Hyperglycemic microenvironment compromises the homeostasis of communication between the bone-brain axis by the epigenetic repression of the osteocalcin receptor, Gpr158 in the hippocampus.

Diabetes mellitus (DM) is a chronic metabolic disease, mainly characterized by increased blood glucose and insulin dysfunction. In response to the persistent systemic hyperglycemic state, numerous metabolic and physiological complications have already been well characterized. However, its relationship to bone fragility, cognitive deficits and increased risk of dementia still needs to be better understood. The impact of chronic hyperglycemia on bone physiology and architecture was assessed in a model of chronic hyperglycemia induced by a single intraperitoneal administration of streptozotocin (STZ; 55 mg/kg) in Wistar rats. In addition, the bone-to-brain communication was investigated by analyzing the gene expression and methylation status of genes that encode the main osteokines released by the bone [Fgf23 (fibroblast growth factor 23), Bglap (bone gamma-carboxyglutamate protein) and Lcn2 (lipocalin 2) and their receptors in both, the bone and the brain [Fgfr1 (fibroblast growth factor receptor 1), Gpr6A (G-protein coupled receptor family C group 6 member A), Gpr158 (G protein-coupled receptor 158) and Slc22a17 (Solute carrier family 22 member 17)]. It was observed that chronic hyperglycemia negatively impacted on bone biology and compromised the balance of the bone-brain endocrine axis. Ultrastructural disorganization was accompanied by global DNA hypomethylation and changes in gene expression of DNA-modifying enzymes that were accompanied by changes in the methylation status of the osteokine promoter region Bglap and Lcn2 (lipocalin 2) in the femur. Additionally, the chronic hyperglycemic state was accompanied by modulation of gene expression of the osteokines Fgf23 (fibroblast growth factor 23), Bglap (bone gamma-carboxyglutamate protein) and Lcn2 (lipocalin 2) in the different brain regions. However, transcriptional regulation mediated by DNA methylation was observed only for the osteokine receptors, Fgfr1(fibroblast growth factor receptor 1) in the striatum and Gpr158 (G protein-coupled receptor 158) in the hippocampus. This is a pioneer study demonstrating that the chronic hyperglycemic state compromises the crosstalk between bone tissue and the brain, mainly affecting the hippocampus, through transcriptional silencing of the Bglap receptor by hypermethylation of Gpr158 gene.

Discerning the Role of the Hydroxyproline Residue in the Structure of Conantokin Rl-B and Its Role in GluN2B Subunit-Selective Antagonistic Activity toward N-Methyl-d-Aspartate Receptors.

Conantokins (con) are short γ-carboxyglutamate (Gla)-containing polypeptides expressed by marine snails that function as antagonists of N-methyl-d-aspartate receptor (NMDAR) ion channels. The Gla residues govern structural conformations and antagonistic activities of the conantokins. In addition to Gla, some conantokins, e.g., conRl-B, also contain a hydroxyproline (HyP or O) residue, which in this case is centrally located in the peptide at position 10. Because conRl-B specifically inhibits ion channels of GluN2B subunit-containing heterotetrameric NMDARs, we evaluated the unusual role of HyP10 in this effect. To accomplish this goal, we examined synthetic variants of conRl-B in which HyP10 was either deleted (conRl-B[ΔO10]) or replaced with alanine (conRl-B[O10A]) or proline (conRl-B[O10P]). The solution structures of these variants were determined by nuclear magnetic resonance spectroscopy. Deletion of HyP10, or replacement of HyP10 with Ala10, attenuated the distortion in the central region of the apo-conRl-B helix and allowed Mg2+-complexed end-to-end α-helix formation. The inhibitory properties of these variants were assessed by measuring NMDA/Gly-stimulated intracellular Ca2+ influx in mice neurons. ConRl-B[O10P] retained its NMDAR ion channel inhibitory activity in wild-type (WT) neurons but lost its GluN2B specificity, whereas conRl-B[ΔO10] showed overall diminished inhibitory function. ConRl-B[O10A] showed attenuated inhibitory function but retained its GluN2B specificity. Thus, HyP10 plays a critical role in maintaining the structural integrity of conRl-B, which can be correlated with its GluN2B subunit-selective inhibition. Weakened inhibition by conRl-B was also observed in neurons lacking either the GluN2C or GluN2D subunit, compared to WT neurons. This suggests that GluN2C and GluN2D are also required for inhibition by conRl-B.

