Activation of TGF-β pathway by areca nut constituents: a possible cause of oral submucous fibrosis

Abstract

Oral submucous fibrosis (OSF) is a chronic inflammatory disease characterized by the accumulation of excess collagen, and areca nut chewing has been proposed as an important etiological factor for disease manifestation. Activation of transforming growth factor-β signaling has been postulated as the main causative event for increased collagen production in OSF. Oral epithelium plays important roles in OSF, and arecoline has been shown to induce TGF-β in epithelial cells. In an attempt to understand the role of areca nut constituents in the manifestation of OSF, we studied the global gene expression profile in epithelial cells (HaCaT) following treatment with areca nut water extract or TGF-β. Interestingly, 64% of the differentially regulated genes by areca nut water extract matches with the TGF-β induced gene expression profile. Out of these, expression of 57% of genes was compromised in the presence of ALK5 (TβRI) inhibitor and 7% were independently induced by areca nut, highlighting the importance of TGF-β in areca nut actions. Areca nut water extract treatment induced p-SMAD2 and TGF-β downstream targets in HaCaT cells but not in human gingival fibroblast cells (hGF), suggesting epithelial cells could be the source of TGF-β in promoting OSF. Water extract of areca nut consists of polyphenols and alkaloids. Both polyphenol and alkaloid fractions of areca nut were able to induce TGF-β signaling and its downstream targets. Also, SMAD-2 was phosphorylated following treatment of HaCaT cells by Catechin, Tannin and alkaloids namely Arecoline, Arecaidine and Guvacine. Moreover, both polyphenols and alkaloids induced TGF-β2 and THBS1 (activator of latent TGF-β) in HaCaT cells suggesting areca nut mediated activation of p-SMAD2 involves up-regulation and activation of TGF-β. These data suggest a major causative role for TGF-β that is induced by areca nut in OSF progression.

Figure 1. Hierarchical clustering of TGF-β and Areca nut induced genes.

A] Hierarchical clustering of commonly regulated genes in HaCaT cells following treatments with areca nut water extract (5H−5 µg/ml) or TGF-β (5 ng/ml) (P≤0.05 and ≥1.5 fold). B] Hierarchical cluster of genes that are commonly regulated by areca nut or TGF-β but compromised in presence of ALK5 inhibitor. Red, green and black colours in A and B represent up, down or un- regulated genes respectively. The vertical axis represents genes and horizontal axis represents treatments. C] i and ii, Venn diagram representation of the genes from A and B respectively.

Figure 2. Areca nut induces TGF-β signaling in epithelial cells.

A & B] Areca nut induced genes by Western blot and by qRT-PCR. HaCaT cells were serum starved for 24 hours and treated with either areca nut water (H) or ethanol (Et) extracts 2.5, 5 µg/ml (2.5H/5H & 2.5Et/5Et) in serum free medium for 48 hours. A) Western blot of HaCaT cell lysates showing induction of p-SMAD2 and its downstream target TGM2. B) qRT-PCR analysis showing expression of TGF-β down-stream target genes TGM2, TMEPAI, THBS1 and TGFBI. UN-Untreated, 2.5H & 5H− 2.5 and 5 µg/ml Areca nut water extract, 2.5 Et,& 5Et- 2.5 and 5 µg/ml, T-TGF-β (_*** = P<0.0001 compared to untreated). C & D] ALK5 inhibitor (SB 431542) reverses the Areca nut induced expression of genes in HaCaT cells. HaCaT cells were treated with areca nut water (H) and ethanol (Et) extracts (5H− 5 µg/ml areca nut water extract and 5Et- 5 µg/ml areca nut ethanol extract) in the presence or absence of ALK5 (TβRI) inhibitor. Western blot of p-SMAD2 and TGM2 (C). qRT-PCR of TGM2,TMEPAI, THBS1 and TGFBI (D). Untreated, 5H− Areca nut water extract (5 µg/ml), 5Et- Areca nut Ethanol extract (5 µg/ml), ALK5- TβRI inhibitor, 5H+ALK5 inhibitor, 5Et+ALK5 inhibitor (_*** = P≤0.0001 compared to untreated).

Figure 3. Alkaloid and Polyphenol fractions of Areca nut induce TGF-β signaling in HaCaT cells.

Treatment of HaCaT cells with both the Alkaloid and Polyphenol fractions of areca nut water extract induced TGF-β signaling (p-SMAD2) and its down-stream target TGM2 as shown by the western blot (Figure 3A). Expression of TGF-β down-stream targets were also studied by Real Time PCR (Figure 3B) and semi quantitative PCR (Figure 3C). Induction of genes by alkaloid and polyphenol fractions of areca nut was compromised in presence of TβRI inhibitor (ALK5 inhibitor). (UN- untreated, WS- water supernatant, POL- Polyphenol supernatant, DCM- Dichloromethane fraction).

Figure 4. Both the pure alkaloids and polyphenols of areca nut induce TGF-β signaling.

Human keratinocytes (HaCaT) cells were serum deprived for 24 hours and treated with areca nut extracts, pure Alkaloids and Polyphenols for 48 hrs. A, shows pSMAD2 by western blot and B, expression of BMP7 by real time PCR; C & D, TGF-β2 expression by real time PCR; E, TGF-β2 protein estimation by ELISA; F, THBS1 expression by real time PCR; G & H, THBS1 western blots. The treatments are depicted in the respective figures. UN- untreated, Arecoline (400 µM), Arecaidine (1000 µM), Guvacine (1000 µM), Catechin (170 µM), Tannin (6 µM), TGF-β,, WS- water supernatant, POL- Polyphenol supernatant.

Figure 5. Areca nut potentiates TGF-β action on hGF cells.

Human gingival fibroblast (hGF) cells were serum deprived by replenishing with medium containing 0.2% serum for 24 h. Subsequently, hGF cells were treated for 72 hours with areca nut water extract (5H− 5 µg/ml) and or TGF-β (5 ng/ml). Real time PCR was used to examine the expression of genes regulated by TGF-β or areca nut extracts (A, B, C, D, E, F, G and H). I] Direct Red 80 stained hGF cells showing the presence of total collagens following treatment with Areca nut water extract and/or TGF-β for 5 days (Figure 5I, 10× magnification images, i-Untreated, ii-5H (5 µg/ml) Areca nut water extract, iii- TGF-β, iv- 5H+TGF-β). Figure 5J shows the quantitation of the Direct Red staining as measured at 550 nm.

Figure 6. Diagrammatic representation of proposed model of OSF pathogenesis by areca nut and its constituents.

Areca nut first comes in contact with epithelial cells where it's both the constituent, alkaloids and polyphenols acts on the epithelial cells and induces TGF-β signaling. This induced TGF-β signaling in the epithelial cells could be source of inflammation and can also diffuse into the connective tissue where it suppresses anti-fibrogenic cytokines like BMP7. In the connective tissue, areca nut acts on fibroblast cells along with TGF-β produced from the epithelium and potentiates its action in activating fibroblast cells responsible for inducing fibrosis.