Gerritsen M E, Shen C P, Perry C A
Institute for Bone and Joint Disease and Cancer, Bayer Corporation, West Haven, Connecticut, USA.
Am J Pathol. 1998 Feb;152(2):505-12.
The activation of sphingomyelinase and the generation of ceramide has been proposed to mediate tumor necrosis factor-alpha (TNF-alpha)-induced nuclear factor (NF)-kappaB activation through its second messenger ceramide. Ceramide may also be an important regulator of cell growth, senescence, and apoptosis. Aberrant cell proliferation and apoptosis have been implicated in the rampant fibroblast proliferation and pannus formation characteristic of rheumatoid arthritis. However, the role of TNF-alpha and the sphingomyelinase pathway in the process have not been determined. The objective of this study was to determine whether TNF-alpha activates the sphingomyelin pathway in human synovial fibroblasts (HSF) and the potential role of ceramide in HSF proliferation and apoptosis. Cultured human synovial fibroblasts were stimulated with exogenous TNF-alpha, sphingomyelinase, and ceramide. Apoptosis was assessed by cell morphology and annexin V labeling. NF-kappaB and stress kinase pathway activation were determined by immunoblotting techniques. Sphingomyelinase activation was determined by quantitation of sphingomyelin and ceramide radioactivity in [14C]serine-prelabeled HSF cells. The addition of TNF-alpha (50 ng/ml) to HSF did not elicit detectable sphingomyelinase activation. TNF-alpha was shown to activate NF-kappaB (p65 translocation and degradation of IkappaBalpha) and the stress kinase pathway (phosphorylation of ATF-2, p38, and c-jun). In contrast, exogenous ceramide had no effect on these signaling pathways nor did ceramide stimulate the generation of interleukin-6 or interleukin-8. High concentrations of ceramide (> or =25 micromol/L) were cytotoxic, whereas lower concentrations of ceramide inhibited cell cycle progression. Thus, although TNF-alpha stimulates the NF-kappaB and stress kinase pathways in HSF, these effects of TNF-alpha are not associated with sphingomyelinase turnover or induction of apoptosis.
鞘磷脂酶的激活和神经酰胺的生成被认为可通过其二信使神经酰胺介导肿瘤坏死因子-α（TNF-α）诱导的核因子（NF）-κB激活。神经酰胺也可能是细胞生长、衰老和凋亡的重要调节因子。异常的细胞增殖和凋亡与类风湿关节炎特有的成纤维细胞过度增殖和血管翳形成有关。然而，TNF-α和鞘磷脂酶途径在该过程中的作用尚未确定。本研究的目的是确定TNF-α是否激活人滑膜成纤维细胞（HSF）中的鞘磷脂途径，以及神经酰胺在HSF增殖和凋亡中的潜在作用。用外源性TNF-α、鞘磷脂酶和神经酰胺刺激培养的人滑膜成纤维细胞。通过细胞形态学和膜联蛋白V标记评估细胞凋亡。通过免疫印迹技术测定NF-κB和应激激酶途径的激活。通过定量[14C]丝氨酸预标记的HSF细胞中鞘磷脂和神经酰胺的放射性来确定鞘磷脂酶的激活。向HSF中添加TNF-α（50 ng/ml）未引起可检测到的鞘磷脂酶激活。TNF-α被证明可激活NF-κB（p65易位和IκBα降解）和应激激酶途径（ATF-2、p38和c-jun磷酸化）。相比之下，外源性神经酰胺对这些信号通路没有影响，神经酰胺也不刺激白细胞介素-6或白细胞介素-8的产生。高浓度的神经酰胺（≥25 μmol/L）具有细胞毒性，而较低浓度的神经酰胺则抑制细胞周期进程。因此，尽管TNF-α刺激HSF中的NF-κB和应激激酶途径，但TNF-α的这些作用与鞘磷脂酶周转或凋亡诱导无关。