Chondrogenic differentiation of human mesenchymal stem cells on fish scale collagen.

Fish collagen has recently been reported to be a novel biomaterial for cell and tissue culture as an alternative to conventional mammalian collagens such as bovine and porcine collagens. Fish collagen could overcome the risk of zoonosis, such as from bovine spongiform encephalopathy. Among fish collagens, tilapia collagen, the denaturing temperature of which is near 37°C, is appropriate for cell and tissue culture. In this study, we investigated chondrogenic differentiation of human mesenchymal stem cells (hMSCs) cultured on tilapia scale collagen fibrils compared with porcine collagen and non-coated dishes. The collagen fibrils were observed using a scanning electronic microscope. Safranin O staining, glycosaminoglycans (GAG) expression, and real-time PCR were examined to evaluate chondrogenesis of hMSCs on each type of collagen fibril. The results showed that hMSCs cultured on tilapia scale collagen showed stronger Safranin O staining and higher GAG expression at day 6. Results of real-time PCR indicated that hMSCs cultured on tilapia collagen showed earlier SOX9 expression on day 4 and higher AGGRECAN and COLLAGEN II expression on day 6 compared with on porcine collagen and non-coated dishes. Furthermore, low mRNA levels of bone gamma-carboxyglutamate, a specific marker of osteogenesis, showed that tilapia collagen fibrils specifically enhanced chondrogenic differentiation of hMSCs in chondrogenic medium, as well as porcine collagen. Accordingly, tilapia scale collagen may provide an appropriate collagen source for hMSC chondrogenesis in vitro.

Restoration of the serum level of SERPINF1 does not correct the bone phenotype in Serpinf1 null mice.

Osteogenesis imperfecta (OI) is a group of genetic disorders characterized by bone fragility and deformity. OI type VI is unique owing to the mineralization defects observed in patient biopsies. Furthermore, it has been reported to respond less well to standard therapy with bisphosphonates [1]. Others and we have previously identified SERPINF1 mutations in patients with OI type VI. SERPINF1 encodes pigment epithelium derived factor (PEDF), a secreted collagen-binding glycoprotein that is absent in the sera of patients with OI type VI. Serpinf1 null mice show increased osteoid and decreased bone mass, and thus recapitulate the OI type VI phenotype. We tested whether restoration of circulating PEDF in the blood could correct the phenotype of OI type VI in the context of protein replacement. To do so, we utilized a helper-dependent adenoviral vector (HDAd) to express human SERPINF1 in the mouse liver and assessed whether PEDF secreted from the liver was able to rescue the bone phenotype observed in Serpinf1(-/-) mice. We confirmed that expression of SERPINF1 in the liver restored the serum level of PEDF. We also demonstrated that PEDF secreted from the liver was biologically active by showing the expected metabolic effects of increased adiposity and impaired glucose tolerance in Serpinf1(-/-) mice. Interestingly, overexpression of PEDF in vitro increased mineralization with a concomitant increase in the expression of bone gamma-carboxyglutamate protein, alkaline phosphatase and collagen, type I, alpha I, but the increased serum PEDF level did not improve the bone phenotype of Serpinf1(-/-) mice. These results suggest that PEDF may function in a context-dependent and paracrine fashion in bone homeostasis.

Carboxylation-dependent conformational changes of human osteocalcin.

Osteocalcin (OCN) is a small noncollagenous protein mainly produced by osteoblasts and is highly represented in bones of most vertebrates. Human OCN contains up to three gamma-carboxyglutamic acid (Gla-OCN) residues at positions 17, 21 and 24 which are thought to increase calcium binding strength, improving mechanical properties of the bone matrix. Recent studies revealed that OCN exerts also important endocrine functions, affecting energy metabolism and male fertility. The latter effect seems to be mediated by the uncarboxylated form of OCN (Glu-OCN). We employed human and mouse OCN as models of fully carboxylated and uncarboxylated OCN forms to investigate, by the use of circular dichroism and molecular dynamics simulations, the respective conformational properties and Ca2+ affinity. Ca2+ binding was found to trigger a similar conformational transition in both Glu-OCN and Gla-OCN, from a disordered structure to a more compact/stable form. Notably, gamma-carboxylation increases the affinity of OCN for Ca2+ by > 30 fold suggesting that, in physiological conditions, Gla-OCN is essentially Ca2+-bound, whereas Glu-OCN circulates mainly in the Ca2+-free form.

