|Year : 2014 | Volume
| Issue : 2 | Page : 75-80
Transforming growth factor beta 1 in oral submucous fibrosis: An immunohistochemical study - Understanding the pathogenesis
V. V. Kamath, K. P. Satelur, Komali Rajkumar, Shruti Krishnamurthy
Department of Oral and Maxillofacial Pathology, Dr. Syamala Reddy Dental College, Hospital and Research Centre, Munnekolala, Marathalli, Bengaluru, Karnataka, India
|Date of Web Publication||5-Jun-2014|
V. V. Kamath
Department of Oral and Maxillofacial Pathology, Dr. Syamala Reddy Dental College, Hospital and Research Centre, Munnekolala, Marathalli, Bengaluru, Karnataka
Source of Support: None, Conflict of Interest: None
Background: The development of fibrosis is pathognomic in the potentially malignant oral disorder, oral submucous fibrosis (OSF). Strong evidence exists to implicate the chewing of areca nut in the pathogenesis of the lesion. The constituents of areca nut activate several pro-fibrotic cytokines, chiefly transforming growth factor beta 1 (TGF-β1), which leads to an increased deposition and decreased degradation of extracellular matrix and collagen. TGF-β1 probably represents the major pathway in the deposition of collagen fibers in this condition. The present study aims to identify and correlate the expressions of TGF-β1 immunohistochemically on paraffin sections of various stages of OSF. Materials and Methods: The expression of TGF-β1 antibody was detected immunohistochemically using the anti-TGF-β1 mouse monoclonal antibodies (8A11-NovusBio USA) on paraffin sections of 58 cases of OSF, 10 cases of normal buccal mucosa tissue and 5 cases of scar tissue. The site, extent, and intensity of expression and quantification of TGF-β1 were noted and a comparative evaluation between various grades of OSF. Scar tissue and normal oral mucosa was made using image analysis software (Jenoptik optical system-ProReg ® Capture Pro 2.8.8 software ). Results: Cells of spinous layer of the epithelium showed more intense staining in all grades of OSF, Grade II showed the highest percentage of expression, same as that of keloid (17%) but less than that of normal mucosa (12%). Positive staining was seen around blood vessels, muscles, fibers in the submucosa and perimuscle fibers. Highest expression was in the muscle in Grade III (80%) compared with normal oral mucosa (37%). Conclusion: These results suggest that the pathogenesis of OSF and scar/keloid could be linked through the TGF-β1 pathway. Interventions directed at the TGF-beta pathway may hold the key in the future management of this oral potentially malignant condition.
Keywords: Immunohistochemistry, keloid, oral submucous fibrosis, scar, transforming growth factor-β1
|How to cite this article:|
Kamath VV, Satelur KP, Rajkumar K, Krishnamurthy S. Transforming growth factor beta 1 in oral submucous fibrosis: An immunohistochemical study - Understanding the pathogenesis. J Dent Res Rev 2014;1:75-80
|How to cite this URL:|
Kamath VV, Satelur KP, Rajkumar K, Krishnamurthy S. Transforming growth factor beta 1 in oral submucous fibrosis: An immunohistochemical study - Understanding the pathogenesis. J Dent Res Rev [serial online] 2014 [cited 2022 May 21];1:75-80. Available from: https://www.jdrr.org/text.asp?2014/1/2/75/133942
| Introduction|| |
Oral submucous fibrosis (OSF) is a collagen disorder induced by areca nut chewing habit. Fibro-elastic changes are seen in sub-epithelial layer due to abnormal accumulation of collagen resulting in dense fibrotic bands in the mouth. A number of factors trigger the disease process by causing a juxta-epithelial inflammatory reaction in the oral mucosa. 
The pathogenesis of the disease is not well-established and the condition is believed to be multifactorial. Contents of arecanut are believed to trigger the deposition of collagen. Exposure of buccal mucosal fibroblasts in cultures to alkaloid extracts of areca nut results in accumulation of collagen. Synthesis of collagen is influenced by a variety of mediators, including growth factors, hormones, cytokines and lymphokines. 
