Characterizing concordance in the secretome of recessive dystrophic epidermolysis bullosa and cellular senescence

About This Grant

Abstract Aging is a natural process resulting in the decline of tissue functionality. In the skin, aging results from degenerative changes in dermal and epidermal compartments. However, the molecular mechanisms behind skin aging are not fully understood. One of the poorly explored characteristics of the aged skin is the reduced number of anchoring fibrils, which connect the dermis to the epidermis, and the reduced level of their main protein component, type VII collagen (COL7A1), which is critical for the stability of the extracellular matrix. Interestingly, the skin of patients with recessive dystrophic epidermolysis bullosa (RDEB), a severe skin fragility condition caused by biallelic mutations in the gene COL7A1, shows clinical similarities with the skin of the elderly. This suggests that COL7A1 deficiency promotes premature aging in RDEB patients. However, it is not clear whether the pro-inflammatory background of chronic non-healing wounds is the only cause of premature skin aging in RDEB patients or functional COL7A1 plays an important, currently unknown role in protecting the skin from aging. My preliminary proteomic analysis of primary patient derived fibroblasts (COL7A1-/- PPF(s)), as well as patient specific organoid derived fibroblasts (iF(s)) generated from primary fibroblasts transitioned through induced pluripotency that were either uncorrected (COL7A1-/-) or genetically corrected using CRISPR/Cas9 (COL7A1+/-), revealed COL7A1 dependent perturbations in insulin-like binding protein 2 (IGFBP2) with COL7A1- /- PPF(s) and COL7A1-/- iF(s) showing marked deficiencies in its expression relative to genetically corrected iF(s). Therefore, I hypothesize that COL7A1 has a moonlighting role regulating fibroblast secretory profiles, and, in its deficiency, leads to accelerated cellular senescence and aberrant perturbations in fibroblast functionality, specifically through the IGF axis. Inhibition of IGFBP2 has been shown to lead to an overexpression of p21, p16, and p19, all powerful promotors of senescence and a potential mechanism by which premature acceleration senescence is induced in COL7A1 deficiency. During my fellowship, I will dissect the role of COL7A1 in senescence of skin cells. In aim 1, I will further analyze COL7A1 dependent perturbations in the proteome and secretome of COL7A1 deficient and COL7A1 corrected lines that are generated from induced pluripotent stem cells. Reprogramming into iPSCs erases aging associated marks in cells that arose from pro-inflammatory and fibrotic background and allows us to generate and characterize COL7A1 deficient and corrected cell lines without the influence of external factors that can mask the role of COL7A1 in skin aging. In aim 2, I will assess other age-associated marks in COL7A1 deficient and corrected cell lines to validate the connection between IGF signaling and COL7A1 functionality. If successful, this proposal will result in our better understanding of the role of COL7A1 in protecting the skin from aging and in developing novel therapies for RDEB and anti-aging interventions.