Baicalein Inhibits Metastasis of Oral Squamous Cell Carcinoma Cells by Regulating ERK/ELK-1/Snail Signaling.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is associated with high recurrence and poor prognosis. Baicalin has multiple pharmacological effects, including anti-inflammatory and anti-proliferative activities. Here, we examine the effect of baicalein on OSCC metastasis and its potential mechanism of action. METHODS: SCC-4 and CAL-27 cells were treated with different concentrations of baicalein. The proliferation of OSCC cells was evaluated by Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. As for migration and metastasis, baicalein-treated OSCC cells were used for wound healing assay and Transwell assay. The levels of epithelial-mesenchymal transition-related proteins (E-cadherin, N-cadherin, vimentin) and extracellular regulated protein kinases (ERK)/ETS Transcription Factor ELK1 (ELK-1)/Snail signaling pathway-related proteins in baicalein-treated OSCC cells were evaluated by western blotting. RESULTS: The rates of cell proliferation and migration, along with the metastatic potential, of baicalein-treated cells were significantly lower than those of the control (p < 0.05), and the effects were concentration-dependent. Furthermore, compared to the control, baicalein significantly decreased the levels of N-cadherin and vimentin in SCC-4 and CAL-27 cells, and increased the E-cadherin level (p < 0.05). Mechanistically, baicalein downregulated the levels of p-ERK1/2, phospho-ETS Transcription Factor ELK1 (p-ELK-1), and Snail (p < 0.05). Finally, the ERK/ELK-1/Snail pathway inhibitor (U0126) promoted the effect of baicalein in inhibiting the migration and invasion of OSCC cells (p < 0.05). CONCLUSION: Baicalein abates the migration, invasion, and metastasis of OSCC cells through the ERK/ELK-1/Snail signaling pathway. This study provides a basis for the development of baicalein as a compound for the treatment of OSCC.

Serum Response Factor Expression in Excess Permits a Dual Contractile-Proliferative Phenotype of Airway Smooth Muscle.

The transcription factors (TFs) MyoCD (myocardin) and Elk-1 (ETS Like-1 protein) competitively bind to SRF (serum response factor) and control myogenic- and mitogenic-related gene expression in smooth muscle, respectively. Their functions are therefore mutually inhibitory, which results in a contractile-versus-proliferative phenotype dichotomy. Airway smooth muscle cell (ASMC) phenotype alterations occur in various inflammatory airway diseases, promoting pathological remodeling and contributing to airflow obstruction. We characterized MyoCD and Elk-1 interactions and their roles in phenotype determination in human ASMCs. MyoCD overexpression in ASMCs increased smooth muscle gene expression, force generation, and partially restored the loss of smooth muscle protein associated with prolonged culturing while inhibiting Elk-1 transcriptional activities and proliferation induced by EGF (epidermal growth factor). However, MyoCD overexpression failed to suppress these responses induced by FBS, as FBS also upregulated SRF expression to a degree that allowed unopposed function of both TFs. Inhibition of the RhoA pathway reversed said SRF changes, allowing inhibition of Elk-1 by MyoCD overexpression and suppressing FBS-mediated contractile protein gene upregulation. Our study confirmed that MyoCD in increased abundance can competitively inhibit Elk-1 function. However, SRF upregulation permits a dual contractile-proliferative ASMC phenotype that is anticipated to exacerbate pathological alterations, whereas therapies targeting SRF may inhibit pathological ASMC proliferation and contractile protein gene expression.

The Mediator Med23 controls a transcriptional switch for muscle stem cell proliferation and differentiation in muscle regeneration.

Muscle stem cells (MuSCs) contribute to a robust muscle regeneration process after injury, which is highly orchestrated by the sequential expression of multiple key transcription factors. However, it remains unclear how key transcription factors and cofactors such as the Mediator complex cooperate to regulate myogenesis. Here, we show that the Mediator Med23 is critically important for MuSC-mediated muscle regeneration. Med23 is increasingly expressed in activated/proliferating MuSCs on isolated myofibers or in response to muscle injury. Med23 deficiency reduced MuSC proliferation and enhanced its precocious differentiation, ultimately compromising muscle regeneration. Integrative analysis revealed that Med23 oppositely impacts Ternary complex factor (TCF)-targeted MuSC proliferation genes and myocardin-related transcription factor (MRTF)-targeted myogenic differentiation genes. Consistently, Med23 deficiency decreases the ETS-like transcription factor 1 (Elk1)/serum response factor (SRF) binding at proliferation gene promoters but promotes MRTF-A/SRF binding at myogenic gene promoters. Overall, our study reveals the important transcriptional control mechanism of Med23 in balancing MuSC proliferation and differentiation in muscle regeneration.

