Emerging evidence indicates that aldosterone and mineralocorticoid receptors (MRs) are associated with the pathogenesis of erectile dysfunction. However, the molecular mechanisms remain largely unknown. In this study, freshly isolated penile corpus cavernosum tissue from rats was treated with aldosterone, with or without MRs inhibitors. Nuclear factor (NF)-kappa B (NF-κB) activity was evaluated by real-time quantitative PCR, luciferase assay, and immunoblot. The results demonstrated that mRNA levels of the NF-κB target genes, including inhibitor of NF-κB alpha (IκB-α), NF-κB1, tumor necrosis factor-alpha (TNF-α), and interleukin 6 (IL-6), were higher after aldosterone treatment. Accordingly, phosphorylation of p65/RelA, IκB-α, and inhibitor of NF-κB kinase-β was markedly increased by aldosterone. Furthermore, knockdown of MRs prevented activation of the NF-κB canonical pathway by aldosterone. Consistent with this finding, ectopic overexpression of MRs enhanced the transcriptional activation of NF-κB by aldosterone. More importantly, the MRs antagonist, spironolactone blocked aldosterone-mediated activation of the canonical NF-κB pathway. In conclusion, aldosterone has an inflammatory effect in the corpus cavernosum penis, inducing NF-κB activation via an MRs-dependent pathway, which may be prevented by selective MRs antagonists. These data reveal the possible role of aldosterone in erectile dysfunction as well as its potential as a novel pharmacologic target for treatment.
Aldosterone/pharmacology* ; Animals ; Cytokines/biosynthesis* ; Gene Knockdown Techniques ; I-kappa B Kinase/antagonists & inhibitors* ; Interleukin-6/genetics* ; Male ; Mineralocorticoid Receptor Antagonists/pharmacology* ; NF-kappa B/genetics* ; Penis/metabolism* ; Protein Serine-Threonine Kinases/antagonists & inhibitors* ; RNA, Messenger/biosynthesis* ; Rats ; Rats, Inbred WKY ; Receptors, Mineralocorticoid/genetics* ; Signal Transduction/drug effects* ; Spironolactone/pharmacology* ; Transcriptional Activation ; Tumor Necrosis Factor-alpha/biosynthesis* ; NF-kappaB-Inducing Kinase

In this study we used two types of cell cultures, i.e., anchorage-dependent basket and full suspension batch cultures of sTNFRII-gAD-expressing CHO cells in the CelliGen 310 bioreactor (7.5 L) to compare their yields in order to optimize the culturing conditions for efficient expression of sTNFRII-gAD fusion protein consisting of soluble tumor necrosis factor receptor II and globular domain of adiponectin. The anchorage-dependent basket culture was performed in 4L 10% serum-containing medium with the final inoculating concentration of 3 x 10(5) to 4 x 10(5) cells/mL of sTNFRII-gAD-expressing CHO cells for 3 days, and then switched to 4 L serum-free LK021 medium to continue the culture for 4 days. The full suspension batch culture was carried out in the 4 L serum-free LK021 medium with the final inoculating concentration of 3 x 10(5) to 4 x 10(5) cells/mL of sTNFRII-gAD-expressing CHO cells for 7 days. The culturing conditions were monitored in real-time to maintain pH and dissolved oxygen stability through the whole process. The supernatants were collected by centrifuge, and the protein was concentrated through Pellicon flow ultrafiltration system and then purified by DEAE anion exchange. The results showed that the yields of sTNFRII-gAD fusion protein were 8.0 mg/L with 95% purity and 7.5 mg/L with 98% purity in the anchorage-dependent basket and the full suspension batch cultures, respectively. The study provided the framework for the pilot production of sTNFRII-gAD fusion protein.
Adiponectin ; biosynthesis ; genetics ; Animals ; Bioreactors ; CHO Cells ; Cell Culture Techniques ; methods ; Cricetinae ; Cricetulus ; Receptors, Tumor Necrosis Factor, Type II ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics

