Research ArticleCELL BIOLOGY

IRE1α kinase–mediated unconventional protein secretion rescues misfolded CFTR and pendrin

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Science Advances  19 Feb 2020:
Vol. 6, no. 8, eaax9914
DOI: 10.1126/sciadv.aax9914
  • Fig. 1 XBP1 splicing and IRE1α endonuclease activity are not involved in UPS of ΔF508-CFTR.

    (A and B) Arf1-Q71L–induced ER-to-Golgi blockade activates ASK1 phosphorylation but not XBP1 splicing. HEK293 cells were transfected with plasmids for Arf1-Q71L (48 hours) or treated with thapsigargin (5 μM, 12 hours) to induce ER stress. Representative immunoblots are shown in (A), and the quantification results of multiple experiments are summarized in (B) (n = 3). The phosphorylation levels of IRE1α and ASK1 were calculated as a ratio of the phospho/total proteins. (C and D) IRE1α, but not XBP1, is required for the Arf1-Q71L–induced UPS of ΔF508-CFTR. Surface biotinylation assays were performed in HEK293 cells transfected with the indicated plasmids and/or small interfering RNAs (siRNAs). Representative immunoblots are shown in (C), and the results of multiple experiments are summarized in (D) (n = 4). Cell surface–specific labeling of proteins was confirmed by the absence of the cytosolic protein aldolase A in the biotinylated fraction. (E and F) Inhibition of IRE1α endonuclease activity by STF-083010 did not block UPS of ΔF508-CFTR. Surface biotinylation assays were performed with STF-083010 treatment (60 μM, 12 hours). Representative results are shown in (E), and the results of multiple experiments are summarized in (F) (n = 4). Bar graph data are shown as the means ± SEM. **P < 0.01; n.s., not significant. Data were analyzed by one-way analysis of variance followed by Tukey’s multiple comparison test.

  • Fig. 2 IRE1α kinase–ASK1 pathway is required for UPS of ΔF508-CFTR.

    (A and B) Effects of IRE1α kinase inhibitor APY29 on the UPS of ΔF508-CFTR. Surface biotinylation assays were performed in HEK293 cells after the induction of ΔF508-CFTR UPS with Arf1-Q71L, followed by the addition of IRE1α kinase inhibitor APY29 (100 μM) or the protein synthesis inhibitor cycloheximide (0.1 mg/ml) for the indicated time periods. Representative immunoblots are shown in (A), and results of multiple experiments are summarized in (B) (n = 5). APY29 reduced surface ΔF508-CFTR in a time-dependent manner without affecting the total protein levels. (C and D) The ASK1 inhibitor MSC2032964A (ASK1-Inh) inhibits Arf1-Q71L–induced UPS of CFTR. Surface biotinylation assays were performed in HEK293 cells transfected with plasmids encoding for WT-CFTR, ΔF508-CFTR, and/or Arf1-Q71L. Some cells were treated with MSC2032964A (10 μM) for the indicated time periods. Representative surface biotinylation results of WT-CFTR and ΔF508-CFTR are presented in (C). The line graph in (D) summarizes the results of multiple experiments (n = 6). The inhibitory effect of MSC2032964A on ASK1 activity was confirmed by decreased ASK1 phosphorylation. b, core-glycosylated CFTR; c, complex-glycosylated CFTR. **P < 0.01, compared to Arf1-Q71L, 0 hours (for both WT-CFTR and ΔF508-CFTRs).

  • Fig. 3 IRE1α kinase activation by CSTMP induces UPS of ΔF508-CFTR.

