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1.
Biochem Biophys Res Commun ; 547: 29-35, 2021 04 02.
Article in English | MEDLINE | ID: mdl-33592376

ABSTRACT

Brown adipose tissue (BAT) and stimulating adaptive thermogenesis have been implicated as anti-obese and anti-diabetic tissues due to their ability to dissipate energy as heat by the expression of UCP1. We have recently demonstrated that TRB3 impairs differentiation of brown preadipocytes via inhibiting insulin signaling. However, the roles of the protein in BAT function and thermogenesis in vivo have not yet been established. For this study we tested the hypothesis that TRB3 mediates obesity- and diabetes-induced impairments in BAT differentiation and function, and that inhibition of TRB3 improves BAT function. TRB3 expression was increased in BAT from high-fat fed mice and ob/ob mice, which was associated with decreased UCP1 expression. Incubation of brown adipocytes with palmitate increased TRB3 expression and decreased UCP1. Knockout of TRB3 in mice displayed higher UCP1 expression in BAT and cold resistance. Incubation of brown adipocytes with ER stressors increased TRB3 but decreased UCP1 and ER stress markers were elevated in BAT from high-fat fed mice and ob/ob mice. Finally, high-fat feeding in TRB3KO mice were protected from obesity-induced glucose intolerance and displayed cold resistance and higher expression of BAT-specific markers. These data demonstrate that high-fat feeding and obesity increase TRB3 in BAT, resulting in impaired tissue function.


Subject(s)
Adipose Tissue, Brown/metabolism , Cell Cycle Proteins/metabolism , Obesity/metabolism , Uncoupling Protein 1/metabolism , Adipose Tissue, Brown/pathology , Adipose Tissue, Brown/physiology , Animals , Cells, Cultured , Diet, High-Fat , Disease Models, Animal , Endoplasmic Reticulum Stress , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Obese , Obesity/pathology , Signal Transduction , Thermogenesis
2.
Biochem Biophys Res Commun ; 519(3): 633-638, 2019 11 12.
Article in English | MEDLINE | ID: mdl-31540695

ABSTRACT

AMP-activated protein kinase (AMPK) is a member of Ser/Thr kinases that has been shown to regulate energy balance. Although recent studies have demonstrated the function of AMPK in adipose tissue using different fat-specific AMPK knockout mouse models, the results were somewhat inconsistent. For this study, we tested the hypothesis that AMPK in adipose tissue regulates whole body glucose metabolism. To determine the role of adipose tissue AMPK in vivo, we generated fat-specific AMPKα1/α2 knockout mice (AMPKFKO) using the Cre-loxP system. Body weights of AMPKFKO mice were not different between 8 and 27 weeks of age. Furthermore, tissue weights (liver, kidney, muscle, heart and white and brown adipose tissue) were similar to wild type littermates and DEXA scan analysis revealed no differences in percentages of body fat and lean mass. Knockout of AMPKα1/α2 in adipose tissue abolished basal and AICAR-stimulated phosphorylation of AMPK and Acetyl-CoA Carboxylase, a downstream of AMPK. Despite of the ablation of AICAR-stimulated AMPK phosphorylation, the blood glucose-lowering effect of AICAR injection (i.p.) was normal in AMPKFKO mice. In addition, AMPKFKO displayed normal fasting blood glucose concentration, glucose tolerance, insulin tolerance, and insulin signaling, indicating normal whole body glucose metabolism. These data demonstrate that adipose tissue AMPK plays a minimum role in whole body glucose metabolism on a chow diet.


Subject(s)
AMP-Activated Protein Kinases/metabolism , Adipose Tissue/metabolism , Aminoimidazole Carboxamide/analogs & derivatives , Glucose/metabolism , Ribonucleotides/metabolism , AMP-Activated Protein Kinases/deficiency , Aminoimidazole Carboxamide/administration & dosage , Aminoimidazole Carboxamide/metabolism , Animals , Diet , Injections, Intraperitoneal , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Ribonucleotides/administration & dosage
3.
Biochem Biophys Res Commun ; 493(3): 1236-1242, 2017 11 25.
Article in English | MEDLINE | ID: mdl-28962861

ABSTRACT

Skeletal muscle atrophy is associated with a disruption in protein turnover involving increased protein degradation and suppressed protein synthesis. Although it has been well studied that the IGF-1/PI3K/Akt pathway plays an essential role in the regulation of the protein turnover, molecule(s) that triggers the change in protein turnover still remains to be elucidated. TRB3 has been shown to inhibit Akt through direct binding. In this study, we hypothesized that TRB3 in mouse skeletal muscle negatively regulates protein turnover via the disruption of Akt and its downstream molecules. Muscle-specific TRB3 transgenic (TRB3TG) mice had decreased muscle mass and fiber size, resulting in impaired muscle function. We also found that protein synthesis rate and signaling molecules, mTOR and S6K1, were significantly reduced in TRB3TG mice, whereas the protein breakdown pathway was significantly activated. In contrast, TRB3 knockout mice showed increased muscle mass and had an increase in protein synthesis rate, but decreases in FoxOs, atrogin-1, and MuRF-1. These findings indicate that TRB3 regulates protein synthesis and breakdown via the Akt/mTOR/FoxO pathways.


