Hypoxia stabilizes the H2 O2 -producing oxidase Nox4 in cardiomyocytes via suppressing autophagy-related lysosomal degradation.

The hydrogen peroxide (H2 O2 )-producing NADPH oxidase Nox4, forming a heterodimer with p22phox , is expressed in a variety of cells including those in the heart to mediate adaptive responses to cellular stresses such as hypoxia. Since Nox4 is constitutively active, H2 O2 production is controlled by its protein abundance. Hypoxia-induced Nox4 expression is observed in various types of cells and generally thought to be regulated at the transcriptional level. Here we show that hypoxia upregulates the Nox4 protein level and Nox4-catalyzed H2 O2 production without increasing the Nox4 mRNA in rat H9c2 cardiomyocytes. In these cells, the Nox4 protein is stabilized under hypoxic conditions in a manner dependent on the presence of p22phox . Cell treatment with the proteasome inhibitor MG132 results in a marked decrease of the Nox4 protein under both normoxic and hypoxic conditions, indicating that the proteasome pathway does not play a major role in Nox4 degradation. The decrease is partially restored by the autophagy inhibitor 3-methyladenine. Furthermore, the Nox4 protein level is upregulated by the lysosome inhibitors bafilomycin A1 and chloroquine. Thus, in cardiomyocytes, Nox4 appears to be degraded via an autophagy-related pathway, and its suppression by hypoxia likely stabilizes Nox4, leading to upregulation of Nox4-catalyzed H2 O2 production.

A basic charge cluster near the C-terminus of the cytoplasmic tail contributes to the molecular stability of human herpesvirus 8 E3 ubiquitin ligases.

Human herpesvirus 8 (HHV8; also known as Kaposi's sarcoma-associated herpesvirus [KSHV]) utilizes the viral E3 ubiquitin ligase family members K3 and K5 for immune evasion. Both K3 and K5 mediate the ubiquitination of host MHC class I (MHC-I) molecules, which play a key role in antigen presentation to cytotoxic T lymphocytes (CTLs). Because ubiquitinated MHC-I is immediately down-regulated from the cell surface, HHV8-infected cells can escape surveillance by CTLs. K3 and K5 have similar domain structures and topologies. They contain an N-terminal RINGv ubiquitin ligase domain, two transmembrane helices, and an intrinsically disordered cytoplasmic tail at the C-terminus. The cytoplasmic tail contains a membrane-proximal "conserved region" involved in ligase activity. On the other hand, the role of the membrane-distal region of the cytoplasmic tail, termed the "C-tail" in this study, remains unclear. Here, we demonstrate that the C-tail contributes to the protein expression of both K3 and K5. The C-tail-truncated K3 and K5 mutants were rapidly reduced in cells. The recombinant C-tail proteins bind to acidic lipids via a basic charge cluster located near the C-terminus of the C-tails. Similar to the C-tail-truncated mutants, the basic charge cluster-substituting mutants showed decreased protein expression of K3 and K5. These findings suggest that the basic charge cluster near the C-terminus of the cytoplasmic tail contributes to the molecular stability of K3 and K5.

Characterization of missense mutations in the NADPH oxidase partner p22phox in the A22° subtype of chronic granulomatous disease.

Defective superoxide production by NADPH oxidase 2 (Nox2) in phagocyte cells results in the development of chronic granulomatous disease (CGD), a hereditary disease characterized by recurrent and life-threatening infections. The partner protein p22phox is a membrane-spanning protein which forms a stable heterodimer with Nox2 in the endoplasmic reticulum. This interaction ensures the stability of each protein and their accurate trafficking to the cell membrane. The present paper describes the characterization of p22phox missense mutations that were identified in a patient with CGD who presented with undetectable levels of p22phox . Using a reconstitution system, it was found that p22phox expression decreased when R90Q, A117E, S118R, A124S, A124V, A125T, or E129K mutations were introduced, suggesting that these mutations destabilize the protein. In contrast, introducing an L105R mutation did not affect protein expression, but did inhibit p22phox binding to Nox2. Thus, the missense mutations discussed here contribute to the development of CGD by either disrupting protein stability or by impairing the interaction between p22phox and Nox2.