    (A and B) Overexpression of IRE1α kinase activates UPS of ΔF508-CFTR. HEK293 cells expressing ΔF508-CFTR were cotransfected with plasmids encoding WT or mutant IRE1α (1 μg/ml, 48 hours). Overexpression of WT and RNase-dead (N906A) IRE1α, but not kinase-dead (K599A) IRE1α, activates UPS of ΔF508-CFTR. Representative surface biotinylation assays are presented in (A), and the results of multiple experiments (n = 3) are summarized in (B). (C and D) Effects of the IRE1α kinase activator CSTMP on ASK1 phosphorylation and cell death signals. HEK293 cells were treated with 3 to 100 μM CSTMP for 48 hours. Activation of cell death signals were analyzed by cleavage of PARP and caspase 3 (arrowhead). Representative immunoblots are shown in (C), and the results of multiple experiments are summarized in (D) (n = 5). CSTMP at a concentration of 10 μM activated ASK1 but not cell death signals (red arrow). (E and F) CSTMP induces UPS of ΔF508-CFTR. Surface biotinylation assays were performed in HEK293 cells expressing ΔF508-CFTR. Cells were treated with CSTMP (10 μM) for the indicated time periods. Representative immunoblots are presented in (E), and a summary of multiple experiments is depicted in (F) (n = 3). Cell surface–specific labeling of proteins was confirmed by the presence of the plasma membrane protein Na- and K-dependent ATPase (Na,K-ATPase) and the absence of the cytosolic protein aldolase A in the biotinylated fraction. (G and H) Comparison of surface expression levels of WT-CFTR and the CSTMP-rescued ΔF508-CFTR. HEK293 cells transfected with WT- and ΔF508-CFTR were incubated with or without CSTMP (10 μM) for 24 hours. Representative surface biotinylation results are shown in (G), and the quantification results of multiple experiments (n = 3) are summarized in (H). The cell surface levels of CFTR (biotin) were normalized to its total protein (lysate) levels as detailed in Materials and Methods. Bar and line graphs data are shown as the means ± SEM. **P < 0.01.

  • Fig. 4 Cell surface expression and functional rescue of ΔF508-CFTR by CSTMP.

    (A and B) Immunofluorescence images of ΔF508-CFTR. HeLa cells were transfected with plasmids encoding for extracellular hemagglutinin (HA)–tagged ΔF508-CFTR. Some cells were incubated with CSTMP (10 μM) for 12 hours before fixation. CFTR at the cell surface was immunostained with anti-HA antibodies before membrane permeabilization (green), and the total CFTR was stained with anti-R4 CFTR antibodies after permeabilization (red). Arrowheads indicate surface CFTR. Morphometric quantification of surface CFTR intensity is shown in (B) (n = 5, each from analyses of 20 to 30 cells). Scale bars, 10 μm. **P < 0.01, compared to no treatment (first lane). (C to F) CSTMP induced discernible CFTR currents in cells expressing ΔF508-CFTR. Whole-cell currents were recorded from HEK293 cells transfected with mock or ΔF508-CFTR plasmids for 48 hours. Currents were evoked by applying a ramp pulse from −100 to +100 mV (0.8 mV/ms; holding potential, 0 mV) at 10-s intervals. CFTR Cl currents were activated by cAMP (5 μM forskolin and 100 μM IBMX) and inhibited by CFTRinh-172 (5 μM). A summary of current densities measured at −80 mV is shown in (C) (n = 8 to 11). In (F), cells were treated with CSTMP (10 μM) for 24 hours. Bar graph data are shown as the means ± SEM; **P < 0.01.

  • Fig. 5 CSTMP rescues the cell surface expression of misfolded pendrin.

    (A and B) Effects of CSTMP on the surface expression of p.H723R-pendrin. PANC-1 cells stably expressing p.H723R-pendrin were transfected with or without plasmids encoding for Arf1-Q71L and incubated with CSTMP (10 or 30 μM) for 48 hours. Representative surface biotinylation assays are shown in (A), and the results of multiple experiments are summarized in (B) (n = 5). *P < 0.05 and **P < 0.01, compared to no treatment (first lane). #P < 0.05 and ##P < 0.01, compared to Arf1-Q71L alone (second lane). (C and D) Functional rescue of p.H723R-pendrin by CSTMP. The Clo/HCO3i exchange activity was measured by recording intracellular pH (pHi) as detailed in Materials and Methods. Representative anion exchange measurements are shown in (C), and the quantitation of multiple experiments is depicted in (D) (n = 7 to 8). CSTMP (30 μM) did not affect the activity of WT-pendrin but significantly increased Cl/HCO3 exchange activity in cells expressing p.H723R-pendrin. Bar graph data are shown as the means ± SEM. **P < 0.01, compared to p.H723R-pendrin alone.

