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TMEM16A chloride channel does not drive mucus production

View ORCID ProfileFilipa B Simões, Margarida C Quaresma, Luka A Clarke, View ORCID ProfileIris AL Silva, Ines Pankonien, Violeta Railean, View ORCID ProfileArthur Kmit, View ORCID ProfileMargarida D Amaral  Correspondence email
Filipa B Simões
University of Lisboa, Faculty of Sciences, BioISI–Biosystems & Integrative Sciences Institute, Lisboa, Portugal
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  • ORCID record for Filipa B Simões
Margarida C Quaresma
University of Lisboa, Faculty of Sciences, BioISI–Biosystems & Integrative Sciences Institute, Lisboa, Portugal
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Luka A Clarke
University of Lisboa, Faculty of Sciences, BioISI–Biosystems & Integrative Sciences Institute, Lisboa, Portugal
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Iris AL Silva
University of Lisboa, Faculty of Sciences, BioISI–Biosystems & Integrative Sciences Institute, Lisboa, Portugal
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Ines Pankonien
University of Lisboa, Faculty of Sciences, BioISI–Biosystems & Integrative Sciences Institute, Lisboa, Portugal
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Violeta Railean
University of Lisboa, Faculty of Sciences, BioISI–Biosystems & Integrative Sciences Institute, Lisboa, Portugal
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Arthur Kmit
University of Lisboa, Faculty of Sciences, BioISI–Biosystems & Integrative Sciences Institute, Lisboa, Portugal
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Margarida D Amaral
University of Lisboa, Faculty of Sciences, BioISI–Biosystems & Integrative Sciences Institute, Lisboa, Portugal
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  • ORCID record for Margarida D Amaral
  • For correspondence: mdamaral@fc.ul.pt
Published 15 November 2019. DOI: 10.26508/lsa.201900462
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  • Figure 1.
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    Figure 1. TMEM16A and MUC5AC expression levels are inversely correlated during differentiation of BCi-NS1.1 cells.

    (A) Time course levels of endogenous expression of TMEM16A protein during differentiation of BCi-NS1.1 cells grown at ALI (days 1–30) detected by WB, showing both its non-glycosylated (∼100 kD) and glycosylated forms (∼120 kD). GAPDH (∼36 kD) was used as a loading control. (A, B) Quantification by densitometry of TMEM16A total protein levels from (A) normalized to the loading control shown as mean ± SEM (n = 3). (C) Fold-change in TMEM16A mRNA expression levels as a time course of differentiation of BCi-NS1.1 cells grown at ALI (days 0, 15, and 30), determined by qRT-PCR. Fold-change values are mean ± SEM, relative to the mean value of day 30 (n = 3). (D) Time course levels of endogenous expression of MUC5AC (>300 kD) analysed by WB during differentiation of BCi-NS1.1 cells. Vinculin was used as a loading control (∼120 kD). Dashed line indicates lanes run on the same gel but noncontiguous. (D, E) Quantification by densitometry of MUC5AC protein levels from (D) normalized to the loading control shown as mean ± SEM (n = 4). (F) Fold-change in MUC5AC mRNA expression levels as a time course of differentiation of BCi-NS1.1 cells grown at ALI (days 0, 15, and 30), determined by qRT-PCR. Fold-change values are mean ± SEM, relative to the mean value of day 30 (n = 4). (G) Correlation of TMEM16A and MUC5AC normalized protein levels during differentiation and TEER measurements. Asterisks and cardinals indicate significant difference compared with day 0 (P-value < 0.05, unpaired t test).

    Source data are available for this figure.

    Source Data for Figure 1[LSA-2019-00462_SdataF1.tif][LSA-2019-00462_SdataF1.1.tif][LSA-2019-00462_SdataF1.2.tif][LSA-2019-00462_SdataF1.3.tif]

  • Figure S1.
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    Figure S1. Tight junction formation of BCi-NS1.1 cells and expression of specific cell-type markers for basal, club, and ciliated cells in BCi-NS1.1 cells cultured at ALI.

