Aurora A regulates the architecture of the Golgi apparatus.

The Golgi apparatus plays roles in cell polarity, directional cell migration, and bipolar spindle assembly, as well as the secretary pathway. In addition, recent studies have suggested the Golgi-dependent control of mitotic entry. We studied the role of the centrosomal kinase Aurora A in maintaining the Golgi apparatus. Knockdown of Aurora A resulted in Golgi dispersal during interphase. Golgi dispersal was also induced by a selective Aurora A inhibitor, MLN8237. Conversely, overexpression of Aurora A led to tightly packed Golgi apparatus during interphase. Knockdown or inhibition of Aurora A had little or no effect on Golgi vesiculation during mitosis. By synchronizing cell division, we studied whether mitosis was required to induce Golgi dispersal during interphase. Aurora A inhibition induced aberrant mitotic spindle and Golgi dispersal only after mitosis. However, the cells treated with the inhibitor MLN8237 at earlier cell cycle stages (wherein the cells remained undivided) had a normal Golgi architecture. Knockdown or inhibition of Aurora A also led to aberrant integrity of centrosome and Golgi apparatus during interphase. These results suggest that Aurora A activity is involved in the maintenance of Golgi architecture and the relationship between the Golgi apparatus and centrosome.

Fate of graft cells: what should be clarified for development of mesenchymal stem cell therapy for ischemic stroke?

Mesenchymal stem cells (MSCs) are believed to be promising for cell administration therapy after ischemic stroke. Because of their advantageous characteristics, such as ability of differentiation into neurovascular lineages, avoidance of immunological problems, and abundance of graft cells in mesodermal tissues, studies regarding MSC therapy have increased recently. However, several controversies are yet to be resolved before a worldwide consensus regarding a standard protocol is obtained. In particular, the neuroprotective effects, the rate of cell migration to the lesion, and differentiation direction differ depending on preclinical observations. Analyses of these differences and application of recent developments in stem cell biology or engineering in imaging modality may contribute to identification of criteria for optimal stem cell therapy in which reliable protocols, which control cell quality and include safe administration procedures, are defined for each recovery phase after cerebral ischemia. In this mini review, we examine controversies regarding the fate of grafts and the prospects for advanced therapy that could be obtained through recent developments in stem cell research as direct conversion to neural cells.

Possible involvement of tumor-producing VEGF-A in the recruitment of lymphatic endothelial progenitor cells from bone marrow.

Lymphatic metastasis of human malignant adenocarcinomas is a critical determinant of prognosis. Lymphangiogenesis, the growth of lymphatic vessels, is closely involved in lymphatic metastasis. However, the mechanisms of tumor lymphangiogenesis are not clearly understood. In a previous study, we showed that human gastric cancer MKN45 cells organize neighboring lymphatic vessels via recruitment of bone marrow-derived lymphatic endothelial progenitor cells in a nude mouse xenograft model. The present results also indicated that human colorectal cancer LS174T and breast cancer SK-BR-3 cells promoted lymphangiogenesis as well as the recruitment of lymphatic endothelial progenitor cells from bone marrow. Among growth factors, which are reported to be involved in lymphangiogenesis, only vascular endothelial growth factor (VEGF)-A was extensively secreted by these three types of adenocarcinoma cells in culture. The well-characterized lymphangiogenic factors VEGF-C and VEGF-D in the culture medium of these three types of adenocarcinoma cells were below the detectable levels in ELISA assay. Secretion of epidermal growth factor (EGF) and hepatocyte growth factor (HGF) was not detected. In in vitro culture assay, VEGF-A directly induced the differentiation of bone marrow mononuclear cells into LYVE-1-positive lymphatic endothelial lineage cells. These data collectively suggest the possibility that VEGF-A-rich human adenocarcinomas induce tumor lymphangiogenesis via recruitment of lymphangiogenic endothelial progenitor cells from bone marrow.

Human gastric cancer organizes neighboring lymphatic vessels via recruitment of bone marrow-derived lymphatic endothelial progenitor cells.

