MicroRNA-145-5p regulates fibrotic features of recessive dystrophic epidermolysis bullosa skin fibroblasts.

BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is a skin fragility disorder caused by mutations in the COL7A1 gene encoding type VII collagen, a cutaneous basement membrane component essential for epidermal-dermal adhesion. Hallmarks of the disease are unremitting blistering and chronic wounds with severe inflammation and fibrosis. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression also implicated in fibrotic processes. However, the role of miRNAs in RDEB fibrosis is almost unexplored. OBJECTIVES: Our study aimed to identify miRNAs deregulated in primary RDEB skin fibroblasts (RDEBFs) and to characterize their function in RDEB fibrosis. METHODS: Real-time quantitative polymerase chain reaction (qRT-PCR) was used to screen RDEBFs for expression levels of a group of miRNAs deregulated in hypertrophic scars and keloids, pathological conditions with abnormal wound healing and fibrosis. Contractility, proliferation and migration rate were evaluated by different in vitro assays in RDEBFs transfected with a miR-145-5p inhibitor. Expression levels of fibrotic markers and miR-145-5p targets were measured using qRT-PCR and western blot. RESULTS: The miR-143/145 cluster was upregulated in RDEBFs compared with fibroblasts from healthy subjects. RDEBFs transfected with a miR-145-5p inhibitor showed attenuated fibrotic traits of contraction, proliferation and migration, accompanied by reduced expression of the contractile proteins α-smooth muscle actin and transgelin. These effects were associated with upregulation of Krüppel-like factor 4 transcriptional repressor and downregulation of Jagged1, a known inducer of fibrosis. CONCLUSIONS: Our results highlight the profibrotic role of miR-145-5p and its regulatory networks in RDEB, shedding light on novel disease pathomechanisms and targets for future therapeutic approaches. What's already known about this topic? Recessive dystrophic epidermolysis bullosa (RDEB) is a highly disabling genetic skin disease caused by mutations in the collagen VII gene and characterized by unremitting blistering and defective wound healing, leading to inflammation and fibrosis. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression in health and disease, and their deregulation has been implicated in fibrotic skin conditions. To date, only miR-29 has been associated with injury-driven fibrosis in RDEB. What does this study add? In patients with RDEB, miR-145-5p is overexpressed in RDEB skin fibroblasts (RDEBFs), where it plays a profibrotic role, as its inhibition reduces RDEBF fibrotic traits (contraction, proliferation and migration). miR-145-5p inhibition in RDEBFs determines the reduction of contractile markers α-smooth muscle actin and transgelin through upregulation of Krüppel-like factor 4, a transcriptional repressor of contractile proteins, and downregulation of Jagged1 (JAG1), an inducer of fibrosis. What is the translational message? Our findings expand the knowledge on miRNA-driven pathomechanisms implicated in RDEB fibrosis. miR-145-5p and its targets (e.g. JAG1) could represent relevant molecules for the development of novel therapeutic strategies to counteract fibrosis progression in patients with RDEB.

Lack of K140 immunoreactivity in junctional epidermolysis bullosa skin and keratinocytes associates with misfolded laminin epidermal growth factor-like motif 2 of the ß3 short arm.

Recessive mutations in the LAMA3, LAMB3 and LAMC2 genes that encode laminin-332 (LM332) (α3a, ß3 and γ2 chains, respectively) cause different junctional epidermolysis bullosa (JEB) subtypes. Biallelic truncating mutations in any of these three genes usually lead to lack of protein expression resulting in the severe generalized JEB subtype, while missense or splice-site mutations in at least one allele lead to reduced expression typical of JEB generalized intermediate (JEB-gen intermed) or localized. Here, we molecularly characterized an adult patient with JEB showing negative skin staining for the anti-ß3 chain monoclonal antibody K140. This antibody recognizes an as yet unidentified epitope within the laminin ß3 short arm. The patient harbours a homozygous splice-site mutation resulting in highly aberrant transcripts with partial skipping of the LAMB3 exon that encodes the laminin epidermal growth factor-like motif 2 of the ß3 short arm (ß3-LE2). At the protein level, mutation consequences predict a misfolded ß3-LE2 motif and, indeed, we found that LM332 is correctly assembled but retained in the endoplasmic reticulum (ER) where it colocalizes with the lumenal ER chaperone protein BiP, leading to dramatically reduced secretion. Lack of K140 reactivity to mutant LM332 was confirmed by immunoprecipitation and Western blot analyses. Our findings not only identify the ß3-LE2 subdomain as the region recognized by K140, but also show that misfolding of LM332 structural motifs and subsequent protein retention in the ER is a common pathomechanism in JEB-gen intermed. In addition to its usefulness in antigen mapping diagnosis of JEB subtypes, this knowledge is relevant to the design of therapeutic strategies aimed at releasing ER-retained LM332 in JEB.