Selective Phosphodiesterase 1 Inhibitor BTTQ Reduces Blood Pressure in Spontaneously Hypertensive and Dahl Salt Sensitive Rats: Role of Peripheral Vasodilation.

Regulation of the peripheral vascular resistance via modulating the vessel diameter has been considered as a main determinant of the arterial blood pressure. Phosphodiesterase enzymes (PDE1-11) hydrolyse cyclic nucleotides, which are key players controlling the vessel diameter and, thus, peripheral resistance. Here, we have tested and reported the effects of a novel selective PDE1 inhibitor (BTTQ) on the cardiovascular system. Normal Sprague Dawley, spontaneously hypertensive (SHR), and Dahl salt-sensitive rats were used to test in vivo the efficacy of the compound. Phosphodiesterase radiometric enzyme assay revealed that BTTQ inhibited all three isoforms of PDE1 in nanomolar concentration, while micromolar concentrations were needed to induce effective inhibition for other PDEs. The myography study conducted on mesenteric arteries revealed a potent vasodilatory effect of the drug, which was confirmed in vivo by an increase in the blood flow in the rat ear arteriols reflected by the rise in the temperature. Furthermore, BTTQ proved a high efficacy in lowering the blood pressure about 9, 36, and 24 mmHg in normal Sprague Dawley, SHR and, Dahl salt-sensitive rats, respectively, compared to the vehicle-treated group. Moreover, additional blood pressure lowering of about 22 mmHg could be achieved when BTTQ was administered on top of ACE inhibitor lisinopril, a current standard of care in the treatment of hypertension. Therefore, PDE1 inhibition induced efficient vasodilation that was accompanied by a significant reduction of blood pressure in different hypertensive rat models. Administration of BTTQ was also associated with increased heart rate in both models of hypertension as well as in the normotensive rats. Thus, PDE1 appears to be an attractive therapeutic target for the treatment of resistant hypertension, while tachycardia needs to be addressed by further compound structural optimization.

Kidney surveillance in the spotlight: contrast-induced acute kidney injury illuminated.

Acute kidney injury comprises a heterogeneous group of conditions characterized by a sudden decrease in renal function over hours to days. Contrast-induced acute kidney injury (CI-AKI) is caused by radiographic contrast agents used in diagnostic imaging. In the current issue of the JCI, Lau et al. use a mouse model of CI-AKI to study the role of resident and infiltrating phagocytes, recruited leukocytes, and tubular cells in the immune surveillance response to contrast agents. This study has the potential to provide innovative therapies for human CI-AKI.

Exogenous Gene Transmission of Isocitrate Dehydrogenase 2 Mimics Ischemic Preconditioning Protection.

Ischemic preconditioning confers organ-wide protection against subsequent ischemic stress. A substantial body of evidence underscores the importance of mitochondria adaptation as a critical component of cell protection from ischemia. To identify changes in mitochondria protein expression in response to ischemic preconditioning, we isolated mitochondria from ischemic preconditioned kidneys and sham-treated kidneys as a basis for comparison. The proteomic screen identified highly upregulated proteins, including NADP+-dependent isocitrate dehydrogenase 2 (IDH2), and we confirmed the ability of this protein to confer cellular protection from injury in murine S3 proximal tubule cells subjected to hypoxia. To further evaluate the role of IDH2 in cell protection, we performed detailed analysis of the effects of Idh2 gene delivery on kidney susceptibility to ischemia-reperfusion injury. Gene delivery of IDH2 before injury attenuated the injury-induced rise in serum creatinine (P<0.05) observed in controls and increased the mitochondria membrane potential (P<0.05), maximal respiratory capacity (P<0.05), and intracellular ATP levels (P<0.05) above those in controls. This communication shows that gene delivery of Idh2 can confer organ-wide protection against subsequent ischemia-reperfusion injury and mimics ischemic preconditioning.

Hydrodynamic Isotonic Fluid Delivery Ameliorates Moderate-to-Severe Ischemia-Reperfusion Injury in Rat Kidneys.

