Changes in type II procollagen isoform expression during chondrogenesis by disruption of an alternative 5′ splice site within Col2a1 exon 2

Abstract

This study describes a new mechanism controlling the production of alternatively spliced isoforms of type II procollagen (Col2a1) in vivo. During chondrogenesis, precursor chondrocytes predominantly produce isoforms containing alternatively spliced exon 2 (type IIA and IID) while Col2a1 mRNA devoid of exon 2 (type IIB) is the major isoform produced by differentiated chondrocytes. We previously identified an additional Col2a1 isoform containing a truncated exon 2 and premature termination codons in exon 6 (type IIC). This transcript is produced by utilization of another 5′ splice site present in exon 2. To determine the role of this IIC splicing event in vivo, we generated transgenic mice containing silent knock-in mutations at the IIC 5′ splice site (Col2a1-mIIC), thereby inhibiting production of IIC transcripts. Heterozygous and homozygous knock-in mice were viable and display no overt skeletal phenotype to date. However, RNA expression profiles revealed that chondrocytes in cartilage from an age range of Col2a1-mIIC mice produced higher levels of IIA and IID mRNAs and decreased levels of IIB mRNAs throughout pre-natal and post-natal development, when compared to chondrocytes from littermate control mice. Immunofluorescence analyses showed a clear increase in expression of embryonic type II collagen protein isoforms (i.e. containing the exon 2-encoded cysteine-rich (CR) protein domain) in cartilage extracellular matrix (ECM). Interestingly, at P14, P28 and P56, expression of embryonic Col2a1 isoforms in Col2a1-mIIC mice persisted in the pericellular domain of the ECM in articular and growth plate cartilage. We also show that persistent expression of the exon 2-encoded CR domain in the ECM of post-natal cartilage tissue may be due, in part, to the embryonic form of type XI collagen (the α3 chain of which is also encoded by the Col2a1 gene). In conclusion, expression of the Col2a1 IIC splice form may have a regulatory function in controlling alternative splicing of exon 2 to generate defined proportions of IIA, IID and IIB procollagen isoforms during cartilage development. Future studies will involve ultrastructural and biomechanical analysis of the collagen ECM to determine the effects of persistent mis-expression of embryonic collagen isoforms in mature cartilage tissue.