Cytokine-inducible SH2 domain containing protein contributes to regulation of adiposity, food intake, and glucose metabolism.

The cytokine-inducible SH2 domain containing protein (CISH) is the founding member of the suppressor of cytokine signaling (SOCS) family of negative feedback regulators and has been shown to be a physiological regulator of signaling in immune cells. This study sought to investigate novel functions for CISH outside of the immune system. Mice deficient in CISH were generated and analyzed using a range of metabolic and other parameters, including in response to a high fat diet and leptin administration. CISH knockout mice possessed decreased body fat and showed resistance to diet-induced obesity. This was associated with reduced food intake, but unaltered energy expenditure and microbiota composition. CISH ablation resulted in reduced basal expression of the orexigenic Agrp gene in the arcuate nucleus (ARC) region of the brain. Cish was basally expressed in the ARC, with evidence of co-expression with the leptin receptor (Lepr) gene in Agrp-positive neurons. CISH-deficient mice also showed enhanced leptin responsiveness, although Cish expression was not itself modulated by leptin. CISH-deficient mice additionally exhibited improved insulin sensitivity on a high-fat diet, but not glucose tolerance despite reduced body weight. These data identify CISH as an important regulator of homeostasis through impacts on appetite control, mediated at least in part by negative regulation of the anorexigenic effects of leptin, and impacts on glucose metabolism.

Correction: ADAMTS5 Is a Critical Regulator of Virus-Specific T Cell Immunity.

[This corrects the article DOI: 10.1371/journal.pbio.1002580.].

ADAMTS5 Is a Critical Regulator of Virus-Specific T Cell Immunity.

The extracellular matrix (ECM) provides physical scaffolding for cellular constituents and initiates biochemical and biomechanical cues that are required for physiological activity of living tissues. The ECM enzyme ADAMTS5, a member of the ADAMTS (A Disintegrin-like and Metalloproteinase with Thrombospondin-1 motifs) protein family, cleaves large proteoglycans such as aggrecan, leading to the destruction of cartilage and osteoarthritis. However, its contribution to viral pathogenesis and immunity is currently undefined. Here, we use a combination of in vitro and in vivo models to show that ADAMTS5 enzymatic activity plays a key role in the development of influenza-specific immunity. Influenza virus infection of Adamts5-/- mice resulted in delayed virus clearance, compromised T cell migration and immunity and accumulation of versican, an ADAMTS5 proteoglycan substrate. Our research emphasises the importance of ADAMTS5 expression in the control of influenza virus infection and highlights the potential for development of ADAMTS5-based therapeutic strategies to reduce morbidity and mortality.

Large and Small Assembly: Combining Functional Macromolecules with Small Peptides to Control the Morphology of Skeletal Muscle Progenitor Cells.

The material properties of natural tissues, such as skeletal muscle, are highly sophisticated and are synthetically challenging to mimic. Using natural biomacromolecules to functionalize self-assembled peptide (SAP) hydrogels has the potential to increase the utility of these materials by more closely reproducing the natural cellular environment. Here, to demonstrate that a conserved co-assembly pathway can retain distinct function, the biocompatible peptide derivative Fmoc-FRGDF was co-assembled with either a sulfated polysaccharide, fucoidan, or the provisional matrix proteoglycan, versican. Our results demonstrate that thermodynamically driven co-assembly with biologically active macromolecules is facile, stable, and does not affect the final assembled nanostructure. Biologically, the incorporation of these functionally distinct molecules had no effect on C2C12 myoblast proliferation and viability but strongly altered their morphology. The surface area of myoblasts cultured on the fucoidan scaffold was reduced at 24 and 72 h post seeding, with a reduction in the formation of multinucleated syncytia. Myoblasts cultured on versican scaffolds were smaller compared to cells grown on the empty vector scaffolds at 24 h but not 72 h post seeding, with multinucleated syncytia formation being unaffected. This work allows programmed and distinct morphological effects of cell behavior, paving the way for further mechanistic studies.

The ADAMTS5 Metzincin Regulates Zebrafish Somite Differentiation.

