Lecithin:retinol Acyltransferase Research Articles

Retinal Gene Expression and Visually Evoked Behavior in Diabetic Long Evans Rats

Patients with diabetic retinopathy may experience severe vision loss due to macular edema and neovascularization secondary to vascular abnormalities. However, before these abnormalities become apparent, there are functional deficits in contrast sensitivity, color perception, and dark adaptation. The goals of this study are to evaluate early changes (up to 3 months) in retinal gene expression, selected visual cycle proteins, and optokinetic tracking (OKT) in streptozotocin (STZ)-induced diabetic rats. Retinal gene expression in diabetic Long Evans rats was measured by whole genome microarray 7 days, 4 weeks, and 3 months after the onset of hyperglycemia. Select gene and protein changes were probed by polymerase chain reaction (PCR) and immunohistochemistry, respectively, and OKT thresholds were measured using a virtual optokinetics system. Microarray analysis showed that the most consistently affected molecular and cellular functions were cell-to-cell signaling and interaction, cell death, cellular growth and proliferation, molecular transport, and cellular movement. Further analysis revealed reduced expression of several genes encoding visual cycle proteins including lecithin/retinol acyltransferase (LRAT), retinal pigment epithelium (RPE)-specific protein 65 kDa (RPE65), and RPE retinal G protein-coupled receptor (RGR). These molecular changes occurred simultaneously with a decrease in OKT thresholds by 4 weeks of diabetes. Immunohistochemistry revealed a decrease in RPE65 in the RPE layer of diabetic rats after 3 months of hyperglycemia. The data presented here are further evidence that inner retinal cells are affected by hyperglycemia simultaneously with blood retinal barrier breakdown, suggesting that glial and neuronal dysfunction may underlie some of the early visual deficits in persons with diabetes.

α-Phenyl-N-tert-butylnitrone (PBN) Prevents Light-induced Degeneration of the Retina by Inhibiting RPE65 Protein Isomerohydrolase Activity

α-Phenyl-N-tert-butylnitrone (PBN), a free radical spin trap, has been shown previously to protect retinas against light-induced neurodegeneration, but the mechanism of protection is not known. Here we report that PBN-mediated retinal protection probably occurs by slowing down the rate of rhodopsin regeneration by inhibiting RPE65 activity. PBN (50 mg/kg) protected albino Sprague-Dawley rat retinas when injected 0.5-12 h before exposure to damaging light at 2,700 lux intensity for 6 h but had no effect when administered after the exposure. PBN injection significantly inhibited in vivo recovery of rod photoresponses and the rate of recovery of functional rhodopsin photopigment. Assays for visual cycle enzyme activities indicated that PBN inhibited one of the key enzymes of the visual cycle, RPE65, with an IC(50) = 0.1 mm. The inhibition type for RPE65 was found to be uncompetitive with K(i) = 53 μm. PBN had no effect on the activity of other visual cycle enzymes, lecithin retinol acyltransferase and retinol dehydrogenases. Interestingly, a more soluble form of PBN, N-tert-butyl-α-(2-sulfophenyl) nitrone, which has similar free radical trapping activity, did not protect the retina or inhibit RPE65 activity, providing some insight into the mechanism of PBN specificity and action. Slowing down the visual cycle is considered a treatment strategy for retinal diseases, such as Stargardt disease and dry age-related macular degeneration, in which toxic byproducts of the visual cycle accumulate in retinal cells. Thus, PBN inhibition of RPE65 catalytic action may provide therapeutic benefit for such retinal diseases.

Lecithin retinol acyltransferase and its S175R mutant have a similar secondary structure content and maximum insertion pressure but different enzyme activities

Recent work on Lecithin:retinol acyltransferase (LRAT) allowed to gather a large amount of information on its secondary structure, enzymatic properties and membrane binding. A truncated form of LRAT (tLRAT) as well as its S175R mutant leading to retinis pigmentosa, a severe form of retinal dystrophy, were studied to understand the role of this mutation on the dysfunction of this protein. Consistently with previous reports, the S175R-tLRAT mutant was shown to lack enzyme activity. However, very similar secondary structures probed by circular dichroism have been obtained with the S175R-tLRAT mutant and tLRAT. Moreover, similar values of maximum insertion pressure of the S175R-tLRAT mutant and tLRAT have been obtained using Langmuir monolayers, thus suggesting that the S175R mutation has no effect on the membrane binding properties of tLRAT. These findings leave open the possibility that the loss of enzymatic activity associated with the S175R mutant is related to loss of an essential nucleophile near the active site, or alternatively, to steric obstruction of the active site that impedes substrate binding.

