Differentiation-induced uroplakin III expression promotes urothelial cell death in response to uropathogenic E. coli

Abstract

Uropathogenic E. coli (UPEC) expressing type 1 pili underlie most urinary tract infections (UTIs). UPEC adherence to the bladder urothelium induces a rapid apoptosis and exfoliation of terminally differentiated urothelial cells, a critical event in pathogenesis. Of the four major uroplakin proteins that are densely expressed on superficial urothelial cells, UPIa serves as the receptor for type 1-piliated UPEC, but the contributions of uroplakins to cell death are not known. We examined the role of differentiation and uroplakin expression on UPEC-induced cell death. Utilizing in vitro models of urothelial differentiation, we demonstrated induction of tissue-specific differentiation markers including uroplakins. UPEC-induced urothelial cell death was shown to increase with enhanced differentiation but required expression of uroplakin III: infection with an adenovirus encoding uroplakin III significantly increased cell death, while siRNA directed against uroplakin III abolished UPEC-induced cell death. In a murine model of UTI where superficial urothelial cells were selectively eroded to expose less differentiated cells, urothelial apoptosis was reduced, indicating a requirement for differentiation in UPEC-induced apoptosis in vivo. These data suggest that induction of uroplakin III during urothelial differentiation sensitizes cells to UPEC-induced death. Thus, uroplakin III plays a pivotal role in UTI pathogenesis.

Figure 1

Culture conditions promote induction of tissue-specific differentiation markers in human urothelial cells in vitro. PD07i human urothelial cells were cultured under serum-free conditions, in medium containing serum and elevated calcium (E medium), or in serum-free medium containing the PPARγ agonist troglitazone and the EGFR antagonist PD153035 (TZ/PD; see Methods for details). A) Immunoblotting detected increased cytokeratin expression in PD07i cells cultured in CK8 in E medium and troglitazone/PD153035 cultures relative to cells cultures in serum-free medium. Cytokeratin CK17 expression was reduced by culture of PD07i cells in E medium or troglitazone/PD153035. B) Immunoblotting revealed uroplakin induction by culture of PD07i cells in either E medium or troglitazone/PD153035. UPIa, UPIb and UPII were induced by culture in troglitazone/PD153035, while E medium induced all four uroplakins. Quantitative RT PCR analysis demonstrated increased expression of UPIII and mRNA (C) and UPIb mRNA (D) in PD07i cells cultured in E medium. Values reflect fold change relative to PD07i cells maintained in serum-free medium (i.e., Day 0).

Figure 2

Differentiation sensitizes urothelial cells cell death from UPEC type 1 pili. A) PD07i cells were cultured in E medium and infected with NU14 or NU14-1 (MOI 500), and cell death was quantified by dye exclusion assay. Cell death induced by FimH^– NU14-1 was significantly attenuated relative to NU14-induced death (*p<0.05). B) PD07i cells were cultured for different time periods in E medium and then infected with NU14 (MOI 500). Dye exclusion assay demonstrated significantly increased cell death (*p<0.05, ** p<0.001) in response to NU14 for cells cultured in E medium for 6 days or longer, relative to cells cultured in serum-free medium (i.e., Day 0). C) PD07i cultures were treated with troglitazone/PD153035 (see Methods) and then infected with NU14. Dye exclusion indicated no difference in NU14-induced cell death between troglitazone/PD153035-treated and control cultures. Bars indicate the mean ± SEM after subtraction of baseline cell counts of dye permeability in untreated cultures.

Figure 3

UPEC-induced urothelial cell death is modulated by UPIII expression. PD07i cells were cultured in serum-free conditions or treated with troglitazone/PD153035. Cultures were then infected with recombinant adenoviruses (MOI 100) encoding luciferase (Luc), UPIII, UPIII plus UPIb, or all four major uroplakins overnight, followed by infection with NU14 (MOI 500) and assay for cell death by dye exclusion. A) PD07i cells cultured in serum-free medium were significantly more sensitive to NU14-induced death when expressing UPIII alone or in combination with other uroplakins (*p<0.05). B) Expression of UPIII alone or in combination with other uroplakins significantly enhanced NU14-induced cell death over control adenovirus infection in UPIII-deficient TZ/PD differentiated cells (*p<0.05). C) PD07i cells were cultured under different conditions and infected with NU14 (MOI 10). After 2 h, cultures washed and total remaining NU14 (cell surface and invasive bacteria) was determined by plating detergent lysates onto selective agar and counting the resulting bacterial colonies. NU14 adherence was not significantly different between cell cultures treated with serum free media, E medium, or troglitazone/PD153035 (TZ/PD). D) PD07i cells were infected with adenoviruses encoding UPIII (III) or UPIII and UPIb (III+Ib) as above and then stained for UPIII expression. Infection with the virus encoding UPIb increased UPIII staining. Bars indicate the mean ± SEM, with each experiment repeated at least three times. Scale bar in D represents 50 μm.

Figure 4

Inhibition of UPIII gene expression abrogates NU14-induced cell death. A) PD70i cells expressing siRNA specific for UPIII (PD07siUPIII cells; labeled “siUPIII”) were cultured in E medium for 10 days and the assessed for UPIII mRNA expression by quantitative RT PCR. PD07siUPIII exhibited less UPIII mRNA than parental PD07i cultures (data are normalized to respective cultures maintained in serum-free medium). B) Immunoblotting revealed less UPIII protein (54% reduction relative to GAPDH by densitometry) in E medium-treated PD07siUPIII cells relative to PD07i cells expressing siRNA specific for TNFR1 (PD07siTNFR). C) Parental PD07i cells (Ctrl), PD07siUPIII, or PD07siTNFR were cultured in E medium for 10 days and assessed for cell death following infection with NU14 (MOI 500) by dye exclusion. NU14-induced cell death was significantly lower in PD07siUPIII cells (*p<0.05) than in PD07i or PD07siTNFR cells. Bars indicate the mean ± SEM, with each experiment repeated at least three times.

Figure 5

Superficial urothelial cells are sensitive to NU14-induced apoptosis. Bladders of female C57BL/6 mice were denuded of superficial urothelial cells by instillation with 10mM HCl, and mice were subsequently infected with NU14 (10⁸ CFU in 10μl). Panels A-C show representative sections from saline-treated control mice, and panels D-F show sections from HCl-treated mice. A and D) HCl-treated mice show eroded urothelium (D) relative to saline-treated mice (A) in H&E stained sections. B and E) HCl-treated mice lack UPIII immunoreactivity (red) in bladder sections (E), but UPIII staining is evident in the superficial epithelial layer of intact bladders (B). C and F) Bladder sections from NU14-infected mice were subjected to TUNEL staining (green) as a marker of apoptosis. Apoptotic cells were evident (green) in the superficial epithelial layer of saline-treated bladders (C, arrowhead and inset) but were absent in sections of HCl-treated bladders (F). G) Bacterial colonization was assessed in tissue homogenates of bladders harvested 2 h after infection with NU14. HCl- and saline-treated bladders exhibited similar levels of colonization. Scale bar represents 50 μm, and the nuclear stain DAPI (blue) was used for epifluorescence imaging.