Cutting edge: the metalloproteinase ADAM17/TNF-alpha-converting enzyme regulates proteolytic shedding of the MHC class I-related chain B protein.

MHC class I-related chain (MIC) A/B are transmembrane proteins expressed in pathological conditions that are ligands for the activating receptor NKG2D found on cytotoxic lymphocytes. Soluble NKG2D ligands are detected in sera of patients suffering from multiple types of cancer where they are associated with reduced levels of receptor expression and compromised function of NK and CTLs. In this study, we report the identification of a metalloproteinase involved in the cleavage process of MIC; inhibition and knockdown of ADAM17/TACE blocks the shedding of these proteins. Strikingly, the recruitment of both enzyme and substrate to detergent-resistant membrane microdomains is crucial for efficient proteolysis. These findings provide a novel insight into the molecular mechanisms of MIC shedding.

The isolated N-terminal domains of TIMP-1 and TIMP-3 are insufficient for ADAM10 inhibition.

ADAM (a disintegrin and metalloproteinase) 10 is a key member of the ADAM family of disintegrin and metalloproteinases which process membrane-associated proteins to soluble forms in a process known as 'shedding'. Among the major targets of ADAM10 are Notch, EphrinA2 and CD44. In many cell-based studies of shedding, the activity of ADAM10 appears to overlap with that of ADAM17, which has a similar active-site topology relative to the other proteolytically active ADAMs. The tissue inhibitors of metalloproteinases, TIMPs, have proved useful in the study of ADAM function, since TIMP-1 inhibits ADAM10, but not ADAM17; however, both enzymes are inhibited by TIMP-3. In the present study, we show that, in comparison with ADAM17 and the MMPs (matrix metalloproteinases), the N-terminal domains of TIMPs alone are insufficient for the inhibition of ADAM10. This knowledge could form the basis for the design of directed inhibitors against different metalloproteinases.

Assessment of gelatinase expression and activity in articular cartilage.

Two methods for the assessment of the expression of gelatinases A and B, MMP-2 and MMP-9, in articular cartilage are described. Immunohistochemical analysis of tissue sections provides information about the precise localization of the enzymes within the tissue, pinpointing the cells that synthesize the proteinases, and zymography of cell/ tissue conditioned culture media allows a semi-quantitative assessment of the gelatinases and their activation status.

MT1-MMP hemopexin domain exchange with MT4-MMP blocks enzyme maturation and trafficking to the plasma membrane in MCF7 cells.

The hemopexin-like domain of membrane-type matrix metalloproteinase-1 (MT1-MMP) enables MT1-MMP to form oligomers that facilitate the activation of pro-matrix metalloproteinase-2 (pro-MMP-2) at the cell surface. To investigate the role of the MT1-MMP hemopexin domain in the trafficking of MT1-MMP to the cell surface we have examined the activity of two MT1-MT4-MMP chimaeras in which the hemopexin domain of MT1-MMP has been replaced with that of human or mouse MT4-MMP. We show that MT1-MMP bearing the hemopexin domain of MT4-MMP was incapable of activating pro-MMP-2 or degrading gelatin in cell based assays. Furthermore, cell surface biotinylation and indirect immunofluorescence show that transiently expressed MT1-MT4-MMP chimaeras failed to reach the plasma membrane and were retained in the endoplasmic reticulum. Functional activity could be restored by replacing the MT4-MMP hemopexin domain with the wild-type MT1-MMP hemopexin domain. Subsequent analysis with an antibody specifically recognising the propeptide of MT1-MMP revealed that the propeptides of the MT1-MT4-MMP chimaeras failed to undergo proper processing. It has previously been suggested that the hemopexin domain of MT4-MMP could exert a regulatory mechanism that prevents MT4-MMP from activating pro-MMP-2. In this report, we demonstrate unambiguously that MT1-MT4-MMP chimaeras do not undergo normal trafficking and are not correctly processed to their fully active forms and, as a consequence, they are unable to activate pro-MMP-2 at the cell surface.

Benefits planning for advanced clinical information systems implementation at Allina hospitals and clinics.

