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PubMed1996

Identification of the cell type origin of odontoma-like cell masses in microphthalmic (mi/mi) mice by in situ hybridization.

Tooth abnormalities occur in microphthalmic (mi/mi) mice. The elongated odontogenic epithelium is interrupted by unresorbed bone at the basal end of the mi/mi incisor, with the epithelium gathered into cell clusters. These clusters develop to odontoma-like masses. To identify the origin of the cell types of these odontoma-like masses, the localization of osteonectin (Osn), osteocalcin (Osc), osteopontin (Osp), matrix Gla protein (MGP) and amelogenin (Am) mRNA in the process of tooth development in mi/mi and +/+ mice was investigated by means of in situ hybridization. Decalcified mandibles of neonatal, 5-, 10-, 14-day-old mice were examined. Osn and Osc mRNA, which localized in osteoblasts and odontoblasts, were also detected in the cells of odontoma-like masses in mi/mi mice. The cells expressing these mRNA were short, columnar and odontoblast-like. Am mRNA was detected in ameloblasts. In mi/mi mice, Am mRNA was also detected in ameloblastic cell clusters, which were formed by the tall columnar cells in the odontoma-like masses. No apparent Osp mRNA expression was detected in the masses. These results indicated that even in odontogenic abnormal cells resulting from physical obstruction in mi/mi mice, the genes that are involved in normal tooth development were still expressed.

Animals↗

PubMed2005

Establishment and characterization of a KIT-positive and stem cell factor-producing cell line, KTHOS, derived from human osteosarcoma.

Osteosarcoma is a malignant bone tumor that commonly affects adolescents and young adults. In the present study a human osteosarcoma cell line, KTHOS, was established from a primary osteosarcoma lesion in the distal femur of a 16-year-old girl. After 106 passages, the KTHOS cell line retained the biological characteristics of osteosarcoma. The KTHOS cells had spindle to pleomorphic cytoplasm with round to ovoid nuclei containing multiple prominent nucleoli, as expected based on the mesodermic origin of osteoblasts. The KTHOS cells were immunoreactive for osteocalcin, osteonectin, stem cell factor (SCF), and KIT (CD117). Reverse transcriptase-polymerase chain reaction indicated that the KTHOS cell line expressed mRNA for SCF and KIT. The KTHOS cells produced relatively high amounts of soluble SCF as determined by enzyme-linked immunosorbent assay. The results suggest that cell proliferation of the KTHOS cell line might be involved in autocrine and/or paracrine loops of the SCF/KIT signaling system. The KTHOS cell line is a novel human osteosarcoma cell line that releases SCF and expresses KIT. This cell line can be used for studies to explore the mechanisms for oncogenesis of human osteosarcomas.

Adolescent↗

PubMed1998

Effects of ethanol on gene expression in rat bone: transient dose-dependent changes in mRNA levels for matrix proteins, skeletal growth factors, and cytokines are followed by reductions in bone formation.

Several studies were performed in female rats to determine dose and time course changes in mRNA levels for matrix proteins in bone after a single administration of ethanol. As expected, dose-dependent transient increases in blood ethanol were measured. Additionally, there was mild hypocalcemia with no change in immunoreactive parathyroid hormone. Coordinated dose-dependent increases in mRNA for type 1 collagen, osteonectin, and osteocalcin were noted in the proximal tibial metaphysis 6 hr after ethanol was given, with the peak values occurring at a dose of 1.2 g/kg (0.4 ml). Similar increases in mRNA levels for matrix proteins were noted in lumbar vertebrae after ethanol treatment. The changes were specific for bone; ethanol had no effect on mRNA levels for matrix proteins in the uterus or liver, although the mRNA concentrations tended to be reduced in uterus. Message levels for several cytokines implicated in the regulation of bone turnover were also assayed; mRNA levels for transforming growth factor-beta1, transforming growth factor-beta2, interferon-gamma, and interleukin-6 were unchanged at doses ranging from 0.14 to 1.7 g/kg. At the highest dose of ethanol, the mRNA level for tumor necrosis factor-alpha was elevated while the level for insulin-like growth factor-1 was reduced. The time course effects of ethanol (0.4 ml dose) were determined in a separate experiment. Ethanol resulted in a transient increase in mRNA levels for the three bone matrix proteins assayed. However, matrix protein synthesis, as determined by incorporation of 3H-proline into the proximal tibial metaphysis, was not changed after 6 hr. The changes in mRNA levels for the matrix proteins were preceded by brief, transient decreases in mRNA levels for interleukin-1beta, interferon-gamma, and migration inhibitory factor, and followed by a more prolonged decrease in the mRNA level for insulin-like growth factor-1. A subsequent study was performed to determine the effects of repetitive daily treatment with ethanol on rat bone. After 7 days, there were highly significant decreases in the mRNA level for type 1 collagen, as well as decreased bone formation. These results suggest that ethanol may alter bone metabolism by disturbing signal transduction pathways that regulate the expression of genes for bone matrix proteins, skeletal growth factors, and cytokines.

