Journal: Cells

Article Title: EF24, a Curcumin Analog, Reverses Interleukin-18-Induced miR-30a or miR-342-Dependent TRAF3IP2 Expression, RECK Suppression, and the Proinflammatory Phenotype of Human Aortic Smooth Muscle Cells

doi: 10.3390/cells13201673

Figure Lengend Snippet: Interleukin-18 (IL-18) upregulates TRAF3IP2 but suppresses RECK expression in primary human aortic smooth muscle cells (ASMC). ( A – C ) IL-18 upregulates TRAF3IP2 expression. ASMCs were grown in complete media, and at 70–80% confluency, made quiescent for 48 h, and then incubated with rhIL-18 (10 ng/mL) for up to 12 h (experimental design in ( A )). TRAF3IP2 mRNA expression was analyzed by RT-qPCR using a TaqMan™ probe ( B ) and its protein levels by Western blotting ( C ), with GAPDH and Tubulin serving as loading controls, respectively. * p < 0.05, ** at least p < 0.01 vs. untreated controls ( n = 4). ( D , E ) Quiescent ASMCs were incubated with IL-18 as in (A) and were analyzed for RECK mRNA expression by RT-qPCR using a TaqMan™ probe ( D ) and protein levels by Western blotting ( E ). * p < 0.05, ** at least p < 0.01 vs. untreated controls ( n = 4). ( F – H ) Specificity of IL-18 on TRAF3IP2 induction ( G ) and RECK suppression ( H ) was verified by incubating with neutralizing IL-18R1 antibody or IL-18BP-Fc chimera for 1 h prior to IL-18 addition for 2 ( G ) or 6 h ( H ), with normal goat IgG or Fc serving as controls. mRNA expressions of TRAF3IP2 and RECK were analyzed by RT-qPCR. ( C , E ) While a representative immunoblot is shown, the intensities of immunoreactive bands from 4 independent experiments were semiquantified by densitometry and are summarized on the right. ( G ) * p < 0.05, ** p < 0.01 vs. untreated controls, † p < 0.01 versus IL-18 (n = 6); ( H ) * p < 0.05, ** p < 0.01 vs. untreated controls, † p < 0.01 versus IL-18 ( n = 4).

Article Snippet: The following antibodies were used: TRAF3IP2 (1:400; NB100–56740, Novus Biologicals, LLC, Centennial, CO, USA), Tubulin (1:1000; #2144, Cell Signaling Technology/CST, Danvers, MA, USA), RECK (1 : 1000; #3433, CST), MMP2 (1 : 500; #ab97779, Abcam, Waltham, MA, USA), MMP9 (1 : 1000; #2270, CST), and GAPDH (1:1000; #NB300-221, Novus Biologicals, LLC).

Techniques: Expressing, Incubation, Quantitative RT-PCR, Western Blot

Journal: Cells

Article Title: EF24, a Curcumin Analog, Reverses Interleukin-18-Induced miR-30a or miR-342-Dependent TRAF3IP2 Expression, RECK Suppression, and the Proinflammatory Phenotype of Human Aortic Smooth Muscle Cells

