ABSTRACT
High-mobility group box-1 (HMGB1) protein acts as a transcription factor in the nucleus and also as a pro-inflammatory cytokine when released into extracellular fluids. The presence of higher levels of HMGB1 is reported in the gingival crevicular fluid from periodontal patients. Since the proliferation of bacteria within the periodontal pocket is closely involved in the exacerbation of periodontal disease, it is hypothesized that the periodontal pocket causes the release of HMGB1. Immunohistochemical staining of inflamed gingiva revealed that HMGB1 is exclusively dislocated from the nucleus to the cytoplasm in the pocket epithelium, whereas it is mainly present in the nucleus in the gingival epithelium. Butyric acid, an extracellular metabolite from periodontopathic bacteria populating the periodontal pocket, induced the passive release of HMGB1 as a result of eliciting necrosis in the human gingival epithelial cell line. Thus, the periodontal epithelium may provide a unique pathological setting for HMGB1 release by bacterial insult.
Subject(s)
Butyric Acid/pharmacology , Gingival Crevicular Fluid/chemistry , HMGB1 Protein/metabolism , Periodontal Pocket/metabolism , Virulence Factors/metabolism , Adult , Aged , Case-Control Studies , Cell Line , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Female , Gingiva/cytology , HMGB1 Protein/analysis , Humans , Male , Middle Aged , Necrosis/metabolism , Periodontal Pocket/microbiology , Periodontal Pocket/pathology , Protein Transport , Reactive Oxygen Species/metabolism , Virulence Factors/analysisABSTRACT
BACKGROUND: Loss of occlusal function has been reported to induce atrophic changes in the periodontal ligament. It is likely that mechanical stress triggers the biological response of periodontal ligament. However, there have been few reports studying the correlation between mechanical stress of varying magnitude and periodontal ligament cell activities such as extracellular matrix (ECM) synthesis. OBJECTIVE: The objective of this study is to clarify the influence of the mechanical stress on changes in mRNA expression levels of type I collagen and decorin genes, as well as alkaline phosphatase (ALP) activity in response to mechanical stress of varying magnitude. METHODS: Bovine periodontal ligament cells were cultured on flexible-bottomed culture plates and placed on the BioFlex Loading Stations. Cells were elongated at 6 cycles/min (5 s on and 5 s off) at each of six levels of stretch (0.2, 1.0, 2.0, 3.0, 10, 18% increase in the surface area of the bottom) for 48 h. We measured mRNA expression levels of type I collagen and decorin genes using quantitative reverse transcription-polymerase chain reaction (RT-PCR), and ALP activity in periodontal ligament cell culture under cyclic mechanical stretching. RESULTS: Mechanical tensional stress of low magnitude induced the increase of both type I collagen and decorin mRNA expression without changing ALP activity in periodontal ligament cells. Mechanical tensional stress of high magnitude induced the increase of type I collagen and decorin mRNA expression while decreasing ALP activity. CONCLUSION: These results suggest that different magnitude of tensional force induces different responses from periodontal ligament cells, and that mechanical stress plays an important role in remodeling and functional regulation of periodontal ligament.
Subject(s)
Alkaline Phosphatase/metabolism , Collagen Type I/metabolism , Periodontal Ligament/enzymology , Proteoglycans/metabolism , Alkaline Phosphatase/genetics , Animals , Cattle , Cell Culture Techniques , Collagen Type I/genetics , Decorin , Extracellular Matrix/enzymology , Extracellular Matrix Proteins , Periodontal Ligament/cytology , Proteoglycans/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methods , Stress, Mechanical , Time FactorsABSTRACT
The biosynthesis of proteoglycans by these cultured pulp cells was investigated by metabolic labelling, using [(35)S]sulphate, [(3)H]glucosamine and [(3)H]leucine as precursors. Versican-like large proteoglycan, decorin- and biglycan-like small proteoglycans and a small amount of sulphated protein were released into the culture medium. Heparan sulphate species were also identified in cell-layer extracts. Versican-like proteoglycan had an average molecular mass of approximately 800kDa. The molecular mass of chondroihnase ABC-digested core protein exhibited heterogeneity, ranging from 250 to 400kDa, and the glycosaminoglycan chains had an average molecular mass of approximately 42kDa. These results indicate the presence of 10-13 glycosaminoglycan chains per core protein, consistent with the characteristics of versican. This glycosaminoglycan chain contained approximately 63% 4-sulphated disaccharides.
