Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Investigating HDAC-MEF2 Roles in Osteoclastogenesis

University of Minnesota Ph.D. dissertation.February 2019. Major: Biology. Advisor: Kim Mansky. 1 computer file (PDF); ix, 115 pages.Bone remodeling is the process of removing damaged or old bone and replacing it with new bone. This process occurs in the adult skeleton in order to maintain structural integrity and accomplish other necessary functions. Osteoclasts degrade bone in a localized and controlled manner, and osteoblasts deposit new bone in response to osteoclast activity. The activity of each cell type can be controlled using multiple mechanisms, including osteoclast-mediated regulation of bone formation. In order to determine how osteoclast activity can be regulated in order to manage pathological bone loss, it is necessary to understand transcriptional mechanisms that control their development. With this goal, I expanded on previous research showing histone deacetylase 7 (HDAC7) repressed osteoclast differentiation. I found that overexpression of a fragment of HDAC7 that interacts with the myocyte enhancer factor 2 (MEF2) family of transcription factors repressed osteoclast differentiation to a similar degree as the full-length HDAC7. This led me to investigate the roles of MEF2 in osteoclast differentiation. Mef2a and Mef2d were more highly expressed than Mef2b and Mef2c. Osteoclast-specific deletion of Mef2a (A-KO) or Mef2d (D-KO) in mice compromised in vitro osteoclast differentiation and activity. A-KO and D-KO male mice and D-KO female mice presented no overt skeletal phenotype, but female A-KO mice were osteopetrotic due to increased trabecular number. To determine whether MEF2A compensated for loss of MEF2D and vice versa, I generated mice with osteoclast-specific deletion of both Mef2a and Mef2d (AD-KO). In vitro assays demonstrated a complete block in osteoclast development prior to fusion. However, AD-KO mice presented with osteopenia from reduced cortical and trabecular bone thickness relative to WT mice but unchanged levels of osteoclast activity markers. These experiments suggest specific signals present in vivo but not in vitro rescue osteoclast differentiation of AD-KO osteoclasts. Additionally, MEF2A/D potentially regulates expression of an osteoclastderived factor that regulates osteoblast activity

Breast cancer cell-derived fibroblast growth factors enhance osteoclast activity and contribute to the formation of metastatic lesions

<div><p>Fibroblast growth factors (FGFs) and their receptors (FGFRs) have been implicated in promoting breast cancer growth and progression. While the autocrine effects of FGFR activation in tumor cells have been extensively studied, little is known about the effects of tumor cell-derived FGFs on cells in the microenvironment. Because FGF signaling has been implicated in the regulation of bone formation and osteoclast differentiation, we hypothesized that tumor cell-derived FGFs are capable of modulating osteoclast function and contributing to growth of metastatic lesions in the bone. Initial studies examining FGFR expression during osteoclast differentiation revealed increased expression of FGFR1 in osteoclasts during differentiation. Therefore, studies were performed to determine whether tumor cell-derived FGFs are capable of promoting osteoclast differentiation and activity. Using both non-transformed and transformed cell lines, we demonstrate that breast cancer cells express a number of FGF ligands that are known to activate FGFR1. Furthermore our results demonstrate that inhibition of FGFR activity using the clinically relevant inhibitor BGJ398 leads to reduced osteoclast differentiation and activity <i>in vitro</i>. Treatment of mice injected with tumor cells into the femurs with BGJ398 leads to reduced osteoclast activity and bone destruction. Together, these studies demonstrate that tumor cell-derived FGFs enhance osteoclast function and contribute to the formation of metastatic lesions in breast cancer.</p></div

FGF ligand expression in MCF10A and BoM-1833 cells.

<p>Real time qPCR of FGF ligands. Experiments were done at least three times and values represent the mean ± SD **p<0.01, comparing MCF10A vs. BoM-1833 conditioned medium.</p

Activation of FGF signaling pathways in osteoclasts.

<p>Representative western blot of day 2 osteoclasts were treated with conditioned media from MCF10A, MDA-MB-231 or BoM-1833 cells. Cell lysates were harvested and analyzed for phosphorylated and total p38, ERK, Akt. alpha-tubulin was used as a loading control for protein lysates.</p

FGF receptor expression stimulated by BoM-1833 media in osteoclasts.

<p>BMMs were harvested from C57Bl/6 mice and differentiated in the presence of M-CSF and RANKL for 5 days in the presence of serum free media (SFM) or BoM-1833 media. (A) Real time qPCR of <i>Dc-stamp</i>, <i>Oscar</i>, <i>Itgb3</i>, and <i>Ctsk</i> as markers of osteoclast differentiation. (B) Expression of <i>Fgf</i> receptors was measured by qPCR. Experiments were done at least three times and values represent the mean ± SD. * p<0.05, **p<0.01, ***p<0.0001 comparing day SFM vs. BoM-1833 medium treated osteoclasts.</p

BGJ398 does not affect proliferation of BoM-1833 cells.

<p>(A) MTS assay of BoM-1833 cells treated with increasing concentrations of BJG398. (B) MTS assay of HC-11/R1 cells treated with increasing concentrations of BJG398. ns = not significant, ***p<0.0001 comparing DMSO vs. BGJ398 treated (A) or EtOH vs. BB treated (B).</p

FGF receptor expression in osteoclasts.

<p>Bone marrow macrophages were cultured from C57Bl/6 mice in M-CSF and/or RANKL (10 ng/mL) for indicated times. (A) Western blot of osteoclast lysates cultured in M-CSF (day 0) or M-CSF and RANKL (day 1–4). Western blot was probed with an antibody recognizing FGFR1 (Cell Signaling) or β-tubulin (Cell Signaling). (B) Measurement of <i>Fgf</i> receptor expression in osteoclasts at day 0 (M-CSF only) or day 2 (M-CSF and RANKL for 48 hours) by qPCR. Experiments were done at least three times and values represent the mean ± SD. ns = not significant **p<0.01, ***p<0.0001 comparing day 0 vs. day 2.</p

Osteoclast differentiation is enhanced by FGF from BoM-1833 medium.

<p>BMMs were harvested from C57Bl/6 mice and differentiated in the presence of M-CSF and RANKL ± BoM-1833 conditioned medium ± BGJ398. (A) Representative TRAP images of different treatments (B) Quantification of osteoclast size (C) Quantification of nuclei per cell (D) Quantification of percent area resorbed by ostoeclasts in growth, MCF10A or BoM-1833 conditioned medium. (E) Quantification of percent area resorbed by osteoclasts in BoM-1833 conditioned medium ± BGJ398. Experiments were done at least three times and values represent the mean ± SD. ns = not significant * p<0.05, **p<0.01, ***p<0.0001.</p