UCSD-Nat. reduces their proliferation; inhibition of JNK signaling or reduction of JNK1 levels restores proliferation. MNP recruitment to inflammatory sites and the corresponding bone marrow response is strongly impaired in Gab2-deficient mice. Our data provide genetic and biochemical evidence that CSF-1R, through Gab2, utilizes different effectors at different stages of MNP development to promote their expansion. INTRODUCTION Mononuclear phagocytes (MNPs) are critical in health to maintain tissue homeostasis and in disease as major effectors of innate immunity (7, 44). In the adult animal, MNPs develop from progenitors in the bone marrow (BM) that differentiate to monocytes (MOs), tissue macrophages (Ms), and specialized cells, including dendritic cells and osteoclasts. The human body makes 109 Butylparaben MOs/day and more under stress (59). Understanding the mechanisms regulating MNP production is essential not only to myelopoiesis but also inflammation. Colony-stimulating factor 1 (CSF-1) acts on the receptor tyrosine kinase CSF-1 receptor (CSF-1R) and is the primary cytokine regulating the proliferation, survival, and differentiation of MOs, Ms, and osteoclasts (44, 52). CSF-1 is also critical to dendritic and Langerhans cell expansion (15, 35). A recently discovered, less potent ligand for the CSF-1R, interleukin-34 (IL-34), has a spatiotemporal expression pattern different from CSF-1 during mouse development (63) and may be involved in microglial development (14). CSF-1-null (values were calculated using the Student’s 2-sided test and indicated on the figures as follows: *, 0.05; **, 0.005; and ns, not significant. Averages are given as the mean standard deviation (SD). RESULTS Gab2 not Gab3 promotes CSF-1-dependent proliferation and Akt Lpar4 or Erk activation. CSF-1 stimulation of 32D.R myeloid progenitors induced Gab2-3 tyrosine phosphorylation and association with known partners, SHP2 and Shc (19, 31). Gab1 expression was undetectable (Fig. 1 a). In BM-derived Ms (BMMs), CSF-1 provoked the tyrosine phosphorylation of Gab1-3 (Fig. 1b). To determine if Gab proteins play overlapping roles in the myeloid lineage, we stably knocked down Gab2 in 32D.R cells. Initially we cotransfected 32D.R cells with pU6 shRNAs and a puromycin selectable marker (36). Gab2 knockdown clones grew more slowly, but cell survival was not noticeably affected. Reconstitution with WT Gab2 cDNA lacking the shRNA target sequence restored proliferation (Fig. 2 a). Since puromycin-resistant clones required 2 to 3 3 weeks of selection and expansion, we also used shRNA lentiviruses for rapid GFP-based isolation. MTS results showed that the EC50 for knockdown clones was 3.1-fold higher than that of the control (Fig. 2b). Reducing Gab3 expression was associated with increased Gab2 levels so that the resulting EC50 was slightly diminished relative to the control (Fig. 2c). When Gab2 and Gab3 were simultaneously silenced, the EC50 was 3.9-fold higher relative to the control. Hence, downregulation of Butylparaben Gab3 in addition to Gab2 had only a small effect. Thus, the CSF-1R uses primarily Gab2 to promote proliferation in 32D.R cells. Open in a Butylparaben separate window Fig. 1. CSF-1 signals to Gab proteins in 32D.R cells (a) and BM-derived macrophages (b), as shown by immunoprecipitation (IP) and immunoblotting (IB) analyses. Previously identified tyrosyl phosphoproteins are indicated. An arrowhead points to the authentic Gab1 band that comigrated with tyrosine-phosphorylated Gab1. PY, phosphotyrosine; M, molecular mass markers (kDa). Open in a separate window Fig. 2. Silencing of Gab2 but not Gab3 reduces CSF-1-dependent proliferation in 32D.R. (a) Gab2 knockdown with pU6 shRNA vectors. WT Gab2 was overexpressed in clone 24. Fifty micrograms was loaded except for WT Gab2 (5 g). Growth curves and D4 viabilities were determined. The results are shown for 3 independent experiments. values were calculated using the one-way analysis of variance (ANOVA) test. (b) Gab2 knockdown with shGab2 lentiviruses. Each CSF-1 MTS response curve is the average of 2 independent experiments. (c) Gab3 knockdown (shGab3) or combined Gab2 and Gab3 knockdown (Double) in 32D.R. Levels were normalized to the averaged control clones. Each CSF-1 response curve is averaged from 3 independent experiments. (b and c) MTS curves normalized to 0 to 1 1 for determination of EC50. Control, thick black lines; shGab3, thin black lines; combined knockdown (Double), gray lines. We and other groups have shown that Gab2 signals through the.