Kenneth H. Gabbay

Identification and Characterization of Multiple Osmotic Response Sequences in the Human Aldose Reductase Gene

Aldose reductase (AR) has been implicated in osmoregulation in the kidney because it reduces glucose to sorbitol, which can serve as an osmolite. Under hyperosmotic stress, transcription of this gene is induced to increase the enzyme level. This mode of osmotic regulation of AR gene expression has been observed in a number of nonrenal cells as well, suggesting that this is a common response to hyperosmotic stress. We have identified a 132-base pair sequence ∼1 kilobase pairs upstream of the transcription start site of the AR gene that enhances the transcription activity of the AR promoter as well as that of the SV40 promoter when the cells are under hyperosmotic stress. Within this 132-base pair sequence, there are three sequences that resemble TonE, the tonicity response element of the canine betaine transporter gene, and the osmotic response element of the rabbit AR gene, suggesting that the mechanism of osmotic regulation of gene expression in these animals is similar. However, our data indicate that cooperative interaction among the three TonE-like sequences in the human AR may be necessary for their enhancer function.

Osmotic Response Element Enhancer Activity REGULATION THROUGH p38 KINASE AND MITOGEN-ACTIVATED EXTRACELLULAR SIGNAL-REGULATED KINASE KINASE

Hypertonicity induces a group of genes that are responsible for the intracellular accumulation of protective organic osmolytes such as sorbitol and betaine. Two representative genes are the aldose reductase enzyme (AR, EC 1.1.1.21), which is responsible for the conversion of glucose to sorbitol, and the betaine transporter (BGT1), which mediates Na+-coupled betaine uptake in response to osmotic stress. We recently reported that the induction of BGT1 mRNA in the renal epithelial Madin-Darby canine kidney cell line is inhibited by SB203580, a specific p38 kinase inhibitor. In these studies we report that the hypertonic induction of aldose reductase mRNA in HepG2 cells as well as the osmotic response element (ORE)-driven reporter gene expression in transfected HepG2 cells are both inhibited by SB203580, suggesting that p38 kinase mediates the activation and/or binding of the transcription factor(s) to the ORE. Electrophoretic gel mobility shift assays with cell extracts prepared from SB203580-treated, hypertonically stressed HepG2 cells further show that the binding of trans-acting factors to the ORE is prevented and is thus also dependent on the activity of p38 kinase. Similarly, treatment of hypertonically stressed cells with PD098059, a mitogen-activated extracellular regulated kinase kinase (MEK1) inhibitor, results in inhibition of the hypertonic induction of aldose reductase mRNA, ORE-driven reporter gene expression, and the binding of trans-acting factors to the ORE. ORE-driven reporter gene expression was not affected by p38 kinase inhibition or MEK1 inhibition in cells incubated in iso-osmotic media. These data indicate that p38 kinase and MEK1 are involved in the regulation of the hyperosmotic stress response.

Ascorbate Synthesis Pathway: DUAL ROLE OF ASCORBATE IN BONE HOMEOSTASIS*

Using mouse gene knock-out models, we identify aldehyde reductase (EC 1.1.1.2, Akr1a4 (GR)) and aldose reductase (EC 1.1.1.21, Akr1b3 (AR)) as the enzymes responsible for conversion of d-glucuronate to l-gulonate, a key step in the ascorbate (ASC) synthesis pathway in mice. The gene knock-out (KO) mice show that the two enzymes, GR and AR, provide ∼85 and ∼15% of l-gulonate, respectively. GRKO/ARKO double knock-out mice are unable to synthesize ASC (>95% ASC deficit) and develop scurvy. The GRKO mice (∼85% ASC deficit) develop and grow normally when fed regular mouse chow (ASC content = 0) but suffer severe osteopenia and spontaneous fractures with stresses that increase ASC requirements, such as pregnancy or castration. Castration greatly increases osteoclast numbers and activity in GRKO mice and promotes increased bone loss as compared with wild-type controls and additionally induces proliferation of immature dysplastic osteoblasts likely because of an ASC-sensitive block(s) in early differentiation. ASC and the antioxidants pycnogenol and resveratrol block osteoclast proliferation and bone loss, but only ASC feeding restores osteoblast differentiation and prevents their dysplastic proliferation. This is the first in vivo demonstration of two independent roles for ASC as an antioxidant suppressing osteoclast activity and number as well as a cofactor promoting osteoblast differentiation. Although humans have lost the ability to synthesize ASC, our mouse models suggest the mechanisms by which suboptimal ASC availability facilitates the development of osteoporosis, which has important implications for human osteoporosis.

