BoneKEy-Osteovision | Commentary
Six (?26) enzymes in search of a substrate (after pirandello): Multicentric osteolysis and decreased function of gelatinase a (MMP-2)
DOI:10.1138/2001034
There are several forms of focal osteolysis in humans that have a genetic basis. Understanding the mechanisms of these disorders could be informative with respect to controls of bone remodeling. For example, familial expansile osteolysis that maps to chromosome 18q21.2-21.3 appears to be explained by a mutation in RANK that results in constitutive activation and excessive ostoclastic bone resorption (). In a provocative paper in the July 28, 2001, issue of Nature Genetics (), Martignetti et al.. describe a form of multicentric osteolysis with striking tarsal and carpal bone resorption, accompanied by severe arthritis, osteoporosis, subcutaneous nodules and a distinctive facies in large, consanguineous Saudi Arabian families (). They localized the gene to 16q12-21 and demonstrated two homoallelic, family-specific, mutations in the gene in the region that encodes one of the matrix metalloproteinases (MMPs), MMP-2, also known as gelatinase A or 72kDa gelatinase. In one family there was a nonsense mutation that predicted the replacement of a tyrosine by a stop codon (Y244X). In another family there was a missense mutation that predicted the substitution of an arginine by a histidine (R101H). In affected members of both families there was no MMP-2 activity detected in serum by gelatin zymography in contrast to its presence in the serum of the unaffected members studied. It is understandable why the individuals with the missense mutation (Y244X) would have no detectable enzyme activity, assayed by gelatin zymography, but it is not clear why those with the R101H mutation would behave similarly, since the zymogen is activated in the process of SDS/PAGE and should be present. The production of unstable, mutant mRNA or protein might account for the observations.
What does MMP-2 do and why should a loss-of-function mutation result in osteolysis as well as other features of the syndrome? Harris and I first described a “gelatinase” in medium conditioned by cultured fragments of synovium from patients with rheumatoid arthritis (). We reasoned that there should be enzymes that could further degrade the large fragments released by collagenase degradation of native collagen and devised an assay to test for such activity using heat-denatured collagen (gelatin) as a substrate in place of native, undenatured collagen used to assay collagenase(s). We could readily separate the “gelatinase” (a zinc-containing proteinase, that required calcium for activity) from the collagenase by gel filtration; the collagenase had a smaller molecular mass. Of course, no one had convincingly demonstrated at the time that there was denatured collagen in vivo that could serve as a substrate for a gelatinase although immunological evidence has since been presented () to show that at least there is “denatured” type II collagen in cartilage. There are now > 20 human MMPs () that have been cloned and characterized including another “gelatinase” (MMP-9, gelatinase B or 92 kDa gelatinase) that is expressed abundantly in osteoclasts. The function in vivo of many of these MMPs is not known. Another shocker is that MMP-2 apparently is a “collagenase” that cleaves collagen at the same limited, specific locus as the others, although the activity can be demonstrated only using recombinant protein or protein purified free of the natural inhibitor, TIMP ().
What is the biological substrate for MMP-2 whose cleavage is critical to normally prevent the excessive osteolysis and other features of the syndrome described? Is it type I or type II collagen, since MMP-2 is a collagenase that can cleave interstitial collagens? Is it type IV collagen, abundant in basement membranes, that must be cleaved for cells to cross tissue boundaries and the walls of blood vessels? Is it type XVIII collagen, whose cleavage releases endostatin (), an inhibitor of angiogenesis? Each of these substrates (and several more) are cleaved in vitro by MMP-2 but it has not been shown whether they are cleaved in vivo only by MMP-2. After all, most of the MMPs were named after they were shown to act on a particular protein substrate, but such proteolysis may not be the biological function of the enzyme. Indeed, the null mutation in MMP-2 engineered in mice resulted in a very mild phenotype, manifested only by decrease in bone length (). T.H. Vu, in her commentary in Nature Genetics () that accompanied the paper by Martignetti et al., speculated that the function of MMP-2 might overlap with that of MMP-14 (MT1-MMP); the engineered loss-of-function mutation of MMP-14 () was associated with skeletal defects that were considered by Vu () to partially resemble those of the patients with the multicentric osteolysis syndrome. Most of the MMP-14 -/- mice die when they are only a few weeks of age, however, and the pathogenesis and nature of the skeletal defects in the mice that survive is not yet clearly established.
Osteolysis implies osteoclastic bone resorption. We must therefore explain how loss of function of MMP-2 leads to increased bone resorption. Interference with function of cathepsin K results in decreased bone resorption () as does inability to cleave helical type I collagen (). Perhaps MMP-2 normally degrades some osteoclast inhibitory protein or releases such a protein from the extracellular matrix. Perhaps MMP-2 acts directly on osteoclasts to shed some critical surface protein, such as a receptor, or acts as an inhibitor on osteoclast precursors. Whatever the mechanism, the observations made in the paper by Martignetti et al..() are important for the field since this is the first demonstration of a spontaneous mutation in an MMP gene resulting in a human disease.
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