Sonic Hedgehog Mediates the Proliferation and Recruitment of Transformed Mesenchymal Stem Cells to the Stomach

Jessica M Donnelly,Jeanmarie Houghton,Yana Zavros,Ambreesh Chawla,Jorge Sans Burns

doi:10.1371/journal.pone.0075225

Abstract

Studies using Helicobacter-infected mice show that bone marrow-derived mesenchymal stem cells (MSCs) can repopulate the gastric epithelium and promote gastric cancer progression. Within the tumor microenvironment of the stomach, pro-inflammatory cytokine interferon-gamma (IFNγ) and Sonic hedgehog (Shh) are elevated. IFNγ is implicated in tumor proliferation via activation of the Shh signaling pathway in various tissues but whether a similar mechanism exists in the stomach is unknown. We tested the hypothesis that IFNγ drives MSC proliferation and recruitment, a response mediated by Shh signaling. The current study uses transplantation of an in vitro transformed mesenchymal stem cell line (stMSCvect), that over-expresses hedgehog signaling, in comparison to non-transformed wild-type MSCs (wtMSCs), wtMSCs transfected to over-express Shh (wtMSCShh), and stMSCs transduced with lentiviral constructs containing shRNA targeting the Shh gene (stMSCShhKO). The effect of IFNγ on MSC proliferation was assessed by cell cycle analysis in vitro using cells treated with recombinant IFNγ (rmIFNγ) alone, or in combination with anti-Shh 5E1 antibody, and in vivo using mice transplanted with MSCs treated with PBS or rmIFNγ. In vitro, IFNγ significantly increased MSC proliferation, a response mediated by Shh that was blocked by 5E1 antibody. The MSC population collected from bone marrow of PBS- or IFNγ-treated mice showed that IFNγ significantly increased the percentage of all MSC cell lines in S phase, with the exception of the stMSCsShhKO cells. While the MSC cell lines with intact Shh expression were recruited to the gastric mucosa in response to IFNγ, stMSCsShhKO were not. Hedgehog signaling is required for MSC proliferation and recruitment to the stomach in response to IFNγ.

Highlights

There is a clearly established role for Sonic Hedgehog (Shh) signaling in the development of cancer [1,2,3]
Using the wild-type MSCs (wtMSCs) and spontaneously transformed mesenchymal stem cell line (stMSC) cell lines both in vivo and in vitro, we demonstrate that IFNc-induced proliferation and recruitment of mesenchymal stem cells (MSCs) cell lines to the stomach is a response that is mediated by Shh secretion and signaling
The role of Hedgehog signaling as a mediator of IFNc-induced stMSC and wtMSC proliferation was identified by flow cytometry and cell cycle progression

Summary

Introduction

There is a clearly established role for Sonic Hedgehog (Shh) signaling in the development of cancer [1,2,3]. Binding of Hedgehog ligand to its receptor, Patched (Ptch), results in removal of the inhibition of Ptch on Smoothened (Smo). This removal of the inhibition on Smo results in the activation of the Gli-family of Hedgehog transcription factors and subsequently tumor growth that can be blocked by Hedgehog neutralizing antibody [1]. While the association between Shh and gastric cancer is clear, the functional role of Shh in the development and progression of gastric cancer is largely unknown. The mechanism that regulates the production of Shh within the tumor microenvironment has yet to be determined

Methods

Results

Conclusion