Estrogen up-regulation of metalloproteinase-2 and -9 expression in rabbit lacrimal glands

https://doi.org/10.1016/j.exer.2007.02.002Get rights and content

Abstract

Increased levels of the matrix metalloproteinases (MMPs)-2 and -9 have been found in tear fluids of patients with dry eye disease, suggesting that these MMPs may be implicated in the pathogenesis of this disease. One of the main causes of dry eye disease is lacrimal gland insufficiency. However, the contribution of the lacrimal gland (LG) to the expression and production of MMP-2 and MMP-9 in tears is not known. Since dry eye disease occurs more frequently in women, sex hormones, especially estrogens, have also been implicated in the pathogenesis of this disease. Estrogens have been shown to regulate the synthesis levels of MMP-2 and MMP-9 in several tissues, Thus, the purpose of these studies was to determine if: (1) rabbit lacrimal glands secrete MMP-2 and MMP-9; (2) MMP-2 and MMP-9 are produced by lacrimal epithelial cells and/or lacrimal lymphocytes; and (3) the expression, activity and level of these enzymes are regulated by sex hormones. Lacrimal epithelial cells (LEC) and lacrimal lymphocytes (LL) from sexually mature New Zealand White female rabbits were isolated, purified and cultured with and without 10−6 M dihydrotestosterone (DHT) or 10−6, 10−8, 10−9 and 10−10 M 17β-estradiol (E2). The culture supernatants were analyzed by zymography and western blotting (WB) using polyclonal anti-human MMP-2 and MMP-9 antibodies. LGs were also collected from rabbits 7 days after being sham-operated, ovariectomized (OVX), OVX treated with 4 mg/kg DHT, and OVX treated with 0.5 mg/kg of E2. LGs were collected and processed for RNA extraction as well as protein determination using WB and immunocytochemistry. The pro-forms of MMP-2 and MMP-9 were detected in primary LEC and LL culture medium by zymography and WB. Pro-MMP-2 and pro-MMP-9 were also detected at the gene and protein levels in the lacrimal glands of all four treatment groups, with the highest levels and gene expression found in the estrogen-treated group. These results suggest that both pro-MMP-2 and pro-MMP-9 are secreted by the lacrimal gland and appear to be up-regulated by estrogen. The role of the lacrimal MMPs in the pathogenesis of dry eye disease needs to be further investigated.

Introduction

The matrix metalloproteinases (MMPs) are a family of structurally and functionally related endopeptidases that are responsible for the proteolytic degradation of extracellular matrix (ECM) components. Individual MMPs have different but overlapping substrate specificities. MMP-9 (gelatinase B, type IV collagenase), and MMP-2 (gelatinase A, type IV collagenase) are enzymes that hydrolyze type IV collagen, the major component of basement membrane. All MMPs are produced in a latent form (pro-MMP) requiring activation for catalytic activity, a process that involves proteolytic removal of the pro-peptide domain.

MMPs are produced by many cell types, including T and B lymphocytes, fibroblasts, macrophages, neutrophils and some epithelial cells (Smith et al., 2001). They are also present in various fluids, including tears. MMP-9 activity found in tears is presumed to be principally derived from the epithelium and polymorphonuclear cells (Sack et al., 2002). However, the contribution of the lacrimal gland to the expression of MMP-2 and MMP-9 is not known.

In addition to their normal roles in tissue remodeling, MMP-2 and MMP-9 are known to be critical extracellular matrix remodeling enzymes in wound healing and diseases of the ocular surface (Li et al., 2001). Both MMP-2 and MMP-9 have been shown to accumulate in tears and saliva of patients with systemic and non-systemic dry eye disease (Konttinen et al., 1998, Smith et al., 2001), as well as in different mouse models of Sjögren’s syndrome (Robinson et al., 1997, Konttinen et al., 1998).

Many factors regulate the expression of MMPs, including hormones, growth factors, cytokines, cell–cell interactions and cellular transformation. Thus, sex steroids such as estrogen and dehydroepiandrosterone (DHEA) have been shown to regulate MMPs. Estrogen is known to increase both MMP-9 mRNA and activity in mesangial cells (Potier et al., 2001), and to increase MMP-2 activity and protein expression in human granulosa lutein cells (Puistola et al., 1995). In addition, 17β-estradiol regulates MMP-2 activity and protein expression in human retinal pigment epithelium (RPE) (Marin-Castano et al., 2003), and increases MMP-2, -7 and -9 mRNAs in immortalized human corneal epithelial cells (Suzuki and Sullivan, 2005). DHEA treatment, on the other hand, has been shown to decrease MMP-2 activity in the ovary (Henmi et al., 2001).