  • Fig. 6 In vivo CSTMP treatment rescues ΔF508-CFTR in the CftrF508del mouse colon.

    (A and B) Surface biotinylation assays were performed using epithelial cells harvested from the colonic mucosa. Protein samples from HEK293 cells were used as controls. WT-CFTR (CftrWT) or ΔF508-CFTR (CftrF508del) mice that were 6 weeks old received vehicle or CSTMP (2.59 mg/kg, per os, once daily) for 5 days. Representative surface biotinylation assay results are shown in (A), and the results of multiple experiments are summarized in (B) (n = 4). (C) Immunohistochemistry of CFTR. Transverse and longitudinal cross sections of mouse colonic crypts were immunostained with anti-CFTR R4 rabbit polyclonal antibodies. Arrowheads indicate CFTR expression at the apical membrane of colonic crypts. The CSTMP treatment induced the cell surface expression of CFTR in the CftrF508del mouse colon. Six independent experiments showed similar results. Scale bars, 10 μm. (D and E) Short-circuit current (Isc) measurements in the mouse colon. The apical side of the mouse colon was treated with amiloride (100 μM) to block epithelial Na+ channels. Application of the adenylyl cyclase activator forskolin (10 μM) to the apical side evoked a lumen-negative Isc, which was fully inhibited by the Na+-K+-2Cl cotransporter inhibitor bumetanide (100 μM) delivered to the basolateral side. The CSTMP treatment induced the CFTR-dependent Isc in the CftrF508del mouse colon. Representative traces of Isc measurements are presented in (D), and the results of multiple experiments are summarized in (E) (n = 6 to 9). b, core-glycosylated CFTR; c, complex-glycosylated CFTR. Bar graph data are shown as the means ± SEM. BLM, basolateral membrane; LM, luminal membrane; **P < 0.01.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/8/eaax9914/DC1

    Fig. S1. XBP1 splicing and IRE1α endonuclease activity are not involved in UPS of ΔF508-CFTR (control experiments of Fig. 1).

    Fig. S2. IRE1α kinase–ASK1 pathway is required for UPS of ΔF508-CFTR (control experiments of Fig. 2).

    Fig. S3. Depletion of ASK1 inhibits Arf1-Q71L–induced UPS of CFTR.

    Fig. S4. CSTMP stimulates the cell surface expression of ΔF508-CFTR by activating IRE1α kinase.

    Fig. S5. CSTMP induces the cell surface expression of ΔF508-CFTR via UPS.

    Fig. S6. CSTMP (10 μM) does not evoke apoptotic signal.

    Fig. S7. CSTMP induces the cell surface expression of ΔF508-CFTR without evoking cellular apoptosis.

    Fig. S8. Measurements of CFTR Cl channel activity (control experiments of Fig. 4, C to F).

    Fig. S9. CSTMP induces the cell surface expression of p.H723R-pendrin via UPS.

    Fig. S10. The LD50 value of CSTMP in mice (per os) is 25.9 mg/kg per day.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. XBP1 splicing and IRE1α endonuclease activity are not involved in UPS of ΔF508-CFTR (control experiments of Fig. 1).
    • Fig. S2. IRE1α kinase–ASK1 pathway is required for UPS of ΔF508-CFTR (control experiments of Fig. 2).
    • Fig. S3. Depletion of ASK1 inhibits Arf1-Q71L–induced UPS of CFTR.
    • Fig. S4. CSTMP stimulates the cell surface expression of ΔF508-CFTR by activating IRE1α kinase.
    • Fig. S5. CSTMP induces the cell surface expression of ΔF508-CFTR via UPS.
    • Fig. S6. CSTMP (10 μM) does not evoke apoptotic signal.
    • Fig. S7. CSTMP induces the cell surface expression of ΔF508-CFTR without evoking cellular apoptosis.
    • Fig. S8. Measurements of CFTR Cl channel activity (control experiments of Fig. 4, C to F).
    • Fig. S9. CSTMP induces the cell surface expression of p.H723R-pendrin via UPS.
    • Fig. S10. The LD50 value of CSTMP in mice (per os) is 25.9 mg/kg per day.

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