ERß-mediated induction of cystatins results in suppression of TGFß signaling and inhibition of triple-negative breast cancer metastasis.

Triple-negative breast cancer (TNBC) accounts for a disproportionately high number of deaths due to a lack of targeted therapies and an increased likelihood of distant recurrence. Estrogen receptor beta (ERß), a well-characterized tumor suppressor, is expressed in 30% of TNBCs, and its expression is associated with improved patient outcomes. We demonstrate that therapeutic activation of ERß elicits potent anticancer effects in TNBC through the induction of a family of secreted proteins known as the cystatins, which function to inhibit canonical TGFß signaling and suppress metastatic phenotypes both in vitro and in vivo. These data reveal the involvement of cystatins in suppressing breast cancer progression and highlight the value of ERß-targeted therapies for the treatment of TNBC patients.

Antitumor activity of Z-endoxifen in aromatase inhibitor-sensitive and aromatase inhibitor-resistant estrogen receptor-positive breast cancer.

BACKGROUND: The tamoxifen metabolite, Z-endoxifen, demonstrated promising antitumor activity in endocrine-resistant estrogen receptor-positive (ER+) breast cancer. We compared the antitumor activity of Z-endoxifen with tamoxifen and letrozole in the letrozole-sensitive MCF7 aromatase expressing model (MCF7AC1), as well as with tamoxifen, fulvestrant, exemestane, and exemestane plus everolimus in a letrozole-resistant MCF7 model (MCF7LR). METHODS: MCF7AC1 tumor-bearing mice were randomized to control (no drug), letrozole (10 µg/day), tamoxifen (500 µg/day), or Z-endoxifen (25 and 75 mg/kg). Treatment in the letrozole arm was continued until resistance developed. MCF7LR tumor-bearing mice were then randomized to Z-endoxifen (50 mg/kg) or tamoxifen for 4 weeks and tumors harvested for microarray and immunohistochemistry analysis. The antitumor activity of Z-endoxifen in the MCF7LR tumors was further compared in a second in vivo study with exemestane, exemestane plus everolimus, and fulvestrant. RESULTS: In the MCF7AC1 tumors, both Z-endoxifen doses were significantly superior to control and tamoxifen in reducing tumor volumes at 4 weeks. Additionally, the 75 mg/kg Z-endoxifen dose was additionally superior to letrozole. Prolonged letrozole exposure resulted in resistance at 25 weeks. In MCF7LR tumor-bearing mice, Z-endoxifen significantly reduced tumor volumes compared to tamoxifen, letrozole, and exemestane, with no significant differences compared to exemestane plus everolimus and fulvestrant. Additionally, compared to tamoxifen, Z-endoxifen markedly inhibited ERα target genes, Ki67 and Akt expression in vivo. CONCLUSION: In endocrine-sensitive and letrozole-resistant breast tumors, Z-endoxifen results in robust antitumor and antiestrogenic activity compared to tamoxifen and aromatase inhibitor monotherapy. These data support the ongoing development of Z-endoxifen.

Periodontal Disease and Senescent Cells: New Players for an Old Oral Health Problem?

The recent identification of senescent cells in periodontal tissues has the potential to provide new insights into the underlying mechanisms of periodontal disease etiology. DNA damage-driven senescence is perhaps one of the most underappreciated delayed consequences of persistent Gram-negative bacterial infection and inflammation. Although the host immune response rapidly protects against bacterial invasion, oxidative stress generated during inflammation can indirectly deteriorate periodontal tissues through the damage to vital cell macromolecules, including DNA. What happens to those healthy cells that reside in this harmful environment? Emerging evidence indicates that cells that survive irreparable genomic damage undergo cellular senescence, a crucial intermediate mechanism connecting DNA damage and the immune response. In this review, we hypothesize that sustained Gram-negative bacterial challenge, chronic inflammation itself, and the constant renewal of damaged tissues create a permissive environment for the abnormal accumulation of senescent cells. Based on emerging data we propose a model in which the dysfunctional presence of senescent cells may aggravate the initial immune reaction against pathogens. Further understanding of the role of senescent cells in periodontal disease pathogenesis may have clinical implications by providing more sophisticated therapeutic strategies to combat tissue destruction.

