The extracellular matrix (ECM) provides a structural and signaling environment that determines cell fate, adhesion, proliferation, differentiation, migration, and communication during development, tissue maintenance and repair, and pathological processes. The composition of the ECM is unique in each organ and consistently includes various types of collagens, noncollagenous glycoproteins, and a range of enzymes essential for ECM assembly and maintenance including lysyl oxidase (LOX). LOX is a copper-dependent amine oxidase that oxidatively deaminates specific peptidyl lysine and hydroxylysine residues within its substrates, the best known of which are fibrillar collagens and elastin. The resulting reactive aldehyde residues then spontaneously condense to form covalent intra- and intermolecular cross-linkages leading to the development of insoluble ECM matrices. Besides the cross-linking activity of LOX in the ECM of skin, lung, cardiovascular, and other systems, novel roles have also been recognized for LOX including significant cellular and nuclear roles and signaling functions.
In mammals, the LOX family has five members, including LOX and four LOX-like proteins (LOXL1-4). The C-terminal region is highly conserved among all five members and includes a copper-binding site, a cytokine receptor-like (CRL) domain, and residues that form the lysine tyrosylquinone (LTQ) cofactor (Csiszar 2001). The N-terminal regions of the LOX-like proteins are more variable, with a proline-rich region in LOXL1 and within LOXL2, LOXL3, and LOXL4, four scavenger receptor cysteine-rich (SRCR) domains. The highly conserved SRCR domains with no defined functions are also present in numerous soluble and membrane-bound proteins. The LOX-like proteins appear catalytically active but with different temporal and tissue-specific expression, different substrates, and different functions.
Aberrant LOX expression and functions have been linked to disorders of the skin, aorta, heart, placenta, diaphragm, lung, pelvic floor and also to pterygium, progressive vision loss in keratoconus, and to tumor development and progression in a wide range of cancer types. Both the normal and pathological LOX-mediated mechanisms involve regulation by and interactions of LOX, including the independently functioning LOX propeptide (LOX-PP), with major signaling pathways including VEGF, TGF-β, ERK, NF-κB, PI3K/AKT, SMAD, MAPK, FGF-2/FGFR, and FAK/AKT in integrin-mediated mechanotransduction pathways. During the catalytic reaction of LOX with various substrates peroxide is produced that additionally contributes to the activation of HIF-1α/LOX and PI3K/AKT signaling and maintains a positive HIF-1α/LOX regulatory loop.
The ECM is a dynamic structural and signaling environment that determines cell fate, adhesion, proliferation, differentiation, migration, and communication during development, tissue maintenance and repair, and pathological processes. LOX, a Cu-dependent enzyme is an essential component of the ECM and plays a primary role in ECM assembly and maintenance. Additionally, LOX has catalytic activity-dependent and independent intracellular and nuclear functions and collectively, important roles in vascular, cardiac, lung, skin, and pelvic floor disorders; fibrotic and inflammatory processes; and tumor development and progression. Both normal and pathological LOX-mediated mechanisms involve regulation by and interactions of LOX, including the independently functioning propeptide, LOX-PP, with major signaling pathways including VEGF, TGF-β, ERK, NF-κB, PI3K/AKT, SMAD, MAPK, FGF-2/FGFR, and FAK/AKT in integrin-mediated mechanotransduction pathways. During the catalytic reactions of LOX with various substrates, peroxide is produced that additionally contributes to the activation of HIF-1α/LOX and PI3K/AKT signaling and maintains a positive HIF-1α/LOX regulatory loop. This review aims to provide an insight into LOX-associated functional contributions to these signaling pathways.
