Expression of TGF-β and MMP-2 in hereditary gingival fibromatosis epithelial cells. A possible contribution of the epithelium to its pathogenesis

Abstract

Background

Although the molecular mechanisms that cause the development of hereditary gingival fibromatosis are not fully understood, multiple theories have been suggested to clarify its pathogenesis. However, the overlying keratinocytes' function is poorly comprehended. This work aimed to investigate the expression of TGF-β and MMP-2 in hereditary gingival fibromatosis epithelial cells compared to the normal gingival epithelium to give an insight into the mechanism of the development of this condition.

Methods

Biopsies were obtained from 20 hereditary gingival fibromatosis patients and 20 healthy controls. Biopsies were stained immunohistochemically and statistically analyzed for MMP-2 and TGF-β expression.

Results

Regarding MMP-2, The hereditary gingival fibromatosis group recorded a higher mean value compared to the normal gingiva, with a mean difference of 3.29 ± 0.34. This difference was statistically significant (p = 0.00). Regarding TGF-β, a higher mean value was recorded in the HGF group compared to the normal gingiva, with a mean difference of 15.88 ± 1.05 The difference was statistically significant (p = 0.00). A strong positive correlation was detected between MMP-2 and TGF-β (R = 0.534, p = 0.015).

Conclusions

In hereditary gingival fibromatosis, the epithelium expresses higher levels of TGF-β and MMP-2 than normal gingival tissue. There was an evident positive correlation between MMP-2 and TGF-β. Our data suggest that the expression of TGF-β and MMP2 by epithelial cells of HGF may play a role in the epithelial-mesenchymal transition pathogenic pathway.

Graphical abstract

1. Introduction

Hereditary gingival fibromatosis (HGF), also known as hereditary gingival hyperplasia, hypertrophic gingiva, and elephantiasis gingivae, is a condition that causes progressive gingival enlargement. This enlargement reveals various severities; sometimes, the whole crowns of the teeth are covered, deforming the palate, causing occlusal and aesthetic problems, and mastication and speech difficulties. Alveolar ridge thickening is uncommon at birth, usually starts with deciduous or permanent dentition eruption, worsens in puberty, and can last during adulthood.¹

The enlargement could be generalized, affecting the whole gingivae of the mandible and maxilla, or localized to a specific region of the mouth, including the labial gingiva, maxillary tuberosities, and the area around the lower molars. Gingival overgrowth (GO) can indeed occur in susceptible persons as a side effect of systemic drugs, such as immunosuppressants, calcium channel blockers, or antiseizure medications. The cause of the enlargement is unclear in some cases.^2,3 HGF may be inherited in an autosomal dominant or recessive manner. The recessive pattern is often associated with systemic diseases or other syndromes.⁴

HGF oral manifestations comprise particularly fibrous and thick gingiva. The enlarged tissues appear nodular, pink, and firm with no tendency for bleeding. Histologically, the overlying epithelium is hyperplastic with prominent, elongated rete ridges that extend into the underlying connective tissue. The underlying connective tissue showed dense and coarse collagen bundles with little vascularization. Inflammatory cells are dispersed between the connective tissue's dense collagen bundles.^3,5

There is no single treatment or medication that can completely prevent gingival fibromatosis, but surgical treatments can help to alleviate this condition if proper oral hygiene is maintained.⁶ In most of the cases, gingivectomy was performed, followed by gingivoplasty.⁴

The discovery of HGF-related genetic alterations could lead to new diagnostic tools, therapy targets, and a better understanding of the molecular mechanisms behind this condition. HGF has been linked to multiple chromosomes and chromosomal regions that may carry mutant genes.⁷

In HGF, chromosomes 2, 4, and 5 are identified with their particular genetic loci, 2p21-p22 (GINGF), 2p13-p16, 2p22.3–23 (GINGF3), 5q13-q22 (GINGF2), 4q21, and 4q which allow genomic aberrations to occur.⁶ One putative etiologic factor in HGF has been reported as a mutant SOS-1 gene, but considering HGF's genetic heterogeneity, mutations in other genes are also expected to occur.² Recently, REST gene mutations were recorded in HGF. However, the involvement of REST in HGF pathogenesis continues to be unknown.⁸

SOS-1 gene (Son of Sevenless 1) is located on chromosome 2p22.1.² It is expressed in various cells and tissues, such as the human gingiva, where it is detected in epithelial cells and different stromal cells. SOS-1 has a central role in growth factors and extracellular matrix signaling.⁷ On the other hand, the REST gene (RE1-silencing transcription factor) is located on chromosome 4q12. The REST gene controls gingival homeostasis by suppression of profibrotic genes and activation of proteolytic genes.⁸

