A procollagen C-proteinase inhibitor diminishes collagen and lysyl oxidase processing but not collagen cross-linking in osteoblastic cultures

The deposition of insoluble functional collagen occurs following extracellular proteolytic processing of procollagens by procollagen N- and C-proteinases, fibril formation, and lysyl oxidase dependent cross-linking. Procollagen C-proteinases in addition process and activate lysyl oxidase. The present study evaluates a possible role for procollagen C-proteinases in controlling different aspects of collagen deposition in vitro. Studies determine whether inhibition of procollagen C-proteinase activity with a specific BMP-1 inhibitor results in perturbations in lysyl oxidase activation, and in collagen processing, deposition, and cross-linking in phenotypically normal cultured murine MC3T3-E1 cells. Data show that BMP-1 Inhibitor dose dependently inhibits lysyl oxidase activation by up to 50% in undifferentiated proliferating cells. In differentiating cultures, BMP-1 inhibitor decreased collagen processing but did not inhibit the accumulation of mature collagen cross-links. Finally, electron microscopy studies show that collagen fibril diameter increased. Thus, inhibition of procollagen C-proteinases results in perturbed collagen deposition primarily via decreased collagen processing.     

Regulation of collagen deposition and lysyl oxidase by tumor necrosis factor-alpha in osteoblasts

Tumor necrosis factor-alpha (TNF-alpha) inhibits osteoblast function in vitro by inhibiting collagen deposition. Studies generally support that TNF-alpha does not inhibit collagen biosynthesis by osteoblasts but that collagen deposition is in some way diminished. The study investigated TNF-alpha regulation of biosynthetic enzymes and proteins crucial for posttranslational extracellular collagen maturation in osteoblasts including procollagen C-proteinases, procollagen C-proteinase enhancer, and lysyl oxidase. The working hypothesis is that such regulation could inhibit collagen deposition by osteoblasts. We report that in phenotypically normal MC3T3-E1 osteoblasts, TNF-alpha decreases collagen deposition without decreasing collagen mRNA levels or procollagen protein synthesis. Analyses of the cell layers revealed that TNF-alpha diminished the levels of mature collagen cross-links, pyridinoline and deoxypyridinoline. Further analyses revealed that the mRNA expression for lysyl oxidase, the determining enzyme required for collagen cross-linking, is down-regulated by TNF-alpha in a concentration- and time-dependent manner by up to 50%. The decrease was accompanied by a significant reduction of lysyl oxidase protein levels and enzyme activity. By contrast, Northern and Western blotting studies revealed that procollagen C-proteinases bone morphogenic protein-1 and mammalians Tolloid and procollagen C-proteinase enhancer were expressed in MC3T3-E1 cells and not down-regulated. The data together demonstrate that TNF-alpha does not inhibit collagen synthesis but does inhibit the expression and activity of lysyl oxidase in osteoblasts, thereby contributing to perturbed collagen cross-linking and accumulation. These studies identify a novel mechanism in which proinflammatory cytokine modulation of an extracellular biosynthetic enzyme plays a determining role in the control of collagen accumulation by osteoblasts.     

The role of lysyl oxidase and collagen crosslinking during sea urchin development

Lysyl oxidase, the only enzyme involved in collagen crosslinking, is shown to be present in embryos of the sea urchin Strongylocentrotus purpuratus. The enzyme specific activity increases over six-fold during development, showing the greatest rise during gastrulation and prism larva formation. The enzyme is inhibited by the specific inhibitor, beta-aminoproprionitrile (BAPN). Continuous BAPN treatment of S. purpuratus and Lytechinus pictus embryos from late cleavage stages onward increases the amount of noncrosslinked collagen present in prism larvae. When BAPN is added at the 128- or 256-cell stage it causes developmental arrest at the mesenchyme blastula stage. Embryos can be maintained in the arrested state for at least 96 h and will resume normal development and morphogenesis following BAPN removal. If BAPN is added after the mesenchyme blastula stage, it has little adverse effect on development; consequently nonspecific toxic effects of the drug are unlikely. The results suggest that lysyl oxidase and collagen crosslinking play a vital role in primary mesenchyme migration, gastrulation, and morphogenesis during sea urchin development and indicate that BAPN may be very useful in studying the extracellular matrix-cell interactions at the cellular and molecular level.     

