more prominent expression of TGFβ, still the weak regional expression of TGFβ type III receptors prove the disordered tissue growth and changed TGFβ signalling pathway in postnatal pathogenesis. In general, expression of TGFβ, BMP 2/4 and FGFR1 is significantly different, giving evidence to the involvement of these mentioned factors in the cleft severity morphopathogenesis.

  18. Epidermal growth factor receptor and KRAS mutations in Brazilian lung cancer patients

    PubMed Central

    Bacchi, Carlos E.; Ciol, Heloísa; Queiroga, Eduardo M.; Benine, Lucimara C.; Silva, Luciana H.; Ojopi, Elida B.

    2012-01-01

    OBJECTIVE: Epidermal growth factor receptor is involved in the pathogenesis of non-small cell lung cancer and has recently emerged as an important target for molecular therapeutics. The KRAS oncogene also plays an important role in the development of lung cancer. The aim of this study was to evaluate the frequency of epidermal growth factor receptor and KRAS mutations in a population of Brazilian patients with non-small cell lung cancer. METHODS: A total of 207 specimens from Brazilian patients with non-small cell lung cancer were analyzed for activating epidermal growth factor receptor and KRAS somatic mutations, and their associations with clinicopathological characteristics (including age, gender, ethnicity, smoking habits, and histological subtype) were examined. RESULTS: We identified 63 cases (30.4%) with epidermal growth factor receptor mutations and 30 cases (14.6%) with KRAS mutations. The most frequent epidermal growth factor receptor mutation we detected was a deletion in exon 19 (60.3%, 38 patients), followed by an L858R amino acid substitution in exon 21 (27%, 17 patients). The most common types of KRAS mutations were found in codon 12. There were no significant differences in epidermal growth factor receptor or KRAS mutations by gender or primary versus metastatic lung cancer. There was a higher prevalence of KRAS mutations in the non-Asian patients. Epidermal growth factor receptor mutations were more prevalent in adenocarcinomas than in non-adenocarcinoma histological types. Being a non-smoker was significantly associated with the prevalence of epidermal growth factor receptor mutations, but the prevalence of KRAS mutations was significantly associated with smoking. CONCLUSIONS: This study is the first to examine the prevalence of epidermal growth factor receptor and KRAS mutations in a Brazilian population sample with non-small cell lung cancer. PMID:22666783

  19. Ganglioside GM3 exerts opposite effects on motility via epidermal growth factor receptor and hepatocyte growth factor receptor-mediated migration signaling.

    PubMed

    Li, Ying; Huang, Xiaohua; Wang, Congcong; Li, Yuzhong; Luan, Mingchun; Ma, Keli

    2015-04-01

    The ganglioside GM3 exerts its different effects via various growth factor receptors. The present study investigated and comparatively analyzed the opposing effects exerted by GM3 on the migration of mouse hepatocellular carcinoma Hepa1‑6 cells via epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (HGFR/cMet). The results demonstrated that GM3 inhibited EGF‑stimulated motility, but promoted HGF‑stimulated motility of the Hepa1‑6 cells via phosphatidylinositol 3‑kinase/Akt‑mediated migration signaling. It is well established that the main cytokines modulating cell proliferation, invasion and metastasis are different in different types of tumor. This difference may, at least in part, explain why GM3 exerted its actions in a tumor‑type specific manner.

  20. Lipo-chitooligosaccharidic nodulation factors and their perception by plant receptors.

    PubMed

    Fliegmann, Judith; Bono, Jean-Jacques

    2015-10-01

    Lipo-chitooligosaccharides produced by nitrogen-fixing rhizobia are signaling molecules involved in the establishment of an important agronomical and ecological symbiosis with plants. These compounds, known as Nod factors, are biologically active on plant roots at very low concentrations indicating that they are perceived by specific receptors. This article summarizes the main strategies developed for the syntheses of bioactive Nod factors and their derivatives in order to better understand their mode of perception. Different Nod factor receptors and LCO-binding proteins identified by genetic or biochemical approaches are also presented, indicating perception mechanisms that seem to be more complicated than expected, probably involving multi-component receptor complexes.

  1. Regulation of ciliary neurotrophic factor receptor alpha in sciatic motor neurons following axotomy.

    PubMed

    MacLennan, A J; Devlin, B K; Neitzel, K L; McLaurin, D L; Anderson, K J; Lee, N

    1999-01-01

    Spinal motor neurons are one of the few classes of neurons capable of regenerating axons following axotomy. Injury-induced expression of neurotrophic factors and corresponding receptors may play an important role in this rare ability. A wide variety of indirect data suggests that ciliary neurotrophic factor receptor alpha may critically contribute to the regeneration of injured spinal motor neurons. We used immunohistochemistry, in situ hybridization and retrograde tracing techniques to study the regulation of ciliary neurotrophic factor receptor alpha in axotomized sciatic motor neurons. Ciliary neurotrophic factor receptor alpha immunoreactivity, detected with two independent antisera, is increased in a subpopulation of caudal sciatic motor neuron soma one, two and six weeks after sciatic nerve transection and reattachment, while no changes are detected at one day and 15 weeks post-lesion. Ciliary neurotrophic factor receptor alpha messenger RNA levels are augmented in the same classes of neurons following an identical lesion, suggesting that increased synthesis contributes, at least in part, to the additional ciliary neurotrophic factor receptor alpha protein. Separating the proximal and distal nerve stumps with a plastic barrier does not noticeably affect the injury-induced change in ciliary neurotrophic factor receptor alpha regulation, thereby indicating that this injury response is not dependent on signals distal to the lesion traveling retrogradely through the nerve or signals generated by axonal growth through the distal nerve. The prolonged increases in ciliary neurotrophic factor receptor alpha protein and messenger RNA found in regenerating sciatic motor neurons contrast with the responses of non-regenerating central neurons, which are reported to display, at most, a short-lived increase in ciliary neurotrophic factor receptor alpha messenger RNA expression following injury. The present data are the first to demonstrate, in vivo, neuronal regulation of

  2. Mechanism of kinase activation in the receptor for colony-stimulating factor 1.

    PubMed Central

    Lee, A W; Nienhuis, A W

    1990-01-01

    Receptor tyrosine kinases remain dormant until activated by ligand binding to the extracellular domain. Two mechanisms have been proposed for kinase activation: (i) ligand binding to the external domain of a receptor monomer may induce a conformational change that is transmitted across the cell membrane (intramolecular model) or (ii) the ligand may facilitate oligomerization, thereby allowing interactions between the juxtaposed kinase domains (intermolecular model). The receptor for colony-stimulating factor 1 was used to test these models. Large insertions at the junction between the external and transmembrane domains of the receptor, introduced by site-directed mutagenesis of the cDNA, were positioned to isolate the external domain and prevent transmembrane conformational propagation while allowing for receptor oligomerization. Such mutant receptors were expressed on the cell surface, bound ligand with high affinity, exhibited ligand-stimulated autophosphorylation, and signaled mitogenesis and cellular proliferation in the presence of ligand. A second experimental strategy directly tested the intermolecular model of ligand activation. A hybrid receptor composed of the external domain of human glycophorin A and the transmembrane and cytoplasmic domains of the colony-stimulating factor 1 receptor exhibited anti-glycophorin antibody-induced kinase activity that supported mitogenesis. Our data strongly support a mechanism of receptor activation based on ligand-induced receptor oligomerization. Images PMID:2169623

  3. Evidence that the modulator of the glucocorticoid-receptor complex is the endogenous molybdate factor.

    PubMed Central

    Bodine, P V; Litwack, G

    1988-01-01

    We have recently purified the modulator of the glucocorticoid-receptor complex from rat liver. Purified modulator inhibits glucocorticoid-receptor complex activation and stabilizes the steroid-binding ability of the unoccupied glucocorticoid receptor. Since these activities are shared by exogenous sodium molybdate, modulator appears to be the endogenous factor that sodium molybdate mimics. In this report, we present additional evidence for the mechanism of action of purified modulator. (i) Molybdate and modulator inhibit receptor activation as measured by DNA-cellulose binding, DEAE-cellulose chromatography, and Sepharose 4B gel filtration. (ii) The ability of molybdate and modulator to inhibit receptor activation and stabilize the unoccupied receptor appears to be additive. (iii) Scatchard analysis of heat-destabilized unoccupied receptors indicates that the number of steroid-binding sites is reduced during destabilization, whereas the steroid dissociation constant remains unchanged. Molybdate and modulator stabilize the receptor by maintaining the number of steroid-binding sites. (iv) Molybdate and modulator do not inhibit alkaline phosphatase-induced destabilization of the unoccupied receptor. However, alkaline phosphatase-induced destabilization is reversed by the addition of dithiothreitol in the presence, but not in the absence, of molybdate or modulator. These results suggest that the mechanism of action for modulator is identical to that of sodium molybdate, and we propose that modulator is the endogenous molybdate factor for the glucocorticoid receptor. PMID:3422744

  4. LysM domain receptor kinases regulating rhizobial Nod factor-induced infection.

    PubMed

    Limpens, Erik; Franken, Carolien; Smit, Patrick; Willemse, Joost; Bisseling, Ton; Geurts, René

    2003-10-24

    The rhizobial infection of legumes has the most stringent demand toward Nod factor structure of all host responses, and therefore a specific Nod factor entry receptor has been proposed. The SYM2 gene identified in certain ecotypes of pea (Pisum sativum) is a good candidate for such an entry receptor. We exploited the close phylogenetic relationship of pea and the model legume Medicago truncatula to identify genes specifically involved in rhizobial infection. The SYM2 orthologous region of M. truncatula contains 15 putative receptor-like genes, of which 7 are LysM domain-containing receptor-like kinases (LYKs). Using reverse genetics in M. truncatula, we show that two LYK genes are specifically involved in infection thread formation. This, as well as the properties of the LysM domains, strongly suggests that they are Nod factor entry receptors.

  5. Lentiviral vector system for coordinated constitutive and drug controlled tetracycline-regulated gene co-expression.

    PubMed

    Stahlhut, Maike; Schwarzer, Adrian; Eder, Matthias; Yang, Min; Li, Zhixiong; Morgan, Michael; Schambach, Axel; Kustikova, Olga S

    2015-09-01

    Constitutive co-expression of cooperating transgenes using retroviral integrating vectors is frequently used for genetic modification of different cell types to establish therapeutic or cancer models. However, such approaches are unable to dissect the influence of dose, order and reversibility of transgene expression on the fate of newly developed therapeutic/malignant phenotypes. We present a modular lentiviral vector system, which provides expression of constitutive and inducible components. To demonstrate its functionality, we constitutively expressed the well-described transcription factor Meis1 followed by inducible co-expression of collaborating partner Hoxa9 under the control of tetracycline responsive promoters in murine fibroblasts and primary hematopoietic progenitor cells (HPCs). Fluorescent markers to track transgene co-expression revealed tightly controlled, efficiently inducible and reversible but cell type dependent gene transfer over time. We demonstrated dose-dependent blockade of myeloid differentiation when both Meis1/Hoxa9 were concomitantly overexpressed in primary HPCs in vitro, but the absence of the transformed phenotype in non-induced samples or when Hoxa9 expression was down-regulated. This system combines the advantages of lentiviral gene transfer and the opportunity for drug-controlled co-expression of multiple transgenes to dissect, among others, gene networks governing complex cell behavior, such as proto-oncogene dose-dependent leukemogenic pathways or collaborating mechanisms of genes enhancing competitive fitness of hematopoietic cells.

  6. Acetylcholine receptor-inducing factor from chicken brain increases the level of mRNA encoding the receptor. alpha. subunit

    SciTech Connect

    Harris, D.A.; Falls, D.L.; Dill-Devor, R.M.; Fischbach, G.D. )

    1988-03-01

    A 42-kDa glycoprotein isolated from chicken brain, referred to as acetylcholine receptor-inducing activity (ARIA), that stimulates the rate of incorporation of acetylcholine receptors into the surface of chicken myotubes may play a role in the nerve-induced accumulation of receptors at developing neuromuscular synapses. Using nuclease-protection assays, the authors have found that ARIA causes a 2- to 16-fold increase in the level of mRNA encoding the {alpha} subunit of the receptor, with little or no change in the levels of {gamma}- and {delta}-subunit messengers. ARIA also increases the amount of a putative nuclear precursor of {alpha}-subunit mRNA, consistent with an activation of gene transcription. These results suggest that the concentration of {alpha} subunit may limit the rate of biosynthesis of the acetylcholine receptors in chicken myotubes. They also indicate that neuronal factors can regulate the expression of receptor subunit genes in a selective manner. Tetrodotoxin, 8-bromo-cAMP, and forskolin also increase the amount of {alpha}-subunit mRNA, with little change in the amount of {gamma}- and {delta}-subunit mRNAs. Unlike ARIA, however, these agents have little effect on the concentration of the {alpha}-subunit nuclear precursor.

  7. The Orphan Nuclear Receptor ERRγ Regulates Hepatic CB1 Receptor-Mediated Fibroblast Growth Factor 21 Gene Expression.

    PubMed

    Jung, Yoon Seok; Lee, Ji-Min; Kim, Don-Kyu; Lee, Yong-Soo; Kim, Ki-Sun; Kim, Yong-Hoon; Kim, Jina; Lee, Myung-Shik; Lee, In-Kyu; Kim, Seong Heon; Cho, Sung Jin; Jeong, Won-Il; Lee, Chul-Ho; Harris, Robert A; Choi, Hueng-Sik

    2016-01-01

    Fibroblast growth factor 21 (FGF21), a stress inducible hepatokine, is synthesized in the liver and plays important roles in glucose and lipid metabolism. However, the mechanism of hepatic cannabinoid type 1 (CB1) receptor-mediated induction of FGF21 gene expression is largely unknown. Activation of the hepatic CB1 receptor by arachidonyl-2'-chloroethylamide (ACEA), a CB1 receptor selective agonist, significantly increased FGF21 gene expression. Overexpression of estrogen-related receptor (ERR) γ increased FGF21 gene expression and secretion both in hepatocytes and mice, whereas knockdown of ERRγ decreased ACEA-mediated FGF21 gene expression and secretion. Moreover, ERRγ, but not ERRα and ERRβ, induced FGF21 gene promoter activity. In addition, deletion and mutation analysis of the FGF21 promoter identified a putative ERRγ-binding motif (AGGTGC, a near-consensus response element). A chromatin immunoprecipitation assay revealed direct binding of ERRγ to the FGF21 gene promoter. Finally, GSK5182, an ERRγ inverse agonist, significantly inhibited hepatic CB1 receptor-mediated FGF21 gene expression and secretion. Based on our data, we conclude that ERRγ plays a key role in hepatic CB1 receptor-mediated induction of FGF21 gene expression and secretion.

  8. The Orphan Nuclear Receptor ERRγ Regulates Hepatic CB1 Receptor-Mediated Fibroblast Growth Factor 21 Gene Expression

    PubMed Central

    Jung, Yoon Seok; Lee, Ji-Min; Kim, Don-Kyu; Lee, Yong-Soo; Kim, Ki-Sun; Kim, Yong-Hoon; Kim, Jina; Lee, Myung-Shik; Lee, In-Kyu; Kim, Seong Heon; Cho, Sung Jin; Jeong, Won-Il; Lee, Chul-Ho; Harris, Robert A.; Choi, Hueng-Sik

    2016-01-01

    Background Fibroblast growth factor 21 (FGF21), a stress inducible hepatokine, is synthesized in the liver and plays important roles in glucose and lipid metabolism. However, the mechanism of hepatic cannabinoid type 1 (CB1) receptor-mediated induction of FGF21 gene expression is largely unknown. Results Activation of the hepatic CB1 receptor by arachidonyl-2’-chloroethylamide (ACEA), a CB1 receptor selective agonist, significantly increased FGF21 gene expression. Overexpression of estrogen-related receptor (ERR) γ increased FGF21 gene expression and secretion both in hepatocytes and mice, whereas knockdown of ERRγ decreased ACEA-mediated FGF21 gene expression and secretion. Moreover, ERRγ, but not ERRα and ERRβ, induced FGF21 gene promoter activity. In addition, deletion and mutation analysis of the FGF21 promoter identified a putative ERRγ-binding motif (AGGTGC, a near-consensus response element). A chromatin immunoprecipitation assay revealed direct binding of ERRγ to the FGF21 gene promoter. Finally, GSK5182, an ERRγ inverse agonist, significantly inhibited hepatic CB1 receptor-mediated FGF21 gene expression and secretion. Conclusion Based on our data, we conclude that ERRγ plays a key role in hepatic CB1 receptor-mediated induction of FGF21 gene expression and secretion. PMID:27455076

  9. Shedding of tumor necrosis factor receptors by activated human neutrophils

    PubMed Central

    1990-01-01

    The capacity of human neutrophils (PMN) to bind tumor necrosis factor (TNF) was rapidly lost when the cells were incubated in suspension with agents that can stimulate their migratory and secretory responses. Both physiological (poly)peptides (FMLP, C5a, CSF-GM) and pharmacologic agonists (PMN, calcium ionophore A23187) induced the loss of TNF receptors (TNF-R) from the cell surface. Half-maximal loss in TNF-R ensued after only approximately 2 min with 10(-7) M FMLP at 37 degrees C, and required only 10(-9) M FMLP during a 30-min exposure. However, there were no such changes even with prolonged exposure of PMN to FMLP at 4 degrees or 16 degrees C. Scatchard analysis revealed loss of TNF- binding sites without change in their affinity (Kd approximately 0.4 nM) as measured at incompletely modulating concentrations of FMLP, C5a, PMA, or A23187. The binding of anti-TNF-R mAbs to PMN decreased in parallel, providing independent evidence for the loss of TNF-R from the cell surface. At the same time, soluble TNF-R appeared in the medium of stimulated PMN. This inference was based on the PMN- and FMLP-dependent generation of a nonsedimentable activity that could inhibit the binding of TNF to fresh human PMN or to mouse macrophages, and the ability of mAbs specific for human TNF-R to abolish inhibition by PMN-conditioned medium of binding of TNF to mouse macrophages. Soluble TNF-R activity was associated with a protein of Mr approximately 28,000 by ligand blot analysis of cell-free supernatants of FMLP-treated PMN. Thus, some portion of the FMLP-induced loss of TNF-R from human PMN is due to shedding of TNF-R. Shedding was unaffected by inhibitors of serine and thiol proteases and could not be induced with phosphatidylinositol- specific phospholipase C. Loss of TNF-R from PMN first stimulated by other agents may decrease their responsiveness to TNF. TNF-R shed by PMN may be one source of the TNF-binding proteins found in body fluids, and may blunt the actions of the

  10. Premature Ligand-Receptor Interaction during Biosynthesis Limits the Production of Growth Factor Midkine and Its Receptor LDL Receptor-related Protein 1*

    PubMed Central

    Sakamoto, Kazuma; Bu, Guojun; Chen, Sen; Takei, Yoshifumi; Hibi, Kenji; Kodera, Yasuhiro; McCormick, Lynn M.; Nakao, Akimasa; Noda, Masaharu; Muramatsu, Takashi; Kadomatsu, Kenji

    2011-01-01

    Protein production within the secretory pathway is accomplished by complex but organized processes. Here, we demonstrate that the growth factor midkine interacts with LDL receptor-related protein 1 (LRP1) at high affinity (Kd value, 2.7 nm) not only at the cell surface but also within the secretory pathway during biosynthesis. The latter premature ligand-receptor interaction resulted in aggregate formation and consequently suppressed midkine secretion and LRP1 maturation. We utilized an endoplasmic reticulum (ER) retrieval signal and an LRP1 fragment, which strongly bound to midkine and the LRP1-specialized chaperone receptor-associated protein (RAP), to construct an ER trapper. The ER trapper efficiently trapped midkine and RAP and mimicked the premature ligand-receptor interaction, i.e. suppressed maturation of the ligand and receptor. The ER trapper also diminished the inhibitory function of LRP1 on platelet-derived growth factor-mediated cell migration. Complementary to these results, an increased expression of RAP was closely associated with midkine expression in human colorectal carcinomas (33 of 39 cases examined). Our results suggest that the premature ligand-receptor interaction plays a role in protein production within the secretory pathway. PMID:21212259

  11. Platelet-derived growth factor receptor: Studies examining synthesis, phosphorylation and degradation of the receptor using an anti-receptor monoclonal antibody

    SciTech Connect

    Hart, C.E.

    1987-01-01

    A monoclonal antibody, designated PR7212 (IgG1), has been developed with specifically recognizes a cell-surface receptor for platelet-derived growth factor (PDGF). The antibody recognizes an extracellular epitope of the receptor, demonstrated by its ability to bind to intact cells. Using this antibody I have detected three forms of the receptor of 180, 164, and 130 kDa. All three forms were detected by Western blot analysis of human dermal fiberblasts. Immunoprecipitates of {sup 32}P-labeled membrane extracts of human dermal fibroblasts demonstrate that phosphorylation of all three forms of the receptor is stimulated by PDGF. In addition, several smaller molecules were detected, ranging in size from 113 to 49 kDa, which are also phosphorylated in response to PDGF addition. These smaller molecules may be either PDGF receptor kinase substrates or partially degraded receptor. Only the 180 and the 164 kDa forms of the receptor are detectable from immunoprecipitates of soluble extracts of {sup 35}S-metabolically labeled cells. Pulse-chase experiments demonstrate that the 164 kDa form is a precursor of the 180 kDa molecule.

  12. Interaction of the. alpha. beta. dimers of the insulin-like growth factor I receptor required for receptor autophosphorylation

    SciTech Connect

    Tollesfsen, S.E.; Stoszek, R.M.; Thompson, K. )

    1991-01-01

    The authors have recently found that association of the two {alpha}{beta} dimers of the insulin-like growth factor I (IGF I) receptor is required for formation of a high-affinity binding site for IGF I. To determine the structural requirements for IGF I activated kinase activity, they have examined the effect of dissociation of the two {alpha}{beta} dimers of the IGF I receptor on {beta} subunit autophosphorylation. The {alpha}{beta} dimers formed after treatment with 2 mM dithiothreitol (DTT) at pH 8.75 for 5 min were separated from IGF I receptor remaining as tetramers after DTT treatment by fast protein liquid chromatography on a Superose 6 gel filtration column. Purification of the {alpha}{beta} dimers was confirmed by Western blot analysis using {sup 125}I-labeled {alpha}IR-3, a monoclonal antibody to the IGF I receptor. Autophosphorylation of the IGF I receptor ({alpha}{beta}){sub 2} tetramer, treated without DTT or remaining after DTT treatment, is stimulated 1.6-2.9-fold by IGF I. In contrast, autophosporylation of the {alpha}{beta} dimers incubated in the presence or absence of IGF I (100 ng/mL) does not occur. Both IGF I receptor dimers and tetramers exhibit similar kinase activities using the synthetic substrate Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly, indicating that the failure to detect autophosphorylation of the IGF I receptor dimers does not result from inactivation of the kinase by DTT treatment. They conclude that autophosphorylation of the IGF I receptor depends upon the interaction of the two {alpha}{beta} dimers.

  13. Tumor necrosis factor receptor-associated factor 3 is a positive regulator of pathological cardiac hypertrophy.

    PubMed

    Jiang, Xi; Deng, Ke-Qiong; Luo, Yuxuan; Jiang, Ding-Sheng; Gao, Lu; Zhang, Xiao-Fei; Zhang, Peng; Zhao, Guang-Nian; Zhu, Xueyong; Li, Hongliang

    2015-08-01

    Cardiac hypertrophy, a common early symptom of heart failure, is regulated by numerous signaling pathways. Here, we identified tumor necrosis factor receptor-associated factor 3 (TRAF3), an adaptor protein in tumor necrosis factor-related signaling cascades, as a key regulator of cardiac hypertrophy in response to pressure overload. TRAF3 expression was upregulated in hypertrophied mice hearts and failing human hearts. Four weeks after aortic banding, cardiac-specific conditional TRAF3-knockout mice exhibited significantly reduced cardiac hypertrophy, fibrosis, and dysfunction. Conversely, transgenic mice overexpressing TRAF3 in the heart developed exaggerated cardiac hypertrophy in response to pressure overload. TRAF3 also promoted an angiotensin II- or phenylephrine-induced hypertrophic response in isolated cardiomyocytes. Mechanistically, TRAF3 directly bound to TANK-binding kinase 1 (TBK1), causing increased TBK1 phosphorylation in response to hypertrophic stimuli. This interaction between TRAF3 and TBK1 further activated AKT signaling, which ultimately promoted the development of cardiac hypertrophy. Our findings not only reveal a key role of TRAF3 in regulating the hypertrophic response but also uncover TRAF3-TBK1-AKT as a novel signaling pathway in the development of cardiac hypertrophy and heart failure. This pathway may represent a potential therapeutic target for this pathological process.

  14. Tumor necrosis factor receptor- associated factor 6 (TRAF6) regulation of development, function, and homeostasis of the immune system.

    PubMed

    Walsh, Matthew C; Lee, JangEun; Choi, Yongwon

    2015-07-01

    Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is an adapter protein that mediates a wide array of protein-protein interactions via its TRAF domain and a RING finger domain that possesses non-conventional E3 ubiquitin ligase activity. First identified nearly two decades ago as a mediator of interleukin-1 receptor (IL-1R)-mediated activation of NFκB, TRAF6 has since been identified as an actor downstream of multiple receptor families with immunoregulatory functions, including members of the TNFR superfamily, the Toll-like receptor (TLR) family, tumor growth factorreceptors (TGFβR), and T-cell receptor (TCR). In addition to NFκB, TRAF6 may also direct activation of mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and interferon regulatory factor pathways. In the context of the immune system, TRAF6-mediated signals have proven critical for the development, homeostasis, and/or activation of B cells, T cells, and myeloid cells, including macrophages, dendritic cells, and osteoclasts, as well as for organogenesis of thymic and secondary lymphoid tissues. In multiple cellular contexts, TRAF6 function is essential not only for proper activation of the immune system but also for maintaining immune tolerance, and more recent work has begun to identify mechanisms of contextual specificity for TRAF6, involving both regulatory protein interactions, and messenger RNA regulation by microRNAs.

