The role of the DAP12 signal in mouse myeloid differentiation

DAP12 is a recently cloned, immunoreceptor tyrosine-based activation motif-bearing transmembrane adapter molecule that is associated with the NK-activating receptors. Previous reports showed that the DAP12 message could be detected not only in NK cells but also in granulocytes, monocytes, dendritic cells, and macrophages. In this study we found a significant level of DAP12 protein expression in macrophage-related cell lines and organs. Additionally, we observed increased expression of DAP12 after LPS-induced differentiation of M1 cells into macrophages. To examine the role of DAP12 in the myeloid cell lineage, we established M1 FLAG-DAP12 transfectants (FDAP-M1) and demonstrated the marked morphological changes in FDAP-M1 cells caused by signaling through DAP12. Cell surface phenotypic analysis showed up-regulation of macrophage markers CD11b, 2.4G2, and adhesion molecule B7-2. Additionally, after stimulation through DAP12, phosphorylated FLAG -DAP12 could be immunoprecipitated using anti-phosphotyrosine mAbs. Collectively, these findings indicate that direct DAP12 signaling has an important role in macrophage differentiation.     

Bone microenvironment specific roles of ITAM adapter signaling during bone remodeling induced by acute estrogen-deficiency

Immunoreceptor tyrosine-based activation motif (ITAM) signaling mediated by DAP12 or Fcepsilon receptor Igamma chain (FcRgamma) have been shown to be critical for osteoclast differentiation and maturation under normal physiological conditions. Their function in pathological conditions is unknown. We studied the role of ITAM signaling during rapid bone remodeling induced by acute estrogen-deficiency in wild-type (WT), DAP12-deficient (DAP12-/-), FcRgamma-deficient (FcRgamma-/-) and double-deficient (DAP12-/-FcRgamma-/-) mice. Six weeks after ovariectomy (OVX), DAP12-/-FcRgamma-/- mice showed resistance to lumbar vertebral body (LVB) trabecular bone loss, while WT, DAP12-/- and FcRgamma-/- mice had significant LVB bone loss. In contrast, all ITAM adapter-deficient mice responded to OVX with bone loss in both femur and tibia of approximately 40%, relative to basal bone volumes. Only WT mice developed significant cortical bone loss after OVX. In vitro studies showed microenvironmental changes induced by OVX are indispensable for enhanced osteoclast formation and function. Cytokine changes, including TGFbeta and TNFalpha, were able to induce osteoclastogenesis independent of RANKL in BMMs from WT but not DAP12-/- and DAP12-/-FcRgamma-/- mice. FSH stimulated RANKL-induced osteoclast differentiation from BMMs in WT, but not DAP12-/- and DAP12-/-FcRgamma-/- mice. Our study demonstrates that although ITAM adapter signaling is critical for normal bone remodeling, estrogen-deficiency induces an ITAM adapter-independent bypass mechanism allowing for enhanced osteoclastogenesis and activation in specific bony microenvironments.     

Cell type-specific regulation of ITAM-mediated NF-kappaB activation by the adaptors, CARMA1 and CARD9

