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황종익,Huong Thi Nguyen,Sunghoon Hurh,Lan Phuong Nguyen,Thai Uy Nguyen,Hee-Kyung Park,Jae Young Seong,Cheol Soon Lee,Byung-Joo Ham 한국분자세포생물학회 2023 Molecules and cells Vol.46 No.5
CXCR3 regulates leukocyte trafficking, maturation, and various pathophysiological conditions. Alternative splicing generates three CXCR3 isoforms in humans. Previous studies investigated the roles of CXCR3 isoforms, and some biochemical data are not correlated with biological relevance analyses. RT-PCR analyses indicate that most cells express all three splicing variants, suggesting that they may mutually affect the chemokine binding and cellular responses of other splicing variants. Here, we performed an integrative analysis of the functional relations among CXCR3 splicing variants and their chemokine-dependent signaling using NanoBiT live cell protein interaction assays. The results indicated that the CXCR3 N-terminal region affected cell surface expression levels and ligand-dependent activation. CXCR3A was efficiently expressed in the plasma membrane and responded to I-TAC, IP-10, and MIG chemokines. By contrast, CXCR3B had low plasma membrane expression and mediated I-TAC–stimulated cellular responses. CXCR3Alt was rarely expressed on the cell surface and did not mediate any cell responses to the tested chemokines; however, CXCR3Alt negatively affected the plasma membrane expression of CXCR3A and CXCR3B and their chemokine-stimulated cellular responses. Jurkat cells express endogenous CXCR3, and exogenous CXCR3A expression enhanced chemotactic activity in response to I-TAC, IP-10, and MIG. By contrast, exogenous expression of CXCR3B and CXCR3Alt eliminated or reduced the CXCR3A-induced chemotactic activity. The PF-4 chemokine did not activate any CXCR3-mediated cellular responses. NanoBiT technology are useful to integrative studies of CXCR3-mediated cell signaling, and expand our knowledge of the cellular responses mediated by molecular interactions among the splicing variants, including cell surface expression, ligand-dependent receptor activation, and chemotaxis.
Generation of Soluble Human Tumor Necrosis Factor-α Receptor 1-Fc Transgenic Pig
Cho, Bumrae,Koo, Ok Jae,Hwang, Jong-Ik,Kim, Hwajung,Lee, Eun Mi,Hurh, Sunghoon,Park, Sol Ji,Ro, Han,Yang, Jaeseok,Surh, Charles D.,d’Apice, Anthony J.,Lee, Byeong Chun,Ahn, Curie Lippincott Williams Wilkins, Inc. 2011 Transplantation Vol.92 No.2
BACKGROUND.: Acute humoral xenograft rejection (AHXR) is an important barrier to xenograft survival. Human tumor necrosis factor-α (hTNF-α) is one of the essential mediators of AHXR and induces activation of porcine endothelial cells (PECs), resulting in upregulation of major histocompatibility complex molecules, adhesion molecules, and proinflammatory chemokines. We investigated whether introduction of a soluble human tumor necrosis factor receptor I-Fc (shTNFRI-Fc) fusion gene can suppress activation of PECs and, more importantly, produced shTNFRI-Fc transgenic pigs. METHODS.: The shTNFRI-Fc gene expression vector was constructed and inserted into PECs. The inhibitory effects of shTNFRI-Fc were tested by luciferase assay, reverse-transcriptase polymerase chain reaction, and flow cytometry. A shTNFRI-Fc transgenic pig was generated by somatic cell nuclear transfer. The expression of shTNFRI-Fc in the transgenic pig was evaluated by PCR, western blot, enzyme-linked immunosorbent assay, and immunohistochemistry. The inhibitory effects of shTNFRI-Fc in the serum obtained from the transgenic pig were also tested. RESULTS.: In comparison with control green fluorescent protein, shTNFRI-Fc protein showed much stronger inhibitory effects on NF-&kgr;B activation in the HEK293-NF-&kgr;B-luciferase reporting cell line, expression of chemokines and adhesion molecules in PECs, and TNF-α-mediated cytotoxicity. We successfully generated shTNFRI-Fc transgenic pig. Sera obtained from the transgenic pig inhibited induction of chemokines, and E-selectin in PECs stimulated with Human TNF-α. CONCLUSIONS.: We have generated transgenic pigs producing shTNFRI-Fc protein that can inhibit TNF-α-mediated activation of PECs. Because TNF-α is an important mediator of xenograft rejection, the use of xenografts that can produce shTNFRI-Fc proteins de novo could be an effective approach in overcoming a considerable component of the xenograft rejection process, especially AHXR.