In Vivo Analysis of Arg-Gly-Asp Sequence/Integrin α5β1-Mediated Signal Involvement in Embryonic Enchondral Ossification by Exo Utero Development System

Abstract

Enchondral ossification is a fundamental mechanism for longitudinal bone growth during vertebrate development. In vitro studies suggested that functional blockade with RGD peptides or with an antibody that interferes with integrin a5b1-ligand interactions inhibited pre-hypertrophic chondrocyte differentiation. The purpose of this study is to elucidate in vivo the roles of the integrin a5b1-mediated signal through the Arg-Gly-Asp (RGD) sequence in the cell-extracellular matrix (ECM) interaction in embryonic enchondral ossification by an exo utero development system. We injected Arg-Gly-Asp-Ser (RGDS) peptides and anti-integrin a5b1 antibody (a5b1 ab) in the upper limbs of mouse embryos at embryonic day (E) 15.5 (RGDS-injected limbs, a5b1 ab-injected limbs), and compared the effects on enchondral ossification with those found in the control limbs (Arg-Gly-Glu-Ser peptide-, mouse IgG-, or vehicle-injected, and no surgery) at E16.5. In the RGDS-injected limbs, the humeri were shorter and there were fewer BrdU-positive cells than in the control limbs. The ratios of cartilage length and area to those of the humerus were higher in the RGDS-injected limbs. The ratios of type X collagen to type 2 collagen mRNA and protein (Coll X/Coll 2) were significantly lower in the RGDS-injected limbs. In those limbs, TUNEL-positive cells were hardly observed, and the ratios of fractin to the Coll X/Coll 2 ratio were lower than in the control limbs. Furthermore, the a5b1 ab-injected limbs showed results similar to those of RGDS-injected limbs. The present in vivo study by exo utero development system showed that RGDS and a5b1 ab injection decreased chondrocyte proliferation, differentiation, and apoptosis in enchondral ossification, and suggested that the integrin a5b1-mediated ECM signal through the RGD sequence is involved in embryonic enchondral ossification.

Angiopoietin-like protein 2 promotes chondrogenic differentiation during bone growth as a cartilage matrix factor

“…We previously reported that a5b1 integrin plays a critical role in ANGPTL2 function in several tissues 11e13 , analyses that did not include chondrocytes. A previous report by others indicated anomalous chondrocyte differentiation and endochondral ossification in long bones of mice in which a5b1 integrin signaling had been blocked, phenotypes similar to those reported here in Angptl2 knockout mice 29,30 . In addition, p38 MAPK signaling is part of a major pathway governing bone development and remodeling 9,10 .…”

ATX-LPA1 axis contributes to proliferation of chondrocytes by regulating fibronectin assembly leading to proper cartilage formation

“…Indeed, mice in which cartilage-specific β1-integrin is conditionally knocked out exhibited dyschondroplasia similar to that of LPA 1 KO mice 33 . Furthermore, topical administration of anti-α5β1-integrin antibody or RGD-containing peptide to the upper limbs of mouse fetuses suppressed chondrocyte proliferation and shortened the upper limbs 34 . Together, these findings indicate that β1-integrin and LPA 1 have closely related roles in the formation of cartilage.…”

Integrins and chondrocyte–matrix interactions in articular cartilage

“…Studies using isolated chick sternal chondrocytes found that blocking α1, α2, or β1 integrins reduced survival and hypertrophic differentiation ( Hirsch et al, 1997 ) while studies in mouse limb organ culture using antibodies to α5β1 found that this integrin also plays a role in chondrocyte differentiation and joint formation ( Garciadiego-Cazares et al, 2004 ). Similar ex vivo experiments used injections into the upper limbs of mouse embryos of antibodies to α5β1 or RGDS peptides to support a role for the α5β1 integrin in endochondral ossification ( Inoue et al, 2014 ).…”