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. 2012 Oct 9;109(41):16648-53.
doi: 10.1073/pnas.1204151109. Epub 2012 Sep 24.

ORMDL3 is an inducible lung epithelial gene regulating metalloproteases, chemokines, OAS, and ATF6

Marina Miller  1 , Arvin B Tam, Jae Youn Cho, Taylor A Doherty, Alexa Pham, Naseem Khorram, Peter Rosenthal, James L Mueller, Hal M Hoffman, Maho Suzukawa, Maho Niwa, David H Broide
Affiliations

ORMDL3 is an inducible lung epithelial gene regulating metalloproteases, chemokines, OAS, and ATF6

Marina Miller et al. Proc Natl Acad Sci U S A. .

Abstract

Orosomucoid like 3 (ORMDL3) has been strongly linked with asthma in genetic association studies, but its function in asthma is unknown. We demonstrate that in mice ORMDL3 is an allergen and cytokine (IL-4 or IL-13) inducible endoplasmic reticulum (ER) gene expressed predominantly in airway epithelial cells. Allergen challenge induces a 127-fold increase in ORMDL3 mRNA in bronchial epithelium in WT mice, with lesser 15-fold increases in ORMDL-2 and no changes in ORMDL-1. Studies of STAT-6-deficient mice demonstrated that ORMDL3 mRNA induction highly depends on STAT-6. Transfection of ORMDL3 in human bronchial epithelial cells in vitro induced expression of metalloproteases (MMP-9, ADAM-8), CC chemokines (CCL-20), CXC chemokines (IL-8, CXCL-10, CXCL-11), oligoadenylate synthetases (OAS) genes, and selectively activated activating transcription factor 6 (ATF6), an unfolded protein response (UPR) pathway transcription factor. siRNA knockdown of ATF-6α in lung epithelial cells inhibited expression of SERCA2b, which has been implicated in airway remodeling in asthma. In addition, transfection of ORMDL3 in lung epithelial cells activated ATF6α and induced SERCA2b. These studies provide evidence of the inducible nature of ORMDL3 ER expression in particular in bronchial epithelial cells and suggest an ER UPR pathway through which ORMDL3 may be linked to asthma.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Allergen challenge induces ORMDL3 expression in epithelium in vitro and in vivo. (A) WT mice were challenged with OVA allergen by inhalation. RNA was extracted from lungs. qPCR for ORMDL3 was normalized to the housekeeping gene GAPDH. No OVA vs. OVA (P < 0.05). A549 lung epithelial cells incubated with tobacco smoke media (TS) (B) or mouse macrophage cell line RAW 264.7 (C) incubated with LPS for 3 h. RNA was extracted. qPCR for ORMDL3 was normalized to GAPDH. P < 0.05 for No TS vs. TS and No LPS vs. LPS. (D) WT mice were challenged with Alternaria allergen by inhalation. RNA was extracted from bronchial brushing derived epithelial cells. qPCR for ORMDL-1, ORMDL-2, and ORMDL3 was normalized to the housekeeping gene GAPDH. Alternaria allergen challenge induced a 127-fold increase in levels of expression of ORMDL3 mRNA (P < 0.01 Alt vs. naïve). Levels of ORMDL-1 did not change, whereas levels of ORMDL-2 increased (P < 0.05 Alt vs. naïve), but less than that noted with ORMDL3 (Alt ORMDL3 vs. Alt ORMDL-2; P < 0.05). Results are from three separate experiments with four mice per group. Double-label immunofluorescence microscopy (E–J) and double-label confocal immunofluorescence microscopy (H–J) was performed on human lung epithelial cells (A549) with an anti-ORMDL Ab and an anti-calreticulin Ab (detects ER). (K) Lung sections from non-OVA challenged WT mice have low levels of ORMDL immunostaining. In contrast, lung sections from OVA challenged WT mice (L) have increased ORMDL immunostaining particularly in airway epithelium and in peribronchial mononuclear cells as quantified (M) by light microscopy and image analysis (P < 0.005 vs. no OVA). Lungs from collagen 1 GFP reporter mice (N) have fibroblasts that fluoresce green but do not colocalize with the anti-ORMDL Ab (red). (O) Lungs from α-smooth muscle actin RFP reporter mice have smooth muscle that fluoresce red but does not colocalize with the anti-ORMDL Ab (green; white arrowheads points to green ORMDL+ cell, likely macrophage). RNA was extracted from either (P) BAL macrophages, (Q) bone marrow derived eosinophils, or (R) peripheral blood neutrophils. qPCR for ORMDL1, 2, 3 was normalized to the housekeeping gene GAPDH. ORMDL3 is significantly induced in BAL macrophages (P < 0.05, No Alternaria vs. Alternaria) (P), but not in neutrophils (R). Bone marrow eosinophils (Q) express higher levels of ORMDL3 compared to ORMDL2 or ORMDL1 (P < 0.05). qPCR results are from two separate experiments with four mice per group.
Fig. 2.
Fig. 2.
ORMDL3 transfection into lung epithelium regulates expression of metalloproteases, chemokines, and OAS genes. (A) Lung epithelial cells (A549) transfected with GFP tagged full-length ORMDL3 (green color; blue nuclei). (B–D) qPCR was performed on primary human bronchial epithelial cells transfected with either full-length ORMDL3 or with the empty vector control. The genes that were highly induced by ORMDL3 transfection include metalloproteases (B), chemokines (C), and OAS (D) genes. Immunohistochemistry was used to demonstrate increased MMP-9 (E) and OAS-2 (F) expression in airway epithelium in vivo in WT mice challenged with OVA.
Fig. 3.
Fig. 3.
Activation of ATF6 branch of UPR in lung epithelial cells transfected with ORMDL3. Lung epithelial cells (A549) were transfected with either full-length ORMDL3 or empty vector and treated with 200 nM thapsigargin (Tg), a known activator of the UPR, for 1 h. (A) Immunofluorescence against ATF6 (red) was performed. Active ATF6 is shown by nuclear localization as depicted by colocalization with DAPI (blue). (B) Lung epithelial cells (A549) were cotransfected with a reporter containing an ATF6 binding site, ERSE fused to a luciferase gene, and either empty vector or full-length ORMDL3 and treated with Tg for 12 h. Luciferase activity was then assayed, indicating ATF6 activity (n = 2 experiments). (C) Activation of PERK is assessed by levels of phosphorylated eIF2α on SDS/PAGE by Western blot. Blot with total elF2α showed overall protein level was not changed. Activation of Ire1 was detected by PCR because it removes the UPR intron from the unspliced form of XBP1 (XBP1u) to generate the spliced form of XBP1 (XBP1s) mRNA. (D–F) A549 lung epithelial cells were transfected with ATF6α or control (scrambeled) siRNA. ATF6α siRNA inhibited ATF6α mRNA expression (D) and SERCA2b expression as assessed by qPCR (E), and Western blot (F). (G and H) A549 lung epithelial cells were transfected with ORMDL3 or control empty vector. ORMDL3 transfection induced SERCA2b expression as assessed by qPCR (G) and Western blot (H).
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