A. Y. Dossa1, A. Roberts1, H. R. Ford1, M. R. Frey1, C. P. Gayer1 1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA
Introduction: Intestinal bacteria can convert taurine-conjugated cholic acid (TCA), a primary bile acid, into the secondary bile acid deoxycholic acid (DCA). Previous work suggests that TCA stimulates cell proliferation while DCA inhibits it. Bile acids can interact with the epidermal growth factor receptor (EGFR) and the farnesoid-X receptor (FXR), which may explain the differential effects of bile acids on cell proliferation. EGFR may promote proliferation through a mechanism involving Src and downstream modulation of the extracellular signal-regulated kinase (ERK), while FXR may inhibit proliferation. Therefore, we hypothesize that TCA and DCA differentially regulate intestinal epithelial cell proliferation by activation of EGFR and FXR, respectively.
Methods: Cell proliferation was measured using nucleic acid incorporation (EdU). Rat intestinal epithelial cells (IEC-6) were treated with TCA with or without the Src kinase inhibitor PP2, the EGFR inhibitor AG1478, the ERK inhibitor PD98059, the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 or the Akt inhibitor IV. The effect of EGFR inactivation was assessed in EGFR-null mouse colon epithelial cells (EGFR-/- MCE) transfected with an empty vector, wild-type human EGFR or kinase inactive EGFR. FXR was either blocked pharmacologically (z-guggulsterone) or decreased with FXR-specific siRNA knockdown prior to treatment with DCA. Src, EGFR and ERK 1/2 phosphorylation was assessed by immunoblotting.
Results: TCA induced intestinal epithelial cell proliferation in a dose-dependent manner, while DCA led to a decrease in proliferation. TCA-induced stimulation was blocked with Src, EGFR or ERK inhibition. Interestingly, blockade of PI3K or Akt had no effect on TCA-induced cell proliferation. TCA treatment of EGFR-/- MCE cells expressing wild-type human EGFR resulted in increased proliferation (48.6% ± 14.7 above baseline). However, EGFR-/- MCE cells expressing an empty vector or kinase inactive EGFR did not respond to TCA administration. TCA treatment resulted in Src, EGFR and ERK 1/2 phosphorylation in a time-dependent fashion. Pre-treatment with EGFR inhibitor abolished TCA-induced phosphorylation of ERK. In DCA-treated cells, pharmacologic inactivation and protein knockdown by siRNA specific to FXR abolished DCA-induced inhibition of proliferation.
Conclusions: These results suggest that TCA-induced intestinal cell proliferation requires EGFR, Src and ERK activation, independent of the PI3K/Akt pathway. This pathway may involve first Src activation with subsequent transactivation of EGFR. In contrast, DCA inhibits proliferation, possibly via FXR activation and inactivation of the Src/EGFR/ERK pathway. Since elevated secondary bile acids levels are the results of specific bacterial modification, this may provide a mechanism by which an altered microbiome contributes to intestinal diseases.