Absence of CFTR impairs the function of plasma membrane Ca2+ pump in pancreatic ductal epithelial cells
Our hypothesis was that the decreased CFTR expression caused by chronic ethanol per se is sufficient to disturb the Ca2+ homeostasis of the gastrointestinal epithelial cells. Previously, we established that acute exposure to ethanol releases Ca2+ from the ER and activates extracellular Ca2+ influx in pancreatic ductal cells 4. To assess whether the decreased CFTR expression disturbs the intracellular Ca2+ homeostasis wild-type (WT) and Cftr KO mice pancreatic ducts were challenged with carbachol. The maximal Ca2+ release was not different between the two groups, the slope of the Ca2+ signal plateau phase –representing the Ca2+ extrusion from the cytosol– was significantly higher in Cftr KO ductal fragments compared to WT (Figure 1.A.). Next, we utilized mouse pancreatic organoids (MPO) generated from WT and Cftr KO mice. WT organoids were treated with 100 mM ethanol (EtOH) and 200 µM palmitic acid (PA) for 12 h, control and Cftr KO MPOs received no treatment. Store operated Ca2+ influx was activated by re-addition of the extracellular Ca2+ after ER depletion (25 µM cyclopiazonic-acid (CPA) in Ca2+-free media) (Figure 1.B.i.). The basal intracellular Ca2+ concentrations were significantly higher in the EtOH-treated and Cftr KO organoids (Figure 1.B.ii.). As expected, the ER Ca2+ release in response to CPA was lower in the EtOH treated organoids and was not changed in Cftr KO organoids (Figure 1.B.iii.), whereas both EtOH treated and Cftr KO organoids showed a significantly decreased Ca2+ extrusion after removal of the extracellular Ca2+ (Figure 1.B.iv.). The same phenomenon was observed in Cftr KO ducts (Supplementary figure 1.A.). Next human pancreatic organoids (HPO) were treated with 100 mM EtOH and 200 µM PA overnight. Importantly, compared to untreated HPOs, Ca2+ extrusion was significantly decreased after pre-incubation with EtOH/PA (Figure 1.C.). Next, to confirm that the observed difference in Ca2+ extrusion was specific to CFTR-expressing cells, we analysed Ca2+ signaling in pancreatic acinar cells, which lack CFTR in general 24 and did not detect difference in the carbachol response (maximal intracellular Ca2+ release or extrusion) between WT and Cftr KO mice acini (Supplementary Figure 1.B.). Moreover, functional inhibition of CFTR with 10 µM CFTR(inh)-172 –which significantly impaired CFTR activity (Supplementary Figure 1.C.) 4– had no effect on the carbachol-induced Ca2+ extrusion in WT ductal cells (Figure 1.D.). Correction of CFTR expression in CFPAC-1 cells 14–derived from liver metastasis of a CF patient’s pancreatic ductal adenocarcinoma–restored Ca2+ extrusion (Supplementary figure 2.A-B.). In contrast, knockdown of CFTR expression in WT ductal fragments with siCFTR impaired Ca2+ extrusion compared to control (Supplementary figure 2.C-D.). Considering that both PMCA and Na+/Ca2+ exchangers (NCX) can forward Ca2+ extrusion in non-excitable cells, we used the pan-NCX inhibitors SEA0400 and CB-DMB to assess the contribution of NCX to the process. None of these inhibitors had any effect on the slope of the decrease (Figure 1.E.;Supplementary Figure 2.E.). Recently, Partner of STIM1 (POST) –an adaptor protein linking STIM1 to other proteins– was shown to enhance the function of PMCA4 25. However, siSTIM1 treatment had no effect on the Ca2+ efflux in WT pancreatic ducts, suggesting that Stim1-POST is not involved in the regulation of PMCA in epithelial cells (Supplementary figure 2.F.). Taken together, these results indicate that attenuation of CFTR expression -rather than the lack of activity- by ethanol treatment is sufficient to alter Ca2+ homeostasis through limiting PMCA activity.
