Bryan D. Crawford

Regulation of matrix metalloproteinase-2 (MMP-2) activity by phosphorylation.

The regulation of matrix metalloprotein‐ases (MMP) has been studied extensively due to the fundamental roles these zinc‐endopeptidases play in diverse physiological and pathological processes. However, phosphorylation has not previously been considered as a potential modulator of MMP activity. The ubiquitously expressed MMP‐2 contains 29 potential phosphorylation sites. Mass spectrometryreveals that at least five of these sites are phosphorylated in hrMMP‐2 expressed in mammalian cells. Treatment of HT1080 cells with an activator of protein kinase C results in a change in MMP‐2 immunoreactivity on 2D immuno‐blots consistent with phosphorylation, and purified MMP‐2 is phosphorylated by protein kinase C in vitro. Furthermore, MMP‐2 from HT1080 cell‐conditioned medium is immunoreactive with antibodies directed against phosphothreonine and phosphoserine, which suggests that it is phosphorylated. Analysis of MMP‐2 activity by zymography, gelatin dequenching assays, and measurement of kinetic parameters shows that the phosphorylation status of MMP‐2 significantly affects its enzymatic properties. Consistent with this, dephos‐phorylation of MMP‐2 immunoprecipitated from HT1080 conditioned medium with alkaline phospha‐tase significantly increases its activity. We conclude that MMP‐2 is modulated by phosphorylation on multiple sites and that protein kinase C may be a regulator of this protease in vivo.—Sariahmetoglu, M., Crawford, B. D., Leon, H., Sawicka, J., Li, L., Ballermann, B. J., Holmes, C., Berthiaume, L. G., Holt, A., Sawicki, G., Schulz, R. Regulation of matrix metalloproteinase‐2 activity by phosphorylation. FASEB J. 21, 2486–2495 (2007)

Mechanisms of cytosolic targeting of matrix metalloproteinase-2

Matrix metalloproteinase‐2 (MMP‐2) is best understood for its biological actions outside the cell. However, MMP‐2 also localizes to intracellular compartments and the cytosol where it has several substrates, including troponin I (TnI). Despite a growing list of cytosolic substrates, we currently do not know the mechanism(s) that give rise to the equilibrium between intracellular and secreted MMP‐2 moieties. Therefore, we explored how cells achieve the unique distribution of this protease. Our data show that endogenous MMP‐2 targets inefficiently to the endoplasmic reticulum (ER) and shows significant amounts in the cytosol. Transfection of canonical MMP‐2 essentially reproduces this targeting pattern, suggesting it is the quality of the MMP‐2 signal sequence that predominantly determines MMP‐2 targeting. However, we also found that human cardiomyocytes express an MMP‐2 splice variant which entirely lacks the signal sequence. Like the fraction of ER‐excluded, full‐length MMP‐2, this variant MMP‐2 is restricted to the cytosol and specifically enhances TnI cleavage upon hypoxia‐reoxygenation injury in cardiomyocytes. Together, our findings describe for the first time a set of mechanisms that cells utilize to equilibrate MMP‐2 both in the extracellular milieu and intracellular, cytosolic locations. Our results also suggest approaches to specifically investigate the overlooked intracellular biology of MMP‐2. J. Cell. Physiol. 227: 3397–3404, 2012.

NAD+ biosynthesis ameliorates a zebrafish model of muscular dystrophy.

NAD+ improves muscle tissue structure and function in dystrophic zebrafish by increasing basement membrane organization.

The Zebrafish Embryo: A Powerful Model System for Investigating Matrix Remodeling

