Daniel G. Fraser

Skeletal Effects of Estrogen Are Mediated by Opposing Actions of Classical and Nonclassical Estrogen Receptor Pathways

ERα acts either through classical (ERE‐mediated) or nonclassical (non‐ERE) pathways. The generation of mice carrying a mutation that eliminates classical ERα signaling presents a unique opportunity to study the relative roles of these pathways in bone. This study defines the skeletal phenotype and responses to ovariectomy and estrogen replacement in these mice.

Identification of Senescent Cells in the Bone Microenvironment.

Cellular senescence is a fundamental mechanism by which cells remain metabolically active yet cease dividing and undergo distinct phenotypic alterations, including upregulation of p16Ink4a, profound secretome changes, telomere shortening, and decondensation of pericentromeric satellite DNA. Because senescent cells accumulate in multiple tissues with aging, these cells and the dysfunctional factors they secrete, termed the senescence‐associated secretory phenotype (SASP), are increasingly recognized as promising therapeutic targets to prevent age‐related degenerative pathologies, including osteoporosis. However, the cell type(s) within the bone microenvironment that undergoes senescence with aging in vivo has remained poorly understood, largely because previous studies have focused on senescence in cultured cells. Thus in young (age 6 months) and old (age 24 months) mice, we measured senescence and SASP markers in vivo in highly enriched cell populations, all rapidly isolated from bone/marrow without in vitro culture. In both females and males, p16Ink4a expression by real‐time quantitative polymerase chain reaction (rt‐qPCR) was significantly higher with aging in B cells, T cells, myeloid cells, osteoblast progenitors, osteoblasts, and osteocytes. Further, in vivo quantification of senescence‐associated distension of satellites (SADS), ie, large‐scale unraveling of pericentromeric satellite DNA, revealed significantly more senescent osteocytes in old compared with young bone cortices (11% versus 2%, p < 0.001). In addition, primary osteocytes from old mice had sixfold more (p < 0.001) telomere dysfunction‐induced foci (TIFs) than osteocytes from young mice. Corresponding with the age‐associated accumulation of senescent osteocytes was significantly higher expression of multiple SASP markers in osteocytes from old versus young mice, several of which also showed dramatic age‐associated upregulation in myeloid cells. These data show that with aging, a subset of cells of various lineages within the bone microenvironment become senescent, although senescent myeloid cells and senescent osteocytes predominantly develop the SASP. Given the critical roles of osteocytes in orchestrating bone remodeling, our findings suggest that senescent osteocytes and their SASP may contribute to age‐related bone loss.

Effects of Chronic Estrogen Treatment on Modulating Age-Related Bone Loss in Female Mice

While female mice do not have the equivalent of a menopause, they do undergo reproductive senescence. Thus, to dissociate the effects of aging versus estrogen deficiency on age‐related bone loss, we sham‐operated, ovariectomized, or ovariectomized and estrogen‐replaced female C57/BL6 mice at 6 months of age and followed them to age 18 to 22 months. Lumbar spines and femurs were excised for analysis, and bone marrow hematopoietic lineage negative (lin–) cells (enriched for osteoprogenitor cells) were isolated for gene expression studies. Six‐month‐old intact control mice were euthanized to define baseline parameters. Compared with young mice, aged/sham‐operated mice had a 42% reduction in lumbar spine bone volume/total volume (BV/TV), and maintaining constant estrogen levels over life in ovariectomized/estrogen‐treated mice did not prevent age‐related trabecular bone loss at this site. By contrast, lifelong estrogen treatment of ovariectomized mice completely prevented the age‐related reduction in cortical volumetric bone mineral density (vBMD) and thickness at the tibial diaphysis present in the aged/sham‐operated mice. As compared with cells from young mice, lin– cells from aged/sham‐operated mice expressed significantly higher mRNA levels for osteoblast differentiation and proliferation marker genes. These data thus demonstrate that, in mice, age‐related loss of cortical bone in the appendicular skeleton, but not loss of trabecular bone in the spine, can be prevented by maintaining constant estrogen levels over life. The observed increase in osteoblastic differentiation and proliferation marker gene expression in progenitor bone marrow cells from aged versus young mice may represent a compensatory mechanism in response to ongoing bone loss.

