SFRP-4 abrogates Wnt-3a-induced beta-catenin and Akt/PKB signalling and reverses a Wnt-3a-imposed inhibition of in vitro mammary differentiation

ABSTRACT: BACKGROUND: Conserved Wnt ligands are critical for signalling during development; however, various factors modulate their activity. Among these factors are the Secreted Frizzled-Related Proteins (SFRP). We previously isolated the SFRP-4 gene from an involuting rat mammary gland and later showed that transgenic mice inappropriately expressing SFRP-4 during lactation exhibited a high level of apoptosis with reduced survival of progeny. RESULTS: In order to address the questions related to the mechanism of Wnt signalling and its inhibition by SFRP-4 which we report here, we employed partially-purified Wnt-3a in a co-culture model system. Ectopic expression of SFRP-4 was accomplished by infection with a pBabepuro construct. The co-cultures comprised Line 31E mouse mammary secretory epithelial cells and Line 30F, undifferentiated, fibroblast-like mouse mammary cells. In vitro differentiation of such co-cultures can be demonstrated by induction of the beta-casein gene in response to lactogenic hormones.We show here that treatment of cells with partially-purified Wnt-3a initiates Dvl-3, Akt/PKB and GSK-3beta hyperphosphorylation and beta-catenin activation. Furthermore, while up-regulating the cyclin D1 and connexin-43 genes and elevating transepithelial resistance of Line 31E cell monolayers, Wnt-3a treatment abrogates differentiation of co-cultures in response to the lactogenic hormones prolactin, insulin and glucocorticoid. Cells which express SFRP-4, however, are largely unaffected by Wnt-3a stimulation. Since a physical association between Wnt-3a and SFRP-4 could be demonstrated with immunoprecipitation/Western blotting experiments, this interaction, presumably owing to the Frizzled homology region typical of all SFRPs, explains the refractory response to Wnt-3a which was observed. CONCLUSION: This study demonstrates that Wnt-3a treatment activates the Wnt signalling pathway and interferes with in vitro differentiation of mammary co-cultures to beta-casein production in response to lactogenic hormones. Similarly, in another measure of differentiation, following Wnt-3a treatment mammary epithelial cells could be shown to up-regulate the cyclin D1 and connexin-43 genes while phenotypically they show increased transepithelial resistance across the cell monolayer. All these behavioural changes can be blocked in mammary epithelial cells expressing SFRP-4. Thus, our data illustrate in an in vitro model a mechanism by which SFRP-4 can modulate a differentiation response to Wnt-3a.

Agents used for chemoprevention of prostate cancer may influence PSA secretion independently of cell growth in the LNCaP model of human prostate cancer progression

BACKGROUND: The aim of this study was to evaluate the inhibitory growth effects of different potential chemopreventive agents in vitro and to determine their influence on PSA mRNA and protein expression with an established screening platform. METHODS: LNCaP and C4-2 cells were incubated with genistein, seleno-L-methionine, lycopene, DL-alpha-tocopherol, and trans-beta-carotene at three different concentrations and cell growth was determined by the MTT assay. PSA mRNA expression was assessed by quantitative real-time RT-PCR and secreted PSA protein levels were quantified by the microparticle enzyme immunoassay. RESULTS: Genistein, seleno-l-methionine and lycopene inhibited LNCaP cell growth, and the proliferation of C4-2 cells was suppressed by seleno-L-methionine and lycopene. PSA mRNA expression was downregulated by genistein in LNCaP but not C4-2 cells. No other compound tested altered PSA mRNA expression. PSA protein expression was downregulated by genistein, seleno-L-methionine, DL-alpha-tocopherol in LNCaP cells. In C4-2 cells only genistein significantly reduced the secretion of PSA protein. CONCLUSIONS: In the LNCaP progression model PSA expression depends on the compound, its concentration and on the hormonal dependence of the cell line used and does not necessarily reflect cell growth or death. Before potential substances are evaluated in clinical trials using PSA as a surrogate end point marker, their effect on PSA mRNA and protein expression has to be considered to correctly assess treatment response by PSA.