Comparison of Ocular Pathologies in Vitamin A–Deficient Mice and RPE65 Gene Knockout Mice

RPE65 gene knockout (Rpe65⁻/⁻) mice showed abolished isomerohydrolase activity in the visual cycle and were considered a model for vitamin A deficiency in the retina. The purpose of this study was to compare the retinal phenotypes between vitamin A-deficient (VAD) mice and Rpe65⁻/⁻ mice under normal diet. The VAD mice were fed with a vitamin A-deprived diet after birth. The age-matched control mice and Rpe65⁻/⁻ mice were maintained under normal diet. The structure of photoreceptor outer segment was compared using electron microscopy. Photoreceptor-specific gene expression was determined using real-time RT-PCR. The isomerohydrolase and lecithin-retinol acyltransferase (LRAT) activities were measured using an in vitro enzymatic activity assay. Endogenous retinoid profiles were analyzed by HPLC in mouse eyecup homogenates. Compared to wild-type mice under normal diet, scanning and transmission electron microscopy showed that the outer segments of photoreceptors were disorganized in VAD mice and were not disorganized in Rpe65⁻/⁻ mice, although they were shortened in the latter. VAD mice showed more prominent downregulation of middle wavelength cone opsin, whereas Rpe65⁻/⁻ mice displayed more suppressed expression of short wavelength cone opsin. In vitro enzymatic activity assay and Western blot analysis showed that vitamin A deprivation downregulated LRAT expression and activity in the eyecup, but Rpe65⁻/⁻ mice showed unchanged LRAT expression and activity. The depressed LRAT activity in VAD mice was partially rescued by the intraperitoneal injection of retinoic acid. VAD and Rpe65⁻/⁻ mice are different in cone photoreceptor degeneration, photoreceptor-specific gene regulation, isomerohydrolase activity, endogenous retinoid profile, and LRAT activity.

Expression of interleukin-4 receptor α in human corneal epithelial cells

We previously reported that human conjunctival epithelial cells expressed functioning interleukin-4 receptor α (IL-4Rα). In this study, we investigated whether human corneal epithelial cells also express functioning IL-4Rα. The presence of IL-4Rα mRNA and protein in human corneal epithelium was examined by reverse-transcriptase polymerase chain reaction (RT-PCR) and immunohistology, respectively. The cell surface expression of IL-4Rα and the transcripts upregulated upon IL-4Rα ligand (IL-4 or IL-13) stimulation were examined by flow cytometry and quantitative RT-PCR, respectively, using immortalized human corneal-limbal epithelial (HCLE) cells. The mRNA and protein of IL-4Rα were detected in human corneal epithelium. Flow cytometry analysis showed the cell surface expression of IL-4Rα protein. Quantitative RT-PCR assay of HCLE cells showed the upregulation of the transcripts tumor necrosis factor alpha-induced protein 6 (TNFAIP6), RAS guanyl-releasing protein 1 (RASGRP1), carbonic anhydrase II (CA2), cytokine-inducible SH2-containing protein (CISH), hyaluronan synthase 3 (HAS3), calpain 14 (CAPN14), endothelin receptor type A (EDNRA), cathepsin C (CTSC), and lecithin retinol acyltransferase (LRAT) as well as human conjunctival epithelial cells. Human corneal epithelial cells expressed functioning IL-4Rα, and stimulation of its ligands, IL-4 and IL-13, could induce the expression of various genes, e.g., antiinflammatory molecule genes such as TNFAIP6 and CISH and cellular differentiation and proliferation-related molecule genes such as RASGRP1, HAS3, EDNRA, and LRAT.

