Wnt3a signaling induces murine dental follicle cells to differentiate into cementoblastic/osteoblastic cells via an osterix-dependent pathway.

BACKGROUND AND OBJECTIVE: Dental follicle cells, putative progenitor cells for cementoblasts, osteoblasts and periodontal ligament cells, interplay with Hertwig's epithelial root sheath (HERS) cells during tooth root formation, in which HERS is considered to have an inductive role in initiating cementogenesis by epithelial-mesenchymal interaction. However, the specific mechanisms controlling the cementoblast/osteoblast differentiation of dental follicle cells are not fully understood. Canonical Wnt signaling has been implicated in increased bone formation by controlling mesenchymal stem cell or osteoblastic cell functions. This study examined the possible expression of canonical Wnt ligand in HERS and the role of Wnt signaling during the cementoblast/osteoblast differentiation of dental follicle cells. MATERIAL AND METHODS: The expression of Wnt3a, a representative canonical Wnt ligand, in HERS was assessed by immunohistochemistry. The differentiation and function of immortalized murine dental follicle cells were evaluated by measuring alkaline phosphatase (ALP, Alpl) activity and osteogenic gene expression. RESULTS: We identified the expression of Wnt3a in HERS during mouse tooth root development by immunohistochemistry as well as in cultured human epithelial rest cells of Malassez by real-time polymerase chain reaction, while no expression of Wnt3a was detected in cultured dental mesenchymal cells. Exposure of immortalized murine dental follicle cells to Wnt3a-induced ALP activity as well as expression of the Alpl gene. Pretreatment of cells with Dickkopf-1, a potent canonical Wnt antagonist, markedly attenuated the effect of Wnt3a on ALP expression. Furthermore, Wnt3a induced transcriptional activity of runt-related transcription factor 2 (Runx2) and expression of osterix at gene and/or protein levels. Treatment with osterix-small interfering RNA significantly inhibited Wnt3a-induced ALP expression at gene and protein levels. CONCLUSION: These findings suggest that HERS has a potential role in stimulating cementoblast/osteoblast differentiation of dental follicle cells via the Wnt/ß-catenin signaling pathway.

Induction of MMP-2 at the interface between epithelial cells and fibroblasts from human periodontal ligament.

BACKGROUND AND OBJECTIVE: MMP-2 can degrade type IV collagen and MMP-14 can activate pro MMP-2. The present study was undertaken to examine the expression of MMP-2 and MMP-14 with respect to interaction between the cells of the epithelial rests of Malassez and fibroblasts from human periodontal ligament. MATERIAL AND METHODS: Explants of human periodontal ligament tissues produced outgrowths containing both putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts after incubation in a modified serum-free medium. The distribution and expression of MMP-2 and MMP-14 were analysed using immunohistochemistry, in situ hybridization and RT-PCR analysis. The conditioned media and cell extracts were collected for western blot analysis for MMP-2. RESULTS: Putative epithelial rests of Malassez cells at the interface between the cells of the epithelial rests of Malassez and fibroblasts expressed MMP-2 and MMP-14 strongly. However, in situ hybridization analysis revealed that human periodontal ligament fibroblasts expressed MMP-2 mRNA while putative epithelial rests of Malassez cells expressed MMP-14 mRNA at the interface. The RT-PCR analysis showed that the expression of MMP-2 mRNA was significantly higher when putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts were cultured together than when cultured alone. Western blot analysis showed that the active form of MMP-2 was detected at higher levels in the conditioned medium of the co-cultured cells. CONCLUSION: These findings indicate that putative epithelial rests of Malassez cells stimulate the production of MMP-2 in human periodontal ligament fibroblasts. Up-regulated proMMP-2 bound by MMP-14 expressed in epithelial rests of Malassez cells can degrade matrix molecules, such as type IV collagen, in the basal membrane between putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts.

Mutual induction of noncollagenous bone proteins at the interface between epithelial cells and fibroblasts from human periodontal ligament.

