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Sample records for protein-4 gene bmp4

  1. Fine mapping of the human bone morphogenetic protein-4 gene (BMP4) to chromosome 14q22-q23 by in situ hybridization

    SciTech Connect

    Wijngaard, A. van den; Boersma, C.J.C.; Olijve, W.

    1995-06-10

    Bone morphogenetic protein-4 (BMP-4) is a member of the transforming growth factor-{beta} (TGF-{beta}) superfamily and is involved in morphogenesis and bone cell differentiation. Recombinant BMP-4 can induce ectopic cartilage and bone formation when implanted subcutaneously or intramuscularly in rodents. This ectopic bone formation process resembles the process of bone formation during embryogenesis and fracture healing. A cosmid clone containing the complete human bone morphogenetic protein-4 gene (BMP4) was isolated (details to be published elsewhere) and used as a probe to determine the precise chromosomal localization of the human BMP4 gene. This cosmid clone was labeled with biotin-14-dATP and hybridized in situ to chromosomal preparations of metaphase cells as described previously. In 20 metaphase preparations, an intense and specific fluorescence signal (FITC) was detected on the q arm of chromosome 14. The DAPI-counterstained chromosomes were computer-converted into GTG-like banding patterns, allowing the regional localization of BMP4 within 14q22-q23. 10 refs., 1 fig.

  2. Cathepsin H indirectly regulates morphogenetic protein-4 (BMP-4) in various human cell lines

    PubMed Central

    Rojnik, Matija; Jevnikar, Zala; Mirkovic, Bojana; Janes, Damjan; Zidar, Nace; Kikelj, Danijel; Kos, Janko

    2011-01-01

    Background Cathepsin H is a cysteine protease considered to play a major role in tumor progression, however, its precise function in tumorigenesis is unclear. Cathepsin H was recently proposed to be involved in processing of bone morphogenetic protein 4 (BMP-4) in mice. In order to clarify whether cathepsin H also regulates BMP-4 in humans, its impact on BMP-4 expression, processing and degradation was investigated in prostate cancer (PC-3), osteosarcoma (HOS) and pro-monocytic (U937) human cell lines. Materials and methods BMP-4 expression was founded to be regulated by cathepsin H using PCR array technology and confirmed by real time PCR. Immunoassays including Western blot and confocal microscopy were used to evaluate the influence of cathepsin H on BMP-4 processing. Results In contrast to HOS, the expression of BMP-4 mRNA in U937 and PC3 cells was significantly decreased by cathepsin H. The different regulation of BMP-4 synthesis could be associated with the absence of the mature 28 kDa cathepsin H form in HOS cells, where only the intermediate 30 kDa form was observed. No co-localization of BMP-4 and cathepsin H was observed in human cell lines and the multistep processing of BMP-4 was not altered in the presence of specific cathepsin H inhibitor. Isolated cathepsin H does not cleave mature recombinant BMP-4, neither with its amino- nor its endopeptidase activity. Conclusions Our results exclude direct proteolytic processing of BMP-4 by cathepsin H, however, they provide support for its involvement in the regulation of BMP-4 expression. PMID:22933963

  3. Comparison of newly developed anti-bone morphogenetic protein 4 llama-derived antibodies with commercially available BMP4 inhibitors

    PubMed Central

    Calpe, Silvia; Correia, Ana C. P.; Sancho-Serra, Maria del Carmen; Krishnadath, Kausilia K.

    2016-01-01

    ABSTRACT Due to improved understanding of the role of bone morphogenetic protein 4 (BMP4) in an increasing number of diseases, the development of selective inhibitors of BMP4 is an attractive therapeutic option. The currently available BMP4 inhibitors are not suitable as therapeutics because of their low specificity and low effectiveness. Here, we compared newly generated anti-BMP4 llama-derived antibodies (VHHs) with 3 different types of commercially available BMP4 inhibitors, natural antagonists, small molecule BMPR inhibitors and conventional anti-BMP4 monoclonal antibodies. We found that the anti-BMP4 VHHs were as effective as the natural antagonist or small molecule inhibitors, but had higher specificity. We also showed that commercial anti-BMP4 antibodies were inferior in terms of both specificity and effectiveness. These findings might result from the fact that the VHHs C4C4 and C8C8 target a small region within the BMPR1 epitope of BMP4, whereas the commercial antibodies target other areas of the BMP4 molecule. Our results show that the newly developed anti-BMP4 VHHs are promising antibodies with better specificity and effectivity for inhibition of BMP4, making them an attractive tool for research and for therapeutic applications. PMID:26967714

  4. The impact of bone morphogenetic protein 4 (BMP4) on breast cancer metastasis in a mouse xenograft model.

    PubMed

    Ampuja, M; Alarmo, E L; Owens, P; Havunen, R; Gorska, A E; Moses, H L; Kallioniemi, A

    2016-06-01

    Bone morphogenetic protein 4 (BMP4) is a key regulator of cell proliferation and differentiation. In breast cancer cells, BMP4 has been shown to reduce proliferation in vitro and interestingly, in some cases, also to induce migration and invasion. Here we investigated whether BMP4 influences breast cancer metastasis formation by using a xenograft mouse model. MDA-MB-231 breast cancer cells were injected intracardially into mice and metastasis formation was monitored using bioluminescence imaging. Mice treated with BMP4 developed metastases slightly earlier as compared to control animals but the overall number of metastases was similar in both groups (13 in the BMP4 group vs. 12 in controls). In BMP4-treated mice, bone metastases were more common (10 vs. 7) but adrenal gland metastases were less frequent (1 vs. 5) than in controls. Immunostaining revealed no differences in signaling activation, proliferation rate, blood vessel formation, EMT markers or the number of cancer-associated fibroblasts between the treatment groups. In conclusion, BMP4 caused a trend towards accelerated metastasis formation, especially in bone. More work is needed to uncover the long-term effects of BMP4 and the clinical relevance of these findings. PMID:26970275

  5. Additive Effects of Retinoic Acid (RA) and Bone Morphogenetic Protein 4 (BMP-4) Apoptosis Signaling in Retinoblastoma Cell Lines

    PubMed Central

    Müller, Patrick; Doliva, Rebekka; Busch, Maike; Philippeit, Claudia; Stephan, Harald; Dünker, Nicole

    2015-01-01

    Retinoids have been shown to serve promising therapeutic agents for human cancers, e.g. the treatment of neuroblastoma. Synthetic retinoids, specific for particular retinoic acid (RA) receptors, are tested as new therapy strategies. In the present study, application of recombinant retinoic acid (RA) lowers retinoblastoma (RB) cell viability and induces apoptosis in RB cell lines. Combined treatment of RA and bone morphogenetic protein 4 (BMP-4) increases the pro-apoptotic effect of RA in the RB cells lines WERI-Rb1, Y-79, RB355, RBL-30 and RBL-15, indicating an additive effect. We could show that in WERI-Rb1 cells RA/BMP-4 mediated cell death is at least partially caspase-dependent, whereby RA and BMP-4 additively increased (i) Apaf-1 mRNA levels, (ii) caspase-9 cleavage activity and (iii) the number of activated, cleaved caspase-3 positive cells. Compared to single application of RA and BMP-4, combined RA/BMP-4 treatment significantly augments mRNA levels of the retinoic acid receptors (RARs) RARα and RARß and the retinoic X receptor (RXR) RXRγ suggesting an interaction in the induction of these RA receptor subtypes in WERI-Rb1 cells. Agonist studies revealed that both, RARs and RXRs are involved in RA/BMP-4 mediated apoptosis in WERI-Rb1 retinoblastoma cells. Employing specific RAR subtype antagonists and a RXRß and RXRγ knockdown, we proved that RA/BMP-4 apoptosis signaling in WERI-Rb1 cells requires the RA receptor subtypes RARα, RARß, RXRß and RXRγ. Deciphering signaling mechanisms underlying apoptosis induction of RA and BMP-4 in WERI-Rb1 cells, our study provides useful starting-points for future retinoid-based therapy strategies in retinoblastoma. PMID:26173116

  6. Molecular characterization, expression and methylation status analysis of BMP4 gene in skin tissue of Liaoning cashmere goat during hair follicle cycle.

    PubMed

    Bai, Wen L; Dang, Yun L; Wang, Jiao J; Yin, Rong H; Wang, Ze Y; Zhu, Yu B; Cong, Yu Y; Xue, Hui L; Deng, Liang; Guo, Dan; Wang, Shi Q; Yang, Shu H

    2016-08-01

    Bone morphogenetic protein 4 (BMP4) is a member of the bone morphogenetic protein family (BMPs). It is involved in the development and cycle of hair follicle, as well as, is thought to be a potential candidate gene for cashmere traits in goats. In the present study, we isolated and characterized a full-length open reading frame (ORF) of BMP4 cDNA from the skin tissue of Liaoning cashmere goat, and investigated the transcriptional pattern and methylation status of BMP4 gene in skin tissue of this breed during different stages of hair follicle cycle. The sequence analysis indicated that the isolated cDNA was 1264-bp in length containing a complete ORF of 1230-bp. It encoded a precursor peptide of 409 amino acids with a signal peptide of 19 amino acids. The structural analysis indicated that goat BMP4 contains typical TGF-β propeptide and TGF-β domains. In skin tissue, BMP4 is generally transcribed in an ascendant pattern from anagen to telogen. The methylation level of 5' flanking regulatory region of BMP4 gene might be involved in its mRNA expression in skin tissue: a higher BMP4 methylation level in skin coincides with a lower expression of BMP4 mRNA. These results from the present work provided a foundation for further insight into the functional and regulatory characteristics of BMP4 in the development and cycle of hair follicle in Liaoning Cashmere goat. PMID:27406581

  7. Haploinsufficiency of BMP4 gene may be the underlying cause of Frías syndrome.

    PubMed

    Martínez-Fernández, María Luisa; Bermejo-Sánchez, Eva; Fernández, Belén; MacDonald, Alexandra; Fernández-Toral, Joaquín; Martínez-Frías, María Luisa

    2014-02-01

    In 2005, we reported on a family as having Frías syndrome (OMIM: 609640), with four affected members displaying a pattern of congenital defects nearly identical to those observed in a mother and son described by Frias [Frías et al. (1975). Birth Defects Orig Artic Ser 11:30-33]. These defects included growth deficiency, facial anomalies, and hand and foot alterations. We had the opportunity to study this family again due to the birth of another affected girl, who presented with similar facial characteristics to those of her elder half-sister and the rest of affected relatives, which consisted of mild exophthalmia, bilateral palpebral ptosis, downslanting palpebral fissures, and hypertelorism. We performed array-CGH, which identified an identical interstitial deletion of chromosome 14q22.1-q22.3 in the mother and two daughters. The deletion is 4.06 Mb in length and includes the BMP4 gene, a member of the bone morphogenetic protein (BMP) family of secreted proteins. A review of the literature showed that deletions or mutations of this gene underlie congenital defects affecting brain, eye, teeth, and digit development. Although the clinical manifestations of the current family correlate with the defects observed in patients having either 14q22-q23 deletions or mutations of BMP4, they show a milder phenotype. In order to understand the clinical variability, we evaluated the already known functional characteristics of the BMP gene members. This gene family plays an important role during early embryogenesis, and the complex synergistic functions and redundancies of the BMPs led us to conclude that haploinsufficiency of BMP4 is likely to be responsible for the clinical expression of Frías syndrome. PMID:24311462

  8. Single-molecule imaging of BMP4 dimerization on human periodontal ligament cells.

    PubMed

    Mi, H-W; Lee, M-C; Chiang, Y-C; Chow, L-P; Lin, C-P

    2011-11-01

    We expressed bone morphogenetic protein 4 (BMP4) fused with enhanced green fluorescent protein (BMP4-EGFP) in the secretory pathways of producer cells. Fluorescent EGFP was acquired only after we interrupted the transport of BMP4-EGFP by culturing cells at a lower temperature (20°C), and the dynamics of BMP4-EGFP could be monitored by single-molecule microscopy. Western blotting analysis confirmed that exposure to low temperature helped the integrated formation of BMP4-EGFP fusion proteins. In this study, for the first time, we could image the fluorescently labeled BMP4 molecules localized on the plasma membrane of living hPDL cells. The one-step photobleaching with EGFP and the "blinking" behavior of quantum dots suggest that the fluorescent spots represent the events of single BMP4 molecules. Single-molecule tracking showed that the BMP receptors (BMPR) dimerize after BMP4 stimulation, or that a complex of one BMP4 molecule and a pre-formed BMPR dimer develops first, followed by the binding of the second BMP4 molecule. Furthermore, BMP4-EGFP enhanced the osteogenic differentiation of hPDL cells via signal transduction involving BMP receptors. This single-molecule imaging technique might be a valuable tool for the future development of BMP4 gene therapy and regenerative medicine mediated by hPDLs. PMID:21841042

  9. Mutations in BMP4 Cause Eye, Brain, and Digit Developmental Anomalies: Overlap between the BMP4 and Hedgehog Signaling Pathways

    PubMed Central

    Bakrania, Preeti; Efthymiou, Maria; Klein, Johannes C.; Salt, Alison; Bunyan, David J.; Wyatt, Alex; Ponting, Chris P.; Martin, Angela; Williams, Steven; Lindley, Victoria; Gilmore, Joanne; Restori, Marie; Robson, Anthony G.; Neveu, Magella M.; Holder, Graham E.; Collin, J Richard O.; Robinson, David O.; Farndon, Peter; Johansen-Berg, Heidi; Gerrelli, Dianne; Ragge, Nicola K.

    2008-01-01

    Developmental ocular malformations, including anophthalmia-microphthalmia (AM), are heterogeneous disorders with frequent sporadic or non-Mendelian inheritance. Recurrent interstitial deletions of 14q22-q23 have been associated with AM, sometimes with poly/syndactyly and hypopituitarism. We identify two further cases of AM (one with associated pituitary anomalies) with a 14q22-q23 deletion. Using a positional candidate gene approach, we analyzed the BMP4 (Bone Morphogenetic Protein-4) gene and identified a frameshift mutation (c.226del2, p.S76fs104X) that segregated with AM, retinal dystrophy, myopia, brain anomalies, and polydactyly in a family and a nonconservative missense mutation (c.278A→G, p.E93G) in a highly conserved base in another family. MR imaging and tractography in the c.226del2 proband revealed a primary brain developmental disorder affecting thalamostriatal and callosal pathways, also present in the affected grandmother. Using in situ hybridization in human embryos, we demonstrate expression of BMP4 in optic vesicle, developing retina and lens, pituitary region, and digits strongly supporting BMP4 as a causative gene for AM, pituitary, and poly/syndactyly. Because BMP4 interacts with HH signaling genes in animals, we evaluated gene expression in human embryos and demonstrate cotemporal and cospatial expression of BMP4 and HH signaling genes. We also identified four cases, some of whom had retinal dystrophy, with “low-penetrant” mutations in both BMP4 and HH signaling genes: SHH (Sonic Hedgehog) or PTCH1 (Patched). We propose that BMP4 is a major gene for AM and/or retinal dystrophy and brain anomalies and may be a candidate gene for myopia and poly/syndactyly. Our finding of low-penetrant variants in BMP4 and HH signaling partners is suggestive of an interaction between the two pathways in humans. PMID:18252212

  10. α1-adrenergic receptor signaling in osteoblasts regulates clock genes and bone morphogenetic protein 4 expression through up-regulation of the transcriptional factor nuclear factor IL-3 (Nfil3)/E4 promoter-binding protein 4 (E4BP4).

    PubMed

    Hirai, Takao; Tanaka, Kenjiro; Togari, Akifumi

    2014-06-13

    Several studies have demonstrated that the α1-adrenergic receptor (AR) plays an important role in regulating cell growth and function in osteoblasts. However, the physiological role of α1-AR signaling in bone metabolism is largely unknown. In this study, the stimulation of phenylephrine (PHE), a nonspecific α1-AR agonist, increased the transcriptional factor Nfil3/E4BP4 and led to the rhythmic expression of bone morphogenetic protein 4 (Bmp4) in MC3T3-E1 osteoblastic cells. We also showed that Bmp4 mRNA expression peaked in bone near zeitgeber time 8 in a 24-h rhythm. Furthermore, the expression of Nfil3 and Bmp4 displayed a circadian pattern with opposing phases, which suggested that Nfil3 repressed the expression of the Bmp4 gene during a circadian cycle. On a molecular level, both loss-of-function and gain-of-function experiments demonstrated that Nfil3/E4BP4 negatively regulated Bmp4 expression in osteoblasts. Furthermore, the systemic administration of PHE increased the expression of Nfil3 mRNA in bone, whereas it decreased that of Bmp4 mRNA. The expression of Bmp4 mRNA was decreased significantly by exposure to PHE, and this was concomitant with the increase in Nfil3 binding to the D-box-containing Bmp4 promoter region in MC3T3-E1 cells, which indicates that the expression of Nfil3 by α1-AR signaling can bind directly to the Bmp4 promoter and inhibit Bmp4 expression in osteoblasts. Our results suggest that α1-AR signaling regulates clock genes and Bmp4 expression in osteoblasts. Moreover, α1-AR signaling negatively regulated Bmp4 expression by up-regulating the transcriptional factor Nfil3/E4BP4 in osteoblasts. PMID:24794868

  11. Identification and analysis of a novel bmp4 enhancer in Fugu genome.

    PubMed

    Wang, Ying; Hutson, Lara D; Chen, Yiping; Guan, Guoqiang

    2015-04-01

    Spatiotemporal expression of bone morphogenetic protein 4 (Bmp4) in epithelial and mesenchymal cells is critical for the development of many organs including teeth. Since Bmp4 has a complex and widespread regulatory area in mammals, the tissue-specific enhancers that are responsible for mesenchymal expression of Bmp4 are difficult to identify in mammals. TakiFugu rubripes (Fugu, pufferfish) has a highly compact genome size and is widely used in comparative genomics studies of gene regulatory mechanisms. In this study, we used the Fugu genome to evaluate the 15kb promoter region upstream of the Fugu bmp4 gene. By DNA segmental cloning and luciferase assay with two dental odontoblast-like cell lines, a dental ameloblast-like cell line, and a kidney fibroblast cell line, we identified a 485bp cis-regulatory enhancer between -4213 and -3728bp of the Fugu bmp4 gene. This enhancer showed strong transcriptional activity in all three dental cell lines and, to a lesser extent, also in kidney fibroblast cells. Though not located in an evolutionary conserved region, the enhancer activity for the DNA segment is intense. This is the first time a bmp4 enhancer sequence with activity in both mesenchymal and epithelial cells has been identified, which will help to decode the mechanism of tooth development in vertebrates. PMID:25594624

  12. Involvement of Ras/Raf/AP-1 in BMP-4 signaling during Xenopus embryonic development.

    PubMed Central

    Xu, R H; Dong, Z; Maeno, M; Kim, J; Suzuki, A; Ueno, N; Sredni, D; Colburn, N H; Kung, H F

    1996-01-01

    Previously, we elucidated the role of bone morphogenetic protein 4 (BMP-4) in the dorsal-ventral patterning of the Xenopus embryo by using a dominant negative mutant of the BMP-4 receptor (DN-BR). The present paper describes the involvement of Ras, Raf, and activator protein 1 (AP-1) in BMP-4 signaling during Xenopus embryonic development. The AP-1 activity was determined by injecting an AP-1-dependent luciferase reporter gene into two-cell-stage Xenopus embryos and measuring the luciferase activity at various developmental stages. We found that injection of BMP-4 mRNA increased AP-1 activity, whereas injection of DN-BR mRNA inhibited AP-1 activity. Similar inhibitory effects were seen with injection of mRNAs encoding dominant negative mutants of c-Ha-Ras, c-Raf, or c-Jun. These results suggest that the endogenous AP-1 activity is regulated by BMP-4/Ras/Raf/Jun signals. We next investigated the effects of Ras/Raf/AP-1 signals on the biological functions of BMP-4. DN-BR-induced dorsalization of the embryo, revealed by the formation of a secondary body axis or dorsalization of the ventral mesoderm explant analyzed by histological and molecular criteria, was significantly reversed by coinjection of [Val12]Ha-Ras, c-Raf, or c-Jun mRNA. Furthermore, the BMP-4-stimulated erythroid differentiation in the ventral mesoderm was substantially inhibited by coinjection with the dominant negative c-Ha-Ras, c-Raf, or c-Jun mutant. Our results suggest the involvement of Ras/Raf/AP-1 in the BMP-4 signaling pathway. Images Fig. 2 Fig. 3 Fig. 4 PMID:8570644

  13. Thrombopoietin induces hematopoiesis from mouse ES cells via HIF-1α-dependent activation of a BMP4 autoregulatory loop.

    PubMed

    Pramono, Andri; Zahabi, Azadeh; Morishima, Tatsuya; Lan, Dan; Welte, Karl; Skokowa, Julia

    2016-07-01

    Understanding the molecular mechanisms underlying hematopoietic differentiation of embryonic stem (ES) cells may help to ascertain the conditions for the in vitro generation of hematopoietic cells. Previously, we found that patients with congenital amegakaryocytic thrombocytopenia (CAMT), who develop pancytopenia early after birth, harbor mutations within the thrombopoietin (TPO) receptor, c-MPL. This knowledge, together with observations in vitro and in vivo, suggests that TPO/c-MPL signaling promotes early hematopoiesis. However, the mechanisms underlying TPO signaling are not fully elucidated. Here, we describe a direct connection between TPO and bone morphogenetic protein 4 (BMP4) signaling pathways in determining the hematopoietic fate of ES cells. Morphogen BMP4 is known to induce early hematopoietic differentiation of ES cells. Treatment of ES cells with TPO induced the autocrine production of BMP4 with concomitant upregulation of the BMP receptor BMPR1A, phosphorylation of SMAD1, 5, 8, and activation of specific BMP4 target genes; this was mediated by TPO-dependent binding of transcription factor HIF-1α to the BMP4 gene promoter. Treatment of ES cells with the BMP antagonist noggin substantially reduced TPO-dependent hematopoietic differentiation of ES cells. Thus, our findings contribute to the establishment of techniques for generating hematopoietic cells from ES cells. PMID:27447537

  14. BMP4/Thrombospondin-1 loop paracrinically inhibits tumor angiogenesis and suppresses the growth of solid tumors.

    PubMed

    Tsuchida, R; Osawa, T; Wang, F; Nishii, R; Das, B; Tsuchida, S; Muramatsu, M; Takahashi, T; Inoue, T; Wada, Y; Minami, T; Yuasa, Y; Shibuya, M

    2014-07-17

    Bone morphogenetic protein 4 (BMP4) has potential as an anticancer agent. Recent studies have suggested that BMP4 inhibits the survival of cancer stem cells (CSCs) of neural and colon cancers. Here, we showed that BMP4 paracrinically inhibited tumor angiogenesis via the induction of Thrombospondin-1 (TSP1), and consequently suppressed tumor growth in vivo. Although HeLa (human cervical cancer), HCI-H460-LNM35 (highly metastatic human lung cancer) and B16 (murine melanoma) cells did not respond to the BMP4 treatment in vitro, the growth of xeno- and allografts of these cells was suppressed via reductions in tumor angiogenesis after intraperitoneal treatment with BMP4. When we assessed the mRNA expression of major angiogenesis-related factors in grafted tumors, we found that the expression of TSP1 was significantly upregulated by BMP4 administration. We then confirmed that BMP4 was less effective in suppressing the tumor growth of TSP1-knockdown cancer cells. Furthermore, we found that BMP4 reduced vascular endothelial growth factor (VEGF) expression in vivo in a TSP1-dependent manner, which indicates that BMP4 interfered with the stabilization of tumor angiogenesis. In conclusion, the BMP4/TSP1 loop paracrinically suppressed tumor angiogenesis in the tumor microenvironment, which subsequently reduced the growth of tumors. BMP4 may become an antitumor agent and open a new field of antiangiogenic therapy. PMID:24013228

  15. Bone morphogenetic protein 4 mediates human embryonic germ cell derivation.

    PubMed

    Hiller, Marc; Liu, Cyndi; Blumenthal, Paul D; Gearhart, John D; Kerr, Candace L

    2011-02-01

    Human primordial germ cells (PGCs) have proven to be a source of pluripotent stem cells called embryonic germ cells (EGCs). Unlike embryonic stem cells, virtually little is known regarding the factors that regulate EGC survival and maintenance. In mice, the growth factor bone morphogenetic protein 4 (BMP4) has been shown to be required for maintaining mouse embryonic stem cells, and disruptions in this gene lead to defects in mouse PGC specification. Here, we sought to determine whether recombinant human BMP4 could influence EGC derivation and/or human PGC survival. We found that the addition of recombinant BMP4 increased the number of human PGCs after 1 week of culture in a dose-responsive manner. The efficiency of EGC derivation and maintenance in culture was also enhanced by the presence of recombinant BMP4 based on alkaline phosphatase and OCT4 staining. In addition, an antagonist of the BMP4 pathway, Noggin, decreased PGC proliferation and led to an increase in cystic embryoid body formation. Quantitative real-time (qRT)-polymerase chain reaction analyses and immunostaining confirmed that the constituents of the BMP4 pathway were upregulated in EGCs versus PGCs. Downstream activators of the BMP4 pathway such as ID1 and phosphorylated SMADs 1 and 5 were also expressed, suggesting a role of this growth factor in EGC pluripotency. PMID:20486775

  16. Delayed BMP4 exposure increases germ cell differentiation in mouse embryonic stem cells.

    PubMed

    Talaei-Khozani, Tahereh; Zarei Fard, Nehleh; Bahmanpour, Soghra; Jaberipour, Mansoureh; Hosseini, Ahmah; Esmaeilpour, Tahereh

    2014-01-01

    Fate mapping studies have revealed that bone morphogenetic protein 4 (BMP4) signaling has a key role in segregation of primordial germ cells from proximal epiblast. Adding BMP4 to the culture media of embryonic stem (ES) cells could induce expression of germ cell markers; however, to provide a desired number of germ cells has remained a challenge. In the current study, we intended to establish an in vitro system to obtain reliable germ cells derived from ES cells. Differentiation was induced in ES cells via embryoid body (EB) and monolayer culture system. Cells were cultured with BMP4 from the beginning (++BMP4) or after 48 hours (+BMP4) of culturing for five days. The cultures were assessed for alkaline phosphatase (ALP) activity, expression of Oct4, Mvh and c-kit. In EB culture protocol, the expression of Mvh, Oct4 and ALP activity significantly increased in +BMP4 culture condition, but a significant down-regulation in the expression of germ cell markers was shown in ++BMP4 condition compared with the control group. Parallel differentiation experiments using monolayer culture system indicated the number of putative germ cells did not change. In the current study, we compared two differentiation methods (EB and monolayer) to achieve an optimal germ cell production. The EBs with a short exposure time period to BMP4, showing typical characteristics of germ cells. Therefore, our approach provides a strategy for the production of germline cells from ES cells. PMID:24969978

  17. Non-virally engineered human adipose mesenchymal stem cells produce BMP4, target brain tumors, and extend survival.

    PubMed

    Mangraviti, Antonella; Tzeng, Stephany Y; Gullotti, David; Kozielski, Kristen L; Kim, Jennifer E; Seng, Michael; Abbadi, Sara; Schiapparelli, Paula; Sarabia-Estrada, Rachel; Vescovi, Angelo; Brem, Henry; Olivi, Alessandro; Tyler, Betty; Green, Jordan J; Quinones-Hinojosa, Alfredo

    2016-09-01

    There is a need for enabling non-viral nanobiotechnology to allow safe and effective gene therapy and cell therapy, which can be utilized to treat devastating diseases such as brain cancer. Human adipose-derived mesenchymal stem cells (hAMSCs) display high anti-glioma tropism and represent a promising delivery vehicle for targeted brain tumor therapy. In this study, we demonstrate that non-viral, biodegradable polymeric nanoparticles (NPs) can be used to engineer hAMSCs with higher efficacy (75% of cells) than leading commercially available reagents and high cell viability. To accomplish this, we engineered a poly(beta-amino ester) (PBAE) polymer structure to transfect hAMSCs with significantly higher efficacy than Lipofectamine™ 2000. We then assessed the ability of NP-engineered hAMSCs to deliver bone morphogenetic protein 4 (BMP4), which has been shown to have a novel therapeutic effect by targeting human brain tumor initiating cells (BTIC), a source of cancer recurrence, in a human primary malignant glioma model. We demonstrated that hAMSCs genetically engineered with polymeric nanoparticles containing BMP4 plasmid DNA (BMP4/NP-hAMSCs) secrete BMP4 growth factor while maintaining their multipotency and preserving their migration and invasion capacities. We also showed that this approach can overcome a central challenge for brain therapeutics, overcoming the blood brain barrier, by demonstrating that NP-engineered hAMSCs can migrate to the brain and penetrate the brain tumor after both intranasal and systemic intravenous administration. Critically, athymic rats bearing human primary BTIC-derived tumors and treated intranasally with BMP4/NP-hAMSCs showed significantly improved survival compared to those treated with control GFP/NP-hAMCSs. This study demonstrates that synthetic polymeric nanoparticles are a safe and effective approach for stem cell-based cancer-targeting therapies. PMID:27240162

  18. BMP4 mediates the interplay between adipogenesis and angiogenesis during expansion of subcutaneous white adipose tissue.

    PubMed

    Tang, Yan; Qian, Shu-Wen; Wu, Meng-Yuan; Wang, Jue; Lu, Ping; Li, Xi; Huang, Hai-Yan; Guo, Liang; Sun, Xia; Xu, Cong-Jian; Tang, Qi-Qun

    2016-08-01

    The expansion of subcutaneous (SC) white adipose tissue (WAT) has beneficial effects on metabolic health. Our previous work showed an increased number of bone morphogenetic protein 4 (BMP4)-activated beige adipocytes in SC WAT, indicating a potential role of BMP4 in adipocyte recruitment. It was also demonstrated that BMP4 committed multipotent mesodermal C3H10T1/2 stem cells to the adipocyte lineage ex vivo However, the mechanism by which BMP4 regulates adipogenesis in vivo has not been clarified. In this study, we found that BMP4 stimulated de novo adipogenesis in SC WAT concomitant with enhanced blood vessel formation, thus promoting adipose tissue angiogenesis. Platelet-derived growth factor receptor-β-positive (PDGFRβ(+)) multipotent stem cells within the neoangiogenic vessels were found to be adipocyte progenitors. Moreover, BMP4 downregulated PDGFRβ by stimulating the lysosome-dependent degradation, which efficiently initiated adipogenic differentiation. These results suggest how BMP4 regulates adipocyte recruitment in SC WAT, and thus promote its beneficial metabolic effects. PMID:27030507

  19. An Evolutionarily Conserved Enhancer Regulates Bmp4 Expression in Developing Incisor and Limb Bud

    PubMed Central

    O’Connell, Daniel J.; Aboukhalil, Anton; Li, Xiao; Choe, Sung E.; Ho, Joshua W. K.; Turbe-Doan, Annick; Robertson, Erin A.; Olsen, Bjorn R.; Bulyk, Martha L.; Amendt, Brad A.; Maas, Richard L.

    2012-01-01

    To elucidate the transcriptional regulation of Bmp4 expression during organogenesis, we used phylogenetic footprinting and transgenic reporter analyses to identify Bmp4 cis-regulatory modules (CRMs). These analyses identified a regulatory region located ∼46 kb upstream of the mouse Bmp4 transcription start site that had previously been shown to direct expression in lateral plate mesoderm. We refined this regulatory region to a 396-bp minimal enhancer, and show that it recapitulates features of endogenous Bmp4 expression in developing mandibular arch ectoderm and incisor epithelium during the initiation-stage of tooth development. In addition, this enhancer directs expression in the apical ectodermal ridge (AER) of the developing limb and in anterior and posterior limb mesenchyme. Transcript profiling of E11.5 mouse incisor dental lamina, together with protein binding microarray (PBM) analyses, allowed identification of a conserved DNA binding motif in the Bmp4 enhancer for Pitx homeoproteins, which are also expressed in the developing mandibular and incisor epithelium. In vitro electrophoretic mobility shift assays (EMSA) and in vivo transgenic reporter mutational analyses revealed that this site supports Pitx binding and that the site is necessary to recapitulate aspects of endogenous Bmp4 expression in developing craniofacial and limb tissues. Finally, Pitx2 chromatin immunoprecipitation (ChIP) demonstrated direct binding of Pitx2 to this Bmp4 enhancer site in a dental epithelial cell line. These results establish a direct molecular regulatory link between Pitx family members and Bmp4 gene expression in developing incisor epithelium. PMID:22701669

  20. Association of BMP4 polymorphisms with non-syndromic cleft lip with or without cleft palate and isolated cleft palate in Latvian and Lithuanian populations.

    PubMed

    Kempa, Inga; Ambrozaitytė, Laima; Stavusis, Janis; Akota, Ilze; Barkane, Biruta; Krumina, Astrida; Matulevičienė, Aušra; Utkus, Algirdas; Kučinskas, Vaidutis; Lace, Baiba

    2014-01-01

    Cleft lip with or without cleft palate (CLP and CL, respectively) and isolated cleft palate (CP) represent one of the most common human birth defects, with a prevalence of approximately 1 in 300-2500 depending on the population. Formation of non-syndromic CL/CLP and CP arises from the interaction of environmental and genetic factors. The objective of this study was to investigate the association between the BMP4 gene (encoding bone morphogenetic protein 4) and non-syndromic CL/CLP and CP in order to clarify the role of this gene in the aetiology of the malformation in Latvian and Lithuanian populations. We genotyped three markers of the BMP4 gene (rs17563, rs2071047 and rs1957860) in order to perform single marker and haplotype association analyses for Latvian and Lithuanian non-syndromic CL/CLP and CP patients and controls. Transmission disequilibrium test was also conducted for Latvian and Lithuanian proband-parent trios. The case-control analysis revealed that SNP rs2071047 allele A was associated with a decreased risk of CL/CLP in the Latvian population, which was confirmed by the haplotype analysis. A modest association was detected between SNP rs1957860 and CP in the Lithuanian population, where allele C was associated with a decreased risk of this cleft phenotype, corroborating haplotype analysis data. Our findings support a role of the BMP4 gene in the aetiology of non-syndromic CL/CLP and CP in the studied populations. PMID:25471993

  1. Chronic psychological stress activates BMP4-dependent extramedullary erythropoiesis.

    PubMed

    Vignjević, Sanja; Budeč, Mirela; Marković, Dragana; Dikić, Dragoslava; Mitrović, Olivera; Mojsilović, Slavko; Durić, Sanja Vranješ; Koko, Vesna; Cokić, Bojana Beleslin; Cokić, Vladan; Jovčić, Gordana

    2014-01-01

    Psychological stress affects different physiological processes including haematopoiesis. However, erythropoietic effects of chronic psychological stress remain largely unknown. The adult spleen contains a distinct microenvironment favourable for rapid expansion of erythroid progenitors in response to stressful stimuli, and emerging evidence suggests that inappropriate activation of stress erythropoiesis may predispose to leukaemic transformation. We used a mouse model to study the influence of chronic psychological stress on erythropoiesis in the spleen and to investigate potential mediators of observed effects. Adult mice were subjected to 2 hrs daily restraint stress for 7 or 14 consecutive days. Our results showed that chronic exposure to restraint stress decreased the concentration of haemoglobin in the blood, elevated circulating levels of erythropoietin and corticosterone, and resulted in markedly increased number of erythroid progenitors and precursors in the spleen. Western blot analysis revealed significantly decreased expression of both erythropoietin receptor and glucocorticoid receptor in the spleen of restrained mice. Furthermore, chronic stress enhanced the expression of stem cell factor receptor in the red pulp. Moreover, chronically stressed animals exhibited significantly increased expression of bone morphogenetic protein 4 (BMP4) in the red pulp as well as substantially enhanced mRNA expression levels of its receptors in the spleen. These findings demonstrate for the first time that chronic psychological stress activates BMP4-dependent extramedullary erythropoiesis and leads to the prolonged activation of stress erythropoiesis pathways. Prolonged activation of these pathways along with an excessive production of immature erythroid cells may predispose chronically stressed subjects to a higher risk of leukaemic transformation. PMID:24283209

  2. Bone morphogenic protein 4 produced in endothelial cells by oscillatory shear stress stimulates an inflammatory response

    NASA Technical Reports Server (NTRS)

    Sorescu, George P.; Sykes, Michelle; Weiss, Daiana; Platt, Manu O.; Saha, Aniket; Hwang, Jinah; Boyd, Nolan; Boo, Yong C.; Vega, J. David; Taylor, W. Robert; Jo, Hanjoong

    2003-01-01

    Atherosclerosis is now viewed as an inflammatory disease occurring preferentially in arterial regions exposed to disturbed flow conditions, including oscillatory shear stress (OS), in branched arteries. In contrast, the arterial regions exposed to laminar shear (LS) are relatively lesion-free. The mechanisms underlying the opposite effects of OS and LS on the inflammatory and atherogenic processes are not clearly understood. Here, through DNA microarrays, protein expression, and functional studies, we identify bone morphogenic protein 4 (BMP4) as a mechanosensitive and pro-inflammatory gene product. Exposing endothelial cells to OS increased BMP4 protein expression, whereas LS decreased it. In addition, we found BMP4 expression only in the selective patches of endothelial cells overlying foam cell lesions in human coronary arteries. The same endothelial patches also expressed higher levels of intercellular cell adhesion molecule-1 (ICAM-1) protein compared with those of non-diseased areas. Functionally, we show that OS and BMP4 induced ICAM-1 expression and monocyte adhesion by a NFkappaB-dependent mechanism. We suggest that BMP4 is a mechanosensitive, inflammatory factor playing a critical role in early steps of atherogenesis in the lesion-prone areas.

  3. Bone morphogenetic protein 4 inhibits liposaccharide-induced inflammation in the airway.

    PubMed

    Li, Zhengtu; Wang, Jian; Wang, Yan; Jiang, Hua; Xu, Xiaoming; Zhang, Chenting; Li, Defu; Xu, Chuyi; Zhang, Kedong; Qi, Yafei; Gong, Xuefang; Tang, Chun; Zhong, Nanshan; Lu, Wenju

    2014-11-01

    Bone morphogenetic protein 4 (BMP4) is a multifunctional growth factor that belongs to the TGF-β superfamily. The role of BMP4 in lung diseases is not fully understood. Here, we demonstrate that BMP4 was upregulated in lungs undergoing lipopolysaccharide (LPS)-induced inflammation, and in airway epithelial cells treated with LPS or TNF-α. BMP4 mutant (BMP4(+/-) ) mice presented with more severe lung inflammation in response to LPS or Pseudomonas aeruginosa, and lower bacterial load compared with that in BMP4(+/+) mice. Knockdown of BMP4 by siRNA increased LPS and TNF-α-induced IL-8 expression in 16HBE human airway epithelial cells and in primary human bronchial epithelial cells. Similarly, peritoneal macrophages from BMP4(+/-) mice produced greater levels of TNF-α and keratinocyte chemoattractant (KC) upon LPS treatment compared with cells from BMP4(+/+) mice. Administration of exogenous BMP4 attenuated the upregulation of TNF-α, IL-8, or KC induced by LPS and/or TNF-α in airway epithelial cells, and peritoneal macrophages. Finally, partial deficiency of BMP4 in BMP4(+/-) mice protected the animals from restrictive lung function reduction upon chronic LPS exposure. These results indicate that BMP4 plays an important anti-inflammatory role, controlling the strength and facilitating the resolution of acute lung inflammation; yet, BMP4 also contributes to lung function impairment during chronic lung inflammation. PMID:25142202

  4. Roles of Bmp4 during tooth morphogenesis and sequential tooth formation

    PubMed Central

    Jia, Shihai; Zhou, Jing; Gao, Yang; Baek, Jin-A; Martin, James F.; Lan, Yu; Jiang, Rulang

    2013-01-01

    Previous studies have suggested that Bmp4 is a key Msx1-dependent mesenchymal odontogenic signal for driving tooth morphogenesis through the bud-to-cap transition. Whereas all tooth germs were arrested at the bud stage in Msx1–/– mice, we show that depleting functional Bmp4 mRNAs in the tooth mesenchyme, through neural crest-specific gene inactivation in Bmp4f/f;Wnt1Cre mice, caused mandibular molar developmental arrest at the bud stage but allowed maxillary molars and incisors to develop to mineralized teeth. We found that expression of Osr2, which encodes a zinc finger protein that antagonizes Msx1-mediated activation of odontogenic mesenchyme, was significantly upregulated in the molar tooth mesenchyme in Bmp4f/f;Wnt1Cre embryos. Msx1 heterozygosity enhanced maxillary molar developmental defects whereas Osr2 heterozygosity partially rescued mandibular first molar morphogenesis in Bmp4f/f;Wnt1Cre mice. Moreover, in contrast to complete lack of supernumerary tooth initiation in Msx1–/–Osr2–/– mice, Osr2–/–Bmp4f/f;Wnt1Cre compound mutant mice exhibited formation and subsequent arrest of supernumerary tooth germs that correlated with downregulation of Msx1 expression in the tooth mesenchyme. In addition, we found that the Wnt inhibitors Dkk2 and Wif1 were much more abundantly expressed in the mandibular than maxillary molar mesenchyme in wild-type embryos and that Dkk2 expression was significantly upregulated in the molar mesenchyme in Bmp4f/f;Wnt1Cre embryos, which correlated with the dramatic differences in maxillary and mandibular molar phenotypes in Bmp4f/f;Wnt1Cre mice. Together, these data indicate that Bmp4 signaling suppresses tooth developmental inhibitors in the tooth mesenchyme, including Dkk2 and Osr2, and synergizes with Msx1 to activate mesenchymal odontogenic potential for tooth morphogenesis and sequential tooth formation. PMID:23250216

  5. Haploinsufficient Bmp4 ocular phenotypes include anterior segment dysgenesis with elevated intraocular pressure

    PubMed Central

    Chang, Bo; Smith, Richard S; Peters, Maureen; Savinova, Olga V; Hawes, Norman L; Zabaleta, Adriana; Nusinowitz, Steven; Martin, Janice E; Davisson, Muriel L; Cepko, Constance L; Hogan, Brigid LM; John, Simon WM

    2001-01-01

    Background Glaucoma is a blinding disease usually associated with high intraocular pressure (IOP). In some families, abnormal anterior segment development contributes to glaucoma. The genes causing anterior segment dysgenesis and glaucoma in most of these families are not identified and the affected developmental processes are poorly understood. Bone morphogenetic proteins (BMPs) participate in various developmental processes. We tested the importance of Bmp4 gene dosage for ocular development and developmental glaucoma. Results Bmp4+/- mice have anterior segment abnormalities including malformed, absent or blocked trabecular meshwork and Schlemm's canal drainage structures. Mice with severe drainage structure abnormalities, over 80% or more of their angle's extent, have elevated IOP. The penetrance and severity of abnormalities is strongly influenced by genetic background, being most severe on the C57BL/6J background and absent on some other backgrounds. On the C57BL/6J background there is also persistence of the hyaloid vasculature, diminished numbers of inner retinal cells, and absence of the optic nerve. Conclusions We demonstrate that heterozygous deficiency of BMP4 results in anterior segment dysgenesis and elevated IOP. The abnormalities are similar to those in human patients with developmental glaucoma. Thus, BMP4 is a strong candidate to contribute to Axenfeld-Rieger anomaly and other developmental conditions associated with human glaucoma. BMP4 also participates in posterior segment development and wild-type levels are usually critical for optic nerve development on the C57BL/6J background. Bmp4+/- mice are useful for studying various components of ocular development, and may allow identification of strain specific modifiers affecting a variety of ocular phenotypes. PMID:11722794

  6. Functional cardiomyocytes derived from Isl1 cardiac progenitors via Bmp4 stimulation.

    PubMed

    Cagavi, Esra; Bartulos, Oscar; Suh, Carol Y; Sun, Baonan; Yue, Zhichao; Jiang, Zhengxin; Yue, Lixia; Qyang, Yibing

    2014-01-01

    As heart failure due to myocardial infarction remains a leading cause of morbidity worldwide, cell-based cardiac regenerative therapy using cardiac progenitor cells (CPCs) could provide a potential treatment for the repair of injured myocardium. As adult CPCs may have limitations regarding tissue accessibility and proliferative ability, CPCs derived from embryonic stem cells (ESCs) could serve as an unlimited source of cells with high proliferative ability. As one of the CPCs that can be derived from embryonic stem cells, Isl1 expressing cardiac progenitor cells (Isl1-CPCs) may serve as a valuable source of cells for cardiac repair due to their high cardiac differentiation potential and authentic cardiac origin. In order to generate an unlimited number of Isl1-CPCs, we used a previously established an ESC line that allows for isolation of Isl1-CPCs by green fluorescent protein (GFP) expression that is directed by the mef2c gene, specifically expressed in the Isl1 domain of the anterior heart field. To improve the efficiency of cardiac differentiation of Isl1-CPCs, we studied the role of Bmp4 in cardiogenesis of Isl1-CPCs. We show an inductive role of Bmp directly on cardiac progenitors and its enhancement on early cardiac differentiation of CPCs. Upon induction of Bmp4 to Isl1-CPCs during differentiation, the cTnT+ cardiomyocyte population was enhanced 2.8±0.4 fold for Bmp4 treated CPC cultures compared to that detected for vehicle treated cultures. Both Bmp4 treated and untreated cardiomyocytes exhibit proper electrophysiological and calcium signaling properties. In addition, we observed a significant increase in Tbx5 and Tbx20 expression in differentiation cultures treated with Bmp4 compared to the untreated control, suggesting a link between Bmp4 and Tbx genes which may contribute to the enhanced cardiac differentiation in Bmp4 treated cultures. Collectively these findings suggest a cardiomyogenic role for Bmp4 directly on a pure population of Isl1 expressing

  7. BMP4 Signaling Is Able to Induce an Epithelial-Mesenchymal Transition-Like Phenotype in Barrett’s Esophagus and Esophageal Adenocarcinoma through Induction of SNAIL2

    PubMed Central

    Kestens, Christine; Siersema, Peter D.; Offerhaus, G. Johan A.; van Baal, Jantine W. P. M.

    2016-01-01

    Background Bone morphogenetic protein 4 (BMP4) signaling is involved in the development of Barrett’s esophagus (BE), a precursor of esophageal adenocarcinoma (EAC). In various cancers, BMP4 has been found to induce epithelial-mesenchymal transition (EMT) but its function in the development of EAC is currently unclear. Aim To investigate the expression of BMP4 and several members of the BMP4 pathway in EAC. Additionally, to determine the effect of BMP4 signaling in a human Barrett’s esophagus (BAR-T) and adenocarcinoma (OE33) cell line. Methods Expression of BMP4, its downstream target ID2 and members of the BMP4 pathway were determined by Q-RT-PCR, immunohistochemistry and Western blot analysis using biopsy samples from EAC patients. BAR-T and OE33 cells were incubated with BMP4 or the BMP4 antagonist, Noggin, and cell viability and migration assays were performed. In addition, expression of factors associated with EMT (SNAIL2, CDH1, CDH2 and Vimentin) was evaluated by Q-RT-PCR and Western blot analysis. Results Compared to squamous epithelium (SQ), BMP4 expression was significantly upregulated in EAC and BE. In addition, the expression of ID2 was significantly upregulated in EAC and BE compared to SQ. Western blot analysis confirmed our results, showing an upregulated expression of BMP4 and ID2 in both BE and EAC. In addition, more phosphorylation of SMAD1/5/8 was observed. BMP4 incubation inhibited cell viability, but induced cell migration in both BAR-T and OE33 cells. Upon BMP4 incubation, SNAIL2 expression was significantly upregulated in BAR-T and OE33 cells while CDH1 expression was significantly downregulated. These results were confirmed by Western blot analysis. Conclusion Our results indicate active BMP4 signaling in BE and EAC and suggest that this results in an invasive phenotype by inducing an EMT-like response through upregulation of SNAIL2 and subsequent downregulation of CDH1. PMID:27191723

  8. Tailbud-derived Bmp4 drives proliferation and inhibits maturation of zebrafish chordamesoderm.

    PubMed

    Esterberg, Robert; Delalande, Jean-Marie; Fritz, Andreas

    2008-12-01

    In zebrafish, BMP signaling establishes cell identity along the dorsoventral (DV) axis during gastrulation. Owing to the early requirements of BMP activity in DV patterning, it has been difficult to assign later roles in cell fate specification to specific BMP ligands. In this study, we have taken advantage of two follistatin-like genes (fstl1 and fstl2), as well as a transgenic zebrafish line carrying an inducible truncated form of the BMP-type 1 receptor to study the role of Bmp4 outside of the context of DV specification. Characterization of fstl1/2 suggests that they exert a redundant role as BMP antagonists during late gastrulation, regulating BMP activity in axial mesoderm. Maintenance of appropriate levels of BMP signaling is crucial for the proper development of chordamesoderm, a subset of axial mesoderm that gives rise to the notochord, but not prechordal mesoderm, which gives rise to the prechordal plate. Bmp4 activity in particular is required during a crucial window beginning at late gastrulation and lasting through early somitogenesis to promote chordamesoderm proliferation. In the absence of Bmp4, the notochord precursor pool is depleted, and the notochord differentiates prematurely. Our results illustrate a role for Bmp4 in the proliferation and timely differentiation of axial tissue after DV axis specification. PMID:18948415

  9. BMP4 density gradient in disk-shaped confinement

    NASA Astrophysics Data System (ADS)

    Bozorgui, Behnaz; Teimouri, Hamid; Kolomeisky, Anatoly B.

    We present a quantitative model that explains the scaling of BMP4 gradients during gastrulation and the recent experimental observation that geometric confinement of human embryonic stem cells is sufficient to recapitulate much of germ layer patterning. Based on a assumption that BMP4 diffusion rate is much smaller than the diffusion rate of it's inhibitor molecules, our results confirm that the length-scale which defines germ layer territories does not depend on system size.

  10. Cartilage Repair in a Rat Model of Osteoarthritis Through Intraarticular Transplantation of Muscle-Derived Stem Cells Expressing Bone Morphogenetic Protein 4 and Soluble Flt-1

    PubMed Central

    Matsumoto, Tomoyuki; Cooper, Gregory M.; Gharaibeh, Burhan; Meszaros, Laura B.; Li, Guangheng; Usas, Arvydas; Fu, Freddie H.; Huard, Johnny

    2016-01-01

    Objective The control of angiogenesis during chondrogenic differentiation is an important issue affecting the use of stem cells in cartilage repair, especially with regard to the persistence of regenerated cartilage. This study was undertaken to investigate the effect of vascular endothelial growth factor (VEGF) stimulation and the blocking of VEGF with its antagonist, soluble Flt-1 (sFlt-1), on the chondrogenesis of skeletal muscle-derived stem cells (MDSCs) in a rat model of osteoarthritis (OA). Methods We investigated the effect of VEGF on cartilage repair in an immunodeficiency rat model of OA after intraarticular injection of murine MDSCs expressing bone morphogenetic protein 4 (BMP-4) in combination with MDSCs expressing VEGF or sFlt-1. Results In vivo, a combination of sFlt-1– and BMP-4–transduced MDSCs demonstrated better repair without osteophyte formation macroscopically and histologically following OA induction, when compared with the other groups. Higher differentiation/proliferation and lower levels of chondrocyte apoptosis were also observed in sFlt-1– and BMP-4–transduced MDSCs compared with a combination of VEGF- and BMP-4– transduced MDSCs or with BMP-4–transduced MDSCs alone. In vitro experiments with mixed pellet co-culture of MDSCs and OA chondrocytes revealed that BMP-4– transduced MDSCs produced the largest pellets, which had the highest gene expression of not only type II collagen and SOX9 but also type X collagen, suggesting formation of hypertrophic chondrocytes. Conclusion Our results demonstrate that MDSC-based therapy involving sFlt-1 and BMP-4 repairs articular cartilage in OA mainly by having a beneficial effect on chondrogenesis by the donor and host cells as well as by preventing angiogenesis, which eventually prevents cartilage resorption, resulting in persistent cartilage regeneration and repair. PMID:19404941

  11. Involvement of over-expressed BMP4 in pentylenetetrazol kindling-induced cell proliferation in the dentate gyrus of adult rats

    SciTech Connect

    Yin Jinbo; Ma Yuxin; Yin Qing; Xu Haiwei . E-mail: haiweixu2001@yahoo.com.cn; An Ning; Liu Shiyong; Fan Xiaotang; Yang Hui . E-mail: huiyang64@yahoo.com

    2007-03-30

    The dentate gyrus (DG) of the hippocampus is one of a few regions in the adult mammalian brain characterized by ongoing neurogenesis. Proliferation of neural precursors in the granule cell layer of the DG has been identified in pentylenetetrazol (PTZ) kindling epilepsy model, however, little is known about the molecular mechanism. We previously reported that the expression pattern of bone morphogenetic proteins-4 (BMP4) mRNA in the hippocampus was developmentally regulated and mainly localized in the DG of the adult. To explore the role of BMP4 in epileptic activity, we detected BMP4 expression in the DG during PTZ kindling process and explore its correlation with cell proliferation combined with bromodeoxyuridine (BrdU) labeling technique. We found that dynamic changes in BMP4 level and BrdU labeled cells dependent on the kindling stage of PTZ induced seizure-prone state. The number of BMP4 mRNA-positive cells and BrdU labeled cells reached the top level 1 day after PTZ kindled, then declined to base level 2 months later. Furthermore, there was a significant correlation between increased BMP4 mRNA expression and increased number of BrdU labeled cells. After effectively blocked expression of BMP4 with antisense oligodeoxynucleotides(ASODN), the BrdU labeled cells in the dentate gyrus subgranular zone(DG-SGZ) and hilus were significantly decreased 16d after First PTZ injection and 1, 3, 7, 14d after kindled respectively. These findings suggest that increased proliferation in the DG of the hippocampus resulted from kindling epilepsy elicited by PTZ maybe be modulated by BMP4 over-expression.

  12. Zebrafish bmp4 functions during late gastrulation to specify ventroposterior cell fates

    PubMed Central

    Stickney, Heather L.; Imai, Yoshiyuki; Draper, Bruce; Moens, Cecilia; Talbot, William S.

    2009-01-01

    Bone morphogenetic proteins (BMPs) are key mediators of dorsoventral patterning in vertebrates and are required for the induction of ventral fates in fish and frogs. A widely accepted model of dorsoventral patterning postulates that a morphogenetic BMP activity gradient patterns cell fates along the dorsoventral axis. Recent work in zebrafish suggests that the role of BMP signaling changes over time, with BMPs required for global dorsoventral patterning during early gastrulation and for tail patterning during late gastrulation and early somitogenesis. Key questions remain about the late phase, including which BMP ligands are required and how the functions of BMPs differ during the early and late gastrula stages. In a screen for dominant enhancers of mutations in the homeobox genes vox and vent, which function in parallel to bmp signaling, we identified an insertion mutation in bmp4. We then performed a reverse genetic screen to isolate a null allele of bmp4. We report the characterization of these two alleles and demonstrate that BMP4 is required during the later phase of BMP signaling for the specification of ventroposterior cell fates. Our results indicate that different bmp genes are essential at different stages. In addition, we present genetic evidence supporting a role for a morphogenetic BMP gradient in establishing mesodermal fates during the later phase of BMP signaling. PMID:17727832

  13. A boost of BMP4 accelerates the commitment of human embryonic stem cells to the endothelial lineage.

    PubMed

    Goldman, Orit; Feraud, Olivier; Boyer-Di Ponio, Julie; Driancourt, Catherine; Clay, Denis; Le Bousse-Kerdiles, Marie-Caroline; Bennaceur-Griscelli, Annelise; Uzan, Georges

    2009-08-01

    Embryoid bodies (EBs) generated during differentiation of human embryonic stem cells (hESCs) contain vascular-like structures, suggesting that commitment of mesoderm progenitors into endothelial cells occurs spontaneously. We showed that bone morphogenetic protein 4 (BMP4), an inducer of mesoderm, accelerates the peak expression of CD133/kinase insert domain-containing receptor (KDR) and CD144/KDR. Because the CD133(+)KDR(+) population could represent endothelial progenitors, we sorted them at day 7 and cultured them in endothelial medium. These cells were, however, unable to differentiate into endothelial cells. Under standard conditions, the CD144(+)KDR(+) population represents up to 10% of the total cells at day 12. In culture, these cells, if sorted, give rise to a homogeneous population with a morphology typical of endothelial cells and express endothelial markers. These endothelial cells derived from the day 12 sorted population were functional, as assessed by different in vitro assays. When EBs were stimulated by BMP4, the CD144(+)KDR(+) peak was shifted to day 7. Most of these cells, however, were CD31(-), becoming CD31(+) in culture. They then expressed von Willebrand factor and were functional. This suggests that, initially, the BMP4-boosted day 7, CD144(+)KDR(+)CD31(-) population represents immature endothelial cells that differentiate into mature endothelial cells in culture. The expression of OCT3/4, a marker of immaturity for hESCs decreases during EB differentiation, decreasing faster following BMP4 induction. We also show that BMP4 inhibits the global expression of GATA2 and RUNX1, two transcription factors involved in hemangioblast formation, at day 7 and day 12. PMID:19544443

  14. Heightened potency of human pluripotent stem cell lines created by transient BMP4 exposure

    PubMed Central

    Yang, Ying; Adachi, Katsuyuki; Sheridan, Megan A.; Alexenko, Andrei P.; Schust, Danny J.; Schulz, Laura C.; Ezashi, Toshihiko; Roberts, R. Michael

    2015-01-01

    Human pluripotent stem cells (PSCs) show epiblast-type pluripotency that is maintained with ACTIVIN/FGF2 signaling. Here, we report the acquisition of a unique stem cell phenotype by both human ES cells (hESCs) and induced pluripotent stem cells (iPSCs) in response to transient (24–36 h) exposure to bone morphogenetic protein 4 (BMP4) plus inhibitors of ACTIVIN signaling (A83-01) and FGF2 (PD173074), followed by trypsin dissociation and recovery of colonies capable of growing on a gelatin substratum in standard medium for human PSCs at low but not high FGF2 concentrations. The self-renewing cell lines stain weakly for CDX2 and strongly for NANOG, can be propagated clonally on either Matrigel or gelatin, and are morphologically distinct from human PSC progenitors on either substratum but still meet standard in vitro criteria for pluripotency. They form well-differentiated teratomas in immune-compromised mice that secrete human chorionic gonadotropin (hCG) into the host mouse and include small areas of trophoblast-like cells. The cells have a distinct transcriptome profile from the human PSCs from which they were derived (including higher expression of NANOG, LEFTY1, and LEFTY2). In nonconditioned medium lacking FGF2, the colonies spontaneously differentiated along multiple lineages, including trophoblast. They responded to PD173074 in the absence of both FGF2 and BMP4 by conversion to trophoblast, and especially syncytiotrophoblast, whereas an A83-01/PD173074 combination favored increased expression of HLA-G, a marker of extravillous trophoblast. Together, these data suggest that the cell lines exhibit totipotent potential and that BMP4 can prime human PSCs to a self-renewing alternative state permissive for trophoblast development. The results may have implications for regulation of lineage decisions in the early embryo. PMID:25870291

  15. High glucose and palmitate increases bone morphogenic protein 4 expression in human endothelial cells

    PubMed Central

    Hong, Oak-Kee; Yoo, Soon-Jib; Son, Jang-Won; Kim, Mee-Kyoung; Baek, Ki-Hyun; Song, Ki-Ho; Cha, Bong-Yun; Jo, Hanjoong

    2016-01-01

    Here, we investigated whether hyperglycemia and/or free fatty acids (palmitate, PAL) aff ect the expression level of bone morphogenic protein 4 (BMP4), a proatherogenic marker, in endothelial cells and the potential role of BMP4 in diabetic vascular complications. To measure BMP4 expression, human umbilical vein endothelial cells (HUVECs) were exposed to high glucose concentrations and/or PAL for 24 or 72 h, and the effects of these treatments on the expression levels of adhesion molecules and reactive oxygen species (ROS) were examined. BMP4 loss-of-function status was achieved via transfection of a BMP4-specific siRNA. High glucose levels increased BMP4 expression in HUVECs in a dose-dependent manner. PAL potentiated such expression. The levels of adhesion molecules and ROS production increased upon treatment with high glucose and/or PAL, but this eff ect was negated when BMP4 was knocked down via siRNA. Signaling of BMP4, a proinflammatory and pro-atherogenic cytokine marker, was increased by hyperglycemia and PAL. BMP4 induced the expression of infl ammatory adhesion molecules and ROS production. Our work suggests that BMP4 plays a role in atherogenesis induced by high glucose levels and/or PAL. PMID:26937213

  16. Molecular cloning and characterization of the protein 4.1O gene, a novel member of the protein 4.1 family with focal expression in ovary.

    PubMed

    Ni, Xiaohua; Ji, Chaoneng; Cao, Gentao; Cheng, Haipeng; Guo, Lingchen; Gu, Shaohua; Ying, Kang; Zhao, Robert C; Mao, Yumin

    2003-01-01

    Protein 4.1 is an important structural protein that is expressed in erythroid and in a variety of nonerythroid tissues. In mammalian erythrocytes, it plays a key role in regulating the physical properties of mechanical stability and deformability in membranes by stabilizing the spectrin-actin interaction. The protein 4.1 family mainly comprises 4.1R, 4.1G (general type), 4.1B (brain type), and 4.1N (neuron type). We identified a novel human 4.1 ( 4.1O) gene that is 2312 bp in length and encodes a protein of 553 amino acid residues. The protein shared homology with mouse protein 4.1B (identity 38%, similarity 55%) with a FERM domain. The expression pattern of the human 4.1O gene in 16 tissues showed that there was a transcript only in ovary, whereas in the remaining 15 tissues, specific bands of the transcript could not be detected. In eight human fetal tissues, the specific bands of the transcript could be detected in skeletal muscle, with lower levels detected in thymus and brain. The 4.1O gene consists of 14 exons and 13 introns and was mapped to Chromosome 9q21-9q22 by bioinformatics analysis. PMID:12601556

  17. Follistatin-like 1 (Fstl1) is a bone morphogenetic protein (BMP) 4 signaling antagonist in controlling mouse lung development.

    PubMed

    Geng, Yan; Dong, Yingying; Yu, Mingyan; Zhang, Long; Yan, Xiaohua; Sun, Jingxia; Qiao, Long; Geng, Huixia; Nakajima, Masahiro; Furuichi, Tatsuya; Ikegawa, Shiro; Gao, Xiang; Chen, Ye-Guang; Jiang, Dianhua; Ning, Wen

    2011-04-26

    Lung morphogenesis is a well orchestrated, tightly regulated process through several molecular pathways, including TGF-β/bone morphogenetic protein (BMP) signaling. Alteration of these signaling pathways leads to lung malformation. We investigated the role of Follistatin-like 1 (Fstl1), a secreted follistatin-module-containing glycoprotein, in lung development. Deletion of Fstl1 in mice led to postnatal lethality as a result of respiratory failure. Analysis of the mutant phenotype showed that Fstl1 is essential for tracheal cartilage formation and alveolar maturation. Deletion of the Fstl1 gene resulted in malformed tracheal rings manifested as discontinued rings and reduced ring number. Fstl1-deficient mice displayed septal hypercellularity and end-expiratory atelectasis, which were associated with impaired differentiation of distal alveolar epithelial cells and insufficient production of mature surfactant proteins. Mechanistically, Fstl1 interacted directly with BMP4, negatively regulated BMP4/Smad1/5/8 signaling, and inhibited BMP4-induced surfactant gene expression. Reducing BMP signaling activity by Noggin rescued pulmonary atelectasis of Fstl1-deficient mice. Therefore, we provide in vivo and in vitro evidence to demonstrate that Fstl1 modulates lung development and alveolar maturation, in part, through BMP4 signaling. PMID:21482757

  18. Phylogenetic relationships and the evolution of BMP4 in triggerfishes and filefishes (Balistoidea).

    PubMed

    McCord, Charlene L; Westneat, Mark W

    2016-01-01

    The triggerfishes (family Balistidae) and filefishes (family Monacanthidae) comprise a charismatic superfamily (Balistoidea) within the diverse order Tetraodontiformes. This group of largely marine fishes occupies an impressive ecological range across the world's oceans, and is well known for its locomotor and feeding diversity, unusual body shapes, small genome size, and ecological and economic importance. In order to investigate the evolutionary history of these important fish families, we used multiple phylogenetic methods to analyze molecular data from 86 species spanning the extant biodiversity of Balistidae and Monacanthidae. In addition to three gene regions that have been used extensively in phylogenetic analyses, we include sequence data for two mitochondrial regions, two nuclear markers, and the growth factor gene bmp4, which is involved with cranial development. Phylogenetic analyses strongly support the monophyly of the superfamily Balistoidea, the sister-family relationship of Balistidae and Monacanthidae, as well as three triggerfish and four filefish clades that are well resolved. A new classification for the Balistidae is proposed based on phylogenetic groups. Bayesian topology, as well as the timing of major cladogenesis events, is largely congruent with previous hypotheses of balistid phylogeny. However, we present a novel topology for major clades in the filefish family that illustrate the genera Aluterus and Stephanolepis are more closely related than previously posited. Molecular rates suggest a Miocene and Oligocene origin for the families Balistidae and Monacanthidae, respectively, and significant divergence of species in both families within the past 5 million years. A second key finding of this study is that, relative to the other protein-coding gene regions in our DNA supermatrix, bmp4 shows a rapid accumulation of both synonymous and non-synonymous substitutions, especially within the family Monacanthidae. Overall substitution patterns in

  19. Regulation of Chondroitin-4-Sulfotransferase (CHST11) Expression by Opposing Effects of Arylsulfatase B on BMP4 and Wnt9A

    PubMed Central

    Bhattacharyya, Sumit; Feferman, Leo; Tobacman, Joanne K.

    2014-01-01

    In this report, the gene regulatory mechanism by which decline in arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) reduces CHST11 (chondroitin-4-sulfotransferase; C4ST) mRNA expression in human colonic epithelial cells and in colonic epithelium of ARSB-deficient mice is presented. ARSB controls the degradation of chondroitin 4-sulfate (C4S) by removing the 4-sulfate group at the non-reducing end of the C4S chain, but has not previously been shown to affect C4S biosynthesis. The decline in CHST11 expression following ARSB reduction is attributable to effects of ARSB on bone morphogenetic protein (BMP)4, since BMP4 expression and secretion declined when ARSB was silenced. Inhibition of BMP4 by neutralizing antibody also reduced CHST11 expression. When C4S was more sulfated due to decline in ARSB, more BMP4 was sequestered by C4S in the cell membrane, and CHST11 expression declined. Exogenous recombinant BMP4, acting through a phospho-Smad3 binding site in the CHST11 promoter, increased the mRNA expression of CHST11. In contrast to the decline in BMP4 that followed decline in ARSB, Wnt9A mRNA expression was previously shown to increase when ARSB was silenced and C4S was more highly sulfated. Galectin-3 bound less to the more highly sulfated C4S, leading to increased nuclear translocation and enhanced galectin-3 interaction with Sp1 in the Wnt9A promoter. Silencing Wnt9A increased the expression of CHST11 in the colonic epithelial cells, and chromatin immunoprecipitation assay demonstrated enhancing effects of Wnt9A siRNA and exogenous BMP4 on the CHST11 promoter through the pSmad3 binding site. These findings suggest that cellular processes mediated by differential effects of Wnt9A and BMP4 can result from opposing effects on CHST11 expression. PMID:25511584

  20. Bone morphogenic protein-4 expression in vascular lesions of calciphylaxis.

    PubMed

    Griethe, Wanja; Schmitt, Roland; Jurgensen, Jan Steffen; Bachmann, Sebastian; Eckardt, Kai-Uwe; Schindler, Ralf

    2003-01-01

    Calciphylaxis is characterized by an extensive media-calcification of cutaneous and subcutaneous arterioles and capillaries. Recent studies have provided evidence that vascular calcification is a process with similarities to bone metabolism. Bone morphogenic protein-4 (BMP-4) is physiologically involved in bone development and repair. The presence of BMP-4 in atherosclerosis and in sclerotic heart valves led us to suggest that BMP-4 is also involved in calciphylaxis. A 47-year-old male patient developed end-stage renal failure due to chronic glomerulonephritis. He has had two kidney transplants with an immunosuppressive regimen consisting of cyclosporine A and steroids. He was admitted to our hospital because of an increase in serum creatinine (Cr) and he subsequently developed progressive dermal ulcerations. A skin biopsy led to the diagnosis of calciphylaxis. Immunohistochemistry for BMP-4 of a skin specimen from our patient showed strong cytoplasmic immunoreactivity of intradermal cells with clear spatial association to arterioles and hair follicles. Whereas there are identified inhibitors and promoters of vascular calcification, the presence of BMP-4 has not been demonstrated in calcific uremic arteriolopathy. In contrast to atherosclerosis, BMP-4 in calciphylaxis cannot be found in vascular media, but in intradermal cells at the border of arterioles and hair follicles. Therefore, in calciphylaxis BMP-4 can play the role of a cytokine, a growth factor or a media-calcification promoter. PMID:14733421

  1. Evolutionarily conserved coupling of transcription and alternative splicing in the EPB41 (protein 4.1R) and EPB41L3 (protein 4.1B) genes.

    PubMed

    Tan, Jeff S; Mohandas, Narla; Conboy, John G

    2005-12-01

    Recent studies have shown that transcription and alternative splicing can be mechanistically coupled. In the EPB41 (protein 4.1R) and EPB41L3 (protein 4.1B) genes, we showed previously that promoter/alternative first exon choice is coupled to downstream splicing events in exon 2. Here we demonstrate that this coupling is conserved among several vertebrate classes from fish to mammals. The EPB41 and EPB41L3 genes from fish, bird, amphibian, and mammal genomes exhibit shared features including alternative first exons and differential splice acceptors in exon 2. In all cases, the 5'-most exon (exon 1A) splices exclusively to a weaker internal acceptor site in exon 2, skipping a fragment designated as exon 2'. Conversely, alternative first exons 1B and 1C always splice to the stronger first acceptor site, retaining exon 2'. These correlations are independent of cell type or species of origin. Since exon 2' contains a translation initiation site, splice variants generate protein isoforms with distinct N-termini. We propose that these genes represent a physiologically relevant model system for mechanistic analysis of transcription-coupled alternative splicing. PMID:16242908

  2. Inhibitory Effect of Bone Morphogenetic Protein 4 in Retinal Pigment Epithelial-Mesenchymal Transition

    PubMed Central

    Yao, Haipei; Li, Hui; Yang, Shuai; Li, Min; Zhao, Chun; Zhang, Jingfa; Xu, Guotong; Wang, Fang

    2016-01-01

    Proliferative vitreoretinopathy (PVR), a serious vision-threatening complication of retinal detachment (RD), is characterized by the formation of contractile fibrotic membranes, in which epithelial-mesenchymal transition (EMT) of the retinal pigment epithelium (RPE) is a major event. Recent studies suggest an important role of bone morphogenetic protein 4 (BMP4) in the suppression of fibrosis. In this study, we aimed to investigate the role of BMP4 in the pathological process of PVR, particularly in the EMT of RPE cells. We found that BMP4 and its receptors were co-labelled with cytokeratin and α-SMA positive cells within the PVR membrane. Moreover, the mRNA and protein expression levels of BMP4 were decreased whereas BMP4 receptors ALK2, ALK3 and ALK6 were increased during TGF-β-induced EMT in primary RPE cells. Exogenous BMP4 inhibited TGF-β-induced epithelial marker down-regulation, as well as mesenchymal marker up-regulation at both the mRNA and protein levels in RPE cells. In addition, BMP4 treatment attenuated the TGF-β-induced gel contraction, cell migration and Smad2/3 phosphorylation. However, knockdown of endogenous BMP4 stimulated changes in EMT markers. Our results confirm the hypothesis that BMP4 might inhibit TGF-β-mediated EMT in RPE cells via the Smad2/3 pathway and suppress contraction. This might represent a potential treatment for PVR. PMID:27586653

  3. Inhibitory Effect of Bone Morphogenetic Protein 4 in Retinal Pigment Epithelial-Mesenchymal Transition.

    PubMed

    Yao, Haipei; Li, Hui; Yang, Shuai; Li, Min; Zhao, Chun; Zhang, Jingfa; Xu, Guotong; Wang, Fang

    2016-01-01

    Proliferative vitreoretinopathy (PVR), a serious vision-threatening complication of retinal detachment (RD), is characterized by the formation of contractile fibrotic membranes, in which epithelial-mesenchymal transition (EMT) of the retinal pigment epithelium (RPE) is a major event. Recent studies suggest an important role of bone morphogenetic protein 4 (BMP4) in the suppression of fibrosis. In this study, we aimed to investigate the role of BMP4 in the pathological process of PVR, particularly in the EMT of RPE cells. We found that BMP4 and its receptors were co-labelled with cytokeratin and α-SMA positive cells within the PVR membrane. Moreover, the mRNA and protein expression levels of BMP4 were decreased whereas BMP4 receptors ALK2, ALK3 and ALK6 were increased during TGF-β-induced EMT in primary RPE cells. Exogenous BMP4 inhibited TGF-β-induced epithelial marker down-regulation, as well as mesenchymal marker up-regulation at both the mRNA and protein levels in RPE cells. In addition, BMP4 treatment attenuated the TGF-β-induced gel contraction, cell migration and Smad2/3 phosphorylation. However, knockdown of endogenous BMP4 stimulated changes in EMT markers. Our results confirm the hypothesis that BMP4 might inhibit TGF-β-mediated EMT in RPE cells via the Smad2/3 pathway and suppress contraction. This might represent a potential treatment for PVR. PMID:27586653

  4. The gene for human erythrocyte protein 4. 2 maps to chromosome 15q15

    SciTech Connect

    Najfeld, V. ); Ballard, S.G.; Menninger, J.; Ward, D.C. ); Bouhassira, E.E.; Schwartz, R.S.; Nagel, R.L.; Rybicki, A.C. )

    1992-01-01

    Protein 4.2 (P4.2), one of the major components of the red-blood-cell membrane, is located on the interior surface, where it binds with high affinity to the cytoplasmic domain of band 3. Individuals whose red blood cells are deficient in P4.2 have osmotically fragile, abnormally shaped cells and moderate hemolytic anemia. cDNA clones from both the 5{prime} and the 3{prime} coding regions of the P4.2 gene were used to map its chromosomal location by fluorescence in situ hybridization. The probes, individually or in combination, gave specific hybridization signals on chromosome 15. The hybridization locus was identified by combining fluorescence images of the probe signals with fluorescence banding patterns generated by Alu-PCR (R-like) probe and by DAPI staining (G-like). The authors results demonstrate that the locus of the P4.2 gene is located within 15q15.

  5. Four paralogous protein 4.1 genes map to distinct chromosomes in mouse and human.

    PubMed

    Peters, L L; Weier, H U; Walensky, L D; Snyder, S H; Parra, M; Mohandas, N; Conboy, J G

    1998-12-01

    Four highly conserved members of the skeletal protein 4.1 gene family encode a diverse array of protein isoforms via tissue-specific transcription and developmentally regulated alternative pre-mRNA splicing. In addition to the prototypical red blood cell 4.1R (human gene symbol EPB41,) these include two homologues that are strongly expressed in the brain (4.1N, EPB41L1; and 4.1B, EPB41L3) and another that is widely expressed in many tissues (4.1G, EPB41L2). As part of a study on the structure and evolution of the 4.1 genes in human and mouse, we have now completed the chromosomal mapping of their respective loci by reporting the localization of mouse 4.1N, 4.1G, and 4.1B, as well as human 4.1B. For the mouse 4.1 genes, Southern blot analysis of RFLPs in The Jackson Laboratory BSS interspecific backcross yielded the following assignments: 4.1N (Epb4.1l1,) chromosome 2; 4.1G (Epb4.1l2,) chromosome 10; and 4.1B (Epb4.1l3,) mouse chromosome 17. Human 4.1B was physically mapped to chromosome 18p11 using fluorescence in situ hybridization. All of the mouse genes mapped within or adjacent to regions of conserved synteny with corresponding human chromosomes. We conclude that a set of four paralogous 4.1 genes has been evolutionarily conserved in rodents and primates. PMID:9828140

  6. Single nucleotide polymorphism of bone morphogenetic protein 4 gene: A risk factor of non-syndromic cleft lip with or without palate

    PubMed Central

    Savitha, Sathyaprasad; Sharma, S. M.; Veena, Shetty; Rekha, R.

    2015-01-01

    Background: The bone morphogenetic protein (BMP) signalling pathway is crucial in a number of developmental processes and is critical in the formation of variety of craniofacial elements including cranial neural crest, facial primordium, tooth, lip and palate. It is an important mediator in regulation of lip and palate fusion, cartilage and bone formation. Aim: To study the role of mutation of BMP4 genes in the aetiology of non-syndromic cleft lip with or without palate (NSCL ± P) and identify it directly from human analyses. Materials and Methods: A case-control study was done to evaluate whether BMP4T538C polymorphism, resulting in an amino acid change of Val=Ala (V152A) in the polypeptide, is associated with NSCL ± P in an Indian paediatric population. Genotypes of 100 patients with NSCL ± P and 100 controls (in whom absence of CL ± P was confirmed in three generations) were detected using a polymerase chain reaction-restriction fragment length polymorphism strategy. Logistic regression was performed to evaluate allele and genotype association with NSCLP. Results: Results showed significant association between homozygous CC genotype with CL ± P (odds ratio [OR]-5.59 and 95% confidence interval [CI] = 2.85-10.99). The 538C allele carriers showed an increased risk of NSCL ± P as compared with 538 T allele (OR - 4.2% CI = 2.75-6.41). Conclusion: This study suggests an association between SNP of BMP4 gene among carriers of the C allele and increased risk for NSCLP in an Indian Population. Further studies on this aspect can scale large heights in preventive strategies for NSCLP that may soon become a reality. PMID:26424979

  7. Intrasplicing coordinates alternative first exons with alternative splicing in the protein 4.1R gene

    SciTech Connect

    Conboy, John G.; Parra, Marilyn K.; Tan, Jeff S.; Mohandas, Narla; Conboy, John G.

    2008-11-07

    In the protein 4.1R gene, alternative first exons splice differentially to alternative 3' splice sites far downstream in exon 2'/2 (E2'/2). We describe a novel intrasplicing mechanism by which exon 1A (E1A) splices exclusively to the distal E2'/2 acceptor via two nested splicing reactions regulated by novel properties of exon 1B (E1B). E1B behaves as an exon in the first step, using its consensus 5' donor to splice to the proximal E2'/2 acceptor. A long region of downstream intron is excised, juxtaposing E1B with E2'/2 to generate a new composite acceptor containing the E1B branchpoint/pyrimidine tract and E2 distal 3' AG-dinucleotide. Next, the upstream E1A splices over E1B to this distal acceptor, excising the remaining intron plus E1B and E2' to form mature E1A/E2 product. We mapped branch points for both intrasplicing reactions and demonstrated that mutation of the E1B 5' splice site or branchpoint abrogates intrasplicing. In the 4.1R gene, intrasplicing ultimately determines N-terminal protein structure and function. More generally, intrasplicing represents a new mechanism whereby alternative promoters can be coordinated with downstream alternative splicing.

  8. mRNA Expression of Ovine Angiopoietin-like Protein 4 Gene in Adipose Tissues.

    PubMed

    Zhang, Jing; Jing, Jiong-Jie; Jia, Xia-Li; Qiao, Li-Ying; Liu, Jian-Hua; Liang, Chen; Liu, Wen-Zhong

    2016-05-01

    Angiopoietin-like protein 4 (ANGPTL4) is involved in a variety of functions, including lipoprotein metabolism and angiogenesis. To reveal the role of ANGPTL4 in fat metabolism of sheep, ovine ANGPTL4 mRNA expression was analyzed in seven adipose tissues from two breeds with distinct tail types. Forty-eight animals with the gender ratio of 1:1 for both Guangling Large Tailed (GLT) and Small Tailed Han (STH) sheep were slaughtered at 2, 4, 6, 8, 10, and 12 months of age, respectively. Adipose tissues were collected from greater and lesser omental, subcutaneous, retroperitoneal, perirenal, mesenteric, and tail fats. Ontogenetic mRNA expression of ANGPTL4 in these adipose tissues from GTL and STH was studied by quantitative real time polymerase chain reaction. The results showed that ANGPTL4 mRNA expressed in all adipose tissues studied with the highest in subcutaneous and the lowest in mesenteric fat depots. Months of age, tissue and breed are the main factors that significantly influence the mRNA expression. These results provide new insights into ovine ANGPTL4 gene expression and clues for its function mechanism. PMID:26954186

  9. mRNA Expression of Ovine Angiopoietin-like Protein 4 Gene in Adipose Tissues

    PubMed Central

    Zhang, Jing; Jing, Jiong-Jie; Jia, Xia-Li; Qiao, Li-Ying; Liu, Jian-Hua; Liang, Chen; Liu, Wen-Zhong

    2016-01-01

    Angiopoietin-like protein 4 (ANGPTL4) is involved in a variety of functions, including lipoprotein metabolism and angiogenesis. To reveal the role of ANGPTL4 in fat metabolism of sheep, ovine ANGPTL4 mRNA expression was analyzed in seven adipose tissues from two breeds with distinct tail types. Forty-eight animals with the gender ratio of 1:1 for both Guangling Large Tailed (GLT) and Small Tailed Han (STH) sheep were slaughtered at 2, 4, 6, 8, 10, and 12 months of age, respectively. Adipose tissues were collected from greater and lesser omental, subcutaneous, retroperitoneal, perirenal, mesenteric, and tail fats. Ontogenetic mRNA expression of ANGPTL4 in these adipose tissues from GTL and STH was studied by quantitative real time polymerase chain reaction. The results showed that ANGPTL4 mRNA expressed in all adipose tissues studied with the highest in subcutaneous and the lowest in mesenteric fat depots. Months of age, tissue and breed are the main factors that significantly influence the mRNA expression. These results provide new insights into ovine ANGPTL4 gene expression and clues for its function mechanism. PMID:26954186

  10. Expression analysis of bone morphogenetic protein 4 between fat and lean birds in adipose tissue and serum.

    PubMed

    Cheng, B H; Leng, L; Wu, M Q; Zhang, Q; Zhang, X Y; Xu, S S; Cao, Z P; Li, Y M; Luan, P; Li, H

    2016-07-01

    The objectives of the present study were to characterize the tissue expression of chicken (Gallus gallus) bone morphogenetic protein 4 (BMP4) and compare differences in its expression in abdominal fat tissue and serum between fat and lean birds and to determine a potential relationship between the expression of BMP4 and abdominal fat tissue growth and development. The results showed that chicken BMP4 messenger RNA (mRNA) and protein were expressed in various tissues, and the expression levels of BMP4 transcript and protein were relatively higher in adipose tissues. In addition, the mRNA and protein expression levels of BMP4 in abdominal fat tissue of fat males were lower than those of lean males at 1, 2, 5, and 7 wk of age (P < 0.05). Furthermore, the serum BMP4 content of fat males was lower than that of lean males at 7 wk of age (P < 0.05). BMP4 mRNA expression levels were significantly higher in preadipocytes than those in mature adipocytes (P < 0.05), and the expression level decreased during differentiation in vitro (P < 0.05). These results suggested that chicken BMP4 might affect abdominal fat deposition through differences in its expression level. The results of this study will provide basic molecular information for studying the role of BMP4 in the regulation of adipogenesis in avian species. PMID:26945137

  11. Wholemount imaging reveals abnormalities of the aqueous outflow pathway and corneal vascularity in Foxc1 and Bmp4 heterozygous mice.

