ALBERT

Description: Article The anti-diabetic drug metformin has been shown to increase lifespan of some model organisms, but results have been conflicting. Here, Martin-Montalvo et al . administer one of two doses of metformin to male mice and show that the lower dose increases healthspan and lifespan, while the higher dose is toxic. Nature Communications doi: 10.1038/ncomms3192 Authors: Alejandro Martin-Montalvo, Evi M. Mercken, Sarah J. Mitchell, Hector H. Palacios, Patricia L. Mote, Morten Scheibye-Knudsen, Ana P. Gomes, Theresa M. Ward, Robin K. Minor, Marie-José Blouin, Matthias Schwab, Michael Pollak, Yongqing Zhang, Yinbing Yu, Kevin G. Becker, Vilhelm A. Bohr, Donald K. Ingram, David A. Sinclair, Norman S. Wolf, Stephen R. Spindler, Michel Bernier, Rafael de Cabo

Description: Ras plays an essential role in lymphocyte development and function. However, in vivo consequence(s) of regulation of Ras activity by guanosine triphosphatase (GTPase)–activating proteins (GAPs) on lymphocyte development and function are not known. In this study we demonstrate that neurofibromin, the protein encoded by theNF1 tumor suppressor gene functions as a GAP for Ras in T cells. Loss of Nf1 in T cells results in enhanced Ras activation, which is associated with thymic and splenic hyperplasia, and an increase in the absolute number of immature and mature T-cell subsets compared with control mice. Interestingly, in spite of a profound T-cell expansion and higher thymidine incorporation in unstimulated Nf1-deficient T cells, T-cell receptor and interleukin-2 receptor–mediated proliferation of thymocytes and mature T cells was substantially reduced compared with control mice. Collectively, these results identify neurofibromin as a GAP for Ras in T cells for maintaining immune homeostasis in vivo.

Description: Mast cells are key participants in allergic diseases via activation of high-affinity IgE receptors (FcϵRI) resulting in release of proinflammatory mediators. The biochemical pathways linking IgE activation to calcium influx and cytoskeletal changes required for intracellular granule release are incompletely understood. We demonstrate, genetically, that Pak1 is required for this process. In a passive cutaneous anaphylaxis experiment, Wsh/Wsh mast cell–deficient mice locally reconstituted with Pak1−/− bone marrow–derived mast cells (BMMCs) experienced strikingly decreased allergen-induced vascular permeability compared with controls. Consistent with the in vivo phenotype, Pak1−/− BMMCs exhibited a reduction in FcϵRI-induced degranulation. Further, Pak1−/− BMMCs demonstrated diminished calcium mobilization and altered depolymerization of cortical filamentous actin (F-actin) in response to FcϵRI stimulation. These data implicate Pak1 as an essential molecular target for modulating acute mast cell responses that contribute to allergic diseases.

Description: Class IA phosphatidylinositol-3 kinase (PI-3K) is a lipid kinase, which is activated in blood cells by hematopoietic growth factors. In vitro experiments using chemical inhibitors of PI-3K suggest that this kinase is potentially important for hematopoietic stem and progenitor cell (HSC/P) function, and recent studies identify PI-3K as a therapeutic target in treating different leukemias and lymphomas. However, the role of PI-3K in regulating fetal liver or adult hematopoiesis in vivo is unknown. Therefore, we examined PI-3K-deficient embryos generated by a targeted deletion of the p85α and p85β regulatory subunits of PI-3K (p85α-/-p85β+/-). The absolute frequency and number of hematopoietic progenitor cells were reduced in p85α-/- p85β+/- fetal livers compared with wild-type (WT) controls. Further, p85α-/-p85β+/- fetal liver hematopoietic stem cells (HSCs) had decreased multilineage repopulating ability in vivo compared with WT controls in competitive repopulation assays. Finally, purified p85α-/-p85β+/- c-kit+ cells had a decrease in proliferation in response to kit ligand (kitL), a growth factor important for controlling HSC function in vivo. Collectively, these data identify PI-3K as an important regulator of HSC function and potential therapeutic target in treating leukemic stem cells.

