PUMA regulation and proapoptotic effects in fibroblast-like synoviocytes
PUMA regulation and proapoptotic effects in fibroblast-like synoviocytes
复制标题
DOI：
10.1002/art.21631
复制
发表时间：
2006-02-01
影响因子：
--
通讯作者：
Firestein, GS
中科院分区：
文献类型：
--
作者：
Cha, HS;Rosengren, S;Firestein, GS
目标。尽管P53在类风湿关节炎(RA)滑膜组织中过表达，但很少有滑膜细胞发生凋亡。这在一定程度上可能是由于促凋亡基因的低表达。P53上调的细胞凋亡调节因子(PUMA)是P53介导的细胞死亡的主要效应因子，可能是导致这种现象的原因之一。为探讨诱导滑膜细胞凋亡的方法，观察PUMA在ST和培养的成纤维样滑膜细胞(FLS)中的表达及功能。免疫组织化学、Western印迹分析和定量聚合酶链式反应分析检测ST段彪马蛋白的表达。用Ad-P53和编码血凝素标记的全长PUMA表达载体(HA-PUMA)、缺失Bcl2同源3区的PUMA或pCEP4分别转染FLS。用台盼蓝拒染法、DNA片段化和caspase 3活性测定细胞凋亡率。在RA ST中检测到Puma蛋白，但大多数免疫反应蛋白定位于亚衬里细胞，而不是内膜滑膜细胞。Western印迹分析显示RA ST与骨性关节炎(OA)ST无差异。在RA和OAST中检测到Puma信使RNA，但其含量明显低于脾和FLS。为了确定PUMA是否是可诱导的，用Ad-p53转导FLS。即使PUMA蛋白表达增加，PUMA表达也不增强。与此一致的是，Ad-P53不能诱导FLS细胞凋亡。而HA-PUMA基因导入FLS后，细胞迅速发生凋亡，caspase3被激活。PUMA可通过FLS诱导细胞凋亡，是RA治疗的一个潜在靶点。
Objective. Although p53 is overexpressed in rheumatoid arthritis (RA) synovial tissue (ST), few synoviocytes undergo apoptosis. This could be partly due to low expression of proapoptotic genes. Deficient p53 upregulated modulator of apoptosis (PUMA), which is a major effector of p53-mediated cell death, could contribute to this phenomenon. To evaluate a method to induce apoptosis, the expression and function of PUMA was investigated in ST and cultured fibroblast-like synoviocytes (FLS).Methods. PUMA expression in ST was measured by immunohistochemistry, Western blot analysis, and quantitative polymerase chain reaction analysis. Ad-p53 and plasmids encoding hemagglutinin-tagged, full-length PUMA expression vector (HA-PUMA), PUMA lacking the Bcl-2 homology 3 domain, or pCEP4 were used to transfect FLS. Apoptosis was quantified by trypan blue exclusion, DNA fragmentation, and caspase 3 activation.Results. PUMA protein was detected in RA ST, although most of the immunoreactive protein was localized to sublining cells rather than the intimal lining synoviocytes. Western blot analysis showed no difference between RA ST and osteoarthritis (OA) ST. PUMA messenger RNA was detected in RA and OA ST, although the amounts were markedly lower than in the spleen and FLS. To determine if PUMA was inducible, FLS were transduced with Ad-p53. Even though p53 protein was produced and p21 expression was increased, PUMA expression was not enhanced. Consistent with this observation, Ad-p53 did not induce apoptosis in FLS. However, HA-PUMA transfection into FLS resulted in rapid apoptosis with the activation of caspase 3.Conclusion. PUMA can induce apoptosis by FLS and represents a potential target in RA.