Production of TGF-β Is Decreased in the Bone Marrow of Double Knockout (DKO) SMAD3^-/- Fancd2^-/- Mice

Homologous deletion recombinant negative DKO mice, missing action of both Fancd2 and SMAD3 proteins were derived to test the hypothesis that abrogation of TGF-β signaling would reverse Fanconi Anemia (FA) stem cell sensitivity to TGF-β. To determine whether DKO mouse tissues demonstrated detectable differences in production of the ligand for TGF-β receptor, tissues from adult six - eight week old DKO mice were compared to those from Fancd2^-/- (C57BL/6J background), SMAD3^-/- (129/Sv genetic background), or wild type F1 mice (129/Sv X B6) for levels of TGF-β. Tissues examined included bone marrow, intestine, spleen, liver, muscle, and brain. Tissues were homogenized, and analyzed for bone marrow protein by Luminex analysis using a TGF beta 1 multispecies kit for Luminex Platform (Life Technologies).

The results demonstrated decreased but not significant production of TGF-β in the bone marrow of DKO mice (3068 ± 753 pg TGF-β/mg protein) compared to 5211 ± 1662 (p = 0.0761) for wild type F1 mice (129/Sv X B6), and 5192 ± 705 (0.1085) for Fancd2^-/- mice. However, TGF-β production in DKO was significantly decreased compared to Smad3-/- (129/Sv) mice (9828 ± 1076, p = 0.0127). The lungs from DKO also had decreased TGF-β production compared to wild type F1 mice (436 ± 125 and 1159 ± 44 pg/mg, respectively, p= 0.0217). Decreased TGF-β production was also seen in the DKO liver compared to F1 wild type liver (13.2 ± 1.7 and 33.5 ± 3.6, respectively, p = 0.0072). Bone marrow stromal cell lines derived from long-term bone marrow cultures of each mouse strain were tested for production of TGF-β. SMAD3-/- bone marrow stromal cells also had an increased production of TGF-β (236 pg/mg) compared to wild type F1, Fancd2-/- and DKO cell lines (117, 136 and 144 pg/ml). Bone marrow CFU-GEMM from each mouse strain was tested for sensitivity to inhibition by increasing concentrations of TGF-β, and both fresh bone marrow from DKO and SMAD3^-/- mice demonstrated no TGF-β mediated abrogation of colony formation. In contrast, fresh marrow from wild type and Fancd2^-/- mice demonstrated TGF-β concentration dependent inhibition of CFU-GEMM colony formation in vitro. These data indicate that TGF-β production in DKO mice is decreased in bone marrow, lung and liver compared to that in F1 wild type, SMAD3^-/- or Fancd2^-/- mice, and suggest that the control of TGF-β production by abrogation of TGF-β signaling in the setting of deletion of Fancd2 may be modulated in an organ specific manner.

Supported by research grant NIAID/NIH, U19A168021.