Ca2+ -binding-region-dependent cell surface localization of NADPH oxidase Nox5.

Six gene splice variants of superoxide-generating NADPH oxidase 5 (Nox5) have been identified in humans, and they differ in the sequence of their N-terminal cytoplasmic domains, which comprise four EF-hand motifs. Here, we demonstrated that the Ca2+ -dependent association and dissociation between the N- and C-terminal cytoplasmic domains of the Nox5ß variant are affected by the alanine substitution of the conserved Ile-113 or Leu-115 at the connecting loop between the third and fourth EF-hand motifs. These substitutions impair the cell surface localization of Nox5ß. In addition, the Nox5ε/S variant, lacking all EF-hand motifs, does not localize to the plasma membrane. Thus, the Ca2+ -sensitive intramolecular interaction determines the Nox5 subcellular localization, that is, whether Nox5 variants generate superoxide in the extracellular or intracellular space.

Inhibition of pancreatic cancer-cell growth and metastasis in vivo by a pyrazole compound characterized as a cell-migration inhibitor by an in vitro chemotaxis assay.

Pancreatic cancer is recalcitrant to treatment as it is highly metastatic and rapidly progressive. While observing the behavior of human pancreatic BxPC-3 cells using an optical assay device called TAXIScan, we found that several synthetic pyrazole and pyrimidine derivatives inhibited cell migration. One such compound, 14-100, inhibited metastasis of fluorescence-labeled BxPC-3 cells, which were transplanted into the pancreas of nude mice as a subcutaneously grown cancer fragment. Surprisingly, despite its low cytotoxicity, the compound also showed an inhibitory effect on cancer cell proliferation in vivo, suggesting that the compound alters cancer cell characteristics needed to grow in situ. Single-cell RNA-sequencing revealed changes in gene expression associated with metastasis, angiogenesis, inflammation, and epithelial-mesenchymal transition. These data suggest that the compound 14-100 could be a good drug candidate against pancreatic cancer.

Importance of accessibility to the extracellular juxtamembrane stalk region of membrane protein for substrate recognition by viral ubiquitin ligase K5.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a carcinogenic virus that latently infects B cells and causes malignant tumors in immunocompromised patients. KSHV utilizes two viral E3 ubiquitin ligases, K3 and K5, in KSHV-infected cells to mediate the polyubiquitination-dependent down-regulation of several host membrane proteins involved in the immune system. Although K3 and K5 are members of the same family and have similar structural topologies, K3 and K5 have different substrate specificities. Hence, K5 may have a different substrate recognition mode than K3; however, the molecular basis of substrate recognition remains unclear. Here, we investigated the reason why human CD8α, which is known not to be a substrate for both K3 and K5, is not recognized by them, to obtain an understanding for molecular basis of substrate specificity. CD8α forms a disulfide-linked homodimer under experimental conditions to evaluate the viral ligase-mediated down-regulation. It is known that two interchain disulfide linkages in the stalk region between each CD8α monomer (Cys164-Cys164 and Cys181-Cys181) mediate homodimerization. When the interchain disulfide linkage of Cys181-Cys181 was eliminated, CD8α was down-regulated by K5 with a functional RING variant (RINGv) domain via polyubiquitination at the cytoplasmic tail. Aspartic acid, located at the stalk/transmembrane interface of CD8α, was essential for K5-mediated down-regulation of the CD8α mutant without a Cys181-Cys181 linkage. These results suggest that disulfide linkage near the stalk/transmembrane interface critically inhibits substrate targeting by K5. Accessibility to the extracellular juxtamembrane stalk region of membrane proteins may be important for substrate recognition by the viral ubiquitin ligase K5.

