Expression and Purification of Human Membrane Progestin Receptor α (mPRα)

Membrane progestin receptors (mPRs) are responsible for mediating the rapid, nongenomic activity of progestins and belong to the G protein-coupled receptor (GPCR) family. mPRs are also considered as attractive proteins to draw a new medicinal approach. In this study, we optimized a procedure for the expression and purification of recombinant human mPRα protein (hmPRα) by a methylotropic yeast, Pichia pastoris, expression system. The protein expressed in crude membrane fractions exhibited a binding affinity of Kd = 3.8 nM and Bmax = 288.8 fmol/mg for progesterone. These results indicated that the hmPRα expressed in yeast was active. Solubilized hmPRα was purified through three column chromatography steps. A nickel-nitrilotriacetic acid (Ni-NTA) column was first used, and the mPRα proteins were then bound to cellulose resin with free amino groups (Cellufine Amino) and finally passed through an SP-Sepharose column. The optimization of expression and purification conditions resulted in a high yield of purified hmPRα (1.3–1.5 mg from 1 L culture). The purified hmPRα protein demonstrated progesterone binding (Kd = 5.2 nM and Bmax = 111.6 fmol/mg). The results indicated that we succeeded in solubilizing and purifying hmPRα in an active form. Sufficient amount of active hmPRα protein will support the establishment of applications for the screening of ligands for mPRα.     

Dual Regulation of Myocardin Expression by Tumor Necrosis Factor-α in Vascular Smooth Muscle Cells

De-differentiation of vascular smooth muscle cells (VSMCs) plays a critical role in the development of atherosclerosis, a chronic inflammatory disease involving various cytokines such as tumor necrosis factor-α (TNFα). Myocardin is a co-factor of serum response factor (SRF) and is considered to be the master regulator of VSMC differentiation. It binds to SRF and regulates the expression of contractile proteins in VSMCs. Myocardin is also known to inhibit VSMC proliferation by inhibiting the NF-κB pathway, whereas TNFα is known to activate the NF-κB pathway in VSMCs. NF-κB activation has also been shown to inhibit myocardin expression and smooth muscle contractile marker genes. However, it is not definitively known whether TNFα regulates the expression and activity of myocardin in VSMCs. The current study aimed to investigate the role of TNFα in regulating myocardin and VSMC function. Our studies showed that TNFα down-regulated myocardin expression and activity in cultured VSMCs by activating the NF-κB pathway, resulting in decreased VSMC contractility and increased VSMC proliferation. Surprisingly, we also found that TNFα prevented myocardin mRNA degradation, and resulted in a further significant increase in myocardin expression and activity in differentiated VSMCs. Both the NF-κB and p44/42 MAPK pathways were involved in TNFα regulation of myocardin, which further increased the contractility of VSMCs. These differential effects of TNFα on myocardin seemingly depended on whether VSMCs were in a differentiated or de-differentiated state. Taken together, our results demonstrate that TNFα differentially regulates myocardin expression and activity, which may play a key role in regulating VSMC functions.     

TNFα Inhibits IGFBP-3 through Activation of p38α and Casein Kinase 2 in Human Retinal Endothelial Cells

