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. ? ? ?
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