Urocortin (Ucn1), a member of the corticotrophin-releasing hormone (CRH) family, has been reported to participate in inflammation. The increased expression of intercellular adhesion molecule 1 (ICAM1) plays important roles in inflammation and immune responses. Our previous results demonstrated that Ucn1 significantly enhanced the expression of ICAM1. However, the underlying mechanisms are still unknown. The purpose of this study is to investigate the detailed mechanisms of Ucn1-induced upregulation of ICAM1. Here, we characterized the mechanisms of Ucn1 usage to regulate ICAM1 expression in human umbilical vein endothelial cells (HUVECs). Our data revealed that Ucn1 increased ICAM1 and cyclooxygenase 2 (COX2) expressions in a time-dependent manner via CRH receptor 2 (CRHR2). In addition, COX2 was involved in ICAM1 upregulation. Furthermore, Ucn1 could increase the expression and phosphorylation of cytosolic phospholipases A2 (cPLA2) in a time-dependent manner via CRHR2 and CRHR1. Moreover, ablation of cPLA2 by the inhibitor pyrrophenone or siRNA attenuated the ICAM1 increase induced by Ucn1. In addition, nuclear factor κB (NF-κB) was activated, indicated by the increase in nuclear p65NF-κB expression and phosphorylation of p65NF-κB, depending on cPLA2 and CRHR2 activation. Pyrrolidinedithiocarbamic acid, an inhibitor of NF-κB, abolished the elevation of ICAM1 but not COX2. Also, Ucn1 increased the production of prostaglandin E₂ (PGE₂) which further activated protein kinase A (PKA)–CREB pathways dependent of cPLA2 via CRHR2. Moreover, the increase in NF-κB phosphorylation was not affected by the selective COX2 inhibitor NS-398 or the PKA inhibitor H89. In conclusion, these data indicate that Ucn1 increase the ICAM1 expression via cPLA2-NF-κB and cPLA2-COX2-PGE₂-PKA-CREB pathways by means of CRHR2.

Peripheral corticotropin-releasing hormone receptors (CRHRs) are G protein-coupled receptors that play different roles depending on tissue types. Previously, we discovered the mechanism of CRHR-mediated apoptosis of mouse prostate cancer cell line (RM-1) to be a change of Bcl-2:Bax ratio, and CRH was found to inhibit transforming growth factor β migration of breast cancer cells via CRHRs. In the present study, we investigated cytosolic calcium-dependent phospholipase A₂ (cPLA₂) bridging CRHR activations and Bcl-2:Bax ratio and the effect of CRHR activation on cell migration. Silencing of cPLA₂ attenuated a CRHR1 agonist, CRH-induced apoptosis, and the decrease of the Bcl-2:Bax ratio, whereas silencing of cPLA₂ aggravated CRHR2 agonist, Urocortin 2 (Ucn2)-inhibited apoptosis, and the increase of the Bcl-2:Bax ratio. CRH in a time- and concentration-dependent manner increased cPLA₂ expression mainly through interleukin 1β (IL1β) upregulation. Ucn2 decreased cPLA₂ expression through neither tumor necrosis factor α nor IL1β. CRH-suppressed decay of cPLA₂ mRNA and Ucn2 merely suppressed its production. Overexpression of CRHR1 or CRHR2 in HEK293 cells correspondingly upregulated or downregulated cPLA₂ expression after CRH or Ucn2 stimulation respectively. In addition, both CRH and Ucn2 induced migration of RM-1 cells. Our observation not only established a relationship between CRHRs and cell migration but also for the first time, to our knowledge, demonstrated that cPLA₂ participates in CRHR1-induced apoptosis and CRHR2-inhibited apoptosis.

Despite all modern advances in medicine, there are few reports of effective and safe drugs to treat obesity. Our objective was to screen anti-obesity natural compounds, and to verify whether they can reduce the body weight gain and investigate their molecular mechanisms. By using drug-screening methods, Phytohemagglutinin (PHA) was found to be the most anti-obesity candidate natural compound. Six-week-old C57BL/6J mice were fed with a high-fat diet (HFD) and intraperitoneally injected with 0.25 mg/kg PHA everyday for 8 weeks. The body weight, glucose homeostasis, oxygen consumption and physical activity were assessed. We also measured the heat intensity, body temperature and the gene expression of key regulators of energy expenditure. Prevention study results showed PHA treatment not only reduced the body weight gain but also maintained glucose homeostasis in HFD-fed mice. Further study indicated energy expenditure and uncoupling protein 1 (UCP-1) expression of brown adipose tissue (BAT) and white adipose tissue (WAT) in HFD-fed mice were significantly improved by PHA. In the therapeutic study, a similar effect was observed. PHA inhibited lipid droplet formation and upregulated mitochondrial-related gene expression during adipogenesis in vitro. UCP-1 KO mice displayed no differences in body weight, glucose homeostasis and core body temperature between PHA and control groups. Our results suggest that PHA prevent and treat obesity by increasing energy expenditure through upregulation of BAT thermogenesis.

