Age-Related Decline in Expression of Molecular Chaperones Induces Endoplasmic Reticulum Stress and Chondrocyte Apoptosis in Articular Cartilage
Li Tan1, Thomas C Register2, Raghunatha R Yammani1,*
1Section of Molecular Medicine, Department of Internal Medicine, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA 2Departments of Pathology and Comparative Medicine, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA
Aging is a major risk factor for the development of osteoarthritis (OA). One hallmark of aging is loss of proteostasis resulting in increased cellular stress and cell death. However, its effect on the development of OA is not clear. Here, using knee articular cartilage tissue from young and old cynomolgus monkeys (Macaca fascicularis), we demonstrate that with aging there is loss of molecular chaperone expression resulting in endoplasmic reticulum (ER) stress and cell death. Chondrocytes from aged articular cartilage showed decreased expression of molecular chaperones, including protein disulfide isomerase, calnexin, and Ero1-like protein alpha, and increased immunohistochemical staining for ER stress markers (phosphorylated IRE1 alpha, spliced X-box binding protein-1, activating transcription factor 4 and C/EBP homologous protein), and apoptotic markers [cleaved caspase 3 and cleaved poly(ADP-ribose) polymerase], suggesting that decreased expression of molecular chaperone during aging induces ER stress and chondrocyte apoptosis in monkey articular cartilage. Apoptosis induced by aging-associated ER stress was further confirmed by TUNEL staining. Aged monkey cartilage also showed increased expression of nuclear protein 1 (Nupr1) and tribbles related protein-3 (TRB3), known regulators of apoptosis and cell survival pathways. Treatment of cultured monkey chondrocytes with a small molecule chemical chaperone, 4-phenylbutyric acid (PBA, a general ER stress inhibitor) or PERK Inhibitor I (an ER stress inhibitor specifically targeting the PERK branch of the unfolded protein response pathway), decreased the expression of ER stress and apoptotic markers and reduced the expression of Nupr1 and TRB3. Consistent with the above finding, knockdown of calnexin expression induces ER stress and apoptotic markers in normal human chondrocytes in vitro. Taken together, our study clearly demonstrates that aging promotes loss of proteostasis and induces ER stress and chondrocyte apoptosis in articular cartilage. Thus, restoring proteostasis using chemical/molecular chaperone or ER stress inhibitor could be a therapeutic option to treat aged-linked OA.
Tan Li,Register Thomas C,Yammani Raghunatha R. Age-Related Decline in Expression of Molecular Chaperones Induces Endoplasmic Reticulum Stress and Chondrocyte Apoptosis in Articular Cartilage[J]. Aging and disease,
2020, 11(5): 1091-1102.
Figure 1. Aging induces ER stress in monkey knee cartilages. Articular cartilage sections of young (6-11 yrs, n=3) or old (20-34 yrs, n=3) monkeys were analyzed immunohistochemically for ER stress markers including BIP, P-IRE1α, XBP1, ATF4, and CHOP. Images on left panels are low magnification (Scale bars: 100 µm). The areas inside the small rectangles were magnified and displayed in right panels (Scale bars: 20 µm).
Figure 2. Aging promotes chondrocyte apoptosis in monkey knee cartilages. (A) Articular cartilage sections of young (6-11 yrs, n=3) or old (20-34 yrs, n=3) monkeys were analyzed immunohistochemically for apoptosis markers CC3 and C-PARP (A). Images on left panels are of low magnification (Scale bars: 100 µm). The areas inside the small rectangles were magnified and displayed in right panels (Scale bars: 20 µm). (B) Monkey knee cartilage sections were evaluated by TUNEL staining. Scale bar, 50 µm.
Figure 3. Aging induces increased expression of Nupr1 and TRB3 in monkey knee cartilages. Articular cartilage sections of young (6-11 yrs, n=3) or old (20-34 yrs, n=3) monkeys were analyzed immunohistochemically for Nupr1 and TRB3. Images on left panels are of low magnification (Scale bars: 100 µm). The areas inside the small rectangles were magnified and displayed in right panels (Scale bars: 20 µm). For the negative control, the cartilage sections were stained with only secondary antibody by replacing a primary antibody with the blocking serum.
Figure 4. Small chemical chaperone PBA and PERK inhibitor alleviate ER stress and decrease chondrocytes apoptosis. Chondrocytes of young (6-11 yrs, n=3) or old (20-34 yrs, n=3) monkeys were treated with PBA or PERKi overnight and probed for P-IRE1α, XBP1, ATF4, CHOP, Nupr1, TRB3, cytochrome c, and CC3 antibodies, respectively (A). Blots were stripped and reprobed with GAPDH as a loading control. Densitometric analysis for protein levels of P-IRE1α (B), XBP1 (C), ATF4 (D), CHOP (E), Nupr1 (F), TRB3 (G), cytochrome c (H) and CC3 (I) were performed on blots obtained in independent experiments similar to the one shown in panel A. Data were shown as mean ± standard deviation of the mean. CytoC, cytochrome c.
Figure 5. Aging reduces the expression of molecular chaperones required for proper protein folding. Chondrocytes of young (6-11 yrs, n=3) or old (20-34 yrs, n=3) monkeys were treated with PBA or PERKi overnight and probed for PDI, calnexin and Ero1-Lα antibodies, respectively (A). Blots were stripped and reprobed with GAPDH as a loading control. Densitometric analysis for protein levels of PDI (B), calnexin (C) and Ero1-Lα (D) were performed on blots obtained in independent experiments similar to the one shown in panel A. PDI concentrations in the young or old samples were further quantitated using a PDI ELISA kit (E). Data were shown as mean ± standard deviation of the mean.
Figure 6. knockdown of calnexin expression induces ER stress and chondrocyte apoptosis in normal human chondrocytes. Human chondrocytes were transfected with control siRNA or siRNA specific for calnexin, and then were cultured in serum-free media for two days and cell lysates were probed for calnexin, P-IRE1α, XBP1, ATF4, CHOP, Nupr1, TRB3, and CC3 antibodies, respectively (A). Blots were stripped and reprobed with GAPDH as a loading control. Densitometry analysis for protein levels of calnexin (B) P-IRE1α (C), XBP1 (D), ATF4 (E), CHOP (F), Nupr1 (G), TRB3 (H), and CC3 (I) were performed on blots obtained in three independent experiments similar to the one shown in panel A. Data were shown as mean ± standard deviation of the mean. CANX, calnexin.
Figure 7. Model for aging inducing ER stress and chondrocyte apoptosis in monkey knee joints. Loss of molecular chaperones, (PDI, calnexin, Ero1-Lα) in aged monkey knee cartilage/chondrocytes induces ER stress. Under ER stress, BIP is dissociated from IRE1 to elicit activation of IRE1 via autophosphorylation (P-IRE1) which further induces the expression of spliced XBP1. Similarly, PERK is autophosphorylated (P-PERK) following the release of BIP thus inducing the ATF4 expression. Both XBP1 and ATF4 induce CHOP expression that further induces the expression of Nupr1 to trigger CC3-mediated chondrocyte apoptosis. Both CHOP and Nupr1 could induce TRB3 expression to inhibit chondrocyte survival. Treatment of aged monkey cartilage/chondrocytes with PBA or PERKi alleviates ER stress and reduces chondrocyte apoptosis. →, one-step stimulation; --->, putative one-step stimulation; → →, multistep stimulations; ---> --->, putative multistep stimulation; ?, one-step inhibition; ? ? , multistep inhibitions.
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