Impaired tumor necrosis factor receptor-1 (TNFR-1) signaling has been found in some malignant tumors with poor prognosis. However, the exact role of TNFR-1 signaling in fibrosarcoma remains unclear. Here, we explored the question by comparing the growth of TNFR-1 deficient (Tnfr1 (-)) and TNFR-1 competent (Tnfr1 (+)) fibrosarcoma FB61 cells (FB61-m and FB61-R1) in mice. TNFR-1 expression on fibrosarcoma cells delayed their growth in vivo but not in vitro. Moreover, reduced FB61-R1 tumor growth was also obtained in TNFR-1 knockout mice. The mechanism relies mainly on the TNFR-1-mediated downregulation of vascular endothelial growth factor (VEGF) production by tumor cells. Importantly, treatment of FB61-m tumors with melphalan resulted in a short delay of tumor growth, followed by a quick remission. However, when FB61-R1 tumors were treated with melphalan, tumor growth was similarly delayed at first and then completely rejected. Our results reveal evidence for TNFR-1 on tumor cells as a prerequisite in chemotherapy for fibrosarcoma, and provide novel insight into the therapeutic approach against some types of tumors using TNFR-1 angonist.
Animals ; Down-Regulation ; drug effects ; Fibrosarcoma ; drug therapy ; genetics ; pathology ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Melphalan ; administration & dosage ; Mice ; Molecular Targeted Therapy ; Receptors, Tumor Necrosis Factor, Type I ; genetics ; Signal Transduction ; drug effects ; Vascular Endothelial Growth Factor A ; biosynthesis

We expressed and prepared the recombinant fusion protein sTNFRII-gAD consisted of soluble TNF receptor II and the globular domain of adiponectin by Adenovirus Vector System in mammalian BHK21c022 cells. First we used the adenovirus vector containing EGFP gene (rAd5-EGFP) to infect BHK21c022 cells at different MOI (from 0 to 1 000), and then evaluated their transduction efficiency and cytotoxicity. Similarly, we constructed the replication-deficient adenovirus type 5-sTNFRII-gAD (rAd5-sTNFRII-gAD). We collected the supernatants for Western blotting to determine the optimal MOI by comparing the expression levels of sTNFRII-gAD fusion protein, 48 h after the BHK21c022 cells were infected by rAd5-sTNFRII-gAD at different MOIs (from 0 to 1 000). Then, we chose rAd5-sTNFRII-gAD at MOI 100 to infect five bottles of BHK21c022 cells in 100 mL of serum-free chemically defined media 100 mL, harvested the supernatant every 48 h for 6 times, and condense and purify sTNFRII-gAD fusion protein by ammonium sulfate salt-out and size-exclusion chromatography, respectively. Finally, we analyzed anti-TNFalpha activity of sTNFRII-gAD fusion protein on L929 cells in vitro. The results showed that the number of BHK21c022 cells expressing EGFP protein was increased significantly with the increase of MOI. However, some cells died at MOI of 1 000 while there was no significant cytotoxicity at MOI from 0 to 100. Western blotting analysis showed that the more adenoviruses, the higher expression of sTNFRII-gAD fusion protein in the supernatant with the highest expression at MOI 1 000. We successfully obtained about 11 mg bioactive and purified sTNFRII-gAD fusion protein at last. The in vitro assay demonstrated that the sTNFRII-gAD fusion protein was potent to antagonize TNFalpha's cytotoxicity to L929 cells. Put together, we established a recombinant adenovirus vector/BHK21 cell expression system, characteristic of the efficient serum-free culture and easy scaling-up.
Adenoviridae ; genetics ; metabolism ; Adiponectin ; biosynthesis ; genetics ; Animals ; Cell Line ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Receptors, Tumor Necrosis Factor, Type II ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics

TNFR-Fc is an important fusion protein that has great potential in therapeutic and diagnostic applications. We developed an efficient fed-batch process for GS-CHO cells to produce TNFR-Fc. The rationale of this fed-batch process relies on the supply of sufficient nutrients to meet the requirements of cell metabolism. The optimal feed medium was designed through ration design. A metabolically responsive feeding strategy was designed and dynamically adjusted based on the residual glucose concentration determined off-line. In this process, the maximal viable cell density and antibody concentration reached above 9.4x10(6) cells/mL and 207 mg/L, respectively. Compared with the batch process, the newly developed fed-batch process increased the cell yield by 3.4 fold and the final antibody concentration by 3 fold. This fed-batch process would therefore facilitate the production of therapeutic antibody by GS-CHO cells.
Animals ; CHO Cells ; Cell Culture Techniques ; methods ; Cricetinae ; Cricetulus ; Culture Media ; Etanercept ; Glucose ; analysis ; Immunoglobulin G ; biosynthesis ; genetics ; Receptors, Tumor Necrosis Factor ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics

