The hetero-oligomeric retinoid oxidoreductase complex (ROC) catalyzes the interconversion of all- trans -retinol and all- trans -retinaldehyde to maintain the steady-state output of retinaldehyde, the precursor of all- trans -retinoic acid that regulates the transcription of numerous genes. The interconversion is catalyzed by two distinct components of the ROC: the NAD(H)-dependent retinol dehydrogenase 10 (RDH10) and the NADP(H)-dependent dehydrogenase reductase 3 (DHRS3). The binding between RDH10 and DHRS3 subunits in the ROC results in mutual activation of the subunits. The molecular basis for their activation is currently unknown. Here, we applied site-directed mutagenesis to investigate the roles of amino acid residues previously implied in subunit interactions in other SDRs to obtain the first insight into the subunit interactions in the ROC. The results of these studies suggest that the cofactor binding to RDH10 subunit is critical for the activation of DHRS3 subunit and vice versa . The C-terminal residues 317–331 of RDH10 are critical for the activity of RDH10 homo-oligomers but not for the binding to DHRS3. The C-terminal residues 291–295 are required for DHRS3 subunit activity of the ROC. The highly conserved C-terminal cysteines appear to be involved in inter-subunit communications, affecting the affinity of the cofactor binding site in RDH10 homo-oligomers as well as in the ROC. Modeling of the ROC quaternary structure based on other known structures of SDRs suggests that its integral membrane-associated subunits may be inserted in adjacent membranes of the endoplasmic reticulum (ER), making the formation and function of the ROC dependent on the dynamic nature of the tubular ER network.

Spermatogenesis is a highly regulated process that involves both mitotic and meiotic divisions, as well as cellular differentiation to yield mature spermatozoa from undifferentiated germinal stem cells. Although Gpat2 was originally annotated as encoding a glycerol-3-phosphate acyltransferase by sequence homology to Gpat1 , GPAT2 is highly expressed in testis but not in lipogenic tissues and is not up-regulated during adipocyte differentiation. New data show that GPAT2 is required for the synthesis of piRNAs (piwi-interacting RNAs), a group of small RNAs that protect the germ cell genome from retrotransposable elements. In order to understand the relationship between GPAT2 and its role in the testis, we focused on Gpat2 expression during the first wave of mouse spermatogenesis. Gpat2 expression was analysed by qPCR (quantitative real-time PCR), in situ hybridization, immunohistochemistry and Western blotting. Gpat2 mRNA content and protein expression were maximal at 15 dpp (days post-partum) and were restricted to pachytene spermatocytes. To achieve this transient expression, both epigenetic mechanisms and trans -acting factors are involved. In vitro assays showed that Gpat2 expression correlates with DNA demethylation and histone acetylation and that it is up-regulated by retinoic acid. Epigenetic regulation by DNA methylation was confirmed in vivo in germ cells by bisulfite sequencing of the Gpat2 promoter. Consistent with the initiation of meiosis at 11 dpp, methylation decreased dramatically. Thus, Gpat2 is expressed at a specific stage of spermatogenesis, consistent with piRNA synthesis and meiosis I prophase, and its on–off expression pattern responds predominantly to epigenetic modifications.

The marine environment has long been known to be species-rich and to be a source of molecules with interesting and beneficial biochemical and clinical properties. However, despite some notable successes the potential of the ‘marine pipeline’ has yet to be fully realized. Recent studies involving members of the nuclear receptor superfamily illustrate the chemical richness of molecules from marine species and helps to re-emphasize the translational possibilities of natural products in drug discovery. In this issue of the Biochemical Journal Wang et al. describe the identification and characterization of such a compound, an agonist for the retinoic acid receptor isolated from the sponges Luffariella sp. and Fascaplysinopsis .

In the present study we provide evidence that SRP-35, a protein we identified in rabbit skeletal muscle sarcoplasmic reticulum, is an all- trans -retinol dehydrogenase. Analysis of the primary structure and tryptic digestion revealed that its N-terminus encompasses a short hydrophobic sequence bound to the sarcoplasmic reticulum membrane, whereas its C-terminal catalytic domain faces the myoplasm. SRP-35 is also expressed in liver and adipocytes, where it appears in the post-microsomal supernatant; however, in skeletal muscle, SRP-35 is enriched in the longitudinal sarcoplasmic reticulum. Sequence comparison predicts that SRP-35 is a short-chain dehydrogenase/reductase belonging to the DHRS7C [dehydrogenase/reductase (short-chain dehydrogenase/reductase family) member 7C] subfamily. Retinol is the substrate of SRP-35, since its transient overexpression leads to an increased production of all- trans -retinaldehyde. Transfection of C2C12 myotubes with a fusion protein encoding SRP-35–EYFP (enhanced yellow fluorescent protein) causes a decrease of the maximal Ca 2+ released via RyR (ryanodine receptor) activation induced by KCl or 4-chloro- m -chresol. The latter result could be mimicked by the addition of retinoic acid to the C2C12 cell tissue culture medium, a treatment which caused a significant reduction of RyR1 expression. We propose that in skeletal muscle SRP-35 is involved in the generation of all- trans -retinaldehyde and may play an important role in the generation of intracellular signals linking Ca 2+ release (i.e. muscle activity) to metabolism.

