Abstract
The endoplasmic reticulum (ER) is an organelle of great importance. It represents the intracellular site for protein synthesis, folding, and post-translational modification. Intricately related processes that oversee the proper folding of newly synthesized polypeptides, secretion of native proteins and degradation of those species that have failed to achieve correct conformations for export, collectively govern the homeostasis of the ER. ER stress reflects the state where there is an imbalance between the influx of polypeptides and the net efflux of proteins from the ER. This may result from an increase in the demand for protein synthesis and resulting cellular indigestion. Accumulation of mutated or unfolded proteins may also cause this phenomenon.
The human unfolded protein response (UPR) is induced by ER stress. The UPR comprises three pathways that initiate mechanisms to overcome the ER stress. Upon detection of stress stimuli, various molecular chaperones and folding enzymes are induced to facilitate and accelerate protein folding. At the same time, global translational attenuation may result in decreasing the protein load into the ER. Some pathways also activate transcriptional factors to up-regulate genes involved in the endoplasmic reticulum quality control (ERQC) and endoplasmic reticulum associated degradation (ERAD) processes. Ultimately, the cell may recover from stress, or apoptosis may be induced where the stress is irrevocable.
Russell bodies (RB) are round eosinophilic intracellular structures that represent distended ER cisternae. They are composed of mutated immunoglobulins, and are commonly observed associated with plasma cells. RBs have been documented in neurodegenerative diseases, chronic inflammatory diseases, as well as in lymphoproliferative conditions. The accumulation of transport-incompetent proteins in the ER has been theorized as the usual cause for this morphological phenomenon. To that end, it had been suggested that RBs originate as a SOS compartment, where abnormal proteins, which cannot be secreted, but have escaped intracellular degradation, can be accumulated without blocking the normal secretory pathway. However, to date, it is not clear precisely which part of the UPR processes are involved in this phenomenon leading to the biogenesis of RB.
In order to elucidate the relationship between inflamed tissues containing RB and ER stress, three hypotheses were addressed in this research; firstly, that ‘RB will be present in association with periodontal inflammation’, secondly that ‘the pattern of GRP-78 expression will differ based on the presence of RB’, and finally, that ‘gene expression patterns of UPR will differ based on the presence of RB’.
To address these hypotheses, chronically inflamed human periodontal tissues were used to identify and quantify RB. RB were identified microscopically and quantified in terms of RB per 1mm2 of a specimen. Histochemical techniques known to stain RB (Haematoxylin and eosin (H&E), periodic-acid Schiff (PAS), and methyl green pyronin (MGP)) were employed for this purpose. The ability of these stains to detect RB, as well as the level of agreement between the stains in RB detection, was statistically analysed. It was found that some, but not all, inflamed periodontal tissues contained RB and that H&E was the most sensitive stain in detecting RB. However, all three stains demonstrated a high level of agreement in RB detection.
In order to examine the GRP-78 expression in inflamed periodontal tissues, the specimens were grouped into three; inflamed tissues containing RB, inflamed tissues without RB and uninflamed control tissues. Immunohistochemical staining characteristics examined included; the dominant positively staining cell type, intensity of staining, site of positivity (inflamed regions and perivascular regions), anatomical level of positivity (lamina propria and/ or submucosa), and the presence of apoptotic and/ or degenerate bodies. The results showed that there was no statistically significant difference in any of the features examined between inflamed tissues, whether or not they contained RB. However, differences were detected between inflamed tissues (both RB-ve and RB+ve) and control tissues.
Cells with histologic morphology consistent with plasma cells represented the dominant positively staining cell type in the inflamed tissues, whereas other GRP-78 positive mononuclear cells predominated in control tissues. Plasma cells in the inflamed tissues also stained more intensely compared to other mononuclear cells in control tissues.
Quantitative real-time reverse transcriptase polymerase chain reaction (qRT2-PCR) was used in examining the UPR gene expression profiles. Tissue groups; inflamed RB+ve, inflamed RB-ve and control tissues were used as per histochemical and immunohistochemical experiments. Fresh samples were obtained from patients undergoing periodontal and oral surgery procedures at the Faculty of Dentistry. The RNA was extracted and purified, then reverse transcribed into cDNA. For gene expression profiling a 96-well human unfolded protein response PCR array was used with SYBR/Rox detection system.
The IRE1 pathway, one of the key upstream components of UPR, was solely up-regulated in association with inflamed tissues which were RB+ve as compared to RB-ve. Furthermore, molecular co-chaperones (DNAJC3, SIL1), as well as those related to ERQC (GANC, UGCGL1) and ERAD (EDEM3) were up-regulated in the inflamed tissue containing RB.
The results of the current research showed for the first time, that RB presentation in inflamed periodontal tissues coincided with the up-regulation of IRE1-induced molecular chaperones, and ERQC and ERAD components. The identification of these genes, in addition to the lack of BIP induction, may be indicative of efforts to restore and maintain protein-processing mechanisms, prior to or concurrently with possible PERK-related apoptosis induced by the accumulation of unfolded or mutated immunoglobulins. It appears that inflamed tissues display this recuperating attempt centred around the CANX cycle, UGCGL1, and EDEM3. Conversely, it may reflect the inability of any of these factors to appropriately target unfolded proteins or that one of these proteins has a yet unknown role in sorting them into RB rather than directing them for ERAD.
With current state of knowledge, the precise mechanisms involved in UPR mechanisms associated with RB formation in inflamed tissues are uncertain. Based on the findings from the current study, we propose that the ERQC site UGCGL1, and ERAD component EDEM3 are strongly related to this process. However, knowledge is rapidly expanding, and the discovery of novel functions and interactions of known proteins identified in our research, will help further our understanding of the relationship between tissue response to RB and ER stress.