Abstract
The small leucine-rich proteoglycan biglycan is involved in several physiological and pathophysiological processes through the ability of its core protein to interact with other extracellular matrix molecules and transforming growth factor-beta (TGF-beta). To learn more about the regulation of biglycan core protein expression, we have cloned and sequenced 1218 base pairs from the 5'-flanking region of the human biglycan gene, demonstrated functional promoter activity, and investigated the molecular mechanisms through which various agents modulate its transcriptional activity. Sequencing revealed the presence of several cis-acting elements including multiple AP-2 sites and interleukin-6 response elements, a NF-kappaB site, a TGF-beta negative element, and an E-box. The TATA and CAAT box-lacking promoter possesses many features of a growth-related gene, e.g. a GC-rich immediate 5' region, many Sp1 sites, and the use of multiple transcriptional start sites. Transient transfections of the tumor cell lines MG-63, SK-UT-1, and T47D with various biglycan 5'-flanking region-luciferase reporter gene constructs showed that the proximal 78 base pairs are sufficient for full promoter activity. Several agents among them interleukin-6, and tumor necrosis factor-alpha. were capable of altering biglycan promoter activity. However, in MG-63 cells, TGF-beta1 failed to increase either activity of the biglycan promoter constructs or specific transcription from the endogenous biglycan gene. Since TGF-beta1 also did not alter the stability of cytoplasmic biglycan mRNA as determined from Northern analysis after inhibition of transcription with 5,6-dichloro-1beta-D-ribofuranosylbenzimidazole, an as yet unidentified nuclear post-transcriptional mechanism was considered responsible for the TGF-beta effect in this cell type. These results might help to elucidate the molecular pathways leading to pathological alterations of biglycan expression observed in atherosclerosis, glomerulonephritis, and fibrosis.
Highlights
The small leucine-rich pericellular proteoglycan (PG)1 biglycan is composed of a protein core of 331 amino acids and two
The largely inverse mode of regulation is in contrast to the great homology that biglycan and decorin share with respect to general gene structure and, together with the observation that changes in core protein and mRNA levels often parallel each other (22, 28 –30), emphasize the importance of regulatory events at the gene and/or mRNA level in the control of their expression
The discovery of two glucocorticoid response elements at Ϫ312 and Ϫ695 is of interest in the light of recent reports showing that dexamethasone can decrease steady state levels of biglycan mRNA in osteoblasts and bone marrow stromal cells [27] and that it can inhibit the TGF--induced increase of biglycan production and mRNA levels in cultured human skin fibroblasts [42]
Summary
(Received for publication, January 18, 1996, and in revised form, February 28, 1996). To learn more about the regulation of biglycan core protein expression, we have cloned and sequenced 1218 base pairs from the 5-flanking region of the human biglycan gene, demonstrated functional promoter activity, and investigated the molecular mechanisms through which various agents modulate its transcriptional activity. Since TGF-1 did not alter the stability of cytoplasmic biglycan mRNA as determined from Northern analysis after inhibition of transcription with 5,6-dichloro-1-D-ribofuranosylbenzimidazole, an as yet unidentified nuclear post-transcriptional mechanism was considered responsible for the TGF- effect in this cell type. These results might help to elucidate the molecular pathways leading to pathological alterations of biglycan expression observed in atherosclerosis, glomerulonephritis, and fibrosis. While regulation by TGF- appears, at least in human decorin, kb, kilobase(s); Luc, luciferase (reporter); RPA, ribonuclease protection assay; SSPE, standard saline phosphate EDTA; TGF-, transforming growth factor-; TNF-␣, tumor necrosis factor-␣; nt, nucleotide(s)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
