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PubMed | Van Andel Research Institute, University of Vermont, Mayo Medical School, University of Melbourne and 2 more.
Type: Journal Article | Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research | Year: 2016

Although there has been extensive characterization of the Wnt signaling pathway in the osteoblast lineage, the effects of Wnt proteins on the osteoclast lineage are less well studied. We found that osteoclast lineage cells express canonical Wnt receptors. Wnt3a reduced osteoclast formation when applied to early bone-marrow macrophage (BMM) osteoclast differentiation cultures, whereas late addition did not suppress osteoclast formation. Early Wnt3a treatment inactivated the crucial transcription factor NFATc1 in osteoclast progenitors. Wnt3a led to the accumulation of nuclear -catenin, confirming activation of canonical Wnt signaling. Reducing low-density lipoprotein receptor-related proteins (Lrp) 5 and Lrp6 protein expression prevented Wnt3a-induced inactivation of NFATc1; however, deletion of -catenin did not block Wnt3a inactivation of NFATc1, suggesting that this effect was mediated by a noncanonical pathway. Wnt3a rapidly activated the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway and pharmacological stimulation of cAMP/PKA signaling suppressed osteoclast differentiation; Wnt3a-induced NFATc1 phosphorylation was blocked by inhibiting interactions between PKA and A-kinase anchoring proteins (AKAPs). These data indicate that Wnt3a directly suppresses osteoclast differentiation through both canonical (-catenin) and noncanonical (cAMP/PKA) pathways in osteoclast precursors. In vivo reduction of Lrp5 and Lrp6 expressions in the early osteoclast lineage via Rank promoter Cre recombination reduced trabecular bone mass, whereas disruption of Lrp5/6 expression in late osteoclast precursors via cathepsin K (Ctsk) promoter Cre recombination did not alter the skeletal phenotype. Surprisingly, reduction of Lrp5/6 in the early osteoclast lineage decreased osteoclast numbers, as well as osteoblast numbers. Published studies have previously noted that -catenin signaling is required for osteoclast progenitor proliferation. Our in vivo data suggest that Rank promoter Cre-mediated deletion of Lrp5/6 may similarly impair osteoclast progenitor proliferation.


PubMed | Health science Research, Endocrine Research Unit and Kogod Center on Aging and Mayo Medical School
Type: | Journal: Bone | Year: 2015

Age-related bone loss in humans is associated with a decrease in bone formation relative to bone resorption, although the mechanisms for this impairment in bone formation with aging are not well understood. It is known that the precursors for the bone-forming osteoblasts reside in the mesenchymal cell population in bone marrow. Thus, in an effort to identify relevant genetic pathways that are altered with aging, we examined the gene expression and DNA methylation patterns from a highly enriched bone marrow mesenchymal cell population from young (mean age, 28.7 years) versus old (mean age, 73.3 years) women. Bone marrow mononuclear cells from these women were depleted of hematopoietic lineage (lin) and endothelial cells using a combination of magnetic- and fluorescence-activated cell sorting, yielding a previously characterized mesenchymal cell population (lin-/CD34-/CD31- cells) that is capable of osteoblast differentiation. Whole transcriptome RNA sequencing (RNAseq) of freshly isolated cells (without in vitro culture) identified 279 differentially expressed genes (p < 0.05, false discovery rate [q]< 0.10) between the young and old subjects. Pathway analysis revealed statistically significant (all p < 0.05) alterations in protein synthesis and degradation pathways, as well as mTOR, gap junction, calcium, melatonin and NFAT signaling pathways. Further, Reduced Representational Bisulphite sequencing (RRBS DNA methylation sequencing) revealed significant differences in methylation between the young and old subjects surrounding the promoters of 1528 target genes that also exhibited significant differences in gene expression by RNAseq. In summary, these studies provide novel insights into potential pathways affected by aging in a highly enriched human mesenchymal cell population analyzed without the confounding effects of in vitro culture. Specifically, our finding of alterations in several genes and pathways leading to impaired protein synthesis and turnover with aging in bone marrow mesenchymal cells points to the need for further studies examining how these changes, as well as the other alterations with aging that we identified, may contribute to the age-related impairment in osteoblast formation and/or function.


