Canonical Wnt signaling is critical to estrogen-mediated uterine growth

Major biological effects of estrogen in the uterus are thought to be primarily mediated by nuclear estrogen receptors, ERalpha and ERbeta. We show here that estrogen in an ER-independent manner rapidly up-regulates the expression of Wnt4 and Wnt5a of the Wnt family and frizzled-2 of the Wnt receptor family in the mouse uterus. One of the mechanisms by which Wnts mediate canonical signaling involves stabilization of intracellular beta-catenin. We observed that estrogen treatment prompts nuclear localization of active beta-catenin in the uterine epithelium. We also found that adenovirus mediated in vivo delivery of SFRP-2, a Wnt antagonist, down-regulates estrogen-dependent beta-catenin activity without affecting some of the early effects (water imbibition and angiogenic markers) and inhibits uterine epithelial cell growth, suggesting that canonical Wnt signaling is critical to estrogen-induced uterine growth. Our present results provide evidence for a novel role of estrogen that targets early Wnt/beta-catenin signaling in an ER-independent manner to regulate the late uterine growth response that is ER dependent.     

The renewal and differentiation of Isl1+ cardiovascular progenitors are controlled by a Wnt/beta-catenin pathway

Isl1(+) cardiovascular progenitors and their downstream progeny play a pivotal role in cardiogenesis and lineage diversification of the heart. The mechanisms that control their renewal and differentiation are largely unknown. Herein, we show that the Wnt/beta-catenin pathway is a major component by which cardiac mesenchymal cells modulate the prespecification, renewal, and differentiation of isl1(+) cardiovascular progenitors. This microenvironment can be reconstituted by a Wnt3a-secreting feeder layer with ES cell-derived, embryonic, and postnatal isl1(+) cardiovascular progenitors. In vivo activation of beta-catenin signaling in isl1(+) progenitors of the secondary heart field leads to their massive accumulation, inhibition of differentiation, and outflow tract (OFT) morphogenic defects. In addition, the mitosis rate in OFT myocytes is significantly reduced following beta-catenin deletion in isl1(+) precursors. Agents that manipulate Wnt signals can markedly expand isl1(+) progenitors from human neonatal hearts, a key advance toward the cloning of human isl1(+) heart progenitors.     

Wnt5a secretion stimulated by the extracellular calcium-sensing receptor inhibits defective Wnt signaling in colon cancer cells

To understand the role of the colonic extracellular calcium-sensing receptor (CaSR) in calcium chemoprotection against colon cancer, we activated the CaSR with 5 mM Ca(2+) on HT-29 cells, an adenocarcinoma cell line. High Ca(2+) stimulated the upregulation (as assessed by RT-PCR) and the secretion of Wnt5a (assessed by Western blot), a noncanonical Wnt family member. Inhibiting CaSR activity with a short interfering RNA (siRNA) duplex against the CaSR reduced CaSR protein and prevented the secretion of Wnt5a. Dominant negative CaSR (R185Q) or siRNA blocked the high Ca(2+)-mediated inhibition of the beta-catenin reporter TOPflash. The CaSR/Wnt5a inhibition of beta-catenin reporter was prevented by dominant negative ubiquitin ligase seven in absentia homolog 2 (Siah2). In low-calcium medium, overexpressing Wnt5a increased Siah2 amplicons and protein. Inducing the expression of full-length adenomatous polyposis coli (APC) prevented CaSRmediated increases of Siah2 and Wnt5a. Overexpressing the receptor tyrosine kinase-like orphan receptor 2 (Ror2) increased Wnt5a and CaSR-mediated inhibition of TOPflash. Conditioned medium from Wnt5a-transfected cells added to HT-29 cells in low-Ca(2+) medium inhibited the beta-catenin reporter. This inhibition was blocked dose responsively by Frizzled-8/Fc chimeric antibody. Overexpression of Ror2 in HT-29 cells in low-Ca(2+) medium increased the inhibition of beta-catenin reporter caused by recombinant Wnt5a protein compared with addition of Wnt5a protein alone. Our findings demonstrate that APC status plays a key role as a determinant of Wnt5a secretion and suggest that CaSR-mediated secretion of Wnt5a will inhibit defective Wnt signaling in APC-truncated cells in an autocrine manner.     

