Person: Silberstein, Leslie
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Silberstein
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Leslie
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Silberstein, Leslie
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Publication G-CSF maintains controlled neutrophil mobilization during acute inflammation by negatively regulating CXCR2 signaling(The Rockefeller University Press, 2016) Bajrami, Besnik; Zhu, Haiyan; Kwak, Hyun-Jeong; Mondal, Subhanjan; Hou, Qingming; Geng, Guangfeng; Karatepe, Kutay; Zhang, Yu C.; Nombela-Arrieta, César; Park, Shin-Young; Loison, Fabien; Sakai, Jiro; Xu, Yuanfu; Silberstein, Leslie; Luo, HongboCytokine-induced neutrophil mobilization from the bone marrow to circulation is a critical event in acute inflammation, but how it is accurately controlled remains poorly understood. In this study, we report that CXCR2 ligands are responsible for rapid neutrophil mobilization during early-stage acute inflammation. Nevertheless, although serum CXCR2 ligand concentrations increased during inflammation, neutrophil mobilization slowed after an initial acute fast phase, suggesting a suppression of neutrophil response to CXCR2 ligands after the acute phase. We demonstrate that granulocyte colony-stimulating factor (G-CSF), usually considered a prototypical neutrophil-mobilizing cytokine, was expressed later in the acute inflammatory response and unexpectedly impeded CXCR2-induced neutrophil mobilization by negatively regulating CXCR2-mediated intracellular signaling. Blocking G-CSF in vivo paradoxically elevated peripheral blood neutrophil counts in mice injected intraperitoneally with Escherichia coli and sequestered large numbers of neutrophils in the lungs, leading to sterile pulmonary inflammation. In a lipopolysaccharide-induced acute lung injury model, the homeostatic imbalance caused by G-CSF blockade enhanced neutrophil accumulation, edema, and inflammation in the lungs and ultimately led to significant lung damage. Thus, physiologically produced G-CSF not only acts as a neutrophil mobilizer at the relatively late stage of acute inflammation, but also prevents exaggerated neutrophil mobilization and the associated inflammation-induced tissue damage during early-phase infection and inflammation.Publication Deactivation of Akt by a Small Molecule Inhibitor Targeting Pleckstrin Homology Domain and Facilitating Akt Ubiquitination(Proceedings of the National Academy of Sciences, 2011) Jo, Hakryul; Lo, Pang-Kuo; Li, Yitang; Loison, Fabien; Green, Sarah; Wang, Jake; Silberstein, Leslie; Ye, Keqiang; Chen, Hexin; Luo, HongboThe phosphatidylinositol-3,4,5-triphosphate (PIP3) binding function of pleckstrin homology (PH) domain is essential for the activation of oncogenic Akt/PKB kinase. Following the PIP3-mediated activation at the membrane, the activated Akt is subjected to other regulatory events, including ubiquitination-mediated deactivation. Here, by identifying and characterizing an allosteric inhibitor, SC66, we show that the facilitated ubiquitination effectively terminates Akt signaling. Mechanistically, SC66 manifests a dual inhibitory activity that directly interferes with the PH domain binding to PIP3 and facilitates Akt ubiquitination. A known PH domain-dependent allosteric inhibitor, which stabilizes Akt, prevents the SC66-induced Akt ubiquitination. A cancer-relevant Akt1 (e17k) mutant is unstable, making it intrinsically sensitive to functional inhibition by SC66 in cellular contexts in which the PI3K inhibition has little inhibitory effect. As a result of its dual inhibitory activity, SC66 manifests a more effective growth suppression of transformed cells that contain a high level of Akt signaling, compared with other inhibitors of PIP3/Akt pathway. Finally, we show the anticancer activity of SC66 by using a soft agar assay as well as a mouse xenograft tumor model. In conclusion, in this study, we not only identify a dual-function Akt inhibitor, but also demonstrate that Akt ubiquitination could be chemically exploited to effectively facilitate its deactivation, thus identifying an avenue for pharmacological intervention in