BACKGROUND: Lymphatic metastasis is a critical determinant of prognosis in human gastrointestinal cancers. Studies suggest that lymphatic metastasis has been linked to lymphangiogenesis, the growth of lymphatic vessels, while the mechanisms of tumor lymphangiogenesis remain poorly characterized. METHODS: Human gastric cancer cells, MKN45, were implanted under the gastric submucosa of nude mice receiving green fluorescent protein-positive bone marrow (BM) transplants. In addition, MKN45 cells were subcutaneously injected into the back of each mouse as a model of human tumor xenografts. The tumor tissue was analyzed 3 weeks after implantation. RESULTS: The mice with MKN45 cells represent recruitment and incorporation of BM-derived lymphatic endothelial progenitor cells (LEPC) into gastric lymphatics. Moreover, in a xenograft model, MKN45 cells induced lymphangiogenesis as well as recruitment of BM-derived LEPC in tumor lymphatics in a xenograft model. CONCLUSIONS: These findings of this study suggest that human gastric adenocarcinoma induces tumor lymphangiogenesis via recruitment of LEPC from BM.

Induction of peroxisomal lipid metabolism in mice fed a high-fat diet.

Peroxisomes catalyze a range of essential metabolic functions, mainly related to lipid metabolism. However, their roles in obesity have yet to be clarified. The aim of this study was to investigate the correlation between obesity and peroxisomal lipid metabolism, particularly very long-chain fatty acid (VLCFA) metabolism, gene expression of peroxisomal ß-oxidation enzymes, peroxisomal ATP-binding cassette (ABC) transporter adrenoleukodystrophy (ABCD1) gene and its related gene, ABCD2, the elongation of the VLCFA (ELOVL) gene family and the transcriptional factors involved in the regulation of these genes, including peroxisome proliferator-activated receptor α (PPARα) and sterol regulatory element-binding protein. These factors were analyzed in livers from mice fed a high-fat diet (HFD) or a regular diet (RD) for 20 weeks. Furthermore, the amounts of plasma saturated and unsaturated fatty acids, including VLCFAs, were measured. A HFD induced hepatic gene expression of not only hydroxysteroid 17-ß dehydrogenase 4 (HSD17b4) and sterol carrier protein 2 (SCP2) in peroxisomal ß-oxidation enzymes but also of ELOVL1, 2, 5 and 6, which are involved in the elongation of saturated and unsaturated VLCFAs. Furthermore, ABCD2 mRNA prominently increased in the HFD mice. The transcriptional regulator of these genes, PPARα, was also up-regulated in the HFD mice. VLCFA ratios including C24:0/C22:0, C25:0/C22:0 and C26:0/C22:0 are the most significant diagnostic markers of inherited peroxisomal diseases. These ratios were found to be low in the plasma of the HFD mice compared with the RD mice. The results suggest that HFD activates hepatic peroxisomal VLCFA metabolism, and may provide useful fundamental information to explain the role of peroxisomal function in obesity and lifestyle-related diseases.

Comparison of mesenchymal stem cells from adipose tissue and bone marrow for ischemic stroke therapy.

BACKGROUND AIMS: Transplantation of mesenchymal stromal cells (MSC) derived from bone marrow (BM) or adipose tissue is expected to become a cell therapy for stroke. The present study compared the therapeutic potential of adipose-derived stem cells (ASC) with that of BM-derived stem cells (BMSC) in a murine stroke model. METHODS: ASC and BMSC were isolated from age-matched C57BL/6J mice. These MSC were analyzed for growth kinetics and their capacity to secrete trophic factors and differentiate toward neural and vascular cell lineages in vitro. For in vivo study, ASC or BMSC were administrated intravenously into recipient mice (1 × 10(5) cells/mouse) soon after reperfusion following a 90-min middle cerebral artery occlusion. Neurologic deficits, the degree of infarction, expression of factors in the brain, and the fate of the injected cells were observed. RESULTS: ASC showed higher proliferative activity with greater production of vascular endothelial cell growth factor (VEGF) and hepatocyte growth factor (HGF) than BMSC. Furthermore, in vitro conditions allowed ASC to differentiate into neural, glial and vascular endothelial cells. ASC administration showed remarkable attenuation of ischemic damage, although the ASC were not yet fully incorporated into the infarct area. Nonetheless, the expression of HGF and angiopoietin-1 in ischemic brain tissue was significantly increased in ASC-treated mice compared with the BMSC group. CONCLUSIONS: Compared with BMSC, ASC have great advantages for cell preparation because of easier and safer access to adipose tissue. Taken together, our findings suggest that ASC would be a more preferable source for cell therapy for brain ischemia than BMSC.

