Glia Maturation Factor Gamma Research Articles

Comprehensive analysis of co-expressed genes with TDP-43: prognostic and therapeutic potential in lung adenocarcinoma

BackgroundTransactivating DNA-binding protein 43 (TDP-43) is intimately associated with tumorigenesis and progression by regulating mRNA splicing, transport, stability, and non-coding RNA molecules. The exact role of TDP-43 in lung adenocarcinoma (LUAD) has not yet been fully elucidated, despite extensive research on its function in various cancer types. An imperative aspect of comprehending the underlying biological characteristics associated with TDP-43 involves investigating the genes that are co-expressed with this protein. This study assesses the prognostic significance of these co-expressed genes in LUAD and subsequently explores potential therapeutic strategies based on these findings.MethodsTranscriptomic and clinical data pertaining to LUAD were retrieved from open-access databases to establish an association between mRNA expression profiles and the presence of TDP-43. A risk-prognosis model was developed to compare patient survival rates across various groups, and its accuracy was also assessed. Additionally, differences in tumor stemness, mutational profiles, tumor microenvironment (TME) characteristics, immune checkpoints, and immune cell infiltration were analyzed in the different groups. Moreover, the study entailed predicting the potential response to immunotherapy as well as the sensitivity to commonly employed chemotherapeutic agents and targeted drugs for each distinct group.ResultsThe TDP-43 Co-expressed Gene Risk Score (TCGRS) model was constructed utilizing four genes: Kinesin Family Member 20A (KIF20A), WD Repeat Domain 4 (WDR4), Proline Rich 11 (PRR11), and Glia Maturation Factor Gamma (GMFG). The value of this model in predicting LUAD patient survival is effectively illustrated by both the Kaplan–Meier (K–M) survival curve and the area under the receiver operating characteristic curve (AUC-ROC). The Gene Set Enrichment Analysis (GSEA) revealed that the high TCGRS group was primarily enriched in biological pathways and functions linked to DNA replication and cell cycle; the low TCGRS group showed primary enrichment in immune-related pathways and functions. The high and low TCGRS groups showed differences in tumor stemness, mutational burden, TME, immune infiltration level, and immune checkpoints. The predictions analysis of immunotherapy indicates that the Tumor Immune Dysfunction and Exclusion (TIDE) score (p < 0.001) and non-response rate (74% vs. 51%, p < 0.001) in the high TCGRS group are higher than those in the low TCGRS group. The Immune Phenotype Score (IPS) in the high TCGRS group is lower than in the low TCGRS group (p < 0.001). The drug sensitivity analysis revealed that the half-maximal inhibitory concentration (IC50) values for cisplatin, docetaxel, doxorubicin, etoposide, gemcitabine, paclitaxel, vincristine, erlotinib, and gefitinib (all p < 0.01) in the high TCGRS group are lower than those in the low TCGRS group.ConclusionsThe TCGRS derived from the model exhibits a reliable biomarker for evaluating both prognosis and treatment effectiveness among patients with LUAD. This study is anticipated to offer valuable insights into developing effective treatment strategies for this patient population. It is believed that this study is anticipated to contribute significantly to clinical diagnostics, the development of therapeutic drugs, and the enhancement of patient care.

Upregulation of lncRNA LANCL1-AS1 inhibits the progression of non-small-cell lung cancer via the miR-3680-3p/GMFG axis.

Patients with non-small-cell lung cancer (NSCLC) have a low survival rate. Long non-coding RNA (LncRNA) LANCL1 antisense RNA 1 (LANCL1-AS1) was indicated to be downregulated in NSCLC; however, its detailed function in NSCLC is unanswered. Real-time quantitative polymerase chain reaction revealed the downregulation of LANCL1-AS1 in NSCLC cell lines and subcellular fractionation assay showed that LANCL1-AS1 was mainly located in the cytoplasm of NSCLC cells. Cell counting kit-8, Transwell, and tube formation assays displayed that overexpression of LANCL1-AS1 suppressed NSCLC cell proliferation, migration, invasiveness, and angiogenesis in vitro. Animal experiments validated the tumor-suppressive role of LANCL1-AS1 in tumor-bearing mice. Mechanistically, LANCL1-AS1 upregulated glia maturation factor gamma (GMFG) expression by competitively binding to miR-3680-3p. GMFG knockdown reversed LANCL1-AS1 overexpression-mediated inhibitory impact on NSCLC malignant behaviors. Collectively, LANCL1-AS1 upregulation inhibits the progression of NSCLC by modulating the miR-3680-3p/GMFG axis.

GMFG (glia maturation factor gamma) inhibits lung cancer growth by activating p53 signaling pathway

ABSTRACT The tumor-promoting or tumor-suppressing functions of Glia maturation factor gamma (GMFG) were described in several cancers. However, how GMFG regulates lung cancer progression is elusive. Bioinformatics analysis was employed to analyze GMFG expression in lung adenocarcinoma (LUAD) and lung squamous cancer (LUSC) as well as its significance in prognosis prediction and diagnosis in lung cancer patients. CCK8 and colony formation assays were adopted to evaluate the impact of GMFG overexpressing and depleting on lung cancer cell proliferation. And in vivo experiments were implemented. Luciferase reporter assays were used to disclose the signaling pathway mediated by GMFG in lung cancer. GMFG expression was lower in LUSC and LUAD tissues compared with normal lung tissues based on TCGA and GTEx databases. Low GMFG expression was associated with lower overall survival and shorter disease specific survival compared high GMFG expression. In vitro loss and gain functions assays demonstrated that ectopically GMFG expression dampened the lung cancer cell proliferation while GMFG knockout escalated the cell proliferation. The promoting effect of GMFG knockout on lung cancer tumorgenesis was also observed in vivo. More interesting, GMFG overexpression reinforced the p53 signaling pathway in lung cancer cells, conversely GMFG deficiency disrupted p53 signaling pathway. In conclusion, we revealed that GMFG is fundamental to p53 signaling pathway to inhibit lung cancer progression, highlighting the importance of GMFG as a p53 inducer for lung cancer patient’s diagnosis and therapy.