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.

Small-molecule α-lipoic acid targets ELK1 to balance human neutrophil and erythrocyte differentiation.

BACKGROUND: Erythroid and myeloid differentiation disorders are commonly occurred in leukemia. Given that the relationship between erythroid and myeloid lineages is still unclear. To find the co-regulators in erythroid and myeloid differentiation might help to find new target for therapy of myeloid leukemia. In hematopoiesis, ALA (alpha lipoic acid) is reported to inhibit neutrophil lineage determination by targeting transcription factor ELK1 in granulocyte-monocyte progenitors via splicing factor SF3B1. However, further exploration is needed to determine whether ELK1 is a common regulatory factor for erythroid and myeloid differentiation. METHODS: In vitro culture of isolated CD34+, CMPs (common myeloid progenitors) and CD34+ CD371- HSPCs (hematopoietic stem progenitor cells) were performed to assay the differentiation potential of monocytes, neutrophils, and erythrocytes. Overexpression lentivirus of long isoform (L-ELK1) or the short isoform (S-ELK1) of ELK1 transduced CD34+ HSPCs were transplanted into NSG mice to assay the human lymphocyte and myeloid differentiation differences 3 months after transplantation. Knocking down of SRSF11, which was high expressed in CD371+GMPs (granulocyte-monocyte progenitors), upregulated by ALA and binding to ELK1-RNA splicing site, was performed to analyze the function in erythroid differentiation derived from CD34+ CD123mid CD38+ CD371- HPCs (hematopoietic progenitor cells). RNA sequencing of L-ELK1 and S-ELK1 overexpressed CD34+ CD123mid CD38+ CD371- HPCs were performed to assay the signals changed by ELK1. RESULTS: Here, we presented new evidence that ALA promoted erythroid differentiation by targeting the transcription factor ELK1 in CD34+ CD371- hematopoietic stem progenitor cells (HSPCs). Overexpression of either the long isoform (L-ELK1) or the short isoform (S-ELK1) of ELK1 inhibited erythroid-cell differentiation, but knockdown of ELK1 did not affect erythroid-cell differentiation. RNAseq analysis of CD34+ CD123mid CD38+ CD371- HPCs showed that L-ELK1 upregulated the expression of genes related to neutrophil activity, phosphorylation, and hypoxia signals, while S-ELK1 mainly regulated hypoxia-related signals. However, most of the genes that were upregulated by L-ELK1 were only moderately upregulated by S-ELK1, which might be due to a lack of serum response factor interaction and regulation domains in S-ELK1 compared to L-ELK1. In summary, the differentiation of neutrophils and erythrocytes might need to rely on the dose of L-ELK1 and S-ELK1 to achieve precise regulation via RNA splicing signals at early lineage commitment. CONCLUSIONS: ALA and ELK1 are found to regulate both human granulopoiesis and erythropoiesis via RNA spliceosome, and ALA-ELK1 signal might be the target of human leukemia therapy.

High expression of RTEL1 predicates worse progression in gliomas and promotes tumorigenesis through JNK/ELK1 cascade.

Gliomas are the most common primary intracranial tumor worldwide. The maintenance of telomeres serves as an important biomarker of some subtypes of glioma. In order to investigate the biological role of RTEL1 in glioma. Relative telomere length (RTL) and RTEL1 mRNA was explored and regression analysis was performed to further examine the relationship of the RTL and the expression of RTEL1 with clinicopathological characteristics of glioma patients. We observed that high expression of RTEL1 is positively correlated with telomere length in glioma tissue, and serve as a poor prognostic factor in TERT wild-type patients. Further in vitro studies demonstrate that RTEL1 promoted proliferation, formation, migration and invasion ability of glioma cells. In addition, in vivo studies also revealed the oncogene role of RTEL1 in glioma. Further study using RNA sequence and phospho-specific antibody microarray assays identified JNK/ELK1 signaling was up-regulated by RTEL1 in glioma cells through ROS. In conclusion, our results suggested that RTEL1 promotes glioma tumorigenesis through JNK/ELK1 cascade and indicate that RTEL1 may be a prognostic biomarker in gliomas.

