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Abstract
Aldose reductase (AR), the first enzyme in the polyol pathway, has been implicated in the pathogenesis of diabetic complications, although its physiological role is unclear. In mice, besides AR, two AR-like proteins, mouse vas deferens protein (MVDP) and fibroblast growth factor-regulated protein (FR-1), have been reported recently. Tissue-specific expression of these two genes was examined using the RNase protection assay method. Contrary to previous reports, MVDP was detected in a variety of tissues besides the vas deferens. High levels of MVDP mRNA were found in the adrenal glands, and low levels of expression were detected in eye, intestine, seminal vesicle, kidney, liver, testis and lung. The major gene expression pattern for FR-1 was slightly different from that of MVDP, with the highest levels of mRNA detected in testis, heart, adrenal gland, and ovary; less was found in the lung and it was barely detectable in eye, intestine, liver and seminal vesicle tissue. Mouse embryos, as early as 10.5 days post coitum, expressed both genes, although the levels of expression were different. Human AR mRNA was found in human vas deferens, although not at the high level found in mice. The localization of both MVDP and FR-1 transcripts in the adrenal cortex by in situ hybridization led to the speculation that these two AR-like proteins could be related to hormone production.
... The Gray shade highlights the necessary 100% identity between 2 identical protein sequences. (1)(2)(3). Aldose reductases are cytosolic monomeric enzymes, belonging to the aldo-keto reductase (AKR) superfamily. This superfamily encompasses more than 150 NAD(P)(H)-dependent oxidoreductases distributed in all prokaryotic and eukaryotic kingdoms, including yeast, plant, invertebrates, and vertebrates. ...
... AKR1B1, Akr1b3, and Akr1b16 are rather ubiquitously expressed (11,21), whereas Akr1b7 and Akr1b8 exhibit a restricted tissue distribution. Indeed, Akr1b7 is detected in vas deferens, adrenal glands, gonads, intestine, white adipose tissue, eye, liver, and kidney (2,22,(24)(25)(26) and Akr1b8 in testis, heart, adrenal glands, intestine, and liver (2,11,23). In studies using murine adrenal cell lines (Y1 adrenocortical cells and MPC862L chromaffin cells), we found that Akr1b3 protein FiGURe 1 | Genomic organization of AKR1B genes in humans and mice. ...
... AKR1B1, Akr1b3, and Akr1b16 are rather ubiquitously expressed (11,21), whereas Akr1b7 and Akr1b8 exhibit a restricted tissue distribution. Indeed, Akr1b7 is detected in vas deferens, adrenal glands, gonads, intestine, white adipose tissue, eye, liver, and kidney (2,22,(24)(25)(26) and Akr1b8 in testis, heart, adrenal glands, intestine, and liver (2,11,23). In studies using murine adrenal cell lines (Y1 adrenocortical cells and MPC862L chromaffin cells), we found that Akr1b3 protein FiGURe 1 | Genomic organization of AKR1B genes in humans and mice. ...
Aldose reductase (AKR1B) proteins are monomeric enzymes, belonging to the aldo-keto reductase (AKR) superfamily. They perform oxidoreduction of carbonyl groups from a wide variety of substrates, such as aliphatic and aromatic aldehydes or ketones. Due to the involvement of human aldose reductases in pathologies, such as diabetic complications and cancer, AKR1B subgroup enzymatic properties have been extensively characterized. However, the issue of AKR1B function in non-pathologic conditions remains poorly resolved. Adrenal activities generated large amount of harmful aldehydes from lipid peroxidation and steroidogenesis, including 4-hydroxynonenal (4-HNE) and isocaproaldehyde (4-methylpentanal), which can both be reduced by AKR1B proteins. More recently, some AKR1B isoforms have been shown to be endowed with prostaglandin F synthase (PGFS) activity, suggesting that, in addition to possible scavenger function, they could instigate paracrine signals. Interestingly, the adrenal gland is one of the major sites for human and murine AKR1B expression, suggesting that their detoxifying/signaling activity could be specifically required for the correct handling of adrenal function. Moreover, chronic effects of ACTH result in a coordinated regulation of genes encoding the steroidogenic enzymes and some AKR1B isoforms. This review presents the molecular mechanisms accounting for the adrenal-specific expression of some AKR1B genes. Using data from recent mouse genetic models, we will try to connect their enzymatic properties and regulation with adrenal functions.
... High levels of MVDP mRNA were found in the mouse vas deferens but not in that of other species studied, suggesting zoological specificity ). Because high levels of MVDP mRNA were recently found in the mouse adrenal (Lau et al. 1995), the first objective of the current study was to determine if adrenals express MVDP at mRNA and protein levels, and if this expression is restricted to the mouse species. The second objective was to investigate whether cAMP can affect MVDP expression, since this nucleotide is directly involved in the control of glucocorticoid secretion by adrenocorticotrophin (ACTH) (Hanukoglu et al. 1990). ...
... High levels of MVDP mRNA have been found in the mouse adrenal gland (Lau et al. 1995). By Western blot analysis using the monoclonal antibody against MVDP, antigens corresponding to this protein could be detected in adrenal glands from mouse and rat (Fig. 1A). ...
... MVDP mRNA were found in the adrenal glands, whereas low levels were detected in several tissues including eye, intestine, seminal vesicle, kidney, liver, testis and lung (Lau et al. 1995). We report here that, using a specific polyclonal antibody, MVDP is present in large amounts in mouse, rat, rabbit and guinea-pig adrenal glands. ...
The MVDP (mouse vas deferens protein) gene encodes an aldose reductase-like protein (AKR1B7) highly expressed in vas deferens epithelium and zona fasciculata of the adrenal cortex. Recombinant MVDP showed kinetic properties distinct from those of aldose reductase, including its spectrum of substrates, cofactor preference and sensitivity to inhibitors. We demonstrate that in adrenocortical cells, MVDP, rather than aldose reductase, is the principal reductase for isocaproaldehyde (a product of side-chain cleavage of cholesterol) and 4-hydroxynonenal (a lipid peroxidation product). In steroidogenic tissues MVDP expression is regulated by pituitary trophic hormones, namely ACTH in adrenals, FSH in ovaries, and LH in testicular Leydig cells.
... AKR1B7 is a 34.5 kDa mouse aldose reductase-like protein with two major expression sites, the vas deferens [36] and adrenal cortex [1,21]. AKR1B7 was first identify in the vas deferens and described as a secretory mouse vas deferens protein (MVDP) [36]. ...
... AKR1B7 is also present in significant amounts in the theca and interstitial cells of the ovary [8], in Leydig cells [4,25], in the pre-adipocytes fraction in white adipose tissue [29,39], in the eyes [43] and the kidney glomerulus and collecting duct [20,35]. A weaker expression is detected in the intestine [42] and in the liver of mouse and rat [17,21]. ...
... Although vas deferens is by far the tissue expressing the greatest amounts of AKR1B7 in mouse species [21], the enzyme appears barely detectable in rat, rabbit and guinea pig vas deferens [1]. By contrast, all these species share in common a strong expression of this protein in the adrenal gland [1]. ...
Aldo-Keto Reductase 1B7 (AKR1B7) is a mouse aldose reductase-like protein with two major sites of expression, the vas deferens and the adrenal cortex. In the adrenal cortex, Akr1b7 is an adrenocorticotropin (ACTH)-responsive-gene whose product scavenges harmful byproducts of steroidogenesis and limits stress response through the biosynthesis of prostaglandin F2α. The purpose of the present study was to explore the possible expression of AKR1B7 in the adrenal glands of two saharan rodents, Libyan jird and Lesser Egyptian gerbil. Western blot analyses demonstrated that a protein related to murine/rat AKR1B7 was highly expressed in adrenals and absent from vas deferens of both saharan species. Based on conserved sequences between mouse and rat, full length cDNA were cloned and sequenced in both species while hormonal regulation and tissue localization were explored in Libyan jird. Both cDNA encoded the expected 316 amino acids protein typical of AKR1B subfamily and contained the highly conserved catalytic tetrad consisting in Asp-44, Tyr-49, Lys-78 and His-111 residues. The deduced proteins shared higher identities with aldose reductase-like, i.e. AKR1B7 (86-94%), AKR1B8 and AKR1B10 (83-86%) than with aldose reductase group, i.e. AKR1B1 and AKR1B3 (70%). Phylogenetic analysis showed that the Libyan jird and gerbil enzymes were more closely related to murine and rat AKR1B7 than to the other AKR1B members. Northern blot analyses of total RNA from Libyan jird adrenals showed a single mRNA transcript of 1.4 kb whose expression was dependent on circulating ACTH levels. In conclusion, we demonstrate here that adrenal glands of Libyan jird and gerbil express both an ortholog of the murine/rat Akr1b7 gene and that ACTH-responsiveness is at least conserved in Libyan jird.
... In addition, the three enzymes differ in their tissue distribution and gene regulation. AKR1B10 is distributed in all human tissues [7,8], whereas the other two mouse enzymes show tissue-specific expression in the testis, ovary, heart and adrenal gland (for AKR1B8) [9,17] and in the male reproductive organs and adrenal gland (for AKR1B7) [17]. While AKR1B8 is induced by FGF-1 and FGF-2 [9], AKR1B7 is up-regulated by androgen and pituitary tropic hormones [10,14]. ...
... In addition, the three enzymes differ in their tissue distribution and gene regulation. AKR1B10 is distributed in all human tissues [7,8], whereas the other two mouse enzymes show tissue-specific expression in the testis, ovary, heart and adrenal gland (for AKR1B8) [9,17] and in the male reproductive organs and adrenal gland (for AKR1B7) [17]. While AKR1B8 is induced by FGF-1 and FGF-2 [9], AKR1B7 is up-regulated by androgen and pituitary tropic hormones [10,14]. ...
... The results suggest that AKR1B13 is expressed in almost all rat tissues, but its contents in rat tissues other than the lung, stomach and adrenal gland are low. The tissue distribution is essentially identical to the previous two-dimensional electrophoresis analysis of AKR1B13 as a tumor-associated protein in some rat tissues [11], but differs from that of the mouse homolog, AKR1B8 [17]. The expression patterns of AKR1B13 in rat tissues are also slightly different from those of AR (AKR1B4), aldehyde reductase (AKR1A3) and AKR1C15 (Fig. 1c-e). ...
... High levels of MVDP mRNA were found in the mouse vas deferens but not in that of other species studied, suggesting zoological specificity ). Because high levels of MVDP mRNA were recently found in the mouse adrenal (Lau et al. 1995), the first objective of the current study was to determine if adrenals express MVDP at mRNA and protein levels, and if this expression is restricted to the mouse species. The second objective was to investigate whether cAMP can affect MVDP expression, since this nucleotide is directly involved in the control of glucocorticoid secretion by adrenocorticotrophin (ACTH) (Hanukoglu et al. 1990). ...
... High levels of MVDP mRNA have been found in the mouse adrenal gland (Lau et al. 1995). By Western blot analysis using the monoclonal antibody against MVDP, antigens corresponding to this protein could be detected in adrenal glands from mouse and rat (Fig. 1A). ...
... MVDP mRNA were found in the adrenal glands, whereas low levels were detected in several tissues including eye, intestine, seminal vesicle, kidney, liver, testis and lung (Lau et al. 1995). We report here that, using a specific polyclonal antibody, MVDP is present in large amounts in mouse, rat, rabbit and guinea-pig adrenal glands. ...
Mouse vas deferens protein (MVDP) is a member of the aldo-keto reductase superfamily. The regulation of MVDP gene expression by activators of the protein kinase A signalling pathway was investigated in human (H295-R) and murine (Y1) adrenocortical carcinoma cells. Immunoblotting with polyclonal antibodies showed that MVDP is expressed in adrenal glands from mouse, rat, rabbit and guinea-pig, probably under the control of ACTH. In both adrenocortical cell lines used, MVDP is constitutively synthesized and its accumulation is increased by treatment with cAMP or forskolin. MVDP mRNA steady-state levels were up-regulated by forskolin in adrenocortical cells by a process that does not require de novo protein synthesis. The results suggest that cAMP is at least one of the key regulators of adrenal MVDP expression and that this effect is direct.
... Among them is the AKR1B family, which comprises several members, including aldo-reductases (AKR1B3, AKR1B8, and AKR1B16) and AKR1B7 (7, 15, 31). AKR1B7 is highly expressed in the deferential duct and also in the adrenal gland (2,24,38). Other organs, including the kidney, show only poor expression of this enzyme (24). ...
... cells and Leydig cells in the testis (2,5,11,24). In the developing kidney the Akr1b7 gene is expressed similarly to the renin gene in mural cells of the preglomerular vasculature, in mesangial cells, and in collecting duct cells (8). ...
Based on evidence that within the adult kidney the aldo-keto reductase AKR1B7 (aldo-keto reductase family 1, member 7; also known as mouse vas deferens protein, MVDP) is selectively expressed in renin producing cells, we aimed to define a possible role of AKR1B7 for the regulation and function of renin cells in the kidney. We could confirm colocalization and corecruitment of renin and of AKR1B7 in wildtype kidneys. Renin cells in AKR1B7 deficient kidneys showed normal morphology, numbers and intrarenal distribution. Plasma renin concentration (PRC) and renin mRNA levels of AKR1B7 deficient mice were normal at standard chow and were lowered by high salt diet directly comparable to wildtype mice. Treatment with low salt diet in combination with an angiotensin-converting enzyme inhibitor strongly increased PRC and renin mRNA in a similar fashion both in AKR1B7 deficient and wildtype mice. Under this condition, we also observed a strong retrograde recruitment of renin expressing cell along the preglomerular vessels, however, without difference between AKR1B7 deficient and wildtype mice. The isolated perfused mouse kidney model was used to study the acute regulation of renin secretion by angiotensin II and by perfusion pressure. Regarding to these parameters, no differences were observed between AKR1B7 deficient and wildtype kidneys. In summary, our data suggest that AKR1B7 is not of major relevance for the regulation of renin production and secretion in spite of its striking coregulation with renin expression.
