Klf4 Transcription Research Articles

Role of KLF4 and SIAT7A interaction accelerates myocardial hypertrophy induced by Ang II.

Sialylation catalysed by sialyltransferase 7A (SIAT7A) plays a role in the development of cardiac hypertrophy. However, the regulatory mechanisms upstream of SIAT7A in this context remain poorly elucidated. Previous study demonstrated that KLF4 activates the SIAT7A gene in ischemic myocardium by binding to its promoter region. Nevertheless, the potential involvement of KLF4 in regulating SIAT7A expression in Ang II-induced hypertrophic cardiomyocytes remains uncertain. This study seeks to deepen the underlying mechanisms of the KLF4 and SIAT7A interaction in the progression of Ang II-induced cardiac hypertrophy. The results showed a concurrent increase in SIAT7A and KLF4 levels in hypertrophic myocardium of essential hypertension patients and in hypertrophic cardiomyocytes stimulated by Ang II. Invitro experiments revealed that reducing KLF4 levels led to a decrease in both SIAT7A synthesis and Sialyl-Tn antigen expression, consequently inhibiting Ang II-induced cardiomyocyte hypertrophy. Intriguingly, reducing SIAT7A levels also resulted in decreased KLF4 expression and suppression cardiomyocyte hypertrophy. Consistent with this, elevating SIAT7A levels increased KLF4 expression and exacerbated cardiomyocyte hypertrophy in both invivo and invitro experiments. Additionally, a time-course analysis indicated that KLF4 expression preceded that of SIAT7A. Luciferase reporter assays further confirmed that modulating SIAT7A levels directly influenced the transcriptional activity of KLF4 in cardiomyocytes. In summary, KLF4 expression is upregulated in cardiomyocytes treated with Ang II, which subsequently induces the expression of SIAT7A. The elevated levels of SIAT7A, in turn, enhance the transcription of KLF4. These findings suggest a positive feedback loop between KLF4 and SIAT7A-Sialyl-Tn, ultimately promoting Ang II-induced cardiac hypertrophy.

Abstract P161: A Single-Short Partial Reprogramming of the Endothelial Cells Decreases Blood Pressure Via Attenuation of Endothelial-to-Mesenchymal Transition in Hypertensive Mice

Small artery remodeling and endothelial dysfunction are hallmarks of hypertension. Growing evidence supports a likely causal association between cardiovascular diseases and the presence of endothelial-to-mesenchymal transition (EndMT). EC reprogramming represents an innovative strategy in regenerative medicine to prevent deleterious effects induced by cardiovascular diseases. Using reprogramming of ECs, via overexpression of Oct-3/4, Sox-2, and Klf-4 (OSK) transcription factors, we aimed to bring ECs back to a youthful phenotype in hypertensive mice. Mouse ECs were infected with lentiviral vectors (LV) containing the specific EC marker cadherin 5 (Cdh5) and the fluorescent reporter enhanced green fluorescence protein (EGFP) with empty vector (LVCO) or with OSK (LV-OSK). Confocal microscopy and western blotting analysis were used to confirm the OSK overexpression. Human aortic ECs (HAoECs) from male and female normotensive and hypertensive patients were analyzed after OSK or control treatments for their endothelial nitric oxide synthase (eNOS) levels, nitric oxide (NO), and genetic profile. Male and female normotensive (BPN/3J) and hypertensive (BPH/2J) mice were treated with an intravenous (i.v.) injection of LVCO or LV-OSK and evaluated 10 days post-infection. The blood pressure, vascular reactivity of small arteries, in vivo EGFP signal and EndMT inhibition were analyzed. Data were analyzed using One-way or Two-way ANOVA, with Tukey post-hoc ( p <0.05). OSK overexpression induced partial EC reprogramming in vitro . OSK treatment of male but not female hypertensive BPH/2J mice normalized blood pressure (BPH/2J LVCO: 105±2mmHg, n=6 versus BPH/2J LV-OSK: 90±2mmHg, n=5) and resistance arteries hypercontractility (BPH/2J LV-CO: 10.50±0.82mN versus BPH/2J LV-OSK: 7.12±0.6mN, n=5), via the attenuation of EndMT (BPH/2J LVCO: 0.041±0.01AU, n=3 versus BPH/2J LV-OSK: 0.007±0.004 AU, n=3) and elastin breaks. EGFP signal was detected in vivo in the prefrontal cortex of BPN/3J and BPH/2J-treated mice, but OSK induced angiogenesis in male BPN/3J mice. OSK-treated human ECs from hypertensive patients showed high eNOS activation and NO production, with low ROS. Single-cell RNA analysis showed that OSK alleviated EC senescence and EndMT, restoring their phenotypes in human ECs from hypertensive patients. Overall, these data indicate that OSK treatment and EC reprogramming can decrease blood pressure and reverse hypertension-induced vascular damage.

