ELMO1 Deficiency Reduces Neutrophil Chemotaxis in Murine Peritonitis.

Peritoneal inflammation remains a major cause of treatment failure in patients with kidney failure who receive peritoneal dialysis. Peritoneal inflammation is characterized by an increase in neutrophil infiltration. However, the molecular mechanisms that control neutrophil recruitment in peritonitis are not fully understood. ELMO and DOCK proteins form complexes which function as guanine nucleotide exchange factors to activate the small GTPase Rac to regulate F-actin dynamics during chemotaxis. In the current study, we found that deletion of the Elmo1 gene causes defects in chemotaxis and the adhesion of neutrophils. ELMO1 plays a role in the fMLP-induced activation of Rac1 in parallel with the PI3K and mTORC2 signaling pathways. Importantly, we also reveal that peritoneal inflammation is alleviated in Elmo1 knockout mice in the mouse model of thioglycollate-induced peritonitis. Our results suggest that ELMO1 functions as an evolutionarily conserved regulator for the activation of Rac to control the chemotaxis of neutrophils both in vitro and in vivo. Our results suggest that the targeted inhibition of ELMO1 may pave the way for the design of novel anti-inflammatory therapies for peritonitis.

Phosphatidic acid generated by PLD2 promotes the plasma membrane recruitment of IQGAP1 and neointima formation.

Very little is known about how lipid signaling regulates intima hyperplasia after vascular injury. Herein, we report that deletion and pharmacological inhibition of phospholipase D (PLD)2, which generates the signaling lipid phosphatidic acid (PA), reduced neointimal formation in the mouse carotid artery ligation model. PLD2 deficiency inhibits migration of vascular smooth muscle cells (VSMCs) into the intima in mice as well as migration and formation of membrane ruffles in primary VSMCs. PA specifically binds to the IQ motif-containing guanosine triphosphatase-activating protein 1 (IQGAP1) scaffold protein. The binding between PA and IQGAP is required for the plasma membrane recruitment of IQGAP1. Similar to PLD2 inhibition, knockdown of IQGAP1 blocks ruffle formation and migration in VSMCs, which are rescued by expression of the exogenous IQGAP1 but not the PA binding-deficient mutant. These data reveal that the PLD2-PA-IQGAP1 pathway plays an important role in VSMC migration and injury-induced vascular remodeling, and implicate PLD2 as a candidate target for therapeutic interventions.-Wang, Z., Cai, M., Tay, L. W. R., Zhang, F., Wu, P., Huynh, A., Cao, X., Di Paolo, G., Peng, J., Milewicz, D. M., Du, G. Phosphatidic acid generated by PLD2 promotes the plasma membrane recruitment of IQGAP1 and neointima formation.

A HIF-1 target, ATIA, protects cells from apoptosis by modulating the mitochondrial thioredoxin, TRX2.

The regulation of apoptosis is critical for controlling tissue homeostasis and preventing tumor formation and growth. Reactive oxygen species (ROS) generation plays a key role in such regulation. Here, we describe a HIF-1 target, Vasn/ATIA (anti-TNFα-induced apoptosis), which protects cells against TNFα- and hypoxia-induced apoptosis. Through the generation of ATIA knockout mice, we show that ATIA protects cells from apoptosis through regulating the function of the mitochondrial antioxidant, thioredoxin-2, and ROS generation. ATIA is highly expressed in human glioblastoma, and ATIA knockdown in glioblastoma cells renders them sensitive to hypoxia-induced apoptosis. Therefore, ATIA is not only a HIF-1 target that regulates mitochondrial redox pathways but also a potentially diagnostic marker and therapeutic target in human glioblastoma.

The adaptor protein TRADD is essential for TNF-like ligand 1A/death receptor 3 signaling.

