Elsevier

Gene

Volume 233, Issues 1–2, 11 June 1999, Pages 67-74
Gene

cDNA cloning of mouse tumor necrosis factor-α converting enzyme (TACE) and partial analysis of its promoter

https://doi.org/10.1016/S0378-1119(99)00155-9Get rights and content

Abstract

Tumor necrosis factor-α (TNFα) is a cytokine that induces pleiotropic inflammatory reactions. Soluble TNFα is released from its membrane-bound precursor by TNFα converting enzyme (TACE)/a disintegrin and metalloproteinase 17 (ADAM17). We have recently cloned the mouse TACE complete cDNA and the 5′ flanking promoter region of the gene. Two versions of the mouse TACE cDNA having a coding region of 2541 bp were obtained: one was about 4.1 kb and the other was 4.4 kb in length, in which only the length of the 3′ UTR was different. Rapid amplification of 5′ cDNA ends suggested that there were multiple transcriptional start sites. From the coding sequence, 827 amino acids were deduced which were 91.9 % identical to those of human TACE. Northern blot analysis indicated that the major transcript was approx. 4.4 kb product in the mouse. The mouse TACE mRNA was ubiquitously expressed, and was particularly high in the lung. The proximal promoter contained multiple AP2 and Sp1 transcription factor binding sites and included a GC box and a CCAAT box, but lacked a consensus TATA box. Reporter gene analysis using RAW264.7 cells showed that the fragment at nt −290 to −1 from the translation start site has a strong promoter activity, and appeared to be essential for transcription of the mouse TACE mRNA. Finally, we found that the mouse TACE promoter at nt −2304 to −1 also worked in NIH3T3, 3T3L1 and C6 cell lines.

Introduction

Tumor necrosis factor-α (TNFα) plays an important role as a key pathogenic mediator of inflammatory and chronic autoimmune disorders, including rheumatoid arthritis and sepsis (Feldmann et al., 1997, Tracey and Cerami, 1994). The gene for human TNFα encodes a precursor that is inserted into the cell membrane as a polypeptide with a molecular mass of 26 kDa. Conversion of the membrane-bound precursor to a secreted mature protein with a molecular mass of 17 kDa has been thought to be conducted by an unidentified protease, termed TNFα convertase or TNFα processing enzyme (Gearing et al., 1994, McGeehan et al., 1994).

Recently, two independent groups succeeded in cloning an enzyme, which was named TNFα converting enzyme (TACE) (Black et al., 1997, Moss et al., 1997). TACE belongs to a member of a disintegrin and metalloprotease (ADAM) family of protease, and thus TACE is called ADAM17 as another name. Another group reported that ADAM10, which was originally described as an enzyme for degradation of myelin basic protein, also has TACE activity (Lunn et al., 1997). However, TACE knockout cells lose TNFα processing activity, indicating that TACE is responsible for the conversion of the TNFα precursor (Black et al., 1997).

Although many membrane-bound proteins, including cytokines, growth factors and their receptors, are known to be released from the cell by proteolysis, the proteases mediating the specific cleavage of these proteins remain undefined except for TNFα (Hooper et al., 1997). Some groups have reported that TACE also plays an important role for shedding other membrane-bound proteins such as TNF receptor, amyloid protein precursor and transforming growth factor-α (Buxbaum et al., 1998, Peschon et al., 1998). Moreover, it was shown that the activity of TACE in shedding the membrane-bound proteins is essential for mammalian development (Peschon et al., 1998).

Since TACE regulates not only converting TNFα precursor to TNFα, but also shedding other important membrane-bound proteins, it is likely that TACE has physiological roles different from TNFα in the onset and/or progression of several diseases. Accordingly, elucidating mechanisms to regulate TACE expression will allow us to understand the physiological functions of TACE, as well as its role in diseased conditions. We here report cloning the mouse TACE complete cDNA and the 5′ flanking promoter region of the gene, and partial characterization of its promoter.

Section snippets

Subcloning of cDNA

We succeeded in amplification of the mouse TACE cDNA by using the primers originally designed based on the human cDNA sequence (GenBank accession number U69611), TACE-14 (5′ AAG GCT GCC CAG AGA GGT GGA GTC GGT A 3′, sense, nt −42 to −15 numbered from the first residue of the translation initiation codon of the human TACE cDNA) and TACE-18R (5′ CAC TCA CAG CTA TGG GAT AC 3′, antisense, nt 1305 to 1324), and TACE-8 (5′ GGA TTA GCT TAT GTT GGC TCT CCC AGA G 3′, sense, nt 1045 to 1072) and TACE-16R

cDNA cloning

The 4406 bp mouse TACE cDNA was determined by sequencing of the PCR products obtained from 5′ and 3′ RACE and RT–PCR, and assembling these fragments (Fig. 1). The cDNA sequence contained an open reading frame of 2484 bp, and a 5′ UTR of 55–161 bp (described below) and a 3′ UTR of 1455 bp with a poly(A) tail. A polyadenylation site was expected at nt 4097 to 4100. We also found a long form of a 3′ UTR of 1806 bp with a poly (A) tail. But the short form was a major 3′ UTR of the mouse TACE mRNA. A

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The nucleotide sequence data reported in this paper have been deposited with DDBJ/EMBL/GenBank nucleotide sequence database with the accession numbers AB021294 and AB021709.

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