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Year : 2014  |  Volume : 6  |  Issue : 2  |  Page : 108-113

Transforming growth factor β2 (TGF-β2) in pathogenesis of oral submucous fibrosis: An immunohistochemical study

Department of Oral and Maxillofacial Pathology, Dr. Syamala Reddy Dental College, Hospital and Research Centre, Munnekolala, Marathalli, Bangalore, Karnataka, India

Date of Web Publication16-Oct-2014

Correspondence Address:
Venkatesh V Kamath
Department of Oral and Maxillofacial Pathology, Dr. Syamala Reddy Dental College, Hospital and Research Centre, Munnekolala, Marathalli, Bangalore - 560 037, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0975-8844.143053

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Background and Objectives: Oral Submucous Fibrosis (OSF) is a potentially malignant oral disorder causing fibrosis of the oral mucosa. Commonly associated with the habit of chewing areca nut in its raw or refined forms, the progressive fibrosis causes intense debility and probable malignant transformation. Arecoline, flavinoids and tannins in the areca nut may activate pro-fibrotic cytokines like transforming growth factor beta (TGF-β) leading to fibrosis. TGF-β and its isoforms probably represent the major pathway in the deposition of collagen fibers in this condition. Very little is known of the role of TGF-β2, as compared withTGF-β1, in OSF. The present study aims to evaluate TGF-β2 immunohistochemically in OSF with a view to understanding its role in the pathogenesis. Materials and Methods: TGF-β2 antibody was detected immunohistochemically on archival paraffin sections of 70 cases of various grades of OSF, 10 cases of normal oral mucosa and five cases of scar tissue. The presence and distribution of the antibody was noted and a quantification of the positive areas was also done using image analyses software and correlated in proportion to the rest of the tissue. Results: Expression of TGF-β2 was more in all grades of OSF when compared with that of normal oral mucosa but less than that expressed in scar tissue. The antibody was detected in epithelium, around the blood vessels, in areas of inflammatory infiltrate, fibroblasts and in muscles. The intensity and proportion of expression paralleled increasing grades of OSF. There was increased expression of the antibody in the epithelium, which is probably the source, but no correlation to epithelial changes (hyperplasia, atrophy or dysplasia) was noted. Conclusion: TGF-β2 is a prominent cytokine in the TGF-β induced pathway of fibrosis but probably plays a contributory role to the main isoform TGF-β1. Its role as a marker of malignant transformation, as seen in other systemic malignant lesions, remains inconclusive in OSF.

Keywords: Immunohistochemistry, oral submucous fibrosis, scar, TGF-β2

How to cite this article:
Kamath VV, Satelur KP, Rajkumar K, Krishnamurthy S. Transforming growth factor β2 (TGF-β2) in pathogenesis of oral submucous fibrosis: An immunohistochemical study . J Orofac Sci 2014;6:108-13

How to cite this URL:
Kamath VV, Satelur KP, Rajkumar K, Krishnamurthy S. Transforming growth factor β2 (TGF-β2) in pathogenesis of oral submucous fibrosis: An immunohistochemical study . J Orofac Sci [serial online] 2014 [cited 2019 Jan 18];6:108-13. Available from:

  Introduction Top

A pathognomic feature of OSF is the progressive fibrosis and the resulting debility. The increased deposition of fibers is probably induced by contents of the areca nut chewed by the afflicted individuals and follows mediation by the cytokine TGF-β pathway. [1]

TGF-β is a member of a large family of structurally related growth and differentiation factors. There are three TGF-β isoforms in humans - TGF-β1, TGF-β2, and TGF-β. The amino acid sequences of the three isoforms are 70-80% homologous and encoded by distinct genes located to 19q13.1-q13.3, 1q41, and 14q24, respectively. [2],[3]

TGF-β2 is an extracellular glycosylated protein, which belongs to the TGF-β family. TGF-β regulates key mechanisms of tumor development, namely immunosuppression, metastasis, angiogenesis and proliferation. This gene encodes a member of the TGF-β family of cytokines, which are multifunctional peptides that regulate proliferation, differentiation, adhesion, migration and other functions in many cell types by transducing their signal through combinations of transmembrane type I and type II receptors (TGF-β1 and TGF-β2) and their downstream effectors, the Sma and Mad Related Family (SMAD) proteins. [4]

TGF-β2 is produced by many cell types and has been found in the highest concentration in porcine platelets and mammalian bone. Latent TGF-β2 is the prominent isoform found in body fluids such as amniotic fluid, breast milk and the aqueous and vitreous humor of the eye. [5]

The role of TGF-β and its prominent isoform TGF-β1, in the pathogenesis of OSF has been discussed in literature. [1] The role of the isoform TGF-β2 surprisingly merits very little mention. The synergy or otherwise of the two isoforms in the TGF-β molecule is of intense interest as both the isoforms have varying effects on tissue especially in EMT (epithelial mediated transformation).

