Search

Bovine alpha-herpesvirus 1 (BoHV-1) is a significant problem for the cattle industry, in part because the virus establishes latency, and stressful stimuli increase the incidence of reactivation from latency. Sensory neurons in trigeminal ganglia and unknown cells in pharyngeal tonsils are important sites for latency. Reactivation from latency can lead to reproductive problems in pregnant cows, virus transmission to young calves, suppression of immune responses, and bacterial pneumonia. BoHV-1 is also a significant cofactor in bovine respiratory disease (BRD). Stress, as mimicked by the synthetic corticosteroid dexamethasone, reproducibly initiates reactivation from latency. Stress-mediated activation of the glucocorticoid receptor (GR) stimulates viral replication and transactivation of viral promoters that drive the expression of infected cell protein 0 (bICP0) and bICP4. Notably, GR and Krüppel-like factor 15 (KLF15) form a feed-forward transcription loop that cooperatively transactivates immediate early transcription unit 1 (IEtu1 promoter). Two pioneer transcription factors, GR and KLF4, cooperatively transactivate the bICP0 early promoter. Pioneer transcription factors bind silent viral heterochromatin, remodel chromatin, and activate gene expression. Thus, we predict that these novel transcription factors mediate early stages of BoHV-1 reactivation from latency. [ABSTRACT FROM AUTHOR]

Halloysite nanotubes (HNTs) are nanomaterials (NMs) derived from natural clays and have been considered as biocompatible NMs for biomedical uses. However, the cardiovascular toxicity of HNTs has not been thoroughly investigated. In this study, we compared the cardiotoxicity of HNTs and multi-walled carbon nanotubes (MWCNTs), focusing on the changes in Kruppel-like factor (KLF)-mediated signaling pathways. Mice were intravenously injected with 50 µg NMs, once a day, for 5 days, and then mouse hearts were removed for experiments. While HNTs or MWCNTs did not induce obvious pathological changes, RNA-sequencing data suggested the alterations of KLF gene expression. We further confirmed an increase of Klf15 positive cells, accompanied by changes in Klf15-related gene ontology (GO) terms. We noticed that most of the changed GO terms are related with the regulation of gene expression, and we confirmed that the NMs increased myoneurin (Mynn) but decreased snail family transcriptional repressor 1 (Snai1), two transcription factors (TFs) related with Klf15. Besides, the changed GO terms also include metal ion binding and positive regulation of glucose import, and we verified an increase of phosphoenolpyruvate carboxykinase 1 (Pck1) and insulin receptor (Insr). However, HNTs and MWCNTs only showed minimal impact on cell death signaling pathways, and no increase in apoptotic sites was observed after NM treatment. We concluded that intravenous administration of HNTs and MWCNTs activated a protective TF, namely Klf15 in mouse aortas, to alter gene expression and signaling pathways related with metal ion binding and glucose import. [ABSTRACT FROM AUTHOR]

Lysine‐specific demethylase 1 (LSD1) inhibitors are being developed for cancer therapy, but their bioeffects on vasculatures are not clear. In this study, we compared the influences of ORY‐1001 (an LSD1 inhibitor being advanced into clinical trials) and 199 (a novel LSD1 inhibitor recently developed by us) to human umbilical vein endothelial cells (HUVECs) in vitro and further verified the bioeffects of ORY‐1001 to zebrafish (Danio rerio) larvae in vivo. The results showed that up to 10 μM ORY‐1001 or 199 did not significantly affect the cellular viability of HUVECs but substantially reduced the release of inflammatory interleukin‐8 (IL‐8) and IL‐6. The signaling molecule in vasculatures, NO, was also increased in HUVECs. As the mechanism, the protein levels of endothelial NO synthase (eNOS) or p‐eNOS, and their regulators Kruppel‐like factor 2 (KLF2) or KLF4, were also increased after drug treatment. In vivo, 24 h treatment with up to 100 nM ORY‐1001 reduced blood speed without changing morphologies or locomotor activities in zebrafish larvae. ORY‐1001 treatment reduced the expression of il8 but promoted the expression of klf2a and nos in the zebrafish model. These data show that LSD1 inhibitors were not toxic but capable to inhibit inflammatory responses and affect the function of blood vessels through the up‐regulation of the NOS‐KLF pathway. It is necessary to investigate the influences of LSD1 inhibitors on vascular systems. This study reported that LSD1 inhibitors ORY‐1001 or 199 were not cytotoxic to HUVECs but reduced inflammatory mediator release and induced NO release through the up‐regulation of the eNOS‐KLF2/4 signaling pathway. However, LSD1 inhibitors also modestly induced oxidative stress. Under in vivo conditions, ORY‐1001 did not induce developmental toxicity but reduced il8 expression and blood speed through the up‐regulation of nos‐klf gene expression. [ABSTRACT FROM AUTHOR]

Recently, we reported that titanium dioxide (TiO2) materials activated endothelial cells via Kruppel‐like factor (KLF)‐mediated nitric oxide (NO) dysfunction, but the roles of physical properties of materials are not clear. In this study, we prepared nanobelts from P25 particles and compared their adverse effects to human umbilical vein endothelial cells (HUVECs). TiO2 nanobelts had belt‐like morphology but comparable surface areas as P25 particles. When applied to HUVECs, P25 particles or nanobelts did not induce cytotoxicity, although nanobelts were much more effective to increase intracellular Ti element concentrations compared the same amounts of P25 particles. Only nanobelts significantly induced THP‐1 adhesion onto HUVECs. Consistently, nanobelts were more significant to induce the expression of intracellular adhesion molecule‐1 (ICAM1) and the release of soluble ICAM‐1 (sICAM‐1), indicating that nanobelts were more potent to induce endothelial activation in vitro. As the mechanisms for endothelial activation, both P25 and nanobelts reduced the generation of intracellular NO as well as the expression of NO regulators KLF2 and KLF4. Combined, the results from this study indicated that the different morphologies of P25 particles and nanobelts only changed their internalization into HUVECs but showed minimal impact on KLF‐mediated NO signaling pathways. Nanobelts of similar surface areas were synthesized from P25 particles. When applied to human umbilical vein endothelial cells (HUVECs), nanobelts were more effective to induce endothelial activation compared with P25. However, both types of materials reduced intracellular NO and decreased the expression of NO regulators KLF2 and KLF4 to similar extent. Since P25 particles and nanobelts may only differ in morphologies, there results suggested that morphologies had minimal impact on KLF‐mediated NO signaling pathways in Ti‐based material‐exposed HUVECs. [ABSTRACT FROM AUTHOR]

