Bruce R. Troen

Molecular mechanisms underlying osteoclast formation and activation

Osteoporosis is one of the leading causes of morbidity in the elderly and is characterized by a progressive loss of total bone mass and bone density. Bone loss in osteoporosis is due to the persistent excess of osteoclastic bone resorption over osteoblastic bone formation. Receptor activator of NFkappaB ligand (RANKL) critically regulates both osteoclast differentiation and activation. TRAFs appear to be central coupling molecules in the signal transduction pathways that regulate osteoclastogenesis, cathepsin K is the major mediator of osteoclastic bone resorption, and sex steroids and aging also affect osteoclastogenesis and osteoclast activity. However, bone homeostasis depends upon the intimate coupling of bone formation and bone resorption, wherein both osteoclasts and osteoblasts exert vital stimulatory and inhibitory effects upon each other via molecules such as RANKL, TGFbeta, PDGF, BMP2, and Mim-1. This review will highlight some of the major features of the complex circuit of cytokines, growth factors, and hormones that underlies the formation and function of osteoclasts and the dynamic equilibrium that marks the interaction between osteoclasts and osteoblasts.

Understanding the Mechanisms of Senile Osteoporosis: New Facts for a Major Geriatric Syndrome

Knowledge of the underlying mechanisms of osteoporosis in older adults has significantly advanced in recent years. There is an acute loss of bone mineral density in the peri‐menopausal period, followed by a more 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, there is also a significant reduction in bone formation. This is mostly due to a shift from osteoblastogenesis to predominant adipogenesis in the bone marrow. This study reviews new evidence on the pathophysiology of senile osteoporosis, with emphasis upon the mechanism of action of current osteoporosis treatments. New potential treatments are also considered, including therapeutic approaches to osteoporosis in elderly people that focus on the pathophysiology and potential reversal of the adipogenic shift in bone.

The Regulation of Cathepsin K Gene Expression

Abstract:u2002 Cathepsin K is essential for normal bone resorption. Osteoclasts synthesize and secrete cathepsin K into the extracellular compartment at the attachment site between osteoclasts and the bone surface, wherein the organic matrix is subsequently degraded by cathepsin K. RANKL, NFAT, Mitf, and various components of AP‐1 enhance osteoclast formation and bone resorption, whereas IFN‐γ, calcitonin, estradiol, and calcium inhibit it. These agents appear to act correspondingly to alter cathepsin K mRNA and protein expression in order to stimulate and suppress the osteoclasts resorbing potential. RANKL signaling via the calcineurin‐calcium‐NFAT signaling cascade plays a significant role in the regulation of cathepsin K expression. Activation via p38 and the micropthalmia transcription factor also enhances cathepsin K expression. Future studies will be needed to elucidate the relative roles of various signaling pathways at different stages of osteoclast formation and activation and to determine whether genetically disrupting these pathways can modulate bone resorption with or without impeding other osteoclast functions.

Transcription of Human Cathepsin B Is Mediated by Sp1 and Ets Family Factors in Glioma

Cathepsin B expression is increased at both the mRNA and protein levels in a wide variety of tumors. The mechanisms responsible for this regulation are not well elucidated. We have isolated a 2.2-kb cathepsin B genomic fragment that contains the 5-flanking region of the cathepsin B gene. Using reporter gene analysis in human glioblastoma U87MG cells, we have mapped a 228-bp fragment (-172 to +56) having high promoter activity. This promoter region has a high G+C content; contains potential Spl, Ets, and USF binding motifs; and lacks canonical TATA and CAAT boxes immediately upstream of the major transcriptional initiation site. Cotransfection experiments demonstrated that Spl and Ets1 could trans-activate cathepsin B transcription, whereas Ets2 could not. Electrophoretic mobility shift assays and supershift assays revealed that three of the four putative Sp1 sites in this promoter region form a specific complex containing the Sp1 transcription factor. Mutating all four of the Spl binding sites individually markedly reduced the promoter activity of transfected reporter genes in U87 cells. Cotransfection of this cathepsin B promoter construct with Spl family expression vectors in Schneiders Drosophila line 2 (SL2) cells demonstrated that Spl and Sp3, but not Sp4, activated cathepsin B transcription. Taken together, these results suggest that Sp1, Sp3, and Ets1 are important factors in cathepsin B transcription. The regulation of cathepsin B transcription by Sp1- and Sp1-related factors is mediated through multiple GC boxes.

