Small Leucine-rich Repeat Proteoglycan Research Articles

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.

Reciprocity in breast cancer progression.

Reciprocity in breast cancer progression.

Tsukushi is involved in the wound healing by regulating the expression of cytokines and growth factors

During the wound-healing process, macrophages, fibroblasts, and myofibroblasts play a leading role in shifting from the inflammation phase to the proliferation phase, although little is known about the cell differentiation and molecular control mechanisms underlying these processes. Previously, we reported that Tsukushi (TSK), a member of the small leucine-rich repeat proteoglycan family, functions as a key extracellular coordinator of multiple signalling networks. In this study, we investigated the contribution of TSK to wound healing. Analysis of wound tissue in heterozygous TSK-lacZ knock-in mice revealed a pattern of sequential TSK expression from macrophages to myofibroblasts. Quantitative PCR and in vitro cell induction experiments showed that TSK controls macrophage function and myofibroblast differentiation by inhibiting TGF-β1 secreted from macrophages. Our results suggest TSK facilitates wound healing by maintaining inflammatory cell quiescence.

The tyrosine sulfate domain of fibromodulin represents a new binding site to collagen enhancing the interaction and fibril formation

Purpose: Fibromodulin is a collagen modulating protein belonging to the family of the small leucine rich repeat proteoglycans. The collagen modulating effect is demonstrated by in vitro fibrillogenesis assay, resulting in retardation of turbidity compared to fibrillogenesis by collagen alone. Fibromodulin binds collagen via the LRR domain, but whether the binding via the LRR has any role in the assembly of the collagen is not clear. Fibromodulin null mice have larger population of thinner collagen fibers in ligaments compared to the wild type mice and develop osteoarthritis due to joint instability, indicating a regulatory role by fibromodulin in vivo as well. The N-terminal domain of fibromodulin has a highly negative charge due to the presence of the posttranslational modification O-sulfation on tyrosine residues that binds heparin binding proteins. The aim of this work is to investigate binding of this domain to collagen and its role in the regulation of collagen fibril assembly. Methods: Different variants of fibromodulin (full length, the N-terminal domain of fibromodulin and N-terminal truncated fibromodulin) were used to study interaction to collagen type I in solid phase assay and surface plasmon resonance assay. Fibrillogenesis assay was used to monitor changes in turbidity over time in the presence of the different variants of fibromodulin. Samples were collected from the fibril formation and visualized by EM negative staining. Specific binding sites for fibromodulin on the collagen monomers by detection with a gold-labeled fibromodulin specific antibody was also identified by EM negative staining. Results: In solid phase assay, all three variants of fibromodulin bind to coated collagen type I, showing a novel binding site located in the N-terminal domain of fibromodulin. By surface plasmon resonance a distinct difference in the dissociation phase is observed. The dissociation of full-length fibromodulin is much slower compared to the N-terminal truncated variant of fibromodulin, indicating contribution of both the N-terminal site and the LRR-domain sites in binding to the collagen molecule. In an in vitro fibrillogenesis assay, full-length and N-terminal truncated fibromodulin delays the fibril formation. Adding the N-terminal tyrosin sulfate rich domain instead accelerates the fibril formation, indicating that this domain binds and facilitates collagen monomer assembly. Samples taken after 24 h of fibril formation in the presence of the different fibromodulin variants or PBS were analysed by electron microscopy. Full-length fibromodulin shows the thinnest fibers, but with the most mature cross-striation. Presence of the N-terminal also results in thinner fibers compared to the control and the N-terminal truncated variant but showed less maturation. Further, all three variants bind to three common sites on the collagen type I molecule, visualized by electron microscopy. An informative observation by the electron microscopy is the finding of full-length fibromodulin binding two collagen molecules at the same time. This shows engagement of the N-terminal and one of the binding sites in the LRR region, concomitantly binding different collagen molecules, which can be important for the assembly. Conclusion: This work shows that the tyrosine sulfate rich domain of fibromodulin contributes to the high affinity binding of the fibromodulin molecule to collagen. It influences and contributes to the arrangement of the collagen molecules, which results in well defined, highly organized collagen fibers.