We recently reported a reciprocal relationship between tumor necrosis factor alpha (TNFα) and insulin-like receptor growth factor binding protein 3 (IGFBP-3) in whole retina of normal and IGFBP-3 knockout mice. A similar relationship was also observed in cultured retinal endothelial cells (REC). We found that TNFα significantly reduced IGFBP-3 levels and vice-versa, IGFBP-3 can lower TNFα and TNFα receptor expression. Since IGFBP-3 is protective to the diabetic retina and TNFα is causative in the development of diabetic retinopathy, we wanted to better understand the cellular mechanisms by which TNFα can reduce IGFBP-3 levels. For these studies, primary human retinal endothelial cells (REC) were used since these cells undergo TNFα-mediated apoptosis under conditions of high glucose conditions and contribute to diabetic retinopathy. We first cultured REC in normal or high glucose, treated with exogenous TNFα, then measured changes in potential signaling pathways, with a focus on P38 mitogen-activated protein kinase alpha (P38α) and casein kinase 2 (CK2) as these pathways have been linked to both TNFα and IGFBP-3. We found that TNFα significantly increased phosphorylation of P38α and CK2. Furthermore, specific inhibitors of P38α or CK2 blocked TNFα inhibition of IGFBP-3 expression, demonstrating that TNFα reduces IGFBP-3 through activation of P38α and CK2. Since TNFα and IGFBP-3 are key mediators of retinal damage and protection respectively in diabetic retinopathy, increased understanding of the relationship between these two proteins will offer new therapeutic options for treatment.     

Bestrophin 3 Ameliorates TNFα-Induced Inflammation by Inhibiting NF-κB Activation in Endothelial Cells

Increasing evidences have suggested vascular endothelial inflammatory processes are the initiator of atherosclerosis. Bestrophin 3 (Best-3) is involved in the regulation of cell proliferation, apoptosis and differentiation of a variety of physiological functions, but its function in cardiovascular system remains unclear. In this study, we investigated the effect of Best-3 on endothelial inflammation. We first demonstrated that Best-3 is expressed in endothelial cells and decreased after tumor necrosis factor-α (TNFα) challenge. Overexpression of Best-3 significantly attenuated TNFα-induced expression of adhesion molecules and chemokines, and subsequently inhibited the adhesion of monocytes to human umbilical vein endothelial cells (HUVECs). Conversely, knockdown of Best-3 with siRNA resulted in an enhancement on TNFα-induced expression of adhesion molecules and chemokines and adhesion of monocytes to HUVECs. Furthermore, overexpression of Best-3 with adenovirus dramatically ameliorated inflammatory response in TNFα-injected mice. Mechanistically, we found up-regulation of Best-3 inhibited TNFα-induced IKKβ and IκBα phosphorylation, IκBα degradation and NF-κB translocation. Our results demonstrated that Best-3 is an endogenous inhibitor of NF-κB signaling pathway in endothelial cells, suggesting that forced Best-3 expression may be a novel approach for the treatment of vascular inflammatory diseases.     

Characterization of golimumab,a human monoclonal antibody specific for human tumor necrosis factor α

We prepared and characterized golimumab, a human IgG1 tumor necrosis factor alpha (TNFα) antagonist monoclonal antibody chosen for clinical development based on its molecular properties. Golimumab was compared with infliximab, adalimumab and etanercept for affinity and in vitro TNFα neutralization. The affinity of golimumab for soluble human TNFα, as determined by surface plasmon resonance, was similar to that of etanercept (18 pM versus 11 pM), greater than that of infliximab (44 pM) and significantly greater than that of adalimumab (127 pM, p = 0.018). The concentration of golimumab necessary to neutralize TNFα-induced E-selectin expression on human endothelial cells by 50% was significantly less than those for infliximab (3.2-fold; p = 0.017) and adalimumab (3.3-fold; p = 0.008) and comparable to that for etanercept. The conformational stability of golimumab was greater than that of infliximab (primary melting temperature [Tm] 74.8°C vs. 69.5°C) as assessed by differential scanning calorimetry. In addition, golimumab showed minimal aggregation over the intended shelf life when formulated as a high concentration liquid product (100 mg/mL) for subcutaneous administration. In vivo, golimumab at doses of 1 and 10 mg/kg significantly delayed disease progression in a mouse model of human TNFα-induced arthritis when compared with untreated mice, while infliximab was effective only at 10 mg/kg. Golimumab also significantly reduced histological scores for arthritis severity and cartilage damage, as well as serum levels of pro-inflammatory cytokines and chemokines associated with arthritis. Thus, we have demonstrated that golimumab is a highly stable human monoclonal antibody with high affinity and capacity to neutralize human TNFα in vitro and in vivo.Key words: TNF, golimumab, neutralization, affinity, bioassay, arthritis, stability, solubility     

