Regulation of extracellular matrix genes by arecoline in primary gingival fibroblasts requires epithelial factors.
OBJECTIVE
Oral submucous fibrosis, a disease of collagen disorder, has been attributed to arecoline present in the saliva of betel quid chewers. However, the molecular basis of the action of arecoline in the pathogenesis of oral submucous fibrosis is poorly understood. The basic aim of our study was to elucidate the mechanism underlying the action of arecoline on the expression of genes in oral fibroblasts.


METHODS
Human keratinocytes (HaCaT cells) and primary human gingival fibroblasts were treated with arecoline in combination with various pathway inhibitors, and the expression of transforming growth factor-beta isoform genes and of collagen isoforms was assessed using reverse transcription-polymerase chain reaction analysis.


RESULTS
We observed the induction of transforming growth factor-beta2 by arecoline in HaCaT cells and this induction was found to be caused by activation of the M-3 muscarinic acid receptor via the induction of calcium and the protein kinase C pathway. Most importantly, we showed that transforming growth factor-beta2 was significantly overexpressed in oral submucous fibrosis tissues (p = 0.008), with a median of 2.13 (n = 21) compared with 0.75 (n = 18) in normal buccal mucosal tissues. Furthermore, arecoline down-regulated the expression of collagens 1A1 and 3A1 in human primary gingival fibroblasts; however these collagens were induced by arecoline in the presence of spent medium of cultured human keratinocytes. Treatment with a transforming growth factor-beta blocker, transforming growth factor-beta1 latency-associated peptide, reversed this up-regulation of collagen, suggesting a role for profibrotic cytokines, such as transforming growth factor-beta, in the induction of collagens.


CONCLUSIONS
Taken together, our data highlight the importance of arecolineinduced epithelial changes in the pathogenesis of oral submucous fibrosis.
Thangjam
G S
GS
Department of Molecular Reproduction Development and Genetics, Indian Institute of Science, Bangalore, India.
Agarwal
P
P
Balapure
A K
AK
Rao
S Girish
SG
Kondaiah
P
P
D001115:Arecoline / Q000494:pharmacology*; D002460:Cell Line; D002614:Chelating Agents / Q000494:pharmacology; D018679:Cholinergic Agonists / Q000494:pharmacology*; D003094:Collagen / Q000187:drug effects / Q000235:genetics; D024042:Collagen Type I / Q000187:drug effects / Q000235:genetics; D000090602:Collagen Type I, alpha 1 Chain; D024061:Collagen Type III / Q000187:drug effects / Q000235:genetics; D024141:Collagen Type IV / Q000187:drug effects / Q000235:genetics; D024142:Collagen Type VI / Q000187:drug effects / Q000235:genetics; D024143:Collagen Type VII / Q000187:drug effects / Q000235:genetics; D015536:Down-Regulation; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005109:Extracellular Matrix / Q000187:drug effects* / Q000235:genetics; D005347:Fibroblasts / Q000187:drug effects*; D005881:Gingiva / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects*; D009061:Mouth Mucosa / Q000473:pathology; D009914:Oral Submucous Fibrosis / Q000235:genetics / Q000473:pathology; D020033:Protein Isoforms / Q000187:drug effects / Q000235:genetics; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000235:genetics; D043587:Receptor, Muscarinic M3 / Q000187:drug effects / Q000235:genetics; D019311:Staurosporine / Q000494:pharmacology; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology; D053781:Transforming Growth Factor beta2 / Q000187:drug effects / Q000235:genetics
C439971:COL3A1 protein, human; D002614:Chelating Agents; D018679:Cholinergic Agonists; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D024061:Collagen Type III; D024141:Collagen Type IV; D024142:Collagen Type VI; D024143:Collagen Type VII; D020033:Protein Isoforms; D043587:Receptor, Muscarinic M3; D053773:Transforming Growth Factor beta1; D053781:Transforming Growth Factor beta2; D001115:Arecoline; D004533:Egtazic Acid; D003094:Collagen; D011493:Protein Kinase C; D019311:Staurosporine; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid