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1.
Life Sci ; 287: 120141, 2021 Dec 15.
Article in English | MEDLINE | ID: mdl-34774620

ABSTRACT

AIMS: SARI (suppressor of activator protein (AP)-1, regulated by interferon (IFN) was identified as a novel tumor suppressor by applying subtraction hybridization to terminally differentiating human melanoma cells. The anti-tumor activity of SARI and the correlation between expression and cancer aggression and metastasis has been examined in multiple cancers, but its potential role in oral squamous cell carcinomas (OSCC) has not been explored. METHODS: SARI expression was monitored in tumor tissues of OSCC patients by performing immunohistochemistry. Ectopic expression of SARI was achieved using a replication defective adenovirus expressing SARI (Ad.SARI). A nude mouse xenograft model was used to evaluate the in vivo efficacy of SARI. Endoplasmic reticulum (ER) stress was monitored in SARI infected OSCC cells by confocal microscopy. KEY FINDING: In this study, we demonstrate that SARI expression is significantly lower in OSCC tumor tissue as compared to normal adjacent tissue. Ectopic expression of SARI induces cancer-specific cell death in human OSCC cell lines and in a paclitaxel plus cisplatin non-responder OSCC patient-derived (PDC1) cell line. Mechanistically, SARI inhibits zinc finger protein GLI1 expression through induction of endoplasmic reticulum (ER) stress. Using a nude mouse xenograft model, we show that intratumoral injections of Ad.SARI significantly reduce PDC1 tumor burden, whereas treatment with an ER stress inhibitor efficiently rescues tumors from growth inhibition. SIGNIFICANCE: Overall, our data provides a link between induction of ER stress and inhibition of the GLI1/Hedgehog signaling pathway and the tumor suppressive activity of SARI in the context of OSCC.


Subject(s)
Basic-Leucine Zipper Transcription Factors/biosynthesis , Carcinoma, Squamous Cell/metabolism , Endoplasmic Reticulum Stress/physiology , Growth Inhibitors/biosynthesis , Mouth Neoplasms/metabolism , Squamous Cell Carcinoma of Head and Neck/metabolism , Tumor Suppressor Proteins/biosynthesis , Animals , Carcinoma, Squamous Cell/pathology , Cell Line, Tumor , Cell Survival/physiology , HEK293 Cells , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Mouth Neoplasms/pathology , Squamous Cell Carcinoma of Head and Neck/pathology , Xenograft Model Antitumor Assays/methods
2.
Cell Cycle ; 19(23): 3260-3276, 2020 12.
Article in English | MEDLINE | ID: mdl-33222607

ABSTRACT

Non-small cell lung cancer (NSCLC) is a leading cause of cancer death in both men and women. microRNAs (miRs) can exert important functions in cancer development. However, the role of miR-877 in NSCLC as it relates to tartrate resistant acid phosphatase 5 (ACP5) is unknown. For this study, the gain-and-loss-of-function experiments were performed to explore the effects of miR-877 and ACP5 on NSCLC. miR-877 expression in LC and paracancerous tissues, lung epithelial cell line and NSCLC cell lines was detected, and the association between miR-877 expression and clinical features of LC patients was analyzed. The levels of ACP5, epithelial-mesenchymal transition (EMT) markers and apoptosis-related proteins were measured. In vivo experiments were conducted for further validation. Consequently, we found that miR-877 expression was lowered in LC tissues and cell lines, and correlated with clinical stage, differentiation, lymph node metastasis and prognosis of NSCLC patients. Additionally, miR-877 was determined to inhibit ACP5 activity, and miR-877 downregulated the PI3K/AKT pathway by silencing ACP5. Furthermore, overexpression of miR-877 inhibited the viability, migration, invasion and EMT of NSCLC cells, but promoted cell apoptosis. In conclusion, miR-877 overexpression inhibited malignant biological behaviors of NSCLC cells by downregulating ACP5 and inactivating the PI3K/AKT pathway.


Subject(s)
Carcinoma, Non-Small-Cell Lung/metabolism , MicroRNAs/biosynthesis , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Tartrate-Resistant Acid Phosphatase/metabolism , A549 Cells , Aged , Animals , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Female , Growth Inhibitors/biosynthesis , Growth Inhibitors/genetics , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , MicroRNAs/genetics , Middle Aged , Phosphatidylinositol 3-Kinases/genetics , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/genetics , Tartrate-Resistant Acid Phosphatase/antagonists & inhibitors , Tartrate-Resistant Acid Phosphatase/genetics , Xenograft Model Antitumor Assays/methods
3.
Metab Eng ; 54: 54-68, 2019 07.
Article in English | MEDLINE | ID: mdl-30851381