Transforming growth factor beta (TGF-β) is a pro-fibrotic growth factor implicated in the development of fibrotic lesions. It causes deposition of extracellular matrix (ECM) by increasing the synthesis of matrix protein like collagen and decreasing the degradation by stimulating various inhibitor mechanisms. TGF-β represents a large family of growth and differentiation factors that mobilize through complex signaling networks to regulate cellular differentiation, proliferation, motility, adhesion, and apoptosis. Although TGF-β is essential for healing, overproduction leads to scar tissue and fibrosis. TGF-β isoform is most implicated in fibrosis.  This peptide plays a critical role not only in synthesis and degradation of ECM, but also in response of cells to ECM mediated through integrin receptors; moreover, specific components of the ECM, in turn, can both deliver TGF-β and regulate its activity. Isoforms of the TGF, TGF-β1, TGF-β2, and TGF-β3 have been linked to variations of protein expression or function. TGF-β1 is a key regulator of ECM assembly and remodeling. The cytokine TGF-β1 is considered to have a central role in inducing myofibroblastic phenotype, and its expression is increased under numerous fibrotic conditions. Thus, TGF-β signaling pathway might be a critical event in the pathogenesis of OSF. TGF-β stimulates fibroblast proliferation and EM elaboration suggesting the importance of this cytokine in fibrotic diseases. 
This study was designed to determine the expression of TGF-β1 in tissue samples of OSF immunohistochemically. The pattern, site, and intensity of expression were also recorded. An image analysis of the samples was done to determine the proportion of the area of TGF-β1 to various components of the tissue sample. A comparison with normal oral mucosa and scar tissue, the two ends of the spectrum of fiber deposition, was also done.
| Materials and Methods|| |
This study involved 58 archival cases of OSF, 10 cases of normal mucosa and 5 cases of skin scar tissue. 4 μ paraffin sections were mounted on silane-coated slides and immunohistochemical staining for TGF-β1 antibody (8A11) (NovusBio USA; mouse monoclonal antibodies) was carried out as per standard protocol.  Scar tissue sections were used as the positive controls and negative control was performed by replacing the primary antibody with mouse negative solution (provided by Biogenex ® ). The sections were then stained with DAB (diaminobenzidine - chromogen reagent) and counterstained with hematoxylin for visual assessment.
Primary grading of the OSF cases was done on hematoxylin and eosin stained sections according to the criteria adopted by Sirsat and Pindborg.  The immunohistochemically stained sections were assessed for the following parameters:
- Positivity of antibody detection (±),
- Site of staining reaction (e.g. epithelial layers, stromal localization-perivascular, perimuscular and subepithelial/submucosal).
Quantitative analysis of the expression of TGF-β1 in the connective tissue was done using Jenoptik Optical System-ProReg Capture Pro 2.8.8 software (2011) ® (Goeschwitzer Strasse, Jena, Germany). We analyzed the following:
- Proportion of expression of TGF-β1 in the epithelium and connective tissue
- Proportion of expression of TGF-β1 in the submucosa to total submucosal area
- Proportion of expression of TGF-β1 in muscle tissue to total muscle tissue area
- Proportion of expression o TGF-β1 in the submucosa and muscle to total tissue area.
The results were statistically analyzed using analysis of variance test to determine the significance.
| Results|| |
The demographic data of the sample is presented in [Table 1].
|Table 1: Data on distribution of OSF cases, control (normal oral mucosa) and scar tissue |
Click here to view
Positivity of expression
Positivity of expression of TGF-β1 antibody was seen in all sections of normal oral mucosa, OSF and scar tissue.
