Uterine dysfunction in biglycan and decorin deficient mice leads to dystocia during parturition

Cesarean birth rates are rising. Uterine dysfunction, the exact mechanism of which is unknown, is a common indication for Cesarean delivery. Biglycan and decorin are two small leucine-rich proteoglycans expressed in the extracellular matrix of reproductive tissues and muscle. Mice deficient in biglycan display a mild muscular dystrophy, and, along with mice deficient in decorin, are models of Ehlers-Danlos Syndrome, a connective tissue anomaly associated with uterine rupture. As a variant of Ehlers-Danlos Syndrome is caused by a genetic mutation resulting in abnormal biglycan and decorin secretion, we hypothesized that biglycan and decorin play a role in uterine function. Thus, we assessed wild-type, biglycan, decorin and double knockout pregnancies for timing of birth and uterine function. Uteri were harvested at embryonic days 12, 15 and 18. Nonpregnant uterine samples of the same genotypes were assessed for tissue failure rate and spontaneous and oxytocin-induced contractility. We discovered that biglycan/decorin mixed double-knockout dams displayed dystocia, were at increased risk of delayed labor onset, and showed increased tissue failure in a predominantly decorin-dependent manner. In vitro spontaneous uterine contractile amplitude and oxytocin-induced contractile force were decreased in all biglycan and decorin knockout genotypes compared to wild-type. Notably, we found no significant compensation between biglycan and decorin using quantitative real time PCR or immunohistochemistry. We conclude that the biglycan/decorin mixed double knockout mouse is a model of dystocia and delayed labor onset. Moreover, decorin is necessary for uterine function in a dose-dependent manner, while biglycan exhibits partial compensatory mechanisms in vivo. Thus, this model is poised for use as a model for testing novel targets for preventive or therapeutic manipulation of uterine dysfunction.     

A decorin-deficient matrix affects skin chondroitin/dermatan sulfate levels and keratinocyte function

Decorin is a small leucine-rich proteoglycan harboring a single glycosaminoglycan chain, which, in skin, is mainly composed of dermatan sulfate (DS). Mutant mice with targeted disruption of the decorin gene (Dcn^−/−) exhibit an abnormal collagen architecture in the dermis and reduced tensile strength, collectively leading to a skin fragility phenotype. Notably, Ehlers–Danlos patients with mutations in enzymes involved in the biosynthesis of DS display a similar phenotype, and recent studies indicate that DS is involved in growth factor binding and signaling. To determine the impact of the loss of DS-decorin in the dermis, we analyzed the glycosaminoglycan content of Dcn^−/− and wild-type mouse skin. The total amount of chondroitin/dermatan sulfate (CS/DS) was increased in the Dcn^−/− skin, but was overall less sulfated with a significant reduction in bisulfated ΔDiS2,X (X = 4 or 6) disaccharide units, due to the reduced expression of uronyl 2-O sulfotransferase (Ust). With increasing age, sulfation declined; however, Dcn^−/− CS/DS was constantly undersulfated vis-à-vis wild-type. Functionally, we found altered fibroblast growth factor (Fgf)-7 and -2 binding due to changes in the micro-heterogeneity of skin Dcn^−/− CS/DS. To better delineate the role of decorin, we used a 3D Dcn^−/− fibroblast cell culture model. We found that the CS/DS extracts of wild-type and Dcn^−/− fibroblasts were similar to the skin sugars, and this correlated with the lack of uronyl 2-O sulfotransferase in the Dcn^−/− fibroblasts. Moreover, Ffg7 binding to total CS/DS was attenuated in the Dcn^−/− samples. Surprisingly, wild-type CS/DS significantly reduced the binding of Fgf7 to keratinocytes in a concentration dependent manner unlike the Dcn^−/− CS/DS that only affected the binding at higher concentrations. Although binding to cell-surfaces was quite similar at higher concentrations, keratinocyte proliferation was differentially affected. Higher concentration of Dcn^−/− CS/DS induced proliferation in contrast to wild-type CS/DS. 3D co-cultures of fibroblasts and keratinocytes showed that, unlike Dcn^−/− CS/DS, wild-type CS/DS promoted differentiation of keratinocytes. Collectively, our results provide novel mechanistic explanations for the reported defects in wound healing in Dcn^−/− mice and possibly Ehlers–Danlos patients. Moreover, the lack of decorin-derived DS and an altered CS/DS composition differentially influence keratinocyte behavior.     

