Search

ObjectiveMonocyte chemotactic protein-1-induced protein 1 (MCPIP1) is highly expressed in inflamed mucosa of inflammatory bowel disease (IBD) and negatively regulates immune response, while the underlying mechanisms regulating mucosal macrophage functions remain unknown. Here, we investigated the roles of MCPIP1 in modulating the differentiation and functions of intestinal macrophages in the pathogenesis of IBD.DesignScRNA-seq was used to cluster the monocyte/macrophage lineage from macrophage-specificMcpip1-deficient (Mcpip1∆Mye) mice andMcpip1fl/fllittermates. The differentially expressed genes were confirmed by RNA-seq, luciferase assay, CUT&Tag assay and Western blotting. Effects of MCPIP1 and the activating transcription factor 3 (ATF3)-AP1S2 axis were assessed in patients with IBD.ResultsMcpip1∆Myemice developed more severe dextran sulfate sodium (DSS)-induced colitis characterised by an increase in macrophage migratory capacity and M1 macrophage polarisation but a decrease in the monocyte-to-macrophage maturation in gut mucosa compared with their littermates. ScRNA-seq unravelled a proinflammatory population (Ccr2+Il-1β+Tlr2+Cx3cr1−Cd163−Mrc1−Ly6c+) of the monocyte/macrophage lineage from lamina propria CD11b+cells and an arrest ofMcpip1∆Myemonocyte-to-macrophage maturation in an Atf3-Ap1s2 axis-dependent manner. Silencing of Ap1s2 or Atf3 markedly suppressedMcpip1∆Myemacrophage migration, M1-like polarisation, and production of proinflammatory cytokines and chemokines. Notably, in vivo blockage of Ap1s2 ameliorated DSS-induced colitis inMcpip1ΔMyemice through enhancing intestinal macrophage maturation. Furthermore, MCPIP1, ATF3 and AP1S2 were highly expressed in inflamed mucosa of active patients with IBD and blockage of ATF3 or AP1S2 significantly suppressed IBD CD14+-derived M1-like macrophage polarisation and proinflammatory cytokine production.ConclusionsMacrophage-specificMcpip1deficiency polarises macrophages towards M1-like phenotype, arrests macrophage maturation and exacerbates intestinal inflammation in an Atf3-Ap1s2-dependent manner, thus providing novel mechanistic insight into intestinal macrophage functions during IBD.

Inflammatory response is a host-protective mechanism against tissue injury or infections, but also has the potential to cause extensive immunopathology and tissue damage, as seen in many diseases, such as cardiovascular diseases, neurodegenerative diseases, metabolic syndrome and many other infectious diseases with public health concerns, such as Coronavirus Disease 2019 (COVID-19), if failure to resolve in a timely manner. Recent studies have uncovered a superfamily of endogenous chemical molecules that tend to resolve inflammatory responses and re-establish homeostasis without causing excessive damage to healthy cells and tissues. Among these, the monocyte chemoattractant protein-induced protein (MCPIP) family consisting of four members (MCPIP-1, -2, -3, and -4) has emerged as a group of evolutionarily conserved molecules participating in the resolution of inflammation. The focus of this review highlights the biological functions of MCPIP-1 (also known as Regnase-1), the best-studied member of this family, in the resolution of inflammatory response. As outlined in this review, MCPIP-1 acts on specific signaling pathways, in particular NFκB, to blunt production of inflammatory mediators, while also acts as an endonuclease controlling the stability of mRNA and microRNA (miRNA), leading to the resolution of inflammation, clearance of virus and dead cells, and promotion of tissue regeneration via its pleiotropic effects. Evidence from transgenic and knock-out mouse models revealed an involvement of MCPIP-1 expression in immune functions and in the physiology of the cardiovascular system, indicating that MCPIP-1 is a key endogenous molecule that governs normal resolution of acute inflammation and infection. In this review, we also discuss the current evidence underlying the roles of other members of the MCPIP family in the regulation of inflammatory processes. Further understanding of the proteins from this family will provide new insights into the identification of novel targets for both host effectors and microbial factors and will lead to new therapeutic treatments for infections and other inflammatory diseases.

Tetramethylpyrazine (TMP), a prominent ingredient of Chinese herb Ligusticum chuanxiong Hort, is known to suppress neuroinflammation and protect blood-brain barrier (BBB) integrity. We investigated whether monocyte chemotactic protein-induced protein 1 (MCPIP1, also known as Regnase-1), a newly identified zinc-finger protein, plays a role in TMP-mediated anti-inflammation and neuroprotection. Male C57BL/6 mice were subjected to focal cerebral ischemia induced by middle cerebral artery occlusion (MCAO) for 2 h, followed by reperfusion for 24 h. TMP (25 mg/kg or 50 mg/kg) or vehicle was administered intraperitoneally 12 h before and post MCAO. The TMP significantly upregulated MCPIP1 in the ischemic brain tissues and effectively inhibited extravasation of fluorescein isothiocyanate (FITC)-dextran, resulting in attenuation of brain edema. These effects of the TMP were associated with a significant reduction in levels of inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and MMP-9 in the ischemic brain tissues. The TMP upregulated the expression of MCPIP1 in primary cultures of neurons and protected against oxygen–glucose deprivation-induced neuron death, while this neuroprotective effect of TMP was abolished by knockdown of MCPIP1 using MCPIP1-specific siRNA. These results suggest that preservation of BBB integrity by TMP is associated with its anti-inflammatory activity. The effect of TMP is mediated, at least in part, via upregulation of MCPIP1 in the ischemic brain.

Potency assays for vaccine products are an important regulatory requirement, and are used to assess product quality and consistency prior to lot release for clinical testing. Ideally they measure an established correlate of efficacy or protection. In cases where there is no known correlate of protection, however, a functional assay that measures a biological response to a vaccine can be applied as a potency assay. Here we describe an in vitro assay which quantitatively measures human T cell activation as a biological response to the TB vaccine candidate H4-IC31. The Cytokine Secretion Assay (CSA) is based on the ability of peripheral blood mononuclear cells (PBMCs) from a Bacillus Calmette-Guérin (BCG)-vaccinated human donor to process and respond to H4-IC31. The ability of H4-IC31 to stimulate a cellular immune response is measured through the quantification of secreted IFNγ and is reported as relative stimulatory activity (RSA) compared to an in-house reference standard. The CSA is specific to the H4-IC31 vaccine, determines the RSA of H4-IC31 in the range of 50% to 150% of the reference standard, and is stability indicating as it detects differences in RSA between intact and heat treated H4-IC31. Although the CSA does not provide a link to clinical efficacy, it fulfills the critical requirements for a biological potency test to assess TB vaccine candidates and can be used along with biochemical and immunochemical assays to define a product profile during clinical development, while eliminating the use of animals for product testing.

