Type IV collagen α1-chain noncollagenous domain blocks MMP-2 activation both in-vitro and in-vivo.

α1(IV)NC1 inhibits angiogenesis by regulating MAPK activation, this biological function was partly attributed α1(IV)NC1 binding to α1ß1-integrin. However, its potent antiangiogenic activity and the molecular targets of α1(IV)NC1 has not been investigated. In the present study, the regulation of MMP-2 activation by α1(IV)NC1 was evaluated. α1ß1-integrin which is required for inhibition of angiogenesis is not playing a role in cellular invasion and inhibition of MMP-2 activation by α1(IV)NC1. We found that α1(IV)NC1 binds the CBD of MMP-2 and forming a stable complex that prevents activation of MMP-2. The antiangiogenic activity of α1(IV)NC1 is mediated, in part, by this binding activity. In addition, up-regulation of TIMP-2 by α1(IV)NC1 led to saturation of MT1-MMP binding sites, which in turn led to inhibition of MMP-2 activation. In-vivo studies using α1-integrin null-mice treated with higher doses of α1(IV)NC1 showed integrin independent inhibition of tumor growth and active-MMP-2, without affecting MMP-9, MMP-7 and angiostatin.

Enhanced activity of an angiotensin-(1-7) neuropeptidase in glucocorticoid-induced fetal programming.

We previously identified angiotensin converting enzyme (ACE) and an endopeptidase activity that degraded angiotensin-(1-7) [Ang-(1-7)] to Ang-(1-5) and Ang-(1-4), respectively, in the cerebrospinal fluid (CSF) of 6-month old male sheep. The present study undertook a more comprehensive analysis of the CSF peptidase that converts Ang-(1-7) to Ang-(1-4) in control and in utero betamethasone-exposed sheep (BMX). Characterization of the Ang-(1-7) peptidase revealed that the thiol agents 4-aminophenylmercuric acetate (APMA) and p-chloromercuribenzoic acid (PCMB), as well as the metallo-chelators o-phenanthroline and EDTA essentially abolished the enzyme activity. Additional inhibitors for serine, aspartyl, and cysteine proteases, as well as selective inhibitors against the endopeptidases neprilysin, neurolysin, prolyl and thimet oligopeptidases did not attenuate enzymatic activity. Competition studies against the peptidase revealed similar IC50s for Ang-(1-7) (5µM) and Ang II (3µM), but lower values for Ala(1)-Ang-(1-7) and Ang-(2-7) of 1.8 and 2.0µM, respectively. In contrast, bradykinin exhibited a 6-fold higher IC50 (32µM) than Ang-(1-7) while neurotensin was a poor competitor. Mean arterial pressure (78±1 vs. 94±2mmHg, N=4-5, P<0.01) and Ang-(1-7) peptidase activity (14.2±1 vs 32±1.5fmol/min/ml CSF, N=5, P<0.01) were higher in the BMX group, and enzyme activity inversely correlated with Ang-(1-7) content in CSF. Lower Ang-(1-7) expression in brain is linked to baroreflex impairment in hypertension and aging, thus, increased activity of an Ang-(1-7) peptidase may contribute to lower CSF Ang-(1-7) levels, elevated blood pressure and impaired reflex function in this model of fetal programming.

Amyloid-ß-induced matrix metalloproteinase-9 secretion is associated with retinal pigment epithelial barrier disruption.

Local and chronic inflammation induced by amyloid-ß (Aß) plays a central role in the development of age-related macular degeneration. The retina is an immune-privileged site due to local tissue barrier. Yet, the manner by which immune cells pass through this barrier and accumulate in the retina remains unclear. Matrix metalloproteinases (MMPs) induce barrier disruption via proteolysis of epithelial tight junction (TJ) proteins. We hypothesized that Aß-induced MMP secretion causes disruption of epithelial barrier integrity. To test this hypothesis, human adult retinal pigment epithelial (haRPE) cells were exposed to Aß, and the expression of MMP-2 and MMP-9 was detected using gelatin zymography. To demonstrate the key role of MMPs in modulating epithelial barrier structure, the MMP agonist 4-aminophenylmercuric acetate (APMA), an MMP inhibitor (GM6001) and siRNA against MMP-9 were employed for comparison. We found that MMP-9, secreted by Aß- or APMA-stimulated cells, mediated low transepithelial electrical resistance (TER) and high transepithelial permeability by disrupting TJ proteins. However, these alterations were reduced by the MMP inhibitor GM6001 or by silencing of the MMP-9 gene. Our findings suggest that the degradation of TJ proteins such as zonula occludens-1, occludin and F-actin by MMP-9 secreted by Aß-stimulated cells constitutes an important mechanism in the breakdown of the barrier which contributes to chronic inflammation in the retina of age-related macular degeneration.

