ERβ Protein Expression Research Articles

Hypoxia drives estrogen receptor β-mediated cell growth via transcription activation in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a highly malignant tumor with a poor prognosis. Hypoxia conditions affect multiple cellular processes promoting the adaptation and progression of cancer cells via the activation of hypoxia-inducible factors (HIF) and subsequent transcription activation of their target genes. Preliminary studies have suggested that estrogen receptor β (ERβ) might play a promoting role in the progression of NSCLC. However, the precise mechanisms, particularly its connection to HIF-1α-mediated modulation under hypoxia, remain unclear. Our findings demonstrated that the overexpression of ERβ, not ERα, increased cell proliferation and inhibition of apoptosis in NSCLC cells and xenografts. Tissue microarray staining revealed a strong correlation between the protein expression of HIF-1α and ERβ. HIF-1α induced ERβ gene transcription and protein expression in CoCl2-induced hypoxia, 1% O2 incubation, or HIF-1α overexpressing cells. ChIP identified HIF-1α binding to a hypoxia response element in the ESR2 promoter. The suppression of HIF-1α and ERβ both in vitro and in vivo effectively reduced the tumor growth, thus emphasizing the promising prospects of targeting HIF-1α and ERβ as a therapeutic approach for the treatment of NSCLC. KEY MESSAGES: ERβ, not ERα, increases cell proliferation and inhibition of apoptosis in NSCLC cells and xenografts. A strong correlation exists between the protein expression of HIF-1α and ERβ. HIF-1α induced ERβ gene transcription and protein expression in hypoxic cells via binding to HRE in the ESR2 promoter. The suppression of HIF-1α and ERβ both in vitro and in vivo effectively reduced the NSCLC tumor growth.

Inhibition of neuroinflammation and neuronal damage by the selective non-steroidal ERβ agonist AC-186.

AC-186 (4-[4-4-Difluoro-1-(2-fluorophenyl) cyclohexyl] phenol) is a neuroprotective non-steroidal selective oestrogen receptor modulator. This study investigated whether inhibition of neuroinflammation contributed to neuroprotective activity of this compound. BV-2 microglia were treated with AC-186 (0.65-5 μM) prior to stimulation with LPS(100 ng/mL). Levels of pro-inflammatory mediators and proteins were then evaluated. Treatment of LPS-activated BV-2 microglia with AC-186 resulted in significant (p < 0.05) reduction in TNFα, IL-6, NO, PGE2, iNOS and COX-2. Further investigations showed that AC-186 decreased LPS-induced elevated levels of phospho-p65, phospho-IκBα and acetyl-p65 proteins, while blocking DNA binding and luciferase activity of NF-κB. AC-186 induced significant (p < 0.05) increase in protein expression of ERβ, while enhancing ERE luciferase activity in BV-2 cells. Effects of the compound on oestrogen signalling in the microglia was confirmed in knockdown experiments which revealed a loss of anti-inflammatory activity following transfection with ERβ siRNA. In vitro neuroprotective activity of AC-186 was demonstrated by inhibition of activated microglia-mediated damage to HT-22 neurons. This study established that AC-186 produces NF-κB-mediated anti-inflammatory activity, which is proposed as a contributory mechanism involved in its neuroprotective actions. It is suggested that the anti-inflammatory activity of this compound is linked to its agonist effect on ERβ.

Homoharringtonine promotes non-small-cell lung cancer cell death via modulating HIF-1α/ERβ/E2F1 feedforward loop.

Hypoxia conditions promote the adaptation and progression of non-small-cell lung cancer (NSCLC) via hypoxia-inducible factors (HIF). HIF-1α may regulate estrogen receptor β (ERβ) and promote the progression of NSCLC. The phytochemical homoharringtonine (HHT) exerts strong inhibitory potency on NSCLC, with molecular mechanism under hypoxia being elusive. The effects of HHT on NSCLC growth were determined by cell viability assay, colony formation, flow cytometry, and H460 xenograft models. Western blotting, molecular docking program, site-directed mutagenesis assay, immunohistochemical assay, and immunofluorescence assay were performed to explore the underlying mechanisms of HHT-induced growth inhibition in NSCLC. HIF-1α/ERβ signaling-related E2F1 is highly expressed and contributes to unfavorable survival and tumor growth. The findings in hypoxic cells, HIF-1α overexpressing cells, as well as ERβ- or E2F1-overexpressed and knockdown cells suggest that the HIF-1α/ERβ/E2F1 feedforward loop promotes NSCLC cell growth. HHT suppresses HIF-1α/ERβ/E2F1 signaling via the ubiquitin-proteasome pathway, which is dependent on the inhibition of the protein expression of HIF-1α and ERβ. Molecular docking and site-directed mutagenesis revealed that HHT binds to the GLU305 site of ERβ. HHT inhibits cell proliferation and colony formation and promotes apoptosis in both NSCLC cells and xenograft models. The formation of the HIF-1α/ERβ/E2F1 feedforward loop promotes NSCLC growth and reveals a novel molecular mechanism by which HHT induces cell death in NSCLC.