Intellectual disability (ID) corresponds to several neurodevelopmental disorders of heterogeneous origin in which cognitive deficits are commonly associated with abnormalities of dendrites and dendritic spines. These histological changes in the brain serve as a proxy for underlying deficits in neuronal network connectivity, mostly a result of genetic factors. Historically, chromosomal abnormalities have been reported by conventional karyotyping, targeted fluorescence in situ hybridization (FISH), and chromosomal microarray analysis. More recently, cytogenomic mapping, whole-exome sequencing, and bioinformatic mining have led to the identification of novel candidate genes, including genes involved in neuritogenesis, dendrite maintenance, and synaptic plasticity. Greater understanding of the roles of these putative ID genes and their functional interactions might boost investigations into determining the plausible link between cellular and behavioral alterations as well as the mechanisms contributing to the cognitive impairment observed in ID. Genetic data combined with histological abnormalities, clinical presentation, and transgenic animal models provide support for the primacy of dysregulation in dendrite structure and function as the basis for the cognitive deficits observed in ID. In this review, we highlight the importance of dendrite pathophysiology in the etiologies of four prototypical ID syndromes, namely Down Syndrome (DS), Rett Syndrome (RTT), Digeorge Syndrome (DGS) and Fragile X Syndrome (FXS). Clinical characteristics of ID have also been reported in individuals with deletions in the long arm of chromosome 10 (the q26.2/q26.3), a region containing the gene for the collapsin response mediator protein 3 (CRMP3), also known as dihydropyrimidinase-related protein-4 (DRP-4, DPYSL4), which is involved in dendritogenesis. Following a discussion of clinical and genetic findings in these syndromes and their preclinical animal models, we lionize CRMP3/DPYSL4 as a novel candidate gene for ID that may be ripe for therapeutic intervention.

Focal cerebral ischemia can cause blood&ndash, brain barrier (BBB) breakdown, which is implicated in neuroinflammation and progression of brain damage. Monocyte chemotactic protein 1&ndash, induced protein 1 (MCPIP1) is a newly identified zinc-finger protein that negatively regulates inflammatory signaling pathways. We aimed to evaluate the impact of genetic MCPIP1 deletion on BBB breakdown and expression of BBB-related matrix metalloproteinases (MMPs) and tight junction proteins after cerebral ischemia/reperfusion (I/R) using MCPIP1-deficient (MCPIP1&ndash, /&ndash, ) mice. Transient middle cerebral artery occlusion was induced in the MCPIP1&ndash, mice and their wild-type littermates for 2 h followed by reperfusion for 24 h. The degree of BBB breakdown was evaluated by injection of fluorescein isothiocyanate (FITC)-dextran. Quantitative real-time polymerase chain reaction, western blot, and immunohistochemistry were performed to compare the expression of MMPs and claudin-5 and zonula occludens-1 (ZO-1). MCPIP1 deficiency in mice resulted in enhanced leakage of FITC-dextran, increased expression of MMP-9/3, and reduced expression of claudin-5 and ZO-1 in the brain compared to that seen in their wild-type littermates subjected to cerebral I/R. These results demonstrate that absence of MCPIP1 exacerbates cerebral I/R-induced BBB disruption by enhancing the expression of MMP-9/3 and the degradation of claudin-5 and ZO-1, providing novel insights into the mechanisms underlying BBB breakdown after cerebral ischemia/reperfusion

MCP-1-induced protein (MCPIP, also known as Zc3h12a or Regnase-1), a newly identified suppressor of cytokine signaling, is expressed in endothelial cells (ECs). To investigate the role of endothelial MCPIP in vascular homeostasis and function, we deleted the MCPIP gene specifically in ECs using the Cre-LoxP system. EC-specific MCPIP deletion resulted in systemic inflammation, increased vessel permeability, edema, thrombus formation, and premature death in mice. Serum levels of cytokines, chemokines, and biomarkers of EC dysfunction were significantly elevated in these mice. Upon lipopolysaccharide (LPS) challenge, mice with EC-specific MCPIP depletion were highly susceptible to LPS-induced death. When subjected to ischemia, these mice showed defective post-ischemic angiogenesis and impaired blood flow recovery in hind limb ischemia. In aortic ring cultures, the MCPIP-deficient ECs displayed significantly impaired vessel sprouting and tube elongation. Mechanistically, silencing of MCPIP by small interfering RNAs in cultured ECs enhanced NF-&kappa, &Beta, activity and dysregulated synthesis of microRNAs linked with elevated cytokines and biomarkers of EC dysfunction. Collectively, these results establish that constitutive expression of MCPIP in ECs is essential to maintaining endothelial homeostasis and function by serving as a key negative feedback regulator that keeps the inflammatory signaling suppressed.

Mycobacterium tuberculosis (Mtb) causes latent tuberculosis infection in one-third of the world population and remains quiescent in the human body for decades. The dormant pathogen accumulates lipid droplets containing triacylglycerol (TAG). In mammals, perilipin regulates lipid droplet homeostasis but no such protein has been identified in Mtb. We identified an Mtb protein (PPE15) that showed weak amino acid sequence identities with mammalian perilipin-1 and was upregulated in Mtb dormancy. We generated a ppe15 gene-disrupted mutant of Mtb and examined its ability to metabolically incorporate radiolabeled oleic acid into TAG, accumulate lipid droplets containing TAG and develop phenotypic tolerance to rifampicin in two in vitro models of dormancy including a three-dimensional human granuloma model. The mutant showed a significant decrease in the biosynthesis and accumulation of lipid droplets containing TAG and in its tolerance of rifampicin. Complementation of the mutant with a wild-type copy of the ppe15 gene restored the lost phenotypes. We designate PPE15 as mycobacterial perilipin-1 (MPER1). Our findings suggest that the MPER1 protein plays a critical role in the homeostasis of TAG -containing lipid droplets in Mtb and influences the entry of the pathogen into a dormant state.

Background: Vascular endothelial cells (ECs) play an important role in angiogenesis and inflammatory responses. MCPIP (also known as Zc3h12a or Regnase-1), a newly identified suppressor of cytokine signaling, is expressed in endothelial cells. To directly test the role of endothelium-derived MCPIP in cardiovascular pathophysiology, we specifically targeted deletion of the murine MCPIP gene in the endothelium by using the loxP/Cre system. Methods and Results: A floxed MCPIP knock in mouse line was crossbred with an endothelial-specific VEcadherin5-Cre driver mouse line to generate offspring with deletion of the MCPIP gene in ECs (ECKO). Ablation of MCPIP in ECs resulted in systemic inflammation, anemia, splenomegaly, increased vessel permeability, muscle wasting, endothelial dysfunction, thrombus formation, impaired blood perfusion, and reduced lifespan in these mice. Serum levels of cytokines, chemokines, and biomarkers of EC dysfunction were significantly elevated in the ECKO mice, suggesting a hypercytokinemia. These mice also were more susceptible to lipopolysaccharide-induced death. When subjected to ischemia, these mice showed defective post-ischemic angiogenesis and impaired blood flow recovery in hind limb ischemia and stroke models, as well as in wound healing. This effect was associated with an increased cellular infiltration, cytokine expression, and decreased angiogenic factors. Moreover, MCPIP-deficient ECs displayed decreased vascular sprouting and tube elongation in ex vivo aortic ring assay. MCPIP-knockdown in cultured ECs enhanced NF-κB activity and dysregulated synthesis of microRNAs linked with elevated cytokines and biomarkers of EC dysfunction. Conclusions: These data show, for the first time, that constitutive expression of MCPIP in ECs is essential to maintain ECs in a quiescent state by serving as an important negative feedback regulator that keeps the inflammatory signaling suppressed. This provides a novel mechanism for maintaining endothelial homeostasis and function.

