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* This work was supported in part by Grant-in-aid for Scientific Research on Priority Area A02 and Grants-in-aid for Scientific Research 10671077 and 11557081 from the Ministry of Education, Science, Sports and Culture and by a grant from Sagawa Science Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Oxidized low density lipoprotein (Ox-LDL) can induce macrophage proliferation in vitro. To explore the mechanisms involved in this process, we reported that activation of protein kinase C (PKC) is involved in its signaling pathway (Matsumura, T., Sakai, M., Kobori, S., Biwa, T., Takemura, T., Matsuda, H., Hakamata, H., Horiuchi, S., and Shichiri, M. (1997) Arterioscler. Thromb. Vasc. Biol. 17, 3013–3020) and that expression of granulocyte/macrophage colony-stimulating factor (GM-CSF) and its subsequent release in the culture medium are important (Biwa, T., Hakamata, H., Sakai, M., Miyazaki, A., Suzuki, H., Kodama, T., Shichiri, M., and Horiuchi, S. (1998) J. Biol. Chem.273, 28305–28313). However, a recent study also demonstrated the involvement of phosphatidylinositol 3-kinase (PI3K) in this process. In the present study, we investigated the role of PKC and PI3K in Ox-LDL-induced macrophage proliferation. Ox-LDL-induced macrophage proliferation was inhibited by 90% by a PKC inhibitor, calphostin C, and 50% by a PI3K inhibitor, wortmannin. Ox-LDL-induced expression of GM-CSF and its subsequent release were inhibited by calphostin C but not by wortmannin, whereas recombinant GM-CSF-induced macrophage proliferation was inhibited by wortmannin by 50% but not by calphostin C. Ox-LDL activated PI3K at two time points (10 min and 4 h), and the activation at the second but not first point was significantly inhibited by calphostin C and anti-GM-CSF antibody. Our results suggest that PKC plays a role upstream in the signaling pathway to GM-CSF induction, whereas PI3K is involved, at least in part, downstream in the signaling pathway after GM-CSF induction.
oxidized low density lipoprotein
bovine serum albumin
enzyme-linked immunosorbent assay
granulocyte/macrophage colony-stimulating factor
protein kinase C
polymerase chain reaction
Macrophage-derived foam cells are the key cellular elements in the early stages of atherosclerosis (
) first demonstrated the Ox-LDL-induced macrophage proliferationin vitro. Subsequent studies showed the growth-stimulating capacity of Ox-LDL for other macrophages, such as mouse resident peritoneal macrophages (
), it is possible that macrophage proliferation may modulate the progression of atherosclerosis. Thus, clarification of the mechanism of macrophage activation, proliferation, and survival process is expected to enhance our understanding of the pathogenesis of atherosclerosis. In this regard, our recent study revealed that Ox-LDL can induce a rise in intracellular calcium concentration and activate protein kinase C (PKC) in mouse peritoneal macrophages (
). Subsequently, it was shown that activation of PKC leads to release into the culture medium of granulocyte/macrophage colony-stimulating factor (GM-CSF), which plays a priming role in the Ox-LDL-induced macrophage proliferation (
) provided evidence that phosphatidylinositol 3-kinase (PI3K) is also involved in the Ox-LDL-induced macrophage proliferation. The present study was undertaken to determine the relationship between PI3K and PKC on one hand and induction of GM-CSF on the other, in the signaling pathway for Ox-LDL-induced macrophage proliferation.
Macrophages and macrophage-derived foam cells are known to proliferate in atherosclerotic lesions (
). However, to our knowledge, the signaling pathway(s) from binding of Ox-LDL to macrophage proliferation has not been fully defined. Since macrophage-derived foam cells are thought to play an important role in the development and progression of atherosclerotic lesions (
), elucidation of the mechanism of Ox-LDL-induced macrophage proliferation would be an interesting project. In this regard, we recently demonstrated that activation of PKC and subsequent release of GM-CSF play an important role in Ox-LDL-induced macrophage proliferationin vitro (
) recently reported the involvement of PI3K in Ox-LDL-induced macrophage proliferation. Therefore, we compared in the present study the role of PKC to that of PI3K. The major conclusions of the present study could be summarized as follows. In the signaling pathway leading to macrophage proliferation, PKC is located before GM-CSF induction, whereas PI3K is located, at least in part, after GM-CSF induction (see Fig. 7). These conclusions were supported by the following findings. (i) Ox-LDL-induced macrophage proliferation was significantly inhibited by a PKC inhibitor, calphostin C, and a PI3K inhibitor, wortmannin (Fig. 1and Table I). (ii) Ox-LDL-induced GM-CSF expression and its subsequent release into the culture medium were inhibited by calphostin C but not by wortmannin (Figs. 2 and 3). (iii) In contrast, recombinant GM-CSF-induced macrophage proliferation was significantly inhibited by wortmannin but not by calphostin C (Fig. 4 and Table II). (iv) PI3K activation by Ox-LDL occurred at two time points (10 min and 4 h after the addition of Ox-LDL); the latter was inhibited by calphostin C and by an anti-GM-CSF antibody, whereas the former was not affected by an anti-GM-CSF antibody or by PKC inhibitor (Fig. 6).
