title: The exocyst gene Sec10 regulates renal epithelial monolayer homeostasis and apoptotic sensitivity.
abstract: The highly conserved exocyst protein complex regulates polarized exocytosis of subsets of secretory vesicles. A previous study reported that shRNA knockdown of an exocyst central subunit, Sec10 (Sec10-KD) in Madin-Darby canine kidney (MDCK) cells disrupted primary cilia assembly and 3D cyst formation. We used three-dimensional collagen cultures of MDCK cells to further investigate the mechanisms by which Sec10 and the exocyst regulate epithelial polarity, morphogenesis, and homeostasis. Sec10-KD cysts initially demonstrated undisturbed lumen formation although later displayed significantly fewer and shorter primary cilia than controls. Later in cystogenesis, control cells maintained normal homeostasis, while Sec10-KD cysts displayed numerous apoptotic cells extruded basally into the collagen matrix. Sec10-KD MDCK cells were also more sensitive to apoptotic triggers than controls. These phenotypes were reversed by restoring Sec10 expression with shRNA-resistant human Sec10. Apico-basal polarity appeared normal in Sec10-KD cysts, whereas mitotic spindle angles differed significantly from controls, suggesting a planar cell polarity defect. In addition, analysis of renal tubules in a newly generated kidney-specific Sec10-knockout mouse model revealed significant defects in primary cilia assembly and in the targeted renal tubules; abnormal epithelial cell extrusion was also observed, supporting our in vitro results. We hypothesize that, in Sec10-KD cells, the disrupted exocyst activity results in increased apoptotic sensitivity through defective primary cilia signaling and that, in combination with an increased basal cell extrusion rate, it affects epithelial barrier integrity and homeostasis.
authors.affiliation: Department of Anatomy, Biochemistry and Physiology, John A. Burns School of Medicine, University of Hawaii at Manoa, Hawaii.
authors.forename: Noemi
authors.initials: N
authors.lastname: Polgar
authors.affiliation: Department of Anatomy, Biochemistry and Physiology, John A. Burns School of Medicine, University of Hawaii at Manoa, Hawaii.
authors.forename: Amanda J
authors.initials: AJ
authors.lastname: Lee
authors.affiliation: Department of Anatomy, Biochemistry and Physiology, John A. Burns School of Medicine, University of Hawaii at Manoa, Hawaii.
authors.forename: Vanessa H
authors.initials: VH
authors.lastname: Lui
authors.affiliation: Department of Anatomy, Biochemistry and Physiology, John A. Burns School of Medicine, University of Hawaii at Manoa, Hawaii.
authors.forename: Josephine A
authors.initials: JA
authors.lastname: Napoli
authors.affiliation: Department of Anatomy, Biochemistry and Physiology, John A. Burns School of Medicine, University of Hawaii at Manoa, Hawaii fogelgre@hawaii.edu.
authors.forename: Ben
authors.initials: B
authors.lastname: Fogelgren
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D049109:Cell Proliferation / Q000502:physiology; D004285:Dogs; D004847:Epithelial Cells / Q000502:physiology*; D006706:Homeostasis / Q000502:physiology*; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000502:physiology*; D061985:Madin Darby Canine Kidney Cells; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D033921:Vesicular Transport Proteins / Q000502:physiology*
chemical_list: C419575:EXOC5 protein, human; D033921:Vesicular Transport Proteins
keywords: apoptosis; cell extrusion; epithelial monolayer; exocyst; primary cilium