Abstract
The endoplasmic reticulum (ER) is a crucial organelle found in every eukaryotic cell. It is primarily responsible for protein synthesis, folding, and post-translational modification. These interconnected processes ensure the correct folding of newly-synthesised polypeptides, the secretion of native proteins, and the degradation of misfolded proteins, thus maintaining ER homeostasis. ER stress arises when cells are subjected to physiological or pathological stress, leading to an increased demand for protein production. When this demand exceeds the cell's capacity, improperly folded or unfolded proteins accumulate within the ER lumen. This leads to activation of a sophisticated compensatory system known as the unfolded protein response (UPR) which is mainly mediated by three main UPR sensor molecules, IRE1, PERK and ATF6.
Oral lichen planus (OLP) is a chronic immune-mediated inflammatory disease, often presenting as whitish lace-like lesions on the buccal mucosa bilaterally. Histologically, OLP lesions in the oral mucosa show basal cell lysis, lymphocytic exocytosis, and a prominent subepithelial band-like infiltrate of lymphocytes and macrophages. Since its discovery, OLP has been extensively studied at the cellular and molecular levels. Research has primarily focused on characterising the immune cells and keratinocytes within the OLP microenvironment, while fibroblasts have received comparatively little attention.
Fibroblasts are the primary cells populating the fibrous connective tissue stroma; they exhibit diverse phenotypes with various functional characteristics in different microenvironments, including in a range of disease states. This diversity largely depends on cell-to-cell communication and the surrounding chemical mediators. Recent studies have revealed previously unexplored aspects of fibroblasts, particularly their intricate involvement in the immune system. In the context of OLP, research has shown that diseased fibroblasts behave differently from normal fibroblasts. OLP fibroblasts contribute significantly to the signalling network within the OLP microenvironment, leading to the prolonged retention of lymphocytes within the connective tissue.
The UPR is extensively interwoven with various systemic diseases. The downstream signalling of UPR was found to induce several important inflammatory cytokines that could promote inflammation. In my series of investigations, it was hypothesised that OLP-associated fibroblasts (OLPF) express higher levels of UPR genes and proteins compared with untreated iii controls, and that the UPR genes will be differentially regulated in OLPF compared with the normal fibroblasts (NF).
To test this hypothesis, experimental ER stress was induced using tunicamycin (Tm) at a concentration of 2.5 µg/ml in six different cell lines, three derived from fibroblasts from normal oral mucosa and three from OLPF at 12 hours and 24 hours. The differential expression of 84 key UPR genes in OLPFs and NFs were compared with and without Tm-induced ER stress using quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, we evaluated the expression of key upstream UPR proteins (phospho-IRE1, phospho-PERK, cleaved ATF6), GRP78, and smooth muscle actin-⍺ (SMA-⍺) in OLP-F and NF under the same conditions using immunocytochemistry.
At the gene level, following Tm treatment, there was notable differential expression of UPRrelated genes between OLPF and NF. Firstly, all three main UPR transducer genes were upregulated in both NF and OLPF after Tm treatment. However, at the protein level, there was a trend of reduced expression of phosphorylated IRE1 and PERK in OLPF compared to NF. In contrast, a heightened expression of cleaved ATF6 was observed in OLPF, suggesting it is the most activated protein among the three following ER stress induction. For the first time, we can now report that following Tm treatment, OLPF showed an upregulation of apoptotic genes, including CEBPB, DDIT3 (CHOP), and GADD34, leading to the downstream activation of TRIB3, a gene known to promote apoptosis and inflammation via several pathways. This pattern of gene expression was not observed in Tm-treated NF when compared to the untreated control group. Additionally, protein quality control system-related genes, such as VCP, DERL1, and OS9, were upregulated in Tm-treated NF but not in Tm-treated OLPF when compared to their respective untreated control groups. Other protein-folding genes, whose dysregulation contributes to altered inflammatory network signalling, were downregulated in Tm-treated OLPF.
For the first time, this study has highlighted the significance and influence of ER stress and the UPR in OLPF within the OLP microenvironment. The identified factors should be further studied and validated ex vivo and, eventually, in vivo, given the potential therapeutic opportunity for improving treatment and management of OLP.