No abstract

Nuclear factor-B (NF-B) is a transcription factor critical for key cellular processes, including immune response, apoptosis, and cell cycle progression. A yeast two-hybrid screening, using the Rel homology domain (RHD) of the p65 subunit (RelA) of NF-B as bait, led to the isolation of PIAS3, previously identified as a specific inhibitor of STAT3. We show that PIAS3 can directly associate with p65 using an in vitro pull-down and in vivo coimmunoprecipitation assays. When overexpressed, PIAS3 inhibits NF-B-dependent transcription induced by treatment with tumor necrosis factor ␣ (TNF-␣) or interleukin-1␤ or by overexpression of TNF family receptors such as RANK, TNFR1, and CD30 or signal transducers of TNF receptor-associated factors (TRAFs), including TRAF2, TRAF5, and TRAF6. Downregulation of PIAS3 by RNA interference reverses its effect on TNF-␣-mediated NF-B activation. We found that an N-terminal region of PIAS3 is necessary for both the interaction with p65 and the transcriptional suppression activity. In addition, we found that an LXXLL coregulator signature motif located within the N-terminal region of PIAS3 is the minimal requirement for the interaction with p65. Furthermore, we demonstrate that PIAS3 interferes with p65 binding to the CBP coactivator, thereby resulting in a decreased NF-B-dependent transcription. Taken together, these data suggest that PIAS3 may function in vivo as a modulator in suppressing the transcriptional activity of p65. NF-B1 is an inducible cellular transcription factor that plays a critical role in the expression of a variety of genes involved in immune and inflammatory responses and cell survival (1-3). There are five known members of the mammalian NF-B/Rel family: p65 (RelA), c-Rel, RelB, p50 (NF-B1), and p52 (NF-B2). These proteins share a conserved 300-amino acid region known as the Rel homology domain (RHD), which is responsible for DNA binding, dimerization, and nuclear translocation of NF-B (4 -6). In most cells, Rel family members form hetero-and homodimers with distinct specificities in various combinations. p65, RelB, and c-Rel are transcriptionally active members of the NF-B family, whereas p50 and p52 primarily serve as the DNA binding subunits (4 -6). The most widely studied and most abundant form of NF-B is the heterodimer of p50 and p65.A common feature of the regulation of NF-B is the sequestration in the cytoplasm as an inactive complex physically associated with a class of inhibitory molecules known as IBs (7). Treatment of cells with a variety of inducers such as phorbol esters, interleukin-1 (IL-1), and tumor necrosis factor-␣ (TNF-␣) results in phosphorylation, ubiquitination, and degradation of the IB proteins (2, 8, 9). The degradation of IB proteins exposes the nuclear localization sequence in the remaining NF-B dimers, which in turn leads to a rapid translocation of NF-B to nucleus where it activates target genes by binding to cognate DNA regulatory elements (4 -6).A recent work has shown that NF-B-dependent transcription requires the function of transc...

Aims Proprotein convertase subtilisin/kexin type-9 (PCSK9), a molecular determinant of low-density lipoprotein (LDL) receptor (LDLR) fate, has emerged as a promising therapeutic target for atherosclerotic cardiovascular diseases. However, the precise mechanism by which PCSK9 regulates the internalization and lysosomal degradation of LDLR is unknown. Recently, we identified adenylyl cyclase-associated protein 1 (CAP1) as a receptor for human resistin whose globular C-terminus is structurally similar to the C-terminal cysteine-rich domain (CRD) of PCSK9. Herein, we investigated the role of CAP1 in PCSK9-mediated lysosomal degradation of LDLR and plasma LDL cholesterol (LDL-C) levels. Methods and results The direct binding between PCSK9 and CAP1 was confirmed by immunoprecipitation assay, far-western blot, biomolecular fluorescence complementation, and surface plasmon resonance assay. Fine mapping revealed that the CRD of PCSK9 binds with the Src homology 3 binding domain (SH3BD) of CAP1. Two loss-of-function polymorphisms found in human PCSK9 (S668R and G670E in CRD) were attributed to a defective interaction with CAP1. siRNA against CAP1 reduced the PCSK9-mediated degradation of LDLR in vitro. We generated CAP1 knock-out mice and found that the viable heterozygous CAP1 knock-out mice had higher protein levels of LDLR and lower LDL-C levels in the liver and plasma, respectively, than the control mice. Mechanistic analysis revealed that PCSK9-induced endocytosis and lysosomal degradation of LDLR were mediated by caveolin but not by clathrin, and they were dependent on binding between CAP1 and caveolin-1. Conclusion We identified CAP1 as a new binding partner of PCSK9 and a key mediator of caveolae-dependent endocytosis and lysosomal degradation of LDLR.

TRANCE/TRAF6 signalling governs osteoclastogenesis in vivo.Only the TRANCE receptor (TRANCE-R) has been shown to induce osteoclastogenesis, even though other immune receptors, including CD40 and IL-1R/Toll-like receptor, use TRAF6 to activate overlapping signalling cascades. These observations led us to question whether qualitative or quantitative differences exist between the TRAF6-mediated signals induced by TRANCE and by other ligand-receptor pairs. Here we show that stimulation by overexpressed wild-type CD40 can induce osteoclastogenesis. Stimulation through modified CD40 containing increased numbers of TRAF6-binding sites in the cytoplasmic tails showed a dosedependent increase in the activation of p38 kinase and more pronounced osteoclastogenesis. Moreover, precursors overexpressing TRAF6 differentiate into osteoclasts in the absence of additional signals from TRANCE. Our results suggest that differences in the osteoclastogenesis-inducing capacity of TRANCE-R versus other TRAF6-associated receptors may in part stem from a quantitative difference in the TRAF6-mediated signals.