Med1 controls thymic T-cell migration into lymph node through enhancer-based Foxo1-Klf2 transcription program.

The migration is the key step for thymic T cells to enter circulation and then lymph nodes (LNs), essential for future immune surveillance. Although promoter-based transcriptional regulation through Foxo1, Klf2, Ccr7, and Sell regulates T-cell migration, it remains largely unexplored whether and how enhancers are involved in this process. Here we found that the conditional deletion of Med1, a component of the mediator complex and a mediator between enhancers and RNA polymerase II, caused a reduction of both CD4+ and CD8+ T cells in LNs, as well as a decrease of CD8+ T cells in the spleen. Importantly, Med1 deletion hindered the migration of thymic αβT cells into the circulation and then into LNs, accompanied by the downregulation of KLF2, CCR7, and CD62L. Mechanistically, Med1 promotes Klf2 transcription by facilitating Foxo1 binding to the Klf2 enhancer. Furthermore, forced expression of Klf2 rescued Ccr7 and Sell expression, as well as αβT-cell migration into LNs. Collectively, our study unveils a crucial role for Med1 in regulating the enhancer-based Foxo1-Klf2 transcriptional program and the migration of αβT cells into LNs, providing valuable insights into the molecular mechanisms underlying T-cell migration.

EphA2 promotes the transcription of KLF4 to facilitate stemness in oral squamous cell carcinoma

Ephrin receptor A2 (EphA2), a member of the Ephrin receptor family, is closely related to the progression of oral squamous cell carcinoma (OSCC). Cancer stem cells (CSCs) play essential roles in OSCC development and occurrence. The underlying mechanisms between EphA2 and CSCs, however, are not yet fully understood. Here, we found that EphA2 was overexpressed in OSCC tissues and was associated with poor prognosis. Knockdown of EphA2 dampened the CSC phenotype and the tumour-initiating frequency of OSCC cells. Crucially, the effects of EphA2 on the CSC phenotype relied on KLF4, a key transcription factor for CSCs. Mechanistically, EphA2 activated the ERK signalling pathway, promoting the nuclear translocation of YAP. Subsequently, YAP was bound to TEAD3, leading to the transcription of KLF4. Overall, our findings revealed that EphA2 can enhance the stemness of OSCC cells, and this study identified the EphA2/KLF4 axis as a potential target for treating OSCC.

METTL3-mediated N6-methyladenosine modification of STAT5A promotes gastric cancer progression by regulating KLF4

N6-methyladenosine (m6A) is the predominant post-transcriptional RNA modification in eukaryotes and plays a pivotal regulatory role in various aspects of RNA fate determination, such as mRNA stability, alternative splicing, and translation. Dysregulation of the critical m6A methyltransferase METTL3 is implicated in tumorigenesis and development. Here, this work showed that METTL3 is upregulated in gastric cancer tissues and is associated with poor prognosis. METTL3 methylates the A2318 site within the coding sequence (CDS) region of STAT5A. IGF2BP2 recognizes and binds METTL3-mediated m6A modification of STAT5A through its GXXG motif in the KH3 and KH4 domains, leading to increased stability of STAT5A mRNA. In addition, both METTL3 and IGF2BP2 are positively correlated with STAT5A in human gastric cancer tissue samples. Helicobacter pylori infection increased the expression level of METTL3 in gastric cancer cells, thereby leading to the upregulation of STAT5A. Functional studies indicated that STAT5A overexpression markedly enhances the proliferation and migration of GC cells, whereas STAT5A knockdown has inhibitory effects. Further nude mouse experiments showed that STAT5A knockdown effectively inhibits the growth and metastasis of gastric cancer in vivo. Moreover, as a transcription factor, STAT5A represses KLF4 transcription by binding to its promoter region. The overexpression of KLF4 can counteract the oncogenic impact of STAT5A. Overall, this study highlights the crucial role of m6A in gastric cancer and provides potential therapeutic targets for gastric cancer.

Enhancing postmenopausal osteoporosis: a study of KLF2 transcription factor secretion and PI3K-Akt signaling pathway activation by PIK3CA in bone marrow mesenchymal stem cells.