Subject(s)
Chondroitin Sulfate Proteoglycans/biosynthesis , Dental Pulp/metabolism , Lectins/biosynthesis , Proteoglycans/biosynthesis , Animals , Biglycan , Cell Culture Techniques , Centrifugation, Density Gradient , Chondroitin ABC Lyase , Chondroitin Sulfate Proteoglycans/isolation & purification , Chondroitin Sulfates/biosynthesis , Chondroitin Sulfates/isolation & purification , Chromatography, Gel , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Decorin , Dental Pulp/cytology , Disaccharides/isolation & purification , Electrophoresis, Polyacrylamide Gel , Extracellular Matrix Proteins , Glucosamine/metabolism , Heparitin Sulfate/biosynthesis , Heparitin Sulfate/isolation & purification , Lectins/isolation & purification , Lectins, C-Type , Leucine/metabolism , Male , Molecular Weight , Proteoglycans/isolation & purification , Radiopharmaceuticals , Rats , Rats, Wistar , Sulfates/metabolism , Sulfur Radioisotopes , Tritium , VersicansABSTRACT
In inflammatory gingival diseases, cytokines have been demonstrated to play critical roles by coordinating the stimulation of immunological and connective tissue cells. The activities of these cells, degrading and remodeling extracellular matrices, constitute the major pathological and repair processes. Thus, elucidating cellular and molecular events occurring in inflamed connective tissues is crucial for the understanding and treatment of inflammation. In order to test a hypothesis that proinflammatory cytokines affect metabolism of major extracellular matrix molecules, we studied metabolism of proteoglycans (PGs) by human gingival fibroblasts (HGF) under the influence of interleukin-4 (IL-4) as a model of gingivitis. HGF in cell culture were metabolically radiolabeled using [3H]glucosamine and [35S]sulfate in the presence or absence of IL-4, and the labeled PGs were analyzed by chromatographic techniques. The incorporation of 35S into PGs increased with IL-4 both in media and cell layer. At 100 ng/ml of IL-4, the increment of 35S incorporation over control culture was 16-39% (p<0.001) in media and 12-35% (p=0.01) in cell layer. The 35S-labeled macromolecules were PGs containing heparan sulfate (HS) and chondroitin sulfate (CS) chains. From the molecular weight and glycosaminoglycan composition analyses, versican and perlecan-type and biglycan and decorin-type were very likely to be the major PG constituents both in media and cell layer. IL-4 stimulated synthesis of versican and perlecan-type more potently than biglycan and decorin-type. With IL-4 treatment, the ratio of CSPG/HSPG decreased in media and increased in cell layer. This ratio suggested that syndecan family HSPGs were also present in HGF. In conclusion, IL-4 stimulated accumulation of CS/HSPGs in human gingival fibroblasts.
Subject(s)
Extracellular Matrix Proteins/metabolism , Fibroblasts/metabolism , Gingiva/metabolism , Interleukin-4/pharmacology , Proteoglycans/metabolism , Adult , Biglycan , Cell Culture Techniques , Chondroitin Sulfate Proteoglycans/analysis , Chondroitin Sulfates/analysis , Chromatography, Agarose , Chromatography, Ion Exchange , Decorin , Extracellular Matrix Proteins/analysis , Female , Gingiva/cytology , Gingivitis/metabolism , Glucosamine , Glycosaminoglycans/analysis , Heparan Sulfate Proteoglycans/analysis , Humans , Lectins , Lectins, C-Type , Membrane Glycoproteins/analysis , Molecular Weight , Proteoglycans/analysis , Radiopharmaceuticals , Sulfur Radioisotopes , Syndecans , Transforming Growth Factor beta/antagonists & inhibitors , Tritium , VersicansABSTRACT
AIM: The main aim of this study was to investigate the developmental changes in the distribution patterns of hyaluronan (HA) and versican in postnatal rat molar dental pulp, in order to confirm the hypothesis that the distribution of both molecules can vary with physiological conditions in the dental pulp. METHODOLOGY: Thirty postnatal Sprague-Dawley rats, 1, 7, 14, 21, 28, 35, 42 and 49 days old, were used for this study. Immunohistochemistry for versican with monoclonal antibodies 12C5 and CS-56 and histochemical staining for HA with HA-binding protein were applied to paraffin sections of the mandibular first molars at each age. RESULTS: At day 1, both molecules were evenly distributed in the interior parts of the pulp, but strong reactions for both molecules appeared in the subodontoblastic layer of the coronal pulp by the completion of crown formation. However, a strong reaction for HA and a weak reaction for versican were seen in the subodontoblastic layer of the radicular pulp. Furthermore, a versican-deficient, low-HA area first appeared in the interior of the coronal pulp at day 42 and expanded at day 49. CONCLUSIONS: Distribution of hyaluronan and versican in the dental pulp varied with age and also showed regional differences between the coronal and the radicular pulp, and this supports the hypothesis described above.
