Research ArticleExperimental Studies
Open Access

Expression of the O-Linked N-Acetylglucosamine-containing Epitope H (O-GlcNAcH) in Human Uterine Cervical Mucosa

FOTIOS ARAPIS, DIMITRA REMPELOU, SOPHIA HAVAKI, DIMITRIOS ARVANITIS, KONSTANTINOS TZELEPIS, ARISTEIDIS ZIBIS, ATHINA A. SAMARA and SOTIRIOS SOTIRIOU
In Vivo May 2024, 38 (3) 1112-1118; DOI: https://doi.org/10.21873/invivo.13545

Abstract

Background/Aim: Epitope H contains an O-linked N-acetylglucosamine (O-GlcNAcH) residue in a specific conformation or environment, recognized by a site-specific monoclonal mouse IgM antibody H. O-GlcNAcH occurs in several normal and pathological cells and in several polypeptides, including keratin-8 and vimentin, on the latter in cells under stress. Materials and Methods: In this work, we studied the distribution of O-GlcNAcH on cells of endocervical mucosa in 60 specimens of endocervical curettings, 10 of which contained 15 inflamed polyps. Results: In our results, expression of O-GlcNAcH was weak in the mucosa with <5% mucin-secreting cells and up to 30% of the polyps staining positively. All non-ciliated, non-mucin-secreting cells, normal and hyperplastic ‘reserve’ cells, as well as the cells of immature squamous metaplasia, showed strong diffuse cytoplasmic staining for O-GlcNAcH. In mature squamous epithelium, fewer than 5% of basal cells and all the intermediate and superficial cells showed cytoplasmic staining for O-GlcNAcH, whereas parabasal cells were negative. All ciliated cells showed patchy or diffuse cytoplasmic staining. Nuclear staining for O-GlcNAcH was weak with fewer than 5% of hyperplastic ‘reserve’ and ciliated cells staining positively. Moreover, mucosal fibroblasts were negative, whereas all stromal cells of the polyps showed strong cytoplasmic staining for O-GlcNAcH. Conclusion: O-GlcNAcH is: a) differentially expressed among the cellular elements of mucosa and polyps, b) upregulated in mucin-secreting cells of polyps, c) induced in stromal cells of inflamed polyps, and d) can be used as a marker to differentiate between ‘reserve’ (positive) and parabasal (negative) cells, which have similar morphology using conventional cytological stains.

Key Words:

Post-translational modification of the serine and threonine residues of polypeptides by the addition of the sugar moiety N-acetylglucosamine (GlcNAc) to hydroxyl groups (O-GlcNAcylation) occurs in specific conformations or environments in many nuclear, cytoplasmic, and mitochondrial proteins (1). These comprise a wide range of functional and structural proteins, such as transcription factors, enzymes, and cytoskeletal proteins, which are involved in regulation of intracellular signaling, including regulation of transcription and translation, cytoskeletal networks, stress responses, and the ubiquitin-proteasome system (1, 2).

O-GlcNAcylation is reversible and the addition of the GlcNAc takes place through the action of the enzyme O-GlcNAc transferase (3) and its removal through the action of beta-N-acetylglucosaminidase (4). The donor of the GlcNAc is UDP-GlcNAc, which is the end-product of the hexosamine biosynthetic pathway, which uses approximately 2-3% of cellular glucose (5).

Epitope H (O-GlcNAcH) contains an O-GlcNAc residue. O-GlcNAcH is expressed in several types of normal and pathological cells, such as in mitochondria-rich normal, metaplastic, and neoplastic cells (6), ductal breast adenocarcinoma cells (7-9), uterine leiomyomas and leiomyosarcomas (10) and in brain astrocytic tumors (11, 12). Fluctuations in the expression of O-GlcNAcH have been noted under certain conditions. For example, in normal human brain tissue, only a minority of fibrous astrocytes express the epitope, whereas the epitope is greatly up-regulated in reactive astrocytes (13); O-GlcNAcH is up-regulated in hypoxia in human ependymal cells (14) and is greatly up-regulated in endometrial decidual cells compared to stromal cells of the proliferative endometrium (15). Epitope H occurs in several polypeptides (7, 10, 13), including keratin 8 (7) and vimentin in cells under stress (16).

In this context, monoclonal antibody H has been produced which is a novel O-GlcNAc-specific IgM mouse antibody recognizing epitope H. This renders monoclonal antibody H a site-specific antibody, thus recognizing a subpopulation of O-GlcNAcylated polypeptides (7, 13).

