You Xin, Boyle David L, Hammaker Deepa, Firestein Gary S
Division of Rheumatology, Allergy and Immunology, University of California at San Diego School of Medicine, 9500 Gilman Drive, La Jolla, California 92093, USA.
Arthritis Res Ther. 2006;8(6):R157. doi: 10.1186/ar2052.
PUMA (p53-upregulated modulator of apoptosis) is a pro-apoptotic gene that can induce rapid cell death through a p53-dependent mechanism. However, the efficacy of PUMA gene therapy to induce synovial apoptosis in rheumatoid arthritis might have limited efficacy if p53 expression or function is deficient. To evaluate this issue, studies were performed to determine whether p53 is required for PUMA-mediated apoptosis in fibroblast-like synoviocytes (FLS). p53 protein was depleted or inhibited in human FLS by using p53 siRNA or a dominant-negative p53 protein. Wild-type and p53-/- murine FLS were also examined to evaluate whether p53 is required. p53-deficient or control FLS were transfected with PUMA cDNA or empty vector. p53 and p21 expression were then determined by Western blot analysis. Apoptosis was assayed by ELISA to measure histone release and caspase-3 activation, or by trypan blue dye exclusion to measure cell viability. Initial studies showed that p53 siRNA decreased p53 expression by more than 98% in human FLS. Loss of p53 increased the growth rate of cells and suppressed p21 expression. However, PUMA still induced apoptosis in control and p53-deficient FLS after PUMA cDNA transfection. Similar results were observed in p53-/- murine FLS or in human FLS transfected with a dominant-negative mutant p53 gene. These data suggest that PUMA-induced apoptosis in FLS does not require p53. Therefore, approaches to gene therapy that involve increasing PUMA expression could be an effective inducer of synoviocyte cell death in rheumatoid arthritis regardless of the p53 status in the synovium.
PUMA（p53上调凋亡调节因子）是一种促凋亡基因，可通过p53依赖性机制诱导细胞快速死亡。然而，如果p53表达或功能存在缺陷，PUMA基因疗法在类风湿性关节炎中诱导滑膜细胞凋亡的疗效可能会受到限制。为了评估这个问题，研究人员进行了一系列实验，以确定在成纤维样滑膜细胞（FLS）中，PUMA介导的细胞凋亡是否需要p53。通过使用p53 siRNA或显性负性p53蛋白，在人FLS中使p53蛋白缺失或受到抑制。同时也检测了野生型和p53基因敲除小鼠的FLS，以评估是否需要p53。将p53缺陷或对照FLS用PUMA cDNA或空载体转染。然后通过蛋白质免疫印迹分析确定p53和p21的表达。通过ELISA检测组蛋白释放和caspase-3激活来测定细胞凋亡，或通过台盼蓝拒染法测定细胞活力。初步研究表明，p53 siRNA可使人类FLS中的p53表达降低98％以上。p53缺失会增加细胞的生长速率并抑制p21的表达。然而，在转染PUMA cDNA后，PUMA仍可在对照和p53缺陷的FLS中诱导细胞凋亡。在p53基因敲除的小鼠FLS或转染显性负性突变p53基因的人FLS中也观察到了类似的结果。这些数据表明，PUMA在FLS中诱导的细胞凋亡不需要p53。因此，无论滑膜中p53的状态如何，增加PUMA表达的基因治疗方法都可能是类风湿性关节炎中滑膜细胞死亡的有效诱导剂。
