Role Of Kruppel-like Factor 2 Research Articles

Renoprotective Effect of KLF2 on Glomerular Endothelial Dysfunction in Hypertensive Nephropathy.

Kruppel-like factor 2 (KLF2) regulates endothelial cell metabolism; endothelial dysfunction is associated with hypertension and is a predictor of atherosclerosis development and cardiovascular events. Here, we investigated the role of KLF2 in hypertensive nephropathy by regulating KLF2 expression in human primary glomerular endothelial cells (hPGECs) and evaluating this expression in the kidney tissues of a 5/6 nephrectomy mouse model as well as patients with hypertension. Hypertension-mimicking devices and KLF2 siRNA were used to downregulate KLF2 expression, while the expression of KLF2 was upregulated by administering simvastatin. After 4 mmHg of pressure was applied on hPGECs for 48 h, KLF2 mRNA expression decreased, while alpha-smooth muscle actin (αSMA) mRNA expression increased. Apoptosis and fibrosis rates were increased under pressure, and these phenomena were aggravated following KLF2 knockdown, but were alleviated after simvastatin treatment; additionally, these changes were observed in angiotensin II, angiotensin type-1 receptor (AT1R) mRNA, and interleukin-18 (IL-18), but not in angiotensin type-2 receptor mRNA. Reduced expression of KLF2 in glomerular endothelial cells due to hypertension was found in both 5/6 nephrectomy mice and patients with hypertensive nephropathy. Thus, our study demonstrates that the pressure-induced apoptosis and fibrosis of glomerular endothelial cells result from angiotensin II, AT1R activation, and KLF2 inhibition, and are associated with IL-18.

Role of KLF2: New insight in inflammatory acne pathogenesis.

Acne is an inflammatory skin condition of pilosebaceous unit. Its pathogenesis is multifactorial with a central role of inflammatory and pro-inflammatory cytokines mediators. Downregulated Kruppel-like factor 2 (KLF2) leads to rapid secretion of many cytokines that are involved in acne pathogenesis. This study aimed at evaluating the level of KLF2 mRNA, clarifying its role in acne pathogenesis and its relation to acne lesion type, degree of severity, and outcome. The level of KLF2 mRNA was measured in 100 patients with acne and 50 age- and sex-matched healthy controls by using quantitative real-time polymerase chain reaction (qRT-PCR). The value of KLF2 mRNA was lower in acne patients than control group (P<.001), being lowest in inflammatory acne group (grades III, IV, and V) than noninflammatory acne group (grades I and II) and highest in the control group (P<.001). KLF2 mRNA was decreased significantly with increased acne severity grade (P<.001). KLF2 mRNA was lower in cases healed by scars than those healed by postinflammatory hyperpigmentation. Decreased serum level of KLF2 is not only a claimed for AV pathogenesis but also a predictor for degree of acne severity and outcome.

The expression of Kruppel-like factor 2 in mice with collagen-induced arthritis and its role in the induction of nitric oxide synthase production

Kruppel-like factor 2 (KLF2) is associated with acute and chronic inflammation. However, the role of KLF2 in rheumatoid arthritis (RA) remains unknown. Here, we investigated the expression of KLF2 in mice with collagen-induced arthritis (CIA) to determine whether KLF2 levels correlate with inducible nitric oxide synthase (iNOS) production in vitro. Hematoxylin and eosin staining was used to assess the synovitis and bone destruction in mice. The concentration of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and nitric oxide (NO) in synovial fluid was determined by enzyme-linked immunosorbent assay, while western blot (WB) analysis was employed to detect the expression of KLF2 and iNOS in the synovium, heart, and kidneys. The expression of iNOS in MH7A cells was analyzed by quantitative polymerase chain reaction (PCR) and WB. The expression of KLF2 and iNOS was significantly elevated in the synovium, heart, and kidneys of CIA mice. This was correlated to an increase in the severity of arthritis and the concentration of inflammatory mediators including TNF-α, IL-6, and NO in joint fluid. KLF2 and iNOS expression in vitro was induced by TNF-α and KLF2 knockdown significantly reduced the TNF-α-induced iNOS expression. These findings indicate that KLF2 influences synovial inflammation in CIA mice by regulating iNOS production in synoviocytes.

