The Experimental Study On Relationship Between ATF4 And Periodontal Tissue Remodeling During Tooth Movement

Background and objectiveIt has been known that a tooth can be moved gradually from one spot in the oral cavity toa more desirable one by the application of mechanical forces to the tooth's crown. Periodontal ligament (PDL) is the connective tissue located between the tooth root and alveolar bone. It functions in bone remodeling during orthodontic tooth movement, has the ability of bone resorption and formation.The rapid and active alveolar bone remodelling followed by tooth movement does not occur unless normal healthy PDL surrounds the tooth root, which suggests that the transmission of the force applied to the teeth to alveolar bone is mediated by the response of periodontal ligament to the force, inducing adaptation of periodontal tissues to the mechanical stress.The tooth movement is mediated by bone resorption on the compression side of the PDL and by bone deposition on the tension side of the PDL. Osteoclasts are the only cells that are responsible for bone resorption, while the formation and activity of osteoclasts are regulated by osteoblasts through expression of osteoclast differentiation factor/ RANKL. So it is believed that osteoblasts play an important role not only in bone formation but in bone remodeling as well. PDL cells are a mixture of mesenchymal cells that have differentiation potential, and mechanical stimulation alone can induce the differentiation of PDL cells to osteoblast-like cells, suggesting that the increased osteoblast differentiation and activity is responsible for the mechanical stress-induced bone remodeling.However, the molecular mechanism by which mechanical stress enhance osteoblast proliferation and differentiation is complex and involves multiple signaling molecules and pathways, but not limited to, nitric oxide, prostaglandin E2 and 12 (PGE2 and PGI2), pertussis toxin-sensitive heterotrimeric G proteins, stretch-activated ion channels, integrins. Recent data indicate that internuclear transcription factors are associated with the intracellular regulatory pathways that convert extracellular physical or mechanical stimuli into a coordinated cellular response.The cAMP-responsible element (CRE) is a well-studied regulatory element, with a consensus sequence of CGTCA. This sequence is located in many viral and cellular promoters, such as the ElA-inducible adenoviruses E2, E3, E4, HTLV-I LTR, somatostatin, VIP, c-fos and HSP-70. This element is recognized by a family of transcription factors, referred to as activating transcription factors (ATF). This family shares closely related basic-leucinezipper (bZip) domains, including ATF2, ATF4, ATF6, B-ATF.ATF4 was first cloned based on the specific recognition of an ATF/CREB binding sequence. ATF4 has also been isolated by several other researchers, and is commonly referred to as TAXREB67, CREB2 or C/ATF. ATF4 binds to the osteoblast cis-acting element 1 (OSE1), and is is a major regulator of osteoblast differentiation and function. It has also been demonstrated that ATF4, like Runx2 and Osterix, has the ability to induce osteoblast-specific gene expression in non-osteoblastic cells. Moreover, ATF4 is the substrate in osteoblasts of RSK2, a gene encoding a kinase that is inactivated in Coffin-Lowry syndrome, which is an inherited, sex-linked disorder associated with craniofacial, dental, and skeletal abnormalities as well as mental retardation. ATF4 Is a Substrate of RSK2 in osteoblasts, and is at least partially dispensable for differentiation of mesenchymal cells into osteoblasts.The aim of the present study has been to examined the relationship between mechanical stress and ATF4 in the PDL by the application of orthodontic force in vivo and in vitro. This study will contribute to a better understanding of mechanism of bone remodeling during tooth movement which may set the basis for clinical work. Methods1. Observe the expression of ATF4 protein in periodontal tissues after orthodontictooth movementA titanium-nickel closed-coil spring was applied to the occlusal surface of the rat maxillary first molar (M1) with a hook and the upper incisors with a ligature wire. The coil spring were kept constant and recorded for 0hr, 1 hr, 2 hrs, 4 hrs, 8 hrs, 12hrs, ldays, 3days or 7days. Upon completion of experiments, the maxillae were removed. The specimens were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 24 hrs and decalcified in 10% ethylene diamine tetraacetic acid (EDTA) at room temperature for 5 wks. After being dehydrated in ascending grades of alcohol, cleared in xylene, and paraffin- embedded, 3-um serial sections were cut parasagittally on a microtome. Immunohistochemical staining was carried out with anti-ATF4 rabbit polyclonal antibody to examine the expression of ATF4. 2.Observe the expression of ATF4 mRNA and protein after application of centrifugal force on PDL cells.Human PDL cells were cultured by sequentialdigestio. Experiments were carried out with cells from the fourth (p4) to sixth (p6) passages. Approximately 5.0×l05cells were seeded onto six-well cell culture plates and cultivated until they reached -80% confluence.The medium was then changed to DMEM supplemented with 2% FBS, to remain quiescent. 