ERK-Induced Activation of TCF Family of SRF Cofactors Initiates a Chromatin Modification Cascade Associated with Transcription.

We investigated the relationship among ERK signaling, histone modifications, and transcription factor activity, focusing on the ERK-regulated ternary complex factor family of SRF partner proteins. In MEFs, activation of ERK by TPA stimulation induced a common pattern of H3K9acS10ph, H4K16ac, H3K27ac, H3K9acK14ac, and H3K4me3 at hundreds of transcription start site (TSS) regions and remote regulatory sites. The magnitude of the increase in histone modification correlated well with changes in transcription. H3K9acS10ph preceded the other modifications. Most induced changes were TCF dependent, but TCF-independent TSSs exhibited the same hierarchy, indicating that it reflects gene activation per se. Studies with TCF Elk-1 mutants showed that TCF-dependent ERK-induced histone modifications required Elk-1 to be phosphorylated and competent to activate transcription. Analysis of direct TCF-SRF target genes and chromatin modifiers confirmed this and showed that H3S10ph required only Elk-1 phosphorylation. Induction of histone modifications following ERK stimulation is thus directed by transcription factor activation and transcription.

Elk1 enhances inflammatory cell infiltration and exacerbates acute lung injury/acute respiratory distress syndrome by suppressing Fcgr2b transcription.

OBJECTIVE: Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are associated with significant mortality rates. The role of Fcgr2b in the pathogenesis of ALI/ARDS is not fully elucidated. This study aimed to investigate the functions of Fcgr2b in ALI/ARDS and explore its underlying mechanisms. METHODS: Methods: In this study, rat models of ARDS and pulmonary microvascular endothelial cell (PMVEC) injury models were established through the administration of lipopolysaccharide (LPS). The expression levels of Fcgr2b and Elk1 were quantified in both LPS-induced ARDS rats and PMVECs. Subsequent gain- and loss-of-function experiments were conducted, followed by comprehensive assessments of lung tissue for pathomorphological changes, edema, glycogen storage, fibrosis, and infiltration of inflammatory cells. Additionally, bronchoalveolar lavage fluid was analyzed for T-helper 17 (Th17) cell infiltration, inflammatory response, and microvascular permeability to evaluate lung injury severity in ARDS models. Furthermore, the activity, cytotoxicity, apoptosis, and angiogenic potential of PMVECs were assessed to gauge cell injury. The interaction between Elk1 and Fcgr2b was also examined to confirm their regulatory relationship. RESULTS: In the context of LPS-induced ARDS and PMVEC injury, Fcgr2b expression was markedly reduced, whereas Elk1 expression was elevated. Overexpression of Fcgr2b led to a decrease in Th17 cell infiltration and mitigated lung tissue damage in ARDS models, in addition to reducing LPS-induced injury in PMVECs. Elk1 was found to suppress Fcgr2b transcription through the recruitment of histone 3 lysine 9 trimethylation (H3K9me3). Knockdown of Elk1 diminished Th17 cell infiltration and lung tissue damage in ARDS models, and alleviated LPS-induced injury in PMVECs, effects that were reversed upon Fcgr2b upregulation. CONCLUSION: Elk1 negatively regulates Fcgr2b transcription, thereby augmenting the inflammatory response and exacerbating lung injury in LPS-induced ALI/ARDS.

SRF Co-factors Control the Balance between Cell Proliferation and Contractility.

The ERK-regulated ternary complex factors (TCFs) act with the transcription factor serum response factor (SRF) to activate mitogen-induced transcription. However, the extent of their involvement in the immediate-early transcriptional response, and their wider functional significance, has remained unclear. We show that, in MEFs, TCF inactivation significantly inhibits over 60% of TPA-inducible gene transcription and impairs cell proliferation. Using integrated SRF ChIP-seq and Hi-C data, we identified over 700 TCF-dependent SRF direct target genes involved in signaling, transcription, and proliferation. These also include a significant number of cytoskeletal gene targets for the Rho-regulated myocardin-related transcription factor (MRTF) SRF cofactor family. The TCFs act as general antagonists of MRTF-dependent SRF target gene expression, competing directly with the MRTFs for access to SRF. As a result, TCF-deficient MEFs exhibit hypercontractile and pro-invasive behavior. Thus, competition between TCFs and MRTFs for SRF determines the balance between antagonistic proliferative and contractile programs of gene expression.

