Elspeth Gold

Bone Mineral Density in Girls with Forearm Fractures

In childhood, the most common site of fracture is the distal forearm. To determine whether young girls with these fractures have low bone density more commonly than fracture‐free controls, we measured bone density at the radius, spine, hip, and whole body and total body bone mineral content, lean tissue mass, and fat mass by dual‐energy X‐ray absorptiometry in 100 Caucasian girls aged 3–15 years with recent distal forearm fractures and 100 age‐ and gender‐matched controls. Bone density (age‐adjusted ratios of all cases:controls with 95% confidence intervals) was lower in cases at the ultradistal radius 0.963 (0.930–0.996), 33% radius 0.972 (0.945–0.999), lumbar spine 0.945 (0.911–0.980), hip trochanter 0.952 (0.918–0.988), and total body 0.978 (0.961–0.995). Moreover, osteopenia (defined as Z score below −1), was more common in cases than controls (p < 0.05) in the forearm, spine, and hip, with one third of fracture cases having low spinal density. Odds ratios (95% confidence intervals) for low bone density were: ultradistal radius, 2.2 (1.1–4.6); lumbar spine, L2‐L4, 2.6 (1.3–4.9); and femur trochanter, 2.0 (1.0–3.9). Fracture patients aged 8‐10 years weighed more (mean ± SD) than age‐matched controls (37.2 ± 8.0 kg vs. 32.5 ± 6.6 kg, p < 0.01) while older patients reported lower current and past calcium intakes than matched controls (p < 0.05). We conclude that low bone density is more common throughout the skeleton in girls with forearm fractures than in those who have never broken a bone, supporting the view that low bone density may contribute to fracture risk in childhood.

Activin C Antagonizes Activin A in Vitro and Overexpression Leads to Pathologies in Vivo

Activin A is a potent growth and differentiation factor whose synthesis and bioactivity are tightly regulated. Both follistatin binding and inhibin subunit heterodimerization block access to the activin receptor and/or receptor activation. We postulated that the activin-beta(C) subunit provides another mechanism regulating activin bioactivity. To test our hypothesis, we examined the biological effects of activin C and produced mice that overexpress activin-beta(C). Activin C reduced activin A bioactivity in vitro; in LNCaP cells, activin C abrogated both activin A-induced Smad signaling and growth inhibition, and in LbetaT2 cells, activin C antagonized activin A-mediated activity of an follicle-stimulating hormone-beta promoter. Transgenic mice that overexpress activin-betaC exhibited disease in testis, liver, and prostate. Male infertility was caused by both reduced sperm production and impaired sperm motility. The livers of the transgenic mice were enlarged because of an imbalance between hepatocyte proliferation and apoptosis. Transgenic prostates showed evidence of hypertrophy and epithelial cell hyperplasia. Additionally, there was decreased evidence of nuclear Smad-2 localization in the testis, liver, and prostate, indicating that overexpression of activin-beta(C) antagonized Smad signaling in vivo. Underlying the significance of these findings, human testis, liver, and prostate cancers expressed increased activin-betaC immunoreactivity. This study provides evidence that activin-beta(C) is an antagonist of activin A and supplies an impetus to examine its role in development and disease.

Vinclozolin Exposure in Utero Induces Postpubertal Prostatitis and Reduces Sperm Production via a Reversible Hormone-Regulated Mechanism

Vinclozolin is an endocrine-disrupting chemical (EDC) that binds with high affinity to the androgen receptor (AR) and blocks the action of gonadal hormones on male reproductive organs. An alternative mechanism of action of Vinclozolin involves transgenerational effects on the male reproductive tract. We previously reported in utero Vinclozolin exposure-induced prostatitis (prostate inflammation) in postpubertal rats concurrent with down-regulation of AR and increased nuclear factor-kappaB activation. We postulated the male reproductive abnormalities induced by in utero Vinclozolin exposure could be reversed by testosterone supplementation, in contrast to the permanent modifications involving DNA methyltransferases (Dnmts) described by others. To test this hypothesis, we administered high-dose testosterone at puberty to Vinclozolin-treated rats and determined the effect on anogenital distance (AGD); testicular germ cell apoptosis, concentration of elongated spermatids, and the onset of prostatitis. Concurrently we examined Dnmt1, -3A, -3B, and -3L mRNA expression. Consistent with previous reports, in utero exposure to Vinclozolin significantly reduced AGD, increased testicular germ cell apoptosis 3-fold, reduced elongated spermatid number by 40%, and induced postpubertal prostatitis in 100% of exposed males. Administration of high-dose testosterone (25 mg/kg) at puberty normalized AGD, reduced germ cell apoptosis, and restored elongated spermatid number. Testosterone restored AR and nuclear factor-kappaB expression in the prostate and abolished Vinclozolin-induced prostatitis. Altered Dnmt expression was evident with in utero Vinclozolin exposure and was not normalized after testosterone treatment. These data demonstrate in utero Vinclozolin-induced male reproductive tract abnormalities are AR mediated and reversible and involve a mechanism independent of Dnmt expression.

