Caffeine induces matrix metalloproteinase‐2 (MMP‐2) and MMP‐9 down‐regulation in human leukemia U937 cells via Ca2+/ROS‐mediated suppression of ERK/c‐fos pathway and activation of p38 MAPK/c‐jun pathway

Caffeine attenuated invasion of human leukemia U937 cells with characteristic of decreased protein expression and mRNA levels of matrix metalloproteinase‐2 (MMP‐2) and MMP‐9. Down‐regulation of MMP‐2 and MMP‐9 in U937 cells was abrogated by abolishment of caffeine‐elicited increase in intracellular Ca²⁺ concentration and ROS generation. Pretreatment with BAPTA‐AM (Ca²⁺ chelator) and N‐acetylcysteine (ROS scavenger) abolished caffeine‐induced ERK inactivation and p38 MPAK activation. Moreover, caffeine treatment led to MAPK phosphatase‐1 (MKP‐1) down‐regulation and protein phosphatase 2A catalytic subunit (PP2Ac) up‐regulation, which were involved in cross‐talk between p38 MAPK and ERK. Transfection of constitutively active MEK1 or pretreatment with SB202190 (p38 MAPK inhibitor) restored MMP‐2 and MMP‐9 protein expression in caffeine‐treated cells. Caffeine treatment repressed ERK‐mediated c‐Fos phosphorylation but evoked p38 MAPK‐mediated c‐Jun phosphorylation. Knock‐down of c‐Fos and c‐Jun by siRNA reflected that c‐Fos counteracted the effect of c‐Jun on MMP‐2/MMP‐9 down‐regulation. Taken together, our data indicate that MMP‐2/MMP‐9 down‐regulation in caffeine‐treated U937 cells is elicited by Ca²⁺/ROS‐mediated suppression of ERK/c‐Fos pathway and activation of p38 MAPK/c‐Jun pathway. J. Cell. Physiol. 224: 775–785, 2010. © 2010 Wiley‐Liss, Inc.     

Effect of arachidonic acid on hypoxia‐induced IL‐6 production in mouse ES cells: Involvement of MAPKs,NF‐κB,and HIF‐1α

This study examined the role of arachidonic acid (AA) in hypoxia‐induced production of interleukin (IL)‐6 and its related signaling pathways in mouse embryonic stem (ES) cells. Hypoxia with AA induced IL‐6 production, which was mediated by reactive oxygen species (ROS). In addition, hypoxia increased the levels of p38 mitogen‐activated protein kinases (MAPKs) and stress‐activated protein kinase/c‐jun NH₂‐terminal kinase (SAPK/JNK) phosphorylation, which were blocked by antioxidant (vitamin C). Inhibition of p38 MAPK and SAPK/JNK blocked hypoxia‐ or hypoxia with AA‐induced nuclear factor‐kappa B (NF‐κB) activation. Furthermore, hypoxia‐induced increase in hypoxia‐inducible factor‐1α (HIF‐1α) expression was regulated by NF‐κB activation. Consequently, the increased HIF‐1α expression induced activation of matrix metalloproteinase (MMP)‐2 and MMP‐9. The expression of each signaling molecule stimulated an increase in IL‐6 production that was greater in hypoxic conditions with AA than with hypoxia alone. Finally, inhibition of IL‐6 production using IL‐6 antibody or soluble IL‐6 receptor attenuated the hypoxia‐induced increases in DNA synthesis of mouse ES cells. In conclusion, AA potentiates hypoxia‐induced IL‐6 production through the MAPKs, NF‐κB, and HIF‐1α pathways in mouse ES cells. J. Cell. Physiol. 222: 574–585, 2010. © 2009 Wiley‐Liss, Inc.     

TNF‐α induces matrix metalloproteinase‐9 expression in A549 cells: Role of TNFR1/TRAF2/PKCα‐dependent signaling pathways