Tumor necrosis factor-alpha upregulates 11beta-hydroxysteroid dehydrogenase type 1 expression by CCAAT/enhancer binding protein-beta in HepG2 cells

The enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the conversion of inactive to active glucocorticoids. 11beta-HSD1 plays a crucial role in the pathogenesis of obesity and controls glucocorticoid actions in inflammation. Several studies have demonstrated that TNF-alpha increases 11beta-HSD1 mRNA and activity in various cell models. Here, we demonstrate that mRNA and activity of 11beta-HSD1 is increased in liver tissue from transgenic mice overexpressing TNF-alpha, indicating that this effect also occurs in vivo. To dissect the molecular mechanism of this increase, we investigated basal and TNF-alpha-induced transcription of the 11beta-HSD1 gene (HSD11B1) in HepG2 cells. We found that TNF-alpha acts via p38 MAPK pathway. Transient transfections with variable lengths of human HSD11B1 promoter revealed highest activity with or without TNF-alpha in the proximal promoter region (-180 to +74). Cotransfection with human CCAAT/enhancer binding protein-alpha (C/EBPalpha) and C/EBPbeta-LAP expression vectors activated the HSD11B1 promoter with the strongest effect within the same region. Gel shift and RNA interference assays revealed the involvement of mainly C/EBPalpha, but also C/EBPbeta, in basal and only of C/EBPbeta in the TNF-alpha-induced HSD11B1 expression. Chromatin immunoprecipitation assay confirmed in vivo the increased abundance of C/EBPbeta on the proximal HSD11B1 promoter upon TNF-alpha treatment. In conclusion, C/EBPalpha and C/EBPbeta control basal transcription, and TNF-alpha upregulates 11beta-HSD1, most likely by p38 MAPK-mediated increased binding of C/EBPbeta to the human HSD11B1 promoter. To our knowledge, this is the first study showing involvement of p38 MAPK in the TNF-alpha-mediated 11beta-HSD1 regulation, and that TNF-alpha stimulates enzyme activity in vivo.

Interferon-beta stabilizes barrier characteristics of the blood-brain barrier in four different species in vitro

BACKGROUND: Blood-brain barrier (BBB) breakdown is an early event in the pathogenesis of multiple sclerosis (MS). In a previous study we have found a direct stabilization of barrier characteristics after treatment of bovine brain capillary endothelial cells (BCECs) with human recombinant interferon-beta-1a (IFN-beta-1a) in an in vitro BBB model. In the present study we examined the effect of human recombinant IFN-beta-1a on the barrier properties of BCECs derived from four different species including humans to predict treatment efficacy of IFN-beta-1a in MS patients. METHODS: We used primary bovine and porcine BCECs, as well as human and murine BCEC cell lines. We investigated the influence of human recombinant IFN-beta-1a on the paracellular permeability for 3H-inulin and 14C-sucrose across monolayers of bovine, human, and murine BCECs. In addition, the transendothelial electrical resistance (TEER) was determined in in vitro systems applying porcine and murine BCECS. RESULTS: We found a stabilizing effect on the barrier characteristics of BCECs after pretreatment with IFN-beta-1a in all applied in vitro models: addition of IFN-beta-1a resulted in a significant decrease of the paracellular permeability across monolayers of human, bovine, and murine BCECs. Furthermore, the TEER was significantly increased after pretreatment of porcine and murine BCECs with IFN-beta-1a. CONCLUSION: Our data suggest that BBB stabilization by IFN-beta-1a may contribute to its beneficial effects in the treatment of MS. A human in vitro BBB model might be useful as bioassay for testing the treatment efficacy of drugs in MS.

Extracts of Lindera obtusiloba induce antifibrotic effects in hepatic stellate cells via suppression of a TGF-beta-mediated profibrotic gene expression pattern

