Possible role of REG Ialpha protein in ulcerative colitis and colitic cancer

Abstract

Background and aims: Although regenerating gene (REG) Ialpha protein may be involved in the inflammation and carcinogenesis in the gastrointestinal tract, its pathophysiological role in ulcerative colitis (UC) and the resulting colitic cancer remains unclear. We investigated expression of the REG Ialpha gene and its protein in UC and colitic cancer tissues. We examined whether cytokines are responsible for REG Ialpha gene expression and whether REG Ialpha protein has a trophic and/or an antiapoptotic effect on colon cancer cells.

Methods: Expression of REG Ialpha mRNA and its gene product in UC tissues was analysed by real time reverse transcription-polymerase chain reaction and immunohistochemistry, respectively. The effects of cytokines on REG Ialpha promoter activity were examined in LoVo cells by luciferase reporter assay. The effects of REG Ialpha protein on growth and H(2)O(2) induced apoptosis were examined in LoVo cells by MTT and TUNEL assays, respectively.

Results: REG Ialpha protein was strongly expressed in inflamed epithelium and in dysplasias and cancerous lesions in UC tissues. The level of REG Ialpha mRNA expression in UC tissues correlated significantly with severity of inflammation and disease duration. REG Ialpha promoter activity was enhanced by stimulation with interferon gamma or interleukin 6. REG Ialpha protein promoted cell growth and conferred resistance to H(2)O(2) induced apoptosis in LoVo cells. REG Ialpha protein promoted Akt phosphorylation and enhanced Bcl-xL and Bcl-2 expression in LoVo cells.

Conclusions: The REG Ialpha gene is inducible by cytokines and its gene product may function as a mitogenic and/or an antiapoptotic factor in the UC-colitic cancer sequence.

Figure 1

REG Iα mRNA expression in ulcerative colitis tissues. (A) REG Iα mRNA expression levels in normal colon (NC), ulcerative colitis (UC), Crohn’s colitis, and proctitis tissues. Comparison of REG Iα mRNA expression levels among groups subdivided by endoscopic appearance (B), histological findings (C), or disease duration (D, E). (F) Correlation between REG Iα expression levels and disease duration. All results are expressed as fold change in REG Iα mRNA/GAPDH mRNA ratio relative to the normal control group. Significant difference between two groups: *p<0.05, **p<0.01. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 2

Immunostaining for REG Iα protein and proliferating cell nuclear antigen (PCNA) in normal colon (A, B), ulcerative colitis (C, D), dysplasia (E, F), and colitic cancer (G, H) tissues. REG Iα: A, C, E, G; PCNA: B, D, F, H. Bars = 100 μm.

Figure 3

Detection of REG Iα mRNA in various human colon cancer cell lines by reverse transcription-polymerase chain reaction. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 4

REG Iα promoter activity in LoVo and SW403 cells in response to various cytokines. Cells were cotransfected with hREG Iα(−1195/+78)-Luc construct and Renilla luciferase plasmid pRL-TK (as a control for transfection efficiency). Forty eight hours later, cells were stimulated with interferon γ (IFN-γ), tumour necrosis factor α (TNF-α), interleukin (IL)-1β, IL-5, IL-6, IL-8, or IL-13. Luciferase activity was measured in extracts from transfected LoVo and SW403 cells and normalised to Renilla luciferase activity. All results are expressed as fold of the activity of the untreated cell group at the 0 hour time point. Vertical lines represent means (SEM) of four independent experiments. *p<0.05 versus control at the same time point.

Figure 5

Effects of REG Iα gene induction or REG Iα conditioned medium on the growth of human colon cancer cells. (A) Effect of REG Iα gene induction on growth of human colon cancer cells. LoVo cells transfected with pIRES2-hREG Iα (LoVo-REG Iα) or pIRES2-EGFP (LoVo-EGFP; control) plasmids were cultured for 24, 48, and 72 hours. WST-8 cleavage in each group was measured by ELISA (absorbance OD450), as described in materials and methods. (B) Effect of anti-REG Iα antibody on cell growth of LoVo-REG Iα and LoVo-EGFP. LoVo-REG Iα and LoVo-EGFP cells were incubated with or without anti-REG Iα antibody (Ab, 50 μg/ml) for 72 hours. (C) Source of REG Iα protein. Human embryonic kidney HEK293T cells were transfected with a human REG Iα cDNA expression plasmid or a control plasmid, and the medium conditioned by these cells was collected. Release of REG Iα protein into the conditioned medium of human REG Iα cDNA transfected cells was confirmed by western blot analysis with an antihuman REG Iα monoclonal antibody. (D) Time course of the effect of REG Iα conditioned medium on growth of human colon cancer cells. LoVo cells were incubated with REG Iα or control medium for 24–72 hours. (E) Effect of anti-REG Iα antibody on growth of colon cancer cells promoted by REG Iα protein. Anti-REG Iα antibody (50 μg/ml) was added to control or REG Iα medium, followed by incubation for 72 hours. All results are expressed as the mean (SEM) of four samples. *p<0.05, **p<0.01 versus control (LoVo-EGFP cells (A, B) or control medium treated cells (D, E)) group at the same time point. ††Significantly lower than LoVo-REG Iα cells (B) or REG Iα medium treated cells (E) (p<0.01).

Figure 6

Effects of REG Iα gene induction or REG Iα conditioned medium on H₂O₂ induced apoptosis of human colon cancer cells. (A) Resistance to H₂O₂ induced apoptosis in human colon cancer cells transfected with REG Iα cDNA. LoVo cells transfected with pIRES2-hREG Iα (LoVo-REG Iα) or pIRES2-EGFP (LoVo-EGFP; control) plasmids were treated with H₂O₂ (0–10 mM) for two hours, followed by incubation in fresh culture medium for 24 hours. The percentage of TUNEL positive cells was evaluated, as described in materials and methods. (B) Effect of anti-REG Iα antibody (Ab, 50 μg/ml) on H₂O₂ (5 mM) induced apoptosis in LoVo-REG Iα and LoVo-EGFP cells. (C) Resistance to H₂O₂ induced apoptosis in human colon cancer cells treated with REG Iα conditioned medium. LoVo cells were incubated with conditioned medium containing recombinant human REG Iα or control medium for 24 hours, followed by incubation for two hours with medium containing H₂O₂ (0–10 mM). (D) Effect of anti-REG Iα antibody on H₂O₂ (5 mM) induced apoptosis in LoVo cells. Anti-REG Iα antibody (50 μg/ml) was added to control or REG Iα medium. All results are expressed as the mean (SEM) of four samples. *p<0.05, ** p<0.01 versus control (LoVo-EGFP cells (A, B)) or control medium treated cells (C, D)) at the same dose point. ††Significantly higher than LoVo-REG Iα cells (B) or REG Iα medium treated cells (D) (p<0.01).

Figure 7

Effects of REG Iα conditioned medium on phosphorylation of Akt and expression of Bcl family proteins in colon cancer cells. Western blotting indicated that phosphorylation of Akt (A) and expression of Bcl-2 and Bcl-xL but not Mcl-1 (B) were enhanced in LoVo cells after 12 hours of incubation with REG Iα conditioned medium.