BACKGROUND: The mechanism by which mutant cartilage oligomeric matrix protein (COMP) induces a pseudoachondroplasia phenotype remains unknown, and the reason why a mutation of a minor protein of the growth plate cartilage causes total disruption of endochondral bone formation has not yet been determined. The current study was performed to investigate the effects of mutated COMP on the synthesis of the cartilage-specific major matrix proteins of Swarm rat chondrosarcoma chondrocytes. METHODS: The Swarm rat chondrosarcoma chondrocytes transfected with a chimeric construct, which consisted of a mutant gene of human COMP and an amino acid FLAG tag sequence, were cultured in agarose gel. Formation of extracellular proteoglycan and type-II collagen by the cells was evaluated by immunohistochemical staining and measuring the (35)S-sulfate incorporation. RESULTS: No difference was observed for the detection of type-II collagen among the cell lines expressing mutant COMP and the control cell lines. Histochemical staining of sulfated proteoglycans with safranin-O showed that lesser amounts of proteoglycans were incorporated into the extracellular matrix of the chondrocytes transfected with the mutant gene. (35)S-sulfate incorporation into the cell/matrix fractions demonstrated markedly lower radiolabel incorporation, as compared to that of the control cells. CONCLUSIONS: Mutation of COMP has an important impact on the processing of proteoglycans, rather than type-II collagen, in the three-dimensional culture of Swarm rat chondrosarcoma chondrocytes.
Aggrecans/analysis/*biosynthesis ; Animals ; Cells, Cultured ; Chondrocytes/*metabolism ; Chondrosarcoma/metabolism ; Collagen Type II/*biosynthesis ; Extracellular Matrix/*metabolism ; Extracellular Matrix Proteins/*genetics ; Glycoproteins/*genetics ; Humans ; Mutation ; Rats ; Transfection

OBJECTIVESTo observe the expression of betaig-h3 in normal cornea and keratoconus and to elucidate the role of extracellular matrix in keratoconus.

METHODSIn situ hybridization was used to detect the expression of betaig-h3 in the cornea. The cDNA library was screened with human betaig-h3 cDNA probe to locate betaig-h3 mRNA in cells.

RESULTSExpression of betaig-h3 was found mainly in the stroma of the normal cornea and keratoconus, but decrease depending on the degree of keratopathy. In some serious cases, no expression signal was detected. The strongest expression was seen at the border of the normal region and keratoconus.

CONCLUSIONSbetaig-h3, the structural component of the extracellular matrix, can affect cell adhensiveness in the development of corneal fibrous interstitial organization. During the development of keratoconus, decreasing levels of betaig-h3 cause the diminution of corneal steadiness, which is related to formation of keratoconus.

Cornea ; metabolism ; Extracellular Matrix Proteins ; Humans ; Keratoconus ; metabolism ; Neoplasm Proteins ; genetics ; RNA, Messenger ; analysis ; Transforming Growth Factor beta ; Wound Healing

No abstract available.
Child, Preschool ; DNA Mutational Analysis ; Diseases in Twins/*genetics/metabolism ; Ectopia Lentis/*genetics/metabolism ; Extracellular Matrix Proteins ; Humans ; Male ; Microfilament Proteins/*genetics/metabolism ; *Mutation ; *Twins, Monozygotic

OBJECTIVETo investigate the effect of chemically synthetic small interfering RNA (siRNA) targeting connective tissue growth factor (CTGF) on the synthesis and secretion of extracellular matrix (ECM) in hepatic stellate cells (HSC).

METHODSChemically synthetic siRNA targeting CTGF was transfected into HSC T6 (an active HSC line in rats) by oligofectamine package, and untreated HSC T6 were used as control. Total RNA and protein of the cells, after their incubation with siRNA for 24, 48 and 72 hours, were extracted, and the supernatants were collected. The expressions of CTGF and type I and III collagen genes were detected by means of reverse transcription-polymerase chain reaction (RT-PCR) and/or Western blot. Contents of hyaluronic acid and type III pro-collagen in the supernatants were determined by radioimmunoassay.

RESULTSThe expression of CTGF at mRNA and protein level and type I and III collagen at mRNA levels were markedly down-regulated in siRNA-transfected HSCs. The contents of hyaluronic acid and type III pro-collagen in the supernatants decreased by 46%+/-7%, 52%+/-7%, 53%+/-7% and 29%+/-18%, 29%+/-7%, 27%+/-5%, compared with those of the blank control at 24, 48 and 72 hours.

CONCLUSIONSChemically synthetic anti-CTGF siRNA can significantly inhibit CTGF gene expression in HSC, and markedly reduce the synthesis and secretion of ECM including type I and III collagen and hyaluronic acid. The siRNA-directed suppression of CTGF gene in HSC was maintained for 72 hours. This suggests that chemically synthetic siRNA may be a potential in preventing and treating liver fibrosis and may have a promising future for development

Cell Line ; Connective Tissue Growth Factor ; Extracellular Matrix ; metabolism ; Gene Targeting ; Humans ; Immediate-Early Proteins ; genetics ; Intercellular Signaling Peptides and Proteins ; genetics ; Liver ; cytology ; metabolism ; RNA, Small Interfering ; genetics

OBJECTIVESTo study cartilage oligomeric matrix protein (COMP) mRNA and protein expression in normal pancreas, chronic pancreatitis (CP), and pancreatic cancer tissues.

