Browsing by Subject "Bone"

Osteoporosis and low bone density affect an estimated 54 million adults of 50 years and over in the United States, resulting in $19 billion in costs for osteoporosis-related bone breaks. Current treatments include the use of antiresorptive and anabolic drugs to decrease the rate of bone resorption and increase the rate of bone formation, respectively. However, these current treatments are unable to completely normalize skeletal integrity. As bone diseases become increasingly prevalent, there is an urgent need to identify novel therapies to improve quality of life and reduce economic burden on the healthcare system.

To identify novel therapeutic targets, we must first begin to understand the cellular complexity of the bone marrow niche and how cellular function is regulated within the bone tissue. Bone-resident cells, such as skeletal progenitors and their descendants, are critically influenced by extrinsic signals derived from the local microenvironment. Previous studies have identified hypoxia as a key microenvironment factor in bone. Thus, the ability to target the hypoxic bone marrow niche presents an attractive and untapped potential for regenerative medicine.

Much of the work investigating the role of hypoxia and HIF signaling have focused on mature osteoblast and chondrocyte populations. In contrast, studies investigating the contribution of HIF signaling on skeletal progenitors and marrow adipocyte populations are scarce. In this dissertation, I investigate the role of hypoxia and HIF signaling in skeletal lineage cells, chiefly skeletal progenitor cells and marrow adipogenic lineage cells. Using cellular, genetic, and pharmacological-based approaches, I characterize the roles of HIF-1α and HIF-2α in both homeostatic and pathological contexts in the aforementioned cell populations.

First, I propose an optimized cell-based system to investigate the function of skeletal progenitors in vitro. Here, I highlight the limitations of current in vitro isolation techniques and introduce a relatively simple method of bone marrow stromal cell purification using hypoxia. Using this system, I assess how skeletal progenitors respond to hypoxic cues and interrogate skeletal progenitor cell differentiation and functional responses in my subsequent research. Next, using genetic and pharmacological approaches, I investigate the role of HIF-2α in bone formation following radiation-injury where I identify HIF-2α as a negative regulator of bone recovery. Additionally, with the assistance of my collaborators, I develop and characterize a bone-targeting nanocarrier to ameliorate radiation-induced bone loss. Lastly, I detail early work I conducted to investigate the role of HIF signaling in marrow adipogenic lineage cells. Here, I establish and characterize animal models to determine how hypoxia and HIF signaling influences adipogenic lineage commitment and expansion in an early and mature marrow adipogenic population.

In summary, this dissertation aims to expand our limited understanding on how the hypoxic bone microenvironment and HIF signaling regulate skeletal lineage cells in vivo, with a special focus on skeletal progenitor and marrow adipogenic populations. In terms of boarder impacts, understanding the signaling networks that regulate bone homeostasis and recovery processes will not only expand our basic understanding of the molecular mechanisms underlying skeletal development, but also provide novel insights for developing therapies to treat bone loss.

Bone development and homeostasis are governed by a number of developmental signals, transcription factors and cellular metabolism. This process is also dependent on the orchestration of multiple cell types including osteoblasts, chondrocytes, skeletal stem cells and osteoclasts. Osteoblasts are the principal bone forming cells responsible for producing and secreting the type I collagen rich extracellular bone matrix. Protein synthesis is an energetically and biosynthetically demanding process. This requires copious amounts of ATP and amino acids amongst other metabolites. However, the precise mechanisms and systems that osteoblasts utilize to meet these synthetic demands are poorly understood. Previous studies have shown amino acid consumption is increased in osteoblasts during differentiation. This process is regulated by transcription factors ATF4 and FOXO. Additionally, osteogenic signals like WNT and PTH can stimulate amino acid uptake. For example, WNT signaling can rapidly stimulate glutamine uptake and metabolism required for osteoblast differentiation. Unfortunately, transporters mediating glutamine uptake in osteoblasts are unknown. Moreover, the mechanism by which WNT stimulates increased glutamine consumption is also unknown. We identified two amino acid transporters, Slc7a7 and Slc1a5, as the primary glutamine transporters in response to WNT. Slc7a7 is responsible for the rapid WNT-induced glutamine uptake via the -catenin dependent pathway. Conversely, Slc1a5 sustains basal glutamine uptake, which is regulated by ATF4 downstream of the mTORC1 pathway. In summary, these data demonstrate the biphasic role of WNT signaling in regulating glutamine consumption, by two amino acid transporters Slc7a7 and Slc1a5, during osteoblast differentiation. While we have shown the importance of glutamine in bone cells, the role of other amino acids is not clear. Proline has long been considered as a critical amino acid due to its enrichment in collagens. Furthermore, PTH stimulates proline consumption in osteoblasts. The transport of proline is characterized by its dependency on sodium and sensitivity to MEAIB. However, the precise transport system responsible for proline import is not known. Here we identified the amino acid transporter Slc38a2, which encodes SNAT2, as the primary proline transporter in osteoblasts. Deletion of Slc38a2 results in defects in both intramembranous and endochondral ossifications. The phenotype is associated with defective osteoblast differentiation highlighted by reduction of proline enriched proteins (e.g. RUNX2, OSX and COL1A1). Slc38a2 provides proline to support osteoblast differentiation through two mechanisms. First, majority of proline is directly incorporated into proteins and does not contribute to amino acid biosynthesis. Second, proline oxidation regulates bioenergetics required for osteoblast differentiation. These findings highlight the multifaceted functions of proline, which is provided by Slc38a2, in osteoblast differentiation and bone formation. Collectively, my work demonstrates the critical role of amino acid transporters in osteoblast differentiation and provides novel insights in their potential applications in treatments of bone diseases like osteoporosis and bone fracture.

