The epidermal growth factor (EGF)-like factors, comprising amphiregulin (AREG), betacellulin (BTC), and epiregulin (EREG), play a critical role in regulating the ovulatory process. Pentraxin 3 (PTX3), an essential ovulatory protein, is necessary for maintaining extracellular matrix (ECM) stability during cumulus expansion. The aim of this study was to investigate the impact of EGF-like factors, AREG, BTC, and EREG on the expression and production of PTX3 in human granulosa-lutein (hGL) cells and the molecular mechanisms involved. Our results demonstrated that AREG, BTC, and EREG could regulate follicular function by upregulating the expression and increasing the production of PTX3 in both primary (obtained from 20 consenting patients undergoing IVF treatment) and immortalized hGL cells. The upregulation of PTX3 expression was primarily facilitated by the activation of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling pathway, induced by these EGF-like factors. In addition, we found that the upregulation of PTX3 expression triggered by the EGF-like factors was completely reversed by either pretreatment with the epidermal growth factor receptor (EGFR) inhibitor, AG1478, or knockdown of EGFR, suggesting that EGFR is crucial for activating the ERK1/2 signaling pathway in hGL cells. Overall, our findings indicate that AREG, BTC, and EREG may modulate human cumulus expansion during the periovulatory stage through the upregulation of PTX3.

Current investigations have illuminated the essential roles played by circular RNAs (circRNAs) in driving breast cancer (BC) tumorigenesis. However, the functional implications and molecular underpinnings of most circRNAs in BC are not well characterized. Here, Circular RNA (circRNA) expression profiles were analyzed in four surgically resected BC cases along with adjacent non-cancerous tissues applying RNA microarray analysis. The levels and prognostic implications of circRREB1 in BC were subjected to quantitative real-time PCR (qRT-PCR) and in situ hybridization (ISH). Experimental manipulation of circRREB1 levels in both in vivo and in vitro settings further delineated its role in BC cell growth, invasion, and metastasis. The mechanical verification of circRREB1\'s interaction with GNB4 was established through RNA pull-down, mass spectrometry, Western blot analysis, RNA immunoprecipitation assays (RIP), fluorescence ISH (FISH), and rescue experiments. We found that circRREB1 exhibited significant upregulation in BC tissues and cells, implicating its association with an unfavorable prognosis in BC patients. CircRREB1 knockdown elicited anti-proliferative, anti-migratory, anti-invasive, and pro-apoptotic effects in BC cells, whereas its upregulation exerted opposing influences. Follow-up mechanistic examinations suggested that circRREB1 might interact with GNB4 directly, inducing the activation of Erk1/2 signaling and driving BC progression. Our findings collectively indicate that the interplay of circRREB1 with GNB4 promotes Erk1/2 signaling, thereby fostering BC progression, and positioning circRREB1 as a candidate therapeutic target for intervention in BC.

Macrophages (MΦ) infected with human immunodeficiency virus (HIV)-1 or activated by its envelope protein gp120 exert neurotoxicity. We found previously that signaling via p38 mitogen-activated protein kinase (p38 MAPK) is essential to the neurotoxicity of HIVgp120-stimulated MΦ. However, the associated downstream pathways remained elusive. Here we show that cysteinyl-leukotrienes (CysLT) released by HIV-infected or HIVgp120 stimulated MΦ downstream of p38 MAPK critically contribute to neurotoxicity. SiRNA-mediated or pharmacological inhibition of p38 MAPK deprives MΦ of CysLT synthase (LTC4S) and, pharmacological inhibition of the cysteinyl-leukotriene receptor 1 (CYSLTR1) protects cerebrocortical neurons against toxicity of both gp120-stimulated and HIV-infected MΦ. Components of the CysLT pathway are differentially regulated in brains of HIV-infected individuals and a transgenic mouse model of NeuroHIV (HIVgp120tg). Moreover, genetic ablation of LTC4S or CysLTR1 prevents neuronal damage and impairment of spatial memory in HIVgp120tg mice. Altogether, our findings suggest a novel critical role for cysteinyl-leukotrienes in HIV-associated brain injury.