Mesenchymal stem cells can develop into osteoblasts, making them a promising cell-based osteoporosis treatment. Despite their therapeutic potential, their molecular processes are little known. Bioinformatics and experimental analysis were used to determine the molecular processes of bone marrow mesenchymal stem cell (BMSC) therapy for postmenopausal osteoporosis (PMO). We used weighted gene co-expression network analysis (WGCNA) to isolate core gene sets from two GEO microarray datasets (GSE7158 and GSE56815). GeneCards found PMO-related genes. GO, KEGG, Lasso regression, and ROC curve analysis refined our candidate genes. Using the GSE105145 dataset, we evaluated KLF2 expression in BMSCs and examined the link between KLF2 and PIK3CA using Pearson correlation analysis. We created a protein-protein interaction network of essential genes involved in osteoblast differentiation and validated the functional roles of KLF2 and PIK3CA in BMSC osteoblast differentiation in vitro. We created 6 co-expression modules from 10 419 differentially expressed genes (DEGs). PIK3CA, the key gene in the PI3K-Akt pathway, was among 197 PMO-associated DEGs. KLF2 also induced PIK3CA transcription in PMO. BMSCs also expressed elevated KLF2. BMSC osteoblast differentiation involved the PI3K-Akt pathway. In vitro, KLF2 increased PIK3CA transcription and activated the PI3K-Akt pathway to differentiate BMSCs into osteoblasts. BMSCs release KLF2, which stimulates the PIK3CA-dependent PI3K-Akt pathway to treat PMO. Our findings illuminates the involvement of KLF2 and the PI3K-Akt pathway in BMSC osteoblast development, which may lead to better PMO treatments.

Activation of the YAP/KLF5 transcriptional cascade in renal tubular cells aggravates kidney injury

The Hippo/YAP pathway plays a critical role in tissue homeostasis. Our previous work demonstrated that renal tubular YAP activation induced by double knockout of the upstream Hippo kinases Mst1 and Mst2 (Mst1/2 dKO) promotes tubular injury and renal inflammation under basal conditions. However, the importance of tubular YAP activation remains to be established in injured kidneys in which many other injurious pathways are simultaneously activated. Here, we show that tubular YAP was already activated 6 h after unilateral ureteral obstruction (UUO). Tubular YAP deficiency greatly attenuated tubular cell over-proliferation, tubular injury and renal inflammation induced by UUO or cisplatin. YAP promoted the transcription of the transcription factor KLF5. Consistently, the elevated expression of KLF5 and its target genes in Mst1/2 dKO or UUO kidneys was blocked by ablation of Yap in tubular cells. Inhibition of KLF5 prevented tubular cell over-proliferation, tubular injury and renal inflammation in Mst1/2 dKO kidneys. Therefore, our results demonstrate that tubular YAP is a key player in kidney injury. YAP and KLF5 form a transcriptional cascade, in which tubular YAP activation induced by kidney injury promotes KLF5 transcription. Activation of this cascade induces tubular cell over-proliferation, tubular injury and renal inflammation.

Regulation of epithelial cell differentiation by the Ubiquitous expressed transcript isoform 1 in ulcerative colitis.

Mucosal healing has emerged as a desirable treatment goal for patients with ulcerative colitis (UC). Healing of mucosal wounds involves epithelial cell proliferation and differentiation, and Y-box transcription factor ZONAB has recently been identified as the key modulator of intestinal epithelial restitution. We studied the characteristics of UXT-V1 expression in UC patients using immunohistochemistry and qPCR. The functional role of UXT-V1 in the colonic epithelium was investigated using lentivirus-mediated shRNA in vitro and ex vivo. Through endogenous Co-immunoprecipitation and LC-MS/MS, we identified ZONAB as a UXT-V1-interactive protein. Herein, we report that UXT-V1 promotes differentiation of intestinal epithelial cells by regulating the nuclear translocation of ZONAB. UXT-V1 was upregulated in the intestinal epithelia of UC patients compared with that of healthy controls. Knocking down UXT-V1 in NCM-460 cells led to the enrichment of pathways associated with proliferation and differentiation. Furthermore, the absence of UXT-V1 in cultured intestinal epithelial cells and colonic organoids inhibited differentiation to the goblet cell phenotype. Mechanistically, the loss of UXT-V1 in the intestinal epithelial cells allowed nuclear translocation of ZONAB, wherein it regulated the transcription of differentiation-related genes, including AML1 and KLF4. Taken together, our study reveals a potential role of UXT-V1 in regulating epithelial cell differentiation, proving a molecular basis for mucosal healing in UC.

Protein Exaptation by Endogenous Retroviral Elements Shapes Tumor Cell Senescence and Downstream Immune Signaling.

