Independent Roles of Estrogen Deficiency and Cellular Senescence in the Pathogenesis of Osteoporosis: Evidence in Young Adult Mice and Older Humans

Abstract

Estrogen deficiency is a seminal mechanism in the pathogenesis of osteoporosis. Mounting evidence, however, establishes that cellular senescence, a fundamental mechanism that drives multiple age-related diseases, also causes osteoporosis. Recently, we systematically identified an accumulation of senescent cells, characterized by increased p16^Ink4a and p21^Cip1 levels and development of a senescence-associated secretory phenotype (SASP), in mouse bone/marrow and human bone with aging. We then demonstrated that elimination of senescent cells prevented age-related bone loss using multiple approaches, eg, treating old mice expressing a "suicide" transgene, INK-ATTAC, with AP20187 to induce apoptosis of p16^Ink4a -senescent cells or periodically treating old wild-type mice with "senolytics," ie, drugs that eliminate senescent cells. Here, we investigate a possible role for estrogen in the regulation of cellular senescence using multiple approaches. First, sex steroid deficiency 2 months after ovariectomy (OVX, n = 15) or orchidectomy (ORCH, n = 15) versus sham surgery (SHAM, n = 15/sex) in young adult (4-month-old) wild-type mice did not alter senescence biomarkers or induce a SASP in bone. Next, in elderly postmenopausal women, 3 weeks of estrogen therapy (n = 10; 74 ± 5 years) compared with no treatment (n = 10; 78 ± 5 years) did not alter senescence biomarkers or the SASP in human bone biopsies. Finally, young adult (4-month-old) female INK-ATTAC mice were randomized (n = 17/group) to SHAM+Vehicle, OVX+Vehicle, or OVX+AP20187 for 2 months. As anticipated, OVX+Vehicle caused significant trabecular/cortical bone loss compared with SHAM+Vehicle. However, treatment with AP20187, which eliminates senescent cells in INK-ATTAC mice, did not rescue the OVX-induced bone loss or alter senescence biomarkers. Collectively, our data establish independent roles of estrogen deficiency and cellular senescence in the pathogenesis of osteoporosis, which has important implications for testing novel senolytics for skeletal efficacy, as these drugs will need to be evaluated in preclinical models of aging as opposed to the current FDA model of prevention of OVX-induced bone loss. © 2019 American Society for Bone and Mineral Research.

Keywords: AGING; ANIMAL MODELS; BONE; ESTROGEN THERAPY; OSTEOCYTE; OSTEOPOROSIS; SEX STEROIDS.

Fig 1.. Comparisons of changes in senescence biomarkers and senescence-associated secretory phenotype (SASP) factors in osteocyte-enriched bone samples from male mice in the context of natural chronological aging and sex steroid deficiency.

(A) rt-qPCR analysis of in vivo age-associated changes in mRNA expression of the senescence effectors, p16^Ink4a (Cdkn2a), p21^Cip1 (Cdkn1a), and p53 (Trp53), in osteocyte-enriched bone samples from young (6-month-old, n=12) versus old (24-month-old, n=10) male C57BL/6 wild-type (WT) mice (data reproduced from J Bone Miner Res 31(11):1920–9, 2016). (B) Experimental design for testing the effects of orchidectomy-induced sex steroid deficiency on biomarkers of cellular senescence in bone; 4-month-old male C57BL/6 WT mice were randomized to undergoing either sham surgery (SHAM, n=15) or orchidectomy (ORCH, n=15) for 2 months; all animals were sacrificed at age 6 months. (C) rt-qPCR analysis of in vivo changes in mRNA expression of the senescence effectors, p16^Ink4a (Cdkn2a), p21^Cip1 (Cdkn1a), and p53 (Trp53), in osteocyte-enriched bone samples from SHAM (n=15) versus ORCH (n=15) male C57BL/6 WT mice. (D) rt-qPCR analysis of in vivo age-associated changes in mRNA expression of 36 established SASP factors in osteocyte-enriched bone samples from young (6-month-old, n=12) versus old (24-month-old, n=10) male C57BL/6 WT mice (data reproduced from J Bone Miner Res 31(11):1920–9, 2016). (E) rt-qPCR analysis of in vivo changes in mRNA expression of 36 established SASP factors in osteocyte-enriched bone samples from SHAM (n=15) versus ORCH (n=15) male C57BL/6 WT mice. Data represent mean ± SEM (error bars); NE = Not expressed (Cycle threshold [Ct] values >35). *p < 0.05; **p < 0.01; ***p < 0.001 (independent samples t-test).