Secukinumab, a fully human monoclonal antibody that selectively neutralizes interleukin-17A (IL-17A), has been shown to have significant efficacy in the treatment of moderate to severe psoriasis, psoriatic arthritis and ankylosing spondylitis. Blocking critical mediators of immunity may carry a risk of increased opportunistic infections. Here we present clinical and in vitro findings examining the effect of secukinumab on Mycobacterium tuberculosis infection. We re-assessed the effect of secukinumab on the incidence of acute tuberculosis (TB) and reactivation of latent TB infection (LTBI) in pooled safety data from five randomized, double-blind, placebo-controlled, phase 3 clinical trials in subjects with moderate to severe plaque psoriasis. No cases of TB were observed after 1 year. Importantly, in subjects with a history of pulmonary TB (but negative for interferon-γ release and receiving no anti-TB medication) or positive for latent TB (screened by interferon-γ release assay and receiving anti-TB medication), no cases of active TB were reported. Moreover, an in vitro study examined the effect of the anti-tumor necrosis factor-α (TNFα) antibody adalimumab and secukinumab on dormant M. tuberculosis H37Rv in a novel human three-dimensional microgranuloma model. Auramine-O, Nile red staining and rifampicin resistance of M. tuberculosis were measured. In vitro, anti-TNFα treatment showed increased staining for Auramine-O, decreased Nile red staining and decreased rifampicin resistance, indicative of mycobacterial reactivation. In contrast, secukinumab treatment was comparable to control indicating a lack of effect on M. tuberculosis dormancy. To date, clinical and preclinical investigations with secukinumab found no evidence of increased M. tuberculosis infections.

Secukinumab, a fully human monoclonal antibody that selectively neutralizes interleukin-17A (IL-17A), has been shown to have significant efficacy in the treatment of moderate to severe psoriasis, psoriatic arthritis and ankylosing spondylitis. Blocking critical mediators of immunity may carry a risk of increased opportunistic infections. Here we present clinical and in vitro findings examining the effect of secukinumab on Mycobacterium tuberculosis infection. We re-assessed the effect of secukinumab on the incidence of acute tuberculosis (TB) and reactivation of latent TB infection (LTBI) in pooled safety data from five randomized, double-blind, placebo-controlled, phase 3 clinical trials in subjects with moderate to severe plaque psoriasis. No cases of TB were observed after 1 year. Importantly, in subjects with a history of pulmonary TB (but negative for interferon-γ release and receiving no anti-TB medication) or positive for latent TB (screened by interferon-γ release assay and receiving anti-TB medication), no cases of active TB were reported. Moreover, an in vitro study examined the effect of the anti-tumor necrosis factor-α (TNFα) antibody adalimumab and secukinumab on dormant M. tuberculosis H37Rv in a novel human three-dimensional microgranuloma model. Auramine-O, Nile red staining and rifampicin resistance of M. tuberculosis were measured. In vitro, anti-TNFα treatment showed increased staining for Auramine-O, decreased Nile red staining and decreased rifampicin resistance, indicative of mycobacterial reactivation. In contrast, secukinumab treatment was comparable to control indicating a lack of effect on M. tuberculosis dormancy. To date, clinical and preclinical investigations with secukinumab found no evidence of increased M. tuberculosis infections.

MTB lysine-ɛ-aminotransferase (LAT) was found to play a crucial role in persistence and antibiotic tolerance. LAT serves as a potential target in the management of latent tuberculosis. In present work we attempted to derivatize the benzothiazole lead identified through high throughput virtual screening of Birla Institute of Technology and Science in house database. For Structure activity relationship purpose 22 derivatives were synthesized and characterized. Among synthesized compounds, eight compounds were found to be more efficacious in terms of LAT inhibition when compared to lead compound (IC50 10.38 ± 1.21 µM). Compound 22 exhibits bactericidal action against nutrient starved Mycobacterium tuberculosis (MTB). It also exhibits significant activity in nutrient starvation model (2.9 log folds) and biofilm model (2.3 log folds).

Semaphorin3A (Sema3A) is a secreted type of axon guidance molecule that regulates axon wiring through complexes of neuropilin-1 (NRP1) with PlexinA protein receptors. Sema3A regulates the dendritic branching through tetrodotoxin (TTX)-sensitive retrograde axonal transport of PlexA proteins and tropomyosin-related kinase A (TrkA) complex. We here demonstrate that Nav1.7 (encoded by SCN9A), a TTX-sensitive Na+ channel, by coupling with collapsin response mediator protein 1 (CRMP1), mediates the Sema3A-induced retrograde transport. In mouse dorsal root ganglion (DRG) neurons, Sema3A increased co-localization of PlexA4 and TrkA in the growth cones and axons. TTX treatment and RNAi knockdown of Nav1.7 sustained Sema3A-induced colocalized signals of PlexA4 and TrkA in growth cones and suppressed the subsequent localization of PlexA4 and TrkA in distal axons. A similar localization phenotype was observed in crmp1-/- DRG neurons. Sema3A induced colocalization of CRMP1 and Nav1.7 in the growth cones. The half maximal voltage was increased in crmp1-/- neurons when compared to that in wild type. In HEK293 cells, introduction of CRMP1 lowered the threshold of co-expressed exogenous Nav1.7. These results suggest that Nav1.7, by coupling with CRMP1, mediates the axonal retrograde signaling of Sema3A.

Inflammatory angiogenesis involves the induction of a novel gene ZC3H12A encoding monocyte chemoattractant protein-1 (MCP-1)-induced protein-1 (MCPIP1) that has deubiquitinase and antidicer RNAse activities. If and how these enzymatic activities of MCPIP1 mediate the biological functions of MCPIP1 are unknown. Present studies with human umbilical vein endothelial cells suggest that MCPIP-induced angiogenesis is mediated via hypoxia-inducible factor (HIF-1α), vascular endothelial growth factor (VEGF), and silent information regulator (SIRT-1) induction that results in the inhibition of angiogenesis inhibitor thrombospondin-1. MCPIP1 expression inhibited the production of the antiangiogenic microRNA (miR)-20b and -34a that repress the translation of HIF-1α and SIRT-1, respectively. The RNase-dead MCPIP mutant D141N not only did not induce angiogenesis but also failed to inhibit the production of miR-20b and -34a suggesting that the antidicer RNase activity of MCPIP1 is involved in MCPIP-mediated angiogenesis. Mimetics of miR-20b and -34a inhibited MCPIP1-induced angiogenesis confirming that MCPIP1 suppresses the biogenesis of miR-20b and -34a. Furthermore, our results indicate that MCPIP expression induces nuclear translocation of HIF-1α. We show that under hypoxia angiogenesis is mediated via induction of MCPIP1 and under normoxia, in vitro, MCPIP deubiquitinates ubiquitinated HIF-1α and the stabilized HIF-1α enters the nucleus to promote the transcription of its target genes, cyclooxygenase-2 and VEGF, suggesting that the deubiquitinase activity of MCPIP may also promote angiogenesis. The present results show for the first time that the antidicer RNase activity of MCPIP1 is critical in mediating a biological function of MCPIP, namely angiogenesis.