Extracellular matrix remodeling in early cardiac fibrosis and diastolic dysfunction

Background: Half of the patients with congestive heart failure suffer from diastolic dysfunction with persistent left ventricular function (HFpEF). Until today different explanations exist, but the exact mechanism of HFpEF is still unclear. From human biopsies and different animal models it is known that in HFpEF the extracellular matrix underlies remodeling processes with increased fibrosis and disturbance of cardiac architecture. Our study aimed to characterize changes of the extracellular matrix in early cardiac fibrosis and diastolic dysfunction. Methods: In C57Bl/6-mice, osmotic minipumps were implanted with a continuous infusion of Angiotensin II (Ang, 0.5μg/kg/min) or NaCl (Sham). After assessment of systolic and diastolic parameters using echocardiography at day 7 or 28, mice were euthanized (Ang7d, n=22/Ang28d, n=27/Sham, n=20). Cardiac tissue was investigated with micro array technology, western blot, qRT-PCR and immunohistochemistry. Results: Echocardiography revealed a reliable induction of diastolic dysfunction with persistent LVEF in the Ang groups (E/E': Sham 25.9±1.0 vs. Ang28d 38.5±4.4; p<0.01). BNP expression was significantly increased (Sham 0.7±0.1 vs. Ang28d 1.9±0.4; p<0.05). Histochemical examination showed a significantly elevated interstitial fibrosis with significant increase of Vimentin positive cardiac fibroblasts. Expressions of Collagen 1, 3, 4, 6 and 14 were increased. By using mRNA micro array technology different targets of the extracellular remodeling process were identified and validated by qRT-PCR. The expressions of the collagen maturation enzyme LOX and of the fibrillogenesis promoting proteins Sparc were significantly increased. Fibromodulin, which is known to regulate collagen fibril diameters, was also elevated (Sham 2.3±0.6 vs. Ang28d 14.5±3.9; p<0.05). Expressions of the Small leucine-rich repeat proteoglycan Decorin and Osteoglycin were reduced after 7 days, while Osteopontin showed significantly elevated expression. Lumican and Asporin were unchanged. The expression of adhesion permitting protein Tenascin C was significantly elevated. Interestingly, expression of the Cartilage Intermediate Layer Protein (CILP), which is only known to take part in the cartilage remodeling process, was clearly increased (Sham 1.9±0.3 vs. Ang28d 8.3±1.9; p<0.01). Conclusion: The Angiotensin induced cardiac fibrosis was composed by an intensified fibrillogenesis and a changed composition of SLRPs. Furthermore, the significantly elevated expression of CILP suggests a contribution of this extracellular protein to the process of cardiac fibrosis and diastolic dysfunction.

Characterization of opticin digestion by proteases involved in osteoarthritis development

ObjectiveOpticin is a class III member of the small leucine-rich repeat proteoglycan (SLRP) family, produced in articular joint tissues. In normal and osteoarthritic (OA) cartilage, opticin is degraded. This study aimed to assess whether human cartilage opticin is degraded by the main proteases involved in OA pathophysiology, and to determine the protease cleavage sites of this SLRP. MethodsWe analyzed the proteolytic activity of matrix metalloproteinases (MMPs)-1, -2, -3, -7, -8 and -9, and ADAMTS-4 and -5 on proteoglycan extracts from normal and moderately fibrillated OA human cartilage, and on recombinant human opticin. Opticin degradation was analyzed by Western blotting and cleavage sites were determined by sequence analysis. ResultsAll eight proteases digested opticin from proteoglycan extracts from both normal and OA samples, as well as recombinant human opticin, MMP-2 and MMP-7 are the proteases that degrade recombinant human opticin most efficiently. The opticin cleavage site determined for these MMPs was between the glycosylation and leucine-rich repeat domains. MMP-7 had two additional digestion sites near the N-terminal end of opticin. ConclusionOpticin is a substrate for several MMPs and aggrecanases involved during OA cartilage degradation, and seems to be a preferential substrate for MMP-7. The role of opticin in cartilage degeneration could be related to decreased levels of intact opticin, followed by its proteolytic degradation, which in turn may stimulate some of the modifications observed in the OA cartilage, such as neovascularisation and changes in the extracellular matrix.

Quantitative analysis of type I collagen fibril regulation by lumican and decorin using AFM

