« The transcription factor NRF2 regulates matrisome gene expression and collagen fibrillogenesis in human skin fibroblasts “

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Maturation in the Golgi apparatus and secretion of the collagen molecule

Procollagen molecules are further translocated to the Golgi apparatus, which ensures accurate procollagen glycosylation and trafficking. Before their transport to the extracellular space, procollagens start to aggregate laterally within the Golgi to plasma membrane carriers. When procollagens reach the plasma membrane, carriers connect to the ECM through deep projections of the plasma membrane, called “fibripositors” (Canty and Kadler, 2005; Kadler, 2017). The precise nature of these vesicles is still a matter of considerable debate. Specifically, the question of how large rod-shape molecules of up to 700 nm in length can fit into secretory vesicles remains puzzling. Collagen trafficking is far from being completely understood and would need further investigations (Banos et al., 2008; Canty and Kadler, 2005; McCaughey and Stephens, 2019).

Supramolecular organization into striated fibrils in the extracellular space

Once secreted in the extracellular space, N- and C-propeptides of fibrillar procollagens are entirely (e.g. collagens I and III) or partially (e.g. collagens V and XI) removed by specific matrix enzymes including BMP-1, mTLD and TLL-1 for the C-propeptide and ADAMTS family members for the N-propeptide. Mature collagens spontaneously organize into fibrils that consist in end-overlap alignment of collagen molecules in a quarter-staggered packing pattern. Fibrils have a starting diameter of 12 nm and can further co-aggregate to form fibers with a diameter to over 500 nm, depending on the tissue and the developmental stage (Craig et al., 1989; Kadler, 2017). Interestingly, it has been shown that fibroblasts in culture are able to α chains in the ER, specific peptidyl lysine residues are hydroxylated to form hydroxylysine ( OH NH2) and, subsequently, specific glycosylated hydroxylysine residues (O linked glycosylation). For the latter, either single galactose (a red hexagon) or a gl ucose galactose (two red hexagons) is attached. After these and other modifications, twopro α 1 chains (solid line) and one pro α 2 chain (dotted line) associate with one another and fold into a triple helical molecule from the C to the N terminus to form a p rocollagen molecule in the ER. This procollagen molecule further undergoes modifications in the Golgi apparatus and is then packaged and secreted into the extracellular space. Then both N and C propeptides are cleaved to release a collagen molecule. Then, collagen molecules spontaneously assemble into a fibril that is stabilized by covalent intra and inter molecular covalent cross linking. During fibrillogenesis, molecules are packed in parallel and are longitudinally staggered by an axial repeat distance , D period (~67 nm) creating two repeated regions, i.e. overlap and hole regions, in the fibril.
synthesize collagens with the characteristic banded pattern, but these in vitro fibrils are not able to form any higher-order organization like the in vivo collagen fibers (Canty and Kadler, 2005). Fibril structures are strengthened by intra- and inter- molecular crosslinks catalyzed by lysyl oxidases between lysine and hydroxylysine residues (Myllyharju and Kivirikko, 2004; Vallet and Ricard-Blum, 2019; Yamauchi and Sricholpech, 2012).
Transglutaminase-2 (TGM2) is another important cross-linker of collagen fibrils. TGM2 catalyzes isopeptide bonds between lysine and glutamate residues in collagens I, III, V and XI (Esposito and Caputo, 2005). These intermolecular bonds are prerequisites for the mechanical resilience ensured by collagen fibrils as well as for the stability of the collagen network (Gelse et al., 2003; Wang et al., 2014).

Formation of elastic fibers, a process called elastogenesis

In humans, elastin is encoded by one single gene (Muiznieks and Keeley, 2013). The protein product of the elastin gene is the elastin precursor, named tropoelastin, which is further bound to a specific chaperone, the elastin binding protein (EBP) before being secreted. EBP stabilizes and prevents tropoelastin from premature aggregation before secretion into the extracellular space (Figure 16). Then, coacervation occurs, a process during which tropoelastin monomers self-assemble and form globular hydrophilic droplets bound to the cell membrane. Tropoelastin monomers then undergo maturation by being cross-linked by the lysyl oxidases LOX, LOXL1 and LOXL2. This enzymatic reticulation enables the formation of larger insoluble aggregates (Liu et al., 2004; Schmelzer et al., 2019). Tropoelastin aggregates then assemble with microfibrils of fibrillins that act as a protein scaffold for further formation of the mature elastic fibers (Figure 16).

