Glucocorticoid hormones (GCs) have profound effects on bone metabolism. Via their nuclear hormone receptor – the GR – they act locally within bone cells and modulate their proliferation, differentiation, and cell death. Consequently, high glucocorticoid levels – as present during steroid therapy or stress – impair bone growth and integrity, leading to retarded growth and glucocorticoid-induced osteoporosis, respectively. Because of their profound impact on the immune system and bone cell differentiation, GCs also affect bone regeneration and fracture healing. The use of conditional-mutant mouse strains in recent research provided insights into the cell-type-specific actions of the GR. However, despite recent advances in system biology approaches addressing GR genomics in general, little is still known about the molecular mechanisms of GCs and GR in bone cells. Here, we review the most recent findings on the molecular mechanisms of the GR in general and the known cell-type-specific actions of the GR in mesenchymal cells and their derivatives as well as in osteoclasts during bone homeostasis, GC excess, bone regeneration and fracture healing.
Yasmine Hachemi, Anna E Rapp, Ann-Kristin Picke, Gilbert Weidinger, Anita Ignatius and Jan Tuckermann
H H Farman, J Wu, K L Gustafsson, S H Windahl, S H Kim, J A Katzenellenbogen, C Ohlsson and M K Lagerquist
Estradiol (E2) signaling via estrogen receptor alpha (ERα) is important for the male skeleton as demonstrated by ERα inactivation in both mice and man. ERα mediates estrogenic effects not only by translocating to the nucleus and affecting gene transcription but also by extra-nuclear actions e.g., triggering cytoplasmic signaling cascades. ERα contains various domains, and the role of activation function 1 (ERαAF-1) is known to be tissue specific. The aim of this study was to determine the importance of extra-nuclear estrogen effects for the skeleton in males and to determine the role of ERαAF-1 for mediating these effects. Five-month-old male wild-type (WT) and ERαAF-1-inactivated (ERαAF-10) mice were orchidectomized and treated with equimolar doses of 17β-estradiol (E2) or an estrogen dendrimer conjugate (EDC), which is incapable of entering the nucleus and thereby only initiates extra-nuclear ER actions or their corresponding vehicles for 3.5 weeks. As expected, E2 treatment increased cortical thickness and trabecular bone volume per total volume (BV/TV) in WT males. EDC treatment increased cortical thickness in WT males, whereas no effect was detected in trabecular bone. In ERαAF-10 males, E2 treatment increased cortical thickness, but did not affect trabecular bone. Interestingly, the effect of EDC on cortical bone was abolished in ERαAF-10 mice. In conclusion, extra-nuclear estrogen signaling affects cortical bone mass in males, and this effect is dependent on a functional ERαAF-1. Increased knowledge regarding estrogen signaling mechanisms in the regulation of the male skeleton may aid the development of new treatment options for male osteoporosis.
Fadil M Hannan, Valerie N Babinsky and Rajesh V Thakker
The extracellular calcium (Ca2+ o)-sensing receptor (CaSR) is a family C G protein-coupled receptor, which detects alterations in Ca2+ o concentrations and modulates parathyroid hormone secretion and urinary calcium excretion. The central role of the CaSR in Ca2+ o homeostasis has been highlighted by the identification of mutations affecting the CASR gene on chromosome 3q21.1. Loss-of-function CASR mutations cause familial hypocalciuric hypercalcaemia (FHH), whereas gain-of-function mutations lead to autosomal dominant hypocalcaemia (ADH). However, CASR mutations are only detected in ≤70% of FHH and ADH cases, referred to as FHH type 1 and ADH type 1, respectively, and studies in other FHH and ADH kindreds have revealed these disorders to be genetically heterogeneous. Thus, loss- and gain-of-function mutations of the GNA11 gene on chromosome 19p13.3, which encodes the G-protein α-11 (Gα11) subunit, lead to FHH type 2 and ADH type 2, respectively; whilst loss-of-function mutations of AP2S1 on chromosome 19q13.3, which encodes the adaptor-related protein complex 2 sigma (AP2σ) subunit, cause FHH type 3. These studies have demonstrated Gα11 to be a key mediator of downstream CaSR signal transduction, and also revealed a role for AP2σ, which is involved in clathrin-mediated endocytosis, in CaSR signalling and trafficking. Moreover, FHH type 3 has been demonstrated to represent a more severe FHH variant that may lead to symptomatic hypercalcaemia, low bone mineral density and cognitive dysfunction. In addition, calcimimetic and calcilytic drugs, which are positive and negative CaSR allosteric modulators, respectively, have been shown to be of potential benefit for these FHH and ADH disorders.