The Small Molecule Antagonist KCI807 Disrupts Association of the Amino-Terminal Domain of the Androgen Receptor with ELK1 by Modulating the Adjacent DNA Binding Domain.

The androgen receptor (AR) is a crucial coactivator of ELK1 for prostate cancer (PCa) growth, associating with ELK1 through two peptide segments (358-457 and 514-557) within the amino-terminal domain (NTD) of AR. The small-molecule antagonist 5-hydroxy-2-(3-hydroxyphenyl)chromen-4-one (KCI807) binds to AR, blocking ELK1 binding and inhibiting PCa growth. We investigated the mode of interaction of KCI807 with AR using systematic mutagenesis coupled with ELK1 coactivation assays, testing polypeptide binding and Raman spectroscopy. In full-length AR, deletion of neither ELK1 binding segment affected sensitivity of residual ELK1 coactivation to KCI807. Although the NTD is sufficient for association of AR with ELK1, interaction of the isolated NTD with ELK1 was insensitive to KCI807. In contrast, coactivation of ELK1 by the AR-V7 splice variant, comprising the NTD and the DNA binding domain (DBD), was sensitive to KCI807. Deletions and point mutations within DBD segment 558-595, adjacent to the NTD, interfered with coactivation of ELK1, and residual ELK1 coactivation by the mutants was insensitive to KCI807. In a glutathione S-transferase pull-down assay, KCI807 inhibited ELK1 binding to an AR polypeptide that included the two ELK1 binding segments and the DBD but did not affect ELK1 binding to a similar AR segment that lacked the sequence downstream of residue 566. Raman spectroscopy detected KCI807-induced conformational change in the DBD. The data point to a putative KCI807 binding pocket within the crystal structure of the DBD and indicate that either mutations or binding of KCI807 at this site will induce conformational changes that disrupt ELK1 binding to the NTD. SIGNIFICANCE STATEMENT: The small-molecule antagonist KCI807 disrupts association of the androgen receptor (AR) with ELK1, serving as a prototype for the development of small molecules for a novel type of therapeutic intervention in drug-resistant prostate cancer. This study provides basic information needed for rational KCI807-based drug design by identifying a putative binding pocket in the DNA binding domain of AR through which KCI807 modulates the amino-terminal domain to inhibit ELK1 binding.

T-cell activation decreases miRNA-15a/16 levels to promote MEK1-ERK1/2-Elk1 signaling and proliferative capacity.

While miRs have been extensively studied in the context of malignancy and tumor progression, their functions in regulating T-cell activation are less clear. In initial studies, we found reduced levels of miR-15a/16 at 3 to 18 h post-T-cell receptor (TCR) stimulation, suggesting a role for decreased levels of this miR pair in shaping T-cell activation. To further explore this, we developed an inducible miR15a/16 transgenic mouse model to determine how elevating miR-15a/16 levels during early stages of activation would affect T-cell proliferation and to identify TCR signaling pathways regulated by this miR pair. Doxycycline (DOX)-induced expression of miR-15a/16 from 0 to 18 h post-TCR stimulation decreased ex vivo T-cell proliferation as well as in vivo antigen-specific T-cell proliferation. We also combined bioinformatics and proteomics approaches to identify the mitogen-activated protein kinase kinase 1 (MEK1) (Map2k1) as a target of miR-15a/16. MEK1 targeting by miR-15a/16 was confirmed using miR mimics that decreased Map2k1 mRNA containing the 3'-UTR target nucleotide sequence (UGCUGCUA) but did not decrease Map2k1 containing a mutated control sequence (AAAAAAAA). Phosphorylation of downstream signaling molecules, extracellular signal-regulated protein kinase 1/2 (ERK1/2) and Elk1, was also decreased by DOX-induced miR-15a/16 expression. In addition to MEK1, ERK1 was subsequently found to be targeted by miR-15a/16, with DOX-induced miR-15a/16 reducing total ERK1 levels in T cells. These findings show that TCR stimulation reduces miR-15a/16 levels at early stages of T-cell activation to facilitate increased MEK1 and ERK1, which promotes the sustained MEK1-ERK1/2-Elk1 signaling required for optimal proliferation.

