NF-kappaB activation for constitutive expression of VCAM-1 and ICAM-1 on B lymphocytes and plasma cells.

Cytokine stimulation can activate NF-kappaB that triggers inducible expression of E-selectin, VCAM-1 (Vascular Cell Adhesion Molecule-1) and ICAM-1 (Intercellular Cell Adhesion Molecule-1) in endothelial cells. In the previous study, we have shown that B lymphocytes and plasma cells can express E-selectin by constitutive activation of NF-kappaB. Here we show that human B lymphocytes and ARH-77 plasma cells expressed VCAM-1 and ICAM-1 in a cytokine dispensable mechanism. NF-kappaB antagonists could inhibit their expressions in ARH-77 cells. The activities of NF-kappaB for VCAM-1 and ICAM-1 promoters prior to cytokine stimulation were detected in ARH-77 cells using electrophoretic mobility shift assays. Again, NF-kappaB antagonists could abrogate these promoter activities. Taken together, our results demonstrate that NF-kappaB activation is the underlying molecular mechanism for constitutive expression of E-selectin, VCAM-1, and ICAM-1 on human B lymphocytes and plasma cells.     

A novel role of the interferon-inducible protein IFI16 as inducer of proinflammatory molecules in endothelial cells

The human IFI16 gene is an interferon-inducible gene implicated in the regulation of endothelial cell proliferation and tube morphogenesis. Immunohistochemical analysis has demonstrated that this gene is highly expressed in endothelial cells in addition to hematopoietic tissues. In this study, gene array analysis of human umbilical vein endothelial cells overexpressing IFI16 revealed an increased expression of genes involved in immunomodulation, cell growth, and apoptosis. Consistent with these observations, IFI16 triggered expression of adhesion molecules such as ICAM-1 and E-selectin or chemokines such as interleukin-8 or MCP-1. Treatment of cells with short hairpin RNA targeting IFI16 significantly inhibited ICAM-1 induction by interferon (IFN)-gamma demonstrating that IFI16 is required for proinflammatory gene stimulation. Moreover, functional analysis of the ICAM-1 promoter by deletion- or site-specific mutation demonstrated that NF-kappaB is the main mediator of IFI16-driven gene induction. NF-kappaB activation appears to be triggered by IFI16 through a novel mechanism involving suppression of IkappaBalpha mRNA and protein expression. Support for this finding comes from the observation that IFI16 targeting with specific short hairpin RNA down-regulates NF-kappaB binding activity to its cognate DNA and inhibits ICAM-1 expression induced by IFN-gamma. Using transient transfection and luciferase assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation, we demonstrate indeed that activation of the NF-kappaB response is mediated by IFI16-induced block of Sp1-like factor recruitment to the promoter of the IkappaBalpha gene, encoding the main NF-kappaB inhibitor. Activation of NF-kappaB accompanied by induction of proinflammatory molecules was also observed when IkappaBalpha expression was down-regulated by specific small interfering RNA, resulting in an outcome similar to that observed with IFI16 overexpression. Taken together, these data implicate IFI16 as a novel regulator of endothelial proinflammatory activity and provide new insights into the physiological functions of the IFN-inducible gene IFI16.     