While the molecular mechanisms that induce this pathological process are not fully known, numerous hypotheses have been suggested to explain its pathogenesis.² Previous researches suggest that gingival fibromatosis (GF) is caused by a change in the proliferation of fibroblasts or disruption in collagen turnover that results in an excessive accumulation of the extracellular matrix constituents.^2,9 On the other hand, the function of the overlying keratinocytes is poorly understood.¹⁰ However, epithelial to mesenchymal transition (EMT) has been recently suggested as another pathogenic pathway that promotes gingival fibrosis in which the basal lamina is disrupted, and epithelial cells penetrate through connective tissue and transform to fibroblast-like cells.^2,11 In oral tissues, type 1 EMT is linked to organ development, type 2 EMT may have an important function in GF and oral submucous fibrosis while the invasion of oral squamous cell carcinoma is related to type 3 EMT.¹¹

Successful EMT is dependent on a combination of cytokines and growth factors involved in the proteolytic degradation of the basement membrane through activation of matrix metalloproteinases (MMPs).¹² MMP-2 is an essential cytokine that mediates type IV collagen proteolysis, an essential constituent of the basement membrane.¹³

Furthermore, transforming growth factor-β (TGF-β) is a multifunctional cytokine that is expressed by almost all types of cells and is known to control a wide range of cellular functions, including differentiation, proliferation, and extracellular matrix formation.¹⁴ This cytokine is essential for MMP-2 expression, which aids in basement membrane breakdown.¹²

As far as we know, no previous studies have evaluated the epithelial expression of MMP-2 and TGF-β in HGF. Thus, we aimed to study the immunohistochemical expression of these markers in HGF epithelium compared to normal gingival epithelium to give an insight into the mechanism of the development of this condition.

2. Materials and methods

2.1. Study population

Participants in this study were recruited from outpatient clinics of the Oral Medicine and periodontology department, Faculty of Oral and Dental Medicine, Al-Azhar University-girls, Cairo, Egypt, (January 2020 to January 2021). The Ethics Committee of Al- Azhar University approved all the procedures. The study's protocol was described to each patient preoperatively. Furthermore, informed written consent was obtained from each participant, indicating his willingness to participate in the planned work. The research was carried out following the Declaration of Helsinki principles.

2.2. Patients' grouping

This study involved a total of 40 patients. They aged from 20 to 37 years and were categorized as follows. The control group (n = 20) consisted of 8 females and 12 males assessed by the modified Cornell medical index¹⁵ as healthy individuals free of systemic disease. The study group (n = 20) included HGF. The study group consisted of 8 females and 12 males.

HGF was diagnosed using previous guidelines.¹⁶ The diagnosis was confirmed by a trained and qualified clinician (HHH) based on the following inclusion criteria; Patients belonged to the same family (Fig. 1 a, b), all participants had no clinical attachment loss (CAL = 0), gingival index range must be from (0) to (1) according to Loe and Silness.¹⁷

Fig. 1.

A clinical photograph of HGF of the same family that shows bulbous, fibrotic enlargement of both maxillary and mandibular gingivae (the son (a), and the father (b)).

Moreover, the clinical appearance revealed keratinized non-inflamed fibrous GO that covers at least one-third of teeth crowns, with no radiographic evidence of alveolar bone loss.

Patients were ruled out if they had a potential cause of GO other than HGF, such as those who are taking one of the three drugs linked to drug-induced GO (Dilantin, calcium channel blockers, or cyclosporine). Lactating or pregnant women, as well as smokers or former smokers, were excluded from the study. Besides, patients who refused to provide informed consent were also excluded from this research. Before the gingival samples were collected, all participants in both groups were clinically monitored for plaque control for two weeks.

Regarding the control group, samples were collected from five patients after crown lengthening, five patients during dental implant surgeries, five patients following tooth extraction for orthodontic purposes, and five patients after operculectomy. HGF samples were collected during gingivoplasty and gingivectomy. Biopsies were examined by an experienced oral pathologist, PHD (NMK).

2.3. Immunohistochemical staining procedure

Three 4 μm thick sections were cut from each formalin-fixed, paraffin-embedded tissue block. The sections were deparaffinized with xylene and rehydrated in graded ethanol for immunohistochemical staining by MMP-2 and TGF-β. For heat-mediated antigen retrieval, a citrate buffer with a pH of (6.0) was used. The sections were then immersed in hydrogen peroxide (H2O2) to suppress endogenous peroxidase activity, washed in phosphate-buffered saline (PBS), and a protein blocking reagent was applied. To remove non-specific staining, a humid chamber was used to incubate the sections at 37 °C for 20 min.