Akt signaling.Publication Focal Adhesion Kinase Regulates Pathogen-Killing Capability and Life Span of Neutrophils via Mediating both Adhesion-Dependent and -Independent Cellular Signals(American Association of Immunologists, 2009) Kasorn, Anongnard; Alcaide, Pilar; Jia, Yonghui; Subramanian, Kulandayan K.; Sarraj, Bara; Li, Yitang; Loison, Fabien; Hattori, Hidenori; Silberstein, Leslie; Luscinskas, William F.; Luo, HongboVarious neutrophil functions such as phagocytosis, superoxide production, and survival are regulated by integrin signaling. Despite the essential role of focal adhesion kinase (FAK) in mediating this signaling pathway, its exact function in neutrophils is ill defined. In this study, we investigated the role of FAK in neutrophils using a myeloid-specific conditional FAK knockout mouse. As reported in many other cell types, FAK is required for regulation of focal adhesion dynamics when neutrophils adhere to fibronectin or ICAM-1. Adhesion on VCAM-1-coated surfaces and chemotaxis after adhesion were not altered in FAK null neutrophils. In addition, we observed significant reduction in NADPH oxidase-mediated superoxide production and complement-mediated phagocytosis in FAK null neutrophils. As a result, these neutrophils displayed decreased pathogen killing capability both in vitro and in vivo in a mouse peritonitis model. In adherent cells, the defects associated with FAK deficiency are likely due to suppression of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) signaling and chemoattractant-elicited calcium signaling. Disruption of FAK also reduced chemoattractant-elicited superoxide production in suspended neutrophils in the absence of cell adhesion. This may be solely caused by suppression of PtdIns(3,4,5)P3 signaling in these cells, because the fMLP-elicited calcium signal was not altered. Consistent with decreased PtdIns(3,4,5)P3/Akt signaling in FAK null neutrophils, we also observed accelerated spontaneous death in these cells. Taken together, our results revealed previously unrecognized roles of FAK in neutrophil function and provided a potential therapeutic target for treatment of a variety of infectious and inflammatory diseases.Publication Fak Depletion in Both Hematopoietic and Nonhematopoietic Niche Cells Leads to Hematopoietic Stem Cell Expansion(Elsevier BV, 2012) Lu, Jiayun; Sun, Yan; Nombela-Arrieta, Cesar; Du, Karrie P.; Park, Shin-Young; Chai, Li; Walkley, Carl; Luo, Hongbo; Silberstein, LeslieHematopoietic stem cells (HSCs) reside in complex bone marrow microenvironments, where niche-induced signals regulate hematopoiesis. Focal adhesion kinase (Fak) is a nonreceptor protein tyrosine kinase that plays an essential role in many cell types, where its activation controls adhesion, motility, and survival. Fak expression is relatively increased in HSCs compared to progenitors and mature blood cells. Therefore, we explored its role in HSC homeostasis. We have used the Mx1-CreLinducible conditional knockout mouse model to investigate the effects of Fak deletion in bone marrow compartments. The total number as well as the fraction of cycling \(Lin^-Sca-1^+c-kit^+\) (LSK) cells is increased in \(Fak^{-/-}\) mice compared to controls, while hematopoietic progenitors and mature blood cells are unaffected. Bone marrow cells from \(Fak^{-/-}\) mice exhibit enhanced, long-term (i.e., 20-week duration) engraftment in competitive transplantation assays. Intrinsic Fak function was assessed in serial transplantation assays, which showed that HSCs (\(Lin^-Sca-1^+c-kit^+CD34^-Flk-2^-\) cells) sorted from \(Fak^{-/-}\) mice have similar self-renewal and engraftment ability on a per-cell basis as wild-type HSCs. When Fak deletion is induced after engraftment of \(Fak^{fl/fl}Mx1-Cre^+\) bone marrow cells into wild-type recipient mice, the number of LSKs is unchanged. In conclusion, Fak inactivation does not intrinsically regulate HSC behavior and is not essential for steady- state hematopoiesis. However, widespread Fak inactivation in the hematopoietic system induces an increased and activated HSC pool size, potentially as a result of altered reciprocal interactions between HSCs and their