BACKGROUND AND OBJECTIVE: Epithelial-mesenchymal interactions are responsible for cell differentiation during periodontal regeneration. The present study was undertaken to examine the expression of alkaline phosphatase and noncollagenous bone proteins, such as osteopontin, osteocalcin and bone sialoprotein, with respect to interaction between the cells of the epithelial rests of Malassez and fibroblasts from human periodontal ligament. MATERIAL AND METHODS: Explants of human periodontal ligament tissues produced outgrowths containing both putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts in a modified serum-free medium. Putative epithelial rests of Malassez cells cultured alone, and human periodontal ligament fibroblasts cultured alone, were used as controls. The expression levels of amelogenin were analyzed by in situ hybridization. The expression and distribution of alkaline phosphatase and noncollagenous bone proteins in both cell populations at the interface between putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts were analyzed by immunohistochemistry, in situ hybridization and reverse transcription-polymerase chain reaction. RESULTS: Amelogenin mRNA was detected at high levels only in putative epithelial rests of Malassez cells at the interface. Alkaline phosphatase and bone sialoprotein mRNAs were detected significantly at the interface between putative epithelial rests of Malassez cells and human periodontal ligament fibroblast cells. In particular, bone sialoprotein and its mRNA were expressed significantly in human periodontal ligament fibroblasts at the interface between putative epithelial rests of Malassez cells and human periodontal ligament fibroblast cells. The expressions of osteopontin and its mRNA were not different between putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts at the interface. Osteocalcin and its mRNA were expressed strongly in putative epithelial rests of Malassez cells at the interface between putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts. CONCLUSION: These findings indicate that the epithelial-mesenchymal interaction modulates the expression of alkaline phosphatase, osteocalcin and bone sialoprotein in putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts, suggesting that epithelial-mesenchymal interactions play a role in the maintenance of periodontal ligament.

In vitro differentiation of epithelial cells cultured from human periodontal ligament.

BACKGROUND AND OBJECTIVE: Alkaline phosphatase and noncollagenous bone proteins are produced prior to cementum formation. While it has been suggested that epithelial rests of Malassez are involved in cementum formation, little is known about the relationship between epithelial rests of Malassez and cementum formation. The purpose of the present study was to determine whether the epithelial rests of Malassez cells cultured from human periodontal ligament can produce alkaline phosphatase and noncollagenous bone proteins, such as osteopontin, osteocalcin and bone sialoprotein. MATERIAL AND METHODS: An outgrowth of putative epithelial rests of Malassez cells was produced from periodontal ligament explant, and second passage cultures were used in the experiments. Human gingival epithelial cells and periodontal ligament fibroblasts were used as controls. The expression levels of amelogenin were analyzed by immunostaining and in situ hybridization. Furthermore, the expression levels of alkaline phosphatase and noncollagenous bone proteins were assessed by immunostaining and reverse transcription-polymerase chain reaction. RESULTS: Amelogenin, alkaline phosphatase and osteopontin proteins and their corresponding mRNAs were detected at high levels in putative epithelial rests of Malassez cells. Osteocalcin and bone sialoprotein were not expressed in putative epithelial rests of Malassez cells. Alkaline phosphatase and noncollagenous bone proteins were seen in periodontal ligament fibroblasts, but not in gingival epithelial cells. CONCLUSION: Our results suggest that putative epithelial rests of Malassez cells cultured alone do not transform into maturing cells to form the cementum, but may play a potential role in the mineralization process.

Prostacyclin-dependent apoptosis mediated by PPAR delta.