Morphology plays a vital role in determining the biological effects of silica nanoparticles (NPs), but its influence on the toxicity of silica NPs in endothelial cells (ECs) is still inconclusive. We synthesized five kinds of Santa Barbara 15 amorphous (SBA‐15) particles with different shapes and added them to human umbilical vein endothelial cells (HUVEC). After 24 After incubation and treatment with 100 ml, more than 80% of the cells are still alive. The microgram/ml of SBA‐15 indicates that SBA‐15 has high biocompatibility. Fibrous SBA‐15 (5) leads to the highest Si element concentration in HUVEC. No NP reduces the release of NO, and NO is an important signaling molecule in the vascular system. Only the aggregated spherical SBA‐15 (3) will moderately reduce the endothelial nitric oxide synthase (eNOS) protein. Regarding transcription factors regulating eNOS, we found that all SBA‐15 types significantly increased Kruppel‐like factor 2 (KLF2) protein, irregular SBA‐15 (1), non‐aggregated spherical SBA‐15 (2) and aggregation The spherical SBA‐15 (3) greatly reduces KLF4 by more than 50%. Overall, our results indicate that SBA‐15 with different morphologies can be internalized into HUVEC and only cause moderate cytotoxicity. All silica NPs have the smallest effect on the NO‐eNOS pathway, but the irregular spherical SBA‐15 reduces the eNOS modifier KLF4. The rod‐shaped SBA‐15 (4) seems to have higher biocompatibility because they are internalized and have negligible adverse effects on HUVEC. These results provide new evidence for the toxic effects of different forms of silica nanoparticles on HUVEC. Synthesize five kinds of Santa Barbara 15 amorphous (SBA‐15) particles with different shapes and add them to human umbilical vein endothelial cells (HUVECs), which induce moderate cytotoxicity, but not reduce NO production. The internalization of fibrous SBA‐15 (5) is higher than that of other particles. The aggregated spherical SBA‐15 (3) moderately reduced the endothelial nitric oxide synthase (eNOS) protein. All SBA‐15 materials increased KLF2 protein, whereas irregular SBA‐15 (1), nonaggregated spherical SBA‐15 (2), and aggregated spherical SBA‐15 (3) greatly reduced KLF4 protein. These results provide new evidence about the effect of silica nanoparticles (NP) morphology on the toxicity of HUVEC. [ABSTRACT FROM AUTHOR]

Nanomaterials (NMs) with tubular structures, such as halloysite nanotubes (HNTs), have potential applications in biomedicine. Although the biocompatibility of HNTs has been investigated before, the toxicity of HNTs to blood vessels is rarely systemically evaluated. Herein, we compared the toxicity of HNTs and multi-walled carbon nanotubes (MWCNTs) to human umbilical vein endothelial cells (HUVECs) in vitro and blood vessels of mice in vivo. HUVECs internalized HNTs and MWCNTs, but the uptake of HNTs was not obviously changed by clathrin inhibitor. Exposure to NMs decreased cellular viability, activated apoptotic proteins and up-regulated adhesion molecules, including soluble vascular cell adhesion molecule 1 (sVCAM-1) and VCAM-1. As the mechanisms, NMs decreased NO levels, eNOS mRNA and eNOS/p-eNOS proteins. Meanwhile, NMs promoted intracellular ROS and autophagy dysfunction, shown as decreased protein levels of LC3, beclin-1 and ATG5. The eNOS regulator Kruppel-like factor 4 (KLF4) was inhibited, but another eNOS regulator KLF4 was surprisingly up-regulated. Under in vivo conditions, ICR mice intravenously injected with NMs (50 μg/mouse, once a day for 5 days) showed an increased percentage of neutrophils, monocytes and basophils. Meanwhile, autophagy dysfunction, eNOS uncoupling, activation of apoptotic proteins and alteration of KLF proteins were also observed in mouse aortas. All of the toxic effects were more pronounced for MWCNTs in comparison with HNTs based on the same mass concentrations. Our results may provide novel insights about the toxicity of NMs with tubular structures to blood vessels. Considering the toxicological data reported here, HNTs are probably safer nanocarriers compared with MWCNTs. [ABSTRACT FROM AUTHOR]

It has been shown before that exposure to nanomaterials (NMs) might promote the formation of foam cells, the key cells involved in all stages of atherosclerosis. However, to our best knowledge, previous studies, particularly in vitro studies, only investigated the transformation of macrophages into foam cells, whereas the importance of smooth muscle cells (SMCs) was overlooked. The present study investigated the toxicity of pristine multi-walled carbon nanotubes (MWCNTs; Code XFM19) and carboxylated MWCNTs (Code XFM21) to human aortic smooth muscle cells (HASMCs). The results showed that exposure to both types of MWCNTs significantly reduced mitochondrial activity but might not damage lysosomes. MWCNT exposure had minimal impact on cytokine release but significantly promoted lipid accumulation, which was significantly inhibited when the cells were pre-incubated with ER stress inhibitors or antioxidants. The mRNA levels of ER stress markers DDIT3 and XBP-1 s and protein levels of chop and p-chop were induced particularly by XFM21, accompanying with increased SREBF1 and SREBF2 mRNA as well as FASN protein, the key regulators involved in de novo lipogenesis. In addition, the mRNA levels of KLF4 and KLF5 and protein levels of KLF were induced after exposure to both types of MWCNTs, associated with an increase of CD68 protein levels. We concluded that MWCNTs might promote lipid accumulation in HASMCs through the induction of ER stress leading to de novo lipogenesis, as well as the activation of KLF pathway resulting in SMC transformation. Unlabelled Image • The cytotoxicity and inflammatory responses of MWCNTs to HASMCs were minimal. • MWCNTs promoted lipid accumulation in HASMCs without external lipid supply. • MWCNTs promoted ER stress leading to de novo lipogenesis. • MWCNTs also activated KLF pathway leading to SMC phenotype switch. [ABSTRACT FROM AUTHOR]

