Regulation of lipopolysaccharide‐induced inflammatory response and endotoxemia by β‐arrestins

β‐Arrestins are scaffolding proteins implicated as negative regulators of TLR4 signaling in macrophages and fibroblasts. Unexpectedly, we found that β‐arrestin‐1 (β‐arr‐1) and ‐2 knockout (KO) mice are protected from TLR4‐mediated endotoxic shock and lethality. To identify the potential mechanisms involved, we examined the plasma levels of inflammatory cytokines/chemokines in the wild‐type (WT) and β‐arr‐1 and ‐2 KO mice after lipopolysaccharide (LPS, a TLR4 ligand) injection. Consistent with lethality, LPS‐induced inflammatory cytokine levels in the plasma were markedly decreased in both β‐arr‐1 and ‐2 KO, compared to WT mice. To further explore the cellular mechanisms, we obtained splenocytes (separated into CD11^b+ and CD11^b? populations) from WT, β‐arr‐1, and ‐2 KO mice and examined the effect of LPS on cytokine production. Similar to the in vivo observations, LPS‐induced inflammatory cytokines were significantly blocked in both splenocyte populations from the β‐arr‐2 KO compared to the WT mice. This effect in the β‐arr‐1 KO mice, however, was restricted to the CD11^b? splenocytes. Our studies further indicate that regulation of cytokine production by β‐arrestins is likely independent of MAPK and IκBα‐NFκB pathways. Our results, however, suggest that LPS‐induced chromatin modification is dependent on β‐arrestin levels and may be the underlying mechanistic basis for regulation of cytokine levels by β‐arrestins in vivo. Taken together, these results indicate that β‐arr‐1 and ‐2 mediate LPS‐induced cytokine secretion in a cell‐type specific manner and that both β‐arrestins have overlapping but non‐redundant roles in regulating inflammatory cytokine production and endotoxic shock in mice. J. Cell. Physiol. 225: 406–416, 2010. © 2010 Wiley‐Liss, Inc.     

The role of calcium in apoptosis induced by 7β‐hydroxycholesterol and cholesterol‐5β,6β‐epoxide

Oxysterols, such as 7β‐hydroxy‐cholesterol (7β‐OH) and cholesterol‐5β,6β‐epoxide (β‐epoxide), may have a central role in promoting atherogenesis. This is thought to be predominantly due to their ability to induce apoptosis in cells of the vascular wall and in monocytes/macrophages. Although there has been extensive research regarding the mechanisms through which oxysterols induce apoptosis, much remains to be clarified. Given that experimental evidence has long associated alterations of calcium (Ca²⁺) homeostasis to apoptotic cell death, the aim of the present study was to determine the influence of intracellular Ca²⁺ changes on apoptosis induced by 7β‐OH and β‐epoxide. Ca²⁺ responses in differentiated U937 cells were assessed by epifluorescence video microscopy, using the ratiometric dye fura‐2. Over 15‐min exposure of differentiated U937 cells to 30 μM of 7β‐OH induced a slow but significant rise in fura‐2 ratio. The Ca²⁺ channel blocker nifedipine and the chelating agent EGTA blocked the increase in cytoplasmic Ca²⁺. Moreover, dihydropyridine (DHP) binding sites identified with BODIPY‐FLX‐DHP were blocked following pretreatment with nifedipine, indicating that the influx of Ca²⁺ occurred through L‐type channels. However, following long‐term incubation with 7β‐OH, elevated levels of cytoplasmic Ca²⁺ were not maintained and nifedipine did not provide protection against apoptotic cell death. Our results indicate that the increase in Ca²⁺ may be an initial trigger of 7β‐OH–induced apoptosis, but following chronic exposure to the oxysterol, the influence of Ca²⁺ on apoptotic cell death appears to be less significant. In contrast, Ca²⁺ did not appear to be involved in β‐epoxide–induced apoptosis. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:324–332, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20295     

Pharmacology and immune modulating properties of 5-androstene-3β,7β,17β-triol, a DHEA metabolite in the human metabolome