Neutrophil elastase inhibitor prevents ischemic brain damage via reduction of vasogenic edema.

Release of neutrophil elastase is one of the harmful inflammatory reactions in acute cerebral ischemia. Therefore, inhibition of elastase released from neutrophils could be a useful strategy for the treatment of acute stroke. To evaluate this hypothesis, the effect of sivelestat, a selective neutrophil elastase inhibitor was examined in a mouse model of focal ischemia. The results obtained indicate that sivelestat reduced brain edema and vascular permeability, and subsequently improved the neurological deficit in an acute focal ischemia. The architecture of microvessels was analyzed by identifying vascular endothelial cells, which were prelabeled by injecting fluorescein-labeled Griffonia simplicifolia lectin I-isolectin B4 into a tail vein. Most of the microvessels in the infarcted area were structurally destroyed in the control group. In sharp contrast, microvessels in the boundary zone were well maintained in the sivelestat-treated group. Moreover, the expression of angiopoietin-1 was elevated at the ischemic margin in the sivelestat-treated group. Furthermore, the neutrophil elastase inhibitor rescued human brain microvascular endothelial cells in culture from neutrophil elastase-induced damage. These results suggest that neutrophil elastase inhibition could protect blood-brain barrier function in acute cerebral ischemia by augmentation of angiopoietin-1 expression and survival of endothelial cells.

The stent-eluting drugs sirolimus and paclitaxel suppress healing of the endothelium by induction of autophagy.

Clinical studies have indicated that the stent-eluting drugs sirolimus and paclitaxel impact restenosis; however, it is still elusive how these drugs affect the vascular endothelium at the molecular and cellular levels. The purpose of this study was to determine whether sirolimus and paclitaxel induce molecular and cellular alterations in the vascular endothelium. Endothelial regrowth was assessed in human aortic endothelial cells and rat aortic endothelium. Molecular and cellular alterations were analyzed in human aortic endothelial cells by Western blot analysis, transmission electron microscopy, and immunofluorescence staining. Green fluorescent protein-LC3 mice were used to analyze autophagic endothelium. Here, we show that sirolimus and paclitaxel differentially induce self-digesting autophagy in vascular endothelial cells with changes in expression of LC3B, p53, and Bcl-2, considerably suppressing re-endothelialization and revascularization. These results suggest that phenotypic alteration in the endothelium by sirolimus or paclitaxel might affect the rates of late stent thrombosis, myocardial infarction, and mortality.

Alzheimer disease-associated peptide, amyloid beta40, inhibits vascular regeneration with induction of endothelial autophagy.

OBJECTIVE: Although the majority of cases of Alzheimer disease (AD) are known to be attributable to the sporadic (nongenetic) form of the disease, the mechanism underlying its cause and progression still remains unclear. METHODS AND RESULTS: We found that vascular beta-amyloid (Abeta), Abeta40, inhibited the proliferative activity of human brain vascular endothelial cells (HBECs) without toxic effects on them. This peptide also inhibited tube formation and migration of HBECs. Moreover, Abeta40 inhibited ex vivo hippocampal revascularization, reendothelialization, and the differentiation of adult endothelial progenitor cells. Importantly, Abeta40 suppressed the proliferative activity of HBECs through the induction of "self-digesting" autophagy. This induction involved the intracellular regulation of class 3 phosphatidylinositol 3-kinase (PI3K) as well as Akt signaling in HBECs. Furthermore, tissue culture of murine brain sections from GFP-LC3 transgenic mice revealed that Abeta40 not only reduced the vessel density in hippocampal lesions, but also induced autophagy in neurovascular ECs. CONCLUSIONS: Our present findings indicate that the initial progression of AD might be in part driven by Abeta40-induced endothelial autophagy and impairment of neurovascular regeneration, suggesting important implications for therapeutic approaches to AD.