Lumican and decorin, two members of the small leucine-rich repeat proteoglycan (SLRP) family, have been implicated as regulators of collagen I fibril structure in different tissues. Both proteoglycans consist of a core protein and a glycosaminoglycan (GAG) chain, but quantitative information regarding the precise role of the protein and GAG moieties in regulating collagen structure is still limited. In this study, we used AFM imaging and a model system of aligned collagen I nanofibrils to investigate the role of lumican and decorin on collagen I fibril structure with high resolution. When co-assembled with collagen I, recombinant lumican or decorin proteins lacking the GAG chains decreased collagen fibril width to values below <100nm and increased interfibrillar spacing in a dose-dependent manner. At lower concentrations, lumican appeared to have a stabilizing effect on newly-formed collagen fibrils, while at higher concentrations both lumican and decorin inhibited collagen fibrillogenesis. GAG-containing decorin also increased interfibrillar spacing, decreased fibril width and ultimately inhibited fibrillogenesis, but these effects required lower concentrations compared to recombinant decorin, indicating that the decorin core protein alone cannot compensate for the full regulatory and structural contribution of the GAG chain during collagen I fibrillogenesis. Using a 2D autocorrelation approach, we furthermore analyzed and compared the effects of recombinant and glycosylated decorin on collagen ultrastructure, providing a quantitative measure for the observed structural differences. AFM analysis of ordered fibrillar collagen arrays in combination with quantitative autocorrelation image analysis thus provides a useful tool for investigating SLRP-dependent nanoscale effects on collagen fibril structure.

Correction: Degradation of small leucine-rich repeat proteoglycans by matrix metalloprotease 13 - identification of a new biglycan cleavage site

Correction: Degradation of small leucine-rich repeat proteoglycans by matrix metalloprotease 13 - identification of a new biglycan cleavage site

Mapping the Differential Distribution of Proteoglycan Core Proteins in the Adult Human Retina, Choroid, and Sclera

To examine the presence and distribution of proteoglycan (PG) core proteins in the adult human retina, choroid, and sclera. Postmortem human eye tissue was dissected into Bruch's membrane/choroid complex, isolated Bruch's membrane, or neurosensory retina. PGs were extracted and partially purified by anion exchange chromatography. Trypsinized peptides were analyzed by tandem mass spectrometry and PG core proteins identified by database search. The distribution of PGs was examined by immunofluorescence microscopy on human macular tissue sections. The basement membrane PGs perlecan, agrin, and collagen-XVIII were identified in the human retina, and were present in the internal limiting membrane, blood vessel walls, and Bruch's membrane. The hyalectans versican and aggrecan were also detected. Versican was identified in Bruch's membrane, while aggrecan was distributed throughout the retina, choroid, and sclera. The cartilage link protein HAPLN1 was abundant in the interphotoreceptor matrix and sclera, while HAPLN4 (brain link protein 2) was found throughout the retina and choroid. The small leucine-rich repeat PG (SLRP) family members biglycan, decorin, fibromodulin, lumican, mimecan, opticin, and prolargin were present, with different patterns of distribution in the retina, choroid, and sclera. A combination of proteomics and immunohistochemistry approaches has provided for the first time a comprehensive analysis of the presence and distribution of PG core proteins throughout the human retina, choroid, and sclera. This complements our knowledge of glycosaminoglycan chain distribution in the human eye, and has important implications for understanding the structure and functional regulation of the eye in health and disease.

Tsukushi controls the hair cycle by regulating TGF-β1 signaling

The hair follicle contains stem/progenitor cells that supply progeny for skin development and the hair cycle. Several signaling molecules belonging to the Wnt, BMP, shh, and transforming growth factor β (TGF-β) signaling cascades are involved in the normal hair follicle cycle. However, the systemic mechanism of how these humoral factors are controlled remains largely unknown. Previously, we reported that Tsukushi (TSK), a member of the small leucine-rich repeat proteoglycan family, functions extracellularly as a key coordinator of multiple signaling networks. Here, we show that TSK is expressed at the restricted areas of hair follicle during the morphogenesis and the hair cycle. Targeted disruption of the TSK gene causes the hair cycle to be delayed with low levels of TGF-β1 and phosphorylated Smad2/3 (pSmad2/3) expression. Biochemical analysis indicates that TSK directly binds to TGF-β1. Our data suggest that TSK controls the hair cycle by regulating TGF-β1 signaling.

Extracellular Matrix Lumican Promotes Bacterial Phagocytosis, and Lum−/− Mice Show Increased Pseudomonas aeruginosa Lung Infection Severity