The Kruppel-like factors (KLF) family of zinc-finger transcriptional regulators control many aspects of cardiomyocyte structure and function. Deletion of Klf15 from the nuclear genome in mice affects cardiac mitochondria. Some become grossly enlarged, extending many sarcomeres in length. These display many sites of incipient pinching, but there is little attenuation of the megamitochondria at these sites; there are no examples of organelles that clearly have reached the point where further membrane encroachment will cause separation into smaller daughter mitochondria. It is clear that deletion of Klf15 interferes with nuclear control of mitochondrial fission, whereas fusion appears to be unaffected. [ABSTRACT FROM AUTHOR]

Abstract: Posttranslational modification (PTM) is a term that implies dynamic modification of proteins after their translation. PTM is involved not only in homeostasis but also in pathologic conditions related to diverse diseases. Histone deacetylases (HDACs), which are known as transcriptional regulators, are one example of posttranslational modifiers with diverse roles in human pathophysiology, including cardiovascular diseases. In experimental models, HDAC inhibitors are beneficial in supraventricular arrhythmia, myocardial infarction, cardiac remodeling, hypertension, and fibrosis. In addition, HDACs are closely related to other vascular diseases such as neointima formation, atherosclerosis, and vascular calcification. Currently, HDACs are classified into four different classes. The class IIa HDACs work as transcriptional regulators mainly by direct association with other transcription factors to their target binding elements in a phosphorylation-dependent manner. Class I HDACs, by contrast, have much greater enzymatic activity than the class II HDACs and target various non-histone proteins as well as the histone-core complex. Class I HDACs undergo PTMs such as phosphorylation, sumoylation, and S-nitrosylation. Considering the growing evidence for the role of HDACs in cardiovascular diseases, the PTMs of the HDACs themselves as well as HDAC-mediated PTM of their targets should be considered for future potential therapeutic targets. In this review, we discuss 1) the roles of each HDAC in specific cardiovascular diseases and 2) the PTM of HDACs, 3) and the implications of such modifications for cardiovascular diseases. [Copyright &y& Elsevier]

Abstract: In this study, we found that Krüppel-like factor (KLF) 9 activate the progression of the early phase of adipocyte differentiation in mouse adipocytic 3T3-L1 cells. KLF9 mRNA was detected in preadipocytes; and its level increased after the initiation of adipocyte differentiation, reached its maximum at 1h, and gradually decreased thereafter. Functional suppression of KLF9 mRNA by its siRNAs repressed the accumulation of the intracellular lipids with a reduction in the expression of CCAAT/enhancer-binding protein (C/EBP) β, but not in that of C/EBPδ. In contrast, C/EBPβ and C/EBPδ did not affect the expression of KLF9 in 3T3-L1 cells. A chromatin immunoprecipitation assay revealed that KLF9 bound the KLF binding element at position −874 of the mouse C/EBPβ promoter. Moreover, the ability of KLF9 to bind to this element was enhanced, with a peak at 1–2h after the initiation of adipogenesis, whose profile well resembled that of the expression of the C/EBPβ gene in 3T3-L1 cells. These results indicate that KLF9 activated the early phase of adipogenesis by enhancing the expression of the C/EBPβ gene in 3T3-L1 cells. [Copyright &y& Elsevier]

Abstract: The Krüppel-like family of transcription factors (KLFs) have been widely studied in proliferating cells, though very little is known about their role in post-mitotic cells, such as neurons. We have recently found that the KLFs play a role in regulating intrinsic axon growth ability in retinal ganglion cells (RGCs), a type of central nervous system (CNS) neuron. Previous KLF studies in other cell types suggest that there may be cell-type specific KLF expression patterns, and that their relative expression allows them to compete for binding sites, or to act redundantly to compensate for another''s function. With at least 15 of 17 KLF family members expressed in neurons, it will be important for us to determine how this complex family functions to regulate the intricate gene programs of axon growth and regeneration. By further characterizing the mechanisms of the KLF family in the nervous system, we may better understand how they regulate neurite growth and axon regeneration. [Copyright &y& Elsevier]

We report the isolation of the human orthologue of the mouse Osterix (Osx/Sp7) gene, a C2H2 zinc finger transcription factor of the SP gene family and putative “master” regulator of bone cell differentiation. The human SP7 cDNA encodes a putative 431 amino acid protein that contains three consecutive C2H2 zinc finger repeats. The SP7 protein is highly conserved between mice and humans with an overall sequence identity of 95%. The expression of a SP7 mRNA transcript of approximately 3.2 kb is restricted to bone-derived cell lines in vitro but undetectable in any adult tissues including mandibular bone by Northern blot hybridization. The specific expression of SP7 mRNA in osteoblasts in vivo was further confirmed by in situ hybridization on human embryonic tissues. The highly restricted expression pattern and the divergence of the sequence outside of the zinc finger region distinguish SP7 as a unique member of the SP family. The SP7 gene consists of two exons, with exon 2 containing most of the protein coding sequence. The gene locus was mapped to chromosome 12q13.13 by fluorescent in situ hybridization (FISH). The identification and initial characterization of the SP7 gene will facilitate the study of the molecular regulation of osteoblast differentiation in humans. [Copyright &y& Elsevier]

Sprouty2 plays a key role in negatively modulating the fibroblast growth factor signaling pathway, which is required for early branching events in embryonic development. The expression of the murine Sprouty2 gene shows a temporally and spatially restricted pattern in developing lung. In order to clarify the molecular mechanisms governing the transcription of the Sprouty2 gene, we first characterized the genomic organization of the human Sprouty2 (hSpry2) gene and mapped its transcription start sites by 5′-rapid amplification of cDNA ends. Subsequently, a 4-kb sequence from the 5′-flanking region of the gene was cloned and determined to contain promoter activity. Detailed truncation analysis of the hSpry2 promoter revealed the presence of context-specific suppressor activity in the distal upstream region. More importantly, we demonstrated that all the elements necessary to achieve strong basal transcription activity were located within the proximal 0.4-kb region. Sequence analysis revealed that this functionally important proximal region contains neither TATA nor CAAT box but an initiator element around the transcription start site. Several cis-acting elements (including AP2, CREB, SP1 and Ets-1) were found to be present in the proximal region, and their interactions with specific nuclear proteins were confirmed by electrophoretic mobility shift assays. To our knowledge, this is the first promoter study of any mammalian Sprouty gene. We propose that the high-level basal expression of hSpry2 is controlled by multiple transcription factors binding to its proximal promoter. [Copyright &y& Elsevier]

