Wnt5a through Noncanonical Wnt/JNK or Wnt/PKC Signaling Contributes to the Differentiation of Mesenchymal Stem Cells into Type II Alveolar Epithelial Cells In Vitro

Airan Liu,Fengmei Guo,Haibo Qiu,Shuling Hu,Song Chen,Liang Dong,Le Liu,Yi Yang,Xiaomin Lu,Shixia Cai,Qihong Chen,Hongli He,Pranela Rameshwar

doi:10.1371/journal.pone.0090229

Abstract

The differentiation of mesenchymal stem cells (MSCs) into type II alveolar epithelial (AT II) cells is critical for reepithelization and recovery in acute respiratory distress syndrome (ARDS), and Wnt signaling was considered to be the underlying mechanisms. In our previous study, we found that canonical Wnt pathway promoted the differentiation of MSCs into AT II cells, however the role of the noncanonical Wnt pathway in this process is unclear. It was disclosed in this study that noncanonical Wnt signaling in mouse bone marrow–derived MSCs (mMSCs) was activated during the differentiation of mMSCs into AT II cells in a modified co-culture system with murine lung epithelial-12 cells and small airway growth media. The levels of surfactant protein (SP) C, SPB and SPD, the specific markers of AT II cells, increased in mMSCs when Wnt5a was added to activate noncanonical Wnt signaling, while pretreatment with JNK or PKC inhibitors reversed the promotion of Wnt5a. The differentiation rate of mMSCs also depends on their abilities to accumulate and survive in inflammatory tissue. We found that the Wnt5a supplement promoted the vertical and horizontal migration of mMSCs, ameliorated the cell death and the reduction of Bcl-2/Bax induced by H2O2. The effect of Wnt5a on the migration of mMSCs and their survival after H2O2 exposure were partially inhibited with PKC or JNK blockers. In conclusion, Wnt5a through Wnt/JNK signaling alone or both Wnt/JNK and Wnt/PKC signaling promoted the differentiation of mMSCs into AT II cells and the migration of mMSCs; through Wnt/PKC signaling, Wnt5a increased the survival of mMSCs after H2O2 exposure in vitro.

Highlights

Despite extensive studies on the pathophysiology and treatment of acute respiratory distress syndrome (ARDS), the mortality remains at 34–44 percent [1]
We found that the activation of the canonical Wnt pathway promoted the differentiation of mice bone marrow-derived mesenchymal stem cells (MSCs) into AT II cells based on a model for the differentiation of mice bone marrow derived MSCs into AT II cells with indirectly co-cultured with murine lung epithelial (MLE)-12 cells plus small airway growth media (SAGM) [10]
The Protein kinase C (PKC) inhibitor, GF109203X, at 2.5 mmol/L or the JNK blocker, SP600125, at 5 mmol/L inhibited the up-regulation of phosphorylation of PKC and/or JNK caused by the 500 ng/ml Wnt5a incubation. (Figs. 1A, 1B) The regulatory effects of Wnt5a, SP600125 and GF109203X on the noncanonical Wnt pathway were observed in marrow–derived MSCs (mMSCs) differentiated into AT II cells. (Fig. 2) we investigated the effect of Wnt5a on canonical Wnt signaling through the detection of nuclear b-catenin in mMSCs by western blotting, and b-catenin was found to be elevated with the incubation of Wnt5a in mMSCs in differentiation conditions but was unchanged in mMSCs in general culture media. (Figs. 1A, 1B, Fig. 2)

Summary

Introduction

Despite extensive studies on the pathophysiology and treatment of acute respiratory distress syndrome (ARDS), the mortality remains at 34–44 percent [1]. Promoting the regeneration and repair of injured AT II cells is critical for patient recovery from ARDS. It was found that MSCs, which can differentiate into AT II cells in vitro and in vivo, were involved in the repair of the alveolar epithelium in ARDS [7,8,9,10]. Their engraftment and differentiation rates in injured lungs were limited [5], and further exploration is needed to improve these rates. It is necessary to explore the mechanisms underlying the differentiation of MSCs into AT II cells and their migration to injured lung tissue

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