The effect of Mongolian Medicine Chagan Gaoyou-4 Powder on bone mineral density induced by retinoic acid in rats

Objective: To investigate the effects of Chagan-Gaoyou-4 powder on spine bone mineral density and serum estrogen and estrogen receptor alpha (ERa) and estrogen receptor beta (ERβ) protein expression in bone tissue induced by retinoic acid in rats. Methods: Sixty 3-month-old SPF female SD rats were randomly divided into model group (retinoin group), normal group (SHAM group), Mongolian medicine group (Chagan-Gaoyou 4 group), and control group (Gushukang granule group). After 14 days of administration, spinal Bone Mineral Density (BMD) was measured, and external manifestations were observed. Results: Compared with the SHAM group, the BMD values of all medication groups decreased (P &lt; 0.05, P &lt; 0.001). Compared with the model group, the spinal BMD value of the Chagan Gaoyou-4 group was increased after 21 days of treatment, and the difference was statistically significant (P &lt; 0.01, P &lt; 0.05). After 21 days, the spinal BMD value of all treatment groups was increased (P &lt; 0.05), the spinal BMD value was also increased, especially Chagan Gaoyou-4 group was more obviously increased (P &lt; 0.05). Compared with the normal group, ERa and ERβ levels in the model group were significantly decreased (P &lt; 0.001), and ERa and ERβ levels in all drug groups were significantly increased (P &lt; 0.001) compared with the model group. After 21 days of treatment, there was no significant difference among the three treatment groups (P &gt; 0.05). ERa and ERβ levels in the Chagan Gaoyou-4 group were higher than those in the model group (P &lt; 0.05), but there was no significant difference between the group and the Gushukang granule group (P &gt; 0.05). The E2 content of the Chagan Gaoyou-4 group was higher than that of the model group (P &lt; 0.05). The mRNA expression of estrogen receptor ERa and ERβ in bone tissue of the left proximal femur was detected by RT-PCR. Compared with the model group, the expression of ERa and ERβ mRNA in the Chagan Gaoyou-4 group and Gushukang granule group was increased. Compared with the model group, ERp mRNA expression was increased in the Chagan Gaoyou-4 powder medium-dose group. Compared with the Gushukang granule group, there was no significant difference in ERm RNA expression in the Chagan Gaoyou-4 dose groups. Chagan Gaoyou-4 can up-regulate the expression of ERa and ERβ induced by retinoic acid in rats, indicating that the Chagan Gaoyou-4 powder group may promote bone formation, regulate bone resorption, improve bone mineral density, and achieve the purpose of preventing and treating OP by increasing estrogen level, stimulating estrogen receptor, and increasing the expression of ERa and ERβ in bone tissue. Conclusion: Chagan-Gaoyu-4 powder may have estrogen-like effects on the bone tissue of rats induced by retinoic acid and may increase the level of serum estrogen, ERa, and ERβ protein expression, thereby improving the spinal BMD of experimental rats. The Chagan-Gaoyou-4 powder group could improve the general condition of osteoporosis induced by retinoic acid in rats.

Neuroprotective effects of resveratrol on retinal ganglion cells in glaucoma in rodents: A narrative review.

Glaucoma, an irreversible optic neuropathy, primarily affects retinal ganglion cells (RGC) and causes vision loss and blindness. The damage to RGCs in glaucoma occurs by various mechanisms, including elevated intraocular pressure, oxidative stress, inflammation, and other neurodegenerative processes. As the disease progresses, the loss of RGCs leads to vision loss. Therefore, protecting RGCs from damage and promoting their survival are important goals in managing glaucoma. In this regard, resveratrol (RES), a polyphenolic phytoalexin, exerts antioxidant effects and slows down the evolution and progression of glaucoma. The present review shows that RES plays a protective role in RGCs in cases of ischemic injury and hypoxia as well as in ErbB2 protein expression in the retina. Additionally, RES plays protective roles in RGCs by promoting cell growth, reducing apoptosis, and decreasing oxidative stress in H2O2-exposed RGCs. RES was also found to inhibit oxidative stress damage in RGCs and suppress the activation of mitogen-activated protein kinase signaling pathways. RES could alleviate retinal function impairment by suppressing the hypoxia-inducible factor-1 alpha/vascular endothelial growth factor and p38/p53 axes while stimulating the PI3K/Akt pathway. Therefore, RES might exert potential therapeutic effects for managing glaucoma by protecting RGCs from damage and promoting their survival.

Cardiomyocyte Notch1 signaling is essential for protection of the maternal heart from peripartum stress

