Collagenase degradation in extracellular matrix in aging

描述(申请人提供)：人的皮肤，像所有其他器官一样，会因老化而发生变化。衰老皮肤的功能减退很大程度上是由于皮肤结缔组织(真皮)中的主要结构蛋白--纤维胶原的不可逆性破坏所致。随着美国人口老龄化，皮肤结缔组织功能下降引起的胶原蛋白丢失的发病率正日益成为公共卫生关注的问题。这项赠款申请的长期目标是了解导致纤维状胶原在衰老过程中降解的分子机制，从而开发预防和治疗措施，以改善人类老化皮肤的健康。我们发现，在活体中，与年轻(18-29岁)人皮肤成纤维细胞相比，老年(80岁)人皮肤成纤维细胞中负责启动纤维状胶原分解的酶-基质金属蛋白酶-1(MMP-1)显著升高。真皮成纤维细胞过度产生基质金属蛋白酶-1会导致胶原纤维的碎裂和解体。这种胶原纤维结构完整性的丧失是人类皮肤结缔组织与年龄相关的功能损害的关键因素。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这种年龄依赖性基质金属蛋白酶-1过度表达的分子机制。根据我们通过对年轻人和老年人皮肤的直接测量获得的初步数据，我们假设老年结缔组织中升高的基质金属蛋白酶-1水平是四种基质金属蛋白酶-1调节因子相互依赖的作用的结果：1)转录因子AP-1，2)α2β1整合素，3)转化生长因子-β1，和4)活性氧。这四种因子通过施加在真皮成纤维细胞上的机械张力和它们所在的皮肤结缔组织的胶原细胞外基质的物理作用来协调调节。基质金属蛋白酶-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基质金属蛋白酶-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。我们的具体目标是通过两种实验方法来测试这一工作模型：1)直接测量不同年龄组人皮肤小样本中的相关转录、蛋白质和酶活性，以及2)在三维胶原晶格成纤维细胞培养模型中对基质金属蛋白酶-1的调节进行分子分析，这概括了在活体中观察到的老年人皮肤结缔组织中基质金属蛋白酶-1过表达的显著特征。通过系统地综合使用这两种实验方法，我们将直接研究人类最大的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，我们的结果很可能直接适用于许多器官系统，包括肺、骨、关节和心脏。除了验证我们关于基质金属蛋白酶-1的年龄依赖性调节机制的假说外，我们提议的研究还将提供直接的定量测量方法，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.