Overexpression of immediate early gene X-1 (IEX-1) enhances gamma-radiation-induced apoptosis of human glioma cell line, U87-MG.

Malignant gliomas are usually incurable even if adjuvant therapy is delivered after neurosurgical treatment. Therefore, to enhance their radiation-induced apoptosis, it is important to detect the mechanism(s) leading to the death of malignant glioma cells. We report that apoptosis was induced in a time-dependent manner after gamma-radiation and that irradiated U87-MG cells (human glioblastoma cell line) expressed immediate early gene X-1 (IEX-1) with p53. We also document that their apoptotic sensitivity to gamma-radiation was enhanced by the overexpression of IEX-1. Our findings suggest that IEX-1 may represent a new factor for the enhancement of radiation-induced apoptosis of human glioma cells.

AKT plays a pivotal role in the acquisition of resistance to 5-fluorouracil in human squamous carcinoma cells.

5-Fluorouracil (5-FU) is a widely used chemotherapeutic agent that inhibits the growth and initiates the apoptosis of epithelial tumors, including squamous cell carcinoma of the head and neck region. However, resistance to this drug is often observed in a clinical setting. The primary mode of action of 5-FU is believed to be the inhibition of thymidylate synthase. Overexpression of the enzymes involved in thymidine synthesis has been shown in some cases to be associated with resistance. However, the detailed mechanisms of resistance of squamous cell carcinoma are not fully understood. In the present study, we examined the involvement of survival signaling pathways in the resistance of squamous carcinoma cells to 5-FU. 5-FU induced the activation of the ERK and Akt kinases in UM-SCC-23 human squamous carcinoma cells, indicating that this anticancer drug activates survival signaling pathways as well as apoptotic signals. In 5-FU-resistant UM-SCC-23 cells established by our group, ERK and Akt signals were constitutively activated. U0126 is an inhibitor of MEK, which is an upstream activator for ERK. U0126 failed to sensitize resistant UM-SCC-23 cells to 5-FU-induced apoptotic cell death. This is in sharp contrast to LY294002, which is an inhibitor of phosphatidylinositol 3-kinase, an upstream activator for Akt. LY294002 drastically enhanced 5-FU-induced apoptotic cell death in resistant UM-SCC-23 cells. These results indicate that the Akt survival signal plays an important role in the resistance of squamous carcinoma cells to 5-FU treatment, and suggest that the modification of Akt activity might provide a new strategy for human 5-FU-resistant squamous carcinoma therapy.

Epidermal growth factor plays a crucial role in mitogenic regulation of human brain tumor stem cells.

A cancer stem cell population in malignant brain tumors takes an essential part in brain tumor initiation, growth, and recurrence. Growth factors, such as epidermal growth factor, fibroblast growth factor-2, vascular endothelial growth factor, platelet-derived growth factor, and hepatocyte growth factor, are shown to support the proliferation of neural stem cells and also may play key roles in gliomagenesis. However, the responsible growth factor(s), which controls maintenance of brain tumor stem cells, is not yet uncovered. We have established three cancer stem cell lines from human gliomas. These cells were immunoreactive with the neuronal progenitor markers, nestin and CD133, and established tumors that closely resembled the features of original tumor upon transplantation into mouse brain. Three cell lines retained their self-renewal ability and proliferation only in the presence of epidermal growth factor (>2.5 ng/ml). In sharp contrast, other growth factors, including fibroblast growth factor-2, failed to support maintenance of these cells. The tyrosine kinase inhibitors of epidermal growth factor signaling (AG1478 and gefitinib) suppressed the proliferation and self-renewal of these cells. Gefitinib inhibited phosphorylation of epidermal growth factor receptor as well as Akt kinase and extracellular signal-regulated kinase 1/2. Flow cytometric analysis revealed that epidermal growth factor concentration-dependently increased the population of CD133-positive cells. Gefitinib significantly reduced CD133-positive fractions and also induced their apoptosis. These results indicate that maintenance of human brain tumor stem cells absolutely requires epidermal growth factor and that tyrosine kinase inhibitors of epidermal growth factor signaling potentially inhibit proliferation and induce apoptosis of these cells.