Since MMP-2 and MMP-9 are up-regulated in the tears of patients with dry eye disease, and sex hormones have been implicated in the pathogenesis of this disease, the purpose of this study was to determine if: (1) rabbit lacrimal glands secrete MMP-2 and MMP-9; (2) MMP-2 and MMP-9 are produced by lacrimal epithelial cells and/or lacrimal lymphocytes; and (3) the expression, activity and level of these enzymes are regulated by sex hormones.

Section snippets

Materials

High glucose Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), Ham's nutrient mixture F-12, low glucose DMEM, penicillin, streptomycin and glutamine were purchased from Irvine Scientific (Irvine, CA). Hank's balanced salt solution (Ca2+ and Mg2+ free), soybean trypsin inhibitor, laminin, carbachol, linoleic acid, bovine serum albumin (BSA), 17β-estradiol, dihydrotestosterone, 2-mercaptoethanol, phenylmethylsulfonyl fluoride (PMSF), leupeptin, 4-(2-aminoethyl) benzenesulfonyl

Cellular sources of MMP-2 and MMP-9 in the lacrimal gland in vitro

Analysis of MMP-2 and MMP-9 using gelatin zymography in the culture supernatant from both lacrimal gland epithelial cells (LEC) and lacrimal lymphocytes (LL) collected after 4 days of culture, revealed bands of protease activity which corresponds to pro-MMP-2 (69 kDa) and pro-MMP-9 (93 kDa) (Fig. 1A). The active forms of MMP-9 or MMP-2 were never observed in this study.

Western blot analyses of LEC and LL showed only two bands corresponding to the 69 kDa pro-MMP-2 and a 93 kDa pro-MMP-9 (Fig. 1B and

Discussion

Semi-quantitative RT-PCR, western blotting, immunocytochemistry and substrate gel zymography analysis showed that the lacrimal glands produce pro-MMP-2 and pro-MMP-9 at both the gene and protein levels. The gelatinase activity and levels of pro-MMP-2 and pro-MMP-9 in the culture supernatant from primary lacrimal epithelial cells (LEC) and lacrimal lymphocytes (LL) culture, suggest both an epithelial and an interstitial origin for these proteins.

Previous research has shown that MMP-2 and MMP-9

Acknowledgements

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

References (53)

  • U. Puistola et al.

    Regulation of 72 kd Type IV collagenase matrix metalloproteinase 2 by estradiol and gonadotropin releasing hormone agonist in human granulosa lutein cells

    Fertil. Steril.

    (1995)
  • S. Silbiger et al.

    Estradiol suppresses type I collagen synthesis in mesangial cells via activation of activator protein-1

    Kidney Int.

    (1999)
  • C.S. Wingrove et al.

    17β-Oestradiol enhances release of matrix metalloproteinase-2 from human vascular smooth muscle cells

    Biochim. Biophys. Acta.

    (1998)
  • P.M. Yao et al.

    Expression of matrix metalloproteinase gelatinases A and B by cultured epithelial cells from human bronchial explants

    J. Biol. Chem.

    (1996)
  • X. Yu et al.

    Involvement of insulin/phosphoinositide 3-kinase/Akt signal pathway in beta-estradiol-mediated neuroprotection

    J. Biol. Chem.

    (2004)
  • F. Acconcia et al.

    Synergism between genomic and non genomic estrogen action mechanisms

    IUBMB Life.

    (2003)
  • A.M. Azzarolo et al.

    Sex-dependent parameters related to electrolyte, water and glycoprotein secretion in rabbit lacrimal glands

    Curr. Eye Res.

    (1993)
  • A.C. Buisson et al.

    Gelatinase B is involved in the in vitro wound repair of human respiratory epithelium

    J. Cell. Physiol.

    (1996)
  • S.W. Cousins et al.

    Female gender, estrogen loss, and Sub-RPE deposit formation in aged mice

    Invest. Ophthalmol. Vis. Sci.

    (2003)
  • S.R. da Costa et al.

    Microtubules facilitate the stimulated secretion of B-hexosaminidase in lacrimal acinar cells

    J. Cell Sci.

    (1998)
  • T.E. Daniels et al.

    Association of patterns of labial salivary gland inflammation with keratoconjunctivitis sicca. Analysis of 618 patients with suspected Sjögren's syndrome

    Arthritis Rheum.

