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ORCID

http://orcid.org/0000-0002-1060-3855

Date of Award

Spring 5-15-2019

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Cell Biology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

The mammalian skeleton is an impressive organ capable of integrating both genetic and environmental signals to ensure its structural integrity. Regulation of bone mass is a highly complex process involving the coordinated actions of osteoblasts (OBs), osteoclasts (OCs), and osteocytes (OCYs). OBs form the bone matrix and OCs resorb this mineralized matrix in a continuous cycle replacing the entire adult human skeleton every 10 years. OCYs are embedded within the bone matrix and serve as mechanosensors that send signals to both OBs and OCs. Input from multiple signaling pathways instructs bone turnover in a time and context-dependent manner. In particular, the nuclear factor kappa-B (NF-kB) family of transcription factors has been shown to regulate both OBs and OCs. NF-kB signaling can be divided into two distinct pathways, classical and alternative. Previous studies have positioned both arms as predominantly negative regulators of osteogenesis. However, interpretation of the OB phenotypes in globally deficient models is complicated by concomitant effects of NF-kB in the OC lineage. While osteolineage-specific models targeting the classical pathway have been reported, to date, the cell-autonomous role of alternative NF-kB in the osteolineage has not been explored. Primary activation of this pathway is mediated through stabilization of NIK protein. NIK then phosphorylates IKKα leading to proteasomal processing of the IkB p100 to p52, allowing nuclear translocation of the active NF-kB dimer RelB/p52.

To better understand the direct role of alternative NF-kB signaling in osteolineage cells, we chose the Osx-Cre driver, which is expressed early in OB differentiation, to modulate key components of this pathway. To activate the pathway, we used NIKΔT3 (NT3), a mutant allele of NIK, placed downstream of a loxP/Neo-STOP/loxP cassette, allowing stabilization of NIK. Thus, mice with both Osx-Cre and NT3 alleles (Osx-Cre;NT3) mice have constitutive activation of alternative NF-kB in osteolineage cells. Conversely, to ablate signaling, we generated Osx-Cre;IKKαf/f (CKO) mice. Collectively, these two models, with lineage-restricted pathway activation or reduction, allow interrogation of the effects of alternative NF-kB signaling during osteogenesis.

Osx-Cre;NT3 mice displayed enhanced bone formation both at baseline (up to 4 months of age) as well as in response to mechanical loading. Mice were not aged further due to development of spontaneous soft-tissue tumors. Interestingly, we also observed enhanced coupling to bone resorption, but overall a net increase in bone mass. Gene expression analysis of both non-loaded and loaded bones uncovered a genomic profile enriched in kB sites both in the NT3 transgene setting and in response to loading.

Conditional deletion of IKKα (CKO mice) did not alter the basal bone phenotype of male mice up to 18 months of age, or alter the response to either ovariectomy or loading. However, we did observe marked reductions in both total fat and lean mass as well as other associated metabolic changes after 1 year of age. Furthermore, younger CKO mice showed attenuated weight gain in response to high-fat diet challenge.

In summary, we found that activation of NIK mobilizes a direct NF-kB responsive gene program important for both basal and mechanically-stimulated bone formation. To our knowledge, this is the first reported instance of a pro-anabolic effect of alternative NF-kB. Unexpectedly, loss of IKKα did not alter either basal or stimulated osteogenesis. However, inhibition of alternative NF-kB signaling in Osx-expressing tissues reshaped body composition and metabolic phenotype with aging and high-fat diet feeding. All told, we have demonstrated that modulation of alternative NF-kB in the osteolineage has important effects both within and outside bone.

Language

English (en)

Chair and Committee

Deborah J. Veis Roberto Civitelli

Committee Members

Yousef Abu-Amer, Clay F. Semenkovich, Matthew J. Silva,

Comments

Permanent URL: https://doi.org/10.7936/z6x1-xn91

Available for download on Thursday, May 13, 2100

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