|Year : 2012 | Volume
| Issue : 1 | Page : 25-29
Immunolocalization of c-Fos, c-Jun and Fra-2 in healthy human marginal gingival tissues
Nurullah Keklikoglu, Sevtap Akinci
Department of Histology and Embryology, Faculty of Dentistry, Istanbul University, Istanbul, Turkey
|Date of Web Publication||22-Mar-2012|
Department of Histology and Embryology, Faculty of Dentistry, Istanbul University, Istanbul
Source of Support: The Research Fund of the Istanbul University (Project number: BYP-762/23082005)., Conflict of Interest: None
| Abstract|| |
Background and Objective: Keeping marginal gingiva healthy is possible with some cellular processes. Activator Protein-1 (AP-1) members Fos and Jun proteins regulate cellular processes including growth, proliferation, differentiation and apoptosis. The aim of this study was to compare of immunoreactivity (IR) of some same or different family member AP-1 proteins in cells forming marginal gingival mucosa. Materials and Methods: IR of Fos family members c-Fos (c-Fos-IR) and Fra-2 (Fra-2-IR) and Jun family member c-Jun (c-Jun-IR) were analysed in epithelial cells (ECs) and lamina propria cells (LPCs) of healthy human marginal gingiva by immunohistochemical method. Results: Both in ECs and LPCs, c-Fos-IR positivity was found higher than the others and Fra-2-IR positivity was found lower. The most postivity was observed in c-Fos-IR in ECs and the least IR was observed in Fra-2-IR in LPCs. Furthermore, the IR positivity percentage of all proteins was found higher in ECs than in LPCs. In the comparision of each of c-Fos-IR, c-Jun-IR and Fra-2-IR in ECs and LPCs, no statistically significant difference in c-Jun-IR was observed; however, there were statistically significant differences in c-Fos-IR and Fra-2-IR. Conclusions: According to these findings, it can be proposed that c-Fos protein in healthy gingiva is more related with physiological processes than other proteins. In this study, establishing a statistically significant difference between c-Fos-IR and Fra-2-IR in both ECs and LPCs has shown that as well as these proteins are within the same family, each one may be related with different cellular processes.
Keywords: Activator protein-1, AP-1, c-Fos, c-Jun, Fra-2, gingiva
|How to cite this article:|
Keklikoglu N, Akinci S. Immunolocalization of c-Fos, c-Jun and Fra-2 in healthy human marginal gingival tissues. J Interdiscip Dentistry 2012;2:25-9
|How to cite this URL:|
Keklikoglu N, Akinci S. Immunolocalization of c-Fos, c-Jun and Fra-2 in healthy human marginal gingival tissues. J Interdiscip Dentistry [serial online] 2012 [cited 2019 Jun 24];2:25-9. Available from: http://www.jidonline.com/text.asp?2012/2/1/25/94188
| Introduction|| |
Gingiva, which is a kind of masticatory mucosa, is divided into attached and marginal (free) gingiva. Attached gingiva is firmly bound to the tooth and alveolar process, whereas free or marginal gingiva is not directly attached to the tooth surface and that composes the outer wall of the gingival sulcus.  Marginal gingival mucosa consists of epithelial cells (ECs) of stratified squamous epithelium and connective tissue cells of lamina propria (LPCs). ,
Gingival tissues are exposed to the effect of external physical and chemical stimuli during mastication and toothbrushing. ,, One of the first exposed tissues to these effects is the marginal gingiva within oral mucosa. Although some of these stimuli can give traumatic effects on the marginal gingiva, mechanical stimuli are regulators of connective tissue homeostasis and affect cell proliferation, development, maintenance and remodeling of the periodontal tissues and may lead to modifications in cell activity. ,,,, However, there is a balance between proliferation, apoptosis and differentiation and break of this balance may cause development of some disorders. 
