Collagenase degradation in extracellular matrix in aging

描述（由申请人提供）：人体皮肤，像所有其他器官一样，由于衰老而发生变化。老化皮肤的功能下降很大程度上是由纤维胶原蛋白的不可逆破坏引起的，纤维胶原蛋白是皮肤结缔组织（真皮）的主要结构蛋白。随着美国人口老龄化，胶原蛋白丧失、皮肤结缔组织功能下降引起的发病率日益成为公众健康关注的问题。这项拨款申请的长期目标是了解在衰老过程中纤维胶原蛋白降解的分子机制，从而开发预防和治疗方法，以改善衰老人类皮肤的健康。我们发现，与体内年轻（18-29岁）的人皮肤真皮成纤维细胞相比，老年人（50 - 80岁）的基质金属蛋白酶-1 (MMP-1)，负责启动纤维性胶原蛋白切割的酶显著升高。真皮成纤维细胞过量产生MMP-1导致胶原原纤维断裂和解体。胶原原纤维结构完整性的丧失是与年龄相关的人类皮肤结缔组织功能损伤的关键因素。这项拨款申请的具体重点是研究导致人类皮肤结缔组织中年龄依赖性MMP-1过表达的分子机制。根据我们通过直接测量年轻人和老年人皮肤获得的初步数据，我们假设衰老结缔组织中MMP-1水平的升高是由四种MMP-1调节因子的相互作用引起的：1)转录因子AP-1, 2) alpha2beta1整合素，3)转化生长因子beta1和4)活性氧。这四个因素通过真皮成纤维细胞与皮肤结缔组织的胶原细胞外基质的物理相互作用而受到机械张力的协调调节。mmp -1介导的胶原纤维断裂导致真皮成纤维细胞内机械张力减弱。这种减弱的机械张力促进了MMP-1的进一步表达，从而启动了皮肤结缔组织破坏的正反馈途径。我们的具体目标是测试这个工作模型，使用两种实验方法；1)直接测量来自不同年龄组个体的人体皮肤小样本中的相关转录本、蛋白质和酶活性；2)在三维胶原晶格成纤维细胞培养模型中对MMP-1调控的分子分析，概括了在体内老年人类皮肤结缔组织中观察到的MMP-1过表达的显著特征。通过系统整合这两种实验方法，我们将直接研究人类最大器官皮肤的衰老过程。鉴于结缔组织生物学在全身都是相似的，我们的结果可能直接适用于许多器官系统，包括肺、骨、关节和心脏。除了测试我们关于年龄依赖性MMP-1调节机制的假设外，我们提出的研究将提供直接的定量测量，以解决“衰老何时开始？”的问题，因为它与人类皮肤结缔组织有关。

DESCRIPTION (provided by applicant): Human skin, like all other organs, undergoes alterations as a consequence of aging. Reduced function of aged skin is largely caused by irreversible destruction of fibrillar collagen, the major structural protein in skin connective tissue (dermis). As the US population ages, morbidity from loss of collagen decline of skin connective tissue function is becoming an increasing public health concern. The long-term objective of this grant application is to understand molecular mechanisms that are responsible for degradation of fibrillar collagen during aging, and thereby develop preventative and therapeutic remedies to improve the health of aged human skin. We have found that matrix metalloproteinase-1 (MMP-1), the enzyme responsible for initiating cleavage of fibrillar collagen is significantly elevated in aged (>80 years old), compared to young (18-29 years old) human skin dermal fibroblasts in vivo. Overproduction of MMP-1 by dermal fibroblasts causes fragmentation and disorganization of collagen fibrils. This loss of structural integrity of collagen fibrils is a critical factor in the age-related functional impairment of human skin connective tissue. The specific focus of this grant application is to investigate molecular mechanisms that cause this age-dependent MMP-1 overexpression in human skin connective tissue. Based on our preliminary data obtained by direct measurements of young and aged human skin, we hypothesize that elevated MMP-1 levels in aged connective tissue results from the interdependent actions of four MMP-1 regulators: 1) transcription factor AP-1, 2) alpha2beta1 integrin, 3) transforming growth factor-beta1, and 4) reactive oxygen species. These four factors are coordinately regulated through mechanical tension exerted on dermal fibroblasts by its physical interactions with the collagenous extracellular matrix of skin connective tissue, in which they reside. MMP-1-mediated collagen fibril fragmentation results in weakened mechanical tension within dermal fibroblasts. This weakened mechanical tension promotes further expression of MMP-1, and thereby sets in motion a positive feedback pathway of skin connective tissue destruction. Our specific aims are designed to test this working model, using two experimental approaches; 1) direct measurements of relevant transcripts, proteins, and enzyme activities in small samples of human skin from individuals of different age groups, and 2) molecular analysis of MMP-1 regulation in a three dimensional collagen lattice fibroblast culture model, which recapitulates the salient features of MMP-1 overexpression observed in aged human skin connective tissue in vivo. By using these two experimental approaches in a systematic integrated manner, we will investigate the aging process directly in human's largest organ, skin. Given that connective tissue biology is similar throughout the body, our results will likely be directly applicable to many organ systems, including lung, bone, joints, and heart. In addition to testing our hypothesis regarding the mechanism of age-dependent regulation of MMP-1, our proposed studies will provide direct quantitative measures that address the question of "when does aging begin?", as it pertains to human skin connective tissue.