CD40-mediated activation of NF-kappa B in airway epithelial cells

We have reported previously that airway epithelial cells (AEC) express CD40 and that activation of this molecule stimulates the expression of inflammatory mediators, including the chemokine RANTES (regulated on activation normal T cell expressed and secreted). Because NF-kappaB regulates the expression of many inflammatory mediators, such as RANTES, we utilized CD40-mediated induction of RANTES expression to investigate the mechanisms that underlie CD40-mediated activation of NF-kappaB in AEC. Results demonstrate that, in AEC, intact NF-kappaB sites were required for CD40-mediated activation of the RANTES promoter. To examine activation of NF-kappaB binding directly, electrophoretic mobility shift analyses were performed. These analyses revealed that CD40 ligation stimulated NF-kappaB binding and that the activated NF-kappaB complexes were composed of p65 subunits. Additional studies focused on the CD40-triggered signaling pathways that facilitate NF-kappaB activation. Findings show that CD40 engagement activated the IkappaB kinases IKK-alpha and IKK-beta and stimulated IkappaBalpha phosphorylation. Analyses also examined the role of tumor necrosis factor-associated factor (TRAF) molecules in CD40-mediated NF-kappaB activation within AEC. Stable transfectants expressing wild-type or mutant forms of the cytoplasmic domain of CD40 suggested that TRAF3, but not TRAF2, binding was essential for CD40-mediated RANTES expression. Further studies indicated that exogenous expression of wild-type TRAF3 enhanced activation of the RANTES promoter, whereas exogenous expression of wild-type TRAF2 inhibited this activation; TRAF3-mediated enhancement was dependent upon NF-kappaB. Together, these findings suggest that, in AEC, ligation of CD40 regulates the expression of inflammatory mediators, such as RANTES, via activation of NF-kappaB. Moreover, these results suggest that CD40-mediated signaling in AEC differs with previously reported findings observed in other cell models, such as B lymphocytes.     

IKKgamma inhibits activation of NF-kappaB by NIK

IKKgamma is a component of the IKK complex, which regulates NF-kappaB activity. To investigate the role of IKKgamma, we expressed wild type IKKgamma containing 412 amino acids, and deletion mutants containing residues 1-312 and 101-412, using murine IKKgamma cDNA. In a co-transfection assay with a CAT reporter plasmid, NIK activated NF-kappaB-dependent gene expression approximately two fold and this expression was inhibited by co-transfection of a wild type IKKgamma expression plasmid. In binding assays IKKgamma inhibited the association of IkappaBalpha with IKKbeta and the subsequent phosphorylation of IkappaBalpha that is activated by NIK. Inhibition by IKKgamma also occurred in an assay with a dominant negative mutant of NIK but not with a C-terminal deletion mutant of IKKgamma, indicating that the C-terminal 100 amino acids of IKKgamma are important for negative regulation of NF-kappaB activation. In addition, the interaction of IKKbeta with IKKgamma was inhibited by co-transfection with a NIK expression plasmid. Our results suggest that overexpression of IKKgamma inhibits activation of NF-kappaB by NIK by competing with NIK for interaction with IKKbeta.     

Basic fibroblast growth factor stimulates fibronectin expression through phospholipase C gamma, protein kinase C alpha, c-Src, NF-kappaB, and p300 pathway in osteoblasts

Fibronectin (Fn) is involved in early stages of bone formation and basic fibroblast growth factor (bFGF) is an important factor regulating osteogenesis. bFGF increased Fn expression, which was attenuated by phosphatidylinositol phospholipase inhibitor (U73122), protein kinase C inhibitor (GF109203X), Src inhibitor (PP2), NF-kappaB inhibitor (PDTC), IkappaBalpha phosphorylation inhibitor (Bay 117082), or IkappaB protease inhibitor (TPCK). bFGF-induced increase of Fn-luciferase activity was antagonized by cells transfected with Fn construct without NF-kappaB regulatory site. Stimulation of osteoblasts with bFGF activated IkappaB kinase alpha/beta (IKK alpha/beta) and increased IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 and p50 translocation from the cytosol to the nucleus, the formation of an NF-kappaB-specific DNA-protein complex and kappaB-luciferase activity. bFGF-mediated an increase of IKKalpha/beta activity and DNA-binding activity was inhibited by U73122, GF109203X, or PP2. The binding of p65 to the NF-kappaB element, as well as the recruitment of p300 and the enhancement of p50 acetylation on the Fn promoter was enhanced by bFGF. Overexpression of constitutively active FGF receptor 2 (FGFR2) increased Fn-luciferase activity, which was inhibited by co-transfection with dominant negative (DN) mutants of PLCgamma2, PKCalpha, c-Src, IKKalpha, or IKKbeta. Our results suggest that bFGF increased Fn expression in rat osteoblasts via the FGFR2/PLCgamma2/PKCalpha/c-Src/NF-kappaB signaling pathway.     

