Comprehensive Analysis of Transcription Dynamics from Brain Samples Following Behavioral Experience

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific ...

The Hebrew University of Jerusalem

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 beta-catenin stabilization. Nuclear localization of beta-catenin is observed in hPar1 transgenic mouse tissue sections but not in the wild-type, age-matched counterparts. PAR1 induces beta-catenin nuclear localization also in established epithelial tumor cell lines of intact beta-catenin system (transformed on the background of mismatch repair system; RKO cells). We propose hereby that PAR1-mediated beta-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 beta-catenin stabilization. Likewise, application of either SFRP2 or SFRP5 on epithelial tumor cells completely abrogated PAR1-induced beta-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 beta-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 beta-catenin stabilization. This link between PAR1 and beta-catenin may bear substantial implications both in developmental and tumor progression processes.

Mammary gland tissue targeted overexpression of human protease-activated receptor 1 reveals a novel link to beta-catenin stabilization.

We have previously shown a novel link between hPar-1 (human protease-activated receptor-1) and beta-catenin stabilization. Although it is well recognized that Wnt signaling leads to beta-catenin accumulation, the role of PAR1 in the process is unknown. We provide here evidence that PAR1 induces beta-catenin stabilization independent of Wnt, Fz (Frizzled), and the co-receptor LRP5/6 (low density lipoprotein-related protein 5/6) and identify selective mediators of the PAR1-beta-catenin axis. Immunohistological analyses of hPar1-transgenic (TG) mouse mammary tissues show the expression of both Galpha(12) and Galpha(13) compared with age-matched control counterparts. However, only Galpha(13) was found to be actively involved in PAR1-induced beta-catenin stabilization. Indeed, a dominant negative form of Galpha(13) inhibited both PAR1-induced Matrigel invasion and Lef/Tcf (lymphoid enhancer factor/T cell factor) transcription activity. PAR1-Galpha(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 beta-catenin accumulation. It is of note that PAR1 also promotes the binding of beta-arrestin-2 to DVL, suggesting a role for beta-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 beta-catenin accumulation, no effect was observed on PAR1-induced beta-catenin stabilization. Collectively, our data show that PAR1 mediates beta-catenin stabilization independent of Wnt. We propose here a novel cascade of PAR1-induced Galpha(13)-DVL axis in cancer and beta-catenin stabilization.

Protease-activated receptor-1 (PAR1) acts via a novel Galpha13-dishevelled axis to stabilize beta-catenin levels.

While protease-activated-receptor 1 (PAR(1)) plays a central role in tumor progression, little is known about the cell signaling involved.

Etk/Bmx regulates proteinase-activated-receptor1 (PAR1) in breast cancer invasion: signaling partners, hierarchy and physiological significance.

Taking the issue of tumor categorization a step forward and establish molecular imprints to accompany histopathological assessment is a challenging task. This is important since often patients with similar clinical and pathological tumors may respond differently to a given treatment. Protease-activated receptor-(1) (PAR(1)), a G protein-coupled receptor (GPCR), is the first member of the mammalian PAR family consisting of four genes. PAR(1) and PAR(2) play a central role in breast cancer. The release of N-terminal peptides during activation and the exposure of a cryptic internal ligand in PARs, endow these receptors with the opportunity to serve as a "mirror-image" index reflecting the level of cell surface PAR(1&2)-in body fluids. It is possible to use the levels of PAR-released peptide in patients and accordingly determine the choice of treatment. We have both identified PAR(1) C-tail as a scaffold site for the immobilization of signaling partners, and the critical minimal binding site. This binding region may be used for future therapeutic modalities in breast cancer, since abrogation of the binding inhibits PAR(1) induced breast cancer. Altogether, both PAR(1) and PAR(2) may serve as molecular probes for breast cancer diagnosis and valuable targets for therapy.

Par genes: molecular probes to pathological assessment in breast cancer progression.