Monocytic cells enhance neovascularization by releasing proangiogenic mediators and/or by transdifferentiating into endothelial-like cells. However, the mechanisms that govern this transdifferentiation process are largely unknown. Recently, monocyte chemotactic protein-1 (MCP-1)-induced protein (MCPIP) has been identified as a novel CCCH-type zinc-finger protein expressed primarily in monocytic cells. Here, we analyzed whether MCPIP might exert angiogenic effects by promoting differentiation of monocytic cells into endothelial cell (EC)-like phenotype. The expression of MCPIP increased during MCP-1-induced transdifferentiation in human bone marrow mononuclear cells (BMNCs). Knockdown of MCPIP with small interfering RNA (siRNA) abolished MCP-1-induced expression of EC markers Flk-1 and Tie-2 in human BMNCs. BMNCs transfected with MCPIP expression vector displayed EC-like morphology accompanied by downregulation of monocytic markers CD14 and CD11b, upregulation of EC markers Flk-1 and Tie-2, induction of cadherin (cdh)-12 and -19, activation of endoplasmic reticulum (ER) stress, and autophagy. Knockdown of cdh-12 or cdh-19 markedly inhibited MCPIP-induced enhancement of cell attachment and EC-marker expression. Inhibition of ER stress by tauroursodeoxycholate abolished MCPIP-induced expression of EC markers. Inhibition of autophagy by knockdown of Beclin-1 with siRNA or by an autophagy inhibitor 3'-methyladenine inhibited MCPIP-induced expression of EC markers. Expression of MCPIP in BMNCs enhanced uptake of acetylated low-density lipoprotein (acLDL), formation of EC-colony, incorporation of cells into capillary-like structure on Matrigel, and exhibited increased neovascularization in the ischemic hindlimb in mice. These results demonstrate that MCPIP may be an important regulator of inflammatory angiogenesis and provide novel mechanistic insights into the link between MCP-1 and cardiovascular diseases.

In this study, a previously uncharacterized gene (Rv0447c) of Mycobacterium tuberculosis, designated as an unknown fatty-acid methyltransferase (ufaA1), was cloned, expressed in Escherichia coli, and purified. The biochemical characterization of the purified protein (UfaA1) showed it to be a methyltransferase that catalyzes biosynthesis of the tuberculostearic acid (10-methylstearic-acid, TSA), a significant constituent lipid of the mycobacterial cell wall and a clinical marker of the disease. Here, we show that UfaA1 transfers the methyl group from S-adenosyl-l-methionine (SAM) to the double bond of oleic acid in phosphatidylethanolamine or phosphatidylcholine to produce TSA. Optimal activity was obtained between pH 7.0 and pH 8.0. The methyltransferase activity of UfaA1 was severely inhibited by S-adenosyl-l-homocysteine. The Km values for dioleyl phosphatidylethanolamine, SAM, and nicotinamide adenine dinucleotide phosphate were 14, 13, and 83 µM, respectively, with Vmax of 1.3-1.6 nmol/Min. These results identify the Rv0447c gene product of M. tuberculosis as the methyltransferase that catalyzes the biosynthesis of TSA. This provides new information in mycobacterial cell wall synthesis.

Collapsin response mediator protein 2, CRMP2, has been identified as an intracellular signaling mediator for Semaphorin 3A (Sema3A). CRMP2 plays a key role in axon guidance, dendritic morphogenesis, and cell polarization. It has been also implicated in a variety of neurological and psychiatric disorders. However, the in vivo functions of CRMP2 remain unknown. We generated CRMP2 gene-deficient (crmp2(-/-) ) mice. The crmp2(-/-) mice showed irregular development of dendritic spines in cortical neurons. The density of dendritic spines was reduced in the cortical layer V pyramidal neurons of crmp2(-/-) mice as well as in those of sema3A(-/-) and crmp1(-/-) mice. However, no abnormality was found in dendritic patterning in crmp2(-/-) compared to wild-type (WT) neurons. The level of CRMP1 was increased in crmp2(-/-) , but the level of CRMP2 was not altered in crmp1(-/-) compared to WT cortical brain lysates. Dendritic spine density and branching were reduced in double-heterozygous sema3A(+/-) ;crmp2(+/-) and sema3A(+/-) ;crmp1(+/-) mice. The phenotypic defects had no genetic interaction between crmp1 and crmp2. These findings suggest that both CRMP1 and CRMP2 mediate Sema3A signaling to regulate dendritic spine maturation and patterning, but through overlapping and distinct signaling pathways.

Cytoskeletal proteins, and the molecules that control their assembly/disassembly, regulate neurite and spine growth and retraction, which are necessary for normal brain function. The neuron begins as a spherical shape, then lamellipodia and filopodia form nascent neurites which differentiate into dendrites and axons. These processes, also involved in neuronal plasticity, require changes in the dynamics of the cytoskeleton proteins and their intracellular binding partners, including collapsin-response mediator proteins (CRMPs). Abnormal changes in CRMP signaling induce structural/ functional abnormalities in neurons which are characteristics of various neurological disorders. Modulation of these pathways may represent unexplored areas for treatment of these diseases.

Major diseases such as cardiovascular diseases, rheumatoid arthritis, diabetes, obesity and tumor growth are known to involve inflammation. Inflammatory molecules such as MCP-1, TNF-α, IL-1β and IL-8 are known to promote angiogenesis. MCP-induced protein (MCPIP), originally discovered as a novel zinc finger protein induced by MCP-1, is also induced by other inflammatory agents. MCPIP was shown to mediate MCP-1-induced angiogenesis. Whether angiogenesis induced by other inflammatory agents is mediated via MCPIP is unknown and the molecular mechanisms involved in angiogenesis induced by MCPIP have not been elucidated. The aim of this study was to bridge this gap and delineate the sequential processes involved in angiogenesis mediated via MCPIP. siRNA knockdown of MCPIP was used to determine whether different inflammatory agents, MCP-1, TNF-α, IL-1β and IL-8, mediate angiogenesis via MCPIP in human umbilical vein endothelial cells (HUVECs). Chemical inhibitors and specific gene knockdown approach were used to inhibit each process postulated. Oxidative stress was inhibited by apocynin or cerium oxide nanoparticles or knockdown of NADPH oxidase subunit, phox47. Endoplasmic reticulum (ER) stress was blocked by tauroursodeoxycholate or knockdown of ER stress signaling protein IRE-1 and autophagy was inhibited by the use of 3'methyl adenine, or LY 294002 or by specific knockdown of beclin1. Matrigel assay was used as a tool to study angiogenic differentiation induced by inflammatory agents or MCPIP overexpression in HUVECs. Tube formation induced by inflammatory agents, TNF-α, IL-1β, IL-8 and MCP-1 was inhibited by knockdown of MCPIP. Forced MCPIP-expression induced oxidative stress, ER stress, autophagy and angiogenic differentiation in HUVECs. Inhibition of each step caused inhibition of each subsequent step postulated. The results reveal that angiogenesis induced by inflammatory agents is mediated via induction of MCPIP that causes oxidative and nitrosative stress resulting in ER stress leading to autophagy required for angiogenesis. The sequence of events suggested to be involved in inflammatory angiogenesis by MCPIP could serve as possible targets for therapeutic intervention of angiogenesis-related disorders.