Subject(s)
Cell Cycle Proteins/metabolism , Muscle, Skeletal/metabolism , Animals , Cell Cycle Proteins/genetics , Female , Forkhead Box Protein O1/metabolism , Forkhead Box Protein O3/metabolism , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Muscle Proteins/genetics , Muscle, Skeletal/physiopathology , Protein Biosynthesis/genetics , Proto-Oncogene Proteins c-akt/metabolism , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , SKP Cullin F-Box Protein Ligases/genetics , TOR Serine-Threonine Kinases/metabolism
4.
Biochem Biophys Res Commun ; 470(4): 783-91, 2016 Feb 19.
Article in English | MEDLINE | ID: mdl-26801556

ABSTRACT

Recent studies have demonstrated that adult humans have substantial amounts of functioning brown adipose tissue (BAT). Since BAT has been implicated as an anti-obese and anti-diabetic tissue, it is important to understand the signaling molecules that regulate BAT function. There has been a link between insulin signaling and BAT metabolism as deletion or pharmaceutical inhibition of insulin signaling impairs BAT differentiation and function. Tribbles 3 (TRB3) is a pseudo kinase that has been shown to regulate metabolism and insulin signaling in multiple tissues but the role of TRB3 in BAT has not been studied. In this study, we found that TRB3 expression was present in BAT and overexpression of TRB3 in brown preadipocytes impaired differentiation and decreased expression of BAT markers. Furthermore, TRB3 overexpression resulted in significantly lower oxygen consumption rates for basal and proton leakage, indicating decreased BAT activity. Based on previous studies showing that deletion or pharmaceutical inhibition of insulin signaling impairs BAT differentiation and function, we assessed insulin signaling in brown preadipocytes and BAT in vivo. Overexpression of TRB3 in cells impaired insulin-stimulated IRS1 and Akt phosphorylation, whereas TRB3KO mice displayed improved IRS1 and Akt phosphorylation. Finally, deletion of IRS1 abolished the function of TRB3 to regulate BAT differentiation and metabolism. These data demonstrate that TRB3 inhibits insulin signaling in BAT, resulting in impaired differentiation and function.


Subject(s)
Adipocytes, Brown/cytology , Adipocytes, Brown/metabolism , Adipogenesis/physiology , Cell Cycle Proteins/metabolism , Insulin/physiology , Animals , Cell Differentiation/physiology , Cells, Cultured , Down-Regulation , Mice , Mice, Inbred C57BL , Signal Transduction/physiology
5.
J Clin Invest ; 123(10): 4195-207, 2013 Oct.
Article in English | MEDLINE | ID: mdl-24051379

ABSTRACT

The embryonic self-renewal factor SALL4 has been implicated in the development of human acute myeloid leukemia (AML). Transgenic mice expressing the human SALL4B allele develop AML, which indicates that this molecule contributes to leukemia development and maintenance. However, the underlying mechanism of SALL4-dependent AML progression is unknown. Using SALL4B transgenic mice, we observed that HoxA9 was significantly upregulated in SALL4B leukemic cells compared with wild-type controls. Downregulation of HoxA9 in SALL4B leukemic cells led to decreased replating capacity in vitro and delayed AML development in recipient mice. In primary human AML cells, downregulation of SALL4 led to decreased HOXA9 expression and enhanced apoptosis. We found that SALL4 bound a specific region of the HOXA9 promoter in leukemic cells. SALL4 overexpression led to enhanced binding of histone activation markers at the HOXA9 promoter region, as well as increased HOXA9 expression in these cells. Furthermore, we observed that SALL4 interacted with mixed-lineage leukemia (MLL) and co-occupied the HOXA9 promoter region with MLL in AML leukemic cells, which suggests that a SALL4/MLL pathway may control HOXA9 expression. In summary, our findings revealed a molecular mechanism for SALL4 function in leukemogenesis and suggest that targeting of the SALL4/MLL/HOXA9 pathway would be an innovative approach in treating AML.


Subject(s)
Carcinogenesis/metabolism , Homeodomain Proteins/metabolism , Leukemia, Myeloid, Acute/metabolism , Myeloid-Lymphoid Leukemia Protein/metabolism , Transcription Factors/metabolism , Animals , Base Sequence , Binding Sites , Gene Expression , Gene Expression Regulation, Leukemic , HEK293 Cells , Hematopoietic Stem Cells , Histone-Lysine N-Methyltransferase , Homeodomain Proteins/genetics , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Mice, Transgenic , Neoplasm Transplantation , Promoter Regions, Genetic , Protein Binding , Protein Isoforms/genetics , Protein Isoforms/metabolism , Signal Transduction , Transcription Factors/genetics , Tumor Cells, Cultured , Up-Regulation
6.
Blood ; 121(8): 1413-21, 2013 Feb 21.
Article in English | MEDLINE | ID: mdl-23287862

