Effect of Interleukin-1beta and Dehydroepiandrosterone on the Expression of Lumican and Fibromodulin in Fibroblast-Like Synovial Cells of the Human Temporomandibular Joint

Several epidemiological studies have reported that temporomandibular disorders (TMDs) are more prevalent in women than in men. It has recently been proposed that sex hormones such as estrogen, testosterone and dehydroepiandrosterone (DHEA) are involved with the pathogenesis of TMDs. Although studies have investigated the relationship between estrogen and testosterone and the restoration of TMDs, the relationship between DHEA and TMDs is unknown. The synovial tissue of the temporomandibular joint (TMJ) is made up of connective tissue with an extracellular matrix (ECM) composed of collagen and proteoglycan. One proteoglycan family, comprised of small leucine-rich repeat proteoglycans (SLRPs), was found to be involved in collagen fibril formation and interaction. In recent years, the participation of SLRPs such as lumican and fibromodulin in the internal derangement of TMJ has been suggested. Although these SLRPs may contribute to the restoration of the synovium, their effect is still unclear. The purpose of this study was to investigate the effect of DHEA, a sex hormone, on the expression of lumican and fibromodulin in human temporomandibular specimens and in cultured human TMJ fibroblast-like synovial cells in the presence or absence of the pro-inflammatory cytokine interleukin-1beta (IL-1beta). In the in vivo study, both normal and osteoarthritic (OA) human temporomandibular synovial tissues were immunohistochemically examined. In the in vitro study, five fibroblast-like synoviocyte (FLS) cell lines were established from human TMJ synovial tissue of patients with osteoarthritis. The subcultured cells were then incubated for 3, 6, 12 or 24 h with/without IL-1beta (1 ng/mL) in the presence or absence of DHEA (10 μM). The gene expression of lumican and fibromodulin was examined using the real-time polymerase chain reaction (PCR) and their protein expression was examined using immunofluorescent staining. We demonstrated that the expression of lumican differs from that of fibromodulin in synovial tissue and furthermore, that IL-1beta induced a significant increase in lumican mRNA and immunofluorescent staining in FLS compared to cells without IL-1beta. DHEA plus IL-1beta induced a significant increase in fibromodulin, but not in lumican mRNA, compared to DHEA alone, IL-1beta alone and in the absence of DHEA and IL-1beta. In immunofluorescent staining, weaker fibromodulin staining of FLS cells was observed in cells cultured in the absence of both DHEA and IL-1beta compared to fibromodulin staining of cells cultured with DHEA alone, with DHEA plus IL-1beta, or with IL-1beta alone. These results indicate that DHEA may have a protective effect on synovial tissue in TMJ by enhancing fibromodulin formation after IL-1beta induced inflammation. DHEA enhancement of fibromodulin expression may also exert a protective effect against the hyperplasia of fibrous tissue that TGF-beta1 induces. In addition lumican and fibromodulin are differentially expressed under different cell stimulation conditions and lumican and fibromodulin may promote regeneration of the TMJ after degeneration and deformation induced by IL-1beta.Key words: Temporomandibular joint, dehydroepiandrosterone, lumican, fibromodulin, small leucine rich repeat proteoglycan     

Lumican: A new inhibitor of matrix metalloproteinase-14 activity

We previously showed that lumican regulates MMP-14 expression. The aim of this study was to compare the effect of lumican and decorin on MMP-14 activity. In contrast to decorin, the glycosylated form of lumican was able to significantly decrease MMP-14 activity in B16F1 melanoma cells. Our results suggest that a direct interaction occurs between lumican and MMP-14. Lumican behaves as a competitive inhibitor which leads to a complete blocking of the activity of MMP-14. It binds to the catalytic domain of MMP-14 with moderate affinity (K_D ∼ 275 nM). Lumican may protect collagen against MMP-14 proteolysis, thus influencing cell–matrix interaction in tumor progression.     

