The Involvement of SMILE/TMTC3 in Endoplasmic Reticulum Stress Response

Abstract

BackgroundThestate of operational tolerance has been detected sporadically in some renal transplanted patients that stopped immunosuppressive drugs, demonstrating that allograft tolerance might exist in humans. Several years ago, a study by Brouard et al. identified a molecular signature of several genes that were significantly differentially expressed in the blood of such patients compared with patients with other clinical situations. The aim of the present study is to analyze the role of one of these molecules over-expressed in the blood of operationally tolerant patients, SMILE or TMTC3, a protein whose function is still unknown.Methodology/Principal FindingsWe first confirmed that SMILE mRNA is differentially expressed in the blood of operationally tolerant patients with drug-free long term graft function compared to stable and rejecting patients. Using a yeast two-hybrid approach and a colocalization study by confocal microscopy we furthermore report an interaction of SMILE with PDIA3, a molecule resident in the endoplasmic reticulum (ER). In accordance with this observation, SMILE silencing in HeLa cells correlated with the modulation of several transcripts involved in proteolysis and a decrease in proteasome activity. Finally, SMILE silencing increased HeLa cell sensitivity to the proteasome inhibitor Bortezomib, a drug that induces ER stress via protein overload, and increased transcript expression of a stress response protein, XBP-1, in HeLa cells and keratinocytes.Conclusion/SignificanceIn this study we showed that SMILE is involved in the endoplasmic reticulum stress response, by modulating proteasome activity and XBP-1 transcript expression. This function of SMILE may influence immune cell behavior in the context of transplantation, and the analysis of endoplasmic reticulum stress in transplantation may reveal new pathways of regulation in long-term graft acceptance thereby increasing our understanding of tolerance.

SMILE silencing and PMA activation gene networks in hela cells: Comparison with kidney transplantation gene networks

“…Arrestin b2 is known to have a significantly reduced expression in monocytes during kidney graft rejection as it has been recently demonstrated in human [Zakrzewicz et al, 2011] (Table I). But most importantly, the network includes the protein XBP-1 already reported by Racapé et al [2011] to be up-regulated in response to SMILE down-regulation, reinforcing these previous findings (Table I). In competition with the binding of Nephrin to the podocin, Arrestin b2 mediates Nephrin endocythosis and therefore its functioning reduction [Quack et al, 2006].…”

“…1). Using this method, we confirmed that indirect neighbors of TMTC3 are related to ER functioning and stress response genes (CANR, CALX, PDIA4, PDIA2, and ERP27) due to TMTC3 interaction with PDIA3 [Racapé et al, 2011]. The resulting map is shown in Figure 2.…”

“…Gene expression microarray data representing three independent experiments from HeLa cells transfected 24 h with negative control or SMILE siRNA, or activated or not with 20 mM PMA during 6 h as previously described [Racapé et al, 2011] were submitted for our analysis. MIAME microarrays data were deposited in Gene Expression Omnibus Database (GEO record: GSE21886).…”

“…The gene SMILE/TMTC3 was one of the genes found to be upregulated in the blood of operationally tolerant patients and whose function was unknown [Racapé et al, 2011]. SMILE silencing was found to directly increase XBP-1 transcript expression after 6 h of Bortezomib treatment, while DNA microarray analysis revealed that SMILE down-regulation in HeLa cells affects secretory pathways, such as vesicle-mediated transport [Racapé et al, 2011]. We showed that siRNA-mediated SMILE knock-down in HeLa cells induces a decrease in several types of transcripts involved in protein catabolism and proteolysis, particularly immunoproteasome subunits, suggesting that SMILE exerts its function via the proteasome pathway [Racapé et al, 2011].…”

Development of Proteasome Inhibitors as Therapeutic Drugs

“…Proteasome inhibition by bortezomib leads to endoplasmic reticular (ER) stress and induces the pro-apoptotic unfolded protein response. Bortezomib has been shown to induce ER stress via protein overload in vitro in SMILE/ TMTC3 silenced HeLa cells and also increases transcript expression of a stress response protein, XBP-1, in HeLa cells and keratinocytes [34]. Other molecular mechanisms of bortezomib-induced sensitization to apoptosis include the generation of reactive oxygen species (ROS) and the induction of JUN N-terminal kinase [14].…”