Cell-specific expression of βC-activin in the rat reproductive tract, adrenal and liver

betaC-activin expression was assessed in rat tissues, using reverse transcription and real-time polymerase chain reaction, Western blotting and immunohistochemistry with a specific monoclonal antibody. betaC-activin mRNA was predominantly expressed in liver, but significant amounts were found in rat whole pituitary extracts (n = 5), and in three of five extracts of ovary, testis, and adrenal gland. Specific betaC-activin immunoreactivity was demonstrated in the cytoplasm of hepatocytes, neurosecretory cell terminals in posterior pituitary, ovarian primordial follicles, theca interna, large luteal cells and rete ovarii, spermatogonia, pachytene spermatocytes and Leydig cells of the testis, uterine endometrium, oviduct epithelium and zona glomerulosa of the adrenal. The observation of stage-specific expression in gonadal cells suggests this activin subunit has specific roles, different from those of other activin/inhibin subunits. Small amounts of mRNA in the presence of significant betaC-activin protein highlights the importance of examining betaC-activin expression at both the mRNA and protein level.

Changing femoral geometry in growing girls: A cross-sectional DEXA study

In elderly women, a long hip axis length has been shown to increase the risk of hip fracture. However, to date, few measurements of hip geometry have been reported in children and adolescents. The present cross-sectional dual-energy X-ray absorptiometry (DEXA) study of 200 girls aged 3-16 years was undertaken to determine at what age adult hip geometry is achieved and to examine possible influences of anthropometry and body composition on the development of femur axis length (FAL) and femur width (FW) during growth. Adult values for FAL and FW were achieved by age 15 years. Age, height, lean tissue mass, total body bone mineral content (BMC), weight, FW, neck of femur bone mineral density (BMD), and fat were each strongly associated with FAL (p < 0.001), the highest correlations being with age (r = 0.917) and height (r = 0.906). However, after adjusting for age and height, only lean tissue mass, weight, and fat mass remained significantly associated with FAL, suggesting that bone mineral accrual does not influence variance in FAL. Our results also suggested that fat mass and weight per se tended to have greater influence on FW than on FAL in age- and height-adjusted data. Twin studies indicate that 20% of adult hip axis length is associated with environmental factors. We therefore conclude that any environmental effects of physical activity or nutrition on hip geometry must occur before early teen-age years.

Changes in activin and activin receptor subunit expression in rat liver during the development of CCl4-induced cirrhosis

Amounts of betaA-activin, betaC-activin, activin receptor subunits ActRIIA and ActRIIB mRNA, and betaA- and betaC-activin subunit protein immunoreactivity were investigated in male Lewis rats, either untreated or after 5 or 10 weeks of CCl(4) treatment to induce cirrhosis. Apoptosis was assessed histologically and with an in situ cell death detection kit (TUNEL). Reverse transcription and polymerase chain reaction were used to evaluate mRNA levels. Activin betaA- and betaC-subunit immunoreactivity was studied by immunohistochemistry using specific monoclonal antibodies. Hepatocellular apoptosis (P<0.001), increased betaA- and betaC-activin mRNAs (three- to fourfold; P<0.01) and increased betaA- and betaC-activin tissue immunoreactivity were evident, whereas ActRIIA mRNA concentrations fell (30%; P<0.01) after 5 weeks of CCl(4) treatment. The mRNA concentrations at 10 weeks were not significantly different from controls, despite extensive hepatic nodule formation. We conclude that the increased activin subunit expression is associated with apoptosis, rather than hepatic fibrosis and nodule formation.