Extracellular matrix (ECM) remodeling is a process that is crucial to the development of embryos, the growth and metastasis of tumors, and wound healing and homeostasis of tissues in adults. As such, it involves dozens of gene products that are regulated by mechanisms operating at transcriptional and multiple posttranslational levels. This complexity of regulation has made the development of a comprehensive understanding of the biology of ECM remodeling in vivo an unusually challenging task, yet such an understanding would be of profound value to our knowledge of and clinical approaches to the treatment of many cancers. The primary effectors of ECM remodeling are the matrix metalloproteinases (MMPs). Homologs of this gene family have been identified in every metazoan examined. We propose that the zebrafish embryo is an ideal system for the study of the regulation of MMP activity, and we present some progress we have made in the development of this organism as a platform for MMP research. We have identified 25 genes encoding MMPs in the zebrafish genome, and 5 genes encoding their endogenous inhibitors, the tissue inhibitors of MMPs. Based on a phylogenetic analysis, we have identified the most probable homologies of these sequences and found that there are two that are of equivocal identity. We have developed 17 antibodies specific to zebrafish MMPs and have begun characterizing the ontogeny of these molecules. Finally, we have developed two novel assays that allow the detection and characterization of active MMPs in vivo (differential in vivo zymography and activity-based protease profiling). In combination with the array of powerful biochemical, genomic, cell, and molecular biological techniques available to zebrafish researchers already, we feel that these new reagents and techniques make the zebrafish the best model system for the study of MMP regulation currently available.

Matrix metalloproteinases in neural development：a phylogenetically diverse perspective

The matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases originally characterized as secreted proteases responsible for degrading extracellular matrix proteins. Their canonical role in matrix remodelling is of significant importance in neural development and regeneration, but emerging roles for MMPs, especially in signal transduction pathways, are also of obvious importance in a neural context. Misregulation of MMP activity is a hallmark of many neuropathologies, and members of every branch of the MMP family have been implicated in aspects of neural development and disease. However, while extraordinary research efforts have been made to elucidate the molecular mechanisms involving MMPs, methodological constraints and complexities of the research models have impeded progress. Here we discuss the current state of our understanding of the roles of MMPs in neural development using recent examples and advocate a phylogenetically diverse approach to MMP research as a means to both circumvent the challenges associated with specific model organisms, and to provide a broader evolutionary context from which to synthesize an understanding of the underlying biology.

Activity-Based Labeling of Matrix Metalloproteinases in Living Vertebrate Embryos

Extracellular matrix (ECM) remodeling is a physiologically and developmentally essential process mediated by a family of zinc-dependent extracellular proteases called matrix metalloproteinases (MMPs). In addition to complex transcriptional control, MMPs are subject to extensive post-translational regulation. Because of this, classical biochemical, molecular and histological techniques that detect the expression of specific gene products provide useful but limited data regarding the biologically relevant activity of MMPs. Using benzophenone-bearing hydroxamate-based probes that interact with the catalytic zinc ion in MMPs, active proteases can be covalently ‘tagged’ by UV cross-linking. This approach has been successfully used to tag MMP-2 in vitro in tissue culture supernatants, and we show here that this probe tags proteins with mobilities consistent with known MMPs and detectable gelatinolytic activity in homogenates of zebrafish embryos. Furthermore, because of the transparency of the zebrafish embryo, UV-photocroslinking can be accomplished in vivo, and rhodamated benzophenone probe is detected in striking spatial patterns consistent with known distributions of active matrix remodeling in embryos. Finally, in metamorphosing Xenopus tadpoles, this probe can be used to biotinylate active MMP-2 by injecting it and cross-linking it in vivo, allowing the protein to be subsequently extracted and biochemically identified.

Laminin and Matrix metalloproteinase 11 regulate Fibronectin levels in the zebrafish myotendinous junction.

BackgroundRemodeling of the extracellular matrix (ECM) regulates cell adhesion as well as signaling between cells and their microenvironment. Despite the importance of tightly regulated ECM remodeling for normal muscle development and function, mechanisms underlying ECM remodeling in vivo remain elusive. One excellent paradigm in which to study ECM remodeling in vivo is morphogenesis of the myotendinous junction (MTJ) during zebrafish skeletal muscle development. During MTJ development, there are dramatic shifts in the primary components comprising the MTJ matrix. One such shift involves the replacement of Fibronectin (Fn)-rich matrix, which is essential for both somite and early muscle development, with laminin-rich matrix essential for normal function of the myotome. Here, we investigate the mechanism underlying this transition.ResultsWe show that laminin polymerization indirectly promotes Fn downregulation at the MTJ, via a matrix metalloproteinase 11 (Mmp11)-dependent mechanism. Laminin deposition and organization is required for localization of Mmp11 to the MTJ, where Mmp11 is both necessary and sufficient for Fn downregulation in vivo. Furthermore, reduction of residual Mmp11 in laminin mutants promotes a Fn-rich MTJ that partially rescues skeletal muscle architecture.ConclusionsThese results identify a mechanism for Fn downregulation at the MTJ, highlight crosstalk between laminin and Fn, and identify a new in vivo function for Mmp11. Taken together, our data demonstrate a novel signaling pathway mediating Fn downregulation. Our data revealing new regulatory mechanisms that guide ECM remodeling during morphogenesis in vivo may inform pathological conditions in which Fn is dysregulated.

