Mi-Yeon Jung

Stabilin-1 mediates phosphatidylserine-dependent clearance of cell corpses in alternatively activated macrophages

Stabilin-1 is specifically expressed in alternatively activated macrophages. These macrophages participate in anti-inflammatory and healing processes, and display a high phagocytic capacity. In this study, we provide evidence that stabilin-1 is a membrane receptor that performs a crucial function in the clearance of cell corpses. Stabilin-1 is expressed on the cell surface of alternatively activated macrophages and is recruited to the sites of recognition and engulfment of apoptotic bodies, as well as to early phagosomes. Blocking stabilin-1 in macrophages results in defective engulfment of aged red blood cells. Ectopic expression of stabilin-1 induces the binding and engulfment of aged cells in mouse fibroblast L cells. The binding and phagocytosis are dependent on phosphatidylserine (PS), which is well known as an engulfing ligand. Furthermore, using PS-coated beads, we demonstrate that PS directly interacts with stabilin-1 and is sufficient for stabilin-1-mediated phagocytosis. EGF-like domain repeat in stabilin-1 is responsible for PS recognition and binding. Thus, our results demonstrate that stabilin-1, found on alternatively activated macrophages, is a phagocytic receptor mediating the clearance of apoptotic cells in a PS-dependent manner. Therefore, this protein might play an important role in the maintenance of tissue homeostasis and prevention of autoimmunity.

Mechanism for phosphatidylserine-dependent erythrophagocytosis in mouse liver.

Aged or damaged RBCs are effectively removed from the blood circulation by Kupffer cells in the liver, but little is known regarding the mechanism of the clearance process. Here we show that stabilin-1 and stabilin-2 in hepatic sinusoidal endothelial cells (HSECs) are critical in effectively clearing damaged RBCs in mouse liver. Damaged RBCs and phosphatidylserine (PS)-coated beads were effectively sequestered in the hepatic sinusoid regardless of the presence of Kupffer cells, suggesting a role for HSECs in PS-dependent sequestration of PS-exposed RBCs in the liver. HSECs mediate tethering of damaged RBCs in a PS-dependent manner via stabilin-1 and stabilin-2. In a sinusoid-mimicked coculture system consisting of macrophages layered over HSECs, there was significant enhancement of the phagocytic capacity of macrophages, and this was mediated by stabilin-1 and stabilin-2 in HSECs. Liver-specific knockdown of stabilin-1 and stabilin-2 inhibited the sequestration of damaged RBCs in the hepatic sinusoid and delayed the elimination of damaged cells in an in vivo animal model. Thus, the roles of stabilin-1 and stabilin-2 in hepatic sequestration of PS-exposed RBCs may represent a potential mechanism for the clearance of damaged RBCs by Kupffer cells and for the control of some pathologic conditions such as hemolytic anemia.

Epidermal Growth Factor-Like Domain Repeat of Stabilin-2 Recognizes Phosphatidylserine during Cell Corpse Clearance

ABSTRACT Exposure of phosphatidylserine (PS) on the cell surface occurs early during apoptosis and serves as a recognition signal for phagocytes. Clearance of apoptotic cells by a membrane PS receptor is one of the critical anti-inflammatory functions of macrophages. However, the PS binding receptors and their recognition mechanisms have not been fully investigated. Recently, we reported that stabilin-2 is a PS receptor that mediates the clearance of apoptotic cells, thus releasing the anti-inflammatory cytokine, transforming growth factor β. In this study, we showed that epidermal growth factor (EGF)-like domain repeats (EGFrp) in stabilin-2 can directly and specifically recognize PS. The EGFrps also competitively impaired apoptotic cell uptake by macrophages in in vivo models. We also showed that calcium ions are required for stabilin-2 to mediate phagocytosis via EGFrp. Interestingly, at least four tandem repeats of EGF-like domains were required to recognize PS, and the second atypical EGF-like domain in EGFrp was critical for calcium-dependent PS recognition. Considering that PS itself is an important target molecule for both apoptotic cells and nonapoptotic cells during various cellular processes, our results should help elucidate the molecular mechanism by which apoptotic cell clearance in the human body occurs and also have implications for targeting PS externalization of nonapoptotic cells.