The ADAMTS5 metzincin, a secreted zinc-dependent metalloproteinase, modulates the extracellular matrix (ECM) during limb morphogenesis and other developmental processes. Here, the role of ADAMTS5 was investigated by knockdown of zebrafish adamts5 during embryogenesis. This revealed impaired Sonic Hedgehog (Shh) signaling during somite patterning and early myogenesis. Notably, synergistic regulation of myod expression by ADAMTS5 and Shh during somite differentiation was observed. These roles were not dependent upon the catalytic activity of ADAMTS5. These data identify a non-enzymatic function for ADAMTS5 in regulating an important cell signaling pathway that impacts on muscle development, with implications for musculoskeletal diseases in which ADAMTS5 and Shh have been associated.

The ADAMTS hyalectanase family: biological insights from diverse species.

The a disintegrin-like and metalloproteinase with thrombospondin type-1 motifs (ADAMTS) family of metzincins are complex secreted proteins that have diverse functions during development. The hyalectanases (ADAMTS1, 4, 5, 8, 9, 15 and 20) are a subset of this family that have enzymatic activity against hyalectan proteoglycans, the processing of which has important implications during development. This review explores the evolution, expression and developmental functions of the ADAMTS family, focusing on the ADAMTS hyalectanases and their substrates in diverse species. This review gives an overview of how the family and their substrates evolved from non-vertebrates to mammals, the expression of the hyalectanases and substrates in different species and their functions during development, and how these functions are conserved across species.

The evolutionary conservation of the A Disintegrin-like and Metalloproteinase domain with Thrombospondin-1 motif metzincins across vertebrate species and their expression in teleost zebrafish.

BACKGROUND: The A Disintegrin-like and Metalloproteinase domain with Thrombospondin-1 motifs (ADAMTS) enzymes comprise 19 mammalian zinc-dependent metalloproteinases (metzincins) with homologues in a wide range of invertebrates. ADAMTS enzymes have a broad range of functions in development and diseases due to their extracellular matrix remodelling activity. Here, we report a detailed characterisation of their evolutionary conservation across vertebrates. RESULTS: Using bioinformatics complemented with de novo sequencing, gene sequences for ADAMTS enzymes were obtained from a variety of organisms. Detailed evolutionary analyses revealed a high level of conservation across vertebrates with evidence of ADAMTS gene expansion during two rounds of whole genome duplication (WGD) in vertebrates, while tandem duplication events and gene loss were also apparent. However, the additional round of teleost-specific WGD did not have a significant effect on ADAMTS gene family members suggesting their conserved roles have remained constant in teleost fish. Quantitative reverse-transcriptase polymerase chain reaction analysis revealed dynamic expression of adamts genes throughout zebrafish embryonic development reflecting the key conserved roles they play in vertebrate embryogenesis. Notably, several adamts mRNAs were maternally expressed with a dramatic increase in mRNA levels coinciding with zygotic expression and organogenesis. Broad adamts mRNA expression was also demonstrated in several adult organs indicating potential roles in adult homeostasis. CONCLUSIONS: Our data highlight the evolution of the ADAMTS gene family through duplication processes across metazoans supplemented by a burst of amplification through vertebrate WGD events. It also strongly posits the zebrafish as a potential model species to further elucidate the function of ADAMTS enzymes during vertebrate development.

Hyaluronan synthesis and myogenesis: a requirement for hyaluronan synthesis during myogenic differentiation independent of pericellular matrix formation.

Exogenous hyaluronan is known to alter muscle precursor cell proliferation, migration, and differentiation, ultimately inhibiting myogenesis in vitro. The aim of the current study was to investigate the role of endogenous hyaluronan synthesis during myogenesis. In quantitative PCR studies, the genes responsible for synthesizing hyaluronan were found to be differentially regulated during muscle growth, repair, and pathology. Although all Has genes (Has1, Has2, and Has3) were differentially regulated in these models, only Has2 gene expression consistently associated with myogenic differentiation. During myogenic differentiation in vitro, Has2 was the most highly expressed of the synthases and increased after induction of differentiation. To test whether this association between Has2 expression and myogenesis relates to a role for Has2 in myoblast differentiation and fusion, C2C12 myoblasts were depleted of Has2 by siRNA and induced to differentiate. Depletion of Has2 inhibited differentiation and caused a loss of cell-associated hyaluronan and the hyaluronan-dependent pericellular matrix. The inhibition of differentiation caused by loss of hyaluronan was confirmed with the hyaluronan synthesis inhibitor 4-methylumbelliferone. In hyaluronan synthesis-blocked cultures, restoration of the pericellular matrix could be achieved through the addition of exogenous hyaluronan and the proteoglycan versican, but this was not sufficient to restore differentiation to control levels. These data indicate that intrinsic hyaluronan synthesis is necessary for myoblasts to differentiate and form syncytial muscle cells, but the hyaluronan-dependent pericellular matrix is not sufficient to support differentiation alone; additional hyaluronan-dependent cell functions that are yet unknown may be required for myogenic differentiation.