Human immunodeficiency virus-1 (HIV-1) infects the central nervous system (CNS) and causes HIV-associated neurocognitive disorders (HAND) in about half of the population living with the virus despite combination anti-retroviral therapy (cART). HIV-1 activates the innate immune system, including the production of type 1 interferons (IFNs) α and β. Transgenic mice expressing HIV-1 envelope glycoprotein gp120 (HIVgp120tg) in the CNS develop memory impairment and share key neuropathological features and differential CNS gene expression with HIV patients, including the induction of IFN-stimulated genes (ISG). Here we show that knocking out IFNβ (IFNβKO) in HIVgp120tg and non-tg control mice impairs recognition and spatial memory, but does not affect anxiety-like behavior, locomotion, or vision. The neuropathology of HIVgp120tg mice is only moderately affected by the KO of IFNβ but in a sex-dependent fashion. Notably, in cerebral cortex of IFNβKO animals presynaptic terminals are reduced in males while neuronal dendrites are reduced in females. The IFNβKO results in the hippocampal CA1 region of both male and female HIVgp120tg mice in an ameliorated loss of neuronal presynaptic terminals but no protection of neuronal dendrites. Only female IFNβ-deficient HIVgp120tg mice display diminished microglial activation in cortex and hippocampus and increased astrocytosis in hippocampus compared to their IFNβ-expressing counterparts. RNA expression for some immune genes and ISGs is also affected in a sex-dependent way. The IFNβKO abrogates or diminishes the induction of MX1, DDX58, IRF7 and IRF9 in HIVgp120tg brains of both sexes. Expression analysis of neurotransmission related genes reveals an influence of IFNβ on multiple components with more pronounced changes in IFNβKO females. In contrast, the effects of IFNβKO on MAPK activities are independent of sex with pronounced reduction of active ERK1/2 but also of active p38 in the HIVgp120tg brain. In summary, our findings show that the absence of IFNβ impairs memory dependent behavior and modulates neuropathology in HIVgp120tg brains, indicating that its absence may facilitate development of HAND. Moreover, our data suggests that endogenous IFNβ plays a vital role in maintaining neuronal homeostasis and memory function.

Nickel, a common environmental hazard, is a risk factor for craniosynostosis. However, the underlying biological mechanism remains unclear. Here, we found that early-life nickel exposure induced craniosynostosis in mice. In vitro, nickel promoted the osteogenic differentiation of human mesenchymal stem cells (hMSCs), and its osteogenic ability in vivo was confirmed by an ectopic osteogenesis model. Further mRNA sequencing showed that ERK1/2 signaling and FGFR2 were aberrantly activated. FGFR2 was identified as a key regulator of ERK1/2 signaling. By promoter methylation prediction and methylation-specific PCR (MSP) assays, we found that nickel induced hypomethylation in the promoter of FGFR2, which increased its binding affinity to the transcription factor Sp1. During pregnancy and postnatal stages, AZD4547 rescued nickel-induced craniosynostosis by inhibiting FGFR2 and ERK1/2. Compared with normal individuals, nickel levels were increased in the serum of individuals with craniosynostosis. Further logistic and RCS analyses showed that nickel was an independent risk factor for craniosynostosis with a nonlinear correlation. Mediated analysis showed that FGFR2 mediated 30.13% of the association between nickel and craniosynostosis risk. Collectively, we demonstrate that early-life nickel exposure triggers the hypomethylation of FGFR2 and its binding to Sp1, thereby promoting the osteogenic differentiation of hMSCs by ERK1/2 signaling, leading to craniosynostosis.

Following the publication of this paper, it was drawn to the Editor\'s attention by a concerned reader that the GAPDH control western blotting data shown in Fig. 2C were strikingly similar to data appearing in different form in other articles written by different authors at different research institutes. Moreover, certain of the western blotting data shown internally within Fig. 4E and F appeared to be strikingly similar, even though the experiments portrayed in these Figure parts were intended to show the results obtained from different cell lines. Owing to the fact that the contentious data in the above article were already under consideration for publication, or had already been published, prior to its submission to Molecular Medicine Reports, and based on an overall lack in the confidence in the data, the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a reply. The Editor apologizes to the readership for any inconvenience caused. [Molecular Medicine Reports 22: 774‑782, 2020; DOI: 10.3892/mmr.2020.11151].

Activated macrophages serve a key role in various inflammatory diseases, such as atherosclerosis and septic shock. Tripartite motif-containing protein 65 (TRIM65) has been previously reported to participate in tumor progression and lung inflammation. However, the molecular mechanisms that controls its expression under inflammatory conditions and its consequences in activated macrophages remain poorly understood. The present study first collected the tissues of C57BL/6J mice, smooth muscle cells, macrophages and endothelial cells to determine the expression and distribution of TRIM65 by reverse transcription-quantitative (RT-q) PCR and western blotting. Mouse and human macrophages were treated with LPS and C57BL/6J mice were intraperitoneally injected with LPS followed by isolation of spleen, lung, aorta and bone marrow. Following treatment, TRIM65 mRNA and protein level was examined by RT-qPCR and western blotting. The results showed that TRIM65 was highly expressed in organs of the immune system, such as the spleen, lymph node and thymus, but lowly expressed in heart, liver, brain and kidneys. TRIM65 was also highly expressed in macrophages and endothelial cells. TRIM65 mRNA and protein expression levels were found to be decreased in LPS-treated macrophages in vitro and in tissues isolated from C57BL/6J mice intraperitoneally injected with LPS in vivo. In addition, to identify the signaling pathways by which LPS regulates TRIM65 expression, inhibitors of MAPK and Akt signaling pathways were used to treat macrophages followed by examination the expression of TRIM65 by western blotting. The results demonstrated that LPS-inhibited TRIM65 expression was blocked by treatment with the ERK1/2 inhibitor U0126. Moreover, the RT-qPCR results showed that TRIM65 knockout potentiated LPS-induced expression of inflammatory cytokines in macrophages. Taken together, data from the present study suggest that LPS decreased TRIM65 expression in macrophages and C57BL/6J mouse by activating the ERK1/2 signaling pathway, whilst TRIM65 knockout promoted macrophage activation. This information may facilitate the development of potential therapeutic strategies for the prevention and treatment of inflammatory diseases, such as atherosclerosis.

