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Summary Background Capsular contracture is the most common complication following breast implant placement. The multiple factors unbalancing the physiological response to the foreign body have not been fully elucidated. The aim of this study was to investigate the role of neo-angiogenesis, inflammation and estrogen receptors in peri-prosthetic tissue development and remodeling. Methods The study enrolled 31 women who underwent expander substitution with definitive implant. Specimens were stained with hematoxylin/eosin, Masson trichrome, immunohistochemistry and immunofluorescence for alpha-smooth muscle actin, estrogen receptor-α (ER-α), estrogen receptor-β (ER-β), Collagen type I and III, CD31 (as a marker of neo-angiogenesis) and vascular endothelial growth factor (VEGF). Inflammatory infiltration was quantified and analyzed. Transmission electron microscopy was performed for ultrastructural evaluation. Results Myofibroblasts, mainly localized in the middle layer of capsular tissue, expressed VEGF, ER-α and ER-β. ER-β expression positively correlated with Collagen type I deposition ( p = 0.025). Neo-angiogenesis was predominant in the middle layer. CD31 expression positively correlated with Collagen type I expression ( p = 0.009 ) and inflammatory infiltration grade ( p = 0.004) . The degree of inflammatory infiltration negatively correlated with the time from implantation ( p = 0.022). Discussion The middle layer is key in the development and remodeling of capsular tissue. Myofibroblasts produce VEGF, that induces neo-angiogenesis. New vessels formation is also correlated to the inflammatory response. Collagen deposition is associated with ER-β expression and neo-angiogenesis. These findings may prelude to targeted pharmacologic therapies able to control such interactions, thus hampering the self-sustaining loop promoting the progression of physiologic fibrosis toward pathologic contracture.

In previous works, we have shown that insulin-like growth factor-binding protein-3 (IGFBP-3), a tissue and circulating protein able to bind to IGFs, decreases drastically in the blood serum of patients with diffuse metastatic melanoma. In agreement with the clinical data, recombinant IGFBP-3 was found to inhibit the motility and invasiveness of cultured metastatic melanoma cells and to prevent growth of grafted melanomas in mice. The present work was aimed at identifying the signal transduction pathways underlying the anti-tumoral effects of IGFBP-3. We show that the anti-tumoral effect of IGFBP-3 is due to inhibition of the Wnt pathway and depends upon the presence of CD44, a receptor protein known to modulate Wnt signaling. Once it has entered the cell, IGFBP-3 binds the Wnt signalosome interacting specifically with its component GSK-3β. As a consequence, the β-catenin destruction complex dissociates from the LRP6 Wnt receptor and GSK-3β is activated through dephosphorylation, becoming free to target cytoplasmic β-catenin which is degraded by the proteasomal pathway. Altogether, the results suggest that IGFBP-3 is a novel and effective inhibitor of Wnt signaling. As IGFBP-3 is a physiological protein which has no detectable toxic effects either on cultured cells or live mice, it might qualify as an interesting new therapeutic agent in melanoma, and potentially many other cancers with a hyperactive Wnt signaling. © 2016 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc.

The aim of the present study was to investigate the role of Cyclic-nucleotide Response Element-Binding (CREB) family members and related nuclear transcription factors in the radiation response of human B lymphoma cell lines (Daudi and Ramos). Unlike the more radiosensitive Daudi cells, Ramos cells demonstrated only a moderate increase in early apoptosis after 3-5 Gy irradiation doses, which was detected with Annexin V/PI staining. Moreover, a significant and dose-dependent G2/M phase accumulation was observed in the same cell line at 24 h after both ionizing radiation (IR) doses. Western blot analysis showed an early increase in CREB protein expression that was still present at 3 h and more evident after 3 Gy IR in Ramos cells, along with the dose-dependent upregulation of p53 and NF-κB. These findings were consistent with real-time RT-PCR analysis that showed an early- and dose-dependent upregulation of NFKB1, IKBKB and XIAP gene expression. Unexpectedly, pre-treatment with SN50 did not increase cell death, but cell viability. Taken together, these findings let us hypothesise that the early induction and activation of NF-κB1 in Ramos cells could mediate necrotic cell death and be linked to other molecules belonging to CREB family and involved in the cell cycle regulation.

