Abstract
Cellular senescence is closely related to tissue aging including bone. Bone homeostasis is maintained by the tight balance between bone-forming osteoblasts and bone-resorbing osteoclasts, but it undergoes deregulation with age, causing age-associated osteoporosis, a main cause of which is osteoblast dysfunction. Oxidative stress caused by the accumulation of reactive oxygen species (ROS) in bone tissues with aging can accelerate osteoblast senescence and dysfunction. However, the regulatory mechanism that controls the ROS-induced senescence of osteoblasts is poorly understood. Here, we identified Peptidyl arginine deiminase 2 (PADI2), a post-translational modifying enzyme, as a regulator of ROS-accelerated senescence of osteoblasts via RNA-sequencing and further functional validations. PADI2 downregulation by treatment with H2O2 or its siRNA promoted cellular senescence and suppressed osteoblast differentiation. CCL2, 5, and 7 known as the elements of the senescence-associated secretory phenotype (SASP) which is a secretome including proinflammatory cytokines and chemokines emitted by senescent cells and a representative feature of senescence, were upregulated by H2O2 treatment or Padi2 knockdown. Furthermore, blocking these SASP factors with neutralizing antibodies or siRNAs alleviated the senescence and dysfunction of osteoblasts induced by H2O2 treatment or Padi2 knockdown. The elevated production of these SASP factors was mediated by the activation of NFκB signaling pathway. The inhibition of NFκB using the pharmacological inhibitor or siRNA effectively relieved H2O2 treatment- or Padi2 knockdown-induced senescence and osteoblast dysfunction. Together, our study for the first time uncover the role of PADI2 in ROS-accelerated cellular senescence of osteoblasts and provide new mechanistic and therapeutic insights into excessive ROS-promoted cellular senescence and aging-related bone diseases.
Highlights
Aging is a high risk factor for osteoporosis, which is characterized predominantly by declines in bone mass and strength
To systematically characterize the biological processes associated in H 2O2-induced senescence of osteoblasts according to the H2O2 treatment periods (Days 1 and 4), we performed Gene Ontology (GO) analysis of differentially expressed genes (DEGs) on days 1 and 4 using the DAVID functional annotation online tool (Fig. 1B and Supplementary files 3, 4, 5 and 6)
Gene Set Enrichment Analysis (GSEA) showed that one gene set (Cell Signaling by Wnt) among upregulated gene sets on Day1 cont group was significant at FDR < 25%, suggesting that gene expression profile of H 2O2-treated group may be negatively associated with this gene set (Supplementary Fig. S2)
Summary
Aging is a high risk factor for osteoporosis, which is characterized predominantly by declines in bone mass and strength. The histomorphometric analysis of bone biopsies obtained from the elderly showed that age-related bone loss is caused by impaired bone formation in comparison with bone resorption, indicating age-related osteoblast dysfunction as the main cause of age-related bone loss [2]. This osteoblast dysfunction can be resulted from extrinsic mechanism mediated by age-related changes in bone microenvironment including levels of hormones and growth factors, and/or intrinsic mechanisms caused by cellular senescence of osteoblasts [3]. It is important to know the mechanism by which oxidative stress caused by the ROS accumulation leads to dysfunctions of osteoblasts during aging in order to refine therapeutic approaches to age-related bone loss
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