    PubMed

    van der Merwe, Elizabeth L; Kidson, Susan H

    2016-05-01

    Mutations in the FOXC1/Foxc1 gene in humans and mice and Bmp4 in mice are associated with congenital anterior segment dysgenesis (ASD) and the development of the aqueous outflow structures throughout the limbus. The aim of this study was to advance our understanding of anterior segment abnormalities in mouse models of ASD using a 3-D imaging approach. Holistic imaging information combined with quantitative measurements were carried out on PECAM-1 stained individual components of the aqueous outflow vessels and corneal vasculature of Foxc1(+/-) on the C57BL/6Jx129 and ICR backgrounds, Bmp4(+/-) ICR mice, and wildtype mice from each background. In both wildtype and heterozygotes, singular, bifurcated and plexus forms of Schlemm's canal were noted. Of note, missing portions of the canal were seen in the heterozygous groups but not in wildtype animals. In general, we found the number of collector channels to be reduced in both heterozygotes. Lastly, we found a significant increase in the complexity of the corneal arcades and their penetration into the cornea in heterozygotes as compared with wild types. In conclusion, our 3-D imaging studies have revealed a more complex arrangement of both the aqueous vessels and corneal arcades in Foxc1(+/-) and Bmp4(+/-) heterozygotes, and further advance our understanding of how such abnormalities could impact on IOP and the aetiology of glaucoma. PMID:27068508

  12. Shh, Bmp-2, Bmp-4 and Fgf-8 are associated with initiation and patterning of mouse tongue papillae.

    PubMed

    Jung, H S; Oropeza, V; Thesleff, I

    1999-03-01

    Spacing patterns are of fundamental importance in various repeated structures which develop at regular intervals such as feathers, teeth and insect ommatidia. The mouse tongue develops a regular papilla pattern and provides a good model to study pattern formation. We examined the expression patterns of the signalling molecules, sonic hedgehog (Shh), bone morphogenetic proteins -2 and -4 (Bmp-2 and Bmp-4), and fibroblast growth factor-8 (Fgf-8) in mouse embryos between E 10.5 and 15. We show that all four genes are expressed uniformly in the tongue epithelium between E 10.5 and 11. At E 13, before morphologically detectable gustatory papillae initiation, Shh, Bmp-2 and Bmp-4 expression segregates into discrete spots, whereas, Fgf-8 is downregulated. At E 14, small eminences in the anterior part of the tongue are the first morphological indications of fungiform papillae, and they express Shh and Bmp-2, whereas, Bmp-4 is almost absent in the tongue. We conclude that these conserved signalling molecules are associated with the initiation and early morphogenesis of the tongue papillae. PMID:10330496

  13. The homing and inhibiting effects of hNSCs-BMP4 on human glioma stem cells

    PubMed Central

    Zhao, Mingming; Zhou, Chunhui; Ren, Junlin; Huang, Qiming; Zhao, Zhongming; Mitra, Ramkrishna; Fan, Wenhong; Fan, Ming

    2016-01-01

    Malignant gliomas patients have a poor survival rate, partially due to the inability in delivering therapeutic agents to the tumors, especially to the metastasis of human glioma stem cells (hGSCs). To explore whether the human neural stem cells (hNSCs) with an over-expression of BMP4 (hNSCs-BMP4) can trace and inhibit hGSCs, in this study, we examined the migration of hNSCs to hGSCs using transwell assay in vitro and performed the fluorescent tracer experiment in vivo. We examined the proliferation, differentiation, apoptosis and migration of hGSCs after co-culturing with hNSCs-BMP4 in vitro and tested the tropism and antitumor effects of hNSCs-BMP4 in the established brain xenograft models of hGSCs. We found that hNSCs-BMP4 could secrete BMP4 and trace hGSCs both in vitro and in vivo. When compared to the normal human astrocytes (NHAs) and hNSCs, hNSCs-BMP4 could significantly inhibit the invasive growth of hGSCs, promote their differentiation and apoptosis by activating Smad1/5/8 signaling, and prolong the survival time of the tumor-bearing nude mice. Collectively, this study suggested that hNSCs-BMP4 may help in developing therapeutic approaches for the treatment of human malignant gliomas. PMID:26908439

  14. Bmp4 Promotes a Brown to White-like Adipocyte Shift.

    PubMed

    Modica, Salvatore; Straub, Leon G; Balaz, Miroslav; Sun, Wenfei; Varga, Lukas; Stefanicka, Patrik; Profant, Milan; Simon, Eric; Neubauer, Heike; Ukropcova, Barbara; Ukropec, Jozef; Wolfrum, Christian

    2016-08-23

    While Bmp4 has a well-established role in the commitment of mesenchymal stem cells into the adipogenic lineage, its role in brown adipocyte formation and activity is not well defined. Here, we show that Bmp4 has a dual function in adipogenesis by inducing adipocyte commitment while inhibiting the acquisition of a brown phenotype during terminal differentiation. Selective brown adipose tissue overexpression of Bmp4 in mice induces a shift from a brown to a white-like adipocyte phenotype. This effect is mediated by Smad signaling and might be in part due to suppression of lipolysis, via regulation of hormone sensitive lipase expression linked to reduced Ppar activity. Given that we observed a strong correlation between BMP4 levels and adipocyte size, as well as insulin sensitivity in humans, we propose that Bmp4 is an important factor in the context of obesity and type 2 diabetes. PMID:27524617

  15. Abrogation of epithelial BMP2 and BMP4 causes Amelogenesis Imperfecta by reducing MMP20 and KLK4 expression.

    PubMed

    Xie, Xiaohua; Liu, Chao; Zhang, Hua; Jani, Priyam H; Lu, Yongbo; Wang, Xiaofang; Zhang, Bin; Qin, Chunlin

    2016-01-01

    Amelogenesis Imperfecta (AI) can be caused by the deficiencies of enamel matrix proteins, molecules responsible for the transportation and secretion of enamel matrix components, and proteases processing enamel matrix proteins. In the present study, we discovered the double deletion of bone morphogenetic protein 2 (Bmp2) and bone morphogenetic protein 4 (Bmp4) in the dental epithelium by K14-cre resulted in hypoplastic enamel and reduced density in X-ray radiography as well as shortened enamel rods under scanning electron microscopy. Such enamel phenotype was consistent with the diagnosis of hypoplastic amelogenesis imperfecta. Histological and molecular analyses revealed that the removal of matrix proteins in the mutant enamel was drastically delayed, which was coincided with the greatly reduced expression of matrix metalloproteinase 20 (MMP20) and kallikrein 4 (KLK4). Although the expression of multiple enamel matrix proteins was down-regulated in the mutant ameloblasts, the cleavage of ameloblastin was drastically impaired. Therefore, we attributed the AI primarily to the reduction of MMP20 and KLK4. Further investigation found that BMP/Smad4 signaling pathway was down-regulated in the K14-cre;Bmp2(f/f);Bmp4(f/f)ameloblasts, suggesting that the reduced MMP20 and KLK4 expression may be due to the attenuated epithelial BMP/Smad4 signaling. PMID:27146352

  16. Abrogation of epithelial BMP2 and BMP4 causes Amelogenesis Imperfecta by reducing MMP20 and KLK4 expression

    PubMed Central

    Xie, Xiaohua; Liu, Chao; Zhang, Hua; Jani, Priyam H.; Lu, Yongbo; Wang, Xiaofang; Zhang, Bin; Qin, Chunlin

    2016-01-01

    Amelogenesis Imperfecta (AI) can be caused by the deficiencies of enamel matrix proteins, molecules responsible for the transportation and secretion of enamel matrix components, and proteases processing enamel matrix proteins. In the present study, we discovered the double deletion of bone morphogenetic protein 2 (Bmp2) and bone morphogenetic protein 4 (Bmp4) in the dental epithelium by K14-cre resulted in hypoplastic enamel and reduced density in X-ray radiography as well as shortened enamel rods under scanning electron microscopy. Such enamel phenotype was consistent with the diagnosis of hypoplastic amelogenesis imperfecta. Histological and molecular analyses revealed that the removal of matrix proteins in the mutant enamel was drastically delayed, which was coincided with the greatly reduced expression of matrix metalloproteinase 20 (MMP20) and kallikrein 4 (KLK4). Although the expression of multiple enamel matrix proteins was down-regulated in the mutant ameloblasts, the cleavage of ameloblastin was drastically impaired. Therefore, we attributed the AI primarily to the reduction of MMP20 and KLK4. Further investigation found that BMP/Smad4 signaling pathway was down-regulated in the K14-cre;Bmp2f/f;Bmp4f/fameloblasts, suggesting that the reduced MMP20 and KLK4 expression may be due to the attenuated epithelial BMP/Smad4 signaling. PMID:27146352

  17. Noggin and BMP4 co-modulate adult hippocampal neurogenesis in the APP{sub swe}/PS1{sub {Delta}E9} transgenic mouse model of Alzheimer's disease

    SciTech Connect

    Tang, Jun; Song, Min; Wang, Yanyan; Fan, Xiaotang; Xu, Haiwei; Bai, Yun

    2009-07-31

    In addition to the subventricular zone, the dentate gyrus of the hippocampus is one of the few brain regions in which neurogenesis continues into adulthood. Perturbation of neurogenesis can alter hippocampal function, and previous studies have shown that neurogenesis is dysregulated in Alzheimer disease (AD) brain. Bone morphogenetic protein-4 (BMP4) and its antagonist Noggin have been shown to play important roles both in embryonic development and in the adult nervous system, and may regulate hippocampal neurogenesis. Previous data indicated that increased expression of BMP4 mRNA within the dentate gyrus might contribute to decreased hippocampal cell proliferation in the APP{sub swe}/PS1{sub {Delta}E9} mouse AD model. However, it is not known whether the BMP antagonist Noggin contributes to the regulation of neurogenesis. We therefore studied the relative expression levels and localization of BMP4 and its antagonist Noggin in the dentate gyrus and whether these correlated with changes in neurogenesis in 6-12 mo old APP{sub swe}/PS1{sub {Delta}E9} transgenic mice. Bromodeoxyuridine (BrdU) was used to label proliferative cells. We report that decreased neurogenesis in the APP/PS1 transgenic mice was accompanied by increased expression of BMP4 and decreased expression of Noggin at both the mRNA and protein levels; statistical analysis showed that the number of proliferative cells at different ages correlated positively with Noggin expression and negatively with BMP4 expression. Intraventricular administration of a chimeric Noggin/Fc protein was used to block the action of endogenous BMP4; this resulted in a significant increase in the number of BrdU-labeled cells in dentate gyrus subgranular zone and hilus in APP/PS1 mice. These results suggest that BMP4 and Noggin co-modulate neurogenesis.

  18. Aerosol delivery of programmed cell death protein 4 using polysorbitol-based gene delivery system for lung cancer therapy.

    PubMed

    Kim, You-Kyoung; Xing, Lei; Chen, Bao-An; Xu, Fengguo; Jiang, Hu-Lin; Zhang, Can

    2014-11-01

    The development of a safe and effective gene delivery system is the most challenging obstacle to the broad application of gene therapy in the clinic. In this study, we report the development of a polysorbitol-based gene delivery system as an alternative gene carrier for lung cancer therapy. The copolymer was prepared by a Michael addition reaction between sorbitol diacrylate (SD) and spermine (SPE); the SD-SPE copolymer effectively condenses with DNA on the nanoscale and protects it from nucleases. SD-SPE/DNA complexes showed excellent transfection with low toxicity both in vitro and in vivo, and aerosol delivery of SD-SPE complexes with programmed cell death protein 4 DNA significantly suppressed lung tumorigenesis in K-ras(LA1) lung cancer model mice. These results demonstrate that SD-SPE has great potential as a gene delivery system based on its excellent biocompatibility and high gene delivery efficiency for lung cancer gene therapy. PMID:24983766

  19. Double homozygous missense mutations in DACH1 and BMP4 in a patient with bilateral cystic renal dysplasia.

    PubMed

    Schild, Raphael; Knüppel, Tanja; Konrad, Martin; Bergmann, Carsten; Trautmann, Agnes; Kemper, Markus J; Wu, Kongming; Yaklichkin, Sergey; Wang, Jing; Pestell, Richard; Müller-Wiefel, Dirk E; Schaefer, Franz; Weber, Stefanie

    2013-01-01

    Renal hypodysplasia (RHD) is characterized by small and/or disorganized kidneys following abnormal organogenesis. Mutations in several genes have been identified recently to be associated with RHD in humans, including BMP4, a member of the transforming growth factor (TGF)-β family of growth factors. DACH1 has been proposed as a candidate gene for RHD because of its involvement in the EYA-SIX-DACH network of renal developmental genes. Here, we present a patient with renal dysplasia carrying homozygous missense mutations in both BMP4 (p.N150K) and DACH1 (p.R684C). The genotype-phenotype correlation in the family hints at an oligogenic mode of inheritance of the disease in this kindred. Functional analyses of the identified DACH1 mutation in HEK293T cells demonstrated enhanced suppression of the TGF-β pathway suggesting that both mutations could act synergistically in the development of the phenotype in this patient. This finding provides a model for RHD as an oligo-/polygenic disorder and supports a role for DACH1 in the development of RHD in humans. PMID:23262432

  20. Characterization and cloning of a receptor for BMP-2 and BMP-4 from NIH 3T3 cells.

    PubMed Central

    Koenig, B B; Cook, J S; Wolsing, D H; Ting, J; Tiesman, J P; Correa, P E; Olson, C A; Pecquet, A L; Ventura, F; Grant, R A

    1994-01-01

    The bone morphogenetic proteins (BMPs) are a group of transforming growth factor beta (TGF-beta)-related factors whose only receptor identified to date is the product of the daf-4 gene from Caenorhabditis elegans. Mouse embryonic NIH 3T3 fibroblasts display high-affinity 125I-BMP-4 binding sites. Binding assays are not possible with the isoform 125I-BMP-2 unless the positively charged N-terminal sequence is removed to create a modified BMP-2, 125I-DR-BMP-2. Cross-competition experiments reveal that BMP-2 and BMP-4 interact with the same binding sites. Affinity cross-linking assays show that both BMPs interact with cell surface proteins corresponding in size to the type I (57- to 62-kDa) and type II (75- to 82-kDa) receptor components for TGF-beta and activin. Using a PCR approach, we have cloned a cDNA from NIH 3T3 cells which encodes a novel member of the transmembrane serine/threonine kinase family most closely resembling the cloned type I receptors for TGF-beta and activin. Transient expression of this receptor in COS-7 cells leads to an increase in specific 125I-BMP-4 binding and the appearance of a major affinity-labeled product of approximately 64 kDa that can be labeled by either tracer. This receptor has been named BRK-1 in recognition of its ability to bind BMP-2 and BMP-4 and its receptor kinase structure. Although BRK-1 does not require cotransfection of a type II receptor in order to bind ligand in COS cells, complex formation between BRK-1 and the BMP type II receptor DAF-4 can be demonstrated when the two receptors are coexpressed, affinity labeled, and immunoprecipitated with antibodies to either receptor subunit. We conclude that BRK-1 is a putative BMP type I receptor capable of interacting with a known type II receptor for BMPs. Images PMID:8065329

  1. BMP2/BMP4 colorectal cancer susceptibility loci in northern and southern European populations.

    PubMed

    Fernandez-Rozadilla, Ceres; Palles, Claire; Carvajal-Carmona, Luis; Peterlongo, Paolo; Nici, Carmela; Veneroni, Silvia; Pinheiro, Manuela; Teixeira, Manuel R; Moreno, Victor; Lamas, Maria-Jesus; Baiget, Montserrat; Lopez-Fernandez, L A; Gonzalez, Dolors; Brea-Fernandez, Alejandro; Clofent, Juan; Bujanda, Luis; Bessa, Xavier; Andreu, Montserrat; Xicola, Rosa; Llor, Xavier; Jover, Rodrigo; Castells, Antoni; Castellvi-Bel, Sergi; Carracedo, Angel; Tomlinson, Ian; Ruiz-Ponte, Clara

    2013-02-01

    Genome-wide association studies have successfully identified 20 colorectal cancer susceptibility loci. Amongst these, four of the signals are defined by tagging single nucleotide polymorphisms (SNPs) on regions 14q22.2 (rs4444235 and rs1957636) and 20p12.3 (rs961253 and rs4813802). These markers are located close to two of the genes involved in bone morphogenetic protein (BMP) signaling (BMP4 and BMP2, respectively). By investigating these four SNPs in an initial cohort of Spanish origin, we found substantial evidence that minor allele frequencies (MAFs) may be different in northern and southern European populations. Therefore, we genotyped three additional southern European cohorts comprising a total of 2028 cases and 4273 controls. The meta-analysis results show that only one of the association signals (rs961253) is effectively replicated in the southern European populations, despite adequate power to detect all four. The other three SNPs (rs4444235, rs1957636 and rs4813802) presented discordant results in MAFs and linkage disequilibrium patterns between northern and southern European cohorts. We hypothesize that this lack of replication could be the result of differential tagging of the functional variant in both sets of populations. Were this true, it would have complex consequences in both our ability to understand the nature of the real causative variants, as well as for further study designs. PMID:23161572

  2. Foxc1 Expression in Early Osteogenic Differentiation Is Regulated by BMP4-SMAD Activity.

    PubMed

    Hopkins, Alexander; Mirzayans, Freda; Berry, Fred

    2016-07-01

    FOXC1 is an important regulator of the initial steps in intramembranous and endochondral ossification processes. As BMP signalling is a key initiator of these processes, we sought to determine whether Foxc1 expression is regulated by such signalling factors. BMP4 treatment of C2C12 cells resulted in an induction in Foxc1 mRNA levels. Chromatin immunoprecipitation assays demonstrated that SMAD proteins interacted with the mouse Foxc1 promoter approximately 300 bp upstream of the transcription start site. This ChIP positive region was cloned into a luciferase reporter and demonstrated to be responsive to BMP4 stimulation. Reduction of Foxc1 levels in C2C12 cells though siRNA impaired BMP4 osteogenic differentiation. In contrast, BMP4 treatment repressed Foxc1 expression in 10T1/2 or D1-ORL mesenchymal cells and MC3T3 preosteoblasts. Finally, siRNA knock-down of Foxc1 in MC3T3 cells resulted in an induction of markers of osteoblast differentiation and an accelerated mineralization. These data indicate that Foxc1 expression is regulated by BMP4 and FOXC1 functions in the commitment of progenitor cells to the osteoblast fate and its expression is reduced when differentiation proceeds. J. Cell. Biochem. 117: 1707-1717, 2016. © 2015 Wiley Periodicals, Inc. PMID:26666591

  3. The Common Variant rs4444235 near BMP4 Confers Genetic Susceptibility of Colorectal Cancer: An Updated Meta-Analysis Based on a Comprehensive Statistical Strategy

    PubMed Central

    Liu, Li; Su, Qinji; Li, Lixia; Lin, Xiaohui; Gan, Yu; Chen, Sidong

    2014-01-01

    Objective We performed an updated meta-analysis, using a comprehensive strategy of a logistic regression and a model-free approach, to evaluate more precisely the role of the rs4444235 variant near the Bone morphogenetic protein-4 (BMP4) gene in susceptibility to colorectal cancer (CRC). Methods A total of 19 studies with 28770 cases and 28234 controls were included. Metagen system with logistic regression was applied to choose the most plausible genetic model for rs4444235. Generalized odds ratio (ORG) metric was used to provide a global test of relationship between rs4444235 and CRC risk. Results Metagen analysis suggested the rs4444235 fitted best to an additive model. In assessment of the additive model, heterogeneity was observed (P = 0.059, I2 = 36.1), and pooled per-allele OR was 1.08 (95% CI = 1.05–1.11). Based on the model-free approach, pooled ORG was 1.09 (95% CI = 1.05–1.14) under a random-effect model. Stratified analyses suggested heterogeneity could be in part explained by population ethnicity, study design, sources of controls, and sample size. Sensitivity analysis further supported the robust stability of the current results, by showing similar pooled estimates before and after sequential removal of each study. Conclusions This meta-analysis provides a robust estimate of the positive association between the rs4444235 and CRC risk and further emphasizes the importance of the rs4444235 in CRC risk prediction. PMID:24932582

  4. Negative feedback in the bone morphogenetic protein 4 (BMP4) synexpression group governs its dynamic signaling range and canalizes development

    PubMed Central

    Paulsen, Malte; Legewie, Stefan; Eils, Roland; Karaulanov, Emil; Niehrs, Christof

    2011-01-01

    What makes embryogenesis a robust and canalized process is an important question in developmental biology. A bone morphogenetic protein (BMP) morphogen gradient plays a key role in embryonic development, and we are beginning to understand how the self-regulating properties of its signaling circuitry ensure robust embryonic patterning. An unexplored question is why the BMP signaling circuit is organized as a modular synexpression group, with a prevalence of feedback inhibitors. Here, we provide evidence from direct experimentation and mathematical modeling that the synexpressed feedback inhibitors BAMBI, SMAD6, and SMAD7 (i) expand the dynamic BMP signaling range essential for proper embryonic patterning and (ii) reduce interindividual phenotypic and molecular variability in Xenopus embryos. Thereby, negative feedback linearizes signaling responses and confers robust patterning, thus promoting canalized development. The presence of negative feedback inhibitors in other growth factor synexpression groups suggests that these properties may constitute a general principle. PMID:21633009

  5. RING finger protein 4 (RNF4) derepresses gene expression from DNA methylation.

    PubMed

    Wang, Yu

    2014-12-01

    RNF4 is an E3 ubiquitin ligase originally identified as a transcription co-activator. The mechanism by which RNF4 promotes transcription remains unclear. In this study, I found that RNF4 antagonizes transcriptional repression mediated by DNA methylation. RNF4 does not promote DNA demethylation, but mediates the ubiquitination of MeCP2, a methyl-CpG-binding domain (MBD) protein. Removal of MeCP2 from gene promoters activates transcription. This study thus not only uncovers how RNF4 functions as a transcription activator, but also reveals the mechanism by which MeCP2 protein stability is regulated. PMID:25355316

  6. TNF-Alpha Represses Transcription of Human Bone Morphogenetic Protein-4 in Lung Epithelial Cells

    PubMed Central

    Zhu, Nian-Ling; Li, Changgong; Huang, Hao Hao; Sebald, Matthew; Londhe, Vedang A.; Heisterkamp, Nora; Warburton, David; Bellusci, Saverio; Minoo, Parviz

    2007-01-01

    Bone Morphogenetic Proteins are key signaling molecules in vertebrate development. Little is known about Bmp gene regulation in any organ. In Drosophila, the Bmp gene, dpp is regulated by Dorsal, the invertebrate homologue of Rel-NFkB. In this study we examined whether TNF-alpha, which activates NF-kB, can regulate Bmp4 gene expression. TNF-alpha reduced Bmp4 mRNA in lung adenocarcinoma A549 cells and repressed transcriptional activity of the human Bmp4 promoter in a dose-dependent manner. Similar repression was observed when the Bmp4 promoter was co-transfected with a p65 (RelA) expression vector in the absence of TNF-alpha treatment, suggesting that RelA mediates the effect of TNF-alpha. In support of this finding, the repressor effect of TNF-alpha on Bmp4 was abrogated by a co-transfected dominant negative mutant of IkB (S32A/S36A). The human Bmp4 promoter contains 3 putative consensus binding sites for NF-kB. Surprisingly, only one of the latter binding sites was capable of binding NFkB. Repressor effect of NFkB was not dependent on any of the three binding sites, but localized to a 122 bp fragment which bound both RelA and SP1. SP1stimulated transcription, whereas increasing doses of RelA opposed this effect. In vivo, TNF-alpha inhibited branching morphogenesis and LacZ gene expression in Bmp4-lacz transgenic lungs. These data support a model in which TNF-alpha-induced RelA interacts with SP1 to bring about transcriptional repression of Bmp4 gene. These findings provide a mechanistic paradigm for interactions between mediators of inflammation and morphogenesis with relevant implications for normal lung development and pathogenesis of disease. PMID:17350185

  7. A Dde I RFLP in exon 21 of human EL1 gene, encoding protein 4.1, detectable by SSCP.

    PubMed

    Maillet, P; Dalla Venezia, N; Bozon, M; Vallier, A; Delaunay, J; Baklouti, F

    1998-01-01

    Protein 4.1 is a major component of the junctional complex at the red cell skeleton. Genomic studies have recently evidenced that the encoding gene (EL1 locus) is present in a single copy per haploid genome. Several RFLPs have already been characterized within intron sequences. Here, we describe the first RFLP found within the coding sequence. This polymorphism (C or T at position 2723, in exon 21) does not affect the amino acid sequence (Thr-->Thr). It can be detected by either Dde I restriction digestion of an appropriate PCR product, or simply by SSCP These findings should facilitate analysis of families with 4.1 deficiencies causing hereditary elliptocytosis. PMID:9554757

  8. Expression of low density lipoprotein receptor-related protein 4 (Lrp4) gene in the mouse germ cells.

    PubMed

    Yamaguchi, Yasuka L; Tanaka, Satomi S; Kasa, Miyuki; Yasuda, Kunio; Tam, Patrick P L; Matsui, Yasuhisa

    2006-08-01

    The low density lipoprotein receptor-related protein 4 gene (Lrp4) was identified by subtractive screening of cDNAs of the migratory primordial germ cells (PGCs) of E8.5-9.5 embryo and E3.5 blastocysts. Lrp4 is expressed in PGCs in the hindgut and the dorsal mesentery of E9.5 embryos, and in germ cells in the genital ridges of male and female E10.5-13.5 embryos. Lrp4 is also expressed in spermatogonia of the neonatal and adult testes and in the immature oocytes and follicular cells of the adult ovary. The absence of Lrp4 expression in the blastocyst, embryonic stem cells and embryonic germ cells suggests the Lrp4 is a molecular marker that distinguishes the germ cells from embryo-derived pluripotent stem cells. PMID:16434236

  9. In Search of Biomarkers for Idiopathic Scoliosis: Leptin and BMP4 Functional Polymorphisms

    PubMed Central

    Nikolova, Svetla; Yablanski, Vasil; Vlaev, Evgeni; Getova, Gergana; Atanasov, Ventseslav; Stokov, Luben; Savov, Alexey Slavkov; Kremensky, Ivo Marinov

    2015-01-01

    Idiopathic scoliosis (IS) is the most common spinal disorder in children and adolescents. The current consensus on IS maintains that it has a multifactorial etiology with genetic predisposition factors. In the present study the association of two functional polymorphisms of leptin (rs7799039) and BMP4 (rs4898820) with susceptibility to IS and curve severity was investigated in a Bulgarian population sample. The molecular detection of the genotypes was performed by amplification followed by restriction technology. The statistical analysis was performed by Pearson's chi-squared test. This case-control study revealed no statistically significant association between the functional polymorphisms of leptin and BMP4 and susceptibility to IS or curve progression (p > 0.05). On the basis of these results the examined polymorphic variants of leptin and BMP4 could not be considered as genetic variants with predisposition effect or as risk factors for the progression of the curve. In addition, these results do not exclude a synergistic effect of the promoter polymorphisms of leptin and BMP4 in the etiology and pathogenesis of IS. The identification of molecular markers for IS could be useful for early detection and prognosis of the risk for a rapid progression of the curve. That would permit early stage treatment of the patient with the least invasive procedures. PMID:26317037

  10. Boldine improves endothelial function in diabetic db/db mice through inhibition of angiotensin II-mediated BMP4-oxidative stress cascade

    PubMed Central

    Lau, Yeh Siang; Tian, Xiao Yu; Mustafa, Mohd Rais; Murugan, Dharmani; Liu, Jian; Zhang, Yang; Lau, Chi Wai; Huang, Yu

    2013-01-01

    BACKGROUND AND PURPOSE Boldine is a potent natural antioxidant present in the leaves and bark of the Chilean boldo tree. Here we assessed the protective effects of boldine on endothelium in a range of models of diabetes, ex vivo and in vitro. EXPERIMENTAL APPROACH Vascular reactivity was studied in mouse aortas from db/db diabetic and normal mice. Reactive oxygen species (ROS) production, angiotensin AT1 receptor localization and protein expression of oxidative stress markers in the vascular wall were evaluated by dihydroethidium fluorescence, lucigenin enhanced-chemiluminescence, immunohistochemistry and Western blot respectively. Primary cultures of mouse aortic endothelial cells, exposed to high concentrations of glucose (30 mmol L−1) were also used. KEY RESULTS Oral treatment (20 mg kg−1day−1, 7 days) or incubation in vitro with boldine (1 μmol L−1, 12 h) enhanced endothelium-dependent aortic relaxations of db/db mice. Boldine reversed impaired relaxations induced by high glucose or angiotensin II (Ang II) in non-diabetic mouse aortas while it reduced the overproduction of ROS and increased phosphorylation of eNOS in db/db mouse aortas. Elevated expression of oxidative stress markers (bone morphogenic protein 4 (BMP4), nitrotyrosine and AT1 receptors) were reduced in boldine-treated db/db mouse aortas. Ang II-stimulated BMP4 expression was inhibited by boldine, tempol, noggin or losartan. Boldine inhibited high glucose-stimulated ROS production and restored the decreased phosphorylation of eNOS in mouse aortic endothelial cells in culture. CONCLUSIONS AND IMPLICATIONS Boldine reduced oxidative stress and improved endothelium-dependent relaxation in aortas of diabetic mice largely through inhibiting ROS overproduction associated with Ang II-mediated BMP4-dependent mechanisms. PMID:23992296

  11. BMP4 and LGL1 are Down Regulated in an Ovine Model of Congenital Diaphragmatic Hernia

    PubMed Central

    Emmerton-Coughlin, Heather M. A.; Martin, K. Kathryn; Chiu, Jacky S. S.; Zhao, Lin; Scott, Leslie A.; Regnault, Timothy R. H.; Bütter, Andreana

    2014-01-01

    Background/Purpose: The molecular pathophysiology of lung hypoplasia in congenital diaphragmatic hernia (CDH) remains poorly understood. The Wnt signaling pathway and downstream targets, such as bone morphogenetic proteins (BMP) 4 and other factors such as late gestation lung protein 1 (LGL1), are essential to normal lung development. Nitrofen-induced hypoplastic CDH rodent lungs demonstrate down regulation of the Wnt pathway including BMP4 and reduced LGL1 expression. The aim of the current study was to examine the molecular pathophysiology associated with a surgically induced CDH in an ovine model. Methods: Left thoracotomy was performed at 80 days in 14 fetal sheep; CDH was created in seven experimental animals. Lungs were harvested at 136 days (term = 145 days). Lung weight (LW) and mean terminal bronchiole density (MTBD) were measured to determine the degree of pulmonary hypoplasia. Quantitative real time PCR was undertaken to analyze Wnt2, Wnt7b, BMP4, and LGL1 mRNA expression. Results: Total LW was decreased while MTBD was increased in the CDH group (p < 0.05), confirming pulmonary hypoplasia. BMP4 and LGL1 mRNA was significantly reduced in CDH lungs (p < 0.05). Wnt2 mRNA was decreased, although not significantly (p < 0.06). Conclusion: For the first time, down regulation of BMP4 and LGL1 are reported in an ovine CDH model. In contrast to other animal models, these changes are persistent to near term. These findings suggest that mechanical compression from herniated viscera may play a more important role in causing pulmonary hypoplasia in CDH, rather than a primary defect in lung organogenesis. PMID:25593968

  12. Comparison of Cell Viability and Embryoid Body Size of Two Embryonic Stem Cell Lines After Different Exposure Times to Bone Morphogenetic Protein 4

    PubMed Central

    Zarei Fard, Nehleh; Talaei-Khozani, Tahereh; Bahmanpour, Soghra; Esmaeilpour, Tahereh

    2015-01-01

    Background Activation of bone morphogenetic protein 4 (BMP4) signaling pathway in embryonic stem (ES) cells plays an important role in controlling cell proliferation, differentiation, and apoptosis. Adverse effects of BMP4 occur in a time dependent manner; however, little is known about the effect of different time exposure of this growth factor on cell number in culture media. In this study, we investigated the role of two different exposure times to BMP4 in cell viability, embryoid body (EB), size, and cavitation of ES cells. Methods Embryonic stem cells (R1 and B1 lines) were released from the feeder cell layers and were cultured using EBs protocol by using the hanging drop method and monolayer culture system. The cells were cultured for 5 days with 100 ng/mL BMP4 from the beginning (++BMP4) or after 48 h (+BMP4) of culture and their cell number were counted by trypan blue staining. The data were analyzed using non-parametric two-tailed Mann-Whitney test. P<0.05 was considered as significant. Results In EB culture protocol, cell number significantly decreased in +BMP4 culture condition with greater cavity size compared to the ++BMP4 condition at day 5 (P=0.009). In contrast, in monolayer culture system, there was no significant difference in the cell number between all groups (P=0.91). Conclusion The results suggest that short-term exposure of BMP4 is required to promote cavitation in EBs according to lower cell number in +BMP4 condition. Different cell lines showed different behavior in cavitation formation. PMID:25821290

  13. BMP4 Is a Peripherally-Derived Factor for Motor Neurons and Attenuates Glutamate-Induced Excitotoxicity In Vitro

    PubMed Central

    Chou, Hui-Ju; Lai, Dar-Ming; Huang, Cheng-Wen; McLennan, Ian S.; Wang, Horng-Dar; Wang, Pei-Yu

    2013-01-01

    Bone morphogenetic proteins (BMPs), members of the transforming growth factor-beta (TGF-β) superfamily, have been shown to play important roles in the nervous system, including neuronal survival and synaptogenesis. However, the physiological functions of BMP signaling in the mammalian neuromuscular system are not well understood. In this study, we found that proteins of the type II bone morphogenetic receptors (BMPRII) were detected at the neuromuscular junction (NMJ), and one of its ligands, BMP4, was expressed by Schwann cells and skeletal muscle fibers. In double-ligated nerves, BMP4 proteins accumulated at the proximal and distal portions of the axons, suggesting that Schwann cell- and muscle fiber-derived BMP4 proteins were anterogradely and retrogradely transported by motor neurons. Furthermore, BMP4 mRNA was down-regulated in nerves but up-regulated in skeletal muscles following nerve ligation. The motor neuron-muscle interactions were also demonstrated using differentiated C2C12 muscle cells and NG108-15 neurons in vitro. BMP4 mRNA and immunoreactivity were significantly up-regulated in differentiated C2C12 muscle cells when the motor neuron-derived factor, agrin, was present in the culture. Peripherally-derived BMP4, on the other hand, promotes embryonic motor neuron survival and protects NG108-15 neurons from glutamate-induced excitotoxicity. Together, these data suggest that BMP4 is a peripherally-derived factor that may regulate the survival of motor neurons. PMID:23472198

  14. BMP4 and FGF strongly induce differentiation of mouse ES cells into oral ectoderm.

    PubMed

    Ochiai, Hiroshi; Suga, Hidetaka; Yamada, Tomiko; Sakakibara, Mayu; Kasai, Takatoshi; Ozone, Chikafumi; Ogawa, Koichiro; Goto, Motomitsu; Banno, Ryoichi; Tsunekawa, Shin; Sugimura, Yoshihisa; Arima, Hiroshi; Oiso, Yutaka

    2015-09-01

    During embryonic development, oral ectoderm differentiates into the adenohypophysis, dental epithelia, salivary glands, and nasal pit. Few reports exist concerning the induction of oral ectoderm from embryonic stem (ES) cells. Generally, any lot differences in fetal bovine serum (FBS) and serum replacer may affect the induction of ES cell-differentiation. Using a previously established culture strategy for differentiation, the proportion of cell aggregates containing Pitx1+ oral ectoderm varied widely between 9-36% when several different lots of FBS or serum replacer were used. We therefore tried to enhance the differentiation method. We found that bone morphogenetic protein (BMP) 4 and fibroblast growth factor (FGF) treatments improved oral ectoderm induction. Such treatment also improved the differentiation of oral ectoderm into the adenohypophysis. Furthermore, increased BMP4 treatment induced dental epithelium and mesenchyme. Such differentiation suggests that the Pitx1+ layer displays similar properties to oral ectoderm, as found in vivo. Differentiation of ES cells into oral ectoderm using different lots of FBS and serum replacer increased 78-90% after treatment with BMP4 and FGF. In summary, we have established a robust strategy for the induction of oral ectoderm differentiation from mouse ES cells. PMID:26209816

  15. The use of real-time PCR technique in the detection of novel protein 4.2 gene mutations that coexist with thalassaemia alpha in a single patient.

    PubMed

    Maciag, Monika; Adamowicz-Salach, Anna; Siwicka, Alicja; Spychalska, Justyna; Burzynska, Beata

    2009-10-01

    Alpha-thalassaemia is a very rare disease in Northern Europe in contrast to hereditary spherocytosis that is associated with red blood cell membrane defects. We report here alpha-thalassaemia case who was also found to bear the erythrocyte membrane protein 4.2 gene mutations. mRNA relative quantification of red cell membrane protein genes in a Polish patient with alpha-thalassaemia trait indicated EPB42 as the gene that could also be involved in anaemia pathogenesis. Sequencing revealed the presence of two novel mutations in the protein 4.2 gene: a G1701A genetic change that predicts an alanine to threonine at position 567 of the protein (A567T) and a T-->A substitution that is located at position +6 of the donor splice site of intron 2 (IVS2nt+6T>A). This is the sixth variant of the erythrocyte membrane protein 4.2 gene mutations identified outside the Japanese population. PMID:19508687

  16. Human carcinoma-associated mesenchymal stem cells promote ovarian cancer chemotherapy resistance via a BMP4/HH signaling loop

    PubMed Central

    Coffman, Lan G.; Choi, Yun-Jung; McLean, Karen; Allen, Benjamin L.; di Magliano, Marina Pasca; Buckanovich, Ronald J.

    2016-01-01

    The tumor microenvironment is critical to cancer growth and therapy resistance. We previously characterized human ovarian carcinoma-associated mesenchymal stem cells (CA-MSCs). CA-MSCs are multi-potent cells that can differentiate into tumor microenvironment components including fibroblasts, myofibroblasts and adipocytes. We previously reported CA-MSCs, compared to normal MSCs, express high levels of BMP proteins and promote tumor growth by increasing numbers of cancer stem-like cells (CSCs). We demonstrate here that ovarian tumor cell-secreted Hedgehog (HH) induces CA-MSC BMP4 expression. CA-MSC-derived BMP4 reciprocally increases ovarian tumor cell HH expression indicating a positive feedback loop. Interruption of this loop with a HH pathway inhibitor or BMP4 blocking antibody decreases CA-MSC-derived BMP4 and tumor-derived HH preventing enrichment of CSCs and reversing chemotherapy resistance. The impact of HH inhibition was only seen in CA-MSC-containing tumors, indicating the importance of a humanized stroma. These results are reciprocal to findings in pancreatic and bladder cancer, suggesting HH signaling effects are tumor tissue specific warranting careful investigation in each tumor type. Collectively, we define a critical positive feedback loop between CA-MSC-derived BMP4 and ovarian tumor cell-secreted HH and present evidence for the further investigation of HH as a clinical target in ovarian cancer. PMID:26755648

  17. Bone morphogenic protein 4 produced in endothelial cells by oscillatory shear stress induces monocyte adhesion by stimulating reactive oxygen species production from a nox1-based NADPH oxidase.

    PubMed

    Sorescu, George P; Song, Hannah; Tressel, Sarah L; Hwang, Jinah; Dikalov, Sergey; Smith, Debra A; Boyd, Nolan L; Platt, Manu O; Lassègue, Bernard; Griendling, Kathy K; Jo, Hanjoong

    2004-10-15

    Atherosclerosis is an inflammatory disease occurring preferentially in arterial regions exposed to disturbed flow conditions including oscillatory shear stress (OS). OS exposure induces endothelial expression of bone morphogenic protein 4 (BMP4), which in turn may activate intercellular adhesion molecule-1 (ICAM-1) expression and monocyte adhesion. OS is also known to induce monocyte adhesion by producing reactive oxygen species (ROS) from reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, raising the possibility that BMP4 may stimulate the inflammatory response by ROS-dependent mechanisms. Here we show that ROS scavengers blocked ICAM-1 expression and monocyte adhesion induced by BMP4 or OS in endothelial cells (ECs). Similar to OS, BMP4 stimulated H2O2 and O2- production in ECs. Next, we used ECs obtained from p47phox-/- mice (MAE-p47-/-), which do not produce ROS in response to OS, to determine the role of NADPH oxidases. Similar to OS, BMP4 failed to induce monocyte adhesion in MAE-p47-/-, but it was restored when the cells were transfected with p47phox plasmid. Moreover, OS-induced O2- production was blocked by noggin (a BMP antagonist), suggesting a role for BMP. Furthermore, OS increased gp91phox (nox2) and nox1 mRNA levels while decreasing nox4. In contrast, BMP4 induced nox1 mRNA expression, whereas nox2 and nox4 were decreased or not affected, respectively. Also, OS-induced monocyte adhesion was blocked by knocking down nox1 with the small interfering RNA (siRNA). Finally, BMP4 siRNA inhibited OS-induced ROS production and monocyte adhesion. Together, these results suggest that BMP4 produced in ECs by OS stimulates ROS release from the nox1-dependent NADPH oxidase leading to inflammation, a critical early atherogenic step. PMID:15388638

  18. Mouse epiblasts change responsiveness to BMP4 signal required for PGC formation through functions of extraembryonic ectoderm.

    PubMed

    Okamura, Daiji; Hayashi, Katsuhiko; Matsui, Yasuhisa

    2005-01-01

    Mouse primordial germ cells (PGCs) are initially identified as a cluster of alkaline phosphatase (AP)-positive cells within the extraembryonic mesoderm near the posterior part of the primitive streak at embryonic day (E) 7.25. Clonal analysis of epiblast cells has revealed that the putative precursors of PGCs are localized in the proximal epiblast, and we demonstrated that the conditions required for PGC formation are induced in the proximal region of epiblasts by extraembryonic ectoderm. Bone morphogenetic protein (BMP) 4 and BMP8b, which belong to the transforming growth factor-beta (TGF-beta) superfamily, might generate induction signals from extraembryonic ectoderm. Smad1 and Smad5, which are intracellular signaling molecules for BMP4, might also play a critical role in stimulating epiblasts to form PGC. However, how pluripotential epiblasts temporally and spatially respond to BMP signals to form PGCs remains unclear. The present study examines changes of responsiveness to BMP4 for PGC formation in epiblasts and their molecular mechanisms. We initially examined the effect of recombinant human (rh) BMP4 upon cultured epiblasts at different developmental stages, and found that they acquire the ability to respond to BMP4 signals for PGC formation between E5.25 and E5.5. In addition, such competence was conferred upon epiblasts by the extraembryonic ectoderm. We also showed that the increased expression of Smad1 and the onset of Smad5 expression induced by extraembryonic ectoderm might be responsible for quick acquisition of this competence. Furthermore, we show that only proximal epiblast cells maintain responsiveness to BMP4 for PGC formation at E6.0, and that this is associated with the proximal epiblast-specific expression of Smad5. These results explain why only the proximal region of epiblasts can sustain the ability to form PGCs. PMID:15515057

  19. Wnt/β-catenin signaling modulates corneal epithelium stratification via inhibition of Bmp4 during mouse development.

    PubMed

    Zhang, Yujin; Yeh, Lung-Kun; Zhang, Suohui; Call, Mindy; Yuan, Yong; Yasunaga, Mayu; Kao, Winston W-Y; Liu, Chia-Yang

    2015-10-01

    The development of organs with an epithelial parenchyma relies on reciprocal mesenchymal-epithelial communication. Mouse corneal epithelium stratification is the consequence of a coordinated developmental process based on mesenchymal-epithelial interactions. The molecular mechanism underlying these interactions remains unclear. The Wnt/β-catenin signaling pathway is involved in fundamental aspects of development through the regulation of various growth factors. Here, we show that conditional ablation of either β-catenin (Ctnnb1(cKO)) or co-receptors Lrp5/6 (Lrp5/6(cKO)) in corneal stromal cells results in precocious stratification of the corneal epithelium. By contrast, ectopic expression of a murine Ctnnb1 gain-of-function mutant (Ctnnb1(cGOF)) retards corneal epithelium stratification. We also discovered that Bmp4 is upregulated in the absence of β-catenin in keratocytes, which further triggers ERK1/2 (Mapk3/1) and Smad1/5 phosphorylation and enhances transcription factor p63 (Trp63) expression in mouse corneal basal epithelial cells and in a human corneal epithelial cell line (HTCE). Interestingly, mouse neonates given a subconjunctival BMP4 injection displayed a phenotype resembling that of Ctnnb1(cKO). Conditional ablation of Bmp4 eradicates the phenotype produced in Ctnnb1(cKO) mice. Furthermore, ChIP and promoter-luciferase assays show that β-catenin binds to and suppresses Bmp4 promoter activity. These data support the concept that cross-talk between the Wnt/β-catenin/Bmp4 axis (in the stromal mesenchyme) and Bmp4/p63 signaling (in the epithelium) plays a pivotal role in epithelial stratification during corneal morphogenesis. PMID:26443636

  20. miR-762 promotes porcine immature Sertoli cell growth via the ring finger protein 4 (RNF4) gene

    PubMed Central

    Ma, Changping; Song, Huibin; Yu, Lei; Guan, Kaifeng; Hu, Pandi; Li, Yang; Xia, Xuanyan; Li, Jialian; Jiang, Siwen; Li, Fenge

    2016-01-01

    A growing number of reports have revealed that microRNAs (miRNAs) play critical roles in spermatogenesis. Our previous study showed that miR-762 is differentially expressed in immature and mature testes of Large White boars. Our present data shows that miR-762 directly binds the 3′ untranslated region (3′UTR) of ring finger protein 4 (RNF4) and down-regulates RNF4 expression. A single nucleotide polymorphism (SNP) in the RNF4 3′UTR that is significantly associated with porcine sperm quality traits leads to a change in the miR-762 binding ability. Moreover, miR-762 promotes the proliferation of and inhibits apoptosis in porcine immature Sertoli cells, partly by accelerating DNA damage repair and by reducing androgen receptor (AR) expression. Taken together, these findings suggest that miR-762 may play a role in pig spermatogenesis by regulating immature Sertoli cell growth. PMID:27596571

  1. miR-762 promotes porcine immature Sertoli cell growth via the ring finger protein 4 (RNF4) gene.

    PubMed

    Ma, Changping; Song, Huibin; Yu, Lei; Guan, Kaifeng; Hu, Pandi; Li, Yang; Xia, Xuanyan; Li, Jialian; Jiang, Siwen; Li, Fenge

    2016-01-01

    A growing number of reports have revealed that microRNAs (miRNAs) play critical roles in spermatogenesis. Our previous study showed that miR-762 is differentially expressed in immature and mature testes of Large White boars. Our present data shows that miR-762 directly binds the 3' untranslated region (3'UTR) of ring finger protein 4 (RNF4) and down-regulates RNF4 expression. A single nucleotide polymorphism (SNP) in the RNF4 3'UTR that is significantly associated with porcine sperm quality traits leads to a change in the miR-762 binding ability. Moreover, miR-762 promotes the proliferation of and inhibits apoptosis in porcine immature Sertoli cells, partly by accelerating DNA damage repair and by reducing androgen receptor (AR) expression. Taken together, these findings suggest that miR-762 may play a role in pig spermatogenesis by regulating immature Sertoli cell growth. PMID:27596571

  2. Differential domain evolution and complex RNA processing in a family of paralogous EPB41 (protein 4.1) genes facilitates expression of diverse tissue-specific isoforms

    SciTech Connect

    Parra, Marilyn; Gee, Sherry; Chan, Nadine; Ryaboy, Dmitriy; Dubchak, Inna; Narla, Mohandas; Gascard, Philippe D.; Conboy, John G.

    2004-07-15

    The EPB41 (protein 4.1) genes epitomize the resourcefulness of the mammalian genome to encode a complex proteome from a small number of genes. By utilizing alternative transcriptional promoters and tissue-specific alternative pre-mRNA splicing, EPB41, EPB41L2, EPB41L3, and EPB41L1 encode a diverse array of structural adapter proteins. Comparative genomic and transcript analysis of these 140kb-240kb genes indicates several unusual features: differential evolution of highly conserved exons encoding known functional domains, interspersed with unique exons whose size and sequence variations contribute substantially to intergenic diversity: alternative first exons, most of which map far upstream of the coding regions; and complex tissue-specific alternative pre-mRNA splicing that facilitates synthesis of functionally different complements of 4.1 proteins in various cells. Understanding the splicing regulatory networks that control protein 4.1 expression will be critical to a full appreciation of the many roles of 4.1 proteins in normal cell biology and their proposed roles in human cancer.

  3. Differential domain evolution and complex RNA processing in a family of paralogous EPB41 (protein 4.1) genes facilitate expression of diverse tissue-specific isoforms.

    PubMed

    Parra, Marilyn; Gee, Sherry; Chan, Nadine; Ryaboy, Dmitriy; Dubchak, Inna; Mohandas, Narla; Gascard, Philippe D; Conboy, John G

    2004-10-01

    The EPB41 (protein 4.1) genes epitomize the resourcefulness of the mammalian genome to encode a complex proteome from a small number of genes. By utilizing alternative transcriptional promoters and tissue-specific alternative pre-mRNA splicing, EPB41, EPB41L2, EPB41L3, and EPB41L1 encode a diverse array of structural adapter proteins. Comparative genomic and transcript analysis of these 140- to 240-kb genes indicates several unusual features: differential evolution of highly conserved exons encoding known functional domains interspersed with unique exons whose size and sequence variations contribute substantially to intergenic diversity; alternative first exons, most of which map far upstream of the coding regions; and complex tissue-specific alternative pre-mRNA splicing that facilitates synthesis of functionally different complements of 4.1 proteins in various cells. Understanding the splicing regulatory networks that control protein 4.1 expression will be critical to a full appreciation of the many roles of 4.1 proteins in normal cell biology and their proposed roles in human cancer. PMID:15475241

  4. Analysis of the gene expression profile in response to human epididymis protein 4 in epithelial ovarian cancer cells.

    PubMed

    Zhu, Liancheng; Guo, Qian; Jin, Shan; Feng, Huilin; Zhuang, Huiyu; Liu, Cong; Tan, Mingzi; Liu, Juanjuan; Li, Xiao; Lin, Bei

    2016-09-01

    Currently, there are emerging multiple studies on human epididymis protein 4 (HE4) in ovarian cancer. HE4 possesses higher sensitivity and specificity than CA125 in the confirmative early diagnosis for ovarian cancer. Although much attention has been given to explore its clinical application, research of the basic mechanisms of HE4 in ovarian cancer are still unclear. In the present study, we provide fundamental data to identify full-scale differentially expressed genes (DEGs) in response to HE4 by use of human whole-genome microarrays in human epithelial ovarian cancer cell line ES-2 following overexpression and silencing of HE4. We found that a total of 717 genes were upregulated and 898 genes were downregulated in the HE4-overexpressing cells vs. the HE4-Mock cells, and 166 genes were upregulated and 285 were downregulated in the HE4-silenced cells vs. the HE4-Mock cells. An overlap of 16 genes consistently upregulated and 8 genes downregulated in response to HE4 were noted. These DEGs were involved in MAPK, steroid biosynthesis, cell cycle, the p53 hypoxia pathway, and focal adhesion pathways. Interaction network analysis predicted that the genes participated in the regulatory connection. Highly differential expression of the FOXA2, SERPIND1, BDKRD1 and IL1A genes was verified by quantitative real-time PCR in 4 cell line samples. Finally, SERPIND1 (HCII) was validated at the protein level by immunohistochemistry in 107 paraffin-embedded ovarian tissues. We found that SERPIND1 may act as a potential oncogene in the development of ovarian cancer. The present study displayed the most fundamental and full-scale data to show DEGs in response to HE4. These identified genes may provide a theoretical basis for investigations of the underlying molecular mechanism of HE4 in ovarian cancer. PMID:27430660

  5. Thymidine kinase-mediated shut down of bone morphogenetic protein-4 expression allows regulated bone production.

    PubMed

    Lombardo, Barbara; Rocco, Teresa; Esposito, Maria T; Cantilena, Bruno; Gargiulo, Sara; Greco, Adelaide; Montanaro, Donatella; Brunetti, Arturo; Pastore, Lucio

    2013-06-01

    Bone morphogenetic Proteins (BMPs) are growth factors also involved in ossification and chondrogenesis that have generated interest for their efficiency in inducing bone neo-synthesis. BMPs expression in engineered cells has been successful in stimulating osteoblastic differentiation and ectopic and orthotopic bone formation in vivo. We have previously shown that an adenoviral vector expressing bone morphogenetic protein type-4 (BMP-4) is able to efficiently drive bone formation in a rabbit model of discontinuous bone lesions. However, unregulated secretion of BMPs has also been implicated in bone overproduction and exostosis. We have constructed a replication-defective first generation adenoviral (FG-Ad) vector containing a cassette for the expression of BMP-4 associated with the Herpes Simplex virus thymidine kinase (TK) gene (FG-B4TK) in order to shut down BMP-4 expression and, therefore, regulate bone production. TK expression does not interfere with BMP-4 ability to induce ectopic bone formation in athymic nude mice. Administration of ganciclovir blocks ectopic bone production in quadriceps muscle transduced with the FG-B4TK with no effect on the contralateral muscle transduced with a vector expressing only BMP-4. Histological findings confirmed the pro-apoptotic activity of TK and the reduction of mineralized areas in the quadriceps transduced with FG-B4TK in mice treated with ganciclovir. We have generated a system to block BMP-4 secretion by inducing apoptosis in transduced cells therefore blocking unwanted bone formation. This system is an additional tool to generate regulated amount of bone in discontinuous bone lesions and can be easily coupled with biomaterials capable of recruiting cells and generating a local bioreactor. PMID:23317056

  6. Gata4 expression in lateral mesoderm is downstream of BMP4 and isactivated directly by Forkhead and GATA transcription factors through adistal enhancer element

    SciTech Connect

    Rojas, Anabel; De Val, Sarah; Heidt, Analeah B.; Xu, Shan-Mei; Bristow, James; Black, Brian L.

    2005-05-20

    The GATA family of zinc-finger transcription factors plays key roles in the specification and differentiation of multiple cell types during development. GATA4 is an early regulator of gene expression during the development of endoderm and mesoderm, and genetic studies in mice have demonstrated that GATA4 is required for embryonic development.Despite the importance of GATA4 in tissue specification and differentiation, the mechanisms by which Gata4 expression is activated and the transcription factor pathways upstream of GATA4 remain largely undefined. To identify transcriptional regulators of Gata4 in the mouse,we screened conserved noncoding sequences from the mouse Gata4 gene for enhancer activity in transgenic embryos. Here, we define the regulation of a distal enhancer element from Gata4 that is sufficient to direct expression throughout the lateral mesoderm, beginning at 7.5 days of mouse embryonic development. The activity of this enhancer is initially broad but eventually becomes restricted to the mesenchyme surrounding the liver. We demonstrate that the function of this enhancer in transgenic embryos is dependent upon highly conserved Forkhead and GATA transcription factor binding sites, which are bound by FOXF1 and GATA4,respectively. Furthermore, the activity of the Gata4 lateral mesoderm enhancer is attenuated by the BMP antagonist Noggin, and the enhancer is not activated in Bmp4-null embryos. Thus, these studies establish that Gata4 is a direct transcriptional target of Forkhead and GATA transcription factors in the lateral mesoderm, and demonstrate that Gata4lateral mesoderm enhancer activation requires BMP4, supporting a model in which GATA4 serves as a downstream effector of BMP signaling in the lateral mesoderm.

  7. The bovine fatty acid binding protein 4 gene is significantly associated with marbling and subcutaneous fat depth in Wagyu x Limousin F2 crosses.

    PubMed

    Michal, J J; Zhang, Z W; Gaskins, C T; Jiang, Z

    2006-08-01

    Fatty acid binding protein 4 (FABP4), which is expressed in adipose tissue, interacts with peroxisome proliferator-activated receptors and binds to hormone-sensitive lipase and therefore, plays an important role in lipid metabolism and homeostasis in adipocytes. The objective of this study was to investigate associations of the bovine FABP4 gene with fat deposition. Both cDNA and genomic DNA sequences of the bovine gene were retrieved from the public databases and aligned to determine its genomic organization. Primers targeting two regions of the FABP4 gene were designed: from nucleotides 5433-6106 and from nucleotides 7417-7868 (AAFC01136716). Direct sequencing of polymerase chain reaction (PCR) products on two DNA pools from high- and low-marbling animals revealed two single nucleotide polymorphisms (SNPs): AAFC01136716.1:g.7516G>C and g.7713G>C. The former SNP, detected by PCR-restriction fragment length polymorphism using restriction enzyme MspA1I, was genotyped on 246 F2 animals in a Waygu x Limousin F2 reference population. Statistical analysis showed that the FABP4 genotype significantly affected marbling score (P = 0.0398) and subcutaneous fat depth (P = 0.0246). The FABP4 gene falls into a suggestive/significant quantitative trait loci interval for beef marbling that was previously reported on bovine chromosome 14 in three other populations. PMID:16879357

  8. Cloning and characterization of 4.1G (EPB41L2), a new member of the skeletal protein 4.1 (EPB41) gene family.

    PubMed

    Parra, M; Gascard, P; Walensky, L D; Snyder, S H; Mohandas, N; Conboy, J G

    1998-04-15

    The prototypical erythrocyte membrane skeletal protein 4.1 (HGMW-approved symbol EPB41), here designated 4.1R, is encoded by a large, complexly spliced gene located on human chromosome 1p32-p33. In this paper we report evidence for a second 4.1 gene, 4.1G (HGMW-approved symbol EPB41L2), which maps to human chromosome 6q23 and is widely expressed among human tissues. The complete nucleotide sequence of 4.1G cDNA predicts a 113-kDa protein that exhibits three regions of high homology to 4.1R, including the membrane binding domain, the spectrinactin binding domain, and the C-terminal domain. Interspersed among the shared domains are unique sequences that may define functional differences between 4.1R and 4.1G. Specific isoforms of 4.1R and 4.1G exhibit differential subcellular localizations. These results expand the 4.1 gene superfamily and demonstrate that the diverse cellular complement of 4.1 isoforms results from both alternative splicing and expression of distinct genes. PMID:9598318

  9. CDC42-Interacting Protein 4 Gene Is Down Trans-Regulated by HBV DNA polymerase Trans Activated Protein 1

    PubMed Central

    LUN, Yongzhi; XU, Chongbo; CHI, Qing; WANG, Xuelei; SUI, Wen; JIANG, Sujuan

    2014-01-01

    Abstract Background Hepatitis B Virus (HBV) DNA polymerase transactivated protein 1 (HBVDNAPTP1) is a novel protein transactivated by HBV DNA polymerase, screened by suppression subtractive hybridization technique (GenBank accession no: AY450389). The biological function of HBVDNAPTP1 was investigated in this study. Methods We constructed a vector pcDNA3.1 (-)/myc-His A-HBVDNAPTP1 and used it to transfect acute monocytic leukemia cell line THP-1. HBVDNAPTP1 expression was detected by western blot analysis in the cells. A cDNA library of genes transactivated by HBVDNAPTP1 in THP-1 cells was made in pGEM-T Easy using suppression subtractive hybridization (SSH). The cDNAs were sequenced and analyzed with BLAST search against the sequences in GenBank. Results Some sequences, such as CIP4, might be involved in apoptosis development. mRNA and protein expression of CIP4 was identified by Real time RT-PCR and western blot in THP-1 cells. HBVDNAPTP1 could down-regulate the expression of CIP4 at both transcription and translation levels. Conclusion HBVDNAPTP1 may be involved in the positive regulation on the initiation of monocyte apoptosis. The result contribute to reveal the HBVDNAPTP1 biological functions and provide new evidences for further exploration of the regulatory mechanism of HBVDNAPTP1. PMID:25988087

  10. BMP4 is increased in the aortas of diabetic ApoE knockout mice and enhances uptake of oxidized low density lipoprotein into peritoneal macrophages

    PubMed Central

    2013-01-01

    Background BMP4, a member of the transforming growth factor-beta superfamily, is upregulated in the aortas of diabetic db/db mice. However, little is known about its role in diabetic atherosclerosis. Therefore, we examined the roles of BMP4 in the formation of diabetic atherosclerosis in apolipoprotein E knockout (ApoE KO) mice and in the uptake of oxidized low density lipoprotein (oxLDL) in peritoneal macrophages of wild-type mice. Methods To induce diabetes, ApoE KO mice were intraperitoneally injected with streptozotocin. Diabetic and non-diabetic ApoE KO mice were then fed a high-fat diet for 4 weeks. Next, to investigate a role of BMP4 in the peritoneal macrophages, we examined the uptake of oxLDL in BMP4-treated macrophages. Results Diabetic ApoE KO mice showed accelerated progression of aortic plaques accompanied by increased luminal plaque area. Western blot analysis showed that BMP4 expression in the whole aorta was greatly increased in diabetic ApoE KO mice, than non-diabetic mice. Western blot analysis showed that the BMP4/SMAD1/5/8 signaling pathway was strongly activated in the aorta from diabetic ApoE KO mice, compared with control ApoE KO mice. Double immunofluorescence staining showed that BMP4 was expressed in MOMA2-labeled macrophage in the aortic lesions of ApoE KO mice. BMP4 significantly increased the uptake of oxLDL into peritoneal macrophages in vitro. Conclusion We show that in the aorta of diabetic ApoE KO mice, BMP4 is increased and activates SMAD1/5/8. Our in vitro findings indicate that BMP4 enhances oxLDL uptake in mouse peritoneal macrophages, suggesting BMP4 may be involved in aortic plaque formation in diabetic ApoE KO mice. Targeting BMP4 may offer a new strategy for inhibition of plaque progression and stabilization of atherosclerotic lesions. PMID:24107300

  11. Cloning and expression of retinoblastoma-binding protein 4 gene in embryo diapause termination and in response to salinity stress from brine shrimp Artemia sinica.

    PubMed

    Wang, Xiaolu; Yao, Feng; Liang, Xiaoyu; Zhu, Xiaolin; Zheng, Ren; Jia, Baolin; Hou, Lin; Zou, Xiangyang

    2016-10-15

    Retinoblastoma binding protein 4 (RBBP4) is a nuclear protein with four WD-repeat sequences and thus belongs to a highly conserved subfamily of proteins with such domains. This retinoblastoma-binding protein plays an important role in nucleosome assembly and histone modification, which influences gene transcription and regulates cell cycle and proliferation. Artemia sinica (brine shrimp) undergoes an unusual diapause process under stress conditions of high salinity and low temperature. However, the role of RBBP4 in diapause termination of embryo development in A. sinica remains unknown. Here, the full-length cDNA of the As-rbbp4 gene was obtained from A. sinica and found to contain 1411 nucleotides, including a 1281 bp open reading frame (ORF), 63 bp 5'-untranslated region (UTR) and a 67-bp 3'-UTR, which encodes a 427 amino acid (48 kDa) protein. Bioinformatic analysis indicated As-RBBP4 to be mainly located in the nucleus, with a theoretical isoelectric point of 4.79. Protein sequence domain analysis showed that As-RBBP4 is a conserved protein, especially in the WD40 domain. No specificity in expression of this gene was observed in tissues or organs by in situ hybridization. Real-time quantitative PCR and Western blot analyses of As-RBBP4 gene and protein expression, respectively, showed notably high levels at 10 h and a subsequent downward trend. Obvious trends in upregulation of As-RBBP4 were observed under conditions of low temperature and high salinity stress. As-E2F1 and As-CyclinE also presented similar trends as that of As-RBBP4 in Western blots. Analysis of the RBBP4 expression in early embryonic development of A. sinica indicated that this protein plays an important role in diapause termination and cell cycle regulation. PMID:27267406

  12. Male germ-like cell differentiation potential of human umbilical cord Wharton’s jelly-derived mesenchymal stem cells in co-culture with human placenta cells in presence of BMP4 and retinoic acid

    PubMed Central

    nejad, Nahid Ataie; Amidi, Fardin; Hoseini, Marziyeh Agha; Nia, Karim Nayer; Habibi, Mehryar; Kajbafzadeh, Abdol Mohammad; Mazaheri, Zohreh; Yamini, Nazila

    2015-01-01

    Objective(s): Mesenchymal stem cells (MSCs) derived from Wharton’s jelly (WJ-MSCs) are now much more appealing for cell-based infertility therapy. Hence, WJ-MSCs differentiation toward germ layer cells for cell therapy purposes is currently under intensive study. Materials and Methods: MSCs were isolated from human Wharton’s jelly and treated with BMP4, retinoic acid (RA) or co-cultured on human amniotic epithelial (HAE) and chorionic plate (HCP) placenta feeder cells. profile of POU5F1, Fragilis, Plzf, DDX4, Piwil2, Stra8, Dazl, β1- and α6-integrins (ITΒ1, ITA6) genes expression as germ cell markers were analyzed using RT-PCR and real-time PCR. Immunocytochemistry of surface markers were conducted. Results: After 3 weeks treatment with different reagents and co-culture system, morphology of WJ-MSCs changed to shiny clusters and germ cell specific markers in mRNA were up-regulated in both placental feeder + RA and BMP4 + RA. Induction of hWJ-MSCs with BMP4 in presence of RA resulted in significant up-regulation (P≤0.05) of all germ cell specific genes (c-Kit; 2.84±0.59, DDX4; 1.69±0.39, Piwil2; 1.14±0.21, Dazl; 0.65±0.25, α6 integrin; 1.26±0.53, β1 integrins; 1.18±0.65) compared to control and placental feeder cells + RA. Our results indicated that HAE and HCP followed by RA treatment were involved in human germ cell development. Conclusion: We demonstrated that under the right conditions, hWJ-MSCs have the ability to differentiate to germ cells and this provides an excellent pattern to study infertility cause and treatment. PMID:26019794

  13. BMP4 Increases Canonical Transient Receptor Potential Protein Expression by Activating p38 MAPK and ERK1/2 Signaling Pathways in Pulmonary Arterial Smooth Muscle Cells

    PubMed Central

    Li, Xiaoyan; Lu, Wenju; Fu, Xin; Zhang, Yi; Yang, Kai; Zhong, Nanshan; Ran, Pixin

    2013-01-01

    Abnormal bone morphogenetic protein (BMP) signaling has been implicated in the pathogenesis of pulmonary hypertension. We previously found that BMP4 elevated basal intracellular Ca2+ ([Ca2+]i) concentrations in distal pulmonary arterial smooth muscle cells (PASMCs), attributable in large part to enhanced store-operated Ca2+ entry through store-operated Ca2+ channels (SOCCs). Moreover, BMP4 up-regulated the expression of canonical transient receptor potential (TRPC) proteins thought to compose SOCCs. The present study investigated the signaling pathways through which BMP4 regulates TRPC expression and basal [Ca2+]i in distal PASMCs. Real-time quantitative PCR was used for the measurement of mRNA, Western blotting was used for the measurement of protein, and fluorescent microscopic for [Ca2+]i was used to determine the involvement of p38 and extracellular regulated kinase (ERK)–1/2 mitogen-activated protein kinase (MAPK) signaling in BMP4–induced TRPC expression and the elevation of [Ca2+]i in PASMCs. We found that the treatment of BMP4 led to the activation of both p38 MAPK and ERK1/2 in rat distal PASMCs. The induction of TRPC1, TRPC4, and TRPC6 expression, and the increases of [Ca2+]i caused by BMP4 in distal PASMCs, were inhibited by treatment with either SB203580 (10 μM), the selective inhibitor for p38 activation, or the specific p38 small interfering RNA (siRNA). Similarly, those responses induced by BMP4 were also abolished by treatment with PD98059 (5 μM), the selective inhibitor of ERK1/2, or by the knockdown of ERK1/2 using its specific siRNA. These results indicate that BMP4 participates in the regulation of Ca2+ signaling in PASMCs by modulating TRPC channel expression via activating p38 and ERK1/2 MAPK pathways. PMID:23526217

  14. Expression of human membrane skeleton protein genes for protein 4.1 and betaIISigma2-spectrin assayed by real-time RT-PCR.

    PubMed

    Taylor-Harris, Pamela M; Felkin, Leanne E; Birks, Emma J; Franklin, Rodney C G; Yacoub, Magdi H; Baines, Anthony J; Barton, Paul J R; Pinder, Jennifer C

    2005-01-01

    The proteins, spectrin and 4.1 confer support and resilience to animal cell membranes, and promote assembly of multimeric, membrane-bound signalling complexes. Protein 4.1 also plays important roles in tumour suppression and the regulation of cell proliferation. To assess relative tissue expression of the four genes encoding human protein 4.1, we measured mRNA levels using quantitative real-time polymerase chain reaction. We compared 4.1 expression with that of a major splice variant of spectrin, betaIISigma2 that has a shortened C-terminus lacking a pleckstrin homology domain. mRNA for 4.1R is four-fold higher in bone marrow than in tissues with the next highest prevalence: cerebellum, lung, testis and thymus. 4.1G mRNA is highly expressed in brain, spinal cord and testis; 4.1N in brain, spinal cord and adrenal gland; 4.1B in testis, brain, spinal cord, and kidney. Thus, 4.1N, 4.1B and 4.1G all show high accumulation in nervous tissues. mRNA for betaIISigma2-spectrin is ubiquitous, but most abundant in cardiac and nervous tissues. Comparative transcript abundance was analysed in heart and brain. betaIISigma2-spectrin was the most abundant transcript in heart with levels 5 fold greater than 4.1G or 4.1N and at least 9 fold greater than 4.1B. In brain, 4.1N was the most abundant transcript, with levels 2.4 fold greater than 4.1B and at least 4 fold greater than 4.1G or betaIISigma2-spectrin. 4.1R abundance was very low in both tissues. Whilst we expected that 4.1 mRNAs would feature highly in muscle and nerve, we note their high abundance in testis, indicating previously unsuspected functions in reproduction. PMID:15809685

  15. Rohon-Beard sensory neurons are induced by BMP4 expressing non-neural ectoderm in Xenopus laevis.

    PubMed Central

    Rossi, Christy Cortez; Hernandez-Lagunas, Laura; Zhang, Chi; Choi, Irene F.; Kwok, Letitia; Klymkowsky, Michael; Artinger, Kristin Bruk

    2008-01-01

    Rohon-Beard mechanosensory neurons (RBs), neural crest cells, and neurogenic placodes arise at the border of the neural- and non-neural ectoderm during anamniote vertebrate development. Neural crest cells require BMP expressing non-neural ectoderm for their induction. To determine if epidermal ectoderm-derived BMP signaling is also involved in the induction of RB sensory neurons, the medial region of the neural plate from donor Xenopus laevis embryos was transplanted into the non-neural ventral ectoderm of host embryos at the same developmental stage. The neural plate border and RBs were induced at the transplant sites, as shown by expression of Xblimp1, and XHox11L2 and XN-tubulin, respectively. Transplantation studies between pigmented donors and albino hosts showed that neurons are induced both in donor neural and host epidermal tissue. Because an intermediate level of BMP4 signaling is required to induce neural plate border fates, we directly tested BMP4's ability to induce RBs; beads soaked in either 1 or 10 ng/ml were able to induce RBs in cultured neural plate tissue. Conversely, RBs fail to form when neural plate tissue from embryos with decreased BMP activity, either from injection of noggin or a dominant negative BMP receptor, was transplanted into the non-neural ectoderm of un-manipulated hosts. We conclude that contact between neural and non-neural ectoderm is capable of inducing RBs, that BMP4 can induce RB markers, and that BMP activity is required for induction of ectopic RB sensory neurons. PMID:18191829

  16. Activated Thyroid Hormone Promotes Differentiation and Chemotherapeutic Sensitization of Colorectal Cancer Stem Cells by Regulating Wnt and BMP4 Signaling.

    PubMed

    Catalano, Veronica; Dentice, Monica; Ambrosio, Raffaele; Luongo, Cristina; Carollo, Rosachiara; Benfante, Antonina; Todaro, Matilde; Stassi, Giorgio; Salvatore, Domenico

    2016-03-01

    Thyroid hormone is a pleiotropic factor that controls many cellular processes in multiple cell types such as cancer stem cells (CSC). Thyroid hormone concentrations in the blood are stable, but the action of the deiodinases (D2-D3) provides cell-specific regulation of thyroid hormone activity. Deregulation of deiodinase function and thyroid hormone status has been implicated in tumorigenesis. Therefore, we investigated the role of thyroid hormone metabolism and signaling in colorectal CSCs (CR-CSC), where deiodinases control cell division and chemosensitivity. We found that increased intracellular thyroid hormone concentration through D3 depletion induced cell differentiation and sharply mitigated tumor formation. Upregulated BMP4 expression and concomitantly attenuated Wnt signaling accompanied these effects. Furthermore, we demonstrate that BMP4 is a direct thyroid hormone target and is involved in a positive autoregulatory feedback loop that modulates thyroid hormone signaling. Collectively, our findings highlight a cell-autonomous metabolic mechanism by which CR-CSCs exploit thyroid hormone signaling to facilitate their self-renewal potential and suggest that drug-induced cell differentiation may represent a promising therapy for preventing CSC expansion and tumor progression. PMID:26676745

  17. Specific Preferences in Lineage Choice and Phenotypic Plasticity of Glioma Stem Cells Under BMP4 and Noggin Influence.

    PubMed

    Videla Richardson, Guillermo Agustín; Garcia, Carolina Paola; Roisman, Alejandro; Slavutsky, Irma; Fernandez Espinosa, Damián Darío; Romorini, Leonardo; Miriuka, Santiago Gabriel; Arakaki, Naomi; Martinetto, Horacio; Scassa, María Elida; Sevlever, Gustavo Emilio

    2016-01-01

    Although BMP4-induced differentiation of glioma stem cells (GSCs) is well recognized, details of the cellular responses triggered by this morphogen are still poorly defined. In this study, we established several GSC-enriched cell lines (GSC-ECLs) from high-grade gliomas. The expansion of these cells as adherent monolayers, and not as floating neurospheres, enabled a thorough study of the phenotypic changes that occurred during their differentiation. Herein, we evaluated GSC-ECLs' behavior toward differentiating conditions by depriving them of growth factors and/or by adding BMP4 at different concentrations. After analyzing cellular morphology, proliferation and lineage marker expression, we determined that GSC-ECLs have distinct preferences in lineage choice, where some of them showed an astrocyte fate commitment and others a neuronal one. We found that this election seems to be dictated by the expression pattern of BMP signaling components present in each GSC-ECL. Additionally, treatment of GSC-ECLs with the BMP antagonist, Noggin, also led to evident phenotypic changes. Interestingly, under certain conditions, some GSC-ECLs adopted an unexpected smooth muscle-like phenotype. As a whole, our findings illustrate the wide differentiation potential of GSCs, highlighting their molecular complexity and paving a way to facilitate personalized differentiating therapies. PMID:25808628

  18. Ameloblasts serum-free conditioned medium: bone morphogenic protein 4-induced odontogenic differentiation of mouse induced pluripotent stem cells.

    PubMed

    Liu, Li; Liu, Ying-Feng; Zhang, Jing; Duan, Yin-Zhong; Jin, Yan

    2016-06-01

    Induced pluripotent stem (iPS) cells possess the ability of self-renewal and can differentiate into cells of the three germ layers, both in vitro and in vivo. Here we report a new method to efficiently induce differentiation of mouse iPS cells into the odontogenic lineage. Using ameloblasts serum-free conditioned medium (ASF-CM), we successfully generated ameloblast-like cells from mouse iPS cells. Importantly, culturing mouse iPS cells in ASF-CM supplemented with BMP4 (ASF-BMP4) promoted odontogenic differentiation, which was evident by the upregulation of ameloblast-specific as well as odontoblast-specific genes. On the other hand, culturing mouse iPS cells in ASF-CM supplemented with noggin (ASF-noggin), an inhibitor of BMP4, abrogated this effect. These results suggest that mouse iPS cells can be induced by ASF-BMP4 to differentiate into ameloblast-like and odontoblast-like cells. The results of our study raise the possibility of using patient-specific iPS cells for tooth regeneration in the future. Copyright © 2016 John Wiley & Sons, Ltd. PMID:23606575

  19. The human insulin-like growth factor-binding protein 4 gene maps to chromosome region 17q12-q21. 1 and is close to the gene for hereditary breast-ovarian cancer

    SciTech Connect

    Tonin, P.; Vivier, A.; Morgan, K.; Narod, S.; Pollack, M. ); Ehrenborg, E.; Zazzi, H.; Luthman, H.; Larsson, C. ); Lenoir, G. )

    1993-11-01

    The gene for insulin-like growth factor-binding protein 4 (IGFBP4) codes for a serum protein that binds to the family of insulin-like growth factors and modulates their activity. It has been mapped by in situ hybridization to chromosome region 17q12-q21.1. The authors have developed a CA-repeat polymorphism from a cosmid clone containing IGFBP4. By linkage analysis, IGFBP4 maps to the chromosome 17q interval THRA1-D17S579. This interval also contains the gene for hereditary breast-ovarian cancer, BRCA1. Genetic recombination between IGFBP4 and BRCA1 places IGFBP4 centromeric to the cancer susceptibility gene and effectively excludes it as a candidate gene for BRCA1. IGFBP4 is, however, one of the closest known centromeric markers for BRCA1; the estimated recombination fraction is 0.015. IGFBP4 and D17S579 together define a 2.8-cM interval that contains BRCA1. 18 refs., 3 figs., 1 tab.

  20. Delayed myelination in an intrauterine growth retardation model is mediated by oxidative stress upregulating bone morphogenetic protein 4.

    PubMed

    Reid, Mary V; Murray, Kaitlin A; Marsh, Eric D; Golden, Jeffrey A; Simmons, Rebecca A; Grinspan, Judith B

    2012-07-01

    Intrauterine growth retardation (IUGR) is associated with neurological deficits including cerebral palsy and cognitive and behavioral disabilities. The pathogenesis involves oxidative stress that leads to periventricular white matter injury with a paucity of mature oligodendrocytes and hypomyelination. The molecular mechanisms underlying this damage remain poorly understood. We used a rat model of IUGR created by bilateral ligation of the uterine artery at embryonic Day 19 that results in fetal growth retardation and oxidative stress in the developing brain. The IUGR rat pups showed significant delays in oligodendrocyte differentiation and myelination that resolved by 8 weeks. Bone morphogenetic protein 4 (BMP4), which inhibits oligodendrocyte maturation, was elevated in IUGR brains at postnatal time points and returned to near normal by adulthood. Despite the apparent recovery, behavioral deficiencies were found in 8-week-old female animals, suggesting that the early transient myelination defects have permanent effects. In support of these in vivo data, oligodendrocyte precursor cells cultured from postnatal IUGR rats retained increased BMP4 expression and impaired differentiation that was reversed with the BMP inhibitor noggin. Oxidants in oligodendrocyte cultures increased BMP expression, which decreased differentiation; however, abrogating BMP signaling with noggin in vitro and in BMP-deficient mice prevented these effects. Together, these findings suggest that IUGR results in delayed myelination through the generation of oxidative stress that leads to BMP4 upregulation. PMID:22710965

  1. Identification of a third protein 4.1 tumor suppressor, protein 4.1R, in meningioma pathogenesis

    SciTech Connect

    Robb, Victoria A.; Li, Wen; Gascard, Philippe; Perry, Arie; Mohandas, Narla; Gutmann, David H.

    2003-06-11

    Meningiomas are common tumors of the central nervous system, however, the mechanisms under lying their pathogenesis are largely undefined. Two members of the Protein 4.1 super family, the neuro fibromatosis 2 (NF2) gene product (merlin/schwannomin) and Protein 4.1B have been implicated as meningioma tumor suppressors. In this report, we demonstrate that another Protein 4.1 family member, Protein 4.1R, also functions as a meningioma tumor suppressor. Based on the assignment of the Protein 4.1R gene to chromosome 1p32-36, a common region of deletion observed in meningiomas, we analyzed Protein 4.1R expression in meningioma cell lines and surgical tumor specimens. We observed loss of Protein 4.1R protein expression in two meningioma cell lines (IOMM-Lee, CH157-MN) by Western blotting as well as in 6 of 15 sporadic meningioma as by immuno histo chemistry (IHC). Analysis of a subset of these sporadic meningiomas by fluorescent in situ hybridization (FISH) with a Protein 4.1R specific probe demonstrated 100 percent concordance with the IHC results. In support of a meningioma tumor suppressor function, over expression of Protein 4.1R resulted in suppression of IOMM-Lee and CH157MN cell proliferation. Similar to the Protein 4.1B and merlin meningioma tumor suppressors, Protein 4.1R localization in the membrane fraction increased significantly under conditions of growth arrest in vitro. Lastly, Protein 4.1R interacted with some known merlin/Protein 4.1B interactors such as CD44 and bII-spectrin, but did not associate with the Protein 4.1B interactors 14-3-3 and PRMT3 or the merlin binding proteins SCHIP-1 and HRS. Collectively, these results suggest that Protein 4.1R functions as an important tumor suppressor important in the molecular pathogenesis of meningioma.

  2. Low-intensity pulsed ultrasound produced an increase of osteogenic genes expression during the process of bone healing in rats.

    PubMed

    Fávaro-Pípi, Elaine; Bossini, Paulo; de Oliveira, Poliani; Ribeiro, Juliana Uema; Tim, Carla; Parizotto, Nivaldo A; Alves, Jose Marcos; Ribeiro, Daniel Araki; Selistre de Araújo, Heloísa Sobreiro; Renno, Ana Claudia Muniz

    2010-12-01

    The aim of this study was to measure the temporal expression of osteogenic genes during the process of bone healing in low-intensity pulsed ultrasound (LIPUS) treated bone defects by means of histopathologic and real-time polymerase chain reaction (PCR) analysis. Animals were randomly distributed into two groups (n = 30): control group (bone defect without treatment) and LIPUS treated (bone defect treated with LIPUS). On days 7, 13 and 25 postinjury, 10 rats per group were sacrificed. Rats were treated with a 30 mW/cm(2) LIPUS. The results pointed out intense new bone formation surrounded by highly vascularized connective tissue presenting a slight osteogenic activity, with primary bone deposition was observed in the group exposed to LIPUS in the intermediary (13 days) and late stages of repair (25 days) in the treated animals. In addition, quantitative real-time polymerase chain reaction (RT-qPCR) showed an upregulation of bone morphogenetic protein 4 (BMP4), osteocalcin and Runx2 genes 7 days after the surgery. In the intermediary period, there was no increase in the expression. The expression of alkaline phosphatase, BMP4 and Runx2 was significantly increased at the last period. Our results indicate that LIPUS therapy improves bone repair in rats and upregulated osteogenic genes, mainly at the late stages of recovery. PMID:20950932

  3. Association of Cytotoxic T-Lymphocyte-Associated Protein 4 (CTLA4) Gene Polymorphisms with Autoimmune Thyroid Disease in Children and Adults: Case-Control Study.

    PubMed

    Ting, Wei-Hsin; Chien, Ming-Nan; Lo, Fu-Sung; Wang, Chao-Hung; Huang, Chi-Yu; Lin, Chiung-Ling; Lin, Wen-Shan; Chang, Tzu-Yang; Yang, Horng-Woei; Chen, Wei-Fang; Lien, Ya-Ping; Cheng, Bi-Wen; Lin, Chao-Hsu; Chen, Chia-Ching; Wu, Yi-Lei; Hung, Chen-Mei; Li, Hsin-Jung; Chan, Chon-In; Lee, Yann-Jinn

    2016-01-01

    Autoimmune thyroid disease (AITD), including Graves disease (GD) and Hashimoto disease (HD), is an organ-specific autoimmune disease with a strong genetic component. Although the cytotoxic T-lymphocyte-associated protein 4 (CTLA4) polymorphism has been reported to be associated with AITD in adults, few studies have focused on children. The aim of our study was to investigate whether the CTLA4 polymorphisms, including -318C/T (rs5742909), +49A/G (rs231775), and CT60 (rs3087243), were associated with GD and HD in Han Chinese adults and children. We studied 289 adult GD, 265 pediatric GD, 229 pediatric HD patients, and 1058 healthy controls and then compared genotype, allele, carrier, and haplotype frequencies between patients and controls. We found that CTLA4 SNPs +49A/G and CT60 were associated with GD in adults and children. Allele G of +49A/G was significantly associated with GD in adults (odds ratio [OR], 1.50; 95% confidence interval [CI], 1.21-1.84; corrected P value [Pc] < 0.001) and children (OR, 1.42; 95% CI, 1.15-1.77; Pc = 0.002). Allele G of CT60 also significantly increased risk of GD in adults (OR, 1.63; 95% CI, 1.27-2.09; Pc < 0.001) and GD in children (OR, 1.58; 95% CI, 1.22-2.04; Pc < 0.001). Significant linkage disequilibrium was found between +49A/G and CT60 in GD and control subjects (D' = 0.92). Our results showed that CTLA4 was associated with both GD and HD and played an equivalent role in both adult and pediatric GD in Han Chinese population. PMID:27111218

  4. Association of Cytotoxic T-Lymphocyte-Associated Protein 4 (CTLA4) Gene Polymorphisms with Autoimmune Thyroid Disease in Children and Adults: Case-Control Study

    PubMed Central

    Lo, Fu-Sung; Wang, Chao-Hung; Huang, Chi-Yu; Lin, Chiung-Ling; Lin, Wen-Shan; Chang, Tzu-Yang; Yang, Horng-Woei; Chen, Wei-Fang; Lien, Ya-Ping; Cheng, Bi-Wen; Lin, Chao-Hsu; Chen, Chia-Ching; Wu, Yi-Lei; Hung, Chen-Mei; Li, Hsin-Jung; Chan, Chon-In; Lee, Yann-Jinn

    2016-01-01

    Autoimmune thyroid disease (AITD), including Graves disease (GD) and Hashimoto disease (HD), is an organ-specific autoimmune disease with a strong genetic component. Although the cytotoxic T-lymphocyte-associated protein 4 (CTLA4) polymorphism has been reported to be associated with AITD in adults, few studies have focused on children. The aim of our study was to investigate whether the CTLA4 polymorphisms, including -318C/T (rs5742909), +49A/G (rs231775), and CT60 (rs3087243), were associated with GD and HD in Han Chinese adults and children. We studied 289 adult GD, 265 pediatric GD, 229 pediatric HD patients, and 1058 healthy controls and then compared genotype, allele, carrier, and haplotype frequencies between patients and controls. We found that CTLA4 SNPs +49A/G and CT60 were associated with GD in adults and children. Allele G of +49A/G was significantly associated with GD in adults (odds ratio [OR], 1.50; 95% confidence interval [CI], 1.21–1.84; corrected P value [Pc] < 0.001) and children (OR, 1.42; 95% CI, 1.15–1.77; Pc = 0.002). Allele G of CT60 also significantly increased risk of GD in adults (OR, 1.63; 95% CI, 1.27–2.09; Pc < 0.001) and GD in children (OR, 1.58; 95% CI, 1.22–2.04; Pc < 0.001). Significant linkage disequilibrium was found between +49A/G and CT60 in GD and control subjects (D’ = 0.92). Our results showed that CTLA4 was associated with both GD and HD and played an equivalent role in both adult and pediatric GD in Han Chinese population. PMID:27111218

  5. BMP2 is superior to BMP4 for promoting human muscle-derived stem cell-mediated bone regeneration in a critical-sized calvarial defect model.

    PubMed

    Gao, Xueqin; Usas, Arvydas; Lu, Aiping; Tang, Ying; Wang, Bing; Chen, Chien-Wen; Li, Hongshuai; Tebbets, Jessica C; Cummins, James H; Huard, Johnny

    2013-01-01

    Muscle-derived cells have been successfully isolated using a variety of different methods and have been shown to possess multilineage differentiation capacities, including an ability to differentiate into articular cartilage and bone in vivo; however, the characterization of human muscle-derived stem cells (hMDSCs) and their bone regenerative capacities have not been fully investigated. Genetic modification of these cells may enhance their osteogenic capacity, which could potentially be applied to bone regenerative therapies. We found that hMDSCs, isolated by the preplate technique, consistently expressed the myogenic marker CD56, the pericyte/endothelial cell marker CD146, and the mesenchymal stem cell markers CD73, CD90, CD105, and CD44 but did not express the hematopoietic stem cell marker CD45, and they could undergo osteogenic, chondrogenic, adipogenic, and myogenic differentiation in vitro. In order to investigate the osteoinductive potential of hMDSCs, we constructed a retroviral vector expressing BMP4 and GFP and a lentiviral vector expressing BMP2. The BMP4-expressing hMDSCs were able to undergo osteogenic differentiation in vitro and exhibited enhanced mineralization compared to nontransduced cells; however, when transplanted into a calvarial defect, they failed to regenerate bone. Local administration of BMP4 protein and cell pretreatment with N-acetylcysteine (NAC), which improves cell survival, did not enhance the osteogenic capacity of the retro-BMP4-transduced cells. In contrast, lenti-BMP2-transduced hMDSCs not only exhibited enhanced in vitro osteogenic differentiation but also induced robust bone formation and nearly completely healed a critical-sized calvarial defect in CD-1 nude mice 6 weeks following transplantation. Herovici's staining of the regenerated bone demonstrated that the bone matrix contained a large amount of type I collagen. Our findings indicated that the hMDSCs are likely mesenchymal stem cells of muscle origin and that BMP2 is

  6. Neuron-specific specificity protein 4 (Sp4) bigenomically regulates the transcription of all mitochondria- and nucleus-encoded cytochrome c oxidase subunit genes in neurons

    PubMed Central

    Johar, Kaid; Priya, Anusha; Dhar, Shilpa; Liu, Qiuli; Wong-Riley, Margaret T. T.

