MCPIP1 Endoribonuclease Activity Negatively Regulates Interleukin-17-Mediated Signaling and Inflammation.

Interleukin-17 (IL-17) induces pathology in autoimmunity and infections; therefore, constraint of this pathway is an essential component of its regulation. We demonstrate that the signaling intermediate MCPIP1 (also termed Regnase-1, encoded by Zc3h12a) is a feedback inhibitor of IL-17 receptor signal transduction. MCPIP1 knockdown enhanced IL-17-mediated signaling, requiring MCPIP1's endoribonuclease but not deubiquitinase domain. MCPIP1 haploinsufficient mice showed enhanced resistance to disseminated Candida albicans infection, which was reversed in an Il17ra(-/-) background. Conversely, IL-17-dependent pathology in Zc3h12a(+/-) mice was exacerbated in both EAE and pulmonary inflammation. MCPIP1 degraded Il6 mRNA directly but only modestly downregulated the IL-6 promoter. However, MCPIP1 strongly inhibited the Lcn2 promoter by regulating the mRNA stability of Nfkbiz, encoding the IκBζ transcription factor. Unexpectedly, MCPIP1 degraded Il17ra and Il17rc mRNA, independently of the 3' UTR. The cumulative impact of MCPIP1 on IL-6, IκBζ, and possibly IL-17R subunits results in a biologically relevant inhibition of IL-17 signaling.

MCPIP1 restrains mucosal inflammation by orchestrating the intestinal monocyte to macrophage maturation via an ATF3-AP1S2 axis.

OBJECTIVE: Monocyte 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. DESIGN: ScRNA-seq was used to cluster the monocyte/macrophage lineage from macrophage-specific Mcpip1-deficient (Mcpip1 ∆Mye) mice and Mcpip1 fl/fl littermates. 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. RESULTS: Mcpip1 ∆Mye mice 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-1ß+Tlr2+Cx3cr1-Cd163-Mrc1-Ly6c+) of the monocyte/macrophage lineage from lamina propria CD11b+ cells and an arrest of Mcpip1 ∆Mye monocyte-to-macrophage maturation in an Atf3-Ap1s2 axis-dependent manner. Silencing of Ap1s2 or Atf3 markedly suppressed Mcpip1 ∆Mye macrophage migration, M1-like polarisation, and production of proinflammatory cytokines and chemokines. Notably, in vivo blockage of Ap1s2 ameliorated DSS-induced colitis in Mcpip1 ΔMye mice 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. CONCLUSIONS: Macrophage-specific Mcpip1 deficiency 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.

Intellectual disability: dendritic anomalies and emerging genetic perspectives.

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.

Monocyte chemotactic protein-induced protein 1 controls allergic airway inflammation by suppressing IL-5-producing TH2 cells through the Notch/Gata3 pathway.

BACKGROUND: Asthmatic and allergic inflammation is mediated by TH2 cytokines (IL-4, IL-5, and IL-13). Although we have learned much about how TH2 cells are differentiated, the TH2 checkpoint mechanisms remain elusive. OBJECTIVES: In this study we investigate how monocyte chemotactic protein-induced protein 1 (MCPIP1; encoded by the Zc3h12a gene) regulates IL-5-producing TH2 cell differentiation and TH2-mediated inflammation. METHODS: The functions of Zc3h12a-/- CD4 T cells were evaluated by checking the expression of TH2 cytokines and transcription factors in vivo and in vitro. Allergic airway inflammation of Zc3h12a-/- mice was examined with murine asthma models. In addition, antigen-specific CD4 T cells deficient in MCPIP1 were transferred to wild-type recipient mice, challenged with ovalbumin (OVA) or house dust mite (HDM), and accessed for TH2 inflammation. RESULTS: Zc3h12a-/- mice have spontaneous severe lung inflammation, with an increase in mainly IL-5- and IL-13-producing but not IL-4-producing TH2 cells in the lung. Mechanistically, differentiation of IL-5-producing Zc3h12a-/- TH2 cells is mediated through Notch signaling and Gata3 independent of IL-4. Gata3 mRNA is stabilized in Zc3h12a-/- TH2 cells. MCPIP1 promotes Gata3 mRNA decay through the RNase domain. Furthermore, deletion of MCPIP1 in OVA- or HDM-specific T cells leads to significantly increased TH2-mediated airway inflammation in OVA or HDM murine models of asthma. CONCLUSIONS: Our study reveals that MCPIP1 regulates the development and function of IL-5-producing TH2 cells through the Notch/Gata3 pathway. MCPIP1 represents a new and promising target for the treatment of asthma and other TH2-mediated diseases.

