HSPA5, also known as GRP78 or BiP, is a central player in ER homeostasis. Under homeostatic conditions, the luminal domain of the proximal sensors ATF6, http://www.selleckchem.com/products/Abiraterone.html IRE1 and PERK1 interacts with HSPA5, inactivating these signaling pathways. Upon accumulation of unfolded or misfolded proteins, HSPA5 dissociates from these molecules, allowing their activation [29], [30], [31]. The transcriptional activation of the HSPA5 promoter is regarded as a reliable measure of ER stress [33]. Literature reports increased HSPA5 mRNA levels in colonic [34], [27], [35] and ileal [27] samples of involved areas of IBD patients. In line with these data, our results demonstrated significant increased HSPA5 transcript and/or protein levels in involved areas of colonic IBD patients.

In contrast to the study of Kaser, we found no differential expression of HSPA5 between ileal samples of healthy controls and active CD patients [27]. However, we suspect that this could be due to the use of a limited sample size in the study of Kaser, along with an important variability in the expression of ileal HSPA5 protein (as we observed in fig. 3B). Nonetheless, our data reflects well activation of the UPR as not only HSPA5 was modulated, but also other transcript and/or protein levels: PDIA4, XBP1s and pEIF2A. These were found to be increased in colonic IBD, while no differential expression of both transcript and protein levels were observed in ileal CD. In this context, we are confident that our results reflect fairly the situation given the reasonable number of biological replicates, the analysis of multiple UPR-related genes and the correlation between transcript and protein levels in our work.

Our investigation of the various UPR-related molecules at the protein levels correlated relatively well the results obtained by qRT-PCR, which is a technique far more sensitive. But the correlation is not perfect and this could be caused by a combination of factors: restricted biopsy samples in immunoblotting, lower sensitivity of this technique, and induction of ER stress by the biopsy technique itself, as it is reported in other tissues [36]. Nonetheless, we consider that the global picture strengthens the findings made by qRT-PCR. An expanded qPCR analysis of 16 UPR-related genes confirmed that a higher basal UPR activity is in place in the ileal mucosa of healthy controls when compared to the colonic mucosa.

In this analysis, twelve genes (HSPA5, XBP1_U, XBP1_S, PDIA4, HMOX1, GADD34, DDIT3, EIF2A, ATF6, ERO1L, ERN1, ATF4, NQO1, PERK, DNAJC3, and ERDJ4) had significantly higher transcript levels in samples of ileal controls than in colonic controls, clearly showing that the two tissues live with a different Carfilzomib basal activation of the UPR. A growing body of evidence suggests that ER stress and inflammation are interconnected. HSPA5 is a reliable marker for ER stress and IL8 is a marker for inflammation.