Six2 Is a Coordinator of LiCl-Induced Cell Proliferation and Apoptosis.

Jianing Liu,Yajun Xie,Yaoshui Long,Yiman Li,Qianya Wan,Qin Zhou,Xiangling Chen,Zhongshi Lyv,Dongsheng Ni,Quist Kanyomse,Junman Zhang,Hui Zhao,Yuru Zhou,Xihan Liu,Yamin Liu,Ruoli Wang,Pan Ju,Jin Hao,Yang Xiang ,Yue Gu ,Yunfeng Zhao ,Zhizhong Mao ,Гэ Ли

doi:10.3390/ijms17091504

Abstract

The metanephric mesenchyme (MM) cells are a subset of kidney progenitor cells and play an essential role in mesenchymal-epithelial transition (MET), the key step of nephron generation. Six2, a biological marker related to Wnt signaling pathway, promotes the proliferation, inhibits the apoptosis and maintains the un-differentiation of MM cells. Besides, LiCl is an activator of Wnt signaling pathway. However, the role of LiCl in cellular regulation of MM cells remains unclear, and the relationship between LiCl and Six2 in this process is also little known. Here, we performed EdU assay and flow cytometry assay to, respectively, detect the proliferation and apoptosis of MM cells treated with LiCl of increasing dosages. In addition, reverse transcription-PCR (RT-PCR) and Western-blot were conducted to measure the expression of Six2 and some maker genes of Wnt and bone-morphogenetic-protein (BMP) signaling pathway. Furthermore, luciferase assay was also carried out to detect the transcriptional regulation of Six2. Then we found LiCl promoted MM cell proliferation at low-concentration (10, 20, 30, and 40 mM). The expression of Six2 was dose-dependently increased in low-concentration (10, 20, 30, and 40 mM) at both mRNA and protein level. In addition, both of cell proliferation and Six2 expression in MM cells declined when dosage reached high-concentration (50 mM). However, Six2 knock-down converted the proliferation reduction at 50 mM. Furthermore, Six2 deficiency increased the apoptosis of MM cells, compared with negative control cells at relative LiCl concentration. However, the abnormal rise of apoptosis at 30 mM of LiCl concentration implies that it might be the reduction of GSK3β that increased cell apoptosis. Together, these demonstrate that LiCl can induce the proliferation and apoptosis of MM cells coordinating with Six2.

Highlights

During renal development, nephrons originate from a population of self-renewing Sine oculis homeobox homolog 2 (Six2) positive nephron progenitor cells, a part of metanephric mesenchyme (MM) cells [1,2,3]
The results Showed that mK3 cells proliferation rate was increased with concentration rising at low-concentration range (0, 10, 20, 30, and 40 mM) compared control cell, while it was partially reduced at high-concentration (50 mM) compared with the highest proliferation at 30 or 40 mM (Figure 1A,B)
In mK4 cells, cell proliferation rate was increased at low concentration of lithium chloride (LiCl) while the increasing was inhibited at 50 mM (Figure 1C,D)

Summary

Introduction

Nephrons originate from a population of self-renewing Six positive nephron progenitor cells, a part of metanephric mesenchyme (MM) cells [1,2,3]. Six regulates the proliferation (self-renewing) and consumption of nephron progenitor cells (a subset of MM cells) [1,6]. In mouse kidney development, Six deficiency promotes abnormal differentiation of mesenchyme cells and depletion of nephron progenitor cells in the cap mesenchyme (CM), leads to renal hypoplasia [1]. Lithium chloride (LiCl) is a classic activator of Wnt signaling by inhibiting GSK3β expression [11]. This lithium salt of hydrochloric acid is an important therapeutic agent and can regulate proliferation and apoptosis in cancer cells [12]. The relationship between LiCl and Six in the cellular regulation of MM cells is unclear

Methods

Results

Conclusion