Ethanol has no effect on the PMCA4 expression in pancreatic ductal cells
Currently, four mammalian PMCA genes have been identified which contribute to cytosolic Ca2+ extrusion 26. Using whole transcriptome analysis we revealed the expression of Pmca1 and Pmca4 in MPO and PMCA1 and PMCA4 in HPO samples, with highest levels of Pmca1 in mouse and highest levels of PMCA4 in humans (Figure 2.A-B.). Of note, expression levels of Pmca2 and Pmca3 were below detection limit in all samples. RT-PCR followed by endpoint analysis confirmed the expression of Pmca1 and Pmca4 in whole pancreatic tissue as well as isolated mouse pancreatic ducts (Supplementary Figure 3.A.). Immunofluoresent staining of PMCA1 and PMCA4 in cross sections of MPOs revealed the apical localization of PMCA4, whereas PMCA1 was evenly distributed over the apical and basolateral membranes (Figure 2.C.). In addition, a strong co-localization of PMCA4 and CFTR at the apical membrane was observed (Mander’s correlation coefficient:0.906, Supplementary Figures 3.A-B.), therefore in the downstream analysis we focused on PMCA4. In intestinal stem cells, loss of CFTR expression results in alkaline pHi deriving Wnt/β-catenin-mediated expression of different genes 27, which may affect the expression of PMCA4 in pancreatic ductal cells. To test this, the relative expression of Pmca4 was compared with qRT-PCR in control, EtOH/PA-treated and Cftr KO MPOs. While, control and EtOH/PA-treated WT MPO showed no significant alteration, Pmca4 expression was moderately increased in Cftr KO ductal organoids compared to WT control suggesting that the difference of Ca2+ efflux is not due to reduced gene expression (Figure 2.D.). Next, we wondered whether loss of CFTR due to EtOH treatment would alter the apical membrane-specific localisation of PMCA4. Whereas immunofluorescent microscopy revealed diminished CFTR levels at the apical membrane in EtOH/PA-treated and Cftr KO MPOs compared to untreated WT, PMCA4 retained its apical localisation in all samples (Figure 2.E.). Subsequently, the presence of CFTR and PMCA4 on the apical plasma membrane of HPOs was confirmed by immunolabelling. Whereas overnight incubation of HPO with EtOH/PA resulted in a diminished, patchy apical expression pattern of CFTR, PMCA4 retained its apical membrane localisation (Figure 2.F.). Notably, alcohol treatment resulted in a detectable cytosolic shift of PMCA4. These results suggest that the lack of CFTR at the apical membrane of pancreatic ductal cells diminishes the activity but not the expression or localization of PMCA4.
iPSC-derived organoids from cystic fibrosis patients recapitulate the alteration of PMCA function
Our results suggest that the diminished CFTR expression caused by genetic mutations in CF may also disturb Ca2+ extrusion of pancreatic ductal cells. Therefore, we assessed the relevance of our findings in human iPSC-derived pancreatic organoids generated from CF patients 20. To establish CF-iPSC lines from donors affected by classical CF, lentiviral reprogramming of patient keratinocytes was used as previously described and performed stepwise in vitro differentiation to direct the iPSCs towards the pancreatic lineage followed by generation of exocrine pancreatic organoids in 3D-suspension culture (Figure 3.A.). First, immunofluorescent analysis revealed that, while CFTR levels were absent in CF patient-derived organoids, which was markedly restored by 12 h incubation with the CFTR-corrector VX-809 (10 μM), PMCA4 expression was present in control- and CF patient-derived iPSC organoids (Figure 3.B.). Then, Ca2+ removal after ER Ca2+ store depletion resulted in a significantly decreased Ca2+ extrusion in CF organoids compared to control, further recapitulating our previous observation obtained in other model systems (Figure 3.C.). Importantly, pre-treatment with 10 μM VX-809 for 12 h significantly improved Ca2+ extrusion indicating that CFTR corrector treatment can restore decreased PMCA activity and thus the Ca2+ extrusion in CF organoids.
