From estrogen-centric to aging and oxidative stress: a revised perspective of the pathogenesis of osteoporosis

Abstract

Estrogen deficiency has been considered the seminal mechanism of osteoporosis in both women and men, but epidemiological evidence in humans and recent mechanistic studies in rodents indicate that aging and the associated increase in reactive oxygen species (ROS) are the proximal culprits. ROS greatly influence the generation and survival of osteoclasts, osteoblasts, and osteocytes. Moreover, oxidative defense by the FoxO transcription factors is indispensable for skeletal homeostasis at any age. Loss of estrogens or androgens decreases defense against oxidative stress in bone, and this accounts for the increased bone resorption associated with the acute loss of these hormones. ROS-activated FoxOs in early mesenchymal progenitors also divert ss-catenin away from Wnt signaling, leading to decreased osteoblastogenesis. This latter mechanism may be implicated in the pathogenesis of type 1 and 2 diabetes and ROS-mediated adverse effects of diabetes on bone formation. Attenuation of Wnt signaling by the activation of peroxisome proliferator-activated receptor gamma by ligands generated from lipid oxidation also contributes to the age-dependent decrease in bone formation, suggesting a mechanistic explanation for the link between atherosclerosis and osteoporosis. Additionally, increased glucocorticoid production and sensitivity with advancing age decrease skeletal hydration and thereby increase skeletal fragility by attenuating the volume of the bone vasculature and interstitial fluid. This emerging evidence provides a paradigm shift from the "estrogen-centric" account of the pathogenesis of involutional osteoporosis to one in which age-related mechanisms intrinsic to bone and oxidative stress are protagonists and age-related changes in other organs and tissues, such as ovaries, accentuate them.

Figure 1

A, Bone loss begins in the third decade of life in both sexes. The data are from the Epidemiological Follow-up Study cohort of the First National Health and Nutrition Examination Survey (NHANES), a nationally representative sample of noninstitutionalized civilians who were followed for a maximum of 22 yr. A cohort of 2879 Caucasian men (1437 in the bone density subsample) aged 45–74 yr at baseline (1971–1975) were observed through 1992. [From A. C. Looker et al.: Osteoporosis Int 8:468–489, 1998 (21). Reproduced with permission of the International Bone & Mineral Society and the IBMS BoneKEy where this graphic depiction of the data is provided online.] B, Age is a more critical determinant of fracture risk than bone mass in humans. Data are from a follow-up of 521 Caucasian women over an average of 6.5 yr with repeated bone mass measurements at the radius. A total of 138 nonspinal fractures in 3388 person-years were detected, and the incident fractures were cross-classified by age and bone mass. The incidence of fracture was then fitted to a log-linear model in age and bone mass. [From S. L. Hui et al.: J Clin Invest 81:1804–1809, 1988 (24). Reproduced with permission from The American Society for Clinical Investigation.]

Figure 2

Schematic illustration of the shuttling of FoxOs between the cytoplasm (in the setting of growth factor stimulation, e.g., insulin) and the nucleus in response to oxidative stress (exemplified by H₂O₂). Note the distinct site of phosphorylation and cytoplasmic retention of FoxOs by the insulin- initiated Ras/PI3K/Akt cascade, as opposed to the sites of phosphorylation and nuclear localization of FoxOs in response to the H₂O₂-induced JNK activation cascade. The structure in the bottom left depicts a mitochondrion and its electron transport chain along with the tricarboxylic acid cycle that is responsible for ATP synthase activation and the generation of ATP.

Figure 3

Both trabecular and cortical bone mass decrease with age in mice. Micro-computed tomography analysis at the distal femoral metaphysis (left) and the femoral midshaft (right) of virgin female mice (n = 6 to 10 per group) was performed at the indicated time points. At the bottom of the right graph is a schematic representation of the changes of the femoral cortex with age, depicting the enlargement and thickening during the growth period (from 1 to 4 months) and the thinning after the attainment of peak bone mass due to endosteal resorption. BV, Bone volume; TV, total volume. [Unpublished data from M. Bouxsein and V. Glatt, Beth Israel Deaconess Medical Center, Harvard Medical School, generously provided for the purpose of this review article.]

