Abstract
BackgroundThe Hedgehog (Hh) signaling pathway regulates a variety of developmental processes, including vasculogenesis, and can also induce the expression of pro-angiogenic factors in fibroblasts postnatally. Misregulation of the Hh pathway has been implicated in a variety of different types of cancer, including pancreatic and small-cell lung cancer. Recently a putative antagonist of the pathway, Hedgehog-interacting protein (HIP), was identified as a Hh binding protein that is also a target of Hh signaling. We sought to clarify possible roles for HIP in angiogenesis and cancer.MethodsInhibition of Hh signaling by HIP was assayed by measuring the induction of Ptc-1 mRNA in TM3 cells treated with conditioned medium containing Sonic hedgehog (Shh). Angiogenesis was assayed in vitro by EC tube formation on Matrigel. Expression of HIP mRNA was assayed in cells and tissues by Q-RT-PCR and Western blot. HIP expression in human tumors or mouse xenograft tumors compared to normal tissues was assayed by Q-RT-PCR or hybridization of RNA probes to a cancer profiling array.ResultsWe show that Hedgehog-interacting protein (HIP) is abundantly expressed in vascular endothelial cells (EC) but at low or undetectable levels in other cell types. Expression of HIP in mouse epithelial cells attenuated their response to Shh, demonstrating that HIP can antagonize Hh signaling when expressed in the responding cell, and supporting the hypothesis that HIP blocks Hh signaling in EC. HIP expression was significantly reduced in tissues undergoing angiogenesis, including PC3 human prostate cancer and A549 human lung cancer xenograft tumors, as well as in EC undergoing tube formation on Matrigel. HIP expression was also decreased in several human tumors of the liver, lung, stomach, colon and rectum when compared to the corresponding normal tissue.ConclusionThese results suggest that reduced expression of HIP, a naturally occurring Hh pathway antagonist, in tumor neo-vasculature may contribute to increased Hh signaling within the tumor and possibly promote angiogenesis.
Highlights
The Hedgehog (Hh) signaling pathway regulates a variety of developmental processes, including vasculogenesis, and can induce the expression of pro-angiogenic factors in fibroblasts postnatally
Hedgehog-interacting protein (HIP) is highly expressed in endothelial cells Ptc-1 and Smo mRNA are expressed in human aortic endothelial cells (HAEC) at levels comparable to that of GAPDH, as determined by comparing the threshold cycle numbers obtained by quantitative RT-PCR (Q-RT-PCR)
When HAEC or human umbilical vein endothelial cells (HUVEC) were treated with conditioned medium containing functional Sonic hedgehog (Shh), the Hh pathway was not activated as determined by measuring the levels of mRNA for Ptc-1 and Gli-1, two genes known to be responsive to Hh signaling
Summary
The Hedgehog (Hh) signaling pathway regulates a variety of developmental processes, including vasculogenesis, and can induce the expression of pro-angiogenic factors in fibroblasts postnatally. All three Hedgehog (Hh) proteins function by binding to the transmembrane receptor, Patched (Ptc-1), leading to the de-repression of the membranebound inhibitor, Smoothened [1,2]. This results in activation of the transcription factor Gli-1, which induces expression of target genes that include Ptc-1 and Gli-1 itself. HIP binds all three Hh proteins with an affinity equal to that of Ptc-1, and in mouse embryos it is expressed in cells adjacent to those expressing Shh, positioning it appropriately for in vivo interactions. It has been shown that overexpression of HIP in cells making Shh reduced the amount of Shh secreted into the media [5], no data has been published demonstrating that expression of HIP in responding cells inhibits the activation of the Shh signaling pathway
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