Synergistic action of GA-binding protein and glucocorticoid receptor in transcription from the mouse mammary tumor virus promoter.

B lymphocytes are among the first cells to be infected by mouse mammary tumor virus (MMTV), and they play a crucial role in its life cycle. To study transcriptional regulation of MMTV in B cells, we have analyzed two areas of the long terminal repeat (LTR) next to the glucocorticoid receptor binding site, fp1 (at position -139 to -146 from the cap site) and fp2 (at -157 to -164). Both showed B-cell-specific protection in DNase I in vitro footprinting assays and contain binding sites for Ets transcription factors, a large family of proteins involved in cell proliferation and differentiation and oncogenic transformation. In gel retardation assays, fp1 and fp2 bound the heterodimeric Ets factor GA-binding protein (GABP) present in B-cell nuclear extracts, which was identified by various criteria: formation of dimers and tetramers, sensitivity to pro-oxidant conditions, inhibition of binding by specific antisera, and comigration of complexes with those formed by recombinant GABP. Mutations which prevented complex formation in vitro abolished glucocorticoid-stimulated transcription from an MMTV LTR linked to a reporter gene in transiently transfected B-cell lines, whereas they did not affect the basal level. Exogenously expressed GABP resulted in an increased level of hormone response of the LTR reporter plasmid and produced a synergistic effect with the coexpressed glucocorticoid receptor, indicating cooperation between the two. This is the first example of GABP cooperation with a steroid receptor, providing the opportunity for studying the integration of their intracellular signaling pathways.

PPARβ/δ activation blocks lipid-induced inflammatory pathways in mouse heart and human cardiac cells.

Owing to its high fat content, the classical Western diet has a range of adverse effects on the heart, including enhanced inflammation, hypertrophy, and contractile dysfunction. Proinflammatory factors secreted by cardiac cells, which are under the transcriptional control of nuclear factor-κB (NF-κB), may contribute to heart failure and dilated cardiomyopathy. The underlying mechanisms are complex, since they are linked to systemic metabolic abnormalities and changes in cardiomyocyte phenotype. Peroxisome proliferator-activated receptors (PPARs) are transcription factors that regulate metabolism and are capable of limiting myocardial inflammation and hypertrophy via inhibition of NF-κB. Since PPARβ/δ is the most prevalent PPAR isoform in the heart, we analyzed the effects of the PPARβ/δ agonist GW501516 on inflammatory parameters. A high-fat diet induced the expression of tumor necrosis factor-α, monocyte chemoattractant protein-1, and interleukin-6, and enhanced the activity of NF-κB in the heart of mice. GW501516 abrogated this enhanced proinflammatory profile. Similar results were obtained when human cardiac AC16 cells exposed to palmitate were coincubated with GW501516. PPARβ/δ activation by GW501516 enhanced the physical interaction between PPARβ/δ and p65, which suggests that this mechanism may also interfere NF-κB transactivation capacity in the heart. GW501516-induced PPARβ/δ activation can attenuate the inflammatory response induced in human cardiac AC16 cells exposed to the saturated fatty acid palmitate and in mice fed a high-fat diet. This is relevant, especially taking into account that PPARβ/δ has been postulated as a potential target in the treatment of obesity and the insulin resistance state.

Optimization and regeneration kinetics of lymphatic-specific photodynamic therapy in the mouse dermis.

Lymphatic vessels transport fluid, antigens, and immune cells to the lymph nodes to orchestrate adaptive immunity and maintain peripheral tolerance. Lymphangiogenesis has been associated with inflammation, cancer metastasis, autoimmunity, tolerance and transplant rejection, and thus, targeted lymphatic ablation is a potential therapeutic strategy for treating or preventing such events. Here we define conditions that lead to specific and local closure of the lymphatic vasculature using photodynamic therapy (PDT). Lymphatic-specific PDT was performed by irradiation of the photosensitizer verteporfin that effectively accumulates within collecting lymphatic vessels after local intradermal injection. We found that anti-lymphatic PDT induced necrosis of endothelial cells and pericytes, which preceded the functional occlusion of lymphatic collectors. This was specific to lymphatic vessels at low verteporfin dose, while higher doses also affected local blood vessels. In contrast, light dose (fluence) did not affect blood vessel perfusion, but did affect regeneration time of occluded lymphatic vessels. Lymphatic vessels eventually regenerated by recanalization of blocked collectors, with a characteristic hyperplasia of peri-lymphatic smooth muscle cells. The restoration of lymphatic function occurred with minimal remodeling of non-lymphatic tissue. Thus, anti-lymphatic PDT allows control of lymphatic ablation and regeneration by alteration of light fluence and photosensitizer dose.