Prostacyclin (PGI(2)) plays important roles in hemostasis both as a vasodilator and an endogenous inhibitor of platelet aggregation. PGI(2) functions in these roles through a specific IP receptor, a G protein-coupled receptor linked to G(s) and increases in cAMP. Here, we report that intracellular prostacyclin formed by expressing prostacyclin synthase in human embryonic kidney 293 cells promotes apoptosis by activating endogenous peroxisome proliferator-activated receptor delta (PPAR delta). In contrast, treatment of cells with extracellular prostacyclin or dibutyryl cAMP actually reduced apoptosis. On the contrary, treatment of the cells with RpcAMP (adenosine 3',5'-cyclic monophosphothioate, Rp-isomer), an antagonist of cAMP, enhanced prostacyclin-mediated apoptosis. The expression of an L431A/G434A mutant of PPAR delta completely blocked prostacyclin-mediated PPAR delta activation and apoptosis. These observations indicate that prostacyclin can act through endogenous PPAR delta as a second signaling pathway that controls cell fate.

Influence of sugar chain on fibrin affinity of recombinant t-PA.

The role of the sugar chain on the fibrin affinity property of tissue plasminogen activator (t-PA) was investigated using two variants of wild type t-PA (WT t-PA I and WT t-PA II) and mutant type t-PA (mt-PA ; Gln117 t-PA I and Gln117 t-PA II), whose sugar chains have different structures. In terms of fibrin affinity, Gln117 t-PA was higher than WT t-PA ; moreover, Type II was higher than Type I. Bindings mediated via finger domain (F mode) and kringle 2 domain (K2 mode) were distinguished using epsilon-amino caproic acid (EACA). Consequently, F mode and K2 mode bindings were inhibited by the sugar chains at Asn117 and 184, respectively. These results were assumed to be due to the steric hindrance of the sugar chains.

Cloning and characterization of 5'-flanking region of mouse prostacyclin synthase gene.

To gain an insight into the mechanisms of prostacyclin expression, a genomic DNA clone harboring 2.0 kb of the 5'-flanking sequence of the mouse prostacyclin synthase (PGIS) gene was isolated. The 5'-flanking region did not possess a TATA box, but contained a GC-rich region and several consensus cis DNA elements. The major product of the primer extension analysis suggested that the transcription of the gene started from 72 bases upstream of the translational initiation codon. To analyze the PGIS promoter activity, the 2.0 kb fragment was fused to the luciferase gene and transient transfection assays were conducted with cultured rat vascular smooth muscle cells (VSMC). The fragment showed significant promoter activity in the cells. Analysis of a series of 5'-deletion constructs showed that the 5'-flanking regions spanning bases -371 to -285 and -229 to -119 were important for the basal transcriptional activity of the mouse PGIS gene. Gel mobility shift assays revealed that DNA-protein complexes were formed with the nuclear extracts from VSMC, and that the formation of these complexes was inhibited by excess consensus Sp1 oligonucleotide. Prior incubation of anti-Sp1 antibody with nuclear extracts in this assay resulted in supershift of the band for the DNA-protein complex. In addition, mutation of two Sp1 recognition motifs residing at bases -297 to -289 and -197 to -192 markedly reduced the basal PGIS promoter activity and retarded the band in a gel mobility shift assay. These results indicated that binding of one Sp1 to two Sp1 sites on the promoter region activated the basal transcription of the PGIS gene.

Gene transfer of human prostacyclin synthase ameliorates monocrotaline-induced pulmonary hypertension in rats.

