Riku Das

Kruppel-like Transcription Factor 6 Regulates Inflammatory Macrophage Polarization

Background: Macrophage polarization regulates human inflammatory disorders. Results: KLF6 is a novel transcriptional regulator of macrophage polarization. Conclusion: KLF6 regulates macrophage inflammatory gene expression by modulating functions of NF-κB and PPARγ. Significance: Pharmacological agents that modulate KLF6 signaling may allow for therapeutic gain in the treatment of inflammatory disorders. Accumulating evidence supports the importance of macrophage plasticity in a broad spectrum of biological processes operative in health and disease. A major locus of control regulating macrophage polarization is at the transcriptional level, and several major pathways have been elucidated in recent years. In this study, we identify the Kruppel-like transcription factor 6 (KLF6) as a molecular toggle controlling macrophage speciation. KLF6 expression was robustly induced by pro-inflammatory M1 stimuli (e.g. LPS and IFN-γ) and strongly suppressed by M2 stimuli (e.g. IL4 and IL-13) in human and murine macrophages. Gain- and loss-of-function studies suggest that KLF6 is required for optimal LPS-induced pro-inflammatory gene expression, acting cooperatively with NF-κB. Furthermore, KLF6 inhibits anti-inflammatory gene expression by negatively regulating peroxisome proliferator-activated receptor γ expression in macrophages. Collectively, these observations identify KLF6 as a novel transcriptional regulator of macrophage polarization.

So Many Plasminogen Receptors: Why?

Plasminogen and plasmin tether to cell surfaces through ubiquitously expressed and structurally quite dissimilar family of proteins, as well as some nonproteins, that are collectively referred to as plasminogen receptors. Of the more than one dozen plasminogen receptors that have been identified, many have been shown to facilitate plasminogen activation to plasmin and to protect bound plasmin from inactivation by inhibitors. The generation of such localized and sustained protease activity is utilized to facilitate numerous cellular responses, including responses that depend on cellular migration. However, many cells express multiple plasminogen receptors and numerous plasminogen receptors are expressed on many different cell types. Furthermore, several different plasminogen receptors can be used to support the same cellular response, such as inflammatory cell migration. Here, we discuss the perplexing issue: why are there so many different Plg-Rs?

Plasminogen and Its Receptors as Regulators of Cardiovascular Inflammatory Responses

In addition to its role in fibrinolysis, plasminogen (Plg) influences inflammatory cell migration and thereby plays a prominent role in cardiovascular pathology. The contribution of Plg to inflammatory cell recruitment depends on its tethering to the surface of responding cells. Plasminogen receptors (Plg-Rs) are heterogeneous and can be classified as tailless, lacking cytoplasmic tails, or tailed (having cytoplasmic tails). In vivo observations implicate several tailless Plg-Rs in inflammatory responses. Tailed Plg-Rs on leukocytes include several integrins, which have also been implicated in Plg-dependent responses. Surface expression of both tailless and tailed Plg-Rs can be modulated in number and/or function. A common mechanism involving intracellular calcium mobilization and calcium channels regulates expression of both classes of Plg-Rs. Data are emerging to indicate that targeting Plg and Plg-Rs may limit inflammation and cardiovascular pathology.

L-Type Calcium Channel Blockers Exert an Antiinflammatory Effect by Suppressing Expression of Plasminogen Receptors on Macrophages

L-type Ca2+ channel (LTCC) blockers, represented by amlodipine and verapamil, are widely used antihypertensive drugs that also have antiinflammatory activities. Plasminogen (Plg) is an important mediator of macrophage recruitment, and this role depends on its interaction with Plg receptors (Plg-Rs). Plg-Rs include histone 2B, α-enolase, annexin 2, and p11, all proteins which lack signal sequences for cell surface export. When human or murine monocytoid cells were induced to differentiate into macrophages, their Plg binding and Plg-R expression increased by 4-fold. These changes were suppressed by pretreatment with verapamil and amlodipine. Expression of the Cav1.2 LTCC pore subunit was induced in differentiated macrophages, and siRNA against this subunit suppressed the upregulation of Plg binding and Plg-Rs. In vivo, amlodipine and verapamil suppressed peritoneal macrophage recruitment in response to thioglycollate by >60% at doses that did not affect blood pressure. In drug-treated animals, macrophages migrated into but not through the peritoneal membrane tissue and showed reduced surface expression of Plg-Rs. These findings demonstrate that Plg-R expression on macrophages is dependent on Cav1.2 LTCC subunit expression. Suppression of Plg-Rs may contribute to the antiinflammatory effects of the widely used LTCC blockers.

Enolase-1 as a plasminogen receptor.