ELK1/MTOR/S6K1 Pathway Contributes to Acquired Resistance to Gefitinib in Non-Small Cell Lung Cancer.

The development of acquired resistance to small molecule tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR) signaling has hindered their efficacy in treating non-small cell lung cancer (NSCLC) patients. Our previous study showed that constitutive activation of the 70 kDa ribosomal protein S6 kinase 1 (S6K1) contributes to the acquired resistance to EGFR-TKIs in NSCLC cell lines and xenograft tumors in nude mice. However, the regulatory mechanisms underlying S6K1 constitutive activation in TKI-resistant cancer cells have not yet been explored. In this study, we recapitulated this finding by taking advantage of a gefitinib-resistant patient-derived xenograft (PDX) model established through a number of passages in mice treated with increasing doses of gefitinib. The dissociated primary cells from the resistant PDX tumors (PDX-R) displayed higher levels of phosphor-S6K1 expression and were resistant to gefitinib compared to cells from passage-matched parental PDX tumors (PDX-P). Both genetic and pharmacological inhibition of S6K1 increased sensitivity to gefitinib in PDX-R cells. In addition, both total and phosphorylated mechanistic target of rapamycin kinase (MTOR) levels were upregulated in PDX-R and gefitinib-resistant PC9G cells. Knockdown of MTOR by siRNA decreased the expression levels of total and phosphor-S6K1 and increased sensitivity to gefitinib in PDX-R and PC9G cells. Moreover, a transcription factor ELK1, which has multiple predicted binding sites on the MTOR promoter, was also upregulated in PDX-R and PC9G cells, while the knockdown of ELK1 led to decreased expression of MTOR and S6K1. The chromatin immunoprecipitation (ChIP)-PCR assay showed the direct binding between ELK1 and the MTOR promoter, and the luciferase reporter assay further indicated that ELK1 could upregulate MTOR expression through tuning up its transcription. Silencing ELK1 via siRNA transfection improved the efficacy of gefitinib in PDX-R and PC9G cells. These results support the notion that activation of ELK1/MTOR/S6K1 signaling contributes to acquired resistance to gefitinib in NSCLC. The findings in this study shed new light on the mechanism for acquired EGFR-TKI resistance and provide potential novel strategies by targeting the ELK1/MTOR/S6K1 pathway.

ANXA3 interference inactivates ERK/ELK1 pathway to mitigate inflammation and apoptosis in sepsis-associated acute lung injury.

Acute lung injury (ALI) is a prevailing and deadly complication of sepsis coupled with increasing incidence and fatality rate. Annexin A3 (ANXA3) has been unraveled to be upregulated during sepsis. This study purposed to assess the role and the mechanism of ANXA3 in sepsis-induced ALI. After the construction of mouse model of sepsis, the pathological changes of mice lung tissues were estimated by H&E staining. ANXA3 expression in mice lung tissues and serum was examined. The degree of pulmonary edema and the levels of inflammatory factors in bronchoalveolar lavage fluid (BALF) were analyzed. In lipopolysaccharide (LPS)-induced mouse ALI model in vitro, CCK-8 assay measured cell viability and flow cytometry analysis detected cell apoptosis. Besides, ELISA assay detected the release of inflammatory cytokines. Western blot analyzed the expression of proteins associated with inflammation, apoptosis and extracellular-signal-regulated kinase (ERK)/ETS-like gene 1 (ELK1) signaling. Results revealed that ANXA3 was overexpressed in the lung tissues and serum of septic mice. Following the knockdown of ANXA3, sepsis-induced lung injury was alleviated, manifested as reduced lung edema, decreased inflammatory cell infiltration and inhibited cell apoptosis. Additionally, ANXA3 silence blocked ERK/ELK1 signaling both in sepsis mouse models and in vitro model of ALI induced by lipopolysaccharide (LPS). Moreover, the inhibitory effects of ANXA3 silencing on ERK/ELK1 signaling activation, the viability damage, inflammation and apoptosis in LPS-induced mouse ALI model in vitro were partially reversed by ERK activator. Collectively, depletion of ANXA3 exerted suppressive effects on the inflammation and apoptosis in sepsis-induced ALI through blocking ERK/ELK1 signaling.

