Macrophages and CSF-1 play important roles in the development of inflammatory diseases. Tcptp−/−
mice die shortly after birth and suffer from splenomegaly, lymphadenopathy, chronic myocarditis, gastritis, and nephritis, which are all symptoms of an inflammatory disorder (12
). Although Tcptp−/−
macrophages are sensitive to activating stimuli, such as IFN-γ and lipopolysaccharide, the mechanism by which Tcptp regulates the growth and development of this lineage remained unclear. In this study, we have identified Tcptp as a novel regulator of CSF-1 signaling and mononuclear phagocyte development.
We have demonstrated that several tissues in Tcptp−/−
mice have increased numbers of F4/80+
macrophages. Since CSF-1 had been implicated as the primary growth factor regulating the growth and development of macrophages in vivo, we investigated the role of Tcptp in CSF-1 signaling and macrophage differentiation. Analysis of the number and type of CFU present in bone marrow revealed a fourfold increase in CFU-M in Tcptp−/−
mice relative to wild-type controls. It has been previously demonstrated that CFU-C are the most primitive cells upon which CSF-1 can act alone to promote differentiation. CSF-1 can also act in concert with other cytokines to promote the proliferation and differentiation of multipotent hematopoietic precursor cells (4
). When we assessed the number of CFU-C in Tcptp+/+
mice, we observed a twofold increase in the number of committed mononuclear phagocyte precursors present in Tcptp−/−
bone marrow relative to the wild-type and increased proliferation of Tcptp−/−
committed mononuclear phagocyte precursors. These data implicate Tcptp in the regulation of the proliferation and differentiation of multipotent cells to committed mononuclear phagocyte precursors during hematopoiesis. Furthermore, when we cultured GMP from Tcptp−/−
mice in the presence of CSF-1 alone, these cells generated fourfold more macrophage colonies relative to Tcptp+/+
GMP. Therefore, the loss of Tcptp results in the sensitivity of early myeloid progenitors to the effects of CSF-1.
The phosphorylation of the activation loop tyrosine in protein tyrosine kinases is critical for the complete activation of kinases. Similarly, the phosphorylation of Y807 in the CSF-1R activation loop is required for proper phosphorylation and activation of the receptor. In fact, Erk activation has been shown to be dependent on the phosphorylation of Y807 (16
). Furthermore, the phosphorylation of Y807 is also an important event in differentiation since FD-Fms cells expressing the Y807F mutant CSF-1R proliferate in response to CSF-1 but have a reduced capacity to differentiate (7
). Our data identify Tcptp as a novel regulator of CSF-1R Y807 phosphorylation and Erk activation in CSF-1 signaling—two known molecular events induced by CSF-1 that are required for effective macrophage differentiation.
Recent studies have also demonstrated that the activation of Erk is important for macrophage differentiation in response to CSF-1 (22
). However, the recruitment of Grb2/Sos complexes to the receptor, which is the “classical” mechanism of Ras and Erk activation, does not play a major role in promoting the prolonged activation of Erk that is required for macrophage differentiation. In fact, the disruption of Grb2/Sos complexes in FD-Fms cells with cell-permeable peptides did not affect Erk activation or the ability of these cells to differentiate in response to CSF-1 (22
). Therefore, these data implicate the recruitment of Gab2/Shp2 as a critical pathway for Erk signaling and differentiation.
