Senescence Of Bone Marrow Stromal Cells Research Articles

Uptake of ox-LDL by binding to LRP6 mediates oxidative stress-induced BMSCs senescence promoting obesity-related bone loss

Obesity has long been thought to be a main cause of hyperlipidemia. As a systemic disease, the impact of obesity on organs, tissues and cells is almost entirely negative. However, the relationship between obesity and bone loss is highly controversial. On the one hand, obesity has long been thought to have a positive effect on bone due to increased mechanical loading on the skeleton, conducive to increasing bone mass to accommodate the extra weight. On the other hand, obesity-related metabolic oxidative modification of low-density lipoprotein (LDL) in vivo causes a gradual increase of oxidized LDL (ox-LDL) in the bone marrow microenvironment. We have reported that low-density lipoprotein receptor-related protein 6 (LRP6) acts as a receptor of ox-LDL and mediates the bone marrow stromal cells (BMSCs) uptake of ox-LDL. We detected elevated serum ox-LDL in obese mice. We found that ox-LDL uptake by LRP6 led to an increase of intracellular reactive oxygen species (ROS) in BMSCs, and N-acetyl-L-cysteine (NAC) alleviated the cellular senescence and impairment of osteogenesis induced by ox-LDL. Moreover, LRP6 is a co-receptor of Wnt signaling. We found that LRP6 preferentially binds to ox-LDL rather than dickkopf-related protein 1 (DKK1), both inhibiting Wnt signaling and promoting BMSCs senescence. Mesoderm development LRP chaperone (MESD) overexpression inhibits ox-LDL binding to LRP6, attenuating oxidative stress and BMSCs senescence, eventually rescuing bone phenotype.

Splicing factor YBX1 regulates bone marrow stromal cell fate during aging.

Senescence and altered differentiation potential of bone marrow stromal cells (BMSCs) lead to age-related bone loss. As an important posttranscriptional regulatory pathway, alternative splicing (AS) regulates the diversity of gene expression and has been linked to induction of cellular senescence. However, the role of splicing factors in BMSCs during aging remains poorly defined. Herein, we found that the expression of the splicing factor Y-box binding protein 1 (YBX1) in BMSCs decreased with aging in mice and humans. YBX1 deficiency resulted in mis-splicing in genes linked to BMSC osteogenic differentiation and senescence, such as Fn1, Nrp2, Sirt2, Sp7, and Spp1, thus contributing to BMSC senescence and differentiation shift during aging. Deletion of Ybx1 in BMSCs accelerated bone loss in mice, while its overexpression stimulated bone formation. Finally, we identified a small compound, sciadopitysin, which attenuated the degradation of YBX1 and bone loss in old mice. Our study demonstrated that YBX1 governs cell fate of BMSCs via fine control of RNA splicing and provides a potential therapeutic target for age-related osteoporosis.

Acute Myeloid Leukaemia Induces p16 Driven Senescence in the Bone Marrow Microenvironment to Support Their Proliferation and Survival

