Regulated protein destruction controls many key cellular processes with aberrant regulation increasingly found during carcinogenesis. Gli proteins mediate the transcriptional effects of the Sonic hedgehog pathway, which is implicated in up to 25% of human tumors. Here we show that Gli is rapidly destroyed by the proteasome and that mouse basal cell carcinoma induction correlates with Gli protein accumulation. We identify two independent destruction signals in Gli1, D N and D C , and show that removal of these signals stabilizes Gli1 protein and rapidly accelerates tumor formation in transgenic animals. These data argue that control of Gli protein accumulation underlies tumorigenesis and suggest a new avenue for antitumor therapy. Factors controlling protein destruction are critical for the timing of key processes such as the cell cycle, apoptosis, and cell fate decisions, with aberrant regulation increasingly found during carcinogenesis (Pickart 2004;Yamasaki and Pagano 2004). Inappropriate Sonic hedgehog (Shh) signaling results in a panoply of birth defects and is implicated in up to 25% of human tumors (Callahan and Oro 2001;Lum and Beachy 2004). While the Gli family of proteins mediates the transcriptional effects of Shh (Methot and Basler 2001;Ruiz i Altaba et al. 2002), the mechanism by which Gli proteins are regulated to achieve changes in pathway output remains poorly understood. Studies in mice and humans show that Shh target gene induction is sufficient to induce a variety of tumors including basal cell carcinomas (BCCs) (Oro et al. 1997;Nilsson et al. 2000;Hutchin et al. 2005). However, there is a wide variability in the onset and severity of phenotypes among patients with mutations in the Shh pathway (Wicking et al. 1997), and a noticeably wide variability of tumor onset in animal models (Oro and Higgins 2003;Hutchin et al. 2005). This suggests the possibility that additional, previously uncharacterized, cellular processes regulate pathway output. Here we show that Gli protein accumulation correlates with tumor formation and stabilizing mutations in Gli protein dramatically accelerate tumor induction. Results and DiscussionWhile expression of either Gli1 or Gli2 in the epidermis of transgenic mice induces BCCs (Fig. 1a), we have observed a considerable delay in the appearance of Gli-dependent tumors. Analysis of transgenic mice expressing Gli2 revealed an average latency of 7 mo before tumor appearance (Fig. 1b). We ruled out changes in transcription of the transgene with age as a cause of the tumors, as similar levels of RNA are seen in both age groups as measured by quantitative PCR (Fig. 1c). This suggested the existence in keratinocytes of additional processes, whose loss or dysregulation is required to permit Gli activity and direct tumor formation. Our previous studies indicated that differential accumulation of Gli protein plays an important role in restricting Shh target gene induction in interfollicular epithelium (Oro and Higgins 2003). Indeed, we detected no transgenic Gli protein in normal...

Objective Inflammation has emerged as an important factor in disease progression in human and transgenic models of amyotrophic lateral sclerosis (ALS). Recent studies demonstrate that the prostaglandin E2 EP2 receptor is a major regulator of inflammatory oxidative injury in innate immunity. We tested whether EP2 signaling participated in disease pathogenesis in the G93A superoxide dismutase (SOD) model of familial ALS. Methods We examined the phenotype of G93A SOD mice lacking the EP2 receptor and performed immunocytochemistry, quantitative reverse transcriptase polymerase chain reaction, and Western analyses to determine the mechanism of EP2 toxicity in this model. Results EP2 receptor is significantly induced in G93A SOD mice in astrocytes and microglia in parallel with increases in expression of proinflammatory enzymes and lipid peroxidation. In human ALS, EP2 receptor immunoreactivity was upregulated in astrocytes in ventral spinal cord. In aging G93A SOD mice, genetic deletion of the prostaglandin E2 EP2 receptor improved motor strength and extended survival. Deletion of the EP2 receptor in G93A SOD mice resulted in significant reductions in levels of proinflammatory effectors, including cyclooxygenase-1, cyclooxygenase-2, inducible nitric oxide synthase, and components of the NADPH oxidase complex. In alternate models of inflammation, including the lipopolysaccharide model of innate immunity and the APPSwe-PS1ΔE9 model of amyloidosis, deletion of EP2 also reduced expression of proinflammatory genes. Interpretation These data suggest that prostaglandin E2 signaling via the EP2 receptor functions in the mutant SOD model and more broadly in inflammatory neurodegeneration to regulate expression of a cassette of proinflammatory genes. Inhibition of EP2 signaling may represent a novel strategy to downregulate the inflammatory response in neurodegenerative disease.