  15. Functional Significance of the Signal Peptides of Corticotropin-Releasing Factor Receptors.

    PubMed

    Schülein, Ralf; Gibert, Arthur; Rutz, Claudia

    2017-02-23

    The corticotropin releasing factor (CRF) receptors belong to the large family of G protein-coupled receptors (GPCRs) and must be transported to the plasma membrane to function properly. The first step of the intracellular transport of GPCRs is their insertion into the membrane of the endoplasmic reticulum (ER). This process is mediated by the translocon complex of the ER membrane and the signal sequences of the receptors. Most GPCRs possess signal sequences which form part of the mature proteins, the so called signal anchor sequences (usually transmembrane domain 1). The CRF receptors possess instead signal sequences at their extreme N tails which were thought to be cleaved off following integration of the receptors into the ER membrane (signal peptides, SPs, also called cleaved signal sequences). Recent work, however, showed that not all subtypes of CRF receptors stick to this rule. Whereas the corticotropin-releasing factor receptor type 1 (CRF1R) and the corticotropin-releasing factor receptor type 2b (CRF2(b)R) possess conventional SPs which are indeed cleaved off following ER insertion, the SP of the cortictropin-releasing factor receptor type 2a (CRF2(a)R) remains uncleaved. It forms a unique N-terminal domain (pseudo signal peptide, PSP) which has surprising functions beyond the ER level. Its presence not only influences expression levels at the plasma membrane but also receptor homodimerisation and, as a consequence, G protein selectivity. In this mini-review, we summarize the progress in understanding the functions of SPs of CRF receptors. Recent data also allow deriving hypotheses for a physiological significance of these sequences. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  16. Transforming growth factor alpha and epidermal growth factor levels in bladder cancer and their relationship to epidermal growth factor receptor.

    PubMed Central

    Mellon, J. K.; Cook, S.; Chambers, P.; Neal, D. E.

    1996-01-01

    We have examined levels of epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) in neoplastic and non-neoplastic bladder tissue using a standard radioimmunoassay technique. Tumour samples had much higher TGF-alpha levels compared with EGF and TGF-alpha levels in malignant tissue were significantly higher than in benign bladder samples. There was, in addition, a difference in mean EGF levels from 'normal' bladder samples from non-tumour bearing areas of bladder in patients with bladder cancer compared with 'normal' bladder tissue obtained at the time of organ retrieval surgery. Levels of EGF and TGF-alpha did not correlate with levels of EGF receptor (EGFR) as determined by a radioligand binding method but levels of TGF-alpha > 10 ng gm-1 of tumour tissue did correlate with EGFR positivity defined using immunohistochemistry. These data suggest that TGF-alpha is the likely ligand for EGFR in bladder tumours. PMID:8605103

  17. Triggering neurotrophic factor actions through adenosine A2A receptor activation: implications for neuroprotection

    PubMed Central

    Sebastião, Ana M; Ribeiro, Joaquim A

    2009-01-01

    G protein coupled receptors and tropomyosin-related kinase (Trk) receptors have distinct structure and transducing mechanisms; therefore, cross-talk among them was unexpected. Evidence has, however, accumulated showing that tonic adenosine A2A receptor activity is a required step to allow synaptic actions of neurotrophic factors, namely upon synaptic transmission at both pre- and post-synaptic level as well as upon synaptic plasticity. An enhancement of A2A receptor tonus upon ageing may partially compensate the loss of TrkB receptors, rescuing to certain degree the facilitatory action of brain derived neurotrophic factor in aged animals, which might prove particularly relevant in the prevention of neurodegeneration upon ageing. A2A receptors also trigger synaptic actions of other neurotrophic factors, such as glial derived neurotrophic factor at dopaminergic striatal nerve endings. The growing evidence that tonic adenosine A2A receptor activity is a crucial step to allow actions of neurotrophic factors in neurones will be reviewed and discussed in the light of therapeutic strategies for neurodegenerative diseases. PMID:19508402

  18. Equine insulin receptor and insulin-like growth factor-1 receptor expression in digital lamellar tissue and insulin target tissues.

    PubMed

    Kullmann, A; Weber, P S; Bishop, J B; Roux, T M; Norby, B; Burns, T A; McCutcheon, L J; Belknap, J K; Geor, R J

    2016-09-01

    Hyperinsulinaemia is implicated in the pathogenesis of endocrinopathic laminitis. Insulin can bind to different receptors: two insulin receptor isoforms (InsR-A and InsR-B), insulin-like growth factor-1 receptor (IGF-1R) and InsR/IGF-1R hybrid receptor (Hybrid). Currently, mRNA expression of these receptors in equine tissues and the influence of body type and dietary carbohydrate intake on expression of these receptors is not known. The study objectives were to characterise InsR-A, InsR-B, IGF-1R and Hybrid expression in lamellar tissue (LT) and insulin responsive tissues from horses and examine the effect of dietary nonstructural carbohydrate (NSC) on mRNA expression of these receptors in LT, skeletal muscle, liver and two adipose tissue (AT) depots of lean and obese ponies. In vivo experiment. Lamellar tissue samples were evaluated by quantitative reverse transcription polymerase chain reaction (RT-qPCR) for receptor mRNA expression (n = 8) and immunoblotting for protein expression (n = 3). Archived LT, skeletal muscle, liver and AT from lean and obese mixed-breed ponies fed either a low (~7% NSC as dry matter; 5 lean, 5 obese) or high NSC diet (~42% NSC as dry matter; 6 lean, 6 obese) for 7 days were evaluated by RT-qPCR to determine the effect of body condition and diet on expression of the receptors in different tissues. Significance was set at P≤0.05. Lamellar tissue expresses both InsR isoforms, IGF-1R and Hybrid. LT IGF-1R gene expression was greater than either InsR isoform and InsR-A expression was greater than InsR-B (P≤0.05). Obesity significantly lowered IGF-1R, InsR-A and InsR-B mRNA expression in LT and InsR-A in tailhead AT. High NSC diet lowered expression of all three receptor types in liver; IGF-1R and InsR-A in LT and InsR-A in tailhead AT. Lamellar tissue expresses IGF-1R, InsR isoforms and Hybrids. The functional characteristics of these receptors and their role in endocrinopathic laminitis warrants further investigation. © 2015 EVJ

  19. The transforming growth factor beta type II receptor can replace the activin type II receptor in inducing mesoderm.

    PubMed Central

    Bhushan, A; Lin, H Y; Lodish, H F; Kintner, C R

    1994-01-01

    The type II receptors for the polypeptide growth factors transforming growth factor beta (TGF-beta) and activin belong to a new family of predicted serine/threonine protein kinases. In Xenopus embryos, the biological effects of activin and TGF-beta 1 are strikingly different; activin induces a full range of mesodermal cell types in the animal cap assay, while TGF-beta 1 has no effects, presumably because of the lack of functional TGF-beta receptors. In order to assess the biological activities of exogenously added TGF-beta 1, RNA encoding the TGF-beta type II receptor was introduced into Xenopus embryos. In animal caps from these embryos, TGF-beta 1 and activin show similar potencies for induction of mesoderm-specific mRNAs, and both elicit the same types of mesodermal tissues. In addition, the response of animal caps to TGF-beta 1, as well as to activin, is blocked by a dominant inhibitory ras mutant, p21(Asn-17)Ha-ras. These results indicate that the activin and TGF-beta type II receptors can couple to similar signalling pathways and that the biological specificities of these growth factors lie in their different ligand-binding domains and in different competences of the responding cells. Images PMID:8196664

  20. Intratumoral Heterogeneity for Expression of Tyrosine Kinase Growth Factor Receptors in Human Colon Cancer Surgical Specimens and Orthotopic Tumors

    PubMed Central

    Kuwai, Toshio; Nakamura, Toru; Kim, Sun-Jin; Sasaki, Takamitsu; Kitadai, Yasuhiko; Langley, Robert R.; Fan, Dominic; Hamilton, Stanley R.; Fidler, Isaiah J.

    2008-01-01

    The design of targeted therapy, particularly patient-specific targeted therapy, requires knowledge of the presence and intratumoral distribution of tyrosine kinase receptors. To determine whether the expression of such receptors is constant or varies between and within individual colon cancer neoplasms, we examined the pattern of expression of the ligands, epidermal growth factor, vascular endothelial growth factor, and platelet-derived growth factor-B as well as their respective receptors in human colon cancer surgical specimens and orthotopic human colon cancers growing in the cecal wall of nude mice. The expression of the epidermal growth factor receptor and the vascular endothelial growth factor receptor on tumor cells and stromal cells, including tumor-associated endothelial cells, was heterogeneous in surgical specimens and orthotopic tumors. In some tumors, the receptor was expressed on both tumor cells and stromal cells, and in other tumors the receptor was expressed only on tumor cells or only on stromal cells. In contrast, the platelet-derived growth factor receptor was expressed only on stromal cells in both surgical specimens and orthotopic tumors. Examination of receptor expression in both individual surgical specimens and orthotopic tumors revealed that the platelet-derived growth factor receptor was expressed only on stromal cells and that the patterns of epidermal growth factor receptor and vascular endothelial growth factor receptor 2 expression differed between tumor cells. This heterogeneity in receptor expression among different tumor cells suggests that targeting a single tyrosine kinase may not yield eradication of the disease. PMID:18202197

  1. Apical Epidermal Growth Factor Receptor Signaling: Regulation of Stretch-dependent Exocytosis in Bladder Umbrella Cells

    PubMed Central

    Balestreire, Elena M.

    2007-01-01

    The apical surface of polarized epithelial cells receives input from mediators, growth factors, and mechanical stimuli. How these stimuli are coordinated to regulate complex cellular functions such as polarized membrane traffic is not understood. We analyzed the requirement for growth factor signaling and mechanical stimuli in umbrella cells, which line the mucosal surface of the bladder and dynamically insert and remove apical membrane in response to stretch. We observed that stretch-stimulated exocytosis required apical epidermal growth factor (EGF) receptor activation and that activation occurred in an autocrine manner downstream of heparin-binding EGF-like growth factor precursor cleavage. Long-term changes in apical exocytosis depended on protein synthesis, which occurred upon EGF receptor-dependent activation of mitogen-activated protein kinase signaling. Our results indicate a novel physiological role for the EGF receptor that couples upstream mechanical stimuli to downstream apical EGF receptor activation that may regulate apical surface area changes during bladder filling. PMID:17287395

  2. Gene expression of growth factors and growth factor receptors for potential targeted therapy of canine hepatocellular carcinoma.

    PubMed

    Iida, Gentoku; Asano, Kazushi; Seki, Mamiko; Sakai, Manabu; Kutara, Kenji; Ishigaki, Kumiko; Kagawa, Yumiko; Yoshida, Orie; Teshima, Kenji; Edamura, Kazuya; Watari, Toshihiro

    2014-03-01

    The purpose of this study was to evaluate the gene expression of growth factors and growth factor receptors of primary hepatic masses, including hepatocellular carcinoma (HCC) and nodular hyperplasia (NH), in dogs. Quantitative real-time reverse transcriptase-polymerase chain reaction was performed to measure the expression of 18 genes in 18 HCCs, 10 NHs, 11 surrounding non-cancerous liver tissues and 4 healthy control liver tissues. Platelet-derived growth factor-B (PDGF-B), transforming growth factor-α, epidermal growth factor receptor, epidermal growth factor and hepatocyte growth factor were found to be differentially expressed in HCC compared with NH and the surrounding non-cancerous and healthy control liver tissues. PDGF-B is suggested to have the potential to become a valuable ancillary target for the treatment of canine HCC.

  3. Corticotropin Releasing Factor (CRF) Receptor Signaling in the Central Nervous System: New Molecular Targets

    PubMed Central

    Hauger, Richard L.; Risbrough, Victoria; Brauns, Olaf; Dautzenberg, Frank M.

    2007-01-01

    Corticotropin-releasing factor (CRF) and the related urocortin peptides mediate behavioral, cognitive, autonomic, neuroendocrine and immunologic responses to aversive stimuli by activating CRF1 or CRF2 receptors in the central nervous system and anterior pituitary. Markers of hyperactive central CRF systems, including CRF hypersecretion and abnormal hypothalamic-pituitary-adrenal axis functioning, have been identified in subpopulations of patients with anxiety, stress and depressive disorders. Because CRF receptors are rapidly desensitized in the presence of high agonist concentrations, CRF hypersecretion alone may be insufficient to account for the enhanced CRF neurotransmission observed in these patients. Concomitant dysregulation of mechanisms stringently controlling magnitude and duration of CRF receptor signaling also may contribute to this phenomenon. While it is well established that the CRF1 receptor mediates many anxiety- and depression-like behaviors as well as HPA axis stress responses, CRF2 receptor functions are not well understood at present. One hypothesis holds that CRF1 receptor activation initiates fear and anxiety-like responses, while CRF2 receptor activation re-establishes homeostasis by counteracting the aversive effects of CRF1 receptor signaling. An alternative hypothesis posits that CRF1 and CRF2 receptors contribute to opposite defensive modes, with CRF1 receptors mediating active defensive responses triggered by escapable stressors, and CRF2 receptors mediating anxiety- and depression-like responses induced by inescapable, uncontrollable stressors. CRF1 receptor antagonists are being developed as novel treatments for affective and stress disorders. If it is confirmed that the CRF2 receptor contributes importantly to anxiety and depression, the development of small molecule CRF2 receptor antagonists would be therapeutically useful. PMID:16918397

  4. Factors That Effect Signal Transduction by the Estrogen Receptor.

    DTIC Science & Technology

    1997-10-01

    Vivat , H. Gronemeyer, R. Losson, and P. Chambon. 1996. Ligand-dependent interaction of nuclear receptors with potential transcriptional... Academy of Sciences 0027-8424/97/9410132-6S2.00/0 PNAS is available online at http://www.pnas.org. inhibitors or CDIs), kinase function (5-8). Because

  5. Module Based Differential Coexpression Analysis Method for Type 2 Diabetes

    PubMed Central

    Yuan, Lin; Zheng, Chun-Hou; Xia, Jun-Feng; Huang, De-Shuang

    2015-01-01

    More and more studies have shown that many complex diseases are contributed jointly by alterations of numerous genes. Genes often coordinate together as a functional biological pathway or network and are highly correlated. Differential coexpression analysis, as a more comprehensive technique to the differential expression analysis, was raised to research gene regulatory networks and biological pathways of phenotypic changes through measuring gene correlation changes between disease and normal conditions. In this paper, we propose a gene differential coexpression analysis algorithm in the level of gene sets and apply the algorithm to a publicly available type 2 diabetes (T2D) expression dataset. Firstly, we calculate coexpression biweight midcorrelation coefficients between all gene pairs. Then, we select informative correlation pairs using the “differential coexpression threshold” strategy. Finally, we identify the differential coexpression gene modules using maximum clique concept and k-clique algorithm. We apply the proposed differential coexpression analysis method on simulated data and T2D data. Two differential coexpression gene modules about T2D were detected, which should be useful for exploring the biological function of the related genes. PMID:26339648

  6. Afr1p regulates the Saccharomyces cerevisiae alpha-factor receptor by a mechanism that is distinct from receptor phosphorylation and endocytosis.

    PubMed Central

    Davis, C; Dube, P; Konopka, J B

    1998-01-01

    The alpha-factor pheromone receptor activates a G protein signaling pathway that induces the conjugation of the yeast Saccharomyces cerevisiae. Our previous studies identified AFR1 as a gene that regulates this signaling pathway because overexpression of AFR1 promoted resistance to alpha-factor. AFR1 also showed an interesting genetic relationship with the alpha-factor receptor gene, STE2, suggesting that the receptor is regulated by Afr1p. To investigate the mechanism of this regulation, we tested AFR1 for a role in the two processes that are known to regulate receptor signaling: phosphorylation and down-regulation of ligand-bound receptors by endocytosis. AFR1 overexpression diminished signaling in a strain that lacks the C-terminal phosphorylation sites of the receptor, indicating that AFR1 acts independently of phosphorylation. The effects of AFR1 overexpression were weaker in strains that were defective in receptor endocytosis. However, AFR1 overexpression did not detectably influence receptor endocytosis or the stability of the receptor protein. Instead, gene dosage studies showed that the effects of AFR1 overexpression on signaling were inversely proportional to the number of receptors. These results indicate that AFR1 acts independently of endocytosis, and that the weaker effects of AFR1 in strains that are defective in receptor endocytosis were probably an indirect consequence of their increased receptor number caused by the failure of receptors to undergo ligand-stimulated endocytosis. Analysis of the ligand binding properties of the receptor showed that AFR1 overexpression did not alter the number of cell-surface receptors or the affinity for alpha-factor. Thus, Afr1p prevents alpha-factor receptors from activating G protein signaling by a mechanism that is distinct from other known pathways. PMID:9504911

  7. Extended Synaptotagmin Interaction with the Fibroblast Growth Factor Receptor Depends on Receptor Conformation, Not Catalytic Activity.

    PubMed

    Tremblay, Michel G; Herdman, Chelsea; Guillou, François; Mishra, Prakash K; Baril, Joëlle; Bellenfant, Sabrina; Moss, Tom

    2015-06-26

    We previously demonstrated that ESyt2 interacts specifically with the activated FGF receptor and is required for a rapid phase of receptor internalization and for functional signaling via the ERK pathway in early Xenopus embryos. ESyt2 is one of the three-member family of Extended Synaptotagmins that were recently shown to be implicated in the formation of endoplasmic reticulum (ER)-plasma membrane (PM) junctions and in the Ca(2+) dependent regulation of these junctions. Here we show that ESyt2 is directed to the ER by its putative transmembrane domain, that the ESyts hetero- and homodimerize, and that ESyt2 homodimerization in vivo requires a TM adjacent sequence but not the SMP domain. ESyt2 and ESyt3, but not ESyt1, selectively interact in vivo with activated FGFR1. In the case of ESyt2, this interaction requires a short TM adjacent sequence and is independent of receptor autophosphorylation, but dependent on receptor conformation. The data show that ESyt2 recognizes a site in the upper kinase lobe of FGFR1 that is revealed by displacement of the kinase domain activation loop during receptor activation.

  8. Gene Sets Net Correlations Analysis (GSNCA): a multivariate differential coexpression test for gene sets

    PubMed Central

    Rahmatallah, Yasir; Emmert-Streib, Frank; Glazko, Galina

    2014-01-01

    Motivation: To date, gene set analysis approaches primarily focus on identifying differentially expressed gene sets (pathways). Methods for identifying differentially coexpressed pathways also exist but are mostly based on aggregated pairwise correlations or other pairwise measures of coexpression. Instead, we propose Gene Sets Net Correlations Analysis (GSNCA), a multivariate differential coexpression test that accounts for the complete correlation structure between genes. Results: In GSNCA, weight factors are assigned to genes in proportion to the genes’ cross-correlations (intergene correlations). The problem of finding the weight vectors is formulated as an eigenvector problem with a unique solution. GSNCA tests the null hypothesis that for a gene set there is no difference in the weight vectors of the genes between two conditions. In simulation studies and the analyses of experimental data, we demonstrate that GSNCA captures changes in the structure of genes’ cross-correlations rather than differences in the averaged pairwise correlations. Thus, GSNCA infers differences in coexpression networks, however, bypassing method-dependent steps of network inference. As an additional result from GSNCA, we define hub genes as genes with the largest weights and show that these genes correspond frequently to major and specific pathway regulators, as well as to genes that are most affected by the biological difference between two conditions. In summary, GSNCA is a new approach for the analysis of differentially coexpressed pathways that also evaluates the importance of the genes in the pathways, thus providing unique information that may result in the generation of novel biological hypotheses. Availability and implementation: Implementation of the GSNCA test in R is available upon request from the authors. Contact: YRahmatallah@uams.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24292935

  9. Evaluation of Gene Association Methods for Coexpression Network Construction and Biological Knowledge Discovery

    PubMed Central

    Kumari, Sapna; Nie, Jeff; Chen, Huann-Sheng; Ma, Hao; Stewart, Ron; Li, Xiang; Lu, Meng-Zhu; Taylor, William M.; Wei, Hairong

    2012-01-01

    Background Constructing coexpression networks and performing network analysis using large-scale gene expression data sets is an effective way to uncover new biological knowledge; however, the methods used for gene association in constructing these coexpression networks have not been thoroughly evaluated. Since different methods lead to structurally different coexpression networks and provide different information, selecting the optimal gene association method is critical. Methods and Results In this study, we compared eight gene association methods – Spearman rank correlation, Weighted Rank Correlation, Kendall, Hoeffding's D measure, Theil-Sen, Rank Theil-Sen, Distance Covariance, and Pearson – and focused on their true knowledge discovery rates in associating pathway genes and construction coordination networks of regulatory genes. We also examined the behaviors of different methods to microarray data with different properties, and whether the biological processes affect the efficiency of different methods. Conclusions We found that the Spearman, Hoeffding and Kendall methods are effective in identifying coexpressed pathway genes, whereas the Theil-sen, Rank Theil-Sen, Spearman, and Weighted Rank methods perform well in identifying coordinated transcription factors that control the same biological processes and traits. Surprisingly, the widely used Pearson method is generally less efficient, and so is the Distance Covariance method that can find gene pairs of multiple relationships. Some analyses we did clearly show Pearson and Distance Covariance methods have distinct behaviors as compared to all other six methods. The efficiencies of different methods vary with the data properties to some degree and are largely contingent upon the biological processes, which necessitates the pre-analysis to identify the best performing method for gene association and coexpression network construction. PMID:23226279

  10. Nerve Growth Factor Receptor TrkA, a New Receptor in Insulin Signaling Pathway in PC12 Cells*

    PubMed Central

    Geetha, Thangiah; Rege, Shraddha D.; Mathews, Salome E.; Meakin, Susan O.; White, Morris F.; Babu, Jeganathan Ramesh

    2013-01-01

    TrkA is a cell surface transmembrane receptor tyrosine kinase for nerve growth factor (NGF). TrkA has an NPXY motif and kinase regulatory loop similar to insulin receptor (INSR) suggesting that NGF→TrkA signaling might overlap with insulin→INSR signaling. During insulin or NGF stimulation TrkA, insulin receptor substrate-1 (IRS-1), INSR (and presumably other proteins) forms a complex in PC12 cells. In PC12 cells, tyrosine phosphorylation of INSR and IRS-1 is dependent upon the functional TrkA kinase domain. Moreover, expression of TrkA kinase-inactive mutant blocked the activation of Akt and Erk5 in response to insulin or NGF. Based on these data, we propose that TrkA participates in insulin signaling pathway in PC12 cells. PMID:23749991

  11. Transactivation of Epidermal Growth Factor Receptor by G Protein-Coupled Receptors: Recent Progress, Challenges and Future Research

    PubMed Central

    Wang, Zhixiang

    2016-01-01

    Both G protein-coupled receptors (GPCRs) and receptor-tyrosine kinases (RTKs) regulate large signaling networks, control multiple cell functions and are implicated in many diseases including various cancers. Both of them are also the top therapeutic targets for disease treatment. The discovery of the cross-talk between GPCRs and RTKs connects these two vast signaling networks and complicates the already complicated signaling networks that regulate cell signaling and function. In this review, we focus on the transactivation of epidermal growth factor receptor (EGFR), a subfamily of RTKs, by GPCRs. Since the first report of EGFR transactivation by GPCR, significant progress has been made including the elucidation of the mechanisms underlying the transactivation. Here, we first provide a basic picture for GPCR, EGFR and EGFR transactivation by GPCR. We then discuss the progress made in the last five years and finally provided our view of the future challenge and future researches needed to overcome these challenges. PMID:26771606

  12. Transactivation of Epidermal Growth Factor Receptor by G Protein-Coupled Receptors: Recent Progress, Challenges and Future Research.

    PubMed

    Wang, Zhixiang

    2016-01-12

    Both G protein-coupled receptors (GPCRs) and receptor-tyrosine kinases (RTKs) regulate large signaling networks, control multiple cell functions and are implicated in many diseases including various cancers. Both of them are also the top therapeutic targets for disease treatment. The discovery of the cross-talk between GPCRs and RTKs connects these two vast signaling networks and complicates the already complicated signaling networks that regulate cell signaling and function. In this review, we focus on the transactivation of epidermal growth factor receptor (EGFR), a subfamily of RTKs, by GPCRs. Since the first report of EGFR transactivation by GPCR, significant progress has been made including the elucidation of the mechanisms underlying the transactivation. Here, we first provide a basic picture for GPCR, EGFR and EGFR transactivation by GPCR. We then discuss the progress made in the last five years and finally provided our view of the future challenge and future researches needed to overcome these challenges.

  13. Receptor-purified, Bolton-Hunter radioiodinated, recombinant, human epidermal growth factor: An improved radioligand for receptor studies

    SciTech Connect

    Kermode, J.C.; Tritton, T.R. )

    1990-01-01

    We report an assessment of the applicability of the Bolton-Hunter method to the radioiodination of epidermal growth factor (EGF). Recombinant human EGF (hEGF) could be radioiodinated successfully by this method, whereas murine EGF could not. Bolton-Hunter {sup 125}I-labeled hEGF was compared with commercial 125I-labeled hEGF prepared by the chloramine-T radioiodination method. Neither radioligand was sufficiently pure for a detailed characterization of the purportedly heterogeneous pattern of binding of EGF to its receptors. A procedure based on receptor adsorption was thus developed for repurification of the Bolton-Hunter 125I-labeled hEGF. This provided a much purer radioligand suitable for detailed studies of receptor-binding heterogeneity.

  14. Tumor necrosis factor receptor superfamily costimulation couples T cell receptor signal strength to thymic regulatory T cell differentiation

    PubMed Central

    Mahmud, Shawn A.; Manlove, Luke S.; Schmitz, Heather M.; Xing, Yan; Wang, Yanyan; Owen, David L.; Schenkel, Jason M.; Boomer, Jonathan S.; Green, Jonathan M.; Yagita, Hideo; Chi, Hongbo; Hogquist, Kristin A.; Farrar, Michael A.

    2014-01-01

    Regulatory T (Treg) cells express tumor necrosis factor receptor superfamily (TNFRSF) members, but their role in thymic Treg development is undefined. We demonstrate that Treg progenitors highly express the TNFRSF members GITR, OX40, and TNFR2. Expression of these receptors correlates directly with T cell receptor (TCR) signal strength, and requires CD28 and the kinase TAK1. Neutralizing TNFSF ligands markedly reduced Treg development. Conversely, TNFRSF agonists enhanced Treg differentiation by augmenting IL-2R/STAT5 responsiveness. GITR-ligand costimulation elicited a dose-dependent enrichment of lower-affinity cells within the Treg repertoire. In vivo, combined inhibition of GITR, OX40 and TNFR2 abrogated Treg development. Thus TNFRSF expression on Treg progenitors translates strong TCR signals into molecular parameters that specifically promote Treg differentiation and shape the Treg repertoire. PMID:24633226

  15. Nerve growth factor receptor TrkA, a new receptor in insulin signaling pathway in PC12 cells.

    PubMed

    Geetha, Thangiah; Rege, Shraddha D; Mathews, Salome E; Meakin, Susan O; White, Morris F; Babu, Jeganathan Ramesh

    2013-08-16

    TrkA is a cell surface transmembrane receptor tyrosine kinase for nerve growth factor (NGF). TrkA has an NPXY motif and kinase regulatory loop similar to insulin receptor (INSR) suggesting that NGF→TrkA signaling might overlap with insulin→INSR signaling. During insulin or NGF stimulation TrkA, insulin receptor substrate-1 (IRS-1), INSR (and presumably other proteins) forms a complex in PC12 cells. In PC12 cells, tyrosine phosphorylation of INSR and IRS-1 is dependent upon the functional TrkA kinase domain. Moreover, expression of TrkA kinase-inactive mutant blocked the activation of Akt and Erk5 in response to insulin or NGF. Based on these data, we propose that TrkA participates in insulin signaling pathway in PC12 cells.