ABSTRACT

Chinese hamster ovary (CHO) cells in fed-batch cultures are known to consume large amounts of nutrients and divert significant portion of them towards the formation of byproducts, some of which, including lactate and ammonia, are known to be growth inhibitory in nature. A major fraction of these inhibitory metabolites are byproducts or intermediates of amino acid catabolism. Limiting the supply of amino acids has been shown to curtail the production of corresponding inhibitory byproducts resulting in enhanced growth and productivities in CHO cell fed-batch cultures (Mulukutla et al., 2017). In the current study, metabolic engineering of CHO cells was undertaken in order to reduce the biosynthesis of these novel growth inhibitors. Phenylalanine-tyrosine (Phe-Tyr) and branched chain amino acid (BCAA) catabolic pathways were engineered as part of this effort. Four genes that encode enzymes in the Phe-Tyr pathway, which were observed to be minimally expressed in CHO cells, were in turn overexpressed. Metabolically engineered cells were prototrophic to tyrosine and had reduced production of the inhibitory byproducts from Phe-Tyr pathway including 3-phenyllactate and 4-hydroxyphenyllactate. In case of BCAA catabolic pathway, branched chain aminotransferase 1 (BCAT1) gene, which encodes the enzyme that catalyzes the first step in the catabolism of BCAAs, was knocked out in CHO cells. Knockout (KO) of BCAT1 function completely eliminated production of inhibitory byproducts from BCAA catabolic pathway, including isovalerate, isobutyrate and 2-methylbutyrate, resulting in significantly enhanced cell growth and productivities in fed-batch cultures. This study is first of its kind to demonstrate that metabolic engineering of essential amino acid metabolism of CHO cells can significantly improve cell culture process performance.


Subject(s)
Batch Cell Culture Techniques , Growth Inhibitors/biosynthesis , Growth Inhibitors/genetics , Metabolic Engineering , Amino Acids/genetics , Amino Acids/metabolism , Animals , CHO Cells , Cricetinae , Cricetulus
4.
Cell Mol Neurobiol ; 37(1): 37-42, 2017 Jan.
Article in English | MEDLINE | ID: mdl-26897357

ABSTRACT

Glioblastoma (GBM) is the most common malignant brain tumor with poor prognosis and limited treatment options. Tumor suppressor candidate 1 (TUSC1) was recently identified as a potential tumor suppressor in human cancers. However, the expression and potential function of TUSC1 in GBM remain unclear. Herein, we report that TUSC1 is significantly decreased in GBM tissues and cell lines. Patients with high levels of TUSC1 displayed a significant better survival compared with those with low levels of TUSC1. Functional experiments demonstrated that exogenous expression of TUSC1 inhibited GBM cell proliferation and induced G1 phase arrest by down-regulating CDK4. Moreover, overexpression of TUSC1 retarded tumor growth in vivo. Together, our findings revealed that TUSC1 might be a crucial tumor suppressor gene and a novel therapeutic target for GBM.


Subject(s)
Brain Neoplasms/metabolism , Glioblastoma/metabolism , Growth Inhibitors/biosynthesis , Tumor Suppressor Proteins/biosynthesis , Animals , Brain Neoplasms/mortality , Brain Neoplasms/prevention & control , Cell Line, Tumor , Cell Proliferation/physiology , Glioblastoma/mortality , Glioblastoma/prevention & control , HEK293 Cells , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Predictive Value of Tests , Survival Rate/trends
5.
Biosci Biotechnol Biochem ; 80(1): 89-94, 2016.
Article in English | MEDLINE | ID: mdl-26168358

ABSTRACT

Isoflavones play important roles in plant-microbe interactions in rhizospheres. Soybean roots secrete daidzein and genistein to attract rhizobia. Despite the importance of isoflavones in plant-microbe interactions, little is known about the developmental and nutritional regulation of isoflavone secretion from soybean roots. In this study, soybeans were grown in hydroponic culture, and isoflavone contents in tissues, isoflavone secretion from the roots, and the expression of isoflavone conjugates hydrolyzing beta-glucosidase (ICHG) were investigated. Isoflavone contents did not show strong growth-dependent changes, while secretion of daidzein from the roots dramatically changed, with higher secretion during vegetative stages. Coordinately, the expression of ICHG also peaked at vegetative stages. Nitrogen deficiency resulted in 8- and 15-fold increases in secretion of daidzein and genistein, respectively, with no induction of ICHG. Taken together, these results suggest that large amounts of isoflavones were secreted during vegetative stages via the hydrolysis of (malonyl)glucosides with ICHG.


Subject(s)
Genistein/metabolism , Glycine max/metabolism , Growth Inhibitors/metabolism , Isoflavones/metabolism , Plant Proteins/genetics , Plant Roots/metabolism , beta-Glucosidase/genetics , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Growth Inhibitors/biosynthesis , Hydrolysis , Hydroponics , Isoflavones/biosynthesis , Nitrogen/deficiency , Nitrogen/pharmacology , Plant Proteins/metabolism , Plant Roots/drug effects , Plant Roots/genetics , Plant Roots/microbiology , Rhizobiaceae/physiology , Rhizosphere , Glycine max/drug effects , Glycine max/genetics , Glycine max/microbiology , Symbiosis/physiology , beta-Glucosidase/metabolism
6.
Cell Mol Neurobiol ; 35(8): 1165-73, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26012781

ABSTRACT

In recent years, microRNA has become a hotspot in research on diseases, especially in the initiation and progression of different types of cancer. In this study, we found that miR-218 could inhibit growth and metabolism in gliomas by directly targeting E2F2. First, we obtained data from the Chinese Glioma Genome Atlas (CGGA) database to analyze miR-218 expression in different grades of gliomas. The effects of miR-218 on cell cycle progression and cell proliferation in U87 and U251 cell lines were investigated by flow cytometry, specifically CCK8 assay and tablet cloning, respectively. Glucose consumption and lactate production of glioma cell lines were measured by correlative test kits. Furthermore, we used Western blot analysis and luciferase reporter assay to identify the direct and functional target of miR-218. Data from the CGGA database and real-time quantitative reverse transcription-PCR demonstrated that miR-218 was obviously reduced in human glioblastoma tissues, as well as in the cell lines. When miR-218 level was elevated in vitro, cell cycle progression was arrested in the G1 phase, and cell proliferation was dramatically inhibited. Both glucose consumption and lactate production of glioma cells were significantly reduced. Western blot analysis and luciferase reporter assay revealed that E2F2 was a direct target of miR-218 in glioma cells. This investigation demonstrated that elevated E2F2 expression could partly weaken the effect of miR-218 in vitro. This study also showed that miR-218 may be a repressor in glioma by directly targeting E2F2, as well as a potential therapeutic target in gliomas.