Liver fibrosis is characterized by high expression of the key profibrogenic cytokine transforming growth factor (TGF)-beta and the natural tissue inhibitor of metalloproteinases (TIMP)-1, leading to substantial accumulation of extracellular matrix. Liver fibrosis originates from various chronic liver diseases, such as chronic viral hepatitis that, to date, cannot be treated sufficiently. Thus, novel therapeutics, for example, those derived from Oriental medicine, have gained growing attention. In Korea, extracts prepared from Lindera obtusiloba are used for centuries for treatment of inflammation, improvement of blood circulation and prevention of liver damage, but experimental evidence of their efficacy is lacking. We studied direct antifibrotic effects in activated hepatic stellate cells (HSCs), the main target cell in the fibrotic liver. L. obtusiloba extract (135 mug/ml) reduced the de novo DNA synthesis of activated rat and human HSCs by about 90%, which was not accompanied by cytotoxicity of HSC, primary hepatocytes and HepG2 cells, pointing to induction of cellular quiescence. As determined by quantitative polymerase chain reaction, simultaneous treatment of HSCs with TGF-beta and L. obtusiloba extract resulted in reduction of TIMP-1 expression to baseline level, disruption of the autocrine loop of TGF-beta autoinduction and increased expression of fibrolytic matrix metalloproteinase (MMP)-3. In addition, L. obtusiloba reduced gelatinolytic activity of HSC by interfering with profibrogenic MMP-2 activity. Since L. obtusiloba extract prevented intracellular oxidative stress experimentally induced by tert-butylhydroperoxide, we concluded that the direct antifibrotic effect of L. obtusiloba extract might be mediated by antioxidant activity. Thus, L. obtusiloba, traditionally used in Oriental medicine, may complement treatment of chronic liver disease.

Beta1 integrins differentially control extravasation of inflammatory cell subsets into the CNS during autoimmunity

Inhibiting the alpha(4) subunit of the integrin heterodimers alpha(4)beta(1) and alpha(4)beta(7) with the monoclonal antibody natalizumab is an effective treatment for multiple sclerosis (MS). However, the pharmacological action of natalizumab is not understood conclusively. Previous studies suggested that natalizumab inhibits activation, proliferation, or extravasation of inflammatory cells. To specify which mechanisms, cell types, and alpha(4) heterodimers are affected by the antibody treatment, we studied MS-like experimental autoimmune encephalomyelitis (EAE) in mice lacking the beta(1)-integrin gene either in all hematopoietic cells or selectively in T lymphocytes. Our results show that T cells critically rely on beta(1) integrins to accumulate in the central nervous system (CNS) during EAE, whereas CNS infiltration of beta(1)-deficient myeloid cells remains unaffected, suggesting that T cells are the main target of anti-alpha(4)-antibody blockade. We demonstrate that beta(1)-integrin expression on encephalitogenic T cells is critical for EAE development, and we therefore exclude alpha(4)beta(7) as a target integrin of the antibody treatment. T cells lacking beta(1) integrin are unable to firmly adhere to CNS endothelium in vivo, whereas their priming and expansion remain unaffected. Collectively, these results suggest that the primary action of natalizumab is interference with T cell extravasation via inhibition of alpha(4)beta(1) integrins.

Cathepsin D specifically cleaves the chemokines macrophage inflammatory protein-1 alpha, macrophage inflammatory protein-1 beta, and SLC that are expressed in human breast cancer

Cathepsin D (Cath-D) expression in human primary breast cancer has been associated with a poor prognosis. In search of a better understanding of the Cath-D substrates possibly involved in cancer invasiveness and metastasis, we investigated the potential interactions between this protease and chemokines. Here we report that purified Cath-D, as well as culture supernatants from the human breast carcinoma cell lines MCF-7 and T47D, selectively degrade macrophage inflammatory protein (MIP)-1 alpha (CCL3), MIP-1 beta (CCL4), and SLC (CCL21). Proteolysis was totally blocked by the protease inhibitor pepstatin A, and specificity of Cath-D cleavage was demonstrated using a large chemokine panel. Whereas MIP-1 alpha and MIP-1 beta degradation was rapid and complete, cleavage of SLC was slow and not complete. Mass spectrometry analysis showed that Cath-D cleaves the Leu(58) to Trp(59) bond of SLC producing two functionally inactive fragments. Analysis of Cath-D proteolysis of a series of monocyte chemoattractant protein-3/MIP-1 beta hybrids indicated that processing of MIP-1 beta might start by cleaving off amino acids located in the C-terminal domain. In situ hybridization studies revealed MIP-1 alpha, MIP-1 beta, and Cath-D gene expression mainly in the stromal compartment of breast cancers whereas SLC transcripts were found in endothelial cells of capillaries and venules within the neoplastic tissues. Cath-D production in the breast carcinoma cell lines MCF-7 and T47D, as assessed by enzyme-linked immunosorbent assay of culture supernatants and cell lysates, was not affected by stimulation with chemokines such as interleukin-8 (CXCL8), SDF-1 (CXCL12), and SLC. These data suggest that inactivation of chemokines by Cath-D possibly influences regulatory mechanisms in the tumoral extracellular microenvironment that in turn may affect the generation of the antitumoral immune response, the migration of cancer cells, or both processes.

