Abstract

Macrophages are integral components of the innate immune system and essential players in inflammation. Enhanced macrophage numbers underlie these pathological states. Colony stimulating factor-1 (CSF-1) is the major physiological regulator of proliferation and survival of cells of the monocyte/macrophage lineage. CSF-1 binds to a receptor tyrosine kinase, the CSF-1 receptor (CSF-1R). CSF-1 and CSF-1R have emerged as drug targets in several diseases where inflammation is a critical component, e.g. breast cancer and rheumatoid arthritis. Multiple pathways are activated downstream of the CSF-1R; however, it is not clear which of these pathways regulate proliferation and survival. Atypical PKCs (aPKCs) are implicated in cell proliferation and survival. They include the isoforms PKCζ and PKCλ/ι. Unlike the classical and novel PKCs, aPKCs are insensitive to Ca2+ and phorbol esters. In this study, we investigated the role of aPKCs in CSF-1-mediated proliferation in myeloid cells. CSF-1 is a proliferation and survival factor for 32D.R cells, a myeloid progenitor cell line transfected with the CSF-1R. Western blotting shows that PKCα, PKCδ, PKCε and PKCζ/λ/I are expressed in 32D.R. Based on studies with PKC inhibitors that have different specificities towards aPKCs (GF109203X, Ro-31-8220, Go6983 and a Myr-PKCζ peptide), maximal CSF-1-dependent proliferation in 32D.R cells appears to depend on the activity of either aPKCs or PKCε. Using phospho-specific antibodies that detect the activation state of PKCζ as well as in vitro kinase assays, we showed that CSF-1 activates aPKCs in 32D.R and bone marrow derived macrophages. In contrast, CSF-1-induced activation of PKCε was not observed. We next asked how aPKC affects CSF-1 signaling. PKCζ promotes activation of the MEK-Erk pathway in different cell types (Corbit, K.C. et al. Mol. Cell. Biol. 20, 5392). In 32D.R cells, treatment with the MEK inhibitor, U0126, reduced CSF-1-provoked proliferation by 60–70%, consistent with the inhibition observed with PKC inhibitors. Previous work from our lab showed that CSF-1 activates the Erk pathway through A-Raf and not Raf-1 (Lee and States, Mol. Cell. Biol. 18, 6779). We found that aPKC inhibitors do not affect CSF-1 induced Ras and A-Raf activity but markedly reduce MEK and Erk activity, implying that aPKC inputs into the CSF-1 Erk pathway at the level of MEK. Transient transfections with dominant-negative and constitutively active (CA) PKCζ confirmed that aPKC promotes CSF-1-induced Erk activation. aPKC inhibition does not affect CSF-1-stimulated Akt activation. To investigate the role of PKCζ in CSF-1-dependent proliferation, we established stable 32D.R mass populations overexpressing wildtype (WT) or CA PKCζ at levels 2-fold above endogenous. Comparing cells expressing CA-PKCζ to WT-PKCζ, the EC50 for CSF-1-dependent proliferation and the cell doubling time at maximal CSF-1 concentration were both reduced, consistent with a role for PKCζ in CSF-1 dependent proliferation. We will use our stable cell lines to elucidate the pathways modulated by PKCζ. Altogether, our results identify atypical PKCs as new targets of CSF-1 signaling.

Disclosure: No relevant conflicts of interest to declare.