The cytochrome P450s (CYP450s) include key oxidative enzymes involved in the metabolism of various carcinogens and anticancer drugs. Bioinformatic studies have demonstrated the association of CYP3A43 with liver cancer and ovarian cancer. However, the biological function of CYP3A43 in tumor progression remains unclear. To further reveal the role of CYP3A43 in tumor progression, we first analyzed the data from the UALCAN database and found that CYP3A43 was negatively correlated to the cancer staging and lymph node metastasis of lung adenocarcinoma (LUAD). We established stable CYP3A43-knockdown LUAD H1299 cell line and found that its knockdown enhanced cell proliferation, colony formation, and migration in vitro, and promoted the growth of tumor xenograft in vivo. Interestingly, when CYP3A43 was ectopically-expressed in the LUAD cell lines, decreased cell proliferation and ERK1/2 phosphorylation level were observed. Lastly, we also identified CYP3A43 co-expressed genes in LUAD from LinkedOmics database followed by GO and KEGG analyses. In conclusion, our results indicate the unprecedented role of CYP3A43 in the suppression of LUAD and provide new possibilities for targeted therapy of this life-threatening disease.

Premature birth is a common obstetric complication but its pathogenesis is unclear. Inflammation at the maternal-fetal interface in preterm labor leads to the infiltration of neutrophils, which promotes inflammatory responses and induces the degradation of extracellular matrix and cell apoptosis, thus contributing to preterm labor. It is unclear whether neutrophil extracellular traps (NETs), a functional form of neutrophils, are involved in preterm labor.
After collecting amniotic membranes from research objects, we localized NETs by immunofluorescence and evaluated the expression of matrix metalloproteinase (MMP)-9 and MMP-2 by western blotting. Primary human amniotic epithelial cells (hAECs) subjected to treatment with NETs, 5-ethynyl-20-deoxyuridine cell proliferation assay, lactate dehydrogenase (LDH) assay, western blotting, and flow cytometry apoptosis assay were used to determine the effects of NETs on hAECs. We also elucidated possible mechanisms underlying the effects.
Compared with normal term women, NETs infiltration and MMP-9 expression in the amniotic membrane from preterm women had increased. Thereafter, NETs might suppress the proliferation and promote the apoptosis of hAECs. Furthermore, after NETs treatment, the mitochondrial membrane potential was significantly decreased, ERK1/2 phosphorylation expression was upregulated and reactive oxygen species (ROS) production was increased in hAECs. Changes in cell proliferation, LDH release, and cell apoptosis level due to NETs could be reversed by ROS inhibitor or ERK phosphorylation inhibitors.
NETs can promote the apoptosis of hAECs via ERK1/2 pathways dependent on ROS release.

Peri-implantitis is characterized by inflammatory cell infiltration and hyperactivation of the osteoclasts surrounding dental implants which can result in bone resorption and ultimately implant failure. Therefore, coordinating the activity of inflammatory response and bone-resorbing osteoclasts is crucial for the prevention of peri-implantitis. Asperuloside (ASP), an iridoid glycoside, has significant anti-inflammatory activities, suggesting the great potential in attenuating peri-implantitis bone resorption. A ligature-induced peri-implantitis model in the maxilla of rats was established, and the effects of ASP on preventing peri-implantitis were evaluated after four weeks of ligation using micro-CT and histological staining. RT-PCR, western blotting, tartrate-resistant acid phosphatase (TRAP), and immunofluorescent staining were conducted on osteoclasts to confirm the mechanisms of ASP on osteoclastogenesis. The results show that ASP could lead to attenuation of alveolar bone resorption in peri-implantitis by inhibiting osteoclast formation and decreasing pro-inflammatory cytokine levels in vivo. Furthermore, ASP could inhibit osteoclastogenesis by downregulating expression levels of transcription factors nuclear factor of activated T-cell (NFATc1) via restraining the activations of nuclear factor kappa beta (NF-κB) and the phosphorylation of extracellular signal-related kinase 1/2 (ERK1/2). In conclusion, ASP could significantly attenuate bone resorption in peri-implantitis via inhibition of osteoclastogenesis by suppressing NF-κB and ERK1/2 signaling pathways activations.