Hypovitaminosis D in the elderly: From bone to brain

ConclusionThere is a growing consensus that vitamin D recommended daily intakes for the elderly are far too low, and that all individuals should take as much vitamin D as needed to raise levels to between 32 to 40 ng/ml (80 to 100 nmol/L) (5, 108, 109). Supplementation will likely be necessary in most elderly, since according to current lifestyles, diet and sunlight alone are inadequate sources of vitamin D (17). We believe that to raise and maintain 25(OH) vitamin D levels at a minimum of 32 ng/ml (80 nmol/L), most elderly will require at least 2,000 IU of cholecalciferol per day.But many questions remain. Are other biological markers preferable to 25(OH) vitamin D to assess repletion? Do the current estimates of optimal serum levels provide health benefits for all conditions, or do optimal vitamin D levels differ depending on the target tissue? How much vitamin D, cholecalciferol, or ergocalciferol, should be given to maintain these levels? What are the molecular mechanisms by which vitamin D influences health and disease?Cross-sectional studies have suggested that low vitamin D levels not only predict nursing home admission but also are associated with increased mortality (1, 2). Further knowledge of the mechanisms of vitamin D action and prospective clinical trials designed to determine if supplementation resulting in vitamin D levels higher than those shown to reduce the risk of falls and fractures is also effective in reducing the burden of various medical conditions could help validate a cost-effective intervention that will provide greater quality of life and longevity and have a major public health impact.

Use of a cloned multidrug resistance gene for coamplification and overproduction of major excreted protein, a transformation-regulated secreted acid protease.

Malignantly transformed mouse fibroblasts synthesize and secrete large amounts of major excreted protein (MEP), a 39,000-dalton precursor to an acid protease (cathepsin L). To evaluate the possible role of this protease in the transformed phenotype, we transfected cloned genes for mouse or human MEP into mouse NIH 3T3 cells with an expression vector for the dominant, selectable human multidrug resistance (MDR1) gene. The cotransfected MEP sequences were efficiently coamplified and transcribed during stepwise selection for multidrug resistance in colchicine. The transfected NIH 3T3 cell lines containing amplified MEP sequences synthesized as much MEP as did Kirsten sarcoma virus-transformed NIH 3T3 cells. The MEP synthesized by cells transfected with the cloned mouse and human MEP genes was also secreted. Elevated synthesis and secretion of MEP by NIH 3T3 cells did not change the nontransformed phenotype of these cells.

Identification of NFAT binding sites that mediate stimulation of cathepsin K promoter activity by RANK ligand

The receptor activator of NFkappaB ligand (RANKL) is a critical mediator of osteoclastogenesis and regulates cathepsin K (CTSK) expression, which is essential for normal bone resorption. RANKL acts, in part, via the Ca(2+)/calmodulin/calcineurin signaling pathway, which in turn, activates NFATc1 (nuclear factor of activated T-cells) and downstream gene expression. We investigated the signals and promoter elements that regulate CTSK gene expression in RAW 264.7 cells, which can be differentiated to osteoclasts by RANKL. Disrupting Ca(2+) signaling, by blocking Ca(2+) channels, thus inhibiting calcineurin or chelation of intracellular Ca(2+), prevented the stimulation of CTSK expression by RANKL. Both RANKL treatment and overexpression of NFATc1 dramatically enhanced CTSK promoter activity, but not in an identical manner. NFATc1 regulates CTSK promoter activity, but the motifs have not been explicitly identified. We found that as few as 238 bp of the CTSK promoter were sufficient to elicit a marked response to both RANKL and NFATc1, truncations of the CTSK promoter illustrated differences in regional responsiveness. Transfection analysis of CTSK promoter-luciferase plasmids revealed that NFATc1 binding sites at 85, 289 and 345 bp upstream of the transcriptional start site mediated responses to RANKL and NFATc1. Deletion of a 4-bp core element from the site at -85 bp dramatically reduced the response of the CTSK promoter to both RANKL and NFATc1, whereas a similar deletion at -345 bp decreased NFATc1- but not RANKL-mediated responses. Mutation of the site at -289 bp did not affect NFAT-mediated stimulation of CTSK on its own, but did decrease responsiveness in combination with either or both of the other two deletions. Electrophoretic mobility shift assays demonstrated NFATc1 binding to oligonucleotides containing the -85-bp and -345-bp sites, while chromatin immunoprecipitation assays demonstrated enhanced in situ binding by NFATc1 to two analogous sites in the mouse CTSK promoter in response to RANKL treatment. Therefore, proximal NFAT binding sites play a significant role in the NFATc1-mediated stimulation of CTSK gene expression by RANKL.

