The lysophosphatidic acid receptor LPA4 regulates hematopoiesis-supporting activity of bone marrow stromal cells

Lysophosphatidic acid (LPA) is a pleiotropic lipid mediator that acts through G protein-coupled receptors (LPA1-6). Although several biological roles of LPA4 are becoming apparent, its role in hematopoiesis has remained unknown. Here, we show a novel regulatory role for LPA4 in hematopoiesis. Lpar4 mRNA was predominantly expressed in mouse bone marrow (BM) PDGFRα+ stromal cells, known as the components of the hematopoietic stem/progenitor cell (HSPC) niche. Compared with wild-type mice, LPA4-deficient mice had reduced HSPC numbers in the BM and spleen and were hypersusceptible to myelosuppression, most likely due to impairments in HSPC recovery and stem cell factor production in the BM. Analysis of reciprocal BM chimeras (LPA4-deficient BM into wild-type recipients and vice versa) indicated that stromal cells likely account for these phenotypes. Consistently, LPA4-deficient BM stromal cells showed downregulated mRNA expression of stem cell factor and tenascin-c in vitro. Taken together, these results suggest a critical and novel role for the LPA/LPA4 axis in regulating BM stromal cells.

Scientific RepoRts | 5:11410 | DOi: 10.1038/srep11410 (http://www.biogps.org) indicates that the mouse Lpar4 gene is expressed in mesenchymal cells such as osteoblasts and C3H10T1/2 cells. Thus, LPA 4 is presumed to play pivotal roles in various cellular processes of mesenchymal cells in multiple tissues. However, this has not yet been thoroughly investigated.
The bone marrow (BM) is the main hematopoietic organ in adult mammals. In the BM, the hematopoietic stem/progenitor cells (HSPCs) give rise to all blood cell lineages. The maintenance, differentiation and proliferation of HSPCs are regulated in both cell-autonomous and non-cell-autonomous fashions 21 . The non-cell-autonomous regulation of HSPCs requires factors important for the proliferation, mobilization, homing and engraftment of hematopoietic stem cells (HSCs); such factors are produced by various cells surrounding HSCs 22 . This local microenvironment is called the HSPC niche. The HSPC niche is subdivided into two types, the osteoblastic and perivascular niches 22 . The cellular components of the perivascular niche have been reported to be mesenchymal stromal cells, such as CXCL12-abundant reticular cells (CAR cells) 23 and nestin + MSCs 24 . Although they are essential for HSPC maintenance, the molecules regulating mesenchymal stromal cells are not yet fully understood 25 .
In this report, we observed that LPA 4 -deficient mice were highly sensitive to myelosuppression and showed a delay in the recovery of HSPC numbers. LPA 4 was predominantly expressed in the BM mesenchymal stromal cells. In addition, LPA 4 in the BM mesenchymal stromal cells was shown to regulate the production of factors involved in HSPC proliferation both in vivo and in vitro. Our present study consistently demonstrates a significant role for LPA 4 in maintaining the HSPC niche.

Methods
Mice. LPA 4 -deficient mice on a C57BL/6 genetic background have been described previously 18 . C57BL/6 mice congenic for the Ly5 locus (B6-Ly5.1) were purchased from Sankyo Labo Service (Tokyo, Japan) by permission of Prof. Hiromitsu Nakauchi (Institute of Medical Science, The University of Tokyo). Male mice of 9-12 weeks old were used for these studies. Mice were housed under specific pathogen-free conditions in an air-conditioned room and fed standard laboratory chow ad libitum (CE-2; CLEA Japan, Tokyo, Japan), in accordance with institutional guidelines. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Akita University.

Myelosuppression models. Mice were intravenously injected with 5-fluorouracil (5-FU; Kyowa
Hakko, Tokyo, Japan) at a dose of 250 mg/kg body weight, and their survival was monitored for 24 days. Some mice were euthanized for analysis of the BM cells and peripheral blood (PB) on days 0, 2, 4, 7 and 10. For experiments with the BM chimeric mice (shown below), mice were intraperitoneally injected with 5-FU (150 mg/kg body weight) twice, at a 1-week interval, and their survival was monitored for 24 days after initial injection. In another model, mice were irradiated with 8 Gy in two split doses with a 3-hr interval using a cabinet X-ray system CP-160 (Faxitron X-ray Corporation, Wheeling, IL). Their survival was then monitored for 24 days. PB collection. Under isoflurane anesthesia, PB was collected from the retro-orbital sinus using capillaries and analyzed using a hematological analyzer (Celltac MEK-5258, Nihon Kohden, Tokyo, Japan).

