HDAC Inhibitors as Epigenetic Regulators

Emerging evidence indicates that in myelodysplastic syndromes (MDS), the bone marrow (BM) microenvironment may also contribute to the ineffective, malignant haematopoiesis in addition to the intrinsic abnormalities of haematopoietic stem precursor cells (HSPCs). decreased expression of FAK or its phosphorylated form in BMSCs from low-risk (LR) MDS directly correlates with BMSCs functional deficiency and is associated with a reduced level of haemoglobin. The downregulation of FAK in HS-5 cells alters their morphology, proliferation, and differentiation capabilities and impairs the expression of several adhesion molecules. In addition, we examine the CD34+ healthy donor (HD)-derived HSPCs properties when co-cultured with FAK-deficient BMSCs. Both abnormal proliferation and the impaired erythroid Pyrimethamine differentiation capacity of HD-HSPCs were observed. Together, these results demonstrate that stromal adhesion mechanisms mediated by FAK are crucial for regulating HSPCs homeostasis. 0.05(*); 0.01(**); 0.001(***); 0.0001(****) were considered statistically significant differences. 3. Results 3.1. Focal Adhesion Kinase (FAK) Deficiency in Bone Marrow Stromal Cells Derived from Patients with Myelodysplastic Syndromes (MDS BMSCs) Impairs Their Normal Function and Correlates with Ineffective Haematopoiesis We have previously reported that, in MDS stromal cells, the expression of total FAK and its phosphorylation at Tyr397 site were abnormal [17,18] and induced abnormal proliferation and differentiation with an increased propensity towards Pyrimethamine adipocyte differentiation to the detriment of osteogenesis [18]. We have also observed the gradual augmentation of FAK expression and activation during MDS progression [18]. Here, we show that, along with abnormal functional capacities (i.e., decreased proliferative and clonogenic capacities, increased propensity towards adipogenic differentiation, and reduced osteogenic differentiation (Physique 1ACD)), the abnormal expression of FAK in MDS-derived MSCs is usually associated with morphological and phenotypic changes (Physique 1E,F). Open in a separate window Physique 1 Intrinsic abnormalities related to focal adhesion kinase (FAK) deficiency in BMSCs from MDS patients correlate with the reduced clonogenic potential of HSPCs and with a degree of anaemia. (A,B) Evaluation of CFU-F and C, proliferative capacities (measured by MTT Cell Proliferation Assay) in BMSCs derived from MDS patients compared with healthy donors as controls (HC). (D) Quantification of oil red (adipogenic differentiation) and alizarin red (osteogenic differentiation) staining at day 14 in MSC derived from HC, LR-MDS (low-risk) and HR-MDS (high-risk) patients. (E) Morphological evaluation of MDS-derived MSCs compared to HC MSCs. (F) Phenotypic differences in BMSCs selected from LR-MDS patients compared to HC. (G) Significant correlation between PTK2 expression in BMSCs and the haemoglobin level in an MDS setting. (H) Evaluation of the clonogenic capacity of HSPCs selected from MDS patients compared to HC. I, SDF-1 mRNA expression in BMSCs isolated from LR-MDS and HR-MDS patients compared to HC. HC, HD controls; LR-MDS, low-risk MDS; HR-MDS, high-risk MDS. 0.05(*); 0.01(**); 0.0001(****). Large, flat, and granular stromal cells were observed in primary cultures Pyrimethamine of BMSCs from MDS patients compared with spindle-shaped cells in cultures from HD BM aspirates. Among the Pyrimethamine phenotypic changes, we observed that this BMSCs deficient in FAK from LR-MDS showed a diminution of expression of the CD106 immunomodulatory molecule, the CD166 osteogenic-related Pyrimethamine marker, and the CD54 (ICAM-1) adhesion molecules (Figure 1F). A common biological characteristic of Rabbit Polyclonal to SFXN4 LR-MDS patients is anaemia. There was a strong positive correlation between the haemoglobin level and the level of PTK2 expression in BMSCs from LR-MDS (Figure 1G). In addition, the clonogenic capacities of HSPCs isolated from LR-MDS patients were significantly reduced (Figure 1H). Moreover, SDF-1 expression, an important cytokine for cell trafficking and the homing of CD34+ HSCs, was decreased in LR-MDS BMSCs (Figure 1I). Thus, these data support the idea that FAK-deficient stroma might contribute to the MDS pathogenesis through abnormal differentiation and the capacity to generate osteoblasts, together with a reduced expression of several haematopoiesis-supporting molecules. 3.2. The Inhibition of Focal Adhesion Kinase (FAK) Phosphorylation or FAK Expression in the HS-5 Cell Line Recapitulates the Morpho-Functional Abnormalities Observed in LR-MDS BMSCs We sought to determine whether the intrinsic deficiencies of LR-MDS BMSCs were related to the abnormal expression of FAK in stromal cells. Therefore, we evaluated the consequences of FAK inhibition in HS-5 cells, a.