    2013-01-01

    Neurons are highly dependent on oxidative metabolism for their energy supply, and cytochrome c oxidase (COX) is a key energy-generating enzyme in the mitochondria. A unique feature of COX is that it is one of only four proteins in mammalian cells that are bigenomically-regulated. Of its thirteen subunits, three are encoded in the mitochondrial genome and ten are nuclear-encoded on nine different chromosomes. The mechanism of regulating this multisubunit, bigenomic enzyme poses a distinct challenge. In recent years, we found that nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) mediate such bigenomic coordination. The latest candidate is the specificity factor (Sp) family of proteins. In N2a cells, we found that Sp1 regulates all 13 COX subunits. However, we discovered recently that in primary neurons, it is Sp4 and not Sp1, that regulates some of the key glutamatergic receptor subunit genes. The question naturally arises as to the role of Sp4 in regulating COX in primary neurons. The present study utilized multiple approaches, including chromatin immunoprecipitation, promoter mutational analysis, knockdown and over-expression of Sp4, as well as functional assays to document that Sp4 indeed functionally regulate all 13 subunits of COX as well as mitochondrial transcription factors A and B. PMID:24032355

  7. Allogeneic Platelet Releasate Preparations Derived via a Novel Rapid Thrombin Activation Process Promote Rapid Growth and Increased BMP-2 and BMP-4 Expression in Human Adipose-Derived Stem Cells

    PubMed Central

    McLaughlin, Michael; Gagnet, Paul; Cunningham, Elizabeth; Yeager, Randi; D'Amico, Michael; Guski, Katie; Scarpone, Michael; Kuebler, Daniel

    2016-01-01

    The administration of human adipose-derived stem cells (ASCs) represents a promising regenerative therapy for the treatment of orthopedic injuries. While ASCs can be easily isolated from liposuction-derived adipose tissue, most clinical applications will likely require in vitro culture expansion of these cells using nonxenogeneic components. In this study, platelet releasate was generated using a novel rapid thrombin activation method (tPR). ASCs grown in media supplemented with tPR proliferated much faster than ASCs grown in media supplemented with 10% fetal bovine serum. The cells also retained the ability to differentiate along chondrogenic, adipogenic, and osteogenic lineages. The tPR cultured ASCs displayed elevated expression of BMP-4 (5.7 ± 0.97-fold increase) and BMP-2 (4.7 ± 1.3-fold increase) and decreased expression of PDGF-B (4.0 ± 1.4-fold decrease) and FGF-2 (33 ± 9.0-fold decrease). No significant changes in expression were seen with TGF-β and VEGF. This pattern of gene expression was consistent across different allogeneic tPR samples and different ASC lines. The use of allogeneic rapidly activated tPR to culture ASCs is associated with both an increased cell yield and a defined gene expression profile making it an attractive option for cell expansion prior to cell-based therapy for orthopedic applications. PMID:26823671

  8. Protein 4.1 in forebrain postsynaptic density preparations: enrichment of 4.1 gene products and detection of 4.1R binding proteins.

    PubMed

    Scott, C; Keating, L; Bellamy, M; Baines, A J

    2001-02-01

    4.1 Proteins are a family of multifunctional cytoskeletal components (4.1R, 4.1G, 4.1N and 4.1B) derived from four related genes, each of which is expressed in the nervous system. Using subcellular fractionation, we have investigated the possibility that 4.1 proteins are components of forebrain postsynaptic densities, cellular compartments enriched in spectrin and actin, whose interaction is regulated by 4.1R. Antibodies to each of 4.1R, 4.1G, 4.1N and 4.1B recognize polypeptides in postsynaptic density preparations. Of these, an 80-kDa 4.1R polypeptide is enriched 11-fold in postsynaptic density preparations relative to brain homogenate. Polypeptides of 150 and 125 kDa represent 4.1B; of these, only the 125 kDa species is enriched (threefold). Antibodies to 4.1N recognize polypeptides of approximately 115, 100, 90 and 65 kDa, each enriched in postsynaptic density preparations relative to brain homogenate. Minor 225 and 200 kDa polypeptides are recognized selectively by specific anti-4.1G antibodies; the 200 kDa species is enriched 2.5-fold. These data indicate that specific isoforms of all four 4.1 proteins are components of postsynaptic densities. Blot overlay analyses indicate that, in addition to spectrin and actin, postsynaptic density polypeptides of 140, 115, 72 and 66 kDa are likely to be 4.1R-interactive. Of these, 72 kDa and 66 kDa polypeptides were identified as neurofilament L and alpha-internexin, respectively. A complex containing 80 kDa 4.1R, alpha-internexin and neurofilament L was immunoprecipitated with anti-4.1R antibodies from brain extract. We conclude that 4.1R interacts with the characteristic intermediate filament proteins of postsynaptic densities, and that the 4.1 proteins have the potential to mediate the interactions of diverse components of postsynaptic densities. PMID:11179975

  9. Simulated Microgravity Regulates Gene Transcript Profiles of 2T3 Preosteoblasts: Comparison of the Random Positioning Machine and the Rotating Wall Vessel Bioreactor

    NASA Technical Reports Server (NTRS)

    Patel, Mamta J.; Liu, Wenbin; Sykes, Michelle C.; Ward, Nancy E.; Risin, Semyon A.; Risin, Diana; Hanjoong, Jo

    2007-01-01

    Microgravity of spaceflight induces bone loss due in part to decreased bone formation by osteoblasts. We have previously examined the microgravity-induced changes in gene expression profiles in 2T3 preosteoblasts using the Random Positioning Machine (RPM) to simulate microgravity conditions. Here, we hypothesized that exposure of preosteoblasts to an independent microgravity simulator, the Rotating Wall Vessel (RWV), induces similar changes in differentiation and gene transcript profiles, resulting in a more confined list of gravi-sensitive genes that may play a role in bone formation. In comparison to static 1g controls, exposure of 2T3 cells to RWV for 3 days inhibited alkaline phosphatase activity, a marker of differentiation, and downregulated 61 genes and upregulated 45 genes by more than two-fold as shown by microarray analysis. The microarray results were confirmed with real time PCR for downregulated genes osteomodulin, bone morphogenic protein 4 (BMP4), runx2, and parathyroid hormone receptor 1. Western blot analysis validated the expression of three downregulated genes, BMP4, peroxiredoxin IV, and osteoglycin, and one upregulated gene peroxiredoxin I. Comparison of the microarrays from the RPM and the RWV studies identified 14 gravi-sensitive genes that changed in the same direction in both systems. Further comparison of our results to a published database showing gene transcript profiles of mechanically loaded mouse tibiae revealed 16 genes upregulated by the loading that were shown to be downregulated by RWV and RPM. These mechanosensitive genes identified by the comparative studies may provide novel insights into understanding the mechanisms regulating bone formation and potential targets of countermeasure against decreased bone formation both in astronauts and in general patients with musculoskeletal disorders.

  10. A single-nucleotide polymorphism in the 3′-UTR region of the adipocyte fatty acid binding protein 4 gene is associated with prognosis of triple-negative breast cancer

    PubMed Central

    Wang, Wenmiao; Yuan, Peng; Yu, Dianke; Du, Feng; Zhu, Anjie; Li, Qing; Zhang, Pin; Lin, Dongxin; Xu, Binghe

    2016-01-01

    Triple-negative breast cancer (TNBC) is a subtype of breast cancer with poor prognosis and high heterogeneity. The aim of this study was to screen patients for single-nucleotide polymorphisms (SNPs) associated with the prognosis of TNBC. Database-derived SNPs (NextBio, Ensembl, NCBI and MirSNP) located in the 3′-untranslated regions (3′-UTRs) of genes that are differentially expressed in breast cancer were selected. The possible associations between 111 SNPs and progression risk among 323 TNBC patients were investigated using a two-step case-control study with a discovery cohort (n=162) and a validation cohort (n=161). We identified the rs1054135 SNP in the adipocyte fatty acid binding protein 4 (FABP4) gene as a predictor of TNBC recurrence. The G allele of rs1054135 was associated with a reduced risk of disease progression as well as a prolonged disease-free survival time (DFS), with a hazard ratio (HR) for recurrence in the combined sample of 0.269 [95%CI: 0.098−0.735;P=0.001]. Notably, for individuals having the rs1054135 SNP with the AA/AG genotype, the magnitude of increased tumour recurrence risk for overweight patients (BMI≥25kg/m2) was significantly elevated (HR2.53; 95%CI: 1.06–6.03). Immunohistochemical staining of adipocytes adjacent to TNBC tissues showed that the expression level of FABP4 was statistically significantly lower in patients with the rs1054135-GG genotype and those in the disease-free group (P=0.0004 and P=0.0091, respectively). These results suggested that the expression of a lipid metabolism-related gene and an important SNP in the 3′-UTR of FABP4 are associated with TNBC prognosis, which may aid in the screening of high-risk patients with TNBC recurrence and the development of novel chemotherapeutic agents. PMID:26959740

  11. Differential proinflammatory and prooxidant effects of bone morphogenetic protein-4 in coronary and pulmonary arterial endothelial cells.

    PubMed

    Csiszar, Anna; Labinskyy, Nazar; Jo, Hanjoong; Ballabh, Praveen; Ungvari, Zoltan

    2008-08-01

    There is increasing evidence that TGF-beta family member cytokine bone morphogenetic protein (BMP)-4 plays different pathophysiological roles in the pulmonary and systemic circulation. Upregulation of BMP-4 has been linked to atherosclerosis and hypertension in the systemic circulation, whereas disruption of BMP-4 signaling is associated with the development of pulmonary hypertension. To test the hypothesis that BMP-4 elicits differential effects in the pulmonary and systemic circulation, we compared the prooxidant and proinflammatory effects of BMP-4 in cultured human coronary arterial endothelial cells (CAECs) and pulmonary arterial endothelial cells (PAECs). We found that BMP-4 (from 0.3 to 10 ng/ml) in CAECs increased O(2)(*-) and H(2)O(2) generation, induced NF-kappaB activation, upregulated ICAM-1, and induced monocyte adhesiveness to ECs. In contrast, BMP-4 failed to induce oxidative stress or endothelial activation in PAECs. Also, BMP-4 treatment impaired acetylcholine-induced relaxation and increased O(2)(*-) production in cultured rat carotid arteries, whereas cultured rat pulmonary arteries were protected from these adverse effects of BMP-4. Thus, we propose that BMP-4 exerts prooxidant, prohypertensive, and proinflammatory effects only in the systemic circulation, whereas pulmonary arteries are protected from these adverse effects of BMP-4. The vascular bed-specific endothelial effects of BMP-4 are likely to contribute to its differential pathophysiological role in the systemic and pulmonary circulation. PMID:18539760

  12. GC Gene Polymorphism and Unbound Serum Retinol-Binding Protein 4 Are Related to the Risk of Insulin Resistance in Patients With Chronic Hepatitis C: A Prospective Cross-Sectional Study.

    PubMed

    Mateos-Muñoz, Beatriz; García-Martín, Elena; Torrejón, María J; Devesa-Medina, María J; Esguevillas, Gara; Cárdenas, María C; Fernández, Cristina; Carballo, Miguel; Agúndez, José A; Ladero, José M

    2016-03-01

    Insulin resistance (IR) is found in chronic hepatitis C (CHC) more frequently than in other chronic liver diseases.Prospective cross-sectional study to evaluate a wide multitest panel to identify factors related with IR in CHC and their possible interactions.In 76 patients with CHC we performed a series of routine laboratory analysis as well as specifically designed serum biochemical tests [retinol, retinol-binding protein 4 (RBP4), 25-OH vitamin D, Vitamin E, lipopolysaccharide-binding protein (LBP), interleukin-6 (IL-6), and cystatin C]. The single nucleotide polymorphisms rs7041 and rs4588 GC-DBP (group-specific component-Vitamin D-binding protein), rs738409 PNPLA3 (patatin-like phospholipase domain containing 3), and rs12979860 IL28B (interleukin-28 B) genes were determined. Insulin sensitivity was established with the HOMA-IR and IR was diagnosed when HOMA-IR > 3. Fibrosis staging was assessed with liver biopsy or transient elastography.After backward logistic regression analysis, independent variables associated with IR were Gc1s/Gc1s DBP phenotype, that results from the homozygous carriage of the rs7041G/rs4588C haplotype (P = 0.033); low retinol/RBP4 ratio, reflecting a greater rate of unbound RBP4 (P = 0.005); older age (P = 0.01); high serum tryglicerides (P = 0.026); and advanced (F3-F4) fibrosis stage. The AUROC provided by the multivariate model was 0.950 (95% CI = 0.906-0.993).In addition to previously known ones, the Gc1s/Gc1s phenotype variant of DBP and the unbound fraction of plasma RBP4 may be considered as factors related with the incidence, and possibly the risk, of IR in CHC patients. PMID:26962819

  13. Bone Morphogenetic Protein 4 Signalling in Neural Stem and Progenitor Cells during Development and after Injury

    PubMed Central

    Cole, Alistair E.; Murray, Simon S.; Xiao, Junhua

    2016-01-01

    Substantial progress has been made in identifying the extracellular signalling pathways that regulate neural stem and precursor cell biology in the central nervous system (CNS). The bone morphogenetic proteins (BMPs), in particular BMP4, are key players regulating neuronal and glial cell development from neural precursor cells in the embryonic, postnatal, and injured CNS. Here we review recent studies on BMP4 signalling in the generation of neurons, astrocytes, and oligodendroglial cells in the CNS. We also discuss putative mechanisms that BMP4 may utilise to influence glial cell development following CNS injury and highlight some questions for further research. PMID:27293450

  14. Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1) expression and possible function in mouse tooth germ development.

    PubMed

    Hasegawa, Kana; Wada, Hiroko; Nagata, Kengo; Fujiwara, Hiroaki; Wada, Naohisa; Someya, Hirotaka; Mikami, Yurie; Sakai, Hidetaka; Kiyoshima, Tamotsu

    2016-08-01

    Abnormal expression of Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1) is involved in the pathogenesis of FSHD. FRG1 is also important for the normal muscular and vascular development. Our previous study showed that FRG1 is one of the highly expressed genes in the mandible on embryonic day 10.5 (E10.5) than on E12.0. In this study, we investigated the temporospatial expression pattern of FRG1 mRNA and protein during the development of the mouse lower first molar, and also evaluated the subcellular localization of the FRG1 protein in mouse dental epithelial (mDE6) cells. The FRG1 expression was identified in the dental epithelial and mesenchymal cells at the initiation and bud stages. It was detected in the inner enamel epithelium at the cap and early bell stages. At the late bell and root formation stages, these signals were detected in ameloblasts and odontoblasts during the formation of enamel and dentin matrices, respectively. The FRG1 protein was localized in the cytoplasm in the mouse tooth germ in vivo, while FRG1 was detected predominantly in the nucleus and faintly in the cytoplasm in mDE6 cells in vitro. In mDE6 cells treated with bone morphogenetic protein 4 (BMP4), the protein expression of FRG1 increased in cytoplasm, suggesting that FRG1 may translocate to the cytoplasm. These findings suggest that FRG1 is involved in the morphogenesis of the tooth germ, as well as in the formation of enamel and dentin matrices and that FRG1 may play a role in the odontogenesis in the mouse following BMP4 stimulation. PMID:27234941

  15. Bone morphogenetic protein-4 strongly potentiates growth factor-induced proliferation of mammary epithelial cells

    SciTech Connect

    Montesano, Roberto Sarkoezi, Rita; Schramek, Herbert

    2008-09-12

    Bone morphogenetic proteins (BMPs) are multifunctional cytokines that elicit pleiotropic effects on biological processes such as cell proliferation, cell differentiation and tissue morphogenesis. With respect to cell proliferation, BMPs can exert either mitogenic or anti-mitogenic activities, depending on the target cells and their context. Here, we report that in low-density cultures of immortalized mammary epithelial cells, BMP-4 did not stimulate cell proliferation by itself. However, when added in combination with suboptimal concentrations of fibroblast growth factor (FGF)-2, FGF-7, FGF-10, epidermal growth factor (EGF) or hepatocyte growth factor (HGF), BMP-4 potently enhanced growth factor-induced cell proliferation. These results reveal a hitherto unsuspected interplay between BMP-4 and growth factors in the regulation of mammary epithelial cell proliferation. We suggest that the ability of BMP-4 to potentiate the mitogenic activity of multiple growth factors may contribute to mammary gland ductal morphogenesis as well as to breast cancer progression.

  16. Regulation of dorsal gene expression in Xenopus by the ventralizing homeodomain gene Vox.

    PubMed

    Melby, A E; Clements, W K; Kimelman, D

    1999-07-15

    Patterning in the vertebrate embryo is controlled by an interplay between signals from the dorsal organizer and the ventrally expressed BMPs. Here we examine the function of Vox, a homeodomain-containing gene that is activated by the ventralizing signal BMP-4. Inhibition of BMP signaling using a dominant negative BMP receptor (DeltaBMPR) leads to the ectopic activation of dorsal genes in the ventral marginal zone, and this activation is prevented by co-injection of Vox. chordin is the most strongly activated of those genes that are up-regulated by DeltaBMPR and is the gene most strongly inhibited by Vox expression. We demonstrate that Vox acts as a transcriptional repressor, showing that the activity of native Vox is mimicked by a Vox-repressor fusion (VoxEnR) and that a Vox-activator fusion (VoxG4A) acts as an antimorph, causing the formation of a partial secondary axis when expressed on the ventral side of the embryo. Although Vox can ectopically activate BMP-4 expression in whole embryos, we see no activation of BMP-4 by VoxG4A, demonstrating that this activation is indirect. Using a hormone-inducible version of VoxG4A, we find that a critical time window for Vox function is during the late blastula period. Using this construct, we demonstrate that only a subset of dorsal genes is directly repressed by Vox, revealing that there are different modes of regulation for organizer genes. Since the major direct target for Vox repression is chordin, we propose that Vox acts in establishing a BMP-4 morphogen gradient by restricting the expression domain of chordin. PMID:10395789

  17. Identification and Location of Brain Protein 4.1

    NASA Astrophysics Data System (ADS)

    Goodman, Steven R.; Casoria, Linda A.; Coleman, Diane B.; Zagon, Ian S.

    1984-06-01

    Protein 4.1 is a membrane skeletal protein that converts the low-affinity interaction between spectrin and actin into a high-affinity ternary complex of spectrin, protein 4.1, and actin that is essential to the structural stability of the erythrocyte. Pig brain was shown to contain an 87-kilodalton immunoreactive analog of protein 4.1 that has partial sequence homology with pig erythrocyte protein 4.1 and the same location as spectrin in the cortical cytoplasm of neuronal and glial cell types of the cerebellum.

  18. Natural bone fragmentation in the blind cave-dwelling fish, Astyanax mexicanus: candidate gene identification through integrative comparative genomics.

    PubMed

    Gross, Joshua B; Stahl, Bethany A; Powers, Amanda K; Carlson, Brian M

    2016-01-01

    Animals that colonize dark and nutrient-poor subterranean environments evolve numerous extreme phenotypes. These include dramatic changes to the craniofacial complex, many of which are under genetic control. These phenotypes can demonstrate asymmetric genetic signals wherein a QTL is detected on one side of the face but not the other. The causative gene(s) underlying QTL are difficult to identify with limited genomic resources. We approached this task by searching for candidate genes mediating fragmentation of the third suborbital bone (SO3) directly inferior to the orbit of the eye. We integrated positional genomic information using emerging Astyanax resources, and linked these intervals to homologous (syntenic) regions of the Danio rerio genome. We identified a discrete, approximately 6 Mb, conserved region wherein the gene causing SO3 fragmentation likely resides. We interrogated this interval for genes demonstrating significant differential expression using mRNA-seq analysis of cave and surface morphs across life history. We then assessed genes with known roles in craniofacial evolution and development based on GO term annotation. Finally, we screened coding sequence alterations in this region, identifying two key genes: transforming growth factor β3 (tgfb3) and bone morphogenetic protein 4 (bmp4). Of these candidates, tgfb3 is most promising as it demonstrates significant differential expression across multiple stages of development, maps close (<1 Mb) to the fragmentation critical locus, and is implicated in a variety of other animal systems (including humans) in non-syndromic clefting and malformations of the cranial sutures. Both abnormalities are analogous to the failure-to-fuse phenotype that we observe in SO3 fragmentation. This integrative approach will enable discovery of the causative genetic lesions leading to complex craniofacial features analogous to human craniofacial disorders. This work underscores the value of cave-dwelling fish as a

  19. Identification of a third Protein 4.1 tumor suppressor, Protein 4.1R, in meningioma pathogenesis.

    PubMed

    Robb, Victoria A; Li, Wen; Gascard, Philippe; Perry, Arie; Mohandas, Narla; Gutmann, David H

    2003-08-01

    Meningiomas are common central nervous system tumors; however, the mechanisms underlying their pathogenesis are largely undefined. In this report, we demonstrate that a third Protein 4.1 family member, Protein 4.1R, functions as a meningioma tumor suppressor. We observed loss of Protein 4.1R expression in two meningioma cell lines (IOMM-Lee, CH157-MN) by Western blotting as well as in 6 of 15 sporadic meningiomas by immunohistochemistry and fluorescence in situ hybridization. In support of a meningioma tumor suppressor function, Protein 4.1R overexpression resulted in reduced IOMM-Lee and CH157-MN cell proliferation. Similar to the Protein 4.1B and merlin tumor suppressors, Protein 4.1R membrane localization increased significantly under conditions of growth arrest in vitro. Lastly, we show that Protein 4.1R interacted with a subset of merlin/Protein 4.1B interactors including CD44 and betaII-spectrin. Collectively, these results suggest that Protein 4.1R functions as an important tumor suppressor in the molecular pathogenesis of meningioma. PMID:12901833

  20. Structural protein 4.1 is located in mammalian centrosomes

    SciTech Connect

    Krauss, S.W.; Chasis, J.A.; Rogers, C.; Mohandas, N.; Krockmalnic, G.; Penman, S.

    1997-07-01

    Structural protein 4.1 was first characterized as an important 80-kDa protein in the mature red cell membrane skeleton. It is now known to be a member of a family of protein isoforms detected at diverse intracellular sites in many nucleated mammalian cells. We recently reported that protein 4.1 isoforms are present at interphase in nuclear matrix and are rearranged during the cell cycle. Here we report that protein 4.1 epitopes are present in centrosomes of human and murine cells and are detected by using affinity-purified antibodies specific for 80-kDa red cell 4.1 and for 4.1 peptides. Immunofluorescence, by both conventional and confocal microscopy, showed that protein 4.1 epitopes localized in the pericentriolar region. Protein 4.1 epitopes remained in centrosomes after extraction of cells with detergent, salt, and DNase. Higher resolution electron microscopy of detergent-extracted cell whole mounts showed centrosomal protein 4.1 epitopes distributed along centriolar cylinders and on pericentriolar fibers, at least some of which constitute the filamentous network surrounding each centriole. Double-label electron microscopy showed that protein 4.1 epitopes were predominantly localized in regions also occupied by epitopes for centrosome-specific autoimmune serum 5051 but were not found on microtubules. Our results suggest that protein 4.1 is an integral component of centrosome structure, in which it may play an important role in centrosome function during cell division and organization of cellular architecture.

  1. Erythrocyte Protein 4.1 Binds and Regulates Myosin

    NASA Astrophysics Data System (ADS)

    Pasternack, Gary R.; Racusen, Richard H.

    1989-12-01

    Myosin was recently identified in erythrocytes and was shown to partition both with membrane and cytosolic fractions, suggesting that it may be loosely bound to membranes [Fowler, V. M., Davis, J. Q. & Bennett, V. (1985) J. Cell Biol. 100, 47-55, and Wong, A. J., Kiehart, D. P. & Pollard, T. D. (1985) J. Biol. Chem. 260, 46-49]; however, the molecular basis for this binding was unclear. The present studies employed immobilized monomeric myosin to examine the interaction of myosin with erythrocyte protein 4.1. In human erythrocytes, protein 4.1 binds to integral membrane proteins and mediates spectrin-actin assembly. Protein 4.1 binds to rabbit skeletal muscle myosin with a Kd = 140 nM and a stoichiometry consistent with 1:1 binding. Heavy meromyosin competes for protein 4.1 binding with Ki = 36-54 nM; however, the S1 fragment (the myosin head) competes less efficiently. Affinity chromatography of partial chymotryptic digests of protein 4.1 on immobilized myosin identified a 10-kDa domain of protein 4.1 as the myosin-binding site. In functional studies, protein 4.1 partially inhibited the actin-activated Mg2+-ATPase activity of rabbit skeletal muscle myosin with Ki = 51 nM. Liver cytosolic and erythrocyte myosins preactivated with myosin light-chain kinase were similarly inhibited by protein 4.1. These studies show that protein 4.1 binds, modulates, and thus may regulate myosin. This interaction might serve to generate the contractile forces involved in Mg2+-ATP-dependent shape changes in erythrocytes and may additionally serve as a model for myosin organization and regulation in non-muscle cells.

  2. Autophagy-related gene 5 and Wnt5 signaling pathway requires differentiation of embryonic stem cells into odontoblast-like cells.

    PubMed

    Ozeki, Nobuaki; Mogi, Makio; Hase, Naoko; Hiyama, Taiki; Yamaguchi, Hideyuki; Kawai, Rie; Kondo, Ayami; Matsumoto, Toru; Nakata, Kazuhiko

    2016-02-01

    We previously confirmed a unique and unanticipated role for an α2 integrin, extracellular matrix metalloproteinase inducer (Emmprin), and matrix metalloproteinase (MMP)-3-mediated signaling cascade, in driving the odontoblast-like differentiation of mouse embryonic stem (ES) cells in a collagen type-I scaffold (CS) combined with bone morphogenetic protein (BMP)-4 (CS/BMP-4). To explore the early signaling cascade for odontoblastic differentiation, we examined the upregulation of autophagy-related gene (Atg) and Wnt signaling by CS/BMP-4 mediated odontoblast differentiation. In a screening experiment, CS/BMP-4 increased the mRNA and protein levels of Atg5, Lrp5/Fzd9 (an Atg5 receptor), and Wnt5, but not microtubule-associated protein 1 light chain (LC3; a mammalian homolog of yeast Atg8), TFE3, Beclin1, and Atg12, together with the amount of autophagosomes and autophagy fluxes. Treatment with siRNAs against Atg5 and Wnt5 individually suppressed the CS/BMP-4-induced increase in odontoblast differentiation. The odontoblastic phenotype, involving dentin matrix protein-1 and dentin sialophosphoprotein expression, decreased when autophagy was inhibited by chloroquine, but increased after treatment with rapamycin (an autophagy enhancer). Taken together with our previous findings, we have revealed a unique sequential cascade involving Atg5, Wnt5a, α2 integrin, Emmprin, and MMP-3. This cascade results in a potent increase in odontoblastic cell differentiation, indicating the unique involvement of Atg5, autophagy and Wnt5 signaling in CS/BMP-4-induced differentiation of ES cells into odontoblast-like cells, at a relatively early stage. PMID:26806855

  3. A haemolytic syndrome associated with the complete absence of red cell membrane protein 4.2 in two Tunisian siblings.

    PubMed

    Ghanem, A; Pothier, B; Marechal, J; Ducluzeau, M T; Morle, L; Alloisio, N; Feo, C; Ben Abdeladhim, A; Fattoum, S; Delaunay, J

    1990-07-01

    We report on the complete absence of protein 4.2 in two Tunisian siblings. The propositus presented with a haemolytic anaemia that evolved in an intermittent fashion until she was cured by splenectomy. Her red cells had a normal morphology, as well as normal deformability upon osmotic gradient ektacytometry. SDS-polyacrylamide gel electrophoresis failed to reveal any protein 4.2. Using anti-protein 4.2 polyclonal antibodies. Western blots were also unable to detect protein 4.2. Preparation of inside out vesicles resulted in no detectable loss of ankyrin. The propositus's sister presented with a haemolytic anaemia but had not undergone splenectomy; she showed the same biochemical features. The two cases presented of missing protein 4.2 are the first ones to be described outside the Japanese population. Considered as homozygotes for some defect that must alter the protein 4.2 gene itself, they exemplify a unique syndrome pertaining neither to elliptocytosis nor to spherocytosis, at least not closely. The parents, who are first cousins and whom we regarded as heterozygotes, were clinically and morphologically normal; they had a normal content of protein 4.2. Therefore, the 4.2 (-) haemolytic anaemia appears as entirely recessive. PMID:2386772

  4. Protein 4.1, a component of the erythrocyte membrane skeleton and its related homologue proteins forming the protein 4.1/FERM superfamily.

    PubMed

    Diakowski, Witold; Grzybek, Michał; Sikorski, Aleksander F

    2006-01-01

    The review is focused on the domain structure and function of protein 4.1, one of the proteins belonging to the membrane skeleton. The protein 4.1 of the red blood cells (4.1R) is a multifunctional protein that localizes to the membrane skeleton and stabilizes erythrocyte shape and membrane mechanical properties, such as deformability and stability, via lateral interactions with spectrin, actin, glycophorin C and protein p55. Protein 4.1 binding is modulated through the action of kinases and/or calmodulin-Ca2+. Non-erythroid cells express the 4.1R homologues: 4.1G (general type), 4.1B (brain type), and 4.1N (neuron type), and the whole group belongs to the protein 4.1 superfamily, which is characterized by the presence of a highly conserved FERM domain at the N-terminus of the molecule. Proteins 4.1R, 4.1G, 4.1N and 4.1B are encoded by different genes. Most of the 4.1 superfamily proteins also contain an actin-binding domain. To date, more than 40 members have been identified. They can be divided into five groups: protein 4.1 molecules, ERM proteins, talin-related molecules, protein tyrosine phosphatase (PTPH) proteins and NBL4 proteins. We have focused our attention on the main, well known representatives of 4.1 superfamily and tried to choose the proteins which are close to 4.1R or which have distinct functions. 4.1 family proteins are not just linkers between the plasma membrane and membrane skeleton; they also play an important role in various processes. Some, such as focal adhesion kinase (FAK), non-receptor tyrosine kinase that localizes to focal adhesions in adherent cells, play the role in cell adhesion. The other members control or take part in tumor suppression, regulation of cell cycle progression, inhibition of cell proliferation, downstream signaling of the glutamate receptors, and establishment of cell polarity; some are also involved in cell proliferation, cell motility, and/or cell-to-cell communication. PMID:17219717

  5. Chordin-Like 1 Suppresses Bone Morphogenetic Protein 4-Induced Breast Cancer Cell Migration and Invasion.

    PubMed

    Cyr-Depauw, Chanèle; Northey, Jason J; Tabariès, Sébastien; Annis, Matthew G; Dong, Zhifeng; Cory, Sean; Hallett, Michael; Rennhack, Jonathan P; Andrechek, Eran R; Siegel, Peter M

    2016-05-15

    ShcA is an important mediator of ErbB2- and transforming growth factor β (TGF-β)-induced breast cancer cell migration, invasion, and metastasis. We show that in the context of reduced ShcA levels, the bone morphogenetic protein (BMP) antagonist chordin-like 1 (Chrdl1) is upregulated in numerous breast cancer cells following TGF-β stimulation. BMPs have emerged as important modulators of breast cancer aggressiveness, and we have investigated the ability of Chrdl1 to block BMP-induced increases in breast cancer cell migration and invasion. Breast cancer-derived conditioned medium containing elevated concentrations of endogenous Chrdl1, as well as medium containing recombinant Chrdl1, suppresses BMP4-induced signaling in multiple breast cancer cell lines. Live-cell migration assays reveal that BMP4 induces breast cancer migration, which is effectively blocked by Chrdl1. We demonstrate that BMP4 also stimulated breast cancer cell invasion and matrix degradation, in part, through enhanced metalloproteinase 2 (MMP2) and MMP9 activity that is antagonized by Chrdl1. Finally, high Chrdl1 expression was associated with better clinical outcomes in patients with breast cancer. Together, our data reveal that Chrdl1 acts as a negative regulator of malignant breast cancer phenotypes through inhibition of BMP signaling. PMID:26976638

  6. Endogenous heparan sulfate and heparin modulate bone morphogenetic protein-4 signaling and activity.

    PubMed

    Khan, Shaukat A; Nelson, Matthew S; Pan, Chendong; Gaffney, Patrick M; Gupta, Pankaj

    2008-06-01

    Bone morphogenetic proteins (BMPs) and their endogenous antagonists are important for brain and bone development and tumor initiation and progression. Heparan sulfate (HS) proteoglycans (HSPG) modulate the activities of BMPs and their antagonists. How glycosaminoglycans (GAGs) influence BMP activity in various malignancies and in inherited abnormalities of GAG metabolism, and the structural features of GAGs essential for modulation of BMP signaling, remain incompletely defined. We examined whether chemically modified soluble heparins, the endogenous HS in malignant cells and the HS accumulated in Hurler syndrome cells influence BMP-4 signaling and activity. We show that both exogenous (soluble) and endogenous GAGs modulate BMP-4 signaling and activity, and that this effect is dependent on specific sulfate residues of GAGs. Our studies suggest that endogenous sulfated GAGs promote the proliferation and impair differentiation of malignant human cells, providing the rationale for investigating whether pharmacological agents that inhibit GAG synthesis or function might reverse this effect. Our demonstration of impairment of BMP-4 signaling by GAGs in multipotent stem cells in human Hurler syndrome identifies a mechanism that might contribute to the progressive neurological and skeletal abnormalities in Hurler syndrome and related mucopolysaccharidoses. PMID:18385288

  7. A comparative examination of odontogenic gene expression in both toothed and toothless amniotes.

    PubMed

    Lainoff, Alexis J; Moustakas-Verho, Jacqueline E; Hu, Diane; Kallonen, Aki; Marcucio, Ralph S; Hlusko, Leslea J

    2015-05-01

    A well-known tenet of murine tooth development is that BMP4 and FGF8 antagonistically initiate odontogenesis, but whether this tenet is conserved across amniotes is largely unexplored. Moreover, changes in BMP4-signaling have previously been implicated in evolutionary tooth loss in Aves. Here we demonstrate that Bmp4, Msx1, and Msx2 expression is limited proximally in the red-eared slider turtle (Trachemys scripta) mandible at stages equivalent to those at which odontogenesis is initiated in mice, a similar finding to previously reported results in chicks. To address whether the limited domains in the turtle and the chicken indicate an evolutionary molecular parallelism, or whether the domains simply constitute an ancestral phenotype, we assessed gene expression in a toothed reptile (the American alligator, Alligator mississippiensis) and a toothed non-placental mammal (the gray short-tailed opossum, Monodelphis domestica). We demonstrate that the Bmp4 domain is limited proximally in M. domestica and that the Fgf8 domain is limited distally in A. mississippiensis just preceding odontogenesis. Additionally, we show that Msx1 and Msx2 expression patterns in these species differ from those found in mice. Our data suggest that a limited Bmp4 domain does not necessarily correlate with edentulism, and reveal that the initiation of odontogenesis in non-murine amniotes is more complex than previously imagined. Our data also suggest a partially conserved odontogenic program in T. scripta, as indicated by conserved Pitx2, Pax9, and Barx1 expression patterns and by the presence of a Shh-expressing palatal epithelium, which we hypothesize may represent potential dental rudiments based on the Testudinata fossil record. PMID:25678399

  8. A comparative examination of odontogenic gene expression in both toothed and toothless amniotes

    PubMed Central

    Lainoff, Alexis J.; Moustakas-Verho, Jacqueline E.; Hu, Diane; Kallonen, Aki; Marcucio, Ralph S.; Hlusko, Leslea J.

    2015-01-01

    A well-known tenet of murine tooth development is that BMP4 and FGF8 antagonistically initiate odontogenesis, but whether this tenet is conserved across amniotes is largely unexplored. Moreover, changes in BMP4-signaling have previously been implicated in evolutionary tooth loss in Aves. Here we demonstrate that Bmp4, Msx1, and Msx2 expression is limited proximally in the red-eared slider turtle (Trachemys scripta) mandible at stages equivalent to those at which odontogenesis is initiated in mice, a similar finding to previously reported results in chicks. To address whether the limited domains in the turtle and the chicken indicate an evolutionary molecular parallelism, or whether the domains simply constitute an ancestral phenotype, we assessed gene expression in a toothed reptile (the American alligator, Alligator mississippiensis) and a toothed non-placental mammal (the gray short-tailed opossum, Monodelphis domestica). We demonstrate that the Bmp4 domain is limited proximally in M. domestica and that the Fgf8 domain is limited distally in A. mississippiensis just preceding odontogenesis. Additionally, we show that Msx1 and Msx2 expression patterns in these species differ from those found in mice. Our data suggest that a limited Bmp4 domain does not necessarily correlate with edentulism, and reveal that the initiation of odontogenesis in non-murine amniotes is more complex than previously imagined. Our data also suggest a partially conserved odontogenic program in T. scripta, as indicated by conserved Pitx2, Pax9, and Barx1 expression patterns and by the presence of a Shh-expressing palatal epithelium, which we hypothesize may represent potential dental rudiments based on the Testudinata fossil record. PMID:25678399

  9. Overexpression of HE4 (human epididymis protein 4) enhances proliferation, invasion and metastasis of ovarian cancer

    PubMed Central

    Wang, Huimin; Tan, Mingzi; Schwab, Carlton L.; Deng, Lu; Gao, Jian; Hao, Yingying; Li, Xiao; Gao, Song; Liu, Juanjuan; Lin, Bei

    2016-01-01

    Overexpression of Human epididymis protein 4 (HE4) related with a role in ovarian cancer tumorigenesis while little is known about the molecular mechanism alteration by HE4 up regulation. Here we reported that overexpressed HE4 promoted ovarian cancer cells proliferation, invasion and metastasis. Furthermore, human whole genome gene expression profile microarrays revealed that 231 differentially expressed genes (DEGs) were altered in response to HE4, in which MAPK signaling, ECM receptor, cell cycle, steroid biosynthesis pathways were involved. The findings suggested that overexpressed HE4 played an important role in ovarian cancer progression and metastasis and that HE4 has the potential to serve as a novel therapeutic target for ovarian cancer. PMID:26575020

  10. Nucleolar protein 4-like has a complex expression pattern in zebrafish embryos.

    PubMed

    Borah, Supriya; Barrodia, Praveen; Swain, Rajeeb K

    2016-01-01

    The nucleolar protein 4-like (NOL4L) gene is present on chromosome 20 (20q11.21) in humans. Parts of this gene have been shown to fuse with RUNX1 and PAX5 in acute myeloid leukemia and acute lymphoblastic leukemia, respectively. The normal function of NOL4L in humans and other organisms is not well understood. The expression patterns and functions of NOL4L homologs during vertebrate development have not been reported. We sought to address these questions by studying the expression pattern of zebrafish nol4l during embryogenesis. Our data show that Znol4l mRNA is expressed in multiple organs in zebrafish embryos. The sites of expression include parts of the brain, spinal cord, pronephros, hematopoietic cells and gut. PMID:26934290

  11. Comprehensive characterization of protein 4.1 expression in epithelium of large intestine.

    PubMed

    Zhang, Jingxin; Yang, Shaomin; An, Chao; Wang, Jie; Yan, Hongxia; Huang, Yumin; Song, Jinlei; Yin, Changcheng; Baines, Anthony J; Mohandas, Narla; An, Xiuli

    2014-11-01

    The protein 4.1 family consists of four members, 4.1R, 4.1N, 4.1B and 4.1G, each encoded by a distinct gene. All 4.1 mRNAs undergo extensive alternative splicing. Functionally, they usually serve as adapters that link actin-based cytoskeleton to plasma membrane proteins. It has been reported that 4.1 proteins are expressed in most animal cell types and tissues including epithelial cells and epithelial tissues. However, the expression of 4.1 proteins in large intestine has not been well characterized. In the present study, we performed RT-PCR, western blot and immunohistochemistry analysis to characterize the transcripts, the protein expression and cellular localization of 4.1 proteins in the epithelia of mouse large intestine. We show that multiple transcripts derive from each gene, including eight 4.1R isoforms, four 4.1N isoforms, four 4.1B isoforms and six 4.1G isoforms. However, at the protein level, only one or two major bands were detected, implying that not all transcripts are translated and/or the proteins do not accumulate at detectable levels. Immunohistochemistry revealed that 4.1R, 4.1N and 4.1B are all expressed at the lateral membrane as well as cytoplasm of epithelial cells, suggesting a potentially redundant role of these proteins. Our findings not only provide new insights into the structure of protein 4.1 genes but also lay the foundation for future functional studies. PMID:24912669

  12. Systems analysis of the role of bone morphogenic protein 4 in endothelial inflammation.

    PubMed

    Yin, Weiwei; Jo, Hanjoong; Voit, Eberhard O

    2010-02-01

    Shear stress is an important factor in the onset and progression of atherosclerosis. High and unidirectional laminar stress is seen as protective, while low and oscillatory shear stress is considered pro-inflammatory and pro-atherogenic. The mechanosensitive response of endothelial cells is governed by a complex system of genes, proteins, and signals that operate at distinctly different time scales. We propose a dynamic mathematical model that quantitatively describes this mechanosensing system and permits novel insights into its functioning. The model, the first of its kind, is constructed within the guidelines of Biochemical Systems Theory and accounts for different time scales by means of approximated delays. Parameter values are obtained directly from biochemical observations in an ad hoc fashion. The model reflects most documented observations well and leads to a number of predictions and novel hypotheses. In particular, it demonstrates the crucial role of Bone Morphogenic Protein 4 and p47(phox)-dependent NADPH oxidases in endothelial inflammation. PMID:19851868

  13. Protein 4.1R–deficient mice are viable but have erythroid membrane skeleton abnormalities

    PubMed Central

    Shi, Zheng-Tao; Afzal, Veena; Coller, Barry; Patel, Dipti; Chasis, Joel A.; Parra, Marilyn; Lee, Gloria; Paszty, Chris; Stevens, Mary; Walensky, Loren; Peters, Luanne L.; Mohandas, Narla; Rubin, Edward; Conboy, John G.

    1999-01-01

    A diverse family of protein 4.1R isoforms is encoded by a complex gene on human chromosome 1. Although the prototypical 80-kDa 4.1R in mature erythrocytes is a key component of the erythroid membrane skeleton that regulates erythrocyte morphology and mechanical stability, little is known about 4.1R function in nucleated cells. Using gene knockout technology, we have generated mice with complete deficiency of all 4.1R protein isoforms. These 4.1R-null mice were viable, with moderate hemolytic anemia but no gross abnormalities. Erythrocytes from these mice exhibited abnormal morphology, lowered membrane stability, and reduced expression of other skeletal proteins including spectrin and ankyrin, suggesting that loss of 4.1R compromises membrane skeleton assembly in erythroid progenitors. Platelet morphology and function were essentially normal, indicating that 4.1R deficiency may have less impact on other hematopoietic lineages. Nonerythroid 4.1R expression patterns, viewed using histochemical staining for lacZ reporter activity incorporated into the targeted gene, revealed focal expression in specific neurons in the brain and in select cells of other major organs, challenging the view that 4.1R expression is widespread among nonerythroid cells. The 4.1R knockout mice represent a valuable animal model for exploring 4.1R function in nonerythroid cells and for determining pathophysiological sequelae to 4.1R deficiency. PMID:9927493

  14. The Neuronal PAS Domain Protein 4 (Npas4) Is Required for New and Reactivated Fear Memories

    PubMed Central

    Nguyen, Tam; DiLeone, Ralph J.; Schafe, Glenn E.

    2011-01-01

    The Neuronal PAS domain protein 4 (Npas4) is a neuronal activity-dependent immediate early gene that has recently been identified as a transcription factor which regulates the transcription of genes that control inhibitory synapse development and synaptic plasticity. The role Npas4 in learning and memory, however, is currently unknown. Here, we systematically examine the role of Npas4 in auditory Pavlovian fear conditioning, an amygdala-dependent form of emotional learning. In our first series of experiments, we show that Npas4 mRNA and protein are regulated in the rat lateral nucleus of the amygdala (LA) in a learning-dependent manner. Further, knockdown of Npas4 protein in the LA via adeno-associated viral (AAV) mediated gene delivery of RNAi was observed to impair fear memory formation, while innate fear and the expression of fear memory were not affected. In our second series of experiments, we show that Npas4 protein is regulated in the LA by retrieval of an auditory fear memory and that knockdown of Npas4 in the LA impairs retention of a reactivated, but not a non-reactivated, fear memory. Collectively, our findings provide the first comprehensive look at the functional role of Npas4 in learning and memory. PMID:21887312

  15. Multidrug Resistance Protein-4 Influences Aspirin Toxicity in Human Cell Line.

    PubMed

    Massimi, Isabella; Ciuffetta, Ambra; Temperilli, Flavia; Ferrandino, Francesca; Zicari, Alessandra; Pulcinelli, Fabio M; Felli, Maria Pia

    2015-01-01

    Overexpression of efflux transporters, in human cells, is a mechanism of resistance to drug and also to chemotherapy. We found that multidrug resistance protein-4 (MRP4) overexpression has a role in reducing aspirin action in patients after bypass surgery and, very recently, we found that aspirin enhances platelet MRP4 levels through peroxisome proliferator activated receptor-α (PPARα). In the present paper, we verified whether exposure of human embryonic kidney-293 cells (Hek-293) to aspirin modifies MRP4 gene expression and its correlation with drug elimination and cell toxicity. We first investigated the effect of high-dose aspirin in Hek-293 and we showed that aspirin is able to increase cell toxicity dose-dependently. Furthermore, aspirin effects, induced at low dose, already enhance MRP4 gene expression. Based on these findings, we compared cell viability in Hek-293, after high-dose aspirin treatment, in MRP4 overexpressing cells, either after aspirin pretreatment or in MRP4 transfected cells; in both cases, a decrease of selective aspirin cell growth inhibition was observed, in comparison with the control cultures. Altogether, these data suggest that exposing cells to low nontoxic aspirin dosages can induce gene expression alterations that may lead to the efflux transporter protein overexpression, thus increasing cellular detoxification of aspirin. PMID:26491233

  16. Protein 4.1 and the control of ion channels.

    PubMed

    Baines, Anthony J; Bennett, Pauline M; Carter, Edward W; Terracciano, Cesare

    2009-01-01

    The classical function of 4.1R in red blood cells is to contribute to the mechanochemical properties of the membrane by promoting the interaction between spectrin and actin. More recently, it has been recognized that 4.1R is required for the stable cell surface accumulation of a number of erythrocyte membrane proteins. 4.1R is one member of the mammalian 4.1 family - the others being 4.1N, 4.1G and 4.1B - and is expressed in many cell types other than erythrocytes. Recently we have examined the phenotype of hearts from 4.1R knockout mice. Although they had a generally normal morphology, these hearts exhibited bradycardia, and prolongation of both action potentials and QT intervals. Electrophysiological analysis revealed anomalies in a range of ion channel activities. In addition, the immunoreactivity of voltage-gated Na(+) channel NaV1.5 was reduced, indicating a role for 4.1R in the cellular accumulation of this ion channel. 4.1 proteins also have roles in the accumulation of at least two other classes of ion channel. In epithelia, 4.1 interacts with the store-operated channel TRPC4. In neurons, the ligand-gated channels GluR1 and GluR4 require 4.1 proteins for cell surface accumulation. The spectrum of transmembrane proteins that bind to 4.1 proteins overlaps with that of ankyrin. A hypothesis to investigate in the future is that differential regulation of 4.1 and ankyrins (e.g. by PIP(2)) allows highly selective control of cell surface accumulation and transport activity of a specific range of ion channels. PMID:19272819

  17. Specificity protein 4 (Sp4) transcriptionally regulates inhibitory GABAergic receptors in neurons.

    PubMed

    Nair, Bindu; Johar, Kaid; Priya, Anusha; Wong-Riley, Margaret T T

    2016-01-01

    Previous studies in our laboratory have shown that the neuron-specific specificity protein 4 (Sp4) transcriptionally regulates many excitatory neurotransmitter receptor subunit genes, such as those for GluN1, GluN2A, and GluN2B of N-methyl-d-aspartate (NMDA) receptors and Gria2 of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. It also regulates Atp1a1 and Atp1b1 subunit genes of Na(+)/K(+)-ATPase, a major energy-consuming enzyme, as well as all 13 subunits of cytochrome c oxidase (COX), an important energy-generating enzyme. Thus, there is a tight coupling between energy consumption, energy production, and excitatory neuronal activity at the transcriptional level in neurons. The question is whether inhibitory neurotransmitter receptors are also regulated by Sp4. In the present study, we tested our hypothesis that Sp4 regulates receptor subunit genes of a major inhibitory neurotransmitter, GABA, specifically GABAA receptors. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, real-time quantitative PCR, chromatin immunoprecipitation, promoter mutational analysis, over-expression and shRNA of Sp4, functional assays, and western blots, we found that Sp4 functionally regulates the transcription of Gabra1 (GABAA α1) and Gabra2 (GABAA α2), but not Gabra3 (GABAA α3) subunit genes. The binding sites of Sp4 are conserved among rats, humans, and mice. Thus, our results substantiate our hypothesis that Sp4 plays a key role in regulating the transcription of GABAA receptor subunit genes. They also indicate that Sp4 is in a position to transcriptionally regulate the balance between excitatory and inhibitory neurochemical expressions in neurons. PMID:26469128

  18. Distinct distribution of specific members of protein 4.1 genefamily in the mouse nephron

    SciTech Connect

    Ramez, Mohamed; Blot-Chabaud, Marcel; Cluzeaud, Francoise; Chanan, Sumita; Patterson, Michael; Walensky, Loren D.; Marfatia, Shirin; Baines, Anthony J.; Chasis, Joel A.; Conboy, John G.; Mohandas, Narla; Gascard, Philippe

    2002-12-11

    Background: Protein 4.1 is an adapter protein which linksthe actin cytoskeleton to various transmembrane proteins. 4.1 proteinsare encoded by four homologous genes, 4.1R, 4.1G, 4.1N, and 4.1B, whichundergo complex alternative splicing. Here we performed a detailedcharacterization of the expression of specific 4.1 proteins in the mousenephron. Methods: Distribution of renal 4.1 proteins was investigated bystaining of paraformaldehyde fixed mouse kidney sections with antibodieshighly specific for each 4.1 protein. Major 4.1 splice forms, amplifiedfrom mouse kidney marathon cDNA, were expressed in transfected COS-7cells in order to assign species of known exon composition to proteinsdetected in kidney. Results: A 105kDa4.1R splice form, initiating atATG-2 translation initiation site and lacking exon 16, but including exon17B, was restricted to thick ascending limb of Henle's loop. A 95kDa 4.1Nspliceform,lacking exons 15 and 17D, was expressed in either descendingor ascending thin limb of Henle'sloop, distal convoluted tubule and allregions of the collecting duct system. A major 108kDa 4.1B spliceform,initiating at a newly characterized ATG translation initiation site, andlacking exons 15, 17B, and 21, was present only in Bowman's capsule andproximal convoluted tubule (PCT). There was no expression of 4.1G inkidney. Conclusion: Distinct distribution of 4.1 proteins along thenephron suggests their involvement in targeting of selected transmembraneproteins in kidney epithelium andtherefore in regulation of specifickidney functions.

  19. The Involvement of hybrid cluster protein 4, HCP4, in Anaerobic Metabolism in Chlamydomonas reinhardtii.

    PubMed

    Olson, Adam C; Carter, Clay J

    2016-01-01

    The unicellular green algae Chlamydomonas reinhardtii has long been studied for its unique fermentation pathways and has been evaluated as a candidate organism for biofuel production. Fermentation in C. reinhardtii is facilitated by a network of three predominant pathways producing four major byproducts: formate, ethanol, acetate and hydrogen. Previous microarray studies identified many genes as being highly up-regulated during anaerobiosis. For example, hybrid cluster protein 4 (HCP4) was found to be one of the most highly up-regulated genes under anoxic conditions. Hybrid cluster proteins have long been studied for their unique spectroscopic properties, yet their biological functions remain largely unclear. To probe its role during anaerobiosis, HCP4 was silenced using artificial microRNAs (ami-hcp4) followed by extensive phenotypic analyses of cells grown under anoxic conditions. Both the expression of key fermentative enzymes and their respective metabolites were significantly altered in ami-hcp4, with nitrogen uptake from the media also being significantly different than wild-type cells. The results strongly suggest a role for HCP4 in regulating key fermentative and nitrogen utilization pathways. PMID:26930496

  20. The Involvement of hybrid cluster protein 4, HCP4, in Anaerobic Metabolism in Chlamydomonas reinhardtii

    PubMed Central

    Olson, Adam C.; Carter, Clay J.

    2016-01-01

    The unicellular green algae Chlamydomonas reinhardtii has long been studied for its unique fermentation pathways and has been evaluated as a candidate organism for biofuel production. Fermentation in C. reinhardtii is facilitated by a network of three predominant pathways producing four major byproducts: formate, ethanol, acetate and hydrogen. Previous microarray studies identified many genes as being highly up-regulated during anaerobiosis. For example, hybrid cluster protein 4 (HCP4) was found to be one of the most highly up-regulated genes under anoxic conditions. Hybrid cluster proteins have long been studied for their unique spectroscopic properties, yet their biological functions remain largely unclear. To probe its role during anaerobiosis, HCP4 was silenced using artificial microRNAs (ami-hcp4) followed by extensive phenotypic analyses of cells grown under anoxic conditions. Both the expression of key fermentative enzymes and their respective metabolites were significantly altered in ami-hcp4, with nitrogen uptake from the media also being significantly different than wild-type cells. The results strongly suggest a role for HCP4 in regulating key fermentative and nitrogen utilization pathways. PMID:26930496

  1. Functional characterization of protein 4.1 homolog in amphioxus: defining a cryptic spectrin-actin-binding site.

    PubMed

    Wang, Lixia; Wang, Yuan; Li, Zhaohe; Gao, Zhan; Zhang, Shicui

    2013-01-01

    Vertebrate 4.1 proteins have a spectrin-actin-binding (SAB) domain, which is lacking in all the invertebrate 4.1 proteins indentified so far, and it was therefore proposed that the SAB domain emerged with the advent of vertebrates during evolution. Here we demonstrated for the first time that amphioxus (an invertebrate chordate) protein 4.1, though lacking a recognizable SAB, was able to bind both spectrin and actin, with a binding capacity comparable to that of human protein 4.1. Detailed structure-activity analyses revealed that the unique domain U2/3 was a newly identified SAB-like domain capable of interacting with spectrin and actin, suggesting the presence of a "cryptic" SAB domain in amphioxus 4.1 protein. We also showed that amphioxus 4.1 protein gene was the common ancestor of vertebrate 4.1 protein genes, from which 4.1R, 4.1N, 4.1G, and 4.1B genes originated. This work will encourage further study on the structure-activity of invertebrate 4.1 protein and its interacting proteins. PMID:24096627

  2. Functional characterization of protein 4.1 homolog in amphioxus: Defining a cryptic spectrin-actin-binding site

    PubMed Central

    Wang, Lixia; Wang, Yuan; Li, Zhaohe; Gao, Zhan; Zhang, Shicui

    2013-01-01

    Vertebrate 4.1 proteins have a spectrin-actin-binding (SAB) domain, which is lacking in all the invertebrate 4.1 proteins indentified so far, and it was therefore proposed that the SAB domain emerged with the advent of vertebrates during evolution. Here we demonstrated for the first time that amphioxus (an invertebrate chordate) protein 4.1, though lacking a recognizable SAB, was able to bind both spectrin and actin, with a binding capacity comparable to that of human protein 4.1. Detailed structure-activity analyses revealed that the unique domain U2/3 was a newly identified SAB-like domain capable of interacting with spectrin and actin, suggesting the presence of a “cryptic” SAB domain in amphioxus 4.1 protein. We also showed that amphioxus 4.1 protein gene was the common ancestor of vertebrate 4.1 protein genes, from which 4.1R, 4.1N, 4.1G, and 4.1B genes originated. This work will encourage further study on the structure-activity of invertebrate 4.1 protein and its interacting proteins. PMID:24096627

  3. Protein 4.1: its association with the human erythrocyte membrane.

    PubMed Central

    Shiffer, K A; Goodman, S R

    1984-01-01

    125I-labeled protein 4.1a and 4.1b have equal ability to reassociate with inside-out erythrocyte vesicles that were depleted of protein 4.1 in addition to other peripheral membrane proteins. The reassociation of 125I-labeled protein 4.1 to protein 4.1-depleted vesicles at 4 degrees C is salt dependent, pH dependent, and saturable with a Kd of 42-50 nM and an extrapolated maximal binding capacity of 120-140 micrograms of protein 4.1 bound per mg of vesicle protein or 60-70 micrograms of protein 4.1 bound per mg of ghost protein, correlating with the protein 4.1 content in the erythrocyte membrane (6-7% of the total membrane protein). Selective proteolytic cleavage of these vesicles with papain (5 micrograms/ml at 4 degrees C) eliminates greater than 60% of the high-affinity binding sites; therefore, we conclude that the interaction of protein 4.1 with the cytoplasmic membrane surface is through a specific high-affinity protein-protein association. Images PMID:6589603

  4. Bovine Rotavirus Nonstructural Protein 4 Produced by Lactococcus lactis Is Antigenic and Immunogenic

    PubMed Central

    Enouf, Vincent; Langella, Philippe; Commissaire, Jacqueline; Cohen, Jean; Corthier, Gérard

    2001-01-01

    Rotavirus nonstructural protein 4 (NSP4) can induce diarrhea in mice. To get insight into the biological effects of NSP4, production of large quantities of this protein is necessary. We first tried to produce the protein in Escherichia coli, but the nsp4 gene proved to be unstable. The capacity of the generally regarded as safe organism Lactococcus lactis to produce NSP4 either intra- or extracellularly was then investigated by using the nisin-controlled expression system. Production of recombinant NSP4 (rNSP4) was observed in L. lactis for both locations. In spite of a very low secretion efficiency, the highest level of production was obtained with the fusion between a lactococcal signal peptide and rNSP4. Cultures of the rNSP4-secreting strain were injected into rabbits, and a specific immune response was elicited. The anti-rNSP4 antibodies produced in these rabbits recognized NSP4 in MA104 cells infected by rotavirus. We showed that L. lactis is able to produce antigenic and immunogenic rNSP4 and thus is a good organism for producing viral antigens. PMID:11282586

  5. Palmitoylation of cytoskeleton associated protein 4 by DHHC2 regulates antiproliferative factor-mediated signaling.

    PubMed

    Planey, Sonia L; Keay, Susan K; Zhang, Chen-Ou; Zacharias, David A

    2009-03-01

    Previously, we identified cytoskeleton-associated protein 4 (CKAP4) as a major substrate of the palmitoyl acyltransferase, DHHC2, using a novel proteomic method called palmitoyl-cysteine identification, capture and analysis (PICA). CKAP4 is a reversibly palmitoylated and phosphorylated protein that links the ER to the cytoskeleton. It is also a high-affinity receptor for antiproliferative factor (APF), a small sialoglycopeptide secreted from bladder epithelial cells of patients with interstitial cystitis (IC). The role of DHHC2-mediated palmitoylation of CKAP4 in the antiproliferative response of HeLa and normal bladder epithelial cells to APF was investigated. Our data show that siRNA-mediated knockdown of DHHC2 and consequent suppression of CKAP4 palmitoylation inhibited the ability of APF to regulate cellular proliferation and blocked APF-induced changes in the expression of E-cadherin, vimentin, and ZO-1 (genes known to play a role in cellular proliferation and tumorigenesis). Immunocytochemistry revealed that CKAP4 palmitoylation by DHHC2 is required for its trafficking from the ER to the plasma membrane and for its nuclear localization. These data suggest an important role for DHHC2-mediated palmitoylation of CKAP4 in IC and in opposing cancer-related cellular behaviors and support the idea that DHHC2 is a tumor suppressor. PMID:19144824

  6. LDL-receptor-related protein 4 is crucial for formation of the neuromuscular junction.

    PubMed

    Weatherbee, Scott D; Anderson, Kathryn V; Niswander, Lee A

    2006-12-01

    Low-density lipoprotein receptor-related protein 4 (Lrp4) is a member of a family of structurally related, single-pass transmembrane proteins that carry out a variety of functions in development and physiology, including signal transduction and receptor-mediated endocytosis. Lrp4 is expressed in multiple tissues in the mouse, and is important for the proper development and morphogenesis of limbs, ectodermal organs, lungs and kidneys. We show that Lrp4 is also expressed in the post-synaptic endplate region of muscles and is required to form neuromuscular synapses. Lrp4-mutant mice die at birth with defects in both presynaptic and postsynaptic differentiation, including aberrant motor axon growth and branching, a lack of acetylcholine receptor and postsynaptic protein clustering, and a failure to express postsynaptic genes selectively by myofiber synaptic nuclei. Our data show that Lrp4 is required during the earliest events in postsynaptic neuromuscular junction (NMJ) formation and suggest that it acts in the early, nerveindependent steps of NMJ assembly. The identification of Lrp4 as a crucial factor for NMJ formation may have implications for human neuromuscular diseases such as myasthenia syndromes. PMID:17119023

  7. Drosophila coracle, a Member of the Protein 4.1 Superfamily, Has Essential Structural Functions in the Septate Junctions and Developmental Functions in Embryonic and Adult Epithelial Cells

    PubMed Central

    Lamb, Rebecca S.; Ward, Robert E.; Schweizer, Liang; Fehon, Richard G.

    1998-01-01

    Although extensively studied biochemically, members of the Protein 4.1 superfamily have not been as well characterized genetically. Studies of coracle, a Drosophila Protein 4.1 homologue, provide an opportunity to examine the genetic functions of this gene family. coracle was originally identified as a dominant suppressor of EgfrElp, a hypermorphic form of the Drosophila Epidermal growth factor receptor gene. In this article, we present a phenotypic analysis of coracle, one of the first for a member of the Protein 4.1 superfamily. Screens for new coracle alleles confirm the null coracle phenotype of embryonic lethality and failure in dorsal closure, and they identify additional defects in the embryonic epidermis and salivary glands. Hypomorphic coracle alleles reveal functions in many imaginal tissues. Analysis of coracle mutant cells indicates that Coracle is a necessary structural component of the septate junction required for the maintenance of the transepithelial barrier but is not necessary for apical–basal polarity, epithelial integrity, or cytoskeletal integrity. In addition, coracle phenotypes suggest a specific role in cell signaling events. Finally, complementation analysis provides information regarding the functional organization of Coracle and possibly other Protein 4.1 superfamily members. These studies provide insights into a range of in vivo functions for coracle in developing embryos and adults. PMID:9843584

  8. Neurobehavioral deficits in mice lacking the erythrocyte membrane cytoskeletal protein 4.1.

    PubMed

    Walensky, L D; Shi, Z T; Blackshaw, S; DeVries, A C; Demas, G E; Gascard, P; Nelson, R J; Conboy, J G; Rubin, E M; Snyder, S H; Mohandas, N

    1998-11-19

    The erythrocyte membrane cytoskeletal protein 4.1 (4.1R) is a structural protein that confers stability and flexibility to erythrocytes via interactions with the cytoskeletal proteins spectrin and F-actin and with the band 3 and glycophorin C membrane proteins. Mutations in 4.1R can cause hereditary elliptocytosis, a disease characterized by a loss of the normal discoid morphology of erythrocytes, resulting in hemolytic anemia [1]. Different isoforms of the 4.1 protein have been identified in a wide variety of nonerythroid tissues by immunological methods [2-5]. The variation in molecular weight of these different 4.1 isoforms, which range from 30 to 210 kDa [6], has been attributed to complex alternative splicing of the 4.1R gene [7]. We recently identified two new 4.1 genes: one is generally expressed throughout the body (4. 1G) [8] and the other is expressed in central and peripheral neurons (4.1N) [9]. Here, we examined 4.1R expression by in situ hybridization analysis and found that 4.1R was selectively expressed in hematopoietic tissues and in specific neuronal populations. In the brain, high levels of 4.1R were discretely localized to granule cells in the cerebellum and dentate gyrus. We generated mice that lacked 4.1R expression; these mice had deficits in movement, coordination, balance and learning, in addition to the predicted hematological abnormalities. The neurobehavioral findings are consistent with the distribution of 4.1R in the brain, suggesting that 4.1R performs specific functions in the central nervous system. PMID:9822582

  9. Immunogenicity of Plasmodium yoelii merozoite surface protein 4/5 produced in transgenic plants.

    PubMed

    Wang, Lina; Webster, Diane E; Campbell, Alison E; Dry, Ian B; Wesselingh, Steve L; Coppel, Ross L

    2008-01-01

    Malaria is a major global health problem for which effective control measures are urgently needed. Considerable effort has been focused on the development of effective vaccines against the causative parasite and protective vaccine trials are now being reported. Due to the relative poverty and lack of infrastructure in malaria-endemic areas, a successful immunisation strategy will depend critically on cheap and scaleable methods of vaccine production, distribution and delivery. One promising technology is transgenic plants, both as a bioreactor for the vaccine-manufacturing process as well as a matrix for oral immunisation. In this study, we investigated the feasibility of using transgenic plants to induce protective immunity against malaria infection using Plasmodium yoelii merozoite surface protein 4/5 (PyMSP4/5) in a mouse model of malaria infection. Our data show that the PyMSP4/5 protein can be produced in plants in a configuration that reacts with protective antibodies. Optimisation of codon usage for the PyMSP4/5 gene resulted in significantly increased antigen expression in plants. PyMSP4/5 protein from the codon-optimised construct accumulated to 0.25% of total soluble protein, a sixfold increase over the native gene sequence. Tobacco-made PyMSP4/5 was able to induce antigen-specific antibodies in mice following parenteral delivery, as well as boost the antibody responses induced by DNA vaccination when delivered parenterally or orally. We believe this is the first report to show that plant-made malaria antigens are immunogenic. However, the antibody levels were not high enough to protect the immunised mice against a lethal challenge with P. yoelii. Further strategies are needed to achieve a protective dose, including improvements to antigen expression levels in plants and strategies to enhance the immunogenicity of the expressed antigen. PMID:17681344

  10. Helicobacter pylori Infection Induces Anemia, Depletes Serum Iron Storage, and Alters Local Iron-Related and Adult Brain Gene Expression in Male INS-GAS Mice

    PubMed Central

    Burns, Monika; Muthupalani, Sureshkumar; Ge, Zhongming; Wang, Timothy C.; Bakthavatchalu, Vasudevan; Cunningham, Catriona; Ennis, Kathleen; Georgieff, Michael; Fox, James G.

    2015-01-01

    Iron deficiency anemia (IDA) affects > 500 million people worldwide, and is linked to impaired cognitive development and function in children. Helicobacter pylori, a class 1 carcinogen, infects about half of the world’s population, thus creating a high likelihood of overlapping risk. This study determined the effect of H. pylori infection on iron homeostasis in INS-GAS mice. Two replicates of INS-GAS/FVB male mice (n = 9-12/group) were dosed with H. pylori (Hp) strain SS1 or sham dosed at 6–9 weeks of age, and were necropsied at 27–29 weeks of age. Hematologic and serum iron parameters were evaluated, as was gene expression in gastric and brain tissues. Serum ferritin was lower in Hp SS1-infected mice than uninfected mice (p < 0.0001). Infected mice had a lower red blood cell count (p<0.0001), hematocrit (p < 0.001), and hemoglobin concentration (p <0.0001) than uninfected mice. Relative expression of gastric hepcidin antimicrobial peptide (Hamp) was downregulated in mice infected with Hp SS1 compared to sham-dosed controls (p<0.001). Expression of bone morphogenic protein 4 (Bmp4), a growth factor upstream of hepcidin, was downregulated in gastric tissue of Hp SS1-infected mice (p<0.001). Hp SS1-infected mice had downregulated brain expression of tyrosine hydroxylase (Th) (p = 0.02). Expression of iron-responsive genes involved in myelination (myelin basic protein (Mbp) and proteolipid protein 2 (Plp2)) was downregulated in infected mice (p = 0.001 and p = 0.02). Expression of synaptic plasticity markers (brain derived neurotrophic factor 3 (Bdnf3), Psd95 (a membrane associated guanylate kinase), and insulin-like growth factor 1 (Igf1)) was also downregulated in Hp SS1-infected mice (p = 0.09, p = 0.04, p = 0.02 respectively). Infection of male INS-GAS mice with Hp SS1, without concurrent dietary iron deficiency, depleted serum ferritin, deregulated gastric and hepatic expression of iron regulatory genes, and altered iron-dependent neural processes. The use

  11. Regulation of dorsal-ventral patterning: the ventralizing effects of the novel Xenopus homeobox gene Vox.

    PubMed

    Schmidt, J E; von Dassow, G; Kimelman, D

    1996-06-01

    The formation of the dorsal-ventral axis in Xenopus laevis is elicited by a signaling cascade on the dorsal side of the embryo initiated by cortical rotation. These early developmental events impart an initial axial polarity to the embryo. By the time gastrulation occurs, the embryo has established opposing dorsal and ventral regulatory regions. Through a dynamic process, the embryo acquires a definitive pattern that reflects the distribution of future cell fates. Here we present a novel homeobox gene, Vox, whose expression reflects this dynamic process. Vox is first expressed throughout the embryo and subsequently eliminated from the notochord and neural plate. Ectopic expression of Vox demonstrates that the normal function of this gene may be to suppress dorsal genes such as Xnot and chordin, and induce ventral and paraxial genes such as Bmp-4 and MyoD. Ectopic expression of BMP-4 ventralizes embryos and positively regulates the expression of Vox, suggesting that these genes are components of a reciprocal regulatory network. PMID:8674411

  12. The Evx1/Evx1as gene locus regulates anterior-posterior patterning during gastrulation

    PubMed Central

    Bell, Charles C.; Amaral, Paulo P.; Kalsbeek, Anton; Magor, Graham W.; Gillinder, Kevin R.; Tangermann, Pierre; di Lisio, Lorena; Cheetham, Seth W.; Gruhl, Franziska; Frith, Jessica; Tallack, Michael R.; Ru, Ke-Lin; Crawford, Joanna; Mattick, John S.; Dinger, Marcel E.; Perkins, Andrew C.

    2016-01-01

    Thousands of sense-antisense mRNA-lncRNA gene pairs occur in the mammalian genome. While there is usually little doubt about the function of the coding transcript, the function of the lncRNA partner is mostly untested. Here we examine the function of the homeotic Evx1-Evx1as gene locus. Expression is tightly co-regulated in posterior mesoderm of mouse embryos and in embryoid bodies. Expression of both genes is enhanced by BMP4 and WNT3A, and reduced by Activin. We generated a suite of deletions in the locus by CRISPR-Cas9 editing. We show EVX1 is a critical downstream effector of BMP4 and WNT3A with respect to patterning of posterior mesoderm. The lncRNA, Evx1as arises from alternative promoters and is difficult to fully abrogate by gene editing or siRNA approaches. Nevertheless, we were able to generate a large 2.6 kb deletion encompassing the shared promoter with Evx1 and multiple additional exons of Evx1as. This led to an identical dorsal-ventral patterning defect to that generated by micro-deletion in the DNA-binding domain of EVX1. Thus, Evx1as has no function independent of EVX1, and is therefore unlikely to act in trans. We predict many antisense lncRNAs have no specific trans function, possibly only regulating the linked coding genes in cis. PMID:27226347

  13. The Evx1/Evx1as gene locus regulates anterior-posterior patterning during gastrulation.

    PubMed

    Bell, Charles C; Amaral, Paulo P; Kalsbeek, Anton; Magor, Graham W; Gillinder, Kevin R; Tangermann, Pierre; di Lisio, Lorena; Cheetham, Seth W; Gruhl, Franziska; Frith, Jessica; Tallack, Michael R; Ru, Ke-Lin; Crawford, Joanna; Mattick, John S; Dinger, Marcel E; Perkins, Andrew C

    2016-01-01

    Thousands of sense-antisense mRNA-lncRNA gene pairs occur in the mammalian genome. While there is usually little doubt about the function of the coding transcript, the function of the lncRNA partner is mostly untested. Here we examine the function of the homeotic Evx1-Evx1as gene locus. Expression is tightly co-regulated in posterior mesoderm of mouse embryos and in embryoid bodies. Expression of both genes is enhanced by BMP4 and WNT3A, and reduced by Activin. We generated a suite of deletions in the locus by CRISPR-Cas9 editing. We show EVX1 is a critical downstream effector of BMP4 and WNT3A with respect to patterning of posterior mesoderm. The lncRNA, Evx1as arises from alternative promoters and is difficult to fully abrogate by gene editing or siRNA approaches. Nevertheless, we were able to generate a large 2.6 kb deletion encompassing the shared promoter with Evx1 and multiple additional exons of Evx1as. This led to an identical dorsal-ventral patterning defect to that generated by micro-deletion in the DNA-binding domain of EVX1. Thus, Evx1as has no function independent of EVX1, and is therefore unlikely to act in trans. We predict many antisense lncRNAs have no specific trans function, possibly only regulating the linked coding genes in cis. PMID:27226347

  14. Limited Global Diversity of the Plasmodium vivax Merozoite Surface Protein 4 Gene

    PubMed Central

    Putaporntip, Chaturong; Jongwutiwes, Somchai; Ferreira, Marcelo U.; Kanbara, Hiroji; Udomsangpetch, Rachanee; Cui, Liwang

    2009-01-01

    Merozoite surface proteins (MSPs) of the malaria parasites are major candidates for vaccine development targeting asexual blood stages. However, the diverse antigenic repertoire of these antigens that induce strain-specific protective immunity in human is a major challenge for vaccine design and often determines the efficacy of a vaccine. Here we further assessed the genetic diversity of Plasmodium vivax MSP4 (PvMSP4) protein using 195 parasite samples collected mostly from Thailand, Indonesia and Brazil. Overall, PvMSP4 is highly conserved with only eight amino acid substitutions. The majority of the haplotype diversity was restricted to the two short tetrapeptide repeat arrays in exon 1 and 2, respectively. Selection and neutrality tests indicated that exon 1 and the entire coding region of PvMSP4 were under purifying selection. Despite the limited nucleotide polymorphism of PvMSP4, significant genetic differentiation among the three major parasite populations was detected. Moreover, microgeographical heterogeneity was also evident in the parasite populations from different endemic areas of Thailand. PMID:19409511

  15. Structural protein 4.1 is located in mammalian centrosomes

    PubMed Central

    Krauss, Sharon Wald; Chasis, Joel Anne; Rogers, Catherine; Mohandas, Narla; Krockmalnic, Gabriela; Penman, Sheldon

    1997-01-01

    Structural protein 4.1 was first characterized as an important 80-kDa protein in the mature red cell membrane skeleton. It is now known to be a member of a family of protein isoforms detected at diverse intracellular sites in many nucleated mammalian cells. We recently reported that protein 4.1 isoforms are present at interphase in nuclear matrix and are rearranged during the cell cycle. Here we report that protein 4.1 epitopes are present in centrosomes of human and murine cells and are detected by using affinity-purified antibodies specific for 80-kDa red cell 4.1 and for 4.1 peptides. Immunofluorescence, by both conventional and confocal microscopy, showed that protein 4.1 epitopes localized in the pericentriolar region. Protein 4.1 epitopes remained in centrosomes after extraction of cells with detergent, salt, and DNase. Higher resolution electron microscopy of detergent-extracted cell whole mounts showed centrosomal protein 4.1 epitopes distributed along centriolar cylinders and on pericentriolar fibers, at least some of which constitute the filamentous network surrounding each centriole. Double-label electron microscopy showed that protein 4.1 epitopes were predominately localized in regions also occupied by epitopes for centrosome-specific autoimmune serum 5051 but were not found on microtubules. Our results suggest that protein 4.1 is an integral component of centrosome structure, in which it may play an important role in centrosome function during cell division and organization of cellular architecture. PMID:9207085

  16. Characterization of zebrafish merlot/chablis as non-mammalian vertebrate models for severe congenital anemia due to protein 4.1 deficiency.

    PubMed

    Shafizadeh, Ebrahim; Paw, Barry H; Foott, Helen; Liao, Eric C; Barut, Bruce A; Cope, John J; Zon, Leonard I; Lin, Shuo

    2002-09-01

    The red blood cell membrane skeleton is an elaborate and organized network of structural proteins that interacts with the lipid bilayer and transmembrane proteins to maintain red blood cell morphology, membrane deformability and mechanical stability. A crucial component of red blood cell membrane skeleton is the erythroid specific protein 4.1R, which anchors the spectrin-actin based cytoskeleton to the plasma membrane. Qualitative and quantitative defects in protein 4.1R result in congenital red cell membrane disorders characterized by reduced cellular deformability and abnormal cell morphology. The zebrafish mutants merlot (mot) and chablis (cha) exhibit severe hemolytic anemia characterized by abnormal cell morphology and increased osmotic fragility. The phenotypic analysis of merlot indicates severe hemolysis of mutant red blood cells, consistent with the observed cardiomegaly, splenomegaly, elevated bilirubin levels and erythroid hyperplasia in the kidneys. The result of electron microscopic analysis demonstrates that mot red blood cells have membrane abnormalities and exhibit a severe loss of cortical membrane organization. Using positional cloning techniques and a candidate gene approach, we demonstrate that merlot and chablis are allelic and encode the zebrafish erythroid specific protein 4.1R. We show that mutant cDNAs from both alleles harbor nonsense point mutations, resulting in premature stop codons. This work presents merlot/chablis as the first characterized non-mammalian vertebrate models of hereditary anemia due to a defect in protein 4.1R integrity. PMID:12183387

  17. Protein 4.1, a multifunctional protein of the erythrocyte membrane skeleton: structure and functions in erythrocytes and nonerythroid cells.

    PubMed

    Takakuwa, Y

    2000-10-01

    Protein 4.1 of red blood cells (4.1R) is a multifunctional protein essential for maintaining erythrocyte shape and membrane mechanical properties, such as deformability and stability, through lateral interactions with spectrin and actin in the skeletal network and vertical interactions with cytoplasmic domains of transmembrane proteins, glycophorin C, and band 3. The primary stucture of the major 80-kd isoform of 4.1R has been elucidated, and on the basis of this identification, the functional domains and sites for binding partners have been clarified. Posttranslational modification of 4.1 R, such as phosphorylation and proteolysis, as well as binding of regulatory proteins including calmodulin-Ca2+ to 4.1R, modulates its interactions with other membrane proteins and, consequently, the membrane functions of red blood cells. Alternative splicing occurs in the 4.1R gene, and various isoforms are expressed not only in erythroid but also in nonerythroid cells. This review introduces current knowledge on biochemical, biophysical, genetic, and functional aspects of 4.1R and its family proteins, 4.1G (general type), 4.1B (brain type), and 4.1N (neuron type), recently identified in nonerythroid cells. PMID:11185985

  18. Protein 4.1 expression in the developing hair cells of the mouse inner ear.

    PubMed

    Okumura, Kazuhiro; Mochizuki, Eiji; Yokohama, Michinari; Yamakawa, Hisashi; Shitara, Hiroshi; Mburu, Philomena; Yonekawa, Hiromichi; Brown, Steve D M; Kikkawa, Yoshiaki

    2010-01-11

    Protein 4.1 (band 4.1 or 4.1R) was originally identified as an abundant protein of the human erythrocyte, in which it stabilizes the spectrin/actin cytoskeleton. Subsequently, several new family members, 4.1N, 4.1G and 4.1B, have been identified, which are expressed in many cell types, in particular at cell-cell junctions. We previously reported that 4.1R and 4.1N are expressed in the inner ear hair cells with specific localization patterns, and that 4.1R forms a complex with the membrane-associated guanylate kinase (MAGUK) protein p55 and two deafness gene products, myosin XV and whirlin. To determine the functions of the other family members, 4.1G and 4.1B, we observed their expression patterns in developing stereocilia in mice inner ear hair cells. 4.1G is expressed in the basal tapers of the stereocilia bundle in early postnatal stages. 4.1B was specifically and constantly expressed in the stereocilia tips during postnatal development. Additionally, we found that 4.1B is ablated in the hair cells of both myosin XV and whirlin mutant mice at all stages in hair cell development. These results suggest that 4.1 family members play important roles in the development and maintenance of the inner ear hair cells, and that 4.1B may be a member of the myosin XV-whirlin complex that is important for stereocilia maturation. PMID:19853587

  19. Fatty Acid Binding Protein 4 Deficiency Protects against Oxygen-Induced Retinopathy in Mice

    PubMed Central

    Saint-Geniez, Magali; Ghelfi, Elisa; Liang, Xiaoliang; Yu, Chenwei; Spencer, Carrie; Abend, Stephanie; Hotamisligil, Gokhan; Cataltepe, Sule

    2014-01-01

    Retinopathy of prematurity (ROP) is a leading cause of blindness in children worldwide due to increasing survival rates of premature infants. Initial suppression, followed by increased production of the retinal vascular endothelial growth factor-A (VEGF) expression are key events that trigger the pathological neovascularization in ROP. Fatty acid binding protein 4 (FABP4) is an intracellular lipid chaperone that is induced by VEGF in a subset of endothelial cells. FABP4 exhibits a pro-angiogenic function in cultured endothelial cells and in airway microvasculature, but whether it plays a role in modulation of retinal angiogenesis is not known. We hypothesized that FABP4 deficiency could ameliorate pathological retinal vascularization and investigated this hypothesis using a well-characterized mouse model of oxygen-induced retinopathy (OIR). We found that FABP4 was not expressed in retinal vessels, but was present in resident macrophages/microglial cells and endothelial cells of the hyaloid vasculature in the immature retina. While FABP4 expression was not required for normal development of retinal vessels, FABP4 expression was upregulated and localized to neovascular tufts in OIR. FABP4−/− mice demonstrated a significant decrease in neovessel formation as well as a significant improvement in physiological revascularization of the avascular retinal tissues. These alterations in retinal vasculature were accompanied by reduced endothelial cell proliferation, but no effect on apoptosis or macrophage/microglia recruitment. FABP4−/− OIR samples demonstrated decreased expression of genes involved in angiogenesis, such as Placental Growth Factor, and angiopoietin 2. Collectively, our findings suggest FABP4 as a potential target of pathologic retinal angiogenesis in proliferative retinopathies. PMID:24802082

  20. Fatty acid binding protein 4 deficiency protects against oxygen-induced retinopathy in mice.

    PubMed

    Saint-Geniez, Magali; Ghelfi, Elisa; Liang, Xiaoliang; Yu, Chenwei; Spencer, Carrie; Abend, Stephanie; Hotamisligil, Gokhan; Cataltepe, Sule

    2014-01-01

    Retinopathy of prematurity (ROP) is a leading cause of blindness in children worldwide due to increasing survival rates of premature infants. Initial suppression, followed by increased production of the retinal vascular endothelial growth factor-A (VEGF) expression are key events that trigger the pathological neovascularization in ROP. Fatty acid binding protein 4 (FABP4) is an intracellular lipid chaperone that is induced by VEGF in a subset of endothelial cells. FABP4 exhibits a pro-angiogenic function in cultured endothelial cells and in airway microvasculature, but whether it plays a role in modulation of retinal angiogenesis is not known. We hypothesized that FABP4 deficiency could ameliorate pathological retinal vascularization and investigated this hypothesis using a well-characterized mouse model of oxygen-induced retinopathy (OIR). We found that FABP4 was not expressed in retinal vessels, but was present in resident macrophages/microglial cells and endothelial cells of the hyaloid vasculature in the immature retina. While FABP4 expression was not required for normal development of retinal vessels, FABP4 expression was upregulated and localized to neovascular tufts in OIR. FABP4-/- mice demonstrated a significant decrease in neovessel formation as well as a significant improvement in physiological revascularization of the avascular retinal tissues. These alterations in retinal vasculature were accompanied by reduced endothelial cell proliferation, but no effect on apoptosis or macrophage/microglia recruitment. FABP4-/- OIR samples demonstrated decreased expression of genes involved in angiogenesis, such as Placental Growth Factor, and angiopoietin 2. Collectively, our findings suggest FABP4 as a potential target of pathologic retinal angiogenesis in proliferative retinopathies. PMID:24802082

  1. Nectin-like molecule 1 is a protein 4.1N associated protein and recruits protein 4.1N from cytoplasm to the plasma membrane.

    PubMed

    Zhou, Yan; Du, Guangwei; Hu, Xiaoyan; Yu, Shun; Liu, Yaobo; Xu, Yaqin; Huang, Xiaowei; Liu, Jin; Yin, Bin; Fan, Ming; Peng, Xiaozhong; Qiang, Boqin; Yuan, Jiangang

    2005-05-20

    Nectins are immunoglobulin superfamily adhesion molecules that participate in the organization of epithelial and endothelial junctions. Sharing high homology with the poliovirus receptor (PVR/CD155), nectins were also named poliovirus receptor-related proteins (PRRs). Four nectins and five nectin-like molecules have been identified. Here we describe the cloning and characterization of human and mouse nectin-like molecular 1 (NECL1). Human and mouse NECL1 share 87.3% identity at the amino acid level. NECL1 contains an ectodomain made of three immunoglobulin-like domains, and a cytoplasmic region homologous to those of glycophorin C and contactin-associated protein. RNA blot and in situ hybridization analysis showed that NECL1 predominantly expressed in the central nervous system, mainly in neuronal cell bodies in a variety of brain regions including the cerebellum, cerebral cortex and hippocampus. In vitro binding assay proved the association of NECL1 with protein 4.1N. NECL1 localizes to the cell-cell junctions and recruits protein 4.1N to the plasma membranes through its C-terminus, thus may regulate the function of the cell-cell junction. We propose that the NECL1 and protein 4.1N complex is involved in the morphological development, stability, and dynamic plasticity of the nervous system. PMID:15893517

  2. Ca(2+)-dependent and Ca(2+)-independent calmodulin binding sites in erythrocyte protein 4.1. Implications for regulation of protein 4.1 interactions with transmembrane proteins.

