Ruan, Ming

ABSTRACT There is an increasing need for biomarkers of senescent cell burden to facilitate the selection of participants for clinical trials. p16 Ink4a is encoded by the CDKN2A locus, which produces five variant transcripts in humans, two of which encode homologous p16 proteins: p16 Inka4a , encoded by p16_variant 1 , and p16ɣ, encoded by p16_variant 5. While distinct quantitative polymerase chain reaction primers can be designed for p16_variant 5, primers for p16_variant 1 also measure p16_variant 5 ( p16_variant 1 + 5 ). In a recent clinical trial evaluating the effects of the senolytic combination, dasatinib + quercetin (D + Q), on bone metabolism in postmenopausal women, we found that women in the highest tertile for T‐cell expression of p16_variant 5 had the most robust skeletal responses to D + Q. Importantly, the assessment of p16_variant 5 was more predictive of these responses than p16_variant 1 + 5 . Here, we demonstrate that in vitro, p16_variant 1 + 5 increased rapidly (Week 1) following the induction of DNA damage, whereas p16_variant 5 increased later (Week 4), suggesting that p16_variant 5 becomes detectable only when the abundance of senescent cells reaches some threshold. Further analysis identified a SASP panel in plasma that performed as well in identifying postmenopausal women with a positive skeletal response to D + Q. Collectively, our findings provide further support for the T‐cell p16_variant 5 assay as a biomarker for selecting participants in clinical trials of senolytic interventions. In addition, our data indicate that correlated plasma SASP markers could be used in lieu of the more technically challenging T‐cell p16 assay. Trial Registration: ClinicalTrials.gov identifier: NCT04313634.

Abstract Extracellular vesicles (EVs) are key mediators of cell–cell communication and are involved in transferring specific biomolecular cargo to recipient cells to regulate their physiological functions. A major challenge in the understanding of EV function in vivo is the difficulty ascertaining the origin of the EV particles. The recent development of the “Snorkel-tag,” which includes EV-membrane-targeted CD81 fused to a series of extra-vesicular protein tags, can be used to mark EVs originating from a specific source for subsequent isolation and characterization. We developed an in vivo mouse model, termed “CAGS-Snorkel,” which expresses the Snorkel-tag under the control of the Cre-lox system, and crossed this mouse with either Prx1-Cre (mesenchymal progenitors) or Ocn-Cre (osteoblasts/osteocytes) and isolated Snorkel-tagged EVs from the mouse bone marrow plasma using a magnetic bead affinity column. miRNA-sequencing was performed on the isolated EVs, and although similar profiles were observed, a few key miRNAs involved in bone metabolism (miR-106b-5p, miRs-19b-3p, and miRs-219a-5p) were enriched in the Ocn-derived relative to the Prx1-derived EV subpopulations. To characterize the effects of these small EVs on a bone cell target, cultured mouse bone marrow stromal cells were treated with Prx1 or Ocn EVs, and mRNA-sequencing was performed. Pathways involved in ossification, bone development, and extracellular matrix interactions were regulated by both EV subpopulations, whereas a few pathways including advanced glycation end-products signaling were uniquely regulated in the Ocn EV subpopulation, underlying important biological effects of specific EV subpopulations within the bone marrow microenvironment. These data demonstrate that EV isolation in vivo using the CAGS-Snorkel mouse model is a useful tool in characterizing the cargo and understanding the biology of tissue-specific EVs. Moreover, while bone mesenchymal cell populations share a common EV secretory profile, we uncover key differences based on the stage of osteoblastic differentiation that may have important biological consequences.

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.

ABSTRACT 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 ERβ 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.

Abstract This abstract is being presented as a short talk in the scientific program. A full abstract is available in the Short Talks from Proffered Abstracts section (PR012) of the Conference Proceedings. Citation Format: Gabriel Alvares Borges, Angelo Jose Guilatco, Christine M. Hachfeld, Ming Ruan, Sonya Royzenblat, Ming Xu, Claire M. Edwards, Marta Diaz-delCastillo, Thomas L. Andersen, Taxiarchis Kourelis, Tamar Tchkonia, James L. Kirkland, Matthew T. Drake, Megan Weivoda. Pre-malignant plasma cells exhibit a senescence-like phenotype and accumulation of transposable elements [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr B020.

