Elucidating the role of YAP in directing mesenchymal stem cell fate
Mesenchymal stem cells (MSCs) are a type of multipotent stem cell capable of differentiating into several cell types, including fat and bone cells. The Hippo pathway effectors TAZ and YAP have been implicated as important regulators of MSC fate, but their roles in this process are poorly understood. The goal of my thesis work was to illuminate the roles of YAP and TAZ in MSC differentiation. I examined how depleting YAP and/or TAZ, or expressing YAP mutants affect the differentiation of C3H10T1/2 cells, which are a multipotent mouse embryo fibroblast cell line capable of forming bone, fat and cartilage. Interestingly, knocking down either YAP or TAZ had different effects on C3H10T1/2 differentiation. YAP knockdown cells that underwent a brown/beige adipogenic protocol showed a significant increase in the amount of lipids produced as compared to control, suggesting that YAP has a role in inhibiting adipogenesis in these cells. YAP knockdown also increased alkaline phosphatase activity in cells subjected to an osteogenic protocol, while simultaneously producing lipid droplets. In contrast, knockdown of TAZ resulted in a decrease in both lipogenesis (in adipogenic differentiation) and alkaline phosphatase activity (in osteogenic differentiation). These observations indicate that depletion of YAP or TAZ leads to defective MSC differentiation, and that without proper YAP and TAZ signaling MSC cells may arrest in an immature state. Bone Morphogenic Proteins (BMPs) are known to play important roles in MSC differentiation, and YAP/TAZ have been implicated in BMP regulation in other systems. I therefore hypothesized that YAP/TAZ may affect MSC differentiation in part by altering BMP signaling. Analysis of activated phosphorylated Smad1 (p-Smad1), a key transcriptional effector of BMP signaling, showed that TAZ and YAP are required to promote p-Smad1 levels. Depletion of TAZ and YAP showed an increase in the expression of Gremlin1 (GREM1), an inhibitor of BMP signaling, and an increase in the expression of BMP4. These observations suggest that TAZ and YAP promote BMP signaling by controlling BMP ligand activity. Given that BMP signaling has important roles early in MSC differentiation, I hypothesized that YAP and TAZ may direct these early processes. To test this, I generated dox-inducible C3H10T1/2 cells capable of expressing wild type YAP or two mutant forms of YAP: a nuclear localized YAP mutant (YAP-5SA), and a transcriptionally inactive YAP mutant (YAP-5SA,S94A). Adipogenic differentiation with these cells showed a reduced level of lipogenesis in the constitutively active nuclear YAP mutant. Analysis of adipogenic markers by qPCR showed a reduction in both PPARγ and UCP1. Osteogenic differentiation experiments in these cells showed that transient expression of all forms of YAP increased the amount of alkaline phosphatase activity, with the 5SA form showing the greatest activity. RNA expression analysis showed that the early marker Runx 2 was highly upregulated in cells overexpressing wild type YAP, while the late markers osteocalcin and osteopontin were greatly reduced in comparison to control. As alkaline phosphatase activity is a mid-stage marker for bone differentiation, the staining results and qPCR data indicate that overexpression of YAP may result in arrest of the differentiation process. A microarray was performed using isolates from 10T1/2 cells transduced with TAZ, YAP or TAZ/YAP siRNA to further evaluate the mechanism through which TAZ and YAP may be acting. The TAZ and YAP double knockdown showed a large change in global gene expression as compared to both the control and the single knockdowns. Among the genes that showed a large change from control was BMP4, the expression of which was reduced more than two-fold over control. Additionally, GREM1 was up-regulated almost two-fold by the double knockdown. Also upregulated was the gene periostin which encodes a protein that enhances BMP incorporation into connective tissues, and may play a role in the mineralization of the extracellular matrix of bone. Based on my observations, along with those of others, I propose that TAZ and YAP are major factors in controlling early cell fate determination of MSCs. There appear to be multiple levels of regulation, and thus the next steps should include clarifying the roles of YAP and TAZ in vivo, and placing their activity chronologically in the differentiation timeline. Such studies will offer insight into stem cell differentiation and may eventually allow for therapeutic opportunities.