Biglycan Core Protein Research Articles

Identification of the effector domain of biglycan that facilitates BMP-2 osteogenic function

We have reported that recombinant biglycan (BGN) core protein accelerates bone formation in vivo by enhancing bone morphogenetic protein (BMP)-2 function. The purpose of the present study was to identify the specific domain (“effector”) within the BGN core protein that facilitates BMP-2 osteogenic function. Thus, we generated various recombinant and synthetic peptides corresponding to several domains of BGN, and tested their effects on BMP-2 functions in vitro. The results demonstrated that the leucine-rich repeats 2–3 domain (LRR2-3) of BGN significantly enhanced the BMP-2 induced Smad1/5/9 phosphorylation, osteogenic gene expression, and alkaline phosphatase activity in myogenic C2C12 cells. Furthermore, addition of LRR2-3 to osteoblastic MC3T3-E1 cells accelerated in vitro mineralization without compromising the quality of the mineral and matrix. These data indicate that LRR2-3 is, at least in part, responsible for BGN’s ability to enhance BMP-2 osteogenic function, and it could be useful for bone tissue regeneration.

The cytoprotective effect of biglycan core protein involves Toll-like receptor 4 signaling in cardiomyocytes

AimsExogenously administered biglycan (core protein with high-molecular weight glycosaminoglycan chains) has been shown to protect neonatal cardiomyocytes against simulated ischemia/reperfusion injury (SI/R), however, the mechanism of action is not clear. In this study we aimed to investigate, which structural component of biglycan is responsible for its cardiocytoprotective effect and to further explore the molecular mechanisms involved in the cytoprotection. Methods and resultsA pilot study was conducted to demonstrate that both native (glycanated) and deglycanated biglycan can attenuate cell death induced by SI/R in a dose-dependent manner in primary neonatal cardiomyocytes isolated from Wistar rats. In separate experiments, we have shown that similarly to glycanated biglycan, recombinant human biglycan core protein (rhBGNc) protects cardiomyocytes against SI/R injury. In contrast, the glycosaminoglycan component dermatan sulfate had no significant effect on cell viability, while chondroitin sulfate further enhanced cell death induced by SI/R. Treatment of cardiomyocytes with rhBGNc reverses the effect of SI/R upon markers of necrosis, apoptosis, mitochondrial membrane potential, and autophagy. We have also shown that pharmacological blockade of Toll-like receptor 4 (TLR4) signaling or its downstream mediators (IRAK1/4, ERK, JNK and p38 MAP kinases) abolished the cytoprotective effect of rhBGNc against SI/R injury. Pretreatment of cardiomyocytes with rhBGNc for 20h resulted in increased Akt phosphorylation and NO production without having significant effect on phosphorylation of ERK1/2, STAT3, and on the production of superoxide. Treatment over 10min and 1h with rhBGNc increased ERK1 phosphorylation, while the SI/R-induced increase in superoxide production was attenuated by rhBGNc. Blockade of NO synthesis also prevented the cardiocytoprotective effect of rhBGNc. ConclusionsThe core protein of exogenous biglycan protects myocardial cells from SI/R injury via TLR4-mediated mechanisms involving activation of ERK, JNK and p38 MAP kinases and increased NO production. The cytoprotective effect of rhBGNc is due to modulation of SI/R-induced changes in necrosis, apoptosis and autophagy.

P148Cardiac myocytes are protected by small leucine rich proteoglycans against simulated ischemia/reperfusion injury

