Krüppel-like Factor 5 Knockdown Research Articles

Influences and mechanism of extracellular vesicles from dermal papilla cells of mice on human hypertrophic scar fibroblasts

Objective: To investigate the influences and mechanism of extracellular vesicles from dermal papilla cells (DPC-EVs) of mice on human hypertrophic scar fibroblasts (HSFs). Methods: The study was an experimental research. The primary dermal papilla cells (DPCs) of whiskers were extracted from 10 6-week-old male C57BL/6J mice and identified successfully. The DPC-EVs were extracted from the 3rd to 5th passage DPCs by ultracentrifugation, and the morphology was observed through transmission electron microscope and the particle diameter was detected by nanoparticle tracking analyzer (n=3) at 24 h after culture. The 3rd passage of HSFs were divided into DPC-EV group and phosphate buffer solution (PBS) group, which were cultured with DPC-EVs and PBS, respectively. The cell scratch test was performed and cell migration rate at 24 h after scratching was calculated (n=5). The cell proliferation levels at 0 (after 12 h of starvation treatment and before adding DPC-EVs or PBS), 24, 48, 72, and 96 h after culture were detected by using cell counting kit 8 (n=4). The protein expressions of α-smooth muscle actin (α-SMA) and collagen typeⅠ (ColⅠ) in cells at 24 h after culture were detected by immunofluorescence method and Western blotting, and the protein expression of Krüppel-like factor 4 (KLF4) in cells at 24 h after culture was detected by Western blotting. After the 3rd passage of HSFs were cultured with DPC-EVs for 24 h, the cells were divided into blank control group, KLF4 knockdown group, and KLF4 overexpression group according to the random number table. The cells in blank control group were only routinely cultured for 48 h. The cells in KLF4 knockdown group and KLF4 overexpression group were incubated with KLF4 knockdown virus for 24 h, then the cells in KLF4 knockdown group were routinely cultured for 24 h while the cells in KLF4 overexpression group were incubated with KLF4 overexpression virus for 24 h. The protein expressions of KLF4, α-SMA, and ColⅠ in cells were detected by Western blotting at 48 h after culture. Results: At 24 h after culture, the extracted DPC-EVs showed vesicular structure with an average particle diameter of 108.8 nm. At 24 h after scratching, the migration rate of HSFs in PBS group was (54±10)%, which was significantly higher than (29±8)% in DPC-EV group (t=4.37, P<0.05). At 48, 72, and 96 h after culture, the proliferation levels of HSFs in DPC-EV group were significantly lower than those in PBS group (with t values of 4.06, 5.76, and 6.41, respectively, P<0.05). At 24 h after culture, the protein expressions of α-SMA and ColⅠ of HSFs in DPC-EV group were significantly lower than those in PBS group, while the protein expression of KLF4 was significantly higher than that in PBS group. At 48 h after culture, compared with those in blank control group, the protein expression of KLF4 of HSFs in KLF4 knockdown group was down-regulated, while the protein expressions of α-SMA and ColⅠ were both up-regulated; compared with those in KLF4 knockdown group, the protein expression of KLF4 of HSFs in KLF4 overexpression group was up-regulated, while the protein expressions of ColⅠ and α-SMA were down-regulated. Conclusions: The DPC-EVs of mice can inhibit the proliferation and migration of human HSFs and significantly inhibit the expressions of fibrosis markers α-SMA and ColⅠ in human HSFs by activating KLF4.

Early growth response 1/Krüppel-like factor 5 pathway inhibitor alleviates lipopolysaccharide-induced lung injury by promoting autophagy

Early transcription factors play critical roles in the development of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Early growth response 1 (EGR1) is a transcription factor essential for various biological processes, including regulation of metabolism, differentiation, and inflammation. However, its role in ALI has been poorly reported. In this study, we aimed to determine the effect of EGR1 on ALI to gain insights into the theoretical basis for further treatment of ALI. By employing concerted molecular biology techniques, we showed that EGR1 protein was upregulated in mice. EGR1 protein was upregulated in mice and human lung epithelial cells in response to lipopolysaccharide (LPS) stimulation. EGR1 knockdown promoted autophagy and reduced LPS-induced pro-inflammatory mediator production. EGR1 was preferentially bound to the GCGTGGGCG motif region and EGR1-binding peak-related genes were mainly enriched in autophagy and injury stress-related pathways. Additionally, EGR1 promoted Krüppel-like factor 5 (KLF5) transcription by binding to the KLF5 promoter region, and KLF5 knockdown significantly decreased inflammatory damage, suggesting that EGR1 promotes ALI progression by regulating KLF5 expression. Furthermore, ML264, an inhibitor of the EGR1/KLF5 pathway axis, displayed a protective role in ALI to reduce inflammation. In conclusion, our findings demonstrate the potential of EGR1 knockdown to inhibit KLF5 and promote autophagy, further reducing the inflammatory response to mitigate ALI/ARDS. The EGR1/KLF5 pathway axis may be a valuable therapeutic target for the treatment of ALI/ARDS.

