Molecular and pharmacological characterization of membrane-associated and soluble forms of Hedgehog interacting protein
Laboratoire de Neurobiologie Cellulaire et Moléculaire
Besides its inductive and proliferative roles in the ventral neuroaxis during embryonic development, the morphogen Sonic Hedgehog (Shh) was recently implicated in tumorigenesis such as in basal cell carcinomas, lung, pancreatic, digestive cancers or primitive neuroectodermal tumors (Berman et al, Science, 2002; Williams et al, Proc Natl Acad Sci, 2002; Berman et al, Nature, 2003; Watkins et al, Nature, 2003 ; Thayer et al, Nature, 2003). Mutations in the genes encoding the different components of the pathway including Patched (Ptc), a transmembrane protein homologous to bacterial transporters, and Smoothened (Smo), a seven-transmembrane protein, have been implicated in basal cell carcinomas and medulloblastomas. Abnormal expression of the Hedgehog pathway which presumably occurs in lung, digestive and pancreatic tumors is believed to be responsible for the growth and malignancy of these tumors. Interestingly, treatment with cyclopamine, a Smo antagonist, results in a reduction of the tumor volume in cancer animal models. These experiments demonstrate the usefulness of antagonizing the Hedgehog pathway for blocking the development of these tumors. Now, we have investigated the pharmacological and biochemical properties of Hedgehog interacting protein (Hip) considered to be a negative regulator of the Shh pathway (Chuang and McMahon, Nature, 1999). Hip has been proposed as a membrane protein implicated in sequestering the Hedgehog morphogens during embryonic development. Here, we demonstrate the presence of membrane-associated and soluble forms of Hip in adult tissues including the brain. Moreover, we show that soluble forms of Hip, present in the conditioned medium of HEK293 cells overexpressing Hip, inhibit Shh-induced differentiation of C3H10T1/2 cells, a well characterised response associated with Shh signalling. After transfection in HEK293 cells, Hip partitions with the raft component ganglioside GM1 during density gradient centrifugation. Analysis of tagged Hip constructs reveals that the putative transmembrane domain of Hip is not cleaved suggesting that other mechanisms are implicated in the release of its soluble forms. Taken together, these data are consistent with the involvement of both membrane-associated and soluble Hip in the regulation of Shh signalling in adult tissues. Further work should delineate the potential role of membrane-associated and soluble forms of HiP for blocking the Shh pathway during tumor development.
Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in oral session 798 (Growth factors & signalling).