In the cells exposed to any of physiological or pathological extracellular stimuli, the genes which respond to these stimuli are immediate early genes (IEGs).  Transcription factor complex Activator Protein-1 (AP-1) members Jun and Fos proteins are nuclear protein products of jun and fos genes of IEGs family.  AP-1 is consist of Jun proteins (c-Jun, JunB and JunD), Fos proteins (c-Fos, FosB, Fra-1 and Fra-2) and some activating transcription factor (ATF) subfamily members (ATFa, ATF-1, ATF-2, and ATF-3). ,, AP-1 regulate cellular processes including growth, proliferation, differentiation and apoptosis. , AP-1 is crucial for cell adaptation to many environmental changes  and is contribute to basal and stimulus-activated gene expression. 
AP-1 plays a pivotal role in physiological processes such as epidermal homeostasis within oral mucosa.  It has been shown that c-Jun and c-Fos expression is present in normal oral mucosa. , However AP-1 could play an important role in the pathogenesis of periodontal disease,  and different stages of oral oncogenesis. ,,
Although Fos and Jun protein families have been extensively studied in oral mucosa in order to understand their role in physiological and pathological mechanisms, there is not enough acknowledgment of the presence of these proteins in marginal gingival tissues in contemporary literature. The aim of this study was to compare of immunoreactivity (IR) of some same or different family member AP-1 proteins which plays important roles in many cellular activities such as growth, proliferation, differentiation and apoptosis in cells forming marginal gingival mucosa. For this purpose, IR of Fos family member c-Fos (c-Fos-IR), Jun family member c-Jun (c-Jun-IR) and also Fos family member Fra-2 (Fra-2-IR) were analysed in ECs and LPCs of healthy human marginal gingiva.
| Materials and Methods|| |
Collection of tissue samples
The study protocol was approved by Istanbul University Istanbul Medical Faculty Ethical Committee (2007-1748). Clinically healthy marginal gingival tissue samples were obtained from eight patients (three women and five men) whose mean age was 37.13±15.13 years. Tissues were collected from different parts of the oral cavity during periodontal surgical treatment (especially clinical crown-lengthening procedures). Only one biopsy was taken per patient. There were no mucosal abnormalities in biopsy regions. None of the patients had systemic disease, and all participants were non-smokers.
Samples processing and immunohistochemistry
Marginal gingival tissue samples were fixed in 10% buffered formalin and embedded in paraffin by routine methods. Paraffin sections (5-μm thick) were mounted on glass slides. Twenty-four slides for each sample, a total of 192 slides were prepared. Twenty-four slides from each sample were divided into three groups and these groups were used for c-Fos-IR, c-Jun-IR and Fra-2-IR. Sections were first deparaffinized in xylene, rehydrated through graded ethanol series and then covered with sodium citrate buffer (at 95°C, pH 6.0) for 15 min, for antigen retrieval. Endogenous peroxidase activity was blocked with 3% H 2 O 2 in absolute methanol for 10 min. After washing, the sections were incubated with 10% goat nonimmune serum (serum-blocking solution, Histostain-Plus Bulk Kit, ZYMED, CA) for 10 min to eliminate nonspecific staining.
Tissues were then incubated with the rabbit polyclonal antibodies against c-Fos (c-Fos (K-25): Sc-253, Santa Cruz Biotechnology, Inc., CA) at dilution 1:80, c-Jun (c-Jun (H-79): Sc-1694, Santa Cruz Biotechnology, Inc.) at dilution 1:80 and Fra-2 (Fra-2 (L-15): Sc-171, Santa Cruz Biotechnology, Inc.) at dilution 1:80 for 6 hours and then in the biotinylated second antibody for 10 min (Histostain-Plus Bulk Kit, ZYMED, CA). Negative controls were performed by using nonimmune rabbit serum. Immunoreactivity was detected by means of horseradish-peroxidase (HRP)-streptavidin complex (Histostain-Plus Bulk Kit, ZYMED) using aminoethyl carbazole substrate (AEC) chromogen as a marker [AEC (red) Substrate Kit, ZYMED]. Sections were counterstained in Mayer's hematoxylin for 2 min and finally mounted. All steps were carried out at room temperature in a humidified chamber. Immunostained-positive cells were counted and evaluated in ECs and LPCs on the whole slide by using a light microscope (Laborlux K, Leitz, Germany) at 400× and 1000× magnification in a blind scoring procedure, and the percentages within cells in the same type were calculated. Thus, percentages within each cell group for each sample were calculated and a single ratio was given.
