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.

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 in vitro. 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

Gene expression of <i>SMAD1/5</i> cKO and WT osteoclast cultures.

qRT-PCR comparing expression of osteoclast genes from WT and SMAD1/5 cKO mice after 3 days of RANKL treatment. (A) c-Fos, (B) Nfatc1, (C) Dcstamp, and (D) Ctsk. Data shown are the mean ± SD of three independent experiments in which gene expression was measured from three wells of each genotype, with each PCR reaction performed in duplicate. Expression of each gene is graphed relative to Hprt. Samples were compared using T-test *** p<0.001 vs. WT.</p

SMAD1/5 signaling negatively regulates osteoclast-osteoblast coupling factors.

Conditioned media was collected from WT or SMAD1/5 cKO osteoclasts that had been treated with RANKL for 4 days. MC3T3 cells were treated with conditioned media and ascorbic acid for 12 days. Von Kossa staining was performed on day 12 and quantitated with NIH Image J. (A) Representative images of von Kossa staining of MC3T3 cells treated with osteoclast conditioned media. (B) Size of mineralization nodules (C) Number of mineralization nodules. (D-F) qRT-PCR comparing expression of osteoclast-osteoblast coupling factors from WT and SMAD1/5 cKO mice. (D) Wnt1, (E) Gja1, and (F) Sphk1. (G-I) Mature osteoclasts from WT mice were treated with 1200 nM dorsomorphin or DMSO for 24 hours, and qRT-PCR comparing expression of the coupling factors. (G) Wnt1, (H) Gja1 and (I) Sphk1. Data shown are the mean ± SD of three independent experiments in which gene expression was measured from three wells of each genotype, with each PCR reaction performed in duplicate. Expression of each gene is graphed relative to Hprt (D-F) and graphed relative to DMSO treatment in (G-I). Samples were compared using T-test * p<0.05 vs. WT, **p<0.001 vs. WT.</p

Proposed model of SMAD1/5 regulation of Wnt1a expression in osteoclasts.

In WT osteoclasts SMAD2/3 and SMAD1/5 compete for binding of the common SMAD4, C-SMAD4 which limits expression of SMAD2/3 target genes such as Wnt1. In SMAD1/5 cKO osteoclasts SMAD2/3 no longer competes with SMAD1/5 for binding to C-SMAD4 and as a result SMAD2/3 target gene such as Wnt1 are enhanced.</p

Bone formation is increased in SMAD1/5 cKO mice.

Three-month-old Smad1fl/fl/Smad5fl/fl;c-Fms Cre male mice were analyzed for cortical bone parameters by micro-CT. (A) Representative μCT scans of cortical bone of femurs from WT and Smad1fl/fl/Smad5fl/fl;c-Fms Cre male mice at 3 months of age. (B) Comparison of cortical thickness (C) ELISA analysis of P1NP as a marker of bone formation. Representative images and MAR of tetracycline/calcein labeling from (D) trabecular or (E) cortical bone from WT and SMAD1/5 cKO mice. Samples were compared using T-test ** p<0.01 vs. WT, **** p<0.0001 vs. WT.</p

SMAD1/5 cKO mice have enhanced osteoclast differentiation and function.

BMMs were flushed from WT or SMAD1/5 cKO mice. BMMs were stimulated with M-CSF and RANKL for indicated days. (A) qRT-PCR was used to measure Smad1 gene expression following 2 days of RANKL stimulation (B) qRT-PCR was used to measure Smad5 gene expression following 2 days of RANKL stimulation (C) TRAP stained images of BMMs differentiated with M-CSF and RANKL for 2, 3, or 4 days (D) Quantification of TRAP stained images measuring number and size of TRAP positive osteoclasts. (E) Demineralization activity of WT and SMAD1/5 cKO osteoclast cultures grown on calcium phosphate surfaces. We quantified (F) pit number (G) average site of the pit (H) percent area demineralized. Scale bar 200 μm. Samples were compared using T-test* p<0.05 vs. WT, ** p<0.01 vs. WT,*** p<0.001 vs. WT, **** p<0.0001 vs. WT.</p