Stabilin-2 is involved in lymphocyte adhesion to the hepatic sinusoidal endothelium via the interaction with αMβ2 integrin

Although lymphocyte recirculation to the endothelium plays a critical role in the movement of immune cells from the blood into tissues and sites of inflammation, the mechanisms involved in lymphocyte trafficking via the hepatic circulation have yet to be elucidated fully. In this study, we investigated the role of stabilin‐2, which is expressed specifically in the sinusoidal endothelium, in the adhesion of lymphocytes to the hepatic endothelium. Stabilin‐2‐expressing cells mediate the adhesion of PBLs. This interaction was attributed specifically to the interaction of stabilin‐2 with αMβ2 integrin. Using mutant stabilin‐2 molecules with deletions in the extracellular domain, we mapped the binding site for αMβ2 integrin to the fasciclin 1 (FAS1) domains of stabilin‐2. The specificity of the interaction between αMβ2 integrin and the FAS1 domain was confirmed further by binding assays using neutralizing antibodies. More physiologically, we showed that the down‐regulation of stabilin‐2 results in the defective binding of lymphocytes to hepatic sinusoidal endothelial cells under conditions of static and physiological flow. Together, these data show that stabilin‐2 can reconstitute the lymphocyte–endothelial adhesion cascade under physiological shear stress. We propose a critical role for stabilin‐2 in lymphocyte adhesion to specialized endothelia, such as that of the hepatic sinusoid.

Combination Therapy and Noninvasive Imaging with a Dual Therapeutic Vector Expressing MDR1 Short Hairpin RNA and a Sodium Iodide Symporter

We investigated the feasibility of using combination gene therapy and noninvasive nuclear imaging after expression of the human sodium iodide symporter (hNIS) and inhibition of the multidrug resistance (MDR1) gene in colon cancer cells. Methods: HCT-15 cells were stably transfected with a dual expression vector, in which the hNIS gene, driven by a constitutive cytomegalovirus promoter, has been coupled to an MDR1 short hairpin RNA (shRNA) cassette. Cell lines stably expressing the hNIS gene and MDR1 shRNA (designated MN-61 and MN-62) were produced, and the expression of the NIS gene and MDR1 shRNA was examined by Western blotting, reverse transcription–polymerase chain reaction, and immunostaining. The functional activities of MDR1 shRNA were determined by paclitaxel uptake and sensitivity to doxorubicin. Functional NIS expression was confirmed by the uptake and efflux of 125I and the cytotoxicity of 131I. The effect of the combination of 131I and doxorubicin was determined by an in vitro clonogenic assay. In vivo NIS expression was examined by small-animal PET with 124I. Results: The shMDR-NIS–expressing cells showed a significant decrease in the expression of MDR1 messenger RNA and its translated product, P-glycoprotein. The inhibition of P-glycoprotein expression by shRNA enhanced the intracellular accumulation of paclitaxel, the cellular retention of which is mediated by P-glycoprotein, thereby increasing sensitivity to the anticancer drug. The shMDR-NIS–expressing cells showed a significant increase of 125I uptake, which was completely inhibited by KClO4. Although the iodide efflux rate was rapid, the cell survival rate was markedly reduced by 131I treatment. Interestingly, the combination of doxorubicin and a radioiodide (131I) displayed synergistic cytotoxicity that correlated with MDR1 inhibition and NIS expression in shMDR-NIS–expressing cells. Furthermore, in mice with shMDR-NIS–expressing tumor xenografts, small-animal PET with 124I clearly visualized shMDR1-NIS–expressing tumors. Conclusion: We developed a dual expression vector with the NIS gene and MDR1 shRNA. This study represents a promising first step in investigations of the potential use of a combination of the NIS gene and MDR1 shRNA as a new therapeutic strategy allowing RNA interference–based gene therapy, NIS-based radioiodine therapy, and in vivo monitoring based on NIS imaging.

