Endothelial Cell Mediated Promotion of Ciliated Cell Differentiation of Human Airway Basal Cells via Insulin and Insulin-Like Growth Factor 1 Receptor Mediated Signaling

Abstract

Human airway basal cells (BC) function as stem/progenitor cells of the human airway epithelium, capable of differentiating into ciliated and secretory cells during turnover and repair. The positioning of BC along the basement membrane allows for potential paracrine signaling from non-epithelial cells in the mesenchyme to regulate BC function. Based on the knowledge that interaction between the airway epithelium and mesenchyme is critical for proper maintenance of both tissues, and that endothelial cells (EC) can regulate multiple functions of BC, the present study was designed to help understand the role of BC and EC cross-talk in regulating BC stem/progenitor function. Using an in vitro co-culture system that mimics the in vivo physical separation of these cell types, we assessed the impact of primary lung microvascular EC on differentiation of primary BC into a mucociliated epithelium. The data demonstrate that co-culture of BC and lung microvasculature EC results in increased ciliated cell differentiation of BC via activation of insulin (INS) and insulin-like growth factor 1 (IGF1) receptor (INSR and IGF1R) mediated signaling in BC. Consistent with this data, siRNA mediated knockdown of INSR and IGF1R in BC suppressed ciliated cell differentiation. Together these findings identify an important signaling pathway required for differentiation of BC into a ciliated cells and demonstrate the importance of BC-EC cross-talk in regulating normal airway epithelial structure.

Keywords: Basal stem/progenitor cells; Ciliated cells; Differentiation; Endothelial cells; Human airway epithelium; Insulin; Insulin-like growth factor 1; Signaling.

Figure 1

Establishment of basal cell (BC) and endothelial cell (EC) co-culture system to study BC differentiation into a mucociliated epithelium. A. Immunofluorescence assessment of a normal human airway biopsy with staining for BC (KRT5, green), EC (CD31, red) and nuclei (DAPI, blue). Scale bar 50 μm. B. Schematic representation of the BC and EC co-culture system to study the impact of EC on BC differentiation into a mucociliated epithelium. C. Immunofluorescence assessment of BC and EC co-culture with staining for BC (KRT5, red), EC (VE-cadherin, green) and nuclei (DAPI, blue). Upper panel, BC cultured alone. Lower panel, BC and EC co-culture. White dashed line outlines the membrane of the Transwell insert. Scale bar 20 μm. D. Immunofluorescence staining of the epithelial layer of ALI day 28 cultures. Basal cells were cultured alone or co-cultured with EC at a 10:1 and 2:1 (BC:EC) ratio and then stained for ciliated cells (β-tubulin IV, green), secretory cells (SCGB1A1, red) and nuclei (DAPI, blue). Scale bar 20 μm. E. Basal cells were cultured alone or co-cultured with EC at a 10:1 and 2:1 (BC:EC) ratio on air-liquid interface (ALI) culture for 7 days to assess the impact of EC on the early stages of BC differentiation into a mucociliated epithelium at the molecular level. TaqMan PCR analysis to assess mRNA expression of BC (KRT5), intermediate (KRT8), secretory (non-mucus producing, SCGB1A1 and mucus producing, MUC5B) and ciliated cell (FOXJ1) markers in BC cultured alone or co-cultured with EC cells at 10:1 and 2:1 (BC:EC) ratio. Bars indicate the mean for n=6 independent experiments each performed in triplicate and error bars indicate standard error of the mean. Asterisks (*) indicate p<0.05 compared to BC alone. The experiments for E were performed with n=4 independent donors of BC and EC.

Figure 2

Endothelial cell (EC) co-culture increases differentiation of basal cells (BC) into ciliated cells. Basal cells were cultured alone or co-cultured with EC at a 10:1 and 2:1 (BC:EC) ratio on air-liquid interface (ALI) culture for 28 days to assess the impact of EC on BC differentiation into a mucociliated epithelium at the histological level via immunofluorescence staining with cell type specific markers. A. KRT5⁺ BC. Sections of cells were stained for KRT5 (red) and DAPI (nuclei, blue). B. KRT8⁺ intermediate cells. Sections of cells were stained for KRT8 (red) and DAPI (nuclei, blue). C. SCGB1A1⁺ secretory cells. Sections of cells were stained for SCGB1A1 (red) and DAPI (nuclei, blue). D. MUC5B⁺ secretory cells. Sections of cells were stained for MUC5B (red) and DAPI (nuclei, blue). E β-tubulin IV⁺ cells. Sections of cells were stained for β-tubulin IV (ciliated, green) and DAPI (nuclei, blue). A–E. Scale bar 20 μm. H. Quantification of KRT5⁺, KRT8⁺, SCGB1A1⁺, MUC5B⁺ and β-tubulin IV⁺ cells. The bars indicate the mean for n=4 independent experiments and error bars indicate standard error of the mean. Asterisks (*) indicate p<0.05 compared to BC cultured alone. The experiments for A–H were performed with n=4 independent donors of BC and EC.

Figure 3

Signaling via the insulin (INS) and insulin-like growth factor 1 (IGF1) receptors (INSR and IGF1R) regulate basal cell (BC) differentiation into ciliated cells. A–C. Basal cells were cultured alone or co-cultured with EC at a 10:1 (BC:EC) ratio on air-liquid interface (ALI) culture for 7 days to assess the activity of specific kinase signaling pathways in BC in response to EC derived factors using the PathScan RTK Proteome Array (Cell signaling). A. Representative images from the same exposure times of the kinase signaling array from BC cultured alone and BC co-cultured with EC. B. Activity of the INS and IGF1 mediated signaling pathways including the receptor tyrosine kinases (ISNR and IGF1R) and downstream signaling nodes (IRS1, SRC, AKT and ERK1/2). “Pos cont” and “neg cont” represent the positive and negative control spots respectively on the array. C. Quantification of the INS and IGF1 signaling pathway activity. The intensities of spots were quantified with ImageJ and all values normalized by the positive control spots on the array (100%) and negative control spots (0%). D. TaqMan PCR analysis to assess mRNA expression of INS, IGF1 and IGF2 in BC (black bars) and EC (grey bars) following co-culture at ALI day 0. Bars indicate the mean expression for n=4 replicate wells and error bars indicate standard error of the mean.

Figure 4

siRNA mediated knockdown of INSR and IGF1R in basal cells (BC) suppresses ciliated cell differentiation. Basal cells with transfected with either control, INSR or IGF1R specific siRNA and cultured on ALI for 7 days to assess the impact of each receptor on the early stages of BC differentiation into a mucociliated epithelium at the molecular level. A. TaqMan PCR analysis to assess INSR mRNA expression at ALI day 0. Bars indicate the mean for n=3 independent experiments each performed in triplicate and error bars indicate standard error of the mean. B. TaqMan PCR analysis to assess IGF1R mRNA expression at ALI day 0. Bars indicate the mean for n=3 independent experiments each performed in triplicate and error bars indicate standard error of the mean. C. TaqMan PCR analysis to assess mRNA expression of BC (KRT5), intermediate (KRT8), secretory (non-mucus producing, SCGB1A1 and mucus producing, MUC5B) and ciliated cell markers (FOXJ1, MYB and DNAI1) in BC at ALI day 7 following siRNA mediated knockdown of INSR (black bars) and IGF1R (grey bars). Bars indicate the mean for n=3 independent experiments each performed in triplicate and error bars indicate standard error of the mean. Asterisks (*) indicate p<0.05 compared to BC transfected with control siRNA. The experiments for A–C were performed with n=3 independent donors of BC.