CD137, which is expressed on activated T cells, plays a critical role in inflammatory responses. However, the exact role that CD137 plays in monocytes is not fully known. Here we studied the expression and function of CD137 in human monocytic THP-1 cells, which we found constitutively expresses CD137 at the mRNA and protein level. Cross-linking of CD137 increased the secretion of IL-8 and TNF-alpha, promoted the expression of CD54 and CD11b, and increased adhesion to extracellular matrix (ECM) proteins. In particular CD137-induced adhesion of THP-1 cells was inhibited by an inhibitor of mitogen-activated protein kinase kinase (MEK), but not by a p38 kinase inhibitor. Taken together, these results show that the adhesion and cytokine production of THP-1 cells induced by CD137 occur via activation of MEK, which results in the activation of ERK-1/2 signaling pathways. Therefore, this study suggests that CD137 induces an activating and migrating signal during inflammatory processes.
Antigens, CD/biosynthesis/*immunology ; Antigens, CD11/biosynthesis ; *Cell Adhesion ; Cell Adhesion Molecules/biosynthesis ; Cell Line ; Cytokines/*biosynthesis ; Enzyme Activation ; Extracellular Matrix Proteins/metabolism ; Flow Cytometry ; Humans ; Immunity, Natural ; Intercellular Adhesion Molecule-1/biosynthesis ; Interleukin-8/biosynthesis ; Mitogen-Activated Protein Kinase 1/metabolism ; Mitogen-Activated Protein Kinase 3/metabolism ; Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors/metabolism ; Monocytes/metabolism/*physiology ; Phosphorylation ; Protein Binding ; Receptors, Nerve Growth Factor/biosynthesis/*immunology ; Receptors, Tumor Necrosis Factor/biosynthesis/*immunology ; Research Support, Non-U.S. Gov't ; Signal Transduction ; Tumor Necrosis Factor-alpha/biosynthesis ; p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors

The principal aim of this study was to determine the effects of antipsychotics (haloperidol, sulpiride, and clozapine) on regulating dopamine (DA) D1 and D2 receptor mRNA levels in the rat caudate putamen (CPu), nucleus accumbens (NAc), and olfactory tubercle (OTu). Twenty male Sprague-Dawley rats (250 g) were treated with haloperidol (1mg/mL), sulpiride (40 mg/mL), clozapine (20 mg/mL), and the control group received only water. Drugs were administered orally for 4 weeks. Antipsychotic drugs had differential effects on DA D1 and D2 receptor gene expression. Haloperidol and sulpiride induced an increase of DA D1 and D2 receptor mRNA levels in the rat CPu, OTu, and NAc; haloperidol caused a greater increase than sulpiride. However, clozapine treatment had less effect on DA receptor mRNAs levels in the same area. Antipsychotic drugs differentially upregulated the expression of DA D1 and D2 receptor mRNAs in the rat brain. These changes may be related, at least in part, to changes of DA concentration following antipsychotics treatment.
Animals ; Antipsychotic Agents* ; Brain* ; Clozapine ; Dopamine* ; Gene Expression ; Haloperidol ; Humans ; In Situ Hybridization ; Male ; Nucleus Accumbens ; Olfactory Pathways ; Putamen ; Rats* ; Rats, Sprague-Dawley ; Receptors, Dopamine ; RNA, Messenger* ; Sulpiride ; Water

Cilostazol is a selective inhibitor of type 3 phosphodiesterase (PDE3) and has been widely used as an antiplatelet agent. Cilostazol mediates this activity through effects on the cyclic adenosine monophosphate (cAMP) signaling cascade. Recently, it has attracted attention as a neuroprotective agent. However, little is known about cilostazol's effect on excitotoxicity induced neuronal cell death. Therefore, this study evaluated the neuroprotective effect of cilostazol treatment against hippocampal neuronal damage in a mouse model of kainic acid (KA)-induced neuronal loss. Cilostazol pretreatment reduced KA-induced seizure scores and hippocampal neuron death. In addition, cilostazol pretreatment increased cAMP response element-binding protein (CREB) phosphorylation and decreased neuroinflammation. These observations suggest that cilostazol may have beneficial therapeutic effects on seizure activity and other neurological diseases associated with excitotoxicity.
Adenosine Monophosphate ; Animals ; Cell Death* ; Cyclic AMP Response Element-Binding Protein ; Hippocampus ; Kainic Acid ; Mice ; Neurons ; Neuroprotective Agents ; Phosphorylation ; Seizures ; Therapeutic Uses

