Sumithra Urs

Selective expression of an aP2/Fatty Acid Binding Protein4-Cre transgene in non-adipogenic tissues during embryonic development

Mouse strains expressing the site-specific Cre recombinase facilitate conditional ablation or activation of genomic sequences when one or several exons of a gene of interest are flanked by loxP sites. Recently, several strains targeting Cre expression to adipocytes have been developed using promoter sequences from the aP2 (Fatty Acid Binding Protein 4, FABP4) gene for adipose tissue-specific gene expression studies. aP2/FABP4 is predominantly expressed in adipose tissue, and while this promoter provides adipocyte-restricted expression postnatally, its expression throughout embryonic development had not been previously characterized. In this report, we demonstrate that the aP2-Cre transgene is expressed and consistently localized within the embryo from mid-gestation stage 9.5 dpc. By 15.5 dpc, β-gal activity was detected primarily in the brown adipose tissue, trigeminal ganglia, dorsal root ganglia, cartilage primordia and vertebrae. Immunofluorescence staining for Cre recombinase and FABP4 protein showed a corresponding staining pattern similar to that of β-gal, confirming that Cre recombinase was produced in the transgenic line at late stages of development, and overlapped with endogenous aP2/FABP4 production. Further, fat-specific oil red O staining of tissue sections validated the presence of lipids in the stained tissues indicating that adipocytes and/or adipocyte-like cells were indeed present in these tissues. This is the first report to our knowledge to describe and confirm aP2/FABP4 promoter expression in this transgenic line during development in the mouse embryo and indicates that aP2/FABP4 expression occurs not only in mature adipocytes, but has a wider embryonic expression pattern than previously appreciated.

Notch and Transforming Growth Factor-β (TGFβ) Signaling Pathways Cooperatively Regulate Vascular Smooth Muscle Cell Differentiation

Notch and transforming growth factor-β (TGFβ) play pivotal roles during vascular development and the pathogenesis of vascular disease. The interaction of these two pathways is not fully understood. The present study utilized primary human smooth muscle cells (SMC) to examine molecular cross-talk between TGFβ1 and Notch signaling on contractile gene expression. Activation of Notch signaling using Notch intracellular domain or Jagged1 ligand induced smooth muscle α-actin (SM actin), smooth muscle myosin heavy chain, and calponin1, and the expression of Notch downstream effectors hairy-related transcription factors. Similarly, TGFβ1 treatment of human aortic smooth muscle cells induced SM actin, calponin1, and smooth muscle protein 22-α (SM22α) in a dose- and time-dependent manner. Hairy-related transcription factor proteins, which antagonize Notch activity, also suppressed the TGFβ1-induced increase in SMC markers, suggesting a general mechanism of inhibition. We found that Notch and TGFβ1 cooperatively activate SMC marker transcripts and protein through parallel signaling axes. Although the intracellular domain of Notch4 interacted with phosphoSmad2/3 in SMC, this interaction was not observed with Notch1 or Notch2. However, we found that CBF1 co-immunoprecipitated with phosphoSmad2/3, suggesting a mechanism to link canonical Notch signaling to phosphoSmad activity. Indeed, the combination of Notch activation and TGFβ1 treatment led to synergistic activation of a TGFβ-responsive promoter. This increase corresponded to increased levels of phosphoSmad2/3 interaction at Smad consensus binding sites within the SM actin, calponin1, and SM22α promoters. Thus, Notch and TGFβ coordinately induce a molecular and functional contractile phenotype by co-regulation of Smad activity at SMC promoters.