Androst-5-ene-3β,7β,17β-triol (βAET) is an anti-inflammatory metabolite of DHEA that is found naturally in humans, but in rodents only after exogenous DHEA administration. Unlike DHEA, C-7-oxidized DHEA metabolites cannot be metabolized into potent androgens or estrogens, and are not peroxisome proliferators in rodents. The objective of our current studies was to characterize the pharmacology of βAET to enable clinical trials in humans. The pharmacology of βAET was characterized by pharmacokinetics, drug metabolism, nuclear hormone receptor interactions, androgenicity, estrogenicity, and systemic toxicity studies. βAET's acute anti-inflammatory activity and immune modulating characteristics were measured in vitro in RAW264.7 cells and in vivo in murine models with parenteral administration. βAET was rapidly metabolized and cleared from circulation in mice and monkeys. βAET was weakly androgenic and estrogenic in immature rodents, but not bound by androgen, estrogen, progesterone, or glucocorticoid nuclear hormone receptors. βAET did not induce peroxisome proliferation, nor was it systemically toxic or trophic for sex hormone responsive tissues in mature rats and monkeys. βAET significantly attenuated acute inflammation both in vitro and in vivo, augmented immune responses in adult mice, and reversed immune senescence in aged mice. βAET may contribute to the anti-inflammatory activity in rodents attributed to DHEA. Unlike DHEA, βAET's anti-inflammatory activity cannot be ascribed to activation of PPARs, androgen, or estrogen nuclear hormone receptors. Exogenous βAET is unlikely to produce untoward toxicity or hormonal perturbations in humans.     

Characterization of an Importin α/β‐recognized nuclear localization signal in β‐dystroglycan

β‐dystroglycan (β‐DG) is a widely expressed transmembrane protein that plays important roles in connecting the extracellular matrix to the cytoskeleton, and thereby contributing to plasma membrane integrity and signal transduction. We previously observed nuclear localization of β‐DG in cultured cell lines, implying the existence of a nuclear targeting mechanism that directs it to the nucleus instead of the plasma membrane. In this study, we delineate the nuclear import pathway of β‐DG, characterizing a functional nuclear localization signal (NLS) in the β‐DG cytoplasmic domain, within amino acids 776–782. The NLS either alone or in the context of the whole β‐DG protein was able to target the heterologous GFP protein to the nucleus, with site‐directed mutagenesis indicating that amino acids R⁷⁷⁹ and K⁷⁸⁰ are critical for NLS functionality. The nuclear transport molecules Importin (Imp)α and Impβ bound with high affinity to the NLS of β‐DG and were found to be essential for NLS‐dependent nuclear import in an in vitro reconstituted nuclear transport assay; cotransfection experiments confirmed the dependence on Ran for nuclear accumulation. Intriguingly, experiments suggested that tyrosine phosphorylation of β‐DG may result in cytoplasmic retention, with Y⁸⁹² playing a key role. β‐DG thus follows a conventional Impα/β‐dependent nuclear import pathway, with important implications for its potential function in the nucleus. J. Cell. Biochem. 110: 706–717, 2010. © 2010 Wiley‐Liss, Inc.     

Basic conformers in β‐peptides

The conformation of oligomers of β‐amino acids of the general type Ac‐[β‐Xaa]_n‐NHMe (β‐Xaa = β‐Ala, β‐Aib, and β‐Abu; n = 1–4) was systematically examined at different levels of ab initio molecular orbital theory (HF/6‐31G*, HF/3‐21G). The solvent influence was considered employing two quantum‐mechanical self‐consistent reaction field models. The results show a wide variety of possibilities for the formation of characteristic elements of secondary structure in β‐peptides. Most of them can be derived from the monomer units of blocked β‐peptides with n = 1. The stability and geometries of the β‐peptide structures are considerably influenced by the side‐chain positions, by the configurations at the C^α‐ and C^β‐atoms of the β‐amino acid constituents, and especially by environmental effects. Structure peculiarities of β‐peptides, in particular those of various helix alternatives, are discussed in relation to typical elements of secondary structure in α‐peptides. © 1999 John Wiley & Sons, Inc. Biopoly 50: 167–184, 1999     

Mutant huntingtin‐impaired degradation of β‐catenin causes neurotoxicity in Huntington's disease

