ATF4与正畸牙周组织改建相关的实验研究
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摘要
背景和目的在正畸治疗过程中，机械力作用于牙齿，引起牙周组织改建，最终使牙齿发生移动而达到矫治目的。这种正畸治疗的生理介质是牙周膜，它是具有成骨能力的骨／牙界面，是一种变异的骨膜，具有明显的骨吸收和骨形成能力。在正畸临床中，没有牙周膜的粘连牙不能发生移动，这也提示我们，在牙槽骨改建的过程中，牙周膜起到至关重要的作用。正畸牙齿受力后的牙周膜组织表现为受压侧破骨细胞聚集、功能活跃，牙槽骨吸收，受牵张侧成骨细胞生成、行使功能，牙槽骨改建，骨形成。破骨细胞来源于造血组织中的破骨细胞前体，是骨吸收的唯一细胞；成骨细胞来源于牙周膜自身，不仅是骨形成的主要细胞，而且在破骨细胞的附着、促进破骨细胞前体分化和合成破骨细胞骨吸收刺激因子等方面起作用。已有研究证实牙周膜细胞中包含具有分化潜能的成纤维细胞群体，正畸机械力能诱导其表达一些成骨细胞的表型和功能蛋白，向成骨样细胞分化。牙周膜细胞分化为成骨细胞，参与骨吸收和骨形成，是正畸骨改建的关键。
牙周膜细胞在机械力作用下向成骨细胞分化的分子机制比较复杂，包含多条信号转导途径。例如，一氧化氮(NO)、前列腺素12(PGI2)、前列腺素E2(PGE2)、百日咳毒素敏感异源三聚体G蛋白(PTX-敏感蛋白)、动力敏感钙离子通道等。近年的研究表明，一些核内转录因子也参与了细胞内调控途径，将细胞外物理或机械刺激转化为协调的细胞反应。
1987年，能以一个共同的核心序列(CGTCA)和腺病毒启动子E2，E3和E4结合的蛋白被命名为ATF，在随后的几年中发现了大量相同的或者类似的能和ATF／CRE位点结合的蛋白，这些蛋白都含有一个命名为bZip的DNA结合区域。基于他们的氨基酸的相似性，ATF家族被分为ATF2，ATF4，ATF6，B-ATF亚类。
1989年，Hai根据ATF／CREB结合序列特异性识别位点，首次克隆了ATF4。其他的学者也相继分离出了ATF4，它通常又被称为TAXREB67、CREB2或C／ATF。研究表明，ATF4与成骨细胞特异性基因骨钙素启动区的成骨细胞特异性元件1(OSE1)相结合，调控成骨细胞的分化。至今为止，只有Runx2和ATF4可诱导非成骨细胞系表达成骨细胞特异型基因骨钙素的表达。另外，有研究证实ATF4是RSK2的磷酸化底物，ATF4(-／-)鼠出现CLS表现型，除精神发育迟缓外，还伴有颅面、牙齿、骨骼发育异常。ATF4和RSK2以一种线性级联的方式调控成骨细胞分化和功能，是间充质细胞向成骨细胞分化所必需的。ATF4作为成骨细胞分化的关键因子，与骨发育和牙齿发育关系的研究正在不断开展。但正畸骨改建与骨发育是两种不同的生理过程，ATF4在正畸牙齿移动牙周组织的表达和分布，在机械力的作用下体外培养的人牙周膜细胞中表达变化的情况，以及在牙周膜细胞向成骨样细胞分化的过程中所起的作用，国内外尚未有报道。
本研究通过建立正畸大鼠牙齿移动动物模型和体外细胞培养，结合细胞力学试验方法和分子生物学测试技术，拟从组织、细胞水平以及分子生物学角度探讨ATF4在正畸牙齿移动过程中对牙周组织改建的影响及作用机制，进一步为正畸牙齿移动提供理论和实践依据。
方法
1．观察正畸牙周组织改建过程中ATF4的表达变化
建立大鼠牙齿移动实验模型，在加力1h，2h，4h，8h，12h，1d，3d，5d，7d后，处死大鼠，固定，脱钙，制作大鼠第一磨牙牙周组织石蜡切片，HE染色观察牙周组织形态学变化，免疫组化进行半定量分析ATF4的表达。
2．观察机械力加载下人牙周膜细胞ATF4mRNA和蛋白的表达变化
组织块法培养原代人牙周膜细胞，并做波形丝蛋白抗体和角蛋白抗体染色鉴定。取4-6代生长旺盛，性质稳定的人牙周膜细胞进行实验。以2.5×10~5／孔将第4代人牙周膜细胞接种于6孔细胞培养板培养24小时，换用含2％胎牛血清的条件培养基继续培养24小时，将六孔板置于离心加力支架中，离心机加力10min，30min，60min，90min，120min，240min(910rev／min，约167g相对离心力)。提取总RNA和核蛋白，以半定量RT-PCR和Western Blotting检测ATF4mRNA和蛋白的表达变化。
3．观察ATF4在牙周膜细胞向成骨样细胞分化过程中的作用
5％CO2，37℃条件下，生长状态良好的4-6代人牙周膜细胞培养于10％胎牛血清的DMEM培养基。按阳离子脂质体Lipofectamine 2000TM说明操作，用美国肿瘤研究所Professor Lee惠赠的重组质粒pMyc-ATF4和空载体质粒pCMV5-myc转染人牙周膜细胞。半定量RT-PCR和Western Blotting检测未转染细胞、转染空质粒(pCMV5-myc)细胞、转染目的基因(pMyc-ATF4)细胞ATF4mRNA和蛋白表达水平。三组细胞均加载30min 167g离心力，检测ALP活性及成骨样基因骨钙素(OCN)、骨桥蛋白(OPN)、胶原Ⅰ(COLI)、骨涎蛋白(BSP)基因的表达变化。
