Molecular and Cellular Effects of Chemical Chaperone-TUDCA on ER-Stressed NHAC-kn Human Articular Chondrocytes Cultured in Normoxic and Hypoxic Conditions.

Osteoarthritis (OA) is considered one of the most common arthritic diseases characterized by progressive degradation and abnormal remodeling of articular cartilage. Potential therapeutics for OA aim at restoring proper chondrocyte functioning and inhibiting apoptosis. Previous studies have demonstrated that tauroursodeoxycholic acid (TUDCA) showed anti-inflammatory and anti-apoptotic activity in many models of various diseases, acting mainly via alleviation of endoplasmic reticulum (ER) stress. However, little is known about cytoprotective effects of TUDCA on chondrocyte cells. The present study was designed to evaluate potential effects of TUDCA on interleukin-1ß (IL-1ß) and tunicamycin (TNC)-stimulated NHAC-kn chondrocytes cultured in normoxic and hypoxic conditions. Our results showed that TUDCA alleviated ER stress in TNC-treated chondrocytes, as demonstrated by reduced CHOP expression; however, it was not effective enough to prevent apoptosis of NHAC-kn cells in either normoxia nor hypoxia. However, co-treatment with TUDCA alleviated inflammatory response induced by IL-1ß, as shown by down regulation of Il-1ß, Il-6, Il-8 and Cox2, and increased the expression of antioxidant enzyme Sod2. Additionally, TUDCA enhanced Col IIα expression in IL-1ß- and TNC-stimulated cells, but only in normoxic conditions. Altogether, these results suggest that although TUDCA may display chondoprotective potential in ER-stressed cells, further analyses are still necessary to fully confirm its possible recommendation as potential candidate in OA therapy.

Short communication: Chemical structure, concentration, and pH are key factors influencing antimicrobial activity of conjugated bile acids against lactobacilli.

Effects of chemical structure, concentration, and pH on antimicrobial activity of conjugated bile acids were investigated in 4 strains of lactobacilli. Considerable differences were observed in the antimicrobial activity between the 6 human conjugated bile acids, including glycocholic acid, taurocholic acid, glycodeoxycholic acid, taurodeoxycholic acid, glycochenodeoxycholic acid, and taurochenodeoxycholic acid. Glycodeoxycholic acid and glycochenodeoxycholic acid generally showed significantly higher antimicrobial activity against the lactobacilli, but glycocholic acid and taurocholic acid exhibited the significantly lower antimicrobial activity. Glycochenodeoxycholic acid was selected for further analysis, and the results showed its antimicrobial activity was concentration-dependent, and there was a significantly negative linear correlation (R2 > 0.98) between bile-antimicrobial index and logarithmic concentration of the bile acid for each strain of lactobacilli. Additionally, the antimicrobial activity of glycochenodeoxycholic acid was also observed to be pH-dependent, and it was significantly enhanced with the decreasing pH, with the result that all the strains of lactobacilli were unable to grow at pH 5.0. In conclusion, chemical structure, concentration, and pH are key factors influencing antimicrobial activity of conjugated bile acids against lactobacilli. This study provides theoretical guidance and technology support for developing a scientific method for evaluating the bile tolerance ability of potentially probiotic strains of lactobacilli.

Evidence for functional selectivity in TUDC- and norUDCA-induced signal transduction via α5ß1 integrin towards choleresis.

Functional selectivity is the ligand-specific activation of certain signal transduction pathways at a receptor and has been described for G protein-coupled receptors. However, it has not yet been described for ligands interacting with integrins without αI domain. Here, we show by molecular dynamics simulations that four side chain-modified derivatives of tauroursodeoxycholic acid (TUDC), an agonist of α5ß1 integrin, differentially shift the conformational equilibrium of α5ß1 integrin towards the active state, in line with the extent of ß1 integrin activation from immunostaining. Unlike TUDC, 24-nor-ursodeoxycholic acid (norUDCA)-induced ß1 integrin activation triggered only transient activation of extracellular signal-regulated kinases and p38 mitogen-activated protein kinase and, consequently, only transient insertion of the bile acid transporter Bsep into the canalicular membrane, and did not involve activation of epidermal growth factor receptor. These results provide evidence that TUDC and norUDCA exert a functional selectivity at α5ß1 integrin and may provide a rationale for differential therapeutic use of UDCA and norUDCA.

Taurochenodeoxycholic Acid Inhibited AP-1 Activation via Stimulating Glucocorticoid Receptor.

