Differential regulation of different human papilloma virus variants by the POU family transcription factor Brn-3a.

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 low-risk 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.

Brn-3a neuronal transcription factor functional expression in human prostate cancer.

Neuroendocrine differentiation has been associated with prostate cancer (CaP). Brn-3a (short isoform) and Brn-3c, transcriptional controllers of neuronal differentiation, were readily detectable in human CaP both in vitro and in vivo. Brn-3a expression, but not Brn-3c, was significantly upregulated in >50% of tumours. Furthermore, overexpression of this transcription factor in vitro (i) potentiated CaP cell growth and (ii) regulated the expression of a neuronal gene, the Nav1.7 sodium channel, concomitantly upregulated in human CaP, in an isoform-specific manner. It is concluded that targeting Brn-3a could be a useful strategy for controlling the expression of multiple genes that promote CaP.

EWS differentially activates transcription of the Brn-3a long and short isoform mRNAs from distinct promoters.

Brn-3a long and short isoforms are known to be encoded by two distinct mRNA transcripts derived from a single gene. Here we report that transcription of the two isoforms is differentially regulated. The short isoform has its own promoter, though many elements in the 5' regulatory region are shared. The protein product of the EWS gene, translocations of which are associated with the Ewing's sarcoma family of tumours, is known to interact with Brn-3a via a direct protein-protein interaction. Here we show that EWS also regulates Brn-3a expression in an isoform-specific manner. The implications of these results are discussed in terms of the functional role of EWS and the distinct functional activities of the two isoforms of Brn-3a.

Transcriptional regulation of serum amyloid A1 gene expression in human aortic smooth muscle cells involves CCAAT/enhancer binding proteins (C/EBP) and is distinct from HepG2 cells.

Regulation of acute-phase serum amyloid A (A-SAA) synthesis by proinflammatory cytokines and steroid hormones in human aortic smooth muscle cells (HASMCs) is distinct from that in HepG2 cells. To study the cis- and trans-activating promoter element involved in the SAA1 gene expression by HASMCs and HepG2 cells, we constructed plasmid vectors for luciferase reporter gene assay with varying lengths of SAA1 upstream regulatory region (up to 1431 bp), and examined their response to proinflammatory cytokines and/or steroid hormones. The corresponding vectors with the SAA4 upstream regulatory region served as controls. The presence of proposed transcriptional regulatory factors binding to these regions was confirmed immunohistochemically. The sequences of 1478 and 1836 bp of the SAA1 and SAA4 5'-flanking regions were determined, respectively. SAA1 promoter transcription in cultured HASMCs was upregulated not by proinflammatory cytokines, but rather by glucocorticoids. This differed from HepG2 cells, in which SAA1 promoter transcription was upregulated synergistically by proinflammatory cytokines and glucocorticoids. The promoter activity of a series of truncated SAA1 promoter constructs measured using the reporter gene assay showed that the 5'-region from -252 to -175, containing a consensus site for CCAAT/enhancer binding proteins alpha,beta (C/EBPalpha,beta), was essential for SAA1 induction in HASMCs. In HepG2 cells, the 5'-region from -119 to -79, containing a nuclear factor kappa-B (NFkappaB) consensus sequence, was essential for the induction. The functional significance of the C/EBP site as indicated by the immunohistochemical result was that in HASMCs anti-C/EBPbeta reactivity was shifted from the cytoplasm to the nuclei. We have, therefore, demonstrated that the region containing the C/EBPalpha,beta consensus binding site between the bases -252 and -175 is important for the glucocorticoid-induced SAA1 gene expression in HASMCs but not in HepG2 cells.

The fifth serum amyloid A-related gene sequence, GSAA4, is SAA3.

The human serum amyloid A (SAA) gene family consists of two acute phase genes, SAA1 and SAA2; a pseudogene, SAA3 and a fourth gene, SAA4. The existence of SAA4 was first described in this laboratory. Subsequently, Sack & Talbot isolated a clone, designated GSAA4, which had homology to SAA3. The clone was described as the same as SAA4 and was characterized as a pseudogene. However, our restriction site and sequence analyses of SAA4 demonstrated that SAA4 and GSAA4 are separate entities. SAA4 encodes a functional, constitutively expressed protein. Recently, the GSAA4 clone has been reported by Sellar & Whitehead as constituting a fifth SAA-related sequence. However, we have demonstrated that GSAA4 has striking homology with the 3' terminus of SAA3 in the reverse orientation. Furthermore, computer analyses strongly indicate that the GSAA4 or fifth SAA-related sequence is in fact, a clone from the SAA3 locus. We have amplified, isolated and sequenced fragments from genomic DNA which demonstrate that the GSAA4 sequence is the correct sequence of the 3' region of SAA3. The SAA family must therefore still be viewed as consisting of four SAA loci at present.

Constitutive and NF-kappa B-like proteins in the regulation of the serum amyloid A gene by interleukin 1.

Serum amyloid A (SAA) is a major acute-phase protein whose chronic production by the liver can lead to the fatal disorder of secondary amyloidosis. Control of SAA is mediated by several inflammatory cytokines, including interleukin 1 (IL-1). To study the cis-acting regulatory elements responsible for constitutive and IL-1-induced expression, DNA constructs containing varying lengths of the promoter region from the human SAA2 beta gene 5' to the bacterial reporter gene, chloramphenicol acetyltransferase (CAT), were generated and transfected into human hepatoma cells, HepG2. Both positive and negative regulatory elements were found in the 5' flanking region of the human SAA2 beta gene. The more proximal region contains an IL-1 enhancer sequence GGGACTTTCC (SAA kappa B1; between -82 and -91), the binding site for the ubiquitous transcription factor NF-kappa B. IL-1 induction of the binding of nuclear factor to this sequence is maximal between 5 min and 30 min after incubation with IL-1 and negative in cells incubated for 60 min or longer. Mutation of the SAA kappa B1 sequence to a nonbinding form of NF-kappa B (CTCACTTTCC) abolishes the IL-1 effect. The SAA 5' region also contained an upstream repressor element, shown by transfection experiments. Within this element, a second NF-kappa B binding site (SAA kappa B2; -626 to -635) was found, and mutation of SAA kappa B2 to a non-NF-kappa B-binding form results in an increase in both constitutive + IL-1 stimulated SAA transcription.(ABSTRACT TRUNCATED AT 250 WORDS)