Thiazolidinediones alter growth and epithelial cell integrity, independent of PPAR-γ and MAPK activation, in mouse M1 cortical collecting duct cells

Abstract

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.