In this study, we have used the following primary antibodies:

Concentrated polyclonal rabbit antibody for MMP-2 (Code No. PA1-16667 at dilution 1:100, Thermo Fisher Scientific USA).

Concentrated polyclonal rabbit antibody for TGF-β (Code No. AB-100-NA at dilution 1:100, Bio-Techne, USA).

The sections were incubated with the primary body overnight, and the bounded antibodies were detected using the streptavidin-biotin complex system. The sections were counterstained with Mayer's Hematoxylin after the immunoreaction.

2.4. Immunohistochemical interpretation (histomorphometric analysis)

The presence of brown immunostaining reaction in the cytoplasm was used to assess the immune-expression of MMP-2 and TGF-β. Ten microscopic fields from each slide with the highest immunopositivity were chosen and photo-micro-graphed with a computerized image analyzer (Leica Qwin-Germany). Two observers select these fields.

In each microscopic field, immunoreactivity for MMP-2 and TGF-β was determined by evaluating the positive immune-stained cells area percentage in relation to the total area examined using a magnification of ×200.

The percentage of positively stained cells and the area were measured.¹⁸ Staining of 10–49% of the tumor cells was considered positive expression. Staining of less than 10% of tumor cells or no detectable staining was considered negative expression.¹⁹ The mean values for the percentage of positively stained cells with standard deviations were then determined.²⁰

2.5. Statistical analysis

Statistical analysis was performed with a commercially accessible software program. (SPSS 18; SPSS, Chicago, IL, USA). The Kolmogorov-Smirnov test of normality was used to analyze the data for normality. Most values were parametric and were compared between groups using an independent t-test.

The Pearson correlation test was used to assess the correlation between MMP-2 and TGF-β results. The significance level was established at P < 0.05.

3. Results

3.1. Patients' characteristics

In this study, the control group consisted of 12 males and 8 females with a mean age of 28.60 ± 5.24 years. For the HGF group, 12 males and 8 females were included with a mean age of 28.45 ± 4.84 years. This showed an insignificant difference for gender and age distribution in the control group compared to HGF patients (P > 0.05) (Table 1).

Table 1.

Characteristics and demographics of the examined groups.

Variables		Demographic data
		Control		Study		p-value
		Mean/n	SD/%	Mean/n	SD/%
Age (Years)		28.60	5.24	28.45	4.84	0.926
Gender (N, %)	Females	8	40%	8	40%	1
	Males	12	60%	12	60%

Significant (P < 0.05); non-significant (P > 0.05).

SD, standard deviation.

3.2. Hematoxylin and eosin stain findings

HGF specimens showed keratinized stratified squamous epithelium with a thickened acanthotic epithelial layer. The rete ridges were broad, long, and extended deep within the underlying connective tissue. The underlying connective tissue revealed dense and coarse collagen bundles with minimal vascularization. A densely and focally distributed inflammatory cells were found between the connective tissue collagen bundles (Fig. 2, a).

Fig. 2.

(a) A photomicrograph of HGF that shows keratinized stratified squamous epithelium with acanthosis and long, broad rete ridges extending deep into the connective tissue (H&E x100). (b) A photomicrograph of normal gingival tissue (control) that shows a keratinized stratified squamous epithelium with a normal arrangement of its layers. The connective tissue underneath showed regular fiber arrangement (H&E x100). (c) A photomicrograph of HGF that shows TGF-β cytoplasmic staining identified by majority of the keratinocytes (TGF-β x100). (d) A photomicrograph of TGF-β immunostaining in normal gingiva shows negative expression (TGF-β x100). (e) A photomicrograph of MMP-2 immunostaining in HGF shows a positive cytoplasmic reaction in the epithelial keratinocytes (MMP-2 x100). (f) A photomicrograph of MMP-2 immunostaining in normal gingiva shows a negative reaction (MMP-2 x100).

The normal gingival tissue (control) showed a keratinized stratified squamous epithelium with a normal arrangement of its layers (6–8 layers). The basement membrane has no extended rete ridges. The connective tissue underneath revealed regular fiber arrangement, blood vessels, and a few chronic inflammatory cells. (Fig. 2, b).

3.3. Immunohistochemical findings

In HGF, cytoplasmic staining for TGF-β was identified by most of the epithelial cells mainly in the basal and parabasal layers (Fig. 2, c). Normal gingival tissue samples were negative for TGF-β (Fig. 2, d).

