T-cell protein tyrosine phosphatase (Tcptp) is a negative regulator of colony-stimulating factor 1 signaling and macrophage differentiation

Abstract

Mice null for the T-cell protein tyrosine phosphatase (Tcptp-/-) die shortly after birth due to complications arising from the development of a systemic inflammatory disease. It was originally reported that Tcptp-/- mice have increased numbers of macrophages in the spleen; however, the mechanism underlying the aberrant growth and differentiation of macrophages in Tcptp-/- mice is not known. We have identified Tcptp as an important regulator of colony-stimulating factor 1 (CSF-1) signaling and mononuclear phagocyte development. The number of CSF-1-dependent CFU is increased in Tcptp-/- bone marrow. Tcptp-/- mice also have increased numbers of granulocyte-macrophage precursors (GMP), and these Tcptp-/- GMP yield more macrophage colonies in response to CSF-1 relative to wild-type cells. Furthermore, we have identified the CSF-1 receptor (CSF-1R) as a physiological target of Tcptp through substrate-trapping experiments and its hyperphosphorylation in Tcptp-/- macrophages. Tcptp-/- macrophages also have increased tyrosine phosphorylation and recruitment of a Grb2/Gab2/Shp2 complex to the CSF-1R and enhanced activation of Erk after CSF-1 stimulation, which are important molecular events in CSF-1-induced differentiation. These data implicate Tcptp as a critical regulator of CSF-1 signaling and mononuclear phagocyte development in hematopoiesis.

FIG. 1.

Tissues from Tcptp^−/− mice are infiltrated with F4/80⁺ macrophages. Tissue sections from Tcptp^+/+ (A, C, E, and G) or Tcptp^−/− (B, D, F, and H) mice were stained with an anti-F4/80 antibody to detect tissue macrophages. Sections from spleen (A, B, C, and D), kidney (E and F), and skin (G and H) are shown. Panels A, B, E, and F were photographed at an original magnification of ×20, while panels C, D, G, and H were photographed at an original magnification of ×40. For each panel, a minimum of three mice of each genotype were analyzed. Arrows indicate staining of deep dermal macrophages.

FIG. 2.

Increased numbers of macrophage precursors in Tcptp^−/− bone marrow. Tcptp^+/+ or Tcptp^−/− bone marrow cells (1 × 10⁴) were plated in methylcellulose containing IL-3, SCF, IL-6, and Epo. At day 12, colonies were scored under high magnification to determine colony constitution and size (means ± SD [error bars]; n = 7; *, P < 0.01).

FIG. 3.

Increased mononuclear phagocytic precursors in Tcptp^−/− bone marrow. (A) Macrophage colonies derived from CFU-C in Tcptp^+/+ or Tcptp^−/− bone marrow cells (1 × 10⁴) cultured in the presence of 100 ng/ml CSF-1. Colonies were scored at day 7 (n = 6; means ± SD [error bars]; *, P < 0.01). (B) Bone marrow cells from Tcptp^+/+ or Tcptp^−/− mice were cultured for 3 days in the presence of IL-3 and CSF-1 to allow proliferation and differentiation of multipotent progenitors to nonadherent mononuclear phagocytic precursors. Cells were stained with anti-CD11b-FITC antibodies (solid line) to determine the percentage of mononuclear phagocytic precursors in both cell populations. Staining with an isotype-matched control antibody is shown as a dashed line (means ± SD [error bars]; n = 4, *, P < 0.01). (C) Increased proliferation of Tcptp^−/− committed mononuclear phagocyte precursor cells. Bone marrow cells were cultured as described for panel A to generate committed mononuclear phagocytes, and cells were labeled with CFSE and allowed to proliferate for 3 days. CFSE fluorescence was measured by flow cytometry, and cell populations were plotted as histograms. (D) Geometric means from histograms in panel C were plotted to quantitate CFSE dilution. Data were analyzed by an unpaired, two-tailed Student t test (means ± SD [error bars]; n = 3; *, P < 0.05). (E) Annexin V-propidium iodide staining of apoptotic cells in the mononuclear phagocyte cultures used in CFSE proliferation assay.

FIG. 4.

Increased sensitivity of GMP from Tcptp^−/− bone marrow to CSF-1. GMP from Tcptp^+/+ (A) and Tcptp^−/− (B) bone marrow were purified by FACS. These cells are defined as Lin⁻/Sca1⁻/CD127(IL-7Rα)⁻, CD117(c-KIT)⁺, FcR⁺, and CD34⁺. (C) Graphical representation of the number of GMP in Tcptp^+/+ and Tcptp^−/− per 10⁶ bone marrow cells. Error bars indicate standard deviations. (n = 4; *, P < 0.01). (D) Colonies derived from 100 GMP in clonogenic assays with 100 ng/ml CSF-1. (means ± SD [error bars]; n = 4, *, P < 0.01).

