The role of CCAAT/enhancer binding protein-α (C/EBPα) in the development of

myeloid and erythroid lineage hematopoietic cells

Hyung Chan Suh

Department of Medicine,The Graduate School, Yonsei University

The role of CCAAT/enhancer binding protein-α (C/EBPα) in the development of

myeloid and erythroid lineage hematopoietic cells

Directed by Professor ; Yun Woong Ko

The Doctoral Dissertation submitted to the Department of Medicine, the Graduate School of Yonsei

University in partial fulfillment of the requirement for the degree of Doctor of Philosophy

Hyung Chan Suh

June, 2004

This certifies that the Doctoral Dissertation of Hyung Chan Suh is

approved.

Thesis Supervisor: Yun Woong Ko

Jonathan R. Keller

In-Hong Choi

Hyun Ok Kim

Yoo Hong Min

The Graduate School,Yonsei University

June, 2004

감사의

이 논문을 완성함에 있어 세심한 배려와 지도를 베풀어 주

신 고윤웅 선생님께 진심으로 감사 드립니다. 또한 연구기간

동안 많은 토론과 의견 교환으로 실험을 이끌어 준 Jonathan R.

Keller 박사에게 심심한 감사를 표하며, 논문을 심사하여 주신

최인홍 선생님, 김현옥 선생님, 민유홍 선생님께 감사드립니다.

아울러 연구기간 내내 함께 일하며, 여러 모양으로 힘이 되

어 준 National Cancer Institute-Frederick의 모든 동료들과 논문

이 나오기까지 궂은 일을 마다하지 않고 도와준 정준원 선생

과 내과학교실 비서실 여러분께 고마운 마음을 드립니다.

항상 믿어주고, 사랑으로 보살펴 주신 어머님과 어려울 때

마다 힘이 되어 준 나의 아내 승연, 큰 아들 유 , 둘째 아들

진 에게 이 논문을 드립니다.

저 자

TABLE OF CONTENTS

ABSTRACT ········································································································ 1

I. INTRODUCTION ··························································································· 3

II. MATERIALS AND METHODS ··································································· 5

1. Mice ········································································································ 5

2. Cells ········································································································ 5

3. Fetal liver cells transplantation ······························································ 5

4. Transplantation of BMCs over-expressing C/EBPα ······························· 6

5. Clonogenic assays ··················································································· 7

6. Antibodies/Flow cytometry analysis ······················································· 7

7. Establishment of C/EBPα over-expressing erythroid cell line ·············· 7

8. Western blot analysis ············································································· 8

9. RT-PCR ··································································································· 8

10. Northern blot analysis and RNase protection assays ···························· 9

11. Electrophoretic mobility shift assay (EMSA) ······································· 10

III. RESULTS ··································································································· 11

1. Effect of HGFs on the proliferation of C/EBPα-/-

FL cells in vitro ·················································································· 11

2. HGF receptor expression on C/EBPα-/- FL cells ································· 13

3. Effect of HGF on the differentiation of C/EBPα-/-

FL cells in vitro ·················································································· 14

4. Expression of C/EBP family members in freshly harvested C/EBPα-/- and C/EBPα+/+ and in HGF-treated FL cells ······································ 14

5. Hyper-proliferation of C/EBPα-/- FL cells in vivo ······························· 16

6. Defect in macrophage development in mice transplanted with C/EBPα-/- FL cells ··············································································· 17

7. Increased BFU-e colony and erythroblast in C/EBPα-/- FL ················ 21

8. Increased erythropoiesis is an intrinsic property of C/EBPα-/- cell ···· 22

9. C/EBPα inhibits erythroid development of BM HPC ························· 24

10. Over-expression of the C/EBPα in MEL cells blocks erythroid differentiation ······················································································· 26

11. Gene expression changes by C/EBP in erythroid differentiation of MEL cells ···························································································· 28

IV. DISCUSSION ····························································································· 32

1. C/EBPα is critical transcription factor for the CMP to differentiate to the GMP ····································································· 32

2. C/EBPα is a negative regulator in erythroid differentiation ··············· 33

V. CONCLUSION ···························································································· 36

REFERENCES ·································································································· 37

ABSTRACT (In Korean) ··················································································· 41

LIST OF FIGURES

Figure 1. Effect of hematopoietic growth factors (HGFs) on the growth of C/EBPα-/- FL cells in vitro ···························································· 12Figure 2. Expression of HGF receptors in C/EBPα-/- FL cells ···················· 13Figure 3. Morphology of C/EBPα-/- FL cells grown in IL-3 and GM-CSF ···· 15Figure 4. C/EBP binding activities in FL extracts and C/EBPα-/- FL cell lines ··················································································· 15Figure 5. Transplantation of C/EBPα-/- FL cells in vivo ······························ 17Figure 6. Role of C/EBPα-/- in macrophage differentiation in vivo ············· 20Figure 7. C/EBPα-/- fetal liver cells have increased erythroid lineage differentiation potential ··································································· 21Figure 8. C/EBPα-/- FL derived BM cells have increased erythroid lineage differentiation potential in vivo ········································· 23Figure 9. Effect of C/EBPα on long term repopulating activity in vivo ····· 25Figure 10. Over-expression of C/EBPα inhibits erythroid differentiation and proliferation of MEL cells ····················································· 27Figure 11. Effect of C/EBPα over-expression on expression of genes related with erythroid differentiation ············································· 29Figure 12. Effect of C/EBPα on the expression of erythropoietin receptor ···· 30Figure 13. GATA-1 and PU.1 protein expression in MEL and αER cells ····· 31Figure 14. Effect of C/EBPα on the expression of Id1 ································· 31Figure 15. Summary of the role of C/EBPα in hematopoiesis ······················ 35

LIST OF TABLES

Table 1. CFU-s activity of C/EBPα-/- fetal liver cells ·································· 17Table 2. Effect of C/EBPα over-expression on M-CSF-induced macrophage differentiation in C/EBPα-/- FL cells ··························· 19

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Abstract

The role of CCAAT/enhancer binding protein-α (C/EBPα) in the development of myeloid and erythroid lineage hematopoietic cells

Hyung Chan Suh

Department of Medicine,The Graduate School, Yonsei University

(Directed by Professor Yun Woong Ko)

The CCAAT enhancer binding protein-α (C/EBPα) is a member of the bZIP family of transcription factors. In the hematopoietic system, C/EBPα plays a role in the growth and development of myeloid cells. Mice that lack C/EBPα show an absence of neutrophils. But it is not clear at which developmental stage, C/EBPα regulates proliferation and induces hematopoietic progenitors to differentiate into neutrophils. Therefore, we performed a detailed progenitor analysis of the proliferative and diffe- rentiative potential of primitive and more committed hematopoietic progenitor cells(HPC) present in the fetal liver (FL) of C/EBPα-/- mice.

In this study we demonstrated that C/EBPα-/- fetal liver (FL) progenitors were hyper- proliferative, and showed decreased differentiation potential to myeloid lineage. Hyper- proliferative spleen colonies were observed in mice reconstituted with C/EBPα-/- FL cells indicating hyper-proliferation of progenitors in vivo, in addition to in vitro. There were fewer committed bi-potential progenitors in C/EBPα-/- FL, while multi- potential progenitors were unaffected. C/EBPα-/- FL lacked macrophage progenitors in vitro and had impaired ability to generate macrophages in vivo. These findings indi- cate that C/EBPα deficiency results in hyper-proliferation of HPC and a block in the ability of multipotential progenitors to differentiate into bi-potential granulocyte/ monocyte progenitors (GMP) and their progeny.

We have found C/EBPα-/- FL progenitors have increased numbers of erythroid

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progenitors (burst forming unit-erythroid; BFU-e) and erythroblasts in the FL, and C/EBPα is known to be expressed in hematopoietic stem cells (HSC) and common myeloid progenitor (CMP). Thus, we hypothesized that C/EBPα may function to promote myeloid differentiation versus erythroid differentiation in primitive hemato- poietic progenitor cells. To test this, we ① evaluated erythroid development in mice transplanted with C/EBPα-/- FL, ② infected 5FU-treated bone marrow cells (BMC) with retroviral vectors that express C/EBPα and evaluated erythroid development in transplanted mice, ③ and established C/EBPα/human estrogen receptor α (hERα)- inducible murine erythroid leukemia (MEL) cell lines. No donor granulocytes were detected in the peripheral blood of mice transplanted with C/EBPα-/- FL, however, we observed an increase in TER119+/CD71+ erythroid cells in the BMC. On the other hand, BMC showed increased granulocyte/macrophage populations (Mac-1+) and decreased erythroid lineage cells in mice transplanted with 5FU-treated BMC infected with retroviral vectors that express C/EBPα. We also found that activated C/EBPα/hERα inhibited erythroid differentiation of MEL cells, even though it did not induce PU. 1 mRNA or protein expression. Activated C/EBPα/hERα induced Id1 expression which is known to inhibit erythroid differentiation of MEL cells, and decreased erythropoietin receptor, SCL, E2A expression that are also important for erythroid differentiation and survival.