The Pro-regions of lysyl oxidase and lysyl oxidase-like 1 are required for deposition onto elastic fibers

These studies were undertaken to determine how lysyl oxidase (LOX) and lysyl oxidase like-1 (LOXL) enzymes are targeted to their substrates in the extracellular matrix. Full-length LOX/LOXL and constructs containing just the pro-regions of each enzyme localized to elastic fibers when expressed in cultured cells. However, the LOXL catalytic domain without the pro-region was secreted into the medium but did not associate with matrix. Ligand blot and mammalian two-hybrid assays confirmed an interaction between tropoelastin and the pro-regions of both LOX and LOXL. Immunofluorescence studies localized both enzymes to elastin at the earliest stages of elastic fiber assembly. Our results showed that the pro-regions of LOX and LOXL play a significant role in directing the deposition of both enzymes onto elastic fibers by mediating interactions with tropoelastin. These findings confirmed that an important element of substrate recognition lies in the pro-domain region of the molecule and that the pro-form of the enzyme is what initially interacts with the matrix substrate. These results have raised the interesting possibility that sequence differences between the pro-domain of LOX and LOXL account for some of the functional differences observed for the two enzymes.     

Changes in collagen cross-linking and lysyl oxidase by estrogen.

Dermal collagen solubility and lysyl oxidase activity of bones were measured in DDD mice of advancing age. Insoluble fractions of the dermal collagen increased more rapidly in females than in males after 5 weeks of age. Activity of the lysyl oxidase extracted from bones was higher in females than in males after 4 weeks of age. After sexual maturation, such sex differences were always observed in skin as well as in bone tissue. In other experimental animals, dermal collagen solubility was markedly decreased by estrogen treatment and lysyl oxidase was remarkably activated by estrogen in both skin and bone. Thus it is clear that estrogen stimulates the enzyme activity and accelerates the maturation of collagen and elastin in extracellular space.     

A micromethod for the purification of lysyl oxidase

A simple, efficient, and rapid procedure for the purification of lysyl oxidase is described. This method utilizes an affinity scheme involving powdered elastin-hydroxyethylmethacrylate hydrogels and high-performance liquid chromatography and permits the study of enzyme from sources which contain limited amounts of enzyme, such as aortic smooth muscle cells in culture.     

Evidence for a functional role for histidine in lysyl oxidase catalysis

The pH-dependent kinetics of lysyl oxidase catalysis was examined for evidence of an ionizable enzyme residue which might function as a general base catalyzing proton abstraction previously shown to be a component of the mechanism of substrate processing by this enzyme. Plots of log Vmax/Km for the oxidation of n-hexylamine versus pH yielded pKa values of 7.0 +/- 0.1 and 10.4 +/- 0.1. The higher pKa varied with different substrates, reflecting ionization of the substrate amino group. A van't Hoff plot of the temperature dependence of the lower pKa yielded a value of 6.1 kcal mol-1 for the enthalpy of ionization. This value as well as the pKa of 7.0 are consistent with those of histidine residues previously implicated as general base catalysts in enzymes. Incubation of lysyl oxidase with low concentrations of diethyl pyrocarbonate, a histidine-selective reagent, at 22 degrees C and pH 7.0 irreversibly inhibited enzyme activity by a pseudo first-order kinetic process. The inactivation of lysyl oxidase correlated with spectral and pH-dependent kinetic evidence for the chemical modification of 1 histidine residue/mol of enzyme, the pKa of which was 6.9 +/- 0.1, within experimental error of that seen in the plot of log Vmax/Km versus pH. Enzyme activity was restored by incubation of the modified enzyme with hydroxylamine, consistent with the ability of this nucleophile to displace the carbethoxy group from N-carbethoxyhistidine. The presence of the n-hexylamine substrate largely protected against enzyme inactivation by diethyl pyrocarbonate. These results thus indicate a functional role for histidine in lysyl oxidase catalysis consistent with that of a general base in proton abstraction.     

