1. Cell Biology

TRPM5-mediated calcium uptake regulates mucin secretion from human colon goblet cells

  1. Sandra Mitrovic
  2. Cristina Nogueira
  3. Gerard Cantero-Recasens
  4. Kerstin Kiefer
  5. José M Fernández-Fernández
  6. Jean-François Popoff
  7. Laetitia Casano
  8. Frederic A Bard
  9. Raul Gomez
  10. Miguel A Valverde
  11. Vivek Malhotra  Is a corresponding author
  1. Centre for Genomic Regulation, Spain
  2. Universitat Pompeu Fabra, Spain
  3. Catalan Institution for Research and Advanced Studies (ICREA), Spain
https://doi.org/10.7554/eLife.00658
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Cite this article
  1. Sandra Mitrovic
  2. Cristina Nogueira
  3. Gerard Cantero-Recasens
  4. Kerstin Kiefer
  5. José M Fernández-Fernández
  6. Jean-François Popoff
  7. Laetitia Casano
  8. Frederic A Bard
  9. Raul Gomez
  10. Miguel A Valverde
  11. Vivek Malhotra
(2013)
TRPM5-mediated calcium uptake regulates mucin secretion from human colon goblet cells
eLife 2:e00658.
https://doi.org/10.7554/eLife.00658
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9 figures, 3 tables and 1 additional file

Figures

Figure 1
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Mucin synthesis and secretion from goblet cells.

(A) Nonstarved and starved N2 cells were fixed and analyzed by immunofluorescence microscopy with an anti-MUC5AC antibody (green). The nuclear DNA was stained with DAPI (blue) to localize the …

https://doi.org/10.7554/eLife.00658.003
Figure 2 with 1 supplement
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Mucin secretion assay.

(A) Illustration of the mucin secretion assay. Starved N2 cells are treated with PMA. Secreted MUC5AC is fixed on the cell surface and labeled with anti-MUC5AC antibodies followed by quantitative …

https://doi.org/10.7554/eLife.00658.004
Figure 2—figure supplement 1
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Time course for MUC5AC secretion.

Starved N2 cells were treated for the indicated times with 2 μM PMA, fixed with formaldehyde and the amount of secreted MUC5AC bound to the cell surface was detected with anti-MUC5AC antibody and …

https://doi.org/10.7554/eLife.00658.005
Figure 3 with 1 supplement
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Identification of proteins required for MUC5AC secretion.

(A) Screening procedure. N2 cells were serum starved for 6 days. The cells were then seeded into the wells of 96-well plates and transfected in triplicates on three sets of plates with siRNAs …

https://doi.org/10.7554/eLife.00658.006
Figure 3—figure supplement 1
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PIMS are not required for constitutive secretion of newly synthesized proteins.

N2 cells were starved for 6 days and transfected with the corresponding siRNA on day 6. The cells were grown for 3 additional days, pulsed with 35S-methionine and chased with cold …

https://doi.org/10.7554/eLife.00658.007
Figure 4
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Expression profile of PIMS.

(A) Total RNA was extracted from nonstarved- and 5-day starved N2 cells. 1 μg of total RNA was used to generate cDNA by reverse transcription. PCR was performed on cDNA with primers specific for the …

https://doi.org/10.7554/eLife.00658.009
Figure 5
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TRPM5 in mucin homeostasis.

(A) Total RNA was extracted from control and TRPM5 stable knockdown (TRPM5 KD) cells and analyzed for knockdown efficiency of TRPM5 on mRNA level by quantitative real-time PCR using primers specific …

https://doi.org/10.7554/eLife.00658.011
Figure 6
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TRPM5 localization and activity.

(A) Nonstarved and starved N2 cells stably transfected with HA-TRPM5 were fixed and analyzed by immunofluorescence microscopy with an anti-HA antibody (green) and an anti-MUC5AC antibody (red). The …

https://doi.org/10.7554/eLife.00658.012
Figure 7 with 1 supplement
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TRPM5 modulates ATP-induced MUC5AC secretion.

(A) Control and TRPM5 stable knockdown cells were starved and incubated for 30 min at 37°C with 100 μM ATP. Secreted MUC5AC was collected and processed for dot blot analysis with an anti-MUC5AC …

https://doi.org/10.7554/eLife.00658.013
Figure 7—figure supplement 1
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Ca2+ dependent MUC5AC secretion.