Statistical analysis was performed using paired-samples t-test. P-values<0.05 were considered significant.
| Results|| |
The results of the immunohistochemical staining mean percentage of c-Fos, c-Jun and Fra-2 proteins in ECs and LPCs of healthy marginal gingival tissue samples has been given in [Table 1]. In both ECs and LPCs, c-Fos-IR positivity was found higher than the others and Fra-2-IR positivity was found lower. Furthermore, the IR positivity percentage of all proteins was found higher in ECs than in LPCs. The most postivity was observed in c-Fos-IR in ECs with a percentage of 35.7±13.5% and the least IR was observed in Fra-2-IR in LPCs with a percentage of 8.9±15.2%.
|Table 1: Percentage of cells with positive c-Fos, c-Jun and Fra-2 immunostaining in epithelial cells and lamina propria cells in healthy human marginal gingival tissues|
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The light microscopical features of immunostaining for each antibody were as following: C-Fos-IR; Both nuclear and cytoplasmic staining were observed in IR-positive cells in ECs and positive cells were more seen in the upper layer of epithelium. In LPCs, nuclear staining was predominant. Positivity was seen in fibroblast and lymphoid cells scattered in lamina propria [Figure 1] c-Jun-IR; Both nuclear and cytoplasmic staining were observed in IR-positive cells in ECs and LPCs. ECs in upper and basale layers of epithelium were intensively positive. Positivity was seen mostly in fibroblast and lymphoid cells scattered in lamina propria. [Figure 2]. Fra-2-IR; In ECs the cytoplasmic staining was predominant and positive cells were more seen in the upper layer of epithelium. Positive LPCs formed randomly distributed regions on lamina propria. Positive cells formed intense stained regions. Both nuclear and cytoplasmic staining were observed in LPCs [Figure 3].
|Figure 1: Immunoreactivity for c-Fos in healthy human marginal gingival tissues. Immunoreactive cells are seen (arrows). Epithelial cells (ECs), lamina propria cells (LPCs). Magnification: ×400|
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|Figure 2: Immunoreactivity for c-Jun in healthy human marginal gingival tissues. Immunoreactive cells are seen (arrows). Epithelial cells (ECs), lamina propria cells (LPCs). Magnification: ×400|
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|Figure 3: Immunoreactivity for Fra-2 in healthy human marginal gingival tissues. Immunoreactive cells are seen (arrows). Epithelial cells (ECs), lamina propria cells (LPCs). Magnification: ×400|
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In the comparision of each of c-Fos-IR, c-Jun-IR and Fra-2-IR in ECs and LPCs no statistically significant difference in c-Jun-IR was observed (P>0.05); however, there were statistically significant differences in c-Fos-IR and Fra-2-IR (P<0.05). Binary comparison of c-Fos-IR, c-Jun-IR and Fra-2-IR in each of ECs and LPCs revealed no significant differences between c-Fos-IR and c-Jun-IR, c-Jun-IR and Fra-2-IR (P>0.05). However, comparison of c-Fos-IR and Fra-2-IR in each of ECs and LPCs revealed significant differences (P<0.05) [Table 2].
|Table 2: Comparing of proteins in each groups of epithelial cells and lamina propria cells|
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| Discussion|| |
In this study, since the most IR is presented by c-Fos protein in both cell groups, it can be proposed that this protein is more related with physiological processes than other proteins used in this study. Also according to this study, Fra-2 protein being less immunoreactive than other proteins in both cell groups shows that this protein may be more effective in regulating pathological processes rather than physiological processes in human marginal gingiva. In a previously performed study, it is been asserted that Fra-2 protein has positive effect in tumor invasion and progression of a great number of tumor types. 