BACKGROUND: Prostacyclin is a potent vasodilator that also inhibits platelet adhesion and cell growth. We investigated whether in vivo gene transfer of human prostacyclin synthase (PGIS) ameliorates monocrotaline (MCT)-induced pulmonary hypertension in rats. METHODS AND RESULTS: The cDNA encoding PGIS was intratracheally transfected into the lungs of rats by the hemagglutinating virus of Japan-liposome method. Rats transfected with control vector lacking the PGIS gene served as controls. Three weeks after MCT injection, mean pulmonary arterial pressure and total pulmonary resistance had increased significantly; the increases were significantly attenuated in PGIS gene-transfected rats compared with controls [mean pulmonary arterial pressure, 31+/-1 versus 35+/-1 mm Hg (-12%); total pulmonary resistance, 0.087+/-0.01 versus 0.113+/-0.01 mm Hg x mL x min(-1) x kg(-1) (-23%), both P:<0.05]. Systemic arterial pressure and heart rate were unaffected. Histologically, PGIS gene transfer inhibited the increase in medial wall thickness of peripheral pulmonary arteries that resulted from MCT injection. PGIS immunoreactivity was intense predominantly in the bronchial epithelium and alveolar cells. Lung tissue levels of 6-keto-PGF(1alpha), a stable metabolite of prostacyclin, were significantly increased for >/=1 week after transfer of PGIS gene. The Kaplan-Meier survival curves demonstrated that repeated transfer of PGIS gene every 2 weeks increased survival rate in MCT rats (log-rank test, P:<0.01). CONCLUSIONS: Intratracheal transfer of the human PGIS gene augmented pulmonary prostacyclin synthesis, ameliorated MCT-induced pulmonary hypertension, and thereby improved survival in MCT rats.

Characterization of free alpha- and beta-chains of recombinant macrophage-stimulating protein.

Human serum macrophage-stimulating protein (MSP) induces motile activity of murine resident peritoneal macrophages and is a growth and motility factor for epithelial cells. It belongs to the plasminogen-related family of kringle proteins, and is secreted as a single-chain, 78-kDa, biologically inactive pro-MSP. Proteolytic cleavage of pro-MSP at a single site yields active MSP, a disulfide-linked alphabeta-chain heterodimer. However cleavage of recombinant pro-MSP yielded not only the disulfide-linked heterodimer, but also free alpha- and beta-chains, indicating that some of the recombinant molecules lacked an alphabeta-chain disulfide. We purified the free chains for characterization. The beta-chain of MSP has three extra cysteines, Cys527, Cys562, and Cys672, which are not found in the plasminogen beta-chain. Disulfide bond analysis showed a Cys527-Cys562, but also a Cys588-Cys672. Coopting Cys588 by Cys672 prevented the expected formation of a disulfide between alpha-chain Cys468 and beta-chain Cys588. Concomitant studies determined structures of oligosaccharides at the three Asn-linked glycosylation sites of MSP. The oligosaccharides at the three Asn loci are heterogeneous; 11 different sugars were identified, all being sialylated fucosyl biantennary structures. We also located the pro-MSP signal peptide cleavage site at Gly18-Gln19 and the scissile bond for formation of mature MSP at Arg483-Val484.

Cloning and expression of the gene for a vanadium-dependent bromoperoxidase from a marine macro-alga, Corallina pilulifera.

The cDNAs for a vanadium-dependent bromoperoxidase were cloned from a marine macro-alga, Corallina pilulifera. The open reading frame of one clone (bpo1) encoded a protein of 598 amino acids with a calculated molecular mass of 65312 Da in good agreement with that of 64 kDa determined for the native enzyme. The deduced amino acid sequence coincided well with partial sequences of peptide fragments of the enzyme. From the same cDNA library we also isolated another cDNA clone (bpo2) encoding a protein of 597 amino acids with an identity of about 90% to BPO1, suggesting a genetic diversity of the bromoperoxidase gene of C. pilulifera growing in a relatively narrow area. The carboxy-terminal 123 residues of the enzyme (BPO1) showed an identity of 45% to that of the marine macro-alga Ascophillum nodosum. The homology search of the sequences of bromoperoxidases from C. pilulifera (this study) and A. nodostum, and chloroperoxidase from the fungus Curvularia inaequalis indicated highly conserved sequences PxYxSGHA and LxxxxAxxRxxxGxHxxxD. Furthermore, it was found that the histidine residue directly bound to vanadium, other residues building up the metal center and catalytic histidine residue forming the active site of the chloroperoxidase from C. inaequalis are conserved in the primary structure of the bromoperoxidase from C. pilulifera. The cloned hpol was introduced into Escherichia coli, and the expressed PO1 was purified from the recombinant strain. The N-terminal amino acid sequence of the purified BPO1 was identical to the deduced sequence from the cDNA except the N-terminal methionine.