Vascular injury activates the ELK1/SND1/SRF pathway to promote vascular smooth muscle cell proliferative phenotype and neointimal hyperplasia.

BACKGROUND: Vascular smooth muscle cell (VSMC) proliferation is the leading cause of vascular stenosis or restenosis. Therefore, investigating the molecular mechanisms and pivotal regulators of the proliferative VSMC phenotype is imperative for precisely preventing neointimal hyperplasia in vascular disease. METHODS: Wire-induced vascular injury and aortic culture models were used to detect the expression of staphylococcal nuclease domain-containing protein 1 (SND1). SMC-specific Snd1 knockout mice were used to assess the potential roles of SND1 after vascular injury. Primary VSMCs were cultured to evaluate SND1 function on VSMC phenotype switching, as well as to investigate the mechanism by which SND1 regulates the VSMC proliferative phenotype. RESULTS: Phenotype-switched proliferative VSMCs exhibited higher SND1 protein expression compared to the differentiated VSMCs. This result was replicated in primary VSMCs treated with platelet-derived growth factor (PDGF). In the injury model, specific knockout of Snd1 in mouse VSMCs reduced neointimal hyperplasia. We then revealed that ETS transcription factor ELK1 (ELK1) exhibited upregulation and activation in proliferative VSMCs, and acted as a novel transcription factor to induce the gene transcriptional activation of Snd1. Subsequently, the upregulated SND1 is associated with serum response factor (SRF) by competing with myocardin (MYOCD). As a co-activator of SRF, SND1 recruited the lysine acetyltransferase 2B (KAT2B) to the promoter regions leading to the histone acetylation, consequently promoted SRF to recognize the specific CArG motif, and enhanced the proliferation- and migration-related gene transcriptional activation. CONCLUSIONS: The present study identifies ELK1/SND1/SRF as a novel pathway in promoting the proliferative VSMC phenotype and neointimal hyperplasia in vascular injury, predisposing the vessels to pathological remodeling. This provides a potential therapeutic target for vascular stenosis.

Mutant p53 and ELK1 co-drive FRA-1 expression to induce metastasis in breast cancer.

Tumor-associated p53 mutations induce activities different from wild-type p53, thus causing loss of the protein's tumor inhibition function. The cells carrying p53 mutations have more aggressive characteristics related to invasion, metastasis, proliferation, and cell survival. By comparing the gene expression profiles of mutant p53 (mutp53) and mutp53 silenced cohorts, we found that FOS-related antigen-1 (FRA-1), which is encoded by FOSL1, is a potential effector of mutp53-mediated metastasis. We demonstrate that the expression of FRA-1, a gatekeeper of mesenchymal-epithelial transition, is elevated in the presence of p53 mutations. Mechanistically, mutant p53 cooperates with the transcription factor ELK1 in binding and activating the promoter of FOSL1, thus fostering lung metastasis. This study reveals new insights into how mutant p53 contributes to metastasis in breast cancer.

BMAL1 inhibits renal fibrosis and renal interstitial inflammation by targeting the ERK1/2/ELK-1/Egr-1 axis.

RATIONALE: Renal fibrosis and renal interstitial inflammation due to hydronephrosis are associated with progressive chronic kidney disease (CKD). The clock gene BMAL1 is thought to be involved in various diseases, including hypertension, diabetes, etc. However, little is known about how BMAL1 regulates renal fibrosis and renal interstitial inflammation in obstructed kidneys. METHODS: The expression level of BMAL1 in UUO was examined using the GEO database. Lentivirus, siRNA and adeno-associated virus were used to modulate BMAL1 levels in HK-2 cells and mouse kidney. qRT-PCR, immunofluorescence staining, histological analysis, ELISA and Western blot were used to determine the level of fibrin deposition and the release of inflammatory factors. Immunofluorescence staining and western blotting were used to examine the interaction between BMAL1 and the ERK1/2/ELK-1/Egr-1 axis. RESULTS: Bioinformatics analysis and in vivo experiments in this study showed that the expression level of BMAL1 in UUO model kidneys was higher than that in normal kidneys. We then found that downregulation of BMAL1 promoted the production of extracellular matrix (ECM) proteins and proinflammatory factors in vivo and in vitro, whereas upregulation inhibited this process. In addition, we demonstrated that the ERK1/2/ELK-1/Egr-1 axis is an important pathway for BMAL1 to play a regulatory role, and the use of PD98059 abolished the promoting effect of down-regulation of BMAL1 on fibrosis and inflammation. CONCLUSIONS: Our findings suggest that BAML1 can target the ERK1/2/ELK-1/Egr-1 axis to suppress fibrotic progression and inflammatory events in obstructed kidneys, thereby inhibiting the development of CKD.

