Xueling Cui

A critical role of activin A in maturation of mouse peritoneal macrophages in vitro and in vivo.

Activin A, a multifunctional factor of the transforming growth factor-beta (TGF-β) superfamily, is mainly produced by microglia and macrophages, and its anti-inflammatory and pro-inflammatory activities are both related to macrophage functions. However the direct effect of activin A on the rest macrophages in vivo remains unclear. In the present study, the results showed that activin A not only increased NO and IL-1β release, but also promoted phagocytic abilities of mouse peritoneal macrophages in vitro and in vivo, whereas it did not influence MHC I and MHC II expression. Moreover, we found that activin A significantly upregulated the expressions of CD14 and CD68, markers of mature macrophages, on the surface of macrophages in vitro and in vivo. These data suggest that activin A can induce primary macrophage maturation in vitro and in vivo, but may not trigger the acquired immune response via regulating expression of MHC molecules involved in presentation of antigen.

The role and mechanism of action of activin A in neurite outgrowth of chicken embryonic dorsal root ganglia

Activin A, a member of the transforming growth factor β (TGFβ) superfamily, plays an essential role in neuron survival as a neurotrophic and neuroprotective factor in the central nervous system. However, the effects and mechanisms of action of activin A on the neurite outgrowth of dorsal root ganglia (DRG) remain unclear. In the present study, we found that activin A is expressed in DRG collected from chicken embryos on embryonic day 8 (E8). Moreover, activin A induced neurite outgrowth of the primary cultured DRG and maintained the survival of monolayer-cultured DRG neurons throughout the observation period of ten days. Follistatin (FS), an activin-binding protein, significantly inhibited activin A-induced neurite outgrowth of DRG, but failed to influence the effect of nerve growth factor (NGF) on DRG neurite outgrowth. Furthermore, the results showed that activin A significantly upregulated mRNA expression of activin receptor type IIA (ActRIIA) and calcitonin gene-related peptide (CGRP) in DRG, and stimulated serotonin (5-HT) production from DRG, indicating that activin A might induce DRG neurite outgrowth by promoting CGRP expression and stimulating 5-HT release. These data suggest that activin A plays an important role in the development of DRG in an autocrine or paracrine manner.

Direct Effects of Activin A on the Activation of Mouse Macrophage RAW264.7 Cells

Macrophages play critical roles in innate immune and acquired immune via secreting pro-inflammatory mediators, phagocytosing microorganisms and presenting antigens. Activin A, a member of transforming growth factor β (TGF-β) superfamily, is produced by macrophages and microglia cells. In this study, we reported a direct effect of activin A as a pro-inflammatory factor on mouse macrophage cell line RAW264.7 cells. Our data revealed that activin A could not only increase IL-1β and IL-6 production from RAW264.7 cells, but also promote pinocytic and phagocytic activities of RAW264.7 cells. In addition, activin A obviously up-regulated MHC II expression on the surface of RAW264.7 cells, whereas did not influence MHC I expression. Activin A also enhanced CD80 expression, which is a marker of activated macrophages, but did not influence RAW264.7 cell proliferation. These data suggest that activin A may regulate primary macrophage-mediated innate and acquired immune response via promoting the activation of rest macrophages.

Activin A inhibits activities of lipopolysaccharide-activated macrophages via TLR4, not of TLR2

Activin A, a member of TGF-β superfamily, is involved in either pro-inflammatory or anti-inflammatory responses. Our previous studies have reported that lipopolysaccharide (LPS) can simulate activin A secretion from macrophage, and activin A can induce rest macrophage activation in mice, but inhibit the activities of the activated macrophages. However, the relationship of activin and LPS actions and their mechanism are not well characterized. In the present study, the results showed that both activin A and LPS promoted the phagocytic activities of mouse peritoneal macrophages in vivo and in vitro, but activin A inhibited the phagocytosis of LPS-activated macrophages. Simultaneously, the results revealed that activin A inhibited the Toll-like receptor 4 (TLR4) expression on LPS-activated mouse peritoneal macrophages in vivo and in vitro, whereas there was no obvious change of TLR2 expression. Moreover, the results showed that activin A obviously reduced the TLR4 mRNA and protein expressions in LPS-activated macrophage cell line RAW264.7 cells, and the inhibitory effect of activin A on the TLR4 expression was significantly attenuated in Smad3 knock-down RAW264.7 cells. Interestingly, LPS promoted the expression of activin type IIA receptor (ActRIIA) on mouse peritoneal macrophages in vivo, and also up-regulated ActRIIA and activin signal molecules Smad2, 3 mRNA expressions. These data suggest that activin A inhibits LPS action on macrophages in vivo via suppressing TLR4 expression, and LPS further augments the negative feedback action of activin A via up-regulating activin signaling transduction.