Proteoglycans decorin and biglycan differentially modulate TGF-beta-mediated fibrotic responses in the lung

Transforming growth factor (TGF)-beta is a key cytokine in the pathogenesis of pulmonary fibrosis, and pharmacological interference with TGF-beta can ameliorate the fibrotic tissue response. The small proteoglycans decorin and biglycan are able to bind and inhibit TGF-beta activity in vitro. Although decorin has anti-TGF-beta properties in vivo, little is known about the physiological role of biglycan in vivo. Adenoviral gene transfer was used to overexpress active TGF-beta, decorin, and biglycan in cell culture and in murine lungs. Both proteoglycans were able to interfere with TGF-beta bioactivity in vitro in a dose-dependant manner. In vivo, overexpression of TGF-beta resulted in marked lung fibrosis, which was significantly reduced by concomitant overexpression of decorin. Biglycan, however, had no significant effect on lung fibrosis induced by TGF-beta. The data suggest that differences in tissue distribution are responsible for the different effects on TGF-beta bioactivity in vivo, indicating that decorin, but not biglycan, has potential therapeutic value in fibrotic disorders of the lung.     

未足月胎膜早破合并绒毛膜羊膜炎孕妇血清淀粉样蛋白A、血小板激活因子水平的表达及临床意义

目的:检测未足月胎膜早破合并绒毛膜羊膜炎(HCA)孕妇血清淀粉样蛋白A(SAA)、血小板激活因子(PAF)水平,并探讨其临床意义。方法:选择从2013年7月到2017年7月,在我院接受治疗的165例胎膜早破孕产妇作为研究对象。165例患者中,未足月胎膜早破者80例(未足月胎膜早破组),足月胎膜早破者85例(足月胎膜早破组),再根据是否合并HCA分为合并HCA胎膜早破组43例和未合并HCA胎膜早破组122例。另选取同期在我院体检的80例健康孕产妇志愿者作为正常组,对比各组血清SAA和PAF水平,分析合并与未合并HCA胎膜早破组的妊娠结局,利用受试者工作特征(ROC)曲线分析血清SAA和PAF对未足月胎膜早破是否合并HCA的诊断价值。结果:未足月胎膜早破组及足月胎膜早破组的血清SAA和PAF水平均明显高于正常组,且未足月胎膜早破组又高于足月胎膜早破组,差异有统计学意义(P0.05)。未足月胎膜早破组80例患者中HCA发生率为35.00%(28/80),明显高于足月胎膜早破组的17.65%(15/85),差异有统计学意义(P0.05)。合并HCA胎膜早破组的血清SAA和PAF水平均明显高于未合并HCA胎膜早破组,差异有统计学意义(P0.05)。合并HCA的未足月胎膜早破患者血清SAA和PAF水平高于未合并HCA的未足月胎膜早破患者(P0.05)。合并HCA的胎膜早破组的产后大出血、剖宫产以及新生儿肺炎的发生率均明显高于未合并HCA胎膜早破组,差异有统计学意义(P0.05)。根据ROC曲线分析可知,血清SAA和PAF对未足月胎膜早破是否合并HCA的诊断价值较高。结论:血清SAA、PAF水平在未足月胎膜早破合并HCA孕妇中明显升高,二者对此种合并症具有较高的诊断价值。临床诊疗过程中可将SAA及PAF纳入到指标监测体系中,从而为临床治疗提供指导。     

Relationship between actions of transforming growth factor (TGF)-β and cell surface expression of its receptors in clonal osteoblastic cells