In order to get soluble TNF receptor (sTNFR) II with good neutralizing activity against TNFalpha, we constructed the fusion gene sTNFRII-gAD, which encoded human sTNFR II and the globular domain of adiponectin (gAD), and then expressed it in mammalian cells and analyzed its anti-TNFalpha activity. First, sTNFRII cDNA was obtained by RT-PCR from the total RNA of human peripheral blood lymphocytes, and fused in frame with gAD gene. Then, the fusion gene sTNFRII-gAD was cloned into the expression vector pAAV2neo to result in the plasmid pAAV2neo-sTNFRII-gAD. By immunofluorescent staining with monoclonal antibody either against TNFRII or against adiponectin, we demonstrated that the pAAV2neo-s7NFRII-gAD-transiently-transfected BHK-21S cells were positive. To obtain G418-resistant BHK-21S/pAAV2neo-sTNFRII-gAD cells, we cultured the transfected BHK-21S cells above in 10% FBS containing DMEM media with 800 microg/mL G418 for 15 days, and changed the serum-containing culture media to a serum-free chemically defined media so as to change the cells culturing style from adhesion to suspension. 24 hours later, we harvested the supernatant of the culture for sTNFRII-gAD fusion protein characterization and anti-TNFalpha activity analysis. With monoclonal antibody either against TNFRII or against adiponectin, the Western blotting analysis showed that the sTNFRII-gAD fusion protein was expressed and existed as monomer, trimer and multimer forms in the supernatant. The bioactivity assay demonstrated that the sTNFRII-gAD fusion protein had the ability to neutralize TNFalpha so as to inhibit the cytotoxicity of TNFalpha on L929 cells. Put together, this study has laid the groundwork for large-scale preparation of sTNFRII-gAD fusion protein.
Adiponectin ; biosynthesis ; genetics ; Animals ; Cells, Cultured ; Cricetinae ; Humans ; Protein Structure, Tertiary ; genetics ; Receptors, Tumor Necrosis Factor, Type II ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Solubility ; Tumor Necrosis Factor-alpha ; antagonists & inhibitors

Tumor necrosis factor receptor-related 2 (TR2, HVEM or TNFRSF-14) plays an important role in immune responses, however, the mechanisms regulating its expression are unclear. To understand the control of TR2 gene expression, we studied the upstream region of the gene. Gel supershift assays revealed inducible binding of nuclear factor of activated T cells (NFAT) to a putative NFAT site within the TR2 promoter. Furthermore, cotransfection of a dominant negative NFAT construct, or siRNA for NFAT, resulted in increased expression of a TR2 reporter gene. Our findings demonstrate that NFAT negatively regulates TR2 expression in activated T cells.
Animals ; Base Sequence ; CD4-Positive T-Lymphocytes/metabolism ; Cells, Cultured ; Down-Regulation ; Mice ; Mice, Inbred C57BL ; Molecular Sequence Data ; NFATC Transcription Factors/*physiology ; Receptors, Tumor Necrosis Factor, Member 14/*biosynthesis ; T-Lymphocytes/*metabolism

OBJECTIVETo investigate the effects of X-ray exposure on the release of soluble tumor necrosis factor receptor-p75 (sTNFR-p75) in hepatocellular carcinoma HepG2 cells in vitro.

METHODSEnzyme-linked immunosorbent assay (ELISA) was used to examine the levels of sTNFR-p75 in the supernatants of HepG2 cells before and after X-ray exposure. The cell apoptosis was analyzed by flow cytometry and transmission electron microscope(TEM), and the morphological changes of the cells were examined under optical microscope and transmission electron microscope(TEM).