Activating NK cell receptors transduce signals through ITAM-containing adaptors, including FcRgamma and DAP12. Although the caspase recruitment domain (CARD)9-Bcl10 complex is essential for FcRgamma/DAP12-mediated NF-kappaB activation in myeloid cells, its involvement in NK cell receptor signaling is unknown. Herein we show that the deficiency of CARMA1 or Bcl10, but not CARD9, resulted in severe impairment of cytokine/chemokine production mediated by activating NK cell receptors due to a selective defect in NF-kappaB activation, whereas cytotoxicity mediated by the same receptors did not require CARMA1-Bcl10-mediated signaling. IkappaB kinase (IKK) activation by direct protein kinase C (PKC) stimulation with PMA plus ionomycin (P/I) was abrogated in CARMA1-deficient NK cells, similar to T and B lymphocytes, whereas CARD9-deficient dendritic cells (DCs) exhibited normal P/I-induced IKK activation. Surprisingly, CARMA1 deficiency also abrogated P/I-induced IKK activation in DCs, indicating that CARMA1 is essential for PKC-mediated NF-kappaB activation in all cell types, although the PKC-CARMA1 axis is not used downstream of myeloid ITAM receptors. Consistently, PKC inhibition abrogated ITAM receptor-mediated activation only in NK cells but not in DCs, suggesting PKC-CARMA1-independent, CARD9-dependent ITAM receptor signaling in myeloid cells. Conversely, the overexpression of CARD9 in CARMA1-deficient cells failed to restore the PKC-mediated NF-kappaB activation. Thus, NF-kappaB activation signaling through ITAM receptors is regulated by a cell type-specific mechanism depending on the usage of adaptors CARMA1 and CARD9, which determines the PKC dependence of the signaling.     

Ly49D-mediated ITAM signaling in immature thymocytes impairs development by bypassing the pre-TCR checkpoint

Activating and inhibitory NK receptors regulate the development and effector functions of NK cells via their ITAM and ITIM motifs, which recruit protein tyrosine kinases and phosphatases, respectively. In the T cell lineage, inhibitory Ly49 receptors are expressed by a subset of activated T cells and by CD1d-restricted NKT cells, but virtually no expression of activating Ly49 receptors is observed. Using mice transgenic for the activating receptor Ly49D and its associated ITAM signaling DAP12 chain, we show in this article that Ly49D-mediated ITAM signaling in immature thymocytes impairs development due to a block in maturation from the double negative (DN) to double positive (DP) stages. A large proportion of Ly49D/DAP12 transgenic thymocytes were able to bypass the pre-TCR checkpoint at the DN3 stage, leading to the appearance of unusual populations of DN4 and DP cells that lacked expression of intracellular (ic) TCRβ-chain. High levels of CD5 were expressed on ic TCRβ(-) DN and DP thymocytes from Ly49D/DAP12 transgenic mice, further suggesting that Ly49D-mediated ITAM signaling mimics physiological ITAM signaling via the pre-TCR. We also observed unusual ic TCRβ(-) single positive thymocytes with an immature CD24(high) phenotype that were not found in the periphery. Importantly, thymocyte development was completely rescued by expression of an Ly49A transgene in Ly49D/DAP12 transgenic mice, indicating that Ly49A-mediated ITIM signaling can fully counteract ITAM signaling via Ly49D/DAP12. Collectively, our data indicate that inappropriate ITAM signaling by activating NK receptors on immature thymocytes can subvert T cell development by bypassing the pre-TCR checkpoint.     

Could a B-1 Cell Derived Phagocyte “Be One” of the Peritoneal Macrophages during LPS-Driven Inflammation?

The inflammatory response is driven by signals that recruit and elicit immune cells to areas of tissue damage or infection. The concept of a mononuclear phagocyte system postulates that monocytes circulating in the bloodstream are recruited to inflamed tissues where they give rise to macrophages. A recent publication demonstrated that the large increase in the macrophages observed during infection was the result of the multiplication of these cells rather than the recruitment of blood monocytes. We demonstrated previously that B-1 cells undergo differentiation to acquire a mononuclear phagocyte phenotype in vitro (B-1CDP), and we propose that B-1 cells could be an alternative origin for peritoneal macrophages. A number of recent studies that describe the phagocytic and microbicidal activity of B-1 cells in vitro and in vivo support this hypothesis. Based on these findings, we further investigated the differentiation of B-1 cells into phagocytes in vivo in response to LPS-induced inflammation. Therefore, we investigated the role of B-1 cells in the composition of the peritoneal macrophage population after LPS stimulation using osteopetrotic mice, BALB/Xid mice and the depletion of monocytes/macrophages by clodronate treatment. We show that peritoneal macrophages appear in op/op((-/-)) mice after LPS stimulation and exhibit the same Ig gene rearrangement (VH11) that is often found in B-1 cells. These results strongly suggest that op/op((-/-)) peritoneal "macrophages" are B-1CDP. Similarly, the LPS-induced increase in the macrophage population was observed even following monocyte/macrophage depletion by clodronate. After monocyte/macrophage depletion by clodronate, LPS-elicited macrophages were observed in BALB/Xid mice only following the transfer of B-1 cells. Based on these data, we confirmed that B-1 cell differentiation into phagocytes also occurs in vivo. In conclusion, the results strongly suggest that B-1 cell derived phagocytes are a component of the LPS-elicited peritoneal macrophage population.     

