IntroductionAs a result of the aging process, the bone deteriorates in composition, structure and function, which predisposes to osteoporosis. Osteoporosis is defined as deterioration in bone mass and micro-architecture, with increasing risk to fragility fractures [Raisz and Rodan, 2003]. Owing to the close relationship between the aging process of bone and the pathogenesis of osteoporosis, research on the mechanisms of age-related bone loss has increased significantly in recent years involving a combination of basic, clinical, observational and translational studies.Bone is a dynamic organ that serves mechanical and homeostatic functions. It undergoes a continual self-regeneration process called remodeling. Remodeling removes old bone and replaces it with new bone. This regenerative process occurs in distinct areas of bone known as bone metabolic units (BMUs) [Riggs et al. 2002]. Within each BMU bone formation by osteoblasts and bone resorption by osteoclasts is coupled tightly in a delicate balance to maintain bone mass and strength to resist deformity. With aging this balance shifts in a negative direction, favoring greater bone resorption and less bone formation. This combination of bone mass deficiency and reduction in strength ultimately results in osteoporosis and fractures.Aging in combination with intrinsic and extrinsic factors accelerates the decline in bone mass that predisposes to fractures. Intrinsic factors include genetics, peak bone mass accrual in youth, alterations in cellular components, hormonal, biochemical and vasculature status. Extrinsic factors include nutrition, physical activity, comorbid medical conditions and drugs. In this article we review the mechanisms of age-related bone deterioration and their impact on the pathogenesis of osteoporosis. In addition, current and future therapeutic approaches focused on the correction of mechanisms associated with aging bone will also be outlined. Bone remodeling in aging boneRemodeling is continuous and coordinated cycle of removal of old bone by osteoclasts followed by the deposition of new bone by osteoblasts in response to micro damage and variable mechanical loadings. Bone remodeling is a continuous process throughout life. In the first three decades of life, bone turnover is coupled tightly to maintain a steady state between bone resorption and bone formation. Although there are variances in turnover rates, peak bone mass and size is achieved around the age of 15-20 years in women and later in men [Raisz and Seeman, 2001]. After this, long before sex steroids deficiency occurs, bone loss Aging and bone loss: new insights for the clinicianOddom Demontiero, Christopher Vidal and Gustavo Duque Abstract: It is well known that the underlying mechanisms of osteoporosis in older adults are different than those associated with estrogen deprivation. Age-related bone loss involves a gradual and progressive decline, which is also seen in men. Markedly increased bone resorption leads to the initial fall in bone mineral density. With increasing age, th...

The mechanisms involved in the anabolic effect of interferon gamma (IFNc) on bone have not been carefully examined. Using microarray expression analysis, we found that IFNc upregulates a set of genes associated with a tryptophan degradation pathway, known as the kynurenine pathway, in osteogenic differentiating human mesenchymal stem cells (hMSC). We, therefore, hypothesized that activation of the kynurenine pathway plays a role in osteoblastogenesis even in the absence of IFNc. Initially, we observed a strong increase in tryptophan degradation during osteoblastogenesis with and without IFNc in the media. We next blocked indoleamine 2,3-dioxygenase-1 (IDO1), the most important enzyme in the kynurenine pathway, using a siRNA and pharmacological approach and observed a strong inhibition of osteoblastogenesis with a concomitant decrease in osteogenic factors. We next examined the bone phenotype of Ido1 knockout (Ido1 2/2 ) mice. Compared to their wild-type littermates, Ido1 2/2 mice exhibited osteopenia associated with low osteoblast and high osteoclast numbers. Finally, we tested whether the end products of the kynurenine pathway have an osteogenic effect on hMSC. We identified that picolinic acid had a strong and dose-dependent osteogenic effect in vitro. In summary, we demonstrate that the activation of the kynurenine pathway plays an important role during the commitment of hMSC into the osteoblast lineage in vitro, and that this process can be accelerated by exogenous addition of IFNc. In addition, we found that mice lacking IDO1 activity are osteopenic. These data therefore support a new role for the kynurenine pathway and picolinic acid as essential regulators of osteoblastogenesis and as potential new targets of bone-forming cells in vivo. STEM CELLS 2015;33:111-121

