Extracellular matrix (ECM) mineralization is a physiological process in bone and a pathological one in soft tissues. The mechanisms determining the spatial restriction of ECM mineralization to bone physiologically are poorly understood. Here we show that a normal extracellular phosphate concentration is required for bone mineralization, while lowering this concentration prevents mineralization of any ECM. However, simply raising extracellular phosphate concentration is not sufficient to induce pathological mineralization, this is because of the presence in all ECMs of pyrophosphate, an inhibitor of mineralization. ECM mineralization occurs only in bone because of the exclusive coexpression in osteoblasts of Type I collagen and Tnap, an enzyme that cleaves pyrophosphate. This dual requirement explains why Tnap ectopic expression in cells producing fibrillar collagen is sufficient to induce pathological mineralization. This study reveals that coexpression in osteoblasts of otherwise broadly expressed genes is necessary and sufficient to induce bone mineralization and provides evidence that pathological mineralization can be prevented by modulating extracellular phosphate concentration.[Keywords: ECM; mineralization; TNAP; pyrophosphate; collagen] Supplemental material is available at http://www.genesdev.org.

Extracellular matrix mineralization (ECMM) is a physiologic process in the skeleton and in teeth and a pathologic one in other organs. The molecular mechanisms controlling ECMM are poorly understood. Inactivation of Matrix gla protein (Mgp) revealed that MGP is an inhibitor of ECMM. The fact that MGP is present in the general circulation raises the question of whether ECMM is regulated locally and/or systemically. Here, we show that restoration of Mgp expression in arteries rescues the arterial mineralization phenotype of Mgp−/− mice, whereas its expression in osteoblasts prevents bone mineralization. In contrast, raising the serum level of MGP does not affect mineralization of any ECM. In vivo mutagenesis experiments show that the anti-ECMM function of MGP requires four amino acids which are γ-carboxylated (gla residues). Surprisingly, another gla protein specific to bone and teeth (osteocalcin) does not display the anti-ECMM function of MGP. These results indicate that ECMM is regulated locally in animals and uncover a striking disparity of function between proteins sharing identical structural motifs.

Large, elastic arteries are composed of cells and a specialized extracellular matrix that provides reversible elasticity and strength. Elastin is the matrix protein responsible for this reversible elasticity that reduces the workload on the heart and dampens pulsatile flow in distal arteries. Here, we summarize the elastin protein biochemistry, self-association behavior, cross-linking process, and multistep elastic fiber assembly that provide large arteries with their unique mechanical properties. We present measures of passive arterial mechanics that depend on elastic fiber amounts and integrity such as the Windkessel effect, structural and material stiffness, and energy storage. We discuss supravalvular aortic stenosis and autosomal dominant cutis laxa-1, which are genetic disorders caused by mutations in the elastin gene. We present mouse models of supravalvular aortic stenosis, autosomal dominant cutis laxa-1, and graded elastin amounts that have been invaluable for understanding the role of elastin in arterial mechanics and cardiovascular disease. We summarize acquired diseases associated with elastic fiber defects, including hypertension and arterial stiffness, diabetes, obesity, atherosclerosis, calcification, and aneurysms and dissections. We mention animal models that have helped delineate the role of elastic fiber defects in these acquired diseases. We briefly summarize challenges and recent advances in generating functional elastic fibers in tissue-engineered arteries. We conclude with suggestions for future research and opportunities for therapeutic intervention in genetic and acquired elastinopathies.

Nidogen 1 is a highly conserved protein in mammals, Drosophila melanogaster, Caenorhabditis elegans, and ascidians and is found in all basement membranes. It has been proposed that nidogen 1 connects the laminin and collagen IV networks, so stabilizing the basement membrane, and integrates other proteins, including perlecan, into the basement membrane. To define the role of nidogen 1 in basement membranes in vivo, we produced a null mutation of the NID-1 gene in embryonic stem cells and used these to derive mouse lines. Homozygous animals produce neither nidogen 1 mRNA nor protein. Surprisingly, they show no overt abnormalities and are fertile, their basement membrane structures appearing normal. Nidogen 2 staining is increased in certain basement membranes, where it is normally only found in scant amounts. This occurs by either redistribution from other extracellular matrices or unmasking of nidogen 2 epitopes, as its production does not appear to be upregulated. The results show that nidogen 1 is not required for basement membrane formation or maintenance.The nidogens form a family of related proteins, which in addition to the original mammalian nidogen, nidogen 1 (16) or entactin 1 (6), also includes a second member, nidogen 2 (14) or entactin 2 (13), and several related species from ascidians (23), Caenorhabditis elegans (12) and Drosophila melanogaster (19).Nidogen 1, the best-described member of this family is, together with perlecan, laminin, and collagen IV, a ubiquitous component of basement membranes (32). First identified from a basement membrane-secreting cell line (4) and the murine EHS tumor (31), nidogen 1 comprises three globular domains, G1 to G3, with G1 and G2 connected by a flexible link and G2 and G3 connected by a rod-like domain (10). When isolated under nondenaturing conditions, nidogen 1 is bound noncovalently to laminin (24) by the G3 domain that has been demonstrated to interact with high affinity with the LE4 module in the short arm of the laminin ␥1 chain (17, 25). As nidogen 1 has also been shown to bind to collagen IV by its G2 domain (3, 10), it has been proposed to be crucial in linking the laminin and collagen IV networks. In vitro nidogen 1 binds to perlecan and fibulins (32) and has therefore been considered to play a key role in the stabilization of the basement membrane. Nidogen 1 is highly susceptible to protease degradation (8,18,28), and its destruction may be the initial step in the breakdown of the basement membrane needed in tissue remodeling (1).Disruption of the laminin-nidogen 1 interaction in organ cultures by use of antibodies against the laminin LE4 domain impaired branching morphogenesis in the kidney or salivary gland and induced a distortion of the basement membrane (9, 11). These effects could be counteracted by epidermal growth factor, which increased the production of nidogen 1 in the mesenchyme (11).Nidogen 2, as described by Kohfeldt et al. (14), conserves all the domains of nidogen 1 and interacts with collagens I and IV as well as perlecan. However, its...