Integrative roles of transforming growth factor-alpha in the cytoprotection mechanisms of gastric mucosal injury

Abstract

Background: Transforming growth factor alpha (TGFalpha) protects against gastric mucosal injury and facilitates wound healing. However, its overexpression is known to induce hypertrophic gastropathy resembling Menetrier's disease in transgenic (TG) mice on an FVB background, as one of the authors reported previously. We studied another TGFalpha-expressing mouse line on a CD1 background, whose gastric mucosa appears normal. Since this TG mouse had a strong resistance to ethanol-induced gastric injury, we considered the long-term effect of TGFalpha on several gastric protection mechanisms.

Methods: TGFalpha-expressing transgenic (TG) mouse lines bearing human TGFalpha cDNA under the control of the mouse metallothionein gene I promoter were generated on a CD1 mouse background, and analyzed their ethanol injury-resistant phenotypes produced by TGFalpha.

Results: In the TG mucosa, blood flow was well maintained after ethanol injury. Further, neural and inducible types of NO synthases were consistently and widely expressed in the TG mucosa, compared with the limited distribution of neural type NO synthase in the luminal pit region of the wild-type (WT) mucosa. COX-2 and its upstream transcription factor NfkB were constitutively elevated in the TG mucosa even before ethanol administration, whereas they were induced in the same region of the WT mucosa only after ethanol injury. Two anti-apoptotic proteins, HSP70 and Bcl-2, were upregulated in the TG mucosa even before ethanol administration, while they were not expressed in the WT mucosa before the injury. Furthermore, pro-caspase 3 activation was inhibited in the TG mucosa, while it was converted to the active form in the WT mucosa following ethanol administration.

Conclusion: We conclude that TGFalpha maintains the gastric mucosal defense against gastric injury by integrating other cytoprotective mechanisms.

Figure 1

Characterization of the TGFα-expressing TG mucosa. Scale in each figure indicates 100 μm. (a) Distribution of TGFα-expressing cells. Brown-colored TGFα-positive cells were visible along the luminal pit region in the WT mucosa, the elongated foveolar pit region, and the lower one-third of the glandular region in the TG mucosa. (b) Overlap of red-colored TGFα-positive cells and green-colored H⁺/K⁺-ATPase-positive parietal cells. Lower TGFα-positive cells in cluster are distinct from the green-colored parietal cells in the TG mucosa. (c) Northern blot of human TGFα mRNA and PCR-amplified mouse TGFα mRNA. Human TGFα mRNA is expressed in the TG mucosa alone (left panel), but PCR-amplified mouse TGFα mRNA is expressed similarly in both WT and TG mucosae (right panel). (d) Western blot of TGFα intermediate forms. TGFα precursor (20 kDa) and its intermediate forms are visualized more intensively in the TG mucosa than in the WT mucosa. (e) Localization of EGF receptor in the gastric mucosa. EGF receptor (EGF-R) was stained with Cy3 (red) and H⁺/K⁺-ATPase was stained with FITC (green). EGF-R was immunostained moderately in the upper pit region and strongly in the lower glandular region similarly in both the WT and TG mucosae. (f) Distribution of metallothionein in the gastric mucosa. Metallothionein is immunostained strongly in the lower glandular region, and moderately along the foveolar region. (g) Elongation of the gastric pit of the TG mucosa. PCNA-positive cells are distributed at the upper-third region in the WT gastric glands, whereas they are distributed more numerously and broadly in the middle glandular region. (h) PAS staining shows elongated pit in the TG mucosa.

