Genomic localization of the human genes TAF1A, TAF1B and TAF1C, encoding TAF(I)48, TAF(I)63 and TAF(I)110 subunits of class I general transcription initiation factor SL1.

Human SL1 is a general transcription initiation factor (GTF) essential for RNA polymerase I to start rRNA synthesis at class I promoters. It is comprised of the TATA box-binding protein (TBP) and three TBP-associated factors (TAF(I)48, TAF(I)63 and TAF(I)110). We have determined that the human genes TAF1A, TAF1B and TAF1C, encoding these three TAF(I) polypeptides, are localized at lq42, 2p25 and 16q24, respectively. All three genes are present as single copies in the human genome and map to different chromosomes, as shown by somatic cell hybrid panel and radiation hybrid panel analysis and FISH. Two of these genes, TAF1C and TAF1B, are transcribed into multiple RNAs, as determined through Northern analysis of mRNA from various human organs and cell lines. If translated into different polypeptides, this could result in production of variant isoforms of SL1 with different activation potentials.

Role of interferon regulatory factor 1 in monocyte/macrophage differentiation.

Interferon regulatory factor-1 (IRF-1) has been recognized as an important tumor suppressor and growth regulatory transcription factor, which is also involved in cell differentiation. In this study we investigated the role of IRF-1 in phorbol 12-myristate 13-acetate (PMA)-induced monocyte/macrophage differentiation of human monoblastic U937 cells. For this purpose U937 cells were stably transfected with a vector overexpressing IRF-1 antisense mRNA (U937 IRF-1A cells) and with the SV-40 empty vector (U937-SV40 e.v. cells). We report here that U937 and U937-SV40 e.v. cells differentiated into macrophage-like cells upon PMA stimulation and showed IRF-1 up-regulation. On the contrary, U937 IRF-1A cells stimulated with PMA kept an undifferentiated phenotype and proliferated actively. A direct correlation between induction of IRF-1 and up-regulation of IRF-1 gene targets such as ornithine decarboxylase (ODC) and WAF-1/CIP-1 was also observed in U937 cells. On the other hand U937 IRF-1A cells down-regulated ODC and did not express WAF-1. Results show that IRF-1 plays a pivotal role in PMA-induced monocyte/macrophage differentiation.

Genomics of the human genes encoding four TAFII subunits of TFIID, the three subunits of TFIIA, as well as CDK8 and SURB7.

By in situ chromosomal hybridization, and by somatic cell and radiation hybrid analysis, we have determined the genomic position of the human genes encoding four TAFII subunits of TFIID (TAFII150, TAFII105, TAFII68, TAFII18), the three subunits of TFIIA (TFIIA35 and TFIIA19, both encoded by the same gene, and TFIIA12), CDK8, and SURB7. All of these proteins are bona fide components of human class II holoenzymes as well as targets of signal transduction pathways that regulate genome expression. The genes encoding them are present in the human genome in a single copy and are localized at 8q23, 18q11.2, 17q11.1-11.2, 1p21, 14q31, 15q21-23, 13q12, and 12p12, respectively. We have mapped all of them to chromosomal regions where hereditary genetic diseases have been localized or which are involved in malignancies, which makes them potential candidates for a causal involvement in these phenotypes.

Genomics and transcription analysis of human TFIID.

TFIID, a multisubunit protein comprised of TBP (TATA box-binding protein) and TAF(II)s (TBP-associated factors), has a central role in transcription initiation at class II promoters. TAF(II)s role as mediators of regulatory transcription factors, such as pRb and p53, and their involvement in signal transduction pathways suggest that some may participate in the control of cell proliferation and differentiation: therefore, they could be considered potential protooncogenes or antioncogenes. With the aim of starting to analyse these potential roles, we have determined the genomic position of nine human TAF(II) genes (TAF[II]250, TAF[II]135, TAF[II]100, TAF[II]80, TAF[II]55, TAF[II]43, TAF[II]31, TAF[II]28, TAF[II]20/15) and of two previously unknown sequences related to TAF(II)250 and TAF(II)31, respectively. Except for those encoding TAF(II)250 and TAF(II)31, these genes are present in a single copy and, with the exclusion of those for TAF(II)43 and TAF(II)28 (both at 6p21), are localized in different segments of the genome. Indeed, six of them map to a chromosomal region commonly altered in specific neoplasias, which defines them as candidates for involvement in oncogenesis. Our experiments also demonstrate that TAF(II) transcripts are synthesized ubiquitously, mostly at low levels similar to those of TBP. Interestingly, the amount of the major mRNA species detected by TAF(II)20/15 cDNA is higher, which suggests that the polypeptide it encodes may also perform functions independently of TFIID. TAF(II) isoforms, indicated by additional bands on Northern blots, may play a role in modulation of TFIID function. These data will be useful for analysing variations of TAF(II) mRNA phenotype during cell proliferation, differentiation and development, both normal and pathological.

Activation of nuclear factor-kappa B by beta-amyloid peptides and interferon-gamma in murine microglia.