RESULTSX-ray exposure of the cells resulted in a strong increase of cell apoptosis (P<0.05) and sTNFR-p75 production in the cells as compared with the those before the exposure (P<0.01). Optical microscopy revealed apoptotic changes of HepG2 cell after the exposure, shown as cell shrinkage, spherical cell morphology, cytoplasmic and nuclear condensation. Apoptotic bodies were detected by TEM.

CONCLUSIONX-ray exposure induces HepG2 cells apoptosis by inhibiting the release of sTNFR-p75 into the supernatant.

Animals ; Apoptosis ; radiation effects ; Carcinoma, Hepatocellular ; pathology ; secretion ; Cell Line, Tumor ; Culture Media, Conditioned ; chemistry ; metabolism ; radiation effects ; Humans ; Liver Neoplasms ; pathology ; secretion ; Microscopy ; Receptors, Tumor Necrosis Factor, Type II ; biosynthesis ; chemistry ; secretion ; Solubility ; X-Rays

A series of aromatic aminoketones were synthesized by Mannich reaction. Structures of these compounds were confirmed by 1H NMR, MS and HRMS or element analysis. Pharmacological screening showed that most target compounds inhibited the release of beta-glucuronidase in polymorphonuclear leucocytes by PAF (platelet activating factor) and compounds MA12, MA13, MA18, MA21 and MA33 were more active. The study suggests that target compounds are potential PAF receptor antagonists and their anti-inflammatory activities are due to the inhibition of release of lysosomal enzyme.
Animals ; Anti-Inflammatory Agents ; chemical synthesis ; chemistry ; pharmacology ; therapeutic use ; Arthritis, Rheumatoid ; drug therapy ; Glucuronidase ; metabolism ; Ketones ; chemical synthesis ; chemistry ; pharmacology ; therapeutic use ; Macrophages, Peritoneal ; metabolism ; Mice ; Neutrophils ; enzymology ; Platelet Membrane Glycoproteins ; antagonists & inhibitors ; Rats ; Receptors, G-Protein-Coupled ; antagonists & inhibitors ; Structure-Activity Relationship ; Tumor Necrosis Factor-alpha ; biosynthesis

Recent studies indicate that several Toll-like receptors (TLRs) are implicated in recognizing viral structures and instigating immune responses against viral infections. The aim of this study is to examine the expression of TLRs and proinflammatory cytokines in viral skin diseases such as verruca vulgaris (VV) and molluscum contagiosum (MC). Reverse transcription-polymerase chain reaction and immunostaining of skin samples were performed to determine the expression of specific antiviral and proinflammatory cytokines as well as 5 TLRs (TLR2, 3, 4, 7, and 9). In normal human skin, TLR2, 4, and 7 mRNA was constitutively expressed, whereas little TLR3 and 9 mRNA was detected. Compared to normal skin (NS), TLR3 and 9 mRNA was clearly expressed in VV and MC specimens. Likewise, immunohistochemistry indicated that keratinocytes in NS constitutively expressed TLR2, 4, and 7; however, TLR3 was rarely detected and TLR9 was only weakly expressed, whereas 5 TLRs were all strongly expressed on the epidermal keratinocytes of VV and MC lesions. In addition, the mRNA expression of IFN-beta and TNF-alpha was upregulated in the VV and MC samples. Immunohistochemistry indicated that IFN-beta and TNF-alpha were predominately localized in the granular layer in the VV lesions and adjacent to the MC bodies. Our results indicated that VV and MC skin lesions expressed TLR3 and 9 in addition to IFN-beta and TNF-alpha. These viral-induced proinflammatory cytokines may play a pivotal role in cutaneous innate immune responses.
Cytokines/metabolism ; *Gene Expression Regulation ; Humans ; Immunohistochemistry/methods ; Inflammation ; Interferon-beta/biosynthesis ; Keratinocytes/cytology ; Models, Biological ; Molluscum Contagiosum/*metabolism ; Toll-Like Receptor 3/biosynthesis ; Toll-Like Receptor 9/biosynthesis ; Toll-Like Receptors/*biosynthesis ; Tumor Necrosis Factor-alpha/biosynthesis ; Warts/*metabolism