Huntington's disease (HD) is a fatal neurodegenerative disorder causing selective neuronal death in the brain. Dysfunction of the ubiquitin–proteasome system may contribute to the disease; however, the exact mechanisms are still unknown. We report here a new pathological mechanism by which mutant huntingtin specifically interferes with the degradation of β‐catenin. Huntingtin associates with the β‐catenin destruction complex that ensures its equilibrated degradation. The binding of β‐catenin to the destruction complex is altered in HD, leading to the toxic stabilization of β‐catenin. As a consequence, the β‐transducin repeat‐containing protein (β‐TrCP) rescues polyglutamine (polyQ)‐huntingtin‐induced toxicity in striatal neurons and in a Drosophila model of HD, through the specific degradation of β‐catenin. Finally, the non‐steroidal anti‐inflammatory drug indomethacin that decreases β‐catenin levels has a neuroprotective effect in a neuronal model of HD and in Drosophila and increases the lifespan of HD flies. We thus suggest that restoring β‐catenin homeostasis in HD is of therapeutic interest.     

Sauchinone prevents IL‐1β‐induced inflammatory response in human chondrocytes

Sauchinone is one of the active lignan isolated from Saururus chinensis, which has been considered to possess various pharmacological activities, such as antitumor, hepatoprotective, antioxidant, and anti‐inflammatory effects. However, the functional roles of sauchinone in interleukin‐1 beta (IL‐1β)‐stimulated human osteoarthritis (OA) chondrocytes are still unknown. Thus, in this study, we investigated the anti‐inflammatory effects of sauchinone in IL‐1β‐stimulated chondrocytes. Our results demonstrated that sauchinone significantly attenuated NO and PGE2 production, as well as inhibited iNOS and COX‐2 expression in IL‐1β‐stimulated OA chondrocytes. In addition, sauchinone efficiently inhibited IL‐1β‐induced MMP‐3 and MMP‐13 release in human OA chondrocytes. Furthermore, sauchinone significantly attenuated the activation of NF‐κB in human OA chondrocytes. In conclusion, we showed for the first time that sauchinone inhibited inflammatory response in IL‐1β‐stimulated human chondrocytes probably through inhibiting the activation of NF‐κB signaling pathway. These data suggest that sauchinone may be a potential agent in the treatment of OA.     

GSKIP,an inhibitor of GSK3β, mediates the N‐cadherin/β‐catenin pool in the differentiation of SH‐SY5Y cells

Emerging evidence has shown that GSK3β plays a pivotal role in regulating the specification of axons and dendrites. Our previous study has shown a novel GSK3β interaction protein (GSKIP) able to negatively regulate GSK3β in Wnt signaling pathway. To further characterize how GSKIP functions in neurons, human neuroblastoma SH‐SY5Y cells treated with retinoic acid (RA) to differentiate to neuron‐like cells was used as a model. Overexpression of GSKIP prevents neurite outgrowth in SH‐SY5Y cells. GSKIP may affect GSK3β activity on neurite outgrowth by inhibiting the specific phosphorylation of tau (ser396). GSKIP also increases β‐catenin in the nucleus and raises the level of cyclin D1 to promote cell‐cycle progression in SH‐SY5Y cells. Additionally, overexpression of GSKIP downregulates N‐cadherin expression, resulting in decreased recruitment of β‐catenin. Moreover, depletion of β‐catenin by small interfering RNA, neurite outgrowth is blocked in SH‐SY5Y cells. Altogether, we propose a model to show that GSKIP regulates the functional interplay of the GSK3β/β‐catenin, β‐catenin/cyclin D1, and β‐catenin/N‐cadherin pool during RA signaling in SH‐SY5Y cells. J. Cell. Biochem. 108: 1325–1336, 2009. © 2009 Wiley‐Liss, Inc.     