Fasudil, a Rho kinase (ROCK) inhibitor, protects against ischemic neuronal damage in vitro and in vivo by acting directly on neurons.

BACKGROUND AND PURPOSE: Recently, fasudil, a Rho kinase (ROCK) inhibitor, was reported to prevent cerebral ischemia in vivo by increasing cerebral blood flow and inhibiting inflammatory responses. However, it is uncertain whether a ROCK inhibitor can directly protect neurons against ischemic damage. Our purpose was to evaluate both the involvement of ROCK activity in ischemic neuronal damage and any direct neuroprotective effect of fasudil against cerebral infarction. METHODS: In vivo, focal cerebral ischemia was induced by permanent middle cerebral artery occlusion in mice, and the resulting infarction was evaluated 24 h later. ROCK expression and activity were assessed using Western blotting and immunohistochemistry. In vitro, the effects of fasudil and hydroxyfasudil (a main metabolite of fasudil) were examined on oxygen-glucose deprivation (OGD)-induced PC12 cell death and on glutamate-induced neurotoxicity in primary cerebral neuronal culture. RESULTS: ROCK expression and activity increased in the striatum, especially in axons, in the early phase of ischemia. Fasudil reduced this ROCK activity and protected against cerebral infarction in vivo. Hydroxyfasudil inhibited OGD-induced PC12 cell death, and fasudil and hydroxyfasudil each attenuated glutamate-induced neurotoxicity in vitro. CONCLUSIONS: These findings indicate that ROCK plays a pivotal role in the mechanism underlying ischemic neuronal damage and that a direct effect of fasudil on neurons may be partly responsible for its protective effects against such damage.

Immediate early gene IEX-1 induces astrocytic differentiation of U87-MG human glioma cells.

The immediate early response gene IEX-1 is involved in the regulation of apoptosis and cell growth. In order to increase the apoptotic sensitivity to chemotherapeutic drugs and gamma-ray, we attempted to establish U87-MG human glioma cell line expressing IEX-1. Unexpectedly, however, transfection of IEX-1 into U87-MG glioma cells resulted in morphological changes to astrocytic phenotype and increase in glial differentiation marker proteins, S-100 and glial fibrillary acidic protein (GFAP). Glial cell differentiation was used to examine in rat C6 glioma cell line, since this cell line express astrocytic phenotypes by increase in intracellular cAMP concentration. Stimulation of human U87-MG glioma cells by membrane-permeable dibutyryl cAMP (dbcAMP) not only elicited their morphological changes but also induced expression of IEX-1 as well as S-100 and GFAP. H89, an inhibitor of protein kinase A (PKA), blocked dbcAMP-induced morphological changes of U87-MG cells and expression of IEX-1. In contrast, morphological changes and expression of S-100 and GFAP induced by IEX-1 were not affected by H89. Morphological changes induced by dbcAMP were totally abolished by functional disruption of IEX-1 expression by anti-sense RNA. These results indicate that IEX-1 plays an important role in astrocytic differentiation of human glioma cells and that IEX-1 functions at downstream of PKA.

Prostaglandin E2 downregulates TNF-alpha-induced production of matrix metalloproteinase-1 in HCS-2/8 chondrocytes by inhibiting Raf-1/MEK/ERK cascade through EP4 prostanoid receptor activation.