Osteoclasts (OCs) are responsible for bone resorption in inflammatory joint diseases. Monocyte chemotactic protein-1 (MCP-1) has been shown to induce differentiation of monocytes to OC precursors, but nothing is known about the underlying mechanisms. Here, we elucidate how MCPIP, induced by MCP-1, mediates this differentiation. Knockdown of MCPIP abolished MCP-1-mediated expression of OC markers, tartrate-resistant acid phosphatase, and serine protease cathepsin K. Expression of MCPIP induced p47(PHOX) and its membrane translocation, reactive oxygen species formation, and induction of endoplasmic reticulum (ER) stress chaperones, up-regulation of autophagy marker, Beclin-1, and lipidation of LC3, and induction of OC markers. Inhibition of oxidative stress attenuated ER stress and autophagy, and suppressed expression of OC markers. Inhibition of ER stress by a specific inhibitor or by knockdown of IRE1 blocked autophagy and induction of OC markers. ER stress inducers, tunicamycin and thapsigargin, induced expression of OC markers. Autophagy inhibition by 3'-methyladenine, LY294002, wortmannin or by knockdown of Beclin-1 or Atg 7 inhibited MCPIP-induced expression of OC markers. These results strongly suggest that MCP-1-induced differentiation of OC precursor cells is mediated via MCPIP-induced oxidative stress that causes ER stress leading to autophagy, revealing a novel mechanistic insight into the role of MCP-1 in OCs differentiation.

Cigarette smoke contains and generates a large amount of reactive oxygen species (ROS) that affect normal cellular function and have pathogenic consequences in the cardiovascular system. Increased oxidative stress and inflammation are considered to be an important mechanism of cardiovascular injury induced by cigarette smoke. Antioxidants may serve as effective therapeutic agents against smoke-related cardiovascular disease. Because of the presence of oxygen vacancies on its surface and self-regenerative cycle of its dual oxidation states, Ce(3+) and Ce(4+), cerium oxide (CeO(2)) nanoparticles offer a potential to quench ROS in biological systems. In this study, we determined the ability of CeO(2) nanoparticles to protect against cigarette smoke extract (CSE)-induced oxidative stress and inflammation in cultured rat H9c2 cardiomyocytes. CeO(2) nanoparticles pretreatment of H9c2 cells resulted in significant inhibition of CSE-induced ROS production and cell death. Pretreatment of H9c2 cells with CeO(2) nanoparticles suppressed CSE-induced phosphorylation of IκBα, nuclear translocation of p65 subunit of nuclear factor-κB (NF-κB), and NF-κB reporter activity in H9c2 cells. CeO(2) nanoparticles pretreatment also resulted in a significant down-regulation of NF-κB-regulated inflammatory genes tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and inducible nitric-oxide synthase and further inhibited CSE-induced depletion of antioxidant enzymes, such as copper zinc superoxide dismutase, manganese superoxide dismutase, and intracellular glutathione content. These results indicate that CeO(2) nanoparticles can inhibit CSE-induced cell damage via inhibition of ROS generation, NF-κB activation, inflammatory gene expression, and antioxidant depletion and may have a great potential for treatment of smoking-related diseases.

It is widely recognized that biofuel production from lignocellulosic materials is limited by inadequate technology to efficiently and economically release fermentable sugars from the complex multi-polymeric raw materials. Therefore, endoglucanases, exoglucanase, pectate lyases, cutinase, swollenin, xylanase, acetyl xylan esterase, beta glucosidase and lipase genes from bacteria or fungi were expressed in Escherichia coli or tobacco chloroplasts. A PCR-based method was used to clone genes without introns from Trichoderma reesei genomic DNA. Homoplasmic transplastomic lines showed normal phenotype and were fertile. Based on observed expression levels, up to 49, 64 and 10, 751 million units of pectate lyases or endoglucanase can be produced annually, per acre of tobacco. Plant production cost of endoglucanase is 3100-fold, and pectate lyase is 1057 or 1480-fold lower than the same recombinant enzymes sold commercially, produced via fermentation. Chloroplast-derived enzymes had higher temperature stability and wider pH optima than enzymes expressed in E. coli. Plant crude-extracts showed higher enzyme activity than E. coli with increasing protein concentration, demonstrating their direct utility without purification. Addition of E. coli extracts to the chloroplast-derived enzymes significantly decreased their activity. Chloroplast-derived crude-extract enzyme cocktails yielded more (up to 3625%) glucose from filter paper, pine wood or citrus peel than commercial cocktails. Furthermore, pectate lyase transplastomic plants showed enhanced resistance to Erwina soft rot. This is the first report of using plant-derived enzyme cocktails for production of fermentable sugars from lignocellulosic biomass. Limitations of higher cost and lower production capacity of fermentation systems are addressed by chloroplast-derived enzyme cocktails.

A previously unappreciated deubiquitinase activity of MCP-induced protein 1 contributes to its role in dampening inflammatory signaling., The intensity and duration of macrophage-mediated inflammatory responses are controlled by proteins that modulate inflammatory signaling pathways. MCPIP1 (monocyte chemotactic protein–induced protein 1), a recently identified CCCH Zn finger–containing protein, plays an essential role in controlling macrophage-mediated inflammatory responses. However, its mechanism of action is poorly understood. In this study, we show that MCPIP1 negatively regulates c-Jun N-terminal kinase (JNK) and NF-κB activity by removing ubiquitin moieties from proteins, including TRAF2, TRAF3, and TRAF6. MCPIP1-deficient mice spontaneously developed fatal inflammatory syndrome. Macrophages and splenocytes from MCPIP1−/− mice showed elevated expression of inflammatory gene expression, increased JNK and IκB kinase activation, and increased polyubiquitination of TNF receptor–associated factors. In vitro assays directly demonstrated the deubiquitinating activity of purified MCPIP1. Sequence analysis together with serial mutagenesis defined a deubiquitinating enzyme domain and a ubiquitin association domain in MCPIP1. Our results indicate that MCPIP1 is a critical modulator of inflammatory signaling.

Many of the major diseases, including cardiovascular disease, are widely recognized as inflammatory diseases. MCP-1 (monocyte chemotactic protein-1) plays a critical role in the development of cardiovascular diseases. MCP-1, by its chemotactic activity, causes diapedesis of monocytes from the lumen to the subendothelial space where they become foam cells, initiating fatty streak formation that leads to atherosclerotic plaque formation. Inflammatory macrophages probably play a role in plaque rupture and the resulting ischaemic episode as well as restenosis after angioplasty. There is strong evidence that MCP-1 plays a major role in myocarditis, ischaemia/reperfusion injury in the heart and in transplant rejection. MCP-1 also plays a role in cardiac repair and manifests protective effects under certain conditions. Such protective effects may be due to the induction of protective ER (endoplasmic reticulum) stress chaperones by MCP-1. Under sustained ER stress caused by chronic exposure to MCP-1, the protection would break down resulting in the development of heart failure. MCP-1 is also involved in ischaemic angiogenesis. The recent advances in our understanding of the molecular mechanisms that might be involved in the roles that MCP-1 plays in cardiovascular disease are reviewed. The gene expression changes induced by the signalling events triggered by MCP-1 binding to its receptor include the induction of a novel zinc-finger protein called MCPIP (MCP-1-induced protein), which plays critical roles in the development of the pathophysiology caused by MCP-1 production. The role of the MCP-1/CCR2 (CC chemokine receptor 2) system in diabetes, which is a major risk factor for cardiovascular diseases, is also reviewed briefly. MCP-1/CCR2- and/or MCPIP-targeted therapeutic approaches to intervene in inflammatory diseases, including cardiovascular diseases, may be feasible.