In vivo effects of a recombinant vaccinia virus expressing a mouse mammary tumor virus superantigen.

Early after infection, the mouse mammary tumor virus (MMTV) expresses a superantigen (SAg) at the surface of B lymphocytes. Interaction with the T-cell receptor Vbeta domain induces a polyclonal proliferative response of the SAg-reactive T cells. Stimulated T cells become anergic and are deleted from the T-cell repertoire. We have used a recombinant vaccinia virus encoding the MMTV(GR) SAg to dissect the effects of the retroviral SAg during an unrelated viral infection. Subcutaneous infection with this recombinant vaccinia virus induces a very rapid increase of Vbeta14 T cells in the draining lymph node. This stimulation does not require a large Plumber of infectious particles and is not strictly dependent on the expression of the major histocompatibility complex class II I-E molecule, as it is required after MMTV(GR) infection. In contrast to MMTV infection during which B cells are infected, we do not observe any clonal deletion of the reactive T cells following the initial stimulation phase. Our data show that contrary to the case with MMTV, macrophages but not B cells are the targets of infection by vaccinia virus in the lymph node, indicating the ability of these cells to present a retroviral SAg. The altered SAg expression in a different target cell observed during recombinant vaccinia virus infection therefore results in significant changes in the SAg response.

"Peptabody": a new type of high avidity binding protein.

A new type of high avidity binding molecule, termed "peptabody" was created by harnessing the effect of multivalent interaction. A short peptide ligand was fused via a semi-rigid hinge region with the coiled-coil assembly domain of the cartilage oligomeric matrix protein, resulting in a pentameric multivalent binding molecule. In the first peptabody (Pab-S) described here, a peptide (S) specific for the mouse B-cell lymphoma BCL1 surface Ig idiotype, was selected from a phage display library. A fusion gene was constructed encoding peptide S, followed by the 24 aa hinge region from camel IgG and a modified 55 aa cartilage oligomeric matrix protein pentamerization domain. The Pab-S fusion protein was expressed in Escherichia coli in a soluble form at high levels and purified in a single step by metal-affinity chromatography. Pab-S specifically bound the BCL1 surface idiotype with an avidity of about 1 nM, which corresponds to a 2 x 10(5)-fold increase compared with the affinity of the synthetic peptide S itself. Biochemical characterization showed that Pab-S is a stable homopentamer of about 85 kDa, with interchain disulfide bonds. Pab-S can be dissociated under denaturing and reducing conditions and reassociated as a pentamer with full-binding activity. This intrinsic feature provides an easy way to combine Pab molecules with two different peptide specificities, thus producing heteropentamers with bispecific and/or chelating properties.

Experimental analysis and computer prediction of CTF/NFI transcription factor DNA binding sites.

Accurate prediction of transcription factor binding sites is needed to unravel the function and regulation of genes discovered in genome sequencing projects. To evaluate current computer prediction tools, we have begun a systematic study of the sequence-specific DNA-binding of a transcription factor belonging to the CTF/NFI family. Using a systematic collection of rationally designed oligonucleotides combined with an in vitro DNA binding assay, we found that the sequence specificity of this protein cannot be represented by a simple consensus sequence or weight matrix. For instance, CTF/NFI uses a flexible DNA binding mode that allows for variations of the binding site length. From the experimental data, we derived a novel prediction method using a generalised profile as a binding site predictor. Experimental evaluation of the generalised profile indicated that it accurately predicts the binding affinity of the transcription factor to natural or synthetic DNA sequences. Furthermore, the in vitro measured binding affinities of a subset of oligonucleotides were found to correlate with their transcriptional activities in transfected cells. The combined computational-experimental approach exemplified in this work thus resulted in an accurate prediction method for CTF/NFI binding sites potentially functioning as regulatory regions in vivo.

Genetic selection system allowing monitoring of myofibrillogenesis in living cardiomyocytes derived from mouse embryonic stem cells