Regulation of LOX Activity
The LOX gene is subject to developmental and age-related regulation and demonstrates aberrant expression patterns in numerous pathologies. The mRNA transcribed from the human LOX gene (chromosome 5q23.3–31.2) encodes a 417 amino acid polypeptide. The human LOX protein is expressed in most cell types and is synthesized as a 48 kDa pre-proenzyme with a 21 amino acid signal sequence at the N-terminus. Following N-terminal glycosylation, the signal peptide is removed and the copper cofactor is incorporated into the protein within the Golgi apparatus, resulting in an intermediary, catalytically inactive 50 kDa proenzyme that is subsequently secreted to the extracellular space. In the ECM, the pro-LOX is proteolytically activated by procollagen C-proteinases BMP-1, TLL1, and TLL2 into a mature ECM enzyme of 30 kDa and an 18 kDa N-terminal propeptide. The activity of LOX is critically dependent on Cu concentrations and redistribution involving the Cu-uptake transporter-1 (CTR1) and the Cu-efflux pump ATP7A. ATP7A, furthermore, also mediates the chaperone function of the Cu-transport protein Antioxidant-1 (Atox-1) that is required for VEGF-induced angiogenesis via the activation of LOX. In addition, LOX and VEGF are coregulated by a Cu-dependent activation of HIF-1alpha, and in turn HIF-1alpha, LOX, and VEGF expression is controlled by histone deacetylase HDAC2.
Initial studies of LOX focused on the essential role of LOX in cross-linking of fibrillar collagen types I, III, basal lamina collagen IV, and elastin during development, tissue remodeling, and maintenance of tissue homeostasis. More recently, diverse biological processes, such as interactions with cell membrane proteins, have been documented to depend on the active LOX. The molecular mechanisms and the range of LOX substrates responsible for these processes have, however, not been fully explored. LOX is also known to participate in activity-independent functions, and among the LOX domains the role of the LOX propeptide (LOX-PP) has been explored in detail. The function(s) of other specific domains and/or mechanisms of LOX activity-independent roles remain largely unknown. The secreted and ECM-processed LOX can reenter cells and has been detected in the cytoplasm, in some cases associated with the cytoskeleton, and also localized within the nuclei in various cell types where it was shown to modify chromatin structure in interactions with histone H1. The mechanism of transport of the LOX protein into the cytoplasm and nucleus and its intracellular functions either related to or independent of its catalytic activity are not fully characterized.
LOX in Caveolae-Compartmentalized Signaling
Caveolae are cholesterol-rich membrane microdomains that serve as signaling platforms to facilitate the temporal and spatial localization of signal transduction events including those stimulated by Ang II both in vascular smooth muscle cells (VSMC) and endothelial cells. Genome-wide transcriptional profiling identified LOX together with ADAM17 and EGFR as central genes among highly ranked gene subnetworks associated with Ang II-induced abdominal aortic aneurysm (AAA) with localization of LOX in VSCM caveolae (Takayanagi et al. 2014). The metalloprotease ADAM17/tumor necrosis factor-α (TNF-α) converting enzyme, also compartmentalized in caveolae, is primarily responsible for the epidermal growth factor receptor (EGFR) trans-activation induced by Ang II. While the role of LOX in this context is not fully understood, recent studies suggest a strong link between caveolae-based signaling and the diverse pathophysiology of cardiovascular diseases including atherosclerosis, dyslipidemia, cardiac fibrosis, insulin resistance, inflammation, oxidative stress, and AAA.