Site and intensity of expression
of spinous layer showed more intense staining in all grades of OSF, Grade II showed the highest percentage of expression similar to scar tissue (17%), but less than that of normal oral mucosa (12%) [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5].
|Figure 1: Transforming growth factor beta 1 expression in normal oral mucosal tissue. Note the intensity of expression in the submucosal areas (×10)|
Click here to view
|Figure 2: Transforming growth factor beta 1 expression in Grade I oral submucous fibrosis. Note the mild expression in the epithelium and the more intense expression in the perivascular region of the submucosa (×10)|
Click here to view
|Figure 3: Transforming growth factor beta 1 expression in Grade II oral submucous fibrosis. The intensity of the expression both in the epithelium and the submucosa has increased (×10)|
Click here to view
|Figure 4: Transforming growth factor beta 1 expression in Grade III oral submucous fibrosis. Note the dense expression of the antibody in the submucosa and muscle region with little expression in the epithelium (×10)|
Click here to view
|Figure 5: Transforming growth factor beta 1 expression in scar tissue. Note the generalized staining pattern of the antibody in comparison with the focal diffuse pattern in oral submucous fibrosis (×10)|
Click here to view
staining was seen around blood vessels, muscles, fibers in the submucosa and perimuscle fibers. Very little or mild intensity of expression was noted in the juxta-epithelial region. Surprisingly, the most expression of the antibody was in the muscle in all cases of OSF, Grade III OSF expressing the highest positivity (38%) as compared with normal oral mucosa (27%). The perivascular expression was generally uniform throughout the grades with the least expression in scar tissue, presumably because of decreased vascularity [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5].
[Table 2] shows the proportionate areas of tissue staining positively with the antibody.
Quantification of expression
The proportion of expression of TGF-β1 in the submucosa to the total submucosal area revealed progressive increase within the grades of OSF with Grade III cases showing the highest expression (0.3295 ± 0.1077), but much less than that of scar tissue (0.576 ± 0.1922) and higher than that of the oral mucosa (0.235 ± 0.0591). This was statistically significant [Table 3].
The proportion of expression of TGF-β1 in the muscle to the total muscle area was analyzed only in OSF cases due to the inadequacy of depth of tissue material in the controls and scar tissue. The results were varied with expression dipping in Grade II cases (0.2759 ± 0.2200) as compared with Grade I (0.3458 ± 0.2443) and again peaking in the increased fibrotic areas of Grade III (0.3773 ± 0.2457) [Table 4].
A final computation of the proportion of expression of TGF-β1 in OSF cases in the submucosa and muscle to the total tissue area within the grades showed almost similar trends to the expression in muscle tissue. A dip in Grade II (0.2812 ± 0.132) cases was seen as compared with Grade I (0.3105 ± 0.174) cases and a peak in the Grade III group (0.3391 ± 0.177) [Table 5].
|Table 5: Proportion of TGF-β1 in the submucosa and muscle to total tissue area |
Click here to view
| Discussion|| |
Oral submucous fibrosis is a chronic inflammatory fibrotic disease and a well-established potentially malignant disorder of the oral cavity. Approximately 5 million people worldwide (0.5% of the Indian population) are afflicted by OSF. It is of great concern since the younger age group, especially in the Indian subcontinent are increasingly being trapped by this disease due to the rising popularity of paan masala, gutkha, and arecanut chewing habit. With a rate of malignant transformation (7.6%)  highest in the group of potentially malignant oral disorders, the burden on healthcare, notwithstanding the affliction to the chewer, cannot be overemphasized.
The etiology of the disease is multifactorial, with many agents having been implicated namely arecanut, ingestion of chilies, genetic predisposition, immunologic processes and nutritional deficiencies. Chewing of arecanut is probably the single most important etiologic factor in the causation of OSF. The various constituents of arecanut chief among them being arecoline and tannins, contribute through various pathways in the disease process by causing a juxta-epithelial inflammatory reaction in the oral mucosa which leads to fibrosis.  Accumulation of collagen causes restricted opening of the mouth, which is pathognomic of the condition. It is due to the enhanced accumulation and impaired degradation of collagen and ECM, that OSF is also considered as a collagen metabolic disorder. 