    (A) Time course values of TEER measurements of BCi-NS1.1 cells cultured at ALI for 30 d. Resistance (Ohm/cm2) is indicated as an average ± SEM of n = 5 independent experiments from day 0 to day 30 of ALI culture. (B) WB showing endogenous expression of different cell type–specific markers during differentiation of BCi-NS1.1 cells—basal cell marker (p63 ∼75 kD), club cell marker (CC16 ∼25 kD), and ciliated cell marker (DNAI1 ∼75 kD). GAPDH (∼36 kD) was used as loading control. (C) Quantification by densitometry of p63, DNAI1, and CC16 WB normalized to the loading control shown as mean ± SEM (n = 3). Asterisks indicate significant difference compared with day 0 (P-value < 0.05, unpaired t test).

    Source data are available for this figure.

    Source Data for Figure S1[LSA-2019-00462_SdataFS1.tif][LSA-2019-00462_SdataFS1.1.tif][LSA-2019-00462_SdataFS1.2.tif][LSA-2019-00462_SdataFS1.3.tif][LSA-2019-00462_SdataFS1.4.tif][LSA-2019-00462_SdataFS1.5.tif]

  • Figure 2.
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    Figure 2. TMEM16A and MUC5AC have distinct spatiotemporal localizations in differentiating BCi-NS1.1 cells.

    (A) Confocal immunofluorescence microscopy images showing TMEM16A localization in permeabilised BCi-NS1.1 cells on days 0 and 30 (upper and lower rows, respectively) of differentiation. Left panels: Nuclei stained with Hoechst stain. Middle panels: endogenous TMEM16A, detected by Alexa Fluor 568 fluorescence. Endogenous ZO-1 was detected by Alexa Fluor 488 fluorescence. Right panels: merged image of the three fluorescent channels: Blue, Alexa 647; Red, Alexa 568; and Green, Alexa 488. Images were acquired with a Leica TCS SP8 confocal microscope (objective 63× oil, NA 1.4). Scale bar = 30 μm. (n = 3). (B) Z-stack of a representative group of cells showing TMEM16A and ZO-1 staining on days 0 and 30 (upper and lower panels, respectively). (C) Confocal immunofluorescence microscopy images showing MUC5AC expression in permeabilised BCi-NS1.1 cells on days 0 and 30 of differentiation (upper and lower rows, respectively). Left panels: nuclei stained with metal green, represented by Alexa Fluor 647 fluorescence. Middle panels: endogenous MUC5AC, detected by Alexa Fluor 488 fluorescence. Right panels: merged image of the two fluorescent channels: Green–Alexa 488; Blue–Hoechst. Images were acquired with a Leica TCS SP8 confocal microscope (objective 63× oil, NA 1.4). Scale bar = 30 μm. (n = 3). (D) Z-stack of a representative group of cells showing MUC5AC stained apically on days 0 and 30 (upper and lower panels, respectively).

  • Figure 3.
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    Figure 3. High TMEM16A expression levels correlate with cell proliferation in BCi-NS1.1 cells after wound healing.

    (A) Time course levels of endogenous expression of Ki-67 protein (∼300 kD) during differentiation of BCi-NS1.1 grown at ALI (days 0-30) analysed by WB. GAPDH (∼36 kD) was used as loading control. (B) Quantification by densitometry of Ki-67 expression detected by WB and normalized to the loading control shown as mean ± SEM (n = 3). (C) Correlation of TMEM16A and Ki-67 normalized protein levels and TEER measurements during differentiation. Summary of TMEM16A total protein levels represents data in Fig 1. (D) Time course (0 h to 5 d) of expression levels of endogenous expression of TMEM16A and Ki-67 during wound closure (0, 8, 24, 48 h, and 5 d after injury) on the 20th day of differentiation at ALI analysed by WB. GAPDH (∼36 kD) was used as loading control. (E) Quantification by densitometry of total TMEM16A and Ki-67 expression detected by WB and normalized to the loading control shown as mean ± SEM (n = 3–5). Asterisks indicate significant difference compared with day 0 (in graph (B)) or before wound (in graph (E)) (P-value < 0.05, unpaired t test).

