Up-regulation of intestinal epithelial cell derived IL-7 expression by keratinocyte growth factor through STAT1/IRF-1, IRF-2 pathway

Abstract

Background: Epithelial cells(EC)-derived interleukin-7 (IL-7) plays a crucial role in control of development and homeostasis of neighboring intraepithelial lymphocytes (IEL), and keratinocyte growth factor (KGF) exerts protective effects on intestinal epithelial cells and up-regulates EC-derived IL-7 expression through KGFR pathway. This study was to further investigate the molecular mechanism involved in the regulation of IL-7 expression by KGF in the intestine.

Methods: Intestinal epithelial cells (LoVo cells) and adult C57BL/6J mice were treated with KGF. Epithelial cell proliferation was studied by flow cytometry for BrdU-incorporation and by immunohistochemistry for PCNA staining. Western blot was used to detect the changes of expression of P-Tyr-STAT1, STAT1, and IL-7 by inhibiting STAT1. Alterations of nuclear extracts and total proteins of IRF-1, IRF-2 and IL-7 following IRF-1 and IRF-2 RNA interference with KGF treatment were also measured with western blot. Moreover, IL-7 mRNA expressions were also detected by Real-time PCR and IL-7 protein level in culture supernatants was measured by enzyme linked immunosorbent assay(ELISA).

Results: KGF administration significantly increased LoVo cell proliferation and also increased intestinal wet weight, villus height, crypt depth and crypt cell proliferation in mice. KGF treatment led to increased levels of P-Tyr-STAT1, RAPA and AG490 both blocked P-Tyr-STAT1 and IL-7 expression in LoVo cells. IRF-1 and IRF-2 expression in vivo and in vitro were also up-regulated by KGF, and IL-7 expression was decreased after IRF-1 and IRF-2 expression was silenced by interfering RNA, respectively.

Conclusion: KGF could up-regulate IL-7 expression through the STAT1/IRF-1, IRF-2 signaling pathway, which is a new insight in potential effects of KGF on the intestinal mucosal immune system.

Figure 1. Detection of cell viability by flow cytometric analysis of BrdU-incorporation.

Incorporation of BrdU was measured with a fluorescein isothiocyanate (FITC, green)-conjugated anti-BrdU antibody and propidium iodid (PI, red), shows a typical example of flow cytometric analysis of LoVo cells proliferation after KGF treatment, where a BrdU+ population is clearly visible. Control (A), KGF treated groups with different concentrations including 80 ng/ml (B) and 150 ng/ml (C).

Figure 2. Alterations in villus height and crypt depth in mice after KGF treatment.

* P<0.05 vs. control group, n = 6 per group.

Figure 3. Alterations in PCNA expression in small intestine of KGF treated mice by immunohistochemistry.

PCNA expression was significantly increased in KGF group (A), as compared to the control group (B). PCNA expression is expressed as means ± SD (C), *P<0.05 vs control group. Original magnification: ×400; n = 6 per group. Scale bar = 25 µm

Figure 4. Changes of P-Tyr-STAT1 and STAT1 expression after KGF treatment in LoVo cells.

Increased expression of P-Tyr-STAT1, but not STAT1, were confirmed by western blot in LoVo cells with KGF (150 ng/ml) treatment. Tubulin was used as internal control.

Figure 5. Changes of P-Tyr-STAT1, STAT1 and IL-7 expression after STAT1 blockade following KGF treatment, by western blot in LoVo cells (A).

Tubulin was used as internal control. Suppressions of P-Tyr-STAT1 and IL-7 expression, but not STAT1, were observed with STAT1 inhibitors including AG490 (50 µmol/l) and RPM (50 ng/ml) following KGF (150 ng/ml) treatment. Changes of IL-7 mRNA expression after STAT1 blockade following KGF treatment were detected by quantitative real-time PCR (B), * indicates significant difference between RPM (or AG490) group and control, ** indicates significant difference between RPM (or AG490)+KGF group and control+KGF group, P<0.05.

Figure 6. KGF administration resulted in an increased IRF-1 and IRF-2 expression of the nuclear extracts and total proteins in vitro.

Dose-dependent increased expression both of IRF-1 (A) and IRF-2 (B) were confirmed by western blot in LoVo cells with KGF treatment. Tubulin and H1 were used as internal control.

Figure 7. Increased expression of IRF-1 and IRF-2 were confirmed by immunofluorenscence staining with KGF treatment in vitro.

Increased expression of IRF-1 and IRF-2 in the nucleus were observed after 6 h with KGF treatment.

Figure 8. Alterations in IRF-1 and IRF-2 expression in small intestine of KGF treated mice by immunohistochemistry.

IRF-1 expression in control group (A) and in KGF group (B), IRF-2 expression in control group (C) and in KGF group (D). Original magnification: ×400; n = 6 per group. Scale bar = 25 µm.

Figure 9. IL-7 is up-regulated by KGF through IRF-1/IRF-2 pathway.

Tubulin and H1 were used as internal control. (A) Reduced the nuclear extracts and total proteins of IRF-1 was confirmed by western blot in LoVo cells following IRF-1 RNA interference. Plasmids 663, 664 and 665 were transfected into LoVo cells and IRF-1 expression was detected. Plasmid 665 can definitely inhibit IRF-1 expression. (B) Reduced the nuclear extracts and total proteins of IRF-2 was confirmed by western blot in LoVo cells following IRF-2 RNA interference. Plasmids 691, 692 and 693 were transfected into LoVo cells and IRF-2 expression was detected. Plasmid 693 can definitely inhibit IRF-2 expression. (C) Reduced expression of IL-7 was confirmed by western blot and quantitative real-time PCR in LoVo cells following IRF-1 RNA interference. Decreased expression of IL-7 was observed in LoVo cells following KGF treatment in response to RNA interference of IRF-1 by plasmid 665 in both mRNA and protein levels. *P<0.05 vs. control group. (D) Reduced expression of IL-7 was confirmed by western blot and quantitative real-time PCR in LoVo cells following IRF-2 RNA interference. Decreased expression of IL-7 was observed in LoVo cells following KGF treatment in response to RNA interference of IRF-2 by plasmid 693 in both mRNA and protein levels. *P<0.05 vs. control group.