Embryonic signalling pathways regulate progenitor cell fates in mammalian epithelial development and cancer. Prompted by the requirement for sonic hedgehog (Shh) signalling in lung development, we investigated a role for this pathway in regeneration and carcinogenesis of airway epithelium. Here we demonstrate extensive activation of the hedgehog (Hh) pathway within the airway epithelium during repair of acute airway injury. This mode of Hh signalling is characterized by the elaboration and reception of the Shh signal within the epithelial compartment, and immediately precedes neuroendocrine differentiation. We reveal a similar pattern of Hh signalling in airway development during normal differentiation of pulmonary neuroendocrine precursor cells, and in a subset of small-cell lung cancer (SCLC), a highly aggressive and frequently lethal human tumour with primitive neuroendocrine features. These tumours maintain their malignant phenotype in vitro and in vivo through ligand-dependent Hh pathway activation. We propose that some types of SCLC might recapitulate a critical, Hh-regulated event in airway epithelial differentiation. This requirement for Hh pathway activation identifies a common lethal malignancy that may respond to pharmacological blockade of the Hh signalling pathway.
We report the cloning and characterization of DANGER, a novel protein which physiologically binds to inositol 1,4,5-trisphosphate receptors (IP 3 R). DANGER is a membrane-associated protein predicted to contain a partial MAB-21 domain. It is expressed in a wide variety of neuronal cell lineages where it localizes to membranes in the cell periphery together with IP 3 R. DANGER interacts with IP 3 R in vitro and co-immunoprecipitates with IP 3 R from cellular preparations. DANGER robustly enhances Ca 2؉ -mediated inhibition of IP 3 R Ca 2؉ release without affecting IP 3 binding in microsomal assays and inhibits gating in single-channel recordings of IP 3 R. DANGER appears to allosterically modulate the sensitivity of IP 3 R to Ca 2؉ inhibition, which likely alters IP 3 R-mediated Ca 2؉ dynamics in cells where DANGER and IP 3 R are co-expressed.The inositol 1,4,5-trisphosphate receptor (IP 3 R) 3 is a large, endoplasmic reticulum (ER) resident protein, which is a key regulator of intracellular Ca 2ϩ signaling (1, 2). Inositol 1,4,5-trisphosphate (IP 3 ) is formed in response to the activation of G protein-coupled receptors or receptor tyrosine kinase receptors located in the plasma membrane (1), which elicit IP 3 Rmediated Ca 2ϩ release from ER stores. The IP 3 recognition site of IP 3 R includes amino acids (aa) 225-578 in the N-terminal portion of the protein, while the Ca 2ϩ channel domain comprises ϳ300 aa in the extreme C terminus (2). The IP 3 binding site and the Ca 2ϩ channel are separated by ϳ2,000 aa, providing a large area for interactions with multiple regulatory proteins including calmodulin, chromogranins, glyceraldehyde-3-phosphate dehydrogenase, RACK1, and caldendrin (3). While these proteins regulate IP 3 R function in diverse ways, only two regulators have been shown to influence Ca 2ϩ sensitivity. Cytochrome c, which binds to the extreme C terminus of the IP 3 R, relieves the inhibitory actions of Ca 2ϩ upon the channel (4), and Bcl-XL binding to the C terminus also influences the Ca 2ϩ dependence (5).We have identified a novel vertebrate protein, designated DANGER, which was isolated by yeast two-hybrid analysis with the regulatory region of the IP 3 R as bait. DANGER physiologically binds to IP 3 R and allosterically enhances the potency of Ca 2ϩ -inhibition of IP 3 R-mediated Ca 2ϩ release without affecting ligand binding. EXPERIMENTAL PROCEDURESYeast Two-hybrid Analysis-The Matchmaker3 yeast-2-hybrid system from Clontech (Palo Alto, CA) was employed. AH109 -galactosidase yeast was used. IP 3 R fragments were cloned into pGADT7 (-galactosidase acceptor domain) vector and were screened against a rat brain and human fetal kidney library (Clontech) as per the manufacturer's specifications. Expression of these fragments was determined by Western blotting using antibodies from Clontech, corresponding to the expression vector. Positive clones grew on minimal SD agar (Clontech) lacking adenine, histidine, leucine, and tryptophan and had -galactosidase activity.Calcium Release Measurements and El...
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