Highly aerobic organs like the kidney are innately susceptible to ischemia-reperfusion (I/R) injury, which can originate from sources including myocardial infarction, renal trauma, and transplant. Therapy is mainly supportive and depends on the cause(s) of damage. In the absence of hypervolemia, intravenous fluid delivery is frequently the first course of treatment but does not reverse established AKI. Evidence suggests that disrupting leukocyte adhesion may prevent the impairment of renal microvascular perfusion and the heightened inflammatory response that exacerbate ischemic renal injury. We investigated the therapeutic potential of hydrodynamic isotonic fluid delivery (HIFD) to the left renal vein 24 hours after inducing moderate-to-severe unilateral IRI in rats. HIFD significantly increased hydrostatic pressure within the renal vein. When conducted after established AKI, 24 hours after I/R injury, HIFD produced substantial and statistically significant decreases in serum creatinine levels compared with levels in animals given an equivalent volume of saline via peripheral infusion (P<0.05). Intravital confocal microscopy performed immediately after HIFD showed improved microvascular perfusion. Notably, HIFD also resulted in immediate enhancement of parenchymal labeling with the fluorescent dye Hoechst 33342. HIFD also associated with a significant reduction in the accumulation of renal leukocytes, including proinflammatory T cells. Additionally, HIFD significantly reduced peritubular capillary erythrocyte congestion and improved histologic scores of tubular injury 4 days after IRI. Taken together, these results indicate that HIFD performed after establishment of AKI rapidly restores microvascular perfusion and small molecule accessibility, with improvement in overall renal function.

A wandering path toward prevention for acute kidney injury.

Acute kidney injury (AKI) is a common cause of hospital-related mortality; therefore, strategies to either prevent or treat this complication are of great interest. In this issue of the JCI, Inoue, Abe, and colleagues have uncovered a targetable neuroimmunomodulatory mechanism that protects mice from ischemia-reperfusion injury (IRI) and subsequent AKI. Specifically, the authors demonstrate that vagus nerve stimulation (VNS) activates the cholinergic antiinflammatory pathway (CAP), resulting in activation of antiinflammatory effects via α7 nicotinic acetylcholine receptor-expressing splenic macrophages. Together, the results of this study have potential clinical implications in the prevention of AKI in at-risk individuals.

A method to facilitate and monitor expression of exogenous genes in the rat kidney using plasmid and viral vectors.

Gene therapy has been proposed as a novel alternative to treat kidney disease. This goal has been hindered by the inability to reliably deliver transgenes to target cells throughout the kidney, while minimizing injury. Since hydrodynamic forces have previously shown promising results, we optimized this approach and designed a method that utilizes retrograde renal vein injections to facilitate transgene expression in rat kidneys. We show, using intravital fluorescence two-photon microscopy, that fluorescent albumin and dextrans injected into the renal vein under defined conditions of hydrodynamic pressure distribute broadly throughout the kidney in live animals. We found injection parameters that result in no kidney injury as determined by intravital microscopy, histology, and serum creatinine measurements. Plasmids, baculovirus, and adenovirus vectors, designed to express EGFP, EGFP-actin, EGFP-occludin, EGFP-tubulin, tdTomato-H2B, or RFP-actin fusion proteins, were introduced into live kidneys in a similar fashion. Gene expression was then observed in live and ex vivo kidneys using two-photon imaging and confocal laser scanning microscopy. We recorded widespread fluorescent protein expression lasting more than 1 mo after introduction of transgenes. Plasmid and adenovirus vectors provided gene transfer efficiencies ranging from 50 to 90%, compared with 10-50% using baculovirus. Using plasmids and adenovirus, fluorescent protein expression was observed 1) in proximal and distal tubule epithelial cells; 2) within glomeruli; and 3) within the peritubular interstitium. In isolated kidneys, fluorescent protein expression was observed from the cortex to the papilla. These results provide a robust approach for gene delivery and the study of protein function in live mammal kidneys.

Dishevelled-associated activator of morphogenesis 1 (Daam1) is required for heart morphogenesis.

