dlk1 specifically interacts with insulin-like growth factor binding protein 1 to modulate adipogenesis of 3T3-L1 cells

dlk1 is an epidermal growth factor (EGF)-like homeotic protein containing an intracellular region, a single transmembrane domain, and an extracellular region possessing six EGF-like repeats and a protease-target sequence. dlk1 functions as a modulator of adipogenesis, and other differentiation processes. The molecular mechanisms by which dlk1 regulates these processes are unclear. It has been reported that different Dlk1 mRNA spliced variants, encoding for isoforms possessing the protease-target sequence or not, determine the production of membrane-associated or soluble, secreted extracellular dlk1 proteins that appear to affect adipogenesis of 3T3-L1 cells differently. In particular, only soluble variants inhibit this process. Some recent evidence suggest that dlk1 may modulate extracellular stimuli inducing differentiation. Thus, an enforced decrease of Dlk1 expression in BALB/c 3T3 cells, which results in an increase of their adipogenic potential in response to insulin-like growth factor 1 (IGF-1), modifies the kinetics and levels of activation of ERK1/2 triggered by it. In this work, we identified a strong and specific interaction between the protease-target dlk1 region and the non-IGF binding region of IGF binding protein 1 (IGFBP1), a protein that binds to IGFs and modulates their action. We also observed that the increased adipogenic potential of 3T3-L1 cells caused by diminishing Dlk1 expression through transfection with an antisense Dlk1 expression construct was inhibited by the presence of IGFBP1 in the differentiation medium. On the other hand, the presence of IGFBP1 in the culture medium slightly increased the adipogenic potential of control 3T3-L1 cells, expressing regular levels of Dlk1. These data suggest that membrane dlk1 variants bind to extracellular IGFBP1/IGF-1 complexes, which may favor the release of IGF-1 and increase the local concentration of free IGF-1 that can enhance IGF receptor signaling, leading to adipogenesis.     

The novel gene EGFL9/Dlk2, highly homologous to Dlk1, functions as a modulator of adipogenesis

The Dlk1 gene appears to function as a regulator of adipogenesis. Adult Dlk1-deficient mice are obese, but adipose tissue still develops in transgenic mice overexpressing an Fc-dlk1 fusion protein, and neither type of genetically modified mice displays serious abnormalities. It was therefore possible that one yet unidentified gene might either compensate or antagonize for the absence or for overexpression, respectively, of Dlk1 in those animals. In database searches, we found a novel gene, EGFL9, encoding for a protein whose structural features are virtually identical to those of dlk1, suggesting it may function in a similar way. As dlk1 does, the protein encoded by EGFL9/Dlk2 affects adipogenesis of 3T3-L1 preadipocytes and mesenchymal C3H10T1/2 cells; however, it does so in an opposite way to that of dlk1. In addition, expression levels of both genes appear to be inversely correlated in both cell lines. Moreover, enforced changes in the expression of one gene affect the expression levels of the other. Our data suggest that adipogenesis may be modulated by the coordinated expression of Dlk1 and EGFL9/Dlk2.     

The EGF-like protein dlk1 inhibits notch signaling and potentiates adipogenesis of mesenchymal cells

The EGF-like homeotic gene Dlk1 appears to function as an inhibitor of adipogenesis. Overexpression of Dlk1 prevents adipogenesis of 3T3-L1 cells. Dlk1-deficient mice are obese; however, adipose tissue still develops in Fc-dlk1 transgenic mice, suggesting that Dlk1 is not a strict inhibitor of adipogenesis. To clarify the role of Dlk1 in adipogenesis, we studied whether Dlk1 could act differently on this process depending upon the differentiation state of the precursor cells. We found that Dlk1 is a potentiator of adipogenesis for mesenchymal C3H10T1/2 cells. This potentiating effect can be triggered by overexpressing the entire protein or the extracellular EGF-like-containing region, but not by overexpressing the intracellular dlk1 sequence. In addition, coculture of C3H10T1/2 cells with other cells expressing Dlk1, but not with cells lacking Dlk1 expression, enhances their adipogenic response. Potentiation of adipogenesis by Dlk1 was associated with changes in the activation of ERK1/2 after IGFI/insulin induction. Finally, as reported with other cells, dlk1 functioned as a Notch signaling inhibitor in C3H10T1/2 cells, but inhibition of Notch1 expression prevented the potentiating effects of Dlk1 in adipogenesis. These data suggest that Dlk1 may potentiate or inhibit adipogenesis depending upon the cellular context, and that Notch1 expression and activation are important factors in this context.     

