Biglycan mRNA Research Articles

Increase in the Expression of Biglycan mRNA Expression Co-localized Closely with that of Type I Collagen mRNA in the Infarct Zone After Experimentally-Induced Myocardial Infarction in Rats

Biglycan, a small dermatan sulphate proteoglycan, has been postulated to interact with other components of the extracellular matrix (ECM), specifically collagens. We hypothesized that biglycan messenger ribonucleic acid (mRNA) is increased in the myocardial infarct zone. Biglycan mRNA expression after acute myocardial infarction (AMI) in rats was determined with the use of Northern blotting andin situhybridization, and its expression pattern was compared to that of type I collagen mRNA [α1(I) collagen]. The left coronary artery was ligated in male Sprague–Dawley rats, and the hearts were excised on days 2 and 7. The Northern blot analysis demonstrated that expression of biglycan mRNA in the infarct on days 2 and 7 were 4.0- and 6.8-fold higher, respectively, compared to the sham-operated hearts. Thein situhybridization revealed intense signals for both biglycan andα1(I) collagen mRNA on day 2 in the spindle-shaped mesenchymal cells located between the surviving myocytes in the infarct peripheral zone. On day 7, biglycan mRNA signals were observed in the interior of the infarct around the infarct granulation tissue, a distribution that was essentially the same as that ofα1(I) collagen. These results demonstrated that the increases in the infarct biglycan mRNA expression produced by mesenchymal cells (presumably myofibroblasts and fibroblasts) was closely co-localized with that of type I collagen mRNA, indicating that biglycan contributes to the infarct healing processes.

Growth factor modulation of mitogenic responses and proteoglycan synthesis by human periodontal fibroblasts.

In order to understand the relationship between specific growth factors and matrix synthesis by periodontal cells, we have investigated the effects of platelet-derived growth factor BB (PDGF-BB), insulin-like growth factor-I (IGF-1), and growth hormone on DNA and proteoglycan synthesis by cultured human gingival and periodontal ligament fibroblasts in vitro. PDGF-BB and IGF-1, but not growth hormone, were mitogenic for both periodontal ligament fibroblasts and gingival fibroblasts, although the periodontal ligament cells responded more strongly. The mitogenic response was accompanied by alterations in expression of matrix proteoglycan mRNA. For both the gingival and periodontal ligament cells, there was a decrease in mRNA for decorin and an increase in mRNA for versican following exposure to IGF-1 and PDGF-BB. Although no change was seen in response to PDGF, biglycan mRNA level was increased by IGF-1 in periodontal ligament fibroblasts. With the gingival fibroblasts, biglycan mRNA levels were unaffected by IGF-1, PDGF-BB, or growth hormone. These findings suggest variable responses of fibroblasts to growth factors depending upon anatomical site within the periodontium. Moreover, there appears to be a correlation between cell proliferation and the types of proteoglycan synthesised with decorin expression being suppressed, and versican being increased during fibroblast proliferation.

Effects of interferon-gamma and tumor necrosis factor alpha on the expression of the genes encoding aggrecan, biglycan, and decorin core proteins in cultured human chondrocytes.

To determine the effects of interferon-gamma (IFN gamma) and tumor necrosis factor alpha (TNF alpha), alone or in combination, on the expression of aggrecan, biglycan, and decorin core protein genes in human chondrocytes. Isolated human chondrocytes were cultured on poly(2-hydroxyethyl methacrylate)-coated plastic dishes to prevent the loss of cartilage-specific phenotype, and the effects of IFN gamma and TNF alpha, alone or in combination, on aggrecan, biglycan, and decorin core protein gene transcription and steady-state messenger RNA (mRNA) levels were examined. The addition of IFN gamma (1.5 pM) or TNF alpha (0.3 pM) caused a decrease in the steady-state level of aggrecan mRNA (-25% and -15%, respectively), and the combination of these low-concentration cytokines caused a potent inhibition (-66%). These effects were the result of a decrease (-50%) in the transcription rate of the corresponding gene. At the concentrations used, IFN gamma did not alter the levels of biglycan mRNA or the transcription rates of the biglycan core protein gene. In contrast, TNF alpha decreased biglycan steady-state mRNA levels (-62%) and the biglycan core protein gene transcription rate (-18%). The combination of IFN gamma and TNF alpha resulted in a potentiation of the inhibitory effects of TNF alpha on biglycan mRNA levels (-79%) and transcription rate of the biglycan core protein gene (-46%). IFN gamma produced a modest decrease in decorin mRNA levels (-23%) and decorin core protein gene transcription rate (-17%). In contrast, TNF alpha resulted in a marked increase in decorin mRNA levels (+260%) that was not the result of transcriptional regulation. Notably, the combination of IFN gamma and TNF alpha potentiated the inhibitory effects of IFN gamma on decorin mRNA (-80%) and on the transcription of the corresponding gene (-43%). Similar results were obtained in fetal and adult articular chondrocytes. These data demonstrate that 1) the expression of the core protein genes encoding the cartilage proteoglycans aggrecan, biglycan, and decorin is differentially regulated by IFN gamma and TNF alpha; 2) these effects are mediated by transcriptional and posttranscriptional mechanisms; and 3) the combination of the 2 cytokines causes a potent inhibitory effect on the expression of the genes for the core proteins of these 3 proteoglycans, which occurs largely at the transcriptional level. The inhibition of aggrecan, decorin, and biglycan core protein gene expression by the combination of IFN gamma and TNF alpha may contribute to the cartilage destruction that is characteristic of inflammatory joint diseases.

