Alpha-Catulin Contributes to Drug-Resistance of Melanoma by Activating NF-κB and AP-1

Melanoma is the most dangerous type of skin cancer accounting for 48,000 deaths worldwide each year and an average survival rate of about 6-10 months with conventional treatment. Tumor metastasis and chemoresistance of melanoma cells are reported as the main reasons for the insufficiency of currently available treatments for late stage melanoma. The cytoskeletal linker protein α-catulin (CTNNAL1) has been shown to be important in inflammation, apoptosis and cytoskeletal reorganization. Recently, we found an elevated expression of α-catulin in melanoma cells. Ectopic expression of α-catulin promoted melanoma progression and occurred concomitantly with the downregulation of E-cadherin and the upregulation of mesenchymal genes such as N-cadherin, Snail/Slug and the matrix metalloproteinases 2 and 9. In the current study we showed that α-catulin knockdown reduced NF-κB and AP-1 activity in malignant melanoma cells. Further, downregulation of α-catulin diminished ERK phosphorylation in malignant melanoma cells and sensitized them to treatment with chemotherapeutic drugs. In particular, cisplatin treatment led to decreased ERK-, JNK- and c-Jun phosphorylation in α-catulin knockdown melanoma cells, which was accompanied by enhanced apoptosis compared to control cells. Altogether, these results suggest that targeted inhibition of α-catulin may be used as a viable therapeutic strategy to chemosensitize melanoma cells to cisplatin by down-regulation of NF-κB and MAPK pathways.     

Synergistic Induction of Endothelin-1 by Tumor Necrosis Factor �� and Interferon �� Is due to Enhanced NF-��B Binding and Histone Acetylation at Specific ��B Sites

Endothelin-1 (ET-1) is a potent vasoconstrictor and co-mitogen for vascular smooth muscle and is implicated in pulmonary vascular remodeling and the development of pulmonary arterial hypertension. Vascular smooth muscle is an important source of ET-1. Here we demonstrate synergistic induction of preproET-1 message RNA and release of mature peptide by a combination of tumor necrosis factor α (TNFα) and interferon γ (IFNγ) in primary human pulmonary artery smooth muscle cells. This induction was prevented by pretreatment with the histone acetyltransferase inhibitor anacardic acid. TNFα induced a rapid and prolonged pattern of nuclear factor (NF)-κB p65 subunit activation and binding to the native preproET-1 promoter. In contrast, IFNγ induced a delayed activation of interferon regulatory factor-1 without any effect on NF-κB p65 nuclear localization or consensus DNA binding. However, we found cooperative p65 binding and histone H4 acetylation at distinct κB sites in the preproET-1 promoter after stimulation with both TNFα and IFNγ. This was associated with enhanced recruitment of RNA polymerase II to the ATG start site and read-through of the ET-1 coding region. Understanding such mechanisms is crucial in determining the key control points in ET-1 release. This has particular relevance to developing novel treatments targeted at the inflammatory component of pulmonary vascular remodeling.Endothelin-1 is a 21-amino acid peptide which is known to be both a potent vasoconstrictor and mitogen for vascular smooth muscle (1, 2). It is released as a 38-amino acid precursor (Big ET-1²) before cleavage to the mature ET-1 form. As such it has been implicated in the pathogenesis of vascular disease and is particularly associated with pulmonary arterial hypertension (3). Indeed, several endothelin receptor antagonists are now approved for the treatment of pulmonary arterial hypertension (4). However, endothelin receptor antagonists as a class are associated with potentially serious side effects (4), making new treatments aimed at blocking ET-1 synthesis an attractive alternative.Although endothelial cells are thought to be the main source of ET-1 release, several groups including our own have shown that ET-1 can be released from the more numerous vascular smooth muscle cells (5–10). The vascular pathology observed in pulmonary arterial hypertension is propagated by inflammation, and circulating levels of cytokines including tumor necrosis factor α (TNFα) are elevated in patients with pulmonary arterial hypertension (11–15). In many cell types cytokines mediate their biological effects at least in part by the activation of the nuclear factor κB (NF-κB) pathway (16), and a role for NF-κB in pulmonary arterial hypertension has been proposed (17). In addition, we have shown previously that a combination of TNFα and interferon γ (IFNγ) stimulates human pulmonary artery smooth muscle (HPASM) cells to release ET-1 (18). However, the mechanisms underlying this effect are unknown.The preproET-1 promoter region has been shown experimentally to possess binding sites for nuclear factor (NF)-1 and phorbol ester-sensitive c-Fos and c-Jun complexes (19), acute phase reactant regulatory proteins, and binding sites for AP-1 and GATA-2 (20–22). In addition, binding sites for interferon regulatory factor-1 (IRF-1) and NF-κB are predicted by Transfac analysis (23). The close proximity of the IRF-1 site and one of the NF-κB sites is characteristic of genes that are regulated by the synergistic action of TNFα and IFNγ, such as interleukin-6 (IL-6) and intercellular adhesion molecule-1 (24, 25), although ET-1 has not previously been recognized in this group.Our aims were, therefore, to investigate the role of NF-κB in ET-1 release by primary HPASM cells. In addition, we were interested in the role of histone acetylation in the epigenetic control of the ET-1 production. Understanding these novel mechanisms will allow a greater understanding of the pathogenesis of vascular remodeling in pulmonary vessels and aid in the development of new treatment strategies aimed at blocking synthesis of ET-1.     