Aberrant expression of CKS2 induced by ELK1 contributes to malignant progression of pancreatic cancer.

Cyclin-dependent kinase subunit 2 (CKS2) has been reported to promote various malignancies. This study investigated the functional role of CKS2 in pancreatic cancer (PC). An analysis of abnormally expressed genes and their prognostic value for PC was performed by using the Gene Expression Profiling Interactive Analysis (GEPIA) database and performing immunohistochemical staining on 64 samples of tumor tissue. CCK-8 assays, EdU staining, colony formation assays, flow cytometry, and a xenograft tumor model were used to analyze the biological function of CKS2 in PC. Our results revealed that CKS2 was expressed at significantly higher levels in PC tissues than in adjacent normal tissues, and a high level of CKS2 expression was associated with a poor prognosis for patients with PC. Moreover, functional assays revealed that CKS2 knockdown suppressed cell proliferation, induced cell cycle S phase, G2/M phase arrest, and apoptosis in vitro, and also reduced tumor growth in vivo. In addition, CKS2 knockdown increased the levels of Bax, caspase-3, P53, P21, and GADD45α expression, but decreased Bcl-2, Cyclin B1, CDK1, Cyclin A, and Cdc25C expression. CKS2 overexpression produced the opposite effects of CKS2 knockdown. Furthermore, we found that ELK1 protein regulated transcription of the CKS2 gene. In conclusion, our findings suggest that CKS2 expression is regulated by ELK1, which could possibly serve as prognostic indicator and therapeutic target for PC.

LncRNA 122049 suppresses apoptosis of renal tubular epithelial cells in ischemic AKI by targeting the miR-330-5p/ELK1 axis.

Several studies have reported that long non-coding RNAs (LncRNAs) were associated with the progression of acute kidney injury (AKI). However, the role and regulation mechanism of lncRNA122049 in ischemic AKI remains unknown. In the present study, we found that lncRNA 122049 protected against the ischemia/reperfusion (I/R) induced apoptosis in BUMPT cells. Mechanistically, the lncRNA 122049 directly sponged miR-330-5p, then increased the expression of ELK1(ETS transcription factor ELK1) to decrease renal cell apoptosis. In addition, miR-330-5p inhibitor completely reversed the pro-apoptotic effect of LncRNA 122049 siRNA on I/R-induced BUMPT cells apoptosis. Finally, overexpression of lncRNA 122049 attenuated ischemic mice AKI via targeting of the miR-330-5p/ELK1 axis. Collectively, the data demonstrated that LncRNA 122049 prevented the I/R-induced renal cell apoptosis via regulation of the miR-330-5p/ELK1 axis, which brings new insights into the pathogenesis and potential targeted treatment of ischemic AKI.

Comparative analysis of protein expression systems and PTM landscape in the study of transcription factor ELK-1.

