Batya Cohen

Modulation of insulin activities by leptin

Leptin mediates its effects on food intake through the hypothalamic form of its receptor OB-R. Variants of OB-R are found in other tissues, but their function is unknown. Here, an OB-R variant was found in human hepatic cells. Exposure of these cells to leptin, at concentrations comparable with those present in obese individuals, caused attenuation of several insulin-induced activities, including tyrosine phosphorylation of the insulin receptor substrate-1 (IRS-1), association of the adapter molecule growth factor receptor-bound protein 2 with IRS-1, and down-regulation of gluconeogenesis. In contrast, leptin increased the activity of IRS-1-associated phosphatidylinositol 3-kinase. These in vitro studies raise the possibility that leptin modulates insulin activities in obese individuals.

The human interferon alpha/beta receptor: characterization and molecular cloning

We describe a universal ligand-binding receptor for human interferons alpha and interferon beta (type I IFNs). A soluble 40 kDa IFN-alpha/beta receptor (p40) that blocks the activity of type I IFNs was purified from urine and sequenced. Antibodies raised against p40 completely block the activity of several type I IFNs and immuno-precipitate both a cellular 102 kDa IFN-alpha/beta receptor and its cross-linked complexes with IFN-alpha 2. The receptor is a disulfide-linked dimer, consisting of 51 kDa subunits. We isolated and expressed a 1.5 kb cDNA, coding for the IFN-alpha/beta receptor. Its 331 amino acid sequence includes a leader and a transmembrane region, while its ectodomain corresponds to p40. IFN-alpha/beta receptor is physically associated with the cytoplasmic Tyr kinase JAK1, hence, in addition to ligand binding, it is directly involved in signal transduction.

Insulin induces transcription of target genes through the hypoxia-inducible factor HIF-1alpha/ARNT.

Hypoxic stress induces the expression of genes associated with increased energy flux, including the glucose transporters Glut1 and Glut3, several glycolytic enzymes, nitric oxide synthase, tyrosine hydroxylase, erythropoietin and vascular endothelial growth factor (VEGF). Induction of these genes is mediated by a common basic helix–loop–helix‐PAS transcription complex, the hypoxia‐inducible factor‐1α (HIF‐1α)/aryl hydrocarbon nuclear translocator (ARNT). Insulin also induces some of these genes; however, the underlying mechanism is unestablished. We report here that insulin shares with hypoxia the ability to induce the HIF‐1α/ARNT transcription complex in various cell types. This induction was demonstrated by electrophoretic mobility shift of the hypoxia response element (HRE), and abolished by specific antisera to HIF‐1α and ARNT, and by transcription activation of HRE reporter vectors. Furthermore, basal and insulin‐induced expression of Glut1, Glut3, aldolase A, phosphoglycerate kinase and VEGF was reduced in cells having a defective ARNT. Similarly, the insulin‐induced activation of HRE reporter vectors and VEGF was impaired in these cells and was rescued by re‐introduction of ARNT. Finally, insulin‐like growth factor‐I (IGF‐I) also induced the HIF‐1α/ARNT transcription complex. These observations establish a novel signal transduction pathway of insulin and IGF‐I and broaden considerably the scope of activity of HIF‐1α/ARNT.

MRI detection of transcriptional regulation of gene expression in transgenic mice.

Ferritin, the iron storage protein, was recently suggested to be a candidate reporter for the detection of gene expression by magnetic resonance imaging (MRI). Here we report the generation of TET:EGFP-HAferritin (tet-hfer) transgenic mice, in which tissue-specific inducible transcriptional regulation of expression of the heavy chain of ferritin could be detected in vivo by MRI. We show organ specificity by mating the tet-hfer mice with transgenic mice expressing tetracycline transactivator (tTA) in liver hepatocytes and in vascular endothelial cells. Tetracycline-regulated overexpression of ferritin resulted in specific alterations of the transverse relaxation rate (R2) of water. Transgene-dependent changes in R2 were detectable by MRI in adult mice, and we also found fetal developmental induction of transgene expression in utero. Thus, the tet-hfer MRI reporter mice provide a new transgenic mouse platform for in vivo molecular imaging of reporter gene expression by MRI during both embryonic and adult life.

Ligand-Induced Association of the Type I Interferon Receptor Components

Two transmembrane polypeptides, IFNAR and IFN-alpha/Beta R, were previously identified as essential components of the type I interferon (IFN) receptor, but their interrelationship and role in ligand binding were not clear. To study these issues, we stably expressed and characterized the two polypeptides in host murine cells. In human cells, native IFN-alpha/beta R is a 102-kDa protein but upon reduction only a 51-kDa protein is detected. In host murine cells human IFN-alpha/beta R was expressed as a 51-kDa protein. Host cells expressing IFN-alpha/beta R bound IFN-alpha 2 with a high affinity (Kd of 3.6 nM), whereas cells expressing IFNAR exhibited no ligand binding. Upon coexpression of IFNAR and the 51-kDa IFN-alpha/beta R, the affinity for IFN-alpha 2 was increased 10-fold, approaching that of the native receptor. We show by cross-linking that both the cloned (51-kDa) and native (102-kDa) IFN-alpha/beta R bind IFN-alpha 2 to form an intermediate product, while IFNAR associates with this product to form a ternary complex. Hence, IFNAR and IFN-alpha/beta R are components of a common type I IFN receptor, cooperating in ligand binding. Ligand-induced association of IFNAR and IFN-alpha/beta R probably triggers transmembrane signaling.

Enhancer-like interferon responsive sequences of the human and murine (2'-5') oligoadenylate synthetase gene promoters.