Khosla S.,Endocrine Research Unit and Kogod Center on Aging | Oursler M.J.,Endocrine Research Unit and Kogod Center on Aging | Monroe D.G.,Endocrine Research Unit and Kogod Center on Aging
Trends in Endocrinology and Metabolism | Year: 2012

Estrogen is the major hormonal regulator of bone metabolism in women and men. Therefore, there is considerable interest in unraveling the pathways by which estrogen exerts its protective effects on bone. Although the major consequence of the loss of estrogen is an increase in bone resorption, estrogen deficiency is associated with a gap between bone resorption and formation, indicating that estrogen is also important for maintaining bone formation at the cellular level. Direct estrogen effects on osteocytes, osteoclasts, and osteoblasts lead to inhibition of bone remodeling, decreased bone resorption, and maintenance of bone formation, respectively. Estrogen also modulates osteoblast/osteocyte and T-cell regulation of osteoclasts. Unraveling these pleiotropic effects of estrogen may lead to new approaches to prevent and treat osteoporosis. © 2012 Elsevier Ltd.


Farr J.N.,Endocrine Research Unit and Kogod Center on Aging | Khosla S.,Endocrine Research Unit and Kogod Center on Aging
Bone | Year: 2016

There is growing evidence that the higher fracture rate observed in patients with type 2 diabetes mellitus (T2DM) is associated with normal, or even increased, areal bone mineral density (aBMD) by DXA. This has led to the hypothesis that patients with T2DM may have abnormalities in bone microarchitecture and/or material composition - i.e., key determinants of bone "quality." Consistent with this hypothesis, several studies using high-resolution peripheral quantitative computed tomography (HRpQCT) have demonstrated preserved indices of trabecular microarchitecture but increased cortical porosity in T2DM patients. In addition, a recent study using a novel in vivo microindentation device found an impairment in a measure of bone material properties (bone material strength index, BMSi) in postmenopausal women with longstanding T2DM; notably, the reduction in BMSi was associated with chronic glycemic control, suggesting that the skeleton should be included as another target organ subject to diabetic complications. The underlying pathogenesis of skeletal fragility in T2DM remains to be defined, although high levels of advanced glycation endproducts (AGEs) may play a role. In addition, T2DM is associated with reduced bone turnover, perhaps with an imbalance between bone resorption and bone formation. Although several studies have found increased serum sclerostin levels in patients with T2DM, the role of these increased levels in mediating the observed increases in cortical porosity or reduction in BMSi remains to be defined. Thus, although bone quality appears to be impaired in T2DM, the pathogenesis of these abnormalities and their relationship to the increased fracture risk observed in these patients needs further study. © 2015 Elsevier Inc.


Roforth M.M.,Endocrine Research Unit and Kogod Center on Aging | Fujita K.,Endocrine Research Unit and Kogod Center on Aging | McGregor U.I.,Endocrine Research Unit and Kogod Center on Aging | Kirmani S.,Endocrine Research Unit and Kogod Center on Aging | And 5 more authors.
Bone | Year: 2014

Although aging is associated with a decline in bone formation in humans, the molecular pathways contributing to this decline remain unclear. Several previous clinical studies have shown that circulating sclerostin levels increase with age, raising the possibility that increased production of sclerostin by osteocytes leads to the age-related impairment in bone formation. Thus, in the present study, we examined circulating sclerostin levels as well as bone mRNA levels of sclerostin using quantitative polymerase chain reaction (QPCR) analyses in needle bone biopsies from young (mean age, 30.0. years) versus old (mean age, 72.9. years) women. In addition, we analyzed the expression of genes in a number of pathways known to be altered with skeletal aging, based largely on studies in mice. While serum sclerostin levels were 46% higher (p. <. 0.01) in the old as compared to the young women, bone sclerostin mRNA levels were no different between the two groups (p. =0.845). However, genes related to notch signaling were significantly upregulated (p. =0.003 when analyzed as a group) in the biopsies from the old women. In an additional analysis of 118 genes including those from genome-wide association studies related to bone density and/or fracture, BMP/TGFβ family genes, selected growth factors and nuclear receptors, and Wnt/Wnt-related genes, we found that mRNA levels of the Wnt inhibitor, SFRP1, were significantly increased (by 1.6-fold, p. =0.0004, false discovery rate [q]. =0.04) in the biopsies from the old as compared to the young women.Our findings thus indicate that despite increases in circulating sclerostin levels, bone sclerostin mRNA levels do not increase in elderly women. However, aging is associated with alterations in several key pathways and genes in humans that may contribute to the observed impairment in bone formation. These include notch signaling, which represents a potential therapeutic target for increasing bone formation in humans. Our studies further identified mRNA levels of SFRP1 as being increased in aging bone in humans, suggesting that this may also represent a viable target for the development of anabolic therapies for age-related bone loss and osteoporosis. © 2013 Elsevier Inc.