Nuclear beta-catenin localization is not sufficient for canonical Wnt signaling activation in human meianoma cell lines

In most cases, advanced stages of melanoma are practically incurable due to high metastatic potential of tumor cells. Multiple observations support the idea that aberrations in Wnt signaling pathway play a significant role in melanoma development and progression. Canonical Wnt signaling activation results in stabilization and accumulation of the major effector molecule called beta-catenin. Mutations promoting beta-catenin stabilization and, thereby, activation of canonical Wnt signaling pathway are frequently found in different cancers, but rarely observed in melanomas. Nevertheless, beta-catenin nuclear and cytoplasmic accumulation is the feature of many human melanoma cell lines and original tumors. That is why, the aim of the investigation was to elucidate the relation between beta-catenin intracellular localization and activity status of Wnt signaling pathway in human melanoma cell lines. Ten human melanoma cell lines were characterized on the basis of the following parameters: canonical Wnt ligand expression, intracellular beta-catenin localization, and activity status of canonical Wnt signaling pathway. Here, it has been demonstrated that nuclear localization of beta-catenin does not always correspond to active status canonical Wnt signaling pathway. Moreover, in the majority of cell lines with nuclear beta-catenin canonical Wnt signaling can't be activated by exogenous expression of an appropriate ligand. Human melanoma cell lines differ in activity of canonical Wnt signaling pathway as well as in mechanisms of its regulation. Therefore, the pathway-targeted potential antineoplastic therapy requires the formation of a "molecular pattern of cancer" for localization of the defect in Wnt signaling cascade in the each case.     

Effects of all-trans retinoic acid on angiotensin II-induced myocyte hypertrophy.

We used cultured neonatal rat cardiac myocytes to test the hypothesis that all-trans retinoic acid (atRA) may act to modulate ANG II actions in inducing myocyte hypertrophy. Our observations were as follows. 1) atRA (10(-7) to approximately 10(-5) M ) inhibited ANG II-induced hyperplasia of fibroblasts in a dose-dependent manner. 2) Treatment of atRA attenuated the ANG II-induced increase in total cell protein content. 3) Treated with ANG II (10(-7) M) for 5 days, the cultured neonatal rat cardiac myocytes demonstrated an apparent accumulation of sarcomeric fiber proteins and Golgi's complex, as well as reorganization of the sarcomeric unit within individual myocytes. atRA (10(-6) M) treatment reduced the accumulation of contractile proteins and Golgi's complex without affecting the ANG II-induced reorganization of the sarcomeric unit. 4) atRA attenuated the ANG II-induced increase in intracellular Ca2+. Our results show that atRA inhibits some effects of ANG II on neonatal rat cardiac myocytes and suggest that atRA may be a therapeutic candidate for the prevention and therapy of cardiac hypertrophy and remodeling.     

Wnt proteins induce dishevelled phosphorylation via an LRP5/6- independent mechanism, irrespective of their ability to stabilize beta-catenin

Wnt glycoproteins play essential roles in the development of metazoan organisms. Many Wnt proteins, such as Wnt1, activate the well-conserved canonical Wnt signaling pathway, which results in accumulation of beta-catenin in the cytosol and nucleus. Other Wnts, such as Wnt5a, activate signaling mechanisms which do not involve beta-catenin and are less well characterized. Dishevelled (Dvl) is a key component of Wnt/beta-catenin signaling and becomes phosphorylated upon activation of this pathway. In addition to Wnt1, we show that several Wnt proteins, including Wnt5a, trigger phosphorylation of mammalian Dvl proteins and that this occurs within 20 to 30 min. Unlike the effects of Wnt1, phosphorylation of Dvl in response to Wnt5a is not concomitant with beta-catenin stabilization, indicating that Dvl phosphorylation is not sufficient to activate canonical Wnt/beta-catenin signaling. Moreover, neither Dickkopf1, which inhibits Wnt/beta-catenin signaling by binding the Wnt coreceptors LRP5 and -6, nor dominant-negative LRP5/6 constructs could block Wnt-mediated Dvl phosphorylation. We conclude that Wnt-induced phosphorylation of Dvl is independent of LRP5/6 receptors and that canonical Wnts can elicit both LRP-dependent (to beta-catenin) and LRP-independent (to Dvl) signals. Our data also present Dvl phosphorylation as a general biochemical assay for Wnt protein function, including those Wnts that do not activate the Wnt/beta-catenin pathway.     

