Jorge Laborda

Identification of a Lipase-linked Cell Membrane Receptor for Pigment Epithelium-derived Factor

Pigment epithelium-derived factor (PEDF) is an extracellular multifunctional protein belonging to the serpin superfamily with demonstrable neurotrophic, gliastatic, neuronotrophic, antiangiogenic, and antitumorigenic properties. We have previously provided biochemical evidence for high affinity PEDF-binding sites and proteins in plasma membranes of retina, retinoblastoma, and CNS cells. This study was designed to reveal a receptor involved in the biological activities of PEDF. Using a yeast two-hybrid screening, we identified a novel gene from pigment epithelium of the human retina that codes for a PEDF-binding partner, which we term PEDF-R. The derived polypeptide has putative transmembrane, intracellular and extracellular regions, and a phospholipase domain. Recently, PEDF-R (TTS-2.2/independent phospholipase A2 (PLA2)ζ and mouse desnutrin/ATGL) has been described in adipose cells as a member of the new calcium-independent PLA2/nutrin/patatin-like phospholipase domain-containing 2 (PNPLA2) family that possesses triglyceride lipase and acylglycerol transacylase activities. Here we describe the PEDF-R gene expression in the retina and its heterologous expression by bacterial and eukaryotic systems, and we demonstrate that its protein product has specific and high binding affinity for PEDF, has a potent phospholipase A2 activity that liberates fatty acids, and is associated with eukaryotic cell membranes. Most importantly, PEDF binding stimulates the enzymatic phospholipase A2 activity of PEDF-R. In conclusion, we have identified a novel PEDF-R gene in the retina for a phospholipase-linked membrane protein with high affinity for PEDF, suggesting a molecular pathway by which ligand/receptor interaction on the cell surface could generate a cellular signal.

Postnatal loss of Dlk1 imprinting in stem cells and niche astrocytes regulates neurogenesis

The gene for the atypical NOTCH ligand delta-like homologue 1 (Dlk1) encodes membrane-bound and secreted isoforms that function in several developmental processes in vitro and in vivo. Dlk1, a member of a cluster of imprinted genes, is expressed from the paternally inherited chromosome. Here we show that mice that are deficient in Dlk1 have defects in postnatal neurogenesis in the subventricular zone: a developmental continuum that results in depletion of mature neurons in the olfactory bulb. We show that DLK1 is secreted by niche astrocytes, whereas its membrane-bound isoform is present in neural stem cells (NSCs) and is required for the inductive effect of secreted DLK1 on self-renewal. Notably, we find that there is a requirement for Dlk1 to be expressed from both maternally and paternally inherited chromosomes. Selective absence of Dlk1 imprinting in both NSCs and niche astrocytes is associated with postnatal acquisition of DNA methylation at the germ-line-derived imprinting control region. The results emphasize molecular relationships between NSCs and the niche astrocyte cells of the microenvironment, identifying a signalling system encoded by a single gene that functions coordinately in both cell types. The modulation of genomic imprinting in a stem-cell environment adds a new level of epigenetic regulation to the establishment and maintenance of the niche, raising wider questions about the adaptability, function and evolution of imprinting in specific developmental contexts.

Notch-1 Controls the Expression of Fatty Acid-activated Transcription Factors and Is Required for Adipogenesis

Notch, a transmembrane receptor member of the homeotic epidermal growth factor-like family of proteins, participates in cell-to-cell signaling to control cell fate during development. Activated Notch-1 constructs lacking the extracellular region prevent differentiation of several mammalian cells in vitro. This effect, however, bypasses the normal mechanisms of cell-to-cell interactions in which Notch-1 participates. We investigated the role of Notch-1 in the hormone-induced adipocyte differentiation of 3T3-L1 fibroblasts, a paradigmatic model of adipogenesis that requires cell-to-cell contact. Unlike other differentiation models, Notch-1 expression and function were necessary conditions for adipogenesis. Impaired Notch-1 expression by antisenseNotch-1 constructs prevented adipocyte differentiation. Strategies aimed at blocking putative Notch/ligand interactions also blocked adipogenesis, implicating Notch as a critical molecule in cell-to-cell signaling necessary for differentiation. Inhibition of Notch-1 expression or function decreased the expression of peroxisomal proliferator-activated receptors δ and γ, transcription factors that control adipocyte differentiation and that are up-regulated at cell confluence. These results implicate Notch in the commitment of 3T3-L1 cells to undergo adipogenesis by controlling the expression of the principal regulators of this process.