Taken together, the data indicate that ① loss of C/EBPα gene expression results in hyper-proliferation of HPC, ② C/EBPα is required for the differentiation of CMP to GMP and their progeny and, ③ C/EBPα is a negative regulator of erythroid development and differentiation.

Key Words: C/EBPα, hematopoiesis, transcription factor, differentiation

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The role of CCAAT/enhancer binding protein-α (C/EBPα) in the development of myeloid and erythroid

lineage hematopoietic cells

Hyung Chan Suh

Department of Medicine,The Graduate School, Yonsei University

(Directed by Professor Yun Woong Ko)

I. INTRODUCTION

Hematopoiesis is a complex cellular process whereby short-lived mature blood cells are continuously replenished by a limited number of hematopoietic stem cells (HSC) through rapidly dividing transit cell populations. Lymphoid cells originate from the common lymphoid progenitors (CLP)1, and non-lymphoid lineages develop from a common myeloid progenitors (CMP) that proliferate and differentiate to restricted progenitors such as granulocyte monocyte (GMP) and megakaryocyte erythrocyte progenitors (MEP)2. This process is regulated, in part, by the hematopoietic microenv- ironment, and hematopoietic growth factor (HGF) that binds to cell surface receptors to activate signal transduction pathways that either positively or negatively regulate transcription factors3.

Transcription factors are essential for the development of hematopoietic cells by regulating the expression of HGF, HGF receptors, other transcription factors, and lineage specific genes4. It is thought that multiple transcription factors are expressed in hematopoietic progenitor cells (HPC) that are not yet committed to a specific diffe- rentiation pathway5. Each committed progenitor has a distinct gene expression pro- gram that is organized and preserved by lineage specific transcription factors. Lineage determination may be controlled through up-regulation of certain lineage

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specific factors and protein-protein interactions that lead either synergistic or inhibi- tory interactions. PU.1, FOG-1 and GATA-1 are good examples to illustrate these process in myeloid, erythroid lineage determination6,7.

The CCAAT enhancer binding protein (C/EBP) family members are basic leucine zipper transcription factors which are important in hematopoietic development. C/ EBPα is expressed in HSC, CMP, and GMP but not CLP or MEP2,5. C/EBPα acti- vates promoters of myeloid specific receptors; G-CSF, macrophage CSF (M-CSF), and granulocytemacrophage CSF (GM-CSF) and synergizes with PU.18-11. Over-expression of C/EBPα in some HPC lines induces granulocyte differentiation12,13, and C/EBPα knock out mice show neutrophil defects due in part to the absence of G-CSF recep- tor and IL-6 receptor expression14. In addition to regulating differentiation, C/EBPα has been shown to inhibit the proliferation of a number of cell types in vitro and in vivo15,16. It has recently been proposed that loss of C/EBPα expression or function may contribute to the differentiation block, enhanced proliferation and development of some acute myelogenous leukemia (AML). For example, the expression of the AML- ETO fusion protein in some patients inhibits C/EBPα transcription, while the over- expression of BCR-ABL prevents C/EBPα translation17,18. Also heterozygous mutations at the C/EBPα locus, which generate dominant negative forms of C/EBPα, have been observed in AML patients19-21. These observations support the hypothesis that C/EBPα plays critical roles in regulating the balance between proliferation and differentiation of hematopoietic progenitors. In this regard, while it is known that C/EBPα is requi- red for the final stages of neutrophil maturation9 and it can promote granulocytic differentiation and block macrophage development in U937 cell line12, it is not known whether C/EBPα functions in more primitive progenitors.

In this study, we asked ① whether C/EBPα might play a role in regulating the proliferation and differentiation of more primitive HPC in vitro and in vivo, and ② if C/EBPα affects the development of erythroid lineage. To address these questions, we performed a detailed progenitor analysis of C/EBPα-/- fetal liver (FL) to define at what stage progenitors are blocked in myeloid differentiation. We also examined the maturation of C/EBPα-/- FL cells or C/EBPα over-expressing congenic bone marrow cells transplanted in vivo. In addition, we established C/EBPα inducible murine erythro- leukemia cell line (MEL) to evaluate the role of C/EBPα in erythroid differentiation.

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II. MATERIALS AND METHODS

1. Mice

The C/EBPα+/- mice, which had been on a mixed background of 129sv and C57Bl/622, were backcrossed nine generations onto a pure C57Bl/6 background (Ly5.1). They were mated to produce C/EBPα+/+, C/EBPα+/-, and C/EBPα-/- embryos for fetal liver transplantation studies. All mice were genotyped by southern blot analysis of geno- mic DNA using a C/EBPα cDNA probe and maintained at the animal production area at National Cancer Institute-Frederick (Frederick, MD, USA). Animal care was provided in accordance with the procedures outlined in the “Guide for Care and Use of Laboratory Animals (publication number 86-23, Bethesda, MD, National Institutes of Health, 1985). Mice were maintained in a pathogen-free environment.

2. Cells

Fetal liver cells were harvested from embryos at 15-16 days after gestation and single cell suspensions were made from individual FLs. Peripheral blood (PB) mononuclear cells were obtained from retro-orbital sinus bleeds, and red cells were lysed by 3 min incubation in 0.15 mol/L ammonium chloride with 0.01 mol/L potassium carbonate (ACK). BM cell (BMC) suspensions were obtained by flushing femurs using phosphate-buffered saline (PBS) and 27 G needle. The suspensions were filtered through nylon mesh. BMCs and PB were washed once and incubated with the mixture of monoclonal antibodies in PBS containing 0.1% BSA for FACS analysis. Peritoneal exudates cells (PEC) were harvested by lavage with 10 ml PBS containing 5mM EDTA before (resident PEC) and 72 hours after (elicited PEC) injection with 2ml of 3% thioglycollate medium (DIFCO, Detroit, MI, USA).

3. Fetal liver cells transplantation

Six- to eight-week-old C57Bl/6 (Ly 5.2) recipient mice were exposed to 1100 rad in a divided dose from a 137Cs source. Donor fetal liver cells (Ly 5.1) were prepared as single cell suspensions in PBS from C/EBPα-/- mice or their littermate controls.

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Five to 50×105 FL cells with or without 1-2×105 host BMCs in 200ul PBS were injected through the tail vein into recipient animals. For the erythroid repopulation assay, FL cells were washed once in PBS and resuspended at a concentration of 1-2×106/ml in complete Isocove Modified Dulbecco's media (IMDM containing 10% FBS, 2 mM L-glutamine, 1% penicillin-streptomycin) with 100 ng/ml murine stem cell factor (mSCF), 100 ng/ml human Tpo, 100 ng/ml human Flt-3L, and 50 ng/ml IL-6(Biosource, Camarillo, CA, USA). The following day, fetal liver cells were co-cultu- red with 65%(v/v) of the retroviral supernatant containing pMSCV-GFP twice 4 hours apart, keeping the identical cytokine concentrations. Two days later, FL cells were sorted according to GFP expression and 3×103 GFP positive FL cells were trans- ferred to recipient mice. After transplantation, the animals were maintained in clean area. Reconstituted donor- and host-derived cells in PB, BM, and peritoneal exudates were analyzed by FACS analysis.

4. Transplantation of BMCs over-expressing C/EBPα

Femoral BM cells were prepared from 4- to 6-week-old inbred female C57Bl/6 mice that had been given 150 mg/kg 5-fluorouracil (5-FU) (Sigma, St Louis, MO, USA) intraperitoneal injection 4 days earlier to enrich for proliferating stem cells. For retroviral infection, nucleated BM cells were pre-stimulated to promote cell division with hIL-6 (50 ng/ml), hTpo (100 ng/ml), hFlt-3L (100 ng/ml) and mSCF (100ng/ml) in a DMEM supplemented with 20% heat-inactivated fetal bovine serum (FBS) plus 1% penicillin and streptomycin (GIBCO-BRL, Rockville, MD, USA) for 48 hours in a humidified atmosphere with 10% CO2 at 37℃. Erythrolysis of spleen and BM cells was done by treating the cells with buffered ammonium chloride for 3 minutes. The cells were then washed 3 times with PBS. Retroviral particles were generated by transfecting the pMSCV-C/EBPα-GFP vectors (kindly provided by Dr. P. Johnson) into the high efficiency Phoenix packaging line. The resulting viruses were used to infect the 5-FU stimulated BM cells. Whole bone marrow cells (5-10 ×105) were transplanted to the recipient mice and sorted GFP positive cells were used for clonogenic assays. GFP expression was used to distinguish donor cells from recipient cells.