The small leucine-rich proteoglycan lumican inhibits melanoma progression

Lumican is a member of the small leucine-rich proteoglycan (SLRP) family. It contributes to the organisation of the collagen network and plays an important role in cell migration and tissue repair. The present study aimed to determine the influence of lumican expression on adhesion, anchorage-dependent and -independent growth, migration, in vitro invasion and in vivo melanoma growth. For that purpose, B16F1 mouse melanoma cells were stably transfected with an expression plasmid containing the complete lumican cDNA. Lumican expression by tumor cells did not change the proliferative activity of mouse melanoma cells in monolayer culture and did not influence either cell adhesion to extracellular matrix gel or type I collagen or cell spreading on these substrates. In contrast, lumican-transfected cells were characterized by a strong reduction of their anchorage-independent proliferation in agarose gel and capacity to invade extracellular matrix gel. After subcutaneous injections of transfected B16F1 cells in syngenic mice, lumican expression significantly decreased subcutaneous tumor formation in vivo, with a concomitant decrease of cyclin D1 expression. Lumican induced and/or increased the apoptosis of B16F1 cells. The results suggest that lumican is involved in the control of melanoma growth and invasion and may be considered, like decorin, as an anti-tumor factor from the extracellular matrix.     

Induced metachromasia in bull spermatozoa

Summary The availability of sequential DNA phosphates to bind toluidine blue molecules after acid hydrolysis was studied in normally shaped and misshaped spermatozoa from subfertile and highly fertile bulls. The aim was to associate induced spermatozoal metachromasia with infertility. Some few normally and abnormally shaped cells from highly fertile bulls exhibited an induced metachromasia after being treated with 4N HCl for 10–30 min at 25°C prior to staining. Subfertile bulls contained 12 times as many metachromatic spermatozoa as highly fertile animals. The induced toluidine blue metachromasia is suggested as a rapid and simple method for detecting bull spermatozoa bearing an anomalous DNA-protein complex. This nucleoprotein complex was found to be more frequent in subfertile bulls.     

Lumican is a major small leucine-rich proteoglycan (SLRP) in Atlantic cod (Gadus morhua L.) skeletal muscle

Knowledge on fish matrix biology is important to ensure optimal fish -quality, -growth and -health in aquaculture. The aquaculture industry face major challenges related to matrix biology, such as inflammations and malformations. Atlantic cod skeletal muscle was investigated for collagen I, decorin, biglycan, and lumican expression and distribution by real-time PCR, immunohistochemical staining and Western blotting. Immunohistochemical staining and Western immunoblotting were also performed using antibodies against glycosaminoglycan side chains of these proteoglycans, in addition to fibromodulin. Real-time PCR showed highest mRNA expression of lumican and collagen I. Collagen I and proteoglycan immunohistochemical staining revealed distinct thread-like structures in the myocommata, with the exception of fibromodulin, which stained in dense structures embedded in the myocommata. Chondroitinase AC-generated epitopes stained more limited than cABC-generated epitopes, indicating a stronger presence of dermatan sulfate than chondroitin sulfate in cod muscle. Lumican and keratan sulfate distribution patterns were strong and ubiquitous in endomysia and myocommata. Western blots revealed similar SLRPs sizes in cod as are known from mammals. Staining of chondroitin/dermatan sulfate epitopes in Western blots were similar in molecular size to those of decorin and biglycan, whereas staining of keratan sulfate epitopes coincided with expected molecular sizes of lumican and fibromodulin. In conclusion, lumican was a major proteoglycan in cod muscle with ubiquitous distribution overlapping with keratan sulfate. Other leucine-rich proteoglycans were also present in cod muscle, and Western blot using antibodies developed for mammalian species showed cross reactivity with fish, demonstrating similar structures and molecular weights as in mammals.     

人颈椎间盘髓核细胞的体外培养和鉴定

目的：建立人颈椎间盘髓核细胞体外培养体系,并对其细胞表型进行鉴定。方法：采用酶消化法分离人颈椎间盘髓核细胞,进行单层培养,倒置相差显微镜观察细胞生长和形态,流式细胞仪测定细胞周期和凋亡率,并行甲苯胺蓝、Ⅱ型胶原及CK8免疫组化染色对其细胞表型进行鉴定。结果：原代髓核细胞凋亡率6.1±1.4%,S期细胞比例7.3±0.5%。贴壁后形态为多角形或短楔形,传代后生长加速。细胞呈甲苯胺蓝异染性;Ⅱ型胶原免疫组化染色阳性;只有少量椭圆形大细胞CK8免疫组化染色阳性。结论：成功建立人颈椎间盘髓核细胞体外培养模型,并证实成年后髓核内仍有少量细胞保持脊索细胞表型。     

Effect of Lumican on the Migration of Human Mesenchymal Stem Cells and Endothelial Progenitor Cells: Involvement of Matrix Metalloproteinase-14

Background

Increasing number of evidence shows that soluble factors and extracellular matrix (ECM) components provide an optimal microenvironment controlling human bone marrow mesenchymal stem cell (MSC) functions. Successful in vivo administration of stem cells lies in their ability to migrate through ECM barriers and to differentiate along tissue-specific lineages, including endothelium. Lumican, a protein of the small leucine-rich proteoglycan (SLRP) family, was shown to impede cell migration and angiogenesis. The aim of the present study was to analyze the role of lumican in the control of MSC migration and transition to functional endothelial progenitor cell (EPC).