In this study, we aimed to calculate the distribution of O-GlcNAcH in the cellular elements of the endocervical mucosa in specimens of endocervical curettings in order to investigate a possible correlation between mucin-secreting cells of inflamed polyps and expression of O-GlcNAcH.

Materials and Methods

The study material comprised 60 specimens from endocervical currettings, including pieces of endocervical mucosa, analyzed at the Pathology Department of General Hospital of Sparta, Greece. Ten out of the 60 specimens contained a total of 15 small polyps; one of the 10 specimens with polyps contained five polyps, another contained two polyps, and eight specimens had one polyp each, with mild to moderate chronic inflammation in the stroma of the polyps.

The indirect immunoperoxidase method was applied to formalin-fixed paraffin-embedded tissues using mouse monoclonal IgM antibody H, which is not commercially available and was produced at the University of Chicago (for details see Acknowledgements) (17) and a rabbit peroxidase-conjugated anti-mouse secondary antibody purchased from POIGI-DAKO (Glostrup, Denmark) as follows. Sections of 4 μm were cut from the formalin-fixed paraffin-embedded tissue blocks. After deparaffinization and blocking of endogenous peroxidase activity by immersing the sections in 3% H2O2 in Tris-buffered saline (pH 7.6) for 30 min, and after washing in Tris, the sections were incubated in 10% normal rabbit serum in Tris buffer for 30 min to inhibit the non-specific binding of antibodies. After discarding the normal serum, the sections were incubated with monoclonal antibody H for 2 h at room temperature. After washing three times for 10 min in Tris, the sections were then incubated for 1 h with the rabbit peroxidase conjugated anti-mouse secondary antibody diluted in 1:50 in Tris. After washing three times for 10 min in Tris, the color was developed by incubating the sections for 8 min in diaminobenzidine-H2O2 Tris-buffer. Then after washing, counterstaining in hematoxylin and dehydrating, the sections were covered with Permount™ (Fisher Chemical, Hampton, NH, USA) for microscopic examination. For negative controls, primary antibody H was omitted.

The following scoring system for the expression of O-GlcNAcH in the cells was used: Negative expression: All cell elements were negative for O-GlcNAcH; low expression: ≤5% of the cells exhibited positive staining for O-GlcNAcH; intermediate expression: 6% to 30% of the cells stained positively; high expression: 31% to 75% of the cells stained positively; extremely high expression: >75% of the cells stained positively.

Results

Low expression of O-GlcNAcH of the mucin-secreting cells of the endocervical mucosa was observed, mainly present as a positive subnuclear cytoplasmic dot, with an even smaller percentage additionally showing a supranuclear reticulated pattern (Figure 1 and Figure 2A and B).

Figure 1.
Figure 1.

Endocervical curettings immunostained for O-linked N-acetylglucosamine-containing epitope H (O-GlcNAcH), counterstained with hematoxylin. A: Mucin-secreting cells negative for O-GlcNAcH counterstained with hematoxylin. ‘reserve’ cells with positive cytoplasmic staining. Desquamated rolled-up superficial mature squamous cells with strong cytoplasmic staining. Original magnification: 630×. B: Hyperplastic ‘reserve’ cells with strong cytoplasmic and weak nuclear staining (arrow heads), with three of them bearing unstained blue nuclei (arrow). Overlying mucin-secreting cells are negative. Original magnification: 630×. C: Stromal fibroblasts negative (asterisk). Hyperplastic ‘reserve’ cells with strong cytoplasmic staining (double arrows). Overlying mucin-secreting cells negative (arrows) or with reticular cytoplasmic staining (arrowheads). Original magnification: 400×. D: ‘reserve’ cells with strong cytoplasmic staining (arrow). Overlying mucin-secreting cells either negative (arrow heads) or positive with reticular cytoplasmic staining (arrows). Original magnification: 630×.

Figure 2.
Figure 2.