Animals↗

PubMed2003

Effects of irradiation on cementum matrix cytokins function during periodontal regeneration.

The influence of gamma-ray irradiation on a cementum-impregnated gelatine membrane (CGM) was analyzed with emphasis on its function during periodontal regeneration. In brief, proteins were extracted from gamma-ray irradiated cementum (gammaC). With the gammaC protein, sample cells (gingival fibroblasts, periodontal ligament cells, and alveolar bone cells) were co-cultured, and cytological parameters (cell attachment, cell differentiation and alkaline phosphatase activity) were analyzed. Additionally, kinetics of some gene expression was analysed using reverse transcript RT-PCR, which included osteoproteogerin (OPG)/osteoclastogenesis inhibitory factor (OCIF) mRNA. BMP-2 and osteonectin were resistant to gamma-rays, and other cytokines involved in regeneration were decreased. Thus, the attachment activity of osteoblasts to gammaC protein was higher than that of non-irradiated cementum (control C). The expression of OPG/OCIF mRNA was lower in co-cultured cells with gammaC protein than those with in control C protein. Together the results imply that some cytokine in intact cementum prevents the attachment (differentiation) of bone cells onto the root surface, which may explain why the introduction of CGM following gingival flap surgery induces new cementum, new ligament and new bone formation, but CGM irradiated with gamma-rays for clinical use causes ankylosis.

Animals↗

PubMed1996

Pasteurella multocida toxin is a mitogen for bone cells in primary culture.

The effect of recombinant Pasteurella multocida toxin (PMT) on primary cultures of embryonic chick bone-derived osteoblastic cells was investigated. It was found that PMT was a potent mitogen for primary derived chicken osteoblasts. The toxin stimulated DNA synthesis and cell proliferation in quiescent osteoblasts at the first passage and accelerated cell growth in subconfluent cultures. Cell viability was not affected by PMT, even at relatively high concentrations. Osteoblast numbers increased in a dose-dependent manner in response to PMT. Intracellular inositol phosphates were elevated in response to PMT, but no elevation in cyclic AMP (cAMP) levels was evident. Indeed, PMT inhibited cAMP elevation in osteoblasts in response to cholera toxin at a stage before other PMT-mediated events take place. In addition to increased cell turnover, PMT down-regulated the expression of several markers of osteoblast differentiation. Both alkaline phosphatase and type I collagen were reduced, but osteonectin was not affected. The in vitro deposition of mineral in cultures of primary osteoblasts and osteoblast-like osteosarcoma cells was also inhibited by the presence of PMT. This suggests that PMT interferes with differentiation at a preosteoblastic stage.

Animals↗

PubMed1991

Diverse gene sequences are overexpressed in werner syndrome fibroblasts undergoing premature replicative senescence.