Gla-rich protein, a new player in tissue calcification?

A novel γ-carboxyglutamate (Gla)-containing protein, named Gla-rich protein (GRP) after its high content in Gla residues or upper zone of growth plate and cartilage matrix associated protein after its preferential expression by cartilage chondrocyte, was recently identified in sturgeon, mice, and humans through independent studies. GRP is the most densely γ-carboxylated protein identified to date and its structure has been remarkably conserved throughout vertebrate evolution but is apparently absent from bird genomes. Several transcript and genomic variants affecting key protein features or regulatory elements were described and 2 paralogs were identified in the teleost fish genome. In the skeleton, most relevant levels of GRP gene expression were observed in cartilaginous tissues and associated with chondrocytes, suggesting a role in chondrogenesis. But GRP expression was also detected in bone cells, indicative of a more widespread role for the protein throughout skeletal formation. Although the molecular function of GRP is yet unknown, the high content of Gla residues and its accumulation at sites of pathological calcification in different human pathologies affecting skin or the vascular system and in breast cancer tumors suggest that GRP may function as a modulator of calcium availability. Because of its association with fibrillar collagens, GRP could also be involved in the organization and/or stabilization of cartilage matrix. Although transgenic mice did not reveal obvious phenotypic alterations in skeletal development or structure, zebrafish morphants lack craniofacial cartilage and exhibit limited calcification, suggesting a role for GRP during skeletal development, but additional functional data are required to understand its function.

Molecular modulation of osteocalcin and its relevance in diabetes (Review).

Osteocalcin (OC) is encoded by the bone γ-carboxyglutamate (Gla) protein (BGLAP) gene, and it is released by osteoblasts during osteogenesis. Its expression can be modulated by growth factors, hormones, cytokines and physical stimuli via signal transduction pathways, binding to the BGLAP gene promoter or interactions with nuclear transcription factors. It was recently demonstrated that uncarboxylated OC improves glucose tolerance and insulin sensitivity in mice by increasing the expression and secretion of insulin in ß-cells and of adiponectin in adipocytes. Humans with type 2 diabetes have significantly lower serum levels of OC than healthy individuals and indeed, serum OC levels have been inversely correlated with fasting plasma glucose, fasting insulin and the homeostasis model assessment of insulin resistance (HOMA-IR) index. Moreover, several drugs have been shown to influence OC expression and its serum concentration. This review summarizes the molecular mechanisms involved in the modulation of OC expression, and discusses the potential relevance of OC in the pathogenesis and treatment of diabetes.

The effects of enamel matrix derivative on the proliferation and differentiation of human mesenchymal stem cells.

PURPOSE: This study was designed to investigate the effect of enamel derivative matrix (EMD) on the proliferation, mineralization, and differentiation of human mesenchymal stem cells (hMSCs). MATERIAL AND METHODS: For the proliferation assay, water-soluble tetrazolium salt-8 tests were carried out after culturing for 24 and 48 h. For the evaluation of mineralization, Alizarin red S (ARS) tests were performed after 21 days of culturing in an osteogenic medium. In order to investigate some of the bone-related proteins, namely type I collagen (Col I A2), bone sialoprotein (BSP), and bone gamma-carboxyglutamate (Gla) protein (BGLAP, osteocalcin), real-time polymerase chain reaction (RT-PCR) tests were carried out after 2, 3, and 4 weeks of culturing, respectively. RESULTS: The activity of proliferation and mineralization increased significantly depending on the concentration of EMD (P<0.05). In the control group, the expression of Col I A2 decreased, but EMD enhanced its expression over time and was correlated to the concentration. The amount of expression of BSP in this group increased over time, but EMD strikingly suppressed its expression in the fourth week. As well, the amount of expression of BGLAP increased as the culture duration lengthened in the control group. However, the expression of BGLAP was suppressed in the experimental group with EMD. CONCLUSION: Within the limits of this study, EMD enhanced the proliferation of hMSCs. After evaluation with ARS staining, EMD seemed to enhance mineralization, and the RT-PCR test revealed that EMD promoted early-stage osteoblast differentiation by enhancing Col I A2 expression, but exerted an inhibitory effect on the mineralization by lowering the gene expression of BSP and BGLAP. Mineralized nodules formed with EMD may be composed of substances other than normal bone. Because most of the organic matrix of bone is type I collagen, which acts as the mineralization site, bone or bone-like mineralized mass might have been formed in spite of the different components of the non-collagenous proteins.