The classical G-protein-coupled receptors (GPCRs) are characterized by their ability to interact with heterotrimeric G proteins upon activation and by structural features such as seven transmembrane spanning domains. Frizzleds (Fzs) are comparable seven transmembrane receptors (7 TMRs) that are activated via Wnts and play a critical role in embryogenesis, tissue hemostasis and oncogenicity. It remains controversial, however, whether they may be considered GPCRs. Hence, the ten members of Fzs constitute a distinct atypical family of seven-transmembrane receptors. Canonical Wnt/β-catenin signaling leads to the core process of β-catenin stabilization and, ultimately, to the translocation of β-catenin to the nucleus where it acts as a co-transcription factor and induces Wnt target gene transcription. we have documented that activation by proteinase-activated receptor1 (PAR(1)), a classical 7TMR, recruits dishevelled (DvL), an upstream Wnt signaling protein, to mediate β-catenin stabilization. DvL is selectively bound to activated G(α13) subunit, coupled to PAR(1) following activation. Formation of the PAR(1)-induced DvL-G(α13) axis is carried out independently of Wnt, Fz and the co-receptor LRP5/6 (low density lipoprotein-related protein 5/6) since neither siRNA-LRP5/6 co-receptors nor the presence of SFRPs; secreted Fz receptor proteins (Wnt antagonists) affect PAR(1)-induced β-catenin stabilization. Similarly, PAR(1) induced placenta cytotrophoblast physiological invasion process was not affected by inhibiting Wnt, but was abrogated by siRNA-DvL. we propose that DvL serves as a central mediator protein that links classical GPCRs to β-catenin stabilization in both pathological (tumor) and physiological (placenta) invasion processes.

DVL as a scaffold protein capturing classical GPCRs.

Protease-activated receptor 1 (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 β-catenin stabilization function. The components connecting PAR(1) to β-catenin stabilization have been determined, assigning at first G(α)(13) as a selective immediate component. The PAR(1)-G(α) (13) 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 β-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 β-catenin stabilization, a core event in both tumorigenesis and developmental processes.

PAR1 plays a role in epithelial malignancies: transcriptional regulation and novel signaling pathway.

Pantothenate Kinase-Associated Neurodegeneration (PKAN) is a neurodegenerative disorder with a poorly understood molecular mechanism. It is caused by mutations in Pantothenate Kinase, the first enzyme in the Coenzyme A (CoA) biosynthetic pathway. Here, we developed a Drosophila model of PKAN (tim-fbl flies) that allows us to continuously monitor the modeled disease in the brain. In tim-fbl flies, downregulation of fumble, the Drosophila PanK homologue in the cells containing a circadian clock results in characteristic features of PKAN such as developmental lethality, hypersensitivity to oxidative stress, and diminished life span. Despite quasi-normal circadian transcriptional rhythms, tim-fbl flies display brain-specific aberrant circadian locomotor rhythms, and a unique transcriptional signature. Comparison with expression data from flies exposed to paraquat demonstrates that, as previously suggested, pathways others than oxidative stress are affected by PANK downregulation. Surprisingly we found a significant decrease in the expression of key components of the photoreceptor recycling pathways, which could lead to retinal degeneration, a hallmark of PKAN. Importantly, these defects are not accompanied by changes in structural components in eye genes suggesting that changes in gene expression in the eye precede and may cause the retinal degeneration. Indeed tim-fbl flies have diminished response to light transitions, and their altered day/night patterns of activity demonstrates defects in light perception. This suggest that retinal lesions are not solely due to oxidative stress and demonstrates a role for the transcriptional response to CoA deficiency underlying the defects observed in dPanK deficient flies. Moreover, in the present study we developed a new fly model that can be applied to other diseases and that allows the assessment of neurodegeneration in the brains of living flies.

Hagit Turm

The Alexander Silberman Institute of Life Sciences & Edmond and Lily Safra Center for Brain Sciences

The Hebrew University of Jerusalem

Comprehensive Analysis of Transcription Dynamics from Brain Samples Following Behavioral Experience

Hagit Turm

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}The Alexander Silberman Institute of Life Sciences & Edmond and Lily Safra Center for Brain Sciences

The Hebrew University of Jerusalem

Comprehensive Analysis of Transcription Dynamics from Brain Samples Following Behavioral Experience

The Alexander Silberman Institute of Life Sciences & Edmond and Lily Safra Center for Brain Sciences