Báo cáo Y học: Acceleration of granulocyte colony-stimulating factor-induced neutrophilic nuclear lobulation by overexpression of Lyn tyrosine kinase

Acceleration of granulocyte colony-stimulating factor-induced neutrophilic nuclear lobulation by overexpression of Lyn tyrosine kinase

Tác giả: Tomomi Omura, Hiroshi Sakai and Hiroshi Murakami

Lĩnh vực: Department of Biotechnology, Faculty of Engineering, Okayama University, Japan

Nội dung tài liệu: Nghiên cứu này khám phá vai trò của tyrosine kinase Lyn trong quá trình biệt hóa tế bào tiền thân tủy thành bạch cầu trung tính do yếu tố kích thích tăng sinh hạt (G-CSF). Các nhà nghiên cứu đã tạo ra các dòng tế bào GM-I62M biểu hiện quá mức Lyn hoặc dạng bất hoạt kinase của nó (LynKN). Kết quả cho thấy việc biểu hiện quá mức Lyn hoặc LynKN đã thúc đẩy nhanh chóng sự biệt hóa thành bạch cầu trung tính và thùy hóa nhân, một dấu hiệu của sự trưởng thành. Điều này gợi ý rằng tương tác protein-protein với Lyn, thay vì hoạt tính kinase của nó, đóng vai trò quan trọng trong hiện tượng này. Nghiên cứu cũng so sánh các tác động này với các nghiên cứu trước đó trên tế bào lympho ở chim, cho thấy sự khác biệt trong các con đường tín hiệu giữa các loài.

Mục lục chi tiết:

• Stimulation with granulocyte colony-stimulating factor (G-CSF) induces myeloid precursor cells to differentiate into neutrophils, and tyrosine phosphorylation of certain cellular proteins is crucial to this process.
• The signaling pathways for neutrophil differentiation are still obscure.
• As the Src-like tyrosine kinase, Lyn, has been reported to play a role in G-CSF-induced proliferation in avian lymphoid cells, we examined its involvement in G-CSF-induced signal transduction in mammalian cells.
• Expression plasmids for wild-type Lyn (Lyn) and kinase-negative Lyn (LynKN) were introduced into a murine granulocyte precursor cell line, GM-I62M, that can respond to G-CSF with neutrophil differentiation, and cell lines that overexpressed these molecules (GM-Lyn, GM-LynKN) were established.
• Upon G-CSF stimulation, both the GM-Lyn and GM-LynKN cells began to differentiate into neutrophils, showing early morphological changes within a few days, much more rapidly than did the parental cells, which started to exhibit nuclear lobulation about 10 days after the cells were transferred to G-CSF-containing medium.
• However, the time course of expression of the myeloperoxidase gene, another neutrophil differentiation marker, was not affected by the overexpression of Lyn or LynKN.
• Therefore, in normal cells, protein interactions with Lyn, but not its kinase activity, are important for the induction of G-CSF-induced neutrophilic nuclear lobulation in mammalian granulopoiesis.
• Keywords: differentiation; granulocyte colony-stimulating factor (G-CSF); granulocyte; lobulation; neutrophil.
• The production of blood cells is regulated by a variety of extracellular stimuli, including a network of hematopoietic growth factors and cytokines [1].
• Among them, granulocyte colony-stimulating factor (G-CSF) is a critical regulator of neutrophilic granulocyte production and stimulates the proliferation, survival, maturation, and functional activation of the cells of the granulocytic lineage [2,3].
• A variety of G-CSF activities are mediated through its interaction with a specific cell-surface receptor [3,4].
• Molecular cloning of the G-CSF receptor cDNA revealed that it is a type I membrane protein consisting of about 800 amino acids and that it belongs to the hematopoietic growth factor receptor family [5,6].
• On ligand binding, the G-CSF receptor forms a homodimer, which induces the signal transduction [7].
• Like other members of the cytokine receptor superfamily, the G-CSF receptor has no intrinsic tyrosine kinase activity, but activates cytoplasmic tyrosine kinases.
• Signaling molecules reported to be activated through the G-CSF receptor include the Janus tyrosine kinases Jak1, Jak2, and Tyk2 [8-11], the signal transducer and activator of transcription (STAT) proteins STAT1, STAT3, and STAT5 [8,12-14], the Src kinases Lyn and Hck [15-17], and components of the Ras, Raf, mitogen-activated protein kinase (MAPK) and MAPK-related pathways [18-22].
• The cytoplasmic region of the G-CSF receptor can be subdivided into a membrane-proximal domain, which has two conserved subdomains designated box 1 and box 2, and a membrane-distal domain, which has four tyrosine residues at positions 703, 728, 743, and 763 of the murine receptor.
• The membrane-proximal domain is known to be a binding site for the Jak family of tyrosine kinases and is essential for mitogenic signaling, whereas both the membrane-proximal domain and the membrane-distal domain are indispensable for the transduction of differentiation signals [23,24].
• Binding of G-CSF to its receptor results in the rapid phosphorylation of these four tyrosine residues in the cytoplasmic domain [25,26], which form potential binding sites for signaling molecules that contain Src homology 2 (SH2) or phosphotyrosine-binding domains [27].
• Indeed, the first (Tyr703) and the third (Tyr743) tyrosines from the membrane-spanning domain have been reported to be the STAT3-docking sites when these residues are phosphorylated [14,28-31].
• In addition, the fourth (Tyr763) tyrosine is necessary for the G-CSF-dependent phosphorylation of Shc and the activation of the p21ras-MAPK signaling pathway [21,32].
• Besides the Jak family of kinases, G-CSF stimulation induces the activation of nonreceptor protein tyrosine kinases, such as the Src-like kinase Lyn and the tandem SH2-containing kinase Syk [16].
• These tyrosine kinases have been reported to be associated with the G-CSF receptor, but their physiological roles are not clearly understood.
• In avian hematopoietic Lyn-deficient cells, ectopic expression of the human G-CSF receptor failed to reconstitute G-CSF-dependent mitotic responses, leading to the conclusion that Lyn is required for G-CSF-induced DNA synthesis [17].
• To investigate the role of Lyn kinase in the G-CSF-induced signaling pathway in mammalian hematopoietic cells, we overexpressed wild-type Lyn (Lyn) and kinase-negative Lyn (LynKN) in murine granulocyte progenitor cells GM-162M and examined their G-CSF responses.
• We found that cells that overexpressed either form of Lyn responded to G-CSF with morphological changes, including nuclear lobulation, much more rapidly than did the control cells.
• Therefore, protein-protein interactions with Lyn, but not its kinase activity, appear to regulate G-CSF-induced nuclear lobulation.