Kurt M. Bohren

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.

Autocatalytic modification of human carbonyl reductase by 2-oxocarboxylic acids

Carbonyl reductase occurs in multiple molecular forms. Sequence analysis has yielded a carboxyethyllysine residue in one of the enzyme forms, suggesting that pyruvate has been incorporated in a posttranslational enzymatic reaction [Krook, M., Ghosh, D., Strömberg, R., Carlquist, M. and Jörnvall, H. (1993) Proc. Natl. Acad. Sci. USA 90,502‐506]. Using highly purified carbonyl reductase from human brain we show that pyruvate and other 2‐oxocarboxylic acids are bound to the enzyme in an autocatalytic reaction. The resulting enzyme forms were indistinguishable from the native enzyme forms by electrophoresis and isoelectric focusing.

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).

Arginine metabolic endotypes in pulmonary arterial hypertension.

Decreased synthesis of nitric oxide (NO) by NO synthases (NOS) is believed to play an important role in the pathogenesis of pulmonary arterial hypertension (PAH). Multiple factors may contribute to decreased NO bioavailability, including increased activity of arginase, the enzyme that converts arginine to ornithine and urea, which may compete with NOS for arginine; inadequate de novo arginine production from citrulline; and increased concentration of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NOS. We hypothesized that PAH patients with the lowest arginine availability secondary to increased arginase activity and/or inadequate de novo arginine synthesis might have a slower rate of NO synthesis and greater pulmonary vascular resistance. Nine patients with group 1 PAH and 10 healthy controls were given primed, constant intravenous infusions of 15N2-arginine, 13C,2H4-citrulline, 15N2-ornithine, and 13C-urea in the postabsorptive state. The results showed that, compared with healthy controls, PAH patients had a tendency toward increased arginine clearance and ornithine flux but no difference in arginine and citrulline flux, de novo arginine synthesis, or NO synthesis. Arginine-to-ADMA ratio was increased in PAH patients. Two endotypes of patients with low and high arginase activity were identified; compared with the low-arginase group, the patients with high arginase had increased arginine flux, slower NO synthesis, and lower plasma concentrations of ADMA. These results demonstrate that increased breakdown of arginine by arginase occurs in PAH and affects NO synthesis. Furthermore, there is no compensatory increase in de novo arginine synthesis to overcome this increased utilization of arginine by arginase.

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.

Indian Women of Childbearing Age Do Not Metabolically Conserve Arginine as Do American and Jamaican Women

BACKGROUND In a previous study in pregnant American women, we reported that arginine flux and nitric oxide synthesis increased in trimester 2. More recently, we reported that Indian women do not increase arginine flux during pregnancy as their American or Jamaican counterparts do. OBJECTIVE The purpose of this study was to determine whether Indian women of childbearing age are producing less arginine and/or catabolizing more arginine and therefore have less available for anabolic pathways than do Jamaican and American women. METHODS Thirty healthy women aged 28.3 ± 0.8 y from the United States, India, and Jamaica (n = 10/group) were given 6 h primed, constant intravenous infusions of guanidino-¹⁵N₂-arginine, 5,5-²H₂-citrulline, ¹⁵N₂-ornithine, and ring-²H₅-phenylalanine, in addition to primed, oral doses of U-¹³C₆-arginine in both the fasting and postprandial states. An oral dose of deuterium oxide was also given to determine fat-free mass (FFM). RESULTS Compared with American women, Indian and Jamaican women had greater ornithine fluxes (μmol · kg fat FFM⁻¹ · h⁻¹) in the fasting and postprandial states (27.3 ± 2.5 vs. 39.6 ± 3.7 and 37.2 ± 2.0, respectively, P = 0.01), indicating greater arginine catabolism. However, Jamaican women had a higher endogenous arginine flux than did Indian and American women in the fasting (66.1 ± 3.1 vs. 54.2 ± 3.1 and 56.1 ± 2.1, respectively, P = 0.01) and postprandial (53.8 ± 2.2 vs. 43.7 ± 4.9 and 42.8 ± 3.1, respectively, P = 0.06) states. As a consequence, Indian women had lower arginine bioavailability (μmol · kg FFM⁻¹ · h⁻¹) in the fasting state (42.0 ± 2.6) than did American (49.9 ± 1.3, P = 0.045) and Jamaican (55.5 ± 3.5, P = 0.004) women, as well as in the postprandial state (40.7 ± 3.5 vs. 51.8 ± 1.2 and 57.5 ± 3.2, respectively, P = 0.001). CONCLUSION Compared with American and Jamaican women, Indian women of childbearing age have a decreased arginine supply because of increased arginine catabolism without an increase in arginine flux.

Characterization of an Element Resembling an Androgen Response Element (are) in the Human Aldose Reductase Promoter

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.