No abstract

A human lumican cDNA sequence was derived by polymerase chain reaction techniques from RNA obtained from intestine, placenta, and articular cartilage. A contiguous sequence of 1729 bases was obtained corresponding to an observed message size of 1.8 kilobases (kb). The cDNA sequence consists of an 80-base pair (bp) 5-untranslated region, a 1014-bp coding sequence, and a 618-bp 3-untranslated region terminating in a 17-bp poly(A) tail. The deduced lumican protein sequence has 338 amino acids, including a putative 18-residue signal peptide. The human lumican gene was shown to be spread over about 7.5 kb of genomic DNA and to be located on chromosome 12q22. The gene consists of 3 exons separated by introns of 2.2 and 3.5 kb. The shorter 5-intron resides 21 bases prior to the translation initiation codon, and the 3-intron resides 152 bases prior to the translation termination codon. The lumican message is expressed at high levels in adult articular chondrocytes but at low levels in the young juvenile. This age-related trend in message level is not, however, common to all tissues in which the lumican gene is expressed. Lumican is present in the extracellular matrix of human articular cartilage at all ages, although its abundance is far greater in the adult. In the adult cartilage lumican exists predominantly in a glycoprotein form lacking keratan sulfate, whereas the juvenile form of the molecule is a proteoglycan.Lumican belongs to the family of relatively small leucine-rich proteoglycans that are present in the extracellular matrix of many tissues. In addition to lumican, the family includes decorin, biglycan, and fibromodulin (1, 2), and each family member has a common structure consisting of a central region of leucine-rich repeats that are flanked at either side by a disulfide-bonded domain. The central leucine-rich region possesses attachment sites for N-linked oligosaccharides, which in fibromodulin and lumican may be modified by sulfation of their polylactosamine units to yield keratan sulfate. Fibromodulin and lumican may therefore also be classed as keratan sulfateproteoglycans (KS-PG).

Splicing variation of the versican message and size heterogeneity of the versican core protein were analyzed in human articular cartilage and intervertebral disc. Splicing variation of the message was studied by PCR analysis to detect the presence or absence of exons 7 and 8, which encode large chondroitin sulfate attachment regions. At all ages in normal cartilage from the third trimester fetus to the mature adult, the presence of the versican isoform possessing exon 8 but not exon 7 ( V , ) could be readily detected. The message isoforms possessing neither exon 7 nor 8 (V,) or both exons 7 and 8 (V,) were only detectable in the fetus, and the isoform possessing only exon 7 (V,) was never detected. In osteoarthritic cartilage and in adult intervertebral disc the versican message pattern was the same as that observed in the normal adult with only the isoform possessing exon 8 being detected. Core protein heterogeneity was studied by immunoblotting following enzymic removal of the glycosaminoglycan chains from the proteoglycan, using an antibody recognizing the globular GI region of versican. All articular cartilage extracts from the fetus to the mature adult contained multiple core protein sizes of greater than 200 kDa. The adult cartilage extracts tended to have an increased proportion of the smaller sized core proteins and osteoarthritic cartilage possessed similar core protein sizes to the normal adult. In contrast, intervertebral disc at all post-natal ages showed a greater range of size heterogeneity with a prominent component of about 50 kDa. The abundance of this component increased if the samples were treated with keratanase prior to analysis, suggesting that the GI region of versican in disc can be substituted with keratan sulfate. The increased presence of versican in the disc relative to articular cartilage may suggest a more pronounced functional role for this proteoglycan, particularly in the nucleus pulposus.

The pathophysiological and biological significance of tissue inhibitor of the metalloproteinases-3 (TIMP-3) gene compared to other TIMPs was investigated in osteoarthritic (OA) human and normal bovine joint tissues. Human OA synovial fibroblasts in culture constitutively expressed TIMP mRNAs. TIMP-3, TIMP-1 and gelatinase A mRNAs were elevated in most human OA synovia over controls, while TIMP-2 expression was similar. TIMP-3 and TIMP-1 mRNAs present in bovine cartilage were inducible by serum factors. Transforming growth factor beta (TGF-beta 1) induced TIMP-3 RNA and protein in human OA and normal bovine chondrocytes. TIMP mRNAs were low (TIMP-1) or undetectable in human fetal chondrocytes but were expressed at all other ages. Thus, the two main joint tissues, synovial membranes and cartilage, express TIMP genes. Due to their matrix protecting activities, the presence of multiple TIMPs may be beneficial for normal joints, while increased TIMP-3 and TIMP-1 expression in arthritic joints may be associated with pathological remodeling.

The chondrocytes in human articular cartilage from subjects of all ages express mRNAs for both of the aggregating proteoglycans aggrecan and versican, although the level of expression of versican mRNA is much lower than that of aggrecan mRNA. Aggrecan shows alternative splicing of the epidermal growth factor (EGF)-like domain within its C-terminal globular region, but there is no evidence for a major difference in situ in the relative expression of this domain with age. At all ages studied from birth to the mature adult, a greater proportion of transcripts lacked the EGF domain. The relative proportions of the two transcripts did not change upon culture and passage of isolated chondrocytes. In contrast, the neighbouring complement regulatory protein (CRP)-like domain was predominantly expressed irrespective of age, but cell culture did result in variation of the splicing of this domain. Versican possesses two EGF-like domains and one CRP-like domain, but at all ages the three domains were predominantly present in all transcripts. This situation persisted upon culture and passage of the chondrocytes. Thus, unlike aggrecan, the versican expressed by human articular cartilage does not appear to undergo alternative splicing of its C-terminal globular region, either in cartilage in situ or in chondrocytes in culture.