Introduction
Monocytes are produced in the bone marrow and circulate in blood for 24–48 hours [1]. In the absence of serum, monocytes die via apoptosis [1], [2]. Macrophage colony-stimulating factor (M-CSF) stimulates mononuclear phagocyte survival and differentiation [3]. Importantly, M-CSF-mediated cell survival and activation is associated with a variety of human diseases, including atherosclerosis, transplant vascular sclerosis and breast cancer metastasis [4], [5], [6].
We previously identified that NF-κB activation is important in M-CSF-induced monocyte survival [7]. In addition to its role in mononuclear phagocyte survival, the transcription factor NF-κB regulates numerous genes that play important roles in cellular signaling, stress response, cell growth, survival, differentiation and inflammation [8]. There are five members in the NF-κB family: RelA/p65, p50, p52, c-Rel and RelB. The most common activating complex is the p50/p65 heterodimer, driven by the activation domain in the NF-κB p65 subunit. NF-κB p65 regulates important developmental processes [9], [10]. Mice lacking NF-κB p65 die in utero and have extensive liver damage via enhanced apoptosis [9]. Embryonic macrophages from NF-κB p65 null mice are susceptible to TNFα-induced apoptosis which is rescued by overexpressing the NF-κB p65 subunit [10]. Moreover, inhibiting NF-κB induces cell death in many cell types and cytokine-independent survival is mediated by constitutively active NF-κB in murine macrophages [11].
In monocytes and macrophages, NF-κB is an important transcriptional factor for expression of cytokines and cell surface receptors [12]. However, unlike resting T-cells, NF-κB is constitutively present in the nuclei of primary monocytes and monocytic cell lines in the absence of exogenous stimuli as demonstrated by mobility shift analysis [13]. Similarly, constitutively active NF-κB was observed in human alveolar macrophages [14].
In the classic/canonical pathway, the NF-κB p50/p65 complex is sequestered in the cytosol by IκBα [15]. Upon stimulation by cytokines or UV radiation, IκBα is phosphorylated, ubiquitinated, and degraded, releasing NF-κB p50/p65 to translocate to the nucleus and transactivate target genes. After its release from IκBα, NF-κB p65 can undergo post-translational modification to activate gene transcription. The role of NF-κB p65 phosphorylation on NF-κB transcriptional activity varies by stimulus, time of stimulation and cell type [16]. Previous research shows that phosphorylation of NF-κB p65 at Ser276, Ser529 or Ser536 plays an important role in regulating transcriptional activity of NF-κB [17]. In TNFα-treated murine fibroblasts, Ser276 of NF-κB p65 is phosphorylated by MSK1 to enhance NF-κB transcriptional activity [18]. In macrophages treated with endotoxin, NF-κB transcription activity is associated with phosphorylation on Ser276 and Ser536 that is regulated through protein kinase A (PKA) and IKKβ respectively[16], [19]. In human T cells, NF-κB p65 is constitutively phosphorylated on Ser536 to facilitate NF-κB p65 nuclear translocation following cellular stimulation [20]. Accumulating evidence reveals that NF-κB p65 phosphorylation at Ser276 is crucial for its transcriptional activity. Upon nuclear translocation, phosphorylation of Ser276 on NF-κB p65 by PKA recruits the transcription co-activator, p300 to potentiate NF-κB-regulated gene transcription [21]. However, other studies show that PKA inhibits NF-κB-regulated gene expression by stabilizing IκBα [22], [23]. Interestingly, the serine/threonine kinase Pim-1 directly phosphorylates NF-κB p65 at Ser276 by stabilizing to prevent ubiquitin-proteasome degradation [24]. Several other phosphorylation sites are also described to enhance NF-κB gene transactivation [25].
Here, we investigate the role of protein kinase C (PKC) in M-CSF-stimulated NF-κB activation. PKC proteins are multifunctional kinases that differ in structure, function and co-factor requirement [26]. PKCs are involved in diverse cell responses, including cell growth, survival, differentiation and development [27]. The 12 closely related enzymes of the PKC family are divided into three classes: conventional (cPKCs: α βI βII and γ require Ca2+ and diacylglycerol (DAG); novel (nPKCs: δ, ε, η, θ and μ) require DAG; and atypical (aPKCs: ξ, ι and λ) require neither Ca2+ nor DAG. Monocytes and macrophages predominantly express conventional PKC isoforms (PKCα PKCβI and PKCβII) and novel PKCs (PKCδ and PKCε). Conventional PKCs regulate differentiation of the human promyelocytic leukemia cell line HL60 to macrophages [28]. PKCα induces IL-1α, iNOS and TNFα mRNA production after lipopolysaccharide (LPS) exposure [29]. In addition, accumulating evidence suggests that conventional PKCs like PKCα have anti-apoptotic functions. For example, PKCα is overexpressed in a variety of tumor cells including gliomas, liver, and lung [30], [31]. In epithelial cells, inhibition of PKCα induces PKCδ-dependent apoptosis [31]. Interestingly, in human monocytes and premonocytic THP-1 leukemia cells, novel PKCs like PKCδ have the opposite effect on cell survival, Voss et al showed that PKCδ directly phosphorylates caspase-3 and promotes etoposide-induced apoptosis [32]. Moreover, knockout mouse studies suggest that another novel PKC, PKCε is critically involved in early LPS-mediated signaling in activated macrophages [33].
Previously, we reported that M-CSF promotes monocyte survival through the activation of the PI3-K/AKT pathway [3]. In addition to AKT activation, M-CSF stimulates PKC in human monocytes and increases NF-κB DNA binding [34]. However, whether PKC and/or NF-κB activation is critical in M-CSF-stimulated mononuclear phagocyte survival and/or differentiation is unclear. In other cells, PKC plays an important role in NF-κB activation and cell survival [35], however the specific mechanisms of this activation and the biological effects on cellular phenotype are not known. Therefore, we focused at understanding the role of PKC in the regulation of NF-κB activation and M-CSF-induced monocyte survival.
Here we demonstrates that M-CSF upregulated the NF-κB transcription and cell survival in human and mouse macrophages. This activity was reduced by the conventional, but not novel, PKC inhibitors, dominant negative PKCα constructs or PKCα siRNA. Conventional PKC regulated NF-κB-regulated gene expression and phosphorylation of Ser276 of NF-κB p65 occurred in an M-CSF-dependent fashion correlating with its maximal transcriptional activity. Furthermore, PKCα-regulated phosphorylation of Ser276 of NF-κB p65 plays an important role in regulating its activity in mononuclear phagocytes and murine embryonic fibroblasts.