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Cell culture & proliferation assays

Ishikawa cells were obtained from the European Collection of Cell Cultures (Cat. No. 99040201) [] and were grown in DMEM/F-12 Ham medium (Cat. No. D6421, Sigma-Aldrich, UK) supplemented with L-glutamine and 10% fetal calf serum in 96-well plates (proliferation assays), 6-well plates (FACS analysis, luciferase reporter assays) or cell culture flasks (RNA extraction, tumour explant preparation). Cells were cultured at 37°C and 5%CO2 with varying doses of fenofibrate (Cat. No. F6020), 9-cis retinoic acid (9-cRA) (Cat. No. R4643) or all-trans retinoic acid (ATRA) (Cat. No. R6265, all Sigma-Aldrich, UK) dissolved in DMSO (Sigma-Aldrich). Control cells were treated with DMSO and the concentration of DMSO was kept the same throughout each experiment, to a maximum of 1% v/v. A minimum of five replicates per dose was performed for automated assays (BrdU and MTS) and all other cell culture experiments were performed independently in duplicate or triplicate. Cell proliferation was measured by the uptake of 5-bromo-2'-deoxyuridine (BrdU) using the BrdU Labelling and Detection Kit (Cat. No. 1444611, Roche, UK) according to the manufacturer's instructions. Relative cell abundance was measured either by cell counting, or using an MTS assay (CellTiter 96 Aqueous Cell Proliferation Assay, Cat. No. TB169, Promega, UK). Absorbance values for each well were measured using a microtiter plate reader (Anthos Labtech Instruments, Salzburg, Austria)

Abstract

The aldehyde dehydrogenase family 1 member A3 (ALDH1A3) catalyzes the oxidation of retinal to the pleiotropic factor retinoic acid using NAD+. The level of ALDHs enzymatic activity has been used as a cancer stem cell marker and seems to correlate with tumour aggressiveness. Elevated ALDH1A3 expression in mesenchymal glioma stem cells highlights the potential of this isozyme as a prognosis marker and drug target. Here we report the first crystal structure of human ALDH1A3 complexed with NAD+ and the product all-trans retinoic acid (REA). The tetrameric ALDH1A3 folds into a three domain-based architecture highly conserved along the ALDHs family. The structural analysis revealed two different and coupled conformations for NAD+ and REA that we propose to represent two snapshots along the catalytic cycle. Indeed, the isoprenic moiety of REA points either toward the active site cysteine, or moves away adopting the product release conformation. Although ALDH1A3 shares high sequence identity with other members of the ALDH1A family, our structural analysis revealed few peculiar residues in the 1A3 isozyme active site. Our data provide information into the ALDH1As catalytic process and can be used for the structure-based design of selective inhibitors of potential medical interest.

REA and NAD+ display different and coupled conformations

Our crystallographic data revealed that both REA and NAD+ bind to the enzyme active site with two different conformations that we observed in different monomers. In particular, in the monomers A and D, REA displays a closed conformation with its carboxyl group pointing toward the catalytic residue C314. In the same monomers, the nicotinic acid moiety of NAD+ is oriented in the direction of the active site. On the other hand, in monomers B and C, the REA adopts what we propose to be the product conformation, with its isoprenic chain moving away from the active site toward the protein surface, so that the entire REA molecule efficiently occupies a tunnel that provides substrate access to and product release from, the catalytic cysteine. In parallel, the NAD+ nicotinic ring also moves away from the active site pointing toward the protein surface (). In summary, we observed two different and linked conformations for the REA-NAD+ couple in two different monomers: the first showing the REA carboxylate pointing toward the C314 and the second showing the product conformation with the REA carboxylate moiety oriented toward the protein surface. The NAD+ and REA coupled conformations do not appear to be determined or influenced by contacts occurring between different monomers within the tetrameric assembly or from crystallographic symmetry mates. Therefore, we can surmise that in human ALDH1A3 structure, the different conformations adopted by the ligands are the consequence of specific interactions with ALDHs conserved residues.