Effect of phosphoric acid on the degradation of human dentin matrix.

This study determined if dentin proteases are denatured by phosphoric acid (PA) used in etch-and-rinse dentin adhesives. Dentin beams were completely demineralized with EDTA for 30 days. We "acid-etched" experimental groups by exposing the demineralized dentin beams to 1, 10, or 37 mass% PA for 15 sec or 15 min. Control beams were not exposed to PA but were incubated in simulated body fluid for 3 days to assay their total endogenous telopeptidase activity, by their ability to solubilize C-terminal crosslinked telopeptides ICTP and CTX from insoluble dentin collagen. Control beams released 6.1 ± 0.8 ng ICTP and 0.6 ± 0.1 ng CTX/mg dry-wt/3 days. Positive control beams pre-incubated in p-aminophenylmercuric acetate, a compound known to activate proMMPs, released about the same amount of ICTP peptides, but released significantly less CTX. Beams immersed in 1, 10, or 37 mass% PA for 15 sec or 15 min released amounts of ICTP and CTX similar to that released by the controls (p > 0.05). Beams incubated in galardin, an MMP inhibitor, or E-64, a cathepsin inhibitor, blocked most of the release of ICTP and CTX, respectively. It is concluded that PA does not denature endogenous MMP and cathepsin activities of dentin matrices.

Activation of MMP-9 by human lung epithelial cells in response to the cystic fibrosis-associated pathogen Burkholderia cenocepacia reduced wound healing in vitro.

Burkholderia cepacia complex is a group of bacterial pathogens that cause opportunistic infections in cystic fibrosis (CF). The most virulent of these is Burkholderia cenocepacia. Matrix metalloproteinases (MMPs) are upregulated in CF patients. The aim of this work was to examine the role of MMPs in the pathogenesis of B. cepacia complex, which has not been explored to date. Real-time PCR analysis showed that B. cenocepacia infection upregulated MMP-2 and MMP-9 genes in the CF lung cell line CFBE41o- within 1 h, whereas MMP-2, -7, and -9 genes were upregulated in the non-CF lung cell line 16HBE14o-. Conditioned media from both cell lines showed increased MMP-9 activation following B. cenocepacia infection. Conditioned media from B. cenocepacia-infected cells significantly reduced the rate of wound healing in confluent lung epithelia (P < 0.05), in contrast to conditioned media from Pseudomonas aeruginosa-infected cells, which showed predominant MMP-2 activation. Treatment of control conditioned media from both cell lines with the MMP activator 4-aminophenylmercuric acetate (APMA) also resulted in clear activation of MMP-9 and to a much lesser extent MMP-2. APMA treatment of control media also delayed the repair of wound healing in confluent epithelial cells. Furthermore, specific inhibition of MMP-9 in medium from cells exposed to B. cenocepacia completely reversed the delay in wound repair. These data suggest that MMP-9 plays a role in the reduced epithelial repair observed in response to B. cenocepacia infection and that its activation following B. cenocepacia infection contributes to the pathogenesis of this virulent pathogen.

MMPs are less efficient than ADAMTS5 in cleaving aggrecan core protein.