Human AKR (aldo–keto reductase) 1C proteins (AKR1C1–AKR1C4) exhibit relevant activity with steroids, regulating hormone signalling at the pre-receptor level. In the present study, investigate the activity of the four human AKR1C enzymes with retinol and retinaldehyde. All of the enzymes except AKR1C2 showed retinaldehyde reductase activity with low K m values (~1 μM). The k cat values were also low (0.18–0.6 min −1 ), except for AKR1C3 reduction of 9- cis -retinaldehyde whose k cat was remarkably higher (13 min −1 ). Structural modelling of the AKR1C complexes with 9- cis -retinaldehyde indicated a distinct conformation of Trp 227 , caused by changes in residue 226 that may contribute to the activity differences observed. This was partially supported by the kinetics of the AKR1C3 R226P mutant. Retinol/retinaldehyde conversion, combined with the use of the inhibitor flufenamic acid, indicated a relevant role for endogenous AKR1Cs in retinaldehyde reduction in MCF-7 breast cancer cells. Overexpression of AKR1C proteins depleted RA (retinoic acid) transactivation in HeLa cells treated with retinol. Thus AKR1Cs may decrease RA levels in vivo. Finally, by using lithocholic acid as an AKR1C3 inhibitor and UVI2024 as an RA receptor antagonist, we provide evidence that the pro-proliferative action of AKR1C3 in HL-60 cells involves the RA signalling pathway and that this is in part due to the retinaldehyde reductase activity of AKR1C3.

Sox3 / SOX3 [SRY (sex determining region Y)-box 3] is considered to be one of the earliest neural markers in vertebrates, playing a role in specifying neuronal fate. We have previously reported characterization of the SOX3 promoter and demonstrated that the general transcription factors NF-Y (nuclear factor-Y), Sp1 (specificity protein 1) and USF (upstream stimulatory factor) are involved in transcriptional regulation of SOX3 promoter activity. In the present study we provide the first evidence that the TALE (three-amino-acid loop extension) transcription factors PBX1 (pre-B-cell leukaemia homeobox 1) and MEIS1 (myeloid ecotropic viral integration site 1 homologue) participate in regulating human SOX3 gene expression in NT2/D1 cells by direct interaction with the consensus PBX/MEIS-binding site, which is conserved in all mammalian orthologue promoters analysed. PBX1 is present in the protein complex formed at this site with nuclear proteins from uninduced cells, whereas both PBX1 and MEIS1 proteins were detected in the complex created with extract from RA (retinoic acid)-induced NT2/D1 cells. By functional analysis we also showed that mutations of the PBX1/MEIS1-binding sites resulted in profound reduction of SOX3 promoter responsiveness to RA. Finally, we demonstrated that overexpressed PBX1 and MEIS1 increased endogenous SOX3 protein expression in both uninduced and RA-induced NT2/D1 cells. With the results of the present study, for the first time, we have established a functional link between the TALE proteins, PBX1 and MEIS1, and expression of the human SOX3 gene. This link is of particular interest since both TALE family members and members of the SOX superfamily are recognized as important developmental regulators.