Blockade of JAK2 activity suppressed accumulation of β‐catenin in leukemic cells

The Wnt/β‐catenin pathway has been implicated in leukemogenesis. We found β‐catenin abnormally accumulated in both human acute T cell leukemia Jurkat cells and human erythroleukemia HEL cells. β‐Catenin can be significantly down‐regulated by the Janus kinase 2 specific inhibitor AG490 in these two cells. AG490 also reduces the luciferase activity of a reporter plasmid driven by LEF/β‐catenin promoter. Similar results were observed in HEL cells infected with lentivirus containing shRNA against JAK2 gene. After treatment with 50 µM AG490 or shRNA, the mRNA expression levels of β‐catenin, APC, Axin, β‐Trcp, GSK3α, and GSK3β were up‐regulated within 12–16 h. However, only the protein levels of GSK3β and β‐Trcp were found to have increased relative to untreated cells. Knockdown experiments revealed that the AG490‐induced inhibition of β‐catenin can be attenuated by shRNA targeting β‐TrCP. Taken together; these results suggest that β‐Trcp plays a key role in the cross‐talk between JAK/STAT and Wnt/β‐catenin signaling in leukemia cells. J. Cell. Biochem. 111: 402–411, 2010. © 2010 Wiley‐Liss, Inc.     

Synthesis of 17,20β,21‐trihydroxypregn‐4‐en‐3‐one by ovaries of reproductively mature Atlantic cod Gadus morhua

Atlantic cod Gadus morhua ovaries were incubated in vitro with tritiated 17‐hydroxypregn‐4‐ene‐3,20‐dione (17‐P) to determine whether 17,20β‐dihydroxypregn‐4‐en‐3‐one (17,20β‐P) or 17,20β, 21‐trihydroxypregn‐4‐en‐3‐one (17,20β,21‐P), or both, were more likely to be the steroid responsible for inducing oocyte final maturation (i.e. resumption of meiosis). Only 17,20β,21‐P was produced, in addition to 11‐deoxycortisol (17,21‐P), which is intermediate between 17‐P and 17,20β,21‐P. Also, the 5β‐reduced forms of 17‐P, 17,21‐P and 17,20β,21‐P were all found. Some sulphation of 21‐hydroxylated steroids was demonstrated. The ability of female G. morhua to make 17,20β,21‐P but not 17,20β‐P was confirmed by radioimmunoassay of plasma samples from spawning fish. Although small amounts of 17,20β‐P immunoreactivity were detected in a few plasma samples, this was shown, by thin‐layer chromatography, to be mostly due to cross‐reaction with other unidentified compounds. The evidence strongly suggests that 17,20β,21‐P is more likely than 17,20β‐P to be the maturation‐inducing steroid in G. morhua.     

Gypenoside inhibits interleukin‐1β‐induced inflammatory response in human osteoarthritis chondrocytes

Gypenoside (GP), the main active ingredient of Gynostemma pentaphyllum, possesses a variety of pharmacological capacities including anti‐inflammation, anti‐oxidation, and anti‐tumor. However, the effects of GP on IL‐1β‐stimulated human osteoarthritis (OA) chondrocytes are still unknown. Therefore, this study aimed to investigate the anti‐inflammatory effects of GP on IL‐1β‐stimulated human OA chondrocytes and explore the possible mechanism. Our results showed that GP dose‐dependently inhibited IL‐1β‐induced NO and PGE2 production in human OA chondrocytes. In addition, treatment of GP inhibited the expression of MMP3 and MMP13, which was increased by IL‐1β. Finally, we found that pretreatment of GP obviously suppressed NF‐κB activation in IL‐1β‐stimulated human OA chondrocytes. Taken together, the results demonstrated that GP has chondro‐protective effects, at least in part, through inhibiting the activation of NF‐κB signaling pathway in human OA chondrocytes. Thus, these findings suggest that GP may be considered as an alternative therapeutic agent for the management of OA patients.     

Selection and optimization of MCF‐7 cell line for screening selective inhibitors of 11β‐hydroxysteroid dehydrogenase 2