Glia maturation factor-gamma inhibits the proliferation of human colorectal cancer LoVo cells and affects the cytoskeletal motion of human umbilical vein endothelial cells

Glia maturation factor-gamma inhibits the proliferation of human colorectal cancer LoVo cells and affects the cytoskeletal motion of human umbilical vein endothelial cells

Glia Maturation Factor-γ Regulates Autophagy in Murine Macrophages

Macrophages are centrally involved in both innate and adaptive immunity by secretion of regulatory immune mediators, phagocytosis, and presentation of antigens. Autophagy is an essential intracellular quality control process with multiple roles in normal physiological homeostasis. In macrophages, autophagy pathway also plays an important role in facilitating proper immunological functions by secretion of diverse proteins involved in inflammation, intercellular signaling, and pathogen infection. Accordingly, perturbations in autophagy have been linked to inflammatory diseases and defective immune responses against pathogens. It has been shown that actin cytoskeleton dynamics and actin regulatory proteins play an important role in the regulation of autophagosome formation and maturation. Here, to determine whether Glia maturation factor gamma (GMFG), a novel regulator of the actin-related protein-2/3 (Arp2/3) complex, regulates autophagy, we silenced GMFG in murine bone marrow-derived macrophages (BMDM) and Raw-264.7 macrophages. We found that GMFG is required for autophagy activation. Immunoblot analysis demonstrated that knockdown of GMFG decreased the LC3-II expression levels in basal condition, starvation- or rapamycin-induced autophagy in murine Raw-264.7 macrophages. Knockdown of GMFG did not prevent autophagic flux, as treatment with lysosomal V-ATPase inhibitor bafilomycin A1 was able to enhance LC3-II levels to comparable level of control siRNA treatment cells. Immunofluorescence analysis also exhibited the number of LC3 puncta reduced per cell during the starvation- or rapamycin-induced autophagy in GMFG knockdown cell compared with control cells, consistent with a defective autophagy process. These observations suggest that GMFG acts as an early stage in autophagosome formation. The serine/threonine kinase mTOR is a central regulator of the canonical autophagic response of cells to nutrient starvation. In addition, AMP-activated protein kinase (AMPK), which is a key sensor of cellular energy status, can trigger autophagy by inhibiting mTOR. Therefore, we decided to investigate the effect of GMFG knockdown on upstream of autophagy pathway. GMFG knockdown decreased the phosphorylation of AMPK and mTOR levels, but did not remarkably decrease Atg5, Atg7, or Atg16L1 levels (autophagy pathway core proteins in the early steps of autophagosome biogenesis) in GMFG knockdown murine macrophage. Moreover, we observed that Atg9 did not colocalization with EEA in both GMFG knockdown and control cells under basal condition. Whereas, after rapamycin treatment for 4 hours, Atg9 translocated to EEA and Atg9 colocalization with EEA decreased in GMFG knockdown macrophages compared with control cells. We also observed increase in the appearance of lysosomes by staining with lysotracker and increased staining for Lamp1-positive compartments by immunofluorescence in GMFG knockdown of macrophages. Furthermore, knockdown of GMFG enhanced the levels of the autophagy substrate p62 and reduced the lysosomal protease cathepsin D and cathepsin C maturation and activity. These observations suggest that GMFG knockdown induced the insufficient autophagy associated with compromised lysosomal function. Importantly, we found that knockdown of GMFG remarkably decreased the phosphorylation of YAP, LATS1 and the levels of RhoA/Rock, suggesting that downregulation of GMFG affected the autophagy associated with YAP-RhoA/Rock signaling pathway. Together, our finding shows that GMFG is an important molecular in regulating autophagy, further study the mechanisms responsible for GMFG's function will be of great value in the understand the autophagy related disease in immune system. Disclosures No relevant conflicts of interest to declare.

Glia maturation factor gamma, is a novel diagnostic marker of leukemia, has TAL1 binding sites in its promoter

Glia maturation factor gamma (GMFG) overexpression increases T-cell migration and adhesion. Its high expression in colorectal and ovarian cancer is associated with chemoresistance and adverse prognosis. However, it remains elusive if GMFG plays a role in the development and progression of leukemia. Tissue invasion in leukemia has long been known as the reason for disease progression as leukemic cells accumulates in bone marrow, spleen and lymph nodes. This tissue invasion requires cell motility and migration. The aim of the present study was to find any association of GMFG expression with leukemia and its potential to be a diagnostic marker. For this purpose, we have carried out expression profiling of GMFG among a cohort of leukemia patients. Liquid biopsies were collected and subjected to GMFG expression analysis. We found that GMFG expression was significantly elevated among leukemia patients of all age groups relative to their age match controls. Further promoter analysis of GMFG reveals binding sites for T-cell Acute Lymphocytic Leukemia 1 (TAL1) and associated transcription factors. TAL1 is master transcription factor which, in coordination with other factors like GATA-binding protein 1 (GATA-1) and LIM domain only 2 (LMO2), regulates gene expression in T-cell leukemia. The induced TAL1 expression coincides with upregulated GMFG expression. Together these data suggest that TAL1 may bind to GMFG promoter to increase its expression. GMFG may play a crucial role in the motility of leukemic cells and facilitates their tissue invasion which leads to therapy resistance.

