ABSTRACT
Innate immune response is critical for the control of Listeria monocytogenes infection. Here, we identified developmentally regulated GTP-binding protein 2 (DRG2) in macrophages as a major regulator of the innate immune response against L. monocytogenes infection. Both whole-body DRG2 knockout (KO) mice and macrophage-specific DRG2 KO mice had low levels of IL-6 during early infection and increased susceptibility to L. monocytogenes infection. Following an initial impaired inflammatory response of macrophages upon i.p. L. monocytogenes infection, DRG2-/- mice showed delayed recruitment of neutrophils and monocytes into the peritoneal cavity, which led to elevated bacterial burden, inflammatory cytokine production at a late infection time point, and liver micro-abscesses. DRG2 deficiency decreased the transcriptional activity of NF-κB and impaired the inflammatory response of both bone marrow-derived and peritoneal macrophages upon L. monocytogenes stimulation. Our findings reveal that DRG2 in macrophages is critical for the initial inflammatory response and protection against L. monocytogenes infection.
Subject(s)
GTP-Binding Proteins , Listeria monocytogenes , Listeriosis , Macrophages , Animals , Mice , Immunity, Innate , Listeriosis/immunology , Macrophages/immunology , Mice, Knockout , Monocytes , GTP-Binding Proteins/metabolismABSTRACT
Endothelial cell senescence is involved in endothelial dysfunction and vascular diseases. However, the detailed mechanisms of endothelial senescence are not fully understood. Here, we demonstrated that deficiency of developmentally regulated GTP-binding protein 2 (DRG2) induces senescence and dysfunction of endothelial cells. DRG2 knockout (KO) mice displayed reduced cerebral blood flow in the brain and lung blood vessel density. We also determined, by Matrigel plug assay, aorta ring assay, and in vitro tubule formation of primary lung endothelial cells, that deficiency in DRG2 reduced the angiogenic capability of endothelial cells. Endothelial cells from DRG2 KO mice showed a senescence phenotype with decreased cell growth and enhanced levels of p21 and phosphorylated p53, γH2AX, senescence-associated ß-galactosidase (SA-ß-gal) activity, and senescence-associated secretory phenotype (SASP) cytokines. DRG2 deficiency in endothelial cells upregulated arginase 2 (Arg2) and generation of reactive oxygen species. Induction of SA-ß-gal activity was prevented by the antioxidant N-acetyl cysteine in endothelial cells from DRG2 KO mice. In conclusion, our results suggest that DRG2 is a key regulator of endothelial senescence, and its downregulation is probably involved in vascular dysfunction and diseases.
Subject(s)
Endothelial Cells , Vascular Diseases , Animals , Cellular Senescence/genetics , Endothelial Cells/metabolism , Mice , Mice, Knockout , Reactive Oxygen Species/metabolism , Vascular Diseases/metabolismABSTRACT
Developmentally regulated GTP-binding protein 2 (DRG2) participates in the regulation of proliferation and differentiation of multiple cells. However, whether DRG2 regulates adipocyte differentiation and related metabolic control remains elusive. This study revealed increases in body weight and adiposity in DRG2 transgenic (Tg) mice overexpressing DRG2. Consistent with these results, DRG2 Tg mice showed increased expression of genes involved in adipogenesis and lipid metabolism in the white adipose tissue. DRG2 was also identified to control adipogenesis by cooperating with peroxisome proliferator activated receptor-γ (PPAR-γ) in cultured adipocytes. Overall, the findings of the current study suggest that DRG2 plays an active role in regulating adipocyte differentiation, and thus participates in the development of obesity during exposure to a fat-rich diet.