Regeneration occurs throughout the animal kingdom and is a well-studied

feature of many model organisms, yet the field lacks a fundamental understanding of

the real-time dynamics of cell behavior during regeneration. I discuss how existing

knowledge of regeneration may be used to inform efforts to translate these remarkable

feats of animals to human regeneration and present research that uses live imaging to

improve understanding of cell origins and diversification during regeneration in the

scale, focusing specifically on osteoblasts the matrix-depositing cells that divide and heal

bone injuries. I developed an imaging platform to monitor and quantify individual and

collective behaviors of osteoblasts in adult zebrafish scales. I show that a founder pool

of osteoblasts emerges through de novo differentiation within one day of scale plucking,

then diversifies across the primordium by two days after injury, with region-specific

changes in proliferation, cell shape, and cell death rates coincident with acquisition of

mature scale morphology. I also demonstrate a role for Fgf signaling in scale

regeneration and present tools for high resolution imaging studies of basal epidermal

cells during skin and scale injury. These findings demonstrate the value of live imaging

in revealing novel biology and gaining a more complete picture of the many complex

processes that must be elegantly choreographed to achieve tissue regeneration.

Zebrafish regenerate amputated caudal fins, restoring the size and shape of the original appendage. Regeneration requires generation of diverse cell types comprising the adult fin tissue. Knowledge of the cellular source of new cells and the molecules involved is fundamental to our understanding of regenerative responses. In this dissertation, the contribution made by the bone cells towards fin regeneration is investigated. Fate mapping of osteoblasts revealed that spared osteoblasts contribute only to regenerating osteoblasts and not to other cell types, thereby suggesting lineage restriction during fin regeneration. The functional significance of osteoblast contribution to fin regeneration is tested by developing an osteoblast ablation tool capable of drug induced loss of bone cells. Normal fin regeneration in the absence of resident osteoblast population suggests that the osteoblast contribution is dispensable and provides evidence for cellular plasticity during fin regeneration. To uncover the genes involved in proliferation of osteoblasts within the fin regenerate, a candidate in-situ screen was carried out and revealed bone specific expression of fgfr4 and twist3. Transgenic tools for visualization of gene expression confirmed the screen results. Knockdown of twist3 by morpholino antisense technology impedes fin regeneration. Mutant heterozygotes for twist3 were generated using genome editing reagents, which will enable loss-of-function study in future.

Enchondroma and chondrosarcoma are common benign and malignant cartilaginous neoplasms. Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) are present in the majority of these tumors. Mutant IDH enzymes gain a neomorphic function of producing D-2-hydroxyglutarate (D-2HG) from ?-ketoglutarate. Expression of a mutant Idh1 gene is sufficient for enchondroma initiation but inhibiting mutant IDH enzymes did not cause a consistent change in the tumorigenic properties of chondrosarcomas. It is unclear how mutations of isocitrate dehydrogenases regulate cartilage tumors from initiation to cancer progression and maintenance. I hypothesize that mutations in IDH enzymes could regulate cartilage tumors through changes in gene expression and cellular metabolism. To address these questions, I examined the transcriptional regulation and metabolic regulations of mutant isocitrate dehydrogenases in chondrocytes and chondrosarcomas and identified cholesterol biosynthesis and glutamine metabolism as two key pathways dictating tumor behavior.