ABSTRACT Estrogen is known to regulate bone metabolism in both women and men, but substantial gaps remain in our knowledge of estrogen and estrogen receptor alpha (ERα) regulation of adult bone metabolism. Studies using global ERα-knockout mice were confounded by high circulating sex-steroid levels, and osteocyte/osteoblast-specific ERα deletion may be confounded by ERα effects on growth versus the adult skeleton. Thus, we developed mice expressing the tamoxifen-inducible CreERT2 in osteocytes using the 8-kilobase (kb) Dmp1 promoter (Dmp1CreERT2). These mice were crossed with ERαfl//fl mice to create ERαΔOcy mice, permitting inducible osteocyte-specific ERα deletion in adulthood. After intermittent tamoxifen treatment of adult 4-month-old mice for 1 month, female, but not male, ERαΔOcy mice exhibited reduced spine bone volume fraction (BV/TV (−20.1%, p = 0.004) accompanied by decreased trabecular bone formation rate (−18.9%, p = 0.0496) and serum P1NP levels (−38.9%, p = 0.014). Periosteal (+65.6%, p = 0.004) and endocortical (+64.1%, p = 0.003) expansion were higher in ERαΔOcy mice compared to control (Dmp1CreERT2) mice at the tibial diaphysis, reflecting the known effects of estrogen to inhibit periosteal apposition and promote endocortical formation. Increases in Sost (2.1-fold, p = 0.001) messenger RNA (mRNA) levels were observed in trabecular bone at the spine in ERαΔOcy mice, consistent with previous reports that estrogen deficiency is associated with increased circulating sclerostin as well as bone SOST mRNA levels in humans. Further, the biological consequences of increased Sost expression were reflected in significant overall downregulation in panels of osteoblast and Wnt target genes in osteocyte-enriched bones from ERαΔOcy mice. These findings thus establish that osteocytic ERα is critical for estrogen action in female, but not male, adult bone metabolism. Moreover, the reduction in bone formation accompanied by increased Sost, decreased osteoblast, and decreased Wnt target gene expression in ERαΔOcy mice provides a direct link in vivo between ERα and Wnt signaling. © 2022 American Society for Bone and Mineral Research (ASBMR).

ABSTRACT 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. © 2017 American Society for Bone and Mineral Research.

ABSTRACT Osteoblast-mediated bone formation is coupled to osteoclast-mediated bone resorption. These processes become uncoupled with age, leading to increased risk for debilitating fractures. Therefore, understanding how osteoblasts are recruited to sites of resorption is vital to treating age-related bone loss. Osteoclasts release and activate TGF-β from the bone matrix. Here we show that osteoclast-specific inhibition of TGF-β receptor signaling in mice results in osteopenia due to reduced osteoblast numbers with no significant impact on osteoclast numbers or activity. TGF-β induced osteoclast expression of Wnt1, a protein crucial to normal bone formation, and this response was blocked by impaired TGF-β receptor signaling. Osteoclasts in aged murine bones had lower TGF-β signaling and Wnt1 expression in vivo. Ex vivo stimulation of osteoclasts derived from young or old mouse bone marrow macrophages showed no difference in TGF-β–induced Wnt1 expression. However, young osteoclasts expressed reduced Wnt1 when cultured on aged mouse bone chips compared to young mouse bone chips, consistent with decreased skeletal TGF-β availability with age. Therefore, osteoclast responses to TGF-β are essential for coupling bone resorption to bone formation, and modulating this pathway may provide opportunities to treat age-related bone loss. © 2015 American Society for Bone and Mineral Research.

In young adults, bone lost through osteoclast-mediated resorption is precisely replaced in both location and amount. Understanding how these two processes are coupled is crucial to advancing treatments for osteoporosis, a disease that progresses when the processes become uncoupled. We documented that osteoclasts secrete the mammalian integration 1 gene that is the homolog of Drosophila Wngless (Wnt) 10b, bone morphogenetic protein 6 (BMP6), and the chemokine sphingosin 1 phosphate (S1P) to promote mesenchymal cell mineralization in vitro. During bone resorption, TGF-β1 is released from the bone extracellular matrix and activated by osteoclasts. Thus, TGF-β1 levels are elevated during the resorption phase of bone turnover. We therefore investigated the influences of TGF-β1 on osteoclast-mediated support of mineralization. TGF-β1 increased osteoclast production of Wnt10b, but not BMP6 or S1P. Blocking Wnt10b activity with the Wnt signaling inhibitor Dickkoph-related protein 1 suppressed the ability of TGF-β–treated osteoclast-conditioned media to promote osteoblast mineralization. Examination of TGF-β signaling in osteoclasts revealed that induction of Wnt10b expression was dependent on Smad2/3 activation and independent from TGF-β1 stimulation of protein kinase B (AKT) or MAPK kinase. TGF-β1–treated osteoclast-conditioned media from cells with blocked Smad signaling exhibited a reduced ability to support mineralization, demonstrating the importance of Smad signaling in this response. Parallel cultures with suppressed TGF-β activation of AKT or MAPK kinase signaling retained their ability to elevate mineralization. These results demonstrate that TGF-β1 stimulates Wnt10b production in osteoclasts, which may enhance restoration of the bone lost during the resorptive phase of bone turnover.