Allina Hospitals and Clinics is implementing an enterprise-wide information system with inpatient and ambulatory clinical documentation and orders, clinical decision support, and revenue cycle applications. Allina has adopted a rigorous approach to planning for and realizing the expected clinical and financial benefits from this investment. Allina's strategies include: Forming a benefits realization team with formal responsibility for analysis, education, facilitation, and measurement; Studying system design to consider requirements for benefits realization; Integrating cultural, organizational and process change plans with system implementation plans; Measuring benefits using a measurement framework that matches organizational reporting, enables multi-level sequential analysis and adjusts for bias in quantifying benefits; Assigning accountability for achieving benefits by matching every benefit with an individual and an operational group; system executives, hospital executives, and department managers are held accountable for benefits within their scope of responsibility, and expected financial benefits are part of their yearly budgets. This article describes Allina's approach for benefits planning, contrasting it with the typical provider's approach to benefits realization. It argues that this approach may greatly increase the likelihood of realizing the value of investments in integrated clinical and business IT

Cellular cholesterol regulates MT1 MMP dependent activation of MMP 2 via MEK-1 in HT1080 fibrosarcoma cells.

Unstimulated human fibrosarcoma cells (HT1080) constitutively secrete matrix metalloproteinase 2 (MMP 2) as a proenzyme requiring proteolytic cleavage by membrane type-1 MMP (MT1 MMP) for activation. Physiological and pharmacological stimuli induce clustering of MT1 MMP/tissue inhibitor of MMP 2 "receptors", promoting binding and activation of MMP 2. We now report that cholesterol depleted HT1080 cells accumulated MT1 MMP on the cell surface and activated MMP 2. A specific inhibitor of mitogen activated protein kinase kinase 1/2 inhibited both MMP 2 activation and extracellular signal-related kinase phosphorylation induced by cholesterol depletion. Our data indicate that the cholesterol content of unstimulated cells is critical for secretion of MMP 2 as an inactive zymogen and control of pericellular proteolysis.

Role of TIMPs (tissue inhibitors of metalloproteinases) in pericellular proteolysis: the specificity is in the detail.

Pericellular proteolysis represents one of the key modes by which the cell can modulate its environment, involving not only turnover of the extracellular matrix but also the regulation of cell membrane proteins, such as growth factors and their receptors. The metzincins are active players in such proteolytic events, and their mode of regulation is therefore of particular interest and importance. The TIMPs (tissue inhibitors of metalloproteinases) are established endogenous inhibitors of the matrix metalloproteinases (MMPs), and some have intriguing abilities to associate with the pericellular environment. It has been shown that TIMP-2 can bind to cell surface MT1-MMP (membrane-type 1 MMP) to act as a 'receptor' for proMMP-2 (progelatinase A), such that the latter can be activated efficiently in a localized fashion. We have examined the key structural features of TIMP-2 that determine this unique function, showing that Tyr36 and Glu192-Asp193 are vital for specific interactions with MT1-MMP and proMMP-2 respectively, and hence activation of proMMP-2. TIMP-3 is sequestered at the cell surface by association with the glycosaminoglycan chains of proteoglycans, especially heparan sulphate, and we have shown that it may play a role in the regulation of some ADAMs (a disintegrin and metalloproteinases), including tumour necrosis factor alpha-converting enzyme (TACE; ADAM17). We have established that key residues in TIMP-3 determine its interaction with TACE. Further studies of the features of TIMP-3 that determine specific binding to both ADAM and glycosaminoglycan are required in order to understand these unique properties.

Cyclic AMP signaling control of action potential firing rate and molecular circadian pacemaking in the suprachiasmatic nucleus.

Circadian pacemaking in suprachiasmatic nucleus (SCN) neurons revolves around transcriptional/posttranslational feedback loops, driven by protein products of "clock" genes. These loops are synchronized and sustained by intercellular signaling, involving vasoactive intestinal peptide (VIP) via its VPAC2 receptor, which positively regulates cAMP synthesis. In turn, SCN cells communicate circadian time to the brain via a daily rhythm in electrophysiological activity. To investigate the mechanisms whereby VIP/VPAC2/cAMP signaling controls SCN molecular and electrical pacemaking, we combined bioluminescent imaging of circadian gene expression and whole-cell electrophysiology in organotypic SCN slices. As a potential direct target of cAMP, we focused on hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels. Mutations of VIP-ergic signaling compromised the SCN molecular pacemaker, diminishing the amplitude and intercellular synchrony of circadian gene expression. These deficits were transiently reversed by elevation of cAMP. Similarly, cellular synchrony in electrical firing rates was lost in SCN slices lacking the VPAC2 receptor for VIP. Whole-cell current-clamp recordings in wild-type (WT) slices revealed voltage responses shaped by the conductance I(h), which is mediated by HCN channel activity. The influence of I(h) on voltage responses showed a modest peak in early circadian day, identifying HCN channels as a putative mediator of cAMP-dependent circadian effects on firing rate. I(h), however, was unaffected by loss of VIP-ergic signaling in VPAC2-null slices, and inhibition of cAMP synthesis had no discernible effect on I(h) but did suppress gene expression and SCN firing rates. Moreover, only sustained but not acute, pharmacological blockade of HCN channels reduced action potential (AP) firing. Thus, our evidence suggests that in the SCN, cAMP-mediated signaling is not a principal regulator of HCN channel function and that HCN is not a determinant of AP firing rate. VIP/cAMP-dependent signaling sustains the SCN molecular oscillator and action potential firing via mechanisms yet to be identified.