Regulation of Derlin-1-mediated degradation of NADPH oxidase partner p22phox by thiol modification.

The transmembrane protein p22phox heterodimerizes with NADPH oxidase (Nox) 1-4 and is essential for the reactive oxygen species-producing capacity of oxidases. Missense mutations in the p22phox gene prevent the formation of phagocytic Nox2-based oxidase, which contributes to host defense. This results in chronic granulomatous disease (CGD), a severe primary immunodeficiency syndrome. In this study, we characterized missense mutations in p22phox (L51Q, L52P, E53V, and P55R) in the A22° type (wherein the p22phox protein is undetectable) of CGD. We demonstrated that these substitutions enhanced the degradation of the p22phox protein in the endoplasmic reticulum (ER) and the binding of p22phox to Derlin-1, a key component of ER-associated degradation (ERAD). Therefore, the L51-L52-E53-P55 sequence is responsible for protein stability in the ER. We observed that the oxidation of the thiol group of Cys-50, which is adjacent to the L51-L52-E53-P55 sequence, suppressed p22phox degradation. However, the suppression effect was markedly attenuated by the serine substitution of Cys-50. Blocking the free thiol of Cys-50 by alkylation or C50S substitution promoted the association of p22phox with Derlin-1. Derlin-1 depletion partially suppressed the degradation of p22phox mutant proteins. Furthermore, heterodimerization with p22phox (C50S) induced rapid degradation of not only Nox2 but also nonphagocytic Nox4 protein, which is responsible for redox signaling. Thus, the redox-sensitive Cys-50 appears to determine whether p22phox becomes a target for degradation by the ERAD system through its interaction with Derlin-1.

Fine definition of the epitopes on the human gp91phox/NOX2 for the monoclonal antibodies CL-5 and 48.

Superoxide-producing NADPH oxidase, gp91phox/NOX2, in phagocytes plays a critical role in the host defenses against pathogens. Moreover, gp91phox/NOX2 contributes to the oxidative stress in endothelial cells. Therefore, investigating the level of gp91phox/NOX2 with immunoblotting is important for estimating the amount of superoxide produced. Here, we showed that the epitopes in human gp91phox/NOX2 recognized by monoclonal antibodies (mAbs) CL-5 and 48 were in amino acids 132-147 and 136-144, respectively. Although the epitopes were close to the N-glycosylation sites, N-glycan maturation did not affect mAbs recognition. When Pro-136 was substituted with Arg, the corresponding mouse residue, human gp91phox/NOX2 was not recognized by mAbs CL-5 and 48; however, the substitution did not affect gp91phox/NOX2-based oxidase activity. This finding explains why these mAbs specifically recognize the human but not mouse gp91phox/NOX2. Hence, these mAbs are useful for investigating the level of gp91phox/NOX2 without amino acid substitutions in the epitopes.

Coculture in vitro with endothelial cells induces cytarabine resistance of acute myeloid leukemia cells in a VEGF-A/VEGFR-2 signaling-independent manner.

