Faculty Bibliography

A human PGHS-2 promoter fragment (300 BP) linked to the luciferase reporter was used to study the regulation of PGHS-2 gene expression in human amnion-derived WISH cells. A cyclic AMP (cAMP) response element (CRE) was found to be important in the induction of PGHS-2 gene expression. This was demonstrated by showing that coexpression of CREB stimulated native but not CRE mutant promoter and that IL-1beta and PMA induced less activity with the mutant promoter as compared to the native promoter. The effect of dexamethasone on IL-1beta and PMA induced promoter activities was further examined. IL-1beta or PMA induced activity was blocked by dexamethasone, whereas IL-1beta or PMA induced mutant activity was not responsive to dexamethasone. Direct activation of CRE by a cAMP elevating agent, isoproterenol, was found to be inhibited significantly dexamethasone. These results suggest that CRE may mediate the induction of PGHS-2 by IL-1beta and PMA as well as the suppression of expression by dexamethasone in amnion-derived cells.

CREB-binding proteins (CBP) and p300 are essential transcriptional coactivators for a large number of regulated DNA-binding transcription factors, including CREB, nuclear receptors, and STATs. CBP and p300 function in part by mediating the assembly of multiprotein complexes that contain additional cofactors such as p300/CBP interacting protein (p/CIP), a member of the p160/SRC family of coactivators, and the p300/CBP associated factor p/CAF. In addition to serving as molecular scaffolds, CBP and p300 each possess intrinsic acetyltransferase activities that are required for their function as coactivators. Here we report that the adenovirus E1A protein inhibits the acetyltransferase activity of CBP on binding to the C/H3 domain, whereas binding of CREB, or a CREB/E1A fusion protein to the KIX domain, fails to inhibit CBP acetyltransferase activity. Surprisingly, p/CIP can either inhibit or stimulate CBP acetyltransferase activity depending on the specific substrate evaluated and the functional domains present in the p/CIP protein. While the CBP interaction domain of p/CIP inhibits acetylation of histones H3, H4, or high mobility group by CBP, it enhances acetylation of other substrates, such as Pit-1. These observations suggest that the acetyltransferase activities of CBP/p300 and p/CAF can be differentially modulated by factors binding to distinct regions of CBP/p300. Because these interactions are likely to result in differential effects on the coactivator functions of CBP/p300 for different classes of transcription factors, regulation of CBP/p300 acetyltransferase activity may represent a mechanism for integration of diverse signaling pathways

Patients with pemphigus foliaceus (PF) have blisters on skin, but not mucous membranes, whereas patients with pemphigus vulgaris (PV) develop blisters on mucous membranes and/or skin. PF and PV blisters are due to loss of keratinocyte cell-cell adhesion in the superficial and deep epidermis, respectively. PF autoantibodies are directed against desmoglein (Dsg) 1; PV autoantibodies bind Dsg3 or both Dsg3 and Dsg1. In this study, we test the hypothesis that coexpression of Dsg1 and Dsg3 in keratinocytes protects against pathology due to antibody-induced dysfunction of either one alone. Using passive transfer of pemphigus IgG to normal and DSG3(null) neonatal mice, we show that in the areas of epidermis and mucous membrane that coexpress Dsg1 and Dsg3, antibodies against either desmoglein alone do not cause spontaneous blisters, but antibodies against both do. In areas (such as superficial epidermis of normal mice) where Dsg1 without Dsg3 is expressed, anti-Dsg1 antibodies alone can cause blisters. Thus, the anti-desmoglein antibody profiles in pemphigus sera and the normal tissue distributions of Dsg1 and Dsg3 determine the sites of blister formation. These studies suggest that pemphigus autoantibodies inhibit the adhesive function of desmoglein proteins, and demonstrate that either Dsg1 or Dsg3 alone is sufficient to maintain keratinocyte adhesion.

Previous observations have established that varicella-zoster virus (VZV) is enveloped in the trans-Golgi network (TGN) in cultures infected with VZV and that the glycoprotein gE is targeted to the TGN by a signal sequence (AYRV) and an acidic TGN signal patch in its cytosolic domain. Neither sequence is present in other VZV glycoproteins. Like gE, gI was targeted to the TGN when it was expressed in transfected cells, suggesting that gI also contains TGN targeting information (colocalized with gE and the AP-1 adaptin complex). In contrast, gB, gC, gH, and gL immunoreactivities were not detected in the TGN when they were expressed individually in transfected cells. In VZV-infected cells, gE, gI, gH, and gL were all concentrated in the TGN. Since VZV glycoproteins that lack targeting sequences (gB, gC, gH, and gL) concentrated in the TGN of infected cells, it is proposed that gE and gI, which have such sequences, serve as navigator glycoproteins, forming complexes that direct the signal-deficient glycoproteins to the TGN

