engleski

Regulation of novel TFFs interacting protein (blottin) gene expression by cytokines

Proper function of gastrointestinal (GI) tract relies on its surface integrity maintained by various factors. Trefoil factor family (TFF) proteins (TFF1, 2 & 3) play multifunctional role in protecting GI mucosa. Although many biological effects of trefoil peptides have already been elucidated the mechanism by which these small peptides exert their biological activity is still largely unknown. However, different tasks within and outside the cell seem to require additional interacting partners for TFFs. Recently, novel TFF1 and TFF2 binding proteins were discovered: TFIZ1 [trefoil factor interactions(z) 1] and its mouse homolog blottin . TFIZ1/blottin protein contains a BRICHOS domain that has been identified in several previously unrelated proteins. These are linked to major diseases as familial dementia, chondrosarcoma, stomach cancer and respiratory distress syndrome, pointing to the importance of this BRICHOS domain. The aim of our study was to see how the blottin gene is regulated by different cytokines. Blottin gene promoter (position – 1906 to -20) was cloned into luciferase reporter gene plasmid (pGL3 basic vector) and introduced into gastrointestinal cell lines (HT-29, KATO III). The expression of reporter gene controlled by blottin promoter was monitored (Dual Luciferase Assay system) upon treatment with various proinflammatory (TNF-α , IL1-β , IL-6, INF-γ ) and anti-inflammatory (TGF-β ) cytokines. To assess direct role of transcription factors in regulating blottin we performed transient co-expression of their expression plasmids (NF-κ B, p65, HNF3-β , hGATA6) and the reporter gene vector. Statistical analysis was performed by using one way ANOVA. Statistically significant down-regulation of blottin transcription was observed by TNF-α , IL1-β and IL-6 (Fig 1a, b). Transcription factor NF-κ B also inhibits transcription of blottin gene (Fig 2)while TGF-β and transcription factors hGATA6 and HNF3-β caused an up-regulation of the gene (Fig 1a and 3). Blottin gene expression is regulated by various cytokines suggesting the crosstalk of blottin as an TFF2 interacting protein with immune response.

GI cell lines; GDDR; cytokines; TFF peptides

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