FIG. 5.

The CSF-1R is hyperphosphorylated in Tcptp^−/− macrophages. (A) BMDMs from Tcptp^+/+ or Tcptp^−/− mice were stimulated with 100 ng/ml CSF-1 for the indicated time points. Equal amounts of lysates were resolved by SDS-PAGE and analyzed by Western blotting with an antiphosphotyrosine antibody (top panel). The blot was reprobed with anti-CSF-1R antibodies (middle panel) and anti-Tcptp antibodies (bottom panel) to demonstrate the correct genotype of the cells. (B) The CSF-1R was immunoprecipitated from Tcptp^+/+ or Tcptp^−/− BMDMs stimulated with 100 ng/ml CSF-1 as indicated. The immunoprecipitates were resolved by SDS-PAGE and analyzed by Western blotting with antiphosphotyrosine (top panel), anti-CSF-1R phospho-Y807 (middle panel), and anti-CSF-1R (bottom panel) antibodies. (C and D) Graphical representation of total tyrosine phosphorylation of the CSF-1R (C) or phosphorylation of Y807 (D) in Tcptp^+/+ or Tcptp^−/− BMDMs at 10 min after CSF-1 stimulation. Western blots were quantitated by densitometry. Data were analyzed by an unpaired, two-tailed Student t test with the results of the wild-type set to 1 (means ± SD [error bars]; n = 3; *, P < 0.05).

FIG. 6.

The CSF-1R coimmunoprecipitates with Tcptp substrate-trapping mutants in vivo. FD-Fms cells were transfected with pEF-Tcptp-WT, pEF-Tcptp-DA, or empty vector, and then stimulated with 100 ng/ml CSF-1. Tcptp immunoprecipitates were analyzed by Western blotting with antiphosphotyrosine antibodies to reveal coimmunoprecipitating substrates (top panel). Immunoprecipitates were then blotted with anti-CSF-1R antibodies (middle panel) and anti-Tcptp antibodies (bottom panel) to ensure overexpression of the indicated constructs. The three right lanes contain whole-cell lysates of vector transfected cells to demonstrate the specificity of Tcptp for the tyrosine phosphorylated CSF-1R.

FIG. 7.

Tcptp^−/− macrophages have increased Erk activation. (A) Lysates from Tcptp^+/+ or Tcptp^−/− BMDMs stimulated with 100 ng/ml CSF-1 were analyzed by Western blotting with anti-phospho-Erk and anti-phospho-Akt antibodies. (B) Graphical representation of total Erk1/2 TEY phosphorylation in Tcptp^+/+ or Tcptp^−/− BMDMs at 10 min CSF-1 stimulation. Western blots were quantitated by densitometry. Data were analyzed by an unpaired, two-tailed Student t test with the results of the wild-type set to 1 (means ± SD [error bars]; n = 3, *, P < 0.05). (C) Increased Erk phosphorylation after CSF-1 sitmulation in Tcptp^−/− BMDMs is not prolonged. PDK1 activity in lysates from Tcptp^+/+ or Tcptp^−/− BMDMs stimulated with 100 ng/ml CSF-1 was also analyzed by Western blotting with anti-phospho-PDK1 antibodies.

FIG. 8.

Increased recruitment and tyrosine phosphorylation of a Grb2/Gab2/Shp2 complex after CSF-1 stimulation in Tcptp^−/− macrophages. (A) Gab2 was immunoprecipitated from CSF-1-stimulated Tcptp^+/+ or Tcptp^−/− BMDMs. Protein complexes were resolved by SDS-PAGE and analyzed by antiphosphotyrosine blotting and then by blotting for total Gab2 protein. (B) Anti-Shp2 immunoprecipitates from CSF-1-stimulated Tcptp^+/+ or Tcptp^−/− BMDMs were analyzed by antiphosphotyrosine immunoblotting as described for panel A. (C) Lysates from unstimulated and CSF-1-stimulated Tcptp^+/+ or Tcptp^−/− BMDMs were incubated with a GST-Grb2-SH2 domain fusion protein. The complexes were resolved by SDS-PAGE and analyzed by Western blotting with an anti-CSF-1R antibody. Input lysates from the experiment were blotted with an anti-CSF-1R antibody to ensure equal amounts of the CSF-1R were used and with an anti-Tcptp antibody to ensure the genotype of the cells used in the experiment.