Proteoglycan Gene Expression Is Decreased in Abdominal Aortic Aneurysms

Background.Abdominal aortic aneurysms (AAA) are characterized by both increases in proteolysis and changes in the biosynthesis of the extracellular matrix (ECM) proteins. Proteoglycans are important components of the ECM, particularly the small proteoglycans, biglycan and decorin. Biglycan and decorin regulate cell proliferation and collagen assembly. Therefore, the purpose of this study is to quantify the levels of mRNA for biglycan and decorin in normal aorta (NA) and AAA.Materials and methods.Northern blot hybridization and competitive polymerase chain reaction using gene-specific external standards were used to quantify mRNA levels of bigylcan and decorin in RNA derived from AAA and NA. Results are expressed as a percentage of glyceraldehyde-3-phosphate dehydrogenase or normalized to ribosomal RNA content and compared using the unpairedttest.Results.A statistically significant 15-fold decrease in biglycan mRNA expression was observed in AAA compared to NA (176.9% vs 11.8%,P< 0.001). In contrast to biglycan, the decorin mRNA expression is unchanged in AAA compared to NA.Conclusions.The marked decrease in biglycan mRNA levels is unique to aneurysmal disease of the aorta. In atherosclerosis and restenosis, biglycan expression is increased in comparison with normal artery. This decrease in biglycan expression may reflect important regulatory changes specific for the AAA. Furthermore, a decrease in biglycan gene expression and biosynthesis could have a broad impact on the physiology and matrix architecture of the aorta.

Elevated expression of proteoglycans in proliferating vascular smooth muscle cells

Smooth muscle cell (SMC) proliferation and increased production of arterial wall proteoglycans (PG) are implicated in atherogenesis. We investigated the effect of SMC proliferation on the biosynthesis of PG and the ability of the newly synthesized PG to bind low density lipoprotein (LDL). Proliferating and quiescent human aortic SMC were pulsed with [ 35S]sulfate for 24 h. Secreted and cell-associated PG were then analyzed. When SMC plated at a low density were induced to proliferate, PG synthesis increased significantly in comparison with quiescent cells. This was the net result of a 2.7-fold increase in secreted PG and a 1.3-fold increase in cell-associated PG. The increased PG synthesis in proliferating SMC correlated with a significant increase in the steady-state level of mRNA for perlecan and biglycan, and a modest increase in the versican-specific mRNA. The mRNA for decorin showed a 40% decrease. The increased PG secretion in proliferating cultures was due to increases in heparan sulfate PG, dermatan sulfate PG, and chondroitin sulfate PG secretion. Quiescent SMC at confluency produced 50% less PG than the corresponding SMC plated at a low density. Although confluent SMC stimulated to proliferate also had increased PG synthesis, this was 50% less than the PG synthesis by proliferating SMC that were initially plated at a low density. The PG synthesized by proliferating and quiescent SMC did not differ in charge density and molecular size. Secreted PG from both quiescent and proliferating cultures contained subfractions that bound LDL with high affinity. However, compared with quiescent cultures, the proliferating cultures produced more of a PG subfraction that exhibited very high affinity to LDL (31.6% in quiescent cultures versus 40.8% in proliferating cultures). These results indicate that PG metabolism is altered significantly in proliferating human SMC which might have implications in the pathophysiology of atherosclerosis.