Post-translational modifications (PTMs) are important for protein folding and activity, and the ability to recreate physiologically relevant PTM profiles on recombinantly-expressed proteins is vital for meaningful functional analysis. The ETS transcription factor ELK-1 serves as a paradigm for cellular responses to mitogens and can synergise with androgen receptor to promote prostate cancer progression, although in vitro protein function analyses to date have largely overlooked its complex PTM landscapes. We expressed and purified human ELK-1 using mammalian (HEK293T), insect (Sf9) and bacterial (E. coli) systems in parallel and compared PTMs imparted upon purified proteins, along with their performance in DNA and protein interaction assays. Phosphorylation of ELK-1 within its transactivation domain, known to promote DNA binding, was most apparent in protein isolated from human cells and accordingly conferred the strongest DNA binding in vitro, while protein expressed in insect cells bound most efficiently to the androgen receptor. We observed lysine acetylation, a hitherto unreported PTM of ELK-1, which appeared highest in insect cell-derived ELK-1 but was also present in HEK293T-derived ELK-1. Acetylation of ELK-1 was enhanced in HEK293T cells following starvation and mitogen stimulation, and modified lysines showed overlap with previously identified regulatory SUMOylation and ubiquitination sites. Our data demonstrate that the choice of recombinant expression system can be tailored to suit biochemical application rather than to maximise soluble protein production and suggest the potential for crosstalk and antagonism between different PTMs of ELK-1.

Smooth Muscle Overexpression of PGC1α Attenuates Atherosclerosis in Rabbits.

[Figure: see text].

Identification of ELK1 interacting peptide segments in the androgen receptor.

Prostate cancer (PCa) growth requires tethering of the androgen receptor (AR) to chromatin by the ETS domain transcription factor ELK1 to coactivate critical cell proliferation genes. Disruption of the ELK1-AR complex is a validated potential means of therapeutic intervention in PCa. AR associates with ELK1 by coopting its two ERK docking sites, through the amino-terminal domain (A/B domain) of AR. Using a mammalian two-hybrid assay, we have now functionally mapped amino acids within the peptide segments 358-457 and 514-557 in the A/B domain as required for association with ELK1. The mapping data were validated by GST (glutathione S-transferase)-pulldown and BRET (bioluminescence resonance energy transfer) assays. Comparison of the relative contributions of the interacting motifs/segments in ELK1 and AR to coactivation of ELK1 by AR suggested a parallel mode of binding of AR and ELK1 polypeptides. Growth of PCa cells was partially inhibited by deletion of the upstream segment in AR and nearly fully inhibited by deletion of the downstream segment. Our studies have identified two peptide segments in AR that mediate the functional association of AR with its two docking sites in ELK1. Identification of the ELK1 recognition sites in AR should enable further structural studies of the ELK1-AR interaction and rational design of small molecule drugs to disrupt this interaction.

ELK1/KIFC1 axis promotes breast cancer cell proliferation by regulating glutathione metabolism.

BACKGROUND: KIFC1 exerts an important function in centrosome aggregation in breast cancer (BC) cells and a variety of other cancer cells, but its potential mechanisms in BC pathogenesis are yet fully elucidated. The aim of this study was to investigate the effects of KIFC1 on BC progression and its underlying mechanisms. METHODS: Expression of ELK1 and KIFC1 in BC was analyzed by The Cancer Genome Atlas database and quantitative real-time polymerase chain reaction. Cell proliferative capacity was examined by CCK-8 and colony formation assays, respectively. Glutathione (GSH)/glutathione disulfide (GSSG) ratio and GSH level were measured using the kit. Expression of GSH metabolism-related enzymes (G6PD, GCLM, and GCLC) was detected by western blot. Intracellular reactive oxygen species (ROS) levels were measured by the ROS Assay Kit. The transcription factor ELK1 upstream of KIFC1 was identified by hTFtarget, KnockTFv2 database and Pearson correlation. Their interaction was validated by dual-luciferase reporter assay and chromatin immunoprecipitation. RESULTS: This study demonstrated the upregulation of ELK1 and KIFC1 in BC and found that ELK1 could bind to the KIFC1 promoter to promote KIFC1 transcription. KIFC1 overexpression increased cell proliferation and intracellular GSH levels, while decreasing intracellular ROS levels. The addition of the GSH metabolism inhibitor BSO attenuated the promotion of BC cell proliferation induced by KIFC1 overexpression. In addition, KIFC1 overexpression reversed the inhibitory effect of knockdown of ELK1 on BC cell proliferation. CONCLUSION: ELK1 was a transcriptional factor of KIFC1. ELK1/KIFC1 axis reduced ROS level by increasing GSH synthesis, thus facilitating BC cell proliferation. Current observations suggest that ELK1/ KIFC1 may be a potential therapeutic target for BC treatment.