Role of ELK1 in regulating colorectal cancer progression: miR-31-5p/CDIP1 axis in CRC pathogenesis.

Background and Objective: Colorectal cancer (CRC) is a malignant tumor that affects the digestive system. With the increased of modernization of society, the incidence of colorectal cancer has increased throughout the world. As a transcription factor, ELK1 has been widely studied in colorectal cancer. However, there are still many unknown factors regarding its specific mechanism of action.This study explored the role of ELK1 and its downstream pathway in CRC pathogenesis. Methods: Based on clinical samples, this study examined miR-31-5p expression in CRC cells and its impact on malignant behaviors (migration, invasion, apoptosis) and autophagy. The promoter sequence of miR-31-5p was obtained from the UCSC database, and ELK1 was identified as its transcription factor. In ELK1-knockdown CRC cells, miR-31-5p was overexpressed, and its response in malignant behaviors and autophagy was analyzed. The target gene CDIP1 was predicted and verified using a dual-luciferase assay. The influence of CDIP1 on malignant behavior in CRC cells was assessed, and CDIP1 siRNA was used as a rescue treatment for miR-31-5p inhibition. The role of ELK1/miR-31-5p in tumor growth was validated in vivo. Results: miR-31-5p expression was upregulated in the colorectal cancer tissues and cells. The knockdown of miR-31-5p markedly inhibited cancer cells' malignant behaviors and mediated autophagy. ELK1 was confirmed to bind with the miR-31-5p promoter and enhance miR-31-5p transcription. miR-31-5p was found to bind with the CDIP1 3'UTR and inhibit CDIP1 expression. CDIP1 siRNA partially rescued the effects of miR-31-5p knockdown on cell metastatic ability, autophagy, and apoptosis. Based on the in vivo experiments, results showed that the ELK1/miR-31-5p axis positively regulated tumor growth in nude mice. Conclusion: Our findings indicate that ELK1 regulates the progression of colorectal cancer via an miR-31-5p/CDIP1 axis, and the ELK1/miR-31-5p/CDIP1 axis could be a therapeutic target for colorectal cancer.

SRF SUMOylation modulates smooth muscle phenotypic switch and vascular remodeling.

Serum response factor (SRF) controls gene transcription in vascular smooth muscle cells (VSMCs) and regulates VSMC phenotypic switch from a contractile to a synthetic state, which plays a key role in the pathogenesis of cardiovascular diseases (CVD). SRF activity is regulated by its associated cofactors. However, it is not known how post-translational SUMOylation regulates the SRF activity in CVD. Here, we show that Senp1 deficiency in VSMCs increased SUMOylated SRF and the SRF-ELK complex, leading to augmented vascular remodeling and neointimal formation in mice. Mechanistically, SENP1 deficiency in VSMCs increased SRF SUMOylation at lysine 143, which reduced its lysosomal localization concomitant with increased nuclear accumulation. SUMOylation of SRF switched its binding with the contractile phenotype-responsive cofactor myocardin to binding with the synthetic phenotype-responsive cofactor phosphorylated ELK1. Both SUMOylated SRF and phosphor-ELK1 were increased in VSMCs from coronary arteries of CVD patients. Importantly, preventing the shift from SRF-myocardin to SRF-ELK complex by AZD6244 inhibited the excessive proliferative, migratory, and synthetic phenotypes, attenuating neointimal formation in Senp1-deficient mice. Therefore, targeting the SRF complex may have a therapeutic potential for the treatment of CVD.

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.

Transcription factor ELK1 regulates the expression of histone 3 lysine 9 to affect developmental potential of porcine preimplantation embryos.

