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eIF2α–ATF4 Pathway Activated by a Change in the Calcium Environment Participates in BCP-Mediated BTE

eIF2α–ATF4 Pathway Activated by a Change in the Calcium Environment Participates in BCP-Mediated Bone Regeneration



Abstract

Biphasic calcium phosphate (BCP) ceramic is a classic bone void filler and a common basis of new materials for bone defect repair. However, the specific mechanism of BCP in osteogenesis has not been fully elucidated. Endoplasmic reticulum stress (ERs) and the subsequent PERK–eIF2α–ATF4 pathway can be activated by various factors, including trauma and intracellular calcium changes, and therefore worth exploring as a potential mechanism in BCP-mediated bone repair. Herein, a rat lateral femoral epicondyle defect model in vivo and a simulated BCP-mediated calcium environment in vitro were constructed for the analysis of BCP-related osteogenesis and the activation of ERs and the eIF2α–ATF4 pathway. An inhibitor of eIF2α dephosphorylation (salubrinal) was also used to explore the effect of the eIF2α–ATF4 pathway on BCP-mediated bone regeneration.



The hypothesis that the eIF2α–ATF4 pathway participates in BCP-mediated bone regeneration. The implanted BCP regulates the local calcium concentration and alters ER homeostasis. Then, the eIF2α–ATF4 pathway is activated and contributes to bone formation.


The results showed that the ERs and eIF2α–ATF4 pathway activation were observed during 4 weeks of bone repair, with a rapid but brief increase immediately after artificial defect surgery and a re-increase after 4 weeks with the resorption of BCP materials. Mild ERs and the activated eIF2α induced by the calcium changes mediated by BCP regulated the expression of osteogenic-related proteins and had an important role during the defect repair. In conclusion, the eIF2α–ATF4 pathway activated by a change in the calcium environment participates in BCP-mediated bone regeneration. eIF2α–ATF4 and ERs could provide new directions for further studies on new materials in bone tissue engineering.


(A) Histological overview showing bone regeneration and material resorption within defects 4 weeks postoperatively by H&E staining. The residual BCP ceramics and the new bone are indicated by blue and yellow arrows, respectively. (B) New bone formation (bone regeneration/defect area).


Biphasic calcium phosphate (BCP) ceramics are a classic material for bone defect repair that are mixed with hydroxyapatite (HAP) and tricalcium phosphate (TCP). Due to its good osteoconductivity, solubility, and high osteoinductivity, BCP has become a common basis for a variety of new materials in bone tissue engineering.Currently, the biological effects of bioceramics are believed to be closely related to their physicochemical properties.The physical structure, roughness, and surface charge of these materials affect cell adhesion and protein adsorption as well as cell recruitment, proliferation, and differentiation. When BCP comes into contact with body fluids, the dissolution–precipitation process will occur. At first, the initial ionic concentration in solution is below the equilibrium concentration. This undersaturation provokes the dissolution until saturation is achieved. Then, the increase in ions leads to saturation and the subsequent precipitation, which causes a decrease of Ca2+ in solution.


BiP and eIF2α-P expression in peridefect tissues 18 h, 3 days, and 4 weeks postsurgery. Light microscopic images taken in the surrounding area of the defect showing BiP (A) and eIF2α-P (C) expression. Red arrows indicate positive cells. The mean density was analyzed by Image-Pro Plus 6.0. (B) BiP and (D) eIF2α-p.


Thus, bioceramics not only partially dissolve and release the calcium and phosphorus ions but also adsorb the ions in the surrounding environment to redeposit on the ceramic surface, regulating the local ionic concentration. Their effect on the extracellular ionic environment is considered to be the core mechanism of their biological activity;however, the specific influence of the ionic changes has not been fully elucidated.


(A) Calcium ion release from BCP into calcium-free SPS at 3, 6, 12, and 24 h. (B) Calcium concentration of α-MEM after 24 h calcium redeposition (* indicates a statistically significant difference: *p < 0.05, **p < 0.005).


The endoplasmic reticulum (ER) is a specialized organelle that orchestrates the synthesis, folding, and transportation of proteins in eukaryotic cells. ER stress (ERs) refers to the stress that responds to the accumulation of unfolded or misfolded proteins in the ER lumen when ER homeostasis is altered due to a wide range of cellular environments and events, including the increased protein synthesis, impaired ubiquitination and proteasomal degradation, deficient autophagy, calcium changes, redox homeostasis, inflammatory challenges, and hypoxia.



