Downregulation of IL-8 and IL-10 by the Activation of Ca2+-Activated K+ Channel KCa3.1 in THP-1-Derived M2 Macrophages

Abstract

THP-1-differentiated macrophages are useful for investigating the physiological significance of tumor-associated macrophages (TAMs). In the tumor microenvironment (TME), TAMs with the M₂-like phenotype play a critical role in promoting cancer progression and metastasis by inhibiting the immune surveillance system. We examined the involvement of Ca²⁺-activated K⁺ channel K_Ca3.1 in TAMs in expressing pro-tumorigenic cytokines and angiogenic growth factors. In THP-1-derived M₂ macrophages, the expression levels of IL-8 and IL-10 were significantly decreased by treatment with the selective K_Ca3.1 activator, SKA-121, without changes in those of VEGF and TGF-β1. Furthermore, under in vitro experimental conditions that mimic extracellular K⁺ levels in the TME, IL-8 and IL-10 levels were both significantly elevated, and these increases were reversed by combined treatment with SKA-121. Among several signaling pathways potentially involved in the transcriptional regulation of IL-8 and IL-10, respective treatments with ERK and JNK inhibitors significantly repressed their transcriptions, and treatment with SKA-121 significantly reduced the phosphorylated ERK, JNK, c-Jun, and CREB levels. These results strongly suggest that the K_Ca3.1 activator may suppress IL-10-induced tumor immune surveillance escape and IL-8-induced tumorigenicity and metastasis by inhibiting their production from TAMs through ERK-CREB and JNK-c-Jun cascades.

Keywords: IL-10; IL-8; K+ channel; KCa3.1; THP-1; tumor microenvironment; tumor-associated macrophage.

Figure 1

Gene expression of M₂ markers, cytokines, chemokines, and growth factors in THP-1-derived M₂-like macrophages. A–G: Real-time PCR examination of CD163 (A), Arg1 (B), IL-10 (C), IL-8 (D), VEGF-A (E), CCL22 (F), and TGF-β1 (G) expression in native THP-1 (‘native’), THP-1-derived M₀-like macrophages (‘M₀’), and THP-1-derived M₂-like macrophages (‘M₂’). Expression levels are shown as a ratio to ACTB (n = 4 for each). **: p < 0.01 vs. ‘native’, ^##: p < 0.01 vs. ‘M₀’.

Figure 2

Functional expression of K_Ca3.1 in THP-1-derived M₂ macrophages. (A,B): Simultaneous measurements of changes in the membrane potential (A) and [Ca²⁺]_i (B) in the three different cells [a (black), b (blue), and c (red)], following the application of a selective K_Ca3.1 activator, SKA-121 (1 μM), and/or the K_Ca3.1 inhibitor, TRAM-34 (1 μM) using DiBAC₄(3) and Fura 2, respectively. The relative time-course changes in fluorescence intensities (1.0 at 0 s) from three different THP-1-derived M₂ macrophages are shown. (C): Real-time PCR examination of K_Ca3.1 expression in THP-1-derived M₀ macrophages (‘M₀’) and M₂ macrophages (‘M₂’). Expression levels are shown as a ratio to ACTB (p > 0.05, n = 4 for each). (D,E): Protein expression levels of K_Ca3.1 in the ‘M₀’ and ‘M₂’ groups were determined by Western blot. Specific band signals for K_Ca3.1 were observed at approximately 50 kDa (D, upper panel). After compensation with the optical density of the ACTB signal (43 kDa) (D, lower panel), the expression level in the ‘M₀’ group was expressed as 1.0 (p > 0.05, n = 4 for each) (E). (F): SKA-121 (1 μM)-induced relative hyperpolarizing responses in the ‘M₀’ and ‘M₂’ groups (p > 0.05, n = 37 and 29, respectively).

