We have used real-time quantitative reverse transcriptase PCR (TaqMan®) to quantify the expression of the four tissue inhibitor of metalloproteinases (Timp) genes in mouse tissues during development and in the adult. Among the four Timp genes, Timp-4 shows the most restricted pattern of expression, with highest RNA levels in brain, heart and testes. These data indicate that in the brain, Timp-4 transcripts are temporally regulated during development, becoming more abundant than those of the other Timps after birth. Cloning of the Timp-4 gene confirmed a five-exon organization resembling that of Timp-2 and Timp-3, and like all Timps, Timp-4 is located within an intron of a synapsin gene. Ribonuclease protection analysis and 5′-rapid amplification of cDNA ends PCR identified multiple transcription starts for Timp-4 from brain and heart mRNA. The promoter region of Timp-4 was functional in transient transfection analysis in mouse C3H10T1/2 fibroblasts, where it directed basal expression that was non-inducible by serum. The TATA-less promoter contains consensus motifs for Sp1 and an inverted CCAAT box upstream of an initiator-like element that is in close proximity to a transcription start site. Mutation of the CCAAT box caused a 2-fold increase in reporter expression. More significantly, mutation of the Sp1 motif or initiator-like element almost completely abolished reporter expression. This first functional characterization of the Timp-4 promoter shows it to be distinct from other members of the Timp family and provides insights into potential mechanisms controlling the tight spatio-temporal expression pattern of the gene.
Abbreviations used: ECM, extracellular matrix; TIMP, tissue inhibitor of metalloproteinases; MMP, matrix metalloproteinase; MT1-MMP, membrane type-1-MMP; RT-PCR, reverse transcriptase PCR; p.c., post-coitum; RPA, ribonuclease protection assay; MEM, minimal essential medium; FCS, fetal calf serum; EMSA, electrophoretic mobility-shift assay; RACE, rapid amplification of cDNA ends; AP-1, activator protein 1; Inr, initiator-like.
These authors contributed equally to this work.