KLF4 regulates skeletal muscle development and regeneration by directly targeting P57 and Myomixer

Krüppel-like factor 4 (KLF4) is an evolutionarily conserved zinc finger-containing transcription factor that regulates diverse cellular processes such as cell proliferation, apoptosis, and differentiation. Our previous study showed that KLF4 expression is upregulated in skeletal muscle ontogeny during embryonic development in pigs, suggesting its importance for skeletal muscle development and muscle function. We revealed here that KLF4 plays a critical role in skeletal muscle development and regeneration. Specific knockout of KLF4 in skeletal muscle impaired muscle formation further affecting physical activity and also defected skeletal muscle regeneration. In vitro, KLF4 was highly expressed in proliferating myoblasts and early differentiated cells. KLF4 knockdown promoted myoblast proliferation and inhibited myoblast fusion, while its overexpression showed opposite results. Mechanically, in proliferating myoblasts, KLF4 inhibits myoblast proliferation through regulating cell cycle arrest protein P57 by directly targeting its promoter; while in differentiated myoblasts, KLF4 promotes myoblast fusion by transcriptionally activating Myomixer. Our study provides mechanistic information for skeletal muscle development, reduced muscle strength and impaired regeneration after injury and unveiling the mechanism of KLF4 in myogenic regulation.

Ursolic Acid Inhibits the Growth of Gastric Cancer by Targeting KLF4/YAP1

Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor which has various mechanisms in different tumors. Ursolic acid (UA), a natural compound that exists in many herbs, is known to prevent tumor progression and has anticancer effects on many human cancers. The present study was to evaluate the antitumor effect of UA on gastric cancer (GC) through KLF4 and the Hippo pathway. Our data showed that UA inhibited the growth of GC in vivo and in vitro. UA treatment significantly increased the expression of KLF4 and decreased the expression of CTGF. The overexpression of KLF4 inhibited the proliferation and cell cycle of GC and decreased the expression of CTGF, whereas the knockdown of KLF4 attenuated the effects of UA. Furthermore, the inhibition of CTGF arrested tumor cells in G2/M which blocked proliferation progress. Confocal laser scanning and molecular simulation software MOE showed that KLF4 combined with YAP1 which may block the formation of the TEADs-YAP1 complex to interrupt the expression of CTGF and the downstream oncogenic process. In conclusion, UA can inhibit GC growth both in vivo and in vitro, and it activated KLF4 which may competitively bind with YAP1 against TEADs and block the oncogenic Hippo pathways.

KLF5/MDM2 Axis Modulates Oxidative Stress and Epithelial-Mesenchymal Transition in Human Lens Epithelial Cells: The Role in Diabetic Cataract

Diabetic cataract (DC) is a common cause of visual loss in older diabetic subjects. Krüppel-like factor 5 (KLF5) plays an essential role in migration and the epithelial-mesenchymal transition (EMT) in diverse cells and is involved in oxidative stress. However, the effects of KLF5 on DC remain unknown. This study aimed to examine the biological function of KLF5 in DC and its underlying mechanism. The expression patterns of KLF5 were detected in vivo and in vitro. Then, KLF5 was knocked down in human lens epithelial cells (HLECs) to explore its functional roles and underlying mechanisms. Dual-luciferase reporter assay and chromatin immunoprecipitation analysis were used to detect whether KLF5 could bind the promoter of E3 ubiquitin ligase mouse double minute 2 (MDM2), a key regulator of EMT. Lastly, the regulation of KLF5 in the biological behaviors of HLECs via MDM2 was analyzed. We found a significant increase of KLF5 in the DC lens anterior capsular, diabetic rat lens, and high glucose (HG)-stimulated HLECs. Knockdown of KLF5 inhibited oxidative stress, inflammation, migration, and EMT of HG-stimulated HLECs. KLF5 silencing impeded MDM2 expression and restricted the activation of MARK1/FAK and NF-κB signaling pathways in HLECs under HG condition. Additionally, KLF5 was found to bind the MDM2 promoter and enhance the transcriptional activity of MDM2. The protective effects by silencing KLF5 on HG-cultured HLECs could be offset by MDM2 overexpression. We demonstrated that knockdown of KLF5 alleviated oxidative stress, migration, and EMT of HG-cultured HLECs by regulating MDM2, suggesting a potential therapeutic strategy for DC.

