Atypical Protein Kinase Cs Promote CSF-1-Dependent Erk Activation and Proliferation in Myeloid Cells.

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.