Stable expression of Escherichia coli beta-glucuronidase A (GusA) in Giardia lamblia: application to high-throughput drug susceptibility testing

OBJECTIVES: In order to create a suitable model for high-throughput drug screening, a Giardia lamblia WB C6 strain expressing Escherichia coli glucuronidase A (GusA) was created and tested with respect to susceptibility to the anti-giardial drugs nitazoxanide and metronidazole. METHODS: GusA, a well-established reporter gene in other systems, was cloned into the vector pPacVInteg allowing stable expression in G. lamblia under control of the promoter from the glutamate dehydrogenase (gdh) gene. The resulting transgenic strain was compared with the wild-type strain in a vitality assay, characterized with respect to susceptibility to nitazoxanide, metronidazole and -- as assessed in a 96-well plate format -- to a panel of 15 other compounds to be tested for anti-giardial activity. RESULTS: GusA was stably expressed in G. lamblia. Using a simple glucuronidase assay protocol, drug efficacy tests yielded results similar to those from cell counting. CONCLUSIONS: G. lamblia WB C6 GusA is a suitable tool for high-throughput anti-giardial drug screening.

Sudden infant death syndrome-associated mutations in the sodium channel beta subunits.

BACKGROUND Approximately 10% of sudden infant death syndrome (SIDS) cases may stem from potentially lethal cardiac channelopathies, with approximately half of channelopathic SIDS involving the Na(V)1.5 cardiac sodium channel. Recently, Na(V) beta subunits have been implicated in various cardiac arrhythmias. Thus, the 4 genes encoding Na(V) beta subunits represent plausible candidate genes for SIDS. OBJECTIVE This study sought to determine the spectrum, prevalence, and functional consequences of sodium channel beta-subunit mutations in a SIDS cohort. METHODS In this institutional review board-approved study, mutational analysis of the 4 beta-subunit genes, SCN1B to 4B, was performed using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing of DNA derived from 292 SIDS cases. Engineered mutations were coexpressed with SCN5A in HEK 293 cells and were whole-cell patch clamped. One of the putative SIDS-associated mutations was similarly studied in adenovirally transduced adult rat ventricular myocytes. RESULTS Three rare (absent in 200 to 800 reference alleles) missense mutations (beta3-V36M, beta3-V54G, and beta4-S206L) were identified in 3 of 292 SIDS cases. Compared with SCN5A+beta3-WT, beta3-V36M significantly decreased peak I(Na) and increased late I(Na), whereas beta3-V54G resulted in a marked loss of function. beta4-S206L accentuated late I(Na) and positively shifted the midpoint of inactivation compared with SCN5A+beta4-WT. In native cardiomyocytes, beta4-S206L accentuated late I(Na) and increased the ventricular action potential duration compared with beta4-WT. CONCLUSION This study provides the first molecular and functional evidence to implicate the Na(V) beta subunits in SIDS pathogenesis. Altered Na(V)1.5 sodium channel function due to beta-subunit mutations may account for the molecular pathogenic mechanism underlying approximately 1% of SIDS cases.

Distinct roles of estrogen receptors alpha and beta mediating acute vasodilation of epicardial coronary arteries.

