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. 2022 Jan 1;322(1):G49-G65.
doi: 10.1152/ajpgi.00246.2021. Epub 2021 Nov 24.

ELAPOR1 is a secretory granule maturation-promoting factor that is lost during paligenosis

Charles J Cho  1 , Dongkook Park  2 , Jason C Mills  1   3   4
Affiliations

ELAPOR1 is a secretory granule maturation-promoting factor that is lost during paligenosis

Charles J Cho et al. Am J Physiol Gastrointest Liver Physiol. .

Abstract

A single transcription factor, MIST1 (BHLHA15), maximizes secretory function in diverse secretory cells (like pancreatic acinar cells) by transcriptionally upregulating genes that elaborate secretory architecture. Here, we show that the scantly studied MIST1 target, ELAPOR1 (endosome/lysosome-associated apoptosis and autophagy regulator 1), is an evolutionarily conserved, novel mannose-6-phosphate receptor (M6PR) domain-containing protein. ELAPOR1 expression was specific to zymogenic cells (ZCs, the MIST1-expressing population in the stomach). ELAPOR1 expression was lost as tissue injury caused ZCs to undergo paligenosis (i.e., to become metaplastic and reenter the cell cycle). In cultured cells, ELAPOR1 trafficked with cis-Golgi resident proteins and with the trans-Golgi and late endosome protein: cation-independent M6PR. Secretory vesicle trafficking was disrupted by expression of ELAPOR1 truncation mutants. Mass spectrometric analysis of co-immunoprecipitated proteins showed ELAPOR1 and CI-M6PR shared many binding partners. However, CI-M6PR and ELAPOR1 must function differently, as CI-M6PR co-immunoprecipitated more lysosomal proteins and was not decreased during paligenosis in vivo. We generated Elapor1-/- mice to determine ELAPOR1 function in vivo. Consistent with in vitro findings, secretory granule maturation was defective in Elapor1-/- ZCs. Our results identify a role for ELAPOR1 in secretory granule maturation and help clarify how a single transcription factor maintains mature exocrine cell architecture in homeostasis and helps dismantle it during paligenosis.NEW & NOTEWORTHY Here, we find the MIST1 (BHLHA15) transcriptional target ELAPOR1 is an evolutionarily conserved, trans-Golgi/late endosome M6PR domain-containing protein that is specific to gastric zymogenic cells and required for normal secretory granule maturation in human cell lines and in mouse stomach.

Keywords: MIST1; trafficking; vesicle; zymogenic cells.

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Conflict of interest statement

No conflicts of interest, financial or otherwise, are declared by the authors.

