Oncostatin M-induced effects on EMT in human proximal tubular cells: differential role of ERK signaling

Abstract

Growing evidence suggests that a proportion of interstitial myofibroblasts detected during renal tubulointerstitial fibrosis originates from tubular epithelial cells by a process called epithelial-mesenchymal transition (EMT). The IL-6-type cytokine oncostatin M (OSM) has been recently implicated in the induction of EMT. We investigated OSM effects on the expression of both cell-cell contact proteins and mesenchymal markers and studied OSM-induced intracellular signaling mechanisms associated with these events in human proximal tubular cells. Human recombinant OSM attenuated the expression of N-cadherin, E-cadherin, and claudin-2 in human kidney-2 (HK-2) cells associated with the induction of HK-2 cell scattering in 3D collagen matrices. Conversely, expression of collagen type I, vimentin, and S100A4 was induced by OSM. OSM-stimulated cell scattering was inhibited by antibodies against gp130. Besides inducing phosphorylation of Stat1 and Stat3, OSM led to a strong concentration-and time-dependent phosphorylation of the mitogen-activated protein kinases ERK1, ERK2, and ERK5. MEK1/2 inhibitor U0126 (10 M) blocked basal and OSM-induced ERK1/2 phosphorylation but not phosphorylation of either ERK5 or Stat1/3. Both synthetic MEK1/2 inhibitors U0126 and Cl-1040, when used at concentrations which inhibit ERK1/2 phosphorylation but not ERK5 phosphorylation, restored N-cadherin expression in the presence of OSM, inhibited basal claudin-2 expression, but did not affect either basal or OSM-inhibited E-cadherin expression or OSM-induced expression of collagen type I and vimentin. These results suggest that in human proximal tubular cells ERK1/2 signaling represents an important component of OSM's inhibitory effect on N-cadherin expression. Furthermore, functional ERK1/2 signaling is necessary for basal claudin-2 expression.

Hypoxia response and VEGF-A expression in human proximal tubular epithelial cells in stable and progressive renal disease

“…Human kidney-2 (HK-2) cells were cultured in keratinocyte-serum free medium containing 10% fetal bovine serum, 5 ng/ml recombinant epidermal growth factor, 0.05 mg/ml bovine pituitary extract, 100 U/ml penicillin, and 100 mg/ml streptomycin. 22 The cells were grown at 5% CO 2 at 371C humidified atmosphere and split in a ratio 1:5 once a week. After growth to subconfluent state, cells were washed once, made quiescent by incubation in serum-and supplement-free medium for 48 h, and then used for the experiments.…”

Neuropilin-1 and neuropilin-2 are differentially expressed in human proteinuric nephropathies and cytokine-stimulated proximal tubular cells

“…[8][9][10] The cells (passages [20][21][22][23][24][25][26][27][28][29][30] were grown at 371C in a humidified 5% CO 2 atmosphere, and split at a 1:10 ratio, once a week. After growth to a subconfluent state, cells were washed once, made quiescent by incubation in serum-and supplementfree medium for 48 h, and then used for experiments.…”

“…10 For the examination of ERK1/2 phosphorylation and ERK2 protein expression, cells were lysed in ice-cold RIPA lysis buffer as described previously. 10 Insoluble material was removed by centrifugation at 12 000 g for 15 min at 41C. The protein concentrations were determined using a microbicinchoninic acid assay (Pierce) or Coomassie protein assay (Pierce) with BSA as a standard.…”

“…[11][12][13][14] Other factors, such as IL-1b, can regulate epithelial-mesenchymal transition (EMT) and fibrosis through modulation of TGF-b1 expression or activity, 15 or in the case of OSM, have potential to act as both a pro-EMT molecule and a pro-MET mediator. 10 As human kidneys with various degrees of tubulointerstitial fibrosis showed robust upregulation of tubular and interstitial NRP2 protein expression, we investigated the effects of pro-fibrotic mediators TGF-b1 and IL-1b when compared with OSM in human PTCs in culture.…”

“…16 Cooperative signaling between TGF-b1 and MEK1/2-ERK1/2 is required for the maintenance of complete EMT in various epithelial cell types, 17,18 and OSM is a strong stimulator of the mitogen-activated protein kinases, ERK1, ERK2, and ERK5, in human PTC. 10 Thus, we investigated the regulatory function of the intracellular signaling molecules ERK1 and ERK2 for NRP1 and NRP2 expression in OSM-when compared with TGF-b1-stimulated human PTC. Preincubation of human PTC with 10 mM of the synthetic MEK1/2 inhibitor U0126 slightly inhibited basal and OSM-induced NRP1 mRNA expression after 24 h (n ¼ 6, Po0.05), but did not affect TGF-b1-and IL-1b-mediated inhibition of NRP1 mRNA expression (Figure 7a).…”