TNFR-associated death domain protein (TRADD) is a key effector protein of TNFR1 signaling. However, the role of TRADD in other death receptor (DR) signaling pathways, including DR3, has not been completely characterized. Previous studies using overexpression systems suggested that TRADD is recruited to the DR3 complex in response to the DR3 ligand, TNF-like ligand 1A (TL1A), indicating a possible role in DR3 signaling. Using T cells from TRADD knockout mice, we demonstrate in this study that the response of both CD4(+) and CD8(+) T cells to TL1A is dependent upon the presence of TRADD. TRADD knockout T cells therefore lack the appropriate proliferative response to TL1A. Moreover, in the absence of TRADD, both the stimulation of MAPK signaling and activation of NF-κB in response to TL1A are dramatically reduced. Unsurprisingly, TRADD is required for recruitment of receptor interacting protein 1 and TNFR-associated factor 2 to the DR3 signaling complex and for the ubiquitination of receptor interacting protein 1. Thus, our findings definitively establish an essential role of TRADD in DR3 signaling.

The role of TRADD in TRAIL-induced apoptosis and signaling.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily. TRAIL is promising for anticancer therapy because it induces apoptosis in cancer cells with little or no toxicity to normal cells; hence, TRAIL-receptor agonists are currently undergoing clinical trials for cancer treatment. However, many molecular signaling mechanisms in TRAIL signaling are not completely characterized. The functions of adaptor proteins, including TNF-receptor-associated death domain protein (TRADD) and receptor-interacting protein-1 (RIP1) in TRAIL signaling have been controversial. We demonstrate that while wild-type mouse embryonic fibroblasts (MEFs) are completely resistant to TRAIL-induced apoptosis, MEFs derived from Tradd(-/-) mice are hypersensitive to TRAIL (IC(50)~0.5 nM rmTRAIL, 24 h), an effect also seen in primary keratinocytes treated with TRAIL/CHX. Restoration of TRADD in Tradd(-/-) MEFs restores TRAIL resistance, indicating that TRADD plays a survival role in TRAIL signaling. We show that TRADD is recruited to the TRAIL-receptor complex, and RIP1 recruitment is mediated by TRADD. While early activation of the MAP kinase ERK is deficient in Tradd(-/-) cells, the main mechanism for enhanced TRAIL sensitivity is likely due to increased recruitment of FADD to the receptor complex, indicating that TRADD may limit FADD binding within the receptor complex and also mediate RIP1-dependent nonapoptotic signaling events, thus reducing caspase activation and subsequent apoptosis. These novel findings have potential implications for cancer therapy using TRAIL-receptor agonists.

ELMO1 Regulates RANKL-Stimulated Differentiation and Bone Resorption of Osteoclasts.

Bone homeostasis is a metabolic balance between the new bone formation by osteoblasts and old bone resorption by osteoclasts. Excessive osteoclastic bone resorption results in low bone mass, which is the major cause of bone diseases such as rheumatoid arthritis. Small GTPases Rac1 is a key regulator of osteoclast differentiation, but its exact mechanism is not fully understood. ELMO and DOCK proteins form complexes that function as guanine nucleotide exchange factors for Rac activation. Here, we report that ELMO1 plays an important role in differentiation and bone resorption of osteoclasts. Osteoclast precursors derived from bone marrow monocytes (BMMs) of Elmo1-/- mice display defective adhesion and migration during differentiation. The cells also have a reduced activation of Rac1, p38, JNK, and AKT in response to RANKL stimulation. Importantly, we show that bone erosion is alleviated in Elmo1-/- mice in a rheumatoid arthritis mouse model. Taken together, our results suggest that ELMO1, as a regulator of Rac1, regulates osteoclast differentiation and bone resorption both in vitro and in vivo.

Isotope dilution LC/ESI--MS-MS quantitation of urinary 1,4-bis(N-acetyl-S-cysteinyl)-2-butanone in mice and rats as the biomarker of 1-chloro-2-hydroxy-3-butene, an in vitro metabolite of 1,3-butadiene.