Phagocytosis is central to bacterial clearance, but the exact mechanism is incompletely understood. Here, we show a novel and critical role for lumican, the connective tissue extracellular matrix small leucine-rich repeat proteoglycan, in CD14-mediated bacterial phagocytosis. In Psuedomonas aeruginosa lung infections, lumican-deficient (Lum(-/-)) mice failed to clear the bacterium from lungs, tissues, and showed a dramatic increase in mortality. In vitro, phagocytosis of nonopsonized gram-negative Escherichia coli and P. aeruginosa was inhibited in Lum(-/-) peritoneal macrophages (MΦs). Lumican co-localized with CD14, CD18, and bacteria on Lum(+/+) MΦ surfaces. Using two different P. aeruginosa strains that require host CD14 (808) or CD18/CR3 (P1) for phagocytosis, we showed that lumican has a larger role in CD14-mediated phagocytosis. Recombinant lumican (rLum) restored phagocytosis in Lum(-/-) MΦs. Surface plasmon resonance showed specific binding of rLum to CD14 (K(A) = 2.15 × 10(6) M(-1)), whereas rLumY20A, and not rLumY21A, where a tyrosine in each was replaced with an alanine, showed 60-fold decreased binding. The rLumY20A variant also failed to restore phagocytosis in Lum(-/-) MΦs, indicating Tyr-20 to be functionally important. Thus, in addition to a structural role in connective tissues, lumican has a major protective role in gram-negative bacterial infections, a novel function for small leucine-rich repeat proteoglycans.

Opticin Exerts Its Anti-angiogenic Activity by Regulating Extracellular Matrix Adhesiveness

Opticin is an extracellular matrix glycoprotein that we identified associated with the collagen network of the vitreous humor of the eye. Recently, we discovered that opticin possesses anti-angiogenic activity using a murine oxygen-induced retinopathy model: here, we investigate the underlying mechanism. Using an ex vivo chick chorioallantoic membrane assay, we show that opticin inhibits angiogenesis when stimulated by a range of growth factors. We show that it suppresses capillary morphogenesis, inhibits endothelial invasion, and promotes capillary network regression in three-dimensional matrices of collagen and Matrigel(TM). We then show that opticin binds to collagen and thereby competitively inhibits endothelial cell interactions with collagen via α(1)β(1) and α(2)β(1) integrins, thereby preventing the strong adhesion that is required for proangiogenic signaling via these integrins.

Abstract 5275: Biglycan is a specific marker and an autocrine angiogenic factor of tumor endothelial cells

Abstract Tumor angiogenesis is necessary for solid tumor progression and metastasis. Tumor blood vessels are morphologically different from their normal counterparts. We isolated tumor endothelial cells (TECs), demonstrated their abnormalities, compared gene expression profiles of TECs and normal endothelial cells (NECs) by microarray analysis and identified several genes upregulated in TECs. We focused on the gene encoding biglycan, a small leucine-rich repeat proteoglycan. Biglycan is overexpressed in inflammation and fibrosis. However, there has been no report about the expression or the function of biglycan in TEC. In this study, we investigated the expression and the role of biglycan in TECs. Real-time PCR, western blotting and immunocytochemistry revealed higher biglycan expression levels in TECs than in NECs. Furthermore, we confirmed that biglycan was secreted from TECs. Biglycan knockdown inhibited cell migration and tube formation in TECs. TLR2 and TLR4 are the biglycan receptors. TLR2 and TLR4 blocking antibodies suppressed biglycan mediated cell migration and tube formation. We isolated TECs from human renal cell carcinoma tissue and NECs from normal renal tissue in the same patients. TECs and NECs were obtained from six patients. Real-time RT-PCR revealed that the biglycan expression levels were significantly higher in four of the six TEC samples than in the corresponding NEC samples. Furthermore, immunostaining revealed strong biglycan expression in vivo in several human tumor vessels, as in mouse TECs. Biglycan was detected in the sera of cancer patients but was hardly detected in those of healthy volunteers. Biglycan is an autocrine angiogenic factor stimulating tumor endothelial cell migration and tube formation. These findings suggested that biglycan is a novel TEC marker and a target for anti-angiogenic therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5275. doi:1538-7445.AM2012-5275

Topological Analysis of Small Leucine-Rich Repeat Proteoglycan Nyctalopin

Nyctalopin is a small leucine rich repeat proteoglycan (SLRP) whose function is critical for normal vision. The absence of nyctalopin results in the complete form of congenital stationary night blindness. Normally, glutamate released by photoreceptors binds to the metabotropic glutamate receptor type 6 (GRM6), which through a G-protein cascade closes the non-specific cation channel, TRPM1, on the dendritic tips of depolarizing bipolar cells (DBCs) in the retina. Nyctalopin has been shown to interact with TRPM1 and expression of TRPM1 on the dendritic tips of the DBCs is dependent on nyctalopin expression. In the current study, we used yeast two hybrid and biochemical approaches to investigate whether murine nyctalopin was membrane bound, and if so by what mechanism, and also whether the functional form was as a homodimer. Our results show that murine nyctalopin is anchored to the plasma membrane by a single transmembrane domain, such that the LRR domain is located in the extracellular space.