Gingival epithelium has a protective effect against the frictional forces of mastication and foreign invaders.  Maintenance of continuous epithelial barrier is materialized through cell proliferation and differentiation.  For example, tooth brushing contributes to the health of gingiva by providing the proliferation of gingival cells by means of mechanical stimulation.  AP-1 plays a very important role during cellular proliferation, differentiation and apoptosis. , Accordingly, it is not surprise that all AP-1 proteins used in this study being more immunoreactive in ECs than LPCs and that most of these proteins being more immunoreactive in upper layers of epithelium than basale layers. It has been stated that toothbrushing induces the activation of c-fos expression in gingival tissues.  Epithelial expression of Fos protein is related to epithelial cell differentiation and cell death.  According to the results of this study, c-Fos-IR has a 35.7±13.5% positivity ratio in ECs, which this value is the highest IR ratio found in this study; indicating that c-Fos is more related with epithelial maintenance in gingiva than other AP-1 proteins used in this study. Additionally, c-Fos-IR and Fra-2-IR in ECs being statistically more than in LPCs has shown that as well as both c-Fos and Fra-2 are members of the Fos protein family, Fos proteins are more related in this process than c-Jun.
According to the results of this study, c-Jun-IR in each one of ECs and LPCs being statistically no different than the IR of other proteins shows that c-Jun may be involved in the processes which other two proteins are related. In a previously performed study on healthy rat small intestine, Fra-2-IR and c-Jun-IR have been researched in ECs and LPCs and established that Fra-2 has been being more expressed than c-Jun.  This difference may be considered as a result of being exposure to different effects in tissues.
It is known that AP-1 member proteins have different transactivation potentials  and each one of these proteins is related with various stages of cellular process.  In addition, it is established that c-Fos and Fra-2 have functionally different properties.  In this study, establishing a statistically significant difference between c-Fos-IR and Fra-2-IR in both ECs and LPCs has shown that as well as these Fos proteins are within the same family, each one may be related with different processes.
Isolation of oral tissues from outer environmental effects is not possible. Oral cavity and especially gingiva are exposed to various mechanical effects during mastication and tooth brushing.  These consistently effects require a precise physiological balance. AP-1 plays an active role in physiological processes in gingiva. Also, stimuli such as hormones, cytokines, growth factors, stress signals, infections, radiation or chemical carcinogens activate AP-1. ,, IEGs and AP-1 are asserted to be the key factors in carcinogenesis. ,, In previously performed studies, it is established that different rates of c-Fos and c-Jun proto-oncogenes and c-Fos and c-Jun proteins are expressed in normal oral mucosa, oral premalignant lesions and oral carcinomas. ,,,, The findings of this study can be used as a base to state the priorities in pathogenity studies resulting from the impairment in the cellular processes controlled by AP-1 in gingiva such as oncogenic transformation, gingival overgrowth (gingival hyperplasia), growth retardation, gingival hypoplasia, tissue destruction in chronic inflammatory periodontal diseases, to delay in tissue formation and tissue remodeling in gingival wound healing and cyst development. The effective factors in the development of such pathologies and their role of possible inductive effect or inhibition of AP-1 expression have very important in aetiology, prevention and treatment of these gingival disorders.
Knowing the expression rates of these proteins under physiological conditions and that, proteins from the same family having different effect mechanism is required for understanding their effects on formation of various pathologies. Maintaining balance of physiological expression ratio of AP-1 proteins protect physiological cellular processes against pathological transformations in gingiva.
| Acknowledgments|| |
This study was supported by the Research Fund of the Istanbul University (Project number: BYP-762/23082005). The author wishes to thank Dr. Humeyra Kocaelli (Istanbul University Faculty of Dentistry Department of Oral Surgery and Medicine, Istanbul, Turkey) for her valuable help in providing the necessary materials for this research, Assoc. Prof. Dr. Vakur Olgac (Istanbul University Institute of Oncology Department of Pathology, Istanbul, Turkey) for his pathology assistance and Biologist Ilker Bolat (Faculty of Dentistry Department of Histology and Embryology Istanbul University, Istanbul, Turkey) for his assistance during experimental work.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]