The non-canonical NOTCH1 ligand Delta-like 1 homolog (DLK1) self interacts in mammals

https://doi.org/10.1016/j.ijbiomac.2017.01.067Get rights and content

Abstract

Delta-like 1 homolog (DLK1) is an imprinted gene, which is widely expressed during mammalian development and plays a pivotal role in differentiation of various tissue types. Most recently, we have shown that DLK1 interacts with NOTCH1, yet several Notch independent mechanisms have previously been suggested as well, but only poorly confirmed in a mammalian context.

In the present study, we employed the mammalian two-hybrid (MTH) system, a genetic in vivo protein–protein interaction system, to show robust DLK1-DLK1, DLK1-FnI (Fibronectin) and DLK1-CFR (cysteine-rich FGF receptor) interactions, whereas the proposed DLK1-IGFBP1 interaction was not supported by MTH. Very little has previously been described on the DLK1 self-interaction. Herein, we showed by immunoprecipitation as well as Sulfo-SBED label transfer that the DLK1-DLK1 interaction likely is part of Dlk1’s function in preadipocytes. Furthermore our data suggest that DLK1 interacts with itself through EGF domain 4 and 5, which is distinct from the recently described NOTCH1-DLK1 interaction, which occurs between EGF domain 5 and 6. This opens up the possibility that Notch independent mechanisms like the DLK1-DLK1 interaction may modulate the non-canonical NOTCH1-DLK1 interaction further complexing this system.

Introduction

Notch signaling is one of the best described, but also most complex pathways in which protein ligands interact with NOTCH proteins on the cell surface, and hereby affect numerous cell functions in organisms ranging from flies to humans [1]. However, while canonical Notch signaling requires the DSL (Delta-Serrate-LAG2) domain for the interaction between NOTCH- membrane proteins and ligands, non-canonical signaling involving DSL lacking ligands is controversial and only poorly described [2], [3]. Most recently, we established that Delta-like 1 homolog (Dlk1) is one such non-canonical Notch ligand in mammals, where it interacts with NOTCH1 during tissue development [4]. Yet, we also observed that Dlk1 must act in a NOTCH independent manner as well [4]. Dlk1 is a paternally expressed imprinted gene that maps to the Dlk1-Dio3 gene cluster on mouse chromosome 12 and human chromosome 14, respectively [5], [6]. Remarkably, Dlk1 is among the few imprinted genes that are translated into protein and therefore subject to a lot of attention, but still its exact function and underlying mechanism of action remains poorly described. Dlk1 encodes a protein comprised of an intracellular region, a single transmembrane domain and an extracellular region containing six epidermal growth factor (EGF)-like repeats [7]. Alternatively spliced Dlk1 transcripts have been described [8], encoding a juxtamembrane protease-recognition site or not. Thus, isoforms lacking the protease-recognition site are membrane-tethered, whereas isoforms encompassing the protease-recognition site can be proteolytically cleaved by ADAM17/TACE generating a large soluble DLK1 protein that is released into circulation [9], [10]. The Dlk1 gene is highly expressed during mammalian embryonic development [11], [12], [13], [14], yet in normal adult tissues Dlk1 expression is highly restricted to cells of neuroendocrine origin and progenitor cells within the stromal vascular fraction (SVF) of adipose tissue [14], [15], [16], [17]. Dlk1 is thought to play a role in maintaining proliferating cells in an undifferentiated state, in turn negatively regulating differentiation processes, such as adipogenesis [7], [18], [19], [20], osteogenesis [21], chondrogenesis [22], angiogenesis [23], neurogenesis [24] and hematopoiesis [25]. Still, it is not known whether DLK1 functions as a receptor or a ligand. Besides interacting with NOTCH1 [26], [27], DLK1 has been suggested to interact extracellularly with: itself [28], IGF binding protein 1 (IGFBP1) [29], cysteine-rich FGF receptor (CFR) [30], FIBRONECTIN [31] and most recently, DLK2 [32]. The majority of these interaction partners were identified in yeast or in aberrant cell systems lacking endogenous Dlk1, and thus still lack confirmation for their direct interaction with DLK1 in mammals. It is intriguing to speculate that these NOTCH independent interactions with DLK1 may interfere or modulate the non-canonical interaction between DLK1-NOTCH1, and therefore have a substantial effect on Notch signalling that is absolutely essential for mammalian function. Thus, to further delineate such issues, we set out to investigate whether DLK1 indeed does interact with DLK1 itself, IGFBP1, CFR and FIBRONECTIN in a mammalian context.