Description: Neurofibromatosis type 1 (NF1), encoded by the NF1 tumor suppressor gene, is one of the most common autosomal dominant genetic disorders. Neurofibromin, the protein product of NF1, functions as a GTPase activating protein (GAP) for p21 Ras (Ras); however, its specific GAP activity towards the different Ras isoforms is not fully understood. We have previously established that Nf1 haploinsufficiency alters mast cell fates both in vitro and in vivo, and modulates the recruitment of mast cells to Nf1-/- Schwann cells, the tumor initiating cell type in neurofibromas (Yang, JCI 2003). We have also found high expression of both K-ras and N-ras, but undetectable levels of H-ras, in mast cells. We examined the role of Nf1 as a GAP for K-ras utilizing fetal liver-derived mast cells. Consistent with previous studies, haploinsufficency of Nf1 results in a two-fold increase in proliferation of mast cells associated with an increase in ERK activity as compared to WT controls. However, genetic inactivation of K-ras rescues the proliferative gain in function of Nf1 +/− mast cells, associated with lower biochemical activation of ERK as compared to WT levels. Additionally, utilizing Boyden chamber assays, as well as videomicroscopy, Nf1 +/− mast cells exhibits increased migration to kit-L, while genetic inactivation of K-ras abrogates this gain in function. Further, degranulation in response to crosslinking the high affinity Fc receptor on the surface of mast cells is markedly reduced in Nf1+/−; K-ras-/- cells as compared to Nf1 +/− mast cells. A key gain in function observed in Nf1+/− and Nf1 -/- hematopoietic cells is increased survival associated with an increase in PI3-K activity. While H-ras has been shown to differentially activate the PI3-K pathway compared to K-ras in other cell systems, we have found that genetic inactivation of K-ras reduces Akt activity, a sensitive measure of PI3-K activity, in Nf1+/− mast cells. This decrease in activity is associated with a two- to three-fold reduction in survival as compared to Nf1 +/− mast cells and is comparable to WT mast cells. Additionally, an intermediate in vitro phenotype is observed in cells that are heterozygous at both the Nf1 and K-ras loci (Nf1 +/−; K-ras +/−). To evaluate the interaction between Nf1 and K-ras in vivo, two model systems were utilized. Since K-ras-/- embryos die in utero, adult K-ras heterozygotes were utilized to examine the in vivo mast cell response to kit-L using microsmotic pumps placed subcutaneously in the dorsal side of the animals. Consistent with previous studies, Nf1 +/− mice have increased mast cell numbers in response to kit-L as compared to WT controls. However, genetic disruption of a single allele of K-ras restores mast cell numbers to WT levels. As a second model system, we are currently evaluating the role of genetic disruption of both alleles of K-ras in mast cells in vivo by adoptively transferring hematopoietic cells of the respective genotypes into lethally irradiated mice that are genetically deficient in the c-kit receptor and lack mast cells (W/Wv mice). Collectively, these findings suggest that Nf1 acts as a GAP for K-ras in mast cells and that K-ras is integral to a range of mast cell functions. Further, these data suggest that K-ras may be a useful molecular target for the treatment of neurofibromas as well as other disorders that involve mast cells in disease pathogenesis.

