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(Investigative Ophthalmology and Visual Science. 2008;49:1797-1808.)
© 2008 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.07-1458
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Estrogen and Progesterone Control of Gene Expression in the Mouse Meibomian Gland

Tomo Suzuki,1,2 Frank Schirra,1,2 Stephen M. Richards,1,2 Roderick V. Jensen,3 and David A. Sullivan1,2

1From the Schepens Eye Research Institute and the 2Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts; and the 3Department of Physics, University of Massachusetts, Boston, Massachusetts.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. The purpose of the study was to test the hypothesis that estrogen and progesterone regulate gene expression in the meibomian gland.

METHODS. Meibomian glands were obtained from young adult, ovariectomized mice that were administered 17β-estradiol, progesterone, 17β-estradiol plus progesterone, or vehicle for 14 days. Glands were pooled according to treatment, processed for the extraction of RNA, and analyzed for differentially expressed mRNAs by using mouse gene microarrays. Bioarray data were evaluated with sophisticated bioinformatics software and statistical programs. The expression of selected genes was confirmed with gene chips and quantitative real-time PCR techniques.

RESULTS. The findings show that 17β-estradiol, progesterone, or both hormones administered together significantly influenced the expression of numerous genes in the mouse meibomian gland. Notable were the effects of 17β-estradiol on genes related to lipid metabolism, tyrosine kinases, immune factors, extracellular matrix components, steroidogenesis, and prolactin dynamics. Also very significant were the actions of progesterone or 17β-estradiol plus progesterone on ribosome or localization gene ontologies, respectively. The various hormone treatments led to many analogous, opposite, or unique effects on gene expression.

CONCLUSIONS. These findings support the study hypothesis that estrogen and progesterone modulate gene expression in the meibomian gland.

The preocular tear film plays a critical role in maintaining ocular surface integrity, defending against microbial challenge, and preserving visual acuity.1 These functions, in turn, are dependent on the composition and stability of the tear film structure, which includes an underlying mucin foundation, a middle aqueous component, and an overlying lipid layer.1 Instability or deficiency of the tear film may severely influence ocular health. If not managed with artificial tear substitutes or tear film conservation therapy, these disorders may lead to intractable desiccation and ulceration of the corneal epithelium, an increased incidence of infectious disease, and ultimately, pronounced visual impairment.1

Throughout the world, countless people have tear film dysfunctions that are collectively diagnosed as dry eye syndromes.1 Most individuals with dry eye syndromes are women (Caffery B et al. IOVS 1996;37:ARVO Abstract 335).1 2 3 4 5 6 7 8 9 10 In fact, being of the female sex has been termed a risk factor for the development of dry eye (Caffery B et al., IOVS, 1996;37:ARVO Abstract S72). However, the mechanism(s) involved in this sex-associated difference in the prevalence of dry eye syndromes is unknown. We hypothesize that this difference is due, at least in part, to the effects of endogenous or exogenous estrogens on the meibomian gland.

This tissue, which is a large sebaceous gland, produces the tear film’s lipid layer and is very important in preventing the evaporation and promoting the stability of the tear film.1 11 12 13 14 Conversely, meibomian gland dysfunction (MGD), and the resulting lipid insufficiency, leads to instability and evaporation of the tear film,1 11 15 16 17 and MGD is believed to be the major cause of dry eye syndromes in the world.18

Recent research suggests that estrogen therapy in postmenopausal women may promote both MGD and evaporative dry eye. Thus, an epidemiologic evaluation of 25,665 postmenopausal women demonstrated that women using estrogen replacement therapy have a significantly higher prevalence of severe dry eye symptoms and clinically diagnosed dry eye syndrome, compared with women who never used the treatment.19 Similarly, an assessment of 44,257 women with dry eye showed that one of the highest prevalences of comorbid conditions was the use of estrogen replacement therapy.20 We hypothesize that this estrogen effect is due primarily to a suppression of meibomian gland function. We also hypothesize that this hormone action is mediated through specific nuclear receptors, which in turn regulate gene expression in the meibomian gland. Consistent with these hypotheses is the finding that the meibomian gland contains estrogen receptor mRNA and protein.21 22 23 In addition, we have discovered that hormone replacement therapy containing estrogens is associated with a significant alteration in the polar lipid profile of meibomian gland secretions in postmenopausal women (Sullivan BD, Sullivan DA, unpublished data, 2002).