Figure 2

Ethanol-induced gastric injury in the gastric mucosae. (a) Macroscopic view of the gastric mucosa. Scale: 5 mm. Both mucosae are 6 h after ethanol administration. Note that the WT mucosa displays extensive hemorrhagic lesions, in contrast, the TG mucosa looks undamaged after ethanol administration. (b) Hematoxylin and eosin staining of ethanol-injured WT mucosa (left) and TG mucosa (right). Note the deep ulcer formation in the WT mucosa. Scale: 100 μm. (c) Time course of ethanol-induced gastric injury. Injured area is plotted against time up to 12 h after the ethanol administration. Each point represents an average of five mice. p < 0.05 (d) TUNEL staining of WT mucosa (left) and TG mucosa (right). Both sections are consecutive to those in 2B, respectively. Apoptotic cells are visible with dark-brown nuclei. Numerous apoptotic cells are noted in the WT mucosa. All photos were 6 h after the ethanol administration. Scale: 100 μm.

Figure 3

Assessment of gastric mucosal blood flow in the gastric mucosae. Gastric mucosal blood flow was assessed by comparing the blood flow ratio before and after the ethanol treatment. The ratio was obtained as a percent against the blood flow before the treatment, which was much larger in the WT mucosa than in the TG mucosa. *p < 0.03.

Figure 4

Immunostaining of NO synthases in the gastric mucosa. Scale: 100 μm. (a) nNOS immunostaining. nNOS was visible along the pit region, resulting in its wider distribution along the elongated pit in the TG mucosa. (b) iNOS immunostaining. iNOS was induced in the lower glandular region after ethanol injury in the WT mucosa, whereas it was constitutively expressed in the same region of the TG mucosa irrespective of ethanol injury.

Figure 5

COX-2 and NFκB expression in the gastric mucosa. (a) Immunostaining of COX-2. COX-2 was intensively induced after the injury at the lower glandular region of the WT mucosa, whereas it was constitutively expressed in the same region of the TG mucosa even before the injury. Ethanol-injured mucosa was stained 6 h after the ethanol administration. Scale: 100 μm. (b) Immunoblotting of COX-2 and COX-1. The blots were measured 0, 3, and 6 h after the injury. Actin filaments were used as a control. Note that COX-2 was intensively expressed before the injury in the TG mucosa. (c) Measurement of prostaglandin E₂. Prostaglandin E₂ levels were measured before the ethanol treatment in the WT and TG mucosae. n = 5, p = 0.013. (d) Immunostaining of NFκB. NFκB was intensively induced after the injury at the same lower glandular region of the WT mucosa as was COX-2. In contrast, NFκB was constitutively expressed in the same region of the TG mucosa even before the injury. Scale: 100 μm.

Figure 6

HSP70 expression in the gastric mucosa. (a) Immunostaining of HSP70. HSP70 was induced along the necrotic region and the lower glandular region of the WT mucosa after ethanol injury, whereas it was constitutively expressed in the lower glandular region of the TG mucosa before and after the injury. Ethanol-injured mucosa was stained 6 h after the ethanol administration. Scale: 100 μm. (b) Immunoblotting of HSP70. HSP70 blot was assessed 0, 3, 6, 12 h after the ethanol injury. HSP was induced after ethanol injury in the WT mucosa, while it was constitutively expressed in the TG mucosa before and after the injury.

Figure 7

Expression of apoptosis-associated proteins. (a) Immunoblot of Bax and Bcl-2. Bax was strongly induced at the 3 h and 6 h time points in the WT mucosa, whereas it was reduced to a negligible level in the TG mucosa after ethanol injury. Bcl-2 was stably expressed similarly before and after the injury in the TG mucosa, whereas it was increased to some extent at 6 h after the injury in the WT mucosa. (b) Immunostaining of Bcl-2. Bcl-2 was not visible in the TG mucosa before the injury, but appeared along the border region of necrotic area after the injury. In contrast, Bcl-2 was constitutively induced over the TG mucosa even before the injury, and the staining was confined to the lower glandular region after the injury. Scale: 100 μm. (c) Immunoblot of caspase 3. After the injury, a 17 kDa active form of caspase 3 appeared in the WT mucosa, whereas it did not in the TG mucosa. A 32 kDa proform was unchanged both in the WT and TG mucosae.