Subject(s)
Adipose Tissue, White/cytology , GTP-Binding Proteins/metabolism , PPAR gamma/metabolism , Adipogenesis , Adipose Tissue, White/metabolism , Animals , Body Weight , Cell Differentiation , Disease Models, Animal , GTP-Binding Proteins/genetics , Lipid Metabolism , Mice , Mice, TransgenicABSTRACT
PURPOSE: This study aimed to confirm the association between developmentally regulated GTP-binding protein 2 (DRG2) expression and docetaxel-induced apoptosis and to determine whether prostate cancer responses to docetaxel treatment differ with DRG2 expression. MATERIALS AND METHODS: PC3, DU145, and LNCaP prostate cancer cell lines were used. The MTT assay was used to determine cell viability. Western blotting analysis was performed using anti-DRG2 antibodies. Cells were transfected with 50 nmol DRG2 siRNA using an siRNA transfection reagent for DRG2 knockdown. The cell cycle was analyzed by using flow cytometry, and apoptosis was detected by using the Annexin V cell death assay. RESULTS: DRG2 expression differed in each prostate cancer cell line. Docetaxel reduced DRG2 expression in a dose-dependent manner. Upon DRG2 knockdown in prostate cancer cells, an increase in the sub-G1 phase was observed without a change in the G1 or G2/M phases. When 4 nM docetaxel was administered to DRG2 knockdown prostate cancer cell lines, an increase in the sub-G1 phase was observed without increasing the G2/M phase, which was similar to that in DU145 cells before DRG2 knockdown. In PC3 and DU145 cell lines, DRG2 knockdown increased docetaxel-induced Annexin V (+) apoptosis by 8.7 and 2.7 times, respectively. CONCLUSIONS: In prostate cancer cells, DRG2 regulates G2/M arrest after docetaxel treatment. In prostate cancer cells with DRG2 knockdown, apoptosis increases without G2/M arrest in response to docetaxel treatment. These results show that inhibition of DRG2 expression can be useful to enhance docetaxel-induced apoptosis despite low-dose administration in castration-resistant prostate cancer.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Docetaxel/pharmacology , GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , Prostatic Neoplasms, Castration-Resistant/genetics , Apoptosis/genetics , Cell Proliferation/drug effects , G1 Phase Cell Cycle Checkpoints/drug effects , G1 Phase Cell Cycle Checkpoints/genetics , G2 Phase Cell Cycle Checkpoints/drug effects , G2 Phase Cell Cycle Checkpoints/genetics , Gene Expression/drug effects , Gene Knockdown Techniques , Humans , M Phase Cell Cycle Checkpoints/drug effects , M Phase Cell Cycle Checkpoints/genetics , Male , PC-3 Cells , Prostatic Neoplasms, Castration-Resistant/drug therapy , RNA, Small Interfering , TransfectionABSTRACT
Malignant metastatic melanoma (MM) is the most lethal of all skin cancers, but detailed mechanisms for regulation of melanoma metastasis are not fully understood. Here, we demonstrated that developmentally regulated GTP-binding protein 2 (DRG2) is required for the growth of primary tumors and for metastasis. DRG2 expression was significantly increased in MM compared with primary melanoma (PM) and dysplastic nevi. A correlation between DRG2 expression and poor disease-specific survival in melanoma patients was also identified. Furthermore, inhibition of DRG2 suppressed the binding of Hypoxia-inducible factor 1α to the VEGF-A promoter region, expression of vascular endothelial growth factor (VEGF)-A, and formation of endothelial cell tubes. In experimental mice, DRG2 depletion inhibited the growth of PM and lung metastases and increased survival. These results identify DRG2 as a critical regulator of VEGF-A expression and of growth of PMs and lung metastases.
Subject(s)
GTP-Binding Proteins/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Lung Neoplasms/genetics , Melanoma/genetics , Vascular Endothelial Growth Factor A/genetics , Adolescent , Adult , Aged , Animals , Cell Line, Tumor , Female , Gene Expression Regulation, Neoplastic/genetics , Humans , Lung Neoplasms/pathology , Lung Neoplasms/secondary , Male , Melanoma/pathology , Melanoma, Experimental/genetics , Melanoma, Experimental/pathology , Mice , Middle Aged , Neoplasm Metastasis , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/pathology , Protein Binding/genetics , Young AdultABSTRACT
Developmentally regulated GTP-binding protein 2 (DRG2) was first identified in the central nervous system of mice. However, the physiological function of DRG2 in the brain remains largely unknown. Here, we demonstrated that knocking out DRG2 impairs the function of dopamine neurons in mice. DRG2 was strongly expressed in the neurons of the dopaminergic system such as those in the striatum (Str), ventral tegmental area (VTA), and substantia nigra (SN), and on neuronal cell bodies in high-density regions such as the hippocampus (HIP), cerebellum, and cerebral cortex in the mouse brain. DRG2 knockout (KO) mice displayed defects in motor function in motor coordination and rotarod tests and increased anxiety. However, unexpectedly, DRG2 depletion did not affect the dopamine (DA) neuron population in the SN, Str, or VTA region or dopamine synthesis in the Str region. We further demonstrated that dopamine release was significantly diminished in the Str region of DRG2 KO mice and that treatment of DRG2 KO mice with l-3,4-dihydroxyphenylalanine (L-DOPA), a dopamine precursor, rescued the behavioral motor deficiency in DRG2 KO mice as observed with the rotarod test. This is the first report to identify DRG2 as a key regulator of dopamine release from dopamine neurons in the mouse brain.