METHODSTissues from 15 cases of normal pancreas, 14 cases of chronic pancreatitis and 14 cases of pancreatic cancer were analyzed by Northern blot, Western blot, in situ hybridization and immunohistochemistry.

RESULTSCOMP mRNA signals and immunoreactivity were strongly present in the cytoplasm of degenerating acinar cells in CP tissues as well as in CP-like lesions in pancreatic cancer tissues. In contrast, COMP expression was weak to absent in the cytoplasm of cancer cells in pancreatic cancer tissues, and in ductal cells and islet cells in normal pancreatic tissues.

CONCLUSIONCOMP is preferentially expressed in degenerating acinar cells in CP and in CP-like areas in pancreatic cancer, suggesting a potential role of this molecular in acinar cell dysfunction in CP.

Blotting, Northern ; Blotting, Western ; Cartilage Oligomeric Matrix Protein ; Dimerization ; Extracellular Matrix Proteins ; chemistry ; genetics ; metabolism ; Glycoproteins ; chemistry ; genetics ; metabolism ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Matrilin Proteins ; Pancreas ; metabolism ; pathology ; Pancreatic Neoplasms ; metabolism ; pathology ; Pancreatitis, Chronic ; metabolism ; pathology ; RNA, Messenger ; genetics ; metabolism

OBJECTIVETo evaluate the expression of dental matrix protein-l (DMP1) in porcine oral mucosa fibroblasts (POMF) transfected by DMP1 and the influences of the transfection.

METHODSThe full length of porcine DMP1 cDNA was linked into an eukaryotic expression vector pEGFP-C1. POMF and mesenchymal stem cells (MSC) were transfected with the pEGFP-DMP1. The expression of DMP1, dental sialoprotein (DSP), amelin and ameloblastin (Ambn) gene of transfected POMF and MSC were detected by RT-PCR. The expression of DMP1 and DSP protein was examined by immunocytochemical staining. The formation ratio of mineralized nodules of transfected cells was compared with un-transfected ones after mineralized induction. The formation of mineralized nodules of three-dimensional pellet transfected cells was compared with un-transfected ones after hematoxylin and eosin staining.

RESULTSThe constructed pEGFP-DMP1 could produce 4.7 kb and 1.5 kb fragments. DMP1 gene, DSP gene and Ambn gene were expressed by POMF after transfection. Immunohistochemical staining and the quantitative analysis of protein showed that DMP1 and DSP protein was positive in transfected POMF and MSC. The formation ratio of mineralized nodules of transfected POMF and MSC was higher than that of un-transfected ones (P < 0.05).

CONCLUSIONSThe expression of porcine DMP1 in POME after gene transfection can induce the expression of tooth-development-associated gene Ambn and DSP and enhance the formation of mineralized nodules.

Animals ; Calcification, Physiologic ; Cell Differentiation ; Cells, Cultured ; Extracellular Matrix Proteins ; genetics ; metabolism ; Fibroblasts ; cytology ; metabolism ; Genetic Vectors ; Mouth Mucosa ; cytology ; Phosphoproteins ; genetics ; metabolism ; Swine ; Transfection

OBJECTIVETo investigate the mineralization capacity of periodontal ligament stem cells (PDLC) by determining the mRNA expressions of alkaline phosphatase (ALP), osteocalcin (OCN) and matrix extracellular phosphoglycoprotein (MEPE) and to explore the potential of MEPE as a differentiation marker for PDLC, and its possible function in PDLC osteogenic differentiation.

METHODSPDLC were digested and cultured by a solution containing collagenase type I and dispase. PDLC were preceded to osteogenic induction for 7, 14 and 21 days respectively, and the cells before induction served as controls. Mineralization nodules and the expression of OCN in PDLC were investigated by alizarin red and immunohistochemistry respectively. The expressions of ALP, OCN and MEPE mRNA were investigated by quantitative real-time RT-PCR analysis. Statistical analysis was performed to compare the differences of mRNA expression levels among cell samples collected at different time points.

RESULTSThe mRNA expressions of ALP, OCN and MEPE in PDLC before induction were 72, 1.1 and 534 respectively, but increased time-dependently in the induction cultures. The mRNA expressions of ALP, OCN and MEPE were 78, 9.56 and 629.6 on day 7; 290, 133 and 638.3 on day 14; 1108, 925 and 2261.1 on day 21 respectively. The relative mRNA levels of OCN, ALP on day 14 and 21, MEPE on day 21 were significantly higher than control group (P < 0.05).