Targeting adenoviruses with factor x-single-chain antibody fusion proteins.

Abstract It has been shown that blood clotting factors, including factor X (FX), bind to the adenovirus serotype 5 (Ad5) hexon protein and target the virus to liver hepatocytes after intravenous injection. These factors bind to hexon via their conserved vitamin K-dependent gamma-carboxyglutamic acid (GLA) domains with subnanomolar affinity. In this work, we have used this strong interaction to retarget Ad to new receptors, using the GLA domain of FX fused to single-chain antibody variable fragment (ScFv). We demonstrate that fusion of the GLA domain of human FX to receptor-specific ScFvs will target Ad5 vectors to cells expressing these receptors. Fusion of an alphaHer2 ScFv to GLA increased in vitro transduction of Her2-positive versus Her2-negative cells when compared with untargeted virus. Similar results were obtained with ScFvs against the epidermal growth factor receptor (EGFR) and against the stem cell marker ATP-binding cassette protein G2 (ABCG2). Direct expression of GLA fusion protein from replication-defective or replication-competent Ad increased infection and killing of cancer cells in vitro and in vivo. These data demonstrate the potential of using GLA domains to bridge secreted ligands with intracellularly produced Ad5 vectors for vector targeting.

Membrane-dependent interaction of factor Xa and prothrombin with factor Va in the prothrombinase complex.

Because all three protein components of prothrombinase, factors (f) Xa and Va and prothrombin, bind to negatively charged membrane phospholipids, the exact role of the membrane in the prothrombinase reaction has not been fully understood. In this study, we prepared deletion derivatives of fXa and prothrombin in which both the Gla and first EGF-like domains of the protease (E2-fXa) as well as the Gla and both kringle domains of the substrate (prethrombin-2) had been deleted. The fVa-mediated catalytic activity of E2-fXa toward prethrombin-2 was analyzed in both the absence and presence of phospholipids composed of 80% phosphatidylcholine (PC) and 20% phosphatidylserine (PS). PCPS markedly accelerated the initial rate of prethrombin-2 activation by E2-fXa, with the cofactor exhibiting saturation only in the presence of phospholipids (apparent K(d) of approximately 60 nM). Competitive kinetic studies in the presence of the two exosite-1-specific ligands Tyr(63)-sulfated hirudin(54-65) and TM456 suggested that while both peptides are highly effective inhibitors of the fVa-mediated activation of prethrombin-2 by E2-fXa in the absence of PCPS, they are ineffective competitors in the presence of phospholipids. Since neither E2-fXa nor prethrombin-2 can interact with membranes, these results suggest that interaction of fVa with PCPS improves the affinity of the activation complex for proexosite-1 of the substrate. Direct binding studies employing OG(488)-EGR-labeled fXa and E2-fXa revealed that the interaction of the Gla domain of fXa with PCPS also induces conformational changes in the protease to facilitate its high-affinity interaction with fVa.

Gas6-mediated signaling is dependent on the engagement of its gamma-carboxyglutamic acid domain with phosphatidylserine.

Gas6 is a vitamin K-dependent protein containing gamma-carboxyglutamic acid (Gla) at its N-terminus and a receptor binding domain at its C-terminus. Gas6-Axl binding is necessary but not sufficient to support endothelial cell survival as decarboxylated gas6 inhibits the pro-survival function of gas6 by binding and inhibiting Axl, even though decarboxylated gas6 cannot support endothelial cell survival itself. It is hypothesized that interactions between the Gla domain of gas6 and phosphatidylserine (PS), though not required for gas6 binding to Axl, are necessary for gas6-Axl function. In support of this hypothesis are results showing that (1) two specific inhibitors of Gla-PS interactions, namely soluble PS and Annexin V, abrogate gas6-mediated endothelial cell survival and (2) Soluble PS inhibits Akt activation, a downstream intracellular event triggered by gas6-Axl binding. In conclusion, we propose a heretofore unknown function of Gla, where Gla-PS binding on the N-terminus of gas6 is necessary for a gas6 function mediated through its binding to Axl via its C-terminus.

Vitamin K intake and atherosclerosis.

