Expression of UNCL during development of periodontal tissue and response of periodontal ligament fibroblasts to mechanical stress in vivo and in vitro

Mutations in two genes, uncoordinated (unc) and uncoordinated-like (uncl), lead to a failure of mechanotransduction in Drosophila. UNCL, the human homolog of unc and uncl, is preferentially expressed in periodontal ligament (PDL) fibroblasts compared with gingival fibroblasts. However, the precise role of UNCL in the PDL remains unclear. The aim of the present study has been to examine whether mechanical stimuli modulate the expression of UNCL in the human PDL in vivo and in vitro and to examine the roles of UNCL in the development, regeneration, and repair of the PDL. We have investigated the expression pattern of UNCL during the development of periodontal tissue and the response of PDL fibroblasts to mechanical stress in vivo and in vitro. The expression of UNCL mRNA and protein increases with PDL fibroblast differentiation from the confluent to multilayer stage but slightly decreases on mineralized nodule formation. UNCL has also been localized in ameloblasts and adjacent cells, differentiating cementoblasts, and osteoblasts of the developing tooth. Strong distinct UNCL expression has further been observed in the differentiating cementoblasts of the tooth periodontium at the site of tension after orthodontic tooth movement. Application of cyclic mechanical stress on PDL fibroblasts increases the expression of UNCL mRNA. These results indicate that UNCL plays important roles in the development, differentiation, and maintenance of periodontal tissues and also suggest a potential role of UNCL in the mechanotransduction of PDL fibroblasts.This work was supported by a grant from the Korea Health 21R&D Project, Ministry of Health & Welfare, Republic of Korea (03-PJ1-PG1-CH08-0001).     

Mechanoregulation of gene expression in fibroblasts

Mechanical loads placed on connective tissues alter gene expression in fibroblasts through mechanotransduction mechanisms by which cells convert mechanical signals into cellular biological events, such as gene expression of extracellular matrix components (e.g., collagen). This mechanical regulation of ECM gene expression affords maintenance of connective tissue homeostasis. However, mechanical loads can also interfere with homeostatic cellular gene expression and consequently cause the pathogenesis of connective tissue diseases such as tendinopathy and osteoarthritis. Therefore, the regulation of gene expression by mechanical loads is closely related to connective tissue physiology and pathology. This article reviews the effects of various mechanical loading conditions on gene regulation in fibroblasts and discusses several mechanotransduction mechanisms. Future research directions in mechanoregulation of gene expression are also suggested.     

Effect of the cyclic stretch on the expression of osteogenesis genes in human periodontal ligament cells

Periodontal ligament cells can potentially differentiate into osteoblast-like cells and influence the remodeling of periodontal tissues under mechanical strain conditions. In the present study, Gene chip technology was adopted to investigate the effect of the cyclic stretch on the expression of osteogenic-related genes in human periodontal ligament cells (HPDLCs). Cultured HPDLCs were subjected to 12% elongation cyclic stretch for 24 h using a Flexercell Strain Unit, and then GEArray Q series human osteogenesis gene expression profile chip with 96 spot array numbers was used to conduct parallel analyses on the change of the related gene expression in the osteogenic differentiation of HPDLCs stimulated by cyclic stretch. The results show that after the HPDLCs were stimulated by the cyclic stretch, the expression of 21 osteogenic-related genes was significantly upregulated, including 10 growth factor genes and their associated molecules, 10 extracellular matrix genes and their associated proteins, and 1 cell adhesion molecule. Two genes were significantly downregulated, including one growth factor gene and one cell adhesion molecule. Then the expressions of 10 candidate genes were validated using Real-time RT-PCR. These results indicate that cyclic stretch with 12% deformation can stimulate or inhibit some gene expression which was associated with the process of HPDLCs differentiation.     

Cytoskeletal changes and the system of regulation of alkaline phosphatase activity in human periodontal ligament cells induced by mechanical stress

The periodontal ligament (PDL) is a specialized, mechanically responsive tissue that adapts via cellular responses to equilibrate the effects of mechanical stress on teeth. However, the mechanism of remodelling by which individual cells in periodontal tissue detect and respond to mechanical stress is not well understood. To identify the cellular mechanisms induced by mechanical stress in the periodontal ligament, we examined the effects of cyclic stretching on periodontal ligament fibroblast-like cells (PDL cells). Furthermore, we investigated the effects of 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), and interaction with peripheral blood mononuclear cells (PBMCs) on mechanically-simulated PDL cells. PDL cells were cultured on type I collagen-coated silicon membranes with 10% FBS alpha-MEM, and then subjected to cyclic mechanical stimulation (1 s stretching/1 s relaxation, 15% maximum elongation). Alkaline phosphatase activity was monitored by cytochemical and spectrophotometric methods. Morphologically, the cells assumed a spindle shape, and the cytoskeletal components, including microtubules and F-actin filaments, were aligned perpendicular to the strain force vector. Cyclic stretching decreased ALPase activity in PDL cells. The anabolic systemic hormone 1,25(OH)(2)D(3) increased ALPase activity, but this effect was suppressed by cyclic stretching. ALPase activities were reduced by co-culture with PBMCs, including lymphocytes and monocytes. This PBMC-induced ALPase reduction was synergistically reduced by cyclic stretching. ALPase activity was decreased by co-culture with PBMCs, and ALPase activity was reduced synergistically by treatment with PBMCs and cyclic stretching. We conclude that PDL cells changed their shape and alignment in response to cyclic stretching. Furthermore, local factors, such as mechanical stress and PBMCs, showed synergistic suppressive effects on ALPase activity.     