Various osteoblastic cell lines were examined for the relationship between the presence of cell-surface transforming growth factor (TGF)-β receptors and the synthesis of matrix proteins with their responsiveness to TGF-β. Treatment with TGF-β1 inhibited proliferation and stimulated proteoglycan and fibronectin synthesis in MC3T3-E1 and MG 63 cells. The major proteoglycans synthesized by these cells were decorin and biglycan, and TGF-β1 markedly stimulated the synthesis of decorin in MC3T3-E1 and of biglycan in MG 63 cells. SaOS 2 and UMR 106 cells synthesized barely detectable amounts of decorin or biglycan, and TGF-β1 did not stimulate the synthesis of these proteoglycans. In SaOS 2 cells, however, TGF-β1 enhanced fibronectin synthesis. TGF-β1 did not show any of these effects in UMR 106 cells. Receptor cross-linking studies revealed that only MC3T3-E1 and MG 63 cells had both types I and II signal-transducing receptors for TGF-β in addition to betaglycan. SaOS 2 cells possessed type I but no type II receptor on the cell surface. In contrast, SaOS 2 as well as MC3T3-E1 and MG 63 cells expressed type II receptor mRNA by Northern blot analysis, and cell lysates contained type II receptor by Western blot analysis. Thus, it appears that type II receptor present in SaOS 2 cells is not able to bind TGF-β1 under these conditions. UMR 106 cells with no response to TGF-β1 had neither of the signal-transducing receptors by any of the analyses. These observations using clonal osteoblastic cell lines demonstrate that the ability of osteoblastic cells to synthesize bone matrix proteoglycans is associated with the responsiveness of these cells to TGF-β1, that the responsiveness of osteoblastic cells to TGF-β1 in cell proliferation and proteoglycan synthesis correlates with the presence of both types I and II receptors, and that the effect of TGF-β1 on fibronectin synthesis can develop with little binding of TGF-β1 to type II receptor if type I receptor is present. It is suggested that the combination of cell-surface receptors for TGF-β determines the responsiveness of osteoblastic cells to TGF-β and that changes in cell-surface TGF-β receptors may play a role in the regulation of matrix protein synthesis and bone formation in osteoblasts. © 1995 Wiley-Liss, Inc.     

Regulation of TGFβ receptor trafficking and signaling by atypical protein kinase C

Transforming growth factor beta (TGFβ) signaling is linked to the membrane trafficking of TGFβ receptors. The Protein Kinase C (PKC) family of serine/threonine kinases have been implicated in modulating the endocytic processes of various receptors. The present study investigated whether PKC activity plays a role in the trafficking, and signaling of TGFβ receptors, and further explored which PKC isoforms may be responsible for altered TGFβ signaling patterns. Using immunofluorescence microscopy and ¹²⁵I-TGFβ internalization assays, we show that the pharmacological inhibition of PKC activity alters TGFβ receptor trafficking and delays TGFβ receptor degradation. Consistent with these findings, we demonstrate that PKC inhibition extends TGFβ-dependent Smad2 phosphorylation. Previous studies have shown that PKCζ associates with TGFβ receptors to modulate cell plasticity. We therefore used siRNA directed at the atypical PKC isoforms to investigate if reducing PKCι and PKCζ protein levels would delay TGFβ receptor degradation and extend TGFβ signaling. Our findings suggest that atypical PKC isoforms regulate TGFβ signaling by altering cell surface TGFβ receptor trafficking and degradation.     

Changes in leucine-rich repeat proteoglycans during maturation of the bovine growth plate

The primary growth plate of the fetal bovine tibia was studied in order to determine whether changes in the structure, abundance and expression of the leucine-rich repeat proteoglycans were occurring during tissue maturation from reserve cartilage to hypertrophic cartilage. The proteoglycans under study were decorin, biglycan, fibromodulin and lumican. Decorin was readily detectable in both the reserve and proliferating zones of the growth plate, but its abundance decreased markedly in the zones of maturation and hypertrophy where it could not be detected under the same conditions of analysis. In contrast to decorin, fibromodulin and biglycan could be detected throughout the growth plate, though their abundance was decreased in the proliferative and hypertrophic zones. Unlike the other proteoglycans, lumican could not be detected throughout the growth plate. At the message level, the expression of decorin shows a similar trend to that of protein abundance in the extracellular matrix, with its expression dropping markedly in the proliferative and hypertrophic zones. In the case of both biglycan and fibromodulin, message expression continued at a similar level throughout the growth plate. Thus, the leucine-rich repeat proteoglycans are different in the way they behave during growth plate maturation.     