Section snippets

Cell culture

Cell lines were cultured at 37 °C in a 5% CO2 humidified atmosphere. Cell lines used were: HEK293T, cultured in Dulbecco’s modified Eagle medium (DMEM, Lonza), supplemented with 10% fetal bovine serum (FBS), 3T3-L1, cultured in Dulbecco’s modified Eagle medium (DMEM, Lonza), supplemented with 10% fetal calf serum (FCS). Cell numbers were determined using a Beckman Coulter Counter Z2 (Ramcon, Birkeroed, Denmark, http://www.ramcon.dk) fitted with a 100 ¼ lm aperture, and counting was performed in

DLK1 interacts with itself, CFR and FIBRONECTIN, but not IGFBP1, in the mammalian two-hybrid system

Recently, we demonstrated that DLK1 interacts with NOTCH1 [4] using the mammalian two-hybrid assay (M2H), which allows one to examine interactions between proteins that may not fold correctly in yeast or require mammalian specific post-translational modification not possible in yeast [34]. In the M2H system, a robust interaction is defined by a significantly higher relative luciferase activity in mammalian cells transfected with the two expression vectors, pACT-X and pBIND-Y, encoding a

Discussion

Herein, we for the first time demonstrate a specific DLK1-DLK1, DLK1-CFR and DLK1-FnI interaction in a mammalian context, while our results using the MH2 system do not support an interaction between DLK1 and IGFBP1 in that mammalian context. Furthermore, our results point out that the DLK1-DLK1 interaction occurs in preadipocytes, and that it is mediated through EGF domains 4 and 5.

The mammalian two-hybrid system allows for posttranslational modifications of hybrid proteins and therefore more

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.

Contributions

GAT, CHJ: Conception and design, Collection of data, Data analysis and interpretation, Manuscript writing. JJGR: Data interpretation, revising manuscript. SPS: Data interpretation, Final approval of manuscript, and Financial support. DCA: Conception and design, Data analysis and interpretation, Manuscript writing, Final approval of manuscript, and Financial support.

Acknowledgements

We would like to thank Charlotte Nielsen, Tonja L. Jørgensen, and Anette Kliem (LMCC, Odense University Hospital) for technical assistance on this study. Moreover, we would like to thank associate professor Victoriano Baladron for help with vector design and Atsushi Miyajima (Tokyo University, Japan) for providing CFR vector. This work was supported by The Danish National Research Council (#09-073648), The Lundbeck Foundation (#R48-A4785 and #R112-A9634), A.P. Møller Foundation, SPAR Nord and

References (44)

  • V. Baladron et al.

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

    Exp. Cell Res.

    (2005)
  • G.A. Traustadottir et al.

    Evidence of non-canonical NOTCH signaling: Delta-like 1 homolog (DLK1) directly interacts with the NOTCH1 receptor in mammals

    Cell Signal

    (2016)
  • M.L. Nueda et al.

    Dlk1 specifically interacts with insulin-like growth factor binding protein 1 to modulate adipogenesis of 3T3-L1 cells

    J. Mol. Biol.

    (2008)
  • B. Sanchez-Solana et al.

    The EGF-like proteins DLK1 and DLK2 function as inhibitory non-canonical ligands of NOTCH1 receptor that modulate each other's activities

    Biochim. Biophys. Acta

    (2011)
  • H. Waller et al.

    The use of AlphaLISA technology to detect interaction between hepatitis C virus-encoded NS5A and cyclophilin A

    J. Virol. Methods

    (2010)
  • Z. Liu et al.

    Proteome-wide prediction of self-interacting proteins based on multiple properties

    Mol. Cell. Proteomics

    (2013)
  • T. Finkel et al.

    Detection and modulation in vivo of helix-loop-helix protein–protein interactions

    J. Biol. Chem.

    (1993)
  • D.C. Andersen et al.

    MicroRNA-15a fine-tunes the level of Delta-like 1 homolog (DLK1) in proliferating 3T3-L1 preadipocytes

    Exp. Cell Res.

    (2010)
  • S. Artavanis-Tsakonas et al.

    Notch signaling: cell fate control and signal integration in development

    Science

    (1999)
  • K.G. Guruharsha et al.

    The Notch signalling system: recent insights into the complexity of a conserved pathway

    Nat. Rev. Genet.

    (2012)
  • E. Gubina et al.

    Assignment of dlk (Dlk1) to mouse chromosome band 12E-F1 by in situ hybridization

    Cytogenet. Cell Genet.

    (2000)
  • E. Gubina et al.

    Assignment of DLK1 to human chromosome band 14q32 by in situ hybridization

    Cytogenet. Cell Genet.

    (1999)
  • Cited by (12)

    • 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

      2019, Cytokine and Growth Factor Reviews
      Citation Excerpt :

      However, the implication of DLK1 in NOTCH signalling is still controversial, and several studies report NOTCH signalling to be unaffected by DLK1 [66,106,126,127] and other interactions partners are also suggested as well (Fig. 1). Regarding the latter, evidence in yeast and mammals show that DLK1 interacts with itself [105,128]. Likewise in yeast, DLK1 has been shown to interact with the cysteine-rich molecules GAS1 and granulin (GRN), involved in the control of cell growth [129], as well as with the IGF binding protein 1 (IGFBP1) [130].

    • Emerging Roles of DLK1 in the Stem Cell Niche and Cancer Stemness

      2022, Journal of Histochemistry and Cytochemistry
    View all citing articles on Scopus
    View full text