... AKRs reduce aldehydes and ketones to their respective alcohols (Penning and Drury, 2007). Among AKR isoforms, the mouse Akr1b7 is highly expressed in vas deferens and adrenal gland, in which its sustained expression is dependent on androgen and adrenocorticotropic hormone, respectively (Lau et al., 1995). It is interesting that Akr1b7 null mice were found to be viable and have no obvious defect in reproduction (Baumann et al., 2007). ...
... It is interesting that Akr1b7 null mice were found to be viable and have no obvious defect in reproduction (Baumann et al., 2007). Besides the steroidogenic tissues, Akr1b7 is also expressed in mouse kidney, eye, intestine, and, at a lower level, in liver (Lau et al., 1995). One of the major functions of Akr1b7 is to detoxify lipid peroxidation. ...
Aldo-keto reductase (AKR) family 1, member 7 (AKR1B7), a member of the AKR superfamily, has been suggested to play an important role in the detoxification of lipid peroxidation by-products. The nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are xenosensors postulated to alleviate xeno- and endobiotic chemical insults. In this study, we show that the mouse Akr1b7 is a shared transcriptional target of PXR and CAR in the liver and intestine. Treatment of wild-type mice with the PXR agonist pregnenolone-16alpha-carbonitrile (PCN) activated Akr1b7 gene expression, whereas the effect was abrogated in PXR(-/-) mice. Similarly, the activation of Akr1b7 gene expression by the CAR agonist 1,4-bis[2-(3,5-dichlorpyridyloxyl)]-benzene, seen in wild-type mice, was abolished in CAR(-/-) mice. The promoter of Akr1b7 gene was activated by PXR and CAR, and this activation was achieved through the binding of PXR-retinoid X receptor (RXR) or CAR-RXR heterodimers to direct repeat-4 type nuclear receptor-binding sites found in the Akr1b7 gene promoter. At the functional level, treatment with PCN in wild-type mice, but not PXR(-/-) mice, led to a decreased intestinal accumulation of malondialdehyde, a biomarker of lipid peroxidation. The regulation of Akr1b7 by PXR was independent of the liver X receptor (LXR), another nuclear receptor known to regulate this AKR isoform. Because a major function of Akr1b7 is to detoxify lipid peroxidation, the PXR-, CAR-, and LXR-controlled regulatory network of Akr1b7 may have contributed to alleviate toxicity associated with lipid peroxidation.
... However, its potential role in reproductive function is not understood. Recently, high levels of MVDP messenger RNA (mRNA) were detected in the adrenal cortex, suggesting that aldose reductase-like proteins might be associated with steroidogenic activity (6). Until now, the hormonal requirements for MVDP gene expres-sion in the adrenal cortex have not been investigated. ...
... The MVDP gene provides a unique model to dissect the regulatory elements controlling cell type-specific and hormonally regulated expression in male genital tract and endocrine tissues, i.e. the vas deferens and the adrenals (2,4,6). Transgenic mice have been used to define the in vivo DNA regions responsible for vas deferens and adrenal-specific expression. ...
The MVDP (mouse vas deferens protein) gene, which encodes an aldose reductase-like enzyme, is mainly expressed in vas deferens epithelium and adrenal cortex. Vas deferens MVDP gene transcription was known to be under androgenic control, we now have evidence for androgen and probable ACTH responsiveness of the MVDP gene in the adrenal. To analyze the role of potential regulatory regions in hormonal, developmental, and tissue-specific aspects of MVDP regulation, we generated transgenic mice harboring MVDP-CAT fusion genes. The constructs carried either -1.8 or -0.5 kb 5'-flanking sequence attached to the chloramphenicol acetyltransferase gene in presence or absence of a 3.5-kb intragenic fragment in a downstream position. We show that at least two regions ensure proper gene regulation in vivo. The first, located within the 1.8-kb promoter fragment, directs tissue specificity; positive elements necessary for vas deferens and adrenal expression lay within positions -1804 to -510 and -510 to +41, respectively. The second, located within the 3.5-kb intragenic fragment spanning intron 1 to intron 2, increases percentage of expressing lines and behaves as a vas deferens-specific enhancer. Hormonal and developmental control of transgenes closely parallel endogenous gene regulation. Androgen and ACTH responsiveness in adrenals is conferred by 0.5-kb promoter, whereas in vas deferens, full androgenic response of the 1.8-kb promoter required the 3.5-kb intragenic fragment. Thus, vas deferens and adrenals use distinct cis-acting elements to direct and regulate the expression of the MVDP gene.
... In accordance with the observed decline in protein carbonylation in treated mice, we noted a reduction in the abundance of proteins involved in carbonyl metabolism subse-quent to the essential oil treatments. Remarkably, the aldose reductase-related protein 2 exhibited reduced levels after the thyme-derived essential oil treatment, an enzyme known to be implicated in the pathogenesis of diabetic complications [55]. Furthermore, alcohol dehydrogenase [NADP (+)], responsible for catalyzing NADPH-dependent reductions in carbonyls into their corresponding alcohols [56], and the mitochondrial delta-1-pyrroline-5-carboxylate dehydrogenase, responsible for facilitating the second step of the proline degradation pathway [57], manifested decreased abundances subsequent to the administration of the oregano-derived essential oil. ...
Essential oils sourced from herbs commonly used in the Mediterranean diet have demonstrated advantageous attributes as nutraceuticals and prebiotics within a model of severe cardiometabolic disorder. The primary objective of this study was to assess the influences exerted by essential oils derived from thyme (Thymus vulgaris) and oregano (Origanum vulgare) via a comprehensive multi-omics approach within a gnotobiotic murine model featuring colonic microbiota acquired from patients diagnosed with coronary artery disease (CAD) and type-2 diabetes mellitus (T2DM). Our findings demonstrated prebiotic and potential antioxidant effects elicited by these essential oils. We observed a substantial increase in the relative abundance of the Lactobacillus genus in the gut microbiota, accompanied by higher levels of short-chain fatty acids and a reduction in trimethylamine N-oxide levels and protein oxidation in the plasma. Moreover, functional enrichment analysis of the cardiac tissue proteome unveiled an over-representation of pathways related to mitochondrial function, oxidative stress, and cardiac contraction. These findings provide compelling evidence of the prebiotic and antioxidant actions of thyme- and oregano-derived essential oils, which extend to cardiac function. These results encourage further investigation into the promising utility of essential oils derived from herbs commonly used in the Mediterranean diet as potential nutraceutical interventions for mitigating chronic diseases linked to CAD and T2DM.
... Le gène akr1b7, codant une protéine appartenant à la famille des aldo-céto réductases réduisant l'isocaproaldéhyde (Lefrancois- Martinez et al., 1999), a été initialement décrit comme un marqueur spécifique du canal déférent de souris (Taragnat et al., 1986) dans lequel, son expression est dépendante des androgènes. Son expression a été ensuite mise en évidence dans la zone fasciculée du cortex surrénalien Lau et al., 1995) sous le contrôle de l'hormone hypophysaire ACTH . Ce contrôle mobilise la voie de l'AMPc comme le montre l'utilisation de la lignée cellulaire corticosurrénalienne Y1 . ...
The use of the adrenocortical specific promoter in the mouse has enabled to establish new cell lines with zona fasciculata adrenocortical cell phenotype (ATC cell line) and to test the capacity of gene candidates in the development of adrenocortical tumors.
... The role of AKR1B7, which acts as a major reductase for isocaproaldehyde, a product of side-chain cleavage of cholesterol generated during steroidogenesis, suggests a similar pattern of expression. Studies from our own and other laboratories have demonstrated that AKR1B7 is expressed in adrenals and ovaries (Lau et al. 1995, Aigueperse et al. 1999, Brockstedt et al. 2000. The bulk of steroid hormone synthesis in the testis is confined to the Leydig cells. ...
The akr1b7 gene encodes an aldose reductase-like protein that is responsible for detoxifying isocaproaldehyde generated by the conversion of cholesterol to pregnenolone. The regulation of gene expression by human chorionic gonadotropin (hCG) was first investigated in the MA-10 Leydig tumor cell line. The akr1b7 gene was constitutively expressed and accumulation of its mRNA was increased in a dose- and time-dependent manner by treatment with hCG. akr1b7 mRNA accumulation was sharply increased in the presence of 0·25 nM hCG and it reached a fivefold increase within 2 h. AKR1B7 protein accumulation was delayed compared with that of the corresponding mRNA. In agreement, hCG significantly increased the levels of mRNA and protein of akr1b7 in primary cultures of adult mouse Leydig cells, thus suggesting that LH potentially regulates akr1b7 gene expression in vivo. Expression of akr1b7 was developmentally regulated in the testis. Unexpectedly, levels of akr1b7 mRNA increased from embryonic day 15 to the day of birth and declined until adulthood while AKR1B7 protein levels followed an inverse pattern, suggesting an important role for translational mechanisms.
... AKR1B7, also known as the mouse vas deferens protein, is expressed in the vas deferens [71], adrenal cortex [72,73], and the ovarian theca cells [74]. It is also expressed in the intestine, eye, kidney and liver in lesser quantities [75]. ...
Extensive research has shown that increased production of reactive oxygen species (ROS) results in tissue injury under a variety of pathological conditions and chronic degenerative diseases. While ROS are highly reactive and can incite significant injury, polyunsaturated lipids in membranes and lipoproteins are their main targets. ROS-triggered lipid peroxidation reactions generate a range of reactive carbonyl species (RCS), and these RCS spread and amplify ROS-related injury. Several RCS generated in oxidizing lipids, such as 4-hydroxy trans-2-nonenal (HNE), 4-oxo-2-(E)-nonenal (ONE), acrolein, malondialdehyde (MDA) and phospholipid aldehydes have been shown to be produced under conditions of oxidative stress and contribute to tissue injury and dysfunction by depleting glutathione and other reductants leading to the modification of proteins, lipids, and DNA. To prevent tissue injury, these RCS are metabolized by several oxidoreductases, including members of the aldo–keto reductase (AKR) superfamily, aldehyde dehydrogenases (ALDHs), and alcohol dehydrogenases (ADHs). Metabolism via these enzymes results in RCS inactivation and detoxification, although under some conditions, it can also lead to the generation of signaling molecules that trigger adaptive responses. Metabolic transformation and detoxification of RCS by oxidoreductases prevent indiscriminate ROS toxicity, while at the same time, preserving ROS signaling. A better understanding of RCS metabolism by oxidoreductases could lead to the development of novel therapeutic interventions to decrease oxidative injury in several disease states and to enhance resistance to ROS-induced toxicity.
... The present data indicates for the first time that Akr1b3 is expressed mainly in brain. Akr1b7 was expressed mainly in intestine, which confirms an earlier published result that was obtained using the RNase protection assay (Lau et al., 1995). In the present study, Akr1b8 was located mainly in testes and stomach. ...
Aldo-keto reductases (Akrs) are a conserved group of NAD(P)H-dependent oxido-reductase enzymes. This study provides a comprehensive examination of the tissue distribution of the 16 substrate metabolizing Akrs in mice, their expression during development and whether they are altered by chemicals that activate distinct transcriptional factor pathways. Akr1c6, 1c14, 1c20, and 1c22 are primarily present in liver; Akr1a4, 1c18, 1c21, and 7a5 in kidney; Akr1d1 in liver and kidney; Akr1b7 in small intestine; Akr1b3 and Akr1e1 in brain; Akr1b8 in testes; Akr1c14 is in ovaries; whereas Akrs1c12, 1c13, and 1c19 are expressed in numerous tissues. Liver expression of Akr1d1 and Akr1c genes are lowest during pre-natal and post-natal development. However, by 20 days of age, liver Akr1d1 increases 120-fold, and Akr1c mRNAs increase as much as 5-fold (Akr1c19) to 1000-fold (Akr1c6). Treatment of mice with chemical activators of transcription factors CAR, PXR, and Nrf2 alters liver mRNAs of Akrs. Specifically, CAR activation by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) increases mRNAs of Akr1b7, Akr1c6, Akr1c19, and Akr1d1; whereas PXR activation by 5-pregnennenolone-16α-carbonitrile (PCN) increase the mRNA of Akrqb7 and suppresses mRNAs of Akr1c13 and Akr1c20. The Nrf2 activator 2-cyano-3,12 dioxooleana-1,9 dien-28-imidazolide (CDDO-Im) induces mRNAs of Akr1c6 and Akr1c19. Moreover, Nrf2-null and Nrf2 over-expressing mice demonstrate that this induction is Nrf2-dependent. This study also examined the ontogeny of Akr mRNAs and the mRNAs of Akr1c6, 1c14, 1c19, 1c20 and 1d1 increase after birth and continue to increase to adulthood.
... Although a recent study indicated that the PGFS activity of Akr1b3 can influence adipogenesis in 3T3-L1 cells (18), no defect in adipose tissue homeostasis was reported in Akr1b3deficient mice (19). Contrasting with the ubiquitous Akr1b3 protein, Akr1b7 isoform is expressed in a restricted set of tissues (i.e., adrenal glands, reproductive organs, and intestine) (20)(21)(22)(23) and was found more recently in the adipose tissue (24). Interestingly, in fat depots, Akr1b7 expression is limited to the preadipocyteenriched stromal fraction and downregulated during the differentiation into adipocytes (24). ...
Negative regulators of white adipose tissue (WAT) expansion are poorly documented in vivo. Prostaglandin F(2α) (PGF(2α)) is a potent antiadipogenic factor in cultured preadipocytes, but evidence for its involvement in physiological context is lacking. We previously reported that Akr1b7, an aldo-keto reductase enriched in adipose stromal vascular fraction but absent from mature adipocytes, has antiadipogenic properties possibly supported by PGF(2α) synthase activity. To test whether lack of Akr1b7 could influence WAT homeostasis in vivo, we generated Akr1b7(-/-) mice in 129/Sv background. Akr1b7(-/-) mice displayed excessive basal adiposity resulting from adipocyte hyperplasia/hypertrophy and exhibited greater sensitivity to diet-induced obesity. Following adipose enlargement and irrespective of the diet, they developed liver steatosis and progressive insulin resistance. Akr1b7 loss was associated with decreased PGF(2α) WAT contents. Cloprostenol (PGF(2α) agonist) administration to Akr1b7(-/-) mice normalized WAT expansion by affecting both de novo adipocyte differentiation and size. Treatment of 3T3-L1 adipocytes and Akr1b7(-/-) mice with cloprostenol suggested that decreased adipocyte size resulted from inhibition of lipogenic gene expression. Hence, Akr1b7 is a major regulator of WAT development through at least two PGF(2α)-dependent mechanisms: inhibition of adipogenesis and lipogenesis. These findings provide molecular rationale to explore the status of aldo-keto reductases in dysregulations of adipose tissue homeostasis.