Previously it has been reported that neural stem cells undergoing apoptotic stress have increased levels of Amyloid precursor protein (APP) and increased APP expression results in glial differentiation. APP activity was also shown to be required for staurosporine induced glial differentiation of neuroprogenitor cells. Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that is expressed early during inflammation. The binding of MCP-1 to its chemokine receptor induces expression of novel transcription factor MCP-1 induced protein (MCPIP). MCPIP expression subsequently leads to cell death. Previous studies have shown that pro apoptotic factors have the ability to induce neural differentiation. Therefore, we investigated if MCPIP expression leads to differentiation of NT2 neuroprogenitor cells. Results showed that MCPIP expression increased glial fibrillary acid protein (GFAP) expression and also caused distinct morphological changes, both indicative of glial differentiation. Similar results were observed with MCP-1 treatment. Interestingly, APP expression decreased in response to MCPIP. Instead, we found APP activity regulates expression of both MCP-1 and MCPIP. Furthermore, inhibition of either p38 MAPK or JAK signaling pathways significantly reduced APP’s effect on MCP-1 and MCPIP. These data demonstrates the role APP has in glial differentiation of NT2 cells through MCP-1/MCPIP signaling. It is possible that increased APP expression after CNS injury could play a role in MCP-1 production, possibly promoting astrocyte activation at injured site.

Doxorubicin (Dox) is known to cause cardiomyopathy and congestive heart failure upon chronic administration. The mechanisms underlying these toxicities remain uncertain but have been attributed, at least in part, by induction of cardiac cell apoptosis. Fas ligation with its cognate ligand (FasL) induces apoptosis and activates cellular inflammatory responses associated with tissue injury. We determined whether interruption of Fas/FasL interaction by cardiac-targeted expression of soluble Fas (sFas), a competitive inhibitor of FasL, would protect against Dox chronic cardiotoxicity in mice. Wild-type (WT) and sFas transgenic mice were administrated intravenously with 4 mg/kg Dox or with an equivalent volume of saline twice a week for a total of 10 injections. There were 25% mortality in WT mice, but no death was observed in sFas mice during the period of Dox treatment. Echocardiographic evaluation revealed a significant decrease in left ventricle fractional shortening after Dox treatment in WT mice but not in sFas mice. WT mice treated with Dox developed extensive myocardial cytoplasmic vacuolization, apoptosis, and interstitial fibrosis, which were much less or absent in sFas mice. The increased inducible nitric oxide synthase expression, nitric oxide production, superoxide generation, and peroxynitrite formation after Dox treatment in WT mice were attenuated by sFas expression. sFas expression also attenuated Dox-mediated induction of proinflammatory cytokines, tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 in the myocardium. These observations indicate that FasL is an important mediator in Dox-associated cardiotoxicity by generating reactive oxygen and nitrogen species.

This is a pre-copy-editing, author-produced PDF of an article accepted for publication in CARDIOVASCULAR RESEARCH 78 (3): 554-562, 2008 following peer review. The definitive publisher-authenticated version is available online at:http://cardiovascres.oxfordjournals.org/cgi/content/abstract/78/3/554., Article, CARDIOVASCULAR RESEARCH. 78(3): 554-562 (2008)

The mechanisms responsible for myocardial dysfunction in the setting of sepsis remain undefined. Fas ligation with its cognate ligand (FasL) induces apoptosis and activates cellular inflammatory responses associated with tissue injury. We determined whether interruption of Fas/FasL interaction by cardiac-specific expression of soluble Fas (sFas), a competitive inhibitor of FasL, would improve myocardial dysfunction and inflammation in a lipopolysaccharide (LPS)-induced mouse model of sepsis. Wild-type (WT) and sFas transgenic mice were injected intraperitoneally with 10 mg/kg LPS or with an equivalent volume of saline. At 18 h after LPS administration, echocardiographic evaluation revealed a significant decrease in left ventricular fractional shortening in the WT mice, whereas the fractional shortening was preserved in the sFas mice. Activation of nuclear factor-kappa B (NF-kappaB) and the increase in the transcript levels of proinflammatory cytokines, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6 resulting from LPS treatment were attenuated in the myocardium of sFas mice. sFas expression also inhibited LPS-induced upregulation of Toll-like receptor 4 (TLR-4) and inducible nitric oxide synthase (iNOS), and formation of peroxynitrite in the myocardium. LPS-induced increase in caspase-3/7 activity and apoptotic cell death were suppressed in sFas mice compared with WT mice. LPS-induced lung injury and increase in lung water content were also significantly reduced in sFas mice. These data indicate that neutralization of FasL by expression of sFas significantly preserves cardiac function and reduces inflammatory responses in the heart, suggesting that Fas/FasL signaling pathway is important in mediating the deleterious effects of LPS on myocardial function.

Collapsin response mediator protein 1 (CRMP1) is one of the CRMP family members that mediates signal transduction of axonal guidance and neuronal migration. We show here evidence that CRMP1 is involved in semaphorin3A (Sema3A)-induced spine development in the cerebral cortex. In the cultured cortical neurons fromcrmp1+/−mice, Sema3A increased the density of clusters of synapsin I and postsynaptic density-95, but this increase was markedly attenuated incrmp1−/−mice. This attenuation was also seen incyclin-dependent kinase 5(cdk5)−/−neurons. Furthermore, the introduction of wild-type CRMP1 but not CRMP1-T509A/S522A, (Thr 509 and Ser 522 were replaced by Ala), a mutant that cannot be phosphorylated by Cdk5, intocrmp1−/−neurons rescued the defect in Sema3A responsiveness. The Golgi-impregnation method showed that thecrmp1−/−layer V cortical neurons showed a lower density of synaptic bouton-like structures and that this phenotype had genetic interaction withsema3A. These findings suggest that Sema3A-induced spine development is regulated by phosphorylation of CRMP1 by Cdk5.

In vitro studies have pointed to the collapsin response mediator proteins (CRMPs) as key regulators of neurite outgrowth and axonal differentiation. CRMP3 is expressed mostly in the nervous system during development but remains at high levels in the hippocampus of adults. To explore CRMP3 function in vivo, we generated mice with targeted disruption of the CRMP3 gene. Immunohistochemistry and Golgi staining of CA1 showed abnormal dendrite and spine morphogenesis in the hippocampus of CRMP3-deficient mice. Apical dendrites displayed an increase in undulation and a reduction in length and branching points. Basal dendrites also exhibited a reduction in length with an alteration in soma stem distribution and an increased number of thick dendrites localized in stratum oriens (SO). Long-term potentiation (LTP) was impaired in this area. These data indicate an important role for CRMP3 in dendrite arborization, guide-posts navigation, and neuronal plasticity.—Quach, T. T., Massicotte, G., Belin, M. F., Honnorat, J...