Subject(s)
Brain Neoplasms/metabolism , E2F2 Transcription Factor/biosynthesis , Glioblastoma/metabolism , Growth Inhibitors/biosynthesis , MicroRNAs/biosynthesis , Brain Neoplasms/genetics , Brain Neoplasms/prevention & control , Cell Line, Tumor , E2F2 Transcription Factor/genetics , Gene Targeting/methods , Glioblastoma/genetics , Glioblastoma/prevention & control , Growth Inhibitors/genetics , Humans , MicroRNAs/genetics
7.
PLoS One ; 10(4): e0123407, 2015.
Article in English | MEDLINE | ID: mdl-25853862

ABSTRACT

The dormancy of tumor cells is a major problem in chemotherapy, since it limits the therapeutic efficacy of anti-tumor drugs that only target dividing cells. One potential way to overcome chemo-resistance is to "wake up" these dormant cells. Here we show that the opioid antagonist methylnaltrexone (MNTX) enhances the effect of docetaxel (Doc) by blocking a cell growth-suppressive pathway. We found that PENK, which encodes opioid growth factor (OGF) and suppresses cell growth, is predominantly expressed in diffuse-type gastric cancers (GCs). The blockade of OGF signaling by MNTX releases cells from their arrest and boosts the effect of Doc. In comparison with the use of Doc alone, the combined use of Doc and MNTX significantly prolongs survival, alleviates abdominal pain, and diminishes Doc-resistant spheroids on the peritoneal membrane in model mice. These results suggest that blockade of the pathways that suppress cell growth may enhance the effects of anti-tumor drugs.


Subject(s)
Naltrexone/analogs & derivatives , Receptors, Opioid/biosynthesis , Stomach Neoplasms/drug therapy , Taxoids/administration & dosage , Animals , Antineoplastic Combined Chemotherapy Protocols , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Proliferation/drug effects , Docetaxel , Drug Synergism , Growth Inhibitors/biosynthesis , Growth Inhibitors/genetics , Humans , Mice , Naltrexone/administration & dosage , Narcotic Antagonists/administration & dosage , Quaternary Ammonium Compounds/administration & dosage , Receptors, Opioid/genetics , Stomach Neoplasms/genetics , Stomach Neoplasms/pathology
8.
J Immunol ; 190(6): 2485-9, 2013 Mar 15.
Article in English | MEDLINE | ID: mdl-23420891

ABSTRACT

During early stages of B-lineage differentiation in bone marrow, signals emanating from IL-7R and pre-BCR are thought to synergistically induce proliferative expansion of progenitor cells. Paradoxically, loss of pre-BCR-signaling components is associated with leukemia in both mice and humans. Exactly how progenitor B cells perform the task of balancing proliferative burst dependent on IL-7 with the termination of IL-7 signals and the initiation of L chain gene rearrangement remains to be elucidated. In this article, we provide genetic and functional evidence that the cessation of the IL-7 response of pre-B cells is controlled via a cell-autonomous mechanism that operates at a discrete developmental transition inside Fraction C' (large pre-BII) marked by transient expression of c-Myc. Our data indicate that pre-BCR cooperates with IL-7R in expanding the pre-B cell pool, but it is also critical to control the differentiation program shutting off the c-Myc gene in large pre-B cells.


Subject(s)
B-Lymphocyte Subsets/immunology , B-Lymphocyte Subsets/metabolism , Down-Regulation/immunology , Interleukin-7/physiology , Proto-Oncogene Proteins c-myc/genetics , Stem Cells/immunology , Stem Cells/metabolism , Animals , B-Lymphocyte Subsets/pathology , Cell Differentiation/genetics , Cell Differentiation/immunology , Cell Line , Cells, Cultured , Coculture Techniques , Down-Regulation/genetics , Gene Knock-In Techniques , Growth Inhibitors/biosynthesis , Growth Inhibitors/genetics , Interleukin-7/antagonists & inhibitors , Interleukin-7/metabolism , Mice , Mice, Knockout , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/immunology , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Proto-Oncogene Proteins c-myc/biosynthesis , Receptors, Interleukin-7/antagonists & inhibitors , Receptors, Interleukin-7/physiology , Signal Transduction/genetics , Signal Transduction/immunology , Stem Cells/pathology , Stromal Cells/immunology , Stromal Cells/metabolism , Stromal Cells/pathology
9.
Microbiology (Reading) ; 159(Pt 1): 36-45, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23125119