Hairy-Related Transcription Factors Inhibit Notch-Induced Smooth Muscle α-Actin Expression by Interfering With Notch Intracellular Domain/CBF-1 Complex Interaction With the CBF-1–Binding Site

Notch signaling regulates smooth muscle cell phenotype and is critical for vascular development. One Notch target is smooth muscle α-actin (SMA), a differentiated smooth muscle cell marker. The Notch intracellular domain (NotchICD) forms a complex with CBF-1 (C-promoter–binding factor-1) and directly induces SMA expression. Using primary human smooth muscle cells, we show that expression of the constitutive active ICD of human Notch1, Notch2, or Notch4 receptors increase SMA levels. NotchICD also induce expression of the transcriptional repressors HRT1 (Hairy-related transcription factor 1) and HRT2, in a CBF-1–dependent manner. However, unlike the activating effects of NotchICD, HRT1 or HRT2 represses basal SMA expression, and both are strong antagonists of NotchICD-induced SMA upregulation. This antagonism does not depend on histone deacetylase activity and occurs at the transcriptional level. Competitive coimmunoprecipitation experiments demonstrate that HRT does not disrupt the association of NotchICD and CBF-1, which form a complex in the presence or absence of HRTs. However, HRT suppresses NotchICD/CBF-1 binding to the SMA promoter, as measured by chromatin immunoprecipitation, and transactivation of an SMA promoter reporter spanning sequences −124/+32. SMA expression was regulated similarly following endogenous Notch activation in smooth muscle cells by coculture with endothelial cells, and this effect was also sensitive to HRT inhibition. Temporally defined HRT activity may constitute a negative feedback mechanism of Notch signaling. Our study presents a novel mechanism by which a balance between Notch signaling and HRT activity determines the expression of smooth muscle differentiation markers including SMA.

RhoA-Mediated Signaling in Notch-Induced Senescence-Like Growth Arrest and Endothelial Barrier Dysfunction

Objective—Notch signaling has a critical role in vascular development and morphogenesis. Activation of Notch in endothelial cells led to a senescence-like phenotype with loss of barrier function. Our objective was to understand the molecular pathways mediating this phenotype. Methods and Results—Human primary endothelial cells increase expression of Notch receptors and ligands during propagation in vitro toward natural senescence. This senescence was induced at low passage with Notch activation. We characterized the pathways activated downstream of Notch signaling. Notch was activated by Delta-like 4 ligand or constitutively active Notch receptors and measured for cell proliferation, migration, and sprouting. Notch signaling triggered early senescence in low-passage cells, characterized by increased p53 and p21 expression. The senescence phenotype was associated with hyperpermeability of the monolayer, with disrupted vascular endothelial cadherin and &bgr;-catenin levels and localization. Consistent with changes in cell shape and contact, we demonstrated that Notch activation increases myosin light chain phosphorylation by activating Rho kinase. Inhibition of Rho abrogated Notch-induced myosin light chain phosphorylation and led to enhanced barrier function by reorganizing F-actin to &bgr;-catenin-containing cell-cell adherens junctions. Conclusion—Our findings show that RhoA/Rho kinase regulation by Notch signaling in endothelial cells triggers a senescence phenotype associated with endothelial barrier dysfunction.

Sprouty1 is a critical regulatory switch of mesenchymal stem cell lineage allocation

Development of bone and adipose tissue are linked processes arising from a common progenitor cell, but having an inverse relationship in disease conditions such as osteoporosis. Cellular differentiation of both tissues relies on growth factor cues, and we focus this study on Sprouty1 (Spry1), an inhibitor of growth factor signaling. We tested whether Spry1 can modify the development of fat cells through its activity in regulating growth factors known to be important for adipogenesis. We utilized conditional expression and genetic‐null mouse models of Spry1 in adipocytes using the fatty acid binding promoter (aP2). Conditional deletion of Spry1 results in 10% increased body fat and decreased bone mass. This phenotype was rescued on Spry1 expression, which results in decreased body fat and increased bone mass. Ex vivo bone marrow experiments indicate Spry1 in bone marrow and adipose progenitor cells favors differentiation of osteoblasts at the expense of adipocytes by suppressing CEBP‐β and PPARγ while up regulating TAZ. Age and gender‐matched littermates expressing only Cre recombinase were used as controls. Spry1 is a critical regulator of adipocyte differentiation and mesenchymal stem cell (MSC) lineage allocation, potentially acting through regulation of CEBP‐β and TAZ.—Urs, S., Venkatesh, D., Tang, Y., Henderson, T., Yang, X., Friesel, R. E., Rosen, C. J., Liaw, L. Sprouty1 is a critical regulatory switch of mesenchymal stem cell lineage allocation. FASEB J. 24, 3264–3273 (2010). www.fasebj.org