Shp2 recruitment through members of the Gab family of scaffold proteins is an important mechanism for Erk activation in numerous signaling pathways, and the function of Gab family members in the activation of Erk is conserved from Drosophila
to mammals (20
). The differentiation of Shp2-null embryonic stem cells using in vitro CFU assays was unable to yield any granulocyte and macrophage precursors, thereby implicating the activation of Shp2 as an important event in macrophage development (43
). In fact, the overexpression of a Gab2 mutant lacking the Shp2 binding sites in FD-Fms cells inhibited macrophage differentiation in response to CSF-1 (30
). In accordance with a role for Gab2 in CSF-1-induced Erk activation, Gab2-null mice have severely reduced Erk activation in mast cells after treatment with both immunoglobulin E and SCF (25
) and the differentiation of mast cells was also found to be severely impaired in Gab2−/−
). Shp2 also can also attenuate Akt activation via the dephosphorylation of PI3K binding sites on Gab proteins in a negative feedback loop mechanism (60
). However, in Tcptp−/−
macrophages we did not observe any significant changes in the level of Akt activation or the activity of PDK1, an upstream activator of Akt, relative to the wild type. This is likely due to the fact that PI3K can bind the CSF-1R directly, which can result in Akt activation independently of Gab2 recruitment. The precise mechanism underlying the increased recruitment of a Grb2/Gab2/Shp2 complex in Tcptp−/−
macrophages is unclear. Although our data imply that hyperphosphorylation of one or both of the known Grb2 binding sites (Y697 or Y921) may be involved, we are unable to test this directly since relevant phosphospecific antibodies are not available. However, the hyperphosphorylation of Y807 in Tcptp−/−
macrophages likely leads to changes in the overall phosphorylation status and kinetics of CSF-1R activation, which could in turn favor the formation of a specific signaling protein complex.
Another protein tyrosine phosphatase family member, Shp1, also plays an important role in myeloid cell differentiation, proliferation, and activation (48
). In fact, motheaten mice, which express either a null or hypomorphic mutant of Shp1, have increased numbers of macrophages in the extremities, resulting in arthritis, and increased levels of macrophages in the lungs, resulting in fatal pneumonitis (23
). The CSF-1R is also hyperphosphorylated in Shp1 mutant macrophages (5
). Shp1 mutant mice also have increases in the numbers of CSF-1-responsive CFU-C and granulocyte-macrophage CFU in bone marrow (36
), indicating that Shp1 plays an important role in myeloid differentiation. However, unlike Tcptp−/−
mice, motheaten mice have increased numbers of both granulocytes and macrophages (53
). We observed changes in only the number of CFU-M in Tcptp−/−
bone marrow, while the number of CFU-G was comparable to the wild-type. Recent studies have also demonstrated that the expression of a dominant-negative Shp1 mutant affects the differentiation of both granulocytes and macrophages in a cell autonomous manner (40
). These data indicate a significant difference between the functions of Tcptp and Shp-1 in myeloid cells. Shp1 most likely acts as a general regulator of myelopoiesis and proliferation, while Tcptp specifically acts to balance mononuclear phagocyte differentiation in vivo through its action upon the CSF-1R.
The lineage commitment of hematopoietic stem cells is thought to be determined by stochastic changes in the levels of lineage-specific transcription factors in these cells (19
), but the effects of the extracellular environment, in the form of cytokines and growth factors, cannot be completely excluded (34
). One model of hematopoiesis proposes that lineage-specific growth factors and cytokines promote the survival and growth of committed cells, while another model implicates these extrinsic signals in commitment decisions. The negative regulation of CSF-1 signaling by Tcptp partly explains the increase in CFU-M that we observed in Tcptp−/−
mice; however, we cannot exclude changes in other growth factor and cytokine signaling pathways involved in early myelopoiesis that are directly affected by the loss of Tcptp. However, the increase in GMP in Tcptp−/−
mice indicates that CSF-1 also plays a role in early myeloid differentiation.
It is possible that the expression levels of lineage-specific transcription factors involved in myelomonocytic differentiation may be affected in Tcptp−/−
hematopoietic progenitor cells, thereby influencing lineage commitment. The level of one such transcription factor, PU.1, has been shown to regulate the development of the myelomonocytic lineage, and its regulation may prove to be an important mechanism underlying the phenotype observed in Tcptp−/−
). However, our results do demonstrate that the loss of a single specific regulator can bias early cell fate decisions during hematopoiesis. Recently, the deletion of Socs3 demonstrated its role as a specific negative regulator of G-CSF signaling and neutrophil proliferation and development (15
In this report, we have identified Tcptp as a novel regulator of CSF-1 signaling, and we have also demonstrated that the production of committed mononuclear phagocyte precursors from Tcptp−/− bone marrow progenitors is increased. These data implicate Tcptp and CSF-1 in the early regulation of myelopoiesis. The identification of specific negative regulators of hematopoiesis, such as Tcptp, is an important step in understanding how cellular responses to extracellular cues affect the intracellular mechanisms of lineage selection.