| Background and ObjectiveIt is known that the teeth can be moved gradually from one point to a more desirable point by applying a mechanical force to the tooth crown. Mechanical force leads to the remodeling of periodontal tissue. Periodontal ligament plays an important role in the transmission of mechanical stimulation from tooth to periodontal tissue. The periodontal ligament is a layer of dense connective tissue which is composed of cells and matrix, located between the tooth root and alveolar bone. The mechanical force applied onto the teeth is transmited by the periodontal ligament, and then acted on the alveolar bone. Periodontal ligament cells (PDLSCs), which are the key to the success of periodontal tissue remodeling, are not only the direct sensory cells, but also the effector cells of mechanical force. At the same time, PDLSCs could secrete and release various cytokines and growth factors under the action of external force and may be involved in the process of bone resorption and bone formation. In addition, they have been proved to be differentiated into osteoblasts and express some of the phenotypic characteristics of osteoblasts under mechanical stress, which is the key to the remodeling of alveolar bone. The molecular mechanism mechanical physical signal into intracellular biochemical signal of PDLSCs under mechanical stimulation is more complex, including a plurality of signal transduction pathways. For example, nitric oxide (NO), bone morphogenetic protein 2 (BMP2), transforming growth factor beta 1 (TGF-beta 1), prostaglandin 12 (PGI2), prostaglandin (PGE2), stretch sensitive calcium channel, etc. Recent studies have shown that internuclear related transcriptional factors are also involved in the intracellular regulatory pathways of periodontal ligament cells, which participated in the transforming the extracellular physical or mechanical stimulation into a coordinated cellular response.Our previous studies have confirmed that activating transcription factor 4 (ATF4) is involved in the process of orthodontic tooth movement, and the expression of ATF4 upregulated under mechanical force, further promoted the osteogenic differentiation of PDLSCs. ATF4, one of the transcription factor family members of cAMP response element binding, could regulate osteoblast differentiation and bone formation. Furthermore, protein kinase R like endoplasmic reticulum kinase (PERK) is an upstream target of ATF4. PERK is a type I transmembrane protein which located in the endoplasmic reticulum (ER). The structure of PERK includes signal peptide and transmembrane domain, and its N terminal is located in the endoplasmic reticulum, C terminal is located in the cytoplasmic side. The ER is one of the most important cellular compartments in eukaryotic cells. ER is the site of protein synthesis and storage of calcium ions. When cells are subjected to hypoxia, the disorder of calcium ion concentration, etc, numerous unfolded or misfolded proteins accumulated in the endoplasmic reticulum, leading to imbalance of endoplasmic reticulum structure and function, trigging endoplasmic reticulum stress (ERS). ERS is mainly mediated by three major transducers-PERK, activating transcription factor 6 (ATF6), and inositol requiring enzyme 1 (Irel). Under the condition of ERS, PERK was activated, resulting in the phosphorylation of a-subunit of eukaryotic initiation factor 2 (eIF2a). Phosphorylated eIF2a specifically promoted the translation of the ATF4 and produced a series of effects. Some scholars have found that the upregulation of ATF4 was related to PERK in the cardiovascular, kidney tissues and the process of proliferation and differentiation of cartilage cells under mechanical force. Studies have shown that PERK-eIF2a-ATF4 signal pathway mediated by ERS was involved in osteoblastic differentiation of osteoblasts. We have known that orthodontic tooth movement is a process of periodontal ligament cells (PDLSCs) osteodifferentiation and alveolar bone remodeling under mechanical force. ATF4 expression was up-regulated in PDLSCs under mechanical force, furthermore, ATF4 and PERK were associated with oxidative folding of intracellular proteins, and were the important signal molecule of endoplasmic reticulum stress. It can be inferred that PERK-eIF2a-ATF4 signaling pathway mediated by ERS was involved in osteogenic differentiation of PDLSCs under