Methodology/Principal Findings

Lumican inhibited tube-like structures formation on Matrigel® by MSC, but not EPC. Since matrix metalloproteinases (MMPs), in particular MMP-14, play an important role in remodelling of ECM and enhancing cell migration, their expression and activity were investigated in the cells grown on different ECM substrata. Lumican down-regulated the MMP-14 expression and activity in MSC, but not in EPC. Lumican inhibited MSC, but not EPC migration and invasion. The inhibition of MSC migration and invasion by lumican was reversed by MMP-14 overexpression.

Conclusion/Significance

Altogether, our results suggest that lumican inhibits MSC tube-like structure formation and migration via mechanisms that involve a decrease of MMP-14 expression and activity.     

The immunoreactivity of a chimeric multi-epitope DNA vaccine against IBV in chickens

Lumican is a major proteoglycans of the human cornea. Lumican knock-out mice have been shown to lose corneal transparency and to display delayed wound healing. The purpose of this study was to define the role of lumican in corneal epithelial cell migration. Over-expression of lumican in human corneal epithelial (HCE-T) cells increased both cell migration and proliferation, and increased levels of integrins α2 and β1. ERK 1/2 was also activated in lumican over-expressed cells. When we treated HCE-T cells with the ERK-specific inhibitor U0126, cell migration and the expression of integrin β1 were completely blocked. These data provide evidence that lumican stimulates cell migration in the corneal epithelium by activating ERK 1/2, and point to a novel signaling pathway implicated in corneal epithelial cell migration.     

人毛乳头细胞组织化学研究

毛乳头细胞是一种高度特殊化的成纤维细胞。本文通过对体外培养的毛乳头细胞进行组织化学染色研究发现,它对阿新蓝、甲苯胺蓝和PAS染色均呈阳性,并对甲苯胺蓝显异染性．与原位时的细胞染色结果相同,表明在体外培养下．毛乳头细胞合成和分泌酸性、中性粘多糖的能力仍能维持较长时间;在细胞聚集区和多层化细胞团中有丰富的细胞外基质,阿新蓝和PAS染色呈强阳性,说明细胞外基质的存在与毛乳头细胞的聚集有很大关系;另外毛囊真皮鞘细胞对阿新蓝、甲苯胺蓝染色呈阳性反应．无甲苯胺蓝的异染性,PAS染色阴性,而真皮成纤维细胞这些染色均阴性,说明它与毛乳头细胞关系密切。     

Role of polyamines in expression of the differentiated phenotype of chondrocytes in culture

Addition of putrescine to cultures of rabbit costal chondrocytes during the log-phase stimulated expression of their differentiated phenotype, as judged by increase in the synthesis of glycosaminoglycans and of metachromasia with toluidine blue staining. Putrescine stimulated glycosaminoglycan synthesis maximally at a concentration of 10(-7) M, and spermidine and spermine were also effective at similar concentration. These polyamines had little effect on DNA synthesis, RNA synthesis, DNA accumulation, or protein accumulation. These findings suggest that polyamines may be important in the expression of the differentiated phenotype of chondrocytes in culture.     