Absence of MCP-induced Protein 1 Enhances Blood-Brain Barrier Breakdown after Experimental Stroke in Mice.

Focal cerebral ischemia can cause blood-brain barrier (BBB) breakdown, which is implicated in neuroinflammation and progression of brain damage. Monocyte chemotactic protein 1-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-/-) mice. Transient middle cerebral artery occlusion was induced in the MCPIP1-/- 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.

Essential Role of Endothelial MCPIP in Vascular Integrity and Post-Ischemic Remodeling.

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-κΒ 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 lysine-ɛ-aminotransferase a potential target in dormancy: Benzothiazole based inhibitors.

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±1.21µM). Compound 22 exhibits bactericidal action against nutrient starved Mycobacterium tuberculosis (MTB). It also exhibits significant activity in nutrient starvation model (2.9log folds) and biofilm model (2.3log folds).

Transcription factors STAT6 and KLF4 implement macrophage polarization via the dual catalytic powers of MCPIP.

Macrophage polarization plays a critical role in tissue homeostasis, disease pathogenesis, and inflammation and its resolution. IL-4-induced macrophage polarization involves induction of STAT6 and Krüppel-like factor 4 (KLF4), which induce each other and promote M2 polarization. However, how these transcription factors implement M2 polarization is not understood. We report that in murine macrophages MCP-1-induced protein (MCPIP), induced by KLF4, inhibits M1 polarization by inhibiting NF-κB activation and implements M2 polarization using both its deubiquitinase and RNase activities that cause sequential induction of reactive oxygen species (ROS), endoplasmic reticulum (ER) stress, and autophagy required for M2 polarization. MCPIP also induces C/EBPß and PPARγ, which promote M2 polarization. Macrophages from mice with myeloid-targeted overexpression of MCPIP show elevated expression of M2 markers and reduced response to LPS, whereas macrophages from mice with myeloid-specific deletion of MCPIP manifest elevated M1 polarization with enhanced phagocytic activity. Thus, both in vivo and in vitro experiments demonstrate that the transcription factors STAT6 and KLF4 implement IL-4-induced M2 polarization via the dual catalytic activities of MCPIP.

Mapping CRMP3 domains involved in dendrite morphogenesis and voltage-gated calcium channel regulation.

Although hippocampal neurons are well-distinguished by the morphological characteristics of their dendrites and their structural plasticity, the mechanisms involved in regulating their neurite initiation, dendrite growth, network formation and remodeling are still largely unknown, in part because the key molecules involved remain elusive. Identifying new dendrite-active cues could uncover unknown molecular mechanisms that would add significant understanding to the field and possibly lead to the development of novel neuroprotective therapy because these neurons are impaired in many neuropsychiatric disorders. In our previous studies, we deleted the gene encoding CRMP3 in mice and identified the protein as a new endogenous signaling molecule that shapes diverse features of the hippocampal pyramidal dendrites without affecting axon morphology. We also found that CRMP3 protects dendrites against dystrophy induced by prion peptide PrP(106-126). Here, we report that CRMP3 has a profound influence on neurite initiation and dendrite growth of hippocampal neurons in vitro. Our deletional mapping revealed that the C-terminus of CRMP3 probably harbors its dendritogenic capacity and supports an active transport mechanism. By contrast, overexpression of the C-terminal truncated CRMP3 phenocopied the effect of CRMP3 gene deletion with inhibition of neurite initiation or decrease in dendrite complexity, depending on the stage of cell development. In addition, this mutant inhibited the activity of CRMP3, in a similar manner to siRNA. Voltage-gated calcium channel inhibitors prevented CRMP3-induced dendritic growth and somatic Ca(2+) influx in CRMP3-overexpressing neurons was augmented largely via L-type channels. These results support a link between CRMP3-mediated Ca(2+) influx and CRMP3-mediated dendritic growth in hippocampal neurons.

MCP-induced protein 1 mediates the minocycline-induced neuroprotection against cerebral ischemia/reperfusion injury in vitro and in vivo.

