Abstract 468: The Role of Ca2+/PKA Signaling Enhancer Protein Phosphatase Inhibitor 1 in the Control of Ca2+ Cycling and Signaling in VSMCs

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Lahouaria Hadri ◽  
Regis Bobe ◽  
Jose J Lopez ◽  
Irene C Turnbull ◽  
Jason C Kovacic ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Protein Phosphatase ◽  
Specific Inhibitor ◽  
Negative Regulator ◽  
Mice Model ◽  
Neointimal Proliferation ◽  
Genetic Deletion ◽  
Vascular Proliferative Diseases ◽  
Deficient Mice

Vascular remodeling is associated with trans-differentiation of contractile vascular smooth muscle cells (VSMC) towards a proliferating/synthetic phenotype. We have recently demonstrated (Bobe et al., 2011) that the Ca2+ cycling in “contractile” VSMC requires the expression of the fast isoform of sarco/endo plasmic ATPase (SERCA2a), whereas the Ca2+ cycling in “proliferating” ones is associated with the expression of the ubiquitous isoform SERCA2b only. Phospholamban (PLB), a negative regulator of SERCA2 activity, is inhibited by PKA phosphorylation and activated by protein phosphatase 1 (PP1) dependent dephosphorylation. Inhibitor-1 (I-1), a highly specific inhibitor of PP1, enhances both PKA-dependent PLB phosphorylation and SERCA activity. The goal of this study was to elucidate the role of PKA signaling enhancer I-1 in the control of VSMC Ca2+ cycling. In humans and rodents, the expression of I-1 was found to be specific for contractile VSMC, whereas its target PP1 was highly expressed in synthetic VSMC. Consequently, PLB phosphorylation was decreased in synthetic VSMC whereas the expression of total PLB remained unchanged. Genetic deletion of I-1 in mice model (I-1 KO) resulted in lack of PLB phosphorylation in VSMC of adult animals. Despite the fact that SERCA2a was expressed, VSMCs from I-1 deficient mice were locked in the synthetic state. Consistent with this, the adult I-1 deficient mice developed a vascular proliferative disorder and excessive neointimal proliferation after vascular injury. Adenovirus-directed gene transfer of constitutively active I-1 (I-1c) significantly increased PLB phosphorylation and Ca2+ uptake in both synthetic and contractile VSMCs, however, there was no effect on the type of Ca2+ transient, which appears to be a SERCA2 isoform-dependent characteristic. Therefore, I-1c prevented proliferation and remodeling of contractile VSMC, but had no effect on synthetic VSMC, which predominantly express SERCA2b. In conclusion, synergistic action of I-1 and SERCA2a is necessary for acquisition of the VSMC contractile phenotype. Gene transfer of I-1c may be considered as a promising therapeutic strategy for preventing vascular proliferative diseases.

scholarly journals B7-H4–deficient mice display augmented neutrophil-mediated innate immunity

Blood ◽  
2009 ◽  
Vol 113 (8) ◽  
pp. 1759-1767 ◽  
Author(s):  
Gefeng Zhu ◽  
Mathew M. Augustine ◽  
Takeshi Azuma ◽  
Liqun Luo ◽  
Sheng Yao ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Negative Regulator ◽  
Immunoglobulin Superfamily ◽  
Cell Responses ◽  
Inhibitory Function ◽  
Genetic Targeting ◽  
Neutrophil Response ◽  
Deficient Mice

Abstract B7-H4 is an immunoglobulin superfamily molecule and shown to be inhibitory for T-cell responses. To explore physiologic roles of B7-H4, we created B7-H4–deficient (KO) mice by genetic targeting. B7-H4KO mice are healthy and their T- and B-cell responses to polyclonal antigens are in normal range. However, B7-H4KO mice are more resistant to infection by Listeria monocytogenes than their littermates. Within 3 days after infection, bacterial colonies in livers and spleens are significantly lower than the controls, suggesting a role of B7-H4 in enhancing innate immunity. Further studies demonstrate that neutrophils increase in peripheral organs of B7-H4KO mice more so than their littermates but their bactericidal functions remain unchanged. Augmented innate resistance is completely dependent on neutrophils, even in the absence of adaptive immunity. In vitro B7-H4 inhibits the growth of bone marrow–derived neutrophil progenitors, suggesting an inhibitory function of B7-H4 in neutrophil expansion. Our results identify B7-H4 as a negative regulator of the neutrophil response to infection and provide a new target for manipulation of innate immunity.