Hyper-O-GlcNAcylation is a general feature of cancer which contributes to various cancer phenotypes, including cell proliferation and cell growth. Quercetin, a naturally occurring dietary flavonoid, has been reported to reduce the proliferation and growth of cancer. Several reports of the anticancer effect of quercetin have been published, but there is no study regarding its effect on O-GlcNAcylation. The aim of this study was to investigate the anticancer effect of quercetin on HeLa cells and compare this with its effect on HaCaT cells. Cell viability and cell death were determined by MTT and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling assays. O-GlcNAcylation of AMP-activated protein kinase (AMPK) was examined by succinylated wheat germ agglutinin pulldown and immunoprecipitation. Immunofluorescence staining was used to detect the immunoreactivitiy of O-linked N-acetylglucosamine transferase (OGT) and sterol regulatory element binding protein 1 (SREBP-1). Quercetin decreased cell proliferation and induced cell death, but its effect on HaCaT cells was lower than that on HeLa cells. O-GlcNAcylation level was higher in HeLa cells than in HaCaT cells. Quercetin decreased the expression of global O-GlcNAcylation and increased AMPK activation by reducing the O-GlcNAcylation of AMPK. AMPK activation due to reduced O-GlcNAcylation of AMPK was confirmed by treatment with 6-diazo-5-oxo-L-norleucine. Our results also demonstrated that quercetin regulated SREBP-1 and its transcriptional targets. Furthermore, immunofluorescence staining showed that quercetin treatment decreased the immunoreactivities of OGT and SREBP-1 in HeLa cells. Our findings demonstrate that quercetin exhibited its anticancer effect by decreasing the O-GlcNAcylation of AMPK. Further studies are needed to explore how quercetin regulates O-GlcNAcylation in cancer.
Adenosine* ; AMP-Activated Protein Kinases ; Cell Death* ; Cell Proliferation ; Cell Survival ; Deoxyuridine ; Diazooxonorleucine ; Fluorescent Antibody Technique ; HeLa Cells ; Humans ; Immunoprecipitation ; Phenotype ; Protein Kinases* ; Quercetin* ; Sterol Regulatory Element Binding Protein 1 ; Transferases ; Triticum ; Uterine Cervical Neoplasms*

BACKGROUND: Macrophages have been known to have diverse roles either after tissue damage or during the wound healing process; however, their roles in flap wound healing are poorly understood. In this study, we aimed to evaluate how macrophages contribute to the flap wound regeneration.METHODS: A murine model of a pedicled flap was generated, and the time-course of the wound healing process was determined. Especially, the interface between the flap and the residual tissue was histopathologically evaluated. Using clodronate liposome, a macrophage-depleting agent, the functional role of macrophages in flap wound healing was investigated. Coculture of human keratinocyte cell line HaCaT and monocytic cell line THP-1 was performed to unveil relationship between the two cell types.RESULTS: Macrophage depletion significantly impaired flap wound healing process showing increased necrotic area after clodronate liposome administration. Interestingly, microscopic evaluation revealed that epithelial remodeling between the flap tissue and residual normal tissue did not occurred under the lack of macrophage infiltration. Coculture and scratch wound healing assays indicated that macrophages significantly affected the migration of keratinocytes.CONCLUSION: Macrophages play a critical role in the flap wound regeneration. Especially, epithelial remodeling at the flap margin is dependent on proper macrophage infiltration. These results implicate to support the cellular mechanisms of impaired flap wound healing.
Cell Line ; Clodronic Acid ; Coculture Techniques ; Humans ; Keratinocytes ; Liposomes ; Macrophages ; Regeneration ; Surgical Flaps ; Wound Healing ; Wounds and Injuries

