Coordination between cytoskeletal organization, cell contraction and extracellular matrix development, is depended on LOX for aneurysm prevention.

Distinct, seemingly independent, cellular pathways affecting intracellular machineries or extracellular matrix (ECM) deposition and organization, have been implicated in aneurysm formation. One of the key genes associated with the pathology in both humans and mice is Lysyl oxidase (LOX), a secreted ECM-modifying enzyme, highly expressed in medial vascular smooth muscle cells. To dissect the mechanisms leading to aneurysm development, we conditionally deleted Lox in smooth muscle cells. We find that cytoskeletal organization is lost following Lox deletion. Cell culture assays and in vivo analyses demonstrate a cell-autonomous role for LOX affecting myosin light chain phosphorylation and cytoskeletal assembly resulting in irregular smooth muscle contraction. These results not only highlight new intracellular roles for LOX, but notably they link between multiple processes leading to aneurysm formation suggesting LOX coordinates ECM development, cytoskeletal organization and cell contraction required for media development and function.

Initiation of fibronectin fibrillogenesis is an enzyme-dependent process.

Fibronectin fibrillogenesis and mechanosensing both depend on integrin-mediated force transmission to the extracellular matrix. However, force transmission is in itself dependent on fibrillogenesis, and fibronectin fibrils are found in soft embryos where high forces cannot be applied, suggesting that force cannot be the sole initiator of fibrillogenesis. Here, we identify a nucleation step prior to force transmission, driven by fibronectin oxidation mediated by lysyl oxidase enzyme family members. This oxidation induces fibronectin clustering, which promotes early adhesion, alters cellular response to soft matrices, and enhances force transmission to the matrix. In contrast, absence of fibronectin oxidation abrogates fibrillogenesis, perturbs cell-matrix adhesion, and compromises mechanosensation. Moreover, fibronectin oxidation promotes cancer cell colony formation in soft agar as well as collective and single-cell migration. These results reveal a force-independent enzyme-dependent mechanism that initiates fibronectin fibrillogenesis, establishing a critical step in cell adhesion and mechanosensing.

Lysyl oxidase interactions with transforming growth factor-ß during angiogenesis are mediated by endothelin 1.

Crosstalk between multiple components underlies the formation of mature vessels. Although the players involved in angiogenesis have been identified, mechanisms underlying the crosstalk between them are still unclear. Using the ex vivo aortic ring assay, we set out to dissect the interactions between two key angiogenic signaling pathways, vascular endothelial growth factor (VEGF) and transforming growth factor ß (TGFß), with members of the lysyl oxidase (LOX) family of matrix modifying enzymes. We find an interplay between VEGF, TGFß, and the LOXs is essential for the formation of mature vascular smooth muscle cells (vSMC)-coated vessels. RNA sequencing analysis further identified an interaction with the endothelin-1 pathway. Our work implicates endothelin-1 downstream of TGFß in vascular maturation and demonstrate the complexity of processes involved in generating vSMC-coated vessels.

Intracellular Role for the Matrix-Modifying Enzyme Lox in Regulating Transcription Factor Subcellular Localization and Activity in Muscle Regeneration.

Integration of extracellular matrix (ECM)-derived cues into transcriptional programs is essential primarily in rapidly morphing environments, such as regenerating tissues. Here, we demonstrate that lysyl oxidase (Lox), known for its ECM-modifying activities, primarily collagen crosslinking, also directly regulates transcription factor (TF) localization. Using genetic and pharmacological strategies, we highlight an intracellular role for Lox in myogenic progenitors essential for muscle regeneration. We show that Lox interacts with, and directly oxidizes, vestigial-like 3 (Vgll3), a transcriptional co-activator acting with Mef2 and transcriptional enhancer factor (TEF) TFs. This enzymatic activity is required for Vgll3 cytoplasmic-to-nuclear translocation in regulation of myogenic differentiation. Our work highlights an additional mechanism for TF subcellular localization facilitating integration of ECM organization with transcriptional output during myogenic differentiation. Modulating this integration mechanism could affect the balance between ECM organization and cell differentiation and serve as a basis for novel therapeutic strategies targeting fibrotic pathologies.

Interactions between lysyl oxidases and ADAMTS proteins suggest a novel crosstalk between two extracellular matrix families.

The extracellular matrix (ECM) regulates numerous cellular events in addition to providing structural integrity. Among several protein and enzyme families implicated in functions of the ECM, the lysyl oxidases and ADAMTS proteins are known to participate in microfibril and elastic fiber formation as well as ECM-associated signaling. A yeast two-hybrid screen to identify lysyl oxidase (LOX) binding proteins identified ADAMTSL4 as a potential interactor. We demonstrate here that several members of the LOX and ADAMTS families interact with one another. Upon investigating the interaction between LOX and ADAMTSL2 we found that the absence or inhibition of Lox affected ADAMTSL2 molecular forms and reduced its tissue levels. Thus, ADAMTSL2 stability and inter-molecular complexes may depend on the activity of lysyl oxidases.