Conclusion

Human ALDH1A3 has a key role in vertebrate development by participating in the synthesis of the differentiation factor retinoic acid. In the present study we report the crystal structure of human ALDH1A3 complexed with NAD+ and the physiological product all-trans retinoic acid. Our structural investigation provides the first description of the REA binding site in a member of the ALDH1A subfamily. Both the REA and NAD+ shows two different conformations that appear to be coupled. Indeed, in the closed-in conformation, both ligands point the reactive like moieties of the molecule toward the active site cysteine. On the other hand, in the product-like conformation both NAD+ and REA move away from the catalytic pocket to the protein surface and occupy a binding pocket located at the entrance of the catalytic tunnel. Remarkably, the conformational changes adopted by the ligands concern the catalytic reactive moieties on both molecules. Indeed, it is the nicotinamide ring of NAD+ and the isoprenic chain of REA that move in a concerted manner either close to the active site cysteine or toward the protein surface, while both the REA β-ionone ring and the ADP moiety of NAD+ maintain the same conformation. Structural superposition with human ALDH1A1 reveals the expected high level of conservation; however, few significantly different residues are observed in structurally equivalent positions in the catalytic pocket. In particular, in ALDH1A3 an asparagine at position 469 and a threonine at position 315, respectively replace a glycine and an isoleucine present in ALDH1A1. Remarkably, both residues contact REA in the closed conformation and therefore play a role in ligand recognition. The last observation suggests that the structure-based design of potent and, most importantly, specific inhibitors of potential medical interest, is achievable.

Experimental design

Thirty male rats (8 weeks old, 172 ± 16 g) were randomly divided into 3 groups: A) sham (SHO) group; B) unilateral ureteral obstruction (UUO) group; C) ATRA treat (ATRA) group, 10 rats per group. Before surgery the rats were performed under general anesthesia (40 mg/kg ip sodium pentobarbital). The left ureter was either completely ligated with two separate silk ties without cutting off the ureter (group B/C) or manipulated similarly but not ligated (group A). ATRA dissolved in corn oil at a dose of 15 mg/kg (group C) or vehicle corn oil (group A/B) was orally administered from 3 days before surgery and on each day until rats were sacrificed after 14 and 28 days (n = 5 for each time point for all groups). Kidneys were removed for histological analysis or Western blot assay.

ARTA improves histopathologic changes in UUO rats

After ureteral ligation, the major signs of renal damage were hydronephrosis and thin renal cortex. From d 14 to d 28, the renal tubular dilation, loss of integrality for tubular brush border, and severity for inflammatory infiltration was significantly enhanced in UUO group when compared with SHO group (). In parallel, the renal interstitial area gradually widened and fibrosis gradually developed in UUO group, and the ATRA group was weaker than UUO group, as showed by the staining of Masson trichrome (). Meanwhile, RIF score was notably up-regulated in UUO rats when compared with that of sham rats. Compared with UUO rats, the RIF score was markedly down-regulated in rats of ATRA group at d 14 and d 28. The values were shown in .

ATRA affects the expressions of angpt-1, TGF-β1, Col-IV and FN in UUO rats

An imbalance of ECM synthesis and degradation was an important event in RIF, including expression of cytokines (e.g., TGF-β1) [,] and mesenchymal markers (e.g., Col-IV and FN) [,]. To assess the effect of ATRA on ECM in the renal interstitial, Angpt-1, TGF-β1, Col-IV and FN protein expression levels were determined by immunohistochemistry in UUO rats treated with or without ATRA, respectively. As shown in , in the group of UUO, TGF-β1, Col-IV and FN expressions were significantly up-regulated, whereas Angpt-1 expression was notably down-regulated than the group of SHO from d 14 to d 28 (p < 0.05). ATRA treatment can reduce TGF-β1, Col-IV and FN expressions and improved Angpt-1 expression at d 14 and d 28 than the UUO group (p < 0.05; ).

ATRA restores angpt-1 expression in UUO rats

To investigate the effect of ATRA on ECM accumulation in the RIF rats, Angpt-1 protein expression was determined using Western-blot method. As shown in , when compared with the group of sham, the expression of Angpt-1 in UUO group was markedly down-regulated from day of d 14 to d 28 (p < 0.05; ). Interestingly, ATRA treatment in group of UUO resulted in increased the expression of Angpt-1 on days 14 and 28 compared to the group of UUO (p < 0.05; ).