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft Background Peripartum cardiomyopathy (PPCM) is a life-threatening disease in women without known cardiovascular disease and is characterized by a sudden onset of heart failure (HF) before, under or after delivery. Previous studies suggest impaired endothelial to cardiomyocyte crosstalk as a major pathomechanism of the disease. Purpose Analyses of the role of Notch1 signaling for the endothelial-cardiomyocyte crosstalk in PPCM. Methods and results Serum from PPCM patients with acute HF reduced Notch1 expression in induced pluripotent stem cell derived cardiomyocytes (-20%, P&amp;lt;0.05) compared to serum from postpartum-matched healthy controls (PP-Ctrl). Beside miRNA-146a previously described to be up-regulated in PPCM plasma (6.8-fold, P&amp;lt;0.01), additional miRNAs targeting Notch1 signaling (miR-30a-3p, miR-34a, miR-200b-3p, miR-200c-3p, miR-429-3p, miR-449a) were up-regulated in PPCM plasma. Overexpression of these miRNAs in cardiomyocytes (CM) reduces Notch1 protein levels. Mice with a CM-restricted knockout of Notch1 (N1-KO) are born at the expected Mendelian ratio and nulli pari (NP) N1-KO female mice do not show a distinct cardiac phenotype at the age of 3 to 4 months compared to WT female mice (%FS WT: 41±5 vs N1-KO: 37±6; n= 9-12). However, they develop mild HF at the age of 6 months (%FS WT: 44±4 vs N1-KO: 31±8; n=11-14; P&amp;lt;0.01). Mated N1-KO females displayed normal cardiac function and LV dimensions in the last week of the first pregnancy. After 3 pregnancies and nursing periods, N1-KO developed severe HF (%FS WT PP: 30±5 vs N1-KO PP: 16±5; n=8-12; P&amp;lt;0.01), and show enhanced mortality (P&amp;lt;0.01). N1-KO-NP and N1-KO-PP displayed significant increase in the cardiac expression of the stress markers ANP and BNP compared to respective WT-NP and WT-PP. RNA-Seq analysis of N1-KO-PP revealed 1380 transcripts (adj. P&amp;lt;0.01) differently regulated genes, among them down-regulation of ErbB4, a known driver in PPCM onset. SiRNA-mediated knockdown of Notch1 in CM were associated with a decrease in ErbB4 protein expression. Treatment with bromocriptin (BR) was not able to improve survival and to rescue cardiac function and LV dimensions in postpartum N1-KO females (WT: 37±4, N1-KO: 23±10; n=7-8). Moreover, RNA-Seq analysis revealed that BR had no effect on the dysregulated cardiac mRNA. In conclusion, the pathophysiology of PPCM is associated with reduced cardiac Notch1 expression, which results in part from 16-kDa-prolactin mediated upregulation of miRNAs targeting Notch1. The reduced cardiac Notch1 expression contributes to PPCM as indicated with severe postpartum HF and high mortality in N1-KO mice. Inhibition of prolactin secretion with BR failed to rescue the development of PPCM in N1-KO mice suggesting that deregulated Notch1 is downstream of the 16-kDa prolactin-miRNA pathophysiology.

Estrogen α and β Receptor Expression in the Various Regions of Resected Glioblastoma Multiforme Tumors and in an In Vitro Model.

Glioblastoma multiforme (GBM) is a malignant tumor with a higher prevalence in men and a higher survival rate in transmenopausal women. It exhibits distinct areas influenced by changing environmental conditions. This study examines how these areas differ in the levels of estrogen receptors (ERs) which play an important role in the development and progression of many cancers, and whose expression levels are often correlated with patient survival. This study utilized two research models: an in vitro model employing the U87 cell line and a second model involving tumors resected from patients (including tumor core, enhancing tumor region, and peritumoral area). ER expression was assessed at both gene and protein levels, with the results validated using confocal microscopy and immunohistochemistry. Under hypoxic conditions, the U87 line displayed a decrease in ERβ mRNA expression and an increase in ERα mRNA expression. In patient samples, ERβ mRNA expression was lower in the tumor core compared to the enhancing tumor region (only in males when the study group was divided by sex). In addition, ERβ protein expression was lower in the tumor core than in the peritumoral area (only in women when the study group was divided by sex). Immunohistochemical analysis indicated the highest ERβ protein expression in the enhancing tumor area, followed by the peritumoral area, and the lowest in the tumor core. The findings suggest that ER expression may significantly influence the development of GBM, exhibiting variability under the influence of conditions present in different tumor areas.

Abstract 6657: Oncolytic Vaccinia Virus Carrying OPCML Tumor Suppressor is active in Epithelial Ovarian Cancer

Abstract Objective: Epithelial Ovarian cancer (EOC) has the highest mortality rate among gynecologic malignancies, necessitating novel therapeutic approaches. Viral oncolytic immunotherapy with recombinant oncolytic vaccinia virus (OVV) offers promise for advanced-stage cancer treatment. OPCML is a GPI-anchored 3 Ig-domain tumor suppressor inactivated by somatic methylation in 87% of EOC. OVV with OPCML (OVV-OPCML) gene insertion may have potential as a therapeutic agent. This study explores the oncolytic, signaling-inhibitory, anti-angiogenic and synergistic immunotherapeutic properties of OVV-OPCML in EOC treatment, evaluating its therapeutic potential. Methods: To create OVV-OPCML, the virulent vaccinia virus Copenhagen strain F3L site was modified with OPCML cDNA, confirmed by qPCR and western blot. Oncolytic potential was assessed through in vitro and in vivo EOC cell infection with OVV-OPCML. Real-time cell analysis (RTCA) assessed cell proliferation. Xenograft tumor models in nude mice were used to evaluate in vivo oncolytic activity, while OVV-OPCML combined with anti-PD-1 treatment was tested using a syngeneic orthotopic EOC mouse model (ID8) in C57BL/6 immunocompetent mice. Serum IFN-γ and TNF-α were quantified via ELISA. Receptor Tyrosine Kinases, angiogenesis and phosphorylation together with immune markers were examined by western blotting and by multiplex immunohistochemistry (mIHC) analysis of tumor sections. Results: OVV-OPCML selectively infected OC cells, leading to viral replication and tumor cell death, while sparing normal ovarian cells in-vitro. OPCML protein expression increased post-infection. OVV-OPCML reduced ERBB2, EGFR, and FGFR1 expression in SKVO3 OV cells. Intratumoral or intraperitoneal (IP) OVV-OPCML administration in human ovarian cancer nude mouse models reduced tumor growth and improved survival. IP in-vivo OVV-OPCML in combination with anti-PD-1 treatment in vivo, in the IP ID8 syngeneic model demonstrated synergy with survival improvement. H&amp;E staining showed tumor cell damage without significant harm to normal tissues. Protein expression of ERBB2, EGFR, FGFR1, and their phosphorylated forms decreased. CD31, VEGFR2, p-VEGFR2 (Tyr1175), and p-VEGFR2 (Tyr1214) downregulation suggested tumor angiogenesis disruption. OVV-OPCML treatment elevated serum TNF-α and INF-γ levels and increased CD8+ T cells and PD-L1 expression in OVV-OPCML treated syngeneic tumors. Conclusions: OPCML gene enhances OVV selectivity against OC cells through direct oncolysis and OPCML protein production, inhibiting specific RTKs, suppressing angiogenesis, and activating antitumor immune responses synergistically with anti-PD-1. OVV-OPCML therapy shows promise as a targeted strategy for ovarian cancer treatment. Citation Format: Yong Tang, Yingzhao Liu, Mingjing Deng, Fei Huang, Jiahu Wang, Paul Blake, Hani Gabra, Qi Wang. Oncolytic Vaccinia Virus Carrying OPCML Tumor Suppressor is active in Epithelial Ovarian Cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6657.