    PubMed

    Nunomura, W; Takakuwa, Y; Parra, M; Conboy, J G; Mohandas, N

    2000-03-01

    In vitro protein binding assays identified two distinct calmodulin (CaM) binding sites within the NH(2)-terminal 30-kDa domain of erythrocyte protein 4.1 (4.1R): a Ca(2+)-independent binding site (A(264)KKLWKVCVEHHTFFRL) and a Ca(2+)-dependent binding site (A(181)KKLSMYGVDLHKAKDL). Synthetic peptides corresponding to these sequences bound CaM in vitro; conversely, deletion of these peptides from a 30-kDa construct reduced binding to CaM. Thus, 4.1R is a unique CaM-binding protein in that it has distinct Ca(2+)-dependent and Ca(2+)-independent high affinity CaM binding sites. CaM bound to 4.1R at a stoichiometry of 1:1 both in the presence and absence of Ca(2+), implying that one CaM molecule binds to two distinct sites in the same molecule of 4.1R. Interactions of 4.1R with membrane proteins such as band 3 is regulated by Ca(2+) and CaM. While the intrinsic affinity of the 30-kDa domain for the cytoplasmic tail of erythrocyte membrane band 3 was not altered by elimination of one or both CaM binding sites, the ability of Ca(2+)/CaM to down-regulate 4. 1R-band 3 interaction was abrogated by such deletions. Thus, regulation of protein 4.1 binding to membrane proteins by Ca(2+) and CaM requires binding of CaM to both Ca(2+)-independent and Ca(2+)-dependent sites in protein 4.1. PMID:10692436

  3. The Protein 4.1 family: hub proteins in animals for organizing membrane proteins.

    PubMed

    Baines, Anthony J; Lu, Hui-Chun; Bennett, Pauline M

    2014-02-01

    Proteins of the 4.1 family are characteristic of eumetazoan organisms. Invertebrates contain single 4.1 genes and the Drosophila model suggests that 4.1 is essential for animal life. Vertebrates have four paralogues, known as 4.1R, 4.1N, 4.1G and 4.1B, which are additionally duplicated in the ray-finned fish. Protein 4.1R was the first to be discovered: it is a major mammalian erythrocyte cytoskeletal protein, essential to the mechanochemical properties of red cell membranes because it promotes the interaction between spectrin and actin in the membrane cytoskeleton. 4.1R also binds certain phospholipids and is required for the stable cell surface accumulation of a number of erythrocyte transmembrane proteins that span multiple functional classes; these include cell adhesion molecules, transporters and a chemokine receptor. The vertebrate 4.1 proteins are expressed in most tissues, and they are required for the correct cell surface accumulation of a very wide variety of membrane proteins including G-Protein coupled receptors, voltage-gated and ligand-gated channels, as well as the classes identified in erythrocytes. Indeed, such large numbers of protein interactions have been mapped for mammalian 4.1 proteins, most especially 4.1R, that it appears that they can act as hubs for membrane protein organization. The range of critical interactions of 4.1 proteins is reflected in disease relationships that include hereditary anaemias, tumour suppression, control of heartbeat and nervous system function. The 4.1 proteins are defined by their domain structure: apart from the spectrin/actin-binding domain they have FERM and FERM-adjacent domains and a unique C-terminal domain. Both the FERM and C-terminal domains can bind transmembrane proteins, thus they have the potential to be cross-linkers for membrane proteins. The activity of the FERM domain is subject to multiple modes of regulation via binding of regulatory ligands, phosphorylation of the FERM associated domain and

  4. Gene therapy for bone healing

    PubMed Central

    Evans, Christopher H.

    2015-01-01

    Clinical problems in bone healing include large segmental defects, nonunion and delayed union of fractures, and spinal fusions. Gene-transfer technologies have the potential to aid healing by permitting the local delivery and sustained expression of osteogenic gene products within osseous lesions. Key questions for such an approach include the choice of transgene, vector and gene-transfer strategy. Most experimental data have been obtained using cDNAs encoding osteogenic growth factors such as bone morphogenetic protein-2 (BMP-2), BMP-4 and BMP-7, in conjunction with both nonviral and viral vectors using in vivo and ex vivo delivery strategies. Proof of principle has been convincingly demonstrated in small-animal models. Relatively few studies have used large animals, but the results so far are encouraging. Once a reliable method has been developed, it will be necessary to perform detailed pharmacological and toxicological studies, as well as satisfy other demands of the regulatory bodies, before human clinical trials can be initiated. Such studies are very expensive and often protracted. Thus, progress in developing a clinically useful gene therapy for bone healing is determined not only by scientific considerations, but also by financial constraints and the ambient regulatory environment. PMID:20569532

  5. Increased expression of fatty acid binding protein 4 and leptin in resident macrophages characterises atherosclerotic plaque rupture

    PubMed Central

    Lee, K.; Santibanez-Koref, M.; Polvikoski, T.; Birchall, D.; Mendelow, A.D.; Keavney, B.

    2013-01-01

    Objective Resident macrophages play an important role in atheromatous plaque rupture. The macrophage gene expression signature associated with plaque rupture is incompletely defined due to the complex cellular heterogeneity in the plaque. We aimed to characterise differential gene expression in resident plaque macrophages from ruptured and stable human atheromatous lesions. Methods and results We performed genome-wide expression analyses of isolated macrophage-rich regions of stable and ruptured human atherosclerotic plaques. Plaques present in carotid endarterectomy specimens were designated as stable or ruptured using clinical, radiological and histopathological criteria. Macrophage-rich regions were excised from 5 ruptured and 6 stable plaques by laser micro-dissection. Transcriptional profiling was performed using Affymetrix microarrays. The profiles were characteristic of activated macrophages. At a false discovery rate of 10%, 914 genes were differentially expressed between stable and ruptured plaques. The findings were confirmed in fourteen further stable and ruptured samples for a subset of eleven genes with the highest expression differences (p < 0.05). Pathway analysis revealed that components of the PPAR/Adipocytokine signaling pathway were the most significantly upregulated in ruptured compared to stable plaques (p = 5.4 × 10−7). Two key components of the pathway, fatty-acid binding-protein 4 (FABP4) and leptin, showed nine-fold (p = 0.0086) and five-fold (p = 0.0012) greater expression respectively in macrophages from ruptured plaques. Conclusions We found differences in gene expression signatures between macrophages isolated from stable and ruptured human atheromatous plaques. Our findings indicate the involvement of FABP4 and leptin in the progression of atherosclerosis and plaque rupture, and suggest that down-regulation of PPAR/adipocytokine signaling within plaques may have therapeutic potential. PMID:23122912

  6. SynCAM1 recruits NMDA receptors via protein 4.1B.

    PubMed

    Hoy, Jennifer L; Constable, John R; Vicini, Stefano; Fu, Zhanyan; Washbourne, Philip

    2009-12-01

    Cell adhesion molecules have been implicated as key organizers of synaptic structures, but there is still a need to determine how these molecules facilitate neurotransmitter receptor recruitment to developing synapses. Here, we identify erythrocyte protein band 4.1-like 3 (protein 4.1B) as an intracellular effector molecule of Synaptic Cell Adhesion Molecule 1 (SynCAM1) that is sufficient to recruit NMDA-type receptors (NMDARs) to SynCAM1 adhesion sites in COS7 cells. Protein 4.1B in conjunction with SynCAM1 also increased the frequency of NMDAR-mediated mEPSCs and area of presynaptic contact in an HEK293 cell/ neuron co-culture assay. Studies in cultured hippocampal neurons reveal that manipulation of protein 4.1B expression levels specifically affects NMDAR-mediated activity and localization. Finally, further experimentation in COS7 cells show that SynCAM1 may also interact with protein 4.1N to specifically effect AMPA type receptor (AMPAR) recruitment. Thus, SynCAM1 may recruit both AMPARs and NMDARs by independent mechanisms during synapse formation. PMID:19796685

  7. CASK and protein 4.1 support F-actin nucleation on neurexins.

    PubMed

    Biederer, T; Sudhof, T C

    2001-12-21

    Rearrangements of the actin cytoskeleton are involved in a variety of cellular processes from locomotion of cells to morphological alterations of the cell surface. One important question is how local interactions of cells with the extracellular space are translated into alterations of their membrane organization. To address this problem, we studied CASK, a member of the membrane-associated guanylate kinase homologues family of adaptor proteins. CASK has been shown to bind the erythrocyte isoform of protein 4.1, a class of proteins that promote formation of actin/spectrin microfilaments. In neurons, CASK also interacts via its PDZ domain with the cytosolic C termini of neurexins, neuron-specific cell-surface proteins. We now show that CASK binds a brain-enriched isoform of protein 4.1, and nucleates local assembly of actin/spectrin filaments. These interactions can be reconstituted on the cytosolic tail of neurexins. Furthermore, CASK can be recovered with actin filaments prepared from rat brain extracts, and neurexins are recruited together with CASK and protein 4.1 into these actin filaments. Thus, analogous to the PDZ-domain protein p55 and glycophorin C at the erythrocyte membrane, a similar complex comprising CASK and neurexins exists in neurons. Our data suggest that intercellular junctions formed by neurexins, such as junctions initiated by beta-neurexins with neuroligins, are at least partially coupled to the actin cytoskeleton via an interaction with CASK and protein 4.1. PMID:11604393

  8. Protein 4.1 tumor suppressors: getting a FERM grip on growth regulation.

    PubMed

    Sun, Chun-Xiao; Robb, Victoria A; Gutmann, David H

    2002-11-01

    Members of the Protein 4.1 superfamily have highly conserved FERM domains that link cell surface glycoproteins to the actin cytoskeleton. Within this large and constantly expanding superfamily, at least five subgroups have been proposed. Two of these subgroups, the ERM and prototypic Protein 4.1 molecules, include proteins that function as tumor suppressors. The ERM subgroup member merlin/schwannomin is inactivated in the tumor-predisposition syndrome neurofibromatosis 2 (NF2), and the prototypic 4.1 subgroup member, Protein 4.1B, has been implicated in the molecular pathogenesis of breast, lung and brain cancers. This review focuses on what is known of mechanisms of action and critical protein interactions that may mediate the unique growth inhibitory signals of these two Protein 4.1 tumor suppressors. On the basis of insights derived from studying the NF2 tumor suppressor, we propose a model for merlin growth regulation in which CD44 links growth signals from plasma membrane to the nucleus by interacting with ERM proteins and merlin. PMID:12356905

  9. A Nonerythroid Isoform of Protein 4.1R Interacts with the Nuclear Mitotic Apparatus (NuMA) Protein

    PubMed Central

    Mattagajasingh, Subhendra N.; Huang, Shu-Ching; Hartenstein, Julia S.; Snyder, Michael; Marchesi, Vincent T.; Benz, Edward J.

    1999-01-01

    Red blood cell protein 4.1 (4.1R) is an 80- kD erythrocyte phosphoprotein that stabilizes the spectrin/actin cytoskeleton. In nonerythroid cells, multiple 4.1R isoforms arise from a single gene by alternative splicing and predominantly code for a 135-kD isoform. This isoform contains a 209 amino acid extension at its NH2 terminus (head piece; HP). Immunoreactive epitopes specific for HP have been detected within the cell nucleus, nuclear matrix, centrosomes, and parts of the mitotic apparatus in dividing cells. Using a yeast two-hybrid system, in vitro binding assays, coimmunolocalization, and coimmunoprecipitation studies, we show that a 135-kD 4.1R isoform specifically interacts with the nuclear mitotic apparatus (NuMA) protein. NuMA and 4.1R partially colocalize in the interphase nucleus of MDCK cells and redistribute to the spindle poles early in mitosis. Protein 4.1R associates with NuMA in the interphase nucleus and forms a complex with spindle pole organizing proteins, NuMA, dynein, and dynactin during cell division. Overexpression of a 135-kD isoform of 4.1R alters the normal distribution of NuMA in the interphase nucleus. The minimal sequence sufficient for this interaction has been mapped to the amino acids encoded by exons 20 and 21 of 4.1R and residues 1788–1810 of NuMA. Our results not only suggest that 4.1R could, possibly, play an important role in organizing the nuclear architecture, mitotic spindle, and spindle poles, but also could define a novel role for its 22–24-kD domain. PMID:10189366

  10. The molecular signature of AML mesenchymal stromal cells reveals candidate genes related to the leukemogenic process.

    PubMed

    Binato, Renata; de Almeida Oliveira, Nathalia Correa; Du Rocher, Barbara; Abdelhay, Eliana

    2015-12-01

    Acute myeloid leukemia (AML) is a heterogeneous disease characterized by myeloid precursor proliferation in the bone marrow, apoptosis reduction and differentiation arrest. Although there are several studies in this field, events related to disease initiation and progression remain unknown. The malignant transformation of hematopoietic stem cells (HSC) is thought to generate leukemic stem cells, and this transformation could be related to changes in mesenchymal stromal cell (hMSC) signaling. Thus, the aim of this work was to analyze the gene expression profile of hMSC from AML patients (hMSC-AML) compared to healthy donors hMSCs (hMSC-HD). The results showed a common molecular signature for all hMSC-AML. Other assays were performed with a large number of patients and the results confirmed a molecular signature that is capable of distinguishing hMSC-AML from hMSC-HD. Moreover, CCL2 and BMP4 genes encode secreted proteins that could affect HSCs. To verify whether these proteins are differentially expressed in AML patients, ELISA was performed with plasma samples. CCL2 and BMP4 proteins are differentially expressed in AML patients, indicating changes in hMSC-AML signaling. Altogether, hMSCs-AML signaling alterations could be an important factor in the leukemic transformation process. PMID:26279521

  11. Bone morphogenetic protein 4 and retinoic acid trigger bovine VASA homolog expression in differentiating bovine induced pluripotent stem cells.

    PubMed

    Malaver-Ortega, Luis F; Sumer, Huseyin; Jain, Kanika; Verma, Paul J

    2016-02-01

    Primordial germ cells (PGCs) are the earliest identifiable and completely committed progenitors of female and male gametes. They are obvious targets for genome editing because they assure the transmission of desirable or introduced traits to future generations. PGCs are established at the earliest stages of embryo development and are difficult to propagate in vitro--two characteristics that pose a problem for their practical application. One alternative method to enrich for PGCs in vitro is to differentiate them from pluripotent stem cells derived from adult tissues. Here, we establish a reporter system for germ cell identification in bovine pluripotent stem cells based on green fluorescent protein expression driven by the minimal essential promoter of the bovine Vasa homolog (BVH) gene, whose regulatory elements were identified by orthologous modelling of regulatory units. We then evaluated the potential of bovine induced pluripotent stem cell (biPSC) lines carrying the reporter construct to differentiate toward the germ cell lineage. Our results showed that biPSCs undergo differentiation as embryoid bodies, and a fraction of the differentiating cells expressed BVH. The rate of differentiation towards BVH-positive cells increased up to tenfold in the presence of bone morphogenetic protein 4 or retinoic acid. Finally, we determined that the expression of key PGC genes, such as BVH or SOX2, can be modified by pre-differentiation cell culture conditions, although this increase is not necessarily mirrored by an increase in the rate of differentiation. PMID:26660942

  12. Antagonizing the Spemann organizer: role of the homeobox gene Xvent-1.

    PubMed Central

    Gawantka, V; Delius, H; Hirschfeld, K; Blumenstock, C; Niehrs, C

    1995-01-01

    We have identified a novel homeobox gene, Xvent-1, that is differentially expressed in the ventral marginal zone of the early Xenopus gastrula. Evidence is presented from mRNA microinjection experiments for a role for this gene in dorsoventral patterning of mesoderm. First, Xvent-1 is induced by BMP-4, a gene known to be a key regulator of ventral mesoderm development. Second, Xvent-1 and the organizer-specific gene goosecoid are able to interact, directly or indirectly, in a cross-regulatory loop suppressing each other's expression, consistent with their mutually exclusive expression in the marginal zone. Third, microinjection of Xvent-1 mRNA ventralizes dorsal mesoderm. The results suggest that Xvent-1 functions in a ventral signaling pathway that maintains the ventral mesodermal state and antagonizes the Spemann organizer. Images PMID:8557046

  13. Loss of DAL-1, a protein 4.1-related tumor suppressor, is an important early event in the pathogenesis of meningiomas.

    PubMed

    Gutmann, D H; Donahoe, J; Perry, A; Lemke, N; Gorse, K; Kittiniyom, K; Rempel, S A; Gutierrez, J A; Newsham, I F

    2000-06-12

    Meningiomas are common nervous system tumors, whose molecular pathogenesis is poorly understood. To date, the most frequent genetic alteration detected in these tumors is loss of heterozygosity (LOH) on chromosome 22q. This finding led to the identification of the neurofibromatosis 2 (NF2) tumor suppressor gene on 22q12, which is inactivated in 40% of sporadic meningiomas. The NF2 gene product, merlin (or schwannomin), is a member of the protein 4.1 family of membrane-associated proteins, which also includes ezrin, radixin and moesin. Recently, we identified another protein 4.1 gene, DAL-1 (differentially expressed in adenocarcinoma of the lung) located on chromosome 18p11.3, which is lost in approximately 60% of non-small cell lung carcinomas, and exhibits growth-suppressing properties in lung cancer cell lines. Given the homology between DAL-1 and NF2 and the identification of significant LOH in the region of DAL-1 in lung, breast and brain tumors, we investigated the possibility that loss of expression of DAL-1 was important for meningioma development. In this report, we demonstrate DAL-1 loss in 60% of sporadic meningiomas using LOH, RT-PCR, western blot and immunohistochemistry analyses. Analogous to merlin, we show that DAL-1 loss is an early event in meningioma tumorigenesis, suggesting that these two protein 4.1 family members are critical growth regulators in the pathogenesis of meningiomas. Furthermore, our work supports the emerging notion that membrane-associated alterations are important in the early stages of neoplastic transformation and the study of such alterations may elucidate the mechanism of tumorigenesis shared by other tumor types. PMID:10888600

  14. Identification and Characterization of a Non-retinoid Ligand for Retinol-binding Protein 4 Which Lowers Serum Retinol-binding Protein 4 Levels in Vivo*

    PubMed Central

    Motani, Alykhan; Wang, Zhulun; Conn, Marion; Siegler, Karen; Zhang, Ying; Liu, Qingxiang; Johnstone, Sheree; Xu, Haoda; Thibault, Steve; Wang, Yingcai; Fan, Pingchen; Connors, Richard; Le, Hoa; Xu, Guifen; Walker, Nigel; Shan, Bei; Coward, Peter

    2009-01-01

    Retinol-binding protein 4 (RBP4) transports retinol from the liver to extrahepatic tissues, and RBP4 lowering is reported to improve insulin sensitivity in mice. We have identified A1120, a high affinity (Ki = 8.3 nm) non-retinoid ligand for RBP4, which disrupts the interaction between RBP4 and its binding partner transthyretin. Analysis of the RBP4-A1120 co-crystal structure reveals that A1120 induces critical conformational changes at the RBP4-transthyretin interface. Administration of A1120 to mice lowers serum RBP4 and retinol levels but, unexpectedly, does not improve insulin sensitivity. In addition, we show that Rpb4-/- mice display normal insulin sensitivity and are not protected from high fat diet-induced insulin resistance. We conclude that lowering RBP4 levels does not improve insulin sensitivity in mice. Therefore, RBP4 lowering may not be an effective strategy for treating diabetes. PMID:19147488

  15. The carboxy-terminus of protein 4.1r resembles Beta-tubulin.

    PubMed

    Fant, Xavier; Merdes, Andreas

    2002-01-01

    The protein 4.1R is an isoform of a larger family of 4.1 proteins. It is known as a component of the plasma membrane skeleton, but it is also found at the centrosomes in interphase and mitosis. To investigate the properties of the carboxy terminal region of protein 4.1R, we raised antibodies against a peptide representing the last 14 amino acids of 4.1R. These antibodies crossreact with an epitope in beta-tubulin and stain the microtubule network by immunofluorescence. Furthermore, sequence comparison of the carboxy terminal 4.1R peptide sequence with tubulin reveals homology with a region at the end of helix 5 in beta-tubulin, but not alpha-tubulin. A potential function of the 4.1R carboxy terminus in regulating the formation of microtubule networks is discussed. PMID:11991667

  16. Protein 4.1R core domain structure and insights into regulation of cytoskeletal organization.

    PubMed

    Han, B G; Nunomura, W; Takakuwa, Y; Mohandas, N; Jap, B K

    2000-10-01

    The crystal structure of the core domain (N-terminal 30 kDa domain) of cytoskeletal protein 4.1R has been determined and shows a cloverleaf-like architecture. Each lobe of the cloverleaf contains a specific binding site for either band 3, glycophorin C/D or p55. At a central region of the molecule near where the three lobes are joined are two separate calmodulin (CaM) binding regions. One of these is composed primarily of an alpha-helix and is Ca 2+ insensitive; the other takes the form of an extended structure and its binding with CaM is dramatically enhanced by the presence of Ca 2+, resulting in the weakening of protein 4.1R binding to its target proteins. This novel architecture, in which the three lobes bind with three membrane associated proteins, and the location of calmodulin binding sites provide insight into how the protein 4.1R core domain interacts with membrane proteins and dynamically regulates cell shape in response to changes in intracellular Ca2+ levels. PMID:11017195

  17. Two distinct domains of protein 4.1 critical for assembly of functional nuclei in vitro.

    PubMed

    Krauss, Sharon Wald; Heald, Rebecca; Lee, Gloria; Nunomura, Wataru; Gimm, J Aura; Mohandas, Narla; Chasis, Joel Anne

    2002-11-15

    Protein 4.1R, a multifunctional structural protein, acts as an adaptor in mature red cell membrane skeletons linking spectrin-actin complexes to plasma membrane-associated proteins. In nucleated cells protein 4.1 is not associated exclusively with plasma membrane but is also detected at several important subcellular locations crucial for cell division. To identify 4.1 domains having critical functions in nuclear assembly, 4.1 domain peptides were added to Xenopus egg extract nuclear reconstitution reactions. Morphologically disorganized, replication deficient nuclei assembled when spectrin-actin-binding domain or NuMA-binding C-terminal domain peptides were present. However, control variant spectrin-actin-binding domain peptides incapable of binding actin or mutant C-terminal domain peptides with reduced NuMA binding had no deleterious effects on nuclear reconstitution. To test whether 4.1 is required for proper nuclear assembly, 4.1 isoforms were depleted with spectrin-actin binding or C-terminal domain-specific antibodies. Nuclei assembled in the depleted extracts were deranged. However, nuclear assembly could be rescued by the addition of recombinant 4.1R. Our data establish that protein 4.1 is essential for nuclear assembly and identify two distinct 4.1 domains, initially characterized in cytoskeletal interactions, that have crucial and versatile functions in nuclear assembly. PMID:12171917

  18. A fucoidan from Nemacystus decipiens disrupts angiogenesis through targeting bone morphogenetic protein 4.

    PubMed

    Wang, Wucheng; Chen, Huanjun; Zhang, Lei; Qin, Yi; Cong, Qifei; Wang, Peipei; Ding, Kan

    2016-06-25

    A sulfated and acetylated fucoidan, named NDH01, was extracted from seaweed Nemacystus decipiens. NDH01 was composed of mannose, glucuronic acid, fucose, sulfate group and acetyl group in the molar ratio of 3.0: 14.4: 82.6: 34.3: 13.9. The backbone of NDH01 was fucose-free core, composed of α-d-1,2-Manp and β-d-1,4-GlcpA disaccharide repeat unit. The branches were attached at the C3, C4 and C6 of α-d-1,2-Manp. The sidechain was composed of α-l-1,3,4-Fucp, α-l-1,4-Fucp, α-l-1,3-Fucp and α-l-1,4-GlcpA. The sulfate group was linked to C4 of α-l-1,3,4-Fucp, whereas, acetyl group was branched on C2 of α-l-1,2,3-Fucp. NDH01 could disrupt tube formation and inhibit the migration as well as cell growth of human microvascular endothelial cells. Besides, phosphorylation of Smad/1/5/8, Erk and FAK was significantly inhibited by NDH01. Further studies uncovered that NDH01 blocked Smad1/5/8 signaling via interacting with bone morphogenetic protein 4 and downregulating bone morphogenetic protein 4 expression. The results suggested that NDH01 might be an angiogenesis inhibitor through targeting bone morphogenetic protein 4. PMID:27083822

  19. Fatty Acid-binding Protein 4, a Point of Convergence for Angiogenic and Metabolic Signaling Pathways in Endothelial Cells*

    PubMed Central

    Harjes, Ulrike; Bridges, Esther; McIntyre, Alan; Fielding, Barbara A.; Harris, Adrian L.

    2014-01-01

    Fatty acid-binding protein 4 (FABP4) is an adipogenic protein and is implicated in atherosclerosis, insulin resistance, and cancer. In endothelial cells, FABP4 is induced by VEGFA, and inhibition of FABP4 blocks most of the VEGFA effects. We investigated the DLL4-NOTCH-dependent regulation of FABP4 in human umbilical vein endothelial cells by gene/protein expression and interaction analyses following inhibitor treatment and RNA interference. We found that FABP4 is directly induced by NOTCH. Stimulation of NOTCH signaling with human recombinant DLL4 led to FABP4 induction, independently of VEGFA. FABP4 induction by VEGFA was reduced by blockade of DLL4 binding to NOTCH or inhibition of NOTCH signal transduction. Chromatin immunoprecipitation of the NOTCH intracellular domain showed increased binding to two specific regions in the FABP4 promoter. The induction of FABP4 gene expression was dependent on the transcription factor FOXO1, which was essential for basal expression of FABP4, and FABP4 up-regulation following stimulation of the VEGFA and/or the NOTCH pathway. Thus, we show that the DLL4-NOTCH pathway mediates endothelial FABP4 expression. This indicates that induction of the angiogenesis-restricting DLL4-NOTCH can have pro-angiogenic effects via this pathway. It also provides a link between DLL4-NOTCH and FOXO1-mediated regulation of endothelial gene transcription, and it shows that DLL4-NOTCH is a nodal point in the integration of pro-angiogenic and metabolic signaling in endothelial cells. This may be crucial for angiogenesis in the tumor environment. PMID:24939870

  20. Hypoxia upregulates Rab11-family interacting protein 4 through HIF-1α to promote the metastasis of hepatocellular carcinoma.

    PubMed

    Hu, F; Deng, X; Yang, X; Jin, H; Gu, D; Lv, X; Wang, C; Zhang, Y; Huo, X; Shen, Q; Luo, Q; Zhao, F; Ge, T; Zhao, F; Chu, W; Shu, H; Yao, M; Fan, J; Qin, W

    2015-12-01

    Hypoxic microenvironment is a powerful driving force for the invasion and metastasis of hepatocellular carcinoma (HCC). Hypoxia-inducible factor 1α (HIF-1α), as a crucial regulator of transcriptional responses to hypoxia, induces the expression of multiple target genes involved in different steps of HCC metastatic process. It is critical to find target genes associated with metastasis under hypoxia for shedding new light on molecular mechanism of HCC metastasis. In this study, we uncovered that hypoxia could induce the upregulation of Rab11-family interacting protein 4 (Rab11-FIP4) and activation of Rab11-FIP4 promoter by HIF-1α. The overexpression of Rab11-FIP4 significantly enhanced the mobility and invasiveness of HCC cells in vitro, also contributed to distant lung metastasis in vivo, whereas silencing of Rab11-FIP4 decreased the ability of migration and invasion in HCC cells in vitro and suppressed lung metastasis in vivo. Rab11-FIP4 facilitated HCC metastasis through the phosphorylation of PRAS40, which was regulated by mTOR. Furthermore, the expression level of Rab11-FIP4 was significantly increased in HCC tissues and high expression of Rab11-FIP4 was closely correlated with vascular invasion and poor prognosis in HCC patients. A markedly positive correlation between the expression of Rab11-FIP4 and HIF-1α was observed in HCC tissues and combination of Rab11-FIP4 and HIF-1α was a more valuable predictor of poor prognosis for HCC patients. In conclusion, Rab11-FIP4 is a target gene of HIF-1α and has a pro-metastatic role in HCC, suggesting that Rab11-FIP4 may be a promising candidate target for HCC treatment. PMID:25745995

  1. Cardiac muscle cell cytoskeletal protein 4.1: analysis of transcripts and subcellular location--relevance to membrane integrity, microstructure, and possible role in heart failure.

    PubMed

    Taylor-Harris, Pamela M; Keating, Lisa A; Maggs, Alison M; Phillips, Gareth W; Birks, Emma J; Franklin, Rodney C G; Yacoub, Magdi H; Baines, Anthony J; Pinder, Jennifer C

    2005-03-01

    The spectrin-based cytoskeleton assembly has emerged as a major player in heart functioning; however, cardiac protein 4.1, a key constituent, is uncharacterized. Protein 4.1 evolved to protect cell membranes against mechanical stresses and to organize membrane microstructure. 4.1 Proteins are multifunctional and, among other activities, link integral/signaling proteins on the plasma and internal membranes with the spectrin-based cytoskeleton. Four genes, EPB41, EPB41L1, EPB41L2, and EPB41L3 encode proteins 4.1R, 4.1N, 4.1G, and 4.1B, respectively. All are extensively spliced. Different isoforms are expressed according to tissue and developmental state, individual function being controlled through inclusion/exclusion of interactive domains. We have defined mouse and human cardiac 4.1 transcripts; other than 4. 1B in humans, all genes show activity. Cardiac transcripts constitutively include conserved FERM and C-terminal domains; both interact with membrane-bound signaling/transport/cell adhesion molecules. Variable splicing within and adjacent to the central spectrin/actin-binding domain enables regulation of cytoskeleton-binding activity. A novel heart-specific exon occurs in human 4.1G, but not in mouse. Immunofluorescence reveals 4.1 staining within mouse cardiomyocytes; thus, both at the plasma membrane and, interdigitated with sarcomeric myosin, across myofibrils in regions close to the sarcoplasmic reticulum. These are all regions to which spectrin locates. 4.1R in human heart shows similar distribution; however, there is limited plasma membrane staining. We conclude that cardiac 4.1s are highly regulated in their ability to crosslink plasma/integral cell membranes with the spectrin-actin cytoskeleton. We speculate that over the repetitive cycles of heart muscle contraction and relaxation, 4.1s are likely to locate, support, and coordinate functioning of key membrane-bound macromolecular assemblies. PMID:15834631

  2. D2 and D3 dopamine receptor cell surface localization mediated by interaction with protein 4.1N.

    PubMed

    Binda, Alicia V; Kabbani, Nadine; Lin, Ridwan; Levenson, Robert

    2002-09-01

    We identified protein 4.1N as a D2-like dopamine receptor-interacting protein in a yeast two-hybrid screen. Protein 4.1N is a neuronally enriched member of the 4.1 family of cytoskeletal proteins, which also includes protein 4.1R of erythrocytes and the 4.1G and 4.1B isoforms. The interaction of protein 4.1N was specific for the D2 and D3 dopamine receptors and was independently confirmed in pulldown and coimmunoprecipitation assays. Deletion mapping localized the site of dopamine receptor/protein 4.1N interaction to the N-terminal segment of the third intracellular domain of D2 and D3 receptors and the carboxyl-terminal domain of protein 4.1N. D2 and D3 receptors were also found to interact with the highly conserved carboxyl-terminal domain of proteins 4.1R, 4.1G, and 4.1B. Immunofluorescence studies show that protein 4.1N and D2 and D3 dopamine receptors are expressed at the plasma membrane of transfected human embryonic kidney 293 and mouse neuroblastoma Neuro2A cells. However, expression of D2 or D3 receptors with a protein 4.1N truncation fragment reduces the level of D2 and D3 receptor expression at the plasma membrane. These results suggest that protein 4.1N/dopamine receptor interaction is required for localization or stability of dopamine receptors at the neuronal plasma membrane. PMID:12181426

  3. Identification of RING finger protein 4 (RNF4) as a modulator of DNA demethylation through a functional genomics screen.

    PubMed

    Hu, Xiaoyi V; Rodrigues, Tânia M A; Tao, Haiyan; Baker, Robert K; Miraglia, Loren; Orth, Anthony P; Lyons, Gary E; Schultz, Peter G; Wu, Xu

    2010-08-24

    DNA methylation is an important epigenetic modification involved in transcriptional regulation, nuclear organization, development, aging, and disease. Although DNA methyltransferases have been characterized, the mechanisms for DNA demethylation remain poorly understood. Using a cell-based reporter assay, we performed a functional genomics screen to identify genes involved in DNA demethylation. Here we show that RNF4 (RING finger protein 4), a SUMO-dependent ubiquitin E3-ligase previously implicated in maintaining genome stability, plays a key role in active DNA demethylation. RNF4 reactivates methylation-silenced reporters and promotes global DNA demethylation. Rnf4 deficiency is embryonic lethal with higher levels of methylation in genomic DNA. Mechanistic studies show that RNF4 interacts with and requires the base excision repair enzymes TDG and APE1 for active demethylation. This activity appears to occur by enhancing the enzymatic activities that repair DNA G:T mismatches generated from methylcytosine deamination. Collectively, our study reveals a unique function for RNF4, which may serve as a direct link between epigenetic DNA demethylation and DNA repair in mammalian cells. PMID:20696907

  4. An Integrative Transcriptomic Analysis for Identifying Novel Target Genes Corresponding to Severity Spectrum in Spinal Muscular Atrophy

    PubMed Central

    Yang, Chung-Wei; Chen, Chien-Lin; Chou, Wei-Chun; Lin, Ho-Chen; Jong, Yuh-Jyh; Tsai, Li-Kai; Chuang, Chun-Yu

    2016-01-01

    Spinal muscular atrophy (SMA) is an inherited neuromuscular disease resulting from a recessive mutation in the SMN1 gene. This disease affects multiple organ systems with varying degrees of severity. Exploration of the molecular pathological changes occurring in different cell types in SMA is crucial for developing new therapies. This study collected 39 human microarray datasets from ArrayExpress and GEO databases to build an integrative transcriptomic analysis for recognizing novel SMA targets. The transcriptomic analysis was conducted through combining weighted correlation network analysis (WGCNA) for gene module detection, gene set enrichment analysis (GSEA) for functional categorization and filtration, and Cytoscape (visual interaction gene network analysis) for target gene identification. Seven novel target genes (Bmp4, Serpine1, Gata6, Ptgs2, Bcl2, IL6 and Cntn1) of SMA were revealed, and are all known in the regulation of TNFα for controlling neural, cardiac and bone development. Sequentially, the differentially expressed patterns of these 7 target genes in mouse tissues (e.g., spinal cord, heart, muscles and bone) were validated in SMA mice of different severities (pre-symptomatic, mildly symptomatic, and severely symptomatic). In severely symptomatic SMA mice, TNFα was up-regulated with attenuation of Bmp4 and increase of Serpine1 and Gata6 (a pathway in neural and cardiac development), but not in pre-symptomatic and mildly symptomatic SMA mice. The severely symptomatic SMA mice also had the elevated levels of Ptgs2 and Bcl2 (a pathway in skeletal development) as well as IL6 and Cntn1 (a pathway in nervous system development). Thus, the 7 genes identified in this study might serve as potential target genes for future investigations of disease pathogenesis and SMA therapy. PMID:27331400

  5. An Integrative Transcriptomic Analysis for Identifying Novel Target Genes Corresponding to Severity Spectrum in Spinal Muscular Atrophy.

    PubMed

    Yang, Chung-Wei; Chen, Chien-Lin; Chou, Wei-Chun; Lin, Ho-Chen; Jong, Yuh-Jyh; Tsai, Li-Kai; Chuang, Chun-Yu

    2016-01-01

    Spinal muscular atrophy (SMA) is an inherited neuromuscular disease resulting from a recessive mutation in the SMN1 gene. This disease affects multiple organ systems with varying degrees of severity. Exploration of the molecular pathological changes occurring in different cell types in SMA is crucial for developing new therapies. This study collected 39 human microarray datasets from ArrayExpress and GEO databases to build an integrative transcriptomic analysis for recognizing novel SMA targets. The transcriptomic analysis was conducted through combining weighted correlation network analysis (WGCNA) for gene module detection, gene set enrichment analysis (GSEA) for functional categorization and filtration, and Cytoscape (visual interaction gene network analysis) for target gene identification. Seven novel target genes (Bmp4, Serpine1, Gata6, Ptgs2, Bcl2, IL6 and Cntn1) of SMA were revealed, and are all known in the regulation of TNFα for controlling neural, cardiac and bone development. Sequentially, the differentially expressed patterns of these 7 target genes in mouse tissues (e.g., spinal cord, heart, muscles and bone) were validated in SMA mice of different severities (pre-symptomatic, mildly symptomatic, and severely symptomatic). In severely symptomatic SMA mice, TNFα was up-regulated with attenuation of Bmp4 and increase of Serpine1 and Gata6 (a pathway in neural and cardiac development), but not in pre-symptomatic and mildly symptomatic SMA mice. The severely symptomatic SMA mice also had the elevated levels of Ptgs2 and Bcl2 (a pathway in skeletal development) as well as IL6 and Cntn1 (a pathway in nervous system development). Thus, the 7 genes identified in this study might serve as potential target genes for future investigations of disease pathogenesis and SMA therapy. PMID:27331400

  6. Morphological and Gene Expression Changes in Cattle Embryos from Hatched Blastocyst to Early Gastrulation Stages after Transfer of In Vitro Produced Embryos

    PubMed Central

    van Leeuwen, Jessica; Berg, Debra K.; Pfeffer, Peter L.

    2015-01-01

    A detailed morphological staging system for cattle embryos at stages following blastocyst hatching and preceding gastrulation is presented here together with spatiotemporal mapping of gene expression for BMP4, BRACHYURY, CERBERUS1 (CER1), CRIPTO, EOMESODERMIN, FURIN and NODAL. Five stages are defined based on distinct developmental events. The first of these is the differentiation of the visceral hypoblast underlying the epiblast, from the parietal hypoblast underlying the mural trophoblast. The second concerns the formation of an asymmetrically positioned, morphologically recognisable region within the visceral hypoblast that is marked by the presence of CER1 and absence of BMP4 expression. We have termed this the anterior visceral hypoblast or AVH. Intra-epiblast cavity formation and the disappearance of the polar trophoblast overlying the epiblast (Rauber’s layer) have been mapped in relation to AVH formation. The third chronological event involves the transition of the epiblast into the embryonic ectoderm with concomitant onset of posterior NODAL, EOMES and BRACHYURY expression. Lastly, gastrulation commences as the posterior medial embryonic ectoderm layer thickens to form the primitive streak and cells ingress between the embryonic ectoderm and hypoblast. At this stage a novel domain of CER1 expression is seen whereas the AVH disappears. Comparison with the mouse reveals that while gene expression patterns at the onset of gastrulation are well conserved, asymmetry establishment, which relies on extraembryonic tissues such as the hypoblast and trophoblast, has diverged in terms of both gene expression and morphology. PMID:26076128

  7. Effect of complete protein 4.1R deficiency on ion transportproperties of murine erythrocytes

    SciTech Connect

    Rivera, Alicia; De Franceschi, Lucia; Peters, Luanne L.; Gascard,Philippe; Mohandas, Narla; Brugnara, Carlo

    2006-06-02

    Moderate hemolytic anemia, abnormal erythrocyte morphology(spherocytosis), and decreased membrane stability are observed in micewith complete deficiency of all erythroid protein 4.1 protein isoforms(4.1-/-; Shi TS et al., J. Clin. Invest. 103:331,1999). We have examinedthe effects of erythroid protein 4.1 (4.1R) deficiency on erythrocytecation transport and volume regulation. 4.1-/- mice exhibited erythrocytedehydration that was associated with reduced cellular K and increased Nacontent. Increased Na permeability was observed in these mice, mostlymediated by Na/H exchange with normal Na-K pump and Na-K-2Cl cotransportactivities. The Na/H exchange of 4.1-/- erythrocytes was markedlyactivated by exposure to hypertonic conditions (18.2+- 3.2 in 4.1 -/- vs.9.8 +- 1.3 mmol/1013 cell x h in control mice), with an abnormaldependence on osmolarity, (K0.5=417 +- 42 in 4.1 -/- vs. 460 +- 35 mOsmin control mice) suggestive of an up-regulated functional state. Whilethe affinity for internal protons was not altered (K0.5= 489.7 +- 0.7 vs.537.0+- 0.56 nM in control mice), the Vmax of the H-induced Na/H exchangeactivity was markedly elevated in 4.1-/- erythrocytes (Vmax 91.47Moderatehemolytic anemia, abnormal erythrocyte morphology (spherocytosis), anddecreased membrane stability are observed in mice with completedeficiency of all erythroid protein 4.1 protein isoforms (4.1-/-; Shi TSet al., J. Clin. Invest. 103:331,1999). We have examined the effects oferythroid protein 4.1 (4.1R) deficiency on erythrocyte cation transportand volume regulation. 4.1-/- mice exhibited erythrocyte dehydration thatwas associated with reduced cellular K and increased Na content.Increased Na permeability was observed in these mice, mostly mediated byNa/H exchange with normal Na-K pump and Na-K-2Cl cotransport activities.The Na/H exchange of 4.1-/- erythrocytes was markedly activated byexposure to hypertonic conditions (18.2 +- 3.2 in 4.1 -/- vs. 9.8 +- 1.3mmol/1013 cell x h in control mice), with an

  8. Protein 4.1B Contributes to the Organization of Peripheral Myelinated Axons

    PubMed Central

    Devaux, Jérôme; Carnaud, Michèle; Levasseur, Grégoire; Niwa-Kawakita, Michiko; Harroch, Sheila; Girault, Jean-Antoine; Giovannini, Marco; Goutebroze, Laurence

    2011-01-01

    Neurons are characterized by extremely long axons. This exceptional cell shape is likely to depend on multiple factors including interactions between the cytoskeleton and membrane proteins. In many cell types, members of the protein 4.1 family play an important role in tethering the cortical actin-spectrin cytoskeleton to the plasma membrane. Protein 4.1B is localized in myelinated axons, enriched in paranodal and juxtaparanodal regions, and also all along the internodes, but not at nodes of Ranvier where are localized the voltage-dependent sodium channels responsible for action potential propagation. To shed light on the role of protein 4.1B in the general organization of myelinated peripheral axons, we studied 4.1B knockout mice. These mice displayed a mildly impaired gait and motility. Whereas nodes were unaffected, the distribution of Caspr/paranodin, which anchors 4.1B to the membrane, was disorganized in paranodal regions and its levels were decreased. In juxtaparanodes, the enrichment of Caspr2, which also interacts with 4.1B, and of the associated TAG-1 and Kv1.1, was absent in mutant mice, whereas their levels were unaltered. Ultrastructural abnormalities were observed both at paranodes and juxtaparanodes. Axon calibers were slightly diminished in phrenic nerves and preterminal motor axons were dysmorphic in skeletal muscle. βII spectrin enrichment was decreased along the axolemma. Electrophysiological recordings at 3 post-natal weeks showed the occurrence of spontaneous and evoked repetitive activity indicating neuronal hyperexcitability, without change in conduction velocity. Thus, our results show that in myelinated axons 4.1B contributes to the stabilization of membrane proteins at paranodes, to the clustering of juxtaparanodal proteins, and to the regulation of the internodal axon caliber. PMID:21966409

  9. Nuclear actin and protein 4.1: essential interactions during nuclear assembly in vitro.

    PubMed

    Krauss, Sharon Wald; Chen, Cynthia; Penman, Sheldon; Heald, Rebecca

    2003-09-16

    Structural protein 4.1, which has crucial interactions within the spectrin-actin lattice of the human red cell membrane skeleton, also is widely distributed at diverse intracellular sites in nucleated cells. We previously showed that 4.1 is essential for assembly of functional nuclei in vitro and that the capacity of 4.1 to bind actin is required. Here we report that 4.1 and actin colocalize in mammalian cell nuclei using fluorescence microscopy and, by higher-resolution detergent-extracted cell whole-mount electron microscopy, are associated on nuclear filaments. We also devised a cell-free assay using Xenopus egg extract containing fluorescent actin to follow actin during nuclear assembly. By directly imaging actin under nonperturbing conditions, the total nuclear actin population is retained and visualized in situ relative to intact chromatin. We detected actin initially when chromatin and nuclear pores began assembling. As nuclear lamina assembled, but preceding DNA synthesis, actin distributed in a reticulated pattern throughout the nucleus. Protein 4.1 epitopes also were detected when actin began to accumulate in nuclei, producing a diffuse coincident pattern. As nuclei matured, actin was detected both coincident with and also independent of 4.1 epitopes. To test whether acquisition of nuclear actin is required for nuclear assembly, the actin inhibitor latrunculin A was added to Xenopus egg extracts during nuclear assembly. Latrunculin A strongly perturbed nuclear assembly and produced distorted nuclear structures containing neither actin nor protein 4.1. Our results suggest that actin as well as 4.1 is necessary for nuclear assembly and that 4.1-actin interactions may be critical. PMID:12960380

  10. Nuclear actin and protein 4.1: Essential interactions during nuclear assembly in vitro

    PubMed Central

    Krauss, Sharon Wald; Chen, Cynthia; Penman, Sheldon; Heald, Rebecca

    2003-01-01

    Structural protein 4.1, which has crucial interactions within the spectrin–actin lattice of the human red cell membrane skeleton, also is widely distributed at diverse intracellular sites in nucleated cells. We previously showed that 4.1 is essential for assembly of functional nuclei in vitro and that the capacity of 4.1 to bind actin is required. Here we report that 4.1 and actin colocalize in mammalian cell nuclei using fluorescence microscopy and, by higher-resolution detergent-extracted cell whole-mount electron microscopy, are associated on nuclear filaments. We also devised a cell-free assay using Xenopus egg extract containing fluorescent actin to follow actin during nuclear assembly. By directly imaging actin under nonperturbing conditions, the total nuclear actin population is retained and visualized in situ relative to intact chromatin. We detected actin initially when chromatin and nuclear pores began assembling. As nuclear lamina assembled, but preceding DNA synthesis, actin distributed in a reticulated pattern throughout the nucleus. Protein 4.1 epitopes also were detected when actin began to accumulate in nuclei, producing a diffuse coincident pattern. As nuclei matured, actin was detected both coincident with and also independent of 4.1 epitopes. To test whether acquisition of nuclear actin is required for nuclear assembly, the actin inhibitor latrunculin A was added to Xenopus egg extracts during nuclear assembly. Latrunculin A strongly perturbed nuclear assembly and produced distorted nuclear structures containing neither actin nor protein 4.1. Our results suggest that actin as well as 4.1 is necessary for nuclear assembly and that 4.1–actin interactions may be critical. PMID:12960380

  11. Nuclear actin and protein 4.1: Essential interactions during nuclear assembly in vitro

    SciTech Connect

    Krauss, Sharon Wald; Chen, Cynthia; Penman, Sheldon; Heald, Rebecca

    2003-06-11

    Structural protein 4.1, which has crucial interactions within the spectin-actin lattice of the human red cell membrane skeleton, also is widely distributed at diverse intracellular sites in nucleated cells. We previously showed that 4.1 is essential for assembly of functional nuclei in vitro and that the capacity of 4.1 to bind actin is required. Here we report that 4.1 and actin colocalize in mammalian cell nuclei using fluorescence microscopy and, by higher resolution cell whole mount electron microscopy, are associated on nuclear filaments. We also devised a cell-free assay using Xenopus egg extract containing fluorescent actin to follow actin during nuclear assembly. By directly imaging actin under non-perturbing conditions, the total nuclear actin population is retained and is visualized in situ relative to intact chromatin. We detected actin initially when chromatin and nuclear pores began assembling. As the nuclear lamina assembled, but preceding DNA synthesis, a discrete actin network formed throughout the nucleus. Protein 4.1 epitopes also were detected when actin began to accumulate in nuclei, producing a diffuse coincident pattern. As nuclei matured, actin was detected both coincident with and also independent of 4.1 epitopes. To test whether acquisition of nuclear actin is required for nuclear assembly, the actin inhibitor latrunculin A was added to Xenopus egg extracts during nuclear assembly. Latrunculin A strongly perturbed nuclear assembly and produced distorted nuclear structures containing neither actin nor protein 4.1. Our results suggest that actin as well as 4.1 is necessary for nuclear assembly and that 4.1-actin interactions may be critical.

  12. Alternative 5' exons and differential splicing regulate expression of protein 4.1R isoforms with distinct N-termini.

    PubMed

    Parra, Marilyn K; Gee, Sherry L; Koury, Mark J; Mohandas, Narla; Conboy, John G

    2003-05-15

    Among the alternative pre-mRNA splicing events that characterize protein 4.1R gene expression, one involving exon 2' plays a critical role in regulating translation initiation and N-terminal protein structure. Exon 2' encompasses translation initiation site AUG1 and is located between alternative splice acceptor sites at the 5' end of exon 2; its inclusion or exclusion from mature 4.1R mRNA regulates expression of longer or shorter isoforms of 4.1R protein, respectively. The current study reports unexpected complexity in the 5' region of the 4.1R gene that directly affects alternative splicing of exon 2'. Identified far upstream of exon 2 in both mouse and human genomes were 3 mutually exclusive alternative 5' exons, designated 1A, 1B, and 1C; all 3 are associated with strong transcriptional promoters in the flanking genomic sequence. Importantly, exons 1A and 1B splice differentially with respect to exon 2', generating transcripts with different 5' ends and distinct N-terminal protein coding capacity. Exon 1A-type transcripts splice so as to exclude exon 2' and therefore utilize the downstream AUG2 for translation of 80-kDa 4.1R protein, whereas exon 1B transcripts include exon 2' and initiate at AUG1 to synthesize 135-kDa isoforms. RNA blot analyses revealed that 1A transcripts increase in abundance in late erythroblasts, consistent with the previously demonstrated up-regulation of 80-kDa 4.1R during terminal erythroid differentiation. Together, these results suggest that synthesis of structurally distinct 4.1R protein isoforms in various cell types is regulated by a novel mechanism requiring coordination between upstream transcription initiation events and downstream alternative splicing events. PMID:12522012

  13. Human epididymis protein 4 (HE4) plays a key role in ovarian cancer cell adhesion and motility

    SciTech Connect

    Lu, Renquan; Sun, Xinghui; Xiao, Ran; Zhou, Lei; Gao, Xiang; Guo, Lin

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer We generated stable transduced HE4 overexpression and knockdown cells. Black-Right-Pointing-Pointer HE4 was associated with EOC cell adhesion and motility. Black-Right-Pointing-Pointer HE4 might have some effects on activation of EGFR-MAPK signaling pathway. Black-Right-Pointing-Pointer HE4 play an important role in EOC tumorigenicity. -- Abstract: Human epididymis protein 4 (HE4) is a novel and specific biomarker for epithelial ovarian cancer (EOC). We previously demonstrated that serum HE4 levels were significantly elevated in the majority of EOC patients but not in subjects with benign disease or healthy controls. However, the precise mechanism of HE4 protein function is unknown. In this study, we generated HE4-overexpressing SKOV3 cells and found that stably transduced cells promoted cell adhesion and migration. Knockdown of HE4 expression was achieved by stable transfection of SKOV3 cells with a construct encoding a short hairpin DNA directed against the HE4 gene. Correspondingly, the proliferation and spreading ability of HE4-expressed cells were inhibited by HE4 suppression. Mechanistically, impaired EGFR and Erk1/2 phosphorylation were observed in cells with HE4 knockdown. The phosphorylation was restored when the knockdown cells were cultured in conditioned medium containing HE4. Moreover, in vivo tumorigenicity showed that HE4 suppression markedly inhibited the growth of tumors. This suggests that expression of HE4 is associated with cancer cell adhesion, migration and tumor growth, which can be related to its effects on the EGFR-MAPK signaling pathway. Our results provide evidence of the cellular and molecular mechanisms that may underlie the motility-promoting role of HE4 in EOC progression. The role of HE4 as a target for gene-based therapy might be considered in future studies.

  14. Alternative 5' exons and differential splicing regulate expression of protein 4.1R isoforms with distinct n-termini

    SciTech Connect

    Parra, Marilyn K.; Gee, Sherry L.; Koury, Mark J.; Mohandas, Narla; Conboy, John G.

    2003-03-25

    Among the alternative pre-mRNA splicing events that characterize protein 4.1R gene expression, one involving exon 2' plays a critical role in regulating translation initiation and N-terminal protein structure. Exon 2' encompasses translation initiation site AUG1 and is located between alternative splice acceptor sites at the 5' end of exon 2; its inclusion or exclusion from mature 4.1R mRNA regulates expression of longer or shorter isoforms of 4.1R protein, respectively. The current study reports unexpected complexity in the 5' region of the 4.1R gene that directly affects alternative splicing of exon 2'. Three mutually exclusive alternative 5' exons, designated 1A, 1B, and 1C, were identified far upstream of exon 2 in both mouse and human genomes; all three are associated with strong transcriptional promoters in the flanking genomic sequence. Importantly, exons 1A and 1B splice differentially with respect to exon 2', generating transcripts with different 5' ends and distinct N-terminal protein coding capacity. Exon 1A-type transcripts splice so as to exclude exon 2' and therefore utilize the downstream AUG2 for translation of 80kD 4.1R protein, whereas exon 1B transcripts include exon 2' and initiate at AUG1 to synthesize 135kD isoforms. RNA blot analyses revealed that 1A transcripts increase in abundance in late erythroblasts, consistent with the previously demonstrated upregulation of 80kD 4.1R during terminal erythroid differentiation. Together these results suggest that synthesis of structurally distinct 4.1R protein isoforms in various cell types is regulated by a novel mechanism requiring coordination between upstream transcription initiation events and downstream alternative splicing events.

  15. Protein 4.1-mediated membrane targeting of human discs large in epithelial cells.

    PubMed

    Hanada, Toshihiko; Takeuchi, Atsuko; Sondarva, Gautam; Chishti, Athar H

    2003-09-01

    Human discs large (hDlg) protein binds to protein 4.1R via a motif encoded by an alternatively spliced exon located between the SH3 and the C-terminal guanylate kinase-like domains. To evaluate the functional significance of protein 4.1R binding for subcellular localization of hDlg in vivo, we expressed full-length recombinant constructs of two naturally occurring isoforms of hDlg termed hDlg-I2 and hDlg-I3. The hDlg-I3 but not the hDlg-I2 isoform binds to the FERM (Four.1-Ezrin-Radixin-Moesin) domain of protein 4.1R in vitro. Upon transient transfection into subconfluent Madine-Darby canine kidney (MDCK) epithelial cells, the hDlg-I3 fused with the green fluorescent protein accumulated predominantly at the plasma membrane of cell-cell contact sites, whereas the hDlg-I2 fusion protein distributed in the cytoplasm. In contrast, in stably transfected confluent MDCK cells, both hDlg-I2 and -I3 isoforms localized efficiently to the lateral membrane, consistent with the previous notion that the N-terminal domain of hDlg mediates its membrane targeting in polarized epithelial cells. We introduced a double mutation (I38A/I40A) into the N-terminal domain of hDlg, which disrupted its interaction with DLG2, a key event in the membrane targeting of hDlg. Interestingly, the hDlg-I2 isoform harboring the I38A/I40A mutation mislocalized from the membrane into cytoplasm. Importantly, the hDlg-I3 isoform with the same mutation localized efficiently to the membrane of confluent MDCK cells. Together, our results demonstrate that in addition to the N-terminal targeting domain, the alternatively spliced I3 insertion plays a critical role in recruiting hDlg to the lateral membrane in epithelial cells via its interaction with protein 4.1R. PMID:12807908

  16. The genetics of the protein 4.1 family: organizers of the membrane and cytoskeleton.

    PubMed

    Hoover, K B; Bryant, P J

    2000-04-01

    Protein 4.1 (also called band 4.1 or simply 4.1) was originally identified as an abundant protein of the human erythrocyte, in which it stabilizes the spectrin/actin cytoskeleton. The protein and its relatives have since been found in many cell types of metazoan organisms and they are often concentrated in the nucleus, as well as in cell-cell junctions. They form multimolecular complexes with transmembrane and membrane-associated proteins, and these complexes may be important for both structural stability and signal transduction at sites of cell contact. PMID:10712924

  17. The cytoskeletal adapter protein 4.1G organizes the internodes in peripheral myelinated nerves

    PubMed Central

    Ivanovic, Aleksandra; Horresh, Ido; Golan, Neev; Spiegel, Ivo; Sabanay, Helena; Frechter, Shahar; Ohno, Shinichi; Terada, Nobuo; Möbius, Wiebke; Rosenbluth, Jack; Brose, Nils

    2012-01-01

    Myelinating Schwann cells regulate the localization of ion channels on the surface of the axons they ensheath. This function depends on adhesion complexes that are positioned at specific membrane domains along the myelin unit. Here we show that the precise localization of internodal proteins depends on the expression of the cytoskeletal adapter protein 4.1G in Schwann cells. Deletion of 4.1G in mice resulted in aberrant distribution of both glial adhesion molecules and axonal proteins that were present along the internodes. In wild-type nerves, juxtaparanodal proteins (i.e., Kv1 channels, Caspr2, and TAG-1) were concentrated throughout the internodes in a double strand that flanked paranodal junction components (i.e., Caspr, contactin, and NF155), and apposes the inner mesaxon of the myelin sheath. In contrast, in 4.1G−/− mice, these proteins “piled up” at the juxtaparanodal region or aggregated along the internodes. These findings suggest that protein 4.1G contributes to the organization of the internodal axolemma by targeting and/or maintaining glial transmembrane proteins along the axoglial interface. PMID:22291039

  18. Two distinct domains of protein 4.1 critical for assembly offunctional nuclei in Vitro

    SciTech Connect

    Krauss, Sharon Wald; Heald, Rebecca; Lee, Gloria; Nunomura, Wataru; Gimm,J. Aura; Mohandas, Narla; Chasis, Joel AnneJ. Aura; Mohandas, Narla; Chasis, Joel Anne

    2002-11-15

    Protein 4.1R, a multifunctional structural protein, acts asan adaptor in mature red cell membrane skeletons linking spectrin-actincomplexes to plasma membrane-associated proteins. In nucleated cellsprotein 4.1 is not associated exclusively with plasma membrane but isalso detected at several important subcellular locations crucial for celldivision. To identify 4.1 domains having critical functions in nuclearassembly, 4.1 domain peptides were added to Xenopus egg extract nuclearreconstitution reactions. Morphologically disorganized, replicationdeficient nuclei assembled when spectrin-actin binding domain orNuMA-binding C-terminal domain peptides were present. However, controlvariant spectrin-actin binding domain peptides incapable of bindingactin, or mutant C-terminal domain peptides with reduced NuMA binding,had no deleterious effects on nuclear reconstitution. To test if 4.1 isrequired for proper nuclear assembly, 4.1 isoforms were depleted withspectrin-actin binding or C-terminal domain-specific antibodies. Nucleiassembled in depleted extracts ha d deranged phenotypes. However, nuclearassembly could be rescued by addition of recombinant 4.1R. Our dataestablishes that protein 4.1 is essential for nuclear assembly andidentifies two distinct 4.1 domains, initially characterized incytoskeletal interactions, that have crucial and versatile functions innuclear assembly.

  19. Downregulation of Protein 4.1R impairs centrosome function,bipolar spindle organization and anaphase

    SciTech Connect

    Spence, Jeffrey R.; Go, Minjoung M.; Bahmanyar, S.; Barth,A.I.M.; Krauss, Sharon Wald

    2006-03-17

    Centrosomes nucleate and organize interphase MTs and areinstrumental in the assembly of the mitotic bipolar spindle. Here wereport that two members of the multifunctional protein 4.1 family havedistinct distributions at centrosomes. Protein 4.1R localizes to maturecentrioles whereas 4.1G is a component of the pericentriolar matrixsurrounding centrioles. To selectively probe 4.1R function, we used RNAinterference-mediated depletion of 4.1R without decreasing 4.1Gexpression. 4.1R downregulation reduces MT anchoring and organization atinterphase and impairs centrosome separation during prometaphase.Metaphase chromosomes fail to properly condense/align and spindleorganization is aberrant. Notably 4.1R depletion causes mislocalizationof its binding partner NuMA (Nuclear Mitotic Apparatus Protein),essential for spindle pole focusing, and disrupts ninein. Duringanaphase/telophase, 4.1R-depleted cells have lagging chromosomes andaberrant MT bridges. Our data provide functional evidence that 4.1R makescrucial contributions to centrosome integrity and to mitotic spindlestructure enabling mitosis and anaphase to proceed with the coordinatedprecision required to avoid pathological events.

  20. Gene and microRNA expression reveals sensitivity to paclitaxel in laryngeal cancer cell line

    PubMed Central

    Xu, Cheng-Zhi; Xie, Jin; Jin, Bin; Chen, Xin-Wei; Sun, Zhen-Feng; Wang, Bao-Xing; Dong, Pin

    2013-01-01

    Paclitaxel is a widely used chemotherapy drug for advanced laryngeal cancer patients. However, the fact that there are 20-40% of advanced laryngeal cancer patients do not response to paclitaxel makes it necessary to figure out potential biomarkers for paclitaxel sensitivity prediction. In this work, Hep2, a laryngeal cancer cell line, untreated or treated with lower dose of paclitaxel for 24 h, was applied to DNA microarray chips for gene and miR expression profile analysis. Expression of eight genes altered significantly following paclitaxel treatment, which was further validated by quantitative real-time PCR. Four up-regulated genes were ID2, BMP4, CCL4 and ACTG2, in which ID2 and BMP4 were implicated to be involved in several drugs sensitivity. While the down-regulated four genes, MAPK4, FASN, INSIG1 and SCD, were mainly linked to the endoplasmic reticulum and fatty acid biosynthesis, these two cell processes that are associated with drug sensitivity by increasing evidences. After paclitaxel treatment, expression of 49 miRs was significantly altered. Within these miRs, the most markedly expression-changed were miR-31-star, miR-1264, miR-3150b-5p and miR-210. While the miRs putatively modulated the mRNA expression of the most significantly expression-altered genes were miR-1264, miR-130a, miR-27b, miR-195, miR-1291, miR-214, miR-1277 and miR-1265, which were obtained by miR target prediction and miRNA target correlation. Collectively, our study might provide potential biomarkers for paclitaxel sensitivity prediction and drug resistance targets in laryngeal cancer patients. PMID:23826416

  1. Angiopoietin-like protein 4 inhibition of lipoprotein lipase: evidence for reversible complex formation.

    PubMed

    Lafferty, Michael J; Bradford, Kira C; Erie, Dorothy A; Neher, Saskia B

    2013-10-01

    Elevated triglycerides are associated with an increased risk of cardiovascular disease, and lipoprotein lipase (LPL) is the rate-limiting enzyme for the hydrolysis of triglycerides from circulating lipoproteins. The N-terminal domain of angiopoietin-like protein 4 (ANGPTL4) inhibits LPL activity. ANGPTL4 was previously described as an unfolding molecular chaperone of LPL that catalytically converts active LPL dimers into inactive monomers. Our studies show that ANGPTL4 is more accurately described as a reversible, noncompetitive inhibitor of LPL. We find that inhibited LPL is in a complex with ANGPTL4, and upon dissociation, LPL regains lipase activity. Furthermore, we have generated a variant of ANGPTL4 that is dependent on divalent cations for its ability to inhibit LPL. We show that LPL inactivation by this regulatable variant of ANGPTL4 is fully reversible after treatment with a chelator. PMID:23960078

  2. Retinol binding protein 4 in relation to diet, inflammation, immunity, and cardiovascular diseases.

    PubMed

    Zabetian-Targhi, Fateme; Mahmoudi, Mohammad J; Rezaei, Nima; Mahmoudi, Maryam

    2015-11-01

    Retinol binding protein 4 (RBP4), previously called retinol binding protein (RBP), is considered a specific carrier of retinol in the blood. It is also an adipokine that has been implicated in the pathophysiology of insulin resistance. RBP4 seems to be correlated with cardiometabolic markers in inflammatory chronic diseases, including obesity, type 2 diabetes, metabolic syndrome, and cardiovascular diseases (CVDs). It has recently been suggested that inflammation produced by RBP4 induces insulin resistance and CVD. The clinical relevance of this hypothesis is discussed in this review. Knowledge concerning the association of RBP4 with inflammation markers, oxidative stress, and CVDs as well as concerning the role of diet and antioxidants in decreasing RBP4 concentrations are discussed. Special attention is given to methodologies used in previously published studies and covariates that should be controlled when planning new studies on this adipokine. PMID:26567199

  3. Human CASK/LIN-2 Binds Syndecan-2 and Protein 4.1 and Localizes to the Basolateral Membrane of Epithelial Cells

    PubMed Central

    Cohen, Alexandra R.; Wood, Daniel F.; Marfatia, Shirin M.; Walther, Zenta; Chishti, Athar H.; Anderson, James Melvin

    1998-01-01

    In Caenorhabditis elegans, mutations in the lin-2 gene inactivate the LET-23 receptor tyrosine kinase/Ras/MAP kinase pathway required for vulval cell differentiation. One function of LIN-2 is to localize LET-23 to the basal membrane domain of vulval precursor cells. LIN-2 belongs to the membrane-associated guanylate kinase family of proteins. We have cloned and characterized the human homolog of LIN-2, termed hCASK, and Northern and Western blot analyses reveal that it is ubiquitously expressed. Indirect immunofluorescence localizes CASK to distinct lateral and/or basal plasma membrane domains in different epithelial cell types. We detect in a yeast two-hybrid screen that the PDZ domain of hCASK binds to the heparan sulfate proteoglycan syndecan-2. This interaction is confirmed using in vitro binding assays and immunofluorescent colocalization. Furthermore, we demonstrate that hCASK binds the actin-binding protein 4.1. Syndecans are known to bind extracellular matrix, and to form coreceptor complexes with receptor tyrosine kinases. We speculate that CASK mediates a link between the extracellular matrix and the actin cytoskeleton via its interaction with syndecan and with protein 4.1. Like other membrane-associated guanylate kinases, its multidomain structure enables it to act as a scaffold at the membrane, potentially recruiting multiple proteins and coordinating signal transduction. PMID:9660868

  4. Deciphering the Nuclear Import Pathway for the Cytoskeletal Red Cell Protein 4.1R

    PubMed Central

    Gascard, Philippe; Nunomura, Wataru; Lee, Gloria; Walensky, Loren D.; Krauss, Sharon Wald; Takakuwa, Yuichi; Chasis, Joel A.; Mohandas, Narla; Conboy, John G.

    1999-01-01

    The erythroid membrane cytoskeletal protein 4.1 is the prototypical member of a genetically and topologically complex family that is generated by combinatorial alternative splicing pathways and is localized at diverse intracellular sites including the nucleus. To explore the molecular determinants for nuclear localization, we transfected COS-7 cells with epitope-tagged versions of natural red cell protein 4.1 (4.1R) isoforms as well as mutagenized and truncated derivatives. Two distant topological sorting signals were required for efficient nuclear import of the 4.1R80 isoform: a basic peptide, KKKRER, encoded by alternative exon 16 and acting as a weak core nuclear localization signal (4.1R NLS), and an acidic peptide, EED, encoded by alternative exon 5. 4.1R80 isoforms lacking either of these two exons showed decreased nuclear import. Fusion of various 4.1R80 constructs to the cytoplasmic reporter protein pyruvate kinase confirmed a requirement for both motifs for full NLS function. 4.1R80 was efficiently imported in the nuclei of digitonin-permeabilized COS-7 cells in the presence of recombinant Rch1 (human importin α2), importin β, and GTPase Ran. Quantitative analysis of protein–protein interactions using a resonant mirror detection technique showed that 4.1R80 bound to Rch1 in vitro with high affinity (KD = 30 nM). The affinity decreased at least 7- and 20-fold, respectively, if the EED motif in exon 5 or if 4.1R NLS in exon 16 was lacking or mutated, confirming that both motifs were required for efficient importin-mediated nuclear import of 4.1R80. PMID:10359596

  5. Comprehensive characterization of expression patterns of protein 4.1 family members in mouse adrenal gland: implications for functions.

    PubMed

    Wang, Hua; Liu, Congrong; Debnath, Gargi; Baines, Anthony J; Conboy, John G; Mohandas, Narla; An, Xiuli

    2010-10-01

    The members of the protein 4.1 family, 4.1R, 4.1G, 4.1N, and 4.1B, are encoded by four genes, all of which undergo complex alternative splicing. It is well established that 4.1R, the prototypical member of the family, serves as an adapter that links the spectrin-actin based cytoskeleton to the plasma membrane in red cells. It is required for mechanical resilience of the membrane, and it ensures the cell surface accumulation of selected membrane proteins. However, the function of 4.1 proteins outside erythrocytes remains under-explored, especially in endocrine tissues. Transcripts of all 4.1 homologs have previously been documented to be abundantly expressed in adrenal gland. In order to begin to decipher the function of 4.1 proteins in adrenal gland, we performed a detailed characterization of the expression pattern of various 4.1 proteins and their cellular localization. We show that 4.1R (~80 and ~135 kDa) splice forms are expressed on the membrane of all cells, while a ~160 kDa 4.1G splice form is distributed in the cytoplasm and the membrane of zona glomerulosa and of medullary cells. Two 4.1N splice forms, ~135 and ~95 kDa, are present in the peri-nuclear region of both zona glomerulosa and medullary cells, while a single ~130 kDa 4.1B splice form, is detected in all layers of adrenal gland in both the cytoplasm and the membrane. The characterization of distinct splice forms of various 4.1 proteins with diverse cellular and sub-cellular localization indicates multiple functions for this family of proteins in endocrine functions of adrenal gland. PMID:20890708

  6. Retinol-Binding Protein 4 Induces Cardiomyocyte Hypertrophy by Activating TLR4/MyD88 Pathway.

    PubMed

    Gao, Wei; Wang, Hao; Zhang, Lin; Cao, Yang; Bao, Ji-Zhang; Liu, Zheng-Xia; Wang, Lian-Sheng; Yang, Qin; Lu, Xiang

    2016-06-01

    Insulin resistance plays a major role in the development and progression of cardiac hypertrophy and heart failure. Heart failure in turn promotes insulin resistance and increases the risk for diabetes. The vicious cycle determines significant mortality in patients with heart failure and diabetes. However, the underlying mechanisms for the vicious cycle are not fully elucidated. Here we show that circulating levels and adipose expression of retinol-binding protein 4 (RBP4), an adipokine that contributes to systemic insulin resistance, were elevated in cardiac hypertrophy induced by transverse aortic constriction and angiotensin-II (Ang-II) infusion. Ang-II increased RBP4 expression in adipocytes, which was abolished by losartan, an Ang-II receptor blocker. The elevated RBP4 in cardiac hypertrophy may have pathophysiological consequences because RBP4 increased cell size, enhanced protein synthesis, and elevated the expression of hypertrophic markers including Anp, Bnp, and Myh7 in primary cardiomyocytes. Mechanistically, RBP4 induced the expression and activity of toll-like receptor 4 (TLR4) and myeloid differentiation primary response gene 88 (MyD88) in cardiomyocytes, resulting in enhanced inflammation and reactive oxygen species production. Inhibition or knockdown of the TLR4/MyD88 pathway attenuated inflammatory and hypertrophic responses to RBP4 stimulation. Importantly, RBP4 also reduced the expression of glucose transporter-4 and impaired insulin-stimulated glucose uptake in cardiomyocytes. This impairment was ameliorated in cardiomyocytes from TLR4 knockout mice. Therefore, RBP4 may be a critical modulator promoting the vicious cycle of insulin resistance and heart failure by activating TLR4/MyD88-mediated inflammatory pathways. Potentially, lowering RBP4 might break the vicious cycle and improve both insulin resistance and cardiac hypertrophy. PMID:27100622

  7. Angiotensin II receptor blockers decrease serum concentration of fatty acid-binding protein 4 in patients with hypertension.

    PubMed

    Furuhashi, Masato; Mita, Tomohiro; Moniwa, Norihito; Hoshina, Kyoko; Ishimura, Shutaro; Fuseya, Takahiro; Watanabe, Yuki; Yoshida, Hideaki; Shimamoto, Kazuaki; Miura, Tetsuji

    2015-04-01

    Elevated circulating fatty acid-binding protein 4 (FABP4/A-FABP/aP2), an adipokine, is associated with obesity, insulin resistance, hypertension and cardiovascular events. However, how circulating FABP4 level is modified by pharmacological agents remains unclear. We here examined the effects of angiotensin II receptor blockers (ARBs) on serum FABP4 level. First, essential hypertensives were treated with ARBs: candesartan (8 mg day(-1); n=7) for 2 weeks, olmesartan (20 mg day(-1); n=9) for 12 weeks, and valsartan (80 mg day(-1); n=94) or telmisartan (40 mg day(-1); n=91) for 8 weeks added to amlodipine (5 mg day(-1)). Treatment with ARBs significantly decreased blood pressure and serum FABP4 concentrations by 8-20% without significant changes in adiposity or lipid variables, though the M value determined by hyperinsulinemic-euglycemic glucose clamp, a sensitive index of insulin sensitivity, was significantly increased by candesartan. Next, alterations in FABP4 secretion from 3T3-L1 adipocytes were examined under several agents. Lipolytic stimulation of the β-adrenoceptor in 3T3-L1 adipocytes by isoproterenol increased FABP4 secretion, and conversely, insulin suppressed FABP4 secretion. However, treatment of 3T3-L1 adipocytes with angiotensin II or ARBs for 2 h had no effect on gene expression or secretion of FABP4 regardless of β-adrenoceptor stimulation. In conclusion, treatment with structurally different ARBs similarly decreases circulating FABP4 concentrations in hypertensive patients as a class effect of ARBs, which is not attributable to blockade of the angiotensin II receptor in adipocytes. Reduction of FABP4 levels by ARBs might be involved in suppression of cardiovascular events. PMID:25672659

  8. BMP signalling in human fetal ovary somatic cells is modulated in a gene-specific fashion by GREM1 and GREM2

    PubMed Central

    Bayne, Rosemary A.; Donnachie, Douglas J.; Kinnell, Hazel L.; Childs, Andrew J.; Anderson, Richard A.

    2016-01-01

    STUDY QUESTION Do changes in the expression of bone morphogenetic proteins (BMPs) 2 and 4, and their antagonists Gremlin1 (GREM1) and Gremlin2 (GREM2) during human fetal ovarian development impact on BMP pathway activity and lead to changes in gene expression that may influence the fate and/or function of ovarian somatic cells? STUDY FINDING BMPs 2 and 4 differentially regulate gene expression in cultured human fetal ovarian somatic cells. Expression of some, but not all BMP target genes is antagonised by GREM1 and GREM2, indicating the existence of a mechanism to fine-tune BMP signal intensity in the ovary. Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), a marker of immature ovarian somatic cells, is identified as a novel transcriptional target of BMP4. WHAT IS KNOWN ALREADY Extensive re-organisation of the germ and somatic cell populations in the feto-neonatal ovary culminates in the formation of primordial follicles, which provide the basis for a female's future fertility. BMP growth factors play important roles at many stages of ovarian development and function. GREM1, an extracellular antagonist of BMP signalling, regulates the timing of primordial follicle formation in the mouse ovary, and mRNA levels of BMP4 decrease while those of BMP2 increase prior to follicle formation in the human fetal ovary. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Expression of genes encoding BMP pathway components, BMP antagonists and markers of ovarian somatic cells were determined by quantitative (q)RT-PCR in human fetal ovaries (from 8 to 21 weeks gestation) and fetal ovary-derived somatic cell cultures. Ovarian expression of GREM1 protein was confirmed by immunoblotting. Primary human fetal ovarian somatic cell cultures were derived from disaggregated ovaries by differential adhesion and cultured in the presence of recombinant human BMP2 or BMP4, with or without the addition of GREM1 or GREM2. MAIN RESULTS AND THE ROLE OF CHANCE We demonstrate that the

  9. [Uterine expression of adenovirus E4 promoter-binding protein 4 (E4BP4) during the embryo implantation period in mice].

    PubMed

    Wang, Jia; Huanh, Zhe Ping; Yang, Zeng Ming; Shen, Wei Xiong; Tso, Jia Ke; Shen, Qing Xiang

    2006-04-01

    Adenovirus E4 promoter-binding protein 4 (E4BP4), a mammalian basic leucine zipper (bZIP) nuclear transcription factor, was identified to be involved in cell survival and proliferation. Previous research data showed that the expression of E4BP4 gene was up-regulated at the implantation sites on day 5 pregnancy of mouse. The aim of the present study was to examine the expression pattern of E4BP4 gene in uterus during pre-implantation period, and at the implantation sites and inter-implantation sites during the implantation process, through the Northern blot, in situ hybridization, Western blot and immunohistochemistry analyses. It was found that, (1) during the pre-implantation period, the expression of E4BP4 gene was gradually increased; (2) its expression was sharply up-regulated at the implantation sites compared with that at the inter-implantation sites during the embryo implantation process; (3) the uterine expression of E4BP4 gene was not embryo-dependent, but observed in artificially induced decidulization of pseudopregnant mouse; (4) both E4BP4 mRNA and E4BP4 protein were localized in stromal cells and decidual cells around the uterine lumen. These results indicated that E4BP4 may play a critical role in embryo implantation process by promoting stromal cell proliferation and inhibiting decidual cell apoptosis. PMID:16944583

  10. Identification of genes related to beak deformity of chickens using digital gene expression profiling.

    PubMed

    Bai, Hao; Zhu, Jing; Sun, Yanyan; Liu, Ranran; Liu, Nian; Li, Dongli; Wen, Jie; Chen, Jilan

    2014-01-01

    Frequencies of up to 3% of beak deformity (normally a crossed beak) occur in some indigenous chickens in China, such as and Beijing-You. Chickens with deformed beaks have reduced feed intake, growth rate, and abnormal behaviors. Beak deformity represents an economic as well as an animal welfare problem in the poultry industry. Because the genetic basis of beak deformity remains incompletely understood, the present study sought to identify important genes and metabolic pathways involved in this phenotype. Digital gene expression analysis was performed on deformed and normal beaks collected from Beijing-You chickens to detect global gene expression differences. A total of >11 million cDNA tags were sequenced, and 5,864,499 and 5,648,877 clean tags were obtained in the libraries of deformed and normal beaks, respectively. In total, 1,156 differentially expressed genes (DEG) were identified in the deformed beak with 409 being up-regulated and 747 down-regulated in the deformed beaks. qRT-PCR using eight genes was performed to verify the results of DGE profiling. Gene ontology (GO) analysis highlighted that genes of the keratin family on GGA25 were abundant among the DEGs. Pathway analysis showed that many DEGs were linked to the biosynthesis of unsaturated fatty acids and glycerolipid metabolism. Combining the analyses, 11 genes (MUC, LOC426217, BMP4, ACAA1, LPL, ALDH7A1, GLA, RETSAT, SDR16C5, WWOX, and MOGAT1) were highlighted as potential candidate genes for beak deformity in chickens. Some of these genes have been identified previously, while others have unknown function with respect to thus phenotype. To the best of our knowledge, this is the first genome-wide study to investigate the transcriptome differences in the deformed and normal beaks of chickens. The DEGs identified here are worthy of further functional characterization. PMID:25198128

  11. New insights into potential functions for the protein 4.1superfamily of proteins in kidney epithelium

    SciTech Connect

    Calinisan, Venice; Gravem, Dana; Chen, Ray Ping-Hsu; Brittin,Sachi; Mohandas, Narla; Lecomte, Marie-Christine; Gascard, Philippe

    2005-06-17

    Members of the protein 4.1 family of adapter proteins are expressed in a broad panel of tissues including various epithelia where they likely play an important role in maintenance of cell architecture and polarity and in control of cell proliferation. We have recently characterized the structure and distribution of three members of the protein 4.1 family, 4.1B, 4.1R and 4.1N, in mouse kidney. We describe here binding partners for renal 4.1 proteins, identified through the screening of a rat kidney yeast two-hybrid system cDNA library. The identification of putative protein 4.1-based complexes enables us to envision potential functions for 4.1 proteins in kidney: organization of signaling complexes, response to osmotic stress, protein trafficking, and control of cell proliferation. We discuss the relevance of these protein 4.1-based interactions in kidney physio-pathology in the context of their previously identified functions in other cells and tissues. Specifically, we will focus on renal 4.1 protein interactions with beta amyloid precursor protein (beta-APP), 14-3-3 proteins, and the cell swelling-activated chloride channel pICln. We also discuss the functional relevance of another member of the protein 4.1 superfamily, ezrin, in kidney physiopathology.

  12. New insights into potential functions for the protein 4.1 superfamily of proteins in kidney epithelium.

    PubMed

    Calinisan, Venice; Gravem, Dana; Chen, Ray Ping-Hsu; Brittin, Sachi; Mohandas, Narla; Lecomte, Marie-Christine; Gascard, Philippe

    2006-01-01

    Members of the protein 4.1 family of adapter proteins are expressed in a broad panel of tissues including various epithelia where they likely play an important role in maintenance of cell architecture and polarity and in control of cell proliferation. We have recently characterized the structure and distribution of three members of the protein 4.1 family, 4.1B, 4.1R and 4.1N, in mouse kidney. We describe here binding partners for renal 4.1 proteins, identified through the screening of a rat kidney yeast two-hybrid system cDNA library. The identification of putative protein 4.1-based complexes enables us to envision potential functions for 4.1 proteins in kidney: organization of signaling complexes, response to osmotic stress, protein trafficking, and control of cell proliferation. We discuss the relevance of these protein 4.1-based interactions in kidney physio-pathology in the context of their previously identified functions in other cells and tissues. Specifically, we will focus on renal 4.1 protein interactions with beta amyloid precursor protein (beta-APP), 14-3-3 proteins, and the cell swelling-activated chloride channel pICln. We also discuss the functional relevance of another member of the protein 4.1 superfamily, ezrin, in kidney physio-pathology. PMID:16368544

  13. α1-Antitrypsin Combines with Plasma Fatty Acids and Induces Angiopoietin-like Protein 4 Expression.

    PubMed

    Frenzel, Eileen; Wrenger, Sabine; Brügger, Britta; Salipalli, Sandeep; Immenschuh, Stephan; Aggarwal, Nupur; Lichtinghagen, Ralf; Mahadeva, Ravi; Marcondes, A Mario Q; Dinarello, Charles A; Welte, Tobias; Janciauskiene, Sabina

    2015-10-15

    α1-Antitrypsin (A1AT) purified from human plasma upregulates expression and release of angiopoietin-like protein 4 (Angptl4) in adherent human blood monocytes and in human lung microvascular endothelial cells, providing a mechanism for the broad immune-regulatory properties of A1AT independent of its antiprotease activity. In this study, we demonstrate that A1AT (Prolastin), a potent inducer of Angptl4, contains significant quantities of the fatty acids (FA) linoleic acid (C18:2) and oleic acid (C18:1). However, only trace amounts of FAs were present in preparations that failed to increase Angplt4 expression, for example, A1AT (Zemaira) or M-type A1AT purified by affinity chromatography. FA pull-down assays with Western blot analysis revealed a FA-binding ability of A1AT. In human blood-adherent monocytes, A1AT-FA conjugates upregulated expression of Angptl4 (54.9-fold, p < 0.001), FA-binding protein 4 (FABP4) (11.4-fold, p < 0.001), and, to a lesser degree, FA translocase (CD36) (3.1-fold, p < 0.001) relative to A1AT devoid of FA (A1AT-0). These latter effects of A1AT-FA were blocked by inhibitors of peroxisome proliferator-activated receptor (PPAR) β/δ (ST247) and PPARγ (GW9662). When compared with controls, cell pretreatment with ST247 diminished the effect of A1AT-LA on Angptl4 mRNA (11.6- versus 4.1-fold, p < 0.001) and FABP4 mRNA (5.4- versus 2.8-fold, p < 0.001). Similarly, preincubation of cells with GW9662 inhibited inducing effect of A1AT-LA on Angptl4 mRNA (by 2-fold, p < 0.001) and FABP4 mRNA (by 3-fold, p < 0.001). Thus, A1AT binds to FA, and it is this form of A1AT that induces Angptl4 and FABP4 expression via a PPAR-dependent pathway. These findings provide a mechanism for the unexplored area of A1AT biology independent of its antiprotease properties. PMID:26363050

  14. Microfibrillar associated protein 4 mfap4 genes in catfish play a novel role in innate immune responses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The lectin pathway of the complement system is characterized by two groups of soluble pattern recognition molecules, mannose-binding lectins (MBLs) and ficolins. These molecules recognize and bind carbohydrates in pathogens and activate complement leading to opsonization, leukocyte activation, and d...