It was previously shown by our group that the overexpression of biglycan in a transgenic mouse model enhances the induction of cardioprotective genes. Ischemia induced cell death was decreased by bigylcan treatment in neonatal cardiomyocytes. In this study we aimed to test whether the biglycan core protein or the glycosaminoglycan is responsible for the cardioprotective effect of biglycan. We also investigated whether decorin, (which has a similar structure to biglycan), has any cardio-protective effects on cardiomyocytes during simulated ischemia/reperfusion (SI/R) injury. Primary cardiomyocyte cultures were isolated from neonatal rat hearts. The 2 day-old cultures were pretreated with 0, 1, 3, 10, 30, and 100 nM decorin, biglycan, biglycan core protein or chondroitin sulfate A for 20 hours followed by an SI/R protocol. The treatment was maintained throughout SI , 240 min, hypoxic solution, hypoxic chamber gassed with 95% N2 es 5% CO2) and reperfusion (120 min, normoxic conditions, medium). At the end of the protocol, a viability assay was performed using calcein staining. Compared to the normoxic untreated control, approximately 40% of the cardiomyocytes died during the simulated ischemia/reperfusion injury. Doses of 10 and 30 nM biglycan increased cell viability significantly, compared to SI/R control (29±4 and 18±5%). The biglycan core protein showed similar cytoprotective effects at 3 and 10 nM doses (21±4 and 24±5%), however, the chondroitin sulfate had no influence on the viability of cardiomyocytes. In the case of decorin, the 3 and 10 nM doses significantly increased the survival of the cells (28±3 and 20±4%). Based on our experiments, we concluded that both decorin and biglycan exert a cyto-protective effect on cardiomyocytes against SI/R injury and this protective effect of biglycan is likely due to the biglycan core protein. Grants: NKTM-OTKA-PD-106001, South-East European Foundation Lykeon Scholarship, GA holds Bolyai Janos Fellowship

Modulation of canonical Wnt signaling by the extracellular matrix component biglycan

Although extracellular control of canonical Wnt signaling is crucial for tissue homeostasis, the role of the extracellular microenvironment in modulating this signaling pathway is largely unknown. In the present study, we show that a member of the small leucine-rich proteoglycan family, biglycan, enhances canonical Wnt signaling by mediating Wnt function via its core protein. Immunoprecipitation analysis revealed that biglycan interacts with both the canonical Wnt ligand Wnt3a and the Wnt coreceptor low-density lipoprotein receptor-related protein 6 (LRP6), possibly via the formation of a trimeric complex. Biglycan-deficient cells treated with exogenous Wnt3a had less Wnt3a retained in cell layers compared with WT cells. Furthermore, the Wnt-induced levels of LRP6 phosphorylation and expression of several Wnt target genes were blunted in biglycan-deficient cells. Both recombinant biglycan proteoglycan and biglycan core protein increased Wnt-induced β-catenin/T cell-specific factor-mediated transcriptional activity, and this activity was completely inhibited by Dickkopf 1. Interestingly, recombinant biglycan was able to rescue impaired Wnt signaling caused by a previously described missense mutation in the extracellular domain of human LRP6 (R611C). Furthermore, biglycan's modulation of canonical Wnt signaling affected the functional activities of osteoprogenitor cells, including the RUNX2-mediated transcriptional activity and calcium deposition. Use of a transplant system and a fracture healing model revealed that expression of Wnt-induced secreted protein 1 was decreased in bone formed by biglycan-deficient cells, further suggesting reduced Wnt signaling in vivo. We propose that biglycan may serve as a reservoir for Wnt in the pericellular space and modulate Wnt availability for activation of the canonical Wnt pathway.

TGF-β stimulates biglycan core protein synthesis but not glycosaminoglycan chain elongation via Akt phosphorylation in vascular smooth muscle

Transforming growth factor-β (TGF-β) can mediate proteoglycan synthesis via Smad and non-Smad signalling pathways in vascular smooth muscle (VSM). We investigated whether TGF-β-mediated proteoglycan synthesis is via PI3K/Akt. TGF-β induced a rapid phosphorylation of Akt that continued upto 4 h. Akt phosphorylation was blocked by Akt1/2 inhibitor SN30978; however, it did not block Smad2 phosphorylation at either the carboxy or linker regions indicating that TGF-β-mediated Akt phosphorylation is independent of Smad2 signalling. The role of Akt in TGF-β-mediated proteoglycan synthesis was investigated. Treatment with SN30978 showed a concentration-dependent decrease in TGF-β-mediated [35S]-sulphate and [35S]-Met/Cys incorporation into secreted proteoglycans; however, SDS-PAGE showed no change in biglycan size. In TGF-β-treated cells, biglycan mRNA levels increased by 40–100% in 24 h and was significantly blocked by SN30978. Our findings demonstrate that Akt is a downstream signalling component of TGF-β-mediated biglycan core protein synthesis but not glycosaminoglycan chain hyper-elongation in VSM.