Ethanol reduces CFTR expression and PMCA activity in cholangiocytes
Although the cholangiocyte secretory function greatly depends on CFTR activity 8, alcohol-related changes in CFTR function or expression were never analysed in alcoholic hepatitis (AH). Immunohistochemistry on formalin-fixed paraffin-embedded liver samples revealed that the apical CFTR distribution in cholangiocytes was significantly impaired in patients with AH compared to controls (Figure 4.A.). Next, we recapitulated this phenomenon in vitro in WT mouse-derived liver organoids (MLO) positive for the epithelial cell lineage marker KRT19 (Supplementary Figure 4.C.). CFTR showed a luminal membrane localisation in untreated MLOs, which was significantly decreased and shifted towards the cytosol in EtOH-treated MLOs without biologically relevant changes in Cftr gene expression levels (Figure 4.B.; Supplementary Figure 4.D.). Subsequent functional analysis of MLOs revealed a significantly impaired apical Cl-/HCO3- exchange activity in EtOH/PA-treated MLOs compared to control (Figure 4.C.). Also, whereas extracellular Cl- removal resulted in CFTR-dependent increase in MQAE fluorescence –used as a marker of intracellular Cl- 17– in control MLOs, alcohol treatment resulted in a significant decrease of CFTR-dependent Cl- extrusion (Figure 4.D). Finally, Ca2+ measurements revealed significantly decreased PMCA activity in ethanol pre-incubated- as well as Cftr KO organoids compared to WT control, suggesting that decreased apical distribution of CFTR impairs PMCA function in cholangiocytes (Figure 4.E.). Changes of Pmca4 gene expression didn’t achieve a biologically relevant level in MLOs (Figure 4.F.). Importantly, these results highlight that EtOH exposure alters CFTR localization and activity in cholangiocytes leading to decreased ion secretion and disturbed intracellular Ca2+ homeostasis.
PMCA4 interacts with CFTR at the apical membrane of pancreatic ductal epithelial cells
Our observations suggesting that proper PMCA4 activity requires a close connection with CFTR. Therefore, we performed Duolink proximity ligation assay (PLA) between endogenous PMCA and CFTR. Of note, to avoid non-specific antibody binding, guinea pig pancreatic ductal fragments were used, which recapitulated the colocalization of PMCA4 and CFTR (Supplementary Figure 5.A.). Duolink PLA suggested that PMCA4 and CFTR are in a proximity of <40 nm (Figure 5.A.). Then, we used dSTORM to visualize this interaction at even higher resolution. First, in HeLa cells co-transfected with plasmids encoding CFTR and PMCA4, we observed a perfect overlap (<20 nm) between the two proteins in the plasma membrane suggesting physical proximity (Figure 5.B., Supplementary Figure 5.B.). Next, we established 2D adherent primary human ductal cell culture from pancreatic ductal organoids, which was suitable for dSTORM imaging. In these cells we confirmed overlapping localisation patterns of endogenously expressed CFTR and PMCA4 with confocal microscope (Figure 5.C.) and dSTORM (Figure 5.D., Supplementary Figure 5.C.). Of note, cluster analysis of the dSTORM images revealed a co-localization of 25.24% between all clusters of endogenously expressed CFTR and PMCA4 in 2D human pancreatic ductal cells (Figure 5.E.).