Extracellular matrix proteoglycans control the fate of bone marrow stromal cells

Extracellular matrix glycoproteins and proteoglycans bind a variety of growth factors and cytokines thereby regulating matrix assembly as well as bone formation. However, little is known about the mechanisms by which extracellular matrix molecules modulate osteogenic stem cells and bone formation. Using mice deficient in two members of the small leucine-rich proteoglycans, biglycan and decorin, we uncovered a role for these two extracellular matrix proteoglycans in modulating bone formation from bone marrow stromal cells. Our studies showed that the absence of the critical transforming growth factor-beta (TGF-beta)-binding proteoglycans, biglycan and decorin, prevents TGF-beta from proper sequestration within the extracellular matrix. The excess TGF-beta directly binds to its receptors on bone marrow stromal cells and overactivates its signaling transduction pathway. Overall, the predominant effect of the increased TGF-beta signaling in bgn/dcn-deficient bone marrow stromal cells is a "switch in fate" from growth to apoptosis, leading to decreased numbers of osteoprogenitor cells and subsequently reduced bone formation. Thus, biglycan and decorin appear to be essential for maintaining an appropriate number of mature osteoblasts by modulating the proliferation and survival of bone marrow stromal cells. These findings underscore the importance of the micro-environment in controlling the fate of adult stem cells and reveal a novel cellular and molecular basis for the physiological and pathological control of bone mass.     

Molecular interactions of biglycan and decorin with elastic fiber components: biglycan forms a ternary complex with tropoelastin and microfibril-associated glycoprotein 1.

The interactions of the dermatan sulfate proteoglycans biglycan and decorin have been investigated with the elastic fiber components, tropoelastin, fibrillin-containing microfibrils, and microfibril-associated glycoproteins (MAGP) 1 and 2. Both proteoglycans were found to bind tropoelastin and fibrillin-containing microfibrils but not MAGPs 1 and 2 in solid phase binding assays. The specificity of the binding of biglycan and decorin to tropoelastin was confirmed by co-immunoprecipitation experiments and by the blocking of the interactions with elastin-derived peptides. Isolated core proteins from biglycan and decorin bound to tropoelastin more strongly than the intact proteoglycans, and there were no differences in the tropoelastin binding characteristics of distinct glucuronate-rich and iduronate-rich glycoforms of biglycan. These findings indicated that the binding sites were contained in the protein cores of the proteoglycans rather than the glycosaminoglycan side chains. Scatchard analysis showed that biglycan bound more avidly than decorin to tropoelastin with K(d) values estimated as 1.95 x 10(-7) m and 5.3 x 10(-7) m, respectively. In blocking experiments each proteoglycan showed extensive inhibition of binding of the other to tropoelastin but was most effective at blocking its own binding. This result suggested that biglycan and decorin had closely spaced but distinct binding sites on tropoelastin. Addition of the elastin-binding protein MAGP-1 to the assays enhanced the binding of biglycan to tropoelastin but had no effect on the decorin-tropoelastin interaction. Co-immunoprecipitation experiments showed that MAGP-1 interacted with biglycan but not decorin in the solution phase. The results indicated that biglycan specifically formed a ternary complex with tropoelastin and MAGP-1. Overall the study supports the concept that biglycan may have a specific role in the elastinogenic phase of elastic fiber formation.     

Evidence for a role of phosphodiesterase 4 in lipopolysaccharide-stimulated prostaglandin E2 production and matrix metalloproteinase-9 activity in human amniochorionic membranes

Chorioamniotic infection is a leading cause of preterm premature rupture of fetal membranes (amnion and chorion). Bacterial infection induces an inflammatory response characterized by elevated production of proinflammatory cytokines; the latter activate the production of both PGs that stimulate uterine contractions, and matrix metalloproteinases (MMPs) that degrade the extracellular matrix of the chorioamniotic membranes. The inflammatory response is under the control of cAMP content, which is partly regulated by phosphodiesterases (PDE). In this study, we investigated the role of the PDE4 family in the inflammatory process triggered by LPS in a model of amniochorionic explants. We found that PDE4 family is the major cAMP-PDE expressed in human fetal membranes and that PDE4 activity is increased by LPS treatment. Selective inhibition of PDE4 activity affected LPS signaling, because PDE4 inhibitors (rolipram and/or cilomilast) reduced the release of the proinflammatory cytokine TNF-alpha and increased the release of the anti-inflammatory cytokine IL-10. PDE4 inhibition reduced cyclooxygenase-2 protein expression and PGE(2) production and also modulated MMP-9, a key mediator of the membrane rupture process, by inhibiting pro-MMP-9 mRNA expression and pro-MMP-9 activity. These results demonstrate that the PDE4 family participates in the regulation of the inflammatory response associated with fetal membrane rupture during infection. The PDE4 family may be an appropriate pharmacological target for the management of infection-induced preterm delivery.     