An interaction between acute myeloid leukemia (AML) cells and endothelial cells in the bone marrow seems to play a critical role in chemosensitivity on leukemia treatment. The endothelial niche reportedly enhances the paracrine action of the soluble secretory proteins responsible for chemoresistance in a vascular endothelial growth factor A (VEGF-A)/VEGF receptor 2 (VEGFR-2) signaling pathway-dependent manner. To further investigate the contribution of VEGF-A/VEGFR-2 signaling to the chemoresistance of AML cells, a biochemical assay system in which the AML cells were cocultured with human endothelial EA.hy926 cells in a monolayer was developed. By coculture with EA.hy926 cells, this study revealed that the AML cells resisted apoptosis induced by the anticancer drug cytarabine. SU4312, a VEGFR-2 inhibitor, attenuated VEGFR-2 phosphorylation and VEGF-A/VEGFR-2 signaling-dependent endothelial cell migration; thus, this inhibitor was observed to block VEGF-A/VEGFR-2 signaling. Interestingly, this inhibitor did not reverse the chemoresistance. When VEGFR-2 was knocked out in EA.hy926 cells using the CRISPR-Cas9 system, the cytarabine-induced apoptosis of AML cells did not significantly change compared with that of wild-type cells. Thus, coculture-induced chemoresistance appears to be independent of VEGF-A/VEGFR-2 signaling. When the transwell, a coculturing device, separated the AML cells from the EA.hy926 cells in a monolayer, the coculture-induced chemoresistance was inhibited. Given that the migration of VEGF-A/VEGFR-2 signaling-dependent endothelial cells is necessary for the endothelial niche formation in the bone marrow, VEGF-A/VEGFR-2 signaling contributes to chemoresistance by mediating the niche formation process, but not to the chemoresistance of AML cells in the niche.

Kaposi's sarcoma-associated herpesvirus ubiquitin ligases downregulate cell surface expression of l-selectin.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic etiological factor for Kaposi's sarcoma and primary effusion lymphoma in immunocompromised patients. KSHV utilizes two immune evasion E3 ubiquitin ligases, namely K3 and K5, to downregulate the expression of antigen-presenting molecules and ligands of natural killer (NK) cells in the host cells through an ubiquitin-dependent endocytic mechanism. This allows the infected cells to evade surveillance and elimination by cytotoxic lymphocytes and NK cells. The number of host cell molecular substrates reported for these ubiquitin ligases is limited. The identification of novel substrates for these ligases will aid in elucidating the mechanism underlying immune evasion of KSHV. This study demonstrated that K5 downregulated the cell surface expression of l-selectin, a C-type lectin-like adhesion receptor expressed in the lymphocytes. Tryptophan residue located at the centre of the E2-binding site in the K5 RINGv domain was essential to downregulate l-selectin expression. Additionally, the lysine residues located at the cytoplasmic tail of l-selectin were required for the K5-mediated downregulation of l-selectin. K5 promoted the degradation of l-selectin through polyubiquitination. These results suggest that K5 downregulates l-selectin expression on the cell surface by promoting polyubiquitination and ubiquitin-dependent endocytosis, which indicated that l-selectin is a novel substrate for K5. Additionally, K3 downregulated l-selectin expression. The findings of this study will aid in the elucidation of a novel immune evasion mechanism in KSHV.

The downregulation of NADPH oxidase Nox4 during hypoxia in hemangioendothelioma cells: a possible role of p22phox on Nox4 protein stability.

NADPH oxidase (Nox) 4 produces H2O2 by forming a heterodimer with p22phox and is involved in hemangioendothelioma development through monocyte chemoattractant protein-1 (MCP-1) upregulation. Here, we show that Nox4 protein levels were maintained by p22phox in hemangioendothelioma cells and Nox4 protein stability was dependent on p22phox coexpression. Conversely, the degradation of Nox4 monomer was enhanced by p22phox knockdown. Under hypoxic conditions in hemangioendothelioma cells, p22phox was downregulated at the mRNA and protein levels. Downregulation of p22phox protein resulted in the enhanced degradation of Nox4 protein in hypoxia-treated hemangioendothelioma cells. In contrast, Nox2, a Nox isoform, was not altered at the protein level under hypoxic conditions. Nox2 exhibited a higher affinity for p22phox compared with Nox4, suggesting that when coexpressed with Nox4 in the same cells, Nox2 acts as a competitor. Nox2 knockdown restored Nox4 protein levels partially reduced by hypoxic treatment. Thus, Nox4 protein levels were attenuated in hypoxia-treated cells resulting from p22phox depletion. MCP-1 secretion was decreased concurrently with hypoxia-induced Nox4 downregulation compared with that under normoxia.