PubMed | Endocrine Research Unit and Kogod Center on Aging
Type: Journal Article | Journal: Nature reviews. Endocrinology | Year: 2015

Age-related fragility fractures are an enormous public health problem. Both acquisition of bone mass during growth and bone loss associated with ageing affect fracture risk late in life. The development of high-resolution peripheral quantitative CT (HRpQCT) has enabled in vivo assessment of changes in the microarchitecture of trabecular and cortical bone throughout life. Studies using HRpQCT have demonstrated that the transient increase in distal forearm fractures during adolescent growth is associated with alterations in cortical bone, which include cortical thinning and increased porosity. Children with distal forearm fractures in the setting of mild, but not moderate, trauma also have increased deficits in cortical bone at the distal radius and in bone mass systemically. Moreover, these children transition into young adulthood with reduced peak bone mass. Elderly men, but not elderly women, with a history of childhood forearm fractures have an increased risk of osteoporotic fractures. With ageing, men lose trabecular bone primarily by thinning of trabeculae, whereas the number of trabeculae is reduced in women, which is much more destabilizing from a biomechanical perspective. However, age-related losses of cortical bone and increases in cortical porosity seem to have a much larger role than previously recognized, and increased cortical porosity might characterize patients at increased risk of fragility fractures.


PubMed | Endocrine Research Unit and Kogod Center on Aging
Type: | Journal: Bone | Year: 2015

There is growing evidence that the higher fracture rate observed in patients with type 2 diabetes mellitus (T2DM) is associated with normal, or even increased, areal bone mineral density (aBMD) by DXA. This has led to the hypothesis that patients with T2DM may have abnormalities in bone microarchitecture and/or material composition - i.e., key determinants of bone quality. Consistent with this hypothesis, several studies using high-resolution peripheral quantitative computed tomography (HRpQCT) have demonstrated preserved indices of trabecular microarchitecture but increased cortical porosity in T2DM patients. In addition, a recent study using a novel in vivo microindentation device found an impairment in a measure of bone material properties (bone material strength index, BMSi) in postmenopausal women with longstanding T2DM; notably, the reduction in BMSi was associated with chronic glycemic control, suggesting that the skeleton should be included as another target organ subject to diabetic complications. The underlying pathogenesis of skeletal fragility in T2DM remains to be defined, although high levels of advanced glycation endproducts (AGEs) may play a role. In addition, T2DM is associated with reduced bone turnover, perhaps with an imbalance between bone resorption and bone formation. Although several studies have found increased serum sclerostin levels in patients with T2DM, the role of these increased levels in mediating the observed increases in cortical porosity or reduction in BMSi remains to be defined. Thus, although bone quality appears to be impaired in T2DM, the pathogenesis of these abnormalities and their relationship to the increased fracture risk observed in these patients needs further study.


PubMed | Endocrine Research Unit and Kogod Center on Aging
Type: | Journal: Bone | Year: 2013

Although aging is associated with a decline in bone formation in humans, the molecular pathways contributing to this decline remain unclear. Several previous clinical studies have shown that circulating sclerostin levels increase with age, raising the possibility that increased production of sclerostin by osteocytes leads to the age-related impairment in bone formation. Thus, in the present study, we examined circulating sclerostin levels as well as bone mRNA levels of sclerostin using quantitative polymerase chain reaction (QPCR) analyses in needle bone biopsies from young (mean age, 30.0years) versus old (mean age, 72.9years) women. In addition, we analyzed the expression of genes in a number of pathways known to be altered with skeletal aging, based largely on studies in mice. While serum sclerostin levels were 46% higher (p<0.01) in the old as compared to the young women, bone sclerostin mRNA levels were no different between the two groups (p=0.845). However, genes related to notch signaling were significantly upregulated (p=0.003 when analyzed as a group) in the biopsies from the old women. In an additional analysis of 118 genes including those from genome-wide association studies related to bone density and/or fracture, BMP/TGF family genes, selected growth factors and nuclear receptors, and Wnt/Wnt-related genes, we found that mRNA levels of the Wnt inhibitor, SFRP1, were significantly increased (by 1.6-fold, p=0.0004, false discovery rate [q]=0.04) in the biopsies from the old as compared to the young women. Our findings thus indicate that despite increases in circulating sclerostin levels, bone sclerostin mRNA levels do not increase in elderly women. However, aging is associated with alterations in several key pathways and genes in humans that may contribute to the observed impairment in bone formation. These include notch signaling, which represents a potential therapeutic target for increasing bone formation in humans. Our studies further identified mRNA levels of SFRP1 as being increased in aging bone in humans, suggesting that this may also represent a viable target for the development of anabolic therapies for age-related bone loss and osteoporosis.