BACKGROUND: Minocycline, a broad-spectrum tetracycline antibiotic, has shown anti-inflammatory and neuroprotective effects in ischemic brain injury. The present study seeks to determine whether monocyte chemotactic protein-induced protein 1 (MCPIP1), a recently identified modulator of inflammatory reactions, is involved in the cerebral neuroprotection conferred by minocycline treatment in the animal model of focal cerebral ischemia and to elucidate the mechanisms of minocycline-induced ischemic brain tolerance. METHODS: Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) for 2 h in male C57BL/6 mice and MCPIP1 knockout mice followed by 24- or 48-h reperfusion. Twelve hours before ischemia or 2 h after MCAO, mice were injected intraperitoneally with 90 mg/kg of minocycline hydrochloride. Thereafter, the animals were injected twice a day, at a dose of 90 mg/kg after ischemia until sacrificed. Transcription and expression of MCPIP1 gene was monitored by quantitative real-time PCR (qRT-PCR), Western blot, and immunohistochemistry. The neurobehavioral scores, infarction volumes, and proinflammatory cytokines in brain and NF-κB signaling were evaluated after ischemia/reperfusion. RESULTS: MCPIP1 protein and mRNA levels significantly increased in mouse brain undergoing minocycline pretreatment. Minocycline treatment significantly attenuated the infarct volume, neurological deficits, and upregulation of proinflammatory cytokines in the brain of wild type mice after MCAO. MCPIP1-deficient mice failed to evoke minocycline-treatment-induced tolerance compared with that of the control MCPIP1-deficient group without minocycline treatment. Similarly, in vitro data showed that minocycline significantly induced the expression of MCPIP1 in primary neuron-glial cells, cortical neurons, and reduced oxygen glucose deprivation (OGD)-induced cell death. The absence of MCPIP1 blocked minocycline-induced protection on neuron-glial cells and cortical neurons treated with OGD. CONCLUSIONS: Our in vitro and in vivo studies demonstrate that MCPIP1 is an important mediator of minocycline-induced protection from brain ischemia.

MCP-1-induced protein attenuates post-infarct cardiac remodeling and dysfunction through mitigating NF-κB activation and suppressing inflammation-associated microRNA expression.

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.

Inflammation, endoplasmic reticulum stress, autophagy, and the monocyte chemoattractant protein-1/CCR2 pathway.

Numerous inflammatory cytokines have been implicated in the pathogenesis of cardiovascular diseases. Monocyte chemoattractant protein (MCP)-1/CCL2 is expressed by mainly inflammatory cells and stromal cells such as endothelial cells, and its expression is upregulated after proinflammatory stimuli and tissue injury. MCP-1 can function as a traditional chemotactic cytokine and also regulates gene transcription. The recently discovered novel zinc-finger protein, called MCPIP (MCP-1-induced protein), initiates a series of signaling events that causes oxidative and endoplasmic reticulum (ER) stress, leading to autophagy that can result in cell death or differentiation, depending on the cellular context. After a brief review of the basic processes involved in inflammation, ER stress, and autophagy, the recently elucidated role of MCP-1 and MCPIP in inflammatory diseases is reviewed. MCPIP was found to be able to control inflammatory response by inhibition of nuclear factor-κB activation through its deubiquitinase activity or by degradation of mRNA encoding a set of inflammatory cytokines through its RNase activity. The potential inclusion of such a novel deubiquitinase in the emerging anti-inflammatory strategies for the treatment of inflammation-related diseases such as cardiovascular diseases and type 2 diabetes is briefly discussed.

MCP-1and IL-1ß expression in the myocardia of two young patients with Type 1 diabetes mellitus and fatal diabetic ketoacidosis.

Convincing evidence exists for the early onset of diabetic cardiomyopathy and coronary artery disease (CAD) as distinct forms of cardiac disease in young patients with Type 1 diabetes mellitus (T1DM) and the pre-stages of T2DM, forms of dysregulated insulin signaling. Progression of both chronic cardiac conditions is mediated by oxidative stress and low grade inflammation. This study reports the expression of monocyte chemotactic protein-1 (MCP-1) chemokine and the interleukin (IL)-1ß inflammatory cytokine in two young patients with suboptimal metabolic control and fatal diabetic ketoacidosis (DKA), two age-matched overweight/obesity cases and two age-matched controls. In addition, markers of oxidative stress, apoptosis, collagen deposition and cardiomyocyte hypertrophy were studied. Significant expression of MCP-1 and IL-1ß was seen in the myocardia of the T1DM/DKA cases, with lesser amounts expressed in the overweight/obesity myocardia. All of the other markers except cardiomyocyte hypertrophy were expressed to a significantly greater extent in the T1DM/DKA and overweight/obesity cases in comparison to the age-matched controls. Cardiomyocyte hypertrophy was significantly greater in the overweight/obesity cases than in the T1DM/DKA or the control cases.