The cathepsin L gene is a direct target of FOXO1 in skeletal muscle

FOXO1 (forkhead box O1), a forkhead-type transcription factor whose gene expression is up-regulated in the skeletal muscle during starvation, appears to be a key molecule of energy metabolism and skeletal muscle atrophy. Cathepsin L, a lysosomal proteinase whose expression is also up-regulated in the skeletal muscle during starvation, is induced in transgenic mice overexpressing FOXO1 relative to wild-type littermates. In the present study, we conducted in vivo and in vitro experiments focusing on FOXO1 regulation of Ctsl (cathepsin L gene; CTSL1 in humans) expression in the skeletal muscle. During fasting and refeeding of C57BL/6 mice, Ctsl was regulated in parallel with FOXO1 in the skeletal muscle. Fasting-induced Ctsl expression was attenuated in transgenic mice overexpressing a dominant-negative form of FOXO1 or in skeletal-muscle-specific Foxo1-knockout mice relative to respective wild-type controls. Using C2C12 mouse myoblasts overexpressing a constitutively active form of FOXO1, we showed that FOXO1 induces Ctsl expression. Moreover, we found FOXO1-binding sites in both the mouse Ctsl and human CTSL1 promoters. The luciferase reporter analysis revealed that the mouse Ctsl and human CTSL1 promoters are activated by FOXO1, which is abolished by mutations in the consensus FOXO1-binding sites. Gel mobility-shift and chromatin immunoprecipiation assays showed that FOXO1 is recruited and binds to the Ctsl promoter. The present study provides in vivo and in vitro evidence that Ctsl is a direct target of FOXO1 in the skeletal muscle, thereby suggesting a role for the FOXO1/cathepsin L pathway in fasting-induced skeletal muscle metabolic change and atrophy.

Differentiating agents regulate cathepsin B gene expression in HL-60 cells

We utilized HL‐60 cells as a model system to examine the regulation of ctsb gene expression by differentiating agents. Inducers of monocytic differentiation [phorbol ester (PMA), calcitriol (D3), and sodium butyrate (NaB)] and inducers of granulocytic differentiation [all‐trans retinoic acid (RA) and 9‐cis retinoic acid (9‐cis RA)] increase ctsb mRNA levels in a dose‐dependent manner as determined by Northern blot hybridization. D3 and retinoids exert additive effects, suggesting that these agents act in part through distinct pathways. Actinomycin D decay experiments indicate that D3, NaB, RA, and 9‐cis RA do not alter mRNA stability. In contrast, PMA markedly increases the half‐life of ctsb mRNA. In transient transfection assays, PMA and NaB both stimulate transcription of the luciferase reporter gene placed under the control of ctsb promoter fragments. Thus, inducers of HL‐60 cell differentiation can regulate the expression of the ctsb gene at both transcriptional and posttranscriptional levels. J. Leukoc. Biol. 66: 609–616; 1999.

Fat and inflammaging: a dual path to unfitness in elderly people?