ELK-1 ubiquitination status and transcriptional activity are modulated independently of F-Box protein FBXO25.

The mitogen-responsive, ETS-domain transcription factor ELK-1 stimulates the expression of immediate early genes at the onset of the cell cycle and participates in early developmental programming. ELK-1 is subject to multiple levels of posttranslational control, including phosphorylation, SUMOylation, and ubiquitination. Recently, removal of monoubiquitin from the ELK-1 ETS domain by the Ubiquitin Specific Protease USP17 was shown to augment ELK-1 transcriptional activity and promote cell proliferation. Here we have used coimmunoprecipitation experiments, protein turnover and ubiquitination assays, RNA-interference and gene expression analyses to examine the possibility that USP17 acts antagonistically with the F-box protein FBXO25, an E3 ubiquitin ligase previously shown to promote ELK-1 ubiquitination and degradation. Our data confirm that FBXO25 and ELK-1 interact in HEK293T cells and that FBXO25 is active toward Hand1 and HAX1, two of its other candidate substrates. However, our data indicate that FBXO25 neither promotes ubiquitination of ELK-1 nor impacts on its transcriptional activity and suggest that an E3 ubiquitin ligase other than FBXO25 regulates ELK-1 ubiquitination and function.

The SETD8/ELK1/bach1 complex regulates hyperglycaemia-mediated EndMT in diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DN), the most common microvascular complication in patients with diabetes, induces kidney failure. Previous research showed that endothelial-to-mesenchymal transition (EndMT) of human glomerular endothelial cells (HGECs) is involved in the progression of DN. Moreover, SET domain-containing protein 8 (SETD8), ETS-domain containing protein (ELK1) and BTB and CNC homology 1 (bach1) all participate in endothelial injury. In this study, we hypothesize that the SETD8/ELK1/bach1 functional axis is involved in mediating EndMT in diabetic nephropathy. METHODS: Immunohistochemistry, Western blotting and qPCR were performed to determine the protein and mRNA levels of genes in HGECs and the kidney tissues of participants and rats. Immunofluorescence, Co-IP and GST pulldown assays were performed to verify the direct interaction between SETD8 and ELK1. ChIP and dual-luciferase assays were performed to determine the transcriptional regulation of bach1 and Snail. AVV-SETD8 injection in rat kidney was used to verify the potential protective effect of SETD8 on DN. RESULTS: Our current study showed that hyperglycaemia triggered EndMT by increasing Snail expression both in vitro and in vivo. Moreover, high glucose increased bach1 expression in HGECs, positively regulating Snail and EndMT. As a transcription factor, ELK1 was augmented and participated in hyperglycaemia-induced EndMT via modulation of bach1 expression. Moreover, ELK1 was found to associate with SETD8. Furthermore, SETD8 negatively regulated EndMT by cooperating with bach1 to regulate Snail transcription. Furthermore, histone H4-Lys-20 monomethylation (H4K20me1), which is downstream of SETD8, was accompanied by ELK1 localization at the same promoter region of bach1. ELK1 overexpression enhanced bach1 promoter activity, which disappeared after specific binding site deletion. Mutual inhibition between ELK1 and SETD8 was found in HGECs. In vivo, SETD8 overexpression decreased ELK1 and bach1 expression, as well as EndMT. Moreover, SETD8 overexpression improved the renal function of rats with DN. CONCLUSIONS: SETD8 cooperates with ELK1 to regulate bach1 transcription, thus participating in the progression of DN. In addition, SETD8 interacts with bach1 to modulate Snail transcription, thus inducing EndMT in DN. SETD8 plays a core role in the SETD8/ELK1/bach1 functional axis, which participates in hyperglycaemia-mediated EndMT in DN, and SETD8 may be a potential therapeutic target for DN. Trial registration ChiCTR, ChiCTR2000029425. 2020/1/31, http://www.chictr.org.cn/showproj.aspx?proj=48548.

