Chronic therapeutic use of steroid hormones to treat a variety of inflammatory conditions carries a high risk of cataracts, even in children. There is no consensus on the mechanism of steroid induced cataracts and disagreement on whether the lens contains classic steroid receptors. The purpose of this project is to exploit a serendipitous observation to provide a fresh approach to exploring the basis of steroid induced cataracts. We discovered a 28 kDa membrane protein present in the epithelium of lenses from several species, including humans, that binds steroids with high affinity. This protein, originally discovered in liver microsomal membrane, is purported to be a new type of steroid receptor and is termed the membrane steroid binding protein (MSBP). We hypothesize that this protein mediates nongenomic actions of steroids on the eye, actions which can contribute to lens opacification. Our broad goals are to clone the genes for this protein (which may be prenylated), described the protein's steroid hormone binding characteristics and the types of steroid actions the protein may mediate. Based upon partial sequence information, primers were used to amplify, clone and sequence a 290 bp segment of the MSBP which was subsequently digoxigenin labeled. This probe will be used to screen cDNA libraries from bovine, rat and human lens for the genes of MSBP. The structure of the coded protein(s) will be deduced, including identification of prenylation sites. The lens epithelial cell membrane fraction responsible for binding steroids will be identified by enzyme analysis and Percoll gradient distribution. Is it plasma or microsomal membrane? Binding studies using radiolabeled steroids will be conducted to establish the binding hierarchy of steroid hormones, principally glucocorticoids, relative to the standard, 3H-progesterone. The capacity of steroids to induce """"""""rapid""""""""-nongenomic effects on lens cells will be evaluated by measuring the capacity of steroids to affect epithelial cell free calcium levels (using Fura-2 fluorescence spectroscopy), Na+/K+- ATPase (using ouabain inhibitable 86Rb uptake), apoptosis measured by the TUNEL assay and in vivo rates of lens epithelial cell migration and differentiation following pulse labeling with 3H-thymidine. Preliminary findings and published data support the possibility that steroids can rapidly elevate intracellular calcium and inhibit Na+/K+-ATPase activity in lens epithelial cells.
| Samadi, Abbas; Carlson, C George; Gueorguiev, Anton et al. (2002) Rapid, non-genomic actions of progesterone and estradiol on steady-state calcium and resting calcium influx in lens epithelial cells. Pflugers Arch 444:700-9 |
| Zhu, X L; Sexton, P S; Cenedella, R J (2001) Characterization of membrane steroid binding protein mRNA and protein in lens epithelial cells. Exp Eye Res 73:213-9 |
