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Parathyroid hormone mediates hematopoietic cell expansion through interleukin-6

PLoS ONE (2010) 5(10)
DOI: 10.1371/journal.pone.0013657
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Abstract

Parathyroid hormone (PTH) stimulates hematopoietic cells through mechanisms of action that remain elusive. Interleukin-6 (IL-6) is upregulated by PTH and stimulates hematopoiesis. The purpose of this investigation was to identify actions of PTH and IL-6 in hematopoietic cell expansion. Bone marrow cultures from C57B6 mice were treated with fms-like tyrosine kinase-3 ligand (Flt-3L), PTH, Flt-3L plus PTH, or vehicle control. Flt-3L alone increased adherent and non-adherent cells. PTH did not directly impact hematopoietic or osteoclastic cells but acted in concert with Flt-3L to further increase cell numbers. Flt-3L alone stimulated proliferation, while PTH combined with Flt-3L decreased apoptosis. Flt-3L increased blasts early in culture, and later increased CD45+ and CD11b+ cells. In parallel experiments, IL-6 acted additively with Flt-3L to increase cell numbers and IL-6-deficient bone marrow cultures (compared to wildtype controls) but failed to amplify in response to Flt-3L and PTH, suggesting that IL-6 mediated the PTH effect. In vivo, PTH increased Lin- Sca-1+c-Kit+ (LSK) hematopoietic progenitor cells after PTH treatment in wild type mice, but failed to increase LSKs in IL-6-deficient mice. In conclusion, PTH acts with Flt-3L to maintain hematopoietic cells by limiting apoptosis. IL-6 is a critical mediator of bone marrow cell expansion and is responsible for PTH actions in hematopoietic cell expansion. © 2010 Pirih et al.