ABSTRACT

The genetic basis of the biosynthesis of the germination-arrest factor (GAF) produced by Pseudomonas fluorescens WH6, and previously identified as 4-formylaminooxyvinylglycine, has been investigated here. In addition to inhibiting the germination of a wide range of grassy weeds, GAF exhibits a selective antimicrobial activity against the bacterial plant pathogen Erwinia amylovora. We utilized the in vitro response of E. amylovora to GAF as a rapid screen for loss-of-function GAF phenotypes generated by transposon mutagenesis. A Tn5 mutant library consisting of 6364 WH6 transformants was screened in this Erwinia assay, resulting in the identification of 18 non-redundant transposon insertion sites that led to loss of GAF production in WH6, as confirmed by TLC analysis. These insertions mapped to five different genes and four intergenic regions. Three of these genes, including two putative regulatory genes (gntR and iopB homologues), were clustered in a 13 kb chromosomal region containing 13 putative ORFs. A GAF mutation identified previously as affecting an aminotransferase also maps to this region. We suggest that three of the genes in this region (a carbamoyltransferase, an aminotransferase and a formyltransferase) encode the enzymes necessary to synthesize dihydroGAF, the putative immediate precursor of GAF in a proposed GAF biosynthetic pathway. RT-qPCR analyses demonstrated that mutations in the gntR and iopB regulatory genes, as well as in a prtR homologue identified earlier as controlling GAF formation, suppressed transcription of at least two of the putative GAF biosynthetic genes (encoding the aminotransferase and formyltransferase) located in this 13 kb region.


Subject(s)
Biosynthetic Pathways/genetics , Glycine/analogs & derivatives , Growth Inhibitors/biosynthesis , Pseudomonas fluorescens/genetics , Anti-Bacterial Agents/biosynthesis , DNA Transposable Elements , Erwinia amylovora/drug effects , Erwinia amylovora/growth & development , Gene Expression Profiling , Glycine/biosynthesis , Multigene Family , Mutagenesis, Insertional , Pseudomonas fluorescens/metabolism , Real-Time Polymerase Chain Reaction
10.
J Immunol ; 189(12): 5590-601, 2012 Dec 15.
Article in English | MEDLINE | ID: mdl-23152566

ABSTRACT

Regulatory T cells play important roles in cancer development and progression by limiting the generation of innate and adaptive anti-tumor immunity. We hypothesized that in addition to natural CD4(+)CD25(+) regulatory T cells (Tregs) and myeloid-derived suppressor cells, tumor Ag-specific Tregs interfere with the detection of anti-tumor immunity after immunotherapy. Using samples from prostate cancer patients immunized with a DNA vaccine encoding prostatic acid phosphatase (PAP) and a trans-vivo delayed-type hypersensitivity (tvDTH) assay, we found that the detection of PAP-specific effector responses after immunization was prevented by the activity of PAP-specific regulatory cells. These regulatory cells were CD8(+)CTLA-4(+), and their suppression was relieved by blockade of CTLA-4, but not IL-10 or TGF-ß. Moreover, Ag-specific CD8(+) Tregs were detected prior to immunization in the absence of PAP-specific effector responses. These PAP-specific CD8(+)CTLA-4(+) suppressor T cells expressed IL-35, which was decreased after blockade of CTLA-4, and inhibition of either CTLA-4 or IL-35 reversed PAP-specific suppression of tvDTH response. PAP-specific CD8(+)CTLA-4(+) T cells also suppressed T cell proliferation in an IL-35-dependent, contact-independent fashion. Taken together, these findings suggest a novel population of CD8(+)CTLA-4(+) IL-35-secreting tumor Ag-specific Tregs arise spontaneously in some prostate cancer patients, persist during immunization, and can prevent the detection of Ag-specific effector responses by an IL-35-dependent mechanism.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , CTLA-4 Antigen/antagonists & inhibitors , Epitopes, T-Lymphocyte/immunology , Growth Inhibitors/antagonists & inhibitors , Interleukins/antagonists & inhibitors , Prostatic Neoplasms/immunology , Protein Tyrosine Phosphatases/physiology , T-Lymphocytes, Regulatory/immunology , Acid Phosphatase , Animals , CD8-Positive T-Lymphocytes/enzymology , CD8-Positive T-Lymphocytes/pathology , CTLA-4 Antigen/biosynthesis , Cancer Vaccines/administration & dosage , Cancer Vaccines/immunology , Cells, Cultured , Clinical Trials as Topic/methods , Coculture Techniques , Growth Inhibitors/biosynthesis , Humans , Interleukins/biosynthesis , Interleukins/metabolism , Male , Mice , Mice, SCID , Prostatic Neoplasms/enzymology , Prostatic Neoplasms/prevention & control , T-Lymphocytes, Regulatory/enzymology , T-Lymphocytes, Regulatory/pathology , Tumor Escape/immunology , Vaccines, DNA/administration & dosage , Vaccines, DNA/immunology
11.
J Immunol ; 189(6): 3168-77, 2012 Sep 15.
Article in English | MEDLINE | ID: mdl-22869907