Extracellular matrix content of ruptured anterior cruciate ligament tissue

Anterior cruciate ligaments (ACLs) can rupture with simple movements, suggesting that structural changes in the ligament may reduce the loading capacity of the ligament. We aimed to investigate if proteoglycan and collagen levels were different between ruptured and non-ruptured ACLs. We also compared changes in ruptured tissue over time. During arthroscopic knee reconstruction surgery 24 ruptured ACLs were collected from participants (10 females; 14 males; mean age 24 years). Four non-ruptured ACLs were obtained from participants undergoing total knee replacement surgery (one female, three males; mean age 66 years). Western blot analysis was used to characterise core proteins of aggrecan, versican, decorin and biglycan and glycosaminoglycan assays were also conducted. Collagen levels were measured by hydroxyproline (OHPr) assays. Significantly lower levels of collagen, were found in ruptured ACL compared to non-ruptured ACL ( p = 0.004). Lower levels of both small and large proteoglycans were found in ruptured than non-ruptured ACLs. No correlation was found between time since rupture and proteoglycan or collagen levels. Ruptured ACLs had less collagen and proteoglycans than non-ruptured ACLs. These changes indicate either extracellular matrix protein levels were reduced prior to rupture or levels decreased immediately after rupture. It is possible that the composition and structure of ACLs that rupture are different to normal ACLs, potentially reducing the tissue's ability to withstand loading. An enhanced understanding of the aetiology of ACL injury could help identify individuals who may be predisposed to rupture.

LRRCE: a leucine-rich repeat cysteine capping motif unique to the chordate lineage.

BackgroundThe small leucine-rich repeat proteins and proteoglycans (SLRPs) form an important family of regulatory molecules that participate in many essential functions. They typically control the correct assembly of collagen fibrils, regulate mineral deposition in bone, and modulate the activity of potent cellular growth factors through many signalling cascades. SLRPs belong to the group of extracellular leucine-rich repeat proteins that are flanked at both ends by disulphide-bonded caps that protect the hydrophobic core of the terminal repeats. A capping motif specific to SLRPs has been recently described in the crystal structures of the core proteins of decorin and biglycan. This motif, designated as LRRCE, differs in both sequence and structure from other, more widespread leucine-rich capping motifs. To investigate if the LRRCE motif is a common structural feature found in other leucine-rich repeat proteins, we have defined characteristic sequence patterns and used them in genome-wide searches.ResultsThe LRRCE motif is a structural element exclusive to the main group of SLRPs. It appears to have evolved during early chordate evolution and is not found in protein sequences from non-chordate genomes. Our search has expanded the family of SLRPs to include new predicted protein sequences, mainly in fishes but with intriguing putative orthologs in mammals. The chromosomal locations of the newly predicted SLRP genes would support the large-scale genome or gene duplications that are thought to have occurred during vertebrate evolution. From this expanded list we describe a new class of SLRP sequences that could be representative of an ancestral SLRP gene.ConclusionGiven its exclusivity the LRRCE motif is a useful annotation tool for the identification and classification of new SLRP sequences in genome databases. The expanded list of members of the SLRP family offers interesting insights into early vertebrate evolution and suggests an early chordate evolutionary origin for the LRRCE capping motif.

Letter by Csont and Ferdinandy Regarding Article, “Biglycan Is Required for Adaptive Remodeling After Myocardial Infarction”

To the Editor: We read with great interest the article by Westermann et al1 in which the authors showed that biglycan expression was strongly increased in the myocardium after permanent coronary occlusion in wild-type mice. In the hearts of bgn −/ 0 mice 21 days after coronary occlusion, however, collagen matrix organization was impaired, and several parameters of left ventricular function were deteriorated. Furthermore, these animals showed increased mortality rates after infarction. The authors conclude that the complete absence of biglycan is deleterious and prevents the establishment of a stable infarct scar and hemodynamic adaptation, suggesting that biglycan is required for proper infarct healing. Although the observations of the authors are most valuable, the major conclusion of the authors1 is …

Biosynthesis of Chondroitin and Heparan Sulfate in Chinese Hamster Ovary Cells Depends on Xylosyltransferase II

