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Sesquiterpene lactones (SLs) have potent anti-inflammatory properties. We have shown previously that they exert this effect in part by inhibiting activation of the transcription factor NF-kappaB, a central regulator of the immune response. We have proposed a molecular mechanism for this inhibition based on computer molecular modeling data. In this model, SLs directly alkylate the p65 subunit of NF-kappaB, thereby inhibiting DNA binding. Nevertheless, an experimental evidence for the proposed mechanism was lacking. Moreover, based on experiments using the SL parthenolide, an alternative mode of action has been proposed by other authors in which SLs inhibit IkappaB-alpha degradation. Here we report the construction of p65/NF-kappaB point mutants that lack the cysteine residues alkylated by SLs in our model. In contrast to wild type p65, DNA-binding of the Cys(38) --> Ser and Cys(38,120) --> Ser mutants is no longer inhibited by SLs. In addition, we provide evidence that parthenolide uses a similar mechanism to other SLs in inhibiting NF-kappaB. Contrary to previous reports, we show that parthenolide, like other SLs, inhibits NF-kappaB most probably by alkylating p65 at Cys(38). Although a slight inhibition of IkappaB degradation was detected for all SLs, the amount of remaining IkappaB was too low to explain the observed NF-kappaB inhibition.

Objective: Polycythemia vera is a clonal stem cell disorder characterized by hyperproliferation of the erythroid, myeloid, and megakaryocytic lineages. While it has been shown that progenitor cells of P. vera patients are hypersensitive to several growth factors including erythropoietin, insulin-like growth factor-1, thrombopoietin, interleukin-3, and granulocyte/monocyte colony-stimulating factor, the molecular pathogenesis of this disease remains unknown. Growth factor hypersensitivity could be mediated by changes in signal transduction pathways. We therefore investigated a common downstream effector of cytokines, the signal transducers and activators of transcription (STATs). A constitutive activation of STAT factors could explain the increased proliferation of P. vera cells even in the absence of growth factor stimulation., Methods: Peripheral granulocytes from patients with P. vera and from healthy volunteers were assayed for STAT1, 3, and 5 DNA binding by electrophoretic mobility shift assay., Results: Four of 14 P. vera patients analyzed showed constitutive STAT3 DNA binding in unstimulated peripheral granulocytes, while none of the 17 healthy volunteers tested did. None of the subjects showed constitutive STAT1 or STAT5 activity. Western blotting demonstrated that, in the three patients, STAT3 is constitutively phosphorylated on Tyr 705, whereas it is unphosphorylated in the other patients and in controls. Interestingly, constitutive STAT3 activity did not correlate with the duration of disease or the treatment regimen. It was observed in a recently diagnosed patient and in two patients treated only with phlebotomy., Conclusion: Our data suggest that constitutive phosphorylation and activation of STAT3 is not a secondary event induced by mutagenizing agents or by prolonged hyperproliferation of hematopoietic cells, but rather represents a primary molecular aberration. Constitutively active STAT3 may contribute to the growth factor hypersensitivity of P. vera cells.

The transcription factor NF-kappa B regulates genes participating in immune and inflammatory responses. In T lymphocytes, NF-kappa B is sequestered in the cytosol by the inhibitor I kappa B-alpha and released after serine phosphorylation of I kappa B-alpha that regulates its ubiquitin-dependent degradation. We report an alternative mechanism of NF-kappa B activation. Stimulation of Jurkat T cells with the protein tyrosine phosphatase inhibitor and T cell activator pervanadate led to NF-kappa B activation through tyrosine phosphorylation but not degradation of I kappa B-alpha. Pervanadate-induced I kappa B-alpha phosphorylation and NF-kappa B activation required expression of the T cell tyrosine kinase p56ick. Reoxygenation of hypoxic cells appeared as a physiological effector of I kappa B-alpha tyrosine phosphorylation. Tyrosine phosphorylation of I kappa B-alpha represents a proteolysis-independent mechanism of NF-kappa B activation that directly couples NF-kappa B to cellular tyrosine kinase.

Post-translational activation of the higher eukaryotic transcription factor NF-kappa B requires both phosphorylation and proteolytic degradation of the inhibitory subunit I kappa B-alpha. Inhibition of proteasome activity can stabilize an inducibly phosphorylated form of I kappa B-alpha in intact cells, suggesting that phosphorylation targets the protein for degradation. In this study, we have identified serines 32 and 36 in human I kappa B-alpha as essential for the control of I kappa B-alpha stability and the activation of NF-kappa B in HeLa cells. A point mutant substituting serines 32 and 36 by alanine residues was no longer phosphorylated in response to okadaic acid (OA) stimulation. This and various other Ser32 and Ser36 mutants behaved as potent dominant negative I kappa B proteins attenuating kappa B-dependent transactivation in response to OA, phorbol 12-myristate 13-acetate (PMA) and tumor necrosis factor-alpha (TNF). While both endogenous and transiently expressed wild-type I kappa B-alpha were proteolytically degraded in response to PMA and TNF stimulation of cells, the S32/36A mutant of I kappa B-alpha remained largely intact under these conditions. Our data suggest that such diverse stimuli as OA, TNF and PMA use the same kinase system to phosphorylate and thereby destabilize I kappa B-alpha, leading to NF-kappa B activation.

The myeloid integrin CD11b is expressed selectively on the surface of mature monocytes, macrophages, granulocytes, and natural killer cells. Tissue-specific and developmentally regulated expression of CD11b is controlled at the level of mRNA transcription, and recent characterization of the human CD11b promoter indicates that the first 92 bp of 5'-flanking DNA are sufficient to direct tissue-specific expression of a reporter gene. Here we show that the sequence AAAAGGAGAAG at base pair -20 of the CD11b promoter binds the proto-oncogene PU.1 in vitro and that mutation of this site significantly reduces the ability of the CD11b promoter to direct expression of a reporter gene in myeloid cells but not in nonmyeloid cells. PU.1 may thus represent a major determinant of the myeloid expression of CD11b.