microenvironment.Publication Deficiency of Lipid Phosphatase SHIP Enables Long-Term Reconstitution of Hematopoietic Inductive Bone Marrow Microenvironment(Elsevier BV, 2013) Liang, Olin Dehui; Lu, Jiayun; Nombela-Arrieta, César; Zhong, Jia; Zhao, Li; Pivarnik, Gregory; Mondal, Subhanjan; Chai, Li; Silberstein, Leslie; Luo, HongboA dysfunctional bone marrow (BM) microenvironment is thought to contribute to the development of hematologic diseases. However, functional replacement of pathologic BM microenvironment through BM transplantation has not been possible. Furthermore, the study of hematopoietic inductive BM microenvironment is hampered by the lack of a functional nonhematopoietic reconstitution system. Here, we show that a deficiency of SH2-containing inositol-5'-phosphatase-1 (SHIP) in a nonhematopoietic host microenvironment enables its functional reconstitution by wild-type donor cells. This microenvironment reconstitution normalizes hematopoiesis in peripheral blood and BM and alleviates pathology of spleen and lung in the SHIP-deficient recipients. SHIP-deficient BM contains a significantly smaller population of multipotent stromal cells with distinct properties, which may contribute to the reconstitution by wild-type cells. We further demonstrate that it is the nonhematopoietic donor cells that are responsible for the reconstitution. Thus, we have established a nonhematopoietic BM microenvironment reconstitution system to functionally study specific cell types in hematopoietic niches.Publication Fgd5 identifies hematopoietic stem cells in the murine bone marrow(The Rockefeller University Press, 2014) Gazit, Roi; Mandal, Pankaj; Ebina, Wataru; Ben-Zvi, Ayal; Nombela-Arrieta, César; Silberstein, Leslie; Rossi, DerrickHematopoietic stem cells (HSCs) are the best-characterized tissue-specific stem cells, yet experimental study of HSCs remains challenging, as they are exceedingly rare and methods to purify them are cumbersome. Moreover, genetic tools for specifically investigating HSC biology are lacking. To address this we sought to identify genes uniquely expressed in HSCs within the hematopoietic system and to develop a reporter strain that specifically labels them. Using microarray profiling we identified several genes with HSC-restricted expression. Generation of mice with targeted reporter knock-in/knock-out alleles of one such gene, Fgd5, revealed that though Fgd5 was required for embryonic development, it was not required for definitive hematopoiesis or HSC function. Fgd5 reporter expression near exclusively labeled cells that expressed markers consistent with HSCs. Bone marrow cells isolated based solely on Fgd5 reporter signal showed potent HSC activity that was comparable to stringently purified HSCs. The labeled fraction of the Fgd5 reporter mice contained all HSC activity, and HSC-specific labeling was retained after transplantation. Derivation of next generation mice bearing an Fgd5-CreERT2 allele allowed tamoxifen-inducible deletion of a conditional allele specifically in HSCs. In summary, reporter expression from the Fgd5 locus permits identification and purification of HSCs based on single-color fluorescence.Publication SOCS3 Protein Developmentally Regulates the Chemokine Receptor CXCR4-FAK Signaling Pathway during B Lymphopoiesis(Elsevier BV, 2007) Le, Yi; Zhu, Bing-Mei; Harley, Brendan; Park, Shin-Young; Kobayashi, Takashi; Manis, John; Luo, Hongbo; Yoshimura, Akihiko; Hennighausen, Lothar; Silberstein, LeslieThe chemokine CXCL12 induces prolonged focal adhesion kinase (FAK) phosphorylation and sustained proadhesive responses in progenitor bone-marrow (BM) B cells, but not in mature peripheral B cells. Here we demonstrate that suppressor of cytokine signaling 3 (SOCS3) regulated CXCL12-induced FAK phosphorylation through the ubiquitin-proteasome pathway. CXCL12 triggered increased FAK ubiquitination in mature B cells, but not in progenitor B cells. Accordingly, SOCS3 expression was low in progenitor B cells, increased in immature B cells, and highest in mature B cells. SOCS3 overexpression in pro-B cells impaired CXCL12-induced FAK phosphorylation and proadhesive responses. Conversely, SOCS3-deficient