Taurochenodeoxycholic acid (TCDCA) as a primary bioactive substance of animal bile has been shown to exert good anti-inflammatory and immunomodulatory functions in adjuvant arthritis in rats. The anti-inflammatory and immunomodulatory properties of TCDCA have exhibited interesting similarities with the effects of glucocorticoids (GCs). To investigate the potential mechanisms of TCDCA in anti-inflammation and immunomodulation, we used a luciferase reporter assay to evaluate the activation of the glucocorticoid receptor (GR) stimulated by TCDCA. Our results showed that GR was activated by TCDCA in a concentration-dependent manner. Moreover, the elevated expressions of c-Fos and phosphorylated c-Jun induced by interleukin-1ß (IL-1ß) were reversed by TCDCA. The inhibition of TCDCA on the transactivation of activator protein-1 (AP-1) was observed as well. However, the suppression of TCDCA on the phosphorylation of c-Jun was blocked incompletely by GR inhibitor RU486. These results have indicated that the anti-inflammatory and immunomodulatory functions of TCDCA involve multiple pathways, with contributions from GR and its related AP-1 signaling pathway.

Review: The bile acids urso- and tauroursodeoxycholic acid as neuroprotective therapies in retinal disease.

Bile acids are produced in the liver and excreted into the intestine, where their main function is to participate in lipid digestion. Ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA) have shown antiapoptotic, anti-inflammatory, and antioxidant effects in various models of neurodegenerative diseases. However, little is known about signaling pathways and molecular mechanisms through which these bile acids act as neuroprotectors, delaying translation to the clinical setting. We review evidence supporting a potentially therapeutic role for bile acids in retinal disorders, and the mechanisms and pathways involved in the cytoprotective effects of bile acids from the liver and the enterohepatic circulation to the central nervous system and the retina. As secondary bile acids are generated by the microbiota metabolism, bile acids might be a link between neurodegenerative retinal diseases and microbiota.

Tauroursodeoxycholic acid (TUDCA) counters osteoarthritis by regulating intracellular cholesterol levels and membrane fluidity of degenerated chondrocytes.

Cholesterol and lipid metabolism are associated with osteoarthritis (OA) in human cartilage. High cholesterol levels in OA chondrocytes leads to decreased membrane fluidity and blocks the signaling cascade associated with the expression of chondrogenic genes. It is known that bile acid plays a role in regulating cholesterol homeostasis and the digestion of fats in the human body. Tauroursodeoxycholic acid (TUDCA), as a member of the bile acid family, also aids in the transport of cellular cholesterol. In this study, we hypothesized that TUDCA might be able to promote the restoration of OA cartilage by reducing membrane cholesterol levels in OA chondrocytes and by stimulating the chondrogenic signaling cascade. To assess this hypothesis, we investigated the effects of TUDCA on degenerated chondrocytes isolated from patients with OA. Importantly, treatment with TUDCA at sub-micellar concentrations (2500 µM) significantly increased cell proliferation and Cyclin D1 expression compared with the controls. In addition, the expression of chondrogenic marker genes (SOX9, COL2, and ACAN), proteins (SOX9 and COL2), and glycosaminoglycan (Chondroitin sulfate) was much higher in the TUDCA-treated group compared to the controls. We also found that TUDCA treatment significantly reduced the intracellular cholesterol levels in the chondrocytes and increased membrane fluidity. Furthermore, the stability of TGF receptor 1 and activity of focal adhesion proteins were also increased following TUDCA treatment. Together, these results demonstrated that TUDCA could be used as an alternative treatment for the restoration of OA cartilage.

A New Drug Delivery System Based on Tauroursodeoxycholic Acid and PEDOT.

Localized drug delivery represents one of the most challenging uses of systems based on conductive polymer films. Typically, anionic drugs are incorporated within conductive polymers through electrostatic interaction with the positively charged polymer. Following this approach, the synthetic glucocorticoid dexamethasone phosphate is often delivered from neural probes to reduce the inflammation of the surrounding tissue. In light of the recent literature on the neuroprotective and anti-inflammatory properties of tauroursodeoxycholic acid (TUDCA), for the first time, this natural bile acid was incorporated within poly(3,4-ethylenedioxythiophene) (PEDOT). The new material, PEDOT-TUDCA, efficiently promoted an electrochemically controlled delivery of the drug, while preserving optimal electrochemical properties. Moreover, the low cytotoxicity observed with viability assays, makes PEDOT-TUDCA a good candidate for prolonging the time span of chronic neural recording brain implants.

