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

基底膜VII型胶原在衰老皮肤异常分化和细胞命运决定中的作用

• 批准号: BB/H016465/1
• 金额: $ 10.61万
• 依托单位: Queen Mary University of London
• 依托单位国家: 英国
• 项目类别: Training Grant
• 财政年份: 2010
• 资助国家: 英国
• 起止时间: 2010 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FH016465%2F1
• 关键词: role; basement; membrane; type; VII

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.

VII型胶原（CollVII）是人上皮中的基底膜（BM）蛋白，其是参与表皮-真皮粘附的锚定纤维（AF）结构的主要组分。在老化皮肤中，累积的紫外线暴露导致异常分化，干细胞标志物表达减少和BM损伤（1，2）。已知来自老年个体的角质形成细胞和成纤维细胞分泌较少的CollVII，并且分泌的CollVII对细胞因子调节的响应性较低（3）。我们最近的工作表明，鳞状细胞癌细胞中CollVII的敲低导致三维培养中上皮细胞的分化明显紊乱，重组CollVII可逆转（4）。已知局部使用维甲酸（维生素A）可能通过抑制胶原酶表达使人AF密度加倍（5）。我们将探讨这一假设，减少ColVII在正常的人类衰老过程中的异常分化和细胞命运的决定，增加内源性维生素A可能会逆转这一过程。原代角质形成细胞将来源于年轻人（n=5< 30岁）和老年人（n=5 >65岁）的多余皮肤，并获得适当的伦理批准。将通过免疫印迹对来自细胞和培养基的CollVII进行定量。器官型3-D表皮培养物（OTC）将在分散酶处理（去除CollVII）的去表皮真皮（DED）上生成。将对增殖、滤泡间干细胞、凋亡以及早期和晚期分化标志物进行免疫染色。将在年轻和年老的细胞中进行靶向siRNA以完全耗尽CollVII，以观察年老的细胞是否更容易发生异常分化。重组CollVII也将被注射到旧细胞OTC的DED中，以观察这是否逆转了该过程。视黄酸代谢结合剂（RAMBA），如利阿唑和他拉罗唑，阻断内源性维生素A的催化，从而增加内源性细胞维生素A分泌。将年轻和年老的原代角质形成细胞与增加剂量的RAMBA一起孵育，以观察这是否在年老角质形成细胞与年轻角质形成细胞中差异性地增加CollVII分泌。视黄酸将用作额外对照。将用DED上的角质形成细胞进行类似的实验，以观察是否存在CollVII在基底膜处的增加的沉积和通过免疫染色评估的改变的分化。由于表观遗传机制可能在类维生素A诱导的分化中起作用，因此将年轻细胞和老年细胞的三份生物学重复暴露于他拉罗唑，并使用Illumina Infinium HumanMethylation 27 BeadChip Array评估全基因组甲基化。在与芯片杂交之前，DNA将被亚硫酸氢盐修饰。同时，将进行全基因组表达阵列。将使用Illumina软件和Inflamity Pathway分析对数据进行分析，专门筛选与胶原蛋白、核激素受体途径和分化相关的变化。定量RT-PCR将用于验证差异表达的基因。这些数据应该阐明CollVII在老化表皮中的功能。1. Craven NM等人，光损伤人类皮肤的临床特征与VII型胶原蛋白减少有关。英国皮肤病学杂志。1997;137（3）：344-50. 2. Giangreco等人，人类皮肤老化与干细胞标记物表达减少有关。《皮肤病学杂志》2009年9月24日Chen YQ等，Type VII collagen gene expression by human skin fibroblasts and keratinocytes in culture：Influence of donor age and cytokine responses.《皮肤病学杂志》1994;102（2）：205-9. 4. Martins VL et al. Increased invasive behavior in skin squamous cell carcinoma with loss of BM type VII collagen.细胞科学杂志2009;122：1788-99. 5. Woodley DT等人，局部用维甲酸治疗光老化皮肤可增加表皮-真皮锚定原纤维。美国医学会杂志1990;263（22）：3057-9.