胶原蛋白/BMP复合材料的制备和成骨性能研究

以胶原膜(含87.5 mg I型胶原蛋白)为载体, 复合3.5 mg rhBMP-2(人基因重组骨形成蛋白-2), 制备胶原蛋白/BMP复合材料。复合材料首先在兔背阔肌中埋置, 预构新生骨组织, 并采用ALP染色、Von Kossa染色和HE染色等观察复合材料的成骨过程和组织形态。然后将形成的新骨组织游离移植修复自体下颌骨体部洞穿性缺损; 并设以胶原为载体的rhBMP-2复合骨修复材料直接修复为对照组, 骨缺损不修复组为空白组。采用X线、抗压强度、硬组织切片、四环素荧光染色、骨形态计量检查, 观察复合材料修复骨缺损的质量和效果。结果表明, 胶原蛋白/BMP复合材料在兔背阔肌中4～6周成骨, 胶原材料于3～5周降解; 成骨过程为是以软骨成骨为主的方式, 新骨形态为编织骨, 可见明显的微血管分布; 游离移植修复自体下颌骨缺损, 6周缺损区为骨性愈合, 与对照组在抗压强度(P = 0.041)、新骨量(P = 0.034)均有显著性差异。胶原蛋白/BMP复合材料在骨骼肌中形成的新生骨组织可作为供骨修复一定范围的骨缺损。     

胶原蛋白/BMP复合材料的制备和成骨性能研究

以胶原膜(含87.5 mg I型胶原蛋白)为载体, 复合3.5 mg rhBMP-2(人基因重组骨形成蛋白-2), 制备胶原蛋白/BMP复合材料。复合材料首先在兔背阔肌中埋置, 预构新生骨组织, 并采用ALP染色、Von Kossa染色和HE染色等观察复合材料的成骨过程和组织形态。然后将形成的新骨组织游离移植修复自体下颌骨体部洞穿性缺损; 并设以胶原为载体的rhBMP-2复合骨修复材料直接修复为对照组, 骨缺损不修复组为空白组。采用X线、抗压强度、硬组织切片、四环素荧光染色、骨形态计量检查, 观察复合材料修复骨缺损的质量和效果。结果表明, 胶原蛋白/BMP复合材料在兔背阔肌中4～6周成骨, 胶原材料于3～5周降解; 成骨过程为是以软骨成骨为主的方式, 新骨形态为编织骨, 可见明显的微血管分布; 游离移植修复自体下颌骨缺损, 6周缺损区为骨性愈合, 与对照组在抗压强度(P = 0.041)、新骨量(P = 0.034)均有显著性差异。胶原蛋白/BMP复合材料在骨骼肌中形成的新生骨组织可作为供骨修复一定范围的骨缺损。     

Lumican Regulates Collagen Fibril Assembly: Skin Fragility and Corneal Opacity in the Absence of Lumican

Lumican, a prototypic leucine-rich proteoglycan with keratan sulfate side chains, is a major component of the cornea, dermal, and muscle connective tissues. Mice homozygous for a null mutation in lumican display skin laxity and fragility resembling certain types of Ehlers-Danlos syndrome. In addition, the mutant mice develop bilateral corneal opacification. The underlying connective tissue defect in the homozygous mutants is deregulated growth of collagen fibrils with a significant proportion of abnormally thick collagen fibrils in the skin and cornea as indicated by transmission electron microscopy. A highly organized and regularly spaced collagen fibril matrix typical of the normal cornea is also missing in these mutant mice. This study establishes a crucial role for lumican in the regulation of collagen assembly into fibrils in various connective tissues. Most importantly, these results provide a definitive link between a necessity for lumican in the development of a highly organized collagenous matrix and corneal transparency.     

Lumican is a major proteoglycan component of the bone matrix.

MC3T3-E1 mouse calvaria cells are a clonal population of committed osteoprogenitors that in the presence of appropriate supplements form a mineralized bone matrix. The development of the MC3T3-E1 cells can be divided into three major stages, namely, proliferation, differentiation, and mineralization. Recently, using the cDNA microarray technology we found lumican to be abundantly expressed during the mineralization and differentiation stages of the MC3T3-E1 development and not during the proliferation stage. Lumican has been shown to play essential roles in regulating collagen fibril formation in different extracellular matrices but its expression in the developing bone matrix remains elusive. By examining the expression profile of this gene during the different stages of MC3T3-E1 development, utilizing the 'real-time' PCR technology, we observed that the expression of lumican increases as the osteoblast culture differentiates and matures, suggesting that lumican may be involved in regulating collagen fibrillogenesis in bone matrices. Using immunostaining, we observed that during the early embryonic development of mouse (E11 to E13), lumican is mainly expressed in the cartilaginous matrices. However, in the older embryos (E14 to E16), the expression of lumican is more prominent in the developing bone matrices. Our data suggest that lumican is a significant proteoglycan component of bone matrix, which is secreted by differentiating and mature osteoblasts only and therefore it can be used as a marker to distinguish proliferating pre-osteoblasts from the differentiating osteoblasts.     