Subject(s)
Corpus Striatum/metabolism , Dopamine/metabolism , GTP-Binding Proteins/genetics , Motor Disorders/genetics , Animals , Anxiety/genetics , Anxiety/metabolism , Corpus Striatum/cytology , Dopaminergic Neurons/cytology , Dopaminergic Neurons/metabolism , GTP-Binding Proteins/analysis , GTP-Binding Proteins/metabolism , Gene Deletion , Mice , Mice, Knockout , Motor Disorders/metabolismABSTRACT
The perinuclear stacks of the Golgi apparatus maintained by dynamic microtubules are essential for cell migration. Activation of Akt (protein kinase B, PKB) negatively regulates glycogen synthase kinase 3ß (GSK3ß)-mediated tau phosphorylation, which enhances tau binding to microtubules and microtubule stability. In this study, experiments were performed on developmentally regulated GTP-binding protein 2 (DRG2)-stably knockdown HeLa cells to determine whether knockdown of DRG2 in HeLa cells treated with epidermal growth factor (EGF) affects microtubule dynamics, perinuclear Golgi stacking, and cell migration. Here, we show that DRG2 plays a key role in regulating microtubule stability, perinuclear Golgi stack formation, and cell migration. DRG2 knockdown prolonged the EGF receptor (EGFR) localization in endosome, enhanced Akt activity and inhibitory phosphorylation of GSK3ß. Tau, a target of GSK3ß, was hypo-phosphorylated in DRG2-knockdown cells and showed greater association with microtubules, resulting in microtubule stabilization. DRG2-knockdown cells showed defects in microtubule growth and microtubule organizing centers (MTOC), Golgi fragmentation, and loss of directional cell migration. These results reveal a previously unappreciated role for DRG2 in the regulation of perinuclear Golgi stacking and cell migration via its effects on GSK3ß phosphorylation, and microtubule stability.
Subject(s)
GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , Glycogen Synthase Kinase 3 beta/metabolism , Golgi Apparatus/metabolism , Microtubules/metabolism , Cell Movement , Gene Knockdown Techniques , HeLa Cells , Humans , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolismABSTRACT
The developmentally regulated GTP binding protein 2 (DRG2) is involved in the control of cell growth and differentiation. Here, we demonstrate that DRG2 regulates microtubule dynamics in HeLa cells. Analysis of live imaging of the plus-ends of microtubules with EB1-EGFP showed that DRG2 deficiency (shDRG2) significantly reduced the growth rate of HeLa cells. Depletion of DRG2 increased 'slow and long-lived' subpopulations, but decreased 'fast and short-lived' subpopulations of microtubules. Microtubule polymerization inhibitor exhibited a reduced response in shDRG2 cells. Using immunoprecipitation, we show that DRG2 interacts with tau, which regulates microtubule polymerization. Collectively, these data demonstrate that DRG2 may aid in affecting microtubule dynamics in HeLa cells.