Mechanical Loading and TGF-β Regulate Proteoglycan Synthesis in Tendon

Fibrocartilage is found in tendon at sites where the tissue is subjected to transverse compressive loadingin vivo.A significant characteristic of the tissue transition from tendon to fibrocartilage in bovine deep flexor tendon is increased gene expression, synthesis, and accumulation of both a large proteoglycan, aggrecan, and a small proteoglycan, biglycan. In order to investigate the cellular events involved in this response, segments of fetal bovine deep flexor tendon were subjectedin vitroto cyclic compressive load for 72 h. Following loading, the level of aggrecan mRNA in cells from loaded tissue was increased 200–450% compared to matched nonloaded tissue segments, as determined by slot-blot analysis. The level of biglycan mRNA increased 100%, and the level of versican mRNA increased 130% in the loaded tissue. The level of decorin mRNA remained virtually unchanged, while expression of α1(I) collagen increased only 40%. When tissue segments were cultured in the presence of transforming growth factor (TGF)-β1 (1 ng/ml), the synthesis and expression of mRNA for both aggrecan and biglycan increased, whereas decorin expression was not affected. Similarity in both the direction and the pattern of the cellular response to mechanical load and TGF-β suggested a causal relationship. Both loading of tendon segments and TGF-β treatment increased expression of mRNA for TGF-β by ∼40% compared to control tissue. In addition, the amount of newly synthesized TGF-β immunoprecipitated from extracts of loaded tissue was several-fold greater than that from nonloaded tissue. The experiments of this study support a hypothesis suggesting that one aspect of the response of cells in fetal tendon to compressive load is increased TGF-β synthesis which, in turn, stimulates synthesis of extracellular matrix proteoglycans and leads toward fibrocartilage formation.

Biglycan Gene Promoter Activity in Osteosarcoma Cells Is Regulated by Cyclic AMP

The pericellular proteoglycan biglycan is among the major secretory products of osteoblasts and articular chondrocytes but the regulatory agents and signal transduction pathways that ultimately lead to alterations in biglycan gene expression are poorly defined. We report here on the transcriptional up-regulation of biglycan in MG-63 osteosarcoma cells by agents that increase intracellular cAMP levels. Transfection of these cells with biglycan promoter luciferase reporter fusion genes and subsequent treatment with forskolin or the cAMP analog 8-Bromo-cAMP resulted in an up to 3.8-fold stimulation of biglycan promoter activity. This effect could be prevented with the compound KT5720, a specific inhibitor of the cAMP-dependent protein kinase. Up-regulation of transcription is also reflected at the level of mRNA expression, since biglycan mRNA steady state levels in MG-63 cells increased ∼2-fold after 24 hours of forskolin treatment. These data suggest that elevated levels of intracellular cAMP increase transcription from the biglycan promoter in bone cells and implicate for the first time the cAMP/protein kinase A signal transduction pathway in the regulation of biglycan gene expression.

Expression of biglycan, decorin and fibromodulin in the hypertrophic phase of experimental osteoarthritis

This study sought to assess the relative levels of the mRNAs of the core proteins of the small proteoglycans (PGs) biglycan, decorin and fibromodulin in the hypertrophic phase of the early osteoarthritis (OA) that follows joint injury. Experimental OA was induced in eight dogs by transection of the anterior cruciate ligament. Articular cartilage was harvested from each joint, the total RNA was extracted and the concentration of DNA in the cartilage was measured. The relative levels of mRNA for biglycan, decorin and fibromodulin were assessed by northern blot analyses. An increase in cartilage mass with no increase in DNA concentration confirmed that the joints were in the hypertrophic phase that follows joint injury. The total RNA per microgram of DNA was increased 2.5 times. Compared with control cartilage, the mRNA levels in osteoarthritic cartilage, when normalized to the concentration of DNA, were increased 3.9 times for biglycan, 1.2 times for decorin and 2.4 times for fibromodulin. Because these small PGs affect collagen fibrillogenesis in vitro, their discoordinate metabolism may contribute to the abnormal collagen formation and deposition that occurs in OA and to the ultimate failure of the articular cartilage.