结果
1．正常大鼠的牙周组织中基本上未见有明显的ATF4的阳性表达，靠近牙骨质和牙槽骨表面的牙周膜部分轻度染色，而中间部分的牙周膜染色更浅。正畸加力组大鼠牙周组织中ATF4表达增强，表达贯穿正畸牙周组织改建的全过程。在压力侧，靠近牙骨质表面的牙周膜强阳性表达；在张力侧，靠近牙骨质和牙槽骨表面的牙周膜区域强阳性表达，中间部分的牙周膜也阳性表达；所有加力组大鼠张力区比压力区阳性染色深，骨形成区域阳性染色较强。
2．正常人牙周膜细胞有ATF4 mRNA表达；加载167g离心力后，ATF4 mR2NA表达发生变化：加力10min时开始增加，但无统计学意义(P＞0.05)；加力30min时达到高峰(P＜0.01)；加力60min，90min时表达开始下降，但仍高于加力前水平(P＜0.01)；加力120min时降至加力前水平(P＞0.05)。
正常人牙周膜细胞ATF4蛋白表达很低；加载167g离心力后，ATF4蛋白表达发生变化：加力10min时开始增加，但无统计学意义(P＞0.05)；加力30min时继续增加(P＜0.01)；加力60min时达到高峰(P＜0.01)，加力90min时表达开始下降，但仍高于加力前水平(P＜0.01)；加力120min时降至加力前水平(P＞0.05)。
3．人牙周膜细胞表达ALP，转染目的基因pMyc-ATF4后，ALP表达活性升高(P＜0.05)，但转染空质粒pCMV5-myc ALP表达未发生明显变化(NS：P＞0.05)。未转染、转染pCMV5-myc、转染pMyc-ATF4人牙周膜细胞受到167g离心力，ALP表达均升高(*P＜0.05，**P＜0.01)；但转染pCMV5-myc与未转染细胞相比，受力后ALP表达无明显差异(NS：P＞0.05)，转染pMyc-ATF4与未转染细胞相比，受力后ALP表达升高(P＜0.01)。
人牙周膜细胞有OPNmRNA、COLImRNA、BSPmRNA表达，没有OCNmRNA表达。转染目的基因pMyc-ATF4后，OPNmRNA、COLImRNA、BSPmRNA、OCNmRNA表达均升高(*P＜0.05，**P＜0.01)，但转染空质粒pCMV5-myc后，OPNmRNA、COLImRNA、BSPmRNA、OCNmRNA表达未发生明显变化(NS：P＞0.05)。未转染、转染pCMV5-myc、转染pMyc-ATF4细胞受到167g离心力，OPNmRNA、COLImRNA、BSPmRNA、OCNmRNA表达均升高(*P＜0.05，**P＜0.01)；但转染pCMV5-myc与未转染细胞相比，受力后OPNmRNA、COLImRNA、BSPmRNA、OCNmRNA表达无明显差异(NS：P＞0.05)，转染pMyc-ATF4与未转染细胞相比，受力后OPNmRNA、COLImRNA、BSPmRNA、OCNmRNA表达升高(P＜0.01)。
结论
1．ATF4在正畸牙齿移动过程中的表达有明显的变化，从而确定其参与了牙齿移动过程中的牙周组织的改建，在正畸牙齿移动的机制中具有重要意义。
2．应力刺激可使ATF4mRNA及蛋白的表达增强，表达量在30分钟到60分钟左右达到峰值，随后逐渐降低至正常水平；表明ATF4可对力学刺激作出快速、一过性的短暂表达反应。从细胞水平证实ATF4参与了正畸牙齿移动牙周组织的改建。
3．ATF4可促进加载下的人牙周膜细胞向成骨细胞分化。
4．此研究有助于更好地理解牙齿移动中的牙周组织改建的机理，并由此指导临床实践。
Background and objective
It has been known that a tooth can be moved gradually from one spot in the oral cavity to
a 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. Methods
1. Observe the expression of ATF4 protein in periodontal tissues after orthodontic
tooth movement
A 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 protein
was measured by Semi-quantitative RT-PCR and Western Blotting respectively.