Cone opsin determines the time course of cone photoreceptor degeneration in Leber congenital amaurosis

Mutations in RPE65 or lecithin-retinol acyltransferase (LRAT) disrupt 11-cis-retinal recycling and cause Leber congenital amaurosis (LCA), the most severe retinal dystrophy in early childhood. We used Lrat(-)(/-), a murine model for LCA, to investigate the mechanism of rapid cone degeneration. Although both M and S cone opsins mistrafficked as reported previously, mislocalized M-opsin was degraded whereas mislocalized S-opsin accumulated in Lrat(-)(/-) cones before the onset of massive ventral/central cone degeneration. As the ventral and central retina express higher levels of S-opsin than the dorsal retina in mice, our results may explain why ventral and central cones degenerate more rapidly than dorsal cones in Rpe65(-)(/-) and Lrat(-)(/-) LCA models. In addition, human blue opsin and mouse S-opsin, but not mouse M-opsin or human red/green opsins, aggregated to form cytoplasmic inclusions in transfected cells, which may explain why blue cone function is lost earlier than red/green-cone function in patients with LCA. The aggregation of short-wavelength opsins likely caused rapid cone degenerations through an endoplasmic reticulum stress pathway, as demonstrated in both the Lrat(-)(/-) retina and transfected cells. Replacing rhodopsin with S-opsin in Lrat(-)(/-) rods resulted in mislocalization and aggregation of S-opsin in the inner segment and the synaptic region of rods, ER stress, and dramatically accelerated rod degeneration. Our results demonstrate that cone opsins play a major role in determining the degeneration rate of photoreceptors in LCA.

Oral synthetic cis-retinoid therapy in subjects with Leber congenital amaurosis (LCA) due to lecithin:retinol acyltransferase (LRAT) or retinal pigment epithelial 65 protein (RPE65) mutations: preliminary results of a phase Ib open label trial

Oral synthetic cis-retinoid therapy in subjects with Leber congenital amaurosis (LCA) due to lecithin:retinol acyltransferase (LRAT) or retinal pigment epithelial 65 protein (RPE65) mutations: preliminary results of a phase Ib open label trial

Abstract 5555: Diethylnitrosamine-induced hepatic injury and hepatocarcinogenesis: A role for hepatic retinoids

Abstract The liver is the main tissue site of retinoid storage in the body and the non-parenchymal hepatic stellate cell (HSC) is the major cellular site of retinoid storage within the liver. Loss of retinoid-containing lipid droplets (LDs) from HSCs is one of the first events in the development of hepatic diseases, leading progressively to liver fibrosis, cirrhosis and hepatocellular carcinoma. Although the loss of LDs is a hallmark of HSC activation, the contribution of hepatic retinoid storage to hepatic injury and hepatocarcinogenesis remains unknown. Lecithin:retinol acyltransferase (LRAT) is the sole enzyme responsible for storing hepatic retinoid and LRAT knockout (KO) mice lack liver retinoid stores. Retinol-binding protein (RBP) is the principal transport protein for retinol in the blood. RBP KO mice are unable to mobilize hepatic retinoid stores and have significantly more retinoid storage in the liver compared to wild type (WT) mice. In this study, we investigated the effects of endogenous retinoids on hepatic injury and hepatocarcinogenesis. We induced hepatic injury and HCC using diethylnitrosamine (DEN) in WT, LRAT KO and RBP KO mice. Both acute (for 24 and 48 hours after DEN administration) and long-term (for 8 months after DEN administration) studies were undertaken. After DEN injection of WT, LRAT KO, and RBP KO mice, serum levels of alanine transaminase (ALT) were significantly lower and Cyclin D1 and Ki-67 mRNA expression levels were decreased in the livers of the KOs compared to WT mice. This suggests that LRAT KO and RBP KO mice are less susceptible to DEN-induced liver injury and following compensatory proliferation. Furthermore, 8-month-old DEN-treated LRAT KO (n = 18) and RBP KO (n = 13) mice showed lower HCC incidence, fewer tumors, and smaller tumors than DEN-treated WT mice (n = 24). Five-month-old male LRAT KO and RBP KO mice showed higher expression levels of retinoic acid (RA) responsive genes, such as Cyp26A1 and RARβ, mRNA in their liver compared to WT, indicating that RA signaling is upregulated in the livers of the KOs’ mice. The expression levels of p21 mRNA in LRAT KO and RBP KO mice liver were higher than WT at several time points in both acute and long-term studies. Together these results suggest that increased RA signaling and associated higher p21 expression play important roles in the lower susceptibility of these mouse models to liver injury and hepatocarcinogenesis. We are presently investigating other possibilities that the presence/absence through which endogenous hepatic retinoids might have effects on hepatic diseases, probably by modulating several cancer signaling pathways, regulating apoptosis of hepatocytes and cancer cells, or influencing the tumor-surrounding microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5555. doi:10.1158/1538-7445.AM2011-5555