Aggrecan degradation in articular cartilage occurs predominantly through proteolysis and has been attributed to the action of members of the matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) families. Both families of enzymes cleave aggrecan at specific sites within the aggrecan core protein. One cleavage site within the interglobular domain (IGD), between Glu(373-374)Ala and five additional sites in the chondroitin sulfate-2 (CS-2) region of aggrecan were characterized as "aggrecanase" (ADAMTS) cleavage sites, while cleavage between Ser(341-342)Phe within the IGD of bovine aggrecan is attributed to MMP action. The objective of this study was to assess the cleavage efficiency of MMPs relative to ADAMTS and their contribution to aggrecan proteolysis in vitro. The analysis of aggrecan IGD degradation in bovine articular cartilage explants treated with catabolic cytokines over a 19-day period showed that MMP-mediated degradation of aggrecan within the IGD can only be observed following day 12 of culture. This delay is associated with the lack of activation of proMMPs during the first 12 days of culture. Analysis of MMP1, 2, 3, 7, 8, 9, 12, 13 and ADAMTS5 efficiencies at cleaving within the aggrecan IGD and CS-2 region in vitro was carried out by the digestion of bovine aggrecan with the various enzymes and Western blot analysis using aggrecan anti-G1 and anti-G3 antibodies. Of these MMPs, MMP12 was the most efficient at cleaving within the aggrecan IGD. In addition to cleavage in the IGD, MMP, 3, 7, 8 and 12 were also able to degrade the aggrecan CS-2 region. MMP3 and MMP12 were able to degrade aggrecan at the very C-terminus of the CS-2 region, cleaving the Glu(2047-2048)Ala bond which was previously shown to be cleaved by ADAMTS5. However, in comparison to ADAMTS5, MMP3 was about 100 times and 10 times less efficient at cleaving within the aggrecan IGD and CS-2 regions, respectively. Collectively, our results showed that the delayed activation of proMMPs and the relatively low cleavage efficiency of MMPs can explain the minor contribution of these enzymes to aggrecan catabolism in vivo. This study also uncovered a potential role for MMPs in the C-terminal truncation of aggrecan.

Utility of combining MMP-9 enzyme-linked immunosorbent assay and MMP-9 activity assay data to monitor plasma enzyme specific activity.

The aim of this study was to combine matrix metalloproteinase-9 (MMP-9) protein (enzyme-linked immunosorbent assay [ELISA]) and MMP-9 activity (fluorescence resonance energy transfer [FRET] assay) data to generate units of specific activity in endogenous and p-aminophenylmercuric acetate (APMA)-activated lithium heparin plasma. The results indicate that specific activity is constant in APMA-activated plasma (mean value=1359.4 pmol/min/microg) and approximately 12% plasma MMP-9 is endogenously active. Exogenous tissue inhibitor of metalloproteinase-1 (TIMP-1) has a greater inhibitory effect on endogenously active MMP-9 than on APMA-activated MMP-9. In conclusion, specific activity can be used as a tool to monitor MMP-9 inhibition. APMA activation affects natural enzyme inhibition, possibly by chemical modification of the C-terminal portion of the enzyme containing the TIMP-1 binding site.

Methods for studying activation of matrix metalloproteinases.

The degradation of the extracellular matrix during development and in disease is thought to result from the combined action of several proteolytic enzyme systems, including the matrix metalloproteinases (MMPs), serine proteinases, and cysteine proteinases. The majority of the soluble MMPs are synthesized as proenzymes which require extracellular activation in order to gain proteolytic activity and the analysis of their activation mechanism is a prerequisite for understanding MMP-mediated proteolysis.The emphasis of this chapter is the provision of the experimental tools to study MMP activation in vitro and in cellular model systems. Hence, we use the activation of procollagenase-3 (proMMP-13) and progelatinase A (proMMP-2) as examples of the methods used.

Assay of matrix metalloproteinases against matrix substrates.

The assays described allow the activity of members of the matrix metalloproteinase (MMP) family that degrade collagen, gelatin and casein substrates to be measured. The protocols described include the preparation of radiolabeled substrates, methods for the separation of degraded product from undegraded substrate, and methods for the activation of MMPs. The advantages and disadvantages of these methods are discussed in relation to immunoassays that measure the amount of individual MMPs.

Walker 256 cancer cells secrete tissue inhibitor of metalloproteinase-free metalloproteinase-9.

