Melanocortins induce interleukin 6 gene expression and secretion through melanocortin receptors 2 and 5 in 3T3-L1 adipocytes

Abstract

Interleukin 6 (IL6) is a pleiotropic cytokine that not only affects the immune system, but also plays an active role in many physiological events in various organs. Notably, 35% of systemic IL6 originates from adipose tissues under noninflammatory conditions. Here, we describe a previously unknown function of melanocortins in regulating Il6 gene expression and production in 3T3-L1 adipocytes through membrane receptors which are called melanocortin receptors (MCRs). Of the five MCRs that have been cloned, MC2R and MC5R are expressed during adipocyte differentiation. alpha-Melanocyte-stimulating hormone (alpha-MSH) or ACTH treatment of 3T3-L1 adipocytes induces Il6 gene expression and production in a time- and concentration-dependent manner via various signaling pathways including the protein kinase A, p38 mitogen-activated protein kinase, cJun N-terminal kinase, and IkappaB kinase pathways. Specific inhibition of MC2R and MC5R expression with short interfering Mc2r and Mc5r RNAs significantly attenuated the alpha-MSH-induced increase of intracellular cAMP and both the level of Il6 mRNA and secretion of IL6 in 3T3-L1 adipocytes. Finally, when injected into mouse tail vein, alpha-MSH dramatically increased the Il6 transcript levels in epididymal fat pads. These results suggest that alpha-MSH in addition to ACTH may function as a regulator of inflammation by regulating cytokine production.

Figure 1

Functional expression of melanocortin receptor subtypes in mouse tissues and 3T3-L1 adipocytes. (A) Real-time quantitative RT-PCR analysis of melanocortin receptor subtypes in mouse metabolic tissues and 3T3-L1 adipocytes. Detection of mRNA corresponding to all five Mcrs in liver, muscle, subcutaneous fat, retroperitoneal fat, epididymal fat, and 3T3-L1 adipocytes. (B) Real-time quantitative RT-PCR analysis of Mc2r and Mc5r during adipogenesis. The cDNAs derived from the 3T3-L1 cells which had undergone differentiation for various time periods were amplified using specific primers. For comparison, five differentiation-specific genes were also analyzed: PPARγ, C/EBPa, Add1, aP2, and Pref1. The relative expression of the transcripts was normalized to acidic ribosomal phosphoprotein P0 (Arbp) mRNA levels, and values represent the mean±s.d. (C) Concentration-dependent effects of melanocortins on cAMP generation. 3T3-L1 adipocytes were treated with various concentrations of α-MSH, β-MSH, γ-MSH, or ACTH for 20 min, and then cAMP production was measured. The data present the means±s.d. of triplicate samples. Each experiment was performed independently at least three times.

Figure 2

Melanocortins increase Il6 gene expression and secretion. (A) Differentiated 3T3-L1 adipocytes were incubated with α-MSH (300 nM) for 0, 1, 3, and 8 h, and various adipocytokines including adiponectin, Il6, Rbp4, resistin, Tnfa, Pref1, Igf1, and Igf2 mRNA levels were analyzed with real-time quantitative RT-PCR using Gapdh as a reference. Values represent the mean±s.d. of triplicate samples. (B) Differentiated 3T3-L1 adipocytes were incubated with various concentrations of ACTH. After 1 h, the cells were harvested, and various adipocytokines including adiponectin, Il6, Pai1, Rbp4, resistin, Tnfa, Igf1, and Igf2 mRNA levels were analyzed with real-time quantitative RT-PCR using Gapdh as a reference. (C) Differentiated 3T3-L1 adipocytes were stimulated with various concentrations of α-MSH. After 1 h, the cells were harvested, and Il6 and Pai1 mRNA levels were determined using real-time quantitative RT-PCR. (D) 3T3-L1 adipocytes were stimulated with α-MSH (300 nM), ACTH (50 nM), and LPS (1 μg/ml) for 1, 3, and 8 h. The supernatants were collected, and IL6 concentrations were determined by ELISA. Values represent the means±s.d. of triplicate samples. *P<0·05; **P<0·001 compared with the zero time point. Each experiment was performed independently at least three times.