Prostaglandin E 2 (PGE 2 ), a potent lipid signaling molecule, modulates inflammatory responses through activation of downstream G-protein coupled EP 1-4 receptors. Here, we investigated the cell-specific in vivo function of PGE 2 signaling through its E-prostanoid 2 (EP2) receptor in murine innate immune responses systemically and in the CNS. In vivo, systemic administration of lipopolysaccharide (LPS) resulted in a broad induction of cytokines and chemokines in plasma that was significantly attenuated in EP2-deficient mice. Ex vivo stimulation of peritoneal macrophages with LPS elicited proinflammatory responses that were dependent on EP2 signaling and that overlapped with in vivo plasma findings, suggesting that myeloid-lineage EP2 signaling is a major effector of innate immune responses. Conditional deletion of the EP2 receptor in myeloid lineage cells in Cd11bCre;EP2 lox/lox mice attenuated plasma inflammatory responses and transmission of systemic inflammation to the brain was inhibited, with decreased hippocampal inflammatory gene expression and cerebral cortical levels of IL-6. Conditional deletion of EP2 significantly blunted microglial and astrocytic inflammatory responses to the neurotoxin MPTP and reduced striatal dopamine turnover. Suppression of microglial EP2 signaling also increased numbers of dopaminergic (DA) neurons in the substantia nigra independent of MPTP treatment, suggesting that microglial EP2 may influence development or survival of DA neurons. Unbiased microarray analysis of microglia isolated from adult Cd11bCre;EP2 lox/lox and control mice demonstrated a broad downregulation of inflammatory pathways with ablation of microglial EP2 receptor. Together, these data identify a cell-specific proinflammatory role for macrophage/microglial EP2 signaling in innate immune responses systemically and in brain.

Store-operated Ca entry occurs through the binding of the endoplasmic reticulum (ER) Ca sensor STIM1 to Orai1, the pore-forming subunit of the Ca release-activated Ca (CRAC) channel. Although the essential steps leading to channel opening have been described, fundamental questions remain, including the functional stoichiometry of the CRAC channel. The crystal structure of Drosophila Orai indicates a hexameric stoichiometry, while studies of linked Orai1 concatemers and single-molecule photobleaching suggest that channels assemble as tetramers. We assessed CRAC channel stoichiometry by expressing hexameric concatemers of human Orai1 and comparing in detail their ionic currents to those of native CRAC channels and channels generated from monomeric Orai1 constructs. Cell surface biotinylation results indicated that Orai1 channels in the plasma membrane were assembled from intact hexameric polypeptides and not from truncated protein products. In addition, the L273D mutation depressed channel activity equally regardless of which Orai1 subunit in the concatemer carried the mutation. Thus, functional channels were generated from intact Orai1 hexamers in which all subunits contributed equally. These hexameric Orai1 channels displayed the biophysical fingerprint of native CRAC channels, including the distinguishing characteristics of gating (store-dependent activation, Ca-dependent inactivation, open probability), permeation (ion selectivity, affinity for Ca block, La sensitivity, unitary current magnitude), and pharmacology (enhancement and inhibition by 2-aminoethoxydiphenyl borate). Because permeation characteristics depend strongly on pore geometry, it is unlikely that hexameric and tetrameric pores would display identical Ca affinity, ion selectivity, and unitary current magnitude. Thus, based on the highly similar pore properties of the hexameric Orai1 concatemer and native CRAC channels, we conclude that the CRAC channel functions as a hexamer of Orai1 subunits.

Ca 2þ release-activated Ca 2þ (CRAC) channels mediate Ca 2þ entry in response