Stromal and hematopoietic cell isolation. Hematopoietic BM cells were obtained from femurs
and tibias by flushing the bones with PBS containing 0.5% BSA (Sigma-Aldrich). For isolation of stromal cells, the flushed bones were minced with scissors. Then, the bone fragments were incubated with DMEM (Sigma-Aldrich) containing 10% FBS (Gibco) and 3 mg/ml type I collagenase (Worthington Biochemical, Lakewood, NJ) for 60 min at 37 °C. The cell suspensions were filtered with a 100-μ m cell strainer. Red blood cells were lysed using BD Pharm Lyse Buffer (BD Biosciences).
Cell culture. The BM stromal cells isolated by collagenase treatment were maintained in α -MEM GlutaMax (Gibco) containing 10% FBS, 10% horse serum (Gibco) and 100 U/ml penicillin/streptomycin (Wako Chemicals, Osaka, Japan) for 7 days. The cells were starved in α -MEM GlutaMax containing 0.1% BSA, 10 μ M HA130 (an inhibitor of autotaxin; Calbiochem) and 100 U/ml penicillin/streptomycin for 12 hr to exclude hematopoietic cells. The starved cells were stimulated with 1-oleoyl LPA (Avanti Polar Lipids, Alabaster, AL) at a final concentration of 10 μ M by adding an equivalent volume of medium containing 20 μ M 1-oleoyl LPA and incubated for 12 hr.
Quantitative reverse transcription-PCR. For preparation of cDNA templates from the cultured cells and sorted cells, total RNA was isolated with the RNAqueous-micro kit (Ambion, Austin, TX) or the RNeasy mini kit (Qiagen, Valencia, CA) and subjected to oligo-dT-and random hexamer-primed reverse transcription with the Primescript enzyme (Takara Bio, Otsu, Japan). Quantitative PCR was performed using a LightCycler 480 instrument (Roche Diagnostics) with the SYBR Premix ExTaq II (Takara Bio) and KAPA SYBR Fast qPCR Kit (Kapa Biosystems, Wilmington, MA). The mRNA levels were normalized to Hprt1 or Rn18s as a standard housekeeping gene. The primer sequences are listed in Supplementary  Table S1. The PCR program was as follows: denaturation at 95 °C for 30 sec and 50 cycles of amplification consisting of denaturation at 95 °C for 10 sec and annealing and extension at 60 °C for 20 sec.

