Tensile strain-induced Ets-2 phosphorylation by CaMKII and the homeostasis of cranial sutures

Background: Mechanotransduction underpins the homeostasis of musculoskeletal tissues, including cranial sutures. Intracellular calcium, [Ca ²⁺]ic, and protein phosphorylation are two intermediate variables in signal relay during mechanotransduction. This project establishes a chain of cause and effect, linking cellular strain to substrate phosphorylation, and identifies the agent and target sites of phosphorylation. Methods: Cyclic tensile force (0.5 N at 1 Hz) was applied to 1-day-old rat sagittal sutures. [Ca]ic was measured by FURA-2. Ets-2 phosphorylation by CaMKII was tested using Western blot autoradiography. Peptide array was constructed to determine the precise sites of phosphorylation. The results were confirmed with mass spectroscopy and Western blots using phospho-specific antibodies. Results: [Ca ²⁺]ic increased rapidly in response to tensile stress. In the presence of Ca, CaMKII caused Ets-2 phosphorylation. Of the three possible sites for phosphorylation of Ets-2 by CaMKII, RVPS, FESF, RLSS, Serine 246, 310, and 313 were the targets. Furthermore, the contiguous sequence modified this effect. Mass spectroscopy showed 80 Da (molecular weight of phosphate group, -PO3) right shifts consistent with phosphorylation. There was cytosolic translocation of Ets-2 on tensile deformation of suture cells. CaMKII binding of Ets-2 occurred within 30 minutes after the onset of tensile strain. Conclusions: Cranial suture cells can respond to tensile forces by increasing [Ca ²⁺]ic, which causes CaMKII to phosphorylate Ets-2, thus altering Ets-2 binding to its downstream promoters. Of note, Ets-2 is at the intersection of three key pathways important in craniosynostosis: fibroblast growth factor-2, transforming growth factor-β, and mechanotransduction.