RDH10 (retinol dehydrogenase 10) was originally identified from the retinal pigment epithelium and retinal Müller cells. It has retinoid oxidoreductase activity and is thought to play a role in the retinoid visual cycle. A recent study showed that RDH10 is essential for generating retinoic acid at early embryonic stages. The present study demonstrated that wild-type RDH10 catalysed both oxidation of all- trans -retinol and reduction of all- trans -retinal in a cofactor-dependent manner In vitro . In cultured cells, however, oxidation is the favoured reaction catalysed by RDH10. Substitution of any of the predicted key residues in the catalytic centre conserved in the RDH family abolished the enzymatic activity of RDH10 without affecting its protein level. Unlike other RDH members, however, replacement of Ser 197 , a key residue for stabilizing the substrate, by glycine and alanine did not abolish the enzymatic activity of RDH10, whereas RDH10 mutants S197C, S197T and S197V completely lost their enzymatic activity. These results suggest that the size of the residue at position 197 is critical for the activity of RDH10. Mutations of the three glycine residues (Gly 43 , Gly 47 and Gly 49 ) in the predicted cofactor-binding motif (Gly-Xaa 3 -Gly-Xaa-Gly) of RDH10 abolished its enzymatic activity, suggesting that the cofactor-binding motif is essential for its activity. Deletion of the two hydrophobic domains dissociated RDH10 from the membrane and abolished its activity. These studies identified the key residues for the activity of RDH10 and will contribute to the further elucidation of mechanism of this important enzyme.

Retinoic acid biosynthesis in vertebrates occurs in two consecutive steps: the oxidation of retinol to retinaldehyde followed by the oxidation of retinaldehyde to retinoic acid. Enzymes of the MDR (medium-chain dehydrogenase/reductase), SDR (short-chain dehydrogenase/reductase) and AKR (aldo-keto reductase) superfamilies have been reported to catalyse the conversion between retinol and retinaldehyde. Estimation of the relative contribution of enzymes of each type was difficult since kinetics were performed with different methodologies, but SDRs would supposedly play a major role because of their low K m values, and because they were found to be active with retinol bound to CRBPI (cellular retinol binding protein type I). In the present study we employed detergent-free assays and HPLC-based methodology to characterize side-by-side the retinoid-converting activities of human MDR [ADH (alcohol dehydrogenase) 1B2 and ADH4), SDR (RoDH (retinol dehydrogenase)-4 and RDH11] and AKR (AKR1B1 and AKR1B10) enzymes. Our results demonstrate that none of the enzymes, including the SDR members, are active with CRBPI-bound retinoids, which questions the previously suggested role of CRBPI as a retinol supplier in the retinoic acid synthesis pathway. The members of all three superfamilies exhibit similar and low K m values for retinoids (0.12–1.1 μM), whilst they strongly differ in their k cat values, which range from 0.35 min −1 for AKR1B1 to 302 min −1 for ADH4. ADHs appear to be more effective retinol dehydrogenases than SDRs because of their higher k cat values, whereas RDH11 and AKR1B10 are efficient retinaldehyde reductases. Cell culture studies support a role for RoDH-4 as a retinol dehydrogenase and for AKR1B1 as a retinaldehyde reductase in vivo .

RALDH3 (retinal dehydrogenase 3) was characterized by kinetic and binding studies, protein engineering, homology modelling, ligand docking and electrostatic-potential calculations. The major recognition determinant of an RALDH3 substrate was shown to be an eight-carbon chain bonded to the aldehyde group whose kinetic influence ( k cat / K m at pH 8.5) decreases when shortened or lengthened. Surprisingly, the β-ionone ring of all- trans -retinal is not a major recognition site. The dissociation constants ( K d ) of the complexes of RALDH3 with octanal, NAD + and NADH were determined by intrinsic tryptophan fluorescence. The similarity of the K d values for the complexes with NAD + and with octanal suggests a random kinetic mechanism for RALDH3, in contrast with the ordered sequential mechanism often associated with aldehyde dehydrogenase enzymes. Inhibition of RALDH3 by tri-iodothyronine binding in competition with NAD + , predicted by the modelling, was established kinetically and by immunoprecipitation. Mechanistic implications of the kinetically influential ionizations with macroscopic p K a values of 5.0 and 7.5 revealed by the pH-dependence of k cat are discussed. Analogies with data for non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans , together with the present modelled structure of the thioacyl RALDH3, suggest (a) that k cat characterizes deacylation of this intermediate for specific substrates and (b) the assignment of the p K a of the major ionization (approximating to 7.5) to the perturbed carboxy group of Glu 280 whose conjugate base is envisaged as supplying general base catalysis to attack of a water molecule. The macroscopic p K a of the minor ionization (5.0) is considered to approximate to that of the carboxy group of Glu 488 .