Antidicer RNAse activity of monocyte chemotactic protein-induced protein-1 is critical for inducing angiogenesis.

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.

Delayed brain ischemia tolerance induced by electroacupuncture pretreatment is mediated via MCP-induced protein 1.

BACKGROUND: Emerging studies have demonstrated that pretreatment with electroacupuncture (EA) induces significant tolerance to focal cerebral ischemia. The present study seeks to determine the involvement of monocyte chemotactic protein-induced protein 1 (MCPIP1), a recently identified novel modulator of inflammatory reactions, in the cerebral neuroprotection conferred by EA pretreatment in the animal model of focal cerebral ischemia and to elucidate the mechanisms of EA pretreatment-induced ischemic brain tolerance. METHODS: Twenty-four hours after the end of the last EA pretreatment, focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) for 90 minutes in male C57BL/6 mice and MCPIP1 knockout mice. Transcription and expression of MCPIP1 gene was monitored by qRT-PCR, Western blot and immunohistochemistry. The neurobehavioral scores, infarction volumes, proinflammatory cytokines and leukocyte infiltration in brain and NF-κB signaling were evaluated after ischemia/reperfusion. RESULTS: MCPIP1 protein and mRNA levels significantly increased specifically in mouse brain undergoing EA pretreatment. EA pretreatment significantly attenuated the infarct volume, neurological deficits, upregulation of proinflammatory cytokines and leukocyte infiltration in the brain of wild-type mice after MCAO compared with that of the non-EA group. MCPIP1-deficient mice failed to evoke EA pretreatment-induced tolerance compared with that of the control MCPIP1 knockout group without EA treatment. Furthermore, the activation of NF-κB signaling was significantly reduced in EA-pretreated wild-type mice after MCAO compared to that of the non-EA control group and MCPIP1-deficient mice failed to confer the EA pretreatment-induced inhibition of NF-κB signaling after MCAO. CONCLUSIONS: Our data demonstrated that MCPIP1 deficiency caused significant lack of EA pretreatment-induced cerebral protective effects after MCAO compared with the control group and that MCPIP1 is involved in EA pretreatment-induced delayed brain ischemia tolerance.

MCP-1-induced protein promotes endothelial-like and angiogenic properties in human bone marrow monocytic cells.

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.

Mycobacterium tuberculosis uses host triacylglycerol to accumulate lipid droplets and acquires a dormancy-like phenotype in lipid-loaded macrophages.

Two billion people are latently infected with Mycobacterium tuberculosis (Mtb). Mtb-infected macrophages are likely to be sequestered inside the hypoxic environments of the granuloma and differentiate into lipid-loaded macrophages that contain triacylglycerol (TAG)-filled lipid droplets which may provide a fatty acid-rich host environment for Mtb. We report here that human peripheral blood monocyte-derived macrophages and THP-1 derived macrophages incubated under hypoxia accumulate Oil Red O-staining lipid droplets containing TAG. Inside such hypoxic, lipid-loaded macrophages, nearly half the Mtb population developed phenotypic tolerance to isoniazid, lost acid-fast staining and accumulated intracellular lipid droplets. Dual-isotope labeling of macrophage TAG revealed that Mtb inside the lipid-loaded macrophages imports fatty acids derived from host TAG and incorporates them intact into Mtb TAG. The fatty acid composition of host and Mtb TAG were nearly identical suggesting that Mtb utilizes host TAG to accumulate intracellular TAG. Utilization of host TAG by Mtb for lipid droplet synthesis was confirmed when fluorescent fatty acid-labeled host TAG was utilized to accumulate fluorescent lipid droplets inside the pathogen. Deletion of the Mtb triacylglycerol synthase 1 (tgs1) gene resulted in a drastic decrease but not a complete loss in both radiolabeled and fluorescent TAG accumulation by Mtb suggesting that the TAG that accumulates within Mtb is generated mainly by the incorporation of fatty acids released from host TAG. We show direct evidence for the utilization of the fatty acids from host TAG for lipid metabolism inside Mtb. Taqman real-time PCR measurements revealed that the mycobacterial genes dosR, hspX, icl1, tgs1 and lipY were up-regulated in Mtb within hypoxic lipid loaded macrophages along with other Mtb genes known to be associated with dormancy and lipid metabolism.

