Ganapati H. Mahabeleshwar

Krüppel-like factor 4 regulates macrophage polarization

Current paradigms suggest that two macrophage subsets, termed M1 and M2, are involved in inflammation and host defense. While the distinct functions of M1 and M2 macrophages have been intensively studied - the former are considered proinflammatory and the latter antiinflammatory - the determinants of their speciation are incompletely understood. Here we report our studies that identify Krüppel-like factor 4 (KLF4) as a critical regulator of macrophage polarization. Macrophage KLF4 expression was robustly induced in M2 macrophages and strongly reduced in M1 macrophages, observations that were recapitulated in human inflammatory paradigms in vivo. Mechanistically, KLF4 was found to cooperate with Stat6 to induce an M2 genetic program and inhibit M1 targets via sequestration of coactivators required for NF-κB activation. KLF4-deficient macrophages demonstrated increased proinflammatory gene expression, enhanced bactericidal activity, and altered metabolism. Furthermore, mice bearing myeloid-specific deletion of KLF4 exhibited delayed wound healing and were predisposed to developing diet-induced obesity, glucose intolerance, and insulin resistance. Collectively, these data identify KLF4 as what we believe to be a novel regulator of macrophage polarization.

Hemizygous deficiency of Krüppel-like factor 2 augments experimental atherosclerosis.

Krüppel-like factor (KLF)2 is a central regulator of endothelial and monocyte/macrophage gene expression and function in vitro. Although the composite effects of KLF2 in these 2 cell types predict that it likely inhibits vascular inflammation, the role of KLF2 in this process in vivo is uncharacterized. In this study, we provide evidence that hemizygous deficiency of KLF2 increased diet-induced atherosclerosis in apolipoprotein E–deficient mice. Our studies highlight an important role for KLF2 in primary macrophage foam cell formation via the potential regulation of the key lipid binding protein adipocyte protein 2/fatty acid–binding protein 4. These novel observations establish that KLF2 is an atheroprotective factor.

Kruppel-like Factor 2 Inhibits Hypoxia-inducible Factor 1α Expression and Function in the Endothelium

Hypoxia-inducible factor 1 (HIF-1) is a central regulator of the hypoxic response in many cell types. In endothelial cells, HIF-1 induces the expression of key proangiogenic factors to promote angiogenesis. Recent studies have identified Kruppel-like factor 2 (KLF2) as a potent inhibitor of angiogenesis. However, the role of KLF2 in regulating HIF-1 expression and function has not been evaluated. KLF2 expression was induced acutely by hypoxia in endothelial cells. Adenoviral overexpression of KLF2 inhibited hypoxia-induced expression of HIF-1α and its target genes such as interleukin 8, angiopoietin-2, and vascular endothelial growth factor in endothelial cells. Conversely, knockdown of KLF2 increased expression of HIF-1α and its targets. Furthermore, KLF2 inhibited hypoxia-induced endothelial tube formation, whereas endothelial cells from mice with haploinsufficiency of KLF2 showed increased tube formation in response to hypoxia. Consistent with this ex vivo observation, KLF2 heterozygous mice showed increased microvessel density in the brain. Mechanistically, KLF2 promoted HIF-1α degradation in a von Hippel-Lindau protein-independent but proteasome-dependent manner. Finally, KLF2 disrupted the interaction between HIF-1α and its chaperone Hsp90, suggesting that KLF2 promotes degradation of HIF-1α by affecting its folding and maturation. These observations identify KLF2 as a novel inhibitor of HIF-1α expression and function. Therefore, KLF2 may be a target for modulating the angiogenic response in disease states.

Kruppel-Like Factor 2 Regulates Endothelial Barrier Function

Objective—A central function of the endothelium is to serve as a selective barrier that regulates fluid and solute exchange. Although perturbation of barrier function can contribute to numerous disease states, our understanding of the molecular mechanisms regulating this aspect of endothelial biology remains incompletely understood. Accumulating evidence implicates the Kruppel-like factor 2 (KLF2) as a key regulator of endothelial function. However, its role in vascular barrier function is unknown. Methods and Results—To assess the role of KLF2 in vascular barrier function in vivo, we measured the leakage of Evans blue dye into interstitial tissues of the mouse ear after treatment with mustard oil. By comparison with KLF2+/+ mice, KLF2+/− mice exhibited a significantly higher degree of vascular leak. In accordance with our in vivo observation, adenoviral overexpression of KLF2 in human umbilical vein endothelial cells strongly attenuated the increase of endothelial leakage by thrombin and H2O2 as measured by fluorescein isothiocyanate dextrans (FITC-dextran) passage. Conversely, KLF2 deficiency in human umbilical vein endothelial cells and primary endothelial cells derived from KLF2+/− mice exhibited a marked increase in thrombin and H2O2-induced permeability. Mechanistically, our studies indicate that KLF2 confers barrier-protection via differential effects on the expression of key junction protein occludin and modification of a signaling molecule (myosin light chain) that regulate endothelial barrier integrity. Conclusion—These observations identify KLF2 as a novel transcriptional regulator of vascular barrier function.