Figure 4

Advancing age (A) and loss of sex steroids (B) cause similar changes in oxidative stress. ROS and GSR activity were measured in the bone marrow aspirates, and the phosphorylation status of p53 and p66^shc was determined by Western blot analysis in vertebral lysates from female or male C57BL6 mice at the indicated ages. Ovariectomy (OVX) or orchidectomy (ORX) was performed at 5 months of age, and analysis was done 6 wk later. Bars indicate mean ± sd; n = 4 mice per group. AFU, Arbitrary fluorescence units; veh, vehicle. *, P < 0.05 compared to 4 months or OVX or ORX + vehicle. [Modified from M. Almeida et al.: J Biol Chem 282:27285–27297, 2007 (113).]

Figure 5

ROS-activated signals affecting the genesis and lifespan of osteoblasts and osteoclasts and the counter-regulatory actions of sex steroids. In osteoclast precursors, RANKL-induced activation of RANK stimulates ROS production, which is essential for osteoclastogenesis. In addition, mitochondria biogenesis coupled with activation of the transferrin receptor (TIR1) by the iron-transferrin (Fe-Tf) complex stimulates mitochondria respiration and ROS production, which are also essential for osteoclast activation. Estrogens and androgens, acting via their respective receptors (ERα and AR), attenuate both osteoclastogenesis and survival by stimulating GSH and Trx reductase in an ERK-dependant manner (113,145,172,176,177). In osteoblastic cells, p66^shc is an essential mediator of the effects of oxidative stress on apoptosis, NF-κB activation, and cytokine production. Estrogens and androgens attenuate these effects by suppressing p66^shc phosphorylation in an ERK-dependent manner (113,169).

Figure 6

Oxidized fatty acids, their degradation products, and bone formation. With advancing age, increased Alox15 expression and increased OS promote lipid peroxidation by adding oxygen to PUFAs, like linoleic acid. The hydroperoxide product of this reaction, 9-HPODE, decomposes into a hydroxy derivative 9-HODE, which binds to and activates PPARγ. Hydroxy radicals generated during this process add to the redox burden of the cell and can further stimulate PUFA peroxidation by nonenzymatic means. Importantly, 9-HPODE is also converted to 4-HNE, itself a potent prooxidant agent. Oxidized PUFAs activate PPARγ and promote its association with β-catenin, resulting in β-catenin degradation. ROS-activated FoxOs divert β-catenin from TCF- to FoxO-mediated transcription and thereby attenuate the restraining effect of β-catenin/TCF on the transcription of PPARγ. The combination of decreased Wnt signaling and increased PPARγ levels as well as PPARγ ligands leads to attenuation of osteoblastogenesis and increased osteoblast/osteocyte apoptosis along with increased adipogenesis (at some sites), thereby suppressing bone formation.

Figure 7

Photomicrograph of a cross-section of a murine, cancellous BMU comprising osteoclasts and osteoblasts along with two capillaries containing erythrocytes [courtesy of Robert S. Weinstein from the University of Arkansas for Medical Sciences]. Osteoclasts are identified by their discrete tartrate-resistant acid phosphatase-positive red granules, and the osteoblasts by their large nuclei with multiple nucleoli and underlying light blue osteoid. An abundance of osteocytes can be seen embedded individually (blue staining cells) within the mineralized bone (green) surrounding the BMU. Methyl green and tartrate-resistant acid phosphatase staining of undecalcified bone viewed with Nomarski differential interference contrast microscopy (X630).

Figure 8

The connection between osteocytes and blood vessels. Left, Low (upper panel) and high (lower panel) magnifications of electron microscopy images demonstrating reliefs of the osteocytes and their canalicular network following acid-etching of murine bone sections [courtesy of Lynda Bonewald from the University of Missouri and Kansas City Dental School]. Please note multiple attachments of the osteocyte processes to the vessels depicted in the center of these images. Right, Cartoon depicting the same connections. [From M. L. Knothe Tate et al.: Bone 22:107–117, 1998 (366). Reproduced with permission of the Journal © Elsevier.]