In this issue of Blood , Wygrecka and colleagues assign a major role to cell-surface enolase-1 as a plasminogen receptor, mediating LPS-induced invasion of monocytes into lungs in mice and humans. Over the past decade, studies conducted in plasminogen-deficient mice have created a compelling case

Plasminogen promotes macrophage phagocytosis in mice

The phagocytic function of macrophages plays a pivotal role in eliminating apoptotic cells and invading pathogens. Evidence implicating plasminogen (Plg), the zymogen of plasmin, in phagocytosis is extremely limited with the most recent in vitro study showing that plasmin acts on prey cells rather than on macrophages. Here, we use apoptotic thymocytes and immunoglobulin opsonized bodies to show that Plg exerts a profound effect on macrophage-mediated phagocytosis in vitro and in vivo. Plg enhanced the uptake of these prey by J774A.1 macrophage-like cells by 3.5- to fivefold Plg receptors and plasmin proteolytic activity were required for phagocytosis of both preys. Compared with Plg(+/+) mice, Plg(-/-) mice exhibited a 60% delay in clearance of apoptotic thymocytes by spleen and an 85% reduction in uptake by peritoneal macrophages. Phagocytosis of antibody-mediated erythrocyte clearance by liver Kupffer cells was reduced by 90% in Plg(-/-) mice compared with Plg(+/+) mice. A gene array of splenic and hepatic tissues from Plg(-/-) and Plg(+/+) mice showed downregulation of numerous genes in Plg(-/-) mice involved in phagocytosis and regulation of phagocytic gene expression was confirmed in macrophage-like cells. Thus, Plg may play an important role in innate immunity by changing expression of genes that contribute to phagocytosis.

Macrophage Gene Expression and Foam Cell Formation Are Regulated by Plasminogen

Background— Deciphering the molecular and cellular processes that govern macrophage foam cell formation is critical to understanding the basic mechanisms underlying atherosclerosis and other vascular pathologies. Methods and Results— Here, we identify a pivotal role of plasminogen (Plg) in regulating foam cell formation. Deficiency of Plg inhibited macrophage cholesterol accumulation on exposure to hyperlipidemic conditions in vitro, ex vivo, and in vivo. Gene expression analysis identified CD36 as a regulated target of Plg, and macrophages from Plg−/− mice had decreased CD36 expression and diminished foam cell formation. The Plg-dependent CD36 expression and foam cell formation depended on conversion of Plg to plasmin, binding to the macrophage surface, and the consequent intracellular signaling that leads to production of leukotriene B4. Leukotriene B4 rescued the suppression of CD36 expression and foam cell formation arising from Plg deficiency. Conclusions— Our findings demonstrate an unanticipated role of Plg in the regulation of gene expression and cholesterol metabolism by macrophages and identify Plg-mediated regulation of leukotriene B4 as an underlying mechanism.

Phosphatidylserine as an anchor for plasminogen and its plasminogen receptor, Histone H2B, to the macrophage surface

Summary.  Background: Plasminogen (Plg) binding to cell surface Plg receptors (Plg‐Rs) on the surface of macrophages facilitates Plg activation and migration of these cells. Histone H2B (H2B) acts as a Plg‐R and its cell surface expression is up‐regulated when monocytes are differentiated to macrophages via a pathway dependent on L‐type Ca2+ channels and intracellular Ca2+. Objectives: We sought to investigate the mechanism by which H2B, a protein without a transmembrane domain, is retained on the macrophage surface. Methods: THP‐1 monocytoid cells were induced to differentiate with interferon gamma + Vitamin D3 or to undergo apoptosis by treatment with camptothecin. Flow cytometry and cell surface biotinylation followed by Western blotting were used to measure the interrelationship between Plg binding, cell surface expression of H2B and outer membrane exposure of phosphatidylserine (PS). Results: H2B interacted directly with PS via an electrostatic interaction. Anti‐PS or PS binding proteins, annexin V and protein S, diminished H2B interaction with PS on the surface of differentiated or apoptotic cells and these same reagents inhibited Plg binding to these cells. L‐type Ca2+ channels played a significant role in PS exposure, H2B surface expression and Plg binding induced either by differentiation or apoptosis. Conclusions: These data suggest that H2B tethers to the surface of cells by interacting with PS on differentiated or apoptotic monocytoid cells. L‐type Ca2+ channels regulate PS exposure on the surface of these cells. The exposed PS interacts directly with H2B and hence provides sites for Plg to bind to.

A new function for old drugs

Das R, et al. Circ Res 2009; 105:167-75.

Plasminogen promotes cholesterol efflux by the ABCA1 pathway

Using genetic and biochemical approaches, we investigated proteins that regulate macrophage cholesterol efflux capacity (CEC) and ABCA1-specific CEC (ABCA1 CEC), 2 functional assays that predict cardiovascular disease (CVD). Macrophage CEC and the concentration of HDL particles were markedly reduced in mice deficient in apolipoprotein A-I (APOA1) or apolipoprotein E (APOE) but not apolipoprotein A-IV (APOA4). ABCA1 CEC was markedly reduced in APOA1-deficient mice but was barely affected in mice deficient in APOE or APOA4. High-resolution size-exclusion chromatography of plasma produced 2 major peaks of ABCA1 CEC activity. The early-eluting peak, which coeluted with HDL, was markedly reduced in APOA1- or APOE-deficient mice. The late-eluting peak was modestly reduced in APOA1-deficient mice but little affected in APOE- or APOA4-deficient mice. Ion-exchange chromatography and shotgun proteomics suggested that plasminogen (PLG) accounted for a substantial fraction of the ABCA1 CEC activity in the peak not associated with HDL. Human PLG promoted cholesterol efflux by the ABCA1 pathway, and PLG-dependent efflux was inhibited by lipoprotein(a) [Lp(a)]. Our observations identify APOA1, APOE, and PLG as key determinants of CEC. Because PLG and Lp(a) associate with human CVD risk, interplay among the proteins might affect atherosclerosis by regulating cholesterol efflux from macrophages.