Fyn positively regulates the activation of DAP12 and FcRγ-mediated costimulatory signals by RANKL during osteoclastogenesis

Osteoclasts (OCs) are the only bone-resorbing cells and are critically involved in various bone-associated diseases, including osteoporosis and rheumatoid arthritis. Differentiation of OCs from bone marrow macrophage cells (BMMs) is regulated by RANK and the adaptor protein (DAP12/FcRγ)-mediated costimulatory signals. However, it is unknown how RANKL/RANK signal stimulates phosphorylation of DAP12/FcRγ to initiate the costimulatory signals. As reported here, we found that OC differentiation and acquisition of bone resorption capacity were suppressed in RANKL-stimulated Fyn(-/-) or Fyn-siRNA-transfected BMMs, but could be restored by overexpression of Fyn kinase in Fyn(-/-) BMMs. However, the RANKL-stimulated proliferation of BMMs was unaffected by the absence of Fyn. In addition, RANKL-stimulated Fyn(-/-) BMMs no longer exhibited the optimal induction of typical OC markers such as NFATc1, c-Fos, c-Src, TRAF6, and cathepsin K or costimulatory signals such as the activating phosphorylations of Syk, PLCγ2, and Gab2. These were restored by overexpression of Fyn in Fyn(-/-) BMMs. Immunoprecipitation studies also indicated that the adaptor proteins DAP12/FcRγ and Syk interacted with RANK during RANKL stimulation in BMMs in a Fyn-dependent manner. Phosphorylation of the DAP12/FcRγ and the recruitment of Syk by DAP12/FcRγ were suppressed in Fyn(-/-) BMMs. This is the first demonstration that Fyn relays the initial RANK/RANKL signal to the ITAM-containing adaptors DAP12/FcRγ for OC differentiation.     

DAP12: a key accessory protein for relaying signals by natural killer cell receptors.

DAP12 is a 12 kDa transmembrane protein recently recognized as a key signal transduction receptor element in Natural Killer (NK) cells. It is a disulfide-linked homodimer that non-covalently associates with several activating receptors expressed on NK cells. Activation signals initiated through DAP12 are predicted to play strategic roles in triggering NK cell cytotoxicity responses toward certain tumor cells and virally infected cells. The cytoplasmic domain of DAP12 contains an Immunoreceptor Tyrosine-based Activation Motif (ITAM). Phosphorylation of ITAM tyrosines mediates associations with protein tyrosine kinases, which is a resonant feature of signalling through these motifs in T and B cell antigen receptors. In addition, its expression in other tissues, including dendritic cells and monocytes, suggests that DAP12 transduces ITAM-mediated activation signals for an extended array of receptors in those cells as well.     

