dlk acts as a negative regulator of Notch1 activation through interactions with specific EGF-like repeats

Abstract

The protein dlk, encoded by the Dlk1 gene, belongs to the Notch epidermal growth factor (EGF)-like family of receptors and ligands, which participate in cell fate decisions during development. The molecular mechanisms by which dlk regulates cell differentiation remain unknown. By using the yeast two-hybrid system, we found that dlk interacts with Notch1 in a specific manner. Moreover, by using luciferase as a reporter gene under the control of a CSL/RBP-Jk/CBF-1-dependent promoter in the dlk-negative, Notch1-positive Balb/c 14 cell line, we found that addition of synthetic dlk EGF-like peptides to the culture medium or forced expression of dlk decreases endogenous Notch activity. Furthermore, the expression of the gene Hes-1, a target for Notch1 activation, diminishes in confluent Balb/c14 cells transfected with an expression construct encoding for the extracellular EGF-like region of dlk. The expression of Dlk1 and Notch1 increases in 3T3-L1 cells maintained in a confluent state for several days, which is associated with a concomitant decrease in Hes-1 expression. On the other hand, the decrease of Dlk1 expression in 3T3-L1 cells by antisense cDNA transfection is associated with an increase in Hes-1 expression. These results suggest that dlk functionally interacts in vivo with Notch1, which may lead to the regulation of differentiation processes modulated by Notch1 activation and signaling, including adipogenesis.

Introduction

The mammalian protein dlk is a cell-surface transmembrane protein containing six EGF-like repeats in the extracellular domain, a transmembrane region, and a short intracellular tail [1], [2], [3], [4], [5], [6], [7]. dlk, encoded by the paternally imprinted gene Dlk1, is a member of the epidermal growth factor (EGF)-like family of homeotic proteins, which includes Notch receptors and their ligands [1], [2]. The proteins of this family mediate protein–protein interactions through their EGF-like repeats and regulate cell fate and differentiation decisions during development in many organisms [1], [8], [9].

The level of expression of Dlk1 is critical to modulate the extracellular signals leading to either cell differentiation or proliferation [10], [11], [12]. dlk is known to participate in several differentiation processes, including adipogenesis [5], [10], [13], [14], hematopoiesis [13], [15], [16], [17], [18], and adrenal gland and neuroendocrine cell differentiation [2], [19], [20], [21], [22]. Recent reports involve dlk in peripheral and central nervous system differentiation [23], [24], in growth arrest and increased malignance of undifferentiated tumors [2], [22], [25], [26], and in wound repair [27]. In addition, mice lacking Dlk1 display growth retardation and accelerated adiposity [28], whereas transgenic mice expressing the complete ectodomain of dlk fused to the human Fc antibody region show both decreased adiposity and glucose tolerance [29]. Several spliced mRNA variants, encoding for dlk proteins possessing or not a protease target sequence, have been reported [5], [30], [31]. The yet unknown protease would promote the release of the extracellular dlk region to the medium as a soluble protein that inhibits adipogenesis [5], [10], [19], [29], [32].

Multiple Notch receptors or ligands have been described in many organisms [8], [9], [33], [34], [35], [36], [37], [38], [39], [40], [41]. These include Drosophila Notch, C. elegans Lin12 and GLP-1, and vertebrate Notch1, 2, 3, and 4. The basic structure of Notch proteins comprises 29–36 epidermal growth factor (EGF)-like motives and three copies of a “Lin-12/Notch repeat” (LNR) in the extracellular region, as well as a RAM (RBP-Jk-associated molecule) domain, a set of six cdc10/Ankyrin repeats, a homopolymer repeat of glutamine (OPA) domain, three nuclear localization signal (NLS) sequences, and a proline–glutamate–serine–threonine-rich (PEST) domain in the intracellular region [9].

Notch ligands include Delta and Serrate in Drosophila, LAG-2 and APX-1 in C. elegans, and the Delta homologs, Dll1, 3, and 4, and the Serrate homologs, Jagged1 and 2, in vertebrates [42], [43]. Notch ligands are transmembrane proteins that share two important extracellular features: a DSL domain at the N-terminus, which interacts with specific EGF-like repeats of Notch proteins [38], [40], [44], [45], and a variable number of EGF-like repeats at their extracellular region. Some of these ligands can be processed by the action of specific proteases that release their extracellular regions to the external medium [46], [47], [48], [49], [50].

Posttranslational modifications and interaction with their ligands trigger a cascade of proteolytic events that finally release the Notch intracellular domain (NICD) from the plasma membrane [47], [51], [52], [53], [54], [55], [56], [57], [58], [59]. NICD translocates then to the nucleus, where it interacts with the DNA-binding protein CSL (CBF-1, suppressor of hairless, LAG-1)/RBP-Jk). NICD displaces a corepressor and binds to CSL/RBP-Jk/CBF-1, generating a transcriptional activation complex that activates several genes [60]. Three of these target genes are Hes-1, Hes-5, and NF-kB2, all of which encode for transcription factors that regulate the expression of other genes involved in differentiation [54], [55], [61]. The CSL/RBP-Jk/CBF-1–Hes-1 pathway is only one among other pathways activated by the action of Notch proteins leading to the regulation of cell differentiation, proliferation, or apoptosis [62], [63]. These alternate pathways involve the p53 family of proteins [64], [65], [66] or the activation of the intracellular protein Deltex [67], [68]. Activation of Deltex inhibits proteins upstream E47 [69], a transcription factor activated by the ras/MAPK pathway, known to be involved in the induction of differentiation or proliferation depending upon extracellular stimuli. A cross-talk between Notch1 and ras/MAPK pathways has been also demonstrated by other investigators [70], [71].

The structure and amino acid sequence of dlk EGF-like repeats are closely related to those present in Dll4 and Delta proteins [1]. However, one important difference between Notch1 ligands and dlk is the lack of a DSL domain at the dlk N-terminus. In this report, we present evidence supporting that, despite lacking a DSL domain, mouse dlk interacts with mouse Notch1 in a specific manner. We also present evidence indicating that these interactions are functional in vivo, supported by the observation of a dlk-dependent inhibition of the Notch signaling pathway leading to the activation of the CSL/RBP-Jk/CBF-1 promoters. The inhibitory effect on Notch signaling was observed in the newly developed Notch1-positive, dlk-negative, Balb/c 14 cell line by enforcing dlk expression through transfection or when synthetic dlk EGF-like peptides were added to the culture medium. Moreover, the expression of Hes-1 decreased in confluent Balb/c 14 cells stably transfected with an extracellular Dlk1 cDNA construct, as well as in 3T3-L1 cells, which show a confluence-related increase in Dlk1 and Notch1 expression levels. Furthermore, confluent 3T3-L1 cells with decreased Dlk1 expression, obtained by transfection with an antisense dlk cDNA expression construct, show an increase in Hes-1 expression compared with nontransfected 3T3-L1 cells.

Our results suggest that dlk may function as a molecule involved in the negative regulation of Notch1 signaling during several differentiation processes regulated by Notch1 activity, including adipogenesis, B and T cell differentiation, and hematopoiesis.