Cultured astrocytes express biglycan, a chondroitin/dermatan sulfate proteoglycan supporting the survival of neocortical neurons

Astrocyte-conditioned medium (ACM) supports the survival of rat E15 neocortical neurons. Using a microtiter assay for neuronal survival, we demonstrated that part of the survival activity is associated with a proteoglycan fraction obtained after two chromatographic steps: (1) preparative Q-Sepharose anion-exchange chromatography under non-denaturating conditions and (2) MonoQ chromatography in the presence of 8 M urea. Analytical SDS-polyacrylamide gradient gel electrophoresis of pooled active MonoQ-fractions (MQ-pool) revealed a broad proteoglycan band migrating with an apparent M r in the range of 150–400 kDa. Digestion of the MQ-pool with chondroitin-ABC-lyase yielded a major core protein of 50 kDa. In Western blots the high molecular weight (150–400 kDa) material as well as the 50 kDa core protein band were immunoreactive to chicken polyclonal antibodies raised against purified biglycan from rat meningeal fibroblasts. Northern blot analysis of total RNA prepared from highly enriched astrocyte cultures revealed a single 2.9 kb biglycan transcript. By using in situ hybridization we demonstrated that essentially all cells in these cultures expressed biglycan mRNA. Furthermore, highly purified biglycan from bovine cartilage was shown to markedly enhance survival of rat neocortical neurons. In conclusion, we have shown that astrocytes synthesize and release the small chondroitin/dermatan sulfate proteoglycan (CS/DSPG) biglycan, a molecule that was found to support survival of neocortical neurons in vitro.

Transcriptional Regulation of the Human Biglycan Gene

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.

Mechanical strain induces constitutive and regulated secretion of glycosaminoglycans and proteoglycans in fetal lung cells.

We have previously shown that an intermittent strain regimen, which simulates fetal breathing movements, enhanced mixed fetal rat lung cell proliferation in organotypic culture. As glycosaminoglycans (GAGs) and proteoglycans (PGs) may modulate growth factor activities, we investigated the effect of intermittent strain on the formation and secretion of GAGs and PGs. Mechanical strain increased the incorporation of [3H]glucosamine and 35SO4 into GAGs and promoted the release of GAGs into the medium. The composition of the individual GAG molecules was not altered by strain. Mixed fetal lung cells subjected to strain secreted more [35S]biglycan into the medium than static controls but biglycan mRNA expression was not significantly altered. As mechanical strain primarily affected the secretion of GAGs and PGs, we then investigated which secretory pathways were stimulated by strain. Fetal lung cells secreted GAGs mainly through a constitutive (basal) pathway which was stimulated by strain. In contrast to static cultures, strain-induced constitutive secretion was partially blocked by the cytoskeletal disruptors colchicine and cytochalasin B, but not by the small G-protein inhibitors N-acetyl-S-farnesyl-L-cysteine and perillic acid. This result suggests that strain-induced constitutive export of GAGs depends on the functional integrity of the cytoskeleton. Strain also triggered the regulated secretion of GAGs. The strain-induced regulatory pathway in fetal lung cells was blocked by ionomycin, BAPTA/AM and gadolinium, suggesting that strain stimulated the regulatory pathway by inducing a rapid calcium influx via a stretch-activated ion channel. We conclude that mechanical strain of mixed fetal lung cells stimulates GAG and PG exocytosis via activation of both the regulated and constitutive pathways.

Marked alteration of proteoglycan metabolism in cholesterol-enriched human arterial smooth muscle cells

To elucidate the correlation between vascular cholesterol metabolism and proteoglycan (PrGl) biosynthesis, we investigated PrGl synthesis in human aortic smooth muscle cells (SMCs) after cholesterol enrichment with cationized low-density lipoproteins (LDL). Compared with normal SMCs, total PrGl synthesis by cholesterol-enriched cells decreased 2.4-fold (11874 +/- 530 d.p.m. per 10(5) cells compared with 4890 +/- 385 d.p.m. per 10(5) cells). This was the net result of a 6.9-fold reduction in medium PrGl (11000 +/- 490 d.p.m. per 10(5) cells compared with 1580 +/- 246 d.p.m. per 10(5) cells) and a 3.8-fold increase in cellular PrGl over controls (874 +/- 27 d.p.m. per 10(5) cells compared with 3310 +/- 193 d.p.m. per 10(5) cells). Prior incubation of SMCs with native LDL had no effect on PrGl synthesis by these cells. The decrease in PrGl synthesis in cholesterol-enriched cells correlated with a 90% and 20% reduction in the steady-state level of mRNA for biglycan and decorin respectively, and a virtual elimination of the steady-state level of mRNA for versican over controls. Despite the down-regulation of PrGl synthesis, cholesterol-loaded cells produced a 2-fold increase in a PrGl subfraction with high affinity for LDL. Compared with the corresponding PrGl subfraction from normal cells, that from the cholesterol-enriched cells exhibited increased charge density and a higher molecular mass and contained relatively larger proportions of chondroitin 6-sulphate and dermatan sulphate. These results show that PrGl metabolism is dramatically altered in cholesterol-enriched human SMCs.