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

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.     

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-kappaB. 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.     

Membrane-bound delta-like 1 homolog (Dlk1) promotes while soluble Dlk1 inhibits myogenesis in C2C12 cells

Delta-like 1 homolog (Dlk1) is important in myogenesis. However, the roles of different Dlk1 isoforms have not been investigated. In C2C12 cell lines producing different Dlk1 isoforms, membrane-bound Dlk1 promoted the hypertrophic phenotype and a higher fusion rate, whereas soluble Dlk1 inhibited myotube formation. Inversed expression patterns of genes related to myogenic differentiation further support these phenotypic changes. In addition, temporal expression and balance between the Dlk1 isoforms have a regulatory role in myogenesis in vivo. Collectively, Dlk1 isoforms have distinctive effects on myogenesis, and its regulation during myogenesis is critical for normal muscle development.     

Adipocyte differentiation is modulated by secreted delta-like (dlk) variants and requires the expression of membrane-associated dlk

Previous studies demonstrate that the delta-like (dlk) and preadipocyte factor 1 (Pref-1) genes encode similar proteins. Pref-1 is downregulated during adipocyte differentiation, and expression of ectopic Pref-1 inhibits adipogenesis. We explored whether dlk functions similarly to Pref-1 and studied the role of alternately spliced dlk variants encoding membrane-associated or -secreted forms. We also studied whether enforced downregulation of dlk/Pref-1 may enhance the differentiation response of non-committed cells. Ectopic expression of a potentially secreted dlk variant, conditioned media from dlk expressing cells or several individual epidermal-growth-factor-dlk peptides inhibited 3T3-L1 differentiation. This demonstrates that dlk and Pref-1 are functionally equivalent. dlk gene mRNA encoding for secreted variants decreased much faster than total dlk gene mRNA during differentiation of 3T3-L1 cells. In fact, total dlk or membrane-associated dlk protein expression increased during the first hours of differentiation. Cells sorted for lowest levels of dlk protein diminished or lost their ability to differentiate. These data suggest that membrane and secreted dlk protein variants play opposite roles in the control of adipogenesis. In addition, enforced downregulation of dlk protein expression in the weakly adipogenic Balb/c 3T3 cell line dramatically enhanced adipogenesis in response to insulin. These results indicate that dlk protein not only participates in processes leading to inhibition of adipogenesis but that the control of its expression and different spliced variants is essential for the adipogenic response to extracellular signals.     

The EGF-like homeotic protein dlk affects cell growth and interacts with growth-modulating molecules in the yeast two-hybrid system

Levels of dlk, an EGF-like homeotic protein, are critical for several differentiation processes. Because growth and differentiation are, in general, exclusive of each other, and increasing evidence indicates that Dlk1 expression changes in tumorigenic processes, we studied whether dlk could also affect cell growth. We found that, in response to glucocorticoids, Balb/c 3T3 cells with diminished levels of dlk expression develop foci-like cells that have lost contact inhibition, display altered morphology, and grow faster than control cell lines. Balb/c 3T3 cells spontaneously growing more rapidly are also dlk-negative cells. Moreover, screening by the yeast two-hybrid system, using Dlk1 constructs as baits, resulted in the isolation of GAS1 and acrogranin cDNAs. Interestingly, these proteins are cysteine-rich molecules involved in the control of cell growth. Taken together, these observations suggest that dlk may participate in a network of interactions controlling how the cells respond to growth or differentiation signals.     

The imprinted gene Delta like non-canonical Notch ligand 1 (Dlk1) is conserved in mammals,and serves a growth modulatory role during tissue development and regeneration through Notch dependent and independent mechanisms