TIEG and estrogen modulate SOST expression in the murine skeleton.

TIEG knockout (KO) mice exhibit a female-specific osteopenic phenotype and altered expression of TIEG in humans is associated with osteoporosis. Gene expression profiling studies identified sclerostin as one of the most highly up-regulated transcripts in the long bones of TIEG KO mice relative to WT littermates suggesting that TIEG may regulate SOST expression. TIEG was shown to substantially suppress SOST promoter activity and the regulatory elements through which TIEG functions were identified using promoter deletion and chromatin immunoprecipitation assays. Knockdown of TIEG in IDG-SW3 osteocyte cells using shRNA and CRISPR-Cas9 technology resulted in increased SOST expression and delayed mineralization, mimicking the results obtained from TIEG KO mouse bones. Given that TIEG is an estrogen regulated gene, and as changes in the hormonal milieu affect SOST expression, we performed ovariectomy (OVX) and estrogen replacement therapy (ERT) studies in WT and TIEG KO mice followed by miRNA and mRNA sequencing of cortical and trabecular compartments of femurs. SOST expression levels were considerably higher in cortical bone compared to trabecular bone. In cortical bone, SOST expression was increased following OVX only in WT mice and was suppressed following ERT in both genotypes. In contrast, SOST expression in trabecular bone was decreased following OVX and significantly increased following ERT. Interestingly, a number of miRNAs that are predicted to target sclerostin exhibited inverse expression levels in response to OVX and ERT. These data implicate important roles for TIEG and estrogen-regulated miRNAs in modulating SOST expression in bone.

Mutual enhancement of differentiation of osteoblasts and osteocytes occurs through direct cell-cell contact.

There is increasing evidence that osteocytes regulate multiple aspects of bone remodeling through bi-directional communication with osteoblasts. This is potentially mediated through cell-cell contact via osteocytic dendritic processes, through the activity of secreted factors, or both. To test whether cell-cell contact affects gene expression patterns in osteoblasts and osteocytes, we used a co-culture system where calvarial osteoblasts and IDG-SW3 osteocytes were allowed to touch through a porous membrane, while still being physically separated to allow for phenotypic characterization. Osteoblast/osteocyte cell-contact resulted in up-regulation of osteoblast differentiation genes in the osteoblasts, when compared to wells where no cell contact was allowed. Examination of osteocyte gene expression when in direct contact with osteoblasts also revealed increased expression of osteocyte-specific genes. These data suggest that physical contact mutually enhances both the osteoblastic and osteocytic character of each respective cell type. Interestingly, Gja1 (a gap junction protein) was increased in the osteoblasts only when in direct contact with the osteocytes, suggesting that Gja1 may mediate some of the effects of direct cell contact. To test this hypothesis, we treated the direct contact system with the gap junction inhibitor 18-alpha-glycyrrhetinic acid and found that Bglap expression was significantly inhibited. This suggests that osteocytes may regulate late osteoblast differentiation at least in part through Gja1. Identification of the specific factors involved in the enhancement of differentiation of both osteoblasts and osteocytes when in direct contact will uncover new biology concerning how these bone cells communicate.

Senotherapeutic drug treatment ameliorates chemotherapy-induced cachexia.

Cachexia is a debilitating skeletal muscle wasting condition for which we currently lack effective treatments. In the context of cancer, certain chemotherapeutics cause DNA damage and cellular senescence. Senescent cells exhibit chronic activation of the transcription factor NF-κB, a known mediator of the proinflammatory senescence-associated secretory phenotype (SASP) and skeletal muscle atrophy. Thus, targeting NF-κB represents a logical therapeutic strategy to alleviate unintended consequences of genotoxic drugs. Herein, we show that treatment with the IKK/NF-κB inhibitor SR12343 during a course of chemotherapy reduces markers of cellular senescence and the SASP in liver, skeletal muscle, and circulation and, correspondingly, attenuates features of skeletal muscle pathology. Lastly, we demonstrate that SR12343 mitigates chemotherapy-induced reductions in body weight, lean mass, fat mass, and muscle strength. These findings support senescent cells as a promising druggable target to counteract the SASP and skeletal muscle wasting in the context of chemotherapy.