Skeletal Consequences of Deletion of Steroid Receptor Coactivator-2/Transcription Intermediary Factor-2

Both estrogen receptor (ER) and peroxisome proliferator-activated receptor γ (PPARγ) regulate bone metabolism, and because steroid receptor coactivator (SRC)-2 (TIF-2) enhances ER and PPARγ activity, we examined the consequences of deletion of SRC-2 on bone using SRC-2 knock out (KO) mice. Loss of SRC-2 resulted in increased bone mass, with SRC-2 KO mice having 80% higher trabecular bone volume as compared with wild type mice. SRC-2 KO mice also had a marked decrease (by 50%) in bone marrow adipocytes. These data suggested that marrow precursor cells in the SRC-2 KO mice may be resistant to the inhibitory effects of endogenous PPARγ ligands on bone formation. Consistent with this, compared with cultures from wild type mice, marrow stromal cultures from SRC-2 KO mice formed significantly more mineralized nodules (by 3-fold) in the presence of the PPARγ agonist, rosiglitazone. Using chromatin immunoprecipitation analysis, we demonstrated that in bone marrow stromal cells, loss of SRC-2 leads to destabilization of the transcription complex at the peroxisome proliferator response elements of a number of PPARγ target genes, resulting in an overall decrease in the expression of adipocyte-related genes and a marked decrease in adipocyte development. Using ovariectomy with or without estrogen replacement, we also demonstrated that SRC-2 KO mice were partially resistant to the skeletal actions of estrogen. Collectively, these findings indicate that loss of SRC-2 leads to partial skeletal resistance to the ER and PPARγ, but resistance to PPARγ is dominant, leading to increased bone mass. Modulating SRC-2 action may, thus, represent a novel therapeutic target for osteoporosis.

Induction of Fracture Repair by Mesenchymal Cells Derived from Human Embryonic Stem Cells or Bone Marrow

Development of novel therapeutic approaches to repair fracture non‐unions remains a critical clinical necessity. We evaluated the capacity of human embryonic stem cell (hESC)‐derived mesenchymal stem/stromal cells (MSCs) to induce healing in a fracture non‐union model in rats. In addition, we placed these findings in the context of parallel studies using human bone marrow MSCs (hBM‐MSCs) or a no cell control group (n = 10–12 per group). Preliminary studies demonstrated that both for hESC‐derived MSCs and hBM‐MSCs, optimal induction of fracture healing required in vitro osteogenic differentiation of these cells. Based on biomechanical testing of fractured femurs, maximum torque, and stiffness were significantly greater in the hBM‐MSC as compared to the control group that received no cells; values for these parameters in the hESC‐derived MSC group were intermediate between the hBM‐MSC and control groups, and not significantly different from the control group. However, some evidence of fracture healing was evident by X‐ray in the hESC‐derived MSC group. Our results thus indicate that while hESC‐derived MSCs may have potential to induce fracture healing in non‐unions, hBM‐MSCs function more efficiently in this process. Additional studies are needed to further modify hESCs to achieve optimal fracture healing by these cells.

Examination of ERα signaling pathways in bone of mutant mouse models reveals the importance of ERE-dependent signaling.

