PERK Contributes To Osteogenic Differentiation Capacity Attenuation Of Periodontal Ligament Stem Cells In Inflammatory Microenvironment

Background: Chronic periodontitis is a kind of oral infection diseases with high morbidity in China even all over the world. Irreversible absorption of alveolar bone is a significant bottleneck in periodontal treatment. Human periodontal ligament stem cells(PDLSCs) are mesenchymal stem cells(MSCs) located in periodontal ligament(PDL), which have osteogenic and cementoblastic differentiation capacity. However, autologous human PDLSCs in periodontitis lesions always display attenuated osteogenic differentiation capacity and fail to facilitate periodontal bone repair. Nowadays, researches on mechanism behind the osteogenic differentiation capacity variation of PDLSCs in inflammatory microenvironment have focused on various signal pathways and molecules,but desired therapeutic effects can’t be achieved by regulating only a few factors in those pathways. Therefore, investigation on upstream regulatory mechanism in organelle level may be more operable and practical. Endoplasmic reticulum(ER) stress is a protective reaction to maintain cellular homeostasis through activating unfolded protein response(UPR). Moderate ER stress possesses preventive effects, whereas excessive ER stress leads to destruction. ER stress/UPR has been identified to participate in initiation and development of various chronic inflammatory diseases in accompany with periodontitis. Besides, ER stress/UPR, especially double-stranded RNA-activated protein kinase(PKR) like endoplasmic reticulum kinase(PERK) pathway is considered to be involved in the osteogenic differentiation of MSCs. However, there is no report on the influence of ER stress/UPR on osteogenesis of PDLSCs under inflammatory conditions.Aim: To investigate the role of ER stress and UPR PERK pathway in the impaired osteogenic differentiation ability of PDLSCs under inflammatory conditions, and offer new perspectives regarding the pathogenesis and treatment of periodontitis.Methods: 1. Healthy and periodontits-affected teeth collected clinically were randomly divided into 3 groups respectively: in one group, teeth were decalcified and embedded in paraffin for immunohistochemistry, before which the presence of periodontal ligament was checked by haematoxylin and eosin(HE) staining, to detect the locational expressions of PERK pathway related factors, glucose regulated protein 78(GRP78), PERK, activating transcription factor 4(ATF4), and transcription factor C/EBP homologous protein(CHOP). In the other groups, PDL or residual PDL and inflamed granulations were scraped from the middle third of roots, then m RNA expressions of PERK pathway related factors were measured by q RT-PCR after tissue homogenate in one of the groups, and protein levels of those factors were measured by Western blot after tissue protein dissociation in the othergroup. The healthy teeth were regarded as control teeth. 2.PDLSCs were isolated from PDL on the middle third of healthy teeth roots by tissue block enzymolytic method in vitro, and purified by limiting dilution method. In order to identify the cells, colony forming capacity was evaluated by colony forming capacity assay, MSC-associated surface makers were detected flow cytometry, and multipotential differentiation capacity was assessed by osteogenic and adipogenic induction. Then ER stress inducers tunicamycin(TM) and thapsigargin(TG) were used to stimulate PDLSCs, and m RNA expressions of PERK pathway related factors in 6 h, 9 h and 12 h were assessed by q RT-PCR. The group in which PDLSCs were untreated with TM or TG was regarded as control group. PDLSCs were also cultured by osteogenic media with TM/TG, and the expression of runt-related transcription factor 2(Runx2) and osteocalcin(OCN), m RNA expression and activity of alkaline phosphatase(ALP), and formation of mineralization nodules were assessed to evaluate the osteogenic differentiation capacity of PDLSCs. 3. PDLSCs were cultured in gradient concentrations(1 ng/ml, 10 ng/ml, and 20 ng/ml) of proinflammatory cytokine tumor necrosis factor-alpha(TNF-α). The m RNA expressions and protein levels of PERK pathway related factors in 6 h, 12 h, 24 h and 72 h were assessed by q RT-PCR and Western blot. The group in which PDLSCs were untreated with TNF-α was regarded as control group. PDLSCs were also cultured by osteogenic media with TNF-α, and their osteogenic differentiation capacity was evaluated. Thus appropriate stimulating concentration and time length of TNF-α were screened. 4. PDLSCs were treated with gradient concentrations(0.5 m M, 1 m M, 2.5 m M, 5 m M and 7.5 m M) of ER stress inhibitor 4-phenylbutyric acid(4-PBA). The cytotoxicity of 4-PBA was evaluated by CCK-8 assay in 12 h and 24 h to confirm the pretreating time. Next, PDLSCs were pretreated by 4-PBA and then stimulated by 10 ng/ml of TNF-α for 12 h, and the m RNA expressions and protein levels of PERK pathway related factors were assessed by q RT-PCR and Western blot. The group in which PDLSCs were only stimulated by TNF-α was regarded as control group. PDLSCs were also cultured byosteogenic media with 4-PBA pretreatment and TNF-α stimulation, and their osteogenic differentiation capacity was evaluated. 