All the samples of HGF showed a cytoplasmic immunopositivity for MMP-2 in the epithelial keratinocytes mainly in the basal layer of the epithelium (Fig. 2, e). Normal gingival tissue samples were negative for MMP-2 (Fig. 2, f).

3.4. Histomorphometric findings

Regarding MMP-2, the greatest mean area percentage of positively stained cells was recorded in the HGF group compared to the normal gingiva. The difference was statistically significant (p < 0.0001) (Table 2).

Table 2.

Percentage of positively stained cells with MMP-2 and TGF-β in HGF and normal gingiva (histomorphometric analysis).

	MMP-2 area percent		TGF-β area percent
	HGF	Normal gingiva	HGF	Normal gingiva
Maximum	15.745	0.144	22.714	1.222
Minimum	10.373	0.12	17.974	0.333
Mean	13.297	0.134	19.719	0.752
SD	1.921	0.009	1.795	0.363
P-value	<0.0001*		<0.0001*

Significance level p < 0.05, *significant.

SD, standard deviation.

Regarding TGF-β, the greatest mean area percentage of positively stained cells was recorded in the HGF group compared to the normal gingiva. The difference was statistically significant (p < 0.0001) (Table 2).

3.5. The correlation between MMP-2 and TGF-β in HGF

A strong positive correlation was detected between MMP-2 and TGF-β (R = 0.534). Pearson correlation test showed that this correlation was statistically significant (p = 0.015), (Fig. 3).

Fig. 3.

Line chart illustrating a correlation between MMP-2 and TGF-β mean area percent of immune-expression.

4. Discussion

HGF is a form of heterogeneous GO characterized by non-hemorrhagic fibrous hyperplasia that is benign and slowly progressive. The underlying mechanisms causing excessive gingival tissue accumulation in GF is unknown, and GF pathogenesis studies have primarily centered on connective tissue changes.²¹

Some authors suggested that EMT could play a role in the pathogenesis of HGF.²² and according to Iwano et al.,²³ EMT is the primary source of fibroblasts in connective tissue fibrosis. The study of Menga et al.,¹⁰ discovered a high level of type 1 collagen expression in fibroblasts derived from mixed cultures of fibroblasts and keratinocytes collected from HGF patients. The authors proposed that keratinocytes have an essential role in the pathogenesis of GF by increasing extracellular matrix synthesis.

The histopathological features of HGF in the current work revealed hyperkeratotic acanthotic epithelium with elongated, thick rete ridges reaching deep within the underlying dense fibrous connective tissue. Baniţă et al.,¹¹ linked similar findings to epithelial proliferation. They also stated that GO may relate to fibrosis by providing a cell pool to replace those cells involved in EMT and transformed into fibroblasts. Furthermore, Smith et al.,²⁴ proposed a potential role of the keratinocyte response in the etiology of drug-induced GO. The data of a previous study by Pascu et al.,²⁵ assisted the EMT origin of gingival fibroblasts in drug-induced GO and identified similar findings in a case of syndromic GO.

Since the main feature of HGF is the accumulation of interstitial collagen, which could be related to EMT and the epithelium is speculated to play a role in HGF pathogenesis, so the current study aimed to evaluate the expression of TGF-β and MMP-2 in the epithelium of HGF compared to the normal gingival epithelium to give an insight into the mechanism of the development of this condition.

To the best of our knowledge, there is no previous data about their expression in the epithelium of HGF.

The formation of fibrotic lesions is indirectly linked to the existence and the histophysiology of epithelial cells. The intervention of oral epithelial cells in extracellular matrix synthesis is supported by the findings of numerous studies that report epithelial morphological changes in addition to connective tissue accumulation. At the same time, epithelial cells or inflammatory cells infiltrating the epithelium produce a variety of biomolecules (cytokines, growth factors, and MMPs) which induce alteration in collagen metabolism and extracellular matrix synthesis.^10,11,24

Many inflammatory cells have been found infiltrating the deep layers of the epithelium in HGF.¹¹ Given that HGF is primarily associated with permanent teeth eruption,¹ it appears that the inflammatory stimulation of eruption increases TGF-β levels.³ TGF-β is a powerful inducer of EMT in epithelial cells from various tissues, and treated cells display typical phenotypic changes such as disassembly of cell junctions and down-regulation of E-cadherin²⁶ and increased synthesis of matrix metalloproteinases including MMP-2 and MMP-9.²⁷ MMP-2 is required for the degradation of type IV collagen and the basement membrane, which is a central factor of EMT. It allows for cell invasion into underlying connective tissue and unusual interactions between the epithelium and connective tissue stroma.^12,27

