Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Molecular Cell Biology


English (en)

Date of Award

Summer 9-1-2014

Degree Type


Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Roberto Civitelli


The main function of parathyroid hormone (PTH) is to regulate serum calcium levels, in part by stimulating osteoclast mediated bone resorption. However, PTH can stimulate bone formation if given in intermittent doses and it is currently used as a bone anabolic agent to treat osteoporosis. The mechanisms by which PTH stimulates new bone formation are not yet completely clarified; PTH targets not only osteoblasts, where PTH receptors are abundantly expressed, but also immune cells. In this thesis, I examined the role of the calcium dependent cell-cell adhesion molecule, N-cadherin, in PTH signaling and bone anabolic responses. The findings presented in this dissertation further our understanding of PTH signaling in the context of bone anabolic responses. More to the point, this work demonstrates novel mechanisms by which N-cadherin intersects Wnt/β-catenin signaling, and how such interactions modulate osteoblast response to extracellular cues leading to bone formation.

N-cadherin is abundantly expressed in cells of the osteoblast lineage, and it has been shown to modulate Wnt/β-catenin signaling by tethering low density lipoprotein receptor-related protein 5/6 (Lrp5/6) and β-catenin at the cell membrane. Lrp5/6 and β-catenin are also important components of PTH signaling and downstream effects. Indeed, formation of a complex between PTH, PTH receptor 1 (PTH1R) and Lrp6 has been linked to PTH anabolic action. Since N-cadherin regulates canonical Wnt/β-catenin pathway by interacting with Lrp5/6 and β-catenin, I hypothesized that N-cadherin may also modulate PTH signaling and bone anabolic response by interfering with Lrp6/ β-catenin signaling.

To test this hypothesis, I examined signaling responses in N-cadherin-ablated primary osteoblasts from mice with a selective deletion of the N-cadherin gene (Cdh2) in osteoprogenitor cells (Cdh2flox/flox::Osx-Cre; Cdh2-cKO). Co-immunoprecipitation experiments using control cells from Cdh2flox/flox mice showed that PTH1R interacts with Lrp6, but not Lrp5 nor N-cadherin. This interaction was enhanced by exposure to PTH1-34 in Cdh2-cKO, but not in control cells. In primary osteoblasts, PTH1-34 promoted protein kinase A (PKA)-dependent activation of β-catenin via C-terminus phosphorylation at serine 675 (S675). This event was via non-canonical mechanisms that do not entail β-catenin's destruction complex. Interestingly, PTH-induced phosphorylation of β-catenin at S675 was accentuated in Cdh2-cKO cells relative to control. Consistent with enhanced β-catenin activation, PTH1-34 and Wnt3a stimulated expression of Tcf/Lef target genes, Lef1 and Axin2, to a larger extent in mutant cells compared to control. In addition, PTH1-34 induced rapid membrane trafficking of N-cadherin and Lrp5 from intracellular pools to the cell surface via cAMP/PKA signaling in osteoblastic cells. These changes in N-cadherin and Lrp5 availability at the cell surface likely influence protein complexes and processes that include adherens and gap junctions and intracellular signaling responses such as Wnt/β-catenin signaling. Hence, these findings suggest that Cdh2-ablated osteoblasts are more responsive to activation of β-catenin signaling by PTH and Wnt stimulators.

Increased PTH and Wnt responsiveness in an N-cadherin-depleted background extends to PTH anabolic effect in vivo. Intermittent treatment with human PTH1-34 (80µg/kg bw, sc, 5x/week, 4 weeks; iPTH) increased trabecular bone mass in Cdh2-cKO mice to a significantly larger extent than in control. Intermittent PTH significantly increased trabecular mineral apposition rate and bone formation rate in Cdh2-cKO mice, contrasting with more modest changes in control mice. On the other hand, in the cortical compartment, iPTH induced bone accrual by increasing bone formation at the periosteal compartment in a similar fashion in Cdh2-cKO and control mice. Osteoclast and osteoblast surface per bone surface were modestly affected by silencing of N-cadherin and/or iPTH therapy. Serum levels of a marker of bone resorption (C-terminal telopeptide; CTX) increased to a similar extent in response to iPTH in mice from both genotypes, while serum levels of a marker of bone formation (procollagen type I N-terminal propeptide; P1NP) were significantly increased in iPTH-treated Cdh2-cKO mice but not in control. Since bone modeling is driven by periosteal bone formation during post-natal life, these findings suggest that N-cadherin specifically regulates bone remodeling rather than modeling in response to iPTH. Based on the results presented in this thesis, I proposed that N-cadherin likely functions as a “buffer” for Lrp5/6 at the cell surface, thus modulating the intensity of PTH-induced activation of β-catenin-mediated responses ultimately restraining osteoblast function.

Therefore, my work identifies N-cadherin as a modulator of signaling and bone anabolic responses of a major endocrine regulator of bone homeostasis. In addition, it supports the notion that N-cadherin may represent a potential pharmacologic target for enhancing the bone forming action of PTH and Wnt signaling stimulators.



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