Notch-1 Up-Regulation and Signaling following Macrophage Activation Modulates Gene Expression Patterns Known to Affect Antigen-Presenting Capacity and Cytotoxic Activity

Notch signaling has been extensively implicated in cell-fate determination along the development of the immune system. However, a role for Notch signaling in fully differentiated immune cells has not been clearly defined. We have analyzed the expression of Notch protein family members during macrophage activation. Resting macrophages express Notch-1, -2, and -4, as well as the Notch ligands Jagged-1 and -2. After treatment with LPS and/or IFN-γ, we observed a p38 MAPK-dependent increase in Notch-1 and Jagged-1 mRNA and protein levels. To study the role of Notch signaling in macrophage activation, we forced the transient expression of truncated, active intracellular Notch-1 (Notch-IC) proteins in Raw 264.7 cells and analyzed their effects on the activity of transcription factors involved in macrophage activation. Notch-IC increased STAT-1-dependent transcription. Furthermore, Raw 264.7 Notch-IC stable transfectants increased STAT1-dependent transcription in response to IFN-γ, leading to higher expression of IFN regulatory factor-1, suppressor of cytokine signaling-1, ICAM-1, and MHC class II proteins. This effect was independent from an increase of STAT1 Tyr or Ser phosphorylation. However, inducible NO synthase expression and NO production decreased under the same conditions. Our results show that Notch up-regulation and subsequent signaling following macrophage activation modulate gene expression patterns known to affect the function of mature macrophages.

Notch1 upregulates LPS-induced macrophage activation by increasing NF-κB activity

Macrophages present different Notch receptors and ligands on their surface. Following macrophage activation by LPS or other TLR ligands, Notch1 expression is upregulated. We report here that Notch signaling increases both basal and LPS‐induced NF‐κB activation, favoring the expression of genes implicated in the inflammatory response, such as the cytokines TNF‐α and IL‐6, or enzymes, such as iNOS. Delta4 seems to be the most effective ligand to induce Notch activation and increasing NF‐κB transcriptional activity in macrophages. We show that Notch1 signaling promotes NF‐κB translocation to the nucleus and DNA binding by increasing both phosphorylation of the IκB kinase α/β complex and the expression of some NF‐κB family members. Treatment of macrophages with the γ‐secretase inhibitor DAPT, which prevents the cleavage and activation of Notch receptors, inhibits all these processes, diminishing NF‐κB activity following LPS stimulation. Additionally, we show that the active intracellular Notch fragment can directly interact with TNF‐α and iNOS promoters. Our results suggest that Notch signaling results in an amplification of the macrophage‐dependent inflammatory response by enhancing NF‐κB signaling.

Predictors of acquired lipodystrophy in juvenile-onset dermatomyositis and a gradient of severity

