Abstract
The NAD+-dependent deacetylase sirtuin-1 (SIRT1) has emerged as an important regulator of chondrogenesis and cartilage homeostasis, processes that are important for physiological skeletal growth and that are dysregulated in osteoarthritis. However, the functional role and underlying mechanism by which SIRT1 regulates chondrogenesis remain unclear. Using cultured rat metatarsal bones and chondrocytes isolated from rat metatarsal rudiments, here we studied the effects of the SIRT1 inhibitor EX527 or of SIRT1 siRNA on chondrocyte proliferation, hypertrophy, and apoptosis. We show that EX527 or SIRT1 siRNA inhibits chondrocyte proliferation and hypertrophy and induces apoptosis. We also observed that SIRT1 inhibition mainly induces the PERK-eIF-2α-CHOP axis of the endoplasmic reticulum (ER) stress response in growth-plate chondrocytes. Of note, EX527- or SIRT1 siRNA-mediated inhibition of metatarsal growth and growth-plate chondrogenesis were partly neutralized by phenylbutyric acid, a chemical chaperone that attenuates ER stress. Moreover, EX527-mediated impairment of chondrocyte function (i.e. of chondrocyte proliferation, hypertrophy, and apoptosis) was partly reversed in CHOP-/- cells. We also present evidence that SIRT1 physically interacts with and deacetylates PERK. Collectively, our findings indicate that SIRT1 deacetylates PERK and attenuates the PERK-eIF-2α-CHOP axis of the unfolded protein response pathway and thereby promotes growth-plate chondrogenesis and longitudinal bone growth.
Highlights
The NAD؉-dependent deacetylase sirtuin-1 (SIRT1) has emerged as an important regulator of chondrogenesis and cartilage homeostasis, processes that are important for physiological skeletal growth and that are dysregulated in osteoarthritis
Western blotting of lysates from cultured chondrocytes treated with EX527 confirmed that graded concentrations of EX527 caused a dose-dependent inhibition of SIRT1 expression, with a lowest inhibitory concentration of 30 M (Fig. S2A)
We found that there were no significant changes in the expression of SIRT2 in metatarsals (Fig. S1C) or chondrocytes (Fig. S2A) treated with 30 M EX527, indicating that 30 M EX527 could act on SIRT1 without hitting other sirtuin
Summary
To determine whether SIRT1 regulates growth-plate chondrogenesis, we cultured fetal rat metatarsals for 3 days in serum-free medium in the presence of EX527 (0 –100 M), a specific SIRT1 inhibitor. To confirm that inhibition of SIRT1 induced ER stress in chondrocytes, we analyzed the expression of ER stress indicators in metatarsal growth plate by Western blotting and RT-qPCR. PBA effectively attenuated ER stress induced by EX527 in chondrocytes, as assessed by expression of p-PERK, p-eIf-2␣, ATF4, and CHOP by Western blotting (Fig. 1f and Fig. S1E) and RT-qPCR (Fig. 1g), respectively, and the addition of PBA partially neutralized the inhibitory effect of EX527 on metatarsal longitudinal growth (Fig. 1, h and i). Our finding indicated that PBA ameliorated ER stress induced by SIRT1 siRNA in chondrocytes as detected by Western blotting and RT-qPCR (Fig. 4, a and b, and Fig. S4A), partially neutralizing the inhibitory effects on chondrocyte proliferation (Fig. 4, c and d, and Fig. S4B) and hypertrophy (Fig. 4g and Fig. S4D) and diminishing the pro-apoptotic effect of SIRT1 siRNA (Fig. 4, e and f, and Fig. S4C). One or more lysine residues and that this acetylation is regulated, at least in part, by SIRT1 to maintain ER homeostasis in chondrocytes
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