Matrix metalloproteinase-1 (MMP-1, collagenase-1) plays a pivotal role in the process of joint destruction in degenerative joint diseases. We have examined the regulation of MMP-1 production in human chondrocytic HCS-2/8 cells stimulated by tumor necrosis factor-alpha (TNF-alpha). In response to TNF-alpha, MMP-1 is induced and actively released from HCS-2/8 cells. The induction of MMP-1 expression correlates with activation of ERK1/2, MEK, and Raf-1, and is potently prevented by U0126, a selective inhibitor of MEK1/2 activation. In contrast, SB203580, a selective p38 mitogen-activated protein kinases (MAPK) inhibitor, had no effects on TNF-alpha-induced MMP-1 release. A serine/threonine kinase, Akt was not activated in TNF-alpha-stimulated HCS-2/8 cells. TNF-alpha stimulated the production of PGE(2) in addition to MMP-1 in HCS-2/8 cells. Exogenously added PGE(2) potently inhibited TNF-alpha-induced both MMP-1 production and activation of ERK1/2. The effects of PGE(2) were mimicked by ONO-AE1-329, a selective EP4 receptor agonist but not by butaprost, a selective EP2 agonist. In contrast, blockade of endogenously produced PGE(2) signaling by ONO-AE3-208, a selective EP4 receptor antagonist, enhanced TNF-alpha-induced MMP-1 production. Furthermore, the suppression of MMP-1 production by exogenously added PGE(2) was reversed by ONO-AE3-208. Activation of EP4 receptor resulted in cAMP-mediated phosphorylation of Raf-1 on Ser259, a negative regulatory site, and blocked activation of Raf-1/MEK/ERK cascade. Taken together, these findings indicate that Raf-1/MEK/ERK signaling pathway plays a crucial role in the production of MMP-1 in HCS-2/8 cells in response to TNF-alpha, and that the produced PGE(2) downregulates the expression of MMP-1 by blockage of TNF-alpha-induced Raf-1 activation through EP4-PGE(2) receptor activation.

Ceramide triggers caspase activation during gamma-radiation-induced apoptosis of human glioma cells lacking functional p53.

We have previously shown that treatment of human glioma U87-MG cells expressing wild-type p53 with a DNA topoisomerase II inhibitor, etoposide resulted in ceramide-dependent apoptotic cell death. However, U87-W E6 cells lacking functional p53 due to the expression of human papilloma virus type 16 (HPV-16) E6 oncoprotein were resistant to etoposide. In order to gain insight into the roles of p53 and ceramide in gamma-radiation-induced glioma cell death, we used U87-W E6 and vector-infected U87-LXSN cells. U87-LXSN glioma cells expressing wild-type p53 were relatively resistant to gamma-radiation. U87-W E6 cells, which lost functional p53, became susceptible to radiation-induced apoptosis. Activation of caspase-3, and formation of ceramide by acid sphingomyelinase, but not by neutral sphingomyelinase, were associated with p53-independent apoptosis. Radiation-induced caspase activation and apoptotic death in U87-W E6 cells were modified by the agents which affected ceramide metabolism. SR33557, an inhibitor of acid sphingomyelinase, suppressed radiation-induced caspase activation and then apoptotic cell death. In contrast, N-oleoylethanolamine (OE) and D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), which inhibit ceramidase and UDP-glucose:ceramide glucosyltransferase-1, respectively, and then augment ceramide formation, enhanced radiation-induced caspase activation. These results indicate that glioma cells with functional p53 were relatively resistant to gamma-radiation, and that ceramide may play an important role in caspase activation during gamma-radiation-induced apoptosis of glioma cells lacking functional p53.