Analysis of Candidate Genes for Susceptibility to Type I Diabetes: A Case-Control and Family-Association Study of Genes on Chromosome 2q31–35

Recent genome searches suggest a putative linkage of many loci to susceptibility to type I diabetes. The chromosome 2q31–35 region is reported to be linked to susceptibility to type I diabetes and is thought to contain several diabetes susceptibility loci. These candidate genes include the HOXD gene cluster, BETA2, CTLA4, CD28, IGFBP2, and IGFBP5. Association studies in populations and families are required to confirm and/or identify the actual susceptibility loci. We hereby report several previously unknown DNA polymorphisms for HOXD8, BETA2, and IGFBP5, which we have used along with previously known polymorphisms of H0XD8 and CTLA4 to test whether these candidate loci are the susceptibility genes on chromosome 2q31–35. Using a casecontrol design with a subsequent family-association approach to confirm associations, we find no evidence that these candidate genes are associated with susceptibility to type I diabetes.

Regulation of aldehyde reductase expression by STAF and CHOP

Aldehyde reductase is involved in the reductive detoxification of reactive aldehydes that can modify cellular macromolecules. To analyze the mechanism of basal regulation of aldehyde reductase expression, we cloned the murine gene and adjacent regulatory region and compared it to the human gene. The mouse enzyme exhibits substrate specificity similar to that of the human enzyme, but with a 2-fold higher catalytic efficiency. In contrast to the mouse gene, the human aldehyde reductase gene has two alternatively spliced transcripts. A fragment of 57 bp is sufficient for 25% of human promoter activity and consists of two elements. The 3 element binds transcription factors of the Sp1 family. Gel-shift assays and chromatin immunoprecipitation as well as deletion/mutation analysis reveal that selenocysteine tRNA transcription activating factor (STAF) binds to the 5 element and drives constitutive expression of both mouse and human aldehyde reductase. Aldehyde reductase thus becomes the fourth protein-encoding gene regulated by STAF. The human, but not the mouse, promoter also binds C/EBP homologous protein (CHOP), which competes with STAF for the same binding site. Transfection of the human promoter into ethoxyquin-treated mouse 3T3 cells induces a 3.5-fold increase in promoter activity and a CHOP-C/EBP band appears on gel shifts performed with the 5 probe from the human aldehyde reductase promoter. Induction is attenuated in similar transfection studies of the mouse promoter. Mutation of the CHOP-binding site in the human promoter abolishes CHOP binding and significantly reduces ethoxyquin induction, suggesting that CHOP mediates stimulated expression in response to antioxidants in the human. This subtle difference in the human promoter suggests a further evolution of the promoter toward responsiveness to exogenous stress and/or toxins.

Linkage Analyses in Type I Diabetes mellitus Using CASPAR, a Software and Statistical Program for Conditional Analysis of Polygenic Diseases

We have developed software and statistical tools for linkage analysis of polygenic diseases. We use type I diabetes mellitus (insulin-dependent diabetes mellitus, IDDM) as our model system. Two susceptibility loci (IDDM1 on 6p21 and IDDM2 on 11p15) are well established, and recent genome searches suggest the existence of other susceptibility loci. We have implemented CASPAR, a software tool that makes it possible to test for linkage quickly and efficiently using multiple polymorphic DNA markers simultaneously in nuclear families consisting of two unaffected parents and a pair of affected siblings (ASP). We use a simulation-based method to determine whether lod scores from a collection of ASP tests are significant. We test our new software and statistical tools to assess linkage of IDDM5 and IDDM7 conditioned on analyses with 1 or 2 other unlinked type I diabetes susceptibility loci. The results from the CASPAR analysis suggest that conditioning of IDDM5 on IDDM1 and IDDM4, and of IDDM7 on IDDM1 and IDDM2 provides significant benefits for the genetic analysis of polygenic loci.

Cys298 is responsible for reversible thiol-induced variation in aldose reductase activity.

Aldose reductase (EC 1.1.1.21) has been purified to apparent homogeneity from a variety of tissues including placenta, brain, nerves, kidney, muscle and lens. Multiple molecular forms of aldose reductase have been claimed to be isolated from bovine lens (Jedziniak et al, 1971) and bovine kidney (Gabbay et al 1974). These forms were subsequently described by some authors whereas others found a single form only (for a review see Wermuth, 1985). Conversion by a reducing agent (s-mercaptoethanol) of one form to a more acidic but activity-retaining form was reported by Wermuth et al (1982), and differential susceptibility to inhibition of different enzyme forms was first described by Maragoudakis et al (1984). Nonlinear kinetics were often attributed to the presence of multiple forms. Thus, two kinetically distinct forms of human erythrocyte aldose reductase were postulated (Srivastava et al 1985) and the presence of bovine aldose reductase oxidized by oxygen radical generating systems was suggested as a possible cause for the nonlinear kinetics (Del Corso et al, 1987). More recently, data seem to firmly establish the existence of different forms of aldose reductase: “activated” and “unactivated” forms were isolated from bovine kidney (Grimshaw (1990) and their persistent peculiar kinetic behavior was then essentially rationalized (Grimshaw, 1991, Grimshaw et al 1990, Kubiseski et al, 1992). Recent advances in molecular biology led to the in vitro expression of rat lens aldose reductase (Old et al 1990) and human aldose reductase (Grundmann et al 1990, Carper et al 1990, Nishimura et al 1990, Bohren et al 1991). Multiple molecular forms of recombinant aldose reductase have so far not been reported with the exception of some charge heterogeneity that is evident upon isoelectric focusing (Bohren et al. 1991).