ELISA.
At days 0 and 9 after 5-FU administration, the femurs were flushed with 500 μ l of ice-cold PBS or 300 μ l of ice-cold PBS containing 1% NP40 and a protease inhibitor cocktail to measure the protein levels of CXCL12 or SCF, respectively. The fluids were centrifuged at 500 × g for 5 min. Then, the supernatant was subjected to ELISAs for CXCL12 and SCF using the DuoSet ELISA kits (R&D systems) according to the manufacturer's instructions.
Statistical analysis. Data are expressed as the mean ± SEM and analyzed using GraphPad Prism 6 software (GraphPad Software). All data were combined from two or three independent experiments. The two-tailed unpaired Welch's t-test, log-rank test or two-way ANOVA followed by Bonferroni's post-hoc test was used for comparisons between 2 groups. One-way ANOVA followed by Tukey's post-hoc test was used for comparisons among 3 groups. Values of P < 0.05 were considered statistically significant.
We also examined mRNA expression levels of other LPA receptors in HSPCs and the three subsets of stromal cells. In HSPCs, Lpar4 mRNA expression level was the lowest among six receptors ( Supplementary Fig. S1A). About the stromal cells, Lpar1 and Lpar6 mRNA were ubiquitously expressed in all subsets, while Lpar2, Lpar3 and Lpar5 mRNA were undetectable under our experimental conditions ( Supplementary Fig. S1B-D). LPA 4 -deficient mice have decreased HSPC number in the BM and spleen. Because the stromal cells form an important constituent of the perivascular niche, we analyzed hematopoietic parameters of LPA 4 -deficient mice under homeostatic conditions, including total BM cellularity and numbers of hematopoietic stem/progenitor cells and mature cells. The total BM cellularity was normal in LPA 4 -deficient mice ( Fig. 2A). The numbers of CD3 + CD4 + , CD3 + CD8 + and B220 + lymphocytes in the BM were also unchanged, but the number of CD11b + Gr-1 + granulocytes/monocytes was significantly higher in the BM of LPA 4 -deficient mice than in that of WT mice (Supplementary Fig. S2A-G). We further observed that the number of HSPCs was significantly lower in LPA 4 -deficient mice than in WT mice (Fig. 2B). However, the number of HSCs, which are defined as CD34 − LSK (Fig. 1A) or CD41 − CD48 − CD150 + LSK cells (Fig. 2C), was normal in LPA 4 -deficient mice ( Fig. 2D and E, respectively). Therefore, we reasoned that the HPC number is decreased in these mice (Fig. 2F). Next, we examined whether the decrease in HPC number was due to impaired cell cycle progression of HSCs and HSPCs. However, their cell cycle status in LPA 4 -deficient mice was normal (Fig. 2G-I).
To further characterize HSPCs of LPA 4 -deficient mice, we assessed the hematopoietic colony-forming capacity of the BM cells cultured with cytokines. The colony numbers of LPA 4 -deficient mice were slightly lower than those of WT mice (Supplementary Fig. S3). However, considering the intrinsically smaller number of HSPCs in the BM of LPA 4 -deficient mice (Fig. 2B), the colony-forming capacity seemed to be comparable between WT and LPA 4 -deficient mice. In addition, the expression levels of various transcripts in HSPCs were measured. Among the genes examined, Cdk2 and Spi1 (encoding PU.1) were down-regulated significantly in HSPCs of LPA 4 -deficient mice compared with those of WT mice (Supplementary Fig. S4).
We next examined the numbers of Lin − IL7Rα + Flt3 + common lymphoid progenitors ( Supplementary  Fig. S2H), Lin − Sca-1 − c-Kit + CD34 + FcRγ II-III high granulocyte and macrophage progenitors and Lin − Sca-1 − c-Kit + CD34 − FcRγ II-III low megakaryocyte and erythrocyte progenitors ( Supplementary Fig. S2I), all of which are differentiated from HPCs. These cell numbers were comparable between the two genotypes ( Supplementary Fig. S2J). However, the ratio of granulocyte and macrophage progenitors to HSPCs was significantly increased in LPA 4 -deficient mice (Supplementary Fig. S2K). In mice, adult hematopoiesis occurs not only in the BM but also in the spleen 27 . By analyzing the spleen, we found that LPA 4 -deficient mice have significantly decreased numbers of both HSCs and HSPCs (Fig. 2J-K).
To investigate the contribution of LPA 4 to BM reconstitution, WT and LPA 4 -deficient mice were lethally irradiated and transplanted with WT or LPA 4 -deficient BM. Three months after transplantation, the chimerism of the recipient mice was determined in PB cells (Fig. 2L). The results showed no significant difference in the percentages of donor cells among these BM chimeras (Fig. 2M-N). Together, these results suggest that LPA 4 regulates the homeostasis of HSPCs in the BM and spleen.

LPA 4 -deficient mice show a delay in hematopoietic recovery after myelosuppression.
To investigate the function of LPA 4 under myelosuppression, mice were injected with 5-FU or were sublethally irradiated. LPA 4 -deficient mice displayed significantly higher lethality than WT mice in both myelosuppression models (Fig. 3A-B). After 5-FU administration, the numbers of red blood cells and platelets in PB were reduced similarly in both genotypes ( Supplementary Fig. S5A-B). However, the numbers of white blood cells in LPA 4 -deficient mice were reduced earlier than those in WT mice ( Supplementary  Fig. S5C). At day 10 after 5-FU administration, LPA 4 -deficient mice had a significantly reduced HSPC number in the BM, although the total BM cellularity was unaffected by the drug (Fig. 3C-D).
As described above, HPCs and stromal cells express Lpar4 mRNA in the BM (Fig. 1C). To determine which cell type is responsible for the hypersusceptibility of LPA 4 -deficient mice to 5-FU, we used the BM chimeric mice shown in Fig. 2M,N. When WT mice transplanted with WT or LPA 4 -deficient BM cells were treated with 5-FU, the lethality was indistinguishable between these two classes of chimeric mice (Fig. 3E). In contrast, when we administered 5-FU to WT and LPA 4 -deficient mice that had undergone transplantation of BM from WT donors, the LPA 4 -deficient recipients were significantly more susceptible to 5-FU than WT recipients (Fig. 3F), recapitulating the results observed with naïve mice. Histological observations of the lung, liver, colon and small intestine 7 days after 5-FU administration revealed that little or no injury occurred in these organs of WT and LPA 4 -deficient mice (data not shown). Thus, these results suggest that LPA 4 expressed in stromal cells is involved in hematopoietic recovery after myelosuppression.

