Efficient replication by herpes simplex virus type 1 involves activation of the IkappaB kinase-IkappaB-p65 pathway

Infection by herpes simplex virus type 1 (HSV-1) induces a persistent nuclear translocation of NFkappaB. To identify upstream effectors of NFkappaB and their effect on virus replication, we employed mouse embryo fibroblast (MEF)-derived cell lines with deletions of either IKK1 or IKK2, the catalytic subunits of the IkappaB kinase (IKK) complex. Infected MEFs were assayed for virus yield, loss of IkappaBalpha, nuclear translocation of p65, and NFkappaB DNA-binding activity. Absence of either IKK1 or IKK2 resulted in an 86 to 94% loss of virus yield compared to that of normal MEFs, little or no loss of IkappaBalpha, and greatly reduced NFkappaB nuclear translocation. Consistent with reduced virus yield, accumulation of the late proteins VP16 and gC was severely depressed. Infection of normal MEFs, Hep2, or A549 cells with an adenovirus vector expressing a dominant-negative (DN) IkappaBalpha, followed by superinfection with HSV, resulted in a 98% drop in virus yield. These results indicate that the IKK-IkappaB-p65 pathway activates NFkappaB after virus infection. Analysis of NFkappaB activation and virus replication in control and double-stranded RNA-activated protein kinase-null MEFs indicated that this kinase plays no role in the NFkappaB activation pathway. Finally, in cells where NFkappaB was blocked because of DNIkappaB expression, HSV failed to suppress two markers of apoptosis, cell surface Annexin V staining and PARP cleavage. These results support a model in which activation of NFkappaB promotes efficient replication by HSV, at least in part by suppressing a host innate response to virus infection.     

Bruton's tyrosine kinase is involved in p65-mediated transactivation and phosphorylation of p65 on serine 536 during NFkappaB activation by lipopolysaccharide

Bruton's tyrosine kinase (Btk) has recently been shown to participate in the induction of nuclear factor kappaB (NFkappaB)-dependent gene expression by the lipopolysaccharide (LPS) receptor Toll-like receptor-4 (TLR4). In this study we have examined the mechanism whereby Btk participates in this response. Treatment of the murine monocytic cell line Raw264.7 with LFM-A13, a specific Btk inhibitor, blocked LPS-induced NFkappaB-dependent reporter gene expression but not IkappaB alpha degradation. Transient transfection of HEK293 cells with Btk had no effect on NFkappaB-dependent reporter gene expression but strongly promoted transactivation of a reporter gene by a p65-Gal4 fusion protein. IkappaB alpha degradation activated by LPS was intact in macrophages from X-linked immunodeficiency (Xid) mice, which contain inactive Btk. Transfection of cells with a dominant negative form of Btk (BtkK430R) inhibited LPS-driven p65 mediated transactivation. Additionally LFM-A13 impaired phosphorylation of serine 536 on p65 induced by LPS in HEK293-TLR4 cells, and in Xid macrophages this response was impaired. This study therefore reveals a novel function for Btk. It is required for the signaling pathway activated by TLR4, which culminates in phosphorylation of p65 on serine 536 promoting transactivation by NFkappaB.     

Endogenous FGF‐2 is critically important in PTH anabolic effects on bone

Parathyroid hormone (PTH) increases fibroblast growth factor receptor‐1 (FGFR1) and fibroblast growth factor‐2 (FGF‐2) expression in osteoblasts and the anabolic response to PTH is reduced in Fgf2?/? mice. This study examined whether candidate factors implicated in the anabolic response to PTH were modulated in Fgf2?/? osteoblasts. PTH increased Runx‐2 protein expression in Fgf2+/+ but not Fgf2?/? osteoblasts. By immunocytochemistry, PTH treatment induced nuclear accumulation of Runx‐2 only in Fgf2+/+ osteoblasts. PTH and FGF‐2 regulate Runx‐2 via activation of the cAMP response element binding proteins (CREBs). Western blot time course studies showed that PTH increased phospho‐CREB within 15 min that was sustained for 24 h in Fgf2+/+ but had no effect in Fgf2?/? osteoblasts. Silencing of FGF‐2 in Fgf2+/+ osteoblasts blocked the stimulatory effect of PTH on Runx‐2 and CREBs phosphorylation. Studies of the effects of PTH on proteins involved in osteoblast precursor proliferation and apoptosis showed that PTH increased cyclinD1‐cdk4/6 protein in Fgf2+/+ but not Fgf2?/? osteoblasts. Interestingly, PTH increased the cell cycle inhibitor p21/waf1 in Fgf2?/? osteoblasts. PTH increased Bcl‐2/Bax protein ratio in Fgf2+/+ but not Fgf2?/? osteoblasts. In addition PTH increased cell viability in Fgf2+/+ but not Fgf2?/? osteoblasts. These data suggest that endogenous FGF‐2 is important in PTH effects on osteoblast proliferation, differentiation, and apoptosis. Reduced expression of these factors may contribute to the reduced anabolic response to PTH in the Fgf2?/? mice. Our results strongly indicate that the anabolic PTH effect is dependent in part on FGF‐2 expression. J. Cell. Physiol. 219: 143–151, 2009. © 2008 Wiley‐Liss, Inc.     