Abstract 4640: Pan-cancer analysis of the influence of ERBB2 alteration on HER2 expression

Abstract Introduction: HER2 (ERBB2) is a target for various anti-cancer therapies, including large (Trastuzumab deruxtecan) or small molecules (Neratinib). Quantitative assessment of HER2 expression at the cellular level may improve therapeutic efficacy prediction. While ERBB2 mutations are known oncogenic drivers, their impact on tumor cell HER2 expression is unclear. In this study, we focused on common ERBB2 mutations, and analyzed HER2 expression via AI-powered image analysis in pan-cancer tumor specimens to identify possible mutation-expression correlations. Methods: We analyzed 183,292 pan-cancer samples from AACR GENIE, and 10,967 from TCGA. A set of 156 cases with both ERBB2 mutation status and HER2 4B5 IHC WSIs was obtained (Neogenomics). An AI-powered HER2 analyzer (Lunit SCOPE HER2) was previously developed using 1,133 HER2 IHC-stained WSIs of breast cancer stained with 4B5. Single-cell HER2 intensity was analyzed and aggregated, reported based on breast ASCO/CAP criteria. ERBB2 RNA levels were analyzed for mutations and amplification. Results: We identified 2,735 cases with ERBB2 mutations (2,603/183,292 [1.4%] in GENIE and 132/10,967 [1.2%] in TCGA). These mutations were categorized for analysis based on their highest frequency and previous characterization: exon 20 insertions (ex20ins, including Y772dup (YVMA) and G776InsVC), S310x, and other pathogenic mutations. In the dataset, ex20ins cases were predominantly non-small cell lung cancer (NSCLC, 381/482 [79.0%]) and S310x cases were enriched in urothelial, NSCLC, and breast cancers (269/683 [39.4%], 64/683 [9.4%], and 47/683 [6.9%], respectively). Of the 156 ERBB2-mutated cases with HER2 IHC images, 59 had ex20ins, 24 had S310x, and 73 had other mutations. AI analysis in each case type indicates a significantly higher proportion of HER2 3+ tumor cells in S310x and ex20ins cases than other mutations (median, 7.5% vs. 6.1% vs. 2.8%, p = 0.003). As such, high HER2 expression was observed in S310x and ex20ins cases compared to other mutation cases (HER2 3+ 10/24 [41.7%], 22/59 [37.3%], and 14/73 [19.2%]). Additionally, in NSCLC, ex20ins and S310x cases exhibited a modest enrichment for high HER2 expression relative to other mutation cases (21/56 [37.5%], 3/9 [33.3%], and 5/20 [27.6%]). RNA analyses from TCGA showed higher levels for ex20ins (median 13.6; interquartile range 13.1-13.7 in log2 scale), compared to S310x (12.4; 11.9-13.6) and other mutations (12.6; 12.1-13.3), but lower than that of amplified ERBB2 (15.5; 13.8-17.0). Conclusion: An AI-powered pan-cancer image analysis of HER2 IHC of tumor cells in conjunction with genomic data reveals a positive correlation between ex20ins and S310x ERBB2 mutation and protein expression. This correlation is also seen at the RNA level, but the lesser levels relative to ERBB2 amplified cases suggests the effect may be mediated at the protein level. Citation Format: Taebum Lee, Sangwon Shin, Heon Song, Seonwook Park, Sergio Pereira, Donggeun Yoo, Juneyoung Ro, Jimin Moon, Soo Ick Cho, Changho Ahn, Chan-Young Ock, Siraj M. Ali. Pan-cancer analysis of the influence of ERBB2 alteration on HER2 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4640.

BRD4-targeting PROTACs Synergize With Chemotherapeutics Against Osteosarcoma Cell Lines.