ABSTRACT

An exciting recent approach to targeting transcription factors in cancer is to block formation of oncogenic complexes. We investigated whether interfering with the interaction of the transcription factor SALL4, which is critical for leukemic cell survival, and its epigenetic partner complex represents a novel therapeutic approach. The mechanism of SALL4 in promoting leukemogenesis is at least in part mediated by its repression of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) through its interaction with a histone deacetylase (HDAC) complex. In this study, we demonstrate that a peptide can compete with SALL4 in interacting with the HDAC complex and reverse its effect on PTEN repression. Treating SALL4-expressing malignant cells with this peptide leads to cell death that can be rescued by a PTEN inhibitor. The antileukemic effect of this peptide can be confirmed on primary human leukemia cells in culture and in vivo, and is identical to that of down-regulation of SALL4 in these cells using an RNAi approach. In summary, our results demonstrate a novel peptide that can block the specific interaction between SALL4 and its epigenetic HDAC complex in regulating its target gene, PTEN. Furthermore, targeting SALL4 with this approach could be an innovative approach in treating leukemia.


Subject(s)
Epigenesis, Genetic/drug effects , Gene Expression Regulation, Leukemic/drug effects , Leukemia, Myeloid, Acute , Transcription Factors/antagonists & inhibitors , Animals , Carcinoma, Hepatocellular , Drug Design , Endometrial Neoplasms , Epigenesis, Genetic/genetics , Female , Gene Expression Regulation, Leukemic/physiology , HL-60 Cells , Histone Deacetylase 1/metabolism , Histone Deacetylase 2/metabolism , Histone Deacetylase Inhibitors/pharmacology , Humans , Hydroxamic Acids/pharmacology , Leukemia, Monocytic, Acute , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/mortality , Liver Neoplasms , Male , Mice , Mice, Inbred NOD , Mice, SCID , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolism , Peptide Fragments/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Xenograft Model Antitumor Assays
7.
PLoS One ; 6(4): e18372, 2011 Apr 19.
Article in English | MEDLINE | ID: mdl-21526180

ABSTRACT

Our previous work shows that the stem cell factor SALL4 plays a central role in embryonic and leukemic stem cells. In this study, we report that SALL4 expression was higher in drug resistant primary acute myeloid leukemic patients than those from drug-responsive cases. In addition, while overexpression of SALL4 led to drug resistance in cell lines, cells with decreased SALL4 expression were more sensitive to drug treatments than the parental cells. This led to our investigation of the implication of SALL4 in drug resistance and its role in side population (SP) cancer stem cells. SALL4 expression was higher in SP cells compared to non-SP cells by 2-4 fold in various malignant hematopoietic cell lines. Knocking down of SALL4 in isolated SP cells resulted in a reduction of SP cells, indicating that SALL4 is required for their self-renewal. The SP phenotype is known to be mediated by members of the ATP-binding cassette (ABC) drug transport protein family, such as ABCG2 and ABCA3. Using chromatin-immunoprecipitation (ChIP), quantitative reverse transcription polymerase chain reaction (qRT-PCR) and electrophoretic mobility shift assay(EMSA), we demonstrated that SALL4 was able to bind to the promoter region of ABCA3 and activate its expression while regulating the expression of ABCG2 indirectly. Furthermore, SALL4 expression was positively correlated to those of ABCG2 and ABCA3 in primary leukemic patient samples. Taken together, our results suggest a novel role for SALL4 in drug sensitivity, at least in part through the maintenance of SP cells, and therefore may be responsible for drug-resistance in leukemia. We are the first to demonstrate a direct link between stem cell factor SALL4, SP and drug resistance in leukemia.


Subject(s)
ATP-Binding Cassette Transporters/genetics , Gene Expression Regulation, Leukemic , Neoplasm Proteins/genetics , Side-Population Cells/metabolism , Stem Cell Factor/metabolism , Transcription Factors/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/metabolism , Adolescent , Adult , Aged , Aged, 80 and over , Base Sequence , Cell Line, Tumor , Drug Resistance, Neoplasm/genetics , Gene Knockdown Techniques , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Middle Aged , Molecular Sequence Data , Neoplasm Proteins/metabolism , Promoter Regions, Genetic/genetics , Protein Binding , Stem Cell Factor/genetics , Transcription Factors/genetics , Transcriptional Activation/genetics , Young Adult
8.
PLoS Pathog ; 5(11): e1000671, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19956712