cyclic stretch. However, whether this signal pathway was involved in cyclic stretch-induced osteogenic differentiation of PDLSCs has not been unclear.In our study, hPDLSCs were primary cultured and subcultured. Then, we applied the cyclic cyclic stretch to hPDLSCs and established a mechanical stimulation model to detect the expression of endoplasmic reticulum stress related factors and osteogenic genes in different time point after loading. Based on this, we constructed the overexpression and knockout of PERK by lentivirus plasmid packaging to study whether PERK-eIF2a-ATF4 signaling pathway mediated by ERS was involved in osteogenic differentiation of PDLSCs under cyclic stretch, which will provide a scientific basis for further study of the molecular biology mechanism of alveolar bone remodeling during the process of orthodontic tooth movement.Materials and Methods1. Cell cultureHuman PDLSCs were cultured with tissue culture method. PDLSCs were observed morphologically and were counted by ordinary counting chamber after 1-10 days of culture. Keratin protein and vimentin protein staining were applied to identify the characteristic of hPDLSCs. PDLSCs of passages three and four were used in this experiment.2. Observe the the expression of ERS-related factors and osteogenic genes after application of cyclic force to hPDLSCsHPDLSCs were seeded into six-well culture plates with 35-mm silicone membrane coated on the bottom. After the cultures reached nearly 80% confluences for 2-3 days, cells were serum-deprived for 24 h. Then, cyclic stretch was applied at 10% deformation and 0.5Hz (30 cycles/min) for lh,3h,6h,12h and 24 h, using Flexcell(?) FX-5000TM Tension System (Flexcell International Corporation). Control cells not submitted to cyclic stretch were cultured on the similar six-well plate and in the same incubator for the maximum stretching period. HPDLSCs were collected at the indicated time, and the total RNA and protein was isolated from each sample. The expressions of ERS-related factors (Bip, Xbpl, PERK, eIF2a, p-eIF2a) and osteogenic genes (ATF4, OCN, BSP) were measured using quantitative real-time RT-PCR and Western blot analysis.3. Observe the role of PERK-eIF2a-ATF4 signaling pathway in cyclic stretch-induced osteogenic differentiation of PDLSCsOverexpression vector of human PERK (PERK+/+) and PERK knockout (PERK-/-) were constructed by Genechem (Shanghai Genechem Co., Ltd.). Empty virus vector GFP served as a negative control respectively. To obtain optimal value of multiplicity of infection (MOI), we first conducted a preliminary experiment. In the formal experiment, the appropriate amounts of lentivirus were added according to the appropriate MOI values (PERK+/+:MOI= 100, PERK-/- vector:MOI= 100, empty vector:MOI= 60). The effects of PERK+/+ and PERK-/- were assessed by quantitative real-time PCR and Western blotting. Alizarin red staining was used to evaluate the degree of mineralization after transfection of lentivirus. Then we applied cyclic stretch on the hPDLSCS after they were transfected by lentvirus. The expression of p-eIF2a and osteogenic genes (ATF4, OCN, BSP) were measured by Quantitative real-time RT-PCR and Western blot analysis.Results1. Primary PDLSCs were isolated through tissue methord. The primary hPDLSCs growing from the scraped PDL tissue in culture after 10 day. Cell morphology was observed by an invert microscope, PDLSCs were generally elongated and spindle shaped; after passage, cells showed a thriving growth and presented the characteristics of fibroblasts. After 2-3 days of subculyure, hPDLSCs started growing exponentially, and reached the peak after 8 days. Immunohistochemical stains were positive for Vimentin, negative for Keratin, which proved that the cultured cells derived from mesenchymal tissue.2. Cyclic stretch resulted in cellular re-orientation of hPDLSCs along with the direction of stretch. While the non-loaded hPDLSCs presented randomly orientiation. During the application of mechanical stress, the mRNA level of Bip did not start to elevate until 6 h, and rose gradually from 12 to 24 h. There were some fluctuations in the expression of Xbpl, it decreased after 3 h and 6 h but increased after 12,18, and 24 h (Fig.1 B). Furthermore, cyclic stretch significantly augmented the expressions of PERK and p-eIF2a in a time-dependent manner, while had very little effect on the