The objective of this study is to understand the role of Type IX collagen in the process of endochondral ossification. Type IX collagen is one of the collagen fragments isolated from pepsin digests of collagenous tissues. Type IX collagen is a heterotrimer consisting of three distinct chains, a1(IX), a2(IX), and a3(IX) which form a molecule with long and short collagenous arms. The long collagenous arm associates along the surface of the Type II collagen molecule and a hinge region allows the short collagenous arm to project out into the surrounding matrix. A chondroitin or dermatan chain is attached at the hinge region. During endochondral ossification, chondrocytes hypertrophy and the cartilage matrix is mineralized before it is degraded and replaced by bone. In this study, expression of the a1X) gene is investigated using in situ hybridization. The first model is 18 day-old embryonic chick sternal cartilage, the superior portion of which undergoes endochondral ossification. The transition zone from small to hypertrophic chondrocytes is examined. A positive control probe, a1(II) (pYN509) stained both types of chondrocytes. the negative control probe (pGEM) stained neither. The short-form a1(IX) probe (IN212) stain primarily the hypertrophic chondrocytes. The long-form al(IX) probe (IN231) stained the small chondrocytes. This localization was confirmed with RNA transfer blot analysis following polymerase chain reaction. To determine whether or not this was a unique or common phemonenon, another model was selected. 12 day-old chick embryonic femur was used to perform in situ hybridization with the long and short form probes of Type IX collagen, a negative control, and a Type X collagen probe. Type X collagen had been demonstrated to occur only in hypertrophic chondrocytes. Similar results were obtained with hypertrophic chondrocytes being labeled by the short-form Type IX probe and the Type X probe. The small chondrocytes were labeled by the long-form probe. There was no specific labeling with the negative control probe. This significant staining of the hypertrophic chondrocytes, in two different tissues, with the short-form Type IX collagen probe, indicates that the downstream promoter becomes active in the hypertrophic chondrocytes. The long-form is produced in the small chondrocytes while the upstream promoter is active. These data suggest that the conversion of cartilage to bone may be regulated, in part, by the alteration at the NC4 domain of Type IX collagen due to a promoter switch in the a1(IX) gene.
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