Fetal Lung Fibroblasts Selectively Down-regulate Proteoglycan Synthesis in Response to Elevated Oxygen

Cell proliferation is in part regulated by extracellular matrix. Therefore, it is possible that elevated O2 may indirectly affect lung fibroblast growth via modulation of extracellular matrix. In the present study, we investigated the effect of elevated O2 on the synthesis of glycosaminoglycans (GAGs) and proteoglycans (PGs) by fetal lung fibroblasts. A 48-h exposure to >/=50% O2 reduced the incorporation of [3H]glucosamine and 35SO4 into GAGs by fetal lung fibroblasts. The relative proportion of the individual GAG molecules was not altered by elevated O2. Fibroblasts exposed to 50% O2 secreted less [35S]proteoglycans into the medium than controls. Specifically, the synthesis of the small soluble PG, biglycan, was decreased by exposure to 50% O2. Fetal lung fibroblasts did not synthesize the small chondroitin/dermatan sulfate PG, decorin. Elevated O2 concentrations also reduced the synthesis of membrane- and matrix-associated PGs. Furthermore, exposure of fetal lung fibroblasts to >/=50% O2 resulted in a decreased mRNA expression for biglycan and versican core protein sequences. In contrast, elevated O2 increased the message for type I collagen and fibronectin without affecting that of beta-actin. The inhibitory effect of elevated O2 on biglycan mRNA and protein expression was overcome by incubating the cells in 3% O2 after the 48-h exposure to 50% O2. The latter treatment also reversed the increased mRNA expression of type I collagen associated with elevated O2 but not that of fibronectin. These results demonstrate that fetal lung fibroblasts, in response to elevated oxygen concentrations, selectively down-regulate their GAG and PG synthesis and that this O2 effect is reversible.

Oxidized lipid-mediated alterations in proteoglycan metabolism in cultured pulmonary endothelial cells

Compared to cholesterol or linoleic acid (18:2), oxidized lipids such as cholestan-3β,5α,6β-triol (triol) and hydroperoxy linoleic acid (HPODE) markedly impair endothelial barrier function in culture [Hennig and Boissonneault, 1987; Hennig et al. 1986]. Because proteoglycans contribute to vascular permeability properties, the effects of cholesterol and 18:2 and their oxidation products, triol and HPODE, on endothelial proteoglycan metabolism were determined. While cholesterol was without effect, a concentration-dependent decrease in cellular proteoglycans (measured by 35S incorporation) was observed after exposure to triol. Compared to control cultures, cholesterol reduced mRNA levels for the proteoglycans, perlecan and biglycan. Triol had a similar effect on biglycan but not on perlecan mRNA levels. Compared to 18:2, 1, 3 and 5 μM HPODE depressed cellular proteoglycans. Perlecan mRNA levels were reduced more by HPODE when compared to 18:2. Biglycan mRNA levels were reduced by 3 μM, but not by 5 μM HPODE. These data demonstrate that oxidized lipids such as triol and HPODE can decrease cellular proteoglycan metabolism in endothelial monolayers and alter mRNA levels of major specific proteoglycans in a concentration-dependent manner. This may have implications in lipid-mediated disruption of endothelial barrier function and atherosclerosis.

Comparison of the characteristics of human gingival fibroblasts and periodontal ligament cells.

