Angela Lang

Stimulation of angiogenesis through cathepsin B inactivation of the tissue inhibitors of matrix metalloproteinases.

The tissue inhibitors of matrix metalloproteinases (MMPs), TIMP‐1 and TIMP‐2, are also angiogenesis inhibitors. Cathepsin B and MMPs are found at sites of neovascularization in pathologies such as cancer and osteoarthritis. Treatment of TIMP‐1, TIMP‐2, and of a mixture of both inhibitors from human articular chondrocytes with cathepsin B resulted in their fragmentation, whereby they lost their MMP‐inhibitory and anti‐angiogenic activities. Our data suggest that, besides directly participating in tissue destruction, cathepsin B can be harmful for two further reasons: it raises the activity of the MMPs also in the absence of mechanisms up‐regulating these enzymes, and it stimulates angiogenesis. This is a prerequisite for blood vessel invasion in a variety of pathological situations of which cancer and osteoarthritis are prominent examples.

Cathepsin B in osteoarthritis: zonal variation of enzyme activity in human femoral head cartilage.

OBJECTIVES--To determine the quantitative topographical distribution of cathepsin B in human femoral head cartilage by measuring the zonal variation of enzyme activity in specimens taken from various anatomical regions of normal and osteoarthritic (OA) tissues, and to correlate this parameter with the severity of the OA lesions. METHODS--OA articular cartilage was obtained at surgery for total hip replacement and control cartilage obtained at postmortem. Cylinders of full thickness cartilage with underlying bone were retrieved with a biopsy trephine. Sections of cartilage were produced by cryocutting the tissue as slices parallel to the articular surface and assayed for cathepsin B with a specific, highly sensitive fluorogenic substrate. The severity of the OA lesions was graded according to the histopathological-histochemical method of Mankin. RESULTS--Zonal cathepsin B activity of normal cartilage was uniform and low in all regions of the femoral head. In apparently intact OA cartilage and in severely degraded tissue the zonal distribution and the amounts of enzyme were similar to control values. At sites with active disease, cathepsin B activity was much greater than in controls and its irregular zonal distribution correlated with tissue degeneration, hypercellularity, or cloning of chondrocytes as determined histochemically. Particularly high enzyme levels were observed at sites with regenerating cartilage, where some zonal peaks attained 20-fold activity with respect to controls. CONCLUSION--Cathepsin B may play a role in sustaining the chronicity of OA, not as an initiator, but rather as a perpetuator of the disease and as an antagonist of regeneration.

Alternative messenger RNA splicing and enzyme forms of cathepsin B in human osteoarthritic cartilage and cultured chondrocytes

OBJECTIVE In previous studies, we suggested that cathepsin B, which is present at sites of cartilage remodeling in osteoarthritis (OA), may act as an antagonist of cartilage repair, an enhancer of the action of metalloproteinases, and a mediator of cartilage neovascularization and mineralization. Alternative splicing of cathepsin B pre-messenger RNA (pre-mRNA) and/or mRNA overexpression is a plausible regulatory mechanism. In the present study, we investigated the abundance of cathepsin B transcripts and the properties of cathepsin B protein in normal and OA cartilage, osteophytes, and cultured chondrocytes. METHODS Cathepsin B mRNA splice variants containing the full-length transcript (CB) and the variants lacking either exon 2 (CB[-2]) or lacking exons 2 and 3 (CB[-2,3]) were measured by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot assays and were localized by in situ RT-PCR. Cathepsin B protein was analyzed by electrophoretic, Western blot, and chromatographic methods. RESULTS The relative content of CB, CB(-2), and CB(-2,3) varied considerably in OA cartilage and osteophytes, with less variation in normal cartilage. The mean cathepsin B mRNA level was significantly higher in OA cartilage and osteophytes than in normal cartilage. Normal cultured chondrocytes attained cathepsin B mRNA levels similar to those in OA cartilage. Enzyme overexpression resulted in the secretion of procathepsin B, followed by activation to the proteolytically active form. CONCLUSION The high levels of CB and CB(-2) are consistent with an overproduction of secreted procathepsin B in OA. Up-regulation of chondrocyte cathepsin B, which takes place at both the transcriptional and the translational level, suggests a leading role of the enzyme in the progression of OA.

Cathepsin B as a marker of the dedifferentiated chondrocyte phenotype.

Rabbit articular cartilage does not secrete cathepsin B in organ culture. By established methods for modulating the chondrocyte phenotype in vitro, however, the synthesis, intracellular storage, and secretion of cathepsin B were followed up over a period of two months. With chondrocytes grown in monolayer cultures both the intracellular pool of the enzyme and its secretion were very small initially, but increased progressively to a factor of 110 after eight weeks. The secretion of cathepsin B was strongly depressed after transferring the cells from monolayer to collagen gel cultures. In contrast, collagenase was secreted in almost the same amounts during the whole period in both monolayer and collagen gel cultures. The cells cultured in collagen gels secreted more collagenase than those grown in monolayers. The reversible switch of cathepsin B secretion suggests that this enzyme, unlike collagenase, is a marker of the dedifferentiated chondrocyte phenotype. Cathepsin B was localised within cultured chondrocytes using antibodies raised against rabbit liver cathepsin B and shared with it many catalytic properties. Its Mr, however, was higher (34,000 compared with 27,000) and showed an unusual resistance to denaturation at neutral-alkaline pH, which may confer on this enzyme an important role in the degradation of cartilage matrix.