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5. Clonogenic assays

FL cells, and BM cells were suspended in 1 ml of complete IMDM and 0.35% sea plaque agarose (FMC, Rockland, ME, USA) and were plated at the indicated cell densities in suspended in 35-mm Lux petri dishes (Miles Scientific, Naperville, IL, USA) in the presence or absence of 50 ng/ml human G-CSF (Amgen, Thousand Oaks, CA, USA), 100 ng/ml M-CSF, 100 ng/ml murine SCF, 50 ng/ml murine GM- CSF, and 30 ng/ml murine IL-3 (Peprotech, Rocky Hill, NJ, USA). Dishes were incubated in a fully humidified atmosphere at 37℃, 5% CO2 and then scored for colony formation after 7-10 days (colony-forming unit-culture, CFU-c).

6. Antibodies/Flow cytometry analysis

Flow cytometry analysis were performed on single cell suspensions by standard methods. Cells were stained with fluorescein isothiocyanate (FITC), phycoerythrin (PE)- conjugated antibodies, or TCⓇ (Caltag Lab, Burlingame, CA, USA) and biotinylated monoclonal antibodies. They were sorted on a dual laser cell sorter (FACSCAN; BD, San Jose, CA, USA). Monoclonal antibodies (MAb) Gr-1, Mac-1, F4/80, TER-119, and CD71 were used according to the instructions of the manufacturer (Pharmingen, San Diego, CA, USA: Biosource, Camarillo, CA, USA for F4/80). GFP positive cells were selected and FACS-sorted on MoFlo (Cytomation, Ft. Collins, CO, USA) for further analysis by cytospin or colony assays. CELLQuest (BD, San Jose, CA, USA) was used for FACS analysis.

7. Establishment of C/EBPα over-expressing erythroid cell line

Murine erythroleukemia cell line, DS-19 (kindly provided by Dr. S. Ruscetti), was cultured in Dulbecco modified Eagle medium (DMEM) without phenol red with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 units/ml) and streptomycin (100μg/ml). To induce differentiation, the cells were washed twice with PBS and trans- ferred to DMEM with 10% FBS and 3 mM HMBA (Sigma, St Louis, MO, USA). High titer replication-free retroviral supernatants were generated as follows; Phoenix packaging cells were plated in 75 T flasks 18 hours prior to transfection and put in

8

15 ml fresh DMEM media 1 hour before transfection. Plasmid (pBabe, 6 μg) containing both C/EBPα/hERα and SV40 early promoter-PuroR cassettes, or the control construct lacking C/EBPα/hERα coding regions (kindly provided by Dr. A Friedman), were co-transfected with pCL-eco (6 μg) using 36 μl Fugene 6 (F-Hoffmann- La Roche, Basel, Switzerland) into proliferating Pheonix cells ( > 50% confluent). The resulting retroviral supernatant was collected 48 hours later and was used to infect DS-19 cells. DS-19 cells were transfected by co-culturing for 2 days with 8 μg/ml poly- brene (Aldrich, Milwaukee, WI). After transduction with pBabe-C/EBPα/hERα-puromy- cin or pBabe-puromycin (mock), subclones were isolated by limiting dilution and selected with 2.0 μg/ml puromycin. For transient C/EBPα expression, pcDNA3.1 cont- aining C/EBPα was transfected into MEL cells with Fugene 6 according to manufactu- rers protocol. The expression of C/EBPα was monitored by Western blot using anti- C/EBPα antibody (SC-61; Santa Cruz Biotechnology, Santa Cruz, CA, USA). For the differentiation assays, cells were seeded at 4×105/ml and incubated at 37℃. At each time point, cells were counted and stained for hemoglobin expression at different time point with benzidine at acid pH. A total of 200-300 cells per sample were evaluated.

8. Western blot analysis

Ten to 30 μg of protein samples were subjected to 12% sodium dodecyl sulfate- polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was then probed with an anti-rabbit C/EBPα antibody (sc-61), anti-rat GATA-1 antibody (sc-265), anti-rabbit EpoR antibody (sc-697), anti- mouse α-actin antibody (sc-8432), or anti-rabbit PU.1 antibody (sc-352) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) Bound antibody was detected by using an ECL kit (Amersham Biosciences UK Limited, Buckinghamshire, England).

9. RT-PCR

Total RNA was purified using RNeasy (Qiagen, Valencia, CA, USA) and converted to cDNA with Moloney murine leukemia virus reverse transcriptase (Applied Biosystems,

9

Fostercity, CA, USA). The PCR was conducted using ampli-Taq DNA polymerase (Perkin Elmer, Wellesley, MA, USA) at four different cycles to confirm that the reacion was in the exponential phase. PCR amplification consisted of 30 seconds at 94℃, 30 seconds at 58℃, and 1 minute at 72℃. The primers used were below; Id1: 5’-GATCATGAAGGTCGCCAGT G-3’, 5’-TCCATCTGGTCCCTCAGTGC-3’; SCL: 5’-TATGAGATGGAGATTTCTGATG-3’, 5’-GCTCCTCTGTGTAACTGTCC-3’; E2A: 5’-CATCCATGTCCTGCGAAGCCA-3’, 5’-TTCTTGTCCTCTTCGGCGTC-3’; EpoR: 5’-ACGAAACAGGGGCGCTGGAG-3’, 5’-ACACGTCCACTTCATATCGG-3’; β-globin: 5’-ATGGTGCACCTGACTGATGCTG-3’, 5’-GGTTTAGTGGTACTTGTGAGCC-3’; β-actin: 5’-GTGGGCCGCTCTAGGCACCAA-3’, 5’-CTCTTTGATGTCACGCACGATTTC-3’. PCR products were resolved in a 1% agarose gel containing ethidium bromide. The inten- sity of the bands was quantified by using the National Institutes of Health Image com- puter program (ImageJ, V1.32) (National Institutes of Health, Bethesda, MD, USA).

10. Northern blot analysis and RNase protection assays

Total RNA was purified using RNeasyTM (Qiagen, Valencia, CA, USA) as outlined by the manufacturer. Separation of RNA samples (10-15 μg) by electrophoresis was performed on 1% agarose, 5.2% formaldehyde (37% solution), 1×MOPS. The running buffer was 1×MOPS, 5% formaldehyde. RNA was transferred to nylon membrane (MSI, Westboro, MA, USA), and hybridized at 60℃ in Church-Gilbert hybridization solution (0.5 M NaPO4, pH 7.0, 1 mM EDTA, 1% BSA, 7% SDS). Murine M-CSFR (gift of Dr. Larry Rorschneider) and GAPDH (Clontech, Palo Alto, CA, USA) cDNA probes were labeled with random primers using Prime-It IITM (Stratagene, La Jolla, CA, USA) following the manufacturers' instructions. For RPA, a probe set contai- ning murine GM-CSFRα, IL-6R, and gp130 (BD-Pharmingen) was labeled with 32P- UTP (Amersham Biosciences UK Limited, Buckinghamshire, England) using the Riboquant In Vitro assay kit (BD-Pharmingen, San Diego, CA, USA). Protection assays were performed on 10 μg of RNA using the Riboquant RNase Protection Assay kit (BD-Pharmingen, San Diego, CA, USA) as recommended by the supplier. GM-CSFRα and IL-6R transcripts were quantitated using a Storm 860 phosphorimager (Amersham Biosciences UK Limited, Buckinghamshire, England).