Pre-α-Inhibitor is a plasma protease inhibitor and a heterodimeric molecule whose one polypeptide chain is encoded by the ITIH3 gene. In order to understand the expression of this protein that is regulated in health and disease, we have analyzed the 5′ flanking region of ITIH3, specifically focussing on its proximal promoter. A combination of methods including wild-type (wt) or mutant promoter linked to a reporter cat gene, co-transfections of cat constructs with expression plasmids for nuclear factors and electrophoretic mobility shift assays revealed that two antagonistic sets of regulatory elements and nuclear proteins are critical for the activity of this promoter. Indeed, several overlapping Sp1/Sp3-binding sites are required for a sustained activity. However, a tripartite complex including CREB-2 and two molecules of the gut-enriched, Kru¨ppel-like factor cooperate to bind to an upstream area whose 3′ end overlaps the Sp1-binding sites. The resulting competition between this tripartite complex and Sp1 results in impaired occupancy of Sp1-binding sites by Sp1 and a consequent reduction in ITIH3 transcription. Competition between Sp1 and a Kru¨ppel-like factor for GC-rich sites has been previously reported, but this is the first description of an elaborate tripartite cooperation of two Kru¨ppel-like factors and CREB as a key step in such a competition. [Copyright &y& Elsevier]

Skeletal muscle dynamically regulates systemic nutrient homeostasis through transcriptional adaptations to physiological cues. In response to changes in the metabolic environment (e.g., alterations in circulating glucose or lipid levels), networks of transcription factors and coregulators are recruited to specific genomic loci to fine-tune homeostatic gene regulation. Elucidating these mechanisms is of particular interest as these gene regulatory pathways can serve as potential targets to treat metabolic disease. The zinc-finger transcription factor Krüppel-like factor 15 (KLF15) is a critical regulator of metabolic homeostasis; however, its genome-wide distribution in skeletal muscle has not been previously identified. Here, we characterize the KLF15 cistrome in vivo in skeletal muscle and find that the majority of KLF15 binding is localized to distal intergenic regions and associated with genes related to circadian rhythmicity and lipid metabolism. We also identify critical interdependence between KLF15 and the nuclear receptor PPARδ in the regulation of lipid metabolic gene programs. We further demonstrate that KLF15 and PPARδ colocalize genome-wide, physically interact, and are dependent on one another to exert their transcriptional effects on target genes. These findings reveal that skeletal muscle KLF15 plays a critical role in metabolic adaptation through its direct actions on target genes and interactions with other nodal transcription factors such as PPARδ., Competing Interests: Conflict of interest S. M. H. is an executive, officer, and shareholder of Amgen and a scientific founder and shareholder of Tenaya Therapeutics. S. M. H. also serves on the Scientific Advisory Board for the German Centre for Cardiovascular Research of the German Ministry of Health (DZHK) on a voluntary and uncompensated basis. Z. J. is an employee of Biomarin. The other authors have declared that no conflict of interest exists., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

A complex network of transcription factors regulates genes involved in establishing and maintaining key biological properties of the human airway epithelium. However, detailed knowledge of the contributing factors is incomplete. Here we characterize the role of Krüppel-like factor 5 (KLF5), in controlling essential pathways of epithelial cell identity and function in the human lung. RNA-seq following siRNA-mediated depletion of KLF5 in the Calu-3 lung epithelial cell line identified significant enrichment of genes encoding chemokines and cytokines involved in the proinflammatory response and also components of the junctional complexes mediating cell adhesion. To determine direct gene targets of KLF5, we defined the cistrome of KLF5 using ChIP-seq in both Calu-3 and 16HBE14o - lung epithelial cell lines. Occupancy site concordance analysis revealed that KLF5 colocalized with the active histone modification H3K27ac and also with binding sites for the transcription factor CCAAT enhancer-binding protein beta (C/EBPβ). Depletion of KLF5 increased both the expression and secretion of cytokines including IL-1β, a response that was enhanced following exposure to Pseudomonas aeruginosa lipopolysaccharide. Calu-3 cells exhibited faster rates of repair after KLF5 depletion compared with negative controls in wound scratch assays. Similarly, CRISPR-mediated KLF5-null 16HBE14o - cells also showed enhanced wound closure. These data reveal a pivotal role for KLF5 in coordinating epithelial functions relevant to human lung disease., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Gold (Au) nanomaterials (NMs), particularly those with PEG surface functionalization, are generally considered to be biocompatible for biomedical applications due to relatively low cytotoxicity. Herein, we investigated the toxicity of PEGylated Au nanorods (NRs) to human umbilical vein endothelial cells (HUVECs), a commonly used in vitro model for human endothelium. We found a previously unknown effect that up to 10 μg/mL Au NRs, albeit not cytotoxic, decreased the mRNA and protein levels of kruppel-like factor 2 (KLF2), a transcription factor with well-documented vasoprotective effects. The results from PCR array showed that a number of genes associated with risk of cardiovascular diseases were altered by Au NRs, and several genes are downstream genes of KLF2 according to ingenuity pathway analysis (IPA). These effects could be observed with or without the presence of inflammatory stimuli lipopolysaccharide (LPS), which suggests a pre-existing inflammatory state is not required for Au NRs to alter KLF2 signaling pathway. We further identified that Au NRs significantly decreased eNOS mRNA/p-eNOS proteins as well as increased MCP-1 mRNA/sMCP-1 release, which are targets of KLF2. Combined, our data revealed a novel pathway that PEGylated Au NPs at non-cytotoxic concentrations might alter KLF leading to the increase of risk of cardiovascular diseases in human endothelial cells. Given the importance of KLF in vascular homeostasis, our data indicate that it is necessary to evaluate the influence of engineered NPs to KLF signaling pathways, especially for NPs with biomedical uses. • Up to 10 μg/mL Au NRs were not cytotoxic to HUVECs with or without the presence of LPS. • The presence of LPS reduced the internalization of Au NRs. • Au NRs down-regulated the levels of KLF2 mRNA and proteins. • eNOS and MCP-1 are likely the downstream events of Au NR-induced KLF2 down-regulation. • LPS is not required for Au NRs to affect KLF2 signaling pathway. [ABSTRACT FROM AUTHOR]

