Involvement of Niemann-Pick Type C2 Protein in Hematopoiesis Regulation - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:05 |只看该作者 |倒序浏览 |打印

作者：Kyu Heoa, Unnati Jariwalaa, Jeongim Wooa, Yuxia Zhana, Kathleen A. Burkea, Lunjian Zhua, W. French Andersona, Yi Zhaob作者单位：a Department of Biochemistry and Molecular Biology, andb Division of Hematology, Department of Medicine, University of Southern California, Keck School of Medicine, Los Angeles, California, USA 2 G3 J- S$ X0 g

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      【摘要】& g$ H/ R! P, X
   Niemann-Pick type C2 (NPC2) protein has been characterized as a cholesterol-binding protein. Its loss leads to NPC2 disease, an inherited neurodegenerative disorder. When analyzing gene expression profile, we noticed high expression of both NPC2 and its receptor, mannose 6-phosphate receptor (MPR), in murine hematopoietic stem cells. NPC2 protein, in the presence of thrombopoietin (TPO), causes an increase in CFU-GEMM (colony-forming unit-granulocyte-erythroid-macrophage-megakaryocyte) and a decrease in CFU-GM (colony-forming unit-granulocyte-macrophage) colony number in colony-forming cell (CFC) assays. This effect is independent of cholesterol binding but does require the presence of MPR. With M07e cells, a TPO-dependent hematopoietic leukemia cell line, NPC2 can inhibit TPO-induced differentiation and enhance TPO-mediated anti-apoptosis effects. Strikingly, these results are not observed under the standard 20% O2 level of the standard incubator, but rather at 7% O2, the physiological oxygen level of bone marrow. Furthermore, NPC2 protein upregulates hypoxia inducible factor 1- protein level at 7% O2, but not at 20% O2. Our results demonstrate that NPC2 protein plays a role in hematopoiesis at the physiologic bone marrow level of O2. 7 c8 F5 q) |/ o; K
      【关键词】 Hematopoietic stem cell Niemann-Pick type C protein Thrombopoietin
   INTRODUCTION. N$ O* Z# O4 z! s& p

Hematopoietic stem cells (HSCs) reside in bone marrow, can renew themselves, differentiate into various specialized cells, and undergo programmed cell death. Murine HSCs consist of long-term reconstituting (LTR) cells and short-term reconstituting (STR) cells based on their repopulation abilities in lethally irradiated animals  to study the gene expression profile of HSC subsets, we found Niemann-Pick type C2 (NPC2) protein and its receptor, mannose 6-phosphate receptor (MPR)/insulin-like growth factor II receptor (IGFII-R), to be highly expressed in both LTR and STR HSC subsets.0 [# t1 z2 @( z2 B0 }: B) |' t

NPC2 protein, originally named epididymic secretory protein, is a small glycoprotein secreted by the epididymis .

Upon glycosylation with mannose, NPC2 protein can bind with MPR. Classically, MPR is considered to be a transport protein that diverts newly synthesized lysosomal enzymes from the secretory pathway to the endolysosomal system. However, recent studies suggest that MPR can interact with other proteins and play various roles. For example, the local level of IGF-II and the activity of transforming growth factor (TGF)-ß-1 can be modified by MPR .

The high level of expression of NPC2 and its receptor in HSCs suggest that these proteins may play a role in the regulation of hematopoiesis. Our initial studies, done at the standard oxygen level of approximately 20%, were negative. However, when the studies were repeated at 7%, the physiological oxygen level of bone marrow, NPC2 was shown to play an active role in the hematopoietic process.
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MATERIALS AND METHODS1 D0 ^# N$ a) m, |; F7 |) ?