Walker 256 (W256) cancer cells, developed as ascites in rats, in response to endogenous unidentified stimuli, secrete a gelatinase of apparent molecular mass of 94 kDa, immunologically homologous to the zymogen of matrix metalloproteinase-9 (proMMP-9). After treatment with the activating agent 4-aminophenylmercuric acetate (APMA), affinity-purified W256 gelatinase is converted to a final processed form of 66 kDa in a similar fashion to TIMP-free human proMMP-9. It is demonstrated that although being capable of binding TIMP-1, W256 proMMP-9 is secreted from W256 cells in TIMP-free forms (monomers or oligomers). Moreover, using biochemical and immunological methods, it is established that the W256 cells do not express or secrete TIMP-1 protein, although RT-PCR analysis indicated low-level TIMP-1 mRNA expression. W256 cancer cells displayed high metastatic ability in rats that may be attributed in part to secretion of TIMP-free proMMP-9.

Shedding of epidermal growth factor receptor is a regulated process that occurs with overexpression in malignant cells.

Soluble isoforms of the epidermal growth factor receptor (sEGFR) previously have been identified in the conditioned culture media (CCM) of the vulvar adenocarcinoma cell line, A431 and within exosomes of the keratinocyte cell line HaCaT. Here, we report that the extracellular domain (ECD) of EGFR is shed from the cell surface of human carcinoma cell lines that express 7x10(5) receptors/cell or more. We purified this proteolytic isoform of EGFR (PI-sEGFR) from the CCM of MDA-MB-468 breast cancer cells. The amino acid sequence of PI-sEGFR was determined by reverse-phase HPLC nano-electrospray tandem mass spectrometry of peptides generated by trypsin, chymotrypsin or GluC digestion. The PI-sEGFR protein is identical in amino acid sequence to the EGFR ECD. The release of PI-sEGFR from MDA-MB-468 cells is enhanced by phorbol 12-myristate 13-acetate, heat-inactivated fetal bovine serum, pervanadate, and EGFR ligands (i.e., EGF and TGF-alpha). In addition, 4-aminophenylmercuric acetate, an activator of metalloproteases, increased PI-sEGFR levels in the CCM of MDA-MB-468 cells. Inhibitors of metalloproteases decreased the constitutive shedding of EGFR while the PMA-induced shedding was inhibited by metalloprotease inhibitors, by the two serine protease inhibitors leupeptin and 3,4-dichloroisocoumarin (DCI), and by the aspartyl inhibitor pepstatin. These results suggest that PI-sEGFR arises by proteolytic cleavage of EGFR via a mechanism that is regulated by both PKC- and phosphorylation-dependent pathways. Our results further suggest that when proteolytic shedding of EGFR does occur, it is correlated with a highly malignant phenotype.

Generation of novel, secreted epidermal growth factor receptor (EGFR/ErbB1) isoforms via metalloprotease-dependent ectodomain shedding and exosome secretion.

Exosomes are small membrane vesicles derived from intracellular multivescicular bodies (MVBs) that can undergo constitutive and regulated secretion from cells. Exosomes can also secrete soluble proteins through metalloprotease-dependent ectodomain shedding. In this study, we sought to determine whether ErbB1 receptors are present within exosomes isolated from the human keratinocyte cell line, HaCaT, and whether exosome-associated ErbB1 receptors can undergo further proteolytic processing. We show that full-length transmembrane ErbB1 is secreted in HaCaT exosomes. EGF treatment and calcium flux stimulated the release of phosphorylated ErbB1 in exosomes but only ligand-stimulated release was blocked by the ErbB1 kinase inhibitor, AG1478, indicating that ligand-dependent ErbB1 receptor activation can initiate ErbB1 secretion into exosomes. In addition, other immunoreactive but truncated ErbB1 isoforms were detected in exosomes suggestive of additional proteolytic processing. We demonstrate that cellular and exosomal ErbB1 receptors can undergo ectodomain shedding to generate soluble N-terminal ectodomains and membrane-associated C-terminal remnant fragments (CTFs). ErbB1 shedding was activated by calcium flux and the metalloprotease activator APMA (4-aminophenylmercuric acetate) and was blocked by a metalloprotease inhibitor (GM6001). Soluble ErbB1 ectodomains shed into conditioned medium retained the ability to bind exogenous ligand. Our results provide new insights into the proteolysis, trafficking and fate of ErbB1 receptors and suggest that the novel ErbB1 isoforms may have functions distinct from the plasma membrane receptor.