Chemical chaperone-conjugated exendin-4 as a cytoprotective agent for pancreatic ß-cells.

Endoplasmic reticulum stress has been considered a major cause of pancreatic ß-cell dysfunction and apoptosis leading to diabetes. Glucagon-like peptide-1 receptor activation and chemical chaperones have been known to reduce endoplasmic reticulum stress and improve ß-cell function and survival. The purpose of this study was to prepare and evaluate the chemical chaperone tauroursodeoxycholic acid-conjugated exendin-4 as a protective agent for pancreatic ß-cells. Mono-tauroursodeoxycholic acid-Lys27-exendin-4 conjugate (TUM1-Ex4) showed better receptor binding affinity than other conjugates with strong in vitro insulinotropic activity in rat pancreatic ß-cells and in vivo hypoglycemic activity in type 2 diabetic db/db mice. In INS-1 cells under endoplasmic reticulum stress induced by thapsigargin, TUM1-Ex4 promoted cell survival in a dose-dependent manner. In western blot analysis, TUM1-Ex4 reduced the expression of the endoplasmic reticulum stress marker GRP78 and phosphorylation of the translation initiation factor eIF2α. These results reveal that TUM1-Ex4 accelerates translational recovery and contributes to ß-cell protection and survival. The present study indicates that the chemical chaperone-coupled glucagon-like peptide-1 receptor agonist is a feasible therapeutic strategy to enhance ß-cell function and survival.

Study on Micelle Formation of Bile Salt Using Nuclear Magnetic Resonance Spectroscopy.

1H-nuclear magnetic resonance (1H-NMR) measurements can identify the specific protons that contribute to interactions between molecules. Using this technique, micelles formed by four bile salts: sodium taurocholate (NaTC), taurodeoxycholate (NaTDC), taurochenodeoxycholate (NaTCDC), and tauroursodeoxycholate (NaTUDC) were measured and compared in viewpoint of molecular interactions. Rotating-frame nuclear Overhauser effect and exchange spectroscopy (ROESY) analysis of the four bile salts showed differences with respect to the type of micelle formation. For all four bile salts, the key protons contributing to hydrophobic interactions were found to be the methyl protons at positions 18 and 19. The cross-peak patterns for the four bile salt species indicated pairs of characteristic proton depending on a bile salt species. The spin-lattice relaxation time (T1) of the alkyl side-chain in NaTC was relatively long compared to that of the three other bile salts, even when the concentration was higher than the critical micelle concentration (cmc). This unique behavior indicates that the hydrophilic region of NaTC molecules is flexible within their micelles. Moreover, T1 values for the typically hydrophobic methyl protons at sites C18 and C19 of NaTC were almost constant above the cmc. These results may suggest that NaTC micelles remain as small primary structures in solution unlike the three other bile salt molecules investigated in the study.

Analysis of the potency of various low molecular weight chemical chaperones to prevent protein aggregation.

Newly translated proteins must undergo proper folding to ensure their function. To enter a low energy state, misfolded proteins form aggregates, which are associated with many degenerative diseases, such as Huntington's disease and chronic kidney disease (CKD). Recent studies have shown the use of low molecular weight chemical chaperones to be an effective method of reducing protein aggregation in various cell types. This study demonstrates a novel non-biased assay to assess the molecular efficacy of these compounds at preventing protein misfolding and/or aggregation. This assay utilizes a thioflavin T fluorescent stain to provide a qualitative and quantitative measure of protein misfolding within cells. The functionality of this method was first assessed in renal proximal tubule epithelial cells treated with various endoplasmic reticulum (ER) stress inducers. Once established in the renal model system, we analyzed the ability of some known chemical chaperones to reduce ER stress. A total of five different compounds were selected: 4-phenylbutyrate (4-PBA), docosahexaenoic acid (DHA), tauroursodeoxycholic acid, trehalose, and glycerol. The dose-dependent effects of these compounds at reducing thapsigargin-induced ER stress was then analyzed, and used to determine their EC50 values. Of the chaperones, 4-PBA and DHA provided the greatest reduction of ER stress and did so at relatively low concentrations. Upon analyzing the efficiency of these compounds and their corresponding structures, it was determined that chaperones with a localized hydrophilic, polar end followed by a long hydrophobic chain, such as 4-PBA and DHA, were most effective at reducing ER stress. This study provides some insight into the use of low molecular weight chemical chaperones and may serve as the first step towards developing new chaperones of greater potency thereby providing potential treatments for diseases caused by protein aggregation.