Exosomes derived from pro‐inflammatory bone marrow‐derived mesenchymal stem cells reduce inflammation and myocardial injury via mediating macrophage polarization

Exosomes are served as substitutes for stem cell therapy, playing important roles in mediating heart repair during myocardial infarction injury. Evidence have indicated that lipopolysaccharide (LPS) pre‐conditioning bone marrow‐derived mesenchymal stem cells (BMSCs) and their secreted exosomes promote macrophage polarization and tissue repair in several inflammation diseases; however, it has not been fully elucidated in myocardial infarction (MI). This study aimed to investigate whether LPS‐primed BMSC‐derived exosomes could mediate inflammation and myocardial injury via macrophage polarization after MI. Here, we found that exosomes derived from BMSCs, in both Exo and L‐Exo groups, increased M2 macrophage polarization and decreased M1 macrophage polarization under LPS stimulation, which strongly depressed LPS‐dependent NF‐κB signalling pathway and partly activated the AKT1/AKT2 signalling pathway. Compared with Exo, L‐Exo had superior therapeutic effects on polarizing M2 macrophage in vitro and attenuated the post‐infarction inflammation and cardiomyocyte apoptosis by mediating macrophage polarization in mice MI model. Consequently, we have confidence in the perspective that low concentration of LPS pre‐conditioning BMSC‐derived exosomes may develop into a promising cell‐free treatment strategy for clinical treatment of MI.     

Identification of DELE,a novel DAP3-binding protein which is crucial for death receptor-mediated apoptosis induction

Death associated protein 3 (DAP3) is known to be a highly conserved protein, and is responsible for regulating apoptosis induced by various stimuli. To understand the molecular mechanism of how DAP3 induces apoptosis, we performed yeast two-hybrid screening, and identified a novel DAP3-binding protein termed death ligand signal enhancer (DELE). In this report, we show that DELE actually binds to DAP3 in mammalian cells. We found that the cells stably expressing DELE are susceptible to apoptosis induction by the stimulation of TNF-α and TRAIL. In addition, knockdown of DELE expression rescued the HeLa cells from apoptosis induction by these stimuli. Moreover, activation of caspase-3, caspase-8 and caspase-9 induced by stimulation of TNF-α, anti-Fas or TRAIL was significantly inhibited by the knockdown of DELE expression. These results demonstrated the biological significance of DELE for apoptosis signal mediated by death receptors.     

Cutting edge: TREM-2 attenuates macrophage activation

The triggering receptor expressed on myeloid cells 2 (TREM-2) delivers intracellular signals through the adaptor DAP12 to regulate myeloid cell function both within and outside the immune system. The role of TREM-2 in immunity has been obscured by the failure to detect expression of the TREM-2 protein in vivo. In this study, we show that TREM-2 is expressed on macrophages infiltrating the tissues from the circulation and that alternative activation with IL-4 can induce TREM-2. TREM-2 expression is abrogated by macrophage maturation with LPS of IFN-gamma. Using TREM-2(-/-) mice, we find that TREM-2 functions to inhibit cytokine production by macrophages in response to the TLR ligands LPS, zymosan, and CpG. Furthermore, we find that TREM-2 completely accounts for the increased cytokine production previously reported by DAP12(-/-) macrophages. Taken together, these data show that TREM-2 is expressed on newly differentiated and alternatively activated macrophages and functions to restrain macrophage activation.     

Regulation of adenylyl cyclase activity by beta-adrenergic agonists in a desensitization-resistant mutant cell line.