Figures

None
Graphical abstract
Figure 1.
Figure 1.
ELAPOR1 (endosome/lysosome-associated apoptosis and autophagy regulator 1) is expressed predominantly in zymogenic chief cells (ZC) of the stomach. A: representative immunohistochemistry staining of ELAPOR1 in gastric units in wild-type mouse. Hematoxylin and Eosin counterstain. ELAPOR1 is expressed in cells in the base of the stomach corpus; cells have the morphology of ZCs. Box is shown in higher magnification to the right. Scale bars: 50 μm (left image), 20 μm (right image). B: base and lower neck zone of a gastric unit; anti-ELAPOR1 (green), anti-gastric intrinsic factor (GIF, red), and nuclei [4′,6-diamidino-2-phenylindole (DAPI), blue]. Scale bars 10 µm. C: base and lower neck zone of a different gastric unit; ELAPOR1 (green), GIF (red), and nuclei (DAPI, blue). Scale bars 10 µm. D: base and neck zone of a gastric unit; ELAPOR1 (green), Griffonia simplicifolia-II lectin (GS-II, red; mucous neck cell marker), and nuclei (DAPI, blue); box highlights the transition between neck and base zones. Scale bars 10 µm. E: base and neck zone of a gastric unit; ELAPOR1 (green), GIF (red), GS-II (white), and nuclei (DAPI, blue). Scale bars 10 µm. (White arrowhead: ELAPOR1, GIF, GS-II copositive pre-ZC, or transitional cell.) Scale bars 10 µm.
Figure 2.
Figure 2.
ELAPOR1 (endosome/lysosome-associated apoptosis and autophagy regulator 1) expression decreases along with expression of other secretory granule-associated genes during paligenosis. A: representative Western blots from gastric corpus for indicated proteins and β-actin loading control at the designated timepoints following high-dose tamoxifen induction of paligenosis. B: mRNA expression of indicated genes at designated days following tamoxifen induction of paligenosis (data from previously published microarray, n = 1). C: as for B but qRT-PCR. Significance by one-way ANOVA and post hoc analysis by Tukey’s test. n = 3 mice per timepoint. ***P < 0.001, **P < 0.01; n.s., not significant. CI-M6PR, cation-independent mannose-6-phosphate receptor; GIF, gastric intrinsic factor.
Figure 3.
Figure 3.
ELAPOR1 (endosome/lysosome-associated apoptosis and autophagy regulator 1) traffics with Golgi network and late endosomes. A: staining patterns in the pancreatic acinar AR42J secretory cell line of endogenous ELAPOR1 (green) and DAPI (blue, nuclei) with various subcellular compartment markers: GM130 (red, cis-Golgi), calnexin (red, rough endoplasmic reticulum), and Giantin (red, cis-golgi). Scale bars 10 µm. B: staining patterns in the AGS gastric cell line of DAPI (blue, nuclei) and transfected, full-length ELAPOR1 (green via GFP tag) with various subcellular compartment markers: CI-M6PR (red, trans-Golgi and late endosomes), RAB7 (red, late endosomes and lysosomes), lysotracker (red, acidified, mature lysosomes). Scale bars 10 µm. CI-M6PR, cation-independent mannose-6-phosphate receptor; FL, full length; GFP, green fluorescent protein.
Figure 4.
Figure 4.
Localization of ELAPOR1 (endosome/lysosome-associated apoptosis and autophagy regulator 1) to Golgi network and late endosomes are lost upon truncation. A: truncated (Δ1-4 and Δ5-6) ELAPOR1-GFP mutants transiently transfected into AGS cells were colabeled with CI-M6PR (red, trans-Golgi, late endosomes); DAPI (blue, nuclei). Scale bars 10 µm. B: as for A but colabeled with RAB7 (red, late endosomes and lysosomes). Scale bars 10 µm. C: as for A but with full-length (FL) ELAPOR1 also transfected along with secretory cargo marker PGC-RFP (red) into secretory AR42J cells. Note that PGC localizes to punctate granules when full-length ELAPOR1 is cotransfected but is more diffused with mutants. Scale bars 10 µm. CI-M6PR, cation-independent mannose-6-phosphate receptor; GFP, green fluorescent protein; PGC, pepsinogen C; RFP, red fluorescent protein.
Figure 5.
Figure 5.
Loss of ELAPOR1 (endosome/lysosome-associated apoptosis and autophagy regulator 1) causes defective secretory granules in vivo in zymogenic cells (ZCs). A: Western blot of ELAPOR1 from two wild-type and two Elapor1–/– mice, β-actin as a loading control. B: representative transmission electron microscopy (TEM) images of ZCs in the bases of representative wild-type and Elapor1–/– mice (scale bar, 2 μm). C: relative secretory granule electro-lucency of ZC granules in wild-type vs. Elapor1–/– mice. Significance by Mann–Whitney U test. Granules were scored in multiple thin sections, n = 2 mice per genotype. D: as for C but secretory granule area was calculated. E: representative TEM image of granules in a wild-type and Elapor1−/− mature ZC at the base of a gastric unit. Null ZCs have granules that rarely reach the maximal electro-density of typical wild-type granules, and they have many more electro-lucent granules. Scale bar, 600 nm. F: a gastric unit base in control (Elapor1+/–) or Elapor1–/– mice with anti-GIF (green, ZC secretory cargo), PGC (red, ZC secretory cargo), and DAPI (blue, nuclei). Note intracellular distribution of PGC and GIF is more diffuse in null ZCs. Scale bar, 10 μm. G: Western blot from two wild-type and two Elapor1–/– mice, β-actin as loading control. H: Western blot showing pro- (higher-mass band, >40 kDa) and mature- (lower-mass, <35 kDa) Cathepsin D and BiP from two wild-type and two Elapor1–/– mice, β-actin as loading control. a.u., arbitrary unit; GIF, gastric intrinsic factor; FL, full length; PGC, pepsinogen C.
Figure 6.
Figure 6.
Loss of ELAPOR1 (endosome/lysosome-associated apoptosis and autophagy regulator 1) causes defective secretory granules in vivo in pre-zymogenic/transitional cells. A: representative transmission electron microscopy (TEM) images of pre-ZCs from wild-type and Elapor1–/– mice (scale bar, 2 μm). Note that null mice harbor frequent cells that are clearly of the pre-ZC/transitional phenotype because they reside in the transition zone between neck and base, have discrete and globoid granules (vs. interloculated ones that characterize neck cells), and have extensive basement membrane contact (right, yellow bracket). However, the granules in these cells are uniformly electro-lucent, without the characteristic mottling of wild-type transitional cells (left, arrowhead). B: representative TEM image of granules in pre-ZC/transitional cells from a wild-type and Elapor1−/− mouse. Note the uniform electro-lucency that typifies the abnormal pattern in null mice. Scale bar, 600 nm. C: representative TEM image showing multiple pre-ZCs at various degree of maturation in a neck-base transition zone of a wild-type mouse. White dotted outline depicts the most neck-like or least mature ZC (“pre-ZC”, <25% of granules mottled); cyan outline shows moderately mature pre-ZC (∼50% mottling), yellow outline shows nearly fully mature ZC with >75% mottling. Scale bar, 2 µm. D: representative TEM image of mucous neck cell in wild-type and Elapor1−/− mouse. Cell of interest was outlined with dotted lines and adjacent cells were darkened for better recognition. Neck cells (which do not express MIST1 or ELAPOR1) appear similar in the two genotypes, with globally electro-lucent, loosely interloculated network of granules. Yellow brackets depict basement membrane contact. Scale bar, 2 µm. E: representative TEM image of a pre-ZC in wild-type mouse. Cell of interest was outlined with dotted lines with adjacent cells shaded. Yellow bracket depicts basement membrane contact. Scale bar, 2 µm. F: representative TEM image of pre-ZC in mouse heterozygous for ELAPOR1. Scale bar, 2 µm. G: parietal cells in wild-type (left) and Elapor1−/−(right) mice show no obvious differences. Scale bar, 2 µm. H: endocrine cells (outlined in white) in wild-type (left) and Elapor1−/− (right) mice show no obvious differences. Scale bar, 2 µm; ZC, zymogenic cell.
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