Elsevier

Experimental Eye Research

Volume 87, Issue 3, September 2008, Pages 184-190
Experimental Eye Research

Sex hormone regulation of tear lipocalin in the rabbit lacrimal gland

https://doi.org/10.1016/j.exer.2008.05.012Get rights and content

Abstract

Tear lipocalin (TL) (∼18 kDa), a member of the lipocalin superfamily, has been identified as one of the major proteins present in rabbit lacrimal fluid. The concentration of TL has been found to be decreased in the tears of patients with dry eye disease. Lacrimal gland insufficiency, one of the major causes of dry eye disease, is known to affect mainly postmenopausal women, where there is a significant decrease in the production of androgen and estrogen. These observations suggest that sex hormones might influence dry eye indirectly by regulating the expression of TL. The purpose of this study was to determine: (1) the effect of sexual maturation on the expression of TL; and (2) if the expression of TL is regulated by the estrogen, 17β-estradiol, and/or the androgen, dihydrotestosterone, in sexually mature female rabbits. Lacrimal fluid (LF) and lacrimal gland soluble fraction (Si) was collected from juvenile (2 kg) and sexually mature (4 kg) male and female New Zealand white (NZW) rabbits. In addition, LF and Si were collected from 4 kg rabbits, 7 days after being either sham operated (control), ovariectomized (OVX), ovariectomized treated with estrogen (OVX + E) or ovariectomized treated with dihydrotestosterone (OVX + DHT). Samples were analyzed for protein levels of TL by SDS-PAGE and Western blotting using a polyclonal rat anti-rabbit TL antibody. Densitometry analysis showed that TL protein levels in both LF and Si increased with age in male and female rabbits. In addition, TL protein levels were significantly higher in the sexually mature 4 kg male compared with the 4 kg female, while no significant difference in TL protein levels were seen among the juvenile male and female rabbits. Furthermore, ovariectomy decreased the protein levels of TL in LF and Si fraction by 50% and 20% respectively, compared with control values. Estrogen treatment increased TL protein levels by 30% and 50% in the LF and Si fraction respectively, compared with the sham operated group. DHT treatment also increased TL protein levels by approximately 150% in both LF and Si fraction compared with control values. These results support the hypothesis that sex hormones influence TL protein levels in rabbit lacrimal glands. The possibility of a role of TL in dry eye needs to be further investigated.

Introduction

Tear lipocalin (TL) (∼18 kDa), a member of the lipocalin superfamily, has been identified as one of the major proteins present in rabbit lacrimal fluid (Azzarolo et al., 2004). Rabbit TL is expressed in the acinar cells, but not in the interstitial cells suggesting that TL in rabbit lacrimal fluid is of epithelial origin (Azzarolo et al., 2004). Homologues of tear lipocalin have been found in other species such as humans, pig, rat, dog and horse (Redl, 2000). In human tears, TL comprises approximately 33% of the protein content (Azen, 1976).

Although many functions have been suggested for TL, its exact biological role is still unclear. TL, in human tears, is the major lipid binding protein and binds to a wide variety of fatty acids, fatty alcohols, phospholipids, glycolipids, and cholesterol (Glasgow et al., 1995). It has been demonstrated that the surface tension of human tears is due to a complex of TL with tear lipids (Nagyova and Tiffany, 1999), suggesting that TL is important in maintaining the integrity of the tear film. TL along with other tear proteins such as lysozyme and lactoferrin has been suggested to be involved in corneal and conjunctival epithelium defence (Gachon and Lacazette, 1998). Recently, it has been shown that human tear lipocalin also exhibits antimicrobial activity by binding with high affinity to microbial siderophores (Fluckinger et al., 2004).

