Abstract
Lysyl oxidase (LO) stabilizes the extracellular matrix by cross-linking 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.
Highlights
In addition to the major function in stabilizing the extracellular matrix (ECM), Lysyl oxidase (LO) exhibits other biological activities
Regions Ϫ410/Ϫ1, Ϫ804/Ϫ411, and Ϫ1,336/Ϫ805 displayed 75.4, 38.6, and 24.8% homology, respectively, in sequences of rat, human, and mouse (Fig. 1). These analyses suggest that the proximal promoter region might play a fundamental role in the regulation of LO transcription in vivo
Regulation of LO transcription is a critical control point for the LO gene expression involved in physiology and pathology of the ECM [1]
Summary
In addition to the major function in stabilizing the ECM, LO exhibits other biological activities. Expression of transfected LO cDNA inhibited Ha-ras-induced cell transformation indicating an anti-tumorigenic effect of LO [2]. Our recent studies indicated that cigarette smoke condensate (CSC), the particulate phase of smoke, inhibited synthesis of nascent LO transcripts leading to reduced levels of LO mRNAs in treated rat fetal lung fibroblasts [16]. Nuclear factor I (NFI) was originally described as a factor required for the replication of adenovirus DNA and shown as a transcription factor widely involved in the regulation of constitutive or inducible gene expression, including both transactivation and repression [20]. NFI has been reported to regulate the expression of a wide range of cellular and viral genes such as collagen [24], cytochrome P450 1A1 [21, 25], metallothionein-I [26], mouse mammary tumor virus, etc. To further understand the control of LO gene transcription, we have cloned the 5Ј-flanking region of the rat LO gene, mapped its transcription start sites and core promoter elements, characterized the regulatory activities in transcription of its different subsegments, and demonstrated NFI as a critical transactivator interacting with the cognate cis-element in the promoter region for LO gene activation
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