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Sonic hedgehog mediates the proliferation and recruitment of transformed mesenchymal stem cells to the stomach.

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 (stMSC(vect)), that over-expresses hedgehog signaling, in comparison to non-transformed wild-type MSCs (wtMSCs), wtMSCs transfected to over-express Shh (wtMSC(Shh)), and stMSCs transduced with lentiviral constructs containing shRNA targeting the Shh gene (stMSC(ShhKO)). 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 stMSCs(ShhKO) cells. While the MSC cell lines with intact Shh expression were recruited to the gastric mucosa in response to IFNγ, stMSCs(ShhKO) were not. Hedgehog signaling is required for MSC proliferation and recruitment to the stomach in response to IFNγ.

SUBMITTER: Donnelly JM

PROVIDER: S-EPMC3777931 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Loss of expression of Sonic Hedgehog (Shh) from parietal cells results in hypergastrinemia in mice, accompanied by increased expression of Indian Hedgehog (Ihh) and hyperproliferation of surface mucous cells. We investigated whether hypergastrinemia induces gastric epithelial proliferation by activating Ihh signaling in mice.We studied mice with parietal cell-specific deletion of Shh (PC-Shh(KO)) and hypergastrinemia, crossed with gastrin-deficient (GKO) mice (PC-Shh(KO)/GKO). When mice were 3-4 months old, gastric tissues were collected and analyzed by histology, for incorporation of bromodeoxyuridine, and for expression of the surface mucous cell marker Ulex europaeus. PC-Shh(KO)/GKO mice were given gastrin infusions for 7 days; gastric surface epithelium was collected and expression of Ihh was quantified by laser capture microdissection followed by quantitative reverse transcriptase polymerase chain reaction. Mouse stomach-derived organoids were incubated with or without inhibitors of WNT (DKK1) or Smoothened (vismodegib) and then cocultured with immortalized stomach mesenchymal cells, to assess proliferative responses to gastrin.Gastric tissues from PC-Shh(KO)/GKO mice with hypergastrinemia had an expanded surface pit epithelium, indicated by a significant increase in numbers of bromodeoxyuridine- and Ulex europaeus-positive cells, but there was no evidence for hyperproliferation. Gastrin infusion of PC PC-Shh(KO)/GKO mice increased expression of Ihh and proliferation within the surface epithelium compared with mice given infusions of saline. In gastric organoids cocultured with immortalized stomach mesenchymal cells, antagonists of WNT and Smoothened inhibited gastrin-induced proliferation and WNT activity. Activity of WNT in media collected from immortalized stomach mesenchymal cells correlated with increased expression of glioma-associated oncogene homolog 1, and was inhibited by DKK1 or vismodegib.Ihh signaling mediates gastrin-induced proliferation of epithelial cells in stomachs of adult mice.

Project description:BackgroundIndian hedgehog (IHH) and Sonic hedgehog (SHH) are important regulators of chondrogenesis. However, activation of IHH and SHH also promotes chondrocyte hypertrophy and ossification during chondrogenesis. The aims of this study were to investigate the effect of microgravity on IHH- and SHH-induced chondrogenic differentiation and to elucidate the role of microgravity in this process.MethodsAdenovirus plasmids encoding the rabbit IHH gene and SHH genes were constructed in vitro and transfected into rabbit bone marrow-derived mesenchymal stem cells (BMSCs). A rotary cell culture system (RCCS), in which a dynamic three-dimensional culture system combines the mechanical environment with a three-dimensional culture surface, was used for cell culture and differentiation. During the induction of differentiation, expression levels of cartilage-related and cartilage hypertrophy-related genes and proteins were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting, respectively. Toluidine blue and collagen II immunohistochemical staining and annexin V-Cy3 staining were used to indicate investigate cartilage matrix synthesis and hypertrophic hypertrophy, respectively, on day 21 after induction of differentiation.ResultsIn this study, IHH and SHH were shown to be equipotent inducers of chondrogenesis in rabbit BMSCs, as evidenced by strong staining for proteoglycans and collagen II, and increased expression of mRNAs and proteins associated with chondrogenesis in an RCCS environment. More importantly, chondrogenic hypertrophy and aging were effectively inhibited in the RCCS environment. In addition, levels of cartilage-related markers in the IHH and SHH transfection groups were initially increased and later decreased in the traditional two-dimensional environment, while cartilage hypertrophy-related factors revealed higher mRNA expression levels during induction.ConclusionsIn summary, microgravity significantly promoted chondrogenic differentiation of BMSCs induced by IHH and SHH and attenuated chondrogenic hypertrophy and aging during chondrogenesis. Furthermore, exogenous IHH and SHH had the same effect on chondrogenic differentiation of BMSCs in the RCCS environment. This study provides further evidence of chondrogenic induction of BMSCs in vitro via IHH and SHH gene delivery.

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Sonic hedgehog mediates the proliferation and recruitment of transformed mesenchymal stem cells to the stomach.

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