    Source data are available for this figure.

    Source Data for Figure 3[LSA-2019-00462_SdataF3.tif][LSA-2019-00462_SdataF3.1.tif][LSA-2019-00462_SdataF3.2.tif][LSA-2019-00462_SdataF3.3.tif][LSA-2019-00462_SdataF3.4.tif]

  • Figure 4.
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    Figure 4. TMEM16A and Ki-67 protein expression during differentiation of primary HBE cells.

    (A) WB of endogenous TMEM16A and Ki-67 proteins during differentiation of primary HBE cells. GAPDH was used as loading control. (B) Quantification by densitometry of total TMEM16A and Ki-67 expression detected by WB and normalized to the loading control shown as mean ± SEM (n = 3). (C) WB of endogenous MUC5AC during differentiation of primary HBE cells. Vinculin was used as loading control. (D) Quantification by densitometry of MUC5AC detected by WB and normalized to the loading control shown as mean ± SEM (n = 3). Asterisks indicate significant difference compared with day 0 (P-value < 0.05, unpaired t test).

    Source data are available for this figure.

    Source Data for Figure 4[LSA-2019-00462_SdataF4.tif][LSA-2019-00462_SdataF4.1.tif][LSA-2019-00462_SdataF4.2.tif][LSA-2019-00462_SdataF4.3.tif][LSA-2019-00462_SdataF4.4.tif]

  • Figure 5.
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    Figure 5. TMEM16A and MUC5AC up-regulation induced by IL-4 are associated with cell proliferation.

    (A) WB indicating up-regulation of TMEM16A, MUC5AC, and Ki-67 by stimulation with 5 ng/ml IL-4 for 48 h in BCi-NS1.1 cells. GAPDH was used as a loading control for TMEM16A and Ki-67 WB. Vinculin was used as a loading control for MUC5AC WB. (B) Quantification by densitometry of WB for total TMEM16A, MUC5AC, and Ki-67 expression normalized to the loading control, data shown as mean ± SEM (n = 3). (C) Original Ussing chamber tracings ± IL-4, obtained for ATP-induced Cl− currents (100 μM) in the presence of the epithelial Na+ channel (ENaC) inhibitor, amiloride (30 μM). Reduction of the ATP-activated Cl− currents was observed under the CaCC-AO1 TMEM16A inhibitor (30 μM). (D) Summary of Isc-eq ATP currents in the presence or absence of IL-4. Values are mean ± SEM (n = 3–7). Asterisk indicates significant difference compared with control; cardinal indicates significant difference compared with ATP alone (P-value < 0.05, unpaired t test). (E) WB showing down-regulation of TMEM16A and Ki-67 expression by treatment with the proliferation blocker mitomycin C (1 μg/ml) and IL-4 (5 ng/μl) for 48 h. Na+/K+ ATPase (∼100 kD)—another protein expressed in the membrane—was detected as a control. GAPDH was used as loading control. Dashed line indicates lanes run on the same gel but noncontiguous. (F) Quantification by densitometry of total TMEM16A, Ki-67, and Na+/K+ ATPase expression detected by WB and normalized to the loading control, shown as mean ± SEM (n = 5–7). (G) WB showing MUC5AC levels upon treatment with mitomycin C (1 μg/ml) ± IL-4 (5 ng/ml) for 48 h. Vinculin was used as loading control. Dashed line indicates lanes run on the same gel but noncontiguous. (H) Quantification by densitometry of MUC5AC detected by WB and normalized to the loading control, shown as mean ± SEM (n = 4–5). Asterisks indicate significant difference compared with control; cardinals indicate significant difference compared with treatment with IL-4 (P-value < 0.05, unpaired t test).

    Source data are available for this figure.

