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 2+]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 Ca2+, 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-beta, and mechanotransduction.
Yu
Jack C
JC
Augusta, Ga.; and Kaohsiung and Taipei, Taiwan, Republic of China From the Center for Regenerative Medicine, Institute of Molecular Medicine and Genetics; Section of Plastic Surgery, Department of Surgery, Medical College of Georgia; Department of Biological Science and Technology, I-Shou University; Faculty of Life Sciences and Institute of Genome Sciences, National Yang Ming University; Driftmier Engineering Center, University of Georgia; Savannah River National Laboratory; Department of Orthopedic Surgery, Medical College of Georgia; College of Dental Medicine, Kaohsiung Medical University; and Department of Oral Biology, School of Dentistry, Medical College of Georgia.
Chen
Jung-Ren
JR
Lin
Chao-Hsiung
CH
Zhang
Guigen
G
Lam
Poh-Sang
PS
Wenger
Karl H
KH
Mozaffari
Farid B
FB
Huang
Shun-Te
ST
Borke
James L
JL
D000818:Animals; D000831:Animals, Newborn; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism*; D003393:Cranial Sutures / Q000378:metabolism*; D006706:Homeostasis; D023421:Models, Animal; D008954:Models, Biological; D009141:Musculoskeletal System / Q000378:metabolism*; D010766:Phosphorylation; D050787:Proto-Oncogene Protein c-ets-2 / Q000378:metabolism / Q000502:physiology*; D051381:Rats; D013718:Tensile Strength
D050787:Proto-Oncogene Protein c-ets-2; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2