Embryonic stem (ES) cell-derived cardiomyocytes recapitulate cardiomyogenesis in vitro and are a potential source of cells for cardiac repair. However, this requires enrichment of mixed populations of differentiating ES cells into cardiomyocytes. Toward this goal, we have generated bicistronic vectors that express both the blasticidin S deaminase (bsd) gene and a fusion protein consisting of either myosin light chain (MLC)-3f or human alpha-actinin 2A and enhanced green fluorescent protein (EGFP) under the transcriptional control of the alpha-cardiac myosin heavy chain (alpha-MHC) promoter. Insertion of the DNase I-hypersensitive site (HS)-2 element from the beta-globin locus control region, which has been shown to reduce transgene silencing in other cell systems, upstream of the transgene promoter enhanced MLC3f-EGFP gene expression levels in mouse ES cell lines. The alpha-MHC-alpha-actinin-EGFP, but not the alpha-MHC-MLC3f-EGFP, construct resulted in the correct incorporation of the newly synthesized fusion protein at the Z-band of the sarcomeres in ES cell-derived cardiomyocytes. Exposure of embryoid bodies to blasticidin S selected for a relatively pure population of cardiomyocytes within 3 days. Myofibrillogenesis could be monitored by fluorescence microscopy in living cells due to sarcomeric epitope tagging. Therefore, this genetic system permits the rapid selection of a relatively pure population of developing cardiomyocytes from a heterogeneous population of differentiating ES cells, simultaneously allowing monitoring of early myofibrillogenesis in the selected myocytes

Inhibition of the Kit Ligand/c-Kit Axis Attenuates Metastasis in a Mouse Model Mimicking Local Breast Cancer Relapse after Radiotherapy.

PURPOSE: Local breast cancer relapse after breast-saving surgery and radiotherapy is associated with increased risk of distant metastasis formation. The mechanisms involved remain largely elusive. We used the well-characterized 4T1 syngeneic, orthotopic breast cancer model to identify novel mechanisms of postradiation metastasis. EXPERIMENTAL DESIGN: 4T1 cells were injected in 20 Gy preirradiated mammary tissue to mimic postradiation relapses, or in nonirradiated mammary tissue, as control, of immunocompetent BALB/c mice. Molecular, biochemical, cellular, histologic analyses, adoptive cell transfer, genetic, and pharmacologic interventions were carried out. RESULTS: Tumors growing in preirradiated mammary tissue had reduced angiogenesis and were more hypoxic, invasive, and metastatic to lung and lymph nodes compared with control tumors. Increased metastasis involved the mobilization of CD11b(+)c-Kit(+)Ly6G(high)Ly6C(low)(Gr1(+)) myeloid cells through the HIF1-dependent expression of Kit ligand (KitL) by hypoxic tumor cells. KitL-mobilized myeloid cells homed to primary tumors and premetastatic lungs, to give rise to CD11b(+)c-Kit(-) cells. Pharmacologic inhibition of HIF1, silencing of KitL expression in tumor cells, and inhibition of c-Kit with an anti-c-Kit-blocking antibody or with a tyrosine kinase inhibitor prevented the mobilization of CD11b(+)c-Kit(+) cells and attenuated metastasis. C-Kit inhibition was also effective in reducing mobilization of CD11b(+)c-Kit(+) cells and inhibiting lung metastasis after irradiation of established tumors. CONCLUSIONS: Our work defines KitL/c-Kit as a previously unidentified axis critically involved in promoting metastasis of 4T1 tumors growing in preirradiated mammary tissue. Pharmacologic inhibition of this axis represents a potential therapeutic strategy to prevent metastasis in breast cancer patients with local relapses after radiotherapy. Clin Cancer Res; 18(16); 4365-74. ©2012 AACR.

Perspective: from molecular endocrinology of mouse to genomic endocrinology of animals.

This short perspective explores some ways in which new genomic methodologies impact the study of endocrine signaling. Emphasis is put on the impact of studying species which are not molecular biology models. This opens the door to using knowledge molecular endocrinology in areas of biology as distant as conservation biology, as well as enriching endocrinology with information from biodiversity and natural variation.

Non-contact microdrilling of mouse zona pellucida with an objective-delivered 1.48-microns diode laser.

BACKGROUND and OBJECTIVE: A non-touch laser-induced microdrilling procedure is studied on mouse zona pellucida (ZP). STUDY DESIGN/MATERIALS and METHODS: A 1.48-microns diode laser beam is focused in a 8-microns spot through a 45x objective of an inverted microscope. Mouse zygotes, suspended in a culture medium, are microdrilled by exposing their ZP to a short laser irradiation and allowed to develop in vitro. RESULTS: Various sharp-edged holes can be generated in the ZP with a single laser irradiation. Sizes can be varied by changing irradiation time (3-100 ms) or laser power (22-55 mW). Drilled zygotes present no signs of thermal damage under light and scanning electron microscopy and develop as expected in vitro, except for a distinct eight-shaped hatching behavior. CONCLUSION: The microdrilling procedure can generate standardized holes in mouse ZP, without any visible side effects. The hole formation can be explained by a local photothermolysis of the protein matrix.