Crystal structure of the biglycan dimer and evidence that dimerization is essential for folding and stability of class I small leucine-rich repeat proteoglycans

Biglycan and decorin are two closely related proteoglycans whose protein cores contain leucine-rich repeats flanked by disulfides. We have previously shown that decorin is dimeric both in solution and in crystal structures. In this study we determined whether biglycan dimerizes and investigated the role of dimerization in the folding and stability of these proteoglycans. We used light scattering to show that biglycan is dimeric in solution and solved the crystal structure of the glycoprotein core of biglycan at 3.40-angstroms resolution. This structure reveals that biglycan dimerizes in the same way as decorin, i.e. by apposition of the concave inner surfaces of the leucine-rich repeat domains. We demonstrate that low concentrations of guanidinium chloride denature biglycan and decorin but that the denaturation is completely reversible following removal of the guanidinium chloride, as assessed by circular dichroism spectroscopy. Furthermore, the rate of refolding is dependent on protein concentration, demonstrating that it is not a unimolecular process. Upon heating, decorin shows a single structural transition at a T(m) of 45-46 degrees C but refolds completely upon cooling to 25 degrees C. This property of decorin enabled us to show both by calorimetry and light scattering that dimer to monomer transition coincided with unfolding and monomer to dimer transition coincided with refolding; thus these processes are inextricably linked. We further conclude that folded monomeric biglycan or decorin cannot exist in solution. This implies novel interrelated functions for the parallel beta sheet faces of these leucine-rich repeat proteoglycans, including dimerization and stabilization of protein folding.     

Increase in decorin and biglycan in Duchenne Muscular Dystrophy: role of fibroblasts as cell source of these proteoglycans in the disease

Fibrosis is a common pathological feature observed in muscles of patients with Duchenne muscular dystrophy (DMD). Biglycan and decorin are small chondroitin/dermatan sulfate proteoglycans in the muscle extracellular matrix (ECM) that belong to the family of structurally related proteoglycans called small leucine-rich repeat proteins. Decorin is considered an anti-fibrotic agent, preventing the process by blocking TGF-beta activity. There is no information about their expression in DMD patients. We found an increased amount of both proteoglycans in the ECM of skeletal muscle biopsies obtained from DMD patients. Both biglycan and decorin were augmented in the perimysium of muscle tissue, but only decorin increased in the endomysium as seen by immunohistochemical analyses. Fibroblasts were isolated from explants obtained from muscle of DMD patients and the incorporation of radioactive sulfate showed an increased synthesis of both decorin and biglycan in cultured fibroblasts compared to controls. The size of decorin and biglycan synthesized by DMD and control fibroblasts seems to be similar in size and anion charge. These findings show that decorin and biglycan are increased in DMD skeletal muscle and suggest that fibroblasts would be, at least, one source for these proteoglycans likely playing a role in the muscle response to dystrophic cell damage.     

Proteoglycan and collagen expression during human air conducting system development

The lung is formed from a bud that grows and divides in a dichotomous way. A bud is a new growth center which is determined by epithelial-mesenchymal interactions where proteins of the extracellular matrix (ECM) might be involved. To understand this protein participation during human lung development, we examined the expression and distribution of proteoglycans in relation to the different types of collagens during the period in which the air conducting system is installed. Using light microscopy and immunohistochemistry we evaluate the expression of collagens (I, III and VI) and proteoglycans (decorin, biglycan and lumican) between 8 to 10 weeks post fertilization and 11 to 14 weeks of gestational age of human embryo and fetus lungs. We show that decorin, lumican and all the collagen types investigated were expressed at the epithelium-mesenchymal interface, forming a sleeve around the bronchiolar ducts. In addition, biglycan was expressed in both the endothelial cells and the smooth muscle of the blood vessels. Thus, the similar distribution pattern of collagen and proteoglycans in the early developmental stages of the human lung may be closely related to the process of dichotomous division of the bronchial tree. This study provides a new insight concerning the participation of collagens and proteoglycans in the epithelial-mesenchymal interface during the period in which the air conducting system is installed in the human fetal lung.Key words: human fetal lung, collagens, proteoglycans, extracellular matrix.     