To elucidate the characteristics of human periodontal ligament cells, we compared these cells with gingival fibroblasts isolated from the periodontal tissues of female human subjects. Human periodontal ligament (HPDL) cells had a sharper spindle shape and exhibited a higher growth rate than human gingival fibroblasts (HGF). HPDL cells had a high level of alkaline phosphatase (ALPase) activity, whereas HGF had a low level of such activity. Northern blot analysis demonstrated that HPDL cells produced ALPase mRNA. Decorin and biglycan mRNA were detected in both HPDL cells and HGF, whereas osteocalcin and bone sialoprotein mRNA was not detected in either cells. Both HPDL cells and HGF responded to prostaglandin E2 (PGE2) and isoproterenol, and produced cyclic AMP (cAMP), but did not respond to human 1-34 parathyroid hormone (PTH). Intracellular Ca2+ ([Ca2+]i) was measured in HPDL cells and HGF, using Fura 2-AM. Bradykinin (BK) and histamine (HIS), which are major chemical mediators, caused a transient rise of [Ca2+]i in the presence of extracellular Ca2+. In HGF, but not HPDL cells, HIS induced a biphasic transient peak in [Ca2+]i. BK and HIS increased PGE2 release in both HPDL cells and HGF. However, HGF released a larger amount of PGE2 than HPDL cells. These results demonstrate that HPDL cells have quite different characteristics from HGF. HPDL cells proliferate at a higher rate than HGF, show higher levels of cAMP production and greater ALPase activity, and respond in a different fashion to chemical mediators (BK and HIS) compared with HGF.

The Murine Biglycan: Complete cDNA Cloning, Genomic Organization, Promoter Function, and Expression

Biglycan is a ubiquitous chondroitin/dermatan sulfate proteoglycan that belongs to a growing family of proteins harboring leucine-rich repeats. We have cloned and sequenced the cDNA containing the complete murine biglycan, elucidated its genomic organization, and demonstrated functional promoter activity of its 5′ flanking region. The deduced biglycan protein core was highly conserved across species. However, the mouse biglycan (Bgn) gene was significantly larger than the human counterpart, primarily because of a large >4.5-kb intron between exons 1 and 2. The mouseBgngene spanned over 9.5 kb of continuous DNA and comprised 8 exons, with a perfectly conserved intron/exon organization vis-á-vis the human counterpart. The promoter region was enriched in GC dinucleotide and contained numerouscis-acting elements including binding sites for SP-1, AP-1, and AP-2 factors. It lacked TATA and CAAT boxes typical of housekeeping genes. In support of this, primer extension analysis showed the existence of multiple transcription start sites. Transient cell transfection assays with a construct comprising the 548 bp upstream of the major transcription start site fused to the chloramphenicol acetyl transferase reporter gene showed functional promoter activity. Internal and 5′ deletion constructs showed that the distal promoter of theBgngene was required for full transcriptional activity. In contrast to the homologous proteoglycan decorin, the highest expression of biglycan mRNA was observed in lung, liver, and spleen of adult mouse and the lowest in skin, heart, and kidney. These results will be useful for the study of biglycan gene regulation and for the generation of mice with targeted null mutation of theBgngene.

The X-chromosomal human biglycan gene BGN is subject to X inactivation but is transcribed like an X-Y homologous gene

We report the mRNA and protein expression levels of human biglycan (BGN) in patients with different numbers of sex chromosomes. BGN maps to the distal long arm of the X chromosome, band Xq28, near the second pseudoautosomal region. BGN expression levels are reduced in 45,X Turner patients and increased in patients with additional sex chromosomes. This is suggestive of a pseudoautosomal gene or a gene that escapes X inactivation and that has an active Y chromosomal copy. However, we also provide evidence from hybrid cell lines that BGN is subject to X inactivation and that there is no homolog on the Y chromosome. This evidence excludes an escape from X inactivation. Moreover, additional Y chromosomes increase BGN expression levels, despite the absence of a Y chromosomal BGN gene. Therefore, another explanation has to be invoked. The "pseudoautosomal expression" of BGN may be attributed to a gene or genes that escape X inactivation and that regulate the transcriptional activity of BGN. This is the first report concerning an X chromosomal gene that does not show the conventional correlation between gene dosage and expression rate known from other X chromosomal genes.

Aberrant expressions of decorin and biglycan genes in the carbohydrate-deficient glycoprotein syndrome.