We describe the clinical features of 28 patients with juvenile dermatomyositis (JDM) and 1 patient with adult-onset dermatomyositis (DM), all of whom developed lipodystrophy (LD) that could be categorized into 1 of 3 phenotypes, generalized, partial, or focal, based on the pattern of fat loss distribution. LD onset was often delayed, beginning a median of 4.6 years after diagnosis of DM. Calcinosis, muscle atrophy, joint contractures, and facial rash were DM disease features found to be associated with LD. Panniculitis was associated with focal lipoatrophy while the anti-p155 autoantibody, a newly described myositis-associated autoantibody, was more associated with generalized LD. Specific LD features such as acanthosis nigricans, hirsutism, fat redistribution, and steatosis/nonalcoholic steatohepatitis were frequent in patients with LD, in a gradient of frequency and severity among the 3 sub-phenotypes. Metabolic studies frequently revealed insulin resistance and hypertriglyceridemia in patients with generalized and partial LD. Regional fat loss from the thighs, with relative sparing of fat loss from the medial thighs, was more frequent in generalized than in partial LD and absent from DM patients without LD. Cytokine polymorphisms, the C3 nephritic factor, insulin receptor antibodies, and lamin mutations did not appear to play a pathogenic role in the development of LD in our patients. LD is an under-recognized sequela of JDM, and certain DM patients with a severe, prolonged clinical course and a high frequency of calcinosis appear to be at greater risk for the development of this complication. High-risk JDM patients should be screened for metabolic abnormalities, which are common in generalized and partial LD and result in much of the LD-associated morbidity. Further study is warranted to investigate the pathogenesis of acquired LD in patients with DM. Abbreviations: CI = confidence interval, CT = computerized tomography, dlk = delta-like, DM= dermatomyositis, DXA = dual-energy X-ray absorptiometry, HDL = high-density lipoprotein, HIV = human immunodeficiency virus, HOMA-IR = homeostasis model assessment of insulin resistance, IL = interleukin, IR = insulin resistance, JDM = juvenile dermatomyositis, LA = lipoatrophy, LD = lipodystrophy, LDL = low-density lipoprotein, LMNA= lamin A, MRI = magnetic resonance imaging, NASH = nonalcoholic steatohepatitis, NIH = National Institutes of Health, OGTT = oral glucose tolerance test, OR = odds ratio, PCR = polymerase chain reaction, TNF = tumor necrosis factor, TTP = tristetraprolin.

dlk1/FA1 Regulates the Function of Human Bone Marrow Mesenchymal Stem Cells by Modulating Gene Expression of Pro-inflammatory Cytokines and Immune Response-related Factors

dlk1/FA1 (delta-like 1/fetal antigen-1) is a member of the epidermal growth factor-like homeotic protein family whose expression is known to modulate the differentiation signals of mesenchymal and hematopoietic stem cells in bone marrow. We have demonstrated previously that Dlk1 can maintain the human bone marrow mesenchymal stem cells (hMSC) in an undifferentiated state. To identify the molecular mechanisms underlying these effects, we compared the basal gene expression pattern in Dlk1-overexpressing hMSC cells (hMSC-dlk1) versus control hMSC (negative for Dlk1 expression) by using Affymetrix HG-U133A microarrays. In response to Dlk1 expression, 128 genes were significantly up-regulated (with >2-fold; p < 0.001), and 24% of these genes were annotated as immune response-related factors, including pro-inflammatory cytokines, in addition to factors involved in the complement system, apoptosis, and cell adhesion. Also, addition of purified FA1 to hMSC up-regulated the same factors in a dose-dependent manner. As biological consequences of up-regulating these immune response-related factors, we showed that the inhibitory effects of dlk1 on osteoblast and adipocyte differentiation of hMSC are associated with Dlk1-induced cytokine expression. Furthermore, Dlk1 promoted B cell proliferation, synergized the immune response effects of the bacterial endotoxin lipopolysaccharide on hMSC, and led to marked transactivation of the NF-κB. Our data suggest a new role for Dlk1 in regulating the multiple biological functions of hMSC by influencing the composition of their microenvironment “niche.” Our findings also demonstrate a role for Dlk1 in mediating the immune response.