MIR-92A/KLF4/P110Δ REGULATES TITANIUM PARTICLE-INDUCED MACROPHAGE INFLAMMATION AND OSTEOLYSIS

As peri-prosthetic aseptic loosening is one of the main causes of implant failure, inhibiting wear particles induced macrophages inflammation is considered as a promising therapy for AL to expand the lifespan of implant. Here, we aim at exploring the role of p110δ, a member of class IA PI3K family, and Krüppel-like factor 4 (KLF4) in titanium particles (TiPs) induced macrophages-inflammation and osteolysis.Firstly, IC87114, the inhibitor of p110δ and siRNA targeting p110δ were applied and experiments including ELISA and immunofluorescence assay were conducted to explore the role of p110δ. Sequentially, KLF4 was predicted as the transcription factor of p110δ and the relation was confirmed by dual luciferase reporter assay. Next, assays including RT-PCR, western blotting and flow cytometry were performed to ensure the specific role of KLF4. Finally, TiPs-induced mice cranial osteolysis model was established, and micro-CT scanning and immunohistochemistry assay were performed to reveal the role of p110δ and KLF4 in vivo.Here, we found that p110δ was upregulated in TiPs-stimulated macrophages. The inhibition of p110δ or knockdown of p110δ could significantly dampen the TiPs-induced secretion of TNFα and IL-6. Further mechanistic studies confirmed that p110δ was responsible for TNFα and IL-6 trafficking out of Golgi complex without affecting their expression in TiPs-treated macrophages. Additionally, we explored the upstream regulators and confirmed that Krüppel-like factor 4 (KLF4) was the transcription repressor of p110δ. Apart from that, KLF4, targeted by miR-92a, could also attenuate TiPs-induced inflammation by mediating NF-κB pathway and M1/M2 polarization. By the establishment of TiPs-induced mice cranial osteolysis model, we found that KLF4 knockdown exacerbated TiPs-induced osteolysis which was strikingly ameliorated by knockdown of p110δ.In summary, our study suggests the key role of miR-92a/KLF4/p110δ signal in TiPs-induced macrophages inflammation and osteolysis.

Extracellular vesicles from 3D cultured dermal papilla cells improve wound healing via Krüppel-like factor 4/vascular endothelial growth factor A -driven angiogenesis.

Non-healing wounds are an intractable problem of major clinical relevance. Evidence has shown that dermal papilla cells (DPCs) may regulate the wound-healing process by secreting extracellular vesicles (EVs). However, low isolation efficiency and restricted cell viability hinder the applications of DPC-EVs in wound healing. In this study, we aimed to develop novel 3D-DPC spheroids (tdDPCs) based on self-feeder 3D culture and to evaluate the roles of tdDPC-EVs in stimulating angiogenesis and skin wound healing. To address the current limitations of DPC-EVs, we previously developed a self-feeder 3D culture method to construct tdDPCs. DPCs and tdDPCs were identified using immunofluorescence staining and flow cytometry. Subsequently, we extracted EVs from the cells and compared the effects of DPC-EVs and tdDPC-EVs on human umbilical vein endothelial cells (HUVECs) in vitro using immunofluorescence staining, a scratch-wound assay and a Transwell assay. We simultaneously established a murine model of full-thickness skin injury and evaluated the effects of DPC-EVs and tdDPC-EVs on wound-healing efficiency in vivo using laser Doppler, as well as hematoxylin and eosin, Masson, CD31 and α-SMA staining. To elucidate the underlying mechanism, we conducted RNA sequencing (RNA-seq) of tdDPC-EV- and phosphate-buffered saline-treated HUVECs. To validate the RNA-seq data, we constructed knockdown and overexpression vectors of Krüppel-like factor 4 (KLF4). Western blotting, a scratch-wound assay, a Transwell assay and a tubule-formation test were performed to detect the protein expression, cell migration and lumen-formation ability of KLF4 and vascular endothelial growth factor A (VEGFA) in HUVECs incubated with tdDPC-EVs after KLF4 knockdown or overexpression. Dual-luciferase reporter gene assays were conducted to verify the activation effect of KLF4 on VEGFA. We successfully cultured tdDPCs and extracted EVs from DPCs and tdDPCs. The tdDPC-EVs significantly promoted the proliferation, lumen formation and migration of HUVECs. Unlike DPC-EVs, tdDPC-EVs exhibited significant advantages in terms of promoting angiogenesis, accelerating wound healing and enhancing wound-healing efficiency both in vitro and in vivo. Bioinformatics analysis and further functional experiments verified that the tdDPC-EV-regulated KLF4/VEGFA axis is pivotal in accelerating wound healing. 3D cultivation can be utilized as an innovative optimization strategy to effectively develop DPC-derived EVs for the treatment of skin wounds. tdDPC-EVs significantly enhance wound healing via KLF4/VEGFA-driven angiogenesis.

