A functional composite cis-element for NF kappa b and RBJ kappa in the rat pregnancy-specific glycoprotein gene.

The rat pregnancy-specific glycoprotein gene rnCGM3 is primarily expressed in the placenta. Previously, three DNase I footprinting sites (FPI, FPII, and FPIII) were identified in the rnCGM3 promoter region, a yeast one-hybrid screen was performed to identify the nuclear factors binding to the FPIII (5'-GCCTGGGAAAAAACTC-3') element, and RBPJ kappa, a downstream effector of the Notch signaling pathway, was identified as one of the FPIII-binding factors. In the present study, the NF kappa B member p65 was identified as another FPIII-binding factor. Electrophoretic mobility shift assays showed that NF kappa B members, including p50 and p65, bound to the FPIII site. The core binding sequence in the FPIII element for p50 and p65 is GGGAAA, which overlaps with that for RBPJ kappa. Competition exists between p50 and RBPJ kappa for binding to the FPIII element. Transient expression analyses revealed that p65 significantly stimulated the expression of a reporter gene directed by the NF kappa B core sequence in the FPIII element. However, RBPJ kappa could block this stimulation. These results suggest that the regulation of rnCGM3 expression involves both NF kappa B and RBPJ kappa, and they are mutually exclusive in the FPIII element.     

Lipopolysaccharide induction of tissue factor gene expression in monocytic cells is mediated by binding of c-Rel/p65 heterodimers to a kappa B-like site.

Exposure of monocytic cells to bacterial lipopolysaccharide (LPS) activates the NF-kappa B/Rel family of proteins and leads to the rapid induction of inflammatory gene products, including tissue factor (TF). TF is the primary cellular initiator of the coagulation protease cascades. Here we report the characterization of a nuclear complex from human monocytic cells that bound to a kappa B-like site, 5'-CGGAGTTTCC-3', in the 5'-flanking region of the human TF gene. This nuclear complex was activated by LPS with kinetics that preceded induction of the TF gene. In vitro binding studies demonstrated that the TF site bound translated c-Rel and p65 homodimers but not p50/p65 heterodimers or p50 homodimers. Base-pair substitutions in the TF site indicated that the presence of a cytosine at position 1 precluded binding of NF-kappa B. In fact, under low-ionic-strength conditions, the TF complex did not migrate with translated p50/p65 dimers but instead comigrated with c-Rel/p65 dimers. Antibodies against the NF-kappa B and Rel proteins and UV cross-linking studies revealed the presence of c-Rel and p65 and the absence of p50 in the TF complex and further showed that c-Rel/p65 heterodimers selectively bound to the TF kappa B-like site. Functional studies indicated that the TF site conferred LPS inducibility on a heterologous promoter and was transactivated by c-Rel or p65. Taken together, our results demonstrated that binding of c-Rel/p65 heterodimers to a novel kappa B-like site mediated LPS induction of TF gene expression in monocytic cells.     

STAT 1 binds to the LPL promoter in vitro

Interferon-gamma (IFNgamma) has been shown to decrease the expression and activity of lipoprotein lipase (LPL). Hence, we searched for IFNgamma sensitive binding sites within the murine LPL promoter. A region of the LPL promoter was identified that specifically binds nuclear, but not cytosolic, extracts isolated from IFNgamma-treated 3T3-L1 adipocytes. EMSA analysis revealed that two protein complexes bind to this site within the LPL promoter and supershift analysis demonstrated that both of these complexes contained STAT 1 proteins. In addition, we have shown that this effect is specific for IFNgamma, since LIF treatment, which also induces STAT 1, did not confer binding to this site. Interestingly, binding to this site within the LPL promoter could be effectively competed with a STAT 1 binding site that we previously identified in the PPARgamma2 promoter. Also, IFNgamma treatment resulted in decreased levels of LPL protein. In summary, we have identified a STAT 1 binding site within the murine LPL promoter which likely plays a role in the IFNgamma induced decrease of LPL expression.