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 3

Transferrin transport in MEFs.

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Transferrin transport in MEFs. (A) Immunofluorescence detection of endog...
(A) Immunofluorescence detection of endogenous SMAP1 in wild-type and Smap1–/– MEFs using an anti-SMAP1 antibody (green). Blue indicates DAPI staining. (B and C) Internalization of transferrin in wild-type and Smap1–/– MEFs. Cells were incubated with fluorescein-transferrin for the indicated times, and then surface-remaining transferrin was stripped off. The cells were then processed for analyses by (B) fluorescence microscopy or (C) flow cytometry. In C, the intensities of intracytoplasmic fluorescence were measured and expressed in relative arbitrary units. Independent cultures were prepared in triplicate from the indicated MEF clones, and averages ± SD are shown (n = 3). “No. 1,” “No. 8,” “No. 5,” and “No. 7” refer to the MEF cell line numbers. (D) Effects of siRNA against SMAP2. Wild-type MEFs were treated with or without siRNA against SMAP2 (2 differentially designed siRNAs, siRNA1 and siRNA2, were used). Protein lysates were prepared and processed for immunoblot analyses using anti-SMAP2 and anti-SMAP1 antibodies. (E) Effects of SMAP2 knockdown on transferrin incorporation. The Smap1+/+ and Smap1–/– MEFs were incubated with siRNA2 against SMAP2 and then with fluorescent transferrin and processed for immunofluorescence detection using an anti-SMAP2 antibody. The arrows indicate the reduction in fluorescence intensity from SMAP2, whereas DAPI staining indicates the location of cell nuclei. Scale bar: 10 μm.