Figure 7. Fully unsaturated, but not partially saturated or fully saturated PAs, induce mineralization, osteogenic differentiation, and ER stress.

(A) Fully saturated PAs are disfavor substrates for LIPIN2. The lysate of HEK293 cells infected with adenovirus containing human LIPIN2 was used as a LIPIN2 recombinant protein. LIPIN2 activity was measured as the conversion of PA to DAG using LC-MS/MS. The fully saturated PAs 18:0/18:0-PA and 16:0/16:0-PA)but not the partially saturated PAs 18:0/18:1-PA and 16:0/18:1-PA or the fully unsaturated PAs 18:1/18:1-PA and 18:2/18:2-PA dose-dependently induced (B–D) mineralization and (E–G) osteogenic differentiation of VSMCs. VSMCs were treated with each PA for 7 days in the presence of 2.0 mM phosphate. (B) Seven days after the treatments, the cells treated with PAs (20 μM) were stained with Alizarin red to identify calcium deposits. (C and D) Calcium was extracted with 0.6 N hydrochloric acid and analyzed using a colorimetric assay. (E–G) Ocn, Opn, and PiT1 were quantified by qPCR. (H–J) Fully saturated PAs such as 18:0/18:0-PA, but not fully unsaturated PAs such as 18:1/18:1-PA, induce ER stress. VSMCs were treated with 10 μM PAs for 6 and 24 hours. Total protein extracts were subjected to immunoblot analysis with ATF4- and CHOP-specific antibodies. Atf4 and Chop mRNA levels were quantified by qPCR. (K) 18:0/18:0-PA induces mineralization of VSMCs through the activation of the ATF4-CHOP axis of the ER stress response. Atf4 and Chop knockdown MOVAS cells were treated with 10 μM 18:0/18:0-PA for 7 days. n = 6; *P < 0.05 (one-way ANOVA).