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ORIGINAL RESEARCH ARTICLE

Protective Role of Connexin 32 in Steady-State Hematopoiesis, Regeneration State, and Leukemogenesis

Yoko Hirabayashi^*, Byung-Il Yoon^*,1, Isao Tsuboi^*, Yan Huo^*, Yukio Kodama^*, Jun Kanno^*, Thomas Ott, James E. Trosko and Tohru Inoue^,2

^* Division of Cellular and Molecular Toxicology, Center for Biological Safety and Research, National Institute of Health Sciences, Tokyo 158-8501, Japan; Service Einrichtung Transgene Tiere, Hertie-Institut für Klinische Hirnforschung, Tübingen 72076 Germany; Department of Pediatrics and Human Development, Michigan State University, College of Human Medicine, East Lansing, Michigan 48824; and Center for Biological Safety and Research, National Institute of Health Sciences, Tokyo 158-8501, Japan

To whom requests for reprints should be addressed at ² 1-18-1 Kamiyohga, Setagayaku, Tokyo 158-8501, Japan;. E-mail: tohru{at}nihs.go.jp

The role of gap junctions formed by connexins (Cxs) has been implicated in the homeostatic regulation of multicellular systems. Primitive hematopoietic progenitor cells form a multicellular system, but a previous report states that Cx32 is not expressed in the bone marrow. Thus, a question arises as to why Cx molecules are not detected in the hematopoietic tissue other than in stromal cells. Based on our preliminary study, which suggested a potential impairment of hematopoiesis in Cx32-knockout (KO) mice, the objectives of the present study were to determine whether Cx32 functions in the bone marrow during steady-state hematopoiesis and to examine its possible protective roles during regeneration after chemical abrasions and during leukemogenesis after the administration of a secondary genotoxic chemical, methyl nitrosourea (MNU). As a result, the Cx32 molecule, functioning in the hematopoietic stem cell (HSC) compartment during steady-state hematopoiesis, was observed for the first time; the expressions of Cx32 at the mRNA level, as determined by polymerase chain reaction analysis, and at the protein level, determined using an anti-Cx32 antibody, were observed only in the lin^–c-kit⁺ HSC fraction, using a combination of immunobead-density gradient and immunomagnetic bead separation. Hematopoiesis was impaired in the absence of Cx32, and it was delayed during regeneration after chemical abrasion with 5-fluorouracil at 150 mg/kg body wt in Cx32-KO mice. Cx32-KO mice showed increased leukemogenicity compared with wild-type mice after MNU injection; furthermore, in a competitive assay for leukemogenicity in mice that had been lethally irradiated and repopulated with a mixed population of bone marrow cells from Cx32-KO mice and wild-type mice, the resulting leukemias originated predominantly from Cx32-KO bone marrow cells. In summary, the role of Cx32 in hematopoiesis was not previously recognized, and Cx32 was expressed only in HSCs and their progenitor cells. The results indicate that Cx32 in wild-type mice protects HSCs from chemical abrasion and leukemogenic impacts.

Key Words: connexin 32 (Cx32) • hematopoietic stem cell • Cx32-knockout mouse • tumor suppressor • experimental leukemogenesis