Endocervical curettings immunostained for O-linked N-acetylglucosamine-containing epitope H (O-GlcNAcH), counterstained with hematoxylin. A: Cells of immature squamous metaplasia with strong cytoplasmic staining (asterisk) and overlying mucin-secreting cells negative (arrow) for O-GlcNAcH. Original magnification: 630×. B: Cells of immature squamous metaplasia with strong cytoplasmic staining (asterisk), overlying mucin-secreting cells either negative or positive with patchy (arrow head) or reticular (arrow) staining. Endothelial cells of a capillary vessel with strong cytoplasmic staining (double arrows). Original magnification: 400×. C: Endocervical glands filled with cells of immature squamous metaplasia with strong cytoplasmic staining (open asterisk). Intervening fibroblasts negative (closed asterisks). Original magnification: 400×. D: Mature squamous epithelium: Basal and parabasal cells negative, intermediate and superficial cells with strong cytoplasmic staining. Stromal fibroblasts negative (asterisk). Original magnification: 200×. E: Mature squamous epithelium: Basal and parabasal cells negative, intermediate and superficial cells with strong cytoplasmic staining. Original magnification: 400×. F: Mature squamous epithelium: Basal cells positive. Parabasal cells negative. Intermediate and superficial cells positive, with the clear areas representing unstained accumulation of glycogen. Original magnification: 400×.

The mucin-secreting cells of small, inflamed polyps showed intermediate expression of O-GlcNAcH, presenting as subnuclear cytoplasmic staining, with a good percentage additionally showing supranuclear cytoplasmic positivity as strong dense patchy staining or as strong dense sub-apical banding (Figure 3A-C). Moreover, a small number of non-ciliated, non-mucin-secreting cells showed strong, diffuse cytoplasmic staining for O-GlcNAcH (Figure 3C and D).

Figure 3.
Figure 3.

Endocervical polyps immunostained for O-linked N-acetylglucosamine-containing epitope H (O-GlcNAcH), counterstained with hematoxylin. A: Endothelial and stromal cells with strong cytoplasmic staining. Overlying mucin-secreting cells either negative or with positive subnuclear or supranuclear positive staining. Original magnification: 200×. B: Parts of adjacent polyps showing the staining spectrum of mucin-secreting cells for O-GlcNAcH: negative cells (thin arrows), positive cells, with either subnuclear (arrow heads) or/and supranuclear patchy staining (thick arrows) or subapical dense band -like staining (the two cells left of the thick arrow). Stained stromal cells (low left). Unstained inflammatory cells (upper polyp). Original magnification: 400×. C: Endocervical polyp with positive endothelial and stromal cells and positive overlying mucin-secreting cells with subnuclear or/and supranuclear staining. Three non-mucin, non-ciliated cells having the shape of a champagne glass can be seen in the box. Original magnification: 200×. D: Higher magnification of the three ‘champagne glass’-shaped cells of the box shown in part C. Original magnification: 630×.

All ciliated cells, whether isolated or in areas of tubal metaplasia, showed cytoplasmic staining for O-GlcNAcH, ranging from moderate to mainly strong in intensity, with 5% or fewer of the cells additionally showing weak nuclear staining (Figure 4).

Figure 4.
Figure 4.

Endocervical polyps immunostained for O-linked N-acetylglucosamine-containing epitope H, counterstained with hematoxylin. A: A row of ciliated-cells with moderate patchy cytoplasmic staining for O-GlcNAcH (arrows). Unstained fibroblasts (asterisk). Original magnification: 630×. B: Pseudostratified ciliated-cells mainly with strong cytoplasmic staining and a smaller number additionally with weak nuclear staining for O-GlcNAcH. Original magnification: 630×.

Furthermore, all normal and hyperplastic ‘reserve’ cells showed strong diffuse cytoplasmic staining for O-GlcNAcH, with fewer than 5% additionally showing weak nuclear staining (Figure 1). All squamous cells in immature squamous metaplasia showed strong diffuse cytoplasmic staining for O-GlcNAcH (Figure 2A-C).

The staining pattern for O-GlcNAcH in mature squamous metaplasia in the overwhelming majority of the cells of the basal layer was negative and the basal cells showed no cytoplasmic staining. All parabasal cells were also negative. All squamous cells above the parabasal cells showed strong cytoplasmic staining, however glycogen-concentrated areas remained unstained (Figure 2D-F).

A result of great interest is that the fibroblasts/fibrocytes of the uninflamed mucosa remained negative for O-GlcNAcH (Figure 1C, Figure 2C and D, and Figure 4A), whereas the stromal cells of the small, inflamed polyps showed strong cytoplasmic staining (Figure 3A-C). Moreover, the endothelial cells of the uninflamed mucosa (Figure 2B) and the inflamed polyps (Figure 3A-C) showed strong cytoplasmic staining for O-GlcNAcH. All negative controls were unstained.

