Development of a high-throughput screening assay for inhibitors of aggrecan cleavage using luminescent oxygen channeling (AlphaScreen ).

Aggrecan is one of the most important structural components of joint cartilage, and members of the metalloprotease (MMP) and ADAM (a disintegrin and metalloproteinase) protease families have been shown to degrade aggrecan in vivo. A robust assay for aggrecan-degrading activity suitable for high-throughput screening (HTS) was set up and measured using AlphaScreen. In this technology, beads brought into proximity through cross-linking and stimulated with laser light generate a signal through luminescent oxygen tunneling, the outcome of which is a time-resolved fluorescent signal. Specific antibodies to the carbohydrate side chains of aggrecan were harnessed to create a scaffold whereby aggrecan could form a cross-link between donor and acceptor AlphaScreen detector beads. Digested aggrecan, which failed to form a cross-link, generated no signal, so that inhibitors of the digestion could be detected as a restoration of signal. The development of this assay and its validation for HTS are described in this report.

Linked deficiencies in extracellular PP(i) and osteopontin mediate pathologic calcification associated with defective PC-1 and ANK expression.

Osteopontin and PP(i) both suppress hydroxyapatite deposition. Extracellular PP(i) deficiency causes spontaneous hypercalcification, yet unchallenged osteopontin knockout mice have only subtle mineralization abnormalities. We report that extracellular PP(i) deficiency promotes osteopontin deficiency and correction of osteopontin deficiency prevents hypercalcification, suggesting synergistic inhibition of hydroxyapatite deposition. Nucleotide pyrophosphatase phosphodiesterase (NPP) isozymes including PC-1 (NPP1) function partly to generate PP(i), a physiologic calcification inhibitor. PP(i) transport is modulated by the membrane channel protein ANK. Spontaneous articular cartilage calcification, increased vertebral cortical bone formation, and peripheral joint and intervertebral ossific ankylosis are associated with both PC-1 deficiency and expression of truncated ANK in ank/ank mice. To assess how PC-1, ANK, and PP(i) regulate both calcification and cell differentiation, we studied cultured PC-1 -/- and ank/ank mouse calvarial osteoblasts. PC-1 -/- osteoblasts demonstrated approximately 50% depressed NPP activity and markedly lowered extracellular PP(i) associated with hypercalcification. These abnormalities were rescued by transfection of PC-1 but not of the NPP isozyme B10/NPP3. PC-1 -/- and ank/ank cultured osteoblasts demonstrated not only comparable extracellular PP(i) depression and hypercalcification but also marked reduction in expression of osteopontin (OPN), another direct calcification inhibitor. Soluble PC-1 (which corrected extracellular PP(i) and OPN), and OPN itself (> or = 15 pg/ml), corrected hypercalcification by PC-1 -/- and ank/ank osteoblasts. Thus, linked regulatory effects on extracellular PP(i) and OPN expression mediate the ability of PC-1 and ANK to regulate calcification.

One of two chondrocyte-expressed isoforms of cartilage intermediate-layer protein functions as an insulin-like growth factor 1 antagonist.

OBJECTIVE: Aging and osteoarthritic (OA) cartilage commonly demonstrate enhanced expression of the large, transforming growth factor beta (TGFbeta)-inducible glycoprotein cartilage intermediate-layer protein (CILP) as well as enhanced extracellular inorganic pyrophosphate (PPi) that promotes the deposition of calcium pyrophosphate dihydrate crystals. In normal chondrocytes, TGFbeta induces elevated chondrocyte extracellular PPi. Insulin-like growth factor 1 (IGF-1) normally blocks this response and reduces extracellular PPi. However, chondrocyte resistance to IGF-1 is observed in OA and aging. Because CILP was reported to chromatographically fractionate with PPi-generating nucleotide pyrophosphatase phosphodiesterase (NPP) activity, it has been broadly assumed that CILP itself has NPP activity. Our objective was to directly define CILP functions and their relationship to IGF-1 in chondrocytes. METHODS: Using primary cultures of articular chondrocytes from the knee, we defined the function of the previously described CILP (CILP-1) and of a recently described 50.6% identical protein that we designated the CILP-2 isoform. RESULTS: Both CILP isoforms were constitutively expressed by primary cultured articular chondrocytes, but only CILP-1 expression was detectable in cultured knee meniscal cartilage cells. Neither CILP isoform had intrinsic NPP activity. But CILP-1 blocked the ability of IGF-1 to decrease extracellular PPi, an activity specific for the CILP-1 N-terminal domain. The CILP-1 N-terminal domain also suppressed IGF-1-induced (but not TGFbeta-induced) proliferation and sulfated proteoglycan synthesis, and it inhibited ligand-induced IGF-1 receptor autophosphorylation. CONCLUSION: Two CILP isoforms are differentially expressed by chondrocytes. Neither CILP isoform exhibits PPi-generating NPP activity. But, increased expression of CILP-1, via N-terminal domain-mediated inhibitory effects of CILP-1 on chondrocyte IGF-1 responsiveness, could impair chondrocyte growth and matrix repair and indirectly promote PPi supersaturation in aging and OA cartilage.