Steroid binding to Autotaxin links bile salts and lysophosphatidic acid signalling.

Autotaxin (ATX) generates the lipid mediator lysophosphatidic acid (LPA). ATX-LPA signalling is involved in multiple biological and pathophysiological processes, including vasculogenesis, fibrosis, cholestatic pruritus and tumour progression. ATX has a tripartite active site, combining a hydrophilic groove, a hydrophobic lipid-binding pocket and a tunnel of unclear function. We present crystal structures of rat ATX bound to 7α-hydroxycholesterol and the bile salt tauroursodeoxycholate (TUDCA), showing how the tunnel selectively binds steroids. A structure of ATX simultaneously harbouring TUDCA in the tunnel and LPA in the pocket, together with kinetic analysis, reveals that bile salts act as partial non-competitive inhibitors of ATX, thereby attenuating LPA receptor activation. This unexpected interplay between ATX-LPA signalling and select steroids, notably natural bile salts, provides a molecular basis for the emerging association of ATX with disorders associated with increased circulating levels of bile salts. Furthermore, our findings suggest potential clinical implications in the use of steroid drugs.

The three-dimensional structure of Clostridium absonum 7α-hydroxysteroid dehydrogenase: new insights into the conserved arginines for NADP(H) recognition.

7α-hydroxysteroid dehydrogenase (7α-HSDH) can catalyse the oxidation of C7 α-OH of the steroid nucleus in the bile acid metabolism. In the paper we determined the crystal structure of 7α-HSDH from Clostridium absonum (CA 7α-HSDH) complexed with taurochenodeoxycholic acid (TCDCA) and NADP(+) by X-ray diffraction, which, as a tetramer, possesses the typical α/ß folding pattern. The four subunits of an asymmetric unit lie in the fact that there are the stable hydrophobic interactions between Q-axis-related subunits. Significantly, we captured an active state of the NADP(+), confirming that nicotinamide moiety of NADP(+) act as electron carrier in the dehydrogenation. On the basis of crystal structure analysis, site-directed mutagenesis and MD simulation, furthermore, we find that the guanidinium of Arg38 can form the stable cation-π interaction with the adenine ring of NADP(+), and the cation-π interaction and hydrogen bonds between Arg38 and NADP(+) have a significant anchor effect on the cofactor binding to CA 7α-HSDH.

Unusual Steroid Constituents from the Tropical Starfish Leiaster sp.

Two polar steroid compounds, taurochenodeoxycholic acid sodium salt (1) and a rare cyclic steroid glycoside luzonicoside A (2), were isolated from the tropical starfish Leiaster sp. and identified by extensive NMR and ESIMS techniques. The isolation of primary bile acid 1 is the first report from a representative of the class Asteroidea and on the whole in invertebrates. Its presence confirms the hypothesis about the digestive role of some polar steroids in starfish and possibly demonstrates the parallel evolution of fat emulsifying agents in vertebrates and some starfish. Compound 2 was also obtained from starfish belonging to this genus for the first time. This finding indicates that cyclic steroid glycosides are more widely distributed in starfish than the two species of the genus Echinaster from which they were isolated earlier.

Two solid forms of tauroursodeoxycholic acid and the effects of milling and storage temperature on solid-state transformations.

Two phase-pure solid forms of tauroursodeoxycholic acid (TUDCA) were prepared and characterized by thermal analysis, vibrational spectroscopy, X-ray diffraction, solid-state nuclear magnetic resonance, and morphological analysis. All solid forms can be produced from solvents and also can be obtained by mechanically and non-mechanically activated polymorph conversion. Near-infrared (NIR) spectroscopy, in combination with chemometrical techniques, was used for the quantitative monitoring of the polymorph conversion of TUDCA in milling process and at different storage temperatures. The NIR spectra in the range of 7139-5488 cm(-1) were considered for multivariate analysis. Results demonstrated that the NIR multivariate chemometric model can predict the percentage of crystal and amorphous TUDCA with the correlation coefficient of 0.9998, root mean square error of calibration of 0.740%, root mean square error of prediction of 0.698%, and root mean square error of cross-validation of 1.49%. In the milling process of crystal TUDCA (Form I), a direct transformation from crystal to glass was observed in 4h. Moreover, the impact of different storage temperatures on the stability of amorphous TUDCA was investigated, and the rate of polymorph transformation was found to be accelerated with increasing temperature.