Mechanical overload-induced apoptosis: A study in cultured neonatal ventricular myocytes and fibroblasts

Apoptosis of cardiac myocytes has been implicated in cardiac dysfunction due to chronic hemodynamic overload. Reports on the role of apoptosis in the transition from hypertrophy to decompensated heart failure are not unequivocal. In this study we analysed the direct relationship between mechanical overload and induction of apoptosis in an in vitro model of cultured heart cells. Cyclic mechanical stretch was applied to cultured neonatal rat ventricular myocytes and fibroblasts. Several indicators of apoptosis were examined, such as morphological features, caspase-3 activity and DNA fragmentation. Mechanical strain did not induce any significant change in these parameters as compared to non-stretched myocytes or fibroblasts. However, administration of staurosporine, a known inducer of apoptosis, induced massive apoptosis in myocytes as well as fibroblasts. We conclude that this in vitro cell model system lacks a direct link between mechanical stretch and apoptosis. The three-dimensional structure-function relationship of myocardial tissue in the intact heart may elicit stretch-induced molecular signaling cascades in a much more complex way than in monolayer cultures of cardiac cells.     

Determination of cell types and numbers during cardiac development in the neonatal and adult rat and mouse

Cardiac fibroblasts, myocytes, endothelial cells, and vascular smooth muscle cells are the major cellular constituents of the heart. The aim of this study was to observe alterations in myocardial cell populations during early neonatal development in the adult animal and to observe any variations of the cardiac cell populations in different species, specifically, the rat and mouse. Whole hearts were isolated from either mice or rats during the neonatal and adult stages of development, and single cell suspensions were prepared via sequential collagenase digestion. Heterogeneous cell populations were immunolabeled for specific cell types and analyzed using fluorescence-activated cell sorting (FACS). In addition, the left ventricle, right ventricle, and septa were isolated, fixed, and sectioned for morphometric analyses. These same cardiac regions were also analyzed using FACS. We observed that the adult murine myocardium is composed of approximately 56% myocytes, 27% fibroblasts, 7% endothelial cells, and 10% vascular smooth muscle cells. Moreover, our morphometric and FACS data demonstrated similar percentages in the three regions examined. During murine neonatal cardiac development, we observed a marked increase in numbers of cardiac fibroblasts and a resultant decrease in percentages of myocytes in late neonatal development (day 15). Finally, FACS analyses of the rat heart during development displayed similar results in relation to increases in cardiac fibroblasts during development; however, cell populations in the rat differed markedly from those observed in the mouse. Taken together, these data enabled us to establish a homeostatic model for the myocardium that can be compared with genetic and cardiac disease models.     

Heparin activates Wnt signaling for neuronal morphogenesis

Wnt factors are secreted ligands that affect different aspects of the nervous system behavior like neurodevelopment, synaptogenesis and neurodegeneration. In different model systems, Wnt signaling has been demonstrated to be regulated by heparan sulfate proteoglycans (HSPGs). Whether HSPGs modulate Wnt signaling in the context of neuronal behavior is currently unknown. Here we demonstrate that activation of Wnt signaling with the endogenous ligand Wnt-7a results in an increased of neurite outgrowth in the neuroblastoma N2a cell line. Interestingly, heparin induces glycogen synthase kinase-3beta (GSK-3beta) inhibition, beta-catenin stabilization and morphological differentiation in both N2a cells and in rat primary hippocampal neuronal cultures. We also show that heparin modulates Wnt-3a-induced stabilization of beta-catenin. Several extracellular matrix and membrane-attached HSPGs were found to be expressed in both in vitro neuronal models. Changes in the expression of specific HSPGs were observed upon differentiation of N2a cells. Taken together, our findings suggest that HSPGs may modulate canonical Wnt signaling for neuronal morphogenesis.