A series of changes occur in the early embryo that are critical for subsequent development, and the pig is an excellent animal model of human disease, so understanding the regulatory mechanisms of early embryonic development in the pig is of very importance. To find key transcription factors regulating pig early embryonic development, we first profiled the transcriptome of pig early embryos, and confirmed that zygotic gene activation (ZGA) in porcine embryos starts from 4 cell stage. Subsequent enrichment analysis of up-regulated gene motifs during ZGA revealed that the transcription factor ELK1 ranked first. The expression pattern of ELK1 in porcine early embryos was analyzed by immunofluorescence staining and qPCR, and the results showed that the transcript level of ELK1 reached the highest at the 8 cell stage, while the protein level reached the highest at 4 cell stage. To further investigate the effect of ELK1 on early embryo development in pigs, we silenced ELK1 in zygotes and showed that ELK1 silencing significantly reduced cleavage rate, blastocyst rate as well as blastocyst quality. A significant decrease in the expression of the pluripotency gene Oct4 was also observed in blastocysts from the ELK1 silenced group by immunofluorescence staining. Silencing of ELK1 also resulted in decreased H3K9Ac modification and increased H3K9me3 modification at 4 cell stage. To investigate the effect of ELK1 on ZGA, we analyzed transcriptome changes in 4 cell embryos after ELK1 silencing by RNA seq, which revealed that ELK1 silencing resulted in significant differences in the expression of a total of 1953 genes at the 4 cell stage compared with their normal counterparts, including 1106 genes that were significantly upregulated and 847 genes that were significantly downregulated. Through GO and KEGG enrichment, we found that the functions and pathways of down-regulated genes were concentrated in protein synthesis, processing, cell cycle regulation, etc., while the functions of up-regulated genes were focused on aerobic respiration process. In conclusion, this study demonstrates that the transcription factor ELK1 plays an important role in regulation of preimplantation embryo development of pigs and deficiency of ELK1 leads to abnormal epigenetic reprogramming as well as zygotic genome activation, thus adversely affecting embryonic development. This study will provide important reference for the regulation of transcription factors in porcine embryo development.

Asiatic acid exhibits antimetastatic activity in human prostate cancer cells by modulating the MZF-1/Elk-1/Snail signaling axis.

Prostate cancer metastasis is associated with poor prognosis and is difficult to treat clinically. Numerous studies have shown that Asiatic Acid (AA) has antibacterial, anti-inflammatory, and antioxidant effects. However, the effect of AA on prostate cancer metastasis is still unclear. This purpose of this study is to investigate the effect of AA on prostate cancer metastasis and to better understand its molecular mechanisms of action. Our results indicate that AA ≤ 30 µM did not influence cell viability and cell cycle distribution in PC3, 22Rv1 and DU145 cells. AA inhibited the migratory and invasive capabilities of three prostate cancer cells to be due to its effects on Snail, but did not have activity on Slug. We observed that AA inhibited the Myeloid zinc finger 1 (MZF-1) and ETS Like-1 (Elk-1) protein interaction and affected the complex's binding capacity to the Snail promoter region, ultimately blocking Snail transcription activity. Kinase cascade analysis revealed that phosphorylation of MEK3/6 and p38MAPK was inhibited by AA treatment. Moreover, knockdown of p38MAPK enhanced AA-suppressed protein levels of MZF-1, Elk-1, and Snail, suggesting that p38MAPK influences prostate cancer cell metastasis. These results provide promise for AA as a future candidate in the development of drug therapies to prevent or treat prostate cancer metastasis.

Hsa_circ_0008500 inhibits apoptosis of adipose-derived stem cells under high glucose through hsa-miR-1273h-5p/ELK1 axis.

Preliminary researches have confirmed that the number of apoptosis of adipose tissue-derived stem cells (ADSCs) in patients with diabetes is significantly increased, leading to a difficult healing wound. Increasing researches revealed that circular RNAs (circRNAs) can control apoptosis. However, it is still unclear whether and how circRNAs are critical for regulating ADSCs apoptosis. In this study, we utilized in vitro model in which ADSCs were cultivated with normal glucose (NG) (5.5 mM) or high glucose (HG) (25 mM) medium, respectively, and found that more apoptotic ADSCs were observed in HG medium comparing to ADSCs in NG medium. Furthermore, we found that hsa_circ_0008500 attenuated HG-mediated ADSCs apoptosis. In addition, Hsa_circ_0008500 could directly interact with hsa-miR-1273h-5p, acting as a miRNA sponge, which subsequently suppressed Ets-like protein-1(ELK1) expression, the downstream target of hsa-miR-1273h-5p. Thus, these results indicated that targeting the hsa_circ_0008500/hsa-miR-1273h-5p/ELK1 signaling pathway in ADSCs may be a potential target for repairing diabetic wounds.

