Response gene to complement 32 protein promotes macrophage phagocytosis via activation of protein kinase C pathway

Abstract

Macrophage phagocytosis plays an important role in host defense. The molecular mechanism, especially factors regulating the phagocytosis, however, is not completely understood. In the present study, we found that response gene to complement 32 (RGC-32) is an important regulator of phagocytosis. Although RGC-32 is induced and abundantly expressed in macrophage during monocyte-macrophage differentiation, RGC-32 appears not to be important for this process because RGC-32-deficient bone marrow progenitor can normally differentiate to macrophage. However, both peritoneal macrophages and bone marrow-derived macrophages with RGC-32 deficiency exhibit significant defects in phagocytosis, whereas RGC-32-overexpressed macrophages show increased phagocytosis. Mechanistically, RGC-32 is recruited to macrophage membrane where it promotes F-actin assembly and the formation of phagocytic cups. RGC-32 knock-out impairs F-actin assembly. RGC-32 appears to interact with PKC to regulate PKC-induced phosphorylation of F-actin cross-linking protein myristoylated alanine-rich protein kinase C substrate. Taken together, our results demonstrate for the first time that RGC-32 is a novel membrane regulator for macrophage phagocytosis.

Keywords: Actin; Macrophage; Phagocytosis; Protein Kinase C (PKC); Response Gene to Complement 32; Signal Transduction.

FIGURE 1.

RGC-32 was induced in differentiated macrophages. A, RGC-32 mRNA was induced during THP-1 to macrophage differentiation by PMA in a dose-dependent manner. B, time-dependent induction of RGC-32 mRNA expression in PMA-induced THP-1-macrophage differentiation. C, RGC-32 protein was expressed in THP-1-derived macrophage. (MO: monocyte; MA: macrophage). D, quantification of protein expression shown in C by normalizing to α-tubulin. E, RGC-32 protein was expressed in bone marrow (BM)-derived macrophage. HSPC: hematopoietic progenitor cell. F, quantification of protein expression shown in E by normalizing to α-tubulin. G, RGC-32 was expressed in spleen macrophage. Macrophages in spleen sections were immunostained with MOMA2 antibody. *, p < 0.05; **, p < 0.01 (n = 3).

FIGURE 2.

RGC-32 was not involved in monocyte-macrophage differentiation. A, RGC-32 deficiency (KO) did not alter peritoneal macrophage (PM) accumulation. B, RGC-32 deficiency did not impact bone marrow hematopoietic progenitor cell differentiation into macrophage (BMDM). C, RGC-32 deficiency did not affect CD68 and dectin-1 expression in differentiated macrophage as detected by flow cytometry. D, RGC-32 deficiency did not alter CD68 protein expression in differentiated macrophage as detected by Western blot. E, quantification of CD68 protein expression shown in D by normalizing to GAPDH. F, knockdown (Ad-shRGC32) or overexpression (Ad-RGC32) of RGC-32 did not affect CD68 or dectin-1 protein expression in differentiated macrophage. G, quantification of protein expression shown in F by normalizing to GAPDH. *, p < 0.01 (n = 3).

FIGURE 3.

RGC-32 deficiency inhibited M2 macrophage polarization. A, RGC-32 deficiency (KO) promoted M1 macrophage marker iNOS expression in LPS/IFN-γ/G-CSF-treated bone marrow hematopoietic progenitor cells. B, quantification of iNOS expression shown in A by normalizing to α-tubulin. C, RGC32 deficiency suppressed M2 macrophage marker arginase-1 expression in M-CSF- and IL-4-treated bone marrow hematopoietic progenitor cells. D, quantification of arginase-1 expression shown in C by normalizing to α-tubulin. *, p < 0.01 (n = 3).

FIGURE 4.

