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 ϳ2.5-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 ϳ90% 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-␤ 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-␣ had no effect and was incapable of counteracting the effects of TGF-␤. Using additional 5 deletions and DNase footprinting analyses, we mapped the TGF-␤ responsive region to a sequence of 177 bp contained between ؊461 and ؊285. This region harbored a 14-bp element similar to a TGF-␤-responsive element present in the promoters of collagen ␣1(I), ␣2(I), elastin, and growth hormone. Electrophoretic mobility shift assays and mutational analyses demonstrated that the perlecan TGF-␤-responsive element bound specifically to TGF-␤-inducible nuclear proteins with high affinity for NF-1 member(s) of transcription factors.

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We report the complete genomic organization of the human WNT-5A gene, which encodes a cysteine-rich growth factor involved in cell-cell signaling during growth and differentiation. The gene comprises five exons with the terminal exon coding for a large 3-untranslated region of Ϸ6.5 kilobase pairs and utilizes multiple polyadenylation signals to generate at least four discrete transcripts. We discovered a new leader exon interrupted by a 411-base pair intron that was retained in our original cDNA cloning. The promoter region was located in a GpC-rich island and harbored numerous cis-acting elements including several GC boxes and Sp1, AP1, and AP2 binding motifs. It lacked TATA or CAAT boxes typical of housekeeping and growth factor genes. In support of this, primer extension revealed two transcription start sites. Transient cell transfection assays showed functional promoter activity for the 3.9-kilobase pair 5-flanking region. Interestingly, internal and 5 deletions revealed that the distal promoter was not required for full transcriptional activity and that the first 631 base pairs of WNT-5A harbored the strongest promoter activity. Using a panel of rodent-human hybrid DNAs carrying portions of chromosome 3p, we mapped the gene to 3p14.2-p21.1, between a constitutional and a familial renal cell carcinoma-associated translocation. In situ hybridization analyses of early human embryos at 28 -42 days of gestation revealed that WNT-5A transcripts were not restricted to the developing brain and limbs but were also observed in the mesenchyme bordering the pharyngeal clefts and pouches and in the developing gonads and kidneys. The relatively high expression in the celomic epithelium and in the precursors of follicles and seminiferous tubules suggest a novel role for WNT-5A in germ-cell differentiation. This study provides the molecular basis for discerning the regulation of the WNT-5A gene and offers the opportunity to investigate genetic disorders linked to this important gene.