Intracellular Ca2+ measurements detected by flow cytometry with the Fluo-3AM probe (* indicates a statistically significant difference: *p < 0.05, **p < 0.005, **p < 0.0005).


The three transmembrane receptor proteins in the ER lumen, protein kinase R-like ER kinase (PERK), inositol-requiring enzyme 1α (IRE1α), and activating transcription factor 6 (ATF6), are in an inactive form due to the binding of glucose-regulated protein 78/binding immunoglobulin (GRP78/BiP). When ERs occurs, GRP78/BiP is bound to the misfolded/unfolded proteins that accumulate in the ER and then dissociate from the three receptors and trigger the following unfolded protein response (UPR), and the IRE1a, ATF6α, and PERK signaling pathways are activated.


Effect of the BCP-mediated Ca2+ environment on ERs and the eIF2α–ATF4 pathway in vitro. The expression of BiP, eIF2α-P, and total eIF2α (A,B) and the quantitative analysis (C,D) showed the influence of calcium on ERs and the eIF2α–ATF4 pathway. The CCK-8 survival assessment (E, F) at day 1, day 3, day 7, and day 14 showed that both BCP-mediated calcium environments had no adverse effect on cell survival (* indicates a statistically significant difference: *p < 0.05, **p < 0.005, ***p < 0.0005).


The UPR induced by mild ERs in an adaptive range helps to restore ER homeostasis and maintains cell survival, while the severe ERs beyond the adaptive range and persistent stress lead to cell apoptosis/death. ERs and the UPR are not only closely related to various injuries and diseases but also participate in cell survival, cell function, and normal organismal physiology.Studies have shown that the PERK–eIF2α–ATF4 pathway plays an important role in the growth and repair of bone tissue.


Salubrinal treatment significantly inhibited the dephosphorylation of eIF2α in the ERs environment simulated by Tm. (A) The expression of BiP, eIF2α-P, total eIF2α, and ATF4 in BMSCs treated with salubrinal in the ERs environment simulated by Tm for 24 h. (B) Quantitative analysis of Western blot (* indicates a statistically significant difference: *p < 0.05, **p < 0.005,***p < 0.0005).


When the PERK pathway is activated, the phosphorylation of downstream eukaryotic translation initiation factor 2α (eIF2α) initiates the nuclear transportation of the activating transcription factor 4 (ATF4), which directly regulates the transcription and expression of osteocalcin (OCN) and bone sialoprotein (BSP).



Expression of BiP, eIF2α-P, total eIF2α, BSP, and OCN in MC3T3-E1 cells treated in a 2.5 mM Ca2+ environment with salubrinal. (A–C) Western blot and quantitative analysis. (D) Alizarin Red staining (* indicates a statistically significant difference: *p < 0.05, **p < 0.005, ***p < 0.0005).


Recently, PERK has been demonstrated to be important for the ALP activity and the expression of related osteogenic genes in human periodontal ligament stem cells. It has also been confirmed that the mild ERs and the PERK–eIF2α–ATF4 pathway contribute to osteoblast differentiation in the process of osteogenesis induced by orthodontic force, material surface morphology, and carbon point materials. Therefore, ERs and the eIF2α–ATF4 pathway could be one of the potential directions to study the specific mechanism by which BCP promotes bone defect repair.



Expression of BiP, eIF2α-P, total eIF2α, Runx2, and OSX in BMSCs treated in a BCP-mediated Ca2+ decreased environment with salubrinal for 24 h and 3 days. (A–C) Western blot and quantitative analysis. (D) ALP staining (* indicates a statistically significant difference: *p < 0.05, **p < 0.005, ***p < 0.0005).


Histological and immunohistochemical analyses of the newly formed bone were performed 4 weeks after BCP implantation and salubrinal injection. (A) Light microscopic images taken in the defect area. The residual BCP ceramics and the new bone are indicated by blue and yellow arrows, respectively. Red arrows indicate positive cells. (B) New bone formation analysis. (C, D) The mean densities of eIF2α-P and BSP were analyzed by Image-Pro Plus 6.0 (* indicates a statistically significant difference: *p < 0.05 **p < 0.005 **p < 0.0005).

  1. eIF2α–ATF4 Pathway Activated by a Change in the Calcium Environment Participates in BCP-Mediated Bone Regeneration Zichao Xiang, Qionghui Wu, Yu Wang, Peng Wang, Yingyou He, and Jihua Li ACS Biomaterials Science & Engineering Article ASAP DOI: 10.1021/acsbiomaterials.0c01802