  16. AFR1 acts in conjunction with the alpha-factor receptor to promote morphogenesis and adaptation.

    PubMed Central

    Konopka, J B

    1993-01-01

    Mating pheromone receptors activate a G-protein signaling pathway that induces changes in transcription, cell division, and morphogenesis needed for the conjunction of Saccharomyces cerevisiae. The C terminus of the alpha-factor pheromone receptor functions in two complex processes, adaptation and morphogenesis. Adaptation to alpha-factor may occur through receptor desensitization, and alpha-factor-induced morphogenesis forms the conjugation bridge between mating cells. A plasmid overexpression strategy was used to isolate a new gene, AFR1, which acts together with the receptor C terminus to promote adaptation. The expression of AFR1 was highly induced by alpha-factor. Unexpectedly, cells lacking AFR1 showed a defect in alpha-factor-stimulated morphogenesis that was similar to the morphogenesis defect observed in cells producing C-terminally truncated alpha-factor receptors. In contrast, AFR1 overexpression resulted in longer projections of morphogenesis, which suggests that this gene may directly stimulate morphogenesis. These results indicate that AFR1 encodes a developmentally regulated function that coordinates both the regulation of receptor signaling and the induction of morphogenesis during conjugation. Images PMID:8413281

  17. Asymmetric Receptor Contact is Required for Tyrosine Autophosphorylation of Fibroblast Growth Factor Receptor in Living Cells

    SciTech Connect

    Bae, J.; Boggon, T; Tomé, F; Mandiyan, V; Lax, I; Schlessinge, J

    2010-01-01

    Tyrosine autophosphorylation of receptor tyrosine kinases plays a critical role in regulation of kinase activity and in recruitment and activation of intracellular signaling pathways. Autophosphorylation is mediated by a sequential and precisely ordered intermolecular (trans) reaction. In this report we present structural and biochemical experiments demonstrating that formation of an asymmetric dimer between activated FGFR1 kinase domains is required for transphosphorylation of FGFR1 in FGF-stimulated cells. Transphosphorylation is mediated by specific asymmetric contacts between the N-lobe of one kinase molecule, which serves as an active enzyme, and specific docking sites on the C-lobe of a second kinase molecule, which serves a substrate. Pathological loss-of-function mutations or oncogenic activating mutations in this interface may hinder or facilitate asymmetric dimer formation and transphosphorylation, respectively. The experiments presented in this report provide the molecular basis underlying the control of transphosphorylation of FGF receptors and other receptor tyrosine kinases.

  18. The CXC Chemokine Receptor 4 Ligands Ubiquitin and Stromal Cell-derived Factor-1α Function through Distinct Receptor Interactions*

    PubMed Central

    Saini, Vikas; Staren, Daniel M.; Ziarek, Joshua J.; Nashaat, Zayd N.; Campbell, Edward M.; Volkman, Brian F.; Marchese, Adriano; Majetschak, Matthias

    2011-01-01

    Recently, we identified extracellular ubiquitin as an endogenous CXC chemokine receptor (CXCR) 4 agonist. However, the receptor selectivity and molecular basis of the CXCR4 agonist activity of ubiquitin are unknown, and functional consequences of CXCR4 activation with ubiquitin are poorly defined. Here, we provide evidence that ubiquitin and the cognate CXCR4 ligand stromal cell-derived factor (SDF)-1α do not share CXCR7 as a receptor. We further demonstrate that ubiquitin does not utilize the typical two-site binding mechanism of chemokine-receptor interactions, in which the receptor N terminus is important for ligand binding. CXCR4 activation with ubiquitin and SDF-1α lead to similar Gαi-responses and to a comparable magnitude of phosphorylation of ERK-1/2, p90 ribosomal S6 kinase-l and Akt, although phosphorylations occur more transiently after activation with ubiquitin. Despite the similarity of signal transduction events after activation of CXCR4 with both ligands, ubiquitin possesses weaker chemotactic activity than SDF-lα in cell migration assays and does not interfere with productive entry of HIV-1 into P4.R5 multinuclear activation of galactosidase indicator cells. Unlike SDF-1α, ubiquitin lacks interactions with an N-terminal CXCR4 peptide in NMR spectroscopy experiments. Binding and signaling studies in the presence of antibodies against the N terminus and extracellular loops 2/3 of CXCR4 confirm that the ubiquitin CXCR4 interaction is independent of the N-terminal receptor domain, whereas blockade of extracellular loops 2/3 prevents receptor binding and activation. Our findings define ubiquitin as a CXCR4 agonist, which does not interfere with productive cellular entry of HIV-1, and provide new mechanistic insights into interactions between CXCR4 and its natural ligands. PMID:21757744

  19. Epidermal growth factor receptor mutation enhances expression of vascular endothelial growth factor in lung cancer.

    PubMed

    Hung, Ming-Szu; Chen, I-Chuan; Lin, Paul-Yann; Lung, Jr-Hau; Li, Ya-Chin; Lin, Yu-Ching; Yang, Cheng-Ta; Tsai, Ying-Huang

    2016-12-01

    Epidermal growth factor receptor (EGFR) activation has been demonstrated to have a critical role in tumor angiogenesis. In the present study, the correlation between EGFR mutations and vascular endothelial growth factor (VEGF) was investigated in lung cancer cell lines and non-small-cell lung cancer (NSCLC) tumor tissues. VEGF levels were significantly increased in culture medium of lung cancer cells and NSCLC tissues with EGFR mutations (H1650 vs. A549, P=0.0399; H1975 vs. A549, P<0.0001). Stable lung cancer cell lines expressing mutant (exon 19 deletion, E746-A750; exon 21 missense mutation, L858R) and wild-type EGFR genes were established. Significantly increased expression of VEGF and stronger inhibitory effects of gefitinib to VEGF expression were observed in exon 19 deletion stable lung cancer cells (exon 19 deletion vs. wild-type EGFR, P=0.0005). The results of the present study may provide an insight into the association of mutant EGFR and VEGF expression in lung cancer, and may assist with further development of targeted therapy for NSCLC in the future.

  20. High expression of Toll-like receptor 4/myeloid differentiation factor 88 signals correlates with poor prognosis in colorectal cancer

    PubMed Central

    Wang, E L; Qian, Z R; Nakasono, M; Tanahashi, T; Yoshimoto, K; Bando, Y; Kudo, E; Shimada, M; Sano, T

    2010-01-01

    Background: The Toll-like receptor (TLR) 4 signalling pathway has been shown to have oncogenic effects in vitro and in vivo. To demonstrate the role of TLR4 signalling in colon tumourigenesis, we examined the expression of TLR4 and myeloid differentiation factor 88 (MyD88) in colorectal cancer (CRC). Methods: The expression of TLR4 and MyD88 in 108 CRC samples, 15 adenomas, and 15 normal mucosae was evaluated by immunohistochemistry, and the correlations between their immunoscores and clinicopathological variables, including disease-free survival (DFS) and overall survival (OS), were analysed. Results: Compared with normal mucosae and adenomas, 20% cancers displayed high expression of TLR4, and 23% cancers showed high expression of MyD88. The high expression of TLR4 and MyD88 was significantly correlated with liver metastasis (P=0.0001, P=0.0054). In univariate analysis, the high expression of TLR4 was significantly associated with shorter OS (hazard ratio (HR): 2.17; 95% confidence interval (95% CI): 1.15–4.07; P=0.015). The high expression of MyD88 expression was significantly associated with poor DFS and OS (HR: 2.33; 95% CI: 1.31–4.13; P=0.0038 and HR: 3.03; 95% CI: 1.67–5.48; P=0.0002). The high combined expression of TLR4 and MyD88 was also significantly associated with poor DFS and OS (HR: 2.25; 95% CI: 1.27–3.99; P=0.0053 and HR: 2.97; 95% CI: 1.64–5.38; P=0.0003). Multivariate analysis showed that high expressions of TLR4 (OS: adjusted HR: 1.88; 95% CI: 0.99–3.55; P=0.0298) and MyD88 (DFS: adjusted HR: 1.93; 95% CI: 1.01–3.67; P=0.0441; OS: adjusted HR: 2.25; 95% CI: 1.17–4.33; P=0.0112) were independent prognostic factors of OS. Furthermore, high co-expression of TLR4/MyD88 was strongly associated with both poor DFS and OS. Conclusion: Our findings suggest that high expression of TLR4 and MyD88 is associated with liver metastasis and is an independent predictor of poor prognosis in patients with CRC. PMID:20145615

  1. Insulin-Like Growth Factor 1 Receptor Is a Prognostic Factor in Classical Hodgkin Lymphoma

    PubMed Central

    Liang, Zheng; Diepstra, Arjan; Xu, Chuanhui; van Imhoff, Gustaaf; Plattel, Wouter; Van Den Berg, Anke; Visser, Lydia

    2014-01-01

    The interaction between the tumor cells in classical Hodgkin lymphoma (cHL) and the microenvironment includes aberrant activity of receptor tyrosine kinases. In this study we evaluated the expression, functionality and prognostic significance of Insulin-like growth factor-1 receptor (IGF-1R) in cHL. IGF-1R was overexpressed in 55% (44/80) of cHL patients. Phosphorylated IGF-1R was detectable in a minority of the IGF-1R positive tumor cells. The overall survival (OS, 98%) and 5-year progression-free survival (PFS, 93%) was significantly higher in IGF-1R positive cHL patients compared to IGF-1R negative patients (OS 83%, p = .029 and PFS 77%, p = .047, respectively). Three cHL cell lines showed expression of IGF-1R, with strong staining especially in the mitotic cells and expression of IGF-1. IGF-1 treatment had a prominent effect on the cell growth of L428 and L1236 cells and resulted in an increased phosphorylation of IGF1R, Akt and ERK. Inhibition of IGF-1R with cyclolignan picropodophyllin (PPP) decreased cell growth and induced a G2/M cell cycle arrest in all three cell lines. Moreover, a decrease in pCcd2 and an increase in CyclinB1 levels were observed which is consistent with the G2/M cell cycle arrest. In conclusion, IGF-1R expression in HRS cells predicts a favorable outcome, despite the oncogenic effect of IGF-1R in cHL cell lines. PMID:24489919

  2. Insulin-like growth factor 1 receptor is a prognostic factor in classical Hodgkin lymphoma.

    PubMed

    Liang, Zheng; Diepstra, Arjan; Xu, Chuanhui; van Imhoff, Gustaaf; Plattel, Wouter; Van Den Berg, Anke; Visser, Lydia

    2014-01-01

    The interaction between the tumor cells in classical Hodgkin lymphoma (cHL) and the microenvironment includes aberrant activity of receptor tyrosine kinases. In this study we evaluated the expression, functionality and prognostic significance of Insulin-like growth factor-1 receptor (IGF-1R) in cHL. IGF-1R was overexpressed in 55% (44/80) of cHL patients. Phosphorylated IGF-1R was detectable in a minority of the IGF-1R positive tumor cells. The overall survival (OS, 98%) and 5-year progression-free survival (PFS, 93%) was significantly higher in IGF-1R positive cHL patients compared to IGF-1R negative patients (OS 83%, p = .029 and PFS 77%, p = .047, respectively). Three cHL cell lines showed expression of IGF-1R, with strong staining especially in the mitotic cells and expression of IGF-1. IGF-1 treatment had a prominent effect on the cell growth of L428 and L1236 cells and resulted in an increased phosphorylation of IGF1R, Akt and ERK. Inhibition of IGF-1R with cyclolignan picropodophyllin (PPP) decreased cell growth and induced a G2/M cell cycle arrest in all three cell lines. Moreover, a decrease in pCcd2 and an increase in CyclinB1 levels were observed which is consistent with the G2/M cell cycle arrest. In conclusion, IGF-1R expression in HRS cells predicts a favorable outcome, despite the oncogenic effect of IGF-1R in cHL cell lines.

  3. SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors.

    PubMed Central

    McGlade, C J; Ellis, C; Reedijk, M; Anderson, D; Mbamalu, G; Reith, A D; Panayotou, G; End, P; Bernstein, A; Kazlauskas, A

    1992-01-01

    The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors. Images PMID:1372092

  4. Coexpression landscape in ATTED-II: usage of gene list and gene network for various types of pathways.

    PubMed

    Obayashi, Takeshi; Kinoshita, Kengo

    2010-05-01

    Gene coexpression analyses are a powerful method to predict the function of genes and/or to identify genes that are functionally related to query genes. The basic idea of gene coexpression analyses is that genes with similar functions should have similar expression patterns under many different conditions. This approach is now widely used by many experimental researchers, especially in the field of plant biology. In this review, we will summarize recent successful examples obtained by using our gene coexpression database, ATTED-II. Specifically, the examples will describe the identification of new genes, such as the subunits of a complex protein, the enzymes in a metabolic pathway and transporters. In addition, we will discuss the discovery of a new intercellular signaling factor and new regulatory relationships between transcription factors and their target genes. In ATTED-II, we provide two basic views of gene coexpression, a gene list view and a gene network view, which can be used as guide gene approach and narrow-down approach, respectively. In addition, we will discuss the coexpression effectiveness for various types of gene sets.

  5. Receptor dimerization is not a factor in the signalling activity of a transforming variant epidermal growth factor receptor (EGFRvIII).

    PubMed Central

    Chu, C T; Everiss, K D; Wikstrand, C J; Batra, S K; Kung, H J; Bigner, D D

    1997-01-01

    The type-III deletion variant of the epidermal growth factor receptor (EGFRvIII) is frequently found in glioblastomas and other malignant human tumours. Although EGFRvIII confers ligand-independent oncogenic transformation of cell lines, the mechanism by which it promotes aberrant cellular proliferation is unknown. Using cell lines expressing comparable numbers of either wild-type receptor (EGFRwt) or EGFRvIII, we compared several parameters of receptor activation: dimerization, tyrosine phosphorylation and activation of intracellular signalling proteins. Like activated EGFRwt, EGFRvIII was phosphorylated and bound constitutively to the Shc adapter protein. Indeed, EGFRvIII-associated Shc had a higher phosphotyrosine content than Shc associated with stimulated EGFRwt. EGFRwt dimerized in response to either EGF or transforming growth factor alpha. Higher cross-linker concentrations and incubation at higher temperatures (37 degrees C) allowed detection of EGFRwt dimers even in the absence of exogenous ligand. In contrast, EGFRvIII failed to dimerize under any conditions studied. Moreover, neither mitogen-activated protein kinase nor phospholipase Cgamma were phosphorylated in EGFRvIII-expressing cells. We conclude that the deletion of 267 amino acids from the 621-amino-acid N-terminal domain of EGFR does not result simply in a constitutively activated receptor, but alters the spectrum of signalling cascades utilized. Furthermore the ligand-independent transforming activity of EGFRvIII is independent of receptor dimerization. PMID:9210410

  6. Heregulin-Induced Growth Factor Receptor Signaling and Breast Carcinogenesis.

    DTIC Science & Technology

    1997-07-01

    heterodimers and receptor cross-phosphorylation [Goldman, et al., 1990; Wada, et al., 1990; Qian, et al., 1992; Spivak -Kroizman, et al., 1992]. The...were obtained from R&D Systems. Recombinant HB-EGF has been provided on a collaborative basis by Michael Klagsbrun (Children’s Hospital, Boston, MA...active peptides expressed in E. coli. We also established collaborations with Dr. Michael Klagsbrun (Children’s Hospital, Boston, MA), who have

  7. Heregulin-Induced Growth Factor Receptor Signaling and Breast Carcinogenesis.

    DTIC Science & Technology

    1996-07-01

    Qian, et al., 1992; Spivak - Kroizman, et al., 1992]. The physiological responses to agonists for erbB family receptors depends on their ability to...established collaborations with Dr. Michael Klagsbrun (Children’s Hospital, Boston, MA), who is supplying us with recombinant heparin-binding EGF-like...FGFR complex exists in a 1:1 stoichiometry [ Spivak -Kroizman, et al., 1994], yet FGFs are monomeric, it has been proposed that HSPG binding

  8. Coexpression systems as models for the analysis of constitutive GPCR activity.

    PubMed

    Schneider, Erich H; Seifert, Roland

    2010-01-01

    The investigation of constitutive activity of GPCRs in transfected mammalian cells is often hampered by the presence of other constitutively active receptors that generate a high background signal. This impairs the measurement of constitutive activity and of inverse agonistic effects, both of which often occur in a relatively small signal range. Moreover, constitutive activity of a GPCR depends on the interacting G-protein. Since the commonly used mammalian cells contain a set of several different G-protein types, it is very difficult to investigate the influence of specific Gα and Gβγ subunits on constitutive activity in more detail in these expression systems. Here, we show that the Sf9 cell/baculovirus expression system provides excellent conditions for the characterization of constitutively active GPCRs. Sf9 cells express a restricted set of G-protein subtypes that show only a limited capability of interacting with mammalian GPCRs. Moreover, the Sf9 cell/baculovirus expression system allows the combined expression of up to four different proteins encoded by the respective genetically modified baculoviruses. Using the highly constitutively active human histamine H₄R (hH₄R) as a paradigm, we demonstrate how the coexpression of hH₄R with different signaling proteins (Gα, Gβγ, and RGS-proteins) in combination with sensitive functional assays (high-affinity agonist binding and steady-state GTPase- and GTPγS-binding assays) allows in-depth studies of constitutive activity. The preparation of Sf9 cell membranes, coexpressing hH₄R and various additional proteins, is described in detail as well as the procedures of the different functional assays. Moreover, we show that coexpression of GPCRs with signal transduction components in Sf9 cells can also be applied to the characterization of other constitutively active receptors, for example, the formyl peptide receptor and β₂-adrenoceptor.

  9. Identification of PLXDC1 and PLXDC2 as the transmembrane receptors for the multifunctional factor PEDF

    PubMed Central

    Cheng, Guo; Zhong, Ming; Kawaguchi, Riki; Kassai, Miki; Al-Ubaidi, Muayyad; Deng, Jun; Ter-Stepanian, Mariam; Sun, Hui

    2014-01-01

    Pigment Epithelium Derived Factor (PEDF) is a secreted factor that has broad biological activities. It was first identified as a neurotrophic factor and later as the most potent natural antiangiogenic factor, a stem cell niche factor, and an inhibitor of cancer cell growth. Numerous animal models demonstrated its therapeutic value in treating blinding diseases and diverse cancer types. A long-standing challenge is to reveal how PEDF acts on its target cells and the identities of the cell-surface receptors responsible for its activities. Here we report the identification of transmembrane proteins PLXDC1 and PLXDC2 as cell-surface receptors for PEDF. Using distinct cellular models, we demonstrate their cell type-specific receptor activities through loss of function and gain of function studies. Our experiments suggest that PEDF receptors form homooligomers under basal conditions, and PEDF dissociates the homooligomer to activate the receptors. Mutations in the intracellular domain can have profound effects on receptor activities. DOI: http://dx.doi.org/10.7554/eLife.05401.001 PMID:25535841

  10. Identification of co-expression gene networks, regulatory genes and pathways for obesity based on adipose tissue RNA Sequencing in a porcine model.

    PubMed

    Kogelman, Lisette J A; Cirera, Susanna; Zhernakova, Daria V; Fredholm, Merete; Franke, Lude; Kadarmideen, Haja N

    2014-09-30

    Obesity is a complex metabolic condition in strong association with various diseases, like type 2 diabetes, resulting in major public health and economic implications. Obesity is the result of environmental and genetic factors and their interactions, including genome-wide genetic interactions. Identification of co-expressed and regulatory genes in RNA extracted from relevant tissues representing lean and obese individuals provides an entry point for the identification of genes and pathways of importance to the development of obesity. The pig, an omnivorous animal, is an excellent model for human obesity, offering the possibility to study in-depth organ-level transcriptomic regulations of obesity, unfeasible in humans. Our aim was to reveal adipose tissue co-expression networks, pathways and transcriptional regulations of obesity using RNA Sequencing based systems biology approaches in a porcine model. We selected 36 animals for RNA Sequencing from a previously created F2 pig population representing three extreme groups based on their predicted genetic risks for obesity. We applied Weighted Gene Co-expression Network Analysis (WGCNA) to detect clusters of highly co-expressed genes (modules). Additionally, regulator genes were detected using Lemon-Tree algorithms. WGCNA revealed five modules which were strongly correlated with at least one obesity-related phenotype (correlations ranging from -0.54 to 0.72, P < 0.001). Functional annotation identified pathways enlightening the association between obesity and other diseases, like osteoporosis (osteoclast differentiation, P = 1.4E-7), and immune-related complications (e.g. Natural killer cell mediated cytotoxity, P = 3.8E-5; B cell receptor signaling pathway, P = 7.2E-5). Lemon-Tree identified three potential regulator genes, using confident scores, for the WGCNA module which was associated with osteoclast differentiation: CCR1, MSR1 and SI1 (probability scores respectively 95.30, 62.28, and 34.58). Moreover, detection

  11. Identification of co-expression gene networks, regulatory genes and pathways for obesity based on adipose tissue RNA Sequencing in a porcine model

    PubMed Central

    2014-01-01

    Background Obesity is a complex metabolic condition in strong association with various diseases, like type 2 diabetes, resulting in major public health and economic implications. Obesity is the result of environmental and genetic factors and their interactions, including genome-wide genetic interactions. Identification of co-expressed and regulatory genes in RNA extracted from relevant tissues representing lean and obese individuals provides an entry point for the identification of genes and pathways of importance to the development of obesity. The pig, an omnivorous animal, is an excellent model for human obesity, offering the possibility to study in-depth organ-level transcriptomic regulations of obesity, unfeasible in humans. Our aim was to reveal adipose tissue co-expression networks, pathways and transcriptional regulations of obesity using RNA Sequencing based systems biology approaches in a porcine model. Methods We selected 36 animals for RNA Sequencing from a previously created F2 pig population representing three extreme groups based on their predicted genetic risks for obesity. We applied Weighted Gene Co-expression Network Analysis (WGCNA) to detect clusters of highly co-expressed genes (modules). Additionally, regulator genes were detected using Lemon-Tree algorithms. Results WGCNA revealed five modules which were strongly correlated with at least one obesity-related phenotype (correlations ranging from -0.54 to 0.72, P < 0.001). Functional annotation identified pathways enlightening the association between obesity and other diseases, like osteoporosis (osteoclast differentiation, P = 1.4E-7), and immune-related complications (e.g. Natural killer cell mediated cytotoxity, P = 3.8E-5; B cell receptor signaling pathway, P = 7.2E-5). Lemon-Tree identified three potential regulator genes, using confident scores, for the WGCNA module which was associated with osteoclast differentiation: CCR1, MSR1 and SI1 (probability scores respectively 95.30, 62.28, and

  12. Altered (/sup 125/I)epidermal growth factor binding and receptor distribution in psoriasis

    SciTech Connect

    Nanney, L.B.; Stoscheck, C.M.; Magid, M.; King, L.E. Jr.

    1986-03-01

    Stimulation of growth and differentiation of human epidermis by epidermal growth factor (EGF) is mediated by its binding to specific receptors. Whether EGF receptors primarily mediate cell division or differentiation in hyperproliferative disease such as psoriasis vulgaris is unclear. To study the pathogenesis of psoriasis, 4-mm2 punch biopsy specimens of normal, uninvolved, and involved psoriatic skin were assayed for EGF receptors by autoradiographic, immunohistochemical, and biochemical methods. Using autoradiographic and immunohistochemical methods, basal keratinocytes were found to contain the greatest number of EGF binding sites and immunoreactive receptors as compared to the upper layers of the epidermis in both normal epidermis and psoriatic skin. No EGF receptor differences between normal and psoriatic epidermis were observed in this layer. In the upper layers of the epidermis, a 2-fold increase in EGF binding capacity was observed in psoriatic skin as compared with normal thin or thick skin. Biochemical methods indicated that (/sup 125/I)EGF binding was increased in psoriatic epidermis as compared with similar thickness normal epidermis when measured on a protein basis. Epidermal growth factor was shown to increase phosphorylation of the EGF receptor in skin. EGF receptors retained in the nonmitotic stratum spinosum and parakeratotic stratum corneum may reflect the incomplete, abnormal differentiation that occurs in active psoriatic lesions. Alternatively, retained EGF receptors may play a direct role in inhibiting cellular differentiation in the suprabasal layers.

  13. Postnatal expression of nerve growth factor receptors in the rat testis.

    PubMed

    Djakiew, D; Pflug, B; Dionne, C; Onoda, M

    1994-08-01

    Because nerve growth factor beta (NGF beta) and its corresponding receptors have been implicated in the paracrine regulation of spermatogenesis, we examined the postnatal developmental expression of the low- and high-affinity NGF receptors in the rat testis, and localized their expression to specific testicular cell types. The neurotropin receptors consist of a low-affinity p75 nerve growth factor receptor (LNGFR) and a family of high-affinity tyrosine receptor kinases (trk). Both the p75 LNGFR gene product and the trk receptor gene product were detected in immature rat testes, with maximal expression in 10- and 20-day-old rats. Expression of the testicular p75 LNGFR and the trk receptor progressively declined in older animals so that they were barely detectable in 90-day-old adult rats. The 75-kDa LNGFR was detected in membrane fractions of Sertoli cells, whereas the p75 LNGFR was not detected by Western blot in membrane fractions of round spermatids and primary spermatocytes. Interestingly, microsomal fractions of peritubular myoid cells were immunoreactive for a 65-kDa band on Western blots with the p75 LNGFR monoclonal antibody. Immunoblot analysis of the trk receptor in cell lysates of isolated cell types was inconclusive. Excess NGF beta and round spermatid protein, which is known to contain a NGF-like protein, were both capable of displacing the binding of 125I-NGF beta from the surface of Sertoli cells.(ABSTRACT TRUNCATED AT 250 WORDS)

  14. Roles of tumor necrosis factor receptor associated factor 3 (TRAF3) and TRAF5 in immune cell functions

    PubMed Central

    Hildebrand, Joanne M.; Yi, Zuoan; Buchta, Claire M.; Poovassery, Jayakumar; Stunz, Laura L.; Bishop, Gail A.