BackgroundAcute myeloid leukaemia (AML) occurs most commonly in older people and depends on the bone marrow microenvironment. The aging bone marrow accumulates senescent cells, characterized by an irreversible arrest of cell proliferation and the secretion of numerous pro-inflammatory cytokines, chemokines and growth factors, termed the secretary associated senescence profile (SASP). Previously we shown that AML is capable of reprogramming bone marrow stromal cells (BMSC) to support blast survival and proliferation. Here, we hypothesis that AML programs a senescent aging phenotype in the BMSC, generating a SASP in the BMSC which then feedback to promote AML blast proliferation in the bone marrow niche.MethodsWe isolated primary AML cells and BMSC from the bone marrow, with informed consent and under approval from the UK National Research Ethics Service (LRCEref07/H0310/146). We characterized BMSC by flow cytometry for expression of CD90+, CD73+, CD105+ and CD45, and assayed cytokines and chemokines secreted by primary AML cells, primary BMSC and AML/BMSC co-cultures using a proteomic array. To assess senescence of BMSC, we measured p16 mRNA and protein levels and senescence associated β-Galactosidase activity. We also transplanted syngeneic AML blasts (generated from lineage negative cells transduced with the oncogene MN1) into p16-3MR mice (Demaria, M., et al. 2014 Cell Development), in which the herpes simplex virus thymidine kinase (HSV-TK) moiety of the p16-3MR transgene allows selective killing of senescent p16-expressing cells by the pro-drug ganciclovir (GCV). We also used the NSG xenograft model in which we subcutaneously transplanted primary human AML cells and BMSC (either control and p16-kockdown).ResultsWe report that human AML cells induce BMSC to secrete a cytokine profile (including interleukin-6, interleukin-8, MIP1a and MIP2a). To examine whether AML cells can induce senescence in BMSC in culture, we co-cultured primary AML-derived BMSC with primary AML blasts and assayed for the senescent phenotype. We found that primary AML cells can induce p16 mRNA and protein and increase senescence associated β-Galactosidase activity in the BMSC after 6 days in culture. Furthermore, primary AML cells cultured on p16 knockdown BMSC had reduced survival compared to control wild type BMSC. In vivo we show that MN1 blasts engraft p16-3MR mice and the depletion of p16 positive stromal cells through treating animals with GVC compared to vehicle control significantly increased survival of MN1 engrafted mice. Finally, subcutaneous injection of human AML cells with human p16-KD BMSC significantly decreased tumour volume compared to injection of human AML cells with control-KD BMSC in mouse xenograft model.ConclusionHere we show that the AML blast induces a senescent phenotype in the bone marrow microenvironment which then feeds back to enhance the growth capacity of the AML blast. Targeting the senescent microenvironment may provide a novel therapeutic approach for the treatment of this disease. DisclosuresCampisi:Unity Biotechnology: Equity Ownership.

Age-related trabecular bone loss is associated with a decline in serum Galectin-1 level

BackgroundSenile osteoporosis with age-related bone loss is diagnosed depending on radiographic changes of bone and bone mineral density (BMD) measurement. However, radiographic alterations are usually signs of medium-late stage osteoporosis. Therefore, biomarkers have been proposed as indicators of bone loss. In the current study, Galectin-1 (Gal-1) showed age-related decline in mice serum. The role of Gal-1 in osteoporosis has not been investigated so far. Hence, the current study illustrated the relationship of serum Gal-1 level with bone loss.MethodsWe employed 6- and 18-month-old mice to establish an animal model of age-related trabecular bone loss, whose bone density and microstructure were investigated by micro-CT. ELISA was used to measure the levels of Gal-1 in serum. The correlation analysis was performed to illustrate the relationship between serum Gal-1 levels and trabecular bone loss. In addition, immunohistochemistry was used to investigate the abundance of Gal-1 in bone marrow of mice. ELISA and western blot were performed to measure the secretion ability and protein expression of Gal-1 in bone marrow stromal cells (BMSC), hematopoietic stem cells (HSC) and myeloid progenitor (MP) respectively. Flow cytometry was used to measure BMSC number in bone marrow. Finally, male volunteers with age-related BMD decrease were recruited and the relationship between serum Gal-1 and BMD was analyzed.ResultsGal-1 showed age-related decline in mice serum. Serum Gal-1 was positively associated with BV/TV of femur, tibia and L1 vertebrae in mice. BMSC secreted more Gal-1 compared with HSC and MP. BMSC number in bone marrow was significantly lower in aged mice compared with young mice. Significant attenuation of Gal-1 protein expression was observed in BMSC and HSC from aged mice compared with young mice. Further, we found a decline in serum Gal-1 levels in men with age-related BMD decrease. There was positive correlation between BMD and serum Gal-1 levels in these men.ConclusionsAge-related trabecular bone loss is associated with a decline in serum Gal-1 level in mice and men. Our study suggested Gal-1 had great potential to be a biomarker for discovering BMSC senescence, diagnosing early osteoporosis and monitoring trabecular bone loss.

Osteoblastic Wntless deletion differentially regulates the fate and functions of bone marrow-derived stem cells in relation to age.

