Doolittle, Madison L.

Cellular senescence is a heterogeneous phenotype characterized primarily in mesenchymal cells, but the extent to which immune cells differ in their senescence phenotype, or “senotype,” is unclear. Here, we applied single-cell approaches alongside both global and cell-specific genetic senolytic mouse models to evaluate the senotype of immune cells in the bone marrow of aging mice. We found that myeloid-lineage cells exhibited the highest expression of p16 and senescence-associated secretory phenotype markers among all immune cell types. In contrast with clearance of p16 + senescent mesenchymal cells, targeted clearance of p16 + myeloid cells in aged mice had only minor effects on age-related bone loss in male mice, with no effects in females. In more detailed analyses, p16 + myeloid cells were only acutely cleared, being repopulated back to basal levels within a short time. This led to a lack of long-lasting reduction in senescent cell burden, unlike when targeting bone mesenchymal cells. In vitro, myeloid-lineage cells differed markedly from mesenchymal cells in the development of a senescent phenotype. Collectively, our findings indicate that aged bone marrow myeloid cells do not achieve the fully developed senescent phenotype originally described in mesenchymal cells, justifying further characterization of senotypes of immune cells across tissues.

ABSTRACT Objective Aging alters mesenchymal stromal cell (MSC) function, leading to dysregulated adipogenesis across tissues through biased lineage commitment. Fat redistribution from adipose depots to skeletal muscle and bone marrow is common in aging, but the underlying mechanisms remain unclear. This study investigates how MSC senescence modulates adipogenesis. Methods Primary MSCs were isolated from mouse skeletal muscle (FAPs), adipose tissue (APCs), and bone marrow (BMSCs). Single‐cell RNA sequencing was performed to compare transcriptional profiles among these populations. In vitro adipogenic differentiation and DNA damage–induced senescence assays were conducted, and the effects of autologous conditioned media from senescent MSCs on adipogenesis were assessed. Results Transcriptional analyses revealed that FAPs and APCs share greater similarity with each other than with BMSCs. All MSC types exhibited adipogenic potential and developed a robust senescence‐associated secretory phenotype (SASP) upon senescence induction. Conditioned media from senescent MSCs enhanced adipogenesis in BMSCs but inhibited adipogenesis in FAPs and APCs, revealing tissue‐specific paracrine effects. Conclusions MSC senescence reprograms adipogenic bias in a tissue‐dependent, non–cell autonomous manner, contributing to age‐related fat redistribution among adipose tissue, skeletal muscle, and bone marrow. Understanding these mechanisms may provide new therapeutic approaches for improving tissue composition and function in the context of aging.

Abstract Germline and osteoblast-directed deletion of G protein-gated inwardly rectifying K+ channel 3 (Girk3) was recently shown to increase bone mass after 18 wk of age in male mice. Here, we show that germline Girk3 deletion also increases trabecular and cortical bone mass and increases the mechanical strength of the femur in female mice after 18 wk of age. Unlike male mice, however, osteoblast-directed Girk3 deletion using 2.3 kb-Col1a1-Cre does not increase bone mass in adult female mice. To discover mechanisms underlying high bone mass in female Girk3−/− mice, bulk RNA-sequencing was performed on 2-d-old calvarial bone, revealing lower expression of proinflammatory cytokines such as IL-1β and IL-6 in Girk3−/− mice. Accordingly, cytokines and chemokines are largely suppressed in the circulation of adult Girk3−/− mice compared to WT littermates. The cytokines GM-CSF, IL-1β, IL-2, and IL-9 are reduced in the serum of both male and female Girk3−/− mice, while eotaxin, IFNγ, MIP-1α, and others are sexually dimorphic. Histomorphometry reveals that osteoclast activity is modestly reduced in Girk3−/− bone, which is supported by in vitro osteoclast resorption assays. However, deletion of Girk3 in myeloid-lineage cells with LysM-Cre is not sufficient to recapitulate high bone mass in either male or female mice. Moreover, female Girk3−/− mice are not protected from ovariectomy-induced bone loss. Finally, single-cell screening using cytometry by time-of-flight in the BM revealed no differences in immune cell abundances due to global Girk3 deletion. Taken together, while Girk3 regulates inflammatory cytokine expression in the bone and serum, deletion of Girk3 in myeloid-lineage cells does not affect bone mass.

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.

AbstractSenescent cells compromise tissue structure and function in older organisms. We recently identified senescent fibroadipogenic progenitors (FAPs) with activated chemokine signaling pathways in the skeletal muscle of old mice, and hypothesized these cells may contribute to the age‐associated accumulation of immune cells in skeletal muscle. In this study, through cell–cell communication analysis of skeletal muscle single‐cell RNA‐sequencing data, we identified unique interactions between senescent FAPs and macrophages, including those mediated by Ccl2 and Spp1. Using mouse primary FAPs in vitro, we verified increased expression of Ccl2 and Spp1 and secretion of their respective proteins in the context of both irradiation‐ and etoposide‐induced senescence. Compared to non‐senescent FAPs, the medium of senescent FAPs markedly increased the recruitment of macrophages in an in vitro migration assay, an effect that was mitigated by preincubation with antibodies to either CCL2 or osteopontin (encoded by Spp1). Further studies demonstrated that the secretome of senescent FAPs promotes polarization of macrophages toward an M2 subtype. These data suggest the unique secretome of senescent FAPs may compromise skeletal muscle homeostasis by recruiting and directing the behavior of macrophages.

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.

The regulation of bone mineral density (BMD) is highly influenced by genetics and age. Although genome‐wide association studies (GWAS) for BMD have uncovered many genes through their proximity to associated variants (variant nearest‐neighbor [VNN] genes), the cell‐specific mechanisms of each VNN gene remain unclear. This is primarily due to the inability to prioritize these genes by cell type and age‐related expression. Using age‐related transcriptomics, we found that the expression of many VNN genes was upregulated in the bone and marrow from aged mice. Candidate genes from GWAS were investigated using single‐cell RNA‐sequencing (scRNA‐seq) datasets to enrich for cell‐specific expression signatures. VNN candidate genes are highly enriched in osteo‐lineage cells, osteocytes, hypertrophic chondrocytes, and Lepr+ mesenchymal stem cells. These data were used to generate a “blueprint” for Cre‐loxp mouse line selection for functional validation of candidate genes and further investigation of their role in BMD maintenance throughout aging. In VNN‐gene‐enriched cells, Sparc , encoding the extracellular matrix (ECM) protein osteonectin, was robustly expressed. This, along with expression of numerous other ECM genes, indicates that many VNN genes likely have roles in ECM deposition by osteoblasts. Overall, we provide data supporting streamlined translation of GWAS candidate genes to potential novel therapeutic targets for the treatment of osteoporosis. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

ABSTRACT 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, H 2 O 2 , 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 H 2 O 2 to induce senescence. Interestingly, these cells exhibited lower p16 Ink4a and p21 Cip1 expression, increased proliferation‐related gene expression, and reduced SA‐β‐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.