    2011-01-01

    Summary A large and diverse group of receptors utilizes the family of cytoplasmic signaling proteins known as tumor necrosis factor receptor (TNFR)-associated factors (TRAFs). In recent years, there has been a resurgence of interest and exploration of the roles played by TRAF3 and TRAF5 in cellular regulation, particularly in cells of the immune system, the cell types of focus in this review. This work has revealed that TRAF3 and TRAF5 can play diverse roles for different receptors even in the same cell type, as well as distinct roles in different cell types. Evidence indicates that TRAF3 and TRAF5 play important roles beyond the TNFR-superfamily (SF) and viral mimics of its members, mediating certain innate immune receptor and cytokine receptor signals, and most recently, signals delivered by the T-cell receptor (TCR) signaling complex. Additionally, much research has demonstrated the importance of TRAF3-mediated cellular regulation via its cytoplasmic interactions with additional signaling proteins. In particular, we discuss below evidence for the participation by TRAF3 in a number of the regulatory post-translational modifications involving ubiquitin that are important in various signaling pathways. PMID:22017431

  15. Platelet-derived growth factor mimics phorbol diester action on epidermal growth factor receptor phosphorylation at threonine-654

    SciTech Connect

    Davis, R.J.; Czech, M.P.

    1985-06-01

    Addition of platelet-derived growth factor (PDGF) to quiescent WI-38 human fetal lung fibroblasts mimics the effect of tumor-promoting phorbol diesters to inhibit the high-affinity binding of SVI-labeled epidermal growth factor ( SVI-EGF). PDGF, like phorbol diesters, was found to increase the phosphorylation state of EGF receptors immunoprecipitated from intact fibroblasts that were labeled to equilibrium with (TSP)phosphate. Phosphoamino acid analysis of the EGF receptors indicated that both PDGF and phorbol diesters increased the level of (TSP)phosphoserine and (TSP)phosphothreonine. Phosphopeptide mapping of the EGF receptor demonstrated that PDGF increased the phosphorylation of several sites and induced the phosphorylation of a site that was not observed to be phosphorylated on EGF receptors isolated from control cells. This latter phosphorylation site on the EGF receptor was identified as threonine-654. These results are consistent with the hypothesis that increases in diacylglycerol and CaS levels caused by addition of PDGF to fibroblasts activate protein kinase C and that this kinase, at least in part, mediates the effect of PDGF on the phosphorylation of the EGF receptor. The data further suggest that protein kinase C may play an important role in the regulation of cellular metabolism and proliferation by PDGF.

  16. Multiscale Embedded Gene Co-expression Network Analysis

    PubMed Central

    Song, Won-Min; Zhang, Bin

    2015-01-01

    Gene co-expression network analysis has been shown effective in identifying functional co-expressed gene modules associated with complex human diseases. However, existing techniques to construct co-expression networks require some critical prior information such as predefined number of clusters, numerical thresholds for defining co-expression/interaction, or do not naturally reproduce the hallmarks of complex systems such as the scale-free degree distribution of small-worldness. Previously, a graph filtering technique called Planar Maximally Filtered Graph (PMFG) has been applied to many real-world data sets such as financial stock prices and gene expression to extract meaningful and relevant interactions. However, PMFG is not suitable for large-scale genomic data due to several drawbacks, such as the high computation complexity O(|V|3), the presence of false-positives due to the maximal planarity constraint, and the inadequacy of the clustering framework. Here, we developed a new co-expression network analysis framework called Multiscale Embedded Gene Co-expression Network Analysis (MEGENA) by: i) introducing quality control of co-expression similarities, ii) parallelizing embedded network construction, and iii) developing a novel clustering technique to identify multi-scale clustering structures in Planar Filtered Networks (PFNs). We applied MEGENA to a series of simulated data and the gene expression data in breast carcinoma and lung adenocarcinoma from The Cancer Genome Atlas (TCGA). MEGENA showed improved performance over well-established clustering methods and co-expression network construction approaches. MEGENA revealed not only meaningful multi-scale organizations of co-expressed gene clusters but also novel targets in breast carcinoma and lung adenocarcinoma. PMID:26618778

  17. Transforming Growth Factor-B Receptors in Human Breast Cancer.

    DTIC Science & Technology

    1998-05-01

    G. S ., Calvert, R. J., and Weghorst, C . M. ’Cold SSCP’: a simple, rapid and non-radioactive method for optimized single-strand conformation...of TGF-beta receptors, J Biol Chem. 271: 13123-9, 1996. 13. Kalkhoven, E., Kwakkenbos-Isbrucker, L., Mummery, C . L., deLaat, S . W., van den...branching morphogenesis in culture, Dev Biol. 180: 242-57, 1996. 23. Fan, D., Chakrabarty, S ., Seid, C , Bell, C . W., Schackert, H., Morikawa, K

  18. Tannic acid, a potent inhibitor of epidermal growth factor receptor tyrosine kinase.

    PubMed

    Yang, Er Bin; Wei, Liu; Zhang, Kai; Chen, Yu Zong; Chen, Wei Ning

    2006-03-01

    Increasing evidence supports the hypothesis that tannic acid, a plant polyphenol, exerts anticarcinogenic activity in chemically induced cancers. In the present study, tannic acid was found to strongly inhibit tyrosine kinase activity of epidermal growth factor receptor (EGFr) in vitro (IC50 = 323 nM). In contrast, the inhibition by tannic acid of p60(c-src) tyrosine kinase (IC50 = 14 microM) and insulin receptor tyrosine kinase (IC50 = 5 microM) was much weaker. The inhibition of EGFr tyrosine kinase by tannic acid was competitive with respect to ATP and non-competitive with respect to peptide substrate. In cultured cells, growth factor-induced tyrosine phosphorylation of growth factor receptors, including EGFr, platelet-derived growth factor receptor, and basic fibroblast growth factor receptor, was inhibited by tannic acid. No inhibition of insulin-induced tyrosine phosphorylation of insulin receptor and insulin-receptor substrate-1 was observed. EGF-stimulated growth of HepG2 cells was inhibited in the presence of tannic acid. The inhibition of serine/threonine-specific protein kinases, including cAMP-dependent protein kinase, protein kinase C and mitogen-activated protein kinase, by tannic acid was only detected at relatively high concentration, IC50 being 3, 325 and 142 microM respectively. The molecular modeling study suggested that tannic acid could be docked into the ATP binding pockets of either EGFr or insulin receptor. These results demonstrate that tannic acid is an in vitro potent inhibitor of EGFr tyrosine kinase.

  19. Regulatory Roles of the Tumor Necrosis Factor Receptor BCMA

    PubMed Central

    Coquery, Christine M.; Erickson, Loren D.

    2012-01-01

    B cell maturation antigen (BCMA) is a tumor necrosis family receptor (TNFR) member that is predominantly expressed on terminally differentiated B cells and, upon binding to its ligands B cell activator of the TNF family (BAFF) and a proliferation inducing ligand (APRIL), delivers pro-survival cell signals. Thus, BCMA is most known for its functional activity in mediating the survival of plasma cells that maintain long-term humoral immunity. The expression of BCMA has been also linked to a number of cancers, autoimmune disorders, and infectious diseases that suggest additional roles for BCMA activity. Despite the recent advances in our understanding of the roles for the related TNFR members BAFF-R and transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), the signaling pathway used by BCMA for mediating plasma cell survival as well as its putative function in certain disease states are not well understood. By examining the expression, regulation, and signaling targets of BCMA we may gain further insight into this receptor and how it operates within cells in both health and disease. This information is important for identifying new therapeutic targets that may be relevant in treating diseases that involve the BAFF/APRIL cytokine network. PMID:23237506

  20. Protein-protein interaction and gene co-expression maps of ARFs and Aux/IAAs in Arabidopsis

    PubMed Central

    Piya, Sarbottam; Shrestha, Sandesh K.; Binder, Brad; Stewart, C. Neal; Hewezi, Tarek

    2014-01-01

    The phytohormone auxin regulates nearly all aspects of plant growth and development. Based on the current model in Arabidopsis thaliana, Auxin/indole-3-acetic acid (Aux/IAA) proteins repress auxin-inducible genes by inhibiting auxin response transcription factors (ARFs). Experimental evidence suggests that heterodimerization between Aux/IAA and ARF proteins are related to their unique biological functions. The objective of this study was to generate the Aux/IAA-ARF protein-protein interaction map using full length sequences and locate the interacting protein pairs to specific gene co-expression networks in order to define tissue-specific responses of the Aux/IAA-ARF interactome. Pairwise interactions between 19 ARFs and 29 Aux/IAAs resulted in the identification of 213 specific interactions of which 79 interactions were previously unknown. The incorporation of co-expression profiles with protein-protein interaction data revealed a strong correlation of gene co-expression for 70% of the ARF-Aux/IAA interacting pairs in at least one tissue/organ, indicative of the biological significance of these interactions. Importantly, ARF4-8 and 19, which were found to interact with almost all Aux-Aux/IAA showed broad co-expression relationships with Aux/IAA genes, thus, formed the central hubs of the co-expression network. Our analyses provide new insights into the biological significance of ARF-Aux/IAA associations in the morphogenesis and development of various plant tissues and organs. PMID:25566309

  1. Evolutionary conserved gene co-expression drives generation of self-antigen diversity in medullary thymic epithelial cells.

    PubMed

    Rattay, Kristin; Meyer, Hannah Verena; Herrmann, Carl; Brors, Benedikt; Kyewski, Bruno

    2016-02-01

    Promiscuous expression of a plethora of tissue-restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs) is essential for central tolerance. This promiscuous gene expression (pGE) is characterized by inclusion of a broad range of TRAs and by its mosaic expression patterns, i.e. each antigen is only expressed in 1-3% of mTECs. It is currently unclear to which extent random and/or deterministic mechanisms are involved in the regulation of pGE. In order to address this issue, we deconstructed the transcriptional heterogeneity in mTEC to minor subsets expressing a particular TRA. We identified six delineable co-expression groups in mouse mTECs. These co-expression groups displayed a variable degree of mutual overlap and mapped to different stages of mTEC development. Co-expressed genes showed chromosomal preference and clustered within delimited genomic regions. Moreover, co-expression groups in mice and humans selected by a pair of orthologous genes preferentially co-expressed sets of orthologous genes attesting to the species conservation of pGE between mouse and human. Furthermore, co-expressed genes were enriched for specific transcription factor binding motifs concomitant with up-regulation of the corresponding transcription factors, implicating additional factors in the regulation of pGE besides the Autoimmune Regulator (Aire). Thus promiscuous transcription of self-antigens in mTECs entails a highly coordinated process, which is evolutionary strictly conserved between species. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Neurosteroid dehydroepiandrosterone interacts with nerve growth factor (NGF) receptors, preventing neuronal apoptosis.

    PubMed

    Lazaridis, Iakovos; Charalampopoulos, Ioannis; Alexaki, Vassilia-Ismini; Avlonitis, Nicolaos; Pediaditakis, Iosif; Efstathopoulos, Paschalis; Calogeropoulou, Theodora; Castanas, Elias; Gravanis, Achille

    2011-04-01

    The neurosteroid dehydroepiandrosterone (DHEA), produced by neurons and glia, affects multiple processes in the brain, including neuronal survival and neurogenesis during development and in aging. We provide evidence that DHEA interacts with pro-survival TrkA and pro-death p75(NTR) membrane receptors of neurotrophin nerve growth factor (NGF), acting as a neurotrophic factor: (1) the anti-apoptotic effects of DHEA were reversed by siRNA against TrkA or by a specific TrkA inhibitor; (2) [(3)H]-DHEA binding assays showed that it bound to membranes isolated from HEK293 cells transfected with the cDNAs of TrkA and p75(NTR) receptors (K(D): 7.4 ± 1.75 nM and 5.6 ± 0.55 nM, respectively); (3) immobilized DHEA pulled down recombinant and naturally expressed TrkA and p75(NTR) receptors; (4) DHEA induced TrkA phosphorylation and NGF receptor-mediated signaling; Shc, Akt, and ERK1/2 kinases down-stream to TrkA receptors and TRAF6, RIP2, and RhoGDI interactors of p75(NTR) receptors; and (5) DHEA rescued from apoptosis TrkA receptor positive sensory neurons of dorsal root ganglia in NGF null embryos and compensated NGF in rescuing from apoptosis NGF receptor positive sympathetic neurons of embryonic superior cervical ganglia. Phylogenetic findings on the evolution of neurotrophins, their receptors, and CYP17, the enzyme responsible for DHEA biosynthesis, combined with our data support the hypothesis that DHEA served as a phylogenetically ancient neurotrophic factor.

  3. Neurosteroid Dehydroepiandrosterone Interacts with Nerve Growth Factor (NGF) Receptors, Preventing Neuronal Apoptosis

    PubMed Central

    Alexaki, Vassilia-Ismini; Avlonitis, Nicolaos; Pediaditakis, Iosif; Efstathopoulos, Paschalis; Calogeropoulou, Theodora; Castanas, Elias; Gravanis, Achille

    2011-01-01

    The neurosteroid dehydroepiandrosterone (DHEA), produced by neurons and glia, affects multiple processes in the brain, including neuronal survival and neurogenesis during development and in aging. We provide evidence that DHEA interacts with pro-survival TrkA and pro-death p75NTR membrane receptors of neurotrophin nerve growth factor (NGF), acting as a neurotrophic factor: (1) the anti-apoptotic effects of DHEA were reversed by siRNA against TrkA or by a specific TrkA inhibitor; (2) [3H]-DHEA binding assays showed that it bound to membranes isolated from HEK293 cells transfected with the cDNAs of TrkA and p75NTR receptors (KD: 7.4±1.75 nM and 5.6±0.55 nM, respectively); (3) immobilized DHEA pulled down recombinant and naturally expressed TrkA and p75NTR receptors; (4) DHEA induced TrkA phosphorylation and NGF receptor-mediated signaling; Shc, Akt, and ERK1/2 kinases down-stream to TrkA receptors and TRAF6, RIP2, and RhoGDI interactors of p75NTR receptors; and (5) DHEA rescued from apoptosis TrkA receptor positive sensory neurons of dorsal root ganglia in NGF null embryos and compensated NGF in rescuing from apoptosis NGF receptor positive sympathetic neurons of embryonic superior cervical ganglia. Phylogenetic findings on the evolution of neurotrophins, their receptors, and CYP17, the enzyme responsible for DHEA biosynthesis, combined with our data support the hypothesis that DHEA served as a phylogenetically ancient neurotrophic factor. PMID:21541365

  4. A Critical Role of the p75 Tumor Necrosis Factor Receptor (p75TNF-R) in Organ Inflammation Independent of  TNF, Lymphotoxin α, or the p55TNF-R

    PubMed Central

    Douni, Eleni; Kollias, George

    1998-01-01

    Despite overwhelming evidence that enhanced production of the p75 tumor necrosis factor receptor (p75TNF-R) accompanies development of specific human inflammatory pathologies such as multi-organ failure during sepsis, inflammatory liver disease, pancreatitis, respiratory distress syndrome, or AIDS, the function of this receptor remains poorly defined in vivo. We show here that at levels relevant to human disease, production of the human p75TNF-R in transgenic mice results in a severe inflammatory syndrome involving mainly the pancreas, liver, kidney, and lung, and characterized by constitutively increased NF-κB activity in the peripheral blood mononuclear cell compartment. This process is shown to evolve independently of the presence of TNF, lymphotoxin α, or the p55TNF-R, although coexpression of a human TNF transgene accelerated pathology. These results establish an independent role for enhanced p75TNF-R production in the pathogenesis of inflammatory disease and implicate the direct involvement of this receptor in a wide range of human inflammatory pathologies. PMID:9763613

  5. Identification of two new hydrophobic residues on basic fibroblast growth factor important for fibroblast growth factor receptor binding.

    PubMed

    Zhu, H; Ramnarayan, K; Menzel, P; Miao, Y; Zheng, J; Mong, S

    1998-10-01

    Basic fibroblast growth factor (bFGF) is implicated in the pathogenesis of several types of vascular and connective diseases. A key step in the discovery of bFGF receptor antagonists to mitigate these actions is to define the functional epitopes required for receptor binding of the growth factor. Using structure-based site-directed mutagenesis, two critical areas on the bFGF surface for the high affinity receptor binding have already been identified [Springer, B.A., Pantoliano, M.W., Barberal, F.A., Gunyuzlu, P.L., Thompson, L.D., Herblin, W.F., Rosenfeld, S.A. and Book, G.W. (1994) J. Biol. Chem., 269, 26879-26884; Zhu, H.Y., Ramnarayan, K., Anchin, J., Miao, Y., Sereno, A., Millman, L., Zheng, J., Balaji, V.N. and Wolff, M.E. (1995) J. Biol. Chem., 270, 21869-21874; Zhu, H.Y., Anchin, J., Ramnarayan, K., Zheng, J., Kawai, T., Mong, S. and Wolff, M.E. (1997) Protein Engng, 10, 417-421]. According to these studies, one receptor binding site includes two polar residues Glu96 and Asn104 on bFGF whereas the other includes four hydrophobic residues Tyr24, Tyr103, Leu140 and Met142. Using a protein modelling technique, we report here the identification of a new hydrophobic patch on bFGF which includes residues Tyr73, Val88 and Phe93. The role of this area on receptor binding affinity was evaluated by mutating each of these residues individually and determining the mutated protein's (mutein's) receptor binding affinity. In addition, we examined the role of two other hydrophobic residues, Phe30 and Leu138, on bFGF for high-affinity receptor binding. These two residues are the neighbors of the hydrophobic residues Tyr24 and Tyr103, respectively. Replacement of Val88 and Phe93 with alanine reduced the receptor binding affinity about 10- and 80-fold, respectively, compared with wild-type bFGF. In contrast, substitution of Phe30 and Leu138 with alanine has no effect on the receptor binding affinities. We conclude that the newly identified hydrophobic residues, Val88 and Phe93

  6. Platelet-activating factor receptor contributes to antileishmanial function of miltefosine.

    PubMed

    Gangalum, Pallavi R; de Castro, Waldionê; Vieira, Leda Q; Dey, Ranadhir; Rivas, Luis; Singh, Shailza; Majumdar, Subrata; Saha, Bhaskar

    2015-06-15

    Miltefosine [hexadecylphosphocholine (HPC)] is the only orally bioavailable drug for the disease visceral leishmaniasis, which is caused by the protozoan parasite Leishmania donovani. Although miltefosine has direct leishmanicidal effects, evidence is mounting for its immune system-dependent effects. The mechanism of such indirect antileishmanial effects of miltefosine remains to be discovered. As platelet-activating factor and HPC share structural semblances and both induce killing of intracellular Leishmania, we surmised that platelet-activating factor (PAF) receptor had a significant role in the antileishmanial function of miltefosine. The proposition was supported by molecular dynamic simulation of HPC docking into PAF receptor and by comparison of its leishmanicidal function on PAF receptor-deficient macrophages and mice under HPC treatment. We observed that compared with wild-type macrophages, the PAF receptor-deficient macrophages showed 1) reduced binding of a fluorescent analog of HPC, 2) decreased TNF-α production, and 3) lower miltefosine-induced killing of L. donovani. Miltefosine exhibited significantly compromised leishmanicidal function in PAF receptor-deficient mice. An anti-PAF receptor Ab led to a significant decrease in miltefosine-induced intracellular Leishmania killing and IFN-γ production in a macrophage-T cell coculture system. These results indicate significant roles for PAF receptor in the leishmanicidal activity of HPC. The findings open new avenues for a more rational understanding of the mechanism of action of this drug as well as for improved therapeutic strategies. Copyright © 2015 by The American Association of Immunologists, Inc.

  7. Involvement of the TP receptor in TNF-α-induced endothelial tissue factor expression.

    PubMed

    Del Turco, Serena; Basta, Giuseppina; Lazzerini, Guido; Chancharme, Laurent; Lerond, Laurence; De Caterina, Raffaele

    2014-08-01

    Thromboxane (TX) A2, prostaglandin endoperoxides and F2-isoprostanes exert their effects through a TX-prostanoid (TP) receptor, also expressed in endothelial cells. We investigated a role of the TP receptor in the endothelial expression of tissue factor (TF), a key trigger to thrombosis. Human umbilical vein endothelial cells (HUVEC) exposed to the TP receptor agonist U46619 featured a concentration-dependent increase in TF surface exposure and procoagulant activity. HUVEC pre-incubation with the TP receptor antagonist S18886, followed by stimulation with either U46619 or tumor necrosis factor-α (TNF-α), attenuated TF surface exposure and activity compared with stimulated control. Aspirin or indomethacin, while inhibiting cyclooxygenase (COX)-1 and -2 activities, did not mimic this effect. Probing of underlying mechanisms by selective pharmacological and gene silencing experiments showed that S18886 reduced U46619- or TNF-α-induced TF expression inhibiting ROS production, NAD(P)H oxidase and PKC activation. In addition, S18886 also inhibited ERK activation in the presence of both U46619 and TNF-α alone, while inhibition of JNK activation only occurred in the presence of U46619. The endothelial TP receptor contributes to TF surface exposure and activity induced not only by known TP receptor agonists, but also by TNF-α. Such findings expand the therapeutic potential of TP receptor inhibition. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Monkey corticotropin-releasing factor1 receptor: Complementary DNA cloning and pharmacological characterization.

    PubMed

    Oshida, Yuichi; Ikeda, Yoko; Chaki, Shigeyuki; Okuyama, Shigeru

    2004-02-27

    The full-length complementary DNA (cDNA) of monkey corticotropin-releasing factor type 1 (CRF1) receptor was isolated from a rhesus monkey (Macaca mulatta) amygdala cDNA library. The cloned monkey CRF1 receptor cDNA has 2,374 bp with an open reading frame encoding a 415-amino acid protein. The sequence of the monkey CRF1 receptor cDNA showed a high degree of sequence identity with other species of CRF1 receptors, and being 99.5% identical to human CRF1 receptors. When monkey CRF1 was expressed into COS-7 cells, high specific binding of [125I]-ovine CRF was observed. CRF and CRF-related peptides inhibited [125I]-ovine CRF binding in a concentration-dependent manner. IC50 values of ovine CRF, human/rat CRF, sauvagine and urotensin I were 23.5 +/- 7.4, 22.7 +/- 10.8, 27.5 +/- 12.3 and 14.2 +/- 7.0 nM, respectively. CRF1 receptor specific antagonists, such as CP-154,526, SC241 and CRA1000, also inhibited the [125I]-ovine CRF binding, with IC50 values of 3.9 +/- 0.4, 43.5 +/- 8.0 and 19.8 +/- 2.0 nM, respectively. GTP and its nonhydrolyzed analogue, GTPgammaS, reduced [125I]-ovine CRF binding, while ATP had a negligible effect, thereby indicating that the monkey CRF1 receptor belongs to a family of G-protein coupled receptors. CRF and its related peptides increased cyclic AMP formation concentration-dependently in COS-7 cells transiently expressing the monkey CRF1 receptor. Monkey CRF1 was expressed abundantly in the pituitary, cerebral cortex, hippocampus, amygdala and cerebellum. Thus the monkey CRF1 receptor and the human CRF1 receptor have similar molecular and pharmacological characteristics.

  9. Ligand-dependent EGFR activation induces the co-expression of IL-6 and PAI-1 via the NFkB pathway in advanced-stage epithelial ovarian cancer.

    PubMed

    Alberti, C; Pinciroli, P; Valeri, B; Ferri, R; Ditto, A; Umezawa, K; Sensi, M; Canevari, S; Tomassetti, A

    2012-09-13

    The epidermal growth factor receptor (EGFR), a member of the ErbB family of receptor tyrosine kinases, is expressed in up to 70% of epithelial ovarian cancers (EOCs), where it correlates with poor prognosis. The majority of EOCs are diagnosed at an advanced stage, and at least 50% present malignant ascites. High levels of IL-6 have been found in the ascites of EOC patients and correlate with shorter survival. Herein, we investigated the signaling cascade led by EGFR activation in EOC and assessed whether EGFR activation could induce an EOC microenvironment characterized by pro-inflammatory molecules. In vitro analysis of EOC cell lines revealed that ligand-stimulated EGFR activated NFkB-dependent transcription and induced secretion of IL-6 and plasminogen activator inhibitor (PAI-1). IL-6/PAI-1 expression and secretion were strongly inhibited by the tyrosine kinase inhibitor AG1478 and EGFR silencing. A significant reduction of EGF-stimulated IL-6/PAI-1 secretion was also obtained with the NFkB inhibitor dehydroxymethylepoxyquinomicin. Of 23 primary EOC tumors from advanced-stage patients with malignant ascites at surgery, 12 co-expressed membrane EGFR, IL-6 and PAI-1 by immunohistochemistry; both IL-6 and PAI-1 were present in 83% of the corresponding ascites. Analysis of a publicly available gene-expression data set from 204 EOCs confirmed a significant correlation between IL-6 and PAI-1 expression, and patients with the highest IL-6 and PAI-1 co-expression showed a significantly shorter progression-free survival time (P=0.028). This suggests that EGFR/NFkB/IL-6-PAI-1 may have a significant impact on the therapy of a particular subset of EOC, and that IL-6/PAI-1 co-expression may be a novel prognostic marker.

  10. Differential trafficking of transforming growth factor-beta receptors and ligand in polarized epithelial cells.

    PubMed

    Murphy, S J; Doré, J J E; Edens, M; Coffey, R J; Barnard, J A; Mitchell, H; Wilkes, M; Leof, E B

    2004-06-01

    Epithelial cells in vivo form tight cell-cell associations that spatially separate distinct apical and basolateral domains. These domains provide discrete cellular processes essential for proper tissue and organ development. Using confocal imaging and selective plasma membrane domain activation, the type I and type II transforming growth factor-beta (TGFbeta) receptors were found to be localized specifically at the basolateral surfaces of polarized Madin-Darby canine kidney (MDCK) cells. Receptors concentrated predominantly at the lateral sites of cell-cell contact, adjacent to the gap junctional complex. Cytoplasmic domain truncations for each receptor resulted in the loss of specific lateral domain targeting and dispersion to both the apical and basal domains. Whereas receptors concentrate basolaterally in regions of direct cell-cell contact in nonpolarized MDCK cell monolayers, receptor staining was absent from areas of noncell contact. In contrast to the defined basolateral polarity observed for the TGFbeta receptor complex, TGFbeta ligand secretion was found to be from the apical surfaces. Confocal imaging of MDCK cells with an antibody to TGFbeta1 confirmed a predominant apical localization, with a stark absence at the basal membrane. These findings indicate that cell adhesion regulates the localization of TGFbeta receptors in polarized epithelial cultures and that the response to TGFbeta is dependent upon the spatial distribution and secretion of TGFbeta receptors and ligand, respectively.