PubMed | Endocrine Research Unit and Kogod Center on Aging
Type: Journal Article | Journal: Biochemical and biophysical research communications | Year: 2013

Control of osteoblastic bone formation involves the cumulative action of numerous transcription factors, including both activating and repressive functions that are important during specific stages of differentiation. The nuclear receptor retinoic acid receptor-related orphan receptor (Ror) has been recently shown to suppress the osteogenic phenotype in cultured osteoblasts, and is highly upregulated in bone marrow-derived osteogenic precursors isolated from aged osteoporotic mice, suggesting Ror is an important regulator of osteoblast function. However the specific gene expression patterns elicited by Ror are unknown. Using microarray analysis, we identified 281 genes regulated by Ror in an MC3T3-E1 mouse osteoblast cell model (MC3T3-Ror-GFP). Pathway analysis revealed alterations in genes involved in MAPK signaling, genes involved in extracellular matrix (ECM) regulation, and cytokine-receptor interactions. Whereas the identified Ror-regulated ECM genes normally decline during osteoblastic differentiation, they were highly upregulated in this non-mineralizing MC3T3-Ror-GFP model system, suggesting that Ror may exert its anti-osteogenic effects through ECM disruption. Consistent with these in vitro findings, the expression of both ROR and a subset of ROR-regulated genes were increased in bone biopsies from postmenopausal women (737 years old) compared to premenopausal women (305 years old), suggesting a role for ROR in human age-related bone loss. Collectively, these data demonstrate that Ror regulates known osteogenic pathways, and may represent a novel therapeutic target for age-associated bone loss.


PubMed | University of California at Irvine and Endocrine Research Unit and Kogod Center on Aging
Type: Journal Article | Journal: PloS one | Year: 2014

The effects of 17--estradiol in osteoblasts are primarily mediated by the nuclear transcription factors, estrogen receptor (ER) and ER. ERs function through three general modes of action: DNA-binding dependent through estrogen response elements (EREs; designated nuclear ERE signaling); nuclear signaling via protein-protein interactions to other transcription factors (nuclear non-ERE signaling); and extra-nuclear signaling (membrane-bound functions of ERs). Identification of the specific transcriptional signatures regulated by each of these modes of action should contribute to an enhanced understanding of estrogen signaling in osteoblasts. To achieve this goal, we utilized specific mutations of ER that eliminate the ability of the receptor to signal through a specific mode of action. The non-classical ER knock-in (NERKI) mutation is incapable of signaling through direct DNA binding to EREs and the nuclear only ER (NOER) mutation eliminates all membrane-localized signaling. Comparison of the gene expression patterns elicited by these mutations with the wild-type ER (WT) pattern provides mode-specific data concerning transcriptional regulation by ER. We expressed these constructs in the ER-negative osteoblastic cell line hFOB (-/+ estrogen) and performed global RNA-sequencing. Using a series of pair-wise comparisons, we generated three lists of genes that were regulated either by the nuclear ERE-dependent, nuclear ERE-independent, or extra-nuclear actions of ER. Pathway and gene ontology analyses revealed that genes regulated through the nuclear ERE and nuclear non-ERE pathways were largely involved in transcriptional regulation, whereas genes regulated through extra-nuclear mechanisms are involved in cytoplasmic signaling transduction pathways. We also intersected our data with genes linked to bone density and fractures from a recent genome-wide association study and found 25 of 72 genes (35%) regulated by estrogen. These data provide a comprehensive list of genes and pathways targeted by these specific modes of ER action and suggest that mode-specific ligands could be developed to modulate specific ER functionality in bone.

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