Transgenic mice overexpressing murine thrombopoietin develop myelofibrosis and osteosclerosis

doi:10.1016/j.leukres.2004.12.009

Leukemia Research

Volume 29, Issue 7, July 2005, Pages 761-769

https://doi.org/10.1016/j.leukres.2004.12.009 Get rights and content

Abstract

Thrombopoietin (TPO) regulates megakaryocytopoiesis and platelet production in vivo and in vitro. Exogenous overexpression of TPO in vivo by viral-mediated gene transfer induced bone marrow (BM) fibrosis and osteosclerosis. On the other hand, transgenic mice (Tg) overexpressing TPO using a liver-specific apolipoprotein E (Apo-E) promoter did not exhibit myelofibrosis or osteosclerosis. These discrepancies in phenotype are not fully understood. Then we have investigated the consequences of long-term in vivo overexpression of TPO in a mouse model. Murine TPO Tg mice driven by the IgH promoter were generated. The number of platelets and neutrophils in peripheral blood, and the number of megakaryocytes and granulocytic immature cells in the BM was elevated, together with the number of progenitor cells for megakaryocyte and myeloid cells. TPO Tg mice demonstrated anemia but the number of progenitor cells for the erythrocyte was increased. TPO Tg mice developed myelofibrosis and osteosclerosis as they aged with extramedullary hematopoiesis in the spleen. As plasma transforming growth factors (TGF)-β1 and osteoprotegerin (OPG) levels were higher in TPO Tg mice than in wild-type mice, the development of myelofibrosis and osteosclerosis depends on local TPO levels in BM and might be due to elevated TGF-β1 and OPG.

Introduction

Thrombopoietin (TPO) is a hematopoietic growth factor that regulates megakaryocytopoiesis and platelet production in vivo and in vitro [1], [2], [3]. The liver and kidneys are the major sites for TPO production [4], [5], although small amounts of TPO mRNA can be detected in a variety of organs and tissues. Administration of recombinant TPO results in elevated circulating platelet levels [2], and mutant mice deficient for TPO or TPO receptor show about an 80% reduction in platelet numbers [6], [7], indicating that TPO is essential for megakaryocytopoiesis. TPO also has effects on hematopoietic stem cells and progenitor cells [8], [9], [10], [11], [12], although no substantial difference in the number of blood cells except for platelets was observed in either TPO or TPO receptor deficient mice. TPO promotes the growth of hematopoietic progenitor cells and augments the number of erythroid burst-forming units (BFU-E), granulocyte-macrophage colony forming units (CFU-GM), and mixed colony forming units (CFU-mix) in collaboration with early or late-acting cytokines [13].

Exogenous overexpression of TPO in vivo by viral-mediated gene transfer induced bone marrow (BM) fibrosis and osteosclerosis. These TPO-overexpressing mice were generated by transplanting bone marrow cells infected with TPO retrovirus into lethally irradiated recipient mice [14], [15], [16] or by intraperitoneal injection of TPO cDNA in an adenovirus vector [17]. Myelofibrosis developed 10–12 weeks after BM transplantation (BMT) or adenovirus infection. On the other hand, transgenic mice overexpressing TPO using a liver-specific apolipoprotein E (Apo-E) promoter did not exhibit myelofibrosis or osteosclerosis [18]. These discrepancies in phenotype are not fully understood.

We have generated a mouse model to study the biological role of TPO in vivo by transgenic expression of the TPO cDNA using the mouse rearranged H chain promoter. These mice are expected to continuously secrete TPO from the bone marrow and spleen for the life of the animal. In contrast to the previous study using the Apo-E promoter, we show here that TPO Tg mice developed myelofibrosis and osteosclerosis.

Section snippets

Mice

Murine TPO (mTPO) cDNA was amplified by polymerase chain reaction (PCR) from murine bone marrow cDNA using oligonucleotides primers specific for the murine TPO sequence: forward 5′-gac tct gcc gaa aga agc ac-3′ and reverse 5′-gct cga gaa gct gca gac gct cac t-3′, and subcloned into the pCR2.1-TOPO vector (Invitrogen, CA, USA). A 1.21-kb XhoI fragment containing the entire mTPO cDNA was cloned into the pEMVhp vector, which contains the mouse H chain enhancer (Eμ), the mouse rearranged H chain

Generation of TPO overexpressing mice (Fig. 1A and B)

Tg mice were generated expressing full-length mTPO under the control of the mouse Ig enhancer and promoter (Fig. 1A). Transgenic founder mice were identified by transgene-specific amplification of genomic DNA using the polymerase chain reaction. Three transgenic lines were established, and the line in which platelet number was highest was analyzed further.

Mice were divided into two age groups (2–4 and 7–9 months of age) for convenience. Plasma levels of TPO were measured by ELISA. Plasma TPO

Discussion

We have shown that IgH promoter-derived murine TPO cDNA overexpression in vivo produces a 2.3-fold elevation in TPO protein compared with wild-type mice, for at least 9 months (Fig. 1B). The elevated TPO levels in TPO Tg mice caused an increase in the number of CFU-Meg and megakaryocytes in bone marrow and of platelets in peripheral blood (Fig. 2, Fig. 3). In addition to the effects on megakaryocytopoiesis, the number of CFU-mix, CFU-GM, and CFU-G in bone marrow was increased in TPO Tg mice (

Acknowledgments

We thank M. Sato and M. Ito for their excellent technical assistance. This work was supported in part by a Grant from the Japan Leukemia Foundation and Grants-in-Aid for Scientific Research (numbers 13218096, 15390302) from the Ministry of Education, Culture, Sports, Science, and Technology in Japan.

H. Kakumitsu contributed to the concept and design, interpreted and analyzed the data, provided drafting of the article, provided study materials, supplied the statistical analysis, collected and

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Transgenic mice overexpressing murine thrombopoietin develop myelofibrosis and osteosclerosis

Abstract

Introduction

Section snippets

Mice

Generation of TPO overexpressing mice (Fig. 1A and B)

Discussion

Acknowledgments

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