The present study aims to assess the expression of TGF-β2 immunohistochemically, in the disorder of OSF with a view to understanding the pathogenesis. The role of TGF-β2 either as a synergist or a molecule working independent of TGF-β1 in the causation of fibrosis in OSF is planned to be evaluated. The establishment of the roles of the two isoforms of TGF-β would be interesting in targeting them in future in attempts to control the lesion.

  Materials and methods Top

Seventy cases of varying grades of OSF, 10 cases of normal oral mucosa and five cases of scar tissue from departmental archives were used for the study. The histological grading of OSF cases was done on hematoxylin and eosin (H&E) stained sections as per criteria of Pindborg and Sirsat (1966). [6]

Four micron sections were mounted on silane coated slides and used for the immunohistochemical staining procedures. Anti TGF-β2 antibody in a dilution of 1 μl/100 μl ((TB21) (NovusBio, USA; Cat No: NBP1-51749) was used following a protocol recommended by the manufacturer. The sections were incubated at 35 o C overnight. This was followed by two washes with xylene of 15 minutes each, two washes with absolute alcohol for one minute each and then washes with 90%, 70% alcohol for a minute. The slides were then washed repeatedly with deionized water before proceeding with antigen retrieval. Antigen retrieval was done in a standard microwave. Slides placed in Tris-buffer were microwaved for 30 minutes prior to antibody staining. Post-retreival the slides were washed in phosphate buffered saline (PBS) and a peroxidase block applied for 15 minutes. Following further washes with PBS thrice for 5 minutes each a power block was applied for 15 minutes. The sections were then incubated in the antibody overnight at 4 o C in a humid chamber. Further washes with PBS were carried out and the slides incubated with super enhancer solution (Dako, Denmark) for 30 minutes followed by secondary antibody (Biogenex Life Sciences Ltd, USA) incubation at room temperature for 1 hour. The slides were then washed with PBS again and 3,3΄-diaminobenzidine tetrahydrochloride (DAB) reagent was applied for 10 minutes. The sections were then washed in tap water and PBS and counterstained with hematoxylin.

The positively stained areas were then quantified using an image analysis software (JenOptix;). A statistical correlation was derived between the normal oral mucosa, OSF and scar tissue expressions using Mann U Whitney and Pearsons' coefficient variation tests.

  Results Top

[Table 1] lists the distribution of the cases involved in the study.
Table 1: Distribution of OSF cases, normal oral mucosa and scar tissues

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Epithelium (TGF-β2)

Cells of spinous layer showed more intense staining in all the grades of OSF, the expression in Grade I OSF being the highest (52.5%) similar to that of normal oral mucosa (50%) and surprisingly more than that of scar tissue (46%) [Table 2] and [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]. The expression of the antibody in the basal layer of the epithelium was uniform in all cases of OSF, and marginally more in the scar tissue.
Table 2: Proportional Expression of TGF-β2 in the different layers of the epithelium

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Figure 1: Immunohistochemical staining with anti-TGF-β2 antibody in normal oral mucosa. Note the intense staining in the sub-epithelial region (×10 original magnification)

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Figure 2: Immunohistochemical staining with anti-TGF-β2 antibody in gr I OSF. Note the positivity in the vascular channels, and epithelium (×10 original magnification)

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Figure 3: IHC staining of TGF-β2 in gr II OSF. Intense positivity is seen in the submucosal collagen fibres especially prominent in the sub-epithelial region (×10 original magnification)

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Figure 4: IHC staining of gr III OSF tissue with anti-TGF-β2 antibody. Note positivity in basal/parabasal areas of epithelium and generalised staining of the submucosa (×10 original magnification)

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Figure 5: IHC anti-TGF-β2 antibody staining in scar tissue. Note the diffuse and generalised positivity of the stain throughout the tissue. (×10 original magnification)

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Connective tissue (TGF-β2)

Positive staining was seen around blood vessels, muscles, fibers in submucosa and peri-muscle fibers [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]. Highest expression was in collagen fibers present in submucosa of all the groups; scar tissue showing the highest expression (66%) compared with OSF and normal oral mucosa [Table 3]. Expression in the muscle was the least, almost negligible.
Table 3: Proportional Expression of TGF-β2 in the different layers of the connective tissue

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A computation by image analysis of the areas expressing TGF-β2 in the submucosa revealed maximum expression in scar tissue followed by Grade III OSF. All cases of OSF and scar tissue showed a higher expression as compared with normal oral mucosa [Table 4]. The statistical comparison was significant at the P level.
Table 4: Proportion of TGF-β2 expression in submucosa as judged by image analysis