High Serum Levels of Follistatin in Patients with Ovarian Cancer

OBJECTIVE: This study investigated the potential use of serum follistatin (FST) as a marker for ovarian cancer alongside serum cancer antigen-125 (CA-125). METHODS: Serum samples were collected from patients with ovarian cancer (n = 45), benign ovarian cysts (n = 40) or other cancers (n = 100) and from healthy subjects (n = 60) for the determination of FST and CA-125 levels using enzyme-linked immunosorbent assays. Expression of FST in ovarian tissue was investigated using immunohistochemical staining. RESULTS: Compared with healthy subjects and patients with benign ovarian cysts, serum FST and CA-125 levels were significantly increased in patients with ovarian cancer. Using the 95% confidence interval for the healthy subjects group as the cut-off value, tumour marker sensitivity and specificity in ovarian cancer were 53.3% and 97% for FST and 77.8% and 84% for CA-125, respectively. Tissue expression of FST protein was more pronounced in ovarian cancer than in normal ovary. CONCLUSIONS: The serum FST level was elevated in the peripheral blood of patients with ovarian cancer and has potential as a tumour marker for ovarian cancer diagnosis. It may be particularly useful when combined with CA-125 detection to reduce the number of false-positive results.

Involvement of TGF-β1/Smad3 Signaling in Carbon Tetrachloride-Induced Acute Liver Injury in Mice

Transforming growth factor-beta1 (TGF-β1) is a major factor in pathogenesis of chronic hepatic injury. Carbon tetrachloride (CCl4) is a liver toxicant, and CCl4-induced liver injury in mouse is a classical animal model of chemical liver injury. However, it is still unclear whether TGF-β1 is involved in the process of CCl4-induced acute chemical liver injury. The present study aimed to evaluate the role of TGF-β1 and its signaling molecule Smad3 in the acute liver injury induce by CCl4. The results showed that CCl4 induced acute liver injury in mice effectively confirmed by H&E staining of liver tissues, and levels of not only liver injury markers serum ALT and AST, but also serum TGF-β1 were elevated significantly in CCl4-treated mice, compared with the control mice treated with olive oil. Our data further revealed that TGF-β1 levels in hepatic tissue homogenate increased significantly, and type II receptor of TGF-β (TβRII) and signaling molecules Smad2, 3, mRNA expressions and Smad3 and phospho-Smad3 protein levels also increased obviously in livers of CCl4-treated mice. To clarify the effect of the elevated TGF-β1/Smad3 signaling on CCl4-induced acute liver injury, Smad3 in mouse liver was overexpressed in vivo by tail vein injection of Smad3-expressing plasmids. Upon CCl4 treatment, Smad3-overexpressing mice showed more severe liver injury identified by H&E staining of liver tissues and higher serum ALT and AST levels. Simultaneously, we found that Smad3-overexpressing mice treated with CCl4 showed more macrophages and neutrophils infiltration in liver and inflammatory cytokines IL-1β and IL-6 levels increment in serum when compared with those in control mice treated with CCl4. Moreover, the results showed that the apoptosis of hepatocytes increased significantly, and apoptosis-associated proteins Bax, cytochrome C and the cleaved caspase 3 expressions were up-regulated in CCl4-treated Smad3-overexpressing mice as well. These results suggested that TGF-β1/Smad3 signaling was activated during CCl4-induced acute liver injury in mice, and Smad3 overexpression aggravated acute liver injury by promoting inflammatory cells infiltration, inflammatory cytokines release and hepatocytes apoptosis. In conclusion, the activation of TGF-β signaling contributes to the CCl4-induced acute liver injury. Thus, TGF-β1/Smad3 may serve as a potential target for acute liver injury therapy.

Expression and localization of activin receptor-interacting protein 2 in mouse tissues

Activin plays important roles in reproductive tissues as a stimulator of follicle-stimulating hormone (FSH) secretion. Activin receptor-interacting protein 2 (ARIP2) has been recently identified in mouse tissues as a regulatory protein of activin signal transduction. However, the localization and function of ARIP2 are not well characterized. In this study, we found that ARIP2 mRNA and protein were widely expressed in mouse tissues by reverse transcription-PCR (RT-PCR) and Western blotting. The immunoreactivities of ARIP2 were mainly localized at myocardial cells of heart, Leydig cells in testis, macrophages and epithelial cells of bronchus in lung, renal tubule and collecting tubule, pancreatic islet, adrenal gland, adenohypophysis and hypothalamus by immunohistochemical staining. Furthermore, ARIP2 overexpression down-regulated signal transduction induced by activin A in pituitary gonadotroph LbetaT2 cells and inhibited FSH secretion from primary cultured anterior pituitary cells induced by activin A. These findings suggest that ARIP2 is widely distributed in various tissues and may be a negative regulator of activin action in pituitary cells.