The activity of a designer tissue inhibitor of metalloproteinases (TIMP)-1 against native membrane type 1 matrix metalloproteinase (MT1-MMP) in a cell-based environment.

The surface-anchored membrane type 1 matrix metalloproteinase (MT1-MMP) degrades a wide range of extracellular matrix components that includes collagens, laminins, fibronectin and the structural proteoglycan aggrecan. The enzyme modulates cell motility and plays an important role in tumour invasion and proliferation. We have previously designed a variant of tissue inhibitor of metalloproteinase (TIMP)-1 bearing a triple mutation (V4A+P6V+T98L, or N-TIMP-1(mt1)) that forms tight binary complex with the soluble catalytic domain of MT1-MMP [M.H. Lee, M. Rapti, G. Murphy, J. Biol. Chem. 278 (2003) 40224-40230]. Here, we report our latest findings on the cellular potency of this mutant against native MT1-MMP in cell-based environment. We show that N-TIMP-1(mt1) is a highly potent inhibitor against the ectodomain form of MT1-MMP (K(i) 9.53nM) with potential for further development as a therapeutic agent. The mutant is devoid of pro-MMP-2-activating capability but is highly effective in blocking MT1-MMP-mediated FITC-labelled collagen and gelatin film degradation in HTC75 fibrosarcoma and MCF7 breast cancer models. Most encouragingly, N-TIMP-1(mt1) is also effective against CD44 shedding in HTC75 cells and able to prevent tubule formation in human umbilical vascular endothelial cells (HUVEC) in a 3D fibrin gel model. We are interested in the development of the TIMPs as therapeutic agents against MT1-MMP related disorders such as cancers. Our findings here indicate the potential for the design of selective TIMPs with refined specificity and possibility for future therapeutic application.

Quality, quantity and harmony: the DataSHaPER approach to integrating data across bioclinical studies.

BACKGROUND: Vast sample sizes are often essential in the quest to disentangle the complex interplay of the genetic, lifestyle, environmental and social factors that determine the aetiology and progression of chronic diseases. The pooling of information between studies is therefore of central importance to contemporary bioscience. However, there are many technical, ethico-legal and scientific challenges to be overcome if an effective, valid, pooled analysis is to be achieved. Perhaps most critically, any data that are to be analysed in this way must be adequately 'harmonized'. This implies that the collection and recording of information and data must be done in a manner that is sufficiently similar in the different studies to allow valid synthesis to take place. METHODS: This conceptual article describes the origins, purpose and scientific foundations of the DataSHaPER (DataSchema and Harmonization Platform for Epidemiological Research; http://www.datashaper.org), which has been created by a multidisciplinary consortium of experts that was pulled together and coordinated by three international organizations: P³G (Public Population Project in Genomics), PHOEBE (Promoting Harmonization of Epidemiological Biobanks in Europe) and CPT (Canadian Partnership for Tomorrow Project). RESULTS: The DataSHaPER provides a flexible, structured approach to the harmonization and pooling of information between studies. Its two primary components, the 'DataSchema' and 'Harmonization Platforms', together support the preparation of effective data-collection protocols and provide a central reference to facilitate harmonization. The DataSHaPER supports both 'prospective' and 'retrospective' harmonization. CONCLUSION: It is hoped that this article will encourage readers to investigate the project further: the more the research groups and studies are actively involved, the more effective the DataSHaPER programme will ultimately be.

Postnatal development of dendritic synaptic integration in rat neocortical pyramidal neurons.

The dendritic tree of layer 5 (L5) pyramidal neurons spans the neocortical layers, allowing the integration of intra- and extracortical synaptic inputs. Here we investigate the postnatal development of the integrative properties of rat L5 pyramidal neurons using simultaneous whole cell recording from the soma and distal apical dendrite. In young (P9-10) neurons, apical dendritic excitatory synaptic input powerfully drove action potential output by efficiently summating at the axonal site of action potential generation. In contrast, in mature (P25-29) neurons, apical dendritic excitatory input provided little direct depolarization at the site of action potential generation but was integrated locally in the apical dendritic tree leading to the generation of dendritic spikes. Consequently, over the first postnatal month the fraction of action potentials driven by apical dendritic spikes increased dramatically. This developmental remodeling of the integrative operations of L5 pyramidal neurons was controlled by a >10-fold increase in the density of apical dendritic Hyperpolarization-activated cyclic nucleotide (HCN)-gated channels found in cell-attached patches or by immunostaining for the HCN channel isoform HCN1. Thus an age-dependent increase in apical dendritic HCN channel density ensures that L5 pyramidal neurons develop from compact temporal integrators to compartmentalized integrators of basal and apical dendritic synaptic input.

