Roles of integrin β1 and discoidin domain receptor 2 in cranial regeneration and skeletal progenitor cell function

Abstract

The two principal collagen receptors in bone, the collagen-binding β1 integrins and discoidin domain receptor 2 (DDR2), each have important roles in development, but their functions in bone regeneration are largely unexplored. Using a critical-size calvarial defect model, we assessed the relative roles of these two receptor classes in BMP2-induced regeneration using a conditional knockout approach where Itgb1 (encodes integrin β1) and/or Ddr2 were selectively inactivated in GLI1⁺ skeletal progenitor cells (SPCs). Inactivating either Itgb1 or Ddr2 partially inhibited calvarial regeneration, while inactivation of both receptors almost completely blocked bone healing. Responses were linked to reduced proliferation and migration of GLI1⁺ SPCs into defects and reduced endochondral and intramembranous bone formation. To examine the consequences of receptor inactivation at the cellular level, calvarial SPCs lacking Itgb1, Ddr2 or both receptors were generated. Loss of either receptor inhibited osteoblast differentiation, migration, cell spreading, focal adhesion formation, and nuclear localization of the mechanotransducer, YAP1. Importantly, inactivation of both receptors inhibited responses to a greater extent than was seen with individual knockouts. Interestingly, while inactivation of one allele of either Itgb1 (Itgb1^fl/+) or Ddr2 (Ddr2^fl/+) did not affect any of the above parameters, inactivation in double heterozygotes (Itgb1^fl/+ ;Ddr2^fl/+) was strongly inhibitory, which is indicative of a genetic interaction between Itgb1 and Ddr2. Lastly, immunofluorescence and immunoprecipitation analysis suggest that DDR2 and ITGB1 physically interact, providing a potential explanation for the observed functional cooperativity. These studies provide a mechanistic basis for bone regeneration strategies involving combined activation of integrin β1 and DDR2.

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Funding

U.S. Department of Health & Human Services | NIH | National Institute of Dental and Craniofacial Research (NIDCR)(DE029465)

U.S. Department of Health & Human Services | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)(P30 AR069620)

U.S. Department of Defense (United States Department of Defense)(CDMRP W81XWH-20-1-0571)

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