Genes that play a role in the senescent arrest of cellular replication are likely to be overexpressed in human diploid fibroblasts (HDF) derived from subjects with Werner syndrome (WS) because these cells have a severely curtailed replicative life span. To identify some of these genes, a cDNA library was constructed from WS HDF after they had been serum depleted and repleted (5 days in medium containing 1% serum followed by 24 h in medium containing 20% serum). Differential screening of 7,500 colonies revealed 102 clones that hybridized preferentially with [32P]cDNA derived from RNA of WS cells compared with [32P]cDNA derived from normal HDF. Cross-hybridization and partial DNA sequence determination identified 18 independent gene sequences, 9 of them known and 9 unknown. The known genes included alpha 1(I) procollagen, alpha 2(I) procollagen, fibronectin, ferritin heavy chain, insulinlike growth factor-binding protein-3 (IGFBP-3), osteonectin, human tissue plasminogen activator inhibitor type I, thrombospondin, and alpha B-crystallin. The nine unknown clones included two novel gene sequences and seven additional sequences that contained both novel segments and the Alu class of repetitive short interspersed nuclear elements; five of these seven Alu+ clones also contained the long interpersed nuclear element I (KpnI) family of repetitive elements. Northern (RNA) analysis, using the 18 sequences as probes, showed higher levels of these mRNAs in WS HDF than in normal HDF. Five selected mRNAs studied in greater detail [alpha 1(I) procollagen, fibronectin, insulinlike growth factor-binding protein-3, WS3-10, and WS9-14] showed higher mRNA levels in both WS and late-passage normal HDF than in early-passage normal HDF at various intervals following serum depletion/repletion and after subculture and growth from sparse to high-density confluent arrest. These results indicate that senescence of both WS and normal HDF is accompanied by overexpression of similar sets of diverse genes which may play a role in the senescent arrest of cellular replication and in the genesis of WS, normal biological aging, and attendant diseases.

Base Sequence↗

PubMed1987

Enamel protein and collagen production by cells subcultured from porcine tooth bud explants.

Fibroblast (F) and epithelial (E) cells were obtained as primary outgrowths from explants of fetal porcine maxillary molars and subcultured up to four passages in monolayers enriched with either cell type. Histology of a tooth bud after 1 day in culture showed intact odontogenic E cell layers which were the probable source of the E cell outgrowths. After 2 months in culture, the fourth passage E cells demonstrated morphological differentiation by an alteration in cell packing and the formation of domes and nodules, when E and F cells were cocultured. Occasionally the nodules grew to considerable size, indicating the potential of these cells to aggregate and reorganize into odontogenic tissues even on culture dishes. The cells were characterized in monolayer culture by immunocytochemical staining. Laminin and type IV collagen staining was distributed diffusely throughout the culture, whereas type I collagen and osteonectin staining was predominantly localized in the F cells. Radiolabelled proteins from both E and F cell media produced similar collagen patterns (95% type I, 4% type V, 1% other), except that the F cells appeared to produce active collagenase. In addition, the E cells produced two radiolabelled proteins (relative masses of 50,000 and 53,000) that reacted with an affinity-purified antibody directed against porcine amelogenin. These experiments show that cells subcultured from tooth buds and grown in monolayer cultures can be used to study tooth organogenesis in vitro, as well as enamel protein biosynthesis.

Amelogenin↗

PubMed1993

Skeletal unloading in rat decreases proliferation of rat bone and marrow-derived osteoblastic cells.

The effects of skeletal unloading on osteoblastic cells were evaluated in tail-suspended rats. Hindlimb elevation for 14 days induced osteopenia, decreased histomorphometric indexes of bone formation in tibial metaphysis, and reduced plasma osteocalcin and alkaline phosphatase (ALP) levels compared with controls. The in vitro proliferation of osteoblastic cells isolated from the endosteal bone surface of suspended tibias was decreased by 42 and 31% at 2 and 4 days of culture, respectively, compared with controls, as shown by [3H]thymidine labeling and cell number. The proliferation of ALP-positive marrow stromal cells was also decreased by 20-24% at 1 and 2 days of culture. However, ALP activity in bone-derived cells and marrow stromal cells was not different in unloaded and control rats, and the number of bone cells synthesizing osteocalcin, osteonectin, and type I or type III collagen was identical in the two groups. The results indicate that the inhibition of bone formation induced by skeletal unloading is related to a decreased proliferation of putative osteoblast precursor cells present along the endosteal bone surface and in the marrow stroma.

Alkaline Phosphatase↗

PubMed1998

Chronic metabolic acidosis reversibly inhibits extracellular matrix gene expression in mouse osteoblasts.