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11. Electrophoretic mobility shift assay (EMSA)

The DNA probe for the wild-type consensus C/EBPα site (bold) was 5'-GATCCA- TATCCCTGATTGCGCAATAGGCTCAAAA-3', 3'-GTATAGGGACTAACGCGTTATCC- GAGTTTTCTAG-5' The probe was end-labeled using [32P]dCTP and Klenow polyme- rase. DNA-binding assays were carried out in a 25-μl reaction containing 5 μg nuclear extract, 20 mM HEPES (pH 7.9), 200 mM NaCl, 5% (w/v) Ficoll, 5% (v/v) glycerol, 1 mM EDTA, 50 mM DTT, 0.01% Nonidet P-40, 1.75 μg of poly(dI-dC), and 7.5×104 cpm probe. After incubation for 20 min at room temperature, 10 μl of the bin- ding reaction was loaded onto a 6% polyacrylamide gel in 1×TBE (90 mM Tris base, 90 mM boric acid, 0.5 mM EDTA) and electrophoresed 160 V for 2 hours. Super shift assays were carried out by preincubating the nuclear extract with 2 μl of rabbit antiserum at 4℃ for 30 min before addition of the binding reaction mixture. Antibodies specific for C/EBP proteins were obtained from Santa Cruz, except for the C/EBPγ antiserum which has been described previously23.

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III. RESULTS

1. Effect of HGFs on the proliferation of C/EBPα-/- FL cells in vitro

To determine whether primitive HPC that respond to multiple HGF combinations and committed HPC that respond to single HGF from C/EBPα-/- mice were hyper-prolife rative and defect in differentiation, FL cells were plated in soft agar colony assays(CFU-c) in the presence of HGF. As expected9, C/EBPα-/- FL cells did not proliferate in response to G-CSF, while C/EBPα+/+ and C/EBPα+/- FL cells showed a normal G- CSF response (Fig. 1A). In contrast to what has been previously observed14, the number of IL-3- and GM-CSF- responsive C/EBPα-/- CFU-c were decreased by 50% and 80% respectively, compared with the controls. Furthermore, there was a 95% decrease in the number of M-CSF-responsive CFU-c, and an absence of SCF- responsive CFU-c in C/EBPα-/- FL. Interestingly, little or no effect on the number of more primitive SCF/ IL-3-responsive CFU-c was observed in the C/EBPα-/- FL cell population.

In addition to the number of CFU-c, the size of the colonies can be readily evaluated in soft agar. As expected, the majority of colonies present in C/EBPα+/+ FL cultures containing IL-3 or GM-CSF consisted of committed progenitor cells that were less than 1 mm in diameter (Fig. 1B). In contrast, the majority of the IL-3- or GM-CSF-responsive CFU-c from C/EBPα-/- FL were large colonies greater than 1mm in diameter and frequently as large as 2 mm; a characteristic of more primitive progenitors with a higher proliferative potential (Fig. 1B). Furthermore, colonies greater than 4-6 mm in diameter were observed when C/EBPα-/- FL cells were grown in multi-HGF conditions (SCF plus IL-3). C/EBPα+/+ FL cells plated in liquid cultures containing GM-CSF or IL-3 contained few mitotic cells (cytocentrifuge preparations) and 2% of the cells were in S phase as determined by propidium iodide staining. In comparison, cytospins of C/EBPα-/- FL cell cultures contained many mitotic cells and the number of cells in S phase was 8.0%±2.4% for GM-CSF and 12.1%±3% for IL-3 cultures (data not shown). Taken together, C/EBPα-/- FL lacked mature unipotential HPC that respond to G-CSF, M-CSF and SCF, and had reduced numbers of committed HPC that respond to GM-CSF or IL-3. However, the remaining GM-CSF-, IL-3- and IL-3/SCF-responsive HPC from C/EBPα-/- FL were hyper proliferative.

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Figure 1. Effect of hematopoietic growth factors (HGFs) on the growth of C/EBPα-/- FL cells in vitro. FL cells were obtained from E16-E17 C/EBPα-/- mice and seeded into soft agar colony assays at 5×104 cells/ml in the presence or absence of 50ng/ml human G-CSF, 100 ng/ml M-CSF, 100 ng/ml murine SCF, 50 ng/ml murine GM-CSF, 30 ng/ml murine IL-3 (Panel A). Bright field photomicrographs (16×) were taken of the 35 mm grided (2 mm×2 mm) plates after 7 days. Dishes were incubated in a fully humidified atmosphere at 37℃, 5% CO2 and then scored for colony formation after 7-10 days. The results are presented as the mean number of colonies plus or minus the standard error, and are representative of at least 3 separate experiments (Panel B).

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2. HGF receptor expression on C/EBPα-/- FL cells

The absence of neutrophils in C/EBPα-/- mice is thought to be due, in part, to the lack of G-CSF and IL-6 receptor expression14. Therefore, we sought to determine whether decreased M-CSF and GM-CSF receptor expression could account for the absence of M-CSF responsive and decrease in GM-CSF-responsive progenitor cells. RNA was obtained from E16-E17 days C/EBPα-/-, C/EBPα+/+, and C/EBPα-/+ FL and analyzed for M-CSFR expression by northern blot hybridization (Fig. 2A). Little or no M-CSFR RNA was detected in the C/EBPα-/- FL by Northern blot analysis, while high levels were observed in C/EBPα+/+ and intermediate levels in C/EBPα+/- FL. In addition, analysis of GM-CSFRα mRNA by RNase protection assay demonstrates that GM-CSFRα expression was reduced by roughly 80% in C/EBPα-/- FL (Fig. 2B and C) compared to C/EBPα+/+ and C/EBPα+/- controls. In agreement with previous findings14, we observed decreased IL-6Rα and gp130 RNA levels in C/EBPα-/- FL compared to C/EBPα+/+ and C/EBPα+/- controls in RNase protection assays (Fig. 2B and C). Taken together, the absence of M-CSF receptor and decreased expression of

Figure 2. Expression of HGF receptors in C/EBPα-/- FL cells. RNA was extracted from E16-17 C/EBPα+/+, +/-, and -/- FL and analyzed by northern blot for the expression of M-CSFR and GAPDH (Panel A) or by RPA for GM-CSFR, IL-6R, and gp130 according to the procedures outlined in the Materials and Methods (Panel B). RPA signals were quantitated, relative to controls, by scanning densitometry (Panel C).

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GM-CSF correlate with decreased growth of C/EBPα-/- FL cells in response to M- CSF and GM-CSF in vitro.

3. Effect of HGF on the differentiation of C/EBPα-/- FL cells in vitro

Both GM-CSF and IL-3 promoted the proliferation and differentiation of granulocytes and macrophages; therefore, we sought to determine the identity of the cells that were present in the hyper proliferating soft agar colonies from C/EBPα-/- FL cells. As predicted, GM-CSF and IL-3 promoted the terminal differentiation of macrophages and granulocytes from C/EBPα+/+ FL cells after 7 days in culture (Fig. 3). IL-3 also induced mast cell differentiation in these cultures. In comparison, there was an increase in the number of primitive myeloid cells and a decrease in the number of differentiated granulocytes and macrophages in C/EBPα-/- FL cultures. Differential cell counts of cytospins showed that 65% of the C/EBPα-/- FL cells cultured in IL-3 were primitive myeloblasts and promyelocytes compared to 15% for the C/EBPα+/+

FL control cell cultures. In addition, only 3% of the cells in C/EBPα-/- FL cultures were more differentiated band and segmented neutrophils compared to 45% for the C/EBPα+/+ FL. Similar results were obtained for FL cells cultured in GM-CSF. Thus, C/EBPα-/- FL progenitor cells showed both increased proliferative potential and reduced differentiation in response to IL-3 or GM-CSF.

4. Expression of C/EBP family members in freshly harvested C/EBPα-/- and C/EBPα+/+ and in HGF-treated FL cells

To determine if other C/EBP proteins are expressed in C/EBPα+/+ and C/EBPα-/- primary FL cells and C/EBPα-/- FL cell lines, we examined nuclear lysates for the presence of C/EBPα, C/EBPβ, C/EBPδ, C/EBPε, and C/EBPγ by EMSA super shift assays. As shown in Fig. 4, DNA: protein complexes containing C/EBPα, C/EBPε, and C/EBPγ were detected in C/EBPα+/+ FL extracts. Two of the complexes appea- red to correspond to heterodimers of C/EBPα or C/EBPε these proteins with C/EBPγ, an inhibitory C/EBP protein that preferentially dimerizes with other C/EBP family members in many cells23-25. C/EBPα-/- FL contained neither C/EBPα nor C/EBPε, but did

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Figure 3. Morphology of C/EBPα-/- FL cells grown in IL-3 and GM-CSF. FL cells were harvested from E16-17 C/EBPα+/+ and -/- mice and plated in soft agar colony assays at 5×104 cells/ml or in liquid cultures at 2×105 cells/ml in cIMDM. Cells were removed from the cultures after 7 days incubation (37℃) in the presence of IL-3 or GM-CSF, cytocentrifuged, and stained with Giemsa, or pelleted and stained with propidium iodide for cell cycle analysis. Bright field photomicrographs were taken at a magnification of 400×.

