It is well established that inducible transcription is essential for the consolidation of salient experiences into long-term memory. However, whether inducible transcription relays information about the identity and affective attributes of the experience being encoded, has not been explored. To this end, we analyzed transcription induced by a variety of rewarding and aversive experiences, across multiple brain regions. Our results describe the existence of robust transcriptional signatures uniquely representing distinct experiences, enabling near-perfect decoding of recent experiences. Furthermore, experiences with shared attributes display commonalities in their transcriptional signatures, exemplified in the representation of valence, habituation and reinforcement. This study introduces the concept of a neural transcriptional code, which represents the encoding of experiences in the mouse brain. This code is comprised of distinct transcriptional signatures that correlate to attributes of the experiences that are being committed to long-term memory.

We have previously shown a novel link between hPar-1 (human protease-activated receptor-1) and ␤-catenin stabilization. Although it is well recognized that Wnt signaling leads to ␤-catenin accumulation, the role of PAR1 in the process is unknown. We provide here evidence that PAR1 induces ␤-catenin stabilization independent of Wnt, Fz (Frizzled), and the coreceptor LRP5/6 (low density lipoprotein-related protein 5/6) and identify selective mediators of the PAR1-␤-catenin axis. Immunohistological analyses of hPar1-transgenic (TG) mouse mammary tissues show the expression of both G␣ 12 and G␣ 13 compared with age-matched control counterparts. However, only G␣ 13 was found to be actively involved in PAR1-induced ␤-catenin stabilization. Indeed, a dominant negative form of G␣ 13 inhibited both PAR1-induced Matrigel invasion and Lef/ Tcf (lymphoid enhancer factor/T cell factor) transcription activity. PAR1-G␣ 13 association is followed by the recruitment of DVL (Dishevelled), an upstream Wnt signaling protein via the DIX domain. Small interfering RNA-Dvl silencing leads to a reduction in PAR1-induced Matrigel invasion, inhibition of Lef/Tcf transcription activity, and decreased ␤-catenin accumulation. It is of note that PAR1 also promotes the binding of ␤-arrestin-2 to DVL, suggesting a role for ␤-arrestin-2 in PAR1-induced DVL phosphorylation dynamics. Although infection of small interfering RNA-LRP5/6 or the use of the Wnt antagonists, SFRP2 (soluble Frizzled-related protein 2) or SFRP5 potently reduced Wnt3A-mediated ␤-catenin accumulation, no effect was observed on PAR1-induced ␤-catenin stabilization. Collectively, our data show that PAR1 mediates ␤-catenin stabilization independent of Wnt. We propose here a novel cascade of PAR1-induced G␣ 13 -DVL axis in cancer and ␤-catenin stabilization.PAR1 (protease-activated receptor-1) is the first identified and prototype member of an established protease-activated receptor family. In addition to the traditional role of PAR1 in thrombosis, hemostasis, and vascular biology, its role in tumor biology is currently emerging (1-3). We have established a novel link between hPar1 and ␤-catenin stabilization, both in transgenic mouse mammary glands and in a wide spectrum of tumor cell lines (4). Protease-activated receptors are part of a large seven-transmembrane-spanning G protein-coupled receptor (GPCR) 2 family (5, 6), shown to couple to G␣ i/o , G␣ q , or G␣ 12/13 within the same cell type (7). Of the 16 G␣ genes found in the mammalian genome, the G␣ 12 subfamily is of particular interest to cancer biologists. G␣ 12 and its sister family member, G␣ 13 , are the only heterotrimeric G proteins that are capable of transforming fibroblasts when overexpressed in their wild-type (WT) form (8 -10). Recent studies have demonstrated that G␣ 12 is markedly up-regulated in adenocarcinoma of the breast and have identified G␣ 12 protein as an important regulator of breast and prostate cancer invasion (11,12). The G␣ 12 protein subunit also plays a role in disrupting cadherin-␤-catenin i...