Subject(s)
Chondroitin Sulfate Proteoglycans/metabolism , Dental Pulp/growth & development , Dental Pulp/metabolism , Hyaluronic Acid/metabolism , Proteoglycans/metabolism , Age Factors , Animals , Chondroitin Sulfate Proteoglycans/analysis , Dental Pulp/chemistry , Histocytochemistry , Hyaluronic Acid/analysis , Lectins, C-Type , Molar , Proteoglycans/analysis , Rats , Rats, Sprague-Dawley , Tooth Crown , Tooth Root , VersicansABSTRACT
Heparan sulfate proteoglycans play important biological roles in cell-cell and cell-matrix adhesion, and are closely associated with growth factor actions. Loss of syndecan-1, a cell surface-bound heparan sulfate proteoglycan, has been reported for advanced head and neck carcinomas, and expression of endoglycosidic heparanase, which cleaves heparan sulfate glycosaminoglycans (HS-GAGs), is associated with invasion and metastatic potential of malignant tumors. Paraffin sections of 103 primary esophageal squamous cell carcinomas were immunohistochemically examined for the expression of syndecan-1 core protein, HS-GAGs and heparanase protein, and the results were compared with various clinicopathological parameters, such as invasion depth. For 16 cases, fresh tumor samples were quantitatively analyzed for heparanase and syndecan-1 mRNA expression by real-time RT-PCR in addition to the immunohistochemical studies. Syndecan-1 core protein and HS-GAGs expression was significantly decreased in pT2 and pT3 cases compared with their pTis and pT1 counterparts. Decreased expression of core protein and HS-GAGs was correlated with the incidence of lymphatic invasion, and venous involvement. Furthermore, decreased expression of HS-GAGs was correlated positively with the incidence of nodal metastasis and distant organ metastasis, and negatively with the grade of tumor cell differentiation. The percentage of cytoplasmic heparanase protein-positive cases increased significantly in pT2 and pT3 cases compared to that in pTis and pT1 cases, and this was associated with lymphatic invasion, and venous and lymph nodal involvement. The level of heparanase mRNA was inversely correlated with the degree of HS-GAGs expression rather than core protein. In conclusion, loss of syndecan-1 and heparanase overexpression in esophageal squamous cell carcinomas are closely associated with malignant potential. Regarding the mechanism of loss of HS-GAGs, heparanase upregulation appears to play an important role.