It is well known that chronic ethanol treatment affects the synthesis of RNA and protein in the brain and the maintenance and function of nervous system. The changes in myelination-related genes are most prominent in human alcoholics. Previously, our cDNA microarray study showed altered Proteolipid protein (PLP), a major protein of central myelin. The present study aimed to gain more understanding of the expression of PLP after chronic ethanol treatment. Male Sprague-Dawley rats were daily treated with ethanol (15% in saline, 3 g/kg, i.p.) or saline for 14 days. Messenger RNAs from hippocampus of each group were subjected to cDNA expression array hybridization to determine the differential gene expressions. Among many ethanol responsive genes, PLP was negatively regulated by ethanol treatment, which is one of the most abundant proteins in the CNS and has an important role in the stabilization of myelin sheath. Using northern blot and immunohistochemical analysis, we showed the change in expression level of PLP mRNA and protein after ethanol treatment. PLP mRNA and protein were decreased in hippocampus of rat with chronic ethanol exposure, suggesting that ethanol may affect the stabilization of myelin sheath through the modulation of PLP expression and induce the pathophysiology of alcoholic brain.
Alcoholics ; Animals ; Blotting, Northern ; Brain ; Chimera ; DNA, Complementary ; Ethanol ; Gene Expression ; Hippocampus ; Humans ; Male ; Myelin Proteolipid Protein ; Myelin Sheath ; Nervous System ; Oligonucleotide Array Sequence Analysis ; Proteins ; Rats ; Rats, Sprague-Dawley ; RNA ; RNA, Messenger

PURPOSE: There are several well-established prognostic factors to predict the course of breast cancer. Recently, a new category of genes that control the process of programmed cell death, also called apoptosis, has been identified. It includes the bcl-2 proto-oncogene which actively blocks apoptosis. The purpose of this retrospective study is to investigate the relationship between Bcl-2 protein expression in primary breast cancers and other markers of prognostic value. MATERIALS AND METHODS: We analyzed 43 cases of invasive ductal carcinoma of breast cancer, which were consecutively operated in the period from January 1990 to December 1994. Analysis of Bcl-2 protein, ER and PgR expression was carried out using immunohistochemistry on the paraffin-embedded tissue section. The following indices were measured.; size of the tumor, number of axillary metastasis, histological grade, menopausal status, ER, PgR, and Bcl-2 status. RESULTS: Expression of the bcl-2 proto-oncogene was found in 28 cases of 43 patients (65.1%). No relationship could be observed between Bcl-2 status and tumor grade, TNM staging and menopausal status. A strong positive relationship was demonstrated between Bcl-2 immunoreactivity and ER status (P 0.001) and PgR status (P=0.014). A favorable prognostic value was demonstrated for Bcl-2 expression on overall survival (P=0.0427), but no prognostic value was demonstrated on disease-free survival (P=0.1587). CONCLUSION: Our results suggest that the Bcl-2 expression may be a favorable prognostic marker and its important role may be a modulator of response to adjuvant therapy in breast cancer.
Apoptosis ; Breast Neoplasms* ; Breast* ; Carcinoma, Ductal ; Cell Death ; Disease-Free Survival ; Humans ; Immunohistochemistry ; Neoplasm Metastasis ; Neoplasm Staging ; Prognosis ; Proto-Oncogenes ; Retrospective Studies