Xylosyltransferase (XylT) catalyzes the initial enzymatic reaction in the glycosaminoglycan assembly pathway for proteoglycan biosynthesis. Its activity is thought to be rate-limiting. Two xylosyltransferases have been found using genomic analyses, and one of these, XylT1, has been shown to have xylosyltransferase activity. On the other hand, the less studied XylT2 in recombinant form lacks xylosyltransferase activity and has no known function. Wild-type Chinese hamster ovary cells express abundant Xylt2 mRNA levels and lack detectable Xylt1 mRNA levels. Analysis of a previously described Chinese hamster ovary cell xylosyltransferase mutant (psgA-745) shows that it harbors an Xylt2 nonsense mutation and fails to assemble glycosaminoglycans onto recombinant biglycan. Transfection of this cell line with a murine Xylt2 minigene results in the production of recombinant chondroitin sulfate-modified biglycan core protein and restoration of fibroblast growth factor binding to cell surface-associated heparan sulfate. Expression analyses on 10 different human transformed cell lines detect exclusive XYLT2 expression in two and co-expression of XYLT1 and XYLT2 in the others but at disparate ratios where XYLT2 expression is greater than XYLT1 in most cell lines. These results indicate that XylT2 has a significant role in proteoglycan biosynthesis and that cell type may control which family member is utilized.

Pre-eclampsia-associated alterations in decorin, biglycan and versican of the umbilical cord vein wall

Objective The role of proteoglycans in the rearrangement of the extracellular matrix of the umbilical cord vein wall in pre-eclampsia is not known. Decorin, biglycan and versican are the main proteoglycans of the umbilical cord vein wall. We decided to test whether the amounts of these proteoglycans alter in pre-eclampsia. Study design Study was performed on the umbilical cord veins taken from 10 newborns delivered by healthy mothers (control group) and from 10 newborns delivered by mothers with pre-eclampsia. Proteoglycans were extracted in dissociative conditions, purified by Q-Sepharose anion exchange chromatography and lyophilised. Decorin, biglycan and versican were analysed by SDS-PAGE followed by Western blotting before and after treatment with chondroitinase ABC. The amounts of decorin, biglycan and versican core proteins were assessed by ELISA method. Results We found that both control and pre-eclamptic umbilical cord vein wall contained all the three proteoglycans. ELISA assay showed the amounts of the core proteins of decorin, biglycan and versican were distinctly higher in pre-eclamptic material in comparison to control vessel. Western blotting confirmed that the expression of all these proteoglycan core proteins increased in pre-eclampsia. They featured in the same electrophoretic mobility—45 and 47 kDa for decorin, 45 kDa for biglycan, and 300 and 320 kDa for versican. Conclusion The content of decorin, biglycan and versican in the umbilical cord vein wall is elevated in pre-eclampsia in comparison to the corresponding control vessel. These alterations may affect the mechanical properties of this vessel and disturb foetal blood circulation.

Decorin Modulates Fibrin Assembly and Structure

Emerging evidence indicates that fibrin clotting is regulated by different external factors. We demonstrated recently that decorin, a regulator of collagen fibrillogenesis and transforming growth factor-beta activity, binds to the D regions of fibrinogen (Dugan, T.A., Yang, V. W.-C., McQuillan, D.J., and Höök, M. (2003) J. Biol. Chem. 278, 13655-13662). We now report that the decorin-fibrinogen interaction alters the assembly, structure, and clearance of fibrin fibers. Relative to fibrinogen, substoichiometric amounts of decorin core protein modulated clotting, whereas an excess of an active decorin peptide was necessary for similar activity. These concentration-dependent effects suggest that decorin bound to the D regions sterically modulates fibrin assembly. Scanning electron microscopy images of fibrin clotted in the presence of increasing concentrations of decorin core protein showed progressively decreasing fiber diameter. The sequestration of Zn(2+) ions from the N-terminal fibrinogen-binding region abrogated decorin incorporation into the fibrin network. Compared with linear thicker fibrin fibers, the curving thin fibers formed with decorin underwent accelerated tissue-type plasminogen activator-dependent fibrinolysis. Collectively, these data demonstrate that decorin can regulate fibrin organization and reveal a novel mechanism by which extracellular matrix components can participate in hemostasis, thrombosis, and wound repair.