ABSTRACT

Macrophage secretion of vascular endothelial growth factor (VEGF) in response to hypoxia contributes to tumor growth and angiogenesis. In addition to VEGF, hypoxic macrophages stimulated with GM-CSF secrete high levels of a soluble form of the VEGF receptor (sVEGFR-1), which neutralizes VEGF and inhibits its biological activity. Using mice with a monocyte/macrophage-selective deletion of hypoxia-inducible factor (HIF)-1α or HIF-2α, we recently demonstrated that the antitumor response to GM-CSF was dependent on HIF-2α-driven sVEGFR-1 production by tumor-associated macrophages, whereas HIF-1α specifically regulated VEGF production. We therefore hypothesized that chemical stabilization of HIF-2α using an inhibitor of prolyl hydroxylase domain 3 (an upstream inhibitor of HIF-2α activation) would increase sVEGFR-1 production from GM-CSF-stimulated macrophages. Treatment of macrophages with the prolyl hydroxylase domain 3 inhibitor AKB-6899 stabilized HIF-2α and increased sVEGFR-1 production from GM-CSF-treated macrophages, with no effect on HIF-1α accumulation or VEGF production. Treatment of B16F10 melanoma-bearing mice with GM-CSF and AKB-6899 significantly reduced tumor growth compared with either drug alone. Increased levels of sVEGFR-1 mRNA, but not VEGF mRNA, were detected within the tumors of GM-CSF- and AKB-6899-treated mice, correlating with decreased tumor vascularity. Finally, the antitumor and antiangiogenic effects of AKB-6899 were abrogated when mice were simultaneously treated with a sVEGFR-1 neutralizing Ab. These results demonstrate that AKB-6899 decreases tumor growth and angiogenesis in response to GM-CSF by increasing sVEGFR-1 production from tumor-associated macrophages. Specific activation of HIF-2α can therefore decrease tumor growth and angiogenesis.


Subject(s)
Antineoplastic Agents/metabolism , Basic Helix-Loop-Helix Transcription Factors/metabolism , Growth Inhibitors/metabolism , Macrophages/immunology , Macrophages/metabolism , Melanoma, Experimental/immunology , Melanoma, Experimental/pathology , Vascular Endothelial Growth Factor Receptor-1/biosynthesis , Animals , Antineoplastic Agents/therapeutic use , Basic Helix-Loop-Helix Transcription Factors/physiology , Cell Line, Tumor , Cells, Cultured , Dioxygenases/antagonists & inhibitors , Dioxygenases/biosynthesis , Disease Models, Animal , Granulocyte-Macrophage Colony-Stimulating Factor/administration & dosage , Growth Inhibitors/biosynthesis , Growth Inhibitors/therapeutic use , Humans , Hypoxia-Inducible Factor-Proline Dioxygenases , Macrophages/pathology , Melanoma, Experimental/prevention & control , Mice , Mice, Inbred C57BL , Mice, SCID , Mice, Transgenic , Neovascularization, Pathologic/immunology , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/prevention & control , Protein Stability , Solubility
12.
J Cell Physiol ; 227(3): 1098-106, 2012 Mar.
Article in English | MEDLINE | ID: mdl-21567397

ABSTRACT

The capacity of human periodontal ligament fibroblasts (PLF) to proliferate in response to mechanical force plays a critical role in orthodontic tooth movement. Extensive research has not fully revealed the mechanisms by which the PLF respond to mechanical force. The responses to force differ according to the origin of cells and the type of stress applied. In this study, we examined the proliferative response of PLF to tensile force. We also explored cellular mechanisms involved in the mechanosignal transduction of tensile force. Application to the force to PLF with 1.5% elongation for 1 h inhibited cell proliferation. This was accompanied by reductions in several cyclins and cyclin-dependent kinases (CDKs) involved in the G(1)/S transition; however, p21 knockdown prevented these events. Pharmacological inhibitor of p38 MAPK suppressed the force-mediated growth inhibition as well as the decrease in p21 expression. Ras inhibitor almost completely blocked the tension-mediated increases in p-p38 MAPK and p-p21, and the attendant increase in PLF proliferation. These findings suggest that tension force activates Ras-p38 MAPK pathways in PLF, which up-regulate p21 and arrest cell cycle progression at the G(1) phase.


Subject(s)
Fibroblasts/cytology , Growth Inhibitors/biosynthesis , Periodontal Ligament/cytology , Proto-Oncogene Proteins p21(ras)/biosynthesis , Tensile Strength/physiology , Up-Regulation/physiology , p38 Mitogen-Activated Protein Kinases/biosynthesis , Adult , Cell Cycle Proteins/metabolism , Cells, Cultured , Fibroblasts/enzymology , G1 Phase/physiology , Growth Inhibitors/physiology , Humans , MAP Kinase Signaling System/physiology , Male , Periodontal Ligament/metabolism , Periodontal Ligament/physiology , Young Adult
13.
J Leukoc Biol ; 91(1): 69-81, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21984745

ABSTRACT

We have previously shown that perturbed bone marrow progenitor development promotes hyporesponsive monocytes following experimental burn sepsis. Clinical and experimental sepsis is associated with monocyte deactivation and depletion of mDCs. Decrease in circulating DCs is reported in burn patients who develop sepsis. In our 15% TBSA scald burn model, we demonstrate a significant reduction in the circulating MHC-II(+) population and mDCs (Gr1(neg)CD11b(+)CD11c(+)) with a corresponding decrease in bone marrow MHC-II(+) cells and mDCs for up to 14 days following burn. We explored the underlying mechanism(s) that regulate bone marrow development of monocytes and DCs following burn injury. We found a robust bone marrow response with a significant increase in multipotential HSCs (LSK) and bipotential GMPs following burn injury. GMPs from burn mice exhibit a significant reduction in GATA-1, which is essential for DC development, but express high levels of MafB and M-CSFRs, both associated with monocyte production. GMPs obtained from burn mice differentiated 1.7 times more into Mϕ and 1.6-fold less into DCs compared with sham. Monocytes and DCs expressed 50% less MHC-II in burn versus sham. Increased monocyte commitment in burn GMPs was a result of high MafB and M-CSFR expressions. Transient silencing of MafB (siRNA) in GMP-derived monocytes from burn mice partially restored DC differentiation deficits and increased GATA-1 expression. We provide evidence that high MafB following burn plays an inhibitory role in monocyte-derived DC differentiation by regulating M-CSFR and GATA-1 expressions.