Small leucine rich proteoglycan family regulates multiple signalling pathways in neural development and maintenance

The small leucine-rich repeat proteoglycan (SLRPs) family of proteins currently consists of five classes, based on their structural composition and chromosomal location. As biologically active components of the extracellular matrix (ECM), SLRPs were known to bind to various collagens, having a role in regulating fibril assembly, organization and degradation. More recently, as a function of their diverse proteins cores and glycosaminoglycan side chains, SLRPs have been shown to be able to bind various cell surface receptors, growth factors, cytokines and other ECM components resulting in the ability to influence various cellular functions. Their involvement in several signaling pathways such as Wnt, transforming growth factor-β and epidermal growth factor receptor also highlights their role as matricellular proteins. SLRP family members are expressed during neural development and in adult neural tissues, including ocular tissues. This review focuses on describing SLRP family members involvement in neural development with a brief summary of their role in non-neural ocular tissues and in response to neural injury.

Biglycan is a specific marker and an autocrine angiogenic factor of tumour endothelial cells

Background:We isolated tumour endothelial cells (TECs), demonstrated their abnormalities, compared gene expression profiles of TECs and normal endothelial cells (NECs) by microarray analysis and identified several genes upregulated in TECs. We focused on the gene encoding biglycan, a small leucine-rich repeat proteoglycan. No report is available on biglycan expression or function in TECs.Methods:The NEC and TEC were isolated. We investigated the biglycan expression and function in TECs. Western blotting analysis of biglycan was performed on sera from cancer patients.Results:Biglycan expression levels were higher in TECs than in NECs. Biglycan knockdown inhibited cell migration and caused morphological changes in TECs. Furthermore, immunostaining revealed strong biglycan expression in vivo in human tumour vessels, as in mouse TECs. Biglycan was detected in the sera of cancer patients but was hardly detected in those of healthy volunteers.Conclusion:These findings suggested that biglycan is a novel TEC marker and a target for anti-angiogenic therapy.

Podocan-like protein: A novel small leucine-rich repeat matrix protein in bone

Recently, significant attention has been drawn to the biology of small leucine-rich repeat proteoglycans (SLRPs) due to their multiple functionalities in various cell types and tissues. Here, we characterize a novel SLRP member, “Podocan-like (Podnl) protein” identified by a bioinformatics approach. The Podnl protein has a signal peptide, a unique cysteine-rich N-terminal cluster, 21 leucine-rich repeat (LRR) motifs, and one putative N-glycosylation site. This protein is structurally similar to podocan in SLRPs. The gene was highly expressed in mineralized tissues and in osteoblastic cells and the high expression level was observed at and after matrix mineralization in vitro. Podnl was enriched in newly formed bones based on immunohistochemical analysis. When Podnl was transfected into osteoblastic cells, the protein with N-glycosylation was detected mainly in the cultured medium, indicating that Podnl is a secreted N-glycosylated protein. The endogenous Podnl protein was also present in bone matrix. These data provide a new insight into our understanding of the emerging SLRP functions in bone formation.

Deficiency of Biglycan Causes Cardiac Fibroblasts to Differentiate into a Myofibroblast Phenotype

Myocardial infarction (MI) is followed by extracellular matrix (ECM) remodeling, which is on the one hand required for the healing response and the formation of stable scar tissue. However, on the other hand, ECM remodeling can lead to fibrosis and decreased ventricular compliance. The small leucine-rich proteoglycan (SLRP), biglycan (bgn), has been shown to be critically involved in these processes. During post-infarct remodeling cardiac fibroblasts differentiate into myofibroblasts which are the main cell type mediating ECM remodeling. The aim of the present study was to characterize the role of bgn in modulating the phenotype of cardiac fibroblasts. Cardiac fibroblasts were isolated from hearts of wild-type (WT) versus bgn(-/0) mice. Phenotypic characterization of the bgn(-/0) fibroblasts revealed increased proliferation. Importantly, this phenotype of bgn(-/0) fibroblasts was abolished to the WT level by reconstitution of biglycan in the ECM. TGF-β receptor II expression and phosphorylation of SMAD2 were increased. Furthermore, indicative of a myofibroblast phenotype bgn(-/0) fibroblasts were characterized by increased α-smooth muscle actin (α-SMA) incorporated into stress fibers, increased formation of focal adhesions, and increased contraction of collagen gels. Administration of neutralizing antibodies to TGF-β reversed the pro-proliferative, myofibroblastic phenotype. In vivo post-MI α-SMA, TGF-β receptor II expression, and SMAD2 phosphorylation were markedly increased in bgn(-/0) mice. Collectively, the data suggest that bgn deficiency promotes myofibroblast differentiation and proliferation in vitro and in vivo likely due to increased responses to TGF-β and SMAD2 signaling.