βig-h3 Interacts Directly with Biglycan and Decorin, Promotes Collagen VI Aggregation, and Participates in Ternary Complexing with These Macromolecules

Recombinant human beta ig-h3 was found to bind 125I-labeled small leucine-rich proteoglycans (SLRPs), biglycan, and decorin, in co-immunoprecipitation experiments. In each instance the binding could be blocked by an excess of the unlabeled proteoglycan, confirming the specificity of the interaction. Scatchard analysis showed that biglycan bound beta ig-h3 more avidly than decorin with Kd values estimated as 5.88 x 10(-8) and 1.02 x 10(-7) M, respectively. In reciprocal blocking experiments both proteoglycans inhibited the others binding to beta ig-h3 indicating that they may share the same binding site or that the two binding sites are in close proximity on the beta ig-h3 molecule. Since beta ig-h3 and the SLRPs are known to be associated with the amino-terminal region of collagen VI in tissue microfibrils, the effects of including collagen VI in the incubations were investigated. Co-immunoprecipitation of 125I-labeled biglycan incubated with equimolar mixtures of beta ig-h3 and pepsin-collagen VI was increased 6-fold over beta ig-h3 alone and 3-fold over collagen VI alone. Similar increases were also observed for decorin. The findings indicate that beta ig-h3 participates in a ternary complex with collagen VI and SLRPs. Static light scattering techniques were used to show that beta ig-h3 rapidly forms very high molecular weight complexes with both native and pepsin-collagen VI, either alone or with the SLRPs. Indeed beta ig-h3 was shown to form a complex with collagen VI and biglycan, which appeared to be much more extensive than that formed by beta ig-h3 with collagen VI and decorin or those formed between the collagen and beta ig-h3, biglycan, or decorin alone. Biglycan core protein was shown to inhibit the extent of complexing of beta ig-h3 with native and pepsin-collagen VI suggesting that the glycosaminoglycan side chains of the proteoglycan were important for the formation of the large ternary complexes. Further studies showed that the direct interaction between beta ig-h3 and biglycan and between biglycan and collagen VI were also important for the formation of these complexes. The globular domains of collagen VI also appeared to have an influence on the interaction of the three components. Overall the results indicate that beta ig-h3 can differentially modulate the aggregation of collagen VI with biglycan and decorin. Thus this interplay is likely to be important in tissues such as cornea where such complexes are considered to occur.

Endostatin binds biglycan and LDL and interferes with LDL retention to the subendothelial matrix during atherosclerosis

Retention of lipoproteins to proteoglycans in the subendothelial matrix (SEM) is an early event in atherosclerosis. We recently reported that collagen XVIII and its proteolytically released fragment endostatin (ES) are differentially depleted in blood vessels affected by atherosclerosis. Loss of collagen XVIII/ES in atherosclerosis-prone mice enhanced plaque neovascularization and increased the vascular permeability to lipids by distinct mechanisms. Impaired endothelial barrier function increased the influx of lipoproteins across the endothelium; however, we hypothesized that enhanced retention might be a second mechanism leading to the increased lipid content in atheromas lacking collagen XVIII. We now demonstrate a novel property of ES that binds both the matrix proteoglycan biglycan and LDL and interferes with LDL retention to biglycan and to SEM. A peptide encompassing the alpha coil in the ES crystal structure mediates the major blocking effect of ES on LDL retention. ES inhibits the macrophage uptake of biglycan-associated LDL indirectly by interfering with LDL retention to biglycan, but it has no direct effect on the macrophage uptake of native or modified lipoproteins. Thus, loss of ES in advanced atheromas enhances lipoprotein retention in SEM. Our data reveal a third protective role of this vascular basement membrane component during atherosclerosis.

Proteoglycans and catabolic products of proteoglycans present in ligament.

The aim of the present study was to characterize the proteoglycans and catabolic products of proteoglycans present in the tensile region of ligament and explant cultures of this tissue, and to compare these with those observed in the tensile region of tendon. Approx. 90% of the total proteoglycans in fresh ligament was decorin, as estimated by N-terminal amino acid sequence analysis. Other species that were detected were biglycan and the large proteoglycans versican (splice variants V(0) and/or V1 and/or V2) and aggrecan. Approx. 23% of decorin detected in the matrix was degraded. Intact decorin and decorin fragments similar to those observed in the matrix that retained the N-terminus were also observed in the medium of ligament cultures. Intact biglycan core protein was detected in the matrix and medium of ligament cultures, and two fragments originating from the N-terminal region of biglycan were observed in the matrix of cultured ligament. Versican and versican fragments that retained the N-terminus of versican core protein were detected in fresh matrix and medium of tendon cultures. Approx. 42% of versican present in the fresh ligament was degraded. Aggrecan catabolites appearing in the culture medium were derived from aggrecanase cleavage of the core protein. An intact link protein and a degradation product from the N-terminal region of type XII collagen were also detected in the medium of the ligament explant.