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The expression of TGF-β2 antibody in the muscle tissue in all grades of OSF was almost similar with marginal increase in Gr II cases. As noted earlier the overall expression of the antibody in the muscle was minimal [Table 5].
Table 5: Proportion of TGF-β2 expression in the muscle as judged by image analysis

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When the total expression of TGF-β2 in the submucosa and muscle were compared with total subepithelial tissue area a progressive increase in expression in the Gr III and Gr II cases was noted. The Gr II cases showed a marginal increase of expression as compared with Gr III [Table 6].
Table 6: Proportion of TGF-β2 expression in submucosa and muscle to total tissue area in OSF cases as judged by image analysis

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  Discussion Top

The tissue pathway of fibrosis in OSF is still under investigation but evidence of implication of the TGF-β cytokine is increasingly being reported. Amongst the isoforms of TGF-β, TGF-β1 is the prominent arbritator with supplementary contribution by the other isoforms. The role of TGF β2 has never been explored in the disorder, most studies concentrating on the TGF molecule as a whole or its major β1 isoform.

The role of TGF-β2 in other human disorders is increasingly being recognized. The role of TGF-β2 in wound healing has been well documented. Of interest are observations in Xenopus froglets (that heal skin wounds without scarring) where induction of injury resulted in upregulation of Xenopus TGF-β2 during the secondary stage of granulation tissue formation. [7] The situation is somewhat different in human skin exposed to solar ultraviolet irradiation. In these circumstances, there is increased expression of TGF-β1 and TGF-β3 but decreased expression of TGF-β2 (the predominant TGF-β isoform in normal skin epithelium); TβR-II is decreased and SMAD 7 is increased. Taken together, the changes are thought to drive extracellular matrix (ECM) elaboration. [8]

TGF-β2 has been implicated as a marker in the progression and metastatic potential of breast cancer and macular cancers. Its role in oral malignant transformation has also been elaborated. Increased production of TGF-β2 has been noted in genetically unstable oral squamous cell carcinoma (GU-OSCC). The latter is a subset of genetically stable oral squamous cell carcinoma (GS-OSCC) possessing wild-type p53. In a study keratinocytes from GU-OSCC lesions were found to lose their senescence and this change was associated with increased production of TGF-β2. Keratinocyte senescence acts as a barrier to malignant transformation and is lost in premalignant and malignant lesions. [9]

Arecanut chewing has always been implicated the most probable causative factor in the etiopathogenesis of OSF. In an interesting study on the effects of areca nut constituents on culture cells to examine their influence on the TGF-β cytokine, the alkaloids (arecoline, arecaidine, guvacine) and the polyphenols (catechins and tannins) were shown to induce TGF-β2 production and stimulation in in vitro studies. The authors found increased expression of the TGF-β2 in cultures of HaCaT cells exposed to areca nut extracts indicating a definitive involvement of the cytokine in the fibrogenetic pathway of the disorder. [10]

In examining the cellular mechanisms of action of the TGF-β in inducing fibrosis it has been reported that the type I plasminogen activator inhibitor (PAI-1), a known Smad-responsive gene, is up-regulated in OSF [11] thus giving indirect evidence for enhanced TGF-β activity in OSF. Furthermore, it has been suggested that polymorphisms in the TGF-β gene may affect an individual's susceptibility to this most debilitating of disorders. [12]

The results of the present study are concurrent with the trends seen in the evaluation of the role of TGF-β2 in OSF in the literature. A persistent increase in the expression of the TGF-β2 antibody in all grades of OSF as compared with normal oral mucosa is reflective of the increased production of this cytokine in the condition. The progressive increase in Gr II and Gr III cases indicates a direct correlation with the fibrotic process. It was interestingly noted that marginally more expressions of the antibody in the connective areas were seen in Gr II OSF as compared with Gr III probably indicating a peaking of the production of the antibody midway through the fibrosis.

The expression of TGF-β2 in the epithelium seemed to be concentrated in the basal-spinous cell region with the spinous cell layer showing the maximum concentration, a feature generally consistent in all the groups. A similar observation has been made for TGF-β and β1 expressions in previous studies.