The purpose of the present investigation was to determine whether estrogen modulates gene expression in the meibomian gland. For comparison, we examined whether progesterone, alone or in combination with estrogen, may also affect gene activity in this tissue.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animals and Hormone Treatment
Age-matched, young adult BALB/c mice, that had been ovariectomized when 8 weeks old, were purchased from Taconic Laboratories (Germantown, NY). The animals were maintained in constant-temperature rooms with a fixed light–dark period of 12 hours’ duration. Ten days after surgery, pellets containing vehicle (cholesterol, methylcellulose, and lactose), 17β-estradiol (0.5 mg), progesterone (10 mg), or a combination of 17β-estradiol and progesterone were implanted subcutaneously (SC) in the ovariectomized mice. The pellets were obtained from Innovative Research of America (Sarasota, FL) and were designed for the constant release of placebo or physiological amounts of sex steroid (i.e., as in pregnancy)24 25 26 27 for 3 weeks. After 14 days of treatment, mice (n = 7–20 mice/condition/experiment) were killed by CO2, inhalation, and meibomian glands were removed from the upper and lower lids under direct visualization with a biomicroscope. This surgical procedure involved making a small incision near the inner corner of the eyelid, separating skin and SC tissue from the inner to outer aspect of the lid, and then removing skin from the meibomian glands by cutting at the mucocutaneous junction. After these steps, the palpebral conjunctiva was removed from the meibomian glands, and the glands were dissected from the remaining tissue by starting at the outer lid corner and carefully avoiding an adjacent vein. The isolated meibomian glands were pooled according to group (n = 14–40 glands/sample) and processed for RNA analysis. All experiments with mice were approved by the Institutional Animal Care and Use Committee of The Schepens Eye Research Institute and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Molecular Biological Procedures
To assess the influence of 17β-estradiol and progesterone on meibomian gland gene expression, we extracted total RNA from the tissues (TRIzol reagent; Invitrogen Corp., Carlsbad, CA). When indicated, samples were exposed to RNase-free DNase (Invitrogen), evaluated spectrophotometrically at 260 nm to determine concentration, and analyzed on 6.7% formaldehyde/1.3% agarose (Invitrogen-Gibco, Grand Island, NY) gels to confirm RNA integrity. The RNA samples were then processed by using several different procedures.

The primary method for evaluating gene expression involved the use of gene microarrays (~10,000 genes; CodeLink Uniset Mouse I Bioarrays; GE Healthcare, Piscataway, NJ). For this procedure, glandular RNA samples were further purified (RNAqueous spin columns; Ambion, Austin, TX), and the integrity of these preparations was verified with a bioanalyzer (RNA 6000 Nano LabChip with a model 2100 Bioanalyzer; Agilent Technologies, Palo Alto, CA). The RNA samples were then processed for the bioarray hybridization, according to reported techniques.28 Briefly, cDNA was synthesized from RNA (2 µg) with a kit (CodeLink Expression Assay Reagent Kit; GE Healthcare) and purified (QIAquick purification kit; Qiagen, Valencia, CA). After the samples were dried, cRNA was generated with a kit (CodeLink Expression Assay Reagent Kit; GE Healthcare), recovered (RNeasy kit; Qiagen), and quantified with a UV spectrophotometer. Fragmented, biotin-labeled cRNA was then incubated and shaken (300 rpm shaker) on a bioarray at 37°C for 18 hours. After this period, the bioarray was washed, exposed to streptavidin-Alexa 647, and scanned (ScanArray Express software and a ScanArray Express HT scanner; Packard BioScience, Meriden, CT), with the laser set at 635 nm, laser power at 100%, and photomultiplier tube voltage at 60%. Scanned image files were examined by using image and data-analysis software (CodeLink; GE Healthcare), which yielded both raw and normalized hybridization signal intensities for each array spot. The spot intensities (~10,000) on the microarray image were normalized to a median of 1. Standardized data, with signal intensities exceeding 0.50, were evaluated (GeneSifter.Net software; VizX Laboratories LLC, Seattle, WA). This comprehensive software program also produced gene ontology and z-score reports. These ontologies included biological processes, molecular functions, and cellular components and were organized according to the guidelines of the Gene Ontology Consortium (http://www.geneontology.org/GO.doc.html).29 Gene expression data were analyzed with and without log transformation, and statistical evaluation of the data was performed with Student’s t-test (two-tailed, unpaired). The data from the individual bioarrays (n = 6) are accessible for download from the National Center for Biotechnology Information’s Gene Expression Omnibus (http://www.genesifter.net/web/dataCenter.html/ NCBI, Bethesda, MD) via series accession number GSE5783.