Chronic metabolic acidosis induces net calcium efflux from bone mineral through an increase in osteoclastic resorption and a decrease in osteoblastic matrix deposition and mineralization. To determine the effects of chronic metabolic acidosis on the expression of genes necessary for mineralization, we grew primary bone cells, which are principally osteoblasts, to confluence in neutral pH (7.5) medium and then switched the cells either to a neutral pH or to an acidic pH (7.1) differentiation medium. Cells were harvested for RNA at 4- to 7-day intervals for up to 44 days. By 36 days, there was extensive bone nodule formation and mineralization in cells cultured in neutral medium; however, there was a substantial decrease in nodule formation and mineralization in cells cultured in acidic medium. There was a marked increase in matrix Gla protein RNA and an increase in osteopontin RNA in neutral cultures; however, acidic medium almost completely prevented any increase. In contrast, RNA levels for osteonectin and transforming growth factor-beta1 were not altered by chronic acidosis. Additional cells were incubated in acid differentiation medium for 1, 2, or 3 wk and then transferred to neutral medium; in each case, there was recovery of matrix Gla protein RNA and osteopontin RNA expression. Still other cells were incubated in neutral differentiation medium for 1, 2, or 3 wk and then transferred to acid medium; in each case there was inhibition of matrix Gla protein RNA and osteopontin RNA expression. Thus metabolic acidosis appears to specifically inhibit RNA accumulation of certain genes whose products may be essential for formation of mature bone matrix.

Acidosis↗

PubMed2006

Ectopic doublecortin gene expression suppresses the malignant phenotype in glioblastoma cells.

Doublecortin (DCX) is one of the three genes found from Affymetrix gene chip analysis related to glioma patient survival. Two other genes (e.g., osteonectin and semaphorin 3B) are well characterized as antioncogenic and tumor suppressor genes. However, there is no report about the involvement of DCX in cancer. Here, we show that gene transfer technology into DCX-deficient glioblastoma cell lines, such as A172, U87, U251N, RG2, and 9L, with DCX cDNA significantly suppressed growth of these glioma cells. U87 cells with ectopic expression of DCX exhibit a marked suppression of the transformed phenotype as growth arrested in the G(2) phase of the cell cycle progression, small colony formation in soft agar, and no tumor formation in nude rats. This transformed phenotype can be restored by knocking down DCX expression with DCX small interfering RNA. DCX was highly phosphorylated in glioma cells. Phosphorylation in the glioma cells was greater than in noncancer cells such as mouse NIH 3T3 and human embryonic kidney 293T cells. Coimmunoprecipitation of the phosphorylated DCX and spinophilin/neurabin II from DCX-synthesizing glioma cells indicated their interaction. This interaction would lead to a block of anchorage-independent growth as neurabin II is a synergistic inhibitor of anchorage-independent growth with p14ARF (ARF). Interaction between phosphorylated DCX and neurabin II may induce the association of the protein phosphatase 1 catalytic subunit (PP1) with neurabin II and inactivate PP1 and block mitosis during G(2) and M phases of the cell cycle progression. Thus, DCX seems to be a tumor suppressor of glioma.

3T3 Cells↗

PubMed2006

The changes in angiogenic gene expression in recurrent multiple chorioangiomas.

OBJECTIVES: To assess the messenger ribonucleic acid expression in placental tissue of growth factors, cytokines, angiogenic and anti-angiogenic factors in one case of recurrent multiple chorioangiomas. METHODS: Complementary deoxyribonucleic acid array analysis was performed on the affected placentae and on normal placentaes (controls) to compare messenger ribonucleic acid levels of 96 genes involved in angiogenesis. RESULTS: Eleven genes presented more than two-fold alteration in expression levels: undetectable (angiopoietin 1, osteonectin, tyrosine kinase endothelial, neuropilin 1), decreased (transcription growth factor beta receptor 3, tissue inhibitor of metalloproteinase type 2, EGF receptor, integrin-alpha V, tyrosine kinase vascular endothelial growth factor receptor 2), increased (angiopoietin 2, osteopontin). CONCLUSIONS: We illustrated the complexity of angiogenic disruption in recurrent multiple chorioangiomas leading to the difficulty to propose a single candidate gene to explain this pathology.

Adult↗

PubMed1991

Somatic cell mapping of bovine clathrin light chain genes: identification of a new bovine syntenic group.