... FR-1 mRNA is abundant in mouse heart, ovary, testis, and adrenal gland, while MVDP mRNA is overexpressed in vas deferens and adrenal gland. 58 In contrast, HARP mRNA is high in human lung, small intestine, colon, and stomach and absent in heart, ovary, testis, and adrenal gland ( Fig. 6 and data not shown). These data suggest that different AR family proteins possibly have different functions in different organs. ...
The multistep process of liver carcinogenesis involves various genetic and phenotypic alterations. To identify genes whose expression is increased during hepatocarcinogenesis, differential-display polymerase chain reaction (DD-PCR) was used to examine differences in the mRNA composition of hepatocellular carcinoma (HCC) versus normal liver (nontumor) tissues. This approach identified 67 cDNAs that were preferentially expressed in HCC tissue. When these cDNAs were analyzed by reverse-Northern analysis, five were reproducibly expressed at high levels in HCC. Interestingly, Northern blot analysis revealed that one of the genes showed significantly increased mRNA levels in all five tested tumor samples, while its mRNA level in the nontumor samples was minimal. BLAST analysis revealed that this gene has high sequence identity with the genes from aldo-keto reductase family of proteins including the mouse fibroblast growth factor-induced gene (FR-1) (80% identity), mouse vas deferens protein (MVDP) (76%), and human aldose reductase (AR) (62%). Expression of this novel AR-related protein in all five tested HCCs suggests that this protein may play an important role in liver carcinogenesis.
... The tissue distributions of the three mouse AKR1B members were previously reported by different laboratories, who worked on different animal strains (Martinez et al., 1990;Donohue et al., 1994;Gui et al., 1995;Lau et al., 1995). To compare the distribution patterns of the three mouse AKR1B homologs, we examined their mRNA levels in the same strain of C57/B6 adult mice, using a semi-quantitative RT-Brought to you by | Stanford Libraries Authenticated Download Date | 1/6/18 11:10 PM Table 2 Kinetic parameters of AKR1B3, AKR1B7, AKR1B8, AKR1B1, and AKR1B10. ...
Aldo-keto reductase family 1 member B10 (AKR1B10), over-expressed in multiple human cancers, might be implicated in cancer development and progression via detoxifying cytotoxic carbonyls and regulating fatty acid synthesis. In the present study, we investigated the ortholog of AKR1B10 in mice, an ideal modeling organism greatly contributing to human disease investigations. In the mouse, there are three aldo-keto reductase family 1 subfamily B (AKR1B) members, i.e., AKR1B3, AKR1B7, and AKR1B8. Among them, AKR1B8 has the highest similarity to human AKR1B10 in terms of amino acid sequence, computer-modeled structures, substrate spectra and specificity, and tissue distribution. More importantly, similar to human AKR1B10, mouse AKR1B8 associates with murine acetyl-CoA carboxylase-α and mediates fatty acid synthesis in colon cancer cells. Taken together, our data suggest that murine AKR1B8 is the ortholog of human AKR1B10.
... Neither 1B7 nor 1B8 is a homolog of the human 1B10, according to their tissue distribution and catalytic properties (Cao et al., 1998). 1B7 has very low enzymatic activity and limited tissue distribution (it is abundant in the adrenal gland) (Lau et al., 1995; Lefrancois-Martinez et al., 1999 ). The FR-1 protein (1B8) has very similar catalytic properties to AR, but it has a higher K m for DL-glyceraldehyde and lacks activity with glucose (Srivastava et al., 1998b). ...
Aldehydes such as acrolein are ubiquitous pollutants present in automobile exhaust, cigarette, wood, and coal smoke. Such aldehydes are also constituents of several food substances and are present in drinking water, irrigation canals, and effluents from manufacturing plants. Oral intake represents the most significant source of exposure to acrolein and related aldehydes. To study the effects of short-term oral exposure to acrolein on lipoprotein levels and metabolism, adult mice were gavage-fed 0.1 to 5 mg acrolein/kg bwt and changes in plasma lipoproteins were assessed. Changes in hepatic gene expression related to lipid metabolism and cytokines were examined by qRT-PCR analysis. Acrolein feeding did not affect body weight, blood urea nitrogen, plasma creatinine, electrolytes, cytokines or liver enzymes, but increased plasma cholesterol and triglycerides. Similar results were obtained with apoE-null mice. Plasma lipoproteins from acrolein-fed mice showed altered electrophoretic mobility on agarose gels. Chromatographic analysis revealed elevated VLDL cholesterol, phospholipids, and triglycerides levels with little change in LDL or HDL. NMR analysis indicated shifts from small to large VLDL and from large to medium-small LDL with no change in the size of HDL particles. Increased plasma VLDL was associated with a significant decrease in post-heparin plasma hepatic lipase activity and a decrease in hepatic expression of hepatic lipase. These observations suggest that oral exposure to acrolein could induce or exacerbate systemic dyslipidemia and thereby contribute to cardiovascular disease risk.
... Indeed, we and others have identified AKR1B7 [8,9] and AKR1B8 [10] as two other murine members of the family and AKR1B10 has been isolated in human small intestine [11,12]. AKR1B7 presents two interesting characteristics: a tissuerestricted expression (vas deferens, steroidogenic tissues, small intestine and adipose tissue) and a tissue-specific hormonal regulation13141516171819. In the murine adrenal cortex, the Akr1b7 gene is up-regulated at the transcriptional level by ACTH [20,21,22] and acts as a major reductase for isocaproaldehyde formed during steroidogenesis [23]. ...
Prostaglandin F(2alpha) (PGF(2alpha)), represses ovarian steroidogenesis and initiates parturition in mammals but its impact on adrenal gland is unknown. Prostaglandins biosynthesis depends on the sequential action of upstream cyclooxygenases (COX) and terminal synthases but no PGF(2alpha) synthases (PGFS) were functionally identified in mammalian cells. In vitro, the most efficient mammalian PGFS belong to aldo-keto reductase 1B (AKR1B) family. The adrenal gland is a major site of AKR1B expression in both human (AKR1B1) and mouse (AKR1B3, AKR1B7). Thus, we examined the PGF(2alpha) biosynthetic pathway and its functional impact on both cortical and medullary zones. Both compartments produced PGF(2alpha) but expressed different biosynthetic isozymes. In chromaffin cells, PGF(2alpha) secretion appeared constitutive and correlated to continuous expression of COX1 and AKR1B3. In steroidogenic cells, PGF(2alpha) secretion was stimulated by adrenocorticotropic hormone (ACTH) and correlated to ACTH-responsiveness of both COX2 and AKR1B7/B1. The pivotal role of AKR1B7 in ACTH-induced PGF(2alpha) release and functional coupling with COX2 was demonstrated using over- and down-expression in cell lines. PGF(2alpha) receptor was only detected in chromaffin cells, making medulla the primary target of PGF(2alpha) action. By comparing PGF(2alpha)-responsiveness of isolated cells and whole adrenal cultures, we demonstrated that PGF(2alpha) repressed glucocorticoid secretion by an indirect mechanism involving a decrease in catecholamine release which in turn decreased adrenal steroidogenesis. PGF(2alpha) may be regarded as a negative autocrine/paracrine regulator within a novel intra-adrenal feedback loop. The coordinated cell-specific regulation of COX2 and AKR1B7 ensures the generation of this stress-induced corticostatic signal.
... Aldo-keto reductase 1B7 (Akr1b7) is a mouse member of the aldose reductase family (AKR1B). It is highly expressed in the vas deferens, in the adrenal cortex where it is involved in the detoxification of harmful aldehydes generated by steroidogenic cytochrome P450 activities (Lefrancois-Martinez et al., 1999) and to a lesser extent in the gonads (Lau et al., 1995; Martinez et al., 2001; Taragnat et al., 1988). Transcription of akr1b7 is acutely regulated by ACTH in the adrenal cortex (Aigueperse et al., 1999), androgens in the vas deferens (Taragnat et al., 1988) and LH in the gonads (Baron et al., 2003; Brockstedt et al., 2000; Martinez et al., 2001). ...
The Cre-loxP system combined with gene targeting strategies has proven to be very useful for gene inactivation in specific tissues and/or cell types. To achieve adrenal cortex specific recombination in vivo, we used a 0.5-kb fragment of the 5'-flanking region of the akr1b7 gene to drive Cre expression in adrenocortical cells. The resulting 0.5 akr1b7-Cre mice express Cre in all steroidogenic zones of the adrenal cortex but not in the gonads. Although recombination of the ROSA26R reporter locus was not observed in all cortical cells, we provide evidence that Cre is expressed in all the cells of the cortex in adult mice. In addition, Cre activity was found in collecting ducts and maturing glomeruli of the kidney. This line is the first to show specific Cre expression in the adrenal cortex in the absence of Cre expression in the gonads. This transgene thus provides a valuable tool for specific gene recombination in the adrenal cortex and kidney.
... Neither 1B7 nor 1B8 is a homolog of the human 1B10, according to their tissue distribution and catalytic properties (Cao et al., 1998). 1B7 has very low enzymatic activity and limited tissue distribution (it is abundant in the adrenal gland) (Lau et al., 1995; Lefrancois-Martinez et al., 1999 ). The FR-1 protein (1B8) has very similar catalytic properties to AR, but it has a higher K m for DL-glyceraldehyde and lacks activity with glucose (Srivastava et al., 1998b). ...
The aldo-keto reductase (AKR) superfamily comprises enzymes that catalyze redox transformations involved in biosynthesis, intermediary metabolism, and detoxification. Substrates of AKRs include glucose, steroids, glycosylation end-products, lipid peroxidation products, and environmental pollutants. These proteins adopt a (beta/alpha)(8) barrel structural motif interrupted by a number of extraneous loops and helixes that vary between proteins and bring structural identity to individual families. The human AKR family differs from the rodent families. Due to their broad substrate specificity, AKRs play an important role in the phase II detoxification of a large number of pharmaceuticals, drugs, and xenobiotics.
... However, FR-1 mRNA and hepatoma-derived AR-like protein were found in new-born mouse liver [14] and in embryonic rat liver, respectively. FR-I mRNA and MVDP mRNA were found in mouse embryos [45], and an AR was found to be expressed in hereditary hepatitis and hepatomas of Long Evans cinnamon-coloured rats [46]. Rat lens AR was expressed distinctly in all tissues investigated, but not in serum, in agreement with recently published results [47]. ...
A protein detected in N-methyl-N-nitrosourea-initiated rat hepatomas by two-dimensional electrophoresis at 35 kDa/pI 7.4 was identified in a previous study by internal amino acid micro sequencing as an aldose-reductase-like protein [Zeindl-Eberhart, E., Jungblut, P. R., Otto, A. & Rabes, H. M. (1994) Identification of tumor-associated protein variants during rat hepatocarcinogenesis, J. Biol. Chem. 269, 14589-14594]. Two-dimensional electrophoresis of rat lens proteins revealed a spot at 37 kDa/pI 6.8 that showed a high degree of identity (98.5%) with rat lens aldose reductase after amino acid sequencing and 80% sequence identity to the rat-hepatoma-derived aldose-reductase-like protein. This suggests that hepatoma-derived aldose-reductase-like protein and rat lens aldose reductase are related proteins encoded by different genes. A different expression profile of these proteins was found in various rat organs. Rat lens aldose reductase is present, in addition to in lens, in heart, brain, muscle, lung, duodenum, kidney, spleen and bone marrow, while the hepatoma-derived aldose-reductase-like protein is found preferentially in hepatomas and in embryonic liver. Though different in organ expression, an identical response was found for both proteins after stimulation with fibroblast growth factor-1 and after exposure to increased glucose concentrations. Since rat hepatoma-derived aldose-reductase-like protein is expressed in embryonic, but not in adult liver, it is assumed that it is expressed in hepatomas as a functionally active embryonal type of aldose reductase during hepatocarcinogenesis. Immunohistochemistry revealed that the hepatoma-derived aldose-reductase-like protein is expressed already in the preneoplastic stage of hepatocarcinogenesis and might potentially serve as a marker enzyme in early hepatic neoplasia.
... Most likely, a local gene duplication event in this region of the chromosome gave rise to these three genes. Previous studies have shown that the major expression patterns for Aldor1, Fgfrp and Avdp mRNAs are different [23], indicating that these three genes may have quite distinct functions. These genes also show different temporal expression and regulation during development and in adult tissues (data not shown). ...
Aldose reductase (AR), best known as the first enzyme in the polyol pathway of sugar metabolism, has been implicated in a wide variety of physiological functions and in the etiology of diabetic complications. We have determined the structures and chromosomal locations of the mouse AR gene (Aldor1) and of two genes highly homologous to Aldor1: the fibroblast growth factor regulated protein gene (Fgfrp) and the androgen regulated vas deferens protein gene (Avdp). The number of introns and their locations in the mouse Aldor1 gene are identical to those of rat and human AR genes and also to those of Fgfrp and Avdp. Mouse Aldor1 gene was found to be located near the Cald1 (Caldesmon) and Ptn (Pleiotropin) loci at the proximal end of chromosome 6. The closely related genes Fgfrp and Avdp were also mapped in this region of the chromosome, suggesting that these three genes may have arisen by a gene duplication event.
... The mouse vas deferens protein (MVDP) belongs to the aldo-keto reductase superfamily ). It has recently been shown that, in addition to the vas deferens, MVDP was expressed in adrenal glands from various rodents (Lau et al, 1995;Aigueperse et al, 1999) and that, in adrenocortical cells, its preferential substrates were isocaproaldehyde (a product of side-chain cleavage of cholesterol) and 4-hydroxynonenal (which results from lipid peroxidation), suggesting a role for MVDP in cell detoxication (Lefrançois-Martinez et al, 1999). In the vas deferens, the protein and its mRNA are triggered by testosterone. ...