Fatty Acid-binding Protein 5 and PPARβ/δ Are Critical Mediators of Epidermal Growth Factor Receptor-induced Carcinoma Cell Growth

Epidermal growth factors and their receptors (EGFRs) promote breast cancer cell proliferation and can drive tumorigenesis. However, the molecular mechanisms that mediate these effects are incompletely understood. We previously showed that mammary tumor development in the mouse model of breast cancer MMTV-neu, a model characterized by amplification of the EGFR ErbB2 in mammary tissue, correlates with a marked up-regulation of fatty acid-binding protein 5 (FABP5). FABP5 functions to deliver ligands to and enhance the transcriptional activity of the nuclear receptor peroxisome proliferator-activated receptor β/δ (PPARβ/δ), a receptor whose target genes include genes involved in cell growth and survival. We show here that in MCF-7 mammary carcinoma cells, EGFR signaling directly up-regulates the expression of FABP5. The data demonstrate that treatment of these cells with the EGFR ligand heregulin-β1 signals through the ERK and the phophatidylinositol-3-kinase cascades, resulting in activation of the transcription factor NF-κB. In turn, NF-κB induces the expression of FABP5 through two cognate response elements in the promoter of this gene. The observations further demonstrate that FABP5 and PPARβ/δ are critical mediators of the ability of EGFR to enhance cell proliferation, indicating that this transcriptional pathway plays a key role in EGFR-induced tumorigenesis. Additional observations indicate that the expression of FABP5 is down-regulated by the Krüppel-like factor KLF2, suggesting a tumor suppressor activity for this factor.

Myeloid Krüppel-Like Factor 4 Deficiency Augments Atherogenesis in ApoE−/− Mice—Brief Report

Objective—To investigate the role of Krüppel-like factor 4 (KLF4), an essential transcriptional regulator of macrophage polarization (M1/M2), in the pathogenesis of atherosclerosis. Methods and Results—Despite the acknowledged importance of macrophages in atherosclerosis, the role of M1 (classically activated or proinflammatory) versus M2 (alternatively activated or anti-inflammatory) macrophages in this process remains incompletely understood. We recently identified KLF4 as a regulator of macrophage subset specification; that is, KLF4 promotes M2 and inhibits M1 phenotype. Here, we provide evidence that KLF4-deficient macrophages exhibit enhanced proinflammatory activation and foam cell formation in response to oxidized lipids. In vivo, myeloid KLF4-deficient mice (ApoE−/− background) develop significantly more vascular inflammation and atherosclerotic lesion formation. Conclusion—Our findings identify myeloid KLF4 as an essential regulator of vascular inflammation and experimental atherogenesis.

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.

A myeloid hypoxia-inducible factor 1α-Krüppel-like factor 2 pathway regulates gram-positive endotoxin-mediated sepsis.

Background: Gram-positive bacterial infections and sepsis are a significant cause of morbidity and mortality. Results: KLF2 inhibits Gram-positive, bacterial endotoxin-induced HIF-1α expression and macrophage activation. Conclusion: Transcription factors KLF2 and HIF-1α are critical regulator of Gram-positive sepsis. Significance: Pharmacological agents that modulate the KLF2/HIF-1 pathway may allow for therapeutic gain in the treatment of bacterial infections and sepsis. Although Gram-positive infections account for the majority of cases of sepsis, the molecular mechanisms underlying their effects remains poorly understood. We investigated how cell wall components of Gram-positive bacteria contribute to the development of sepsis. Experimental observations derived from cultured primary macrophages and the cell line indicate that Gram-positive bacterial endotoxins induce hypoxia-inducible factor 1α (HIF-1α) mRNA and protein expression. Inoculation of live or heat-inactivated Gram-positive bacteria with macrophages induced HIF-1 transcriptional activity in macrophages. Concordant with these results, myeloid deficiency of HIF-1α attenuated Gram-positive bacterial endotoxin-induced cellular motility and proinflammatory gene expression in macrophages. Conversely, Gram-positive bacteria and their endotoxins reduced expression of the myeloid anti-inflammatory transcription factor Krüppel-like transcription factor 2 (KLF2). Sustained expression of KLF2 reduced and deficiency of KLF2 enhanced Gram-positive endotoxins induced HIF-1α mRNA and protein expression in macrophages. More importantly, KLF2 attenuated Gram-positive endotoxins induced cellular motility and proinflammatory gene expression in myeloid cells. Consistent with these results, mice deficient in myeloid HIF-1α were protected from Gram-positive endotoxin-induced sepsis mortality and clinical symptomatology. By contrast, myeloid KLF2-deficient mice were susceptible to Gram-positive sepsis induced mortality and clinical symptoms. Collectively, these observations identify HIF-1α and KLF2 as critical regulators of Gram-positive endotoxin-mediated sepsis.

Kruppel-like factor 2 is a transcriptional regulator of chronic and acute inflammation.

Although myeloid cell activation is requisite for an optimal innate immune response, this process must be tightly controlled to prevent collateral host tissue damage. Kruppel-like factor 2 (KLF2) is a potent regulator of myeloid cell proinflammatory activation. As an approximately 30% to 50% reduction in KLF2 levels has been observed in human subjects with acute or chronic inflammatory disorders, we studied the biological response to inflammation in KLF2(+/-) mice. Herein, we show that partial deficiency of KLF2 modulates the in vivo response to acute (sepsis) and subacute (skin) inflammatory challenge. Mechanistically, we link the anti-inflammatory effects of KLF2 to the inhibition of NF-κB transcriptional activity. Collectively, the observations provide biologically relevant insights into KLF2-mediated modulation of these inflammatory processes that could potentially be manipulated for therapeutic gain.

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.