The mechanisms of estrogen receptor (ER)-α activity can be categorized into those involving direct (classical) or indirect (nonclassical) DNA binding. Although various mouse models have demonstrated the importance of ERα in bone, the specific gene expression patterns affected by these modes of ERα action are unknown. In this report, the gene expression patterns of ERα-deficient (ERKO) mice and nonclassical ER knock-in (NERKI) mice, which can function only by nonclassical means, were analyzed. Three-month-old mice were ovariectomized and implanted with estrogen pellets for 1 month to normalize estrogen levels. Microarray analysis of flushed cortical bone revealed 28% (210 of 763) of the genes differentially expressed in ERKO mice were altered in NERKI mice, suggesting estrogen response element-dependent regulation of these genes in bone. Pathway analysis revealed alterations in genes involved in focal adhesion and extracellular matrix interactions. However, the majority of genes regulated in ERKO mice (72%) were unique (i.e. not altered in NERKI mice), suggesting these are regulated by nonclassical mechanisms. To further explore the pathways affected in ERKO mice, we performed focused quantitative PCR arrays for genes involved in various aspects of bone physiology. Genes involved in bone formation, senescence, apoptosis, and autophagy were significantly regulated. Overall, the majority of the genes regulated by ERα in bone are via nonclassical pathways. However, because NERKI mice display an osteoporotic phenotype, it can be deduced that the minority of the estrogen response element-dependent genes/pathways play critical roles in the regulation of bone physiology. These data demonstrate the importance of classical ERα signaling in regulating bone metabolism.

Deletion of Estrogen Receptor Beta in Osteoprogenitor Cells Increases Trabecular but Not Cortical Bone Mass in Female Mice

Although the role of ERα in regulating bone metabolism has been extensively studied, ERβ has been largely dismissed as a relevant modulator of bone mass. Previous studies examining ERβ utilized a germline knockout mouse expressing transcript variants of ERβ and displaying systemic hormonal changes that confounded interpretation of the skeletal phenotype. Thus, we used a conditional ERβ mouse model to achieve deletion of ERβ specifically in early osteoprogenitor cells using the Prx1-Cre driver. We observed marked increases in the trabecular bone volume fraction (of 58% [p < 0.003] and 93% [p < 0.0003] in 6- and 12-week-old female ERβ(Prx1-CKO) mice, respectively) but no changes in cortical bone. Serum estradiol and IGF-I levels were unaltered in ERβ(Prx1-CKO) mice. Bone formation and resorption indices by histomorphometry and serum assays were unchanged in these mice, suggesting that alterations in bone turnover may have occurred early in development. However, the ratio of colony-forming unit-osteoblasts (CFU-OBs) to CFU-fibroblasts (CFU-Fs) was increased in bone marrow cultures from ERβ(Prx1-CKO) compared with control mice, indicating increased differentiation of osteoblast precursor cells into osteoblasts in ERβ(Prx1-CKO) mice. Detailed quantitative polymerase chain reaction analyses of 128 genes in 16 prespecified pathways revealed significant downregulation of 11 pathways in ERβ(Prx1-CKO) mice. Thus, deletion of ERβ specifically in osteoblast lineage cells, in the absence of all splice variants, increases trabecular bone mass and modulates multiple pathways related to bone metabolism. These findings suggest that pharmacological inhibition of ERβ in bone may provide a novel approach to treat osteoporosis.

Senolytics improve physical function and increase lifespan in old age

Physical function declines in old age, portending disability, increased health expenditures, and mortality. Cellular senescence, leading to tissue dysfunction, may contribute to these consequences of aging, but whether senescence can directly drive age-related pathology and be therapeutically targeted is still unclear. Here we demonstrate that transplanting relatively small numbers of senescent cells into young mice is sufficient to cause persistent physical dysfunction, as well as to spread cellular senescence to host tissues. Transplanting even fewer senescent cells had the same effect in older recipients and was accompanied by reduced survival, indicating the potency of senescent cells in shortening health- and lifespan. The senolytic cocktail, dasatinib plus quercetin, which causes selective elimination of senescent cells, decreased the number of naturally occurring senescent cells and their secretion of frailty-related proinflammatory cytokines in explants of human adipose tissue. Moreover, intermittent oral administration of senolytics to both senescent cell–transplanted young mice and naturally aged mice alleviated physical dysfunction and increased post-treatment survival by 36% while reducing mortality hazard to 65%. Our study provides proof-of-concept evidence that senescent cells can cause physical dysfunction and decreased survival even in young mice, while senolytics can enhance remaining health- and lifespan in old mice.Transfer of senescent cells into naive, young mice can induce physical dysfunction, and a senolytic can reverse this dysfunction and potently increase lifespan in aged mice.