Transforming growth factor beta 1 is among the major fibrotic cytokines in the fibrotic pathway. This pro-inflammatory, multifunctional cytokine belongs to the TGF-β super family. TGF-β controls diverse cellular mechanisms that maintain the proliferation, growth, homeostasis, development and repair of the tissues. The main source of secretion of TGF-β1 is platelets, monocytes, lymphocytes, and macrophages. They direct functions of monocytes, endothelial cells, fibroblasts and keratinocytes such as arresting the proliferation of keratinocytes, blood vessels, regulation of ECM formation, differentiation of mesenchymal tissue, migration of inflammatory cells, control of wound healing, and embryogenesis. 
Transforming growth factor beta signaling pathway is initiated by attachment of ligand to trans membranous serine/threonine kinase Type I and II receptors, which in turn activates the cascade of SMAD (Sma and Mad Related Family) signal transduction pathway. , Attachment of TGF-β ligand to Type II receptor phosphorylates Type I receptor which further activates the intracellular SMAD cascade. SMAD2 and SMAD3 initiates the series of phosphorylation of receptor regulated SMADs. Finally, SMAD2, SMAD3, and SMAD4 are imported to the nucleus which causes regulation of the genes and activates transcription. Inhibitory SMAD7 inhibits the recruitment and phosphorylation of receptor regulated SMADs. ,,,
The chronic microtrauma due to chewing arecanut probably initiates the inflammation and juxta-epithelial fibrosis. TGF-β plays a prominent role in the pathogenesis of OSF, by ECM increased deposition of collagen and its decreased degradation. Here, TGF-β1 plays a dual role of stimulation as well as inhibition. TGF-β isoforms exhibit overlapping, but distinct temporal and spatial patterns of expression in vivo.
Transforming growth factor beta 1 is a key mediator of tissue fibrosis resulting from the accumulation of extra cellular matrix activator protein which induces transcription of COL1A1 procollagen gene, increases levels and activities of the N- and C-procollagen proteinases and promotes the expression of lysyl oxidases, an essential enzyme for final processing of collagen fibers into a stabilized covalently cross-linked mature fibillar form that is resistant to proteolysis. It decreases the collagen degradation by activating tissue inhibitor of matrix metalloproteinase gene and plasminogen activator inhibitor gene. TGF-β causes induction of connective tissue growth factor which further mediates stimulatory actions of TGF-β on ECM synthesis. 
In this study, TGF-β1 showed intense positivity in OSF and scar tissue whereas mild to moderate reactivity was seen in normal oral mucosa. Both epithelium and the connective tissue showed positivity in OSF. This is consistent with previously reported studies. ,,,
Positivity was seen in all the layers epithelium, negligible positivity was noted in the keratinized layer. Fibroblasts, inflammatory cell, endothelial cells showed the positivity in the connective tissue. TGF-β1 showed positivity in muscle fibroblasts.
Staining of TGF-β1 was seen in all the layers of epithelium, with more intense expression in the spinous layer and its intercellular junctions than in the basal and parabasal layer. This may be due to exaggerated appearance of the surface area of the cells in the spinous layer. Very minimal expression was seen in the keratinized layer in our group of tissues. Interestingly, Khan et al.  found no expression in their study in the keratinized layers of OSF, this may be due to structural variations in the parakeratinized layer of buccal mucosa in OSF.
The percentage of expression of TGF-β1 in the epithelium was maximum in Grade I OSF and a gradual decrease was noted with the increasing grades and was almost equal to that of normal in Grade III. Predictably, the expression in scar tissue remained higher than that of all grades OSF. It has been postulated that the inflammatory cells in the early stages of OSF stimulate keratinocytes to produce inflammatory cytokines thereby accounting for the presence of TGF-β1 in the epithelium. ,
Intensity of staining of TGF-β1 in the connective tissue was prominent in perivascular regions, fibroblasts in collagen fibers and in the areas of inflammatory infiltrate.