Matrix metalloproteinases (MMPs), in particular MMP‐9, have been shown to be induced by cytokines, including TNF‐α and contributes to airway inflammation. However, the mechanisms underlying TNF‐α‐induced MMP‐9 expression in human A549 cells remain unclear. Here, we report that TNF‐α‐induced MMP‐9 gene expression was mediated through the TNFR1/TRAF2/PKCα‐dependent signaling pathways in A549 cells, determined by zymographic, RT‐PCR, and Western blotting analyses. TNF‐α‐induced MMP‐9 expression was reduced by pretreatment with a TNFR Ab. Furthermore, TNF‐α‐induced TNFR1 and TRAF2 complex formation was revealed by immunoprecipitation using an anti‐TNFR1 Ab followed by Western blot analysis against an anti‐TRAF2 or anti‐TNFR1 Ab. In addition, TNF‐α‐induced MMP‐9 expression was also reduced by pretreatment with the inhibitor of PKCα (Gö6983), c‐Src (PP1), EGFR (AG1478), or PI3K (LY294002) or transfection with siRNAs of PKCα, Src, EGFR, Akt, p65, p300, and c‐Jun. On the other hand, TNF‐α stimulated the phosphorylation of c‐Src, EGFR, Akt, JNK1/2, and c‐Jun, which were inhibited by pretreatment with Gö6983. We also showed that TNF‐α induced Akt translocation and the formation of an Akt/p65/p300 complex. Pretreatment with the inhibitor of JNK1/2 (SP600125) but not the inhibitor of MEK1/2 (U0126), p38 MAPK (SB202190), or PI3K (LY294002), markedly inhibited TNF‐α‐induced c‐Jun mRNA levels. Taken together, these data suggest that in A549 cells, TNF‐α induces MMP‐9 expression via the TNFR1/TRAF2/PKCα‐dependent JNK1/2/c‐Jun and c‐Src/EGFR/PI3K/Akt pathways. J. Cell. Physiol. 454–464, 2010. © 2010 Wiley‐Liss, Inc.     

Amyloid‐β‐induced occludin down‐regulation and increased permeability in human brain endothelial cells is mediated by MAPK activation

Vascular dysfunction is emerging as a key pathological hallmark in Alzheimer’s disease (AD). A leaky blood–brain barrier (BBB) has been described in AD patient tissue and in vivo AD mouse models. Brain endothelial cells (BECs) are linked together by tight junctional (TJ) proteins, which are a key determinant in restricting the permeability of the BBB. The amyloid β (Aβ) peptides of 1–40 and 1–42 amino acids are believed to be pivotal in AD pathogenesis. We therefore decided to investigate the effect of Aβ 1–40, the Aβ variant found at the highest concentration in human plasma, on the permeability of an immortalized human BEC line, hCMEC/D3. Aβ 1–40 induced a marked increase in hCMEC/D3 cell permeability to the paracellular tracer 70 kD FITC‐dextran when compared with cells incubated with the scrambled Aβ 1–40 peptide. Increased permeability was associated with a specific decrease, both at the protein and mRNA level, in the TJ protein occludin, whereas claudin‐5 and ZO‐1 were unaffected. JNK and p38MAPK inhibition prevented both Aβ 1–40‐mediated down‐regulation of occludin and the increase in paracellular permeability in hCMEC/D3 cells. Our findings suggest that the JNK and p38MAPK pathways might represent attractive therapeutic targets for preventing BBB dysfunction in AD.     

Suppression of ERK signaling evokes autocrine Fas‐mediated death in arachidonic acid‐treated human chronic myeloid leukemia K562 cells

Arachidonic acid (AA)‐induced apoptotic death of K562 cells (human chronic myeloid leukemic cells) was characteristic of reactive oxygen species (ROS) generation and mitochondrial depolarization. N‐Acetylcysteine pretreatment rescued viability of AA‐treated cells and abolished mitochondrial depolarization. In contrast to no significant changes in phospho‐JNK and phospho‐ERK levels, AA evoked notable activation of p38 MAPK. Unlike that of JNK and p38 MAPK, ERK suppression further reduced the viability of AA‐treated cells. Increases in Fas/FasL protein expression, caspase‐8 activation, the production of tBid and the loss of mitochondrial membrane potential were noted with K562 cells that were treated with a combination of U0126 and AA. Down‐regulation of FADD attenuated U0126‐evoked degradation of procaspase‐8 and Bid. Abolition of p38 MAPK activation abrogated U0126‐elicited Fas/FasL up‐regulation in AA‐treated cells. U0126 pretreatment suppressed c‐Fos phosphorylation but increased p38 MAPK‐mediated c‐Jun phosphorylation. Knock‐down of c‐Fos and c‐Jun protein expression by siRNA suggested that c‐Fos counteracted the effect of c‐Jun on Fas/FasL up‐regulation. Taken together, our data indicate that AA induces the ROS/mitochondria‐dependent death pathway and blocks the ERK pathway which enhances the cytotoxicity of AA through additionally evoking an autocrine Fas‐mediated apoptotic mechanism in K562 cells. J. Cell. Physiol. 222: 625–634, 2010. © 2009 Wiley‐Liss, Inc.     