In vitro irradiation of basement membrane enhances the invasiveness of breast cancer cells.

Following removal of the primary breast tumour by conservative surgery, patients may still have additional malignant foci scattered throughout the breast. Radiation treatments are not designed to eliminate all these residual cancer cells. Rather, the radiation dose is calculated to optimise long-term results with minimal complications. In a tumour, cancer cells are surrounded by a basement membrane, which plays an important role in the regulation of gene expression. Using an invasion chamber, we have shown that irradiation before cell plating of a reconstituted basement membrane (Matrigel; Becton Dickinson, Bedford, MA, USA) increased the invasiveness of the breast cancer cells MDA-MB-231. This radiation enhancement of invasion was associated with the upregulation of the pro-invasive gene matrix metalloproteinase (MMP)-2. The expression of membrane type 1 matrix metalloproteinase (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP), which are required to activate the MMP-2, were also increased. Confirming the role of MMP-2 and MT1-MMP, radiation enhancement of cancer cell invasion was prevented by an MMP-2 inhibitor and an anti-MT1-MMP antibody. This study also demonstrated that radiation can potentially enhance the invasion ability by inducing the release of pro-invasive factors stored in the Matrigel. Conversely, no enhancement of invasiveness was observed with the low metastatic cell line MCF-7. This lack of invasiveness correlated with the absence of the MMP-2 activator MT1-MMP in the MCF-7 cells. Radiotherapy is an efficient modality to treat breast cancer which could be further improved by inhibiting the pro-invasive gene upregulated by radiation.

Reduced nonprotein thiols inhibit activation and function of MMP-9: implications for chemoprevention.

Clinical studies demonstrate a positive correlation between the extent of matrix metalloproteinase (MMP) activation and malignant progression of precancerous lesions. Therefore, identification of effective, well-tolerated MMP inhibitors represents a rational chemopreventive strategy. A variety of agents, including proteinases and thiol-oxidizing compounds, activate MMPs by initiating release of the propeptide's cysteine sulfur "blockage" of the MMP active site. Despite the importance of the propeptide's cysteine thiol in preserving MMP latency, limited studies have evaluated the effects of reduced thiols on MMP function. This study investigated the effects of two naturally occurring nonprotein thiols, i.e., glutathione (GSH) and N-acetylcysteine (NAC), on activation, function, and cellular-extracellular matrix interactions of the basement-membrane-degrading gelatinase, MMP-9. Our results reveal that NAC and GSH employ protein S-thiolation to inhibit organomercurial activation of pro-MMP-9. Gelatinase activity assays showed that GSH and NAC significantly inhibited MMP-9 but not MMP-2 function, implying isoform structural specificity. Immunoblot analyses, which suggested GSH interacts with MMP-9's active-site Zn, were corroborated by computational molecular modeling. Cell invasion assays revealed that NAC enhanced endostatin's ability to inhibit human cancer cell invasion. Collectively, these data demonstrate that nonprotein thiols suppress MMP-9 activation and function and introduce the prospect for their use in chemopreventive applications.

Differential MMP-2 activity of ligament cells under mechanical stretch injury: an in vitro study on human ACL and MCL fibroblasts.