Mice and Progenitor Cell Isolation

C57 BL/6J (Ly5.2) female mice were obtained from the Jackson Laboratory (Bar Harbor, ME, http://www.jax.org) and the animal facility at the University of Southern California (USC; Los Angeles). All animals were housed under specific pathogen-free conditions and were given acidified drinking water and autoclaved food ad libitum. Mice used in the experiments were 8¨C12 weeks of age. The study protocol was approved by the USC Animal Care and Use Committee. Stem and progenitor cells were isolated as previously described .
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Cell Culture and Filipin Staining' ^* ?0 r) N9 c* C
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293T cells were cultured as previously described  and used at a final concentration of 50 µg/ml) for 1 hour at room temperature. After four washes with PBS, Filipin fluorescence was detected by fluorescence microscopy using a Nikon Eclipse E800 with x 100 magnification (Melville, NY, http://www.nikonusa.com).
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Production of Polyclonal Antibody to NPC2 Protein( ]) p! e  y, E9 ]

Based on the structural similarity and alignment between NPC2 protein and Derf2 protein , three peptides were synthesized as follows: (a) NH2-PSIKLVVEWKLEDDKKNNL-COOH, (b) NH2-NCPIQKDKTTSYLNKLPVK-COOH, and (c) NH2-PCQLHKGQSYSVNIT-COOH. Each peptide was injected into two rabbits, and serum was collected and tested by Western blot. The serum from rabbit no. 3 using peptide no. 2 was shown to give the best results. Aliquots of the serum were stored at ¨C80¡ãC., Y2 @( U7 [/ z, I0 V, A& |
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NPC2 cDNA Cloning, Expression, and Purification0 ^3 ?5 j- P! k" [6 d, C6 N! L
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NPC2 cDNA was cloned from a mouse bone marrow low-density cDNA library using the following primers, 5'-GC-GAATTCCTATTAGCTTGTGATCTGAACTGG-3' and 5'G-CTCTAGACTATTAATGGTGATGGTGATGATGGCTTGTG-ATCTGAACTGGGATCTC-3' (the 6-histidine tag is italicized). The 450-bp fragment of the PCR product was subcloned into a protein expression vector, pTT3 , to form an NPC2 expression plasmid: pTT3/NPC2. NPC2 protein was expressed in 293T cells with transfection using Lipofectamine Plus reagent according to the manufacturer¡¯s instructions (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). The medium was replaced with 293 SFM II medium (Invitrogen) 24 hours after transfection. At 72 hours after transfection, the supernatants were collected and concentrated by using Amicon Centricon Plus-80 (Millipore, Billerica, MA, http://www.millipore.com). NPC2 protein was purified from concentrated supernatants using Ni -NTA affinity column system (Qiagen, Valencia, CA, http://www1.qiagen.com) and desalted using Centricon Plus-80 according to the manufacturer¡¯s instructions. Purified proteins were characterized by Filipin staining to confirm cholesterol-binding activity. To perform reverse transcription (RT)-PCR analysis of NPC2 expression in bone marrow cell subsets, we used the following primers: forward 5'-ATGCGTTTTCTGGC-CGCCACG-3', reverse 5'-GCTTGTGATCTGAACTGG-GATCTC-3' (450 bp).

Immunoblotting

Western blots were performed as previously described . Hypoxia inducible factor (HIF)-1- and ARNT (aryl hydrocarbon receptor nuclear translocator) antibodies were purchased from BD Pharmingen (San Diego, http://www.bdbiosciences.com/pharmingen).
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Detection of Apoptotic Cells% H1 T: i& s* k; @9 r! ~8 w5 P

An apoptosis assay was performed with TACS apoptosis detection kit according to the manufacturer¡¯s instructions (R&D Systems). In brief, the cells were cultured for 4 days with thrombopoietin (TPO) in the presence or absence of NPC2. Then, cells were transferred to serum-free medium for 4 hours and collected to determine the percentage of apoptotic cells. Five x 105 MO7e cells were collected and washed in PBS. Then, cells were resuspended in 100 µl of incubation reagent and incubated in the dark for 15 minutes at room temperature. After 400 µl of binding buffer was added, cells were analyzed by flow cytometry within 1 hour for maximal signal.# h, h* }4 @* C

