Shou-Ih Hu

Inhibition of interleukin‐1α‐induced cartilage oligomeric matrix protein degradation in bovine articular cartilage by matrix metalloproteinase inhibitors: Potential role for matrix metalloproteinases in the generation of cartilage oligomeric matrix protein fragments in arthritic synovial fluid

OBJECTIVE To determine whether matrix metalloproteinases (MMPs) degrade cartilage oligomeric matrix protein (COMP) to produce fragments similar to those found in synovial fluid (SF) from patients with arthritis. METHODS COMP fragments were generated in vitro by treating (a) bovine articular cartilage with interleukin-1alpha (IL-1alpha), (b) purified bovine COMP with MMPs, and (c) articular cartilage with MMPs. The fragments generated in each case were analyzed by Western blot, using an antibody to the C-terminal heptadecapeptide of COMP. RESULTS IL-1alpha stimulation of cartilage resulted in a fragmentation of COMP, which was inhibited by MMP inhibitors CGS 27023A and BB-94. Isolated, recombinant MMPs rapidly degraded purified COMP, as well as COMP residing in cartilage. Several COMP fragments produced in vitro had similar electrophoretic mobility to those in SF of patients with arthritis. CONCLUSION MMPs may contribute to the COMP fragments found in vivo. Quantitation of MMP-specific fragments may be useful in the evaluation of MMP inhibitors in patients with arthritis.

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. (Journal of Biomolecular Screening 2003:136-148)

MNK2 Inhibits eIF4G Activation Through a Pathway Involving Serine-Arginine–Rich Protein Kinase in Skeletal Muscle

In muscle, the kinase-dependent and -independent functions of MNK2 inhibit the protein translation protein eIF4G under conditions that promote atrophy. Limiting Muscle Mass Skeletal muscle mass changes in response to activity, the availability of nutrients, and pathologic loss of muscle mass are associated with long-term bed rest, cachexia-inducing diseases, and starvation. Hu et al. used overexpression, knockout, and knockdown studies to show that the kinase MNK2 had a negative regulatory role on proteins involved in protein synthesis under atrophy-promoting conditions. This was unexpected because MNK2 is also part of a eukaryotic translation initiation complex and is one of the two isoforms capable of phosphorylating a component of that complex on a site associated with promotion of protein synthesis. The negative regulatory effects on protein synthesis machinery appeared to involve a kinase-independent interaction with mTOR, a master regulator of protein synthesis, and a kinase-dependent regulation of another kinase, SRPK, which had not been previously implicated in regulation of protein synthesis. Skeletal muscle mass is regulated by activity, metabolism, and the availability of nutrients. During muscle atrophy, MNK2 expression increases. We found that MNK2 (mitogen-activated protein kinase–interacting kinase 2), but not MNK1, inhibited proteins involved in promoting protein synthesis, including eukaryotic translation initiation factor 4G (eIF4G) and mammalian target of rapamycin (mTOR). Phosphorylation at serine 1108 (Ser1108) of eIF4G, which is associated with enhanced protein translation, is promoted by insulin-like growth factor 1 and inhibited by rapamycin or starvation, suggesting that phosphorylation of this residue is regulated by mTOR. In cultured myotubes, small interfering RNA (siRNA) knockdown of MNK2 increased eIF4G Ser1108 phosphorylation and overcame rapamycin’s inhibitory effect on this phosphorylation event. Phosphorylation of Ser1108 in eIF4G, in gastrocnemius muscle, was increased in mice lacking MNK2, but not those lacking MNK1, and this increased phosphorylation was maintained in MNK2-null animals under atrophy conditions and upon starvation. Conversely, overexpression of MNK2 decreased eIF4G Ser1108 phosphorylation. An siRNA screen revealed that serine-arginine–rich protein kinases linked increased MNK2 activity to decreased eIF4G phosphorylation. In addition, we found that MNK2 interacted with mTOR and inhibited phosphorylation of the mTOR target, the ribosomal kinase p70S6K (70-kD ribosomal protein S6 kinase), through a mechanism independent of the kinase activity of MNK2. These data indicate that MNK2 plays a unique role, not shared by its closest paralog MNK1, in limiting protein translation through its negative effect on eIF4G Ser1108 phosphorylation and p70S6K activation.

Identification of a splice variant of neutrophil collagenase (MMP-8)

We have identified a splice variant of human neutrophil collagenase (MMP‐8) transcript (MMP‐8alt) that has a 91 bp insertion between codons for amino acid residues 34 and 35 of MMP‐8 cDNA. This splice variant encodes an open reading frame for a 444 residue protein, lacking a secretory signal sequence. Our data suggested that, as opposed to the original MMP‐8, the translation product of MMP‐8alt is not a secreted protein; nevertheless, it is enzymatically active. Further studies aimed at identifying the physiological substrates of MMP‐8alt protein may lead to uncover novel roles it plays in cellular physiology.

A stromelysin assay for the assessment of metalloprotease inhibitors on human aggregated proteoglycan

Human proteoglycan was aggregated to an immobilized hyaluronan solid phase on a 96-well ELISA plate. This complex was then degraded by recombinant human stromelysin. The remaining proteoglycan fragments were detected using a monoclonal antibody probe directed against the chondroitin sulfate (CS) region of the core protein. Stromelysin degraded the aggregate in a time and dose dependent manner as reflected by the loss of the CS epitope. Assay sensitivity was 0.125 U/well with total loss of the CS epitope occurring at 4 U/well.o-phenanthroline (IC50=52 μM) and U24522 (IC50=9 μM) inhibted degradation, while phosphoramidon did not. Serine and cysteine protease inhibitors had no effect. A comparative analysis of this assay with a reference method, substance P assay, gave similar inhibitor profiles. The use of aggregated human proteoglycan (native conformation) as a substrate, may better reflect how stromelysin inhibitors behave in the presence of complex substrates such as cartilage matrix.

Intra-articular injection of stromelysin into rabbit knees as a model to evaluate matrix metalloprotease inhibitors

Mat r ix me ta l lopro teases ( M M P s ) such as s t romelysin , col lagenase and gela t inase are e levated in the synovial fluids o f pa t ien ts wi th arthri t is . The ext racel lu lar ma t r ix o f car t i lage is suscept ible to deg rada t ion by these proteases . A desi rable p r o p e r t y o f a ma t r ix me ta l lop ro tease inh ib i to r ( M M P I s ) is one tha t inhibi ts ma t r ix me ta l lopro teases f rom degrad ing car t i lage in the joint . W e repor t here our f indings of a mode l tha t tests the d u r a t i o n o f ac t ion and oral b ioava i lab i l i ty o f M M P I s to inhibi t M M P s in the synovia l fluid.