Differentially expressed mRNAs were also examined on gene chips (GEM 1, >8000 genes; GEM 2, >9500 genes; Incyte Genomics, Inc., St. Louis, MO), which have over 4700 sequences in common with the CodeLink arrays. For these studies RNA samples were further purified on spin columns (RNAqueous; Ambion, Inc.), and poly(A) mRNA was isolated from meibomian gland RNA samples (400 µg; MicroPoly(A)Pure mRNA Isolation Kit; Ambion, Inc.). The mRNA concentration was determined (RiboGreen RNA Quantitation Kit; Invitrogen-Molecular Probes, Eugene, OR), according to Incyte’s instructions. After assigning mRNA samples (750 ng) for use with either the cy3 or cy5 probes, preparations were suspended in TE buffer, put in siliconized microfuge tubes (RNase-Free; Ambion), and shipped on dry ice to Incyte for hybridization. Gene chip data were sent electronically to the Harvard Center for Genomic Research (Cambridge, MA), downloaded into a data analysis system (Resolver Gene Expression Data Analysis System, ver. 2.0; Rosetta Inpharmatics, Kirkland, WA), normalized, and analyzed as previously described.28

Real-Time PCR Procedures
The differential expression of selected genes was confirmed by using quantitative real-time PCR (qPCR). Sense and antisense primers (Table 1) were designed on computer (Primer Express Software, ver. 1.5a; Applied Biosystems, Inc. [ABI], Foster City, CA). The qPCR reactions were conducted according to the manufacturer’s protocol, with aliquots of meibomian gland cDNA (0.01–0.3 µL cDNA), optimal primer concentrations, and master mix (Applied Biosystems’ SYBR Green PCR Master Mix, MicroAmp Optical 96-Well Reaction Plates, ABI PRISM Optical Adhesive Covers, and the GeneAmp 7900 HT Sequence Detection System). Gene expression was calculated according to the relative standard curve method as outlined by the manufacturer (User Bulletin 2 ABI Prism 7700 Sequence Detection System; Applied Biosystems), and then standardizing data to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Dissociation curves were monitored to verify the absence of secondary PCR products.


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  		TABLE 1. Oligonucleotide Primers for Real-Time PCR Confirmation of Selected Gene Expression

 

   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
To determine the effect of "female" sex steroids on gene expression in the meibomian gland, we isolated tissues from ovariectomized mice (n = 7/treatment group) who had been treated for 14 days with 17β-estradiol, progesterone, both hormones in combination or placebo. Glands were processed for evaluation (Uniset Mouse I Bioarrays; CodeLink; GeneSifter software; VizX Labs).

Analysis of non- and log-transformed data from three different studies showed that 17β-estradiol and progesterone have a highly significant influence on gene expression in the meibomian gland. As demonstrated in Table 2 , these hormones, whether administered individually or together, significantly modified the expression of numerous genes. The nature of these sex steroids’ effects was dependent on the treatment, with 17β-estradiol up (48.3%)- and down (51.7%)-regulating approximately the same number of genes, whereas progesterone predominantly decreased gene expression (i.e., with 17β-estradiol: 63% of genes {downarrow}; without 17β-estradiol: 83% of genes {downarrow}; Table 2 ). Those genes that demonstrated the greatest hormone-induced differences in terms of ratios are listed in Tables 3 4 and 5 . Genes that showed the greatest alterations in statistical significance included those increased or decreased by 17β-estradiol (neurotrophic tyrosine kinase, receptor, type 2 {uparrow}, P < 0.00002; arginine vasopressin receptor 1A {downarrow}, P < 0.0003), progesterone (gastric intrinsic factor {uparrow}, P < 0.0003; desmoplakin {downarrow}, P < 0.006), and 17β-estradiol plus progesterone ({alpha}1 microglobulin/bikunin{uparrow}, P < 0.00002; cell division cycle 6 homologue {downarrow}, P < 0.0004).