Cisplatin activates survival signals in UM-SCC-23 squamous cell carcinoma and these signal pathways are amplified in cisplatin-resistant squamous cell carcinoma.

cis-Diaminodichloroplatinum (II) (cisplatin) is one of the most effective anticancer drugs and is widely used for the treatment of squamous cell carcinoma (SCC). However, its efficacy is often limited due to the development of resistance. Although several factors implicated in cisplatin resistance have been identified, the resistance mechanisms in detail are not fully understood yet. In the present study, we have examined the implication of survival signaling pathways in cisplatin-resistance. Cisplatin induced activation of Ras and its downstream effector kinases, Raf/MEK/ERK in UM-SCC-23 human squamous cell carcinoma, suggesting that this anticancer drug activates survival signal pathway in addition to apoptosis signals. In cisplatin-resistant UM-SCC-23 in culture, which we have established, the protein levels of Ras, Raf-1 and MEK were drastically elevated compared to parent UM-SCC-23, and ERK and Akt signals were constitutively activated. U0126, an inhibitor for MEK and LY294002, an inhibitor for phosphatidylinositol 3-kinase (PI3K), sensitized resistant UM-SCC-23 to cisplatin-induced cell death. These results indicate that Raf/MEK/ERK and PI3K/Akt signal cascades may play a considerable role in cisplatin resistance in SCC.

Effect of high-NaCl or high-KCl diet on hepatic Na+- and K+-receptor sensitivity and NKCC1 expression in rats.

We previously reported that the bumetanide-sensitive Na(+)-K(+)-2Cl- cotransporter (NKCC1) is involved in the hepatic Na+ and K+ sensor mechanism. In the present study, we examined the effects of a high-NaCl or high-KCl diet on hepatic Na+ and K+ receptor sensitivity and NKCC1 expression in the liver of Sprague-Dawley rats. RT-PCR and Western blots were used to measure NKCC1 mRNA and protein expression, respectively. Infusion of hypertonic NaCl or isotonic KCl + NaCl solutions into the portal vein increased hepatic afferent nerve activity (HANA) in a Na+ or K+ dose-dependent manner. After 4 wk on a high-NaCl or high-KCl diet, HANA responses were attenuated compared with animals fed a normal diet, and NKCC1 expression was reduced. These results show that a high-NaCl or high-KCl diet decreases NKCC1 expression in the liver, and it might cause a reduction in hepatic Na(+)- and K(+)-receptor sensitivity.

Expression of the NF-kappa B target gene X-ray-inducible immediate early response factor-1 short enhances TNF-alpha-induced hepatocyte apoptosis by inhibiting Akt activation.

Using a cDNA microarray analysis, we identified x-ray-inducible immediate early response factor-1 (IEX-1) as a proapoptotic gene which was induced by TNF-alpha and also depend on NF-kappaB activation in Hc human hepatocytes. In these cells only the original form of IEX-1, termed IEX-1S, but not its longer transcript IEX-1L, was expressed. Overexpression of IEX-1S resulted in promotion of TNF-alpha-induced apoptosis in Hc cells expressing a mutant form of IkappaB. This proapoptotic action can be explained by its inhibitory findings on survival signals; inhibition of TNF-alpha-induced activation and expression of phosphatidylinositol 3-kinase (PI3K)/Akt, and also blockage of expression of Mcl-1, an antiapoptotic Bcl-2 family member which is located downstream of Akt, was inhibited by IEX-1S. LY 294002, an inhibitor of PI3K, increased IEX-1S expression induced by TNF-alpha and accelerated TNF-alpha-induced apoptosis in IkappaB-treated Hc cells. Overexpression of the dominant-negative Akt enhanced, but the constitutively active Akt suppressed, TNF-alpha-induced IEX-1S expression, suggesting that PI3K/Akt negatively regulated IEX-1S expression. These results demonstrate that NF-kappaB-dependent recruitment of IEX-1S may play a proapoptotic role in TNF-alpha-stimulated hepatocytes through blockage of the PI3K/Akt pathway. Moreover, the reciprocal cross-talk between IEX-1S and PI3K/Akt may closely be involved in the regulation of TNF-alpha-induced hepatocyte apoptosis.