Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Developmental, Regenerative and Stem Cell Biology

Language

English (en)

Date of Award

Summer 9-1-2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Fanxin Long

Abstract

Differentiation and cell-specific functions are coupled with metabolic alterations to meet the needs of the cell. In this thesis, I have investigated the alterations of cellular metabolism in osteoblast-lineage cells in response to two different bone anabolic signals, WNT and PTH. I have further elucidated the mechanism underlying the metabolic changes, and have explored the functional importance of such changes for bone anabolism.

Osteoblasts, the principal bone-forming cells, are differentiated from mesenchymal progenitor cells through sequential stages. These stages are identifiable by molecular markers, cell morphology and location. Transcription factors and developmental signals important for osteoblast differentiation have been studied in detail. One such developmental signal is the WNT family of proteins. WNTs are a large family of glycoproteins that activate beta-catenin-dependent or -independent intracellular pathways, both of which are involved in bone formation. However, the mechanism through which WNT signaling stimulates osteoblast differentiation is not well understood.

Early studies demonstrated that bone cells consume a large amount of glucose, producing lactate as the major end product even in aerobic conditions, a phenomenon known as aerobic glycolysis. However, the significance of increased aerobic glycolysis for bone formation was not known. Based on the link between metabolic abnormalities and the genetic mutations in WNT pathway components, I hypothesized that WNT regulates cellular metabolism and that such regulation contributes to osteoblast differentiation. I tested this hypothesis in vitro by using ST2 cells, and showed that WNT signaling increased glucose utilization, stimulated aerobic glycolysis via induction of glycolytic enzymes, and suppressed glucose entry to TCA cycle. This process was mostly regulated by a signaling cascade dependent on Lrp5-Rac1-mTORC2 and independent of beta-catenin. Increased glycolysis was important for in vitro osteoblast differentiation and correlated with increased bone formation in WNT hyperactivation mouse models. I tested the functional importance of enhanced aerobic glycolysis in vivo by two different models. First, I showed that pharmacological enhancement of pyruvate entering the TCA cycle attenuated the high-bone mass phenotype caused by hyperactive WNT signaling in the mouse. Second, I showed that genetic deletion of LDHA, the enzyme catalyzing the last step of glycolysis, from osteoblast-lineage cells suppressed normal postnatal bone accrual due to reduced osteoblast number and function. Thus, WNT signaling reprograms glucose metabolism, and WNT-induced metabolic reprogramming contributes to osteoblast differentiation both in vitro and in vivo. Moreover, LDHA is required for optimal bone formation in postnatal mice.

Parathyroid hormone (PTH) has been an effective bone anabolic drug in the clinic by targeting osteoblasts and stimulating bone formation. However, it is not well understood how PTH signaling stimulates bone formation. In early studies, PTH was shown to alter cellular metabolism towards lactate production. In light of the role of metabolic regulation in WNT-induced bone formation, I examined the potential role of metabolic alterations in mediating the anabolic effect of PTH. In MC3T3-E1 cells and neonatal calvarial cells, I showed that PTH enhanced glucose uptake and aerobic glycolysis, activated pentose phosphate pathway but reduced contribution of glucose to TCA cycle. PTH-induced glucose utilization required IGF-PI3K-SGK1 signaling. Importantly, pharmacological enhancement of pyruvate entering the TCA cycle attenuated the bone anabolic effect by PTH. Thus, changes in cellular glucose metabolism may be an important mechanism mediating the anabolic effect of PTH.

This thesis confirms the earlier findings that lactate-producing glycolysis is an important feature of osteoblasts, and further characterizes the alterations of cellular metabolism during osteoblast differentiation in response to both WNT and PTH pathways. More importantly, this thesis shows for the first time that metabolic alterations are functionally important for the differentiation process.

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Permanent URL: http://dx.doi.org/10.7936/K7348HCQ

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