Remodeling of the ECM

ECM is a highly dynamic structure that is synthesized during development and then undergoes continuous remodeling in response to external stimuli, such as applied forces, injuries or during wound healing in healthy and pathological conditions but also in physiological situations as diverse as during exercise or pregnancy. ECM remodeling is tightly controlled by diverse ECM degrading proteases, including the sheddases a disintegrin and metalloproteinases (ADAMs), ADAM with thrombospondin motifs (ADAMTS) and matrix metalloproteinases (MMPs). MMPS activity is counterbalanced by the tissue inhibitor metalloproteinases (TIMPS), but also by other enzymes, such as the lysyl oxidase (LOX) and transglutaminases (TGM), which cross-link ECM fibers and control the stiffness of the matrix (Frantz et al., 2010; Karamanos et al., 2019). In healthy conditions, the degraded ECM can be renewed and this remodeling is indeed crucial to maintain a proper tissue integrality and functionality.
Dysregulation of ECM remodeling appears to initiate and promote the progression of various diseases, including fibrosis and cancers as examples (Coentro et al., 2019; Kass et al., 2007) but it also represents an aging hallmark, especially in the skin (Figure 19) (Freitas-Rodríguez et al., 2017; Phillip et al., 2015).

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Fibroblasts are heterogeneous and plastic cells

Fibroblasts are abundant cells throughout the human body and among the most studied cell types as they are easily isolated and cultivated in vitro. Nevertheless, they still remain poorly defined and enigmatic cells. Fibroblasts are mesenchymal cells that have been firstly described as spindle-shaped cells synthetizing and organizing extracellular matrix proteins into the extracellular space (Figure 20A). Notably, fibroblasts are the major cells responsible for the production of collagen molecules. Therefore, they are responsible for the integrity and homeostasis of most connective tissues such as cartilage, tendons and dermis (Kalluri and Zeisberg, 2006). However, this definition does not take into account the remarkable heterogeneity and plasticity of the fibroblast population. The heterogeneity of fibroblasts can be illustrated by the vast array of biomarkers, the expression of which can vary according to the tissue and even to the location within a given tissue (Figure 20B) (Chang et al., 2002). This is actually perfectly illustrated with the dermis, age in intracellular components over time generate an imbalance between synthetic and degradation pathways involved in skin ECM remodeling by promoting protease production, leading to a loss of ECM structure and organization. From Freitas Rodríguez et al, 2017.
which comprises different fibroblast subtypes that will be presented in section 2.2.3. Despite intensive investigations in the last decade, there is still no specific fibroblast biomarker identified and even less for each subpopulation of fibroblasts, since most of the biomarkers are also expressed in other cell types, such as perivascular, epithelial or immune cells (LeBleu and Neilson, 2020). This is a very limiting step that complicates fibroblast characterization. As an example, collagen I was first believed to be specific for fibroblasts, but it can also be synthesized by bone marrow-derived cells (Buchtler et al., 2018; Florin et al., 2004). So far, fibroblast-specific-protein 1 (FSP1) has been shown to provide the best specificity for fibroblast detection in vivo (Strutz et al., 1995). Therefore, isolation of fibroblasts from tissues still uses the combination of both inclusive and exclusive biomarkers (LeBleu and Neilson, 2020).

Table of contents :

I. Introduction and objectives
1.4.1. Structure and main components
1.4.2. Dermal vascular, lymphatic and nervous systems
1.4.3. Skin appendages
The dermis: a highly dynamic and complex compartment .
2.1.1. An introduction to the extracellular matrix components: the matrisome
2.1.2. Composition and structure of the dermal interstitial matrix
2.1.3. Remodeling of the ECM
2.2.1. Fibroblasts are heterogeneous and plastic cells
2.2.2. Fibroblast activation and regulation of ECM deposition ………..
2.2.3. The dynamic interplay between dermal fibroblasts, ECM and surrounding cells
2.2.4. Heterogeneity of dermal fibroblasts
3.2.1. History of the central transcription factor, NRF2
3.2.2. NRF2-KEAP1 structures and stress response pathway
3.2.3. NRF2 target genes and main functions
3.2.4. NRF2 additional functions through crosstalk with other signaling pathways.
3.2.5. Regulation of NRF2 activity
4.3.1. NRF2 contributes to the maintenance of the epidermal barrier function
4.3.2. NRF2 activity is associated with skin fibrosis
4.3.3. The dual role of NRF2 in skin cancer
II. Results
1. Part 1 « The transcription factor NRF2 regulates matrisome gene expression and collagen fibrillogenesis in human skin fibroblasts “
Manuscript in preparation
Part 2 “Role of AMPK in dermis”
Additional results
III. General discussion and perspectives
References ….

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