Description: Endothelial progenitor cells (EPCs) can be isolated from adult peripheral and umbilical cord blood. EPCs are thought to originate from bone marrow, circulate in peripheral blood, and migrate to sites of angiogenesis. However, the number of circulating EPCs in peripheral blood is remarkably low, and recent genetic studies show that the contribution of bone marrow derived EPCs to newly formed vascular networks is minimal. Further, while endothelial cells (ECs) derived from vessel walls are widely considered to be differentiated mature ECs, these cells retain extensive proliferative potential and can be passaged for at least 40 population doubling in vitro. Based on these observations, we tested whether EPCs potentially reside in vessel walls utilizing a newly developed single cell deposition assay (Blood, 2004). Analogous to a paradigm established in the hematopoietic cell system, we can utilize this assay to reproducibly identify the following EPCs: (1) high proliferative potential - endothelial colony forming cells (HPP-ECFC), which form macroscopic colonies that form secondary and tertiary colonies upon replating, (2) low proliferative potential - endothelial colony forming cells (LPP-ECFC), which form colonies greater than 50 cells, but do not form secondary colonies upon replating, (3) endothelial cell clusters (EC-clusters) that contain less than 50 cells, and (4) mature terminally differentiated endothelial cells (EC), which do not divide. Utilizing this assay, we compared the clonogenic potential of 1000 single adult human dermal microvascular endothelial cells (HMVECds), human umbilical vein endothelial cells (HUVECs), human umbilical artery endothelial cells (HUAECs), human coronary artery endothelial cells (HCAECs), and human aortic endothelial cells (HAECs) to the potential of adult peripheral and umbilical cord blood derived EPCs. We conducted four independent experiments. Remarkably, we demonstrate that a complete hierarchy of EPCs can be identified in EC populations derived from every vessel wall tested (Table I and n=4). Further, we show that ECs derived from each vessel wall cell population tested contain more proliferative EPCs (LPP-ECFCs and HPP-ECFCs) compared to EPCs derived from adult peripheral blood. Percent of 1,000 Single Cells Plated Mature EC EC-Cluster LPP-ECFC HPP-ECFC HUVEC 42±6 18±2 29±9 11±5 HAEC 37±3 23±8 21±4 20±6 HMVECd 65±9 21±6 12±4 2±0.6 HCAEC 46±2 18±2 20±2 16±2 HUAEC 41±1 10±1 27±4 21±2 Adult EPC 81±9 9±1 12±8 0.2±0.2 Cord EPC 50±20 7±2 20±10 23±9 Thus, this study provides evidence that a diversity of EPCs exists in human vessels and provides a new conceptual framework for determining both the origin and function of EPCs in maintaining vessel integrity and contributing to new sites of angiogenesis.

Description: While it has been established that c-Kit/stem cell factor (SCF) interactions are indispensable for normal mast cell development and functions, the intracellular signals that regulate the development and function of these cells downstream from c-Kit are not fully understood. Here, we demonstrate that p85 regulatory subunits of class IA PI-3Kinase (p85α and p85β) play an essential role in regulating mast cell growth and maturation. We show that deficiency of p85α in mast cells results in reduced growth and survival in response to SCF stimulation, which is associated with impaired activation of Akt, Rac and JNK MAP kinase. Furthermore, we demonstrate that in addition to regulating growth and survival, loss of p85α also impairs the maturation of mast cells as seen by significantly reduced expression of c-Kit and the IgE receptor on p85α−/− mast cells compared to wildtype controls. To determine the extent to which Akt contributes to the growth, survival and maturation of p85α−/− mast cells, we reconstituted p85α−/− mast cells with a retrovirus encoding an activated form of Akt. Remarkably, p85α−/− mast cells expressing an activated Akt completely rescued the proliferative defect associated with p85α deficient mast cells in response to IL-3 as well as IL-3 plus SCF, and a 70% correction in response to SCF alone. The phenotypic abnormalities seen in p85α−/− mast cells were surprising in light of the sequence homology and continued expression in these cells of p85β subunit of class IA PI-3Kinase, and suggested that p85α might function with specificity in mast cells in regulating c-Kit functions. To examine the relationship between p85α and p85β in regulating mast cell functions, we generated mast cells from p85β−/− mice and compared them with p85α−/− mast cells. We found that p85α and p85β perform qualitatively distinct functions in regulating mast cell growth and differentiation. We found that although deficiency of p85α in mast cells consistently results in reduced growth and impaired maturation, deficiency of p85β in these same cells results in enhanced growth and no significant defect in maturation. To further demonstrate that p85α and p85β play qualitatively distinct functions in regulating mast cell growth and differentiation, a genetic intercross between p85α+/− and p85β+/− mice was performed. Since mice completely deficient in both the isoforms of p85 (α & β) die early in embryonic development, we analyzed mast cell growth and differentiation in mice that were deficient in the expression of p85α(p85α−/−) and heterozygous at the p85β (p85β+/−) locus, along with the appropriate controls. We found that heterozygosity of p85β (p85β+/−) in the setting of p85α deficiency (p85α−/−) significantly corrected the mast cell defect associated with p85α deficiency (p85α−/−) alone. Finally, we reconstituted p85α−/− mast cells with a bicistronic retrovirus encoding EGFP and a cDNA for p85β, and found that p85β could not rescue the mast cell defect associated with p85α deficiency. In contrast, reconstituting p85α−/− mast cells with a retrovirus encoding the p85α cDNA completely rescued the mast cell growth defect associated with p85α deficiency. The level of expression of p85α and p85β in p85α−/− reconstituted mast cells was similar as determined by western blot analysis. Taken together, our results demonstrate qualitatively distinct functions for two closely related regulatory subunits of class IA PI-3Kinase in controlling mast cell growth and maturation.