A Novel Role of Coagulation Proteases on Viral-Based Gene Transfer Efficacy.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 691-691
Author(s):  
Joerg Schuettrumpf ◽  
Jianxiang Zou ◽  
Shin Jen Tai ◽  
Alexander Schlachterman ◽  
Kian Tian ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Transfer ◽  
Knockout Mice ◽  
Viral Vectors ◽  
Hemophilia B ◽  
Gene Copy ◽  
Dependent Manner ◽  
Deficient Mice

Abstract Coagulation proteases are crucial for hemostasis and have also been implicated in inflammatory responses, blood vessel formation, and tumor cell metastasis. Cellular responses triggered by proteases are mediated by protease-activated receptors (PAR). Adeno-associated virus (AAV)-2 vectors hold promise for the treatment of several diseases and were already tested in Phase I studies for hemophilia B following intramuscular or hepatic artery deliveries. Previously, we determined an unexpected inhibitory effect (60–70% downregulation) on AAV-2 and adenovirus mediated gene transfer by thrombin- or FXa inhibitors. These results were independent of mouse strain, transgene product, or vector promoter, and gene expression by vectors of alternate serotypes AAV-5 or -8, which do not share cellular receptors with AAV-2, were not affected by any drug. Here we present in vivo evidence of a novel role of coagulation proteases and PARs in modulating gene transfer by viral vectors. We tested AAV-2 gene transfer efficacy in (a) animal models for proteases deficiency [FX and FIX deficient animals], (b) PAR-1 or PAR-2 deficient mice, (c) and following in vivo activation of PARs. FX knockout mice with residual activity of only 1–3% of normal (n=9) were injected with AAV-2-human(h)FIX vector and compared to littermates with FX levels of 50% (n=4). FIX expression levels were 2-fold lower among FX-deficient mice compared to controls (p<0.03). The second model, FIX deficient mice, received AAV expressing α1-antitrypsin (AAT-1). Severe hemophilia B models due to large-gene deletion (n=5) or missense mutation (R180T) in the FIX gene (n=3, <1% FIX) were compared to littermate controls with normal FIX levels (n=6). The results showed that AAT-1 levels among hemophilia B mice were 2-fold lower than in controls (24 vs 48 ng/ml, p<0.05, respectively). Because PAR activation by thrombin enhances αVβ5 (co-receptor for AAV-2 and adenovirus)-dependent cellular function (JBC 276:10952) we hypothesized that PAR modulates AAV-2 gene transfer. Homozygous (−/−) or heterozygous deficient (+/−) PAR-1 (n=24) or PAR-2 (n=25) mice received AAV-2-hF.IX and were compared to littermate controls (+/+). FIX levels among PAR-1 controls (1.9 μg/ml) were comparable to levels obtained among heterozygotes but higher than in homozygotes (1.1 μg/ml, p<0.02). Similarly, PAR-2 deficient mice presented 2-fold lower FIX levels than controls (0.7 vs 1.3 μg/ml, p<0.02) whereas heterozygous mice presented intermediate levels. To further confirm the role of PARs in AAV-2 gene transfer we activated PARs prior to AAV-2 injection. C57BL/6 mice received specific peptide agonists at doses ranging from 10 to 60 μM/kg (n=4 per dose and per peptide) and were compared to controls receiving scramble peptide. FIX levels increased 1.5 to 5-fold in a dose-dependent manner and the activation of PAR-1 and -2 simultaneously was superior to single peptide. Gene copy monitoring revealed low vector uptake by livers of PAR knockout mice while activation of PARs increased uptake. In conclusion, these data demonstrated a novel in vivo role of coagulation proteases and PARs on viral vectors (AAV-2 and adenovirus)-mediated gene expression and provide an alternative target to modulate gene therapy strategies.

scholarly journals Role of Toll Interleukin-1 Receptor (IL-1R) 8, a Negative Regulator of IL-1R/Toll-Like Receptor Signaling, in Resistance to Acute Pseudomonas aeruginosa Lung Infection

2011 ◽  
Vol 80 (1) ◽  
pp. 100-109 ◽  
Author(s):  
Tania Véliz Rodriguez ◽  
Federica Moalli ◽  
Nadia Polentarutti ◽  
Moira Paroni ◽  
Eduardo Bonavita ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Interleukin 1 ◽  
Bacterial Load ◽  
Lung Infection ◽  
Negative Regulator ◽  
Current Evidence ◽  
Toll Like Receptor ◽  
Content Type ◽  
Deficient Mice

