Activin A regulates activities of peripheral blood natural killer cells of mouse in an autocrine and paracrine manner.

Activin A, a multifunctional cytokine of transforming growth factor-ß (TGF-ß) superfamily, can be produced by the diverse immune cells. NK cells in peripheral blood are one of the major immune cells applied to cancer therapy in recent years. However, whether activin A can be produced by natural killer (NK) cells and be involved in regulation of peripheral blood NK cells activities of mouse are not well characterized. Here, we found that activin type IIA and IIB receptors and signaling molecules Smad2, 3 were expressed in peripheral blood NK cells of mouse by flow cytometry and RT-PCR. The cultured blood NK cells of mouse not only produced activin ßA chain protein by intracellular cytokine staining, but also secreted mature activin A protein by enzyme-linked immunosorbent assay (ELISA), and the production was promoted by IL-2. In addition, IL-2 as a positive control obviously promoted IFNγ production of mouse blood NK cells in vitro. However, activin A suppressed IFNγ production, but enhanced IL-2 synthesis and did not alter IL-10 production. Moreover, we found that activin A significantly suppressed the ability of NK cells to lyse target cells. These data revealed that blood NK cells of mouse were not only the target cells in response to activin A, but also the source of activin A, suggesting that activin A may play an important role in regulation of NK cells activities of mouse in an autocrine / paracrine manner.

Effect of Collagen Hydrolysates from Silver Carp Skin (Hypophthalmichthys molitrix) on Osteoporosis in Chronologically Aged Mice: Increasing Bone Remodeling.

Osteoporosis is a common skeletal disorder in humans and gelatin hydrolysates from mammals have been reported to improve osteoporosis. In this study, 13-month-old mice were used to evaluate the effects of collagen hydrolysates (CHs) from silver carp skin on osteoporosis. No significant differences were observed in mice body weight, spleen or thymus indices after daily intake of antioxidant collagen hydrolysates (ACH; 200 mg/kg body weight (bw) (LACH), 400 mg/kg bw (MACH), 800 mg/kg bw (HACH)), collagenase hydrolyzed collagen hydrolysates (CCH) or proline (400 mg/kg body weight) for eight weeks, respectively. ACH tended to improve bone mineral density, increase bone hydroxyproline content, enhance alkaline phosphatase (ALP) level and reduce tartrate-resistant acid phosphatase 5b (TRAP-5b) activity in serum, with significant differences observed between the MACH and model groups (p < 0.05). ACH exerted a better effect on osteoporosis than CCH at the identical dose, whereas proline had no significant effect on repairing osteoporosis compared to the model group. Western blotting results demonstrated that CHs mainly increased bone remodeling by stimulating the transforming growth factor ß1 (TGF-ß1)/Smad signaling pathway and improving the interaction between collagen and α2ß1 integrin. The results indicated that CHs from fish could be applied to alleviate osteoporosis or treat bone loss.

Immunoexpression of TGF-ß/Smad and VEGF-A proteins in serum and BAL fluid of sarcoidosis patients.

BACKGROUND: The chronic course of pulmonary sarcoidosis can lead to lung dysfunction due to fibrosis, in which the signalling pathways TGF-ß/Smad and VEGF-A may play a key role. METHODS: We evaluated immunoexpression of TGF-ß1, Smad2, 3, and 7, and VEGF-A in serum and bronchoalveolar lavage (BAL) fluid of patients (n = 57) classified according to the presence of lung parenchymal involvement (radiological stage I vs. II-III), acute vs. insidious onset, lung function test (LFT) results, calcium metabolism parameters, percentage of BAL lymphocytes (BAL-L%), BAL CD4(+)/CD8(+) ratio, age, and gender. Immunoexpression analysis of proteins was performed by ELISA. RESULTS: The immunoexpression of all studied proteins were higher in serum than in BAL fluid of patients (p >0.05). The serum levels of TGF-ß1 (p = 0.03), Smad2 (p = 0.01), and VEGF-A (p = 0.0002) were significantly higher in sarcoidosis patients compared to healthy controls. There were no differences within the sarcoidosis group between patients with vs. without parenchymal involvement, acute vs. insidious onset, or patients with normal vs. abnormal spirometry results. In patients with abnormal spirometry results a negative correlation was found between forced vital capacity (FVC) % predicted value and TGF-ß1 immunoexpression in BAL fluid, and positive correlations were observed between the intensity of lung parenchymal changes estimated by high-resolution computed tomography (HRCT scores) and Smad 2 level in serum. CONCLUSIONS: TGF-ß/Smad signalling pathway and VEGF-A participate in the pathogenesis of sarcoidosis. BAL TGF-ß1, and Smad 2 in serum seem to be promising biomarkers with negative prognostic value, but further studies are required to confirmed our observations.

BMP signaling is responsible for serum-induced Id2 expression.

