Up-regulation of Human myo-Inositol Oxygenase by Hyperosmotic Stress in Renal Proximal Tubular Epithelial Cells

K Sandeep Prabhu,Ryan J Arner,Hema Vunta,C Channa Reddy

doi:10.1074/jbc.m502621200

K Sandeep Prabhu, Ryan J Arner + Show 2 more

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https://doi.org/10.1074/jbc.m502621200

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Abstract

myo-Inositol oxygenase (MIOX) catalyzes the oxidative cleavage of myo-inositol (MI) to give d-glucuronic acid, a committed step in MI catabolism. d-Glucuronic acid is further metabolized to xylitol via the glucuronate-xylulose pathway. Although accumulation of polyols such as xylitol and sorbitol is associated with MI depletion in diabetic complications, no causal relationship has been established. Therefore we are examining the role of MIOX in diabetic nephropathy. Here we present evidence that the basis for the depletion of MI in diabetes is likely to be mediated by the increased expression of MIOX, which is induced by sorbitol, mannitol, and xylitol in a porcine renal proximal tubular epithelial cell line, LLC-PK1. To understand the molecular mechanism of regulation of MIOX expression by polyols, we have cloned the human MIOX gene locus of 10 kb containing 5.6 kb of the 5' upstream sequence. Analysis of the 5' upstream sequence led to the identification of an osmotic response element (ORE) in the promoter region, which is present approximately 2 kb upstream of the translation start site. Based on luciferase reporter and electrophoretic mobility shift assays, polyols increased the ORE-dependent expression of MIOX. In addition, we demonstrate that the activity of the promoter is dependent on the binding of the transcription factor, tonicity element-binding protein, or osmotic response element-binding protein, to the ORE site. These results suggest that the expression of MIOX is up-regulated by a positive feedback mechanism where xylitol, one of the products of MI catabolism via the glucuronate-xylulose pathway, induces an overexpression of MIOX.

Highlights

Myo-Inositol (MI),1 the dominant form of the physiological inositol isomers, is utilized in many tissues and cell types as a precursor for the synthesis of second messengers and as an organic osmolyte [1]
We present evidence that the basis for the depletion of MI in diabetes is likely to be mediated by the increased expression of myo-Inositol oxygenase (MIOX), which is induced by sorbitol, mannitol, and xylitol in a porcine renal proximal tubular epithelial cell line, LLC-PK1
We have employed the porcine renal proximal tubular epithelial cell line (LLC-PK1) that expresses MIOX to test the inducibility of MIOX by hyperosmolar stress

Summary

Introduction

Myo-Inositol (MI), the dominant form of the physiological inositol isomers, is utilized in many tissues and cell types as a precursor for the synthesis of second messengers and as an organic osmolyte [1]. We have recently reported the cloning and expression of MIOX, where we demonstrated that D-chiro-inositol, a MI isomer that exhibits insulin-like signaling properties [4], is a substrate for MIOX [2]. In diabetic complications, increased glucose levels are associated with high sorbitol accumulation in the kidney, retina, nerve, and lens, which is followed by depletion of MI [5,6,7,8,9]. Depletion of tissue MI and accumulation of sorbitol and xylitol seems to be a common feature observed in most of the pathologies associated with diabetes; the mechanism of depletion of MI in diabetes remains largely unknown. Our studies reported here are based on the hypothesis that in the kidney cortex, osmotic gradients of metabolites, including polyols, lead to the activation of MIOX expression. It is well established that the expression of several renal-specific genes is under the influence of osmotic stress, via a plethora of cellular signaling events, including the protein kinase pathways and

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