The postnatal evolution of the ciliary processes in the Mongolian gerbil (Meriones unguiculatus) was examined, with particular regard to their morphological organization and to the structural and ultrastructural differentiation of both inner and outer epithelial layers. Gerbil eyes at postnatal (P) 3, 9, 12, 15, 18, 21, and 26 days and at 6 months were fixed with glutaraldehyde 2.5% and examined with the light microscopy on semithin sections and with the transmission electron microscopy. Masked histologists evaluated the samples and performed a morphometric exam on ciliary processes and epithelial cells layers height using the ANOVA test for comparison among groups. From P3 to P15, ciliary processes were formed by rectilinear folds, which progressively elongated at their free extremities. From P18, the processes branched dichotomously and reached their adult zig-zag appearance in cross section at P26. Inner epithelial cells were at first arranged on many layers, then on two layers and, from P15, on a single layer. Outer cells were arranged on a single row from P3. Both cell types showed linear contacts with occasional interdigitations till P9, then complex intercellular junctions. The morphometric analysis demonstrated that while epithelial folds elongated, the ciliary epithelium, and particularly the inner cells, reduced its height. The ciliary processes development of gerbil starts in utero, undergoes significant morphological changes soon after eyelid opening and is completed at P26, when adult organization is reached.

The fragile X syndrome (FXS), the most common form of inherited intellectual disability, is due to the absence of FMRP, a protein regulating RNA metabolism. Recently, an unexpected function of FMRP in modulating the activity of Adenosine Deaminase Acting on RNA (ADAR) enzymes has been reported both in Drosophila and Zebrafish. ADARs are RNA-binding proteins that increase transcriptional complexity through a post-transcriptional mechanism called RNA editing. To evaluate the ADAR2-FMRP interaction in mammals we analyzed several RNA editing re-coding sites in the fmr1 knockout (KO) mice. Ex vivo and in vitro analysis revealed that absence of FMRP leads to an increase in the editing levels of brain specific mRNAs, indicating that FMRP might act as an inhibitor of editing activity. Proximity Ligation Assay (PLA) in mouse primary cortical neurons and in non-neuronal cells revealed that ADAR2 and FMRP co-localize in the nucleus. The ADAR2-FMRP co-localization was further observed by double-immunogold Electron Microscopy (EM) in the hippocampus. Moreover, ADAR2-FMRP interaction appeared to be RNA independent. Because changes in the editing pattern are associated with neuropsychiatric and neurodevelopmental disorders, we propose that the increased editing observed in the fmr1-KO mice might contribute to the FXS molecular phenotypes.

Many smokers have recently turned to electronic cigarettes (e-cigarettes) because they have been marketed as a cheaper, safer smokeless alternative to traditional cigarettes and a possible smoking cessation tool. Although the safety of these electronic devices is still not fully known, there is evidence of their cytotoxicity on cells belonging to the oral cavity. In a previous study by the authors, the increase of reactive oxygen production and Bax expression, followed by the occurrence of apoptosis, was demonstrated in human gingival fibroblasts (HGFs). The aim of this paper is to further investigate the effects of the e-cigarette liquids (with and without nicotine) on the same experimental model.HGFs were treated with e-cigarette fluids containing nicotine (final concentration 1 mg/mL) and the equivalent volume of a fluid without nicotine, for periods ≤48 hours. Lactate dehydrogenase assay (LDH), electronic microscopy analysis, collagen I production, flow cytometry lysosome compartment evaluation, and western blotting light chain 3 (microtubule-associated protein 1A/1B-LC3) expression were performed.Fluids containing nicotine exerted cytotoxicity as demonstrated by increased levels of LDH, in parallel to the presence of numerous vacuoles in the cytoplasm, a decrease in collagen I production, and augmented LC3 II expression. Autophagic vesicles and more procollagen I molecules were present in the cytoplasm of fibroblasts exposed to nicotine-free fluids. In the same samples, time-dependent activation of the lysosomal compartment with no changes in LC3 expression was detected.E-cigarette fluids (with and without nicotine) trigger molecular and morphologic responses in oral fibroblasts, raising concerns about their role in the pathogenesis of oral diseases.