Figure 4. C/EBP binding activities in FL extracts and C/EBPα-/- FL cell lines. Nu- clear extracts prepared from wild type FL (left panel), mutant FL (middle) or the C/EBPα-/- FL cell line cultured in IL-3 (right) were analyzed by EMSA using a con- sensus C/EBP site probe. Binding reactions included either non-immune serum (NI) or the indicated C/EBP antibodies. DNA-protein complexes were resolved on non- denaturing polyacrylamide gels. The identities of various dimeric C/EBP species are shown by arrows; asterisks denote complexes that are supershifted by the antibody.

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have detectable levels of C/EBPβ and C/EBPδ binding species that occur as hetero dimeric complexes with C/EBPγ. Examination of nuclear extracts from the SLF-IL- 3-2 cell line (Fig. 4, right panel) revealed only C/EBPγ complexes, with little or no expression of the other 4 known C/EBP family members. Thus, C/EBPα and C/EBPγ were the major isoforms present in FL hematopoietic cells, and other C/EBPα acti- vators did not appear to significantly compensate for the lack of C/EBPα in C/EBP-/- FL cells.

5. Hyper-proliferation of C/EBPα-/- FL cells in vivo

To determine whether primitive hematopoietic progenitors in the C/EBPα-/- FL cell population showed hyper-proliferation in vivo, we transplanted C/EBPα-/- FL cells into lethally irradiated recipients at increasing cell doses and looked for the ability of these cells to give rise to macroscopic spleen colonies (CFU-s). Radioprotection was not observed in animals receiving up to 1×106/cells C/EBPα-/- FL cells per mouse, as these cells are unable to give rise to mature neutrophils (Fig. 5A). In comparison, C/EBPα+/+ FL cells were able to radioprotect all recipient mice and promote their short term survival for 60-90 days with only 2×105 cells/mouse. Even though mice transplanted with C/EBPα-/- FL cells did not survive lethal irradiation, there was a 4-fold increase in the number of progenitors that gave rise to macro- scopic spleen colonies (CFU-s) in mice transplanted with C/EBPα-/- FL compared with their littermate controls (Fig. 5B and Table 1). In addition, the size of the CFU-s derived from C/EBPα-/- transplanted FL cells was much larger than those CFU-s derived from the C/EBPα+/+ transplanted FL cells, indicating that the C/EBPα-/- CFU- s were hyper-proliferating in vivo (Fig. 5B). Thus, despite the fact that C/EBPα-/- FL cells gave rise to increased numbers and size of CFU-s, they could not confer radio- protection. This was presumably due to an inability of these cells differentiate into functionally mature cells.

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Figure 5. Transplantation of C/EBPα-/- FL cells in vivo. C/EBPα+/+ and -/- FL cells were transplanted into lethally irradiated mouse recipients at the indicated cell doses and monitored for survival according to the procedures described in the Materials and Methods (Panel A). In other groups, spleens were removed 10-12 days after transplan- tation and fixed in Tellesniczky's solution to visualize spleen colonies (CFU-s) and photographed (Panel B).

Table 1. CFU-s activity of C/EBPα-/- fetal liver cellsCFU-s Cells Injected CFU-s per liver Mean CFU-s per Liver

C/EBPα+/+ 8±210±310±2

2×105

2×105

2×105

384 650 603 546±116

C/EBPα-/- 9±210±1 8±1

5×104

5×104

5×104

164722102240 2032±273

FL cells were harvested from E16-17 days C/EBPα+/+ and -/- mice and transplanted into lethally irradiated mouse recipients at the indicated cell doses according to the procedures described in the Materials and Methods. Total CFU-s per liver was calculated using the number of CFU-s/injected cell number and total liver cellularity. These data are represen- tative of three separate experiments.

6. Defect in macrophage development in mice transplanted with C/EBPα-/- FL cells

Because C/EBPα-/- FL could not differentiate into macrophages in response to M- CSF in vitro, we evaluated whether C/EBPα-/- FL could give rise to macrophages in vivo. In addition, we wanted to determine whether this developmental defect was

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intrinsic to the HPC or was due to their environment. Therefore, C/EBPα-/- and C/ EBPα+/+ FL cells (Ly5.1 donor) were transplanted into lethally irradiated mice toge- ther with normal BMC (Ly5.2 host cells) to provide radioprotection and track the development of donor-derived macrophages in vivo. First, we examined PB cells for the presence of donor-derived monocytes/macrophages and granulocytes by flow cyto- metry (Fig. 6 A). As expected, few if any donor-derived granulocytes (Gr-1Br Mac- 1Br) (0.1%) were detected in the PBC of mice transplanted with C/EBPα-/- FL cells, while 4.7% of the PBC in mice transplanted with C/EBPα+/+ were Gr-1Br Mac-1Br granulocytes. Also, mice transplanted with C/EBPα-/- FL cells showed low levels(0.6%) of Mac-1br F4.80br macrophages compared to mice transplanted with C/EBPα+/+ FL cells (1.7%). This defect in PBC macrophages was also observed with the Mac-1 and Gr-1 antibodies (Fig. 6A).

To confirm the deficit in macrophages present in PBC of mice transplanted with C/EBPα-/- FL cells, we analyzed donor-derived macrophages in resident PEC and thioglycollate elicited PEC populations. We found that 21.7% of the donor-derived resident PEC were Mac-1br F4.80br -macrophages in mice transplanted with C/EBPα+/+ FL cells, in contrast to 7.8% for C/EBPα-/- FL cells (Fig. 6B). In addition, 20.7% of the donor-derived PEC population were Mac-1br F4.80br -macrophages 3 days after thioglycollate treatment in mice transplanted with C/EBPα+/+ FL cells compared to 0.8% for C/EBPα-/- FL cells (Fig. 6 B and C). Similarly, C/EBPα+/+ FL cells gave rise to more macrophages (22%) than C/EBPα-/- FL cells (1.5%) in transplanted animals when Mac-1 and Gr-1 markers were assayed (Fig. 6B and C).

To evaluate whether mice transplanted with C/EBPα-/- FL cells lacked progenitors that proliferate in response to M-CSF, we obtained donor-derived (Ly-5.1) HPC by FACS and plated them in soft agar colony assays in the presence of M-CSF, GM-CSF, and the combination of SCF and IL-3. Donor-derived BMC from mice transplanted with C/EBPα-/- FL cells lacked progenitors responsive to M-CSF in contrast to BMC from mice transplanted with C/EBPα+/+ FL cells, which showed significant colony formation in response to M-CSF (Fig. 6D). Mice transplanted with C/EBPα-/- FL cells also lacked progenitors responsive to GM-CSF and contained fewer colonies that responded to the combination of SCF/IL-3 in comparison to mice transplanted with C/EBPα+/+ FL cells. We would expect that over-expression of C/

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EBPα in C/EBPα-/- FL would result in increased M-CSF response and macrophage development. Therefore, we infected C/EBPα-/- FL cells with retroviral vectors that express C/EBPα and measured macrophage development in soft agar in response to M-CSF or M-CSF/SCF. M-CSF and M-CSF/SCF induced macrophage colony forma- tion in C/EBPα-/- FL cells infected with C/EBPα-expressing retroviral vectors (Table 2). Although we observed a decrease in the number of colonies that responded to SCF/ IL-3 in C/EBPα-/- FL transplants, the remaining colonies in the plates were large hyper-proliferative colonies similar to those observed when C/EBPα-/- FL was directly cultured in SCF/IL-3. Collectively, these data showed that C/EBPα-/- FL cells had an intrinsic defect in macrophage development in vivo.

Table 2. Effect of C/EBP over-expression on M-CSF-induced macrophage differen- tiation in C/EBPα-/- FL cells

Growth Factor MSCV-GFP MSCV-C/EBPα-GFP

MediumM-CSF

M-CSF/SCF

00.5±0.5

4±1

07±18±2

FL cells were harvested from E16-17 C/EBPα-/- mice and cultured in SCF/TPO/ Flt3L/ IL-6 for 48 hours. The cells were washed and pelleted and then resuspended in medium containing C/EBPα-GFP, and GFP-expressing retroviral particles (3×). GFP+ cells were sorted by flow cytometry 48 hours after the last infection. Cells were plated in soft agar colony assays at 1.25×104 cells/plate according to the procedures described in the Mate- rials and Methods. Colony formation was evaluated after 7 days, and results are presented as the mean number of colonies plus or minus the standard error.

