Increased expression of transforming growth factor alpha precursors in acute experimental colitis in rats

Abstract

Background and aim: Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha), members of the EGF family of growth factors, protect rat gastric and colonic mucosa against injury. Having shown previously that exogenously applied EGF protects rat colonic mucosa against injury, the aim of the present study was to evaluate the endogenously expressed ligand mediating the protective effect of EGF/TGF-alpha in vivo.

Methods: In an experimental model of trinitrobenzene sulphonic acid (TNBS)/ethanol induced colitis in rats EGF and TGF-alpha expression was evaluated using a ribonuclease protection assay, northern blot analysis, western blot analysis, and immunohistochemistry.

Results: TGF-alpha mRNA increased 3-4 times at 4-8 hours after induction of colitis and returned to control levels within 24 hours. TGF-alpha immunoreactive protein with a molecular size of about 28 kDa representing TGF-alpha precursors increased markedly after induction of colitis with a peak at 8-12 hours. No fully processed 5.6 kDa TGF-alpha protein was detected in normal or inflamed colon tissue. Only a weak signal for EGF mRNA expression was detected in the rat colon and no EGF protein was observed by immunohistochemistry or western blot analysis.

Conclusions: TGF-alpha precursors are the main ligands for the EGF receptor in acute colitis. It is hypothesised that TGF-alpha precursors convey the biological activity of endogenous TGF-alpha peptides during mucosal defence and repair.

Figure 1

: Myeloperoxidase (MPO) activity in U/mg protein before and up to 24 hours after induction of colitis (n=6).

Figure 2

: Nuclease protection assays analysing expression of TGF-α mRNA before (control) and at 2, 4, 8, 12 and 24 hours after induction of colitis. 15 µg of total RNA per sample was loaded in each lane. In lanes 1 and 2 the synthesised TGF-α and β-actin probes were loaded which were slightly longer due to extension of the probe by parts of the copied polycloning sites of the host plasmids. Note the marked increase in TGF-α mRNA expression at 4 and 8 hours after induction of colitis and the increase in β-actin expression after induction of colitis.

Figure 3

: Comparison of β-actin and 18 rRNA expression before and at 2-12 hours after induction of colitis on northern blot analysis. A marked increase in β-actin mRNA is seen during colitis.

Figure 4

: Nuclease protection assay analysing expression of EGF mRNA in normal and inflamed colonic tissue. Weak protected bands were detected in both normal and inflamed tissue. Abundant EGF mRNA was present in kidney extracts.

Figure 5

: TGF-α-like immunoreactive staining in colonic tissue before and at different time points after mucosal injury. (A) In uninflamed colon TGF-α-like immunoreactivity was predominantly found in the upper half of the crypts and in the luminal surface epithelium. (B) Within the first two hours after mucosal injury the upper half of the mucosa became necrotic and TGF-α-like immunoreactive staining disappeared. (C) While some of the damaged areas remained necrotic and presumably will turn into ulcers, TGF-α-like immunoreactivity reappeared in remaining epithelial cells at the bottom of the crypts and in epithelial cells of areas adjacent to necrotic areas. Between 8 and 12 hours TGF-α-like immunoreactivity was detected in epithelial cells along the entire crypts (C) and in migrating epithelial cells (D). (E) Twenty four hours after induction of colitis TGF-α-like immunoreactivity was again more pronounced in the upper half of the crypts. (F, G) The staining with both antibodies was completely abolished after overnight preabsorption of either antibody with excess rat TGF-α.

Figure 6

: TGF-α protein expression on western blot analysis before and after induction of colitis. The same polyclonal antibody was used as for immunohistochemical analysis. No immunoreactive band was found in the molecular size of mature processed 5.6 kDa TGF-α. Immunoreactive bands were found with a higher molecular weight of about 28 kDa. Additional weak non-specific bands were seen which also appeared after incubation of corresponding blots with different non-immune rabbit sera.

Figure 7

: Comparison of TGF-α protein (n = 6) and mRNA (n = 3) expression during the first 24 hours of colitis by western blot analysis and nuclease protection assays, respectively.