Proteoglycans of human umbilical cord arteries.

Proteoglycans (PGs) were dissociatively extracted from human umbilical cord arteries (UCAs) with 4 M guanidine hydrochloride containing Triton X-100 and protease inhibitors, purified by Q-Sepharose anion exchange chromatography and lyophilized. They were analysed by gel filtration, SDS/PAGE and agarose gel electrophoresis before and after treatment with chondroitinase ABC. It was found that the PG preparation was especially enriched in chondroitin/dermatan sulphate PGs. The predominant PG fraction included small PGs that emerged from Sepharose CL-2B with Kav = 0.74. Their molecular mass, estimated by SDS/PAGE, was 160-200 kDa and 90-150 kDa, i.e. it was typical for biglycan and decorin, respectively. Treatment with chondroitinase ABC yielded the core proteins of 45 and 47 kDa, characteristic for both small PGs. Remarkable amounts of the 45 kDa protein were detected in non-treated PG samples, suggesting the presence of free core proteins of biglycan and decorin. Large PGs were present in lower amounts. In intact form they were eluted from Sepharose CL-2B with Kav = 0.17 and 0.43. Digestion with chondroitinase ABC yielded the core proteins with a molecular mass within the range of 180-360 kDa but predominant were the bands of 200, 250 and 360 kDa. The large PGs probably represent various forms of versican or perlecan bearing chondroitin sulphate chains.

Gene expression and immunohistochemical localization of biglycan in association with mineralization in the matrix of epiphyseal cartilage.

This study has used in situ hybridization, Northern blot analysis, and immunohistochemistry at the light and electron microscope levels to localize mRNAs and core proteins of biglycan in developing tibial epiphyseal cartilage of 10-day old Wistar rats. The expression of mRNAs and core proteins of biglycan appeared prominent in hypertrophic and degenerative chondrocytes associated with the epiphyseal ossification centre and the growth plate cartilage, but was not seen in the rest of epiphyseal cartilage. Northern blot analysis confirmed biglycan mRNA expression in the epiphyseal cartilage. Ultrastructural immunogold cytochemistry of the growth plate revealed that prominent immunolabelling was confined to the Golgi apparatus and cisternae of rough-surfaced endoplasmic reticulum of the hypertrophic and the degenerating chondrocytes, the early mineralized cartilage matrices of the longitudinal septum of the lower hypertrophic and the calcifying zones, and fully mineralized cartilage mitrices, which were present in the metaphyseal bone trabeculae. Furthermore, Western blot analysis of biglycan in extracts of fresh epiphyseal cartilage revealed that an EDTA extract, after chondroitinase ABC digestion, contains core proteins of biglycan, indicating the presence of biglycan in mineralized cartilage matrices. These results indicate that the distribution of biglycan is associated with cartilage matrix mineralization.

Selective Increase in Decorin Core mRNA Level in Cultured Vascular Smooth Muscle Cells after Exposure to Advanced Glycation End products.

Although advanced glycation endproducts (AGEs) have been proved to be involved in the pathogenesis of vascular complications, such as atherosclerosis in diabetic patients, little is known about the functional damage to vascular cells caused by AGEs. In early and late atherosclerosis, excess proteoglycans (PGs) derived from vascular smooth muscle and endothelial cells deposit in the subendothelial extracellular matrix of the vascular wall. To address the question whether AGEs affect the synthesis of PGs in vascular cels or not, the effect of AGEs on steady-state levels of PG core mRNAs in cultured human aortic smooth muscle and endothelial cells was investigated. AGEs were prepared by incubation of bovine serum albumin with glucose and the mRNAs coding for the core proteins of heparan sulfate PGs (perlecan and syndecan-1) and chondroitin/dermatan sulfate PGs (versican, biglycan and decorin) were determined by quantitative reverse transcription-polymerase chain reactions. In vascular smooth muscle cells, the steady-state level of mRNAs coding for perlecan, syndecan-1, versican and biglycan core proteins was unchanged whereas that of mRNA coding for decorin core protein was increased by AGEs in a dose- and time-dependent manner. On the other hand, in vascular endothelial cells, mRNAs coding for perlecan, syndecan-1 and biglycan were observed, although their steady-state levels were unaffected by AGEs. The present data suggest that AGEs may be involved in the accumulation of decorin derived from vascular smooth muscle cells in the atherosclerotic vascular wall of diabetic patients.

