Jonathan H. Tobias

Friend or foe: high bone mineral density on routine bone density scanning, a review of causes and management

A finding of high BMD on routine DXA scanning is not infrequent and most commonly reflects degenerative disease. However, BMD increases may also arise secondary to a range of underlying disorders affecting the skeleton. Although low BMD increases fracture risk, the converse may not hold for high BMD, since elevated BMD may occur in conditions where fracture risk is increased, unaffected or reduced. Here we outline a classification for the causes of raised BMD, based on identification of focal or generalized BMD changes, and discuss an approach to guide appropriate investigation by clinicians after careful interpretation of DXA scan findings within the context of the clinical history. We will also review the mild skeletal dysplasia associated with the currently unexplained high bone mass phenotype and discuss recent advances in osteoporosis therapies arising from improved understanding of rare inherited high BMD disorders.

Estrogen‐induced osteogenesis in mice is associated with the appearance of Cbfa1‐expressing bone marrow cells

Cbfa1 is a transcription factor recognised as being involved in early osteoblast differentiation during embryonic skeletogenesis. To determine whether Cbfa1 plays a similar role in bone formation in the adult, we analysed whether its expression is altered during estrogen‐induced osteogenesis, following our recent studies which suggest that this response involves the generation of early osteoblast precursors within bone marrow. To facilitate identification of Cbfa1‐expressing cells, these studies were performed in mice heterozygous for a cbfa1 gene deletion (cbfa1+/−) using β‐galactosidase (lacZ) as a genetic marker. Cbfa1‐expressing cells were identified by lacZ staining of longitudinal sections of the proximal tibial metaphysis. Treatment of cbfa1+/− mice with 17β‐estradiol 0.5 mg/week for 24 days led to the appearance of new cancellous bone surfaces. This response was associated with a marked increase in number of Cbfa1‐expressing cells within the metaphysis, consisting not only of osteoblasts on bone surfaces but also of cells within the adjacent bone marrow. We subsequently enumerated Cbfa1‐expressing cells at earlier time‐points following estrogen, in sections co‐stained for ALP activity. After 4 days of estrogen treatment, a population of cells appeared within the marrow cavity which expressed Cbfa1, but were negative for ALP. At later time‐points, large numbers of Cbfa1u2009+ bone marrow cells were still present, but the majority of these were close to new trabecular bone surfaces at sites which showed high levels of ALP activity. An equivalent distribution of Cbfa1‐expressing cells was observed in further studies where Cbfa1 expression was analysed in wild‐type mice by immunohistochemistry. We conclude that estrogen‐induced osteogenesis is associated with the appearance of a population of Cbfa1‐expressing cells within bone marrow, which we hypothesize to represent the osteoblast precursor population responsible for subsequent new bone formation. J. Cell. Biochem. 84: 285–294, 2002.

The social patterning of fat and lean mass in a contemporary cohort of children

Studies of the social patterning of obesity in children using body mass index have reported inconsistent results. We explored the association of social class with fat mass and lean mass in a contemporary cohort of children measured using dual energy X-ray absorptiometry. We observed a clear social gradient of fat mass (with children of higher social class having a lower fat mass), but no gradient in lean mass or trunk fat mass. Our data show that inequalities in adiposity are present in primary school children and suggest that social inequalities in childhood obesity may have been underestimated in previous studies.

A novel member of the SAF (scaffold attachment factor)-box protein family inhibits gene expression and induces apoptosis