Fig 2.. Comparisons of changes in senescence biomarkers and senescence-associated secretory phenotype (SASP) factors in osteocyte-enriched bone samples from female mice in the context of natural chronological aging and sex steroid deficiency.

(A) rt-qPCR analysis of in vivo age-associated changes in mRNA expression of the senescence effectors, p16^Ink4a (Cdkn2a), p21^Cip1 (Cdkn1a), and p53 (Trp53), in osteocyte-enriched bone samples from young (6-month-old, n=15) versus old (24-month-old, n=9) female C57BL/6 wild-type (WT) mice (data reproduced from J Bone Miner Res 31(11):1920–9, 2016). (B) Experimental design for testing the effects of ovariectomy-induced sex steroid deficiency on biomarkers of cellular senescence in bone; 4-month-old female C57BL/6 WT mice were randomized to undergoing either sham surgery (SHAM, n=15) or ovariectomy (OVX, n=15) for 2 months; all animals were sacrificed at age 6 months. (C) rt-qPCR analysis of in vivo changes in mRNA expression of the senescence effectors, p16^Ink4a (Cdkn2a), p21^Cip1 (Cdkn1a), and p53 (Trp53), in osteocyte-enriched bone samples from SHAM (n=15) versus OVX (n=15) female C57BL/6 WT mice. (D) rt-qPCR analysis of in vivo age-associated changes in mRNA expression of 36 established SASP factors in osteocyte-enriched bone samples from young (6-month-old, n=15) versus old (24-month-old, n=9) female C57BL/6 WT mice. (E) rt-qPCR analysis of in vivo changes in mRNA expression of 36 established SASP factors in osteocyte-enriched bone samples from SHAM (n=15) versus OVX (n=15) female C57BL/6 WT mice. Data represent mean ± SEM (error bars); NE = Not expressed (Cycle threshold [Ct] values >35). *p < 0.05; **p < 0.01; ***p < 0.001 (independent samples t-test).

Fig. 3.. Comparisons of changes in senescence biomarkers and senescence-associated secretory phenotype (SASP) factors in human bone biopsies obtained from healthy women in the context of aging and in response to short-term estrogen (STE) therapy.

(A) rt-qPCR analysis of in vivo age-associated changes in mRNA expression of the senescence effectors, p16^INK4A (CDKN2A), p21^CIP1 (CDKN1A), and p53 (TP53), in human bone biopsies isolated from healthy younger premenopausal (n=10; mean age ± SD, 27±3 yrs) versus healthy older postmenopausal (n=10; mean age ± SD, 78±5 yrs) women (data reproduced from J Bone Miner Res 31(11):1920–9, 2016). (B) Experimental design for testing the effects of STE therapy on biomarkers of cellular senescence in bone; human bone biopsies were obtained from 20 healthy postmenopausal women who received either no treatment (NT, n=10; mean age ± SD, 78±5 yrs) or short-term estrogen (STE, n=10; mean age ± SD, 74±5 yrs) therapy for 3 weeks. (C) rt-qPCR analysis of in vivo changes in mRNA expression of the senescence effectors, p16^INK4A (CDKN2A), p21^CIP1 (CDKN1A), and p53 (TP53), in human bone biopsies isolated from 20 healthy postmenopausal women following either NT or STE therapy for 3 weeks. (D) rt-qPCR analysis of in vivo age-associated changes in mRNA expression of 36 established SASP factors in human bone biopsies isolated from younger premenopausal versus older postmenopausal women who received NT (data reproduced from J Bone Miner Res 31(11):1920–9, 2016). (E) rt-qPCR analysis of in vivo changes in mRNA expression of 36 established SASP factors in human bone biopsies isolated from 20 healthy postmenopausal women following either NT or STE therapy for 3 weeks. Data represent mean ± SEM (error bars); NE = Not expressed (Cycle threshold [Ct] values >35). *p < 0.05; **p < 0.01; ***p < 0.001 (independent samples t-test).