The IRF2BP2-KLF2 axis regulates osteoclast and osteoblast differentiation

Kruppel-like factor 2 (KLF2) has been implicated in the regulation of cell proliferation, differentiation, and survival in a variety of cells. Recently, it has been reported that KLF2 regulates the p65-mediated transactivation of NF-κB. Although the NF-κB pathway plays an important role in the differentiation of osteoclasts and osteoblasts, the role of KLF2 in these bone cells has not yet been fully elucidated. In this study, we demonstrated that KLF2 regulates osteoclast and osteoblast differentiation. The overexpression of KLF2 in osteoclast precursor cells inhibited osteoclast differentiation by downregulating c-Fos, NFATc1, and TRAP expression, while KLF2 overexpression in osteoblasts enhanced osteoblast differentiation and function by upregulating Runx2, ALP, and BSP expression. Conversely, the downregulation of KLF2 with KLF2-specific siRNA increased osteoclast differentiation and inhibited osteoblast differentiation. Moreover, the overexpression of interferon regulatory protein 2-binding protein 2 (IRF2BP2), a regulator of KLF2, suppressed osteoclast differentiation and enhanced osteoblast differentiation and function. These effects were reversed by downregulating KLF2. Collectively, our data provide new insights and evidence to suggest that the IRF2BP2/KLF2 axis mediates osteoclast and osteoblast differentiation, thereby affecting bone homeostasis.

Abstract WP443: KLF2 Protects Against Ischemic Stroke by Regulating Endothelial Blood Brain Barrier Function

Background During an ischemic stroke normal brain endothelial function is abrogated resulting in blood brain barrier (BBB) breakdown with subsequent infiltration of activated inflammatory blood cells, ultimately leading to neuronal cell death. Kruppel-like factor 2 (KLF2) is regulated by flow, is highly expressed in vascular endothelial cells, and serves as a key molecular switch regulating endothelial function and promoting vascular health. Purpose To determine the role of KLF2 in ischemic stroke. Methods and Results —To examine the specific role of KLF2 in the brain and cerebrovascular disease, we performed ischemic strokes and conducted BBB assays in KLF2 deficient (K2-/-), KLF2 overexpressing (K2tg), and control mice. K2 -/- mice exhibited larger strokes in the transient middle cerebral artery occlusion model (95% greater stroke volume vs. control; n=10-20/group; P=0.001) and significant impairment in BBB function as assessed by in vivo real time quantitative PET analysis and evans blue dye (EBD) assays (86% greater EBD permeability vs. control; n=3/group; P=0.002). In contrast, K2tg mice were protected against ischemic stroke (49% smaller stroke volume vs. control; n=8-9/group, P=0.004) and demonstrated less impairment in BBB function (30% less EBD permeability vs control; n=3/group; P=0.003). In concordance, gain and loss of function studies in primary brain microvascular ECs using transwell assays revealed KLF2 to be BBB protective (32% less permeability in adenoviral KLF2 overexpression vs. control; P=0.005 and 107% greater permeability in siRNA mediated KLF2 knockdown vs. control; P&lt;0.0005). Mechanistically, KLF2 was demonstrated to regulate the critical BBB tight junction factor occludin, both in vitro (7.0±0.3 fold expression in adenoviral KLF2 overexpression vs. control; P&lt;0.0001 and 0.43±0.03 fold expression in siRNA mediated KLF2 knockdown vs. control; P&lt;0.0005) and in vivo (0.56±0.11 fold expression in K2 -/- vs. control; n=4-7/group; P=0.01 and 2.5±0.5 fold expression in K2tg vs. control; n=3-4/group; P&lt;0.05). Conclusions These data are first to identify KLF2 as a key regulator of brain endothelial function and cerebrovascular homeostasis, thereby serving as a novel neuroprotective factor in ischemic stroke.

Kruppel-Like Factor 2 (KLF2) Regulates Monocyte Differentiation and Functions in mBSA and IL-1&amp;#946;-Induced Arthritis

