The Involvement of SMILE/TMTC3 in Endoplasmic Reticulum Stress Response

The state 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.We 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.In 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

Primary cilia of odontoblasts: possible role in molar morphogenesis

International audienceA primary cilium, a sensory organelle present in almost every vertebrate cell, is regularly described in odontoblasts, projecting from the surfaces of the cells. Based on the hypothesis that the primary cilium is crucial both for dentin formation and possibly in tooth pain transmission, we have investigated the expression and localization of the main cilium components and involvement of the OFD1 gene in tooth morphogenesis. Odontoblasts in vitro express tubulin, inversin, rootletin, OFD1, BBS4, BBS6, ALMS1, KIF3A, PC1, and PC2. In vivo, cilia are aligned parallel to the dentin walls, with the top part oriented toward the pulp core. Close relationships between cilium and nerve fibers are evidenced. Calcium channels are concentrated in the vicinity of the basal body. Analysis of these data suggests a putative role of cilia in sensing the microenvironment, probably related to dentin secretion. This hypothesis is enhanced by the huge defects observed on molars from Ofd1 knockout mice, showing undifferentiated dentin-forming cells

Human pulp-derived cells immortalized with Simian Virus 40 T-antigen

Primary cells in culture have a limited capacity to divide and soon reach a non-proliferative state. This cellular senescence limits the investigation of cells derived from human pulp concerning cellular pathways, gene regulation, mechanisms of dentin formation, or responses to material exposure. To overcome this problem, primary human pulp-derived cells were established and transfected with a plasmid containing coding sequences of Simian Virus 40 (SV40) large T-antigen. This resulted in the establishment of several cell clones showing an extension of life span. Expression of T-antigen transcripts and protein was verified by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Primary human pulp cells were cultured until senescence (i.e. up to passage 7) and transfected cells could be cultured to passage 18 after transfection, when a cellular crisis with massive cell death occurred. One clone escaped from crisis and has been maintained in culture for 55 wk. Experiments were performed to characterize transfected cells in comparison to primary cells. Cell morphology and proliferation were analyzed, and expression of cell-specific gene transcripts and proteins (including collagen types I and III, alkaline phosphatase, bone sialoprotein, osteocalcin, and dentin sialophosphoprotein and dentin matrix protein I) was detected by RT-PCR and immunohistochemistry. Transfection of human pulp-derived cells resulted in an immortalized cell line retaining many of the phenotypic characteristics observed in primary cells

Calcium ions promote osteogenic differentiation and mineralization of human dental pulp cells: implications for pulp capping materials

Calcium (Ca) is the main element of most pulp capping materials and plays an essential role in mineralization. Different pulp capping materials can release various concentrations of Ca ions leading to different clinical outcomes. The purpose of this study was to investigate the effects of various concentrations of Ca ions on the growth and osteogenic differentiation of human dental pulp cells (hDPCs). Different concentrations of Ca ions were added to growth culture medium and osteogenic inductive culture medium. A Cell Counting Kit-8 (CCK-8) was used to determine the proliferation of hDPCs in growth culture medium. Osteogenic differentiation and mineralization were measured by alkaline phosphatase (ALP) assay, Alizarin red S/von kossa staining, calcium content quantitative assay. The selected osteogenic differentiation markers were investigated by quantitative real-time polymerase chain reaction (qRT-PCR). Within the range of 1.8–16.2 mM, increased concentrations of Ca ions had no effect on cell proliferation, but led to changes in osteogenic differentiation. It was noted that enhanced mineralized matrix nodule formation was found in higher Ca ions concentrations; however, ALP activity and gene expression were reduced. qRT-PCR results showed a trend towards down-regulated mRNA expression of type I collagen (COL1A2) and Runx2 at elevated concentrations of Ca ions, whereas osteopontin (OPN) and osteocalcin (OCN) mRNA expression was significantly up-regulated. Ca ions content in the culture media can significantly influence the osteogenic properties of hDPCs, indicating the importance of optimizing Ca ions release from dental pulp capping materials in order to achieve desirable clinical outcomes