Discussion

The results of the present work clearly show that there is differential expression of O-GlcNAcH in the various cellular elements of the endocervical mucosa and endocervical polyps. In a given cell with a given status, the level of global O-GlcNAcylation depends upon: a) the expression and activity of O-GlcNAc transferase, b) the expression and activity of beta-N-acetylglucosaminidase, c) the concentration of UDP-GlcNAc, d) the availability of substrates, and e) the enzymatic targeting of the substrates (18).

According to the above factors, the most obvious reason for the differential expression of O-GlcNAcH, taking into account that monoclonal antibody H is a site-specific antibody recognizing a subpopulation of O-GlcNAcylated polypeptides, is the differential availability of the substrates among the population of cells present. Previous work has shown that there is striking differential expression of O-GlcNAcH among adjacent cells in the same organ, such as strong cytoplasmic expression of O-GlcNAcH in parietal cells of the mucosa of the gastric body and its absence from chief cells (6), and its cytoplasmic expression in a minority of fibrous astrocytes in the human brain but strong up-regulation in reactive astrocytes, with adjacent neurons being negative for O-GlcNAcH (13), primarily reflecting the different availability of one or more substrates bearing O-GlcNAcH among the different types of cells in both of the above organs. One of the most interesting findings of the present work is the strong cytoplasmic staining of normal as well as hyperplastic ‘reserve’ cells for O-GlcNAcH, and the fact that this strong cytoplasmic staining also existed in the squamous cells of immature squamous metaplasia. Based on this finding, we can argue that O-GlcNAcH might be considered a biochemical link between ‘reserve’ cells and immature squamous metaplastic cells and that further isolation and characterization of the polypeptides bearing O-GlcNAcH might shed light on the biology of the conversion of hyperplastic ‘reserve’ cells into immature squamous cells. The pattern of O-GlcNAcH changes drastically from the immature to mature squamous epithelium. In the mature squamous epithelium, the overwhelming majority of cells of the basal layer were negative for O-GlcNAcH and only few foci showed basal cells with strong cytoplasmic staining. The parabasal cells were always negative, even in the foci above strongly stained basal cells, and the squamous cells of the intermediate and superficial layers showed strong cytoplasmic staining for O-GlcNAcH. Previous work has shown that out of all keratins, keratins 8, 18 (19) and 13 (20) are O-GlcNAcylated. Other work has shown that O-GlcNAcH occurs in keratin 8 but not in keratin 18 (7), and this led to the notion of GlcNAc being located in a specific conformation or environment within epitope H. Keratin 13 exists in the squamous cells of all layers of mature non-keratinizing epithelium of the internal organs, including the uterine cervix, except the cells of the basal layer (21). Since our findings show that parabasal cells, which contain keratin 13, were negative for O-GlcNAcH, we can argue that we have immunohistochemical evidence that keratin 13 does not bear O-GlcNAcH and that the presence of O-GlcNAcH in the cells of the intermediate and superficial layers derives from other polypeptides and not keratin. Taking into account that ‘reserve’ cells and parabasal cells are morphologically similar, O-GlcNAcH can be used as an immunohistochemical marker to distinguish ‘reserve’ cells (positive) from parabasal cells (negative) in cytological smears or in other cell preparations.

Concerning the mucin-producing cells of the uninflamed endocervical mucosa, 5% or fewer cells showed either subnuclear dot-like staining or supranuclear reticular staining for O-GlcNAcH. The paucity of O-GlcNAcH staining in these cells might be attributed to the filling of the cytoplasm with mucin which is negative for O-GlcNAcH, leaving almost nothing of the true cytoplasmic matrix which might contain some substrates for O-GlcNAcH, or to keratin 8 not bearing the O-GlcNAcH epitope in the majority of normal mucin-secreting cells. On the contrary, mucin-secreting cells of the inflamed endocervical polyps showed low expression of O-GlcNAcH, although staining was increased compared with the other mucin-secreting cells. More specifically, ≤30% of the cells showed subnuclear cytoplasmic staining and a small percentage additionally exhibited supranuclear staining, which appeared as a subapical dense band or a subapical dense patchily stained area, leaving an underlying, unstained mucin-secreting area above the nucleus. Interestingly, among the mucin-secreting cells of the polyps, a small number of non-ciliated cells was noted whose whole cytoplasm stained strongly for O-GlcNAcH but which had no-mucin content; these cells had a ‘champagne glass’ shape, finally taking the form of non-ciliated, non-mucin-secreting cells.