Up‐regulation of TRPM6 transcriptional activity by AP‐1 in renal epithelial cells

Transient receptor potential melastatin 6 (TRPM6) channel is involved in the reabsorption of magnesium in the kidney. We recently found that TRPM6 expression is up‐regulated by EGF, but the regulatory mechanism has not been clear. TRPM6 mRNA was endogenously expressed in HEK293 cells. TRPM6 mRNA expression was increased by EGF, which was inhibited by U0126, an MEK inhibitor. Promoter activity of human TRPM6 was observed in the TRPM6 5′‐flanking region from ?1,214 to ?718. This promoter activity was enhanced by EGF and inhibited by U0126. Three putative AP‐1 binding sites were identified within the region of ?1,214/?718. The mutation of the putative AP‐1 binding site (?741/?736) completely inhibited the EGF‐induced promoter activity. EGF increased p‐ERK1/2, c‐Fos, c‐Jun, and p‐c‐Jun levels, which were inhibited by U0126. The introduction of c‐Fos or c‐Jun siRNA inhibited the EGF‐induced promoter activity. A chromatin immunoprecipitation assay revealed that c‐Fos and c‐Jun bind to the AP‐1 binding site within the region of ?1,214/?718. These results suggest that EGF up‐regulates TRPM6 mRNA expression mediate via the activation of ERK/AP‐1‐dependent pathway. J. Cell. Physiol. 222: 481–487, 2010. © 2009 Wiley‐Liss, Inc.     

Hypoxia‐induced apoptosis of astrocytes is mediated by reduction of Dicer and activation of caspase‐1

Hypoxia is a condition in which the whole body or a region of the body is deprived of oxygen supply. The brain is very sensitive to the lack of oxygen and cerebral hypoxia can rapidly cause severe brain damage. Astrocytes are essential for the survival and function of neurons. Therefore, protecting astrocytes against cell death is one of the main therapeutic strategies for treating hypoxia. Hence, the mechanism of hypoxia‐induced astrocytic cell death should be fully elucidated. In this study, astrocytes were exposed to hypoxic conditions using a hypoxia work station or the hypoxia mimetic agent cobalt chloride (CoCl₂). Both the hypoxic gas mixture (1% O₂) and chemical hypoxia‐induced apoptotic cell death in T98G glioblastoma cells and mouse primary astrocytes. Reactive oxygen species were generated in response to the hypoxia‐mediated activation of caspase‐1. Active caspase‐1 induced the classical caspase‐dependent apoptosis of astrocytes. In addition, the microRNA processing enzyme Dicer was cleaved by caspase‐3 during hypoxia. Knockdown of Dicer using antisense oligonucleotides induced apoptosis of T98G cells. Taken together, these results suggest that astrocytic cell death during hypoxia is mediated by the reactive oxygen species/caspase‐1/classical caspase‐dependent apoptotic pathway. In addition, the decrease in Dicer levels by active caspase‐3 amplifies this apoptotic pathway via a positive feedback loop. These findings may provide a new target for therapeutic interventions in cerebral hypoxia.     

During Human Melanoma Progression AP‐1 Binding Pairs are Altered with Loss of c‐Jun In Vitro

We demonstrated previously that c‐Jun, JunB and c‐Fos RNA were dysregulated in metastatic melanoma cells compared with normal human melanocytes. The purpose of this study was to evaluate the distribution in composition of AP‐1 dimers in human melanoma pathogenesis. We investigated AP‐1 dimer pairing in radial growth phase‐like (RGP) (w3211) and vertical growth phase‐like (VGP) (w1205) human melanoma cells and metastatic cell lines (cloned from patients, c83‐2c, c81‐46A, A375, respectively) compared with melanocytes using electrophoretic mobility shift assay (EMSA), Western blot and transfection analyses. There are progressive variations in AP‐1 composition in different melanoma cell lines compared with normal melanocytes, in which c‐Jun, JunD and FosB were involved in AP‐1 complexes. In w3211, c‐Jun, JunD and Fra‐1 were involved in AP‐1 binding, while in w1205, overall AP‐1 binding activity was decreased significantly and supershift binding was detected only with JunD antibodies. In metastatic c81‐46A and A375 cells, only JunD was involved in AP‐1 binding activity, but in a third (c83‐2c) c‐Jun, JunD and Fra‐1 were present. Western blot evaluation detected c‐Jun in melanocytes and w3211, but this component was decreased significantly or was not detectable in w1205, c81‐46A and A375 cells. In contrast, JunD protein was elevated in c81‐46A and c83‐2c cells compared with melanocytes and RGP and VGP cell lines. Normal melanocytes and c83‐2c cells (which have c‐Jun involved in AP‐1 binding), transfected with c‐Jun antisense and treated with cisplatin, showed higher viability compared with untransfected cells, while in c81‐46A cells (in which only JunD is detectable) no change in cell viability was observed following treatment with cisplatin and c‐jun antisense transfection. A dominant‐negative c‐Jun mutant (TAM67) significantly increased the soft agar colony formation of w3211 and c83‐2c cells. These results suggest that components of AP‐1, especially c‐Jun, may offer a new target for the prevention or treatment of human melanoma progression.     