In Vitro Colony-Forming Cell Assays

Assays for colony-forming unit-granulocyte-macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and colony-forming unit-granulocyte-erythroid-macrophage-megakaryocyte (CFU-GEMM) were performed using methylcellulose-based medium (M3334; StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com). This medium contains 3 U/ml erythropoietin (EPO), 100 ng/ml stem cell factor (SCF), and 100 ng/ml TPO (R&D Systems). Sorted murine stem cells (Lin¨C Sca  Kit ) were mixed with methylcellulose-containing supplements and plated (500 cells/plate) in a 35-mm dish. Cells were incubated at 37¡ãC with 10% CO2 and 7% O2 for 12 days.
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Statistical Analysis

Student¡¯s t test was used for data analysis.8 U3 o; |3 W  h9 E! S; g0 O* j5 Z- \
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RESULTS

Murine NPC2 Protein Expression, Purification, and Characterization

NPC2 mRNA and protein levels in murine HSC subsets and bone marrow cells were detected by RT-PCR (Fig. 1A, left) and Western blot (Fig. 1B, right). Secreted NPC2 protein from 293T cells was purified, and the purity was determined with silver staining (Fig. 1B). To examine the cholesterol-binding activity of the protein, Filipin staining was performed. Functional NPC2 protein can rescue the mutant NPC2 fibroblasts from intracellular cholesterol accumulation, resulting in the decrease of intracellular Filipin staining. The intensity of Filipin staining is significantly decreased in NPC2 fibroblasts treated with NPC2, thereby indicating that the purified NPC2 protein retains active cholesterol-binding ability and prevents intracellular cholesterol accumulation in the cell line (Fig. 1C).

Figure 1. NPC2 protein expression, purification, and characterization. (A): Reverse transcription-polymerase chain reaction (RT-PCR) (left): negative control (lane 1), mRNA from Lin¨C Sca  Kit  CD34  CD38¨C cells (lane 2), Lin¨C Sca  Kit  CD34  CD38  cells (lane 3), and Lin¨C Sca  Kit  CD34¨C CD38  cells (lane 4). PCR was performed with this primer set; forward 5'-ATGCGTTTTCTGGCCGCCACG-3' and reverse 5'-GCTTGTGATCTGAACTGGGATCTC-3' (450 bp). HPRT gene was used as internal control. Western blot of NPC2 protein (right): secreted NPC2 protein in the medium from 293T cells (lane 1), NPC2 protein in whole bone marrow cells (lane 2), and Lin¨C Sca  cells (lane 3). NPC2 from 293T supernatant shows slower migration due to glycosylation. After treatment with deglycosidase (PNGase F), the NPC2 from 293T supernatants migrates at the same position as that isolated from the bone marrow (data not shown). Arrow indicates 16-kDa molecular weight. (B): Silver staining of an SDS-polyacrylamide gel containing purified recombinant NPC2 protein; 10 and 20 µg of purified NPC2 protein were loaded in lanes 1 and 2, respectively. (C): Filipin staining: NPC2 fibroblasts accumulate intracellular cholesterol, which is stained by Filipin (top). In the presence of NPC2 protein, the defect is corrected and the cells do not accumulate cholesterol, as shown by the absence of Filipin staining (bottom). Abbreviations: HPRT, hypoxanthine-guanine phosphoribosyl transferase; NPC2, Niemann-Pick type C2.
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Effect of NPC2 Protein in Hematopoietic Colony-Forming Cell Assays

Colony-forming cell (CFC) assays were used to examine whether NPC2 protein plays a role in the process of hematopoiesis. When the assays were performed in normal air oxygen (~20%), no effect was seen. However, when the assays were repeated at the physiological oxygen concentration of bone marrow, 7%, the results shown in Figure 2A were obtained. The plates with NPC2 protein gave 35% more CFU-GEMM colonies and 25% fewer CFU-GM colonies compared with control plates. Besides the shift in colony type and number, most of the CFU-GEMM colonies in plates with NPC2 were high-potential proliferation colonies, as the diameter of most these colonies was larger than 0.5 mm, whereas the CFU-GEMM colonies in control plates were significantly smaller (Fig. 2B). The BFU-E colony number in these assays was less than five, and there was no significant difference between control and NPC2-treated groups.! p# `8 p+ K  B- p- `- a