Cancer cell senescence in lung squamous cell carcinoma (LUSC) is associated with a poor response to chemotherapies and immunotherapies due to promotion of an immunosuppressive tumor microenvironment. This environment is shaped by the senescence-associated secretory pathway, which recruits suppressive immune cell populations. In a recent study, Attig and colleagues identified a transcription factor-activated molecular switch that circumvents cellular senescence through increased expression of the calbindin protein. A human endogenous retrovirus (HERV) sequence upstream of the calbindin gene, CALB1, promotes the transcription of an HERVH-CALB1 transcript through a splice event at the third CALB1 exon in a process known as protein exaptation. The KLF5 transcription factor mediates this transcriptional activity by binding at the HERVH sequence, subsequently initiating the chimeric HERVH-CALB1 transcription. This increased expression of calbindin reduces CXCL8 chemokine production and downstream neutrophil recruitment in LUSC tumor cells. CALB1 exaptation by HERVH is one example by which endogenous retroelements (ERE) regulate immunity in human cancers, highlighting the emerging role of EREs in tumor immunity.

Abstract A053: Epigenomic analyses of circulating tumor DNA in patients with metastatic castration resistant prostate cancer (mCRPC) treated with immune checkpoint blockade and PARP inhibition

Abstract Background: Molecular characterization of mCRPC has revealed disruption of DNA damage repair genes that may improve sensitivity to PARP inhibitors such as olaparib (O), which in turn may further complement the activity of the PD-L1 immune checkpoint inhibitor, durvalumab (D), through mechanisms such as activation of the STING pathway. We hypothesized that (epi)genomic analyses of circulating tumor DNA (ctDNA) would identify mechanisms of treatment response in patients with mCRPC treated with O plus D (NCT02484404). Methods: Eligible patients for this study had mCRPC with prior treatment by abiraterone and/or enzalutamide. Study participants were administered 300 mg of O twice daily and 1500 mg of D every 4 weeks. We obtained blood samples in Streck tubes from 38 (out of 60) study participants. Plasma samples were acquired at baseline and at progression. Buffy coat was isolated from each sample. Genomic DNA and cell-free DNA (cfDNA) were separately isolated and assembled into sequencing libraries. Low-pass whole-genome sequencing (3-4 × coverage) was performed for somatic copy number and fragmentomic analyses. In a subset of samples with the greatest cfDNA content, chromatin immunoprecipitation sequencing (ChIP-seq) with antibodies against H3K4me2 and H3K4me3 was performed directly in additional aliquots of plasma. Results: Amongst the 38 patients with at least one cfDNA sample, 5 patients had a radiographic partial response (PR) per RECIST v1.1, 28 patients had stable disease (SD), and 6 patients had progressive disease (PD) during the first two months of treatment. At baseline, ctDNA content in plasma was significantly lower in patients who went on to have PR or SD versus PD (p=0.02). However, upon progression, ctDNA content in plasma was not significantly different between groups. Losses to PTEN and NKX3-1 were significantly enriched in ctDNA from patients with PD (p=0.041 and p=0.047, respectively). By contrast, biallelic combined losses to BRCA2, RB1, BRCA1 and TP53 were observed in all PRs at baseline but not in any patient with PD (p=0.048). In patients with SD, longitudinal fragmentomic analysis inferred that the activity of the mitogenic KLF4 transcription factor (TF) was the most increased upon progression, while activity of the protective HIF2A TF was the most decreased. However, the TF binding motifs predicted by H3K4me2 and H3K4me3 cfDNA ChIP-seq did not significantly change in patients with PD. Conclusions: Distinctive genomic and epigenomic profiles were observed in plasma from patients with responsive vs. progressive disease, and the profiles may predict responses to combination D+O therapy. Deeper whole-genome sequencing of these samples currently in progress will reveal the contribution of germline and somatic point mutations to clinical response. Ongoing efforts to estimate the phylogenetic architecture of patients’ tumors from these plasma samples will enable the inference of drug response phenotypes as a function of evolutionary potential and potentially identify new opportunities to target treatment-resistant disease. Citation Format: Anna Baj, Clara C. Y. Seo, Nicholas T. Terrigino, John R. Bright, S. Thomas Hennigan, Isaiah M. King, Shana Y. Trostel, William D. Figg, William L. Dahut, Jung-Min Lee, David Y. Takeda, Fatima Karzai, Adam G. Sowalsky. Epigenomic analyses of circulating tumor DNA in patients with metastatic castration resistant prostate cancer (mCRPC) treated with immune checkpoint blockade and PARP inhibition [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A053.