Cloning, expression, and characterization of a cDNA encoding a novel human growth factor for primitive hematopoietic progenitor cells.

Multiple growth factors synergistically stimulate proliferation of primitive hematopoietic progenitor cells. A human myeloid cell line, KPB-M15, constitutively produces a novel hematopoietic cytokine, termed stem cell growth factor (SCGF), possessing species-specific proliferative activities. Here we report the molecular cloning, expression, and characterization of a cDNA encoding human SCGF using a newly developed lambdaSHDM vector that is more efficient for differential and expression cloning. cDNA for SCGF encodes a 29-kDa polypeptide without N-linked glycosylation. SCGF transiently produced by COS-1 cells supports growth of hematopoietic progenitor cells through a short-term liquid culture of bone marrow cells and exhibits promoting activities on erythroid and granulocyte/macrophage progenitor cells in primary semisolid culture with erythropoietin and granulocyte/macrophage colony-stimulating factor, respectively. Expression of SCGF mRNA is restricted to myeloid cells and fibroblasts, suggesting that SCGF is a growth factor functioning within the hematopoietic microenvironment. SCGF could disclose some human-specific mechanisms as yet unidentified from studies on the murine hematopoietic system.

Analysis of the structure and expression of the augmenter of liver regeneration (ALR) gene.

BACKGROUND: The gene encoding the hepatotrophic factor Augmenter of Liver Regeneration (ALR) has recently been cloned in the rat. The availability of the mouse form of ALR would allow the analysis of the role of this factor in the physiology of liver and other organs, while the identification of the human homolog would allow the transfer of the great wealth of information that has been generated in animal models to clinically oriented pilot trials, and eventually the therapeutic application of this information. MATERIALS AND METHODS: Standard molecular biology approaches have been used to determine the genomic structure of the ALR gene in the mouse, and to characterize the ALR transcript and its protein product. The human ALR cDNA was also isolated and the amino acid sequence of the human gene product deduced. The mapping of mouse and human ALR genes on mouse and human chromosomes was then completed. RESULTS: The protein coding portion of the mouse ALR gene is comprised of three exons, the first containing the 5' untranslated sequence and the initial 18 bases after the ATG translation initiation codon, the second exon encompasses 198 bases, and the third exon contains the remaining portion of the protein coding sequence. Rat, mouse, and human ALR genes (and protein products) were found to be highly conserved and preferentially expressed in the testis and in the liver. The ALR gene maps to the mouse chromosome 17, in a region syntenic with human chromosome 16, where the T/t region has also been mapped. CONCLUSIONS: ALR appears to be a protein with important physiologic properties, not exclusively limited to liver regeneration, with roles that are involved in the synthesis or stability of the nuclear and mitochondrial transcripts that are present in actively regenerating cells, particularly the germ cells of the testes.

Cloning and sequence analysis of the rat augmenter of liver regeneration (ALR) gene: expression of biologically active recombinant ALR and demonstration of tissue distribution.

A full-length cDNA clone encoding a purified augmenter of liver regeneration (ALR) factor prepared from the cytosol of weanling rat livers was isolated. The 1.2-kb cDNA included a 299-bp 5' untranslated region, a 375-bp coding region, and a 550-bp 3' untranslated region. It encoded a protein consisting of 125 amino acids. The molecular weight of ALR calculated from the cDNA was 15,081, which is consistent with the size estimated by SDS/PAGE under reducing conditions. The molecular weight of the purified native ALR estimated by SDS/PAGE under nonreducing conditions was approximately 30,000; thus ALR apparently has a homodimeric structure. The recombinant ALR produced by expression of the cDNA in COS cells was tested in vivo in the canine Eck fistula model and found to have potency equivalent to the purified native ALR. The 125-aa sequence deduced from the rat ALR cDNA shows 50% homology to the amino acid sequence of the gene for oxidative phosphorylation and vegetative growth in the yeast Saccharomyces cerevisiae.