Figure 2. NPC2 effects in colony-forming cell (CFC) assays. (A): NPC2 results in shift of colony type in CFC assay. Five hundred Lin¨C Sca  Kit  cells were cultured in the presence of stem cell factor, erythropoietin, and thrombopoietin under 7% oxygen condition. Compared with control plate, there are more CFU-GEMM colonies and less CFU-GM colonies when 1 µg/ml of purified NPC2 protein is added to the plate. Results are a summary of three individual experiments, with a total of six samples (*, p

Because different cytokines and growth factors play different roles in hematopoiesis regulation, to determine whether the effects of NPC2 protein in the CFC assay are growth factor/cytokine-specific, CFC assays were performed with different growth factor/cytokine combinations. In the experiment, both SCF and EPO were kept constant with a third factor added (100 ng/plate): TPO, interleukin (IL)-3, IL-6, IL-11, or Flt-3 ligand. The NPC2 protein resulted in an increase of CFU-GEMM colony number and size only when TPO was present in the assay system (Table 1) and not with any other tested factors.# }1 G0 V! E  e! W2 ^* P; r

Table 1. Effects of growth factors on Niemann-Pick type C2 (NPC2) protein function$ W! Z# q  U7 n0 ?

NPC2 Protein Function in Hematopoiesis Is Independent of Cholesterol-Binding Activity but Dependent on M6P Receptor
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Because NPC2 is known to be a cholesterol-binding protein, we used different NPC2 mutant proteins to determine whether the observed NPC2 effects on hematopoiesis depend on the protein¡¯s cholesterol-binding activity . Plasmids containing the gene for four different mutant proteins were kindly provided by Drs. M.P. Scott and D.C. Ko (Stanford University, Stanford, CA). The mutant proteins were expressed and purified, and their cholesterol-binding activities were determined with Filipin staining. F66A and V96F are mutants with abolished cholesterol-binding activity, whereas D72A and D75A are mutants with normal cholesterol-binding activity. In the CFC assays, all four of the mutant proteins showed the same effects as the wild-type protein: increased CFU-GEMM colony number and size, strongly suggesting that the effects of NPC2 protein on hematopoiesis are independent of its cholesterol-binding activity (Fig. 3A).
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Figure 3. NPC2 function relies on mannose 6-phosphate receptor but is independent of cholesterol binding. (A): Effects of different mutant NPC2 proteins in the CFC assay. WT and mutant NPC2 proteins (1 µg) were added in the colony-forming cell assay in the presence of stem cell factor, erythropoietin, and thrombopoietin under 7% oxygen. Mutants D72A and D75A retain cholesterol-binding activity; F66A and V96F are defective in cholesterol binding. There is no significant difference in colony number and type between WT NPC2 protein and mutant proteins. Both WT and mutant proteins induce significant colony-type shift compared with control plates. Results are a summary of two individual experiments, with a total of four samples. (B): Mannose 6-phosphate (M6P) inhibits NPC2 function. The NPC2-dependent colony-type shift is reversed by M6P in a dose-dependent manner. There is no change in total colony numbers. One microgram of NPC2 and different doses of M6P were used. *, p * [/ n- v) W5 @' }1 Y
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Next, we determined whether mannose 6-phosphate (M6P) could block the effects of NPC2 protein via MPR. As shown in Figure 3B, increased CFU-GEMM and decreased CFU-GM colony numbers are gradually diminished as increased amounts of M6P (1¨C7 mM) are added to the assay system. In the absence of NPC2 protein, M6P (5 mM) itself does not show any effect (Fig. 3B), suggesting that NPC2 plays its role via MPR.. y% ]8 q$ X. L. H+ P" ]5 Y% ~2 c
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NPC2 Protein Regulates Survival and Differentiation in MO7e Cells
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To determine whether NPC2 promotes MO7e cell survival via inhibiting apoptosis, the Annexin V staining assay was used. After 4 days of culture with 7% oxygen, apoptosis was induced by switching cells to serum-free medium for 4 hours. Cells were then collected to determine the percentage of apoptotic cells. As shown in Figure 4A and 4B, there were significantly fewer apoptotic cells in TPO-plus-NPC2-treated cells than TPO-only-treated cells (30.9% vs. 39.4%, p = .00235). Next, the effect of NPC2 protein on cell proliferation was determined. After 24 hours of serum starvation, MO7e cells were cultured with TPO only or TPO plus NPC2 protein at both 7% and 20% oxygen conditions. As shown in Figure 4B, there was no significant cell proliferation difference under either condition for the first 5 days. However, at day 10, under 7% oxygen, the viable cell number declined in the TPO-only condition, whereas there was still a slight increase in the TPO-plus-NPC2 condition. The difference in the total viable cell number at 10 days was significant (2.1 vs. 1.7 x 106, p = .022). These results suggest that NPC2 protein, together with TPO, may play roles in MO7e cell survival but have less effect on cell proliferation.