Lipocalins have also been used as biomarkers for disease, such as breast cancer (Lea et al., 1987) diabetes and coronary heart disease (Schmidt et al., 1999). Similarly, tear lipocalin could be used as a marker to study lacrimal gland performance since the concentration of TL has been found to be decreased in tears of patients with dry eye disease or keratoconjunctivitis sicca when compared to controls (Janssen and Van Bijsterveld, 1983, Xu and Venge, 2000).

One of the most common causes of dry eye disease is lacrimal gland insufficiency (Serrander and Peek, 1993). Lacrimal gland insufficiency affects women much more frequently than men. Of those women, most are postmenopausal, whose production of sex hormones is significantly decreased (Serrander and Peek, 1993), suggesting that sex hormones play a role in the regulation of the lacrimal gland secretory functions. Indeed, androgens have been shown to play a major role in supporting the secretory function (Azzarolo et al., 1997) and regulating various aspects of the lacrimal gland, including epithelial cell morphology, gene expression, synthesis of proteins, and immune function (Sullivan, 2004).

On the other hand, the role of estrogen in the lacrimal gland is still controversial. Previous research suggested that estrogen treatment alone had no effect in rat lacrimal glands (Cornell-Bell et al., 1985, Sullivan and Allansmith, 1986). However, we have subsequently reported that the synthetic estrogen, diethylstilbestrol (DES) influenced biochemical parameters that correlate to secretory function in the rabbit lacrimal gland (Azzarolo et al., 1997). In addition, we have found that 17β-estradiol prevented lymphocytic infiltration and lacrimal gland cell death in ovariectomized (OVX) rabbits (Azzarolo et al., 2003). Messenger RNAs (mRNAs) for both alpha and beta estrogen receptors have been identified in the lacrimal glands (Wickham et al., 1998, Spelsberg et al., 2004).

Since the concentration of TL decreases in patients with dry eye, a condition, which develops primarily during menopause, research suggests that sex hormones might influence dry eye indirectly by regulating the protein levels of TL. Thus, the purpose of this study was to: (1) determine the protein levels of tear lipocalin in lacrimal fluid (LF) and lacrimal gland soluble fraction (Si) in both juvenile and sexually mature male and female rabbits; and (2) determine the effect of androgen and estrogen treatment on the protein levels of TL in LF and Si of OVX rabbits, used as a model for menopause.

Section snippets

Materials

17β-estradiol, dihydrotestosterone, corn oil, phenylmethylsulphonyl fluoride (PMSF), leupeptin, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), trans-epoxysuccinyl-l-leucylamido(4-guanidino) butane (E-64), bestatin, aprotinin, mouse anti-actin IgG antibody, goat anti-mouse IgG antibody, sheep anti-rat IgG antibody, bovine serum albumin (BSA), 2-mercaptoethanol and brilliant blue R-250 were obtained from Sigma (St. Louis, MO). Precision plus protein dual color and nitrocellulose membranes

Tear lipocalin protein levels in the lacrimal fluid of juvenile and sexually mature female and male rabbits

Western blot analyses of TL in the lacrimal fluid showed increased protein levels with age in male and female rabbits (Fig. 1). Protein levels of TL increased by 53% and 28%, respectively, in the 4 kg males and 4 kg females compared with their respective juvenile rabbits. In addition, TL levels were higher in sexually mature 4 kg male rabbits (159.75 ± 7.81) compared with sexually mature 4 kg female rabbits (127.66 ± 8.54), while no significant difference was found between the juvenile 2 kg male (106.72 ±

Discussion

Our results indicate that sex hormones, estrogen and androgen, regulate the protein levels of TL in the rabbit lacrimal gland. Ovariectomy, a procedure, which removes ovarian androgen and estrogens, decreased the levels of TL in both the LF and total gland Si. On the other hand, treatment of ovariectomized rabbits with DHT or 17β-estradiol increased the levels of TL compared to sham operated rabbits.

The regulation of TL by sex hormones agrees with previous observations in which other members of

Acknowledgements

The authors thank Dr. Keith Brew for his suggestions and helpful comments in the preparation of this manuscript.

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