Glia Maturation Factor-Gamma Modulates M2 Activation of Macrophage Via Suppression of Mitochondria Function

Macrophages are innate immune cells of dynamic phenotype as they can acquire distinct phenotypes and biological functions depending on the microenvironment and the metabolic state. In response to various signals, macrophages can undergo M1 activation (stimulated by TLR ligand and interferon-gamma) or M2 activation (stimulated by IL-4/IL-13, immunocomplexes, TGFbeta, and IL-10). M1 and M2 macrophages have distinct metabolic phenotypes that differ from those of resting macrophages. M1 macrophages rely on aerobic glycolysis, while M2 macrophages use fatty acid oxidation to fuel mitochondrial oxidative phosphorylation. IL-4/IL13 induced M2 macrophages polarization has an enhanced mitochondrial oxygen-consumption rate, whereas M1 has a significantly decreased oxygen-consumption rate. This indicates that macrophage phenotypes can be regulated by the different aspects of cellular metabolism. Thus, the identification of molecules and mechanisms associated with the phenotypic switch of them provides a molecular basis for macrophage-centered therapeutic strategies. Glia maturation factor gamma (GMFG), a novel regulator of the actin-related protein-2/3 (Arp2/3) complex, is predominantly expressed in inflammatory cells. We have previously found that GMFG-knockdown macrophages enhanced IL-4/IL-13-stimulated M2 markers and promoted iron metabolism change to iron recycling states, but its underlying molecular mechanism remains unclear. In this study, to explore the possible role of GMFG on mitochondrial function, we measured mitochondrial respiration by determining the oxygen consumption rate (OCR) and glycolytic flux in GMFG-knockdown M0 (unactivated), M1 and M2 activated bone marrow-derived macrophages using an extracellular flux analyzer. We observed that decreased basal oxygen consumption (ORC), diminished maximal respiratory capacity (MRC) and reduced cellular ATP levels in GMFG-knockdown M0 and M2 macrophages compared with control siRNA transfected macrophages. Whereas, there were no detectable changes in the rate of extracellular acidification (ECAR) in GMFG-knockdown cells. Importantly, oxygen consumption in GMFG-knockdown cells were reduced to the same level of TGFbeta-stimulated cells, but not to the level of M1 macrophages, reflecting the loss of mitochondria fitness similar to TGFbeta-stimulated M2 macrophages. Additionally, we observed a moderately increased mitochondrial ROS levels in GMFG-knockdown cells which is similar to increased levels in M2 macrophages, but significantly lower than the increased level in M1 macrophages. We did not observe decreased mitochondrial membrane potential, but found moderately increased mitochondria mass in the GMFG-knockdown cells, which may be due to the accumulation of dysfunctional mitochondria. Moreover, we found that suppressed OCR in GMFG-knockdown cells were accompanied by suppressed mitochondrial biogenesis as shown by decreased protein expression levels of mitochondrial complex I, ISCU, MnSOD2 and CuZnSOD1 by immunoblotting analysis. We also found that GMFG expression was downregulated by treatment with hydrogen peroxide at a concentration of 0.25 mM, which accompanied with increased protein levels of TfR1 and IRP1 in macrophages. Increased mROS, TfR1 and decreased MnSOD2, CuZnSOD1 in GMFG-knockdown cells can be attenuated by treatment of 5 mM NAC and 0.01 mM MitoTEMPO for 1 hour. Furthermore, we found that the remarkable decreased protein level of Dynamin-related protein 1(Drp1), which promoted mitochondria fission, in GMFG-knockdown cells. Consistent with this, confocal microscope analysis exhibited predominantly punctate mitochondria in GMFG-knockdown cells compared with the elongated tubules mitochondria in control cells. Finally, immunoblotting analysis showed that GMFG-knockdown macrophages showed an increase of phosphorylated-CREB, which might be contributed to enhanced M2 markers in GMFG-knockdown cells. Together, these results suggest that downregulation of GMFG skewing macrophages toward a M2 phenotype through suppression of mitochondrial function, which associated with impaired mitochondria fission/fusion balance. Our results indicate that GMFG plays an important role in maintaining mitochondrial function and dynamic, which is necessary for macrophage reprogramming. DisclosuresNo relevant conflicts of interest to declare.

