Garoby-Salom Sandra, Rouahi Myriam, Mucher Elodie, Auge Nathalie, Salvayre Robert, Negre-Salvayre Anne
INSERM UMR-1048, BP 84225, 31432 Toulouse Cedex 4, France; University of Toulouse, Toulouse, France.
INSERM UMR-1048, BP 84225, 31432 Toulouse Cedex 4, France; University of Toulouse, Toulouse, France.
Redox Biol. 2015;4:118-26. doi: 10.1016/j.redox.2014.12.004. Epub 2014 Dec 16.
The neutral type 2 sphingomyelinase (nSMase2) hydrolyzes sphingomyelin and generates ceramide, a major bioactive sphingolipid mediator, involved in growth arrest and apoptosis. The role of nSMase2 in apoptosis is debated, and apparently contradictory results have been observed on fibroblasts isolated from nSMase2-deficient fragilitas ossium (homozygous fro/fro) mice. These mice exhibit a severe neonatal dysplasia, a lack of long bone mineralization and delayed apoptosis patterns of hypertrophic chondrocytes in the growth plate. We hypothesized that apoptosis induced by nutrient deprivation, which mimics the environmental modifications of the growth plate, requires nSMase2 activation. In this study, we have compared the resistance of fro/fro fibroblasts to different death inducers (oxidized LDL, hydrogen peroxide and nutrient starvation). The data show that nSMase2-deficient fro/fro cells resist to apoptosis evoked by nutrient starvation (fetal calf serum/glucose/pyruvate-free DMEM), whereas wt fibroblasts die after 48h incubation in this medium. In contrast, oxidized LDL and hydrogen peroxide are similarly toxic to fro/fro and wt fibroblasts, indicating that nSMase2 is not involved in the mechanism of toxicity evoked by these agents. Interestingly, wt fibroblasts treated with the SMase inhibitor GW4869 were more resistant to starvation-induced apoptosis. The resistance of fro/fro cells to starvation-induced apoptosis is associated with an increased expression of hyaluronan synthase 2 (HAS2) mRNAs and protein, which is inhibited by ceramide. In wt fibroblasts, this HAS2 rise and its protective effect did not occur, but exogenously added HA exhibited a protective effect against starvation-induced apoptosis. The protective mechanism of HAS2 involves an increased expression of the heat-shock protein Hsp72, a chaperone with antiapoptotic activity. Taken together, these results highlight the role of nSMase2 in apoptosis evoked by nutrient starvation that could contribute to the delayed apoptosis of hypertrophic chondrocytes in the growth plate, and emphasize the antiapoptotic properties of HAS2.
中性2型鞘磷脂酶（nSMase2）可水解鞘磷脂并生成神经酰胺，神经酰胺是一种主要的生物活性鞘脂介质，参与生长停滞和细胞凋亡。nSMase2在细胞凋亡中的作用存在争议，在从nSMase2缺陷型骨脆症（纯合子fro/fro）小鼠分离出的成纤维细胞上观察到了明显相互矛盾的结果。这些小鼠表现出严重的新生儿发育不良、长骨矿化缺失以及生长板中肥大软骨细胞凋亡模式延迟。我们推测，模拟生长板环境变化的营养剥夺诱导的细胞凋亡需要nSMase2激活。在本研究中，我们比较了fro/fro成纤维细胞对不同死亡诱导剂（氧化型低密度脂蛋白、过氧化氢和营养饥饿）的抗性。数据表明，缺乏nSMase2的fro/fro细胞对营养饥饿（无胎牛血清/葡萄糖/丙酮酸钠的DMEM）诱发的细胞凋亡具有抗性，而野生型成纤维细胞在此培养基中培养48小时后死亡。相反，氧化型低密度脂蛋白和过氧化氢对fro/fro和野生型成纤维细胞的毒性相似，表明nSMase2不参与这些试剂诱发的毒性机制。有趣的是，用鞘磷脂酶抑制剂GW4869处理的野生型成纤维细胞对饥饿诱导的细胞凋亡更具抗性。fro/fro细胞对饥饿诱导的细胞凋亡的抗性与透明质酸合酶2（HAS2）mRNA和蛋白表达增加有关，而神经酰胺可抑制这种增加。在野生型成纤维细胞中，这种HAS2的升高及其保护作用并未出现，但外源性添加的透明质酸对饥饿诱导的细胞凋亡具有保护作用。HAS2的保护机制涉及热休克蛋白Hsp72表达增加，Hsp72是一种具有抗凋亡活性的伴侣蛋白。综上所述，这些结果突出了nSMase2在营养饥饿诱发细胞凋亡中的作用，这可能导致生长板中肥大软骨细胞凋亡延迟，并强调了HAS2的抗凋亡特性。