Figure 4. Anti-apoptosis effect of NPC2 protein in MO7e cells. (A): After 4 days of culture in the presence of either TPO alone or TPO with NPC2 protein, MO7e cells were switched to serum-free medium for 4 hours to induce apoptosis, followed by Annexin V staining to determine the percentage of apoptotic cells by fluorescence-activated cell sorting analysis (*, p = .00235). (B): MO7e cell proliferation and survival curve. Twenty-four-hour serum-starved cells were transferred into 24-well plates and cultured under various conditions (no treatment: control; NPC2 only: 0.5 µg/ml; TPO only: 10 ng/ml; and NPC2 plus TPO: 0.5 µg/ml and 10 ng/ml, respectively). Cell number was determined every 24 hours using a hemacytometer with trypan blue staining. Results are a summary of two individual experiments, with total of six samples (*, p = .022). Abbreviations: NPC2, Niemann-Pick type C2; TPO, thrombopoietin.2 ~2 N0 M9 ^3 {6 }) x8 r; F

Because NPC2 protein results in more CFU-GEMM in the CFC assay, an indication of a possible anti-differentiation effect, we tested whether NPC2 protein could play a similar role with MO7e cells. As determined by the appearance of CD41 expression on cell surface, MO7e cells undergo partial megakaryocyte differentiation when cultured in the presence of TPO . Ten thousand cells were mixed with methylcellulose in the presence of SCF and TPO (100 ng/ml each) under 7% oxygen conditions. After 7 days of culture, a difference in colony phenotype was observed: most of the colonies in the NPC2-treated plates were more condensed, with cells in high density, whereas colonies in the control plates were more diffuse, with cells at a low density (Fig. 5A). These cells were recovered and stained with anti-CD41 antibody. There were fewer CD41  cells from NPC2-treated plates compared with plates without NPC2 protein (Fig. 5B, 4.99% vs. 8.85%, p = .0319). NPC2 mutants (both cholesterol-binding-defective and nondefective mutants) showed the same effects as wild-type protein (data not shown). These results suggest that NPC2 may have an anti-differentiation effect in MO7e cells and that these effects are independent of cholesterol-binding activity.
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Figure 5. Antidifferentiation effect of NPC2 in MO7e cells. (A): NPC2 induces MO7e colony phenotype change. At day 7, in the presence of NPC2, MO7e cells made more compacted colonies, whereas MO7e cells without NPC2 made more diffuse colonies. Magnification, x 4. (B): NPC2 inhibits TPO-induced differentiation. MO7e cells were stained with anti-CD41 antibody and analyzed with fluorescence-activated cell sorting to measure CD41 expression. Results are a summary of two individual experiments, with total of six samples (*, p = .0319). Abbreviations: NPC2, Niemann-Pick type C2; TPO, thrombopoietin.6 f3 s5 C6 P8 @: k