β‐Carotene and its cleavage enzyme β‐carotene‐15,15′‐oxygenase (CMOI) affect retinoid metabolism in developing tissues

The mammalian embryo relies on maternal circulating retinoids (vitamin A derivatives) for development. β-Carotene is the major human dietary provitamin A. β-Carotene-15,15'-oxygenase (CMOI) has been proposed as the main enzyme generating retinoid from β-carotene in vivo. CMOI is expressed in embryonic tissues, suggesting that β-carotene provides retinoids locally during development. We performed loss of CMOI function studies in mice lacking retinol-binding protein (RBP), an established model of embryonic vitamin A deficiency (VAD). We show that, unexpectedly, lack of CMOI in the developing tissues further exacerbates the severity of VAD and thus the embryonic malformations of RBP(-/-) mice. Since β-carotene was not present in any of the mouse diets, we unveiled a novel action of CMOI independent from its β-carotene cleavage activity. We also show for the first time that CMOI exerts an additional function on retinoid metabolism by influencing retinyl ester formation via modulation of lecithin:retinol acyltransferase (LRAT) activity, at least in developing tissues. Finally, we demonstrate unequivocally that β-carotene can serve as an alternative vitamin A source for the in situ synthesis of retinoids in developing tissues by the action of CMOI.

Absence of hepatic stellate cell retinoid lipid droplets does not enhance hepatic fibrosis but decreases hepatic carcinogenesis

ObjectiveHepatic stellate cells (HSCs) contain a number of bioactive metabolites or their precursors including retinoids in their characteristic lipid droplets. The loss of lipid droplets and retinoids is a hallmark...

Altered Expression of Both β-Carotene 15,15' Monooxygenase and Lecithin:Retinol Acyltransferase in Obese Zucker Rats

Retinol and its active derivative retinoic acid have an important role in development, reproduction, immunity, and cell proliferation/differentiation. Obesity and dyslipidemia are risk factors for cardiovascular disease that may affect hepatic homeostasis. It is unclear whether the expression of retinol-related proteins is affected and influences the retinol status in obesity and dyslipidemia. The aim of this study was to evaluate the retinol status and expression of retinol-metabolizing enzymes and binding protein in obese and dyslipidemic fa/fa (Zucker) rats. We examined the expression of genes for β-carotene 15,15' monooxygenase (BCM), lecithin:retinol acyltransferase (LRAT), cellular retinol binding protein-I (CRBP-I), and cytochrome P450 26A1 (CYP26A1) in fa/fa rats and lean control rats. We also measured the retinol level in plasma and liver samples from both groups. Plasma retinol levels in fa/fa rats were increased compared with lean rats, while hepatic retinol levels were similar in both groups. In obese and dyslipidemic fa/fa rats, intestinal BCM gene expression was increased, whereas hepatic LRAT gene expression was deceased. There was no difference in hepatic CRBP-I and CYP26A1 gene expression between fa/fa rats and lean rats. Altered expression of BCM and LRAT genes may affect plasma retinol status in obesity and dyslipidemia.