GPX4 suppresses ferroptosis to promote malignant progression of endometrial carcinoma via transcriptional activation by ELK1.

BACKGROUND: Glutathione Peroxidase 4 (GPX4) is a key protein that inhibits ferroptosis. However, its biological regulation and mechanism in endometrial cancer (EC) have not been reported in detail. METHODS: The expression of GPX4 in EC tissues was determined by TCGA databases, qRT-PCR, Western blot, and immunohistochemistry (IHC). The effects of GPX4 on EC cell proliferation, migration, apoptosis, and tumorigenesis were studied in vivo and in vitro. In addition, ETS Transcription Factor ELK1 (ELK1) was identified by bioinformatics methods, dual-luciferase reporter assay, and chromatin immunoprecipitation (ChIP). Pearson correlation analysis was used to evaluate the association between ELK1 and GPX4 expression. RESULTS: The expression of GPX4 was significantly up-regulated in EC tissues and cell lines. Silencing GPX4 significantly inhibited the proliferation, migration ability, induced apoptosis, and arrested the cell cycle of Ishikawa and KLE cells. Knockdown of GPX4 accumulated intracellular ferrous iron and ROS, disrupted MMP, and increased MDA levels. The xenograft tumor model also showed that GPX4 knockdown markedly reduced tumor growth in mice. Mechanically, ELK1 could bind to the promoter of GPX4 to promote its transcription. In addition, the expression of ELK1 in EC was positively correlated with GPX4. Rescue experiments confirmed that GPX4 knockdown could reverse the strengthens of cell proliferation and migration ability and the lower level of Fe2+ and MDA caused by upregulating ELK1. CONCLUSION: The results of the present study suggest that ELK1 / GPX4 axis plays an important role in the progress of EC by promoting the malignant biological behavior and inducing ferroptosis of EC cells, which provides evidence for investigating the potential therapeutic strategies of endometrial cancer.

Early epigenomic and transcriptional changes reveal Elk-1 transcription factor as a therapeutic target in Huntington's disease.

Huntington's disease (HD) is a chronic neurodegenerative disorder characterized by a late clinical onset despite ubiquitous expression of the mutant Huntingtin gene (HTT) from birth. Transcriptional dysregulation is a pivotal feature of HD. Yet, the genes that are altered in the prodromal period and their regulators, which present opportunities for therapeutic intervention, remain to be elucidated. Using transcriptional and chromatin profiling, we found aberrant transcription and changes in histone H3K27acetylation in the striatum of R6/1 mice during the presymptomatic disease stages. Integrating these data, we identified the Elk-1 transcription factor as a candidate regulator of prodromal changes in HD. Exogenous expression of Elk-1 exerted beneficial effects in a primary striatal cell culture model of HD, and adeno-associated virus-mediated Elk-1 overexpression alleviated transcriptional dysregulation in R6/1 mice. Collectively, our work demonstrates that aberrant gene expression precedes overt disease onset in HD, identifies the Elk-1 transcription factor as a key regulator linked to early epigenetic and transcriptional changes in HD, and presents evidence for Elk-1 as a target for alleviating molecular pathology in HD.

E3 ubiquitin ligase PARK2, an inhibitor of melanoma cell growth, is repressed by the oncogenic ERK1/2-ELK1 transcriptional axis.