Subject(s)
Burns/immunology , Burns/metabolism , Dendritic Cells/immunology , Growth Inhibitors/biosynthesis , Hematopoietic Stem Cells/immunology , MafB Transcription Factor/biosynthesis , Monocytes/immunology , Sepsis/immunology , Animals , Burns/pathology , Cell Differentiation/immunology , Dendritic Cells/cytology , Disease Models, Animal , Down-Regulation/immunology , GATA1 Transcription Factor/genetics , GATA1 Transcription Factor/physiology , Growth Inhibitors/genetics , Growth Inhibitors/physiology , Hematopoietic Stem Cells/cytology , MafB Transcription Factor/genetics , MafB Transcription Factor/physiology , Male , Mice , Mice, Inbred Strains , Monocytes/cytology , Monocytes/metabolism , Receptor, Macrophage Colony-Stimulating Factor/genetics , Receptor, Macrophage Colony-Stimulating Factor/physiology , Sepsis/metabolism , Sepsis/pathology , Up-Regulation/immunology
14.
Dev Neurobiol ; 71(9): 733-46, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21714103

ABSTRACT

During development, axons elongate vigorously, carefully controlling their speed, to connect with their targets. In general, rapid axon growth is correlated with active growth cones driven by dynamic actin filaments. For example, when the actin-driven tip is collapsed by repulsive guidance molecules, axon growth is severely impaired. In this study, we report that axon growth can be suppressed, without destroying the actin-based structure or motility of the growth cones, when antibodies bind to the four-transmembrane glycoprotein M6a concentrated on the growth cone edge. Surprisingly, M6a-deficient axons grow actively but are not growth suppressed by the antibodies, arguing for an inductive action of the antibody. The binding of antibodies clusters and displaces M6a protein from the growth cone edge membrane, suggesting that the spatial rearrangement of this protein might underlie the unique growth cone behavior triggered by the antibodies. Molecular dissection of M6a suggested involvement for the N-terminal intracellular domain in this antibody-induced growth cone arrest.


Subject(s)
Actin Cytoskeleton/physiology , Axons/metabolism , Growth Cones/metabolism , Growth Inhibitors/biosynthesis , Membrane Glycoproteins/physiology , Nerve Tissue Proteins/physiology , Peptide Fragments/biosynthesis , Actin Cytoskeleton/metabolism , Animals , Antibodies, Monoclonal, Murine-Derived , Binding Sites, Antibody/physiology , Cell Line , Cricetinae , Female , Growth Cones/physiology , Growth Inhibitors/physiology , Membrane Glycoproteins/immunology , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Mice, Knockout , Nerve Tissue Proteins/immunology , Nerve Tissue Proteins/metabolism , Peptide Fragments/immunology , Peptide Fragments/physiology , Primary Cell Culture
15.
J Immunol ; 187(1): 266-73, 2011 Jul 01.
Article in English | MEDLINE | ID: mdl-21622862

ABSTRACT

IL-27 is a cytokine that regulates Th function during autoimmune and pathogen-induced immune responses. Although previous studies have shown that regulatory T cells (Tregs) express the IL-27R, and that IL-27 inhibits forkhead box P3 upregulation in vitro, little is known about how IL-27 influences Tregs in vivo. The studies presented in this article show that mice that overexpress IL-27 had decreased Treg frequencies and developed spontaneous inflammation. Although IL-27 did not cause mature Tregs to downregulate forkhead box P3, transgenic overexpression in vivo limited the size of a differentiating Treg population in a bone marrow chimera model, which correlated with reduced production of IL-2, a vital cytokine for Treg maintenance. These data identify an indirect role for IL-27 in shaping the Treg pool.


Subject(s)
Cell Differentiation/immunology , Growth Inhibitors/physiology , Interleukins/physiology , Protein Subunits/physiology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Animals , Bone Marrow Transplantation/immunology , Bone Marrow Transplantation/pathology , Cell Differentiation/genetics , Cells, Cultured , Female , Growth Inhibitors/biosynthesis , Growth Inhibitors/genetics , Inflammation/genetics , Inflammation/immunology , Inflammation/pathology , Interleukin-2/antagonists & inhibitors , Interleukin-2/biosynthesis , Interleukins/biosynthesis , Interleukins/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Minor Histocompatibility Antigens , Protein Subunits/biosynthesis , Protein Subunits/genetics , Radiation Chimera/immunology , Receptors, Cytokine/biosynthesis , Receptors, Cytokine/genetics , Receptors, Cytokine/physiology , T-Lymphocytes, Regulatory/pathology
17.
J Immunol ; 186(7): 4244-52, 2011 Apr 01.
Article in English | MEDLINE | ID: mdl-21368228