Tingyuan Ren, Yuping Zhu, Xuejuan Xia, Yongbo Ding, Jing Guo and Jianquan Kan
This study aimed to evaluate the protein metabolism effect of Zanthoxylum alkylamides and to explore the potential mechanism in streptozotocin (STZ)-induced diabetic rats. Diabetic rats were orally treated with 2, 4 and 8 mg per kg bw of alkylamides daily for 28 days. Alkylamides decreased the relative weight of the liver and food intake, significantly increased the relative skeletal muscle weight and significantly decreased the blood urea nitrogen levels. Insulin, insulin-like growth factor 1, total protein (TP) and albumin (ALB), globular proteins and ALB proteins/globulin protein levels in serum significantly increased. TP, RNA content and RNA/DNA ratio significantly increased in the skeletal muscle of diabetic rats. Real-time quantitative polymerase chain reaction results indicated that alkylamides significantly increased the mRNA expression of insulin receptor (InR), IGF1 and insulin-like growth factor 1 receptor (IGF1R) in the liver and skeletal muscle. Moreover, the mRNA and protein expression levels of PI3K, PKB and mTOR significantly increased, whereas those of atrogin-1, muscle ring finger 1 and FOXO in the skeletal muscle significantly decreased. Alkylamides may advance protein synthesis by the PI3K/PKB/mTOR signalling pathway and attenuate the catabolism of protein through the ubiquitin–proteasome pathway. Therefore, it was possible that alkylamides ameliorate protein metabolism disorders in diabetic rats by activating the mTOR pathway.
Jian-Hua Chen, Maria Segni, Felicity Payne, Isabel Huang-Doran, Alison Sleigh, Claire Adams, UK10K Consortium, David B Savage, Stephen O'Rahilly, Robert K Semple and Inês Barroso
We describe a female proband with primordial dwarfism, skeletal dysplasia, facial dysmorphism, extreme dyslipidaemic insulin resistance and fatty liver associated with a novel homozygous frameshift mutation in POC1A, predicted to affect two of the three protein products of the gene. POC1A encodes a protein associated with centrioles throughout the cell cycle and implicated in both mitotic spindle and primary ciliary function. Three homozygous mutations affecting all isoforms of POC1A have recently been implicated in a similar syndrome of primordial dwarfism, although no detailed metabolic phenotypes were described. Primary cells from the proband we describe exhibited increased centrosome amplification and multipolar spindle formation during mitosis, but showed normal DNA content, arguing against mitotic skipping, cleavage failure or cell fusion. Despite evidence of increased DNA damage in cells with supernumerary centrosomes, no aneuploidy was detected. Extensive centrosome clustering both at mitotic spindles and in primary cilia mitigated the consequences of centrosome amplification, and primary ciliary formation was normal. Although further metabolic studies of patients with POC1A mutations are warranted, we suggest that POC1A may be added to ALMS1 and PCNT as examples of centrosomal or pericentriolar proteins whose dysfunction leads to extreme dyslipidaemic insulin resistance. Further investigation of links between these molecular defects and adipose tissue dysfunction is likely to yield insights into mechanisms of adipose tissue maintenance and regeneration that are critical to metabolic health.
Richard Barbuto and Jane Mitchell
Osterix (Osx, Sp7) is a zinc-finger transcription factor belonging to the specificity protein (Sp) family expressed in cells of the osteoblast lineage in the developing skeleton where it regulates expression of a number of osteoblastic genes. We previously reported inhibition of osterix mRNA and protein by parathyroid hormone (PTH) stimulation of cAMP in osteoblasts. We here show that Osx expression in osteoblasts is regulated by Sp proteins as demonstrated by mithramycin A inhibition of Osx mRNA and OSX protein levels. Mutation of putative transcription factor binding sites within the Osx promoter demonstrated a tandem repeat sequence that selectively binds OSX but not other Sp factors expressed in osteoblasts (Sp1, Sp3, or Tieg (Klf10)). Mutation of either or both the repeat sequences inhibited 90% of the promoter activity and also abrogated some of the PTH-mediated inhibition of the promoter. Previous studies have shown growth factor regulation of Osx expression by MAPK proteins, particularly p38 phosphorylation of OSX that increases its transcriptional activity. PTH stimulation of osteoblasts inhibits MAPK components (ERK, JNK, and p38) but inhibition of Osx mRNA and protein expression by PTH was selectively mimicked by p38 inhibition and expression of constitutively active MKK6, which stimulates p38, blocked PTH inhibition of OSX. Together, our studies suggest that OSX autoregulation is a major mechanism in osteoblasts and that PTH stimulation inhibits osterix by inhibition of p38 MAPK regulation of OSX.