Malignant melanoma, the most aggressive form of skin cancer, is characterized by high prevalence of BRAF/NRAS mutations and hyperactivation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), mitogen-activated protein kinases (MAPK), leading to uncontrolled melanoma growth. Efficacy of current targeted therapies against mutant BRAF or MEK1/2 have been hindered by existence of innate or development of acquired resistance. Therefore, a better understanding of the mechanisms controlled by MAPK pathway driving melanogenesis will help develop new treatment approaches targeting this oncogenic cascade. Here, we identify E3 ubiquitin ligase PARK2 as a direct target of ELK1, a known transcriptional effector of MAPK signaling in melanoma cells. We show that pharmacological inhibition of BRAF-V600E or ERK1/2 in melanoma cells increases PARK2 expression. PARK2 overexpression reduces melanoma cell growth in vitro and in vivo and induces apoptosis. Conversely, its genetic silencing increases melanoma cell proliferation and reduces cell death. Further, we demonstrate that ELK1 is required by the BRAF-ERK1/2 pathway to repress PARK2 expression and promoter activity in melanoma cells. Clinically, PARK2 is highly expressed in WT BRAF and NRAS melanomas, but it is expressed at low levels in melanomas carrying BRAF/NRAS mutations. Overall, our data provide new insights into the tumor suppressive role of PARK2 in malignant melanoma and uncover a novel mechanism for the negative regulation of PARK2 via the ERK1/2-ELK1 axis. These findings suggest that reactivation of PARK2 may be a promising therapeutic approach to counteract melanoma growth.

c-KIT-ERK1/2 signaling activated ELK1 and upregulated carcinoembryonic antigen expression to promote colorectal cancer progression.

Carcinoembryonic antigen (CEA) is highly expressed in embryo and colorectal cancer (CRC) and has been widely used as a marker for CRC. Emerging evidence has demonstrated that elevated CEA levels promote CRC progression. However, the mechanism of the increased CEA expression in patients with primary and recurrent CRC is still an open question. In this study, we showed that c-KIT, ELK1, and CEA were hyperexpressed in patients with CRC, especially patients with recurrent disease. From bioinformatics analysis, we picked ELK1 as a candidate transcription factor (TF) for CEA; the binding site of ELK1 within the CEA promoter was confirmed by chromatin immunoprecipitation and dual luciferase reporter assays. Overexpression of ELK1 increased CEA expression in vitro, while knockdown of ELK1 decreased CEA. Upregulated ELK1 promoted the adhesion, migration, and invasion of CRC cells, however knockdown of CEA blocked the activities of ELK1-overexpressed CRC cells. Furthermore, we explored the role of c-KIT-ERK1/2 signaling in activation of ELK1. Blocking c-KIT signaling using Imatinib or ISCK03 reduced p-ELK1 expression and consequently decreased CEA levels in CRC cells, as did blocking the ERK1/2 pathway by U0126. Compared with wild type littermates, the c-kit loss-of-functional Wadsm/m mice showed lowered c-KIT, ELK1, and CEA expression. In conclusion, our study revealed that ELK1, which was activated by c-KIT-ERK1/2 signaling, was a key TF for CEA expression. Blocking ELK1 or its upstream signaling could be an alternative way to decelerate CRC progression. Besides being a biomarker for CRC, CEA could be used for guiding targeted therapy.

Long noncoding RNA HBBP1 enhances γ-globin expression through the ETS transcription factor ELK1.

ß-Thalassemia is an autosomal recessive genetic disease caused by defects in the production of adult hemoglobin (HbA, α2ß2), which leads to an imbalance between α- and non-α-globin chains. Reactivation of γ-globin expression is an effective strategy to treat ß-thalassemia patients. Previously, it was demonstrated that hemoglobin subunit beta pseudogene 1 (HBBP1) is associated with elevated fetal hemoglobin (HbF, α2γ2) in ß-thalassemia patients. However, the mechanism underlying HBBP1-mediated HbF production is unknown. In this study, using bioinformatics analysis, we found that HBBP1 is involved in γ-globin production, and then preliminarily confirmed this finding in K562 cells. When HBBP1 was overexpressed, γ-globin expression was increased at the transcript and protein levels in HUDEP-2 cells. Next, we found that ETS transcription factor ELK1 (ELK1) binds to the HBBP1 proximal promoter and significantly promotes its activity. Moreover, the synthesis of γ-globin was enhanced when ELK1 was overexpressed in HUDEP-2 cells. Surprisingly, ELK1 also directly bound to and activated the γ-globin proximal promoter. Furthermore, we found that HBBP1 and ELK1 can interact with each other in HUDEP-2 cells. Collectively, these findings suggest that HBBP1 can induce γ-globin by enhancing ELK1 expression, providing some clues for γ-globin reactivation in ß-thalassemia.