SRF and SRF Cofactor mRNA Expression Is Differentially Regulated by BDNF Stimulation in Cortical Neurons.

Serum response factor (SRF) is a transcription factor that plays essential roles in multiple brain functions in concert with SRF cofactors such as ternary complex factor (TCF) and megakaryoblastic leukemia (MKL)/myocardin-related transcription factor (MRTF), which comprises MKL1/MRTFA and MKL2/MRTFB. Here, we stimulated primary cultured rat cortical neurons with brain-derived neurotrophic factor (BDNF) and investigated the levels of SRF and SRF cofactor mRNA expression. We found that SRF mRNA was transiently induced by BDNF, whereas the levels of SRF cofactors were differentially regulated: mRNA expression of Elk1, a TCF family member, and MKL1/MRTFA were unchanged, while in contrast, mRNA expression of MKL2/MRTFB was transiently decreased. Inhibitor experiments revealed that BDNF-mediated alteration in mRNA levels detected in this study was mainly due to the extracellular signal-regulated protein kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway. Collectively, BDNF mediates the reciprocal regulation of SRF and MKL2/MRTFB at the mRNA expression level through ERK/MAPK, which may fine-tune the transcription of SRF target genes in cortical neurons. Accumulating evidence regarding the alteration of SRF and SRF cofactor levels detected in several neurological disorders suggests that the findings of this study might also provide novel insights into valuable therapeutic strategies for the treatment of brain diseases.

Glucose Homeostasis and Pancreatic Islet Size Are Regulated by the Transcription Factors Elk-1 and Egr-1 and the Protein Phosphatase Calcineurin.

Pancreatic ß-cells synthesize and secrete insulin. A key feature of diabetes mellitus is the loss of these cells. A decrease in the number of ß-cells results in decreased biosynthesis of insulin. Increasing the number of ß-cells should restore adequate insulin biosynthesis leading to adequate insulin secretion. Therefore, identifying proteins that regulate the number of ß-cells is a high priority in diabetes research. In this review article, we summerize the results of three sophisticated transgenic mouse models showing that the transcription factors Elk-1 and Egr-1 and the Ca2+/calmodulin-regulated protein phosphatase calcineurin control the formation of sufficiently large pancreatic islets. Impairment of the biological activity of Egr-1 and Elk-1 in pancreatic ß-cells leads to glucose intolerance and dysregulation of glucose homeostasis, the process that maintains glucose concentration in the blood within a narrow range. Transgenic mice expressing an activated calcineurin mutant also had smaller islets and showed hyperglycemia. Calcineurin induces dephosphorylation of Elk-1 which subsequently impairs Egr-1 biosynthesis and the biological functions of Elk-1 and Egr-1 to regulate islet size and glucose homeostasis.

An allosteric potentiator of metabotropic glutamate (mGlu) 2 receptors reduces the cocaine-stimulated ERK1/2 phosphorylation in the mouse striatum.

Metabotropic glutamate (mGlu) receptors are involved in the experience-dependent neuroplasticity in the mesolimbic reward circuit. A Gαi/o-coupled mGlu2 subtype is distributed presynaptically in the striatum. These autoreceptors may have a significant influence over striatal neurons in their intracellular signaling pathways in response to a psychostimulant. Here we explored the effect of pharmacological potentiation of mGlu2 receptors on cocaine-stimulated phosphorylation (activation) of extracellular signal-regulated kinases (ERK) in the mouse striatum in vivo. We found that an mGlu2 selective positive allosteric modulator (PAM) LY487379 after a systemic injection did not alter basal phosphorylation of ERK1/2 or c-Jun N-terminal kinases in the striatum. However, pretreatment with LY487379 blocked the ERK1/2 phosphorylation induced by cocaine in the two subdivisions of the striatum, i.e., the caudate putamen and nucleus accumbens. LY487379 also blocked the cocaine-induced phosphorylation of Elk-1, a transcription factor downstream to the ERK pathway. Additionally, LY487379 reduced locomotor behavioral responses to cocaine. These results demonstrate that the mGlu2 PAM LY487379 possesses the ability to attenuate the activation of the ERK1/2 pathway in striatal neurons and reduce locomotor activity in response to cocaine in vivo.