Sepp R. Jansen

β-Catenin regulates airway smooth muscle contraction

beta-Catenin is an 88-kDa member of the armadillo family of proteins that is associated with the cadherin-catenin complex in the plasma membrane. This complex interacts dynamically with the actin cytoskeleton to stabilize adherens junctions, which play a central role in force transmission by smooth muscle cells. Therefore, in the present study, we hypothesized a role for beta-catenin in the regulation of smooth muscle force production. beta-Catenin colocalized with smooth muscle alpha-actin (sm-alpha-actin) and N-cadherin in plasma membrane fractions and coimmunoprecipitated with sm-alpha-actin and N-cadherin in lysates of bovine tracheal smooth muscle (BTSM) strips. Moreover, immunocytochemistry of cultured BTSM cells revealed clear and specific colocalization of sm-alpha-actin and beta-catenin at the sites of cell-cell contact. Treatment of BTSM strips with the pharmacological beta-catenin/T cell factor-4 (TCF4) inhibitor PKF115-584 (100 nM) reduced beta-catenin expression in BTSM whole tissue lysates and in plasma membrane fractions and reduced maximal KCl- and methacholine-induced force production. These changes in force production were not accompanied by changes in the expression of sm-alpha-actin or sm-myosin heavy chain (MHC). Likewise, small interfering RNA (siRNA) knockdown of beta-catenin in BTSM strips reduced beta-catenin expression and attenuated maximal KCl- and methacholine-induced contractions without affecting sm-alpha-actin or sm-MHC expression. Conversely, pharmacological (SB-216763, LiCl) or insulin-induced inhibition of glycogen synthase kinase-3 (GSK-3) enhanced the expression of beta-catenin and augmented maximal KCl- and methacholine-induced contractions. We conclude that beta-catenin is a plasma membrane-associated protein in airway smooth muscle that regulates active tension development, presumably by stabilizing cell-cell contacts and thereby supporting force transmission between neighboring cells.

TGF-β-induced profibrotic signaling is regulated in part by the WNT receptor Frizzled-8.

TGF‐β is important in lung injury and remodeling processes. TGF‐β and Wingless/integrase‐1 (WNT) signaling are interconnected; however, the WNT ligand‐receptor complexes involved are unknown. Thus, we aimed to identify Frizzled (FZD) receptors that mediate TGF‐β‐induced profibrotic signaling. MRC‐5 and primary human lung fibroblasts were stimulated with TGF‐β1, WNT‐5A, or WNT‐5B in the presence and absence of specific pathway inhibitors. Specific small interfering RNA was used to knock down FZD8. In vivo studies using bleomycin‐induced lung fibrosis were performed in wild‐type and FZD8‐deficient mice. TGF‐β 1 induced FZD8 specifically via Smad3‐dependent signaling in MRC‐5 and primary human lung fibroblasts. It is noteworthy that FZD8 knockdown reduced TGF‐β1‐induced collagen Iα1, fibronectin, versican, α‐smooth muscle (sm)‐actin, and connective tissue growth factor. Moreover, bleomycin‐induced lung fibrosis was attenuated in FZD8‐deficient mice in vivo. Although inhibition of canonical WNT signaling did not affect TGF‐β 1‐induced gene expression in vitro, noncanonical WNT‐5B mimicked TGF‐β1‐induced fibroblast activation. FZD8 knockdown reduced both WNT‐5B‐induced gene expression of fibronectin and α‐sm‐actin, as well as WNT‐5B‐induced changes in cellular impedance. Collectively, our findings demonstrate a role for FZD8 in TGF‐β‐induced profibrotic signaling and imply that WNT‐5B may be the ligand for FZD8 in these responses.—Spanjer, A. I. R., Baarsma, H. A., Oostenbrink, L. M., Jansen, S. R., Kuipers, C. C., Lindner, M., Postma, D. S., Meurs, H., Heijink, I. H., Gosens, R., Königshoff, M. TGF‐β‐induced profibrotic signaling is regulated in part by the WNT receptor Frizzled‐8. FASEB J. 30, 1823–1835 (2016). www.fasebj.org

Prostaglandin E2 promotes MYCN non-amplified neuroblastoma cell survival via β-catenin stabilization

Amplification of MYCN is the most well‐known prognostic marker of neuroblastoma risk classification, but still is only observed in 25% of cases. Recent evidence points to the cyclic adenosine monophosphate (cAMP) elevating ligand prostaglandin E2 (PGE2) and β‐catenin as two novel players in neuroblastoma. Here, we aimed to define the potential role of PGE2 and cAMP and its potential interplay with β‐catenin, both of which may converge on neuroblastoma cell behaviour. Gain and loss of β‐catenin function, PGE2, the adenylyl cyclase activator forskolin and pharmacological inhibition of cyclooxygenase‐2 (COX‐2) were studied in two human neuroblastoma cell lines without MYCN amplification. Our findings show that PGE2 enhanced cell viability through the EP4 receptor and cAMP elevation, whereas COX‐2 inhibitors attenuated cell viability. Interestingly, PGE2 and forskolin promoted glycogen synthase kinase 3β inhibition, β‐catenin phosphorylation at the protein kinase A target residue ser675, β‐catenin nuclear translocation and TCF‐dependent gene transcription. Ectopic expression of a degradation‐resistant β‐catenin mutant enhances neuroblastoma cell viability and inhibition of β‐catenin with XAV939 prevented PGE2‐induced cell viability. Finally, we show increased β‐catenin expression in human high‐risk neuroblastoma tissue without MYCN amplification. Our data indicate that PGE2 enhances neuroblastoma cell viability, a process which may involve cAMP‐mediated β‐catenin stabilization, and suggest that this pathway is of relevance to high‐risk neuroblastoma without MYCN amplification.