This study investigated the contribution of estrogen receptors (ERs) alpha and beta for epicardial coronary artery function, vascular NO bioactivity, and superoxide (O(2)(-)) formation. Porcine coronary rings were suspended in organ chambers and precontracted with prostaglandin F(2alpha) to determine direct effects of the selective ER agonists 4,4',4''-(4-propyl-[(1)H]pyrazole-1,3,5-triyl)tris-phenol (PPT) or 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) or the nonselective ER agonist 17beta-estradiol. Indirect effects on contractility to U46619 and relaxation to bradykinin were assessed and effects on NO, nitrite, and O(2)(-) formation were measured in cultured cells. Within 5 minutes, selective ERalpha activation by PPT, but not 17beta-estradiol or the ERbeta agonist DPN, caused rapid, NO-dependent, and endothelium-dependent relaxation (49+/-5%; P<0.001 versus ethanol). PPT also caused sustained endothelium- and NO-independent vasodilation similar to 17beta-estradiol after 60 minutes (72+/-3%; P<0.001 versus ethanol). DPN induced endothelium-dependent NO-independent relaxation via endothelium-dependent hyperpolarization (40+/-4%; P<0.01 versus ethanol). 17beta-Estradiol and PPT, but not DPN, attenuated the responses to U46619 and bradykinin. All of the ER agonists increased NO and nitrite formation in vascular endothelial but not smooth muscle cells and attenuated vascular smooth muscle cell O(2)(-) formation (P<0.001). ERalpha activation had the most potent effects on both nitrite formation and inhibiting O(2)(-) (P<0.05). These data demonstrate novel and differential mechanisms by which ERalpha and ERbeta activation control coronary artery vasoreactivity in males and females and regulate vascular NO and O(2)(-) formation. The findings indicate that coronary vascular effects of sex hormones differ with regard to affinity to ERalpha and ERbeta, which will contribute to beneficial and adverse effects of hormone replacement therapy.

Clostridium perfringens beta-toxin induces necrostatin-inhibitable, calpain-dependent necrosis in primary porcine endothelial cells

Clostridium perfringens β-toxin (CPB) is a β-barrel pore-forming toxin and an essential virulence factor of C. perfringens type C strains, which cause fatal hemorrhagic enteritis in animals and humans. We have previously shown that CPB is bound to endothelial cells within the intestine of affected pigs and humans, and that CPB is highly toxic to primary porcine endothelial cells (pEC) in vitro. The objective of the present study was to investigate the type of cell death induced by CPB in these cells, and to study potential host cell mechanisms involved in this process. CPB rapidly induced lactate dehydrogenase (LDH) release, propidium iodide uptake, ATP depletion, potassium efflux, a marked rise in intracellular calcium [Ca(2+)]i, release of high-mobility group protein B1 (HMGB1), and caused ultrastructural changes characteristic of necrotic cell death. Despite a certain level of caspase-3 activation, no appreciable DNA fragmentation was detected. CPB-induced LDH release and propidium iodide uptake were inhibited by necrostatin-1 and the two dissimilar calpain inhibitors PD150606 and calpeptin. Likewise, inhibition of potassium efflux, chelation of intracellular calcium and treatment of pEC with cyclosporin A also significantly inhibited CPB-induced LDH release. Our results demonstrate that rCPB primarily induces necrotic cell death in pEC, and that necrotic cell death is not merely a passive event caused by toxin-induced membrane disruption, but is propagated by host cell-dependent biochemical pathways activated by the rise in intracellular calcium and inhibitable by necrostatin-1, consistent with the emerging concept of programmed necrosis ("necroptosis").

Beta 1-integrins are required for hippocampal AMPA receptor-dependent synaptic transmission, synaptic plasticity, and working memory.

Integrins comprise a large family of cell adhesion receptors that mediate diverse biological events through cell-cell and cell-extracellular matrix interactions. Recent studies have shown that several integrins are localized to synapses with suggested roles in synaptic plasticity and memory formation. We generated a postnatal forebrain and excitatory neuron-specific knock-out of beta1-integrin in the mouse. Electrophysiological studies demonstrated that these mutants have impaired synaptic transmission through AMPA receptors and diminished NMDA receptor-dependent long-term potentiation. Despite the impairment in hippocampal synaptic transmission, the mutants displayed normal hippocampal-dependent spatial and contextual memory but were impaired in a hippocampal-dependent, nonmatching-to-place working memory task. These phenotypes parallel those observed in animals carrying knock-outs of the GluR1 (glutamate receptor subunit 1) subunit of the AMPA receptor. These observations suggest a new function of beta1-integrins as regulators of synaptic glutamate receptor function and working memory.

