Cloning and Characterization of the Rat Lysyl Oxidase Gene Promoter

Abstract

Lysyl oxidase (LO) stabilizes the extracellular matrix by crosslinking collagen and elastin. To assess the transcriptional regulation of LO, we cloned the 5-flanking region with 3,979 bp of the rat LO gene. LO transcription started at multiple sites clustered at the region from ؊78 to ؊51 upstream of ATG. The downstream core promoter element functionally independent of the initiator predominantly activated the TATA-less LO gene. 5 Deletion assays illustrated a sequence of 804 bp upstream of ATG sufficient for eliciting the maximal promoter activity and the region ؊709/؊598 exhibiting strongly enhancing effects on the reporter gene expression in transiently transfected RFL6 cells. DNase I footprinting assays showed a protected pattern existing in the fragment ؊612/؊580, which contains a nuclear factor I (NFI)-binding site at the region ؊594/؊580 confirmed by electrophoretic mobility supershift assays. Mutations on this acting site decreased both NFI binding affinity in gel shift assays and stimulation of SV40 promoter activities in cells transfected with the NFI-binding site-SV40 promoter chimeric construct. Furthermore, at least two functional NFI-binding sites, including another one located at ؊147/؊133, were identified in the LO promoter region ؊804/ ؊1. Only NFI-A and NFI-B were expressed in rat lung fibroblasts, and their interaction with the LO gene was sensitively modulated by exogenous stimuli such as cigarette smoke condensate. In conclusion, the isolated rat LO gene promoter contains functionally independent initiator and downstream core promoter elements, and the conserved NFI-binding sites play a critical role in the LO gene activation. Lysyl oxidase (LO)2 (EC 1.4.3.13) is a copper-dependent enzyme secreted by fibrogenic cells such as fibroblasts (1). This enzyme catalyzes the initiation of cross-linking of collagen and elastin, major structural components of the extracellular matrix (ECM), by oxidizing peptidyl lysine residues within these proteins to peptidyl ␣-aminoadipic-␦-semialdehyde, leading to the formation of condensation products stabilizing polymeric collagen or elastin as insoluble fibers. Thus, LO plays a central role in ECM morphogenesis and tissue repair (1).In addition to the major function in stabilizing the ECM, LO also exhibits other biological activities. As reported, expression of transfected LO cDNA inhibited Ha-ras-induced cell transformation indicating an anti-tumorigenic effect of LO (2). LO can oxidize lysine residues in various globular proteins other than collagen and elastin (1). Oxidation of basic fibroblast growth factor (bFGF) by LO blocks the proliferation of bFGFstimulated cells and highly tumorigenic bFGF autocrine-transformed cells (3). Purified mature bovine LO displays chemotactic activity for monocytes and vascular smooth muscle cells (4, 5). LO and its oxidized substrates exist within the nuclei, potentially using histone H1 as a substrate and modulating the promoter activity of the collagen type III gene (6 -8). Increased LO activity is associated with fibrotic...

Lysyl Oxidase, A Critical Intra- and Extra-Cellular Target in the Lung for Cigarette Smoke Pathogenesis

“…Comparative LO gene sequence alignment across species of rat, human and mouse showed that the proximal promoter region is highly conserved whereas the distal promoter region was more divergent among three species suggesting a fundamental role of the proximal promoter region in the regulation of LO transcription. The maximal promoter activities were detected in the 804, 808 and 924 base pair regions, respectively, immediately upstream of ATG in rat, mouse and human LO genes [59]. Thus, the LO transcription control is likely to be regulated by similar elements in these species.…”

“…The INR and the DPE conserved in rat, human and mouse species coordinately function as a single core promoter unit for the RNA-Poly II-directed gene transcription [61]. It should be noted that the DPE in the rat LO gene promoter also worked as an independent core promoter module playing a key role in transactivation of the TATA-less LO gene at multiple transcription starts sites [59]. …”

“…, the cloned rat LO promoter also contains the hypoxia response element (HRE), the metal response element (MRE) and antioxidant response element (ARE), etc. [59]. The cloned rat LO promoter −804/−1 yielding the maximal promoter activity contains three putative NFI-binding sites [59].…”

“…[59]. The cloned rat LO promoter −804/−1 yielding the maximal promoter activity contains three putative NFI-binding sites [59]. NFI binds to the consensus sequence TTGGC(N5)GCCAA (N = any nucleotides) on duplex DNA as a dimer.…”

“…Four cysteine residues are conserved in the DNA binding domain of all rat NFI isoforms sensitive to oxidative damage [63,64]. In addition, the rat LO promoter region −804/−1 also contains four putative hypoxia response elements (HRE, core sequence = RCGTG, R = purine) [65,66] including one on the coding strand and three on the noncoding strand at the region −457/−453, −387/−383, −194/−190 and −37/−33 (relative to ATG) [59], two putative metal response elements (MRE, core sequence = TGCRCNC, R = purine, N = any nucleotide) [67–69] located at −269/−263 and −248/−241, and one antioxidant response element (ARE, core sequence = RTGACNNNGC, R = purine, N = any nucleotides) [70] at the region −581/−572 [59]. The HIF1 is composed of HIF1α and HIF1β subunits [71].…”