ABSTRACT

Although the physiological consequences of Notch signaling in hematopoiesis have been extensively studied, the differential effects of individual notch cleavage products remain to be elucidated. Given that ADAM10 is a critical regulator of Notch and that its deletion is embryonically lethal, we generated mice that overexpress ADAM10 (ADAM10 transgenic [A10Tg]) at early stages of lympho- and myeloid development. Transgene expression resulted in abrogated B cell development, delayed T cell development in the thymus, and unexpected systemic expansion of CD11b(+)Gr-1(+) cells, also known as myeloid-derived suppressor cells. Mixed bone marrow reconstitution assays demonstrated that transgene expression altered hematopoiesis via a cell-intrinsic mechanism. Consistent with previously reported observations, we hypothesized that ADAM10 overexpression dysregulated Notch by uncoupling the highly regulated proteolysis of Notch receptors. This was confirmed using an in vitro model of hematopoiesis via culturing A10Tg hematopoietic Lineage(-)Sca-1(+)c-Kit(+) cells with OP-9 stromal cells in the presence or absence of Delta-like 1, a primary ligand for Notch. Blockade of the site 2 (S2) and site 3 (S3) cleavage of the Notch receptor demonstrated differential effects on hematopoiesis. OP9-DL1 cultures containing the ADAM10 inhibitor (S2 cleavage site) enhanced and rescued B cell development from wild-type and A10Tg Lineage(-)Sca-1(+)c-Kit(+) cells, respectively. In contrast, blockade of γ-secretase at the S3 cleavage site induced accumulation of the S2 product and consequently prevented B cell development and resulted in myeloid cell accumulation. Collectively, these findings indicate that the differential cleavage of Notch into S2 and S3 products regulated by ADAM10 is critical to hematopoietic cell-fate determination.


Subject(s)
ADAM Proteins/genetics , Amyloid Precursor Protein Secretases/genetics , Lymphopoiesis/genetics , Lymphopoiesis/immunology , Membrane Proteins/genetics , Myelopoiesis/genetics , Myelopoiesis/immunology , Receptors, Notch/antagonists & inhibitors , Receptors, Notch/metabolism , ADAM Proteins/biosynthesis , ADAM Proteins/physiology , ADAM10 Protein , Amyloid Precursor Protein Secretases/biosynthesis , Amyloid Precursor Protein Secretases/physiology , Animals , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , B-Lymphocytes/pathology , Cell Lineage/genetics , Cell Lineage/immunology , Cell Proliferation , Cells, Cultured , Growth Inhibitors/biosynthesis , Growth Inhibitors/genetics , Growth Inhibitors/physiology , Hematopoietic Stem Cells/immunology , Hematopoietic Stem Cells/metabolism , Hydrolysis , Membrane Proteins/biosynthesis , Membrane Proteins/physiology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Multipotent Stem Cells/immunology , Multipotent Stem Cells/metabolism , Myeloid Progenitor Cells/immunology , Myeloid Progenitor Cells/metabolism , Receptors, Notch/physiology , Signal Transduction/genetics , Signal Transduction/immunology , Thymus Gland/immunology , Thymus Gland/metabolism , Thymus Gland/pathology
18.
J Immunol ; 186(7): 4183-90, 2011 Apr 01.
Article in English | MEDLINE | ID: mdl-21368233

ABSTRACT

Angiopoietin 2 (ANGPT2) is a proangiogenic cytokine whose expression is often upregulated by endothelial cells in tumors. Expression of its receptor, TIE2, defines a highly proangiogenic subpopulation of myeloid cells in circulation and tumors called TIE2-expressing monocytes/macrophages (TEMs). Genetic depletion of TEMs markedly reduces tumor angiogenesis in various tumor models, emphasizing their essential role in driving tumor progression. Previously, we demonstrated that ANGPT2 augments the expression of various proangiogenic genes, the potent immunosuppressive cytokine, IL-10, and a chemokine for regulatory T cells (Tregs), CCL17 by TEMs in vitro. We now show that TEMs also express higher levels of IL-10 than TIE2(-) macrophages in tumors and that ANGPT2-stimulated release of IL-10 by TEMs suppresses T cell proliferation, increases the ratio of CD4(+) T cells to CD8(+) T cells, and promotes the expansion of CD4(+)CD25(high)FOXP3(+) Tregs. Furthermore, syngeneic murine tumors expressing high levels of ANGPT2 contained not only high numbers of TEMs but also increased numbers of Tregs, whereas genetic depletion of tumor TEMs resulted in a marked reduction in the frequency of Tregs in tumors. Taken together, our data suggest that ANGPT2-stimulated TEMs represent a novel, potent immunosuppressive force in tumors.


Subject(s)
Angiopoietin-2/physiology , Cell Cycle Proteins/physiology , Cell Differentiation/immunology , DNA-Binding Proteins/physiology , Lymphocyte Activation/immunology , Monocytes/immunology , Neovascularization, Pathologic/immunology , Repressor Proteins/physiology , T-Lymphocytes, Regulatory/immunology , Transcription Factors/physiology , Animals , Apoptosis Regulatory Proteins , Carcinoma, Lewis Lung/immunology , Carcinoma, Lewis Lung/pathology , Cell Cycle Proteins/biosynthesis , Cell Line, Tumor , Coculture Techniques , DNA-Binding Proteins/biosynthesis , Growth Inhibitors/biosynthesis , Growth Inhibitors/physiology , Humans , Interleukin-10/biosynthesis , Interleukin-10/metabolism , Interleukin-10/physiology , Macrophages/immunology , Macrophages/metabolism , Mammary Neoplasms, Experimental/immunology , Mammary Neoplasms, Experimental/metabolism , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Inbred BALB C , Mice, Transgenic , Monocytes/metabolism , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Repressor Proteins/biosynthesis , T-Lymphocytes, Regulatory/cytology , Transcription Factors/biosynthesis
19.
J Immunol ; 186(7): 4388-95, 2011 Apr 01.
Article in English | MEDLINE | ID: mdl-21357259

