Interaction of IL-3 with its receptor is known to activate STAT-3 via phosphorylation of Tyrosine 701, which facilitates its dimerization and translocation to the nucleus, leading to the transcription of its target genes. In this communication, we have investigated the nature of tyrosine kinases that mediate STAT-3 phosphorylation during IL-3-mediated activation of myeloid cell proliferation. Our results show that interaction of IL-3 with its receptor leads to the activation of c-Src kinase activity, which in turn facilitates the binding of c-Src to STAT-3. This association leads to the phosphorylation of STAT-3, allowing this transcription factor to translocate to the nucleus. Expression of a dominant negative mutant of src (AMSrc) in these cells results in a block to IL-3 mediated phosphorylation of STAT-3, and its ability to bind to DNA. On the other hand, expression of a dominant negative mutant of JAK2 (JAK2KE) had no eect on IL-3-mediated activation of STAT-3. Our results also show that AMSrc does not aect the phosphorylation of JAK2, suggesting that JAK and STAT phosphorylation events are mediated by two independent pathways. Inhibition of c-Src activation by AMSrc, which leads to a block to STAT-3 activation, results in a dramatic inhibition of cell proliferation mediated by IL-3. However, expression of AMSrc does not activate apoptotic pathways. In contrast, expression of JAK2KE results in accelerated apoptosis of 32Dcl3 cells grown in the absence of IL-3 with concomitant down-regulation of Erk-2 kinase activity. These results suggest that Src family kinases mediate the phosphorylation of STATs and play a critical role in signal transduction pathways associated with myeloid cell proliferation while JAK kinases mediate the activation of Erk-2 pathway which appears to provide antiapoptotic signals. Thus the activation of JAKs and STATs appear to be two independent but related events, which dictate two separate biological outcomes, the combination of which results in proliferation and survival of myeloid precursor cells.

Cyclooxygenase-2 (COX-2) inhibitors are promising anticancer agents but their long-term use at high doses is associated with adverse cardiovascular events. The molecular mechanisms underlying the anticancer or toxic cardiovascular effects of COX-2 inhibitors remain unknown. Here we report that COX-2-selective celecoxib and a novel COX-2 inhibitor ON09310 upregulate death receptor 5 (DR5) and cooperate with tumor necrosis factor-related apoptosisinducing ligand (TRAIL), the ligand for DR5, to induce apoptosis in COX-2-positive and -negative cancer cells. We also show that both agents engage GADD153/CHOP to transcriptionally upregulate DR5 expression; GADD153/ CHOP is a C/EBP homologous transcription factor implicated in cellular stress response and apoptosis. Based on our results, we propose that (1) these agents appear to mediate their effects, at least in part, by engaging GADD153/CHOP to activate DR5-dependent apoptotic pathway and (2) their regulation of GADD153/CHOP and DR5 expression appears to occur independent of their COX-2 inhibitory effects. Our results also indicate that ON09310 is generally more potent than celecoxib and, at lower concentration, strongly cooperates with TRAIL to induce apoptosis. Taken together, our findings form the basis for future in-depth studies to further explore the utility of TRAIL and/or agonistic anti-DR5 antibodies in combination with low-dose COX-2 inhibitors as a rational approach for cancer prevention and treatment.

Rheumatoid Arthritis (RA) is a chronic, autoimmune disease that affects a vast population worldwide with women being afflicted three times more than men. There is evidence for an increased risk of cardiovascular events in RA patients compared to the general population. These cardiovascular events may be associated with the chronic inflammatory state in which activation of coagulation leads to thrombin generation. Our laboratory has evidence that thrombospondin-1 (TSP1), an adhesive molecule that plays a major role in RA, promotes thrombin generation on the surface of a monocytic cell line (Isordia-Salas et al, Thromb Res.2005;116(6)). We also have documented that disrupting the TSP1 interaction on human neutrophils prevents the development of erosive arthritis in an experimental animal model (Manns et al, Arthritis and Rheumatism54(8), 2006). This observation is mediated by a novel pathway whereby TSP1 induces the up-regulation of Connective Tissue Growth Factor (CTGF). Therefore, to assess whether our in vitro and in vivo observations can be extrapolated to human disease, blood samples were collected from 20 patients afflicted with rheumatoid arthritis and 13 from healthy donors which served as the negative control. Plasma samples were separated and analyzed by Enzyme-Linked Immunosorbent Assay (ELISA) to determine the levels of transforming growth factor beta (TGF-β), protrombin F1+2 fragments (F1+2) and thrombospondin-1, and by multiplexed cytokine protein profiling on microarray by rolling-circle amplification (RCA) to determine cytokine levels. The F1+2 plasma levels showed an elevated trend in the RA group (p=0.06). TSP1 plasma levels were significantly increased in the RA group compared to the normal control (p=0.0004). Pro-inflammatory cytokine levels including IL-1β (p=0.0365), IL-6 (p=0.0029), TNF-α (p=0.0339), interferon-inducible protein 10 (p=0.0003) and macrophage inflammatory protein -1α (p=0.0012) were found elevated in the RA group compared to the normal control group. Some regulatory cytokines such as transforming growth factor-β (0.06), interferon-gamma (p=0.06) and IL-13 (p=0.22) showed no statistically significance between groups, but all of them showed a trend for higher circulation levels in plasma in the RA group. In summary, comparison between normal individuals and RA patients showed an increase in the levels of cytokines in the RA afflicted patients confirming what has been reported in the literature. We were able to correlate an increase in proteolytic factors and TSP1 levels in the RA patients with an increase of pro-inflammatory cytokines. Further studies are needed to elucidate why TSP1 acts as a pro-inflammatory molecule on the neutrophil surface of the RA patients.