Primary myelofibrosis (PMF) is characterized by fibrosis, ineffective hematopoiesis in marrow, and hematopoiesis in extramedullary sites and is associated with abnormal megakaryocyte (MK) development and increased transforming growth factor (TGF)-β1 release. To clarify the role of TGF-β1 in the pathogenesis of this disease, the TGF-β1 signaling pathway of marrow and spleen of the Gata1(low) mouse model of myelofibrosis (MF) was profiled and the consequences of inhibition of TGF-β1 signaling on disease manifestations determined. The expression of 20 genes in marrow and 36 genes in spleen of Gata1(low) mice was altered. David-pathway analyses identified alterations of TGF-β1, Hedgehog, and p53 signaling in marrow and spleen and of mammalian target of rapamycin (mTOR) in spleen only and predicted that these alterations would induce consequences consistent with the Gata1(low) phenotype (increased apoptosis and G1 arrest both in marrow and spleen and increased osteoblast differentiation and reduced ubiquitin-mediated proteolysis in marrow only). Inhibition of TGF-β1 signaling normalized the expression of p53-related genes, restoring hematopoiesis and MK development and reducing fibrosis, neovascularization, and osteogenesis in marrow. It also normalized p53/mTOR/Hedgehog-related genes in spleen, reducing extramedullary hematopoiesis. These data identify altered expression signatures of TGF-β1 signaling that may be responsible for MF in Gata1(low) mice and may represent additional targets for therapeutic intervention in PMF.

GATA1 is a member of the GATA family of transcription factors that exerts a concentration/dependent control on the differentiation of erythroid, megakaryocytic (MK), eosinophil and mast cells. Changes of GATA1 content also controls hematopoietic stem/progenitor cell proliferation and commitment. In fact, in addition to X-linked inherited disorders of the erythroid and/or MK lineage, GATA1 mutations are found in inherited and acquired MK leukaemia and myeloproliferative disorders. To study the regulation of the human β-globin locus, the Stamatoyannopoulos laboratory generated transgenic mice carrying human GATA1 driven by the human μLCR coupled with the β-globin promoter (hGATA1) (Li Q et al. PNAS 1997, 18;94:2444). These transgenic mice are born viable with normal phenotype and express hGATA1 at high levels in embryonic and fetal erythroblasts and barely detectable in the adult ones, raising the question whether in adult cells expression of the endogenous Gata1 may suppress the activity of the human μLCR- β-globin promoter. The aim of this study was to determine the effects of Gata1 on the activity of hGATA1 in mice. Since the Gata1null mutation is embryonically lethal, the hypothesis was tested by generating with standard genetic approaches double mutants carrying hGATA1 and the hypomorphic Gata1low mutation, a mutation that strongly reduces expression of Gata1 in adult hematopoietic cells (erythroblasts, MK, hematopoietic progenitors, others). If successful, these experiments would also indirectly assess the extent by which hGATA1 is capable to rescue the complex Gata1low phenotype. Gata1low mice, in fact, are born anemic and thrombocytopenic but recover from their anemia at 1 month by activating extramedullary hematopoiesis in spleen (Vannucchi et al Blood 2001;97:3040). Spleen erythropoiesis is however mostly ineffective since many of the erythroblasts present in this organ are in apoptosis (Tunel+). Gata1low mice remain thrombocytopenic all their life and with age develop myelofibrosis, a phenotype that resemble primary myelofibrosis, the most severe of the Phyladelphia-negative myeloproliferative neoplasms (Vannucchi et al Blood 2002;100:1123). In these mice, hematopoietic stem cells are detected in spleen, and not in bone marrow, and generate bipotent MK/erythroid progenitors (MEP) with a commitment program screwed toward the mast cell lineage and capable to generate in vitro at the single cell basis mast cell lines (Ghinassi et al. Blood 2007;109:1460-71). Double hGATA1Gata1low mutants were easily generated and were not anemic at birth. At 4 months, they expressed normal haematocrit but reduced blood platelet counts (Fig. 1A,B). RT-PCR analyses of prospectively isolated cell populations confermed that the transgene mRNA is barely detectable in cells from single hGATA1 mutants. By contrast, in the double mutants, hGATA1 mRNA was expressed by common myeloid progenitors, its expression increased by 10-fold in MEP, remained at the same levels expressed by MEP in erythroid cells (CD71+/Ter119+) but was barely detectable in MK (CD41+/CD61+). Expression of hGATA1 did not alter the levels of the endogenous Gata1 expressed by these populations that remained low. MEP purified from double hGATA1/Gata1low mice generated in culture more cells (fold increase=250 in Gata1low/hGATA1 cultures vs 50 in single hGATA1 or Gata1low cultures) which were mostly composed by erythroid precursors unable to generate cell lines. By contrast with single Gata1low mutants, by confocal microscopy, GATA1 was readily detectable in erythroid cells from bone marrow and spleen that these cells were not Tunel+ (Fig. 1C), indicating that hGATA1 reduced ineffective erythropoiesis. GATA1 protein remained not detectable in MK from the same tissues (Fig. 1C) and these cells displayed the abnormal morphology characteristic of the retarded maturation of Gata1low MK. Furthermore, double hGATA1/Gata1low mice, as the single Gata1low ones, developed myelofibrosis in bone marrow (by Gomori staining) at 8 months, confirming that myelofibrosis is driven by MK abnormalities. In conclusion, hGATA1 is expressed by adult CMP, MEP and erythroblasts in a genetic Gata1low background rescuing the MEP and erythroid phenotype induced by the hypomorphic mutation. hGATA1 is not expressed and does not rescue the MK phenotype of Gata1low mice, highlighting the specificity of the β-globin promoter for erythroid cells. Disclosures No relevant conflicts of interest to declare.