Osteosarcoma at an advanced stage has a poor outcome, and novel targeted therapies are needed, especially for metastatic disease. Bromodomain inhibitors (BETi) are epigenetic modulators that broadly impair the expression of oncogenic proteins and exert antitumor effects. BETi can be combined with chemotherapeutics to increase therapeutic responses with superior effects in the form of proteolysis targeting chimeras (PROTACs) that degrade proteins of interest (POI) in multiple cycles. This work aimed to investigate the efficacy of BETi, such as JQ1, dBET57, and MZ1 PROTACs in combination with cytotoxic drugs against osteosarcoma cell lines. Chemosensitivity of the osteosarcoma cell lines HOS, Saos-2, MG-63, and G292 were tested with BET-directed agents alone or in combination with cytotoxic drugs comprising cisplatin, doxorubicin, topotecan, and gemcitabine using cell viability assays. The BET degraders exhibited highest toxicity to HOS cells and showed synergistic activity in combination with the chemotherapeutics, except for the degrader - topotecan/gemcitabine combinations. Highest synergy between BET agents and chemotherapeutics were found for the more chemoresistant Saos-2 cells and potentiation of toxicity in MG-63 cells for the BET agents - doxorubicin combinations and the MZ1-topotecan pair. HOS and Saos-2 cell lines had reduced protein expression of AXL, BCL-X, e-cadherin, CAIX, EpCAM, ErbB2, and vimentin in response to JQ1, MZ1, and BET57. The study suggests that the application of novel BET PROTACs in combination with chemotherapeutics could represent a new therapeutic option to improve the therapy of osteosarcomas. First orally available PROTACs have reached clinical trials.

Effects and mechanisms of frehmaglutin D and rehmaionoside C improve LPS-induced acute kidney injury through the estrogen receptor-mediated TLR4 pathway in vivo and in vitro

BackgroundSepsis-induced acute kidney injury (S-AKI) is an inflammatory disease with sex differences and there has no effective drugs to cure it. Frehmaglutin D (Fre D) and rehmaionoside C (Reh C) are two violetone compounds with estrogenic activity isolated from Rehmannia glutinosa. However, whether these two drugs exert protective effects on S-AKI through their estrogen-like activity are unclear. PurposeThis study aimed to explore the effects and mechanisms of Fre D and Reh C on lipopolysaccharide (LPS)-induced S-AKI through the estrogen receptor pathway in vivo and in vitro and to explore the interaction between ER and TLR4 for the first time. MethodsThe LPS-induced female BALB/c mice S-AKI mouse model was established by adding the estrogen receptor antagonist ICI182,780. Renal function, inflammation, oxidative stress, apoptosis, immune cells, and expression of key proteins of the ER-TLR4-IL-1β pathway were tested. The affinity of Fre D and Reh C for the ER was investigated by molecular docking. Then, an in vitro S-AKI model was established, and ERα/ERβ antagonists (MPP/PHTPP) were added and combined with gene overexpression techniques. The interaction between ER and TLR4 was further explored by Co-IP, GST pull-down and SPR techniques. ResultsFre D and Reh C ameliorated LPS-induced renal damage, inflammation in mice, regulated the immune cells, decreased ROS levels, increased ERα and ERβ protein expression, and decreased TLR4, caspase 11 and IL-1β protein expression. These effects were blocked by ICI182,780. Molecular docking results showed that Fre D and Reh C bound ERα and ERβ with similar potency. The results of in vitro suggested that Fre D and Reh C reduced the levels of inflammation, ROS and apoptosis, TLR4, caspase 11, and IL-1β protein expression and increased ERα/ERβ protein expression in cells. All of these effects were reversed by the addition of MPP/PHTPP and further enhanced after ERα/ERβ gene overexpression with no significant difference in effects. Moreover, there was an indirect or direct interaction between ER and TLR4, and the binding of ERα and ERβ to TLR4 was concentration dependent. ConclusionFre D and Reh C may improve S-AKI through the ER-TLR4-IL-1β pathway and may act on both ERα and ERβ receptors. Moreover, ERα and ERβ may interact directly or indirectly with TLR4, which was studied for the first time.

The ERβ-cAMP signaling pathway regulates estradiol-induced ovine oocyte meiotic arrest

Although 17β-estradiol (E2) and its receptors (ERs) are reported to play important roles in regulating oocyte maturation, the specific mechanism remains unclear. First, we performed immunohistochemistry analyses to determine the expression of the ERα and ERβ proteins in ovine ovarian tissue. Second, E2 (0.5 ng/mL and 1 μg/mL) were added to pre-IVM medium for 0 h, 1 h and 2 h. The effects of E2 (0.5 ng/mL and 1 μg/mL) on cyclic adenosine monophosphate (cAMP) level in cumulus-oocyte complexes (COCs) and on oocyte meiotic progression were evaluated by ELISA and DAPI staining respectively. Third, the effects of E2 on the gene and protein expression of ERα and ERβ in COCs were investigated by Western blotting and real-time PCR. Afterward, ERβ and cAMP regulators were added to the 2-h pretreatment medium with or without E2 (0.5 ng/mL) to explore the possible interactions among E2, cAMP and ERβ. The results showed that both ERα and ERβ proteins were expressed in ovine cumulus layers and oocytes. E2 significantly increased intra-COC cAMP levels, maintained oocyte meiotic arrest, and promoted ERβ transcript and protein expression. E2 treatment increased the cAMP concentration, which was enhanced by ERβ agonist treatment and remarkably attenuated by ERβ inhibitor treatment. Forskolin plus IBMX treatment increased ERβ protein expression in COCs (P < 0.05), and this was attenuated by Rp-cAMP treatment. In conclusion, E2 (0.5 ng/mL) increased intra-COC cAMP levels by promoting ERβ expression, thereby maintaining oocyte meiotic arrest. cAMP in COCs has a positive feedback effect on ERβ expression, which provides a novel explanation for the positive role of E2 in regulating ovine follicle development and oocyte maturation.