Excessive immune responses induced by ischemia-reperfusion injury (IRI) are known to lead to necrotic and apoptotic cell death, and calcineurin plays a major role in this process. Calcineurin dephosphorylates the nuclear factor of activated T-cells (NFAT), permitting its translocation into the nucleus. As a result, calcineurin promotes the release of pro-inflammatory cytokines, such as tumor necrosis factor-alpha. The overproduction of pro-inflammatory cytokines causes renal cell death. Calcineurin activity is regulated by calpain, a cysteine protease present in the nucleus. Calpain-mediated proteolysis increases the phosphatase activity of calcineurin, resulting in NFAT dephosphorylation. This process has been studied in cardiomyocytes but its role in renal IRI is unknown. Thus, we examined whether calpain regulates calcineurin in renal tubule nuclei. We established an in vivo renal IRI model in mice and identified the protective role of a calcineurin inhibitor, FK506, in this process. Calcineurin is expressed in the nucleus, where it is present in its calpain-cleaved form. FK506 reduced nuclear expression of calcineurin and prevented calcineurin-mediated NFAT activation. Our study shows clearly that FK506 reduces calpain-mediated calcineurin activity. Consequently, calcineurin could not maintain NFAT activation. FK506 reduced renal cell death by suppressing the transcription of pro-inflammatory cytokine genes. This study provides evidence that FK506 protects against inflammation in a renal IRI mouse model. We also provided a mechanism of calcineurin action in the nucleus. Therefore, FK506 could improve renal function by decreasing calcineurin activity in both the cytoplasm and the nucleus of renal tubule cells.
Animals ; Calcineurin* ; Calpain ; Cell Death ; Cysteine Proteases ; Cytokines ; Cytoplasm* ; Inflammation ; Mice ; Myocytes, Cardiac ; Proteolysis ; Reperfusion Injury ; T-Lymphocytes ; Tacrolimus* ; Tumor Necrosis Factor-alpha

Excessive immune responses induced by ischemia-reperfusion injury (IRI) are known to lead to necrotic and apoptotic cell death, and calcineurin plays a major role in this process. Calcineurin dephosphorylates the nuclear factor of activated T-cells (NFAT), permitting its translocation into the nucleus. As a result, calcineurin promotes the release of pro-inflammatory cytokines, such as tumor necrosis factor-alpha. The overproduction of pro-inflammatory cytokines causes renal cell death. Calcineurin activity is regulated by calpain, a cysteine protease present in the nucleus. Calpain-mediated proteolysis increases the phosphatase activity of calcineurin, resulting in NFAT dephosphorylation. This process has been studied in cardiomyocytes but its role in renal IRI is unknown. Thus, we examined whether calpain regulates calcineurin in renal tubule nuclei. We established an in vivo renal IRI model in mice and identified the protective role of a calcineurin inhibitor, FK506, in this process. Calcineurin is expressed in the nucleus, where it is present in its calpain-cleaved form. FK506 reduced nuclear expression of calcineurin and prevented calcineurin-mediated NFAT activation. Our study shows clearly that FK506 reduces calpain-mediated calcineurin activity. Consequently, calcineurin could not maintain NFAT activation. FK506 reduced renal cell death by suppressing the transcription of pro-inflammatory cytokine genes. This study provides evidence that FK506 protects against inflammation in a renal IRI mouse model. We also provided a mechanism of calcineurin action in the nucleus. Therefore, FK506 could improve renal function by decreasing calcineurin activity in both the cytoplasm and the nucleus of renal tubule cells.
Animals ; Calcineurin* ; Calpain ; Cell Death ; Cysteine Proteases ; Cytokines ; Cytoplasm* ; Inflammation ; Mice ; Myocytes, Cardiac ; Proteolysis ; Reperfusion Injury ; T-Lymphocytes ; Tacrolimus* ; Tumor Necrosis Factor-alpha

Oxidative stress-induced cell death leads to phosphorylation of 14-3-3zeta at serine 58. 14-3-3zeta is detected at significant levels in cerebrospinal fluid after kainic acid (KA)-induced seizures. Here we examined temporal changes in 14-3-3zeta phosphorylation in the hippocampus and amygdala of mice after KA treatment. Mice were killed at 2, 6, 24, or 48 h after KA (30 mg/kg) injection. We observed an increase in TUNEL and Fluoro-Jade B (FJB)-stained neurons in the hippocampus and amygdala of KA-treated mice. Phospho (p)-14-3-3zeta and p-JNK expression was increased in the hippocampus 2 and 6 h after KA treatment, respectively. In immunohistochemical analysis, p-14-3-3zeta-positive cells were present in the CA3 region of the hippocampus and the central nucleus of amygdala (CeA) of KA-treated mice. Thus, phosphorylation of 14-3-3zeta at serine 58 may play an important role in KA-induced hippocampal and amygdaloid neuronal damage.
Amygdala ; Animals ; Cell Death ; Fluoresceins ; Hippocampus ; In Situ Nick-End Labeling ; Kainic Acid ; Mice ; Neurons ; Phosphorylation ; Seizures ; Serine