CONCLUSIONSPDLC showed analogously temporal expression of ALP, OCN and MEPE mRNA while differentiating into cementoblast/osteoblast-like cells in vitro. MEPE may play a regulatory role in PDLC osteogenic differentiation, and may be a potential osteogenic differentiation marker along with ALP and OCN.

Alkaline Phosphatase ; genetics ; metabolism ; Cell Differentiation ; Cells, Cultured ; Extracellular Matrix Proteins ; genetics ; metabolism ; Glycoproteins ; genetics ; metabolism ; Humans ; Osteoblasts ; cytology ; metabolism ; Osteocalcin ; genetics ; metabolism ; Periodontal Ligament ; cytology ; metabolism ; Phosphoproteins ; genetics ; metabolism ; RNA, Messenger ; genetics

OBJECTIVETo examine the expression of periodontal ligament-associated protein-1 (PLAP-1) in the periodontal tissues and periodontal ligament cells (PDLC).

METHODSThe PLAP-1 expression in normal periodontal tissue was examined by immunohistochemistry. The protein expression and mRNA transcription of PLAP-1 in PDLC were investigated by immunocytochemistry and reverse transcription-polymerase chain reaction.

RESULTSPLAP-1 was expressed in periodontium but not in cementum, alveolar bone and gingival tissues. PLAP-1 expression was observed in cell plasma, but not in nuclei. There was a 350 bp electrophoresis band representing PLAP-1 mRNA.

CONCLUSIONSPLAP-1 may play a role in physiology of periodontal tissues and cells in normal adult rats.

Animals ; Extracellular Matrix Proteins ; genetics ; metabolism ; Immunohistochemistry ; Male ; Periodontal Ligament ; cytology ; metabolism ; Periodontium ; metabolism ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction

The blood-brain barrier (BBB) is a tight boundary formed between endothelial cells and astrocytes, which separates and protects brain from most pathogens as well as neural toxins in circulation. However, detailed molecular players involved in formation of BBB are not completely known. Dentin matrix protein 1 (DMP1)-proteoglycan (PG), which is known to be involved in mineralization of bones and dentin, is also expressed in soft tissues including brain with unknown functions. In the present study, we reported that DMP1-PG was expressed in brain astrocytes and enriched in BBB units. The only glycosylation site of DMP1 is serine89 (S89) in the N-terminal domain of the protein in mouse. Mutant mice with DMP1 point mutations changing S89 to glycine (S89G), which completely eradicated glycosylation of the protein, demonstrated severe BBB disruption. Another breed of DMP1 mutant mice, which lacked the C-terminal domain of DMP1, manifested normal BBB function. The polarity of S89G-DMP1 astrocytes was disrupted and cell-cell adhesion was loosened. Through a battery of analyses, we found that DMP1 glycosylation was critically required for astrocyte maturation both in vitro and in vivo. S89G-DMP1 mutant astrocytes failed to express aquaporin 4 and had reduced laminin and ZO1 expression, which resulted in disruption of BBB. Interestingly, overexpression of wild-type DMP1-PG in mouse brain driven by the nestin promoter elevated laminin and ZO1 expression beyond wild type levels and could effectively resisted intravenous mannitol-induced BBB reversible opening. Taken together, our study not only revealed a novel element, i.e., DMP1-PG, that regulated BBB formation, but also assigned a new function to DMP1-PG.
Animals ; Astrocytes ; cytology ; metabolism ; Blood-Brain Barrier ; cytology ; metabolism ; Cells, Cultured ; Extracellular Matrix Proteins ; genetics ; metabolism ; Female ; Glycosylation ; Male ; Mice ; Proteoglycans ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

Articular cartilage is a connective tissue consisting of a specialized extracellular matrix (ECM) that dominates the bulk of its wet and dry weight. Type II collagen and aggrecan are the main ECM proteins in cartilage. However, little attention has been paid to less abundant molecular components, especially minor collagens, including type IV, VI, IX, X, XI, XII, XIII, and XIV, etc. Although accounting for only a small fraction of the mature matrix, these minor collagens not only play essential structural roles in the mechanical properties, organization, and shape of articular cartilage, but also fulfil specific biological functions. Genetic studies of these minor collagens have revealed that they are associated with multiple connective tissue diseases, especially degenerative joint disease. The progressive destruction of cartilage involves the degradation of matrix constituents including these minor collagens. The generation and release of fragmented molecules could generate novel biochemical markers with the capacity to monitor disease progression, facilitate drug development and add to the existing toolbox for in vitro studies, preclinical research and clinical trials.
Aggrecans ; chemistry ; genetics ; metabolism ; Animals ; Biomarkers ; metabolism ; Cartilage, Articular ; chemistry ; metabolism ; pathology ; Collagen ; chemistry ; classification ; genetics ; metabolism ; Extracellular Matrix Proteins ; chemistry ; genetics ; metabolism ; Gene Expression ; Humans ; Osteoarthritis ; diagnosis ; genetics ; metabolism ; pathology ; Protein Isoforms ; chemistry ; classification ; genetics ; metabolism