1-Chloro-2-hydroxy-3-butene (CHB) is a possible metabolite of 1,3-butadiene, a carcinogenic air pollutant. To demonstrate its formation in vivo, it is desirable to develop a practical biomarker and the corresponding analysis method. CHB can undergo alcohol dehydrogenase- and cytochromes P450 enzymes (P450)-mediated oxidation to yield 1-chloro-3-buten-2-one (CBO), which readily forms glutathione conjugates. We hypothesized that CBO-derived mercapturic acids, which are the expected biotransformed products of CBO-glutathione conjugates, could be used as CHB biomarkers. Thus, in the present study, we investigated the in vivo biotransformation of CHB into CBO-derived mercapturic acids. Because the reaction of CBO with N-acetyl-l-cysteine yields two products, 1,4-bis(N-acetyl-S-cysteinyl)-2-butanone (NC1) and 1-chloro-4-(N-acetyl-S-cysteinyl)-2-butanone (NC2), we first developed an isotope dilution LC/ESI--MS-MS method to quantitate urinary NC1 and NC2, and then determined their concentrations in urine of C57BL/6 mice and Sprague-Dawley rats administered CHB. Since no NC2 was detected in samples, the LC/ESI--MS-MS method was optimized specifically for NC1. NC1 was enriched through solid phase extraction with the recovery being 75-82%. The limits of detection and quantitation were 6.8 and 34 fmol/0.1 mL for mouse urine, and 4.5 and 7.1 fmol/0.1 mL for rat urine, respectively. In urine of animals before CHB administration, no NC1 was detected; in mice administered CHB at 10 and 30 mg/kg, and rats at 5 and 15 mg/kg, NC1 was detected and its concentrations in urine from animals given higher doses were 3-6 fold higher than those given lower doses. Moreover, the NC1 concentrations in urine during 0-8 h were 4-6 fold and 10-11 fold higher than those during 8-24 h for mice and rats, respectively. The results demonstrated that CHB could be in vivo biotransformed into NC1, which could be used as a practical CHB biomarker.

Involvement of mitogen-activated protein kinase (MAPK) pathway in LH- and meiosis-activating sterol (MAS)-induced maturation in rat and mouse oocytes.

Gonadotropic stimulation of meiotic resumption in mice is dependent upon mitogen-activated protein kinase (MAPK) activation in the somatic compartment of the follicle. By contrast, spontaneous resumption of meiosis is independent of MAPK activation. In view of the suggested role of meiosis-activating sterol (MAS) in oocyte maturation we have (i) compared MAPK activation in rat preovulatory follicles stimulated by LH or by accumulation of endogenous MAS by using an inhibitor of MAS conversion, AY9944; (ii) examined whether stimulation of meiosis by MAS is dependent upon MAPK activation using denuded oocytes (DO) of Mos- null mice (hereafter Mos(-/-)) with oocytes unable to activate MAPK. Rat preovulatory follicles responded to LH or AY9944 stimulation by MAPK activation. Inhibition of MAPK phosphorylation blocked both LH- and AY9944 triggered resumption of meiosis. In mouse cumulus-enclosed oocytes (CEOs) and DOs AY9944 stimulated GVB in wild-type and Mos(-/-) mouse CEOs cultured with hypoxanthine (Hx). Addition of MAS or AY9944 to mouse DOs cultured with Hx induced resumption of meiosis only in wild-type and Mos(+/-) oocytes, but they were ineffective in Mos(-/-) oocytes. The observed sluggish activation of MAPK induced by AY9944 in rat follicle-enclosed oocytes (FEO) may cause the delay in meiotic resumption in response to MAS and AY9944 stimulation. Further, it is incompatible with the suggested role of MAS as an obligatory mediator of LH in the induction of meiotic maturation. MAPK/MOS activation, whether in the somatic compartment or in denuded oocytes, is required for MAS- like LH-, FSH-, or EGF-induced resumption of meiosis.

Responsiveness of voltage-gated calcium channels in SH-SY5Y human neuroblastoma cells on micropillar substrates.