Adipose tissue stores energy and plays an important role in energy homeostasis. CCAAT/enhancer-binding protein β (C/EBPβ) is an important early transcription factor for 3T3-L1 preadipocyte differentiation, facilitating mitotic clonal expansion (MCE) and transactivating C/EBPα and peroxisome proliferator-activated receptor-γ (PPARγ) to promote adipogenesis. C/EBPβ is induced early, but the expression of antimitotic C/EBPα and PPARγ is not induced until ∼48 h. The delayed expression of C/EBPα and PPARγ is thought to ensure MCE progression, but the molecular mechanism for this delay remains elusive. Here, we show that the zinc-finger transcription factor Krüppel-like factor 10 (KLF10) is induced after adipogenic induction and that its expression positively correlates with that of C/EBPβ but inversely correlates with expression of C/EBPα and PPARγ. C/EBPβ bound to the KLF10 promoter and transactivated its expression during MCE. KLF10 overexpression in 3T3-L1 preadipocyte repressed adipogenesis and decreased C/EBPα and PPARγ expression, whereas siRNA-mediated down-regulation of KLF10 enhanced adipogenesis and increased C/EBPα and PPARγ expression. Luciferase assays revealed an inhibitory effect of KLF10 on C/EBPα promoter activity. Using promoter deletion and mutation analysis, we identified a KLF10-binding site within the proximal promoter region of C/EBPα. Furthermore, KLF10 interacted with and recruited histone deacetylase 1 (HDAC1) to the C/EBPα promoter, decreasing acetylated histone H4 on the C/EBPα promoter and inactivating C/EBPα transcription. Because C/EBPα can transactivate PPARγ, our results suggest a mechanism by which expression of C/EBPα and PPARγ is delayed via KLF10 expression and shed light on the negative feedback loop for C/EBPβ-regulated adipogenesis in 3T3-L1 preadipocyte., (© 2018 Liu et al.)

