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. 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 impartial pathway (Lee and Says 18 6779 PKC inhibitors did not impact CSF-1 induced Ras and A-Raf activity but markedly reduced MEK and Erk Roscovitine (Seliciclib) 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 impartial 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 unfavorable regulatory step upstream of MEK. Our results exhibited that CSF-1 dependent Erk activation and proliferation are regulated differentially in progenitors and differentiated cells. Introduction Colony stimulating factor-1 (CSF-1 or M-CSF) is usually a growth factor secreted by numerous cell types whose synthesis is usually often increased in response to different stimuli such as those causing inflammation [1]. It promotes the proliferation survival and differentiation Roscovitine (Seliciclib) of cells of the mononuclear phagocyte (MNP) lineage and their myeloid progenitors [1] [2]. CSF-1 functions around the CSF-1R a receptor tyrosine kinase (RTK) of the platelet-derived growth factor (PDGF) receptor family that also includes c-and the Flt3/Flk2 receptor. CSF-1R c-Kit and Flt3 all play pivotal functions in hematopoiesis. The importance of CSF-1-CSF-1R signaling is Roscovitine (Seliciclib) usually revealed by the pleiotropic functional defects of the CSF-1 null (autokinase activity of a catalytic fragment of PKCδ but activated PKCζ was not detected in that assay [27]. In another study PKCζ activation by CSF-1 was assessed by membrane translocation [28] but that may not be an adequate indication of PKCζ activation since atypical PKCs are not dependent on diacylglcyerol generated at the membrane for activation. Yet in a third study PKCζ knockdown was found to reduce CSF-1 induced macrophage migration [29]. Herein we tested the hypothesis that PKCζ may mediate the A-Raf impartial pathway to activate MEK-Erk in response to CSF-1 in myeloid cells: 32D.R myeloid progenitors and main bone marrow derived macrophages (BMMs). We found that CSF-1 increased PKCζ Thr 410 phosphorylation and kinase activity in 32D.R cells. Pharmacologic inhibition and transfection studies exhibited that atypical PKCs but not standard or novel PKCs contributed towards CSF-1 induced MEK-Erk activity in a c-Raf-1 and A-Raf-independent fashion. While PKCζ kinase inhibition reduced CSF-1 supported mitogenesis in 32D.R cells overexpression of PKCζ increased CSF-1 mitogenic responsiveness. However PKCζ’s promotion of mitogenic signaling in 32D.R cells was indie of NF-κB. In BMMs PKCζ inhibition experienced a more modest effect on CSF-1 dependent mitogenesis and pan-PKC inhibition experienced a paradoxically enhancing effect on MEK-Erk phosphorylation. Thus the importance of PKCζ in the control of CSF-1 mediated MEK-Erk activity and mitogenesis depends on differentiation stage. Methods Rabbit Polyclonal to TACD1. Antibodies and reagents Cell culture reagents and media were from Life Technologies (Carlsbad CA) or Sigma-Aldrich (St. Louis MO). GF109203X was from EMD Chemicals (Rockland MA) or Enzo Life Sciences (Plymouth Meting PA) Ro-31-8220 was from Axxora (San Diego CA) and Go 6983 was from EMD Chemicals. Myelin basic protein (MBP) was from Life Technologies PKCε pseudosubstrate peptide (residues 149-164 Ala to Ser 159) as phosphorylation substrate and myristoylated PKCζ pseudosubstrate peptide were from Enzo Life Sciences..