doi: 10.3390/cells13201673

Figure Lengend Snippet: Targeting TRAF3IP2 or RECK overexpression blunts IL-18-induced ASMC proliferation and migration. ( A – E ) Silencing TRAF3IP2 inhibits IL-18-induced ASMC proliferation ( B ) and migration ( C ). ASMC were transduced with adenovirus-expressing shRNA targeting human TRAF3IP2 (moi 10 for 48 h), made quiescent, and then exposed to IL-18 (10 ng/mL; experimental design in ( A )). ASMC proliferation was assessed after 48 h of IL-18 addition using the CyQUANT Cell proliferation assay ( B ), and migration after 18 h using Boyden chamber assay ( C ). ASMCs migrating to the lower surface of the membrane were counted in 10 different fields and summarized as mean ± SEM. ( B , C ) * p < at least 0.01 vs. Untreated; † p < 0.01 vs. IL-18 or IL-18+GFP (n = 6). Knockdown of TRAF3IP2 was confirmed by RT-qPCR using a TaqMan™ probe ( D ) and Western blotting ( E ). ( D , E ) * p < 0.01 vs. untreated (n = 3). ( F , G ) Dose-dependent effects of Ad.RECK on RECK expression (experimental design in ( F )). Induction of RECK following adenoviral transduction was confirmed by Western blotting with tubulin serving as an internal control ( G ). ( H – K ) Forced expression of RECK inhibits IL-18-stimulated ASMC proliferation and migration. ASMCs were transduced with adenovirus-expressing human RECK cDNA (moi 10 for 24 h), made quiescent, and then treated with IL-18 (experimental design in ( H )) and analyzed for proliferation ( I ) and migration ( J ) as in ( B , C ). ( C , J ) The insets show representative images of Matrigel™ Transwell invasion. Scale bar: 20 μM. ( E , G , K ) While a representative immunoblot is shown, the intensities of immunoreactive bands from three ( E ), four ( G ) and three ( K ) independent experiments were semiquantified by densitometry and are summarized on the right. ( I ) * p < at least 0.01 vs. Untreated; † p < 0.01 vs. IL-18 or IL-18+GFP (n = 6); ( J ) * p < at least 0.01 vs. Untreated; † p < 0.01 vs. IL-18 or IL-18+eGFP (n = 4); ( K ) * p < 0.01 vs. Untreated; † p < 0.05 vs. IL-18 or IL-18+eGFP (n = 3).

Article Snippet: The following antibodies were used: TRAF3IP2 (1:400; NB100–56740, Novus Biologicals, LLC, Centennial, CO, USA), Tubulin (1:1000; #2144, Cell Signaling Technology/CST, Danvers, MA, USA), RECK (1 : 1000; #3433, CST), MMP2 (1 : 500; #ab97779, Abcam, Waltham, MA, USA), MMP9 (1 : 1000; #2270, CST), and GAPDH (1:1000; #NB300-221, Novus Biologicals, LLC).

Techniques: Over Expression, Migration, Transduction, Expressing, shRNA, CyQUANT Assay, Proliferation Assay, Boyden Chamber Assay, Membrane, Knockdown, Quantitative RT-PCR, Western Blot, Control

Journal: Cells

Article Title: EF24, a Curcumin Analog, Reverses Interleukin-18-Induced miR-30a or miR-342-Dependent TRAF3IP2 Expression, RECK Suppression, and the Proinflammatory Phenotype of Human Aortic Smooth Muscle Cells

doi: 10.3390/cells13201673

Figure Lengend Snippet: Schematic showing that EF24, a curcumin analog with better bioavailability and biologic activity, inhibits the proinflammatory IL-18-induced primary human aortic smooth muscle cells’ (ASMCs) proliferation, migration, and proinflammatory phenotype changes. EF24 inhibits the stress-activated kinase-dependent miR-30a and miR-342 inhibition, TRAF3IP2 upregulation and RECK suppression. While miR-30a mimic reverses IL-18-induced TRAF3IP2 upregulation, the miR-342 mimic restores IL-18-mediated RECK suppression, potentially via reduced DNMT1 expression and promoter demethylation (dashed purple box). While IL-18 promotes ASMC migration and proliferation, these effects were reversed by TRAF3IP2 knockdown or the ectopic expression of RECK. Further, while IL-18 induced ASMC migration in part via induction of the gelatinases MMP2 and MMP9, these effects were inhibited by EF24. Moreover, EF24 restores IL-18-mediated suppression in SMC markers and blunts the expression of the proinflammatory phenotype markers. These results suggest that the curcumin analog EF24 reverses IL-18-induced ASMC proliferation and migration and proinflammatory phenotypic changes by targeting TRAF3IP2 and restoring RECK expression. These results suggest the therapeutic potential of EF24 in vascular inflammatory and proliferative diseases, including atherosclerosis.

Article Snippet: The following antibodies were used: TRAF3IP2 (1:400; NB100–56740, Novus Biologicals, LLC, Centennial, CO, USA), Tubulin (1:1000; #2144, Cell Signaling Technology/CST, Danvers, MA, USA), RECK (1 : 1000; #3433, CST), MMP2 (1 : 500; #ab97779, Abcam, Waltham, MA, USA), MMP9 (1 : 1000; #2270, CST), and GAPDH (1:1000; #NB300-221, Novus Biologicals, LLC).

Techniques: Activity Assay, Migration, Inhibition, Expressing, Knockdown