Analysis of KLF7 and KLF5 transcription factors gene variants in coronary artery disease

Introduction and objectiveGenetic susceptibility has a key role in the pathogenesis of coronary artery disease (CAD). KLF5 and KLF7 are transcriptional factors essential to cell development and differentiation. Their genetic variants have been associated with the risk of metabolic disorders. The present study aimed to evaluate the possible correlation of KLF5 (rs3812852) and KLF7 (rs2302870) single nucleotide polymorphisms (SNPs) with the risk of CAD for the first time in the world. MethodsThe clinical trial study comprised 150 patients with CAD and 150 control subjects without CAD from the Iranian population. After blood sampling, deoxyribonucleic acid was extracted and genotyped using the Tetra Primer ARMS-PCR method and confirmed by Sanger sequencing. ResultsThe KLF7 A/C genotypes and C allele frequency were meaningfully higher in the control group compared to the CAD+ group (p<0.05). No obvious association has been observed between KLF5 variants and CAD risk. However, the distribution of the AG genotype of KLF5 was statistically lower in CAD+ patients with diabetes than in CAD+ patients without diabetes (p<0.05). ConclusionThis study identified KLF7 SNP as a causative gene contributing to CAD, which presents novel insight into the molecular pathogenesis of the disease. It is, however, unlikely that KLF5 SNP has an essential role in the risk of CAD in the studied population.

Wall shear stress activation of EGR1 and KLF2 transcription in valvular cells is mediated by the ERK1/2 mitogen-activated protein kinase pathway

The aortic valve leaflet is composed of 2 cell types, an interstitial layer of fibroblast-like cells named valve interstitial cells (VICs), and an outer-layer of valve endothelial cells (VECs). The VECs are exposed to different stresses, in particular wall shear stress (WSS). Alteration of hemodynamics can lead to remodeling of the valve leaflet, which eventually result in calcified or degenerative valve. EGR1 and KLF2 are known as flow responsive transcription factors. However, the pathway activating these factors in response to WSS in the valve is not well understood. Our study aims to decipher how EGR1 and KLF2 are activated in response to the WSS in valvular cells. We used a unique fluid activation device that applies physiologically relevant pulsatile WSS to identify the signal required to activate EGR1 and KLF2. We used in vitro and ex vivo approaches to assess the downstream effect of EGR1 and KLF2. VECs exposed to the WSS were treated with U0126 (ERK1/2 inhibitor). Interestingly, treatment of the VECs with U0126 shows a downregulation of EGR1 and KLF2 transcription factors suggesting that the MAPK pathway and more precisely ERK1/2 is required to the activation of these factors. We found that addition of EGR1 adenovirus in the VECs results in the transcriptional activation of NOS3. Using luciferase assay we identified that both EGR1 and KLF2 activate NOS3 promoter. Our ex vivo model using explanted valves confirmed these data. In addition, we investigate which receptor could activate the MAPK pathway in response to the WSS. VECs exposed to the WSS and treated with different recombinant molecules to identify receptor involved in the upregulation of EGR1. Altogether, our data suggest a link between the MAPK pathway and the remodeling and/or proliferation of valve cells. In conclusion, EGR1 and KLF2 are upregulated in VECs exposed to WSS and this activation dependents on the MAP kinase pathway. EGR1 activates the transcription of NOS3 an essential regulator of valve homeostasis.

Klf4 protects thymus integrity during late pregnancy.

Pregnancy causes abrupt thymic atrophy. This atrophy is characterized by a severe decrease in the number of all thymocyte subsets and qualitative (but not quantitative) changes in thymic epithelial cells (TECs). Pregnancy-related thymic involution is triggered by progesterone-induced functional changes affecting mainly cortical TECs (cTECs). Remarkably, this severe involution is rapidly corrected following parturition. We postulated that understanding the mechanisms of pregnancy-related thymic changes could provide novel insights into signaling pathways regulating TEC function. When we analyzed genes whose expression in TECs was modified during late pregnancy, we found a strong enrichment in genes bearing KLF4 transcription factor binding motifs. We, therefore, engineered a Psmb11-iCre : Klf4lox/lox mouse model to study the impact of TEC-specific Klf4 deletion in steady-state conditions and during late pregnancy. Under steady-state conditions, Klf4 deletion had a minimal effect on TEC subsets and did not affect thymic architecture. However, pregnancy-induced thymic involution was much more pronounced in pregnant females lacking Klf4 expression in TECs. These mice displayed a substantial ablation of TECs with a more pronounced loss of thymocytes. Transcriptomic and phenotypic analyses of Klf4 -/- TECs revealed that Klf4 maintains cTEC numbers by supporting cell survival and preventing epithelial-to-mesenchymal plasticity during late pregnancy. We conclude that Klf4 is essential for preserving TEC's integrity and mitigating thymic involution during late pregnancy.