ABSTRACTToll interleukin-1 receptor (IL-1R) 8 (TIR8), also known as single Ig IL-1 receptor (IL-R)-related molecule, or SIGIRR, is a member of the IL-1R-like family, primarily expressed by epithelial cells. Current evidence suggests that TIR8 plays a nonredundant role as a negative regulatorin vivounder different inflammatory conditions that are dependent on IL-R and Toll-like receptor (TLR) activation. In the present study, we examined the role of TIR8 in innate resistance to acute lung infections caused byPseudomonas aeruginosa, a Gram-negative pathogen responsible for life-threatening infections in immunocompromised individuals and cystic fibrosis patients. We show that Tir8 deficiency in mice was associated with increased susceptibility to acuteP. aeruginosainfection, in terms of mortality and bacterial load, and to exacerbated local and systemic production of proinflammatory cytokines (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], IL-1β, and IL-6) and chemokines (CXCL1, CXCL2, and CCL2). It has been reported that host defense againstP. aeruginosaacute lung infection can be improved by blocking IL-1 since exaggerated IL-1β production may be harmful for the host in this infection. In agreement with these data, IL-1RI deficiency rescues the phenotype observed in Tir8-deficient mice: in Tir8−/−IL-1RI−/−double knockout mice we observed higher survival rates, enhanced bacterial clearance, and reduced levels of local and systemic cytokine and chemokine levels than in Tir8-deficient mice. These results suggest that TIR8 has a nonredundant effect in modulating the inflammation caused byP. aeruginosa, in particular, by negatively regulating IL-1RI signaling, which plays a major role in the pathogenesis of this infectious disease.

scholarly journals S100A8/A9 modulates inflammatory collateral tissue damage during intraperitoneal origin systemic candidiasis

2020 ◽  
Author(s):  
Madhu Shankar ◽  
Nathalie Uwamahoro ◽  
Sandra Holmberg ◽  
Maria Joanna Niemiec ◽  
Johannes Roth ◽  
...  
Keyword(s):  
Tissue Damage ◽  
Critical Role ◽  
Specific Inhibitor ◽  
Surgical Intensive Care ◽  
Inflammatory Protein ◽  
Systemic Level ◽  
Combating Infection ◽  
Deficient Mice

AbstractPeritonitis is a leading cause of severe sepsis in surgical intensive care units, as over 70% of patients diagnosed with peritonitis develop septic shock. A critical role of the immune system is to return to homeostasis after combating infection. S100A8/A9 (calprotectin) is an antimicrobial, pro-inflammatory protein complex often used as a biomarker for diagnosis of disease activities in many inflammatory disorders. Here we describe the role of S100A8/A9 on inflammatory collateral tissue damage (ICTD).We performed an in vivo Candida albicans disseminated peritonitis mouse model using WT and S100A9-deficient mice and stimulated primary macrophages with recombinant S100A8/A9 in the presence or absence of the compound paquinimod, a specific inhibitor of S100A9. In addition, the effects on ICTD and fungal clearance were investigated. S100A9-deficient mice developed less ICTD than wildtype mice. Restoration of S100A8/A9 in S100A9 knockout mice resulted in increased ICTD and fungal clearance comparable to wildtype levels. Treatment with paquinimod abolished ICTD.The data indicated that S100A8/A9 controls ICTD levels and host antimicrobial modulation at a systemic level during intra-abdominal candidiasis (IAC).

Insight in Protein S Deficiency From Mouse Models

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 529-529
Author(s):  
Sara Calzavarini ◽  
François Saller ◽  
Jose A. Fernandez ◽  
Linda Kadi ◽  
Anne C. Brisset ◽  
...  
Keyword(s):  
Mouse Models ◽  
Purpura Fulminans ◽  
Protein S ◽  
Negative Regulator ◽  
Thrombotic Risk ◽  
Increased Risk ◽  
Deficient Mice

