Ulrich Pessara

Monoclonal antibodies to NF-Y define its function in MHC class II and albumin gene transcription.

NF‐Y is a sequence‐specific DNA‐binding protein which, as a heterodimer, recognizes CCAAT motifs in a variety of transcriptional promoters. We have generated a panel of monoclonal and affinity‐purified polyclonal antibodies directed against various epitopes of NF‐Y. These reagents are highly specific for either of the A or B subunits; we have mapped the epitopes recognized by the monoclonal antibodies to the glutamine‐rich activation domain of NF‐YA. The antibodies inhibit in vitro transcription from the promoters of the albumin gene and of Ea, a class II gene of the major histocompatibility complex. These data definitively demonstrate the role of NF‐Y in regulating the transcription of two tissue‐specific genes whose expression patterns do not overlap. Interestingly, the antibodies cannot inhibit a formed pre‐initiation complex, but do block reinitiation of subsequent rounds of transcription from the same templates.

Urokinase-catalysed cleavage of the urokinase receptor requires an intact glycolipid anchor.

Urokinase (uPA) has the striking ability to cleave its receptor, uPAR, thereby inactivating the binding potential of this molecule. Here we demonstrate that the glycosylphosphatidylinositol (GPI) anchor of uPAR, which is attached to the third domain, is an important determinant in governing this reaction, even though the actual cleavage occurs between the first and second domains. Purified full-length GPI-anchored uPAR (GPI-uPAR) proved much more susceptible to uPA-mediated cleavage than recombinant truncated soluble uPAR (suPAR), which lacks the glycolipid anchor. This was not a general difference in proteolytic susceptibility since GPI-uPAR and suPAR were cleaved with equal efficiency by plasmin. Since the amino acid sequences of GPI-uPAR and suPAR are identical except for the C-terminal truncation, the different cleavage patterns suggest that the two uPAR variants differ in the conformation or the flexibility of the linker region between domains 1 and 2. This was supported by the fact that an antibody to the peptide AVTYSRSRYLE, amino acids 84-94 in the linker region, recognizes GPI-uPAR but not suPAR. This difference in the linker region is thus caused by a difference in a remote hydrophobic region. In accordance with this model, when the hydrophobic lipid moiety was removed from the glycolipid anchor by phospholipase C, low concentrations of uPA could no longer cleave the modified GPI-uPAR and the reactivity to the peptide antibody was greatly decreased. Naturally occurring suPAR, purified from plasma, was found to have a similar resistance to uPA cleavage as phospholipase C-treated GPI-uPAR and recombinant suPAR.

Inhibition of cytosolic phospholipase A2 attenuates activation of mitogen-activated protein kinases in human monocytic cells

Eicosanoids and platelet-activating factor generated upon activation of cytosolic phospholipase A(2) enhance activity of transcription factors and synthesis of proinflammatory cytokines. Here, we show that selective inhibitors and antisense oligonucleotides against this enzyme suppressed expression of the interleukin-1beta gene at the level of transcription and promoter activation in human monocytic cell lines. This inhibitory effect was due to failure of activation of mitogen-activated protein kinases (MAPK) through phosphorylation by upstream mitogen-activated protein kinase kinases (MKK). Consequently, phosphorylation and degradation of inhibitor-kappaBalpha (I-kappaBalpha) and subsequent cytoplasmic mobilization, DNA-binding and the transactivating potential of nuclear factor-kappaB (NF-kB), nuclear factor-interleukin-6 (NF-IL6), activation protein-1 (AP-1) and signal-transducer-and-activator-of-transcription-1 (STAT-1) were impaired. It is concluded, that lipid mediators promote activation of MAPKs, which in turn lead to phosphorylation and liberation of active transcription factors. Since inhibition of cytosolic phospholipase A(2) ameliorates inflammation in vivo, this potency may reside in interference with the MAPK pathway.