Predictive and Regenerative Medicine for Humans

In human history, finding a cure for the devastating disease of cancer has been a long, ongoing battle. Recently, scientific research has shown several feasible bio-medicinal ways for the treatment of cancer. The first method is through predictive regenerative medicine, which converts (breast) cancer cells back into phenotypically benign cells using continually updated predictions from Bayesian inference models. Another common method is the rewriting of damaged coordinating genes, but this has underlying moral issues — who can authorize the right to alter human genes? To investigate these methods, this paper will discuss elementary point-set topologies, physical gene mapping, and gene sequencing. The main purpose of this paper is to establish a statistical method with the help of mathematics in our biological life science. The proposed method can be applied in the case of manipulating genetic transcription and expression with a suitable Bayesian inference model (for genetic reprogramming). The peak value of cancer cell conversion is its Bayesian regression (based on the model’s parameters) from the corresponding Bayesian inference (for the normal posterior distribution, prior and likelihood). The relevant predictive conditions can be determined from the respective model, which could be used to reveal elements that help cancer cell conversion maximization/minimization. Practically, we may apply both of the Bayesian optimization and the related attribute adjustment (to the Oct4, SoX2, Klf4 and c-Myc transcription factors) that can induce the reprogramming of cancer cells. The expected outcome is that we can biomedical-engine the cancer cells conversion. To go a further step, one may apply my proposed Net-Seizing Theory to capture the genetic mutations that underly cancer disease. It is hope that we can design the corresponding universal strategy to heal cancer. Finally, one may establish an intelligent warning mechanism for those high risked cancer candidates but NOT for the thinking control in most of the dictatorship countries.

Generation of an induced pluripotent stem cell lines NSHDMUi001-A from patients with type 2 diabetes

Type 2 diabetes mellitus (T2DM) is common in China, and its aetiology and pathogenesis are still unclear. We reprogrammed pEP4EO2SEN2K and pEP4EO2SET2K, pCEP4-M2L was electrotransfected in T2DM patients with pEP4EO2SEN2K, and pCEP4-M2L was electrotransfected in T2DM patients expressing the OCT4, SOX2, NANOG, LIN28, c-MYC, KLF4, and SV40LT transcription factors to obtain induced pluripotent stem cells (iPSCs). The obtained iPSCs have been verified to have pluripotency, normal karyotype and differentiation potential; therefore, these cells can be used in the study of disease pathophysiology and drug development to create new therapeutic targets for T2DM and associated central nervous system damage.

Vascular smooth muscle cell contractile proteins as universal markers of vessels of microcirculatory bed

Highlights. The use of vascular smooth muscle cell markers, e.g. smooth muscle myosin heavy chain (SM-MHC) and alpha smooth muscle actin (α-SMA) for immunodetection of adventitial and perivascular microvessels (vasa vasorum) is preferrable over endothelial markers (CD31 and VE-cadherin) as it allows to define vascular geometry regardless of sectioning artifacts and provides ideal signal-to-noise ratio.Aside from elastic laminae which discriminate arterioles from venules and capillaries, we were unable to confirm any specific markers of arterial, venous, and capillary differentiation, although KLF2 and PROX1 transcription factors indicated venous specification and HEY1 suggested capillary identity in rat aortas.Aim. To develop an optimal approach to detection of microvessels and to evaluate the techniques for the differential immunostaining of arterioles, venules, and capillaries in human saphenous veins and rat aortas.Methods. Saphenous veins excised during the coronary artery bypass graft surgery were used for the study. Serial cryosections were analyzed by means of haematoxylin and eosin and Russell-Movat’s pentachrome stainings and by immunofluorescent staining for endothelial cell markers (CD31 and VE-cadherin), vascular smooth muscle cell markers (SM-MHC and α-SMA), mechanosensitive transcription factors (KLF2 and KLF4), transcription factors of arterial specification (HES1, HEY1, ERG), transcription factors and markers of venous identity (NR2F2, NRP2), and transcription factors and markers of lymphatic lineage (PROX1, LYVE1, VEGFR3). Samples were visualized by light and confocal microscopy.Results. In comparison with endothelial cell markers (CD31 and VE-cadherin), vascular smooth muscle cell markers (SM-MHC and α-SMA) permitted objective evaluation of vascular geometry and maximized signal-to-noise ratio irrespective of specific marker, microvessel specification or antibody used. Autofluorescence and specific histological pattern of elastic membranes at Russell-Movat’s pentachrome staining allowed to discriminate arterioles from venules and capillaries. Albeit immunostaining of rat aortas found specific markers of venous endothelial cells (KLF2 and PROX1) and capillary endothelial cells (HEY1), these findings have not been confirmed in saphenous veins. We were unable to find specific markers of human venules and capillaries among the saphenous vein vasa vasorum despite an extensive screening of multiple markers.Conclusion. Immunodetection of microvessels (e.g., vasa vasorum) should be performed by using vascular smooth muscle cell markers (SM-MHC and α-SMA) rather than endothelial cell markers (CD31 and VE-cadherin). Lack of specific markers to discern microvessels of different lineages suggests Russell-Movat’s pentachrome staining as an optimal option for the machine learning of neural networks to analyse the microvessels including vasa vasorum.