To understand the transcriptional regulation of IDH1 mutation in cartilage tumors, I performed RNA-sequencing analysis in chondrocytes from Col2a1Cre;Idh1LSL/+ mutant animals and their littermate wildtype controls and identified that cholesterol biosynthesis pathway was upregulated. Genetic inhibition of cholesterol biosynthesis in an enchondroma mouse model and pharmacological inhibition of cholesterol biosynthesis in human patient chondrosarcoma samples suppressed tumor development in vivo. Taken together, these data suggest that intracellular cholesterol synthesis is a potential therapeutic target for enchondromas and chondrosarcomas.

From a metabolic perspective, I found that chondrocytes and chondrosarcomas with mutations in IDH1/2 genes had enhanced glutamine utilization for downstream metabolism. Using genetic and pharmacological approaches, I demonstrated that glutaminase-mediated glutamine metabolism played distinct roles in enchondromas and chondrosarcomas with IDH1/2 mutations. Genetic ablation of glutaminase in chondrocytes with Idh1 mutation increased the number and size of enchondroma-like lesions. Pharmacological inhibition of glutaminase led to decreased tumor weight of chondrosarcoma xenograft. During enchondroma development, glutamine-derived -ketoglutarate plays important roles in regulating chondrocyte differentiation and proliferation. In chondrosarcoma, glutamine-derived non-essential amino acids are important in preventing cell apoptosis.

In summary, findings in this dissertation described transcriptional and metabolic regulations by mutations in isocitrate dehydrogenases in cartilage tumors enchondroma and chondrosarcoma and provide novel insights for developing therapies for these diseases.

Estrogens and the estrogen receptor (ER) have been implicated in the etiology of breast cancer and osteoporosis. However, the mechanisms by which this receptor-ligand complex manifest their regulatory activities in these processes is not completely understood. The development and subsequent definition of the molecular mechanism of action of selective ER modulators (SERMs), compounds with differential relative agonist/antagonist activity, has uncovered an unanticipated complexity in this signaling pathway. Furthermore, these analyses indicat that it is likely that in addition to the classical steroidal estrogens, which exhibit agonist properties, endogenous compounds exist that interact with ER and function as physiological SERMs. Recently, 27-hydroxycholesterol (27HC) was identified as an endogenous ER ligand with tissue-specific estrogenic/anti-estrogenic activities. Indeed, we determined that 27HC exhibited the three basic properties of a SERM: 1) it bound competitively with estradiol (E2) to both genetic subtypes of ER, ERα and ERβ; 2) it induced a unique conformation of ER that is likely related to its biological activity; and 3) it displayed tissue-specific ER modulatory activity in the cardiovascular system, breast, and bone. In particular, we undertook a series of in vivo studies to show that a pathological elevation of 27HC was associated with decreased bone quantity, an effect that was partially rescued by E2 supplementation. The ability of 27HC to decrease bone density in the absence of endogenous estrogens suggests that the circulating level of 27HC may be of critical importance in determining osteoporosis risk in post-menopausal women. Interestingly, cholesterol-lowering statins have been shown to improve bone density; thus, given the stoichiometric relationship between circulating cholesterol and 27HC, our data provide a possible explanation for the observed bone sparing actions of this class of drugs.

In general, it is considered that SERM activity can be explained by the ability to induce differential alterations in ER structure and the impact that this has on the recruitment of functionally distinct cofactors. The results of our studies reveal a much more complex picture and suggest that some SERM pharmacology can be ascribed to actions in pathways that do not include ER. Specifically, we have determined that the SERM 4-hydroxy-tamoxifen (4OHT) can bind to and activate the aryl hydrocarbon receptor (AHR). Given that AHR controls the expression of E2-metabolizing enzymes, our finding that 4OHT regulates AHR in the context of breast cancer could have important pharmacological and pathological implications. Interestingly, our preliminary in vitro data indicate that the ability of 4OHT to inhibit osteoclast (OC) differentiation, and thus aid in preserving bone density in post-menopausal women, is primarily dependent on expression of AHR, not ER. Conversely, the inhibitory activity of raloxifene (RAL), another SERM, on OC differentiation was absolutely dependent on ER. Thus, the activity of 4OHT in bone is likely to be a composite response requiring its actions on both ER and AHR.

Many new aspects of the estrogen and ER signaling pathways have been uncovered as we learn more about ligands that modulate ER by altering its conformation and thus its ability to engage in protein-protein interactions. Collectively, our findings demonstrate that the intersection between cholesterol metabolism, ER signaling, and the AHR pathway will have important consequences in regulating cellular function, and may be involved in the development or progression of multiple disease states.