Transcriptional Regulation of the Human Biglycan Gene

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.

Smad4/DPC4-dependent Regulation of Biglycan Gene Expression by Transforming Growth Factor-β in Pancreatic Tumor Cells

“…The extent of TGF-␤ induction of BGN expression was extremely high in PANC-1 but comparatively low in COLO-357 and Smad4/DPC4-transduced CFPAC-1 cells, the latter exhibiting response rates previously found in other cell types (41). This raised the possibility that the TGF-␤/Smad pathway in COLO-357 and CFPAC-1 cells was not fully active, which could be due to either down-regulation of TGF-␤ receptors and/or overexpression of inhibitory Smads (59,60).…”

“…2 An interesting issue concerns the mechanism of Smad4/DPC4-mediated induction of BGN expression. In contrast to PAI-1 (26, 27) and various collagens (61), the BGN gene is likely not to be a direct Smad target, since 1) no SBE (the consensus sequence is the 8-bp palindrome, GTCTAGAC (38)) is present within the available 1218 bp of human BGN promoter sequence previously reported by us (41) and others (62,63), 2) transfected BGN promoterreporter fusion genes were essentially unresponsive to TGF-␤, 3) the increase in BGN mRNA in PANC-1 by far exceeded the overall TGF-␤-mediated transcriptional activity on the TGF-␤-responsive reporter plasmids p3TP-lux and p6SBE, and 4) the rise in BGN mRNA levels occurred relatively late (10). Stabilization of cytoplasmic mRNA would represent another potential mechanism through which TGF-␤ could increase BGN mRNA.…”

“…We therefore consider it more likely that TGF-␤1 exerts its effect in PANC-1 cells at a nuclear post-transcriptional level (e.g. pre-mRNA processing and/or nuclear export rather than on mature mRNA in the cytoplasm) (41). Regarding the lack of transcriptional induction of the BGN gene, Smad4/DPC4 may initially induce another gene(s) whose protein product(s) then translocates to the nucleus to mediate the BGN mRNA increase.…”

“…PANC-1 and CFPAC-1 cells stably transduced with retroviral expression vectors were cultured in the presence of 700 and 250 g/ml Geneticin (biologically active concentration; Invitrogen), respectively. (41). The following oligonucleotide primers were used: BGNsense (nt 124 -143), 5Ј-CCCTCTCCAGGTCCATCCGC-3Ј; BGN-antisense (nt 623-604), 5Ј-GAGCTGGGTAGGTTGGGCGGG-3Ј; PAI-1-sense (nt 357-378; GenBank TM accession number X04744), 5Ј-CTTCT-TCAGGCTGTTCCGGAGC-3Ј; PAI-1-antisense (nt 1164 -1143), 5Ј-GG-GTCAGGGTTCCATCACTTGG-3Ј; GAPDH-sense, 5Ј-GGCGTCTTCA-CCACCATGGAG-3Ј; GAPDH-antisense, 5Ј-AAGTTGTCATGGATGAC-CTTGGC-3Ј.…”

Regulation of Biglycan Gene Expression by Transforming Growth Factor-β Requires MKK6-p38 Mitogen-activated Protein Kinase Signaling Downstream of Smad Signaling

“…PANC-1 and CFPAC-1 cells stably transduced with recombinant retrovirus were cultured in the presence of 700 g/ml (PANC-1) or 350 g/ml (CFPAC-1) geneticin (biologically active concentration; Invitrogen) or 2.5 g/ml puromycin (Sigma RNA Isolation and Semiquantitative RT-PCR-Total RNA was isolated from cells with RNA Clean (AGS, Heidelberg, Germany) according to the manufacturer's instructions. The general RT-PCR protocol and the oligonucleotide primers used for amplification of BGN and GAPDH mRNAs were described in detail earlier (33,36). For semiquantification of BGN, PAI-1, and GAPDH mRNAs we carried out a competitive approach using gene-specific internal standards (33).…”