LPA 4 deficiency in BM stromal cells impairs the production of HSPC proliferation
factors. CXCL12 and SCF, important cytokines for the HSPC maintenance and proliferation, are produced predominantly by BM stromal cells 28,29 . Because LPA 4 -deficient mice displayed reduced numbers of HSPCs and impaired extramedullary hematopoiesis, we evaluated the CXCL12 and SCF protein levels in the BM of LPA 4 -deficient mice. Under homeostatic conditions, there was no significant difference in the protein levels of CXCL12 between LPA 4 -deficient and WT mice (Fig. 4A). The protein level of SCF was too low to be detected. At day 9 after 5-FU administration, LPA 4 -deficient mice had a significantly lower level of SCF protein in the BM than did WT mice (Fig. 4B), and the production of CXCL12 protein tended to be impaired in LPA 4 -deficient mice (Fig. 4C). These in vivo data suggest that LPA 4 in the stromal cells regulates the SCF protein expression under myelosuppression. Consistent results were obtained with cultures of primary BM stromal cells. The LPA 4 -deficient stromal cells expressed a significantly lower level of Scf mRNA than did WT cells (Fig. 4D). In addition, the LPA 4 -deficient stromal cells also showed a significant reduction in the mRNA level of tenascin-c (TN-C) (Fig. 4F), an extracellular matrix protein that promotes HSPC proliferation 30 . When we stimulated stromal cells with LPA, upregulation of the TN-C mRNA level was observed in both WT and LPA 4 -deficient cells (Fig. 4F). In contrast, the expression levels of Scf and Cxcl12 mRNA were unaffected by LPA stimulation (Fig. 4D-E). Together, these results suggest that LPA 4 in stromal cells regulates the production of proliferation-promoting factors for HSPCs.