Figure 3

Whole-cell patch-clamp recordings of SKA-121-activated K⁺ currents in THP-1-derived M₂ macrophages. (A): Typical current density and voltage-relationships after addition of vehicle (black), 1 μM SKA-121 (red), and 1 μM SKA-121 plus 1 μM TRAM-34 (blue). Currents were elicited by ramp depolarization from −120 to +40 mV from a holding potential of −80 mV every 10 s. (B): Typical current density and voltage-relationship of SKA-121-sensitive component. (C): Summarized results of current densities (pA/pF) at +40 mV in three groups (n = 9 for each). *: p < 0.05 vs. vehicle control (−/−); ^## p < 0.01 vs. SKA-121 alone (+/−).

Figure 4

Effects of treatment with SKA-121 on IL-10, IL-8, VEGF-A, and TGF-β1 expression and on IL-10 and IL-8 secretion in THP-1-derived M₂ macrophages. A–D: Real-time PCR examination of IL-10 (A), IL-8 (B), VEGF-A (C), and TGF-β1 (D) expression in THP-1-derived M₂ macrophages treated (+) or untreated (−) with 1 μM SKA-121 and 10 μM TRAM-34 for 24 h. Relative mRNA expression in the vehicle control (‘−’ for SKA and ‘−’ for TRAM) is expressed as 1.0 (n = 4 for each). (E,F): Quantitative detection of IL-10 (E) and IL-8 (F) secretion by an ELISA assay in THP-1-derived M₂ macrophages treated and untreated with SKA-121 and TRAM-34. Relative cytokine secretion in the vehicle control (−/−) is expressed as 1.0 (n = 4 for each). **: p < 0.01 vs. the vehicle control (−/−), ^##: p < 0.01 vs. SKA-121 alone (+/−).

Figure 5

Effects of treatment with SKA-121 on high [K⁺]_e-enhanced IL-10 and IL-8 expression and secretion in THP-1-derived M₂ macrophages. A,B: Real-time PCR examination of IL-10 (A) and IL-8 (B) expression in normal [K⁺]_e (5 mM)- and high [K⁺]_e (25 mM)-treated THP-1-derived M₂ macrophages for 24 h in the presence (+) or absence (−) of SKA-121 (1, 10 μM). Relative mRNA expression in normal [K⁺]_e is expressed as 1.0 (n = 4 for each). (C,D): Quantitative detection of IL-10 (C) and IL-8 (D) secretion by an ELISA assay in THP-1-derived M₂ macrophages treated and untreated with SKA-121. Relative cytokine secretion in normal [K⁺]_e is expressed as 1.0 (n = 4 for each). **: p < 0.01 vs. normal [K⁺]_e, ^##: p < 0.01 vs. the vehicle control (−/−) of high [K⁺]_e.

Figure 6

Effects of a 24 h treatment with various signaling pathway inhibitors on IL-10 and IL-8 expression and effects of the 24 h treatment with the ERK1/2 inhibitor, SCH772984 and JNK inhibitor, SP600125 on IL-10 and IL-8 secretion in THP-1-derived M₂ macrophages. (A,D): Real-time PCR examination of IL-10 (A) and IL-8 (D) expression in SCH772964 (1 μM)-, SP600125 (10 μM)-, PD169316 (10 μM)-, LY294002 (10 μM)-, AZD5363 (2 μM)-, everolimus (10 nM)-, LY364947 (10 μM)-, ciclosporin A (CsA, 1 μM)-, T-5224 (10 μM)-, and Bay11-7082 (10 μM)-treated THP-1-derived M₂ macrophages for 24 h. Relative mRNA expression in the vehicle control is expressed as 1.0 (n = 4 for each). (B,C,E,F): Quantitative detection of IL-10 (B,C) and/or IL-8 (E,F) secretion by an ELISA assay in SCH772964 (B,E)- and SP600125 (C,F)-treated THP-1-derived M₂ macrophages. Relative cytokine secretion in the vehicle control is expressed as 1.0 (n = 4 for each). **: p < 0.01 vs. the vehicle control.

