Smap1 deficiency perturbs receptor trafficking and predisposes mice to myelodysplasia
Shunsuke Kon, … , Takuro Nakamura, Masanobu Satake
Shunsuke Kon, … , Takuro Nakamura, Masanobu Satake
Published February 22, 2013
Citation Information: J Clin Invest. 2013;123(3):1123-1137. https://doi.org/10.1172/JCI63711.
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Research Article Hematology

Smap1 deficiency perturbs receptor trafficking and predisposes mice to myelodysplasia

Abstract

The formation of clathrin-coated vesicles is essential for intracellular membrane trafficking between subcellular compartments and is triggered by the ARF family of small GTPases. We previously identified SMAP1 as an ARF6 GTPase-activating protein that functions in clathrin-dependent endocytosis. Because abnormalities in clathrin-dependent trafficking are often associated with oncogenesis, we targeted Smap1 in mice to examine its physiological and pathological significance. Smap1-deficent mice exhibited healthy growth, but their erythroblasts showed enhanced transferrin endocytosis. In mast cells cultured in SCF, Smap1 deficiency did not affect the internalization of c-KIT but impaired the sorting of internalized c-KIT from multivesicular bodies to lysosomes, resulting in intracellular accumulation of undegraded c-KIT that was accompanied by enhanced activation of ERK and increased cell growth. Interestingly, approximately 50% of aged Smap1-deficient mice developed anemia associated with morphologically dysplastic cells of erythroid-myeloid lineage, which are hematological abnormalities similar to myelodysplastic syndrome (MDS) in humans. Furthermore, some Smap1-deficient mice developed acute myeloid leukemia (AML) of various subtypes. Collectively, to our knowledge these results provide the first evidence in a mouse model that the deregulation of clathrin-dependent membrane trafficking may be involved in the development of MDS and subsequent AML.

Authors

Shunsuke Kon, Naoko Minegishi, Kenji Tanabe, Toshio Watanabe, Tomo Funaki, Won Fen Wong, Daisuke Sakamoto, Yudai Higuchi, Hiroshi Kiyonari, Katsutoshi Asano, Yoichiro Iwakura, Manabu Fukumoto, Motomi Osato, Masashi Sanada, Seishi Ogawa, Takuro Nakamura, Masanobu Satake

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Figure 1

Establishment of Smap1-targeted mice and SMAP1 expression.

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Establishment of Smap1-targeted mice and SMAP1 expression. (A) Physica...
(A) Physical maps of the SMAP1 gene locus and its targeting vector. Features of the recombined and targeted SMAP1 alleles are also depicted. Horizontal lines indicate the genomic sequences. The thick lines indicate the sequence incorporated into the targeting vector. Exon 1, neomycin resistance gene, and diphtheria toxin subunit A gene are indicated. Black and white arrowheads indicate the loxP and frt sequences, respectively. The small rectangle under the line corresponds to the probe that was used for Southern blot hybridization. B indicates a BamHI restriction site. (B) Southern blot analysis of genomic DNA prepared from Smap1+/+ (+/+), Smap1+/– (+/–), and Smap1–/– (–/–) mice. DNA was digested by BamHI and processed for Southern blotting using the hybridization probe shown in A. The wild-type and targeted alleles gave rise to 2.7-kb and 3.3-kb bands, respectively. (C) Immunoblot analysis of protein lysates prepared from bone marrow cells of Smap1+/+, Smap1+/–, and Smap1–/– genotypes. The 50-kDa band represents SMAP1, whereas SMAP2 served as a control. Three independent experiments were performed, and one representative reproducible result is shown. (D) RT-PCR analyses of Smap1 transcripts in bone marrow cells from Smap1+/+ and Smap1–/– mice. Primers were set between exons 1 or 3 and exon 9. HPRT served as a control. (E) SMAP1 expression in hematopoietic cells. Fractions of various hematopoietic lineages were sorted from bone marrow cells of wild-type mice by flow cytometry, and RNA was prepared from each and processed for semiquantitative RT-PCR analyses. DDW, distilled deionized water; -RT, without reverse transcription.