Macrophages are the predominant innate immune cells recruited to tissues following injury or infection. These early-responding, pro-inflammatory macrophages play an essential role in the amplification of inflammation. However, macrophage pro-inflammatory gene expression should be tightly regulated to avert host tissue damage. In this study, we identify the Kruppel-like transcription factor 6 (KLF6)-B cell leukemia/lymphoma 6 (BCL6) signaling axis as a novel regulator of macrophage inflammatory gene expression and function. Utilizing complementary gain- and loss-of-function studies, we observed that KLF6 is essential for macrophage motility under ex vivo and in vivo conditions. Concordant with these observations, myeloid-specific deficiency of KLF6 significantly attenuates macrophage pro-inflammatory gene expression, recruitment, and progression of inflammation. At the molecular level, KLF6 suppresses BCL6 mRNA and protein expression by elevating PR domain-containing 1 with ZNF domain (PRDM1) levels in macrophages. Interestingly, pharmacological or genetic inhibition of BCL6 in KLF6-deficient macrophages completely abrogated the attenuation of pro-inflammatory cytokine/chemokine expression and cellular motility. Collectively, our observations reveal that KLF6 repress BCL6 to enhance macrophage inflammatory gene expression and function., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Glucocorticoid receptor (GR) signaling has recently been shown to play a direct role in the regulation of cardiomyocyte function. In this study, we investigated the potential role of KLF13 as a downstream effector of GR action utilizing both in vivo and in vitro approaches. Our data show that KLF13 mRNA and protein levels are significantly diminished in the hearts of mice lacking GR in cardiomyocytes. Glucocorticoid administration up-regulated Klf13 mRNA in the mouse heart, in isolated primary cardiomyocytes, and in immortal cardiomyocyte cell lines. Glucocorticoid Klf13 gene expression was abolished by treatment with a GR antagonist (RU486) or by knockdown of GR in cardiomyocytes. Moreover, glucocorticoid induction of Klf13 mRNA was resistant to de novo protein synthesis inhibition, demonstrating that Klf13 is a direct glucocorticoid receptor gene target. A glucocorticoid responsive element (GRE) was identified in the Klf13 gene and its function was verified by chromatin immunoprecipitation in HL-1 cells and mouse hearts. Functional studies showed that GR regulation of Klf13 is critical to protect cardiomyocytes from DNA damage and cell death induced by cobalt(II) chloride hexahydrate (CoCl2·6H2O) and the antineoplastic drug doxorubicin. These results established a novel role for GR and KLF13 signaling in adult cardiomyocytes with potential clinical implications for the prevention of cardiotoxicity induced heart failure., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Regenerative medicine holds great promise for the treatment of degenerative retinal disorders. Krüppel-like factors (KLFs) are transcription factors that have recently emerged as key tools in regenerative medicine because some of them can function as epigenetic reprogrammers in stem cell biology. Here, we show that KLF16, one of the least understood members of this family, is a POU4F2 independent transcription factor in retinal ganglion cells (RGCs) as early as embryonic day 15. When overexpressed, KLF16 inhibits RGC neurite outgrowth and enhances RGC growth cone collapse in response to exogenous ephrinA5 ligands. Ephrin/EPH signaling regulates RGC connectivity. The EphA5 promoter contains multiple GC- and GT-rich KLF-binding sites, which, as shown by ChIP-assays, bind KLF16 in vivo In electrophoretic mobility shift assays, KLF16 binds specifically to a single KLF site near the EphA5 transcription start site that is required for KLF16 transactivation. Interestingly, methylation of only six of 98 CpG dinucleotides within the EphA5 promoter blocks its transactivation by KLF16 but enables transactivation by KLF2 and KLF15. These data demonstrate a role for KLF16 in regulation of RGC neurite outgrowth and as a methylation-sensitive transcriptional regulator of EphA5 expression. Together, these data identify differential low level methylation as a novel mechanism for regulating KLF16-mediated EphA5 expression across the retina. Because of the critical role of ephrin/EPH signaling in patterning RGC connectivity, understanding the role of KLFs in regulating neurite outgrowth and Eph receptor expression will be vital for successful restoration of functional vision through optic nerve regenerative therapies., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The Lgals3 gene encodes a multifunctional β-galactoside-binding protein, galectin-3. Galectin-3 has been implicated in a broad range of biological processes from chemotaxis and inflammation to fibrosis and apoptosis. The role of galectin-3 as a modulator of inflammation has been studied intensively, and recent evidence suggests that it may serve as a protective factor in obesity and other metabolic disorders. Despite considerable interest in galectin-3, little is known about its physiological regulation at the transcriptional level. Here, using knockout mice, chromatin immunoprecipitations, and cellular and molecular analyses, we show that the zinc finger transcription factor Krüppel-like factor 3 (KLF3) directly represses galectin-3 transcription. We find that galectin-3 is broadly up-regulated in KLF3-deficient mouse tissues, that KLF3 occupies regulatory regions of the Lgals3 gene, and that KLF3 directly binds its cognate elements (CACCC boxes) in the galectin-3 promoter and represses its activation in cellular assays. We also provide mechanistic insights into the regulation of Lgals3, demonstrating that C-terminal binding protein (CtBP) is required to drive optimal KLF3-mediated silencing. These findings help to enhance our understanding of how expression of the inflammatory modulator galectin-3 is controlled, opening up avenues for potential therapeutic interventions in the future., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Erythroid Kruppel-like factor (EKLF or KLF1) is a transcription factor crucial for red cell development that is directly involved in regulation of a large number of erythroid genes. EKLF serves mostly as an activator of expression of these genes; however, it can act also as a repressor. Here, we present evidence that EKLF interacts with proteins from the PIAS (protein inhibitor of activated STAT) family that convey repressive activity to EKLF in the absence of sumoylation. Our studies identify PIAS3 as a transcriptional corepressor of EKLF for at least a subset of its target genes during erythropoiesis (e.g. β-globin, α-hemoglobin stabilizing protein). We demonstrate an interaction between EKLF and PIAS proteins confirmed by in vivo coimmunoprecipitation assays with both exogenous and endogenous proteins. We identified an LXXLL signature motif located near the N terminus of PIAS proteins that, although not involved in the EKLF-PIAS3 interaction, is required for the transrepression activity. Knockdown of endogenous PIAS3 accelerates differentiation of both murine erythroleukemia cells, as well as fetal liver cells, whereas an increase in PIAS3 levels inhibits this increase. Using chromatin immunoprecipitation assays, we show that PIAS3 preferentially occupies the β-globin promoter in undifferentiated murine erythroleukemia cells. Together these results demonstrate that an interaction between EKLF and PIAS3 provides a novel mode of regulation of EKLF activity in the absence of sumolylation and furthermore shows an important involvement of PIAS proteins in erythropoiesis., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Krüppel-like factor 3 (KLF3/BKLF), a member of the Krüppel-like factor (KLF) family of transcription factors, is a widely expressed transcriptional repressor with diverse biological roles. Although there is considerable understanding of the molecular mechanisms that allow KLF3 to silence the activity of its target genes, less is known about the signal transduction pathways and post-translational modifications that modulate KLF3 activity in response to physiological stimuli. We observed that KLF3 is modified in a range of different tissues and found that the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) can both bind and phosphorylate KLF3. Mass spectrometry identified serine 249 as the primary phosphorylation site. Mutation of this site reduces the ability of KLF3 to bind DNA and repress transcription. Furthermore, we also determined that HIPK2 can phosphorylate the KLF3 co-repressor C-terminal binding protein 2 (CtBP2) at serine 428. Finally, we found that phosphorylation of KLF3 and CtBP2 by HIPK2 strengthens the interaction between these two factors and increases transcriptional repression by KLF3. Taken together, our results indicate that HIPK2 potentiates the activity of KLF3., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