An increasing body of evidence suggests that amyloid-beta (A beta) peptides and microglia are crucially involved in the pathogenesis of Alzheimer's disease. In an effort to further elucidate the biological effects of A beta towards microglia, we investigated the ability of A beta peptides to activate nuclear factor (NF)-kappa B in the N9 murine microglial cell line. Co-stimulation of microglia with suboptimal concentrations of A beta(25-35) and 100 U/ml IFN gamma resulted in the detection of a specific NF-kappa B DNA-binding activity in nuclear extracts, as determined in gel mobility shift assays. This response required at least 120 min to be evident and supershift experiments revealed that the NF-kappa B complex contains both RelA and p50. Accordingly, immunoblot experiments showed that amongst NF-kappa B/Rel proteins, RelA and p50 are mobilized to the nucleus following microglial cell stimulation with A beta(25-35) plus IFN gamma. Higher concentrations of A beta(25-35) were effective by themselves in inducing NF-kappa B activation, both in the N9 microglial cell line and in rat primary microglia, as well as in human monocytes. For purposes of comparison, microglia were also stimulated with bacterial LPS, a known NF-kappa B inducer. As expected, LPS strongly induced the formation of two NF-kappa B DNA-binding activities, one of which was identified as RelA/p50. The LPS response was also more rapid, as it was already evident by 40 min and remained sustained for up to 3 h. Collectively, these findings indicate that NF-kappa B activation might constitute one of the mechanisms underlying the inducible expression of kappa B-dependent genes in microglia stimulated by A beta peptides and IFN gamma, or by LPS.

Nuclear factor-kappaB activation in human monocytes stimulated with lipopolysaccharide is inhibited by fibroblast conditioned medium and exogenous PGE2.

The nuclear factor kappaB (NF-kappaB) is thought to be crucially involved in the gene activation of several cytokines, including tumor necrosis factor alpha (TNF). Previously, we showed that fibroblast conditioned medium (FCM) is able to inhibit both TNF mRNA accumulation and protein release in peripheral blood-derived human monocytes (PBM) stimulated with lipopolysaccharide (LPS). In this study we have investigated the effect of FCM on the LPS-induced DNA-binding activity of NF-kappaB, by means of electrophoretic shift assay (EMSA). We provide evidence that FCM strongly inhibits the LPS-induced NF-kappaB activation in PBM. Furthermore, we show that exogenous PGE2 mimics the NF-kappaB inhibitory effect of FCM. On the other hand, FCM produced in the presence of indomethacin does not inhibit NF-kappaB activation by LPS. Our results lend further support to the hypothesis that inflammatory and immune responses of monocytes/macrophages may be modulated at the molecular level by signals originating from tissue structural cells such as fibroblasts.

Priming of monocyte respiratory burst by beta-amyloid fragment (25-35).

In the present study we have examined the ability of the beta-amyloid peptide (A beta(25-35)) to modulate the respiratory burst activity of human monocytes in vitro. Incubation of the cells for 24 h with A beta(25-35) as well as with A beta(1-42) resulted in an enhanced production of reactive oxygen radicals (ROI) in response to phorbol 12-myristate 13-acetate (PMA). Such effect was additively increased by coincubation with interferon-gamma (IFN gamma), and was paralleled by modulation of gene and protein expression of some components of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system. Since the effects of A beta(25-35) were also reproduced in primary rat microglia, our findings indicate that A beta(25-35) can potentiate the ability of mononuclear phagocytes to produce ROI, and add further insights into its biological effects.

Beta-amyloid (25-35) peptide and IFN-gamma synergistically induce the production of the chemotactic cytokine MCP-1/JE in monocytes and microglial cells.

The molecular mechanisms involved in the development of senile plaques characteristic of aging and Alzheimer's disease are poorly understood. In this study, we examined whether human monocytes and murine microglial cells stimulated with the active fragment of amyloid beta-protein (Abeta(25-35)) express the monocyte chemotactic protein-1 (MCP-1)/JE. We show that upon incubation with Abeta(25-35), human monocytes accumulate MCP-1 mRNA and produce significant amounts of MCP-1. The effect of Abeta(25-35) on MCP-1 secretion was neither mimicked by a scrambled analogue nor affected by polymyxin B sulfate, even though the latter almost completely abolished the effect of LPS on MCP-1 expression. Murine microglial cells stimulated with Abeta(25-35) also expressed high levels of JE mRNA (the murine counterpart of MCP-1) and released bioactive chemotactic factors. In addition, we report that IFN-gamma significantly synergizes with Abeta(25-35) either in human monocytes or in murine microglial cells, and that Abeta(25-35) plus/minus IFN-gamma-mediated early induction of MCP-1 mRNA does not require new protein synthesis. Finally, we provide evidence that the Abeta(25-35)- and Abeta plus IFN-gamma-induced production of MCP-1 is, in large part, mediated in an autocrine fashion by endogenous TNF-alpha. Taken together, our findings uncover another novel biologic action of Abeta(25-35) and might help in better understanding the mechanisms underlying mononuclear phagocyte recruitment and activation into amyloid deposits.

beta-Amyloid(25-35) induces the production of interleukin-8 from human monocytes.

In an effort to unravel some of the cellular actions of beta-amyloid protein (A beta), we investigated its effects on interleukin-8 (IL-8) production from human monocytes. Supernatants harvested from cultured monocytes stimulated with the neurotoxic fragment 25-35 of beta-amyloid [A beta(25-35)] contained significant amounts of IL-8. Northern blot analysis demonstrated that A beta(25-35) also induced IL-8 mRNA accumulation. The effect of A beta(25-35) on IL-8 mRNA accumulation and secretion was not mimicked by a scrambled A beta(25-35) peptide, and was not affected by polymyxin B sulphate, which, on the other hand, almost completely abolished the effect of lipopolysaccharide. Our results uncover a new biological action of beta-amyloid: that of stimulating the production of a chemokine from monocytes.