The SLTM [SAF (scaffold attachment factor)-like transcription modulator] protein contains a SAF-box DNA-binding motif and an RNA-binding domain, and shares an overall identity of 34% with SAFB1 {scaffold attachment factor-B1; also known as SAF-B (scaffold attachment factor B), HET [heat-shock protein 27 ERE (oestrogen response element) and TATA-box-binding protein] or HAP (heterogeneous nuclear ribonucleoprotein A1-interacting protein)}. Here, we show that SLTM is localized to the cell nucleus, but excluded from nucleoli, and to a large extent it co-localizes with SAFB1. In the nucleus, SLTM has a punctate distribution and it does not co-localize with SR (serine/arginine) proteins. Overexpression of SAFB1 has been shown to exert a number of inhibitory effects, including suppression of oestrogen signalling. Although SLTM also suppressed the ability of oestrogen to activate a reporter gene in MCF-7 breast-cancer cells, inhibition of a constitutively active beta-galactosidase gene suggested that this was primarily the consequence of a generalized inhibitory effect on transcription. Measurement of RNA synthesis, which showed a particularly marked inhibition of [(3)H]uridine incorporation into mRNA, supported this conclusion. In addition, analysis of cell-cycle parameters, chromatin condensation and cytochrome c release showed that SLTM induced apoptosis in a range of cultured cell lines. Thus the inhibitory effects of SLTM on gene expression appear to result from generalized down-regulation of mRNA synthesis and initiation of apoptosis consequent upon overexpressing the protein. While indicating a crucial role for SLTM in cellular function, these results also emphasize the need for caution when interpreting phenotypic changes associated with manipulation of protein expression levels.

At the crossroads of skeletal responses to estrogen and exercise

The recent observation that mice deficient in estrogen receptor alpha (ERalpha) have an impaired response to mechanical strain suggests that ERalpha plays an important role in mediating the response of the skeleton to mechanical loading as well as to estrogen. In view of previous findings that estrogen deficiency leads to a fall in ERalpha numbers, postmenopausal bone loss might result from the impaired response of bone to mechanical strain caused by deficient ERalpha signalling.

High bone mass is associated with an increased prevalence of joint replacement: a case–control study

Objective. Epidemiological studies have shown an association between OA and increased BMD. To explore the nature of this relationship, we examined whether the risk of OA is increased in individuals with high bone mass (HBM), in whom BMD is assumed to be elevated due to a primary genetic cause. Methods. A total of 335 115 DXA scans were screened to identify HBM index cases (defined by DXA scan as an L1 Z-score of ≥+3.2 and total hip Z-score ≥+1.2, or total hip Z-score ≥+3.2 and L1 Z-score ≥+1.2). In relatives, the definition of HBM was L1 Z-score plus total hip Z-score ≥+3.2. Controls comprised unaffected relatives and spouses. Clinical indicators of OA were determined by structured assessment. Analyses used logistic regression adjusting for age, gender, BMI and social deprivation. Results. A total of 353 HBM cases (mean age 61.7 years, 77% female) and 197 controls (mean age 54.1 years, 47% female) were included. Adjusted NSAID use was more prevalent in HBM cases versus controls [odds ratio (OR) 2.17 (95% CI 1.10, 4.28); P = 0.03]. The prevalence of joint replacement was higher in HBM cases (13.0%) than controls (4.1%), with an adjusted OR of 2.42 (95% CI 1.06, 5.56); P = 0.04. Adjusted prevalence of joint pain and knee crepitus did not differ between cases and controls. Conclusion. HBM is associated with increased prevalence of joint replacement surgery and NSAID use compared with unaffected controls.

Neridronate Preferentially Suppresses the Urinary Excretion of Peptide-Bound Deoxypyridinoline in Postmenopausal Women

SUMMARYnELISAs for measuring the urinary excretion of collagen crosslinks and related peptides appear to show marked differences in sensitivity to anti-resorptive therapy. This presumably reflects variations in specificity of the anylate being detected in these assays, and the way in which they respond to treatment. To clarify these points, we used HPLC analysis to assess the effect of four weeks treatment with the amino-bisphosponate, neridronate, on free and peptide-bound fractions of the collagen cross-links deoxypyridinoline (Dpd) and pyridinoline (Pyd). Six postmenopausal women, in whom two hour morning urine samples were obtained at baseline (x2), and one, two and four weeks after commencing treatment, were included. We found that neridronate had relatively little effect on peptide-bound or free urinary Pyd, but markedly reduced peptide-bound urinary Dpd. However, urinary excretion of free Dpd was not significantly affected. As a consequence of these differential effects on collagen cross-link excretion, neridronate led to a striking increase in the free/total Dpd ratio, and in the peptide-bound Pyd/Dpd ratio. We conclude that neridronate, and presumably other bisphosphonates, selectively suppresses peptide-bound Dpd excretion, possibly reflecting altered processing of collagen crosslinks released during bone resorption.