Cyp26A1 encodes an RA (retinoic acid)-catabolizing CYP (cytochrome P450) protein that plays a critical role in regulating RA distribution in vivo . Cyp26A1 expression is inducible by RA, and the locus has previously been shown to contain a RARE (RA response element), R1, within the minimal promoter [Loudig, Babichuk, White, Abu-Abed, Mueller and Petkovich (2000) Mol. Endocrinol. 14 , 1483–1497]. In the present study, we report the identification of a second functional RARE (R2) located 2.0 kb upstream of the Cyp26A1 transcriptional start site. Constructs containing murine sequences encompassing both R1 and R2 showed that these elements work together to generate higher transcriptional activity upon treatment with RA than those containing R1 alone. Inclusion of R2 also dramatically enhanced the sensitivity of reporter constructs to RA, as even treatment with 10 −8 M RA resulted in a 5-fold induction of reporter activity. Mutational analysis identified R2 as the functional element responsible for the increased RA inducibility of promoter constructs. The element was shown to bind RARγ (RA receptor γ)/RXRα (retinoid X receptor α) heterodimers in vitro , and inclusion of nuclear receptors in transfections boosted the transcriptional response. A construct containing both R1 and R2 was used to generate a stable luciferase reporter cell line that can be used as a tool to identify factors regulating Cyp26A1 expression. The analysis of R1 and R2 has led to the proposal that the two elements work synergistically to provide a maximal response to RA and that R2 is an upstream enhancer.

We have investigated effects of neuronal differentiation on hormone-induced Ca 2+ entry. Fura-2 fluorescence measurements of undifferentiated SH-SY5Y neuroblastoma cells, stimulated with methacholine, revealed the presence of voltage-operated Ca 2+ -permeable, Mn 2+ -impermeable entry pathways, and at least two voltage-independent Ca 2+ - and Mn 2+ -permeable entry pathways, all of which apparently contribute to both peak and plateau phases of the Ca 2+ signal. Similar experiments using 9- cis retinoic acid-differentiated cells, however, revealed voltage-operated Ca 2+ -permeable, Mn 2+ -impermeable channels, and, more significantly, the absence or down-regulation of the most predominant of the voltage-independent entry pathways. This down-regulated pathway is probably due to CCE (capacitative Ca 2+ entry), since thapsigargin also stimulated Ca 2+ and Mn 2+ entry in undifferentiated but not differentiated cells. The Ca 2+ entry components remaining in methacholine-stimulated differentiated cells contributed to only the plateau phase of the Ca 2+ signal. We conclude that differentiation of SH-SY5Y cells results in a mechanistic and functional change in hormone-stimulated Ca 2+ entry. In undifferentiated cells, voltage-operated Ca 2+ channels, CCE and NCCE (non-CCE) pathways are present. Of the voltage-independent pathways, the predominant one appears to be CCE. These pathways contribute to both peak and plateau phases of the Ca 2+ signal. In differentiated cells, CCE is either absent or down-regulated, whereas voltage-operated entry and NCCE remain active and contribute to only the plateau phase of the Ca 2+ signal.

We report a sensitive LC (liquid chromatography)/MS/MS assay using selected reaction monitoring to quantify RA (retinoic acid), which is applicable to biological samples of limited size (10–20 mg of tissue wet weight), requires no sample derivatization, provides mass identification and resolves atRA (all- trans -RA) from its geometric isomers. The assay quantifies over a linear range of 20 fmol to 10 pmol, and has a 10 fmol limit of detection at a signal/noise ratio of 3. Coefficients of variation are: instrumental, 0.5–2.9%; intra-assay, 5.4±0.4%; inter-assay 8.9±1.0%. An internal standard (all- trans -4,4-dimethyl-RA) improves accuracy by confirming extraction efficiency and revealing handling-induced isomerization. Tissues of 2–4-month-old C57BL/6 male mice had atRA concentrations of 7–9.6 pmol/g and serum atRA of 1.9±0.6 pmol/ml (±S.E.M.). Tissue 13- cis -RA ranged from 2.9 to 4.2 pmol/g, and serum 13- cis -RA was 1.2±0.3 pmol/ml. CRBP (cellular retinol-binding protein)-null mouse liver had atRA ∼30% lower than wild-type ( P <0.05), but kidney, testis, brain and serum atRA were similar to wild-type. atRA in brain areas of 12-month-old female C57BL/6 mice were (±S.E.M.): whole brain, 5.4±0.4 pmol/g; cerebellum, 10.7±0.3 pmol/g; cortex, 2.6±0.4 pmol/g; hippocampus, 8.4±1.2 pmol/g; striatum, 15.3±4.7 pmol/g. These data provide the first analytically robust quantification of atRA in animal brain and in CRBP-null mice. Direct measurements of endogenous RA should have a substantial impact on investigating target tissues of RA, mechanisms of RA action, and the relationship between RA and chronic disease.

