Effect of orthodontic force on the expression of PI3K,Akt, and P70S6 K in the human periodontal ligament during orthodontic loading

The mammalian target of rapamycin (mTOR) is an atypical serine/threonine protein kinases involved in the regulation of cell growth, proliferation, and differentiation through the PI3K/Akt/mTOR/P70S6 K signalling pathway. P70S6 K as a downstream molecule of mTOR is activated by phosphorylation and subsequently promotes the synthesis of ribosomal and translational proteins. In this study, we investigated the role of PI3K, Akt, and P70S6 K in human periodontal tissue remodelling during orthodontic loading. The prepared tissue specimens taken from 4 extracted premolars were processed for immunolabelling. The changes in the expression of PI3K, Akt, and P70S6 K in the periodontal tissues were detected by real‐time quantitative‐polymerase chain reaction and Western blot analysis. The results from real‐time quantitative‐polymerase chain reaction and Western blot both showed that the expression of PI3K, Akt, and P70S6 K in the experimental group began to increase at 3 days and increased significantly at 10 days, then decreased approaching the control group level at 28 days. Our findings showed that the expression of PI3K, Akt, and P70S6 K in human periodontal ligament demonstrated a variability during the orthodontic loading, which suggested that the PI3K/Akt/mTOR/P70S6 K signal pathway was involved in orthodontic tooth movement and played a role in the process of periodontium remodelling.     

Tissue changes in the rabbit periodontal ligament during orthodontic tooth movement

In this (semi) quantitative animal study the reaction of the periodontal ligament (PDL) to experimental tooth movement is described. To this end, rabbit first incisors were moved sideways with helical torsion springs for periods varying from 3-24 hours. The initial force of the springs was 50 gf. The histomorphology of the PDL was studied in 5 microns thick plastic sections. Comparison with control animals and animals wearing passive springs showed that tooth movement leads to an increased trauma in the PDL within only a few hours. This trauma is characterized by hyalinization, tears and ruptures in the fibres and blood vessels, and by the presence of extravascular erythrocytes and pyknosis. Tissue damage significantly increased with time. After 24 hours of tooth movement, the PDL fibers are compressed or stretched in 68% of the sections and the blood vessels in the PDL are compressed or stretched in 62% of the sections. Even in the controls, more than 15% of the sections displayed slightly stretched or compressed fibers, and about 10% showed slightly compressed or stretched blood vessels. This indicates that some damage is regularly present in a normally functioning PDL. Increases in the percentage of sections with blood vessel compression are found in all groups wearing passive springs, especially after 6 hours. A high concordancy in compression and tension patterns of blood vessels and fibers is present in 83% of the sections. Pyknotic cells are practically confined to areas with compressed PDL fibers in rabbits wearing active springs. Extravascular erythrocytes were found in sections with all types of fiber patterns. A significant majority of extravascular erythrocytes, however, was found in areas with compressed fibers.     

Functions of beta2-adrenergic receptor in human periodontal ligament cells

Adrenergic receptors (ARs) are receptors of noradrenalin and adrenalin, of which there are nine different subtypes. In particular, β2 adrenergic receptor (β2-AR) is known to be related to the restoration and maintenance of homeostasis in bone and cardiac tissues; however, the functional role of signaling through β2-AR in periodontal ligament (PDL) tissue has not been fully examined. In this report, we investigated that β2-AR expression in PDL tissues and their features in PDL cells. β2-AR expressed in rat PDL tissues and human PDL cells (HPDLCs) derived from two different patients (HPDLCs-2G and -3S). Rat PDL tissue with occlusal loading showed high β2-AR expression, while its expression was downregulated in that without loading. In HPDLCs, β2-AR expression was increased exposed to stretch loading. The gene expression of PDL-related molecules was investigated in PDL clone cells (2-23 cells) overexpressing β2-AR. Their gene expression and intracellular cyclic adenosine monophosphate (cAMP) levels were also investigated in HPDLCs treated with a specific β2-AR agonist, fenoterol (FEN). Overexpression of β2-AR significantly promoted the gene expression of PDL-related molecules in 2 to 23 cells. FEN led to an upregulation in the expression of PDL-related molecules and increased intracellular cAMP levels in HPDLCs. In both HPDLCs, inhibition of cAMP signaling by using protein kinase A inhibitor suppressed the FEN-induced gene expression of α-smooth muscle actin. Our findings suggest that the occlusal force is important for β2-AR expression in PDL tissue and β2-AR is involved in fibroblastic differentiation and collagen synthesis of PDL cells. The signaling through β2-AR might be important for restoration and homeostasis of PDL tissue.     