  11. Epidermal growth factor binding and receptor distribution in the mouse reproductive tract during development

    SciTech Connect

    Bossert, N.L.; Nelson, K.G.; Ross, K.A.; Takahashi, T.; McLachlan, J.A. )

    1990-11-01

    The ontogeny of the epidermal growth factor (EGF) receptor in the different cell types in the neonatal and immature mouse uterus and vagina was examined. Immunohistochemical examination of prenatal and neonatal reproductive tracts with a polyclonal antibody to the EGF receptor shows immunoreactive EGF receptors as early as Day 13 of gestation. Autoradiographic analysis of tissue sections at 3 to 17 days of age (the day of birth is Day 1) demonstrates that both uterine and vaginal epithelial and stromal cells are capable of binding 125I-labeled EGF. Both the 125I-labeled EGF autoradiography and immunohistochemistry in whole tissue show higher EGF receptor levels in the uterine epithelium than the uterine stroma. The presence of EGF receptors was also confirmed by affinity labeling and Scatchard analysis of isolated uterine cell types at 7 and/or 17 days of age. However, in contrast to the autoradiography and immunohistochemistry data of intact tissue, the affinity labeling and Scatchard data of isolated cells indicate that the uterine stroma contains higher levels of EGF receptor than that of the uterine epithelium. The reason for this discrepancy between the different techniques is, as yet, unknown. Regardless of the differences in the actual numbers of EGF receptors obtained, our data demonstrate that the developing mouse reproductive tract contains immunoreactive EGF receptors that are capable of binding 125I-labeled EGF.

  12. Tumor necrosis factor inhibits ligand-stimulated EGF receptor activation through a TNF receptor 1-dependent mechanism

    PubMed Central

    McElroy, Steven J.; Frey, Mark R.; Yan, Fang; Edelblum, Karen L.; Goettel, Jeremy A.; John, Sutha; Polk, D. Brent

    2008-01-01

    Tumor necrosis factor (TNF) and epidermal growth factor (EGF) are key regulators in the intricate balance maintaining intestinal homeostasis. Previous work from our laboratory shows that TNF attenuates ligand-driven EGF receptor (EGFR) phosphorylation in intestinal epithelial cells. To identify the mechanisms underlying this effect, we examined EGFR phosphorylation in cells lacking individual TNF receptors. TNF attenuated EGF-stimulated EGFR phosphorylation in wild-type and TNFR2−/−, but not TNFR1−/−, mouse colon epithelial (MCE) cells. Reexpression of wild-type TNFR1 in TNFR1−/− MCE cells rescued TNF-induced EGFR inhibition, but expression of TNFR1 deletion mutant constructs lacking the death domain (DD) of TNFR1 did not, implicating this domain in EGFR downregulation. Blockade of p38 MAPK, but not MEK, activation of ERK rescued EGF-stimulated phosphorylation in the presence of TNF, consistent with the ability of TNFR1 to stimulate p38 phosphorylation. TNF promoted p38-dependent EGFR internalization in MCE cells, suggesting that desensitization is achieved by reducing receptor accessible to ligand. Taken together, these data indicate that TNF activates TNFR1 by DD- and p38-dependent mechanisms to promote EGFR internalization, with potential impact on EGF-induced proliferation and migration key processes that promote healing in inflammatory intestinal diseases. PMID:18467504

  13. Inter-Tissue Gene Co-Expression Networks between Metabolically Healthy and Unhealthy Obese Individuals.

    PubMed

    Kogelman, Lisette J A; Fu, Jingyuan; Franke, Lude; Greve, Jan Willem; Hofker, Marten; Rensen, Sander S; Kadarmideen, Haja N

    2016-01-01

    Obesity is associated with severe co-morbidities such as type 2 diabetes and nonalcoholic steatohepatitis. However, studies have shown that 10-25 percent of the severely obese individuals are metabolically healthy. To date, the identification of genetic factors underlying the metabolically healthy obese (MHO) state is limited. Systems genetics approaches have led to the identification of genes and pathways in complex diseases. Here, we have used such approaches across tissues to detect genes and pathways involved in obesity-induced disease development. Expression data of 60 severely obese individuals was accessible, of which 28 individuals were MHO and 32 were metabolically unhealthy obese (MUO). A whole genome expression profile of four tissues was available: liver, muscle, subcutaneous adipose tissue and visceral adipose tissue. Using insulin-related genes, we used the weighted gene co-expression network analysis (WGCNA) method to build within- and inter-tissue gene networks. We identified genes that were differentially connected between MHO and MUO individuals, which were further investigated by homing in on the modules they were active in. To identify potentially causal genes, we integrated genomic and transcriptomic data using an eQTL mapping approach. Both IL-6 and IL1B were identified as highly differentially co-expressed genes across tissues between MHO and MUO individuals, showing their potential role in obesity-induced disease development. WGCNA showed that those genes were clustering together within tissues, and further analysis showed different co-expression patterns between MHO and MUO subnetworks. A potential causal role for metabolic differences under similar obesity state was detected for PTPRE, IL-6R and SLC6A5. We used a novel integrative approach by integration of co-expression networks across tissues to elucidate genetic factors related to obesity-induced metabolic disease development. The identified genes and their interactions give more

  14. [Co-expression of beta-subunit with other subunits of Qbeta replicase].

    PubMed

    Wang, Dong

    2004-12-01

    In researches involving in vitro protein synthesis and self-replication system, Qbeta replicase is one of the key enzymes, which are demanded for the high availability. Qbeta replicase is a RNA-dependent RNA polymerase of Qbeta coliphage. It consists of four subunits (alpha, beta, gamma, and delta subunit), where the beta-subunit is encoded by the viral genome, while the other three subunits are host proteins normally involved in protein synthesis, namely, ribosomal protein S1 (alpha), elongation factors EF-Tu (gamma) and EF-Ts (delta). To increase the production of the Qbeta replicase holoenzyme, several types of expression vectors, including pKK, pET and others, were employed to produce Qbeta replicase. However, the beta-subunit was almost in the precipitate fraction. Considering that the four subunits of Qbeta replicase holoenzyme are in equivalent molar ratio and the amount of the subunits, ribosomal S1 and EF-Ts, being produced by the host cells is relatively low, co-expression of beta-subunit with the other three subunits was performed to know whether the availability of the host subunits is the contributing factor for the solubility of the Qbeta replicase. pBAD33-rep was constructed by cloning the beta-subunit gene into pBAD 33, a pACYC derivative, and pET21a(+) was employed as expression vector for the three other subunits. Among the different combinations of co-expression experiments, solubility was found to slightly increase by SDS-PAGE analysis when the beta-subunit was co-expressed with EF-Tu-Ts. And the replicase activity assay showed this soluble enzyme is in active form. The expression of beta-subunit was enhanced by decreasing the level of inducer IPTG in co-expression, and more soluble enzyme were obtained.

  15. Inter-Tissue Gene Co-Expression Networks between Metabolically Healthy and Unhealthy Obese Individuals

    PubMed Central

    Kogelman, Lisette J. A.; Fu, Jingyuan; Franke, Lude; Greve, Jan Willem; Hofker, Marten; Rensen, Sander S.; Kadarmideen, Haja N.

    2016-01-01

    Background Obesity is associated with severe co-morbidities such as type 2 diabetes and nonalcoholic steatohepatitis. However, studies have shown that 10–25 percent of the severely obese individuals are metabolically healthy. To date, the identification of genetic factors underlying the metabolically healthy obese (MHO) state is limited. Systems genetics approaches have led to the identification of genes and pathways in complex diseases. Here, we have used such approaches across tissues to detect genes and pathways involved in obesity-induced disease development. Methods Expression data of 60 severely obese individuals was accessible, of which 28 individuals were MHO and 32 were metabolically unhealthy obese (MUO). A whole genome expression profile of four tissues was available: liver, muscle, subcutaneous adipose tissue and visceral adipose tissue. Using insulin-related genes, we used the weighted gene co-expression network analysis (WGCNA) method to build within- and inter-tissue gene networks. We identified genes that were differentially connected between MHO and MUO individuals, which were further investigated by homing in on the modules they were active in. To identify potentially causal genes, we integrated genomic and transcriptomic data using an eQTL mapping approach. Results Both IL-6 and IL1B were identified as highly differentially co-expressed genes across tissues between MHO and MUO individuals, showing their potential role in obesity-induced disease development. WGCNA showed that those genes were clustering together within tissues, and further analysis showed different co-expression patterns between MHO and MUO subnetworks. A potential causal role for metabolic differences under similar obesity state was detected for PTPRE, IL-6R and SLC6A5. Conclusions We used a novel integrative approach by integration of co-expression networks across tissues to elucidate genetic factors related to obesity-induced metabolic disease development. The identified

  16. Crosstalk between G-protein-coupled receptors and epidermal growth factor receptor in cancer.

    PubMed

    Bhola, Neil E; Grandis, Jennifer R

    2008-01-01

    EGFR and its respective ligands are overexpressed in various tumors and this over-expression correlates with poor prognosis in selected cancers. In addition to direct activation by EGFR autocrine ligands, the large family of G-protein-coupled receptors (GPCRs) has been reported to transactivate EGFR via both ligand-dependent and independent mechanisms. GPCRs can induce the cleavage of membrane-bound EGFR-ligand precursors or directly activate the juxtamembrane tyrosine kinase domain of EGFR. Due to the heterogenous expression of GPCRs in tumors, this form of receptor crosstalk may contribute to the modest clinical responses to EGFR-targeted therapies observed to date. Studies, so far, have indicated that the signaling mechanisms involved in transactivation are specifically influenced by the activated GPCR and the tumor type in question. The progression of colon, lung, breast, head and neck, prostate and ovarian cancers have all been reported to be mediated, at least in part, by GPCR-EGFR crosstalk. Increased understanding of the specific signaling pathways involved in EGFR transactivation by GPCR will facilitate the identification of new biomarkers for molecular targeting strategies.

  17. Deregulation of Flk-1/vascular endothelial growth factor receptor-2 in fibroblast growth factor receptor-1-deficient vascular stem cell development.

    PubMed

    Magnusson, Peetra; Rolny, Charlotte; Jakobsson, Lars; Wikner, Charlotte; Wu, Yan; Hicklin, Daniel J; Claesson-Welsh, Lena

    2004-03-15

    We have employed embryoid bodies derived from murine embryonal stem cells to study effects on vascular development induced by fibroblast growth factor (FGF)-2 and FGF receptor-1, in comparison to the established angiogenic factor vascular endothelial growth factor (VEGF)-A and its receptor VEGF receptor-2. Exogenous FGF-2 promoted formation of morphologically distinct, long slender vessels in the embryoid bodies, whereas VEGF-A-treated bodies displayed a compact plexus of capillaries. FGF-2 stimulation of embryonal stem cells under conditions where VEGF-A/VEGFR-2 function was blocked, led to formation of endothelial cell clusters, which failed to develop into vessels. FGFR-1(-/-) embryoid bodies responded to VEGF-A by establishment of the characteristic vascular plexus, but FGF-2 had no effect on vascular development in the absence of FGFR-1. The FGFR-1(-/-) embryoid bodies displayed considerably increased basal level of vessel formation, detected by immunohistochemical staining for platelet-endothelial cell adhesion molecule (PECAM)/CD31. This basal vascularization was blocked by neutralizing antibodies against VEGFR-2 or VEGF-A and biochemical analyses indicated changes in regulation of VEGFR-2 in the absence of FGFR-1 expression. We conclude that VEGF-A/VEGFR-2-dependent vessel formation occurs in the absence of FGF-2/FGFR-1, which, however, serve to modulate vascular development.

  18. Interactions between Type III receptor tyrosine phosphatases and growth factor receptor tyrosine kinases regulate tracheal tube formation in Drosophila.

    PubMed

    Jeon, Mili; Scott, Matthew P; Zinn, Kai

    2012-06-15

    The respiratory (tracheal) system of the Drosophila melanogaster larva is an intricate branched network of air-filled tubes. Its developmental logic is similar in some ways to that of the vertebrate vascular system. We previously described a unique embryonic tracheal tubulogenesis phenotype caused by loss of both of the Type III receptor tyrosine phosphatases (RPTPs), Ptp4E and Ptp10D. In Ptp4E Ptp10D double mutants, the linear tubes in unicellular and terminal tracheal branches are converted into bubble-like cysts that incorporate apical cell surface markers. This tube geometry phenotype is modulated by changes in the activity or expression of the epidermal growth factor receptor (Egfr) tyrosine kinase (TK). Ptp10D physically interacts with Egfr. Here we demonstrate that the Ptp4E Ptp10D phenotype is the consequence of the loss of negative regulation by the RPTPs of three growth factor receptor TKs: Egfr, Breathless and Pvr. Reducing the activity of any of the three kinases by tracheal expression of dominant-negative mutants suppresses cyst formation. By competing dominant-negative and constitutively active kinase mutants against each other, we show that the three RTKs have partially interchangeable activities, so that increasing the activity of one kinase can compensate for the effects of reducing the activity of another. This implies that SH2-domain downstream effectors that are required for the phenotype are likely to be able to interact with phosphotyrosine sites on all three receptor TKs. We also show that the phenotype involves increases in signaling through the MAP kinase and Rho GTPase pathways.

  19. Interactions between Type III receptor tyrosine phosphatases and growth factor receptor tyrosine kinases regulate tracheal tube formation in Drosophila

    PubMed Central

    Jeon, Mili; Scott, Matthew P.; Zinn, Kai

    2012-01-01

    Summary The respiratory (tracheal) system of the Drosophila melanogaster larva is an intricate branched network of air-filled tubes. Its developmental logic is similar in some ways to that of the vertebrate vascular system. We previously described a unique embryonic tracheal tubulogenesis phenotype caused by loss of both of the Type III receptor tyrosine phosphatases (RPTPs), Ptp4E and Ptp10D. In Ptp4E Ptp10D double mutants, the linear tubes in unicellular and terminal tracheal branches are converted into bubble-like cysts that incorporate apical cell surface markers. This tube geometry phenotype is modulated by changes in the activity or expression of the epidermal growth factor receptor (Egfr) tyrosine kinase (TK). Ptp10D physically interacts with Egfr. Here we demonstrate that the Ptp4E Ptp10D phenotype is the consequence of the loss of negative regulation by the RPTPs of three growth factor receptor TKs: Egfr, Breathless and Pvr. Reducing the activity of any of the three kinases by tracheal expression of dominant-negative mutants suppresses cyst formation. By competing dominant-negative and constitutively active kinase mutants against each other, we show that the three RTKs have partially interchangeable activities, so that increasing the activity of one kinase can compensate for the effects of reducing the activity of another. This implies that SH2-domain downstream effectors that are required for the phenotype are likely to be able to interact with phosphotyrosine sites on all three receptor TKs. We also show that the phenotype involves increases in signaling through the MAP kinase and Rho GTPase pathways. PMID:23213447

  20. Endocannabinoid receptor deficiency affects maternal care and alters the dam's hippocampal oxytocin receptor and brain-derived neurotrophic factor expression.

    PubMed

    Schechter, M; Weller, A; Pittel, Z; Gross, M; Zimmer, A; Pinhasov, A

    2013-10-01

    Maternal care is the newborn's first experience of social interaction, and this influences infant survival, development and social competences throughout life. We recently found that postpartum blocking of the endocannabinoid receptor-1 (CB1R) altered maternal behaviour. In the present study, maternal care was assessed by the time taken to retrieve pups, pups' ultrasonic vocalisations (USVs) and pup body weight, comparing CB1R deleted (CB1R KO) versus wild-type (WT) mice. After culling on postpartum day 8, hippocampal expression of oxytocin receptor (OXTR), brain-derived neurotrophic factor (BDNF) and stress-mediating factors were evaluated in CB1R KO and WT dams. Comparisons were also performed with nulliparous (NP) CB1R KO and WT mice. Compared to WT, CB1R KO dams were slower to retrieve their pups. Although the body weight of the KO pups did not differ from the weight of WT pups, they emitted fewer USVs. This impairment of the dam-pup relationship correlated with a significant reduction of OXTR mRNA and protein levels among CB1R KO dams compared to WT dams. Furthermore, WT dams exhibited elevated OXTR mRNA expression, as well as increased levels of mineralocorticoid and glucocorticoid receptors, compared to WT NP mice. By contrast, CB1R KO dams showed no such elevation of OXTR expression, alongside lower BDNF and mineralocorticoid receptors, as well as elevated corticotrophin-releasing hormone mRNA levels, when compared to CB1R KO NP. Thus, it appears that the disruption of endocannabinoid signalling by CB1R deletion alters expression of the OXTR, apparently leading to deleterious effects upon maternal behaviour.

  1. USP17 is required for clathrin mediated endocytosis of epidermal growth factor receptor

    PubMed Central

    Jaworski, Jakub; de la Vega, Michelle; Fletcher, Sarah J.; McFarlane, Cheryl; Greene, Michelle K.; Smyth, Andrew W.; Van Schaeybroeck, Sandra; Johnston, James A.; Scott, Christopher J.; Rappoport, Joshua Z.; Burrows, James F.

    2014-01-01

    Previously we have shown that expression of the deubiquitinating enzyme USP17 is required for cell proliferation and motility. More recently we reported that USP17 deubiquitinates RCE1 isoform 2 and thus regulates the processing of ‘CaaX’ motif proteins. Here we now show that USP17 expression is induced by epidermal growth factor and that USP17 expression is required for clathrin mediated endocytosis of epidermal growth factor receptor. In addition, we show that USP17 is required for the endocytosis of transferrin, an archetypal substrate for clathrin mediated endocytosis, and that USP17 depletion impedes plasma membrane recruitment of the machinery required for clathrin mediated endocytosis. Thus, our data reveal that USP17 is necessary for epidermal growth factor receptor and transferrin endocytosis via clathrin coated pits, indicate this is mediated via the regulation of the recruitment of the components of the endocytosis machinery and suggest USP17 may play a general role in receptor endocytosis. PMID:25026282

  2. How glucocorticoid receptors modulate the activity of other transcription factors: a scope beyond tethering.

    PubMed

    Ratman, Dariusz; Vanden Berghe, Wim; Dejager, Lien; Libert, Claude; Tavernier, Jan; Beck, Ilse M; De Bosscher, Karolien

    2013-11-05

    The activity of the glucocorticoid receptor (GR), a nuclear receptor transcription factor belonging to subclass 3C of the steroid/thyroid hormone receptor superfamily, is typically triggered by glucocorticoid hormones. Apart from driving gene transcription via binding onto glucocorticoid response elements in regulatory regions of particular target genes, GR can also inhibit gene expression via transrepression, a mechanism largely based on protein:protein interactions. Hereby GR can influence the activity of other transcription factors, without contacting DNA itself. GR is known to inhibit the activity of a growing list of immune-regulating transcription factors. Hence, GCs still rule the clinic for treatments of inflammatory disorders, notwithstanding concomitant deleterious side effects. Although patience is a virtue when it comes to deciphering the many mechanisms GR uses to influence various signaling pathways, the current review is testimony of the fact that groundbreaking mechanistic work has been accumulating over the past years and steadily continues to grow.

  3. An Integrated Model of Epidermal Growth Factor Receptor Trafficking and Signal Transduction

    PubMed Central

    Resat, Haluk; Ewald, Jonathan A.; Dixon, David A.; Wiley, H. Steven

    2003-01-01

    Endocytic trafficking of many types of receptors can have profound effects on subsequent signaling events. Quantitative models of these processes, however, have usually considered trafficking and signaling independently. Here, we present an integrated model of both the trafficking and signaling pathway of the epidermal growth factor receptor (EGFR) using a probability weighted-dynamic Monte Carlo simulation. Our model consists of hundreds of distinct endocytic compartments and ∼13,000 reactions/events that occur over a broad spatio-temporal range. By using a realistic multicompartment model, we can investigate the distribution of the receptors among cellular compartments as well as their potential signal transduction characteristics. Our new model also allows the incorporation of physiochemical aspects of ligand-receptor interactions, such as pH-dependent binding in different endosomal compartments. To determine the utility of this approach, we simulated the differential activation of the EGFR by two of its ligands, epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-α). Our simulations predict that when EGFR is activated with TGF-α, receptor activation is biased toward the cell surface whereas EGF produces a signaling bias toward the endosomal compartment. Experiments confirm these predictions from our model and simulations. Our model accurately predicts the kinetics and extent of receptor downregulation induced by either EGF or TGF-α. Our results suggest that receptor trafficking controls the compartmental bias of signal transduction, rather than simply modulating signal magnitude. Our model provides a new approach to evaluating the complex effect of receptor trafficking on signal transduction. Importantly, the stochastic and compartmental nature of the simulation allows these models to be directly tested by high-throughput approaches, such as quantitative image analysis. PMID:12885624

  4. Tumor Necrosis Factor Inhibits Glucocorticoid Receptor Function in Mice

    PubMed Central

    Van Bogaert, Tom; Vandevyver, Sofie; Dejager, Lien; Van Hauwermeiren, Filip; Pinheiro, Iris; Petta, Ioanna; Engblom, David; Kleyman, Anna; Schütz, Günther; Tuckermann, Jan; Libert, Claude

    2011-01-01

    As glucocorticoid resistance (GCR) and the concomitant burden pose a worldwide problem, there is an urgent need for a more effective glucocorticoid therapy, for which insights into the molecular mechanisms of GCR are essential. In this study, we addressed the hypothesis that TNFα, a strong pro-inflammatory mediator in numerous inflammatory diseases, compromises the protective function of the glucocorticoid receptor (GR) against TNFα-induced lethal inflammation. Indeed, protection of mice by dexamethasone against TNFα lethality was completely abolished when it was administered after TNFα stimulation, indicating compromised GR function upon TNFα challenge. TNFα-induced GCR was further demonstrated by impaired GR-dependent gene expression in the liver. Furthermore, TNFα down-regulates the levels of both GR mRNA and protein. However, this down-regulation seems to occur independently of GC production, as TNFα also resulted in down-regulation of GR levels in adrenalectomized mice. These findings suggest that the decreased amount of GR determines the GR response and outcome of TNFα-induced shock, as supported by our studies with GR heterozygous mice. We propose that by inducing GCR, TNFα inhibits a major brake on inflammation and thereby amplifies the pro-inflammatory response. Our findings might prove helpful in understanding GCR in inflammatory diseases in which TNFα is intimately involved. PMID:21646349

  5. Rhodopsin coexpression in UV photoreceptors of Aedes aegypti and Anopheles gambiae mosquitoes.

    PubMed

    Hu, Xiaobang; Leming, Matthew T; Whaley, Michelle A; O'Tousa, Joseph E

    2014-03-15

    Differential rhodopsin gene expression within specialized R7 photoreceptor cells divides the retinas of Aedes aegypti and Anopheles gambiae mosquitoes into distinct domains. The two species express the rhodopsin orthologs Aaop8 and Agop8, respectively, in a large subset of these R7 photoreceptors that function as ultraviolet receptors. We show here that a divergent subfamily of mosquito rhodopsins, Aaop10 and Agop10, is coexpressed in these R7 photoreceptors. The properties of the A. aegypti Aaop8 and Aaop10 rhodopsins were analyzed by creating transgenic Drosophila expressing these rhodopsins. Electroretinogram recordings, and spectral analysis of head extracts, obtained from the Aaop8 strain confirmed that Aaop8 is an ultraviolet-sensitive rhodopsin. Aaop10 was poorly expressed and capable of eliciting only small and slow light responses in Drosophila photoreceptors, and electroretinogram analysis suggested that it is a long-wavelength rhodopsin with a maximal sensitivity near 500 nm. Thus, coexpression of Aaop10 rhodopsin with Aaop8 rhodopsin has the potential to modify the spectral properties of mosquito ultraviolet receptors. Retention of Op10 rhodopsin family members in the genomes of Drosophila species suggests that this rhodopsin family may play a conserved role in insect vision.

  6. The ligand specificities of the insulin receptor and the insulin-like growth factor I receptor reside in different regions of a common binding site

    SciTech Connect

    Kjeldsen, T.; Andersen, A.S.; Wiberg, F.C.; Rasmussen, J.S.; Schaeffer, L.; Balschmidt, P.; Moller, K.B.; Moller, N.P.H. )

    1991-05-15

    To identify the region(s) of the insulin receptor and the insulin-like growth factor I (IGF-I) receptor responsible for ligand specificity (high-affinity binding), expression vectors encoding soluble chimeric insulin/IGF-I receptors were prepared. The chimeric receptors were expressed in mammalian cells and partially purified. Binding studies revealed that a construct comprising an IGF-I receptor in which the 68 N-terminal amino acids of the insulin receptor {alpha}-subunit had replaced the equivalent IGF-I receptor segment displayed a markedly increased affinity for insulin. In contrast, the corresponding IGF-I receptor sequence is not critical for high-affinity IGF-I binding. It is shown that part of the cysteine-rich domain determines IGF-I specificity. The authors have previously shown that exchanging exons 1, 2, and 3 of the insulin receptor with the corresponding IGF-I receptor sequence results in loss of high affinity for insulin and gain of high affinity for IGF-I. Consequently, it is suggested that the ligand specificities of the two receptors (i.e., the sequences that discriminate between insulin and IGF-I) reside in different regions of a binding site with common features present in both receptors.

  7. Genome-Wide Tissue-Specific Gene Expression, Co-expression and Regulation of Co-expressed Genes in Adult Nematode Ascaris suum

    PubMed Central

    Rosa, Bruce A.; Jasmer, Douglas P.; Mitreva, Makedonka

    2014-01-01

    Background Caenorhabditis elegans has traditionally been used as a model for studying nematode biology, but its small size limits the ability for researchers to perform some experiments such as high-throughput tissue-specific gene expression studies. However, the dissection of individual tissues is possible in the parasitic nematode Ascaris suum due to its relatively large size. Here, we take advantage of the recent genome sequencing of Ascaris suum and the ability to physically dissect its separate tissues to produce a wide-scale tissue-specific nematode RNA-seq datasets, including data on three non-reproductive tissues (head, pharynx, and intestine) in both male and female worms, as well as four reproductive tissues (testis, seminal vesicle, ovary, and uterus). We obtained fundamental information about the biology of diverse cell types and potential interactions among tissues within this multicellular organism. Methodology/Principal Findings Overexpression and functional enrichment analyses identified many putative biological functions enriched in each tissue studied, including functions which have not been previously studied in detail in nematodes. Putative tissue-specific transcriptional factors and corresponding binding motifs that regulate expression in each tissue were identified, including the intestine-enriched ELT-2 motif/transcription factor previously described in nematode intestines. Constitutively expressed and novel genes were also characterized, with the largest number of novel genes found to be overexpressed in the testis. Finally, a putative acetylcholine-mediated transcriptional network connecting biological activity in the head to the male reproductive system is described using co-expression networks, along with a similar ecdysone-mediated system in the female. Conclusions/Significance The expression profiles, co-expression networks and co-expression regulation of the 10 tissues studied and the tissue-specific analysis presented here are a

  8. Transient receptor potential cation channel subfamily V member 1 expressing corneal sensory neurons can be subdivided into at least three subpopulations.