The role of 5‐HT2c receptor on corticosterone‐mediated food intake

Corticosterone plays an important role in feeding behavior. However, its mechanism remains unclear. Therefore, the present study aimed to investigate the effect of corticosterone on feeding behavior. In this study, cumulative food intake was increased by acute corticosterone administration in a dose‐dependent manner. Administration of the 5‐HT_2c receptor agonist m‐chlorophenylpiperazin (mCPP) reversed the effect of corticosterone on food intake. The anorectic effects of mCPP were also blocked by the 5‐HT_2c receptor antagonist RS102221 in corticosterone‐treated mice. Both corticosterone and mCPP increased c‐Fos expression in hypothalamic nuclei, but not the nucleus of the solitary tract. RS102221 inhibited c‐Fos expression induced by mCPP, but not corticosterone. In addition, mCPP had little effect on TH and POMC levels in the hypothalamus. Furthermore, mCPP antagonized decreasing effect of the leptin produced by corticosterone. Taken together, our findings suggest that 5‐HT_2c receptors and leptin may be involved in the effects of corticosterone‐induced hyperphagia.     

Mutual regulation between butyrate and hypoxia‐inducible factor‐1α in epithelial cell promotes expression of tight junction proteins

Inflammatory bowel disease is a kind of multi‐aetiological chronic disease that is driven by multidimensional factors. Hypoxia‐inducible factor‐1α (HIF‐1α) plays an important role in anti‐inflammatory and cellular responses to hypoxia. Previous studies have found that B or T‐cell‐specific HIF‐1α knock out mice exhibit severe colonic inflammation. However, we know very little about other functions of HIF‐1α in intestinal epithelial cells (IECs). In our study, HIF‐1α^ΔIEC mice were used to study the function of HIF‐1α in IECs. HIF‐1α was knocked down in Caco‐2 cells by transfection with a small interfering (si) RNA. Immunohistochemical staining and western blotting were used to detect the expression of zonula occluden‐1 (ZO‐1) and Occludin. The content of colon was harvested for high‐performance liquid chromatography analysis to examine the levels of butyrate in the gut. Our research found that HIF‐1α played a protective role in dextran sulphate sodium‐induced colitis, which was partly due to its regulation of tight junction (TJ) protein expression. Further study revealed that HIF‐1α mediated TJ proteins levels by moderating the content of butyrate. Moreover, we found that butyrate regulated TJ protein expression, which is dependent on HIF‐1α. These results indicated that there is a mutual regulatory mechanism between butyrate and HIF‐1α, which has an important role in the maintenance of barrier function of the gastrointestinal tract.     

12‐O‐tetradecanoylphorbol‐13‐acetate‐induced invasion/migration of glioblastoma cells through activating PKCα/ERK/NF‐κB‐dependent MMP‐9 expression

An increase in MMP‐9 gene expression and enzyme activity with stimulating the migration of GBM8401 glioma cells via wound healing assay by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) was detected in glioblastoma cells GBM8401. TPA‐induced translocation of protein kinase C (PKC)α from the cytosol to membranes, and migration of GBM8401 elicited by TPA was suppressed by adding the PKCα inhibitors, GF109203X and H7. Activation of extracellular signal‐regulated kinase (ERK) and c‐Jun‐N‐terminal kinase (JNK) by TPA was identified, and TPA‐induced migration and MMP‐9 activity was significantly blocked by ERK inhibitor PD98059 and U0126, but not JNK inhibitor SP600125. Activation of NF‐κB protein p65 nuclear translocation and IκBα protein phosphorylation with increased NF‐κB‐directed luciferase activity by TPA were observed, and these were blocked by the PD98059 and IkB inhibitor BAY117082 accompanied by reducing migration and MMP‐9 activity induced by TPA in GBM8401 cells. Transfection of GBM8401 cells with PKCα siRNA specifically reduced PKCα protein expression with blocking TPA‐induced MMP‐9 activation and migration. Additionally, suppression of TPA‐induced PKCα/ERK/NK‐κB activation, migration, and MMP‐9 activation by flavonoids including kaempferol (Kae; 3,5,7,4′‐tetrahydroxyflavone), luteolin (Lut; 5,7,3′4′‐tetrahydroxyflavone), and wogonin (Wog; 5,7‐dihydroxy‐8‐methoxyflavone) was demonstrated, and structure–activity relationship (SAR) studies showed that hydroxyl (OH) groups at C4′ and C8 are critical for flavonoids' action against MMP‐9 enzyme activation and migration/invasion of glioblastoma cells elicited by TPA. Application of flavonoids to prevent the migration/invasion of glioblastoma cells through blocking PKCα/ERK/NF‐κB activation is first demonstrated herein. J. Cell. Physiol. 225: 472–481, 2010. © 2010 Wiley‐Liss, Inc.