Serum response factor controls CYLD expression via MAPK signaling pathway

Tumor suppressor gene CYLD is a deubiquitinating enzyme which negatively regulates various signaling pathways by removing the lysine 63-linked polyubiquitin chains from several specific substrates. Loss of CYLD in different types of tumors leads to either cell survival or proliferation. In this study we demonstrate that lack of CYLD expression in CYLD-/- MEFs increases proliferation rate of these cells compared to CYLD+/+ in a serum concentration dependent manner without affecting cell survival. The reduced proliferation rate in CYLD+/+ in the presence of serum was due to the binding of serum response factor (SRF) to the serum response element identified in the CYLD promoter for the up-regulation of CYLD levels. The serum regulated recruitment of SRF to the CYLD promoter was dependent on p38 mitogen-activated protein kinase (MAPK) activity. Elimination of SRF by siRNA or inhibition of p38 MAPK reduced the expression level of CYLD and increased cell proliferation. These results show that SRF acts as a positive regulator of CYLD expression, which in turn reduces the mitogenic activation of serum for aberrant proliferation of MEF cells.     

Identification of TNF-alpha-responsive NF-kappaB p65-binding element in the distal promoter of the mouse serine protease inhibitor SerpinE2

Serine protease inhibitor SerpinE2 is known as a cytokine-inducible gene. Here, we investigated whether tumor necrosis factor alpha-(TNF-alpha)-induced expression of SerpinE2 is mediated by the nuclear factor-kappaB (NF-kappaB) p65 subunit. Both steady state and TNF-alpha-induced expression of SerpinE2 mRNA were abrogated in p65-/- murine embryonic fibroblasts (MEFs). Reconstitution with wild-type p65 rescued SerpinE2 mRNA expression in an IkappaB kinase beta-dependent manner. Electrophoresis mobility shift assay and ChIP assay demonstrated that p65 bound to the kappaB-like DNA sequence located at approximately -9 kbp in the SerpinE2 promoter. In addition, TNF-alpha stimulated luciferase gene expression driven by the kappaB-like element in the reconstituted MEFs, but not in p65-/- MEFs. These results indicated that activation of NF-kappaB p65 plays an important role in TNF-alpha-induced expression of SerpinE2.     

Up-regulation of NFkappaB-responsive gene expression by DeltaNp73alpha in p53 null cells

Transactivation domain (TAD)-truncated p73, DeltaNp73, associates with p53, resulting in suppression of p53's functions. Using p53 null cell lines, we examined whether or not DeltaNp73 can regulate gene expression in a p53-independent manner. When DeltaNp73alpha was co-transfected with a luciferase reporter plasmid with various enhancer elements, NFkappaB-responsive luciferase gene expression was selectively up-regulated by DeltaNp73alpha, but not by other p73-isoforms with TAD and DeltaNp73beta. Deletion of the TAD endowed p73alpha with the ability to enhance the responsive gene's expression, but deletion of the N-terminal proline-rich domain (PRD) rendered the TAD-deleted p73alpha inactive. Considering the inability of DeltaNp73beta, which is the C-terminus-truncated form of DeltaNp73alpha, to function, these results indicate that both the PRD and C-terminus are necessary for DeltaNp73alpha to can activate NFkappaB-responsive luciferase expression. Over-expression of p53 suppressed the TAD-truncated p73alpha-mediated luciferase expression, suggesting that p53 interferes with the TAD-truncated p73alpha-mediated activation of NFkappaB. Inhibitors for NFkappaB activation reduced the TAD-truncated p73alpha-dependent NFkappaB-responsive gene expression, indicating that TAD-truncated p73alpha activates NFkappaB as does TNFalpha. In addition to the results obtained in the reporter gene assay, TAD-truncated p73alpha stimulated the translocation of NFkappaB to the nucleus and the expression of an endogenous NFkappaB-responsive gene, Bcl-XL. Taken together, these results demonstrate that TAD-truncated p73alpha can activate NFkappaB.     

In mouse embryonic fibroblasts, neither caspase-8 nor cellular FLICE-inhibitory protein (FLIP) is necessary for TNF to activate NF-κB, but caspase-8 is required for TNF to cause cell death, and induction of FLIP by NF-κB is required to prevent it

Binding of TNF to TNF receptor-1 can give a pro-survival signal through activation of p65/RelA NF-κB, but also signals cell death. To determine the roles of FLICE-inhibitory protein (FLIP) and caspase-8 in TNF-induced activation of NF-κB and apoptosis, we used mouse embryonic fibroblasts derived from FLIP and caspase-8 gene-deleted mice, and treated them with TNF and a smac-mimetic compound that causes degradation of cellular inhibitor of apoptosis proteins (cIAPs). In cells treated with smac mimetic, TNF and Fas Ligand caused wild-type and FLIP(-/-) MEFs to die, whereas caspase-8(-/-) MEFs survived, indicating that caspase-8 is necessary for death of MEFs triggered by these ligands when IAPs are degraded. By contrast, neither caspase-8 nor FLIP was required for TNF to activate p65/RelA NF-κB, because IκB was degraded, p65 translocated to the nucleus, and an NF-κB reporter gene activated normally in caspase-8(-/-) or FLIP(-/-) MEFs. Reconstitution of FLIP(-/-) MEFs with the FLIP isoforms FLIP-L, FLIP-R, or FLIP-p43 protected these cells from dying when treated with TNF or FasL, whether or not cIAPs were depleted. These results show that in MEFs, caspase-8 is necessary for TNF- and FasL-induced death, and FLIP is needed to prevent it, but neither caspase-8 nor FLIP is required for TNF to activate NF-κB.