Abstract
The bone marrow microenvironment, including osteolineage cells, regulates hematopoietic stem cell (HSC) fate choices. Intermittent pharmacologic treatment of mice with parathyroid hormone, PTH (1-34), indirectly increases HSCs through their niche, as HSCs do not express the PTH receptor (PTH1R). Osteocytes, the most abundant osteolineage cells in bone, are a critical target of the skeletal actions of PTH and coordinate multiple cell types that are components of the HSC niche including osteoblasts, osteoclasts and resident macrophages. While osteocytes express the PTH1R, the role of osteocytes in HSC regulation is unclear. Therefore, we studied the role of osteocyte-mediated PTH regulation of HSCs, using cre recombinase driven by the 8kb-DMP1 promoter to conditionally delete PTH1R in osteocytes (OCyPTHRko mice).
OCyPTHRko mice were viable, fertile, did not exhibit any significant skeletal defect as juveniles or at 6 months of age, had no significant difference in serum PTH levels, and had no significant difference in osteoblastic or mesenchymal stem cell numbers compared to WT mice. In juvenile OCyPTH1Rko mice there was a decrease in long-term HSCs as measured by flow cytometric analysis (0.0029 ± 0.00028 vs. 0.0021 ± 0.00021 % of cells, WT vs. OCyPTH1Rko p≤0.05 N≥19 mice/group). OCyPTH1Rko mice had 4 fold lower long-term engraftment capacity as measured by secondary competitive transplantation over 16 weeks (WT vs. OCyPTH1Rko donors, 2-way ANOVA p≤0.001, N≥10 mice/group) that was evident in all hematopoietic lineages. Short-term engraftment however was increased in OCyPTH1Rko mice as measured by primary competitive transplantation (WT vs. OCyPTH1Rko donors, 2-way ANOVA p≤0.01, N≥9 mice/group). These data demonstrate that physiologic PTH signaling in osteocytes regulates the balance of long-term and short-term HSC potential in juvenile, growing mice. Adult OCyPTH1Rko mice also had 5 fold lower long-term engraftment as measured by secondary competitive transplantation over 16 weeks (WT vs. OCyPTH1Rko donors, 2-way ANOVA p≤0.001, N≥15 mice/group). These findings demonstrate a previously unrecognized physiologic role of PTH signaling in HSC regulation. Having demonstrated a role for PTH signaling in HSC homeostasis, we investigated if sustained PTH elevations (as are found in vitamin D deficiency and in hyperparathyroidism) alter HSC function. Therefore, we utilized a murine model of secondary hyperparathyroidism caused by a low calcium (LCa) diet. In juvenile mice placed on the LCa diet immediately upon weaning, serum PTH levels were significantly elevated. Fourteen days on the LCa diet caused a significant reduction in long-term engraftment potential as measured by secondary competitive transplants over 22 weeks (Normal vs. LCa diet donors, 2-way ANOVA p≤0.001, N≥20 mice/group), while there was no decrease in HSCs when adult mice were placed on the LCa diet. These data suggest that sustained PTH signaling decreases microenvironmental support for HSCs in juvenile mice. We utilized the OCyPTHRko mice to study the role of osteocytes in hyperparathyroidism-induced loss of functional HSCs. In juvenile mice the lack of PTH signaling in osteocytes rescued the long-term engraftment defects, suggesting that PTH signaling in osteocytes mediates the loss of long-term HSC support caused by the LCa diet. In further support of a deleterious effect mediated by the PTH1R in osteocytes in the setting of continuous PTH, adult OCyPTH1Rko mice placed on LCa diet had superior long term HSC function.
Our findings demonstrate a physiologic role for PTH in HSC regulation and identify osteocytes as a critical constituent of the HSC niche that, either directly or indirectly, contribute to maintenance of the long-term repopulating HSC pool. In addition, we show that continuous exposure to elevated levels of PTH in a model of secondary hyperparathyroidism leads to osteocyte-mediated loss of long-term engraftment potential of HSCs in juvenile mice. We speculate that removing the effect of continuous PTH from osteocytes uncovers additional HSC-supportive effects of continuous PTH, mediated by non-osteocyte HSC niche cellular populations. Together these data establish PTH as a critical regulatory signal in the HSC niche, and show that the relative contributions of niche populations to HSC regulation are modulated by age.
Disclosures
Calvi: Fate Therapeutics: Patents & Royalties.
The parathyroid hormone-dependent activation of osteoblasts enhances hematopoietic stem cell migration and reduces their engraftment abilities