Multiple cytochromeP-450 genes are concomitantly regulated by vitamin A under steady-state conditions and by retinoic acid during hepatic first-pass metabolism

Vitamin A (retinol) is an essential precursor for the production of retinoic acid (RA), which in turn is a major regulator of gene expression, affecting cell differentiation throughout the body. Understanding how vitamin A nutritional status, as well as therapeutic retinoid treatment, regulates the expression of retinoid homeostatic genes is important for improvement of dietary recommendations and therapeutic strategies using retinoids. This study investigated genes central to processes of retinoid uptake and storage, release to plasma, and oxidation in the liver of rats under steady-state conditions after different exposures to dietary vitamin A (deficient, marginal, adequate, and supplemented) and acutely after administration of a therapeutic dose of all-trans-RA. Over a very wide range of dietary vitamin A, lecithin:retinol acyltransferase (LRAT) as well as multiple cytochrome P-450s (CYP26A1, CYP26B1, and CYP2C22) differed by diet and were highly correlated with one another and with vitamin A status assessed by liver retinol concentration (all correlations, P < 0.05). After acute treatment with RA, the same genes were rapidly and concomitantly induced, preceding retinoic acid receptor (RAR)β, a classical direct target of RA. CYP26A1 mRNA exhibited the greatest dynamic range (change of log 2(6) in 3 h). Moreover, CYP26A1 increased more rapidly in the liver of RA-primed rats than naive rats, evidenced by increased CYP26A1 gene expression and increased conversion of [(3)H]RA to polar metabolites. By in situ hybridization, CYP26A1 mRNA was strongly regulated within hepatocytes, closely resembling retinol-binding protein (RBP)4 in location. Overall, whether RA is produced endogenously from retinol or administered exogenously, changes in retinoid homeostatic gene expression simultaneously favor both retinol esterification and RA oxidation, with CYP26A1 exhibiting the greatest dynamic change.

Inhibition of the visual cycle by A2E through direct interaction with RPE65 and implications in Stargardt disease

Stargardt disease (STGD) is the major form of inherited juvenile macular degeneration. Pyridinium bis-retinoid A2E is a major component of lipofuscin which accumulates in retinal pigment epithelium (RPE) cells in STGD and contributes to the disease pathogenesis. However, the precise role of A2E in vision loss is unclear. Here we report that A2E efficiently inhibits RPE65 isomerohydrolase, a key enzyme in the visual cycle. Subretinal injection of A2E significantly inhibited retinoid isomerohydrolase activity in mice. Likewise, A2E also inhibited isomerohydrolase activity in cells coexpressing RPE65, lecithin retinol acyltransferase (LRAT), and cellular retinaldehyde-binding protein. In vitro isomerohydrolase activity assays confirmed that A2E inhibited enzymatic activity of recombinant RPE65 in a concentration-dependent manner, but did not inhibit LRAT activity. The inhibition type for isomerohydrolase was found to be reversible and competitive with K(i)=13.6μM. To determine the direct interaction of A2E with RPE65 protein, fluorescence binding assays were performed. As shown by fluorimetric titration, binding of purified RPE65 with A2E enhanced the bis-retinoid fluorescence. Consistently, the fluorescence of RPE65 decreased upon incubation with A2E. Both of these experiments suggest a direct, specific binding of A2E to RPE65. The binding constant for A2E and purified RPE65 was calculated to be 250nM. These results demonstrate that A2E inhibits the regeneration of 11-cis retinal, the chromophore of visual pigments, which represents a unique mechanism by which A2E may impair vision in STGD.

Solution structure of the N-terminal catalytic domain of human H-REV107 – A novel circular permutated NlpC/P60 domain

H-REV107 is a Ca 2+-independent phospholipase A 1/2, and it is also a pro-apoptosis protein belonging to the novel class II tumor suppressor family, H-REV107-like family. Here we report the solution structure of the N-terminal catalytic domain of human H-REV107, which has a similar architecture to classical NlpC/P60 domains, even though their fold topologies are different due to circular permutation in the primary sequence. The phospholipase active site possesses a structurally conserved Cys–His–His catalytic triad as found in NlpC/P60 peptidases, indicating H-REV107 should adopt a similar catalytic mechanism towards phospholipid substrates to that of NlpC/P60 peptidases towards peptides. As H-REV107 is highly similar to lecithin retinol acyltransferase, our study also provides structural insight to this essential enzyme in retinol metabolism.