Expression and characterization of Rv0447c product, potentially the methyltransferase involved in tuberculostearic acid biosynthesis in Mycobacterium tuberculosis.

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.

Huoluo Xiaoling Pellet promotes microglia M2 polarization through increasing MCPIP1 expression for ischemia stroke alleviation.

Huoluo Xiaoling Pellet (HXP), a Chinese patent medicine, is commonly administered for the treatment of treat ischemic strokes. MCPIP1, an inducible suppressor of the inflammatory response, is a regulator of microglial M2 polarization. This study aimed to explore whether HXP can promote microglial M2 polarization by upregulating MCPIP1 expression, consequently mitigating cerebral ischemic injury. Our study involved 85 Sprague-Dawley rats (weighing 250-280 g). We established middle cerebral artery occlusion (MCAO) and oxygen-glucose deprivation-reoxygenation (OGD/R) models with MCPIP1 knockdown to assess the effects of HXP on ischemic strokes. Our findings show that HXP reduced brain water content, improved neurological function, and inhibited the expression of inflammatory factors in the brain tissues of MCAO rats. The neuroprotective effects of HXP on cerebral ischemic injuries were compromised by MCPIP1 knockdown. Immunofluorescence results indicated that the expression of microglia marker Iba1 and M2 phenotypic marker CD206 was upregulated in MCAO rats and OGD/R-treated microglia. Administration of HXP significantly reduced Iba1 expression and facilitated CD206 expression, an effect that was counteracted by sh-MCPIP1 transfection. Western blotting revealed that HXP treatment augmented the expression of MCPIP1, microglial M2 marker proteins (CD206 and Arg1), and PPARγ, while reducing the expression of microglial M1 marker proteins (CD16 and iNOS) in MCAO rats and OGD/R-induced microglia. MCPIP1 knockdown suppressed HXP-mediated upregulation of MCPIP1, CD206, Arg1, and PPARγ, as well as the downregulation of CD16 and iNOS. Our findings suggest that HXP primarily ameliorates ischemic stroke through the upregulation of MCPIP1, which in turn induces microglial M2 polarization.

Fruit-specific overexpression of wound-induced tap1 under E8 promoter in tomato confers resistance to fungal pathogens at ripening stage.

Based on high economic importance and nutritious value of tomato fruits and as previous studies employed E8 promoter in fruit ripening-specific gene expression, we have developed transgenic tomato plants overexpressing tomato anionic peroxidase cDNA (tap1) under E8 promoter. Stable transgene integration was confirmed by polymerase chain reaction (PCR) and Southern analysis for nptII. Northern blotting confirmed elevated tap1 levels in the breaker- and red-ripe stages of T(1) transgenic fruits, whereas wild-type (WT) plants did not show tap1 expression in these developmental stages. Further, tap1 expression levels were significantly enhanced in response to wounding in breaker- and red-ripe stages of transgenic fruits, whereas wound-induced expression of tap1 was not detected in WT fruits. Confocal microscopy revealed high accumulation of phenolic compounds at the wound site in transgenic fruits suggesting a role of tap1 in wound-induced phenolic polymerization. Total peroxidase activity has increased remarkably in transgenic pericarp tissues in response to wounding, while very less or minimal levels were recorded in WT pericarp tissues. Transgenic fruits also displayed reduced post-harvest decay and increased resistance toward Alternaria alternata and Fusarium solani infection with noticeable inhibition in lesion formation. Conidiospore germination and mycelial growth of F. solani were severely inhibited when treated with E8-tap1 fruit extracts compared to WT fruits. 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay showed reduced spore viability when incubated in E8-tap1 fruit extracts. Thus, fruit-specific expression of tap1 using E8 promoter is associated with enhanced total peroxidase activity and high phenolic accumulation in fruits with minimized post-harvest deterioration caused by wounding and fungal attack in tomato fruits.