Involvement of miRNA-34a regulated Krüppel-like factor 4 expression in hyperoxia-induced senescence in lung epithelial cells.

Premature infants, subjected to supplemental oxygen and mechanical ventilation, may develop bronchopulmonary dysplasia, a chronic lung disease characterized by alveolar dysplasia and impaired vascularization. We and others have shown that hyperoxia causes senescence in cultured lung epithelial cells and fibroblasts. Although miR-34a modulates senescence, it is unclear whether it contributes to hyperoxia-induced senescence. We hypothesized that hyperoxia increases miR-34a levels, leading to cellular senescence. We exposed mouse lung epithelial (MLE-12) cells and primary human small airway epithelial cells to hyperoxia (95% O2/5% CO2) or air (21% O2/5% CO2) for 24h. Newborn mice (< 12h old) were exposed to hyperoxia (> 95% O2) for 3 days and allowed to recover in room air until postnatal day 7. Lung samples from premature human infants requiring mechanical ventilation and control subjects who were not mechanically ventilated were employed. Hyperoxia caused senescence as indicated by loss of nuclear lamin B1, increased p21 gene expression, and senescence-associated secretory phenotype factors. Expression of miR-34a-5p was increased in epithelial cells and newborn mice exposed to hyperoxia, and in premature infants requiring mechanical ventilation. Transfection with a miR-34a-5p inhibitor reduced hyperoxia-induced senescence in MLE-12 cells. Additionally, hyperoxia increased protein levels of the oncogene and tumor-suppressor Krüppel-like factor 4 (KLF4), which were inhibited by a miR-34a-5p inhibitor. Furthermore, KLF4 knockdown by siRNA transfection reduced hyperoxia-induced senescence. Hyperoxia increases miR-34a-5p, leading to senescence in lung epithelial cells. This is dictated in part by upregulation of KLF4 signaling. Therefore, inhibiting hyperoxia-induced senescence via miR-34a-5p or KLF4 suppression may provide a novel therapeutic strategy to mitigate the detrimental consequences of hyperoxia in the neonatal lung.

Lysophosphatidic Acid Promotes Epithelial-Mesenchymal Transition in Kidney Epithelial Cells via the LPAR1/MAPK-AKT/KLF5 Signaling Pathway in Diabetic Nephropathy.

The epithelial–mesenchymal transition (EMT) is a differentiation process associated with fibrogenesis in diabetic nephropathy (DN). Lysophosphatidic acid (LPA) is a small, naturally occurring glycerophospholipid implicated in the pathogenesis of DN. In this study, we investigated the role of LPA/LPAR1 signaling in the EMT of tubular cells as well as the underlying mechanisms. We observed a decrease in E-cadherin and an increase in vimentin expression levels in the kidney tubules of diabetic db/db mice, and treatment with ki16425 (LPAR1/3 inhibitor) inhibited the expression of these EMT markers. Ki16425 treatment also decreased the expression levels of the fibrotic factors fibronectin and alpha-smooth muscle actin (α-SMA) in db/db mice. Similarly, we found that LPA decreased E-cadherin expression and increased vimentin expression in HK-2 cells, which was reversed by treatment with ki16425 or AM095 (LPAR1 inhibitor). In addition, the expression levels of fibronectin and α-SMA were increased by LPA, and this effect was reversed by treatment with ki16425 and AM095 or by LPAR1 knockdown. Moreover, LPA induced the expression of the transcription factor, Krüppel-like factor 5 (KLF5), which was decreased by AM095 treatment or LPAR1 knockdown. The expression levels of EMT markers and fibrotic factors induced by LPA were decreased upon KLF5 knockdown in HK-2 cells. Inhibition of the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and serine-threonine kinase (AKT) pathways decreased LPA-induced expression of KLF5 and EMT markers. In conclusion, these data suggest that LPA contributes to the pathogenesis of diabetic nephropathy by inducing EMT and renal tubular fibrosis via regulation of KLF5 through the LPAR1.

Krüppel-like factor 4 promotes the proliferation and osteogenic differentiation of BMSCs through SOX2/IGF2 pathway.

Human bone marrow mesenchymal stem cells (BMSCs) have multi-lineage differentiation potential and have been widely researched in regenerative medicine. The purpose of this research was to explore whether Krüppel-like factor 4 (KLF4) can regulate the osteogenic differentiation of BMSCs. We transfected human BMSCs with KLF4 overexpression plasmid and si-KLF4 to study the effects of KLF4. We performed cell proliferation assay, flow cytometry and Alizarin Red staining on BMSCs. Quantitative real-time PCR and western blot was performed to determined mRNA and protein expression of osteogenic differentiation markers, KLF4, SOX2 and IGF2. Bone defect animal model was created and the adenovirus containing KLF4 overexpression or knockdown plasmid was injected. Finally, HE staining was performed on tibia to assess the new bone formation. Our results showed that KLF4 promotes not only the growth of BMSCs, but also their osteogenic differentiation. Also, it mediated these effects through SOX2/IGF2 signaling pathway. In addition, KLF4 overexpression could increase the bone regeneration in in-vivo model, whereas KLF4 knockdown decreased the bone regeneration. KLF4 regulates BMSC's osteogenic differentiation via SOX2/IGF2 pathway.