Ids function as negative regulators of basic helix-loop-helix transcription factors and their expression is rapidly induced by serum stimulation in various cell types. In this study, we investigated the molecular basis of serum-induced expression of the mouse Id2 gene in NIH3T3 cells. A small-molecule inhibitor of bone morphogenetic protein (BMP) type I receptor kinases blocked the serum induction of Id2 mRNA. The chemical compound and several inhibitory proteins specific for BMP signaling suppressed the serum-induced activation of the luciferase construct with the mouse Id2 4.6-kb promoter region. Importantly, serum stimulation evoked rapid phosphorylation of Smad1/5/8 and significant activation of the reporter plasmid containing the recently identified BMP-responsive element (BRE) of the mouse Id2. Mutation analysis demonstrated that the binding sites for Smad proteins in the Id2 BRE were critical for serum response of the 4.6-kb whole construct. Gel shift and chromatin immunoprecipitation (ChIP) assays confirmed the serum-inducible binding of Smad1/5/8 and Smad4 to the Id2 BRE in vitro and in vivo. Finally, a knockdown experiment revealed the functional importance of Smad1 in the serum induction of Id2 expression. Thus, we concluded that BMP signaling is primarily responsible for the serum-induced Id2 expression. Our results also suggest that some of the cellular effects caused by serum are mediated through BMP signaling.

Cytochrome P450 epoxygenase CYP2J2 attenuates nephropathy in streptozotocin-induced diabetic mice.

Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid into epoxyeicosatrienoic acids (EETs), which play important and diverse roles in the cardiovascular system. The anti-inflammatory, anti-apoptotic, pro-angiogenic, and anti-hypertensive properties of EETs in the cardiovascular system suggest a beneficial role for EETs in diabetic nephropathy. This study investigated the effects of endothelial specific overexpression of CYP2J2 epoxygenase on diabetic nephropathy in streptozotocin-induced diabetic mice. Endothelial CYP2J2 overexpression attenuated renal damage as measured by urinary microalbumin and glomerulosclerosis. These effects were associated with inhibition of TGF-ß/Smad signaling in the kidney. Indeed, overexpression of CYP2J2 prevented TGF-ß1-induced renal tubular epithelial-mesenchymal transition in vitro. These findings highlight the beneficial roles of the CYP epoxygenase-EET system in the pathogenesis of diabetic nephropathy.

TGF-beta signalling-related markers in cancer patients with bone metastasis.

We measured transforming growth factor (TGF)-beta-dependent biomarkers in plasma and in peripheral blood mononuclear cells (PBMCs) to identify suitable pharmacodynamic markers for future clinical trials with TGF-beta inhibitors. Forty-nine patients with bone metastasis were enrolled in the study, including patients with breast (n=23) and prostate cancer (n=15). Plasma TGF-beta1 levels were elevated in more than half of the cancer patients (geometric mean 2.63 ng ml(-1)) and positively correlated with increased platelet factor 4 (PF4) levels, parathyroid-related protein (PTHrP), von Willebrand Factor (vWF) and interleukin (IL)-10. PBMC were stimulated ex vivo to determine the individual biological variability of an ex vivo assay measuring pSMAD expression. This assay performed sufficiently well to allow its future use in a clinical trial of a TGF-beta inhibitor.

Distinct expression profiles of TGF-beta1 signaling mediators in pathogenic SIVmac and non-pathogenic SIVagm infections.

BACKGROUND: The generalized T-cell activation characterizing HIV-1 and SIVmac infections in humans and macaques (MACs) is not found in the non-pathogenic SIVagm infection in African green monkeys (AGMs). We have previously shown that TGF-beta1, Foxp3 and IL-10 are induced very early after SIVagm infection. In SIVmac-infected MACs, plasma TGF-beta1 induction persists during primary infection 1. We raised the hypothesis that MACs are unable to respond to TGF-beta1 and thus cannot resorb virus-driven inflammation. We therefore compared the very early expression dynamics of pro- and anti-inflammatory markers as well as of factors involved in the TGF-beta1 signaling pathway in SIV-infected AGMs and MACs. METHODS: Levels of transcripts encoding for pro- and anti-inflammatory markers (tnf-alpha, ifn-gamma, il-10, t-bet, gata-3) as well as for TGF-beta1 signaling mediators (smad3, smad4, smad7) were followed by real time PCR in a prospective study enrolling 6 AGMs and 6 MACs. RESULTS: During primary SIVmac infection, up-regulations of tnf-alpha, ifn-gamma and t-bet responses (days 1-16 p.i.) were stronger whereas il-10 response was delayed (4th week p.i.) compared to SIVagm infection. Up-regulation of smad7 (days 3-8 p.i.), a cellular mediator inhibiting the TGF-beta1 signaling cascade, characterized SIV-infected MACs. In AGMs, we found increases of gata-3 but not t-bet, a longer lasting up-regulation of smad4 (days 1-21 p.i), a mediator enhancing TGF-beta1 signaling, and no smad7 up-regulations. CONCLUSION: Our data suggest that the inability to resorb virus-driven inflammation and activation during the pathogenic HIV-1/SIVmac infections is associated with an unresponsiveness to TGF-beta1.