Roles of lumican and keratocan on corneal transparency

Lumican and keratocan are members of the small leucine-rich proteoglycan (SLRP) family, and are the major keratan sulfate (KS) proteoglycans in corneal stroma. Both lumican and keratocan are essential for normal cornea morphogenesis during embryonic development and maintenance of corneal topography in adults. This is attributed to their bi-functional characteristic (protein moiety binding collagen fibrils to regulate collagen fibril diameters, and highly charged glycosaminoglycan (GAG) chains extending out to regulate interfibrillar spacings) that contributes to their regulatory role in extracellular matrix assembly. The absence of lumican leads to formation of cloudy corneas in homozygous knockout mice due to altered collagenous matrix characterized by larger fibril diameters and disorganized fibril spacing. In contrast, keratocan knockout mice exhibit thin but clear cornea with insignificant alteration of stromal collaegenous matrix. Mutations of keratocan cause cornea plana in human, which is often associated with glaucoma. These observations suggest that lumican and keratocan have different roles in regulating formation of stromal extracellular matrix. Experimental evidence indicates that lumican may have additional biological functions, such as modulation of cell migration and epithelium-mesenchyme transition in wound healing and tumorgenesis, besides regulating collagen fibrillogenesis. Published in 2003.     

A novel role of the lumican core protein in bacterial lipopolysaccharide-induced innate immune response

Lumican is an extracellular matrix protein modified as a proteoglycan in some tissues. The core protein with leucine-rich repeats, characteristic of the leucine-rich-repeat superfamily, binds collagen fibrils and regulates its structure. In addition, we believe that lumican sequestered in the pericellular matrix interacts with cell surface proteins for specific cellular functions. Here we show that bacterial lipopolysaccharide sensing by the Toll-like receptor 4 signaling pathway and innate immune response is regulated by lumican. Primary cultures of lumican-deficient (Lum(-/-)) macrophages show impaired innate immune response to lipopolysaccharides with lower induction of tumor necrosis factor alpha (TNFalpha) and interleukin-6. Macrophage response to other pathogen-associated molecular patterns is not adversely affected by lumican deficiency, suggesting a specific role for the lumican core protein in the Toll-like receptor 4 pathway. An exogenous recombinant lumican core protein increases lipopolysaccharide-mediated TNFalpha induction and partially rescues innate immune response in Lum(-/-) macrophages. We further show that the core protein binds lipopolysaccharide. Immunoprecipitation of lumican from peritoneal lavage co-precipitates CD14, a cell surface lipopolysaccharide-binding protein that is involved in its presentation to Toll-like receptor 4. The Lum(-/-) mice are hypo-responsive to lipopolysaccharide-induced septic shock, with poor induction of pro-inflammatory cytokines, TNFalpha, and interleukins 1beta and 6 in the serum. Taken together, the data indicates a novel role for lumican in the presentation of bacterial lipopolysaccharide to CD14 and host response to this bacterial endotoxin.     

Lumican inhibits collagen deposition in tissue engineered cartilage

Lumican is a glycoprotein that is found in the extracellular matrix of many connective tissues, including cartilage. It is a member of the small leucine-rich repeat proteoglycans family and along with two others, decorin and fibromodulin, has the capacity to bind to fibrillar collagens and limit their growth. Cartilage tissue engineering provides a potential method for the production of three-dimensional tissue for implantation into eroded joints. Many studies have demonstrated the growth of cartilage in vitro. However in all cases, biochemical analysis of the tissue revealed a significant deficit in the collagen content. We have now tested the hypothesis that the reduced collagen accumulation in engineered cartilage is a result of over-expression of decorin, fibromodulin or lumican. We have found that the lumican gene and protein are both over-expressed in engineered compared to natural cartilage whereas this is not the case for decorin or fibromodulin. Using a small hairpin lumican antisense sequence we were able to knockdown the lumican gene and protein expression in chondrocytes being used for tissue engineering. This resulted in increased accumulation of type II collagen (the major collagen of cartilage) whilst there was no significant alteration in the proteoglycan content. Furthermore, the antisense knockdown of lumican resulted in an increase in the average collagen fibril diameter measured by transmission electron microscopy. These results suggest that lumican plays a pivotal role in the development of tissue engineered cartilage and that regulation of this protein may be important for the production of high-quality implants.