Notch and BCR signaling synergistically promote the proliferation of Raji B-lymphoma cells
Introduction
The Notch signaling pathway has multiple and essential roles in cell fate decisions in development and is conserved during evolution from worm through man [1]. The Notch pathway in mammals includes four receptors, Notch1-4, and five ligands including Jagged1, 2 and Delta-like1, 3, 4. When Notch receptor is triggered by direct interaction with its ligands, expressed on neighboring cells, the intracellular domain of the Notch receptor (NIC) is released from the membrane after sequential receptor cleavages executed by ADAM/TACE protease and γ-secretase complex [2], [3]. NIC translocates into nucleus and associates with the transcription factor RBP-J, and transactivates promoters harboring RBP-J-binding sites through recruiting co-activators of the Mastermind-like (MAML) family [4], [5]. The extracellular domains, including an N-terminal Delta-Serrate-Lag2 (DSL) domain that is essential for Notch-binding, and varying number of EGF-like repeats, of Notch ligands are evolutionarily conserved. The human Delta-like1 (hDll1) is a homolog of the Drosophila Delta, and is composed of 723 amino acids, with one DSL domain and eight EGF-like repeats [1].
During mammalian hematopoiesis and lymphopoiesis, Notch signaling shows multiple functions, such as the regulation of T-cell commitment from a common lymphoid precursor at the expense of B-cell development in thymus [6], and the control of marginal zone B-cell differentiation in spleen [7]. Notch signaling must be precisely regulated, as constitutive Notch signaling leads to T-cell malignancies [8]. In the B-cell compartment, the effect of de-regulating Notch signaling in the B-cell lineage is more complicated and controversial [9]. Although there has been no report showing that constitutive activation of Notch signaling lead to B-cell malignancies, activation of the transcription factor RBP-J by EBNA2 is essential for B-cell transformation by Epstein-Barr (EB) virus [10], [11].
B-cell antigen receptor (BCR) plays an important role in the regulation of proliferation, apoptosis, and differentiation of B-cells at distinct stages during B-cell development. It has been shown that Notch signaling synergizes with BCR to enhance B-cell activation [12]. But, it is not clear how BCR and Notch signaling interacts in B-cell malignancies. In this report, using a human Burkitt’s lymphoma, the Raji cells, we showed that BCR-mediated proliferation was enhanced by co-stimulating Notch receptors with a recombinant hDll1 protein, and that BCR and Notch signaling might interact at the level of c-myc expression.
Section snippets
Expression and purification of the recombinant hDll1 fusion protein
The coding sequence of the N-terminal DSL domain of human Dll1 (hDll1DSL, amino acids 127–225) was amplified from a human liver cDNA library by PCR, and was cloned, as described previously [13]. To construct a expression vector, the inserted fragment from a correct clone was inserted in-frame into the cloning sites of pET32a(+) (Novagen, Madison, WI) using routine DNA recombination technology, to generate pET32a-hDll1DSL.
E. coli BL21 (DE3) transformed with pET32a(+) or pET32a-Dll1DSL were
Notch and BCR signaling synergistically enhanced Raji proliferation
To trigger Notch signaling in Raji cells, we expressed the hDll1 fragment (hDll1DSL) in E. coli. We previously have expressed GST-hDll1DSL in E. coli[13]. To get better expression and purification, we inserted in frame hDll1DSL (amino acids 127–225 into another E. coli expression vector, pET32a(+), to generate pET32a-hDll1DSL. Upon induction, the Trx-hDll1DSL fusion protein with a His tag was successfully expressed in E. coli transformed with pET32a-hDll1DSL, and was purified to a single band
Discussion
In this study, we expressed an hDll1 truncation containing the DSL domain (Trx-hDll1DSL) in E. coli. The N-terminus of the Delta extracellular domain is necessary and sufficient for interaction with Notch receptors [18]. We proved the bioactivity of recombinant Trx-hDll1DSL by reporter assay and the induction of the downstream Hes1 expression. Some researchers have assumed that a soluble form of hDll1 plays a dominant negative role in Notch signaling [19], [20], [21], and only the immobilized
Confliction of interest statement
The authors declare no conflict of interest.
Acknowledgements
We thank Ms Hui Wang for English editing of the manuscript. This work was supported by grants from the National Natural Science Foundation of China (30370598, 30425015, 30400079, 30600544), the PCSIRT program (IRT0459) of the Ministry of Education of China, and the Ministry of Science and Technology of China (2006AA02A111).
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These authors contributed equally to this study.