Paving the Rho in cancer metastasis: Rho GTPases and beyond

Malignant carcinomas are often characterized by metastasis, the movement of carcinoma cells from a primary site to colonize distant organs. For metastasis to occur, carcinoma cells first must adopt a pro-migratory phenotype and move through the surrounding stroma towards a blood or lymphatic vessel. Currently, there are very limited possibilities to target these processes therapeutically. The family of Rho GTPases is an ubiquitously expressed division of GTP-binding proteins involved in the regulation of cytoskeletal dynamics and intracellular signaling. The best characterized members of the Rho family GTPases are RhoA, Rac1 and Cdc42. Abnormalities in Rho GTPase function have major consequences for cancer progression. Rho GTPase activation is driven by cell surface receptors that activate GTP exchange factors (GEFs) and GTPase-activating proteins (GAPs). In this review, we summarize our current knowledge on Rho GTPase function in the regulation of metastasis. We will focus on key discoveries in the regulation of epithelial-mesenchymal-transition (EMT), cell-cell junctions, formation of membrane protrusions, plasticity of cell migration and adaptation to a hypoxic environment. In addition, we will emphasize on crosstalk between Rho GTPase family members and other important oncogenic pathways, such as cyclic AMP-mediated signaling, canonical Wnt/β-catenin, Yes-associated protein (YAP) and hypoxia inducible factor 1α (Hif1α) and provide an overview of the advancements and challenges in developing pharmacological tools to target Rho GTPase and the aforementioned crosstalk in the context of cancer therapeutics.

Epac1 links prostaglandin E2 to β-catenin-dependent transcription during epithelial-to-mesenchymal transition.

In epithelial cells, β-catenin is localized at cell-cell junctions where it stabilizes adherens junctions. When these junctions are disrupted, β-catenin can translocate to the nucleus where it functions as a transcriptional cofactor. Recent research has indicated that PGE2 enhances the nuclear function of β-catenin through cyclic AMP. Here, we aim to study the role of the cyclic AMP effector Epac in β-catenin activation by PGE2 in non-small cell lung carcinoma cells. We show that PGE2 induces a down-regulation of E-cadherin, promotes cell migration and enhances β-catenin translocation to the nucleus. This results in β-catenin-dependent gene transcription. We also observed increased expression of Epac1. Inhibition of Epac1 activity using the CE3F4 compound or Epac1 siRNA abolished the effects of PGE2 on β-catenin. Further, we observed that Epac1 and β-catenin associate together. Expression of an Epac1 mutant with a deletion in the nuclear pore localization sequence prevents this association. Furthermore, the scaffold protein Ezrin was shown to be required to link Epac1 to β-catenin. This study indicates a novel role for Epac1 in PGE2-induced EMT and subsequent activation of β-catenin.

Catecholamines facilitate VEGF-dependent angiogenesis via β 2 -adrenoceptor-induced Epac1 and PKA activation

Chronic stress has been associated with the progression of cancer and antagonists for β-adrenoceptors (βAR) are regarded as therapeutic option. As they are also used to treat hemangiomas as well as retinopathy of prematurity, a role of endothelial β2AR in angiogenesis can be envisioned. We therefore investigated the role of β2AR-induced cAMP formation by analyzing the role of the cAMP effector molecules exchange factor directly activated by cAMP 1 (Epac1) and protein kinase A (PKA) in endothelial cells (EC). Epac1-deficient mice showed a reduced amount of pre-retinal neovascularizations in the model of oxygen-induced retinopathy, which is predominantly driven by vascular endothelial growth factor (VEGF). siRNA-mediated knockdown of Epac1 in human umbilical vein EC (HUVEC) decreased angiogenic sprouting by lowering the expression of the endothelial VEGF-receptor-2 (VEGFR-2). Conversely, Epac1 activation by β2AR stimulation or the Epac-selective activator cAMP analog 8-p-CPT-2’-O-Me-cAMP (8-pCPT) increased VEGFR-2 levels and VEGF-dependent sprouting. Similar to Epac1 knockdown, depletion of the monomeric GTPase Rac1 decreased VEGFR-2 expression. As Epac1 stimulation induces Rac1 activation, Epac1 might regulate VEGFR-2 expression through Rac1. In addition, we found that PKA was also involved in the regulation of angiogenesis in EC since the adenylyl cyclase (AC) activator forskolin (Fsk), but not 8-pCPT, increased sprouting in Epac1-depleted HUVEC and this increase was sensitive to a selective synthetic peptide PKA inhibitor. In accordance, β2AR- and AC-activation, but not Epac1 stimulation increased VEGF secretion in HUVEC. Our data indicate that high levels of catecholamines, which occur during chronic stress, prime the endothelium for angiogenesis through a β2AR-mediated increase in endothelial VEGFR-2 expression and VEGF secretion.