The protective effects of butorphanol tartrate against homocysteine-induced blood-brain barrier dysfunction

ABSTRACT A high concentration of homocysteine (Hcy) has been recently reported to be closely associated with the development of stroke, which is related to the Hcy-induced blood-brain barrier (BBB) dysfunction. Butorphanol tartrate is a promising analgesic agent that targets the opiate receptor and shows promising protective effects on ischemia/reperfusion injury. The present research proposes to investigate the protective effect of butorphanol tartrate on Hcy-induced BBB disruption to explore the potential application of butorphanol tartrate in treating Hcy-induced stroke. Hcy was utilized to establish both an in vivo animal model and in vitro human brain vascular endothelial cells (HBVECs) injury model. We found that the increased diffusion of sodium fluorescein and Evan’s blue, declined expression of Claudin-5, and increased production of interleukin- 6 (IL-6) and tumor necrosis factor-α (TNF-α) were observed in Hcy-treated mice, which were all significantly reversed by butorphanol tartrate. In Hcy-stimulated HBVECs, increased endothelial permeability and reduced expression levels of Claudin-5 and Krüppel-like factor 5 (KLF5) were observed, all of which were dramatically rescued by 2 and 5 µM butorphanol tartrate. Lastly, the protective function of butorphanol tartrate in Hcy-stimulated HBVECs was dramatically abolished by the knockdown of KLF5. Collectively, butorphanol tartrate showed protective effects on Hcy-induced BBB disruption by upregulating the KLF5/Claudin-5 axis.

Krüppel-like factor 5 -mediated Sirtuin6 promotes osteogenic differentiation and inhibits inflammatory injury of lipopolysaccharide-induced periodontal membrane stem cells by inhibiting nuclear factor kappa-B pathway

ABSTRACT Periodontitis is a chronic infectious disease that causes inflammation and immune response and has an ultimate impact on the health of the whole body. Sirtuin6 (SIRT6) and Krüppel-like factor 5 (KLF5) have been reported to regulate the inflammatory response and play an important role in the development of periodontitis. LPS was adopted to induce periodontal ligament stem cells (PDLSCs) to construct a periodontitis cell model. SIRT6 expression was assayed through RT-qPCR and Western blot. Subsequently, after SIRT6 was overexpressed, CCK8 was to appraise cell viability. ELISA analysis was used to estimate inflammatory response. ALP staining, ARS staining, and Western blot were used to detect osteogenic differentiation. The JASPAR website then predicts the binding of transcription factor KLF5 to SIRT6 promoter. The interaction between KLF5 and SIRT6 was verified by a luciferase reporter and ChIP assays. Additionally, the osteogenic differentiation and inflammation in LPS-induced PDLSCs transfected with Ov-SIRT6 and si-KIF5 were also explored. Finally, the protein levels of the nuclear factor kappa-B (NF-κB) pathway-related factors were detected by Western blot to further explore the mechanism. There was a marked decrease in SIRT6 expression in LPS-induced PDLSCs. SITR6 overexpression prevented LPS-induced cell viability loss and inflammation, while promoting osteogenic differentiation. In addition, KLF5 could transcriptionally activate SIRT6. Further, KLF5 knockdown reversed the impacts of SIRT6 on the proliferation, inflammation, and osteogenic differentiation of LPS-induced PDLSCs via mediating NF-κB pathway. Overall, KLF5-mediated SIRT6 promoted the viability and osteogenic differentiation, while inhibiting the inflammatory response of LPS-induced PDLSCs by inhibiting NF-κB pathway.

MicroRNA-92a-3p Enhances Cisplatin Resistance by Regulating Krüppel-Like Factor 4-Mediated Cell Apoptosis and Epithelial-to-Mesenchymal Transition in Cervical Cancer.

