Abstract:
We analyzed the role of DLK1 protein, an inhibitor of NOTCH receptors, in the osteogenic differentiation of MC3T3-E1 pre-osteoblastic cells, which lack endogenous Dlk1 expression. We have generated pools of cells stably overexpressing DLK1 protein by plasmid transfection. Dlk1 expression levels were analyzed by RT-qPCR and DLK1 protein levels by Western Blot. We performed osteogenic differentiation assays by treatment with culture medium supplemented with 10 mM β-glycerophosphate and 50 μg/ml ascorbic acid. The differentiation capacity of the DLK1-overexpressing cells was assessed by alkaline phosphatase staining and by the analysis of osteogenic differentiation markers by RT-qPCR. These assays were also performed using DAPT, a pharmacological inhibitor of NOTCH receptors. We have observed that the cells overexpressing Dlk1 showed a lower rate of osteogenic differentiation with respect to control cells, as shown by the lower degree of alkaline phosphatase staining and lower mRNA expression of some osteogenic markers, such as Alpl, Col1A1, Runx2 and Osteocalcin. The NOTCH receptor inhibitor, DAPT, also caused the decrease of both alkaline phosphatase staining and osteogenic markers in MC3T3-E1 cells treated with the osteogenic inducers. The results obtained indicate that Dlk1 overexpression or treatment with DAPT exert an inhibitory effect on the osteoblastic differentiation of MC3T3-E1 cells. This indicates that NOTCH signaling is required for osteogenesis of these cells and that DLK1 could exert its inhibitory effect on this process through inhibition of NOTCH signaling.
Biography:
I graduated in Biological Sciences at the University of Salamanca, Spain. My PhD Thesis was defended in April 1997 with honors. I started my postdoctoral stage at CBER (FDA, NIH Campus, Bethesda, MD, USA), where I applied my knowledge about yeast field to the study of the interaction of the mouse membrane protein DLK1 with the NOTCH1 receptor. In October 2000, I joined the Department of Biochemistry and Molecular Biology of the Faculty of Medicine of Albacete, UCLM, Spain. Now, I am working as a Full-Time Associate Professor, and I teach the first cycle of Biochemistry and Molecular Biology in the Degrees of Medicine, Pharmacy and Biotechnology. In my research work, I have mainly continued with the study of the function and mechanism of action of NOTCH receptors and DLK proteins in cell differentiation and cell proliferation. I have published several articles in international journals, whose papers have been presented in various national and international congresses. I have co-directed 3 PhD Theses, and I actively participate in several academic committees.

X