TGF-β Regulation of and Interactions with LOX
There is a strong association between LOX activity and lung, arterial, heart, skin, and kidney fibrosis and pathological conditions leading to fibrosis including epithelial-mesenchymal transition and fibroblast to myofibroblast trans-differentiation within wounds that undergo repeated cycles of injury and healing. During fibrosis, the activation of transforming growth factor-β1 (TFG-β1)/SMAD3 signaling is a driving force that prominently upregulates LOX mRNA, protein levels, and activity in fibroblasts, osteoblasts, epithelial, and aortic smooth muscle cells among other cell types. In a model of TGF-β-induced synovial fibrosis and in end-stage human osteoarthritis, TGF-β upregulated LOX expression proved disease stage-dependent. While increased TGF-β and TGFβR1 played a role in the maintenance of symptoms, TGFβ/TGFβR1-induced LOX upregulation occurred only in the initial phases (Belangero et al. 2016). LOX and the COL1A1 and COL1A2 genes are coregulated by their similar TGF-β response promoter elements, and consistent with this mechanism, upregulated TGF-β/p38 mitogen-activated protein kinase (p38MAPK) signaling increases both LOX levels and collagen synthesis shown in obesity-associated fetal muscle fibrogenesis. In adult rat cardiac fibroblasts, TGF-β1 upregulates LOX mRNA, protein, and activity with concomitant increases in collagen type I, III, and BMP-1 expression. TGF-β1 induction of LOX proved to involve PI3K, SMAD3, p38-MAPK, JNK, and ERK1/2 and suggested the integration of the PI3K/AKT and SMAD pathways (Voloshenyuk et al. 2011). SMAD4, a key component of the TGF-β/BMP pathway, regulates LOX during skeletal growth, and consequently, mice with ablated Smad4 present a combination of features associated with osteogenesis imperfecta, cleidocranial dysplasia, and Wnt deficiency syndromes with fully differentiated osteoblasts that, however, lack Lox regulated by BMP-2-responsive Smad4 and Runx2 (Salazar et al. 2013).
A LOX-TGF-β feedback loop was identified during skeletal muscle development that maintained homeostasis between muscle components and muscle connective tissue. In a mouse model, Lox secreted by myofibers was shown to regulate the balance between the amount of myofibers and the muscle connective tissue by attenuating TGF-β1 signaling that acted as an inhibitor for myofiber differentiation and a promoter of muscle connective tissue development (Kutchuk et al. 2015). A similar LOX regulation of TGF-β was reported in idiopathic pulmonary fibrosis where inhibition of LOX in the inflammatory stage, but not in the fibrogenic stage, impaired inflammatory cell infiltration, TGF-β signaling, myofibroblast accumulation, and reduced collagen deposition.
A direct interaction between the LOX protein and TGF-β1 has also been identified and shown to suppress TGF-β1-induced SMAD3 phosphorylation. This effect proved to be due to the BAPN-sensitive amine oxidase activity of LOX, but not to peroxide production, a LOX-associated signaling mechanism active in multiple tumor types. In interactions with TGF-β1, LOX was proposed to be involved in oxidative deamination of lysine residues within the lysine-rich terminus in TGF-β1, a process that could either change charges or covalently stabilize the conformation of TGF-β1 and interfere with binding of TGF-β1 to its receptor leading to diminished signaling via SMAD3 in a cross-talk with PI3K and AKT (Atsawasuwan et al. 2008).
LOX in Integrin-Mediated Mechanotransduction Pathways
Coordinated chemical and physical regulators, including the mechanical properties of the ECM, orchestrate the behavior and fate of tissue resident cells. The mechanical environment is shaped by reciprocal cell-ECM interactions involving cell-generated endogenous (cytoskeletal contractility) and exogenous (gravity, shear stress, tensile, and compressive) forces. Translation of the ECM stiffness into intracellular signals is mainly mediated by transmembrane receptor integrin α and β heterotrimers with an integrin β subunit tail linking to the cytoskeleton through adaptor proteins. During ECM remodeling, wound healing, fibrotic diseases, or tumor progression, following binding to ECM substrates, integrin clustering initiates the assembly of adaptor molecules, focal adhesion kinase (FAK) vinculin, paxilin, tensin, and tallin to form ECM-cell focal adhesions, induce reorganization of active filaments into stress fibers, and activate signaling pathways, such as FAK-ERK1/2, to regulate cell activities. LOX-dependent cross-linking and deposition of collagen fibers is an integral part of ECM remodeling with elevated LOX activity increasing tissue stiffness. Tissue microarray analysis of genes associated with increased stromal stiffness identified a positive correlation between increased LOX, osteopontin, and COL1 expression via a mechanism that involved the integrin β1/GSK-3β/β-catenin pathway associated with hepatocellular carcinoma development and progression (You et al. 2015).
