Paola Gasperini

Impaired Recruitment of Grk6 and β-Arrestin2 Causes Delayed Internalization and Desensitization of a WHIM Syndrome-Associated CXCR4 Mutant Receptor

WHIM (warts, hypogammaglobulinemia, infections, and myelokatexis) syndrome is a rare immunodeficiency syndrome linked to heterozygous mutations of the chemokine receptor CXCR4 resulting in truncations of its cytoplasmic tail. Leukocytes from patients with WHIM syndrome display impaired CXCR4 internalization and enhanced chemotaxis in response to its unique ligand SDF-1/CXCL12, which likely contribute to the clinical manifestations. Here, we investigated the biochemical mechanisms underlying CXCR4 deficiency in WHIM syndrome. We report that after ligand activation, WHIM-associated mutant CXCR4 receptors lacking the carboxy-terminal 19 residues internalize and activate Erk 1/2 slower than wild-type (WT) receptors, while utilizing the same trafficking endocytic pathway. Recruitment of β-Arrestin 2, but not β-Arrestin 1, to the active WHIM-mutant receptor is delayed compared to the WT CXCR4 receptor. In addition, while both kinases Grk3 and Grk6 bind to WT CXCR4 and are critical to its trafficking to the lysosomes, Grk6 fails to associate with the WHIM-mutant receptor whereas Grk3 associates normally. Since β-Arrestins and Grks play critical roles in phosphorylation and internalization of agonist-activated G protein-coupled receptors, these results provide a molecular basis for CXCR4 dysfunction in WHIM syndrome.

Kaposi Sarcoma Herpesvirus Promotes Endothelial-to-Mesenchymal Transition through Notch-Dependent Signaling

Endothelial-to-mesenchymal transition (EndMT) is now widely considered a pivotal contributor to cancer progression. In this study, we show that the Kaposis sarcoma (KS)-associated herpesvirus (KSHV) is a sufficient cause of EndMT, potentially helping to explain the aggressiveness of KS that occurs commonly in AIDS patients. Upon KSHV infection, primary dermal microvascular endothelial cells lost expression of endothelial markers and acquired expression of mesenchymal markers, displaying new invasive and migratory properties along with increased survival. KSHV activated Notch-induced transcription factors Slug and ZEB1, and canonical Notch signaling was required for KSHV-induced EndMT. In contrast, KSHV did not utilize the TGFβ signaling pathway, which has also been linked to EndMT. Within KS lesions, KSHV-infected spindle cells displayed features compatible with KSHV-induced EndMT including a complex phenotype of endothelial and mesenchymal properties, Notch activity, and nuclear ZEB1 expression. Our results show that KSHV engages the EndMT program to increase the invasiveness and survival of infected endothelial cells, traits that likely contribute to viral persistence and malignant progression. One important implication of our findings is that therapeutic approaches to disrupt the Notch pathway may offer novel approaches for KS treatment.

MicroRNA126 contributes to granulocyte colony-stimulating factor-induced hematopoietic progenitor cell mobilization by reducing the expression of vascular cell adhesion molecule 1.

Background Mobilization of hematopoietic stem/progenitor cells from the bone marrow to the peripheral blood by granulocyte colony-stimulating factor is the primary means to acquire stem cell grafts for hematopoietic cell transplantation. Since hematopoietic stem/progenitor cells represent a minority of all blood cells mobilized by granulocyte colony-stimulating factor, the underlying mechanisms need to be understood in order to develop selective drugs. Design and Methods We analyzed phenotypic, biochemical and genetic changes in bone marrow cell populations from granulocyte colony-stimulating factor-mobilized and control mice, and linked such changes to effective mobilization of hematopoietic stem/progenitor cells. Results We show that granulocyte colony-stimulating factor indirectly reduces expression of surface vascular cell adhesion molecule 1 on bone marrow hematopoietic stem/progenitor cells, stromal cells and endothelial cells by promoting the accumulation of microRNA-126 (miR126)-containing microvescicles in the bone marrow extracellular compartment. We found that hematopoietic stem/progenitor cells, stromal cells and endothelial cells readily incorporate these miR126-loaded microvescicles, and that miR126 represses vascular cell adhesion molecule 1 expression on bone marrow hematopoietic stem/progenitor cells, stromal cells and endothelial cells. In line with this, miR126-null mice displayed a reduced mobilization response to granulocyte colony-stimulating factor. Conclusions Our results implicate miR126 in the regulation of hematopoietic stem/progenitor cell trafficking between the bone marrow and peripheral sites, clarify the role of vascular cell adhesion molecule 1 in granulocyte colony-stimulating factor-mediated mobilization, and have important implications for improved approaches to selective mobilization of hematopoietic stem/progenitor cells.

