Raf-independent, PP2A-dependent MEK activation in response to ERK silencing

Biological roles of ERK and MEK in signal transduction have been controversial. The aim of the current study was to determine the role of ERK1/2 in signaling through the ERK-MAPK cascade by using RNAi methodology. Transient transfection of erk1 or erk2 siRNA decreased the respective protein level to 3-8% in human lung fibroblasts. Interestingly, individual ERK isoform silencing resulted in a 2-fold reciprocal increase in phosphorylation of the alternate ERK isoform, with no change in respective total protein expression. Moreover, MEK was hyperphosphorylated as a result of combined ERK1 and ERK2 silencing, but was unaffected in individual ERK1 or ERK2 silenced cells. This hyperactivation of MEK was not due to activation of Raf family members, but rather was associated with PP2A downregulation. These data highlight the existence of a feedback loop in normal cells whereby ERK silencing is associated with decreased PP2A activity and consequent MEK activation. © 2009 Elsevier Inc. All rights reserved

Comparative binding of bile acids to serum lipoproteins and albumin

Characteristics of the binding of lithocholic acid (LC), chenodeoxycholic acid (CDC), and cholic acid to human plasma proteins were studied. Affinity of the different plasma protein fractions for the bile acids studied decreased with increased polarity of the steroid nucleus of the bile acid. Binding of LC, CDC, and cholic acid to the lipoprotein-free, albumin-rich plasma fraction was characterized by two classes of binding sites with respective K(D)s of 2, 5, and 51 μM, and of 39, 2,387, and 5,575 μM, while corresponding B(max) values were similar for the different bile acids, at around 6 and 100 nmol/mg protein. Bile acid binding to the different lipoprotein fractions was characterized by a single population of binding sites, with a K(D) ranging from 47 to 66 μM for LC, 695 to 1010 μM for CDC, and 2,511 to 2,562 μM for cholic acid. B(max) values, at 416-913 nmol/mg protein, were similar among the different bile acids studied. Both glycine- and taurine-conjugated, as well as unconjugated LC competitively inhibited [24-14C]LC binding to low density (LDL) and to high density lipoproteins (HDL) to the same extent, while the more polar LC-3-sulfate, CDC, and cholic acid were increasingly less potent in displacing LC binding from the respective lipoproteins. Furthermore, all bile acids studied shared the same lipoprotein binding site. The lipoprotein fluorescence at 330-334 nm, following excitation at 280 nm, was diminished after incubation with LC, suggesting that the bile acid masks the tryptophan residues of the protein moiety. Finally, the initial rate of uptake of 1 μM LC, in isolated hamster hepatocytes, at around 0.045 nmol · sec-1 · mg cell wt-1, was not affected by the protein carrier. However, for the same concentration of LC, bound to either LDL or HDL, LC binding resulted in 75-77% of the total [24- 14C]LC nonspecifically bound to the hepatocyte, compared to 65% when bound to albumin, and 45% in the absence of protein. The studies show that, under conditions when the serum bile acid concentration exceeds the capacity of the high affinity class of albumin binding sites for bile acids, lipoproteins have similar or greater affinity to bind bile acids than does albumin. The ability of lipoproteins to increase the nonspecific association of lithocholic acid with liver cells may also facilitate bile acid association with extrahepatic tissues. As lipoproteins, in contrast to albumin, are targeted to most cells, they may play a major role in the transport of potentially toxic bile acids to peripheral cells

Isolation and identification of Δ6-lithocholenic acid (3α-hydroxy-5β-6-cholen-24-oic acid) as an intestinal bacterial metabolite of chenodeoxycholic acid in man

Fecal bacterial biotransformation studies of chenodeoxycholic acid were performed. Incubations were carried out for 30-s to 12-h time intervals. Δ6-Lithocholenic acid was isolated by thin-layer chromatography. Its structure was confirmed by gas-liquid chromatography and mass spectrometry. The proportion of chenodeoxycholic acid biotransformed to Δ6-lithocholenic acid consistently ranged from 5.5 to 14.0%

Extrahepatic deposition and cytotoxicity of lithocholic acid: studies in two hamster models of hepatic failure and in cultured human fibroblasts

Effects of bile acids on tissues outside of the enterohepatic circulation may be of major pathophysiological significance under conditions of elevated serum bile acid concentrations, such as in hepatobiliary disease. Two hamster models of hepatic failure, namely functional hepatectomy (HepX), and 2-day bile duct ligation (BDL), as well as cultured human fibroblasts, were used to study the comparative tissue uptake, distribution, and cytotoxicity of lithocholic acid (LCA) in relation to various experimental conditions, such as binding of LCA to low-density lipoprotein (LDL) or albumin as protein carriers. Fifteen minutes after iv infusion of [24- 14C]LCA, the majority of LCA in sham-operated control animals was recovered in liver, bile, and small intestine. After hepatectomy, a significant increase in LCA was found in blood, muscle, heart, brain, adrenals, and thymus. In bile duct-ligated animals, significantly more LCA was associated with blood and skin, and a greater than twofold increase in LCA was observed in the colon. In the hepatectomized model, the administration of LCA bound to LDL resulted in a significantly higher uptake in the kidneys and skin. The comparative time- and concentration-dependent uptake of [14C]LCA, [14C]chenodeoxycholic acid (CDCA), and [14C]cholic acid (CA) in cultured human fibroblasts was nonsaturable and remained a function of concentration. Initial rates of uptake were significantly increased by approximately tenfold, with decreasing hydroxylation of the respective bile acid. After 1 hour of exposure of fibroblasts to LCA, there was a significant, dose- dependent decrease in mitochondrial dehydrogenase activity from 18% to 34% of the control, at LCA concentrations ranging from 1 to 20 μmol/L. At a respective concentration of 100 and 700 μmol/L, CDCA caused a 35% and 99% inhibition of mitochondrial dehydrogenase activity. None of the bile acids tested, with the exception of 700 μmol/L CDCA, caused a significant release of cytosolic lactate dehydrogenase into the medium. In conclusion, we show that bile acids selectively accumulate in nonhepatic tissues under two conditions of impaired liver function. Furthermore, the extrahepatic tissue distribution of bile acids during cholestasis may be affected by serum lipoprotein composition. At a respective concentration of 1 and 100 μmol/L, LCA and CDCA induced mitochondrial damage in human fibroblasts, after just i hour of exposure. Therefore, enhanced extrahepatic uptake of hydrophobic bile acids during liver dysfunction, or disorders of lipoprotein metabolism, may have important implications for bile-acid induced cytotoxic effects in tissues of the systemic circulation