Matrix metalloproteinase activity synergizes with alpha2beta1 integrins to enhance collagen remodeling

Cell-matrix interactions transmit a wealth of information about the extracellular environment. In return, a variety of responses from the cell are initiated by changes in the matrix. One such response involves the positive regulation of matrix metalloproteinases (MMPs) by alpha2beta1 integrin attaching to a specific extracellular matrix component, collagen. This study explores the relationship between mechanical and biochemical functions of alpha2beta1 integrins as it pertains to regulating matrix remodeling. To understand this relationship, the individual influences of MMP activity and alpha2beta1 integrin function on collagen gel contraction were studied. We have observed little evidence of mutual participation in matrix remodeling by the alpha2beta1 integrin and MMP activity in cell models where alpha2 is minimally expressed. In cells expressing high levels of alpha2, we see an increase in gel contraction that is enhanced by MMP activity. Measuring tension as it builds within the gel reveals that alpha2beta1 integrin presence correlates with force output but is insensitive to MMP activity. These data strongly suggest that alpha2beta1 regulates collagen gel remodeling through multiple simultaneous mechanisms including force generation and modulation of MMP activity.     

Effect of vitamin on 2-deoxy-d-glucose uptake in human fibroblast cultures

Summary 2-Deoxy-d-glucose (2-DOG) uptake was tested in human fibroblast cultures in the presence and absence of vitamin E. Addition of 10 μg/ml vitamin E to the culture medium significantly reduced this uptake for 2-DOG concentrations of 0.005, to 10 mmol/liter (P≤0.01). The decrease of 2-DOG uptake was inversely proportional to the rise in 2-DOG concentration (P≤0.01). The presence of vitamin E reduced by 71% the average cellular level of lipid peroxides (expressed as thiobarbituric acid reactive substances) and caused a small but significant decrease in the cholesterol concentration (P≤0.01). These last results might explain the decrease in 2-DOG uptake observed in the presence of vitamin E.     

Cyclic tensile force stimulates BMP9 synthesis and in vitro mineralization by human periodontal ligament cells

Periodontal ligament (PDL) cells are mechanosensitive and have the potential to differentiate into osteoblast-like cells under the influence of cyclic tensile force (CTF). CTF modulates the expression of regulatory proteins including bone morphogenetic proteins (BMPs), which are essential for the homeostasis of the periodontium. Among the BMPs, BMP9 is one of the most potent osteogenic BMPs. It is yet unknown whether CTF affects the expression of BMP9 and mineralization. Here, we demonstrated that continuously applied CTF for only the first 6 hr stimulated the synthesis of BMP9 and induced mineral deposition within 14 days by human PDL cells. Stimulation of BMP9 expression depended on ATP and P2Y ₁ receptors. Apyrase, an ecto-ATPase, inhibited CTF-mediated ATP-induced BMP9 expression. The addition of ATP increased the expression of BMP9. Loss of function experiments using suramin (a broad-spectrum P2Y antagonist), MRS2179 (a specific P2Y ₁ receptor antagonist), MRS 2365 (a specific P2Y ₁ agonist), U-73122 (a phospholipase C [PLC] inhibitor), and thapsigargin (enhancer of intracytosolic calcium) revealed the participation of P2Y ₁ in regulating the expression of BMP9. This was mediated by an increased level of intracellular Ca ²⁺ through the PLC pathway. A neutralizing anti-BMP9 antibody decreased mineral deposition, which was stimulated by CTF for almost 45% indicating a role of BMP9 in an in vitro mineralization. Collectively, our findings suggest an essential modulatory role of CTF in the homeostasis and regeneration of the periodontium.     