Expression of lumican and fibromodulin following interleukin-1 beta stimulation of disc cells of the human temporomandibular joint

Small leucine-rich repeat proteoglycans (SLRP) are present in the extracellular matrix of the temporomandibular joint (TMJ) disc. Lumican and fibromodulin, classified as class 2 SLRPs, play important roles in TMJ assembly, proliferation and inflammation. Degenerative change in the TMJ disc gives rise to the process of internal derangement (ID). In this study, we immunohistochemically examined the expression of lumican and fibromodulin in nine human TMJ specimens and examined the gene expression of both proteoglycans in cultured human TMJ disc cells under interleukin-1 beta (IL-1 β)-stimulated conditions. An articular disc cell line was established by collagenase treatment of a TMJ disc. The subcultured cells were then incubated for 1, 3, 6, 12, 24 or 48 h under both normal and IL-1 β (1 ng/mL) conditions. The gene expression of lumican and fibromodulin was examined using the reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR. We demonstrated that the expression of lumican significantly differs from that of fibromodulin in the deformed disc and that IL-1 β induces a significant increase in lumican mRNA, but not in fibromodulin mRNA, after 24∼48 h culture compared to cells cultured in the absence of IL-1 β (P<0.05). These results indicate that lumican and fibromodulin display different behaviors and that lumican may promote regeneration of the TMJ after degeneration and deformation induced by IL-1 β.

Abstract P4-06-18: Asporin Is Associated with Invasive Growth and Bone Metastasis of Breast Cancer

Abstract Background: In an earlier publication we identified asporin as a novel cancer related protein (BMC Cancer 2007, 7:55), also reported among bone metastasis specific genes (Cancer Letters 2009, 276:212-220). Asporin is a member of the class I small leucine-rich repeat proteoglycan family and besides affinity to collagen, it binds calcium and initiates collagen mineralization. Asporin is abundantly expressed in tissues with high remodeling rate (e.g. peridontal ligament) whereas extremely high turnover of matrix proteins is also associated with invasive cancer. Material and methods: Formalin-fixed paraffin-embedded tissues [invasive ductal (101) and lobular (39) carcinomas classified according to hormone receptors and Her-2; 5 bone metastases from autopsies] were immunohistochemically stained with in-house polyclonal peptide antibody against asporin. The antibody was validated on uterus tissue as a positive control together with a commercial antibody (Abcam). Data were evaluated by non-parametric Mann-Whitney U test and Spearman correlation coefficient. Expression of asporin in both tissues and cell lines was also queried in databases Gene Expression Omnibus, ArrayExpress and Oncomine. Expression of asporin was further tested by RT-PCR in selected breast cancer cell lines. Migration and invasion of MDA-MB-231 cells were tested using real-time cell analysis Xcelligence system (Roche). Transwells were coated by solidifying collagen for 6 hours (with or without recombinant asporin). Starved cells were seeded on the collagen in transwells while FBS was used as a chemoattractant in bottom wells. Results: Levels of asporin were significantly higher in invasive lobularcarcinomas than ductal ones (P&amp;lt;0.001). Asporin was also highly expressed in all bone metastases. Asporin expression positively correlated with tumor grade within the triple negative subgroup (Rs 0.4, P&amp;lt;0.05) but not in luminal and Her-2 subtypes. Expression of asporin in invasive breast carcinomas was also confirmed in other public datasets (e.g. Karnoub breast and Hess breast in Oncomine database). Surprisingly, we found no breast cancer cell line overexpressing asporin, which underlines the importance of the tumor microenvironment in vivo. MDA-MB-231 cells migrated faster through collagen I with recombinant asporin. Conclusion: Asporin is expressed by both invasive breast carcinomas and bone metastases. Mechanistically, asporin may affect extracellular matrix structure and promote invasive growth of breast cancer cells. Further investigation of the role of asporin in metastasizing to bone is additionally justified given its affinity for calcium. Acknowledgements This work was supported by grants NS 9956-4 from the Czech Ministry of Health and MSM 6198959216 from the Czech Ministry of Education. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-18.