CGRP-CRLR/RAMP1 signal is important for stress-induced hematopoiesis

Ecotropic viral integration site-1 (EVI1) has a critical role in normal and malignant hematopoiesis. Since we previously identified high expression of calcitonin receptor like receptor (CRLR) in acute myeloid leukemia (AML) with high EVI1 expression, we here characterized the function of CRLR in hematopoiesis. Since higher expression of CRLR and receptor activity modifying protein 1 (RAMP1) was identified in immature hematopoietic bone marrow (BM) cells, we focused on calcitonin gene-related peptide (CGRP), a specific ligand for the CRLR/RAMP1 complex. To elucidate the role of CGRP in hematopoiesis, Ramp1-deficient (Ramp1−/−) mice were used. The steady-state hematopoiesis was almost maintained in Ramp1−/− mice; however, the BM repopulation capacity of Ramp1−/− mice was significantly decreased, and the transplanted Ramp1−/− BM mononuclear cells had low proliferation capacity with enhanced reactive oxygen species (ROS) production and cell apoptosis. Thus, CGRP is important for maintaining hematopoiesis during temporal exposures with proliferative stress. Moreover, continuous CGRP exposure to mice for two weeks induced a reduction in the number of BM immature hematopoietic cells along with differentiated myeloid cells. Since CGRP is known to be increased under inflammatory conditions to regulate immune responses, hematopoietic exhaustion by continuous CGRP secretion under chronic inflammatory conditions is probably one of the important mechanisms of anti-inflammatory responses.

SCienTiFiC REPORts | (2019) 9:429 | DOI: 10.1038/s41598-018-36796-0 in C fibers and upstream, with mechanical stimulation, infection, ischemia, and pain [28][29][30] . Binding of CGRP to the CRLR/RAMP1 receptor activates adenylate cyclase, which elevates intracellular cAMP and activates protein kinase A (PKA), leading to phosphorylation of multiple targets, such as potassium-sensitive ATP channels, extracellular signal-related kinases (ERKs), and cAMP response element-binding protein (CREB) 28 . Although the ability of CGRP to regulate immune cells, such as dendritic cells and lymphocytes, under inflammation has been reported 29 , the function of CGRP in HSCs and/or LSCs has not been clearly elucidated. Therefore, here, we investigated how CGRP-mediated activation of the CRLR/RAMP1 signaling pathway affects hematopoiesis using Ramp1 −/− mice. The CRLR/RAMP1 complex was highly expressed in hematopoietic progenitor cells. The hematopoiesis of Ramp1 −/− mice under steady-state conditions was not affected, which was revealed by examining their hematopoietic progenitor cells in the BM and differentiated blood cells in the peripheral blood (PB). However, the BM repopulation capacity of Ramp1 −/− mice was significantly decreased, and the transplanted Ramp1 −/− bone marrow mononuclear cells (BMMNCs) had low proliferation capacity with enhanced ROS production and cell apoptosis. Therefore, the CGRP-CRLR/RAMP1 signaling pathway might play an important role in hematopoiesis under proliferative stress conditions. Moreover, given that long-term CGRP administration to mice decreased the numbers of most of immature hematopoietic cells and differentiated blood cells, it is suggested that continuous CGRP secretion under inflammation and chronic disease may be involved in the hematopoietic exhaustion.

CGRP is a ligand of Crlr/Ramp1 in murine hematopoietic cells and the hematopoiesis of Ramp1deficient mice is maintained under steady-state condition.