Changes in the expression of lamin A/C, a fibrilar protein of the nuclear envelope, are associated with the cellular features of age-related bone loss. Reduced expression of lamin A/C inhibits osteoblastogenesis while facilitating adipogenic differentiation of mesenchymal stem cells (MSC) in vitro and in vivo. In this study we investigated the regulatory role that lamin A/C plays on the essential elements of the Wnt/β-catenin pathway, which are pivotal in MSC differentiation. Initially, we assessed the effect of lamin A/C gene (LMNA) overexpression on MSC differentiation while compared it to lamin A/C depleted MSC. Osteogenesis and gene expression of osteogenic factors were higher in LMNA-transfected MSC as compared to control. Conversely, adipogenesis and expression of adipogenic factors were significantly lower in LMNA transfected cells. Nuclear β-catenin was significantly higher (∼two fold) in MSC expressing higher levels of LMNA as compared to control with nuclear β-catenin levels being significantly lower (∼ -42%) in siRNA-treated MSC. Luciferase activity for β-catenin-mediated transcriptional activation was significantly higher in cells overexpressing LMNA. These data indicate that MSC overexpressing LMNA have higher osteogenic and lower adipogenic differentiation potential. In conclusion, our studies demonstrate that lamin A/C plays a significant role in the differentiation of both osteoblasts and adipocytes by regulating some of the elements of Wnt/β-catenin signaling during early MSC differentiation.

The interest in the relationship between fat and bone has increased steadily during recent years. Fat could have a lipotoxic effect on bone cells through the secretion of fatty acids. Palmitate is the most prevalent fatty acid secreted by adipocytes in vitro. Considering that palmitate has shown a high lipotoxic effect in other tissues, here we characterized the lipotoxic effect of palmitate on human osteoblasts (Obs). Initially we tested for changes in palmitoylation in this model. Subsequently we compared the capacity of Obs to differentiate and form bone nodules in the presence of palmitate. From a mechanistic approach, we assessed changes in nuclear activity of β-catenin and runt-related transcription factor 2 (Runx2)/phosphorylated mothers against decapentaplegic (Smad) complexes using Western blotting and confocal microscopy. Quantitative real-time PCR showed negative changes in gene expression of palmitoyltransferase genes. Furthermore, palmitate negatively affected differentiation and bone nodule formation and mineralization by Obs. Although the expression of β-catenin in palmitate-treated cells was not affected, there was a significant reduction in the transcriptional activities of both β-catenin and Runx2. Confocal microscopy showed that whereas Runx2 and Smad-4 and -5 complex formation was increased in bone morphogenetic protein-2-treated cells, palmitate had a negative effect on protein expression and colocalization of these factors. In summary, in this study we identified potential mechanisms of palmitate-induced lipotoxicity, which include changes in palmitoylation, defective mineralization, and significant alterations in the β-catenin and Runx2/Smad signaling pathways. Our evidence facilitates the understanding of the relationship between fat and bone and could allow the development of new potential therapies for osteoporosis in older persons.

Age-related bone loss is associated with changes in bone cellularity with characteristically low levels of osteoblastogenesis. The mechanisms that explain these changes remain unclear. Although recent in vitro evidence has suggested a new role for proteins of the nuclear envelope in osteoblastogenesis, the role of these proteins in bone cells differentiation and bone metabolism in vivo remains unknown. In this study, we used the lamin A/C null (Lmna −/−) mice to identify the role of lamin A/C in bone turnover and bone structure in vivo. At three weeks of age, histological and micro computed tomography measurements of femurs in Lmna −/− mice revealed a significant decrease in bone mass and microarchitecture in Lmna −/− mice as compared with their wild type littermates. Furthermore, quantification of cell numbers after normalization with bone surface revealed a significant reduction in osteoblast and osteocyte numbers in Lmna −/− mice compared with their WT littermates. In addition, Lmna −/− mice have significantly lower osteoclast number, which show aberrant changes in their shape and size. Finally, mechanistic analysis demonstrated that absence of lamin A/C is associated with increase expression of MAN-1 a protein of the nuclear envelope closely regulated by lamin A/C, which also colocalizes with Runx2 thus affecting its capacity as osteogenic transcription factor. In summary, these data clearly indicate that the presence of lamin A/C is necessary for normal bone turnover in vivo and that absence of lamin A/C induces low bone turnover osteopenia resembling the cellular changes of age-related bone loss.