Description: Neurofibromin, the protein encoded by the NF1 tumor-suppressor gene, negatively regulates the output of p21ras proteins by accelerating the hydrolysis of active Ras-guanosine triphosphate to inactive Ras-guanosine diphosphate. Children with neurofibromatosis type 1 (NF1) are predisposed to juvenile myelomonocytic leukemia (JMML) associated with loss of the normal NF1 allele. Genetically engineered mice containing nullizygous loss of Nf1 in the hematopoietic system develop a leukemia with complete penetrance that is similar to JMML, including hepatosplenomegaly, elevated peripheral blood cell counts, and elevated numbers of myeloid progenitors that are hypersensitive to multiple cytokines, particularly GM-CSF. Though we and others have found that p21ras is hyperactivated in Nf1−/− myeloid progenitors, inhibiting Ras specifically as a therapeutic target has been challenging. Therefore, identification of alterations in distinct p21ras effector pathways that control leukemia progression in Nf1-deficient cells is critical for understanding disease pathogenesis and identifying therapeutic targets. Here we intercrossed MxCre; Nf1flox/flox mice with mice that are deficient in the small Rho GTPases Rac1 or Rac2 to generate syngeneic progeny that were MxCre; Nf1flox/flox, MxCre; Nf1flox/flox;Rac1flox flox or MxCre; Nf1flox/flox; Rac2 −/−. Consistent with previous studies (Le, Blood 2004) MxCre; Nf1flox/flox mice develop a progressive myeloproliferative disease with 100% penetrance 6 months following interferon inducible induction of the MxCre transgene to disrupt the Nf1flox alleles. Eighty percent of MxCre; Nf1flox/flox die by 9 months after inactivation. Genetic disruption of Rac2 was not sufficient to diminish the onset or severity of the characteristic myeloproliferative disease of MxCre; Nf1flox/flox mice. In contrast, MxCre; Nf1flox/flox; Rac1flox/flox mice followed for 11 months all survived and had normal bone marrow cellularity, spleen weight and splenic architecture. MxCre; Nf1flox/flox mice have elevated numbers of both HPP-CFC and LPP-CFC in the bone marrow and spleen. In contrast, MxCre; Nf1flox/flox; Rac1flox/flox mice had myeloid progenitor numbers that were comparable to wildtype, age-matched controls. Further, though myeloid progenitors from MxCre; Nf1flox/flox mice are hypersensitive to GM-CSF, myeloid progenitors from MxCre; Nf1flox/flox; Rac1flox/flox have a sensitivity to GM-CSF comparable to wildtype controls. The correction in the myeloproliferative phenotype isolated from MxCre; Nf1flox/flox; Rac1flox/flox mice was associated with the reduction in Rac-GTP and prolonged Erk phosphorylation, a MAPK effector that is characteristically elevated in Nf1−/− myeloid progenitors. Collectively, these genetic data identify Ras-Rac1 signaling pathway as a key axis in the genesis of juvenile myelomonocytic leukemia and provide evidence that Rac1 is a therapeutic molecular target for this myeloproliferative disease that currently has no effective therapies.