HIF-1- Is Upregulated by NPC2 Protein in MO7e Cells

All results showing NPC2 effects in hematopoiesis were obtained under the condition of 7% O2, the oxygen level of bone marrow, but not under normal oxygen levels (~20% O2). This observation led us to examine whether NPC2 might also modulate HIF-1- expression in MO7e cells. MO7e cells were collected at 4, 8, and 12 hours of growth in medium with TPO or TPO/NPC2 protein to detect HIF-1- protein by Western blot. There was no change in HIF-1- protein level under 20% oxygen. However, under 7% oxygen, an increase of HIF-1- at 8 and 12 hours was observed. Cells with TPO and NPC2 protein showed a higher HIF-1- level compared with cells with TPO only (Fig. 6).' c( z7 P" a$ T1 s0 C& a& y

Figure 6. NPC2 upregulates HIF-1- level in MO7e cells. Western blot analysis assessing changes of HIF-1- protein level with or without NPC2 protein in MO7e cells at different times. Results represent three individual experiments. Oxygen level was 7%. ARNT, a binding partner of HIF-1- expressed constitutively, was used as loading control. Density of band was determined by using Quantity one (Bio-Rad, Hercules, CA, http://www.bio-rad.com). Abbreviations: ARNT, aryl hydrocarbon receptor nuclear translocator; HIF, hypoxia inducible factor; NPC2, Niemann-Pick type C2; TPO, thrombopoietin.

DISCUSSION* J3 u' I3 ~/ O; v% }7 }
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The high expression of NPC2 protein and its receptor, MPR, in HSCs led us to study their potential function in hematopoiesis. Using in vitro colony assays, we showed that NPC2 could shift the colony type in the CFC assay: increasing early progenitor cell colony formation (CFU-GEMM) at the expense of later progenitor cell colony formation (CFU-GM). This observation occurred only at the oxygen level of bone marrow (7%), with no effect being seen in air (~20% oxygen). Further study also showed that NPC2¡¯s effect occurs specifically in the presence of TPO in the assay system, because NPC2 did not show any effect when TPO is replaced by IL-3, IL-6, or Flt-3 ligand, factors commonly used in CFC assays. This observation suggests that NPC2 may play its role by influencing the TPO/Mpl system. If NPC2 functions via a general mechanism such as by influencing cholesterol content in the cell, its effect should be observed when TPO is replaced by other factors.
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NPC2 has been characterized as a cholesterol-binding protein. Cholesterol is an important biological molecule with multiple functions, including modifying the permeability and fluidity of lipid membranes, serving as a precursor for steroid hormone and bile acid synthesis, and modifying proteins by covalent binding in the lipid membrane . We first attempted to examine whether both NPC2 and Mpl are located in lipid rafts, which might suggest that the function of the TPO/Mpl system may be facilitated by the presence of NPC2 protein. However, we could detect neither NPC2 nor Mpl in lipid raft fractions (data not shown).

Further experiments demonstrated that the NPC2 function is independent of cholesterol-binding activity (Fig. 3A). NPC2 mutants, whether maintaining cholesterol-binding activity or not, can function as well as wild-type protein in the CFC assay (Fig. 2A). Thus, NPC2 effects on HSCs are not dependent on cholesterol-binding activity.