Osteoprotection Through the Deletion of the Transcription Factor Rorβ in Mice: OSTEOPROTECTION THROUGH DELETION OF TRANSCRIPTION FACTOR RORβ IN MICE

There is a clinical need to identify new molecular targets for the treatment of osteoporosis, particularly those that simultaneously inhibit bone resorption while stimulating bone formation. We have previously shown in overexpression studies that retinoic acid receptor‐related orphan receptor β (Rorβ) suppresses in vitro osteoblast differentiation. In addition, the expression of Rorβ is markedly increased in bone marrow–derived mesenchymal stromal cells with aging in both mice and humans. Here we establish a critical role for Rorβ in regulating bone metabolism using a combination of in vitro and in vivo studies. We used Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene editing to demonstrate that loss of Rorβ in osteoblasts enhances Wnt signaling, specifically through increased recruitment of β‐catenin to T‐cell factor/lymphoid enhancer factor (Tcf/Lef) DNA binding sites in the promoters of the Wnt target genes Tcf7 and Opg. This resulted in increased osteogenic gene expression and suppressed osteoclast formation through increased osteoprotegerin (OPG) secretion in Rorβ‐deficient cells. Consistent with our in vitro data, genetic deletion of Rorβ in both female and male mice resulted in preserved bone mass and microarchitecture with advancing age due to increased bone formation with a concomitant decrease in resorption. The improved skeletal phenotype in the Rorβ–/– mice was also associated with increased bone protein levels of TCF7 and OPG. These data demonstrate that loss of Rorβ has beneficial skeletal effects by increasing bone formation and decreasing bone resorption, at least in part through β‐catenin–dependent activation of the Wnt pathway. Thus, inhibition of Rorβ represents a novel approach to potentially prevent or reverse osteoporosis.

Ability of Circulating Human Hematopoietic Lineage Negative Cells to Support Hematopoiesis

Hematopoietic stem cell (HSC) self‐renewal is regulated by osteoblast and/or endothelial cells within the hematopoietic niche. However, the true identity of the supporting cells and the nature of the secreted factors remain uncertain. We developed a novel mouse model and analyzed whether circulating human peripheral hematopoietic lineage negative/AP+ (lin−/AP+) cells support hematopoiesis in vivo. Thus, immunocompromised (Rag) mice expressing thymidine kinase (Tk) under the control of the 3.6Col1α1 promoter (Tk‐Rag) were treated with ganciclovir, resulting in osteoblast progenitor cell ablation and subsequent loss of hematopoiesis (evaluated by measuring mouse Ter119+ erythroid cells). Following hematopoietic cell depletion, human bone marrow‐derived marrow stromal cells (MSCs) or lin−/AP+ cells were infused into Tk‐Rag mice and compared with saline infusions. Ganciclovir significantly reduced (7.4‐fold) Ter119+ cells in the bone marrow of Tk‐Rag mice compared to saline injections. Infusion of either MSCs or lin−/AP+ cells into ganciclovir‐treated mice resulted in a 3.3‐fold and 2.7‐fold increase (P < 0.01), respectively, in Ter119+ cells compared to mice receiving saline. Relative to lin−/AP− cells, lin−/AP+ cells expressed high levels of mesenchymal, endothelial, and hematopoiesis supporting genes. Thus, human peripheral blood lin−/AP+ cells represent a novel cell type capable of supporting hematopoiesis in a manner comparable to MSCs. J. Cell. Biochem. 116: 58–66, 2015.