Effect of thymopentin on the production of cytokines, heat shock proteins, and NF-κB signaling proteins

In vivo effects of thymopentin, an active fragment of the naturally occurring thymic hormone thymopoietin, on the production of cytokines, nitric oxide, heat shock proteins, and signaling proteins NF-κB, phNF-κB, and IκB-α in lymphoid cells of male NMRI mice was studied. Activation of production of several cytokines (IL-1α, IL-2, IL-6, IL-10, and IFN-γ), nitric oxide, and heat shock proteins (HSP70 and HSP90) was observed in peritoneal macrophages and spleen lymphocytes of mice that received intraperitoneal injections of thymopentin (15μg per 100 g body weight). Thymopentin apparently produces stress-like rather than damaging effects. A probable action mechanism of this hormone is activation of the NF-κB signaling pathway, which is most pronounced at the NF-κB phosphorylation stage.     

Association of PARP-1, NF-κB,NF-κBIA and IL-6, IL-1β and TNF-α with Graves Disease and Graves Ophthalmopathy

Background

Graves Disease (GD) is an autoimmune disorder affected by an interaction of multiple genes such as Nuclear Factor-κB (NF-κB), Nuclear Factor-κB Inhibitor (NF-κBIA), Poly (ADP-ribose) polymerase-1 (PARP-1) and cytokines like Interleukin-1β (IL-1β), Interleukin-6 (IL-6) and Tumor Necrosis Factor-α (TNF-α) and mostly accompanied by an ocular disorder, Graves Ophthalmopathy (GO). We hypothesize that there is a relationship between GD, GO, polymorphisms of inflammatory related genes and their association with cytokines, which may play important roles in autoimmune and inflammatory processes.

Subjects and methods

To confirm our hypothesis, we studied the polymorphisms and cytokine levels of 120 patients with GD and GO using PCR-RFLP and ELISA methods, respectively.

Results

We found that patients with GG genotype and carriers of G allele of PARP-1 G1672A polymorphism are at risk in the group having GD (p = 0.0007) while having GA genotype may be protective against the disease. PARP-1 C410T polymorphism was found to be associated with GO by increasing the risk by 1.7 times (p = 0.004). Another risk factor for development of GO was the polymorphism of del/ins of NFkB1 gene (p = 0.032) that increases the risk by 39%. Levels of cytokines were also elevated in patients with GD, but no association was found between levels of cytokines and the development of GO as there was no change in levels of cytokines.

Conclusions

We suggest that, PARP-1 and NFkB1 gene polymorphisms may be risk factors for developing Graves Disease and Ophthalmopathy.     

Coordinate Regulation of TPL-2 and NF-κB Signaling in Macrophages by NF-κB1 p105

The role of IκB kinase (IKK)-induced proteolysis of NF-κB1 p105 in innate immune signaling was investigated using macrophages from Nfkb1(SSAA/SSAA) mice, in which the IKK target serines on p105 are mutated to alanines. We found that the IKK/p105 signaling pathway was essential for TPL-2 kinase activation of extracellular signal-regulated kinase (ERK) mitogen-activate protein (MAP) kinase and modulated the activation of NF-κB. The Nfkb1(SSAA) mutation prevented the agonist-induced release of TPL-2 from its inhibitor p105, which blocked activation of ERK by lipopolysaccharide (LPS), tumor necrosis factor (TNF), CpG, tripalmitoyl-Cys-Ser-Lys (Pam(3)CSK), poly(I · C), flagellin, and R848. The Nfkb1(SSAA) mutation also prevented LPS-induced processing of p105 to p50 and reduced p50 levels, in addition to decreasing the nuclear translocation of RelA and cRel. Reduced p50 in Nfkb1(SSAA/SSAA) macrophages significantly decreased LPS induction of the IκBζ-regulated Il6 and Csf2 genes. LPS upregulation of Il12a and Il12b mRNAs was also impaired although specific blockade of TPL-2 signaling increased expression of these genes at late time points. Activation of TPL-2/ERK signaling by IKK-induced p105 proteolysis, therefore, induced a negative feedback loop to downregulate NF-κB-dependent expression of the proinflammatory cytokine interleukin-12 (IL-12). Unexpectedly, TPL-2 promoted soluble TNF production independently of IKK-induced p105 phosphorylation and its ability to activate ERK, which has important implications for the development of anti-inflammatory drugs targeting TPL-2.     