Dishevelled-associated activator of morphogenesis 1 (Daam1), a member of the formin protein family, plays an important role in regulating the actin cytoskeleton via mediation of linear actin assembly. Previous functional studies of Daam1 in lower species suggest its essential role in Drosophila trachea formation and Xenopus gastrulation. However, its in vivo physiological function in mammalian systems is largely unknown. We have generated Daam1-deficient mice via gene-trap technology and found that Daam1 is highly expressed in developing murine organs, including the heart. Daam1-deficient mice exhibit embryonic and neonatal lethality and suffer multiple cardiac defects, including ventricular noncompaction, double outlet right ventricles and ventricular septal defects. In vivo genetic rescue experiments further confirm that the lethality of Daam1-deficient mice results from the inherent cardiac abnormalities. In-depth analyses have revealed that Daam1 is important for regulating filamentous actin assembly and organization, and consequently for cytoskeletal function in cardiomyocytes, which contributes to proper heart morphogenesis. Daam1 is also found to be important for proper cytoskeletal architecture and functionalities in embryonic fibroblasts. Biochemical analyses indicate that Daam1 does not regulate cytoskeletal organization through RhoA, Rac1 or Cdc42. Our study highlights a crucial role for Daam1 in regulating the actin cytoskeleton and tissue morphogenesis.

RhoA/Rho kinase (ROCK) alters fetuin-A uptake and regulates calcification in bovine vascular smooth muscle cells (BVSMC).

RhoA/Rho kinases (ROCK) play a critical role in vascular smooth muscle cell (VSMC) actin cytoskeleton organization, differentiation, and function and are implicated in the pathogenesis of cardiovascular disease. We have previously determined that an important step in the regulation of calcification is fetuin-A endocytosis, a process that is dependent on changes in the cytoskeleton, which, in turn, is known to be affected by the RhoA/ROCK signaling pathway. In the present study, bovine VSMC (BVSMC) were treated with the ROCK inhibitor Y-27632 or transfected with ROCK small interfering (si) RNA to knock down ROCK expression. Both conditions resulted in reduced actin stress fibers and increased Cy5-labeled fetuin-A uptake. Inhibition of ROCK by Y-27632 or siRNA also significantly increased BVSMC alkaline phosphatase (ALP) activity and calcification of BVSMC and rat aorta organ cultures. Cells were then incubated in calcification media in the presence or absence of Y-27632 and matrix vesicles (MV) isolated by collagenase digestion. These MV, isolated from BVSMC incubated with Y-27632, had increased ALP activity and increased ability of MV to subsequently calcify collagen by 66%. In contrast, activation of RhoA, which is upstream of ROCK, by transfecting plasmids encoding the dominant active Rho GTPase mutant (Rho-L63) led to decreased fetuin-A uptake and reduced calcification in BVSMC. These results demonstrate that the RhoA/ROCK signaling pathway is an important negative regulator of vascular calcification.

siRNA targeted to p53 attenuates ischemic and cisplatin-induced acute kidney injury.

Proximal tubule cells (PTCs), which are the primary site of kidney injury associated with ischemia or nephrotoxicity, are the site of oligonucleotide reabsorption within the kidney. We exploited this property to test the efficacy of siRNA targeted to p53, a pivotal protein in the apoptotic pathway, to prevent kidney injury. Naked synthetic siRNA to p53 injected intravenously 4 h after ischemic injury maximally protected both PTCs and kidney function. PTCs were the primary site for siRNA uptake within the kidney and body. Following glomerular filtration, endocytic uptake of Cy3-siRNA by PTCs was rapid and extensive, and significantly reduced ischemia-induced p53 upregulation. The duration of the siRNA effect in PTCs was 24 to 48 h, determined by levels of p53 mRNA and protein expression. Both Cy3 fluorescence and in situ hybridization of siRNA corroborated a short t(1/2) for siRNA. The extent of renoprotection, decrease in cellular p53 and attenuation of p53-mediated apoptosis by siRNA were dose- and time-dependent. Analysis of renal histology and apoptosis revealed improved injury scores in both cortical and corticomedullary regions. siRNA to p53 was also effective in a model of cisplatin-induced kidney injury. Taken together, these data indicate that rapid delivery of siRNA to proximal tubule cells follows intravenous administration. Targeting siRNA to p53 leads to a dose-dependent attenuation of apoptotic signaling, suggesting potential therapeutic benefit for ischemic and nephrotoxic kidney injury.

p21-activated kinase regulates mast cell degranulation via effects on calcium mobilization and cytoskeletal dynamics.