To determine whether Crlr is expressed in normal murine BMMNCs, several BM cell fractions [lineage marker (Lin) − Sca-1 + c-kit + (LSK), common myeloid progenitors (CMP) (Lin − Sca-1 − c-kit + CD16/32 − CD34 + ), granulocyte/monocyte progenitor (GMP) (Lin − Sca-1 − c-kit + CD16/32 + CD34 + ), megakaryocyte/erythroid progenitor (MEP) (Lin − Sca-1 − c-kit + CD16/32 + CD34 + ), myeloid (Gr-1 + CD11b + ), and lymphoid (B220 + for B lineage and CD3e + for T lineage)] were separated from mouse BM by cell sorting using specific antibodies (Methods), and levels of Crlr mRNA and protein expression were determined in the BM fractions by real-time PCR (Fig. 1a,b) and flow cytometry (Fig. 1c), respectively. The highest Crlr mRNA expression was detected in the LSK fraction, and the expression levels were gradually decreased during progression of differentiation to myeloid and lymphoid lineages (Fig. 1a). Moreover, Ramp1 mRNA was highly expressed in the LSK fraction, but Ramp2 and Ramp3 mRNAs expression was not detected (Fig. 1b), suggesting that the ligand of Crlr is possibly CGRP. The expression patterns of Crlr and Ramp1 proteins were almost same tendency as those of the respective mRNAs (Fig. 1c). To assess whether CGRP is really a ligand for Crlr in hematopoietic progenitor cells, LSK cells were treated with 100 nM CGRP and intracellular cAMP responsiveness was measured (Fig. 1d). The CGRP treatment significantly increased intracellular cAMP concentrations in LSK cells, suggesting that CGRP is a ligand for Crlr and Ramp1 in hematopoietic progenitor cells.
To determine whether the Crlr/Ramp1 signaling pathway affects hematopoietic cell growth and differentiation, Ramp1 −/− mice were used 31 . Ramp1 −/− mice were born and grew similarly to wild-type (WT) mice. When mice were 8 to 12 weeks old, we determined the number of blood cells in the PB and BM of the mice. The numbers of white blood cells (WBCs), red blood cells (RBCs), and platelets (Fig. 1e), and percentages of population of myeloid cells (granulocytes and monocytes), and B and T lymphocytes (Fig. 1f) in the PB of Ramp1 −/− mice were not significantly different from those in WT mice. In addition, percentages of population of myeloid cells, B and T lymphocytes in the BM (Fig. 1g) and the colony-forming ability of BMMNCs (Fig. 1h) were not significantly different between Ramp1 −/− and WT mice. Moreover, the numbers of BMMNCs and fractionated immature hematopoietic cells with various lineage cell fractions [HSC (Lin − Sca-1 + c-kit + CD150 + CD48 − ), transiently reconstituting multipotent progenitor (MPP) (Lin − Sca-1 + c-kit + CD150 − CD48 − ), CMP, GMP, and MEP] were almost the same between Ramp1 −/− and WT mice (Fig. 1i), suggesting that Crlr/Ramp1 signaling might not an important factor for the regulation of hematopoiesis under steady-state conditions. Activation of the Crlr/Ramp1 signaling pathway through CGRP stimulation is important for maintenance of murine hematopoietic stem cells under proliferative stress conditions. Since CGRP is known to be released from sensory nerves in response to several stress conditions, such as irradiation 32 or 5-fluorouracil (5-FU) treatment, we here investigated whether Crlr/Ramp1 signaling has an important function in hematopoietic cells under stress conditions using BM transplantation. Initially, to investigate whether radiation therapy induces CGRP secretion in the BM, the BM cells were analyzed for the expression of CGRP protein and proCGRP mRNA four days after irradiation with 10 Gy. The levels of CGRP protein and proCGRP mRNA were significantly increased after the irradiation treatment (Fig. 2a,b). To evaluate short-term stress hematopoiesis, a single sublethal dose of 150 mg/kg 5-FU was administered to WT (n = 4) and Ramp1 −/− (n = 4) mice. Since all of the mice survived at 30 days after the 5-FU treatment (Supplemental Fig. S2a), the mice were sacrificed at seven days after 5-FU injection and determined the percentages and absolute numbers of immature BM cells and differentiated myeloid cells. The percentages and total numbers of LSK cells in the BM of Ramp1 −/− mice were significantly decreased as compared to WT mice (Fig. 2c,d). The numbers of the myeloid progenitor cells (MP; Lin − Sca1 − c-kit + ) and the proportion of GMP were also significantly decreased in the BM of Ramp1 −/− mice (Fig. 2d) whereas the differentiated myeloid cell population was largely unaffected (Supplemental Fig. S2b). To evaluate hematopoiesis under exposure to temporal proliferative stress, we performed a competitive repopulation assay. Briefly, after 5.0 × 10 5 of BMMNCs from WT (CD45.2 + ) or Ramp1 −/− (CD45.2 + ) mice together with the same number of competitor BMMNCs from WT mice (CD45.1 + ) were transplanted into irradiated recipient WT mice (CD45.1 + ), percentages of donor-derived cells in the recipient mice were determined monthly for four months. In the first transplantation, percentages of donor-derived cells in BMMNCs or each PB cell fraction  (Fig. 3a), although the homing abilities of the transplanted BMMNCs were not significantly different between Ramp1 −/− and WT mice (Fig. 3b). Moreover, the rate of ROS production and the percentage of apoptotic cells were significantly increased in Lin − cells from Ramp1 −/− mice as compared to those in WT mice ( Fig. 3c and d). To evaluate whether intrinsic CGRP secretion from mesenchymal cells affect the growth and differentiation of BM cells under stress conditions, the BMMNCs isolated from WT mice were cultured alone or co-cultured with BM stromal cell fraction in the presence or absence of CGRP 8-36 , a peptide antagonist for CGRP1 receptor. After co-culturing with BM stromal cells, the numbers of LSK cells were significantly increased, while LSK numbers were unchanged in the presence of CGRP 8-36 (Fig. 3e), suggesting that the CGRP signaling from BM stromal cells is essential for the increase of LSK pool in the BM. Additionally, the numbers of differentiated myeloid cells were not significantly different between the three groups under these conditions (Supplemental Fig. S3). Therefore, these results suggest that the secretion of CGRP from mesenchymal cells might promote the hematopoiesis through the enhancement of cell proliferation with suppression of ROS production and apoptosis in BM cells under proliferative stress conditions.

Sustained CGRP treatment leads to the reduction in the number of hematopoietic cells in vivo.
To evaluate whether continuous CGRP treatment affects hematopoietic cells, in vitro replating assays were performed in culture medium with or without CGRP. The colony-forming ability and the percentage of Gr-1 + CD11b + myeloid cell population in BMMNCs from WT mice were significantly increased in culture medium with CGRP as compared to those of the medium without CGRP in the first replating (Fig. 4a). However, the colony-forming ability was significantly reduced in the presence of CGRP after the second replating (Fig. 4a, left). In addition, the colony-forming ability and the percentage of myeloid cell population in BMMNCs from Ramp1 −/− mice were not significantly different between CGRP-treated and untreated groups (Fig. 4b). These results suggest that CGRP treatment enhances the colony-forming ability of BMMNCs with myeloid cell differentiation, and that continuous stimulation with CGRP may reduce the hematopoietic proliferation and differentiation of BM cells.
To examine whether continuous CGRP exposure affects hematopoietic cells in vivo, high-dose CGRP (0.2 mg/ two weeks) or phosphate-buffered saline (PBS) as a control were subcutaneously and continuously administered into WT mice for two weeks using an osmotic infusion pump, and the numbers of blood cells and the percentages of population of each PB cell fraction (myeloid, B cells, and T cells) were determined at one and two weeks after CGRP treatment. One week after the CGRP treatment, the numbers of WBCs, myeloid cells, RBCs, and PLTs were not significantly different between the control and CGRP-treated groups; however, the numbers of myeloid cells and PLTs were significantly reduced two weeks after the CGRP treatment (Fig. 4c). In addition, within the WBC population, the percentages of myeloid cell population were significantly decreased (Fig. 4d), suggesting that the cells, CD45.2) from Ramp1 −/− and WT mice in BMMNCs, myeloid cells, B lymphocytes, and T lymphocytes from the PB of lethally irradiated WT recipients (CD45.1) at each indicated time point after transplantation (1, 2, 3, and 4 months). Donor/recipient ratio: 50:50. Data are shown as means ± SD of four mice. *p < 0.05, **p < 0.01 compared with WT mice (Student's t test). (f) The proportion of donor-derived myeloid, B lymphocytes, and T lymphocytes (Ramp1 −/− and WT mice) among donor-derived WBCs in the PB of lethally irradiated WT recipients at four months after transplantation. Data are shown as means ± SD of four mice. (g) Chimerism (percent donor-derived cells, CD45.2) from Ramp1 −/− and WT mice in BMMNCs, Lin + , Lin − , MP, and LSK cells in BMMNCs from lethally irradiated WT recipients (CD45.1). Donor/recipient ratio: 50:50. Data are shown as means ± SD of four mice. *p < 0.05, **p < 0.01 compared with WT mice (Student's t test). Moreover, among BMMNCs, the numbers of all immature hematopoietic cell fraction including HSC and CMP, were significantly decreased in CGRP-treated mice as compared to those of the controls (Fig. 4e, left), while the percentage of CMP was significantly decreased in CGRP-treated mice than in controls (Fig. 4e, right). To study