    Source Data for Figure 5[LSA-2019-00462_SdataF5.tif][LSA-2019-00462_SdataF5.1.tif][LSA-2019-00462_SdataF5.2.tif][LSA-2019-00462_SdataF5.3.tif][LSA-2019-00462_SdataF5.4.tif][LSA-2019-00462_SdataF5.5.tif][LSA-2019-00462_SdataF5.6.tif][LSA-2019-00462_SdataF5.7.tif][LSA-2019-00462_SdataF5.8.tif][LSA-2019-00462_SdataF5.9.tif][LSA-2019-00462_SdataF5.10.tif][LSA-2019-00462_SdataF5.11.tif]

  • Figure S2.
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    Figure S2. Original Ussing chamber tracing from differentiated control cells (ALI day 30) obtained for ATP-induced Cl− currents (100 μM) in the presence of the epithelial Na+ channel (ENaC) inhibitor, amiloride (30 μM) ± CaCC-AO1 TMEM16A inhibitor (30 μM).
  • Figure 6.
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    Figure 6. TMEM16A is not essential for mucus production in BCi-NS1.1 cells.

    (A) WB of endogenous TMEM16A and Ki-67 expression in the presence or absence of 400 ng/μl DLL4 for 30 d. Samples from undifferentiated cells were used as a positive control for TMEM16A and Ki-67 expression. GAPDH was used as loading control. (B) Quantification by densitometry of total TMEM16A and Ki-67 expression detected by WB and normalized to the loading control shown as mean ± SEM (n = 3). (C) WB of endogenous MUC5AC in the presence or absence of 400 ng/μl DLL4. Vinculin was used as a loading control. (D) Quantification by densitometry of MUC5AC expression detected by WB and normalized to the loading control shown mean ± SEM (n = 3). Asterisks indicate significant difference compared with control (P-value < 0.05, unpaired t test).

    Source data are available for this figure.

    Source Data for Figure 6[LSA-2019-00462_SdataF6.tif][LSA-2019-00462_SdataF6.1.tif][LSA-2019-00462_SdataF6.2.tif][LSA-2019-00462_SdataF6.3.tif][LSA-2019-00462_SdataF6.4.tif]

  • Figure 7.
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    Figure 7. TMEM16A contributes for fluid secretion in differentiated BCi-NS1.1 cells.

    (A) Representative confocal images of ASL labelled with FITC-Dextran obtained 0.5 and 4 h after apical exposure to DMSO or 10 μm Ani9. (B) Quantification of overtime (0.5, 1, 2, 3, and 4 h) ASL height measurements shown as mean ± SEM (n = 7–9). Asterisks indicate significant difference compared with 0.5 h (P-value < 0.05, unpaired t test).

  • Figure 8.
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    Figure 8. Relationship between TMEM16A, MUC5AC, and Ki-67 during differentiation and GCH.

    (A) Undifferentiated basal cells are proliferating and, thus, have high levels of TMEM16A and Ki-67. On the contrary, because of the absence of goblet cells, MUC5AC is not expressed. (B) When cells are differentiated, proliferation stops (low TMEM16A and Ki-67) and mucus production is high, mostly secreted from goblet cells. (C) Induction of GCH by pro-inflammatory cytokines such as IL-4/13 switches the human airway epithelium from a non-proliferative to a proliferative state, inducing TMEM16A and Ki-67 expression. As a consequence of inflammation, basal cells proliferate and differentiate into mucus-producing cells, increasing MUC5AC levels.

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TMEM16A and mucus production
Filipa B Simões, Margarida C Quaresma, Luka A Clarke, Iris AL Silva, Ines Pankonien, Violeta Railean, Arthur Kmit, Margarida D Amaral
Life Science Alliance Nov 2019, 2 (6) e201900462; DOI: 10.26508/lsa.201900462

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TMEM16A and mucus production
Filipa B Simões, Margarida C Quaresma, Luka A Clarke, Iris AL Silva, Ines Pankonien, Violeta Railean, Arthur Kmit, Margarida D Amaral
Life Science Alliance Nov 2019, 2 (6) e201900462; DOI: 10.26508/lsa.201900462
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Volume 2, No. 6
December 2019
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