In this study, poly-L-lactic acid micropillar substrates were fabricated to evaluate the influence of topographic substrates on cell morphological and functional characteristics, such as spreading area, voltage-gated calcium channels (VGCCs) and membrane potential. The proliferation, spreading area, perimeter and circularity of SH-SY5Y cells interfaced with different substrates were first investigated. In addition, the cytoskeleton and focal adhesion of a cell as important manifestations of cell morphology were analyzed by immunofluorescence. VGCC responsiveness was evaluated by measuring the dynamic changes in intracellular Ca2+ evoked by 50 mM extracellular K+. To determine study whether the differences in VGCC responsiveness were caused by the differences in VGCC gene expression, the expression of N/L- type VGCCs was determined by qPCR and fluorescence staining. Notably, improved measurement of the membrane potential with potentiometric fluorescent dye TMRM was applied to determine the membrane potential of SH-SY5Y cells. Results indicated that the SH-SY5Y cells were deformed significantly to adapt to the substrates; however, no distinct effect on the proliferative ability of SH-SY5Y cells was observed. The micropillar substrates markedly influenced VGCC responsiveness, which correlated strongly with cell spreading but not with VGCC expression. The resting membrane potential of SH-SY5Y cells cultured on different substrates also changed, but no effect on responsiveness of VGCC was observed. These results suggest that the effect of the micropillar substrates on cell VGCC responsiveness may be attributed to changes in the functionality of the ion channel itself. Thus, topographic substrates can be used to engineer cell functionality in cell-based drug screening.

Identification of Associated Proteins by Immunoprecipitation and Mass Spectrometry Analysis.

Protein-protein interactions play central roles in intercellular and intracellular signal transduction. Impairment of protein-protein interactions causes many diseases such as cancer, cardiomyopathies, diabetes, microbial infections, and genetic and neurodegenerative disorders. Immunoprecipitation is a technique in which a target protein of interest bound by an antibody is used to pull down the protein complex out of cell lysates, which can be identified by mass spectrometry. Here, we describe the protocol to immunoprecipitate and identify the components of the protein complexes of ElmoE in Dictyostelium discoideum cells.

Is meiosis activating sterol (MAS) an obligatory mediator of meiotic resumption in mammals.

In-vitro studies of mouse oocytes have provided evidence that two closely related sterols, subsequently named meiosis-activating sterols (MAS), can overcome the inhibitory effect of hypoxanthine on resumption of meiosis. These sterols are synthesized by cytochrome P450 (CYP) lanosterol 14alpha-demethylase (LDM), a key enzyme in cholesterol biosynthesis. Our studies in the rat with specific inhibitors and molecular approaches did not support the hypothesis that MAS is an obligatory step in the stimulation of the resumption of meiosis. (i) Specific inhibitors of MAS synthesizing enzymes did not prevent spontaneous or LH-stimulated meiosis at doses that have previously been shown to effectively suppress LDM activity. At higher doses, they caused degeneration of oocytes. (ii) The timing of LDM expression in the ovary was incompatible with a role for MAS in meiosis. (iii) The preferential localization of LDM protein in the oocytes suggests MAS production in oocytes, rather than its transport from the somatic compartment as expected by the suggested role of MAS in the regulation of meiosis as a putative cumulus-oocyte signal molecule. (iv) AY-9944, which supposedly increases MAS levels by inhibiting its metabolism, induced the maturation of follicle-enclosed oocytes that was much delayed as compared with gonadotropic stimulation. Thus, the resumption of meiosis induced by added MAS [Biol. Reprod. 61 (1999) 1362, Biol. Reprod. 64 (2001) 418] or presumed endogenous MAS accumulation by AY-9944, resulted in oocyte maturation with remarkably slower kinetics than observed with LH stimulation. This delay in meiosis after MAS stimulation, the studies with LDM inhibitors and its spatial and temporal expression, cast serious doubts whether MAS is indeed mediating the meiosis inducing action of the gonadotropins, as suggested.

Arrestins function in cAR1 GPCR-mediated signaling and cAR1 internalization in the development of Dictyostelium discoideum.