Endothelial differentiation in multipotent cells derived from mouse and human white mature adipocytes

White mature adipocytes give rise to multipotent cells, so-called de-differentiated fat (DFAT) cells, when losing their fat in culture. The objective of this study was to examine the ability of DFAT cells to give rise to endothelial cells (ECs) in vitro and vivo. We demonstrate that mouse and human DFAT cells, derived from adipose tissue and lipospirate, respectively, initially lack expression of CD34, CD31, CD146, CD45 and pericyte markers, distinguishing them from progenitor cells previously identified in adipose stroma. The DFAT cells spontaneously differentiate into vascular ECs in vitro, as determined by real-time PCR, fluorescence activated cell sorting, immunostaining, and formation of tube structures. Treatment with bone morphogenetic protein (BMP)4 and BMP9, important in regulating angiogenesis, significantly enhances the EC differentiation. Furthermore, adipocyte-derived cells from Green Fluorescent Protein-transgenic mice were detected in the vasculature of infarcted myocardium up to 6 weeks after ligation of the left anterior descending artery in mice. We conclude that adipocyte-derived multipotent cells are able to spontaneously give rise to ECs, a process that is promoted by BMPs and may be important in cardiovascular regeneration and in physiological and pathological changes in fat and other tissues.

Soluble Forms of the Notch Ligands Delta1 and Jagged1 Promote in Vivo Tumorigenicity in NIH3T3 Fibroblasts with Distinct Phenotypes

We previously found that soluble forms of the Notch ligands Jagged1 and Delta1 induced fibroblast growth factor receptor-dependent cell transformation in NIH3T3 fibroblasts. However, the phenotypes of these lines differed, indicating distinct functional differences among these Notch ligands. In the present study, we used allografts to test the hypothesis that NIH3T3 fibroblasts that express soluble forms of Delta1 and Jagged1 accelerate tumorigenicity in vivo. With the exception of the full-length Jagged1 transfectant, all other cell lines, including the control, generated tumors when injected subcutaneously in athymic mice. Suppression of Notch signaling by the soluble ligands significantly increased tumor onset and growth, whereas full-length Jagged1 completely suppressed tumor development. In addition, there were striking differences in tumor pathology with respect to growth kinetics, vascularization, collagen content, size and number of necrotic foci, and invasiveness into the underlying tissue. Further, the production of angiogenic factors, including vascular endothelial growth factor, also differed among the tumor types. Lastly, both Jagged1- and Delta1-derived tumors contained phenotypically distinct populations of lipid-filled cells that corresponded with increased expression of adipocyte markers. The divergence of tumor phenotype may be attributed to ligand-specific alterations in Notch receptor responses in exogenous and endogenous cell populations within the allographs. Our findings demonstrate distinct functional properties for these Notch ligands in the promotion of tumorigenicity in vivo.

Effect of soluble Jagged1-mediated inhibition of Notch signaling on proliferation and differentiation of an adipocyte progenitor cell model

