Abstract
Perlecan, a modular heparan sulfate proteoglycan of basement membranes and cell surfaces, plays a crucial role in regulating the assembly of extracellular matrices and the binding of nutrients and growth factors to target cells. To achieve a molecular understanding of perlecan gene regulation, we isolated the 5'-flanking region and investigated its functional promoter activity and its response to cytokines. Transient cell transfection assays, using plasmid constructs harboring the perlecan promoter linked to the chloramphenicol acetyltransferase reporter gene, demonstrated that the largest approximately 2.5-kilobase construct contained maximal promoter activity. This promoter region was functionally active in a variety of cells of diverse histogenetic origin, thus corroborating the widespread expression of this gene product. Stepwise 5' deletion analyses demonstrated that the -461-base pair (bp) proximal promoter retained approximately 90% of the total activity, and internal deletions confirmed that the most proximal sequence was essential for proper promoter activity. Nanomolar amounts of transforming growth factor-beta induced 2-3-fold perlecan mRNA and protein core levels in normal human skin fibroblasts, and this induction was transcriptionally regulated; in contrast, tumor necrosis factor-alpha had no effect and was incapable of counteracting the effects of TGF-beta. Using additional 5' deletions and DNase footprinting analyses, we mapped the TGF-beta responsive region to a sequence of 177 bp contained between -461 and -285. This region harbored a 14-bp element similar to a TGF-beta-responsive element present in the promoters of collagen alpha1(I), alpha2(I), elastin, and growth hormone. Electrophoretic mobility shift assays and mutational analyses demonstrated that the perlecan TGF-beta-responsive element bound specifically to TGF-beta-inducible nuclear proteins with high affinity for NF-1 member(s) of transcription factors.
Highlights
Perlecan, a modular heparan sulfate proteoglycan of basement membranes and cell surfaces, plays a crucial role in regulating the assembly of extracellular matrices and the binding of nutrients and growth factors to target cells
Because most of the synthesized perlecan (Ͼ90%) is released as soluble proteoglycan [3], these results indicate that the transcriptional effects of TGF- are seen at the protein level, albeit at a degree lower than that observed at the mRNA level
The ubiquitous distribution of perlecan correlates well with functional promoter activity detected in cells with disparate histogenetic origin and of different species
Summary
TRANSCRIPTIONAL ACTIVATION BY TRANSFORMING GROWTH FACTOR- VIA A NUCLEAR FACTOR 1-BINDING ELEMENT*. Perlecan binds to fibronectin [16], ␥-interferon [17], and TGF-1 [18], and recent evidence indicates that perlecan can be adhesive for fibroblasts, endothelial cells [19], and chondrocytes [5] while being anti-adhesive for hematopoietic cells [19] These opposing functions are not surprising given the multipurpose structure of the protein core and the complexity of its potential post-translational modifications. In the current investigation we cloned and sequenced 2.5 kb of the upstream region, and, by using transient cell transfection assays and stepwise 5Ј or internal deletions, we demonstrate functional promoter activity of the perlecan gene. Our results indicate that perlecan is up-regulated by TGF- via a transcriptionally mediated control of perlecan promoter and that these effects could be important in the modulation of this proteoglycan during angiogenesis, tissue remodeling, and tumor formation
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