24 hour later, human PDL cells were centrifuged at 910 rev/min for 0, 10, 30, 60, 90, 120, and 240 min (910rev/min, about 167gRCF)by horizontal microplate rotor. Total RNA and nuclear extracts were isolated. The expression of ATF4 mRNA and proteinwas measured by Semi-quantitative RT-PCR and Western Blotting respectively.3. Observe the role of ATF4 in the mechanical stress-induced human PDL cells differentiationCells from the fourth (p4) to sixth (p6) passages were cultured at 37℃in a humidified atmosphere of 95% air and 5% CO2. Expression vector of human ATF4 (pMyc-ATF4) and its control vector (pCMV5-myc vector) were generous gifts from Dr A. S. Lee. Transient transfection of pMyc-ATF4 was carried out using Lipofectamine TM2000 (Invitrogen). The expression of ATF4 mRNA and protein in untransfected cells, pCMV5-myc transfected cells, or pMyc-ATF4 transfected cells was measured by Semi-quantitative RT-PCR and Western Blotting respectively. Cells in three groups were centrifuged at 910 rev/min for 30 min, and the changes of alkaline phosphatase(ALP) activity and osteocalcin(OCN), osteopontin(OCN), collagen I (COLI) , bone sialoprotein(BSP) genes were measured to assess the differentiation of human PDL cells. Results1. In the untreated control teeth, ATF4 was expressed at a low level in the rat periodontal tissues, mostly located near the alveolar bone or cementum, much less in the middle of periodontal tissues. Application of orthodontic loading simultaneously induced a significant increase of of periodontal ligamentcells positive for ATF4. Strong direct ATF4 expression was observed in the differentiating cementoblasts of the tooth periodontium near the root surface at the site of the applied compression. ATF4 immunoreactivity was stronger in differentiating cementoblasts at the tension side compared to that at the compression side.2. As early as 10 min after mechanical stimuli, ATF4 mRNA levels (compared to control and normalised by p-actin) increased, but has no stastistical significance (P> 0.05). The mRNA levels increased significantly to a peak level of more than two-fold (P < 0.001) at 30 min, remained at more than one half-fold (P < 0.001) at 60 min, and decreased but remained high (P < 0.01) at 90 min. After 240 min, the mRNA levels decreased to control level (P> 0.05).There is very low level of ATF4 protein in unloaded cells, but simulation of orthodontic force induces a rapid up-regulation of ATF4. The results show increased ATF4 protein expression in loaded versus unloaded periodontal ligament cells at 30 min (P < 0.001), 60 min (P < 0.001) and 90 min (P < 0.01) after the load episode. The ATF4 protein expression was up regulated, with a peak at 60 min, and then down regulated after 30 min. 3.An increase in the specific activity of cellular ALP has frequently been used as an index for osteoblast differentiation. The ALP activity of human PDL cells transfected with pMyc-ATF4 markedly increased (P<0.05), and it remained unchanged in untransfected and pCMV5-myc transfected cells (P>0.05).The ALP activity of human PDL cells increased (P<0.05) in the empty vector pCMV5-myc transfected cells as well as in the untransfected cells in response to the centrifugal force.Overexpression of pMyc-ATF4 exhibited a greater increase compared to the untransfected cells (P < 0.01) in response to the centrifugal force. The osteogenic genes assessed included osteocalcin (OCN), osteopontin (OPN), collagen I (COLI) and bone sialoprotein (BSP). There was no difference in mRNA expression of all four osteogenic genes between pCMV5-myc transfected human PDL cells and nontransfected cells, and up-regulated mRNA expression of all four osteogenic genes was observed in pMyc-ATF4 transfected human PDL cells (*P < 0.05**P < 0.01). A 30-min centrifugal force at a magnitude of 33.5 g/cm~2 significantly enhanced mRNA expression of all four osteogenic genes (approximately threefold each) in the empty vector pCMV5-myc transfected cells as well as in the untransfected cells (*P < 0.05**P < 0.01). This confirms that this level of centrifugal force significantly increased human PDL cells differentiation and indicates that the Lipofectamine TM2000 transfection did not alter the mitogenic response of human PDL cells to the centrifugation. Overexpression of pMyc-ATF4 exhibited a greater increase (P < 0.01 for each) in mRNA expression of all four osteogenic genes in response to the centrifugal force compared to the untransfected cells. Conclusion1. ATF4 is highly expressed by mechanical stimuli during tooth movement. ATF4 participated in the periodontal tissue remodeling during orthodontic tooth movement, and probably plays a key role in this process.2. The ATF4 mRNA and protein expression increased dramatically after force application, and decreased rapidly towards its pre-treatment level, but with a maximum at 30 min and 60 min after application of mechanical force, respectively. These observations suggest that ATF4 could make a rapid and temporal response to mechanical stress. ATF4 plays a rival role in remodeling of periodontal tissue in vitro.3. It is suggested that ATF4 plays a rival role in the diferentiation process from human periodontal ligament cells to osteoblast-like cells.4. This study will contribute to a better understanding of mechanism of bone remodeling during tooth movement which may set the basis for clinical work.