Effect of simvastatin on collagen I deposition in non-infarcted myocardium: role of NF-κB and osteopontin

The novel biological effect of statins in alleviating myocardium fibrosis following infarction has been increasingly recognized, yet the underlying mechanisms are not fully understood. The purpose of this study was to characterize the effect of simvastatin on myocardial fibrosis and collagen I deposition in the non-infarcted region after myocardial infarction (MI) and to identify the role of NF-κB and osteopontin in simvastatin-mediated inhibition of post-MI collagen over-expression. A rat model of MI was generated by ligating the left anterior descending coronary artery. The rats surviving the MI operation were randomly divided into the following 3 groups: myocardial infarction (MI, vehicle), simvastatin (Sim, 30 mg·kg-1·day-1), and pyrrolidine dithiocarbamate (PDTC, an inhibitor of NF-κB, 100 mg·kg-1·day-1). Four weeks after MI, cardiac function, mRNAs, and protein expression in non-infarcted myocardium were analyzed. Myocardial fibrosis and collagen I over-expression were observed following MI, accompanied by an increase of NF-κB and osteopontin. Simvastatin improved post-MI left ventricular dysfunction and ameliorated post-MI associated changes to several cardiac parameters, including the left ventricular end diastolic pressure (LVEDP), the maximal rate of pressure development (+dP/dtmax), and the maximal rate of pressure decline (-dP/dtmax). Concurrently, simvastatin significantly suppressed the over-expression of NF-κB, osteopontin, and collagen I in the non-infarcted region following MI. Inhibition of NF-κB by PDTC also reduced osteopontin over-expression and excessive collagen I production and improved the above functional myocardial parameters. These results show that post-MI myocardial fibrosis and collagen I over-expression in the non-infarcted region is associated with activation of NF-κB and osteopontin up-regulation. The anti-fibrotic effect of simvastatin following MI is associated with the attenuation of the expression of osteopontin and NF-κB. The inhibition of NF-κB activation could be the process upstream of osteopontin suppression in the simvastatin-mediated effect.     

Binding of NFκB Appears to Twist the Ankyrin Repeat Domain of IκBα

Total internal reflection fluorescence-based single-molecule Förster resonance energy transfer (FRET) measurements were previously carried out on the ankyrin repeat domain (ARD) of IκBα, the temporally regulated inhibitor of canonical NFκB signaling. Under native conditions, most of the IκBα molecules showed stable, high FRET signals consistent with distances between the fluorophores estimated from the crystal structures of the NFκB(RelA/p50)-IκBα complex. Similar high FRET efficiencies were found when the IκBα molecules were either free or in complex with NFκB(RelA/p50), and were interpreted as being consistent with the crystallographically observed ARD structure. An exception to this was observed when the donor and acceptor fluorophores were attached in AR3 (residue 166) and AR6 (residue 262). Surprisingly, the FRET efficiency was lower for the bound IκBα molecules (0.67) than for the free IκBα molecules (0.74), apparently indicating that binding of NFκB(RelA/p50) stretches the ARD of IκBα. Here, we conducted confocal-based single-molecule FRET studies to investigate this phenomenon in greater detail. The results not only recapitulated the apparent stretching of the ARD but also showed that the effect was more pronounced when the N-terminal domains (NTDs) of both RelA and p50 were present, even though the interface between NFκB(RelA/p50) and IκBα encompasses only the dimerization domains. We also performed mass spectrometry-detected amide hydrogen/deuterium exchange (HDXMS) experiments on IκBα as well as IκBα bound to dimerization-domain-only constructs or full-length NFκB(RelA/p50). Although we expected the stretched IκBα to have regions with increased exchange, instead the HDXMS experiments showed decreases in exchange in AR3 and AR6 that were more pronounced when the NFκB NTDs were present. Simulations of the interaction recapitulated the increased distance between residues 166 and 262, and also provide a plausible mechanism for a twisting of the IκBα ARD induced by interactions of the IκBα proline-glutamate-serine-threonine-rich sequence with positively charged residues in the RelA NTD.