Starved N2 cells were treated for 30 min with the indicated concentrations of thapsigargin and ionomycin. Secreted MUC5AC was collected and processed for dot blot analysis with an anti-MUC5AC …

https://doi.org/10.7554/eLife.00658.014
Figure 8
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TRPM5 modulates ATP-induced Ca2+ entry.

(A) Time course of mean Ca2+ responses (Fura-2 ratio) obtained in starved N2 cells treated with 100 μM ATP in the presence (n = 138) or absence of 1.2 mM Ca2+ (n = 118) in the extracellular …

https://doi.org/10.7554/eLife.00658.015
Figure 9 with 1 supplement
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Effect of inhibiting the NCX on MUC5AC secretion and Ca2+ entry.

(A) Starved N2 cells were preincubated for 15 min with or without KB-R7943 (50 μM) followed by incubation with 100 μM ATP in the presence or absence of KB-R7943. Secreted MUC5AC was analyzed by dot …

https://doi.org/10.7554/eLife.00658.016
Figure 9—figure supplement 1
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Voltage-gated Ca2+ channels are not expressed or functional in N2 cells.

(A) Current traces (bottom) obtained in a starved N2 cell held at −80 mV and pulsed from −60 mV to +60 mV in 5 mV voltage steps (top). Traces are representative of six cells recorded under identical …

https://doi.org/10.7554/eLife.00658.017

Tables

Table 1

Thirty-four proteins involved in MUC5AC secretion

https://doi.org/10.7554/eLife.00658.008
Gene IDSymbolMUC5AC secretionTypeKnown localizationExpression in N2 cells
6833SUR1HyposecretoryTransporterPMDetected
2900GRIK4HyposecretoryIon channelPMDetected
29,850TRPM5HyposecretoryIon channelPMDetected
6326SCN2AHyposecretoryIon channelPMNot detected
6328SCN3AHyposecretoryIon channelPMNot detected
6915TBXA2RHyposecretoryGPCRPMNot detected
6753SSTR3HyposecretoryGPCRPMNot detected
122042RXFP2HyposecretoryGPCRPMNot detected
1394CRHR1HyposecretoryGPCRPMNot detected
10,803CCR9HyposecretoryGPCRPMDetected
524CX3CR1HyposecretoryGPCRPMNot detected
8484GALR3HyposecretoryGPCRPMNot detected
83,550GPR101HyposecretoryGPCRPMNot detected
118442GPR62HyposecretoryGPCRPMDetected
4914NTRK1HyposecretoryTM receptorPMNot detected
9437NCR1HyposecretoryTM receptorPMNot detected
55,824PAG1HyposecretoryOtherPMDetected
259215LY6G6FHyposecretoryTM receptorPMNot detected
56,140PCDHA8HyposecretoryCadherinPMNot detected
225689MAPK15HyposecretoryKinaseCytosolDetected
23,542MAPK8IP2HyposecretoryOtherCytosolNot detected
10,454TAB1HyposecretoryScaffoldCytosolDetected
6490SILVHyposecretoryMelanosome biogenesisMelanosomeDetected
6494SIPA1HyposecretoryGTPase activatorCytosol, nucleusDetected
30,818KCNIP3HyposecretoryCa2+ bindingPalmitoylated: membrane, cytosolDetected
22,926ATF6HyposecretoryTranscriptionERDetected
26,499PLEK2HyposecretoryActin bindingCytoskeletonDetected
4790NFKB1HyposecretoryTranscriptionNucleus, cytosolDetected
6720SREBF1HyposecretoryTranscriptionERDetected
1238CCBP2HypersecretoryGPCRPMDetected
84,033OBSCNHypersecretoryKinaseCytosolNT
9373PLAAHypersecretoryOtherCytosolNT
4843NOS2HypersecretoryOtherCytosolNT
4893NRASHypersecretoryG-proteinCytosolNT

  1. ER: endoplasmic reticulum; GPCR: G protein coupled receptor; PM: plasma membrane; TM receptor: transmembrane receptor; NT: not tested.