Vitamin A homoeostasis requires the gene encoding cellular retinol-binding protein-1 ( Crbp1 ) which stimulates conversion of retinol into retinyl esters that serve as a storage form of vitamin A. The gene encoding alcohol dehydrogenase-1 ( Adh1 ) greatly facilitates degradative metabolism of excess retinol into retinoic acid to protect against toxic effects of high dietary vitamin A. Crbp1 −/− / Adh1 −/− double mutant mice were generated to explore whether the stimulatory effect of CRBP1 on retinyl ester formation is due to limitation of retinol oxidation by ADH1, and whether ADH1 limits retinyl ester formation by opposing CRBP1. Compared with wild-type mice, liver retinyl ester levels were greatly reduced in Crbp1 −/− mice, but Adh1 −/− mice exhibited a significant increase in liver retinyl esters. Importantly, relatively normal liver retinyl ester levels were restored in Crbp1 −/− / Adh1 −/− mice. During vitamin A deficiency, the additional loss of Adh1 completely prevented the excessive loss of liver retinyl esters observed in Crbp1 −/− mice for the first 5 weeks of deficiency and greatly minimized this loss for up to 13 weeks. Crbp1 −/− mice also exhibited increased metabolism of a dose of retinol into retinoic acid, and this increased metabolism was not observed in Crbp1 −/− / Adh1 −/− mice. Our findings suggest that opposing actions of CRBP1 and ADH1 enable a large fraction of liver retinol to remain esterified due to CRBP1 action, while continuously allowing some retinol to be oxidized to retinoic acid by ADH1 for degradative retinoid turnover under any dietary vitamin A conditions.

Q (coenzyme Q or ubiquinone) is reported to be a cofactor obligatory for proton transport by UCPs (uncoupling proteins) in liposomes [Echtay, Winkler and Klingenberg (2000) Nature (London) 408 , 609–613] and for increasing the binding of the activator retinoic acid to UCP1 [Tomás, Ledesma and Rial (2002) FEBS Lett. 526 , 63–65]. In the present study, yeast ( Saccharomyces cerevisiae ) mutant strains lacking Q and expressing UCP1 were used to determine whether Q was required for UCP function in mitochondria. Wild-type yeast strain and two mutant strains (CENΔCOQ3 and CENΔCOQ2), both not capable of synthesizing Q, were transformed with the mouse UCP1 gene. UCP1 activity was measured as fatty acid-dependent, GDP-sensitive proton conductance in mitochondria isolated from the cells. The activity of UCP1 was similar in both Q-containing and -deficient yeast mitochondria. We conclude that Q is neither an obligatory cofactor nor an activator of proton transport by UCP1 when it is expressed in yeast mitochondria.

Aldo–keto reductases (AKRs) are NAD(P)H-dependent oxidoreductases that catalyse the reduction of a variety of carbonyl compounds, such as carbohydrates, aliphatic and aromatic aldehydes and steroids. We have studied the retinal reductase activity of human aldose reductase (AR), human small-intestine (HSI) AR and pig aldehyde reductase. Human AR and HSI AR were very efficient in the reduction of all- trans -, 9- cis - and 13- cis -retinal ( k cat / K m =1100–10300 mM −1 ·min −1 ), constituting the first cytosolic NADP(H)-dependent retinal reductases described in humans. Aldehyde reductase showed no activity with these retinal isomers. Glucose was a poor inhibitor ( K i =80 mM) of retinal reductase activity of human AR, whereas tolrestat, a classical AKR inhibitor used pharmacologically to treat diabetes, inhibited retinal reduction by human AR and HSI AR. All- trans -retinoic acid failed to inhibit both enzymes. In this paper we present the AKRs as an emergent superfamily of retinal-active enzymes, putatively involved in the regulation of retinoid biological activity through the assimilation of retinoids from β-carotene and the control of retinal bioavailability.