Biosynthesis and expression of a disintegrin-like and metalloproteinase domain with thrombospondin-1 repeats-15: a novel versican-cleaving proteoglycanase.

The proteoglycanase clade of the ADAMTS superfamily shows preferred proteolytic activity toward the hyalectan/lectican proteoglycans as follows: aggrecan, brevican, neurocan, and versican. ADAMTS15, a member of this clade, was recently identified as a putative tumor suppressor gene in colorectal and breast cancer. However, its biosynthesis, substrate specificity, and tissue expression are poorly described. Therefore, we undertook a detailed study of this proteinase and its expression. We report propeptide processing of the ADAMTS15 zymogen by furin activity, identifying RAKR(212)↓ as a major furin cleavage site within the prodomain. ADAMTS15 was localized on the cell surface, activated extracellularly, and required propeptide processing before cleaving V1 versican at position (441)E↓A(442). In the mouse embryo, Adamts15 was expressed in the developing heart at E10.5 and E11.5 days post-coitum and in the musculoskeletal system from E13.5 to E15.5 days post-coitum, where it was co-localized with hyaluronan. Adamts15 was also highly expressed in several structures within the adult mouse colon. Our findings show overlapping sites of Adamts15 expression with other members of ADAMTS proteoglycanases during embryonic development, suggesting possible cooperative roles during embryogenesis, consistent with other ADAMTS proteoglycanase combinatorial knock-out mouse models. Collectively, these data suggest a role for ADAMTS15 in a wide range of biological processes that are potentially mediated through the processing of versican.

Versican processing by a disintegrin-like and metalloproteinase domain with thrombospondin-1 repeats proteinases-5 and -15 facilitates myoblast fusion.

Skeletal muscle development and regeneration requires the fusion of myoblasts into multinucleated myotubes. Because the enzymatic proteolysis of a hyaluronan and versican-rich matrix by ADAMTS versicanases is required for developmental morphogenesis, we hypothesized that the clearance of versican may facilitate the fusion of myoblasts during myogenesis. Here, we used transgenic mice and an in vitro model of myoblast fusion, C2C12 cells, to determine a potential role for ADAMTS versicanases. Versican processing was observed during in vivo myogenesis at the time when myoblasts were fusing to form multinucleated myotubes. Relevant ADAMTS genes, chief among them Adamts5 and Adamts15, were expressed both in developing embryonic muscle and differentiating C2C12 cells. Reducing the levels of Adamts5 mRNA in vitro impaired myoblast fusion, which could be rescued with catalytically active but not the inactive forms of ADAMTS5 or ADAMTS15. The addition of inactive ADAMTS5, ADAMTS15, or full-length V1 versican effectively impaired myoblast fusion. Finally, the expansion of a hyaluronan and versican-rich matrix was observed upon reducing the levels of Adamts5 mRNA in myoblasts. These data indicate that these ADAMTS proteinases contribute to the formation of multinucleated myotubes such as is necessary for both skeletal muscle development and during regeneration, by remodeling a versican-rich pericellular matrix of myoblasts. Our study identifies a possible pathway to target for the improvement of myogenesis in a plethora of diseases including cancer cachexia, sarcopenia, and muscular dystrophy.

Altered versican cleavage in ADAMTS5 deficient mice; a novel etiology of myxomatous valve disease.

In fetal valve maturation the mechanisms by which the relatively homogeneous proteoglycan-rich extracellular matrix (ECM) of endocardial cushions is replaced by a specialized and stratified ECM found in mature valves are not understood. Therefore, we reasoned that uncovering proteases critical for 'remodeling' the proteoglycan rich (extracellular matrix) ECM may elucidate novel mechanisms of valve development. We have determined that mice deficient in ADAMTS5, (A Disintegrin-like And Metalloprotease domain with ThromboSpondin-type 1 motifs) which we demonstrated is expressed predominantly by valvular endocardium during cardiac valve maturation, exhibited enlarged valves. ADAMTS5 deficient valves displayed a reduction in cleavage of its substrate versican, a critical cardiac proteoglycan. In vivo reduction of versican, in Adamts5(-/-) mice, achieved through Vcan heterozygosity, substantially rescued the valve anomalies. An increase in BMP2 immunolocalization, Sox9 expression and mesenchymal cell proliferation were observed in Adamts5(-/-) valve mesenchyme and correlated with expansion of the spongiosa (proteoglycan-rich) region in Adamts5(-/-) valve cusps. Furthermore, these data suggest that ECM remodeling via ADAMTS5 is required for endocardial to mesenchymal signaling in late fetal valve development. Although adult Adamts5(-/-) mice are viable they do not recover from developmental valve anomalies and have myxomatous cardiac valves with 100% penetrance. Since the accumulation of proteoglycans is a hallmark of myxomatous valve disease, based on these data we hypothesize that a lack of versican cleavage during fetal valve development may be a potential etiology of adult myxomatous valve disease.

