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Background The two isoforms of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), 1 with a long cytoplasmic domain (CEACAM1-L) and 1 with a short (CEACAM1-S), are involved in different signaling pathways. β2-spectrin (β2SP) is an adaptor protein that plays critical roles in the proper control of Smad access to activate receptors involved in regulation of TGF-β signaling. In this study, we examined the association between CEACAM1 isoform balance and hepatocellular carcinoma (HCC) malignant potential and investigated the possibility of a molecular interaction between CEACAM1 and β2SP. Methods Immunohistochemical analysis was carried out with CEACAM1-L or CEACAM1-S antibodies on 154 HCC tissues to correlate with the factors of malignancy. Invasion assay was performed for the effect of CEACAM1 expression on HCC cell lines. Moreover, immunohistochemical analysis and immunoprecipitation analysis were performed to investigate the association between CEACAM1 isoform balance and β2SP. Results In immunohistochemical analysis, CEACAM1-L expression dominance was a risk factor for HCC recurrence (p = 0.04) and was significantly associated with a shorter survival compared with CEACAM1-S expression dominance. Invasion assay indicated that CEACAM1-4L-transfected HLF and PLC/PRF/5 cells showed significantly increased invasion (p<0.0001) and CEACAM1-4S-transfected HLF cells showed significantly decreased invasion. Immunohistochemical analysis of β2SP suggested that the HCCs with CEACAM1-L-dominant expression were more strongly stained with β2SP than the HCCs with CEACAM1-S-dominant expression (p = 0.013), and coprecipitation assays indicated that CEACAM1-L could bind to β2SP. Conclusions CEACAM1-L may enhance the HCC invasiveness through an interaction with β2SP and subsequent effects on TGF-β signaling.

We previously reported that the responsiveness of hepatocytes to thyroid hormone is markedly attenuated when they were cultured as monolayers rather than spheroids. To elucidate the mechanisms underlying the altered responsiveness, thyroid hormone receptor auxiliary proteins in the hepatocytes were analyzed by electrophoretic mobility shift assay. The major thyroid hormone receptor auxiliary protein was identified as 9-cisretinoic acid receptor ␣ (RXR␣) in the hepatocytes regardless of the culture conditions. The cytoplasmic fraction was shown to contain a protease(s) that cleaves RXR␣ at its amino terminus. The presence of the protease in the cytosol, but not in the nucleus, was ascertained by incubating full-length 35 S-labeled RXR␣ with each fraction. Using various protease inhibitors, it was shown that cathepsin L-type protease could participate in the cleavage of the RXR␣. The enzyme activity was much higher in the monolayers than the spheroids. Inhibition of this enzyme activity in the monolayer hepatocyte resulted in the increase of nuclear RXR␣ protein and the augmentation of T 3 -dependent induction of spot 14 mRNA. These results suggest that the changes in cathepsin L-type protease activity in the cytosol may alter the turnover of RXR␣ in the nucleus and modify the function of steroid receptor superfamilies that heterodimerize with RXR␣.Thyroid hormones are essential for normal growth and development as well as for the regulation of a variety of metabolic pathways (1). These effects are mediated by thyroid hormone receptors (TRs), 1 which activate or repress the transcription of specific target genes in a ligand-dependent manner (2). TRs belong to members of the family of nuclear receptors which also include several steroid, retinoic acid, and vitamin D 3 receptors. TRs are encoded by two genes, TR␣ and TR␤, as well as alternative splicing products of each (3). For transcriptional regulation by thyroid hormones, TRs bind to a specific DNA sequence (thyroid-hormone responsive element, TRE) of target genes by forming a monomer, a homodimer, and a heterodimer with TR auxiliary proteins (TRAPs) (4). TREs are composed of the arrangement of two common hexameric DNA half-site sequences (AGGTCA). The orientation of two half-sites is headto-head (palindrome, Pal) or in an opposite orientation as an inverted palindrome (Lap), with the half-sites arranged headto-tail as a direct repeat with 4 spacing (DR4). The heterodimer formation (TR/TRAP) on these TREs is suggested to be more stable and important for transcriptional regulation (5). Several studies (6 -8) identified 9-cis-retinoic acid receptors (RXRs), which are also members of the nuclear receptor superfamily, as TRAPs in most tissues. Also demonstrated was that preferential expression of RXR isoforms in certain cell lines or tissues results in the apparent heterogeneity of TRAP on EMSA (9).One of the major target organs for thyroid hormones is the liver, in which a number of genes in the metabolic pathways such as spot 14 (10), malic enzyme (11), and type I...

We have previously demonstrated that dexamethasone (DEX) enhances the T 3 -dependent increase in type I 5 -deiodinase (5 DI) mRNA in primary cultured rat hepatocytes grown as spheroids. Here we report that DEX-enhanced T 3 -responsiveness also occurs in two other T 3 -regulated hepatic genes, Spot 14 and malic enzyme. This enhancement was inhibited by pretreatment with cycloheximide and the stability of 5 DI and Spot 14 mRNAs was not affected by DEX. We thus hypothesized that a factor(s) that augments T 3 -responsiveness is induced by DEX. Among the possible candidates examined, retinoid-X receptor alpha (RXR ), which is a main heterodimer partner with T 3 receptor, appeared to be involved. Whereas DEX increased the amount of RXR mRNA, it did not affect the expression of other possible factors such as steroid receptor coactivator-1 and the binding protein of cAMP response element-binding protein.Northern and Western blot analysis, and electrophoretic mobility shift assay revealed that DEX increased RXR expression at both the mRNA and protein levels. Maximal increase in RXR protein was achieved with the addition of physiological concentrations of DEX (10 8 M). To test whether the DEX-induced increase in RXR affects liganddependent transcriptional activation through other receptors that form heterodimer with RXR, we examined its effect on the retinoic acid (RA)/RA receptor (RAR) system. Indeed, DEX also enhanced the RA-dependent increase in RAR mRNA in a cycloheximide-sensitive manner. Increase in the level of RXR in hepatocytes by infection with the RXR -expressing adenovirus resulted in enhancement of the T 3 -dependent increase in 5 DI mRNA. These results strongly suggest that the DEX-induced augmentation of T 3 -responsiveness in cultured hepatocytes is mediated, in part, by the increased expression of RXR .

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