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ERK Signaling Is Essential for Macrophage Development.

Richardson ET, Shukla S, Nagy N, Boom WH, Beck RC, Zhou L, Landreth GE, Harding CV - PLoS ONE (2015)

Bottom Line: Collectively, these data indicate that the defect in production of macrophages is not due to a reduced number of progenitors, but rather due to reduced ability of progenitors to proliferate and produce macrophages in response to M-CSF-triggered ERK signaling.Macrophages from Erk1(-/-) Erk2(flox/flox) Lyz2(Cre/Cre) bone marrow showed reduced induction of M-CSF-regulated genes that depend on the ERK pathway for their expression.These data demonstrate that ERK1/ERK2 play a critical role in driving M-CSF-dependent proliferation of bone marrow progenitors for production of macrophages.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Case Western Reserve University and University Hospitals Case Medical Center, Cleveland, Ohio, United States of America; Medical Scientist Training Program, Case Western Reserve University, Cleveland, Ohio, United States of America.

ABSTRACT
Macrophages depend on colony stimulating factor 1 (also known as M-CSF) for their growth and differentiation, but the requirements for intracellular signals that lead to macrophage differentiation and function remain unclear. M-CSF is known to activate ERK1 and ERK2, but the importance of this signaling pathway in macrophage development is unknown. In these studies, we characterized a novel model of Erk1(-/-) Erk2(flox/flox) Lyz2(Cre/Cre) mice in which the ERK2 isoform is deleted from macrophages in the background of global ERK1 deficiency. Cultures of M-CSF-stimulated bone marrow precursors from these mice yielded reduced numbers of macrophages. Whereas macrophages developing from M-CSF-stimulated bone marrow of Erk2(flox/flox) Lyz2(Cre/Cre) mice showed essentially complete loss of ERK2 expression, the reduced number of macrophages that develop from Erk1(-/-) Erk2(flox/flox) Lyz2(Cre/Cre) bone marrow show retention of ERK2 expression, indicating selective outgrowth of a small proportion of precursors in which Cre-mediated deletion failed to occur. The bone marrow of Erk1(-/-) Erk2(flox/flox) Lyz2(Cre/Cre) mice was enriched for CD11b+ myeloid cells, CD11b(hi) Gr-1(hi) neutrophils, Lin- c-Kit+ Sca-1+ hematopoietic stem cells, and Lin- c-Kit+ CD34+ CD16/32+ granulocyte-macrophage progenitors. Culture of bone marrow Lin- cells under myeloid-stimulating conditions yielded reduced numbers of monocytes. Collectively, these data indicate that the defect in production of macrophages is not due to a reduced number of progenitors, but rather due to reduced ability of progenitors to proliferate and produce macrophages in response to M-CSF-triggered ERK signaling. Macrophages from Erk1(-/-) Erk2(flox/flox) Lyz2(Cre/Cre) bone marrow showed reduced induction of M-CSF-regulated genes that depend on the ERK pathway for their expression. These data demonstrate that ERK1/ERK2 play a critical role in driving M-CSF-dependent proliferation of bone marrow progenitors for production of macrophages.

No MeSH data available.