Additional mechanistic details of LOX-mediated matrix stiffness signal stimulation were shown to involve gradually upregulated LOX and VEGF indicating positive correlation between matrix rigidity and angiogenesis with integrin β1 expression as the most distinctive mediator activating the PI3K/AKT pathway (Dong et al. 2014). In three-dimensional organotypic skin cultures of recessive dystrophic epidermolysis bullosa with elevated risk for early onset cutaneous squamous cell carcinoma, preexisting injury-driven alteration of the microenvironment including TFG-β availability, increased ECM cross-linking, and tissue stiffening identified integrin β1/FAK/AKT mechanosignaling that proved to be TGF-β and LOX dependent (Mittapalli et al. 2016).
In stiffness-driven inflammatory activation of pulmonary endothelial cells, increased LOX activation and increased expression of the Rho activator GEF-H1 signaling was noted that was effectively reversed by LOX suppression leading to reduced GEF-H1 and inflammation (Mambetsariev et al. 2014). Increased LOX was found to also contribute to matrix-mediated mechanotransduction in posttraumatic osteoarthritis a process involving age- and mechanical stress-related increase in advanced glycation products, increased matrix stiffness, and disrupted homeostatic balance between chondrocyte catabolism and anabolism via the Rho-kinase myosin light chain axis (Kim et al. 2015).
LOX in Tumor Development and Invasion
Studies focused on LOX-dependent mechanisms during tumor development, invasion, and metastasis identified distinct functions including tissue stiffness contributing to tumor cell adhesion and invasion involving LOX/integrin β1/FAK/CAS and PI3K/EGF/AKT signaling, tumor angiogenesis via LOX-VEGF interactions, and a role for primary tumor cell secreted LOX at metastatic sites in promoting tumor cell extravasation and establishment of the metastatic niche. Tumor epithelial cell migration and invasion depends on FAK/SRC signaling activated by H2O2 generated during the catalytic reaction of the active LOX with its substrates. In astrocytoma and glioma cell lines and tissues high levels of LOX expression correlated with increased tumor grade and increased activation of FAK/paxillin signaling (Laczko et al. 2007). In tumor cells, the LOX-associated invasive mechanism further involved increased lamellipodia formation by affecting signaling downstream of FAK and involving the CAS/CRK/DOCK signaling complex, activation of RAC GTPase, and a direct activation of the PI3K/AKT pathway as demonstrated in human colorectal carcinoma cell lines (Pez et al. 2011).
In solid tumors, angiogenesis, increased glycolysis, and cellular adaptation to hypoxic environment are representative features that correlate with invasion and metastasis, and resistance to therapy. HIF-1α is a central player in these processes with distinct branching of the activated pathways including LOX activation and the VEGF axis that also activate/induce each other as shown in prostate cancer (Baker et al. 2013). An additional positive regulatory loop was identified between LOX and HIF-1α by which the HIF-1α-inducible LOX activated HIF-1α via the PI3K/AKT pathway in human colorectal carcinoma cell lines. HIF-1α protein upregulation occurred via peroxide (the byproduct of LOX catalytic activity) leading to tumor cell proliferation and in vivo tumor growth (Pez et al. 2011).
The LOX Propeptide in RAF, NF-κB, FGF-2, and FAK Signaling
The 18 kDa LOX propeptide (LOX-PP) is required for optimal LOX secretion and is proteolytically removed in the ECM as part of the activation process by BMP-1. The LOX-PP can reenter cells via cell-type-dependent primary macropinocytosis or a secondary clathrin-dependent pathway. The LOX-PP has a putative nuclear localization signal and has been detected in the nucleus where it was proposed to function as a signaling molecule. Nuclear LOX-PP proved to directly impair DNA repair regulator MRE11 by forming a protein complex detected in prostate cancer cell lines. The phosphorylation of DNA repair molecules ATM and CHK2, typically elevated in prostate cancer, was also inhibited by recombinant LOX-PP expression resulting in inhibited tumor xenograft growth (Bais et al. 2015).