mature B cells from \(Cre^{MMTV}Socs3^{fl/fl}\) mice exhibited prolonged FAK phosphorylation and adhesion to VCAM-1. In contrast to wild-type mice, \(Cre^{MMTV}Socs3^{fl/fl}\) mice had a 2-fold increase in immature B cells, which were evenly distributed in endosteal and perisinusoidal BM compartments. We propose that the developmental regulation of CXCR4-FAK signaling by SOCS3 is an important mechanism to control the lodgement of B cell precursors in the BM microenvironment.Publication Inositol Trisphosphate 3-Kinase B (InsP3KB) as a Physiological Modulator of Myelopoiesis(Proceedings of the National Academy of Sciences, 2008) Jia, Yonghui; Loison, Fabien; Hattori, Hidenori; Li, Yitang; Erneux, Christophe; Park, Shin-Young; Gao, Chong; Chai, Li; Silberstein, Leslie; Schurmans, Stephane; Luo, HongboInositol trisphosphate 3-kinase B (InsP3KB) belongs to a family of kinases that convert inositol 1,4,5-trisphosphate (Ins(1,4,5)P3 or IP3) to inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4). Previous studies have shown that disruption of InsP3KB leads to impaired T cell and B cell development as well as hyperactivation of neutrophils. Here, we demonstrate that InsP3KB is also a physiological modulator of myelopoiesis. The InsP3KB gene is expressed in all hematopoietic stem/progenitor cell populations. In InsP3KB null mice, the bone marrow granulocyte monocyte progenitor (GMP) population was expanded, and GMP cells proliferated significantly faster. Consequently, neutrophil production in the bone marrow was enhanced, and the peripheral blood neutrophil count was also substantially elevated in these mice. These effects might be due to enhancement of PtdIns(3,4,5)P3/Akt signaling in the InsP3KB null cells. Phosphorylation of cell cycle-inhibitory protein \(p21^{cip1}\), one of the downstream targets of Akt, was augmented, which can lead to the suppression of the cell cycle-inhibitory effect of p21.Publication Relevant Mouse Model for Human Monocytic Leukemia through Cre/lox-Controlled Myeloid-Specific Deletion of PTEN(Nature Publishing Group, 2010) Yu, H.; Li, Yili; Gao, Chuanyun; Fabien, L.; Jia, Y.; Lu, Junjie; Silberstein, Leslie; Pinkus, Geraldine; Ye, K.; Chai, Li; Luo, HongboPublication Pretreatment with Phosphatase and Tensin Homolog Deleted on Chromosome 10 (PTEN) Inhibitor SF1670 Augments the Efficacy of Granulocyte Transfusion in a Clinically Relevant Mouse Model(American Society of Hematology, 2011) Li, Yitang; Prasad, Amit; Jia, Yonghui; Roy, Saurabh Ghosh; Loison, Fabien; Mondal, Subhanjan; Kocjan, Paulina; Silberstein, Leslie; Ding, Sheng; Luo, HongboThe clinical outcome of granulocyte transfusion therapy is often hampered by short ex vivo shelf life, inefficiency of recruitment to sites of inflammation, and poor pathogen-killing capability of transplanted neutrophils. Here, using a recently developed mouse granulocyte transfusion model, we revealed that the efficacy of granulocyte transfusion can be significantly increased by elevating intracellular phosphatidylinositol (3,4,5)-trisphosphate signaling with a specific phosphatase and tensin homolog deleted on chromosome 10 (PTEN) inhibitor SF1670. Neutrophils treated with SF1670 were much sensitive to chemoattractant stimulation. Neutrophil functions, such as phagocytosis, oxidative burst, polarization, and chemotaxis, were augmented after SF1670 treatment. The recruitment of SF1670-pretreated transfused neutrophils to the inflamed peritoneal cavity and lungs was significantly elevated. In addition, transfusion with SF1670-treated neutrophils led to augmented bacteria-killing capability (decreased bacterial burden) in neutropenic recipient mice in both peritonitis and bacterial pneumonia. Consequently, this alleviated the severity of and decreased the mortality of neutropenia-related pneumonia. Together, these observations demonstrate that the innate immune responses can be enhanced and the severity of neutropenia-related infection can be alleviated by augmenting phosphatidylinositol (3,4,5)-trisphosphate in transfused neutrophils with PTEN inhibitor SF1670, providing a therapeutic strategy for improving the efficacy of granulocyte transfusion.