The effect of conditional inactivation of beta 1 integrins using twist 2 Cre, Osterix Cre and osteocalcin Cre lines on skeletal phenotype

Abstract

Skeletal development and growth are complex processes regulated by multiple microenvironmental cues, including integrin-ECM interactions. The β1 sub-family of integrins is the largest integrin sub-family and constitutes the main integrin binding partners of collagen I, the major ECM component of bone. As complete β1 integrin integrin knockout results in embryonic lethality, studies of β1 integrin function in vivo rely on tissue-specific gene deletions. While multiple in vitro studies indicate that β1 integrins are crucial regulators of osteogenesis and mineralization, in vivo osteoblast-specific perturbations of β1 integrins have resulted in mild and sometimes contradictory skeletal phenotypes. To further investigate the role of β1 integrins on skeletal phenotype, we used the Twist2-Cre, Osterix-Cre and Osteocalcin-Cre lines to generate conditional β1 integrin deletions, where cre is expressed primarily in mesenchymal condensation, pre-osteoblast, and mature osteoblast lineage cells respectively within these lines. Mice with Twist2-specific β1 integrin disruption were smaller, had impaired skeletal development, especially in the craniofacial and vertebral tissues at E19.5, and did not survive beyond birth. Osterix-specific β1 integrin deficiency resulted in viable mice which were normal at birth but displayed early defects in calvarial ossification, incisor eruption and growth as well as femoral bone mineral density, structure, and mechanical properties. Although these defects persisted into adulthood, they became milder with age. Finally, a lack of β1 integrins in mature osteoblasts and osteocytes resulted in minor alterations to femur structure but had no effect on mineral density, biomechanics or fracture healing. Taken together, our data indicate that β1 integrin expression in early mesenchymal condensations play an important role in skeletal ossification, while β1 integrin-ECM interactions in pre-osteoblast, odontoblast- and hypertrophic chondryocyte- lineage cells regulate incisor eruption and perinatal bone formation in both intramembranously and endochondrally formed bones in young, rapidly growing mice. In contrast, the Osteocalcin-specific β1 integrin deletion had only minor effects on skeletal phenotype.

Multiple essential MT1-MMP functions in tooth root formation, dentinogenesis, and tooth eruption

“…While the Osx-Cre transgene has been reported to cause delayed or defective skeletal and craniofacial mineralization resulting from Osterix loss-of-function [35-37], studies including analysis of molar teeth have not identified similar dental defects [30, 38, 39]. To rule out dental alterations from the Osx-Cre transgene, several control genotypes were analyzed (Supplementary Figure 6).…”

Physiological mechanisms and therapeutic potential of bone mechanosensing

“…Third, activation of integrin α5β1 or the extracellular matrix (ECM)-integrin α5β1 interactions via RGD binding have been linked to the control of osteoblastogenesis through activation of TRPV4 ion channels and FAK/RhoA-ROCK/ERK [40–42, 158–160]. Fourth, integrin α5β1 affects bone formation in osteoblasts and osteocytes in vivo [161–164]. …”

Integrin-Linked Kinase Regulates Bone Formation by Controlling Cytoskeletal Organization and Modulating BMP and Wnt Signaling in Osteoprogenitors

“…The role of ILK has been less profoundly characterized, and especially, information on its functioning early in the osteoblast lineage is lacking. Yet previous transgenic expression of a dominant‐negative β1 integrin subunit or conditional inactivation of β1 integrin, FAK, or ILK in mature osteoblasts was without discernible effects or only mildly or transiently affected the skeleton in basal conditions, whereas recent studies in which β1 integrin or FAK were targeted in osteogenic progenitors revealed important functions for these molecules in the control of bone formation.…”