Elsevier

Biochimie

Volume 165, October 2019, Pages 48-56
Biochimie

Research paper
Upregulation of JHDM1D-AS1 protects PDLSCs from H2O2-induced apoptosis by decreasing DNAJC10 via phosphorylation of eIF2α

https://doi.org/10.1016/j.biochi.2019.06.018Get rights and content

Highlights

  • The expression of JHDM1D-AS1 is decreasing when PDLSCs treated with hydrogen peroxide.

  • JHDM1D-AS1 regulates PDLSC apoptosis through inhibiting DNAJC10.

  • JHDM1D-AS1/DNAJC10/p-eIF2α/Bcl-2 regulatory axis that controls apoptosis in PDLSC.

Abstract

Periodontal ligament stem cells (PDLSCs) are a promising tool for regenerative medicine in clinical periodontal ligament repair. However, clinical maintenance of high quality and large quantity of PDLSCs faces multiple obstacles. One of them is how PDLSCs respond to environmental stimuli such as reactive oxygen species. We aim to elucidate how PDLSCs react to oxidative stress and the underlying mechanisms. We utilized hydrogen peroxide-induced oxidative stress to mimic ROS increase in rat PDLSCs. Our data indicated a rapid downregulation of a long non-coding RNA, lncRNA JHDM1D antisense 1 (JHDM1D-AS1), when PDLSCs were treated with hydrogen peroxide, which was negatively associated with PDLSC apoptosis. Moreover, our data showed that JHDM1D-AS1 regulated PDLSC apoptosis via inhibition of DNAJC10, a heat shock protein 40 family member. Moreover, overexpressed DNAJC10 inhibited Bcl-2 protein level and eIF2α phosphorylation level, which, in turn, contributed to PDLSC apoptosis. Our results revealed a protective role of JHDM1D-AS1 in ROS-induced apoptosis, and validated that JHDM1D-AS1/DNAJC10/phosphorylated-eIF2α/Bcl-2 pathway works as an anti-apoptotic signaling axis in PDLSCs.These findings will facilitate the in vitro culturing of PDLSCs for clinical usage and promote stem cell-based therapy for periodontal tissue regeneration.

Introduction

Almost 90% people worldwide are suffering from periodontal related diseases [1]. Recently, stem cell based regeneration of periodontal tissues has showed promising potential [2]. Periodontal ligament stem cells (PDLSCs) are multipotent stem cells which can be differentiated into cementum, peripheral nerves, and other type of cells [[3], [4], [5], [6], [7]]. The in situ PDLSCs are limited and in vitro cultured PDLSCs endure apoptotic death which restricts the amount of PDLSCs that can be utilized in regenerative medicine. Previous studies have shown that genetic background affects the quality and function of PDLSCs [8,9]. External stimuli, such as hypoxia, are also well known factors that regulate PDLSC apoptosis [[10], [11], [12], [13], [14]]. Accumulating evidence has indicated that non-coding RNA, especially long non-coding RNA (lncRNA) response to external stimuli regulates cellular processes [15,16]. Oxidative stress is characterized by dysregulated levels of reactive oxygen species (ROS) which are primarily generated from mitochondrial complexes I and III. It has been previously demonstrated that oxidative stress is critical for stem cell aging and survival [[17], [18], [19]]; however, the underlying mechanisms are still unclear.

LncRNAs are a class of non-protein coding RNAs consisting of more than 200 nucleotides [20]. Studies have illustrated that lncRNAs are critical in multiple biological processes, including the regulation of stem cell and osteogenic differentiation [21]. Moreover, previous studies have shown that lncRNA HIF1α-anti-sense 1 (HIF11α-AS1) regulates osteogenic differentiation of bone marrow stromal cells and lncRNA ANCR modulates Wnt signaling to regulate osteogenic differentiation of PDLSCs [9,22]. Recently, large scale screening for lncRNAs that are critical for PDLSC osteogenic differentiation has been performed [23]. Since non-coding RNAs exhibit huge potentials in stem cell regulation, and disease diagnosis and treatment [24,25], it is important to further analyze the role of lncRNAs in PDLSC homeostasis. Histone demethylase Jumonji C domain containing histone demethylase 1 homolog D (JHDM1D) was initially identified to harbor a plant homeodomain and a Jumonji domain that binds to specific histone modification sites [26]. In addition, JHDM1D inhibited tumor growth by suppressing angiogenesis [27]. Importantly, a previous study has shown that the lncRNA JHDM1D antisense 1 promotes pancreatic tumorigenesis by regulating angiogenesis in response to nutrient starvation. In addition, JHDM1D-AS1 increases blood vessel formation and macrophage infiltration [28]. However, whether JHDM1D-AS1 is active in stem cells is unknown.