    PubMed

    Alamri, Abdulhakeem; Bron, Romke; Brock, James A; Ivanusic, Jason J

    2015-01-01

    The cornea is innervated by three main functional classes of sensory neurons: polymodal nociceptors, pure mechano-nociceptors and cold-sensing neurons. Here we explored transient receptor potential cation channel subfamily V member 1 (TRPV1) expression in guinea pig corneal sensory neurons, a widely used molecular marker of polymodal nociceptors. We used retrograde tracing to identify corneal afferent neurons in the trigeminal ganglion (TG) and double label in situ hybridization and/or immunohistochemistry to determine their molecular profile. In addition, we used immunohistochemistry to reveal the neurochemistry and structure of TRPV1 expressing nerve endings in the corneal epithelium. Approximately 45% of corneal afferent neurons expressed TRPV1, 28% expressed Piezo2 (a marker of putative pure mechano-nociceptors) and 8% expressed the transient receptor potential cation channel subfamily M member 8 (TRPM8; a marker of cold-sensing neurons). There was no co-expression of TRPV1 and Piezo2 in corneal afferent neurons, but 6% of TRPV1 neurons co-expressed TRPM8. The TRPV1 expressing corneal afferent neurons could be divided into three subpopulations on the basis of calcitonin gene-related peptide (CGRP) and/or or glial cell line-derived neurotrophic factor family receptor alpha3 (GFRα3) co-expression. In the corneal epithelium, the TRPV1 axons that co-expressed CGRP and GFRα3 ended as simple unbranched endings in the wing cell layer. In contrast, those that only co-expressed GFRα3 had ramifying endings that branched and terminated in the squamous cell layer, whereas those that only co-expressed CGRP had simple endings in the basal epithelium. This study shows that the majority of TRPV1 expressing corneal afferent neurons (>90%) are likely to be polymodal nociceptors. Furthermore, TRPV1 expressing corneal afferent neurons can be subdivided into specific subpopulations based on their molecular phenotype, nerve terminal morphology and distribution in the corneal

  9. Transient receptor potential cation channel subfamily V member 1 expressing corneal sensory neurons can be subdivided into at least three subpopulations

    PubMed Central

    Alamri, Abdulhakeem; Bron, Romke; Brock, James A.; Ivanusic, Jason J.

    2015-01-01

    The cornea is innervated by three main functional classes of sensory neurons: polymodal nociceptors, pure mechano-nociceptors and cold-sensing neurons. Here we explored transient receptor potential cation channel subfamily V member 1 (TRPV1) expression in guinea pig corneal sensory neurons, a widely used molecular marker of polymodal nociceptors. We used retrograde tracing to identify corneal afferent neurons in the trigeminal ganglion (TG) and double label in situ hybridization and/or immunohistochemistry to determine their molecular profile. In addition, we used immunohistochemistry to reveal the neurochemistry and structure of TRPV1 expressing nerve endings in the corneal epithelium. Approximately 45% of corneal afferent neurons expressed TRPV1, 28% expressed Piezo2 (a marker of putative pure mechano-nociceptors) and 8% expressed the transient receptor potential cation channel subfamily M member 8 (TRPM8; a marker of cold-sensing neurons). There was no co-expression of TRPV1 and Piezo2 in corneal afferent neurons, but 6% of TRPV1 neurons co-expressed TRPM8. The TRPV1 expressing corneal afferent neurons could be divided into three subpopulations on the basis of calcitonin gene-related peptide (CGRP) and/or or glial cell line-derived neurotrophic factor family receptor alpha3 (GFRα3) co-expression. In the corneal epithelium, the TRPV1 axons that co-expressed CGRP and GFRα3 ended as simple unbranched endings in the wing cell layer. In contrast, those that only co-expressed GFRα3 had ramifying endings that branched and terminated in the squamous cell layer, whereas those that only co-expressed CGRP had simple endings in the basal epithelium. This study shows that the majority of TRPV1 expressing corneal afferent neurons (>90%) are likely to be polymodal nociceptors. Furthermore, TRPV1 expressing corneal afferent neurons can be subdivided into specific subpopulations based on their molecular phenotype, nerve terminal morphology and distribution in the corneal

  10. A fibronectin scaffold approach to bispecific inhibitors of epidermal growth factor receptor and insulin-like growth factor-I receptor

    PubMed Central

    Emanuel, Stuart L; Engle, Linda J; Chao, Ginger; Zhu, Rong-Rong; Cao, Carolyn; Lin, Zheng; Yamniuk, Aaron; Hosbach, Jennifer; Brown, Jennifer; Fitzpatrick, Elizabeth; Gokemeijer, Jochem; Morin, Paul; Morse, Brent; Carvajal, Irvith M; Fabrizio, David; Wright, Martin C; Das Gupta, Ruchira; Gosselin, Michael; Cataldo, Daniel; Ryseck, Rolf P; Doyle, Michael L; Wong, Tai W; Camphausen, Raymond T; Cload, Sharon T; Marsh, H Nicholas; Gottardis, Marco M

    2011-01-01

    Engineered domains of human fibronectin (Adnectins™) were used to generate a bispecific Adnectin targeting epidermal growth factor receptor (EGFR) and insulin-like growth factor-I receptor (IGF-IR), two transmembrane receptors that mediate proliferative and survival cell signaling in cancer. Single-domain Adnectins that specifically bind EGFR or IGF-IR were generated using mRNA display with a library containing as many as 1013 Adnectin variants. mRNA display was also used to optimize lead Adnectin affinities, resulting in clones that inhibited EGFR phosphorylation at 7 to 38 nM compared to 2.6 µM for the parental clone. Individual optimized Adnectins specific for blocking either EGFR or IGF-IR signaling were engineered into a single protein (EI-Tandem Adnectin). The EI-Tandems inhibited phosphorylation of EGFR and IGF-IR, induced receptor degradation and inhibited down-stream cell signaling and proliferation of human cancer cell lines (A431, H292, BxPC3 and RH41) with IC50 values ranging from 0.1 to 113 nM. Although Adnectins bound to EGFR at a site distinct from those of anti-EGFR antibodies cetuximab, panitumumab and nimotuzumab, like the antibodies, the anti-EGFR Adnectins blocked the binding of EGF to EGFR. PEGylated EI-Tandem inhibited the growth of both EGFR and IGF-IR driven human tumor xenografts, induced degradation of EGFR and reduced EGFR phosphorylation in tumors. These results demonstrate efficient engineering of bispecific Adnectins with high potency and desired specificity. The bispecificity may improve biological activity compared to monospecific biologics as tumor growth is driven by multiple growth factors. Our results illustrate a technological advancement for constructing multi-specific biologics in cancer therapy. PMID:21099371

  11. ErbB2 resembles an autoinhibited invertebrate epidermal growth factor receptor

    SciTech Connect

    Alvarado, Diego; Klein, Daryl E.; Lemmon, Mark A.

    2009-09-25

    The orphan receptor tyrosine kinase ErbB2 (also known as HER2 or Neu) transforms cells when overexpressed, and it is an important therapeutic target in human cancer. Structural studies have suggested that the oncogenic (and ligand-independent) signalling properties of ErbB2 result from the absence of a key intramolecular 'tether' in the extracellular region that autoinhibits other human ErbB receptors, including the epidermal growth factor (EGF) receptor. Although ErbB2 is unique among the four human ErbB receptors, here we show that it is the closest structural relative of the single EGF receptor family member in Drosophila melanogaster (dEGFR). Genetic and biochemical data show that dEGFR is tightly regulated by growth factor ligands, yet a crystal structure shows that it, too, lacks the intramolecular tether seen in human EGFR, ErbB3 and ErbB4. Instead, a distinct set of autoinhibitory interdomain interactions hold unliganded dEGFR in an inactive state. All of these interactions are maintained (and even extended) in ErbB2, arguing against the suggestion that ErbB2 lacks autoinhibition. We therefore suggest that normal and pathogenic ErbB2 signalling may be regulated by ligands in the same way as dEGFR. Our findings have important implications for ErbB2 regulation in human cancer, and for developing therapeutic approaches that target novel aspects of this orphan receptor.

  12. Developmentally Regulated Expression of the Nerve Growth Factor Receptor Gene in the Periphery and Brain

    NASA Astrophysics Data System (ADS)

    Buck, C. R.; Martinez, Humberto J.; Black, Ira B.; Chao, Moses V.

    1987-05-01

    Nerve growth factor (NGF) regulates development and maintenance of function of peripheral sympathetic and sensory neurons. A potential role for the trophic factor in brain has been detected only recently. The ability of a cell to respond to NGF is due, in part, to expression of specific receptors on the cell surface. To study tissue-specific expression of the NGF receptor gene, we have used sensitive cRNA probes for detection of NGF receptor mRNA. Our studies indicate that the receptor gene is selectively and specifically expressed in sympathetic (superior cervical) and sensory (dorsal root) ganglia in the periphery, and by the septum-basal forebrain centrally, in the neonatal rat in vivo. Moreover, examination of tissues from neonatal and adult rats reveals a marked reduction in steady-state NGF receptor mRNA levels in sensory ganglia. In contrast, a 2- to 4-fold increase was observed in the basal forebrain and in the sympathetic ganglia over the same time period. Our observations suggest that NGF receptor mRNA expression is developmentally regulated in specific areas of the nervous system in a differential fashion.

  13. The epidermal growth factor receptor family as a central element for cellular signal transduction and diversification.

    PubMed

    Prenzel, N; Fischer, O M; Streit, S; Hart, S; Ullrich, A

    2001-03-01

    Homeostasis of multicellular organisms is critically dependent on the correct interpretation of the plethora of signals which cells are exposed to during their lifespan. Various soluble factors regulate the activation state of cellular receptors which are coupled to a complex signal transduction network that ultimately generates signals defining the required biological response. The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases represents both key regulators of normal cellular development as well as critical players in a variety of pathophysiological phenomena. The aim of this review is to give a broad overview of signal transduction networks that are controlled by the EGFR superfamily of receptors in health and disease and its application for target-selective therapeutic intervention. Since the EGFR and HER2 were recently identified as critical players in the transduction of signals by a variety of cell surface receptors, such as G-protein-coupled receptors and integrins, our special focus is the mechanisms and significance of the interconnectivity between heterologous signalling systems.

  14. Harnessing tumor necrosis factor receptors to enhance antitumor activities of drugs.

    PubMed

    Muntané, Jordi

    2011-10-17

    Cancer is the second-leading cause of death in the U.S. behind heart disease and over stroke. The hallmarks of cancer comprise six biological capabilities acquired during the multistep development of human tumors. The inhibition of cell death pathways is one of these tumor characteristics which also include sustained proliferative signaling, evading growth suppressor signaling, replicative immortality, angiogenesis, and promotion of invasion and metastasis. Cell death is mediated through death receptor (DR) stimulation initiated by specific ligands that transmit signaling to the cell death machinery or through the participation of mitochondria. Cell death involving DR is mediated by the superfamily of tumor necrosis factor receptor (TNF-R) which includes TNF-R type I, CD95, DR3, TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 (TRAIL-R1) and -2 (TRAIL-R2), DR6, ectodysplasin A (EDA) receptor (EDAR), and the nerve growth factor (NGF) receptor (NGFR). The expression of these receptors in healthy and tumor cells induces treatment side effects that limit the systemic administration of cell death-inducing therapies. The present review is focused on the different therapeutic strategies such as targeted antibodies or small molecules addressed to selective stimulated DR-mediated apoptosis or reduce cell proliferation in cancer cells.

  15. Targeting fibroblast growth factor receptor signaling inhibits prostate cancer progression

    PubMed Central

    Feng, Shu; Shao, Longjiang; Yu, Wendong; Gavine, Paul; Ittmann, Michael

    2015-01-01

    Purpose Extensive correlative studies in human prostate cancer (PCa) as well as studies in vitro and in mouse models indicate that FGF receptor (FGFR) signaling plays an important role in PCa progression. In this study, we employed a probe compound for an FGFR inhibitor which potently inhibits FGFR1-3 and significantly inhibits FGFR-4. The purpose of this study is to determine if targeting FGFR signaling from all four FGFRs will have in vitro activities consistent with inhibition of tumor progression and will inhibit tumor progression in vivo. Experimental Design Effects of AZ8010 on FGFR signaling and invasion were analyzed using immortalized normal prostate epithelial (PNT1a) cells and PNT1a overexpressing FGFR-1 or FGFR-4. The effect of AZ8010 on invasion and proliferation in vitro was also evaluated in PCa cell lines. Finally, the impact of AZ8010 on tumor progression in vivo was evaluated using a VCaP xenograft model. Results AZ8010 completely inhibits FGFR-1 and significantly inhibits FGFR-4 signaling at 100 nM, which is an achievable in vivo concentration. These results in marked inhibition of ERK phosphorylation and invasion in PNT1a cells expressing FGFR-1 and FGFR-4 and all PCa cell lines tested. Treatment in vivo completely inhibited VCaP tumor growth and significantly inhibited angiogenesis and proliferation and increased cell death in treated tumors. This was associated with marked inhibition of ERK phosphorylation in treated tumors. Conclusions Targeting FGFR signaling is a promising new approach to treating aggressive PCa. PMID:22573348

  16. Platelet-activating factor (PAF) receptor as a promising target for cancer cell repopulation after radiotherapy

    PubMed Central

    da Silva-Jr, I A; Chammas, R; Lepique, A P; Jancar, S

    2017-01-01

    A major drawback of radiotherapy is the accelerated growth of the surviving tumor cells. Radiotherapy generates a variety of lipids that bind to the receptor for platelet-activating factor, expressed by cells in the tumor microenvironment. In the present study, using the TC-1 tumor cell line, we found that irradiation induced a twofold increase in receptor expression and generated agonists of receptor. Irradiated cells induced a 20-fold increase in live TC-1 proliferation in vitro. Furthermore, subcutaneous co-injection of irradiated TC-1 cells with TC-1 expressing luciferase (TC-1 fluc+) markedly increased TC-1 fluc+ proliferation in a receptor-dependent way. Moreover we used a human carcinoma cell line not expressing the PAF receptor (KBM) and the same cell transfected with the receptor gene (KBP). Following co-injection of live KBP cells with irradiated KBM in RAG mice, the tumor growth was significantly increased compared with tumor formed following co-injection of live KBM with irradiated KBM. This tumor cell repopulation correlated with increased infiltration of tumor-promoting macrophages (CD206+). We propose that receptor represents a possible target for improving the efficacy of radiotherapy through inhibition of tumor repopulation. PMID:28134937

  17. ADP-ribosylation factor-dependent phospholipase D activation by the M3 muscarinic receptor.

    PubMed

    Mitchell, Rory; Robertson, Derek N; Holland, Pamela J; Collins, Daniel; Lutz, Eve M; Johnson, Melanie S

    2003-09-05

    G protein-coupled receptors can potentially activate phospholipase D (PLD) by a number of routes. We show here that the native M3 muscarinic receptor in 1321N1 cells and an epitope-tagged M3 receptor expressed in COS7 cells substantially utilize an ADP-ribosylation factor (ARF)-dependent route of PLD activation. This pathway is activated at the plasma membrane but appears to be largely independent of G, phospholipase C, Ca2+ q/11, protein kinase C, tyrosine kinases, and phosphatidyl inositol 3-kinase. We report instead that it involves physical association of ARF with the M3 receptor as demonstrated by co-immunoprecipitation and by in vitro interaction with a glutathione S-transferase fusion protein of the receptor's third intracellular loop domain. Experiments with mutant constructs of ARF1/6 and PLD1/2 indicate that the M3 receptor displays a major ARF1-dependent route of PLD1 activation with an additional ARF6-dependent pathway to PLD1 or PLD2. Examples of other G protein-coupled receptors assessed in comparison display alternative pathways of protein kinase C- or ARF6-dependent activation of PLD2.

  18. Endoglin structure and function: Determinants of endoglin phosphorylation by transforming growth factor-beta receptors.

    PubMed

    Koleva, Rositsa I; Conley, Barbara A; Romero, Diana; Riley, Kristin S; Marto, Jarrod A; Lux, Andreas; Vary, Calvin P H

    2006-09-01

    Determination of the functional relationship between the transforming growth factor-beta (TGFbeta) receptor proteins endoglin and ALK1 is essential to the understanding of the human vascular disease, hereditary hemorrhagic telangiectasia. TGFbeta1 caused recruitment of ALK1 into a complex with endoglin in human umbilical vein endothelial cells (HUVECs). Therefore, we examined TGFbeta receptor-dependent phosphorylation of endoglin by the constitutively active forms of the TGFbeta type I receptors ALK1, ALK5, and the TGFbeta type II receptor, TbetaRII. Of these receptors, TbetaRII preferentially phosphorylated endoglin on cytosolic domain serine residues Ser(634) and Ser(635). Removal of the carboxyl-terminal tripeptide of endoglin, which comprises a putative PDZ-liganding motif, dramatically increased endoglin serine phosphorylation by all three receptors, suggesting that the PDZ-liganding motif is important for the regulation of endoglin phosphorylation. Constitutively active (ca)ALK1, but not caALK5, phosphorylated endoglin on cytosolic domain threonine residues. caALK1-mediated threonine phosphorylation required prior serine phosphorylation, suggesting a sequential mechanism of endoglin phosphorylation. Wild-type, but not a threonine phosphorylation-defective endoglin mutant blocked cell detachment and the antiproliferative effects of caALK1 expressed in HUVECs. These results suggest that ALK1 is a preferred TGFbeta receptor kinase for endoglin threonine phosphorylation in HUVECs and indicate a role for endoglin phosphorylation in the regulation of endothelial cell adhesion and growth by ALK1.

  19. Direct visualization of the phosphorylated epidermal growth factor receptor during its internalization in A-431 cells

    PubMed Central

    1987-01-01

    Epidermal growth factor (EGF) rapidly stimulates receptor autophosphorylation in A-431 cells. After 1 min the phosphorylated receptor can be identified at the plasma membrane using an anti- phosphotyrosine antibody. With further incubation at 37 degrees C, approximately 50% of the phosphorylated EGF receptor was internalized (t1/2 = 5 min) and associated with the tubulovesicular system and later with multivesicular bodies, but not the nucleus. During this period, there was no change in the extent or sites of phosphorylation. At all times the phosphotyrosine remained on the cytoplasmic side of the membrane, opposite to the EGF ligand identified by anti-EGF antibody. These data indicate that (a) the tyrosine-phosphorylated EGF receptor is internalized in its activated form providing a mechanism for translocation of the receptor kinase to substrates in the cell interior; (b) the internalized receptor remains intact for at least 60 min, does not associate with the nucleus, and does not generate any tyrosine-phosphorylated fragments; and (c) tyrosine phosphorylation alone is not the signal for receptor internalization. PMID:2447100

  20. Transforming Growth Factor β Can Stimulate Smad1 Phosphorylation Independently of Bone Morphogenic Protein Receptors*

    PubMed Central

    Wrighton, Katharine H.; Lin, Xia; Yu, Paul B.; Feng, Xin-Hua

    2009-01-01

    Transforming growth factor-β (TGFβ) superfamily ligands control a diverse set of cellular processes by activating type I and type II serine-threonine receptor kinases. Canonical TGFβ signaling is mediated via the TβRI/ALK5 type I receptor that phosphorylates Smad2 and Smad3 in their SXS motif to facilitate their activation and subsequent role in transcriptional regulation. Canonical bone morphogenic protein (BMP) signaling is mediated via the ALK1/2/3/6 type I receptors that phosphorylate Smad1, Smad5, and Smad8 in their SXS motif. However, studies in endothelial cells have shown that TGFβ can also lead to the phosphorylation of Smad1, dependent on ALK1 receptor activity. Here we present data showing that TGFβ can significantly induce Smad1 phosphorylation in several non-endothelial cell lineages. Additionally, by using chemical inhibitors specific for the TGFβ/activin/nodal (ALK4/5/7) and BMP (ALK1/2/3/6) type I receptors, we show that in some cell types TGFβ induces Smad1 phosphorylation independently of the BMP type I receptors. Thus, TGFβ-mediated Smad1 phosphorylation appears to occur via different receptor complexes in a cell type-specific manner. PMID:19224917

  1. Transforming Growth Factor {beta} Can Stimulate Smad1 Phosphorylation Independently of Bone Morphogenic Protein Receptors.

    PubMed

    Wrighton, Katharine H; Lin, Xia; Yu, Paul B; Feng, Xin-Hua

    2009-04-10

    Transforming growth factor-beta (TGFbeta) superfamily ligands control a diverse set of cellular processes by activating type I and type II serine-threonine receptor kinases. Canonical TGFbeta signaling is mediated via the TbetaRI/ALK5 type I receptor that phosphorylates Smad2 and Smad3 in their SXS motif to facilitate their activation and subsequent role in transcriptional regulation. Canonical bone morphogenic protein (BMP) signaling is mediated via the ALK1/2/3/6 type I receptors that phosphorylate Smad1, Smad5, and Smad8 in their SXS motif. However, studies in endothelial cells have shown that TGFbeta can also lead to the phosphorylation of Smad1, dependent on ALK1 receptor activity. Here we present data showing that TGFbeta can significantly induce Smad1 phosphorylation in several non-endothelial cell lineages. Additionally, by using chemical inhibitors specific for the TGFbeta/activin/nodal (ALK4/5/7) and BMP (ALK1/2/3/6) type I receptors, we show that in some cell types TGFbeta induces Smad1 phosphorylation independently of the BMP type I receptors. Thus, TGFbeta-mediated Smad1 phosphorylation appears to occur via different receptor complexes in a cell type-specific manner.

  2. Defective lysosomal targeting of activated fibroblast growth factor receptor 3 in achondroplasia

    PubMed Central

    Cho, Jay Y.; Guo, Changsheng; Torello, Monica; Lunstrum, Gregory P.; Iwata, Tomoko; Deng, Chuxia; Horton, William A.

    2004-01-01

    Mutations of fibroblast growth factor receptor 3 (FGFR3) are responsible for achondroplasia (ACH) and related dwarfing conditions in humans. The pathogenesis involves constitutive activation of FGFR3, which inhibits proliferation and differentiation of growth plate chondrocytes. Here we report that activating mutations in FGFR3 increase the stability of the receptor. Our results suggest that the mutations disrupt c-Cbl-mediated ubiquitination that serves as a targeting signal for lysosomal degradation and termination of receptor signaling. The defect allows diversion of actively signaling receptors from lysosomes to a recycling pathway where their survival is prolonged, and, as a result, their signaling capacity is increased. The lysosomal targeting defect is additive to other mechanisms proposed to explain the pathogenesis of ACH. PMID:14699054

  3. Activation of the epidermal growth factor receptor by hydrogels in artificial tears

    PubMed Central

    PALUS, JENNIFER S.; CHAY, EDWARD Y.; HEALEY, JEFFREY; SULLENBERGER, REBECCA; KLARLUND, JES K.

    2008-01-01

    Most formulations of artificial tears include high-molecular weight hydrophilic polymers (hydrogels) that are usually thought to serve to enhance viscosity and to act as demulcents. A few reports have indicated that application of some of the polymers accelerates healing of wounds in epithelia. Since activation of the epidermal growth factor (EGF) receptor is critical for spontaneous corneal epithelial wound healing, we tested commonly used hydrogels for their ability to activate the EGF receptor and enhance closure of wounds. Five structurally unrelated hydrogels used in artificial tears were found to activate the EGF receptor. Importantly, two of the hydrogels enhanced wound healing in an organ culture model. We propose that the efficacy of hydrogels in treating dry eye may be related to their ability to activate the EGF receptor, and that hydrogels are inexpensive, safe agents to promote healing of wounds in the cornea and possibly in other tissues. PMID:18242602

  4. Epidermal Growth Factor-Dependent Transformation by a Human EGF Receptor Proto-Oncogene

    NASA Astrophysics Data System (ADS)

    Velu, Thierry J.; Beguinot, Laura; Vass, William C.; Willingham, Mark C.; Merlino, Glenn T.; Pastan, Ira; Lowy, Douglas R.

    1987-12-01

    The epidermal growth factor (EGF) receptor gene EGFR has been placed in a retrovirus vector to examine the growth properties of cells that experimentally overproduce a full-length EGF receptor. NIH 3T3 cells transfected with the viral DNA or infected with the corresponding rescued retrovirus developed a fully transformed phenotype in vitro that required both functional EGFR expression and the presence of EGF in the growth medium. Cells expressing 4 × 105 EGF receptors formed tumors in nude mice, while control cells did not. Therefore, the EGFR retrovirus, which had a titer on NIH 3T3 cells that was greater than 107 focus-forming units per milliliter, can efficiently transfer and express this gene, and increased numbers of EGF receptors can contribute to the transformed phenotype.

  5. Heparin stimulates epidermal growth factor receptor-mediated phosphorylation of tyrosine and threonine residues.

    PubMed

    Revis-Gupta, S; Abdel-Ghany, M; Koland, J; Racker, E

    1991-07-15

    We have described previously that in extracts of A431 cells epidermal growth factor (EGF) stimulates the phosphorylation of tyrosine as well as of threonine residues in the EGF receptor and in lipocortin 1. We now report that heparin at low concentrations also stimulates the autophosphorylation of the EGF receptor and of the recombinant 56-kDa domain of the EGF receptor that lacks the EGF binding site. To study the stimulations of phosphorylation of threonine residues, a fusion protein was prepared with glutathione S-transferase (GST) and an EGF receptor fragment, TK8 (residues 647-688), that contains the threonine phosphorylation site but no tyrosine. We show that the phosphorylation of threonine residues in GST-TK8 by extracts of A431 cells is stimulated by heparin but not by EGF. These and other results suggest that heparin acts as a chaperone, a substrate modulator, that enhances the susceptibility of the substrate to phosphorylation by protein kinases.

  6. Key regulators in prostate cancer identified by co-expression module analysis.

    PubMed

    Jiang, Junfeng; Jia, Peilin; Zhao, Zhongming; Shen, Bairong

    2014-11-24

    Prostate cancer (PrCa) is the most commonly diagnosed cancer in men in the world. Despite the fact that a large number of its genes have been investigated, its etiology remains poorly understood. Furthermore, most PrCa candidate genes have not been rigorously replicated, and the methods by which they biologically function in PrCa remain largely unknown. Aiming to identify key players in the complex prostate cancer system, we reconstructed PrCa co-expressed modules within functional gene sets defined by the Gene Ontology (GO) annotation (biological process, GO_BP). We primarily identified 118 GO_BP terms that were well-preserved between two independent gene expression datasets and a consequent 55 conserved co-expression modules within them. Five modules were then found to be significantly enriched with PrCa candidate genes collected from expression Quantitative Trait Loci (eQTL), somatic copy number alteration (SCNA), somatic mutation data, or prognostic analyses. Specifically, two transcription factors (TFs) (NFAT and SP1) and three microRNAs (hsa-miR-19a, hsa-miR-15a, and hsa-miR-200b) regulating these five candidate modules were found to be critical to the development of PrCa. Collectively, our results indicated that genes with similar functions may play important roles in disease through co-expression, and modules with different functions could be regulated by similar genetic components, such as TFs and microRNAs, in a synergistic manner.