Table 2

Identification of proteins involved in MUC5AC secretion (PIMS)

https://doi.org/10.7554/eLife.00658.010
GenePIMSMUC5AC secretionTypeKnown localizationExpression in starved N2 cellsDisease
SUR11HyposecretoryTransporterPMUnchangedDiabetes mellitus, insulin secretion (Aittoniemi et al., 2009)
GRIK42HyposecretoryIon channelPMUpregulated
TRPM53HyposecretoryIon channelPMUnchangedInsulin secretion (Colsoul et al., 2010)
KCNIP34HyposecretoryCa2+ bindingPalmitoylated membrane, cytosolUpregulated
SIPA15HyposecretoryGTPase activatorCytosol, nucleusUnchanged
PLEK26HyposecretoryActin bindingCytoskeletonUnchanged
TAB17HyposecretoryScaffoldCytosolUnchanged
MAPK158HyposecretoryKinaseCytosolUpregulated
SILV9HyposecretoryMelanosome biogenesisMelanosomeUnchanged
SREBF110HyposecretoryTranscription regulatorERUnchanged
ATF611HyposecretoryTranscription regulatorERUnchanged
NFKB112HyposecretoryTranscription regulatorCytosol, nucleusUnchangedMUC5AC biosynthesis (Fujisawa et al., 2009); asthma (Hart et al., 1998); COPD (di Stefano et al., 2002)
PAG113HyposecretoryOtherPMUnchanged
CCR914HyposecretoryGPCRPMUpregulatedInflammatory bowel disease (Nishimura et al., 2009)
GPR6215HyposecretoryGPCRPMUnchanged
CCBP216HypersecretoryGPCRPMUnchanged

  1. ER: endoplasmic reticulum; GPCR: G protein-coupled receptor; PM: plasma membrane; TM receptor: transmembrane receptor.

Table 3

Primer sequences used for detecting mRNA for the respective PIMS in N2 cells

https://doi.org/10.7554/eLife.00658.018
GeneForward primer 5′–3′Reverse primer 5′–3′
TRPM5GTGGCCATCTTCCTGTTCATCTTCATCATGCGCTCTACCA
CCR9GCCAGCCTTGGCCCTGTTGTTCCAGCAAGGCCTGCGCTTC
PLEK2AGAACAGGCCAGTGGGTGGGTGCTCGCTCAGCCTTGCTGCT
TAB1TCAATCATCGCAGCAATCTCGGCTACACGGACATTGACCT
KCNIP3CCACCACCTATGCACACTTCCGTCGTAGAGATTAAAGGCCCAC
SILVGGGCTACAAAAGTACCCAGAAACCCTTGAGGGACACTTGACCAC
SIPA1CTCCTTTCTGCCACGTACCTTTTTTGGAGTTCCCTTAGGGTCT
HPRT1TGACACTGGCAAAACAATGCAGGTCCTTTTCACCAGCAAGCT
MUC5ACCAACCCCTCCTACTGCTACGCTGGTGCTGAAGAGGGTCAT
GPR62GGTGGTTTCCGTGGGGGCTCTGGGCCCAGACCGCAGGATT
PAG1TGGACGGCAGCCATGCATCCACTGTTGGTGTGGGCAGCGG
ATF6AGGTGGGTAGCGGTTGGGAGGGCGGCACCTTACAGGCACCC
SREBF1CCACGGCAGCCCCTGTAACGGGGACTGAGACCTGCCGCCT
MAPK15TACAACAGGTCCCTCCCCGGCCCCAGTGCCGAGTGGCAGAC
SUR1GCCTTCGCAGACCGCACTGTCTGCACGGACGAAGGAGGCG
NFKB1CGCCACCCGGCTTCAGAATGGGTATGGGCCATCTGCTGTTGGCA
CCBP2CGGCGGGCATGGGACCATTTAAGGCCACCACCAAGGCTGC
GRIK4CGTGGCTCGTGATGGTCGCCGCCTCTCAGGAGCGCGGTTG
GAPDHTGCACCACCAACTGCTTAGCGGCATGGACTGTGGTCATGAG

Additional files

Supplementary file 1

List of positive hits from primary screen.

Lists positive hits from the primary screen, their reason for exclusion, the B-score, the ranking product and the E-value.

https://doi.org/10.7554/eLife.00658.019

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