Although functional association between Wnt signaling and bone homeostasis has been well described through genetic ablation of Wntless (Wls), the mechanisms of how osteoblastic Wls regulates the fate of bone marrow stromal cells (BMSCs) and hematopoietic stem cells (HSCs) in relation to age are not yet understood. Here, we generated Col2.3-Cre;Wlsfl/fl mice that were free from premature lethality and investigated age-related impacts of osteoblastic Wls deficiency on hematopoiesis, BM microenvironment, and maintenance of BMSCs (also known as BM-derived mesenchymal stem/stromal cells) and HSCs. Ablation of osteoblastic Wls deteriorated BM microenvironment and bone mass accrual along with age-independent effects on functions of BMSCs. Osteoblastic Wls deletion impaired HSC repopulation and progeny with skewing toward myeloid lineage cells only at old stage. As proven by hallmarks of stem cell senescence, osteoblastic Wls ablation differentially induced senescence of BMSCs and HSCs in relation to age without alteration in their BM frequency. Our findings support that deletion of Wls in Col2.3-expressing cells induces senescence of BMSCs and impairs BM microenvironment in age-independent manner. Overall, long-term deterioration in BM microenvironment contributes to age-related HSC senescence with impaired progeny and hematopoiesis, which also suggests possible roles of osteoblastic Wls on the maintenance of BM HSCs.

Myeloma Derived Extracellular Vesicles Containing Endoplasmic Reticulum Remodel the Bone Marrow Microenvironment Towards a Pro-Tumoral Senescent Phenotype

Multiple myeloma (MM) is a tumor characterised by the generation of large quantities of immunoglobulin which undergoes protein folding and secretion through the endoplasmic reticulum (ER). Many studies have shown that primary MM cells have high ER stress (Lee et al, 2003 PNAS, Obeng et al 2006 Blood and Mimura et al, 2012 Blood,). Moreover, it has also been shown that serum from MM contains significantly more extracellular vesicles (EVs) than serum from normal patients (Caivano et al, 2015 Tumour Biol). Here we explore if these two observations are connected and determine whether primary MM cells export ER inside EVs and the impact this has on the tumor microenvironment. Primary MM and primary MM bone marrow stromal cells (BMSC) were isolated from the bone marrow of patients. To determine if MM secrete ER inside EV, patient derived MM and MM cell lines were transduced with rLV.EF.mCherry-ER lentivirus which fluorescently tags the ER. High-resolution imaging combined with image cytometry shows that CD38+ vesicles containing ER are formed by budding from the MM cell surface. Analysis of MM derived EVs using a combination of proteomics, confocal microscopy, image cytometry and dynamic light scattering shows that MM actively export ER in the large EVs, approximately 0.6micron-1.5micron in diameter. To track the recipient cell for large EV packaged ER in vivo, U266 MM cell line (transduced with rLV.EF.mCherry-ER9 lentivirus) was injected into the tail of NSG mice. BM was extracted from engrafted mice and various cell populations were analysed for increases in mCherry fluorescence, as evidence of MM derived ER uptake. Murine CD45-/CD31-Ter119-/CD105+/CD140a+ BMSC had increased mCherry fluorescence but not F4/80+/GR1-/CD115int BM macrophages or CD45-/Ter119-/CD31+ endothelial cells or CD45+ leukocytes. Two proteins detected at high levels in the proteomics analysis of large EV were endoplasmic reticulum oxidoreductin 1 (ERO1) and protein disulfide-isomerase (P4B4) which functionally catalyze the formation, breakage and rearrangement of disulfide bonds resulting in the production of superoxide. Next, we analysed large EVs containing ER for superoxide. MM derived ER+ and ER- large EVs were isolated by sorting for mCherry fluorescence (MM transduced with rLV.EF.mCherry-ER9 lentivirus) and Amplex Red assay confirmed that ER+ large EVs had increased levels of H2O2. In vivo analysis of the BMSC from MM engrafted NSG mice confirmed high oxidative stress as measured by increased H2DCFDA fluorescence. To determine the impact of MM derived ER containing EVs on the function of the BMSC isolated ER containing EVs were incubated with BMSC repeatedly for up to 7 days and senescent markers were assessed. Beta-galactosidase staining, p16ink4a gene expression and a senescence associated secretary phenotype (SASP) were all upregulated in BMSC cultured with MM derived ER+ large EV and not ER- large EV. To determine if MM induced BMSC senescence in vivo we injected U266 and primary MM into NSG mice, humanised NSG mice were used as a control. Post MM engraftment, animals were sacrificed and BMSC were isolated by cell sorting for CD45-/CD31-Ter119-/CD105+/CD140a+ cells and senescent markers were analysed by real-time PCR. p16ink4a and p21 were both upregulated in BMSC from U266 and primary MM engrafted NSG mice and not from humanised NSG mice. Knockdown of p16 in BMSC prevents ER+ large EV from inducing a SASP and conditioned media had no effect on MM proliferation compared to conditioned media from ER+ large EV treated BMSC. Finally, we used an NSG mouse model whereby we transplanted p16ink4a KD BMSC or control KD BMSC with MM cells subcutaneously into the flank. MM combined with p16ink4a KD BMSC has reduced tumor volume compared with animals with control KD BMSC. Data indicates that MM secrete ER in large EV and that MM derived EVs containing ER function as a signal which then changes the physiology of BMSC, towards a senescent phenotype which in turn promotes malignant plasma cell survival and proliferation. Disclosures Bowles: Janssen: Research Funding; Abbvie: Research Funding. Rushworth:Abbvie: Research Funding; Janssen: Research Funding.