Two bovine DNA probes (LCa and LCb) complementary to the clathrin light chain genes were hybridized to DNAs from bovine hamster hybrid somatic cell lines retaining different combinations of bovine chromosomes. Concordancy of retention of the clathrin genes was compared to existing syntenic data for the domestic cow. LCb identified a single locus. CLTB, concordant with the genes encoding bovine anti-Müllerian hormone (AMH) and bovine osteonectin from bovine syntenic group U22. LCa recognized two loci. CLTAL1 from a previously unidentified bovine syntenic group. U25, and CLTAL2 which is concordant with GGTB2, a gene marker for bovine syntenic group U18.

Animals↗

PubMed1999

Medial localization of mineralization-regulating proteins in association with Mönckeberg's sclerosis: evidence for smooth muscle cell-mediated vascular calcification.

BACKGROUND: Calcification of the media of peripheral arteries is referred to as Mönckeberg's sclerosis (MS) and occurs commonly in aged and diabetic individuals. Its pathogenesis is unknown, but its presence predicts risk of cardiovascular events and leg amputation in diabetic patients. Several studies have documented expression of bone-associated genes in association with intimal atherosclerotic calcification, leading to the suggestion that vascular calcification may be a regulated process with similarities to developmental osteogenesis. Therefore, we examined gene expression in vessels with MS to determine whether there was evidence for a regulated calcification process in the vessel media. METHODS AND RESULTS: In situ hybridization, immunohistochemistry, and semiquantitative reverse-transcription polymerase chain reaction were used to examine the expression of mineralization-regulating proteins in human peripheral arteries with and without MS. MS occurred in direct apposition to medial vascular smooth muscle cells (VSMCs) in the absence of macrophages or lipid. These VSMCs expressed the smooth muscle-specific gene SM22alpha and high levels of matrix Gla protein but little osteopontin mRNA. Compared with normal vessels, vessels with MS globally expressed lower levels of matrix Gla protein and osteonectin, whereas alkaline phosphatase, bone sialoprotein, bone Gla protein, and collagen II, all indicators of osteogenesis/chondrogenesis, were upregulated. Furthermore, VSMCs derived from MS lesions exhibited osteoblastic properties and mineralized in vitro. CONCLUSIONS: These data indicate that medial calcification in MS lesions is an active process potentially orchestrated by phenotypically modified VSMCs.

Adult↗

PubMed1994

Age- and gender-related changes in the distribution of osteocalcin in the extracellular matrix of normal male and female bone. Possible involvement of osteocalcin in bone remodeling.

With increasing age, bone undergoes changes in remodeling that ultimately compromise the structural integrity of the skeleton. The presence of osteocalcin in bone matrix may alter bone remodeling by promoting osteoclast activity. Whether age- and/or gender-related differences exist in the distribution of osteocalcin within individual bone remodeling units is not known. In this study, we determined the immunohistochemical distribution of osteocalcin in the extracellular matrix of iliac crest bone biopsies obtained from normal male and female volunteers, 20-80 yr old. Four different distribution patterns of osteocalcin within individual osteons were arbitrarily defined as types I, II, III, or IV. The frequency of appearance of each osteon type was determined as a percent of the total osteons per histologic section. The proportion of osteons that stained homogeneously throughout the concentric lamellae (type I) decreased in females and males with increasing age. The proportion of osteons that lack osteocalcin in the matrix immediately adjacent to Haversian canals (type III) increased in females and males with age. Osteons staining intensely in the matrix adjacent to Haversian canals (type II) increased in females and was unchanged in aging males. Osteons that contained osteocalcin-positive resting lines (type IV) increased in bone obtained from males with increasing age but were unchanged in females. Sections of bone immunostained for osteopontin (SPP-I), osteonectin, and decorin did not reveal multiple patterns or alterations in staining with gender or increasing age. We suggest that the morphology of individual bone remodeling units is heterogeneous and the particular morphologic pattern of osteocalcin distribution changes with age and gender. These results suggest that differences in the distribution of osteocalcin in bone matrix may be responsible, in part, for the altered remodeling of bone associated with gender and aging.

Adult↗

PubMed1994

TGF-beta 1 and 25-hydroxycholesterol stimulate osteoblast-like vascular cells to calcify.