Figures

  • Figure 1. PTH augments Flt-3L cell expansion. Whole bone marrow was isolated from C57B6 mice and seeded at 1.86105 cells/cm2 and treated with vehicle, Flt-3L (100 ng/ml), PTH (10 nM), or a combination of Flt-3L and PTH. Non-adherent (A), and adherent (B), cell populations were harvested at days 2, 4, 6, and 8, then enumerated using trypan blue exclusion. Data shown are mean 6 SEM of 2 experiments performed in duplicate. Error bars are present on all data points. * p,0.05 versus vehicle ** p,0.05 versus all other groups. doi:10.1371/journal.pone.0013657.g001
  • Figure 2. Lack of direct effect of PTH on cells of the osteoclast lineage. (A) Non-adherent cells expanded in the presence or absence of Flt-3L (100 ng/ml) or PTH (10 nM), a combination of both, or vehicle (control), were counted and plated at equal numbers and allowed to differentiate in osteoclastogenic media, then induced via RANK-L (0–30 ng/ml) and M-CSF (50 ng/ml). Five days later, multi-nucleated TRAP+ cells were counted. Data are mean 6 SEM of 2 experiments performed in duplicate. * p,0.05 versus respective vehicle or PTH alone ** p,0.05 versus all other groups in their respective RANK-L concentrations. The 0 ng/ml RANK-L resulted in no osteoclasts; therefore, the data was not plotted. (B) Cells were expanded in Flt-3L (5 ng/ml) media for 8 days with or without 10 nM PTH. Cells were counted and plated at equal numbers, then induced via RANK-L (30 ng/ml) and M-CSF (50 ng/ml) to differentiate with additional treatments of PTH, forskolin or tetrahydrofuryladenine (THFA). Multinucleated TRAP+ cells were enumerated 5 days later. Data are mean 6 SEM of 2 experiments performed in duplicate. *p,0.05 and **p,0.01 versus vehicle. (C) Visualization of the cytoskeleton of actin, by confocal microscopy in mature osteoclasts seeded on coverslips or ACC and stained for actin and vinculin at different time points. All images are the same magnification. (D) Osteoclast transmigration assay: osteoclasts were seeded on MC3T3-E1 cell layers, treated with 0–10 nM PTH then fixed. Cells were stained with phalloidin to visualize actin under confocal microscopy. Data are mean 6 SEM of number of osteoclasts that transmigrated compared to the total number of osteoclasts. Experiments were performed a minimum of 3 times. * p,0.05 versus vehicle or PTH. (E) Osteoclast functional assay: Cells were expanded in the presence of Flt-3L (100 ng/ml). At day 8, they were seeded onto ACC (TRAP staining) or osteologic disks (resorption pit assay) and induced to differentiate in the presence of 50 ng/ml M-CSF and 30 ng/ml RANK-L. When osteoclasts started to form, PTH, calcitonin or vehicle (control) were added to the medium. Data are mean 6 SEM of the area of the pit divided by the total area. Experiments were performed a minimum of 3 times in duplicate. * p,0.05 versus vehicle. doi:10.1371/journal.pone.0013657.g002
  • Figure 3. Increased cell proliferation by Flt-3L but not PTH. Whole bone marrow was isolated from C57B6 mice and seeded at 1.86105 cells/ cm2 in the presence or absence of Flt-3L (100 ng/ml), PTH (10 nM), a combination of both, or vehicle only control. (A) cyclin D1 mRNA levels of nonadherent cells at days 2, 4, 6, and 8 as determined by real-time PCR. Data are mean 6 SEM of at least 3 experiments, performed in duplicate, normalized to GAPDH, and represented as treatment over control (T/C). *p,0.05 versus vehicle. (B) Representative western blot analyses of cyclin D1 and b-actin, and graphs of cyclin D1 protein normalized for b-actin in ex vivo cultures at days 2, 4, 6 and 8. Data are mean 6 SEM of 3 experiments performed in duplicate, and represented as treatment over control (T/C). * p,0.05 vs. vehicle. (C) Flow cytometric analyses of cell cycle ex vivo. Graphs represent the percentage of non-adherent cells stained for propidium iodide at days 2, 4, 6 and 8 to demonstrate G1, S and G2 phases. Experiments were performed 4 times in duplicate. *p,0.05 versus vehicle for G1, S and G2 phases. doi:10.1371/journal.pone.0013657.g003
  • Figure 4. PTH decreased cell apoptosis in a Flt-3L expanded population. Whole bone marrow was isolated from wild-type mice and seeded at 1.86105 cells/cm2 in the presence or absence of Flt-3L (100 ng/ml), PTH (10 nM), a combination of both, or vehicle only, (A) Flow cytometric analyses of Annexin V+ Propidium Iodide- (early apoptosis) cells performed on non-adherent cells. Representative Annexin V histogram from day 8. (B) Graph of the fold induction for percentage of Annexin V+ cells, (lower right quadrant from histograms represented in A) *p,0.05 versus Flt-3L, ** p,0.01 for vehicle and PTH versus Flt-3L. (C) Graph of the fold induction of active caspase 3+ cells. Data are mean 6 SEM of 4 experiments performed in duplicate. *p,0.05 versus Flt-3L. (D) Four-day-old wild-type C57B6 mice (n$9/group) were treated daily with 50 mg/kg PTH or vehicle for 3 weeks. Bone marrow was isolated and flow cytometric analyses of Annexin V+ cells were performed. Graph of the percentage of Annexin V+ cells, *p,0.05 versus vehicle. doi:10.1371/journal.pone.0013657.g004
  • Figure 6. IL-6 mediates the ex vivo and in vivo PTH effects. (A–B) Whole bone marrow was isolated from wild-type (WT) mice and seeded at 1.86105 cells/cm2 in the presence or absence of Flt-3L (100 ng/ml) or PTH (10 nM), a combination of both, or vehicle only control, with and without IL-6 (10 ng/ml). Non-adherent (A), and adherent (B), cells were harvested and enumerated using trypan blue exclusion at day 8. Data are mean 6 SEM of 2 experiments performed in duplicate *p,0.05 versus vehicle/vehicle, ** p,0.05 versus vehicle/Flt-3L. (C–D) Whole bone marrow was isolated from wild-type or IL-6 deficient mice (IL-6 KO) and seeded at 1.86105 cells/cm2 in the presence of Flt-3L (100 ng/ml), PTH (10 nM), a combination of PTH and Flt-3L, or vehicle only control. Non-adherent (C) and adherent (D) cells were harvested and enumerated using trypan blue exclusion at day 8 of culture. Data are mean 6 SEM of 2 experiments performed in duplicate. *p,0.05 versus vehicle (wild-type cells) **p,0.05 versus Flt-3L (wild-type cells) ***p,0.05 IL6-KO cells versus wild-type cells of the respective treatment group. Whole bone marrow was isolated from wild-type mice and seeded at 1.86105 cells/cm2 in the presence or absence of Flt-3L (100 ng/ml) or PTH (10 nM), a combination of both. One hour after cells were plated vehicle (control) or a STAT-3 inhibitor, cucurbitacin (20 nM) was added to the culture. Non-adherent cells were harvested and enumerated using trypan blue exclusion at day 8 of culture (E). Data are mean 6 SEM, from one of two experiments performed with similar results, *p,0.05 versus vehicle of the respective group, **p,0.05 versus Flt-3L of the respective group, ***p,0.05 vehicle versus cucurbitacin in the combined Flt-3L and PTH groups. (F) Four-day-old wild-type and IL-6-deficient mice (n$5/group) were treated daily with 50 mg/kg PTH or vehicle for 3 weeks. Bone marrow was isolated and flow cytometric analyses of Annexin V+ cells were performed. Fold induction of Annexin V+ cells measured as treatment over vehicle (control) of the respective phenotype. *p,0.05 versus vehicle of the respective phenotype. doi:10.1371/journal.pone.0013657.g006
  • Figure 7. PTH failed to increase LSK cells in IL-6 deficient mice. A) Bone marrow was isolated from four-day-old wild-type (WT) and IL-6deficient mice IL-6 KO) (n$9/group) and flow cytometric analyses of Lin- Sca-1+ c-Kit+ (LSK) cells were performed. Graph represents the percentage of LSK cells out of total bone marrow cells. B) Bone marrow was isolated from 26-day-old wild-type and IL-6 deficient mice (n$7/group) and flow cytometric analyses of Lin- Sca-1+ c-Kit+ (LSK) cells were performed. Graph represents the percentage of LSK cells per total bone marrow cells. C) Fourday-old wild-type and IL-6-deficient mice (n$6/group) were treated daily with 50 mg/kg PTH or vehicle for 3 weeks. Bone marrow was isolated and flow cytometric analyses of Lin- Sca-1+ c-Kit+ (LSK) cells were performed. Fold induction of LSK cells measured as treatment over vehicle (control) of the respective phenotype, *p,0.05 versus wildtype vehicle. doi:10.1371/journal.pone.0013657.g007
  • Figure 8. Working Model: IL-6 mediates the PTH anti-apoptotic effect on cells of the hematopoietic lineage. PTH induces IL-6 expression in stromal cells. Flt-3L increases hematopoietic cell proliferation. IL-6 acts in conjunction with Flt-3L reducing apoptosis in the hematopoietic cell compartment. doi:10.1371/journal.pone.0013657.g008

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Pirih, F. Q., Michalski, M. N., Cho, S. W., Koh, A. J., Berry, J. E., Ghaname, E., … McCauley, L. K. (2010). Parathyroid hormone mediates hematopoietic cell expansion through interleukin-6. PLoS ONE, 5(10). https://doi.org/10.1371/journal.pone.0013657

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