ABSTRACT

Foxp3(+)CD4(+) regulatory T (Treg) cells inhibit immune responses and temper inflammation. IL-17(+)CD4(+) T (Th17) cells mediate inflammation of autoimmune diseases. A small population of IL-17(+)Foxp3(+)CD4(+) T cells has been observed in peripheral blood in healthy human beings. However, the biology of IL-17(+)Foxp3(+)CD4(+) T cells remains poorly understood in humans. We investigated their phenotype, cytokine profile, generation, and pathological relevance in patients with ulcerative colitis. We observed that high levels of IL-17(+)Foxp3(+)CD4(+) T cells were selectively accumulated in the colitic microenvironment and associated colon carcinoma. The phenotype and cytokine profile of IL-17(+)Foxp3(+)CD4(+) T cells was overlapping with Th17 and Treg cells. Myeloid APCs, IL-2, and TGF-ß are essential for their induction from memory CCR6(+) T cells or Treg cells. IL-17(+)Foxp3(+)CD4(+) T cells functionally suppressed T cell activation and stimulated inflammatory cytokine production in the colitic tissues. Our data indicate that IL-17(+)Foxp3(+) cells may be "inflammatory" Treg cells in the pathological microenvironments. These cells may contribute to the pathogenesis of ulcerative colitis through inducing inflammatory cytokines and inhibiting local T cell immunity, and in turn may mechanistically link human chronic inflammation to tumor development. Our data therefore challenge commonly held beliefs of the anti-inflammatory role of Treg cells and suggest a more complex Treg cell biology, at least in the context of human chronic inflammation and associated carcinoma.


Subject(s)
Inflammation Mediators/physiology , Interleukin-17/physiology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/pathology , Carcinoma, Renal Cell/immunology , Carcinoma, Renal Cell/metabolism , Carcinoma, Renal Cell/pathology , Cells, Cultured , Chronic Disease , Coculture Techniques , Colitis, Ulcerative/immunology , Colitis, Ulcerative/metabolism , Colitis, Ulcerative/pathology , Colonic Neoplasms/immunology , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Cytokines/biosynthesis , Female , Forkhead Transcription Factors/biosynthesis , Forkhead Transcription Factors/physiology , Growth Inhibitors/biosynthesis , Growth Inhibitors/physiology , Humans , Immune Tolerance/immunology , Inflammation Mediators/metabolism , Interleukin-17/biosynthesis , Kidney Neoplasms/immunology , Kidney Neoplasms/metabolism , Kidney Neoplasms/pathology , Melanoma/immunology , Melanoma/metabolism , Melanoma/pathology , Ovarian Neoplasms/immunology , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , T-Lymphocytes, Regulatory/metabolism
20.
Circ Res ; 108(8): 960-70, 2011 Apr 15.
Article in English | MEDLINE | ID: mdl-21350213

ABSTRACT

RATIONALE: Stem cell therapies to regenerate damaged cardiac tissue represent a novel approach to treat heart disease. However, the majority of adoptively transferred stem cells delivered to damaged myocardium do not survive long enough to impart protective benefits, resulting in modest functional improvements. Strategies to improve survival and proliferation of stem cells show promise for significantly enhancing cardiac function and regeneration. OBJECTIVE: To determine whether injected cardiac progenitor cells (CPCs) genetically modified to overexpress nuclear Akt (CPCeA) increase structural and functional benefits to infarcted myocardium relative to control CPCs. METHODS AND RESULTS: CPCeA exhibit significantly increased proliferation and secretion of paracrine factors compared with CPCs. However, CPCeA exhibit impaired capacity for lineage commitment in vitro. Infarcted hearts receiving intramyocardial injection of CPCeA have increased recruitment of endogenous c-kit cells compared with CPCs, but neither population provides long-term functional and structural improvements compared with saline-injected controls. Pharmacological inhibition of Akt alleviated blockade of lineage commitment in CPCeA. CONCLUSIONS: Although overexpression of nuclear Akt promotes rapid proliferation and secretion of protective paracrine factors, the inability of CPCeA to undergo lineage commitment hinders their capacity to provide functional or structural benefits to infarcted hearts. Despite enhanced recruitment of endogenous CPCs, lack of functional improvement in CPCeA-treated hearts demonstrates CPC lineage commitment is essential to the regenerative response. Effective stem cell therapies must promote cellular survival and proliferation without inhibiting lineage commitment. Because CPCeA exhibit remarkable proliferative potential, an inducible system mediating nuclear Akt expression could be useful to augment cell therapy approaches.


Subject(s)
Cell Nucleus/enzymology , Gene Expression Regulation, Enzymologic , Growth Inhibitors/physiology , Myocardial Infarction/enzymology , Myocytes, Cardiac/enzymology , Proto-Oncogene Proteins c-akt/biosynthesis , Stem Cells/enzymology , Animals , Cell Lineage/genetics , Cell Nucleus/pathology , Cell Proliferation , Cells, Cultured , Female , Growth Inhibitors/biosynthesis , Growth Inhibitors/genetics , Male , Mice , Myocardial Infarction/pathology , Myocardial Infarction/surgery , Myocardium/cytology , Myocardium/enzymology , Myocardium/pathology , Myocytes, Cardiac/cytology , Myocytes, Cardiac/pathology , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/physiology , Stem Cell Transplantation/methods , Stem Cells/cytology , Stem Cells/pathology , Structure-Activity Relationship
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