Glia Maturation Factor-Gamma Regulates Alternative Activation of Macrophage By the Modulation of Iron Homeostasis through HO-1 in Ex Vivo

Macrophages play a key role at the crossroad of iron metabolism and immune function. They store and recycle iron derived from the phagocytosis of senescent erythrocytes. Macrophages iron homeostasis is coupled to their remarkable heterogeneity and functional plasticity. It is well known that the macrophage polarization process dictates expression profiles of genes involved in iron metabolism. M1 macrophages are characterized by increased iron retention, whereas, M2 macrophages showed increased iron recycling. However, the molecular mechanisms underlying iron metabolism link to regulation of macrophage-polarized phenotype are not fully understood. Glia maturation factor gamma (GMFG), a novel regulator of the actin-related protein-2/3 (Arp2/3) complex, is predominantly expressed in inflammatory cells. We have previously found that GMFG mediated macrophage resistance to Salmonella infection, but its function in iron metabolism and macrophage phenotype remains unclear. In this study, we explored the important role of GMFG in the regulation of iron metabolism and macrophages phenotypes interlinked. We found that GMFG expression was downregulated in a dose-dependent manner in murine bone marrow-derived macrophages (BMDM) and RAW-264.7 cells by treated with iron or heme. Immunoblotting analysis demonstrated that knockdown of GMFG in BMDM and RAW-264.7 cells lead to remarkable increased the protein levels of ferroportin (Fpn), transferrin receptor 1 (TfR1), as well as heme oxygenase 1 (HO-1), whereas decreased the ferritin light chain 1 (FtL1) compared with control siRNA transfected BMDM or Raw264.7 cells. Knockdown of GMFG display higher iron export capacity and elevated intracellular labile iron pool (LIP) compared with control macrophages. These results suggest that GMFG is the crucial regulator in macrophages iron metabolism because its downregulation caused an alteration in iron-handling proteins similar to IL-4 induced M2 polarization phenotype. Quantitative PCR analysis showed that M2 alternative activation markers Arg1, Mrc1, and Ym1 were noted to be induced in GMFG knockdown macrophages in the absence of M2-induceer cytokine treatment, confirming the skewing of these macrophages toward M2 alternative activation. Moreover, treatment of GMFG-knockdown BMDM or RAW-264.7 cells with Th2 cytokines IL-4 or IL-13 markedly enhanced the induction of several genes characteristic of M2 alternative activation, including Arg1, Mrc1, and Ym1 compared with control macrophages. Furthermore, M2 skewing was confirmed by the enhancement of the IL-4-induced Arg1 protein levels in GMFG knockdown macrophages relative to control macrophages by immunoblotting analysis. Interestingly, GMFG knockdown macrophages further markedly enhanced the IL-4-induced protein levels of HO-1 and TfR1 in M2 phenotypes, but there was no marked altered in protein level of Fpn or FtL1 compared with control macrophages. These results indicated that knockdown of GMFG might enhance the M2 phenotypes through modulation of HO-1. Finally, we observed that GMFG knockdown macrophages showed more accumulation of transcriptional factor Nrf2 in nuclear without alternation of its transcriptional expression levels compared with control macrophages, suggesting that downregulation of GMFG skewing macrophages toward a M2 phenotype might be through regulation of Nrf2-mediated HO-1 expression. Our results indicate that GMFG plays an important role in the regulation of M2 alternative activation through modulation of iron metabolism and act as a negative feedback loop in macrophages. DisclosuresNo relevant conflicts of interest to declare.

Glia Maturation Factor-Gamma Regulates the Intracellular Growth of Salmonella Via Modulation of Ferroportin in Murine Macrophages

Salmonella is an intracellular bacterial pathogen that replicates within membrane-bound compartment and alters host iron metabolism for its own survival. Persistent survival and replication within phagocytes is central to the pathogenesis of Salmonella infections. Macrophages play a critical role in regulating iron metabolism for securing body iron sufficiency and controlling the availability of iron for intracellular proliferation of pathogens. However, the relationship of Salmonella-induced changes of macrophage iron metabolism to the survival and replication mechanism of this pathogen within macrophages remains poorly understood. Thus, it is critical to identify the host factors involved in the intracellular survival and replication of Salmonella in order to design more-efficient antimicrobial therapeutics. Glia maturation factor gamma (GMFG), a novel regulator of the actin-related protein-2/3 (Arp2/3) complex, is predominantly expressed in inflammatory cells. We have previously demonstrated that GMFG negatively regulate TLR4-induced proinflammatory signaling, but its function in macrophage response to intracellular bacteria infection remains unclear. In this study, we investigated the role of GMFG in Salmonella-infected murine macrophages by using small-interfering RNA (RNAi) techniques to knockdown GMFG. We found that knockdown of GMFG significantly enhanced the numbers of intracellular Salmonella growth (>3-fold, p< 0.008) at 24 hr postinfection compared with control siRNA transfected Raw264.7 macrophages. However, there was no significant difference in growth numbers of bacteria observed at 4 hr postinfection, indicating that GMFG does not influence bacterial phagocytosis. Immunofluorescence microscopy also revealed an accumulation of Salmonella, in GMFG knockdown macrophages at 24 hr postinfection. Knockdown of GMFG results in marked decreased the iron exporter ferroportin protein levels and increased iron storage ferritin-L protein levels in Raw264.7 macrophages. Further, the intracellular iron content was elevated in GMFG-knockdown macrophages compared with control macrophages (1.9-fold, p< 0.05). These observations indicate that regulatory impact of GMFG in Salmonella intracellular growth may be through modulation of macrophage iron metabolism. Moreover, consistent with previous studies, we found that Raw264.7 macrophage infections with Salmonella increase the expression of the iron transporter ferroportin and ferritin, indicating this is the host defense strategy against infection with intracellular microbes by limiting their access to iron. Although there was no marked altered in GMFG protein level after 24hr infection with Salmonella, GMFG knockdown macrophage infected with Salmonella displayed increased the intracellular iron content and iron storage protein ferritin compared with control macrophages. Further analysis of cytokines expression in Salmonella-infected GMFG-knockdown macrophage revealed enhanced the proinflammatory TNF-alpha mRNA (1.86-fold, p<0.05) and anti-inflammatory IL-10 mRNA (2.2-fold, p< 0.0) at 24 hr postinfection, which is paralleled with increased intracellular Salmonella replication. Our results suggest that GMFG modulation of crucial pathways of macrophage iron metabolism and immune function in murine macrophages infected with Salmonella. DisclosuresNo relevant conflicts of interest to declare.