Osteochondroprogenitor cells and neutrophils expressing p21 and senescence markers modulate fracture repair.

Cells expressing features of senescence, including upregulation of p21 and p16, appear transiently following tissue injury, yet the properties of these cells or how they contrast with age-induced senescent cells remains unclear. Here, we used skeletal injury as a model and identified the rapid appearance following fracture of p21+ cells expressing senescence markers, mainly as osteochondroprogenitors (OCHs) and neutrophils. Targeted genetic clearance of p21+ cells suppressed senescence-associated signatures within the fracture callus and accelerated fracture healing. By contrast, p21+ cell clearance did not alter bone loss due to aging; conversely, p16+ cell clearance, known to alleviate skeletal aging, did not affect fracture healing. Following fracture, p21+ neutrophils were enriched in signaling pathways known to induce paracrine stromal senescence, while p21+ OCHs were highly enriched in senescence-associated secretory phenotype factors known to impair bone formation. Further analysis revealed an injury-specific stem cell-like OCH subset that was p21+ and highly inflammatory, with a similar inflammatory mesenchymal population (fibro-adipogenic progenitors) evident following muscle injury. Thus, intercommunicating senescent-like neutrophils and mesenchymal progenitor cells were key regulators of tissue repair in bone and potentially across tissues. Moreover, our findings established contextual roles of p21+ versus p16+ senescent/senescent-like cells that may be leveraged for therapeutic opportunities.

Osteochondroprogenitor cells and neutrophils expressing p21 and senescence markers modulate fracture repair.

Cells expressing features of senescence, including upregulation of p21 and p16, appear transiently following tissue injury, yet the properties of these cells or how they contrast with age-induced senescent cells remains unclear. Here, we used skeletal injury as a model and identified the rapid appearance following fracture of p21+ cells expressing senescence markers, mainly as osteochondroprogenitors (OCHs) and neutrophils. Targeted genetic clearance of p21+ cells suppressed senescence-associated signatures within the fracture callus and accelerated fracture healing. By contrast, p21+ cell clearance did not alter bone loss due to aging; conversely, p16+ cell clearance, known to alleviate skeletal aging, did not affect fracture healing. Following fracture, p21+ neutrophils were enriched in signaling pathways known to induce paracrine stromal senescence, while p21+ OCHs were highly enriched in senescence-associated secretory phenotype factors known to impair bone formation. Further analysis revealed an injury-specific stem cell-like OCH subset that was p21+ and highly inflammatory, with a similar inflammatory mesenchymal population (fibro-adipogenic progenitors) evident following muscle injury. Thus, intercommunicating senescent-like neutrophils and mesenchymal progenitor cells are key regulators of tissue repair in bone and potentially across tissues. Moreover, our findings establish contextual roles of p21+ vs p16+ senescent/senescent-like cells that may be leveraged for therapeutic opportunities.

Effects of intermittent senolytic therapy on bone metabolism in postmenopausal women: a phase 2 randomized controlled trial.

Preclinical evidence demonstrates that senescent cells accumulate with aging and that senolytics delay multiple age-related morbidities, including bone loss. Thus, we conducted a phase 2 randomized controlled trial of intermittent administration of the senolytic combination dasatinib plus quercetin (D + Q) in postmenopausal women (n = 60 participants). The primary endpoint, percentage changes at 20 weeks in the bone resorption marker C-terminal telopeptide of type 1 collagen (CTx), did not differ between groups (median (interquartile range), D + Q -4.1% (-13.2, 2.6), control -7.7% (-20.1, 14.3); P = 0.611). The secondary endpoint, percentage changes in the bone formation marker procollagen type 1 N-terminal propeptide (P1NP), increased significantly (relative to control) in the D + Q group at both 2 weeks (+16%, P = 0.020) and 4 weeks (+16%, P = 0.024), but was not different from control at 20 weeks (-9%, P = 0.149). No serious adverse events were observed. In exploratory analyses, the skeletal response to D + Q was driven principally by women with a high senescent cell burden (highest tertile for T cell p16 (also known as CDKN2A) mRNA levels) in which D + Q concomitantly increased P1NP (+34%, P = 0.035) and reduced CTx (-11%, P = 0.049) at 2 weeks, and increased radius bone mineral density (+2.7%, P = 0.004) at 20 weeks. Thus, intermittent D + Q treatment did not reduce bone resorption in the overall group of postmenopausal women. However, our exploratory analyses indicate that further studies are needed testing the hypothesis that the underlying senescent cell burden may dictate the clinical response to senolytics. ClinicalTrials.gov identifier: NCT04313634 .