Recent studies have revealed that following injuries, ligament tissues such as anterior cruciate ligaments (ACL), release large amounts of matrix metalloproteinases (MMPs). These enzymes have a devastating effect on the healing process of the injured ligaments. Although these enzymes are produced following ligament injuries, because of different healing capacities seen between the medial collateral ligament (MCL) and ACL, we were curious to find if the MMP activity was expressed and modulated differently in these tissues. For this purpose ACL and MCL fibroblasts were seeded on equi-biaxial stretch chambers and were stretched in different levels. The stretched cells were assayed using Zymography, Western Blot and global MMP activity assays. The results showed that within 72 h after injurious stretch, production of 72 kD pro-MMP-2 increased in both ACL and MCL. However, the ACL fibroblasts generated significantly more pro-MMP-2 than the MCL fibroblasts. Furthermore we found in ACL pro-MMP-2 was converted more into active form. With 4-aminophenyl mercuric acetate (APMA) treatment, large amounts of pro-MMP-2 were converted into active form in both. This indicates that there is no significant difference between ACL and MCL fibroblasts in post-translational modification of MMP-2. The fluorescent MMP activity assays revealed that the MMP family activities were higher in the injured ACL fibroblasts than the MCL. Since the MMPs are critically involved in extracellular matrix (ECM) turnover, these findings may explain one of the reasons why the injured ACL hardly repairs. The higher levels of active MMP-2 seen in the ACL injuries may disrupt the delicate balance of ECM remodeling process. These results suggest that the generation and modulation of MMP-2 may be directly involved in the different responses seen in ACL and MCL injuries.

The organomercurial 4-aminophenylmercuric acetate, independent of matrix metalloproteinases, induces dose-dependent activation/inhibition of platelet aggregation.

Matrix metalloproteinases (MMPs) play an important role in many biological and pathological processes including tissue remodeling, wound healing, inflammation, atherosclerosis, and cancer. Numerous publications have supported the concept that activated MMP-2 enhances agonist-induced platelet aggregation and activated MMP-9 inhibits platelet aggregation. In this study, we demonstrated that the organomercurial compound, 4-aminophenyl mercuric acetate (APMA), which is routinely employed to activate latent MMPs at a concentration of 1000 microM, induces platelet aggregation at low concentration (5 microM) and inhibits agonist-induced platelet aggregation at concentrations >or= 50 microM. Activated MMP-2, MMP-1, and MMP-9, following removal of APMA by ultrafiltration through an anisotropic membrane, exert no independent effect on platelet aggregation. Acetylsalicylic acid and BAPTA inhibited APMA-induced platelet aggregation indicating that the APMA mediated pathway of platelet activation is dependent upon thromboxane and calcium signaling. Zinc chelation with 1,10-phenanthroline, which inhibits zinc-dependent proteins including metalloproteinases, also abrogated platelet functional responses to APMA. Additional studies will be required to clarify the mechanism of the biphasic effect of APMA on platelet aggregation.

ADAM10 mediates ectodomain shedding of the betacellulin precursor activated by p-aminophenylmercuric acetate and extracellular calcium influx.

Betacellulin belongs to the family of epidermal growth factor-like growth factors that are expressed as transmembrane precursors and undergo proteolytic ectodomain shedding to release a soluble mature growth factor. In this study, we investigated the ectodomain shedding of the betacellulin precursor (pro-BTC) in conditionally immortalized wild-type (WT) and ADAM-deficient cell lines. Sequential ectodomain cleavage of the predominant cell-surface 40-kDa form of pro-BTC generated a major (26-28 kDa) and two minor (20 and 15 kDa) soluble forms and a cellular remnant lacking the ectodomain (12 kDa). Pro-BTC shedding was activated by calcium ionophore (A23187) and by the metalloprotease activator p-aminophenylmercuric acetate (APMA), but not by phorbol esters. Culturing cells in calcium-free medium or with the protein kinase Cdelta inhibitor rottlerin, but not with broad-based protein kinase C inhibitors, blocked A23187-activated pro-BTC shedding. These same treatments were without effect for constitutive and APMA-induced cleavage events. All pro-BTC shedding was blocked by treatment with a broad-spectrum metalloprotease inhibitor (GM6001). In addition, constitutive and activated pro-BTC shedding was differentially blocked by TIMP-1 or TIMP-3, but was insensitive to treatment with TIMP-2. Pro-BTC shedding was functional in cells from ADAM17- and ADAM9-deficient mice and in cells overexpressing WT or catalytically inactive ADAM17. In contrast, overexpression of WT ADAM10 enhanced constitutive and activated shedding of pro-BTC, whereas overexpression of catalytically inactive ADAM10 reduced shedding. These results demonstrate, for the first time, activated pro-BTC shedding in response to extracellular calcium influx and APMA and provide evidence that ADAM10 mediates constitutive and activated pro-BTC shedding.

