Department of Cellular and Molecular Medicine, and Kidney Research Centre, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada.
Am J Physiol Renal Physiol. 2010 May;298(5):F1105-12. doi: 10.1152/ajprenal.00735.2009. Epub 2010 Feb 17.
Peroxisome proliferator-activated receptor (PPAR)-γ is highly expressed in the collecting duct (CD), yet little is known about the effects of PPAR-γ ligands, thiazolidinediones (TZDs), on CD cell structure and function. M1 mouse cortical CD cells were treated with 5 μM troglitazone (TRO) and rosiglitazone (ROSI). First, growth was measured by [(3)H]thymidine and [(3)H]leucine incorporation, as well as analysis of cyclin D1 and the CDK inhibitor p27 by Western blot. [(3)H]thymidine incorporation was reduced by 56 and 24% by TRO and ROSI at 6 h, and [(3)H]leucine by 21 and 10%. A similar growth inhibition was also observed after 24 h for thymidine, but leucine was reduced by 48 and 24%, respectively. Likewise, cyclin D1 was diminished 60% by TRO, and p27 was elevated 1.6- and 1.7-fold in response to TRO and ROSI. Next, epithelial cell integrity was assessed by measuring different markers by Western blot analysis. While fibronectin and α-smooth muscle actin levels were unchanged, by 24 h E-cadherin was decreased by 50%, and β-catenin levels were reduced 2- and 1.5-fold in response to TRO and ROSI, respectively. GW9662, a PPAR-γ antagonist, did not reverse any of the TZD responses in M1 cells. Of interest, phosho-p38 levels were also elevated 2-fold in response to TRO and 2.3-fold to ROSI, but MAPK inhibition by PD98059 or SB203580 caused an additive inhibition of cell growth and did not alter E-cadherin or β-catenin in response to TZDs. Finally, apoptotic death was assessed by Western blot, but cleaved caspase-3 levels were unchanged from 15 min to 24 h in response to TZDs, and TRO did not affect cell viability or reactive oxygen species generation. Our data suggest that TZDs cause a disruption of M1 cell integrity that is preceded by an inhibition of cell growth. This response is independent of p38 or PPAR-γ activation.
过氧化物酶体增殖物激活受体 (PPAR)-γ 在集合管 (CD) 中高度表达，但对于 PPAR-γ 配体噻唑烷二酮 (TZD) 对 CD 细胞结构和功能的影响知之甚少。用 5 μM 曲格列酮 (TRO) 和罗格列酮 (ROSI) 处理 M1 小鼠皮质 CD 细胞。首先，通过 [(3)H]胸腺嘧啶核苷和 [(3)H]亮氨酸掺入以及 Western blot 分析 cyclin D1 和 CDK 抑制剂 p27 来测量生长。TRO 和 ROSI 在 6 小时时将 [(3)H]胸腺嘧啶核苷掺入减少了 56%和 24%，将 [(3)H]亮氨酸掺入减少了 21%和 10%。24 小时后，胸腺嘧啶核苷的生长抑制也相似，但亮氨酸分别减少了 48%和 24%。同样，TRO 使 cyclin D1 减少了 60%，而 TRO 和 ROSI 分别使 p27 增加了 1.6 倍和 1.7 倍。接下来，通过 Western blot 分析测量不同标记物来评估上皮细胞完整性。虽然纤连蛋白和 α-平滑肌肌动蛋白水平不变，但 E-钙粘蛋白在 24 小时时减少了 50%，而 TRO 和 ROSI 分别使 β-连环蛋白水平降低了 2 倍和 1.5 倍。PPAR-γ 拮抗剂 GW9662 并未逆转 M1 细胞中任何 TZD 反应。有趣的是，TRO 和 ROSI 还使磷酸化 p38 水平增加了 2 倍，但 PD98059 或 SB203580 的 MAPK 抑制导致细胞生长的附加抑制，并且不改变 TZDs 对 E-钙粘蛋白或 β-连环蛋白的影响。最后，通过 Western blot 评估细胞凋亡死亡，但 TZDs 作用 15 分钟至 24 小时后，cleaved caspase-3 水平没有变化，TRO 也不影响细胞活力或活性氧的产生。我们的数据表明，TZD 导致 M1 细胞完整性的破坏，这是细胞生长抑制的前奏。这种反应与 p38 或 PPAR-γ 激活无关。
