Molecular insights in the pathogenesis of classical Ehlers-Danlos syndrome from transcriptome-wide expression profiling of patients’ skin fibroblasts

Abstract

Classical Ehlers-Danlos syndrome (cEDS) is a dominant inherited connective tissue disorder mainly caused by mutations in the COL5A1 and COL5A2 genes encoding type V collagen (COLLV), which is a fibrillar COLL widely distributed in a variety of connective tissues. cEDS patients suffer from skin hyperextensibility, abnormal wound healing/atrophic scars, and joint hypermobility. Most of the causative variants result in a non-functional COL5A1 allele and COLLV haploinsufficiency, whilst COL5A2 mutations affect its structural integrity. To shed light into disease mechanisms involved in cEDS, we performed gene expression profiling in skin fibroblasts from four patients harboring haploinsufficient and structural mutations in both disease genes. Transcriptome profiling revealed significant changes in the expression levels of different extracellular matrix (ECM)-related genes, such as SPP1, POSTN, EDIL3, IGFBP2, and C3, which encode both matricellular and soluble proteins that are mainly involved in cell proliferation and migration, and cutaneous wound healing. These gene expression changes are consistent with our previous protein findings on in vitro fibroblasts from other cEDS patients, which exhibited reduced migration and poor wound repair owing to COLLV disorganization, altered deposition of fibronectin into ECM, and an abnormal integrin pattern. Microarray analysis also indicated the decreased expression of DNAJB7, VIPAS39, CCPG1, ATG10, SVIP, which encode molecular chaperones facilitating protein folding, enzymes regulating post-Golgi COLLs processing, and proteins acting as cargo receptors required for endoplasmic reticulum (ER) proteostasis and implicated in the autophagy process. Patients’ cells also showed altered mRNA levels of many cell cycle regulating genes including CCNE2, KIF4A, MKI67, DTL, and DDIAS. Protein studies showed that aberrant COLLV expression causes the disassembly of itself and many structural ECM constituents including COLLI, COLLIII, fibronectin, and fibrillins. Our findings provide the first molecular evidence of significant gene expression changes in cEDS skin fibroblasts highlighting that defective ECM remodeling, ER homeostasis and autophagy might play a role in the pathogenesis of this connective tissue disorder.

Cellular and Molecular Mechanisms in the Pathogenesis of Classical, Vascular, and Hypermobile Ehlers‒Danlos Syndromes

“…Our in vitro findings demonstrated that cEDS patients’ fibroblasts show disassembly of many ECM components, including collagen V and III, fibronectin, and fibrillins, and disorganization of collagen- and fibronectin-specific α2β1 and α5β1 integrin receptors [44,45,46,47]. cEDS cells also exhibit a reduced in vitro migration capability, an abnormal wound healing response, and a crosstalk involving the αvβ3 integrin and epidermal growth factor (EGF) receptor that rescues them from anoikis [44,45,46,47,48,49,50]. In line with these in vitro findings, Col5a1 and Col5a2 deficient mice show a defective wound healing response and reduced cell migration [51,52].…”

“…Transcriptome profiling of cEDS fibroblasts added new insights into the complex molecular mechanisms involved in the maintenance of ECM homeostasis and proper wound healing, since patients’ cells showed the dysregulated expression of many genes encoding matricellular and soluble proteins with prominent functions in cell proliferation and migration, collagen assembly and ECM remodeling during wound healing, i.e., SPP1 , POSTN , EDIL3 , IGFBP2 , and C3 [47]. Wound healing is a highly controlled multistep process involving several growth factors, cytokines, matrix metalloproteases, and cellular receptors, as well as proper crosstalk of different ECM constituents essential for ensuring tissue regeneration [53].…”

“…In cEDS fibroblasts, a possible unbalance of ER homeostasis and autophagy was assumed given the decreased expression of many associated genes such as DNAJB7 , ATG10 , CCPG1 , and SVIP [47]. DNAJB7 encodes a member of the J protein/heat shock protein family acting as ER chaperones in the quality control of aggregate protein [81].…”

Hypermobile Ehlers‐Danlos syndromes: Complex phenotypes, challenging diagnoses, and poorly understood causes

“…Various types of EDS have shown changes in αvβ3, α5β1, and α2β1 integrin expression in dermal fibroblasts isolated from patients. 231,[251][252][253][254] The αvβ3 integrin is widely expressed in endothelial cells, which interacts with several proteins including vitronectin, laminin, fibronectin, fibrillin as well as collagen. Recruitment of αvβ3 integrin is also an indicator of fibroblast activation.…”

“…They link the ECM proteins to the cytoskeleton and serve as mechanotransducers for various cellular process, including development, cell proliferation, cell movement and tissue homeostasis. Various types of EDS have shown changes in αvβ3, α5β1, and α2β1 integrin expression in dermal fibroblasts isolated from patients 231,251‐254 . The αvβ3 integrin is widely expressed in endothelial cells, which interacts with several proteins including vitronectin, laminin, fibronectin, fibrillin as well as collagen.…”