Inflammatory regulation of the metalloproteinase ADAM17 by the pseudoprotease iRhom2 in epithelial cells

Giese, Anja Adelina; Ludwig, Andreas (Thesis advisor); van Dongen, Joost Thomas (Thesis advisor); Fabry, Martha Elisabeth (Thesis advisor)

Aachen : RWTH Aachen University (2022, 2023)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2022


Initiation and progression of chronic intestinal diseases are significantly influenced by the a disintegrin and metalloproteinase 17 (ADAM17). By shedding membrane-bound substrates, this protease is able to regulate inflammatory as well as regenerative processes. Furthermore, it influences the paracellular permeability of the epithelial intestinal barrier. The activation and localization of ADAM17 to the plasma membrane (PM) is regulated by the pseudoproteases inactive Rhomboid 1 (iRhom1) and inactive Rhomboid 2 (iRhom2). These adapter proteins bind to the proform of ADAM17 in the endoplasmic reticulum, assist in its transport during maturation through the Golgi apparatus, and ultimately in the incorporation of the active form of ADAM17 into the plasma membrane. According to the literature, iRhom1 is ubiquitously expressed and iRhom2 is mainly expressed in immune cells. In this dissertation, transcriptome analysis demonstrated that iRhom2 mRNA is also detectable in healthy human colon tissue. The analysis also revealed induced expression of iRhom2 in epithelial cells of acutely inflamed tissues from patients with inflammatory bowel diseases (IBDs). This regulation of expression could be reproduced in vivo in a mouse model, as well as in vitro in different human cell lines, demonstrating that this is a general and not a cell-specific mechanism. Next, iRhom2 was shown to be basally expressed and synergistically induced by interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) in the human epithelial colon carcinoma cell line HT-29. Here, it could be shown that the induction of iRhom2 transcription is a strictly time-limited process with a maximum at 6 h. By defining a potential promoter region, 3 signal transduction pathways involved in the induction of iRhom2 could be identified. Pharmacological inhibition of key proteins of the respective pathways (Janus kinases 1/2 (JAK1/2), nuclear factor-kappa B (NF-κB), or activator protein 1 (AP-1)) confirmed the influence of each signal transduction pathway. Furthermore, a luciferase assay demonstrated that after co-stimulation with IFNγ and TNFα transcription is initiated starting from the defined promoter region. An ADAM17 activity assay was used to show the direct dependence of this metalloprotease on iRhom2. It was demonstrated that inhibition of iRhom2 by blocking a signal transduction pathway via JAK1/2, NF-κB, or AP-1 led to reduced shedding of transforming growth factor alpha (TGFα). Thus, it is clear that the inflammatory activity of ADAM17 is directly dependent on iRhom2 expression. To consolidate this finding, it was investigated whether inflammatory ADAM17 activity can be induced via overexpression of iRhoms. This was found not to be possible with short-term overexpression of iRhom2. The overexpressed iRhom2 protein had only low stability as there was no concomitant increase in ADAM17, its stabilizing factor. In contrast, stable overexpression of iRhom2 resulted in accumulation of active ADAM17 on the cell surface over time. Basal increased shedding of TGFα and junctional adhesion molecule-A (JAM-A) was detected, which could be further enhanced by co-stimulation with IFNγ and TNFα. In addition, it was shown that induction of iRhom2 tended to reduce iRhom1, suggesting a compensatory mechanism. In the last section, the influence of missing iRhoms was investigated. Here, it was shown that a knockdown of iRhom1, iRhom2, or both iRhoms had drastic consequences for the epithelial cell lines examined. Knockdown resulted in severe loss of viability and death of HT-29 cells and the human epithelial lung carcinoma cell line A549. This result underscores the findings of other groups that iRhom1 and iRhom2 are vital proteins for epithelial cancer cellsThese findings suggest that targeted blockade of iRhom2 in inflammatory and/or malignant bowel diseases may be an approach for future treatments.