We used cultured vas deferens epithelial cells (VDECs) as a model system to determine the conditions that allow mouse vas deferens protein (MVDP) gene expression and acquisition of androgen responsiveness. On the basis of Northern blot analysis, the mvdp gene is constitutively expressed at very low levels in prepubertal VDECs grown on collagen-coated plastic or on microporous membrane inserts. In the presence of dihydrotestosterone (DHT), mvdp messenger RNA levels dramatically increased in cells cultured on microporous membrane inserts and stayed unchanged in cells grown on matrix-coated plastic. Epithelial cells derived from fetal vas deferens were able to synthesize MVDP in response to DHT, and the presence of fetal mesenchymal cells did not influence MVDP production. Providing the cells with a culture procedure that permits access to the basolateral membranes and caters to the polarity requirements of the cell is a prerequisite for androgen induction of MVDP gene expression. The results also point to a role for epidermal growth factor, insulin, and tyrosine kinase activity in mediating the action of androgen on mvdp gene expression. In vivo studies show that the first expression of the mvdp gene between 5 and 7 days postpartum is not associated with major structural changes in the epithelium. The acquisition of a mature phenotype by epithelial and peritubular contractile cells, between 10 and 20 days, correlates with androgen dependency of the mvdp gene. We propose that cell differentiation and polarization on a matrix-coated microporous membrane reproduces some of the events that are necessary for acquisition of androgenic responsiveness of the mvdp gene during postnatal development.
... Although MVDP represents a major secretory component of the vas deferens, this expression profile is restricted to the mouse species (29). More recently, MVDP was shown to be highly expressed in the adrenals from various rodents under the control of the hypothalamo-pituitary-adrenal axis (30,31). This evolutionary conserved expression of MVDP is restricted to the zona fasciculata and, in human and murine adrenocortical cells, the MVDP gene is induced by cAMP at both mRNA and protein levels (31). ...
The MVDP (mouse vas deferens protein) gene encodes an aldose reductase-like protein (AKR1B7) that is responsible for detoxifying isocaproaldehyde generated by steroidogenesis. In adrenocortical cell cultures, hormonal regulation of MVDP gene occurs through the cAMP pathway. We show that in adrenals, the pituitary hormone ACTH regulates MVDP gene expression in a coordinate fashion with steroidogenic genes. Cell transfection and DNA-binding studies were used to investigate the molecular mechanisms underlying MVDP gene regulation in Y1 adrenocortical cells. Progressive deletions of upstream regulatory regions identified a -121/+41 fragment that was sufficient for basal and cAMP-mediated transcriptional activities. Gel shift assays showed that CTF1/nuclear factor 1 (NF1), CCAAT enhancer binding protein-ss (C/EBPss), and selective promoter factor 1 (Sp1) factors bound to cis-acting elements at positions -76, -61, and -52, respectively. We report that the cell-specific steroidogenic factor-1 (SF-1) interacts specifically with a novel regulatory element located in the downstream half-site of the proximal androgen response element (AREp) at position -102. Functional analysis of SF-1 and NF1 sites in the -121/+41 promoter showed that mutation of one of them decreases both constitutive and forskolin-stimulated promoter activity without affecting the fold induction (forskolin stimulated/basal). Individual mutations of C/EBP and Sp1 sites resulted in a loss of more than 50% of the cAMP-dependent induction. When both sites were mutated simultaneously, cAMP responsiveness was nearly abolished. Thus, in adrenocortical cells, both SF-1 and NF1 are required for high expression of the MVDP promoter while Sp1 and C/EBPss functionally interact in an additive manner to mediate cAMP-dependent regulation. Furthermore, we report that MVDP gene regulation is impaired in stably transfected Y1 clones expressing DAX-1. Taken together, our findings suggest that detoxifying enzymes of the aldose reductase family may constitute new potential targets for regulators of adrenal and gonadal differentiation and function, e.g. SF-1 and DAX-1.
... The gene expression spectrum is not the same. MVDP gene was primarily expressed in the vas deferens and the adrenal gland [30][31][32][33][34][35][36][37] . AR, was different. ...
To prepare and characterize polyclonal antibodies against aldose reductase-like (ARL-1) protein.
ARL-1 gene was inserted into the E. coli expression vector pGEX-4T-1(His)(6)C and vector pQE-30. Recombinant ARL-1 proteins named ARL-(His)(6) and ARL-GST were expressed. They were purified by affinity chromatography. Sera from domestic rabbits immunized with ARL-(His) (6) were purified by CNBr-activated sepharose 4B coupled ARL-GST. Polyclonal antibodies were detected by Western blotting.
Recombinant proteins of ARL-(His)(6) with molecular weight of 35.7 KD and ARL-GST with molecular weight of 60.8 KD were highly expressed. The expression levels of ARL-GST and ARL-(His)(6) were 15.1 % and 27.7 % among total bacteria proteins, respectively. They were soluble, predominantly in supernatant. After purification by non-denatured way, SDS-PAGE showed one band. In the course of polyclonal antibodies purification, only one elution peak could be seen. Western blotting showed positive signals in the two purified proteins and the bacteria transformed with pGEX-4T-1(His) (6) C-ARL and pQE-30-ARL individually.
Polyclonal antibodies are purified and highly specific against ARL-1 protein. ARL-GST and ARL-(His) (6) are highly expressed and purified.
Polycystic ovary syndrome (PCOS) is a complex gynecological endocrine and metabolic disease. Orlistat as a lipase inhibitor may improve the pathological characteristics of PCOS and is the sole antiobesity agent available in various countries. In this study, the PCOS rat models were established using letrozole and high-fat diet. Tandem Mass Tag labeling peptide coupled with liquid chromatography with tandem mass spectrometry (LC-MS/MS) approach was employed to investigate the differentially expressed ovarian proteins (DEPs) in the PCOS and control rats for the effect of PCOS, and in the PCOS and orlistat-treated PCOS rats for the effect of orlistat in PCOS. The orlistat attenuated the body weight gain; decreased the levels of testosterone, luteinizing hormone, a ratio of luteinizing/follicle-stimulating hormones; increased the level of estradiol; and recovered the estrous cycle in PCOS rats. In addition, 795 and 119 DEPs were found in PCOS and orlistat-treated PCOS groups, respectively. Based on the Gene Ontology and Kyoto Encyclopedia of Gene and Genomes pathway analysis of DEPs, orlistat restored the disturbed metabolism of linoleic acid, arachidonic acid, galactose, and glycerolipids, and then improved the chronic inflammation in PCOS rats. This study analyzed the ovarian proteome of orlistat-treated PCOS rats and identified targeted proteins, which explored the pathogenesis of PCOS and the potential effects of orlistat in PCOS rats.
Aldo-keto reductases (AKRs) are distributed in three families and multiple subfamilies in mammals. The mouse Akr1b3 gene is clearly orthologous to human AKR1B1, both coding for aldose reductase, and their gene products show similar tissue distribution, regulation by osmotic stress and kinetic properties. In contrast, no unambiguous orthologs of human AKR1B10 and AKR1B15.1 have been identified in rodents. Although two more AKRs, AKR1B7 and AKR1B8, have been identified and characterized in mouse, none of them seems to exhibit properties similar to the human AKRs. Recently, a novel mouse AKR gene, Akr1b16, was annotated and the respective gene product, AKR1B16 (sharing 83% and 80% amino acid sequence identity with AKR1B10 and AKR1B15.1, respectively), was expressed as insoluble and inactive protein in a bacterial expression system. Here we describe the expression and purification of a soluble and enzymatically active AKR1B16 from E. coli using three chaperone systems. A structural model of AKR1B16 allowed the estimation of its active-site pocket volume, which was much wider (402 ų) than those of AKR1B10 (279 ų) and AKR1B15.1 (60 ų). AKR1B16 reduced aliphatic and aromatic carbonyl compounds, using NADPH as a cofactor, with moderate or low activity (highest kcat values around 5 min⁻¹). The best substrate for the enzyme was pyridine-3-aldehyde. AKR1B16 showed poor inhibition with classical AKR inhibitors, tolrestat being the most potent. Kinetics and inhibition properties resemble those of rat AKR1B17 but differ from those of the human enzymes. In addition, AKR1B16 catalyzed the oxidation of 17β-hydroxysteroids in a NADP⁺-dependent manner. These results, together with a phylogenetic analysis, suggest that mouse AKR1B16 is an ortholog of rat AKR1B17, but not of human AKR1B10 or AKR1B15.1. These human enzymes have no counterpart in the murine species, which is evidenced by forming a separate cluster in the phylogenetic tree and by their unique activity with retinaldehyde.
White adipose tissue development is tightly regulated by pro-and anti-adipogenic factors. In obesity, its increased development leads to many metabolic complications. To date, little is known about the factors that control negatively its growth. In this context, the laboratory has focused researches on the murine aldose reductase Akr1b7 role in white adipose tissue. Akr1b7 is expressed in stromal vascular fraction of white adipose tissue and exhibits an anti-adipogenic action on a preadipocyte cell line. Generation and study of Akr1b7-/- knockout mice allows us to demonstrate that lack of Akr1b7 leads to adipose tissue expansion due to hypertrophy and hyperplasia of adipose cells associated to insulin resistance. Akr1b7-/- mice are not hyperphagic but show reduced basal metabolic rate. This phenotype confirms Akr1b7 involvement in adipose tissue physiology. Akr1b7 regulates development of adipose tissue by a PGF2α-dependent inhibition of both adipogenesis and lipogenesis. On the other hand, we have developed a transgenic murine model over-expressing the human aldose reductase AKR1B1 in adipose tissue. Against all odds and contrary to Akr1b7, this model shows a pro-adipogenic effect of AKR1B1. These in vivo data reveal new and opposed activities of different aldose reductase isoforms and open new avenues to understand the mecanisms regulating fat homeostasis and its disturbances.
The murine AKR1B7 protein belongs to the aldose reductase family. By its tissuerestricted expression and its tissue-specific hormonal regulation, it is a good model for the study of aldose reductase physiological functions. On the one hand, these doctoral work demonstrated ex vivo the role of murine and human aldose reductase prostaglandin F synthase activity in the adrenal gland. We highlighted a new feed-back loop between the cortex and the medulla using prostaglandin F2alpha as paracrine and/or autocrine signal. On the other hand, to achieve adrenal cortex specific inactivation of genes, we have developed the first transgenic mouse line showing specific Cre recombinase expression in this tissue. This line is a very powerful tool to create animal models of adrenal tumors.
We have identified a novel human protein that is highly homologous to aldose reductase (AR). This protein, which we called
ARL-1, consists of 316 amino acids, the same size as AR, and its amino acid sequence is 71% identical to that of AR. It is
more closely related to the AR-like proteins such as mouse vas deferens protein, fibroblast growth factor-regulated protein,
and Chinese hamster ovary reductase, with 81, 82, and 83%, respectively, of its amino acid sequence identical to the amino
acid sequence of these proteins. The cDNA of ARL-1 was expressed in Escherichia coli to obtain recombinant protein for characterization of its enzymatic activities. For comparison, the cDNA of human AR was
also expressed in E. coli and analyzed in parallel. These two enzymes differ in their pH optima and salt requirement, but they act on a similar spectrum
of substrates. Similar to AR, ARL-1 can efficiently reduce aliphatic and aromatic aldehydes, and it is less active on hexoses.
While AR mRNA is found in most tissues studied,ARL-1 is primarily expressed in the small intestines and in the colon, with a low level of its mRNA in the liver. The ability of
ARL-1 to reduce various aldehydes and the locations of expression of this gene suggest that it may be responsible for detoxification
of reactive aldehydes in the digested food before the nutrients are passed on to other organs. Interestingly, ARL-1 and ARare overexpressed in some liver cancers, but it is not clear if they contribute to the pathogenesis of this disease.
The MVDP (mouse vas deferens protein) gene encodes an aldose reductase-like protein (AKR1B7) that is responsible for detoxifying isocaproaldehyde generated by steroidogenesis. In adrenocortical cell cultures, hormonal regulation of MVDP gene occurs through the cAMP pathway. We show that in adrenals, the pituitary hormone ACTH regulates MVDP gene expression in a coordinate fashion with steroidogenic genes. Cell transfection and DNA-binding studies were used to investigate the molecular mechanisms underlying MVDP gene regulation in Y1 adrenocortical cells. Progressive deletions of upstream regulatory regions identified a -121/+41 fragment that was sufficient for basal and cAMP-mediated transcriptional activities. Gel shift assays showed that CTF1/nuclear factor 1 (NF1), CCAAT enhancer binding protein-beta (C/EBP beta), and selective promoter factor 1 (Sp1) factors bound to cis-acting elements at positions -76, -61, and -52, respectively. We report that the cell-specific steroidogenic factor-1 (SF-1) interacts specifically with a novel regulatory element located in the downstream half-site of the proximal androgen response element (AREp) at position -102. Functional analysis of SF-1 and NF1 sites in the -121/+41 promoter showed that mutation of one of them decreases both constitutive and forskolin-stimulated promoter activity without affecting the fold induction (forskolin stimulated/basal). Individual mutations of C/EBP and Spl sites resulted in a loss of more than 50% of the cAMP-dependent induction. When both sites were mutated simultaneously, cAMP responsiveness was nearly abolished. Thus, in adrenocortical cells, both SF-1 and NF1 are required for high expression of the MVDP promoter while Spl and C/EBP beta functionally interact in an additive manner to mediate cAMP-dependent regulation. Furthermore, we report that MVDP gene regulation is impaired in stably transfected Y1 clones expressing DAX-1. Taken together, our findings suggest that detoxifying enzymes of the aldose reductase family may constitute new potential targets for regulators of adrenal and gonadal differentiation and function, e.g. SF-1 and DAX-1.