Dehydroepiandrosterone (DHEA) may be a promising agent for postmenopausal osteoporosis (PMO), but its mechanism to modulate osteoblasts (OBs) is yet to be explained. To elucidate the effects of DHEA treatment on the ovariectomized (OVX) mice and its mechanisms, we evaluated the morphology of mice bone tissue and expression of proliferating cell nuclear antigen (PCNA) in the vertebrae-derived OB after having treated the OVX animals with DHEA. The results showed that DHEA administration increased the expression of PCNA in OB and changed the bone tissue morphometry of the PMO model. To further investigate this mechanism, the OB was isolated from neonatal mice calvariae by the enzyme-digested assay, exposed to DHEA, and then analyzed for ultrastructure, DNA content, early apoptotic cells, and phosphorylation of extracellular signal-regulated kinase 1/2. It was found that DHEA promoted proliferation and inhibited apoptosis of OB significantly, via mitogen-activated protein kinase signaling pathway independent of either androgen receptor or estrogen receptor, suggesting that it may exert roles via a DHEA-specific receptor directly, not by way of conversion to androgens or estrogens.

This study aimed to identify circular RNAs differentially expressed in the islets of type 2 diabetes (T2DM) models and clarify their roles in the control of β-cell functions. Circular RNAs dysregulated in the islets of diabetic db/db mice were identified by high-throughput RNA sequencing. Then, the expression level of the selected circular RNA circ-Tulp4 was confirmed by real-time PCR in the islets of diabetic models and Min6 cells. MTS, EdU, western blot, flow cytometric analysis, and luciferase assay were performed to investigate the impact of circ-Tulp4 on β-cell functions. This study identified thousands of circular RNAs in mouse pancreatic islets. The circ-Tulp4 level significantly decreased in the diabetic models and altered in the Min6 cells under lipotoxic condition. The modulation of circ-Tulp4 level in Min6 cells regulated cell proliferation. Furthermore, an interaction was demonstrated between circ-Tulp4 and miR-7222-3p, which suppressed the expression of cholesterol esterification-related gene, sterol O-acyltransferase 1 (SOAT1). The accumulation of soat1 activated cyclin D1 expression, thus promoting cell cycle progression. These findings showed that circ-Tulp4 regulated β-cell proliferation via miR-7222-3p/soat1/cyclin D1 signaling. Our research suggested that circ-Tulp4 might be a potential therapeutic intervention for T2DM. Besides, soat1 might be important for β-cell adaptation to lipotoxicity.

RANTES (C–C chemokine, regulated on activation, normal T cell expressed and secreted) is involved in progression of endometriosis, but the precise mechanism is understood inadequately. This study is to elucidate the roles of RANTES in macrophage recruitment and tolerance in the endometriotic milieu. The expression of RANTES was analyzed by immunohistochemistry. The cell co-cultures were applied to simulate the endometriotic milieu to investigate the regulation of RANTES secretion and its receptor CCR1 expression. Transwell migration assay was used for chemotaxis of U937 cells (macrophage line) to endometrial stromal cells (ESCs) and/or human pelvic mesothelial cells. The expression of CCR1 was analyzed by RT-PCR and qPCR in transcription and by western blot in translation respectively. Concentrations of RANTES, IL10, and IL12p70 were determined by ELISA. The phenotype of U937 cells and apoptosis of ESCs were analyzed by flow cytometry. We have found that the expression of RANTES is significantly higher in the endometriotic tissue and eutopic endometrium than that of the normal endometrium without endometriosis. The combination of 17β-estradiol and dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin increases significantly RANTES secretion in the endometriosis-associated cell co-culture which can recruit more macrophages, upregulate CCR1 expression, and induce tolerant phenotype, which inhibits the apoptosis of ESC in the milieu. In conclusion, the higher levels of RANTES in the ectopic milieu facilitate the onset and progression of endometriosis by macrophage recruitment and tolerance that in turn inhibits apoptosis and enhances growth of ESC.

Tetraspanin CD82 is a wide-spectrum tumor metastasis suppressor that inhibits motility and invasiveness of cancer cells. Endometriosis is a benign gynecological disorder, but appears malignant behaviors including invasion, ectopic implantation and recurrence. This study is to elucidate the role of CD82 expression regulation in the pathogenesis of endometriosis. The short interfering RNA silence was established to analyze the roles of CD82, chemokine CCL2, and its receptor CCR2 in the invasiveness of endometrial stromal cells (ESCs). We have found that the mRNA and protein levels of CD82 in the primary normal ESCs from endometrium without endometriosis are significantly higher than that of the primary ESCs from eutopic endometrium and ectopic tissue. CD82 inhibits the invasiveness of ESCs by downregulating CCL2 secretion and CCR2 expression via mitogen-activated protein kinase (MAPK) and integrinβ1 signal pathway, and in turn upregulating the expression of TIMP1 and TIMP2 in an autocrine manner. The combination of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with 17β-estradiol can promote the invasion of ESCs via suppressing CD82 expression and stimulating CCL2 secretion and CCR2 expression, and the enhanced interaction of CCL2–CCR2 recruits more macrophages into the ectopic milieu in a paracrine manner, which further downregulates CD82 expression in the ectopic ESCs. Our study has demonstrated for the first time that the abnormal lower CD82 expression in ESCs induced by TCDD and estrogen may be an important molecular basis of endometriosis pathogenesis through enhancing the CCL2 secretion and CCR2 expression and the invasion of ESCs via MAPK and integrinβ1 signal pathway.