Inhibition by heparin of the oxidation of lysine in collagen by lysyl oxidase

The generation of covalent cross-linkages in collagen is initiated by the deamination by lysyl oxidase of specific lysine residues in this connective tissue protein. Since lysyl oxidase activity is influenced by ionic ligands bound to its protein substrates, the effect of heparin, an anionic glycosaminoglycan known to bind to collagen, was explored by using collagen and elastin substrates and highly purified lysyl oxidase. Concentrations of heparin up to 1 mg mL-1 had little effect on the enzymatic rate of oxidation if it was added prior to the addition of enzyme to a preformed fibrillar collagen substrate or to an insoluble elastin substrate. However, collagen oxidation was inhibited by 85% if this glycosaminoglycan was present at 0.4 mg mL-1 during collagen fibril formation before addition of the enzyme. Similarly, the rate and extent of collagen fibrillogenesis in the absence of lysyl oxidase were each markedly inhibited in the presence of 0.4 mg mL-1 heparin. Heparin also inhibited the extent of tight binding of lysyl oxidase to preformed fibrils by about 40% under conditions where enzyme activity against preformed fibrils was hardly affected. These results suggest that heparin may modulate the oxidation and thus the insolubilization of extracellular collagen fibers, possibly under conditions where elastin fiber synthesis is not affected, and that the tight binding of lysyl oxidase to collagen is not completely related to the expression of enzyme activity toward this substrate. These results also have mechanistic implications for the retarding effect of heparin on postoperative wound healing.     

A microassay for lysyl oxidase activity.

Pinnell and Martin's [(1968) Proc. Nat. Acad. Sci. USA61, 708–716] standard assay for lysyl oxidase is modified to determine activity in small samples. Data collected by both methods are comparable, and the microassay has the advantages of being economical and rapid.     

Effects of L-ascorbic acid on lysyl oxidase in the formation of collagen cross-links

To clarify the role of L-ascorbic acid (AsA) in the formation of pyridinoline, we examined the effects of AsA in vitro using soluble collagen and partially purified lysyl oxidase from bovine aorta. The concentration of dehydrodihydroxylysinonorleucine decreased when AsA was added in the early stage of pyridinoline formation. However, when AsA was added in a later stage of pyridinoline formation, the concentration of pyridinoline was not affected. These findings indicated that AsA was involved in the initial enzymatic reaction in pyridinoline synthesis. We purified lysyl oxidase to confirm its association of AsA. AsA inhibited the enzyme activity. Erythorbic acid and 3,4-dihydroxybenzoate suppressed the enzyme activity as well as AsA did. The inhibition by AsA of the lysyl oxidase activity arose from characteristics of AsA structure. AsA might be important in the regulation of the oxidative reaction of lysine.     

Deficient production of lysyl oxidase in cultures of malignantly transformed human cells

Lysyl oxidase activity in the culture medium of eight malignantly transformed human cell lines was very low compared with that in four control fibroblast lines, being 9-16% in five sarcoma cell lines and 7-11% in three other tumour cell lines. The low enzyme activity was probably due to deficient enzyme synthesis rather than impaired secretion into the cell medium, as low activity was also found in urea extracts of the cell pellets. Lysyl oxidase production thus appears to be closely regulated with deficient collagen gene expression in malignant transformation.     