Subject(s)
Esophageal Neoplasms/chemistry , Esophageal Neoplasms/pathology , Glucuronidase/metabolism , Membrane Glycoproteins/metabolism , Neoplasm Invasiveness , Proteoglycans/metabolism , Adult , Aged , Aged, 80 and over , Carcinoma, Squamous Cell/chemistry , Carcinoma, Squamous Cell/enzymology , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Esophageal Neoplasms/enzymology , Esophageal Neoplasms/genetics , Esophagus/chemistry , Esophagus/cytology , Female , Glucuronidase/genetics , Glucuronidase/immunology , Heparan Sulfate Proteoglycans/metabolism , Humans , Immunohistochemistry , Male , Membrane Glycoproteins/analysis , Membrane Glycoproteins/genetics , Membrane Glycoproteins/immunology , Middle Aged , Proteoglycans/analysis , Proteoglycans/genetics , Proteoglycans/immunology , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Syndecan-1 , Syndecans , Tumor Cells, CulturedABSTRACT
The biological functions of proteoglycans and glycosaminoglycans are closely associated with mechanical stress on the tissue. In order to reveal the relationship between proteoglycans in the periodontal ligament and mechanical stress such as occlusal stimuli, occlusal hypofunction of rat unilateral mandibular molars was induced by extraction of the opposing first, second and third maxillary molars. Immunohistochemical analyses were performed using antibodies for chondroitin sulfate, decorin, biglycan, heparan sulfate and keratan sulfate, and hyaluronic acid-binding protein. Chondroitin sulfate, observed more strongly in the cervical side than in the apical side of the periodontal ligament of the unextracted sides of mandible, and uniformly present in the extracellular matrix of the periodontal ligament, decreased significantly from 1 wk post-extraction of the antagonists, with a decrease in thickness and disarrangement in fibrous components. Decorin core protein, uniformly present in the periodontal ligament of the unextracted sides, decreased as early on as 2 d post-extraction. Heparan sulfate, mainly localized on the cell surface of vascular endothelial cells and osteoclastic cells as well as in the extracellular matrix of the unextracted sides, decreased significantly in association with the decreased number of blood vessels and osteoclastic cells as early on as 2 d post-extraction. Biglycan, keratan sulfate and hyaluronic acid, uniformly distributed in the periodontal ligament of the unextracted sides, showed little change after the extraction. These results demonstrate that occlusal hypofunction causes tissue remodeling of the periodontal ligament, with a significant decrease of chondroitin sulfate, decorin and heparan sulfate.
Subject(s)
Dental Occlusion, Traumatic/metabolism , Periodontal Ligament/metabolism , Proteoglycans/biosynthesis , Animals , Extracellular Matrix Proteins/biosynthesis , Glycosaminoglycans/biosynthesis , Histocytochemistry , Male , Mandible/physiopathology , Periodontal Ligament/pathology , Rats , Rats, WistarABSTRACT
In the process of metastasis, cancer cells secrete several enzymes which degrade extracellular matrices (ECMs) and basement membranes (BMs) of blood vessels. One of them, heparanase, has been reported to be an important enzyme when metastatic cancer cells invade blood vessels. The enzyme cleaves heparan sulfate (HS), a main component of ECM and BM. In the present study, HS-degrading ability of several human oral cancer cell lines (HSC2, HSC3, HSC4, Ca9-22, NA, ACC3 and Ab-J) and tissues derived from human oral squamous cell carcinomas (both metastatic and non-metastatic) were investigated by measuring heparanase activities and levels of heparanase mRNA by a quantitative reverse transcriptase-polymerase chain reaction. The catalytic activities and the mRNA levels of heparanase showed a good agreement. Clinical demonstration of cancer metastasis generally correlated with high levels of heparanase activity and its mRNA. The results suggest that heparanase activity and its mRNA level are good diagnostic parameters for evaluating the metastatic properties of human oral cancer cells.
Subject(s)
Carcinoma, Squamous Cell/enzymology , Glucuronidase/metabolism , Mouth Neoplasms/enzymology , Neoplasm Proteins/metabolism , Adolescent , Adult , Aged , Aged, 80 and over , Basement Membrane/enzymology , Basement Membrane/metabolism , Carcinoma, Squamous Cell/secondary , Extracellular Matrix/enzymology , Extracellular Matrix/metabolism , Female , Glucuronidase/genetics , Humans , Male , Middle Aged , Mouth Neoplasms/pathology , Neoplasm Metastasis , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured/enzymologyABSTRACT
Proteoglycans are suggested to regulate cell adhesion, differentiation and mineralization of hard tissues. In vitro studies have shown that many humoral and local factors regulate proteoglycan synthesis. Among them, parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) have potent stimulating effects on proteoglycan synthesis. However, the exact role of PTHrP on the biosynthesis and metabolism of proteoglycans during skeletal development is not clear. To clarify this point, we examined bony and cartilaginous explants of newborn mice with disrupted PTHrP alleles. Ribs of homozygous PTHrP-knockout mice and wildtype littermates were dissected into bony and cartilaginous regions and metabolically labeled with [35S]sulfate in culture. Radiolabeled proteoglycans were analyzed by column chromatography. The elution profiles of [35S]-labeled proteoglycan from cartilaginous explants did not differ between homozygous PTHrP-knockout mice and wild-type littermates. However, the amount of labeled proteoglycan in homozygous PTHrP-knockout mice was only 4%-5% that of wild-type littermates. In contrast with cartilaginous explants, the amount of labeled proteoglycans in bony explants did not differ between the two genotypes. Interestingly, besides the common major peak (Kd = 0.10-0.16) observed in the bony explants of both genotypes, a minor peak (Kd = 0.42) was specifically present in homozygous PTHrP-knockout mice. This minor peak was earlier than that of free glycosaminoglycan (GAG) chains, suggesting that the core protein, but not GAG chain, was cleaved in the bony explants of homozygous PTHrP. These findings demonstrate a crucial nonredundant role of PTHrP in the regulation of proteoglycan synthesis and metabolism during skeletal development.