3. Observe the role of ATF4 in the mechanical stress-induced human PDL cells differentiation
Cells 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.
Results
1. 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.
Conclusion
1. 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.
牙周膜细胞在机械力作用下向成骨细胞分化的分子机制比较复杂，包含多条信号转导途径。例如，一氧化氮(NO)、前列腺素12(PGI2)、前列腺素E2(PGE2)、百日咳毒素敏感异源三聚体G蛋白(PTX-敏感蛋白)、动力敏感钙离子通道等。近年的研究表明，一些核内转录因子也参与了细胞内调控途径，将细胞外物理或机械刺激转化为协调的细胞反应。
1987年，能以一个共同的核心序列(CGTCA)和腺病毒启动子E2，E3和E4结合的蛋白被命名为ATF，在随后的几年中发现了大量相同的或者类似的能和ATF／CRE位点结合的蛋白，这些蛋白都含有一个命名为bZip的DNA结合区域。基于他们的氨基酸的相似性，ATF家族被分为ATF2，ATF4，ATF6，B-ATF亚类。
1989年，Hai根据ATF／CREB结合序列特异性识别位点，首次克隆了ATF4。其他的学者也相继分离出了ATF4，它通常又被称为TAXREB67、CREB2或C／ATF。研究表明，ATF4与成骨细胞特异性基因骨钙素启动区的成骨细胞特异性元件1(OSE1)相结合，调控成骨细胞的分化。至今为止，只有Runx2和ATF4可诱导非成骨细胞系表达成骨细胞特异型基因骨钙素的表达。另外，有研究证实ATF4是RSK2的磷酸化底物，ATF4(-／-)鼠出现CLS表现型，除精神发育迟缓外，还伴有颅面、牙齿、骨骼发育异常。ATF4和RSK2以一种线性级联的方式调控成骨细胞分化和功能，是间充质细胞向成骨细胞分化所必需的。ATF4作为成骨细胞分化的关键因子，与骨发育和牙齿发育关系的研究正在不断开展。但正畸骨改建与骨发育是两种不同的生理过程，ATF4在正畸牙齿移动牙周组织的表达和分布，在机械力的作用下体外培养的人牙周膜细胞中表达变化的情况，以及在牙周膜细胞向成骨样细胞分化的过程中所起的作用，国内外尚未有报道。
本研究通过建立正畸大鼠牙齿移动动物模型和体外细胞培养，结合细胞力学试验方法和分子生物学测试技术，拟从组织、细胞水平以及分子生物学角度探讨ATF4在正畸牙齿移动过程中对牙周组织改建的影响及作用机制，进一步为正畸牙齿移动提供理论和实践依据。
方法
1．观察正畸牙周组织改建过程中ATF4的表达变化
建立大鼠牙齿移动实验模型，在加力1h，2h，4h，8h，12h，1d，3d，5d，7d后，处死大鼠，固定，脱钙，制作大鼠第一磨牙牙周组织石蜡切片，HE染色观察牙周组织形态学变化，免疫组化进行半定量分析ATF4的表达。
2．观察机械力加载下人牙周膜细胞ATF4mRNA和蛋白的表达变化
组织块法培养原代人牙周膜细胞，并做波形丝蛋白抗体和角蛋白抗体染色鉴定。取4-6代生长旺盛，性质稳定的人牙周膜细胞进行实验。以2.5×10~5／孔将第4代人牙周膜细胞接种于6孔细胞培养板培养24小时，换用含2％胎牛血清的条件培养基继续培养24小时，将六孔板置于离心加力支架中，离心机加力10min，30min，60min，90min，120min，240min(910rev／min，约167g相对离心力)。提取总RNA和核蛋白，以半定量RT-PCR和Western Blotting检测ATF4mRNA和蛋白的表达变化。
3．观察ATF4在牙周膜细胞向成骨样细胞分化过程中的作用
5％CO2，37℃条件下，生长状态良好的4-6代人牙周膜细胞培养于10％胎牛血清的DMEM培养基。按阳离子脂质体Lipofectamine 2000TM说明操作，用美国肿瘤研究所Professor Lee惠赠的重组质粒pMyc-ATF4和空载体质粒pCMV5-myc转染人牙周膜细胞。半定量RT-PCR和Western Blotting检测未转染细胞、转染空质粒(pCMV5-myc)细胞、转染目的基因(pMyc-ATF4)细胞ATF4mRNA和蛋白表达水平。三组细胞均加载30min 167g离心力，检测ALP活性及成骨样基因骨钙素(OCN)、骨桥蛋白(OPN)、胶原Ⅰ(COLI)、骨涎蛋白(BSP)基因的表达变化。
结果
1．正常大鼠的牙周组织中基本上未见有明显的ATF4的阳性表达，靠近牙骨质和牙槽骨表面的牙周膜部分轻度染色，而中间部分的牙周膜染色更浅。正畸加力组大鼠牙周组织中ATF4表达增强，表达贯穿正畸牙周组织改建的全过程。在压力侧，靠近牙骨质表面的牙周膜强阳性表达；在张力侧，靠近牙骨质和牙槽骨表面的牙周膜区域强阳性表达，中间部分的牙周膜也阳性表达；所有加力组大鼠张力区比压力区阳性染色深，骨形成区域阳性染色较强。