Tensile force inhibits the proliferation of human periodontal ligament fibroblasts through Ras-p38 MAPK up-regulation

The capacity of human periodontal ligament fibroblasts (PLF) to proliferate in response to mechanical force plays a critical role in orthodontic tooth movement. Extensive research has not fully revealed the mechanisms by which the PLF respond to mechanical force. The responses to force differ according to the origin of cells and the type of stress applied. In this study, we examined the proliferative response of PLF to tensile force. We also explored cellular mechanisms involved in the mechanosignal transduction of tensile force. Application to the force to PLF with 1.5% elongation for 1 h inhibited cell proliferation. This was accompanied by reductions in several cyclins and cyclin-dependent kinases (CDKs) involved in the G(1)/S transition; however, p21 knockdown prevented these events. Pharmacological inhibitor of p38 MAPK suppressed the force-mediated growth inhibition as well as the decrease in p21 expression. Ras inhibitor almost completely blocked the tension-mediated increases in p-p38 MAPK and p-p21, and the attendant increase in PLF proliferation. These findings suggest that tension force activates Ras-p38 MAPK pathways in PLF, which up-regulate p21 and arrest cell cycle progression at the G(1) phase.     

Tissue origin and extracellular matrix control neutral proteinase activity in human fibroblast three-dimensional cultures

Remodeling of the extracellular matrix by fibroblasts is an important step in the process of wound healing and tissue repair. We compared the behavior of fibroblasts from two different tissues, dermis and gingiva, in three-dimensional lattices made of two different extracellular matrix macromolecules, collagen and fibrin. Cells were grown in monolayer cultures from normal skin or gingiva and seeded in three-dimensional lattices made of either collagen or fibrin. Photonic and scanning electron microscopy did not reveal any morphological differences between the two types of fibroblasts in both sets of lattices. Both types of fibroblasts retracted collagen lattices similarly and caused only a slight degradation of the collagen substratum. By contrast, when seeded in fibrin lattices, gingival fibroblasts completely digested their substratum in less than 8 days, whereas only a slight fibrin degradation was observed with dermal fibroblasts. The ability of gingival but not dermal fibroblasts to express high levels of tissue plasminogen activators (tPA) when cultured in fibrin lattices was assessed on an immunological basis. Also, deprivation of plasminogen-contaminating fibrinogen preparations or use of tPA inhibitors markedly inhibited both fibrinolysis and retraction rates of fibrin lattices by gingival fibroblasts. Casein-zymography confirmed the intense proteolytic activity induced by fibrin in gingival fibroblasts. It was inhibited by aprotinin and phenyl methylsulfonyl fluoride (PMSF), two non-specific inhibitors of serine proteinases, and by η-amino-caproic acid (ηACA), an inhibitor of plasminogen activators. Monolayer cultures exhibited only trace amounts of caseinolytic activity. Our results demonstrate that the expression of proteinases by fibroblasts is dependent not only on their tissue origin but also on the surrounding extracellular matrix. The intense fibrinolytic activity of gingival fibroblasts in fibrin lattices may explain partially the high rate of healing clinically observed in gingiva. © 1996 Wiley-Liss, Inc.     

Reduction of alkaline phosphatase activity in aged human osteogenic periodontal ligament fibroblasts exhibiting short telomeres

The osteogenic cell type of human periodontal ligament fibroblasts (PDLF) undergoes senescence at finite population doubling numbers unrelated to donor ages. This study investigated telomere lengths of osteogenic PDLF from differently aged donors and alterations of the osteoblast-like properties in the aged PDLF with short telomeres. Telomere lengths of osteogenic PDLF were biased towards long or short among all 15- to 51-year-old individuals, and did not show a normal distribution by Pearsons test or a correlation to donor age by simple regression analysis. In osteogenic PDLF, senescence-associated -galactosidase was expressed in 78.5% of cells in the clones with short telomeres (mean 3.02 kbp), and in 9.4% of cells in the clones with long telomeres (mean 13.06 kbp). These results suggest that human periodontium comprises aged osteogenic PDLF without correlation to age. Osteogenic PDLF with long telomeres strongly expressed alkaline phosphatase (ALPase) activity whereas cells with short telomeres expressed ALPase activity to a weaker extent. Total activity of ALPase in the clones of osteogenic PDLF with long telomeres was significantly higher than that in the clones with short telomeres. The produced amounts of both osteopontin and osteocalcin in the clones of osteogenic PDLF with long telomeres were slightly but statistically significantly smaller than those in the clones with short telomeres. These findings suggest that aged osteogenic PDLF reduce the expression of ALPase activity but that there is not a critical alteration in bone-associated protein production. Aged osteogenic PDLF may impair the ability to induce ALPase-dependent calcification.This work was supported by a grant-in-aid for Scientific Research (B) (2) from the Ministry of Education, Science, Sports, and Culture of Japan (No. 12470379).     