Glia Maturation Factor-Gamma Mediates Human Monocytes Migration Through Regulating β1/β2-Integrin Recycling

Recruitment of monocytes is essential for effective control and clearance of invading pathogens by migrating to the sites of infection, but recruited monocytes also contribute to the pathogenesis of chronic inflammatory and degenerative diseases, such as rheumatoid arthritis, multiple sclerosis, atherosclerosis and cancer. Thus, understanding the mechanisms controlling monocytes migration within different environments is of paramount importance. Although it is clear that adhesion signaling via integrin receptors and the surrounding ECM play a significant role in regulating migration of monocytes to site of inflammation, the underlying cellular and molecular mechanisms responsible for these process is still not fully characterized. Defining the molecular circuits through which integrins regulate monocytes motility is therefore important for gaining a better understanding of monocytes function. Glia maturation factor gamma (GMFG), a novel ADF/cofilin superfamily protein that is predominantly expressed in inflammatory cells, has been implicated in regulating actin reorganization. We have previously demonstrated that GMFG plays a role in regulating neutrophil chemotaxis and migration. We now examine whether GMFG has similar effects on monocytes and the cellular mechanism for these effects by using small-interfering RNA to knockdown GMFG in human primary monocytes. Knockdown of endogenous GMFG results in significantly reduced (220.6 ± 9.4 to 89.0 ± 3.2, p<0.003) chemotactic migration toward SDF-1 and enhanced adhesion on fibronectin, VCAM-1 and ICAM-1 compared with control siRNA transfected cells. Flow cytometry analysis shows that knockdown of GMFG enhances the expression of β1-, β2−integrin on the cell surface compared with control cells. Confocal microscopy analysis exhibited that GMFG is colocalized with internalized β1-, β2-integrin in early endosomes in primary monocytes. However, an internalization assay shows that β1- and β2-integrin were internalized with similar kinetics during the initial uptake time points both in control or GMFG knockdown primary monocytes. These data demonstrated that internalization of β1- and β2-integrin was not impaired in GMFG knockdown cells, suggesting that GMFG regulates β integrins recycling. Antibody-based recycling assays showed that GMFG knockdown cells resulted in enhanced plasma membrane exocytosis of β1- and β2-integrin compared with control cells. Western blot analysis revealed that SDF-1 stimulated the phosphoryalation of FAK (Tyr397) is moderately (∼ 40%) increased in GMFG knockdown monocytes compared with control cells. These results suggest that impaired monocytes migrated toward SDF-1 stimulation in GMFG depletion monocytes is due to enhanced adhesion, which is caused by accelerated recycling of β1- and β2-integrin to the surface. Taken together, these results indicate that GMFG is required to maintain the proper expression levels of β1- and β2-integrin on the plasma membrane and is fundamental for integrin-mediated monocytes motility. Disclosures:No relevant conflicts of interest to declare.

Glia Maturation Factor-Gamma Regulates Macrophage Migration Through Control Recycling of alpha5beta1 Integrin in Endosome

AbstractAbstract 3273Macrophages play an essential role in defending against invading pathogens by migrating to the sites of infection, removing apoptotic cells, and secreting inflammatory cytokines. Macrophage migration is a critical step in the regulation of immune response and is also associated with many chronic inflammatory diseases such as rheumatoid arthritis, multiple sclerosis, atherosclerosis and cancer. Thus, understanding the mechanisms controlling macrophage migration within different environments is of paramount importance. Although it is clear that adhesion signaling via integrin receptors and the surrounding ECM play a significant role in regulating migration of macrophages to site of inflammation, the underlying cellular and molecular mechanisms responsible for these process is still not fully characterized. Defining the molecular circuits through which integrins regulate macrophage motility is therefore important for gaining a better understanding of macrophage function. Glia maturation factor gamma (GMFG), a novel ADF/cofilin superfamily protein that is predominantly expressed in inflammatory cells, has been implicated in regulating actin reorganization. We have previously demonstrated that GMFG plays a role in regulating neutrophil chemotaxis and migration. We now examine whether GMFG has similar effects on macrophage and the cellular mechanism for these effects by using small-interfering RNA technic to knockdown GMFG in human macrophage like THP-1cells. Knockdown of endogenous GMFG results in significantly reduced chemotactic migration toward fMLP and enhanced adhesion on fibronectin compared with control siRNA transfected cells. Flow cytometry analysis shows that GMFG-knockdown cells expresses significant amount of α5β1 integrin on the surface compared with control cells. Confocal microscopy analysis exhibited that GMFG was distributed throughout the cytosol and colocalized with the multiple endocytic compartment such as early endosome marker EEA1, Rab5, Rab4 and Rab11. Importantly, we found that most of the α5β1 integrins accumulated in clusters at the rear of the GMFG-knockdown cells, along with colocalization with endosome marker EEA1, rather than localization toward the leading edge in response to fMLP stimulation in control cells. These results suggest that impaired efficient α5β1 integrin recycling cause to increased adhesion, which is responsible for reduced macrophage migration on fibronectin in GMFG depletion macrophages. Accordingly, GMFG is required for proper trafficking of endosomal α5β1 integrin to the plasma membrane and is fundamental for integrin-mediated macrophage motility. Disclosures:No relevant conflicts of interest to declare.

