ABSTRACT Osteoarthritis (OA) is the most prevalent joint disease. Although aging represents one of the most important risk factors for OA, mechanisms leading to the aging-related cartilage degeneration remain to be determined. We reported that cellular homeostasis mechanisms such as autophagy and oxidant defenses are compromised in aging and OA-affected cartilage. In our recent studies we investigated FoxO transcription factors, which regulate expression of autophagy proteins and autophagy activation. FoxO3a in particular is known as molecular gatekeeper of cellular aging. We observed a reduction in FoxO mRNA and protein expression in aging and OA-affected cartilage in humans and mice. These findings support the hypothesis that ?Aging-related reduction of FoxO expression impairs protective cellular homeostasis mechanisms, compromises chondrocyte survival and biosynthetic capacity and leads to accelerated joint aging and initiation of OA pathogenesis?. This hypothesis will be tested in the following aims. Aim 1: Regulation of FoxO expression and function in chondrocytes. We will examine the extracellular regulators and mechanisms of FoxO suppression or activation and identify the responsible cis-elements in the FoxO promoters. Using in vitro models of FoxO knock down and overexpression in normal and OA human chondrocytes and cartilage explants from FoxO deficient mice we will examine the role of FoxO in regulating chondrocyte functions. Aim 2: The FoxO signaling network in cartilage. FoxO target genes are tissue and context specific. We will establish FoxO target genes in mouse cartilage and human chondrocytes by using RNA-seq, ChIP-seq and ChIP-seq for histone marks. Integrative analysis of these datasets will identify pathways and signaling mechanisms that are regulated by FoxO. Aim 3: Role of FoxO in cartilage homeostasis, aging and experimental OA. Our preliminary studies show that conditional cartilage specific deletion of FoxO1 leads to abnormal cartilage growth postnatally and spontaneous OA-like degradation by 6 months. We will generate postnatal triple and single knock out mice using Acan-CreER and test them for aging related changes and severity of experimental OA. Aim 4: Protective effects of FoxO overexpression and activation We will generate mice that overexpress active forms of FoxO1 or FoxO3 in cartilage to balance the OA associated suppression and determine outcomes with respect to homeostasis mechanisms and OA severity in the aging and surgical models. This project will establish that aging-related reduction in FoxO expression is an early and critical event in OA pathogenesis. This will provide the foundation for therapeutic approaches aimed at modulating FoxO expression and/or activity to prevent age-related and injury-induced onset and progression of OA. !