Effects of transforming growth factor-beta 1 and fibronectin on SPARC expression in cultures of human periodontal ligament cells

Transforming growth factor-beta(1) (TGF-beta(1)) increases synthesis of secreted protein, acidic and rich in cysteine (SPARC), as well as fibronectin (FN) and type I collagen. However, little is known about the regulatory mechanism of SPARC expression. We examined the effect of FN on SPARC expression by TGF-beta(1) in cultures of human periodontal ligament cells (HPL cells). TGF-beta(1) increased the SPARC and SPARC mRNA levels in HPL cells. Extracellular matrix (ECM) produced by HPL cells in the presence of TGF-beta(1) also increased the SPARC levels. Contents of FN and type I collagen in the ECM were increased by TGF-beta(1). HPL cells cultured on FN-coated plates secreted more SPARC than those on non-coated plates. However, type I collagen had little effect on SPARC levels. The addition of anti-alpha5 antibody to the cultures abolished the increase in SPARC mRNA expression by TGF-beta(1). This study demonstrated that FN may be partly involved in the increase in SPARC expression by TGF-beta(1) in HPL cells.     

The in vitro life-span of human periodontal ligament fibroblasts

The in vitro life-span of human periodontal ligament fibroblasts (PDLF) was studied on clones from periodontium of teeth extracted due to periodontitis and dental caries (69 clones/192 individuals, aged 20-80 years) and from periodontium of teeth extracted for orthodontic reasons (23 clones/26 individuals, aged 15-19 years). In the primary cultures the ratio of the number of cells expressing senescence-associated beta-galactosidase (SA-beta-Gal) to the total number of cells is significantly larger in PDLF (92 clones; 11.1+/-4.9%) than in human gingival fibroblasts (GF) (10 clones; 0.5+/-0.1 %). The finite population doubling numbers (PD) of PDLF are not age-matched and the mean PD of PDLF (7.1+/-2.9) is significantly smaller than GF (28.5+/-3.2), IMR-90 (human lung fibroblasts, 5 clones; 44.3 +/- 2.2), and human osteoblasts (5 clones; 19.7+/-1.4). Comparing the ratio of the number of SA-beta-Gal positive cells to the total number of cells in primary culture, and the finite PD in PDLF cultures: 1) the ratio of 15-19 years old donor group is significantly smaller than in the other donor groups (20-29, 30-39, 40-49, 50-59 and 60-80 years old), and 2) there were no statistically significant differences among the 20-29, 30-39, 40-49 and 50-59 year old donor groups, and the 30-39, 40-49, 50-59 and 60-80 year old donor groups. These findings suggest that the in vitro life-span of PDLF is shorter than other fibroblasts in the connective tissues and that PDLF may undergo senescence in adult clones without relation to donor's age. There may be more aged fibroblasts in periodontium than in other tissues, such as gingiva and lung.     

Latent TGF-beta binding protein LTBP-2 decreases fibroblast adhesion to fibronectin

We have analyzed the effects of latent TGF-beta binding protein 2 (LTBP-2) and its fragments on lung fibroblast adhesion. Quantitative cell adhesion assays indicated that fibroblasts do not adhere to full-length LTBP-2. Interestingly, LTBP-2 had dominant disrupting effects on the morphology of fibroblasts adhering to fibronectin (FN). Fibroblasts plated on LTBP-2 and FN substratum exhibited less adherent morphology and displayed clearly decreased actin stress fibers than cells plated on FN. These cells formed, instead, extensive membrane ruffles. LTBP-2 had no effects on cells adhering to collagen type I. Fibroblasts adhered weakly to the NH2-terminal fragment of LTBP-2. Unlike FN, this fragment did not augment actin stress fiber formation. Interestingly, the adhesion-mediating and cytoskeleton-disrupting effects were localized to the same NH2-terminal proline-rich region of LTBP-2. LTBP-2 and its antiadhesive fragment bound to FN in vitro, and the antiadhesive fragment associated with the extracellular matrix FN fibrils. These observations reveal a potentially important role for LTBP-2 as an antiadhesive matrix component.     