Glia maturation factor gamma regulates the migration and adherence of human T lymphocytes

BackgroundLymphocyte migration and chemotaxis are essential for effective immune surveillance. A critical aspect of migration is cell polarization and the extension of pseudopodia in the direction of movement. However, our knowledge of the underlying molecular mechanisms responsible for these events is incomplete. Proteomic analysis of the isolated leading edges of CXCL12 stimulated human T cell lines was used to identify glia maturation factor gamma (GMFG) as a component of the pseudopodia. This protein is predominantly expressed in hematopoietic cells and it has been shown to regulate cytoskeletal branching. The present studies were undertaken to examine the role of GMFG in lymphocyte migration.ResultsMicroscopic analysis of migrating T-cells demonstrated that GMFG was distributed along the axis of movement with enrichment in the leading edge and behind the nucleus of these cells. Inhibition of GMFG expression in T cell lines and IL-2 dependent human peripheral blood T cells with shRNAmir reduced cellular basal and chemokine induced migration responses. The failure of the cells with reduced GMFG to migrate was associated with an apparent inability to detach from the substrates that they were moving on. It was also noted that these cells had an increased adherence to extracellular matrix proteins such as fibronectin. These changes in adherence were associated with altered patterns of β1 integrin expression and increased levels of activated integrins as detected with the activation specific antibody HUTS4. GMFG loss was also shown to increase the expression of the β2 integrin LFA-1 and to increase the adhesion of these cells to ICAM-1.ConclusionsThe present studies demonstrate that GMFG is a component of human T cell pseudopodia required for migration. The reduction in migration and increased adherence properties associated with inhibition of GMFG expression suggest that GMFG activity influences the regulation of integrin mediated adhesion.

Glia Maturation Factor-Gamma Negatively Modulates Fatty Acid-Induced Inflammation in Macrophages Via PI3K/AKT Pathway

Abstract 1096The macrophage-mediated inflammatory response plays a critical role in the development of obesity-related tissue inflammation and insulin resistance. Free-fatty acids (FFAs) are well-characterized factors causing production of inflammatory factors and insulin resistance. Glia maturation factor-gamma (GMFG), a member of the ADF/cofilin family of proteins that regulate actin cytoskeleton reorganization, is preferentially expressed in inflammatory cells, but its function in the macrophages immune response remains unclear. In this study, we investigated whether GMFG may affect FFAs-induced inflammatory reaction in macrophages. We show here that silencing of GMFG (80% inhibition of the endogenous expression levels) by transfected with GMFG siRNA significantly enhanced FFAs-induced production of proinflammatory cytokines and chemokines, including TNF-α, IL-6 and IL-8 compared to transfected non-targeting silencing siRNA in human peripheral blood monocytes-derived macrophage (1.2 × 104 ± 224.2 vs. 4.6 × 103 ± 98.4 molecules/ng, P < 0.001; 1.2 × 103 ± 56.1 vs. 2.3 × 102 ± 8.4 molecules/ng, P =0.001; 1.3 × 107 ± 5.8 × 105 vs. 4.9 × 106 ± 2.8 × 105 molecules/ng, P < 0.001) as determined by quantitative real time-PCR and confirmed by enzyme-linked immunosorbent assay (TNF-α: 9.4 × 102 ± 38.6 vs. 5.6 × 102 ± 31.1 ng/mL, P < 0.01; IL-6: 4.5 × 102 ± 31.3 vs. 2.2 × 102 ± 22.7 ng/mL, P < 0.01; IL-8: 2.2 × 103 ± 205.5 vs. 1.3 × 103 ± 113.9 ng/mL, P < 0.01). These increased inflammatory cytokines resulted from an increased activation of NF-κB and the ERK1/2 MAPK signaling pathway (based on increased NF-κB p65 phosphorylation, IκBα loss and enhanced phosphorylation of ERK1/2 in Western blot analysis) and a reduced phosphorylation of the PI3K/Akt/GSK3-β pathway following FFAs stimulation. Overexpression of GFP-GMFG in GMFG-silenced cells abrogated enhanced phosphorylation of NF-κB p65, ERK1/2 MAPK and reduced phosphorylation of Akt and GSK3-β. Furthermore, in cells in which GMFG was knockdown, FFAs induced an increase in the expression of SHIP1, a phosphatase that negatively regulates the PI3K signaling pathway, suggesting increased SHIP1 expression may be responsible for the augmentation of inflammatory cytokines following FFAs stimulation. We also show that silencing of GMFG enhances the oxidized low density lipoprotein (oxLDL) induced expression of TNF-α and IL-6 (2.9 × 104 ± 204.8 vs. 1.3 × 104 ± 153.9 molecules/ng, P < 0.01; 2.5 × 103 ± 31.3 vs. 9.2 × 102 ± 22.7 ng/mL, P < 0.01) in MDM. Taking together with the data that GMFG is constitutively expressed in macrophages and its expression is down-regulated by FFAs stimulation, GMFG might function as a novel negative regulator through participating in the PI3K/Akt signaling pathway, suggesting that macrophage-specific modulation of GMFG may be beneficial in the treatment of FFAs induced inflammation. Disclosures:No relevant conflicts of interest to declare.

Glia Maturation Factor-Gamma Negatively Modulates TLR4 Signaling In Macrophages Induced by Lipopolysaccharide (LPS).