Mast cells are key participants in allergic diseases via activation of high-affinity IgE receptors (FcepsilonRI) 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, W(sh)/W(sh) 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 FcepsilonRI-induced degranulation. Further, Pak1(-/-) BMMCs demonstrated diminished calcium mobilization and altered depolymerization of cortical filamentous actin (F-actin) in response to FcepsilonRI stimulation. These data implicate Pak1 as an essential molecular target for modulating acute mast cell responses that contribute to allergic diseases.

Cytokine-induced F-actin reorganization in endothelial cells involves RhoA activation.

Acute ischemic kidney injury results in marked increases in local and systemic cytokine levels. IL-1alpha, IL-6, and TNF-alpha orchestrate various inflammatory reactions influencing endothelial permeability by altering cell-to-cell and cell-to-extracellular matrix attachments. To explore the role of actin and the regulatory proteins RhoA and cofilin in this process, microvascular endothelial cells (MS1) were exposed to individual cytokines or a cytokine cocktail. Within minutes, a marked, time-dependent redistribution of the actin cytoskeleton occurred with the formation of long, dense F-actin basal stress fibers. The concentration of F-actin, normalized to nuclear staining, significantly increased compared with untreated cells (up 20%, P < or = 0.05). Western blot analysis of MS1 lysates incubated with the cytokine cocktail for 4 h showed an increase in phosphorylated/inactive cofilin (up 25 +/- 15%, P < or = 0.05) and RhoA activation (up to 227 +/- 26% increase, P < or = 0.05) compared with untreated cells. Decreasing RhoA levels using small interfering RNA blocked the effect of cytokines on stress fiber organization. Treatment with Y-27632, an inhibitor of the RhoA effector p160-ROCK, decreased levels of phosphorylated cofilin and reduced stress fiber fluorescence by 22%. In cells treated with Y-27632 followed by treatment with the cytokine cocktail, stress fiber levels were similar to control cells and cofilin phosphorylation was 55% of control levels. Taken together, these studies demonstrate cytokine stimulation of RhoA, which in turn leads to cofilin phosphorylation and formation of numerous basal actin stress fibers. These results suggest cytokines signal through the Rho-ROCK pathway, but also through another pathway to affect actin dynamics.

Fc gamma R-stimulated activation of the NADPH oxidase: phosphoinositide-binding protein p40phox regulates NADPH oxidase activity after enzyme assembly on the phagosome.

The phagocyte NADPH oxidase generates superoxide for microbial killing, and includes a membrane-bound flavocytochrome b(558) and cytosolic p67(phox), p47(phox), and p40(phox) subunits that undergo membrane translocation upon cellular activation. The function of p40(phox), which binds p67(phox) in resting cells, is incompletely understood. Recent studies showed that phagocytosis-induced superoxide production is stimulated by p40(phox) and its binding to phosphatidylinositol-3-phosphate (PI3P), a phosphoinositide enriched in membranes of internalized phagosomes. To better define the role of p40(phox) in FcgammaR-induced oxidase activation, we used immunofluorescence and real-time imaging of FcgammaR-induced phagocytosis. YFP-tagged p67(phox) and p40(phox) translocated to granulocyte phagosomes before phagosome internalization and accumulation of a probe for PI3P. p67(phox) and p47(phox) accumulation on nascent and internalized phagosomes did not require p40(phox) or PI3 kinase activity, although superoxide production before and after phagosome sealing was decreased by mutation of the p40(phox) PI3P-binding domain or wortmannin. Translocation of p40(phox) to nascent phagosomes required binding to p67(phox) but not PI3P, although the loss of PI3P binding reduced p40(phox) retention after phagosome internalization. We conclude that p40(phox) functions primarily to regulate FcgammaR-induced NADPH oxidase activity rather than assembly, and stimulates superoxide production via a PI3P signal that increases after phagosome internalization.

Protein phosphatase 2A plays an important role in stromal cell-derived factor-1/CXC chemokine ligand 12-mediated migration and adhesion of CD34+ cells.