Identification of Rorß targets in cultured osteoblasts and in human bone.

Control of osteoblastic bone formation involves the cumulative action of numerous transcription factors, including both activating and repressive functions that are important during specific stages of differentiation. The nuclear receptor retinoic acid receptor-related orphan receptor ß (Rorß) has been recently shown to suppress the osteogenic phenotype in cultured osteoblasts, and is highly upregulated in bone marrow-derived osteogenic precursors isolated from aged osteoporotic mice, suggesting Rorß is an important regulator of osteoblast function. However the specific gene expression patterns elicited by Rorß are unknown. Using microarray analysis, we identified 281 genes regulated by Rorß in an MC3T3-E1 mouse osteoblast cell model (MC3T3-Rorß-GFP). Pathway analysis revealed alterations in genes involved in MAPK signaling, genes involved in extracellular matrix (ECM) regulation, and cytokine-receptor interactions. Whereas the identified Rorß-regulated ECM genes normally decline during osteoblastic differentiation, they were highly upregulated in this non-mineralizing MC3T3-Rorß-GFP model system, suggesting that Rorß may exert its anti-osteogenic effects through ECM disruption. Consistent with these in vitro findings, the expression of both RORß and a subset of RORß-regulated genes were increased in bone biopsies from postmenopausal women (73±7 years old) compared to premenopausal women (30±5 years old), suggesting a role for RORß in human age-related bone loss. Collectively, these data demonstrate that Rorß regulates known osteogenic pathways, and may represent a novel therapeutic target for age-associated bone loss.

MicroRNA-19a-3p Decreases with Age in Mice and Humans and Inhibits Osteoblast Senescence.

Aging is a major risk factor for most chronic diseases, including osteoporosis, and is characterized by an accumulation of senescent cells in various tissues. MicroRNAs (miRNAs) are critical regulators of bone aging and cellular senescence. Here, we report that miR-19a-3p decreases with age in bone samples from mice as well as in posterior iliac crest bone biopsies of younger versus older healthy women. miR-19a-3p also decreased in mouse bone marrow stromal cells following induction of senescence using etoposide, H2O2, or serial passaging. To explore the transcriptomic effects of miR-19a-3p, we performed RNA sequencing of mouse calvarial osteoblasts transfected with control or miR-19a-3p mimics and found that miR-19a-3p overexpression significantly altered the expression of various senescence, senescence-associated secretory phenotype-related, and proliferation genes. Specifically, miR-19a-3p overexpression in nonsenescent osteoblasts significantly suppressed p16 Ink4a and p21 Cip1 gene expression and increased their proliferative capacity. Finally, we established a novel senotherapeutic role for this miRNA by treating miR-19a-3p expressing cells with H2O2 to induce senescence. Interestingly, these cells exhibited lower p16 Ink4a and p21 Cip1 expression, increased proliferation-related gene expression, and reduced SA-ß-Gal+ cells. Our results thus establish that miR-19a-3p is a senescence-associated miRNA that decreases with age in mouse and human bones and is a potential senotherapeutic target for age-related bone loss. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Multiparametric senescent cell phenotyping reveals CD24 osteolineage cells as targets of senolytic therapy in the aged murine skeleton.

Senescence drives organismal aging, yet the deep characterization of senescent cells in vivo remains incomplete. Here, we applied mass cytometry by time-of-flight (CyTOF) using carefully validated antibodies to analyze senescent cells at single-cell resolution. We used multiple criteria to identify senescent mesenchymal cells that were growth arrested and resistant to apoptosis (p16+/Ki67-/BCL-2+; "p16KB" cells). These cells were highly enriched for senescence-associated secretory phenotype (SASP) and DNA damage markers and were strongly associated with age. p16KB cell percentages were also increased in CD24+ osteolineage cells, which exhibited an inflammatory SASP in aged mice and were robustly cleared by both genetic and pharmacologic senolytic therapies. Following isolation, CD24+ skeletal cells exhibited growth arrest, SA-ßgal positivity, and impaired osteogenesis in vitro . These studies thus provide a new approach using multiplexed protein profiling by CyTOF to define senescent mesenchymal cells in vivo and identify a highly inflammatory, senescent CD24+ osteolineage population cleared by senolytics.