ErbB4 promotes M2 activation of macrophages in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is the most common and fatal diffuse fibrotic lung disease accompanied by macrophage M2 activation. ErbB4 is involved in and affects the process of inflammation. In this study, we determined that the mRNA level and protein expression of ErbB4 and M2 cytokine members were increased in the serum of IPF patients. In mouse alveolar macrophage MH-S cells, after knocking down ErbB4 by siRNA, the mRNA level and protein expression of M2 activator induced by interleukin (IL)-4 were decreased compared with the control group. Activating by ErbB4 agonist neuromodulatory protein (NRG)-1, IL-4-induced M2 program was promoted. Mechanistically, treated with NRG-1 in MH-S cells, the phosphorylation level of Akt did not change, while the phosphorylation level of ERK increased. Using SCH772984 to inhibit ERK pathway, the increasing IL-4-induced M2 activation by NRG-1 was inhibited, and the high level of M2 activator protein expression and mRNA expression was restored. Collectively, our data support that ErbB4 and M2 programs are implicated in IPF, and ErbB4 participates in the regulation of M2 activation induced by IL-4 through the ERK pathway.

The Mechanism of Bushen Zhuyun Prescription in Regulating Luteal Phase Deficiency via Intervention of the Kiss1/GPR54 System in the Hypothalamus

Luteal phase deficiency (LPD) is a significant contributor to infertility and miscarriage. Bushen Zhuyun Prescription (BSZY) is a traditional Chinese medicine formula that has potential to treat LPD. To investigate the effect of BSZY in treating LPD, SD rats with complete estrous cycles were divided into blank, model, dydrogesterone (DYD), high-dose BSZY (BSZY-HD), and low-dose BSZY (BSZY-LD) groups. All the groups were received mifepristone gavage except for the blank group. The pathological changes were observed in the hypothalamus, and the analysis of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), progesterone (P), gonadotropin-releasing hormone (GnRH), and cyclic adenosine monophosphate (cAMP) levels in serum. Additionally, the mRNA and their protein expression levels of estrogen receptor α (ERα), ERβ, G protein-coupled receptor 30 (GPR30), kisspeptin 1 (Kiss1), and GPR54 were assessed; and the key molecules expression in cAMP/protein kinase A (PKA) pathway were analyzed. The results revealed that mifepristone treatment caused a decrease in FSH and cAMP levels and an increase in E2 and LH levels, which were normalised in the BSZY treatment groups. The expression of ERα and ERβ mRNA and protein in the hypothalamus was higher in the BSZY groups compared to the blank group. BSZY also resulted in increased expression of Kiss-1, GPR54, and PKA, as well as decreased expression of cAMP-response element binding protein (CREB), c-Fos, phospho-CREB (p-CREB), and p-c-Fos, compared to model group. No significant difference observed in GPR30 expression between the mifepristone and BSZY groups. The mRNA and protein of Kiss1 and GPR54 were decreased in model group and increased after BSZY treatment, while the mRNA and protein expression of PKA, CREB, c-Fos, p-CREB, and p-c-Fos were also decreased significantly. In conclusion, the findings demonstrate that BSZY could effectively regulate hypothalamic ER, interfere with the Kiss1/GPR54 signal transduction system, activate the cAMP/PKA pathway, and regulate key transduction molecules expression of PKA, CREB, c-Fos, there by effecting the level of reproductive hormones and has the potential to treat LPD-related infertility.

Jiawei Shoutai Pill promotes decidualization by regulating the SGK1/ENaC pathway in recurrent spontaneous abortion

Ethnopharmacological relevanceJiawei Shoutai Pill (JWSTW) is a traditional herbal formula for recurrent spontaneous abortion (RSA). Although JWSTW significantly improves the clinical symptoms of RSA patients, its molecular mechanism remains unclear. Aim of studyThis study evaluated the expression and function of the serum/glucocorticoid regulated kinase 1/epithelial sodium channel (SGK1/ENaC) pathway and decidualization level in RSA patients and mice. It also investigated the therapeutic effects and potential mechanisms of JWSTW. Materials and methods30 early RSA patients and 30 normal pregnant women undergoing induced abortion during the same period were included in the study. Decidual tissues were collected, and HE staining, immunohistochemistry, Western blot, and RT-PCR were used to detect protein and mRNA expression levels of SGK1, ENaC-a, estrogen Rreceptor β (ERβ), and progesterone receptor (PR) in patients’ decidual tissues. Protein expression levels of prolactin receptor (PRLR) and insulin-like growth factor binding protein 1 (IGFBP-1) were also detected. A classical RSA mouse model was constructed, and the mice were randomly divided into four groups: normal, model, dydrogesterone (DQYT) (0.33 g/kg/d), and JWSTW (1.66 g/kg/d). The normal and model groups received the same volume of distilled water by gavage for 8 and 14 days after pregnancy. On the 14th day of pregnancy, the embryonic loss rate of each group, the number of offspring born to naturally delivered mice, and the protein or mRNA expression levels of key factors of the SGK1/ENaC pathway (SGK1, ENaC-a, ERβ, and PR), decidual proliferation marker (Ki67), mesenchymal-epithelial transition (E-cadherin and Vimentin), and decidualization markers (PRLR and IGFBP-1) in mouse decidual tissue on the eighth day of pregnancy were observed. ResultsThe decidual tissue structure of RSA patients was abnormal. Immunohistochemical analysis revealed significantly reduced positive expression of SGK1, ENaC-a, ERβ, and PR proteins in the decidual tissue of RSA patients (P < 0.001). Western blot and RT-PCR analyses demonstrated significantly decreased protein and mRNA expression of SGK1, ENaC-a, ERβ, and PR in the decidual tissue of RSA patients (all P < 0.05). Additionally, protein expression of PRLR and IGFBP-1 was significantly reduced (both P < 0.001). The RSA mouse model exhibited a significant increase in embryo loss rate and decreased litter size (both P < 0.001). Treatment with DQYT and JWSTW rescued the embryo loss rate and litter size to varying extents (all P < 0.05). The protein or mRNA expression levels of SGK1, ENaC-a, ERβ, PR, Ki67, E-cadherin, vimentin, PRLR, and IGFBP-1 in RSA mice were improved to different degrees after treatment with DQYT and JWSTW (all P < 0.05). ConclusionsAbnormal SGK1/ENaC signaling pathway regulation is closely associated with early endometrial damage in RSA patients. JWSTW promotes endometrial proliferation and mesenchymal-epithelial transition through the SGK1/ENaC signaling pathway, improving endometrial shedding. Consequently, JWSTW is a potential treatment for RSA.