) and those of the present study clearly showed that the Ox-LDL-induced GM-CSF release is mediated by activation of PKC. Extensive studies using T-lymphocytes showed that GM-CSF induction following PKC activation is mainly regulated at a transcriptional level (
). The PKC family is known to comprise at least 11 different isoforms of serine/threonine protein kinase, such as conventional PKC (α, β1, β2, and γ), novel PKC (δ, ε, θ, and η), and atypical PKC (ζ and λ) (
). The C1 region is composed of two tandem repeats of a cysteine-rich, zinc finger-like motif, which serves as a binding site for diacylglycerol and phorbol 12-myristate 13-acetate, and the C2 region is required for calcium sensitivity (
). Therefore, it is possible to assume that conventional PKC containing both C1 and C2 regions is a candidate signal mediator of Ox-LDL-induced GM-CSF induction, but the involvement of novel PKC having only C1 region cannot be ruled out. With regard to downstream signaling pathways from PKC activation to GM-CSF expression, our recent study using gel shift and luciferase assays showed that a putative AP-2 binding site from −169 to −160 of the murine GM-CSF promoter was a positive responsive site and GM-κB/GC box (−95 to −73) was a negative responsive site for Ox-LDL-induced GM-CSF expression in mouse peritoneal macrophages (
). Binding of GM-CSF to its receptor in various types of cells generates several intracellular tyrosine phosphorylation pathways, such as Janus kinase/signal transducers and activators of transcription, Jun NH2-terminal kinase/stress-activated protein kinase, Ras-Raf mitogen-activated protein kinase, PI3K-protein kinase B, and protein kinase A (
). In the present study, we demonstrated that Ox-LDL enhanced PI3K activity at 10 min and 4 h after the addition of Ox-LDL (Fig. 5), and that the latter was significantly inhibited by an anti-GM-CSF antibody (Fig. 6). Moreover, macrophage proliferation induced by Ox-LDL or GM-CSF was significantly inhibited by wortmannin (Tables I and II). Furthermore, an anti-GM-CSF antibody significantly inhibited Ox-LDL-induced macrophage proliferation (
). These findings strongly suggest that activation of PI3K at the late time point is involved in Ox-LDL-induced macrophage proliferation after GM-CSF expression. However, we also demonstrated that 50 nm wortmannin produced 50% inhibition of Ox-LDL-induced macrophage proliferation (Fig. 1 and Table I). Moreover, under identical conditions, 20 μm LY294002, another PI3K inhibitor, also showed 50–55% inhibition when assessed by both thymidine incorporation and cell counting assays (data not shown). The concentrations of these PI3K inhibitors used in our experiments have been reported to be high enough to completely inhibit PI3K in human macrophages (
) when cells were stimulated by GM-CSF. Thus, it is likely that the PI3K pathway is involved in GM-CSF-mediated macrophage proliferation. However, since PI3K inhibitors produce only 50% inhibition of macrophage proliferation, it is likely that another pathway is also involved, which is activated by GM-CSF but is PI3K-independent (see Fig. 7).
Although PI3K inhibitor had no effect on Ox-LDL-mediated GM-CSF induction as well as its release into the medium (Figs. 2 and 3), it significantly inhibited Ox-LDL-induced macrophage proliferation (Figs.1 and Table I). These findings suggest that PI3K activation at 10 min after the addition of Ox-LDL may be partly responsible for macrophage proliferation. However, this pathway is GM-CSF independent. Since GM-CSF-independent pathway accounted for < 20% of macrophage proliferation, Ox-LDL-induced PI3K activation at 10 min may play a minor, if any, role in Ox-LDL-induced macrophage proliferation (Fig. 7).