RESUMO
Ca2+ plays a significant role in linking the induction of apoptosis. The key anti-apoptotic protein, Bcl-2, has been reported to regulate the movement of Ca2+ across the ER membrane, but the exact effect of Bcl-2 on Ca2+ levels remains controversial. Store-operated Ca2+ entry (SOCE), a major mode of Ca2+ uptake in non-excitable cells, is activated by depletion of Ca2+ in the ER. Depletion of Ca2+ in the ER causes translocation of the SOC channel activator, STIM1, to the plasma membrane. Thereafter, STIM1 binds to Orai1 or/and TRPC1 channels, forcing them to open and thereby allow Ca2+ entry. In addition, several anti-cancer drugs have been reported to induce apoptosis of cancer cells via the SOCE pathway. However, the detailed mechanism underlying the regulation of SOCE by Bcl-2 is not well understood. In this study, a three-amino acid mutation within the Bcl-2 BH1 domain was generated to verify the role of Bcl-2 in Ca2+ handling during ER stress. The subcellular localization of the Bcl-2 mutant (mt) is similar to that in the wild-type Bcl-2 (WT) in the ER and mitochondria. We found that mt enhanced thapsigargin and tunicamycin-induced apoptosis through ER stress-mediated apoptosis but not through the death receptor- and mitochondria-dependent apoptosis, while WT prevented thapsigargin- and tunicamycin-induced apoptosis. In addition, mt depleted Ca2+ in the ER lumen and also increased the expression of SOCE-related molecules. Therefore, a massive Ca2+ influx via SOCE contributed to caspase activation and apoptosis. Furthermore, inhibiting SOCE or chelating either extracellular or intracellular Ca2+ inhibited mt-mediated apoptosis. In brief, our results explored the critical role of Bcl-2 in Ca2+ homeostasis and the modulation of ER stress.
RESUMO
OBJECTIVES: To evaluate the collagen cross-linkers, riboflavin-ultraviolet-A (RF/UVA) and glutaraldehyde, with regard to their efficacy in cross-linking the dentinal collagen and improving dentin bonding. METHODS: Glutaraldehyde and different RF/UVA protocols (0.1%RF/1-minUV, 0.1%RF/2-minUV, and 1%RF/1-minUV) were first evaluated by gel electrophoresis to determine their abilities of collagen cross-linking. The mechanical properties of acid-etched dentin receiving these cross-linking treatments were examined in either dry or wet condition by a nanoindentation test. Fifteen teeth with exposed occlusal dentin received the microtensile bond strength (µTBS) test. The teeth were primed either with RF/UVA or glutaraldehyde, followed by adhesive treatment and composite restorations, and then cut into resin-dentin microbeams. Half of the microbeams received the µTBS test after 24h, and the other half received test after 5000 thermocycles. Nanoleakage at the bond interface was examined under TEM. The alignments of collagen fibrils in the hybrid layers were also defined by an image analysis. RESULTS: Gel electrophoresis showed that glutaraldehyde induced strong collagen gelation, while RF/UVA generated milder collagen cross-linking. Glutaraldehyde, 0.1%RF/2-min-UVA, and 1%RF/1-minUV showed higher stiffness compared to untreated and 0.1%RF/1-minUV in wet condition. All the crosslinking treatments improved early µTBS, but 0.1%RF/2-minUVA treatment maintained high µTBS after theromocycles. Under TEM, glutaraldehyde-treated dentin showed dense and enclosed collagen network on the adhesive interface. 0.1%RF/2-minUVA showed the least nanoleakage, and this could be associated with the suspended collagen fibrils in the hybrid layer. SIGNIFICANCE: 0.1%RF/2-minUVA treatment enhanced resin-dentin bond possibly through enhancing the stiffness and maintaining the expanding collagen matrix in the hybrid layer.