Variegated translocation mosaicism in human skin fibroblast cultures.

The term "variegated translocation mosaicism" is used to describe the repeated occurrence, within cultures of human skin fibroblasts, of a multiplicity of chromosomal rearrangements. With respect to the frequencies of such cytogenetically aberrant clones we found that they (1) were not detectable in routine diagnostic skin fibroblast cultures from 29 subjects with a wide variety of indications for biopsy; (2) were not detectable during in vitro aging of diploid strains with four normal individuals; (3) could be detected after rescue from bacterial contamination of a culture from an otherwise normal diploid male; (4) occurred with high frequencies in independent cultures from another apparently normal subject; (5) occurred with high frequencies in multiple biopsies obtained at autopsy from a patient with Werner's syndrome who died of sepsis; (6) were of pseudodiploid nature; and (7) involved a different spectrum of chromosomes in different individuals. A consistent association with mycoplasma contamination could not be made.     

Fibronectin matrix: antibody-induced reorganization in human fibroblast cultures

Fibronectin, a major pericellular glycoprotein of adherent cells, was predominantly present in fibrillar structures in human fibroblast cultures as shown by indirect immunofluorescence. In conventional "patching experiments" where one day old cells were exposed to anti-fibronectin IgG in the cold, washed, and reincubated at 37 degrees no redistribution was seen. However, continuous exposure of the cultures to IgG at 37 degrees resulted in redistribution. The fibrillar structures were lost and fibronectin aggregates (patches) were found. Fab-fragments had no such effect. These results support the findings that fibronectin is predominantly a matrix protein and show that matrix components may be redistributed in cell culture conditions.     

Matrix metalloproteinase expression and activity following prostaglandin F(2 alpha)-induced luteolysis

Luteal tissue contains matrix metalloproteinases (MMPs) that cleave specific components of the extracellular matrix (ECM) and are inhibited by tissue inhibitors of metalloproteinases (TIMPs). We previously reported a decrease in luteal TIMP-1 within 15 min of prostaglandin F(2 alpha) (PGF(2 alpha))-induced luteolysis. An increase in the MMP:TIMP ratio may promote ECM degradation and apoptosis, as observed in other tissues that undergo involution. The objectives of these experiments were to determine whether 1) PGF(2 alpha) affects expression of mRNA encoding fibrillar collagenases (MMP-1 and -13), gelatinases A and B (MMP-2 and -9), membrane type (mt)-1 MMP (MMP-14), stromelysin (MMP-3), and matrilysin (MMP-7), and 2) PGF(2 alpha) increases MMP activity during PGF(2 alpha)-induced luteolysis in sheep. Corpora lutea (n = 3-10/time point) were collected at 0, 15, and 30 min and 1, 2, 4, 6, 12, 24, and 48 h after PGF(2 alpha) administration. Northern blot analysis confirmed the presence of all MMPs except MMP-9. Expression of mRNA for the above MMPs (except MMP-2) increased significantly (P < 0.05) by 30 min, and all MMPs increased significantly (P < 0.05) by 6 h after PGF(2 alpha) administration. Expression of MMP-14 mRNA increased significantly (P < 0.05) by 15 min post-PGF(2 alpha) and remained elevated through 48 h. MMP activity in luteal homogenates (following proenzyme activation and inactivation of inhibitors) was increased significantly (P < 0.05) by 15 min and remained elevated through 48 h post-PGF(2 alpha). MMP activity was localized (in situ zymography) to the pericellular area of various cell types in the 0-h group and was markedly increased by 30 min post-PGF(2 alpha). MMP mRNA expression and activity were significantly increased following PGF(2 alpha) treatment. Increased MMP activity may promote ECM degradation during luteolysis.