Related in: MedlinePlus

ERK signaling is required for some macrophage responses to M-CSF.(A) Wild-type bone marrow-derived macrophages were treated with 2 ng/ml M-CSF for the indicated times, lysed, and analyzed by Western blotting. Data represent two independent experiments. (B) Bone marrow-derived macrophages were pre-treated for 30 min with U0126 or DMSO vehicle (all others) and then treated with 2 ng/ml M-CSF for 10 min, lysed, and analyzed by Western blotting. Densitometric values (in arbitrary units) for LysM, E1 and ERK1/2 respectively were 33.7, 31.9 and 32.3 for β-actin; 43.7, 36.1 and 6.81 for ERK2; and 31.3, 54.9 and 14.0 for p-ERK2. Data represent two independent experiments. (C) and (D) Bone marrow-derived macrophages were pre-treated for 30 min with U0126 or DMSO and then treated with 2 ng/ml M-CSF for 24 h. RNA expression was measured by qRT-PCR. Each data point represents mean ± standard deviation of triplicate samples. Data are representative of three independent experiments. (E) Bone marrow macrophages were pre-treated with U0126 or DMSO, treated with 2 ng/ml M-CSF for 24 h, collected by detaching the cells using PBS with 10 mM EDTA, stained with 2 μg/ml anti-CD115, and analyzed by flow cytometry. Data represent two independent experiments. Labels: -, untreated; +, treated with M-CSF; B6, C57BL/6J; E1, Erk1-/-; E2, Erk2flox/floxLyz2Cre/Cre; U, U0126-treated C57BL/6J. **: P<0.01, *: P<0.05, NS: P>0.05.
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pone.0140064.g002: ERK signaling is required for some macrophage responses to M-CSF.(A) Wild-type bone marrow-derived macrophages were treated with 2 ng/ml M-CSF for the indicated times, lysed, and analyzed by Western blotting. Data represent two independent experiments. (B) Bone marrow-derived macrophages were pre-treated for 30 min with U0126 or DMSO vehicle (all others) and then treated with 2 ng/ml M-CSF for 10 min, lysed, and analyzed by Western blotting. Densitometric values (in arbitrary units) for LysM, E1 and ERK1/2 respectively were 33.7, 31.9 and 32.3 for β-actin; 43.7, 36.1 and 6.81 for ERK2; and 31.3, 54.9 and 14.0 for p-ERK2. Data represent two independent experiments. (C) and (D) Bone marrow-derived macrophages were pre-treated for 30 min with U0126 or DMSO and then treated with 2 ng/ml M-CSF for 24 h. RNA expression was measured by qRT-PCR. Each data point represents mean ± standard deviation of triplicate samples. Data are representative of three independent experiments. (E) Bone marrow macrophages were pre-treated with U0126 or DMSO, treated with 2 ng/ml M-CSF for 24 h, collected by detaching the cells using PBS with 10 mM EDTA, stained with 2 μg/ml anti-CD115, and analyzed by flow cytometry. Data represent two independent experiments. Labels: -, untreated; +, treated with M-CSF; B6, C57BL/6J; E1, Erk1-/-; E2, Erk2flox/floxLyz2Cre/Cre; U, U0126-treated C57BL/6J. **: P<0.01, *: P<0.05, NS: P>0.05.

Mentions: We next explored how ERK1/2 macrophages would respond to M-CSF, which is known to trigger ERK phosphorylation among its multiple downstream signaling mechanisms. We compared results from ERK1/2 macrophages to control macrophages with intact ERK1 and ERK2 expression (from wild-type C57BL/6J or LysM mice), macrophages with single knockout of ERK1 or ERK2, or macrophages incubated with the highly specific MEK inhibitor U0126 [25] to comprehensively inhibit ERK signaling. We observed that M-CSF triggered rapid phosphorylation of ERK (peak at 5–15 min, Fig 2A). Densitometric analyses revealed that the ratios of p-ERK2 to ERK2 were 0.72 for LysM macrophages, 1.52 for E1 macrophages and 2.05 for ERK1/2 macrophages. However, due to the reduced levels of total ERK2, ERK1/2 macrophages had decreased total phosphorylated ERK levels relative to wild-type or single knockout macrophages (Fig 2B); as expected, U0126 fully blocked ERK phosphorylation. We studied the induction of two genes that are known to be induced by M-CSF and regulated by ERK, Cd33 [22] and Dusp5 [26]. Induction of these genes by M-CSF was reduced in ERK1/2 macrophages relative to LysM macrophages and in wild-type macrophages by treatment with U0126 (Fig 2C and 2D). M-CSF receptor was similarly expressed under all these conditions (Fig 2E), indicating that the explanation for altered gene induction by M-CSF was due to altered intracellular signal transduction as a consequence of reduced ERK expression rather than altered M-CSF receptor expression. Thus, ERK signaling is required for M-CSF induction of some genes.


