The transcription factor NF-κB is regulated by the IκB family of proteins. The nonphosphorylatable, nondegradable superrepressor IκBα (srIκBα) mutant is a potent inhibitor of NF-κB activity when expressed in cells. We generated a form of srIκBα in which its N terminus is fused to the protein transduction domain of HIV TAT (TAT-srIκBα). Purified TAT-srIκBα protein rapidly and efficiently entered HeLa or Jurkat T cells. TAT-srIκBα, when exogenously added to HeLa cells, inhibited in a dose-dependent manner TNF-α- or IL-1β-induced NF-κB activation and binding of NF-κB to its consensus DNA sequence. TAT-srIκBα was coimmunoprecipitated with the p65 subunit of NF-κB, and this interaction was resistant to stimulation with IL-1β. Therefore, TAT-srIκBα-mediated inhibition could result from its nonreversible binding and sequestration of endogenous NF-κB. In contrast, exogenously added TAT-srIκBα did not inhibit IL-1β-induced activation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, or p38 mitogen-activated protein kinases or the phosphorylation and degradation of endogenous IκBα. These results identify a novel way for direct regulation of NF-κB activity in diverse cell types that may be useful for therapeutic purposes.

Using an ELISA approach, we demonstrate that recombinant human IL-12 (rhIL-12) binds strongly to an immobilized heparin-BSA complex. This binding is completely displaceable with soluble heparin, IC50∼ 0.1 μg/ml, corresponding to ∼ 10 nM. By interpolation with our previous findings, this indicates an affinity for heparin greater than that of antithrombin III and comparable with that of FGF-2, two high-affinity heparin-binding proteins. Recombinant murine IL-12 also binds strongly to heparin. The binding of rhIL-12 to heparin shows specificity because chondroitin sulfates A and C fail to compete, whereas chondroitin B inhibits weakly. A highly sulfated heparan sulfate is a strong competitor, whereas other heparan sulfates show weak or no activity. Small heparin fragments inhibit binding, although activity decreases with size. An octasaccharide pool derived by cleavage of heparin with nitrous acid is a significantly stronger inhibitor than its heparinase I-derived counterpart, further indicating structural specificity in the interaction between rhIL-12 and heparin. The binding of recombinant p40 to heparin appears indistinguishable from that of the IL-12 heterodimer, implying that the heparin binding site is largely if not solely located in this subunit. These results show for the first time that IL-12 is a heparin-binding cytokine, a property common to the other Th1-response-inducing cytokines, IFN-γ and IL-2. Our findings strongly suggest that IL-12 will tend to be retained close to its sites of secretion in the tissues by binding to heparin-like glycosaminoglycans, thus favoring a paracrine role for IL-12.