Distinct Secondary Structures of the Leucine-rich Repeat Proteoglycans Decorin and Biglycan: GLYCOSYLATION-DEPENDENT CONFORMATIONAL STABILITY

Biglycan and decorin have been overexpressed in eukaryotic cells and two major glycoforms isolated under native conditions: a proteoglycan substituted with glycosaminoglycan chains; and a core protein form secreted devoid of glycosaminoglycans (Hocking, A. M., Strugnell, R. A., Ramamurthy, P., and McQuillan, D. J. (1996) J. Biol. Chem. 271, 19571-19577; Ramamurthy, P., Hocking, A. M., and McQuillan, D. J. (1996) J. Biol. Chem. 271, 19578-19584). Far-UV CD spectroscopy of decorin and biglycan proteoglycans indicates that, although they are predominantly beta-sheet, biglycan has a significantly higher content of alpha-helical structure. Decorin proteoglycan and core protein are very similar, whereas the biglycan core protein exhibits closer similarity to the decorin glycoforms than to the biglycan proteoglycan form. However, enzymatic removal of the chondroitin sulfate chains from biglycan proteoglycan does not induce a shift to the core protein structure, suggesting that the final form is influenced by polysaccharide addition only during biosynthesis. Fluorescence emission spectroscopy demonstrated that the single tryptophan residue, which is at a conserved position at the C-terminal domain of both biglycan and decorin, is found in similar microenvironments. This indicates that in this specific domain the different glycoforms do exhibit apparent conservation of structure. Exposure of decorin and biglycan to 10 M urea resulted in an increase in fluorescent intensity, which indicates that the emission from tryptophan in the native state is quenched. Comparison of urea-induced protein unfolding curves provide further evidence that decorin and biglycan assume different structures in solution. Decorin proteoglycan and core protein unfold in a manner similar to a classic two-state model, in which there is a steep transition to an unfolded state between 1 and 2 M urea. The biglycan core protein also shows a similar steep transition. However, biglycan proteoglycan shows a broad unfolding transition between 1 and 6 M urea, probably indicating the presence of stable unfolding intermediates.

PDGF-induced glycosaminoglycan synthesis is mediated via phosphatidylinositol 3-kinase.

Platelet-derived growth factor (PDGF)-BB has been shown previously to increase glycosaminoglycan (GAG) synthesis but not DNA synthesis in freshly isolated fetal lung fibroblasts. In the present study, we found that PDGF-BB also enhanced 35SO4 incorporation into the small, soluble proteoglycan biglycan without affecting biglycan's core protein mRNA expression, suggesting that PDGF-BB mainly affects GAG chain elongation and/or sulfation. PDGF-BB-stimulated GAG synthesis was abrogated by tyrphostin 9, a PDGF receptor-associated tyrosine kinase inhibitor, implying that the stimulatory effect is mediated via the PDGF beta-receptor (PDGFR). The intracellular signal transduction pathways that mediate PDGF-BB-stimulated GAG synthesis in fetal lung fibroblasts were investigated. On ligand-induced tyrosine phosphorylation, PDGFR associated with phospholipase C (PLC)-gamma 1, Ras GTPase activating protein (RasGAP), and phosphatidylinositol 3-kinase (PI3K) but not with the Syp-growth factor receptor-bound protein 2-Son of Sevenless complex. Association of PDGFR with PLC-gamma 1 and RasGAP followed by their tyrosine phosphorylation failed, however, to activate PLC-gamma 1, protein kinase C (PKC), and Ras. Neither a PLC-gamma inhibitor, U-73122; a PKC inhibitor, calphostin C; nor a mitogen-activated protein kinase kinase inhibitor, PD-98059, inhibited PDGF-BB-induced GAG synthesis. In contrast, PDGF-BB stimulation triggered PDGFR-associated PI3K activity. Both PDGF-BB-induced PI3K activation and GAG synthesis were abolished by the PI3K inhibitors wortmannin and LY-294002. The results suggest that PI3K is a downstream mediator of PDGF-BB-stimulated GAG synthesis in fetal rat lung fibroblasts.

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.