Trim28 citrullination maintains mouse embryonic stem cell pluripotency via regulating Nanog and Klf4 transcription.

Protein citrullination, including histone H1 and H3 citrullination, is important for transcriptional regulation, DNA damage response, and pluripotency of embryonic stem cells (ESCs). Tripartite motif containing 28 (Trim28), an embryonic development regulator involved in ESC self-renewal, has recently been identified as a novel substrate for citrullination by Padi4. However, the physiological functions of Trim28 citrullination and its role in regulating the chromatin structure and gene transcription of ESCs remain unknown. In this paper, we show that Trim28 is specifically citrullinated in mouse ESCs (mESCs), and that the loss of Trim28 citrullination induces loss of pluripotency. Mechanistically, Trim28 citrullination enhances the interaction of Trim28 with Smarcad1 and prevents chromatin condensation. Additionally, Trim28 citrullination regulates mESC pluripotency by promoting transcription of Nanog and Klf4 which it does by increasing the enrichment of H3K27ac and H3K4me3 and decreasing the enrichment of H3K9me3 in the transcriptional regulatory region. Thus, our findings suggest that Trim28 citrullination is the key for the epigenetic activation of pluripotency genes and pluripotency maintenance of ESCs. Together, these results uncover a role Trim28 citrullination plays in pluripotency regulation and provide novel insight into how citrullination of proteins other than histones regulates chromatin compaction.

Krüppel-like factor 5 promotes the progression of oral squamous cell carcinoma via the baculoviral IAP repeat containing 5 gene.

BackgroundKrüppel-like factor 5 (KLF5) is highly expressed in a variety of tumors, and our study aimed to investigate the role of KLF5 in oral squamous cell carcinoma (OSCC).MethodsTo explore the differential expression of KLF5, next-generation sequencing (NGS) and further analyses were conducted in paired premalignant and tumor tissues and adjacent normal mucosa. We then analyzed the mRNA expression data from The Cancer Genome Atlas (TCGA) and performed gene set enrichment analysis (GSEA) to predict the function of KLF5. Small interfering RNA (siRNA) targeting KLF5 was used to knock down its expression in cells and further evaluate the changes in cell apoptosis, proliferation, and migration. We predicted whether baculoviral inhibitor of apoptosis protein (IAP) repeat containing 5 (BIRC5) was the potential target gene of KLF5 via the NCBI and JASPAR databases. Furthermore, we analyzed BIRC5 expression after KLF5 knockdown and explored its function in athymic BALB/c nude mice.ResultsKLF5 expression in clinical samples gradually increased from normal mucosa tissues to premalignant and then to OSCC tissues. Analysis of TCGA data and GSEA also suggested that KLF5 was expressed at higher levels in OSCC and involved apoptosis and the protein 53 (P53), transforming growth factor-β (TGF-β), and wingless/integrated (Wnt) signaling pathways. Cell apoptosis was promoted, whereas proliferation and migration were inhibited after KLF5 knockdown. Furthermore, we found KLF5 transcription binding sites on the BIRC5 promoter and BIRC5 expression was inhibited after suppressing KLF5 in vitro and in vivo.ConclusionsOur findings indicate that KLF5 promotes the development of OSCC via BIRC5, and could be a potential diagnostic and therapeutic target for OSCC.

