Etk/Bmx Regulates Proteinase-Activated-Receptor1 (PAR1) in Breast Cancer Invasion: Signaling Partners,Hierarchy and Physiological Significance

Background

While protease-activated-receptor 1 (PAR₁) plays a central role in tumor progression, little is known about the cell signaling involved.

Methodology/Principal Findings

We 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/Significance

This 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.     

Small‐scale spatial variability in the distribution of ectomycorrhizal fungi affects plant performance and fungal diversity

The effects of spatial heterogeneity in negative biological interactions on individual performance and species diversity have been studied extensively. However, little is known about the respective effects involving positive biological interactions, including the symbiosis between plants and ectomycorrhizal (EM) fungi. Using a greenhouse bioassay, we explored how spatial heterogeneity of natural soil inoculum influences the performance of pine seedlings and composition of their root‐associated EM fungi. When the inoculum was homogenously distributed, a single EM fungal taxon dominated the roots of most pine seedlings, reducing the diversity of EM fungi at the treatment level, while substantially improving pine seedling performance. In contrast, clumped inoculum allowed the proliferation of several different EM fungi, increasing the overall EM fungal diversity. The most dominant EM fungal taxon detected in the homogeneous treatment was also a highly beneficial mutualist, implying that the trade‐off between competitive ability and mutualistic capacity does not always exist.     

A critical role for macrophages in promotion of urethane-induced lung carcinogenesis

Macrophages have established roles in tumor growth and metastasis, but information about their role in lung tumor promotion is limited. To assess the role of macrophages in lung tumorigenesis, we developed a method of minimally invasive, long-term macrophage depletion by repetitive intratracheal instillation of liposomal clodronate. Compared with controls treated with repetitive doses of PBS-containing liposomes, long-term macrophage depletion resulted in a marked reduction in tumor number and size at 4 mo after a single i.p. injection of the carcinogen urethane. After urethane treatment, lung macrophages developed increased M1 macrophage marker expression during the first 2-3 wk, followed by increased M2 marker expression by week 6. Using a strategy to reduce alveolar macrophages during tumor initiation and early promotion stages (weeks 1-2) or during late promotion and progression stages (weeks 4-16), we found significantly fewer and smaller lung tumors in both groups compared with controls. Late-stage macrophage depletion reduced VEGF expression and impaired vascular growth in tumors. In contrast, early-stage depletion of alveolar macrophages impaired urethane-induced NF-κB activation in the lungs and reduced the development of premalignant atypical adenomatous hyperplasia lesions at 6 wk after urethane injection. Together, these studies elucidate an important role for macrophages in lung tumor promotion and indicate that these cells have distinct roles during different stages of lung carcinogenesis.     

Experimental Myocardial Infarction Induces Altered Regulatory T Cell Hemostasis,and Adoptive Transfer Attenuates Subsequent Remodeling

Background

Ischemic cardiac damage is associated with upregulation of cardiac pro-inflammatory cytokines, as well as invasion of lymphocytes into the heart. Regulatory T cells (Tregs) are known to exert a suppressive effect on several immune cell types. We sought to determine whether the Treg pool is influenced by myocardial damage and whether Tregs transfer and deletion affect cardiac remodeling.

Methods and Results

The number and functional suppressive activity of Tregs were assayed in mice subjected to experimental myocardial infarction. The numbers of splenocyte-derived Tregs in the ischemic mice were significantly higher after the injury than in the controls, and their suppressive properties were significantly compromised. Compared with PBS, adoptive Treg transfer to mice with experimental infarction reduced infarct size and improved LV remodeling and functional performance by echocardiography. Treg deletion with blocking anti-CD25 antibodies did not influence infarct size or echocardiographic features of cardiac remodeling.

Conclusion

Treg numbers are increased whereas their function is compromised in mice with that underwent experimental infarction. Transfer of exogeneous Tregs results in attenuation of myocardial remodeling whereas their ablation has no effect. Thus, Tregs may serve as interesting potential interventional targets for attenuating left ventricular remodeling.     