Retinyl ester, the most abundant form of vitamin A (retinol), is synthesized by the enzyme lecithin:retinol acyltransferase (LRAT). Previously, we cloned a 2.5kb LRAT cDNA from rodent liver which codes for functional LRAT activity. However, Northern blots of tissues probed with the 2.5kb cDNA revealed the presence of a larger transcript of 5kb as well as several smaller transcripts. To elucidate the nature of the large LRAT transcript, a high-molecular-mass adrenal gland cDNA library was screened. Two similar clones of 3962 and 3187nt were identified which appeared to be part of the 3′-untranslated region (UTR) of a 5358nt LRAT mRNA. The 5.3kb cDNA was then amplified from liver by reverse transcriptase PCR (RT-PCR) and demonstrated to encode functional LRAT activity. The 3′-UTR of the 5.3kb cDNA contains several AAUAAA polyadenylation signals. Analysis of the 3′ ends of LRAT mRNA transcripts from liver, intestine and testis showed the usage of both canonical and non-canonical polyadenylation signals. To further analyse the LRAT mRNAs expressed in vivo , Northern blot analysis was performed using four probes spanning sections from the 5′ end to the distal 3′ end of the 5.3kb LRAT cDNA. The results show that the major 5.3kb transcript uses the canonical signal AAUAAA located at nt 5308, and the major short transcript of 1.5kb uses the non-canonical signal AUUAAA located at nt 1330. The 5.3kb LRAT transcript was predominant in the liver of retinoic acid-repleted vitamin A-deficient rats, coincident with increased quantitative expression of LRAT mRNA and enzyme activity. The differential usage of these polyadenylation signals can explain the presence of multiple LRAT mRNA transcripts which are expressed in different tissue-specific patterns.

Gall-bladder epithelial cells (GBEC) are exposed to high concentrations of cholesterol in bile. Whereas cholesterol absorption by GBEC is established, the fate of this absorbed cholesterol is not known. The aim of this study was to determine whether ABCA1 (ATP-binding cassette transporter A1) mediates cholesterol efflux in GBEC. Polarized canine GBEC were cultured on porous membrane filters allowing separate access to apical (AP) and basolateral (BL) compartments. After AP loading of cells with model bile and [ 14 C]cholesterol, cholesterol efflux was measured. Cholesterol loading together with 8-bromo-cAMP treatment, which increased ABCA1 expression, led to a significant increase in cholesterol efflux with apolipoprotein A-I (apoA-I) as the acceptor. Cholesterol efflux was observed predominantly into the BL compartment. Similar results were found for phospholipid efflux. Confocal immunofluorescence microscopy showed a predominantly BL ABCA1 localization. Interestingly, apoA-I added to either the AP or the BL compartments elicited BL lipid efflux with cAMP treatment. No paracellular or transcellular passage of 125 I-apoA-I occurred. Ligands for the nuclear hormone receptors liver X receptor α (LXRα) and retinoid X receptor (RXR) elicited AP and BL cholesterol efflux, suggesting the involvement of both ABCA1- and non-ABCA1-mediated pathways. In summary, BL cholesterol/phospholipid efflux consistent with an ABCA1-mediated mechanism occurs in GBEC. This efflux pathway is stimulated by cAMP and by LXRα/RXR ligands, and in the case of the cAMP pathway appears to involve a role for biliary apoA-I.

Data presented previously suggest that release of components of the cartilage matrix, in response to catabolic agents, cannot be accounted for by proteolytic mechanisms alone. In the present study, the release of glycosaminoglycan-containing components from bovine nasal cartilage cultured in the presence of interleukin-1β, and from bovine nasal, fetal bovine epiphyseal and adult human articular cartilage cultured in the presence of retinoic acid, was accompanied by the loss of link protein and hyaluronate into the culture medium. Chromatographic analysis of the released hyaluronate showed it to be markedly reduced in size relative to that extracted from the corresponding tissue. It is proposed that, under stimulation by catabolic agents, two independent, but concurrent, mechanisms act to promote the release of aggrecan from the cartilage matrix. First, proteolytic cleavage of the aggrecan core protein results in the production of glycosaminoglycan-containing fragments that are free to diffuse from the tissue. Secondly, cleavage of hyaluronate renders portions of the proteoglycan aggregate small enough so that complexes of aggrecan (or fragments containing its G1 domain) and link protein are released from the tissue. It is likely that both mechanisms contribute to cartilage metabolism in normal physiology and pathology.

The mechanisms of aggrecan degradation in adult human articular, adult bovine nasal and fetal bovine epiphyseal cartilage in response to either interleukin-1β (IL-1β) or retinoic acid were compared using an explant culture system. Bovine nasal cartilage cultured with either IL-1β or retinoic acid exhibited significant release of glycosaminoglycan (GAG). For both factors, aggrecan proteolysis occurred predominantly at the ‘aggrecanase’ site, with no evidence for the action of matrix metalloproteinases, and resulted in the appearance of the corresponding G1 fragment in tissue extracts and in culture media. In human cartilage, little effect of IL-1β was seen, but abundant release of GAG occurred in the presence of retinoic acid, with evidence of aggrecanase action. Treatment of fetal epiphyseal cartilage with retinoic acid resulted in significant GAG release, whereas treatment with IL-1β did not. In the retinoic acid-treated tissue, however, no evidence for the cleavage of aggrecan in the interglobular region was apparent. Thus, in the fetal system, agents in addition to aggrecanase and matrix metalloproteinases appear to be active. Taken together, these data demonstrate that the pathways utilized for aggrecan catabolism may vary between different cartilages for a given stimulatory agent, and that, for a given tissue, different factors may elicit aggrecan release via different pathways.

