Increased TGF-β1 expression Cooperates with the Gata1^low Mutation in Determining the Gata1^low Phenotype in CD1 Mice

Abstract 3462

The Gata1^low mutation impairs megakaryocytic and erythroid development. Gata1^low mice die at birth with thrombocytopenia and anemia but in the CD1 background, which readily activates spleen hematopoiesis, they recover from anemia at 1 month living up to 2 years but die from fatal anemia within 3-weeks following splenectomy. The increased levels of TGF-β1 expressed by these mice suggests that TGF-β1 may determine some of the traits of their phenotype. This hypothesis was tested by profiling the TGF-β signaling pathway of bone marrow (BM) and spleen of Gata1^low mice and by determining the effects of loss of TGF-β1 function on phenotype. Differences (³two-fold) in TGF-β1 signaling gene expression were determined with the Mouse TGF-β RT²Profiler™ PCR array kit (SABiosciences, Frederick, MD, USA) and their biological effects predicted with the David Bioinformatic Database (David Bioinformatics Resources 6.7 NIAID/NIH). Loss of TGF- β1 function was achieved by treatment for 2 months with SB431542, or vehicle as control. During treatment, mice were phlebotomized (50 μL) every 3-days for blood evaluation.

Differences in the TGF- β pathway genes abnormally expressed in BM and spleen from Gata1^low mice were observed. In BM, 6 genes were up-regulated and 14 down-regulated (Bmp2, Bmp5, Acvrl1, Tgfblil and Igf1 with a p<0.05) with respect to wild-type. In spleen, seven genes were up-regulated (Cdkn1a and Ltbp1 with a p<0.05) and 29 down-regulated (Gdf2 and Nodal with a p<0.05). David analyses identified activation of TGF- β, Hedgehog and p53 pathways both in BM and spleen and predicted biological effects consistent with the Gata1^low phenotype. TGF- β signaling alterations predicted increased osteoblast maturation in BM but not in spleen (mice are osteopetrotic), increased apoptosis and G1 arrest in BM and spleen (erythroblast apoptosis increases in both organs) and reduced ubiquitin-mediated proteolysis, a process necessary for erythroid maturation, in BM but not in spleen (erythropoiesis is ineffective in BM). The Hedgehog pathway, required for BMP4 (the stress-erythroid growth factor) expression, was active in both BM and spleen. Also the p53 pathway was activated in both organs but mTOR signaling was activated only in spleen.

SB431542–treatment had profound effects on TGF- β expression profile and on the phenotype of Gata1^low mice. SB431542- treatment only significantly reduced TGF- β1 mRNA (∼20%) and protein (>80%) levels in BM and spleen. However, numerous TGF- β pathway genes were abnormally expressed in BM and spleen of both vehicle- and SB431542-treated mice. In BM, 48 genes were up-regulated and 2 down-regulated by vehicle-treatment while 46 genes were up-regulated and 2 down-regulated by SB431542-treatment. Thirty-nine of the alterations were common to the two groups and included BMP4 activation, indicating that they were induced by phlebotomy. No gene was abnormally expressed only in vehicle-treated mice but David analyses identified that p53 signaling was normal in SB431542-treated mice, predicting reduced apoptosis and activation of hematopoiesis in their BM. In spleen, 38 genes were up-regulated and 11 down-regulated in vehicle-treated animals while 37 were up-regulated and 6 down-regulated in SB431542-treated animals. Thirty genes were altered in both groups but these common genes did not include BMP4 which was activated in vehicle-treated mice only, suggesting that SB431542-treated mice are unable to activate stress-erythropoiesis in spleen. In addition, Id2 and Stat1 were down-regulated in vehicle-treated animals but expressed at normal levels in SB431542-treated mice, suggesting that these genes may be involved in the activation of splenic erythropoiesis in Gata1^low mice. David analyses identified that expression of genes involved in ubiquitin-mediated proteolysis was reduced in spleen from SB431542-treated mice, predicting that inhibition of TGF- β signaling would reduce erythroid maturation in their spleen. Both predictions were confirmed by comparing the phenotype of vehicle- and SB431542-treated mice which indicated that inhibition of TGF-β1 signaling restores hematopoiesis, including megakaryocyte development, in BM and reduces hematopoiesis in spleen. In conclusion, these results suggest that TGF-β1 signaling cooperates with the Gata1^low mutation in determining the phenotype of Gata1^low mice in the CD1 background.