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  		TABLE 2. Effect of 17β-Estradiol and/or Progesterone on Gene Expression in the Meibomian Gland

 

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  		TABLE 3. 17β-Estradiol’s Effect on Gene Expression Ratios in the Mouse Meibomian Gland

 

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  		TABLE 4. Progesterone’s Impact on Gene Expression Ratios in the Mouse Meibomian Gland

 

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  		TABLE 5. 17β-Estradiol+Progesterone’s Impact on Gene Expression Ratios in the Mouse Meibomian Gland

 
Exposure to these sex steroids had striking effects on many biological process, molecular function, and cellular component ontologies in the meibomian gland. For instance, 17β-estradiol, progesterone, and the combined sex steroid administration all significantly influenced the expression of more than 60 genes associated with cellular metabolism, binding, and intracellular organelles (Table 6) .


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  		TABLE 6. Influence of Sex Steroid Treatment on the Expression of Gene Ontologies in the Meibomian Gland

 
Quite notable was the impact of 17β-estradiol on genes related to lipid metabolism and/or lipid binding (Tables 7 8) , as well as to tyrosine kinases (e.g., {uparrow} growth arrest specific 6), immune factors (e.g., {uparrow} interleukin 1 receptor, type II), extracellular matrix (ECM) components (e.g., {downarrow} secreted acidic cysteine rich glycoprotein), steroidogenesis (e.g., {uparrow} cytochrome P450, family 7, subfamily b, polypeptide 1; CYP7B1), and prolactin dynamics (e.g., {uparrow} prolactin receptor, {uparrow} signal transducer and activator of transcription 5A; STAT5A; Tables 3 7 8 ). Estrogen treatment also exerted significant influences on genes in a variety of KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways. These actions included an overall suppression of genes in pathways linked to fatty acid metabolism (e.g., {downarrow} hydroxyacyl-coenzyme A dehydrogenase; z-score = 2.77), cell cycle (e.g., {downarrow} TGF-b3; z-score = 3.02) and cytokine-cytokine receptor interactions (e.g., {downarrow} TGF-b3, {downarrow} vascular endothelial growth factor A; z-score = 2.16), an upregulation of genes in the Jak-Stat signaling pathway ({uparrow} growth hormone receptor, {uparrow} STAT5A; z-score = 2.68), and an alteration of genes in the MAPK signaling pathway ({uparrow} fibroblast growth factor receptor 1, {downarrow} TGF-b3).


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  		TABLE 7. Highest and Lowest Expression of Gene Ontologies in the Meibomian Glands of Placebo- or 17β-Estradiol-Treated Mice

 

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  		TABLE 8. Influence of 17β-Estradiol on the Expression of Meibomian Gland Genes Related to Lipid Metabolism and/or Lipid Binding

 
Progesterone stimulated the expression of genes involved in transport, ATP binding and organelle membranes, and reduced the activity of genes linked to protein biosynthesis, structural molecules, and ribosomes (Table 9) . The most dramatic effect of progesterone was the suppression of genes associated with ribosome biogenesis, assembly, and structure. For example, progesterone downregulated 29 genes related to structural constituents of ribosomes and upregulated none (Table 10) .


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  		TABLE 9. Highest and Lowest Expression of Gene Ontologies in the Meibomian Glands of Placebo- or Progesterone-Treated Mice

 

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  		TABLE 10. Progesterone Downregulation of Meibomian Gland Genes Encoding Structural Constituents of Ribosomes

 
Combined 17β-estradiol and progesterone treatment exerted a significant effect on many ontologies that were similar to, or different from, those modulated by 17β-estradiol or progesterone alone (Table 11) . A distinctive result was the influence of combined hormone administration on 71 genes in the localization ontology. Of these genes, 39 were also affected by either 17β-estradiol or progesterone treatment. However, the remaining 42 genes were altered only by combined hormone exposure (Table 12) .


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  		TABLE 11. Highest and Lowest Expression of Gene Ontologies in the Meibomian Glands of Placebo- or 17β-Estradiol+Progesterone-Treated Mice

 

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  		TABLE 12. Effects of Combined 17β-Estradiol and Progesterone on the Expression of Meibomian Gland Genes in the Localization Ontology

 
In these experiments the administration of 17β-estradiol, progesterone, or both steroids together led to many analogous, opposite, or unique effects on gene expression (Table 13) . There were genes significantly upregulated by 17β-estradiol (nuclear FMRP interacting protein 1, EIA) and 17β-estradiol plus progesterone (EIA) that were downregulated by progesterone. Moreover, there were genes that were only stimulated (e.g., cytosolic phosphoenolpyruvate carboxykinase 1, programed cell death 4) or suppressed (e.g., RAR-related orphan receptor gamma, chemokine [C-C motif] ligand 17) by combined 17β-estradiol and progesterone treatment.