LY2109761, a novel transforming growth factor beta receptor type I and type II dual inhibitor, as a therapeutic approach to suppressing pancreatic cancer metastasis.

Most pancreatic cancer patients present with inoperable disease or develop metastases after surgery. Conventional therapies are usually ineffective in treating metastatic disease. It is evident that novel therapies remain to be developed. Transforming growth factor beta (TGF-beta) plays a key role in cancer metastasis, signaling through the TGF-beta type I/II receptors (TbetaRI/II). We hypothesized that targeting TbetaRI/II kinase activity with the novel inhibitor LY2109761 would suppress pancreatic cancer metastatic processes. The effect of LY2109761 has been evaluated on soft agar growth, migration, invasion using a fibroblast coculture model, and detachment-induced apoptosis (anoikis) by Annexin V flow cytometric analysis. The efficacy of LY2109761 on tumor growth, survival, and reduction of spontaneous metastasis have been evaluated in an orthotopic murine model of metastatic pancreatic cancer expressing both luciferase and green fluorescence proteins (L3.6pl/GLT). To determine whether pancreatic cancer cells or the cells in the liver microenvironment were involved in LY2109761-mediated reduction of liver metastasis, we used a model of experimental liver metastasis. LY2109761 significantly inhibited the L3.6pl/GLT soft agar growth, suppressed both basal and TGF-beta1-induced cell migration and invasion, and induced anoikis. In vivo, LY2109761, in combination with gemcitabine, significantly reduced the tumor burden, prolonged survival, and reduced spontaneous abdominal metastases. Results from the experimental liver metastasis models indicate an important role for targeting TbetaRI/II kinase activity on tumor and liver microenvironment cells in suppressing liver metastasis. Targeting TbetaRI/II kinase activity on pancreatic cancer cells or the cells of the liver microenvironment represents a novel therapeutic approach to prevent pancreatic cancer metastasis.

Mutations affecting beta-tubulin folding and degradation.

Revertants of a colcemid-resistant Chinese hamster ovary cell line with an altered (D45Y) beta-tubulin have allowed the identification of four cis-acting mutations (L187R, Y398C, a 12-amino acid in-frame deletion, and a C-terminal truncation) that act by destabilizing the mutant tubulin and preventing it from incorporating into microtubules. These unstable beta-tubulins fail to form heterodimers and are predominantly found in association with the chaperonin CCT, suggesting that they cannot undergo productive folding. In agreement with these in vivo observations, we show that the defective beta-tubulins do not stably interact with cofactors involved in the tubulin folding pathway and, hence, fail to exchange with beta-tubulin in purified alphabeta heterodimers. Treatment of cells with MG132 causes an accumulation of the aberrant tubulins, indicating that improperly folded beta-tubulin is degraded by the proteasome. Rapid degradation of the mutant tubulin does not elicit compensatory changes in wild-type tubulin synthesis or assembly. Instead, loss of beta-tubulin from the mutant allele causes a 30-40% decrease in cellular tubulin content with no obvious effect on cell growth or survival.

14-3-3 proteins interact with the beta-thymosin repeat protein Csp24.

Conditioned stimulus pathway protein 24 (Csp24) is a beta-thymosin-like protein that is homologous to other members of the family of beta-thymosin repeat proteins that contain multiple actin binding domains. Actin co-precipitates with Csp24 and co-localizes with it in the cytosol of type-B photoreceptor cell bodies. Several signal transduction pathways have been shown to regulate the phosphorylation of Csp24 and contribute to cellular plasticity. Here, we report the identification of the adapter protein 14-3-3 in lysates of the Hermissenda circumesophageal nervous system and its interaction with Csp24. Immunoprecipitation experiments using an antibody that is broadly reactive with several isoforms of the 14-3-3 family of proteins showed that Csp24 co-precipitates with 14-3-3 protein, and nervous systems stimulated with 5-HT exhibited a significant increase in co-precipitated Csp24 probed with a phosphospecific antibody as compared with controls. These results indicate that post-translational modifications of Csp24 regulate its interaction with 14-3-3 protein, and suggest that this mechanism may contribute to the control of intrinsic enhanced excitability.