The aldose reductase (AR; human AKR1B1/mouse Akr1b3) has been the focus of many research because of its role in diabetic complications. The starting point of these alterations is the massive entry of glucose in polyol pathway where it is converted into sorbitol by this enzyme. However, the issue of AR function in non-diabetic condition remains unresolved. AR-like enzymes (AKR1B10, Akr1b7, and Akr1b8) are highly related isoforms often co-expressed with bona fide AR, making functional analysis of one or the other isoform a challenging task. AKR1B/Akr1b members share at least 65% protein identity and the general ability to reduce many redundant substrates such as aldehydes provided from lipid peroxidation, steroids and their by-products, and xenobiotics in vitro. Based on these properties, AKR1B/Akr1b are generally considered as detoxifying enzymes. Considering that divergences should be more informative than similarities to help understanding their physiological functions, we chose to review specific hallmarks of each human/mouse isoforms by focusing on tissue distribution and specific mechanisms of gene regulation. Indeed, although the AR shows ubiquitous expression, AR-like proteins exhibit tissue-specific patterns of expression. We focused on three organs where certain isoforms are enriched, the adrenal gland, enterohepatic, and adipose tissues and tried to connect recent enzymatic and regulation data with endocrine and metabolic functions of these organs. We presented recent mouse models showing unsuspected physiological functions in the regulation of glucido-lipidic metabolism and adipose tissue homeostasis. Beyond the widely accepted idea that AKR1B/Akr1b are detoxification enzymes, these recent reports provide growing evidences that they are able to modify or generate signal molecules. This conceptually shifts this class of enzymes from unenviable status of scavenger to upper class of messengers.
Aldo-keto reductase 1B7 (AKR1B7) is proposed to play a role in detoxification of by-products of lipid peroxidation. In this article, we show that activation of the nuclear receptor farnesoid X receptor (FXR) induces AKR1B7 expression in the liver and intestine, and reduces the levels of malondialdehyde (MDA), the end product of lipid peroxidation, in the intestine but not in the liver. To determine whether AKR1B7 regulates MDA levels in vivo, we overexpressed AKR1B7 in the liver. Overexpression of AKR1B7 in the liver had no effect on hepatic or plasma MDA levels. Interestingly, hepatic expression of AKR1B7 significantly lowered plasma glucose levels in both wild-type and diabetic db/db mice, which was associated with reduced hepatic gluconeogenesis. Hepatic expression of AKR1B7 also significantly lowered hepatic triglyceride and cholesterol levels in db/db mice. These data reveal a novel function for AKR1B7 in lipid and glucose metabolism and suggest that AKR1B7 may not play a role in detoxification of lipid peroxides in the liver. AKR1B7 may be a therapeutic target for treatment of fatty liver disease associated with diabetes mellitus.
NADP(H)-dependent cytosolic aldo-keto reductases (AKR) are mostly monomeric enzymes which fold into a typical (α/β)(8)-barrel structure. Substrate specificity and inhibitor selectivity are determined by interaction with residues located in three highly variable loops (A, B, and C). Based on sequence identity, AKR have been grouped into families, namely AKR1-AKR15, containing multiple subfamilies. Two human enzymes from the AKR1B subfamily (AKR1B1 and AKR1B10) are of special interest. AKR1B1 (aldose reductase) is related to secondary diabetic complications, while AKR1B10 is induced in cancer cells and is highly active with all-trans-retinaldehyde. Residues interacting with all-trans-retinaldehyde and differing between AKR1B1 and AKR1B10 are Leu125Lys and Val131Ala (loop A), Leu301Val, Ser303Gln, and Cys304Ser (loop C). Recently, we demonstrated the importance of Lys125 as a determinant of AKR1B10 specificity for retinoids. Residues 301 and 304 are also involved in interactions with substrates or inhibitors, and thus we checked their contribution to retinoid specificity. We also extended our study with retinoids to rodent members of the AKR1B subfamily: AKR1B3 (aldose reductase), AKR1B7 (mouse vas deferens protein), AKR1B8 (fibroblast-growth factor 1-regulated protein), and AKR1B9 (Chinese hamster ovary reductase), which were tested against all-trans isomers of retinaldehyde and retinol. All enzymes were active with retinaldehyde, but with k(cat) values (0.02-0.52 min(-1)) much lower than that of AKR1B10 (27 min(-1)). None of the enzymes showed oxidizing activity with retinol. Since these enzymes (except AKR1B3) have Lys125, other residues should account for retinaldehyde specificity. Here, by using site-directed mutagenesis and molecular modeling, we further delineate the contribution of residues 301 and 304. We demonstrate that besides Lys125, Ser304 is a major structural determinant for all-trans-retinaldehyde specificity of AKR1B10.
A recent rat genomic sequencing predicts a gene Akr1b10 that encodes a protein with 83% sequence similarity to human aldo-keto reductase (AKR) 1B10. In this study, we isolated the cDNA for the rat AKR1B10 (R1B10) from rat brain, and examined the enzymatic properties of the recombinant protein. R1B10 utilized NADPH as the preferable coenzyme, and reduced various aldehydes (including cytotoxic 4-hydroxy-2-hexenal and 4-hydroxy- and 4-oxo-2-nonenals) and α-dicarbonyl compounds (such as methylglyoxal and 3-deoxyglucosone), showing low K(m) values of 0.8-6.1μM and 3.7-67μM, respectively. The enzyme also reduced glyceraldehyde and tetroses (K(m)=96-390μM), although hexoses and pentoses were inactive and poor substrates, respectively. Among the substrates, 4-oxo-2-nonenal was most efficiently reduced into 4-oxo-2-nonenol, and its cytotoxicity against bovine endothelial cells was decreased by the overexpression of R1B10. R1B10 showed low sensitivity to aldose reductase inhibitors, and was activated to approximately two folds by valproic acid, and alicyclic and aromatic carboxylic acids. The mRNA for R1B10 was expressed highly in rat brain and heart, and at low levels in other rat tissues and skin fibroblasts. The results suggest that R1B10 functions as a defense system against oxidative stress and glycation in rat tissues.
The aldo-keto reductase (AKR) proteins catalyze reduction of diverse aldehydes and play detoxification roles in many organisms. Since many substrates are shared among AKR, it is generally accepted that these enzymes can functionally compensate each other in response to oxidative stress. Their overall abundances are the important factor that partially reflects the capacity of antioxidant and detoxification in tissues. In this study, the strategy was proposed for generation of Pan-AKR antibodies to recognize most AKR proteins in mouse tissues. Derived from bioinformatic analysis, several consensus peptides with different potential antigenicities were synthesized, conjugated to hemocyanin from keyhole limpets and further delivered to rabbits to generate polyclonal antibodies. Three Pan-AKR antibodies exhibited the immune specificities and immune sensitivities, Pan-AKR-P1 for AKR1B and AKR1C, Pan-AKR-P3 for AKR1C and Pan-AKR-P4 for all the AKR proteins. Pan-AKR-P4 antibody was employed to 2-DE Western blot to examine the AKR abundances in mouse liver and kidney, resulting in seven immune-reactive spots from each tissue. Protein identification with MS revealed that most immune-positive spots were the members of AKR superfamily. Furthermore, Pan-AKR-P4 antibody was implemented to compare the different abundances of the AKR proteins in liver and kidney between normal and diabetic mice, suggesting that diabetes did cause some abnormal changes in the AKR protein abundances.
Mouse vas deferens protein/aldo-keto reductase 1B7 (AKR1B7) is involved in the detoxification of isocaproaldehyde, a steroidogenesis byproduct, and of 4-hydroxynonenal formed by lipid peroxidation. The rat orthologue of AKR1B7 has recently been named AKR1B14 in the AKR superfamily. Recombinant AKR1B14 was expressed in a bacterial system and purified to homogeneity. The purified protein was crystallized from polyethylene glycol solutions using the hanging-drop vapour-diffusion method and an X-ray diffraction data set was collected to 1.86 A resolution. The crystals belonged to space group P2(1), with unit-cell parameters a = 50.66, b = 69.14, c = 72.27 A, beta = 96.4 degrees. This is the first crystallization report of a rodent AKR1B7 orthologue.
Here, we show that three enzymes belonging to the 1B group of the aldo–keto reductase (AKR) superfamily, i.e., human placental
aldose reductase (AKR1B1), mouse kidney aldose reductase (AKR1B3) and mouse vas deferens protein (AKR1B7), catalyse the reduction
of prostaglandin (PG) H2, a common intermediate of various prostanoids, to form PGF2α in the presence of NADPH. AKR1B1, AKR1B3 and AKR1B7 displayed higher affinities for PGH2 (Km = 1.9, 9.3 and 3.8 μM, respectively) and Vmax values (26, 53 and 44 nmol/min/mg protein, respectively) than did the human lung PGF2α synthase (AKR1C3; 18 μM and 4 nmol/min/mg protein, respectively). The PGF2α synthase activity of AKR1B1 and AKR1B3 was efficiently inhibited by two AKR inhibitors, tolrestat (Ki = 3.6 and 0.26 μM, respectively) and sorbinil (Ki = 21.7 and 0.89 μM, respectively), in a non-competitive or mixed-type manner, whereas that of AKR1B7 was not sensitive to
these inhibitors (Ki = 9.2 and 18 mM, respectively). These data provide a molecular basis for investigating novel functional roles for AKR1B members
and PGF2α as mediators of physiological and pathological processes in mammalian organisms.
Aldose reductase has been implicated in a number of diabetic complications including background retinopathy (microaneurysms) and cataracts. The formation of microaneurysms is independently related to duration of disease and hyperglycemia, and is found in post-pubertal patients (Murphy et al., 1990). They are thought to be caused by the loss of pericytes resulting from the activity of aldose reductase. The mechanisms regulating the expression of the enzyme in specific tissues and in pathological conditions are unknown.
Treatment of Chinese hamster ovary (CHO) cells by the aldehyde containing calpain inhibitor I resulted in the induction of a 35-kDa protein that was partially sequenced and shown to be a member of the aldo-keto reductase superfamily (Inoue, S., Sharma, R. C., Schimke, R. T., and Simoni, R. D. (1993) J. Biol. Chem. 268, 5894-5898). Using rapid amplification of cDNA ends polymerase chain reaction, we have sequenced the cDNA for this protein (CHO reductase). This enzyme is a new member of the aldo-keto reductase superfamily and shows greatest amino acid sequence identity to mouse fibroblast growth factor-regulated protein and mouse vas deferens protein (92 and 80% sequence identity, respectively). The enzyme exhibits about 70% sequence identity with the aldose reductases (ALR2; EC 1.1.1.21) and about 47% with the aldehyde reductases (ALR1; EC 1.1.1.2). Northern analysis showed that it is induced in preference to either ALR1 or ALR2 and RNase protection assays showed gene expression in bladder, testis, jejunum, and ovary in descending order of expression. The cDNA for this inducible reductase was cloned into the pET16b vector and expressed in BL21(DE3) cells. Expressed CHO reductase showed kinetic properties distinct from either ALR1 or ALR2 including the ability to metabolize ketones. This protein joins a growing number of inducible aldo-keto reductases that may play a role in cellular regulation and protection.
Transcription of the mouse vas deferens protein (MVDP) gene is stimulated by androgens and we have previously shown that in a 162 bp fragment, located at position -121 to +41, a TGAAGTtccTGTTCT sequence functions as an androgen-dependent enhancer. To determine which factors are involved in the hormonally regulated MVDP gene transcription, we have used DNase I footprinting and band-shift assays to examine in vitro binding of proteins to the enhancer and promoter sequences and have determined the functional significance of the recognized DNA sequences in transient transfection assays. Studies using recombinant proteins such as the DNA binding domain of the androgen receptor (AR-DBD) and Sp1, and crude cellular extracts from T47D and vas deferens epithelial cells (VDEC) showed that in addition to AR-DBD, the transcriptional activators NF1 and Sp1 interact with the -121/+41 fragment in a specific manner. Transient transfection assays revealed that site-directed mutations in the NF1 and Sp1 binding elements strongly reduced (NF1) or abolished (Sp1) androgen induced expression. The results demonstrate that the -121/+41 sequence is a composite site for the androgen receptor mediated transactivation of the MVDP gene.
Aldose reductase (AR) is an NADPH-dependent aldo-keto reductase implicated in cellular osmoregulation and detoxification. Two distinct murine genes have been identified that are predicted to encode proteins with significant amino acid sequence identity with mouse AR: mouse vas deferens protein and fibroblast growth factor (FGF)-regulated-1 protein (FR-1). Here we report that the AR and FR-1 genes are differentially regulated in NIH 3T3 fibroblasts. FGF-1 stimulation of quiescent cells induces both AR and FR-1 mRNA levels, but the effect on FR-1 mRNA expression is significantly greater. FGF-1 treatment also increases FR-1 protein expression, as determined by Western-blot analysis using FR-1-specific polyclonal antiserum. Calf serum stimulation of quiescent cells increases AR mRNA expression but not FR-1 mRNA expression. Finally, when NIH 3T3 cells are grown in hypertonic medium, AR mRNA levels are significantly increased whereas FR-1 mRNA levels are only slightly up-regulated. These results indicate that the AR and FR-1 genes are differentially regulated in murine fibroblasts by two different growth-promoting agents and by hyperosmotic stress. Therefore these structurally related enzymes may have at least some distinct cellular functions; for example, although both AR and FR-1 activity may be important for the metabolic changes associated with cellular proliferation, AR may be the primary aldo-keto reductase involved in cellular osmoregulation.
Kinetic studies on the aldose reductase protein (AR2) have shown that it does not behave as a classical enzyme in relation to ring aldose sugars. These results have been confirmed by X-ray crystallography studies, which have pinpointed binding sites for pharmacological “aldose reductase inhibitors” (ARIs). As with non-enzymic glycation reactions, there is probably a free-radical element involved derived from monosaccharide autoxidation. In the case of AR2, there is free radical oxidation of NADPH by autoxidising monosaccharides, enhanced in the presence of the NADPH-binding protein. Whatever the behaviour of AR2, many studies have showed that sorbitol production is not an initiating aetiological factor in the development of diabetic complications in humans. Vitamin E (α-tocopherol), other antioxidants and high fat diets can delay or prevent cataract in diabetic animals even though sorbitol and fructose levels are not modified; vitamin C acts as an ARI in humans. Protein post-translational modification by glyc-oxidation or other events is probably the key factor in the aetiology of diabetic complications.
We have isolated a human cDNA clone from small intestine that represents a new member of the aldo-keto reductase family. This new member showed 70% identity at the protein level to human aldose reductase and around 80% identity to other Chinese hamster and mouse reductases. The expression pattern shows that this message is located primarily in the adrenal gland, thus suggesting an involvement in steroid metabolism. It is also strongly expressed in the intestinal tract and has been called human small intestine reductase.