The present study results revealed increased levels of TGF-β expression in the epithelium of HGF compared to normal gingival tissue. The difference was statistically significant. These results could be supported by previous work of Pisoschi et al.²⁸ They performed immunohistochemical studies on phenytoin-induced GO samples in which many TGF-β positive cells were detected in large areas of the basal and parabasal layers of the epithelium, in epithelial rete ridges, as well as in some mesenchymal cells lining the epithelium. In addition, their findings revealed a decrease in E-cadherin expression in the basal layer of the epithelium near the basement membrane, implying that these cells underwent EMT.

In this regard, Sume et al.,²⁹ stated that EMT is a biological mechanism that relates to drug-induced GO and reported TGF-β as a potent inducer of EMT in primary cultured gingival epithelial cells. Their study showed that TGF-β could alter the phenotype of human primary gingival epithelial cells in primary culture. In addition, they detected a loss of cellular integrity of the epithelial cells.

It was found that the TGF-β pathway undergoes a profibrotic cascade that is activated in the epithelium and influences the underlying submucosa for a fibrotic response. Among the transcription factors directly contributing to EMT includes the snail superfamily of zinc-finger transcription factors, Snail1 and Snail2 (also known as Slug).³⁰ Adding to this, TGF-β/Smad/Snail is a key signaling pathway involved in different subtypes of EMT.³¹

In this context, Snail expression is triggered in response to a variety of growth factors. In cells undergoing TGF-β Induced EMT, the expression of Snail is regulated by Smad2/3, which forms complexes with Smad4 and stimulates transcription via binding to the Snail promoter.¹¹ Snail suppresses E-cadherin expression, which results in reduced cell junctions, disruption of intercellular adhesion, and activation of fibronectin, N-cadherin, and vimentin expression resulting in a complete EMT phenotype.³² Also, Pisoschi et al.,²⁸ discovered an up-regulation of Smad3 and Snail transcription factors in cells from deep epithelial layers in phenytoin induced GO.

In the present study, we report that in HGF the epithelium expresses increased levels of MMP-2 than normal gingival tissue, the difference was statistically significant. This finding can be justified by Min et al.,³³ in which MMPs are expressed in the epithelium undergoing EMT and relate to extracellular matrix remodeling, thus facilitating cell migration. Additionally, Sume et al.,²⁹ suggested that MMP-2 may play a role in gingival tissue remodeling and the initiation of gingival fibrosis.

Therefore, the present outcomes regarding the increased epithelial expression of TGF-β and MMP-2 in HGF compared to the normal gingiva may denote their possible role in EMT and initiation of HGF.

The results of the current work revealed a strong positive correlation between MMP-2 and TGF-β, this correlation was statistically significant. In support of these results, a study by Sume et al.,²⁹ investigated whether MMPs expressions could be increased by the action of TGF-β in cell cultures of primary human gingival epithelium. In their study, TGF-β treated epithelial cells of the gingiva displayed a significant increase in mRNA levels of MMP-2, MMP-9, and MMP-13. These increased levels of MMPs lead to basement membrane degradation. In the absence of regular cell-to-cell junctions and a disrupted basement membrane, epithelial cells then acquire the ability for migration. The damaged basement membrane allows unusual contact between the epithelium and underlying stroma, leading to TGF-β stimulation of fibronectin, which correlates to connective tissue fibrosis.²⁹

Collectively, the current study's finding supports the role of the epithelium in developing HGF through the expression of TGF-β and MMP2 by epithelial cells that facilitate cell migration (an essential step in epithelial-mesenchymal transition).

Ultimately, one possible limitation of the current work is the scarcity of data concerning the epithelial expression of MMP2 in fibrotic gingival lesions. Further studies are still needed to support our findings and to clarify the precise interactions between the epithelium and stroma. Even though this study could provide objective evidence, this appears to be an interesting area for future research, for a possible link between the genetic and molecular basis to determine whether all keratinocytes behave in the same manner, or some population of cells have this criterion.

5. Conclusion

Our preliminary findings show that the epithelium in HGF expresses higher levels of TGF-β, and MMP-2 than normal gingival tissue. A strong positive correlation between MMP-2 and TGF-β was evident. Therefore, our clinical study could be considered as new evidence supporting the role of the epithelium in the pathogenesis of HGF.

Funding

This study was not funded by any agencies in the commercial, public, or non-profit organizations.

Declaration of competing interest

There is no conflict of interest.