ADAMTS-15 Has a Tumor Suppressor Role in Prostate Cancer.

Extracellular matrix remodeling has emerged as an important factor in many cancers. Proteoglycans, including versican (VCAN), are regulated via cleavage by the proteolytic actions of A Disintegrin-like And Metalloproteinase domain with Thrombospondin-1 motif (ADAMTS) family members. Alterations in the balance between Proteoglycans and ADAMTS enzymes have been proposed to contribute to cancer progression. Here, we analyzed the expression of ADAMTS-15 in human prostate cancer, and investigated the effects of enforced expression in prostate cancer cell lines. ADAMTS-15 was found to be expressed in human prostate cancer biopsies with evidence of co-localization with VCAN and its bioactive cleavage fragment versikine. Enforced expression of ADAMTS-15, but not a catalytically-inactive version, decreased cell proliferation and migration of the 'castrate-resistant' PC3 prostate cancer cell line in vitro, with survival increased. Analysis of 'androgen-responsive' LNCaP prostate cancer cells in vivo in NOD/SCID mice revealed that ADAMTS-15 expression caused slower growing tumors, which resulted in increased survival. This was not observed in castrated mice or with cells expressing catalytically-inactive ADAMTS-15. Collectively, this research identifies the enzymatic function of ADAMTS-15 as having a tumor suppressor role in prostate cancer, possibly in concert with androgens, and that VCAN represents a likely key substrate, highlighting potential new options for the clinic.

Genetic reduction of the extracellular matrix protein versican attenuates inflammatory cell infiltration and improves contractile function in dystrophic mdx diaphragm muscles.

There is a persistent, aberrant accumulation of V0/V1 versican in skeletal muscles from patients with Duchenne muscular dystrophy and in diaphragm muscles from mdx mice. Versican is a provisional matrix protein implicated in fibrosis and inflammation in various disease states, yet its role in the pathogenesis of muscular dystrophy is not known. Here, female mdx and male hdf mice (haploinsufficient for the versican allele) were bred. In the resulting F1 mdx-hdf male pups, V0/V1 versican expression in diaphragm muscles was decreased by 50% compared to mdx littermates at 20-26 weeks of age. In mdx-hdf mice, spontaneous physical activity increased by 17% and there was a concomitant decrease in total energy expenditure and whole-body glucose oxidation. Versican reduction improved the ex vivo strength and endurance of diaphragm muscle strips. These changes in diaphragm contractile properties in mdx-hdf mice were associated with decreased monocyte and macrophage infiltration and a reduction in the proportion of fibres expressing the slow type I myosin heavy chain isoform. Given the high metabolic cost of inflammation in dystrophy, an attenuated inflammatory response may contribute to the effects of versican reduction on whole-body metabolism. Altogether, versican reduction ameliorates the dystrophic pathology of mdx-hdf mice as evidenced by improved diaphragm contractile function and increased physical activity.

Erratum: Treatment of Dystrophic mdx Mice with an ADAMTS-5 Specific Monoclonal Antibody Increases the Ex Vivo Strength of Isolated Fast Twitch Hindlimb Muscles. Biomolecules 2020, 10, 416.

The authors wish to make a change to this published paper [...].

Treatment of Dystrophic mdx Mice with an ADAMTS-5 Specific Monoclonal Antibody Increases the Ex Vivo Strength of Isolated Fast Twitch Hindlimb Muscles.

