Expression of the transmembrane mucins, MUC1, MUC4 and MUC16, in normal endometrium and in endometriosis

Abstract

Study question: Are the transmembrane mucins, MUC1, MUC4 and MUC16, differentially expressed in endometriosis compared with normal endometrium?

Summary answer: This study revealed that transmembrane mucin expression does not vary significantly in normal endometrium during the menstrual cycle and is not altered in endometriosis relative to the epithelial marker, cytokeratin-18 (KRT18).

What is known already: Increased serum levels of the transmembrane mucin fragments MUC1, MUC4 and MUC16 that normally dominate the apical surface of simple epithelia are found in several pathological conditions, including endometriosis. Altered mucin expression in gynecologic diseases may promote infertility or endometrial pathologies.

Study design, size, duration: This was a laboratory-based study of samples from 12 endometriosis patients as well as non-endometriosis control samples obtained from 31 patients.

Participants/materials, setting, methods: Total RNA was isolated from endometrial biopsies of ectopic and eutopic endometrium from women with endometriosis and control patients from different stages of the menstrual cycle. Quantitative (q)-RT-PCR analyses were performed for the mucins, MUC1, MUC4 and MUC16, relative to the epithelial marker, cytokeratin-18 (KRT18), or β-actin (ACTB). Frozen sections from endometrial biopsies of proliferative and mid-secretory stage women with endometriosis were immunostained for MUC1, MUC4 and MUC16.

Main results and the role of chance: qRT-PCR analyses of MUC1 and MUC16 mRNA revealed that these mucins do not vary significantly during the menstrual cycle nor are they altered in women with endometriosis relative to the epithelial marker, KRT18. MUC4 mRNA is expressed at very low levels relative to MUC1 and MUC16 under all conditions. There was little difference in MUC1 and MUC16 expression between eutopic endometrial and ectopic endometriotic tissues. MUC4 expression also was not significantly higher in the ectopic endometriotic tissues. Immunostaining for all three mucins reveals robust expression of MUC1 and MUC16 at the apical surfaces of endometrial epithelia, but little to no staining for MUC4.

Limitations, reasons for caution: qRT-PCR analysis was the main method used for mucin detection. Additional studies with stage III-IV endometriotic tissue would be useful to determine if changes in MUC1 and MUC16 expression occur, or if MUC4 expression increases, at later stages of endometriosis.

Wider implications of the findings: We report a comprehensive comparative profile of the major transmembrane mucins, MUC1, MUC4 and MUC16, relative to the epithelial marker, KRT18, in normal cycling endometrium and in endometriosis, and indicate constitutive expression. Previous studies have profiled the expression of individual mucins relative to β-actin and indicate accumulation in the luteal phase. Thus, these differences in interpretation appear to reflect the increased epithelial content of endometrium during the luteal phase.

Study funding: This study was supported by: NIH R01HD29963 to D.D.C.; NIH U54HD007495 to S.M.H.; and NIH R01HD067721 to S.L.Y. and B.A.L. The authors have no competing interests to declare.

Keywords: cytokeratin-18; endometriosis; endometrium; mucins.

Figure 1

Mucin mRNA profile in normal endometrium. Box plots were used to express levels of transmembrane mucin mRNA levels in the cycling human endometrium. Mucin mRNA expression was determined relative to that of epithelial marker, cytokeratin-18 (KRT18) by qRT–PCR as described in Materials and Methods. Mucins were expressed at similar levels at all stages of the cycle with very low expression of MUC4. The box plot shades the second and third quartiles of the data. The black line in the box is drawn at the median value. The whiskers above and below show the maximum and minimum values, respectively.

Figure 2

Mucin mRNA levels relative to those of KRT18 (A) and ACTB (B). MUC1, MUC16, KRT18 and ACTB mRNA levels were determined by qRT–PCR as described in Materials and Methods. Box plots were used to express these data as described in the legend to Fig. 1. No significant differences in mucin expression were observed across the cycle when compared with KRT18 mRNA levels.

Figure 3

Relative abundance of mucin mRNAs in normal endometrium and in endometrium of women with endometriosis. MUC1, MUC4 and MUC16 mRNA in normal endometria at different stages of the menstrual cycle and in endometria of women with endometriosis relative to KRT18 mRNA were determined as described in Materials and Methods. Box plots were used to express the data as described in the legend to Fig. 1. No significant differences were observed between these conditions.

Figure 4

A comparison of MUC4 mRNA detection by primers against tandem repeat (TR) and cytoplasmic tail (CT) regions of the MUC4 gene. MUC4 mRNA in normal endometria at different stages of the menstrual cycle and in endometria of women with endometriosis relative to KRT18 mRNA using primers to the TR and CT regions. Box plots were used to express these data as described in the legend to Fig. 1. No significant differences were observed using either set of primers.

Figure 5

(A) MUC1, (B) MUC4 and (C) MUC16 mRNA expression in stage-matched eutopic and ectopic endometria of endometriosis patients. Six menstrual stage-matched samples, either from secretory or proliferative stage of the menstrual cycle and stage II or III (one sample) endometriosis were analyzed by qRT–PCR for the different mucins as described in Materials and Methods. Box plots were used to express these data as described in the legend to Fig. 1. Changes in levels of MUC4 expression was observed in endometriotic tissue when compared with eutopic endometriotic tissue.

Figure 6

MUC1, MUC4 and MUC16 expression in normal endometrium and in endometriosis. Frozen sections were stained with antibodies to the indicated mucins as described in Materials and Methods. Pan-cytokeratin staining was used to detect epithelia in each case (F, H, J and L). DAPI staining was used to detect nuclei as described in Materials and Methods. Robust staining for both MUC1 (B and D) and MUC16 (C, D, K and L) were observed in all cytokeratin positive (epithelial) cells. No MUC4 staining was detected (G and H). DAPI: blue; pan-cytokeratin or MUC1: green; MUC4 or MUC16: red. Magnification for all fields is as indicated in (L), 50 μm.