Subject(s)
Bone and Bones/metabolism , Cartilage/metabolism , Proteins/genetics , Proteins/physiology , Proteoglycans/biosynthesis , Animals , Animals, Newborn , Bone and Bones/anatomy & histology , Chromatography, Agarose , Chromatography, Ion Exchange , Culture Techniques , Glycosaminoglycans/analysis , Mice , Mice, Knockout , Parathyroid Hormone-Related Protein , Proteoglycans/chemistry , Ribs/anatomy & histology , Ribs/metabolismABSTRACT
Versican is a large interstitial proteoglycan that is believed to be able to bind hyaluronan to form large aggregate structures, but no study has isolated native versican aggregates from any tissue. In this study, ternary aggregate structures consisting of versican, hyaluronan, and link protein were isolated from rat dental pulp by associative extractions followed by caesium sulphate rate zonal sedimentation centrifugation. Fractions from the centrifugation were analysed by dot blot and Western blot using monoclonal antibodies and hyaluronan-binding protein. About 60% of the hexuronic acid was extracted by associative extractions. Positive reactions for versican, hyaluronan and link protein were clearly detected in the bottom fractions from the centrifugation, but were barely detectable in the top fractions. These results suggest that the majority of the versican, hyaluronan, and link protein forms ternary aggregate structures in the rat dental pulp.
Subject(s)
Chondroitin Sulfate Proteoglycans/isolation & purification , Dental Pulp/metabolism , Extracellular Matrix Proteins , Proteoglycans/isolation & purification , Animals , Blotting, Western/methods , Chondroitin Sulfate Proteoglycans/analysis , Chondroitin Sulfate Proteoglycans/metabolism , Dental Pulp/chemistry , Electrophoresis, Polyacrylamide Gel/methods , Hyaluronic Acid/analysis , Hyaluronic Acid/isolation & purification , Hyaluronic Acid/metabolism , Immunoblotting/methods , Lectins, C-Type , Proteins/analysis , Proteins/isolation & purification , Proteins/metabolism , Proteoglycans/analysis , Proteoglycans/metabolism , Rats , Rats, Sprague-Dawley , VersicansABSTRACT
Retinoic acid is a potent inducer of tissue-non-specific alkaline phosphatase (TNSALP) expression in various osteoblastic and fibroblastic cells, and may be involved in morphogenesis, cellular growth and differentiation. This study investigates the effects of retinoic acid on alkaline phosphatase activity and TNSALP gene expression in human dental pulp cells. Cultured cells were treated with various concentrations of retinoic acid (0, 10(-7), 10(- 6), 10 (-5) M) in 0.5% bovine serum albumin without serum. Alkaline phosphatase activity was determined by the rate of p-nitrophenyl phosphate hydrolysis and was also assayed in the presence of various inhibitors and under thermal inactivation. A set of specific oligonucleotide primers was selected, based on the nucleotide sequences of two human TNSALP mRNA (bone and liver) types, and reverse transcription-polymerase chain reaction (RT-PCR) performed. Inhibitory and thermal inactivation experiments revealed that the elevated alkaline phosphatase activity had properties of the TNSALP type. RT-PCR showed that retinoic acid enhanced the expression of bone-type TNSALP mRNA in pulp cells. However, the liver-type TNSALP mRNA was not detected. These findings suggest that the high alkaline phosphatase activity of retinoic acid-treated dental pulp cells is associated with increased transcription of the bone-type mRNA of the TNSALP gene and not with liver-type.
