Type IIA procollagen in development of the human intervertebral disc: Regulated expression of the NH2‐propeptide by enzymic processing reveals a unique developmental pathway

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Abstract
Type II collagen can be synthesized in two forms generated by alternative splicing of the precursor mRNA. Type IIA procollagen, which contains a cysteine-rich domain in the NH2-propeptide (exon 2), is produced by precartilage and noncartilage epithelial and mesenchymal cells, and type IIB procollagen, without the cysteine-rich domain, is characteristic of chondrocytes. Mice lacking type II collagen fail to develop intervertebral discs. We have previously shown that the human intervertebral disc and notochord synthesize primarily the type IIA form of procollagen. Therefore, we investigated the distribution of type IIA procollagen during early disc development in humans. By processes of radioactive in situ hybridization and fluorescence immunohistochemistry, we localized mRNA and protein of type IIA procollagen, type I collagen, and type III collagen in fetal intervertebral disc specimens ranging from day 42 (embryonic stage 17) to day 101 (week 14.5) of gestation. Antibodies to the three distinct domains of type IIA procollagen: the NH2-propeptide, the fibrillar domain, and the COOH-propeptide were used. The earliest stage of developing intervertebral disc (42 days, stage 17) was characterized by diffuse synthesis of types I and III collagens in the dense zone (intervertebral area) and synthesis of type IIA procollagen by the chondrocyte progenitor cells surrounding the disc. The notochord cells synthesized and deposited into the notochordal sheath all three fibrillar collagens. By 54 days (stage 22), the developing disc was clearly divided into three regions: 1.) the outer annulus, characterized by synthesis and deposition of types I and III collagens; 2.) the inner annulus, characterized by synthesis and deposition of type IIA collagen containing the NH2-propeptide but devoid of the COOH-propeptide (pN-procollagen); and 3.) the notochord, the cells of which synthesized and deposited of all three fibrillar collagens. In later stages of fetal development (72–101 days), a change in type IIA procollagen processing was observed in the cells of the inner annulus: even though these cells continued to synthesize type IIA procollagen, they deposited into the extracellular matrix (ECM) only the processed fibrillar domain, with the NH2-propeptide removed. This finding indicates that there is a developmentally regulated change in the processing of type IIA procollagen NH2-propeptide in the cells of the inner annulus. This mechanism is in contrast to previously shown developmental regulation of the cysteine-rich domain of the NH2-propeptide by alternative splicing of the precursor mRNA. Although the cells of the inner annulus have been identified as chondrocytes, based on their shape and synthesis of characteristic ECM components, they appear to represent a distinct developmental pathway characterized by their synthesis and differential processing of type IIA procollagen. This developmental pattern may prove important for disc regeneration.

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