  15. AKAP-9 promotes colorectal cancer development by regulating Cdc42 interacting protein 4.

    PubMed

    Hu, Zhi-Yan; Liu, Yan-Ping; Xie, Lin-Ying; Wang, Xiao-Yan; Yang, Fang; Chen, Shi-You; Li, Zu-Guo

    2016-06-01

    Our previous studies have shown that PRKA kinase anchor protein 9 (AKAP-9) is involved in colorectal cancer (CRC) cell proliferation and migration in vitro. However, whether or not AKAP-9 is important for CRC development or metastasis in vivo remains unknown. In the present study, we found that AKAP-9 expression was significantly higher in human colorectal cancer tissues than the paired normal tissues. In fact, AKAP-9 level correlated with the CRC infiltrating depth and metastasis. Moreover, the higher AKAP-9 expression was associated with the lower survival rate in patients. In cultured CRC cells, knockdown of AKAP-9 inhibited cell proliferation, invasion, and migration. AKAP-9 deficiency also attenuated CRC tumor growth and metastasis in vivo. Mechanistically, AKAP-9 interacted with cdc42 interacting protein 4 (CIP4) and regulated its expression. CIP4 levels were interrelated to the AKAP-9 level in CRC cells. Functionally, AKAP-9 was essential for TGF-β1-induced epithelial-mesenchymal transition of CRC cells, and CIP4 played a critical role in mediating the function of AKAP-9. Importantly, CIP4 expression was significantly up-regulated in human CRC tissues. Taken together, our results demonstrated that AKAP-9 facilitates CRC development and metastasis via regulating CIP4-mediated epithelial-mesenchymal transition of CRC cells. PMID:27039663

  16. Targeting bromodomain-containing protein 4 (BRD4) benefits rheumatoid arthritis.

    PubMed

    Zhang, Qing-gang; Qian, Jing; Zhu, Yu-chang

    2015-08-01

    We aimed to explore the effects of bromodomain-containing protein 4 (BRD4) inhibition on tumor necrosis factor (TNF)-α-stimulated human rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) behavior and the therapeutic implications using BRD4 inhibitor JQ1 were explored in vivo. The levels of interleukin (IL)-1β, IL-6, IL-17 and IL-18 in cultural supernatants from TNFα-stimulated RA-FLS were measured by ELISA. RA-FLS migration and invasion in vitro were investigated using wound healing and Matrigel assay. Expression of signaling pathway proteins was measured by Western blot. The in vivo effects of BRD4 inhibitor JQ1 were elucidated using collagen-induced arthritis (CIA) mice. We found BRD4 silencing reduced the secretion of IL-1β, IL-6, IL-17 and IL-18 from TNFα-stimulated human RA-FLS. Downregulation of BRD4 inhibited FBS-induced migration and invasion of human RA-FLS. BRD4 silencing decreased the phosphorylation of c-Jun and activation of NFκB in TNFα-stimulated RA-FLS. In vivo, BRD4 inhibitor JQ1 reduced the inflammatory response, autoantibody production and joint damage of CIA model. Our data suggest for the first time that BRD4 inhibition has anti-inflammatory property in RA. PMID:26093279

  17. The Pharmacological and Physiological Role of Multidrug-Resistant Protein 4.

    PubMed

    Wen, Jiagen; Luo, Jianquan; Huang, Weihua; Tang, Jie; Zhou, Honghao; Zhang, Wei

    2015-09-01

    Multidrug-resistant protein 4 (MRP4), a member of the C subfamily of ATP-binding cassette transporters, is distributed in a variety of tissues and a number of cancers. As a drug transporter, MRP4 is responsible for the pharmacokinetics and pharmacodynamics of numerous drugs, especially antiviral drugs, antitumor drugs, and diuretics. In this regard, the functional role of MRP4 is affected by a number of factors, such as genetic mutations; tissue-specific transcriptional regulations; post-transcriptional regulations, including miRNAs and membrane internalization; and substrate competition. Unlike other C family members, MRP4 is in a pivotal position to transport cellular signaling molecules, through which it is tightly connected to the living activity and physiologic processes of cells and bodies. In the context of several cancers in which MRP4 is overexpressed, MRP4 inhibition shows striking effects against cancer progression and drug resistance. In this review, we describe the role of MRP4 more specifically in both healthy conditions and disease states, with an emphasis on its potential as a drug target. PMID:26148856

  18. Plasma Fatty Acid Binding Protein 4 and Risk of Sudden Cardiac Death in Older Adults

    PubMed Central

    Djoussé, Luc; Maziarz, Marlena; Biggs, Mary L.; Ix, Joachim H.; Zieman, Susan J.; Kizer, Jorge R.; Lemaitre, Rozenn N.; Mozaffarian, Dariush; Tracy, Russell P.; Mukamal, Kenneth J.; Siscovick, David S.; Sotoodehnia, Nona

    2013-01-01

    Although fatty acid binding protein 4 (FABP4) may increase risk of diabetes and exert negative cardiac inotropy, it is unknown whether plasma concentrations of FABP4 are associated with incidence of sudden cardiac death (SCD). We prospectively analyzed data on 4,560 participants of the Cardiovascular Health Study. FABP4 was measured at baseline using ELISA, and SCD events were adjudicated through review of medical records. We used Cox proportional hazards to estimate effect measures. During a median followup of 11.8 years, 146 SCD cases occurred. In a multivariable model adjusting for demographic, lifestyle, and metabolic factors, relative risk of SCD associated with each higher standard deviation (SD) of plasma FABP4 was 1.15 (95% CI: 0.95–1.38), P = 0.15. In a secondary analysis stratified by prevalent diabetes status, FABP4 was associated with higher risk of SCD in nondiabetic participants, (RR per SD higher FABP4: 1.33 (95% CI: 1.07–1.65), P = 0.009) but not in diabetic participants (RR per SD higher FABP4: 0.88 (95% CI: 0.62–1.27), P = 0.50), P for diabetes-FABP4 interaction 0.049. In summary, a single measure of plasma FABP4 obtained later in life was not associated with the risk of SCD in older adults overall. Confirmation of our post-hoc results in nondiabetic people in other studies is warranted. PMID:24455402

  19. Insulin-Like Growth Factor Binding Protein-4 as a Marker of Chronic Lupus Nephritis

    PubMed Central

    Han, Jie; Ye, Yujin; Singh, Sandeep; Zhou, Jinchun; Li, Yajuan; Ding, Huihua; Li, Quan-zhen; Zhou, Xin; Putterman, Chaim; Saxena, Ramesh; Mohan, Chandra

    2016-01-01

    Kidney biopsy remains the mainstay of Lupus Nephritis (LN) diagnosis and prognostication. The objective of this study is to identify non-invasive biomarkers that closely parallel renal pathology in LN. Previous reports have demonstrated that serum Insulin-like growth factor binding protein 4 (IGFBP-4) was increased in diabetic nephropathy in both animal models and patients. We proceeded to assess if IGFBP4 could be associated with LN. We performed ELISA using the serum of 86 patients with LN. Normal healthy adults (N = 23) and patients with other glomerular diseases (N = 20) served as controls. Compared to the healthy controls or other glomerular disease controls, serum IGFBP-4 levels were significantly higher in the patients with LN. Serum IGFBP-4 did not correlate well with systemic lupus erythematosus disease activity index (SLEDAI), renal SLEDAI or proteinuria, but it did correlate with estimated glomerular filtration rate (R = 0.609, P < 0.0001). Interestingly, in 18 patients with proliferative LN whose blood samples were obtained at the time of renal biopsy, serum IGFBP-4 levels correlated strongly with the chronicity index of renal pathology (R = 0.713, P < 0.001). IGFBP-4 emerges a potential marker of lupus nephritis, reflective of renal pathology chronicity changes. PMID:27019456

  20. Structure of human monocyte chemoattractant protein 4 (MCP-4/CCL13)

    SciTech Connect

    Barinka, Cyril; Prahl, Adam; Lubkowski, Jacek

    2008-04-02

    Monocyte chemoattractant proteins (MCPs) belong to the CC chemokine family and are involved in many (patho)physiological processes characterized by mononuclear cell infiltration, including tissue remodeling, atherosclerosis and cancer metastasis. Here, the crystal structure of human monocyte chemoattractant protein 4 (MCP-4) refined at 1.70 {angstrom} resolution is reported with crystallographic values R = 0.180 and R{sub free} = 0.212. The overall MCP-4 fold reveals the typical tertiary features of the CC chemokine family. A central three-stranded antiparallel {beta}-sheet is C-terminally flanked by an overlaying {alpha}-helix, while the N-terminal part of the molecule forms an extended loop that is anchored to the rest of the molecule via two disulfide bridges, Cys11-Cys35 and Cys12-Cys51. The crystal packing suggests the existence of MCP-4 dimers with a dimerization interface similar to those previously reported for the X-ray structures of MCP-1 and MCP-2.

  1. Human epididymis protein 4 (HE4) in laboratory medicine and an algorithm in renal disorders.

    PubMed

    Kappelmayer, János; Antal-Szalmás, Péter; Nagy, Béla

    2015-01-01

    Over the past three decades, cancer antigen (CA) 125 has been utilized for monitoring women who were treated for ovarian cancer. However, this tumor marker showed several limitations such as false elevation in benign pelvic diseases and, in turn, no alterations in ovarian tumors at early stages with a relatively high ratio. For more than ten years, the human epididymis protein 4 (HE4) has become available for the routine laboratory repertoire, showing a higher sensitivity and specificity compared to that of CA125 in ovarian malignancies, but also in other types of tumors based on recently accumulated clinical data. Despite its remarkable diagnostic characteristics, in certain cases, the evaluation of HE4 results may be problematic when patients suffer from additional conditions that may alter HE4 level. Besides the direct effects of age and smoking, menopause status and decreased renal function also show a substantial impact on HE4 values, which should be considered in each patient. For this purpose, we attempted to create a new formula and an algorithm that may be helpful to predict the probability of the presence of ovarian tumor by using the concentrations of HE4 and CA125. PMID:25127713

  2. Moesin: a member of the protein 4.1-talin-ezrin family of proteins.

    PubMed Central

    Lankes, W T; Furthmayr, H

    1991-01-01

    Moesin (membrane-organizing extension spike protein, pronounced mó ez in) has previously been isolated from bovine uterus and characterized as a possible receptor protein for heparan sulfate. We now have cloned and sequenced its complete cDNA, which represents a single 4.2-kilobase mRNA encoding a protein of 577 amino acids. It contains no apparent signal peptide or transmembrane domain. In addition, the protein shows significant sequence identity (72%) to ezrin (cytovillin, p81), as well as similarity to protein 4.1 and talin. All of the latter proteins have been postulated to serve as structural links between the plasma membrane and the cytoskeleton. A similar role for moesin is implied by structure and domain predictions derived from the cDNA-deduced peptide sequence. Furthermore, our data indicate that moesin is identical to the 77-kDa band that copurifies with ezrin in its isolation from human placenta [Bretscher, A. (1989) J. Cell Biol. 108, 921-930]. Images PMID:1924289

  3. Antibodies against low-density lipoprotein receptor-related protein 4 induce myasthenia gravis.

    PubMed

    Shen, Chengyong; Lu, Yisheng; Zhang, Bin; Figueiredo, Dwight; Bean, Jonathan; Jung, Jiung; Wu, Haitao; Barik, Arnab; Yin, Dong-Min; Xiong, Wen-Cheng; Mei, Lin

    2013-12-01

    Myasthenia gravis (MG) is the most common disorder affecting the neuromuscular junction (NMJ). MG is frequently caused by autoantibodies against acetylcholine receptor (AChR) and a kinase critical for NMJ formation, MuSK; however, a proportion of MG patients are double-negative for anti-AChR and anti-MuSK antibodies. Recent studies in these subjects have identified autoantibodies against low-density lipoprotein receptor-related protein 4 (LRP4), an agrin receptor also critical for NMJ formation. LRP4 autoantibodies have not previously been implicated in MG pathogenesis. Here we demonstrate that mice immunized with the extracellular domain of LRP4 generated anti-LRP4 antibodies and exhibited MG-associated symptoms, including muscle weakness, reduced compound muscle action potentials (CMAPs), and compromised neuromuscular transmission. Additionally, fragmented and distorted NMJs were evident at both the light microscopic and electron microscopic levels. We found that anti-LRP4 sera decreased cell surface LRP4 levels, inhibited agrin-induced MuSK activation and AChR clustering, and activated complements, revealing potential pathophysiological mechanisms. To further confirm the pathogenicity of LRP4 antibodies, we transferred IgGs purified from LRP4-immunized rabbits into naive mice and found that they exhibited MG-like symptoms, including reduced CMAP and impaired neuromuscular transmission. Together, these data demonstrate that LRP4 autoantibodies induce MG and that LRP4 contributes to NMJ maintenance in adulthood. PMID:24200689

  4. Antibodies against low-density lipoprotein receptor–related protein 4 induce myasthenia gravis

    PubMed Central

    Shen, Chengyong; Lu, Yisheng; Zhang, Bin; Figueiredo, Dwight; Bean, Jonathan; Jung, Jiung; Wu, Haitao; Barik, Arnab; Yin, Dong-Min; Xiong, Wen-Cheng; Mei, Lin

    2013-01-01

    Myasthenia gravis (MG) is the most common disorder affecting the neuromuscular junction (NMJ). MG is frequently caused by autoantibodies against acetylcholine receptor (AChR) and a kinase critical for NMJ formation, MuSK; however, a proportion of MG patients are double-negative for anti-AChR and anti-MuSK antibodies. Recent studies in these subjects have identified autoantibodies against low-density lipoprotein receptor–related protein 4 (LRP4), an agrin receptor also critical for NMJ formation. LRP4 autoantibodies have not previously been implicated in MG pathogenesis. Here we demonstrate that mice immunized with the extracellular domain of LRP4 generated anti-LRP4 antibodies and exhibited MG-associated symptoms, including muscle weakness, reduced compound muscle action potentials (CMAPs), and compromised neuromuscular transmission. Additionally, fragmented and distorted NMJs were evident at both the light microscopic and electron microscopic levels. We found that anti-LRP4 sera decreased cell surface LRP4 levels, inhibited agrin-induced MuSK activation and AChR clustering, and activated complements, revealing potential pathophysiological mechanisms. To further confirm the pathogenicity of LRP4 antibodies, we transferred IgGs purified from LRP4-immunized rabbits into naive mice and found that they exhibited MG-like symptoms, including reduced CMAP and impaired neuromuscular transmission. Together, these data demonstrate that LRP4 autoantibodies induce MG and that LRP4 contributes to NMJ maintenance in adulthood. PMID:24200689

  5. Identification of a Novel Determinant for Membrane Association in Hepatitis C Virus Nonstructural Protein 4B▿

    PubMed Central

    Gouttenoire, Jérôme; Castet, Valérie; Montserret, Roland; Arora, Naveen; Raussens, Vincent; Ruysschaert, Jean-Marie; Diesis, Eric; Blum, Hubert E.; Penin, François; Moradpour, Darius

    2009-01-01

    Nonstructural protein 4B (NS4B) plays an essential role in the formation of the hepatitis C virus (HCV) replication complex. It is a relatively poorly characterized integral membrane protein predicted to comprise four transmembrane segments in its central portion. Here, we describe a novel determinant for membrane association represented by amino acids (aa) 40 to 69 in the N-terminal portion of NS4B. This segment was sufficient to target and tightly anchor the green fluorescent protein to cellular membranes, as assessed by fluorescence microscopy as well as membrane extraction and flotation analyses. Circular dichroism and nuclear magnetic resonance structural analyses showed that this segment comprises an amphipathic α-helix extending from aa 42 to 66. Attenuated total reflection infrared spectroscopy and glycosylation acceptor site tagging revealed that this amphipathic α-helix has the potential to traverse the phospholipid bilayer as a transmembrane segment, likely upon oligomerization. Alanine substitution of the fully conserved aromatic residues on the hydrophobic helix side abrogated membrane association of the segment comprising aa 40 to 69 and disrupted the formation of a functional replication complex. These results provide the first atomic resolution structure of an essential membrane-associated determinant of HCV NS4B. PMID:19357161

  6. The new platinum-based anticancer agent LA-12 induces retinol binding protein 4 in vivo

    PubMed Central

    2011-01-01

    Background The initial pharmacokinetic study of a new anticancer agent (OC-6-43)-bis(acetato)(1-adamantylamine)amminedichloroplatinum (IV) (LA-12) was complemented by proteomic screening of rat plasma. The objective of the study was to identify new LA-12 target proteins that serve as markers of LA-12 treatment, response and therapy monitoring. Methods Proteomic profiles were measured by surface-enhanced laser desorption-ionization time-of-flight mass spectrometry (SELDI-TOF MS) in 72 samples of rat plasma randomized according to LA-12 dose and time from administration. Correlation of 92 peak clusters with platinum concentration was evaluated using Spearman correlation analysis. Results We identified Retinol-binding protein 4 (RBP4) whose level correlated with LA-12 level in treated rats. Similar results were observed in randomly selected patients involved in Phase I clinical trials. Conclusions RBP4 induction is in agreement with known RBP4 regulation by amantadine and cisplatin. Since retinol metabolism is disrupted in many cancers and inversely associates with malignancy, these data identify a potential novel mechanism for the action of LA-12 and other similar anti-cancer drugs. PMID:22040120

  7. Gene Expression of Rat Alveolar Type II Cells during Hyperoxia Exposure and Early Recovery

    PubMed Central

    Chen, Zhongming; Chintagari, Narendranath Reddy; Guo, Yujie; Bhaskaran, Manoj; Chen, Jiwang; Gao, Li; Jin, Nili; Weng, Tingting; Liu, Lin

    2007-01-01

    Alveolar epithelial cell (AEC) injury and repair during hyperoxia exposure and recovery have been investigated for decades, but the molecular mechanisms of these processes are not clear. To identify potentially important genes involved in lung injury and repair, we studied the gene expression profiles of isolated AEC II from control, 48-hour hyperoxia-exposed (>95% O2) and 1-7 day recovering rats using a DNA microarray containing 10,000 genes. Fifty genes showed significant differential expression between two or more time points (p<0.05, fold change >2). These genes can be classified into 8 unique gene expression patterns. Real-time PCR verified 14 selected genes in three patterns related to hyperoxia exposure and early recovery. The change in the protein level for two of the selected genes, bmp-4 and retnla, paralleled that of the mRNA level. Many of these genes were found to be involved in cell proliferation and differentiation. In an in vitro AEC trans-differentiation culture model using AEC II isolated from control and 48 hrs hyperoxia-exposed rats, the expression of the cell proliferation and differentiation genes identified above were consistent with their predicted roles in the trans-differentiation of AEC. These data indicate that a coordinated mechanism may control AEC differentiation during in vivo hyperoxia exposure and recovery as well as during in vitro AEC culture. PMID:17640573

  8. Exogenous fatty acid binding protein 4 promotes human prostate cancer cell progression.

    PubMed

    Uehara, Hisanori; Takahashi, Tetsuyuki; Oha, Mina; Ogawa, Hirohisa; Izumi, Keisuke

    2014-12-01

    Epidemiologic studies have found that obesity is associated with malignant grade and mortality in prostate cancer. Several adipokines have been implicated as putative mediating factors between obesity and prostate cancer. Fatty acid binding protein 4 (FABP4), a member of the cytoplasmic fatty acid binding protein multigene family, was recently identified as a novel adipokine. Although FABP4 is released from adipocytes and mean circulating concentrations of FABP4 are linked with obesity, effects of exogenous FABP4 on prostate cancer progression are unclear. In this study, we examined the effects of exogenous FABP4 on human prostate cancer cell progression. FABP4 treatment promoted serum-induced prostate cancer cell invasion in vitro. Furthermore, oleic acid promoted prostate cancer cell invasion only if FABP4 was present in the medium. These promoting effects were reduced by FABP4 inhibitor, which inhibits FABP4 binding to fatty acids. Immunostaining for FABP4 showed that exogenous FABP4 was taken up into DU145 cells in three-dimensional culture. In mice, treatment with FABP4 inhibitor reduced the subcutaneous growth and lung metastasis of prostate cancer cells. Immunohistochemical analysis showed that the number of apoptotic cells, positive for cleaved caspase-3 and cleaved PARP, was increased in subcutaneous tumors of FABP4 inhibitor-treated mice, as compared with control mice. These results suggest that exogenous FABP4 might promote human prostate cancer cell progression by binding with fatty acids. Additionally, exogenous FABP4 activated the PI3K/Akt pathway, independently of binding to fatty acids. Thus, FABP4 might be a key molecule to understand the mechanisms underlying the obesity-prostate cancer progression link. PMID:24740818

  9. A novel neuron-enriched homolog of the erythrocyte membrane cytoskeletal protein 4.1.

    PubMed

    Walensky, L D; Blackshaw, S; Liao, D; Watkins, C C; Weier, H U; Parra, M; Huganir, R L; Conboy, J G; Mohandas, N; Snyder, S H

    1999-08-01

    We report the molecular cloning and characterization of 4.1N, a novel neuronal homolog of the erythrocyte membrane cytoskeletal protein 4.1 (4.1R). The 879 amino acid protein shares 70, 36, and 46% identity with 4.1R in the defined membrane-binding, spectrin-actin-binding, and C-terminal domains, respectively. 4.1N is expressed in almost all central and peripheral neurons of the body and is detected in embryonic neurons at the earliest stage of postmitotic differentiation. Like 4.1R, 4.1N has multiple splice forms as evidenced by PCR and Western analysis. Whereas the predominant 4.1N isoform identified in brain is approximately 135 kDa, a smaller 100 kDa isoform is enriched in peripheral tissues. Immunohistochemical studies using a polyclonal 4.1N antibody revealed several patterns of neuronal staining, with localizations in the neuronal cell body, dendrites, and axons. In certain neuronal locations, including the granule cell layers of the cerebellum and dentate gyrus, a distinct punctate-staining pattern was observed consistent with a synaptic localization. In primary hippocampal cultures, mouse 4.1N is enriched at the discrete sites of synaptic contact, colocalizing with the postsynaptic density protein of 95 kDa (a postsynaptic marker) and glutamate receptor type 1 (an excitatory postsynaptic marker). By analogy with the roles of 4.1R in red blood cells, 4.1N may function to confer stability and plasticity to the neuronal membrane via interactions with multiple binding partners, including the spectrin-actin-based cytoskeleton, integral membrane channels and receptors, and membrane-associated guanylate kinases. PMID:10414974

  10. Multidrug resistance-associated protein 4 is a determinant of arsenite resistance.

    PubMed

    Yuan, Bo; Yoshino, Yuta; Fukushima, Hisayo; Markova, Svetlana; Takagi, Norio; Toyoda, Hiroo; Kroetz, Deanna L

    2016-01-01

    Although arsenic trioxide (arsenite, As(III)) has shown a remarkable efficacy in the treatment of acute promyelocytic leukemia patients, multidrug resistance is still a major concern for its clinical use. Multidrug resistance-associated protein 4 (MRP4), which belongs to the ATP-binding cassette (ABC) superfamily of transporters, is localized to the basolateral membrane of hepatocytes and the apical membrane of renal proximal tubule cells. Due to its characteristic localization, MRP4 is proposed as a candidate in the elimination of arsenic and may contribute to resistance to As(III). To test this hypothesis, stable HEK293 cells overexpressing MRP4 or MRP2 were used to establish the role of these two transporters in As(III) resistance. The IC50 values of As(III) in MRP4 cells were approximately 6-fold higher than those in MRP2 cells, supporting an important role for MRP4 in resistance to As(III). The capacity of MRP4 to confer resistance to As(III) was further confirmed by a dramatic decrease in the IC50 values with the addition of MK571, an MRP4 inhibitor, and cyclosporine A, a well-known broad-spectrum inhibitor of ABC transporters. Surprisingly, the sensitivity of the MRP2 cells to As(III) was similar to that of the parent cells, although insufficient formation of glutathione and/or Se conjugated arsenic compounds in the MRP2 cells might limit transport. Given that MRP4 is a major contributor to arsenic resistance in vitro, further investigation into the correlation between MRP4 expression and treatment outcome of leukemia patients treated with arsenic-based regimens is warranted. PMID:26497925

  11. Retinol Binding Protein-4 and Adiponectin Levels in Thyroid Overt and Subclinical Dysfunction.

    PubMed

    Kokkinos, S; Papazoglou, D; Zisimopoulos, A; Papanas, N; Tiaka, E; Antonoglou, C; Maltezos, E

    2016-02-01

    Thyroid dysfunction is accompanied by numerous changes in intermediary metabolism. Retinol binding protein-4 (RBP-4) and adiponectin are 2 adipocytokines that have multiple metabolic functions. The aim of our study was to examine serum RBP4 and adiponectin levels in clinical (before and after therapy) and subclinical hyperthyroid and hypothyroid subjects as compared to controls.150 patients with thyroid dysfunction were recruited (65 hyperthyroid and 85 hypothyroid) while 28 euthyroid subjects served as a control group. We measured anthropometric, biochemical and hormonal (free T4, free T3, TSH, insulin) parameters in all participants. RBP-4 and adiponectin were measured using commercial ELISA kits.Mean baseline levels of RBP-4 were higher in patients with clinical hypothyroidism (29.0±10.2 ng/ml, 25.1±12.6 ng/ml, 38.8±16.5 ng/ml, 31.9±13.2 ng/ml, 20.4±8.2 ng/ml in patients with hyperthyroidism, subclinical hyperthryrodism, hypothyroidism, subclinical hypothyroidism and controls respectively, F=4.86, P<0.001) and decreased significantly in patients with clinical hyperthyroidism and hypothyroidism after normalization of thyroid hormones' levels (from 29.0±10.2 to 24.9±8.4 ng/ml, p=0.003 and from 38.8±16.5 to 29.0±10.8 ng/ml, p=0.001 respectively). We did not observe analogous changes in adiponectin levels in any of the studied groups.RBP-4 levels are higher in patients with clinical hypothyroidism and exhibit a marked decrease after normalization of thyroid function in both hyper and hypothyroid patients. We suggest that RBP-4 may play a role in the metabolic disturbances which accompany thyroid dysfunction. PMID:26575118

  12. Circulating Retinol-Binding Protein-4 Concentration Might Reflect Insulin Resistance–Associated Iron Overload

    PubMed Central

    Fernández-Real, José Manuel; Moreno, José María; Ricart, Wifredo

    2008-01-01

    OBJECTIVES—The mechanisms behind the association between retinol-binding protein-4 (RBP4) and insulin resistance are not well understood. An interaction between iron and vitamin A status, of which RBP4 is a surrogate, has long been recognized. We hypothesized that iron-associated insulin resistance could be behind the impaired insulin action caused by RBP4. RESEARCH DESIGN AND METHODS—Serum ferritin and RBP4 concentration and insulin resistance were evaluated in a sample of middle-aged men (n = 132) and in a replication independent study. Serum RBP4 was also studied before and after iron depletion in patients with type 2 diabetes. Finally, the effect of iron on RBP4 release was evaluated in vitro in adipose tissue. RESULTS—A positive correlation between circulating RBP4 and log serum ferritin (r = 0.35 and r = 0.61, respectively; P < 0.0001) was observed in both independent studies. Serum RBP4 concentration was higher in men than women in parallel to increased ferritin levels. On multiple regression analyses to predict serum RBP4, log serum ferritin contributed significantly to RBP4 variance after controlling for BMI, age, and homeostasis model assessment value. Serum RBP4 concentration decreased after iron depletion in type 2 diabetic patients (percent mean difference −13.7 [95% CI −25.4 to −2.04]; P = 0.024). The iron donor lactoferrin led to increased dose-dependent adipose tissue release of RBP4 (2.4-fold, P = 0.005) and increased RBP4 expression, while apotransferrin and deferoxamine led to decreased RBP4 release. CONCLUSIONS—The relationship between circulating RBP4 and iron stores, both cross-sectional and after iron depletion, and in vitro findings suggest that iron could play a role in the RBP4–insulin resistance relationship. PMID:18426863

  13. A new marker for breast cancer diagnosis, human epididymis protein 4: A preliminary study

    PubMed Central

    Gündüz, Umut Riza; Gunaldi, Meral; Isiksacan, Nilgun; Gündüz, Seyda; Okuturlar, Yildiz; Kocoglu, Hakan

    2016-01-01

    Breast cancer is the most frequently diagnosed cancer type in women. Tumor markers have been widely used for assessing the treatment response and early diagnosis of recurrence. Human epididymis protein 4 (HE4) is expressed in ductal carcinoma of the breast tissue; however, its serum levels and their diagnostic and prognostic potential in breast cancer have not been investigated, which was therefore the aim of the present study. The serum levels of HE4 were determined in 36 breast cancer patients, 11 ovarian cancer patients and 16 healthy volunteers. The association between clinicopathological characteristics of breast cancer and serum HE4 levels was investigated. A significant difference in the median serum levels of HE4 was identified between breast cancer patients, ovarian cancer patients and healthy volunteers (P=0.013). The cutoff value for the prediction of breast cancer was determined at >13.24 pmol/l for HE4, with a sensitivity of 61.11%, specificity of 68.75%, positive predictive value of 81.48%, negative predictive value of 44.0% and accuracy of 63.46%. Furthermore, a positive correlation between the serum levels of HE4 and cancer antigen 15–3 was determined (r=0.399, P=0.026). To the best of our knowledge, the present study was the first to determine the diagnostic value of serum HE4 for breast cancer. A significant elevation of serum HE4 levels in patients with breast cancer compared with that in healthy controls was identified. HE4 may serve as a novel biomarker for the diagnosis of breast cancer. PMID:27446579

  14. Regulation of angiopoietin‐like protein 4 production during and after exercise

    PubMed Central

    Norheim, Frode; Hjorth, Marit; Langleite, Torgrim M.; Lee, Sindre; Holen, Torgeir; Bindesbøll, Christian; Stadheim, Hans K.; Gulseth, Hanne L.; Birkeland, Kåre I.; Kielland, Anders; Jensen, Jørgen; Dalen, Knut T.; Drevon, Christian A.

    2014-01-01

    Abstract Angiopoietin‐like protein 4 (ANGPTL4) may regulate lipoprotein lipase‐dependent plasma clearance of triacylglycerol from skeletal muscle during exercise. The aim of this study was to examine the importance of muscle in regulating ANGPTL4 in response to exercise. We sampled muscle biopsies and serum before, immediately after, and 2 h after 45 min of ergometer cycling. Sampling was done before and after a 12‐week training intervention in controls and dysglycemic subjects. Moreover, fat biopsies were taken before and after the training intervention. The regulation of ANGPTL4 was also investigated in several tissues of exercising mice, and in cultured myotubes. ANGPTL4 levels in serum and expression in muscle were highest 2 h after exercise in both groups. Whereas ANGPTL4 was higher in muscle of exercising controls as compared to dysglycemic subjects, the opposite was observed in serum. In exercising mice, Angptl4 mRNA showed both higher basal expression and induction in liver compared to muscle. Angptl4 mRNA was much higher in adipose tissue than muscle and was also induced by exercise. We observed two mRNA isoforms of ANGPTL4 in muscle and fat in humans. Both were induced by exercise in muscle; one isoform was expressed 5‐ to 10‐fold higher than the other. Studies in mice and cultured myotubes showed that both fatty acids and cortisol have the potential to increase ANGPTL4 expression in muscle during exercise. In conclusion, ANGPTL4 is markedly induced in muscle in response to exercise. However, liver and adipose tissue may contribute more than muscle to the exercise‐induced increase in circulating ANGPTL4. PMID:25138789

  15. Phylogenetic Relationships and the Evolution of Regulatory Gene Sequences in the Parrotfishes

    PubMed Central

    Smith, Lydia L.; Fessler, Jennifer L.; Alfaro, Michael E.; Streelman, J. Todd; Westneat, Mark W.

    2008-01-01

    Regulatory genes control the expression of other genes and are key components of developmental processes such as segmentation and embryonic construction of the skull in vertebrates. Here we examine the variability and evolution of three vertebrate regulatory genes, addressing issues of their utility for phylogenetics and comparing the rates of genetic change seen in regulatory loci to the rates seen in other genes in the parrotfishes. The parrotfishes are a diverse group of colorful fishes from coral reefs and seagrasses worldwide and have been placed phylogenetically within the family Labridae. We tested phylogenetic hypotheses among the parrotfishes, with a focus on the genera Chlorurus and Scarus, by analyzing eight gene fragments for 42 parrotfishes and eight outgroup species. We sequenced mitochondrial 12s rRNA (967 bp), 16s rRNA (577 bp), and cytochrome b (477 bp). From the nuclear genome, we sequenced part of the protein-coding genes rag2 (715 bp), tmo4c4 (485 bp), and the developmental regulatory genes otx1 (672 bp), bmp4 (488 bp), and dlx2 (522 bp). Bayesian, likelihood, and parsimony analyses on the resulting 4903 bp of DNA sequence produced similar topologies that confirm the monophyly of the scarines and provide a phylogeny at the species level for portions of the genera Scarus and Chlorurus. Four major clades of Scarus were recovered, with three distributed in the Indo-Pacific and one containing Caribbean/Atlantic taxa. Molecular rates suggest a Miocene origin of the parrotfishes (22 mya) and a recent divergence of species within Scarus and Chlorurus, within the past 5 million years. Developmentally important genes made a significant contribution to phylogenetic structure, and rates of genetic evolution were high in bmp4, similar to other coding nuclear genes, but low in otx1 and the dlx2 exons. Synonymous and nonsynonymous substitution patterns in developmental regulatory genes support the hypothesis of stabilizing selection during the history of

  16. Retinol-binding protein 4 and its potential roles in hypercholesterolemia revealed by proteomics

    PubMed Central

    Jugnam-ang, Watcharapong; Pannengpetch, Supitcha; Isarankura-Na-Ayudhya, Patcharee; Thippakorn, Chadinee; Isarankura-Na-Ayudhya, Chartchalerm; Lawung, Ratana; Prachayasittiku, Virapong

    2015-01-01

    Effects of hypercholesterolemia on alterations of serum proteins have not been fully elucidated. Herein, using two-dimensional gel electrophoresis (2-DE) in conjunction with LC-MS searching has successfully been carried out to investigate the change of protein expression profiles as consequences of raised blood cholesterol at different levels (normal group: total cholesterol 200 mg/dL; borderline high group: total cholesterol 200-239 mg/dL; and high group: total cholesterol ≥ 240 mg/dL) (n = 45). Results revealed that down-regulation of retinol-binding protein 4 (RBP4) (-2.26 fold), transthyretin (-1.25 fold) and gelsolin (-1.47 fold) was observed in the high group. Meanwhile, the other proteins such as haptoglobin, complement factor B and CD5 antigen-like protein were up-regulated upto +3.24, +1.96 and +2.04 fold, respectively. Confirmation by Western blotting revealed a significant reduction of RBP4 (approximately 50 %) in individual samples derived from the high group. Presumptive conclusion can be drawn that down-regulation of RBP4 might be attributable to the inflammation of adipocytes caused by the release of proinflammatory cytokines (e.g. tumor necrosis factor α and interleukin-1β) from adipose tissues. Moreover, the decrease of transthyretin might also be taken into accounts since it is known that the transthyretin usually forms complex with RBP4 to prevent glomerular filtration and excretion through the kidney. The suppressing effect on RBP4 should be potentiated by the increase of complement factor B and CD5 antigen-like protein, which rendered the adipose tissues to overwhelm the liberation of RBP4 to blood circulation by metabolic and inflammatory processes. Such inflammation could further modulate the induction of cytokine release (e.g. IL-6 and IL-1β), resulting in the synthesis of acute phase protein, in particular, haptoglobin and C-reactive proteins from hepatocytes. However, the mechanism of gelsolin reduction remains unclear. Among these

  17. Retinoblastoma Binding Protein 4 Modulates Temozolomide Sensitivity in Glioblastoma by Regulating DNA Repair Proteins

    PubMed Central

    Kitange, Gaspar J.; Mladek, Ann C.; Schroeder, Mark A.; Pokorny, Jenny C.; Carlson, Brett L.; Zhang, Yuji; Nair, Asha A.; Lee, Jeong-Heon; Yan, Huihuang; Decker, Paul A.; Zhang, Zhiguo; Sarkaria, Jann N.

    2016-01-01

    Summary Here we provide evidence that RBBP4 modulates temozolomide (TMZ) sensitivity through coordinate regulation of 2 key DNA repair genes critical for recovery from TMZ-induced DNA damage: methylguanine-DNA-methyltransferase (MGMT) and RAD51. Disruption of RBBP4 enhanced TMZ sensitivity, induced synthetic lethality to PARP inhibition and increased DNA damage signaling in response to TMZ. Moreover, RBBP4 silencing enhanced TMZ-induced H2AX phosphorylation and apoptosis in GBM cells. Intriguingly, RBBP4 knockdown suppressed the expression of MGMT, RAD51 and other genes in association with decreased promoter H3K9 acetylation (H3K9Ac) and increased H3K9 tri-methylation (H3K9me3). Consistent with these data, RBBP4 interacts with CBP/p300 to form a chromatin modifying complex that binds within the promoter of MGMT, RAD51 and perhaps other genes. Globally, RBBP4 positively and negatively regulates genes involved in critical cellular functions including tumorigenesis. RBBP4/CBP/p300 complex may provide an interesting target for developing therapy sensitizing strategies for GBM and other tumors. PMID:26972001

  18. Purmorphamine induces osteogenesis by activation of the hedgehog signaling pathway.

    PubMed

    Wu, Xu; Walker, John; Zhang, Jie; Ding, Sheng; Schultz, Peter G

    2004-09-01

    Previously, a small molecule, purmorphamine, was identified that selectively induces osteogenesis in multipotent mesenchymal progenitor cells. In order to gain insights into the mechanism of action of purmorphamine, high-density oligonucleotide microarrays were used to profile gene expression in multipotent mesenchymal progenitor cells treated with either purmorphamine or bone morphogenetic protein-4 (BMP-4). In contrast to BMP-4 treatment, purmorphamine activates the Hedgehog (Hh) signaling pathway, resulting in the up- and downregulation of its downstream target genes, including Gli1 and Patched. Moreover, the known Hh signaling antagonists, cyclopamine and forskolin, completely block the osteogenesis and Glimediated transcription induced by purmorphamine. These results demonstrate that purmorphamine is a small molecule agonist of Hedgehog signaling, and it may ultimately be useful in the treatment of bone-related disease and neurodegenerative disease. PMID:15380183

  19. Eye development genes and known syndromes.

    PubMed

    Slavotinek, Anne M

    2011-12-01

    Anophthalmia and microphthalmia (A/M) are significant eye defects because they can have profound effects on visual acuity. A/M is associated with non-ocular abnormalities in an estimated 33-95% of cases and around 25% of patients have an underlying genetic syndrome that is diagnosable. Syndrome recognition is important for targeted molecular genetic testing, prognosis and for counseling regarding recurrence risks. This review provides clinical and molecular information for several of the commonest syndromes associated with A/M: Anophthalmia-Esophageal-Genital syndrome, caused by SOX2 mutations, Anophthalmia and pituitary abnormalities caused by OTX2 mutations, Matthew-Wood syndrome caused by STRA6 mutations, oculofaciocardiodental syndrome and Lenz microphthalmia caused by BCOR mutations, Microphthalmia Linear Skin pigmentation syndrome caused by HCCS mutations, Anophthalmia, pituitary abnormalities, polysyndactyly caused by BMP4 mutations and Waardenburg anophthalmia caused by mutations in SMOC1. In addition, we briefly discuss the ocular and extraocular phenotypes associated with several other important eye developmental genes, including GDF6, VSX2, RAX, SHH, SIX6 and PAX6. PMID:22005280

  20. An Integrated Gene Regulatory Network Controls Stem Cell Proliferation in Teeth

    PubMed Central

    Felszeghy, Szabolcs; Zelarayan, Laura C; Alonso, Maria T; Plikus, Maksim V; Maas, Richard L; Chuong, Cheng-Ming; Schimmang, Thomas; Thesleff, Irma

    2007-01-01

    Epithelial stem cells reside in specific niches that regulate their self-renewal and differentiation, and are responsible for the continuous regeneration of tissues such as hair, skin, and gut. Although the regenerative potential of mammalian teeth is limited, mouse incisors grow continuously throughout life and contain stem cells at their proximal ends in the cervical loops. In the labial cervical loop, the epithelial stem cells proliferate and migrate along the labial surface, differentiating into enamel-forming ameloblasts. In contrast, the lingual cervical loop contains fewer proliferating stem cells, and the lingual incisor surface lacks ameloblasts and enamel. Here we have used a combination of mouse mutant analyses, organ culture experiments, and expression studies to identify the key signaling molecules that regulate stem cell proliferation in the rodent incisor stem cell niche, and to elucidate their role in the generation of the intrinsic asymmetry of the incisors. We show that epithelial stem cell proliferation in the cervical loops is controlled by an integrated gene regulatory network consisting of Activin, bone morphogenetic protein (BMP), fibroblast growth factor (FGF), and Follistatin within the incisor stem cell niche. Mesenchymal FGF3 stimulates epithelial stem cell proliferation, and BMP4 represses Fgf3 expression. In turn, Activin, which is strongly expressed in labial mesenchyme, inhibits the repressive effect of BMP4 and restricts Fgf3 expression to labial dental mesenchyme, resulting in increased stem cell proliferation and a large, labial stem cell niche. Follistatin limits the number of lingual stem cells, further contributing to the characteristic asymmetry of mouse incisors, and on the basis of our findings, we suggest a model in which Follistatin antagonizes the activity of Activin. These results show how the spatially restricted and balanced effects of specific components of a signaling network can regulate stem cell proliferation in

  1. Xenopus tropicalis nodal-related gene 3 regulates BMP signaling: an essential role for the pro-region.

    PubMed

    Haramoto, Yoshikazu; Tanegashima, Kousuke; Onuma, Yasuko; Takahashi, Shuji; Sekizaki, Hiroyuki; Asashima, Makoto

    2004-01-01

    In vertebrates, nodal-related genes are crucial for specifying mesendodermal cell fates. Six nodal-related genes have been identified in Xenopus, but only one, nodal, has been identified in the mouse. The Xenopus nodal-related gene 3 (Xnr3), however, lacks the mesoderm-inducing activity of the other five nodal-related genes in Xenopus, and can directly induce neural tissue in animal caps by antagonizing BMP signals. In this study, we isolated three clones of the Xenopus (Silurana) tropicalis nodal-related gene 3 (Xtnr3) and analyzed their function. The Xtnr3 genes show high homology to Xnr3 and have the same activity. Southern blot and genomic PCR analyses indicate that the X. tropicalis genome has duplications in the Xtnr3 gene sequences and our three clones represent separate gene loci. We also found a partial clone of Xtnr3 that coded for the N-terminal part of its pro-region. Surprisingly, this sequence also induced neural tissue by antagonizing BMP signals, and its coded protein physically associated with BMP4 mature protein. Furthermore, we showed that the pro-region of Xnr5 has the same activity. Together, these findings indicate that the pro-region of nodal-related genes acts antagonistically towards BMP signals, which identifies a novel mechanism for the inhibition of BMP signaling. PMID:14697360

  2. Genotype-Specific Interaction of Latent TGFβ Binding Protein 4 with TGFβ

    PubMed Central

    Lamar, Kay-Marie; Miller, Tamari; Dellefave-Castillo, Lisa; McNally, Elizabeth M.

    2016-01-01

    Latent TGFβ binding proteins are extracellular matrix proteins that bind latent TGFβ to form the large latent complex. Nonsynonymous polymorphisms in LTBP4, a member of the latent TGFβ binding protein gene family, have been linked to several human diseases, underscoring the importance of TGFβ regulation for a range of phenotypes. Because of strong linkage disequilibrium across the LTBP4 gene, humans have two main LTBP4 alleles that differ at four amino acid positions, referred to as IAAM and VTTT for the encoded residues. VTTT is considered the “risk” allele and associates with increased intracellular TGFβ signaling and more deleterious phenotypes in muscular dystrophy and other diseases. We now evaluated LTBP4 nsSNPs in dilated cardiomyopathy, a distinct disorder associated with TGFβ signaling. We stratified based on self-identified ethnicity and found that the LTBP4 VTTT allele is associated with increased risk of dilated cardiomyopathy in European Americans extending the diseases that associate with LTBP4 genotype. However, the association of LTBP4 SNPs with dilated cardiomyopathy was not observed in African Americans. To elucidate the mechanism by which LTBP4 genotype exerts this differential effect, TGFβ’s association with LTBP4 protein was examined. LTBP4 protein with the IAAM residues bound more latent TGFβ compared to the LTBP4 VTTT protein. Together these data provide support that LTBP4 genotype exerts its effect through differential avidity for TGFβ accounting for the differences in TGFβ signaling attributed to these two alleles. PMID:26918958

  3. Protein 4.1 R-135 Interacts with a Novel Centrosomal Protein (CPAP) Which Is Associated with the γ-Tubulin Complex

    PubMed Central

    Hung, Liang-Yi; Tang, Chieh-Ju C.; Tang, Tang K.

    2000-01-01

    Using a yeast two-hybrid system, we isolated a novel human centrosomal protein, CPAP (centrosomal P4.1-associated protein), which specifically interacts with the head domain of the 135-kDa protein 4.1R isoform (4.1R-135). Sequence analysis revealed that the carboxyl terminus of CPAP has 31.3% amino acid identity with human Tcp-10 (a t-complex responder gene product). Interestingly, most of the sequence identity is restricted to two conserved regions. One carries a leucine zipper, which may form a series of heptad repeats involved in coiled-coil formation; the other contains unusual glycine repeats with unknown function. Immunofluorescence analysis revealed that CPAP and γ-tubulin are localized within the centrosome throughout the cell cycle. CPAP cosediments with γ-tubulin in sucrose gradients and coimmunoprecipitates with γ-tubulin, indicating that CPAP is a part of the γ-tubulin complex. Furthermore, functional analysis revealed that CPAP is localized within the center of microtubule asters and may participate in microtubule nucleation. The formation of microtubule asters was significantly inhibited by anti-CPAP antibody. Together, these observations indicate that CPAP may play an important role in cell division and centrosome function. PMID:11003675

  4. Protein 4.1 R-135 interacts with a novel centrosomal protein (CPAP) which is associated with the gamma-tubulin complex.

    PubMed

    Hung, L Y; Tang, C J; Tang, T K

    2000-10-01

    Using a yeast two-hybrid system, we isolated a novel human centrosomal protein, CPAP (centrosomal P4.1-associated protein), which specifically interacts with the head domain of the 135-kDa protein 4.1R isoform (4.1R-135). Sequence analysis revealed that the carboxyl terminus of CPAP has 31.3% amino acid identity with human Tcp-10 (a t-complex responder gene product). Interestingly, most of the sequence identity is restricted to two conserved regions. One carries a leucine zipper, which may form a series of heptad repeats involved in coiled-coil formation; the other contains unusual glycine repeats with unknown function. Immunofluorescence analysis revealed that CPAP and gamma-tubulin are localized within the centrosome throughout the cell cycle. CPAP cosediments with gamma-tubulin in sucrose gradients and coimmunoprecipitates with gamma-tubulin, indicating that CPAP is a part of the gamma-tubulin complex. Furthermore, functional analysis revealed that CPAP is localized within the center of microtubule asters and may participate in microtubule nucleation. The formation of microtubule asters was significantly inhibited by anti-CPAP antibody. Together, these observations indicate that CPAP may play an important role in cell division and centrosome function. PMID:11003675

  5. The linker histone in Saccharomyces cerevisiae interacts with actin-related protein 4 and both regulate chromatin structure and cellular morphology.

    PubMed

    Georgieva, Milena; Staneva, Dessislava; Uzunova, Katya; Efremov, Toni; Balashev, Konstantin; Harata, Masahiko; Miloshev, George

    2015-02-01

    Chromatin structure promotes important epigenetic mechanisms that regulate cellular fate by organizing, preserving and controlling the way by which the genetic information works. Our understanding of chromatin and its functions is sparse and not yet well defined. The uncertainty comes from the complexity of chromatin and is induced by the existence of a large number of nuclear proteins that influence it. The intricate interaction among all these structural and functional nuclear proteins has been under extensive study in the recent years. Here, we show that Saccharomyces cerevisiae linker histone physically interacts with Arp4p (actin-related protein 4) which is a key subunit of three chromatin modifying complexes - INO80, SWR1 and NuA4. A single - point mutation in the actin - fold domain of Arp4p together with the knock-out of the gene for the linker histone in S. cerevisiae severely abrogates cellular and nuclear morphology and leads to complete disorganizing of the higher levels of chromatin organization. PMID:25542182

  6. Homozygous deletion of EPB41 genuine AUG-containing exons results in mRNA splicing defects, NMD activation and protein 4.1R complete deficiency in hereditary elliptocytosis.

    PubMed

    Baklouti, Faouzi; Morinière, Madeleine; Haj-Khélil, Amel; Fénéant-Thibault, Madeleine; Gruffat, Henri; Couté, Yohann; Ninot, Alain; Guitton, Corinne; Delaunay, Jean

    2011-10-15

    Complete loss of protein 4.1R in red blood cell membrane is a very rare condition in humans. We here explore the third case. The morphological and biochemical observations suggested that the proband suffers from homozygous hereditary elliptocytosis. Both parents, who are consanguineous, have an elliptocytosis with no cell fragmentation, typical of a heterozygous 4.1R deficiency with a silent allele. A genomic deletion was found; it encompasses about 50 kb of genomic DNA, and suppresses the two key exons 2 and 4, which contain the two functional AUG translation initiation sites in erythroid and nonerythroid cells. The alternative first exons are intact, hence preserving the transcription potential of the altered gene. Extensive analysis of 4.1R transcripts revealed multiple splicing defects upstream of the deleted sequences. Importantly, we found that most of the transcripts generated from the altered gene are intercepted by the nonsense-mediated mRNA decay mechanism, suggesting that the massive degradation of the mRNA species jeopardizes the production of shortened but functional protein 4.1R from an alternative translation initiation site downstream of the deletion. PMID:21839655

  7. Secreted Frizzled-Related Protein 4 (SFRP4) is Elevated in Patients with Diabetes Mellitus.

    PubMed

    Brix, J M; Krzizek, E C; Hoebaus, C; Ludvik, B; Schernthaner, G; Schernthaner, G H

    2016-05-01

    Recently, SFRP4 was identified as a molecular link between islet inflammation and defective insulin secretion. Gene co-expression analysis detected a molecule associated with type 2 diabetes mellitus (T2D), elevated HbA1c, and reduced insulin secretion in mice as well as in a pilot sample of humans. To our knowledge SFRP4 has never been investigated in patients with different types of diabetes. We included 179 patients: 46 with type 1 diabetes (T1D), 30 age matched healthy controls for patients with T1D (CO-T1D), 55 with T2D, 37 with latent autoimmune diabetes of the adult (LADA) and 30 healthy controls (CO) for patients with T2D and LADA. Apart from anthropometric data, lipids and renal parameters were assessed. SFRP4 levels were measured by a commercial ELISA. Patients with diabetes had significant higher SFRP4 levels than CO: T2D vs. CO: 37.1±26.7 vs. 8.8±3.0 ng/ml, p<0.001; LADA vs. CO: 15.6±6.2 vs. 8.7±3.0 ng/ml, p<0.001; T1D vs. CO-T1D: 24.6±17.9 vs. 16.9±4.5 ng/ml, p=0.011. SFRP4 levels were correlated with age, BMI, HbA1c, HDL-cholesterol, and triglycerides. A multivariate model revealed HDL-cholesterol, triglycerides and BMI as predictors for SFRP4. This is the first study demonstrating that SFRP4 is significantly increased in patients with different types of diabetes suggesting that this protein is generally involved in islet dysfunction and potentially subclinical inflammation irrespective of type of diabetes. PMID:26882051

  8. Overexpression of Latent TGFβ Binding Protein 4 in Muscle Ameliorates Muscular Dystrophy through Myostatin and TGFβ.

    PubMed

    Lamar, Kay-Marie; Bogdanovich, Sasha; Gardner, Brandon B; Gao, Quan Q; Miller, Tamari; Earley, Judy U; Hadhazy, Michele; Vo, Andy H; Wren, Lisa; Molkentin, Jeffery D; McNally, Elizabeth M

    2016-05-01

    Latent TGFβ binding proteins (LTBPs) regulate the extracellular availability of latent TGFβ. LTBP4 was identified as a genetic modifier of muscular dystrophy in mice and humans. An in-frame insertion polymorphism in the murine Ltbp4 gene associates with partial protection against muscular dystrophy. In humans, nonsynonymous single nucleotide polymorphisms in LTBP4 associate with prolonged ambulation in Duchenne muscular dystrophy. To better understand LTBP4 and its role in modifying muscular dystrophy, we created transgenic mice overexpressing the protective murine allele of LTBP4 specifically in mature myofibers using the human skeletal actin promoter. Overexpression of LTBP4 protein was associated with increased muscle mass and proportionally increased strength compared to age-matched controls. In order to assess the effects of LTBP4 in muscular dystrophy, LTBP4 overexpressing mice were bred to mdx mice, a model of Duchenne muscular dystrophy. In this model, increased LTBP4 led to greater muscle mass with proportionally increased strength, and decreased fibrosis. The increase in muscle mass and reduction in fibrosis were similar to what occurs when myostatin, a related TGFβ family member and negative regulator of muscle mass, was deleted in mdx mice. Supporting this, we found that myostatin forms a complex with LTBP4 and that overexpression of LTBP4 led to a decrease in myostatin levels. LTBP4 also interacted with TGFβ and GDF11, a protein highly related to myostatin. These data identify LTBP4 as a multi-TGFβ family ligand binding protein with the capacity to modify muscle disease through overexpression. PMID:27148972

  9. Overexpression of Latent TGFβ Binding Protein 4 in Muscle Ameliorates Muscular Dystrophy through Myostatin and TGFβ

    PubMed Central

    Gardner, Brandon B.; Gao, Quan Q.; Hadhazy, Michele; Vo, Andy H.; Wren, Lisa; Molkentin, Jeffery D.; McNally, Elizabeth M.

    2016-01-01

    Latent TGFβ binding proteins (LTBPs) regulate the extracellular availability of latent TGFβ. LTBP4 was identified as a genetic modifier of muscular dystrophy in mice and humans. An in-frame insertion polymorphism in the murine Ltbp4 gene associates with partial protection against muscular dystrophy. In humans, nonsynonymous single nucleotide polymorphisms in LTBP4 associate with prolonged ambulation in Duchenne muscular dystrophy. To better understand LTBP4 and its role in modifying muscular dystrophy, we created transgenic mice overexpressing the protective murine allele of LTBP4 specifically in mature myofibers using the human skeletal actin promoter. Overexpression of LTBP4 protein was associated with increased muscle mass and proportionally increased strength compared to age-matched controls. In order to assess the effects of LTBP4 in muscular dystrophy, LTBP4 overexpressing mice were bred to mdx mice, a model of Duchenne muscular dystrophy. In this model, increased LTBP4 led to greater muscle mass with proportionally increased strength, and decreased fibrosis. The increase in muscle mass and reduction in fibrosis were similar to what occurs when myostatin, a related TGFβ family member and negative regulator of muscle mass, was deleted in mdx mice. Supporting this, we found that myostatin forms a complex with LTBP4 and that overexpression of LTBP4 led to a decrease in myostatin levels. LTBP4 also interacted with TGFβ and GDF11, a protein highly related to myostatin. These data identify LTBP4 as a multi-TGFβ family ligand binding protein with the capacity to modify muscle disease through overexpression. PMID:27148972

  10. Genetic and epigenetic mutations of tumor suppressive genes in sporadic pituitary adenoma.

    PubMed

    Zhou, Yunli; Zhang, Xun; Klibanski, Anne

    2014-04-01

    Human pituitary adenomas are the most common intracranial neoplasms. Approximately 5% of them are familial adenomas. Patients with familial tumors carry germline mutations in predisposition genes, including AIP, MEN1 and PRKAR1A. These mutations are extremely rare in sporadic pituitary adenomas, which therefore are caused by different mechanisms. Multiple tumor suppressive genes linked to sporadic tumors have been identified. Their inactivation is caused by epigenetic mechanisms, mainly promoter hypermethylation, and can be placed into two groups based on their functional interaction with tumor suppressors RB or p53. The RB group includes CDKN2A, CDKN2B, CDKN2C, RB1, BMP4, CDH1, CDH13, GADD45B and GADD45G; AIP and MEN1 genes also belong to this group. The p53 group includes MEG3, MGMT, PLAGL1, RASSF1, RASSF3 and SOCS1. We propose that the tumor suppression function of these genes is mainly mediated by the RB and p53 pathways. We also discuss possible tumor suppression mechanisms for individual genes. PMID:24035864

  11. Methionine restriction decreases endogenous oxidative molecular damage and increases mitochondrial biogenesis and uncoupling protein 4 in rat brain.

    PubMed

    Naudí, Alba; Caro, Pilar; Jové, Mariona; Gómez, José; Boada, Jordi; Ayala, Victoria; Portero-Otín, Manuel; Barja, Gustavo; Pamplona, Reinald

    2007-12-01

    Aging plays a central role in the occurrence of neurodegenerative diseases. Caloric restriction (CR) mitigates oxidative stress by decreasing the rate of generation of endogenous damage, a mechanism that can contribute to the slowing of the aging rate induced by this intervention. Various reports have recently linked methionine to aging, and methionine restriction (MetR) without energy restriction also increases life span. We have thus hypothesized that MetR can be responsible, at least in part, for the decrease in endogenous oxidative damage in CR. In this investigation we subjected male rats to exactly the same dietary protocol of MetR that is known to increase their life span. We have found that MetR: (1) decreases the mitochondrial complex I content and activity, as well as complex III content, while the complex II and IV, the mitochondrial flavoprotein apoptosis-inducing factor (AIF) and ATP content are unchanged; (2) increases the mitochondrial biogenesis factor PGC-1alpha; (3) increases the resistance of brain to metabolic and oxidative stress by increasing mitochondrial uncoupling protein 4 uncoupling protein 4 (UCP4); and (4) decreases mitochondrial oxidative DNA damage and all five different markers of protein oxidation measured and lowers membrane unsaturation in rat brain. No changes were detected for protein amino acid composition. These beneficial MetR-induced changes likely derived from metabolic reprogramming at the cellular and tissue level can play a key role in the protection against aging-associated neurodegenerative disorders. PMID:17716000

  12. Gene expression responses for detecting sublethal effects of xenobiotics and whole effluents on a Xenopus laevis embryo assay.

    PubMed

    San Segundo, Laura; Martini, Federica; Pablos, M Victoria

    2013-09-01

    In the present study, the authors investigated the effects of bisphenol A, chlorpyrifos, methylparaben, and 2 effluent samples from wastewater treatment plants located in the province of Madrid, Spain, on the messenger RNA expression of specific genes involved in early development (ESR1, pax6, bmp4, and myf5) and a gene involved in the general stress response (hsp70) during Xenopus laevis embryo development. Gene expression was analyzed after 4 h, 24 h, and 96 h of exposure by semiquantitative reverse-transcriptase-polymerase chain reaction. Concentration ranges of the compounds and dilutions for the samples were selected to cause morphological alterations in embryos after 96 h of exposure. Transcript levels of ESR1, pax6, and hsp70 were differentially altered at early developmental stages with patterns specific to the contaminant and the exposure time. However, further studies are needed to establish transcript levels of specific genes as biomarkers of sublethal effects in an environmental risk-assessment framework. Besides, studies including more generic responses, such as genes encoding antioxidant enzymes, together with genes related to embryonic development have to be developed to look for a battery of mechanistic endpoints for the evaluation of chemical exposure at the molecular level in a first-tier assessment. PMID:23637088

  13. Activation of Mitochondrial Uncoupling Protein 4 and ATP-Sensitive Potassium Channel Cumulatively Decreases Superoxide Production in Insect Mitochondria.

    PubMed

    Slocińska, Malgorzata; Rosinski, Grzegorz; Jarmuszkiewicz, Wieslawa

    2016-01-01

    It has been evidenced that mitochondrial uncoupling protein 4 (UCP4) and ATP-regulated potassium channel (mKATP channel) of insect Gromphadorhina coqereliana mitochondria decrease superoxide anion production. We elucidated whether the two energy-dissipating systems work together on a modulation of superoxide level in cockroach mitochondria. Our data show that the simultaneous activation of UCP4 by palmitic acid and mKATP channel by pinacidil revealed a cumulative effect on weakening mitochondrial superoxide formation. The inhibition of UCP4 by GTP (and/or ATP) and mKATP channel by ATP elevated superoxide production. These results suggest a functional cooperation of both energy-dissipating systems in protection against oxidative stress in insects. PMID:26548865

  14. Computer aided screening of secreted frizzled-related protein 4 (SFRP4): a potential control for diabetes mellitus.

    PubMed

    Bukhari, Shazia Anwer; Shamshari, Waseem Akhtar; Ur-Rahman, Mahmood; Zia-Ul-Haq, Muhammad; Jaafar, Hawa Z E

    2014-01-01

    Diabetes mellitus is a life threatening disease and scientists are doing their best to find a cost effective and permanent treatment of this malady. The recent trend is to control the disease by target base inhibiting of enzymes or proteins. Secreted frizzled-related protein 4 (SFRP4) is found to cause five times more risk of diabetes when expressed above average levels. This study was therefore designed to analyze the SFRP4 and to find its potential inhibitors. SFRP4 was analyzed by bio-informatics tools of sequence tool and structure tool. A total of three potential inhibitors of SFRP4 were found, namely cyclothiazide, clopamide and perindopril. These inhibitors showed significant interactions with SFRP4 as compared to other inhibitors as well as control (acetohexamide). The findings suggest the possible treatment of diabetes mellitus type 2 by inhibiting the SFRP4 using the inhibitors cyclothiazide, clopamide and perindopril. PMID:25019556

  15. Fatty acid binding protein 4 is a target of VEGF and a regulator of cell proliferation in endothelial cells

    PubMed Central

    Elmasri, Harun; Karaaslan, Cagatay; Teper, Yaroslav; Ghelfi, Elisa; Weng, MeiQian; Ince, Tan A.; Kozakewich, Harry; Bischoff, Joyce; Cataltepe, Sule

    2009-01-01

    Fatty acid binding protein 4 (FABP4) plays an important role in maintaining glucose and lipid homeostasis. FABP4 has been primarily regarded as an adipocyte- and macrophage-specific protein, but recent studies suggest that it may be more widely expressed. We found strong FABP4 expression in the endothelial cells (ECs) of capillaries and small veins in several mouse and human tissues, including the heart and kidney. FABP4 was also detected in the ECs of mature human placental vessels and infantile hemangiomas, the most common tumor of infancy and ECs. In most of these cases, FABP4 was detected in both the nucleus and cytoplasm. FABP4 mRNA and protein levels were significantly induced in cultured ECs by VEGF-A and bFGF treatment. The effect of VEGF-A on FABP4 expression was inhibited by chemical inhibition or short-hairpin (sh) RNA-mediated knockdown of VEGF-receptor-2 (R2), whereas the VEGFR1 agonists, placental growth factors 1 and 2, had no effect on FABP4 expression. Knockdown of FABP4 in ECs significantly reduced proliferation both under baseline conditions and in response to VEGF and bFGF. Thus, FABP4 emerged as a novel target of the VEGF/VEGFR2 pathway and a positive regulator of cell proliferation in ECs.—Elmasri, H., Karaaslan, C., Teper, Y., Ghelfi, E., Weng, M., Ince, T. A., Kozakewich, H., Bischoff, J., Cataltepe, S. Fatty acid binding protein 4 is a target of VEGF and a regulator of cell proliferation in endothelial cells. PMID:19625659

  16. Fatty acid binding protein 4 expression marks a population of adipocyte progenitors in white and brown adipose tissues

    PubMed Central

    Shan, Tizhong; Liu, Weiyi; Kuang, Shihuan

    2013-01-01

    Adipose tissues regulate metabolism, reproduction, and life span. The development and growth of adipose tissue are due to increases of both adipocyte cell size and cell number; the latter is mediated by adipocyte progenitors. Various markers have been used to identify either adipocyte progenitors or mature adipocytes. The fatty acid binding protein 4 (FABP4), commonly known as adipocyte protein 2 (aP2), has been extensively used as a marker for differentiated adipocytes. However, whether aP2 is expressed in adipogenic progenitors is controversial. Using Cre/LoxP-based cell lineage tracing in mice, we have identified a population of aP2-expressing progenitors in the stromal vascular fraction (SVF) of both white and brown adipose tissues. The aP2-lineage progenitors reside in the adipose stem cell niche and express adipocyte progenitor markers, including CD34, Sca1, Dlk1, and PDGFRα. When isolated and grown in culture, the aP2-expressing SVF cells proliferate and differentiate into adipocytes upon induction. Conversely, ablation of the aP2 lineage greatly reduces the adipogenic potential of SVF cells. When grafted into wild-type mice, the aP2-lineage progenitors give rise to adipose depots in recipient mice. Therefore, the expression of aP2 is not limited to mature adipocytes, but also marks a pool of undifferentiated progenitors associated with the vasculature of adipose tissues. Our finding adds to the repertoire of adipose progenitor markers and points to a new regulator of adipose plasticity.—Shan, T., Liu, W., Kuang, S. Fatty acid-binding protein 4 expression marks a population of adipocyte progenitors in white and brown adipose tissues. PMID:23047894

  17. Loss of Secreted Frizzled-Related Protein 4 Correlates with an Aggressive Phenotype and Predicts Poor Outcome in Ovarian Cancer Patients

    PubMed Central

    Nixdorf, Sheri; Ford, Caroline E.; Olivier, Jake; Caduff, Rosmarie; Scurry, James P.; Guertler, Rea; Hornung, Daniela; Mueller, Renato; Fink, Daniel A.; Hacker, Neville F.; Heinzelmann-Schwarz, Viola A.

    2012-01-01

    Background Activation of the Wnt signaling pathway is implicated in aberrant cellular proliferation in various cancers. In 40% of endometrioid ovarian cancers, constitutive activation of the pathway is due to oncogenic mutations in β-catenin or other inactivating mutations in key negative regulators. Secreted frizzled-related protein 4 (SFRP4) has been proposed to have inhibitory activity through binding and sequestering Wnt ligands. Methodology/Principal Findings We performed RT-qPCR and Western-blotting in primary cultures and ovarian cell lines for SFRP4 and its key downstream regulators activated β-catenin, β-catenin and GSK3β. SFRP4 was then examined by immunohistochemistry in a cohort of 721 patients and due to its proposed secretory function, in plasma, presenting the first ELISA for SFRP4. SFRP4 was most highly expressed in tubal epithelium and decreased with malignant transformation, both on RNA and on protein level, where it was even more profound in the membrane fraction (p<0.0001). SFRP4 was expressed on the protein level in all histotypes of ovarian cancer but was decreased from borderline tumors to cancers and with loss of cellular differentiation. Loss of membrane expression was an independent predictor of poor survival in ovarian cancer patients (p = 0.02 unadjusted; p = 0.089 adjusted), which increased the risk of a patient to die from this disease by the factor 1.8. Conclusions/Significance Our results support a role for SFRP4 as a tumor suppressor gene in ovarian cancers via inhibition of the Wnt signaling pathway. This has not only predictive implications but could also facilitate a therapeutic role using epigenetic targets. PMID:22363760

  18. Silencing Angiopoietin-Like Protein 4 (ANGPTL4) Protects Against Lipopolysaccharide-Induced Acute Lung Injury Via Regulating SIRT1 /NF-kB Pathway.

    PubMed

    Guo, Liang; Li, Shaoying; Zhao, Yunfeng; Qian, Pin; Ji, Fuyun; Qian, Lanlan; Wu, Xueling; Qian, Guisheng

    2015-10-01

    Lung inflammation and alveolar epithelial cell death are critical events in the development and progression of acute lung injury (ALI). Although angiopoietin-like protein 4 (ANGPTL4) participates in inflammation, whether it plays important roles in ALI and alveolar epithelial cell inflammatory injury remains unclear. We therefore investigated the role of angptl4 in lipopolysaccharide (LPS)-induced ALI and the associated mechanisms. Lentivirus-mediated short interfering RNA targeted to the mouse angptl4 gene (AngsiRNA) and a negative control lentivirus (NCsiRNA) were intranasally administered to mice. Lung inflammatory injury and the underlying mechanisms for regulation of angptl4 on the LPS-induced ALI were subsequently determined. We reported that angptl4 levels were increased both in human alveolar epithelial A549 cells and lung tissues obtained from a mouse model of LPS-induced ALI. Angptl4 expression was induced by LPS in alveolar epithelial cells, whereas LPS-induced lung inflammation (neutrophils infiltration in the lung tissues, tumor necrosis factor α, interleukin 6), lung permeability (lung wet/dry weight ratio and bronchoalveolar lavage fluid (BALF) protein concentration), tissue damage (caspase3 activation), and mortality rates were attenuated in AngsiRNA-treated mice. The inflammatory reaction (tumor necrosis factor α, interleukin 6) and apoptosis rates were reduced in AngsiRNA(h)-treated A549 cells. Moreover, angptl4 promoted NF-kBp65 expression and suppressed SIRT1 expression both in mouse lungs and A549 cells. Additionally, SIRT1 antagonist nicotinamide (NAM) attenuated the inhibitory effects of AngsiRNA both on LPS-induced NF-kBp65 expression and IL6 expression. These findings suggest that silencing angptl4 protects against LPS-induced ALI via regulating SIRT1/NF-kB signaling pathway. PMID:25727991

  19. Identification of novel GH-regulated genes in C2C12 cells.

    PubMed

    Resmini, E; Morte, B; Sorianello, E; Gallardo, E; de Luna, N; Illa, I; Zorzano, A; Bernal, J; Webb, S M

    2011-12-01

    Growth hormone (GH) is the main regulator of longitudinal growth before puberty, and treatment with human recombinant (rh) GH can increase muscle strength. Nevertheless, molecular mechanisms responsible remain mostly unknown. Many physiological effects of GH require hormone-mediated changes in gene expression. In an attempt to gain insight into the mechanism of GH action in muscle cells we evaluated the effects of rhGH on gene expression profile in a murine skeletal muscle cell line C2C12. The objective of the work was to identify changes in gene expression in the murine skeletal muscle cell line C2C12 after rGH treatment using microarray assays. C2C12 murine skeletal muscle cell cultures were differentiated during 4 days. After 16 h growing in serum-free medium, C2C12 myotubes were stimulated during 6 h with 500 ng/ml rhGH. Four independent sets of experiments were performed to identify GH-regulated genes. Total RNA was isolated and subjected to analysis. To validate changes candidate genes were analyzed by real-time quantitative polymerase chain reaction. One hundred and fifty-four differentially expressed genes were identified; 90 upregulated and 64 downregulated. Many had not been previously identified as GH-responsive. Real-time PCR in biological replicates confirmed the effect of rGH on 15 genes: Cish, Serpina3g, Socs2, Bmp4, Tnfrsf11b, Rgs2, Tgfbr3, Ugdh, Npy1r, Gbp6, Tgfbi, Tgtp, Btc, Clec3b, and Bcl6. This study shows modifications in the gene expression profile of the C2C12 cell line after rhGH exposure. In vitro and gene function analysis revealed genes involved in skeletal and muscle system as well as cardiovascular system development and function. PMID:22072432

  20. Analysis of Gene Expression in PTHrP−/− Mammary Buds Supports a Role for BMP Signaling and MMP2 in the Initiation of Ductal Morphogenesis

    PubMed Central

    Hens, Julie; Dann, Pamela; Hiremath, Minoti; Pan, Tien-Chi; Chodosh, Lewis; Wysolmerski, John

    2010-01-01

    Parathyroid hormone-related protein (PTHrP) acts on the mammary mesenchyme and is required for proper embryonic mammary development. In order to understand PTHrP’s effects on mesenchymal cells, we profiled gene expression in WT and PTHrP−/− mammary buds, and in WT and K14-PTHrP ventral skin at E15.5. By cross-referencing the differences in gene expression between these groups, we identified 35 genes potentially regulated by PTHrP in the mammary mesenchyme, including 6 genes known to be involved in BMP signaling. One of these genes was MMP2. We demonstrated that PTHrP and BMP4 regulate MMP2 gene expression and MMP2 activity in mesenchymal cells. Using mammary bud cultures, we demonstrated that MMP2 acts downstream of PTHrP to stimulate ductal outgrowth. Future studies on the functional role of other genes on this list should expand our knowledge of how PTHrP signaling triggers the onset of ductal outgrowth from the embryonic mammary buds. PMID:19795511

  1. Role for the nuclear receptor-binding SET domain protein 1 (NSD1) methyltransferase in coordinating lysine 36 methylation at histone 3 with RNA polymerase II function

    PubMed Central

    Lucio-Eterovic, Agda Karina; Singh, Melissa M.; Gardner, Jeffrey E.; Veerappan, Chendhore S.; Rice, Judd C.; Carpenter, Phillip B.

    2010-01-01

    The NSD (nuclear receptor-binding SET domain protein) family encodes methyltransferases that are important in multiple aspects of development and disease. Perturbations in NSD family members can lead to Sotos syndrome and Wolf–Hirschhorn syndrome as well as cancers such as acute myeloid leukemia. Previous studies have implicated NSD1 (KMT3B) in transcription and methylation of histone H3 at lysine 36 (H3-K36), but its molecular mechanism in these processes remains largely unknown. Here we describe an NSD1 regulatory network in human cells. We show that NSD1 binds near various promoter elements and regulates multiple genes that appear to have a concerted role in various processes, such as cell growth/cancer, keratin biology, and bone morphogenesis. In particular, we show that NSD1 binding is concentrated upstream of gene targets such as the bone morphogenetic protein 4 (BMP4) and zinc finger protein 36 C3H type-like 1 (ZFP36L1/TPP). NSD1 regulates the levels of the various forms of methylation at H3-K36 primarily, but not exclusively, within the promoter proximal region occupied by NSD1. At BMP4 we find that this reduces the levels of RNAP II recruited to the promoter, suggesting a role for NSD1-dependent methylation in initiation. Interestingly, we also observe that the RNAP II molecules that lie within BMP4 have inappropriate persistence of serine-5 phosphorylation and reduced levels of serine-2 phosphorylation within the C-terminal domain (CTD) of the large subunit of RNAP II. Our findings indicate that NSD1 regulates RNAP II recruitment to BMP4, and failure to do so leads to reduced gene expression and abrogated levels of H3K36Me and CTD phosphorylation. PMID:20837538

  2. Making teeth to order: conserved genes reveal an ancient molecular pattern in paddlefish (Actinopterygii)

    PubMed Central

    Smith, Moya M.; Johanson, Zerina; Butts, Thomas; Ericsson, Rolf; Modrell, Melinda; Tulenko, Frank J.; Davis, Marcus C.; Fraser, Gareth J.

    2015-01-01

    Ray-finned fishes (Actinopterygii) are the dominant vertebrate group today (+30 000 species, predominantly teleosts), with great morphological diversity, including their dentitions. How dental morphological variation evolved is best addressed by considering a range of taxa across actinopterygian phylogeny; here we examine the dentition of Polyodon spathula (American paddlefish), assigned to the basal group Acipenseriformes. Although teeth are present and functional in young individuals of Polyodon, they are completely absent in adults. Our current understanding of developmental genes operating in the dentition is primarily restricted to teleosts; we show that shh and bmp4, as highly conserved epithelial and mesenchymal genes for gnathostome tooth development, are similarly expressed at Polyodon tooth loci, thus extending this conserved developmental pattern within the Actinopterygii. These genes map spatio-temporal tooth initiation in Polyodon larvae and provide new data in both oral and pharyngeal tooth sites. Variation in cellular intensity of shh maps timing of tooth morphogenesis, revealing a second odontogenic wave as alternate sites within tooth rows, a dental pattern also present in more derived actinopterygians. Developmental timing for each tooth field in Polyodon follows a gradient, from rostral to caudal and ventral to dorsal, repeated during subsequent loss of teeth. The transitory Polyodon dentition is modified by cessation of tooth addition and loss. As such, Polyodon represents a basal actinopterygian model for the evolution of developmental novelty: initial conservation, followed by tooth loss, accommodating the adult trophic modification to filter-feeding. PMID:25788604

  3. Prickle1 stunts limb growth through alteration of cell polarity and gene expression

    PubMed Central

    Yang, Tian; Bassuk, Alexander G.; Fritzsch, Bernd

    2014-01-01

    Background Wnt/PCP signaling plays a critical role in multiple developmental processes, including limb development. Wnt5a, a ligand of the PCP pathway, signals through the Ror2/Vangl2 or the Vangl2/Ryk complex to regulate limb development along the proximal-distal axis in mice. Based on the interaction between Van Gogh and Prickle in Drosophila, we hypothesized the vertebrate Prickle1 have similar function as Vangl2 in limb development. Results We show Prickle1 is expressed in the skeletal condensates that will differentiate into chondrocytes and later form bones. Disrupted Prickle1 function in Prickle1C251X/C251X mouse mutants alters expression of genes such as Bmp4, Fgf8, Vangl2 and Wnt5a. These expression changes correlate with shorter and wider bones in the limbs and loss of one phalangeal segment in digits 2-5 of Prickle1C251X mutants. These growth defects along the proximal-distal axis are also associated with increased cell death in the growing digit tip, reduced cell death in the interdigital membrane and disrupted chondrocyte polarity. Conclusions We suggest Prickle1 is part of the Wnt5a/PCP signaling, regulating cell polarity and affecting expression of multiple factors to stunt limb growth through altered patterns of gene expression, including the PCP genes Wnt5a and Vangl2. PMID:23913870

  4. Expression of Cartilage Developmental Genes in Hoxc8- and Hoxd4-Transgenic Mice

    PubMed Central

    Kruger, Claudia; Kappen, Claudia

    2010-01-01

    Hox genes encode transcription factors, which regulate skeletal patterning and chondrocyte differentiation during the development of cartilage, the precursor to mature bone. Overexpression of the homeobox transcription factors Hoxc8 and Hoxd4 causes severe cartilage defects due to delay in cartilage maturation. Matrix metalloproteinases (MMPs), bone morphogenetic proteins (BMPs) and fibroblastic growth factors (FGFs) are known to play important roles in skeletal development and endochondral bone formation and remodeling. In order to investigate whether these molecules are aberrantly expressed in Hoxc8- and/or Hoxd4-transgenic cartilage, we performed quantitative RT-PCR on chondrocytes from Hox-transgenic mice. Gene expression levels of Bmp4, Fgf8, Fgf10, Mmp9, Mmp13, Nos3, Timp3, Wnt3a and Wnt5a were altered in Hoxc8-transgenic chondrocytes, and Fgfr3, Ihh, Mmp8, and Wnt3a expression levels were altered in Hoxd4-transgenic chondrocytes, respectively. Notably, Wnt3a expression was elevated in Hoxc8- and reduced in Hoxd4-transgenic cartilage. These results suggest that both transcription factors affect cartilage maturation through different molecular mechanisms, and provide the basis for future studies into the role of these genes and possible interactions in pathogenesis of cartilage defects in Hoxc8- and Hoxd4-transgenic mice. PMID:20126390

  5. The membrane-cytoskeletal protein 4.1N is involved in the process of cell adhesion, migration and invasion of breast cancer cells.

    PubMed

    Ji, Zhenyu; Shi, Xiaofang; Liu, Xin; Shi, Yu; Zhou, Qingqing; Liu, Xilong; Li, Li; Ji, Xiang; Gao, Yanfeng; Qi, Yuanming; Kang, Qiaozhen

    2012-10-01

    Protein 4.1N belongs to the protein 4.1 superfamily that links transmembrane proteins to the actin cytoskeleton. Recent evidence has shown that protein 4.1 is important in tumor suppression. However, the functions of 4.1N in the metastasis of breast cancer are largely unknown. In the present study, MCF-7, T-47D and MDA-MB-231 breast cancer cell lines with various metastatic abilities were employed. Protein 4.1N was found to be expressed in poorly metastatic MCF-7 and middle metastatic T-47D cell lines, and was predominantly associated with cell-cell junctions. However, no 4.1N expression was detected in the highly metastatic MDA-MB-231 cells. Moreover, re-expression of 4.1N in MDA-MB-231 cells inhibited cell adhesion, migration and invasion. The results suggest that protein 4.1N is a negative regulator of cell metastasis in breast cancer. PMID:23170136

  6. The membrane-cytoskeletal protein 4.1N is involved in the process of cell adhesion, migration and invasion of breast cancer cells

    PubMed Central

    JI, ZHENYU; SHI, XIAOFANG; LIU, XIN; SHI, YU; ZHOU, QINGQING; LIU, XILONG; LI, LI; JI, XIANG; GAO, YANFENG; QI, YUANMING; KANG, QIAOZHEN

    2012-01-01

    Protein 4.1N belongs to the protein 4.1 superfamily that links transmembrane proteins to the actin cytoskeleton. Recent evidence has shown that protein 4.1 is important in tumor suppression. However, the functions of 4.1N in the metastasis of breast cancer are largely unknown. In the present study, MCF-7, T-47D and MDA-MB-231 breast cancer cell lines with various metastatic abilities were employed. Protein 4.1N was found to be expressed in poorly metastatic MCF-7 and middle metastatic T-47D cell lines, and was predominantly associated with cell-cell junctions. However, no 4.1N expression was detected in the highly metastatic MDA-MB-231 cells. Moreover, re-expression of 4.1N in MDA-MB-231 cells inhibited cell adhesion, migration and invasion. The results suggest that protein 4.1N is a negative regulator of cell metastasis in breast cancer. PMID:23170136

  7. Lack of Protein 4.1G Causes Altered Expression and Localization of the Cell Adhesion Molecule Nectin-Like 4 in Testis and Can Cause Male Infertility ▿

    PubMed Central

    Yang, Shaomin; Weng, Haibo; Chen, Lixiang; Guo, Xinhua; Parra, Marilyn; Conboy, John; Debnath, Gargi; Lambert, Amy J.; Peters, Luanne L.; Baines, Anthony J.; Mohandas, Narla; An, Xiuli

    2011-01-01

    Protein 4.1G is a member of the protein 4.1 family, which in general serves as adaptors linking transmembrane proteins to the cytoskeleton. 4.1G is thought to be widely expressed in many cells and tissues, but its function remains largely unknown. To explore the function of 4.1G in vivo, we generated 4.1G−/− mice and bred the mice in two backgrounds: C57BL/6 (B6) and 129/Sv (129) hybrids (B6-129) and inbred B6. Although the B6 4.1G−/− mice showed no obvious abnormalities, deficiency of 4.1G in B6-129 hybrids was associated with male infertility. Histological examinations of these 4.1G−/− mice revealed atrophy, impaired cell-cell contact and sloughing off of spermatogenic cells in seminiferous epithelium, and lack of mature spermatids in the epididymis. Ultrastructural examination revealed enlarged intercellular spaces between spermatogenic and Sertoli cells as well as the spermatid deformities. At the molecular level, 4.1G is associated with the nectin-like 4 (NECL4) adhesion molecule. Importantly, the expression of NECL4 was decreased, and the localization of NECL4 was altered in 4.1G−/− testis. Thus, our findings imply that 4.1G plays a role in spermatogenesis by mediating cell-cell adhesion between spermatogenic and Sertoli cells through its interaction with NECL4 on Sertoli cells. Additionally, the finding that infertility is present in B6-129 but not on the B6 background suggests the presence of a major modifier gene(s) that influences 4.1G function and is associated with male infertility. PMID:21482674

  8. Dictyostelium calcium-binding protein 4a interacts with nucleomorphin, a BRCT-domain protein that regulates nuclear number.