Objective: Cerium oxide (CeO2) nanoparticles have been shown to protect cells in culture from lethal stress, but no protection in vivo has been reported. Cardiac-specific expression of monocyte chemoattractant protein (MCP)-1 in mice causes ischemic cardiomyopathy associated with activation of endoplasmic reticulum (ER) stress. The aim of this study was to assess the effects of CeO2 nanoparticles on cardiac function and remodeling as well as ER stress response in this murine model of cardiomyopathy. Methods: MCP-1 transgenic mice (MCP mice) and wild-type controls were administered intravenously 15 nmol of CeO2 nanoparticles or vehicle only twice a week for 2 weeks. Cardiac function, myocardial histology, nitrotyrosine formation, expression of cytokines, and ER stress-associated genes were evaluated. Results: Treatment with CeO2 nanoparticles markedly inhibited progressive left ventricular dysfunction and dilatation in MCP mice and caused a significant decrease in serum levels of MCP-1, C-reactive protein, and total nitrated proteins. The infiltration of monocytes/macrophages, accumulation of 3-nitrotyrosine, apoptotic cell death, and expression of proinflammatory cytokines, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in the myocardium were markedly inhibited by CeO2 nanoparticles. Expression of the key ER stress-associated genes, including glucose-regulated protein 78 (Grp78), protein disulfide isomerase (PDI), and heat shock proteins (HSP25, HSP40, HSP70), were also suppressed by CeO2 nanoparticles. Conclusions: CeO2 nanoparticles protect against the progression of cardiac dysfunction and remodeling by attenuation of myocardial oxidative stress, ER stress, and inflammatory processes probably through their autoregenerative antioxidant properties.

Macrophages respond to environmental cues by changing gene expression to assume different states of activation. The activator-dependent states include M1, induced by LPS and IFNγ and M2, induced by IL-4 and IL-13. Macrophages exist in a full spectrum of states of activation between M1 and M2. M1 macrophages produce inflammatory cytokines whereas M2 macrophages produce anti-inflammatory IL-10 and TGFβ. A variety of transcription factors regulate transcription programs that control M1/M2 polarization. Recent findings indicate that IL-4 induces KLF4 via activation of STAT6. KLF4 and STAT6 induce each other and cooperatively induce M2 markers. Transcription factors must use catalytic proteins to implement their biological functions. We discovered that KLF4 induce a novel zinc finger protein with dual catalytic activities, called MCPIP, which mediates M2 polarization. The role of MCPIP in vivo was demonstrated with macrophages from mice with myeloid cell targeted knockout or over expression of MCPIP. ROS production, ER stress and autophagy, required for M2 polarization, was sequentially induced by MCPIP expression in murine macrophages. With MCPIP mutants, that have only ubiquitinase activity or RNase activity, it was demonstrated that both catalytic activities are involved in M2 polarization. MCPIP modulation of the miRome and ubiquitome involved in macrophage polarization remains obscure. Involvement of macrophage polarization in many major diseases suggest the possibilities of novel therapeutics that can modulate MCPIP functions to control macrophage polarization.

Collapsin response mediator protein 3 increases the dendritic arborization of hippocampal neurons

MCP-1-induced protein (MCPIP, also known as ZC3H12A) has recently been uncovered to act as a negative regulator of inflammation. Expression of MCPIP was elevated in the ventricular myocardium of patients with ischemic heart failure. However, the role of MCPIP in the development of post-infarct cardiac inflammation and remodeling is unknown. The objective of the present study was to investigate whether MCPIP exerts an inhibitory effect on the cardiac inflammatory response and adverse remodeling after myocardial infarction (MI). Mice with cardiomyocyte-specific expression of MCPIP and their wild-type littermates (FVB/N) were subjected to permanent ligation of left coronary artery. The levels of MCPIP were significantly increased in the ischemic myocardium and sustained for 4 weeks after MI. Acute infarct size was comparable between groups. However, constitutive overexpression of MCPIP in the murine heart resulted in improved survival rate, decreased cardiac hypertrophy, less of fibrosis and scar formation, and better cardiac performance at 28 days after MI, along with a markedly reduced monocytic cell infiltration, less cytokine expression, decreased caspase-3/7 activities and apoptotic cell death compared to the wild-type hearts. Cardiomyocyte-specific expression of MCPIP also attenuated activation of cardiac NF-κB signaling and expression of inflammation-associated microRNAs (miR-126, -146a, -155, and -199a) when compared with the post-infarct wild-type hearts. In vitro, MCPIP expression suppressed hypoxia-induced NF-κB-luciferase activity in cardiomyocytes. In conclusion, MCPIP expression in the ischemic myocardium protects against adverse cardiac remodeling and dysfunction following MI by modulation of local myocardial inflammation, possibly through mitigating NF-κB signaling and suppressing inflammation-associated microRNA expression.

Collapsin response mediator protein 1 (CRMP1) is one of the CRMP family members that mediates signal transduction of axon guidance molecules. Here, we show evidence that CRMP1 is involved in Reelin (Reln) signaling to regulate neuronal migration in the cerebral cortex. Incrmp1−/−mice, radial migration of cortical neurons was retarded. This phenotype was not observed in thesema3A−/−andcrmp1+/−;sema3A+/−cortices. However, CRMP1 was colocalized with disabled-1 (Dab1), an adaptor protein in Reln signaling. In theRelnrl/rlcortex, CRMP1 and Dab1 were expressed at a higher level, yet tyrosine phosphorylated at a lower level. Loss ofcrmp1in adab1heterozygous background led to the disruption of hippocampal lamination, a Reeler-like phenotype. In addition to axon guidance, CRMP1 regulates neuronal migration by mediating Reln signaling.

Collapsin response mediator proteins (CRMPs) consist of five homologous cytosolic proteins that participate in signal transduction involved in a variety of physiological events. CRMP1 is highly expressed during brain development; however, its functions remains unclear. To gain insight into its function, we generated CRMP1(-/-) mice with a knock-in LacZ gene. No gross anatomical changes or behavioral alterations were observed. Expression of CRMP1 was examined by the expression of the knocked-in LacZ gene, in situ hybridization with riboprobes and by imunohistochemistry. CRMP1 was found to be highly expressed in the developing the cerebellum, olfactory bulbs, hypothalamus and retina. In adults, expression level was high in the olfactory bulbs and hippocampus but very low in the retina and cerebellum and undetectable in hypothalamus. To study potential roles of CRMP1, we focused on cerebellum development. CRMP1(-/-) mice showed a decrease in the number of granule cells migrating out of explants of developing cerebellum, as did treatment of the explants from normal mice with anti-CRMP1 specific antibodies. CRMP1(-/-) mice showed a decrease in granule cell proliferation and apoptosis in external granule cell layers in vivo. Adult cerebellum of CRMP1(-/-) did not show any abnormalities.

Endoplasmic reticulum (ER) stress has been found to be associated with neurodegenerative diseases and diabetes mellitus. Whether ER stress is involved in the development of heart disease is not known. Cardiac-specific expression of monocyte chemoattractant protein-1 (MCP-1) in mice causes the development of ischemic heart disease. Here we report that microarray analysis of gene expression changes in the heart of these transgenic mice revealed that a cluster of ER stress-related genes was transcriptionally activated in the heart during the development of ischemic heart disease. The gene array results were verified by quantitative real-time PCR that showed highly elevated transcript levels of genes involved in unfolded protein response such as ER and cytoplasmic chaperones, oxidoreductases, protein disulfide isomerase (PDI) family, and ER-associated degradation system such as ubiquitin. Immunoblot analysis confirmed the expression of chaperones, PDI, and ubiquitin. Immunohistochemical analyses showed that ER stress proteins were associated mainly with the degenerating cardiomyocytes. A novel ubiquitin fold modifier (Ufm1) that has not been previously associated with ER stress and not found to be induced under any condition was also found to be upregulated in the hearts of MCP mice (transgenic mice that express MCP-1 specifically in the heart). The present results strongly suggest that activation of ER stress response is involved in the development of ischemic heart disease in this murine model.

