The osteocyte is the terminally differentiated state of the osteogenic mesenchymal progenitor immobilized in the bone matrix. Despite their numerical prominence, little is known about osteocytes and their formation. Osteocytes are physically separated in the bone matrix but seemingly compensate for their seclusion from other cells by maintaining an elaborate network of cell processes through which they interact with other osteocytes and bone-lining cells at the periosteal and endosteal surfaces of the bone. This highly organized architecture suggests that osteocytes make an active contribution to the structure and maintenance of their environment rather than passively submitting to random embedding during bone growth or repair. The most abundant matrix protein in the osteocyte environment is type-I collagen and we demonstrate here that, in the mouse, osteocyte phenotype and the formation of osteocyte processes is highly dependent on continuous cleavage of type-I collagen. This collagenolytic activity and formation of osteocyte processes is dependent on matrix metalloproteinase activity. Specifically, a deficiency of membrane type-1 matrix metalloproteinase leads to disruption of collagen cleavage in osteocytes and ultimately to the loss of formation of osteocyte processes. Osteocytogenesis is thus an active invasive process requiring cleavage of collagen for maintenance of the osteocyte phenotype.

Skeletal tissues develop either by intramembranous ossification, where bone is formed within a soft connective tissue, or by endochondral ossification. The latter proceeds via cartilage anlagen, which through hypertrophy, mineralization, and partial resorption ultimately provides scaffolding for bone formation. Here, we describe a novel and essential mechanism governing remodeling of unmineralized cartilage anlagen into membranous bone, as well as tendons and ligaments. Membrane-type 1 matrix metalloproteinase (MT1-MMP)–dependent dissolution of unmineralized cartilages, coupled with apoptosis of nonhypertrophic chondrocytes, mediates remodeling of these cartilages into other tissues. The MT1-MMP deficiency disrupts this process and uncouples apoptotic demise of chondrocytes and cartilage degradation, resulting in the persistence of “ghost” cartilages with adverse effects on skeletal integrity. Some cells entrapped in these ghost cartilages escape apoptosis, maintain DNA synthesis, and assume phenotypes normally found in the tissues replacing unmineralized cartilages. The coordinated apoptosis and matrix metalloproteinase-directed cartilage dissolution is akin to metamorphosis and may thus represent its evolutionary legacy in mammals.

Nonsteroidal anti-inflammatory drug-activated gene (NAG-1) or GDF15 is a divergent member of the transforming growth factor beta (TGF-β) superfamily and mice expressing hNAG-1/hGDF15 have been shown to be resistant to HFD-induced obesity and inflammation. This study investigated if hNAG-1 increases lifespan in mice and its potential mechanisms. Here we report that female hNAG-1 mice had significantly increased both mean and median life spans in two transgenic lines, with a larger difference in life spans in mice on a HFD than on low fat diet. hNAG-1 mice displayed significantly reduced body and adipose tissue weight, lowered serum IGF-1, insulin and glucose levels, improved insulin sensitivity, and increased oxygen utilization, oxidative metabolism and energy expenditure. Gene expression analysis revealed significant differences in conserved gene pathways that are important regulators of longevity, including IGF-1, p70S6K, and PI3K/Akt signaling cascades. Phosphorylation of major components of IGF-1/mTOR signaling pathway was significantly lower in hNAG-1mice. Collectively, hNAG-1 is an important regulator of mammalian longevity and may act as a survival factor. Our study suggests that hNAG-1 has potential therapeutic uses in obesity-related diseases where life span is frequently shorter.

Matrix metalloproteinase-14 is required for degradation of fibrillar collagen by mesenchymal cells. Here we show that keratinocytes use an alternative plasminogen and matrix metalloproteinase-13-dependent pathway for dissolution of collagen fibrils. Primary keratinocytes displayed an absolute requirement for serum to dissolve collagen. Dissolution of collagen was abolished in plasminogen-depleted serum and could be restored by the exogenous addition of plasminogen. Both plasminogen activator inhibitor-1 and tissue inhibitor of metalloproteinase blocked collagen dissolution, demonstrating the requirement of both plasminogen activation and matrix metalloproteinase activity for degradation. Cell surface plasmin activity was critical for the degradation process as aprotinin, but not ␣ 2 -antiplasmin, prevented collagen dissolution. Keratinocytes with single deficiencies in either urokinase or tissue plasminogen activator retained the ability to dissolve collagen. However, collagen fibril dissolution was abolished in keratinocytes with a combined deficiency in both urokinase and tissue plasminogen activator. Combined, but not single, urokinase and tissue plasminogen activator deficiency also completely blocked the activation of the fibrillar collagenase, matrix metalloproteinase-13, by keratinocytes. The activation of matrix metalloproteinase-13 in normal keratinocytes was prevented by plasminogen activator inhibitor-1 and aprotinin but not by tissue inhibitor of metalloproteinase-1 and -2, suggesting that plasmin activates matrix metalloproteinase-13 directly. We propose that plasminogen activation facilitates keratinocyte-mediated collagen breakdown via the direct activation of matrix metalloproteinase-13 and possibly other fibrillar collagenases.