An 11β‐hydroxysteroid dehydrogenase type 1 (11β‐HSD1) produces glucocorticoid (GC) from 11‐keto metabolite, and its modulation has been suggested as a novel approach to treat metabolic diseases. In contrast, type 2 isozyme 11β‐HSD2 is involved in the inactivation of glucocorticoids (GCs), protecting the non‐selective mineralocorticoid receptor (MR) from GCs in kidney. Therefore, when 11β‐HSD1 inhibitors are pursued to treat the metabolic syndrome, preferential selectivity of inhibitors for type 1 over type 2 isozyme is rather important than inhibitory potency. Primarily, to search for cell lines with 11β‐HSD2 activity, we investigated the expression profiles of enzymes or receptors relevant to GC metabolism in breast, colon, and bone‐derived cell lines. We demonstrated that MCF‐7 cells had high expression for 11β‐HSD2, but not for 11β‐HSD1 with its cognate receptor. Next, for the determination of enzyme activity indirectly, we adopted homogeneous time resolved fluorescence (HTRF) cortisol assay. Obviously, the feasibility of HTRF to cellular 11β‐HSD2 was corroborated by constructing inhibitory response to an 11b‐HSD2 inhibitor glycyrrhetinic acid (GA). Taken together, MCF‐7 that overexpresses type 2 but not type 1 enzyme is chosen for cellular 11β‐HSD2 assay, and our results show that a nonradioactive HTRF assay is applicable for type 2 as well as type 1 isozyme. Copyright © 2010 John Wiley & Sons, Ltd.     

β‐arrestin2/miR‐155/GSK3β regulates transition of 5′‐azacytizine‐induced Sca‐1‐positive cells to cardiomyocytes

Stem‐cell antigen 1–positive (Sca‐1+) cardiac stem cells (CSCs), a vital kind of CSCs in humans, promote cardiac repair in vivo and can differentiate to cardiomyocytes with 5′‐azacytizine treatment in vitro. However, the underlying molecular mechanisms are unknown. β‐arrestin2 is an important scaffold protein and highly expressed in the heart. To explore the function of β‐arrestin2 in Sca‐1+ CSC differentiation, we used β‐arrestin2–knockout mice and overexpression strategies. Real‐time PCR revealed that β‐arrestin2 promoted 5′‐azacytizine‐induced Sca‐1+ CSC differentiation in vitro. Because the microRNA 155 (miR‐155) may regulate β‐arrestin2 expression, we detected its role and relationship with β‐arrestin2 and glycogen synthase kinase 3 (GSK3β), another probable target of miR‐155. Real‐time PCR revealed that miR‐155, inhibited by β‐arrestin2, impaired 5′‐azacytizine‐induced Sca‐1+ CSC differentiation. On luciferase report assay, miR‐155 could inhibit the activity of β‐arrestin2 and GSK3β, which suggests a loop pathway between miR‐155 and β‐arrestin2. Furthermore, β‐arrestin2‐knockout inhibited the activity of GSK3β. Akt, the upstream inhibitor of GSK3β, was inhibited in β‐arrestin2‐Knockout mice, so the activity of GSK3β was regulated by β‐arrestin2 not Akt. We transplanted Sca‐1+ CSCs from β‐arrestin2‐knockout mice to mice with myocardial infarction and found similar protective functions as in wild‐type mice but impaired arterial elastance. Furthermore, low level of β‐arrestin2 agreed with decreased phosphorylation of AKT and increased phophorylation of GSK3β, similar to in vitro findings. The β‐arrestin2/miR‐155/GSK3β pathway may be a new mechanism with implications for treatment of heart disease.     

Distinct roles for β‐arrestin2 and arrestin‐domain‐containing proteins in β2 adrenergic receptor trafficking

β‐arrestin 1 and 2 (also known as arrestin 2 and 3) are homologous adaptor proteins that regulate seven‐transmembrane receptor trafficking and signalling. Other proteins with predicted ‘arrestin‐like’ structural domains but lacking sequence homology have been indicated to function like β‐arrestin in receptor regulation. We demonstrate that β‐arrestin2 is the primary adaptor that rapidly binds agonist‐activated β₂ adrenergic receptors (β₂ARs) and promotes clathrin‐dependent internalization, E3 ligase Nedd4 recruitment and ubiquitin‐dependent lysosomal degradation of the receptor. The arrestin‐domain‐containing (ARRDC) proteins 2, 3 and 4 are secondary adaptors recruited to internalized β₂AR–Nedd4 complexes on endosomes and do not affect the adaptor roles of β‐arrestin2. Rather, the role of ARRDC proteins is to traffic Nedd4–β₂AR complexes to a subpopulation of early endosomes.     