Kruppel-like factor 2 (KLF2) plays an important role in the regulation of a variety of immune cells, including monocytes. We have previously shown that KLF2 inhibits proinflammatory activation of monocytes. However, the role of KLF2 in arthritis is yet to be investigated. In the current study, we show that recruitment of significantly greater numbers of inflammatory subset of CD11b(+)F4/80(+)Ly6C+ monocytes to the inflammatory sites in KLF2 hemizygous mice compared to the wild type littermate controls. In parallel, inflammatory mediators, MCP-1, Cox-2 and PAI-1 were significantly up-regulated in bone marrow-derived monocytes isolated from KLF2 hemizygous mice, in comparison to wild-type controls. Methylated-BSA and IL-1β-induced arthritis was more severe in KLF2 hemizygous mice as compared to the littermate wild type controls. Consistent with this observation, monocytes isolated from KLF2 hemizygous mice showed an increased number of cells matured and differentiated towards osteoclastic lineage, potentially contributing to the severity of cartilage and bone damage in induced arthritic mice. The severity of arthritis was associated with the higher expression of proteins such as HSP60, HSP90 and MMP13 and attenuated levels of pPTEN, p21, p38 and HSP25/27 molecules in bone marrow cells of arthritic KLF2 hemizygous mice compared to littermate wild type controls. The data provide new insights and evidences of KLF2-mediated transcriptional regulation of arthritis via modulation of monocyte differentiation and function.

Crippling of Krüppel (-Like Factor 2) by Bad Flow Portends a miRky Day for Endothelial Function

Kruppel-like Factor 2 (KLF2) is a 38-kDa transcription factor that is highly expressed in the vascular endothelium. The enormous attention that KLF2 has received in recent years is well deserved, because experimental evidence has shown that it is a vital protein that, via transcriptional and nontranscriptional targets, mediates a host of endothelial functions.1 These include inhibition of vascular inflammation and attendant atherosclerosis, maintenance of an antithrombotic endothelial surface, stimulation of endothelial nitric oxide synthase expression and vascular nitric oxide production, inhibition of hypoxia-stimulated angiogenesis, and promotion of prenatal vasculogenesis, among others. It is not surprising, therefore, that regulation of endothelial KLF2 expression has been the subject of intense investigation. Article see p 633 Systole, diastole, and the intrinsic elasticity of the arterial tree results in a blood flow that is pulsatile in fashion. This blood flow, in straight parts of the arterial tree, imparts a laminar stress on the endothelium. Laminar flow stimulates endothelial KLF2 expression.2 Segments of the arterial tree that face pulsatile laminar flow are less prone to the development of atherosclerotic plaque than those that are subject to oscillatory flow (which results in nonlaminar shear), such as at branch points and curvatures.3 Parallel with the susceptibility of arterial regions exposed to oscillatory flow to atheromatous changes, endothelial KLF2 expression is decreased in such regions.2 Consistent with the role of KLF2 in stimulating endothelial nitric oxide synthase expression and nitric oxide production, laminar flow promotes endothelium-dependent vasorelaxation,4,5 whereas oscillatory flow impairs it.6 Differential regulation of endothelial KLF2 expression in response …

Abstract 4888: A Novel Role of Kruppel-like Factor 2 (KLF2) in Regulating Endothelial Barrier Function

Background : Selective permeability is a life sustaining function of endothelial cells. Defects in permeability are implicated in several diseases such as sepsis, ischemia reperfusion injury and diabetes. Previous studies by our group and others have identified KLF2 as a molecular switch regulating endothelial gene expression and function. However the role of KLF2 in vascular permeability remains unknown. Methods and Results : Adenoviral overexpression of KLF2 in HUVECs strongly attenuated the increase of endothelial permeability by thrombin (1U/ml), histamine (200 μ M) and hydrogen peroxide (500 μ M) as measured by two independent assays – transendothelial electrical resistance (TER) and FITC-Dextran passage using a transwell system. Conversely, KLF2 deficiency in HUVECs (by siRNA knockdown) and primary mouse endothelial cells (derived from KLF2 +/+ and KLF2+/− mice) exhibited a marked increase in thrombin-induced permeability. To assess the effect of KLF2 in vascular permeability in vivo, we measured the leakage of Evans Blue dye into interstitial tissues of the mouse ear (expressed as μ g of dye/mg of ear tissue) after treatment with mustard oil. Consistent with our in vitro observation, by comparison to KLF2+/+ mice, KLF2 +/− mice exhibited a significantly higher degree of vascular leak (53.4±2.68 μ g/mg in KLF2+/+ versus 72.36±4.71 μ g/mg in KLF2+/−, p&lt;0.05). Mechanistically, our gain and loss-of-function studies indicate that KLF2 differentially affects key endothelial targets that regulate barrier function. Specifically, KLF2 markedly attenuated agonist- induced increase in Myosin Light Chain (MLC) phosphorylation (a key signaling molecule that promotes endothelial barrier permeability), increased Rac1 activation (a small GTPase that has been shown to confer barrier protection properties), and increased the expression of occludin and zona occludens-1 (tight junction proteins that promote barrier function). Conclusions : These observations identify KLF2 as an essential regulator of endothelial barrier function. Targeting of KLF2 function may be beneficial in disorders characterized by excessive vascular permeability such as sepsis. This research has received full or partial funding support from the American Heart Association, Founders Affiliate (Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont).