A definitive role for many members of the aldo-keto reductase (AKR) superfamily has been elusive despite evidence of the involvement of these enzymes in the metabolism of steroids, biogenic amines and even the products of lipid peroxidation. It is generally believed that the primary role of AKR’s is the detoxication of aldehydes but there is also evidence for the involvement of one of them, aldose reductase (ADR), in pathological processes i.e., the development of diabetic complications.
Mouse vas deferens protein (MVDP) is an aldose reductase-like protein that is highly expressed in the vas deferens and adrenal glands and whose physiological functions were unknown. We hereby describe the enzymatic characteristics of MVDP and its role in murine adrenocortical Y1 cells. The murine aldose reductase (AR) and MVDP cDNAs were expressed in bacteria to obtain recombinant proteins and to compare their enzymatic activities. Recombinant MVDP was functional and displayed kinetic properties distinct from those of murine AR toward various substrates, a preference for NADH, and insensitivity to AR inhibitors. For MVDP, isocaproaldehyde, a product of side-chain cleavage of cholesterol generated during steroidogenesis, is the best natural substrate identified so far. In Y1 cells, we found that NADH-linked isocaproaldehyde reductase (ICR) activity was much higher than NADPH-linked ICR activity and was not abolished by AR inhibitors. We demonstrate that in Y1 cells, forskolin-induced MVDP expression enhanced NADH-linked ICR activity by 5-6-fold, whereas no variation in ICR-linked NADPH activity was observed in the same experiment. In cells stably transfected with MVDP antisense cDNA, NADH-linked ICR activity was abolished even in the presence of forskolin, and the isocaproaldehyde toxicity was increased compared with that of intact Y1 cells, as measured by isocaproaldehyde LD(50). In Y1 cells transfected with MVDP antisense cDNA, forskolin-induced toxicity was abolished by aminoglutethimide. These results indicate that in adrenocortical cells, MVDP is responsible for detoxifying isocaproaldehyde generated by steroidogenesis.
Vas deferens epithelial cell subcultures were used to study the sequential regulation of jun/fos proto-oncogene expression and AP1 activities during cell proliferation, polarization and androgen-induced expression of a terminal differentiation marker, i. e. the mvdp gene. Proliferation of epithelial cells is associated with a high expression in the nucleus of most Jun and Fos oncoproteins. After cell seeding on an extracellular matrix which allows polarization and expression of the mvdp gene in response to androgens, AP1 protein accumulation is greatly altered and consists in a loss of JunB, Fra1, FosB and a decrease in c-Fos, c-Jun and Fra2, while JunD remained at the same level. This was correlated with a drop in AP1 binding activity as evaluated by gel shift assay using either AP1 consensus sequence or AP1 binding sites of the mvdp gene promoter region, and in AP1 transactivating activity, as estimated by stable transfection experiments using an AP1 responsive promoter (TRE-TK-luc). Androgens did not significantly influence AP1 activities. On the contrary, stimulation of AP1 proteins by the tumor-promoting phorbol ester caused a decrease in androgen-induced mvdp mRNA accumulation, and this effect was reversed by staurosporine, a potent inhibitor of PKC. Our data suggest that a down-regulation of AP1 activities induced by epithelial cell differentiation is a prerequisite to androgen-induced mvdp gene expression. The high AP1 activities observed during proliferative state or induced in TPA-treated polarized cells, exert a repressive effect on androgen action.
Studies of ACTH functions in adrenal steroidogenesis have been facilitated by the availability of immortalized mouse adrenocortical Y1 cells. In order to obtain alternative cell lines with a more differentiated zona fasciculata (ZF) phenotype we used targeted tumorigenesis strategy. We have generated transgenic mice expressing the SV40 T antigen under the control of the ACTH-dependent promoter for the AKR1B7/MVDP gene (aldo-keto reductase 1B7/mouse vas deferens protein), which encodes an enzyme responsible for detoxifying isocaproaldehyde, the product of side-chain cleavage of cholesterol generated by steroidogenesis. Our previous data indicated that in the mouse adrenal, AKR1B7 expression was restricted to the ZF and that a 0.5-kb promoter region was able to target specific adrenal expression in transgenic mice. In situ hybridization analyses indicate that AKR1B7 expression during fetal and post-natal periods paralleled the onset of glucocorticoid synthesis and the development of ZF. In transgenic mice, ACTH control and developmental programming of the CAT gene driven by the 0.5-kb promoter followed endogenous gene regulation. Then transgenic mice harboring the 0.5-kb/SV40 T antigen construct were generated and two founders out of three developed adrenal tumors. Cells derived from the tumor of founder 1 (ATC1) were grown in presence of forskolin to maintain ACTH receptor expression and were tested for ACTH responsiveness by immunocychemistry and northern blot analyses. Even after several passages, the ACTH induced AKR1B7 and P450c11beta mRNAs accumulations were similar to that observed in mouse primary adrenocortical cell cultures. Our findings suggest that ATC1 cells have conserved essential features of ZF cells. In order to achieve complete characterization of these cells further analyses are currently performed to investigate their steroidogenic activity.
The structure and sequence of the human gene for aldose reductase (AR) was determined by analysis of cDNA and genomic clones. The AR gene was independently isolated from two different cosmid libraries and the clones were characterized by restriction mapping, Southern blotting, and DNA sequencing. The gene extends over approximately 18 kilobases and consists of 10 exons giving rise to a 1,384 nucleotide mRNA (excluding the poly(A) tail). The human aldose reductase gene codes for a 316-amino acid protein with a molecular mass of 35,858 daltons. The size range for the exons is 82-168 base pairs (bp), whereas that for the introns is 325 to about 7,160 bp. A major site of transcription initiation in liver was mapped to an A residue 31 nucleotides upstream from the A of the ATG initiation codon. The promotor region of the gene contains a TATA (TATTTA) box and a CCAAT box which are located 37 and 104 nucleotides upstream, respectively, from the transcription initiation site. We have found four Alu elements in the AR gene; two are found in intron 1 and one each in intron 4 and intron 9
We report the cloning and sequencing of a 1225-base pair cDNA encoding an abundant protein from mouse vas deferens. An open reading frame of 948 nucleotides encodes a protein of 316 amino acids with a calculated mass of 35,965 Da. A high degree of homology was found between this protein and members of the aldo-keto reductase superfamily, especially aldose reductase from human placenta (82%) and from rat lens (80%), suggesting that it could be an aldose reductase. Castration resulted in a marked decrease in the level of the 1.4 kilobase mRNA coding for the 35,965 protein, whereas administration of testosterone to castrated males resulted in a marked increase. Southern hybridization analysis of mouse genomic DNA revealed relatively simple patterns of bands.
Aldose reductase was purified from human skeletal and heart muscle by a rapid and efficient scheme involving Red Sepharose chromatography, chromatofocusing on Pharmacia PBE 94, and hydroxylapatite high pressure liquid chromatography. The scheme afforded homogeneous enzyme, 65% recovery, in 2 days. All muscle samples express aldose reductase but not the closely related aldehyde reductase. Aldose reductase is isolated in one of two forms that are distinguishable by their kinetic patterns with glyceraldehyde as substrate and which are interconvertible by treatment with dithiothreitol. Both forms are capable of catalyzing the reduction of glucose (Km = 68 mM), and both are highly sensitive to inhibition by aldose reductase inhibitors. The reduction of glucose was shown to be nearly stoichiometric with production of sorbitol (92 +/- 2%). Dialysis of aldose reductase in the absence of thiols or NADP converts it into a form that shows markedly different kinetic properties, including very weak catalytic activity toward glucose and insensitivity to aldose reductase inhibitors. This modified form can be converted back into the native form by dithiothreitol. Thiol titration of the two forms of aldose reductase with Ellman's reagent indicated that two thiol groups were lost when the enzyme was dialyzed in the absence of dithiothreitol or NADP.
The human aldose reductase gene has been cloned by screening a human placental cDNA library with antibodies against bovine lens aldose reductase. The nucleotide sequence of the entire coding region has been determined. The deduced amino acid sequence indicates that the human enzyme is 84% identical to the bovine lens aldose reductase and 85% identical to the rat lens aldose reductase. It is also very similar to the human aldehyde reductase, the bovine prostaglandin F synthase, and to the European common frog rho-crystallin. The deduced amino acid sequence also indicates that maturation of aldose reductase involves removal of the N-terminal methionine.
Cells generally respond to long-term hyperosmotic stress by accumulating nonperturbing organic osmolytes. Unlike bacteria, in which molecular mechanisms involved in the increased accumulation of osmolytes have been identified, those in multicellular organisms are virtually unknown. In mammals, during antidiuresis, cells of the renal inner medulla are exposed to high and variable extracellular NaCl. Under these conditions, the cells contain a high level of sorbitol and other osmolytes which help balance the high extracellular osmolality. PAP-HT25 is a continuous line of cells derived from rabbit renal inner medulla. When medium osmolality is increased by raising the NaCl concentration, these cells accumulate sorbitol. The sorbitol is synthesized from glucose in a reaction catalyzed by aldose reductase. When the medium is made hyperosmotic, aldose reductase activity increases because of a larger increase in the amount of enzyme. This increase is produced by the accelerated rate of synthesis of aldose reductase protein. The purpose of the present studies was to examine the mechanism of this increase in aldose reductase protein by measuring the relative abundance of aldose reductase mRNA. A cDNA clone coding for rabbit kidney aldose reductase was isolated. Antisense RNA probes transcribed from this clone hybridized specifically with a 1.5-1.6 kilobase mRNA in Northern blots. Cells grown chronically in hyperosmotic medium had a relative abundance of this specific mRNA which was six times that of cells grown in isoosmotic medium. When cells grown in isoosmotic medium were switched to hyperosmotic medium, the level of aldose reductase mRNA peaked (18-fold) at 18-24 h. The induction of aldose reductase mRNA by osmotic stress was reversible. Our finding of increased abundance of a specific mRNA in direct response to hyperosmotic stress represents the first report of such an effect in animals.
Using a modification of the procedure of Kormann et al. (Kormann, A. W., Hurst, R. O., and Flynn, T. G. (1972) Biochim. Biophys. Acta 258, 40-55) for the purification of glycerol dehydrogenase, two enzymes have been purified from the skeletal muscle of male rabbits. From a consideration of their properties these enzymes have been named aldose reductase 1 and aldose reductase 2, respectively. Both enzymes are monomeric by the criteria of gel filtration and polyacrylamide gel electrophoresis in sodium dodecyl sulfate and both reductases are immunologically identical as shown by double immunodiffusion and rocket immunoelectrophoresis. Aldose reductases 1 and 2 have almost identical amino acid compositions, their NH2 termini are blocked and the COOH termini of both enzymes are apparently identical. The enzymes differ, however, in molecular weight with aldose reductase 2 having Mr = 41,500 and aldose reductase 1 Mr 40,200. Both enzymes have the broad substrate specificity typical of the aldehyde reductase family of enzymes; Km values of aldose reductase 1 for aldo sugars were similar to those reported for rabbit lens aldose reductase, and both aldose reductase 1 and 2 were inhibited by the commercial aldose reductase inhibitors Alrestatin and Sorbinil. Two aldose reductases, immunologically and electrophoretically identical to the muscle enzymes, were found in rabbit lens. Two aldose reductases were also detected in the skeletal muscle of male rats and pigs and in pig and bovine lens. The presence of relatively large amounts of aldose reductase in muscle identifies a new and rich source of the enzyme.
The addition of polypeptide mitogens to quiescent cell lines induces the expression of various gene products, some of which are likely to perform functions critical for cell cycle progression, DNA synthesis, and mitosis. We have used a differential display approach to identify fibroblast growth factor (FGF)-1-inducible genes in NIH-3T3 cells. One of these genes, termed FGF-regulated (FR)-1, encodes a 316-amino acid protein with approximately 82% amino acid sequence identity to an abundant protein expressed in mouse vas deferens and approximately 70% identity to human aldose reductase. The function of the vas deferens protein is unknown; however, aldose reductase is an NADPH-dependent monomeric oxidoreductase implicated in the pathogenesis of diabetic complications. FGF-1 induction of FR-1 mRNA expression is first detectable at 4 h after mitogen addition and is dependent on de novo RNA and protein synthesis. FGF-2 or phorbol ester treatment can also increase FR-1 mRNA levels; in contrast, whole blood serum or individual growth factors present in serum have only minimal effects on FR-1 mRNA expression. FR-1 mRNA is detectable in a number of mouse tissues but is most abundant in newborn liver and in adult intestine, ovary, and testis. These results raise the possibility that aldose reductase-related proteins may play a role in FGF-1- and FGF-2-stimulated mitogenesis.
Diverse organisms accumulate organic osmolytes to adapt to hyperosmotic stress. The molecular basis of eukaryotic gene osmoregulation remains obscure. Aldose reductase [AR; alditol:NAD(P)+ 1-oxidoreductase, EC 1.1.1.21], which catalyzes the conversion of glucose to sorbitol (an organic osmolyte), is induced in renal medullary cells under hyperosmotic conditions. Elevated extracellular NaCl increases AR mRNA transcription in PAP-HT25 cells, a cell line derived from the rabbit renal papilla. We have cloned and characterized the rabbit AR gene to determine how it is regulated by hyperosmolality. The length of the gene, not including 5' or 3' flanking regions, is approximately 14.7 kilobases (kb) organized into 10 exons and 9 introns. The transcription start site is 36 base pairs upstream of the initiator methionine codon. A 5-kb fragment containing approximately 3.5 kb of 5' flanking region was isolated. The 3.5-kb sequence was examined for basal promoter activity and hyperosmotic response in luciferase reporter gene constructs. A 235-base-pair fragment (base pairs -208 to +27) was able to drive the downstream reporter gene in transfected PAP-HT25 cells under isoosmotic conditions (300 mosmol/kg of H2O). When this fragment plus the remaining upstream sequence (from approximately base pair -3429 to base pair +27) was used, cells in hyperosmotic medium (500 mosmol/kg of H2O) showed about 40-fold induction of luciferase expression compared with cells in isoosmotic medium. The upstream fragment (from approximately base pair -3429 to base pair -192) also conferred osmotic response to a heterologous promoter (B19). This finding evidences putative osmotic response element(s) (OREs) within a specific DNA fragment in a eukaryotic genome. Identification and characterization of OREs within this fragment and their associated trans-acting factors should reveal the molecular mechanisms of gene regulation in osmotic stress.