Constitutive activity of NADPH oxidase 1 (Nox1) that promotes its own activity suppresses the colon epithelial cell migration.

Superoxide producing NADPH oxidase 1 (Nox1), abundantly expressed in the colon epithelium, plays a crucial role in mucosal host defenses. In this study, we found that pre-treatment of cells with edaravone, a free radical scavenger, inhibited Nox1 constitutive activity even after washout without affecting Nox1 trafficking to the plasma membrane and membrane recruitment of the cytosolic regulators Noxo1 and Noxa1. These results suggest that a Nox1-derived product is involved in the step that initiates the electron transfer reaction after the formation of the Nox1-Noxo1-Noxa1 complex. Furthermore, we show that the mean migration directionality and velocity of epithelial cells were significantly enhanced by the inhibition of constitutive Nox1 activity. Thus, the constitutive Nox1 activity limits undesired cell migration in resting cells while participating in a positive feedback loop toward its own oxidase activity.

The rRNA synthesis inhibitor CX-5461 may induce autophagy that inhibits anticancer drug-induced cell damage to leukemia cells.

Autophagy induced in cancer cells during chemotherapy is classified into two types, which differ depending on the kind of cells or anticancer drugs. The first type of autophagy contributes to the death of cells treated with drugs. In contrast, the second type plays a crucial role in preventing anticancer drug-induced cell damages; the use of an autophagy inhibitor is considered effective in improving the efficacy of chemotherapy. Thus, it is important to determine which type of autophagy is induced during chemotherapy. Here, we showed that a novel inhibitor of RNA polymerase I, suppresses growth, induces cell cycle arrest and promotes apoptosis in leukemia cell lines. The number of apoptotic cells induced by co-treatment with CX-5461 and chloroquine, an autophagy inhibitor, increased compared with CX-5461 alone. Thus, the autophagy which may be induced by CX-5461 was the second type.

The NADPH oxidase NOX4 promotes the directed migration of endothelial cells by stabilizing vascular endothelial growth factor receptor 2 protein.

Directed migration of endothelial cells (ECs) is an important process during both physiological and pathological angiogenesis. The binding of vascular endothelial growth factor (VEGF) to VEGF receptor-2 (VEGFR-2) on the EC surface is necessary for directed migration of these cells. Here, we used TAXIScan, an optically accessible real-time horizontal cell dynamics assay approach, and demonstrate that reactive oxygen species (ROS)-producing NADPH oxidase 4 (NOX4), which is abundantly expressed in ECs, mediates VEGF/VEGFR-2-dependent directed migration. We noted that a continuous supply of endoplasmic reticulum (ER)-retained VEGFR-2 to the plasma membrane is required to maintain VEGFR-2 at the cell surface. siRNA-mediated NOX4 silencing decreased the ER-retained form of VEGFR-2, resulting in decreased cell surface expression levels of the receptor. We also found that ER-localized NOX4 interacts with ER-retained VEGFR-2 and thereby stabilizes this ER-retained form at the protein level in the ER. We conclude that NOX4 contributes to the directed migration of ECs by maintaining VEGFR-2 levels at their surface.

Intramolecular interaction in LGN, an adaptor protein that regulates mitotic spindle orientation.