Stabilin-2 mediates homophilic cell–cell interactions via its FAS1 domains

Stabilin‐2 was recently shown to mediate a heterophilic interaction with integrin alphaMbeta2 via its FAS1 domain. Here, we demonstrate that stabilin‐2 also mediates homophilic cell–cell interactions. L cells expressing stabilin‐2 mediate a significant level of cell aggregation, and this aggregation is significantly inhibited by anti‐stabilin‐2 antibody. Stabilin‐2‐mediated aggregation is mediated by homophilic interactions and enhanced in the presence of Ca2+ and Mg2+. Interestingly, exogenous addition of FAS1 domains but not EGF‐like domains enhances stabilin‐2‐mediated cell aggregation, suggesting that exogenous FAS1 domains may form polymeric structure with FAS1 domains of stabilin‐2. Together, these data show the participation of stabilin‐2 in homophilic cell adhesion and role of FAS1 domains.

T-CAM, a fastatin-FIII 9-10 fusion protein, potently enhances anti-angiogenic and anti-tumor activity via alphavbeta3 and alpha5beta1 integrins.

We made fusion protein of fastatin and FIII 9-10, termed tetra-cell adhesion molecule (T-CAM) that can interact simultaneously with αvβ3 and α5β1 integrins, both playing important roles in tumor angiogenesis. T-CAM can serve as a cell adhesion substrate mediating adhesion and migration of endothelial cells in αvβ3 and α5β1 integrin-dependent manner. T-CAM showed pronounced anti-angiogenic activities such as inhibition of endothelial cell tube formation, endothelial cell proliferation, and induction of endothelial cell apoptosis. T-CAM also inhibited angiogenesis and tumor growth in mouse xenograft model. The anti-angiogenic and anti-tumoral activity of molecule like fastatin could be improved by fusing it with integrin-recognizing cell adhesion domain from other distinct proteins. The strategy of combining two distinct anti-angiogenic molecules or cell adhesion domains could facilitate designing improved anticancer agent of therapeutic value.

T-CAM, a fastatin-FIII 9-10 fusion protein, potently enhances anti-angiogenic and anti-tumor activity αvβ3 and α5β1 integrins

We made fusion protein of fastatin and FIII 9-10, termed tetra-cell adhesion molecule (T-CAM) that can interact simultaneously with αvβ3 and α5β1 integrins, both playing important roles in tumor angiogenesis. T-CAM can serve as a cell adhesion substrate mediating adhesion and migration of endothelial cells in αvβ3 and α5β1 integrin-dependent manner. T-CAM showed pronounced anti-angiogenic activities such as inhibition of endothelial cell tube formation, endothelial cell proliferation, and induction of endothelial cell apoptosis. T-CAM also inhibited angiogenesis and tumor growth in mouse xenograft model. The anti-angiogenic and anti-tumoral activity of molecule like fastatin could be improved by fusing it with integrin-recognizing cell adhesion domain from other distinct proteins. The strategy of combining two distinct anti-angiogenic molecules or cell adhesion domains could facilitate designing improved anticancer agent of therapeutic value.

T-CAM, a fastatin-FIII 9-10 fusion protein, potently enhances anti-angiogenic and anti-tumor activity via αvβ3 and α5β1 integrins

We made fusion protein of fastatin and FIII 9-10, termed tetra-cell adhesion molecule (T-CAM) that can interact simultaneously with αvβ3 and α5β1 integrins, both playing important roles in tumor angiogenesis. T-CAM can serve as a cell adhesion substrate mediating adhesion and migration of endothelial cells in αvβ3 and α5β1 integrin-dependent manner. T-CAM showed pronounced anti-angiogenic activities such as inhibition of endothelial cell tube formation, endothelial cell proliferation, and induction of endothelial cell apoptosis. T-CAM also inhibited angiogenesis and tumor growth in mouse xenograft model. The anti-angiogenic and anti-tumoral activity of molecule like fastatin could be improved by fusing it with integrin-recognizing cell adhesion domain from other distinct proteins. The strategy of combining two distinct anti-angiogenic molecules or cell adhesion domains could facilitate designing improved anticancer agent of therapeutic value.