Eukaryotic DNAJ family proteins are conserved with the first J protein identified in Escherichia coli [29]. It was proposed that DNAJ is required for the loading of Hsc70 onto a nascent polypeptide to form a proper folding complex [30]. Previous studies have demonstrated that DNAJ family proteins are responsive to environmental stimuli, such as reactive oxygen species (ROS) [31,32]. However, how DNAJ proteins regulate PDLSCs, especially under environmental stress, is largely unknown.

Eukaryotic Translation Initiation Factor 2 Subunit Alpha (EIF2α) is a key regulator of apoptosis in multiple cell types [33]. Recently, it has been reported that EIF2α is responsive to endoplasmic reticulum stress and regulates the osteoblast differentiation [34]. These reports suggested that eIF2α may function as a link between stress response and apoptosis.

Hence, we explored the expression level of JHDM1D-AS1 in PDLSCs treated with H2O2 and examined the apoptotic status of PDLSCs. In addition, we assessed the changes in apoptotic signaling in PDLSCs overexpressing JHDM1D-AS1. Interestingly, our results revealed a regulatory axis involving JHDM1D-AS1, DNAJC10, and apoptotic inhibitor Bcl-2. Besides, we also examined the involvement of phosphorylated-eIF2α in JHDM1D-AS1-dependent regulation of PDLSC apoptosis.

Section snippets

Periodontal ligament stem cell isolation and culture

The rat PDLSCs were isolated by modifying the method described previously [35]. Briefly, PDLSCs were collected from the periodontal ligament by cutting periodontal ligament into small pieces and digesting in 0.3% collagenase (Sigma) at 37 °C for 4 h. The cells obtained were then seeded in culture dishes and cultured in DMEM (Dulbecco's modified Eagle's medium, Hyclone), containing 10% fetal bovine serum (Gibco), 100 IU/mL penicillin, and 100 IU/mL streptomycin (Gibco), in a humidified

H2O2 treatment transiently reduces JHDM1D-AS1 expression in human PDLSCs

To study the role of lncRNA JHDM1D-AS1 in human PDLSCs, we first established H2O2-induced apoptosis in human PDLSCs. We then treated the isolated PDLSCs with 100 μM, 200 μM, and 400 μM H2O2, and then, assessed the cell viability via MTT experiment. Our data showed that H2O2 treatment significantly affected the cell viability at the concentrations of 200 μM and 400 μM (Fig. 1A). Furthermore, we monitored apoptosis of PDLSCs treated with H2O2, and found that 200 μM H2O2 significantly induced

Discussion

PDLSCs are a promising tool for regenerative medicine [36]. Since PDLSCs potentially reside in extreme environment, it is of huge significance to monitor how PDLSCs response to external stimuli. Our data first revealed that under hydrogen peroxide treatment, a long non-coding RNA, JHDM1D-AS1, is rapidly downregulated. It has been reported that lncRNAs, MALAT1 and Sox2ot, were protective non-coding RNAs against hydrogen peroxide exposure [16,37]. These data suggested a promising role of these

Conclusion

Our data showed that lncRNA JHDM1D-AS1 is inhibited in PDLSCs by hydrogen peroxide treatment, which, in turn, leads to upregulation of DNAJC10. Upregulated DNAJC10 promoted the de-phosphorylation of phosphorylated eIF2α, which, in turn, caused the inhibition of anti-apoptotic gene, Bcl-2, and induction of pro-apoptotic gene, Bax, and thus, promoted apoptosis. Our data illustrated an alternative pathway of how hydrogen peroxide promoted PDLSC apoptosis, which provided insights into PDLSCs

Funding

This work was supported by grants from the Science and Technology Project of Yubei District, Chongqing (grant No. 2017 (agriculture society) 45), and the Administration of traditional Chinese Medicine of Zhejiang Province (2014ZA069), the Department of Education of Zhejiang Province(Y201534720).

Availability of supporting data

The datasets used and/or analyzed in the current study are available from the corresponding author based on a reasonable request.

Author contributions

BS and SB performed statistical analysis, evaluated the results, and drafted the manuscript. GN, BS, and SB participated in the conception and design of the study. YC, GY, XM, and FX contributed to laboratory measurements and data assurance. All authors have read and approved the final version of the manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Acknowledgements

The authors thank all current and previous members of the laboratory for valuable discussions and support.

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