RGC-32 was essential for macrophage phagocytosis. A, RGC-32 deficiency (KO) in peritoneal macrophage (PM) attenuated phagocytosis of FITC-labeled zymosan-A particles as shown by flow cytometry analysis. B, overexpression of RGC-32 (Ad-RGC) increased while knockdown of RGC-32 (Ad-shRGC) decreased the phagocytosis of THP-1-derived macrophages as shown by flow cytometry analysis. C, RGC-32 overexpression increased THP-1-derived macrophage phagocytosis of PKH26-labeled Hep3D tumor cells. D, RGC-32 deficiency suppressed expression of phagosome markers Rab5 and Rab7 in bone marrow-derived macrophages. E, quantification of protein expression shown in D by normalizing to GAPDH. F, overexpression of RGC-32 increased while knockdown of RGC-32 decreased Rab5 expression in THP-1-derived macrophage. G, quantification of Rab5 protein expression shown in F by normalizing to GAPDH. *, p < 0.01 (n = 3).

FIGURE 5.

RGC-32 co-localized with F-actin on macrophage cell membrane. Mouse peritoneal macrophages were cultured with or without (0 min) zymosan-A (ZA) particles for 5 and 10 min as indicated. RGC-32 expression and F-actin formation were examined by immunostaining with RGC-32 and F-actin antibody, respectively, as indicated. DAPI was used to stain nuclei. Arrows indicate that RGC-32 membrane expression preceded (5 min, middle panel) the F-actin membrane formation (10 min, top panel) after the zymosan-A addition.

FIGURE 6.

RGC-32 was essential for F-actin assembly during macrophage phagocytosis. A, adenoviral vector-mediated overexpression of RGC-32 (RGC32) increased while knockdown of RGC-32 (shRGC32) decreased F-actin expression in THP-1-derived macrophages. B, quantification of F-actin expression shown in A by normalizing to GAPDH. C, RGC-32 deficiency (KO) suppressed F-actin protein expression in bone marrow-derived macrophages without (Ctrl) or with zymosan-A treatment (ZA 2 h). D, quantification of F-actin expression shown in C by normalizing to GAPDH. E, RGC-32 deficiency suppressed F-actin assembly on cell membrane of bone marrow-derived macrophage during the phagocytosis of zymosan-A particles. F-actin was stained with phalloidin. *, p < 0.01 (n = 3).

FIGURE 7.

PKC interacted with RGC-32 and mediated its role in phagocytosis. A, PKCα and PKCϵ co-immunoprecipitated (IP) with RGC-32 (RGC) during bone marrow-derived macrophage phagocytosis of zymosan-A (ZA) particle. IB, immunoblot; Ctrl, control. B, RGC-32 co-immunoprecipitated with PKCα during bone marrow-derived macrophage phagocytosis. C, RGC-32 co-immunoprecipitated with PKCϵ during bone marrow-derived macrophage phagocytosis. D, RGC-32 deficiency (KO) blocked PKC activity as shown by the reduced activation (phosphorylation) of its downstream target MARCKS (phospho-MARCKS (p-MARCKS)) in bone marrow-derived macrophages. E, quantification of phospho-MARCKS and total MARCKS expression shown in D by normalizing to GAPDH. F, PKC inhibitor Go6976 (GO) blocked RGC-32-induced F-actin expression in THP-1-derived macrophage. G, quantification of F-actin expression shown in F by normalizing to GAPDH. H, PKC inhibitor Go6976 blocked RGC-32-induced F-actin expression in bone marrow-derived macrophages with RGC32 deficiency. I, quantification of protein expression shown in H by normalizing to GAPDH. J, PKC activator PMA pretreatment restored the RGC-32 deficiency-caused attenuation of phagocytosis of bone marrow-derived macrophages as shown by flow cytometry analysis. K, PKC inhibitor Go6976 pretreatment suppressed RGC-32 (AdRGC)-rescued phagocytosis of BMDM with RGC-32 deficiency. *, p < 0.01 (n = 3).

FIGURE 8.

The mechanism whereby RGC-32 regulates macrophage phagocytosis. Foreign particles stimulate PKC activation while recruiting RGC32 to macrophage membrane where RGC-32 directly binds to PKC and facilitates PKC-induced F-actin assembly, which promotes foreign particle internalization and phagocytosis.