Cell shedding from the intestinal villus is a key element of normal tissue turnover. The shedding rate must be tightly coordinated with cell division in the stem cell compartment to maintain homeostasis, and deregulated cell extrusion is associated with disorders such as inflammatory bowel diseases. However, the signals regulating this process are not well understood. In this study, we asked whether shedding is controlled by epidermal growth factor (EGF) receptor (R), an important driver of intestinal growth and differentiation. METHODS: Cell extrusion events in vitro (IEC-6 rat ileal, MDCK canine kidney, or IPEC-J2 pig jejunal cells) were identified by rhodamine-phalloidin labeling of condensed actin rings and by counting extruded cells in the culture media over time. Cultures were treated with EGF and/or inhibitors to MEK/ERK signaling (U0126), caspases (Z-VAD-FMK), ROCK (Y27632), or Piezo1/2 (gadolinium (III) chloride) and fixed or collected after 2 h. Cell extrusion events in vivo (zebrafish midgut) were identified by rhodamine-phalloidin labeling on cryosections; fish were anaesthetized by perfusion with 0.6 mM tricaine and given EGF and/or U0126 by intraperitoneal injection, then euthanized after 4 h. All zebrafish experiments were approved and monitored by the Children’s Hospital Los Angeles Institutional Animal Care and Use Committee. All results are from at least 5 independent cultures or fish per condition. Data were analyzed by either t test or ANOVA with Tukey post-test as appropriate. RESULTS: EGF reduced detectable shedding events from IEC-6 (42% decrease, p<0.01), MDCK (49% decrease, p<0.01), and IPEC-J2 (28% decrease, p<0.001) cell monolayers. Pharmacological blockade of MEK/ERK reversed these effects. In contrast, inhibitors to other EGF-stimulated pathways such as PI3K/Akt or PKC had no effect. Interestingly, EGF suppression of shedding did not affect the percentage of shed cells which were viable and could be recultured. Furthermore, MEK blockade promoted shedding even in the presence of caspase, ROCK, and Piezo1 inhibitors, which normally suppress cell extrusion. In vivo, EGF reduced detectable shedding events on zebrafish villi (34% decrease, p<0.05) in a MEK/ERK dependent manner. CONCLUSIONS: EGFR suppresses cell shedding in the intestinal epithelium through a specific MEK/ERK dependent pathway, independent of known extrusion pathways including apoptosis, ROCK, and Piezo1. These results may point the way to new therapeutic avenues for targeting excessive cell turnover in conditions such as inflammatory bowel diseases.