Monitoring Brain Tumor Vascular Heamodynamic following Anti-Angiogenic Therapy with Advanced Magnetic Resonance Imaging in Mice

Advanced MR imaging methods have an essential role in classification, grading, follow-up and therapeutic management in patients with brain tumors. With the introduction of new therapeutic options, the challenge for better tissue characterization and diagnosis increase, calling for new reliable non-invasive imaging methods. In the current study we evaluated the added value of a combined protocol of blood oxygen level dependent (BOLD) imaging during hyperoxic challenge (termed hemodynamic response imaging (HRI)) in an orthotopic mouse model for glioblastoma under anti-angiogenic treatment with B20-4.1.1, an anti-VEGF antibody. In glioblastoma tumors, the elevated HRI indicated progressive angiogenesis as further confirmed by histology. In the current glioblastoma model, B20-treatment caused delayed tumor progression with no significant changes in HRI yet with slightly reduced tumor vascularity as indicated by histology. Furthermore, fewer apoptotic cells and higher proliferation index were detected in the B20-treated tumors compared to control-treated tumors. In conclusion, HRI provides an easy, safe and contrast agent free method for the assessment of the brain hemodynamic function, an additionally important clinical information.     

Synthesis and possible role of proteoglycans during Volvox development

During the embryonic development of Volvox the cells synthesize proteoglycans of relatively small molecular size up to 14 h after inversion. In the following stages there is a gradual transition towards larger forms, due to an increase in sulfated sugar moieties. The presence of sulfate in the medium was obligatory for this transition. None of these molecules could be dissociated in 4.0 M GuHCl, neither could they be digested by glycosidases. Of all proteases examined subtilisin, a bacterial protease, digested the proteoglycans. The possible role of proteoglycans during Volvox morphogenesis has been examined. Somatic proteoglycans caused spheroids to start their swimming earlier, although they inhibited their expansion. Spheroids cultured in sulfate-free medium start their swimming 24 h later than controls, but the addition of somatic proteoglycans to the sulfate-free medium could induce motility almost as well as in the normal forms. Embryonic weight proteoglycans caused aggregation of the spheroids, an effect that also appeared in the sulfate-free medium.     

Structural Dynamics of the Activation of Elongation Factor 2 Kinase by Ca2 +-Calmodulin

Eukaryotic elongation factor 2 kinase (eEF-2K), the only known calmodulin (CaM)-activated α-kinase, phosphorylates eukaryotic elongation factor 2 (eEF-2) on a specific threonine (Thr-56) diminishing its affinity for the ribosome and reducing the rate of nascent chain elongation during translation. Despite its critical cellular role, the precise mechanisms underlying the CaM-mediated activation of eEF-2K remain poorly defined. Here, employing a minimal eEF-2K construct (TR) that exhibits activity comparable to the wild-type enzyme and is fully activated by CaM in vitro and in cells, and using a variety of complimentary biophysical techniques in combination with computational modeling, we provide a structural mechanism by which CaM activates eEF-2K. Native mass analysis reveals that CaM, with two bound Ca^2 + ions, forms a stoichiometric 1:1 complex with TR. Chemical crosslinking mass spectrometry and small-angle X-ray scattering measurements localize CaM near the N-lobe of the TR kinase domain and the spatially proximal C-terminal helical repeat. Hydrogen/deuterium exchange mass spectrometry and methyl NMR indicate that the conformational changes induced on TR by the engagement of CaM are not localized but are transmitted to remote regions that include the catalytic site and the functionally important phosphate binding pocket. The structural insights obtained from the present analyses, together with our previously published kinetics data, suggest that TR, and by inference, wild-type eEF-2K, upon engaging CaM undergoes a conformational transition resulting in a state that is primed to efficiently auto-phosphorylate on the primary activating T348 en route to full activation.     

Thiol status in human sperm

The passage of spermatozoa along the epididymis is characterized by a gradual stabilization of intracellular organelles mainly through the oxidation of thiol groups. In this study, we examined the relationship between the thiol-disulfide status of human spermatozoa (using a specific fluorescent probe, monobromobimane) and routine semen analysis parameters. Fluorescence intensity was measured by spectrofluorimeter and its frequency distribution within samples, using a fluorescence-activated cell sorter. The mean proportion of reactive thiols SH/(SS + SH) in 29 semen samples was 29.8% +/- 2.5%. When comparing thiol labeling patterns, oligozoospermic samples differed from normozoospermic ones (P less than 0.05). However, within the normozoospermic group, no correlation was found between thiol-labeling patterns and routine sperm parameters or fertilizing capacity in vitro. No difference in thiol labeling patterns was found between "swim-up" and "whole semen" preparations.