Aldose reductase modulates cardiac glycogen synthase kinase-3β phosphorylation during ischemia-reperfusion

Earlier studies have demonstrated that aldose reductase (AR) plays a key role in mediating ischemia-reperfusion (I/R) injury. Our objective was to investigate if AR mediates I/R injury by influencing phosphorylation of glycogen synthase kinase-3β (p-GSK3β). To investigate this issue, we used three separate models to study the effects of stress injury on the heart. Hearts isolated from wild-type (WT), human expressing AR transgenic (ARTg), and AR knockout (ARKO) mice were perfused with/without GSK3β inhibitors (SB-216763 and LiCl) and subjected to I/R. Ad-human AR (Ad-hAR)-expressing HL-1 cardiac cells were exposed to hypoxia (0.5% O(2)) and reoxygenation (20.9% O(2)) conditions. I/R in a murine model of transient occlusion and reperfusion of the left anterior descending coronary artery (LAD) was used to study if p-GSK3β was affected through increased AR flux. Lactate dehydrogenase (LDH) release and left ventricular developed pressure (LVDP) were measured. LVDP was decreased in hearts from ARTg mice compared with WT and ARKO after I/R, whereas LDH release and apoptotic markers were increased (P < 0.05). p-GSK3β was decreased in ARTg hearts compared with WT and ARKO (P < 0.05). In ARKO, p-GSK3β and apoptotic markers were decreased compared with WT (P < 0.05). WT and ARTg hearts perfused with GSK3β inhibitors improved p-GSK3β expression and LVDP and exhibited decreased LDH release, apoptosis, and mitochondrial pore opening (P < 0.05). Ad-hAR-expressing HL-1 cardiac cells, exposed to hypoxia (0.5% O(2)) and reoxygenation (20.9% O(2)), had greater LDH release compared with control HL-1 cells (P < 0.05). p-GSK3β was decreased and correlated with increased apoptotic markers in Ad-hAR HL-1 cells (P < 0.05). Treatment with phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) inhibitor increased injury demonstrated by increased LDH release in ARTg, WT, and ARKO hearts and in Ad-hAR-expressing HL-1 cells. Cells treated with protein kinase C (PKC) α/β inhibitor displayed significant increases in p-Akt and p-GSK3β expression, and resulted in decreased LDH release. In summary, AR mediates changes in p-GSK3β, in part, via PKCα/β and Akt during I/R.

Characterization of a Novel Murine Aldo-Keto Reductase

The aldo-keto reductase superfamily (Bohren et al., 1989) consists of many reductases that differ in their primary structure, substrate specficities and catalytic properties. Many subfamilies have been recognized, including the aldose reductase, aldehyde reductase, 3α-hydroxysteroid dehydrogenase, androgen regulated protein from the mouse vas deferens, and many more. In the course of cloning murine liver aldo-keto reductases, we discovered yet another aldo-keto reductase with some unique properties. We hereby describe the cloning, sequencing, over-expression and characterization of this novel murine aldo-keto reductase which appears to be uniquely different from any hitherto described member of the superfamily.

Pulsatility of luteinizing hormone during puberty is dependent on recent glycemie control

Abstract We prospectively studied an adolescent girl with insulin-dependent diabetes over a 22-month period. She initially presented with poor glycemie control and arrested pubertal development. Despite an immature luteinizing hormone (LH) secretory profile, she exhibited a mature LH response to injection of a bolus of synthetic gonado-tropin-releasing hormone (GnRH) at the time of initial evaluation. Improvement of her glycemic control was associated with maturation of LH pulsatility, improved estrogen production, and menarche. Subsequent deterioration of her glycemic control resulted in immaturity of pulsatile LH secretory pattern, whereas her response to GnRH remained mature. We hypothesize that the ovarian hypo-function observed was due to inhibition of the hypothalamic GnRH pulse generator and was influenced by the prevailing level of glycemia. In all cases, short-term glycemic control (as measured by fructosamine) was a better predictor of hormonal secretion than was long-term glycemic control (as measured by glycohemoglobin).