Migration of hemopoietic stem and progenitor cells (HSPC) is required for homing to bone marrow following transplantation. Therefore, it is critical to understand signals underlying directional movement of HSPC. Stromal cell-derived factor-1 (SDF-1)/CXCL12 is a potent chemoattractant for HSPC. In this study, we demonstrate that the serine-threonine protein phosphatase (PP)2A plays an important role in regulation of optimal level and duration of Akt/protein kinase B activation (a molecule important for efficient chemotaxis), in response to SDF-1. Inhibition of PP2A, using various pharmacological inhibitors of PP2A including okadaic acid (OA) as well as using genetic approaches including dominant-negative PP2A-catalytic subunit (PP2A-C) or PP2A-C small interfering RNA, in primary CD34(+) cord blood (CB) cells led to reduced chemotaxis. This was associated with impairment in polarization and slower speed of movement in response to SDF-1. Concomitantly, SDF-1-induced Akt phosphorylation was robust and prolonged. Following SDF-1 stimulation, Akt and PP2A-C translocate to plasma membrane with enhanced association of PP2A-C with Akt observed at the plasma membrane. Inhibition of PI3K by low-dose LY294002 partially recovered chemotactic activity of cells pretreated with OA. In addition to chemotaxis, adhesion of CD34(+) cells to fibronectin was impaired by OA pretreatment. Our study demonstrates PP2A plays an important role in chemotaxis and adhesion of CD34(+) CB cells in response to SDF-1. CD34(+) CB cells pretreated with OA showed impaired ability to repopulate NOD-SCID mice in vivo, suggesting physiological relevance of these observations.

aPKC-PAR complex dysfunction and tight junction disassembly in renal epithelial cells during ATP depletion.

Renal ischemia and in vitro ATP depletion result in disruption of the epithelial tight junction barrier, which is accompanied by breakdown of plasma membrane polarity. Tight junction formation is regulated by evolutionarily conserved complexes, including that of atypical protein kinase C (aPKC), Par3, and Par6. The aPKC signaling complex is activated by Rac and regulated by protein phosphorylation and associations with other tight junction regulatory proteins, for example, mLgl. In this study, we examined the role of aPKC signaling complex during ATP depletion and recovery in Madin-Darby canine kidney cells. ATP depletion reduced Rac GTPase activity and induced Par3, aPKCzeta, and mLgl-1 redistribution from sites of cell-cell contact, which was restored following recovery from ATP depletion. Zonula occludens (ZO)-1 and Par3 phosphorylation was reduced and association of aPKCzeta with its substrates Par3 and mLgl-1 was stabilized in ATP-depleted Madin-Darby canine kidney cells. ATP depletion also induced a stable association of Par3 with Tiam-1, a Rac GTPase exchange factor, which explains how aPKCzeta and Rac activities were suppressed. Experimental inhibition of aPKCzeta during recovery from ATP depletion interfered with reassembly of ZO-1 and Par3 at cell junctions. These data indicate that aPKC signaling is impaired during ATP depletion, participates in tight junction disassembly during cell injury and is important for tight junction reassembly during recovery.

Functional intravital imaging of leukocytes in animal models of renal injury.

An important emerging paradigm in the understanding of renal disease is the recognition of the central role of inflammation in the initiation and progression of acute and chronic kidney injury. These advances have led to an increasing awareness of the importance of leukocytes (white blood cells (WBC)) in the pathogenesis of renal disease, and the necessity for a greater understanding of the specific roles of different WBC lineages. All aspects of WBC function have been implicated in aspects of renal disease. In many cases soluble factors derived from these cells (cytokines, complement, immunoglobulins, etc.) having effects remote from the secreting cells are involved, while in other cases there is apparently more direct involvement of infiltrating cells themselves acting on their immediate surroundings. This highlights the importance of understanding the dynamic behavior of specific WBC cell types and their interactions with the intrinsic cells of the kidney during injury. New insight into this question is promised by recent developments in imaging technology that allow WBC movement and interactions with endothelial or epithelial cells or with the extracellular matrix to be visualized within tissues, even in the relatively unperturbed setting of intact organs in the live animal.