S Das, I Sepahi, A Duthie, S Clark and J C Crockett
The interaction of receptor activator of NFκB (RANK), a member of the tumour necrosis factor receptor superfamily, with RANK ligand is crucial for the formation, function and survival of osteoclasts. The role of the cytoplasmic oligomerisation domain (pre-ligand assembly domain; PLAD or ‘IVVY’ motif) in the ligand-dependent activation of downstream NFκB signalling has not been studied previously. The discovery of truncating mutations of TNFRSF11A (W434X and G280X that lack the PLAD) as the cause of rare cases of osteoclast-poor osteopetrosis offered the opportunity for functional study of this region. Recapitulating the W434X mutation by transcription activator-like effector nuclease (TALEN)-mediated targeted disruption of Tnfrsf11a within the region homologous to W434X in the mouse macrophage-like cell line RAW264.7 impaired formation of osteoclast-like cells. Using overexpression studies, we demonstrated that, in contrast to WT-RANK, the absence of the PLAD in G280X-RANK and W434X-RANK prevented ligand-independent but not ligand-dependent oligomerisation. Cells expressing W434X-RANK, in which only two of the three TRAF6-binding motifs are present, continued to exhibit ligand-dependent NFκB signalling. Hence, the absence of the PLAD did not prevent ligand-induced trimerisation and subsequent NFκB activation of RANK, demonstrating that therapeutic targeting of the PLAD in the prevention of osteoporosis may not be as effective as proposed previously.
Edith Bonnelye, Frédéric Saltel, Anne Chabadel, Ralph A Zirngibl, Jane E Aubin and Pierre Jurdic
The orphan nuclear receptor, estrogen receptor-related receptor α (ERRα) is expressed in osteoblasts and osteoclasts (OCs) and has been proposed to be a modulator of estrogen signaling. To determine the role of ERRα in OC biology, we knocked down ERRα activity by transient transfection of an siRNA directed against ERRα in the RAW264.7 monocyte–macrophage cell line that differentiates into OCs in the presence of receptor activator of nuclear factor κB-ligands and macrophage colony-stimulating factor. In parallel, stable RAW cell lines expressing a dominant-negative form of ERRα and green fluorescent protein (RAW-GFP-ERRαΔAF2) were used. Expression of OC markers was assessed by real-time PCR, and adhesion and transmigration tests were performed. Actin cytoskeletal organization was visualized using confocal microscopy. We found that RAW264.7 cells expressing siRNA directed against ERRα and RAW-GFP-ERRαΔAF2 OCs displayed abnormal spreading, and decreased osteopontin and β3 integrin subunit expression compared with the corresponding control cells. Decreased adhesion and the absence of podosome belts concomitant with abnormal localization of c-src were also observed in RAW-GFP-ERRαΔAF2-derived OCs. In addition, RAW-GFP-ERRαΔAF2-derived OCs failed to transmigrate through osteoblast cell layers. Our data show that the impairment of ERRα function does not alter OC precursor proliferation and differentiation but does alter the adhesion/spreading and migration capacities of mature OCs.
Daniela Nasteska and David J Hodson
It is becoming increasingly apparent that not all insulin-secreting beta cells are equal. Subtle differences exist at the transcriptomic and protein expression levels, with repercussions for beta cell survival/proliferation, calcium signalling and insulin release. Notably, beta cell heterogeneity displays plasticity during development, metabolic stress and type 2 diabetes mellitus (T2DM). Thus, heterogeneity or lack thereof may be an important contributor to beta cell failure during T2DM in both rodents and humans. The present review will discuss the molecular and cellular features of beta cell heterogeneity at both the single-cell and islet level, explore how this influences islet function and insulin release and look into the alterations that may occur during obesity and T2DM.
Jo E Lewis, John M Brameld, Phil Hill, Dana Wilson, Perry Barrett, Francis J P Ebling and Preeti H Jethwa
The Siberian hamster (Phodopus sungorus) survives winter by decreasing food intake and catabolizing abdominal fat reserves, resulting in a sustained, profound loss of body weight. Hypothalamic tanycytes are pivotal for this process. In these cells, short-winter photoperiods upregulate deiodinase 3, an enzyme that regulates thyroid hormone availability, and downregulate genes encoding components of retinoic acid (RA) uptake and signaling. The aim of the current studies was to identify mechanisms by which seasonal changes in thyroid hormone and RA signaling from tanycytes might ultimately regulate appetite and energy expenditure. proVGF is one of the most abundant peptides in the mammalian brain, and studies have suggested a role for VGF-derived peptides in the photoperiodic regulation of body weight in the Siberian hamster. In silico studies identified possible thyroid and vitamin D response elements in the VGF promoter. Using the human neuroblastoma SH-SY5Y cell line, we demonstrate that RA increases endogenous VGF expression (P<0.05) and VGF promoter activity (P<0.0001). Similarly, treatment with 1,25-dihydroxyvitamin D3 increased endogenous VGF mRNA expression (P<0.05) and VGF promoter activity (P<0.0001), whereas triiodothyronine (T3) decreased both (P<0.01 and P<0.0001). Finally, intra-hypothalamic administration of T3 blocked the short day-induced increase in VGF expression in the dorsomedial posterior arcuate nucleus of Siberian hamsters. Thus, we conclude that VGF expression is a likely target of photoperiod-induced changes in tanycyte-derived signals and is potentially a regulator of seasonal changes in appetite and energy expenditure.