ERK Signaling Is Essential for Macrophage Development.

Richardson ET, Shukla S, Nagy N, Boom WH, Beck RC, Zhou L, Landreth GE, Harding CV - PLoS ONE (2015)

ERK signaling is required for some macrophage responses to M-CSF.(A) Wild-type bone marrow-derived macrophages were treated with 2 ng/ml M-CSF for the indicated times, lysed, and analyzed by Western blotting. Data represent two independent experiments. (B) Bone marrow-derived macrophages were pre-treated for 30 min with U0126 or DMSO vehicle (all others) and then treated with 2 ng/ml M-CSF for 10 min, lysed, and analyzed by Western blotting. Densitometric values (in arbitrary units) for LysM, E1 and ERK1/2 respectively were 33.7, 31.9 and 32.3 for β-actin; 43.7, 36.1 and 6.81 for ERK2; and 31.3, 54.9 and 14.0 for p-ERK2. Data represent two independent experiments. (C) and (D) Bone marrow-derived macrophages were pre-treated for 30 min with U0126 or DMSO and then treated with 2 ng/ml M-CSF for 24 h. RNA expression was measured by qRT-PCR. Each data point represents mean ± standard deviation of triplicate samples. Data are representative of three independent experiments. (E) Bone marrow macrophages were pre-treated with U0126 or DMSO, treated with 2 ng/ml M-CSF for 24 h, collected by detaching the cells using PBS with 10 mM EDTA, stained with 2 μg/ml anti-CD115, and analyzed by flow cytometry. Data represent two independent experiments. Labels: -, untreated; +, treated with M-CSF; B6, C57BL/6J; E1, Erk1-/-; E2, Erk2flox/floxLyz2Cre/Cre; U, U0126-treated C57BL/6J. **: P<0.01, *: P<0.05, NS: P>0.05.
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getmorefigures.php?uid=PMC4596867&req=5

pone.0140064.g002: ERK signaling is required for some macrophage responses to M-CSF.(A) Wild-type bone marrow-derived macrophages were treated with 2 ng/ml M-CSF for the indicated times, lysed, and analyzed by Western blotting. Data represent two independent experiments. (B) Bone marrow-derived macrophages were pre-treated for 30 min with U0126 or DMSO vehicle (all others) and then treated with 2 ng/ml M-CSF for 10 min, lysed, and analyzed by Western blotting. Densitometric values (in arbitrary units) for LysM, E1 and ERK1/2 respectively were 33.7, 31.9 and 32.3 for β-actin; 43.7, 36.1 and 6.81 for ERK2; and 31.3, 54.9 and 14.0 for p-ERK2. Data represent two independent experiments. (C) and (D) Bone marrow-derived macrophages were pre-treated for 30 min with U0126 or DMSO and then treated with 2 ng/ml M-CSF for 24 h. RNA expression was measured by qRT-PCR. Each data point represents mean ± standard deviation of triplicate samples. Data are representative of three independent experiments. (E) Bone marrow macrophages were pre-treated with U0126 or DMSO, treated with 2 ng/ml M-CSF for 24 h, collected by detaching the cells using PBS with 10 mM EDTA, stained with 2 μg/ml anti-CD115, and analyzed by flow cytometry. Data represent two independent experiments. Labels: -, untreated; +, treated with M-CSF; B6, C57BL/6J; E1, Erk1-/-; E2, Erk2flox/floxLyz2Cre/Cre; U, U0126-treated C57BL/6J. **: P<0.01, *: P<0.05, NS: P>0.05.
Mentions: We next explored how ERK1/2 macrophages would respond to M-CSF, which is known to trigger ERK phosphorylation among its multiple downstream signaling mechanisms. We compared results from ERK1/2 macrophages to control macrophages with intact ERK1 and ERK2 expression (from wild-type C57BL/6J or LysM mice), macrophages with single knockout of ERK1 or ERK2, or macrophages incubated with the highly specific MEK inhibitor U0126 [25] to comprehensively inhibit ERK signaling. We observed that M-CSF triggered rapid phosphorylation of ERK (peak at 5–15 min, Fig 2A). Densitometric analyses revealed that the ratios of p-ERK2 to ERK2 were 0.72 for LysM macrophages, 1.52 for E1 macrophages and 2.05 for ERK1/2 macrophages. However, due to the reduced levels of total ERK2, ERK1/2 macrophages had decreased total phosphorylated ERK levels relative to wild-type or single knockout macrophages (Fig 2B); as expected, U0126 fully blocked ERK phosphorylation. We studied the induction of two genes that are known to be induced by M-CSF and regulated by ERK, Cd33 [22] and Dusp5 [26]. Induction of these genes by M-CSF was reduced in ERK1/2 macrophages relative to LysM macrophages and in wild-type macrophages by treatment with U0126 (Fig 2C and 2D). M-CSF receptor was similarly expressed under all these conditions (Fig 2E), indicating that the explanation for altered gene induction by M-CSF was due to altered intracellular signal transduction as a consequence of reduced ERK expression rather than altered M-CSF receptor expression. Thus, ERK signaling is required for M-CSF induction of some genes.