Oscillation of chemical signals is a common biological phenomenon, but its regulation is poorly understood. At the aggregation stage of Dictyostelium discoideum development, the chemoattractant cAMP is synthesized and released at 6-min intervals, directing cell migration. Although the G protein-coupled cAMP receptor cAR1 and ERK2 are both implicated in regulating the oscillation, the signaling circuit remains unknown. Here we report that D. discoideum arrestins regulate the frequency of cAMP oscillation and may link cAR1 signaling to oscillatory ERK2 activity. Cells lacking arrestins (adcB(-)C(-)) display cAMP oscillations during the aggregation stage that are twice as frequent as for wild- type cells. The adcB(-)C(-) cells also have a shorter period of transient ERK2 activity and precociously reactivate ERK2 in response to cAMP stimulation. We show that arrestin domain-containing protein C (AdcC) associates with ERK2 and that activation of cAR1 promotes the transient membrane recruitment of AdcC and interaction with cAR1, indicating that arrestins function in cAR1-controlled periodic ERK2 activation and oscillatory cAMP signaling in the aggregation stage of D. discoideum development. In addition, ligand-induced cAR1 internalization is compromised in adcB(-)C(-) cells, suggesting that arrestins are involved in elimination of high-affinity cAR1 receptors from cell surface after the aggregation stage of multicellular development.

MicroRNA-31 suppresses medulloblastoma cell growth by inhibiting DNA replication through minichromosome maintenance 2.

Medulloblastoma is an aggressive childhood brain tumor with poor prognosis. Recent studies indicate that dys-regulation of microRNA expression plays important roles in tumorigenesis. By comparing microRNA levels between mouse medulloblastoma and normal cerebellar tissues, we identified a set of down-regulated microRNAs including miR-31. Here, we show that the genomic region surrounding human miR-31 at 9p21.3 is frequently deleted in many solid tumor cell lines, and reintroducing miR-31 into DAOY cells, a line of human medulloblastoma cells devoid of miR-31, strongly suppresses cell growth, causes cell cycle arrest at the G1/S boundary, and inhibits colony formation in vitro and xenograft tumorigenesis in nude mice. Global gene expression profiling of mouse medulloblastomas and bioinformatics analyses of microRNA targets suggest that minichromosome maintenance complex component 2 (MCM2) is a likely target gene of miR-31 in suppressing cell growth. We demonstrate that miR-31 inhibits MCM2 expression via its 3'-untranslated region, that knockdown of MCM2 in DAOY cells leads to a degree of growth inhibition comparable to that by miR-31 restoration, and that overexpression of miR-31 reduces the chromatin loading of MCM2 at the point of G1/S transition. Taken together, these data indicate that miR-31 suppresses medulloblastoma tumorigenesis by negatively regulating DNA replication via MCM2.

GnRH actions on rat preovulatory follicles are mediated by paracrine EGF-like factors.

Gonadotropin releasing hormone (GnRH) has been shown to mimic the actions of LH/hCG on oocyte maturation and ovulation. Recent studies demonstrated that induction of ovulation by LH/hCG is mediated, at least in part, by transactivation of epidermal growth factor receptors (EGFR) by autocrine/paracrine EGF-like factors activated by metalloproteases. Here we have examined whether the action of GnRH on the preovulatory follicles is exerted through similar mechanisms involving activation of EGFR. The EGFR kinase inhibitor, AG1478, inhibited GnRH-induced oocyte maturation in explanted follicles in vitro. Its inactive analog, AG43, did not affect GnRH-stimulated resumption of meiosis. GnRH, like LH, stimulated transient follicular expression of EGF-like agents, as well as rat cycloxygenase-2 (rCOX-2), rat hyaluronan synthase-2 (rHAS-2), and rat tumor necrosis factor-alpha-stimulated gene 6 (rTSG-6) mRNAs, known ovulatory enzymes. Likewise, GnRH stimulated follicular progesterone synthesis. Conversely AG1478 inhibited all these actions of GnRH. Furthermore, Galardin, a broad-spectrum metalloprotease inhibitor, blocked GnRH-induced oocyte maturation and follicular progesterone synthesis. In conclusion, we have demonstrated that follicular EGF-like factors mediate also the GnRH-stimulation of ovulatory changes, like these of LH/hCG.

Meiosis-activating sterol synthesis in rat preovulatory follicle: is it involved in resumption of meiosis?