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  		TABLE 13. Analogous and Opposite Effects of 17β-Estradiol and Progesterone on Gene Expression in the Meibomian Gland

 
To verify in part the CodeLink Bioarray data, additional meibomian gland mRNA samples (n = 20 mice/group/experiment) were processed for GEM-1 and -2 gene chip analyses. These methods identified genes on GEM chips that were influenced by 17β-estradiol (n = 42 genes), progesterone (n = 20 genes), or combined hormone (n = 14 genes) exposure, and that met our evaluation criteria (i.e., GEM signal intensity ≥ 100; expression ratio ≥ 1.8 or ≤ -1.8). However, only seven of these genes were common to both the GEM and CodeLink platforms. Consequently, to confirm further the CodeLink results, various genes were examined by qPCR. This experimental approach verified the action of hormones on the specified genes (Table 14) .


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  		TABLE 14. Confirmation of Selected CodeLink Microarray and GEM Chip Results

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In our study, 17β-estradiol, progesterone, or both hormones combined significantly altered the expression of numerous genes in the mouse meibomian gland. The various hormone treatments induced analogous, opposite, or unique effects on gene activity. These data support our hypothesis that estrogen and progesterone modulate gene transcription in the meibomian gland.

Estrogen treatment had a significant impact on a wide variety of meibomian gland genes. For example, 17β-estradiol upregulated genes encoding epoxide hydrolase 2, cytoplasmic, which plays a role in xenobiotic metabolism30 and glutathione peroxidase 3, a secreted protein that protects cells and enzymes from oxidative damage.30 In addition, 17β-estradiol downregulated genes for carbonic anhydrase 6, which is involved in the reversible hydration of carbon dioxide.30 and arginine vasopressin receptor 1A, which stimulates glycogenolysis and suppresses fluid secretion.30

Of particular interest was the effect of 17β-estradiol on genes related to tyrosine kinases, immune factors, ECM components, steroidogenesis, prolactin dynamics, and lipid metabolism. With regard to tyrosine kinases, 17β-estradiol upregulated genes for fibroblast growth factor receptor 1 (a member of the tyrosine kinase superfamily),30 neurotrophic tyrosine kinase receptor type 2 (a membrane tyrosine-protein kinase receptor involved in nervous system development and/or maintenance),30 and growth arrest specific 6 (a ligand for tyrosine-protein kinase receptors whose signaling is implicated in cell growth, migration, adhesion, and survival).30 In contrast, 17β-estradiol downregulated the gene for neural precursor cell expressed developmentally downregulated gene 9 (coordinates tyrosine-kinase-based signaling of cell adhesion).30

As concerns immune factors, estradiol increased the expression of genes for (1) interleukin 1 receptor type II, which is the receptor for IL-1{alpha}, IL-1β, and IL-1 receptor antagonist protein30 ; and (2) IL-4 receptor {alpha}, which is the receptor for both IL-4 and -13. IL-4 promotes Th2 cell differentiation, and both IL-4 and -13 modulate IgE, chemokine, and mucus production at sites of allergic inflammation.30 Estrogen is also known to enhance IL4 receptor mRNA levels in the rat uterus.32 (3) {alpha}1 microglobulin/bikunin, which was the most highly upregulated (>280-fold), is a lipocalin with immunosuppressive properties.33 It also functions as an inter-{alpha}-trypsin inhibitor and blocks trypsin, plasmin, and lysosomal granulocytic elastase.30 Bikunin is a small chondroitin sulfate proteoglycan that occurs as the light chain of inter-{alpha}-trypsin inhibitor family members.30 (4) STAT5A, which is a transcription factor mediating the action of specific cytokines, growth factors, and hormones on gene expression and has been implicated in breast cancer development.34 Similarly, 17β-estradiol upregulated the gene for stanniocalcin 2, which is known to be estrogen-responsive and coexpressed with the estrogen receptor in human breast cancer.35