Multiparametric senescent cell phenotyping reveals targets of senolytic therapy in the aged murine skeleton.

Senescence drives organismal aging, yet the deep characterization of senescent cells in vivo remains incomplete. Here, we apply mass cytometry by time-of-flight using carefully validated antibodies to analyze senescent cells at single-cell resolution. We use multiple criteria to identify senescent mesenchymal cells that are growth-arrested and resistant to apoptosis. These p16 + Ki67-BCL-2+ cells are highly enriched for senescence-associated secretory phenotype and DNA damage markers, are strongly associated with age, and their percentages are increased in late osteoblasts/osteocytes and CD24high osteolineage cells. Moreover, both late osteoblasts/osteocytes and CD24high osteolineage cells are robustly cleared by genetic and pharmacologic senolytic therapies in aged mice. Following isolation, CD24+ skeletal cells exhibit growth arrest, senescence-associated ß-galactosidase positivity, and impaired osteogenesis in vitro. These studies thus provide an approach using multiplexed protein profiling to define senescent mesenchymal cells in vivo and identify specific skeletal cell populations cleared by senolytics.

Modest Effects of Osteoclast-Specific ERα Deletion after Skeletal Maturity.

Estrogen regulates bone mass in women and men, but the underlying cellular mechanisms of estrogen action on bone remain unclear. Although both estrogen receptor (ER)α and ERß are expressed in bone cells, ERα is the dominant receptor for skeletal estrogen action. Previous studies using either global or cell-specific ERα deletion provided important insights, but each of these approaches had limitations. Specifically, either high circulating sex steroid levels in global ERα knockout mice or the effects of deletion of ERα during growth and development in constitutive cell-specific knockout mice have made it difficult to clearly define the role of ERα in specific cell types in the adult skeleton. We recently generated and characterized mice with tamoxifen-inducible ERα deletion in osteocytes driven by the 8-kb Dmp1 promoter (ERαΔOcy mice), revealing detrimental effects of osteocyte-specific ERα deletion on trabecular bone volume (-20.1%) and bone formation rate (-18.9%) in female, but not male, mice. Here, we developed and characterized analogous mice with inducible ERα deletion in osteoclasts using the Cathepsin K promoter (ERαΔOcl mice). In a study design identical to that with the previously described ERαΔOcy mice, adult female, but not male, ERαΔOcl mice showed a borderline (-10.2%, p = 0.084) reduction in trabecular bone volume, no change in osteoclast numbers, but a significant increase in serum CTx levels, consistent with increased osteoclast activity. These findings in ERαΔOcl mice differ from previous studies of constitutive osteoclast-specific ERα deletion, which led to clear deficits in trabecular bone and increased osteoclast numbers. Collectively, these data indicate that in adult mice, estrogen action in the osteocyte is likely more important than via the osteoclast and that ERα deletion in osteoclasts from conception onward has more dramatic skeletal effects than inducible osteoclastic ERα deletion in adult mice. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Distinct secretomes in p16- and p21- positive senescent cells across tissues.

Senescent cells drive age-related tissue dysfunction via the induction of a chronic senescenceassociated secretory phenotype (SASP). The cyclin-dependent kinase inhibitors p21Cip1 and p16Ink4a have long served as markers of cellular senescence. However, their individual roles remain incompletely elucidated. Thus, we conducted a comprehensive examination of multiple single-cell RNA sequencing (scRNA-seq) datasets spanning both murine and human tissues during aging. Our analysis revealed that p21Cip1 and p16Ink4a transcripts demonstrate significant heterogeneity across distinct cell types and tissues, frequently exhibiting a lack of co-expression. Moreover, we identified tissue-specific variations in SASP profiles linked to p21Cip1 or p16Ink4a expression. Our study underscores the extraordinary diversity of cellular senescence and the SASP, emphasizing that these phenomena are inherently cell- and tissue-dependent. However, a few SASP factors consistently contribute to a shared "core" SASP. These findings highlight the need for a more nuanced investigation of senescence across a wide array of biological contexts.

