Defining Vitamin C–Dependent Pathways in Extracellular Matrix Synthesis and Cell-Fate Transitions

About This Grant

Project Summary: Nutrients are important regulators of cell states and cell fate transitions. In this context, Vitamin C (VitC), an essential nutrient, is an important regulator of extracellular matrix (ECM) synthesis and of multiple cell fate transitions, including somatic cell to induced pluripotent stem cell (iPSC) reprogramming. However, the molecular mechanisms underlying these effects remain poorly understood. Prior studies have largely attributed VitC function to the regulation of Fe2+/α-ketoglutarate-dependent dioxygenases (αKGDDs). Yet, several studies report only minimal VitC-dependent changes in collagen hydroxylation, and our preliminary data indicate that VitC- dependent enhancement of iPSC reprogramming occurs independently of αKGDD activity. Instead, we discovered that VitC rapidly activates the oxidative pentose phosphate pathway (PPP), increasing NADPH and ribose-5-phosphate (R5P) levels, and enhances nucleotide sugar synthesis. We also found that VitC increases total ECM glycosylation, including N-linked glycosylation of collagen I (Col I) and P4HA1, the prolyl hydroxylase required for collagen secretion. We hypothesize that VitC-dependent metabolic rewiring promotes glycosylation- dependent ECM synthesis, and regulates transcriptional programs to facilitate iPSC reprogramming. The proposed research aims to understand the molecular mechanisms by which VitC regulates ECM synthesis and cell-fate transitions. AIM 1 will elucidate the molecular mechanisms of VitC-dependent metabolic rewiring and provide training in genetic manipulation and metabolite tracing techniques to enable mechanistic studies of metabolic pathway regulation. AIM 2 will determine the role of VitC-driven metabolic changes in regulating ECM synthesis, and in regulating transcriptional programs during iPSC reprogramming, and provide necessary training in single-cell RNA-seq approaches to define how metabolic and ECM changes influence transcriptional trajectories and cell fate transitions. Finally, the independent phase of this award, AIM 3, will elucidate the mechanisms by which VitC-dependent metabolic rewiring regulates ECM protein glycosylation and secretion. This work will reveal novel pathways linking nutrient-dependent metabolic rewiring to ECM remodeling and cell state regulation, with broad implications for understanding ECM-related diseases such as fibrosis and for nutrient-driven control of cell fate. The mentored phase of this award provides the opportunity to work with leaders in the fields of cell-fate regulation and metabolism, to acquire the training needed to achieve my long- term goal of becoming an independent investigator at the interface of both fields. Together, the proposed research, mentorship team, and resources provided for career development at University of California, Los Angeles, will provide the training needed to achieve the defined career goals.