AbstractAbstract 1482Toll-like receptor 4 (TLR4) plays a critical role in innate immunity that recognize pathogenic molecules and trigger inflammatory response. However, excessive activation of TLR4 activation may contribute to pathogenesis of autoimmune and inflammatory diseases. Therefore, the negative regulation of TLR4-triggered inflammatory response attracts much attention in recent years. Activation of TLR4 signaling pathways by lipopolysaccharide (LPS) leads to the production of a broad array of cytokines and mediators that coordinate the immune response in macrophages. Glia maturation factor gamma (GMFG), a member of the ADF/cofilin family of proteins that regulate actin cytoskeleton reorganization, is preferentially expressed in inflammatory cells, but its function in macrophages immune response remains unclear. In this study, we investigated whether GMFG participates in the molecular events underlying the inflammatory reaction to LPS in macrophages by knockdown of GMFG using small-interfering RNA approach. We show here that knockdown of GMFG significantly enhanced LPS-induced production of proinflammatory cytokines and chemokines, including TNF-alpha, IL-1beta, IL-8, and MCP-1 in human peripheral blood monocytes-derived macrophage as determined by quantitative real time-PCR and confirmed by enzyme-linked immunosorbent assay. Silencing of GMFG expression potentiates LPS-induced activation of p38, ERK1/2 and NF-kappaB signaling pathways by Western blot analysis. Moreover, luciferase assay revealed that gene silencing of GMFG promoted LPS-induced NF-kappaB activity for ∼2.5- to 4-fold. Furthermore, we found that TLR4 protein expression level were higher in GMFG-silenced macrophage compared with that of the control siRNA-transfected macrophages after stimulated with LPS for 1 hour. These results suggest that GMFG negatively regulation of TLR4 signaling-induced inflammatory cytokines by modulation of TLR4 expression levels and its down-stream NF-kappaB and p38 MAPK signaling pathway. In summary, we report that GMFG, in macrophage, function as a novel negative regulator that participates in the regulation of TLR4-signaling pathway, implicating that macrophage-specific modulation of GMFG may be beneficial in the treatment of inflammation as well as autoimmune disease. Disclosures:No relevant conflicts of interest to declare.

Glia Maturation Factor-Gamma Mediates Lipopolysaccharide-Induced Inflammation Via p38 MAPK and NF-Kappab.

Abstract 3598Poster Board III-535Neutrophils are the most abundant leukocytes in the circulation and provide a primary innate immune defense function against bacterial pathogens before the development of a specific immune response. Activation of TLR4 signaling pathways by lipopolysaccharide (LPS) leads to the production of a broad array of cytokines and mediators that coordinate the immune response. The TLR4-mediated inflammatory response is tightly regulated, since excessive activation of TLR4 may induce a variety of inflammatory diseases, so the negative regulation of TLR-triggered inflammatory response attracts much attention. Glia maturation factor gamma (GMFG), a novel factor in actin cytoskeleton reorganization, is preferentially expressed in inflammatory cells, but its function in leucocytes is undefined. In this study, we investigated whether GMFG participates in the molecular events underlying the inflammatory reaction and how GMFG mediated neutrophils inflammatory response to LPS. We show here that small interfering RNA-mediated knockdown of GMFG expression in leukocytes exhibited greater than 80% inhibition of the endogenous expression levels of proinflammatory cytokines and chemokines, including TNF-alpha, IL-1beta, IL-6, and IL-8 using quantitative RT-PCR. These decreased cytokine productions could be partially restored by stimulation with TLR4 agonist LPS, but exhibit significantly lower level in silencing of GMFG cells compared to non-targeting silencing control cells (inhibition by 71%+/−3.9% of TNF-alpha, 68%+/−6.9% of IL-1beta, 50%+/−4.3% of IL-6 and 40%+/−3.1% of IL-8). Knockdown of GMFG expression results in decreased phospho-p38 MAPK levels as well as suppressed NF-kappaB by Western blot analysis. These data suggest that endogenous expression of GMFG is required for basal cytokine and chemokine response and their induced response to LPS stimulation. Moreover, immunofluorescence analysis revealed that gene silencing of GMFG inhibited LPS-induced nuclear translocation of NF-kappaB in myeloid HL-60 and THP-1 cells. In contrast, transient overexpression of GMFG significantly enhanced endogenous level of proinflammatory cytokines by 1.8∼2.2 fold, phosphor-p38 MAPK and nuclear translocation of NF-kappaB in the absence of LPS stimulation. These results suggest that GMFG-mediated regulation of inflammatory cytokines occurs through activation of p38 MAPK and NF-kappaB signaling pathway. Taken together, our findings identify GMFG as a novel factor that participates in the regulation of TLR4-mediated control of cytokine and chemokine expression through the p38 MAPK and NF-kappaB pathway. These data, if confirmed in in vivo experiments, implicates GMFG as a potential target for therapeutic intervention in neutrophil-mediated autoimmune disease. Disclosures:No relevant conflicts of interest to declare.

