Transactivation of EGFR by LPS induces COX-2 expression in enterocytes

Necrotizing enterocolitis (NEC) is the leading cause of gastrointestinal morbidity and mortality in preterm infants. NEC is characterized by an exaggerated inflammatory response to bacterial flora leading to bowel necrosis. Bacterial lipopolysaccharide (LPS) mediates inflammation through TLR4 activation and is a key molecule in the pathogenesis of NEC. However, LPS also induces cyclooxygenase-2 (COX-2), which promotes intestinal barrier restitution through stimulation of intestinal cell survival, proliferation, and migration. Epidermal growth factor receptor (EGFR) activation prevents experimental NEC and may play a critical role in LPS-stimulated COX-2 production. We hypothesized that EGFR is required for LPS induction of COX-2 expression. Our data show that inhibiting EGFR kinase activity blocks LPS-induced COX-2 expression in small intestinal epithelial cells. LPS induction of COX-2 requires Src-family kinase signaling while LPS transactivation of EGFR requires matrix metalloprotease (MMP) activity. EGFR tyrosine kinase inhibitors block LPS stimulation of mitogen-activated protein kinase ERK, suggesting an important role of the MAPK/ERK pathway in EGFR-mediated COX-2 expression. LPS stimulates proliferation of IEC-6 cells, but this stimulation is inhibited with either the EGFR kinase inhibitor AG1478, or the selective COX-2 inhibitor Celecoxib. Taken together, these data show that EGFR plays an important role in LPS-induction of COX-2 expression in enterocytes, which may be one mechanism for EGF in inhibition of NEC

LPS induces COX-2 in IEC-6 cells.

<p>A) IEC-6 cells were treated with the indicated concentrations of LPS for 24 hours. B) IEC-6 cells were treated with 2 µg/mL LPS for the indicated time. C) IEC-6 cells were treated with LPS (2 µg/mL) for the indicated times. D) Cells were treated with LPS (2 µg/mL), EGF (10 ng/mL), or co-treated with LPS and EGF for 24 hours. Protein expression was determined by Western blot analysis and densitometry. Treatment with LPS did not induce COX-1 expression in IEC-6 cells. In contrast, treatments with LPS, EGF, or both significantly increased COX-2 expression compared to control (p<0.001,  = 0.05, and <0.001 respectively). Cells treated with LPS and EGF had significantly greater COX-2 expression than by either EGF (p = 0.002) or LPS (p = 0.006) alone. Single asterisks indicate significant differences from control. Double asterisks indicate significant differences between two bracketed conditions.</p

ERK and Src, but not p38, are required for EGFR-mediated induction of COX-2.

<p>A) IEC-6 cells were treated with LPS (2 µg/mL) for 15 minutes or with EGF (10 ng/mL) for 5 minutes in the presence or absence of the EGFR kinase inhibitor AG1478 (1 µM). Western blot analysis of P-p38 MAPK showed no significant difference in p38 activation in the presence of EGFR inhibition. B) IEC-6 cells stimulated with LPS (2 µg/mL) for 15 minutes or with EGF (10 ng/mL) for 5 minutes in the presence or absence of AG1478 (1 µM). C) IEC-6 cells were treated with LPS (2 µg/mL) for 24 hours or with EGF (10 ng/mL) for 5 minutes in the presence or absence of the ERK1/2 inhibitor U0126 (10 µM) or the p38 inhibitor SB202190 (10 µM) as shown, and COX-2 expression was determined using Western blot analysis. D) IEC-6 cells were treated with EGF (10 ng/mL) for 5 minutes in the presence or absence of the Src family kinase inhibitor CGP77675 (2 µM) and analyzed for P-ERK activation using Western blot analysis. Src inhibition had no effect on EGF-induced P-ERK (p = 0.7). IEC-6 cells were also treated with EGF (10 ng/mL) for 5 minutes in the presence or absence of the ERK kinase inhibitor U1026 (10 µM) and analyzed for P-Src activation using Western blot analysis. ERK inhibition had no effect on EGF-induced P-Src activation (p = 0.17). Single asterisks indicate significant differences from control. Double asterisks indicate significant differences between two bracketed conditions.</p

Stimulation of IEC-6 cell proliferation by LPS requires COX-2 activity.

<p>IEC-6 cells were treated with LPS (2 µg/mL) for 48 hours in the presence or absence of A) Celecoxib (10 µM) or B) AG1478 (1 µM). Cell numbers were determined by a Nucleocassette counter. Celecoxib and AG1478 treatment significantly blocked LPS-induced proliferation (p = 0.03 and 0.001 respectively).</p