Implicación de la Cx43 y el cilio primario en la actividad de los osteocitos

The bone tissue has the ability to adapt to the stimuli of the environment by altering its morphology and metabolism. The development and maintenance of this tissue are dynamic processes, regulated by the different bone cells communicated with each other through Gap Junctions (GJs). Connexin 43 (Cx43), which is the most abundant protein in GJs, has key roles in the signal transduction of bone tissue and in the response to hormonal and mechanical stimuli. Other mechanosensing elements in the osteocytes are: the primary cilium, mainly formed of microtubules and developed when the cells are in the G0 phase of the cell cycle; and the integrins, a family of glycoproteins forming heterodimers composed of two subunits (the alpha and beta chain) and located in the cell membrane. The involvement of Cx43, the primary cilium and integrins in the mouse osteocyte cell line MLO-Y4 control and deficient in Cx43 will be studied in this project. The response of this cell line to hormonal stimuli (ligand of the parathyroid hormone receptor type 1, the protein related to parathyroid hormone, PTHrP) and mechanical stimuli (by fluid flow of culture medium) will also be analyzed. In addition, the possible functional interaction between these three mechanical elements will be studied. For protein expression analyses of IFT88 (homolog of the intraflagelar transport protein 88), Cx43, P-ERK (Kinases regulated by extracellular signal phosphorylated) and P-GSK3 (Glycogen synthase kinase 3 beta phosphorylated), Western blotting was performed. The expression of the integrins was determined by PCR (Polymerase Chain Reaction) and, to characterize the possible colocalization between the primary cilium and Cx43 an immunofluorescence was performed. The results obtained show that Cx43 and the primary cilium do not collocate, and that the presence of Cx43 is not necessary for ciliogenesis. On the other hand, it was determined that fluid flow and PTHrP increase the phosphorylation of ERK and GSK3. Therefore, these stimuli regulate cell survival pathways in osteocytes. Finally, it was observed that integrin α2 increased its expression in control cells (Cx43 +/+). However, integrins α6, β1 and β3 increased in Cx43 deficient cells (Cx43 -/-), suggesting an interaction between these two protein families