Contribution of viral and cellular cytokines to Kaposi’s sarcoma-associated herpesvirus pathogenesis

Kaposis sarcoma (KS)‐associated herpesvirus is associated with the proliferative/malignant disorders KS, primary effusion lymphoma (PEL), and multicentric Castlemans disease (MCD) in patients with AIDS. In spite of recent advances in the treatment of KS, PEL and MCD represent therapeutic challenges. Recent advances in dissecting the pathogenesis of these diseases have indicated that the viral cytokine IL‐6 and the cellular cytokines/growth factors IL‐10, IL‐6, stromal cell‐derived factor 1, and vascular endothelial growth factor are important contributors to the growth, survival, and spread of PEL and MCD and are therefore potential targets for drug development.

The transcription factor Gfi1 regulates G-CSF signaling and neutrophil development through the Ras activator RasGRP1

The transcription factor growth factor independence 1 (Gfi1) and the growth factor granulocyte colony-stimulating factor (G-CSF) are individually essential for neutrophil differentiation from myeloid progenitors. Here, we provide evidence that the functions of Gfi1 and G-CSF are linked in the regulation of granulopoiesis. We report that Gfi1 promotes the expression of Ras guanine nucleotide releasing protein 1 (RasGRP1), an exchange factor that activates Ras, and that RasGRP1 is required for G-CSF signaling through the Ras/mitogen-activated protein/extracellular signal-regulated kinase (MEK/Erk) pathway. Gfi1-null mice have reduced levels of RasGRP1 mRNA and protein in thymus, spleen, and bone marrow, and Gfi1 transduction in myeloid cells promotes RasGRP1 expression. When stimulated with G-CSF, Gfi1-null myeloid cells are selectively defective at activating Erk1/2, but not signal transducer and activator of transcription 1 (STAT1) or STAT3, and fail to differentiate into neutrophils. Expression of RasGRP1 in Gfi1-deficient cells rescues Erk1/2 activation by G-CSF and allows neutrophil maturation by G-CSF. These results uncover a previously unknown function of Gfi1 as a regulator of RasGRP1 and link Gfi1 transcriptional control to G-CSF signaling and regulation of granulopoiesis.

Targeting the mammalian target of Rapamycin to inhibit VEGF and cytokines for the treatment of primary effusion lymphoma

Primary effusion lymphoma (PEL) is a fatal malignancy, which typically presents as a lymphomatous effusion that later disseminates. Rapamycin (Rapa), which targets mTOR (mammalian target of Rapa), is currently evaluated as a treatment for PEL, but the recent development of PEL in Rapa-treated post-transplant recipients questions the drugs use in PEL. Here, we used a murine model of PEL effusion that mimics the human disease to investigate the anti-PEL activity of Rapa. We found that Rapa reduces ascites accumulation and extends mouse survival. Initially, Rapa reduced PEL load compared with control mice, but most mice rapidly showed PEL progression. Levels of VEGF, which promotes vascular permeability contributing to effusion formation, were significantly reduced in ascites of Rapa-treated mice compared with controls. Expression of IL-10, the principal autocrine growth factor for PEL, was initially reduced in PEL from Rapa-treated mice but rapidly increased despite treatment. We found that the hypoxic environment of ascites and Rapa cooperate in stimulating IL-10 expression in PEL, which likely contributes to the emergence of drug resistance. These results identify Rapa an effective drug to reduce PEL effusions but illustrate the rapid development of drug resistance, which likely limits the efficacy of Rapa in PEL.