Splenomegaly is a major manifestation of primary myelofibrosis (PMF) contrib- uting to clinical symptoms and hematologic abnormalities. The spleen from PMF patients contains increased numbers of hematopoietic stem cells (HSC) and megakar- yocytes. These megakaryocytes express high levels of P-selectin (P-sel) that, by trig- gering neutrophil emperipolesis, may cause TGF-β release and disease progression. This hypothesis was tested by deleting the P-sel gene in the myelofibrosis mouse model carrying the hypomorphic Gata1low mutation that induces megakaryocyte abnormalities that recapitulate those observed in PMF. P-selnullGata1low mice survived splenectomy and lived three months longer than P-selWTGata1low littermates and did not express fibrosis and osteosclerosis in the marrow or splenomegaly. Furthermore, deletion of P-sel disrupted megakaryocyte/neutrophil interactions in spleen, reduced TGF-β content and corrected the HSC distribution that in Gata1low mice, as in PMF patients, is abnormally expanded in spleen. Conversely, pharmacological inhibition of TGF-β reduced P-sel expression in megakaryocytes and corrected HSC distribution. Spleens, but not marrow, of Gata1low mice contained numerous cKITpos activated fibro- cytes, probably of dendritic cell origin, whose membrane protrusions interacted with megakaryocytes establishing niches hosting immature cKITpos hematopoietic cells. These activated fibrocytes were not detected in spleens from P-selnullGata1low or TGF-β-inhibited Gata1low littermates and were observed in spleen, but not in marrow, from PMF patients. Therefore in Gata1low mice, and possibly in PMF, abnormal P-sel expres- sion in megakaryocytes may mediate the pathological cell interactions that increase TGF-β content in MK and favor establishment of a microenvironment that supports myelofibrosis-related HSC in spleen., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