Description: Patients with neurofibromatosis type 1 (NF1) have mutations in the NF1 tumor-suppressor gene that functions as a GTPase activating protein (GAP) for p21ras. Individuals with NF1 display a variety of skeletal defects including lytic bone lesions. However, the effect of loss of NF1 on osteoclasts or osteoblasts, the two principal cells involved in bone remodeling, is not understood. Osteoclasts are specialized myeloid cells that adhere to bone matrix, and secrete lytic enzymes that degrade bone. Given that we have previously identified haploinsufficient phenotypes in Nf1 +/− mast cells, we defined the role of neurofibromin in regulating various osteoclast functions in vitro and in vivo in Nf1 +/− mice. Strikingly, histologic sections from femurs of Nf1 +/− mice, revealed large numbers of multinucleated osteoclasts. This phenotype is reminiscent of the osteoclasts in patients with Paget’s disease and of osteoclasts isolated from SHIP deficient mice, which have elevated levels of PI-3K activity. Additionally, osteoclast progenitors from Nf1 +/− mice were hyperresponsive to limiting concentrations of M-CSF and RANKL and formed higher numbers of OCL progenitors compared to +/+ controls (P

Description: Cutaneous neurofibromas are a hallmark of neurofibromatosis type 1 (NF1), a common genetic disorder that is caused by mutations in the NF1 gene, which functions as a GAP for p21ras. Though the pathogenesis of neurofibroma formation is not completely known, haploinsufficiency of the nonneuronal lineages (fibroblasts, mast cells and endothelial cells) in the tumor microenvironment are required for neurofibroma formation (Zhu, Science 2002). These tumors are characterized by a high concentration of degranulating mast cells (MC) closely associated with fibroblasts, endothelial cells and Schwann cells. We have recently shown that Nf1−/− Schwann cells secrete kit-ligand to recruit Nf1+/− MCs to the tumor microenvironment via hyperactivation of a p21ras-PI3K-Rac dependent pathway (Yang, JCI 2003). Further, Nf1+/− MC also promote Schwann cell invasion and proliferation. Given that collagen is synthesized by fibroblasts and is approximately 80% of the weight of neurofibromas, we tested whether Nf1+/− MC promote the proliferation and collagen synthesis of fibroblasts. Strikingly, the proliferation of Nf1+/− fibroblasts in response to Nf1+/− MC conditioned media (MCCM) was 3 fold higher than any other group tested. In a wound healing assay Nf1+/− MCCM provided potent stimulus for the migration of Nf1+/− but not WT fibroblasts. Similarly, MCCM from primary human NF1+/− MC stimulated the proliferation, migration, and collagen synthesis of human NF1+/− fibroblasts, validating that our observations in Nf1+/− murine cells faithfully phenocopy the biology of human NF1 heterozygous cells. We next established three dimensional collagen lattices containing MC and fibroblasts of the respective genotypes to evaluate extracellular matrix (ECM) reorganization given that remodeling of the ECM by inflammatory cells promotes tumorigenesis. Histological examination revealed that while MC and fibroblasts of both genotypes localized to each other, there was a 2–3 fold quantitative increase in the localization or attachment of Nf1+/− MC to Nf1+/− fibroblasts. Further, Nf1+/− MC preferentially promoted a 2–3 fold increase in the lattice contraction indicative of alteration of the ECM in lattices containing either Nf1+/− or WT fibroblasts. Given that c-kit/kit-ligand interactions between MC and fibroblasts contribute to MC-fibroblast interactions, c-kit blocking antibodies or Gleevec, an antitumor drug that inhibits both BCR/ABL and c-kit tyrosine kinases, were added to MC-fibroblast cultures. Both of these agents blocked the activity of Nf1+/− MC on fibroblast proliferation, collagen remodeling and fibroblast migration. Collectively, these studies demonstrate that murine and human NF1 (Nf1) haploinsufficient MC stimulate the proliferation, migration, collagen synthesis of Nf1+/− fibroblasts as well as remodeling the ECM. This study provides strong evidence that Gleevec may be a candidate therapy for the treatment or prevention of neurofibromas.