The CBP and p300 co-activators play a key role in many aspects of gene regulation being recruited to the DNA via transcription factors that are targets for specific signaling pathways. It has previously been demonstrated that in neuronal cells the ability of CBP and p300 to activate transcription can be directly stimulated by nerve growth factor or calcium-activated signaling pathways. Here we demonstrate that, in cardiac cells, the activity of CBP and p300 is stimulated by phenylephrine (PE) treatment and that they are required for the activation of atrial naturetic factor (ANF) gene expression by PE. Activation of CBP/p300 by PE involves the p42/p44 MAPK pathway and targets primarily the N terminus of p300 and the C terminus of CBP, which are not homologous to one another. To our knowledge, this is the first report of a specific stimulus modulating the activity of CBP and p300 in cardiac cells and it suggests that these factors play an important role in the hypertrophic effect of PE.The CBP 1 co-activator protein was originally identified as a factor that interacts with the CREB transcription factor only following phosphorylation of CREB on serine 133 (for review see Ref. 1). Thus, following exposure to cAMP, the CREB factor, which is already bound to the cAMP-response element (CRE) in its target genes, is phosphorylated on serine 133 and can then recruit the CBP co-activator, resulting in cAMP-dependent activation of gene expression (1, 2).Although originally identified in the cAMP pathway, it is now clear that CBP and the closely related p300 protein play a key role as co-activators for a wide variety of transcription factors involved in various different pathways, including the steroid/thyroid hormone receptors, AP1, STAT factors, and muscle-specific transcription factors such as MyoD and MEF2

In breast cancer, overexpression of the small heat shock protein, HSP-27, is associated with increased anchorage-independent growth, increased invasiveness, and resistance to chemotherapeutic drugs and is associated with poor prognosis and reduced disease-free survival. Therefore, factors that increase the expression of HSP-27 in breast cancer are likely to affect the prognosis and outcome of treatment. In this study, we show a strong correlation between elevated levels of the Brn-3b POU transcription factor and high levels of HSP-27 protein in manipulated MCF-7 breast cancer cells as well as in human breast biopsies. Conversely, HSP-27 is decreased on loss of Brn-3b. In cotransfection assays, Brn-3b can strongly transactivate the HSP-27 promoter, supporting a role for direct regulation of HSP-27 expression. Brn-3b also cooperates with the estrogen receptor (ER) to facilitate maximal stimulation of the HSP-27 promoter, with significantly enhanced activity of this promoter observed on coexpression of Brn-3b and ER compared with either alone. RNA interference and site-directed mutagenesis support the requirement for the Brn-3b binding site on the HSP-27 promoter, which facilitates maximal transactivation either alone or on interaction with the ER. Chromatin immunoprecipitation provides evidence for association of Brn-3b with the HSP-27 promoter in the intact cell. Thus, Brn-3b can, directly and indirectly (via interaction with the ER), activate HSP-27 expression, and this may represent one mechanism by which Brn-3b mediates its effects in breast cancer cells.

In the antiphospholipid syndrome (APS), antiphospholipid Abs (aPL) bind to anionic phospholipids (PL) and various associated proteins, especially β2-glycoprotein I (β2GPI) and prothrombin. In the present study, we show that altering specific Arg residues in the H chain of a human pathogenic β2GPI-dependent aPL, IS4, has major effects on its ability to bind these clinically important Ags. We expressed whole human IgG in vitro by stable transfection of Chinese hamster ovary cells with expression plasmids containing different VH and VL sequences. VH sequences were derived from IS4 by altering the number of Arg residues in CDR3. VL sequences were those of IS4, B3 (anti-nucleosome Ab), and UK4 (β2GPI-independent aPL). Binding of the expressed H/L chain combinations to a range of anionic, neutral, and zwitterionic PL, as well as prothrombin, β2GPI, dsDNA, and chicken OVA, was determined by ELISA. Of four Arg residues in IS4VH CDR3 substituted to Ser, two at positions 100 and 100g, reduced binding to all Ags, while two at positions 96 and 97 reduced binding to β2GPI but increased or decreased binding to different PL. Eleven of 14 H/L chain combinations displayed weak binding to OVA with Arg to Ser replacements of all four Arg residues enhancing binding to this Ag. Only one H/L chain combination bound neutral PL and none bound dsDNA; hence, these effects are particularly relevant to Ags important in antiphospholipid syndrome. We hypothesize that these four Arg residues have developed as a result of somatic mutations driven by an Ag containing both PL and β2GPI.

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The Brn-3a POU family transcription factor is overexpressed in human cervical carcinoma biopsies and is able to activate expression of the human papilloma virus type 16 (HPV-16) upstream regulatory region (URR), which drives the expression of the E6 and E7 oncoproteins. Inhibition of Brn-3a expression in human cervical cancer cells inhibits HPV gene expression and reduces cellular growth and anchorage independence in vitro as well as the ability to form tumours in vivo. Here, we show that Brn-3a differentially regulates different HPV-16 variants that have previously been shown to be associated with different risks of progression to cervical carcinoma. In human cervical material, Brn-3a levels correlate directly with HPV E6 levels in individuals infected with a high risk variant of HPV-16, whereas this is not the case for a low-risk variant. Moreover, the URRs of high-and intermediate-risk variants are activated by Brn-3a in transfection assays, whereas the URR of a low-risk variant is not. The change of one or two bases in a lowrisk variant URR to their equivalent in a higher-risk URR can render the URR responsive to Brn-3a and vice versa. These results help explain why the specific interplay between viral and cellular factors necessary for the progression to cervical carcinoma only occurs in a minority of those infected with HPV-16.