    PubMed

    Myre, Michael A; O'Day, Danton H

    2004-09-17

    Nucleomorphin from Dictyostelium discoideum is a nuclear calmodulin-binding protein that is a member of the BRCT-domain containing cell cycle checkpoint proteins. Two differentially expressed isoforms, NumA and NumB, share an extensive acidic domain (DEED) that when deleted produces highly multinucleated cells. We performed a yeast two-hybrid screen of a Dictyostelium cDNA library using NumA as bait. Here we show that nucleomorphin interacts with calcium-binding protein 4a (CBP4a) in a Ca(2+)-dependent manner. Further deletion analysis suggests this interaction requires residues found within the DEED domain. NumA and CBP4a mRNAs are expressed at the same stages of development. CBP4a belongs to a large family of Dictyostelium CBPs, for which no cellular or developmental functions had previously been determined. Since the interaction of CBP4a with nucleomorphin requires the DEED domain, this suggests that CBP4a may respond to Ca(2+)-signalling through modulating factors that might function in concert to regulate nuclear number. PMID:15325281

  9. Urinary Kininogen-1 and Retinol binding protein-4 respond to Acute Kidney Injury: predictors of patient prognosis?

    PubMed Central

    Gonzalez-Calero, Laura; Martin-Lorenzo, Marta; Ramos-Barron, Angeles; Ruiz-Criado, Jorge; Maroto, Aroa S.; Ortiz, Alberto; Gomez-Alamillo, Carlos; Arias, Manuel; Vivanco, Fernando; Alvarez-Llamas, Gloria

    2016-01-01

    Implementation of therapy for acute kidney injury (AKI) depends on successful prediction of individual patient prognosis. Clinical markers as serum creatinine (sCr) have limitations in sensitivity and early response. The aim of the study was to identify novel molecules in urine which show altered levels in response to AKI and investigate their value as predictors of recovery. Changes in the urinary proteome were here investigated in a cohort of 88 subjects (55 AKI patients and 33 healthy donors) grouped in discovery and validation independent cohorts. Patients’ urine was collected at three time points: within the first 48 h after diagnosis(T1), at 7 days of follow-up(T2) and at discharge of Nephrology(T3). Differential gel electrophoresis was performed and data were confirmed by Western blot (WB), liquid chromatography/mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA). Retinol binding protein 4 (RBP4) and kininogen-1 (KNG1) were found significantly altered following AKI. RBP4 increased at T1, and progressively decreased towards normalization. Maintained decrease was observed for KNG1 from T1. Individual patient response along time revealed RBP4 responds to recovery earlier than sCr. In conclusion, KNG1 and RBP4 respond to AKI. By monitoring RBP4, patient’s recovery can be anticipated pointing to a role of RBP4 in prognosis evaluation. PMID:26792617

  10. Fatty Acid-Binding Protein 4 (FABP4): Pathophysiological Insights and Potent Clinical Biomarker of Metabolic and Cardiovascular Diseases

    PubMed Central

    Furuhashi, Masato; Saitoh, Shigeyuki; Shimamoto, Kazuaki; Miura, Tetsuji

    2014-01-01

    Over the past decade, evidences of an integration of metabolic and inflammatory pathways, referred to as metaflammation in several aspects of metabolic syndrome, have been accumulating. Fatty acid-binding protein 4 (FABP4), also known as adipocyte FABP (A-FABP) or aP2, is mainly expressed in adipocytes and macrophages and plays an important role in the development of insulin resistance and atherosclerosis in relation to metaflammation. Despite lack of a typical secretory signal peptide, FABP4 has been shown to be released from adipocytes in a non-classical pathway associated with lipolysis, possibly acting as an adipokine. Elevation of circulating FABP4 levels is associated with obesity, insulin resistance, diabetes mellitus, hypertension, cardiac dysfunction, atherosclerosis, and cardiovascular events. Furthermore, ectopic expression and function of FABP4 in several types of cells and tissues have been recently demonstrated. Here, we discuss both the significant role of FABP4 in pathophysiological insights and its usefulness as a biomarker of metabolic and cardiovascular diseases. PMID:25674026

  11. Identification of bromodomain-containing protein-4 as a novel marker and epigenetic target in mast cell leukemia.

    PubMed

    Wedeh, G; Cerny-Reiterer, S; Eisenwort, G; Herrmann, H; Blatt, K; Hadzijusufovic, E; Sadovnik, I; Müllauer, L; Schwaab, J; Hoffmann, T; Bradner, J E; Radia, D; Sperr, W R; Hoermann, G; Reiter, A; Horny, H-P; Zuber, J; Arock, M; Valent, P

    2015-11-01

    Advanced systemic mastocytosis (SM) is a life-threatening neoplasm characterized by uncontrolled growth and accumulation of neoplastic mast cells (MCs) in various organs and a poor survival. So far, no curative treatment concept has been developed for these patients. We identified the epigenetic reader bromodomain-containing protein-4 (BRD4) as novel drug target in aggressive SM (ASM) and MC leukemia (MCL). As assessed by immunohistochemistry and PCR, neoplastic MCs expressed substantial amounts of BRD4 in ASM and MCL. The human MCL lines HMC-1 and ROSA also expressed BRD4, and their proliferation was blocked by a BRD4-specific short hairpin RNA. Correspondingly, the BRD4-targeting drug JQ1 induced dose-dependent growth inhibition and apoptosis in HMC-1 and ROSA cells, regardless of the presence or absence of KIT D816V. In addition, JQ1 suppressed the proliferation of primary neoplastic MCs obtained from patients with ASM or MCL (IC50: 100-500 nm). In drug combination experiments, midostaurin (PKC412) and all-trans retinoic acid were found to cooperate with JQ1 in producing synergistic effects on survival in HMC-1 and ROSA cells. Taken together, we have identified BRD4 as a promising drug target in advanced SM. Whether JQ1 or other BET-bromodomain inhibitors are effective in vivo in patients with advanced SM remains to be elucidated. PMID:26055303

  12. An alternative domain containing a leucine-rich sequence regulates nuclear cytoplasmic localization of protein 4.1R.

    PubMed

    Luque, Carlos M; Pérez-Ferreiro, Carmen M; Pérez-Gonzalez, Alicia; Englmeier, Ludwig; Koffa, Maria D; Correas, Isabel

    2003-01-24

    In red blood cells, protein 4.1 (4.1R) is an 80-kDa protein that stabilizes the spectrin-actin network and anchors it to the plasma membrane. The picture is more complex in nucleated cells, in which many 4.1R isoforms, varying in size and intracellular location, have been identified. To contribute to the characterization of signals involved in differential intracellular localization of 4.1R, we have analyzed the role the exon 5-encoded sequence plays in 4.1R distribution. We show that exon 5 encodes a leucine-rich sequence that shares key features with nuclear export signals (NESs). This sequence adopts the topology employed for NESs of other proteins and conserves two hydrophobic residues that are shown to be critical for NES function. A 4.1R isoform expressing the leucine-rich sequence binds to the export receptor CRM1 in a RanGTP-dependent fashion, whereas this does not occur in a mutant whose two conserved hydrophobic residues are substituted. These two residues are also essential for 4.1R intracellular distribution, because the 4.1R protein containing the leucine-rich sequence localizes in the cytoplasm, whereas the mutant protein predominantly accumulates in the nucleus. We hypothesize that the leucine-rich sequence in 4.1R controls distribution and concomitantly function of a specific set of 4.1R isoforms. PMID:12427749

  13. Phosphorylation of serine residues affects the conformation of the calmodulin binding domain of human protein 4.1.

    PubMed

    Vetter, S W; Leclerc, E

    2001-08-01

    We have previously characterized the calcium-dependent calmodulin (CaM)-binding domain (Ser76-Ser92) of the 135-kDa human protein 4.1 isoform using fluorescence spectroscopy and chemically synthesized nonphosphorylated or serine phosphorylated peptides [Leclerc, E. & Vetter, S. (1998) Eur. J. Biochem. 258, 567-671]. Here we demonstrate that phosphorylation of two serine residues within the 17-residue peptide alters their ability to adopt alpha helical conformation in a position-dependent manner. The helical content of the peptides was determined by CD-spectroscopy and found to increase from 36 to 45% for the Ser80 phosphorylated peptide and reduce to 28% for the Ser84 phosphorylated peptide; the di-phosphorylated peptide showed 32% helical content. Based on secondary structure prediction methods we propose that initial helix formation involves the central residues Leu82-Phe86. The ability of the peptides to adopt alpha helical conformations did not correlate with the observed binding affinities to CaM. We suggest that the reduced CaM-binding affinities observed for the phosphorylated peptides are more likely to be the result of unfavorable sterical and electrostatic interactions introduced into the CaM peptide-binding interface by the phosphate groups, rather than being due to the effect of phosphorylation on the secondary structure of the peptides. PMID:11488924

  14. Effect of Omega-3 Supplementation on Lipocalin 2 and Retinol-Binding Protein 4 in Type 2 Diabetic Patients

    PubMed Central

    FARAHBAKHSH-FARSI, Payam; DJAZAYERY, Abolghassem; ESHRAGHIAN, Mohammad Reza; KOOHDANI, Fariba; ZAREI, Mahnaz; JAVANBAKHT, Mohammad Hassan; DERAKHSHANIAN, Hoda; DJALALI, Mahmoud

    2016-01-01

    Background: Serum levels of lipocalin 2 (LCN 2) and retinol-binding protein-4 (RBP 4), increase in type 2 diabetes mellitus (T2DM). We sought to determine whether serum LCN 2 and RBP 4 change after an intervention with omega-3 fatty acids supplementation in diabetic patients. Methods: Forty-five type 2 diabetic patients from Iranian Diabetic Association in Tehran, Iran in 2013 were randomly recruited into two groups: one group received 4 g/d omega-3 for 10 wk; and the control group received placebo. Blood samples, food intake records, anthropometric measurements were obtained from all participants at the beginning and end of the study. Results: Fasting RBP 4 plasma levels significantly changed after 10 wk supplementation (P = 0.01). The LCN 2 concentrations decreased in omega-3 group, but the changes were not statistically significant. Omega-3 supplementation had no noticeable effect on anthropometric factors. Conclusion: These findings provide a rationale for omega-3 supplements aimed at lowering serum RBP 4 levels in T2DM. PMID:27114982

  15. Effect of Omega-3 Supplementation on Lipocalin 2 and Retinol-Binding Protein 4 in Type 2 Diabetic Patients

    PubMed Central

    FARAHBAKHSH-FARSI, Payam; DJAZAYERY, Abolghassem; ESHRAGHIAN, Mohammad Reza; KOOHDANI, Fariba; ZAREI, Mahnaz; JAVANBAKHT, Mohammad Hassan; DERAKHSHANIAN, Hoda; DJALALI, Mahmoud

    2016-01-01

    Background: Serum levels of lipocalin 2 (LCN 2) and retinol-binding protein-4 (RBP 4), increase in type 2 diabetes mellitus (T2DM). We sought to determine whether serum LCN 2 and RBP 4 change after an intervention with omega-3 fatty acids supplementation in diabetic patients. Methods: Forty-five type 2 diabetic patients from Iranian Diabetic Association in Tehran, Iran in 2013 were randomly recruited into two groups: one group received 4 g/d omega-3 for 10 wk; and the control group received placebo. Blood samples, food intake records, anthropometric measurements were obtained from all participants at the beginning and end of the study. Results: Fasting RBP 4 plasma levels significantly changed after 10 wk supplementation (P = 0.01). The LCN 2 concentrations decreased in omega-3 group, but the changes were not statistically significant. Omega-3 supplementation had no noticeable effect on anthropometric factors. Conclusions: These findings provide a rationale for omega-3 supplements aimed at lowering serum RBP 4 levels in T2DM. PMID:27057523

  16. Endothelial cell-fatty acid binding protein 4 promotes angiogenesis: role of stem cell factor/c-kit pathway.

    PubMed

    Elmasri, Harun; Ghelfi, Elisa; Yu, Chen-wei; Traphagen, Samantha; Cernadas, Manuela; Cao, Haiming; Shi, Guo-Ping; Plutzky, Jorge; Sahin, Mustafa; Hotamisligil, Gokhan; Cataltepe, Sule

    2012-09-01

    Fatty acid binding protein 4 (FABP4) plays an important role in regulation of glucose and lipid homeostasis as well as inflammation through its actions in adipocytes and macrophages. FABP4 is also expressed in a subset of endothelial cells, but its role in this cell type is not known. We found that FABP4-deficient human umbilical vein endothelial cells (HUVECs) demonstrate a markedly increased susceptibility to apoptosis as well as decreased migration and capillary network formation. Aortic rings from FABP4(-/-) mice demonstrated decreased angiogenic sprouting, which was recovered by reconstitution of FABP4. FABP4 was strongly regulated by mTORC1 and inhibited by Rapamycin. FABP4 modulated activation of several important signaling pathways in HUVECs, including downregulation of P38, eNOS, and stem cell factor (SCF)/c-kit signaling. Of these, the SCF/c-kit pathway was found to have a major role in attenuated angiogenic activity of FABP4-deficient ECs as provision of exogenous SCF resulted in a significant recovery in cell proliferation, survival, morphogenesis, and aortic ring sprouting. These data unravel a novel pro-angiogenic role for endothelial cell-FABP4 and suggest that it could be exploited as a potential target for diseases associated with pathological angiogenesis. PMID:22562362

  17. Fatty acid binding protein 4 is a target of VEGF and a regulator of cell proliferation in endothelial cells.

    PubMed

    Elmasri, Harun; Karaaslan, Cagatay; Teper, Yaroslav; Ghelfi, Elisa; Weng, Meiqian; Ince, Tan A; Kozakewich, Harry; Bischoff, Joyce; Cataltepe, Sule

    2009-11-01

    Fatty acid binding protein 4 (FABP4) plays an important role in maintaining glucose and lipid homeostasis. FABP4 has been primarily regarded as an adipocyte- and macrophage-specific protein, but recent studies suggest that it may be more widely expressed. We found strong FABP4 expression in the endothelial cells (ECs) of capillaries and small veins in several mouse and human tissues, including the heart and kidney. FABP4 was also detected in the ECs of mature human placental vessels and infantile hemangiomas, the most common tumor of infancy and ECs. In most of these cases, FABP4 was detected in both the nucleus and cytoplasm. FABP4 mRNA and protein levels were significantly induced in cultured ECs by VEGF-A and bFGF treatment. The effect of VEGF-A on FABP4 expression was inhibited by chemical inhibition or short-hairpin (sh) RNA-mediated knockdown of VEGF-receptor-2 (R2), whereas the VEGFR1 agonists, placental growth factors 1 and 2, had no effect on FABP4 expression. Knockdown of FABP4 in ECs significantly reduced proliferation both under baseline conditions and in response to VEGF and bFGF. Thus, FABP4 emerged as a novel target of the VEGF/VEGFR2 pathway and a positive regulator of cell proliferation in ECs. PMID:19625659

  18. Endothelial cell-fatty acid binding protein 4 promotes angiogenesis: role of stem cell factor/c-kit pathway

    PubMed Central

    Elmasri, Harun; Ghelfi, Elisa; Yu, Chen-wei; Traphagen, Samantha; Cernadas, Manuela; Cao, Haiming; Shi, Guo-Ping; Plutzky, Jorge; Sahin, Mustafa; Hotamisligil, Gokhan; Cataltepe, Sule

    2013-01-01

    Fatty acid binding protein 4 (FABP4) plays an important role in regulation of glucose and lipid homeostasis as well as inflammation through its actions in adipocytes and macrophages. FABP4 is also expressed in a subset of endothelial cells, but its role in this cell type is not known. We found that FABP4-deficient human umbilical vein endothelial cells (HUVECs) demonstrate a markedly increased susceptibility to apoptosis as well as decreased migration and capillary network formation. Aortic rings from FABP4−/− mice demonstrated decreased angiogenic sprouting, which was recovered by reconstitution of FABP4. FABP4 was strongly regulated by mTORC1 and inhibited by Rapamycin. FABP4 modulated activation of several important signaling pathways in HUVECs, including downregulation of P38, eNOS, and stem cell factor (SCF)/c-kit signaling. Of these, the SCF/c-kit pathway was found to have a major role in attenuated angiogenic activity of FABP4-deficient ECs as provision of exogenous SCF resulted in a significant recovery in cell proliferation, survival, morphogenesis, and aortic ring sprouting. These data unravel a novel pro-angiogenic role for endothelial cell-FABP4 and suggest that it could be exploited as a potential target for diseases associated with pathological angiogenesis. PMID:22562362

  19. RBFOX2 Promotes Protein 4.1R Exon 16 Selection via U1 snRNP Recruitment

    PubMed Central

    Ou, Alexander C.; Park, Jennie; Yu, Faye; Yu, Brian; Lee, Angela; Yang, Guang; Zhou, Anyu; Benz, Edward J.

    2012-01-01

    The erythroid differentiation-specific splicing switch of protein 4.1R exon 16, which encodes a spectrin/actin-binding peptide critical for erythrocyte membrane stability, is modulated by the differentiation-induced splicing factor RBFOX2. We have now characterized the mechanism by which RBFOX2 regulates exon 16 splicing through the downstream intronic element UGCAUG. Exon 16 possesses a weak 5′ splice site (GAG/GTTTGT), which when strengthened to a consensus sequence (GAG/GTAAGT) leads to near-total exon 16 inclusion. Impaired RBFOX2 binding reduces exon 16 inclusion in the context of the native weak 5′ splice site, but not the engineered strong 5′ splice site, implying that RBFOX2 achieves its effect by promoting utilization of the weak 5′ splice site. We further demonstrate that RBFOX2 increases U1 snRNP recruitment to the weak 5′ splice site through direct interaction between its C-terminal domain (CTD) and the zinc finger region of U1C and that the CTD is required for the effect of RBFOX2 on exon 16 splicing. Our data suggest a novel mechanism for exon 16 5′ splice site activation in which the binding of RBFOX2 to downstream intronic splicing enhancers stabilizes the pre-mRNA–U1 snRNP complex through interactions with U1C. PMID:22083953

  20. RUN and FYVE domain–containing protein 4 enhances autophagy and lysosome tethering in response to Interleukin-4

    PubMed Central

    Terawaki, Seigo; Camosseto, Voahirana; Prete, Francesca; Wenger, Till; Papadopoulos, Alexia; Rondeau, Christiane; Combes, Alexis; Rodriguez Rodrigues, Christian; Vu Manh, Thien-Phong; Fallet, Mathieu; English, Luc; Santamaria, Rodrigo; Soares, Ana R.; Weil, Tobias; Hammad, Hamida; Desjardins, Michel; Gorvel, Jean-Pierre; Santos, Manuel A.S.

    2015-01-01

    Autophagy is a key degradative pathway coordinated by external cues, including starvation, oxidative stress, or pathogen detection. Rare are the molecules known to contribute mechanistically to the regulation of autophagy and expressed specifically in particular environmental contexts or in distinct cell types. Here, we unravel the role of RUN and FYVE domain–containing protein 4 (RUFY4) as a positive molecular regulator of macroautophagy in primary dendritic cells (DCs). We show that exposure to interleukin-4 (IL-4) during DC differentiation enhances autophagy flux through mTORC1 regulation and RUFY4 induction, which in turn actively promote LC3 degradation, Syntaxin 17–positive autophagosome formation, and lysosome tethering. Enhanced autophagy boosts endogenous antigen presentation by MHC II and allows host control of Brucella abortus replication in IL-4–treated DCs and in RUFY4-expressing cells. RUFY4 is therefore the first molecule characterized to date that promotes autophagy and influences endosome dynamics in a subset of immune cells. PMID:26416964

  1. Delayed Cytotoxic T Lymphocyte-Associated Protein 4-Immunoglobulin Treatment Reverses Ongoing Alloantibody Responses and Rescues Allografts From Acute Rejection.

    PubMed

    Young, J S; Chen, J; Miller, M L; Vu, V; Tian, C; Moon, J J; Alegre, M-L; Sciammas, R; Chong, A S

    2016-08-01

    Antibody-mediated rejection has emerged as the leading cause of late graft loss in kidney transplant recipients, and inhibition of donor-specific antibody production should lead to improved transplant outcomes. The fusion protein cytotoxic T lymphocyte-associated protein 4-immunoglobulin (CTLA4-Ig) blocks T cell activation and consequently inhibits T-dependent B cell antibody production, and the current paradigm is that CTLA4-Ig is effective with naïve T cells and less so with activated or memory T cells. In this study, we used a mouse model of allosensitization to investigate the efficacy of continuous CTLA4-Ig treatment, initiated 7 or 14 days after sensitization, for inhibiting ongoing allospecific B cell responses. Delayed treatment with CTLA4-Ig collapsed the allospecific germinal center B cell response and inhibited alloantibody production. Using adoptively transferred T cell receptor transgenic T cells and a novel approach to track endogenous graft-specific T cells, we demonstrate that delayed CTLA4-Ig minimally inhibited graft-specific CD4(+) and T follicular helper responses. Remarkably, delaying CTLA4-Ig until day 6 after transplantation in a fully mismatched heart transplant model inhibited alloantibody production and prevented acute rejection, whereas transferred hyperimmune sera reversed the effects of delayed CTLA4-Ig. Collectively, our studies revealed the unexpected efficacy of CTLA4-Ig for inhibiting ongoing B cell responses even when the graft-specific T cell response was robustly established. PMID:26928966

  2. Suppression of Poly(rC)-Binding Protein 4 (PCBP4) reduced cisplatin resistance in human maxillary cancer cells

    PubMed Central

    Ito, Yumi; Narita, Norihiko; Nomi, Nozomi; Sugimoto, Chizuru; Takabayashi, Tetsuji; Yamada, Takechiyo; Karaya, Kazuhiro; Matsumoto, Hideki; Fujieda, Shigeharu

    2015-01-01

    Cisplatin plays an important role in the therapy for human head and neck cancers. However, cancer cells develop cisplatin resistance, leading to difficulty in treatment and poor prognosis. To analyze cisplatin-resistant mechanisms, a cisplatin-resistant cell line, IMC-3CR, was established from the IMC-3 human maxillary cancer cell line. Flow cytometry revealed that, compared with IMC-3 cells, cisplatin more dominantly induced cell cycle G2/M arrest rather than apoptosis in IMC-3CR cells. That fact suggests that IMC-3CR cells avoid cisplatin-induced apoptosis through induction of G2/M arrest, which allows cancer cells to repair damaged DNA and survive. In the present study, we specifically examined Poly(rC)-Binding Protein 4 (PCBP4), which reportedly induces G2/M arrest. Results showed that suppression of PCBP4 by RNAi reduced cisplatin-induced G2/M arrest and enhanced apoptosis in IMC-3CR cells, resulting in the reduction of cisplatin resistance. In contrast, overexpression of PCBP4 in IMC-3 cells induced G2/M arrest after cisplatin treatment and enhanced cisplatin resistance. We revealed that PCBP4 combined with Cdc25A and suppressed the expression of Cdc25A, resulting in G2/M arrest. PCBP4 plays important roles in the induction of cisplatin resistance in human maxillary cancers. PCBP4 is a novel molecular target for the therapy of head and neck cancers, especially cisplatin-resistant cancers. PMID:26196957

  3. Identification of bromodomain-containing protein 4 (BRD4) as a novel marker and epigenetic target in mast cell leukemia

    PubMed Central

    Wedeh, Ghaith; Cerny-Reiterer, Sabine; Eisenwort, Gregor; Herrmann, Harald; Blatt, Katharina; Hadzijusufovic, Emir; Sadovnik, Irina; Müllauer, Leonhard; Schwaab, Juliana; Hoffmann, Thomas; Bradner, James E.; Radia, Deepti; Sperr, Wolfgang R.; Hoermann, Gregor; Reiter, Andreas; Horny, Hans-Peter; Zuber, Johannes; Arock, Michel; Valent, Peter

    2015-01-01

    Advanced systemic mastocytosis (SM) is a life-threatening neoplasm characterized by uncontrolled growth and accumulation of neoplastic mast cells (MCs) in various organs and a poor survival. So far, no curative treatment concept has been developed for these patients. We identified the epigenetic reader bromodomain-containing protein-4 (BRD4) as novel drug target in aggressive SM (ASM) and MC leukemia (MCL). As assessed by immunohistochemistry and PCR, neoplastic MCs expressed substantial amounts of BRD4 in ASM and MCL. The human MCL lines HMC-1 and ROSA also expressed BRD4, and their proliferation was blocked by a BRD4-specific shRNA. Correspondingly, the BRD4-targeting drug JQ1 induced dose-dependent growth inhibition and apoptosis in HMC-1 cells and ROSA cells, regardless of the presence or absence of KIT D816V. In addition, JQ1 suppressed the proliferation of primary neoplastic MCs obtained from patients with ASM or MCL (IC50: 100-500 nM). In drug combination experiments, midostaurin (PKC412) and all-trans retinoic acid (ATRA) were found to cooperate with JQ1 in producing synergistic effects on survival in HMC-1 and ROSA cells. Together, we have identified BRD4 as a promising drug target in advanced SM. Whether JQ1 or other BET-bromodomain inhibitors are effective in vivo in patients with advanced SM remains to be elucidated. PMID:26055303

  4. Rapid quantitation of human epididymis protein 4 in human serum by amplified luminescent proximity homogeneous immunoassay (AlphaLISA).

    PubMed

    Zhao, Hui; Lin, Guanfeng; Liu, Tiancai; Liang, Junyu; Ren, Zhiqi; Liang, Rongliang; Chen, Baihong; Huang, Wenhua; Wu, Yingsong

    2016-10-01

    A sensitive, rapid and homogeneous reaction measurement method for quantitation of human epididymis protein 4 (HE4) in human serum by amplified luminescent proximity homogeneous immunoassay (AlphaLISA) was described. Built on a sandwich-type immunoassay format, analytes in samples were captured by one biotinylated monoclonal antibody combining on the surface of streptavidin coated donor beads, and "sandwiched" by another monoclonal antibody coated on acceptor beads. The coefficient variations of the method were lower than 10%, and the recoveries were in the range of 90-110% for serum samples. A value of 0.88pmol/l was identified as the minimum detectable dose of the present method for HE4. Compared with the results from electrochemiluminescence immunoassay kit (Roche) in 170 serum samples, there was a satisfied correlation coefficient of 0.984. The present assay demonstrated high sensitivity, wider effective detection range and excellent reproducibility for quantitation of HE4 can be useful for early screening and prognosis evaluation of patients with ovarian cancer. PMID:27568283

  5. Sorting nexin 27 interacts with multidrug resistance-associated protein 4 (MRP4) and mediates internalization of MRP4.

    PubMed

    Hayashi, Hisamitsu; Naoi, Sotaro; Nakagawa, Takayuki; Nishikawa, Toru; Fukuda, Hiroyuki; Imajoh-Ohmi, Shinobu; Kondo, Ayano; Kubo, Kiyotaka; Yabuki, Takashi; Hattori, Asami; Hirouchi, Masakazu; Sugiyama, Yuichi

    2012-04-27

    Multidrug resistance-associated protein 4 (MRP4/ABCC4) makes a vital contribution to the bodily distribution of drugs and endogenous compounds because of its cellular efflux abilities. However, little is known about the mechanism regulating its cell surface expression. MRP4 has a PDZ-binding motif, which is a potential sequence that modulates the membrane expression of MRP4 via interaction with PDZ adaptor proteins. To investigate this possible relationship, we performed GST pull-down assays and subsequent analysis with matrix-assisted laser desorption/ionization-time of flight mass spectrometry. This method identified sorting nexin 27 (SNX27) as the interacting PDZ adaptor protein with a PDZ-binding motif of MRP4. Its interaction was confirmed by a coimmunoprecipitation study using HEK293 cells. Knockdown of SNX27 by siRNA in HEK293 cells raised MRP4 expression on the plasma membrane, increased the extrusion of 6-[(14)C]mercaptopurine, an MRP4 substrate, and conferred resistance against 6-[(14)C]mercaptopurine. Cell surface biotinylation studies indicated that the inhibition of MRP4 internalization was responsible for these results. Immunocytochemistry and cell surface biotinylation studies using COS-1 cells showed that SNX27 localized to both the early endosome and the plasma membrane. These data suggest that SNX27 interacts with MRP4 near the plasma membrane and promotes endocytosis of MRP4 and thereby negatively regulates its cell surface expression and transport function. PMID:22411990

  6. Oestrogen receptors and small nuclear ring finger protein 4 (RNF4) in malignant ovarian germ cell tumours.

    PubMed

    Salonen, Jonna; Butzow, Ralf; Palvimo, Jorma J; Heikinheimo, Markku; Heikinheimo, Oskari

    2009-08-13

    The peak incidence of malignant ovarian germ cell tumours occurs soon after puberty. Thus, gonadal steroids may play a role in their development. Oestrogen receptors (ERalpha and ERbeta) and their co-regulators, including small nuclear ring finger protein 4 (SNURF/RNF4) mediate oestrogen actions. While ERbeta and SNURF are down-regulated in testicular germ cell tumours, their role in the ovarian germ cell tumours remains unknown. We herein studied the different subtypes of malignant ovarian germ cell tumours, and found that they all express ERalpha, ERbeta, and SNURF. Stimulation with oestradiol (E2), ERalpha, ERbeta and SNURF significantly up-regulated mRNA expression in the human germinoma derived NCC-IT cells. Further, the effects of E2 were counteracted by an anti-oestrogen (ICI 182,780). Neither E2 nor ICI 182,780 had an effect on the proliferation of NCC-IT cells as assessed by flow cytometric analysis. Our results suggest that oestrogen signalling has a role in malignant ovarian germ cell tumours. PMID:19524139

  7. Molecular cloning and expression analysis of prostaglandin E receptor 2 gene in cashmere goat (Capra hircus) skin during hair follicle development.

    PubMed

    Geng, Rong-Qing; Yuan, Chao; Chen, Yu-Lin

    2014-04-01

    As a member of the four subtypes of receptors for prostaglandin E2 (PGE2), prostaglandin E receptor 2 (PTGER2) is in the family of G-protein coupled receptors and has been characterized to be involved in the development and growth of hair follicles. In this study, we cloned and characterized the full-length coding sequence (CDS) of PTGER2 gene from cashmere goat skin. The entire open reading frame (ORF) of PTGER2 gene was 1047 bp and encoded 348 amino acid residues. The deduced protein contained one G-protein coupled receptors family 1 signature, seven transmembrane domains, and other potential sites. Tissue expression analysis showed that PTGER2 gene was expressed strongly in the skin. The general expression tendency of PTGER2 gene at different hair follicle developmental stages in the skin was gradually decreased from anagen to catagen to telogen. After comparing with the expression of BMP4 gene and related reports, we further presume that it seems to have a relationship between the hair follicle cycle and the expression level of PTGER2 gene in cashmere goat skin. PMID:24555795

  8. Dual-stage growth factor release within 3D protein-engineered hydrogel niches promotes adipogenesis

    PubMed Central

    Greenwood-Goodwin, Midori; Teasley, Eric S.; Heilshorn, Sarah C.

    2014-01-01

    Engineered biomimetic microenvironments from hydrogels are an emerging strategy to achieve lineage-specific differentiation in vitro. In addition to recapitulating critical matrix cues found in the native three-dimensional (3D) niche, the hydrogel can also be designed to deliver soluble factors that are present within the native inductive microenvironment. We demonstrate a versatile materials approach for the dual-stage delivery of multiple soluble factors within a 3D hydrogel to induce adipogenesis. We use a Mixing-Induced Two-Component Hydrogel (MITCH) embedded with alginate microgels to deliver two pro-adipogenic soluble factors, fibroblast growth factor 1 (FGF-1) and bone morphogenetic protein 4 (BMP-4) with two distinct delivery profiles. We show that dual-stage delivery of FGF-1 and BMP-4 to human adipose-derived stromal cells (hADSCs) significantly increases lipid accumulation compared with the simultaneous delivery of both growth factors together. Furthermore, dual-stage growth factor delivery within a 3D hydrogel resulted in substantially more lipid accumulation compared to identical delivery profiles in 2D cultures. Gene expression analysis shows upregulation of key adipogenic markers indicative of brown-like adipocytes. These data suggest that dual-stage release of FGF-1 and BMP-4 within 3D microenvironments can promote the in vitro development of mature adipocytes. PMID:25309741

  9. Activity-dependent expression of neuronal PAS domain-containing protein 4 (npas4a) in the developing zebrafish brain

    PubMed Central

    Klarić, Thomas; Lardelli, Michael; Key, Brian; Koblar, Simon; Lewis, Martin

    2014-01-01

    In rodents, the Npas4 gene has recently been identified as being an important regulator of synaptic plasticity and memory. Homologs of Npas4 have been found in invertebrate species though their functions appear to be too divergent for them to be studied as a proxy for the mammalian proteins. The aim of this study, therefore, was to ascertain the suitability of the zebrafish as a model organism for investigating the function of Npas4 genes. We show here that the expression and regulation of the zebrafish Npas4 homolog, npas4a, is remarkably similar to that of the rodent Npas4 genes. As in mammals, expression of the zebrafish npas4a gene is restricted to the brain where it is up-regulated in response to neuronal activity. Furthermore, we also show that knockdown of npas4a during embryonic development results in a number of forebrain-specific defects including increased apoptosis and misexpression of the forebrain marker genes dlx1a and shha. Our work demonstrates that the zebrafish is a suitable model organism for investigating the role of the npas4a gene and one that is likely to provide valuable insights into the function of the mammalian homologs. Furthermore, our findings highlight a potential role for npas4a in forebrain development. PMID:25538572

  10. Genes and Gene Therapy

    MedlinePlus

    ... a child can have a genetic disorder. Gene therapy is an experimental technique that uses genes to ... prevent disease. The most common form of gene therapy involves inserting a normal gene to replace an ...

  11. Genes and Gene Therapy

    MedlinePlus

    ... correctly, a child can have a genetic disorder. Gene therapy is an experimental technique that uses genes to ... or prevent disease. The most common form of gene therapy involves inserting a normal gene to replace an ...

  12. Spherical Body Protein 4 Is a New Serological Antigen for Global Detection of Babesia bovis Infection in Cattle ▿

    PubMed Central

    Terkawi, Mohamad Alaa; Huyen, Nguyen Xuan; Wibowo, Putut Eko; Seuseu, Faasoa Junior; Aboulaila, Mahmoud; Ueno, Akio; Goo, Youn-Kyoung; Yokoyama, Naoaki; Xuan, Xuenan; Igarashi, Ikuo

    2011-01-01

    Five Babesia bovis recombinant proteins, including merozoite surface antigen 2c (BbMSA-2c), C-terminal rhoptry-associated protein 1 (BbRAP-1/CT), truncated thrombospondin-related anonymous protein (BbTRAP-T), spherical body protein 1 (BbSBP-1), and spherical body protein 4 (BbSBP-4), were evaluated as diagnostic antigens to detect the infection in cattle. The recombinant proteins were highly antigenic when tested with experimentally B. bovis-infected bovine serum in Western blot analysis. Furthermore, five antisera that had been raised against each of the recombinant proteins reacted specifically with the corresponding authentic protein, as determined in Western blot analysis. Next, enzyme-linked immunosorbent assays (ELISAs) using these recombinant proteins were evaluated for diagnostic use, and the sensitivity and specificity of each protein were demonstrated with a series of serum samples from experimentally B. bovis-infected cattle. Furthermore, a total of 669 field serum samples collected from cattle in regions of B. bovis endemicity in seven countries were tested with the ELISAs, and the results were compared to those of an indirect fluorescent antibody test (IFAT), as a reference. Among five recombinant antigens, recombinant BbSBP-4 (rBbSBP-4) had the highest concordance rate (85.3%) and kappa value (0.705), indicating its reliability in the detection of specific antibodies to B. bovis in cattle, even in different geographical regions. Overall, we have successfully developed an ELISA based on rBbSBP-4 as a new serological antigen for a practical and sensitive test which will be applicable for epidemiologic survey and control programs in the future. PMID:21123520

  13. [Knockdown of angiopoietin-like protein 4 inhibits proliferation and promotes apoptosis in cervical cancer SiHa cells].

    PubMed

    Nie, Dan; Liu, Ling; Xia, Jiyi; Wang, Chunyan; Zhan, Ping; Mao, Xiguang

    2016-04-01

    Objective To investigate the effect of lentivirus-mediated shRNA silencing of angiopoietin-like protein 4 (ANGPTL4) on the proliferation and apoptosis of cervical cancer SiHa cells. Methods The ANGPTL4 lentiviral vectors were used to transfect SiHa cells. Real-time quantitative PCR (qRT-PCR) and Western blotting were respectively used to detect ANGPTL4 expression at mRNA and protein levels. The proliferation ability of SiHa cells after transfection was assessed by MTT assay and colony formation assay. The cell cycle was examined by flow cytometry. The annexin V-phycoerythrin/7-aminoactinomycin D (annexin V-PE/7-AAD) staining combined with flow cytometry was used to examine the effect of ANGPTL4 silence on the apoptosis of SiHa cells. Results After the ANGPTL4 lentiviral vectors were transfected into SiHa cells, qRT-PCR and Western blotting showed that the expression of ANGPTL4 mRNA and protein were significantly inhibited in LV3-ANGPTL4 group. The MTT assay showed that the proliferation ability of SiHa cells in LV3-ANGPTL4 group was also inhibited. Colony formation assay revealed that the colony number in LV3-ANGPTL4 group was reduced. The cells in G0/G1 phase and the apoptosis rate increased in LV3-ANGPTL4 group. Conclusion The lentivirus-mediated ANGPTL4 shRNA can inhibit the proliferation, induce the cell cycle arrest in G0/G1 phase, and promote the apoptosis in SiHa cells. PMID:27053616

  14. Inflammation increases plasma angiopoietin-like protein 4 in patients with the metabolic syndrome and type 2 diabetes

    PubMed Central

    Tjeerdema, Nathanja; Georgiadi, Anastasia; Jonker, Jacqueline T; van Glabbeek, Marjolijn; Dehnavi, Reza Alizadeh; Tamsma, Jouke T; Smit, Johannes W A; Kersten, Sander; Rensen, Patrick C N

    2014-01-01

    Background Angiopoietin-like protein 4 (ANGPTL4) inhibits lipoprotein lipase and associates with dyslipidemia. The expression of ANGPTL4 is regulated by free fatty acids (FFA) that activate lipid-sensing peroxisome proliferator-activated receptors (PPARs), but FFA can also activate pattern recognition receptors including Toll-like receptor 4 (TLR4) in macrophages. Objective To assess whether systemic low-grade inflammation is a determinant for plasma ANGPTL4 levels in patients with the metabolic syndrome (MetS) and type 2 diabetes mellitus (T2DM). Design We studied 335 male participants: healthy controls (Controls), patients with the MetS without inflammation (MetS−I) and with low-grade inflammation (MetS+I), and patients with T2DM. All patients without diabetes included in the present study were initially matched for waist circumference. In plasma, ANGPTL4, C reactive protein (CRP) and metabolic parameters were determined. Underlying mechanisms were examined using human macrophages in vitro. Results As compared with Controls, plasma ANGPTL4 levels were increased in patients with MetS−I, MetS+I, and T2DM. Furthermore, ANGPTL4 was increased in T2DM compared with MetS−I. In fact, plasma CRP correlated positively with plasma ANGPTL4. In vitro studies showed that TLR 3/4 activation largely increased the expression and release of ANGPTL4 by macrophages. Conclusions Plasma ANGPTL4 levels in humans are predicted by CRP, a marker of inflammation, and ANGPTL4 expression by macrophages is increased by inflammatory stimuli. PMID:25512873

  15. Serum Fatty Acid-Binding Protein 4 Is a Predictor of Cardiovascular Events in End-Stage Renal Disease

    PubMed Central

    Furuhashi, Masato; Ishimura, Shutaro; Ota, Hideki; Hayashi, Manabu; Nishitani, Takahiro; Tanaka, Marenao; Yoshida, Hideaki; Shimamoto, Kazuaki; Hotamisligil, Gökhan S.; Miura, Tetsuji

    2011-01-01

    Background Fatty acid-binding protein 4 (FABP4/A-FABP/aP2), a lipid chaperone, is expressed in both adipocytes and macrophages. Recent studies have shown that FABP4 is secreted from adipocytes and that FABP4 level is associated with obesity, insulin resistance, and atherosclerosis. However, little is known about the impact of FABP4 concentrations on prognosis. We tested the hypothesis that FABP4 level predicts prognosis of patients with end-stage renal disease (ESRD), a group at high risk for atherosclerosis-associated morbidity and mortality. Methods and Results Biochemical markers including FABP4 were determined in 61 ESRD patients on chronic hemodialysis (HD). Serum FABP4 level in females (404.2±30.5 ng/ml) was significantly higher than that in males (315.8±30.0 ng/ml), and the levels in ESRD patients were about 20-times higher than those in age-, gender- and body mass index (BMI)-matched control subjects with normal renal function. FABP4 level was decreased by 57.2% after HD and was positively correlated with blood pressure, BMI, and levels of lipids and insulin. Multiple regression analysis indicated that HD duration, BMI, and triglycerides level were independent determinants for FABP4 level. ESRD patients with high FABP4 levels had higher cardiovascular mortality during the 7-year follow-up period. Cox proportional hazard regression analysis showed that logarithmically transformed FABP4 level was an independent predictor of cardiovascular death adjusted for age, gender, HD duration, BMI, and triglycerides level (hazard ratio, 7.75; 95% CI, 1.05–25.31). Conclusion These findings suggest that FABP4 level, being related to adiposity and metabolic disorders, is a novel predictor of cardiovascular mortality in ESRD. PMID:22102888

  16. Fatty Acid Binding Protein 4 Regulates VEGF-Induced Airway Angiogenesis and Inflammation in a Transgenic Mouse Model

    PubMed Central

    Ghelfi, Elisa; Yu, Chen-Wei; Elmasri, Harun; Terwelp, Matthew; Lee, Chun G.; Bhandari, Vineet; Comhair, Suzy A.; Erzurum, Serpil C.; Hotamisligil, Gökhan S.; Elias, Jack A.; Cataltepe, Sule

    2014-01-01

    Neovascularization of the airways occurs in several inflammatory lung diseases, including asthma. Vascular endothelial growth factor (VEGF) plays an important role in vascular remodeling in the asthmatic airways. Fatty acid binding protein 4 (FABP4 or aP2) is an intracellular lipid chaperone that is induced by VEGF in endothelial cells. FABP4 exhibits a proangiogenic function in vitro, but whether it plays a role in modulation of angiogenesis in vivo is not known. We hypothesized that FABP4 promotes VEGF-induced airway angiogenesis and investigated this hypothesis with the use of a transgenic mouse model with inducible overexpression of VEGF165 under a CC10 promoter [VEGF-TG (transgenic) mice]. We found a significant increase in FABP4 mRNA levels and density of FABP4-expressing vascular endothelial cells in mouse airways with VEGF overexpression. FABP4−/− mouse airways showed a significant decrease in neovessel formation and endothelial cell proliferation in response to VEGF overexpression. These alterations in airway vasculature were accompanied by attenuated expression of proinflammatory mediators. Furthermore, VEGF-TG/FABP4−/− mice showed markedly decreased expression of endothelial nitric oxide synthase, a well-known mediator of VEGF-induced responses, compared with VEGF-TG mice. Finally, the density of FABP4-immunoreactive vessels in endobronchial biopsy specimens was significantly higher in patients with asthma than in control subjects. Taken together, these data unravel FABP4 as a potential target of pathologic airway remodeling in asthma. PMID:23391391

  17. Decreased retinol-binding protein 4 in the sera of patients with end-stage renal disease after kidney transplantation.

    PubMed

    Zhang, W X; Zhou, W; Zhang, Z M; Zhang, Z Q; He, J F; Shi, B Y

    2014-01-01

    Retinol-binding protein 4 (RBP4) is a novel adipokine that has been associated with insulin resistance and type 2 diabetes. Patients with end-stage renal disease (ESRD) have very high serum RBP4 levels. However, whether successful kidney transplantation alleviates these elevated serum RBP4 levels is unclear. The serum RBP4 levels of 24 ESRD patients were determined before transplantation and at 1 day, 1 week, and 1 month after kidney transplantation. The control group included 22 healthy subjects. Serum RBP4 concentrations were measured using a commercial kit via the immunologic turbidimetric method, and were related to biomarkers for renal and liver function. The serum RBP4 level of ESRD patients before kidney transplantation (160.8 ± 29.1 mg/L) was approximately 7-fold higher than that of normal controls (22.6 ± 11.0 mg/L; P = 0.000). The serum RBP4 level before transplantation was significantly higher than that at 1 day (65.3 ± 28.4 mg/L), 1 week (48.3 ± 22.9 mg/L), and 1 month after transplantation (53.1 ± 25.5 mg/L; P = 0.000). However, these values were still higher than those of controls (P = 0.000). Univariate regression analysis showed that the percent changes in serum RBP4 concentration before and after kidney transplantation were positively correlated with serum creatinine, blood urea nitrogen, phosphate, and pre-albumin concentrations and negatively correlated with the estimated glomerular filtration rate. The serum RBP4 concentration of patients with ESRD decreased significantly after kidney transplantation; therefore, we found that serum RBP4 concentration was related to renal function. PMID:25299197

  18. Metformin reduces lipid accumulation in macrophages by inhibiting FOXO1-mediated transcription of fatty acid-binding protein 4

    SciTech Connect

    Song, Jun; Ren, Pingping; Zhang, Lin; Wang, Xing Li; Chen, Li; Shen, Ying H.

    2010-02-26

    Objective: The accumulation of lipids in macrophages contributes to the development of atherosclerosis. Strategies to reduce lipid accumulation in macrophages may have therapeutic potential for preventing and treating atherosclerosis and cardiovascular complications. The antidiabetic drug metformin has been reported to reduce lipid accumulation in adipocytes. In this study, we examined the effects of metformin on lipid accumulation in macrophages and investigated the mechanisms involved. Methods and results: We observed that metformin significantly reduced palmitic acid (PA)-induced intracellular lipid accumulation in macrophages. Metformin promoted the expression of carnitine palmitoyltransferase I (CPT-1), while reduced the expression of fatty acid-binding protein 4 (FABP4) which was involved in PA-induced lipid accumulation. Quantitative real-time PCR showed that metformin regulates FABP4 expression at the transcriptional level. We identified forkhead transcription factor FOXO1 as a positive regulator of FABP4 expression. Inhibiting FOXO1 expression with FOXO1 siRNA significantly reduced basal and PA-induced FABP4 expression. Overexpression of wild-type FOXO1 and constitutively active FOXO1 significantly increased FABP4 expression, whereas dominant negative FOXO1 dramatically decreased FABP4 expression. Metformin reduced FABP4 expression by promoting FOXO1 nuclear exclusion and subsequently inhibiting its activity. Conclusions: Taken together, these results suggest that metformin reduces lipid accumulation in macrophages by repressing FOXO1-mediated FABP4 transcription. Thus, metformin may have a protective effect against lipid accumulation in macrophages and may serve as a therapeutic agent for preventing and treating atherosclerosis in metabolic syndrome.

  19. Phosphorylation of Transcription Factor Specificity Protein 4 Is Increased in Peripheral Blood Mononuclear Cells of First-Episode Psychosis

    PubMed Central

    Fusté, Montserrat; Meléndez-Pérez, Iria; Villalta-Gil, Victoria; Haro, Josep Maria; Gill, Grace; Ramos, Belén

    2015-01-01

    Background Altered expression of transcription factor specificity protein 4 (SP4) has been found in the postmortem brain of patients with psychiatric disorders including schizophrenia and bipolar disorder. Reduced levels of SP4 protein have recently been reported in peripheral blood mononuclear cells in first-episode psychosis. Also, SP4 levels are modulated by lithium treatment in cultured neurons. Phosphorylation of SP4 at S770 is increased in the cerebellum of bipolar disorder subjects and upon inhibition of NMDA receptor signaling in cultured neurons. The aim of this study was to investigate whether SP4 S770 phosphorylation is increased in lymphocytes of first-episode psychosis patients and the effect of lithium treatment on this phosphorylation. Methods A cross-sectional study of S770 phosphorylation relative to total SP4 immunoreactivity using specific antibodies in peripheral blood mononuclear cells in first-episode psychosis patients (n = 14, treated with lithium or not) and matched healthy controls (n = 14) by immunoblot was designed. We also determined the effects of the prescribed drugs lithium, olanzapine or valproic acid on SP4 phosphorylation in rat primary cultured cerebellar granule neurons. Results We found that SP4 S770 phosphorylation was significantly increased in lymphocytes in first-episode psychosis compared to controls and decreased in patients treated with lithium compared to patients who did not receive lithium. Moreover, incubation with lithium but not olanzapine or valproic acid reduced SP4 phosphorylation in rat cultured cerebellar granule neurons. Conclusions The findings presented here indicate that SP4 S770 phosphorylation is increased in lymphocytes in first-episode psychosis which may be reduced by lithium treatment in patients. Moreover, our study shows lithium treatment prevents this phosphorylation in vitro in neurons. This pilot study suggests that S770 SP4 phosphorylation could be a peripheral biomarker of psychosis, and may

  20. Gender difference in plasma fatty-acid-binding protein 4 levels in patients with chronic obstructive pulmonary disease

    PubMed Central

    Zhang, Xue; Li, Diandian; Wang, Hao; Pang, Caishuang; Wu, Yanqiu; Wen, Fuqiang

    2016-01-01

    COPD (chronic obstructive pulmonary disease) is characterized by airway inflammation and increases the likelihood of the development of atherosclerosis. Recent studies have indicated that FABP4 (fatty-acid-binding protein 4), an intracellular lipid chaperone of low molecular mass, plays an important role in the regulation of inflammation and atherosclerosis. We carried out a preliminary clinical study aiming at investigating the relationships between circulating FABP4 levels in patients with COPD and inflammation and lung function. We enrolled 50 COPD patients and 39 healthy controls in the study. Lung function tests were performed in all subjects. Plasma levels of FABP4 and adiponectin, TNFα (tumour necrosis factor α) and CRP (C-reactive protein) were measured. The correlations between FABP4 and lung function, adipokine (adiponectin), inflammatory factors and BMI (body mass index) were analysed. Compared with both males with COPD and healthy females, plasma FABP4 levels in females with COPD were significantly increased. Adiponectin and CRP levels were significantly higher in patients with COPD. Furthermore, we found that FABP4 levels were inversely correlated with FEV1% predicted (FEV1 is forced expiratory volume in 1 s) and positively correlated with adiponectin and TNFα in COPD patients. In addition, a positive correlation between plasma FABP4 and CRP was found in females with COPD. However, FABP4 levels were not correlated with BMI. Our results underline a gender difference in FABP4 secretion in stable COPD patients. Further studies are warranted to clarify the exact role of FABP4 in the pathogenesis of COPD. PMID:26823558

  1. Acute-phase protein α1-antitrypsin--a novel regulator of angiopoietin-like protein 4 transcription and secretion.

    PubMed

    Frenzel, Eileen; Wrenger, Sabine; Immenschuh, Stephan; Koczulla, Rembert; Mahadeva, Ravi; Deeg, H Joachim; Dinarello, Charles A; Welte, Tobias; Marcondes, A Mario Q; Janciauskiene, Sabina

    2014-06-01

    The angiopoietin-like protein 4 (angptl4, also known as peroxisome proliferator-activated receptor [PPAR]γ-induced angiopoietin-related protein) is a multifunctional protein associated with acute-phase response. The mechanisms accounting for the increase in angptl4 expression are largely unknown. This study shows that human α1-antitrypsin (A1AT) upregulates expression and release of angplt4 in human blood adherent mononuclear cells and in primary human lung microvascular endothelial cells in a concentration- and time-dependent manner. Mononuclear cells treated for 1 h with A1AT (from 0.1 to 4 mg/ml) increased mRNA of angptl4 from 2- to 174-fold, respectively, relative to controls. In endothelial cells, the maximal effect on angptl4 expression was achieved at 8 h with 2 mg/ml A1AT (11-fold induction versus controls). In 10 emphysema patients receiving A1AT therapy (Prolastin), plasma angptl4 levels were higher relative to patients without therapy (nanograms per milliliter, mean [95% confidence interval] 127.1 [99.5-154.6] versus 76.8 [54.8-98.8], respectively, p = 0.045) and correlated with A1AT levels. The effect of A1AT on angptl4 expression was significantly diminished in cells pretreated with a specific inhibitor of ERK1/2 activation (UO126), irreversible and selective PPARγ antagonist (GW9662), or genistein, a ligand for PPARγ. GW9662 did not alter the ability of A1AT to induce ERK1/2 phosphorylation, suggesting that PPARγ is a critical mediator in the A1AT-driven angptl4 expression. In contrast, the forced accumulation of HIF-1α, an upregulator of angptl4 expression, enhanced the effect of A1AT. Thus, acute-phase protein A1AT is a physiological regulator of angptl4, another acute-phase protein. PMID:24760148

  2. Adenovirus-Mediated Efficient Gene Transfer into Cultured Three-Dimensional Organoids

    PubMed Central

    Wang, Ning; Zhang, Hongyu; Zhang, Bing-Qiang; Liu, Wei; Zhang, Zhonglin; Qiao, Min; Zhang, Hongmei; Deng, Fang; Wu, Ningning; Chen, Xian; Wen, Sheng; Zhang, Junhui; Liao, Zhan; Zhang, Qian; Yan, Zhengjian; Yin, Liangjun; Ye, Jixing; Deng, Youlin; Luu, Hue H.; Haydon, Rex C.; Liang, Houjie; He, Tong-Chuan

    2014-01-01

    Three-dimensional organoids have been recently established from various tissue-specific progenitors (such as intestinal stem cells), induced pluripotent stem cells, or embryonic stem cells. These cultured self-sustaining stem cell–based organoids may become valuable systems to study the roles of tissue-specific stem cells in tissue genesis and disease development. It is thus conceivable that effective genetic manipulations in such organoids may allow us to reconstruct disease processes and/or develop novel therapeutics. Recombinant adenoviruses are one of the most commonly used viral vectors for in vitro and in vivo gene deliveries. In this study, we investigate if adenoviruses can be used to effectively deliver transgenes into the cultured “mini-gut” organoids derived from intestinal stem cells. Using adenoviral vectors that express fluorescent proteins, we demonstrate that adenoviruses can effectively deliver transgenes into the cultured 3-D “mini-gut” organoids. The transgene expression can last at least 10 days in the cultured organoids. As a proof-of-principle experiment, we demonstrate that adenovirus-mediated noggin expression effectively support the survival and self-renewal of mini-gut organoids, while adenovirus-mediated expression of BMP4 inhibits the self-sustainability and proliferation of the organoids. Thus, our results strongly suggest that adenovirus vectors can be explored as effective gene delivery vehicles to introduce genetic manipulations in 3-D organoids. PMID:24695466

  3. Adenovirus-mediated efficient gene transfer into cultured three-dimensional organoids.

    PubMed

    Wang, Ning; Zhang, Hongyu; Zhang, Bing-Qiang; Liu, Wei; Zhang, Zhonglin; Qiao, Min; Zhang, Hongmei; Deng, Fang; Wu, Ningning; Chen, Xian; Wen, Sheng; Zhang, Junhui; Liao, Zhan; Zhang, Qian; Yan, Zhengjian; Yin, Liangjun; Ye, Jixing; Deng, Youlin; Luu, Hue H; Haydon, Rex C; Liang, Houjie; He, Tong-Chuan

    2014-01-01

    Three-dimensional organoids have been recently established from various tissue-specific progenitors (such as intestinal stem cells), induced pluripotent stem cells, or embryonic stem cells. These cultured self-sustaining stem cell-based organoids may become valuable systems to study the roles of tissue-specific stem cells in tissue genesis and disease development. It is thus conceivable that effective genetic manipulations in such organoids may allow us to reconstruct disease processes and/or develop novel therapeutics. Recombinant adenoviruses are one of the most commonly used viral vectors for in vitro and in vivo gene deliveries. In this study, we investigate if adenoviruses can be used to effectively deliver transgenes into the cultured "mini-gut" organoids derived from intestinal stem cells. Using adenoviral vectors that express fluorescent proteins, we demonstrate that adenoviruses can effectively deliver transgenes into the cultured 3-D "mini-gut" organoids. The transgene expression can last at least 10 days in the cultured organoids. As a proof-of-principle experiment, we demonstrate that adenovirus-mediated noggin expression effectively support the survival and self-renewal of mini-gut organoids, while adenovirus-mediated expression of BMP4 inhibits the self-sustainability and proliferation of the organoids. Thus, our results strongly suggest that adenovirus vectors can be explored as effective gene delivery vehicles to introduce genetic manipulations in 3-D organoids. PMID:24695466

  4. Reproducibility of Retinol Binding Protein 4 and Omentin-1 Measurements over a Four Months Period: A Reliability Study in a Cohort of 207 Apparently Healthy Participants

    PubMed Central

    Wittenbecher, Clemens; di Giuseppe, Romina; Biemann, Ronald; Menzel, Juliane; Arregui, Maria; Hoffmann, Juliane; Aleksandrova, Krasimira; Boeing, Heiner; Isermann, Berend; Schulze, Matthias B.; Weikert, Cornelia

    2015-01-01

    The reliability of single time point measurements of the novel adipokines retinol-binding protein 4 and omentin-1 in the blood has not been evaluated in large samples yet. The present study aimed to assess the amount of biological variation of these two adipokines within individuals. The study sample comprised 207 participants (124 women and 83 men) from Potsdam (Germany) and surrounding areas, with an average age of 56.5 years (SD 4.2). Blood samples were collected from each participant twice, approximately four months apart. Using enzyme linked immunosorbent assays, the concentrations of retinol-binding protein 4 and omentin-1 were determined in EDTA plasma. As indicators of reliability, intraclass correlation coefficients (ICCs) were calculated from the repeated biomarker measurements. The ICCs for repeated retinol-binding protein 4 and omentin-1 measurements were 0.77 (95% CI 0.71, 0.82) and 0.83 (95% CI 0.78, 0.87), respectively, indicating for both adipokines excellent reliability. ICCs were stable across strata according to sex, age, BMI, and blood pressure. Thus, for epidemiological studies it seems reasonable to rely on concentrations of retinol-binding protein 4 and omentin-1 in samples from a single time point if repeated measurements are not available. PMID:26402656

  5. Regulation of the glycophorin C-protein 4.1 membrane-to-skeleton bridge and evaluation of its contribution to erythrocyte membrane stability.

    PubMed

    Chang, S H; Low, P S

    2001-06-22

    The band 3-ankyrin-spectrin bridge and the glycophorin C-protein 4.1-spectrin/actin bridge constitute the two major tethers between the erythrocyte membrane and its spectrin skeleton. Although a structural requirement for the band 3-ankyrin bridge is well established, the contribution of the glycophorin C-protein 4.1 bridge to red cell function remains to be defined. In order to explore this latter bridge further, we have identified and/or characterized five stimuli that sever the linkage in intact erythrocytes and have examined the impact of this rupture on membrane mechanical properties. We report here that elevation of cytosolic 2,3-bisphosphoglycerate, an increase in intracellular Ca(2+), removal of cell O(2), a decrease in intracellular pH, and activation of erythrocyte protein kinase C all promote dissociation of protein 4.1 from glycophorin C, leading to reduced retention of glycophorin C in detergent-extracted spectrin/actin skeletons. Significantly, where mechanical studies could be performed, we also observe that rupture of the membrane-to-skeleton bridge has little or no impact on the mechanical properties of the cell, as assayed by ektacytometry and nickel mesh filtration. We, therefore, suggest that, although regulation of the glycophorin C-protein 4.1-spectrin/actin bridge likely occurs physiologically, the role of the tether and the associated regulatory changes remain to be established. PMID:11294862

  6. ADP-ribosylation factor-like protein 4C (ARL4C) interacts with galectin-3 during oocyte development and embryogenesis in rainbow trout (Oncorhynchus mykiss)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ADP-ribosylation factor-like protein 4 (ARL4) is a GTP-binding protein which belongs to the ADP-ribosylation factor protein (ARF) superfamily of small GTPases. ARL4 has been shown to be mainly related to the development of male germ cells and embryogenesis in mouse. To investigate the role of ARL4 i...

  7. Usefulness of human epididymis protein 4 in predicting cytoreductive surgical outcomes for advanced ovarian tubal and peritoneal carcinoma

    PubMed Central

    Tang, Zhijian; Chang, Xiaohong; Ye, Xue; Li, Yi; Cheng, Hongyan

    2015-01-01

    Objective Human epididymis protein 4 (HE4) is a promising biomarker of epithelial ovarian cancer (EOC). But its role in assessing the primary optimal debulking (OD) of EOC remains unknown. The purpose of this study is to elucidate the ability of preoperative HE4 in predicting the primary cytoreductive outcomes in advanced EOC, tubal or peritoneal carcinoma. Methods We reviewed the records of 90 patients with advanced ovarian, tubal or peritoneal carcinoma who underwent primary cytoreduction at the Department of Obstetrics and Gynecology of Peking University People’s Hospital between November 2005 and October 2010. Preoperative serum HE4 and CA125 levels were detected with EIA kit. A receiver operating characteristic (ROC) curve was used to determine the most useful HE4 cut-off value. Logistic regression analysis was performed to identify significant preoperative clinical characteristics to predict optimal primary cytoreduction. Results OD was achieved in 47.7% (43/48) of patients. The median preoperative HE4 level for patients with OD vs. suboptimal debulking was 423 and 820 pmol/L, respectively (P<0.001). The areas under the ROC curve for HE4 and CA125 were 0.716 and 0.599, respectively (P=0.080). The most useful HE4 cut-off value was 473 pmol/L. Suboptimal cytoreduction was obtained in 66.7% (38/57) of cases with HE4 ≥473 pmol/L compared with only 27.3% (9/33) of cases with HE4 <473 pmol/L. At this threshold, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for diagnosing suboptimal debulking were 81%, 56%, 67%, and 73%, respectively. Logistic regression analysis showed that the patients with HE4 ≥473 pmol/L were less likely to achieve OD (odds ratio =5.044, P=0.002). Conclusions Preoperative serum HE4 may be helpful to predict whether optimal cytoreductive surgery could be obtained or whether extended cytoreduction would be needed by an interdisciplinary team. PMID:26157328

  8. Identification of high-risk patients by human epididymis protein 4 levels during follow-up of ovarian cancer

    PubMed Central

    STEFFENSEN, KARINA DAHL; WALDSTRØM, MARIANNE; BRANDSLUND, IVAN; LUND, BENTE; SØRENSEN, SARAH MEJER; PETZOLD, MAX; JAKOBSEN, ANDERS

    2016-01-01

    The majority of ovarian cancer patients with advanced disease at diagnosis will relapse following primary treatment, with a dismal prognosis. Monitoring the levels of serum markers in patients under follow-up may be essential for the early detection of relapse, and for distinguishing high-risk patients from those with less aggressive disease. The aim of the present study was to investigate the possible predictive value of human epididymis protein 4 (HE4) and carbohydrate antigen 125 (CA125) in relation to recurrence of epithelial ovarian cancer by measuring the two markers during follow-up subsequent to surgery and adjuvant first-line carboplatin/paclitaxel chemotherapy. Serum HE4 and CA125 were analyzed in 88 epithelial ovarian cancer patients at the end of treatment and consecutively during follow-up. The patients were divided into a high-risk and a low-risk group based on having an increase in HE4 and CA125 levels above or below 50% during follow-up, relative to the baseline (end-of-treatment) level. Disease recurrence was detected in 55 patients during follow-up. Patients with an increase in HE4 of >50% at 3- and 6-month follow-up compared to the end-of-treatment sample had significantly poorer progression-free survival (PFS) [hazard ratio (HR), 2.82 (95% CI, 0.91–8.79; P=0.0052) and HR, 7.71 (95% CI, 3.03–19.58; P<0.0001), respectively]. The corresponding 3- and 6-month biomarker assessments for increased CA125 levels (>50%) showed HRs of 1.86 (95% CI, 0.90–3.80; P=0.0512) and 2.55 (95% CI, 1.39–4.68; P=0.0011), respectively. Multivariate analysis confirmed HE4 as a predictor of short PFS, with an HR of 8.23 (95% CI, 3.28–20.9; P<0.0001) at 6-month follow-up. The increase of CA125 was not a significant prognostic factor in multivariate analysis for PFS. In conclusion, HE4 appears to be a sensitive marker of recurrence and instrumental in risk assessment during the first 6 months of follow-up. PMID:27313725

  9. Upregulation of Cyclooxygenase-2/Prostaglandin E2 (COX-2/PGE2) Pathway Member Multiple Drug Resistance-Associated Protein 4 (MRP4) and Downregulation of Prostaglandin Transporter (PGT) and 15-Prostaglandin Dehydrogenase (15-PGDH) in Triple-Negative Breast Cancer

    PubMed Central

    Kochel, Tyler J.; Goloubeva, Olga G.; Fulton, Amy M.

    2016-01-01

    Elevated levels of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) are indicators of a poor prognosis in breast cancer. Using several independent publicly available breast cancer gene expression databases, we investigated other members of the PGE2 pathway. PGE2 is produced by COX-2 and actively exported by multiple drug resistance-associated protein 4 (MRP4) into the extracellular microenvironment, where PGE2 can bind four cognate EP receptors (EP1–EP4) and initiate diverse biological signaling pathways. Alternatively, PGE2 is imported via the prostaglandin transporter (PGT) and metabolized by 15-prostaglandin dehydrogenase (15-PGDH/HPGD). We made the novel observation that MRP4, PGT, and 15-PGDH are differentially expressed among distinct breast cancer molecular subtypes; this finding was confirmed in independent datasets. In triple-negative breast cancer, the observed gene expression pattern (high COX-2, high MRP4, low PGT, and low 15-PGDH) would favor high levels of tumor-promoting PGE2 in the tumor microenvironment that may contribute to the overall poor prognosis of triple-negative breast cancer. PMID:27257388

  10. Specificity protein 4 (Sp4) regulates the transcription of AMPA receptor subunit GluA2 (Gria2).

    PubMed

    Priya, Anusha; Johar, Kaid; Nair, Bindu; Wong-Riley, Margaret T T

    2014-06-01

    The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are important glutamatergic receptors mediating fast excitatory synaptic transmission in the brain. The regulation of the four subunits of AMPA receptors, GluA1-4, is poorly understood. Excitatory synaptic transmission is highly energy-demanding, and this energy is derived mainly from the oxidative pathway. Recently, we found that specificity factor regulates all subunits of cytochrome c oxidase (COX), a critical energy-generating enzyme. COX is also regulated by nuclear respiratory factor 1 (NRF-1), which transcriptionally controls the Gria2 (GluA2) gene of AMPA receptors. The goal of the present study was to test our hypothesis that Sp-factors (Sp1, Sp3, and/or Sp4) also regulate AMPA subunit genes. If so, we wish to determine if Sp-factors and NRF-1 function via a complementary, concurrent and parallel, or a combination of complementary and concurrent/parallel mechanism. By means of multiple approaches, including electrophoretic mobility shift and supershift assays, chromatin immunoprecipitation, promoter mutations, real-time quantitative PCR, and western blot analysis, we found that Sp4, but not Sp1 or Sp3, regulates the Gria2, but not Gria1, 3, or 4, subunit gene of the AMPA receptor in a concurrent and parallel manner with NRF-1. Thus, Sp4 and NRF-1 both mediate the tight coupling between neuronal activity and energy metabolism at the transcriptional level. PMID:24576410

  11. Expression of programmed cell death protein 4 (PDCD4) and miR-21 in urothelial carcinoma

    SciTech Connect

    Fischer, Nicolas; Goeke, Friederike; Splittstoesser, Vera; Lankat-Buttgereit, Brigitte; Mueller, Stefan C.; Ellinger, Joerg

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer The tumor suppressor gene PDCD4 is down-regulated in many tumorous entities. Black-Right-Pointing-Pointer We investigate the impact of PDCD4 and its regulating factor miR-21 in urothelial carcinoma. Black-Right-Pointing-Pointer We confirm PDCD4 as a tumor suppressor gene and it could be a diagnostic marker for this tumor. -- Abstract: Background: We investigated the role of the programmed cell death 4 (PDCD4) tumor suppressor gene in specimens of transitional cell carcinoma and of healthy individuals. Methods: PDCD4 immunohistochemical expression was investigated in 294 cases in histologically proven transitional cell carcinoma in different tumorous stages (28 controls, 122 non-muscle invasive urothelial carcinoma, stages Tis-T1, 119 invasive transitional cell carcinoma stages T2-T4 and 25 metastases). MiR-21 expression, an important PDCD4 regulator, was assessed with real-time PCR analysis and showed inverse correlation to tissue PDCD4 expression. Results: Nuclear and cytoplasmatic PDCD4 immunostaining decreased significantly with histopathological progression of the tumor (p < 0001). Controls showed strong nuclear and cytoplasmatic immunohistochemical staining. MiR-21 up regulation in tissue corresponded to PDCD4 suppression. Conclusions: These data support a decisive role for PDCD4 down regulation in transitional cell carcinoma and confirm miR-21 as a negative regulator for PDCD4. Additionally, PDCD4 immunohistochemical staining turns out to be a possible diagnostic marker for transitional cell carcinoma.

  12. Multivalent interactions of the SUMO-interaction motifs in RING finger protein 4 determine the specificity for chains of the SUMO

    PubMed Central

    Keusekotten, Kirstin; Bade, Veronika N.; Meyer-Teschendorf, Katrin; Sriramachandran, Annie Miriam; Fischer-Schrader, Katrin; Krause, Anke; Horst, Christiane; Schwarz, Günter; Hofmann, Kay; Dohmen, R. Jürgen; Praefcke, Gerrit J. K.

    2013-01-01

    RNF4 (RING finger protein 4) is a STUbL [SUMO (small ubiquitin-related modifier)-targeted ubiquitin ligase] controlling PML (promyelocytic leukaemia) nuclear bodies, DNA double strand break repair and other nuclear functions. In the present paper, we describe that the sequence and spacing of the SIMs (SUMO-interaction motifs) in RNF4 regulate the avidity-driven recognition of substrate proteins carrying SUMO chains of variable length. PMID:24151981

  13. Protein 4.1N does not interact with the inositol 1,4,5-trisphosphate receptor in an epithelial cell line.

    PubMed

    Sehgal, Sona; Guerra, Mateus T; Kruglov, Emma A; Wang, Jun; Nathanson, Michael H

    2005-11-01

    Cytosolic Ca2+ regulates a variety of cell functions, and the spatial patterns of Ca2+ signals are responsible in part for the versatility of this second messenger. The subcellular distribution of the inositol 1,4,5-trisphosphate receptor (IP3R) is thought to regulate Ca2+-signaling patterns but little is known about how the distribution of the IP3R itself is regulated. Here we examined the relationship between the IP3R and the cytoskeletal linker protein 4.1N in the polarized WIF-B cell line because protein 4.1N regulates targeting of the type I IP3R in neurons, but WIF-B cells do not express this cytoskeletal protein. WIF-B cells expressed all three isoforms of the IP3R, and each isoform was distributed throughout the cell. These cells did not express the ryanodine receptor. Photorelease of microinjected, caged IP3 induced a rapid rise in cytosolic Ca2+, but the increase began uniformly throughout the cell rather than at a specific initiation site. Expression of protein 4.1N was not associated with redistribution of the IP3R or changes in Ca2+-signaling patterns. These findings are consistent with the hypothesis that the subcellular distribution of IP3R isoforms regulates the formation of Ca2+ waves, and the finding that interactions between protein 4.1N and the IP3R vary among cell types may provide an additional, tissue-specific mechanism to shape the pattern of Ca2+ waves. PMID:16122796

  14. Multivalent interactions of the SUMO-interaction motifs in RING finger protein 4 determine the specificity for chains of the SUMO.

    PubMed

    Keusekotten, Kirstin; Bade, Veronika N; Meyer-Teschendorf, Katrin; Sriramachandran, Annie Miriam; Fischer-Schrader, Katrin; Krause, Anke; Horst, Christiane; Schwarz, Günter; Hofmann, Kay; Dohmen, R Jürgen; Praefcke, Gerrit J K

    2014-01-01

    RNF4 (RING finger protein 4) is a STUbL [SUMO (small ubiquitin-related modifier)-targeted ubiquitin ligase] controlling PML (promyelocytic leukaemia) nuclear bodies, DNA double strand break repair and other nuclear functions. In the present paper, we describe that the sequence and spacing of the SIMs (SUMO-interaction motifs) in RNF4 regulate the avidity-driven recognition of substrate proteins carrying SUMO chains of variable length. PMID:24151981

  15. Protein 4.1R links E-cadherin/beta-catenin complex to the cytoskeleton through its direct interaction with beta-catenin and modulates adherens junction integrity.

    PubMed

    Yang, Shaomin; Guo, Xinhua; Debnath, Gargi; Mohandas, Narla; An, Xiuli

    2009-07-01

    Protein 4.1R (4.1R) is the prototypical member of the protein 4.1 superfamily comprising of the protein 4.1 family (4.1R, 4.1B, 4.1G and 4.1N) and ERM family (ezrin, radixin and meosin). These proteins in general serve as adaptors between the membrane and the cytoskeleton. Here we show that 4.1R expressed in the gastric epithelial cells associates with adherens junction protein beta-catenin. Biochemical examination of 4.1R-deficient stomach epithelia revealed a selective reduction of beta-catenin which is accompanied by a weaker linkage of E-cadherin to the cytoskeleton. In addition, organization of actin cytoskeleton was altered in 4.1R-deficient cells. Moreover, histological examination revealed that cell-cell contacts are impaired and gastric glands are disorganized in 4.1R null stomach epithelia. These results demonstrate an important and previously unidentified role of 4.1R in linking the cadherin/catenin complex to the cytoskeleton through its direct interaction with beta-catenin and in regulating the integrity of adherens junction. PMID:19376086

  16. Organization of the human protein 4.1 genomic locus: New insights into the tissue-specific alternative splicing of the pre-mRNA

    SciTech Connect

    Baklouti, F. ||; Huang, Shu-Ching; Benz, E.J. Jr. |

    1997-02-01

    Protein 4.1 is a globular 80-kDa component of the erythrocyte membrane skeleton that enhances spectrin-actin interaction via its internal 10-kDa domain. Previous studies have shown that protein 4.1 mRNA is expressed as multiple alternatively spliced isoforms, resulting from the inclusion or exclusion of small cassette sequences called motifs. By tissue screening for protein 4.1 isoforms, we have observed new features of an already complex pattern of alternative splicing within the spectrin/actin binding domain. In particular, we found a new 51-nt exon that is present almost exclusively in muscle tissue. In addition, we have isolated multiple genomic clones spanning over 200 kb, containing the entire erythroid and nonerythroid coding sequence of the human locus. The exon/intron structure has now been characterized; with the exception of a 17-nt motif, all of the alternatively spliced motifs correspond to individual exons. The 3{prime}-untranslated region (UTR) has also been completely sequenced using various PCR and genomic-sequencing methods. The 3{prime} UTR, over 3 kb, accounts for one-half of the mature mRNA. 83 refs., 8 figs., 1 tab.

  17. The protein 4.1 tumor suppressor, DAL-1, impairs cell motility, but regulates proliferation in a cell-type-specific fashion.

    PubMed

    Gutmann, D H; Hirbe, A C; Huang, Z Y; Haipek, C A

    2001-04-01

    The neurofibromatosis 2 (NF2) tumor suppressor belongs to the Protein 4.1 family of molecules that link the actin cytoskeleton to cell surface glycoproteins. We have previously demonstrated that the NF2 protein, merlin, can suppress cell growth in vitro and in vivo as well as impair actin cytoskeleton-associated processes, such as cell spreading, attachment, and motility. Recently, we determined that expression of a second Protein 4.1 tumor suppressor, DAL-1, was lost in 60% of sporadic meningiomas, but not schwannomas. In this report, we demonstrate that DAL-1 suppresses cell proliferation in meningioma, but not schwannoma cells. Similar to merlin, DAL-1 interacts with other ERM proteins and betaII-spectrin, but not the merlin interactor protein, SCHIP-1. In addition, we report the identification of the full-length DAL-1 tumor suppressor, termed KIAA0987. Collectively, these results suggest that the two Protein 4.1 meningioma tumor suppressors, merlin and DAL-1, may be functionally distinct proteins with different mechanisms of action. PMID:11300722

  18. Misexpression of BRE gene in the developing chick neural tube affects neurulation and somitogenesis

    PubMed Central

    Wang, Guang; Li, Yan; Wang, Xiao-Yu; Chuai, Manli; Yeuk-Hon Chan, John; Lei, Jian; Münsterberg, Andrea; Lee, Kenneth Ka Ho; Yang, Xuesong

    2015-01-01

    The brain and reproductive expression (BRE) gene is expressed in numerous adult tissues and especially in the nervous and reproductive systems. However, little is known about BRE expression in the developing embryo or about its role in embryonic development. In this study, we used in situ hybridization to reveal the spatiotemporal expression pattern for BRE in chick embryo during development. To determine the importance of BRE in neurogenesis, we overexpressed BRE and also silenced BRE expression specifically in the neural tube. We established that overexpressing BRE in the neural tube indirectly accelerated Pax7+ somite development and directly increased HNK-1+ neural crest cell (NCC) migration and TuJ-1+ neurite outgrowth. These altered morphogenetic processes were associated with changes in the cell cycle of NCCs and neural tube cells. The inverse effect was obtained when BRE expression was silenced in the neural tube. We also determined that BMP4 and Shh expression in the neural tube was affected by misexpression of BRE. This provides a possible mechanism for how altering BRE expression was able to affect somitogenesis, neurogenesis, and NCC migration. In summary, our results demonstrate that BRE plays an important role in regulating neurogenesis and indirectly somite differentiation during early chick embryo development. PMID:25568339

  19. Dragon (repulsive guidance molecule b, RGMb) is a novel gene that promotes colorectal cancer growth

    PubMed Central

    Shi, Ying; Chen, Guo-Bin; Huang, Xiao-Xiao; Xiao, Chuan-Xing; Wang, Huan-Huan; Li, Ye-Sen; Zhang, Jin-Fang; Li, Shao; Xia, Yin; Ren, Jian-Lin; Guleng, Bayasi

    2015-01-01

    Colorectal cancer (CRC) is one of the most commonly diagnosed cancers and a major cause of cancer death. However, the molecular mechanisms underlying CRC initiation, growth and metastasis are poorly understood. Dragon (RGMb), a member of the repulsive guidance molecule (RGM) family, has been recently identified as a co-receptor for bone morphogenetic protein (BMP) signaling, but the role of Dragon in CRC development is undefined. Here, we show that Dragon expression was increased in colon cancer tissues compared to control tissues in CAC mouse model and in human patients. Dragon promoted proliferation of CT26.WT and CMT93 colon cancer cells and accelerated tumor growth in the xenograft mouse model. Dragon's action on colon cancer development was mediated via the BMP4-Smad1/5/8 and Erk1/2 pathways. Therefore, our results have revealed that Dragon is a novel gene that promotes CRC growth through the BMP pathway. Dragon may be exploited as a potential therapeutic target for CRC treatment. PMID:26029998

  20. Mature parasite-infected erythrocyte surface antigen (MESA) of Plasmodium falciparum binds to the 30-kDa domain of protein 4.1 in malaria-infected red blood cells.

    PubMed

    Waller, Karena L; Nunomura, Wataru; An, Xiuli; Cooke, Brian M; Mohandas, Narla; Coppel, Ross L

    2003-09-01

    The Plasmodium falciparum mature parasite-infected erythrocyte surface antigen (MESA) is exported from the parasite to the infected red blood cell (IRBC) membrane skeleton, where it binds to protein 4.1 (4.1R) via a 19-residue MESA sequence. Using purified RBC 4.1R and recombinant 4.1R fragments, we show MESA binds the 30-kDa region of RBC 4.1R, specifically to a 51-residue region encoded by exon 10 of the 4.1R gene. The 3D structure of this region reveals that the MESA binding site overlaps the region of 4.1R involved in the p55, glycophorin C, and 4.1R ternary complex. Further binding studies using p55, 4.1R, and MESA showed competition between p55 and MESA for 4.1R, implying that MESA bound at the IRBC membrane skeleton may modulate normal 4.1R and p55 interactions in vivo. Definition of minimal binding domains involved in critical protein interactions in IRBCs may aid the development of novel therapies for falciparum malaria. PMID:12730097

  1. Fatty acid transport protein 1 can compensate for fatty acid transport protein 4 in the developing mouse epidermis.

    PubMed

    Lin, Meei-Hua; Miner, Jeffrey H

    2015-02-01

    Fatty acid transport protein (FATP) 4 is one of a family of six FATPs that facilitate long- and very-long-chain fatty acid uptake. Mice lacking FATP4 are born with tight, thick skin and a defective barrier; they die neonatally because of dehydration and restricted movements. Mutations in SLC27A4, the gene encoding FATP4, cause ichthyosis prematurity syndrome (IPS), characterized by premature birth, respiratory distress, and edematous skin with severe ichthyotic scaling. Symptoms of surviving patients become mild, although atopic manifestations are common. We previously showed that suprabasal keratinocyte expression of a Fatp4 transgene in Fatp4 mutant skin rescues the lethality and ameliorates the skin phenotype. Here we tested the hypothesis that FATP1, the closest FATP4 homolog, can compensate for the lack of FATP4 in our mouse model of IPS, as it might do postnatally in IPS patients. Transgenic expression of FATP1 in suprabasal keratinocytes rescued the phenotype of Fatp4 mutants, and FATP1 sorted to the same intracellular organelles as endogenous FATP4. Thus, FATP1 and FATP4 likely have overlapping substrate specificities, enzymatic activities, and biological functions. These results suggest that increasing expression of FATP1 in suprabasal keratinocytes could normalize the skin of IPS patients and perhaps prevent the atopic manifestations. PMID:25184958

  2. RM-04RETINOBLASTOMA BINDING PROTEIN 4 (RBBP4) MODULATES TEMOZOLOMIDE RESPONSE THROUGH REGULATION OF MGMT EXPRESSION IN GLIOBLASTOMA

    PubMed Central

    Kitange, Gaspar; Schroeder, Mark; Sarkaria, Jann

    2014-01-01

    Through shRNA library screen we identified RBBP4 as a modulator of TMZ response in glioblastoma (GBM). Consequently, we investigated the mechanisms whereby RBBP4 modulates TMZ response using shRNA to silence this gene in MGMT-expressing T98G and U138 GBM cells. The cytotoxicity was evaluated using fluorescence-based CYQUANT proliferation assay. A total of 4 shRNA constructs significantly suppressed RBBP4 in both T98G and U138. Cells expressing non-specific targeting shRNA (NT-shRNA) were used as control. RBBP4 knockdown significantly sensitized TMZ both in T98G and U138 cells; the relative fluorescence for the TMZ-treated (100 µM) control T98NT-shRNA cells was 1.17 ± 0.15, whereas for T98RBBP4-shRNA clones were 0.54 ± 0.02, 0.29 ± 0.03, 0.36 ± 0.05, and 0.34 ± 0.03, respectively (p < 0.001). Similar sensitization was observed in U138 cells; relative fluorescence for the TMZ-treated (300 µM) control U138NT-shRNA cells was 0.70 ± 0.05 and for U138RBBP4-shRNA clones were 0.42 ± 0.06, 0.27 ± 0.01, 0.28 ± 0.02, and 0.30 ± 0.01, respectively (p < 0.001). Interestingly, knockdown of RBBP4 in T98G was accompanied with a synthetic lethality to PARP inhibition and increased response to TMZ-induced DNA damage, as evidenced by increased phosphorylation of KAP1, CHK1 and CHK2. Moreover, phosphorylation of H2AX in response to TMZ treatment was significantly higher in T98RBBP4-shRNA clones. Consistent with deficient homologous recombination (HR), T98RBBP4-shRNA clones significantly expressed less RAD51 compared with the control T98NT-shRNA cells. Even more interesting, RBBP4 knockdown silenced MGMT expression in both T98G and U138, which was accompanied by decreased recruitment of acetylated H3K9 coupled with increased recruitment of tri-methylated H3K9. Moreover, RBBP4 knockdown was coupled with loss of p300 recruitment to bind MGMT promoter region. Collectively, these findings suggest that RBBP4 modulates TMZ response in GBM cells through epigenetic regulation of

  3. Protein 4.1N acts as a potential tumor suppressor linking PP1 to JNK-c-Jun pathway regulation in NSCLC.

    PubMed

    Wang, Zi; Ma, Bianyin; Li, Hui; Xiao, Xiaojuan; Zhou, Weihua; Liu, Feng; Zhang, Bin; Zhu, Min; Yang, Qin; Zeng, Yayue; Sun, Yang; Sun, Shuming; Wang, Yanpeng; Zhang, Yibin; Weng, Haibo; Chen, Lixiang; Ye, Mao; An, Xiuli; Liu, Jing

    2016-01-01

    Protein 4.1N is a member of protein 4.1 family and has been recognized as a potential tumor suppressor in solid tumors. Here, we aimed to investigate the role and mechanisms of 4.1N in non-small cell lung cancer (NSCLC). We confirmed that the expression level of 4.1N was inversely correlated with the metastatic properties of NSCLC cell lines and histological grade of clinical NSCLC tissues. Specific knockdown of 4.1N promoted tumor cell proliferation, migration and adhesion in vitro, and tumor growth and metastasis in mouse xenograft models. Furthermore, we identified PP1 as a novel 4.1N-interacting molecule, and the FERM domain of 4.1N mediated the interaction between 4.1N and PP1. Further, ectopic expression of 4.1N could inactivate JNK-c-Jun signaling pathway through enhancing PP1 activity and interaction between PP1 and p-JNK. Correspondingly, expression of potential downstream metastasis targets (ezrin and MMP9) and cell cycle targets (p53, p21 and p19) of JNK-c-Jun pathway were also regulated by 4.1N. Our data suggest that down-regulation of 4.1N expression is a critical step for NSCLC development and that repression of JNK-c-Jun signaling through PP1 is one of the key anti-tumor mechanisms of 4.1N. PMID:26575790

  4. The type I inositol 1,4,5-trisphosphate receptor interacts with protein 4.1N to mediate neurite formation through intracellular Ca waves.

    PubMed

    Fiedler, Michael J; Nathanson, Michael H

    2011-01-01

    Ca(2+) waves are an important mechanism for encoding Ca(2+) signaling information, but the molecular basis for wave formation and how this regulates neuronal function is not entirely understood. Using nerve growth factor-differentiated PC12 cells as a model system, we investigated the interaction between the type I inositol 1,4,5-trisphosphate receptor (IP3R1) and the cytoskeletal linker, protein 4.1N, to examine the relationship between Ca(2+) wave formation and neurite development. This was examined using RNAi and overexpressed dominant negative binding regions of each protein. Confocal microscopy was used to monitor neurite formation and Ca(2+) waves. Knockdown of IP3R1 or 4.1N attenuated neurite formation, as did binding regions of IP3R1 and 4.1N, which colocalized with endogenous 4.1N and IP3R1, respectively. Upon stimulation with the IP3-producing agonist carbachol, both RNAi and dominant negative molecules shifted signaling events from waves to homogeneous patterns of Ca(2+) release. These findings provide evidence that IP3R1 localization, via protein 4.1N, is necessary for Ca(2+) wave formation, which in turn mediates neurite formation. PMID:21389686

  5. Protein 4.1R regulates cell adhesion, spreading, migration and motility of mouse keratinocytes by modulating surface expression of β1 integrin

    PubMed Central

    Chen, Lixiang; Hughes, Richard A.; Baines, Anthony J.; Conboy, John; Mohandas, Narla; An, Xiuli

    2011-01-01

    Protein 4.1R is a membrane-cytoskeleton adaptor protein that has diverse roles in controlling the cell surface expression and/or function of transmembrane proteins, and in organizing F-actin. 4.1R is expressed in keratinocytes, but its role in these cells has not been explored. Here, we have investigated the role of 4.1R in skin using 4.1R−/− mice. Cell adhesion, spreading, migration and motility were significantly impaired in 4.1R−/− keratinocytes, and 4.1R−/− mice exhibited defective epidermal wound healing. Cultured 4.1R−/− keratinocytes on fibronectin failed to form actin stress fibres and focal adhesions. Furthermore, in the absence of 4.1R, the surface expression, and consequently the activity of β1 integrin were reduced. These data enabled the identification of a functional role for protein 4.1R in keratinocytes by modulating the surface expression of β1 integrin, possibly through a direct association between 4.1R and β1 integrin. PMID:21693581

  6. Protein 4.1N binding to nuclear mitotic apparatus protein in PC12 cells mediates the antiproliferative actions of nerve growth factor.

    PubMed

    Ye, K; Compton, D A; Lai, M M; Walensky, L D; Snyder, S H

    1999-12-15

    Protein 4.1N is a neuronal selective isoform of the erythrocyte membrane cytoskeleton protein 4.1R. In the present study, we demonstrate an interaction between 4.1N and nuclear mitotic apparatus protein (NuMA), a nuclear protein required for mitosis. The binding involves the C-terminal domain of 4.1N. In PC12 cells treatment with nerve growth factor (NGF) elicits translocation of 4. 1N to the nucleus and promotes its association with NuMA. Specific targeting of 4.1N to the nucleus arrests PC12 cells at the G1 phase and produces an aberrant nuclear morphology. Inhibition of 4.1N nuclear translocation prevents the NGF-mediated arrest of cell division, which can be reversed by overexpression of 4.1N. Thus, nuclear 4.1N appears to mediate the antiproliferative actions of NGF by antagonizing the role of NuMA in mitosis. PMID:10594058

  7. The Type I Inositol 1,4,5-Trisphosphate Receptor Interacts with Protein 4.1N to Mediate Neurite Formation through Intracellular Ca2+ Waves

    PubMed Central

    Fiedler, Michael J.; Nathanson, Michael H.

    2011-01-01

    Ca2+ waves are an important mechanism for encoding Ca2+ signaling information, but the molecular basis for wave formation and how this regulates neuronal function is not entirely understood. Using nerve growth factor-differentiated PC12 cells as a model system, we investigated the interaction between the type I inositol 1,4,5-trisphosphate receptor (IP3R1) and the cytoskeletal linker, protein 4.1N, to examine the relationship between Ca2+ wave formation and neurite development. This was examined using RNAi and overexpressed dominant negative binding regions of each protein. Confocal microscopy was used to monitor neurite formation and Ca2+ waves. Knockdown of IP3R1 or 4.1N attenuated neurite formation, as did binding regions of IP3R1 and 4.1N, which colocalized with endogenous 4.1N and IP3R1, respectively. Upon stimulation with the IP3-producing agonist carbachol, both RNAi and dominant negative molecules shifted signaling events from waves to homogeneous patterns of Ca2+ release. These findings provide evidence that IP3R1 localization, via protein 4.1N, is necessary for Ca2+ wave formation, which in turn mediates neurite formation. PMID:21389686

  8. Protein 4.1N acts as a potential tumor suppressor linking PP1 to JNK-c-Jun pathway regulation in NSCLC

    PubMed Central

    Zhou, Weihua; Liu, Feng; Zhang, Bin; Zhu, Min; Yang, Qin; Zeng, Yayue; Sun, Yang; Sun, Shuming; Wang, Yanpeng; Zhang, Yibin; Weng, Haibo; Chen, Lixiang; Ye, Mao; An, Xiuli; Liu, Jing

    2016-01-01

    Protein 4.1N is a member of protein 4.1 family and has been recognized as a potential tumor suppressor in solid tumors. Here, we aimed to investigate the role and mechanisms of 4.1N in non-small cell lung cancer (NSCLC). We confirmed that the expression level of 4.1N was inversely correlated with the metastatic properties of NSCLC cell lines and histological grade of clinical NSCLC tissues. Specific knockdown of 4.1N promoted tumor cell proliferation, migration and adhesion in vitro, and tumor growth and metastasis in mouse xenograft models. Furthermore, we identified PP1 as a novel 4.1N-interacting molecule, and the FERM domain of 4.1N mediated the interaction between 4.1N and PP1. Further, ectopic expression of 4.1N could inactivate JNK-c-Jun signaling pathway through enhancing PP1 activity and interaction between PP1 and p-JNK. Correspondingly, expression of potential downstream metastasis targets (ezrin and MMP9) and cell cycle targets (p53, p21 and p19) of JNK-c-Jun pathway were also regulated by 4.1N. Our data suggest that down-regulation of 4.1N expression is a critical step for NSCLC development and that repression of JNK-c-Jun signaling through PP1 is one of the key anti-tumor mechanisms of 4.1N. PMID:26575790

  9. Protein 4.1R regulates cell adhesion, spreading, migration and motility of mouse keratinocytes by modulating surface expression of beta1 integrin.