Mouse vas deferens protein (MVDP), a member of the aldo-keto reductase superfamily, is exclusively produced in the epithelial cells of the deferent duct under androgenic regulation. To better understand androgenregulated MVDP gene expression, the location and sequences of androgen response elements (AREs) in the 5'-flanking DNA were determined. Sequence analysis revealed two putative AREs as follows: one between positions -1186 and -1171 (distal ARE) and the other between -111 and -97 (proximal ARE). To study hormonal regulation, fragments of the MVDP promoter region, extending from residue -1804 to +41, were linked to the chloramphenicol acetyltransferase (CAT) reporter gene and cotransfected with a human androgen receptor expression vector into T47D cells in a transient expression assay. A minimal region (-121 to +41) was identified as being sufficient for androgen-regulated gene expression. A mutation in proximal ARE almost completely abolished androgen induction of CAT. One copy of the sequence TGAAGT tcc TGTTCT, cloned in the opposite orientation in front of the thymidine kinase promoter, confers androgen responsiveness to the CAT reporter gene. Androgen transcriptional activity was not detected with the distal ARE. The data provide strong evidence that transcriptional regulation of the MVDP gene occurs via the sequence TGAAGT tcc TGTTCT.
Tumor-associated protein variants were detected by two-dimensional gel electrophoresis (2-DE) in soluble proteins from chemically induced rat hepatomas and transformed rat liver cell lines. Among them, a series of 8 protein variants was found localized at similar sites in 2-DE gels (33-35 kDa, with different pI values). We characterized four of them. In situ peptide mapping with limited proteolysis disclosed a significant relationship between these four individual protein spots. Their different position in 2-DE gels might be due to posttranslational modifications: one of the variants was phosphorylated, three others were modified by glycosylation. The most prominent tumor-associated protein variant of this series (spot 17) was further studied by amino acid analysis and internal amino acid microsequencing. It became evident that this variant is identical to aldose reductase (EC 1.1.1.21). This enzyme of the sorbitol pathway is expressed in the liver during embryogenesis, but is absent in adult rat liver. Our results suggest that it is reexpressed and functionally active during liver carcinogenesis.
Structural models of human aldose reductase complexed with NADPH have revealed the apposition of C4 of the nicotinamide ring with tyrosine 48 and histidine 110, suggesting that either of these residues could function as the proton donor in the reaction mechanism. Tyrosine 48 is also part of a hydrogen-bonding network that includes lysine 77 and aspartate 43. In order to study the potential catalytic roles of these 4 residues, we evaluated the kinetic properties of mutants containing structurally conservative replacements at these sites. Enzymatic activity was undetectable when Tyr-48 was mutated to phenylalanine (Y48F) although affinity for NADPH was unchanged. In contrast, a mutant containing asparagine substituted for His-110 (H110N) was characterized by an almost 80,000-fold increase in Km, but only about a 14-fold reduction in kcat measured with D-glyceraldehyde. Modest changes in catalytic properties were observed in the mutant containing aspartate 43 substituted with asparagine (D43N): Km for aldehyde substrates was elevated up to 17-fold, and kcat decreased less than 16-fold. However, the Kd(NADP) values for D43N were about 5 times higher than those for wild type. Mutant enzyme containing methionine substituted for lysine 77 (K77M) was up to 1,460-fold less active than the wild type. These results are consistent with Tyr-48 acting as the acid-base catalyst in human aldose reductase and confirm the importance of Asp-43, Lys-77, and His-110 to the structure and function of the active site.
Complementary DNA (cDNA) clones encoding bovine testicular 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) have been isolated from a bovine testicular lambdagt11 library using polyclonal antibodies against 20alpha-HSD and DNA probe hybridization. Nucleotide sequencing of three independently isolated clones was used to establish a composite cDNA sequence that encodes the enzyme. It contains a coding sequence of 921 nucleotides, a stop codon, and a 264-nucleotide 3'-noncoding segment which allowed deduction of the amino acid sequence of the enzyme. A computer homology search of the 20alpha-HSD cDNA performed against the GenBank DNA sequence database revealed it to be identical with bovine lens aldose reductase (alditol:NADPH oxidoreductase; EC 1.1.1.21), and a literature search reveals the deduced amino acid sequence to be identical with that reported for the bovine enzyme. Sequences obtained from the N-terminus of purified testicular 20alpha-HSD and from random peptides obtained by treatment with endopeptidase Lys-C are all identical with regions of the deduced amino acid sequence of 20alpha-HSD and/or the published sequence of aldose reductase. Further, the enzyme purified to homogeneity by following activity with 17-hydroxyprogesterone as a substrate was shown to reduce glucose, glyceraldehyde, and benzaldehyde (all classic aldose reductase substrates). Finally, 17-hydroxyprogesterone inhibited the reduction of benzaldehyde and glyceraldehyde. Because aldose reductase has been implicated in the etiology of diabetic complications, acceptance of steroid substrates may offer new implications for therapy.
Although the enzyme aldose reductase (AR) is implicated in the development of tissue pathology in diabetes, the exact mechanism of this involvement remains unclear. To better understand the role that expression of the aldose reductaseencoding gene (ALR) may play in diabetic complications, we have begun to analyze the gene and its regulatory regions, and we present here the sequence of four ALR genes in the rat. The putative functional gene is 14,1 kb long, has ten exons which show perfect sequence identity to the rat lens AR RNA sequence, and nine introns with classical splice-site consensus sequences. Potential regulatory elements in the 5'-flanking region of this gene include a TATA box and two CCAAT boxes. Probing rat genomic Southern blots with a fragment from the first intron indicates that there is probably only one copy of this gene in the rat genome. The other three genes are processed pseudogenes which show approx. 90% identity to the rat lens AR RNA sequence, contain no introns, and have poly(A) regions at their 3' ends. Chromosomal localization studies show the presence of ALR genes on chromosomes 3, 4 and 6 in the rat with the putative functional gene mapped on chromosome 4.
Aldose reductase (EC 1.1.1.21) has been implicated in a variety of diabetic complications. Here we present the first primary sequence data for the rat lens enzyme, obtained by amino acid and cDNA analysis. We have found structural similarities with another NADPH-dependent oxidoreductase: human liver aldehyde reductase (EC 1.1.1.2). The identity between these two enzymes is 50%. Both enzymes share approx. 40–50% homology with ϱ-crystallin, a major lens protein present only in the frog, Rana pipiens. We propose that aldose reductase, aldehyde reductase and ϱ-crystallin are members of a superfamily of related proteins.
Six aldo-keto reductases from rat adrenal gland have been highly purified to apparent homogeneity. These enzymes were identified as carbonyl reductases (CR-K1, CR-K2, CR-A, and CR-B), aldehyde reductase (AR-H), and aldose reductase (AR-L) in terms of substrate specificity, molecular weight (33,000–39,000), inhibitor susceptibility, cofactor requirement, and immunochemical properties. Both CR-K1 and CR-K2 were characterized as possessing high affinity towards 13,14-dihydro-15-ketoprostaglandin F 2α and were localized immunohistochemically in the zona glomerulosa and the zona reticularis of adrenal cortex, and in the ganglion cell of adrenal medulla. Immunoreactive proteins to anti-CR-K2 antibody were observed in male and female reproductive tissues of rats. Positive immunoreactive protein to anti-CR-A antibody was found in mouse, hamster, and rabbit adrenal gland, whereas that to anti-CR-K2 antibody was present only in rat adrenal gland. AR-H and AR-L mainly reduced aromatic and aliphatic aldehydes. All the aldo-keto reductases from rat adrenal gland were completely inhibited by p -chloromercuribenzoate. Barbiturate and 3,3'-tetramethylene glutarate potently inhibited AR-H, and quercitrin significantly decreased the activity of CR-K1, CR-K2, and AR-L. We propose that these aldo-keto reductases may play important roles in the rat adrenal functions.
The response of aldose reductase (AR) to crush injury was studied in normal rat sciatic nerve. Enzyme activity and immunoreactivity of AR were determined at intervals of 1, 5, 14, 28, and 35 days after crush and correlated with histologic and immunocytochemical observations. During nerve degeneration in the distal segments of crushed nerves, a significant reduction in AR activity was detected. At 5 and 14 days, coincident with Schwann cell proliferation, enzyme activity decreased by nearly two- and fourfold, respectively. Although activity of AR increased by 28 days during nerve regeneration, it was not restored to normal levels at 35 days. Similar reductions were observed with the immunoblotting of the enzyme. Quantitative analysis of immunogold labelling on electron micrographs confirmed that proliferating as well as remyelinating Schwann cells contained reduced gold particle density compared to Schwann cells of noncrushed myelinated fibers. Immunoblots of P0, a marker for the degree of Schwann cell differentiation or myelination, showed that the temporal sequence of changes in P0 paralleled that of AR. Thus expression of AR is a function of differentiated or mature Schwann cells. The putative volume regulatory role of AR in Schwann cells may become superfluous during Wallerian degeneration.
The gene for mouse vas deferens protein (MVDP) is expressed, under androgenic control, exclusively in the epithelial cells of the deferent duct. As a first step in correlating cell-specific and hormonal regulations with the structure of the gene, the complete sequence of the MVDP gene (11 kb) and 0.5 kb of the 5' flanking region have been determined. The size range for the 10 exons is 78 to 168 bp, whereas that of introns is 292 to 2833 bp. A major site of transcription is located on an A residue 46 nucleotides upstream from the A of the ATG initiation codon. A TATA (CATAA) box, a CAAT box, a GC-rich motif and a (5'-TGTTCT-3') element that closely resembles the consensus sequence of the androgen response elements are present in the 5' flanking region of the MVDP gene.
In most higher plants a period of desiccation is the terminal event in embryogenesis. Excised barley embryos acquire desiccation tolerance at a precise developmental stage and cDNA clones have been isolated which are temporally linked with desiccation tolerance. One such clone (pG22-69) with a putative gene product of 34 kd displays high structural homology to mammalian genes encoding an NADPH dependent aldose reductase involved in the synthesis of sorbitol. This first aldose reductase gene of plants is expressed constitutively during embryo maturation and is modulated by the plant hormones abscisic acid (ABA) and gibberellic acid (GA). Immunohistochemistry showed that the protein is preferentially expressed in tissues formed at early stages in embryogenesis. Measurements of enzymatic activity indicate that pG22-69 encodes an active aldose reductase. The finding of this reductase activity and the cloning of the corresponding gene supports the existence of a metabolic pathway in plants playing a role in the synthesis of osmolytes like sorbitol. The significance of this work is that genes of related structure and functions are being used in diverse organisms to fulfil stress related biological requirements.
The mRNA encoding a major protein of the mouse vas deferens (MVDP) was first detected in 10-day-old males and its concentration increased sharply between 10 and 20 days, reaching adult levels at 40 days. This increase was not associated with an increase in tissular androgen concentrations. In 30-day-old mice castrated at birth or treated with cyproterone acetate over 29 days, MVDP mRNA levels were not abolished and were similar to those measured in 10- and 20-day-old controls. These results suggest that the neonatal expression of MVDP gene is independent of androgens. In addition, precocious accumulation of MVDP mRNA could be induced by injection of excess amounts of androgens in 20- but not in 10-day-old animals. The prepubertal increase in MVDP mRNA levels is androgen-dependent but other factors may be necessary for MVDP expression.
Possible estrogen influences on oxytocin mRNA expression were studied in preoptic and anterior hypothalamic regions as might be important for behavioral as well as neuroendocrine controls. In situ hybridization for oxytocin mRNA determination was supported by immunocytochemical identification and was compared with vasopressin mRNA in situ hybridization. With these techniques, oxytocin-expressing neurons were identified in medial preoptic, anterior commissural, periventricular, paraventricular, supraoptic, and perifornical nuclei as well as the bed nucleus of the stria terminalis and intersupraopticoparaventricular (internuclear) islands. Distribution and number of oxytocin mRNA-containing neurons and oxytocin mRNA levels were compared between ovariectomized control rats given cholesterol implants and ovariectomized rats given short-term (2 days) or long-term (2 months) estradiol treatment (10% estradiol, subcutaneous silastic implants). Effectiveness of long-term estrogen treatment was confirmed behaviorally. While there was a trend in several cell groups for a larger number of oxytocin-mRNA-containing neurons to be observed following 2 days of estrogen treatment, this was not statistically significant. Moreover, additional oxytocin-mRNA containing cell groups were not seen after short or long estradiol treatment. With computer-aided analysis, mean pixels per oxytocin-mRNA expressing neuron (reflecting oxytocin mRNA content) were compared between groups: In the supraoptic nucleus and the anterior commissural nucleus, these were increased both by 2 days and 2 months of estradiol treatment. These differences may be important in modulating female reproductive behavior. Present findings also suggest that estradiol can affect the oxytocinergic system via an indirect route since the cell groups influenced here by estradiol do not contain estrogen receptors. Oxytocinergic neurons may serve as a good system to compare direct transcriptional with indirect effects of hormones.
We have resolved and characterized isoforms of aldose reductase from bovine and porcine lenses by preparative isoelectric focusing with narrow pH gradients using the Rotofor. Both bovine and porcine lens aldose reductases were resolved as two enzyme isoforms. The bovine isoforms were Mr40400 +/- 445 polypeptides of pI4.71 and 5.19. Porcine isoforms were Mr41500 +/- 450 polypeptides of pI 4.90 and 5.30. Staphylococcus aureus V-8 protease digestion patterns for each set of isoforms were essentially identical and all isoforms probably contain blocked amino terminal amino acids. Antiserum to bovine lens aldose reductase cross-reacted with porcine lens aldose reductase. Each isoform displayed substrate preferences characteristic of mammalian aldose reductases. With purification, both bovine and porcine lens aldose reductases became less sensitive to inhibition by 6-fluoro-spiro-(chroman-4.4'-imidazolidine)-2',5'-dione (sorbinil).