Meiosis-activating sterol (MAS) was shown to overcome the inhibitory effect of hypoxanthine on spontaneous maturation of mouse oocytes and was suggested to mediate the stimulation of meiosis by gonadotropins. Follicular fluid (FF)-MAS is synthesized by cytochrome P450 lanosterol 14alpha-demethylase (LDM). Follicular LDM was preferentially localized in oocytes by immunohistochemistry. Using [3H]acetate or R-[5-3H]mevalonate as precursors as well as high-performance liquid chromatographic and thin-layer chromatographic separation, we have measured the concentrations of de novo-synthesized lanosterol, FF-MAS, and cholesterol in rat graafian follicles, cumulus-oocyte complexes (COCs), and denuded oocytes (DOs) treated with LH, AY-9944 (an inhibitor of Delta14-reductase, which was anticipated to increase FF-MAS levels by inhibiting its metabolism), or both after 8 h of culture. In follicles, both LH and AY-9944 increased the accumulation of FF-MAS as compared to controls. In COCs, AY-9944 caused a marked increase in FF-MAS, but we were unable to detect accumulation of FF-MAS in DOs. Neither the endogenous increases in FF-MAS accumulation nor the addition of FF-MAS to the culture medium could overcome the inhibition on resumption of meiosis by phosphodiesterase inhibitors. Compared to LH-induced resumption of meiosis in follicles, that induced by AY-9944 was much delayed. These results call into question any role of FF-MAS as an obligatory mediator of LH activity on germinal vesicle breakdown. The discrepancy between the positive staining for LDM in oocytes and our inability to detect de novo synthesized FF-MAS in DOs may relate to the sensitivity of the methodology employed and either the number of oocytes used or a deficiency in LDM synthetic activity in such oocytes. Further studies are required to confirm any of these alternatives.

Expression of matrix metalloproteinase -2, -9 and tissue inhibitors of metalloproteinase -1, -2, -3 mRNAs in rat uterus during early pregnancy.

Zymography and in situ hybridizition were used to investigate matrix metalloproteinase-2, -9 (MMP-2, -9) activities, and expression of mRNAs for MMP-2, -9 and tissue inhibitors of matrix metalloproteinases (TIMP-1, -2, -3) in the rat uterus during early pregnancy (day 1-7). The zymography results showed two forms of MMP-2 (64 and 67 kDa) in the rat uteri during early pregnancy. The 64-kDa MMP-2 activity was the highest on day 2 (P < 0.01) and higher on day 5 and 6 (P < 0.05). The 67-kDa MMP-2 activity reached the highest on day 5 and 6 (P < 0.01). The 64-kDa MMP-2 activity at the implantation sites was higher than those at interimplantation sites (P < 0.05). Furthermore, the 67 kDa MMP-2 can be converted to 64 kDa forms by incubation with p-aminophenylmercuric acetate (APMA) and trypsin in vitro. The 92-kDa MMP-9 activity was only detected on day 5 and 6 of pregnancy (P < 0.01). In situ hybridization showed that on day 1-4 of pregnancy, both MMP-2 and TIMP-2 mRNAs were evidently localized in the basal stromal cells. On day 5, MMP-2 mRNA signals were decreased in the basal stromal cells and mRNA for TIMP-2 was expressed in the epithelial cells and subepithelial stromal cells. The mRNAs for MMP-9, TIMP-1, and -3 were mainly expressed in epithelial cells on day 1-5. At the implantation site on day 6, the mRNAs for MMP-2, -9, TIMP-1, -2, and -3 were highly expressed in the primary decidual zone surrounding the implanting embryo, and in the whole decidualized stromal cells (the primary and secondary decidual zones) at the implantation site on day 7. The intensities of mRNAs for the TIMPs in decidualized stromal cells at the implantation site on day 6 and 7 were stronger than those for the MMPs. The weak mRNAs for MMP-2, -9, TIMP-1, and -3 but not TIMP-2 were also observed in the ectoplacental cone/trophoblastic cells of the implanting embryos. However, at the interimplantation sites on day 6 and 7, MMP-2, -9, TIMP-1, -2, and -3 mRNAs were weakly expressed in the epithelial cells, subepithelial stromal cells, and myometrium. The results suggested that the implanting rat embryo strongly induced MMP-2 and -9 proteins and gene expression for decidulization and embryo invasion, which were strictly controlled and balanced by the simultaneous expression of TIMP-1, -2 and -3.