Figure 3

PKA, p38 MAPK, JNK, and IKK are involved in α-MSH-induced Il6 gene expression and secretion. Differentiated 3T3-L1 adipocytes were pretreated with A, SB203580 (10 μM); B, SP600125 (10 μM); C, Bay 11-7085 (10 μM); and D, H89 (1 μM) and 6-Bnz-cAMP (50 μM) for 30 min prior to treatment with α-MSH (300 nM) for 1 h. (D) 3T3-L1 adipocytes were also treated with forskolin (3 μM), and isoproterenol (3 μM) alone for 1 h. (E) In the presence of each inhibitor, cells were treated with α-MSH for indicated time intervals, and culture medium was collected to measure secreted IL6. Data represent the means±s.d. of triplicate samples. *P<0·05; **P<0·001 compared with the α-MSH-treated group. ^#P<0·001 compared with the control group. Each experiment was performed independently at least three times.

Figure 4

Phosphorylation of JNK, p38 MAPK, and IKK in response to α-MSH. Serum-starved (12 h) 3T3-L1 adipocytes were treated with α-MSH (300 nM) for the indicated time periods. Whole-cell extracts were analyzed by western blots of phospho-JNK, phospho-cJUN, phospho-p38 kinases, and phospho-IKK protein. Equal loading of protein was checked by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. Each experiment was performed independently at least three times.

Figure 5

PKA, p38 MAPK, JNK, and IKK are involved in ACTH-induced Il6 gene expression. Differentiated 3T3-L1 adipocytes were pretreated with A, SB203580 (10 μM); B, SP600125 (10 μM); C, Bay 11-7085 (10 μM); and D, H89 (1 μM) for 30 min prior to treatment with ACTH (50 nM) for 1 h. Data represent the means±s.d. of triplicate samples. *P<0·05; **P<0·001 compared with the ACTH-treated group. Each experiment was performed independently at least three times.

Figure 6

α-MSH increases Il6 gene expression through MC5R, and ACTH increases Il6 gene expression through MC2R. 3T3-L1 adipocytes were transfected for 48 h with siRNAs targeted to the MC5Rs (siMC5Rs), or scrambled control siRNAs (siControl), and transfected for 24 h with siRNAs targeted to the MC2Rs (siMC2Rs), or scrambled control siRNA (siControl). (A) The MC5R-specific siRNA reduced endogenous Mc5r expression as detected by real-time quantitative RT-PCR. Gapdh served as an internal control. (B) 3T3-L1 adipocytes in which MC5R had been knocked down were stimulated with α-MSH for 1 h. Il6 gene expression was evaluated by real-time quantitative RT-PCR. (C) The MC2R-specific siRNA reduced endogenous Mc2r expression as detected by real-time quantitative RT-PCR. Gapdh served as an internal control. (D) 3T3-L1 adipocytes in which MC2R had been knocked down were stimulated with ACTH for 1 h. Il6 gene expression was evaluated by real-time quantitative RT-PCR. Values represent the means±s.d. of triplicate samples. *P<0·05; **P<0·001 compared with the siControl-transfected group. Each experiment was performed independently at least three times.

Figure 7

I.v. injection of α-MSH induces Il6 gene expression in mouse epididymal fat tissue. Mice were i.v. injected via tail vein with 200 μl sterile PBS containing 1 mg/kg α-MSH. Epididymal fat tissue was collected at 1, 3, and 6 h after α-MSH injection. Il6 transcript levels were determined by real-time quantitative RT-PCR. Values represent the means±s.d., n=3 for each time point. ^##P<0·001 compared with the control group. Each experiment was performed independently at least three times.