Next, we did experiments to determine whether NPC2 protein functions via MPR. It has been demonstrated that M6P can block the NPC2 effect in NPC2 mutant fibroblasts .  w4 ~& H# H3 n. M& L7 m
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How NPC2 functions in HSC regulation is unknown. One speculation is that upon binding and internalization via MPR, NPC2 is released into the cytoplasm and interacts with other molecules to regulate HSCs. Recently, it was demonstrated that NPC2 interacts with dehydrodolichyl diphosphate synthase, which is involved in the regulation of dolichol biosynthesis . This finding opens the possibility that NPC2 may have different binding partners in the cytoplasm to perform different functions. By immunofluorescence staining, we also observed that exogenous NPC2 protein can accumulate in the nucleus (data not shown). We plan to examine whether NPC2 protein may play a role in transcriptional regulation or in the transportation of other molecules between the nucleus and cytoplasm.* e( ~6 G$ G2 s1 N. n  D4 A

It has been reported that TPO induces cell differentiation. TPO also shows an anti-apoptosis function . In both HSCs (Lin¨C Sca  Kit ) and MO7e cells, NPC2 plays a role in cell differentiation and apoptosis. When NPC2 is present with TPO, we observed increased CFU-GEMM in the CFC assay (Fig. 2) and decreased CD41  cells in MO7e culture (Fig. 5B). These data suggest NPC2 may inhibit TPO-induced differentiation. In addition, NPC2 increases the TPO-mediated anti-apoptosis effect (Fig. 4A, 4B). Because MAPK and/or PI3K/Akt signaling pathways are important for cell survival and apoptosis regulation, protein phosphorylation of signaling molecules such as JAK2, STAT3, STAT5, p38, and ERK1/2 was examined in normal oxygen conditions. There was no significant difference in protein phosphorylation between NPC2/TPO-treated and TPO-treated cells under these conditions (data not shown). We could not perform the same experiment under 7% oxygen, because protein phosphorylation occurs very rapidly and we did not have apparatus available to carry out the experiment in low oxygen. However, considering that all NPC2 effects on hematopoiesis were observed under low oxygen conditions, it is possible that NPC2-regulated phosphorylation might also occur in HSCs under low oxygen.

Following this line of thinking, we tested the hypothesis that NPC2 may affect molecules regulated by oxygen level. HIF-1- is one of the best-studied proteins with such a property. By regulating its target genes, HIF-1- is involved in multiple cellular functions, including cell survival, proliferation, and differentiation via multiple factors such as vascular endothelial growth factor (VEGF), EPO, IGF2, and Nix protein . NPC2 may play its role together with TPO in influencing HIF-1- level in HSCs. It remains to be determined whether NPC2 functions via the TPO signaling pathway.. p2 }! Q9 l* E3 X: D, b
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NPC2 is a cholesterol-binding protein related to neuron-degenerative disease. Our report is the first to show the involvement of NPC2 protein in hematopoiesis. Furthermore, our data demonstrate that NPC2 functions via TPO/Mpl system on 7% oxygen. Because of the high expression of endogenous NPC2 protein, the observed effects from the exogenous NPC2 in our experimental system are not dramatic but show that the exogenous NPC2-mediated effects are statistically significant. In NPC2 patients, the major pathological change occurs in the nervous system. The lack of a hematopoietic phenotype in NPC2 disease may be from the presence of other molecules that can compensate for the loss of functional NPC2 protein. A similar observation is seen in the TPO/Mpl knockout mice: normal platelets are produced in these animals, suggesting a compensatory mechanism . Our study suggests that NPC2 may function in hematopoietic tissue differently than in the central nervous system.

CONCLUSION
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We reported in this study that NPC2 protein is involved in hematopoiesis regulation. However, this function can be detected only under physiological oxygen level in the bone marrow. Using different methods, we show NPC2 protein has anti-differentiation and anti-apoptosis effects via the TPO/Mpl system. The hematopoiesis regulation effect is independent of cholesterol-binding activity but relies on MPR/IGFII-R.

DISCLOSURES6 P* Y! i# P) z  V+ `9 b
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The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS7 `3 D5 J: M! T" k7 H5 C
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We thank Dr. Y. Durocher for pTT3 vector, Drs. M.P. Scott and D.C. Ko for the NPC2 mutants, Dr. D.S. Ory for NPC2 fibroblasts, and Drs. T.E. Adams and A. Ciarletta for MO7e cells. This study was supported by the G. Harold & Leila Y. Mathers Charitable Foundation and Farmal Biomedicines LLC.
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