Estrogen-regulated miRs in bone enhance osteoblast differentiation and matrix mineralization.

Estrogen signaling is critical for the development and maintenance of healthy bone, and age-related decline in estrogen levels contributes to the development of post-menopausal osteoporosis. Most bones consist of a dense cortical shell and an internal mesh-like network of trabecular bone that respond differently to internal and external cues such as hormonal signaling. To date, no study has assessed the transcriptomic differences that occur specifically in cortical and trabecular bone compartments in response to hormonal changes. To investigate this, we employed a mouse model of post-menopausal osteoporosis (ovariectomy, OVX) and estrogen replacement therapy (ERT). mRNA and miR sequencing revealed distinct transcriptomic profiles between cortical and trabecular bone in the setting of OVX and ERT. Seven miRs were identified as likely contributors to the observed estrogen-mediated mRNA expression changes. Of these, four miRs were prioritized for further study and decreased predicted target gene expression in bone cells, enhanced the expression of osteoblast differentiation markers, and altered the mineralization capacity of primary osteoblasts. As such, candidate miRs and miR mimics may have therapeutic relevance for bone loss resulting from estrogen depletion without the unwanted side effects of hormone replacement therapy and therefore represent novel therapeutic approaches to combat diseases of bone loss.

Local senolysis in aged mice only partially replicates the benefits of systemic senolysis.

Clearance of senescent cells (SnCs) can prevent several age-related pathologies, including bone loss. However, the local versus systemic roles of SnCs in mediating tissue dysfunction remain unclear. Thus, we developed a mouse model (p16-LOX-ATTAC) that allowed for inducible SnC elimination (senolysis) in a cell-specific manner and compared the effects of local versus systemic senolysis during aging using bone as a prototype tissue. Specific removal of Sn osteocytes prevented age-related bone loss at the spine, but not the femur, by improving bone formation without affecting osteoclasts or marrow adipocytes. By contrast, systemic senolysis prevented bone loss at the spine and femur and not only improved bone formation, but also reduced osteoclast and marrow adipocyte numbers. Transplantation of SnCs into the peritoneal cavity of young mice caused bone loss and also induced senescence in distant host osteocytes. Collectively, our findings provide proof-of-concept evidence that local senolysis has health benefits in the context of aging, but, importantly, that local senolysis only partially replicates the benefits of systemic senolysis. Furthermore, we establish that SnCs, through their senescence-associated secretory phenotype (SASP), lead to senescence in distant cells. Therefore, our study indicates that optimizing senolytic drugs may require systemic instead of local SnC targeting to extend healthy aging.

In vitro and in vivo effects of zoledronate on senescence and senescence-associated secretory phenotype markers.

In addition to reducing fracture risk, zoledronate has been found in some studies to decrease mortality in humans and extend lifespan and healthspan in animals. Because senescent cells accumulate with aging and contribute to multiple co-morbidities, the non-skeletal actions of zoledronate could be due to senolytic (killing of senescent cells) or senomorphic (inhibition of the secretion of the senescence-associated secretory phenotype [SASP]) actions. To test this, we first performed in vitro senescence assays using human lung fibroblasts and DNA repair-deficient mouse embryonic fibroblasts, which demonstrated that zoledronate killed senescent cells with minimal effects on non-senescent cells. Next, in aged mice treated with zoledronate or vehicle for 8 weeks, zoledronate significantly reduced circulating SASP factors, including CCL7, IL-1ß, TNFRSF1A, and TGFß1 and improved grip strength. Analysis of publicly available RNAseq data from CD115+ (CSF1R/c-fms+) pre-osteoclastic cells isolated from mice treated with zoledronate demonstrated a significant downregulation of senescence/SASP genes (SenMayo). To establish that these cells are potential senolytic/senomorphic targets of zoledronate, we used single cell proteomic analysis (cytometry by time of flight [CyTOF]) and demonstrated that zoledronate significantly reduced the number of pre-osteoclastic (CD115+/CD3e-/Ly6G-/CD45R-) cells and decreased protein levels of p16, p21, and SASP markers in these cells without affecting other immune cell populations. Collectively, our findings demonstrate that zoledronate has senolytic effects in vitro and modulates senescence/SASP biomarkers in vivo . These data point to the need for additional studies testing zoledronate and/or other bisphosphonate derivatives for senotherapeutic efficacy.