Activin-βc reduces reproductive tumour progression and abolishes cancer-associated cachexia in inhibin-deficient mice

Activins are involved in the regulation of a diverse range of physiological processes including development, reproduction, and fertility, and have been implicated in the progression of cancers. Bioactivity is regulated by the inhibin α‐subunit and by an activin‐binding protein, follistatin. The activin‐βC subunit was not considered functionally significant in this regard due to an absence of phenotype in knockout mice. However, activin‐βC forms heterodimers with activin‐βA and activin‐C antagonizes activin‐A in vitro. Thus, it is proposed that overexpression, rather than loss of activin‐βC, regulates activin‐A bioactivity. In order to prove biological efficacy, inhibin α‐subunit knockout mice (α‐KO) were crossed with mice overexpressing activin‐βC (ActC++). Deletion of inhibin leads to Sertoli and granulosa cell tumours, increased activin‐A, and cancer‐associated cachexia. Therefore, cachexia and reproductive tumour development should be modulated in α‐KO/ActC++ mice, where excessive activin‐A is the underlying cause. Accordingly, a reduction in activin‐A, no significant weight loss, and reduced incidence of reproductive tumours were evident in α‐KO/ActC++ mice. Overexpression of activin‐βC antagonized the activin signalling cascade; thus, the tumourigenic effects of activin‐A were abrogated. This study provides proof of the biological relevance of activin‐βC. Being a regulator of activin‐A, it is able to abolish cachexia and modulate reproductive tumour development in α‐KO mice.

Activins and activin antagonists in the prostate and prostate cancer.

Activins are members of the TGF-β super-family. There are 4 mammalian activin subunits (β(A), β(B), β(C) and β(E)) that combine to form functional proteins. The role of activin A (β(A)β(A)) is well characterized and known to be a potent growth and differentiation factor. Two of the activin subunits (β(C) and β(E)) were discovered more recently and little is known about their biological functions. In this review the evidence that activin-β(C) is a significant regulator of activin A bioactivity is presented and discussed. It is concluded that activin-β(C), like other antagonists of activin A, is an important growth regulator in prostate health and disease.

Buserelin-mediated osteoporosis: Effects of restoring estrogen on bone resorption and whole body calcium content in the rat

SummaryIn the rat, prolonged administration of the luteinizing, hormone-releasing hormone agonist buserelin (25 μg/kg body wt/day s.c.) lowers blood estradiol, raises bone resorption, and induces osteopenia. The present study was undertaken to determine whether withdrawal of buserelin normalizes blood estradiol, slows bone resorption, and corrects buserelin-mediated osteopenia. Four groups of female rats with45Ca-labeled bones were studied: group 1A received 0.2 ml saline s.c. daily for 4 weeks; group 2A received 0.2 ml buserelin s.c. daily for 4 weeks; group 1B received 0.2 ml saline s.c. daily for 8 weeks; group 2B received 0.2 ml buserelin s.c. daily for 4 weeks followed by 0.2 ml saline s.c. daily for 4 weeks. Bone resorption was monitored by measuring urinary45Ca and hydroxyproline. The rats in groups 1A and 2A were killed after 4 weeks and those in groups 1B and 2B after 8 weeks. The mineral contents of the femoral bones and the whole skeletons were measured. Buserelin lowered blood estradiol, elevated urinary45Ca and urinary hydroxyproline, and lowered femur and total body calcium and45Ca in group 2A vs. 1A (P<0.05). By contrast all these measurements became similar in groups 2B and 1B. Thus, osteopenia generated by a 4-week period of buserelin-mediated hypo-estrogenism is reversible by withdrawing buserelin for 4 weeks. Consequently, buserelin administration and withdrawal may be used to study effects of inducing and reversing estrogen-deficiency bone loss in the rat.

Activin-βC modulates cachexia by repressing the ubiquitin-proteasome and autophagic degradation pathways

Cancer‐associated cachexia and muscle wasting are considered key determinants of cancer‐related death and reduction in the quality of life of cancer patients. A crucial link has been established between activin signaling and skeletal muscle atrophy‐hypertrophy.