Identification of the extracellular matrix (ECM) binding motifs of tissue inhibitor of metalloproteinases (TIMP)-3 and effective transfer to TIMP-1.

Tissue inhibitor of metalloproteinases (TIMPs) are the endogenous inhibitors of the zinc-dependent endopeptidases of the matrix metalloproteinase families. There are four mammalian TIMPs (TIMP-1 to -4) but only TIMP-3 is sequestered to the extracellular matrix (ECM). The molecular basis for the TIMP-3:ECM association has never been fully investigated until now. In this report, we identify the unique amino acid configuration that constitutes the basis of the ECM binding motif in TIMP-3. By systematically exchanging the subdomains of the TIMPs and exhaustive mutation of TIMP-3, we have identified the surface residues directly responsible for ECM association. Contrary to the accepted view, we have found that TIMP-3 interacts with the ECM via both its N- and C-terminal domains. The amino acids involved in ECM binding are all basic in nature: Lys-26, Lys-27, Lys-30, Lys-76 of the N-terminal domain and Arg-163, Lys-165 of the C-terminal domain. Replacement of these residues with glutamate (E) and glutamine (Q) (K26/27/30/76E + R163/K165Q) resulted in a soluble TIMP-3 devoid of ECM-adhering ability. Using the ECM binding motif derived from TIMP-3, we have also created a TIMP-1 mutant (K26/27/30 + K76 transplant) capable of ECM association. This is the first instance of TIMPs being intentionally rendered soluble or ECM-bound. The ability to prepare TIMPs in soluble or ECM-bound forms also opens new avenues for future TIMP research.

Pathway-specific use-dependent dynamics of excitatory synaptic transmission in rat intracortical circuits.

Information processing in neuronal networks is determined by the use-dependent dynamics of synaptic transmission. Here we characterize the dynamic properties of excitatory synaptic transmission in two major intracortical pathways that target the output neurons of the neocortex, by recording unitary EPSPs from layer 5 pyramidal neurons evoked in response to action potential trains of increasing complexity in presynaptic layer 2/3 or layer 5 pyramidal neurons. We find that layer 2/3 to layer 5 synaptic transmission is dominated by frequency-dependent depression when generated at fixed frequencies of > 10 Hz. Synaptic depression evolved on a spike-by-spike basis in response to action potential trains that possessed a broad range of interspike intervals, but a low mean frequency (10 Hz). Layer 2/3 to layer 2/3 and layer 2/3 to layer 5 synapses were incapable of sustained release during prolonged, complex trains of presynaptic action potential firing (mean frequency, 48 Hz). By contrast, layer 5 to layer 5 synapses operated effectively across a wide range of frequencies, exhibiting increased efficacy at frequencies > 10 Hz. Furthermore, layer 5 to layer 5 synapses sustained release throughout the duration of prolonged, complex spike trains. The use-dependent properties of synaptic transmission could be modulated by pharmacologically changing the probability of release and by induction of long-term depression. The dynamic properties of intracortical excitatory synapses are therefore pathway-specific. We suggest that the synaptic output of layer 5 pyramidal neurons is ideally suited to control the neocortical network across a wide range of frequencies and for sustained periods of time, a behaviour that helps to explain the pivotal role played by layer 5 neurons in the genesis of periods of network 'up' states and epileptiform activity in the neocortex.

Androgens influence expression of matrix proteins and proteolytic factors during cutaneous wound healing.