The LOX-PP, in interactions with cellular-binding partners, acts as a tumor growth inhibitor via several distinct signal transduction pathways stimulated by oncogenic RAS including the RAF/MAPK/ERK with RAF as the proposed primary target, the interrelated nuclear factor-κB (NF-κB), and the PI3K/AKT pathways. In prostate cancer cell lines LOX-PP was also shown to target FGF-2 signaling mediated by FGRF1 and AKT activation, and in breast cancer cells, fibronectin-stimulated FAK signaling and ERK1/2 activation (Vora et al. 2010). Within LOX-PP the G473A polymorphism resulting in an Arg-to-Gln substitution impairs its ability to inhibit the invasive phenotype and tumor formation of breast cancer cells, and a link of this polymorphism to an increased risk of ER- breast cancer confirmed a prominent role for LOX-PP in in vivo signaling.
Diverse molecular targets affecting cell proliferation and migration have been identified for LOX-PP in various cell lines including AKT-dependent induction of RAS in c-Ha-RAS-transformed NIH3T3 cells, DNA synthesis in androgen-independent prostate cell lines, and action via the MEK/ERK and PI3K/AKT pathways in DU145 cells, but through a different mechanism in PC-3 cells (Palamakumbura et al. 2009). In lung cancer cells, RAS/c-RAF/AP-1-mediated migration is reduced by LOX-PP physically binding to c-RAF, a downstream kinase of RAS. LOX-PP also inhibits regulatory elements and represses expression of Blymphocyte-induced maturation protein 1 (Blimp1) an upstream AP-1 activator commonly upregulated in epithelia-derived tumors and linked to increased migration and invasion (Yu et al. 2012). Interference of LOX-PP with FGF-2 and FGFR binding and signaling were noted in DU145 prostate cancer cell lines to result in cell growth inhibition, however, the same mechanism was not active in PC-3 cells (Palamakumbura et al. 2009). Signaling of FGF-2 that induces ERK/MAPK phosphorylation was similarly inhibited in MC3T3 and osteoblast cultures by LOX-PP through reducing FGF-2 binding to its receptors (Vora et al. 2010).
The LOX-PP was also reported in connection with NF-κB. LOX-PP significantly reduced levels of NF-κB and its tissue-specific target BCL-2, decreased the migrating ability of pancreatic and lung cancer cell lines, and inhibited transformation of murine breast cancer cells driven by Her-2/neu, an upstream activator of RAS via activation of NF-κB. In these cells LOX-PP reversed Her-2/neu-induced migration and epithelial to mesenchymal transition as measured by reduced levels of Snail and vimentin, upregulation of E-cadherin, and inhibited tumor formation (Min et al. 2007).
LOX is an essential component of the ECM that contributes to the assembly and maintenance of the ECM and the structural and signaling microenvironment that controls the phenotype of tissue resident cell types during normal and pathological processes. LOX has additional catalytic activity-dependent and independent cellular and nuclear functions and collectively important roles in fibrotic, vascular, lung, and pelvic floor disorders, in inflammation, and during tumor development and progression. Both the normal and pathological LOX-mediated processes involve regulation by and interactions of LOX and its propeptide (LOX-PP), with major signaling pathways. A novel regulatory detail of LOX signaling was recently identified with its localization within caveolae, cholesterol-rich membrane microdomains, that serve as platforms for temporal and spatial control of signal transduction and with LOX being part of a caveolae-associated network involved in Ang II-induced AAA.