Mutant clones resistant to ACTH-induced desensitization of adenylyl cyclase (Y1DR) were previously isolated from the Y1 mouse adrenocortical tumor cell line. In this study, both parental Y1 cells (Y1DS) and a Y1DR mutant were transfected with a gene encoding the mouse beta 2-adrenergic receptor, and transfectants isolated from both Y1DS and Y1DR cells were shown to express beta 2-adrenergic receptors. These transfectants responded to the beta-adrenergic agonist isoproterenol with increases in adenylyl cyclase activity and steroidogenesis and changes in cell shape. The transfectants were analyzed to determine whether the Y1DR mutation was specific for ACTH-induced desensitization of adenylyl cyclase or also affected desensitization of adenylyl cyclase via the beta 2-adrenergic receptor. Treatment of intact Y1DS transfectants with isoproterenol caused a rapid desensitization of the adenylyl cyclase system to further stimulation by the beta-adrenergic agonist. Treatment of intact cells with isoproterenol did not affect ACTH-stimulated adenylyl cyclase activity, indicating that desensitization was agonist specific or homologous. Y1DR transfectants were resistant to the desensitizing effects of isoproterenol in intact cells as well as in cell homogenates. These results indicate that the mutation in Y1DR transfectants affects a component that is common to the pathways of isoproterenol-induced desensitization and ACTH-induced desensitization of adenylyl cyclase. As determined using the hydrophilic beta-receptor antagonist CGP-12177, isoproterenol caused a rapid sequestration of cell surface receptors in both Y1DS and Y1DR transfectants. From these results we infer that the DR phenotype does not arise from mutations affecting receptor sequestration and that receptor number does not limit the response to isoproterenol in these transfectants.     

Death-associated protein 5 (DAP5/p97/NAT1) contributes to retinoic acid-induced granulocytic differentiation and arsenic trioxide-induced apoptosis in acute promyelocytic leukemia

All-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) induce differentiation and apoptosis in acute promyelocytic leukemia (APL) cells. Here we investigated the role and regulation of death-associated protein-5 (DAP5/p97/NAT1), a novel inhibitor of translational initiation, in APL cell differentiation and apoptosis. We found that ATRA markedly induced DAP5/p97 protein and gene expression and nuclear translocation during terminal differentiation of APL (NB4) and HL60 cells but not differentiation-resistant cells (NB4.R1 and HL60R), which express very low levels of DAP5/p97. At the differentiation inducing concentrations, ATO (<0.5 μM), dimethyl sulfoxide, 1,25-dihydroxy-vitamin-D3, and phorbol-12-myristate 13-acetate also significantly induced DAP5/p97 expression in NB4 cells. However, ATO administered at apoptotic doses (1–2 μM) induced expression of DAP5/p86, a proapoptotic derivative of DAP5/p97. ATRA and ATO-induced expression of DAP5/p97 was associated with inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Furthermore, DAP5/p97 expression was upregulated by inhibition of the PI3K/Akt/mammalian target of rapamycin (mTOR) pathway via LY294002 and via rapamycin. Finally, knockdown of DAP5/p97 expression by small interfering RNA inhibited ATRA-induced granulocytic differentiation and ATO-induced apoptosis. Together, our data reveal new roles for DAP5/p97 in ATRA-induced differentiation and ATO-induced apoptosis in APL and suggest a novel regulatory mechanism by which PI3K/Akt/mTOR pathway inhibition mediates ATRA- and ATO-induced expression of DAP5/p97. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. B. Ozpolat and U. Akar contributed equally.     

Down-regulation of beta2-adrenergic receptor expression by exercise training increases IL-12 production by macrophages following LPS stimulation

Three-week exercise training decreased the steady state level of beta(2)-adrenergic receptor (beta(2)AR) mRNA in peritoneal macrophages from BALB/c mice. When peritoneal macrophages from both exercise-trained and sedentary control mice were stimulated with lipopolysaccharide (LPS), interleukin (IL)-12 mRNA and protein expression was markedly higher in trained mice than in control mice. To determine whether enhanced production of IL-12 was associated with decreased expression of beta(2)AR, we transfected the macrophage cell line, RAW264, with a eukaryotic expression vector containing beta(2)ar cDNA, establishing a cell line overexpressing beta(2)AR (RAWar). Following LPS stimulation, IL-12 mRNA and protein expression was significantly lower in RAWar cells than in RAW264 cells transfected with vector alone (RAWvec). Furthermore, when the expression of transfected beta(2)AR in RAWar cells was down-regulated by a tetracycline repressor-regulated mammalian expression system, expression of IL-12 mRNA and protein following LPS stimulation tended to return to the levels in RAWvec cells. These findings indicate that macrophage production of IL-12 following LPS stimulation is regulated by the expression level of beta(2)AR, suggesting that the down-regulation of beta(2)AR expression associated with exercise training improves IL-12-induced type 1 helper T cell-mediated immune responses.     