Long-Term Clearance of Senescent Cells Prevents the Hematopoietic Stem Cell Aging in Naturally Aged Mice

Senescent cells (SnCs) accumulate with age. Genetic or pharmacological clearance of SnCs delays several age-associated disorders and extends the healthspan of both progeroid and wild-type (WT) mice (Baker DJ et al. Nature, 479: 232, 2011; Baker DJ et al. Nature, 530: 184, 2016). In addition, our previous study showed that transient depletion of SnCs could rejuvenated aged hematopoietic stem cells (HSCs) and muscle stem cells in normally aged mice (Chang J et al. Nat Med, 22:78, 2016). These findings suggest that SnCs play a causative role in aging and certain aging-related pathologies, and that selective clearance of SnCs with a senolytic agent that can selectively kill SnCs can be beneficial in part by rejuvenating aged tissue stem cells. Thus, senolytic drugs are potential novel anti-aging agents. However, SnCs can re-accumulate within a few weeks after depletion in mice, suggesting that long-term and intermittent clearance of SnCs is required to extend healthspan, as shown in previous studies (Baker DJ et al. Nature, 479: 232, 2011; Baker DJ et al. Nature, 530: 184, 2016). It is not known how long-term and intermittent clearance of SnCs affects tissue stem cells such as HSCs because HSCs are mostly quiescent in order to self-renew and prevent exhaustion. We used p16-3MR transgenic mice to evaluate the effect on HSCs of long-term and intermittent clearance of SnCs by ganciclovir (GCV), which selectively kills SnCs in these mice. We treated WT and p16-3MR mice with GCV (25 mg/kg/day x 5 days/per cycle with a 2-week interval between the cycles by ip injection) or saline (vehicle control) from 12 to 24 months of age. GCV effectively prevented SnC accumulation in p16-3MR mice but had no effect in WT mice. In general, aged p16-3MR mice treated with GCV looked healthier than old vehicle-treated p16-3MR mice and old WT mice treated with vehicle or GCV as expected (Baker DJ et al. Nature, 530: 184, 2016). Importantly, GCV abrogated aging-induced increases in p16 expression, p38MAPK activation, and accumulation of DNA double-strand breaks in HSCs in p16-3MR mice. This abrogation was associated with a significant improvement in HSC clonogenicity, suggesting that long-term and intermittent clearance of SnCs prevents HSC aging in naturally aged mice. This suggestion is confirmed by competitive serial and limited dilution HSC transplantation assays, which revealed that HSCs from GCV-treated old p16-3MR mice were functionally indistinguishable from those of young mice. To elucidate the mechanism by which SnC clearance prevents HSC aging, we evaluated the clonogenicity of Lin−Sca1+c-Kit+ (LSK) cells from young or vehicle- or GCV-treated old p16-3MR mice in co-cultures with bone marrow stromal cells (BMSCs) from these mice. We found that clearance of SnCs not only prevented the decline in HSC clonogenic function with aging but also preserved the hematopoietic supporting function of BMSCs. This preservation was associated with reduced levels of Cdkn2a and Cdkn1a mRNAs and senescence-associated secretory phenotype (SASP) factors (such as Il1β, Tnfα and Ccl5), which can adversely affect HSC self-renewal, and decrease of adipogenic and osteoclastic differentiation. In addition, the levels of the HSC self-renewal promoting factor Spp1 mRNA was higher in BMSCs from GCV-treated old p16-3MR mice than in the cells from vehicle-treated old p16-3MR mice. These findings suggest that long-term and intermittent clearance of SnCs can also inhibit BMSC senescence, which contributes to the prevention of HSC aging. Disclosures He: University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Chang:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Kim:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Almeida:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Campisi:Unity Biotechnology: Equity Ownership, Other: co-founder of Unity Biotechnology which develops small-molecule senolytic drugs. Zhou:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents; Unity Biotechnology: Equity Ownership, Other: Co-founder of Unity Biotechnology which develops small-molecule senolytic drugs; Dialectic Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: co-founders of Dialectic Therapeutics that develops Bcl-xl PROTACs as anti-cancer agents.