Recent studies have confirmed the existence and key roles of microRNA (miRNAs) in cancer drug resistance, including cervical cancer (CC). The present study aims to establish a novel role for miR-92a-3p and its associated gene networks in cisplatin (DDP) resistance of CC. First, the disparities in miRNA expression between CC tissues and adjacent normal tissues were screened based on GSE19611 microarray data that retrieved from Gene Expression Omnibus (GEO), and we identified several miRs that were significantly downregulated or upregulated in CC tissues including miR-92a-3p. Moreover, miR-92a-3p was significantly up-regulated in DDP-resistant cells and was the most differently expressed miRNA. Functionally, knockdown of miR-92a-3p increased the sensitivity of DDP-resistant cells to DDP via inhibiting cell proliferation, migration and invasion, and promoting apoptosis. Conversely, overexpression of miR-92a-3p significantly induced DDP resistance in CC parental cells including HeLa and SiHa cells. Moreover, Krüppel-like factor 4 (KLF4) was identified as a direct target of miR-92a-3p, and an obvious inverse correlation was observed between the expression of miR-92a-3p and KLF4 in 40 pairs of cancer tissues. Furthermore, KLF4 knockdown reversed the promoting effect of miR-92a-3p inhibition on DDP sensitivity in DDP-resistant CC cells. Besides, high expression of miR-92a-3p was associated with DDP resistance, as well as a short overall survival in clinic. Taken together, these findings provide important evidence that miR-92a-3p targets KLF4 and is significant in DDP resistance in CC, indicating that miR-92a-3p may be an attractive target to increase DDP sensitivity in clinical CC treatment.

Salidroside Ameliorates Vascular Endothelial Cell Senescence through Downregulation of KLF4

Salidroside is extensively used as a herbal medicine worldwide, and it has been shown to protect against disruption of endothelial homeostasis and act as an anti-aging agent. The present study aimed to investigate the ameliorative effects of salidroside on homocysteine (Hcy)-induced cell senescence in human umbilical vein endothelial cells (HUVECs) that were mediated via inhibition of Krüppel-like factor 4 (KLF4). An endothelial cell senescence model was induced by Hcy. The cell viability, activities of telomerase and lactate dehydrogenase (LDH), and the level of reactive oxygen species were determined using commercial kits. The expression levels of KLF4, p53 and p21 were determined via western blot analysis, whereas the mRNA expression levels of KLF4 were detected by reverse transcription-quantitative PCR. Small interfering RNA-mediated knockdown of KLF4 was found to reverse Hcy-induced cell senescence. Hcy treatment led to an accelerated cell senescence, as evidenced by decreases in both cell viability and telomerase activity, whereas increases were noted in the leakage of LDH and the level of reactive oxygen species, in addition to an up-regulation of the protein levels of p53 and p21, and up-regulation of KLF4 at both the mRNA and protein level. Treatment with salidroside ameliorated Hcy-induced cell senescence in a dose-dependent manner. Taken together, these results suggested that Hcy may induce cell senescence through upregulation of KLF4, and this may be reversed by treatment with salidroside. Therefore, salidroside was shown to inhibit Hcy-induced cell senescence through KLF4 inhibition.

KLF4 down-regulation resulting from TLR4 promotion of ERK1/2 phosphorylation underpins inflammatory response in sepsis.

Sepsis is a systemic inflammatory response to invading pathogens, leading to high mortality rates in intensive care units worldwide. Krüppel‐like factor 4 (KLF4) is an important anti‐inflammatory transcription factor. In this study, we investigate the anti‐inflammatory role of KLF4 in caecal ligation and puncture (CLP)‐induced septic mice and lipopolysaccharide (LPS)‐induced RAW264.7 cells and its potential mechanism. We found that KLF4 was down‐regulated in CLP‐induced septic mice and in LPS‐induced RAW264.7 cells, and that its overexpression led to increased survival rates of septic mice along with inhibited inflammatory response in vivo and in vitro. ITGA2B was up‐regulated in the setting of sepsis and was inhibited by KLF4 overexpression. ITGA2B knock‐down mimicked the effects of KLF4 overexpression on septic mice and LPS‐induced RAW264.7 cells. TLR4 promoted the phosphorylation of ERK1/2 and then up‐regulated the ubiquitination and the degradation of KLF4, thereby elevating the expression of ITGA2B. Moreover, TLR4 knock‐down or treatment with PD98059 (a MEK inhibitor) inhibited inflammatory response in the setting of sepsis in vivo and in vitro. Furthermore, this effect of PD98059 treatment was lost upon KLF4 knock‐down. Collectively, these results explain the down‐regulation of KLF4 in sepsis, namely via TLR4 promotion of ERK1/2 phosphorylation, and identify ITGA2B as the downstream gene of KLF4, thus highlighting the anti‐inflammatory role of KLF4 in sepsis.