Vav1 controls DAP10-mediated natural cytotoxicity by regulating actin and microtubule dynamics

The NK cell-activating receptor NKG2D recognizes several MHC class I-related molecules expressed on virally infected and tumor cells. Human NKG2D transduces activation signals exclusively via an associated DAP10 adaptor containing a YxNM motif, whereas murine NKG2D can signal through either DAP10 or the DAP12 adaptor, which contains an ITAM sequence. DAP10 signaling is thought to be mediated, at least in part, by PI3K and is independent of Syk/Zap-70 kinases; however, the exact mechanism by which DAP10 induces natural cytotoxicity is incompletely understood. Herein, we identify Vav1, a Rho GTPase guanine nucleotide exchange factor, as a critical signaling mediator downstream of DAP10 in NK cells. Specifically, using mice deficient in Vav1 and DAP12, we demonstrate an essential role for Vav1 in DAP10-induced NK cell cytoskeletal polarization involving both actin and microtubule networks, maturation of the cytolytic synapse, and target cell lysis. Mechanistically, we show that Vav1 interacts with DAP10 YxNM motifs through the adaptor protein Grb2 and is required for activation of PI3K-dependent Akt signaling. Based on these findings, we propose a novel model of ITAM-independent signaling by Vav downstream of DAP10 in NK cells.     

TLR4-mediated survival of macrophages is MyD88 dependent and requires TNF-alpha autocrine signalling

Modulation of macrophage survival is a critical factor in the resolution of inflammatory responses. Exposure to LPS protects innate immune cells against apoptosis, although the precise pathways responsible for prolongation of macrophage survival remain to be fully established. The goal of this study was to characterize the mechanism of TLR4-mediated survival of murine bone marrow-derived macrophages upon M-CSF withdrawal in more detail. Using a combination of knockout mice and pharmacological inhibitors allowed us to show that TLR4 and TLR2 stimulation promotes long-term survival of macrophages in a MyD88-, PI3K-, ERK-, and NF-kappaB-dependent manner. LPS-induced long-term, but not short-term, survival requires autocrine signaling via TNF-alpha and is facilitated by a general cytoprotective program, similar to that mediated by M-CSF. TLR4-mediated macrophage survival is accompanied by a remarkable up-regulation of specific cell surface markers, suggesting that LPS stimulation leads to the differentiation of macrophages toward a mixed macrophage/dendritic cell-like phenotype.     

Inactivation of DAP12 in PMN Inhibits TREM1-Mediated Activation in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is an autoimmune disease characterized by dysregulated and chronic systemic inflammatory responses that affect the synovium, bone, and cartilage causing damage to extra-articular tissue. Innate immunity is the first line of defense against invading pathogens and assists in the initiation of adaptive immune responses. Polymorphonuclear cells (PMNs), which include neutrophils, are the largest population of white blood cells in peripheral blood and functionally produce their inflammatory effect through phagocytosis, cytokine production and natural killer-like cytotoxic activity. TREM1 (triggering receptor expressed by myeloid cells) is an inflammatory receptor in PMNs that signals through the use of the intracellular activating adaptor DAP12 to induce downstream signaling. After TREM crosslinking, DAP12’s tyrosines in its ITAM motif get phosphorylated inducing the recruitment of Syk tyrosine kinases and eventual activation of PI3 kinases and ERK signaling pathways. While both TREM1 and DAP12 have been shown to be important activators of RA pathogenesis, their activity in PMNs or the importance of DAP12 as a possible therapeutic target have not been shown. Here we corroborate, using primary RA specimens, that isolated PMNs have an increased proportion of both TREM1 and DAP12 compared to normal healthy control isolated PMNs both at the protein and gene expression levels. This increased expression is highly functional with increased activation of ERK and MAPKs, secretion of IL-8 and RANTES and cytotoxicity of target cells. Importantly, based on our hypothesis of an imbalance of activating and inhibitory signaling in the pathogenesis of RA we demonstrate that inhibition of the DAP12 signaling pathway inactivates these important inflammatory cells.     