Mice Rendered Severely Deficient in Megakaryocytes through Targeted Gene Deletion of the Thrombopoietin Receptor c-Mpl Have a Normal Skeletal Phenotype

To explore whether a functional relationship exists between megakaryocytes and the cellular processes responsible for bone formation, we examined if Mpl−/− mice, which are severely megakaryocyte-deficient through c-Mpl gene deletion, have an abnormal skeletal phenotype compared to Mpl+/− and wild-type littermates. We also analyzed whether the osteogenic response to high-dose estrogen treatment is altered in Mpl−/− mice. Megakaryocyte numbers and skeletal indices were compared between Mpl−/− mice and littermate Mpl+/− and wild-type 12-week-old mice (six per group). Dual-energy X-ray absorbtiometry of whole body, excised tibias, and femurs was performed. Histomorphometric analyses of the proximal metaphysis and mid-diaphysis were carried out on longitudinal and transverse sections, respectively. Histomorphometry was performed on the proximal tibial metaphysis of four Mpl−/− and four wild-type mice following high-dose estrogen treatment (0.5 mg/animal/week) for 4 weeks. Mpl−/− mice had 10% the megakaryocyte number of Mpl+/− and wild-type littermates. Bone mineral density values in Mpl−/− mice were identical to those in Mpl+/− and wild-type mice for whole body, femur, and tibia. Histomorphometric analysis demonstrated that cancellous and cortical tibial bone parameters were similar across all genotypes. The osteogenic response to estrogen treatment was indistinguishable between Mpl−/−and wild-type mice. We found that mice severely deficient in megakaryocytes have a normal skeletal phenotype. Additionally, the deficiency did not diminish the osteogenic marrow response to high-dose estrogen treatment. These results represent the first in vivo evidence that severe megakaryocyte deficiency does not affect bone formation, suggesting that this process is not dependent on normal megakaryocyte number.

Genome-wide mapping identifies beta-1,4-N-acetyl-galactosaminyl-transferase as a novel determinant of sclerostin levels and bone mineral density

ABSTRACT In bone, sclerostin is mainly osteocyte-derived and plays an important local role in adaptive responses to mechanical loading. Sclerostin is also present at detectable concentrations within the circulation. Our genome wide association study (GWAS) meta-analysis of 10,584 European-descent individuals identified two novel serum sclerostin loci, B4GALNT3 (standard deviation (SD) change in sclerostin per A allele β=0.20, P=4.6x10-49), and GALNT1 (β=0.11 per G allele, P=4.4x10-11), of which the former is a known locus for BMD estimated by heel ultrasound (eBMD). Common variants across the genome explained 16% of the phenotypic variation of serum sclerostin. Mendelian randomization revealed an inverse causal relationship between serum sclerostin and femoral neck BMD and eBMD, and a positive relationship with fracture risk. Colocalization analysis demonstrated common genetic signals within the B4GALNT3 locus for higher sclerostin, lower BMD, and greater B4GALNT3 expression in arterial tissue (Probability>99%). Renal and cortical bone tissue, and osteoblast cultures, were found to express high levels of B4GALNT3, an N-acetylgalactosaminyltransferase which adds a terminal LacdiNAc disaccharide to target glycocoproteins. Together, these findings raise the possibility that sclerostin is a substrate for B4GALNT3, such that its modification leads to higher levels, possibly through greater stability. GALNT1, an enzyme causing mucin-type O-linked glycosylation, may act in a similar capacity. We conclude that genetic variation in glycosylation enzymes represents a novel determinant of BMD and fracture risk, acting via alterations in levels of circulating sclerostin.