Promoter Structure and Transcriptional Activation of the Murine TSG-14 Gene Encoding a Tumor Necrosis Factor/Interleukin-1-inducible Pentraxin Protein

Abstract

Human TNF-stimulated gene 14 (TSG-14) encodes a secreted 42-kDa glycoprotein that shows significant homology to proteins of the pentraxin family, which includes the acute phase reactants C-reactive protein and serum amyloid P component. Levels of TSG-14 protein (also termed PTX-3) become elevated in the serum of mice and humans after injection with bacterial lipopolysaccharide, but in contrast to conventional acute phase proteins, the bulk of TSG-14 synthesis in the intact organism occurs outside the liver. In the present study we cloned and partially sequenced murine genomic TSG-14 DNA. Analysis of the coding region predicts a high degree of amino acid sequence homology between murine and human TSG-14 (88 and 75% identity in the first and second exons, respectively). The promoter of the TSG-14 gene lacks consensus sequences for either a TATA box or CCAAT box. Primer extension analysis and S1 nuclease protection assay revealed one major transcription start site, situated within a consensus sequence for an initiator element. Sequence analysis of a ϳ1.

Differential regulation of TSG-14 expression in murine fibroblasts and peritoneal macrophages

“…Induction of TSG-14 mRNA expression by LPS in cultures of primary human macrophages and by both TNF and IL-1 in several human myelomonocytic cell lines was reported [12]. TSG-14 expression could not be demonstrated in several murine macrophage cell lines [14], but the regulation of TSG-14 expression by cytokines in primary murine macrophages has not been investigated. TSG-14 is not readily detectable in tissues from healthy adult mice, but upon LPS injection TSG-14 protein levels in the serum rise with kinetics similar to other acute phase proteins [3,11].…”

“…The B site in the human and murine TSG-14 promoters was previously shown to be necessary for TSG-14 expression in reporter assays in transiently transfected fibroblasts [14,34]. To assess the requirement for NF-B activation in the induction of TSG-14 in murine macrophages, cells were stimulated with LPS and simultaneously treated with the antioxidant PDTC, which prevents NF-B activation by inhibiting degradation of I B, the negative regulator of NF-B [35].…”

“…Protein concentrations were quantified using the Bradford assay (Bio-Rad). Oligonucleotides 5'AAT CCA GGG GAA CTC CCT CGC GC3' and 5'GCG CGA GGG AGT TCC CCT GGA TT3' (GeneLink, Thornwood, NY), corresponding to the previously identified NF-B site in the murine TSG-14 promoter at position Ϫ40 to Ϫ30 [14], were synthesized and annealed to each other. Probes were labeled by T4 kinase (New England BioLabs, Beverly, MA) with [␥-32 P]ATP (New England Nuclear, Boston, MA) and purified in a native acrylamide gel before use.…”

“…A murine TSG-14 probe was generated using a portion of the second exon previously isolated during genomic cloning [14] or with the full-length murine cDNA obtained from Alberto Mantovani, Istituto Mario Negri, Milan, Italy. Murine guanylate binding protein (GBP) cDNA was obtained from Donna Paulnock, University of Wisconsin-Madison Medical School, Madison, WI [19].…”

Characterization of the Promoter for the Human Long Pentraxin PTX3

“…While this article was in preparation, the sequence of the mPTX3 became available (23), and the alignment shows an overall 50.1% conservation, with the last 380 nucleotides showing a 66% conservation (Fig. 1A).…”