PURPOSE OF REVIEW: It has been hypothesized that insufficient intake of vitamin K may increase soft-tissue calcification owing to impaired gamma-carboxylation of the vitamin K-dependent protein matrix gamma-carboxyglutamic acid. The evidence to support this putative role of vitamin K intake in atherosclerosis is reviewed. RECENT FINDINGS: In animal models, multiple forms of vitamin K have been shown to reverse the arterial calcification created by vitamin K antagonists. The human data, however, are less consistent. Phylloquinone, the primary dietary form, has not been associated consistently with the risk of cardiovascular diseases. High menaquinone intake may be associated with lower risk of coronary heart disease mortality, but this needs to be confirmed. SUMMARY: The role of vitamin K in calcification remains controversial. Although biologically plausible, results from the human studies have not consistently supported this hypothesis.

Molecular dynamics calculations of wild type vs. mutant protein C: relationship between binding affinity to endothelial cell protein C receptor and hereditary disease.

Molecular dynamics simulations of the protein C gamma-carboxyglutamic acid (Gla) domain and endothelial cell protein C receptor (EPCR) complex were performed to determine the effect of a hereditary disease, which results in a mutation (Gla 25 --> Lys) in the protein C Gla domain. Our results suggest that the Gla 25 --> Lys mutation causes a significant reduction in the binding force between protein C Gla domain and EPCR due to destabilization of the helix structure of EPCR and displacement of a Ca2+ ion.

The first gamma-carboxyglutamate-containing neuropeptide.

A key factor in the characterization of peptide transmitters used in neuronal signaling is the correct elucidation of post-translational modifications, especially as they are often required to confer biological activity. A rare carboxylation modification is described on the D-peptide from the insulin prohormone in the sea slug, Aplysia californica. Using liquid chromatography purification coupled with electrospray ionization and nanoelectrospray ionization-ion trap-mass spectrometry (ESI- and nanoESI-MS), the presence of this D-peptide within Aplysia insulin (AI)-producing neurons is confirmed. Further detailed mass spectrometric analyses demonstrate that the Aplysia insulin D-peptide is carboxylated on the single glutamate residue within the sequence. This gamma-carboxy D-peptide, along with other identified AI-related peptides, is secreted from the central nervous system in response to ionophore stimulation, thus suggesting a signaling role within the nervous system. Although carboxylated peptides have been described previously, the Aplysia gamma-carboxy D-peptide appears to be the first reported carboxylated neuropeptide.

Kinetics of factor Xa inhibition by recombinant tick anticoagulant peptide: both active site and exosite interactions are required for a slow- and tight-binding inhibition mechanism.

Recombinant tick anticoagulant peptide (rTAP) is a competitive slow- and tight-binding inhibitor of factor Xa (FXa) with a reported equilibrium dissociation constant (K(I)) of approximately 0.2 nM. The inhibitory characteristics and the high selectivity of rTAP for FXa are believed to arise from the ability of the inhibitor to specifically interact with the residues of both the active site as well as those remote from the active site pocket of the protease. To localize the rTAP-interactive sites on FXa, the kinetics of inhibition of wild-type and 18 different mutants of recombinant FXa by the inhibitor were studied by either a discontinuous assay method employing the tight-binding quadratic equation or a continuous assay method employing the slow-binding kinetic approach. It was discovered that K(I) values for the interaction of rTAP with four FXa mutants (Tyr(99) --> Thr, Phe(174) --> Asn, Arg(143) --> Ala, and a Na(+)-binding loop mutant in which residues 220-225 of FXa were replaced with the corresponding residues of thrombin) were elevated by 2-3 orders of magnitude for each mutant. Further studies revealed that the characteristic slow type of inhibition by rTAP was also eliminated for the mutants. These findings suggest that the interaction of rTAP with the P2-binding pocket, the autolysis loop, and the Na(+)-binding loop is primarily responsible for its high specificity of FXa inhibition by a slow- and tight-binding mechanism.

Identification of a gene encoding a typical gamma-carboxyglutamic acid domain in the tunicate Halocynthia roretzi.

We report the identification of a gene capable of encoding a novel Gla (gamma-carboxyglutamic acid) protein from the tunicate Halocynthia roretzi, a primitive member of the phylum Chordata. We call this new hypothetical protein Gla-RTK; it has a Gla domain typical of human vitamin K-dependent coagulation factors, a transmembrane domain, and a receptor tyrosine kinase domain. The receptor tyrosine kinase domain is very similar to the ARK (adhesion-related kinase) family of receptor tyrosine kinases. The ARK family includes Axl, Tyro3, and c-Mer. This gene also encodes a propeptide that binds to the human gamma-glutamyl carboxylase within a range of affinities observed for mammalian propeptides. The cDNA for this putative protein is found distributed throughout the oocyte and embryo but the cDNA is apparently not transcribed except during oogenesis. One of the most interesting aspects of this hypothetical protein is that its Gla domain is highly homologous to the Gla domain of Gas6, a ligand for Axl, while its receptor tyrosine kinase domain is highly homologous to Axl.