PTPN12 down-regulated by miR-146b-3p gene affects the malignant progression of laryngeal squamous cell carcinoma.

Laryngeal squamous cell carcinoma (LSCC) is a common malignancy among men in the anatomical position of head and neck. Hoarseness, pharyngalgia, and dyspnea are common symptoms. LSCC is a complex polygenic carcinoma that is caused by many factors involving polygenic alteration, environmental pollution, tobacco, and human papillomavirus. Classical protein tyrosine phosphatase nonreceptor type 12 (PTPN12) has been extensively studied to decipher its mechanism as a tumor suppressor gene in various human carcinomas; however, there is no comprehensive elucidation of the PTPN12 expression and its regulatory mechanisms in LSCC. As such, we expect to provide new insights for finding new biomarkers and effective therapeutic targets in LSCC. Immunohistochemical staining, western blot (WB), and quantitative real-time RT-PCR (qRT-PCR) were used for the messenger RNA (mRNA) and protein expression analyses of PTPN12, respectively. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, clone formation, transwell migration, and transwell invasion assays were used to assess the proliferation, migration, and invasion ability of LSCC cells. Online prediction and design software tools (http://www.targetscan.org/ and http://www.microRNA.org) were used to predict associated miRNA. Studying the targeted regulatory relationship between miR-146b-3p and PTPN12 was based on dual luciferase reporter gene analysis. qRT-PCR was used to assess miR-146b-3p expression in LSCC. miR-146b-3p inhibitor and mimic were transfected, followed by qRT-PCR and WB assays to detect the expression of PTPN12. The gain and loss functional experiments were used to investigate the effects of miR-146b-3p transfection on the proliferation, migration, and invasion of tumor cells. Online bioinformatics prediction software (https://cn.string-db.org/ and https://www.genecards.org/) was used to determine potential downstream target genes of PTPN12. qRT-PCR and WB analyses were used to assess the mRNA and protein expression levels of target genes. Our study showed significantly decreased mRNA and protein expression levels of PTPN12 in LSCC compared with the adjacent normal tissues. The lower PTPN12 mRNA expression was correlated with pathological differentiation, and lower PTPN12 protein expression was correlated with the TNM stage in LSCC tissues. The subsequent in vitro functional analyses showed the inhibitory effect of PTPN12 over-expression on the proliferation, migration, and invasiveness abilities of LSCC cell line. Using online prediction and design software, miR-146b-3p was searched to target PTPN12. The miR-146b-3p was expressed at a high level in LSCC tissues and cell lines. Luciferase reporter assay exhibited that miR-146b-3p inhibited the luciferase activity of PTPN12 markedly. The functional analyses showed the tumor-promoting role of miR-146b-3p on the proliferation, migration, and invasiveness abilities of LSCC cell. Furthermore, co-transfection of cells with miR-146b-3p and PTPN12 significantly restored the inhibitory effect of PTPN12 on LSCC cell growth, migration, and invasiveness. This phenomenon unveiled that miR-146b-3p regulated the proliferation, migration, and invasion of LSCC cells by targeting PTPN12. EGFR and ERBB2 were selected as the downstream-regulation target genes. Up-regulation of PTPN12 significantly suppressed EGFR expression. Accordingly, the miR-146b-3p mimic significantly up-regulated the EGFR expression. However, up-regulation of PTPN12 and miR-146b-3p mimic suppressed ERBB2 protein expression but induced its gene expression. Down-regulation of PTPN12 is associated with up-regulation of miR-146b-3p in LSCC. Moreover, PTPN12 serves as a tumor suppressor gene through regulating the proliferation, migration, and invasion of LSCC cells. miR-146b-3p/PTPN12 axis is expected to be a novel therapeutic target in LSCC.

Therapeutic Role of Tamoxifen for Triple-Negative Breast Cancer: Leveraging the Interaction Between ERβ and Mutant p53.