In vitro and in vivo effects of zoledronic acid on senescence and senescence-associated secretory phenotype markers.

In addition to reducing fracture risk, zoledronic acid has been found in some studies to decrease mortality in humans and extend lifespan and healthspan in animals. Because senescent cells accumulate with aging and contribute to multiple co-morbidities, the non-skeletal actions of zoledronic acid could be due to senolytic (killing of senescent cells) or senomorphic (inhibition of the secretion of the senescence-associated secretory phenotype [SASP]) actions. To test this, we first performed in vitro senescence assays using human lung fibroblasts and DNA repair-deficient mouse embryonic fibroblasts, which demonstrated that zoledronic acid killed senescent cells with minimal effects on non-senescent cells. Next, in aged mice treated with zoledronic acid or vehicle for 8 weeks, zoledronic acid significantly reduced circulating SASP factors, including CCL7, IL-1ß, TNFRSF1A, and TGFß1 and improved grip strength. Analysis of publicly available RNAseq data from CD115+ (CSF1R/c-fms+) pre-osteoclastic cells isolated from mice treated with zoledronic acid demonstrated a significant downregulation of senescence/SASP genes (SenMayo). To establish that these cells are potential senolytic/senomorphic targets of zoledronic acid, we used single cell proteomic analysis (cytometry by time of flight [CyTOF]) and demonstrated that zoledronic acid significantly reduced the number of pre-osteoclastic (CD115+/CD3e-/Ly6G-/CD45R-) cells and decreased protein levels of p16, p21, and SASP markers in these cells without affecting other immune cell populations. Collectively, our findings demonstrate that zoledronic acid has senolytic effects in vitro and modulates senescence/SASP biomarkers in vivo. These data point to the need for additional studies testing zoledronic acid and/or other bisphosphonate derivatives for senotherapeutic efficacy.

A DNA binding mutation in estrogen receptor-α leads to suppression of Wnt signaling via ß-catenin destabilization in osteoblasts.

Estrogen receptors (ERs) play vital roles in the function and remodeling of bone. Their cellular mechanisms can broadly be categorized into those involving direct DNA binding (classical) or indirect DNA binding (non-classical). The generation of non-classical ER knock-in (ERα(-/NERKI) ) mice provides a unique opportunity to define these pathways in bone. We previously demonstrated that ERα(-/NERKI) mice exhibit an osteoporotic phenotype; however, the mechanism(s) for this remain unresolved. Gene expression analyses of cortical bone from ERα(-/NERKI) mice revealed suppression of lymphoid enhancer factor-1 (Lef1), a classic Wnt-responsive transcription factor that associates with ß-catenin. Since Wnt signaling is generally considered bone anabolic, this observation leads to the hypothesis that NERKI-induced suppression of Wnt signaling may contribute to the low bone mass phenotype. We generated ERα(-/NERKI) mice crossed with the Wnt-responsive TOPGAL transgenic mouse model and observed significantly less ß-galactosidase activity in ERα(-/NERKI) mice, confirming suppression of Wnt activity in vivo. Adenoviral expression of the NERKI receptor using an in vitro cell system resulted in the induction of several secreted antagonists of Wnt signaling. Furthermore, expression of NERKI abrogated Wnt10b-dependent Wnt activation using a lentiviral-mediated reporter assay. Finally, expression of NERKI destabilized ß-catenin cellular protein levels and disrupted ER/ß-catenin interactions. Collectively, these data suggest the osteoporotic phenotype of ERα(-/NERKI) mice may involve the suppression of Lef1-mediated Wnt signaling through both the stimulation of secreted Wnt inhibitors and/or disruption of normal ß-catenin function.