17β-estradiol exerted effects on several genes associated with the ECM. This hormone downregulated genes for matrix metallopeptidase 3 (degrades fibronectin, laminin, gelatins and proteoglycans30 ), cathepsin K (degrades extracellular matrix30 ), secreted acidic cysteine rich glycoprotein (regulates the ECM27 ), and procollagen, type I, -{alpha}1, and -{alpha}2 and upregulated the gene for tissue inhibitor of metalloproteinase 4 (inhibits matrix metalloproteinases). For comparison, estrogen is also known to decrease cathepsin K in mouse osteoclasts,36 and ovariectomy is associated with an increased expression of genes for secreted acidic cysteine rich glycoprotein; procollagen, type I, -{alpha}1 and -{alpha}2; as well as glutathione peroxidase 3, in mouse adipose tissue.37

17β-Estradiol upregulated a variety of genes involved in steroidogenic pathways, including cytochrome P450, family 17, subfamily a, polypeptide 1 (CYP17A1), CYP7B1, 11β-hydroxysteroid dehydrogenase 1, and 17β-hydroxysteroid dehydrogenase 7 (17β-HSD1). The CYP17A1 promotes DHEA formation,30 whereas CYP7B1 diverts DHEA from the sex hormone biosynthetic pathway.30 17β-HSD1 converts estrone to biologically active estradiol38 and also associates with the prolactin receptor.39 Estrogen downregulated the gene for androgen-binding protein {eta}, a secretoglobin originally identified in the mouse lacrimal gland.40

Estrogen also modulated genes associated with prolactin dynamics. 17β-Estradiol significantly increased the mRNA levels of the prolactin receptor, as well as those for STAT5A, which is known to activate prolactin-induced transcription.30 Prolactin, in turn, regulates estrogen receptor expression, and this action requires both intact STAT5 binding sites and functional STAT5.41 In addition, prolactin may directly repress expression of fatty acid synthase through STAT5A binding.42

A prominent effect of 17β-estradiol was the regulation of genes related to lipid metabolism. 17β-Estradiol treatment enhanced the expression of genes encoding monoacylglycerol O-acyltransferase 1, which catalyzes the formation of diacylglycerol,30 and phosphatidylcholine transfer protein, which replenishes the plasma membrane with phosphatidylcholines.43 Estrogen also decreased the genes encoding acyl-Coenzyme A oxidase 2, branched chain, which is involved in the degradation of long-branched fatty acids30 and carboxylesterase 3, a lipase.44 Given these hormone actions, one might conclude that 17β-estradiol promotes lipid elaboration.

However, most of estrogen’s effects were not consistent with this conclusion. Rather, 17β-estradiol appeared to have an overall negative influence on lipid production. Thus, estrogen administration stimulated the expression of several genes involved in lipid and/or fatty acid catabolism, including the anti-lipogenic STAT5A,36 growth hormone receptor (growth hormone decreases fatty acid synthase activity45 ), WNT1 inducible signaling pathway protein 2, phospholipase A2 (group VII), and adiponectin. Further, 17β-estradiol downregulated genes involved in lipid synthesis (i.e., acyl-CoA synthetase bubblegum family member 1),30 lipid mobilization (i.e., arylacetamide deacetylase),46 lipid processing (i.e., hydroxyacyl-coenzyme A dehydrogenase), lipid formation, and membrane trafficking (phospholipase D1).47 48 A decline in phospholipase D1 may also lead to a reduction in the generation of phosphatidic acid, which is an intracellular lipid mediator of many biological functions.48

Given these antagonistic effects, it might be anticipated that estrogen administration would suppress lipid synthesis in the meibomian gland and promote both MGD and evaporative dry eye. In support of this hypothesis, estrogens have been demonstrated to cause a significant decrease in the size, activity and lipid synthesis of sebaceous glands in multiple species.49 50 51 52 53 54 55 56 In fact, estrogen has been termed the prototype agent for the suppression of sebum production,55 and for years estrogen treatment was used to reduce sebaceous gland function and secretion in humans.50 53 54 57 58 59 Such an estrogen action in the meibomian gland could account for the increased prevalence of dry eye syndromes in postmenopausal women taking estrogen replacement therapy.19 60 61 62 63 64 In addition, estrogen–meibomian gland interactions may explain why estrogen treatment of women in several studies led to tear film instability, foreign body sensation, contact lens intolerance, and ocular surface dryness.61 65