Excessive proteolytic activity is a feature of chronic wounds such as venous ulcers, in which resolution of the inflammatory response fails and restorative matrix accumulation is delayed as a consequence. The inflammatory actions of native androgens during the healing of acute skin wounds have lately been characterized. We have now investigated the hypothesis that such activities may impact upon the balance between anabolic and catabolic processes during wound healing. We report that wound deposition of both type I collagen and fibronectin is increased in castrated rats compared with control animals. This response is accompanied by early increases and later decreases in overall wound levels of the key collagenolytic enzymes, matrix metalloproteinase (MMP)-1 and MMP-13. Moreover, the activities of MMP-2 and MMP-9, two further enzymes that contribute to collagen digestion during venous ulceration, were significantly decreased in the wounds of castrated rats. Additional analyses provide evidence that androgens directly stimulate dermal fibroblast collagen production, which supports the suggestion that increased wound collagen deposition in androgen-deprived rats results from reduced matrix degradation (as opposed to enhanced matrix protein biosynthesis). Androgen-mediated dysregulation of the parallel processes of collagen deposition and turnover may underscore the delayed healing of cutaneous wounds in elderly male patients and further contribute to the increased incidence of non-healing wounds in this population.

Individual Timp deficiencies differentially impact pro-MMP-2 activation.

Membrane-type matrix metalloproteinases (MT-MMPs) have emerged as key enzymes in tumor cell biology. The importance of MT1-MMP, in particular, is highlighted by its ability to activate pro-MMP-2 at the cell surface through the formation of a trimolecular complex comprised of MT1-MMP/tissue inhibitor of metalloproteinase-2 (TIMP-2)/pro-MMP-2. TIMPs 1-4 are physiological MMP inhibitors with distinct roles in the regulation of pro-MMP-2 processing. Here, we have shown that individual Timp deficiencies differentially affect MMP-2 processing using primary mouse embryonic fibroblasts (MEFs). Timp-3 deficiency accelerated pro-MMP-2 activation in response to both cytochalasin D and concanavalin A. Exogenous TIMP-2 and N-TIMP-3 inhibited this activation, whereas TIMP-3 containing matrix from wild-type MEFs did not rescue the enhanced MMP-2 activation in Timp-3(-/-) cells. Increased processing of MMP-2 did not arise from increased expression of MT1-MMP, MT2-MMP, or MT3-MMP or altered expression of TIMP-2 and MMP-2. To test whether increased MMP-2 processing in Timp-3(-/-) MEFs is dependent on TIMP-2, double deficient Timp-2(-/-)/-3(-/-) MEFs were used. In these double deficient cells, the cleavage of pro-MMP-2 to its intermediate form was substantially increased, but the subsequent cleavage of intermediate-MMP-2 to fully active form, although absent in Timp-2(-/-) MEFs, was detectable with combined Timp-2(-/-)/-3(-/-) deficiency. TIMP-4 associates with MMP-2 and MT1-MMP in a manner similar to TIMP-3, but its deletion had no effect on pro-MMP-2 processing. Thus, TIMP-3 provides an inherent regulation over the kinetics of pro-MMP-2 processing, serving at a level distinct from that of TIMP-2 and TIMP-4.

A new development of matrix metalloproteinase inhibitors: twin hydroxamic acids as potent inhibitors of MMPs.

Starting from the observation that the CbzNH(CH2)2 side chain of the potent MMP-2/MMP-14 inhibitor, benzyl-(3R)-4-(hydroxyamino)-3-[isopropoxy(1,1'-biphenyl-4-yl-sulfonyl)amino]-4-oxobutylcarbamate, (R)-1 lies in a hydrophobic region (S1) exposed to the solvent of the protease active site, we hypothesized that an aminoethylcarboxamido chain structurally related to that of (R)-1 might be an useful tool to bind another linker stretching out from the protein. This would be able to interact either with a enzyme region adjacent to the active site, or with other molecules of matrix metalloproteinases (MMPs), or other proteins of the extracellular matrix (ECM) that may be involved in the enzyme activation. On these basis we describe new dimeric compounds of type 2, twin hydroxamic acids, obtained by the joint of two drug entities of (R)-1 linked in P1 by extendable semirigid linkers. Type 2 compounds are potentially able to undergo more complex inhibitor-enzyme interactions than those occurring with monomeric compounds of type 1, thus influencing positively the potency, selectivity and/or cytotoxicity of the new compounds.

Matrix metalloproteinases in arthritic disease.

The role of matrix metalloproteinases in the degradative events invoked in the cartilage and bone of arthritic joints has long been appreciated and attempts at the development of proteinase inhibitors as potential therapeutic agents have been made. However, the spectrum of these enzymes orchestrating connective tissue turnover and general biology is much larger than anticipated. Biochemical studies of the individual members of the matrix metalloproteinase family are now underway, ultimately leading to a more detailed understanding of the function of their domain structures and to defining their specific role in cellular systems and the way that they are regulated. Coupled with a more comprehensive and detailed study of proteinase expression in different cells of joint tissues during the progress of arthritic diseases, it will be possible for the future development and application of highly specific proteinase inhibitors to be directed at specific key cellular events.