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a phytol-derived branched-chain fatty acid present in dietary products. Phytanic acid increased uncoupling protein-1 (UCP1) mRNA expression in brown adipocytes differentiated in culture. Phytanic acid induced the expression of the UCP1 gene promoter, which was enhanced by co-transfection with a retinoid X receptor (RXR) expression vector but not with other expression vectors driving peroxisome proliferator-activated receptor (PPAR) α, PPARγ or a form of RXR devoid of ligand-dependent sensitivity. The effect of phytanic acid on the UCP1 gene required the 5′ enhancer region of the gene and the effects of phytanic acid were mediated in an additive manner by three binding sites for RXR. Moreover, phytanic acid activates brown adipocyte differentiation: long-term exposure of brown preadipocytes to phytanic acid promoted the acquisition of the brown adipocyte morphology and caused a co-ordinate induction of the mRNAs for gene markers of brown adipocyte differentiation, such as UCP1, adipocyte lipid-binding protein aP2, lipoprotein lipase, the glucose transporter GLUT4 or subunit II of cytochrome c oxidase. In conclusion, phytanic acid is a natural product of phytol metabolism that activates brown adipocyte thermogenic function. It constitutes a potential nutritional signal linking dietary status to adaptive thermogenesis.

We reported previously that the expression of the gene encoding MUC5AC mucin in human airway epithelial cells is controlled by retinoic acid via the retinoic acid receptor (RAR)-α and that 3,3′,5-tri-iodothyronine (T 3 ) inhibits the expression of MUC5AC . The purpose of the present study was to identify mechanisms mediating the effect of T 3 . T 3 has been shown to inhibit gene expression via several mechanisms, either by enhancing or repressing the transcription of target genes or by the regulation of post-transcriptional events. Results showed that T 3 strongly inhibited MUC5AC -driven luciferase activity in normal human tracheobronchial epithelial cells that had been transiently transfected with a MUC5AC –luciferase reporter construct; however, it did not affect MUC5AC mRNA stability. These results indicate that T 3 suppresses MUC5AC expression at the transcriptional level. An analysis of deletion constructs showed that deletion of the region downstream of 3kb resulted in markedly decreased levels of MUC5AC transcription in the absence of T 3 (i.e. under control conditions) as well as a loss of responsiveness to the inhibitory effects of T 3 . This suggests that this region might contain elements important for the activation as well as the repression of MUC5AC transcription. To determine whether T 3 modulates retinoic-acid-dependent MUC5AC transcription via an alteration in the abundance of retinoid receptor proteins, we examined the type and abundance of these receptors in nuclear extracts of airway epithelial cells grown in the presence or absence of T 3 . Western blots showed that T 3 markedly decreased several types of retinoid receptor while not affecting T 3 receptor proteins. Consistent with this finding were gel-shift assays revealing a decrease in RAR–retinoic acid response element complexes obtained from T 3 -treated cells. We propose that T 3 might inhibit retinoid-dependent MUC5AC expression by decreasing retinoid receptor levels and thereby decreasing the transcriptional activation of this gene for mucins.

Binding sites for transcription factor Sp1have been implicated in the transcriptional regulation of several genes by hormones or vitamins, and here we show that a GC-rich element contributes to the retinoic acid response of the interleukin 1β promoter. To explain such observations, it has been proposed that nuclear receptors can interact with Sp1 bound to GC-rich DNA. However, evidence supporting this model has remained indirect. So far, nuclear receptors have not been detected in a complex with Sp1 and GC-rich DNA, and the expected ternary complexes in non-denaturing gels were not seen. In search for these missing links we found that nuclear receptors [retinoic acid receptor (RAR), thyroid hormone receptor (TR), vitamin D 3 receptor, peroxisome-proliferator-activated receptor and retinoic X receptor] induce an electrophoretic mobility increase of Sp1ŐGC-rich DNA complexes. Concomitantly, binding of Sp1 to the GC-box is enhanced. It is proposed that nuclear receptors may partially replace Sp1 in homo-oligomers at the GC-box. RARs and Sp1 can also combine into a complex with a retinoic acid-response element. The presence of RAR and Sp1 in complexes with either cognate site was revealed in supershift experiments. The C-terminus of Sp1 interacts with nuclear receptors. Both the ligand- and DNA-binding domains of the receptor are important for complex formation with Sp1 and GC-rich DNA. In spite of similar capacity to form ternary complexes, RAR but not TR up-regulated an Sp1-driven reporter in a ligand-dependent way. Thus additional factors limit the transcriptional response mediated by nuclear receptors and Sp1.

