Department of Obstetrics and Gynecology, Cellular and Molecular Biology Program, 6428 Medical Science I Box 0617, University of Michigan Medical Center, Ann Arbor, MI 48109-0617, USA
Received on August 6, 2003; revised on October 3, 2003; accepted on October 6, 2003
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Abstract |
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Key words: endocervical glands / fucosyltransferase / glandular stomach / mucus
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Introduction |
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Classes of fucosyltransferase are divided by the glycosidic linkage catalyzed (Breton et al., 1998). FUT2, the secretor gene, encodes an
(1,2)fucosyltransferase (E.C. 2.4.1.69) found in mucosal epithelium of buccal, gastrointestinal, respiratory, breast, and genitourinary tracts of humans and other mammals. Polymorphism of FUT2 is well recognized and is associated with a number of inactivating mutations (Oriol et al., 2000
). Approximately 20% of various populations are homozygous for enzyme-inactivating mutations, called nonsecretors, lack
(1,2)fucose residues on secreted mucins. The function(s) of FUT2 remains controversial, but polymorphism of this gene has been proposed to be of medical importance because absence of a functional
(1,2)fucosyltransferase in mucosal tissues is associated with differences in susceptibility to microbial infections, inflammation, and malignancy (D'Adamo and Kelly, 2001
; Ishitoya et al., 2002
; Lindesmith et al., 2003
; Marionneau et al., 2002
; Ronchetti et al.,2001
).
In the reproductive tract, expression of specific uterine glycans vary during the ovulatory cycle (Carson, 2002; Kimber et al., 2001
). Correlated with alterations in the hydrodynamic properties of cervical mucus, cervical mucins show changes in oligosaccharide structures during the ovulatory cycle (Argueso et al., 2002
; Yurewicz et al., 1987
). In women, nonsecretor status is associated with recurrent vulvovaginal candidiasis (Chaim et al., 1997
), whereas absence of a functional FUT2 gene was associated with reduced risk of HIV-1 infection (Ali et al., 2000
) suggesting that alterations in vaginal mucosal immunity are associated with expression of FUT2, either increased or decreased susceptibility depending on the pathogen.
Despite the large number of fucosylated glycans and proposed biological functions, little is know about the regulation of their expression. Because FUT2 is expressed in both gastrointestinal and reproductive tracts, we investigated the hormonal regulation and cell type of expression of Fut2 in a mutant lacZ reporter mouse expressing the bacterial reporter gene lacZ under the endogenous promoter for Fut2.
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Results |
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The effects of estradiol and estrogen-primed progesterone treatments on Fut2 and Fut1 steady-state mRNA levels were compared in ovariectomized mice. Analogous to X-gal staining in Fut2-LacZ mice, estradiol treatment of ovariectomized wild-type mice increases Fut2 expression 15-fold in uterus with no significant change by estrogen-primed progesterone treatment or estrogen plus progesterone (Figure 6A). Levels of Fut1 were tested because Fut1 was previously reported to be stimulated by estrogen in mouse uterus (Sidhu and Kimber, 1999) (those studies were published prior to the cloning of Fut2 and likely used a probe nonselective between Fut1 and Fut2). Using DNA probes selective for Fut1 versus Fut2, Fut1 mRNA levels do not significantly vary with hormone replacement (Figure 6B).
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Discussion |
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Localization of X-gal staining to glandular mucosa of the endocervix throughout pregnancy is an unexpected finding. In studies focusing on a potential role of fucosyltransferase genes in implantation, decreased fucosyltransferase expression was noted in early pregnancy (Sidhu and Kimber, 1999; White and Kimber, 1994
). In this study, minimal levels of X-gal staining are seen in the gravid uterus. Prominent X-gal expression, however, is present in the endocervix. Because endocervical glands represent only a small portion of the total uterus on homogenization, previous northern blot analyses showing an overall decrease in expression during pregnancy are consistent with the data presented here.
Although Fut2-null mice were noted to display normal fertility eliminating an essential role for this gene in implantation, the endocervix was not studied (Domino et al., 2001a). Endocervical expression that varies during the estrous cycle and prominent expression during pregnancy suggests a potential protective role against ascending infection analogous to the hypothesized role of glycosyltransferases genes in the gastrointestinal tract (Henry, 2001
). In humans, expression of fucosylated blood group antigens in cervical mucus and on the cell surface of vaginal cells vary cyclically in both secretor and nonsecretor women (Schaeffer et al., 1994
). Because hostpathogen interactions frequently involve cell surface glycans as adhesion molecules, polymorphism of the secretor gene have been proposed to be associated with susceptibility to microbial infections (Varki, 1993
). A role for FUT2 in modifying cervical mucus barrier properties is supported by epidemiological studies of woman showing variation with the nonsecretor polymorphism in the prevalence of recurrent vulvovaginal candidiasis with Candida albicans, and urinary tract infection by some strains of Escherichia coli (Chaim et al., 1997
; Stapleton et al., 1995
).