Protease-activated receptor 1 (PAR1) is emerging with distinct assignments in tumor biology. We show that tissue targeted overexpression of hPar1 in mice mammary glands results in precocious hyperplasia, characterized by a dense network of ductal side branching and accelerated proliferation. These glands exhibit increased levels of wnt-4 and wnt-7b and a striking B-catenin stabilization. Nuclear localization of B-catenin is observed in hPar1 transgenic mouse tissue sections but not in the wild-type, age-matched counterparts. PAR1 induces B-catenin nuclear localization also in established epithelial tumor cell lines of intact B-catenin system (transformed on the background of mismatch repair system; RKO cells). We propose hereby that PAR1-mediated B-catenin stabilization is taking place primarily via the increase of Wnt expression. Enforced expression of a specific Wnt antagonist family member, secreted frizzled receptor protein 5 (SFRP5), efficiently inhibited PAR1-induced B-catenin stabilization. Likewise, application of either SFRP2 or SFRP5 on epithelial tumor cells completely abrogated PAR1-induced B-catenin nuclear accumulation. This takes place most likely via inhibition of Wnt signaling at the level of cell surface ( forming a neutralizing complex of ''Receptors-SFRP-Wnt ''). Furthermore, depletion of hPar1 by small interfering RNA (siRNA) vectors markedly inhibited PAR1-induced Wnt-4. The striking stabilization of B-catenin, inhibited by SFRPs on one hand and Wnt-4 silencing by hPar1 siRNA on the other hand, points to a novel role of hPar1 in Wnt-mediated B-catenin stabilization. This link between PAR1 and B-catenin may bear substantial implications both in developmental and tumor progression processes. (Cancer Res 2006; 66(10): 5224-33)

SummaryProtease-activated receptor1 (PAR 1 ) is the first and prototype member of an established PAR family comprising four members. The role of PAR 1 in tumor biology has been established, and is characterized by a consistent direct correlation between overexpression of its levels and epithelial tumor aggressiveness. We have found that high expression of the human Par 1 (hPar 1 ) gene in epithelial tumors is controlled largely at the transcriptional level. This led us to assign Egr-1, a transcription activator, as an inducer of hPar 1 , and p53, a tumor suppressor gene, as an inhibitor, both acting to achieve fine tuning of hPar 1 in prostate carcinoma. High PAR 1 levels maintain prosurvival signals in tumor cells while silencing or ablation of the gene induce apoptosis. Studies of our hPar 1 transgenic mice, which overexpress hPar 1 in the mammary glands, revealed a novel PAR 1 -induced b-catenin stabilization function. The components connecting PAR 1 to b-catenin stabilization have been determined, assigning at first G a13 as a selective immediate component. The PAR 1 -G a13 axis recruits disheveled (DVL), an upstream signaling partner of the canonical Wnt signaling pathway. Silencing of DVL by siRNA-DVL potently abrogates PAR 1 -induced b-catenin stabilization, demonstrating its critical role in the process. We, thus, propose that transcriptional regulation of hPar 1 gene over expression in epithelia malignancies initiates a novel signaling pathway, directly connecting to b-catenin stabilization, a core event in both tumorigenesis and developmental processes.

BackgroundWhile protease-activated-receptor 1 (PAR1) plays a central role in tumor progression, little is known about the cell signaling involved.Methodology/Principal FindingsWe show here the impact of PAR1 cellular activities using both an orthotopic mouse mammary xenograft and a colorectal-liver metastasis model in vivo, with biochemical analyses in vitro. Large and highly vascularized tumors were generated by cells over-expressing wt hPar1, Y397Z hPar1, with persistent signaling, or Y381A hPar1 mutant constructs. In contrast, cells over-expressing the truncated form of hPar1, which lacks the cytoplasmic tail, developed small or no tumors, similar to cells expressing empty vector or control untreated cells. Antibody array membranes revealed essential hPar1 partners including Etk/Bmx and Shc. PAR1 activation induces Etk/Bmx and Shc binding to the receptor C-tail to form a complex. Y/A mutations in the PAR1 C-tail did not prevent Shc-PAR1 association, but enhanced the number of liver metastases compared with the already increased metastases obtained with wt hPar1. We found that Etk/Bmx first binds via the PH domain to a region of seven residues, located between C378-S384 in PAR1 C-tail, enabling subsequent Shc association. Importantly, expression of the hPar1-7A mutant form (substituted A, residues 378-384), which is incapable of binding Etk/Bmx, resulted in inhibition of invasion through Matrigel-coated membranes. Similarly, knocking down Etk/Bmx inhibited PAR1-induced MDA-MB-435 cell migration. In addition, intact spheroid morphogenesis of MCF10A cells is markedly disrupted by the ectopic expression of wt hPar1. In contrast, the forced expression of the hPar1-7A mutant results in normal ball-shaped spheroids. Thus, by preventing binding of Etk/Bmx to PAR1 -C-tail, hPar1 oncogenic properties are abrogated.Conclusions/SignificanceThis is the first demonstration that a cytoplasmic portion of the PAR1 C-tail functions as a scaffold site. We identify here essential signaling partners, determine the hierarchy of binding and provide a platform for therapeutic vehicles via definition of the critical PAR1 -associating region in the breast cancer signaling niche.