Glucocorticoids protect and enhance recovery of cultured murine podocytes via actin filament stabilization.

BACKGROUND: Nephrotic syndrome is a common kidney disease in both children and adults that is characterized by dramatic structural changes in the actin-rich foot processes of glomerular podocytes. Although glucocorticoids are the primary treatment for nephrotic syndrome, neither the target cell nor mechanism of action of glucocorticoids in nephrotic syndrome is known. For the last 30 years glucocorticoids have been presumed to act by reducing the release of soluble mediators of disease by circulating lymphocytes. In contrast, we hypothesized that glucocorticoids exert their beneficial effects in nephrotic syndrome by direct action on podocytes. METHODS: Cultured murine podocytes were treated with glucocorticoids in the presence and absence of mifepristone (to inhibit glucocorticoid-induced transcriptional activation) and challenged using our previously reported in vitro model of puromycin aminonucleoside (PAN)-induced podocyte injury, as well as by direct disruption of actin filaments with latrunculin and cytochalasin. Cell viability, actin filament distribution, total polymerized actin content, and actin-regulating guanine triphosphatase (GTPase) activities were measured. RESULTS: We demonstrated that treatment of cultured murine podocytes with the glucocorticoid dexamethasone both protected and enhanced recovery from PAN-induced injury. Dexamethasone also increased total cellular polymerized actin, stabilized actin filaments against disruption by PAN, latrunculin, or cytochalasin, and induced a significant increase in the activity of the actin-regulating GTPase RhoA. CONCLUSION: These data suggest that, contrary to the current therapeutic paradigm, the beneficial effects of glucocorticoids in nephrotic syndrome may result, at least in part, from direct effects on podocytes leading to enhanced actin filament stability.

Rac GTPase isoform-specific regulation of NADPH oxidase and chemotaxis in murine neutrophils in vivo. Role of the C-terminal polybasic domain.

The Rho family GTPase Rac acts as a molecular switch for signal transduction to regulate various cellular functions. Mice deficient in the hematopoietic-specific Rac2 isoform exhibit agonist-specific defects in neutrophil chemotaxis and superoxide production, despite expression of the highly homologous Rac1 isoform. To examine whether functional defects in rac2(-/-) neutrophils reflect effects of an overall decrease in total cellular Rac or an isoform-specific role for Rac2, retroviral vectors were used to express exogenous Rac1 or Rac2 at levels similar to endogenous. In rac2(-/-) neutrophils differentiated from transduced myeloid progenitors in vitro, increasing cellular Rac levels by expression of either exogenous Rac1 or Rac2 increased formylmethionylleucylphenylalanine- or phorbol ester-stimulated NADPH oxidase activity. Of note, placement of an epitope tag on the N terminus of Rac1 or Rac2 blunted reconstitution of responses in rac2(-/-) neutrophils. In rac2(-/-) neutrophils isolated from mice transplanted with Rac-transduced bone marrow cells, superoxide production and chemotaxis were fully reconstituted by expression of exogenous Rac2, but not Rac1. A chimeric Rac1 protein in which the Rac1 C-terminal polybasic domain, which contains six lysines or arginines, was replaced with that of the human Rac2 polybasic domain containing only three basic residues, also reconstituted superoxide production and chemotaxis, whereas expression of a Rac2 derivative in which the polybasic domain was replaced with that of Rac1 did not and resulted in disoriented cell motility. Thus, the composition of the polybasic domain is sufficient for determining Rac isoform specificity in the production of superoxide and chemotaxis in murine neutrophils in vivo.

Rac2-deficient murine macrophages have selective defects in superoxide production and phagocytosis of opsonized particles.