TNFα-exposed Bone Marrow-derived Mesenchymal Stem Cells Promote Locomotion of MDA-MB-231 Breast Cancer Cells through Transcriptional Activation of CXCR3 Ligand Chemokines

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are often recruited to solid tumors, integrate into the tumor stroma, and contribute to tumor development. TNFα is a major inflammatory cytokine present in the tumor microenvironment and has a profound influence on the progression of tumor development. This study was aimed to investigate the role of BM-MSCs in tumor promotion in response to TNFα. Quantitative real-time PCR arrays show that diverse cytokines/chemokines were induced in TNFα-treated BM-MSCs; in particular, CXCR3 ligand chemokines, including CXCL9, CXCL10, and CXCL11, were potently induced. A serial and site-directed mutation analysis in the CXCL9, CXCL10, and CXCL11 promoters revealed that NF-κB binding elements were responsible for TNFα-induced promoter activation of CXCR3 ligand chemokines. TNFα stimulated NF-κB activity, and ectopic expression of NF-κB enhanced TNFα-induced promoter activities of the CXCR3 ligand chemokines. Gel shift and supershift assays showed that NF-κB was associated with CXCR3 ligand chemokine promoters in response to TNFα treatment. All three CXCR3 ligand chemokines enhanced the migration and invasive motility of MDA-MB-231 breast cancer cells expressing CXCR3. Treatment of MDA-MB-231 cells with CXCL10 activated small GTPase of Rho family proteins, such as RhoA and Cdc42. CXCL9-, CXCL10-, or CXCL11-induced invasive capability of MDA-MB-231 cells was completely abrogated in the presence of a neutralizing anti-CXCR3 antibody in the culture medium. Moreover, CXCL9, CXCL10, and CXCL11 stimulated the expression of MMP-9, but not MMP-2, in MDA-MB-231 cells. These results suggest that BM-MSCs promote the locomotion of breast cancer cells through CXCR3 ligand-mediated actin rearrangement by TNFα in the tumor microenvironment.     

β-Transducin Repeat-containing Protein 1 (β-TrCP1)-mediated Silencing Mediator of Retinoic Acid and Thyroid Hormone Receptor (SMRT) Protein Degradation Promotes Tumor Necrosis Factor α (TNFα)-induced Inflammatory Gene Expression

Cytokine modulation of the endothelium is considered an important contributor to the inflammation response. TNFα is an early response gene during the initiation of inflammation. However, the detailed mechanism by which TNFα induces proinflammatory gene expression is not completely understood. In this report, we demonstrate that silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) represses the expression of a subset of TNFα target genes in human umbilical vein endothelial cells. Upon TNFα stimulation, we observed an increase in the E3 ubiquitin ligase β-TrCP1 and a decrease in SMRT protein levels. We show that β-TrCP1 interacts with SMRT in a phosphorylation-independent manner and cooperates with the E2 ubiquitin-conjugating enzyme E2D2 to promote ubiquitination-dependent SMRT degradation. Knockdown of β-TrCP1 increases SMRT protein accumulation, increases SMRT association with its targeted promoters, and decreases SMRT target gene expression. Taken together, our results support a model in which TNFα-induced β-TrCP1 accumulation promotes SMRT degradation and the subsequent induction of proinflammatory gene expression.     