PB1969: FUNCTIONAL CHARACTERIZATION OF SUSCEPTIBILITY LOCI TO MULTIPLE MYELOMA

Background: Multiple myeloma (MM) is the second most common blood malignancy, caused by an uncontrolled growth of plasma cells in the bone marrow, accounting for 20% of all newly diagnosed hematological cancers. Although the current 5-year survival rate is ranging between 40-60%, MM is still considered an incurable disease since most of the patients eventually relapse. While the causes of MM are incompletely understood, several genome-wide association studies (GWAS) have been conducted to identify germline variants that predispose to MM. Up to date, a total of 24 loci were found to be associated with MM risk, but very little information is available about their functional role. Aims: The principal goal is to explore in silico the function of the germline variants associated with MM risk. As we do not know if the GWAS-identified SNPs are the causal variants or just markers of risk, the functional characterization of the causal risk variants would lead to a better understanding of disease development. Methods: GWAS design takes advantage of the linkage disequilibrium (LD) structure of the human genome, thus the main GWAS findings are single-nucleotide polymorphisms (SNPs) that show the strongest association with MM risk (measured as the lowest p-values), but they are not necessarily the functionally causal variants. In this project, we used bioinformatics tools (GTEx, HaploReg v4.1, Roadmap, LDlinke, SNPnexus, RegulomeDB 2.0.3, SNP2TFBS, miRNASNP v3, GeneMANIA) to perform fine mapping of all GWAS-identified loci and to prioritize in each locus the polymorphism with the highest chance of being functionally relevant. In particular, we focused on the loci with the smallest number of SNPs in high LD (r2>0.8) in order to maximize the probability to capture the casual variant. Results: Four of the 24 MM risk loci had a relatively small number of SNPs in high LD and within them we found that the locus located at chromosome 16 contained the greatest number of functionally annotated SNPs. Of particular functional interest was rs3747481 (chr16:30666367 C/T) due to the following reasons: it is a missense variant (protein change: P359L), it is located in the PRR14 gene, that contributes to chromatin hierarchical organization and has a role in gene regulation, has a high CADD PHRED score (22.1). Additionally, according to GTEx portal, rs3747481 is associated with the expression level of the RNF40 gene (which plays a central role in histone code and gene regulation) in whole blood cells (p=3.02-12). It has a score of “1d” in RegulomeDB (eQTL+ TF binding + any motif + DNase peak), meaning that this variant has a high likelihood to affect binding of transcription factors. Some other SNPs (like rs35629860 and rs67128646) in the same LD block show the co-occurrence of H3K4me3 and H3K27me3 histone marks (associated with gene activation and repression, respectively) in promoters and enhancers in B lymphocytes. rs6565197 is predicted to affect the binding of the KLF4 and KLF5 transcription factors which play key roles in cell cycle regulation. Summary/Conclusion: Through a fine mapping of MM risk loci by bioinformatics tools, we found a variant in the locus 16p11.2 that shows in silico a very high probability to have biological role in the risk disease.

Role of lncRNA TUG1 in Adenomyosis and its Regulatory Mechanism in Endometrial Epithelial Cell Functions.

Adenomyosis (AM) is a common gynecological disorder that can cause pelvic pain. The regulatory role of long noncoding RNAs (lncRNAs) in AM progression has been widely reported. This study investigated the effect and mechanism of lncRNA taurine-upregulated gene 1 (TUG1) on endometrial epithelial cells (EECs) in AM. Endometrial tissues of AM patients and controls were collected. A murine model of AM was established by tamoxifen induction. TUG1 expression in endometrial tissues of AM patients and mice was determined. In vivo, the effect of TUG1 on AM mice was measured through H&E staining, Masson's staining, uterine weight, and estradiol concentration. EECs isolated from AM patients were transfected with sh-TUG1. In vitro, the effect of TUG1 on the proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), and angiogenesis of EECs was evaluated by CCK8, colony formation, immunofluorescence, wound healing, and Transwell assays. The binding relationship among TUG1, E2F4, and KLF5 was confirmed using RNA immunoprecipitation and RNA pull-down assays. A function rescue experiment was designed to verify the effect of KLF5 on EECs. TUG1 expression was elevated in AM mice and patients. Downregulation of TUG1 promoted the recovery of AM mice. Downregulation of TUG1 suppressed proliferation, migration, invasion, EMT, and angiogenesis of EECs. Mechanically, TUG1 suppressed KLF5 transcription by binding to E2F4. Downregulation of KLF5 reversed the inhibitory effect of TUG1 silencing on the functions of EECs. TUG1 expression was elevated in AM, and TUG1 facilitated proliferation, migration, invasion, EMT, and angiogenesis of EECs via E2F4/KLF5, thereby aggravating AM.