The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induced megakaryoblastic differentiation and c-sis expression in the human hematopoietic stem cell line K-562. This induction occurred at the transcriptional level, as determined by a nuclear runoff transcriptional assay, and was not a generalized effect of TPA, since the treatment of other hematopoietic cell lines and normal peripheral blood lymphocytes with TPA did not result in the appearance of c-sis mRNA.The study of cellular oncogenes (c-onc) has become an important tool in exploring the mechanisms of neoplasia (2). Many proto-oncogenes are also expressed in normal cells during growth and differentiation (7,13,21). Simian sarcoma virus bears in its structure the viral oncogene sis (v-sis) which has its homologous cellular counterpart, c-sis (7). Expression of this oncogene has been reported in glioblastoma and sarcoma cell lines (10,22,28), but it is also expressed in normal endothelial and placental cells (1,13,15). c-sis codes for the B chain of platelet-derived growth factor (4, 9, 16, 23, 27), a potent mitogen for connective tissue cells which is released from platelet alpha granules during clot formation (17). Recently, the sequence of a cDNA clone (from normal endothelial cells) coding for c-sis was reported (6). However, the expression of this oncogene has not been studied in megakaryocytes, the main source of platelet-derived growth factor in the organism, because it is extremely difficult to obtain enough megakaryocytes from normal bone marrow. We report here the expression of c-sis in the stem cell line K-562 during phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA])-induced megakaryocytic differentiation of these cells (12,24,25). The c-sis transcript can be detected as early as 4 h after adding TPA to cultures, even though megakaryocytic differentiation could only be detected after day 2 of treatment. We demonstrate that c-sis expression may occur concomitantly with the megakaryocytic differentiation of K-562 cells rather than as result of nonspecific effects of TPA, since other cell lines and normal peripheral blood lymphocytes treated with TPA do not show c-sis-related transcripts. c-sis induction occurs at the transcriptional level as determined by a nuclear runoff assay.The K-562 cell line was grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillinstreptomycin. Log-phase K-562 cells were treated with TPA (Sigma Chemical Co., St. Louis, Mo.) (final concentration, 10-9 M) and kept in culture for variable lengths of time ranging from 1 h to 6 days. Megakaryoblastic differentiation was assessed by flow cytometric analysis with the monoclonal antibody lOES (5; kindly provided by B. Coller, State University of New York at Stony Brook) which recognizes the GpIIb-IIIa complex, a specific megakaryocytic marker expressed early in the differentiation of this lineage (19,26). Cells positive for the GpIIb-IIIa complex were detected after '* Corresponding author.2 days of TPA treatment, and by 4 days, the majori...

Infiltration of bone marrow with fat is a prevalent feature in people with age-related bone loss and osteoporosis, which correlates inversely with bone formation and positively with high expression levels of peroxisomal proliferator-activated receptor gamma (PPARg). Inhibition of PPARg thus represents a potential therapeutic approach for age-related bone loss. In this study, we examined the effect of PPARg inhibition on bone in skeletally mature C57BL/6 male mice. Nine-month-old mice were treated with a PPARg antagonist, bisphenol-A-diglycidyl ether (BADGE), alone or in combination with active vitamin D (1,25[OH] 2 D 3 ) for 6 weeks. Micro-computed tomography and bone histomorphometry indicated that mice treated with either BADGE or BADGE þ 1,25(OH) 2 D 3 had significantly increased bone volume and improved bone quality compared with vehicle-treated mice. This phenotype occurred in the absence of alterations in osteoclast number. Furthermore, the BADGE þ 1,25(OH) 2 D 3 -treated mice exhibited higher levels of unmineralized osteoid. All of the treated groups showed a significant increase in circulating levels of bone formation markers without changes in bone resorption markers, while blood glucose, parathyroid hormone, and Ca þ remained normal. Furthermore, treatment with BADGE induced higher levels of expression of vitamin D receptor within the bone marrow. Overall, treated mice showed higher levels of osteoblastogenesis and bone formation concomitant with decreased marrow adiposity and ex vivo adipogenesis. Taken together, these observations demonstrate that pharmacological inhibition of PPARg may represent an effective anabolic therapy for osteoporosis in the near future. ß

SummaryLipotoxicity is an overload of lipids in non-adipose tissues that affects function and induces cell death. Lipotoxicity has been demonstrated in bone cells in vitro using osteoblasts and adipocytes in coculture. In this condition, lipotoxicity was induced by high levels of saturated fatty acids (mostly palmitate) secreted by cultured adipocytes acting in a paracrine manner. In the present study, we aimed to identify the underlying mechanisms of lipotoxicity in human osteoblasts. Palmitate induced autophagy in cultured osteoblasts, which was preceded by the activation of autophagosomes that surround palmitate droplets. Palmitate also induced apoptosis though the activation of the Fas/Jun kinase (JNK) apoptotic pathway. In addition, osteoblasts could be protected from lipotoxicity by inhibiting autophagy with the phosphoinositide kinase inhibitor 3-methyladenine or by inhibiting apoptosis with the JNK inhibitor SP600125. In summary, we have identified two major molecular mechanisms of lipotoxicity in osteoblasts and in doing so we have identified a new potential therapeutic approach to prevent osteoblast dysfunction and death, which are common features of age-related bone loss and osteoporosis.