The expression of TGF-β1 in the fibroblasts, endothelial cells, and inflammatory cells in OSF was more than in normal mucosa. This trend has been generally observed in previous studies. ,
Expression of TGF-β1 in scar tissue was increased compared to OSF. We deliberately included scar tissue in our study for two reasons. The development of fibrosis in scars has been much studied and tabulated. In scars, fibrosis is due to upregulation of SMAD3 and downregulation of SMAD 7 through the TGF-β1 pathway.  The pathogenesis of scar tissue follows the development of fibrosis in OSF.
Significant increase in the expression of TGF-β1 with the increasing grades of OSF may be due infiltration of chronic inflammatory infiltrate such as lymphocytes, monocytes, and macrophages, which are seen in the earlier stages of OSF and are the main source of inflammatory cytokines that lead to progressive fibrosis of the submucosa due to deposition the collagen. 
Significantly TGF-β1 stained muscle bundles, a feature seen in all grades of OSF. This may probably be due to reparative and degenerative changes in the muscle due to the disease process. Rooban et al. in 2005  studied muscle in OSF and observed that muscle are invaded by cytokines which leads to deposition of the mature collagen fibers in the advanced stage of OSF. Due to fibrosis, the striated muscle is probably strained and traumatized leading to pathological changes like necrosis and degeneration. Normally, TGF-β1 is present in mature muscle fibers and can be used as a marker for injured muscle. The higher percentage of expression of TGF-β1 seen in the Grade III OSF is suggestive of possible repair mechanism of the muscle. ,
The establishment of increased levels of TGF-β1 in progressive grades of OSF, in the present study, suggests the involvement of this cytokine as one of the possible main pathways in the pathogenesis of the disorder. A parallel correlation with scar tissue emphasizes the fact that increased fibrosis in OSF is probably a result of exuberant repair induced by ingredients of the areca nut. The only difference between the two disorders is that fibrosis in scar is a localized phenomenon, while OSF graduates into a generalized deposition of collagen resulting in the involvement of major portions of the oral cavity. Interventions in this pathway, chemically or genetically, may possibly be the answer to controlling the lesion.
| References|| |
|1.||Rajalalitha P, Vali S. Molecular pathogenesis of oral submucous fibrosis-a collagen metabolic disorder. J Oral Pathol Med 2005;34:321-8. |
|2.||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. |
|3.||Prime SS, Pring M, Davies M, Paterson IC. TGF-beta signal transduction in oro-facial health and non-malignant disease (part I). Crit Rev Oral Biol Med 2004;15:324-36. |
|4.||Prabin S, Isha S, Kaoru K. Immunohistochemistry: A review of practical procedure. Nepal J Neurosci 2009;6:38-41. |
|5.||Sirsat SM, Pindborg JJ. Subepithelial changes in oral submucous fibrosis. Acta Pathol Microbiol Scand 1967;70:161-73. |
|6.||Murti PR, Bhonsle RB, Gupta PC, Daftary DK, Pindborg JJ, Mehta FS. Etiology of oral submucous fibrosis with special reference to the role of areca nut chewing. J Oral Pathol Med 1995;24:145-52. |
|7.||Tilakaratne WM, Klinikowski MF, Saku T, Peters TJ, Warnakulasuriya S. Oral submucous fibrosis: Review on aetiology and pathogenesis. Oral Oncol 2006;42:561-8. |
|8.||Massagué J. TGF-beta signal transduction. Annu Rev Biochem 1998;67:753-91. |