Fibrotic disorders have strong association with LOX including the activation of TFG-β1/SMAD3 signaling as a driving force that prominently upregulates LOX mRNA, protein levels, and activity in various cell types. There is a disease-stage control of upregulated LOX expression by TGF-β and TGFβR1 that appears to be specific for the initial phases of synovial fibrosis and end-stage human osteoarthritis. TGF-β1 induction of LOX involves a TGF-β responsive LOX promoter element, PI3K, SMAD3, p38-MAPK, JNK, and ERK1/2 signaling and integration of the PI3K/AKT and SMAD pathways. A LOX-TGF-β feedback loop also exists and was identified in idiopathic pulmonary fibrosis and during skeletal muscle development where attenuated TGF-β1 signaling maintained a balance between myofiber differentiation and connective tissue development. Interaction between the LOX protein and TGF-β1 suppresses TGF-β1-induced SMAD3 phosphorylation attributed to either active LOX-mediated oxidative deamination of lysine residues within TGF-β1 or covalent stabilization of its conformation interfering with TGF-β1 receptor binding and diminished SMAD3 signaling involving PI3K and AKT.
LOX is an essential contributor to ECM stiffness that via mechanotransduction pathways orchestrate the behavior of resident cells involving the integrin β1/GSK-3β/β-catenin pathway. The gradually upregulated LOX and VEGF with integrin β1 as the distinctive mediator also activate the PI3K/AKT signaling in positive correlation between matrix rigidity and angiogenesis. Preexisting injury-driven alteration of the microenvironment contributes to the activation of TGF-β/LOX-dependent integrin β1/FAK/AKT mechanosignaling a pathomechanism relevant to recessive dystrophic epidermolysis bullosa. In stiffness-driven inflammation, LOX activation and increased expression of the Rho activator GEF-H1 signaling is present, and in posttraumatic osteoarthritis with age- and mechanical stress-related increase in advanced glycation products, LOX contributes to disrupted homeostatic balance between catabolism and anabolism of chondrocytes via the Rho-Rho kinase myosin light chain axis.
LOX plays significant and distinct roles during tumor development including LOX-mediated increase in tissue stiffness with the involvement of the integrin β1/FAK/CAS and PI3K/EGF/AKT signaling pathways and promotion of tumor angiogenesis via LOX-VEGF interactions. LOX-mediated peroxide, additionally, activates FAK/SRC and FAK/paxillin in astrocytoma and glioma, promoting migratory and invasive tumor cell phenotypes. LOX-associated invasive mechanisms also involve the CAS/CRK/DOCK signaling complex, activation of RAC GTPase, and a direct activation of the PI3K/AKT pathway resulting in increased lamellipodia formation. A hypoxic environment in solid tumors correlates with invasion and metastasis with HIF-1α as a central player and distinct branching of the activated pathways including activation of LOX and the VEGF axis that subsequently induce each other. A positive regulatory loop between LOX and HIF-1α involves LOX activation of HIF-1α via PI3K/AKT and LOX activity-derived peroxide leading to tumor cell proliferation and tumor growth.
The LOX-PP has independent and highly cell type-specific roles. In interactions with cellular-binding partners it acts as a tumor growth inhibitor via several distinct signal transduction pathways characterized in osteoblasts and prostate, pancreatic, lung, and breast cancer cell lines. The LOX-PP proved to modify the RAF/MAPK/ERK, the interrelated NF-κB and PI3K/AKT pathways, FGF-2 via reduced binding to FGFR that also mediates ERK/MAPK activation, and fibronectin-stimulated FAK signaling and ERK1/2 activation. LOX-PP also inhibits expression of Blimp1 an activator of AP-1 upregulated in epithelial tumors and reverses Her-2/neu-induced epithelial to mesenchymal transition.
Collectively LOX and the LOX-PP have significant signaling functions active under normal and pathological conditions that include the VEGF, TGF-β, ERK, NF-κB, PI3K/AKT, SMAD, MAPK, FGF-2/FGFR, and FAK/AKT in integrin-mediated mechanotransduction pathways. LOX catalytic reaction-derived peroxide additionally activates HIF-1α/ LOX and PI3K/AKT signaling and maintains a positive HIF-1α/LOX regulatory loop.
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