    PubMed

    Chen, Lixiang; Hughes, Richard A; Baines, Anthony J; Conboy, John; Mohandas, Narla; An, Xiuli

    2011-07-15

    Protein 4.1R is a membrane-cytoskeleton adaptor protein that has diverse roles in controlling the cell surface expression and/or function of transmembrane proteins, and in organizing F-actin. 4.1R is expressed in keratinocytes, but its role in these cells has not been explored. Here, we have investigated the role of 4.1R in skin using 4.1R(-/-) mice. Cell adhesion, spreading, migration and motility were significantly impaired in 4.1R(-/-) keratinocytes, and 4.1R(-/-) mice exhibited defective epidermal wound healing. Cultured 4.1R(-/-) keratinocytes on fibronectin failed to form actin stress fibres and focal adhesions. Furthermore, in the absence of 4.1R, the surface expression, and consequently the activity of β1 integrin were reduced. These data enabled the identification of a functional role for protein 4.1R in keratinocytes by modulating the surface expression of β1 integrin, possibly through a direct association between 4.1R and β1 integrin. PMID:21693581

  10. CHIR99021 promotes self-renewal of mouse embryonic stem cells by modulation of protein-encoding gene and long intergenic non-coding RNA expression

    SciTech Connect

    Wu, Yongyan; Ai, Zhiying; Yao, Kezhen; Cao, Lixia; Du, Juan; Shi, Xiaoyan; Guo, Zekun; Zhang, Yong

    2013-10-15

    Embryonic stem cells (ESCs) can proliferate indefinitely in vitro and differentiate into cells of all three germ layers. These unique properties make them exceptionally valuable for drug discovery and regenerative medicine. However, the practical application of ESCs is limited because it is difficult to derive and culture ESCs. It has been demonstrated that CHIR99021 (CHIR) promotes self-renewal and enhances the derivation efficiency of mouse (m)ESCs. However, the downstream targets of CHIR are not fully understood. In this study, we identified CHIR-regulated genes in mESCs using microarray analysis. Our microarray data demonstrated that CHIR not only influenced the Wnt/β-catenin pathway by stabilizing β-catenin, but also modulated several other pluripotency-related signaling pathways such as TGF-β, Notch and MAPK signaling pathways. More detailed analysis demonstrated that CHIR inhibited Nodal signaling, while activating bone morphogenetic protein signaling in mESCs. In addition, we found that pluripotency-maintaining transcription factors were up-regulated by CHIR, while several developmental-related genes were down-regulated. Furthermore, we found that CHIR altered the expression of epigenetic regulatory genes and long intergenic non-coding RNAs. Quantitative real-time PCR results were consistent with microarray data, suggesting that CHIR alters the expression pattern of protein-encoding genes (especially transcription factors), epigenetic regulatory genes and non-coding RNAs to establish a relatively stable pluripotency-maintaining network. - Highlights: • Combined use of CHIR with LIF promotes self-renewal of J1 mESCs. • CHIR-regulated genes are involved in multiple pathways. • CHIR inhibits Nodal signaling and promotes Bmp4 expression to activate BMP signaling. • Expression of epigenetic regulatory genes and lincRNAs is altered by CHIR.

  11. Genes and gene regulation

    SciTech Connect

    MacLean, N.

    1988-01-01

    Genetics has long been a central topic for biologists, and recent progress has captured the public imagination as well. This book addresses questions that are at the leading edge of this continually advancing discipline. In tune with the increasing emphasis on molecular biology and genetic engineering, this text emphasizes the molecular aspects of gene expression, and the evolution of gene sequence organization and control. It reviews the genetic material of viruses, bacteria, and of higher organisms. Cells and organisms are compared in terms of gene numbers, their arrangements within a cell, and the control mechanisms which regulate the activity of genes.

  12. Identification and functional analysis of novel facial patterning genes in the duplicated beak chicken embryo.

    PubMed

    Nimmagadda, Suresh; Buchtová, Marcela; Fu, Katherine; Geetha-Loganathan, Poongodi; Hosseini-Farahabadi, Sara; Trachtenberg, Alexander J; Kuo, Winston Patrick; Vesela, Iva; Richman, Joy M

    2015-11-15

    Cranial neural crest cells form the majority of the facial skeleton. However exactly when the pattering information and hence jaw identity is established is not clear. We know that premigratory neural crest cells contain a limited amount of information about the lower jaw but the upper jaw and facial midline are specified later by local tissue interactions. The environmental signals leading to frontonasal identity have been explored by our group in the past. Altering the levels of two signaling pathways (Bone Morphogenetic Protein) and retinoic acid (RA) in the chicken embryo creates a duplicated midline on the side of the upper beak complete with egg tooth in place of maxillary derivatives (Lee et al., 2001). Here we analyze the transcriptome 16 h after bead placement in order to identify potential mediators of the identity change in the maxillary prominence. The gene list included RA, BMP and WNT signaling pathway genes as well as transcription factors expressed in craniofacial development. There was also cross talk between Noggin and RA such that Noggin activated the RA pathway. We also observed expression changes in several poorly characterized genes including the upregulation of Peptidase Inhibitor-15 (PI15). We tested the functional effects of PI15 overexpression with a retroviral misexpression strategy. PI15 virus induced a cleft beak analogous to human cleft lip. We next asked whether PI15 effects were mediated by changes in expression of major clefting genes and genes in the retinoid signaling pathway. Expression of TP63, TBX22, BMP4 and FOXE1, all human clefting genes, were upregulated. In addition, ALDH1A2, ALDH1A3 and RA target, RARβ were increased while the degradation enzyme CYP26A1 was decreased. Together these changes were consistent with activation of the RA pathway. Furthermore, PI15 retrovirus injected into the face was able to replace RA and synergize with Noggin to induce beak transformations. We conclude that the microarrays have generated a

  13. Characterization of Microfibrillar-associated Protein 4 (MFAP4) as a Tropoelastin- and Fibrillin-binding Protein Involved in Elastic Fiber Formation.

    PubMed

    Pilecki, Bartosz; Holm, Anne T; Schlosser, Anders; Moeller, Jesper B; Wohl, Alexander P; Zuk, Alexandra V; Heumüller, Stefanie E; Wallis, Russell; Moestrup, Soren K; Sengle, Gerhard; Holmskov, Uffe; Sorensen, Grith L

    2016-01-15

    MFAP4 (microfibrillar-associated protein 4) is an extracellular glycoprotein found in elastic fibers without a clearly defined role in elastic fiber assembly. In the present study, we characterized molecular interactions between MFAP4 and elastic fiber components. We established that MFAP4 primarily assembles into trimeric and hexameric structures of homodimers. Binding analysis revealed that MFAP4 specifically binds tropoelastin and fibrillin-1 and -2, as well as the elastin cross-linking amino acid desmosine, and that it co-localizes with fibrillin-1-positive fibers in vivo. Site-directed mutagenesis disclosed residues Phe(241) and Ser(203) in MFAP4 as being crucial for type I collagen, elastin, and tropoelastin binding. Furthermore, we found that MFAP4 actively promotes tropoelastin self-assembly. In conclusion, our data identify MFAP4 as a new ligand of microfibrils and tropoelastin involved in proper elastic fiber organization. PMID:26601954

  14. Compartmentalized Accumulation of cAMP near Complexes of Multidrug Resistance Protein 4 (MRP4) and Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Contributes to Drug-induced Diarrhea*

    PubMed Central

    Moon, Changsuk; Zhang, Weiqiang; Ren, Aixia; Arora, Kavisha; Sinha, Chandrima; Yarlagadda, Sunitha; Woodrooffe, Koryse; Schuetz, John D.; Valasani, Koteswara Rao; de Jonge, Hugo R.; Shanmukhappa, Shiva Kumar; Shata, Mohamed Tarek M.; Buddington, Randal K.; Parthasarathi, Kaushik; Naren, Anjaparavanda P.

    2015-01-01

    Diarrhea is one of the most common adverse side effects observed in ∼7% of individuals consuming Food and Drug Administration (FDA)-approved drugs. The mechanism of how these drugs alter fluid secretion in the gut and induce diarrhea is not clearly understood. Several drugs are either substrates or inhibitors of multidrug resistance protein 4 (MRP4), such as the anti-colon cancer drug irinotecan and an anti-retroviral used to treat HIV infection, 3′-azido-3′-deoxythymidine (AZT). These drugs activate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated fluid secretion by inhibiting MRP4-mediated cAMP efflux. Binding of drugs to MRP4 augments the formation of MRP4-CFTR-containing macromolecular complexes that is mediated via scaffolding protein PDZK1. Importantly, HIV patients on AZT treatment demonstrate augmented MRP4-CFTR complex formation in the colon, which defines a novel paradigm of drug-induced diarrhea. PMID:25762723

  15. Assembly of juxtaparanodes in myelinating DRG culture: Differential clustering of the Kv1/Caspr2 complex and scaffolding protein 4.1B.

    PubMed

    Hivert, Bruno; Pinatel, Delphine; Labasque, Marilyne; Tricaud, Nicolas; Goutebroze, Laurence; Faivre-Sarrailh, Catherine

    2016-05-01

    The precise distribution of ion channels at the nodes of Ranvier is essential for the efficient propagation of action potentials along myelinated axons. The voltage-gated potassium channels Kv1.1/1.2 are clustered at the juxtaparanodes in association with the cell adhesion molecules, Caspr2 and TAG-1 and the scaffolding protein 4.1B. In the present study, we set up myelinating cultures of DRG neurons and Schwann cells to look through the formation of juxtaparanodes in vitro. We showed that the Kv1.1/Kv1.2 channels were first enriched at paranodes before being restricted to distal paranodes and juxtaparanodes. In addition, the Kv1 channels displayed an asymmetric expression enriched at the distal juxtaparanodes. Caspr2 was strongly co-localized with Kv1.2 whereas the scaffolding protein 4.1B was preferentially recruited at paranodes while being present at juxtaparanodes too. Kv1.2/Caspr2 but not 4.1B, also transiently accumulated within the nodal region both in myelinated cultures and developing sciatic nerves. Studying cultures and sciatic nerves from 4.1B KO mice, we further showed that 4.1B is required for the proper targeting of Caspr2 early during myelination. Moreover, using adenoviral-mediated expression of Caspr-GFP and photobleaching experiments, we analyzed the stability of paranodal junctions and showed that the lateral stability of paranodal Caspr was not altered in 4.1B KO mice indicating that 4.1B is not required for the assembly and stability of the paranodal junctions. Thus, developing an adapted culture paradigm, we provide new insights into the dynamic and differential distribution of Kv1 channels and associated proteins during myelination. GLIA 2016;64:840-852. PMID:26840208

  16. A 130-kDa Protein 4.1B Regulates Cell Adhesion, Spreading, and Migration of Mouse Embryo Fibroblasts by Influencing Actin Cytoskeleton Organization*

    PubMed Central

    Wang, Jie; Song, Jinlei; An, Chao; Dong, Wenji; Zhang, Jingxin; Yin, Changcheng; Hale, John; Baines, Anthony J.; Mohandas, Narla; An, Xiuli

    2014-01-01

    Protein 4.1B is a member of protein 4.1 family, adaptor proteins at the interface of membranes and the cytoskeleton. It is expressed in most mammalian tissues and is known to be required in formation of nervous and cardiac systems; it is also a tumor suppressor with a role in metastasis. Here, we explore functions of 4.1B using primary mouse embryonic fibroblasts (MEF) derived from wild type and 4.1B knock-out mice. MEF cells express two 4.1B isoforms: 130 and 60-kDa. 130-kDa 4.1B was absent from 4.1B knock-out MEF cells, but 60-kDa 4.1B remained, suggesting incomplete knock-out. Although the 130-kDa isoform was predominantly located at the plasma membrane, the 60-kDa isoform was enriched in nuclei. 130-kDa-deficient 4.1B MEF cells exhibited impaired cell adhesion, spreading, and migration; they also failed to form actin stress fibers. Impaired cell spreading and stress fiber formation were rescued by re-expression of the 130-kDa 4.1B but not the 60-kDa 4.1B. Our findings document novel, isoform-selective roles for 130-kDa 4.1B in adhesion, spreading, and migration of MEF cells by affecting actin organization, giving new insight into 4.1B functions in normal tissues as well as its role in cancer. PMID:24381168

  17. Elliptocytosis in patients with C-terminal domain mutations of protein 4.1 correlates with encoded messenger RNA levels rather than with alterations in primary protein structure.

    PubMed

    Morinière, M; Ribeiro, L; Dalla Venezia, N; Deguillien, M; Maillet, P; Cynober, T; Delhommeau, F; Almeida, H; Tamagnini, G; Delaunay, J; Baklouti, F

    2000-03-01

    Early biochemical studies defined 4 functional domains of the erythroid protein 4.1 (4.1R). From amino-terminal to carboxy-terminal, these are 30 kd, 16 kd, 10 kd, and 22/24 kd in size. Although the functional properties of both the 30-kd and the 10-kd domain have been demonstrated in red cells, no functional activities have been assigned to either the 16-kd or the 22/24-kd domain in these cells. We here describe new mutations in the sequence encoding the C-terminal 22/24-kd domain that are associated with hereditary elliptocytosis. An unusually mild phenotype observed in heterozygous and homozygous members of 1 family suggested heterogeneity in the pattern of expression of 4.1R deficiency. Using a variety of protein and messenger RNA (mRNA) quantification strategies, we showed that, regardless of the alteration in the C-terminal primary sequence, when the protein is produced, it assembles at the cell membrane. In addition, we found that alterations in red cell morphologic features and membrane function correlate with the amount of membrane-associated protein-and therefore with the amount of mRNA accumulated-rather than with the primary structure of the variant proteins. These data suggest that an intact sequence at exons 19 through 21 encoding part of the C-terminal 22/24-kd region is not required for proper protein 4.1R assembly in mature red cells. (Blood. 2000;95:1834-1841) PMID:10688845

  18. MAB21L2, a vertebrate member of the Male-abnormal 21 family, modulates BMP signaling and interacts with SMAD1

    PubMed Central

    Baldessari, Danila; Badaloni, Aurora; Longhi, Renato; Zappavigna, Vincenzo; Consalez, G Giacomo

    2004-01-01

    Background Through in vivo loss-of-function studies, vertebrate members of the Male abnormal 21 (mab-21) gene family have been implicated in gastrulation, neural tube formation and eye morphogenesis. Despite mounting evidence of their considerable importance in development, the biochemical properties and nature of MAB-21 proteins have remained strikingly elusive. In addition, genetic studies conducted in C. elegans have established that in double mutants mab-21 is epistatic to genes encoding various members of a Transforming Growth Factor beta (TGF-beta) signaling pathway involved in the formation of male-specific sensory organs. Results Through a gain-of-function approach, we analyze the interaction of Mab21l2 with a TGF-beta signaling pathway in early vertebrate development. We show that the vertebrate mab-21 homolog Mab21l2 antagonizes the effects of Bone Morphogenetic Protein 4 (BMP4) overexpression in vivo, rescuing the dorsal axis and restoring wild-type distribution of Chordin and Xvent2 transcripts in Xenopus gastrulae. We show that MAB21L2 immunoprecipitates in vivo with the BMP4 effector SMAD1, whilst in vitro it binds SMAD1 and the SMAD1-SMAD4 complex. Finally, when targeted to an heterologous promoter, MAB21L2 acts as a transcriptional repressor. Conclusions Our results provide the first biochemical and cellular foundation for future functional studies of mab-21 genes in normal neural development and its pathological disturbances. PMID:15613244

  19. Studying Genes

    MedlinePlus

    ... Area What are genes? Genes are sections of DNA that contain instructions for making the molecules—many ... material in an organism. This includes genes and DNA elements that control the activity of genes. Does ...

  20. The ability of mouse nuclear transfer embryonic stem cells to differentiate into primordial germ cells

    PubMed Central

    Mansouri, Vahid; Salehi, Mohammad; Nourozian, Mohsen; Fadaei, Fatemeh; Farahani, Reza Mastery; Piryaei, Abbas; Delbari, Ali

    2015-01-01

    Nuclear transfer embryonic stem cells (ntESCs) show stem cell characteristics such as pluripotency but cause no immunological disorders. Although ntESCs are able to differentiate into somatic cells, the ability of ntESCs to differentiate into primordial germ cells (PGCs) has not been examined. In this work, we examined the capacity of mouse ntESCs to differentiate into PGCs in vitro. ntESCs aggregated to form embryoid bodies (EB) in EB culture medium supplemented with bone morphogenetic protein 4(BMP4) as the differentiation factor. The expression level of specific PGC genes was compared at days 4 and 8 using real time PCR. Flow cytometry and immunocytochemical staining were used to detect Mvh as a specific PGC marker. ntESCs expressed particular genes related to different stages of PGC development. Flow cytometry and immunocytochemical staining confirmed the presence of Mvh protein in a small number of cells. There were significant differences between cells that differentiated into PGCs in the group treated with Bmp4 compared to non-treated cells. These findings indicate that ntESCs can differentiate into putative PGCs. Improvement of ntESC differentiation into PGCs may be a reliable means of producing mature germ cells. PMID:26273226

  1. Design, Synthesis, and Evaluation of Nonretinoid Retinol Binding Protein 4 Antagonists for the Potential Treatment of Atrophic Age-Related Macular Degeneration and Stargardt Disease

    PubMed Central

    2015-01-01

    Accumulation of lipofuscin in the retina is associated with pathogenesis of atrophic age-related macular degeneration and Stargardt disease. Lipofuscin bisretinoids (exemplified by N-retinylidene-N-retinylethanolamine) seem to mediate lipofuscin toxicity. Synthesis of lipofuscin bisretinoids depends on the influx of retinol from serum to the retina. Compounds antagonizing the retinol-dependent interaction of retinol-binding protein 4 (RBP4) with transthyretin in the serum would reduce serum RBP4 and retinol and inhibit bisretinoid formation. We recently showed that A1120 (3), a potent carboxylic acid based RBP4 antagonist, can significantly reduce lipofuscin bisretinoid formation in the retinas of Abca4–/– mice. As part of the NIH Blueprint Neurotherapeutics Network project we undertook the in vitro exploration to identify novel conformationally flexible and constrained RBP4 antagonists with improved potency and metabolic stability. We also demonstrate that upon acute and chronic dosing in rats, 43, a potent cyclopentyl fused pyrrolidine antagonist, reduced circulating plasma RBP4 protein levels by approximately 60%. PMID:25210858

  2. Downregulation of protein 4.1R, a mature centriole protein, disrupts centrosomes, alters cell cycle progression, and perturbs mitotic spindles and anaphase.

    PubMed

    Krauss, Sharon Wald; Spence, Jeffrey R; Bahmanyar, Shirin; Barth, Angela I M; Go, Minjoung M; Czerwinski, Debra; Meyer, Adam J

    2008-04-01

    Centrosomes nucleate and organize interphase microtubules and are instrumental in mitotic bipolar spindle assembly, ensuring orderly cell cycle progression with accurate chromosome segregation. We report that the multifunctional structural protein 4.1R localizes at centrosomes to distal/subdistal regions of mature centrioles in a cell cycle-dependent pattern. Significantly, 4.1R-specific depletion mediated by RNA interference perturbs subdistal appendage proteins ninein and outer dense fiber 2/cenexin at mature centrosomes and concomitantly reduces interphase microtubule anchoring and organization. 4.1R depletion causes G(1) accumulation in p53-proficient cells, similar to depletion of many other proteins that compromise centrosome integrity. In p53-deficient cells, 4.1R depletion delays S phase, but aberrant ninein distribution is not dependent on the S-phase delay. In 4.1R-depleted mitotic cells, efficient centrosome separation is reduced, resulting in monopolar spindle formation. Multipolar spindles and bipolar spindles with misaligned chromatin are also induced by 4.1R depletion. Notably, all types of defective spindles have mislocalized NuMA (nuclear mitotic apparatus protein), a 4.1R binding partner essential for spindle pole focusing. These disruptions contribute to lagging chromosomes and aberrant microtubule bridges during anaphase/telophase. Our data provide functional evidence that 4.1R makes crucial contributions to the structural integrity of centrosomes and mitotic spindles which normally enable mitosis and anaphase to proceed with the coordinated precision required to avoid pathological events. PMID:18212055

  3. Inhibition of lectin-like oxidized low-density lipoprotein receptor-1 reduces cardiac fibroblast proliferation by suppressing GATA Binding Protein 4.

    PubMed

    Liu, Bin; Liu, Ning-Ning; Liu, Wei-Hua; Zhang, Shuang-Wei; Zhang, Jing-Zhi; Li, Ai-Qun; Liu, Shi-Ming

    2016-07-01

    Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and GATA Binding Protein 4 (GATA4) are important for the growth of cardiac fibroblasts (CFs). When deregulated, LOX-1 and GATA4 can cause cardiac remodeling. In the present study, we found novel evidence that GATA4 was required for the LOX-1 regulation of CF proliferation. The inhibition of LOX-1 by RNA interference LOX-1 lentivirus resulted in the loss of PI3K/Akt activation and GATA4 protein expression. The overexpression of LOX-1 by lentivirus rescued CF proliferation, PI3K/Akt activation, and GATA4 protein expression. Moreover, GATA4 overexpression enhanced CF proliferation with LOX-1 inhibition. We also found that the inhibition of PI3K/Akt activation by LY294002, a PI3K inhibitor, reduced cell proliferation and protein level of GATA4. In summary, GATA4 may play an important role in the LOX-1 and PI3K/Akt regulation of CF proliferation. PMID:27216460

  4. Downregulation of Protein 4.1R, a Mature Centriole Protein, Disrupts Centrosomes, Alters Cell Cycle Progression, and Perturbs Mitotic Spindles and Anaphase▿

    PubMed Central

    Krauss, Sharon Wald; Spence, Jeffrey R.; Bahmanyar, Shirin; Barth, Angela I. M.; Go, Minjoung M.; Czerwinski, Debra; Meyer, Adam J.

    2008-01-01

    Centrosomes nucleate and organize interphase microtubules and are instrumental in mitotic bipolar spindle assembly, ensuring orderly cell cycle progression with accurate chromosome segregation. We report that the multifunctional structural protein 4.1R localizes at centrosomes to distal/subdistal regions of mature centrioles in a cell cycle-dependent pattern. Significantly, 4.1R-specific depletion mediated by RNA interference perturbs subdistal appendage proteins ninein and outer dense fiber 2/cenexin at mature centrosomes and concomitantly reduces interphase microtubule anchoring and organization. 4.1R depletion causes G1 accumulation in p53-proficient cells, similar to depletion of many other proteins that compromise centrosome integrity. In p53-deficient cells, 4.1R depletion delays S phase, but aberrant ninein distribution is not dependent on the S-phase delay. In 4.1R-depleted mitotic cells, efficient centrosome separation is reduced, resulting in monopolar spindle formation. Multipolar spindles and bipolar spindles with misaligned chromatin are also induced by 4.1R depletion. Notably, all types of defective spindles have mislocalized NuMA (nuclear mitotic apparatus protein), a 4.1R binding partner essential for spindle pole focusing. These disruptions contribute to lagging chromosomes and aberrant microtubule bridges during anaphase/telophase. Our data provide functional evidence that 4.1R makes crucial contributions to the structural integrity of centrosomes and mitotic spindles which normally enable mitosis and anaphase to proceed with the coordinated precision required to avoid pathological events. PMID:18212055

  5. Positive immunostaining of Sal-like protein 4 is associated with poor patient survival outcome in the large and undifferentiated Korean hepatocellular carcinoma

    PubMed Central

    Jung, Yun Kyung; Jang, Kiseok; Paik, Seung Sam; Kwon, Yong Jin; Kim, Han Jun; Lee, Kyeong Geun; Park, Hwon Kyum

    2016-01-01

    Purpose Previous studies have shown the role of Sal-like protein 4 (SALL4) as a biomarker in hepatocellular carcinoma (HCC), and some studies have shown the relationship between SALL4 and prognosis. Given the debates in study groups differences in terms of etiologic causes between Western and Asian HCC and detection methods, we attempted to verify the features of SALL4 immunoreactivity and its clinical correlation in Korean HCC patients. Methods Immunohistochemical staining of SALL4 of tissue microarrays (TMAs) consisting of 213 surgically resected HCC patients' tissue were scored in a semiquantitative scoring system with immunoreactive score and the results analyzed with clinical outcome, in addition to general demographics and clinical characteristics. Results SALL4 immunoreactivity was expressed in 50 cases. Relevance between SALL4 and α-FP correlated significantly (P = 0.002). Also, the SALL4-positive patients had considerably higher tumor grade (P < 0.001). The survival analysis showed negative correlation with SALL4 immunoreactivity in all HCC patient groups, but SALL4 immunoreactivity in T3 and T4 HCC correlated with poor prognosis. Conclusion Here, we found that positive immunostaining of SALL4 is correlated with poor patient survival outcome in large and undifferentiated Korean HCC. SALL4 expression showed close relationship with clinical outcomes of HCCs in Korean patients. PMID:27433461

  6. High fat diet induced insulin resistance and elevated retinol binding protein 4 in female rats; treatment and protection with Berberis vulgaris extract and vitamin A.

    PubMed

    El-Sayed, Mohamed Mohammed; Ghareeb, Doaa Ahmad; Talat, Heba Allah; Sarhan, Eman Mohammed

    2013-11-01

    This research was conducted to investigate two main aims; the first aim was to find if there is a relationship between insulin resistance (IR) and retinol binding protein 4 (RBP4). The second aim was to use berberis vulgaris extract and vitamin A as protective and/or curative agents against insulin resistance. IR was developed by feeding the female rats a high fat diet (HFD) for six weeks then treating or protecting them with b. vulgaris extract (0.2 g/Kg body weight) or vitamin A (12.8μg/Kg/day) for two weeks. HFD intake elevated insulin level and RBP4 expression that associated with hyperglycemia and hyperlipidemia. Co-administration of vitamin A and B. vulgaris extracts reduced blood glucose level, insulin, body weight and RBP4 expression before, during and after HFD. Furthermore, vitamin A reduced the blood glucose, triglycerides (TG) and cholesterol levels. IR syndrome associated with the RBP 4 alteration that gives high indication about the role of RBP4 expression in the IR progression and development. Furthermore, the treatment with vitamin A and/or b. vulgaris alleviated the IR syndrome through the action on RBP4 and Insulin secretion. On the other hand, vitamin A must be avoided for the predisposed IR and prediabetic patients. PMID:24191325

  7. Dendritic Cell-Secreted Cytotoxic T-Lymphocyte-Associated Protein-4 Regulates the T-cell Response by Downmodulating Bystander Surface B7.

    PubMed

    Halpert, Matthew M; Konduri, Vanaja; Liang, Dan; Chen, Yunyu; Wing, James B; Paust, Silke; Levitt, Jonathan M; Decker, William K

    2016-05-15

    The remarkable functional plasticity of professional antigen-presenting cells (APCs) allows the adaptive immune system to respond specifically to an incredibly diverse array of potential pathogenic insults; nonetheless, the specific molecular effectors and mechanisms that underpin this plasticity remain poorly characterized. Cytotoxic T-lymphocyte-associated protein-4 (CTLA-4), the target of the blockbuster cancer immunotherapeutic ipilimumab, is one of the most well-known and well-studied members of the B7 superfamily and negatively regulates T cell responses by a variety of known mechanisms. Although CTLA-4 is thought to be expressed almost exclusively among lymphoid lineage hematopoietic cells, a few reports have indicated that nonlymphoid APCs can also express the CTLA-4 mRNA transcript and that transcript levels can be regulated by external stimuli. In this study, we substantially build upon these critical observations, definitively demonstrating that mature myeloid lineage dendritic cells (DC) express significant levels of intracellular CTLA-4 that they constitutively secrete in microvesicular structures. CTLA-4(+) microvesicles can competitively bind B7 costimulatory molecules on bystander DC, resulting in downregulation of B7 surface expression with significant functional consequences for downstream CD8(+) T-cell responses. Hence, the data indicate a previously unknown role for DC-derived CTLA-4 in immune cell functional plasticity and have significant implication for the design and implementation of immunomodulatory strategies intended to treat cancer and infectious disease. PMID:26979751

  8. Insights into the Function of the Unstructured N-Terminal Domain of Proteins 4.1R and 4.1G in Erythropoiesis.

    PubMed

    Nunomura, Wataru; Gascard, Philippe; Takakuwa, Yuichi

    2011-01-01

    Membrane skeletal protein 4.1R is the prototypical member of a family of four highly paralogous proteins that include 4.1G, 4.1N, and 4.1B. Two isoforms of 4.1R (4.1R(135) and 4.1R(80)), as well as 4.1G, are expressed in erythroblasts during terminal differentiation, but only 4.1R(80) is present in mature erythrocytes. One goal in the field is to better understand the complex regulation of cell type and isoform-specific expression of 4.1 proteins. To start answering these questions, we are studying in depth the important functions of 4.1 proteins in the organization and function of the membrane skeleton in erythrocytes. We have previously reported that the binding profiles of 4.1R(80) and 4.1R(135) to membrane proteins and calmodulin are very different despite the similar structure of the membrane-binding domain of 4.1G and 4.1R(135). We have accumulated evidence for those differences being caused by the N-terminal 209 amino acids headpiece region (HP). Interestingly, the HP region is an unstructured domain. Here we present an overview of the differences and similarities between 4.1 isoforms and paralogs. We also discuss the biological significance of unstructured domains. PMID:21904552

  9. Insights into the Function of the Unstructured N-Terminal Domain of Proteins 4.1R and 4.1G in Erythropoiesis

    PubMed Central

    Nunomura, Wataru; Gascard, Philippe; Takakuwa, Yuichi

    2011-01-01

    Membrane skeletal protein 4.1R is the prototypical member of a family of four highly paralogous proteins that include 4.1G, 4.1N, and 4.1B. Two isoforms of 4.1R (4.1R135 and 4.1R80), as well as 4.1G, are expressed in erythroblasts during terminal differentiation, but only 4.1R80 is present in mature erythrocytes. One goal in the field is to better understand the complex regulation of cell type and isoform-specific expression of 4.1 proteins. To start answering these questions, we are studying in depth the important functions of 4.1 proteins in the organization and function of the membrane skeleton in erythrocytes. We have previously reported that the binding profiles of 4.1R80 and 4.1R135 to membrane proteins and calmodulin are very different despite the similar structure of the membrane-binding domain of 4.1G and 4.1R135. We have accumulated evidence for those differences being caused by the N-terminal 209 amino acids headpiece region (HP). Interestingly, the HP region is an unstructured domain. Here we present an overview of the differences and similarities between 4.1 isoforms and paralogs. We also discuss the biological significance of unstructured domains. PMID:21904552

  10. Impaired Intestinal Calcium Absorption in Protein 4.1R-deficient Mice Due to Altered Expression of Plasma Membrane Calcium ATPase 1b (PMCA1b)*

    PubMed Central

    Liu, Congrong; Weng, Haibao; Chen, Lixiang; Yang, Shaomin; Wang, Hua; Debnath, Gargi; Guo, Xinhua; Wu, Liancheng; Mohandas, Narla; An, Xiuli

    2013-01-01

    Protein 4.1R was first identified in the erythrocyte membrane skeleton. It is now known that the protein is expressed in a variety of epithelial cell lines and in the epithelia of many tissues, including the small intestine. However, the physiological function of 4.1R in the epithelial cells of the small intestine has not so far been explored. Here, we show that 4.1R knock-out mice exhibited a significantly impaired small intestinal calcium absorption that resulted in secondary hyperparathyroidism as evidenced by increased serum 1,25-(OH)2-vitamin D3 and parathyroid hormone levels, decreased serum calcium levels, hyperplasia of the parathyroid, and demineralization of the bones. 4.1R is located on the basolateral membrane of enterocytes, where it co-localizes with PMCA1b (plasma membrane calcium ATPase 1b). Expression of PMCA1b in enterocytes was decreased in 4.1−/− mice. 4.1R directly associated with PMCA1b, and the association involved the membrane-binding domain of 4.1R and the second intracellular loop and C terminus of PMCA1b. Our findings have enabled us to define a functional role for 4.1R in small intestinal calcium absorption through regulation of membrane expression of PMCA1b. PMID:23460639

  11. Characterization of the interaction between protein 4.1R and ZO-2. A possible link between the tight junction and the actin cytoskeleton.

    PubMed

    Mattagajasingh, S N; Huang, S C; Hartenstein, J S; Benz, E J

    2000-09-29

    Multiple isoforms of the red cell protein 4.1R are expressed in nonerythroid cells, including novel 135-kDa isoforms. Using a yeast two-hybrid system, immunocolocalization, immunoprecipitation, and in vitro binding studies, we found that two 4.1R isoforms of 135 and 150 kDa specifically interact with the protein ZO-2 (zonula occludens-2). 4.1R is colocalized with ZO-2 and occludin at Madin-Darby canine kidney (MDCK) cell tight junctions. Both isoforms of 4.1R coprecipitated with proteins that organize tight junctions such as ZO-2, ZO-1, and occludin. Western blot analysis also revealed the presence of actin and alpha-spectrin in these immunoprecipitates. Association of 4.1R isoforms with these tight junction and cytoskeletal proteins was found to be specific for the tight junction and was not seen in nonconfluent MDCK cells. The amino acid residues that sustain the interaction between 4.1R and ZO-2 reside within the amino acids encoded by exons 19-21 of 4.1R and residues 1054-1118 of ZO-2. Exogenously expressed 4.1R containing the spectrin/actin- and ZO-2-binding domains was recruited to tight junctions in confluent MDCK cells. Taken together, our results suggest that 4.1R might play an important role in organization and function of the tight junction by establishing a link between the tight junction and the actin cytoskeleton. PMID:10874042

  12. Structural and functional characterization of protein 4.1R-phosphatidylserine interaction: potential role in 4.1R sorting within cells.

    PubMed

    An, X L; Takakuwa, Y; Manno, S; Han, B G; Gascard, P; Mohandas, N

    2001-09-21

    Erythrocyte protein 4.1R is a multifunctional protein that binds to various membrane proteins and to phosphatidylserine. In the present study, we report two important observations concerning 4.1R-phosphatidylserine interaction. Biochemically, a major finding of the present study is that 4.1R binding to phosphatidylserine appears to be a two-step process in which 4.1R first interacts with serine head group of phosphatidylserine through the positively charged amino acids YKRS and subsequently forms a tight hydrophobic interaction with fatty acid moieties. 4.1R failed to dissociate from phosphatidylserine liposomes under high ionic strength but could be released specifically by phospholipase A(2) but not by phospholipase C or D. Biochemical analyses showed that acyl chains were associated with 4.1R released by phospholipase A(2). Importantly, the association of acyl chains with 4.1R impaired its ability to interact with calmodulin, band 3, and glycophorin C. Removal of acyl chains restored 4.1R binding. These data indicate that acyl chains of phosphatidylserine play an important role in its interaction with 4.1R and on 4.1R function. In terms of biological significance, we have obtained evidence that 4.1R-phosphatidylserine interaction may play an important role in cellular sorting of 4.1R. PMID:11423550

  13. Protein 4.1B associates with both Caspr/paranodin and Caspr2 at paranodes and juxtaparanodes of myelinated fibres.

    PubMed

    Denisenko-Nehrbass, Natalia; Oguievetskaia, Ksénia; Goutebroze, Laurence; Galvez, Thierry; Yamakawa, Hisashi; Ohara, Osamu; Carnaud, Michèle; Girault, Jean-Antoine

    2003-01-01

    Caspr/paranodin, a neuronal transmembrane glycoprotein, is essential for the structure and function of septate-like paranodal axoglial junctions at nodes of Ranvier. A closely related protein, Caspr2, is concentrated in juxtaparanodal regions where it associates indirectly with the shaker-type potassium channels. Although ultrastructural studies indicate that paranodal complexes are linked to the cytoskeleton, the intracellular partners of Caspr/paranodin, as well as those of Caspr2, are poorly characterized. We show that the conserved intracellular juxtamembrane regions (GNP motif) of Caspr/paranodin and Caspr2 bind proteins 4.1R and 4.1B. 4.1B is known to be enriched in paranodal and juxtaparanodal regions. 4.1B immunoreactivity accumulates progressively at paranodes and juxtaparanodes during postnatal development, following the concentration of Caspr/paranodin and Caspr2, respectively, in central and peripheral myelinated axons. These two proteins coimmunoprecipitated with 4.1B in brain homogenates. Our results provide strong evidence for the association of 4.1B with Caspr/paranodin at paranodes and with Caspr2 at juxtaparanodes. We propose that 4.1B anchors these axonal proteins to the actin-based cytoskeleton in these two regions. PMID:12542678

  14. Sitagliptin downregulates retinol-binding protein 4 and upregulates glucose transporter type 4 expression in a type 2 diabetes mellitus rat model.

    PubMed

    Hu, Honglin; Xu, Min; Qi, Renjuan; Wang, Youmin; Wang, Changjiang; Liu, Jiongjiong; Luo, Li; Xia, Li; Fang, Zhaohui

    2015-01-01

    The present study was designed to investigate the effects of sitagliptin on metabolic parameters as well as the expression levels of retinol-binding protein 4 (RBP4) and glucose transporter type 4 (GLUT4) in a rat model of type 2 diabetes mellitus. A rat model of type 2 diabetes mellitus was established by a combination of a high-fat diet and intraperitoneal injection of low-dose streptozotocin. Rats were divided into three groups: normal control group, diabetes group, and diabetes + sitagliptin group. Body weight, glycemic parameters, lipid profiles, fasting insulin (FINS) and serum RBP4 levels were assessed at baseline and after 6 weeks of therapy. Western blotting was used to detect the tissue RBP4 and GLUT4 expression levels. After treatment for 6 weeks, the diabetes + sitagliptin group displayed significantly improve levels of blood sugar, blood grease, and insulin sensitizing functions (P < 0.05) than the diabetes group. Sitagliptin markedly down regulated RBP4 expression levels and up-regulated GLUT4 expression levels in adipose tissue and skeletal muscle. The results indicate that sitagliptin can modulate the RBP4-GLUT4 system in adipose tissue and skeletal muscle. Modulation of the RBP4-GLUT4 system may be one of the mechanisms by which sitagliptin ameliorates the symptoms of type 2 diabetes mellitus. PMID:26770384

  15. Fatty acid binding protein 4 regulates VEGF-induced airway angiogenesis and inflammation in a transgenic mouse model: implications for asthma.

    PubMed

    Ghelfi, Elisa; Yu, Chen-Wei; Elmasri, Harun; Terwelp, Matthew; Lee, Chun G; Bhandari, Vineet; Comhair, Suzy A; Erzurum, Serpil C; Hotamisligil, Gökhan S; Elias, Jack A; Cataltepe, Sule

    2013-04-01

    Neovascularization of the airways occurs in several inflammatory lung diseases, including asthma. Vascular endothelial growth factor (VEGF) plays an important role in vascular remodeling in the asthmatic airways. Fatty acid binding protein 4 (FABP4 or aP2) is an intracellular lipid chaperone that is induced by VEGF in endothelial cells. FABP4 exhibits a proangiogenic function in vitro, but whether it plays a role in modulation of angiogenesis in vivo is not known. We hypothesized that FABP4 promotes VEGF-induced airway angiogenesis and investigated this hypothesis with the use of a transgenic mouse model with inducible overexpression of VEGF165 under a CC10 promoter [VEGF-TG (transgenic) mice]. We found a significant increase in FABP4 mRNA levels and density of FABP4-expressing vascular endothelial cells in mouse airways with VEGF overexpression. FABP4(-/-) mouse airways showed a significant decrease in neovessel formation and endothelial cell proliferation in response to VEGF overexpression. These alterations in airway vasculature were accompanied by attenuated expression of proinflammatory mediators. Furthermore, VEGF-TG/FABP4(-/-) mice showed markedly decreased expression of endothelial nitric oxide synthase, a well-known mediator of VEGF-induced responses, compared with VEGF-TG mice. Finally, the density of FABP4-immunoreactive vessels in endobronchial biopsy specimens was significantly higher in patients with asthma than in control subjects. Taken together, these data unravel FABP4 as a potential target of pathologic airway remodeling in asthma. PMID:23391391

  16. Bicyclic [3.3.0]-Octahydrocyclopenta[c]pyrrolo Antagonists of Retinol Binding Protein 4: Potential Treatment of Atrophic Age-Related Macular Degeneration and Stargardt Disease.

    PubMed

    Cioffi, Christopher L; Racz, Boglarka; Freeman, Emily E; Conlon, Michael P; Chen, Ping; Stafford, Douglas G; Schwarz, Daniel M C; Zhu, Lei; Kitchen, Douglas B; Barnes, Keith D; Dobri, Nicoleta; Michelotti, Enrique; Cywin, Charles L; Martin, William H; Pearson, Paul G; Johnson, Graham; Petrukhin, Konstantin

    2015-08-13

    Antagonists of retinol-binding protein 4 (RBP4) impede ocular uptake of serum all-trans retinol (1) and have been shown to reduce cytotoxic bisretinoid formation in the retinal pigment epithelium (RPE), which is associated with the pathogenesis of both dry age-related macular degeneration (AMD) and Stargardt disease. Thus, these agents show promise as a potential pharmacotherapy by which to stem further neurodegeneration and concomitant vision loss associated with geographic atrophy of the macula. We previously disclosed the discovery of a novel series of nonretinoid RBP4 antagonists, represented by bicyclic [3.3.0]-octahydrocyclopenta[c]pyrrolo analogue 4. We describe herein the utilization of a pyrimidine-4-carboxylic acid fragment as a suitable isostere for the anthranilic acid appendage of 4, which led to the discovery of standout antagonist 33. Analogue 33 possesses exquisite in vitro RBP4 binding affinity and favorable drug-like characteristics and was found to reduce circulating plasma RBP4 levels in vivo in a robust manner (>90%). PMID:26181715

  17. Secreted Frizzled-Related Protein 4 Inhibits Glioma Stem-Like Cells by Reversing Epithelial to Mesenchymal Transition, Inducing Apoptosis and Decreasing Cancer Stem Cell Properties

    PubMed Central

    G, Bhuvanalakshmi; Arfuso, Frank; Millward, Michael; Dharmarajan, Arun; Warrier, Sudha

    2015-01-01

    The Wnt pathway is integrally involved in regulating self-renewal, proliferation, and maintenance of cancer stem cells (CSCs). We explored the effect of the Wnt antagonist, secreted frizzled-related protein 4 (sFRP4), in modulating epithelial to mesenchymal transition (EMT) in CSCs from human glioblastoma cells lines, U87 and U373. sFRP4 chemo-sensitized CSC-enriched cells to the most commonly used anti-glioblastoma drug, temozolomide (TMZ), by the reversal of EMT. Cell movement, colony formation, and invasion in vitro were suppressed by sFRP4+TMZ treatment, which correlated with the switch of expression of markers from mesenchymal (Twist, Snail, N-cadherin) to epithelial (E-cadherin). sFRP4 treatment elicited activation of the Wnt-Ca2+ pathway, which antagonizes the Wnt/ß-catenin pathway. Significantly, the chemo-sensitization effect of sFRP4 was correlated with the reduction in the expression of drug resistance markers ABCG2, ABCC2, and ABCC4. The efficacy of sFRP4+TMZ treatment was demonstrated in vivo using nude mice, which showed minimum tumor engraftment using CSCs pretreated with sFRP4+TMZ. These studies indicate that sFRP4 treatment would help to improve response to commonly used chemotherapeutics in gliomas by modulating EMT via the Wnt/ß-catenin pathway. These findings could be exploited for designing better targeted strategies to improve chemo-response and eventually eliminate glioblastoma CSCs. PMID:26030909

  18. Ubiquitin-specific Protease 11 (USP11) Deubiquitinates Hybrid Small Ubiquitin-like Modifier (SUMO)-Ubiquitin Chains to Counteract RING Finger Protein 4 (RNF4)*

    PubMed Central

    Hendriks, Ivo A.; Schimmel, Joost; Eifler, Karolin; Olsen, Jesper V.; Vertegaal, Alfred C. O.

    2015-01-01

    Ring finger protein 4 (RNF4) is a SUMO-targeted ubiquitin E3 ligase with a pivotal function in the DNA damage response (DDR). SUMO interaction motifs (SIMs) in the N-terminal part of RNF4 tightly bind to SUMO polymers, and RNF4 can ubiquitinate these polymers in vitro. Using a proteomic approach, we identified the deubiquitinating enzyme ubiquitin-specific protease 11 (USP11), a known DDR-component, as a functional interactor of RNF4. USP11 can deubiquitinate hybrid SUMO-ubiquitin chains to counteract RNF4. SUMO-enriched nuclear bodies are stabilized by USP11, which functions downstream of RNF4 as a counterbalancing factor. In response to DNA damage induced by methyl methanesulfonate, USP11 could counteract RNF4 to inhibit the dissolution of nuclear bodies. Thus, we provide novel insight into cross-talk between ubiquitin and SUMO and uncover USP11 and RNF4 as a balanced SUMO-targeted ubiquitin ligase/protease pair with a role in the DDR. PMID:25969536

  19. Sitagliptin downregulates retinol-binding protein 4 and upregulates glucose transporter type 4 expression in a type 2 diabetes mellitus rat model

    PubMed Central

    Hu, Honglin; Xu, Min; Qi, Renjuan; Wang, Youmin; Wang, Changjiang; Liu, Jiongjiong; Luo, Li; Xia, Li; Fang, Zhaohui

    2015-01-01

    The present study was designed to investigate the effects of sitagliptin on metabolic parameters as well as the expression levels of retinol-binding protein 4 (RBP4) and glucose transporter type 4 (GLUT4) in a rat model of type 2 diabetes mellitus. A rat model of type 2 diabetes mellitus was established by a combination of a high-fat diet and intraperitoneal injection of low-dose streptozotocin. Rats were divided into three groups: normal control group, diabetes group, and diabetes + sitagliptin group. Body weight, glycemic parameters, lipid profiles, fasting insulin (FINS) and serum RBP4 levels were assessed at baseline and after 6 weeks of therapy. Western blotting was used to detect the tissue RBP4 and GLUT4 expression levels. After treatment for 6 weeks, the diabetes + sitagliptin group displayed significantly improve levels of blood sugar, blood grease, and insulin sensitizing functions (P < 0.05) than the diabetes group. Sitagliptin markedly down regulated RBP4 expression levels and up-regulated GLUT4 expression levels in adipose tissue and skeletal muscle. The results indicate that sitagliptin can modulate the RBP4-GLUT4 system in adipose tissue and skeletal muscle. Modulation of the RBP4-GLUT4 system may be one of the mechanisms by which sitagliptin ameliorates the symptoms of type 2 diabetes mellitus. PMID:26770384

  20. Characterizing the interaction of the mammalian eIF4E-related protein 4EHP with 4E-BP1.

    PubMed

    Tee, Andrew R; Tee, Jennifer A; Blenis, John

    2004-04-23

    Eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) represses translation initiation by binding to eukaryotic initiation factor 4E (eIF4E). 4E-BP1 also binds to the eIF4E homologous protein (4EHP). We show that eIF4E-binding mutants of 4E-BP1 (Y54A and L59A) fail to form heterodimeric complexes with wild-type 4EHP. In addition, the W95A mutant of 4EHP, similar to a homologous mutation in eIF4E, inhibits its binding to wild-type 4E-BP1. Interestingly, 4EHP over-expression instigates a negative feedback loop that inhibits upstream signaling to 4E-BP1 and ribosomal protein S6 kinase 1 (S6K1) whereas the 4E-BP1-binding-deficient mutant of 4EHP(W95A) was unable to trigger this feedback loop. Thus, the interaction of 4EHP with 4E-BP1 is necessary for this observed impaired signaling to 4E-BP1 and S6K1. PMID:15094042

  1. Ubiquitin-specific Protease 11 (USP11) Deubiquitinates Hybrid Small Ubiquitin-like Modifier (SUMO)-Ubiquitin Chains to Counteract RING Finger Protein 4 (RNF4).

    PubMed

    Hendriks, Ivo A; Schimmel, Joost; Eifler, Karolin; Olsen, Jesper V; Vertegaal, Alfred C O

    2015-06-19

    Ring finger protein 4 (RNF4) is a SUMO-targeted ubiquitin E3 ligase with a pivotal function in the DNA damage response (DDR). SUMO interaction motifs (SIMs) in the N-terminal part of RNF4 tightly bind to SUMO polymers, and RNF4 can ubiquitinate these polymers in vitro. Using a proteomic approach, we identified the deubiquitinating enzyme ubiquitin-specific protease 11 (USP11), a known DDR-component, as a functional interactor of RNF4. USP11 can deubiquitinate hybrid SUMO-ubiquitin chains to counteract RNF4. SUMO-enriched nuclear bodies are stabilized by USP11, which functions downstream of RNF4 as a counterbalancing factor. In response to DNA damage induced by methyl methanesulfonate, USP11 could counteract RNF4 to inhibit the dissolution of nuclear bodies. Thus, we provide novel insight into cross-talk between ubiquitin and SUMO and uncover USP11 and RNF4 as a balanced SUMO-targeted ubiquitin ligase/protease pair with a role in the DDR. PMID:25969536

  2. Control of the Normal and Pathological Development of Neural Stem and Progenitor Cells by the PC3/Tis21/Btg2 and Btg1 Genes.

    PubMed

    Micheli, Laura; Ceccarelli, Manuela; Farioli-Vecchioli, Stefano; Tirone, Felice

    2015-12-01

    The PC3/Tis21/Btg2 and Btg1 genes are transcriptional cofactors belonging to the Btg/Tob family, which regulate the development of several cell types, including neural precursors. We summarize here the actions of these genes on neural precursors in the adult neurogenic niches and the cognitive defects associated when their expression is altered. We consider also recent findings implicating them in neural and non-neural tumors, since common developmental mechanisms are involved. PC3/Tis21 is required for the regulation of the maturation of stem and progenitor cells in the adult dentate gyrus and subventricular zone (SVZ), by controlling both their exit from the cell cycle and the ensuing terminal differentiation. Such actions are effected by regulating the expression of several genes, including cyclin D1, BMP4, Id3. In cerebellar precursors, however, PC3/Tis21 regulates chiefly their migration rather than proliferation or differentiation, with important implications for the onset of medulloblastoma, the cerebellar tumor. In fact PC3/Tis21 is a medulloblastoma-suppressor, as its overexpression in cerebellar precursors inhibits this tumor; PC3/Tis21 shows anti-tumor activity also in non-neural tumors. Btg1 presents a different functional profile, as it controls proliferation in adult stem/progenitor cells of dentate gyrus and SVZ, where is required to maintain their self-renewal and quiescence, but is apparently devoid of a direct control of their terminal differentiation or migration. Notably, physical exercise in Btg1-null mice rescues the loss of proliferative capability occurring in older stem cells. Both genes could be further investigated as therapeutical targets, namely, Btg1 in the process of aging and PC3/Tis21 as a tumor-suppressor. PMID:25967096

  3. Regulatory elements of the EKLF gene that direct erythroid cell-specific expression during mammalian development.

    PubMed

    Xue, Li; Chen, Xiaoyong; Chang, Yanjie; Bieker, James J

    2004-06-01

    Erythroid Krüppel-like factor (EKLF) plays an essential role in enabling beta-globin expression during erythroid ontogeny. It is first expressed in the extraembryonic mesoderm of the yolk sac within the morphologically unique cells that give rise to the blood islands, and then later within the hepatic primordia. The BMP4/Smad pathway plays a critical role in the induction of EKLF, and transient transfection analyses demonstrate that sequences located within less than 1 kb of its transcription initiation site are sufficient for high-level erythroid-specific transcription. We have used transgenic analyses to verify that 950 bp located adjacent to the EKLF start site of transcription is sufficient to generate lacZ expression within the blood islands as well as the fetal liver during embryonic development. Of particular importance are 3 regions, 2 of which overlap endogenous erythroid-specific DNase hypersensitive sites, and 1 of which includes the proximal promoter region. The onset of transgene expression mimics that of endogenous EKLF as it begins by day 7.5 (d7.5) to d8.0. In addition, it exhibits a strict hematopoietic specificity, localized only to these cells and not to the adjacent vasculature at all stages examined. Finally, expression is heterocellular, implying that although these elements are sufficient for tissue-specific expression, they do not shield against the position effects of adjacent chromatin. These analyses demonstrate that a surprisingly small DNA segment contains all the information needed to target a linked gene to the hematopoietic compartment at both early and later stages of development, and may be a useful cassette for this purpose. PMID:14764531

  4. Identification of novel osteochondrosis--Associated genes.

    PubMed

    Mirams, Michiko; Ayodele, Babatunde A; Tatarczuch, Liliana; Henson, Frances M; Pagel, Charles N; Mackie, Eleanor J

    2016-03-01

    During the early stages of articular osteochondrosis, cartilage is retained in subchondral bone, but the pathophysiology of this condition of growing humans and domestic animals is poorly understood. A subtractive hybridization study was undertaken to compare gene expression between the cartilage of early experimentally induced equine osteochondrosis lesions and control cartilage. Of the many putative differentially expressed genes identified, eight were confirmed by quantitative PCR analysis as differentially expressed, in addition to those already known to be associated with early lesions. Genes encoding vacuolar H(+)-ATPase V0 subunit d2 (ATP6V0D2), cathepsin K, integrin-binding sialoprotein, integrin αV, low density lipoprotein receptor-related protein 4, lumican, osteopontin, and thymosin β4 (TMSB4) were expressed at higher levels in lesions than in control cartilage. These genes included 34 genes not previously identified in cartilage. Some genes identified as associated with early lesions are known chondrocyte hypertrophy-associated genes, and in transmission electron microscopy studies normal hypertrophic chondrocytes were observed in lesions. Differential expression of ATP6V0D2 and TMSB4 in the cartilage of early naturally occurring osteochondrosis lesions was confirmed by immunohistochemistry. These results identify novel osteochondrosis-associated genes and provide evidence that articular osteochondrosis does not necessarily result from failure of chondrocytes to undergo hypertrophy. PMID:26296056

  5. Novel exons in the tbx5 gene locus generate protein isoforms with distinct expression domains and function.

    PubMed

    Yamak, Abir; Georges, Romain O; Sheikh-Hassani, Massomeh; Morin, Martin; Komati, Hiba; Nemer, Mona

    2015-03-13

    TBX5 is the gene mutated in Holt-Oram syndrome, an autosomal dominant disorder with complex heart and limb deformities. Its protein product is a member of the T-box family of transcription factors and an evolutionarily conserved dosage-sensitive regulator of heart and limb development. Understanding TBX5 regulation is therefore of paramount importance. Here we uncover the existence of novel exons and provide evidence that TBX5 activity may be extensively regulated through alternative splicing to produce protein isoforms with differing N- and C-terminal domains. These isoforms are also present in human heart, indicative of an evolutionarily conserved regulatory mechanism. The newly identified isoforms have different transcriptional properties and can antagonize TBX5a target gene activation. Droplet Digital PCR as well as immunohistochemistry with isoform-specific antibodies reveal differential as well as overlapping expression domains. In particular, we find that the predominant isoform in skeletal myoblasts is Tbx5c, and we show that it is dramatically up-regulated in differentiating myotubes and is essential for myotube formation. Mechanistically, TBX5c antagonizes TBX5a activation of pro-proliferative signals such as IGF-1, FGF-10, and BMP4. The results provide new insight into Tbx5 regulation and function that will further our understanding of its role in health and disease. The finding of new exons in the Tbx5 locus may also be relevant to mutational screening especially in the 30% of Holt-Oram syndrome patients with no mutations in the known TBX5a exons. PMID:25623069

  6. Transcription regulation of the vegf gene by the BMP/Smad pathway in the angioblast of zebrafish embryos

    SciTech Connect

    He Chen; Chen Xiaozhuo . E-mail: chenx@ohiou.edu

    2005-04-01

    Vascular endothelial growth factor (VEGF) is a mitogen that is critically involved in vasculogenesis, angiogenesis, and hematopoiesis. However, what and how transcription factors participate in the regulation of vegf gene expression are not fully understood. Here we report the cloning and sequencing of the zebrafish vegf promoter which revealed that the promoter contains a number of bone morphogenetic protein (BMP)-activated Smad binding elements (SBE), implicating Smad1 and Smad5 in the regulation of BMP-induced expression of vegf. Electrophoretic mobility shift assays of adding recombinant Smad proteins to the SBE-containing DNA oligonucleotides that represent portions of zebrafish vegf promoter resulted in mobility shift of the oligonucleotides. These changes demonstrate potential interactions between Smad1/5 and the vegf promoter. Reporter activity assays using the wild-type or SBE-deleted vegf promoters to drive the luciferase reporter gene expression revealed that Smad1 stimulated while Smad5 repressed the vegf promoter activity in zebrafish embryos. These data indicate that the BMP/Smad signaling pathway is involved in the regulation of zebrafish vegf transcription. In addition, we demonstrate that transgenic expression of human BMP4 in zebrafish embryos induced an expansion of the posterior intermediate cell mass (ICM, also commonly called blood island), a population of cells containing endothelial and hematopoietic precursors. In the expanded ICM, vegf and VEGF receptor 2 (flk-1) were ectopically co-expressed, suggesting that an autocrine/paracrine regulation of vegf expression may exist and contribute to the BMP-induced hemangiogenic cell proliferation.

  7. Cytoprotective role of the fatty acid binding protein 4 against oxidative and endoplasmic reticulum stress in 3T3-L1 adipocytes.

    PubMed

    Kajimoto, Kazuaki; Minami, Yoshitaka; Harashima, Hideyoshi

    2014-01-01

    The fatty acid binding protein 4 (FABP4), one of the most abundant proteins in adipocytes, has been reported to have a proinflammatory function in macrophages. However, the physiological role of FABP4, which is constitutively expressed in adipocytes, has not been fully elucidated. Previously, we demonstrated that FABP4 was involved in the regulation of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) production in 3T3-L1 adipocytes. In this study, we examined the effects of FABP4 silencing on the oxidative and endoplasmic reticulum (ER) stress in 3T3-L1 adipocytes. We found that the cellular reactive oxygen species (ROS) and 8-nitro-cyclic GMP levels were significantly elevated in the differentiated 3T3-L1 adipocytes transfected with a small interfering RNA (siRNA) against Fabp4, although the intracellular levels or enzyme activities of antioxidants including reduced glutathione (GSH), superoxide dismutase (SOD) and glutathione S-transferase A4 (GSTA4) were not altered. An in vitro evaluation using the recombinant protein revealed that FABP4 itself functions as a scavenger protein against hydrogen peroxide (H2O2). FABP4-knockdown resulted in a significant lowering of cell viability of 3T3-L1 adipocytes against H2O2 treatment. Moreover, four kinds of markers related to the ER stress response including the endoplasmic reticulum to nucleus signaling 1 (Ern1), the signal sequence receptor α (Ssr1), the ORM1-like 3 (Ormdl3), and the spliced X-box binding protein 1 (Xbp1s), were all elevated as the result of the knockdown of FABP4. Consequently, FABP4 might have a new role as an antioxidant protein against H2O2 and contribute to cytoprotection against oxidative and ER stress in adipocytes. PMID:25161868

  8. Therapeutic approach to target mesothelioma cancer cells using the Wnt antagonist, secreted frizzled-related protein 4: Metabolic state of cancer cells.

    PubMed

    Perumal, Vanathi; Pohl, Sebastian; Keane, Kevin N; Arfuso, Frank; Newsholme, Philip; Fox, Simon; Dharmarajan, Arun

    2016-02-15

    Malignant mesothelioma (MM) is an aggressive cancer, characterized by rapid progression, along with late metastasis and poor patient prognosis. It is resistant to many forms of standard anti-cancer treatment. In this study, we determined the effect of secreted frizzled-related protein 4 (sFRP4), a Wnt pathway inhibitor, on cancer cell proliferation and metabolism using the JU77 mesothelioma cell line. Treatment with sFRP4 (250 pg/ml) resulted in a significant reduction of cell proliferation. The addition of the Wnt activator Wnt3a (250 pg/ml) or sFRP4 had no significant effect on ATP production and glucose utilisation in JU77 cells at both the 24 and 48 h time points examined. We also examined their effect on Akt and Glycogen synthase kinase-3 beta (GSK3β) phosphorylation, which are both important components of Wnt signalling and glucose metabolism. We found that protein phosphorylation of Akt and GSK3β varied over the 24h and 48 h time points, with constitutive phosphorylation of Akt at serine 473 (pAkt) decreasing to its most significant level when treated with Wnt3a+sFRP4 at the 24h time point. A significant reduction in the level of Cytochrome c oxidase was observed at the 48 h time point, when sFRP4 and Wnt3a were added in combination. We conclude that sFRP4 may function, in part, to reduce/alter cancer cell metabolism, which may lead to sensitisation of cancer cells to chemotherapeutics, or even cell death. PMID:26868304

  9. Fox-2 Splicing Factor Binds to a Conserved Intron Motif to PromoteInclusion of Protein 4.1R Alternative Exon 16

    SciTech Connect

    Ponthier, Julie L.; Schluepen, Christina; Chen, Weiguo; Lersch,Robert A.; Gee, Sherry L.; Hou, Victor C.; Lo, Annie J.; Short, Sarah A.; Chasis, Joel A.; Winkelmann, John C.; Conboy, John G.

    2006-03-01

    Activation of protein 4.1R exon 16 (E16) inclusion during erythropoiesis represents a physiologically important splicing switch that increases 4.1R affinity for spectrin and actin. Previous studies showed that negative regulation of E16 splicing is mediated by the binding of hnRNP A/B proteins to silencer elements in the exon and that downregulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This paper demonstrates that positive regulation of E16 splicing can be mediated by Fox-2 or Fox-1, two closely related splicing factors that possess identical RNA recognition motifs. SELEX experiments with human Fox-1 revealed highly selective binding to the hexamer UGCAUG. Both Fox-1 and Fox-2 were able to bind the conserved UGCAUG elements in the proximal intron downstream of E16, and both could activate E16 splicing in HeLa cell co-transfection assays in a UGCAUG-dependent manner. Conversely, knockdown of Fox-2 expression, achieved with two different siRNA sequences resulted in decreased E16 splicing. Moreover, immunoblot experiments demonstrate mouse erythroblasts express Fox-2, but not Fox-1. These findings suggest that Fox-2 is a physiological activator of E16 splicing in differentiating erythroid cells in vivo. Recent experiments show that UGCAUG is present in the proximal intron sequence of many tissue-specific alternative exons, and we propose that the Fox family of splicing enhancers plays an important role in alternative splicing switches during differentiation in metazoan organisms.

  10. Cytoprotective role of the fatty acid binding protein 4 against oxidative and endoplasmic reticulum stress in 3T3-L1 adipocytes

    PubMed Central

    Kajimoto, Kazuaki; Minami, Yoshitaka; Harashima, Hideyoshi

    2014-01-01

    The fatty acid binding protein 4 (FABP4), one of the most abundant proteins in adipocytes, has been reported to have a proinflammatory function in macrophages. However, the physiological role of FABP4, which is constitutively expressed in adipocytes, has not been fully elucidated. Previously, we demonstrated that FABP4 was involved in the regulation of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) production in 3T3-L1 adipocytes. In this study, we examined the effects of FABP4 silencing on the oxidative and endoplasmic reticulum (ER) stress in 3T3-L1 adipocytes. We found that the cellular reactive oxygen species (ROS) and 8-nitro-cyclic GMP levels were significantly elevated in the differentiated 3T3-L1 adipocytes transfected with a small interfering RNA (siRNA) against Fabp4, although the intracellular levels or enzyme activities of antioxidants including reduced glutathione (GSH), superoxide dismutase (SOD) and glutathione S-transferase A4 (GSTA4) were not altered. An in vitro evaluation using the recombinant protein revealed that FABP4 itself functions as a scavenger protein against hydrogen peroxide (H2O2). FABP4-knockdown resulted in a significant lowering of cell viability of 3T3-L1 adipocytes against H2O2 treatment. Moreover, four kinds of markers related to the ER stress response including the endoplasmic reticulum to nucleus signaling 1 (Ern1), the signal sequence receptor α (Ssr1), the ORM1-like 3 (Ormdl3), and the spliced X-box binding protein 1 (Xbp1s), were all elevated as the result of the knockdown of FABP4. Consequently, FABP4 might have a new role as an antioxidant protein against H2O2 and contribute to cytoprotection against oxidative and ER stress in adipocytes. PMID:25161868

  11. Molecular-weight-dependent, anionic-substrate-preferential transport of β-lactam antibiotics via multidrug resistance-associated protein 4.

    PubMed

    Akanuma, Shin-Ichi; Uchida, Yasuo; Ohtsuki, Sumio; Kamiie, Jun-ichi; Tachikawa, Masanori; Terasaki, Tetsuya; Hosoya, Ken-ichi

    2011-01-01

    β-Lactam antibiotics have cerebral and peripheral adverse effects. Multidrug resistance-associated protein 4 (MRP4) has been reported to transport several β-lactam antibiotics, and its expression at the blood-brain barrier also serves to limit their distribution to the brain. Therefore, the purpose of this study was to clarify the structure-activity relationship of MRP4-mediated transport of β-lactam antibiotics using MRP4-expressing Sf9 membrane vesicles. The transport activity was evaluated as MRP4-mediated transport per MRP4 protein [nL/(min·fmol MRP4 protein)] based on measurement of MRP4 protein expression by means of liquid chromatography-tandem mass spectrometry. Cefotiam showed the greatest MRP4-mediated transport activity [8.90 nL/(min·fmol MRP4 protein)] among the β-lactam antibiotics examined in this study. Measurements of differential transport activity of MRP4 for various β-lactam antibiotics indicated that (i) cephalosporins were transported via MRP4 at a greater rate than were penams, β-lactamase inhibitors, penems, or monobactams; (ii) MRP4-mediated transport activity of anionic cephalosporins was greater than that of zwitterionic cephalosporins; and (iii) higher-molecular-weight anionic β-lactam antibiotics showed greater MRP4-mediated transport activity than lower-molecular-weight ones, whereas zwitterionic β-lactam antibiotics did not show molecular weight dependency of MRP4-mediated transport. These quantitative data should prove useful for understanding MRP-related adverse effects of β-lactam antibiotics and their derivatives. PMID:21897051

  12. The linoleic acid derivative DCP-LA increases membrane surface localization of the α7 ACh receptor in a protein 4.1N-dependent manner.

    PubMed

    Kanno, Takeshi; Tsuchiya, Ayako; Tanaka, Akito; Nishizaki, Tomoyuki

    2013-03-01

    In yeast two-hybrid screening, protein 4.1N, a scaffolding protein, was identified as a binding partner of the α7 ACh (acetylcholine) receptor. For rat hippocampal slices, the linoleic acid derivative DCP-LA {8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid} increased the association of the α7 ACh receptor with 4.1N, and the effect was inhibited by GF109203X, an inhibitor of PKC (protein kinase C), although DCP-LA did not induce PKC phosphorylation of 4.1N. For PC-12 cells, the presence of the α7 ACh receptor in the plasma membrane fraction was significantly suppressed by knocking down 4.1N. DCP-LA increased the presence of the α7 ACh receptor in the plasma membrane fraction, and the effect was still inhibited by knocking down 4.1N. In the monitoring of α7 ACh receptor mobilization, DCP-LA enhanced signal intensities for the α7 ACh receptor at the membrane surface in PC-12 cells, which was clearly prevented by knocking down 4.1N. Taken together, the results of the present study show that 4.1N interacts with the α7 ACh receptor and participates in the receptor tethering to the plasma membrane. The results also indicate that DCP-LA increases membrane surface localization of the α7 ACh receptor in a 4.1N-dependent manner under the control of PKC, but without phosphorylating 4.1N. PMID:23256752

  13. Structural protein 4.1R is integrally involved in nuclear envelope protein localization, centrosome–nucleus association and transcriptional signaling

    PubMed Central

    Meyer, Adam J.; Almendrala, Donna K.; Go, Minjoung M.; Krauss, Sharon Wald

    2011-01-01

    The multifunctional structural protein 4.1R is required for assembly and maintenance of functional nuclei but its nuclear roles are unidentified. 4.1R localizes within nuclei, at the nuclear envelope, and in cytoplasm. Here we show that 4.1R, the nuclear envelope protein emerin and the intermediate filament protein lamin A/C co-immunoprecipitate, and that 4.1R-specific depletion in human cells by RNA interference produces nuclear dysmorphology and selective mislocalization of proteins from several nuclear subcompartments. Such 4.1R-deficiency causes emerin to partially redistribute into the cytoplasm, whereas lamin A/C is disorganized at nuclear rims and displaced from nucleoplasmic foci. The nuclear envelope protein MAN1, nuclear pore proteins Tpr and Nup62, and nucleoplasmic proteins NuMA and LAP2α also have aberrant distributions, but lamin B and LAP2β have normal localizations. 4.1R-deficient mouse embryonic fibroblasts show a similar phenotype. We determined the functional effects of 4.1R-deficiency that reflect disruption of the association of 4.1R with emerin and A-type lamin: increased nucleus–centrosome distances, increased β-catenin signaling, and relocalization of β-catenin from the plasma membrane to the nucleus. Furthermore, emerin- and lamin-A/C-null cells have decreased nuclear 4.1R. Our data provide evidence that 4.1R has important functional interactions with emerin and A-type lamin that impact upon nuclear architecture, centrosome–nuclear envelope association and the regulation of β-catenin transcriptional co-activator activity that is dependent on β-catenin nuclear export. PMID:21486941

  14. Protein 4.1R binding to eIF3-p44 suggests an interaction between the cytoskeletal network and the translation apparatus.

    PubMed

    Hou, C L; Tang, C j; Roffler, S R; Tang, T K

    2000-07-15

    Erythroid protein 4.1 (4.1R) is an 80-kd cytoskeletal protein that stabilizes the membrane-skeletal network structure underlying the lipid bilayer. Using the carboxyl terminal domain (22/24 kd) of 4.1R as bait in a yeast 2-hybrid screen, we isolated cDNA clones encoding a polypeptide of eIF3-p44, which represents a subunit of a eukaryotic translation initiation factor 3 (eIF3) complex. The eIF3 complex consists of at least 10 subunits that play an essential role in the pathway of protein translation initiation. Northern blot analysis revealed that eIF3-p44 (approximately 1.35 kb) is constitutively expressed in many tissues. The essential sequence for this interaction was mapped to the carboxyl-terminus of 4.1R (residues 525-622) and a region (residues 54-321) of eIF3-p44. The direct association between 4.1R and eIF3-p44 was further confirmed by in vitro binding assays and coimmunoprecipitation studies. To characterize the functions of eIF3-p44, we depleted eIF3-p44 from rabbit reticulocyte lysates either by anti-eIF3-p44 antibody or by GST/4.1R-80 fusion protein. Our results show that the eIF3-p44 depleted cell-free translation system was unable to synthesize proteins efficiently. The direct association between 4.1R and elF3-p44 suggests that 4.1R may act as an anchor protein that links the cytoskeleton network to the translation apparatus. (Blood. 2000;96:747-753) PMID:10887144

  15. The 13-kD FK506 Binding Protein, FKBP13, Interacts with a Novel Homologue of the Erythrocyte Membrane Cytoskeletal Protein 4.1

    PubMed Central

    Walensky, Loren D.; Gascard, Philippe; Field, Michael E.; Blackshaw, Seth; Conboy, John G.; Mohandas, Narla; Snyder, Solomon H.

    1998-01-01

    We have identified a novel generally expressed homologue of the erythrocyte membrane cytoskeletal protein 4.1, named 4.1G, based on the interaction of its COOH-terminal domain (CTD) with the immunophilin FKBP13. The 129-amino acid peptide, designated 4.1G–CTD, is the first known physiologic binding target of FKBP13. FKBP13 is a 13-kD protein originally identified by its high affinity binding to the immunosuppressant drugs FK506 and rapamycin (Jin, Y., M.W. Albers, W.S. Lane, B.E. Bierer, and S.J. Burakoff. 1991. Proc. Natl. Acad. Sci. USA. 88:6677– 6681); it is a membrane-associated protein thought to function as an ER chaperone (Bush, K.T., B.A. Henrickson, and S.K. Nigam. 1994. Biochem. J. [Tokyo]. 303:705–708). We report the specific association of FKBP13 with 4.1G–CTD based on yeast two-hybrid, in vitro binding and coimmunoprecipitation experiments. The histidyl-proline moiety of 4.1G–CTD is required for FKBP13 binding, as indicated by yeast experiments with truncated and mutated 4.1G–CTD constructs. In situ hybridization studies reveal cellular colocalizations for FKBP13 and 4.1G–CTD throughout the body during development, supporting a physiologic role for the interaction. Interestingly, FKBP13 cofractionates with the red blood cell homologue of 4.1 (4.1R) in ghosts, inside-out vesicles, and Triton shell preparations. The identification of FKBP13 in erythrocytes, which lack ER, suggests that FKBP13 may additionally function as a component of membrane cytoskeletal scaffolds. PMID:9531554

  16. Human Immunodeficiency Virus Protease Inhibitors Interact with ATP Binding Cassette Transporter 4/Multidrug Resistance Protein 4: A Basis for Unanticipated Enhanced Cytotoxicity

    PubMed Central

    Fukuda, Yu; Takenaka, Kazumasa; Sparreboom, Alex; Cheepala, Satish B.; Wu, Chung-Pu; Ekins, Sean; Ambudkar, Suresh V.

    2013-01-01

    Human immunodeficiency virus (HIV) pharmacotherapy, by combining different drug classes such as nucleoside analogs and HIV protease inhibitors (PIs), has increased HIV-patient life expectancy. Consequently, among these patients, an increase in non-HIV–associated cancers has produced a patient cohort requiring both HIV and cancer chemotherapy. We hypothesized that multidrug resistance protein 4/ATP binding cassette transporter 4 (MRP4/ABCC4), a widely expressed transporter of nucleoside-based antiviral medications as well as cancer therapeutics might interact with PIs. Among the PIs evaluated (nelfinavir, ritonavir, amprenavir, saquinavir, and indinavir), only nelfinavir both effectively stimulated MRP4 ATPase activity and inhibited substrate-stimulated ATPase activity. Saos2 and human embryonic kidney 293 cells engineered to overexpress MRP4 were then used to assess transport and cytotoxicity. MRP4 expression reduced intracellular accumulation of nelfinavir and consequently conferred survival advantage to nelfinavir cytotoxicity. Nelfinavir blocked Mrp4-mediated export, which is consistent with its ability to increase the sensitivity of MRP4-expressing cells to methotrexate. In contrast, targeted inactivation of Abcc4/Mrp4 in mouse cells specifically enhanced nelfinavir and 9-(2-phosphonylmethoxyethyl) adenine cytotoxicity. These results suggest that nelfinavir is both an inhibitor and substrate of MRP4. Because nelfinavir is a new MRP4/ABCC4 substrate, we developed a MRP4/ABCC4 pharmacophore model, which showed that the nelfinavir binding site is shared with chemotherapeutic substrates such as adefovir and methotrexate. Our studies reveal, for the first time, that nelfinavir, a potent and cytotoxic PI, is both a substrate and inhibitor of MRP4. These findings suggest that HIV-infected cancer patients receiving nelfinavir might experience both enhanced antitumor efficacy and unexpected adverse toxicity given the role of MRP4/ABCC4 in exporting nucleoside

  17. Deletion of collapsin response mediator protein 4 results in abnormal layer thickness and elongation of mitral cell apical dendrites in the neonatal olfactory bulb.

    PubMed

    Tsutiya, Atsuhiro; Watanabe, Hikaru; Nakano, Yui; Nishihara, Masugi; Goshima, Yoshio; Ohtani-Kaneko, Ritsuko

    2016-05-01

    Collapsin response mediator protein 4 (CRMP4), a member of the CRMP family, is involved in the pathogenesis of neurodevelopmental disorders such as schizophrenia and autism. Here, we first compared layer thickness of the olfactory bulb between wild-type (WT) and CRMP4-knockout (KO) mice. The mitral cell layer (MCL) was significantly thinner, whereas the external plexiform layer (EPL) was significantly thicker in CRMP4-KO mice at postnatal day 0 (PD0) compared with WTs. However, differences in layer thickness disappeared by PD14. No apoptotic cells were found in the MCL, and the number of mitral cells (MCs) identified with a specific marker (i.e. Tbx21 antibody) did not change in CRMP4-KO neonates. However, DiI-tracing showed that the length of mitral cell apical dendrites was greater in CRMP4-KO neonates than in WTs. In addition, expression of CRMP4 mRNA in WT mice was most abundant in the MCL at PD0 and decreased afterward. These results suggest that CRMP4 contributes to dendritic elongation. Our in vitro studies showed that deletion or knockdown of CRMP4 resulted in enhanced growth of MAP2-positive neurites, whereas overexpression of CRMP4 reduced their growth, suggesting a new role for CRMP4 as a suppressor of dendritic elongation. Overall, our data suggest that disruption of CRMP4 produces a temporary alteration in EPL thickness, which is constituted mainly of mitral cell apical dendrites, through the enhanced growth of these dendrites. PMID:26739921

  18. Flow Cytofluorimetric Analysis of Anti-LRP4 (LDL Receptor-Related Protein 4) Autoantibodies in Italian Patients with Myasthenia Gravis

    PubMed Central

    Marino, Mariapaola; Scuderi, Flavia; Samengo, Daniela; Saltelli, Giorgia; Maiuri, Maria Teresa; Shen, Chengyong; Mei, Lin; Sabatelli, Mario; Pani, Giovambattista; Antonini, Giovanni; Evoli, Amelia; Bartoccioni, Emanuela

    2015-01-01

    Background Myasthenia gravis (MG) is an autoimmune disease in which 90% of patients have autoantibodies against the muscle nicotinic acetylcholine receptor (AChR), while autoantibodies to muscle-specific tyrosine kinase (MuSK) have been detected in half (5%) of the remaining 10%. Recently, the low-density lipoprotein receptor-related protein 4 (LRP4), identified as the agrin receptor, has been recognized as a third autoimmune target in a significant portion of the double sero-negative (dSN) myasthenic individuals, with variable frequency depending on different methods and origin countries of the tested population. There is also convincing experimental evidence that anti-LRP4 autoantibodies may cause MG. Methods The aim of this study was to test the presence and diagnostic significance of anti-LRP4 autoantibodies in an Italian population of 101 myasthenic patients (55 dSN, 23 AChR positive and 23 MuSK positive), 45 healthy blood donors and 40 patients with other neurological diseases as controls. All sera were analyzed by a cell-based antigen assay employing LRP4-transfected HEK293T cells, along with a flow cytofluorimetric detection system. Results We found a 14.5% (8/55) frequency of positivity in the dSN-MG group and a 13% frequency of co-occurrence (3/23) in both AChR and MuSK positive patients; moreover, we report a younger female prevalence with a mild form of disease in LRP4-positive dSN-MG individuals. Conclusion Our data confirm LRP4 as a new autoimmune target, supporting the value of including anti-LRP4 antibodies in further studies on Myasthenia gravis. PMID:26284792

  19. Fatty acid binding protein 4 and 5 play a crucial role in thermogenesis under the conditions of fasting and cold stress.

    PubMed

    Syamsunarno, Mas Rizky A A; Iso, Tatsuya; Yamaguchi, Aiko; Hanaoka, Hirofumi; Putri, Mirasari; Obokata, Masaru; Sunaga, Hiroaki; Koitabashi, Norimichi; Matsui, Hiroki; Maeda, Kazuhisa; Endo, Keigo; Tsushima, Yoshito; Yokoyama, Tomoyuki; Kurabayashi, Masahiko

    2014-01-01

    Hypothermia is rapidly induced during cold exposure when thermoregulatory mechanisms, including fatty acid (FA) utilization, are disturbed. FA binding protein 4 (FABP4) and FABP5, which are abundantly expressed in adipose tissues and macrophages, have been identified as key molecules in the pathogenesis of overnutrition-related diseases, such as insulin resistance and atherosclerosis. We have recently shown that FABP4/5 are prominently expressed in capillary endothelial cells in the heart and skeletal muscle and play a crucial role in FA utilization in these tissues. However, the role of FABP4/5 in thermogenesis remains to be determined. In this study, we showed that thermogenesis is severely impaired in mice lacking both FABP4 and FABP5 (DKO mice), as manifested shortly after cold exposure during fasting. In DKO mice, the storage of both triacylglycerol in brown adipose tissue (BAT) and glycogen in skeletal muscle (SkM) was nearly depleted after fasting, and a biodistribution analysis using 125I-BMIPP revealed that non-esterified FAs (NEFAs) are not efficiently taken up by BAT despite the robustly elevated levels of serum NEFAs. In addition to the severe hypoglycemia observed in DKO mice during fasting, cold exposure did not induce the uptake of glucose analogue 18F-FDG by BAT. These findings strongly suggest that DKO mice exhibit pronounced hypothermia after fasting due to the depletion of energy storage in BAT and SkM and the reduced supply of energy substrates to these tissues. In conclusion, FABP4/5 play an indispensable role in thermogenesis in BAT and SkM. Our study underscores the importance of FABP4/5 for overcoming life-threatening environments, such as cold and starvation. PMID:24603714

  20. Toward predicting drug-induced liver injury: parallel computational approaches to identify multidrug resistance protein 4 and bile salt export pump inhibitors.

    PubMed

    Welch, Matthew A; Köck, Kathleen; Urban, Thomas J; Brouwer, Kim L R; Swaan, Peter W

    2015-05-01

    Drug-induced liver injury (DILI) is an important cause of drug toxicity. Inhibition of multidrug resistance protein 4 (MRP4), in addition to bile salt export pump (BSEP), might be a risk factor for the development of cholestatic DILI. Recently, we demonstrated that inhibition of MRP4, in addition to BSEP, may be a risk factor for the development of cholestatic DILI. Here, we aimed to develop computational models to delineate molecular features underlying MRP4 and BSEP inhibition. Models were developed using 257 BSEP and 86 MRP4 inhibitors and noninhibitors in the training set. Models were externally validated and used to predict the affinity of compounds toward BSEP and MRP4 in the DrugBank database. Compounds with a score above the median fingerprint threshold were considered to have significant inhibitory effects on MRP4 and BSEP. Common feature pharmacophore models were developed for MRP4 and BSEP with LigandScout software using a training set of nine well characterized MRP4 inhibitors and nine potent BSEP inhibitors. Bayesian models for BSEP and MRP4 inhibition/noninhibition were developed with cross-validated receiver operator curve values greater than 0.8 for the test sets, indicating robust models with acceptable false positive and false negative prediction rates. Both MRP4 and BSEP inhibitor pharmacophore models were characterized by hydrophobic and hydrogen-bond acceptor features, albeit in distinct spatial arrangements. Similar molecular features between MRP4 and BSEP inhibitors may partially explain why various drugs have affinity for both transporters. The Bayesian (BSEP, MRP4) and pharmacophore (MRP4, BSEP) models demonstrated significant classification accuracy and predictability. PMID:25735837

  1. Toward Predicting Drug-Induced Liver Injury: Parallel Computational Approaches to Identify Multidrug Resistance Protein 4 and Bile Salt Export Pump Inhibitors

    PubMed Central

    Welch, Matthew A.; Köck, Kathleen; Urban, Thomas J.; Brouwer, Kim L. R.

    2015-01-01

    Drug-induced liver injury (DILI) is an important cause of drug toxicity. Inhibition of multidrug resistance protein 4 (MRP4), in addition to bile salt export pump (BSEP), might be a risk factor for the development of cholestatic DILI. Recently, we demonstrated that inhibition of MRP4, in addition to BSEP, may be a risk factor for the development of cholestatic DILI. Here, we aimed to develop computational models to delineate molecular features underlying MRP4 and BSEP inhibition. Models were developed using 257 BSEP and 86 MRP4 inhibitors and noninhibitors in the training set. Models were externally validated and used to predict the affinity of compounds toward BSEP and MRP4 in the DrugBank database. Compounds with a score above the median fingerprint threshold were considered to have significant inhibitory effects on MRP4 and BSEP. Common feature pharmacophore models were developed for MRP4 and BSEP with LigandScout software using a training set of nine well characterized MRP4 inhibitors and nine potent BSEP inhibitors. Bayesian models for BSEP and MRP4 inhibition/noninhibition were developed with cross-validated receiver operator curve values greater than 0.8 for the test sets, indicating robust models with acceptable false positive and false negative prediction rates. Both MRP4 and BSEP inhibitor pharmacophore models were characterized by hydrophobic and hydrogen-bond acceptor features, albeit in distinct spatial arrangements. Similar molecular features between MRP4 and BSEP inhibitors may partially explain why various drugs have affinity for both transporters. The Bayesian (BSEP, MRP4) and pharmacophore (MRP4, BSEP) models demonstrated significant classification accuracy and predictability. PMID:25735837

  2. Aminoterminal amphipathic α-helix AH1 of hepatitis C virus nonstructural protein 4B possesses a dual role in RNA replication and virus production.

    PubMed

    Gouttenoire, Jérôme; Montserret, Roland; Paul, David; Castillo, Rosa; Meister, Simon; Bartenschlager, Ralf; Penin, François; Moradpour, Darius

    2014-10-01

    Nonstructural protein 4B (NS4B) is a key organizer of hepatitis C virus (HCV) replication complex formation. In concert with other nonstructural proteins, it induces a specific membrane rearrangement, designated as membranous web, which serves as a scaffold for the HCV replicase. The N-terminal part of NS4B comprises a predicted and a structurally resolved amphipathic α-helix, designated as AH1 and AH2, respectively. Here, we report a detailed structure-function analysis of NS4B AH1. Circular dichroism and nuclear magnetic resonance structural analyses revealed that AH1 folds into an amphipathic α-helix extending from NS4B amino acid 4 to 32, with positively charged residues flanking the helix. These residues are conserved among hepaciviruses. Mutagenesis and selection of pseudorevertants revealed an important role of these residues in RNA replication by affecting the biogenesis of double-membrane vesicles making up the membranous web. Moreover, alanine substitution of conserved acidic residues on the hydrophilic side of the helix reduced infectivity without significantly affecting RNA replication, indicating that AH1 is also involved in virus production. Selective membrane permeabilization and immunofluorescence microscopy analyses of a functional replicon harboring an epitope tag between NS4B AH1 and AH2 revealed a dual membrane topology of the N-terminal part of NS4B during HCV RNA replication. Luminal translocation was unaffected by the mutations introduced into AH1, but was abrogated by mutations introduced into AH2. In conclusion, our study reports the three-dimensional structure of AH1 from HCV NS4B, and highlights the importance of positively charged amino acid residues flanking this amphipathic α-helix in membranous web formation and RNA replication. In addition, we demonstrate that AH1 possesses a dual role in RNA replication and virus production, potentially governed by different topologies of the N-terminal part of NS4B. PMID:25392992

  3. Affinity Map of Bromodomain Protein 4 (BRD4) Interactions with the Histone H4 Tail and the Small Molecule Inhibitor JQ1*

    PubMed Central

    Jung, Marie; Philpott, Martin; Müller, Susanne; Schulze, Jessica; Badock, Volker; Eberspächer, Uwe; Moosmayer, Dieter; Bader, Benjamin; Schmees, Norbert; Fernández-Montalván, Amaury; Haendler, Bernard

    2014-01-01

    Bromodomain protein 4 (BRD4) is a member of the bromodomain and extra-terminal domain (BET) protein family. It binds to acetylated histone tails via its tandem bromodomains BD1 and BD2 and forms a complex with the positive transcription elongation factor b, which controls phosphorylation of RNA polymerase II, ultimately leading to stimulation of transcription elongation. An essential role of BRD4 in cell proliferation and cancer growth has been reported in several recent studies. We analyzed the binding of BRD4 BD1 and BD2 to different partners and showed that the strongest interactions took place with di- and tetra-acetylated peptides derived from the histone 4 N-terminal tail. We also found that several histone 4 residues neighboring the acetylated lysines significantly influenced binding. We generated 10 different BRD4 BD1 mutants and analyzed their affinities to acetylated histone tails and to the BET inhibitor JQ1 using several complementary biochemical and biophysical methods. The impact of these mutations was confirmed in a cellular environment. Altogether, the results show that Trp-81, Tyr-97, Asn-140, and Met-149 play similarly important roles in the recognition of acetylated histones and JQ1. Pro-82, Leu-94, Asp-145, and Ile-146 have a more differentiated role, suggesting that different kinds of interactions take place and that resistance mutations compatible with BRD4 function are possible. Our study extends the knowledge on the contribution of individual BRD4 amino acids to histone and JQ1 binding and may help in the design of new BET antagonists with improved pharmacological properties. PMID:24497639

  4. Multi-drug Resistance Protein 4 (MRP4)-mediated Regulation of Fibroblast Cell Migration Reflects a Dichotomous Role of Intracellular Cyclic Nucleotides*

    PubMed Central

    Sinha, Chandrima; Ren, Aixia; Arora, Kavisha; Moon, Chang-Suk; Yarlagadda, Sunitha; Zhang, Weiqiang; Cheepala, Satish B.; Schuetz, John D.; Naren, Anjaparavanda P.