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-1alpha 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-1alpha 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-1alpha degradation in a von Hippel-Lindau protein-independent but proteasome-dependent manner. Finally, KLF2 disrupted the interaction between HIF-1alpha and its chaperone Hsp90, suggesting that KLF2 promotes degradation of HIF-1alpha by affecting its folding and maturation. These observations identify KLF2 as a novel inhibitor of HIF-1alpha expression and function. Therefore, KLF2 may be a target for modulating the angiogenic response in disease states.

The role of Kruppel-like factor 2 (KLF2) in thymic emigration and trafficking of non-conventional T cell lineages (82.16)

Abstract Immune function is dependent on the proper development of T cells in the thymus and emigration of these cells to the periphery. We recently showed the transcription factor KLF2 is required for emigration via regulation of S1P1 (sphingosine 1-phosphate receptor 1) and CD62L in conventional αβ T cells. However, the role of KLF2 and S1P1 in trafficking of non-conventional T cell subsets (γδ T cells, Treg, NKT, and Gut intraepithelial lymphocytes -IEL) has yet to be described. We show here that KLF2 is differentially expressed within the T cells subsets studied using both mRNA expression via quantitative PCR and/or a novel KLF2-GFP reporter mouse model. While KLF2 and S1P1 can be detected in sorted non-conventional thymic T cells and Gut IEL αβ T cells, we report no detectable KLF2 in Gut IEL γδ T cells. Using several KLF2 deficient mouse models, trafficking defects were observed in Treg, NKT, and Gut-IEL (αβ and γδ) populations. Finally, the data suggest that lack of KLF2 alters both the distribution and the phenotype of γδ T cells in secondary lymphoid organs. Overall, our results suggest that KLF2 is a common means by which different T cell lineages regulate trafficking and/or entry. Funding provided by the National Institutes of Health (AI038903 to SCJ and AI039560 to KAH).

Abstract 5440: Klf2 Inhibits Hif-1α Expression and Function in the Endothelium

Introduction: 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 pro-angiogenic factors such as vascular endothelial growth factor (VEGF) to induce new blood vessel formation. Recent studies have identified Kruppel-like Factor 2 (KLF2) as a potent inhibitor of angiogenesis. However, the role of KLF2 in regulating the expression and function of HIF-1 has not been evaluated. Hypothesis: KLF2 inhibits HIF-1α expression and function. Methods and Results: Adenoviral overexpression of KLF2 in human umbilical vein endothelial cells (HUVECs) inhibited hypoxia-induced expression of HIF-1α, VEGF secretion, and matrigel tube formation. Conversely, siRNA-mediated knockdown of KLF2 in HUVECs increased HIF-1α expression and VEGF secretion. Consistent with this observation, KLF2−/− mouse embryonic fibroblasts (MEFs) showed accelerated HIF-1α accumulation in response to hypoxia and a marked induction of VEGF secretion (90.4±7.4 pg/ml in KLF2+/+ versus 234.6±6.1 pg/ml in KLF2−/−, p&lt;0.01). From a mechanistic standpoint, KLF2-mediated reduction of HIF-1α level was attenuated by three distinct proteasome inhibitors (MG132, ALLN, or lactacystin), demonstrating that KLF2 mediated reduction of HIF-1α protein was proteasome-dependent. In addition, KLF2 maintained its ability to inhibit HIF-1α protein levels in VHL−/− cells, thermo-sensitive E1 deficient cells, and p53−/− cells. Finally, co-immunoprecipitation experiments revealed that KLF2 overexpression disrupted the interaction between HIF-1α and its chaperone Hsp90, suggesting that KLF2 promotes degradation of HIF-1α by affecting its folding and maturation. Conclusions: These observations identify KLF2 as a novel inhibitor of HIF-1α expression and function through a VHL/p53 independent but proteasome dependent pathway. As such, KLF2 may be a target for modulating the angiogenic response in disease states.