Organization of the human hepatocyte growth factor-encoding gene.

Human genomic phage libraries were screened for the human hepatocyte growth factor (HGF)-encoding gene (HGF) using a cDNA encoding the human protein as a probe. Characterization of the clones revealed that this gene is composed of 18 exons interrupted by 17 introns spanning approx. 70 kb. The first exon contains the 5'-untranslated region and the signal peptide. The next ten exons encode the alpha-chain which contains four kringle structures. Each kringle domain is encoded by two exons as observed in other kringle-containing proteins. The twelfth exon contains the short spacer region between the alpha- and beta-chains and the remaining six exons comprise the beta-chain. The beta-chain is structurally similar to the catalytic domains of serine proteases; amino acid substitutions in the active site were found. The organization of the HGF gene is highly homologous to those of the serine proteases involved in blood coagulation and fibrinolysis, especially with that of plasminogen. This suggests that the human HGF gene is evolutionally related to these genes.

Isolation and expression of cDNA for different forms of hepatocyte growth factor from human leukocyte.

Human leukocyte cDNA library was screened to isolate cDNA clones coding for hepatocyte growth factor using cDNA from human liver as a probe. Nucleotide and deduced amino acid sequences were analyzed for two of four clones obtained. One of them contained an open reading frame coding for a polypeptide chain of 728 amino acid residues like that of cDNA clone derived from human liver. In another clone a spontaneous deletion of 15 base pairs was found within the coding sequence. When expressed transiently using COS-1 cells both clones produced protein with similar biological activity against rat hepatocyte in vitro.

Deduced primary structure of rat hepatocyte growth factor and expression of the mRNA in rat tissues.

The primary structure of rat hepatocyte growth factor (HGF) was elucidated by determining the base sequence of the complementary DNA (cDNA) of HGF. The cDNA for rat HGF was isolated by screening a liver cDNA library with oligonucleotides based on the partial N-terminal amino acid sequence of the beta subunit of purified rat HGF. HGF is encoded in an mRNA of about 6 kilobases. Both alpha and beta subunits of HGF are specified in a single open reading frame for a 728-amino acid protein with a calculated molecular weight of 82,904. The N-terminal part of HGF has a signal sequence and a prosequence with 30 and 25 amino acid residues, respectively. The mature heterodimer structure is derived proteolytically from this single pre-pro precursor polypeptide. The calculated molecular weights of the alpha and beta subunits are 50,664 and 25,883, respectively, and each subunit has two potential N-linked glycosylation sites. The amino acid sequence of HGF is 38% identical with that of plasminogen. The alpha subunit of HGF contains four "kringle" structures, and the beta subunit has 37% amino acid identity with the serine protease domain of plasmin. Northern blot analysis revealed that HGF mRNA was expressed in rat various tissues, including the liver, kidney, lung, and brain.

Molecular cloning and expression of human hepatocyte growth factor.

Hepatocyte growth factor (HGF) is the most potent mitogen for mature parenchymal hepatocytes in primary culture, and seems to be a hepatotrophic factor that acts as a trigger for liver regeneration after partial hepatectomy and liver injury. The partial purification and characterization of HGF have been reported. We have demonstrated that pure HGF from rat platelets is a new growth factor effective at concentrations as low as 1 ng ml-1. The effects of HGF and epidermal growth factor (EGF) are additive. The activity of HGF is not species-specific, although it does not stimulate growth in Swiss 3T3 fibroblasts. HGF has a relative molecular mass (Mr) of 82,000 and is a heterodimer composed of a large alpha-subunit of Mr 69,000 and a small beta-subunit of Mr 34,000. Here we report the amino-acid sequence of human HGF determined by complementary DNA cloning and the expression of biologically active human HGF from COS-1 cells transfected with cloned cDNA. The nucleotide sequence of the human HGF cDNA reveals that both alpha- and beta-chains are contained in a single open reading frame coding for a pre-pro precursor protein of 728 amino acids.