Lipopolysaccharide-induced monocyte chemotactic protein-1 is enhanced by suppression of nitric oxide production, which depends on poor CD14 expression on the surface of skeletal muscle

It is known that lipopolysaccharide (LPS)-induced monocyte chemotactic protein (MCP)-1 secretion from tissues recruits monocytes from the circulation, but the mechanism of the LPS-induced MCP-1 production in skeletal muscle is largely unexplained. To clarify the effect of LPS on MCP-1 production in skeletal muscle cells, C2C12 cells from a mouse skeletal muscle cell line, and RAW 264.7 cells from a mouse macrophage cell line, were used to assess production of LPS-induced MCP-1, nitric oxide (NO) and interferon (IFN)-beta. In addition, we evaluated inducible NO synthases (iNOS) mRNA expression using RT-PCR, and cell surface expression of CD14 and toll-like receptor (TLR) 4 using flow cytometry. In C2C12 cells, LPS stimulation increased MCP-1 production (p < 0.01), but combined treatment with LPS and NO inducer, diethylammonium (Z)-1-(N,N-diethylamino) diazen-1-ium-1,2-diolate (NONOate), significantly inhibited its production (p < 0.01). LPS stimulation neither induced production of NO nor of IFN-beta, which is an NO inducer. Recombinant IFN-beta stimulation, on the other hand, enhanced LPS-induced NO production (p < 0.01). Interestingly, we found that surface expression of CD14, which regulates IFN-beta production, in C2C12 cells was much lower than that in RAW 264.7 cells, although TLR4 expression on C2C12 cells was similar to that on RAW 264.7 cells. These data suggest that the reduced NO production in response to LPS may depend on low expression of CD14 on the cell surface of skeletal muscle, and that it may enhance LPS-induced MCP-1 production. Together, these functions of skeletal muscle could decrease the risk of bacterial infection by recruitment of monocytes.     

The ras suppressor, RSU-1, enhances nerve growth factor-induced differentiation of PC12 cells and induces p21CIP expression.

The Rsu-1 Ras suppressor gene was isolated based on its ability to inhibit v-Ras transformation. Using Rsu-1 transfectants of the pheochromocytoma cell line PC12, we demonstrated previously that Rsu-1 expression inhibited Jun kinase activation but enhanced Erk2 activation in response to epidermal growth factor. In the present study, the Rsu-1 PC12 transfectants were used to investigate the role of Rsu-1 in nerve growth factor (NGF)- and v-Ki-ras-mediated neuronal differentiation. NGF-induced neurite extension was enhanced, not inhibited, by the expression of Rsu-1 in PC12 cells. The activation of Erk kinase activity in response to NGF was sustained longer in the Rsu-1 transfectants compared with the vector control cells. During NGF-mediated differentiation, an increase in the expression of specific mRNAs for the early response genes Fos, cJun, and NGF1a was detected in both the vector control and Rsu-1 transfectants. The expression of the differentiation-specific genes VGF8 and SCG10 was similar in Rsu-1 transfectants compared with the vector control cells. The induction of Rsu-1 expression in these cell lines did not inhibit v-Ki-ras-induced differentiation, as measured by neurite extension. These data suggest that although Rsu-1 blocked some Ras-dependent response(s), these responses were not required for differentiation. Moreover, the induction of Rsu-1 expression in the PC12 clones resulted in growth inhibition and p21(WAF/CIP) expression. Hence, Rsu-1 expression enhances NGF-induced differentiation while inhibiting the growth of cells.