Tanshinone prevents alveolar bone loss in ovariectomized osteoporosis rats by up-regulating phosphoglycerate dehydrogenase

Osteoporosis is manifested by reduced bone mass. Tanshinone has been shown to affect osteoclast differentiation, but its role in osteoporosis remains less clear. This study aimed to investigate the effects and molecular mechanisms of tanshinone on osteoporosis. Osteoporosis was induced by bilateral ovariectomy (OVX) in adult female rats treated with or without tanshinone. Trabecular bone structure was assessed by micro-computed tomography (micro-CT). Bone marrow stromal cells (BMSCs) were isolated for analysis of stemness and senescence. mRNA levels of age related genes were examined and the role of the gene that was upregulated by tanshinone treatment was suppressed to determine its involvement in tanshinone mediated effects. Finally, the mechanism underlying tanshinone induced gene upregulation was explored. We found that tanshinone treatment restored alveolar bone structure in OVX rats as well as the stemness and senescence status of BMSCs isolated from OVX rats. Tanshinone upregulated Phgdh mRNA levels and inhibition of phosphoglycerate dehydrogenase Phgdh, the protein encoded by the Phgdh gene, abolished the effects of tanshinone on BMSC stemness and senescence. Finally, we found that OVX lead to hypermethylation of the promoter region of Phgdh which was suppressed by tanshinone treatment. Our study shows that tanshinone potently suppress OVX induced osteoporosis and BMSC senescence through upregulation of PHGDH.

NOX2 Derived Superoxide Induces Pro-Tumoral p16INK4a Driven Senescence in the AML Bone Marrow Microenvironment