Previous studies in our laboratory demonstrated messenger RNA for bone morphogenetic protein-2a in human calcified plaque, suggesting that arterial calcification is a regulated process, similar to osteogenesis. To further test this hypothesis, we have isolated and cloned a subpopulation of cells from bovine aortic media that show osteoblastic potential. These novel cells are primarily distinguished from smooth muscle cells by expression of a surface marker preliminarily identified as a modified form of the ganglioside sialyl-lactosylceramide (GM3). Osteoblastic potential was indicated by high levels of alkaline phosphatase and collagen I, expression of osteopontin and osteonectin (SPARC), and production of bone-specific osteocalcin and hydroxyapatite. Cultures of these cells were stimulated to form increased numbers of calcium-mineral-producing nodules by the oxysterol 25-hydroxycholesterol as well as by transforming growth factor-beta 1, both known to be present in atherosclerotic lesions. The stimulation of calcifying vascular cells in the artery wall by these two factors suggests a possible mechanism for the colocalization of calcification with atherosclerosis in vivo.

Alkaline Phosphatase↗

PubMed1995

A compilation of partial sequences of randomly selected cDNA clones from the rat incisor.

The formation of tooth organs is regulated by a series of developmental programs. We have initiated a genome project with the ultimate goal of identifying novel genes important for tooth development. As an initial approach, we constructed a unidirectional cDNA library from the non-calcified portion of incisors of 3- to 4-week-old rats, sequenced cDNA clones, and classified their sequences by homology search through the GenBank data base and the PIR protein data base. Here, we report partial DNA sequences obtained by automated DNA sequencing on 400 cDNA clones randomly selected from the library. Of the sequences determined, 51% represented sequences of new genes that were not related to any previously reported gene. Twenty-six percent of the clones strongly matched genes and proteins in the data bases, including amelogenin, alpha 1(I) and alpha 2(I) collagen chains, osteonectin, and decorin. Nine percent of clones revealed partial sequence homology to known genes such as transcription factors and cell surface receptors. A significant number of the previously identified genes were expressed redundantly and were found to encode extracellular matrix proteins. Identification and cataloging of cDNA clones in these tissues are the first step toward identification of markers expressed in a tissue- or stage-specific manner, as well as the genetic linkage study of tooth anomalies. Further characterization of the clones described in this paper should lead to the discovery of novel genes important for tooth development.

Amelogenesis↗

PubMed1994

Gingival crevicular fluid: biomarkers of periodontal tissue activity.

The lack of precise clinical criteria for assessment of periodontal disease has led to a search for alternative means of determining active disease sites, prognosis of future sites of breakdown, and response to therapy. This review highlights the potential array of biomarkers present in gingival crevicular fluid and which may relate to existing or predicted tissue regions undergoing metabolic change and derived from bacterial or host-cell-derived products. Among the former may be listed endotoxin, amines, butyrate, and a variety of enzymes and their inhibitors, such as trypsin-like proteases and bacterial collagenase. Arising from host cells is a variety of leucocytic hydrolase enzymes, lactoferrin, and lysozyme. These appear to be useful inflammatory markers and may be distinguished from products of connective tissue breakdown which include collagenous and non-collagenous products, including collagen peptides, osteonectin, and fibronectin. The proteoglycans have found particular favor as biomarkers of possible bone-resorptive activity. Attention has also been directed at the immune response, including comment on immunoglobulins, complement, eicosanoids, and cytokines. This review lists available information on the presence of these in gingival sulcus fluid and wherever possible relates their presence to disease activity.

Biomarkers↗

PubMed2005

Hyper-expression of osteocalcin mRNA in odontoblasts of Hyp mice.

The Hyp mouse is a murine homologue of human X-linked hypophosphatemia that displays hypo-mineralization in bone and dentin. In this study, we tested the hypothesis that the defect in Hyp mice leads to alterations in the expression of dentin matrix proteins that may be associated with the hypo-mineralization changes in the tissues. Quantitative RT-PCR analyses showed that expression of the osteocalcin gene in Hyp mice tooth germ samples was significantly higher than in wild-type mice, whereas the gene expressions of osteonectin, osteopontin, dentin matrix protein 1, and type I collagen in both types of mice were similar. Further, cultured Hyp mice tooth germ samples exhibited a higher expression of the osteocalcin gene than did those from wild-type mice, which was in accord with the results of our in vivo analysis. These findings suggest that osteocalcin mRNA is highly expressed in Hyp mice odontoblasts and may be associated with dentin hypo-mineralization.

Animals↗