Abstract
Colony stimulating factor-1 (CSF-1 or M-CSF) is the major physiological regulator of the proliferation, differentiation and survival of cells of the mononuclear phagocyte lineage. CSF-1 binds to a receptor tyrosine kinase, the CSF-1 receptor (CSF-1R). Multiple pathways are activated downstream of the CSF-1R; however, it is not clear which pathways regulate proliferation and survival. Here, we investigated the role of atypical protein kinase Cs (PKCζ) in a myeloid progenitor cell line that expressed CSF-1R (32D.R) and in primary murine bone marrow derived macrophages (BMMs). In 32D.R cells, CSF-1 induced the phosphorylation of PKCζ and increased its kinase activity. PKC inhibitors and transfections with mutant PKCs showed that optimal CSF-1-dependent Erk activation and proliferation depended on the activity of PKCζ. We previously reported that CSF-1 activated the Erk pathway through an A-Raf-dependent and an A-Raf independent pathway (Lee and States, Mol. Cell. Biol. 18, 6779). PKC inhibitors did not affect CSF-1 induced Ras and A-Raf activity but markedly reduced MEK and Erk activity, implying that PKCζ regulated the CSF-1-Erk pathway at the level of MEK. PKCζ has been implicated in activating the NF-κB pathway. However, CSF-1 promoted proliferation in an NF-κB independent manner. We established stable 32D.R cell lines that overexpressed PKCζ. Overexpression of PKCζ increased the intensity and duration of CSF-1 induced Erk activity and rendered cells more responsive to CSF-1 mediated proliferation. In contrast to 32D.R cells, PKCζ inhibition in BMMs had only a modest effect on proliferation. Moreover, PKCζ -specific and pan-PKC inhibitors induced a paradoxical increase in MEK-Erk phosphorylation suggesting that PKCs targeted a common negative regulatory step upstream of MEK. Our results demonstrated that CSF-1 dependent Erk activation and proliferation are regulated differentially in progenitors and differentiated cells.
Highlights
Colony stimulating factor-1 (CSF-1 or M-CSF) is a growth factor secreted by numerous cell types, whose synthesis is often increased in response to different stimuli such as those causing inflammation [1]
32D.R cells Since PKCf appeared to play a role in CSF-1 mediated mitogenesis and PKCf has been shown to activate the NF-kB pathway which regulates genes involved in cell proliferation [48], we examined if CSF-1 stimulated NF-kB activity
We addressed two questions using the 32D.R myeloid progenitor cell line and primary mouse bone marrow derived macrophages (BMMs) : 1) Is protein kinase C (PKC) an activator of the MEK-Erk pathway; 2) Does PKC contribute towards CSF-1 dependent proliferation? In 32D.R cells, we presented evidence in support of a role for the atypical PKC, PKCf, in CSF-1 dependent activation of MEK-Erk and mitogenesis
Summary
Colony stimulating factor-1 (CSF-1 or M-CSF) is a growth factor secreted by numerous cell types, whose synthesis is often increased in response to different stimuli such as those causing inflammation [1]. It promotes the proliferation, survival and differentiation of cells of the mononuclear phagocyte (MNP) lineage and their myeloid progenitors [1,2]. CSF-1 acts on the CSF-1R, a receptor tyrosine kinase (RTK) of the platelet-derived growth factor (PDGF) receptor family that includes c-kit and the Flt3/Flk receptor. Oncogenic signals emanating from an aberrantly expressed CSF-1R in tumor cells promote tumor growth and metastasis in breast and ovarian cancers [3]. The existence of IL-34 explains the milder phenotype of CSF-1 null mice compared to mice lacking CSF-1R
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