2．正常人牙周膜细胞有ATF4 mRNA表达；加载167g离心力后，ATF4 mR2NA表达发生变化：加力10min时开始增加，但无统计学意义(P＞0.05)；加力30min时达到高峰(P＜0.01)；加力60min，90min时表达开始下降，但仍高于加力前水平(P＜0.01)；加力120min时降至加力前水平(P＞0.05)。
正常人牙周膜细胞ATF4蛋白表达很低；加载167g离心力后，ATF4蛋白表达发生变化：加力10min时开始增加，但无统计学意义(P＞0.05)；加力30min时继续增加(P＜0.01)；加力60min时达到高峰(P＜0.01)，加力90min时表达开始下降，但仍高于加力前水平(P＜0.01)；加力120min时降至加力前水平(P＞0.05)。
3．人牙周膜细胞表达ALP，转染目的基因pMyc-ATF4后，ALP表达活性升高(P＜0.05)，但转染空质粒pCMV5-myc ALP表达未发生明显变化(NS：P＞0.05)。未转染、转染pCMV5-myc、转染pMyc-ATF4人牙周膜细胞受到167g离心力，ALP表达均升高(*P＜0.05，**P＜0.01)；但转染pCMV5-myc与未转染细胞相比，受力后ALP表达无明显差异(NS：P＞0.05)，转染pMyc-ATF4与未转染细胞相比，受力后ALP表达升高(P＜0.01)。
人牙周膜细胞有OPNmRNA、COLImRNA、BSPmRNA表达，没有OCNmRNA表达。转染目的基因pMyc-ATF4后，OPNmRNA、COLImRNA、BSPmRNA、OCNmRNA表达均升高(*P＜0.05，**P＜0.01)，但转染空质粒pCMV5-myc后，OPNmRNA、COLImRNA、BSPmRNA、OCNmRNA表达未发生明显变化(NS：P＞0.05)。未转染、转染pCMV5-myc、转染pMyc-ATF4细胞受到167g离心力，OPNmRNA、COLImRNA、BSPmRNA、OCNmRNA表达均升高(*P＜0.05，**P＜0.01)；但转染pCMV5-myc与未转染细胞相比，受力后OPNmRNA、COLImRNA、BSPmRNA、OCNmRNA表达无明显差异(NS：P＞0.05)，转染pMyc-ATF4与未转染细胞相比，受力后OPNmRNA、COLImRNA、BSPmRNA、OCNmRNA表达升高(P＜0.01)。
结论
1．ATF4在正畸牙齿移动过程中的表达有明显的变化，从而确定其参与了牙齿移动过程中的牙周组织的改建，在正畸牙齿移动的机制中具有重要意义。
2．应力刺激可使ATF4mRNA及蛋白的表达增强，表达量在30分钟到60分钟左右达到峰值，随后逐渐降低至正常水平；表明ATF4可对力学刺激作出快速、一过性的短暂表达反应。从细胞水平证实ATF4参与了正畸牙齿移动牙周组织的改建。
3．ATF4可促进加载下的人牙周膜细胞向成骨细胞分化。
4．此研究有助于更好地理解牙齿移动中的牙周组织改建的机理，并由此指导临床实践。
Background and objective
It has been known that a tooth can be moved gradually from one spot in the oral cavity to
a 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. Methods
1. Observe the expression of ATF4 protein in periodontal tissues after orthodontic
tooth movement
A 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 protein
was measured by Semi-quantitative RT-PCR and Western Blotting respectively.
3. Observe the role of ATF4 in the mechanical stress-induced human PDL cells differentiation
Cells 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.
Results
1. 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.
Conclusion
1. 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.
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