Induction of Bcl-xL Expression by Human T-Cell Leukemia Virus Type 1 Tax through NF-κB in Apoptosis-Resistant T-Cell Transfectants with Tax

Human T-cell leukemia virus type 1 (HTLV-1) Tax is thought to play a pivotal role in immortalization of T cells. We have recently shown that the expression of Tax protected the mouse T-cell line CTLL-2 against apoptosis induced by interleukin-2 (IL-2) deprivation and converted its growth from being IL-2 dependent to being IL-2 independent. In this study, we demonstrate that constitutive expression of bcl-xl but not bcl-2, bcl-xs, bak, bad, or bax was associated with apoptosis resistance after IL-2 deprivation in CTLL-2 cells that expressed Tax. Transient-transfection assays showed that bcl-x promoter was transactivated by wild-type Tax. Similar effects were observed in mutant Tax retaining transactivating ability through NF-kappaB. Deletion or substitution of a putative NF-kappaB binding site identified in the bcl-x promoter significantly decreased Tax-induced transactivation. This NF-kappaB-like element was able to form a complex with NF-kappaB family proteins in vitro. Furthermore, Tax-induced transactivation of the bcl-x promoter was also diminished by the mutant IkappaBalpha, which specifically inhibits NF-kappaB activity. Our findings suggest that constitutive expression of Bcl-x(L) induced by Tax through the NF-kappaB pathway contributes to the inhibition of apoptosis in CTLL-2 cells after IL-2 deprivation.     

Modulation of NF-kappa B activity and apoptosis in chronic lymphocytic leukemia B cells

Chronic lymphocytic leukemia (CLL) is an indolent malignancy of CD5+ B lymphocytes. CLL cells express CD40, a key regulator of B cell proliferation, differentiation, and survival. In nonmalignant B cells, CD40 ligation results in nuclear translocation and activation of NF-kappaB proteins. Based on observations that in some CLL cases, the tumor cells express both CD40 and its ligand, CD154 (CD40 ligand), we proposed a model for CLL pathogenesis due to CD40 ligation within the tumor. To evaluate this issue, we used freshly isolated CLL B cells to examine constitutive and inducible NF-kappaB activity by electrophoretic mobility shift assay. We consistently observed high levels of nuclear NF-kappaB-binding activity in unstimulated CLL B cells relative to that detected in nonmalignant human B cells. In each case examined, CD40 ligation further augmented NF-kappaB activity and prolonged CLL cell survival in vitro. The principle NF-kappaB proteins in stimulated CLL cells appear to be quite similar to those in nonmalignant human B cells and include p50, p65, and c-Rel. In a CD154-positive case, blocking CD154 engagement by mAb to CD154 resulted in inhibition of NF-kappaB activity in the CLL cells. The addition of anti-CD154 mAb resulted in accelerated CLL cell death to a similar degree as was observed in cells exposed to dexamethasone. These data indicate that CD40 engagement has a profound influence on NF-kappaB activity and survival in CLL B cells, and are consistent with a role for CD154-expressing T and B cells in CLL pathogenesis. The data support the development of novel therapies based on blocking the CD154-CD40 interaction in CLL.     

Phosphorylation by the protein kinase CK2 promotes calpain-mediated degradation of IkappaBalpha.

Rapid IkappaBalpha turnover has been implicated in the high basal NF-kappaB activity in WEHI 231 B immature IgM(+) B cells. Here we show that treatment of WEHI 231 cells with apigenin, a selective inhibitor of the protein kinase CK2, decreased the rate of IkappaBalpha turnover and nuclear levels of NF-kappaB. Turnover of IkappaBalpha in these cells is mediated in part by the protease calpain. Since both CK2 and calpain target the proline-glutamic acid-serine-threonine (PEST) domain, we investigated the role of CK2 in the degradation of IkappaBalpha by calpain using an in vitro phosphorylation/degradation assay. CK2 phosphorylation enhanced mu-calpain-mediated degradation of wild-type IkappaBalpha, but not of mutant 3CIkappaBalpha, with S283A, T291A, and T299A mutations in phosphorylation sites within the PEST domain. Roles for CK2 and calpain in IkappaBalpha turnover were similarly shown in CH31 immature and CH12 mature IgM(+) B cells, but not in A20 and M12 IgG(+) B cells. These findings demonstrate for the first time that CK2 phosphorylation of serine/threonine residues in the PEST domain promotes calpain-mediated degradation of IkappaBalpha and thereby increases basal NF-kappaB levels in IgM(+) B cells.     