Objective: Infiltrating inflammatory cells within the myocardium have been shown to be apoptotic, but the significance of apoptotic inflammatory cells to the development of cardiomyopathy remains undefined. Transgenic mice with cardiac-specific expression of MCP-1 exhibit extensive apoptosis of infiltrating mononuclear cells and develop heart failure. Here, we tested the hypothesis that in vivo selective inhibition of apoptosis of infiltrating mononuclear cells would preserve cardiac structure and function and improve survival in this murine model. Methods: Mice with cardiac-specific expression of MCP-1 and monocyte-specific expression of Bcl-2 were generated by cross-breeding MCP-1 transgenic mice with hMRP8-Bcl-2 mice that over-express Bcl-2 in the monocytes. Structural and functional parameters and the inflammatory response of the heart were evaluated and compared between the wild-type and transgenic mice. Results: Expression of Bcl-2 in monocytes results in superior preservation of myocardial structure, cardiac function and a significant prolongation of survival of MCP-1 transgenic mice. The beneficial effects of monocyte-specific Bcl-2 expression are associated with inhibition of apoptosis of infiltrating mononuclear cells, normalization of circulating C-reactive protein levels, attenuation of cellular infiltrates, macrophage activation and production of proinflammatory cytokines, tumor necrosis factor (TNF-a), interleukin (IL)-1h and IL-6 in the hearts. Conclusions: These results demonstrate that apoptosis of infiltrating mononuclear cells plays a detrimental role in the development of heart failure in this murine model, suggesting that modulation of apoptosis of infiltrating mononuclear cells may be of clinical benefit in heart failure.

Monocyte chemoattractant protein-1 (MCP-1; CCL2)–mediated inflammation plays a critical role in the development of ischemic heart disease (IHD). However, the gene expression changes caused by signal transduction, triggered by MCP-1 binding to its receptor CCR2, and their possible role in the development of IHD are not understood. We present evidence that MCP-1 binding to CCR2 induces a novel transcription factor (MCP-induced protein [MCPIP]) that causes cell death. Gene microarray analysis showed that when expressed in hiuman embryonic kidney 293 cells, MCPIP induced apoptotic gene families before causing cell death. Mutagenesis studies showed that the structural features required for transcription factor–like activity were also required for causing cell death. Activation of caspase-3 was detected after MCPIP transfection and Z-VAD-fmk partially inhibited cell death. Cardiomyocyte-targeted expression of MCP-1 in mice caused death by heart failure at 6 months of age. MCPIP expression increased in parallel with the development of ventricular dysfunction. In situ hybridization showed the presence of MCPIP transcripts in the cardiomyocytes and immunohistochemistry showed that MCPIP was associated with the cardiomyocyte nuclei of apoptotic cardiomyocytes. CCR2 expression in cardiomyocytes increased with the development of IHD. MCPIP production induced by MCP-1 binding to CCR2 in the cardiomyocytes is probably involved in the development of IHD in this murine model. MCPIP transcript levels were much higher in the explanted human hearts with IHD than with nonischemic heart disease. These results provide a molecular insight into how chronic inflammation and exposure to MCP-1 contributes to heart failure and suggest that MCPIP could be a potential target for therapeutic intervention.

We show that the disruption of one of the mycocerosic acid synthase ( mas )-like genes, msl5 ( pks8 plus pks17 ) in Mycobacterium tuberculosis H37Rv generates a mutant incapable of producing monomethyl branched unsaturated C 16 to C 20 fatty acids that are minor constituents of acyltrehaloses and sulfolipids. The msl5 mutation did not cause any significant change in the acyl lipid composition and also did not affect growth in culture, in mouse alveolar macrophage cell line MH-S, or in the murine lung.

Lipids that are found only in the cell envelope of pathogenic mycobacteria, such as those containing multiple methyl-branched fatty acids, have long been thought to play a role in pathogenesis. Among these complex lipids, sulfolipids have been the most extensively studied over the last 50 years. The numerous biological effects exhibited by purified sulfolipids on phagocytic cells led to the idea that these molecules are probably important virulence factors facilitating the intracellular survival ofMycobacterium tuberculosis. However, definitive evidence to support this concept has been lacking. The recent construction of an isogenic sulfolipid-deficient mutant ofM. tuberculosisH37Rv (Sirakova et al., J. Biol. Chem.276:16833-16839, 2001) has for the first time provided the opportunity to directly assess the contribution of these complex lipids to pathogenesis. In the present study, we show that against all expectations, sulfolipid deficiency does not significantly affect the replication, persistence, and pathogenicity ofM. tuberculosisH37Rv in mice and guinea pigs or in cultured macrophages.

The cell wall lipids in Mycobacterium tuberculosis are probably involved in pathogenesis. The largest open reading frame in the genome of M. tuberculosis H37Rv, pks12 , is unique in that it encodes two sets of domains needed to produce fatty acids. A pks12 -disrupted mutant was produced, and disruption was confirmed by both PCR analysis and Southern blotting. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that a 430-kDa protein band present in the wild type was missing in the mutant. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MS) and liquid chromatography (LC)-MS analysis of tryptic peptides showed that 54 peptides distributed throughout this protein matched the pks12- encoded sequence. Biochemical analysis using [1- 14 C]propionate as the radiotracer showed that the pks12 mutant was deficient in the synthesis of dimycocerosyl phthiocerol (DIM). SDS-PAGE, immunoblot analysis of proteins, and analysis of fatty acids showed that the mutant can produce mycocerosic acids. Thus, the pks12 gene is probably involved in the synthesis of phthiocerol, the diol required for DIM synthesis. Growth of the pks12 mutant was attenuated in mouse alveolar macrophage cell line MH-S, and the virulence of the mutant in vivo was highly attenuated in a murine model. Thus, pks12 probably participates in DIM production and its expression is involved in pathogenesis.

The cell envelope of pathogenic mycobacteria is highly distinctive in that it contains a large number of structurally related very long multiple methyl-branched fatty acids. These complex molecules are thought to play important roles in cell envelope organization and virulence. The genetic and enzymic characterization of the polyketide synthase Mas, which is responsible for the synthesis of one such family of fatty acids (the mycocerosic acids), paved the way towards the identification of other enzymes involved in the synthesis of methyl-branched fatty acids in M. tuberculosis. In an effort to elucidate the origin of these complex fatty acids and their possible involvement in pathogenesis, the two mas-like polyketide genes pks5 and pks7 were disrupted in M. tuberculosis and the effects of their inactivation on fatty acid composition and virulence were analysed. While the disruption of pks7 resulted in a mutant deficient in the production of phthiocerol dimycocerosates, the cell envelope composition of the pks5 mutant was found to be identical to that of the wild-type parental strain M. tuberculosis H37Rv. Interestingly, both the pks5 and pks7 mutants displayed severe growth defects in mice.