ABSTRACT

Host-adapted strains of Salmonella enterica cause systemic infections and have the ability to persist systemically for long periods of time despite the presence of a robust immune response. Chronically infected hosts are asymptomatic and transmit disease to naïve hosts via fecal shedding of bacteria, thereby serving as a critical reservoir for disease. We show that the bacterial effector protein SseI (also called SrfH), which is translocated into host cells by the Salmonella Pathogenicity Island 2 (SPI2) type III secretion system (T3SS), is required for Salmonella typhimurium to maintain a long-term chronic systemic infection in mice. SseI inhibits normal cell migration of primary macrophages and dendritic cells (DC) in vitro, and such inhibition requires the host factor IQ motif containing GTPase activating protein 1 (IQGAP1), an important regulator of cell migration. SseI binds directly to IQGAP1 and co-localizes with this factor at the cell periphery. The C-terminal domain of SseI is similar to PMT/ToxA, a bacterial toxin that contains a cysteine residue (C1165) that is critical for activity. Mutation of the corresponding residue in SseI (C178A) eliminates SseI function in vitro and in vivo, but not binding to IQGAP1. In addition, infection with wild-type (WT) S. typhimurium suppressed DC migration to the spleen in vivo in an SseI-dependent manner. Correspondingly, examination of spleens from mice infected with WT S. typhimurium revealed fewer DC and CD4(+) T lymphocytes compared to mice infected with Delta sseI S. typhimurium. Taken together, our results demonstrate that SseI inhibits normal host cell migration, which ultimately counteracts the ability of the host to clear systemic bacteria.


Subject(s)
Bacterial Proteins/physiology , Cell Movement , Host-Pathogen Interactions , Membrane Proteins/physiology , Salmonella Infections/etiology , Salmonella enterica/pathogenicity , Animals , Dendritic Cells/microbiology , Dendritic Cells/physiology , Macrophages/microbiology , Macrophages/physiology , Mice , Spleen/immunology , Time Factors
9.
PLoS One ; 4(5): e5577, 2009.
Article in English | MEDLINE | ID: mdl-19440552

ABSTRACT

BACKGROUND: The embryonic stem cell (ESC) factor, SALL4, plays an essential role in both development and leukemogenesis. It is a unique gene that is involved in self-renewal in ESC and leukemic stem cell (LSC). METHODOLOGY/PRINCIPAL FINDINGS: To understand the mechanism(s) of SALL4 function(s), we sought to identify SALL4-associated proteins by tandem mass spectrometry. Components of a transcription repressor Mi-2/Nucleosome Remodeling and Deacetylase (NuRD) complex were found in the SALL4-immunocomplexes with histone deacetylase (HDAC) activity in ESCs with endogenous SALL4 expression and 293T cells overexpressing SALL4. The SALL4-mediated transcriptional regulation was tested on two potential target genes: PTEN and SALL1. Both genes were confirmed as SALL4 downstream targets by chromatin-immunoprecipitation, and their expression levels, when tested by quantitative reverse transcription polymerase chain reaction (qRT-PCR), were decreased in 293T cells overexpressing SALL4. Moreover, SALL4 binding sites at the promoter regions of PTEN and SALL1 were co-occupied by NuRD components, suggesting that SALL4 represses the transcriptions of PTEN and SALL1 through its interactions with the Mi-2/NuRD complex. The in vivo repressive effect(s) of SALL4 were evaluated in SALL4 transgenic mice, where decreased expressions of PTEN and SALL1 were associated with myeloid leukemia and cystic kidneys, respectively. CONCLUSIONS/SIGNIFICANCE: In summary, we are the first to demonstrate that stem cell protein SALL4 represses its target genes, PTEN and SALL1, through the epigenetic repressor Mi-2/NuRD complex. Our novel finding provides insight into the mechanism(s) of SALL4 functions in kidney development and leukemogenesis.


Subject(s)
Histone Deacetylases/metabolism , PTEN Phosphohydrolase/genetics , Transcription Factors/genetics , Transcription Factors/physiology , Animals , Binding Sites , Cell Line , Chromatin Immunoprecipitation , Humans , Immunohistochemistry , Immunoprecipitation , Magnetic Resonance Imaging , Mass Spectrometry , Mi-2 Nucleosome Remodeling and Deacetylase Complex , Mice , Mice, Transgenic , Promoter Regions, Genetic/genetics , Protein Binding , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/metabolism
10.
J Biol Chem ; 283(2): 1008-17, 2008 Jan 11.
Article in English | MEDLINE | ID: mdl-17981797

ABSTRACT

The scaffold protein IQGAP1 integrates signaling pathways and participates in diverse cellular activities. IQGAP1 is overexpressed in a number of human solid neoplasms, but its functional role in tumorigenesis has not been previously evaluated. Here we report that IQGAP1 contributes to neoplastic transformation of human breast epithelial cells. The amount of IQGAP1 in breast carcinoma is greater than that in normal tissue, with highly metastatic breast epithelial cells expressing the highest levels. Overexpression of IQGAP1 enhances proliferation of MCF-7 breast epithelial cells. Reduction of endogenous IQGAP1 by RNA interference impairs both serum-dependent and anchorage-independent growth of MCF-7 cells. Consistent with these in vitro observations, immortalized MCF-7 cells overexpressing IQGAP1 form invasive tumors in immunocompromised mice, whereas tumors derived from MCF-7 cells with stable knockdown of IQGAP1 are smaller and less invasive. In vitro analysis with selected IQGAP1 mutant constructs and a chemical inhibitor suggests that actin, Cdc42/Rac1, and the mitogen-activated protein kinase pathway contribute to the mechanism by which IQGAP1 increases cell invasion. Collectively, our data reveal that IQGAP1 enhances mammary tumorigenesis, suggesting that it may be a target for therapeutic intervention.