A possible mechanism for affect of FUT2 on microbe adhesion is alteration of cervical mucin glycosylation. Multiple mucin forms are expressed in endocervix and MUC5B expression varies with the ovulatory cycle (Gipson et al., 2001). Microbe binding could be altered by shifting the balance of mucin glycosylation between fucosylation and sulfation (Scharfman et al., 1996
) similar to cystic fibrosis mice, which were found to have elevated Fut2 expression in intestine (Thomsson et al., 2002
). Expression of the Fut2 cannot always be considered protective for mucosal immunity, however, because the risk of HIV-1 infection was increased in secretor women relative to nonsecretors (Ali et al., 2000
). Other data suggest a positive effect on hostmicrobe interaction from the loss of FUT2, such as a lower incidence of Helicobacter pylori exposure in secretor-null individuals (Ikehara et al., 2001
). An effect of hormonally regulated genes on sexually transmitted infections, whether protective or deleterious, appears to depend on the specific infecting organism (Brabin, 2002
).
This study did not address whether regulation of Fut2 expression by estrogen is direct or indirect. Estrogen receptor- (ER-
) has been reported to be present in mouse uterus throughout gestation, but with decreasing levels as gestation progressed (Spong et al., 2000
). Both epithelial and stromal ER-
were found to be necessary for the production of uterine secretory proteins (Buchanan et al., 1999
). ER-ß subtype ligands also exerted an effect on some markers of estrogen action (Frasor et al., 2003
). To consider potential transcriptional regulation, the 5' genomic regions of mouse and human Fut2 were searched for estrogen response consensus sequences. Although annotation of the human and mouse Fut2 genes are currently provisional, mouse Fut2 mRNA (NCBI RefSeq NM_018876) and human FUT2 mRNA (NCBI RefSeq NM_000511) were aligned to the respective genome sequence from the Mouse and Human Genome Consortiums using the UCSC Genome Bioinformatics Human Genome Browser Gateway (Karolchik et al., 2003
). The corresponding genomic regions (6000 bp) 5' to exon 1 from mouse (chr7:3430735334313352 minus strand, February 2003 freeze) and human (chr19:53853281 to 53859280 plus strand, April 2003 freeze) were scanned with the MacInspector program V2.2 using TRANFAC 4.0 matrixes (Wingender et al., 2000
). No consensus estrogen response elements were found, although multiple weak partial matches to the half palindrome site GGTCA are present.
ER-mediated transcriptional regulation may, however, proceed through nonclassical ER-/Sp1 binding, where estrogen binds its receptor followed by binding to Sp1 transcription factors, which then directs the complex to a binding motif in the 5' region (Khan et al., 2003
). Multiple potential Sp1 binding motifs were present in the Fut2 5' region along with dozens of potential transcription factor sites. Alternatively to transcriptional control, the 3' untranslated region of human Fut2 mRNA may form a large stem-and-loop structure (1.2 kb) potentially regulating the stability and level of the Fut2 transcript (Koda et al., 1997
). Study of cells from Fut2-LacZ tissues would benefit from the relative ease of assay for LacZ and may help elucidate molecular mechanism of Fut2 hormonal regulation. The Fut2-LacZ mouse may also serve as a model of the human nonsecretor for testing the importance of cell surface
(1,2)fucosylated glycans in hostmicrobe interactions.
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Materials and methods |
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X-gal staining
Control experiments for X-gal staining using wild-type strain 129X1/SvJ and strain C57BL/6 J (stock no. 000664) showed no background staining in the tissues tested. The experimental mutant mice, Fut2-LacZ, were on a hybrid 129X1/SvJ and C57BL/6 J background. To prepare tissues for X-gal staining, 89-week-old Fut2-LacZ and wild-type mice were terminally perfused with ice-cold phosphate buffered saline followed by freshly thawed 4% paraformaldehyde (Turkay et al., 1999). Tissues were excised, rinsed in phosphate buffered saline, then partially fixed in ice-cold 4% paraformaldyde for 20 min on ice with occasional shaking. Tissues were washed with 0.1 M sodium phosphate, pH 7.3, three times and stained with X-gal overnight at 37°C as described (Saunders, 2002
). To preserve the blue X-gal precipitant, tissues were postfixed in 10% formalin and 0.2% glutaraldehyde overnight, then processed for paraffin sectioning and nuclear fast red staining avoiding all organic solvents (Histoserv, Germantown, MD). Whole-organ pictures were photographed with a 35 mm macrolens.
Northern blot analysis
Total RNA was isolated by the guanidine isothiocyanate/phenol method (TRIzol; GibcoBRL, Grand Island, NY) followed by isolation of polyadenylated RNA by Oligotex midi mRNA columns (Qiagen, Valencia, CA). Colon samples were taken from the mid-portion of the large intestine of each animal. Independently isolated samples from individual mice containing 5 µg polyadenylated RNA were run on formaldehyde agarose gels, transferred to nylon, and hybridized with 32P-dCTP random primed DNA probes specific for Fut1 and Fut2 exactly as previously described (Domino et al., 2001b). Blots were stripped and hybridized with probes for either ribosomal protein L32 (113-bp probe purified from a Hind III digestion of mouse L32 template; BD Biosciences Pharmingen, San Diego, CA; no. 45181P) or cyclophillin-A (Ambion, Austin, TX; mouse DECAprobe template no.7375). A PhosphorImager Model SP was used to visualize and quantify northern blot radioactivity (Amersham Biosciences, Piscataway, NJ). Northern blot data were normalized to the level of L32 in each lane. One-way analysis of variance with Dunnett's multiple comparison test was performed using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, CA).
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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