How do fibroblasts translate mechanical signals into changes in extracellular matrix production?

Mechanical forces are important regulators of connective tissue homeostasis. Our recent experiments in vivo indicate that externally applied mechanical load can lead to the rapid and sequential induction of distinct extracellular matrix (ECM) components in fibroblasts, rather than to a generalized hypertrophic response. Thus, ECM composition seems to be adapted specifically to changes in load. Mechanical stress can regulate the production of ECM proteins indirectly, by stimulating the release of a paracrine growth factor, or directly, by triggering an intracellular signalling pathway that activates the gene. We have evidence that tenascin-C is an ECM component directly regulated by mechanical stress: induction of its mRNA in stretched fibroblasts is rapid both in vivo and in vitro, does not depend on prior protein synthesis, and is not mediated by factors released into the medium. Fibroblasts sense force-induced deformations (strains) in their ECM. Findings by other researchers indicate that integrins within cell-matrix adhesions can act as 'strain gauges', triggering MAPK and NF-kappaB pathways in response to changes in mechanical stress. Our results indicate that cytoskeletal 'pre-stress' is important for mechanotransduction to work: relaxation of the cytoskeleton (e.g. by inhibiting Rho-dependent kinase) suppresses induction of the tenascin-C gene by cyclic stretch, and hence desensitizes the fibroblasts to mechanical signals. On the level of the ECM genes, we identified related enhancer sequences that respond to static stretch in both the tenascin-C and the collagen XII promoter. In the case of the tenascin-C gene, different promoter elements might be involved in induction by cyclic stretch. Thus, different mechanical signals seem to regulate distinct ECM genes in complex ways.     

Cell survival and gene expression under compressive stress in a three-dimensional in vitro human periodontal ligament-like tissue model

This study investigated cell survival and gene expression under various compressive stress conditions mimicking orthodontic force by using a newly developed in vitro model of human periodontal ligament-like tissue (HPdLLT). The HPdLLT was developed by three-dimensional culturing of human periodontal ligament fibroblasts in a porous poly-l-lactide matrix with threefold increased culture media permeability due to hydrophilic modification. In vitro HPdLLTs in experimental groups were subjected to 5, 15, 25 and 35 g/cm² compressive stress for 1, 3, 7 or 14 days; controls were cultured over the same periods without compressive stress. Cell morphology and cell apoptosis in the experimental and control groups were investigated using scanning electron microscopy and caspase-3/7 detection. Real-time polymerase chain reaction was performed for seven osteogenic and osteoclastic genes. Similar extracellular matrix and spindle-shaped cells were observed inside or on the surface of in vitro HPdLLTs, with no relation to compressive stress duration or intensity. Similar caspase-3/7 activity indicating comparable apoptosis levels was observed in all samples. Receptor activator of nuclear factor kappa-B ligand and bone morphogenetic protein 2 genes showed characteristic “double-peak” expression at 15 and 35 g/cm² on day 14, and alkaline phosphatase and periodontal ligament-associated protein 1 expression peaked at 5 g/cm² on day 14; other genes also showed time-dependent and load-dependent expression patterns. The in vitro HPdLLT model system effectively mimicked the reaction and gene expression of the human periodontal ligament in response to orthodontic force. This work provides new information on the effects of compressive stress on human periodontal ligament tissue.     

Role of regulator of G-protein signaling 2 (RGS2) in periodontal ligament cells under mechanical stress

Mechanical stress is thought to regulate the expression of genes in the periodontal ligament (PDL) cells. Using a microarray approach, we recently identified a regulator of G-protein signaling 2 (RGS2) as an up-regulated gene in the PDL cells under compressive force. The RGS protein family is known to turn off G-protein signaling. G-protein signaling involves the production of cAMP, which is thought to be one of the biological mediators in response to mechanical stress. Here, we investigated the role of RGS2 in the PDL cells under mechanical stress. PDL cells derived from the ligament tissues of human premolar teeth were cultured in collagen gels and subjected to static compressive force. Compressive force application time-dependently enhanced RGS2 expression and intracellular cAMP levels. To examine the interrelationship between RGS2 and cAMP, the PDL cells were treated with 2',5'-dideoxyadenosine (DDA), an inhibitor of adenyl cyclase, or antisense S-oligonucleotide (S-ODN) to RGS2 under compressive force. DDA dose-dependently inhibited RGS2 stimulated by compressive force. Blockage of RGS2 by antisense S-ODN elevated the cAMP levels compared with controls. These results indicate that cAMP stimulates RGS2 expression, which in turn leads to a decrease in the cAMP production by inactivating the G-protein signaling in the mechanically stressed PDL cells.     