NMR solution structures of actin depolymerizing factor homology domains

Actin is one of the most conserved proteins in nature. Its assembly and disassembly are regulated by many proteins, including the family of actin-depolymerizing factor homology (ADF-H) domains. ADF-H domains can be divided into five classes: ADF/cofilin, glia maturation factor (GMF), coactosin, twinfilin, and Abp1/drebrin. The best-characterized class is ADF/cofilin. The other four classes have drawn much less attention and very few structures have been reported. This study presents the solution NMR structure of the ADF-H domain of human HIP-55-drebrin-like protein, the first published structure of a drebrin-like domain (mammalian), and the first published structure of GMF beta (mouse). We also determined the structures of mouse GMF gamma, the mouse coactosin-like domain and the C-terminal ADF-H domain of mouse twinfilin 1. Although the overall fold of the five domains is similar, some significant differences provide valuable insights into filamentous actin (F-actin) and globular actin (G-actin) binding, including the identification of binding residues on the long central helix. This long helix is stabilized by three or four residues. Notably, the F-actin binding sites of mouse GMF beta and GMF gamma contain two additional beta-strands not seen in other ADF-H structures. The G-actin binding site of the ADF-H domain of human HIP-55-drebrin-like protein is absent and distorted in mouse GMF beta and GMF gamma.

Regulation of granulosa and theca cell transcriptomes during ovarian antral follicle development.

Coordinated interactions between ovarian granulosa and theca cells are required for female endocrine function and fertility. To elucidate these interactions the regulation of the granulosa and theca cell transcriptomes during bovine antral follicle development were investigated. Granulosa cells and theca cells were isolated from small (<5 mm), medium (5-10 mm), and large (>10 mm) antral bovine follicles. A microarray analysis of 24,000 bovine genes revealed that granulosa cells and theca cells each had gene sets specific to small, medium and large follicle cells. Transcripts regulated (i.e., minimally changed 1.5-fold) during antral follicle development for the granulosa cells involved 446 genes and for theca cells 248 genes. Only 28 regulated genes were common to both granulosa and theca cells. Regulated genes were functionally categorized with a focus on growth factors and cytokines expressed and regulated by the two cell types. Candidate regulatory growth factor proteins mediating both paracrine and autocrine cell-cell interactions include macrophage inflammatory protein (MIP1 beta), teratocarcinoma-derived growth factor 1 (TDGF1), stromal derived growth factor 1 (SDF1; i.e., CXCL12), growth differentiation factor 8 (GDF8), glia maturation factor gamma (GMFG), osteopontin (SPP1), angiopoietin 4 (ANGPT4), and chemokine ligands (CCL 2, 3, 5, and 8). The current study examined granulosa cell and theca cell regulated genes associated with bovine antral follicle development and identified candidate growth factors potentially involved in the regulation of cell-cell interactions required for ovarian function.

Glia Maturation Factor Gamma (GMFG): A Cytokine-Responsive Protein During Hematopoietic Lineage Development and Its Functional Genomics Analysis

Human hematopoiesis was evaluated using the techniques of controlled stem cell differentiation, two-dimensional gel electrophoresis-based proteomics, and functional genomics. We provide the first report that glia maturation factor gamma (GMFG) is a cytokine-responsive protein in erythropoietin-induced and granulocyte-colony stimulating factor-induced hematopoietic lineage development. Results from global functional genomics analysis indicate that GMFG possesses several other features: hematopoietic tissue-specific gene expression, a promoter concentrated with high-score hematopoiesis-specific transcription factors, and possible molecular coevolution with a rudimentary blood/immune system. On the basis of our findings, we hypothesize that GMFG is a hematopoietic-specific protein that may mediate the pluripotentiality and lineage commitment of human hematopoietic stem cells.

Analysis of mRNAs that are enriched in the post-synaptic domain of the neuromuscular junction

The identity of synaptically-enriched genes was investigated by comparing the abundance of various mRNAs in the synaptic and extra-synaptic regions of the same muscle fibers. The mRNAs for several known synaptic proteins were significantly elevated in the synaptic region when measured by real-time PCR. The synaptic mRNAs were then further analyzed using microarrays and real-time PCR to identify putative regulators of the neuromuscular junction (NMJ). MRF4 was the only member of the MyoD family that was concentrated at the mature NMJ, suggesting that it may have a unique role in the maintenance of post-synaptic specialization. Three potential regulators of the NMJ were identified and confirmed by real-time PCR: glia maturation factor gamma was concentrated at the NMJ whereas Unr protein and protein tyrosine phosphatase were repressed synaptically. The identification of synaptically-repressed genes may indicate that synaptic specialization is created by a combination of positive and negative signals.

Gene Expression Versus Sequence for Predicting Function: Glia Maturation Factor Gamma Is Not A Glia Maturation Factor

It is standard practice, whenever a researcher finds a new gene, to search databases for genes that have a similar sequence. It is not standard practice, whenever a researcher finds a new gene, to search for genes that have similar expression (co-expression). Failure to perform co-expression searches has lead to incorrect conclusions about the likely function of new genes, and has lead to wasted laboratory attempts to confirm functions incorrectly predicted. We present here the example of Glia Maturation Factor gamma (GMF-gamma). Despite its name, it has not been shown to participate in glia maturation. It is a gene of unknown function that is similar in sequence to GMF-beta. The sequence homology and chromosomal location led to an unsuccessful search for GMF-gamma mutations in glioma. We examined GMF-gamma expression in 1432 human cDNA libraries. Highest expression occurs in phagocytic, antigen-presenting and other hematopoietic cells. We found GMF-gamma mRNA in almost every tissue examined, with expression in nervous tissue no higher than in any other tissue. Our evidence indicates that GMF-gamma participates in phagocytosis in antigen presenting cells. Searches for genes with similar sequences should be supplemented with searches for genes with similar expression to avoid incorrect predictions.