Subject(s)
Alkaline Phosphatase/drug effects , Alkaline Phosphatase/genetics , Dental Pulp/drug effects , Dental Pulp/enzymology , Gene Expression Regulation, Enzymologic/drug effects , RNA, Messenger/drug effects , RNA, Messenger/genetics , Tretinoin/pharmacology , Alkaline Phosphatase/analysis , Alkaline Phosphatase/biosynthesis , Analysis of Variance , Base Sequence , Cells, Cultured , DNA Primers , Dental Pulp/cytology , Dose-Response Relationship, Drug , Enzyme Induction/drug effects , Gene Expression Regulation, Enzymologic/physiology , Humans , Molecular Sequence Data , RNA, Messenger/metabolism , Reference Values , Reverse Transcriptase Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/statistics & numerical data , Substrate Specificity/drug effects , Time FactorsABSTRACT
The distribution of hyaluronan and versican in the dental pulp of the young rat was mapped histochemically. The pattern of staining showed considerable variation between different teeth and different specimens. The most common pattern was a strong reaction for hyaluronan and a weak reaction for versican in the subodontoblastic region, with the reverse deeper in the pulp. This was not an entirely consistent pattern and there was considerable regional variation in the staining intensity for both molecules. The localization of these molecules at similar sites could thus indicate related roles in the connective tissue matrix rather than any chemical bonding between them.
Subject(s)
Chondroitin Sulfate Proteoglycans/analysis , Dental Pulp/cytology , Hyaluronic Acid/analysis , Lectins/analysis , Proteoglycans/analysis , Animals , Coloring Agents , Connective Tissue Cells/cytology , Connective Tissue Cells/ultrastructure , Dental Pulp/ultrastructure , Histocytochemistry , Immunoenzyme Techniques , Lectins, C-Type , Male , Molar , Odontoblasts/cytology , Odontoblasts/ultrastructure , Rats , Rats, Sprague-Dawley , VersicansABSTRACT
Tissue-nonspecific-type alkaline phosphatase (TNSALP) is found in the bone, liver, kidney, and other tissues, and its gene consists of 12 exons with the coding sequence beginning in the second exon. Recently, a noncoding first exon was identified in the liver message (liver type) which differed from that of the previously known osteoblast-derived cDNA sequence (bone type). Although these two mRNAs produce an identical protein, they have different promoter regions. It is known that ALPs in dental pulp and periodontal ligament are classified into TNSALP by their enzymatic and immunological properties, but little is known about the expression of ALP mRNAs and the transcriptional mechanisms. In order to examine the expression of their mRNA type, specific oligonucleotide primers corresponding to two types of mRNAs of human TNSALP were designed and amplified by reverse transcription-polymerase chain reaction (RT-PCR). It was found that bone-type mRNA was expressed in the human dental tissues such as dental pulp, periodontal ligament, and dental sac, whereas liver-type mRNA was not expressed. Thus, it was concluded that the human dental tissues express the bone-type isozymes and are regulated by the same transcriptional mechanism as in the bone.
Subject(s)
Alkaline Phosphatase/genetics , Dental Pulp/enzymology , Dental Sac/enzymology , Isoenzymes/genetics , Periodontal Ligament/enzymology , Cells, Cultured , Dental Pulp/cytology , Dental Sac/cytology , Gene Expression , Humans , Periodontal Ligament/cytology , RNA, Messenger , Tissue DistributionABSTRACT
Hypophosphatasia (HOPS) is an inherited disorder characterized by defects in skeletal mineralization due to the deficiency of tissue-nonspecific alkaline phosphatase (TNSALP). To date, various mutations in the TNSALP gene have been identified. Especially, a deletion of T at position 1735 (1735T-del) located in exon 12 has been detected in three genetically unrelated Japanese patients, which seems to be one of the hot spots among the causative mutations in Japanese HOPS patients. 1735T-del causes a frame shift downstream from codon 503 (Leu), and consequently the normal termination codon at 508 is eliminated. Since a new inframe termination codon appears at codon 588 in the mutant DNA, the resultant protein is expected to have 80 additional amino acids. Expression of the mutant TNSALP gene using COS-1 cells demonstrated that the protein translated from the mutant 1735T-del had undetectable ALP activity, and its molecule size was larger than normal, as expected. Interestingly, an immunoprecipitation study of patients' sera using antibody against TNSALP revealed an abnormal protein which corresponded in size to the mutated TNSALP expressed by COS-1 cells, suggesting that the abnormal TNSALP is made by HOPS patients. The detection of TNSALP in cells transfected with 1735T-del using an immunofluorescent method exhibited only a faint signal on the cell surface, but an intense intracellular fluorescence after permeabilization.