Inhibition of NF-kappaB/Rel induces apoptosis of murine B cells.

Apoptosis of the WEHI 231 immature B cell lymphoma line following membrane interaction with an antibody against the surface IgM chains (anti-IgM) is preceded by dramatic changes in Nuclear Factor-kappaB (NF-kappaB)/ Rel binding activities. An early transient increase in NF-kappaB/Rel binding is followed by a significant decrease in intensity below basal levels. Here we have explored the role of these changes in Rel-related factors in B cell apoptosis. Treatment of WEH1 231 cells with N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), a protease inhibitor which prevents degradation of the inhibitor of NF-kappaB (IkappaB)-alpha, or with low doses of pyrrolidinedithiocarbamate (PDTC) selectively inhibited NF-kappaB/Rel factor binding and induced apoptosis. Bcl-XL expression protected WEHI 231 cells from apoptosis induced by these agents. Microinjection of WEHI 231 cells with either IkappaB-alpha-GST protein or a c-Rel affinity-purified antibody induced apoptosis. Ectopic c-Rel expression ablated apoptosis induced by TPCK or anti-IgM. Treatment of BALENLM 17 and A20 B lymphoma cells or normal murine splenic B lymphocytes with either TPCK or PDTC also resulted in apoptosis. These findings indicate that the drop in NF-kappaB/Rel binding following anti-IgM treatment activates apoptosis of WEHI 231 cells; furthermore, they implicate the NF-kappaB/Rel family in control of apoptosis of normal and transformed B cells.     

A functional role of I kappa B-epsilon in endothelial cell activation

The NF-kappa B inhibitor I kappa B-epsilon is a new member of the I kappa B protein family, but its functional role in regulating NF-kappa B-mediated induction of adhesion molecule expression is unknown. In vascular endothelial cells, I kappa B-epsilon associates predominantly with the NF-kappa B subunit Rel A and to a lesser extent with c-Rel, whereas I kappa B-alpha and I kappa B-beta associate with Rel A only. Following stimulation with TNF-alpha, pyrrolidine dithiocarbamate (PDTC), N-acetylcysteine, and dexamethasone prevented I kappa B kinase-induced I kappa B-alpha, but not I kappa B-beta or I kappa B-epsilon phosphorylation and degradation. Since the activation of NF-kappa B is required for the induction of adhesion molecule expression, we examined the role of I kappa B-epsilon in the transactivation of promoters from VCAM-1, ICAM-1, and E-selectin. Using reporter gene constructs of adhesion molecule promoters, PDTC inhibited VCAM-1 and E-selectin, but to a lesser extent, ICAM-1 promoter activity. Subcloning of kappa B cis-acting elements of VCAM-1, E-selectin, and ICAM-1 into a heterologous promoter construct revealed that PDTC inhibited VCAM-1 and E-selectin, but to a lesser extent, ICAM-1 kappa B promoter activity. By electrophoretic mobility shift assay, NF-kappa B heterodimers containing c-Rel specifically bind to the kappa B motif in the ICAM-1, but not VCAM-1 or E-selectin promoter. Indeed, overexpression of c-Rel induced ICAM-1 kappa B promoter activity to a greater extent than that of E-selectin and overexpression of I kappa B-epsilon inhibited ICAM-1 and VCAM-1 promoter activity in endothelial cells. These findings indicate that c-Rel-associated I kappa B-epsilon is involved in the induction of ICAM-1 expression.