Figure 7

Protein expression levels of phosphorylated ERK1/2 (P-ERK1/2) and P-JNK in THP-1-derived M₂ macrophages following the SKA-121 treatment and high [K⁺]_e exposure. A–D: Western blot showing P-ERK1/2, total ERK1/2 (ERK1/2) (A,B), P-JNK, and total JNK (JNK) (C,D) in 1 μM SKA-121-treated (A,C) and high [K⁺]_e-exposed (B,D) THP-1-derived M₂ macrophages. Specific band signals were observed at 42 (P-ERK2), 42 (ERK2), 43/50 (P-JNK), and 43/50 (JNK) kDa. E–H: Summarized results of the relative protein expression of P-ERK2/ERK2 (E,F) and P-JNK/JNK (G,H) in 1 μM SKA-121-treated (E,G) and high [K⁺]_e-exposed (F,H) THP-1-derived M₂ macrophages. After compensation with the optical density of the ACTB signal (43 kDa), the expression level in the vehicle control or 5 mM K⁺ is expressed as 1.0 (n = 4 for each). **: p < 0.01 vs. the vehicle control and 5 mM K⁺.

Figure 8

Protein expression levels of phosphorylated c-Jun (P-c-Jun) following the SKA-121 treatment and high [K⁺]_e exposure in THP-1-derived M₂ macrophages. (A,B): Western blot showing P-c-Jun, and unphosphorylated c-Jun (c-Jun) in 1 μM SKA-121-treated (A) and high [K⁺]_e-exposed (B) THP-1-derived M₂ macrophages. Specific band signals were observed at 42-46 (P-c-Jun) and 40 (c-Jun) kDa. (C,D): Summarized results of the relative protein expression of P-c-Jun/c-Jun in 1 μM SKA-121-treated (C) and high [K⁺]_e-exposed (D) THP-1-derived M₂ macrophages. After compensation with the optical density of the ACTB signal (43 kDa), the expression level in the vehicle control is expressed as 1.0 (n = 4 for each). **: p < 0.01 vs. the vehicle control and 5 mM K⁺.

Figure 9

Effects of treatment with a CREB inhibitor on expression levels of IL-10, IL-8, VEGF-A, and TGF-β1 transcripts and on phosphorylated P-CREB protein levels following the SKA-121 treatment and high [K⁺]_e exposure in THP-1-derived M₂ macrophages. (A–D): Real-time PCR examination of IL-10 (A), IL-8 (B), VEGF-A (C), and TGF-β1 (D) expression in THP-1-derived M₂ macrophages treated with vehicle and 1 μM 666-15 for 24 h. Relative mRNA expression in the vehicle control is expressed as 1.0 (n = 4 for each). E-F: Western blot showing P-CREB and total CREB (CREB) in 1 μM SKA-121-treated (E) and high [K⁺]_e-exposed (F) THP-1-derived M₂ macrophages. Specific band signals for P-CREB and CREB were observed at approximately 40 kDa. G, H: Summarized results of the relative protein expression of P-CREB/CREB in 1 μM SKA-121-treated (G) and high [K⁺]_e-exposed (H) THP-1-derived M₂ macrophages. After compensation with the optical density of the ACTB signal, the expression level in the vehicle control or 5 mM K⁺ is expressed as 1.0 (n = 4 for each). **: p < 0.01 vs. the vehicle control or 5 mM K⁺.

Figure 10

Effects of treatment with SKA-121 on expression levels of IL-10, IL-8, VEGF-A, and TGF-β1 transcripts and IL-10 and IL-8 secretion in human prostate cancer PC-3 cell-cultured medium-treated THP-1-derived M₂ macrophages. (A–D): Real-time PCR examination of IL-10 (A), IL-8 (B), VEGF-A (C), and TGF-β1 (D) expression in PC-3 media-treated THP-1-derived M₂ macrophages treated (+) or untreated (−) with 1 or 10 μM SKA-121 for 24 h. Relative mRNA expression in the group untreated with PC-3 medium is expressed as 1.0 (n = 4 for each). (E,F): Quantitative detection of IL-10 (E) and IL-8 (F) secretion by an ELISA assay in THP-1-derived M₂ macrophages treated or untreated with SKA-121. Relative secretion in the group untreated with PC-3 medium is expressed as 1.0 (n = 4 for each). **: p < 0.01 vs. the vehicle control (−/−).