Regulation of pre-adipocyte proliferation and apoptosis by the small leucine-rich proteoglycans, biglycan and decorin

Evidence for a functional role for extracellular matrix (ECM) proteins in adipose tissue is demonstrated in dynamic changes in expression of ECM genes during adipocyte differentiation and in obesity. Components of the ECM may regulate adipose cell number expansion by restricting pre-adipocyte proliferation, regulating apoptosis and inhibiting adipogenesis. Although pre-adipocytes express multiple proteoglycans, their role in pre-adipocyte proliferation up to now has remained unknown. The study described here was conducted to characterize roles of small leucine-rich proteoglycans (SLRPs) in adipocyte proliferation. Pre-adipocytes were seeded on plates coated with biglycan and decorin and were allowed to differentiate. In addition, pre-adipocytes were incubated on plates coated with biglycan, decorin, or fibronectin and measurements were made of cell proliferation and apoptosis. We are able to report that SLRPs decorin and biglycan did not affect differentiation of our 3T3-L1 cells; however, biglycan and decorin did reduce proliferation of pre-adipocytes, partly by induction of apoptosis. Furthermore, anti-proliferative capabilities of decorin and biglycan were nullified with removal of GAG side-chains suggesting that the chains played key roles in anti-proliferative effects of the SLRPs. We also found that co-treatment of decorin or biglycan with the proteoglycan fibronectin restored normal proliferation, an indication that multiple ECM proteins may act in concert to regulate overall proliferation rates of pre-adipocytes. These studies indicate that SLRPs may compose a regulatory factor in adipose tissue expansion, through hyperplasia.     

Fibrils of different collagen types containing immobilised proteoglycans (PGs) as coatings: characterisation and influence on osteoblast behaviour

Collagen, the main organic component of bone, is used as a coating on titanium implants and as a scaffold material in bone tissue engineering. Surface modifications of titanium which promote osteoblast adhesion, proliferation and synthesis of collagen by osteoblasts are desirable. One biomimetic approach is the coating of titanium with collagen in fibrillar form. Other organic components of bone may be bound to fibrils and exert additional effects. In this study, the collagen types I-III were compared regarding their ability to bind the proteoglycans decorin and biglycan, which are found in bone. More collagen was bound to collagen II fibrils than to those of types I and III. Therefore, titanium surfaces were coated with fibrils of collagen type II containing biglycan or decorin or neither to investigate the effect of the proteoglycans on human primary osteoblast behaviour. In addition, the growth factor TGF-beta1 was adsorbed onto surfaces coated with fibrils of collagen type II containing biglycan or decorin or neither to investigate the influence of decorin and biglycan on the effect of TGF-beta1 on osteoblasts. Fibril-bound biglycan and decorin influence primary osteoblast behaviour by themselves. The presence of substrate-bound biglycan or decorin influences the effect of TGF-beta1. These results may be important when designing collagen-based coatings or scaffolds for tissue engineering, including those loaded with growth factors.     

Differential regulation of extracellular matrix proteoglycan (PG) gene expression. Transforming growth factor-beta 1 up-regulates biglycan (PGI), and versican (large fibroblast PG) but down-regulates decorin (PGII) mRNA levels in human fibroblasts in culture

Proteoglycans (PGs) comprise a group of extracellular matrix macromolecules which play an important role in matrix biology. In this study, normal human skin and gingival fibroblast cultures were incubated with transforming growth factor-beta 1 (TGF-beta 1), and the expression of three PGs, viz. biglycan (PGI), decorin (PGII), and versican (a large fibroblast proteoglycan) was examined. The results indicate that TGF-beta 1 (5 ng/ml) markedly increased the expression of biglycan (up to 24-fold) and versican (up to 6-fold) mRNAs and the enhancement of biglycan expression was coordinate with elevated type I procollagen gene expression in the same cultures. In contrast, the expression of decorin mRNA was markedly (up to approximately 70%) inhibited by TGF-beta 1. The response to TGF-beta 1 was similar in both skin and gingival fibroblasts, although the gingival cells were clearly more responsive to stimulation by TGF-beta 1 with respect to biglycan gene expression. Analysis of 35S-labeled proteoglycans in the culture media of skin and gingival fibroblasts also revealed stimulation of biglycan and versican production, and reduction in decorin production. Quantitation of both [35S]sulfate and [3H]leucine-labeled decorin in cell culture media by immunoprecipitation revealed a 50% reduction in decorin production in cell cultures treated with TGF-beta 1. This TGF-beta 1-elicited reduction was accompanied by an apparent increase in the size of the decorin molecules, although the size of the core protein was not altered, as judged by Western immunoblotting following chondroitinase ABC digestion. Analysis of the proteoglycans in the matrix and membrane fractions also revealed increased amounts of versican in cultures treated with TGF-beta 1. These results indicate differential regulation of PG gene expression in fibroblasts by TGF-beta 1, and these observations emphasize the role of PGs in the extracellular matrix biology and pathology.     