The human (2′‐5′) oligo(A) synthetase gene contains two independent cis‐acting DNA elements, A and B, which act as transcriptional enhancers. Element A alone is not activated by IFN treatment. Element B alone confers IFN‐inducibility to the herpes tk promoter. Two murine (2′‐5′) oligo(A) synthetase genes were isolated and their promoter sequences show high conservation of element A and B. A synthetic oligonucleotide, containing 16 bp of the human element B, or 14 bp of the homologue murine element B, was linked to a TK‐CAT construct. These oligonucleotides were shown to be sufficient to activate the TK promoter in the presence of IFN. When multiple repeats of the interferon‐responsive sequence (E‐IRS) were cloned in 5′ of the TK promoter, the activation ratio was increased. In vitro, specific binding of nuclear protein(s) is observed to the radiolabelled synthetic human E‐IRS. This binding is competed by the addition of cold synthetic mouse E‐IRS or fragments of genomic DNA containing the E‐IRS.

Leptin Induces Angiopoietin-2 Expression in Adipose Tissues

Adipose tissues consisting of adipocytes, microvasculature, and stroma are completely ablated upon over-expression of leptin in rats. This tissue regression is mediated by enhanced lipid beta-oxidation, adipocyte dedifferentiation, and apoptosis. To further characterize this phenomenon, we studied the possible effect of leptin on the adipose microvasculature. Tissue microvasculature is maintained by the interplay between positive and negative signals mediated by factors including vascular endothelial growth factor (VEGF), basic fibroblast growth factor, angiopoietin-1 (Ang-1), and Ang-2. Expression of the negative signal Ang-2 was reported in fetal tissues and in the adult ovary, which undergoes vascular remodeling or regression. We demonstrate that leptin induces the expression of Ang-2 in adipose tissue without a concomitant increase in VEGF. Induction of Ang-2 occurred in an autocrine manner, as demonstrated in cultured adipocytes but not in several other cell types. This tissue-specific induction of Ang-2 coincided with initiation of apoptosis in adipose endothelial cells. We propose that induction of Ang-2 by leptin in adipose cells is one of the events leading to adipose tissue regression.

Mammalian type I interferon receptors consists of two subunits: IFNaR1 and IFNaR2

The human type I interferon (IFN) receptor consists of two essential subunits, huIFNaR1 and huIFNaR2; however, so far only IFNaR1 has been identified in other species. Furthermore, it has been suggested that in some species the type I IFN receptor may consist of a single subunit, since expression of murine IFNaR1 in human cells rendered them responsive to several type I murine IFNs. To resolve this issue, we screened a mouse cDNA library with a probe derived from huIFNaR2 cDNA. A cDNA clone, coding for a transmembrane protein which has 49% identity with huIFNaR2 was isolated. This level of identity suggests that this cDNA codes for a muIFNaR2. In addition, several cDNA clones, coding for two distinct soluble variants of muIFNaR2 were identified. To test whether muIFNaR2 is a functional component of the receptor, we co-expressed it with muIFNaR1 in human cells and with an IFN-responsive luciferase reporter vector. Treatment of these cells with muIFN-beta induced high levels of luciferase, whereas no induction was obtained in cells expressing only one of the two subunits. We therefore conclude that the murine type I IFN receptor consists of two different subunits--a configuration shared by humans, and probably all other mammals.

Molecular imaging of angiogenesis

Angiogenesis (the growth of new blood vessels) is a complex multistep process that involves multiple cell types, numerous growth factors, and complex regulatory checks and balances. Tight control of vascular remodeling evolved to ensure stability of the vasculature while maintaining the bodys ability to rapidly mount an angiogenic response requiring a high degree of plasticity. Angiogenesis is critical not only for physiological development, but also for the progression of pathologies, and is thus a target for therapeutic intervention. The importance of the process coupled with the ease of access for delivery of contrast agents makes the vasculature at large, and angiogenesis in particular, a favorable target of functional and molecular imaging. Recent developments in molecular imaging tools have expanded our views to encompass many components of the process. Functional imaging of blood volume, vessel permeability, and vasoreactivity is complemented by novel contrast agents that reveal specific targets on endothelial cells. Methods have been developed to label vascular cells so as to track their recruitment to sites of angiogenesis, and new “smart” contrast agents have been designed to reveal the activity of enzymatic reactions in altering the extracellular matrix (ECM) during angiogenesis. J. Magn. Reson. Imaging 2007.

MRI reporter genes: applications for imaging of cell survival, proliferation, migration and differentiation

Molecular imaging strives to detect molecular events at the level of the whole organism. In some cases, the molecule of interest can be detected either directly or with targeted contrast media. However many genes and proteins and particularly those located in intracellular compartments are not accessible for targeted agents. The transcriptional regulation of these genes can nevertheless be detected, although indirectly, using reporter gene encoding for readily detectable proteins. Such reporter proteins can be expressed in the tissue of interest by genetically introducing the reporter gene in the target cells. Imaging of reporter genes has become a powerful tool in modern biomedical research. Typically, expression of fluorescent and bioluminescent proteins and the reaction product of expressed enzymes and exogenous substrates were examined using in vitro histological methods and in vivo whole body imaging methods. Recent advances in MRI reporter gene methods raised the possibility that MRI could become a powerful tool for concomitant high‐resolution anatomical and functional imaging and for imaging of reporter gene activity. An immediate application of MRI reporter gene methods was by monitoring gene expression patterns in gene therapy and in vivo imaging of the survival, proliferation, migration and differentiation of pluripotent and multipotent cells used in cell‐based regenerative therapies for cancer, myocardial infarction and neural degeneration. In this review, we characterized a variety of MRI reporter gene methods based on their applicability to report cell survival/proliferation, migration and differentiation. In particular, we discussed which methods were best suited for translation to clinical use in regenerative therapies. Copyright