ß-Catenin-independent activation of TCF1/LEF1 in human hematopoietic tumor cells through interaction with ATF2 transcription factors.

The role of Wnt signaling in embryonic development and stem cell maintenance is well established and aberrations leading to the constitutive up-regulation of this pathway are frequent in several types of human cancers. Upon ligand-mediated activation, Wnt receptors promote the stabilization of ß-catenin, which translocates to the nucleus and binds to the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors to regulate the expression of Wnt target genes. When not bound to ß-catenin, the TCF/LEF proteins are believed to act as transcriptional repressors. Using a specific lentiviral reporter, we identified hematopoietic tumor cells displaying constitutive TCF/LEF transcriptional activation in the absence of ß-catenin stabilization. Suppression of TCF/LEF activity in these cells mediated by an inducible dominant-negative TCF4 (DN-TCF4) inhibited both cell growth and the expression of Wnt target genes. Further, expression of TCF1 and LEF1, but not TCF4, stimulated TCF/LEF reporter activity in certain human cell lines independently of ß-catenin. By a complementary approach in vivo, TCF1 mutants, which lacked the ability to bind to ß-catenin, induced Xenopus embryo axis duplication, a hallmark of Wnt activation, and the expression of the Wnt target gene Xnr3. Through generation of different TCF1-TCF4 fusion proteins, we identified three distinct TCF1 domains that participate in the ß-catenin-independent activity of this transcription factor. TCF1 and LEF1 physically interacted and functionally synergized with members of the activating transcription factor 2 (ATF2) family of transcription factors. Moreover, knockdown of ATF2 expression in lymphoma cells phenocopied the inhibitory effects of DN-TCF4 on the expression of target genes associated with the Wnt pathway and on cell growth. Together, our findings indicate that, through interaction with ATF2 factors, TCF1/LEF1 promote the growth of hematopoietic malignancies in the absence of ß-catenin stabilization, thus establishing a new mechanism for TCF1/LEF1 transcriptional activity distinct from that associated with canonical Wnt signaling.

Canonical and noncanonical Wnts use a common mechanism to activate completely unrelated coreceptors.

Wnt ligands signal through ß-catenin and are critically involved in cell fate determination and stem/progenitor self-renewal. Wnts also signal through ß-catenin-independent or noncanonical pathways that regulate crucial events during embryonic development. The mechanism of noncanonical receptor activation and how Wnts trigger canonical as opposed to noncanonical signaling have yet to be elucidated. We demonstrate here that prototype canonical Wnt3a and noncanonical Wnt5a ligands specifically trigger completely unrelated endogenous coreceptors-LRP5/6 and Ror1/2, respectively-through a common mechanism that involves their Wnt-dependent coupling to the Frizzled (Fzd) coreceptor and recruitment of shared components, including dishevelled (Dvl), axin, and glycogen synthase kinase 3 (GSK3). We identify Ror2 Ser 864 as a critical residue phosphorylated by GSK3 and required for noncanonical receptor activation by Wnt5a, analogous to the priming phosphorylation of low-density receptor-related protein 6 (LRP6) in response to Wnt3a. Furthermore, this mechanism is independent of Ror2 receptor Tyr kinase functions. Consistent with this model of Wnt receptor activation, we provide evidence that canonical and noncanonical Wnts exert reciprocal pathway inhibition at the cell surface by competition for Fzd binding. Thus, different Wnts, through their specific coupling and phosphorylation of unrelated coreceptors, activate completely distinct signaling pathways.

Novel role for aldose reductase in mediating acute inflammatory responses in the lung.

Exaggerated inflammatory responses and the resultant increases in alveolar-capillary permeability underlie the pathogenesis of acute lung injury during sepsis. This study examined the functions of aldose reductase (AR) in mediating acute lung inflammation. Transgenic mice expressing human AR (ARTg) were used to study the functions of AR since mice have low intrinsic AR activity. In a mild cecal ligation and puncture model, ARTg mice demonstrated an enhanced AR activity and a greater inflammatory response as evaluated by circulating cytokine levels, neutrophil accumulation in the lungs, and activation of Rho kinase in lung endothelial cells (ECs). Compared with WT lung cells, ARTg lung cells produced more IL-6 and showed augmented JNK activation in response to LPS stimulation ex vivo. In human neutrophils, AR activity was required for fMLP-included CD11b activation and up-regulation, respiratory burst, and shape changes. In human pulmonary microvascular ECs, AR activity was required for TNF-alpha-induced activation of the Rho kinase/MKK4/JNK pathway and IL-6 production, but not p38 activation or ICAM-1 expression. Importantly, AR activity in both human neutrophils and ECs was required for neutrophil adhesion to TNF-alpha-stimulated ECs. These data demonstrate a novel role for AR in regulating the signaling pathways leading to neutrophil-EC adhesion during acute lung inflammation.

Activation of Rho kinase isoforms in lung endothelial cells during inflammation.