Delta like non-canonical Notch ligand 1 (Dlk1) is an imprinted gene, mainly known for its involvement in adipogenesis, although it has been associated with many other stem cells/progenitors and is known to be widely expressed during organism development and tissue regeneration. In a systematic manner, we have outlined the overall expression pattern of Dlk1 in both man and mouse, and found Dlk1 to be expressed in tissues from all three germ layers. Yet, Dlk1 expression decreases along with increased differentiation as gestation proceeds and in most tissues Dlk1 is absent around birth. Thus, in adults, expression of Dlk1 is restricted to a few tissues and progenitor cells, but is re-expressed during disease and regeneration. Although diffferences exist, we found an overall conservation of Dlk1 expression between mouse and man, and conclude in that sense that the mouse is an appropiate model to study Dlk1. In agreement with the observed Dlk1 expression pattern, we found that the majority of published Dlk1 studies, report Dlk1 to have an inhibitory effect on both cell proliferation and differentiation, but the levels of the different DLK1 isoforms may be critical and have an impact on the overall outcome. This may also be an issue during tissue regeneration where several studies have reported Dlk1′s impact during skeletal muscle and liver regeneration without establishing the exact role. Likewise, the underlying mechanism of Dlk1 action is unknown, and seems to depend on both Notch dependent and independent pathways. However, from our data it is intriguing to speculate that the actual role of DLK1 may be to function as a checkpoint to slow down proliferation while forcing cells into the process of differentiation, and thus switch the cell/organ to a state of growth and hypertrophy. This may fit well with its reported impact on growth restiction and body size. Thus, our study which for the first time summarizes reported knowledge on Dlk1 in tissue development and regeneration as well as on the Dlk1 mechanism may provide novel insight to the general role of this remarkable imprinted gene in controlling cell growth, from which new hypotheses can be made in the field of stem cell biology and regenerative medicine.     

Adipogenic genes on induction and stabilization of commitment to adipose conversion

Most late events of adipose conversion are known, but those early events that lead to cell commitment, and important aspects of its mechanism remain unknown. We recently described that, in the absence of any other adipogenic factor, 4 h incubation with staurosporine promotes commitment of 3T3-F442A cells to adipogenesis. This commitment consists of two stages; a first stage of 4 h induction by staurosporine, and, in the absence of this drug, a second stage of stabilization which becomes completed after 40-48 h from staurosporine treatment. Here, we demonstrate that pparg2 gene is expressed early after induction stage but before commitment is stabilized, whereas cebpa is highly expressed during the last part of stabilization stage. A decrease of dlk1 expression, whose down-regulation is indispensable for adipogenesis, began to take place between 24 and 48 h of St-Dex incubation started, reaching the lowest levels well into the end of stabilization stage.     

Differential Expression and Function of Stamp Family Proteins in Adipocyte Differentiation

Six transmembrane protein of prostate (Stamp) proteins play an important role in prostate cancer cell growth. Recently, we found that Stamp2 has a critical role in the integration of inflammatory and metabolic signals in adipose tissue where it is highly expressed and regulated by nutritional and metabolic cues. In this study, we show that all Stamp family members are differentially regulated during adipogenesis: whereas Stamp1 expression is significantly decreased upon differentiation, Stamp2 expression is increased. In contrast, Stamp3 expression is modestly changed in adipocytes compared to preadipocytes, and has a biphasic expression pattern during the course of differentiation. Suppression of Stamp1 or Stamp2 expression both led to inhibition of 3T3-L1 differentiation in concert with diminished expression of the key regulators of adipogenesis - CCAAT/enhancer binding protein alpha (C/ebpα) and peroxisome proliferator-activated receptor gamma (Pparγ). Upon Stamp1 knockdown, mitotic clonal expansion was also inhibited. In contrast, Stamp2 knockdown did not affect mitotic clonal expansion, but resulted in a marked decrease in superoxide production that is known to affect adipogenesis. These results suggest that Stamp1 and Stamp2 play critical roles in adipogenesis, but through different mechanisms.     

The role of the epidermal growth factor-like protein dlk in cell differentiation

This review focuses on the current knowledge about the function of the EGF-like homeotic protein dlk. dlk is a transmembrane protein that possesses six Epidermal Growth Factor-like sequences at the extracellular domain, a single transmembrane domain and a short intracellular tail. Because of its overall structure and amino acid homology, dlk belongs to the EGF-like homeotic protein family. This family includes proteins such as the Notch receptor and its homologues, as well as Notch ligands, such as Delta, Serrate, and their mammalian homologues Dll1, Dll2 and Dll3 and Jagged 1 and Jagged 2. (For a recent review see Fleming, 1998). dlk is highly expressed by preadipose cell lines, and neuroendocrine tumors, such as pheochromocytomas and neuroblastomas. dlk has been involved in several differentiation processes, such as adipogenesis, hematopoiesis and B cell lymphopoiesis, and neuroendocrine differentiation, including the differentiation of pancreas and the adrenal gland. The extracellular region of dlk can be released by action of an unknown protease and this soluble dlk variant accumulates in the amniotic fluid and is able to inhibit adipocyte differentiation in vitro. Recent evidence indicates, however, that membrane-associated dlk variants play a positive role in the differentiation process. These findings suggest that dlk plays an important role in differentiation and tumorigenesis of several cellular types.     