Water-soluble fraction of particulate matter

Ambient fine particulate matter of <2.5 μm (PM2.5) has been linked to morbidity and mortality from respiratory and cardiovascular diseases. Lung epithelial cells bear the brunt of PM2.5 exposure. In the present study, we found that exposure of A549 cells to the water-soluble fraction of PM2.5 (WS-PM2.5) promoted the expression and internalization of caveolin-1. Caveolin-1 knockdown restrained the endocytosis of WS-PM2.5. In addition, WS-PM2.5 accumulation in the cells induced the phosphorylation of serine/threonine protein kinase B (AKT) and nuclear factor κ-light-chain enhancer of activated B cells (NFκB), as well as the expression of Krüppel-like factor 5 (KLF5). Inhibiting activation of AKT and NFκB also partly reduced WS-PM2.5 concentration in cells, but KLF5 knockdown did not affect the intracellular accumulation of WS-PM2.5. KLF5 knockdown suppressed cytochrome P450 family 1 subfamily A member 1 (CYP1A1) expression and activated caspase 3. Luciferase reporter assay and chromatin immunoprecipitation assay showed that KLF5 positively regulated the transcription of KLF5. These results suggested that caveolin-1 was required for the endocytosis of WS-PM2.5. Intracellular accumulation of WS-PM2.5 activated AKT and NFκB, which facilitated WS-PM2.5 endocytosis. WS-PM2.5 accumulation also induced KLF5 expression, increasing the transcriptional expression of CYP1A1, which contributed to activate caspase 3.

Cyanidin-3-O-glucoside inhibits epithelial-to-mesenchymal transition, and migration and invasion of breast cancer cells by upregulating KLF4.

BackgroundAnthocyanins (ACNs) are capable of suppressing breast cancer growth; however, investigation on the effect and mechanism of ACNs on epithelial-to-mesenchymal transition (EMT), and cell migration and invasion in breast cancer cells is limited. A complete understanding of those properties may provide useful information on of how to use these natural compounds for cancer prevention and treatment.ObjectivesThe aim of this work was to investigate the role of cyanidin-3-O-glucoside (Cy3G), one of the most widely distributed ACNs in edible fruits, in the EMT process, and cell migration and invasion of breast cancer cells, and its underlying molecular mechanisms of how Cy3G establishes these functional roles in these cells.MethodsMDA-MB-231 and MDA-MB-468 breast cancer cells were treated with Cy3G (20 μM) for 24 h, and then the cells were used for cell migration and invasion assay. Western blotting, luciferase assay, ubiquitination assay, gene knockdown, and cycloheximide chase assay were performed to analyze the molecular mechanisms of Cy3G in suppressing EMT, and cell migration and invasion.ResultsCy3G inhibited the EMT process in these cells and significantly suppressed the migration and invasion of breast cancer cells (P ≤ 0.05) by upregulating Krüppel-like factor 4 (KLF4) expression at protein level. KLF4 knockdown in MDA-MB-231 cells did not reveal any change in EMT marker expression, and cell migration and invasion upon treatment with Cy3G (P ≥ 0.05), which strongly indicated that the effects of Cy3G were mediated by KLF4. Furthermore, we determined that Cy3G indirectly upregulated KLF4 expression by downregulating FBXO32, which is the E3 ligase of KLF4.ConclusionCy3G is a potential anticancer reagent as it can inhibit EMT and breast cancer cell migration and invasion by upregulating KLF4.

High expression of Krüppel-like factor 5 is associated with poor prognosis in patients with colorectal cancer.

Krüppel‐like factor 5 (KLF5) plays an oncogenic role and has diverse functions in cancer cells. However, correlation between KLF5 and clinical outcome has not been determined in patients with colorectal cancer and colorectal liver metastasis. Herein, we analyzed 65 patients with colorectal cancer who developed colorectal liver metastasis. Clinical effects were assessed through immunohistochemical analysis of primary colorectal cancer lesions and metastatic liver lesions. High expression of KLF5 in these tissues correlated with the presence of vascular invasion, elevated serum carbohydrate antigen 19‐9 levels, large diameters of metastatic liver tumors, and poor prognosis following surgery. Multivariate analyses revealed that high expression of KLF5 was an independent prognostic factor. Increased expression of KLF5 in both colorectal cancer primaries and colorectal liver metastasis was significantly associated with shorter overall survival time and time to surgical failure. Krüppel‐like factor 5 expression positively correlated with Ki‐67 and c‐Myc expression in colorectal cancer tissues. In vitro experiments with colon cancer cell lines showed that siRNA knockdown of KLF5 inhibited cell proliferation. Western blot analyses revealed that knockdown of KLF5 expression reduced cyclin D1 and c‐Myc expression. It also impaired the stem cell‐like properties of cancer cells in tumorsphere formation assays. Furthermore, anoikis assay indicated that KLF5 contributed to anoikis resistance. High KLF5 expression is associated with poor prognosis in patients with colorectal cancer and liver metastasis by promoting cell proliferation and cancer stem cell‐like properties.