Subject(s)
Alkaline Phosphatase/genetics , Frameshift Mutation , Hypophosphatasia/genetics , Alkaline Phosphatase/immunology , Amino Acid Sequence , Animals , COS Cells , Child , Child, Preschool , Codon , Electrophoresis, Polyacrylamide Gel , Female , Fluorescent Antibody Technique, Direct , Gene Expression , Humans , Hypophosphatasia/enzymology , Infant , Male , Molecular Sequence Data , Pedigree , Protein Biosynthesis , Sequence Deletion , TransfectionABSTRACT
Alkaline phosphatase (ALP) in human periodontal ligament (HPDL) cells is classified as a tissue-non-specific alkaline phosphatase (TNSALP) by its enzymatic and immunological properties. Since retinoic acid (RA) has been shown as a potent inducer of TNSALP expression in various osteoblastic and fibroblastic cells, we investigated the effects of RA on the level of ALP activity and expression of TNSALP mRNAs in HPDL cells. Cultured cells were treated with desired RA concentrations (0, 10(-7), 10(-6), 10(-5) M) in medium containing 1% bovine serum albumin without serum. ALP activity was determined by the rate of hydrolysis of p-nitrophenyl phosphate and was also assayed in the presence of specific inhibitors. In order to identify the TNSALP mRNA type expressed by HPDL, a set of oligonucleotide primers corresponding to 2 types of human TNSALP mRNA (i.e. bone-type and liver-type) were designed, and mRNA isolated from HPDL was amplified by means of reverse transcription-polymerase chain reaction (RT-PCR). After treatment with RA (10(-6) M) for 4 d, there was a significant increase in the ALP activity of HPDL cells. The use of inhibitors and thermal inactivation experiments showed that the increased ALP activity had properties of the TNSALP type. RT-PCR analysis revealed that bone-type mRNA was highly stimulated in HPDL cells by RA treatment, but the expression of liver-type mRNA was not detected. These results indicated that the upregulation of ALP activity in HPDL cells by RA was due to the increased transcription of bone-type mRNA of the TNSALP gene.
Subject(s)
Alkaline Phosphatase/biosynthesis , Periodontal Ligament/drug effects , Periodontal Ligament/enzymology , Tretinoin/pharmacology , Alkaline Phosphatase/antagonists & inhibitors , Alkaline Phosphatase/metabolism , Animals , Bone and Bones/enzymology , Cattle , Cells, Cultured , Enzyme Induction/drug effects , Fibroblasts/drug effects , Fibroblasts/enzymology , Gingiva/cytology , Gingiva/enzymology , Humans , Liver/enzymology , Organ Specificity , Periodontal Ligament/cytology , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Cartilage-derived morphogenetic proteins-1 and -2 (CDMP-1 and CDMP-2) are members of the bone morphogenetic protein (BMP) family, which play important roles in embryonic skeletal development. We studied the biological activities of recombinant CDMP-1 and CDMP-2 in chondrogenic and osteogenic differentiation and investigated their binding properties to type I and type II serine/threonine kinase receptors. In vivo, CDMP-1 and CDMP-2 were capable of inducing dose-dependently de novo cartilage and bone formation in an ectopic implantation assay. In vitro studies using primary chondrocyte cultures showed that both CDMP-1 and CDMP-2 stimulated equally de novo synthesis of proteoglycan aggrecan in a concentration-dependent manner. This activity was equipotent when compared with osteogenic protein-1 (OP-1). In contrast, CDMPs were less stimulatory than OP-1 in osteogenic differentiation as evaluated by alkaline phosphatase activity and expression levels of bone markers in ATDC5, ROB-C26, and MC3T3-E1 cells. CDMP-2 was the least osteogenic in these assays. Receptor binding studies of CDMP-1 and CDMP-2 revealed that both have affinity for the BMP receptor type IB (BMPR-IB) and BMPR-II, and weakly for BMPR-IA. Moreover, using a promoter/reporter construct, transcriptional activation signal was transduced by BMPR-IB in the presence of BMPR-II upon CDMP-1 and CDMP-2 binding. Our data show that distinct members of the BMP family differentially regulate the progression in the osteogenic lineage, and this may be due to their selective affinity for specific receptor complexes.