the reduction mechanism of blood cells after two weeks of continuous CGRP treatment, the expression of Crlr in BMMNCs cells was determined by immunoblot analysis. As shown in Fig. 4f, the Crlr expression was decreased in BMMNCs after two-weeks treatment with CGRP. Moreover, after separation of BMMNCs from CGRP-treated mice, we determined the colony-forming abilities of BMMNCs in the presence or absence of CGRP in in vitro culture. In the absence of CGRP, the colony-forming abilities of BMMNCs from CGRP-treated mice were significantly decreased compared to those of WT-mice (Fig. 4g). While the CGRP treatment significantly increased the colony-forming abilities of BMMNCs from the PBS-treated mice, it had no obvious effect on the colony-forming abilities in BMMNCs from the CGRP-treated mice (Fig. 4g). These results suggest that continuous exposure of CGRP induces reduction of myeloid/erythroid/megakaryocyte immature hematopoietic progenitor cells with downregulation of the Crlr expression in BM cells, leading to the depletion of immature and differentiated blood cells, particularly myeloid cells.

Discussion
In this study, we identified CGRP as the specific ligand for CRLR/RAMP1 in HSPCs. We found that the CGRP-CRLR signaling is not an important factor for the regulation of murine hematopoiesis under steady-state conditions, and that the BM repopulation ability of BMMNCs from Ramp1-deficient mice is significantly inhibited following BM transplantation in mice. Moreover, the transplanted BMMNCs from Ramp1-deficient mice displayed a decreased cell proliferation with enhanced ROS accumulation and apoptosis, while CGRP treatment of BMMNCs from wild-type mice but not Ramp1-deficient mice increased the colony forming ability along with production of differentiated progenitor cells. Surprisingly, however, continuous treatment with CGRP induced a reduction of hematopoietic proliferation and differentiation in vivo in mice as well as in in vitro experiments. Therefore, we propose that one of the important functions of CGRP in HSPCs is possibly to enhance the hematopoiesis under temporal exposures to proliferative stress; however, under pathological conditions of persistent proliferative stress via continuous CGRP secretion, HSPCs lose Crlr expression, which may promote the depletion of hematopoietic progenitors and differentiated cells. CGRP is one of the main neurotransmitters involved in immune function and is released in response to activation of TRPV1 through sensory neurons and C fibers and is a key responder to tissue damage that is perceived as "pain". The sensory C fiber neurons innervate every organ in the body and particularly TRPV1 and CGRP have been identified in the subset of neuron fibers within the trabecular bone in the BM 33 . When TRPV1 is activated by external factors, such as heat, pH, ischemia, bone fracture, irradiation, or toxins, CGRP is released from the sensory neurons, resulting in the expansion of the numbers of circulating granulocytes, macrophages, mast cells, and other immune cells at various locations to defend the body and heal the damage. The CGRP signaling has been implicated in the cAMP, PKC, ERK, and p38 signal transduction pathways 28 . In particular, cAMP-dependent activation of CREB transcription factor has been shown to play a central role in granulopoiesis directly from CD34 + BM cells 34 . In this study, we demonstrated for the first time that cells of the BM stromal fraction secretes CGRP to stimulate proliferation and differentiation of BM cells, and that the CGRP-CRLR/Ramp1 signaling plays a critical role in stress-induced hematopoiesis.
CGRP has an important function that connects nervous and immune systems, and directly or indirectly targets peripheral immune cells such as dendritic cells, mast cells, T cells and macrophages in peripheral tissues 29 . In acute stages of inflammation, CGRP that is present in peripheral tissues is known to enhance inflammatory responses by dilation of blood vessels, extravasation of inflammatory cells, activation of secretion of inflammatory cytokines 35 , and, in the later stage of inflammation, the proinflammatory activities of macrophages and lymphocytes can be inhibited by the action of CGRP, leading to the suppression of inflammatory responses 36 . Consistent with this, CGRP stimulates hematopoiesis in the BM after initiation of inflammation, which may compensate proliferating hematopoietic cells including monocytes, which are recruited to the local inflammatory tissues. On the other hand, in chronic inflammation, expression of the CGRP receptor CRLR may be reduced and the loss of CGRP function may inhibit hematopoiesis with reduction of myeloid cells, which could terminate the inflammatory responses by reduction of tissue-invaded microphages.