Expression of genes for gelatinases and tissue inhibitors of metalloproteinases in periodontal tissues during orthodontic tooth movement

Orthodontic tooth movement progresses by a combination of periodontal ligament (PDL) tissue and alveolar bone remodeling processes. Besides the remodeling of alveolar bone around the moving teeth, the major extracellular matrix (ECM) components of PDLs, collagens, are degenerated, degraded, and restructured. Matrix metalloproteinases (MMPs) and their specific inhibitors, tissue inhibitors of metalloproteinases (TIMPs), act in a co-ordinated fashion to regulate the remodeling of periodontal tissues. We hypothesized that the expression levels of the genes for MMP-2, MMP-9, and TIMPs 1–3 are increased transiently in the periodontal tissue during orthodontic tooth movement. To test this hypothesis, we employed an animal model of tooth movement using rats, as well as in situ hybridization to analyze the expression levels of Mmp-2, Mmp-9, and Timps 1-3. The expression levels of these genes increased transiently in cells of periodontal tissues, which include cementoblasts, fibroblasts, osteoblasts, and osteoclasts, at the compression side of the moving teeth. The transient increases in gene expression at the tension side were mainly limited to osteoblasts and cementoblasts. In conclusion, the expression levels of Mmp-2, Mmp-9, and Timps 1-3 increase transiently during orthodontic tooth movement at both the tension and compression sides. The expression of these genes is regulated differentially in the periodontal tissue of the tension side and compression side. This altered pattern of gene expression may determine the rate and extent of remodeling of the collagenous ECM in periodontal tissues during orthodontic tooth movement.     

Cyclic compression of chondrocytes modulates a purinergic calcium signalling pathway in a strain rate- and frequency-dependent manner

Mechanical loading modulates cartilage homeostasis through the control of matrix synthesis and catabolism. However, the mechanotransduction pathways through which chondrocytes detect different loading conditions remain unclear. The present study investigated the influence of cyclic compression on intracellular Ca2+ signalling using the well-characterised chondrocyte-agarose model. Cells labelled with Fluo4 were visualised using confocal microscopy following a period of 10 cycles of compression between 0% and 10% strain. In unstrained agarose constructs, not subjected to cyclic compression, a subpopulation of approximately 45% of chondrocytes exhibited spontaneous global Ca2+ transients with mean transient rise and fall times of 19.4 and 29.4 sec, respectively. Cyclic compression modulated global Ca2+ signalling by increasing the percentage of cells exhibiting Ca2+ transients (population modulation) and/or reducing the rise and fall times of these transients (transient shape modulation). The frequency and strain rate of compression differentially modulated these Ca2+ signalling characteristics providing a potential mechanism through which chondrocytes may distinguish between different loading conditions. Treatment with apyrase, gadolinium and the P2 receptor blockers, suramin and basilen blue, significantly reduced the percentage of cells exhibiting Ca2+ transients following cyclic compression, such that the mechanically induced upregulation of Ca2+ signalling was completely abolished. Thus cyclic compression appears to activate a purinergic pathway involving the release of ATP followed by the activation of P2 receptors causing a combination of extracellular Ca2+ influx and intracellular Ca2+ release. Knowledge of this fundamental cartilage mechanotransduction pathway may lead to improved therapeutic strategies for the treatment of cartilage damage and disease.     

Mechanical stretch stimulates integrin αVβ3-mediated collagen expression in human anterior cruciate ligament cells

Biomechanical stimuli have fundamental roles in the maintenance and remodeling of ligaments including collagen gene expressions. Mechanical stretching signals are mainly transduced by cell adhesion molecules such as integrins. However, the relationships between stress-induced collagen expressions and integrin-mediated cellular behaviors are still unclear in anterior cruciate ligament cells. Here, we focused on the stretch-related responses of different cells derived from the ligament-to-bone interface and midsubstance regions of human anterior cruciate ligaments. Chondroblastic interface cells easily lost their potential to produce collagen genes in non-stretched conditions, rather than fibroblastic midsubstance cells. Uni-axial mechanical stretches increased the type I collagen gene expression of interface and midsubstance cells up to 14- and 6-fold levels of each non-stretched control, respectively. Mechanical stretches also activated the stress fiber formation by shifting the distribution of integrin αVβ3 to the peripheral edges in both interface and midsubstance cells. In addition, integrin αVβ3 colocalized with phosphorylated focal adhesion kinase in stretched cells. Functional blocking analyses using anti-integrin antibodies revealed that the stretch-activated collagen gene expressions on fibronectin were dependent on integrin αVβ3-mediated cellular adhesions in the interface and midsubstance cells. These findings suggest that the integrin αVβ3-mediated stretch signal transduction might have a key role to stimulate collagen gene expression in human anterior cruciate ligament, especially in the ligament-to-bone interface.