Proper mitotic spindle orientation requires that astral microtubules are connected to the cell cortex by the microtubule-binding protein NuMA, which is recruited from the cytoplasm. Cortical recruitment of NuMA is at least partially mediated via direct binding to the adaptor protein LGN. LGN normally adopts a closed conformation via an intramolecular interaction between its N-terminal NuMA-binding domain and its C-terminal region that contains four GoLoco (GL) motifs, each capable of binding to the membrane-anchored Gαi subunit of heterotrimeric G protein. Here we show that the intramolecular association with the N-terminal domain in LGN involves GL3, GL4, and a region between GL2 and GL3, whereas GL1 and GL2 do not play a major role. This conformation renders GL1 but not the other GL motifs in a state easily accessible to Gαi To interact with full-length LGN in a closed state, NuMA requires the presence of Gαi; both NuMA and Gαi are essential for cortical recruitment of LGN in mitotic cells. In contrast, mInsc, a protein that competes with NuMA for binding to LGN and regulates mitotic spindle orientation in asymmetric cell division, efficiently binds to full-length LGN without Gαi and induces its conformational change, enhancing its association with Gαi In nonpolarized symmetrically dividing HeLa cells, disruption of the LGN-NuMA interaction by ectopic expression of mInsc results in a loss of cortical localization of NuMA during metaphase and anaphase and promotes mitotic spindle misorientation and a delayed anaphase progression. These findings highlight a specific role for LGN-mediated cell cortex recruitment of NuMA.

Soluble Regulatory Proteins for Activation of NOX Family NADPH Oxidases.

NOX family NADPH oxidases deliberately produce reactive oxygen species and thus contribute to a variety of biological functions. Of seven members in the human family, the three oxidases NOX2, NOX1, and NOX3 form a heterodimer with p22phox and are regulated by soluble regulatory proteins: p47phox, its related organizer NOXO1; p67phox, its related activator NOXA1; p40phox; and the small GTPase Rac. Activation of the phagocyte oxidase NOX2 requires p47phox, p67phox, and GTP-bound Rac. In addition to these regulators, p40phox plays a crucial role when NOX2 is activated during phagocytosis. On the other hand, NOX1 activation prefers NOXO1 and NOXA1, although Rac is also involved. NOX3 constitutively produces superoxide, which is enhanced by regulatory proteins such as p47phox, NOXO1, and p67phox. Here we describe mechanisms for NOX activation with special attention to the soluble regulatory proteins.

Differential cell surface recruitment of the superoxide-producing NADPH oxidases Nox1, Nox2 and Nox5: The role of the small GTPase Sar1.

Transmembrane glycoproteins, synthesized at the endoplasmic reticulum (ER), generally reach the Golgi apparatus in COPII-coated vesicles en route to the cell surface. Here, we show that the bona fide nonglycoprotein Nox5, a transmembrane superoxide-producing NADPH oxidase, is transported to the cell surface in a manner resistant to co-expression of Sar1 (H79G), a GTP-fixed mutant of the small GTPase Sar1, which blocks COPII vesicle fission from the ER. In contrast, Sar1 (H79G) effectively inhibits ER-to-Golgi transport of glycoproteins including the Nox5-related oxidase Nox2. The trafficking of Nox2, but not that of Nox5, is highly sensitive to over-expression of syntaxin 5 (Stx5), a t-SNARE required for COPII ER-to-Golgi transport. Thus, Nox2 and Nox5 mainly traffic via the Sar1/Stx5-dependent and -independent pathways, respectively. Both participate in Nox1 trafficking, as Nox1 advances to the cell surface in two differentially N-glycosylated forms, one complex and one high mannose, in a Sar1/Stx5-dependent and -independent manner, respectively. Nox2 and Nox5 also can use both pathways: a glycosylation-defective mutant Nox2 is weakly recruited to the plasma membrane in a less Sar1-dependent manner; N-glycosylated Nox5 mutants reach the cell surface in part as the complex form Sar1-dependently, albeit mainly as the high-mannose form in a Sar1-independent manner.

TRPC3-GEF-H1 axis mediates pressure overload-induced cardiac fibrosis.