Myelofibrosis (MF) is characterized by hyperactivation of thrombopoietin (TPO) signaling, which induces a RPS14 deficiency that de-regulates GATA1 in megakaryocytes by hampering its mRNA translation. As mice carrying the hypomorphic Gata1low mutation, which reduces the levels of Gata1 mRNA in megakaryocytes, develop MF, we investigated whether the TPO axis is hyperactive in this model. Gata1low mice contained two times more Tpo mRNA in liver and TPO in plasma than wild-type littermates. Furthermore, Gata1low LSKs expressed levels of Mpl mRNA (five times greater than normal) and protein (two times lower than normal) similar to those expressed by LSKs from TPO-treated wild-type mice. Gata1low marrow and spleen contained more JAK2/STAT5 than wild-type tissues, an indication that these organs were reach of TPO-responsive cells. Moreover, treatment of Gata1low mice with the JAK inhibitor ruxolitinib reduced their splenomegaly. Also in Gata1low mice activation of the TPO/MPL axis was associated with a RSP14 deficiency and a discordant microarray ribosome signature (reduced RPS24, RPS26 and SBDS expression). Finally, electron microscopy revealed that Gata1low megakaryocytes contained poorly developed endoplasmic reticulum with rare polysomes. In summary, Gata1low mice are a bona fide model of MF, which recapitulates the hyperactivation of the TPO/MPL/JAK2 axis observed in megakaryocytes from myelofibrotic patients.

Patients with primary myelofibrosis have increased risk for bleeding and thrombosis. It is debated whether propensity to thrombosis is due to increased numbers of platelet microparticles and/or to pathological platelet-neutrophil interactions. Platelet neutrophil interactions are mediated by P-selectin and even though the megakaryocytes of myelofibrosis patients express normal levels of P-selectin, it remains abnormally localized to the demarcation membrane system rather than being assembled into the α-granules in platelets. Mice carrying the hypomorphic Gata1(low) mutation express the same megakaryocyte abnormalities presented by primary myelofibrosis patients, including abnormal P-selectin localization to the DMS and develop with age myelofibrosis, a disease that closely resembles human primary myelofibrosis. Whether these mice would also develop thrombosis has not been investigated as yet. The aim of this study was to determine whether Gata1(low) mice would develop thrombosis with age and, in this case, the role played by P-selectin in the development of the trait. To this aim, Gata1(low) mice were crossed with P-sel(null) mice according to standard genetic protocols and Gata1(low)P-sel(wt), Gata1(low)P-sel(null) and Gata1(WT)P-sel(null) or Gata1(wt)P-sel(wt) (as controls) littermates obtained. It was shown that platelet counts, but not hematocrit, are reduced in Gata1(low) mice. Moreover, platelet microparticles are reduced in Gata1(low) mice and P-selectin positive platelet microparticles were not found. To determine the phenotypic implications of the different mutations, bleeding time was estimated by a tail cut procedure. Mutant mice were sacrificed and presence of thrombosis was determined by immunohistological staining of organs. Gata1(low) mice with or without the P-selectin null trait had a prolonged bleeding time compared to wild type mice. However, in Gata1(low) mice significantly higher frequency of thrombotic events was seen in adult and old Gata1(low) mice compared to Gata1(low)P-sel(null) mice. Thus, presence of the P-selectin null trait rescued Gata1(low) mice from the thrombotic phenotype, but did not change the level of platelet microparticles. Taken together these data indicate that abnormal localization of P-selectin, induced by the Gata1(low) mutation, and thus, increased pathological interactions with leucocytes, is responsible for the increased presence of thrombosis seen in these mice.