  7. Co-expression analysis identifies CRC and AP1 the regulator of Arabidopsis fatty acid biosynthesis.

    PubMed

    Han, Xinxin; Yin, Linlin; Xue, Hongwei

    2012-07-01

    Fatty acids (FAs) play crucial rules in signal transduction and plant development, however, the regulation of FA metabolism is still poorly understood. To study the relevant regulatory network, fifty-eight FA biosynthesis genes including de novo synthases, desaturases and elongases were selected as "guide genes" to construct the co-expression network. Calculation of the correlation between all Arabidopsis thaliana (L.) genes with each guide gene by Arabidopsis co-expression dating mining tools (ACT) identifies 797 candidate FA-correlated genes. Gene ontology (GO) analysis of these co-expressed genes showed they are tightly correlated to photosynthesis and carbohydrate metabolism, and function in many processes. Interestingly, 63 transcription factors (TFs) were identified as candidate FA biosynthesis regulators and 8 TF families are enriched. Two TF genes, CRC and AP1, both correlating with 8 FA guide genes, were further characterized. Analyses of the ap1 and crc mutant showed the altered total FA composition of mature seeds. The contents of palmitoleic acid, stearic acid, arachidic acid and eicosadienoic acid are decreased, whereas that of oleic acid is increased in ap1 and crc seeds, which is consistent with the qRT-PCR analysis revealing the suppressed expression of the corresponding guide genes. In addition, yeast one-hybrid analysis and electrophoretic mobility shift assay (EMSA) revealed that CRC can bind to the promoter regions of KCS7 and KCS15, indicating that CRC may directly regulate FA biosynthesis. © 2012 Institute of Botany, Chinese Academy of Sciences.

  8. Sequestration of human muscarinic acetylcholine receptor hm1-hm5 subtypes: effect of G protein-coupled receptor kinases GRK2, GRK4, GRK5 and GRK6.

    PubMed

    Tsuga, H; Okuno, E; Kameyama, K; Haga, T

    1998-03-01

    Sequestration of porcine muscarinic acetylcholine receptor m2 subtypes (m2 receptors) expressed in COS-7 cells is facilitated by coexpression of G protein-coupled receptor kinases 2 (GRK2). We examined the effect of coexpression of GRK2, GRK4 delta, GRK5 and GRK6 on sequestration of human m1-m5 receptors expressed in COS-7 cells, which was assessed as loss of [3H]N-methylscopolamine binding activity from the cell surface. Sequestration of m4 receptors as well as m2 receptors was facilitated by coexpression of GRK2 and attenuated by coexpression of the dominant negative form of GRK2 (DN-GRK2). Sequestration of m3 and m5 receptors also was facilitated by coexpression of GRK2 but not affected by coexpression of DN-GRK2. On the other hand, proportions of sequestered m1 receptors were not significantly different with coexpression of GRK2 and DN-GRK2. GRK4 delta, GRK5 and GRK6 did not facilitate sequestration of m1-m5 receptors in COS-7 cells, except that the sequestration of m2 receptors tended to be facilitated by coexpression of GRK4 delta, GRK5 and GRK6. However, coexpression of GRK4 delta, GRK5, but not GRK6, in BHK-21 cells facilitated sequestration of m2, but not m3, receptors. These results indicate that the effect of GRK2 to facilitate receptor sequestration is not restricted to m2 receptors but is generalized to other muscarinic receptors except m1 receptors and that other kinases, including GRK4 delta, GRK5 and endogenous kinase(s) in COS-7 cells, also contribute to sequestration of m2 and m4 receptors.

  9. Regulation of fibroblast growth factor receptor signalling and trafficking by Src and Eps8.

    PubMed

    Auciello, Giulio; Cunningham, Debbie L; Tatar, Tulin; Heath, John K; Rappoport, Joshua Z

    2013-01-15

    Fibroblast growth factor receptors (FGFRs) mediate a wide spectrum of cellular responses that are crucial for development and wound healing. However, aberrant FGFR activity leads to cancer. Activated growth factor receptors undergo stimulated endocytosis, but can continue to signal along the endocytic pathway. Endocytic trafficking controls the duration and intensity of signalling, and growth factor receptor signalling can lead to modifications of trafficking pathways. We have developed live-cell imaging methods for studying FGFR dynamics to investigate mechanisms that coordinate the interplay between receptor trafficking and signal transduction. Activated FGFR enters the cell following recruitment to pre-formed clathrin-coated pits (CCPs). However, FGFR activation stimulates clathrin-mediated endocytosis; FGF treatment increases the number of CCPs, including those undergoing endocytosis, and this effect is mediated by Src and its phosphorylation target Eps8. Eps8 interacts with the clathrin-mediated endocytosis machinery and depletion of Eps8 inhibits FGFR trafficking and immediate Erk signalling. Once internalized, FGFR passes through peripheral early endosomes en route to recycling and degredative compartments, through an Src- and Eps8-dependent mechanism. Thus Eps8 functions as a key coordinator in the interplay between FGFR signalling and trafficking. This work provides the first detailed mechanistic analysis of growth factor receptor clustering at the cell surface through signal transduction and endocytic trafficking. As we have characterised the Src target Eps8 as a key regulator of FGFR signalling and trafficking, and identified the early endocytic system as the site of Eps8-mediated effects, this work provides novel mechanistic insight into the reciprocal regulation of growth factor receptor signalling and trafficking.

  10. Regulation of fibroblast growth factor receptor signalling and trafficking by Src and Eps8

    PubMed Central

    Auciello, Giulio; Cunningham, Debbie L.; Tatar, Tulin; Heath, John K.; Rappoport, Joshua Z.

    2013-01-01

    Summary Fibroblast growth factor receptors (FGFRs) mediate a wide spectrum of cellular responses that are crucial for development and wound healing. However, aberrant FGFR activity leads to cancer. Activated growth factor receptors undergo stimulated endocytosis, but can continue to signal along the endocytic pathway. Endocytic trafficking controls the duration and intensity of signalling, and growth factor receptor signalling can lead to modifications of trafficking pathways. We have developed live-cell imaging methods for studying FGFR dynamics to investigate mechanisms that coordinate the interplay between receptor trafficking and signal transduction. Activated FGFR enters the cell following recruitment to pre-formed clathrin-coated pits (CCPs). However, FGFR activation stimulates clathrin-mediated endocytosis; FGF treatment increases the number of CCPs, including those undergoing endocytosis, and this effect is mediated by Src and its phosphorylation target Eps8. Eps8 interacts with the clathrin-mediated endocytosis machinery and depletion of Eps8 inhibits FGFR trafficking and immediate Erk signalling. Once internalized, FGFR passes through peripheral early endosomes en route to recycling and degredative compartments, through an Src- and Eps8-dependent mechanism. Thus Eps8 functions as a key coordinator in the interplay between FGFR signalling and trafficking. This work provides the first detailed mechanistic analysis of growth factor receptor clustering at the cell surface through signal transduction and endocytic trafficking. As we have characterised the Src target Eps8 as a key regulator of FGFR signalling and trafficking, and identified the early endocytic system as the site of Eps8-mediated effects, this work provides novel mechanistic insight into the reciprocal regulation of growth factor receptor signalling and trafficking. PMID:23203811

  11. Cross-talk between the calcium-sensing receptor and the epidermal growth factor receptor in Rat-1 fibroblasts

    SciTech Connect

    Tomlins, Scott A.; Bollinger, Nikki; Creim, Jeffrey A.; Rodland, Karin D.

    2005-08-15

    The calcium-sensing receptor (CaR) is a G-protein coupled receptor that is activated by extracellular calcium (Ca2+o). Rat-1 fibroblasts have been shown to proliferate and increase ERK activity in response to elevation of [Ca2+]o, and these responses are dependent on functional CaR expression. In this report, we examined the role of cross-talk between the CaR and the epidermal growth factor receptor (EGFR) in mediating these responses in Rat-1 cells. This report shows that AG1478, a specific inhibitor of the EGFR kinase, significantly inhibits the increase in proliferation induced by elevated Ca2+o. Further, we show that AG1478 acts downstream or separately from G-protein subunit activation of phospholipase C. AG1478 significantly inhibits Ca2+o-stimulated ERK phosphorylation and in vitro kinase activity. A similar inhibition of ERK phosphorylation was observed in response to the inhibitor AG494. In addition, treatment with inhibitors of metalloproteases involved in shedding of membrane anchored EGF family ligands substantially inhibited the increase in ERK activation in response to elevated Ca2+o. This is consistent with the known expression of TGFa by Rat-1 cells. These results indicate that EGFR transactivation is an important component of the CaR mediated response to increased Ca2+o in Rat-1 fibroblasts, and most likely involves CaR-mediated induction of regulated proteolysis and ligand shedding.

  12. Functional annotation of novel lineage-specific genes using co-expression and promoter analysis

    PubMed Central

    2010-01-01

    Background The diversity of placental architectures within and among mammalian orders is believed to be the result of adaptive evolution. Although, the genetic basis for these differences is unknown, some may arise from rapidly diverging and lineage-specific genes. Previously, we identified 91 novel lineage-specific transcripts (LSTs) from a cow term-placenta cDNA library, which are excellent candidates for adaptive placental functions acquired by the ruminant lineage. The aim of the present study was to infer functions of previously uncharacterized lineage-specific genes (LSGs) using co-expression, promoter, pathway and network analysis. Results Clusters of co-expressed genes preferentially expressed in liver, placenta and thymus were found using 49 previously uncharacterized LSTs as seeds. Over-represented composite transcription factor binding sites (TFBS) in promoters of clustered LSGs and known genes were then identified computationally. Functions were inferred for nine previously uncharacterized LSGs using co-expression analysis and pathway analysis tools. Our results predict that these LSGs may function in cell signaling, glycerophospholipid/fatty acid metabolism, protein trafficking, regulatory processes in the nucleus, and processes that initiate parturition and immune system development. Conclusions The placenta is a rich source of lineage-specific genes that function in the adaptive evolution of placental architecture and functions. We have shown that co-expression, promoter, and gene network analyses are useful methods to infer functions of LSGs with heretofore unknown functions. Our results indicate that many LSGs are involved in cellular recognition and developmental processes. Furthermore, they provide guidance for experimental approaches to validate the functions of LSGs and to study their evolution. PMID:20214810

  13. Functional annotation of novel lineage-specific genes using co-expression and promoter analysis.

    PubMed

    Kumar, Charu G; Everts, Robin E; Loor, Juan J; Lewin, Harris A

    2010-03-09

    The diversity of placental architectures within and among mammalian orders is believed to be the result of adaptive evolution. Although, the genetic basis for these differences is unknown, some may arise from rapidly diverging and lineage-specific genes. Previously, we identified 91 novel lineage-specific transcripts (LSTs) from a cow term-placenta cDNA library, which are excellent candidates for adaptive placental functions acquired by the ruminant lineage. The aim of the present study was to infer functions of previously uncharacterized lineage-specific genes (LSGs) using co-expression, promoter, pathway and network analysis. Clusters of co-expressed genes preferentially expressed in liver, placenta and thymus were found using 49 previously uncharacterized LSTs as seeds. Over-represented composite transcription factor binding sites (TFBS) in promoters of clustered LSGs and known genes were then identified computationally. Functions were inferred for nine previously uncharacterized LSGs using co-expression analysis and pathway analysis tools. Our results predict that these LSGs may function in cell signaling, glycerophospholipid/fatty acid metabolism, protein trafficking, regulatory processes in the nucleus, and processes that initiate parturition and immune system development. The placenta is a rich source of lineage-specific genes that function in the adaptive evolution of placental architecture and functions. We have shown that co-expression, promoter, and gene network analyses are useful methods to infer functions of LSGs with heretofore unknown functions. Our results indicate that many LSGs are involved in cellular recognition and developmental processes. Furthermore, they provide guidance for experimental approaches to validate the functions of LSGs and to study their evolution.

  14. Co-expression of midkine and pleiotrophin predicts poor survival in human glioma.

    PubMed

    Ma, Jinyang; Lang, Bojuan; Wang, Xiongwei; Wang, Lei; Dong, Yuanxun; Hu, Huojun

    2014-11-01

    The aim of this study was to investigate whether co-expression of midkine (MK) and pleiotrophin (PTN) has prognostic relevance in human gliomas. Immunohistochemistry was used to investigate the expression of MK and PTN proteins in 168 patients with gliomas. The levels of MK and PTN mRNA in glioma tissues and paratumor tissues were evaluated in 45 paired cases by quantitative real-time polymerase chain reaction (qRT-PCR). Kaplan-Meier survival analysis was performed to assess prognostic significance. The expression levels of MK and PTN proteins in glioma tissue were both significantly higher (both p<0.001) than those in paratumor tissues on immunohistochemistry analysis, which was confirmed by qRT-PCR analysis. Additionally, the overexpression of either MK or PTN was significantly associated with the World Health Organization Grade (p=0.001 and 0.034, respectively), low Karnofsky Performance Status (KPS) score (p=0.022 and 0.001, respectively), time to recurrence (p=0.043 and 0.011, respectively) and poor overall survival (p=0.018 and 0.001, respectively). Multivariate Cox proportional-hazards regression analysis revealed that increased expressions of MK and PTN were both independent prognostic factors for poor overall survival (p=0.030 and 0.022, respectively). Furthermore, the co-expression of MK and PTN was more significantly (p=0.003) associated with adverse prognosis in patients with gliomas than the respective expression of MK or PTN alone. To our knowledge, these findings are the first to indicate that the co-expression of MK and PTN is significantly correlated with prognosis in glioma patients, suggesting that the co-expression of these proteins may be used as both an early diagnostic and independent prognostic marker. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Expression of endothelial cell-specific receptor tyrosine kinases and growth factors in human brain tumors.

    PubMed Central

    Hatva, E.; Kaipainen, A.; Mentula, P.; Jääskeläinen, J.; Paetau, A.; Haltia, M.; Alitalo, K.

    1995-01-01

    Key growth factor-receptor interactions involved in angiogenesis are possible targets for therapy of CNS tumors. Vascular endothelial growth factor (VEGF) is a highly specific endothelial cell mitogen that has been shown to stimulate angiogenesis, a requirement for solid tumor growth. The expression of VEGF, the closely related placental growth factor (PIGF), the newly cloned endothelial high affinity VEGF receptors KDR and FLT1, and the endothelial orphan receptors FLT4 and Tie were analyzed by in situ hybridization in normal human brain tissue and in the following CNS tumors: gliomas, grades II, III, IV; meningiomas, grades I and II; and melanoma metastases to the cerebrum. VEGF mRNA was up-regulated in the majority of low grade tumors studied and was highly expressed in cells of malignant gliomas. Significantly elevated levels of Tie, KDR, and FLT1 mRNAs, but not FLT4 mRNA, were observed in malignant tumor endothelia, as well as in endothelia of tissues directly adjacent to the tumor margin. In comparison, there was little or no receptor expression in normal brain vasculature. Our results are consistent with the hypothesis that these endothelial receptors are induced during tumor progression and may play a role in tumor angiogenesis. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7856749

  16. Human microvascular endothelial cells express receptors for platelet-derived growth factor

    SciTech Connect

    Beitz, J.G.; Kim, Insoon; Calabresi, P.; Frackelton, A.R. Jr. )

    1991-03-01

    Endothelial cells have been widely thought to be unresponsive to platelet-derived growth factor (PDGF, a major growth factor released from stimulated platelets at the sites of vascular insults) and devoid of PDGF receptors. Nevertheless, in examining the growth-factor responses of microvascular endothelial cells isolated from human omental adipose tissue, the authors were surprised to detect PDGF-induced tyrosine phosphorylation of a 180-kDa glycoprotein, subsequently identified as the cellular receptor for PDGF by specific immunoprecipitation. Scatchard analysis of {sup 125}I-labeled PDGF binding to human microvascular endothelial cells revealed 30,000 PDGF receptors per cell with a K{sub d} of 0.14 nM. Normal cellular consequences of receptor activation were also observed, including tyrosine phosphorylation of a 42-kDa protein and serine phosphorylation of ribosomal protein S6. Furthermore, PDGF was mitogenic for these cells. Microvascular endothelial cells play a central role in neovascularization required for wound healing and solid tumor growth. Thus, the discovery of functional PFDG receptors on human microvascular endothelial cells suggests a direct role for PDGF in this process.

  17. Self-renewing and differentiating properties of cortical neural stem cells are selectively regulated by basic fibroblast growth factor (FGF) signaling via specific FGF receptors.

    PubMed

    Maric, Dragan; Fiorio Pla, Alessandra; Chang, Yoong Hee; Barker, Jeffery L

    2007-02-21

    Developmental processes mediating the initiation of lineage commitment from self-renewing neural stem cells (NSCs) remain mostly unclear because of the persisting ambiguity in identifying true NSCs from proliferative lineage-restricted progenitors (LRPs), which are directly or indirectly derived from NSCs. Our multilineage immunohistochemical analyses of early embryonic rat telencephalon at the onset of neurogenesis revealed clear dorsoventral gradients in the emergence of two types of neuronal progenitors (NPs) from multilineage-negative NSCs. Enumeration of NSCs using comprehensive flow cytometric analysis demonstrated that their precipitous decline in vivo involved both active differentiation into NPs and an increased propensity toward apoptosis. Both processes paralleled the dorsoventral changes in fibroblast growth factor receptor (FGFR) expressions. NSCs residing in the dorsal telencephalon coexpressed FGFR1 and FGFR3, whereas those residing in the ventral telencephalon also expressed FGFR2. NSCs exposed to basic fibroblast growth factor (bFGF) in vitro generated four stereotypical clonal expansion states: efficiently self-renewing, inefficiently self-renewing limited by apoptosis, exclusively neurogenic, and multipotential, generating up to five types of LRPs. The plasticity among these expansion states depended on ambient [bFGF], telencephalic developmental stage, and differential activation/inactivation of specific FGFRs. Coactivation of FGFR1 and FGFR3 promoted symmetrical divisions of NSCs (self-renewal), whereas inactivation of either triggered asymmetrical divisions and neurogenesis from these cells. Developmental upregulation of FGFR2 expression correlated with a shift of NSCs into a multipotential state or apoptosis. These results provide new insights regarding the roles of FGFRs in diversification of NSC properties and initiation of neural lineage-restricted differentiation.

  18. Co-expression and co-localization of hub proteins and their partners are encoded in protein sequence.

    PubMed

    Feiglin, Ariel; Ashkenazi, Shaul; Schlessinger, Avner; Rost, Burkhard; Ofran, Yanay

    2014-04-01

    Spatiotemporal coordination is a critical factor in biological processes. Some hubs in protein-protein interaction networks tend to be co-expressed and co-localized with their partners more strongly than others, a difference which is arguably related to functional differences between the hubs. Based on numerous analyses of yeast hubs, it has been suggested that differences in co-expression and co-localization are reflected in the structural and molecular characteristics of the hubs. We hypothesized that if indeed differences in co-expression and co-localization are encoded in the molecular characteristics of the protein, it may be possible to predict the tendency for co-expression and co-localization of human hubs based on features learned from systematically characterized yeast hubs. Thus, we trained a prediction algorithm on hubs from yeast that were classified as either strongly or weakly co-expressed and co-localized with their partners, and applied the trained model to 800 human hub proteins. We found that the algorithm significantly distinguishes between human hubs that are co-expressed and co-localized with their partners and hubs that are not. The prediction is based on sequence derived features such as "stickiness", i.e. the existence of multiple putative binding sites that enable multiple simultaneous interactions, "plasticity", i.e. the existence of predicted structural disorder which conjecturally allows for multiple consecutive interactions with the same binding site and predicted subcellular localization. These results suggest that spatiotemporal dynamics is encoded, at least in part, in the amino acid sequence of the protein and that this encoding is similar in yeast and in human.

  19. Positive and Negative Cross-Talk between Lysophosphatidic Acid Receptor 1, Free Fatty Acid Receptor 4, and Epidermal Growth Factor Receptor in Human Prostate Cancer Cells.

    PubMed

    Hopkins, Mandi M; Liu, Ze; Meier, Kathryn E

    2016-10-01

    Lysophosphatidic acid (LPA) is a lipid mediator that mediates cellular effects via G protein-coupled receptors (GPCRs). Epidermal growth factor (EGF) is a peptide that acts via a receptor tyrosine kinase. LPA and EGF both induce proliferation of prostate cancer cells and can transactivate each other's receptors. The LPA receptor LPA1 is particularly important for LPA response in human prostate cancer cells. Previous work in our laboratory has demonstrated that free fatty acid 4 (FFA4), a GPCR activated by ω-3 fatty acids, inhibits responses to both LPA and EGF in these cells. One potential mechanism for the inhibition involves negative interactions between FFA4 and LPA1, thereby suppressing responses to EGF that require LPA1 In the current study, we examined the role of LPA1 in mediating EGF and FFA4 agonist responses in two human prostate cancer cell lines, DU145 and PC-3. The results show that an LPA1-selective antagonist inhibits proliferation and migration to both LPA and EGF. Knockdown of LPA1 expression, using silencing RNA, blocks responses to LPA and significantly inhibits responses to EGF. The partial response to EGF that is observed after LPA1 knockdown is not inhibited by FFA4 agonists. Finally, the role of arrestin-3, a GPCR-binding protein that mediates many actions of activated GPCRs, was tested. Knockdown of arrestin-3 completely inhibits responses to both LPA and EGF in prostate cancer cells. Taken together, these results suggest that LPA1 plays a critical role in EGF responses and that FFA4 agonists inhibit proliferation by suppressing positive cross-talk between LPA1 and the EGF receptor. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  20. The transcription factors Nur77 and retinoid X receptors participate in amphetamine-induced locomotor activities.

    PubMed

    Bourhis, Emmanuelle; Maheux, Jérôme; Paquet, Brigitte; Kagechika, Hiroyuki; Shudo, Koichi; Rompré, Pierre-Paul; Rouillard, Claude; Lévesque, Daniel

    2009-03-01

    The major substrate underlying amphetamine (AMPH)-induced locomotor activity is associated with dopamine forebrain circuits. Brain regions associated with AMPH-induced locomotor activity express high levels of retinoid receptors. However, the role of these transcription factors in dopamine-mediated effects remains poorly understood. Two nuclear receptor families, the retinoic acid receptors (RAR) and the retinoid X receptors (RXR), transduce retinoic acid signal. RARs are specifically involved in retinoid signaling, whereas RXRs also participate in other signaling pathways as partners for other nuclear receptors such as Nur77, an orphan member of the nuclear receptor family expresses in dopamine system. To explore the role of retinoid receptors and Nur77 in AMPH-induced locomotor activity, we administered selective retinoid receptor drugs in combination with AMPH in adult wild-type and Nur77-deficient mice. At a low dose, AMPH similarly increased ambulatory activity in wild-type and Nur77-deficient mice, while it did not alter non-ambulatory activity. At a high dose, AMPH did not alter ambulatory activity anymore, while non-ambulatory activity strongly increased in wild-type mice. Nur77-deficient mice still displayed a higher ambulatory activity with no change in non-ambulatory activity. HX531, a synthetic RXR antagonist, blocks AMPH-induced ambulatory activity, whereas RAR drugs tested remained without effect. Interestingly, the effect of HX531 was abolished in Nur77-deficient mice, suggesting that this orphan nuclear receptor is essential for the action of the RXR drug. This study shows that RXR and Nur77 participate in AMPH-induced locomotor activity and prompts for further investigations on the role of Nur77 and RXR in addiction and reward-related behaviors.

  1. Prostaglandin E2 regulates angiogenesis via activation of fibroblast growth factor receptor-1.

    PubMed

    Finetti, Federica; Solito, Raffaella; Morbidelli, Lucia; Giachetti, Antonio; Ziche, Marina; Donnini, Sandra

    2008-01-25

    Prostaglandin E(2) (PGE(2)) behaves as a mitogen in epithelial tumor cells as well as in many other cell types. We investigated the actions of PGE(2) on microvascular endothelial cells (capillary venular endothelial cells) with the purpose of delineating the signaling pathway leading to the acquisition of the angiogenic phenotype and to new vessel formation. PGE(2) (100 nM) produced activation of the fibroblast growth factor receptor 1 (FGFR-1), as measured by its phosphorylation, but not of vascular endothelial growth factor receptor 2. PGE(2) stimulated the EP3 subtype receptor, as deduced by abrogation of EP3 Galpha(i) subunit activity through pertussis toxin. Consistent with this result, in human umbilical venular endothelial cells missing the EP3 receptor, PGE(2) did not phosphorylate FGFR-1. Upon binding to its receptor, PGE(2) initiated an autocrine/paracrine signaling cascade involving the intracellular activation of c-Src, activation of matrix metalloproteinase (predominantly MMP2), which in turn caused the mobilization of membrane-anchored fibroblast growth factor-2 (FGF-2). In fact, in cells unable to release FGF-2 the transfection with both FGFR-1 and EP3 did not result in FGFR-1 phosphorylation in response to PGE(2). Relevance for the FGF2-FGFR-1 system was highlighted by confocal analysis, showing receptor internalization after cell exposure to the prostanoid. ERK1/2 appeared to be the distal signal involved, its phosphorylation being sensitive to either cSrc inhibitor or FGFR-1 blocker. Finally, PGE(2) stimulated cell migration and capillary formation in aortic rings, which were severely reduced by inhibitors of signaling molecules or by receptor antagonist. In conclusion, this study provides evidence for the involvement of FGFR-1 through FGF2 in eliciting PGE(2) angiogenic responses. This signaling pattern is similar to the autocrine-paracrine mechanism which operates in endothelial cells to support neovascular growth.

  2. Co-Expression Network and Pathway Analyses Reveal Important Modules of miRNAs Regulating Milk Yield and Component Traits.