[Structure determination of three novel bile acids from bear bile powder].

A method of LC-QTOF/MS combining with chemical synthesis has been used to determine the structures of three novel bile acids from bear bile powder. Reference substances of tauroursodeoxycholic acid and taurochenodeoxycholic acid were oxidized by pyridinium chlorochromate. The products were analyzed by LC-QTOF/MS. Total 4 products including 3 isomers were predicted and identified according to the PCC oxidation theory and LC-QTOF/MS results. Bear bile powder samples were dissolved by methanol and analyzed by LC-QTOF/MS. Three unknown peaks were found and identified as 2-[[(3beta, 5beta)-3-hydroxy-7, 24-dioxocholan-24-yl]amino]-ethanesulfonic acid, 2-[[(5beta)-3, 7, 24-trioxocholan-24-yl]amino]-ethanesulfonic acid and 2-[[(5beta, 7beta)-7-hydroxy-3, 24-dioxocholan-24-yl]amino]-ethanesulfonic acid, separately, by matching their results with that of oxidation products above.

Cytotoxic bile acids, but not cytoprotective species, inhibit the ordering effect of cholesterol in model membranes at physiologically active concentrations.

Submillimolar concentrations of cytotoxic bile acids (BAs) induce cell death via apoptosis. On the other hand, several cytoprotective BAs were shown to prevent apoptosis in the same concentration range. Still, the mechanisms by which BAs trigger these opposite signaling effects remain unclear. This study was aimed to determine if cytotoxic and cytoprotective BAs, at physiologically active concentrations, are able to modulate the biophysical properties of lipid membranes, potentially translating into changes in the apoptotic threshold of cells. Binding of BAs to membranes was assessed through the variation of fluorescence parameters of suitable derivatized BAs. These derivatives partitioned with higher affinity to liquid disordered than to the cholesterol-enriched liquid ordered domains. Unlabeled BAs were also shown to have a superficial location upon interaction with the lipid membrane. Additionally, the interaction of cytotoxic BAs with membranes resulted in membrane expansion, as concluded from FRET data. Moreover, it was shown that cytotoxic BAs were able to significantly disrupt the ordering of the membrane by cholesterol at physiologically active concentrations of the BA, an effect not associated with cholesterol removal. On the other hand, cytoprotective bile acids had no effect on membrane properties. It was concluded that, given the observed effects on membrane rigidity, the apoptotic activity of cytotoxic BAs could be potentially associated with changes in plasma membrane organization (e.g. modulation of lipid domains) or with an increase in mitochondrial membrane affinity for apoptotic proteins.

Structure-function analysis of the tertiary bile acid TUDCA for the resolution of endoplasmic reticulum stress in intestinal epithelial cells.

Inflammatory bowel diseases (IBD) are chronically relapsing and immune-mediated disorders of the gastrointestinal tract. Endoplasmic reticulum (ER) stress mechanisms in the epithelium have been demonstrated to be implemented into the pathogenesis of intestinal inflammation. Chemical chaperones have been demonstrated to exhibit beneficial effects in various diseases associated with ER stress mechanisms by prohibiting the unfolded protein response (UPR). In a structure-function analysis, we tested the potential of the conjugated bile salt sodium tauroursodeoxycholate (TUDCA), naturally present in the small bowel, to resolve ER stress in intestinal epithelial cells. TUDCA efficiently inhibited the expression of UPR dependent genes like GRP78 triggered by the ER stressor tunicamycin in the small intestinal epithelial cell line Mode-K. TUDCA inhibited upstream signaling events in all three branches of the UPR cascade and diminished binding of UPR activated transcription factors to the grp78 promoter. A structure-function analysis revealed that UDCA but not its conjugation partner taurine, known as a chemical chaperone, is responsible for the inhibition of GRP78 induction and that UDCA is 10 times more effective than its taurine conjugate. This inhibitory effect was confirmed in a cell free assay, where TUDCA and UDCA but not taurine effectively inhibited the aggregation of thermally denatured BSA. We conclude that TUDCA and UDCA are potent anti-aggregants for the resolution of ER stress in intestinal epithelial cells and should be considered as a potential drug target to resolve ER stress mechanisms underlying the pathology of IBD.