Insect molting and metamorphosis are regulated by two hormones: 20-hydroxyecdysone (20E) and juvenile hormone (JH). The hormone 20E regulates gene transcription via the nuclear receptor EcR to promote metamorphosis, whereas JH regulates gene transcription via its intracellular receptor methoprene-tolerant (Met) to prevent larval–pupal transition. However, the function and mechanism of Met in various insect developments are not well understood. We propose that Met1 plays a key role in maintaining larval status not only by promoting JH-responsive gene transcription but also by repressing 20E-responsive gene transcription in the Lepidopteran insect Helicoverpa armigera. Met1 protein is increased during feeding stage and decreased during molting and metamorphic stages. Met1 is upregulated by JH III and a low concentration of 20E independently, but is downregulated by a high concentration of 20E. Knockdown of Met1 in larvae causes precocious pupation, decrease in JH pathway gene expression, and increase in 20E pathway gene expression. Met1 interacts with heat shock protein 90 and binds to JH response element to regulate Krüppel homolog 1 transcription in JH III induction. Met1 interacts with ultraspiracle protein 1 (USP1) to repress 20E transcription complex EcRB1/USP1 formation and binding to ecdysone response element. These data indicate that JH via Met1 regulates JH pathway gene expression and represses 20E pathway gene expression to maintain the larval status.

Progranulin (PGRN), a multifunctional protein implicated in embryonic development and immune response, was recently introduced as a novel marker of chronic inflammation related with insulin resistance in obesity and type 2 diabetes mellitus. However, the potential mechanisms of PGRN on insulin signaling pathways are poorly understood. In this study, PGRN mediated the chemotaxis of RAW264.7, impaired insulin action and stimulated production of inflammatory factors in adipocytes, which was accompanied by increased c-Jun N-terminal kinase (JNK) activation and serine phosphorylation of insulin receptor substrate-1. PGRN knockdown partially led to an increase in insulin action as well as a decrease in the JNK activation and extracellular signal-regulated kinase phosphorylation in cells exposed to tumor-necrosis factor-α (TNF-α). Meanwhile, PGRN treatment resulted in an elevation of transcription factor nuclear factor κB (NF-κB) nuclear translocation and acetylation, and increased Il-1b, Il6, Tnf-a expression, whereas NF-κB inhibition reversed PGRN-induced insulin action impairment and inflammatory gene expression. Finally, we showed that sirtuin 1 (SIRT1) expression was downregulated by PGRN treatment, whereas SIRT1 overexpression improved PGRN-induced insulin resistance, NF-κB activation, and inflammatory gene expression. Our results suggest that PGRN regulates adipose tissue inflammation possibly by controlling the gain of proinflammatory transcription in a SIRT1-NF-κB dependent manner in response to inducers such as fatty acids and endoplasmic reticulum stress.

The pathogenesis of nonalcoholic steatohepatitis (NASH), a severe stage of nonalcoholic fatty liver disease, is complex and implicates multiple cell interactions. However, therapies for NASH that target multiple cell interactions are still lacking. Melatonin (MEL) alleviates NASH with mechanisms not yet fully understood. Thus, we herein investigate the effects of MEL on key cell types involved in NASH, including hepatocytes, macrophages, and stellate cells. In a mouse NASH model with feeding of a methionine and choline-deficient (MCD) diet, MEL administration suppressed lipid accumulation and peroxidation, improved insulin sensitivity, and attenuated inflammation and fibrogenesis in the liver. Specifically, MEL reduced proinflammatory cytokine expression and inflammatory signal activation and attenuated CD11C⁺CD206^– M1-like macrophage polarization in the liver of NASH mice. The reduction of proinflammatory response by MEL was also observed in the lipopolysaccharide-stimulated Raw264.7 cells. Additionally, MEL increased liver fatty acid β-oxidation, leading to reduced lipid accumulation, and restored the oleate-loaded primary hepatocytes. Finally, MEL attenuated hepatic stellate cell (HSC) activation and fibrogenesis in the liver of MCD-fed mice and in LX-2 human HSCs. In conclusion, MEL acts on multiple cell types in the liver to mitigate NASH-associated phenotypes, supporting MEL or its analog as potential treatment for NASH.