Juergen Mollenhauer

Human Articular Chondrocytes Express Osteogenic Protein-1

This study demonstrates for the first time that human articular chondrocytes express osteogenic protein-1 (OP-1). OP-1 was originally purified from bone matrix and was shown to induce cartilage and bone formation. Both OP-1 protein and message were present in human normal and osteoarthritic (OA) cartilages. OP-1 mRNA was upregulated in OA cartilage compared with normal adult tissues. However, the level of mature OP-1 protein in the same OA tissues was downregulated, whereas the pro-OP-1 remained high. Moreover, these two forms of OP-1 were localized in an inverted manner. Mature OP-1 was primarily detected in the superficial layer, whereas the pro-form was mostly in the deep layer of cartilage. The presence of pro- and mature OP-1 in extracts of normal and OA cartilages was confirmed by Western blotting. These findings imply that articular chondrocytes continue to express and synthesize OP-1 throughout adulthood. The observed patterns of the distribution of pro- and mature OP-1 also suggest differences in the processing of this molecule by normal and OA chondrocytes and by the cells in the superficial and deep layers. Distinct distribution of OP-1 and its potential activation in deep zones and regions of cloning in OA cartilages may provide clues to the potential involvement of endogenous OP-1 in repair mechanisms.

The Effects of Collagen Fragments on the Extracellular Matrix Metabolism of Bovine and Human Chondrocytes

Cartilage matrix degradation generates collagen type II fragments. The objective of this study is to explore the possibility that these collagen fragments may be part of an endogenous metabolic feedback. Initially, collagen fragments were extracted from normal or osteoarthritic cartilage, as part of a matrix fragment preparation. Later, collagen fragments were generated by digestion of bovine collagen type II with bacterial collagenase (col2f). These fragments were added to cultures of isolated chondrocytes (bovine and human) and cartilage explants (human). In a dose-dependent manner, col2f caused inhibition of cell attachment to collagen, inhibition of collagen synthesis, and induction of matrix degradation. In addition, when col2f were added to human cartilage explants, an induction of gelatinase activity was detected in the media. These data sets present first evidence that degradation products of collagen may be directly involved in the regulation of cartilage homeostasis.

Cathepsins B, K, and L Are Regulated by a Defined Collagen Type II Peptide via Activation of Classical Protein Kinase C and p38 MAP Kinase in Articular Chondrocytes

Degradation of the extracellular matrix (ECM) is a prominent feature in osteoarthritis (OA), which is mainly because of the imbalance between anabolic and catabolic processes in chondrocytes resulting in cartilage and bone destruction. Various proteases act in concert to degrade matrix components, e.g. type II collagen, MMPs, ADAMTS, and cathepsins. Protease-generated collagen fragments may foster the destructive process. However, the signaling pathways associated with the action of collagen fragments on chondrocytes have not been clearly defined. The present data demonstrate that the N-terminal telopeptide of collagen type II enhances expression of cathepsins B, K, and L in articular chondrocytes at mRNA, protein, and activity levels, mediated at least in part through extracellular calcium. We also demonstrate that the induction is associated with the activation of protein kinase C and p38 MAP kinase.

N-Telopeptide of type II collagen interacts with annexin V on human chondrocytes

Type II collagen binds to chondrocytes through integrins and annexin V. While the potential integrin binding sites have been identified, it is unclear which domains bind to annexin V. Proteolytic fragments of collagen are known to modulate cell signaling pathways resulting in degradation of articular cartilage; it is unknown whether annexin V binds to the fragments. The focus of our study was to determine the binding of type II collagen and its fragments to chondrocytes using flow cytometry and fluorescence microscopy. The N-telopeptide binds to annexin V, whereas the C-telopeptide and triple helical peptides do not. These data suggest that the binding of the N-telopeptide of type II collagen is through annexin V, whereas binding of the C-telopeptide and the triple helical peptide to the surface of chondrocytes are potentially facilitated through other collagen receptors, such as integrins or cell-associated matrix proteins.

Expression of bioactive bone morphogenetic proteins in the subacromial bursa of patients with chronic degeneration of the rotator cuff

Degeneration of the rotator cuff is often associated with inflammation of the subacromial bursa and focal mineralization of the supraspinatus tendon. Portions of the supraspinatus tendon distant from the insertion site could transform into fibrous cartilage, causing rotator-cuff tears owing to mechanical instability. Indirect evidence is presented to link this pathology to ectopic production and secretion of bioactive bone morphogenetic proteins (BMPs) from sites within the subacromial bursa. Surgically removed specimens of subacromial bursa tissue from patients with chronic tears of the rotator cuff were analyzed by immunohistochemistry and reverse transcription-PCR. Bioactive BMP was detected in bursa extracts by a bioassay based on induction of alkaline phosphatase in the osteogenic/myogenic cell line C2C12. Topical and differential expression of BMP-2/4 and BMP-7 mRNA and protein was found in bursa tissue. The bioassay of C2C12 cells revealed amounts of active BMP high enough to induce osteogenic cell types, and blocking BMP with specific antibodies or soluble BMP receptors Alk-3 and Alk-6 abolished the inductive properties of the extract. Sufficient information was gathered to explain how ectopic expression of BMP might induce tissue transformation into ectopic bone/cartilage and, therefore, promote structural degeneration of the rotator cuff. Early surgical removal of the subacromial bursa might present an option to interrupt disease progression.

Absence of Cell-Surface Annexin V Is Accompanied by Defective Collagen Matrix Binding in the Swarm Rat Chondrosarcoma

Annexin V has been characterized as a major collagen type II binding cell‐surface component of normal chondrocytes and is also called anchorin CII in chondrogenic populations. Herein we present evidence that in vitro cultured Swarm rat chondrosarcoma cells are not capable of binding collagen type II in significant quantities to their surfaces, as compared to normal rat chondrocytes. This finding coincides with a deficiency of annexin V on the surface of these cells. A small quantity of an intracellular polypeptide could be detected which is immunologically cross‐reactive with annexin V but displayed a mobility in SDS‐PAGE of less than 34 kD compared to the Mr 36 kD of intact rat annexin V. By immunohistochemistry the protein could be localized in the cytoplasm of in vitro and in vivo grown tumor cells. By reverse transcription‐polymerase chain reaction and Northern blot analysis, a regular‐sized mRNA for annexin V could be detected in the chondrosarcoma cells that is expressed in only slightly lower quantities than in normal chondrocytes. Taken together, the data suggest a modified processing or turnover for annexin V in the chondrosarcoma excluding it from being a functionally active collagen type II binding protein. The findings support the hypothesis of cell‐surface annexin V as a key component for the formation of the pericellular matrix of chondrocytes. J. Cell. Biochem. 65:131–144.