Faculty Bibliography

Interleukin (IL)-18, a newly discovered cytokine produced primarily by macrophages, has been shown to induce gamma interferon (IFN-gamma) production by natural killer cells, to induce the T helper type 1 response. To further elucidate the role of this cytokine in uncomplicated malaria caused by Plasmodium falciparum, serum levels of IL-18, and gamma interferon (IFN-gamma), determined by an immunoenzymatic assay, were analyzed in 40 adult patients, and in 15 healthy control subjects. A significant increase in serum levels of IL-18 was observed in patients with uncomplicated P. falciparum malaria on admission, whereas serum levels of IFN-gamma tended to increase although not significantly. Serum levels of IL-18 decreased three days later, but still remained significantly high, whereas IFN-gamma levels returned to normal levels compared to the controls. No significant correlation was found between parasitemia and serum levels of IL-18 and IFN-gamma. The increase of IL-18 levels during acute and recovery phases of uncomplicated P. falciparum malaria may reflect a proinflammatory role of IL-18 in these patients. An early and effective immune response regulated by proinflammatory Th1 cytokines, including tumor necrosis factor (TNF), interleukin (IL)-12, and possibly IFN-gamma may limit the progression from uncomplicated malaria to severe and life-threatening complications

The highly conserved homeodomains and HMG domains are components of a large number of proteins that play a role in the transcriptional regulation of gene expression during embryogenesis. Both the HMG domain and the homeodomain serve as interfaces for factor interactions with DNA, as well as with other proteins, and it is likely that the high degree of structural and sequence conservation within these domains reflects the conservation of basic aspects of these interactions. Classical HMG domain proteins have an ancient origin, being found in all eukaryotes, and are thought to have given rise to the metazoan-specific class of HMG domain proteins called the Sox proteins. Similarly, the metazoan-specific POU domain proteins are thought to have arisen from genes encoding ancestral homeodomain proteins. In this review, we summarize several examples of different HMG-homeodomain interactions that illustrate not only the ancient origin of each of these protein families, but also their relationship to each other, and discuss how coevolution of HMG and homeodomains may have lead to creation of the specialized Sox/POU protein complexes. Using the FGF-4 gene as an example, we also speculate on how coevolution of regulatory Sox/POU target DNA sequences may have occurred, and how the summation of these changes may have lead to the emergence of new developmental pathways

Kaposi's sarcoma (KS) is an angioproliferative disease characterized by proliferation of neoplastic cells (spindle cells) mixed with endothelial and inflammatory cells. In this study we evaluated the role of the adhesive glycoprotein, fibronectin (FN) and its receptor alpha(5)beta(1) (FNR), and the proto-oncogene bcl-2, an anti-apoptotic protein. Significantly decreased serum levels of FN were noted in HIV-1-infected patients with KS, whereas serum levels of FNR were significantly increased in the same patients. Furthermore, increased FNR expression was observed on CD4 cells from KS patients. Serum levels of bcl-2 protein were significantly decreased in asymptomatic seropositive patients, whereas HIV-1-infected patients with KS showed increased serum levels of bcl-2. These results provide further information about interaction between integrins and the extracellular matrix and bcl-2 protein that can support cell survival either of neoplastic cells or endothelial and inflammatory cells

The Fgf4 gene encodes an important signaling molecule which is expressed in specific developmental stages, including the inner cell mass of the blastocyst, the myotomes, and the limb bud apical ectodermal ridge (AER). Using a transgenic approach, we previously identified overlapping but distinct enhancer elements in the Fgf4 3' untranslated region necessary and sufficient for myotome and AER expression. Here we have investigated the hypothesis that Fgf4 is a target of myogenic bHLH factors. We show by mutational analysis that a conserved E box located in the Fgf4 myotome enhancer is required for Fgf4-lacZ expression in the myotomes. A DNA probe containing the E box binds MYF5, MYOD, and bHLH-like activities from nuclear extracts of differentiating C2-7 myoblast cells, and both MYF5 and MYOD can activate gene expression of reporter plasmids containing the E-box element. Analyses of Myf5 and MyoD knockout mice harboring Fgf4-lacZ transgenes show that Myf5 is required for Fgf4 expression in the myotomes, while MyoD is not, but MyoD can sustain Fgf4 expression in the ventral myotomes in the absence of Myf5. Sonic hedgehog (Shh) signaling has been shown to have an essential inductive function in the expression of Myf5 and MyoD in the epaxial myotomes, but not in the hypaxial myotomes. We show here that expression of an Fgf4-lacZ transgene in Shh-/- embryos is suppressed not only in the epaxial but also in the hypaxial myotomes, while it is maintained in the AER. This suggests that Shh mediates Fgf4 activation in the myotomes through mechanisms independent of its role in the activation of myogenic factors. Thus, a cascade of events, involving Shh and bHLH factors, is responsible for activating Fgf4 expression in the myotomes in a spatial- and temporal-specific manner.