Aberrant extracellular matrix synthesis and remodeling contributes to muscle degeneration and weakness in Duchenne muscular dystrophy (DMD). ADAMTS-5, a secreted metalloproteinase with catalytic activity against versican, is implicated in myogenesis and inflammation. Here, using the mdx mouse model of DMD, we report increased ADAMTS-5 expression in dystrophic hindlimb muscles, localized to regions of regeneration and inflammation. To investigate the pathophysiological significance of this, 4-week-old mdx mice were treated with an ADAMTS-5 monoclonal antibody (mAb) or IgG2c (IgG) isotype control for 3 weeks. ADAMTS-5 mAb treatment did not reduce versican processing, as protein levels of the cleaved versikine fragment did not differ between hindlimb muscles from ADAMTS-5 mAb or IgG treated mdx mice. Nonetheless, ADAMTS-5 blockade improved ex vivo strength of isolated fast extensordigitorumlongus, but not slow soleus, muscles. The underpinning mechanism may include modulation of regenerative myogenesis, as ADAMTS-5 blockade reduced the number of recently repaired desmin positive myofibers without affecting the number of desmin positive muscle progenitor cells. Treatment with the ADAMTS-5 mAb did not significantly affect makers of muscle damage, inflammation, nor fiber size. Altogether, the positive effects of ADAMTS-5 blockade in dystrophic muscles are fiber-type-specific and independent of versican processing.

Adamts5, the gene encoding a proteoglycan-degrading metalloprotease, is expressed by specific cell lineages during mouse embryonic development and in adult tissues.

The secreted metalloprotease ADAMTS5 is implicated in destruction of the cartilage proteoglycan aggrecan in arthritis, but its physiological functions are unknown. Its expression profile during embryogenesis and in adult tissues is therefore of considerable interest. beta-Galactosidase (beta-gal) histochemistry, enabled by a LacZ cassette inserted in the Adamts5 locus, and validated by in situ hybridization with an Adamts5 cRNA probe and ADAMTS5 immunohistochemistry, was used to profile Adamts5 expression during mouse embryogenesis and in adult mouse tissues. Embryonic expression was scarce prior to 11.5 days of gestation (E11.5) and noted only in the floor plate of the developing brain at E 9.5. After E11.5 there was continued expression in brain, especially in the choroid plexus, peripheral nerves, dorsal root ganglia, cranial nerve ganglia, spinal and cranial nerves, and neural plexuses of the gut. In addition to nerves, developing limbs have Adamts5 expression in skeletal muscle (from E13.5), tendons (from E16.5), and inter-digital mesenchyme of the developing autopod (E13.5-15.5). In adult tissues, there is constitutive Adamts5 expression in arterial smooth muscle cells, mesothelium lining the peritoneal, pericardial and pleural cavities, smooth muscle cells in bronchi and pancreatic ducts, glomerular mesangial cells in the kidney, dorsal root ganglia, and in Schwann cells of the peripheral and autonomic nervous system. Expression of Adamts5 during neuromuscular development and in smooth muscle cells coincides with the broadly distributed proteoglycan versican, an ADAMTS5 substrate. These observations suggest the major contexts in which developmental and physiological roles could be sought for this protease.

The metalloproteinase-proteoglycans ADAMTS7 and ADAMTS12 provide an innate, tendon-specific protective mechanism against heterotopic ossification.

Heterotopic ossification (HO) is a significant clinical problem with incompletely resolved mechanisms. Here, the secreted metalloproteinases ADAMTS7 and ADAMTS12 are shown to comprise a unique proteoglycan class that protects against a tendency toward HO in mouse hindlimb tendons, menisci, and ligaments. Adamts7 and Adamts12 mRNAs were sparsely expressed in murine forelimbs but strongly coexpressed in hindlimb tendons, skeletal muscle, ligaments, and meniscal fibrocartilage. Adamts7-/- Adamts12-/- mice, but not corresponding single-gene mutants, which demonstrated compensatory upregulation of the intact homolog mRNA, developed progressive HO in these tissues after 4 months of age. Adamts7-/- Adamts12-/- tendons had abnormal collagen fibrils, accompanied by reduced levels of the small leucine-rich proteoglycans (SLRPs) biglycan, fibromodulin, and decorin, which regulate collagen fibrillogenesis. Bgn-/0 Fmod-/- mice are known to have a strikingly similar hindlimb HO to that of Adamts7-/- Adamts12-/- mice, implicating fibromodulin and biglycan reduction as a likely mechanism underlying HO in Adamts7-/- Adamts12-/- mice. Interestingly, degenerated human biceps tendons had reduced ADAMTS7 mRNA compared with healthy biceps tendons, which expressed both ADAMTS7 and ADAMTS12. These results suggest that ADAMTS7 and ADAMTS12 drive an innate pathway protective against hindlimb HO in mice and may be essential for human tendon health.