Abstract Abstract 529 Protein S (ProS) is an important negative regulator of blood coagulation. Its physiological importance is evident in purpura fulminans and other life-threatening thrombotic disorders typical of ProS deficient patients. Our previous characterization of ProS deficiency in mouse models has shown similarities with the human phenotypes: heterozygous ProS-deficient mice (Pros+/−) had increased thrombotic risk whereas homozygous deficiency in ProS (Pros−/−) was incompatible with life (Blood 2009; 114:2307-2314). In tissues, ProS exerts cellular functions by binding to and activating tyrosine kinase receptors of the Tyro3 family (TAM) on the cell surface. To extend the analysis of coagulation defects beyond the Pros−/− phenotype and add new insights into the sites of synthesis ProS and its action, we generated mice with inactivated ProS in hepatocytes (Proslox/loxAlbCre+) as well as in endothelial and hematopoietic cells (Proslox/loxTie2Cre+). Both models resulted in significant reduction of circulating ProS levels and in a remarkable increased thrombotic risk in vivo. In a model of tissue factor (TF)-induced venous thromboembolism (VTE), only 17% of Proslox/loxAlbCre+ mice (n=12) and only 13% of Proslox/loxTie2Cre+ mice (n=14) survived, compared with 86% of Proslox/lox mice (n=14; P<0.001). To mimic a severe acquired ProS deficiency, ProS gene was inactivated at the adult stage using the polyI:C-inducible Mx1-Cre system (Proslox/loxMx1Cre+). Ten days after polyI:C treatment, Proslox/loxMx1Cre+ mice developed disseminated intravascular coagulation with extensive lung and liver thrombosis. It is worth noting that no skin lesions compatible with purpura fulminans were observed in any of the above-described models of partial ProS deficiency. In order to shed light on the pathogenesis of purpura fulminans, we exposed the different ProS-deficient mice to warfarin (0.2 mg/day). We observed that Pros+/−, Proslox/loxAlbCre+ and Proslox/loxTie2Cre+ mice developed retiform purpura (characterized by erythematous and necrotic lesions of the genital region and extremities) and died after 3 to 5 days after the first warfarin administration. In human, ProS is also synthesized by megakaryocytes and hence stored at high concentrations in circulating platelets (pProS). The role of pProS has been investigated by generating megakaryocyte ProS-deficient model using the PF4 promoter as Cre driver (Proslox/loxPf4Cre+). In the TF-induced VTE model, Proslox/loxPf4Cre+ (n=15) mice showed a significant increased risk of thrombosis compared to Proslox/lox controls (n=14; survival rate 47% and 86%, respectively; P<0.05). Furthermore, preliminary results suggest survival to be associated with higher circulating ProS levels. In order to evaluate the potential role of pProS in thrombus formation, we investigated the thrombotic response to intravenous injection of collagen-epinephrine in vivo and platelet function in vitro. Both in vivo and in vitro experiments showed similar results between Proslox/loxPf4Cre+ and Proslox/lox, indicating that platelet reactivity was not influenced by the absence of pProS. These data suggest that pProS is delivered at the site of thrombosis to inhibit thrombin generation. We further investigated the ability of ProS to function as a ligand of TAM receptors, by using homozygous and heterozygous deficient mice for both the TAM ligands ProS and Gas6. Gas6−/−Pros−/− mice died in utero and showed comparable dramatic bleeding and thrombotic phenotype as described for Pros−/− embryos. In conclusion, like complete ProS deficiency, double deficiency in ProS and Gas6 was lethal, whereas partial ProS deficiency was not. Mice partially deficient in ProS displayed a prothrombotic phenotype, including those with only deficiency in pProS. Purpura fulminans did not occur spontaneously in mice with partial Pros deficiency but developed upon warfarin administration. Thus, the use of different mice models of ProS deficiency can be instrumental in the study of its highly variable thrombotic phenotype and in the investigation of additional roles of ProS in inflammation and autoimmunity through TAM signaling. Disclosures: No relevant conflicts of interest to declare.

scholarly journals C4b binding protein negatively regulates TLR1/2 response

2016 ◽  
Vol 23 (1) ◽  
pp. 11-19 ◽  
Author(s):  
Naoko Morita ◽  
Ikuko Yamai ◽  
Koichiro Takahashi ◽  
Yutaka Kusumoto ◽  
Takuma Shibata ◽  
...  
Keyword(s):  
Inflammatory Cytokine ◽  
Cytokine Production ◽  
Binding Protein ◽  
Negative Regulation ◽  
Negative Regulator ◽  
Wild Type ◽  
Inflammatory Cytokine Production ◽  
Inflammatory Signals ◽  
Deficient Mice

