DOP16 Endoplasmic reticulum stress in subepithelial myofibroblasts increases the TGF-β1 activity that regulates fibrosis in Crohn’s disease
Bibliographic record
Abstract
Endoplasmic reticulum (ER) Stress is an essential response of epithelial and immune cells to inflammation in Crohn’s disease. ER stress sensors, GRP-78, ATF-6α, and XBP1, can influence the expression and activity of the pro-fibrotic cytokine TGF-β1 in addition to initiating the unfolded protein response. In addition to the genetically mediated components that lead to fibrosis Crohn’s disease, epigenetic changes also influence the development of fibrosis in susceptible patients. GRP78 is a key factor for two reasons: (1) it has an RGD-binding domain complementary to latent TGF-β1 and (2) GRP78 expression is regulated by miR-199a. Transcription of miR-199a is silenced by DNMT-1 mediated methylation. The presence and regulation of the ER stress in subepithelial myofibroblasts (SEMF), and its role in the development of fibrosis in patients with Crohn’s disease have not been reported yet. SEMF were isolated from the affected ileum and normal ileum of patients with each Montreal phenotype of Crohn’s disease (B1 inflammatory, B2 fibrostenotic, and B3 penetrating) and from non-Crohn’s subjects. Isolated SEMF were used to prepare RNA, cell lysates or initiate primary cell cultures. ER stress was induced by treatment with thapsigargin. Binding of GRP78 to latent TGF-β1 and it subcellular trafficking was examined using proximity ligation–hybridisation assay (PLA). The effects of GRP78, ATF-6α, XBP1 on TGF-β1 were measured using siRNA-mediated knockdown and DNA-ChIP. Transcriptional activity of TGF-β1 was measured by dual luciferase (Firefly–Renilla) assay system after transfection of cells with TGFB1-SBE reporter. Latent-TGF-β1 activation was quantified by ELISA. In SEMF of strictured intestine from patients with B2 Crohn’s disease expression of ER stress sensors GRP78 and ATF-6α increased 3.1 ± 0.2-fold and 2.5 ± 0.1-fold, respectively, compared with normal intestine in the same patient and compared with other Crohn’s phenotypes. ER stress induced by thapsigargin elicited time-dependent and concentration-dependent increase in GRP78 protein levels, direct interaction with latent-TGF-β1, and translocation of the complex to the cell surface where TGF-β1 is activated. The process was abolished after siRNA-mediated knockdown of GRP78. TGF-β1 DNA-binding activity of ATF-6α and XBP1 were similarly increased by 3.2 ± 0.16- and 8.5 ± 0.43-fold, respectively, in SEMF of strictured intestine compared with normal intestine. Latent-TGF-β1 activation was significantly blocked even in the presence of tunicamycin by GRP78 siRNA. Collagen production was further reduced by 2.5 ± 0.2-fold by GRP78 siRNA compared with control. ER stress-mediated pathway presents a novel therapeutic intervention for the patients with fibrostenotic Crohn’s disease.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.005 | 0.002 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".