Calmodulin binding by CFTR regulates PMCA4 activity in pancreatic ductal cells and in cholangiocytes
Next, we wanted to provide mechanistic insight into the regulation of PMCA4 activity by CFTR. The recently described alternative calmodulin binding of CFTR has been suggested to allow the regulation of other proteins 12. Thus, we hypothesized that such type of calmodulin-CFTR interaction might subsequently influence the activity of the calmodulin-regulated PMCA4. First, we evidenced strong co-localization of calmodulin with CFTR and PMCA4 at the apical membrane of ductal epithelial cells with dSTORM on cross-sections of MPOs (Supplementary Figure 6.). Next, whereas calmodulin strongly associated with the apical membrane in WT MPOs and MLOs, it dissociated from the apical membrane and diffused throughout the cytosol –as suggested by the line intensity profiles– in EtOH-treated or Cftr KO MPOs and MLOs (Figure 6.A-B.). A similar localisation pattern was observed in Cftr KO ductal fragments (Supplementary Figure 7.). Then, we wanted to analyse the effect of impaired calmodulin-CFTR interaction on PMCA4 activity in epithelial cells. As general knockdown or inhibition of calmodulin can have multiple downstream effects, we co-transfected HEK-293 cells with PMCA4 and CFTR or CFTR harboring a mutation in the calmodulin binding site (CFTR(S768A)). Of note, both CFTR and CFTR(S768A) localised to the plasma membrane and co-localized with PMCA4 (Supplementary Figure 8.A-B.). While co-transfection of PMCA4 and CFTR markedly increased the slope of Ca2+ extrusion, PMCA4 alone showed moderate activity (Figure 6.C.). However, more importantly, cells transfected with CFTR(S768A) showed a significantly impaired PMCA4 activity compared to cells transfected with CFTR. Moreover, dSTORM cluster analysis revealed a 34% reduction of the co-localization ratio between PMCA4-CFTR(S768A) compared to PMCA4-CFTR (Figure 6.D-E., Supplementary Figure 8.C.) suggesting that the lack of calmodulin/CFTR interaction is sufficient to decrease PMCA4 activity as well as the stability of the protein nanodomain on the apical plasma membrane.
Inhibition of PMCA4 impairs mitochondrial function, increases apoptosis, and results in more severe ethanol-induced acute pancreatitis
Sustained intracellular Ca2+ elevation is known to impair mitochondrial function and trigger apoptosis 28. In the next step we wanted to assess the role of impaired CFTR expression in this phenomenon. Transmission electron microscopy showed no difference in the mitochondrial volume/cell ratio between Cftr KO and WT pancreatic ductal cells (Supplementary figure 9.A.). Next, administration of 100 µM carbachol resulted in a significant decrease in mitochondrial membrane potential (Δψm) in EtOH/PA pre-treated and Cftr KO–but not in WT– MPOs, suggesting that a sustained intracellular Ca2+ elevation impairs mitochondrial function (Figure 7.A.). To function properly, the ATPase PMCA4 relies on ATP generated by oxidative phosphorylation and glycolysis. As EtOH decreases the mitochondrial ATP production 4, we inhibited the F1F0-ATPase by oligomycin, which had no effects on the PMCA function in ductal cells (Supplementary Figure 9.B.). However, the intracellular distribution of cytochrome c released from the mitochondria –a hallmark of apoptosis– significantly increased in Cftr KO compared to WT pancreatic ductal cells, suggesting that sustained Ca2+ elevation and disturbed mitochondrial function leads to apoptosis (Supplementary Figure 9.C.). Additionally, Cftr KO pancreatic ductal cells had higher cytoplasmic levels of the initiator caspase 9 compared to WT pancreatic ductal cells, further confirming the increased rate of apoptosis (Supplementary Figure 9.D.). Finally, by using the PMCA4 inhibitor aurintricarboxylic acid (ATA) in an alcohol-induced pancreatitis mouse model, we aimed to analyse if impaired PMCA4 function could independently enhance the severity of pancreatic and liver diseases. Incubation of pancreatic ductal organoids with 10 µM ATA for 30 min before in vitro Ca2+ measurements resulted in significantly decreased PMCA4 activity compared to controls, confirming the inhibitory effect of ATA on PMCA4 function (Figure 7.B.). Next, a single injection of ATA (intraperitoneally, 5 mg/kg) was administered to WT FVB/N mice 90 min before the first EtOH/POA injection 23. Compared to vehicle control, ATA pre-treated animals had significantly elevated pancreatic oedema and necrosis scores paralleled with significantly elevated serum amylase activities (Figure 7.C.). Taken together, these results indicate that impaired PMCA4 activity diminish mitochondrial function, augments apoptosis, and potentially increases the severity of CFTR-related pancreatic- and presumably liver diseases.