Cell Type-independent Manner Research Articles

Mapping the Unique Activation Function 3 in the Progesterone B-receptor Upstream Segment: TWO LXXLL MOTIFS AND A TRYPTOPHAN RESIDUE ARE REQUIRED FOR ACTIVITY

Progesterone receptors (PR) contain three activation functions (AFs) that together define the extent to which they regulate transcription. AF1 and AF2 are common to the two isoforms of PR, PR-A and PR-B, whereas AF3 lies within the N-terminal 164 amino acids unique to PR-B, termed the "B-upstream segment" (BUS). To define the BUS regions that contribute to AF3 function, we generated a series of deletion and amino acid substitution mutants and tested them in three backgrounds as follows: BUS alone fused to the PR DNA binding domain (BUS-DBD), the entire PR-B N terminus linked to its DBD (NT-B), and full-length PR-B. Analyses of these mutants identified two regions in BUS whose loss reduces AF3 activity by more than 90%. These are associated with amino acids 54-90 (R1) and 120-154 (R2). R1 contains a consensus (55)LXXLL(59) motif (L1) identical to ones found in nuclear receptor co-activators. R2 is adjacent to a second nuclear receptor box (L2) at (115)LXXLL(119) and contains a conserved tryptophan (Trp-140). Their mutation completely disrupts AF3 activity in a promoter and cell type-independent manner. Critical mutations elicited similar effects on all three B-receptor backgrounds. This underscores the probability that these mutations alter a process linking BUS structure to the function of full-length PR-B in a fundamental way.

Cell type-dependent and -independent control of HER-2/ neu translation

Overexpression of the HER-2 oncogene occurs in a variety of human tumors, including 25–30% of breast carcinomas, and has been associated with an adverse prognosis. Amplification of the HER-2 gene is frequently detected in tumors, but by itself may not fully account for HER-2 overexpression since transcriptional and post-transcriptional mechanisms also regulate HER-2 protein synthesis. Our studies reveal that the efficiency of HER-2 translation differs between primary and transformed cells. In primary human fibroblasts and human mammary epithelial cells, the HER-2 mRNA is associated with monosome and small polysome fractions. In contrast, in BT474 and MCF-7 human breast cancer cell lines and in COS-7 cells the mRNA co-sedimented with larger polysomes, indicating that it is more efficiently translated in these transformed cells. Northern analysis revealed no detectable mRNA size difference, and nuclease S1 protection and sequence analyses showed no differences between the HER-2 transcript leader in primary cells compared to transformed human cells. The transcript leader in all cell types contains a short upstream open reading frame that is also conserved in other mammalian species. Transient transfection assays revealed that the HER-2 transcript leader repressed downstream translation approximately five-fold in both primary and transformed cells and mutation of the upstream initiation codon alleviated most of the inhibitory effect. These results indicate that HER2 expression is translationally controlled both by a short upstream open reading frame that represses HER-2 translation in a cell type-independent manner, and by a distinct cell type-dependent mechanism that increases translational efficiency of HER-2 in transformed cells.

Molecular analysis of regulation of gene expression of the human erythroid anion exchanger (AE) 1

The anion exchange protein AE1 is the most abundant membrane protein in human erythrocytes mediating the electroneutral chloride/bicarbonate exchange. We identified a promoter region in the 5′ flanking region of the human AE1 gene which controls transcription in a cell type independent manner. In addition a second, distal promoter element mediates gene expression only in erythroid cells and in dependence upon differentiation. Within this distal promoter region we defined a 44 bp sequence containing a novel CT-rich motif with very strong promoter activity whereas a second 28 bp segment suppresses gene expression.

Functional Domains of the Nuclear Receptor Hepatocyte Nuclear Factor 4

The hepatocyte nuclear factor 4 (HNF-4) is a member of the nuclear receptor superfamily and participates in the regulation of several genes involved in diverse metabolic pathways and developmental processes. To date, the functional domains of this nuclear receptor have not been identified, and it is not known whether its transcriptional activity is regulated by a ligand or other signals. In this report, we show that HNF-4 contains two transactivation domains, designated AF-1 and AF-2, which activate transcription in a cell type-independent manner. AF-1 consists of the extreme N-terminal 24 amino acids and functions as a constitutive autonomous activator of transcription. This short transactivator belongs to the class of acidic activators, and it is predicted to adopt an amphipathic alpha-helical structure. In contrast, the AF-2 transactivator is complex, spanning the 128-366 region of HNF-4, and it cannot be further dissected without impairing activity. The 360-366 region of HNF-4 contains a motif that is highly conserved among transcriptionally active nuclear receptors, and it is essential for AF-2 activity, but it is not necessary for dimerization and DNA binding of HNF-4. Thus, HNF-4 deletion mutants lacking the 361-465 region bind efficiently to DNA as homo- and heterodimers and behave as dominant negative mutants. Remarkably, the full transactivation potential of AF-2 is inhibited by the region spanning residues 371-465 (region F). The inhibitory effect of region F on the HNF-4 AF-2 activity is a unique feature among members of the nuclear receptor superfamily, and we propose that it defines a distinct regulatory mechanism of transcriptional activation by HNF-4.

Differential effects of the bicyclic imidazoles on cytokine biosynthesis in human monocytes and endothelial cells

The effects of bicyclic imidazoles on human monocyte and endothelial cell cytokine production were examined. These compounds constitute the CSAIDTM class of anti-inflammatories and are inhibitors of cytokine biosynthesis. The bicyclic imidazoles differ from glucocorticoids and phosphodiesterase inhibitors in their chemical structure as well as pharmacological profile. At optimal concentrations of LPS (50 ng/ml), SK&F 86002, a prototypic compound, inhibited IL-1 and TNF but not g-CSF or IRAP production in human monocytes. At suboptimal concentrations of LPS (50 pg/ml), IL-6 and IL-8 production were also inhibited. Inhibition of cytokine biosynthesis was stimulus independent. For example, induction of IL-1 or TNF expression by phosphatase 1 and 2A inhibitors (Okadaic acid or Calyculin A) and Vitamin D3-dependent induction of IL-1 or TNF was also inhibited. In addition, IL-8 production, but not ICAM/E-Selectin expression in IL-1-stimulated HUVEC, was inhibited at similar IC50s. Taken together, the bicyclic imidazoles inhibit cytokine production selectively in a stimulus and cell type independent manner.