Adiponectin regulates vascular endothelial growth factor-C expression in macrophages via Syk-ERK pathway.

Adiponectin is exclusively expressed in adipose tissues and exhibits protective effects against cardiovascular and metabolic diseases. It enhances AMP-activated kinase (AMPK) and peroxisome proliferator-activated receptor α (PPARα) signaling in the liver and skeletal muscles, however, its signaling pathways in macrophages remain to be elucidated. Here, we show that adiponectin upregulated the expression of vascular endothelial growth factor (VEGF)-C, and induced phosphorylation of extracellular signal-regulated kinase (ERK) in macrophages. Inhibition of Syk abrogated adiponectin-induced VEGF-C expression and ERK phosphorylation. Furthermore, inhibition of ERK blocked the induction of VEGF-C gene. Inhibition of Syk, but not that of ERK, abrogated adiponectin-induced expression of cyclooxygenase (COX)-2, tissue inhibitor of metalloproteinase (TIMP)-1, and interleukin (IL)-6. These results indicate that adiponectin regulates VEGF-C expression via Syk-ERK pathway in macrophages

Effects of Src inhibitor on adiponectin-induced changes in peripheral blood monocyte-derived macrophages (PBDMs).

<p>PBDMs were preincubated with 10 µM PP1 for 30 min and then incubated with adiponectin for 6 hours (A–D). The mRNA expression levels of COX-2 (A), TIMP-1 (B), IL-6 (C), and VEGF-C (D) were quantified by real-time PCR. Values are normalized to the level of GAPDH mRNA and expressed as mean ± SEM (n = 3). ***P<0.001. N.S not significant.</p

Effects of Syk inhibitors on the adiponectin-induced changes in peripheral blood monocyte-derived macrophages (PBDMs).

<p>PBDMs were preincubated with 10 µM BAY 61-3606 (BAY) (A, B, C, D), or 100 µM piceatannol (E, F, G, H) for 30 min followed by treatment with 10 µg/ml of adiponectin for 6 hours. The mRNA expression levels of COX-2 (A, E), TIMP-1 (B, F), IL-6 (C, G), and VEGF-C (D, H) were quantified by real-time PCR. Values are normalized to the level of GAPDH mRNA and expressed as mean ± SEM (n = 3). ***P<0.001.</p

Effects of adiponectin on mRNA expression levels of various genes in peripheral blood monocyte-derived macrophages (PBDMs).

<p>After incubation for 7 days, mature human PBDMs were incubated for 6 hours with 10 µg/ml of adiponectin protein. The mRNA expression levels of COX-2 (A), TIMP-1 (B), IL-6 (C), and VEGF-C (D) were quantified by real-time PCR. Values are normalized to the level of GAPDH mRNA and expressed as mean ± SEM (n = 3). **P<0.01, ***P<0.001.</p

Adiponectin-induced phosphorylation of ERK in peripheral blood monocyte-derived macrophages (PBDMs).

<p>PBDMs were incubated for 20 min with 10 µg/ml of adiponectin protein. Cell lysates were subjected to SDS-PAGE followed by western blotting with anti-phosphorylated ERK1/2, and anti-ERK1/2 antibodies (A). PBDMs were preincubated with 10 µM BAY 61-3606 (BAY) for 30 min and then treated for 20 min with 10 µg/ml of adiponectin protein. Cell lysates were subjected to SDS-PAGE followed by western blotting with anti-phosphorylated ERK1/2, and anti-ERK1/2 antibodies (B). Equal protein loading and transfer were confirmed with Ponceau staining.</p