Myelofibrosis (MF) is caused by genetic abnormalities involving the thrombopoietin (TPO) axis (mutations in either the TPO receptor, MPL, MPL-signaling, JAK2, or MPL partners, Calreticulin). Furthermore MF patients have elevated serum TPO levels. By contrast with other MPNs, MF is also associated with reduced GATA1 content in MKs (Vannucchi et al, Am J Pathol 2005;167:849). In 2014, we suggested that the "element" that specifically links TPO signaling with de-regulated Gata1 expression in MKs represents the phenotypic modifier leading to MF development and presented data suggesting that this "element" is a ribosomal deficiency, which hampers GATA1 mRNA translation (Gilles et al, Blood 2014; 124:4585 abstract). Support for GATA1 deficiency in MKs as a "phenotypic modifier" in MF was provided by the observation that mice carrying a hypomorphic mutation (Gata1low) which develop MF have reduced transcription of Gata1 in MKs (Vannucchi et al, Blood 2002;100:1123). These mice are subjected to continuous "erythroid stress" due to reduced RBC half-life and contain increased numbers of bipotent Ery/MK precursors with a unique phenotype (Vannucchi et al, Blood 2000; 95:2559) similar to that of the bipotent Ery/Mk precursors induced in vitro from cord blood by a chemical MPL stimulator (Belay et al, Blood 2015; 125:1025). These considerations led us to investigate the activation state of the TPO/Mpl axis in this model. In Gata1low and wild-type mice, TPO mRNA was expressed by bone marrow (BM), spleen and liver. The greatest levels (by 300-fold) were detected in the liver. Gata1low livers expressed TPO mRNA at 6-fold greater levels than wild-type livers. TPO protein was detected in BM, spleen, liver, peritoneum and plasma. Gata1low plasma contained TPO levels 2-times greater than plasma from mice with similar levels of thrombocytosis (778±101 vs 451±51pg/mL in bleed wild-type mice and 351±31pg/mL in Mplnull mice) suggesting that the TPO axis is activated in Gata1low mice. This hypothesis was further tested by determining that Gata1low LSK expressed levels of Mpl mRNA 3-times greater than wild-type cells, but expressed on their surface levels of MPL 2-times lower than wild-type LSK. This reduction was similar to the level of MPL in LSK from wild-type mice treated with TPO, suggesting that TPO stimulation down-modulated MPL. The MPL signaling partners JAK2 and STAT5 were barely detectable in wild-type BM but were detected at robust levels in Gata1low BM and spleen (Fig 1) suggesting TPO stimulated greater JAK/STAT signaling. The role of the TPO/Mpl axis in Gata1low mice was further investigated by determining the effects of the JAK/STAT inhibitor ruxolitinib (90mg/Kg/Day or vehicle for 2 weeks, three females per group). This treatment induced a slight anemia (38±1 vs 41±4), reduced spleen size (240±60 vs 380±80 mg in vehicle and 350±80 in untreated Gata1low mice), did not increase BM cellularity (10±1.3x106 vs 9.6±1.0x106 in vehicle and 18±2x106 in wild type cells/femur), reduced BM fibrosis (2.7±0.3 vs 7.9±0.1 arbitrary units of Gomori staining) and reduced the abnormal TGF-β content present in the spleen but not that of BM (Fig 2). The discrepancy between the modestly reduced Gata1 mRNA levels and the undetectable GATA1 protein observed in Gata1low MKs (Fig 3) led us to investigate the expression of ribosomal related genes in these mice. Microarray analyses of BM and spleen from Gata1low and wild-type mice indeed identified a discordant ribosome signature solely in BM. This signature included reduced expression of RPS24 (-2-fold, p=0.04) and RPS26 (-3-fold, p=0.03), two genes mutated in a subset of patients with Diamond Blackfan Anemia associated with ribosomapathies leading to reduced GATA1 mRNA translation (Danolova et al, Disease Model&Mechanism 2015;8:1013) and SBDS (-2-fold, p=0.01), another hypomorphic gene in the inherited BM failure state Schwachman-Diamond syndrome which regulates ribosome biogenesis (Zambetti et al, Haematologica 2015;100:1285). In addition, electron microscopy revealed that Gata1low MKs contain poorly developed endoplasmic reticulum with rare polysomes. We conclude that Gata1low mice that develop MF have an activated TPO/MPL axis and an abnormal ribosomal signature which may lead to reduced Gata1 translation. Disclosures No relevant conflicts of interest to declare.

Abstract 3462 The Gata1low mutation impairs megakaryocytic and erythroid development. Gata1low 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 Gata1low 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 Gata1low mice were observed. In BM, 6 genes were up-regulated and 14 down-regulated (Bmp2, Bmp5, Acvrl1, Tgfblil and Igf1 with a p SB431542–treatment had profound effects on TGF- β expression profile and on the phenotype of Gata1low 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 Gata1low 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 Gata1low mutation in determining the phenotype of Gata1low mice in the CD1 background. Disclosures: No relevant conflicts of interest to declare.