Experimental Dissection of Metalloproteinase Inhibition-Mediated and Toxic Effects of Phenanthroline on Zebrafish Development

Metalloproteinases are zinc-dependent endopeptidases that function as primary effectors of tissue remodeling, cell-signaling, and many other roles. Their regulation is ferociously complex, and is exquisitely sensitive to their molecular milieu, making in vivo studies challenging. Phenanthroline (PhN) is an inexpensive, broad-spectrum inhibitor of metalloproteinases that functions by chelating the catalytic zinc ion, however its use in vivo has been limited due to suspected off-target effects. PhN is very similar in structure to phenanthrene (PhE), a well-studied poly aromatic hydrocarbon (PAH) known to cause toxicity in aquatic animals by activating the aryl hydrocarbon receptor (AhR). We show that zebrafish are more sensitive to PhN than PhE, and that PhN causes a superset of the effects caused by PhE. Morpholino knock-down of the AhR rescues the effects of PhN that are shared with PhE, suggesting these are due to PAH toxicity. The effects of PhN that are not shared with PhE (specifically disruption of neural crest development and angiogenesis) involve processes known to depend on metalloproteinase activity. Furthermore these PhN-specific effects are not rescued by AhR knock-down, suggesting that these are bona fide effects of metalloproteinase inhibition, and that PhN can be used as a broad spectrum metalloproteinase inhibitor for studies with zebrafish in vivo.

Mammalian fibroblast cells show strong preference for laser-generated hybrid amorphous silicon-SiO2 textures.

Background In this study, we investigated a method to produce bioactive hybrid amorphous silicon and silicon oxide patterns using nanosecond laser pulses. Methods Microscale line patterns were made by laser pulses on silicon wafers at different frequencies (25, 70 and 100 kHz), resulting in ablation patterns with frequency-dependent physical and chemical properties. Results Incubating the laser-treated silicon substrates with simulated body fluid demonstrated that the physicochemical properties of the laser-treated samples were stable under these conditions, and favored the deposition of bone-like apatite. More importantly, while NIH 3T3 fibroblasts did colonize the untreated regions of the silicon wafers, they showed a strong preference for the laser-treated regions, and further discriminated between substrates treated with different frequencies. Conclusions Taken together, these data suggest that laser materials processing of silicon-based devices is a promising avenue to pursue in the production of biosensors and other bionic devices.

Zebrafish Xenograft: An Evolutionary Experiment in Tumour Biology

Though the cancer research community has used mouse xenografts for decades more than zebrafish xenografts, zebrafish have much to offer: they are cheap, easy to work with, and the embryonic model is relatively easy to use in high-throughput assays. Zebrafish can be imaged live, allowing us to observe cellular and molecular processes in vivo in real time. Opponents dismiss the zebrafish model due to the evolutionary distance between zebrafish and humans, as compared to mice, but proponents argue for the zebrafish xenograft’s superiority to cell culture systems and its advantages in imaging. This review places the zebrafish xenograft in the context of current views on cancer and gives an overview of how several aspects of this evolutionary disease can be addressed in the zebrafish model. Zebrafish are missing homologs of some human proteins and (of particular interest) several members of the matrix metalloproteinase (MMP) family of proteases, which are known for their importance in tumour biology. This review draws attention to the implicit evolutionary experiment taking place when the molecular ecology of the xenograft host is significantly different than that of the donor.