Subject(s)
Breast Neoplasms/genetics , Breast/cytology , Epithelial Cells/cytology , ras GTPase-Activating Proteins/physiology , Breast Neoplasms/pathology , Cell Division/drug effects , Cell Line, Tumor , Cell Transformation, Neoplastic , Epithelial Cells/drug effects , Female , Gene Expression Regulation, Neoplastic , Humans , Signal Transduction , ras GTPase-Activating Proteins/genetics
11.
Cell Signal ; 19(9): 1857-65, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17544257

ABSTRACT

Cell migration, a highly complex physiological phenomenon that requires the co-ordinated and tightly regulated function of several proteins, is mediated by a number of signalling pathways. Elucidation of the molecular mechanisms of cell migration impacts our comprehension of numerous cell functions, ranging from development and immune surveillance to angiogenesis and metastasis. The scaffold protein IQGAP1, which binds multiple proteins and regulates their functions, promotes cell motility. Many of the IQGAP1 binding proteins have been implicated in cell migration. In this study, we employed a multifaceted strategy to identify proteins that contribute to IQGAP1-stimulated cell migration. Using specific IQGAP1 point mutant constructs, an interaction with actin was shown to be essential for IQGAP1 to increase cell migration. In contrast, eliminating the binding of Ca(2+)/calmodulin, but not Ca(2+)-free calmodulin, augmented the ability of IQGAP1 to stimulate cell migration. Consistent with these findings, selective inhibition of calmodulin function at the plasma membrane with a specific peptide inhibitor enhanced cell migration mediated by IQGAP1. Interestingly, immunofluorescence staining and confocal microscopy suggest that localization of Cdc42 at the leading edge is not necessary for maximal migration of epithelial cells. Coupled with the observations that Cdc42 and Rac1 contribute to IQGAP1-stimulated cell migration, these data suggest that IQGAP1 serves as a junction to integrate multiple signalling molecules to facilitate cell migration.


Subject(s)
Cell Movement , Proteins/metabolism , ras GTPase-Activating Proteins/metabolism , Actins/metabolism , Calmodulin/metabolism , Cell Line, Tumor , Humans , Protein Binding , cdc42 GTP-Binding Protein/metabolism , rac1 GTP-Binding Protein/metabolism
12.
J Biol Chem ; 282(28): 20752-62, 2007 Jul 13.
Article in English | MEDLINE | ID: mdl-17517894

ABSTRACT

IQGAP1 is a scaffolding protein involved in multiple fundamental cellular activities, including transcription, cell-cell attachment, and regulation of the cytoskeleton. To function in these pathways, IQGAP1 associates with numerous proteins such as actin, calmodulin, E-cadherin, beta-catenin, CLIP-170, and components of the mitogen-activated protein kinase pathway. Moreover, IQGAP1 binds to active Cdc42 and Rac1 but not RhoA or Ras. Here we show that IQGAP1 also binds to the small GTPase Rap1. In vitro analysis demonstrates a direct interaction between Rap1 and IQGAP1, which is augmented by activation (GTP loading) of Rap1. Cdc42 does not modulate the interaction between Rap1 and IQGAP1. In contrast, the association is eliminated by calmodulin both in the absence and presence of Ca(2+). The binding of Rap1 to a point mutant IQGAP1 construct that is unable to interact with calmodulin is 2.5-fold more than to wild type IQGAP1. Consistent with these findings, Rap1 binds to the IQ region of IQGAP1. Confocal microscopy demonstrates that Rap1 and IQGAP1 co-localize at the periphery of human epithelial cells but not in the cytoplasm. The interaction has functional sequelae. Overexpression of IQGAP1 substantially reduces adhesion-mediated activation of Rap1. In addition, Rap1 activation by cAMP is attenuated in cells that overexpress IQGAP1 and enhanced in cells lacking IQGAP1. These findings reveal that the interaction of IQGAP1 with Rap1 differs in several respects from its interaction with other small GTPases. Furthermore, our data suggest that IQGAP1 may link the calmodulin and Rap1 signaling pathways.


Subject(s)
Calmodulin/metabolism , Epithelial Cells/metabolism , Signal Transduction/physiology , rap1 GTP-Binding Proteins/metabolism , ras GTPase-Activating Proteins/metabolism , Cell Adhesion/physiology , Cell Line, Tumor , Cyclic AMP/metabolism , Cytoskeletal Proteins/metabolism , Epithelial Cells/cytology , GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , Gene Expression , Humans , Point Mutation , Protein Binding/physiology , Transcription, Genetic/physiology , rap1 GTP-Binding Proteins/genetics , ras GTPase-Activating Proteins/genetics
13.
Cancer Res ; 65(10): 4153-61, 2005 May 15.
Article in English | MEDLINE | ID: mdl-15899806