Fig. 4.. Treatment with AP20187, which eliminates senescent cells in old INK-ATTAC mice, does not rescue ovariectomy (OVX)-induced bone loss in young mice.

(A) Experimental design for testing the effects of AP20187 treatment on OVX-induced bone loss; 4-month-old (Baseline) female INK-ATTAC mice were randomized to either SHAM+Vehicle (n=17), OVX+Vehicle (n=17), or OVX+AP20187 (n=17) for 8 weeks; all mice were sacrificed at age 6 months (Endpoint). Uterine weights (g) at study Endpoint in INK-ATTAC SHAM+Vehicle (n=17), INK-ATTAC OVX+Vehicle (n=17), and INK-ATTAC OVX+AP20187 mice. (C) Changes in total body mass (g) throughout the duration of the 8 wk study. (D) Changes in fat mass (g) from Baseline to study Endpoint. (E) Changes in lean mass (g) from Baseline to study Endpoint. (F) Representative micro-computed tomography (µCT) images of bone microarchitecture at the lumbar spine of INK-ATTAC SHAM+Vehicle (n=17), INK-ATTAC OVX+Vehicle (n=17), and INK-ATTAC OVX+AP20187 (n=17) mice. (G–J) Quantification of µCT-derived bone volume fraction (BV/TV; %) (G), trabecular number (Tb.N; 1/mm) (H), trabecular thickness (Tb.Th; mm) (I), and trabecular separation (Tb.Sp; mm) (J) at the lumbar spine. (K) Representative µCT images of bone microarchitecture at the femur diaphysis of INK-ATTAC SHAM+Vehicle (n=17), INK-ATTAC OVX+Vehicle (n=17), and INK-ATTAC OVX+AP20187 (n=17) mice. (L–N) Quantification of µCT-derived cortical thickness (Ct.Th; mm) (L), endocortical circumference (EC; mm) (M), and periosteal circumference (PC; mm) (N) at the femur diaphysis. (O–P) Quantification of plasma levels of markers of bone formation (amino-terminal propeptide of type I collagen [P1NP]) (O) and bone resorption (cross-linked C-telopeptide of type I collagen [CTx]) (P) in INK-ATTAC SHAM+Vehicle (n=17), INK-ATTAC OVX+Vehicle (n=17), and INK-ATTAC OVX+AP20187 (n=17) mice at study Endpoint. Data represent mean ± SEM (error bars); ns = not significant (p > 0.05). *p < 0.05; **p < 0.01; ***p < 0.001 (one-way analysis of variance [ANOVA] followed by Tukey post-hoc test to adjust for multiple comparisons). ^ap < 0.05 vs OVX+Vehicle vs SHAM+Vehicle (repeated measures ANOVA, followed by the Tukey post-hoc test to adjust for multiple comparisons); ^bp < 0.05 vs OVX+Vehicle vs SHAM+Vehicle (repeated measures ANOVA, followed by the Tukey post-hoc test to adjust for multiple comparisons).

Fig. 5.. Treatment with AP20187 in young female INK-ATTAC mice does not alter senescence biomarkers in bone following OVX.

(A–D) rt-qPCR analysis of in vivo changes in mRNA expression of enhanced green fluorescent protein (EGFP) (A), which is encoded by the INK-ATTAC transgene, as well as the senescence effectors, p16^Ink4a (Cdkn2a) (B), p21^Cip1 (Cdkn1a) (C), and p53 (Trp53) (D), in osteocyte-enriched samples from 6-month-old female INK-ATTAC SHAM+Vehicle (n=17), INK-ATTAC OVX+Vehicle (n=17), and INK-ATTAC OVX+AP20187 (n=17) mice (see experimental design in Fig. 4A). (E) Quantification of the percentage of senescent osteocytes (OCYs) in 6-month-old female INK-ATTAC SHAM+Vehicle (n=17), INK-ATTAC OVX+Vehicle (n=17), and INK-ATTAC OVX+AP20187 (n=17) mice according to the SADS assay performed on cortical bone diaphyses (n > 30 images per animal, n=8 mice/group). Data represent mean ± SEM (error bars); ns = not significant (p > 0.05; one-way analysis of variance [ANOVA] followed by Tukey post-hoc test to adjust for multiple comparisons).