Rho kinase (ROCK) is a downstream effector of Rho family GTPases, and two highly homologous isoforms, ROCK1 and ROCK2, are similarly inhibited by the widely used pharmacologic inhibitors. In endothelial cells (ECs), activation of ROCK regulates myosin L chain (MLC) phosphorylation, stress fiber formation and permeability increases during inflammation. This study examined isoform-specific ROCK activation in lung ECs in vitro using human pulmonary microvascular ECs and ex vivo using freshly isolated lung ECs from mice. In unstimulated human as well as mouse lung ECs, ROCK2 activity was greater than ROCK1 activity. TNF-alpha stimulation induced activation of both ROCK1 and ROCK2 in cultured human ECs. Studies using lung ECs freshly isolated from mice showed that intratracheal instillation of LPS induced ROCK activation in lung ECs that was inhibited by treating animals with fasudil, a pharmacologic ROCK inhibitor, and that both ROCK1 and ROCK2 were activated. Small interference RNA targeting ROCK1 or ROCK2 was used to examine their functions in regulating MLC phosphorylation and permeability increases induced by TNF-alpha in human ECs. TNF-alpha-induced MLC phosphorylation required ROCK activation. Inhibition of ROCK1 alone was not sufficient to prevent TNF-alpha-induced MLC phosphorylation, whereas inhibition of ROCK2 prevented TNF-alpha-induced late MLC phosphorylation at 24 h. Although ROCK1 was dispensable for TNF-alpha-induced MLC phosphorylation, ROCK1 was required for TNF-alpha-induced early permeability increases. Therefore, ROCK1 and ROCK2 are both activated by TNF-alpha and can be functionally separated in the signaling pathways leading to TNF-alpha-induced MLC phosphorylation and permeability increases.

Intrarectal vaccination with recombinant vaccinia virus expressing carcinoembronic antigen induces mucosal and systemic immunity and prevents progression of colorectal cancer.

The gastrointestinal mucosa contains an intact immune system that protects the host from pathogens and communicates with the systemic immune system. Absorptive epithelial cells in the mucosa give rise to malignant tumors although the interaction between tumor cells and the mucosal immune system is not well defined. The pathophysiology of colorectal cancer has been elucidated through studies of hereditary syndromes, such as familial adenomatous polyposis, a cancer predisposition syndrome caused by germline mutations in the adenomatous polyposis coli tumor suppressor gene. Patients with FAP develop adenomas and inevitably progress to invasive carcinomas by the age of 40. To better delineate the role of mucosal immunity in colorectal cancer, we evaluated the efficacy of intrarectal recombinant vaccinia virus expressing the human carcinoembryonic Ag (CEA) in a murine FAP model in which mice are predisposed to colorectal cancer and also express human CEA in the gut. Mucosal vaccination reduced the incidence of spontaneous adenomas and completely prevented progression to invasive carcinoma. The therapeutic effects were associated with induction of mucosal CEA-specific IgA Ab titers and CD8(+) CTLs. Mucosal vaccination was also associated with an increase in systemic CEA-specific IgG Ab titers, CD4(+) and CD8(+) T cell responses and resulted in growth inhibition of s.c. implanted CEA-expressing tumors suggesting communication between mucosal and systemic immune compartments. Thus, intrarectal vaccination induces mucosal and systemic antitumor immunity and prevents progression of spontaneous colorectal cancer. These results have implications for the prevention of colorectal cancer in high-risk individuals.

Activation of Rho kinase by TNF-alpha is required for JNK activation in human pulmonary microvascular endothelial cells.

TNF-alpha induces complex signaling events in endothelial cells (ECs), leading to inflammatory gene transcription and junctional permeability increases. This study examined the activation of RhoA and Rho kinase induced by TNF-alpha in primary human pulmonary microvascular ECs and its role in regulating EC responses to TNF-alpha. TNF-alpha induced a time-dependent activation of RhoA and Rho kinase in these ECs. TNF-alpha also induced activation of JNK that peaked at 15 min and lasted for at least 3 h. Inhibition of Rho kinase using a specific pharmacological inhibitor, Y27632, prevented TNF-alpha-induced early and late JNK activation. Inhibition of RhoA protein expression using small-interfering RNA, however, did not prevent TNF-alpha-induced Rho kinase activation or JNK activation. Studies using MAPK kinase 4 (MKK4) small-interfering RNA showed that MKK4 was not required for TNF-alpha-induced early JNK activation and that Rho kinase modulated early JNK activation through MKK4-independent mechanisms. Rho kinase, however, modulated TNF-alpha-induced late JNK activation mainly through MKK4-dependent mechanisms. Activation of Rho kinase was required for JNK-dependent IL-6 secretion induced by TNF-alpha. Moreover, inhibition of Rho kinase prevented TNF-alpha-induced cytoskeletal changes and permeability increases. Inhibition of JNK activation, however, did not prevent TNF-alpha-induced cytoskeletal changes, suggesting that Rho kinase did not modulate cytoskeletal changes through JNK activation. Therefore, Rho kinase plays important roles in EC responses to TNF-alpha by regulating permeability increases and JNK-dependent IL-6 production during pulmonary inflammation.

Interaction of prion protein with small highly structured RNAs: detection and characterization of PrP-oligomers.

Conformational modification of normal prion protein (PrP(c)) to protease-resistant, beta-sheet rich, aggregates (PrP(sc)) is commonly accepted cause for prion diseases. On the other hand, several studies in recent years implicate soluble, protease-sensitive, oligomers of PrP(c) in neuronal damage. Previously, our group has shown that small, highly structured RNAs (shsRNAs), in conjunction with a serum factor, facilitated the conversion of hrPrP to a protease resistant, high molecular weight isoform. In the current study we demonstrate that shsRNAs, in the absence of the serum factor, generate soluble, protease-sensitive, and potentially toxic oligomers of ovrPrP. We have isolated a 500 kD oligomer by size exclusion chromatography of the reaction mixture and identified the accessible epitopes. The soluble PrP-oligomers were present in enhanced amounts in scrapie infected sheep brain and treating extracts of normal sheep brain with shsRNA resulted in oligomerization of endogenous PrP. Isolation, characterization of PrP-oligomers and their possible implication in prion diseases is discussed.