Subject(s)
GTP-Binding Proteins/deficiency , Microtubules/metabolism , Cell Proliferation/physiology , Gene Knockdown Techniques , HeLa Cells , Humans , Phosphorylation , Transfection , tau Proteins/metabolismABSTRACT
Previously we have reported that developmentally regulated GTP-binding protein 2 (DRG2) localizes on Rab5 endosomes and plays an important role in transferrin (Tfn) recycling. We here identified DRG2 as a key regulator of membrane tubule stability. At 30 min after Tfn treatment, DRG2 localized to membrane tubules which were enriched with phosphatidylinositol 4-monophosphate [PI(4)P] and did not contain Rab5. DRG2 interacted with Rac1 more strongly with GTP-bound Rac1 and tubular localization of DRG2 depended on Rac1 activity. DRG2 depletion led to destabilization of membrane tubules, while ectopic expression of DRG2 rescued the stability of the membrane tubules in DRG2-depleted cells. Our results reveal a novel mechanism for regulation of membrane tubule stability mediated by DRG2.
Subject(s)
Cell Membrane/metabolism , Endosomes/metabolism , GTP-Binding Proteins/metabolism , Neuropeptides/metabolism , Phospholipids/metabolism , rac1 GTP-Binding Protein/metabolism , Animals , Cells, Cultured , Fibroblasts , Humans , MCF-7 Cells , MiceABSTRACT
Mitochondrial dynamics, including constant fusion and fission, play critical roles in maintaining mitochondrial morphology and function. Here, we report that developmentally regulated GTP-binding protein 2 (DRG2) regulates mitochondrial morphology by modulating the expression of the mitochondrial fission gene dynamin-related protein 1 (Drp1). shRNA-mediated silencing of DRG2 induced mitochondrial swelling, whereas expression of an shRNA-resistant version of DRG2 decreased mitochondrial swelling in DRG2-depleted cells. Analysis of the expression levels of genes involved in mitochondrial fusion and fission revealed that DRG2 depletion significantly decreased the level of Drp1. Overexpression of Drp1 rescued the defect in mitochondrial morphology induced by DRG2 depletion. DRG2 depletion reduced the mitochondrial membrane potential, oxygen consumption rate (OCR), and amount of mitochondrial DNA (mtDNA), whereas it increased reactive oxygen species (ROS) production and apoptosis. Taken together, our data demonstrate that DRG2 acts as a regulator of mitochondrial fission by controlling the expression of Drp1.
Subject(s)
GTP Phosphohydrolases/metabolism , GTP-Binding Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Mitochondria/metabolism , Mitochondria/pathology , Mitochondrial Dynamics/physiology , Mitochondrial Proteins/metabolism , Down-Regulation/physiology , Dynamins , HeLa Cells , HumansABSTRACT
Developmentally regulated GTP-binding protein 2 (DRG2) plays an important role in cell growth. Here we explored the linkage between DRG2 and G2/M phase checkpoint function in cell cycle progression. We observed that knockdown of DRG2 in HeLa cells affected growth in a wound-healing assay, and tumorigenicity in nude mice xenografts. Flow cytometry assays and [(3)H] incorporation assays indicated that G2/M phase arrest was responsible for the decreased proliferation of these cells. Knockdown of DRG2 elicited down-regulation of the major mitotic promoting factor, the cyclin B1/Cdk1 complex, but up-regulation of the cell cycle arresting proteins, Wee1, Myt1, and p21. These findings identify a novel role of DRG2 in G2/M progression.
Subject(s)
Cyclin B1/physiology , Cyclin-Dependent Kinases/physiology , G2 Phase Cell Cycle Checkpoints/physiology , GTP-Binding Proteins/physiology , Animals , CDC2 Protein Kinase , Cell Proliferation/physiology , Cyclin B1/genetics , Cyclin B1/metabolism , Cyclin-Dependent Kinases/genetics , Cyclin-Dependent Kinases/metabolism , GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , Gene Knockdown Techniques , HeLa Cells , Heterografts , Humans , Male , Mice , Mice, Nude , Mitosis/physiologyABSTRACT
Developmentally regulated GTP-binding protein 2 (DRG2) represents a novel subclass of GTP-binding proteins. We here report that transgenic overexpression of DRG2 in mice ameliorates experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS). The protective effect of DRG2 in EAE was mediated by the inhibition of the development of T(H)17 cells. DRG2 enhanced the activity of PPARγ, which led to an inhibition of the nuclear factor kappa B (NF-κB) activity and IL-6 production in antigen presenting cells and an inhibition of the development of T(H)17 cells. Our results demonstrate that DRG2 is an essential modulator of EAE.