Decorin biology, expression, function and therapy in the cornea

Decorin is a small leucine-rich proteoglycan (SLRP) that plays a vital role in many important cellular processes in several tissues including the cornea. A normal constituent of the corneal stroma, decorin is also found in the majority of connective tissues and is related structurally to other small proteoglycans. It interacts with various growth factors such as epidermal growth factor (EGF) and transforming growth factor beta (TGFβ) to regulate processes like collagen fibrillogenesis, extracellular matrix (ECM) compilation, and cell-cycle progression. Studies have linked decorin dysregulation to delayed tissue healing in patients with various diseases including cancer. In the cornea, decorin is involved in the regulation of transparency, a key function for normal vision. It has been reported that mutations in the decorin gene are associated with congenital stromal dystrophy, a disease that leads to corneal opacity and visual abnormalities. Decorin also antagonizes TGFβ in the cornea, a central regulatory cytokine in corneal wound healing. Following corneal injury, increased TGFβ levels induce keratocyte transdifferentiation to myofibroblasts and, subsequently, fibrosis (scarring) in the cornea. We recently reported that decorin overexpression in corneal fibroblasts blocks TGFβ-driven myofibroblast transformation and fibrosis development in the cornea in vitro suggesting that decorin gene therapy can be used for the treatment of corneal scarring in vivo.     

Biglycan and decorin bind close to the n-terminal region of the collagen VI triple helix

The binding of native biglycan and decorin to pepsin-extracted collagen VI from human placenta was examined by solid phase assay and by measurement of surface plasmon resonance in the BIAcore(TM)2000 system. Both proteoglycans exhibited a strong affinity for collagen VI with dissociation constants (K(D)) of approximately 30 nm. Removal of the glycosaminoglycan chains by chondroitinase ABC digestion did not significantly affect binding. In coprecipitation experiments, biglycan and decorin bound to collagen VI and equally competed with the other, suggesting that biglycan and decorin bind to the same binding site on collagen VI. This was confirmed by electron microscopy after negative staining of complexes between gold-labeled proteoglycans and collagen VI, demonstrating that both biglycan and decorin bound exclusively to a domain close to the interface between the N terminus of the triple helical region and the following globular domain. In solid phase assay using recombinant collagen VI fragments, it was shown that the alpha2(VI) chain probably plays a role in the interaction.     

Decorin and biglycan expression: its relation with endothelial heterogeneity

Decorin and biglycan proteoglycans play important roles in the organization of the extracellular matrix, and in the regulation of cell adhesion and migration. Given morphological and functional endothelial heterogeneity, information is needed regarding whether endothelial cells (ECs) from different vascular beds possess different profiles of proteoglycan constituents of the basement membranes. Here, we report that endothelia from different murine organs and EC lines derived thereof produce and secrete different patterns of proteoglycans. A faint colocalization between decorin and PECAM/CD31 was found on tissue sections from mouse heart, lung and kidney by immunofluorescence. Three EC lines derived from these organs produced decorin (100-kDa) and its core protein (45-kDa). Extracellular decorin recognition in culture supernatant was only possible after chondroitin lyase digestion suggesting that the core protein of secreted proteoglycan is more encrypted by glycosaminoglycans than the intracellular one. Heart and lung ECs were able to produce and release decorin. Kidney ECs synthesized the proteoglycan and its core protein but no secretion was detected in culture supernatants. Although biglycan production was recorded in all EC lines, secretion was almost undetectable, consistent with immunofluorescence results. In addition, no biglycan secretion was detected after EC growth supplement treatment, indicating that biglycan is synthesized, secreted and quickly degraded extracellularly by metalloproteinase-2. Low molecular-mass dermatan sulfate was the predominant glycosaminoglycan identified bound to the core protein. ECs from different vascular beds, with differences in morphology, physiology and cell biology show differences in the proteoglycan profile, extending their heterogeneity to potential differences in cell migration capacities.