Loss of Bicc1 impairs tubulomorphogenesis of cultured IMCD cells by disrupting E-cadherin-based cell-cell adhesion

Abstract

The Bicaudal-C (Bic-C) gene was originally discovered in Drosophila melanogaster. The gene product Bic-C is thought to serve as an RNA-binding molecule targeting diverse proteins at the post-transcriptional level. Recent research has shown this gene to be conserved in many species, from Caenorhabditis elegans to humans. Disruption of this protein can disturb the normal migration direction of the anterior follicle cell of Drosophila oocytes, while mutation of a mouse Bicc1 (a mouse homologue of Bic-C) results in phenotypes mimicking human hereditary polycystic kidney disease (PKD). However, the cellular function of Bicc1 gene products in mammalian systems remains largely unknown. In this study, we established stable IMCD (mouse inner medullary collecting duct) cell lines, in which Bicc1 was silenced by short hairpin RNA inhibition (shRNA). We show that inhibition of Bicc1 disrupted normal tubulomorphogenesis and induced cystogenesis of IMCD cells grown in three dimensional cultures. To determine what factors contributed to the defect, we systematically examined biological changes of Bicc1-silenced IMCD cells. We found that the cells had significant defects in E-cadherin-based cell-cell adhesion, along with abnormalities in actin cytoskeleton organization, cell-extracellular matrix interactions, cell proliferation, and apoptosis. These findings suggest that lack of Bicc1 leads to disruption of normal cell-cell junctions, which in turn impedes establishment of epithelial polarity. These cellular defects may initiate abnormal tubulomorphogenesis and cystogenesis of IMCD cells grown in vitro. The observation of aberrant cellular behaviors in Bicc1-silenced IMCD cells reveal functions for Bicc1 in renal epithelial cells and provides insight into a potential pathogenic mechanism of polycystic kidney disease.

Figure 1

Establishment of stable Bicc1-silenced IMCD cell lines. (A) Bicc1-shRNA-C was inserted into the pRS-shRNA vector. The pRS-Hush-GFP vector was used to establish a stable control cell line, IMCD^shGFP1. (B) Quantitative PCR was used to test Bicc1 mRNA levels in wildtype (IMCD^WT), control (IMCD^shGFP1) and Bicc1-silenced (IMCD^shC1C and IMCD^shC4C) cell lines (*P<0.05). The bars and error lines represent the mean and standard error of three experiments performed in triplicate. (C) Wildtype (IMCD^WT), control (IMCD^shGFP1) and Bicc1-silenced (IMCD^shC1C and IMCD^shC4C) cell lines were subjected to western blotting analysis with mBic-Np. The Bicc1-silenced cell lines showed a significant reduction of Bicc1 protein expression when compared with IMCD^WT and IMCD^shGFP1 cells. Equivalent protein loading was shown using anti-β-actin antibody on the same western blots. (D) Statistical examination of normalized Bicc1 expression levels indicates the Bicc1-silenced IMCD cell lines (IMCD^shC1C and IMCD^shC4C) have a significant decrease in Bicc1 expression compared to wild-type and control cell lines (IMCD^WT and IMCD^shGFP1) (*P<0.05).

Figure 2

Tubulomorphogenesis is inhibited in 3-D cultured Bicc1-silenced IMCD cell lines. Phase contrast photomicrographs were taken of examples of wild-type (A) and Bicc1-silenced IMCD cells (B-D) cultured for 7 days in 3-D MG gels. (E) A significantly higher rate of cyst formation and cell aggregation was observed for the Bicc1-silenced IMCD cell lines (IMCD^shC1C and IMCD^shC4C) when compared to either wild-type or controls cell lines (IMCD^WT and IMCD^shGFP1). White bars indicate no tubular branching; gray and black bars show 1-3 and >3 tubular branches respectively. Bar: 20 μm in (A-D).