It is increasingly important to understand the molecular basis for the plasticity of neoplastic cells and their capacity to transition between differentiated and stemlike phenotypes. Kruppel-like factor-9 (KLF9), a member of the large KLF transcription factor family, has emerged as a regulator of oncogenesis, cell differentiation, and neural development; however, the molecular basis for the diverse contextual functions of KLF9 remains unclear. This study focused on the functions of KLF9 in human glioblastoma stemlike cells. We established for the first time a genome-wide map of KLF9-regulated targets in human glioblastoma stemlike cells and show that KLF9 functions as a transcriptional repressor and thereby regulates multiple signaling pathways involved in oncogenesis and stem cell regulation. A detailed analysis of one such pathway, integrin signaling, showed that the capacity of KLF9 to inhibit glioblastoma cell stemness and tumorigenicity requires ITGA6 repression. These findings enhance our understanding of the transcriptional networks underlying cancer cell stemness and differentiation and identify KLF9-regulated molecular targets applicable to cancer therapeutics., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The histone deacetylase inhibitor (HDACi) sodium butyrate promotes differentiation of colon cancer cells as evidenced by induced expression and enzyme activity of the differentiation marker intestinal alkaline phosphatase (ALPi). Screening of a panel of 33 colon cancer cell lines identified cell lines sensitive (42%) and resistant (58%) to butyrate induction of ALP activity. This differential sensitivity was similarly evident following treatment with the structurally distinct HDACi, MS-275. Resistant cell lines were significantly enriched for those harboring the CpG island methylator phenotype (p = 0.036, Chi square test), and resistant cell lines harbored methylation of the ALPi promoter, particularly of a CpG site within a critical KLF/Sp regulatory element required for butyrate induction of ALPi promoter activity. However, butyrate induction of an exogenous ALPi promoter-reporter paralleled up-regulation of endogenous ALPi expression across the cell lines, suggesting the presence or absence of a key transcriptional regulator is the major determinant of ALPi induction. Through microarray profiling of sensitive and resistant cell lines, we identified KLF5 to be both basally more highly expressed as well as preferentially induced by butyrate in sensitive cell lines. KLF5 overexpression induced ALPi promoter-reporter activity in resistant cell lines, KLF5 knockdown attenuated butyrate induction of ALPi expression in sensitive lines, and butyrate selectively enhanced KLF5 binding to the ALPi promoter in sensitive cells. These findings demonstrate that butyrate induction of the cell differentiation marker ALPi is mediated through KLF5 and identifies subsets of colon cancer cell lines responsive and refractory to this effect., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Sphingosine kinase 1 (SK1) is an FGF-inducible gene responsible for generation of sphingosine-1-phosphate, a critical lipid signaling molecule implicated in diverse endothelial cell functions. In this study, we identified SK1 as a target of the canonical FGF2/FGF receptor 1 activation pathway in endothelial cells and sought to identify novel transcriptional pathways that mediate lipid signaling. Studies using the 1.9-kb SK1 promoter and deletion mutants revealed that basal and FGF2-stimulated promoter activity occurred through two GC-rich regions located within 633 bp of the transcription start site. Screening for GC-rich binding transcription factors that could activate this site demonstrated that KLF14, a gene implicated in obesity and the metabolic syndrome, binds to this region. Congruently, overexpression of KLF14 increased basal and FGF2-stimulated SK1 promoter activity by 3-fold, and this effect was abrogated after mutation of the GC-rich sites. In addition, KLF14 siRNA transfection decreased SK1 mRNA and protein levels by 3-fold. Congruently, SK1 mRNA and protein levels were decreased in livers from KLF14 knock-out mice. Combined, luciferase, gel shift, and chromatin immunoprecipitation assays showed that KLF14 couples to p300 to increase the levels of histone marks associated with transcriptional activation (H4K8ac and H3K14ac), while decreasing repressive marks (H3K9me3 and H3K27me3). Collectively, the results demonstrate a novel mechanism whereby SK1 lipid signaling is regulated by epigenetic modifications conferred by KLF14 and p300. Thus, this is the first description of the activity and mechanisms underlying the function of KLF14 as an activator protein and novel regulator of lipid signaling., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The transcription factor NF-κB plays a pivotal role in a broad range of physiological and pathological processes, including development, inflammation, and immunity. How NF-κB integrates activating signals to expression of specific sets of target genes is of great interest. Here, we identified Krüppel-like factor 6 (KLF6) as a co-activator of NF-κB after TNFα and IL-1β stimulation. Overexpression of KLF6 enhanced TNFα- and IL-1β-induced activation of NF-κB and transcription of a subset of downstream genes, whereas knockdown of KLF6 had opposite effects. KLF6 interacted with p65 in the nucleus and bound to the promoters of target genes. Upon IL-1β stimulation, KLF6 was recruited to promoters of a subset of NF-κB target genes in a p65-dependent manner, which was in turn required for the optimal binding of p65 to the target gene promoters. Our findings thus identified KLF6 as a previously unknown but essential co-activator of NF-κB and provided new insight into the molecular regulation of p65-dependent gene expression.

The S100 protein family represents the largest subgroup of calcium binding EF-hand type proteins. These proteins have been reported to be involved in a wide range of biological functions that are related to normal cell development and tumorigenesis. S100A14 is a recently identified member of the S100 protein family and differentially expressed in a number of different human malignancies. However, the transcriptional regulation of S100A14 and its role in breast cancer needs to be further investigated. Here, we determined that 12-O-tetradecanoylphorbol-13-acetate (TPA) up-regulated the expression of KLF4 and facilitated its binding directly to two conserved GC-rich DNA segments within the S100A14 promoter, which is essential for the transactivation of KLF4 induced S100A14 expression. Furthermore, stable silencing of KLF4 significantly suppressed breast cancer cell migration induced by TPA. Collectively, these results offer insights into the fact that TPA provokes cell motility through regulating the expression and function of S100A14 in a KLF4-dependent manner.

The mammalian heart, the body's largest energy consumer, has evolved robust mechanisms to tightly couple fuel supply with energy demand across a wide range of physiologic and pathophysiologic states, yet, when compared with other organs, relatively little is known about the molecular machinery that directly governs metabolic plasticity in the heart. Although previous studies have defined Kruppel-like factor 15 (KLF15) as a transcriptional repressor of pathologic cardiac hypertrophy, a direct role for the KLF family in cardiac metabolism has not been previously established. We show in human heart samples that KLF15 is induced after birth and reduced in heart failure, a myocardial expression pattern that parallels reliance on lipid oxidation. Isolated working heart studies and unbiased transcriptomic profiling in Klf15-deficient hearts demonstrate that KLF15 is an essential regulator of lipid flux and metabolic homeostasis in the adult myocardium. An important mechanism by which KLF15 regulates its direct transcriptional targets is via interaction with p300 and recruitment of this critical co-activator to promoters. This study establishes KLF15 as a key regulator of myocardial lipid utilization and is the first to implicate the KLF transcription factor family in cardiac metabolism.

Rosiglitazone, a well known insulin sensitizer, stimulates adipocyte differentiation via the activation of peroxisome proliferator-activated receptor γ (PPARγ). Previous two-dimensional proteomics studies using C3H10T1/2 murine mesenchymal pluripotent stem cells revealed that prolyl hydroxylase domain protein (PHD) levels significantly increased during rosiglitazone-induced adipocyte differentiation (RIAD). In this study, we investigated the functional role played by PHD during RIAD. Three PHD isoforms (PHD1, 2, and 3) were found to be up-regulated in C3H10T1/2 cells during RIAD, whereas PHD knockdown and treatment with PHD inhibitors (dimethyloxalyl glycine or ethyl-3,4-dihydroxybenzoate) blocked RIAD. PHD inhibition was found to be associated with increases in the levels of anti-adipogenic proteins such as GATA-3, KLF-2, and transcriptional coactivator with PDZ binding motif (TAZ), with their reduced ubiquitination, suggesting that PHDs evoke the ubiquitination/proteasomal degradation of anti-adipogenic proteins. On the other hand, MG-132 (a proteasomal inhibitor) prevented the degradation of anti-adipogenic proteins and retarded RIAD. PPARγ antagonists (bisphenol A diglycidyl ether or GW9662) blunted the effects of rosiglitazone on PHD regulation. Furthermore, putative PPARγ binding sites were identified in the promoter region of PHDs by ChIP-PCR, implying that rosiglitazone may induce PHD up-regulation directly by PPARγ activation. Consistent with in vitro results, oral administration of rosiglitazone to ob/ob mice for 2 weeks increased adipose PHD levels and decreased anti-adipogenic protein levels by increasing their ubiquitination. These results suggest that rosiglitazone increases PHD expression in a PPARγ-dependent manner and that this leads to the commitment of anti-adipogenic proteins to the ubiquitination-proteasomal pathway and to the subsequent induction of adipocyte differentiation.