OBJECTIVEThe NLRP3 inflammasome plays an important regulatory role in obesity-induced insulin resistance. NSAID activated gene-1 (NAG-1) is a divergent member of the TGF-β superfamily. NAG-1 Tg mice are resistant to dietary- and genetic-induced obesity and have improved insulin sensitivity. Our objective was to examine whether NLRP3 inflammasome activity is associated with this observed phenotype in NAG-1 Tg mice.DESIGN AND METHODSKey components of the NLRP3 inflammasome were examined in NAG-1 Tg mice on both regular and high fat diet (HFD) conditions.RESULTSThe expression of caspase-1 and ASC, key components of the NLRP3 inflammasome, is significantly reduced at mRNA and protein levels in white adipose tissue (WAT) of NAG-1 Tg mice. HFD increases the expression of caspase-1 and ASC in WT mice, but their expression is reduced in NAG-1 Tg mice. Furthermore, there is reduced IL-18, IL-1β, and TNF-α expression in the WAT of NAG-1 Tg mice. NAG-1 Tg mice have significantly lower serum leptin and insulin levels and reduced expression of macrophage infiltration markers (F4/80, CD11b, and CD11c) in WAT.CONCLUSIONSOur study suggests the lower NLRP3 inflammasome activity may play a role in the resistance of NAG-1 Tg mice to diet-induced obesity and improved insulin sensitivity.

Background Nonsteroidal anti-inflammatory drug-activated gene (NAG-1), a divergent member of the transforming growth factor beta superfamily, has been implicated in many cellular processes, including inflammation, early bone formation, apoptosis, and tumorigenesis. Recent clinical studies suggests that a C to G single nucleotide polymorphism at position 6 (histidine to aspartic acid substitution, or H6D) of the NAG-1 protein is associated with lower human prostate cancer incidence. The objective of the current study is to investigate the activity of NAG-1 H6D variant in prostate cancer tumorigenesis in vivo. Methods Human prostate cancer DU145 cells expressing the H6D NAG-1 or wild-type NAG-1 were injected subcutaneously into nude mice and tumor growth was monitored. Serum and tumor samples were collected for subsequent analysis. Results The H6D variant was more potent than the wild-type NAG-1 and inhibited tumor growth significantly compared to control mice. Mice with tumors expressing the wild-type NAG-1 have greater reduced both body weight and abdominal fat than mice with H6D variant tumors suggesting different activities of the wild-type NAG-1 and the H6D NAG-1. A significant reduction in adiponectin, leptin and IGF-1 serum levels was observed in the tumor bearing mice with a more profound reduction observed with expression of H6D variant. Cyclin D1 expression was suppressed in the tumors with a dramatic reduction observed in the tumor expressing the H6D variant. Conclusion Our data suggest that the H6D variant of NAG-1 inhibits prostate tumorigenesis by suppressing IGF-1 and Cyclin D1 expression but likely additional mechanisms are operative.

Non-steroidal anti-inflammatory drug (NSAID)-activated gene-1 (NAG-1) and COX-2 are involved in cellular processes such as inflammation, apoptosis, and tumorigenesis. To address the relationship between COX-2 and NAG-1 expression, we investigated the expression of NAG-1 and COX-2 in normal and tumor tissue from human patients, ApcMin/+ mice, and COX-2(−/−) mice. While COX-2 expression is highly induced in tumor tissue, NAG-1 expression is reduced. Furthermore, PGE2 reduces NAG-1 while celebrex induces NAG-1 expression. The results suggest that a possible inverse relationship exists between the expression of NAG-1 and COX-2 in tumor formation of colon tissue.

15‐LOX‐1 and its metabolites are involved in colorectal cancer. Recently, we reported that 15‐LOX‐1 overexpression in HCT‐116 human colorectal cancer cells inhibited cell growth by induction of p53 phosphorylation (4). To determine whether the 15‐LOX‐1 protein or its metabolites are responsible for phosphorylation of p53 in HCT‐116 cells, we used HCT‐116 cells that expressed a mutant 15‐LOX‐1. The mutant 15‐LOX‐1 enzyme, with a substitution of Leu at residue His361, was devoid of enzymatic activity. HCT‐116 cells transiently transfected with either native or mutant 15‐LOX‐1 showed an increase in p53 phosphorylation and an increase in the expression of downstream genes. Thus, 15‐LOX‐1 induces p53 phosphorylation independent of enzymatic activity. Treatment of A549 human lung carcinoma cells with IL‐4 increased the expression of 15‐LOX‐1 and also increased the expression of downstream targets of p53. This confirmed that the activation of p53 was also observed in wild‐type cells expressing physiological 15‐LOX‐1. Immunoprecipitation experiments revealed that 15‐LOX‐1 interacts with, and binds to, DNA‐dependent protein kinase (DNA‐PK). The binding of 15‐LOX‐1 to DNA‐PK caused an approximate 3.0‐fold enhancement in kinase activity, resulting in increased p53 phosphorylation at Ser15. Knockdown of DNA‐PK by small interfering RNA (siRNA) significantly reduced p53 phosphorylation. Furthermore, confocal microscopy demonstrated a colocalization of 15‐LOX and DNA‐PK in the cells. We propose that the 15‐LOX‐1 protein binds to DNA‐PK, increasing its kinase activity and results in downstream activation of the tumor suppressor p53, thus revealing a new mechanism by which lipoxygenases (LOX) may influence the phenotype of tumor cells. Published 2008 Wiley‐Liss, Inc.