TLR2 associates with TLR1 and recognizes microbial lipoproteins. Pam3CSK4, a triacylated lipoprotein, is anchored to the extracellular domain of TLR1 and TLR2 and induces pro-inflammatory signals. Here we show that C4b binding protein (C4BP), which is a complement pathway inhibitor, is a TLR2-associated molecule. Immunoprecipitation assay using anti-TLR2 mAb shows that C4BP binds to TLR2. In C4BP-deficient mice, Pam3CSK4-induced IL-6 levels were increased compared with wild type mice. In C4BP-expressing cells, Pam3CSK4-induced IL-8 production was reduced depending on the C4BP expression levels. These results reveal the important role of C4BP in negative regulation of TLR1/2-dependent pro-inflammatory cytokine production. Furthermore, using a fluorescent conjugated Pam3CSK4, we show that C4BP blocks the binding of Pam3CSK4 to TLR1/2. Finally, we show that exogenous C4BP also inhibits Pam3CSK4-induced signaling leading to IL-8 production. Our results indicate C4BP binding to TLR2 and consequent neutralization of its activity otherwise inducing pro-inflammatory cytokine production. C4BP is a negative regulator of TLR1/2 activity.

scholarly journals Induction and Suppression of Collagen-Induced Arthritis Is Dependent on Distinct Fcγ Receptors

2000 ◽  
Vol 191 (9) ◽  
pp. 1611-1616 ◽  
Author(s):  
Sandra Kleinau ◽  
Pernilla Martinsson ◽  
Birgitta Heyman
Keyword(s):  
Cell Activation ◽  
Negative Regulator ◽  
Human Rheumatoid Arthritis ◽  
Collagen Induced Arthritis ◽  
Effector Functions ◽  
Ig G ◽  
Anticollagen Antibodies ◽  
Deficient Mice

Receptors for immunoglobulin (Ig)G (FcγRs) are important for the antibody-mediated effector functions of the immune system. FcγRI and FcγRIII trigger cell activation through a common γ chain, whereas FcγRII acts as a negative regulator of antibody production and immune complex–triggered activation. Here we describe the in vivo consequences of FcγR deficiency in a mouse model of human rheumatoid arthritis. FcRγ chain–deficient mice on arthritis-susceptible DBA/1 background were immunized with collagen for induction of collagen-induced arthritis. The DBA/1 mice lacking FcRγ chain were protected from collagen-induced arthritis in contrast to wild-type mice, although both groups produced similar levels of IgG anticollagen antibodies. In comparison, DBA/1 mice lacking FcγRII developed an augmented IgG anticollagen response and arthritis. These observations suggest a crucial role of FcγRI and FcγRIII in triggering autoimmune arthritis.

scholarly journals Role of Protein Phosphatase-2A (PP-2A) in the Control of Mitosis and Meiosis.

2020 ◽  
Author(s):  
H.Y. Lim Tung ◽  
H.Y. Lim Tung ◽  
H.Y. Lim Tung ◽  
H.Y. Lim Tung ◽  
H.Y. Lim Tung ◽  
...  
Keyword(s):  
Meiotic Division ◽  
Protein Phosphatase ◽  
Xenopus Oocytes ◽  
Protein Phosphatase 2A ◽  
Scientific Evidence ◽  
Specific Inhibitor ◽  
Phosphatase 2A ◽  
Greatwall Kinase ◽  
Mitosis And Meiosis

A specific form of Protein Phosphatase-2A (PP-2A), namely PP2A-B55δ was proposed to occupy a central role in the control of mitosis entry and exit, and meiosis in Xenopus oocytes [1,3]. It was held that PP2A-B55δ is responsible for dephosphorylating substrates of cdc2/Cdk1 and that inhibition of PP2A-B55δ by Arpp-19 phosphorylated at serine 67 by Greatwall kinase triggers entry of both mitosis and meiosis in Xenopus oocytes. It was further declared that the phosphorylation of Arpp19 at serine 109 by PKA underlies the blockade of meiotic division and that dephosphorylation of serine 109 of Arpp19 triggers resumption of meiotic division in Xenopus oocytes [4]. Recently two groups have stated that PP2A-B55δ is the protein phosphatase that is responsible for dephosphorylating both serine 67 and serine 109 of Arpp19 [4,5] However, unfortunately for the authors concerned [1-5], no verifiable scientific evidence exists that shows that Arpp19 is a specific inhibitor of PP-2AB55ɗ when Arpp19 is phosphorylated at serine 67 by Greatwall kinase and that Arpp-19 phosphorylated at serine 67 and Arpp19 phosphorylated at 109 are both specifically dephosphorylated by PP-2AB55ɗ Arpp19. The idea that Arpp-19 phosphorylated at serine 67 is both an inhibitor and a substrate of PP-2AB55ɗ has more to do with science fiction than science. The role of other Protein Phosphatases, including, PP-2A-B'56ɗ and Protein Phosphatase-1 I (PP-1 I ) cannot be ignored. Evidenceis presented and discussed here..

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