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Figure 6. Role of C/EBPα-/- in macrophage differentiation in vivo. C/EBPα+/+ and -/- FL cells were transplanted into lethally irradiated mouse recipients in combination with normal BMCs. Four to six months after transplantation, peripheral blood (Panel A), PEC (Panel B) and thioglycollate-elicited PEC (Panel C) were analyzed by three color flow cytometry for donor-derived (Ly-5.1) granulocyte and macrophage using Gr-1, F4/80 and Mac-1 according to the procedures outlined in the Materials and Methods. BMC were obtained from transplanted mice and FACS sorted for donor- derived cells and plated in colony assays in the presence of the indicated cytokines according to the procedures outlined in the Materials and Methods (Panel D). This data is presented as the mean colony formation plus or minus the S.E. and is repre- sentative of two separate experiments.

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7. Increased BFU-e colony and erythroblast in C/EBPα-/- FL

In addition to effects on myeloid cell growth, we evaluated whether erythroid development was impaired in C/EBPα-/- mice. Therefore we examined erythroid develop- ment by morphology in cytocentrifuge preparations of FL cells and colony assays (BFU-e). Differential cell count from C/EBPα-/- FL cells showed a higher mean erythroblast number (C/EBPα-/- = 394, C/EBPα+/- = 315, and C/EBPα+/+ = 274 per 400 hematopoietic cells) as expected, there was concomitant decrease in neutrophils (Fig. 7A). To test whether the differences might be due to the difference of erythroid progenitor activity, C/EBPα-/-, C/EBPα+/-, and C/EBPα+/+ FL cells were grown in methylcellulose culture in the presence of mSCF (100 ng/ml), mIL-3 (50 ng/ml) and hEpo (3 IU/ml). BFU-e colonies were scored after 7 days. Mean of 207.4 BFU-e was detected per fetal liver in C/EBPα-/- FL cells contained about two fold more in comparison to C/EBPα+/+ and C/EBPα+/- FL cells (Fig. 7B). Collectively, C/EBPα-/- FL showed increased number of erythroid progenitors and their progeny.

Figure 7. C/EBPα-/- fetal liver cells have increased erythroid lineage differentiation potential. Differential count was done with 400 hematopoietic cells and C/EBPα-/- FL cells shows increased number of erythroblasts in addition to decreased numbers of granulocytes. (Panel A) The FL cells from C/EBPα+/+, +/-, and C/EBPα-/- embryos at E15.5 stage (2.5×104 cells) were cultured in IMDM-based methylcellulose with erythropoietin (EPO) for 7 days. BFU-e colonies were then scored. The mean and SD of triplicate plates is shown (Panel B).

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8. Increased erythropoiesis is an intrinsic property of C/EBPα-/- cell

The differences observed in erythroid development could be due either to an intrinsic defect in erythroid progenitor cells or to the fetal liver microenvironment. Therefore we transplanted C/EBPα-/- FL cells into irradiated recipient mice to answer this question. Since erythroid lineage cells do not express CD45, GFP tagged FL cells were used to distinguish donor cells from recipient cells. FL cells were infec- ted with retroviral vectors containing MSCV-GFP. The infection efficiency of hema- topoietic progenitor cells with MSCV-GFP ranged from 10% to 50%. Normal mouse bone marrow cells were transplanted with GFP positive fetal liver cells of each genotype and then analyzed 120 days after transplantation. Differentiation of these progenitors in vivo was assessed by flow cytometry with antibodies that recognize myeloid markers Gr-1 in comparison with Mac-1 (granulocyte and monocytes), B cell marker (B220), T cell marker (CD3), erythroid marker TER119 in comparison with CD71 versus GFP expression in BM cells of the transplanted mice. As shown above with CD45 expression, C/EBPα-/- FL cells showed a defect in myeloid differentiation including macrophages and granulocytes. In comparison, there was a significant increase in percentage of TER119+ /CD71+ erythroid cells present in the BM cells of mice transplanted with C/EBPα-/- FL cells (68.1%) compared with C/EBPα+/+ control (45.3%) (Fig. 8A). To evaluate whether erythroid progenitors were also increased, we sorted GFP+ BMC and plated them in colony assays. Consistent with results obtained with FL cells, there was an increase in the number of BFU-e colonies in C/EBPα-/- FL transplanted mice (C/EBPα-/- group = 15.7, C/EBPα+/+ group = 7.3 per 7.5×104 cells) (Fig. 8B). Thus, C/EBPα-/- HPC showed increased erythropoiesis in vivo and it was not dependent on interactions with the microenvironment, but was an intrinsic pro- perty of C/EBPα-/- HPC itself.

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Figure 8. C/EBPα-/- FL derived BM cells have increased erythroid lineage diffe- rentiation potential in vivo. C/EBPα-/-, and C/EBPα+/+ FL cells were infected with retroviral vectors that express GFP to track donor cells and transplanted to lethally irradiated mice with host bone marrow cells. Bone marrow cells were harvested from mice 5 months after transplantation then analyzed for the expression of the erythroid markers (TER119 and CD71) and myeloid cell markers (Gr-1 and Mac-1) (Panel A) or cells were placed in soft agar for colony assay (Panel B).

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9. C/EBPα inhibits erythroid development of BM HPC

Since we observed that C/EBPα-/- mice had defect in myeloid progenitor develop- ment and showed increased numbers of erythroid progenitors in comparison to C/ EBPα+/+, we reasoned that up-regulating C/EBPα levels could promote myeloid development but negatively regulate erythroid development in primitive hematologic progenitors. To address this question, we infected BMC with retroviral vectors that express C/EBPα. Specifically, BM cells were obtained from 5-FU (150 mg/kg, IP) treated mice 3 days after treatment, and were infected with a retrovirus containing MSCV-C/EBPα-GFP or MSCV-GFP (control) (Fig. 9A). Two days after infection, 5 to 10×105 cells were injected into lethally irradiated mice with host normal bone marrow. Four months after transplantation, BMC, spleen, and PB were obtained from mice of each group to evaluate what effect C/EBPα had on myeloid and erythroid lineage development. Flow cytometry analysis with various monoclonal antibodies revealed that expression of Mac-1 was greatly increased, whereas expression of the TER119 was decreased in C/EBPα over-expressing BM cells compared with mock control (Fig. 9B) and in some mice, both the expression of Gr-1 and Mac-1 were increased (data not shown). The PB, spleen cells showed a similar pattern to that of BM cells (data not shown). Collectively over-expression of C/EBPα in HSC showed that C/EBPα promotes myeloid development while inhibiting erythroid development in vivo.

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Figure 9. Effect of C/EBPα on long term repopulating activity in vivo. C/EBPα was cloned into a retroviral expression vector (Panel A) 5FU stimulated BM cells from C57BL/6 mice were harvested and then infected with retrovirus containing MSCV- GFP or MSCV-C/EBPα-GFP. These cells were transplanted into lethally irradiated mice with host bone marrow cells. Four months after transplantation, the presence of donor-derived (GFP) granulocytes and monocytes (Gr-1 and Mac-1), and erythroid cells (CD71 and TER119) were determined by flow cytometry (Panel B).

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10. Over-expression of the C/EBPα in MEL cells blocks erythroid differentiation

To evaluate the effect of C/EBPα on erythroid differentiation, we transfected MEL cells with retrovirus vector that expresses C/EBPα/hERα fusion gene (Fig. 10A and D) and established stable C/EBPα/hERα inducible cell lines (αER-D6, αER-E4, αER-5F4). MEL cells are transformed erythroid precursors that are blocked roughly at the proery- throblast stage of differentiation26,27. Treatment of the cells with a variety of compo- unds, including dimethylsulf-oxide (DMSO) and hexamethylene bisacetamide (HMBA), induces cell differentiation program resulting in terminal cell differentiation and accumulation of hemoglobin and other erythrocyte-specific proteins. To determine whe- ther C/EBPα could inhibit MEL cell differentiation, the cells were treated with β- estradiol to activate C/EBPα and then treated with HMBA to induce differentiation. MEL and mock infected MEL cells differentiated into hemoglobin-positive cells (76%) within 96 hours. By contrast, the αER clone showed delayed kinetics of hemoglobin expression and exhibited only 17% positive cells after 96 hours (Fig. 10A). This was confirmed by Northern blot analysis, which showed reduced levels and delayed kinetics of β-globin mRNA expression in αER clones compared with control clones (Fig. 10B and C). Furthermore, Activation of C/EBPα in three αER clones showed little to no hemoglobinization compared to MEL or mock infected cells 4 days after stimulation with HMBA (Fig. 10B). There was an inverse relationship between C/ EBPα/hERα fusion protein expression and terminal erythroid differentiation, suggesting that C/EBPα inhibits terminal differentiation of MEL cells. C/EBPα/hERα also inhi- bited proliferation significantly compared with parent MEL and mock cells (data not shown). This observation is consistent with previous reports that C/EBPα can inhibit cell cycle by interacting with proteins that regulate the cell cycle28. Taken together, our data showed that C/EBPα/hERα inhibited erythroid differentiation of MEL cells, suggesting C/EBPα is negative regulator for erythroid differentiation.