ABSTRACT

We previously reported that the FAS1 domains of betaig-h3 bear motifs that mediate endothelial cell adhesion and migration via interactions with alphavbeta3 integrin and regulate angiogenesis. In the present study, we show that the fourth FAS1 domain, designated fastatin, inhibits endothelial adhesion and migration, not only to betaig-h3, but also fibronectin and vitronectin, in a RGD-dependent manner. Fastatin and other FAS1 domains suppress endothelial cell tube formation and in vivo neovascularization in a Matrigel plug assay. The antiangiogenic activity of fastatin is associated with antitumor activity in mouse tumor models. Fastatin additionally induces apoptosis in several cells expressing alphavbeta3 integrin, including endothelial cells. Binding of fastatin to alphavbeta3 integrin inhibits phosphorylation of focal adhesion kinase, Raf, extracellular signal-regulated kinase, Akt, and mammalian target of rapamycin. Fastatin is thus the first endogenous angiogenesis regulator identified that inhibits both endothelial cell migration and growth by binding to alphavbeta3 integrin. Our data suggest that FAS1 domains from all possible forms of the four human FAS1 family proteins are potential endogenous regulators for pathologic angiogenesis. Moreover, FAS1 domains such as fastatin may be developed into drugs for blocking tumor angiogenesis.


Subject(s)
Endothelial Cells/drug effects , Extracellular Matrix Proteins/pharmacology , Integrin alphaVbeta3/metabolism , Neoplasms/blood supply , Neovascularization, Pathologic/pathology , Receptors, Tumor Necrosis Factor/metabolism , Transforming Growth Factor beta/pharmacology , Animals , Apoptosis/drug effects , Apoptosis/physiology , Cell Adhesion/drug effects , Cell Movement/drug effects , Endothelial Cells/cytology , Extracellular Matrix Proteins/genetics , Humans , Male , Melanoma, Experimental/blood supply , Melanoma, Experimental/genetics , Mice , Mice, Inbred C57BL , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/genetics , Peptide Fragments/genetics , Peptide Fragments/pharmacology , Protein Structure, Tertiary , Receptors, Tumor Necrosis Factor/genetics , Transfection , Transforming Growth Factor beta/genetics , fas Receptor
14.
Cancer Res ; 65(2): 507-15, 2005 Jan 15.
Article in English | MEDLINE | ID: mdl-15695393

ABSTRACT

R-Ras has a high degree of sequence homology with Ras and other members of the Ras subfamily, including Rap, TC21, and M-Ras. Although R-Ras has been suggested to regulate cell adhesion, migration, and invasion, the biological mechanism has not been well assessed. In this report, we show that constitutively active R-Ras (38V) induces a more rounded cell shape and redistribution of focal adhesion, and enhances the phosphorylation of focal adhesion kinase and paxillin. Active R-Ras (38V) induces cell adhesion to type I collagen, but inhibits cell motility. In active R-Ras (38V) cells, the activity of RhoA is increased and accompanied with translocation to plasma membrane, but not that of Rac1 or Cdc42. In parallel, dominant-negative RhoA (N19RhoA) and Y27632, a specific inhibitor of Rho-associated kinase, dramatically reverse the rounded cell morphology to a spread cell shape and enhance motility. Furthermore, coincident with the formation of cortical actin filaments in active R-Ras (38V) cells, myosin light chain and Ser-19-phosphorylated myosin light chain mainly accumulate at the peripheral region, which is inhibited by the treatment of Y27632. Using H-Ras/R-Ras and R-Ras/H-Ras hybrid constructs, we show that the COOH-terminal region of R-Ras contains the specific signal for inducing changes in motility and morphology. Our results suggest that R-Ras in breast epithelial cells disrupts cell polarity and motility through the Rho/Rho-associated kinase pathway triggered by a signal from the COOH-terminal end of R-Ras.


Subject(s)
Breast/cytology , Breast/enzymology , GTP Phosphohydrolases/metabolism , Protein Serine-Threonine Kinases/metabolism , ras Proteins/metabolism , Cell Movement/physiology , Cell Polarity/physiology , Epithelial Cells/cytology , Epithelial Cells/enzymology , Focal Adhesions/physiology , Humans , Intracellular Signaling Peptides and Proteins , Signal Transduction/physiology , rho-Associated Kinases , rhoA GTP-Binding Protein/metabolism
15.
J Cell Biochem ; 92(4): 770-80, 2004 Jul 01.
Article in English | MEDLINE | ID: mdl-15211574

ABSTRACT

betaig-h3 is an extracellular matrix (ECM) protein whose expression is highly induced by transforming growth factor beta1 (TGF-beta1). We previously demonstrated that betaig-h3 has two alpha3beta1 integrin-interacting motifs, which promote adhesion, migration, and proliferation of human keratinocytes. Both betaig-h3 and TGF-beta1 have been suggested to play important roles in the healing of skin wounds. In this study, we demonstrate that TGF-beta1 enhances keratinocyte adhesion and migration toward betaig-h3 through the alpha3beta1 integrin. TGF-beta1 did not increase the amount of the alpha3beta1 integrin on the cell surface, but rather increased its affinity for betaig-h3. LY294002, an inhibitor of PI3K, blocked the basal and TGF-beta1-enhanced cell migration but not adhesion to betaig-h3. A constitutively active mutant of PI3K stimulated cell migration but not adhesion to betaig-h3. The PI3K pathway is also not associated with the affinity of the alpha3beta1 integrin to betaig-h3. TGF-beta1 induced phosphorylation of AKT and FAK. Taken together, these data suggest that TGF-beta1 increases affinity of the alpha3beta1 integrin to betaig-h3, resulting in enhanced adhesion and migration of keratinocytes toward betaig-h3. TGF-beta1 also enhances migration through PI3K, but PI3K is not associated with either the binding affinity of the alpha3beta1 integrin or its adhesion to betaig-h3.