Carbohydrate-deficient glycoprotein syndrome (CDGS) is a congenital disorder characterized by neurological and developmental defects. We have examined the expressions of the small proteoglycans decorin and biglycan in cultured skin fibroblasts from a patient with CDGS Type-I. Northern blotting analysis identified a marked reduction in decorin mRNA and an increase in biglycan mRNA levels. The decorin protein in the culture medium was decreased. Responses to interleukin-1 beta (IL-1 beta) and transforming growth factor-beta 1 (TGF-beta 1) were apparently abnormal; decorin was only slightly up-regulated by IL-1 beta, while biglycan was markedly down-regulated by IL-1 beta and significantly up-regulated by TGF-beta 1. The constitutional and developmental abnormalities characteristic of CDGS may be associated with such derangements in the expression of proteoglycan genes.

Expression of the Small Proteoglycans Biglycan and Decorin in the Adult Human Testis1

The genes coding for the core proteins of the small chondroitin sulfate/dermatan sulfate proteoglycans (PGs) biglycan and decorin are both expressed in the adult human testis. Northern hybridization of human testicular mRNA indicated the presence of one specific transcript for biglycan at 2.6 kb and two specific transcripts for decorin at 1.6 kb and 1.9 kb. In situ hybridization localized the mRNA for biglycan to the peritubular tissue as well as to the tunica muscularis and adventitia of arteries. Leydig cells and cells of the seminiferous epithelium both proved to be negative. For decorin mRNA, strong signals were found over single, dispersed interstitial cells (possibly fibroblasts), the adventitious layer of large arteries, and the perivascular tissue cells of small arteries, arterioles, and capillaries. In the peritubular cell layers, decorin gene expression was comparably lower and was restricted to the outer fibroblast layers. Germ cells and Sertoli cells were devoid of any positive signal. Immunoblot analysis using specific antisera directed against the core proteins of biglycan and decorin confirmed their presence in chondroitin ABC lyase-treated proteinaceous extracts of the human testis. Immunohistochemistry for biglycan showed a good spatial correlation with the in situ hybridization data and revealed in addition a nonuniform distribution of the antigenic material, which was located predominantly over peritubular myoid cells and the adventitious layer of arteries. Together these results indicate that the expression of both genes in the human testis is differentially regulated, despite the overall similarity of their protein products, and suggest distinct roles for these PGs in testicular function.

The effect of glucocorticoid on the synthesis of biglycan and decorin in human osteoblasts and bone marrow stromal cells.

We have previously demonstrated that glucocorticoids induce differentiation of human bone marrow stromal cells (BMSC) into cells expressing mature osteoblast phenotype. As glucocorticoids have marked effects on extracellular matrix protein synthesis in bone, and because proteoglycans are important components of bone matrix and may condition the differentiation and biological activities of osteoblasts, we studied the effects of dexamethasone (Dex) on the synthesis of small proteoglycans [decorin (DCN) and biglycan (BGN)] in adult human BMSC and human osteoblasts (HOB). First passaged HOB and BMSC were treated with either ethanol or 10(-7) M Dex for 7 days. After treatment, the cells were metabolically labeled with either [35S]SO4 alone or [35S]SO4 and [3H]leucine together for 24 h. Conditioned media were collected, and cell layers were extracted with 4 M guanidine HCl. The extracts and conditioned media were subjected to gel filtration and ion exchange chromatography. Fractions containing radiolabeled proteoglycans were analyzed either directly or after immunoprecipitation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Dex treatment resulted in a dramatic increase in DCN and an associated decrease in BGN in both the conditioned medium and the cell layer of HOB cultures. In Dex-treated BMSC cultures, BGN was decreased in both the conditioned medium and cell layer, whereas DCN was stimulated in the majority of cultures. Northern blot analysis indicated that steady state messenger RNA (mRNA) concentration of DCN was increased by Dex in all of the HOB cultures and in seven of eight BMSC cultures analyzed. The steady state mRNA level of BGN was decreased by Dex in both HOB and BMSC cultures. The regulation of DCN and BGN mRNA by Dex in both HOB and BMSC (when responsive) was dose dependent. Time-course analysis indicated that as little as 1 day of treatment with Dex was sufficient to decrease BGN mRNA and increase DCN mRNA (when observed) levels in BMSC; the regulation spanned a 4-week interval, during which the extracellular matrix of BMSC was mineralized. The effect of Dex on the steady state mRNA levels of DCN and BGN in HOB was also apparent after 1 day of treatment. These accumulated results suggest that Dex modulates the synthesis of small proteoglycans in both human bone marrow stromal osteoprogenitor cells and mature osteoblasts.