In summary, the CGRP-CRLR/RAMP1 signaling acts to promote hematopoiesis under proliferative stress conditions, but the sustained CGRP secretion conversely reduces undifferentiated BM cells together with differentiated myeloid cells. Therefore, these results suggest that CGRP may play an important role in biological defenses including infection and inflammation by integrating nervous system, hematopoiesis, and immunity.

Methods
Mice. All animal experiments were performed in accordance with protocols approved by the Animal Experiment Review Board of the University of Miyazaki. Ramp1-deficient mice were described elsewhere 31 . C57BL/6 CD45.1 congenic mice (B6-CD45.1) were purchased from Sankyo-Lab Service (Tokyo, Japan). For analysis of blood counts, PB from the tail vein was collected in EDTA-coated microtubes and analyzed with a hematology analyzer (CellTac, NIHON KOHDEN, Tokyo, Japan).

5-FU treatment in mice.
A single dose of 5-fluorouracil (5-FU) at 150 mg/kg was administered by intraperitoneal injection to 8-12 weeks old WT (n = 3) and Ramp1-deficient (n = 3) mice and the mice were followed up until 30 days. In the second experiment, the treated mice (n = 4 in each group) were sacrificed at seven days after injection and populations of LSK cells (Lin − Sca-1 + c-kit + ), myeloid progenitor cells (MP; Lin − Sca-1 − c-kit + ) including CMP, GMP, and MEP, and BMMNCs were analyzed by flow cytometry.
CGRP administration in mice. A total of 0.2 mg of CGRP in physiological saline was infused into the subcutaneous tissues of the backs of mice using osmotic pumps (Alzat, Cupertino, CA, USA) at 0.25 l/h rate, and the administration was continued for 14 days.
Long-term competitive repopulation assay. C57BL/6-CD45.1 mice and Ramp1 −/− or WT/Ramp1 +/+ -CD45.2 mice were used for the long-term competitive repopulation assay, according to the procedure previously described 37 . BM cells were isolated from tibia and femoral bones of 8-10 weeks old Ramp1 −/− or WT/Ramp1 +/+ -CD45.2 mice. Eight to 10 weeks old C57BL/6-CD45.1 mice were irradiated individually with a three mice for each group were analyzed. The full-length gels/blots are presented in Supplemental Fig. S4. (g) The colony forming abilities of BMMNCs of PBS-treated (PBS + ) or CGRP-treated (CGRP + ) mice for two weeks were determined in the in vitro cell culture with (CGRP) or without CGRP (Control). BMMNCs from three mice for each group were analyzed. ns; not significant, *p < 0.05, **p < 0.01 compared with PBS-treated mice (Ordinary one-way ANOVA and Dunnett's multiple comparisons test).
Co-culture of BMNNCs with BM stromal cells. BM stromal cells were isolated from tibia and femoral bones of 8-10 weeks old mice, according to the procedure previously described 38

Analysis of CGRP expression after irradiation in BM stromal cells. Four days after 10 Gy total body
irradiation to 8-10 weeks old mice, the BM stromal cells were isolated from tibia and femoral bones of the irradiated mice 38  Western blotting. For protein extraction from tissues, excised organs were homogenized in NP-40 lysis buffer (50 mM Tris-HCl, pH8.0, 150 mM NaCl, 5 mM EDTA, and 1% NP-40) supplemented with a proteinase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and phosphatase inhibitor tablet (Roche, Penzberg, Germany). The lysate was centrifuged for 10 minutes at 15000 × g (maximum) and 4 °C, and the supernatant was then collected. The protein concentration was determined with a BCA protein assay (Thermo Scientific, Waltham, MA, USA), with bovine serum albumin (BSA) as the standard. Equal amounts of protein samples were loaded into SDS-polyacrylamide gels for electrophoresis, and then, the separated proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Billerrica, MA, USA). The membranes were blocked in PBS-Tween (0.1%) (PBST) with 1% BSA or 5% nonfat dried milk and were then probed with the primary antibodies diluted PBST-BSA, 5%