Bottom Line: Collectively, these data indicate that the defect in production of macrophages is not due to a reduced number of progenitors, but rather due to reduced ability of progenitors to proliferate and produce macrophages in response to M-CSF-triggered ERK signaling.Macrophages from Erk1(-/-) Erk2(flox/flox) Lyz2(Cre/Cre) bone marrow showed reduced induction of M-CSF-regulated genes that depend on the ERK pathway for their expression.These data demonstrate that ERK1/ERK2 play a critical role in driving M-CSF-dependent proliferation of bone marrow progenitors for production of macrophages.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Case Western Reserve University and University Hospitals Case Medical Center, Cleveland, Ohio, United States of America; Medical Scientist Training Program, Case Western Reserve University, Cleveland, Ohio, United States of America.

ABSTRACT
Macrophages depend on colony stimulating factor 1 (also known as M-CSF) for their growth and differentiation, but the requirements for intracellular signals that lead to macrophage differentiation and function remain unclear. M-CSF is known to activate ERK1 and ERK2, but the importance of this signaling pathway in macrophage development is unknown. In these studies, we characterized a novel model of Erk1(-/-) Erk2(flox/flox) Lyz2(Cre/Cre) mice in which the ERK2 isoform is deleted from macrophages in the background of global ERK1 deficiency. Cultures of M-CSF-stimulated bone marrow precursors from these mice yielded reduced numbers of macrophages. Whereas macrophages developing from M-CSF-stimulated bone marrow of Erk2(flox/flox) Lyz2(Cre/Cre) mice showed essentially complete loss of ERK2 expression, the reduced number of macrophages that develop from Erk1(-/-) Erk2(flox/flox) Lyz2(Cre/Cre) bone marrow show retention of ERK2 expression, indicating selective outgrowth of a small proportion of precursors in which Cre-mediated deletion failed to occur. The bone marrow of Erk1(-/-) Erk2(flox/flox) Lyz2(Cre/Cre) mice was enriched for CD11b+ myeloid cells, CD11b(hi) Gr-1(hi) neutrophils, Lin- c-Kit+ Sca-1+ hematopoietic stem cells, and Lin- c-Kit+ CD34+ CD16/32+ granulocyte-macrophage progenitors. Culture of bone marrow Lin- cells under myeloid-stimulating conditions yielded reduced numbers of monocytes. Collectively, these data indicate that the defect in production of macrophages is not due to a reduced number of progenitors, but rather due to reduced ability of progenitors to proliferate and produce macrophages in response to M-CSF-triggered ERK signaling. Macrophages from Erk1(-/-) Erk2(flox/flox) Lyz2(Cre/Cre) bone marrow showed reduced induction of M-CSF-regulated genes that depend on the ERK pathway for their expression. These data demonstrate that ERK1/ERK2 play a critical role in driving M-CSF-dependent proliferation of bone marrow progenitors for production of macrophages.

No MeSH data available.


Related in: MedlinePlus