The Induction of Lipocalin-2 Protein Expression in Vivo and in Vitro

Lipocalin-2 (LCN2) is secreted from adipocytes, and its expression is up-regulated in obese and diabetic mice and humans. LCN2 expression and secretion have been shown to be induced by two proinflammatory cytokines, IFNγ and TNFα, in cultured murine and human adipocytes. In these studies, we demonstrated that IFNγ and TNFα induced LCN2 expression and secretion in vivo. Although we observed a strong induction of LCN2 expression and secretion from white adipose tissue (WAT) depots, the induction of LCN2 varied among different insulin-sensitive tissues. Knockdown experiments also demonstrated that STAT1 is required for IFNγ-induced lipocalin-2 expression in murine adipocytes. Similarly, knockdown of p65 in adipocytes demonstrated the necessity of the NF-κB signaling pathway for TNFα-mediated effects on LCN2. Activation of ERKs by IFNγ and TNFα also affected STAT1 and NF-κB signaling through modulation of serine phosphorylation. ERK activation-induced serine phosphorylation of both STAT1 and p65 mediated the additive effects of IFNγ and TNFα on LCN2 expression. Our results suggest that these same mechanisms occur in humans as we observed STAT1 and NF-κB binding to the human LCN2 promoter in chromatin immunoprecipitation assays performed in human fat cells. These studies substantially increase our knowledge regarding the requirements and mechanisms used by proinflammatory cytokines to induce LCN2 expression.     

Neurotropin Suppresses Inflammatory Cytokine Expression and Cell Death through Suppression of NF-κB and JNK in Hepatocytes

Inflammatory response and cell death in hepatocytes are hallmarks of chronic liver disease, and, therefore, can be effective therapeutic targets. Neurotropin® (NTP) is a drug widely used in Japan and China to treat chronic pain. Although NTP has been demonstrated to suppress chronic pain through the descending pain inhibitory system, the action mechanism of NTP remains elusive. We hypothesize that NTP functions to suppress inflammatory pathways, thereby attenuating disease progression. In the present study, we investigated whether NTP suppresses inflammatory signaling and cell death pathways induced by interleukin-1β (IL-1β) and tumor necrosis factor-α (TNFα) in hepatocytes. NTP suppressed nuclear factor-κB (NF-κB) activation induced by IL-1β and TNFα assessed by using hepatocytes isolated from NF-κB-green fluorescent protein (GFP) reporter mice and an NF-κB-luciferase reporter system. The expression of NF-κB target genes, Il6, Nos2, Cxcl1, ccl5 and Cxcl2 induced by IL-1β and TNFα was suppressed after NTP treatment. We also found that NTP suppressed the JNK phosphorylation induced by IL-1β and TNFα. Because JNK activation contributes to hepatocyte death, we determined that NTP treatment suppressed hepatocyte death induced by IL-1β and TNFα in combination with actinomycin D. Taken together, our data demonstrate that NTP attenuates IL-1β and TNFα-mediated inflammatory cytokine expression and cell death in hepatocytes through the suppression of NF-κB and JNK. The results from the present study suggest that NTP may become a preventive or therapeutic strategy for alcoholic and non-alcoholic fatty liver disease in which NF-κB and JNK are thought to take part.     

TNFα‐induced abnormal activation of TNFR/NF‐κB/FTH1 in endometrium is involved in the pathogenesis of early spontaneous abortion

Early spontaneous abortion (ESA) is one of the most common complications during pregnancy and the inflammation condition in uterine environment such as long‐term exposure to high TNFα plays an essential role in the aetiology. Ferritin heavy chain (FTH1) is considered to be closely associated with inflammation and very important in normal pregnancy, yet the underlying mechanism of how TNFα induced abortion and its relationship with FTH1 remain elusive. In this study, we found that TNFα and FTH1 were positively expressed in decidual stromal cells and increased significantly in the ESA group compared with the normal pregnancy group (NP group). Besides, TNFα expression was positively correlated with FTH1 expression. Furthermore, in vitro cell model demonstrated that high TNFα could induce the abnormal signals of TNFR/NF‐κB/FTH1 and activate apoptosis both in human endometrium stromal cells (hESCs) and in local decidual tissues. Taken together, the present findings suggest that the excessive apoptosis in response to TNFα‐induced upregulation of FTH1 may be responsible for the occurrence of ESA, and thus provide a possible therapeutic target for the treatment of ESA.