Chemical genetics suggests a critical role for lysyl oxidase in zebrafish notochord morphogenesis

As a result of a chemical genetic screen for modulators of metalloprotease activity, we report that 2-mercaptopyridine-N-oxide induces a conspicuous undulating notochord defect in zebrafish embryos, a phenocopy of the leviathan mutant. The location of the chemically-induced wavy notochord correlated with the timing of application, thus defining a narrow chemical sensitivity window during segmentation stages. Microscopic observations revealed that notochord undulations appeared during the phase of notochord cell vacuolation and notochord elongation. Notochord cells become swollen as well as disorganized, while electron microscopy revealed disrupted organization of collagen fibrils in the surrounding sheath. We demonstrate by assay in zebrafish extracts that 2-mercaptopyridine-N-oxide inhibits lysyl oxidase. Thus, we provide insight into notochord morphogenesis and reveal novel compounds for lysyl oxidase inhibition. Taken together, these data underline the utility of small molecules for elucidating the dynamic mechanisms of early morphogenesis and provide a potential explanation for the recently established role of copper in zebrafish notochord formation.     

Evidence for the role of vitamin C-6 as a cofactor of lysyl oxidase.

At 24 h after injection of 16-day chick embryos with [C-3H]pyridoxine hydrochloride, some of this label appears in the epiphysial cartilage. Over 35% of this radioactivity appears in the form of [G-3H]pyridoxal and a further 30% as other vitamin B-6 compounds. Partial purification of lysyl oxidase from the labelled epiphysial cartilage reveals a single peak of radioactivity coinciding with a single peak of enzyme activity. On dialysis against phosphate-buffered saline, 75% of this radioactivity is found to be non-diffusible. After incubation with isonicotinic acid hydrazide, a carbonyl reagent that appears to inhibit lysyl oxidase both in vivo and in vitro, a further 70% of the radioactivity is lost, with a roughly corresponding loss of enzyme activity. It is suggested that a form of vitamin B-6 is required as a cofactor of lysyl oxidase, and that this may have important implications in terms of connective-tissue metabolism.     

Multiple bone morphogenetic protein 1-related mammalian metalloproteinases process pro-lysyl oxidase at the correct physiological site and control lysyl oxidase activation in mouse embryo fibroblast cultures

Lysyl oxidase catalyzes the final enzymatic step required for collagen and elastin cross-linking in extracellular matrix biosynthesis. Pro-lysyl oxidase is processed by procollagen C-proteinase activity, which also removes the C-propeptides of procollagens I-III. The Bmp1 gene encodes two procollagen C-proteinases: bone morphogenetic protein 1 (BMP-1) and mammalian Tolloid (mTLD). Mammalian Tolloid-like (mTLL)-1 and -2 are two genetically distinct BMP-1-related proteinases, and mTLL-1 has been shown to have procollagen C-proteinase activity. The present study is the first to directly compare pro-lysyl oxidase processing by these four related proteinases. In vitro assays with purified recombinant enzymes show that all four proteinases productively cleave pro-lysyl oxidase at the correct physiological site but that BMP-1 is 3-, 15-, and 20-fold more efficient than mTLL-1, mTLL-2, and mTLD, respectively. To more directly assess the roles of BMP-1 and mTLL-1 in lysyl oxidase activation by connective tissue cells, fibroblasts cultured from Bmp1-null, Tll1-null, and Bmp1/Tll1 double null mouse embryos, thus lacking BMP-1/mTLD, mTLL-1, or all three enzymes, respectively, were assayed for lysyl oxidase enzyme activity and for accumulation of pro-lysyl oxidase and mature approximately 30-kDa lysyl oxidase. Wild type cells or cells singly null for Bmp1 or Tll1 all produced both pro-lysyl oxidase and processed lysyl oxidase at similar levels, indicating apparently normal levels of processing, consistent with enzyme activity data. In contrast, double null Bmp1/Tll1 cells produced predominantly unprocessed 50-kDa pro-lysyl oxidase and had lysyl oxidase enzyme activity diminished by 70% compared with wild type, Bmp1-null, and Tll1-null cells. Thus, the combination of BMP-1/mTLD and mTLL-1 is shown to be responsible for the majority of processing leading to activation of lysyl oxidase by murine embryonic fibroblasts, whereas in vitro studies identify pro-lysyl oxidase as the first known substrate for mTLL-2.