Subject(s)
Bone Morphogenetic Proteins/pharmacology , Cartilage/physiology , Extracellular Matrix Proteins , Growth Substances/pharmacology , Osteogenesis/drug effects , Protein Serine-Threonine Kinases , Receptors, Transforming Growth Factor beta , Aggrecans , Alkaline Phosphatase/metabolism , Animals , Bone Development/drug effects , Bone Morphogenetic Protein 7 , Bone Morphogenetic Protein Receptors, Type I , Cell Differentiation/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Growth Differentiation Factor 5 , Growth Substances/biosynthesis , Humans , Lectins, C-Type , Mice , Proteoglycans/biosynthesis , RNA/genetics , RNA/isolation & purification , Receptors, Growth Factor/metabolism , Recombinant Proteins/pharmacology , Signal Transduction/drug effects , Transforming Growth Factor beta/pharmacologySubject(s)
Proteoglycans/physiology , Cell Division/physiology , Decorin , Epidermal Growth Factor/physiology , Extracellular Matrix/metabolism , Extracellular Matrix Proteins , Fibroblast Growth Factors/physiology , Heparan Sulfate Proteoglycans/physiology , Heparin-binding EGF-like Growth Factor , Heparitin Sulfate/physiology , Humans , Intercellular Signaling Peptides and Proteins , Proteoglycans/metabolism , Receptors, Fibroblast Growth Factor/physiology , Transforming Growth Factor beta/physiologyABSTRACT
UNLABELLED: Osteogenic protein-1 (also known as bone morphogenetic protein-7) is a member of the bone morphogenetic protein family. Bone morphogenetic proteins and related members of the TGF-beta (transforming growth factor-beta) superfamily are involved in the development and repair of bone. Recombinant bone morphogenetic proteins induce the formation of new cartilage and bone at heterotopic sites. We investigated the influence of recombinant osteogenic protein-1 (at doses of three, ten, thirty, or 100 nanograms per milliliter) on the synthesis and release of proteoglycans and the maintenance of a steady-state concentration of proteoglycans in explants of porcine articular cartilage that were maintained in chemically defined serum-free medium. We found a dose-dependent stimulation of proteoglycan synthesis and a concurrent decrease in the rate of release of proteoglycans from the explants. The size of the proteoglycan monomers and the composition of the glycosaminoglycan chains in the untreated articular cartilage were similar to those in the articular cartilage treated with osteogenic protein-1. The capacity of the newly synthesized proteoglycan monomers to form aggregates with exogenous hyaluronic acid was found to be similar to that of proteoglycans in bovine nasal cartilage. Our results demonstrated that osteogenic protein-1 stimulated the synthesis of proteoglycans and diminished the release of proteoglycans from explants of porcine articular cartilage. CLINICAL RELEVANCE: The maintenance and repair of articular cartilage is a formidable challenge in clinical orthopaedics. The stimulation of proteoglycan synthesis by osteogenic protein-1 (bone morphogenetic protein-7) in explants of cartilage maintained in chemically defined serum-free medium implies that recombinant osteogenic protein-1 may play a role in the maintenance of a steady-state concentration of proteoglycans in articular cartilage, a desirable prerequisite for optimum repair of cartilage. Osteogenic protein-1 can initiate the formation of cartilage from mesenchymal cells. Once new cartilage has formed at the site of repair, osteogenic protein-1 also may maintain the synthesis of proteoglycans.