Hedgehog-interacting protein is highly expressed in endothelial cells but down-regulated during angiogenesis and in several human tumors.
BACKGROUND
The 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.


METHODS
Inhibition 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.


RESULTS
We 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.


CONCLUSIONS
These 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.
Department of Gene Therapy, Berlex Laboratories, Inc, Richmond, CA 94806, USA. catherine_olsen@berlex.com
Catherine L
CL
Olsen
Pin-Pin
PP
Hsu
Jens
J
Glienke
Gabor M
GM
Rubanyi
Alan R
AR
Brooks
D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002455:Cell Division / Q000235:genetics; D015536:Down-Regulation; D004730:Endothelium, Vascular / Q000378:metabolism*; D053823:Hedgehog Proteins; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007985:Leydig Cells / Q000378:metabolism; D008297:Male; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008565:Membrane Proteins; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000235:genetics* / Q000378:metabolism; D009389:Neovascularization, Pathologic / Q000235:genetics*; D018919:Neovascularization, Physiologic / Q000235:genetics; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011956:Receptors, Cell Surface; D015534:Trans-Activators / Q000378:metabolism; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; C117596:HHIP protein, human; D053823:Hedgehog Proteins; C498447:Hhip protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; D011506:Proteins; C000606240:Ptch1 protein, mouse; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; C509239:SHH protein, human; D015534:Trans-Activators