    PubMed

    Do, Duy N; Dudemaine, Pier-Luc; Li, Ran; Ibeagha-Awemu, Eveline M

    2017-07-18

    Co-expression network analyses provide insights into the molecular interactions underlying complex traits and diseases. In this study, co-expression network analysis was performed to detect expression patterns (modules or clusters) of microRNAs (miRNAs) during lactation, and to identify miRNA regulatory mechanisms for milk yield and component traits (fat, protein, somatic cell count (SCC), lactose, and milk urea nitrogen (MUN)) via miRNA target gene enrichment analysis. miRNA expression (713 miRNAs), and milk yield and components (Fat%, Protein%, lactose, SCC, MUN) data of nine cows at each of six different time points (day 30 (D30), D70, D130, D170, D230 and D290) of an entire lactation curve were used. Four modules or clusters (GREEN, BLUE, RED and TURQUOISE) of miRNAs were identified as important for milk yield and component traits. The GREEN and BLUE modules were significantly correlated (|r| > 0.5) with milk yield and lactose, respectively. The RED and TURQUOISE modules were significantly correlated (|r| > 0.5) with both SCC and lactose. In the GREEN module, three abundantly expressed miRNAs (miR-148a, miR-186 and miR-200a) were most significantly correlated to milk yield, and are probably the most important miRNAs for this trait. DDR1 and DDHX1 are hub genes for miRNA regulatory networks controlling milk yield, while HHEX is an important transcription regulator for these networks. miR-18a, miR-221/222 cluster, and transcription factors HOXA7, and NOTCH 3 and 4, are important for the regulation of lactose. miR-142, miR-146a, and miR-EIA17-14144 (a novel miRNA), and transcription factors in the SMAD family and MYB, are important for the regulation of SCC. Important signaling pathways enriched for target genes of miRNAs of significant modules, included protein kinase A and PTEN signaling for milk yield, eNOS and Noth signaling for lactose, and TGF β, HIPPO, Wnt/β-catenin and cell cycle signaling for SCC. Relevant enriched gene ontology (GO)-terms related to

  3. Co-Expression Network and Pathway Analyses Reveal Important Modules of miRNAs Regulating Milk Yield and Component Traits

    PubMed Central

    Do, Duy N.; Dudemaine, Pier-Luc; Li, Ran; Ibeagha-Awemu, Eveline M.

    2017-01-01

    Co-expression network analyses provide insights into the molecular interactions underlying complex traits and diseases. In this study, co-expression network analysis was performed to detect expression patterns (modules or clusters) of microRNAs (miRNAs) during lactation, and to identify miRNA regulatory mechanisms for milk yield and component traits (fat, protein, somatic cell count (SCC), lactose, and milk urea nitrogen (MUN)) via miRNA target gene enrichment analysis. miRNA expression (713 miRNAs), and milk yield and components (Fat%, Protein%, lactose, SCC, MUN) data of nine cows at each of six different time points (day 30 (D30), D70, D130, D170, D230 and D290) of an entire lactation curve were used. Four modules or clusters (GREEN, BLUE, RED and TURQUOISE) of miRNAs were identified as important for milk yield and component traits. The GREEN and BLUE modules were significantly correlated (|r| > 0.5) with milk yield and lactose, respectively. The RED and TURQUOISE modules were significantly correlated (|r| > 0.5) with both SCC and lactose. In the GREEN module, three abundantly expressed miRNAs (miR-148a, miR-186 and miR-200a) were most significantly correlated to milk yield, and are probably the most important miRNAs for this trait. DDR1 and DDHX1 are hub genes for miRNA regulatory networks controlling milk yield, while HHEX is an important transcription regulator for these networks. miR-18a, miR-221/222 cluster, and transcription factors HOXA7, and NOTCH 3 and 4, are important for the regulation of lactose. miR-142, miR-146a, and miR-EIA17-14144 (a novel miRNA), and transcription factors in the SMAD family and MYB, are important for the regulation of SCC. Important signaling pathways enriched for target genes of miRNAs of significant modules, included protein kinase A and PTEN signaling for milk yield, eNOS and Noth signaling for lactose, and TGF β, HIPPO, Wnt/β-catenin and cell cycle signaling for SCC. Relevant enriched gene ontology (GO)-terms related to

  4. Polymorphism in the tumour necrosis factor receptor II gene is associated with circulating levels of soluble tumour necrosis factor receptors in rheumatoid arthritis

    PubMed Central

    Glossop, John R; Dawes, Peter T; Nixon, Nicola B; Mattey, Derek L

    2005-01-01

    Levels of soluble tumour necrosis factor receptors (sTNFRs) are elevated in the circulation of patients with rheumatoid arthritis (RA). Although these receptors can act as natural inhibitors of tumour necrosis factor-α, levels of sTNFRs in RA appear to be insufficient to prevent tumour necrosis factor-α induced inflammation. The factors that regulate circulating levels of sTNFRs are unclear, but polymorphisms in the tumour necrosis factor receptor genes may play a role. We investigated the relationship between polymorphisms in the tumour necrosis factor receptor I (TNF-RI) and II (TNF-RII) genes and levels of sTNFRs in two groups of Caucasian RA patients: one with early (disease duration ≤2 years; n = 103) and one with established disease (disease duration ≥5 years; n = 151). PCR restriction fragment length polymorphism analysis was used to genotype patients for the A36G polymorphism in the TNF-RI gene and the T676G polymorphism in TNF-RII. Levels of sTNFRs were measured using ELISA. We also isolated T cells from peripheral blood of 58 patients with established RA with known TNF-R genotypes, and release of sTNFRs into the culture medium was measured in cells incubated with or without phytohaemagglutinin. Serum levels of the two sTNFRs (sTNF-RI and sTNF-RII) were positively correlated in both populations, and the level of each sTNFR was significantly higher in the patients with established disease (P < 0.0001). Multiple regression analyses corrected for age, sex and disease duration revealed a significant trend toward decreasing sTNF-RI and sTNF-RII levels across the TNF-RII genotypes (TT > TG > GG) of patients with established disease (P for trend = 0.01 and P for trend = 0.03, respectively). A similar nonsignificant trend was seen for early disease. No relationship with the TNF-RI A36G polymorphism was observed. sTNFRs released by isolated T cells exhibited a similar trend toward decreasing levels according to TNF-RII genotype, although only the association

  5. Kinetics of the Attachment of Intrinsic Factor-Bound Cobamides to Ileal Receptors

    PubMed Central

    Mathan, V. I.; Babior, Bernard M.; Donaldson, Robert M.

    1974-01-01

    To determine whether the molecular configuration of vitamin B12 influences the attachment of intrinsic factor-vitamin B12 complex to ileal microvillous membrane receptor sites, we have examined the kinetics of uptake of intrinsic factor-bound cyanocobalamin by brush borders and microvillous membranes isolated from guinea pig ileum, and have compared this uptake with that of intrinsic factor alone and with that of intrinsic factor complexed with various analogs of cyanocobalamin. We first studied the kinetics of binding of cyanocobalamin and other cobamides to human gastric intrinsic factor. The binding of cyanocobalamin showed saturation kinetics and, at relatively high concentrations of cyanocobalamin, a Scatchard plot of binding was linear. The dissociation constant for the intrinsic factor-cyanocobalamin complex was 0.066 nM. When the binding of various vitamin B12 analogs to intrinsic factor was determined by competition experiments, the analogs could be separated into two categories: those with affinities similar to that of cyanocobalamin and those with affinities much lower than that of cyanocobalamin. The affinity of cyanocobalamin for intrinsic factor was not altered by various substitutions at the -CN position, while removal of a single amido group on the corrin ring of substitution of the dimethylbenzimidazole base greatly reduced affinity. Removal of the base totally abolished binding. These findings, confirming those reported by others, are consistent with the concept that the cyanocobalamin molecule fits into a “pocket” in the intrinsic factor molecule, with the nucleotide base facing inward and the -CN side of the planar corrin ring facing outward. We then investigated the attachment of intrinsic factor-bound cyanocobalamin to ileal receptor. Attachment to microvillous membranes showed saturation kinetics with a dissociation constant of 0.25 nM. Attachment was rapid and was 70% complete within 5 min; the second-order rate constant for attachment

  6. Epidermal growth factor and hepatocyte growth factor receptors collaborate to induce multiple biological responses in bovine mammary epithelial cells.

    PubMed

    Accornero, P; Martignani, E; Miretti, S; Starvaggi Cucuzza, L; Baratta, M

    2009-08-01

    The aim of this work was to explore whether epidermal growth factor (EGF) and hepatocyte growth factor (HGF) could increase the biological responses of a mammary epithelial cell line of bovine origin when added simultaneously. We also investigated a possible molecular mechanism underlying this cooperation. The development of mammary gland requires several circulating and locally produced hormones. Hepatocyte growth factor and its tyrosine kinase receptor, mesenchymal-epithelial transition factor (MET), are expressed and temporally regulated during mammary development and differentiation. Epidermal growth factor receptor and its ligands have also been implicated in the growth and morphogenesis of the mammary epithelium. Both EGF and HGF seem to exert a morphogenic program in this tissue; therefore, we hypothesized that these cytokines could act cooperatively in bovine mammary epithelial cells. We have already shown that the bovine BME-UV cell line, a nontumorigenic mammary epithelial line, expresses both MET and EGF receptor. Simultaneous treatment with HGF and EGF elicited an increase in proliferation, dispersion, degradation of extracellular matrix, and motility. Following EGF treatment, BME-UV mammary cells exhibited an increase in MET expression at both the mRNA and protein levels. Long-term treatment of BME-UV cells with HGF and EGF together increased the level of activation of the extracellular signal-regulated kinase 1/2 and protein kinase B signaling pathways when compared with HGF or EGF alone. These data outline a possible cooperative role of the EGF and HGF pathways and indicate that cross-talk between their respective receptors may modulate mammary gland development in the cow.

  7. Selective opioid growth factor receptor antagonists based on a stilbene isostere.

    PubMed

    Stockdale, David P; Titunick, Michelle B; Biegler, Jessica M; Reed, Jessie L; Hartung, Alyssa M; Wiemer, David F; McLaughlin, Patricia J; Neighbors, Jeffrey D

    2017-08-15

    As part of an ongoing drug development effort aimed at selective opioid receptor ligands based on the pawhuskin natural products we have synthesized a small set of amide isosteres. These amides were centered on lead compounds which are selective antagonists for the delta and kappa opioid receptors. The amide isomers revealed here show dramatically different activity from the parent stilbene compounds. Three of the isomers synthesized showed antagonist activity for the opioid growth factor (OGF)/opioid growth factor receptor (OGFR) axis which is involved in cellular and organ growth control. This cellular signaling mechanism is targeted by "low-dose" naltrexone therapy which is being tested clinically for multiple sclerosis, Crohn's disease, cancer, and wound healing disorders. The compounds described here are the first selective small molecule ligands for the OGF/OGFR system and will serve as important leads and probes for further study. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Predicting response to epidermal growth factor receptor-targeted therapy in colorectal cancer.

    PubMed

    Adams, Richard; Maughan, Tim

    2007-04-01

    The discovery over 20 years ago by the Nobel Laureate Stanley Cohen of epidermal growth factor and its receptor, followed by the recognition that this receptor is overexpressed in multiple cancer types, has been of phenomenal significance. From these events the 'Holy Grail' of targeted therapy has looked increasingly realistic. Over the last 5 years this work has come of age with the licensing of multiple agents targeting this important mitogenic pathway in multiple tumor types. However, these agents and the technology behind them, while impressive, have resulted in lower clinical response rates than anticipated. In this review we will focus on the epidermal growth factor receptor-targeted therapies in colorectal cancer, why our expectations from these therapies have not yet been fulfilled and how we may predict those cancers that are likely to respond or be resistant to these therapies through a greater appreciation of the intricacy, diversity and dynamism of cellular signaling mechanisms.

  9. The epidermal growth factor receptor (EGFR) in head and neck cancer: its role and treatment implications

    PubMed Central

    Zimmermann, Michel; Zouhair, Abderrahim; Azria, David; Ozsahin, Mahmut

    2006-01-01

    Epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptors. Its stimulation by endogenous ligands, EGF or transforming growth factor-alpha (TGF-α) results in activation of intracellular tyrosine kinase, therefore, cell cycle progression. High levels of EGFR expression are correlated with poor prognosis and resistance to radiation therapy in a variety of cancers, mostly in squamous-cell carcinoma of the head and neck (SCCHN). Blocking the EGFR by a monoclonal antibody results in inhibition of the stimulation of the receptor, therefore, in inhibition of cell proliferation, enhanced apoptosis, and reduced angiogenesis, invasiveness and metastases. The EGFR is a prime target for new anticancer therapy in SCCHN, and other agents in development include small molecular tyrosine kinase inhibitors and antisense therapies. PMID:16722544

  10. The interaction between the Drosophila secreted protein argos and the epidermal growth factor receptor inhibits dimerization of the receptor and binding of secreted spitz to the receptor.

    PubMed

    Jin, M H; Sawamoto, K; Ito, M; Okano, H

    2000-03-01

    Drosophila Argos (Aos), a secreted protein with an epidermal growth factor (EGF)-like domain, has been shown to inhibit the activation of the Drosophila EGF receptor (DER). However, it has not been determined whether Aos binds directly to DER or whether regulation of the DER activation occurs through some other mechanism. Using DER-expressing cells (DER/S2) and a recombinant DER extracellular domain-Fc fusion protein (DER-Fc), we have shown that Aos binds directly to the extracellular domain of DER with its carboxyl-terminal region, including the EGF-like domain. Furthermore, Aos can block the binding of secreted Spitz (sSpi), a transforming growth factor alpha-like ligand of DER, to the extracellular domain of DER. We observed that sSpi stimulates the dimerization of both the soluble DER extracellular domain (sDER) and the intact DER in the DER/S2 cells and that Aos can block the sSpi-induced dimerization of both sDER and intact DER. Moreover, we have shown that, by directly interacting with DER, Aos and SpiAos (a chimeric protein that is composed of the N-terminal region of Spi and the C-terminal region of Aos) inhibit the dimerization and phosphorylation of DER that are induced by DER's overexpression in the absence of sSpi. These results indicate that Aos exerts its inhibitory function through dual molecular mechanisms: by blocking both the receptor dimerization and the binding of activating ligand to the receptor. This is the first description of this novel inhibitory mechanism for receptor tyrosine kinases.

  11. Cytoplasmic domains determine signal specificity, cellular routing characteristics and influence ligand binding of epidermal growth factor and insulin receptors.

    PubMed Central

    Riedel, H; Dull, T J; Honegger, A M; Schlessinger, J; Ullrich, A

    1989-01-01

    The cell surface receptors for insulin and epidermal growth factor (EGF) both employ a tyrosine-specific protein kinase activity to fulfil their distinct biological roles. To identify the structural domains responsible for various receptor activities, we have generated chimeric receptor polypeptides consisting of major EGF and insulin receptor structural domains and examined their biochemical properties and cellular signalling activities. The EGF-insulin receptor hybrids are properly synthesized and transported to the cell surface, where they form binding competent structures that are defined by the origin of their extracellular domains. While their ligand binding affinities are altered, we find that these chimeric receptors are fully functional in transmitting signals across the plasma membrane and into the cell. Thus, EGF receptor and insulin receptor cytoplasmic domain signalling capabilities are independent of their new heterotetrameric or monomeric environments respectively. Furthermore, the cytoplasmic domains carry the structural determinants that define kinase specificity, mitogenic and transforming potential, and receptor routing. Images PMID:2583088

  12. Learning from Co-expression Networks: Possibilities and Challenges

    PubMed Central

    Serin, Elise A. R.; Nijveen, Harm; Hilhorst, Henk W. M.; Ligterink, Wilco

    2016-01-01

    Plants are fascinating and complex organisms. A comprehensive understanding of the organization, function and evolution of plant genes is essential to disentangle important biological processes and to advance crop engineering and breeding strategies. The ultimate aim in deciphering complex biological processes is the discovery of causal genes and regulatory mechanisms controlling these processes. The recent surge of omics data has opened the door to a system-wide understanding of the flow of biological information underlying complex traits. However, dealing with the corresponding large data sets represents a challenging endeavor that calls for the development of powerful bioinformatics methods. A popular approach is the construction and analysis of gene networks. Such networks are often used for genome-wide representation of the complex functional organization of biological systems. Network based on similarity in gene expression are called (gene) co-expression networks. One of the major application of gene co-expression networks is the functional annotation of unknown genes. Constructing co-expression networks is generally straightforward. In contrast, the resulting network of connected genes can become very complex, which limits its biological interpretation. Several strategies can be employed to enhance the interpretation of the networks. A strategy in coherence with the biological question addressed needs to be established to infer reliable networks. Additional benefits can be gained from network-based strategies using prior knowledge and data integration to further enhance the elucidation of gene regulatory relationships. As a result, biological networks provide many more applications beyond the simple visualization of co-expressed genes. In this study we review the different approaches for co-expression network inference in plants. We analyse integrative genomics strategies used in recent studies that successfully identified candidate genes taking advantage of

  13. Metabotropic glutamate receptor 1 mediates melanocyte transformation via transactivation of insulin-like growth factor 1 receptor.

    PubMed

    Teh, Jessica L F; Shah, Raj; Shin, Seung-Shick; Wen, Yu; Mehnert, Janice M; Goydos, James; Chen, Suzie

    2014-07-01

    Our laboratory previously described the oncogenic properties of metabotropic glutamate receptor 1 (mGluR1) in melanocytes. mGluR1 transformed immortalized mouse melanocytes in vitro and induced vigorous tumor formation in vivo. Subsequently, we observed the activation of PI3K/AKT in mGluR1-mediated melanocytic tumorigenesis in vivo. In particular, we identified AKT2 being the predominant isoform contributing to the activation of AKT. Suppression of Grm1 or AKT2 using an inducible Tet-R siRNA system resulted in a 60 or 30% reduction, respectively, in in vivo tumorigenesis. We show that simultaneous downregulation of Grm1 plus AKT2 results in a reduction of approximately 80% in tumor volumes, suggesting that both mGluR1 and AKT2 contribute to the tumorigenic phenotype in vivo. The discrepancy between the mild in vitro transformation characteristics and the aggressive in vivo tumorigenic phenotypes of these stable mGluR1-melanocytic clones led us to investigate the possible involvement of other growth factors. Here, we highlight a potential crosstalk network between mGluR1 and tyrosine kinase, insulin-like growth factor 1 receptor (IGF-1R). © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. KRÜPPEL-LIKE FACTOR 9 AND REGULATION OF ENDOMETRIAL ESTROGEN RECEPTOR-ALPHA SIGNALING

    USDA-ARS?s Scientific Manuscript database

    Endometrial cancer risk is linked to aberrant estrogen receptor-alpha (ER alpha) signaling caused by increased ER alpha activation due to hyper-estrogenic environments or mutations in growth-regulatory factors. We had shown that ER alpha signaling is attenuated by the Sp1-related transcription facto...

  15. MECHANISMS OF ZN-INDUCED SIGNAL INITIATION THROUGH THE EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR)

    EPA Science Inventory

    MECHANISMS OF Zn-INDUCED SIGNAL INITIATION THROUGH THE EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR)
    James M. Samet*, Lee M. Graves? and Weidong Wu?. *Human Studies Division, NHEERL, ORD, Research Triangle Park, NC 27711, and ?Center for Environmental Medicine, University of North C...

  16. Brain-derived neurotrophic factor in human subjects with function-altering melanocortin-4 receptor variants

    USDA-ARS?s Scientific Manuscript database

    In rodents, hypothalamic brain-derived neurotrophic factor (BDNF) expression appears to be regulated by melanocortin-4 receptor (MC4R) activity. The impact of MC4R genetic variation on circulating BDNF in humans is unknown. The objective of this study is to compare BDNF concentrations of subjects wi...

  17. Epidermal Growth Factor Receptor Overexpression as a Target for Auger Electron Radiotherapy of Breast Cancer

    DTIC Science & Technology

    2002-08-01

    perfused rat liver: ligand and receptor dynamics. J . Cell Biol. 1984; 98: 2148-2159. 5. Fisher DA, Salido EC, Barajas L. Epidermal growth factor and the...Appendices .................................... .................................------------------------------------------------------ 21 Preprint 1. Wang J ...tumor uptake. Cancer Biother. and Radiopharm. (in press) 2002. Reprint 2. Wang J , Chen P, Su Z-F et al. Amplified delivery of indium- 111 to EGFR

  18. An Analysis of Trends and Growth Factor Receptor Expression of GI Carcinoid Tumors

    PubMed Central

    Bowen, Kanika A.; Silva, Scott R.; Johnson, Jessica N.; Doan, Hung Q.; Jackson, Lindsey N.; Gulhati, Pat; Qiu, Suimin; Riall, Taylor S.; Evers, B. Mark

    2009-01-01

    The purpose of our study was twofold: 1) to determine the incidence, patient and tumor characteristics, and outcome of patients with GI carcinoid tumors using the Surveillance, Epidemiology and End Results (SEER) database, and 2) to delineate the expression pattern of growth factor receptors (GFRs) in carcinoid tumors. The SEER database search provided information on patients diagnosed with carcinoid tumors from 1990–2002. Carcinoid tumor sections (n = 46) were stained for the GFRs: epidermal growth factor receptor (EGFR), insulin-like growth factor receptor (IGFR), vascular endothelial growth factor receptor (VEGFR), and HER-2/neu. Over the 12 year analysis period, 18,180 patients were identified with carcinoid tumors of the foregut, midgut, and hindgut; the incidence of carcinoid tumors increased ~2-fold during this time period. Of the patients with carcinoid tumors, there was a trend of increased expression of VEGF-R and IGF-R, particularly in the foregut and midgut carcinoids. Analysis of the SEER database confirms that the incidence of carcinoid tumors is increasing with an approximate doubling in the number of carcinoid cases from 1990–2002. Furthermore, an increase in VEGF-R and IGF-R expression suggests that GFR inhibitors may be effective adjuvant therapy for carcinoid cancer. PMID:19582519

  19. Immunohistochemical expression of Type IV Collagen and Autocrine Motility Factor Receptor in Odontogenic Tumours

    PubMed Central

    Sethi, Sneha

    2014-01-01

    Background: Autocrine motility factor receptor (AMFR) is a tumour motility stimulating protein secreted by tumour cells. The protein encoded by this gene is a glycosylated transmembrane protein and a receptor for autocrine motility factor. It has been known to play a role in progression of neoplastic lesions. Basement membranes are specialized extracellular matrices that serve as structural barriers as well as substrates for cellular interactions. The network of type IV collagen is thought to define the scaffold integrating other components such as laminins and perlecan into highly organized supramolecular architecture. The aim of this study was to determine and evaluate the immunohistochemical expression of Type IV Collagen and Autocrine motility factor receptor in odontogenic lesions. Materials and Methods: Immunohistochemical expression of Type IV Collagen and Autocrine motility factor receptor was evaluated in 31 odontogenic lesions, including unicystic ameloblastoma, multicystic ameloblastoma, keratocystic odontogenic tumour and ameloblastic carcinoma. Normal follicular tissue formed the control. Results: Maximum expression for Type IV Collagen was seen in multicystic ameloblastoma and minimum expression in keratocystic odontogenic tumour. The maximum expression of AMFR was seen in ameloblastic carcinoma and minimum expression in multicystic ameloblastoma. Conclusion: The results of this study suggested an association of loss of expression of type IV Collagen with progression of lesion. AMFR expression was found to be associated with the aggressive potential of tumours. PMID:25478440

  20. MECHANISMS OF ZN-INDUCED SIGNAL INITIATION THROUGH THE EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR)

    EPA Science Inventory

    MECHANISMS OF Zn-INDUCED SIGNAL INITIATION THROUGH THE EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR)
    James M. Samet*, Lee M. Graves? and Weidong Wu?. *Human Studies Division, NHEERL, ORD, Research Triangle Park, NC 27711, and ?Center for Environmental Medicine, University of North C...

  1. Dynamic expression of neurotrophic factor receptors in postnatal spinal motoneurons and in mouse model of ALS.

    PubMed

    Zhang, Jiasheng; Huang, Eric J

    2006-07-01

    Neurotrophic factors support the survival of spinal motoneurons (MNs) and have been considered as strong candidates for treating motoneuron diseases. However, it is unclear if the right combination of neurotrophic factor receptors is present in postnatal spinal MNs. In this study, we show that the level of c-ret expression remains relatively stable in embryonic and postnatal spinal MNs. In contrast, the mRNA and protein of GFRalpha1 and -2 are progressively down-regulated in postnatal life. By 3 and 6 months of age, both receptors are barely detectable in spinal MNs. The down-regulation of GFRalpha1 appears accelerated in transgenic mice expressing mutant SOD1(G93A). Despite the progressive loss of GFRalpha1 and -2, phosphorylation of c-ret shows no detectable reduction on tyrosine residues or on serine 696. In addition to the GFRalpha subunits, expression of TrkB also shows a dynamic change. During embryogenesis, there is twice as much full-length TrkB as the truncated TrkB isoform. However, this ratio is reversed in postnatal spinal cord. Expression of the mutant SOD1(G93A) appears to have no effect on the TrkB receptor ratio. Taken together, our data indicate that the expression of neurotrophic factor receptors, GFRalpha1, -2, and TrkB, is not static, but undergoes dynamic changes in postnatal spinal MNs. These results provide insights into the use of neurotrophic factors as therapeutic agents for ALS.

  2. Homozygous nonsense mutation in the insulin receptor gene of a patient with severe congenital insulin resistance: leprechaunism and the role of the insulin-like growth factor receptor.

    PubMed

    Jospe, N; Kaplowitz, P B; Furlanetto, R W

    1996-08-01

    Severe congenital insulin resistance in the syndrome of leprechaunism is caused by mutations in the insulin receptor gene. We report a patient with leprechaunism who was homozygous for a mutation resulting in the absence of cell surface insulin receptors. To determine whether the receptor for Insulin-like growth factor-I (IGF-I) is involved in the phenotype of leprechaunism, we studied the effect of insulin and of IGF-I on cells from this patient. The patient had a homozygous C-->T substitution at base pair 8212 in exon 12 of the insulin receptor gene, creating a premature stop codon. This nonsense mutation is in the extracellular portion of the receptor and truncates the insulin receptor proximal to its transmembrane anchor, resulting in the absence of cell surface insulin receptors. This finding indicates that complete absence of the insulin receptor is compatible with life. Secondly, DNA synthesis was studied in skin derived fibroblasts in response to increasing concentrations of either insulin or Insulin-like growth factor-I (IGF-I), and was assessed by 3H-thymidine incorporation. In this patient's cells, both of these hormones increased 3H-thymidine incorporation, and the effect was blocked by alpha-IR3, a monoclonal antibody that blocks activation of the IGF-I receptor. These findings confirmed the absence of the insulin receptor and indicated that insulin acts here through activation of the IGF-I receptor. These data support the contention that the phenotypic and metabolic abnormalities of leprechaunism result from the combination of lack of insulin receptor action and over-activation by insulin of the type 1 IGF receptor.

  3. Synthesis and Evaluation of Candidate PET Radioligands for Corticotropin-Releasing Factor Type-1 Receptors

    PubMed Central

    Lodge, Nicholas J.; Li, Yu-Wen; Chin, Frederick T.; Dischino, Douglas D.; Zoghbi, Sami S.; Deskus, Jeffrey A.; Mattson, Ronald J.; Imaizumo, Masao; Pieschl, Rick; Molski, Thaddeus F.; Fujita, Masahiro; Dulac, Heidi; Zaczek, Robert; Bronson, Joanne J.; Macor, John E.; Innis, Robert B.; Pike, Victor W.

    2014-01-01

    Introduction A radioligand for measuring the density of corticotrophin-releasing factor subtype-1 receptors (CRF1 receptors) in living animal and human brain with positron emission tomography (PET) would be a useful tool for neuropsychiatric investigations and the de