The Rho family GTPase Rac is a crucial participant in numerous cellular functions and acts as a molecular switch for signal transduction. Mice deficient in hemopoietic-specific Rac2 exhibited agonist-specific defects in neutrophil functions including chemoattractant-stimulated filamentous actin polymerization and chemotaxis, and superoxide production elicited by phorbol ester, fMLP, or IgG-coated particles, despite expression of the highly homologous Rac1 isoform. In this study, functional responses of Rac2-null murine macrophages were characterized to examine whether Rac2 also has nonredundant functions in this phagocytic lineage. In contrast to murine neutrophils, in which Rac1 and Rac2 are present in similar amounts, Rac1 was approximately 4-fold more abundant than Rac2 in both bone marrow-derived and peritoneal exudate macrophages, and macrophage Rac1 levels were unchanged by the absence of Rac2. Accumulation of exudate macrophages during peritoneal inflammation was reduced in rac2(-/-) mice. FcgammaR-mediated phagocytosis of IgG-coated SRBC was also significantly decreased in Rac2-null macrophages, as was NADPH oxidase activity in response to phorbol ester or FcgammaR stimulation. However, phagocytosis and oxidant production stimulated by serum-opsonized zymosan was normal in rac2(-/-) macrophages. Macrophage morphology was also similar in wild-type and Rac2-null cells, as was actin polymerization induced by FcgammaR-mediated phagocytosis or M-CSF. Hence, Rac2-null macrophages have selective defects paralleling many of the observed functional defects in Rac2-null neutrophils. These results provide genetic evidence that although Rac2 is a relatively minor isoform in murine macrophages, it plays a nonoverlapping role with Rac1 to regulate host defense functions in this phagocyte lineage.

Mechanism of actin polymerization in cellular ATP depletion.

Cellular ATP depletion in diverse cell types results in the net conversion of monomeric G-actin to polymeric F-actin and is an important aspect of cellular injury in tissue ischemia. We propose that this conversion results from altering the ratio of ATP-G-actin and ADP-G-actin, causing a net decrease in the concentration of thymosinactin complexes as a consequence of the differential affinity of thymosin beta4 for ATP- and ADP-G-actin. To test this hypothesis we examined the effect of ATP depletion induced by antimycin A and substrate depletion on actin polymerization, the nucleotide state of the monomer pool, and the association of actin monomers with thymosin and profilin in the kidney epithelial cell line LLC-PK1. ATP depletion for 30 min increased F-actin content to 145% of the levels under physiological conditions, accompanied by a corresponding decrease in G-actin content. Cytochalasin D treatment did not reduce F-actin formation during ATP depletion, indicating that it was predominantly not because of barbed end monomer addition. ATP-G-actin levels decreased rapidly during depletion, but there was no change in the concentration of ADP-G-actin monomers. The decrease in ATP-G-actin levels could be accounted for by dissociation of the thymosin-G-actin binary complex, resulting in a rise in the concentration of free thymosin beta4 from 4 to 11 microm. Increased detection of profilin-actin complexes during depletion indicated that profilin may participate in catalyzing nucleotide exchange during depletion. This mechanism provides a biochemical basis for the accumulation of F-actin aggregates in ischemic cells.

Neurofibromin-deficient Schwann cells secrete a potent migratory stimulus for Nf1+/- mast cells.

The NF1 tumor suppressor gene encodes a GTPase-activating protein called neurofibromin that negatively regulates Ras signaling. Mutations in NF1 cause neurofibromatosis type 1 (NF1). The development of neurofibromas, which are complex tumors composed of multiple cell types, is a hallmark of NF1. Somatic inactivation of murine Nf1 in Schwann cells is necessary, but not sufficient, to initiate neurofibroma formation. Neurofibromas occur with high penetrance in mice in which Nf1 is ablated in Schwann cells in the context of a heterozygous mutant (Nf1+/-) microenvironment. Mast cells infiltrate neurofibromas, where they secrete proteins that can remodel the ECM and initiate angiogenesis. Thus, identification of mechanisms responsible for mast cell migration to tumor microenvironments is important for understanding tumorigenesis and for designing potential therapies. Here, we show that homozygous Nf1 mutant (Nf1-/-) Schwann cells secrete Kit ligand (KitL), which stimulates mast cell migration, and that Nf1+/- mast cells are hypermotile in response to KitL. Furthermore, we link hyperactivation of the Ras-class IA-PI3K-Rac2 pathway to increased Nf1+/- mast cell migration. Thus, these studies identify a novel interaction between Nf1-/- Schwann cells and Nf1+/- mast cells that is likely to be important in neurofibroma formation.