Progesterone had a unique effect on the meibomian gland, which is known to contain progesterone receptors and respond to this hormone with an apparent change in morphology.21 22 66 Most genes influenced by this progesterone were downregulated and were different from those modulated by estradiol. In addition, the magnitude of progesterone’s action was moderate, with changes in gene expression predominately less than threefold. Progesterone upregulated a variety of genes related to transcription (e.g., jun-B oncogene and MAP kinase-interacting serine/threonine kinase 1), cytokines (e.g., interleukin 8 receptor, β), peroxisomes (e.g., acyl-coenzyme A dehydrogenase, short chain), and desmosomes (e.g., desmoplakin). This effect on desmoplakin, which helps to anchor intermediate filaments,30 is also elicited by progesterone in breast cancer cells.67 68 In contrast, progesterone downregulated a diverse array of genes, such as those associated with immune processes (e.g., nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 4, and β2 microglobulin), gluconeogenesis (e.g., glucose-6-phosphatase catalytic), and energy transduction (e.g., creatine kinase, brain). Creatine kinase is also found in sebaceous glands and many different epithelia, presumably because of the manifold energy requirements of these cells (e.g., high proliferation rates, active ion pumps, and transport processes).69

By far, the most impressive effect of progesterone in the meibomian gland was the downregulation of all genes related to ribosome biogenesis, assembly, and structure. The z-score for progesterone’s action on ribosome structural constituents alone exceeded 20, which is extraordinarily high. This hormone effect suggests that progesterone may have an overall suppressive impact on protein, macromolecule, and cellular biosynthesis in the meibomian gland. For comparison, progesterone was once thought to be the tropic hormone responsible for sebaceous gland secretion in women (i.e., analogous to androgens in men),50 given that sebum production was significantly increased by progestin treatment.50 70 71 However, other groups have reported that progestin administration has no effect on sebaceous gland output,70 72 and yet others found that these hormones decrease sebaceous gland function by inhibiting local androgen metabolism and action.73 74 75 76 One explanation for these conflicting results is that progestin’s effects on different types of sebaceous glands appear to be significantly influenced by dose, endocrine environment, and sex.76 77 78 79

Many of the effects of combined estradiol and progesterone treatment on meibomian gland gene expression duplicated those of estradiol or progesterone treatment alone. Unique actions included downregulation of certain immune-related genes (e.g., RAR-related orphan receptor gamma and chemokine [C-C motif] ligand 17), and upregulation of genes associated with gluconeogenesis (e.g., phosphoenolpyruvate carboxykinase 1, cytosolic), and topoisomerase inhibition (e.g., programmed cell death 4; PDCD4). The PDCD4 gene transcribes a tumor suppressor protein that inhibits protein synthesis, AP-1-dependent transcription and cell proliferation and promotes apoptosis.80 81 82 83 Combined estrogen and progestin administration also induced a unique upregulation of genes involved with the localization ontology. The reason for this influence is unclear.

In summary, our study demonstrates that 17β-estradiol and progesterone have a significant impact on meibomian gland gene expression. We believe that these sex steroid effects, and in particular those of estrogen, contribute to the sex-associated difference (i.e., female predominance) in the prevalence of dry eye syndromes.


   Acknowledgements
 
The authors thank Michael J. Lombardi, Patricia Rowely, and Nathaniel S. Treister (Schepens Eye Research Institute) for technical assistance and the Harvard Center for Genomic Research for help with processing GEM-1 and -2 data.


   Footnotes
 
Supported by National Eye Institute Grants EY05612, K16, and EY12523; Allergan; Advanced Medical Optics; Bausch & Lomb; German Research Society (Deutsche Forschungsgemeinschaft) Grant SCHI 562/1-1 and 1-2); and the Japanese Eye Bank Association.

Submitted for publication November 13, 2007; revised December 21, 2007; accepted March 4, 2008.

Disclosure: T. Suzuki, Advanced Medical Optics (F), Allergan (F), Bausch & Lomb (F); F. Schirra, None; S.M. Richards, None; R.V. Jensen, None; D.A. Sullivan, Allergan (C)

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: David A. Sullivan, Schepens Eye Research Institute, 20 Staniford Street, Boston, MA 02114; david.sullivan{at}schepens.harvard.edu.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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