Cholesterol-derived bile acids enhance the chaperone activity of α-crystallins.

Human lens membranes contain the highest cholesterol concentration of any known biological membranes, but it significantly decreases with age. Oxygenation of cholesterol generates numerous forms of oxysterols (bile acids). We previously showed that two forms of the bile acid components--ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA)--suppressed lens epithelial cell death and alleviated cataract formation in galactosemic rat lenses. We investigated whether these compounds also suppress the thermal aggregation of human lens crystallins. Total water-soluble (WS) proteins were prepared from human lenses, and recombinant human crystallins (αA-, αB-, ßB2-, and γC-crystallin) were generated by a prokaryotic expression system and purified by liquid chromatography. The light scattering of proteins in the presence or absence of UDCA or TUDCA was measured using a spectrofluorometer set at Ex/Em = 400/400 nm. Protein blot analysis was conducted for detection of α-crystallins in the human lens WS proteins. High concentrations of UDCA and TUDCA significantly suppressed thermal aggregation of total lens WS proteins, which contained a low level of αA-/αB-crystallin. Spectroscopic analysis with each recombinant human lens crystallin indicated that the bile acids did not suppress the thermal aggregation of γC-, ßB2-, αA-, or αB-crystallin. Combination of α-crystallin and bile acid (either UDCA or TUDCA) suppressed thermal aggregation of each individual crystallin as well as a non-crystallin protein, insulin. These results suggest that UDCA or TUDCA protects the chaperone activity of α-crystallin. It is believed that these two naturally occurring intermediate waste products in the lens enhance the chaperone activity of α-crystallin. This finding may lead to the development of UDCA and TUDCA as anticataract agents.

Quantitative analysis of cholesterol nucleation with time in supersaturated model bile.

The amount of cholesterol (Ch) crystals formed in supersaturated taurochenodeoxycholate (TCDC) - lecithin (L) solutions of the same Ch saturation index (CSI) but at different Ch thermodynamic activities (Ch A(T)) was quantified at different time intervals. The initial Ch nucleation rate (i.e., amount of Ch crystals formed with respect to time) in a Ch A(T) = 1.73 and TCDC to L molar ratio (TCDC:L) = 5.1 system was faster than that in a Ch A(T) = 1.42 and TCDC:L = 3.4 system. Shaking could enhance the early appearance of Ch crystals and cause the fast initial Ch nucleation rates for the TCDC:L = 5.1 and the TCDC:L = 3.4 systems. The final Ch nucleation rates were faster than the initial Ch nucleation rates for the TCDC:L = 5.1 and the TCDC:L = 3.4 systems. According to a light scattering analysis of vesicle concentration in supersaturated TCDC-L solutions, vesicles provide nucleation sites only in the Ch nucleation process and the vesicle concentration may not be an important factor for the Ch nucleation rate. A model of a mixed TCDC-L micelle releasing Ch molecules together with the surface area of Ch crystals formed was used in the interpretation of the Ch nucleation.

Inhibition of interferon-α-induced signaling by hyperosmolarity and hydrophobic bile acids.

Apart from viral conditions, host factors such as elevated bile acid concentrations are determinants of successful interferon-α (IFN-α) treatment in patients with chronic hepatitis C or B. The present study demonstrates that hydrophobic bile acids inhibit Jak1- and Tyk2-phosphorylation, which lead to blockade of STAT1-mediated IFN-α-signaling in the sodium-taurocholate cotransporting peptide (NTCP)-transfected human hepatoma cell line HepG2, resulting in a decreased mRNA and protein expression of IFN-stimulated genes such as myxovirus resistance protein A (MxA) or dsRNA-activated protein kinase (PKR). In addition, hyperosmotic stress leads to an inhibition of IFN-α-induced Jak1- and Tyk2-phosphorylation, and STAT1/STAT2-phosphorylation and gene expression. This inhibitory effect of hydrophobic bile acids or hyperosmolarity is not due to caspase-mediated cleavage or lysosomal degradation of the cognate receptors or to the generation of oxidative stress, activation of p38- or Erk-mediated MAPK pathways or phosphatase activity. Preincubation with the organic osmolyte betaine blocked the inhibitory effect of bile acids or hyperosmolarity on MxA protein expression, but had no effect on transcript levels or activation of STAT1, suggesting that betaine mediates its effects on MxA expression at a translational or post-translational level. Our findings could provide a rationale for betaine use in cholestatic HBV/HCV patients undergoing interferon therapy.