KLF5 promotes proliferation in gastric cancer via regulating p21 and CDK4.

This study aims to investigate the expression characteristics of Krüppel-like factor 5 (KLF5) in gastric cancer (GC) and its potential correlation to pathological indexes in GC patients. Molecular mechanisms underlying the regulatory effect of KLF5 on GC progression are explored. KLF5 expressions in GC tissues were analyzed through GEPIA dataset and those collected in our hospital. By analyzing the clinical data of GC patients, the correlation between KLF5 level and pathological characteristics of GC was determined. The regulatory effects of KLF5 on proliferative ability and cell cycle progression of SGC7901 and MGC803 cells were assessed by Cell Counting Kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU) assay, and flow cytometry. The regulation of KLF5 on expression levels of p21 and CDK4 in GC cells was determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot. KLF5 was markedly upregulated in GC tissues. KLF5 level was positively correlated to TNM staging, tumor size, and metastasis of GC. Meanwhile, high level of KLF5 predicted poor prognosis in GC patients. The knockdown of KLF5 in SGC7901 and MGC803 cells attenuated proliferative ability and arrested cell cycle in G0/G1 phase. Both mRNA and the protein levels of p21 were upregulated, and CDK4 levels were downregulated in SGC7901 and MGC803 cells transfected with si-KLF5. KLF5 is upregulated in GC and closely linked to pathological characteristics of GC patients. High level of KLF5 indicates poor prognosis of GC. It is believed that KLF5 may be a potential therapeutic target for GC.

Kruppel‐like Factor 4 is Critical to the Maintenance of Glomerular Endothelial Integrity

BackgroundThrombotic microangiopathy (TMA) represents the most severe manifestation of glomerular endothelial injury consisting of endothelial cell (EC) swelling, subendothelial expansion, inflammation and microthrombi. TMA occurs subsequent to various etiologies and is associated with dysregulation of thrombotic and inflammatory transcripts and complement activation. Expression of membrane bound (CD55, CD59, CD46) complement regulators, is altered in many subtypes of TMA and it is unclear if this is a driver or consequence of EC injury. To investigate transcriptional mediators involved in TMA, we reviewed expression arrays of both global and single cell RNA‐seq from human kidney biopsies with complement mediated renal microvascular injury and observed that Krüppel‐Like Factor 4 (KLF4), a zinc finger transcription factor, is both differentially expressed in ECs and predicted to regulate other differentially expressed genes. Previous studies demonstrate that KLF4 is a critical mediator of anti‐thrombotic and anti‐inflammatory phenotype in systemic vascular beds. We aimed to investigate its role in modulating renal microvascular injury and complement activation in TMA.MethodsEndothelial‐specific Klf4 knockout mice (Klf4ΔEC) were generated by crossing Klf4fl/fl mice withCdh5‐Cre mice. As two separate models of endothelial dysfunction, Klf4ΔEC mice were both aged to 1 year and crossed with Nos3−/− mice to generate double knockout mice (DKO). Periodic acid‐Schiff, electron microscopy, immunofluorescence, ELISA and RT‐PCR were performed to investigate the effects of endothelial Klf4 on the renal microvasculature. Renal histology and ultrastructure were evaluated, blinded, by renal pathology (MPR).ResultsAt 12 weeks of age, while no overt histologic changes were observed, Klf4ΔEC mice exhibited increased glomerular Vcam‐1 and Pai‐1 expression and complement activation (increased C3 and C5b‐9 deposition) as compared to Klf4fl/fl mice. To investigate the basis for this increased complement activation, we measured the expression of the membrane‐bound complement regulators and noted reduced glomerular Cd55 and Cd59 expression in Klf4ΔEC as compared with Klf4fl/fl mice. Interestingly, 1‐year‐old Klf4ΔEC mice exhibited pathology consistent with subacute TMA (glomerular capillary congestion, EC swelling, subendothelial expansion and mesangiolysis as well as increased intraglomerular von Willebrand Factor and albuminuria) as compared to age‐matched Klf4fl/fl mice. In addition, Klf4ΔEC mice crossed with nitric oxide synthase knockout (Nos3−/−) mice also exhibited increased glomerular complement activation and histological and ultrastructural features consistent with subacute TMA (loss of fenestrations, glomerular capillary congestion, tuft collapse with basement membrane wrinkling) as compared to all other groups (12 weeks). Finally, knockdown of KLF4 in cultured human microvascular endothelial cells reduced the expression of CD55, whereas induction of KLF4 increased CD55 expression in HMEC‐1s.ConclusionsLoss of endothelial Klf4 in both aged mice and in Nos3−/− mice recapitulates many pathologic features of renal‐TMA, including complement activation and is potentially mediated through its regulation of Cd55.Support or Funding InformationNIDDK