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Figure 10. Over-expression of C/EBPα inhibits erythroid differentiation and proli- feration of MEL cells. MEL, mock, and αER-E4 clone were incubated in the absence or presence of HMBA (3 mM) and/or β-estradiol (1 uM) for 96 hours, and differentiated cells were identified by benzidine staining for hemoglobin (Panel A) Gross color of cell pellet after 4 days stimulation with HMBA indicates hemoglobinization. Over-expre- ssion of C/EBPα protein was confirmed by Western blot analysis (Panel B) β-globin mRNA levels were measured in HMBA treated MEL and αER cells by Northern blot analysis using 447bp fragment of mouse β-globin cDNA. Control lane shows 18S and 28S ribosomal RNA (Panel C) C/EBPα/hERα cDNA was subcloned into a pBabe vector and was transfected into Pheonix packaging cells The resulting retroviral supernatant was collected 48 hours later and was used to infect MEL cells MEL cells were transduced by co-culture for 2 days with 8 ug/ml polybrene After transduction with C/EBPα/hERα-puromycin or puromycin (mock), subclones were isolated by limiting dilution and selected with 2.0 ug/ml puromycin (Panel D).

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11. Gene expression changes by C/EBPα in erythroid differentiation of MEL cells

To determine the molecular mechanisms that C/EBPα inhibits erythroid differentiation, we first examined the expression of transcription factors and other genes that are known to be required for erythroid differentiation in αER cells. In particular, we found that erythropoietin receptor (EpoR) mRNA expression was decreased in HMBA and β-estradiol treated αER cells by RT-PCR (Fig. 11). These results were confirmed by Northern blot analysis (Fig. 12A). To confirm this inverse relationship between EpoR and C/EBPα expression, we determined EpoR protein levels by Western blot analysis in C/EBPα-/-, C/EBPα+/-, and C/EBPα+/+ FL cell protein lysates. C/EBPα-/- FL lysates showed an increase in EpoR expression in comparison to C/EBPα+/- and C/EBPα+/+ suggesting higher levels of EpoR are expressed in FL in the absence of C/EBPα, and that C/EBPα may suppress EpoR expression (Fig. 12B). We next determined if the expression of transcription factors that play important roles in erythropoiesis including GATA-1, PU.1, E2A, SCL, and Id1 were affected. HMBA treatment of MEL cells resulted in significant decrease of PU.1 protein and increase Id1 protein in 24 hours, while GATA-1 protein expression was not significantly changed during differentiation (Fig. 13 and Fig. 14). Finally, we found that expression of E2A, and SCL mRNA was down-regulated while Id1, which is known to inhibit erythro- poiesis in MEL cells29,30, was up-regulated by activated C/EBPα/hERα protein. These results suggest C/EBPα may inhibit erythropoiesis through multiple pathways by directly inhibiting EpoR or indirectly through transcription factors that inhibit erythroid differentiation like Id1.

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Figure 11. Effect of C/EBPα over-expression on expression of genes related with erythroid differentiation. Cells were harvested 10 hours after treatment with HMBA (3 mM) and β-estradiol (1 uM) Expression of each gene indicated was evaluated by RT-PCR using different cycles; i.e., from left to right, 24, 26, 28, and 30 cycles (Panel A) The line graph represents densitometry results of each band on Panel A (Panel B).

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Figure 12. Effect of C/EBPα on the expression of erythropoietin receptor. EpoR mRNA expression in mock and αER cells before and after stimulation with HMBA by Northern blot analysis (Panel A) EpoR protein expression in fetal liver cells from C/EBPα-/-, C/EBPα+/-, C/EBPα+/+ mice was measured by Western blot analysis (Panel B).

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Figure 13. GATA-1 and PU.1 protein expression in MEL and αER cells. MEL cells and αER clones were stimulated with 3 mM HMBA and 1.0 uM β-estradiol. At the times indicated, total cellular extract was prepared and 30 ug was loaded in each lane Western blot were probed with antibodies that recognize GATA-1, PU.1 and α-Actin according to procedure described in the Materials and Methods.

Figure 14. Effect of C/EBPα on the expression of Id1. Mock and αER cell clone were cultured in 3 mM HMBA and 1.0 uM β-estradiol as indicated. After 1 day culture, Id1 protein expression was determined by Western blot analysis (Panel A) Analysis of Id1 protein levels during HMBA and β-estradiol treatment of αER clone. αER cells were grown in the presence of 3 mM HMBA and 1.0 uM β-estradiol Total cellular protein was prepared at the indicated times and analyzed by western blot with Id1 antibody (Panel B).

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IV. DISCUSSION

1. C/EBPα is critical transcription factor for the CMP to differentiate to the GMP

Our data showed that HPC that lack C/EBPα were hyper-proliferative, and were unable to differentiate into myeloid lineage cells beyond the CMP stage of development as indicated by the loss in GM-CSF- and IL-3-responsive bi-potential GM-progenitors (CFU-GM or GMP). This maturation block was intrinsic to the hematopoietic cell compartment since BM progenitors derived from C/EBPα-/- FL cells were blocked at the same stage in differentiation when transplanted in vivo. Therefore, we propose that C/EBPα is required for the CMP to differentiate into the GMP.

In addition to the absence of G-CSF-responsive progenitors, we found that C/EBPα-/- FL lacked M-CSF-responsive macrophage progenitors. This novel macrophage defect was intrinsic to the hematopoietic compartment since the development of donor-derived macrophage progenitors and their progeny was severely impaired in mice transplanted with C/EBPα-/- FL. The defect in macrophage development in C/EBPα-/- FL progenitors was not observed in a previous study9. However, in that study, multiple HGF were used to stimulate colony formation rather than M-CSF alone. It is possible that multiple HGF may act differently in combination than M-CSF alone to drive macrophage proliferation and differentiation from more primitive progenitors and this would bypass the requirement for C/EBPα. Furthermore, in the previous study, PBC monocyte counts were used to determine the effect of C/EBPα on the macrophage lineage. In this regard, accurate measurements of PBC monocytes are difficult due to the low percentage of these cells in the PB even in C/EBPα+/+ mice ( <2%). Finally, our results suggest that defects in macrophage development might be due to a defect in M- CSFR expression, which was nearly absent in C/EBPα-/- FL cells. While defects in M-CSFR were not observed in the previous study, C/EBPα in combination with PU.1 and AML1 has been shown to regulate M-CSFR expression11,31. It is also possible that the absence of detectable G-CSFR and M-CSFR in C/EBPα-/- FL might reflect the severe reduction observed in the CFU-GM (GMP) population, thus resulting in the absence of M-CSF- and G-CSF-responsive progenitors and their differentiated progeny

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that would normally express M-CSFR and G-CSFR receptors.Our results indicate that C/EBPα is required for the development of CMP and

GMP but also suppressed the proliferation of the HSC and primitive progenitor cells. The data that increased numbers of spleen colonies (CFU-s) were observed when C/EBPα-/- FL cells were transplanted in vivo and proliferation assay results which shows that decreased proliferation by activation of C/EBPα/hERα in MEL cells (data not shown), suggests that C/EBPα may also act to limit the proliferation of more primitive progenitors. This observation is supported by the experiments showing enhanced secondary plating potential. These findings are also consistent with previous studies demonstrating that loss of C/EBPα function is related to the development of leukemia, and give clues why functional loss of C/EBPα is related with the development of monocytic leukemia (AML M4 or M5)20,21. It is currently not known whether C/EBPα plays a role in limiting the proliferation of HSC and whether there are increased numbers of HSC in C/EBPα-/- FL. However, this question can be addressed using the competitive repopulation assay with C/EBPα-/- FL and BMC to more preci- sely determine the number of stem cells present.

