Increased longevity of hematopoiesis in continuous bone marrow cultures and adipocytogenesis in marrow stromal cells derived from Smad3(-/-) mice.
OBJECTIVE
To determine the role of Smad3 in modulating hematopoiesis, continuous bone marrow cultures were established from Smad-/- mice, and the longevity of hematopoiesis and extent of adipogenesis in the supportive hematopoietic microenvironment were compared to those from cultures of control, Smad3+/+ or heterozygous Smad3+/- mice.


METHODS
Long-term bone marrow cultures (LTBMCs) were established from Smad3+/+, Smad3+/-, or Smad3-/- mice. On a weekly basis, the number of cobblestone islands, number of nonadherent cells, confluence of the adherent cells, or CFU-GEMM colonies was determined. Bone marrow stromal cell lines were established and cobblestone island production on these cell lines determined in the presence of nonadherent cells from week-42 Smad3-/- or week-4 C57BL/6J LTBMCs.


RESULTS
Initial proliferative capacity of the LTBMCs was similar in all groups through week 20, at which time there was an increase in cobblestone islands and production of nonadherent cells and CFU-GEMM colonies in the Smad3-/- group. By week 28, only the Smad3-/- LTBMCs had significantly maintained increased production of these parameters. Maintenance of cobblestone islands indicative of the most primitive hematopoietic progenitor cells persisted past 45 weeks in Smad3-/- cultures. The Smad3-/- stromal cell line also demonstrated increased support of cobblestone island production when incubated with nonadherent cells from week-42 Smad3-/- or week-4 C57BL/6J LTBMCs. Evaluation of adipocytogenesis in stromal cells showed significantly greater accumulation of adipocytes in lines from Smad3-/- than from Smad3+/+ mice.


CONCLUSIONS
These data provide evidence for a significant effect of deletion of the Smad3 signaling pathway in increased hematopoiesis in LTBMCs and support the negative regulatory influence of TGFbeta signaling on adipocytogenesis and long-term hematopoiesis in vitro.
Epperly
Michael W
MW
Department of Radiation Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, USA.
Cao
Shaonan
S
Goff
Julie
J
Shields
Donna
D
Zhou
Shuanhu
S
Glowacki
Julie
J
Greenberger
Joel S
JS
D017667:Adipocytes / Q000502:physiology*; D000818:Animals; D001853:Bone Marrow / Q000502:physiology*; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006410:Hematopoiesis / Q000235:genetics / Q000502:physiology*; D006412:Hematopoietic Stem Cells / Q000502:physiology*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D051900:Smad3 Protein; D017154:Stromal Cells / Q000502:physiology; D013997:Time Factors; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000502:physiology
D004268:DNA-Binding Proteins; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; D015534:Trans-Activators; D016212:Transforming Growth Factor beta