Structural cardiac remodeling, accompanying cytoskeletal reorganization of cardiac cells, is a major clinical outcome of diastolic heart failure. A highly local Ca2+ influx across the plasma membrane has been suggested to code signals to induce Rho GTPase-mediated fibrosis, but it is obscure how the heart specifically decodes the local Ca2+ influx as a cytoskeletal reorganizing signal under the conditions of the rhythmic Ca2+ handling required for pump function. We found that an inhibition of transient receptor potential canonical 3 (TRPC3) channel activity exhibited resistance to Rho-mediated maladaptive fibrosis in pressure-overloaded mouse hearts. Proteomic analysis revealed that microtubule-associated Rho guanine nucleotide exchange factor, GEF-H1, participates in TRPC3-mediated RhoA activation induced by mechanical stress in cardiomyocytes and transforming growth factor (TGF) ß stimulation in cardiac fibroblasts. We previously revealed that TRPC3 functionally interacts with microtubule-associated NADPH oxidase (Nox) 2, and inhibition of Nox2 attenuated mechanical stretch-induced GEF-H1 activation in cardiomyocytes. Finally, pharmacological TRPC3 inhibition significantly suppressed fibrotic responses in human cardiomyocytes and cardiac fibroblasts. These results strongly suggest that microtubule-localized TRPC3-GEF-H1 axis mediates fibrotic responses commonly in cardiac myocytes and fibroblasts induced by physico-chemical stimulation.

TRPC3 positively regulates reactive oxygen species driving maladaptive cardiac remodeling.

Reactive oxygen species (ROS) produced by NADPH oxidase 2 (Nox2) function as key mediators of mechanotransduction during both physiological adaptation to mechanical load and maladaptive remodeling of the heart. This is despite low levels of cardiac Nox2 expression. The mechanism underlying the transition from adaptation to maladaptation remains obscure, however. We demonstrate that transient receptor potential canonical 3 (TRPC3), a Ca2+-permeable channel, acts as a positive regulator of ROS (PRROS) in cardiomyocytes, and specifically regulates pressure overload-induced maladaptive cardiac remodeling in mice. TRPC3 physically interacts with Nox2 at specific C-terminal sites, thereby protecting Nox2 from proteasome-dependent degradation and amplifying Ca2+-dependent Nox2 activation through TRPC3-mediated background Ca2+ entry. Nox2 also stabilizes TRPC3 proteins to enhance TRPC3 channel activity. Expression of TRPC3 C-terminal polypeptide abolished TRPC3-regulated ROS production by disrupting TRPC3-Nox2 interaction, without affecting TRPC3-mediated Ca2+ influx. The novel TRPC3 function as a PRROS provides a mechanistic explanation for how diastolic Ca2+ influx specifically encodes signals to induce ROS-mediated maladaptive remodeling and offers new therapeutic possibilities.

DOCK2 and DOCK5 act additively in neutrophils to regulate chemotaxis, superoxide production, and extracellular trap formation.

Neutrophils are highly motile leukocytes that play important roles in the innate immune response to invading pathogens. Neutrophils rapidly migrate to the site of infections and kill pathogens by producing reactive oxygen species (ROS). Neutrophil chemotaxis and ROS production require activation of Rac small GTPase. DOCK2, an atypical guanine nucleotide exchange factor (GEF), is one of the major regulators of Rac in neutrophils. However, because DOCK2 deficiency does not completely abolish fMLF-induced Rac activation, other Rac GEFs may also participate in this process. In this study, we show that DOCK5 acts with DOCK2 in neutrophils to regulate multiple cellular functions. We found that fMLF- and PMA-induced Rac activation were almost completely lost in mouse neutrophils lacking both DOCK2 and DOCK5. Although ß2 integrin-mediated adhesion occurred normally even in the absence of DOCK2 and DOCK5, mouse neutrophils lacking DOCK2 and DOCK5 exhibited a severe defect in chemotaxis and ROS production. Similar results were obtained when human neutrophils were treated with CPYPP, a small-molecule inhibitor of these DOCK GEFs. Additionally, we found that DOCK2 and DOCK5 regulate formation of neutrophil extracellular traps (NETs). Because NETs are involved in vascular inflammation and autoimmune responses, DOCK2 and DOCK5 would be a therapeutic target for controlling NET-mediated inflammatory disorders.