    2013-01-01

    It has long been known that cyclic nucleotides and cyclic nucleotide-dependent signaling molecules control cell migration. However, the concept that it is not just the absence or presence of cyclic nucleotides, but a highly coordinated balance between these molecules that regulates cell migration, is new and revolutionary. In this study, we used multidrug resistance protein 4 (MRP4)-expressing cell lines and MRP4 knock-out mice as model systems and wound healing assays as the experimental system to explore this unique and emerging concept. MRP4, a member of a large family of ATP binding cassette transporter proteins, localizes to the plasma membrane and functions as a nucleotide efflux transporter and thus plays a role in the regulation of intracellular cyclic nucleotide levels. Here, we demonstrate that mouse embryonic fibroblasts (MEFs) isolated from Mrp4−/− mice have higher intracellular cyclic nucleotide levels and migrate faster compared with MEFs from Mrp4+/+ mice. Using FRET-based cAMP and cGMP sensors, we show that inhibition of MRP4 with MK571 increases both cAMP and cGMP levels, which results in increased migration. In contrast to these moderate increases in cAMP and cGMP levels seen in the absence of MRP4, a robust increase in cAMP levels was observed following treatment with forskolin and isobutylmethylxanthine, which decreases fibroblast migration. In response to externally added cell-permeant cyclic nucleotides (cpt-cAMP and cpt-cGMP), MEF migration appears to be biphasic. Altogether, our studies provide the first experimental evidence supporting the novel concept that balance between cyclic nucleotides is critical for cell migration. PMID:23264633

  5. Expression of retinol binding protein 4 and nuclear factor-κB in diabetic rats with atherosclerosis and the intervention effect of pioglitazone

    PubMed Central

    Zhou, Wan; Ye, Shandong; Li, Jing

    2016-01-01

    This study aims to investigate the expression of retinol binding protein 4 (RBP4) and the activity of nuclear factor-kappa B (NF-κB) in diabetic rats with atherosclerosis, and to evaluate the intervention effect of pioglitazone. A total of 75 Wistar rats were randomly divided into four groups: Normal control (NC), diabetic rats (DM1), diabetic rats with atherosclerosis (DM2) and diabetic rats treated with pioglitazone (DM + Pio). The activity of NF-κB, the levels of serum and adipose tissue RBP4, fasting plasma glucose (FPG), fasting insulin (FINS), low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c), triglycerides (TG) and arteria caudilis systolic blood pressure (SBP) were measured. Percentage of fat mass (PFM), atherogenic index of plasma (AIP) and homeostasis model assessment of insulin resistance (HOMA-IR) were calculated. Compared with the NC and DM + Pio groups, all the parameters mentioned above increased significantly in the DM1 and DM2 groups, with the exception that HDL-c decreased. Pearson analysis showed that RBP4 in serum and adipose tissue were positively associated with TG, LDL-c, FINS, PFM, AIP, HOMA-IR, NF-κB, SBP and negatively associated with HDL-c. Multivariable logistic regression analysis showed that serum RBP4 and TG were predictors for the presence of diabetic atherosclerosis. In conclusion, RBP4 may be an effective predictor for diabetic atherosclerosis; pioglitazone is able to decrease RBP4 and NF-κB, which may partly contribute to its protective effect against diabetic atherosclerosis. PMID:27446311

  6. Comparison of syncytiotrophoblast generated from human embryonic stem cells and from term placentas

    PubMed Central

    Yabe, Shinichiro; Alexenko, Andrei P.; Amita, Mitsuyoshi; Yang, Ying; Schust, Danny J.; Ezashi, Toshihiko; Roberts, R. Michael

    2016-01-01

    Human embryonic stem cells (ESCs) readily commit to the trophoblast lineage after exposure to bone morphogenetic protein-4 (BMP-4) and two small compounds, an activin A signaling inhibitor and a FGF2 signaling inhibitor (BMP4/A83-01/PD173074; BAP treatment). During differentiation, areas emerge within the colonies with the biochemical and morphological features of syncytiotrophoblast (STB). Relatively pure fractions of mononucleated cytotrophoblast (CTB) and larger syncytial sheets displaying the expected markers of STB can be obtained by differential filtration of dispersed colonies through nylon strainers. RNA-seq analysis of these fractions has allowed them to be compared with cytotrophoblasts isolated from term placentas before and after such cells had formed syncytia. Although it is clear from extensive gene marker analysis that both ESC- and placenta-derived syncytial cells are trophoblast, each with the potential to transport a wide range of solutes and synthesize placental hormones, their transcriptome profiles are sufficiently dissimilar to suggest that the two cell types have distinct pedigrees and represent functionally different kinds of STB. We propose that the STB generated from human ESCs represents the primitive syncytium encountered in early pregnancy soon after the human trophoblast invades into the uterine wall. PMID:27051068

  7. Comparison of syncytiotrophoblast generated from human embryonic stem cells and from term placentas.

    PubMed

    Yabe, Shinichiro; Alexenko, Andrei P; Amita, Mitsuyoshi; Yang, Ying; Schust, Danny J; Sadovsky, Yoel; Ezashi, Toshihiko; Roberts, R Michael

    2016-05-10

    Human embryonic stem cells (ESCs) readily commit to the trophoblast lineage after exposure to bone morphogenetic protein-4 (BMP-4) and two small compounds, an activin A signaling inhibitor and a FGF2 signaling inhibitor (BMP4/A83-01/PD173074; BAP treatment). During differentiation, areas emerge within the colonies with the biochemical and morphological features of syncytiotrophoblast (STB). Relatively pure fractions of mononucleated cytotrophoblast (CTB) and larger syncytial sheets displaying the expected markers of STB can be obtained by differential filtration of dispersed colonies through nylon strainers. RNA-seq analysis of these fractions has allowed them to be compared with cytotrophoblasts isolated from term placentas before and after such cells had formed syncytia. Although it is clear from extensive gene marker analysis that both ESC- and placenta-derived syncytial cells are trophoblast, each with the potential to transport a wide range of solutes and synthesize placental hormones, their transcriptome profiles are sufficiently dissimilar to suggest that the two cell types have distinct pedigrees and represent functionally different kinds of STB. We propose that the STB generated from human ESCs represents the primitive syncytium encountered in early pregnancy soon after the human trophoblast invades into the uterine wall. PMID:27051068

  8. Phosphatidylinositol-4,5-biphosphate (PIP2) differentially regulates the interaction of human erythrocyte protein 4.1 (4.1R) with membrane proteins.

    PubMed

    An, Xiuli; Zhang, Xihui; Debnath, Gargi; Baines, Anthony J; Mohandas, Narla

    2006-05-01

    Human erythrocyte protein 4.1 (4.1R) participates in organizing the plasma membrane by linking several surface-exposed transmembrane proteins to the internal cytoskeleton. In the present study, we characterized the interaction of 4.1R with phosphatidylinositol-4,5-bisphosphate (PIP2) and assessed the effect of PIP2 on the interaction of 4.1R with membrane proteins. We found that 4.1R bound to PIP2-containing liposomes through its N-terminal 30 kDa membrane-binding domain and PIP2 binding induced a conformational change in this domain. Phosphatidylinositol-4-phosphate (PIP) was a less effective inducer of this conformational change, and phosphatidylinositol (PI) and inositol-1,4,5-phosphate (IP3) induced no change. Replacement of amino acids K63,64 and K265,266 by alanine abolished the interaction of the membrane-binding domain with PIP2. Importantly, binding of PIP2 to 4.1R selectively modulated the ability of 4.1R to interact with its different binding partners. While PIP2 significantly enhanced the binding of 4.1R to glycophorin C (GPC), it inhibited the binding of 4.1R to band 3 in vitro. PIP2 had no effect on 4.1R binding to p55. Furthermore, GPC was more readily extracted by Triton X-100 from adenosine triphosphate (ATP)-depleted erythrocytes, implying that the GPC-4.1R interaction may be regulated by PIP2 in situ. These findings define an important role for PIP2 in regulating the function of 4.1R. Because 4.1R and its family members (4.1R, 4.1B, 4.1G, and 4.1N) are widely expressed and the PIP2-binding motifs are highly conserved, it is likely that the functions of other 4.1 proteins are similarly regulated by PIP2 in many different cell types. PMID:16669616

  9. Associations between retinol-binding protein 4 and cardiometabolic risk factors and subclinical atherosclerosis in recently postmenopausal women: cross-sectional analyses from the KEEPS study

    PubMed Central

    2012-01-01

    Background The published literature regarding the relationships between retinol-binding protein 4 (RBP4) and cardiometabolic risk factors and subclinical atherosclerosis is conflicting, likely due, in part, to limitations of frequently used RBP4 assays. Prior large studies have not utilized the gold-standard western blot analysis of RBP4 levels. Methods Full-length serum RBP4 levels were measured by western blot in 709 postmenopausal women screened for the Kronos Early Estrogen Prevention Study. Cross-sectional analyses related RBP4 levels to cardiometabolic risk factors, carotid artery intima-media thickness (CIMT), and coronary artery calcification (CAC). Results The mean age of women was 52.9 (± 2.6) years, and the median RBP4 level was 49.0 (interquartile range 36.9-61.5) μg/mL. Higher RBP4 levels were weakly associated with higher triglycerides (age, race, and smoking-adjusted partial Spearman correlation coefficient = 0.10; P = 0.01), but were unrelated to blood pressure, cholesterol, C-reactive protein, glucose, insulin, and CIMT levels (all partial Spearman correlation coefficients ≤0.06, P > 0.05). Results suggested a curvilinear association between RBP4 levels and CAC, with women in the bottom and upper quartiles of RBP4 having higher odds of CAC (odds ratio [95% confidence interval] 2.10 [1.07-4.09], 2.00 [1.02-3.92], 1.64 [0.82-3.27] for the 1st, 3rd, and 4th RBP4 quartiles vs. the 2nd quartile). However, a squared RBP4 term in regression modeling was non-significant (P = 0.10). Conclusions In these healthy, recently postmenopausal women, higher RBP4 levels were weakly associated with elevations in triglycerides and with CAC, but not with other risk factors or CIMT. These data using the gold standard of RBP4 methodology only weakly support the possibility that perturbations in RBP4 homeostasis may be an additional risk factor for subclinical coronary atherosclerosis. Trial registration ClinicalTrials.gov number NCT00154180 PMID:22587616

  10. Assessment of Neutrophil Gelatinase-Associated Lipocalin (NGAL) and Retinol-Binding Protein 4 (RBP4) in Type 2 Diabetic Patients with Nephropathy

    PubMed Central

    Mahfouz, Mohamed H.; Assiri, Adel M.; Mukhtar, Mohammed H.

    2016-01-01

    Diabetic nephropathy (DN) is one of the most serious microvascular complications of diabetes. The study aims to evaluate the diagnostic value of serum neutrophil gelatinase-associated lipocalin (NGAL) and retinol-binding protein 4 (RBP4) as biomarkers for early detection of nephropathy in type 2 diabetic patients. The current study was performed on 150 type 2 diabetic patients. These patients were classified into three equal groups according to their albumin/creatinine ratio (ACR), including patients with normoalbuminuria (ACR <30 mg/g creatinine), patients with microalbuminuria (ACR = 30–300 mg/g creatinine), and patients with macroalbuminuria (ACR >300 mg/g creatinine). Fifty apparently healthy subjects matching the same age and socioeconomic status with diabetic subjects were selected as a control group. The plasma glucose, insulin, glycosylated hemoglobin (HbA1c), homeostasis model assessment of insulin resistance (HOMA-IR), lipid profile, urea, creatinine, cystatin C, glomerular filtration rate (GFR), NGAL, and RBP4 were measured in the studied groups. Significantly elevated NGAL and RBP4 levels were observed in micro- and macroalbuminuric diabetic groups when compared to the control and normoalbuminuric diabetic groups. NGAL and RBP4 were found to correlate positively with duration of diabetes, systolic and diastolic blood pressure, glucose, HbA1c, HOMA-IR, triacylglycerol, and ACR, but correlate inversely with GFR in DN groups. Receiver operating characteristic curves revealed that for early detection of DN, the best cutoff values to discriminate DN and diabetic without nephropathy groups were 91.5 ng/mL for NGAL with 87% sensitivity, 74% specificity, and area under the curve (AUC) = 0.881; 24.5 ng/mL for RBP4 with 84% sensitivity, 90% specificity, and AUC = 0.912; and 37.5 mg/g creatinine for ACR with 89% sensitivity, 72% specificity, and AUC = 0.819. RBP4 is more specific (90% specificity) than NGAL (74% specificity) and ACR (72% specificity). Therefore

  11. Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 4 Antagonizes Beta Interferon Expression by Targeting the NF-κB Essential Modulator

    PubMed Central

    Huang, Chen; Zhang, Qiong; Guo, Xue-kun; Yu, Zhi-bin; Xu, Ao-Tian; Tang, Jun

    2014-01-01

    ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly infectious pathogen that causes severe diseases in pigs and great economic losses to the swine industry worldwide. Type I interferons (IFNs) play a crucial role in antiviral immunity. In the present study, we demonstrated that infection with the highly pathogenic PRRSV strain JXwn06 antagonized type I IFN expression induced by poly(I·C) in both porcine alveolar macrophages (PAMs) and blood monocyte-derived macrophages (BMo). Subsequently, we showed that the inhibition of poly(I·C)-induced IFN-β production by PRRSV was dependent on the blocking of NF-κB signaling pathways. By screening PRRSV nonstructural and structural proteins, we demonstrated that nonstructural protein 4 (nsp4), a viral 3C-like serine protease, significantly suppressed IFN-β expression. Moreover, we verified that nsp4 inhibited NF-κB activation induced by signaling molecules, including RIG-I, VISA, TRIF, and IKKβ. nsp4 was shown to target the NF-κB essential modulator (NEMO) at the E349-S350 site to mediate its cleavage. Importantly, nsp4 mutants with defective protease activity abolished its ability to cleave NEMO and inhibit IFN-β production. These findings might have implications for our understanding of PRRSV pathogenesis and its mechanisms for evading the host immune response. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is a major agent of respiratory diseases in pigs. Like many other viruses, PRRSV has evolved a variety of strategies to evade host antiviral innate immunity for survival and propagation. In this study, we show that PRRSV nsp4 is a novel antagonist of the NF-κB signaling pathway, which is responsible for regulating the expression of type I interferons and other crucial cytokines. We then investigated the underlying mechanism used by nsp4 to suppress NF-κB-mediated IFN-β production. We found that nsp4 interfered with the NF-κB signaling pathway through the

  12. The Vitis vinifera C-repeat binding protein 4 (VvCBF4) transcriptional factor enhances freezing tolerance in wine grape

    PubMed Central

    Tillett, Richard L.; Wheatley, Matthew D.; Tattersall, Elizabeth A.R.; Schlauch, Karen A.; Cramer, Grant R.; Cushman, John C.

    2014-01-01

    Summary Chilling and freezing can reduce significantly vine survival and fruit set in Vitis vinifera wine grape. To overcome such production losses, a recently identified grapevine C-repeat binding factor (CBF) gene, VvCBF4, was overexpressed in grape vine cv. “Freedom” and found to improve freezing survival and reduced freezing-induced electrolyte leakage by up to 2°C in non-cold-acclimated vines. In addition, overexpression of this transgene caused a reduced growth phenotype similar to that observed for CBF overexpression in Arabidopsis and other species. Both freezing tolerance and reduced growth phenotypes were manifested in a transgene dose-dependent manner. To understand the mechanistic basis of VvCBF4 transgene action, one transgenic line (9–12) was genotyped using microarray-based mRNA expression profiling. Forty-seven and 12 genes were identified in unstressed transgenic shoots with either a greater than 1.5-fold increase or decrease in mRNA abundance, respectively. Comparison of mRNA changes with characterized CBF regulons in woody and herbaceous species revealed partial overlaps suggesting that CBF-mediated cold acclimation responses are widely conserved. Putative VvCBF4-regulon targets included genes with functions in cell wall structure, lipid metabolism, epicuticular wax formation, and stress-responses suggesting that the observed cold tolerance and dwarf phenotypes are the result of a complex network of diverse functional determinants. PMID:21914113

  13. Phosphatidylethanolamine-binding protein 4 promotes lung cancer cells proliferation and invasion via PI3K/Akt/mTOR axis

    PubMed Central

    Yu, Guiping; Chen, Guoqiang; Mi, Yedong

    2015-01-01

    Background While phosphatidylethanolamine-binding protein 4 (PEBP4) is a key factor in the malignant proliferation and metastasis of tumor cells, the exact regulatory network governing its roles remains unclear. This study was designed to investigate the effect of PEBP4 on PI3K/Akt/mTOR pathway and explore its molecular network that governs the proliferation and metastasis of tumor cells. Methods After the recombinant plasmid pcDNA3.1-PEBP4 was constructed, the recombinant plasmid pcDNA3.1-PEBP4 and PEBP4-targeting siRNA were transfected into lung cancer HCC827 cell line. The expressions of PI3K/Akt/mTOR pathway components in HCC827 cells in each group were determined using Western blotting. In the HCC827 cells, the effect of PI3K pathway inhibitor LY294002 on the expressions of PI3K/Akt/mTOR pathway components under the effect of PEBP4 was determined using Western blotting, and the effects of LY294002 on the cell viability, proliferation, and migration capabilities under the overexpression of PEBP4 were determined using MTT method, flow cytometry, and Transwell migration assay. Furthermore, the effect of mTOR inhibitor rapamycin (RAPA) on the expressions of PI3K/Akt/mTOR pathway components under the effect of PEBP4 was determined using Western blotting, and the effects of RAPA on the cell viability, proliferation, and migration capabilities under the overexpression of PEBP4 were determined using MTT method, flow cytometry, and Transwell migration assay. Results As shown by Western blotting, the protein expressions of p-Akt and phosphorylated mTOR (p-mTOR) were significantly higher in the pcDNA3.1-PEBP4-transfected group than in the normal control group and PEBP4 siRNA group (P<0.05); furthermore, the protein expressions of p-Akt and p-mTOR significantly decreased in the PEBP4 targeting siRNA-transfected group (P<0.05). Treatment with LY294002 significantly inhibited the protein expressions of p-Akt and p-mTOR in HCC827 cells (P<0.05). In contrast, treatment with

  14. Osteogenic gene expression of murine osteoblastic (MC3T3-E1) cells under cyclic tension

    NASA Astrophysics Data System (ADS)

    Kao, C. T.; Chen, C. C.; Cheong, U.-I.; Liu, S. L.; Huang, T. H.

    2014-08-01

    Low-level laser therapy (LLLT) can promote cell proliferation. The remodeling ability of the tension side of orthodontic teeth affects post-orthodontic stability. The purpose of the present study was to investigate the osteogenic effects of LLLT on osteoblast-like cells treated with a simulated tension system that provides a mechanical tension regimen. Murine osteoblastic (MC3T3-E1) cells were cultured in a Flexcell strain unit with programmed loads of 12% elongation at a frequency of 0.5 Hz for 24 and 48 h. The cultured cells were treated with a low-level diode laser using powers of 5 J and 10 J. The proliferation of MC3T3-E1 cells was determined using the Alamar Blue assay. The expression of osteogenic genes (type I collagen (Col-1), osteopontin (OPN), osteocalcin (OC), osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), bone morphologic protein (BMP-2), and bone morphologic protein (BMP-4)) in MC3T3-E1 cells was analyzed using reverse transcription polymerase chain reaction (RT-PCR). The data were analyzed using one-way analysis of variance. The proliferation rate of tension-cultured MC3T3-E1 cells under 5 J and 10 J LLLT increased compared with that of the control group (p < 0.05). Prominent mineralization of the MC3T3-E1 cells was visible using a von Kossa stain in the 5 J LLLT group. Osteogenic genes (Col-1, OC, OPG and BMP-2) were significantly expressed in the MC3T3-E1 cells treated with 5 J and 10 J LLLT (p < 0.05). LLLT in tension-cultured MC3T3-E1 cells showed synergistic osteogenic effects, including increases in cell proliferation and Col-1, OPN, OC, OPG and BMP-2 gene expression. LLLT might be beneficial for bone remodeling on the tension side of orthodontics.

  15. Mechanistic Analysis of the Role of Bromodomain-containing Protein 4 (BRD4) in BRD4-NUT Oncoprotein-induced Transcriptional Activation*

    PubMed Central

    Wang, Ranran; You, Jianxin

    2015-01-01

    NUT midline carcinoma (NMC) is a rare but highly aggressive cancer typically caused by the translocation t(15;19), which results in the formation of the BRD4-NUT fusion oncoprotein. Previous studies have demonstrated that fusion of the NUT protein with the double bromodomains of BRD4 may significantly alter the cellular gene expression profile to contribute to NMC tumorigenesis. However, the mechanistic details of this BRD4-NUT function remain poorly understood. In this study, we examined the NUT function in transcriptional regulation by targeting it to a LacO transgene array integrated in U2OS 2-6-3 cells, which allow us to visualize how NUT alters the in situ gene transcription dynamic. Using this system, we demonstrated that the NUT protein tethered to the LacO locus recruits p300/CREB-binding protein (CBP), induces histone hyperacetylation, and enriches BRD4 to the transgene array chromatin foci. We also discovered that, in BRD4-NUT expressed in NMC cells, the NUT moiety of the fusion protein anchored to chromatin by the double bromodomains also stimulates histone hyperacetylation, which causes BRD4 to bind tighter to chromatin. Consequently, multiple BRD4-interacting factors are recruited to the NUT-associated chromatin locus to activate in situ transgene expression. This gene transcription function was repressed by either expression of a dominant negative inhibitor of the p300-NUT interaction or treatment with (+)-JQ1, which dissociates BRD4 from the LacO chromatin locus. Our data support a model in which BRD4-NUT-stimulated histone hyperacetylation recruits additional BRD4 and interacting partners to support transcriptional activation, which underlies the BRD4-NUT oncogenic mechanism in NMC. PMID:25512383

  16. SMAD1/5 Signaling in the Early Equine Placenta Regulates Trophoblast Differentiation and Chorionic Gonadotropin Secretion

    PubMed Central

    Cabrera-Sharp, Victoria; Read, Jordan E.; Richardson, Stephanie; Kowalski, Alycia A.; Antczak, Douglas F.; Cartwright, Judith E.; Mukherjee, Abir

    2014-01-01

    TGFβ superfamily proteins, acting via SMAD (Sma- and Mad-related protein)2/3 pathways, regulate placental function; however, the role of SMAD1/5/8 pathway in the placenta is unknown. This study investigated the functional role of bone morphogenetic protein (BMP)4 signaling through SMAD1/5 in terminal differentiation of primary chorionic gonadotropin (CG)-secreting trophoblast. Primary equine trophoblast cells or placental tissues were isolated from day 27–34 equine conceptuses. Detected by microarray, RT-PCR, and quantitative RT-PCR, equine chorionic girdle trophoblast showed increased gene expression of receptors that bind BMP4. BMP4 mRNA expression was 20- to 60-fold higher in placental tissues adjacent to the chorionic girdle compared with chorionic girdle itself, suggesting BMP4 acts primarily in a paracrine manner on the chorionic girdle. Stimulation of chorionic girdle-trophoblast cells with BMP4 resulted in a dose-dependent and developmental stage-dependent increase in total number and proportion of terminally differentiated binucleate cells. Furthermore, BMP4 treatment induced non-CG-secreting day 31 chorionic girdle trophoblast cells to secrete CG, confirming a specific functional response to BMP4 stimulation. Inhibition of SMAD2/3 signaling combined with BMP4 treatment further enhanced differentiation of trophoblast cells. Phospho-SMAD1/5, but not phospho-SMAD2, expression as determined by Western blotting was tightly regulated during chorionic girdle trophoblast differentiation in vivo, with peak expression of phospho-SMAD1/5 in vivo noted at day 31 corresponding to maximal differentiation response of trophoblast in vitro. Collectively, these experiments demonstrate the involvement of BMP4-dependent pathways in the regulation of equine trophoblast differentiation in vivo and primary trophoblast differentiation in vitro via activation of SMAD1/5 pathway, a previously unreported mechanism of TGFβ signaling in the mammalian placenta. PMID:24848867

  17. Characterization of gene-environment interactions for colorectal cancer susceptibility loci

    PubMed Central

    Hutter, Carolyn M.; Chang-Claude, Jenny; Slattery, Martha L.; Pflugeisen, Bethann M.; Lin, Yi; Duggan, David; Nan, Hongmei; Lemire, Mathieu; Rangrej, Jagadish; Figueiredo, Jane C.; Jiao, Shuo; Harrison, Tabitha A.; Liu, Yan; Chen, Lin S.; Stelling, Deanna L.; Warnick, Greg S.; Hoffmeister, Michael; Küry, Sébastien; Fuchs, Charles S.; Giovannucci, Edward; Hazra, Aditi; Kraft, Peter; Hunter, David J.; Gallinger, Steven; Zanke, Brent W.; Brenner, Hermann; Frank, Bernd; Ma, Jing; Ulrich, Cornelia M.; White, Emily; Newcomb, Polly A.; Kooperberg, Charles; LaCroix, Andrea Z.; Prentice, Ross L.; Jackson, Rebecca D.; Schoen, Robert E.; Chanock, Stephen J.; Berndt, Sonja I.; Hayes, Richard B.; Caan, Bette J.; Potter, John D.; Hsu, Li; Bézieau, Stéphane; Chan, Andrew T.; Hudson, Thomas J.; Peters, Ulrike

    2012-01-01

    Genome-wide association studies (GWAS) have identified over a dozen loci associated with colorectal cancer (CRC) risk. Here we examined potential effect-modification between single nucleotide polymorphisms (SNPs) at 10 of these loci and probable or established environmental risk factors for CRC in 7,016 CRC cases and 9,723 controls from nine cohort and case-control studies. We used meta-analysis of an efficient empirical-Bayes estimator to detect potential multiplicative interactions between each of the SNPs [rs16892766 at 8q23.3 (EIF3H/UTP23); rs6983267 at 8q24 (MYC); rs10795668 at 10p14 (FLJ3802842); rs3802842 at11q23 (LOC120376); rs4444235 at 14q22.2 (BMP4); rs4779584 at15q13 (GREM1); rs9929218 at16q22.1 (CDH1); rs4939827 at18q21 (SMAD7); rs10411210 at19q13.1 (RHPN2); and rs961253 at 20p12.3 (BMP2)] and select major CRC risk factors (sex, body mass index, height, smoking status, aspirin/non-steroidal anti-inflammatory drug use, alcohol use, and dietary intake of calcium, folate, red meat, processed meat, vegetables, fruit, and fiber). The strongest statistical evidence for a gene-environment interaction across studies was for vegetable consumption and rs16892766, located on chromosome 8q23.3, near the EIF3H and UTP23 genes (nominal p-interaction =1.3×10–4; adjusted p-value 0.02). The magnitude of the main effect of the SNP increased with increasing levels of vegetable consumption. No other interactions were statistically significant after adjusting for multiple comparisons. Overall, the association of most CRC susceptibility loci identified in initial GWAS appears to be invariant to the other risk factors considered; however, our results suggest potential modification of the rs16892766 effect by vegetable consumption. PMID:22367214

  18. Gene Therapy

    PubMed Central

    Baum, Bruce J

    2014-01-01

    Applications of gene therapy have been evaluated in virtually every oral tissue, and many of these have proved successful at least in animal models. While gene therapy will not be used routinely in the next decade, practitioners of oral medicine should be aware of the potential of this novel type of treatment that doubtless will benefit many patients with oral diseases. PMID:24372817

  19. Trichoderma genes

    DOEpatents

    Foreman, Pamela; Goedegebuur, Frits; Van Solingen, Pieter; Ward, Michael

    2012-06-19

    Described herein are novel gene sequences isolated from Trichoderma reesei. Two genes encoding proteins comprising a cellulose binding domain, one encoding an arabionfuranosidase and one encoding an acetylxylanesterase are described. The sequences, CIP1 and CIP2, contain a cellulose binding domain. These proteins are especially useful in the textile and detergent industry and in pulp and paper industry.

  20. [Language gene].

    PubMed

    Takahashi, Hiroshi

    2006-11-01

    The human capacity for acquiring speech and language must derive, at least in part, from the genome. Recent advance in the field of molecular genetics finally discovered 'Language Gene'. Disruption of FOXP2 gene, the firstly identified 'language gene' causes severe speech and language disorder. To elucidate the anatomical basis of language processing in the brain, we examined the expression pattern of FOXP2/Foxp2 genes in the monkey and rat brains through development. We found the preferential expression of FOXP2/Foxp2 in the striosomal compartment of the developing striatum. Thus, we suggest the striatum, particularly striosomal system may participate in neural information processing for language and speech. Our suggestion is consistent with the declarative/ procedural model of language proposed by Ullman (1997, 2001), which the procedural memory-dependent mental grammar is rooted in the basal ganglia and the frontal cortex, and the declarative memory-dependent mental lexicon is rooted in the temporal lobe. PMID:17432197

  1. Genes V.

    SciTech Connect

    Lewin, B.

    1994-12-31

    This fifth edition book encompasses a wide range of topics covering 1,272 pages. The book is arranged into nine parts with a total of 36 chapters. These nine parts include Introduction; DNA as a Store of Information; Translation; Constructing Cells; Control of Prokaryotypic Gene Expression; Perpetuation of DNA; Organization of the Eukaryotypic Genome; Eukaryotypic Transcription and RNA Processing; The Dynamic Genome; and Genes in Development.

  2. Enhanced haemolysis with beta-thalassaemia trait due to the unstable beta chain variant, Hb Gunma, accompanied by hereditary elliptocytosis due to protein 4.1 deficiency in a Japanese family.

    PubMed

    Maehara, Tadashi; Tsukamoto, Norifumi; Nojima, Yoshihisa; Karasawa, Masamitsu; Murakami, Hirokazu; Hattori, Yukio; Ideguchi, Hiroshi

    2002-04-01

    We identified a Japanese family with a beta-thalassaemia trait and hereditary elliptocytosis (HE). We studied five members of this family. One was normal, one had only the beta-thalassaemia trait, one had heterozygous HE, and two had compound heterozygous beta-thalassaemia trait and HE. The last two had already undergone splenectomy. The molecular profile of beta-thalassaemia was consistent with that of Hb Gunma: codon 127/128CAGGCT(Gln-Ala)--> CCT(Pro). Analysis of erythrocyte membrane proteins revealed a partial deficiency of protein 4.1 in all those with HE, whereas the spectrin content was within the normal range. Each heterozygous family member with either the beta-thalassaemia trait or HE was asymptomatic, whereas the two with both beta-thalassaemia and HE had marked red blood cell deformities and haemolysis. The abnormalities of the red blood cells in patients with the beta-thalassaemia trait might be enhanced by association with HE owing to a protein 4.1 deficiency. PMID:11918554

  3. The protein 4.1, ezrin, radixin, moesin (FERM) domain of Drosophila Coracle, a cytoplasmic component of the septate junction, provides functions essential for embryonic development and imaginal cell proliferation.

    PubMed Central

    Ward, R E; Schweizer, L; Lamb, R S; Fehon, R G

    2001-01-01

    Coracle is a member of the Protein 4.1 superfamily of proteins, whose members include Protein 4.1, the Neurofibromatosis 2 tumor suppressor Merlin, Expanded, the ERM proteins, protein tyrosine phosphatases, and unconventional myosins. Recent evidence suggests that members of this family participate in cell signaling events, including those that regulate cell proliferation and the cytoskeleton. Previously, we demonstrated that Coracle protein is localized to the septate junction in epithelial cells and is required for septate junction integrity. Loss of coracle function leads to defects in embryonic development, including failure in dorsal closure, and to proliferation defects. In addition, we determined that the N-terminal 383 amino acids define an essential functional domain possessing membrane-organizing properties. Here we investigate the full range of functions provided by this highly conserved domain and find that it is sufficient to rescue all embryonic defects associated with loss of coracle function. In addition, this domain is sufficient to rescue the reduced cell proliferation defect in imaginal discs, although it is incapable of rescuing null mutants to the adult stage. This result suggests the presence of a second functional domain within Coracle, a notion supported by molecular characterization of a series of coracle alleles. PMID:11560899

  4. The protein 4.1, ezrin, radixin, moesin (FERM) domain of Drosophila Coracle, a cytoplasmic component of the septate junction, provides functions essential for embryonic development and imaginal cell proliferation.

    PubMed

    Ward, R E; Schweizer, L; Lamb, R S; Fehon, R G

    2001-09-01

    Coracle is a member of the Protein 4.1 superfamily of proteins, whose members include Protein 4.1, the Neurofibromatosis 2 tumor suppressor Merlin, Expanded, the ERM proteins, protein tyrosine phosphatases, and unconventional myosins. Recent evidence suggests that members of this family participate in cell signaling events, including those that regulate cell proliferation and the cytoskeleton. Previously, we demonstrated that Coracle protein is localized to the septate junction in epithelial cells and is required for septate junction integrity. Loss of coracle function leads to defects in embryonic development, including failure in dorsal closure, and to proliferation defects. In addition, we determined that the N-terminal 383 amino acids define an essential functional domain possessing membrane-organizing properties. Here we investigate the full range of functions provided by this highly conserved domain and find that it is sufficient to rescue all embryonic defects associated with loss of coracle function. In addition, this domain is sufficient to rescue the reduced cell proliferation defect in imaginal discs, although it is incapable of rescuing null mutants to the adult stage. This result suggests the presence of a second functional domain within Coracle, a notion supported by molecular characterization of a series of coracle alleles. PMID:11560899

  5. L’inducteur céphalique Cerberus est un inhibiteur multivalent extracellulaire

    PubMed Central

    Agius, Par Eric; Piccolo, Stefano; De Robertis, E. M.

    2008-01-01

    SUMMARY The head inducer Cerberus is a multivalent extracellular inhibitor Cerberus encodes for a secreted protein which when overexpressed ventrally in a Xenopus embryo induces head differentiation without trunk (Bouwmeester et al., 1996). We have recently shown that Cerberus can bind BMP-4 (Bone Morphogenetic Protein-4), Xnr-1 (Xenopus Nodal-related 1) and Xwnt-8 in the extracellular space (Piccolo et al., 1999). We present here studies showing that Cerberus does not have a receptor nor a dedicated transduction pathway but rather acts as an extracellular inhibitor. Our results suggest that the action of Cerberus in head induction can be explained by an inhibitory activity upstream of the Nodal-related and BMP-4 receptors. In addition, using dominant negative receptor mutants which block both the Xnr-1 and BMP-4 transduction pathways, we show that this double inhibition is sufficient for head induction in ventral mesoderm explants. PMID:10689616

  6. The immediate early gene of canine herpesvirus is transcribed through early and late phases.

    PubMed

    Miyoshi, Masahiro; Okazaki, Katsunori; Takiguchi, Mitsuyoshi; Kida, Hiroshi; Hashimoto, Akira

    2002-07-01

    The immediate early (IE) gene of canine herpesvirus (CHV), homologue of the infected cell protein 4 (ICP4) gene of herpes simplex virus 1, is transcribed as a 4.9kb mRNA during IE phase. The IE gene was further transcribed as a 4.8kb mRNA through early (E) and late (L) phases of productive infection. Transcription of the 4.8kb mRNA initiated from downstream of the TATA box in an intron which was spliced out during IE phase. The reverse transcription-polymerase chain reaction revealed that the IE promoter was turned off during L phase at a permissive temperature. We, thus, propose to redesignate the IE gene of CHV as CICP4 gene. PMID:12185320

  7. Attention Genes

    ERIC Educational Resources Information Center

    Posner, Michael I.; Rothbart, Mary K.; Sheese, Brad E.

    2007-01-01

    A major problem for developmental science is understanding how the cognitive and emotional networks important in carrying out mental processes can be related to individual differences. The last five years have seen major advances in establishing links between alleles of specific genes and the neural networks underlying aspects of attention. These…

  8. Designer Genes.

    ERIC Educational Resources Information Center

    Miller, Judith; Miller, Mark

    1983-01-01

    Genetic technologies may soon help fill some of the most important needs of humanity from food to energy to health care. The research of major designer genes companies and reasons why the initial mad rush for biotechnology has slowed are reviewed. (SR)

  9. Loss of collapsin response mediator protein 4 suppresses dopaminergic neuron death in an 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced mouse model of Parkinson's disease.

    PubMed

    Tonouchi, Aine; Nagai, Jun; Togashi, Kentaro; Goshima, Yoshio; Ohshima, Toshio

    2016-06-01

    Parkinson's disease (PD) is a progressive neurodegenerative disorder that is characterized by the selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNc). Several lines of evidence suggest that neurodegeneration in PD is accelerated by a vicious cycle in which apoptosis in dopaminergic neurons triggers the activation of microglia and harmful inflammatory processes that further amplify neuronal death. Recently, we demonstrated that the deletion of collapsin response mediator protein 4 (CRMP4) suppresses inflammatory responses and cell death in a mouse model of spinal cord injury, leading to improved functional recovery. We thus hypothesized that Crmp4-/- mice may have limited inflammatory responses and a decrease in the loss of SNc dopaminergic neurons in an 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. We observed CRMP4 expression in neurons, astrocytes, and microglia/macrophages following the injection of 25 mg/kg MPTP. We compared the number of dopaminergic neurons and the inflammatory response in SNc between Crmp4+/+ and Crmp4-/- mice after MPTP injection. Limited loss of SNc dopaminergic neurons and decreased activations of microglia and astrocytes were observed in Crmp4-/- mice. These results suggest that CRMP4 is a novel therapeutic target in the treatment of PD patients. We demonstrated that genetic CRMP4 deletion delays a vicious cycle of inflammation and neurodegeneration in a Parkinson's disease mouse model. MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) injection to wild-type mice induces collapsin response mediator protein 4 (CRMP4) up-regulation in neurons, astrocytes, and microglia. CRMP4-deficient mice show reduced inflammation and suppressed dopaminergic neuronal death after MPTP injection. These findings suggest that CRMP4 deletion may be a new therapeutic strategy against Parkinson's diseases. PMID:26991935

  10. Similarities and differences in the structure and function of 4.1G and 4.1R135, two protein 4.1 paralogues expressed in erythroid cells.

    PubMed

    Nunomura, Wataru; Kinoshita, Kengo; Parra, Marilyn; Gascard, Philippe; An, Xiuli; Mohandas, Narla; Takakuwa, Yuichi

    2010-12-01

    Membrane skeletal protein 4.1R is the prototypical member of a family of four highly paralogous proteins that include 4.1G, 4.1N and 4.1B. Two isoforms of 4.1R (4.1R135 and 4.1R80), as well as 4.1G, are expressed in erythroblasts during terminal differentiation, but only 4.1R80 is present in mature erythrocytes. Although the function of 4.1R isoforms in erythroid cells has been well characterized, there is little or no information on the function of 4.1G in these cells. In the present study, we performed detailed characterization of the interaction of 4.1G with various erythroid membrane proteins and the regulation of these interactions by calcium-saturated calmodulin. Like both isoforms of 4.1R, 4.1G bound to band 3, glycophorin C, CD44, p55 and calmodulin. While both 4.1G and 4.1R135 interact with similar affinity with CD44 and p55, there are significant differences in the affinity of their interaction with band 3 and glycophorin C. This difference in affinity is related to the non-conserved N-terminal headpiece region of the two proteins that is upstream of the 30 kDa membrane-binding domain that harbours the binding sites for the various membrane proteins. The headpiece region of 4.1G also contains a high-affinity calcium-dependent calmodulin-binding site that plays a key role in modulating its interaction with various membrane proteins. We suggest that expression of the two paralogues of protein 4.1 with different affinities for band 3 and glycophorin C is likely to play a role in assembly of these two membrane proteins during terminal erythroid differentiation. PMID:20812914

  11. Similarities and differences in the structure and function of 4.1G and 4.1R135, two protein 4.1 paralogs expressed in erythroid cells

    PubMed Central

    Nunomura, Wataru; Kinoshita, Kengo; Parra, Marilyn; Gascard, Philippe; An, Xiuli; Mohandas, Narla; Takakuwa, Yuichi

    2015-01-01

    Summary Membrane skeletal protein 4.1R is the prototypical member of a family of four highly paralogous proteins that include 4.1G, 4.1N and 4.1B. Two isoforms of 4.1R (4.1R135 and 4.1R80) as well as 4.1G are expressed in erythroblasts during terminal differentiation, but only 4.1R80 is present in mature erythrocytes. While the function of 4.1R isoforms in erythroid cells has been well characterized, there is little or no information on the function of 4.1G in these cells. In the present study, we performed detailed characterization of the interaction of 4.1G with various erythroid membrane proteins and the regulation of these interactions by calcium-saturated calmodulin. Like both isoforms of 4.1R, 4.1G bound to band 3, glycophorin C, CD44, p55 and calmodulin. While both 4.1G and 4.1R135 interact with similar affinity with CD44 and p55, there are significant differences in the affinity of their interaction with band 3 and glycophorin C. This difference in affinity is related to the non-conserved N-terminal headpiece region of the two proteins that is upstream of the 30kDa membrane binding domain that harbors the binding sites for the various membrane proteins. The headpiece region of 4.1G also contains a high affinity calcium-dependent calmodulin-binding site that plays a key role in modulating its interaction with various membrane proteins. We suggest that expression of the two paralogs of protein 4.1 with different affinities for band 3 and glycophorin C is likely to play a role in assembly of these two membrane proteins during terminal erythroid differentiation. PMID:20812914

  12. A Bmp/Admp regulatory circuit controls maintenance and regeneration of dorsal-ventral polarity in planarians.

    PubMed

    Gaviño, Michael A; Reddien, Peter W

    2011-02-22

    Animal embryos have diverse anatomy and vary greatly in size. It is therefore remarkable that a common signaling pathway, BMP signaling, controls development of the dorsoventral (DV) axis throughout the Bilateria. In vertebrates, spatially opposed expression of the BMP family proteins Bmp4 and Admp (antidorsalizing morphogenetic protein) can promote restoration of DV pattern following tissue removal. bmp4 orthologs have been identified in all three groups of the Bilateria (deuterostomes, ecdysozoans, and lophotrochozoans). By contrast, the absence of admp orthologs in ecdysozoans such as Drosophila and C. elegans has suggested that a regulatory circuit of oppositely expressed bmp4 and admp genes represents a deuterostome-specific innovation. Here we describe the existence of spatially opposed bmp and admp expression in a protostome. An admp ortholog (Smed-admp) is expressed ventrally and laterally in adult Schmidtea mediterranea planarians, opposing the dorsal-pole expression of Smed-bmp4. Smed-admp is required for regeneration following parasagittal amputation. Furthermore, Smed-admp promotes Smed-bmp4 expression and Smed-bmp4 inhibits Smed-admp expression, generating a regulatory circuit that buffers against perturbations of Bmp signaling. These results suggest that a Bmp/Admp regulatory circuit is a central feature of the Bilateria, used broadly for the establishment, maintenance, and regeneration of the DV axis. PMID:21295483

  13. Genes and Hearing Loss

    MedlinePlus

    ... Meeting Calendar Find an ENT Doctor Near You Genes and Hearing Loss Genes and Hearing Loss Patient ... mutation may only have dystopia canthorum. How Do Genes Work? Genes are a road map for the ...

  14. Hirudo medicinalis: a platform for investigating genes in neural repair.

    PubMed

    Wang, W Z; Emes, R D; Christoffers, K; Verrall, J; Blackshaw, S E

    2005-03-01

    We have used the nervous system of the medicinal leech as a preparation to study the molecular basis of neural repair. The leech central nervous system, unlike mammalian CNS, can regenerate to restore function, and contains identified nerve cells of known function and connectivity. We have constructed subtractive cDNA probes from whole and regenerating ganglia of the ventral nerve cord and have used these to screen a serotonergic Retzius neuron library. This identifies genes that are regulated as a result of axotomy, and are expressed by the Retzius cell. This approach identifies many genes, both novel and known. Many of the known genes identified have homologues in vertebrates, including man. For example, genes encoding thioredoxin (TRX), Rough Endoplasmic Reticulum Protein 1 (RER-1) and ATP synthase are upregulated at 24 h postinjury in leech nerve cord. To investigate the functional role of regulated genes in neuron regrowth we are using microinjection of antisense oligonucleotides in combination with horseradish peroxidase to knock down expression of a chosen gene and to assess regeneration in single neurons in 3-D ganglion culture. As an example of this approach we describe experiments to microinject antisense oligonucleotide to a leech isoform of the structural protein, Protein 4.1. Our approach thus identifies genes regulated at different times after injury that may underpin the intrinsic ability of leech neurons to survive damage, to initiate regrowth programs and to remake functional connections. It enables us to determine the time course of gene expression in the regenerating nerve cord, and to study the effects of gene knockdown in identified neurons regenerating in defined conditions in culture. PMID:16047550

  15. Functional Study of Ectodysplasin-A Mutations Causing Non-Syndromic Tooth Agenesis

    PubMed Central

    Liu, Yang; Liu, Haochen; Zhao, Hongshan; Zhang, Guozhong; Snead, Malcolm L.; Han, Dong; Feng, Hailan

    2016-01-01

    Recent studies have demonstrated that ectodysplasin-A (EDA) mutations are associated with non-syndromic tooth agenesis. Indeed, we were the first to report three novel EDA mutations (A259E, R289C and R334H) in sporadic non-syndromic tooth agenesis. We studied the mechanism linking EDA mutations and non-syndromic tooth agenesis in human embryonic kidney 293T cells and mouse ameloblast-derived LS8 cells transfected with mutant isoforms of EDA. The receptor binding capability of the mutant EDA1 protein was impaired in comparison to wild-type EDA1. Although the non-syndromic tooth agenesis-causing EDA1 mutants possessed residual binding capability, the transcriptional activation of the receptor’s downstream target, nuclear factor κB (NF-κB), was compromised. We also analyzed the changes of selected genes in other signaling pathways, such as WNT and BMP, after EDA mutation. We found that non-syndromic tooth agenesis-causing EDA1 mutant proteins upregulate BMP4 (bone morphogenetic protein 4) mRNA expression and downregulate WNT10A and WNT10B (wingless-type MMTV integration site family member 10A and 10B) mRNA expression. Our results indicated that non-syndromic tooth agenesis causing EDA mutations (A259E, R289C and R334H) were loss-of-function, and suggested that EDA may regulate the expression of WNT10A, WNT10B and BMP4 via NF-κB during tooth development. The results from our study may help to understand the molecular mechanism linking specific EDA mutations with non-syndromic tooth agenesis. PMID:27144394

  16. The Structure of an LIM-Only Protein 4 (LMO4) and Deformed Epidermal Autoregulatory Factor-1 (DEAF1) Complex Reveals a Common Mode of Binding to LMO4

    PubMed Central

    Joseph, Soumya; Kwan, Ann H.; Stokes, Philippa H.; Mackay, Joel P.; Cubeddu, Liza; Matthews, Jacqueline M.

    2014-01-01

    LIM-domain only protein 4 (LMO4) is a widely expressed protein with important roles in embryonic development and breast cancer. It has been reported to bind many partners, including the transcription factor Deformed epidermal autoregulatory factor-1 (DEAF1), with which LMO4 shares many biological parallels. We used yeast two-hybrid assays to show that DEAF1 binds both LIM domains of LMO4 and that DEAF1 binds the same face on LMO4 as two other LMO4-binding partners, namely LIM domain binding protein 1 (LDB1) and C-terminal binding protein interacting protein (CtIP/RBBP8). Mutagenic screening analysed by the same method, indicates that the key residues in the interaction lie in LMO4LIM2 and the N-terminal half of the LMO4-binding domain in DEAF1. We generated a stable LMO4LIM2-DEAF1 complex and determined the solution structure of that complex. Although the LMO4-binding domain from DEAF1 is intrinsically disordered, it becomes structured on binding. The structure confirms that LDB1, CtIP and DEAF1 all bind to the same face on LMO4. LMO4 appears to form a hub in protein-protein interaction networks, linking numerous pathways within cells. Competitive binding for LMO4 therefore most likely provides a level of regulation between those different pathways. PMID:25310299

  17. Phosphorylation of the Plant Immune Regulator RPM1-INTERACTING PROTEIN4 Enhances Plant Plasma Membrane H+-ATPase Activity and Inhibits Flagellin-Triggered Immune Responses in Arabidopsis[OPEN

    PubMed Central

    Lee, DongHyuk; Bourdais, Gildas; Yu, Gang; Robatzek, Silke; Coaker, Gitta

    2015-01-01

    The Pseudomonas syringae effector AvrB targets multiple host proteins during infection, including the plant immune regulator RPM1-INTERACTING PROTEIN4 (RIN4) and RPM1-INDUCED PROTEIN KINASE (RIPK). In the presence of AvrB, RIPK phosphorylates RIN4 at Thr-21, Ser-160, and Thr-166, leading to activation of the immune receptor RPM1. Here, we investigated the role of RIN4 phosphorylation in susceptible Arabidopsis thaliana genotypes. Using circular dichroism spectroscopy, we show that RIN4 is a disordered protein and phosphorylation affects protein flexibility. RIN4 T21D/S160D/T166D phosphomimetic mutants exhibited enhanced disease susceptibility upon surface inoculation with P. syringae, wider stomatal apertures, and enhanced plasma membrane H+-ATPase activity. The plasma membrane H+-ATPase AHA1 is highly expressed in guard cells, and its activation can induce stomatal opening. The ripk knockout also exhibited a strong defect in pathogen-induced stomatal opening. The basal level of RIN4 Thr-166 phosphorylation decreased in response to immune perception of bacterial flagellin. RIN4 Thr166D lines exhibited reduced flagellin-triggered immune responses. Flagellin perception did not lower RIN4 Thr-166 phosphorylation in the presence of strong ectopic expression of AvrB. Taken together, these results indicate that the AvrB effector targets RIN4 in order to enhance pathogen entry on the leaf surface as well as dampen responses to conserved microbial features. PMID:26198070

  18. Benzyl [(11)C]Hippurate as an Agent for Measuring the Activities of Organic Anion Transporter 3 in the Brain and Multidrug Resistance-Associated Protein 4 in the Heart of Mice.

    PubMed

    Kikuchi, Tatsuya; Okamura, Toshimitsu; Okada, Maki; Ogawa, Masanao; Suzuki, Chie; Wakizaka, Hidekatsu; Yui, Joji; Fukumura, Toshimitsu; Gee, Antony D; Zhang, Ming-Rong

    2016-06-23

    Multidrug resistance-associated protein 4 (MRP4) and organic anion transporter 3 (OAT3) mediate the efflux of organic anions from the brain and heart. In this study, we have developed a probe for estimating the activity of these transporters in these tissues using positron emission tomography. Several (11)C-labeled hippuric acid ester derivatives were screened with the expectation that they would be hydrolyzed in situ to form the corresponding (11)C-labeled organic acids in target tissues. Among the compounds screened, benzyl [(11)C]hippurate showed favorable hydrolysis rates and uptake properties in the target tissues of mice. Subsequent evaluation using transporter knockout mice revealed that radioactivity was retained in the brain and heart of Oat3(-/-) and Mrp4(-/-) mice, respectively, compared with that of control mice after the intravenous administration of benzyl [(11)C]hippurate. Benzyl [(11)C]hippurate could therefore be used as a probe for estimating the activities of OAT3 and MRP4 in mouse brain and heart, respectively. PMID:27232368

  19. Phosphorylation of the Plant Immune Regulator RPM1-INTERACTING PROTEIN4 Enhances Plant Plasma Membrane H⁺-ATPase Activity and Inhibits Flagellin-Triggered Immune Responses in Arabidopsis.

    PubMed

    Lee, DongHyuk; Bourdais, Gildas; Yu, Gang; Robatzek, Silke; Coaker, Gitta

    2015-07-01

    The Pseudomonas syringae effector AvrB targets multiple host proteins during infection, including the plant immune regulator RPM1-INTERACTING PROTEIN4 (RIN4) and RPM1-INDUCED PROTEIN KINASE (RIPK). In the presence of AvrB, RIPK phosphorylates RIN4 at Thr-21, Ser-160, and Thr-166, leading to activation of the immune receptor RPM1. Here, we investigated the role of RIN4 phosphorylation in susceptible Arabidopsis thaliana genotypes. Using circular dichroism spectroscopy, we show that RIN4 is a disordered protein and phosphorylation affects protein flexibility. RIN4 T21D/S160D/T166D phosphomimetic mutants exhibited enhanced disease susceptibility upon surface inoculation with P. syringae, wider stomatal apertures, and enhanced plasma membrane H(+)-ATPase activity. The plasma membrane H(+)-ATPase AHA1 is highly expressed in guard cells, and its activation can induce stomatal opening. The ripk knockout also exhibited a strong defect in pathogen-induced stomatal opening. The basal level of RIN4 Thr-166 phosphorylation decreased in response to immune perception of bacterial flagellin. RIN4 Thr166D lines exhibited reduced flagellin-triggered immune responses. Flagellin perception did not lower RIN4 Thr-166 phosphorylation in the presence of strong ectopic expression of AvrB. Taken together, these results indicate that the AvrB effector targets RIN4 in order to enhance pathogen entry on the leaf surface as well as dampen responses to conserved microbial features. PMID:26198070

  20. Compare Gene Profiles

    SciTech Connect

    2014-05-31

    Compare Gene Profiles (CGP) performs pairwise gene content comparisons among a relatively large set of related bacterial genomes. CGP performs pairwise BLAST among gene calls from a set of input genome and associated annotation files, and combines the results to generate lists of common genes, unique genes, homologs, and genes from each genome that differ substantially in length from corresponding genes in the other genomes. CGP is implemented in Python and runs in a Linux environment in serial or parallel mode.

  1. Global gene expression profiling of JMJD6- and JMJD4-depleted mouse NIH3T3 fibroblasts

    PubMed Central

    Hu, Yu-Jie; Imbalzano, Anthony N.

    2016-01-01

    Emerging evidence suggests Jumonji domain-containing proteins are epigenetic regulators in diverse biological processes including cellular differentiation and proliferation. RNA interference-based analyses combined with gene expression profiling can effectively characterize the cellular functions of these enzymes. We found that the depletion of Jumonji domain-containing protein 6 (JMJD6) and its paralog protein Jumonji domain-containing protein 4 (JMJD4) individually by small hairpin RNAs (shRNAs) slowed cell proliferation of mouse NIH3T3 fibroblasts. We subsequently performed gene expression profiling on both JMJD6- and JMJD4-depleted mouse NIH3T3 fibroblasts using the Affymetrix GeneChip Mouse Exon 1.0 ST Array. Here we report the gene profiling datasets along with the experimental procedures. The information can be used to further investigate how JMJD6 and JMJD4 affect gene expression and cellular physiology. PMID:27071056

  2. The effects of nonyl phenoxypolyethoxyl ethanol on cell damage pathway gene expression in SK-NSH cells

    PubMed Central

    Park, Samel; Hwang, Il-woong; Kim, Jin-sheon; Kang, Hyo-chul; Park, Su-Yeon; Gil, Hyo-wook; Song, Ho-yeon; Hong, Sae-yong

    2015-01-01

    Background/Aims: Most pesticide formulations contain both chief and additive ingredients. But, the additives may not have been tested as thoroughly as the chief ingredients. The surfactant, nonyl phenoxypolyethoxylethanol (NP40), is an additive frequently present in pesticide formulations. We investigated the effects of NP40 and other constituents of a validamycin pesticide formulation on cell viability and on the expression of genes involved in cell damage pathways. Methods: The effects of validamycin pesticide ingredients on cell viability and of NP40 on the mRNA expression of 80 genes involved in nine key cellular pathways were examined in the human neuroblastoma SK-N-SH cell line. Results: The chemicals present in the validamycin pesticide formulation were cytotoxic to SK-N-SH cells and NP40 showed the greatest cytotoxicity. A range of gene expression changes were identified, with both up- and down-regulation of genes within the same pathway. However, all genes tested in the necrosis signaling pathway were down-regulated and all genes tested in the cell cycle checkpoint/arrest pathway were up-regulated. The median fold-change in gene expression was significantly higher in the cell cycle checkpoint/arrest pathway than in the hypoxia pathway category (p = 0.0064). The 70 kDa heat shock protein 4 gene, within the heat shock protein/unfolded protein response category, showed the highest individual increase in expression (26.1-fold). Conclusions: NP40 appeared to be particularly harmful, inducing gene expression changes that indicated genotoxicity, activation of the cell death (necrosis signaling) pathway, and induction of the 70 kDa heat shock protein 4 gene. PMID:26552463

  3. Gene gymnastics

    PubMed Central

    Vijayachandran, Lakshmi S; Thimiri Govinda Raj, Deepak B; Edelweiss, Evelina; Gupta, Kapil; Maier, Josef; Gordeliy, Valentin; Fitzgerald, Daniel J; Berger, Imre

    2013-01-01

    Most essential activities in eukaryotic cells are catalyzed by large multiprotein assemblies containing up to ten or more interlocking subunits. The vast majority of these protein complexes are not easily accessible for high resolution studies aimed at unlocking their mechanisms, due to their low cellular abundance and high heterogeneity. Recombinant overproduction can resolve this bottleneck and baculovirus expression vector systems (BEVS) have emerged as particularly powerful tools for the provision of eukaryotic multiprotein complexes in high quality and quantity. Recently, synthetic biology approaches have begun to make their mark in improving existing BEVS reagents by de novo design of streamlined transfer plasmids and by engineering the baculovirus genome. Here we present OmniBac, comprising new custom designed reagents that further facilitate the integration of heterologous genes into the baculovirus genome for multiprotein expression. Based on comparative genome analysis and data mining, we herein present a blueprint to custom design and engineer the entire baculovirus genome for optimized production properties using a bottom-up synthetic biology approach. PMID:23328086

  4. Protein 4.1N is required for translocation of inositol 1,4,5-trisphosphate receptor type 1 to the basolateral membrane domain in polarized Madin-Darby canine kidney cells.

    PubMed

    Zhang, Songbai; Mizutani, Akihiro; Hisatsune, Chihiro; Higo, Takayasu; Bannai, Hiroko; Nakayama, Tomohiro; Hattori, Mitsuharu; Mikoshiba, Katsuhiko

    2003-02-01

    Protein 4.1N was identified as a binding molecule for the C-terminal cytoplasmic tail of inositol 1,4,5-trisphosphate receptor type 1 (IP(3)R1) using a yeast two-hybrid system. 4.1N and IP(3)R1 associate in both subconfluent and confluent Madin-Darby canine kidney (MDCK) cells, a well studied tight polarized epithelial cell line. In subconfluent MDCK cells, 4.1N is distributed in the cytoplasm and the nucleus; IP(3)R1 is localized in the cytoplasm. In confluent MDCK cells, both 4.1N and IP(3)R1 are predominantly translocated to the basolateral membrane domain, whereas 4.1R, the prototypical homologue of 4.1N, is localized at the tight junctions (Mattagajasingh, S. N., Huang, S. C., Hartenstein, J. S., and Benz, E. J., Jr. (2000) J. Biol. Chem. 275, 30573-30585), and other endoplasmic reticulum marker proteins are still present in the cytoplasm. Moreover, the 4.1N-binding region of IP(3)R1 is necessary and sufficient for the localization of IP(3)R1 at the basolateral membrane domain. A fragment of the IP(3)R1-binding region of 4.1N blocks the localization of co-expressed IP(3)R1 at the basolateral membrane domain. These data indicate that 4.1N is required for IP(3)R1 translocation to the basolateral membrane domain in polarized MDCK cells. PMID:12444087

  5. Expression levels of multidrug resistance-associated protein 4 (MRP4) in human leukemia and lymphoma cell lines, and the inhibitory effects of the MRP-specific inhibitor MK-571 on methotrexate distribution in rats

    PubMed Central

    TAKEUCHI, KENJI; SHIBATA, MASAKAZU; KASHIYAMA, EIJI; UMEHARA, KEN

    2012-01-01

    In the development of anti-blood cancer drugs, the chronic myelocytic leukemia (KU812), acute myelocytic leukemia (KG-1) and lymphoma (U937) cell lines are commonly used in preclinical pharmacology studies as human cancer xenograft models in mice. In the present study, mRNA expression levels of typical human ATP-binding cassette (ABC) transporters in these human blood cancer cell lines were analyzed by real-time polymerase chain reaction (RT-PCR). Based on the results, the expression level of multidrug resistance-associated protein 4 (MRP4) was found to be extremely high in KU812 cells compared with those of other transporters. Additionally, MRP4 expression levels were found to be relatively high in U937, KG-1 and a blood cell line derived from a healthy subject (RPMI 1788). In addition, to elucidate the contribution of MRP4 to the methotrexate (MTX) distribution in normal blood cells and tissues, [3H]MTX was intravenously (i.v.) administered to two groups of rats. Animals in one group received [3H]MTX only; the other group was concomitantly administered i.v. MK-571, a typical inhibitor of MRP transporters. No marked difference was observed between the two groups; the Kp values (tissue concentration/plasma concentration) of the concomitant group showed slightly higher values compared with those of the MTX alone group in erythrocytes (1.4 times, P<0.001), spleen (1.3 times, P<0.05) and thymus (1.2 times, P<0.05), respectively. Although in the present study we could not evaluate the direct involvement of MRP4 in blood cancer cells in which MRP4 expression was excessively high, these results suggest a possible functional role of MRP4 in blood cancer cells and albeit only slightly in normal blood cells/tissues. PMID:23181130

  6. Complex regulation of voltage-dependent activation and inactivation properties of retinal voltage-gated Cav1.4 L-type Ca2+ channels by Ca2+-binding protein 4 (CaBP4).

    PubMed

    Shaltiel, Lior; Paparizos, Christos; Fenske, Stefanie; Hassan, Sami; Gruner, Christian; Rötzer, Katrin; Biel, Martin; Wahl-Schott, Christian A

    2012-10-19

    Cav1.4 L-type Ca(2+) channels are crucial for synaptic transmission in retinal photoreceptors and bipolar neurons. Recent studies suggest that the activity of this channel is regulated by the Ca(2+)-binding protein 4 (CaBP4). In the present study, we explored this issue by examining functional effects of CaBP4 on heterologously expressed Cav1.4. We show that CaBP4 dramatically increases Cav1.4 channel availability. This effect crucially depends on the presence of the C-terminal ICDI (inhibitor of Ca(2+)-dependent inactivation) domain of Cav1.4 and is absent in a Cav1.4 mutant lacking the ICDI. Using FRET experiments, we demonstrate that CaBP4 interacts with the IQ motif of Cav1.4 and that it interferes with the binding of the ICDI domain. Based on these findings, we suggest that CaBP4 increases Cav1.4 channel availability by relieving the inhibitory effects of the ICDI domain on voltage-dependent Cav1.4 channel gating. We also functionally characterized two CaBP4 mutants that are associated with a congenital variant of human night blindness and other closely related nonstationary retinal diseases. Although both mutants interact with Cav1.4 channels, the functional effects of CaBP4 mutants are only partially preserved, leading to a reduction of Cav1.4 channel availability and loss of function. In conclusion, our study sheds new light on the functional interaction between CaBP4 and Cav1.4. Moreover, it provides insights into the mechanism by which CaBP4 mutants lead to loss of Cav1.4 function and to retinal disease. PMID:22936811

  7. Retinol-Binding Protein 4 Inhibits Insulin Signaling in Adipocytes by Inducing Proinflammatory Cytokines in Macrophages through a c-Jun N-Terminal Kinase- and Toll-Like Receptor 4-Dependent and Retinol-Independent Mechanism

    PubMed Central

    Norseen, Julie; Hosooka, Tetsuya; Hammarstedt, Ann; Yore, Mark M.; Kant, Shashi; Aryal, Pratik; Kiernan, Urban A.; Phillips, David A.; Maruyama, Hiroshi; Kraus, Bettina J.; Usheva, Anny; Davis, Roger J.; Smith, Ulf

    2012-01-01

    Retinol-binding protein 4 (RBP4), the sole retinol transporter in blood, is secreted from adipocytes and liver. Serum RBP4 levels correlate highly with insulin resistance, other metabolic syndrome factors, and cardiovascular disease. Elevated serum RBP4 causes insulin resistance, but the molecular mechanisms are unknown. Here we show that RBP4 induces expression of proinflammatory cytokines in mouse and human macrophages and thereby indirectly inhibits insulin signaling in cocultured adipocytes. This occurs through activation of c-Jun N-terminal protein kinase (JNK) and Toll-like receptor 4 (TLR4) pathways independent of the RBP4 receptor, STRA6. RBP4 effects are markedly attenuated in JNK1−/− JNK2−/− macrophages and TLR4−/− macrophages. Because RBP4 is a retinol-binding protein, we investigated whether these effects are retinol dependent. Unexpectedly, retinol-free RBP4 (apo-RBP4) is as potent as retinol-bound RBP4 (holo-RBP4) in inducing proinflammatory cytokines in macrophages. Apo-RBP4 is likely to be physiologically significant since RBP4/retinol ratios are increased in serum of lean and obese insulin-resistant humans compared to ratios in insulin-sensitive humans, indicating that higher apo-RBP4 is associated with insulin resistance independent of obesity. Thus, RBP4 may cause insulin resistance by contributing to the development of an inflammatory state in adipose tissue through activation of proinflammatory cytokines in macrophages. This process reveals a novel JNK- and TLR4-dependent and retinol- and STRA6-independent mechanism of action for RBP4. PMID:22431523

  8. Effect of Diabetes Mellitus on Adipocyte-Derived Stem Cells in Rat.

    PubMed

    Jumabay, Medet; Moon, Jeremiah H; Yeerna, Huwate; Boström, Kristina I

    2015-11-01

    Diabetes mellitus affects the adipose tissue and mesenchymal stem cells derived from the adipose stroma and other tissues. Previous reports suggest that bone morphogenetic protein 4 (BMP4) is involved in diabetic complications, at the same time playing an important role in the maintenance of stem cells. In this study, we used rats transgenic for human islet amyloid polypeptide (HIP rats), a model of type 2 diabetes, to study the effect of diabetes on adipocyte-derived stem cells, referred to as dedifferentiated fat (DFAT) cells. Our results show that BMP4 expression in inguinal adipose tissue is significantly increased in HIP rats compared to controls, whereas matrix Gla protein (MGP), an inhibitor of BMP4 is decreased as determined by quantitative PCR, and immunofluorescence. In addition, adipose vascularity and expression of multiple endothelial cell markers was increased in the diabetic tissue, visualized by immunofluorescence for endothelial markers. The endothelial markers co-localized with the enhanced BMP4 expression, suggesting that vascular cells play a role BMP4 induction. The DFAT cells are multipotent stem cells derived from white mature adipocytes that undergo endothelial and adipogenic differentiation. DFAT cells prepared from the inguinal adipose tissue in HIP rats exhibited enhanced proliferative capacity compared to wild type. In addition, their ability to undergo both endothelial cell and adipogenic lineage differentiation was enhanced, as well as their response to BMP4, as assessed by lineage marker expression. We conclude that the DFAT cells are affected by diabetic changes and may contribute to the adipose dysfunction in diabetes. PMID:25854185

  9. Data Mining in Networks of Differentially Expressed Genes during Sow Pregnancy

    PubMed Central

    Wang, Ligang; Zhang, Longchao; Li, Yong; Li, Wen; Luo, Weizhen; Cheng, Duxue; Yan, Hua; Ma, Xiaojun; Liu, Xin; Song, Xin; Liang, Jing; Zhao, Kebin; Wang, Lixian

    2012-01-01

    Small to moderate gains in Pig fertility can mean large returns in overall efficiency, and developing methods to improve it is highly desirable. High fertility rates depend on completion of successful pregnancies. To understand the molecular signals associated with pregnancy in sows, expression profiling experiments were conducted to identify differentially expressed genes in ovary and myometrium at different pregnancy periods using the Affymetrix Porcine GeneChipTM. A total of 974, 1800, 335 and 710 differentially expressed transcripts were identified in the myometrium during early pregnancy (EP) and late pregnancy (LP), and in the ovary during EP and LP, respectively. Self-Organizing Map (SOM) clusters indicated the differentially expressed genes belonged to 7 different functional groups. Based on BLASTX searches and Gene Ontology (GO) classifications, 129 unique genes closely related to pregnancy showed differential expression patterns. GO analysis also indicated that there were 21 different molecular function categories, 20 different biological process categories, and 8 different cellular component categories of genes differentially expressed during sow pregnancy. Gene regulatory network reconstruction provided us with an interaction model of known genes such as insulin-like growth factor 2 (IGF2) gene, estrogen receptor (ESR) gene, retinol-binding protein-4 (RBP4) gene, and several unknown candidate genes related to reproduction. Several pitch point genes were selected for association study with reproduction traits. For instance, DPPA5 g.363 T>C was found to associate with litter born weight at later parities in Beijing Black pigs significantly (p < 0.05). Overall, this study contributes to elucidating the mechanism underlying pregnancy processes, which maybe provide valuable information for pig reproduction improvement. PMID:22532788

  10. Gene doping: gene delivery for olympic victory

    PubMed Central

    Gould, David

    2013-01-01

    With one recently recommended gene therapy in Europe and a number of other gene therapy treatments now proving effective in clinical trials it is feasible that the same technologies will soon be adopted in the world of sport by unscrupulous athletes and their trainers in so called ‘gene doping’. In this article an overview of the successful gene therapy clinical trials is provided and the potential targets for gene doping are highlighted. Depending on whether a doping gene product is secreted from the engineered cells or is retained locally to, or inside engineered cells will, to some extent, determine the likelihood of detection. It is clear that effective gene delivery technologies now exist and it is important that detection and prevention plans are in place. PMID:23082866

  11. CMKLR1 deficiency maintains ovarian steroid production in mice treated chronically with dihydrotestosterone

    PubMed Central

    Tang, Mi; Huang, Chen; Wang, Yu-Fei; Ren, Pei-Gen; Chen, Li; Xiao, Tian-Xia; Wang, Bao-Bei; Pan, Yan-Fei; Tsang, Benjamin K.; Zabel, Brian A; Ma, Bao-Hua; Zhao, Hui-Ying; Zhang, Jian V.

    2016-01-01

    Elevated serum chemerin levels correlate with increased severity of polycystic ovary syndrome (PCOS). However, the role of CMKLR1 signaling in ovarian biology under conditions of excess DHT remains unclear. In this study we compared the effects of continuous 90-day high dose DHT exposure (83.3 □g/day) on wild type and CMKLR1-deficient mice. DHT induced PCOS-like clinical signs in wild type mice as well as significant changes in the expression of hormone receptors, steroid synthesis enzymes, and BMPs and their receptors. In contrast, CMKLR1-deficient mice significantly attenuated DHT-induced clinical signs of PCOS and alterations in ovarian gene expression. To determine whether the BMP4 signaling pathway was involved in the pathogenic effects of CMKLR1 signaling in DHT-induced ovarian steroidogenesis, antral follicles were isolated from wild type and CMKLR1 knockout (KO) mice and treated in vitro with combinations of hCG, DHT, and BMP4 inhibitors. BMP4 inhibition attenuated the induction effects of hCG and DHT on estrogen and progesterone secretion in CMKLR1 KO mice, but not in WT mice, implicating the BMP4 signaling pathway in the CMKLR1-dependent response to DHT. In conclusion, CMKLR1 gene deletion attenuates the effects of chronic DHT treatment on ovarian function in experimental PCOS, likely via BMP4 signaling. PMID:26893072

  12. Rad53 homologue forkhead-associated kinase A (FhkA) and Ca2+-binding protein 4a (CBP4a) are nucleolar proteins that differentially redistribute during mitosis in Dictyostelium

    PubMed Central

    2013-01-01

    Background During mitosis most nucleolar proteins redistribute to other locales providing an opportunity to study the relationship between nucleolar protein localization and function. Dictyostelium is a model organism for the study of several fundamental biological processes and human diseases but only two nucleolar proteins have been studied during mitosis: NumA1 and Snf12. Both of them are linked to the cell cycle. To acquire a better understanding of nucleolar protein localization and dynamics in Dictyostelium we studied the nucleolar localization of two additional proteins during mitosis: Snf12-linked forkhead-associated kinase A (FhkA), which is involved in the cell cycle, and Ca2+-binding protein 4a (CBP4a), which is a binding partner of NumA1. Methods Polyclonal antibodies were produced in-house. Cells were fixed and probed with either anti-FhkA or anti-CBP4a in order to determine cellular localization during interphase and throughout the stages of mitosis. Colocalization with DAPI nuclear stain allowed us to determine the location of the nucleus and nucleolus while colocalization with anti-α-tubulin allowed us to determine the cell cycle stage. Results Here we verify two novel nucleolar proteins, Rad53 homologue FhkA which localized around the edge of the nucleolus and CBP4a which was detected throughout the entire nucleolus. Treatment with the Ca2+ chelator BAPTA (5 mM) showed that the nucleolar localization of CBP4a is Ca2+-dependent. In response to actinomycin D (0.05 mg/mL) CBP4a disappeared from the nucleolus while FhkA protruded from the nucleus, eventually pinching off as cytoplasmic circles. FhkA and CBP4a redistributed differently during mitosis. FhkA redistributed throughout the entire cell and at the nuclear envelope region from prometaphase through telophase. In contrast, during prometaphase CBP4a relocated to many large, discrete “CBP4a islands” throughout the nucleoplasm. Two larger “CBP4a islands” were also detected specifically

  13. Isoforms of protein 4.1 are differentially distributed in heart muscle cells: relation of 4.1R and 4.1G to components of the Ca2+ homeostasis system.

    PubMed

    Pinder, Jennifer C; Taylor-Harris, Pamela M; Bennett, Pauline M; Carter, Edward; Hayes, Nandini V L; King, Mikayala D A; Holt, Mark R; Maggs, Alison M; Gascard, Philippe; Baines, Anthony J

    2012-08-01

    The 4.1 proteins are cytoskeletal adaptor proteins that are linked to the control of mechanical stability of certain membranes and to the cellular accumulation and cell surface display of diverse transmembrane proteins. One of the four mammalian 4.1 proteins, 4.1R (80 kDa/120 kDa isoforms), has recently been shown to be required for the normal operation of several ion transporters in the heart (Stagg MA et al. Circ Res, 2008; 103: 855-863). The other three (4.1G, 4.1N and 4.1B) are largely uncharacterised in the heart. Here, we use specific antibodies to characterise their expression, distribution and novel activities in the left ventricle. We detected 4.1R, 4.1G and 4.1N by immunofluorescence and immunoblotting, but not 4.1B. Only one splice variant of 4.1N and 4.1G was seen whereas there are several forms of 4.1R. 4.1N, like 4.1R, was present in intercalated discs, but unlike 4.1R, it was not localised at the lateral plasma membrane. Both 4.1R and 4.1N were in internal structures that, at the level of resolution of the light microscope, were close to the Z-disc (possibly T-tubules). 4.1G was also in intracellular structures, some of which were coincident with sarcoplasmic reticulum. 4.1G existed in an immunoprecipitable complex with spectrin and SERCA2. 80 kDa 4.1R was present in subcellular fractions enriched in intercalated discs, in a complex resistant to solubilization under non-denaturing conditions. At the intercalated disc 4.1R does not colocalise with the adherens junction protein, β-catenin, but does overlap with the other plasma membrane signalling proteins, the Na/K-ATPase and the Na/Ca exchanger NCX1. We conclude that isoforms of 4.1 proteins are differentially compartmentalised in the heart, and that they form specific complexes with proteins central to cardiomyocyte Ca(2+) metabolism. PMID:22429617

  14. Autism and Genes

    ERIC Educational Resources Information Center

    National Institutes of Health, 2005

    2005-01-01

    This document defines and discusses autism and how genes play a role in the condition. Answers to the following questions are covered: (1) What are genes? (2) What is autism? (3) What causes autism? (4) Why study genes to learn about autism? (5) How do researchers look for the genes involved in autism? (screen the whole genome; conduct cytogenetic…

  15. Compare Gene Profiles

    Energy Science and Technology Software Center (ESTSC)

    2014-05-31

    Compare Gene Profiles (CGP) performs pairwise gene content comparisons among a relatively large set of related bacterial genomes. CGP performs pairwise BLAST among gene calls from a set of input genome and associated annotation files, and combines the results to generate lists of common genes, unique genes, homologs, and genes from each genome that differ substantially in length from corresponding genes in the other genomes. CGP is implemented in Python and runs in a Linuxmore » environment in serial or parallel mode.« less

  16. Comparative analysis of zebrafish bone morphogenetic proteins 2, 4 and 16: molecular and evolutionary perspectives.

    PubMed

    Marques, Cátia L; Fernández, Ignacio; Viegas, Michael N; Cox, Cymon J; Martel, Paulo; Rosa, Joana; Cancela, M Leonor; Laizé, Vincent

    2016-02-01

    BMP2, BMP4 and BMP16 form a subfamily of bone morphogenetic proteins acting as pleiotropic growth factors during development and as bone inducers during osteogenesis. BMP16 is the most recent member of this subfamily and basic data regarding protein structure and function, and spatio-temporal gene expression is still scarce. In this work, insights on BMP16 were provided through the comparative analysis of structural and functional data for zebrafish BMP2a, BMP2b, BMP4 and BMP16 genes and proteins, determined from three-dimensional models, patterns of gene expression during development and in adult tissues, regulation by retinoic acid and capacity to activate BMP-signaling pathway. Structures of Bmp2a, Bmp2b, Bmp4 and Bmp16 were found to be remarkably similar; with residues involved in receptor binding being highly conserved. All proteins could activate the BMP-signaling pathway, suggesting that they share a common function. On the contrary, stage- and tissue-specific expression of bmp2, bmp4 and bmp16 suggested the genes might be differentially regulated (e.g. different transcription factors, enhancers and/or regulatory modules) but also that they are involved in distinct physiological processes, although with the same function. Retinoic acid, a morphogen known to interact with BMP-signaling during bone formation, was shown to down-regulate the expression of bmp2, bmp4 and bmp16, although to different extents. Taxonomic and phylogenetic analyses indicated that bmp16 diverged before bmp2 and bmp4, is not restricted to teleost fish lineage as previously reported, and that it probably arose from a whole genomic duplication event that occurred early in vertebrate evolution and disappeared in various tetrapod lineages through independent events. PMID:26341094

  17. Evolution by gene loss.

    PubMed

    Albalat, Ricard; Cañestro, Cristian

    2016-07-01

    The recent increase in genomic data is revealing an unexpected perspective of gene loss as a pervasive source of genetic variation that can cause adaptive phenotypic diversity. This novel perspective of gene loss is raising new fundamental questions. How relevant has gene loss been in the divergence of phyla? How do genes change from being essential to dispensable and finally to being lost? Is gene loss mostly neutral, or can it be an effective way of adaptation? These questions are addressed, and insights are discussed from genomic studies of gene loss in populations and their relevance in evolutionary biology and biomedicine. PMID:27087500

  18. Human Gene Therapy: Genes without Frontiers?

    ERIC Educational Resources Information Center

    Simon, Eric J.

    2002-01-01

    Describes the latest advancements and setbacks in human gene therapy to provide reference material for biology teachers to use in their science classes. Focuses on basic concepts such as recombinant DNA technology, and provides examples of human gene therapy such as severe combined immunodeficiency syndrome, familial hypercholesterolemia, and…

  19. Gene Expression Dynamics During Bone Healing and Osseointegration

    PubMed Central

    Lin, Zhao; Rios, Hector F.; Volk, Sarah L.; Sugai, James V.; Jin, Qiming; Giannobile, William V.

    2012-01-01

    Background Understanding the molecular features of bone repair and osseointegration may aid in the development of therapeutics to improve implant outcomes. The purpose of this investigation is to determine the gene expression dynamics during alveolar bone repair and implant osseointegration. Methods An implant osseointegration preclinical animal model was used whereby maxillary defects were created at the time of oral implant placement, while a tooth extraction socket healing model was established on the contralateral side of each animal. The surrounding tissues in the zone of the healing defects were harvested during regeneration for temporal evaluation using histology, immunohistochemistry, laser capture microdissection, and quantitative reverse transcription–polymerase chain reaction for the identification of a panel of 17 putative genes associated with wound repair. Results In both models, three distinct expression patterns were displayed: 1) genes that are slowly increased during the healing process, such as bone morphogenetic protein 4, runt-related transcription factor 2, and osteocalcin; 2) genes that are upregulated at the early stage of healing and then downregulated at later stages, such as interleukin and chemokine (C-X-C motif) ligands 2 and 5; and 3) genes that are constitutively expressed over time, such as scleraxis. Although some similarities between osseointegration and tooth extraction socket were seen, distinct features developed and triggered a characteristic coordinated expression and orchestration of transcription factors, growth factors, extracellular matrix molecules, and chemokines. Conclusions Characterization of these events contributes to a better understanding of cooperative molecular dynamics in alveolar bone healing, and highlights potential pathways that could be further explored for the enhancement of osseous regenerative strategies. PMID:21142982

  20. Evolution of gene expression after gene amplification.

    PubMed

    Garcia, Nelson; Zhang, Wei; Wu, Yongrui; Messing, Joachim

    2015-05-01

    We took a rather unique approach to investigate the conservation of gene expression of prolamin storage protein genes across two different subfamilies of the Poaceae. We took advantage of oat plants carrying single maize chromosomes in different cultivars, called oat-maize addition (OMA) lines, which permitted us to determine whether regulation of gene expression was conserved between the two species. We found that γ-zeins are expressed in OMA7.06, which carries maize chromosome 7 even in the absence of the trans-acting maize prolamin-box-binding factor (PBF), which regulates their expression. This is likely because oat PBF can substitute for the function of maize PBF as shown in our transient expression data, using a γ-zein promoter fused to green fluorescent protein (GFP). Despite this conservation, the younger, recently amplified prolamin genes in maize, absent in oat, are not expressed in the corresponding OMAs. However, maize can express the oldest prolamin gene, the wheat high-molecular weight glutenin Dx5 gene, even when maize Pbf is knocked down (through PbfRNAi), and/or another maize transcription factor, Opaque-2 (O2) is knocked out (in maize o2 mutant). Therefore, older genes are conserved in their regulation, whereas younger ones diverged during evolution and eventually acquired a new repertoire of suitable transcriptional activators. PMID:25912045

  1. Evolution of Gene Expression after Gene Amplification

    PubMed Central

    Garcia, Nelson; Zhang, Wei; Wu, Yongrui; Messing, Joachim

    2015-01-01

    We took a rather unique approach to investigate the conservation of gene expression of prolamin storage protein genes across two different subfamilies of the Poaceae. We took advantage of oat plants carrying single maize chromosomes in different cultivars, called oat–maize addition (OMA) lines, which permitted us to determine whether regulation of gene expression was conserved between the two species. We found that γ-zeins are expressed in OMA7.06, which carries maize chromosome 7 even in the absence of the trans-acting maize prolamin-box-binding factor (PBF), which regulates their expression. This is likely because oat PBF can substitute for the function of maize PBF as shown in our transient expression data, using a γ-zein promoter fused to green fluorescent protein (GFP). Despite this conservation, the younger, recently amplified prolamin genes in maize, absent in oat, are not expressed in the corresponding OMAs. However, maize can express the oldest prolamin gene, the wheat high-molecular weight glutenin Dx5 gene, even when maize Pbf is knocked down (through PbfRNAi), and/or another maize transcription factor, Opaque-2 (O2) is knocked out (in maize o2 mutant). Therefore, older genes are conserved in their regulation, whereas younger ones diverged during evolution and eventually acquired a new repertoire of suitable transcriptional activators. PMID:25912045

  2. Reading and Generalist Genes

    ERIC Educational Resources Information Center

    Haworth, Claire M. A.; Meaburn, Emma L.; Harlaar, Nicole; Plomin, Robert

    2007-01-01

    Twin-study research suggests that many (but not all) of the same genes contribute to genetic influence on diverse learning abilities and disabilities, a hypothesis called "generalist genes". This generalist genes hypothesis was tested using a set of 10 DNA markers (single nucleotide polymorphisms [SNPs]) found to be associated with early reading…

  3. Accepting Foreign Genes.

    PubMed

    Boto, Luis

    2016-05-01

    Three recent papers underline the importance of the host genomic background in allowing the stable maintenance of horizontally acquired genes. These studies suggest that post-transfer changes in both host genome and acquired genes contribute to the stable integration of foreign genes. PMID:27075565

  4. Rampant Gene Loss in the Underground Orchid Rhizanthella gardneri Highlights Evolutionary Constraints on Plastid Genomes

    PubMed Central

    Delannoy, Etienne; Fujii, Sota; Colas des Francs-Small, Catherine; Brundrett, Mark; Small, Ian

    2011-01-01

    Since the endosymbiotic origin of chloroplasts from cyanobacteria 2 billion years ago, the evolution of plastids has been characterized by massive loss of genes. Most plants and algae depend on photosynthesis for energy and have retained ∼110 genes in their chloroplast genome that encode components of the gene expression machinery and subunits of the photosystems. However, nonphotosynthetic parasitic plants have retained a reduced plastid genome, showing that plastids have other essential functions besides photosynthesis. We sequenced the complete plastid genome of the underground orchid, Rhizanthella gardneri. This remarkable parasitic subterranean orchid possesses the smallest organelle genome yet described in land plants. With only 20 proteins, 4 rRNAs, and 9 tRNAs encoded in 59,190 bp, it is the least gene-rich plastid genome known to date apart from the fragmented plastid genome of some dinoflagellates. Despite numerous differences, striking similarities with plastid genomes from unrelated parasitic plants identify a minimal set of protein-encoding and tRNA genes required to reside in plant plastids. This prime example of convergent evolution implies shared selective constraints on gene loss or transfer. PMID:21289370

  5. Bypassing Iron Storage in Endodermal Vacuoles Rescues the Iron Mobilization Defect in the natural resistance associated-macrophage protein3natural resistance associated-macrophage protein4 Double Mutant.

    PubMed

    Mary, Viviane; Schnell Ramos, Magali; Gillet, Cynthia; Socha, Amanda L; Giraudat, Jérôme; Agorio, Astrid; Merlot, Sylvain; Clairet, Colin; Kim, Sun A; Punshon, Tracy; Guerinot, Mary Lou; Thomine, Sébastien

    2015-09-01

    To improve seed