Skeletal muscle mass and strength decrease dramatically with advancing age. This decrease in muscle mass, also known as sarcopenia, may contribute to the development of functional limitations and disability in elderly people. Sarcopenia is a major contributor to frailty, falls, and loss of independence. Physical disability occurs frequently in older adults and rises steadily with age in people aged 65 and older. An estimated 20% to 30% of community-dwelling adults aged 70 and older report disability in mobility, tasks of household management (instrumental activities of daily living, IADLs), or self-care tasks (activities of daily living, ADLs). Among risk factors associated with disability and frailty, obesity is becoming a major contributor, which is particularly troubling because of the rise in obesity prevalence in elderly people. Although recent studies have shown that overweight and mildly obese older patients may live longer than their normal-weight counterparts, obese elderly people may live a greater portion of their life with some disability. Furthermore, there is a high prevalence of sarcopenia in obese older adults, which leads to mobilityand strength-based disability. Obese patients have a lower percentage of body weight as fat-free mass and can exert lower force per unit of cross-sectional muscle area than ageand sex-matched lean, nonfrail adults. Therefore obese elderly people are at higher risk for frailty and disability. Obesity and aging are both associated with metabolic, physiological, and functional changes. Increase in fat mass leads to higher production of cytokines that may exert a catabolic effect on muscle and enhances the risk of functional decline and frailty in elderly people. This association between fat mass and inflammation appears to account for most of the relationship between inflammation and sarcopenia, suggesting that obesity-associated inflammation may precede sarcopenia. A chronic inflammatory state that has been called ‘‘inflammaging,’’ which results from an upregulation of cellular and molecular processes in response to a variety of stressors, often accompanies aging. Inflammaging appears to be an important driving force of several age-related pathologies, such as neurodegeneration, atherosclerosis, diabetes mellitus, and sarcopenia. Two studies in this month’s Journal of the American Geriatrics Society contribute to our understanding of the potential role that inflammation and associated strength loss have in the epidemic of mobility disability. In the first study, Stenholm et al. used data from the Health 2000 Survey, a comprehensive nationwide health interview and examination survey in Finland, to show that low-grade inflammation and muscle strength mediate the association between obesity and walking limitation in communitydwelling older adults. Body fat percentage, C-reactive protein (CRP), and grip strength were associated with slow walking, independent of health behavioral factors and the presence of comorbidities. A previous cross-sectional study of community-dwelling older women revealed the association between obesity and slow walking and low physical activity. Obesity was also independently associated with poorer lower extremity physical performance in homebound older adults. In these two studies, however, the role of inflammation in the functional decline was not clear. A recent analysis from the Cardiovascular Health Study showed that high CRP is associated with incident frailty, and interleukin (IL)-6 levels were also found to be associated with frailty in community-dwelling older women. In the second study, Bautman et al. demonstrate that inflammatory markers are related to poorer muscle endurance (i.e., poorer strength and worse fatigue resistance) and poorer mobility in frail elderly nursing home residents. Correlations were found with higher levels of tumor necrosis factor alpha (TNF-a), IL-6, and heat shock protein 70 (Hsp 70). In addition, better fatigue resistance and higher grip work were both associated with less self-perceived tiredness and better balance and mobility. Recently Bautman et al. have shown that independently living elderly subjects with lower grip strength and less hand-muscle fatigue resistance experience more tiredness during their daily activities. Body weight appeared to play a role in the relationship between muscle performance and fatigue perception. Daily activities might be more challenging for obese persons than for leaner subjects with the same muscle performance and might thus be more likely to be accompanied by fatigue. Because poorer muscle fatigue resistance and greater fatigue perception are both related to inflammation, adipokines and cytokines may be playing an important pathophysiological role (Figure 1). The evaluation of inflammatory biomakers (CRP, IL-6, TNF-a, Hsp70) together with the assessment of grip strength, walking speed, and fatigue resistance offer the possibility of developing specific interventions for frail elderly people complaining of fatigue to improve muscle endurance and reduce mobility disability. Inflammatory factors contribute to the onset and progression of loss of muscle mass and strength and mobility decline. Intentional weight loss leads to significant reductions of inflammatory biomarkers in younger obese adults DOI: 10.1111/j.1532-5415.2007.01584.x