Table 1.
Quantitative-PCR primer gene identification, sequences and product sizes.
Figure 1.
Activation of CMKLR1 by adipocyte media is chemerin-specific.
Twenty-four hour conditioned media from 3T3-L1 (A, C) or BMSC (B) adipocytes treated with 20 ng mL−1 TNFα or an equivalent volume of the 0.1% BSA/PBS vehicle control were either incubated for 1 hour with 10 µg mL−1 of goat anti-mouse chemerin neutralization antibody or 10 µg mL−1 IgG control antibody (A, B), or separated by size using exclusion column with a 10 kDa molecular weight cutoff (C) prior to analysis by CMKLR1 bioassay. All bars represent the mean ± s.e.m. of 3 samples and are representative of 3 independent experiments. *P<0.05 compared to the goat IgG, 0.1% PBS/BSA control (A, B) or the 0.1% BSA/PBS control (C) and † P<0.05 compared to the within group goat IgG control (A, B) and the respective >10 kDa group (C), two-way ANOVA, followed by Bonferroni’s post hoc test.
Figure 2.
3T3-L1 adipocytes express immunocyte and fibrinolytic associated enzymes.
The mRNA expression of neutrophil elastase (A), mast cell tryptase (B), angiotensin converting enzyme (C), tissue plasminogen activator (tPA) (D), tissue plasminogen activator urokinase (uPA) (E) and cathepsin K (F) were analyzed in 3T3-L1 adipocytes throughout differentiation. All bars represent the mean ± s.e.m. of 3 samples and are representative of 3 independent experiments. * P<0.05 compared to the control (D0, preadipocytes), one-way ANOVA, followed by Tukey’s post hoc test.
Figure 3.
Proteolytic inhibition increases active chemerin concentrations in adipocyte media.
3T3-L1 adipocytes were treated for 24 hours with 20 ng mL−1 TNFα or 0.1% BSA/PBS vehicle control in combination with a 1∶200 dilution of a protease inhibitor cocktail (PIC) or its respective vehicle (1∶200 diluted DMSO) prior to analysis by CMKLR1 bioassay (A, B) or western blot (C, D). All bars represent the mean ± s.e.m. of 3 samples, and are representative of 3 independent experiments. Western blot analysis using an R&D anti-chemerin antibody is representative of 4 samples per group and 3 independent experiments. *P<0.05 compared to the 0.1% BSA/PBS, 1∶200 DMSO vehicle control, two-way ANOVA, followed by Bonferroni’s post hoc test.
Figure 4.
Serine and cysteine protease inhibitors attenuate TNFα-dependent increases in the apparent concentration of active chemerin in adipocyte media.
3T3-L1 or BMSC adipocytes were treated with TNFα or 0.1% BSA/PBS vehicle control in combination with 0–30 µM of the serine protease inhibitor aprotinin or 0–100 µM of the cysteine protease inhibitor E-64 or an equivalent volume of their respective vehicle controls, water or 0.9% NaCl. The effect of these treatments on the apparent concentration of active chemerin in adipocyte media was measured by the CMKLR1 bioassay (A–C). The effect of these treatments on the immunodetectable levels of total chemerin in adipocyte media was measured by western blot and quantified by densitometry (D–F). For densitometry analysis, the dual vehicle treatment (i.e. 0.1% BSA/PBS with H20 or 0.9% NaCl) served as the reference control and was assigned a value of 100%. All bars represent the mean ± s.e.m. of 3 samples and are representative of 3 independent experiments. Western blot using an R&D anti-chemerin antibody is representative of 4 samples per group and 3 independent experiments. * P<0.05 compared to the within group 0.1% BSA/PBS vehicle control, † P<0.05 compared to the TNFα/vehicle control groups, two-way ANOVA, followed by Bonferroni’s post hoc test (A–C).
Figure 5.
Elastase and tryptase are responsible for the TNFα-mediated increase in the apparent concentration of active chemerin in adipocyte media.
The concentrations of elastase, tryptase, and tPA in 24 hour conditioned media from 3T3-L1 and BMSC adipocytes treated with 20 ng mL−1 of TNFα or equivalent volume of 0.1% BSA/PBS vehicle control were measured by western blot analysis (A). The apparent concentration of active chemerin in 24 h conditioned media from 3T3-L1 adipocytes treated with 20 ng mL−1 of TNFα (B) or equivalent volume of 0.1% BSA/PBS (B, Inset) together with neutralizing antibodies for elastase and tryptase (alone or in combination) or IgG control was measured using the CMKLR1 bioassay. All bars represent the mean ± s.e.m. of 3 samples, and are representative of 2 independent experiments. Western blots are representative of 4 samples per group and 3 independent experiments. * P<0.05 compared to the TNFα/IgG or the 0.1% BSA/PBS/IgG (Inset) treated cells, † P<0.05 compared to the TNFα+anti-elastase or anti-tryptase treated cells, two-way ANOVA, followed by Bonferroni’s post hoc test.
Figure 6.
Bestatin heightens the apparent adipocyte media concentration of active chemerin.
CMKLR1 bioassay and western blot analysis were used to identify the effect of bestatin, an inhibitor of aminopeptidases, alone and in combination with TNFα or a vehicle control on the apparent (A) and total (B) media chemerin concentration of 3T3-L1 adipocytes. All bars represent the mean ± s.e.m. of 3 samples and are representative of 3 independent experiments. Western blot using an R&D anti-chemerin antibody is representative of 4 samples per group and 3 independent experiments. † P<0.05 compared to the TNFα/vehicle control, two-way ANOVA followed by Bonferroni’s post hoc test (A).
Figure 7.
Working model of adipocyte-derived proteolytic control of active chemerin under basal conditions and following treatment with TNFα.
Our findings together support a model of adipocyte-derived proteolytic control of chemerin activity. Under basal conditions the activity of adipocyte-secreted chemerin (1) at CMKLR1 is determined by a precise balance between activation by serine and cysteine protease (2) and deactivation by aminopeptidases (3). Following treatment with TNFα, elevated secretion of chemerin (4) and production of elastase and tryptase (5) alter this balance resulting in increased concentration of a chemerin product(s) with high activity towards CMKLR1 (6).