Discussion
In this study, we found that LPA 4 -deficient mice showed hypersusceptibility to myelosuppressive stresses, likely due to impaired stress recovery of the HSPC number in the BM. This impairment was associated with reduced production of SCF in the BM of LPA 4 -deficient mice. Bone marrow chimeric mice showed that the target cells of LPA 4 signaling were of non-hematopoietic origin. Therefore, it was consistent that the LPA 4 deficiency in the BM stromal cell cultures suppressed the expression levels of Scf and also TN-C. SCF potently regulates HSPC proliferation and differentiation through the receptor c-Kit 31 . Previously, PDGFRα + Sca-1 − cells were shown to produce SCF in the BM and contribute to the formation of the perivascular niche 23 . TN-C is an extracellular matrix protein that is predominantly produced by PDGFRα + stromal cells in the BM and regulates HSPC proliferation through integrin α 9 30 . The expression of TN-C was reportedly upregulated after myelosuppressive stress 30 . Similarly to LPA 4 -deficient mice, TN-C-deficient mice were hypersensitive to lethal myelosuppression and showed a delay of hematopoietic recovery 30 . Thus, we assume that the lethality to myelosuppressive stress was dependent, at least in part, on LPA 4 from the stromal cells. Because Lpar4 mRNA was predominantly expressed in PDGFRα + Sca-1 + / PDGFRα + Sca-1 − stromal cells in the BM, LPA 4 may regulate the function of PDGFRα + cells by affecting the production of HSPC proliferation factors, although the underlying molecular mechanisms remain to be elucidated.
To date, BM mesenchymal stromal cells have been reported to express at least two G protein-coupled receptors (GPCRs), parathyroid hormone/parathyroid hormone-related peptide receptor (PTH 1 ) 32 and prostaglandin E receptor 4 (EP 4 ) 33 , that support HSPC proliferation. Activation of PTH 1 resulted in the upregulation of the protein expression of Jag1 in α 1(I) collagen-expressing osteoblastic cells and then induced HSPC proliferation through Notch1. Meanwhile, EP 4 activation enhanced the mRNA expression of various mitogenic protein genes, as well as Jagged1, in ALCAM -Sca-1 + mesenchymal progenitor cells. It is interesting to note that both GPCRs are coupled to Gs protein for these phenotypes 25 . However, we observed that LPA 4 is coupled to G12/13 protein not but to Gs, Gi/o or Gq protein in mouse C3H10T1/2 mesenchymal cells (Keisuke Yanagida and S.I., manuscript in preparation). Consistently, LPA 4 -deficient stromal cells showed no change in Jagged1 expression level (Supplementary Fig. S6). Thus, our results suggest that LPA 4 controls the HSPC niche via a novel GPCR signaling pathway in BM stromal cells. Because Lpar1 and Lpar6 mRNA were also expressed in PDGFRα + stromal cells in the BM, LPA 1 , LPA 4 and LPA 6 may exert redundant functions in the hematopoiesis-supporting activity. Indeed, these three LPA receptors were reported to couple to G12/13 protein 15 .
In steady-state hematopoiesis, LPA 4 -deficient mice showed mildly reduced numbers of HPCs in the BM. Moreover, we detected a severe reduction in HSPCs in the spleen. It is likely, therefore, that the hypersusceptibility to myelosuppression of LPA 4 -deficient mice was also partly caused by these "basal" impairments of steady-state hematopoiesis in addition to the attenuated emergency hematopoiesis. As far as we know, no gene-targeted knockout mouse lines have been reported to have hematopoietic phenotypes similar to that of LPA 4 -deficient mice, although gene targeting in mice has allowed investigators to reveal hematopoietic functions of various genes.
In relation to the abnormal BM hematopoiesis in LPA 4 -deficient mice, we would like to note that both the ratios of granulocyte and macrophage progenitors to HSPCs and the absolute number of granulocyte/monocytes were mildly increased in the BM of LPA 4 -deficient mice compared with WT mice. In line with the phenotypes, we found that Spi1 gene (encoding PU.1) was down-regulated significantly in HSPCs of LPA 4 -deficient mice compared with those of WT mice. PU.1 is the transcription factor that suppresses early granulocytic development in adult mice 34 . Together, these results suggest that LPA 4 deficiency promotes the differentiation of HPCs into granulocyte and macrophage progenitors, also contributing to the reduced numbers of HPCs.
Hematopoiesis during embryonic development occurs in the fetal liver, and then, postnatally, the HSCs in the fetal liver migrate to the BM 35 , where CXCL12 is abundant and acts as an essential factor for HSC retention 36 . Indeed, the in vivo deletion of CXCL12 decreased and increased the number of HSCs in the BM and of that in the spleen and PB, respectively 36 . It is worth mentioning that compromised HSC homing was observed in the spleen of naïve LPA 4 -deficient mice, although the BM of these mice contains the normal number of HSCs as well as a normal level of CXCL12 protein. These results suggest that the total absolute number of HSCs in the LPA 4 -deficient fetal liver may be intrinsically reduced, resulting in the compromised extramedullary hematopoiesis observed in the adult. Alternatively, the abnormal angiogenesis during the embryogenesis of LPA 4 -deficient mice 18 may inhibit the homing of HSCs from the liver to the spleen.
The LPA 4 -deficient stromal cells showed significant decreases in the basal mRNA expression levels of Scf and TN-C in vitro. These significant decreases were observed even after LPA treatment, although the expression of TN-C was comparably induced by LPA treatment in both genotypes, likely via LPA receptor(s) other than LPA 4 . Thus, LPA 4 signaling in stromal cells may indirectly regulate the intrinsic capacity for the production of SCF and TN-C by affecting unknown processes such as cellular differentiation.
Myelosuppression is a life-threatening adverse event observed during anticancer treatments such as chemotherapy and radiotherapy 37 . Currently, granulocyte colony-stimulating factor is used to promote granulocytic recovery after such anticancer treatments 38 . In the present study, we reveal that LPA 4 facilitated the regeneration of HSPCs after myelosuppression in mice. It is possible that selective agonism of LPA 4 could promote multi-lineage hematopoietic recovery and protect cancer patients from myelosuppression.