Macrophage activation is a central event in immune responses. Macrophages undergoing classical activation (M1 macrophages) are proinflammatory, whereas alternatively activated macrophages (M2 macrophages) are generally anti-inflammatory. miRNAs play important regulatory roles in inflammatory response. However, the manner in which miRNAs regulate macrophage activation in response to different environmental cues has not been well defined. In this study, we found that M-BMM macrophages (M2) express greater levels of miR-125a-5p than do GM-BMM macrophages (M1). Stimulation of macrophages through TLR2 and TLR4 but not through TLR3 enhanced miR-125a-5p expression. Up-regulation of miR-125a-5p after TLR2/4 activation requires the adaptor MYD88 but not TRIF. Overexpression of miR-125a-5p diminished M1 phenotype expression induced by LPS but promoted M2 marker expression induced by IL-4. In contrast, knockdown of miR-125a-5p promoted M1 polarization and diminished IL-4-induced M2 marker expression. We found that miR-125a-5p targets KLF13, a transcriptional factor that has an important role in T lymphocyte activation and inflammation. KLF13 knockdown had similar effects on M1 activation as did miR-125a-5p overexpression. In addition, miR-125a-5p regulates phagocytic and bactericidal activities of macrophages. Our data suggest that miR-125a-5p has an important role in suppressing classical activation of macrophages while promoting alternative activation.

The initiation and maintenance of the immune response require a coordinated regulation of signal transduction pathways. Identifying the mechanisms by which these pathways are controlled and modulated is a significant goal of immunology. In the present report, we show a novel role for the zinc finger transcription factor Kruppel-like factor 4 (KLF4) in the modulation of the inflammatory immune response via its regulation of IL-6. We analyzed the role of KLF4 in the production of IL-6 by dendritic cells. Our data indicate that KLF4 can act in a dual function manner. It acts as a transcription factor in that it can bind to and activate the IL-6 promoter at specific binding sites. KLF4 also has a role in the chromatin remodeling of the IL-6 promoter in that cells deficient in KLF4 exhibited a relative hypoacetylation. These results indicate a molecular role for KLF4 in modulating the intensity of the inflammatory response and help to explain its pleiotropic role in different settings.

The function of Krüppel-like factor 11 (KLF11) in the regulation of metabolic pathways is conserved from flies to human. Alterations in KLF11 function result in maturity onset diabetes of the young 7 (MODY7) and neonatal diabetes; however, the mechanisms underlying the role of this protein in metabolic disorders remain unclear. Here, we investigated how the A347S genetic variant, present in MODY7 patients, modulates KLF11 transcriptional activity. A347S affects a previously identified transcriptional regulatory domain 3 (TRD3) for which co-regulators remain unknown. Structure-oriented sequence analyses described here predicted that the KLF11 TRD3 represents an evolutionarily conserved protein domain. Combined yeast two-hybrid and protein array experiments demonstrated that the TRD3 binds WD40, WWI, WWII, and SH3 domain-containing proteins. Using one of these proteins as a model, guanine nucleotide-binding protein β2 (Gβ2), we investigated the functional consequences of KLF11 coupling to a TRD3 binding partner. Combined immunoprecipitation and biomolecular fluorescence complementation assays confirmed that activation of three different metabolic G protein-coupled receptors (β-adrenergic, secretin, and cholecystokinin) induces translocation of Gβ2 to the nucleus where it directly binds KLF11 in a manner that is disrupted by the MODY7 A347S variant. Using genome-wide expression profiles, we identified metabolic gene networks impacted upon TRD3 disruption. Furthermore, A347S disrupted KLF11-mediated increases in basal insulin levels and promoter activity and blunted glucose-stimulated insulin secretion. Thus, this study characterizes a novel protein/protein interaction domain disrupted in a KLF gene variant that associates to MODY7, contributing to our understanding of gene regulation events in complex metabolic diseases.

KLF4 is a member of the Kruppel-like factor family of transcriptional regulators. KLF4 has been shown to be required for normal terminal differentiation of keratinocytes, but the molecular mechanism whereby KLF4 regulates genes associated with the differentiation process has not been studied. In the present study, we explore the impact of KLF4 on expression of involucrin, a gene that is specifically expressed in differentiated keratinocytes. KLF4 overexpression and knockdown studies show that involucrin mRNA and protein level correlates directly with KLF4 level. Moreover, studies of mutant KLF4 proteins indicate that transcriptionally inactive forms do not increase involucrin expression. PKCδ is a regulator of keratinocyte differentiation that increases expression of differentiation-associated target genes, including involucrin. Overexpression of KLF4 augments the PKCδ-dependent increase in involucrin expression, whereas KLF4 knockdown attenuates this response. The KLF4 induction of human involucrin (hINV) promoter activity is mediated via KLF4 binding to a GC-rich element located in the hINV promoter distal regulatory region, a region of the promoter required for in vivo involucrin expression. Mutation of the GC-rich element, an adjacent AP1 factor binding site, or both sites severely attenuates the response. Moreover, loss of KLF4 in an epidermal equivalent model of differentiation results in loss of hINV expression. These studies suggest that KLF4 is part of a multiprotein complex that interacts that the hINV promoter distal regulatory region to drive differentiation-dependent hINV gene expression in epidermis.