Assays of proteoglycan and collagen degradation in cultures of rabbit cartilage explants.

Cultures of cartilage explants have long been used to study the effects of modulators of extracellular matrix degradation. We present a simple and rapid assay system, based on culture of rabbit cartilage explants, which permits study of the effects of protease inhibitors on proteoglycan degradation (caused by either aggrecanases or matrix metalloproteinases [MMPs]), and on collagen degradation. The assay is based on the ability of interleukin-1 to stimulate both aggrecanase activity and synthesis of inactive MMPs, which are then activated by p-aminophenylmercuric acetate for the study of MMP-mediated proteoglycan degradation or by plasmin for the study of collagen degradation. Proteoglycan degradation is quantified as percent release of radioactivity from cartilage explants previously labeled with (35)SO4(2-). Collagen degradation is calculated as percent release of collagen, measured by colorimetric assay of hydroxyproline.

The role of ADAM10 and ADAM17 in the ectodomain shedding of angiotensin converting enzyme and the amyloid precursor protein.

Numerous transmembrane proteins, including the blood pressure regulating angiotensin converting enzyme (ACE) and the Alzheimer's disease amyloid precursor protein (APP), are proteolytically shed from the plasma membrane by metalloproteases. We have used an antisense oligonucleotide (ASO) approach to delineate the role of ADAM10 and tumour necrosis factor-alpha converting enzyme (TACE; ADAM17) in the ectodomain shedding of ACE and APP from human SH-SY5Y cells. Although the ADAM10 ASO and TACE ASO significantly reduced (> 81%) their respective mRNA levels and reduced the alpha-secretase shedding of APP by 60% and 30%, respectively, neither ASO reduced the shedding of ACE. The mercurial compound 4-aminophenylmercuric acetate (APMA) stimulated the shedding of ACE but not of APP. The APMA-stimulated secretase cleaved ACE at the same Arg-Ser bond in the juxtamembrane stalk as the constitutive secretase but was more sensitive to inhibition by a hydroxamate-based compound. The APMA-activated shedding of ACE was not reduced by the ADAM10 or TACE ASOs. These results indicate that neither ADAM10 nor TACE are involved in the shedding of ACE and that APMA, which activates a distinct ACE secretase, is the first pharmacological agent to distinguish between the shedding of ACE and APP.

Equine laminitis: glucose deprivation and MMP activation induce dermo-epidermal separation in vitro.

REASONS FOR PERFORMING STUDY: Acute laminitis is characterised by hoof lamellar dermal-epidermal separation at the basement membrane (BM) zone. Hoof lamellar explants cultured in vitro can also be made to separate at the basement membrane zone and investigating how this occurs may give insight into the poorly understood pathophysiology of laminitis. OBJECTIVES: To investigate why glucose deprivation and metalloproteinase (MMP) activation in cultured lamellar explants leads to dermo-epidermal separation. METHODS: Explants, cultured without glucose or with the MMP activator p-amino-phenol-mercuric acetate (APMA), were subjected to tension and processed for transmission electron microscopy (TEM). RESULTS: Without glucose, or with APMA, explants under tension separated at the dermo-epidermal junction. This in vitro separation occurred via 2 different ultrastructural processes. Lack of glucose reduced hemidesmosomes (HDs) numbers until they disappeared and the basal cell cytoskeleton collapsed. Anchoring filaments (AFs), connecting the basal cell plasmalemma to the BM, were unaffected although they failed under tension. APMA activation of constituent lamellar MMPs did not affect HDs but caused AFs to disappear, also leading to dermo-epidermal separation under tension. CONCLUSIONS: Natural laminitis may occur in situations where glucose uptake by lamellar basal cells is compromised (e.g. equine Cushing's disease, obesity, hyperlipaemia, ischaemia and septicaemia) or when lamellar MMPs are activated (alimentary carbohydrate overload). POTENTIAL RELEVANCE: Therapies designed to facilitate peripheral glucose uptake and inhibit lamellar MMP activation may prevent or ameliorate laminitis.