A cDNA encoding the major mouse vas deferens protein (MVDP) has been cloned and characterized. Using in situ hybridization we have identified the epithelial cells of the vas deferens as the site of synthesis of MVDP mRNA. Northern blot analysis suggests that a high level of an mRNA corresponding to the MVDP gene is present in the mouse vas deferens whereas the amount of MVDP mRNA in vas deferens of other species studied, or in other mouse tissues, even if present, is undetectable. Steady-state levels of MVDP mRNA are decreased by approximately 42% 3 days after castration but a significant hybridization signal is still observed even 50 days after castration. Testosterone treatment for 2 weeks is necessary to completely reverse the effect of castration. In vitro transcription assays on isolated nuclei showed that the hormonal induction of the MVDP gene is achieved mainly at transcriptional level.
The ductus deferens of the mouse contains a major protein with a molecular weight of 34.5 kd called mouse vas deferens protein (MVDP). Immunofluorescence histochemistry and immunoblotting with monoclonal antibodies have been used to investigate the localization, tissue, and species distribution, androgen-regulation, and developmental expression of this protein. Consistent positive immunoreaction was achieved in the ductus deferens epithelium, and immunofluorescence revealed that spermatozoa from the deferent duct were coated with MVDP. Western blot analysis showed the organ specificity of MVDP, which could not be detected in several organs in the mouse. Furthermore, MVDP appeared to be species specific since the proteins extracted from the ductus deferens of man, rat, guinea-pig, rabbit, and hamster did not react with the anti-MVDP probe. MVDP, whose expression is regulated by testosterone, was detectable at 20 days of age and its concentration increased rapidly from 20-30 days.
Aldose reductase (AR), an enzyme which converts glucose to sorbitol, has been implicated in the pathogenesis of diabetic cataracts and retinopathy. The normal physiological role of this enzyme in ocular tissue, however, remains unclear. In a developmental study in the rat using in situ and Northern hybridization analyses, we have found that there is a high level of AR mRNA expression in optic cup and lens as early as embryonic day 13. Serial sections through whole embryos at this stage showed that the eye was the only site of AR mRNA hybridization. Levels of AR mRNA declined in the retina as differentiation proceeded and were very sparse there postnatally. As lens development progressed, epithelial AR mRNA levels remained high, especially in the germinative zone, which is the source of the cells that will become lens fibers, and in the bow region, where these cells undergo a dramatic morphogenetic differentiation into lens fibers. AR mRNA was undetectable in terminally differentiated lens fibers. Since it has been suggested that AR-catalyzed sorbitol production could be an osmoprotective device of lens epithelium during systemic hyperosmolar stress, AR mRNA levels from dehydrated hyperosmolar rats were compared with euvolemic control values, and no difference was found. In summary, AR appears to be of particular importance in the development of the eye, with its retinal role receding relative to lens as differentiation is completed. A continued high level of expression in lens epithelium in adulthood may be explained by the fact that lens tissue, unlike retina, normally continues to proliferate and differentiate after birth. The temporal and spatial pattern of distribution of AR mRNA is strongly suggestive of a role for this enzyme in lens fiber morphogenesis.
The action of aldose reductase has been implicated in the etiology of a variety of diabetic complications affecting the visual system. However, very little is known regarding the structure and functional organization of the genes encoding this key enzyme. In the present study, we have isolated and characterized complementary DNA clones encoding bovine lens aldose reductase. Nucleotide sequencing of four independently isolated clones was used to establish a 1154 nucleotide composite cDNA sequence. The cDNA sequence encodes 296 amino acids of the aldose reductase primary structure, and contains an additional 261 nucleotides of apparently untranslated sequence downstream from the coding region. No nucleotide sequence differences were found among the four independently isolated aldose reductase cDNA clones. The aldose reductase amino acid sequence deduced from the cDNA shows high homology to that reported for aldose reductase of the rat lens. Significant similarities are also evident between bovine lens aldose reductase and both human liver aldehyde reductase and frog lens rho-crystallin.
Glycerophosphorylcholine, inositol, and sorbitol were measured in rat kidney homogenates and tubules from inner medulla and papilla by enzymatic spectrophotometric techniques. Organic osmolytes exhibited their highest concentrations in the papillary tip. In contrast to glycerophosphorylcholine and sorbitol, inositol was of similar high concentrations in inner and outer medulla. Freshly prepared inner medullary tubules maintained tissue osmolyte concentrations under control, antidiuretic, and furosemide diuretic conditions. When tubules were incubated in vitro over 90 min, tubular organic osmolyte concentrations decreased as a function of extracellular NaCl, but not urea concentrations. Organic osmolyte disappearance from cells was quantitatively recovered from the medium. In contrast, medium lactate dehydrogenase activity did not rise in parallel and tubular ATP remained constant. Glucose up to a concentration of 200 mM increased tubule and medium sorbitol. The results obtained indicate that glycerophosphorylcholine, sorbitol, and inositol rapidly adapt their intracellular concentrations to extracellular NaCl osmolality by a change in tubular plasma membrane permeability. In addition sorbitol levels are regulated by the extracellular glucose concentration.
When total RNA and poly (A) ⁺ RNA extracted from the adult mouse vas deferens were translated in the rabbit reticulocyte lysate protein synthesizing system, they showed a similar electrophoretic pattern of about 20 protein bands of widely varying molecular weights. Within the total mRNA population, three functional mRNAs coding for proteins with molecular weights of 24, 34·5 and 36 kDa showed marked changes after 1 month of castration. The time-course of the response of the translatable proteins showed that while 3 days after castration the 24 and 36 kDa bands were missing, the mRNA coding for the 34·5 kDa band was markedly reduced only 20 days after castration. The negative effect of castration on the three mRNAs could be completely reversed by treatment with testosterone. When total RNA extracted from the immature mouse vas deferens was translated, the 24, 34·5 and 36 kDa protein bands were detectable in 10-day-old males and were synthesized in significant amounts between 10 and 20 days (24 and 36 kDa bands) or between 20 and 30 days (34·5 kDa band). Based on its electrophoretic and immunological properties, the 34·5 kDa protein band, which is predominant in the translation products, was identified as the major androgen-dependent protein previously described in vivo .
Journal of Endocrinology (1989) 120, 67–74
A general method for the identification and purification of aldose reductase (alditohNADP oxidoreductase, EC 1.1.1.21) and NADP-L-hexonate dehydrogenase (L-gulonate:NADP oxidoreductase, EC 1.1.1.19) from tissues is described. Two aldose reductase isoenzymes, AR-A and AR-B, were isolated from renal papilla and were characterized. These two forms were also found in brain, retina, pancreas, lens and optic nerve. AR-A and AR-B differed primarily in glucuronate-reducing activity. All tissues, with the exception of lens and optic nerve, also contained NADP-L-hexonate dehydrogenase activity. Human red blood cells contained NADP-L-hexonate dehydrogenase activity but not aldose reductase.
The aldose reductases and the two isoenzyme forms isolated from these tissues were immunologically identical as demonstrated with a specific antiserum prepared in rabbits against papillary AR-B. The antiserum inhibited AR-A and AR-B activity equally in vitro. The extent of enzyme inhibition by the antiserum was similar when glucuronate, glucuronolactone, xylose and glyceraldehyde were used as substrates. The antiserum did not react with NADP-L-hexonate dehydrogenase. Therefore, the method provides a specific means for identifying aldose reductase in crude tissue homogenates.
A permanent human cell line, EA . hy 926, has been established that expresses at least one highly differentiated function of vascular endothelium, factor VIII-related antigen. This line was derived by fusing human umbilical vein endothelial cells with the permanent human cell line A549. Hybrid cells that survived in selective medium had more chromosomes than either progenitor cell type and included a marker chromosome from the A549 line. Factor VIII-related antigen can be identified intracellularly in the hybrids by immunofluorescence and accumulates in the culture fluid. Expression of factor VIII-related antigen by these hybrid cells has been maintained for more than 100 cumulative population doublings, including more than 50 passages and three cloning steps. This is evidence that EA . hy 926 represents a permanent line.
The University of Wisconsin Genetics Computer Group (UWGCG) has been organized to develop computational tools for the analysis
and publication of biological sequence data. A group of programs that will interact with each research-article has been developed
for the Digital Equipment Corporation VAX computer using the VMS operating system. The programs available and the conditions
for transfer are described.
A procedure is described for the large-scale purification of light (L) and heavy (H) chain mRNAs from plasmacytomas produced in mice. Intact RNA is selectively precipitated in high yield from frozen tumors homogenized in 3 M LiCl and 6 M urea. L and H-chain mRNAs were purified by oligo(dT)-cellulose chromatography and either sucrose gradient centrifugation in conditions preventing aggregation or by means of high-resolution preparative gel electrophoresis under non-denaturing conditions. gamma 2a and alpha H-chain mRNAs sedimented as major components at 15.5 S and 16.5 S respectively, when L-chain mRNAs sedimented as 12-S species. H-chain mRNAs isolated by continuous elution during preparative gel electrophoresis were completely separated from both L-chain mRNA and residual 18-S rRNA, and migrated as single components of 1900 +/- 50 nucleotides on analytical denaturing gels. The partially purified H-chain mRNAs were translated into major components of molecular weights of 56,000 (gamma 2a) and 60,000 (alpha) in an mRNA-dependent rabbit reticulocyte lysate, whereas L-chain mRNAs yielded polypeptides of molecular weights of 25,000 (gamma) and 27,000 (chi). Up to 95% of the translation products directed by the purified mRNAs were immunoprecipitated using specific antisera. The purity of L and H-chain mRNAs was assessed by hybridization of corresponding cDNAs with excess recombinant plasmid DNA. The results indicated a minimum purity of 47% (gamma 2a), 62% (alpha), for H-chain mRNAs and 60% (chi), for L-chain mRNAs.
Aldose reductase (AR) has been implicated in the etiology of diabetic cataract, as well as in other complications. However, the role of AR in these complications remains controversial because the strongest supporting evidence is drawn from the use of AR inhibitors for which specificity in vivo cannot be ascertained. To settle this issue we developed transgenic mice that overexpress AR in their lens epithelial cells and found that they become susceptible to the development of diabetic and galactose cataracts. When the sorbitol dehydrogenase-deficient mutation is also present in these transgenic mice, greater accumulation of sorbitol and further acceleration of diabetic cataract develop. These genetic studies demonstrated convincingly that accumulation of polyols from the reduction of hexose by AR leads to the formation of sugar cataracts.
Aldose reductase (alditol:NAD(P)+ 1-oxidoreductase), an enzyme implicated in the pathogenesis of various diabetic complications, catalyzes the reduction of a variety of aldehydes. From a mouse kidney library, we isolated aldose reductase cDNA that encodes a 316-amino-acid protein with approximately 97% identity to rat lens aldose reductase, approximately 69% identity to the mouse vas deferens protein and also approximately 69% identity to mouse fibroblast growth-factor-1-regulated protein. RNA-blot analysis demonstrated abundant expression of the enzyme transcript in the testis, skeletal muscle and kidney. However, a very low level of the transcript was detected in the sciatic nerve and lens, where abundant expression and involvement of the enzyme in diabetic complications were documented in other animals species. The isolated cDNA was expressed in Escherichia coli and the recombinant protein was purified to homogeneity by affinity chromatography and chromatofocusing. The expressed enzyme demonstrated reductase activity for various aldo sugars but not for the steroids. The enzyme reaction with DL-glyceraldehyde was, however, competitively inhibited by progesterone or 17 alpha-hydroxyprogesterone. The results not only indicate a unique tissue distribution and enzyme attribute of mouse aldose reductase, but also the presence of a closely related subgroup within the aldo-oxidoreductase superfamily in mouse tissues.
Mouse vas deferens protein (MVDP), a member of the aldo-keto reductase superfamily, is exclusively produced in the vas deferens. To better understand androgen-regulated MVDP gene expression we have used RNA hybridization to study the effects of androgens on the steady-state levels of MVDP mRNA in vas deferens epithelial cell subcultures. Northern blot analysis revealed that these cells only express MVDP mRNA in the presence of androgens. There was a close relationship between MVDP mRNA levels and dihydrotestosterone concentrations. MVDP mRNA is induced over a period of 24h and maximal induction is about 25-fold. Treatment of cells with cycloheximide completely abolished the observed androgen effect suggesting that the induction of the MVDP gene by androgens depends on continuous protein synthesis. Transient transfection of vas deferens epithelial cells with MMTV-CAT vector showed that these cells contained functional androgen receptors and that they are a suitable system to study androgen effect on MVDP gene regulatory elements.
1. Aldose reductase, aldehyde reductase and high-Km aldose reductase were purified from the inner medulla of dog kidney. 2. Compared with aldose reductase, high-Km aldose reductase had a lower isoelectric point, a lower activity for aldo-sugars and a lower sensitivity for aldose reductase inhibitors, and it was not activated by sulfate ions. Both reductases had the same molecular weight (38,500) and immunochemical properties. 3. High-Km aldose reductase was easily converted into an aldose reductase-like enzyme, namely a generated reductase upon incubation in neutral buffer solution. 4. The generated reductase was identical with aldose reductase with respect to the isoelectric point, substrate specificity, activation by sulfate ions and IC50 values for aldose reductase inhibitors. The generated reductase revealed immunochemical identity with aldose reductase as well as high-Km aldose reductase.
The RNAse protection assay is a highly sensitive assay which is commonly used to detect specific hybridization between complementary RNAs and to determine exon sizes in gene characterization studies. Unfortunately, each of the numerous steps involved in the assay could give artifacts depending on the probe used. In this study, common causes of artifacts have been identified using riboprobes which identify exons of known sizes. The RNAse concentration and duration of digestion used were found to be critical factors affecting exon size estimations. Five different riboprobes were tested to obtain a consensus optimum RNAse condition--10 micrograms/ml RNAse A, 0.5 microgram/ml RNAse T1--enabling the correct determination of exon sizes. This condition was further analyzed for its specificity when RNAse protection assays were performed between highly homologous RNA fragments from two different species. Results show that this concentration of RNAse would efficiently cleave a minimum of two nucleotide mismatches. Single nucleotide mismatches were frequently not cleaved by the same RNAse concentration making it possible to detect the correct exon size regardless of such sequence polymorphisms in gene sequences.
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