Adipose tissue development is dependent on multiple signaling mechanisms and cell-cell interactions that regulate adipogenesis, angiogenesis and extracellular remodeling. The Notch signaling pathway is an important cell-fate determinant whose role in adipogenesis is not clearly defined. To address this issue, we examined the effect of inhibition of Notch signaling by soluble-Jagged1 in the 3T3-L1 preadipocyte line. In vitro, soluble-Jagged1 expression in 3T3-L1 cells altered cell morphology, increased the rate of cell proliferation and induced an early transcriptional response to differentiation stimuli. However, these cells did not form mature adipocytes due to their inability to exit the cell-cycle in response to serum-starvation and glucocorticoid-induced cell-cycle arrest. In contrast, subcutaneous allografts of soluble-Jagged1 cells formed larger fat pads containing lipid-filled adipocytes with improved neovascularization compared with controls. Since adipogenesis is tightly associated with angiogenesis, we evaluated the influence of soluble-Jagged1 on endothelial cells by culturing them in cell-free conditioned media from preadipocytes. Soluble Jagged1-mediated inhibition of Notch signaling increased levels of secreted cytokines, potentially contributing to the improved cell growth and proliferation observed in these cultures. Our findings demonstrate an initial requirement of Notch signaling inactivation for preadipocyte cell commitment and support the hypothesis that cell-to-cell crosstalk between the preadipocytes and endothelial cells is required for neovascularization and remodeling of the tissue to promote hyperplasia and hypertrophy of differentiating adipocytes.

Notch1 activation in embryonic VE-cadherin populations selectively blocks hematopoietic stem cell generation and fetal liver hematopoiesis.

Hematopoietic stem cells (HSC) are found in several independent sites embryonically. Loss-of-function studies indicated that Notch1, but not Notch2 signaling was required for HSC emergence from the aortic-gonado-mesonephros (AGM) region. We previously showed that constitutive Notch1 activation impaired primitive erythroid differentiation, but its effects on HSC emergence from the AGM region were not studied. To further define specific roles of Notch receptors, we characterized HSC in mouse embryos expressing either Notch1 intracellular domain (ICD) or Notch4ICD in VE-cadherin or SM22α expressing populations. Although embryonic Notch1 activation in VE-cadherin populations led to lethality after E13.5, earlier defects in the fetal liver were observed. Embryos were analyzed at E12.5 to assess hematopoiesis and the phenotype of developing cells in the AGM region. We found that activation of Notch1 in the endothelial compartment in VE-cadherin expressing cells resulted in the absence of intra-aortic clusters and defects in fetal liver hematopoiesis. In contrast, although Notch4 expression is regulated during fetal hematopoiesis, activation of Notch4 in VE-cadherin expressing populations did not affect HSC phenotype, although later vascular remodeling was impaired. Likewise, activation of Notch1 in SM22α positive populations had no significant effect on hematopoiesis. Our results indicate a cell type-dependent activity and distinct features of Notch1 versus Notch4 signaling and their impact on HSC generation.

Diseases of Energy and Lipid Metabolism and Bone: Emerging Therapeutics

Lipids constitute a broad group of naturally occurring molecules that function as energy storage organs, as components of cell membranes, and as signaling molecules. Lipids and fatty acids are stored as triglycerides and are the most important source of energy for most organisms. When the body needs energy, lipolysis is initiated in adipocytes, and the triglycerides are hydrolyzed to glycerol and nonesterified fatty acids (NEFAs) [64]. The capacity to store triglycerides makes it possible to maintain energy homeostasis and prevent an abnormal toxic increase in plasma NEFAs. Under normal conditions, fat cells fine-tune the balance between lipogenesis and lipolysis of triglycerides in response to physiological needs. However, changes in lipid storage and metabolism, whether anabolic or catabolic, modulate whole body homeostasis. When fat mass is lacking as in lipoatrophy, there arise complications similar to those in type 2 diabetes, i.e., insulin resistance, dyslipidemia, hyperphagia, and liver steatosis. On the other hand, excessive lipid storage leads to obesity, along with a multitude of manifestations and clinical complications including altered insulin production. Fatty tissue accumulation is a major risk factor for chronic diseases, including the metabolic syndrome, also known as “syndrome X,” a cluster of phenotypes that contribute to elevating the risk of disorders. Symptoms of the metabolic syndrome include insulin resistance, impaired glucose regulation, dyslipidemia, hypertension, central obesity, and microalbuminuria, with insulin resistance the major feature. Between 60% and 90% of type 2 diabetes cases are thought to be due to obesity. Altogether, adipose tissue is essential to maintaining health and in glucose and lipid homeostasis [64].