Lack of CFTR alters the ferret pancreatic ductal epithelial secretome and cellular proteome: Implications for exocrine/endocrine signaling

Abstract

Background: Cystic fibrosis (CF) related diabetes is the most common comorbidity for CF patients and associated with islet dysfunction. Exocrine pancreas remodeling in CF alters the microenvironment in which islets reside. Since CFTR is mainly expressed in pancreatic ductal epithelium, we hypothesized altered CF ductal secretions could impact islet function through paracrine signals.

Method: We evaluated the secretome and cellular proteome of polarized WT and CF ferret ductal epithelia using quantitative ratiometric mass spectrometry. Differentially secreted proteins (DSPs) or expressed cellular proteins were used to mine pathways, upstream regulators and the CFTR interactome to map candidate CF-associated alterations in ductal signaling and phenotype. Candidate DSPs were evaluated for their in vivo pancreatic expression patterns and their functional impact on islet hormone secretion.

Results: The secretome and cellular proteome of CF ductal epithelia was significantly altered relative to WT and implicated dysregulated TGFβ, WNT, and BMP signaling pathways. Cognate receptors of DSPs from CF epithelia were equally distributed among endocrine, exocrine, and stromal pancreatic cell types. IGFBP7 was a downregulated DSP in CF ductal epithelia in vitro and exhibited reduced CF ductal expression in vivo. IGFBP7 also altered WT islet insulin secretion in response to glucose. Many CFTR-associated proteins, including SLC9A3R1, were differentially expressed in the CF cellular proteome. Upstream regulators of the differential CF ductal proteome included TGFβ, PDX1, AKT/PTEN, and INSR signaling. Data is available via ProteomeXchange with identifier PXD025126.

Conclusion: These findings provide a proteomic roadmap for elucidating disturbances in autocrine and paracrine signals from CF pancreatic ducts and how they may alter islet function and maintenance.

Keywords: Cystic Fibrosis; Cystic Fibrosis Related Diabetes; ductal cells; pancreas; proteome; secretome.

Fig. 1.. Mass spectrometry evaluation of pancreatic ductal secretions and whole cell proteome.

(A) Expression of pancreatic ductal cell-specific markers Sox9 and KRT7 in P10 WT and CF ferret ductal cells by immunofluorescence. Scale bars, 50 μm. (B,C) Short circuit current (Isc) analysis of CFTR-mediated currents in polarized WT and CF ductal epithelia. Cultures were sequentially treated with amiloride (to inhibit ENaC), DIDS (to inhibit non-CFTR chloride channels), IBMX/Forskolin (I/F, to activate CFTR), and GlyH101 (to inhibit CFTR). Shown is a representative Isc tracing (B) and the average change in response to each antagonist or agonist (C). Data depicts the Mean+/−SEM for N=6 transwells per genotype (6 WT and 6 CF donors). (D) Baseline apical surface pH measurements on WT and CF polarized duct epithelia. Data depicts the Mean+/−SEM for N=12 transwells per genotype (4 WT and 4 CF donors). (E) Schematic protocol for the mass spectrometry analysis of the IBMX/forskolin (I/F)-stimulated secretome and unstimulated whole cell proteome of WT and CF pancreatic duct epithelia. (F) Schematic protocol for quantitative mass spectrometry using isotope labelling to calculate the fold change (FC) of proteins in the secretome and whole cell proteome in CF relative to WT (CF/WT). Statistical analysis in (C, D) was performed using a two-tailed Student’s t-test, **p<0.01, ***p<0.001.

Fig 2.. Predicted alteration to upstream regulators and paracrine signaling in CF pancreatic ductal epithelia.

(A) Volcano plot showing cAMP-stimulated differentially secreted proteins (DSPs) from the apical or basolateral surface of WT (N=4 donors) and CF (N=4 donors) pancreatic duct epithelia. Proteins labelled in the plot have a Log₂FC>5 (p<0.5) or Log₂FC<−5 (p<0.05). (B) Heatmap of predicted upstream regulators of the differential secretomes from the basolateral (Baso) and apical surface (p<0.05) of CF and WT pancreatic duct epithelium. The activation Z-score for each of the upstream regulators are shown in the heatmap to the right. (C) Chordplot showing putative functional interaction between the DSPs from the CF duct epithelium with exocrine, endocrine, and stromal cell types of the pancreas.

Fig. 3.. IGFBP7 expression is altered in the CF pancreas and treatment of islets with IGFBP7 alters glucose-stimulated insulin secretion.

(A,B) smFISH localization of IGFBP7 (A) and LGALS3 (B) mRNA in neonate WT and CF ferret pancreata. Boxed regions in the main panels are shown below as enlarged single grey channels images. Micron bars, 50 μm. (C) Percent insulin and glucagon secretion from WT islets in low glucose and high glucose in the presence (+) and absence (−) of IGFBP7 exposure. Data shows the Mean+/−SEM for N=3 measurements from three independent donor islet preparations normalized to the mean hormone secreted at low glucose. Statistical analysis in (C) was performed using a two-tailed Student’s t-test, *p<0.05, **p<0.01.

Fig 4.. CF pancreatic duct epithelia have altered expression of proteins that interact with CFTR.

(A) Schematic of the CFTR interactome in the cytoplasmic, cell membrane, and extracellular compartments of epithelial cells. (B) Differentially expressed proteins from whole cell proteomic analysis of CF and WT pancreatic ductal epithelia cross-referenced to the CFTR interactome. Shown are heatmaps of the Log₂(FC) for differentially expressed proteins (CF/WT) within the various domains of interaction with CFTR.

Fig. 5.. Pathway analysis of differentially expressed proteins within CF ductal epithelia.

(A) IPA analysis of disease pathways associated with the differential CF ductal epithelial proteome. (B) IPA analysis of canonical biologic processes and functions associated with the differential CF ductal epithelial proteome. (C) Upstream regulators of the differential CF ductal epithelial proteome related to proinflammatory and redox-dependent signaling pathways. Heatmaps show the Log₂(FC) of target molecules of each upstream regulator that were differentially expressed in CF ductal epithelia. (D) Upstream regulators of the differential CF ductal epithelial proteome that are known to influence specification of endocrine cells from ductal progenitors during pancreatic development. Heatmaps show the Log₂(FC) of target molecules of each upstream regulator that were differentially expressed in CF ductal epithelia.