Mouse Resistin Modulates Adipogenesis and Glucose Uptake in 3T3-L1 Preadipocytes Through the ROR1 Receptor

Mouse resistin, a cysteine-rich protein primarily secreted from mature adipocytes, is involved in insulin resistance and type 2 diabetes. Human resistin, however, is mainly secreted by immune mononuclear cells, and it competes with lipopolysaccharide for the binding to Toll-like receptor 4, which could mediate some of the well-known proinflammatory effects of resistin in humans. In addition, resistin has been involved in the regulation of many cell differentiation and proliferation processes, suggesting that different receptors could be involved in mediating its numerous effects. Thus, a recent work identifies an isoform of Decorin (Δ Decorin) as a functional resistin receptor in adipocyte progenitors that may regulate white adipose tissue expansion. Our work shows that the mouse receptor tyrosine kinase-like orphan receptor (ROR)1 could mediate some of the described functions of resistin in 3T3-L1 adipogenesis and glucose uptake. We have demonstrated an interaction of mouse resistin with specific domains of the extracellular region of the ROR1 receptor. This interaction results in the inhibition of ROR1 phosphorylation, modulates ERK1/2 phosphorylation, and regulates suppressor of cytokine signaling 3, glucose transporter 4, and glucose transporter 1 expression. Moreover, mouse resistin modulates glucose uptake and promotes adipogenesis of 3T3-L1 cells through ROR1. In summary, our results identify mouse resistin as a potential inhibitory ligand for the receptor ROR1 and demonstrate, for the first time, that ROR1 plays an important role in adipogenesis and glucose homeostasis in 3T3-L1 cells. These data open a new line of research that could explain important questions about the resistin mechanism of action in adipogenesis and in the development of insulin resistance.

Dlk1 influences differentiation and function of B lymphocytes.

The Dlk1 (delta-like-1) gene is a member of the epidermal growth factor (EGF)-like homeotic gene family. It influences cell-cell interactions between stromal cells and pro-B cells in vitro. To define the in vivo role of the dlk protein in B cell development, we established a Dlk1-/- mouse model. In spleens of Dlk1-/- mice, transitional B cell numbers were increased and the ratio between transitional B cell subsets was altered. Numbers of follicular B cells decreased, while the number of marginal zone B cells and the size of the marginal zone were increased. Loss of dlk resulted in increased immunoglobulin G1 (IgG1) and IgG3 in preimmune sera. Furthermore, there was an exaggerated primary T-dependent antigen-specific humoral immune response. In bone marrow, the lack of dlk led to increased numbers of the earliest B lineage cells in young mice without affecting numbers of later B lineage cells. In vitro experiments showed that lack of dlk on either stromal cells or pro-B cells caused changes in differentiation and proliferation of pro-B cells, suggesting that lack of dlk leads to changes in cell-cell interactions in the bone marrow microenvironment. These results show that dlk expression is essential for normal B cell development.

Modulated Expression of the Epidermal Growth Factor-Like Homeotic Protein dlk Influences Stromal-Cell–Pre-B-Cell Interactions, Stromal Cell Adipogenesis, and Pre-B-Cell Interleukin-7 Requirements

ABSTRACT A close relationship exists between adipocyte differentiation of stromal cells and their capacity to support hematopoiesis. The molecular basis for this is unknown. We have studied whether dlk, an epidermal growth factor-like molecule that intervenes in adipogenesis and fetal liver hematopoiesis, affects both stromal cell adipogenesis and B-cell lymphopoiesis in an established pre-B-cell culture system. Pre-B-cell cultures require both soluble interleukin-7 (IL-7) and interactions with stromal cells to promote cell growth and prevent B-cell maturation or apoptosis. We found that BALB/c 3T3 fibroblasts express dlk and function as stromal cells. Transfection of these cells with antisense dlk decreased dlk expression and increased insulin-induced adipocytic differentiation. When antisense transfectants were used as stroma, IL-7 was no longer required to support the growth of pre-B cells and prevent maturation or apoptosis. Antisense dlk transfectants of S10 stromal cells also promoted pre-B-cell growth in the absence of IL-7. These results show that modulation of dlk on stromal cells can influence their adipogenesis and the IL-7 requirements of the pre-B cells growing in contact with them. These results indicate that dlk influences differentiation signals directed both to the stromal cells and to the lymphocyte precursors, suggesting that dlk may play an important role in the bone marrow hematopoietic environment.