Subject(s)
Cell Movement/drug effects , Extracellular Matrix Proteins/metabolism , Integrin alpha3beta1/metabolism , Keratinocytes/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta/pharmacology , Cells, Cultured , Enzyme Inhibitors/pharmacology , Extracellular Matrix , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Humans , Keratinocytes/drug effects , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation/drug effects , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Transforming Growth Factor beta1
16.
Oncogene ; 22(13): 2045-53, 2003 Apr 03.
Article in English | MEDLINE | ID: mdl-12673209

ABSTRACT

Beta ig-h3 is a transforming growth factor-beta (TGF-beta)-induced cell-adhesive molecule and has an RGD sequence at its C-terminus. A previous report suggested that beta ig-h3 normally undergoes carboxy-terminal processing that results in the loss of the RGD sequence. RGD peptides appear to play various roles in cell function. Here we show that the RGD peptides released from beta ig-h3 may facilitate TGF-beta-induced apoptosis. We found that carboxy-terminal cleavage of beta ig-h3 occurred after its secretion, and that overexpression of the wild-type beta ig-h3 induced apoptosis, unlike the C-terminal deleted but RGD-containing mutant beta ig-h3, which is resistant to C-terminal processing. The beta ig-h3-induced apoptosis was abolished by either deletion of the RGD sequence or mutation of RGD to RAE. Synthetic peptides of ERGDEL and GRGDSP derived from beta ig-h3 and fibronectin, respectively, also induced apoptosis, unlike ERGEEL and GRGESP. Culture supernatants of cells overexpressing beta ig-h3 filtered to isolate molecules smaller than 3 kDa also induced apoptosis. A fusion protein composed of the N-terminal 100 amino acids of fibronectin and the RGD-containing C-terminal part of beta ig-h3 was also subjected to C-terminal cleavage and overexpression resulted in apoptosis. The anti-beta ig-h3 antibody blocks TGF-beta-induced apoptosis. Thus, beta ig-h3 may be important in regulating cell apoptosis by providing soluble RGD peptides.


Subject(s)
Apoptosis/physiology , Extracellular Matrix Proteins , Neoplasm Proteins/physiology , Oligopeptides/physiology , Transforming Growth Factor beta/pharmacology , Amino Acid Motifs , Animals , Antibodies, Monoclonal/pharmacology , Apoptosis/drug effects , CHO Cells , Cell Adhesion/drug effects , Cricetinae , Cricetulus , Fibronectins/chemistry , Humans , Monensin/pharmacology , Neoplasm Proteins/chemistry , Neoplasm Proteins/immunology , Neoplasm Proteins/metabolism , Oligopeptides/chemistry , Oligopeptides/pharmacology , Peptide Fragments/chemical synthesis , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Peptide Fragments/physiology , Protein Processing, Post-Translational , Protein Sorting Signals , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/physiology , Sequence Deletion , Transfection
17.
J Biol Chem ; 278(28): 25902-9, 2003 Jul 11.
Article in English | MEDLINE | ID: mdl-12704192

ABSTRACT

betaig-h3 is an extracellular matrix protein that mediates adhesion and migration of several cell types through interaction with integrins. In the present study, we tested whether betaig-h3 mediates endothelial cell adhesion and migration, thereby regulating angiogenesis. In this study, we demonstrate that not only betaig-h3 itself but also all four fas-1 domains of betaig-h3 mediate endothelial cell adhesion and migration through interaction with the alphavbeta3 integrin. We found that the alphavbeta3 integrin-interacting motif of the four fas-1 domains of betaig-h3 is the same YH motif that we reported previously to interact with alphavbeta5 integrin. The YH peptide inhibited endothelial cell adhesion and migration in a dose-dependent manner. We demonstrate that the YH peptide has anti-angiogenic activity in vitro and in vivo using an endothelial cell tube formation assay and a Matrigel plug assay, respectively. Our results reveal that betaig-h3 bears alphavbeta3 integrin-interacting motifs that mediate endothelial cell adhesion and migration and, therefore, may regulate angiogenesis.