The absence of effective therapeutic targets and aggressive nature of triple-negative breast cancer (TNBC) renders this disease subset difficult to treat. Although estrogen receptor beta (ERβ) is expressed in TNBC, studies on its functional role have yielded inconsistent results. However, recently, our preclinical studies, along with other observations, have shown the potential therapeutic utility of ERβ in the context of mutant p53 expression. The current case study examines the efficacy of the selective estrogen receptor modulator tamoxifen in p53-mutant TNBC with brain metastases. Significant increase in ERβ protein expression and anti-proliferative interaction between mutant p53 and ERβ were observed after cessation of tamoxifen therapy, with significant regression of brain metastases. This case study provides supporting evidence for the use of tamoxifen in p53-mutant, ERβ+TNBC, especially in the setting of brain metastasis.

Metformin alleviates ethanol-induced cardiomyocyte injury by activating AKT/Nrf2 signaling in an ErbB2-dependent manner.

Metformin, a first-line oral anti-diabetic drug, has recently been reported to exert protective effect on various cardiovascular diseases. However, the potential role of metformin in ethanol-induced cardiomyocyte injury is still unknown. Therefore, this study was aimed to investigate the effect of metformin on ethanol-induced cardiomyocyte injury and its underlying mechanism. H9c2 cardiomyocytes were exposed to ethanol for 24h to establish an ethanol-induced cardiomyocyte injury model, and followed by treatment with metformin in the presence or absence of Lapatinib (an ErbB2 inhibition). CCK8 and LDH assays demonstrated that metformin improved cell viability in cardiomyocytes exposed to ethanol. Furthermore, metformin suppressed cardiomyocyte apoptosis and reduced the expressions of apoptosis-related proteins (Bax and C-CAS-3). In addition, our results showed that metformin activated the AKT/Nrf2 pathway, and then promoted Nrf2 nuclear translocation and the transcription of its downstream antioxidant genes (HO-1, CAT and SOD2), thereby inhibiting oxidative stress. Interestingly, we found that ErbB2 protein expression was significantly inhibited in ethanol-treated cardiomyocytes, which was markedly reversed by metformin. In contrast, Lapatinib largely abrogated the activation of AKT/Nrf2 signaling by metformin, accompanied by the increases in oxidative stress and cardiomyocyte apoptosis, indicating that metformin prevented ethanol-induced cardiomyocyte injury in an ErbB2-dependent manner. In summary, our study provides the first evidence that metformin protects cardiomyocyte against ethanol-induced oxidative stress and apoptosis by activating ErbB2-mediated AKT/Nrf2 signaling. Thus, metformin may be a potential novel treatment approach for alcoholic cardiomyopathy.

Zearalenone Promotes Uterine Development of Weaned Gilts by Interfering with Serum Hormones and Up-Regulating Expression of Estrogen and Progesterone Receptors.

In this study, we aimed to assess the effect of diet ZEA on serum hormones, the location and expression of estrogen receptor ERα/β and progesterone receptor (PR) of the uterus in weaned piglets and to reveal the mechanism underneath. A total of 40 healthy weaned gilts were randomly allocated to basal diet supplemented with 0 (Control), 0.5 (ZEA0.5), 1.0 (ZEA1.0) and 1.5 (ZEA1.5) mg ZEA/kg and fed individually for 35 days. Meanwhile, the porcine endometrial epithelial cells (PECs) were incubated for 24 h with ZEA at 0 (Control), 5 (ZEA5), 20 (ZEA20) and 80 (ZEA80) μmol/L, respectively. The results showed that nutrient apparent digestibility (CP and GE), nutrient apparent availability (ME/GE, BV and NPU), the uterine immunoreactive integrated optic density (IOD), relative mRNA and protein expression of ER-α, ER-β and PR and the relative mRNA and protein expression of ER-α and ER-β in PECs all increased linearly (p < 0.05) with ZEA. Collectively, ZEA can interfere with the secretion of some reproductive hormones in the serum and promote the expression of estrogen/progesterone receptors in the uterus and PECs. All these indicate that ZEA may promote the development of the uterus in weaned gilts through estrogen receptor pathway.

Antitumor and radiosensitizing effects of Anagallis arvensis hydromethanolic extract on breast cancer cells through upregulating FOXO3, Let-7, and mir-421 Expression

This study aimed to observe the anticancer, as well as radiosensitizing, effect of Anagallis arvensis extract (AAE) on the growth of breast cancer cells (BCCs). This effect was evaluated through measured cell cycle arrest plus apoptosis through flowcytometry analyses associated with a protein level of estrogen receptors (ER-α and ER-β), Fork head box O3a (FOXO3), and phosphorylated histone H2AX (γ-H2AX) by western blotting analysis. Further, the gene expression of mir-18, mir-101, mir-421, and let-7 was detected via a real-time polymerase chain reaction (RTPCR). AAE and/or γ-radiation (4Gy) inhibited the mediator-induced growth activation and enhanced radiosensitivity in the BCC lines (MCF-7 and MDA-MB-231). AAE reduced both cell cycle progression and cell growth via induced apoptosis. Additionally, the protein expression of ERα and ERβ was decreased, while the protein expression of FOXO3 and γ-H2AX was increased. Moreover, the gene expression of mir-18 and mir-101 was decreased, while the gene expression of let-7 and mir-421 was stimulated in BCCs. AAE has an anticancer with a radiosensitizing effect and is recommended for BC treatment.