Phosphorylation of PPP(S/T)P motif of the free LRP6 intracellular domain is not required to activate the Wnt/β‐catenin pathway and attenuate GSK3β activity

The canonical Wnt/β‐catenin signaling pathway plays a critical role in numerous physiological and pathological processes. LRP6 is an essential co‐receptor for Wnt/β‐catenin signaling; as transduction of the Wnt signal is strongly dependent upon GSK3β‐mediated phosphorylation of multiple PPP(S/T)P motifs within the membrane‐anchored LRP6 intracellular domain. Previously, we showed that the free LRP6 intracellular domain (LRP6‐ICD) can activate the Wnt/β‐catenin pathway in a β‐catenin and TCF/LEF‐1 dependent manner, as well as interact with and attenuate GSK3β activity. However, it is unknown if the ability of LRP6‐ICD to attenuate GSK3β activity and modulate activation of the Wnt/β‐catenin pathway requires phosphorylation of the LRP6‐ICD PPP(S/T)P motifs, in a manner similar to the membrane‐anchored LRP6 intracellular domain. Here we provide evidence that the LRP6‐ICD does not have to be phosphorylated at its PPP(S/T)P motif by GSK3β to stabilize endogenous cytosolic β‐catenin resulting in activation of TCF/LEF‐1 and the Wnt/β‐catenin pathway. LRP6‐ICD and a mutant in which all 5 PPP(S/T)P motifs were changed to PPP(A)P motifs equivalently interacted with and attenuated GSK3β activity in vitro, and both constructs inhibited the in situ GSK3β‐mediated phosphorylation of β‐catenin and tau to the same extent. These data indicate that the LRP6‐ICD attenuates GSK3β activity similar to other GSK3β binding proteins, and is not a result of it being a GSK3β substrate. Our findings suggest the functional and regulatory mechanisms governing the free LRP6‐ICD may be distinct from membrane‐anchored LRP6, and that release of the LRP6‐ICD may provide a complimentary signaling cascade capable of modulating Wnt‐dependent gene expression. J. Cell. Biochem. 108: 886–895, 2009. © 2009 Wiley‐Liss, Inc.     

Molecular characterization of the β‐amyloid(4‐10) epitope of plaque specific Aβ antibodies by affinity‐mass spectrometry using alanine site mutation

Alzheimer disease is a neurodegenerative disease affecting an increasing number of patients worldwide. Current therapeutic strategies are directed to molecules capable to block the aggregation of the β‐amyloid(1‐42) (Aβ) peptide and its shorter naturally occurring peptide fragments into toxic oligomers and amyloid fibrils. Aβ‐specific antibodies have been recently developed as powerful antiaggregation tools. The identification and functional characterization of the epitope structures of Aβ antibodies contributes to the elucidation of their mechanism of action in the human organism. In previous studies, the Aβ(4‐10) peptide has been identified as an epitope for the polyclonal anti‐Aβ(1‐42) antibody that has been shown capable to reduce amyloid deposition in a transgenic Alzheimer disease mouse model. To determine the functional significance of the amino acid residues involved in binding to the antibody, we report here the effects of alanine single‐site mutations within the Aβ‐epitope sequence on the antigen‐antibody interaction. Specific identification of the essential affinity preserving mutant peptides was obtained by exposing a Sepharose‐immobilized antibody column to an equimolar mixture of mutant peptides, followed by analysis of bound peptides using high‐resolution MALDI‐Fourier transform‐Ion Cyclotron Resonance mass spectrometry. For the polyclonal antibody, affinity was preserved in the H6A, D7A, S8A, and G9A mutants but was lost in the F4, R5, and Y10 mutants, indicating these residues as essential amino acids for binding. Enzyme‐linked immunosorbent assays confirmed the binding differences of the mutant peptides to the polyclonal antibody. In contrast, the mass spectrometric analysis of the mutant Aβ(4‐10) peptides upon affinity binding to a monoclonal anti‐Aβ(1‐17) antibody showed complete loss of binding by Ala‐site mutation of any residue of the Aβ(4‐10) epitope. Surface plasmon resonance affinity determination of wild‐type Aβ(1‐17) to the monoclonal Aβ antibody provided a binding constant K_D in the low nanomolar range. These results provide valuable information in the elucidation of the binding mechanism and the development of Aβ‐specific antibodies with improved therapeutic efficacy.