Objective. To evaluate the anabolic activity of osteoarthritic chondrocytes in situ by investigating the messenger RNA (mRNA) expression of 3 major cartilage components, type I1 collagen, aggrecan, and link protein.Methods. In situ hybridization experiments and histochemical analysis for proteoglycan content were performed on parallel sections of normal and osteoarthritic (OA) cartilage specimens.Results. Most chondrocytes in the deeper zones of OA cartilage showed an increase in mRNA expression, in particular, of type I1 collagen and to a lesser extent, aggrecan, compared with normal specimens. However, chondrocytes of the upper zone were largely negative for aggrecan or type I1 collagen mRNA. The expression of link protein mRNA was low in normal and OA specimens.Conclusion. These observations suggest that suppression of the anabolic activity of chondrocytes in the upper zones contributes to the metabolic imbalance observed in OA cartilage. Stimulation of matrix anabolism in superficial chondrocytes might be a suitable target for therapeutic intervention.The unique biomechanical properties of articular cartilage are provided by its extracellular matrix, and the failure of cartilage in joint disease is a consequence of the progressive destruction of this matrix. The extracelMar matrix of articular cartilage consists of 2 major Supported by the Deutsche Forschungsgemeinschaft (DFG Grant Ai 20/1-1).T. Aigner, MD, S. I. Vornehm, BsC, K. von der Mark, PhD: University of Erlangen-Nurnberg, Erlangen, Germany; G. Zeiler, MD: Orthopedic Hospital Wichernhaus, Rummelsberg, Schwarzenbruck, Germany; J. Dudhia, PhD, M. T. Bayliss, PhD: Kennedy Institute of Rheumatology, London, UK.Address reprint requests to T. Aigner, MD, Institute of Pathology, University of Erlangen-Nurnberg, Krankenhausstrasse 8-10, D-91054 Erlangen, Germany.Submitted for publication June 17, 1996; accepted in revised form September 3, 1996. components: the network of types 11, IX, and XI collagen (l), which provides the tensile strength and stiffness of articular cartilage, and the large aggregating proteoglycan, aggrecan, which is responsible for the osmotic swelling capacity, and thus the elasticity, of the cartilage matrix (2,3). Aggrecan associates with hyaluronic acid, and this interaction is stabilized by link protein (4), which shares homology with the hyaluronan-binding (Gl) domain of aggrecan. Other cartilage proteoglycans such as decorin, biglycan, fibromodulin, and versican may be present in the cartilage matrix in equimolar amounts, but have other important functions, such as control of collagen fibril diameter and binding of growth factors (2,5,6). Similarly, type VI collagen, which is specifically found in the pericellular matrix of articular cartilage, presumably plays a crucial role in the establishment of the microenvironment of the chondrocytes (7).In normal adult articular cartilage, the turnover of collagen fibrils is very low (8-lo), whereas a relatively high turnover rate for aggrecan has been measured (11,12). In osteoarthritic...

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Collagen type X is a short, network-forming collagen expressed temporally and spatially tightly controlled in hypertrophic chondrocytes during endochondral ossification. Studies on chicken chondrocytes indicate that the regulation of type X collagen gene expression is regulated at the transcriptional level. In this study, we have analyzed the regulatory elements of the human type X collagen (Col10a1) by reporter gene constructs and transient transfections in chondrogenic and nonchondrogenic cells. Four different promoter fragments covering up to 2,864 bp of 5'-flanking sequences, either including or lacking the first intron, were linked to luciferase reporter gene and transfected into 3T3 fibroblasts, HT1080 fibrosarcoma cells, prehypertrophic chondrocytes from the resting zone, hypertrophic chondrocytes, and chondrogenic cell lines. The results indicated the presence of three regulatory elements in the human Col10a1 gene besides the proximal promoter. First, a negative regulatory element located between 2.4 and 2.8 kb upstream of the transcription initiation site was active in all nonchondrogenic cells and in prehypertrophic chondrocytes. Second, a positive, but also non-tissue-specific positive regulatory element was present in the first intron. Third, a cell-type-specific enhancer element active only in hypertrophic chondrocytes was located between -2.4 and -0.9 kb confirming a previous report by Thomas et al. [(1995): Gene 160:291-296]. The enhancing effect, however, was observed only when calcium phosphate was either used for transfection or included in the culture medium after lipofection. These findings demonstrate that the rigid control of human Col10a1 gene expression is achieved by both positive and negative regulatory elements in the gene and provide the basis for the identification of factors binding to those elements.

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Abstract. The effect of parathyroid hormone (PTH) in vivo after secretion by the parathyroid gland is mediated by bioactive fragments of the molecule. To elucidate their possible role in the regulation of cartilage matrix metabolism, the influence of the amino-terminal (NH2-terminal), the central, and the carboxyl-terminal (COOH-terminal) portion of the PTH on collagen gene expression was studied in a serum free cell culture system of fetal bovine and human chondrocytes. Expression of al (I), oil (II), al (III), and otl (X) mRNA was investigated by in situ hybridization and quantified by Northern blot analysis. NH2-terminal and mid-regional fragments containing a core sequence between amino acid residues 28-34 of PTH induced a significant rise in etl (II) mRNA in proliferating chondrocytes. In addition, the COOH-terminal portion (aa 52-84) of the PTH molecule was shown to exert a stimulatory effect on etl(II) and etl (X) mRNA expression in chondrocytes from the hypertrophic zone of bovine epiphyseal cartilage. PTH peptides harboring either the functional domain in the central or COOH-terminal region of PTH can induce cAMP independent Ca 2+ signaling in different subsets of chondrocytes as assessed by microfluorometry of Fura-2/AM loaded cells. These results support the hypothesis that different hormonal effects of PTH on cartilage matrix metabolism are exerted by distinct effector domains and depend on the differentiation stage of the target cell.p ARATHYROID hormone plays a predominant role in the regulation of calcium homeostasis by acting mainly on its target tissues in the renal cortex and bone (15,42). Soon after secretion the parathyroid hormone (PTH) I molecule undergoes rapid proteolysis in the liver resulting in multiple fragments (7). Since most of the calcium regulatory functions could be mapped to the NH2-terminal portion (PTH 1-34) of PTH, it was thought that this fragment contains all structural requirements for biological activity of the entire molecule (43, 52). The other fragments were regarded as inactive metabolites whose functional importance was confined to processing and intracellular transport events during hormone secretion by the cells of the parathyroid gland (PTH 53-84, references 35, 46). However, there is now increasing evidence for a broader spectrum of target tissues, including cartilage (26,33,34), and of hormone action in growth (30,48) ferentiation processes (8, 13) which are mediated by additional functional domains on the mid-regional (23) and COOH-terminal portion (39, 40, 44) of PTH. For example, PTH (53-84) increases alkaline phosphatase activity in osteoblastic cell lines (39) and more recent studies showed that PTH (39-84) and PTH (53-84) dose dependently stimulate the differentiation of osteoclast precursors into osteoclast-like cells (25). Moreover, for two domains of PTH their functional role in the induction of second messenger pathways has been elucidated: the first two NH2-terminal amino acids of PTH are needed for adenylate cyclase stimulation via the "classical" ...