Cathepsin B secretion by rabbit articular chondrocytes : modulation by cycloheximide and glycosaminoglycans

SummaryRabbit articular chondrocytes in monolayer culture are modulated away from their differentiated state and undergo morphological and biochemical changes. One of the characteristics of the modulated state is an abnormally high production of the cysteine endopeptidase cathepsin B. Addition to chondrocyte cultures of the protein biosynthesis inhibitor, cycloheximide, resulted in a concentration-dependent reduction of cathepsin B secretion, which was fully restored after removal of cycloheximide. Glycosaminoglycans added to the culture medium of modulated chondrocytes partially reduced the rate of secretion of cathepsin B, this effect being dependent on their structure, the degree of sulfation, and concentration. The age of the chondrocytes and the duration of the treatment also influenced this response. The switching off of cathepsin B release was apparently best favored by a high concentration of negatively charged sulfate groups attached to a polymeric glycosaminoglycan chain; this simulates the natural environment of the chondrocytes in articular cartilage.

Effect of interleukin-1β on the production of cathepsin B by rabbit articular chondrocytes

Rabbit articular chondrocytes in monolayer culture were stimulated with human recombinant interleukin‐1β. Under the influence of the cytokine the intracellular pool of the cysteine endopeptidase cathepsin B was increased by a 2‐4‐fold factor, while enzyme secretion was not stimulated at a significant level. Under the same conditions, the secretion of collagenase, measured as an internal control, was stimulated about 6‐fold. The effects of interieukin‐1β were compared to those caused by phenotypic modulation. Chondrocytes modulated by serial subcultures in monolayer secreted more cathepsin B, but less collagenase than differentiated cells (cultured within collagen gels). Thus, interleukin‐1β and phenotypic modulation affected differently two endopeptidases which are relevant in the pathogenesis of osteoarthritis.

Differential expression of mRNAs for endopeptidases in phenotypically modulated (`dedifferentiated') human articular chondrocytes

Human articular chondrocytes modulated away from their original phenotype by serial subcultures in monolayer differentially express mRNAs for endopeptidases. The mRNAs for the cathepsins B and L are extremely low in differentiated cells, but are soon expressed in parallel with the loss of the differentiated state. In contrast, the mRNA for collagenase‐1 is strongly expressed by differentiated chondrocytes and declines rapidly following phenotypic modulation. The mRNA for stromelysin‐1 and the tissue inhibitor of metalloproteinases‐2 is high and does not appreciably change after modulation. Chondrocyte activation induced by alteration of its original phenotype leads to the expression of endopeptidases in a way that markedly differs from that induced by cytokines. The results are relevant to cartilage catabolism in osteoarthritis and suggest a prominent role of fibroblastic metaplasia on the part of the chondrocytes as a mechanism of expressing catabolic endopeptidases.

Erratum to: Stimulation of angiogenesis through cathepsin B inactivation of the tissue inhibitors of matrix metalloproteinases (FEBS 22347)

The mature form of cathepsin B was a¤nity-puri¢ed from human kidney [18] and was free of other enzymes as ascertained by electrophoresis, speci¢city tests with substrates and inhibitors, and N-terminal sequencing. Cathepsin B (containing the pro-form and the single-chain form) was also prepared from conditioned media of dedi¡erentiated human articular chondrocytes by gel chromatography [12] followed by ion exchange chromatography on DEAE-Sephacel (Pharmacia). Human recombinant, nonglycosylated TIMP-1 whose Asn-30 and Asn-78 were mutated to Ala was expressed in CHO K1 cells and puri¢ed on MacroPrep 50Q anion exchange resin (Bio-Rad), Green A Dyematrex (Amicon) and Sephacryl S-200 (Pharmacia). Recombinant human, glycosylated TIMP-1 was expressed in CHO K1 cells and puri¢ed using anti-(human TIMP-1) a¤nity chromatography [19]. Human TIMP-2 was puri¢ed from the medium of uterine cervical ¢broblasts [20]. The incubation solutions in 50 mM sodium acetate bu¡er, 2 mM dithiothreitol, 2 mM EDTA, pH 5.50, contained cathepsin B (5 Wg/ml ¢nal concentration) and recombinant wild-type or non-glycosylated TIMP-1 (¢nal concentrations in di¡erent experiments 100^160 Wg/ml), or the same cathepsin B concentration and TIMP-2 (¢nal concentrations in di¡erent experiments 25^57 Wg/ml). Incubation was performed at 303C, reaction was stopped at time intervals by making the solution 2 mM with iodoacetic acid, and the progressive disappearance of the original protein band followed by SDS-PAGE. After 5 h of incubation the original proteins were completely digested giving a mixture of peptides. Control experiments included the addition of the cathepsin B-speci¢c inactivator CA-074 at a ¢nal concentration of 1 WM. MALDI-TOF spectra of TIMP-1 and TIMP-2 peptides were accumulated using a Voyager Elite instrumentation (PerSeptive Biosystems, Framingham, MA, USA) with K-cyano-4-hydroxy-cinnamic acid (5 mg/ml in 50% acetonitrile and 0.1% tri£uoroacetic acid in water) as the matrix. Samples were analyzed in pulsed extraction re£ectron mode using an acceleration voltage of 20 kV, a pulse delay time of 75 ns, a grid voltage of 55% and a guide voltage of 0.05%. Peptide sequences matching the determined molecular masses were calculated with the software PAWS (Protein Analysis WorkSheet) of ProteoMetrics (http://www.proteometrics.com).