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. 2015 Nov;12(6):692-9.
doi: 10.1038/cmi.2014.108. Epub 2014 Nov 24.

Response gene to complement 32 (RGC-32) expression on M2-polarized and tumor-associated macrophages is M-CSF-dependent and enhanced by tumor-derived IL-4

Peng Zhao  1   2 , Daiqing Gao  2 , Qingjie Wang  1 , Bingfeng Song  1 , Qianqian Shao  1 , Jintang Sun  1 , Chunyan Ji  1 , Xingang Li  3 , Peng Li  1 , Xun Qu  1
Affiliations

Response gene to complement 32 (RGC-32) expression on M2-polarized and tumor-associated macrophages is M-CSF-dependent and enhanced by tumor-derived IL-4

Peng Zhao et al. Cell Mol Immunol. 2015 Nov.

Abstract

Response gene to complement 32 (RGC-32) is a cell cycle regulator involved in the proliferation, differentiation and migration of cells and has also been implicated in angiogenesis. Here we show that RGC-32 expression in macrophages is induced by IL-4 and reduced by LPS, indicating a link between RGC-32 expression and M2 polarization. We demonstrated that the increased expression of RGC-32 is characteristic of alternatively activated macrophages, in which this protein suppresses the production of pro-inflammatory cytokine IL-6 and promotes the production of the anti-inflammatory mediator TGF-β. Consistent with in vitro data, tumor-associated macrophages (TAMs) express high levels of RGC-32, and this expression is induced by tumor-derived ascitic fluid in an M-CSF- and/or IL-4-dependent manner. Collectively, these results establish RGC-32 as a marker for M2 macrophage polarization and indicate that this protein is a potential target for cancer immunotherapy, targeting tumor-associated macrophages.

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Figures

Figure 1
Figure 1
RGC-32 is expressed at high levels in M2 macrophages. (a) RGC-32 expression was assayed using quantitative RT-PCR in THP-1 cells after induction for the indicated hours. (b) RGC-32 protein was detected using western blotting. (c) THP-1 cells and PMA-treated macrophages were stained with DAPI (blue) and TRITC-conjugated mAb (red) against RGC-32 and examined using a fluorescence microscope. (d) Quantitative RT-PCR analysis of RGC-32 mRNA in M1- and M2-polarized THP-1 macrophages. (e) RGC-32 expression in M1- and M2-polarized THP-1 macrophages was confirmed through western blotting. The graphs are representative of three separate experiments (*P<0.05, **P<0.01). RGC-32, response gene to complement 32; TAM, tumor-associated macrophage.
Figure 2
Figure 2
RGC-32 expression during macrophage polarization was induced by IL-4 or LPS. (a) PMA-treated macrophages were polarized to M1 using a standard dose of LPS (100 ng/ml) in combination with graded doses of IFN-γ (0, 5, 10 and 20 ng/ml) for 42 h. (b) PMA-treated macrophages were stimulated using a standard dose of IFN-γ (20 ng/ml) in combination with graded doses of LPS (0, 25, 50 and 100 ng/ml) for 42 h. (c) PMA-treated macrophages were polarized to M2 using graded doses of IL-4 (0, 5, 10 and 20 ng/ml) for 42 h. PMA, phorbol 12-myristate 13-acetate; RGC-32, response gene to complement 32.
Figure 3
Figure 3
Effect of RGC-32 on inflammatory cytokines. THP-1 cells were transfected with RGC-32 siRNA or two siRNA controls (a) and PBMN-GFP or PBMN-GFP-RGC-32 (c). The transfected cells were treated with PMA (100 ng/ml) for 48 h. The culture supernatants were collected from PMA-treated THP-1 macrophages after 48 h. (b and d) The cell-free supernatants obtained from different cultures were collected and analyzed for IL-6, IL-1β, TNF-α and TGF-β using ELISA. *P<0.05, **P<0.01. PMA, phorbol 12-myristate 13-acetate; RGC-32, response gene to complement 32.
Figure 4
Figure 4
PI3K signaling is important for the RGC-32-mediated induction of IL-6 production. (a) THP-1 cells were exposed to 100 ng/ml PMA for the indicated times. Whole-cell lysates were prepared and subjected to western blot analysis to detect phosphorylated Akt. (b) RGC-32-silenced or control THP-1 cells were treated with LY294002 for 1 h prior to further stimulation with PMA. After 48 h, the production of IL-1β, IL-6, TNF-α and TGF-β in the cell-free supernatants was assayed by ELISA. (c) RGC-32-silenced or control THP-1 cells were exposed to PMA for 48 h. Whole-cell lysates were subjected to western blot analysis to detect phosphorylated Akt. (d) RGC-32 physically interacts with AKT in THP-1 cells and PMA-treated macrophages. THP-1 cells were treated with 0 or 100 ng/ml PMA for 48 h, and subsequently coimmunoprecipitation was performed. Control IgG and AKT antibodies were used for coimmunoprecipitation, and anti-RGC-32 was used for immunoblotting. *P<0.05, **P<0.01. PI3K, phosphoinositide 3-kinase; PMA, phorbol 12-myristate 13-acetate; RGC-32, response gene to complement 32.
Figure 5
Figure 5
Induction of RGC-32 by tumor-conditioned medium is dependent on M-CSF and IL-4. (a) RGC-32 mRNA expression in monocytes obtained from peripheral blood and TAMs from ascitic fluid was assayed by quantitative RT-PCR (n=12). (b) RGC-32 mRNA expression in monocytes exposed for 72 h to M-CSF, IL-4 and conditioned medium from the ascitic fluid of three colon carcinomas (CM1, CM2 and CM3), as determined by quantitative RT-PCR. (c) Inhibitory effect of anti–M-CSF and anti-IL-4 on RGC-32 mRNA levels induced by ascitic fluid obtained from metastatic colon carcinoma. The results are depicted as the RGC-32 mRNA levels detected in the presence of the anti-M-CSF and anti-IL-4 antibody relative to the levels observed in control cells treated with an isotype-matched control Ab. *P<0.05, **P<0.01. Ab, antibody; RGC-32, response gene to complement 32.
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