    (1994)
  • G.H. Di et al.

    Over expression of aromatase protein is highly related to MMPs levels in human breast carcinomas

    J. Exp. Clin. Cancer Res.

    (2005)
  • D. Ferrara et al.

    Androgen and estrogen receptors expression in the rat exorbital lacrimal gland in relation to “harderianization”

    J. Exp. Zool. Part A Comp. Exp. Biol.

    (2004)
  • E.J. Filardo et al.

    Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptors through release of HB-EGF

    Mol. Endocrinol.

    (2000)
  • D. Greenbaum et al.

    Comparing protein abundance and mRNA expression levels on a genomic scale

    Genome Biol.

    (2003)
  • R.X. Guo et al.

    Activation of phosphatidylinositol 3-kinase-protein kinase B (PI3K-PK) induced by 17beta-estradiol in endometrial carcinoma cell (Ishikawa)

    Zhonghua Fu Chan Ke Za Zhi.

    (2004)

    • Cited by (24)

    • TFOS DEWS II Sex, Gender, and Hormones Report

      2017, Ocular Surface
      Citation Excerpt :

      Although the work did not clearly define the criteria to label DED individuals or the possible causes of DED in the non TAO group, it indicated a higher expression of pro-inflammatory proteins in both TAO groups, suggesting possible biomarkers and unique mechanisms of disease in DED caused by TAO [960]. Animal studies reveal sex-based differences that are rooted in biology and appear to affect risk for DED and could shed light on the mechanisms behind the disease and its presentation [89,98,263,265,527,671]. For example, Zylberberg et al. [671] found increased levels of the pro-MMPs-2 and -9 in cell cultures obtained from female rabbits' lacrimal glands that were exposed to estradiol but not to DHT.

    • Does estrogen deficiency cause lacrimal gland inflammation and aqueous-deficient dry eye in mice?

      2014, Experimental Eye Research
      Citation Excerpt :

      Consistent with this possibility are some (Sato and Sullivan, 1994; Sullivan, 2004; Toda et al., 1999), but not other (Affinito et al., 2003; Arakaki et al., 2010; Azzarolo et al., 2003; Forsblad-d'Elia et al., 2009; Guaschino et al., 2003; Hayashi et al., 2004; Ishimaru et al., 2003, 1999; Kuscu et al., 2003; Mostafa et al., 2012; Pelit et al., 2003; Shim et al., 2004; Sullivan, 2004; Takahashi et al., 1997; Taner et al., 2004), research findings. Indeed, with the possible exception of specific genes (Suzuki et al., 2006), no consensus exists concerning the cellular targets for, or the cellular processes that may be regulated by, estrogens in lacrimal tissue (Affinito et al., 2003; Azzarolo et al., 2003; Imbert et al., 2009; Ishimaru et al., 2008; Kuscu et al., 2003; Paliwal and De, 2006; Pelit et al., 2003; Sato and Sullivan, 1994; Seamon et al., 2008; Srikantan and De, 2008; Srikantan et al., 2007; Sullivan, 2004; Suzuki et al., 2006; Taner et al., 2004; Zylberberg et al., 2007). In fact, it has not yet even been established whether estrogens have functional receptors in the lacrimal gland.

    • Ocular surface immunity: Homeostatic mechanisms and their disruption in dry eye disease

      2012, Progress in Retinal and Eye Research
      Citation Excerpt :

      Androgen can stimulate meibomian gland genes related to lipid metabolic pathways. Its deficiency in human may promote meibomian gland dysfunction and evaporative dry eye (Sullivan et al., 2009, 2002) while estrogen may up-regulate metalloproteinase-2 and -9 expression by rabbit lacrimal glands (Zylberberg et al., 2007). The effect of sex hormones on the immuno-inflammatory responses in DED is not widely investigated.

    • Is dry eye disease the same in young and old patients? A narrative review of the literature

      2022, BMC Ophthalmology

    • Once-Daily Topical Phosphosulindac Is Efficacious in the Treatment of Dry Eye Disease: Studies in Rabbit Models of Its Main Clinical Subtypes

      2022, Journal of Ocular Pharmacology and Therapeutics

    • Transforming growth factor-β1 in intrauterine adhesion

      2020, American Journal of Reproductive Immunology
    View all citing articles on Scopus
    1

    These authors contributed equally to this work.

    View full text
    Copyright © 2007 Elsevier Ltd. All rights reserved.