Figure 1.
Regulation of furin and serpinB8 during monocyte transformation.
(A) Monocyte transformation (THP-1 cells (upper left), primary monocytes (upper right)) to macrophages (THP-1/ϕ) was evident by vimentin expression. Furin was increased and serpinB8 was decreased in macrophages (upper panel). Furin was detected in its pro- and active form (actin-reblotting for protein loading). Immunoblotting of supernatants demonstrated that furin was time-dependently shed from THP-1/ϕ, whereas serpinB8 was primarily released from monocytic cells (both at 24h; lower panel). (B) QRT-PCR demonstrated significant upregulation of furin and MT1-MMP accompanying vimentin in THP-1/ϕ, whereas decreases in serpinB8 were non-significant. (C) Cell-transformation increased furin-like PCSK activity (#p<0.05 THP-1/ϕ vs. THP-1 cells), which was concentration-dependently inhibited by dec-CMK (CMK, 50 and 100 µmol/L; 12h), or transfection of THP-1/ϕ with serpinB8 protein (5 µg/mL; 12h). TM = transfection medium. *p<0.05 vs. THP-1/ϕ. n=3.
Figure 2.
Subcellular localization and interaction of furin, MT1-MMP and serpinB8.
(A) Subcellular localization of furin, serpinB8 and MT1-MMP was compared in THP-1 and THP-1/ϕ using cell fractionation. SerpinB8 was mostly found in THP-1 cells in the cytosolic fraction (F1). Furin and MT1-MMP were highly expressed in THP-1/ϕ in the organelle fraction (F2), and to a lesser extent in the membrane fraction (F3). (B) Dec-CMK (CMK, 50 µmol/L; 12h) or BFA (10 µg/mL; 30 min) inhibited pro-MT1-MMP activation in THP-1/ϕ, evident by increases in its 65 kDa pro-form (upper panel). THP-1/ϕ were transfected with serpinB8 (5 µg/mL; 12h) or treated with transfection medium (TM) alone, and further processed for immunoblotting. SerpinB8 inhibited pro-MT1-MMP activation, evident by increases in the pro-form of MT1-MMP (lower panel). (C) Monocyte (THP-1)/macrophage (THP-1/ϕ) transformation was accompanied by increases of TIMP-1 and TIMP-2, whereas TIMP-3 was downregulated (actin-reblotting for protein loading). (D) Macrophage chemotaxis towards MCP-1 (10 ng/mL) was inhibited by the furin-like PCSK inhibitor dec-CMK (CMK, 50 µmol/L), serpinB8 (5 µg/mL), and the MMP-inhibitors GM6001 (50 µmol/L), TIMP-2 and TIMP-3 (both 200 ng/mL). TIMP-1 (200 ng/mL) and transfection medium (TM) had no effect (*p<0.05 vs. controls, co.; #p<0.05 vs. MCP-1 alone). n=3.
Figure 3.
Obese white adipose tissue (WAT) gene expression and impact on macrophage migration.
(A) WAT derived from wild-type (wt) and obese ob/ob mice was subjected to qRT-PCR. Increased gene expression of CD68, MCP-1, furin, MT1-MMP, and PCSK5 was found in ob/ob mice (*p<0.05; **p<0.01 vs. wt). (B) Supernatants from WAT cultures of ob/ob mice increased THP-1/ϕ migration (##p<0.05 vs. wt), comparable to MCP-1 (*p<0.05 vs. control, co.; **p<0.01 vs. 50 ng/mL MCP-1). (C) Supernatant-facilitated migration was inhibited by dec-CMK (CMK, 50 µmol/L; 12h) or the MMP-inhibitor GM6001 (50 µmol/L; 12h) (both #p<0.05 vs. controls, co.). n=3.
Figure 4.
BMI-based gene expression in monocytes.
(A) Monocytes isolated from patients were subjected to qRT-PCR. Furin, MT1-MMP, and resistin gene expression was higher in patients with a BMI>30 kg/m2 compared to patients with a BMI<25 kg/m2, whereas higher serpinB8 transcript levels did not reach statistical significance. (B) Furin levels correlated with BMI (left) and MT1-MMP (right) gene expression.
Figure 5.
Impact of resistin on monocyte furin, MT1-MMP and serpinB8 expression.
(A) THP-1 monocytes were stimulated with resistin or MCP-1 and subjected to qRT-PCR. Resistin increased furin and MT1-MMP, whereas MCP-1 had no effect (*p<0.05 vs. control, co.). (B) Immunoblotting revealed that resistin (100 ng/mL) increased furin and MT1-MMP. In contrast, protein levels of serpinB8 were not affected (actin-reblotting for protein loading). n=3.