2. C/EBPα is a negative regulator in erythroid differentiation

In this study, we analyzed the negative role of C/EBPα in the development of HSC in vivo and in vitro. We identified the transcription factor C/EBPα as a negative regulator of erythroid differentiation. We found that C/EBPα-/- mouse FL cells have increased numbers of BFU-e progenitors and increased EpoR expression. Over-expression of C/EBPα in 5-FU BM cells significantly increased the proportion of progenitor cells that developed along the myeloid lineage at the expense of the erythroid lineage. Furthermore, activation of C/EBPα/hERα inhibits erythropoiesis in MEL cells. Altogether, this suggests that C/EBPα may involve in regulations of red blood cell development and differentiation.

Our results showed that C/EBPα-/- FL cells which showed increased EpoR expression, had increased BFU-e colony number than C/EBPα+/+ FL cells. Since hematopoietic growth factors play a role in proliferation and survival of HPC, if HGF receptor expression is abolished or suppressed, HPC that depend on HGF signal transduction

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can not proliferate and survive32. In this context, the fact that C/EBPα could suppress the expression of EpoR while inducing G-CSF receptor, GM-CSF receptor, and M-CSF receptor might explain one mechanism to inhibit erythroid development while promoting myeloid differentiation.

PU.1 is known as functional antagonist of GATA-1 and can block erythroid differentia- tion6,7. But in our experiment we showed that even though PU.1 expression was decreased by HMBA, C/EBPα inhibited erythroid differentiation of MEL. This means C/EBPα plays a role as an erythroid differentiation inhibitor independent to PU.1. Although we found that GATA-1 expression was not affected by C/EBPα in MEL cells, β-globin and erythropoietin receptor is known to be transactivated by GATA-133, therefore it remains to be determined whether inhibition of erythroid differentiation by C/EBPα might be a consequence of the repression of GATA-1 activity by preventing the binding of GATA-1 to target DNA site or it's interaction with other proteins. Of note is the existence of a possible C/EBPα binding site from nucleotide -1129 to -1120 that was contained in repressor segment of EpoR expression in the mouse EpoR promoter34. It is also possible that expression of high levels of C/EBPα in hematopoietic cells might inhibit GATA-1 activity and then, activate myeloid cell-specific genes, thus establishing its own auto-regulatory loop. It is known that PU.1 synergy with C/EBPα to induce myeloid HGF receptor expression. In this regards, we proposed that C/EBPα might drive myeloid development in MEL cells.

The results reported here also provide insight in another mechanism of C/EBPα to inhibit erythroid differentiation. Id1 is able to interact with the products of E2A and other bHLH proteins like SCL, and function as dominant negative regulators for them35-38. By the mechanism, over-expression of Id1 has been shown to block MEL cell differentiation29,30. Our data shows that over-expression of C/EBPα increased Id1 expression while it decreased E2A, SCL transcription which are important for erythro- poiesis. This result suggests C/EBPα inhibits erythroid differentiation by regulating Id1, E2A, SCL expression.

In summary, these observations showed that C/EBPα plays a critical role in regulating the balance between proliferation and growth arrest/differentiation in hematopoietic progenitors. Our study supports a model in which lineage choice in hematopoietic system is determined by a reciprocal relationship between the activation

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of a one lineage and the repression of another. The differentiation along the myeloid versus the erythroid lineage thus may not only require a distinct combination of transc- riptional activators but also the activity of an appropriate set of inhibitors, one of which would be C/EBPα.

Figure 15. Summary of the role of C/EBPα in hematopoiesis. C/EBPα is a repressor for erythroid development and differentiation and promote myeloid maturation of proge- nitor cells It may act as a switch to drive hematopoietic stem/progenitor cells toward myeloid lineage at the expense of other lineages.

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V. CONCLUSION

1) Loss of C/EBPα gene expression results in hyper-proliferation of HPC in vivo and in vitro.

2) C/EBPα suppresses the expression of EpoR which is important for erythroid proliferation and survival while inducing GM-CSF and M-CSF receptor expression.

3) C/EBPα plays a critical role in the differentiation of CMP to GMP and their progeny.

4) C/EBPα is a negative regulator of erythroid development and differentiation.

5) C/EBPα induced Id1, which is known to inhibit erythroid differentiation of MEL, and suppressed the expression of E2A and SCL, which are required for erythroid development.

6) This study shows that C/EBPα plays a critical role in regulating the balance between proliferation and growth arrest/differentiation in hematopoietic progenitors.

7) This study supports a model in which lineage choice in the hematopoietic system is determined by a reciprocal relationship between the activation of a parti- cular lineage and the suppression of another. The differentiation along the myeloid versus the erythroid lineage thus may not only require a distinct combination of transcriptional activators but also the activity of an appropriate set of inhibitors, one of which would be C/EBPα.

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18. Perrotti D, Cesi V, Trotta R, Guerzoni C, Santilli G, Campbell K, et al. BCR- ABL suppresses C/EBPalpha expression through inhibitory action of hnRNP E2. Nat Genet 2002;30:48-58.

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국문요약

골수구와 적혈구 조혈세포 발생과정에서의 CCAAT/인핸서 결합 단백-α (C/EBPα)의 기능

서 형 찬

연세대학교 의학과

(지도교수 고 윤 웅)

CCAAT/인핸서 결합 단백-α (C/EBPα)은 bZip 계열의 전사인자로 조혈계에서 골수구의 증식과 발생에 기능한다. C/EBPα가 소실된 마우스는 중성구의 소실을 보이지만, 어느 조혈단계에서 중성구의 증식과 발생에 향을 미치는지는 명확

하지 않다. 이에 연구자는 C/EBPα 결손 마우스의 태아 간에 존재하는 원시조상세포(primitive hematopoietic progenitor cells)와 계열특이 조혈조상세포 (committed hematopoietic progenitor cells)의 증식능을 조사하고, 조혈작용에 미치는 C/EBPα 작용을 연구하고자 하 다.본 연구를 통하여 C/EBPα 결손 마우스의 태아 간 유래 조혈세포는 과증식과 분화능의 감소를 보이는 것이 확인되었다. 이들 세포의 과증식은 실험실 세포 배양을 통해서 뿐 아니라, 비장 콜로니 측정을 통한 마우스 생체 실험을 통해서도 확인되었다. 분화 연구를 통해서 골수구로의 분화과정에서 특히, common myeloid progenitor (CMP)로의 분화는 적은 감소를 보인 반면, granulocytemonocyte progenitor (GMP)로의 분화는 심각한 장애를 보여 중성구 뿐 아니라, 단핵구/거식세포의 발생장애를 유발하 다. 이러한 결과는 C/EBPα가 CMP에서 GMP로의 분화에 결정적으로 관여하여 기존에 알려진 중성구 발생 뿐 아니라, 단핵구/거식세포의 발생에도 관여함을 증명하 다.본 연구자는 또한, C/EBPα 결손 마우스의 태아 간 유래 조혈세포에서 적혈구 조혈세포의 분획이 증가한 것을 확인하 다. 이에 C/EBPα 결손 태아 간 조혈모세포 (hematopoietic stem cell) 이식, C/EBPα 과발현 골수 조혈모세포 이식 연구를 통해서 조혈모세포에서의 C/EBPα의 결손이 골수구의 조혈능을 상실시키는 한편 적혈구 조혈능을 향상시키고, C/EBPα의 과발현이 반대로 적혈구의 조혈능

42

을 감소시키면서 조혈모세포를 골수구로 분화를 유도함을 확인하 다. C/EBPα 유도성 마우스 적백혈병 (erythroleukemia) 세포주를 이용한 실험에서는 적백혈병 세포에 과발현된 C/EBPα가 PU.1 전사인자와 무관하게 적혈구 분화를 억제함을 확인하 다. C/EBPα는 적백혈병 세포주에서 적혈구 분화억제인자로 알려진, Id1의 발현을 증가시키고, 적혈구 분화와 관련된 SCL, E2A, erythropoietin receptor의 발현을 감소시켜, 이들 단백의 발현 변화가 C/EBPα에 의한 적혈구 분화억제의 기전이 될 수 있음을 제시하 다.이상과 같은 결과를 통하여 ① 조혈세포에서의 C/EBPα 결손은 조혈조상 세포의 과증식을 초래하며, ② CMP에서 GMP로의 분화단계에 C/EBPα가 중요한 인자이며, ③ C/EBPα는 적혈구의 발생과 분화에 억제인자로 작용함을 확인하 다. 본 연구 결과는 조혈세포의 계열분화가 특정 계열로의 분화 촉진과 다른 계열

로의 분화억제의 상호작용에 의해 이루어짐을 증명하며, C/EBPα가 계열세포 특이적 분화유도 및 분화억제의 두 가지 기능을 가지고 있음을 보여 주고 있다.

핵심되는 말: C/EBPα, hematopoiesis, transcription factor, differentiation