Proprotein Convertase Subtilisin/Kexin Type 3 Promotes Adipose Tissue-Driven Macrophage Chemotaxis and Is Increased in Obesity

<div><p>Background</p><p>Matrix metalloproteinase (MMP)-dependent extracellular matrix (ECM) remodeling is a key feature in cardiometabolic syndrome-associated adipogenesis and atherosclerosis. Activation of membrane-tethered (MT) 1-MMP depends on furin (PCSK3). However, the regulation and function of the natural furin-inhibitor serpinB8 and thus furin/MT1-MMP-activity in obesity-related tissue inflammation/remodeling is unknown. Here we aimed to determine the role of serpinB8/furin in obesity-associated chronic inflammation.</p> <p>Methods and Results</p><p>Monocyte → macrophage transformation was characterized by decreases in serpinB8 and increases in furin/MT1-MMP. Rescue of serpinB8 by protein overexpression inhibited furin-dependent pro-MT1-MMP activation in macrophages, supporting its role as a furin-inhibitor. Obese white adipose tissue-facilitated macrophage migration was inhibited by furin- and MMP-inhibition, stressing the importance of the furin-MMP axis in fat tissue inflammation/remodeling. Monocytes from obese patients (body mass index (BMI) >30kg/m²) had higher furin, MT1-MMP, and resistin gene expression compared to normal weight individuals (BMI<25kg/m²) with significant correlations of BMI/furin and furin/MT1-MMP. <i>In vitro</i>, the adipocytokine resistin induced furin and MT1-MMP in mononuclear cells (MNCs), while MCP-1 had no effect.</p> <p>Conclusions</p><p>Acquisition of the inflammatory macrophage phenotype is characterized by an imbalance in serpinB8/furin, leading to MT1-MMP activation, thereby enhancing migration. Increases in MT1-MMP and furin are present in MNCs from obese patients. Dissecting the regulation of furin and its inhibitor serpinB8 should facilitate targeting inflammation/remodeling in cardiometabolic diseases.</p> </div

Subcellular localization and interaction of furin, MT1-MMP and serpinB8.

<p>(<b>A</b>) 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>B</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 (<i>lower panel</i>). (<b>C</b>) 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). (<b>D</b>) 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.</p

Impact of resistin on monocyte furin, MT1-MMP and serpinB8 expression.

<p>(<b>A</b>) 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>B</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.</p

BMI-based gene expression in monocytes.

<p>(<b>A</b>) 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>B</b>) Furin levels correlated with BMI (<i>left</i>) and MT1-MMP (<i>right</i>) gene expression.</p

Obese white adipose tissue (WAT) gene expression and impact on macrophage migration.

<p>(<b>A</b>) WAT derived from wild-type (wt) and obese ob<i>/</i>ob mice was subjected to qRT-PCR. Increased gene expression of CD68, MCP-1, furin, MT1-MMP, and PCSK5 was found in ob<i>/</i>ob mice (*p<0.05; **p<0.01 vs. wt). (<b>B</b>) Supernatants from WAT cultures of ob<i>/</i>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). (<b>C</b>) 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.</p

Regulation of furin and serpinB8 during monocyte transformation.

<p>(<b>A</b>) Monocyte transformation (THP-1 cells (<i>upper left</i>), primary monocytes (<i>upper right</i>)) to macrophages (THP-1/ϕ) was evident by vimentin expression. Furin was increased and serpinB8 was decreased in macrophages (<i>upper panel</i>). 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; <i>lower panel</i>). (<b>B</b>) QRT-PCR demonstrated significant upregulation of furin and MT1-MMP accompanying vimentin in THP-1/ϕ, whereas decreases in serpinB8 were non-significant. (<b>C</b>) 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.</p

ERα and lipolysis.

<p>A: Ex-vivo lipolysis assay in murine gonadal-AT explants from wild-type (WT) and estrogen receptor alpha knock out mice (KO) expressed as percent of WT FFA-release after stimulation with forskolin. Bonferroni posttest showed a significant difference between WT and KO in females [n = 4−5 mice/group, two-way ANOVA]. <b>B</b>: Analysis of ERα mRNA expression in gonadal-AT from female/male mice before/after weight reduction. Data are presented as <i>x</i>-fold of females (DIO) [n = 9−10 mice/group, two-way ANOVA]. The black/dark grey columns and symbols represent male mice; white/light grey: females. DIO: before weight reduction, –20%: after weight reduction. *p≤0.05 DIO vs. -20% or WT vs. KO.</p

Animal model.

<p>A: Scheme of the feeding protocol to induce body weight changes (DIO =  diet-induced obesity). <b>B</b>: Original BW data of female/male mice throughout the feeding protocol.</p

Lipolytic activity.

<p>A: Ex-vivo lipolysis assay in murine gonadal adipose tissue explants as a marker of fat-tissue specific lipolytic activity, measured as release of FFA after stimulation with forskolin. Shown is the release of FFA in female/male mice before/after weight reduction [n = 7 mice/group, two-way ANOVA] <b>B</b>: Serum concentration of free glycerol in female/male mice before/during weight loss (day 3 of CR) [n = 10 mice/group, two-way ANOVA] ] (factor interaction: p_{sex/weight loss}<0.05). <b>C</b>: Mean RER during day time (6 a.m.–6 p.m.) in females/males measured during weight loss [n = 10 mice/group, unpaired <i>t</i>-test]. <b>D</b>: Analysis of ATGL, HSL, and LPL mRNA expression in gonadal-AT from female/male mice after weight reduction. Data are presented as <i>x</i>-fold expression of females [n = 8−10 mice/group, unpaired <i>t</i>-test, genes were analyzed separately]. Black columns and symbols represent male mice; white: females. DIO: before weight reduction; restriction: during restrictive feeding phase; -20%: after weight reduction, at target weight. *p≤0.05; **p≤0.01 DIO vs. −20% or vs. other sex.</p

Weight maintenance and regain.

<p>A: Stability of body weight of female/male mice during 16 days of adaptive feeding. Shown are the means ±SEM of body weight, measured daily. [n = 10 mice/group] <b>B</b>: During adaptive feeding the amount of food was individually adapted to maintain the target weight over 16 days. Shown are the mean amount of given food ±SEM normalized to the BW of female/male mice [n = 10 mice/group, two-way ANOVA with repeated measures] (factor interaction: p_sex/time<0.001). <b>C</b>: Sex-specific differences during weight regain expressed as percent change of body weight before re-feeding. Shown is the BW-development in female and male mice during 6 weeks ad libitum re-feeding. [n = 10 mice/group, two-way ANOVA with repeated measures] (factor interaction: p_sex/time<0.05). Black symbols represent male mice; white: females. *p≤0.05; **p≤0.01; # p<0.001 vs. other sex.</p