טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentYehezkely Ravit
SubjectIntracellular Role for the Matrix-Modifying Enzyme Lox
in Regulating Transcription Factor
Subcellular Localization and Activity in
Muscle Regeneration
DepartmentDepartment of Medicine
Supervisor Professor Peleg Hasson
Full Thesis textFull thesis text - English Version


Abstract

Integration of extracellular matrix (ECM)-derived cues into transcriptional programs is essential primarily in rapidly morphing environments such as regenerating tissues. Both stromal and parenchymal components should be orchestrated to generate a functional tissue. Imbalance between these components underlies diseases such as chronic lung disease, liver cirrhosis, lung fibrosis and muscle dystrophies.  Both stromal and parenchymal tissues have autonomic regulation but are also able to finely tune and regulate each other. This fine tuning accounts for the exact amounts, morphologies and relations between each component and is crucial for generating functional tissues.

The skeletal muscle is a tissue in which this balance is crucial for normal function. It is the largest tissue in our body, constituting 40% of total body mass. In the muscle, each myofiber, fascicle as well as the whole muscle itself are tightly sheathed by a layer of muscle connective tissue (MCT). This tightly organized morphology is necessary for generating a functioning contractile tissue and for transmitting the contractile force to the skeleton to generate movement. Disturbance to this balance can tilt the whole tissue towards a more fibrotic and less functional phenotypes. This principle underlies muscle changes occurring normally during development and aging but also pathologically during traumatic muscle injuries, congenital or degenerative muscle diseases.

Lysyl oxidase (Lox) is an enzyme classically known for its ECM modifying activities, primarily collagen crosslinking. Previous work from our lab has demonstrated that Lox is a key player in regulating coordinated homeostatic growth of the muscle and its surrounding ECM. Deletion of Lox uncouples the balance between myofibers and the muscle connective tissue (MCT), resulting in reduced muscle content and increased ECM.

In this work, we demonstrate that, in addition to its extracellular role as ECM modifier, Lox also intracellularly modulate muscle progenitor differentiation by regulating transcription factor (TF) localization. Using genetic and pharmacological strategies, we highlight a novel intracellular role for Lox in myogenic progenitors essential for muscle regeneration. We demonstrate that Lox overexpression during muscle regeneration results in a better regeneration dynamics and muscle fiber formation without generating excessive fibrosis, while its pharmacologic and specific genetic inhibition in muscle satellite cells (SC) both result in aberrant muscle fiber formation. We show that these effects are due to Lox’s interaction and direct oxidation of Vestigial-Like 3 (Vgll3), a transcriptional co-activator acting with Mef2 and TEF TFs. This enzymatic activity is required for Vgll3 cytoplasmic to nuclear translocation in regulation of myogenic differentiation. Our work highlights a novel mechanism for TF sub-cellular localization and suggests a mechanism for integrating ECM organization with transcriptional output during myogenic differentiation. In addition, we examine Lox contribution to the DMD pathology and find that its inhibition improves the dystrophic phenotype, by promoting better regeneration dynamics and better muscle function, and not only through inhibition of fibrosis formation

Further work should focus on modulating this integration mechanism and by that affect the balance between ECM organization and muscle tissue formation. This concept can serve as a basis for novel therapeutic strategies targeting fibrotic pathologies.