Figure 3

Reduction of Bicc1 disrupts normal cell-cell contact. (A) Wildtype (IMCD^WT), control (IMCD^shGFP1), and Bicc1-silenced cells (IMCD^shC1C and IMCD^shC4C) were stained with an antibody to E-cadherin. Microscopic images from cells stained with anti-E-cadherin antibody revealed a more diffuse E-cadherin distribution (arrows) in Bicc1-silenced cells (c-d) versus wildtype (IMCD^WT) and control cells (IMCD^shGFP1) (a-b). (B) Tight junction integrity was assessed in wildtype (IMCD^WT), control (IMCD^shGFP1), and Bicc1-silenced cells (IMCD^shC1C and IMCD^shC4C) using an anti-ZO-1 antibody. Similar results to A were obtained with a more diffuse ZO-1 distribution (arrows) observed in Bicc1-silenced cells (c-d) versus wildtype (IMCD^WT) and control cells (IMCD^shGFP1) (a-b). Arrows point to site of magnification in each panel. (C) Western blots of duplicate protein lysates from wildtype (IMCD^WT), control (IMCD^shGFP1), and Bicc1-silenced IMCD cells (IMCD^shC1C and IMCD^shC4C) cells did not show significant differences in expression of E-cadherin or ZO-1. Anti-β-actin antibodies were used for protein loading control. (D) Transepithalial resistance (TER) of the same cell penal was measured over a six day period. Values shown represent the mean and SD of at least three independent experiments. Significant differences of TER in Bicc1-silenced IMCD cells were observed after 3 days of culture (*P<0.05). Bars, 2.5 μm in (A) and (B).

Figure 4

The actin cytoskeleton distribution in IMCD^WT, IMCD^shGFP1, IMCD^shC1C and IMCDshC4C cell lines. (A) Phase micrographs of sub-confluent cells stained with rhodamine-phalloidin (F-actin) showed a cortical band of actin filaments and few stress fibers in wildtype (IMCD^WT) and control (IMCD^shGFP1) cells whereas Bicc1-silenced cells (IMCD^shC1C and IMCD^shC4C) have thick and enriched stress fibers. (B) Confocal micrographs of the same cells, confluent for 5 days, showed a peripheral distribution of F-actin filaments at cell-cell borders in wildtype (IMCD^WT) and control (IMCD^shGFP1) cells, while Bicc1-silenced cells (IMCD^shC1C and IMCD^shC4C) demonstrated prominent stress fibers and loss of F-actin peripheral distribution pattern. Bars: 2.5 μm in (A-B).

Figure 5

Reduction of Bicc1 decreases integrin-dependent adhesion on collagen I (CI) and shows aberrant cell motility. (A) Adhesion assays were performed on CI coated plates as described in the Materials and Methods. The values shown represent the mean and SD of at least three independent experiments. Significant differences of CI-dependent cell adhesion were seen between the Bicc1-silenced cells (IMCD^shC1C and IMCD^shC4C) and wild-type/control cells (IMCD^WT and IMCD^shGFP1) (*P<0.05). (B) Results from transwell migration assays indicating the absolute number of cells that migrate to the underside of the transwell are provided. There were significant differences between Bicc1-silenced cells (IMCD^shC1C and IMCD^shC4C) and wild-type/control cells (IMCD^WT and IMCD^shGFP1) (*P<0.05).

Figure 6

Downregulation of Bicc1 alters the proliferation and apoptosis of IMCD cells. (A) Wildtype (IMCD^WT), control (IMCD^shGFP1) and Bicc1-silenced (IMCD^shC1C and IMCD^shC4C) cells were incubated with ³H-thymidine after which the rate of ³H-thymidine incorporation was determined as described in Materials and Methods for at least three times. ³H-thymidine values were significantly different between cells with and without Bicc1-silencing (*P<0.05). (B) The same panel of cell lines was used for Phospho-Histone H3 staining to evaluate cell proliferation. The percentage of cells found to be positive for Phospho-Histone H3 was significantly different between cells with and without Bicc1-silencing (*P<0.05). (C) The same panel of cell lines was cultured in 24-well plates; then fixed and subjected to TUNEL assays to assess apoptosis for three times. The values represent the percentage of apoptotic cells relative to the total number of cells. Cells with Bicc1-silencing were significantly different then wildtype/control IMCD cells (*P<0.05). (D) The same panel of cell lines was cultured in 24-well plates with the same number of cells per well for 24 hrs. A PE Caspase-3 Active Apoptosis kit was used to measure the apoptosis rate, according to the manufacturer's instructions. The values represent the percentage of apoptotic cells relative to the total number of cells. An increase in the percentage of cells positive for active apoptosis was observed with the Bicc1-silenced cells (*P<0.05).