Endothelin-3 stimulates survival of goblet cells in organotypic cultures of fetal human colonic epithelium

Abstract

Cells within the normal human colonic epithelium undergo a dynamic cycle of growth, differentiation, and death. The organotypic culture system of human fetal colonic epithelial cells seeded on top of collagen gels with embedded colonic fibroblasts allowed prolonged culture of the colonic epithelial cells (Kalabis J, Patterson MJ, Enders GM, Marian B, Iozzo RV, Rogler G, Gimotty PA, Herlyn M. FASEB J 17: 1115-1117, 2003). Herein, we have evaluated the role of endothelin-3 (ET3) and both cognate endothelin receptors (ETRA, ETRB) for human colonic epithelial cell growth and survival. ET3 was produced continuously by the fibroblasts as a result of adenovirus-mediated gene transfer. The presence and function of the endothelin receptors (ETRs) in epithelial cells was evaluated by [(3)H]thymidine incorporation using primary epithelial cells in monoculture and by immunohistochemistry on human fetal and adult paraffin-embedded tissues. In organotypic culture, ET3 increased the number of goblet cells but not of enteroendocrine cells. The increase in goblet cells was caused by prolonged cell survival and differentiation. The inhibition of both ETRA and ETRB significantly decreased the number of goblet cells and proliferation in epithelial cells, whereas the number of enteroendocrine cells remained unchanged. ET3 induced activation of IkappaB and MAPK in the epithelial cells, suggesting that these signaling pathways mediate its proproliferation and prosurvival activities. Our results demonstrate that ET3 is involved in regulating human colonic epithelial cell proliferation and survival, particularly for goblet cells, and may be an important component of colonic homeostasis.

Autocrine Endothelin-3/Endothelin Receptor B Signaling Maintains Cellular and Molecular Properties of Glioblastoma Stem Cells

“…A previous study has shown that stimulation of the EDNRB in astrocyte induces cAMP response element-binding protein (CREB) and c-fos expression via multiple MAPK signaling pathways, including the extracellular regulated kinase (ERK) 2, c-Jun N-terminal kinase 1 (JNK1), and p38 kinase (38). EDN3 stimulation also rapidly increased phosphorylation of ERK2 in neural progenitor cells (39) and activated IkappaB and MAPK in the human colonic epithelial cells (40). Likewise, EDN3 treatment resulted in the activation of MAPK-p90 ribosomal S6 kinase-CREB and cAMP-protein kinase A-CREB pathways in melanocyte culture (41, 42) and induction of ERK1/2 and focal adhesion kinase (FAK) phosphorylation in malenoma cells (43).…”

Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3

“…In recent years, it has also been applied to the isolation of many other highyield homogenous and viable cells (27). Because of the persistent renewal of human fetal intestinal cells, continuous epithelial cell cultures can be generated relatively easily from the human fetal intestine in a reproducible manner (17,19). Furthermore, the thermolysin method of generating viable HIEC cultures has proven to be particularly useful in the study of normal function and of metabolic and growth mechanisms (7,17,28).…”

“…Furthermore, the thermolysin method of generating viable HIEC cultures has proven to be particularly useful in the study of normal function and of metabolic and growth mechanisms (7,17,28). Since ET-3 affects the growth and differentiation of gastrointestinal epithelial cells by mediating epithelial-mesenchymal interactions, which control intestinal epithelial growth in the developing mammalian fetus (19), ET-3 was added to stimulate the growth of HIEC. By adding ET-3, the achievement ratio was enhanced again to 60% from only 38% in a previous study (6).…”

“…Thermolysin treatment, which was previously used for dermo-epidermal separation and keratinocyte isolation, could improve the yield of HIEC (6). Furthermore, it was also reported that endothelin-3 (ET-3) could affect the growth and differentiation of gastrointestinal epithelial cells by mediating the epithelial-mesenchymal interaction, a process that controls intestinal epithelial growth in developing mammalian fetuses (19). Consequently, ET-3 could be added to stimulate the growth of HIEC.…”

IMP-1 Displays Cross-Talk with K-Ras and Modulates Colon Cancer Cell Survival through the Novel Proapoptotic Protein CYFIP2

“…SW480 cells were transiently transfected with either scramble control or IMP-1 siRNAs, seeded on Type I Collagen embedded with human fetal colonic fibroblasts (ATCC), grown in organotypic culture (OTC), harvested, and fixed as previously described (34). …”