Subject(s)
Extracellular Matrix Proteins , Integrin alphaVbeta3/chemistry , Neoplasm Proteins/chemistry , Transforming Growth Factor beta , Amino Acid Motifs , Amino Acid Sequence , Animals , Cell Adhesion , Cell Movement , Cells, Cultured , Collagen/pharmacology , DNA/metabolism , Dose-Response Relationship, Drug , Drug Combinations , Endothelium, Vascular/cytology , Flow Cytometry , Humans , Laminin/pharmacology , Male , Mice , Mice, Inbred C57BL , Models, Genetic , Molecular Sequence Data , Neovascularization, Pathologic , Peptides/chemistry , Protein Binding , Protein Structure, Tertiary , Proteoglycans/pharmacology , Sequence Homology, Amino Acid , Time Factors , Umbilical Veins/cytology
18.
Biochem Pharmacol ; 65(8): 1343-50, 2003 Apr 15.
Article in English | MEDLINE | ID: mdl-12694875

ABSTRACT

Cinnamaldehydes have been shown to have inhibitory effects on farnesyl protein transferase (FPTase; EC 2.5.1.29) in vitro, angiogenesis, cell-cell adhesion, and tumor cell growth and to be immunomodulators. However, the mechanisms responsible for these effects remain unknown. To elucidate the molecular mechanism of the cinnamaldehyde derivative CB403 for growth inhibition, CB403 was synthesized from 2'-hydroxycinnamaldehyde. CB403-treated cells were weakly adherent to the culture dishes. In addition, CB403 inhibited tumor growth in these cells in a concentration-dependent manner. FACS analysis using human cancer cells treated with this compound showed cell cycle arrest in mitosis, which was correlated with a marked increase in the amount of cyclin B1. Furthermore, CB403 blocked in vivo growth of human colon and breast tumor xenografts without loss of body weight in nude mice. These results support the hypothesis that the cinnamaldehyde derivative CB403 exerts cytostatic properties by inducing mitotic arrest in cancer cells.


Subject(s)
Acrolein/analogs & derivatives , Acrolein/toxicity , Antineoplastic Agents/toxicity , Breast Neoplasms/pathology , Cell Cycle/drug effects , Colonic Neoplasms/pathology , Phenyl Ethers/toxicity , Animals , Breast Neoplasms/drug therapy , Cell Division/drug effects , Colonic Neoplasms/drug therapy , Female , G2 Phase/drug effects , Humans , Mice , Mice, Nude , Mitosis/drug effects , Tumor Cells, Cultured
19.
J Biol Chem ; 277(48): 46159-65, 2002 Nov 29.
Article in English | MEDLINE | ID: mdl-12270930

ABSTRACT

betaig-h3 is a TGF-beta-induced matrix protein known to mediate the adhesion of several cell types. In this study, we found that all four of the fas-1 domains in betaig-h3 mediate MRC-5 fibroblast adhesion and that this was specifically inhibited by a function-blocking monoclonal antibody specific for the alphavbeta5 integrin. Using deletion mutants of the fourth fas-1 domain revealed the MRC-5 cell adhesion motif (denoted the YH motif) is located in amino acids 548-614. Experiments with substitution mutants showed that tyrosine 571, histidine 572, and their flanking leucine and isoleucine amino acids, which are all highly conserved in many fas-1 domains, are essential for mediating MRC-5 cell adhesion. A synthetic 18-amino acid peptide encompassing these conserved amino acids could effectively block MRC-5 cell adhesion to betaig-h3. Using HEK293 cells stably transfected with the beta5 integrin cDNA, we confirmed that the alphavbeta5 integrin is a functional receptor for the YH motif. In conclusion, we have identified a new alphavbeta5 integrin-interacting motif that is highly conserved in the fas-1 domains of many proteins. This suggests that fas-1 domain-containing proteins may perform their biological functions by interacting with integrins.


Subject(s)
Amino Acid Motifs , Elapid Venoms/metabolism , Extracellular Matrix Proteins , Integrins/metabolism , Neoplasm Proteins/metabolism , Receptors, Vitronectin/metabolism , Transforming Growth Factor beta , Amino Acid Sequence , Cells, Cultured , Elapid Venoms/chemistry , Flow Cytometry , Humans , Molecular Sequence Data , Protein Binding , Sequence Homology, Amino Acid
20.
Bioorg Med Chem ; 10(10): 3129-34, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12150857

ABSTRACT

Sesquicillin, isolated from fungal fermentation broth, strongly induced G1 phase arrest in human breast cancer cells. During G1 phase arrest, the expression level of cyclin D1, cyclin A, and cyclin E was decreased, and the expression of CDK (cyclin-dependent-kinase) inhibitor, protein p21(Waf1/Cip1), was increased in a time-dependent manner in a breast cancer cell MCF-7. Interestingly, the G1 phase arrest induced by sesquicillin also occurred independently of the tumor suppressor protein, p53. Sesquicillin inhibits the proliferation of MCF-7 via G1 phase arrest in association with the induction of CDK inhibitor protein, p21(Waf1/Cip1), and the reduction of G1 phase related-cyclin proteins.


Subject(s)
Breast Neoplasms/pathology , G1 Phase/drug effects , Naphthalenes/pharmacology , Breast Neoplasms/drug therapy , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/drug effects , Cyclins/metabolism , Female , Fungi/chemistry , Humans , Naphthalenes/isolation & purification , Phosphorylation/drug effects , Tumor Cells, Cultured , Tumor Suppressor Protein p53/pharmacology
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