An Acyl-covalent Enzyme Intermediate of Lecithin:Retinol Acyltransferase*[S

Synthesis of fatty acid retinyl esters determines systemic vitamin A levels and provides substrate for production of visual chromophore (11-cis-retinal) in vertebrates. Lecithin:retinol acyltransferase (LRAT), the main enzyme responsible for retinyl ester formation, catalyzes the transfer of an acyl group from the sn-1 position of phosphatidylcholine to retinol. To delineate the catalytic mechanism of this reaction, we expressed and purified a fully active, soluble form of this enzyme and used it to examine the possible formation of a transient acyl-enzyme intermediate. Detailed mass spectrometry analyses revealed that LRAT undergoes spontaneous, covalent modification upon incubation with a variety of phosphatidylcholine substrates. The addition of an acyl chain occurs at the Cys(161) residue, indicating formation of a thioester intermediate. This observation provides the first direct experimental evidence of thioester intermediate formation that constitutes the initial step in the proposed LRAT catalytic reaction. Additionally, we examined the effect of increasing fatty acyl side chain length in phosphatidylcholine on substrate accessibility in this reaction, which provided insights into the function of the single membrane-spanning domain of LRAT. These observations are critical to understanding the catalytic mechanism of LRAT protein family members as well as other lecithin:acyltransferases wherein Cys residues are required for catalysis.

Expression of the interleukin-4 receptor α in human conjunctival epithelial cells

AimTo investigate the expression and function of interleukin-4 receptor α (IL-4Rα) in human conjunctival epithelial cells (HCjECs).MethodsThe presence of IL-4Rα mRNA and protein was examined by reverse transcription (RT) PCR...

Abstract 83: A role of hepatic retinoid storage in diethynitrosamine-induced hepatic injury and hepatocarcinogenesis

Abstract The liver is the main tissue site of retinoid (vitamin A and its derivatives) storage in the body and the non-parenchymal hepatic stellate cell (HSC) is the major cellular site of retinoid storage within the liver. Lipid droplets of HSCs contain a number of bioactive metabolites including retinoids. Following liver injury, HSCs undergo a process known as activation. Loss of retinyl esters, the retinoid storage form, and lipid droplets from HSCs is one of the first events in the development of hepatic disease, leading progressively to liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC). Although the loss of lipid droplets is a hallmark of HSC activation, whether it is a cause or consequence of the HSC activation process is unclear. Moreover, the contribution of the loss of hepatic retinoid storage to hepatic injury and hepatocarcinogenesis remains unknown. To investigate the role of hepatic retinoid storage and retinoid-containing lipid droplets in hepatic injury and hepatocarcingenesis, we studied diethynitrosamine (DEN)-induced HCC in lecithin:retinol acyltransferase (LRAT) knockout (KO) mice, which lack retinoid-containing lipid droplets in their HSCs, and in retinol-binding protein (RBP) KO mice, which are unable to mobilize hepatic retinoid stores. We measured a number of factors implicated in hepatic injury and cancer initiation. Using an acute hepatic injury model for investigating cancer initiation, assessment of mRNA levels by qRT-PCR of TNF alpha and TGF beta in livers of LRAT KO mice indicated that these mice were more susceptible to cancer initiation than WT mice. On the other hand RBP KO mice showed less susceptibility. LRAT KO mice showed higher levels of CYP26A1, a gene whose expression is known to be highly and directly responsive to retinoic acid level, suggesting that hepatic retinoic acid levels in LRAT KO mice were higher than that of WT, and that this may influence hepatic injury and carcinogenesis. Overall, our data suggest that hepatic retinoid storage can influence the progression of hepatic disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 83.

Multiple dosings wtih acidic retinoids increase retinol stores and expression of retinoid homeostatic genes in the lung of neonatal rats

Previously we have shown that vitamin A (VA) combined with acidic retinoids synergistically increases retinol uptake and retinyl ester (RE) formation in neonatal rat lung, concomitant with the expression of several retinoid homeostatic genes: LRAT (lecithin:retinol acyltransferase), CYP26B1 (a cytochrome P450), and Stra6 (stimulated by retinoic acid gene 6). In the present study we compared the response to VA alone or combined with retinoic acid (RA) or a analog, Am580, in two timing protocols: single early dosing (d 4) vs. multiple dosings throughout the period of lung septation (d 4, 7, 11, 14). Multiple dosing resulted in a higher, cumulative increase in lung RE content for all treatments: vehicle&lt;RA≅VA&lt;Am580&lt;VARA&lt;VAAm580. However, most of the retinoid homeostatic genes were up‐regulated by acidic retinoids to the same extent after a single vs. multiple dosings. Lung functional gene, surfactant protein‐A (SPA1) mRNA remained nearly constant with both treatment and timing. In conclusion, multiple treatments of VA and acidic retinoids in combination during the septation period greatly increased neonatal lung RE content in a synergistic and cumulative manner. Repeated but transient induction of retinoid homeostatic genes by acidic retinoids at the time of each dosing may explain the observed cumulative synergistic enhancement of RE formation.Support: NIH CA90214.Grant Funding Source: NIH CA90214

Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity

Regeneration of visual chromophore in the vertebrate visual cycle involves the retinal pigment epithelium-specific protein RPE65, the key enzyme catalyzing the cleavage and isomerization of all-trans-retinyl fatty acid esters to 11-cis-retinol. Although RPE65 has no predicted membrane spanning domains, this protein predominantly associates with microsomal fractions isolated from bovine retinal pigment epithelium (RPE). We have re-examined the nature of RPE65 interactions with native microsomal membranes by using extraction and phase separation experiments. We observe that hydrophobic interactions are the dominant forces that promote RPE65 association with these membranes. These results are consistent with the crystallographic model of RPE65, which features a large lipophilic surface that surrounds the entrance to the catalytic site of this enzyme and likely interacts with the hydrophobic core of the endoplasmic reticulum membrane. Moreover, we report a critical role for phospholipid membranes in preserving the retinoid isomerization activity and physical properties of RPE65. Isomerase activity measured in bovine RPE was highly sensitive to phospholipase A(2) treatment, but the observed decline in 11-cis-retinol production did not directly reflect inhibition by products of lipid hydrolysis. Instead, a direct correlation between the kinetics of phospholipid hydrolysis and retinoid isomerization suggests that the lipid membrane structure is critical for RPE65 enzymatic activity. We also provide evidence that RPE65 operates in a multiprotein complex with retinol dehydrogenase 5 and retinal G protein-coupled receptor in RPE microsomes. Modifications in the phospholipid environment affecting interactions with these protein components may be responsible for the alterations in retinoid metabolism observed in phospholipid-depleted RPE microsomes. Thus, our results indicate that the enzymatic activity of native RPE65 strongly depends on its membrane binding and phospholipid environment.

Oral carcinogenesis induced by 4-nitroquinoline 1-oxide in lecithin:retinol acyltransferase gene knockout mice

Lecithin:retinol acyltransferase ( LRAT) regulates retinol (vitamin A) metabolism by esterifying retinol. LRAT expression is decreased in cultured human squamous cell carcinoma cells of the head and neck relative to normal epithelial cells. We investigated whether the carcinogen 4-nitroquinoline 1-oxide (4-NQO) induced a higher incidence of oral cancer in LRAT knockout ( LRAT −/− ) than in wild-type (Wt) mice. We also investigated retinol deprivation during 4-NQO treatment in LRAT −/− mice as a model for rapid retinol deficiency. We observed higher levels of secreted frizzled-related protein ( Sfrp) 2, an inhibitor of WNT signaling, in tongue tumors in LRAT −/− versus Wt. LRAT −/− embryonic stem cells also expressed higher Sfrp2 transcripts, indicating an interaction between retinol and WNT signaling. Cox-2, Cyclin D1, p21, Trop2 and RARβ 2 were not differentially expressed in Wt versus LRAT −/− tongue tumors. Wt and LRAT −/− mice fed a retinol-sufficient diet showed the same oral tumor incidence after 4-NQO treatment. In contrast, tongue tumors developed in 60% of Wt mice and in 100% of LRAT −/− mice fed a retinol-deficient diet during 4-NQO treatment ( P=.22); moreover, the bromodeoxyuridine labeling index was 21.0±2.4% in LRAT −/− normal tongue epithelium as compared to 9.9±0.8% in Wt normal tongue epithelium ( P<.001). Thus, partial retinol deficiency during carcinogen treatment (achieved in LRAT −/− ) resulted in more proliferating cells in tongue epithelia from LRAT −/− mice and, ultimately, a greater probability of carcinogenesis.