ELK1-induced upregulation lncRNA LINC02381 accelerates the osteosarcoma tumorigenesis through targeting CDCA4 via sponging miR-503-5p.

Long noncoding RNAs (lncRNAs) have been identified as functional modulators in human tumors. The purpose of our study was to determine the expressing trend, clinical significance and functions of lncRNA LINC02381(LINC02381) in osteosarcoma. We observed that the expression of LINC02381 and cell division cycle-associated protein 4 (CDCA4) were distinctly increased in osteosarcoma specimens and cells, while miR-503-5p expression was decreased. Additionally, ETS transcription factor ELK1 (ELK1) could bind directly to the LINC02381 promoter region and activate its transcription. Clinical assays revealed that high LINC02381 was associated with advanced clinical progress and poor clinical outcome. Functionally, knockdown of LINC02381 suppressed the proliferation, migration and invasion of osteosarcoma cells. What's more, LINC02381 could down-regulate CDCA4 via sponging miR-503-5p, and there existed a negative correlation between LINC02381 expression and miR-503-5p expression in 92 osteosarcoma samples. Rescue experiments proved the carcinogenic role of LINC02381/miR-503-5p/CDCA4 axis in osteosarcoma progression. Overall, our data illustrated how LINC02381 played an oncogenic role in osteosarcoma and might offer a novel diagnostic and prognostic biomarker and potential therapeutic target for osteosarcoma.

14-3-3 epsilon is an intracellular component of TNFR2 receptor complex and its activation protects against osteoarthritis.

OBJECTIVES: Osteoarthritis (OA) is the most common joint disease; however, the indeterminate nature of mechanisms by which OA develops has restrained advancement of therapeutic targets. TNF signalling has been implicated in the pathogenesis of OA. TNFR1 primarily mediates inflammation, whereas emerging evidences demonstrate that TNFR2 plays an anti-inflammatory and protective role in several diseases and conditions. This study aims to decipher TNFR2 signalling in chondrocytes and OA. METHODS: Biochemical copurification and proteomics screen were performed to isolate the intracellular cofactors of TNFR2 complex. Bulk and single cell RNA-seq were employed to determine 14-3-3 epsilon (14-3-3ε) expression in human normal and OA cartilage. Transcription factor activity screen was used to isolate the transcription factors downstream of TNFR2/14-3-3ε. Various cell-based assays and genetically modified mice with naturally occurring and surgically induced OA were performed to examine the importance of this pathway in chondrocytes and OA. RESULTS: Signalling molecule 14-3-3ε was identified as an intracellular component of TNFR2 complexes in chondrocytes in response to progranulin (PGRN), a growth factor known to protect against OA primarily through activating TNFR2. 14-3-3ε was downregulated in OA and its deficiency deteriorated OA. 14-3-3ε was required for PGRN regulation of chondrocyte metabolism. In addition, both global and chondrocyte-specific deletion of 14-3-3ε largely abolished PGRN's therapeutic effects against OA. Furthermore, PGRN/TNFR2/14-3-3ε signalled through activating extracellular signal-regulated kinase (ERK)-dependent Elk-1 while suppressing nuclear factor kappa B (NF-κB) in chondrocytes. CONCLUSIONS: This study identifies 14-3-3ε as an inducible component of TNFR2 receptor complex in response to PGRN in chondrocytes and presents a previously unrecognised TNFR2 pathway in the pathogenesis of OA.