The role of basement membrane type VII collagen in aberrant differentiation and cell fate determination in ageing skin

Type VII collagen (CollVII) is a basement membrane (BM) protein in human epithelia that is the main component of anchoring fibrils (AF) structures involved in epidermal-dermal adherence. In ageing skin, cumulative ultraviolet light exposure results in aberrant differentiation, reduced expression of stem cell markers and damage to the BM(1,2). Keratinocytes and fibroblasts from older individuals are known to secrete less CollVII and secreted CollVII is less responsive to cytokine modulation(3). Our recent work showed that knock-down of CollVII in squamous cell carcinoma cells results in markedly disorganized differentiation of epithelium in 3-D cultures reversible by recombinant CollVII(4). Topical tretinoin (Vitamin A) is known to double human AF density, possibly by inhibiting collagenase expression(5). We will explore the hypothesis that reduction of ColVII during normal human ageing results in aberrant differentiation and cell fate determination and that increasing endogenous vitamin A might reverse this process. Primary keratinocytes will be derived from redundant skin from young (n=5< 30 years) and older individuals (n=5 >65 years), with appropriate ethical approval. CollVII from cells and media will be quantitated by immunoblotting. Organotypic 3-D epidermal cultures (OTC) will be generated on dispase-treated (removes CollVII) de-epidermalised dermis (DED). Immunostaining will be performed for proliferation, interfollicular stem cell, apoptotic and early and late differentiation markers. Targeted siRNA to completely deplete CollVII will be performed in young and old cells to see if older cells are more susceptible to aberrant differentiation. Recombinant CollVII will also be injected into the DED of old cell OTCs to see if this reverses the process. Retinoic acid metabolism binding agents (RAMBAs), such as liarozole and talarozole, block the catabolism of endogenous vitamin A, thus increasing endogenous cellular vitamin A secretion. Young and old primary keratinocytes will be incubated with increasing doses of the RAMBAs to see if this increases CollVII secretion differentially in old versus young keratinocytes. Retinoic acid will be used as an additional control. Similar experiments will be performed with keratinocytes on DED to see if there is increased deposition of CollVII at the basement membrane and altered differentiation as assessed by immunostaining. As epigenetic mechanisms may play a role in retinoid-induced differentiation, triplicate biological replicates of young and old cells will be exposed to talarozole and whole-genome methylation will be assessed using the Illumina Infinium HumanMethylation 27 BeadChip Array. DNA will be bisulphite modified prior to hybridization to the chip. In parallel, whole genome expression arrays will be performed. Data will be analysed using Illumina software and Ingenuity Pathway analyses, screening specifically for changes related to collagens, nuclear hormone receptor pathways and differentiation. Quantitative RT-PCR will be used to verify differentially expressed genes. These data should shed light on the function of CollVII in ageing epidermis. 1.Craven NM et al. Clinical features of photodamaged human skin are associated with a reduction in collagen VII. Br J Dermatol. 1997;137(3):344-50. 2.Giangreco et al. Human Skin Aging Is Associated with Reduced Expression of the Stem Cell Markers. J Invest Dermatol. 2009 Sep 24. in press 3.Chen YQ et al. Type VII collagen gene expression by human skin fibroblasts and keratinocytes in culture: influence of donor age and cytokine responses. J Invest Dermatol. 1994;102(2):205-9. 4.Martins VL et al. Increased invasive behaviour in cutaneous squamous cell carcinoma with loss of BM type VII collagen. J Cell Sci. 2009;122:1788-99. 5.Woodley DT et al. Treatment of photoaged skin with topical tretinoin increases epidermal-dermal anchoring fibrils. JAMA. 1990;263(22):3057-9.