|9.||Sporn MB. The early history of TGF-beta, and a brief glimpse of its future. Cytokine Growth Factor Rev 2006;17:3-7. |
|10.||Faler BJ, Macsata RA, Plummer D, Mishra L, Sidawy AN. Transforming growth factor-beta and wound healing. Perspect Vasc Surg Endovasc Ther 2006;18:55-62. |
|11.||Moustakas A, Souchelnytskyi S, Heldin CH. Smad regulation in TGF-beta signal transduction. J Cell Sci 2001;114:4359-69. |
|12.||Moustakas A. Smad signalling network. J Cell Sci 2002;115:3355-6. |
|13.||Sebestyén A, Barna G, Nagy K, Jánosi J, Paku S, Kohut E, et al. Smad signal and TGFbeta induced apoptosis in human lymphoma cells. Cytokine 2005;30:228-35. |
|14.||Fuchs O, Provazníková D, Peslová G. Promyelocytic leukaemia protein and defect in transforming growth factor-beta signal pathway in acute promyelocytic leukaemia. Cas Lek Cesk 2005;144:90-4. |
|15.||Illeperuma RP, Ryu MH, Kim KY, Tilakaratne WM, Kim J. Relationship of fibrosis and the expression of TGF-β1, MMP-1, and TIMP-1 with epithelial dysplasia in oral submucous fibrosis. Oral Med Pathol 2010;15:21-4. |
|16.||Gao Y, Ling T, Wu H. Expression of transforming growth factor beta 1 in keratinocytes of oral submucous fibrosis tissue. Zhonghua Kou Qiang Yi Xue Za Zhi 1997;32:239-41. |
|17.||Haque MF, Harris M, Meghji S, Barrett AW. Immunolocalization of cytokines and growth factors in oral submucous fibrosis. Cytokine 1998;10:713-9. |
|18.||Khan I, Agarwal P, Thangjam GS, Radhesh R, Rao SG, Kondaiah P. Role of TGF-ß and BMP7 in the pathogenesis of oral submucous fibrosis. Growth Factors 2011;29:119-27. |
|19.||Kale AD, Mane DR, Shukla D. Expression of transforming growth factor ß and its correlation with lipodystrophy in oral submucous fibrosis: An immunohistochemical study. Med Oral Patol Oral Cir Bucal 2013;18:e12-8. |
|20.||Rooban T, Saraswathi TR, Al Zainab FH, Devi U, Eligabeth J, Ranganathan K. A light microscopic study of fibrosis involving muscle in oral submucous fibrosis. Indian J Dent Res 2005;16:131-4. |
|21.||Heupel K, Sargsyan V, Plomp JJ, Rickmann M, Varoqueaux F, Zhang W, et al. Loss of transforming growth factor-beta 2 leads to impairment of central synapse function. Neural Dev 2008;3:25. |
|22.||Smith CA, Stauber F, Waters C, Alway SE, Stauber WT. Transforming growth factor-beta following skeletal muscle strain injury in rats. J Appl Physiol (1985) 2007;102:755-61. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]
|This article has been cited by|
||The Role of Increased Connective Tissue Growth Factor in the Pathogenesis of Oral Submucous Fibrosis and its Malignant Transformation—An Immunohistochemical Study
| ||Aakruti Mahendra Shah,Kejal Jain,Rajiv S. Desai,Shivani Bansal,Pankaj Shirsat,Pooja Prasad,Kshitija Bodhankar |
| ||Head and Neck Pathology. 2021; |
|[Pubmed] | [DOI]|
||Hyalinization as a histomorphological risk predictor in oral pathological lesions
| ||Dominic Augustine,Roopa S. Rao,Shankargouda Patil |
| ||Journal of Oral Biology and Craniofacial Research. 2021; 11(3): 415 |
|[Pubmed] | [DOI]|
||Oral Submucous Fibrosis: A Review on Biomarkers, Pathogenic Mechanisms, and Treatments
| ||Yen-Wen Shen,Yin-Hwa Shih,Lih-Jyh Fuh,Tzong-Ming Shieh |
| ||International Journal of Molecular Sciences. 2020; 21(19): 7231 |
|[Pubmed] | [DOI]|
||Signaling pathways promoting epithelial mesenchymal transition in oral submucous fibrosis and oral squamous cell carcinoma
| ||Smitha Sammith Shetty,Mohit Sharma,Felipe Paiva Fonseca,Pradyumna Jayaram,Ankit Singh Tanwar,Shama Prasada Kabekkodu,Satyamoorthy Kapaettu,Raghu Radhakrishnan |
| ||Japanese Dental Science Review. 2020; 56(1): 97 |
|[Pubmed] | [DOI]|