The mouse disabled 2 ( mDab2 ) gene is a mouse homologue of the Drosophila disabled gene and is alternatively spliced to form two isoforms, p96 and p67. Although p96 has been known to regulate the RasŐSos G-protein signal transduction pathway by interacting with Grb2, little is known about the biological function of p67. Recent studies have shown that the expression of mDab2 is markedly up-regulated during the retinoic acid (RA)-induced differentiation of F9 cells, suggesting another role for mDab2 in cell differentiation [Cho, Lee and Park (1999) Mol. Cells 9, 179Ő184). In the present study, we first elucidated the biological function of p67 isoform of mDab2 and identified its binding partner. Unlike p96, p67 largely resides in RA-treated F9 cell nuclei. In this system, p67 interacts with mouse androgen-receptor interacting protein 3, termed the mDab2 interacting protein, which acts as a transcriptional co-regulator. By using a fusion protein with a heterologous DNA-binding domain (GAL4), we showed that p67 had an intrinsic transcriptional activation function. These results suggest that mDab2 p67 may function as a transcriptional co-factor for certain complexes of transcriptional regulatory elements involved in the RA-induced differentiation of F9 cells.

Development of the cellular complexity of the vertebrate neural retina relies on an intricate interplay between extracellular signals and intracellular factors. In particular, transcription factors play a key role in determining the competence of cells to respond to extracellular signals. We have previously shown that, in the developing chick neural retina, expression of the retinoid X receptor-γ (RXR-γ2) nuclear receptor gene is restricted to photoreceptors. To characterize the mechanisms that regulate expression of this gene in the neural retina, we isolated a chicken RXR-γ genomic clone containing the RXR-γ2 promoter and mapped the transcription initiation site by means of ribonuclease protection. We analysed promoter activity by transient transfection of luciferase reporter gene constructs into cultured cells isolated from embryonic-chick neural retina or facial mesenchyme, which does not normally express detectable RXR-γ2 transcripts. The DNA fragment lying between nucleotides -657 and +37 with respect to the transcription initiation site had basal promoter activity in both cell types. The fragment lying between nucleotides -1198 and -991 directed 10-20-fold higher levels of luciferase activity in neural retina cells, but only basal levels in facial mesenchyme cells. This 208 bp fragment also enhanced the activity of the simian-virus-40 promoter, when placed upstream in either orientation. Electrophoretic-mobility-shift assays using this 208 bp fragment demonstrated the formation of four neural retina-specific protein-DNA complexes. These results indicate that regulation of RXR-γ2 transcription in the developing chick neural retina involves the binding of one or more neural retina-specific protein factors to an enhancer element located approx. 1 kbp upstream of the transcription initiation site.

The effect of mannosamine, an inhibitor of glycosylphosphatidylinositol (GPI) anchor formation, on chondrocyte-mediated cartilage proteoglycan breakdown was investigated using cartilage explant cultures. Mannosamine inhibited interleukin 1α-, tumour necrosis factor α- and retinoic acid-stimulated proteoglycan release from bovine nasal and articular cartilage, and retinoic acid-stimulated proteoglycan release from human cartilage. Its effects on two GPI-anchored proteins [the urokinase receptor, which binds urokinase-type plasminogen activator (uPA) to cell surfaces, and alkaline phosphatase] were also studied using bovine chondrocytes. Enzyme histochemistry and zymography demonstrated cell-associated uPA-like serine proteinase activity and PA activity respectively which was not reduced by treatment of chondrocytes with mannosamine at concentrations effective at inhibiting cartilage proteoglycan breakdown. Similarly, the activity of cell-associated alkaline phosphatase was not reduced, except at mannosamine concentrations much higher than those used to inhibit proteoglycan breakdown. These results demonstrate that inhibition of proteoglycan breakdown by mannosamine is too potent to be explained by an effect on GPI-anchor formation.