The evaluation of TGF-β2 independently in the OSF tissues was done to establish its role in the fibrosis development of the lesion. An increase in TGF-β expression in all grades of OSF has been well-documented. The contribution of the two isoforms in the increased overall expression of the TGF-β has not been assessed until now. The results of our present study clearly indicate an increased expression of TGF-β2 in all grades of OSF with a peaking in the second grade. This demonstrates a significant contribution of the isoform in the pathogenesis of the lesion. The pattern of expression also demonstrates a peak in Grade II OSF tissues indicating a controlled liberation of the isoform during the progression of the disease. It is also well established that the stimulation of fibroblasts by arecoline of the areca nut induces a phenotypic change towards a population of fibroblasts that produce more type I collagen. The development of this population may be reflected in the increased expression of TGF-β2 as a possible pathway in the deposition of the excess collagen.

We could not detect any dysplastic changes in the epithelium of our samples and no correlation could be drawn with the expression of the antibody in such conditions. This is specially interesting in view of two facts; one being the potentially malignant nature of OSF and other being the increasing acceptance of TGF-β2 as a marker for the progression of malignancy in breast and oral carcinomas.

The exact role of TGF-β2 in the fibrotic process in the disorder of OSF still remains to be ascertained but it is increasingly being recognized that this isoform of the TGF-β cytokine is synergistic and additive in the cellular pathway. The mode of initiation of fibrosis also seems to follow a similar pattern to the mother cytokine.

Of increasing interest to the researcher involved in understanding the pathogenesis of the disorder of OSF is the possible relationship of this cytokine to the premalignant potential of the lesion. An establishment of a direct correlation between this molecule and the premalignant status of the tissue and its possible malignant transformation would help in identifying TGF-β2 as a possible marker of such a change, similar to the recognition in some types of oral carcinomas and breast cancer.

  Conclusion Top

TGF-β2 was found to be increased in all grades of OSF with a peak in the Grade II as compared with normal mucosa. The histological location of the antibody was found to be concentrated in the basal-spinous layer of the epithelium with increased concentration in the spinous layer. The role of this isoform seems to be important in the pathogenesis of the disorder and it appears that the isoform synergistically acts with its counterpart in the deposition of a more stable form of collagen in the OSF tissues.

  References Top

Rajalalitha P, Vali S. Molecular pathogenesis of oral submucous fibrosis - A collagen metabolic disorder. J Oral Pathol Med 2005;34:321-8.  Back to cited text no. 1
Fujii D, Brissenden JE, Derynck R, Francke U. Transforming growth factor- β gene maps to human chromosome 19 long arm and to mouse chromosome 7. Somat Cell Mol Genet 1986;12:281-8.  Back to cited text no. 2
Barton DE, Foellmer BE, Du J, Tamm J, Derynck R, Francke U. Chromosomal mapping of genes for transforming growth factors- β2 and - β3 in man and mouse: Dispersion of the TGF-β gene family. Oncogene Res 1988;3:323-31.  Back to cited text no. 3
Prime SS, Pring M, Davies M, Paterson IC. TGF-β2 signal transduction in oro-facial health and non-malignant disease (part I). Crit Rev Oral Biol Med 2004;15:324-36.  Back to cited text no. 4
Massague J. TGF-β2 signal transduction. Annu Rev Biochem 1998;67:753-91.  Back to cited text no. 5
Sirsat SM, Pindborg JJ. Subepithelial changes in oral submucous fibrosis. Acta Pathol Microbiol Scand 1967;78:161-73.  Back to cited text no. 6
Bertolotti E, Malagoli D, Franchini A. Skin wound healing in differently aged Xenopuslaevis. J Morphol 2013;274:956-64.  Back to cited text no. 7
Quan T, He T, Kang S, Voorhees JJ, Fisher GJ. Ultraviolet irradiation alters transforming growth factor -β/Smad pathway in human skin in vivo. J Invest Dermatol 2002;119:499-506.  Back to cited text no. 8
Hassona Y, Cirillo N, Lim KP, Hermann A, Mellone M, Thomas GJ, et al. Progression of genotype specific oral cancer leads to senescence of cancer associated fibroblasts and is mediated by oxidative stress and TGF-β. Carcinogenesis 2013;34:1286-95.  Back to cited text no. 9
Khan I, Kumar N, Pant I, Narra S, Kondaiah P. Activation of TGF-β pathway by areca nut constituents: A possible cause of oral submucous fibrosis. PLoS One 2012;7:e51806.  Back to cited text no. 10
Dong C, Li Z, Alvarez R Jr, Feng XH, Goldschmidt-Clermont PJ. Microtubule binding to Smads may regulate TGF-β activity. Mol Cell 2000;5:27-34.  Back to cited text no. 11
Chiu CJ, Chang ML, Chiang CP, Hahn LJ, Hsieh LL, Chen CJ. Interaction of collagen-related genes and susceptibility to betel quid-induced oral submucous fibrosis. Cancer Epidemiol Biomarkers Prev 2002;11:646-53.  Back to cited text no. 12


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]


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