BackgroundAcute myeloid leukaemia (AML) is an age-related disease that is highly dependent on the bone marrow (BM) microenvironment. Senescence is the irreversible arrest of cell proliferation and is associated with the senescence associated secretary phenotype (SASP). The senescent phenotype has been associated with pro-tumoral changes that support proliferation and survival of malignant cells. Because AML is an age-related disease and highly dependent on the BM microenvironment, which naturally becomes senescent with age, we hypothesized, and subsequently tested the idea, that AML not only favors a senescent microenvironment but might actively shape a senescent microenvironment to promote tumor proliferation and survival.MethodsPrimary AML and bone marrow stroma was isolated from bone marrow samples obtained from patients following informed consent and non-malignant CD34+ cells were isolated from cord blood following informed consent. All primary tissue was used under approval from the UK Health Research Authority. NSG, p16-3MR(Demaria et al. 2014 Cell Development) and wildtype C57Bl/6 animals were used and experiments were conducted with approval from the UK Home Office and University of East Anglia Animal Welfare and Ethical Review Board. Primary AML and non-malignant human CD34+ cells were engrafted into NSG mice (AML-NSG and hu-NSG). Bone marrow stromal cells (BMSCs) were isolated by cell sorting for mouse CD45-, Ter119-, CD31-, CD105+, CD140a+. Syngeneic AML cells were generated from lineage negative cells transduced with the oncogene MN1. Senescence was determined by p16, p21 mRNA expression, SASP expression (IL-6 and MIP3a), senescence associated β-Galactosidase activity and using themonomeric red fluorescent protein (mRFP) component of the p16-3MR mouse model. Oxidative stress and superoxide levels were measured using 2‘,7‘-dichlorofluorescein (DCF) fluorescence and the Amplex Red assay. NOX-2 in AML was inhibited by RNAi.ResultsFirst, we isolated and analysed BMSC from AML-NSG and hu-NSG. Using real-time PCR we detected high expression of p16 and p21 in the BMSC isolated from AML-NSG but not hu-NSG. Moreover, we detected high mouse IL-6 and MIP3a cytokine levels from serum isolated from AML-NSG but not hu-NSG. MN1 engrafted into the p16-3MR model induced mRFP expression in the BMSC population. This shows that in both a xenograft and syngeneic model of AML the BMSC population becomes senescent. Using the p16-3MR model we selectively depleted senescent BMSC via the viral thymidine kinase death switch. Treatment of p16-3MR mice engrafted with MN1 with ganciclovir reduced tumour volume and prolonged animal survival. Since hydrogen peroxide has been shown to induce senescence we hypothesised that NOX2 derived superoxide from AML drives the BMSC senescence. BMSCs isolated from the MN1 engrafted p16-3MR model isolated BMSC show increased DCF fluorescence and isolated BM shows elevated superoxide compared to BMSC and BM isolated from control mice. NOX2 knock down resulted in significant reduction in BM derived superoxide and BMSC DCF fluorescence and this resulted in a reduced BMSC senescent phenotype.ConclusionHere we show that in AML, NOX2 derived superoxide induces a p16INK4a drivensenescent phenotype in the BMSC. Moreover, we find that this process forms a fundamental part of the pro-tumoral pathophysiology of the disease. The senescent bone marrow microenvironment may therefore present new treatment targets in AML. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2449: Superoxide regulates protumoral p16-driven senescence in the bone marrow microenvironment of acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is primarily a disease of the elderly and depends on the bone marrow microenvironment. The aging bone marrow accumulates senescent cells, characterized by an irreversible arrest of cell proliferation and the secretion of numerous pro-inflammatory cytokines, chemokines and growth factors, termed the secretary associated senescence profile (SASP). Previously we showed that AML is capable of reprogramming bone marrow stromal cells (BMSC) and induces oxidative stress in the BMSC (via superoxide) to support blast survival and proliferation. Here, we hypothesize that AML derived superoxide induces a senescent aging phenotype in the BMSC, generating a SASP in the BMSC which then feed back to promote AML blast proliferation in the bone marrow niche. We isolated primary AML cells and BMSCs from the bone marrow of patients attending our clinic. To assess senescence of BMSC we measured p16 mRNA and protein levels and senescence associated β-Galactosidase activity. Primary AML was engrafted into NSG mice and CD105+ / CD45- cells (BMSC) were sorted and analysed for SASP. We transplanted syngeneic AML blasts (generated from lineage negative cells transduced with the oncogene MN1) into p16-3MR, WT C57Bl/6 and NOX2 (CYBB) deficient mice. Patient samples were taken in accordance with the Declaration of Helsinki and animal studies performed under approval of the UK Home Office. We report that human AML cells induce SASP in vivo as measured by RT-PCR of sorted BMSC cells from NSG mice engrafted with human AML. Next, we engrafted p16-3MR mice with MN1 cells and flow cytometry analysis of isolated BSMC (CD105+ CD45-) showed AML cells induce micro-environment senescence in vivo. Furthermore, depletion of p16 positive stromal cells through treating MN1 engrafted p16-3MR animals with ganciclovir significantly increased survival of MN1 engrafted mice compared with animals treated with vehicle control. In addition, we show that MN1 AML cells, transplanted into NOX2 deficient mice, generate high levels of superoxide in the bone marrow, which results in oxidative stress in the BMSC. Finally NOX2-KD in MN1 AML reduces BMSC senescence in the BM of p16-3MR recipient mice and results in improved animal survival. Here we show that the AML derived superoxide induces a senescent phenotype in the bone marrow micro-environment which then feeds back to enhance the growth capacity of AML. Targeting the senescent microenvironment may therefore provide novel therapeutic approaches for the treatment of this disease in the future. Citation Format: Charlotte Hellmich, Amina Abdul-Aziz, Christopher Marlein, Yu Sun, Manar Shafat, Angela Collins, Rachel Piddock, Judith Campisi, Kristian Bowles, Stuart Rushworth. Superoxide regulates protumoral p16-driven senescence in the bone marrow microenvironment of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2449.