Fibroblast growth factor (FGF)-4 gene expression in the inner cell mass of the blastocyst and in EC cells requires the combined activity of two transcriptional regulators, Sox2 and Oct-3, which bind to adjacent sites on the FGF-4 enhancer DNA and synergistically activate transcription. Sox2 and Oct-3 bind cooperatively to the enhancer DNA through their DNA-binding, high mobility group and POU domains, respectively. These two domains, however, are not sufficient to activate transcription. We have analyzed a number of Sox2 and Oct-3 deletion mutants to identify the domains within each protein that contribute to the activity of the Sox2 x Oct-3 complex. Within Oct-3, we have identified two activation domains, the N-terminal AD1 and the C-terminal AD2, that play a role in the activity of the Sox2 x Oct-3 complex. AD1 also displays transcriptional activation functions in the absence of Sox2 while AD2 function was only detected within the Sox2 x Oct-3 complex. In Sox2, we have identified three activation domains within its C terminus: R1, R2, and R3. R1 and R2 can potentiate weak activation by Sox2 in the absence of Oct-3 but their deletion has no effect on the Sox2 x Oct-3 complex. In contrast, R3 function is only observed when Sox2 is complexed with Oct-3. In addition, analysis of Oct-1/Oct-3 chimeras indicates that the Oct-3 homeodomain also plays a critical role in the formation of a functional Sox2 x Oct-3 complex. Our results are consistent with a model in which the synergistic action of Sox2 and Oct-3 results from two major processes. Cooperative binding of the factors to the enhancer DNA, mediated by their binding domains, stably tethers each factor to DNA and increases the activity of intrinsic activation domains within each protein. Protein-protein and protein-DNA interactions then may lead to reciprocal conformational changes that expose latent activation domains within each protein. These findings define a mechanism that may also be utilized by other Sox x POU protein complexes in gene activation

Fibroblast growth factors (FGF) play a critical role in bone growth and development affecting both chondrogenesis and osteogenesis. During the process of intramembranous ossification, which leads to the formation of the flat bones of the skull, unregulated FGF signaling can produce premature suture closure or craniosynostosis and other craniofacial deformities. Indeed, many human craniosynostosis disorders have been linked to activating mutations in FGF receptors (FGFR) 1 and 2, but the precise effects of FGF on the proliferation, maturation and differentiation of the target osteoblastic cells are still unclear. In this report, we studied the effects of FGF treatment on primary murine calvarial osteoblast, and on OB1, a newly established osteoblastic cell line. We show that FGF signaling has a dual effect on osteoblast proliferation and differentiation. FGFs activate the endogenous FGFRs leading to the formation of a Grb2/FRS2/Shp2 complex and activation of MAP kinase. However, immature osteoblasts respond to FGF treatment with increased proliferation, whereas in differentiating cells FGF does not induce DNA synthesis but causes apoptosis. When either primary or OB1 osteoblasts are induced to differentiate, FGF signaling inhibits expression of alkaline phosphatase, and blocks mineralization. To study the effect of craniosynostosis-linked mutations in osteoblasts, we introduced FGFR2 carrying either the C342Y (Crouzon syndrome) or the S252W (Apert syndrome) mutation in OB1 cells. Both mutations inhibited differentiation, while dramatically inducing apoptosis. Furthermore, we could also show that overexpression of FGF2 in transgenic mice leads to increased apoptosis in their calvaria. These data provide the first biochemical analysis of FGF signaling in osteoblasts, and show that FGF can act as a cell death inducer with distinct effects in proliferating and differentiating osteoblasts