expressions of t-eIF2a (Fig.1 C and D). The level of PERK peaked after 3 h and 6 h of cyclic stretch, and slightly decreased but remained at a relatively stable level after 12,18, and 24 h. Expressions of p-eIF2a have not obvious change in early period, then increased sharply after 6 h, and maintained at a higher level after 12,18, and 24 h. These results indicated that mechanical stimulation would induce ERS.On the other hand, the expressions of osteogenic genes up-regulated in a time-dependent manner after mechanical stimulation application in hPDLSCs. After 1 h of mechanical stimulation, the mRNA levels of ATF4 increased significantly to a peak, then decreased rapidly at 6 h, and then started to increase from 12 h to 24 h. The level of OCN and BSP showed a stable increase up to 24 h. 3. HPDLSCs were transfected by lentivirus containing PERK (PERK+/+), PERK knockout (PERK-/-), and GFP (LV-GFP). The transfection effiency of them was very high after 72 h of transfection. Real-time PCR and Western blot results confirmed the upregulated expressions of PERK mRNA and protein in PERK+/+ infected hPDLSCs and the downregulated expressions of PERK mRNA and protein in PERK-/- infected hPDLSCs. For the mineralization analysis, Alizarin red staining was performed. The results revealed that PERK+/+ significantly promoted matrix mineralization after 21 days of culture, while PERK-/- significantly inhibited matrix mineralization.The levels of osteogenic genes were determined by quantitative PCR and Western blot. The expressions of p-eIF2α, ATF4, OCN, and BSP were elevated by overexpression of PERK and cyclic stretch greatly enhanced this augmentation. Moreover, the group of PERK+/+ cells loaded by cyclic stretch exhibited the highest expressions of these genes among all groups. However, the PERK-/- group without application of cyclic stretch showed the lowest expressions of these genes. The levels of p-eIF2α, ATF4, OCN, and BSP decreased significantly in PERK-/- group. Similarly, cyclic stretch also enhanced the expression of p-eIF2α, ATF4, OCN, and BSP in PERK-/- group. Additionally, between PERK+/+ group and PERK-/- groups, those genes expressions were always higher in cells loaded by CMF than that of non-loading cells. PERK-eIF2α-ATF4 signaling pathway mediated by ERS involved in PDLSCs osteodifferentiation under cyclic stretch.Conclusions1. Cyclic stretch could intensify some ERS-related genes (Bip, Xbp1, PERK, and p-eIF2α) and several osteoblast marker osteo-related genes (ATF4, BSP and OCN) expression. Mechanical stimulation would induce ERS and futher activated PERK-eIF2α-ATF4 pathway.2. Overexpression of PERK significantly promoted matrix mineralization and osteodifferentiation of hPDLSCs, while knockout of PERK significantly inhibited matrix mineralization and osteodifferentiation of hPDLSCs. PERK plays a role in the process of hPDLSCs diferentiatiated into osteoblast-like cells.3. PERK-eIF2a-ATF4 signaling pathway mediated by ERS exerts an influence on periodontal remodeling and osteogenesis differentiation of hPDLSCs during the process of orthodontic tooth movement. ERS involved in osteoblast differentiation of PDLSCs under cyclic stretch. PERK overexpression increased eIF2a phosphorylation and expression of ATF4, furthermore induced BSP, OCN expression, thus it will promote osteodifferentiation of hPDLSCs; cyclic stretch could promote this effect. However, PERK-/- cells showed the opposite changes, which will inhibit osteodifferentiation of hPDLSCs. Taken together, overexpression of PERK and mechanical stimulation have a synergistic effect on the promotion of osteogenic differentiation.Significance and innovationThis study firstly explored the role of PERK-eIF2a-ATF4 signaling pathway mediated by ERS in the osteogenic differentiation process of hPDLSCs under cyclic stretch. We demonstrated that cyclic tension force acted as a stress and induced endoplasmic reticulum stress. Additionally, we constructed an external force stimulation model and the cyclic stretch stimulated PDLSCs model. It proved that PERK-eIF2a-ATF4 signaling pathway is involved in the osteogenic differentiation of PDLSCs induced by cyclic stretch. This will contribute to a better understanding about the mechanism of cyclic stretch-induced periodontal remodeling via PERK-eIF2a-ATF4 pathway and supply a basis for clinical work during orthodontic tooth movement. |