Leptin receptor signaling via Janus kinase 2/Signal transducer and activator of transcription 3 impacts on ovarian cancer cell phenotypes.

Ovarian cancer is a leading cause of cancer mortality in women world-wide. Considerable progress has been made to characterize the different subtypes of ovarian cancer, but specific therapies remain limited and prognosis poor. Cytokine signaling via the interleukin-6 receptor (IL-6R) family and related receptors has been implicated in a number of cancers, including those with an ovarian origin. The leptin receptor (LEPR) is structurally related to these receptors and utilizes similar downstream pathways. LEPR has diverse roles in metabolism, appetite and bone formation with obesity linked to both elevated levels of leptin and increased cancer incidence. This study investigated a potential role for LEPR signaling in ovarian cancer. Leptin stimulation led to increased proliferation, survival and migration of LEPR-expressing ovarian cancer cell lines, with the effects shown to be mediated by the downstream Janus kinase 2/Signal transducer and activator of transcription 3 (JAK2/STAT3) pathway. A significant correlation was identified between high co-expression of leptin and LEPR and decreased patient survival. This study collectively suggests that leptin/LEPR signaling via JAK2/STAT3 has the potential to significantly impact on pathogenesis in a subset of ovarian cancer patients who may benefit from strategies that dampen this pathway.

The extracellular matrix in cancer progression: Role of hyalectan proteoglycans and ADAMTS enzymes.

Remodelling of the extracellular matrix (ECM) has emerged as a key factor in cancer progression. Proteoglycans, including versican and other hyalectans, represent major structural elements of the ECM where they interact with other important molecules, including the glycosaminoglycan hyaluronan and the CD44 cell surface receptor. The hyalectan proteoglycans are regulated through cleavage by the proteolytic actions of A Disintegrin-like And Metalloproteinase domain with Thrombospondin-1 motif (ADAMTS) family members. Alteration in the balance between hyalectan proteoglycans and ADAMTS enzymes has been proposed to be a crucial factor in cancer progression either in a positive or negative manner depending on the context. Further complexity arises due to the formation of bioactive cleavage products, such as versikine, which may also play a role, and non-enzymatic functions for ADAMTS proteins. This research is providing fresh insights into cancer biology and opportunities for the development of new diagnostic and treatment strategies.

Upregulated MT1-MMP/TIMP-2 axis in the TSU-Pr1-B1/B2 model of metastatic progression in transitional cell carcinoma of the bladder.

Muscle invasive transitional cell carcinoma (TCC) of the bladder is associated with a high frequency of metastasis, resulting in poor prognosis for patients presenting with this disease. Models that capture and demonstrate step-wise enhancement of elements of the human metastatic cascade on a similar genetic background are useful research tools. We have utilized the transitional cell carcinoma cell line TSU-Pr1 to develop an in vivo experimental model of bladder TCC metastasis. TSU-Pr1 cells were inoculated into the left cardiac ventricle of SCID mice and the development of bone metastases was monitored using high resolution X-ray. Tumor tissue from a single bone lesion was excised and cultured in vitro to generate the TSU-Pr1-B1 subline. This cycle was repeated with the TSU-Pr1-B1 cells to generate the successive subline TSU-Pr1-B2. DNA profiling and karyotype analysis confirmed the genetic relationship of these three cell lines. In vitro, the growth rate of these cell lines was not significantly different. However, following intracardiac inoculation TSU-Pr1, TSU-Pr1-B1 and TSU-Pr1-B2 exhibited increasing metastatic potential with a concomitant decrease in time to the onset of radiologically detectable metastatic bone lesions. Significant elevations in the levels of mRNA expression of the matrix metalloproteases (MMPs) membrane type 1-MMP (MT1-MMP), MT2-MMP and MMP-9, and their inhibitor, tissue inhibitor of metalloprotease-2 (TIMP-2), across the progressively metastatic cell lines, were detected by quantitative PCR. Given the role of MT1-MMP and TIMP-2 in MMP-2 activation, and the upregulation of MMP-9, these data suggest an important role for matrix remodeling, particularly basement membrane, in this progression. The TSU-Pr1-B1/B2 model holds promise for further identification of important molecules.