Previous studies have shown that any kind of stress and noxious stimuli provoke a global increase of O-GlcNAcylation in cells (22), and these stress-induced changes of O-GlcNAcylation are thought to reprogram cellular pathways promoting survival (23). O-GlcNAc has been demonstrated to activate proteins/pathways leading to cell survival through heat-shock protein expression and to inhibit proteins that promote cell death, e.g., through the activation of C/EBP homologous protein, finally leading to increased cellular protection (24).

Based on the above studies, we hypothesize that the up-regulation of the O-GlcNAcH in mucin-secreting cells of inflamed polyps takes place in order to protect them from the stressful and noxious influence of the underlying inflamed stroma. One mechanism protecting from stress involves keratin 8, which confers pro-survival protection in simple epithelia cells through the activation of AKT serine/threonine kinase 1 (AKT1) under cellular stress or noxious stimuli (25). AKT1 links to keratin 8 and simultaneously to keratin 18, which forms obligate heterodimers with keratin 8 in simple epithelia (21), becomes O-GlcNAcylated and this leads to phosphorylation of AKT1 at threonine 308, which then activates anti-apoptotic biochemical cascades (25).

Inflammation can modify the expression of keratin 8 and in intestinal epithelial cells, the inflammatory cytokine interleukin 6 increases the production of keratin 8, forming mainly a sub-apical dense band enhancing thus the resistance of the cells and increasing the sealing of the intercellular barrier (26). Our findings show that O-GlcNAcH was more highly expressed on mucin-secreting cells of the polyps (where inflammation and stress are present) compared to mucin-secreting cells of the normal mucosa. Based on the above findings, we can hypothesize that, compared to mucin-secreting cells of the normal mucosa without inflammation, the inflammation of polyps increases the production and redistribution of keratin 8 in mucin-secreting cells and that cellular stress increases total O-GlcNAcylation of polypeptides, including the O-GlcNAcH epitope, of keratin 8 and in other polypeptides in order to protect the mucin-secreting cells.

Concerning mesenchymal cells, the endothelial cells in the mucosa and in the polyps showed strong cytoplasmic staining for O-GlcNAcH. The stromal fibroblasts of the uninflamed mucosa were negative, immunohistochemically confirming that vimentin in fibroblasts bears the O-GlcNAcH epitope only under stress cellular conditions (16). The stromal cells of the polyps showed induction of O-GlcNAcH, since all of them showed strong cytoplasmic staining for the epitope. This strong induction may be attributed either to O-GlcNAcylation of vimentin under the stress condition of inflammation, or to the conversion of fibroblasts to myofibroblasts, thus acquiring some polypeptides bearing O-GlcNAcH, as has been shown to occur in normal smooth muscle and leiomyoma cells (10). Further biochemical characterization of the polypeptides which bear the O-GlcNAcH epitope might shed light on all these processes.

In conclusion, this study shows: a) that the O-GlcNAcH epitope, exhibits differential expression in the cellular elements of the endocervical mucosa, b) there is up-regulation of O-GlcNAcH in mucin-secreting cells of endocervical polyps and c) there is strong induction of O-GlcNAcH in the stroma cells of the endocervical inflamed polyps.

Acknowledgements

The Authors are grateful to Dr. Sara Szuchet, Professor of Neurology at the University of Chicago, and to the late Dr. Gladys Mori de Moro a former post-doctoral Research Fellow at the Department of Neurology of the University of Chicago for their generous donation of monoclonal antibody H. This work was partially funded by Grant No 3326 from the Research Committee of the University of Thessaly.

Footnotes

  • Authors’ Contributions

    Study conception and design: S. Sotiriou and F. Arapis. Acquisition of data: D. Rempelou, S. Havaki, and A. Samara. Analysis and interpretation of data: D. Arvanitis, A. Zibis, and A. Samara. Drafting of article: F. Arapis and K. Tzelepis. Critical revision: A. Zibis, D. Arvanitis, and S. Sotiriou.

  • Conflicts of Interest

    The Authors declare that they have no conflicts of interest.

  • Received December 17, 2023.
  • Revision received January 28, 2024.
  • Accepted February 7, 2024.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Expression of the O-Linked N-Acetylglucosamine-containing Epitope H (O-GlcNAcH) in Human Uterine Cervical Mucosa
FOTIOS ARAPIS, DIMITRA REMPELOU, SOPHIA HAVAKI, DIMITRIOS ARVANITIS, KONSTANTINOS TZELEPIS, ARISTEIDIS ZIBIS, ATHINA A. SAMARA, SOTIRIOS SOTIRIOU
In Vivo May 2024, 38 (3) 1112-1118; DOI: 10.21873/invivo.13545
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