5. PDLSCs were transfected by PERK si RNA, and silencing efficiency of gene and protein expression were evaluated. PDLSCs were stimulated by 10 ng/ml of TNF-α for 12 h after transfection, and m RNA expressions and protein levels of PERK, ATF4, and CHOP were assessed by q RT-PCR and Western blot. The group in which PDLSCs were not transfected but stimulated by TNF-α was regarded as control group. PDLSCs were also cultured by osteogenic media with TNF-α stimulation, and their osteogenic differentiation capacity was evaluated.Results: 1. In inflamed PDL and granulations of periodontits-affected teeth, positive rate in immumohistochemical staining, m RNA expressions, and protein levels of PERK pathway related factors GRP78, PERK, ATF4, and CHOP were significantly higher than those of healthy PDL. 2. PDLSCs could be isolated from human healthy PDL successfully. Purified PDLSCs displayed a fibroblast-like morphology and were able to form colony units as MSCs. They were staining positively for CD146, CD105, CD90, and CD44, but negatively for endothelial cell marker CD31 and hematopoietic marker CD34 by flow cytometry. PDLSCs also exhibited osteogenic and adipogenic differentiation potential. The m RNA expressions of PERK related factors in PDLSCs after treated with 1 μg/ml of TM for 6 h and 9 h were significantly higher than those of control group, and they were down-regulated in 12 h. When PDLSCs were treated by 0.1 μM of TG, m RNA expressions of GRP78, PERK and ATF4 were significantly increased only in 9 h, and CHOP m RNA expressions were higher in both 9 h and 12 h compared with untreated control group. The m RNA expression of PERK related factors in TM-treated group was significantly higher than those in TG-treated group in 9 h. After osteogenic induction for 1, 2, or 4 weeks, TM/TG treatment resulted in reduced m RNA expression of ALP, Runx2, and OCNtogether with protein levels of Runx2 and OCN of PDLSCs compared with untreated cells. Furthermore, TM/TG treatment was associated with decreased ALP staining and formation of mineralized nodules. 3. When stimulated for 12 h, all the gradient concentrations of TNF-α increased the m RNA and protein expressions of PERK related factors to the peak significantly, whereas PERK, ATF4 and CHOP m RNA and protein expressions of PDLSCs stimulated by 1 ng/ml of TNF-α were lower than the other two concentrations of TNF-α. ATF4 m RNA and protein expressions and protein level of PERK were significantly higher in PDLSCs stimulated by 10 ng/ml of TNF-α compared with those by 20 ng/ml of TNF-α. All the concentrations of TNF-α suppressed osteogenic differentiation capacity of PDLSCs. ALP activity, Runx2 m RNA expression, OCN m RNA and protein expressions, and formation of mineralized nodules of PDLSCs stimulated by 10 ng/ml and 20 ng/ml of TNF-α were significantly higher than those by 1 ng/ml of TNF-α. Hence 10 ng/ml of TNF-α were selected to mimic inflammatory microenvironment, and cells were stimulated for 12 hours. 4. Treatment of PDLSCs with 7.5 m M of 4-PBA for 24 hours significantly suppressed cell proliferation as cytotoxicity was exhibited. Therefore, we pretreated cells with 4-PBA for 12 hours in all subsequent studies. When PDLSCs were stimulated by 10 ng/ml of TNF-α for 12 h after 4-PBA pretreatment, lower concentrations(0.5, 1, and 2.5 m M) of 4-PBA blocked the induction of GRP78, PERK, and ATF4 m RNA and protein levels by TNF-α comparing with the control group without 4-PBA pretreatment, and there was no statistical different among the three concentrations of 4-PBA. However, unlike gene expression, 4-PBA failed to suppress the induction of CHOP protein levels. Additionally, a higher concentration of 4-PBA(7.5 m M) even enhanced PERK expression and ATF4 protein level. During osteogenic induction, lower concentrations of 4-PBA(0.5, 1, 2.5 m M) were associated with significant increases in the m RNA expressions of ALP and Runx2, protein levels of Runx2 and OCN, along with ALP staining and the formation of mineralized nodules. OCN gene expression was restored by 4-PBA at concentrations of 1 and 2.5 m M.However, 7.5 m M of 4-PBA aggravated the negative effects of TNF-α on Runx2 m RNA expression, protein levels of osteogenic-related factors and ALP staining. 5. A 56% knockdown in m RNA expressions and 51% knockdown in protein levels were confirmed after PERK si RNA transfection. PERK, ATF4, and CHOP m RNA and protein expressions of untransfected PDLSCs were still significantly increased by TNF-α comparing with those of cells not stimulated by TNF-α, but m RNA and protein expressions of those factors were decreased robustly by PERK knockdown even PDLSCs had been stimulated by TNF-α for 12 h. Meanwhile, PERK depletion reversed the attenuate osteogenic differentiation capacity of PDLSCs caused by TNF-α treatment.Conclusion: 1. ER stress and PERK pathway are activated in inflamed PDL and granulations of periodontits-affected teeth. 2. ER stress activators could activate ER stress and PERK pathway robustly but suppress osteogenic differentiation capacity of PDLSCs. 3.TNF-α attenuates the osteogenic differentiation ability of PDLSCs through activation of ER stress and PERK pathway in inflammatory microenvironment. In addition, this effect could be reversed by ER stress inhibitor 4-PBA or PERK knockdown.