PCLAF promotes neuroblastoma G1/S cell cycle progression via the E2F1/PTTG1 axis

PCLAF (PCNA clamp-associated factor), also known as PAF15/ KIAA0101, is overexpressed in most human cancers and is a predominant regulator of tumor progression. However, its biological function in neuroblastoma remains unclear. PCLAF is extremely overexpressed in neuroblastoma and is associated with poor prognosis. Through the analysis of various data sets, we found that the high expression of PCLAF is positively correlated with increased stage and high risk of neuroblastoma. Most importantly, knocking down PCLAF could restrict the proliferation of neuroblastoma cells in vitro and in vitro. By analyzing RNA-seq data, we found that the enrichment of cell cycle-related pathway genes was most significant among the differentially expressed downregulated genes after reducing the expression of PCLAF. In addition, PCLAF accelerated the G1/S transition of the neuroblastoma cell cycle by activating the E2F1/PTTG1 signaling pathway. In this study, we reveal the mechanism by which PCLAF facilitates cell cycle progression and recommend that the PCLAF/E2F1/PTTG1 axis is a therapeutic target in neuroblastoma.Subject terms: Cancer epigenetics, Cell growth     

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

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.     

The FBXL10/KDM2B Scaffolding Protein Associates with Novel Polycomb Repressive Complex-1 to Regulate Adipogenesis

Polycomb repressive complex 1 (PRC1) plays an essential role in the epigenetic repression of gene expression during development and cellular differentiation via multiple effector mechanisms, including ubiquitination of H2A and chromatin compaction. However, whether it regulates the stepwise progression of adipogenesis is unknown. Here, we show that FBXL10/KDM2B is an anti-adipogenic factor that is up-regulated during the early phase of 3T3-L1 preadipocyte differentiation and in adipose tissue in a diet-induced model of obesity. Interestingly, inhibition of adipogenesis does not require the JmjC demethylase domain of FBXL10, but it does require the F-box and leucine-rich repeat domains, which we show recruit a noncanonical polycomb repressive complex 1 (PRC1) containing RING1B, SKP1, PCGF1, and BCOR. Knockdown of either RING1B or SKP1 prevented FBXL10-mediated repression of 3T3-L1 preadipocyte differentiation indicating that PRC1 formation mediates the inhibitory effect of FBXL10 on adipogenesis. Using ChIP-seq, we show that FBXL10 recruits RING1B to key specific genomic loci surrounding the key cell cycle and the adipogenic genes Cdk1, Uhrf1, Pparg1, and Pparg2 to repress adipogenesis. These results suggest that FBXL10 represses adipogenesis by targeting a noncanonical PRC1 complex to repress key genes (e.g. Pparg) that control conversion of pluripotent cells into the adipogenic lineage.     

Conditional deletions refine the embryonic requirement for Dlk1

Numerous studies have implicated Delta-like 1 (DLK1), a transmembrane protein that shares homology with Notch ligands, in embryonic growth and differentiation. Dlk1 expression is widespread, though not ubiquitous, during early development, but is confined to a few specific cell types in adults. Adult Dlk1-expressing tissues include the Insulin-producing β-cells of the pancreas and the Growth hormone-producing somatotrophs of the pituitary gland. Previously generated Dlk1 null mice (Dlk1^Sul-pat), display a partially penetrant neonatal lethality and a complex pattern of developmental and adult phenotypes. Here we describe the generation of a conditional Dlk1 mouse line (Dlk1^flox) to facilitate cell type-specific deletion of the Dlk1 gene, providing a powerful system to explore each aspect of the Dlk1 null phenotype. Four tissue-specific Cre mouse lines were used to produce individual Dlk1 deletions in pancreatic β-cells, pituitary somatotrophs and the endothelial cells of the embryo and placenta, key candidates for the Dlk1 phenotype. Contrary to expectations, all of these conditional mice were fully viable, and none recapitulated any aspect of the Dlk1^Sul-pat null mice. Dlk1 expression is therefore not essential for the normal development of β-cells, somatotrophs and endothelial cells, and the tissues responsible for the Dlk1 null phenotype remain to be identified. Dlk1^flox mice will continue to provide an important tool for further research into the function of Dlk1.