Follistatin Is a Novel Biomarker for Lung Adenocarcinoma in Humans

Background Follistatin (FST), a single chain glycoprotein, is originally isolated from follicular fluid of ovary. Previous studies have revealed that serum FST served as a biomarker for pregnancy and ovarian mucinous tumor. However, whether FST can serve as a biomarker for diagnosis in lung adenocarcinoma of humans remains unclear. Methods and Results The study population consisted of 80 patients with lung adenocarcinoma, 40 patients with ovarian adenocarcinoma and 80 healthy subjects. Serum FST levels in patients and healthy subjects were measured using ELISA. The results showed that the positive ratio of serum FST levels was 51.3% (41/80), which was comparable to the sensitivity of FST in 40 patients with ovarian adenocarcinoma (60%, 24/40) using the 95th confidence interval for the healthy subject group as the cut-off value. FST expressions in lung adenocarcinoma were examined by immunohistochemical staining, we found that lung adenocarcinoma could produce FST and there was positive correlation between the level of FST expression and the differential degree of lung adenocarcinoma. Furthermore, the results showed that primary cultured lung adenocarcinoma cells could secrete FST, while cells derived from non-tumor lung tissues almost did not produce FST. In addition, the results of CCK8 assay and flow cytometry showed that using anti-FST monoclonal antibody to neutralize endogenous FST significantly augmented activin A-induced lung adenocarcinoma cells apoptosis. Conclusions These data indicate that lung adenocarcinoma cells can secret FST into serum, which may be beneficial to the survival of adenocarcinoma cells by neutralizing activin A action. Thus, FST can serve as a promising biomarker for diagnosis of lung adenocarcinoma and a useful biotherapy target for lung adenocarcinoma.

Role of activin A in carbon tetrachloride-induced acute liver injury

AIM To investigate the expression and role of activin A in a mouse model of acute chemical liver injury. METHODS Acute liver injury in C57BL/6 male mice was induced by intraperitoneal injection with carbon tetrachloride (CCl4) (0.5 mL/kg, body weight) dissolved in olive oil (1:19 v/v). Mice were sacrificed 1, 3, 5 and 7 d after the treatment. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were examined and pathological changes of liver observed by hematoxylin and eosin staining to evaluate the liver injury. Activin A protein levels in serum and hepatic tissue homogenate of mice were detected by enzyme-linked immunosorbent assay, and the expression pattern of activin A protein in livers of mice was examined by immunohistochemistry. Activin type IIA receptor (ActRIIA) and Smad3 expressions in the liver were analyzed by real-time quantitative reverse transcription-polymerase chain reaction. In order to further investigate the role of activin A, we also utilized activin A blocking experiment by anti-activin A antibody (500 μg/kg, body weight) injection into mouse tail vein. RESULTS In CCl4-treated mice, serum ALT and AST levels were significantly increased, compared with that in control mice (P < 0.01). Furthermore, the serious necrosis was observed around hepatic portal areas in CCl4-treated mice. Simultaneously, activin A levels in serum and hepatic tissue homogenate of mice treated with CCl4 for 1, 3 and 5 d increased significantly, compared with that in control mice (P < 0.01). Activin A protein expression in hepatocytes not within the necrotic area was also upregulated in mice following CCl4 treatment. Not only activin A, but also ActRIIA and activin signaling molecule Smad3 mRNA expressions in injury liver induced by CCl4 were significantly higher than that in control liver. In addition, levels of serum ALT and AST in CCl4-treated mice were significantly decreased by injection of anti-activin A antibody to block endogenous activin A action, compared with that in CCl4-treated mice by injection of immunoglobulin G instead of anti-activin A antibody (P < 0.01), and the severity of liver injury was also reduced remarkably. CONCLUSION These data show that activin A is involved in CCl4-induced acute liver injury. Blocking activin A actions may be a therapeutic approach for acute liver injury.

Co-expression of activin receptor-interacting protein 1 and 2 in mouse nerve cells

Activin is a neurotrophic and neuroprotective factor in the central nervous system. Activin receptor-interacting protein 1 and 2 (ARIP1 and ARIP2) are identified as activin signal proteins in mouse brain. However, whether ARIP1 and ARIP2 are co-expressed in nerve cells and the differences of their biological activities are not well characterized. In the present study, we found that ARIP1 and ARIP2 mRNA expressions were detectable in mouse brain and their proteins were co-localized at the hypothalamus of cerebrum and granular layers in cerebellum, especially in Purkinje cells. Furthermore, ARIP1 and ARIP2 were co-expressed in mouse Neuro-2a cells, which is similar to the co-localization of ARIP1 and ARIP2 in hypothalamus neurons and Purkinje cells. Overexpression of ARIP1 in Neuro-2a cells inhibited activin signal transduction induced by activin A and Smad3, and activin A-induced voltage-gated Na(+) current (INa), while ARIP2 was only a negative regulator of signal transduction induced by activin A and did not alter activin A-induced INa. Taken together, these data demonstrate that ARIP1 and ARIP2 are co-expressed in some nerve cells and their biological activities are distinct.