Gla domain-mutated human protein C exhibiting enhanced anticoagulant activity and increased phospholipid binding.

Protein C is a member of the vitamin K- dependent protein family. Proteins in this family have similar gamma-carboxyglutamic acid (Gla)-rich domains, but their affinities for negatively charged phospholipid membranes vary more than 1000-fold. We have shown that it is possible to enhance anticoagulant activity and membrane affinity of protein C by selective mutagenesis of the Gla domain. In this study, 3 new mutants, Q10G11N12 (QGN), S23E32D33Y44 (SEDY), and Q10G11N12S23E32D33Y44 (QGNSEDY), were created. In plasma-based coagulation assays, the activated form of QGNSEDY (QGNSEDY-APC) demonstrated approximately 20-fold higher anticoagulant activity than wild-type activated protein C (WT APC), while QGN-APC and SEDY-APC did not. Both normal activated factor V (FVa) and FVa Leiden (Arg506Gln) were degraded much more efficiently by QGNSEDY-APC than by WT APC in the presence as well as in the absence of protein S. Binding of protein C variants to negatively charged phospholipid membranes was investigated using light scattering and the BIAcore technique. QGNSEDY demonstrated 3- to 7-fold enhanced binding as compared with WT protein C, suggesting the membrane affinity to be influenced by several residues located at different parts of the Gla domain. The anticoagulant activity as well as phospholipid binding ability was only enhanced when multiple regions of the Gla domain were modified. The results provide insights into the molecular mechanisms that are involved in determining the binding affinity of the interaction between Gla domains and phospholipid membranes. The unique properties of QGNSEDY-APC suggest this APC variant possibly to have greater therapeutic potential than WT APC.

Mutagenesis of the gamma-carboxyglutamic acid domain of human factor VII to generate maximum enhancement of the membrane contact site.

Site-directed mutagenesis of the 40 N-terminal residues (gamma-carboxyglutamic acid domain) of blood clotting factor VII was carried out to identify sites that improve membrane affinity. Improvements and degree of change included P10Q (2-fold), K32E (13-fold), and insertion of Tyr at position 4 (2-fold). Two other beneficial changes, D33F (2-fold) and A34E (1.5-fold), may exert their impact via influence of K32E. The modification D33E (5.2-fold) also resulted in substantial improvement. The combined mutant with highest affinity, (Y4)P10Q/K32E/D33F/A34E, showed 150-296-fold enhancement over wild-type factor VIIa, depending on the assay used. Undercarboxylation of Glu residues at positions 33 and 34 may result in an underestimate of the true contributions of gamma-carboxyglutamic acid at these positions. Except for the Tyr(4) mutant, all other beneficial mutations were located on the same surface of the protein, suggesting a possible membrane contact region. An initial screening assay was developed that provided faithful evaluation of mutants in crude mixtures. Overall, the results suggest features of membrane binding by vitamin K-dependent proteins and provide reagents that may prove useful for research and therapy.

The induction of c-fos mRNA expression by mechanical stress in human periodontal ligament cells.

The periodontal ligament is subjected to mechanical loading during occlusion and mastication. Although internuclear transcription factors are associated with the regulatory pathway that converts these extracellular mechanical stimuli into a cellular response, there are no reports on these in human periodontal ligament fibroblasts. In this study, the amounts of c-fos mRNA in human periodontal ligament fibroblasts were investigated shortly after subjecting them to a cyclic tension force in vitro. The mRNA of alkaline phosphatase and the matrix proteins type I collagen, type III collagen, matrix Gla-protein, osteonectin, osteopontin, and osteocalcin were also examined. A significant, rapid, transient increase in c-fos mRNA was detected, which peaked 30 min after the application of mechanical force. However, there was no significant change in the mRNA for alkaline phosphatase or the matrix proteins. These results provide evidence that periodontal ligament fibroblasts and c-fos may play a critical part in the response of periodontal tissue to mechanical stimulation.