Interleukin-1beta (IL-1beta) stimulated PGE2 synthesis in human amnion derived WISH cells, whereas dexamethasone blocked IL-1beta-mediated stimulation of PGE2 production. Sequence analysis of the 5'-flanking region of the human prostaglandin H synthase-2 (PGHS-2) gene indicates two putative NF-kB binding sites. Mutation of a single site or both sites resulted in significantly decreased activity of the PGHS-2 promoter. IL-1beta treatment increased significantly the native promoter activity and this increase was attenuated by using the NF-kB-mutant promoter. Dexamethasone treatment also decreased the IL-1beta mediated stimulation of the PGHS-2 native promoter but not the NF-kB mutant promoter. Furthermore, the involvement of the NF-kB was supported by electrophoretic mobility shift assay which revealed an increased nuclear binding of the NF-kB probe upon IL-1beta induction and a decreased nuclear binding of the NF-kB probe upon dexamethasone pre-treatment. These results provide convincing evidence that NF-kB may mediate the IL-1beta stimulation of PGHS-2 gene expression as well as the dexamethasone inhibition of the IL-1beta induction process in WISH cells.

To explore the antitumor mechanism of bacterial cytosine deaminase plus 5-fluorocytosine (CD/5-FCyt) in combination with interferons (IFNs), glioma cells were transduced with recombinant retroviruses expressing CD. The transduced glioma cells become sensitive to the nontoxic prodrug 5-FCyt. Apoptosis, DNA damage, bystander effect, and inhibition of thymidylate synthase (TS) and DNA synthesis are associated with CD/5-FCyt-mediated glioma cell killing. Furthermore, IFNs enhance this effect by increasing DNA damage and further inhibiting TS activity. The bystander effect is mediated by the release of cytotoxic metabolites of 5-FCyt into the extracellular milieu triggering apoptosis and DNA damage. Our data indicate that the use of CD/5-FCyt in combination with IFNs may provide a more effective approach for the treatment of brain tumors

To explore the antitumor effect of insulin-like growth factor 1 (IGF-I) antisense RNA and the interaction of IGF-I with insulin-like growth factor-binding proteins (IGFBPs) in glioma cells, a recombinant retrovirus expressing IGF-I antisense RNA was constructed and introduced into C6 glioma cells. IGF-I antisense RNA reverses the transformed phenotype in glioma cells and inhibits glioma cell growth by blocking overexpression of endogenous IGF-I. Expression of IGFBP-2 is increased in glioma cells as compared with normal adult glial cells. IGF-I antisense RNA also inhibits expression of IGFBP-2 in glioma cells, but does not influence expression of the other IGFBPs. Although IGFBP-2 in conditioned medium from wild-type C6 cell cultures itself does not directly influence glioma cell growth, it synergistically enhances exogenous IGF-I-mediated DNA synthesis in IGF-I-negative C6 cells. These findings indicate the inhibitory effect of IGF-I antisense RNA on growth and development of glioma cells. IGF-I-dependent glioma cell growth may, in some circumstances, require IGFBP-2 as a cofactor. The antitumor effect of IGF-I antisense RNA is also associated with inhibition of IGFBP-2 expression

Human amnion-derived WISH cells synthesize little prostaglandin E2 at the basal state. However, the cells were stimulated greatly to synthesize prostaglandin E2 by interleukin-1beta in a dose-dependent manner. Stimulation by interleukin-1beta was synergistically increased by pretreatment of the cells with interferon alpha or gamma, which alone were inactive. Synergism by interferons was dose-dependent. Stimulation by interleukin-1beta, on the contrary, was inhibited by preincubation of the cells with interleukin-4. Inhibition by interleukin-4 was also dose-dependent. Regulation of prostaglandin E2 synthesis by cytokines was further examined at the m-RNA level of cyclooxygenase-2. Quantitative reverse transcription-polymerase chain reaction indicated that the m-RNA level was not increased by interferon-gamma but was synergistically increased by interferon-gamma plus interleukin-1beta. Furthermore, the m-RNA level increased by interleukin-1beta was attenuated by interleukin-4. These results indicate that regulation of interleukin-1beta-stimulated prostaglandin E2 synthesis by interferons and interleukin-4 is controlled at the m-RNA level of cyclooxygenase-2.