Estrogen regulates stemness and senescence of bone marrow stromal cells to prevent osteoporosis via ERβ‐SATB2 pathway

Decline of pluripotency in bone marrow stromal cells (BMSCs) associated with estrogen deficiency leads to a bone formation defect in osteoporosis. Special AT-rich sequence binding protein 2 (SATB2) is crucial for maintaining stemness and osteogenic differentiation of BMSCs. However, whether SATB2 is involved in estrogen-deficiency associated-osteoporosis is largely unknown. In this study, we found that estrogen mediated pluripotency and senescence of BMSCs, primarily through estrogen receptor beta (ERβ). BMSCs from the OVX rats displayed increased senescence and weaker SATB2 expression, stemness, and osteogenic differentiation, while estrogen could rescue these phenotypes. Inhibition of ERβ or ERα confirmed that SATB2 was associated with ERβ in estrogen-mediated pluripotency and senescence of BMSCs. Furthermore, estrogen mediated the upregulation of SATB2 through the induction of ERβ binding to estrogen response elements (ERE) located at -488 of the SATB2 gene. SATB2 overexpression alleviated senescence and enhanced stemness and osteogenic differentiation of OVX-BMSCs. SATB2-modified BMSCs transplantation could prevent trabecular bone loss in an ovariectomized rat model. Collectively, our study revealed the role of SATB2 in stemness, senescence, and osteogenesis of OVX-BMSCs. These results indicate that estrogen prevents osteoporosis by promoting stemness and osteogenesis, and inhibiting senescence of BMSCs through an ERβ-SATB2 pathway. Therefore, SATB2 is a novel anti-osteoporosis target gene.

Transplantation of osteoporotic bone marrow stromal cells rejuvenated by the overexpression of SATB2 prevents alveolar bone loss in ovariectomized rats

Estrogen-deficient osteoporosis is an aging-related disease with high morbidity that not only significantly increases a woman's risk of fragility fracture but is also associated with tooth and bone loss in the supporting alveolar bone of the jaw. Emerging evidence suggests that the aging of bone marrow stromal cells (BMSCs) contributes to the development of osteoporosis. In this study, we aimed to investigate the role of the special AT-rich sequence-binding protein 2 (SATB2), a stemness and senescence regulator of craniofacial BMSCs, in rat ovariectomy-induced alveolar osteoporosis. We also sought to determine whether transplantation of SATB2-modified BMSCs could ameliorate estrogen deficient alveolar bone loss. Our data revealed that BMSCs from ovariectomy-induced alveolar bone exhibited typical senescence phenotypes such as diminished stemness and osteogenic capacity, increased expression of senescence or osteoclastic markers and enhanced adipogenic potential. These phenotypic changes are a result of SATB2-mediated senescence dysregulation as evidenced by nuclear γH2AX foci formation. Moreover, overexpression of SATB2 significantly alleviated the senescence of osteoporotic BMSCs in vitro. Importantly, transplantation of SATB2-modified BMSCs significantly attenuated ovariectomy-induced alveolar bone loss in vivo. Together, our results revealed that SATB2 is a critical regulator of alveolar BMSC senescence, and its overexpression decreases these senescent changes both in vitro and in vivo. SATB2-modified BMSC delivery could be a viable and promising therapeutic strategy for alveolar bone loss induced by estrogen-deficient osteoporosis.

Inhibition of phosphatidylcholine‐specific phospholipase C prevents bone marrow stromal cell senescence in vitro

Bone marrow stromal cells (BMSCs) can proliferate in vitro and can be transplanted for treating many kinds of diseases. However, BMSCs become senescent with long-term culture, which inhibits their application. To understand the mechanism underlying the senescence, we investigated the activity of phosphatidylcholine-specific phospholipase C (PC-PLC) and levels of integrin beta4, caveolin-1 and ROS with BMSC senescence. The activity of PC-PLC and levels of integrin beta4, caveolin-1 and ROS increased greatly during cell senescence. Selective inhibition of increased PC-PLC activity with D609 significantly decreased the number of senescence-associated beta galactosidase positive cells in BMSCs. Furthermore, D609 restored proliferation of BMSCs and their differentiation into adipocytes. Moreover, D609 suppressed the elevated levels of integrin beta4, caveolin-1 and ROS. The data suggest that PC-PLC is involved in senescence of BMSCs, and its function is associated with integrin beta4, caveolin-1 and ROS.