Proceedings of The Physiological Society

Physiology 2016 (Dublin, Ireland) (2016) Proc Physiol Soc 37, PCA119

Poster Communications

The ErbB3 receptor tyrosine kinase restricts intestinal Paneth cell numbers

D. Almohazey4,5, C. V. Vossler1, J. J. Hsieh1, K. S. Lau3, M. R. Frey1,2

1. Pediatrics, Children's Hospital Los Angeles, Los Angeles, California, United States. 2. Biochemistry and Molecular Biology, University of Southern California Keck School of Medicine, Los Angeles, California, United States. 3. Vanderbilt University Medical Center, Nashville, Tennessee, United States. 4. University of Southern California Ostrow School of Dentistry, Los Angeles, California, United States. 5. University of Dammam, Dammam, Saudi Arabia.

Paneth cells (PCs), a secretory population located at the base of the intestinal crypt, support the intestinal stem cells (ISC) with growth factors and participate in innate immunity by releasing antimicrobial peptides (AMPs), including lysozyme. Loss of or functional defects in PCs are seen in disorders such as inflammatory bowel disease and necrotizing enterocolitis. We recently showed that activation of the neuregulin receptor ErbB4 protects PCs against injury. However, the role of ErbB3, the other neuregulin receptor, in PCs is unknown. In this study we tested the effects of ErbB3 signaling on PC numbers, AMP production, and the ISC niche. METHODS: Ileal tissues from ErbB3flox/flox (functionally wild type) and ErbB3flox/flox; Villin-Cre (E3KOIE; intestinal epithelium specific deletion) mice were characterized by immunofluorescence, qPCR, and disaggregation/cytometry time-of-flight (CyTOF) analyses. In vitro, ErbB3 was activated with NRG1β (10ng/ml, 24 h exposure) or downstream signaling was inhibited (PI 3-kinase/Akt with LY294002; MEK/ERK with U0126) in matrigel-embedded ileal enteroids and human HT-29 colon epithelial cells, and RNA was extracted and analyzed by qPCR. RESULTS: Adult E3KOIE intestinal crypts had more lysozyme+ Paneth cells per crypt (9.9 vs. 5.6% of epithelial cells, p=0.008), as well as increased Lyz1 (by 67.53%, p<0.0001) and ISC marker Lgr5 (by 46.65%, p=0.012) RNA expression. This appeared to be developmental, as lysozyme+ cells were readily detectable in crypts of 7 day-old E3KOIE but not ErbB3flox/flox animals. Conversely, activation of ErbB3 with NRG1β resulted in reduced expression of Lyz1 in WT enteroids (by 29.47%, p=0.03) or HT-29 cells (by 31.0%, p=0.02). We did not observe differences in expression of other ErbB family members (Egfr, ErbB2, and ErbB4) or other secretory cell markers (Muc2 and Chga). Interestingly, E3KOIE mice had a significant increase in expression of the secretory regulator Atoh1 (by 59.20%, p=0.0009), which is required for PC development. With regard to the underlying mechanisms, CyTOF analysis of disaggregated crypts showed loss of basal Akt (1.8-fold decrease, p=0.01) and ERK MAPK (3.7-fold reduction, p=0.02) phosphorylation specifically in CK20-/Lyz- transit amplifying/progenitor cells. Furthermore, in HT-29 cells, exposure to PI 3-kinase or MEK inhibitors resulted in a dose-dependent increase in Lyz1 expression. Interestingly, NRG1β exposure could overcome MAPK but not PI 3-kinase inhibition, suggesting differential requirement for these cascades. CONCLUSIONS: ErbB3 restricts PC numbers by a mechanism involving PI 3-kinase/Akt, MAPK, and Atoh1. Understanding the role of ErbB3 receptor could identify new therapeutic targets for regulating PCs and the ISC niche.

Where applicable, experiments conform with Society ethical requirements