Gary M. Leong

Estrogen inhibits GH signaling by suppressing GH-induced JAK2 phosphorylation, an effect mediated by SOCS-2

Oral estrogen administration attenuates the metabolic action of growth hormone (GH) in humans. To investigate the mechanism involved, we studied the effects of estrogen on GH signaling through Janus kinase (JAK)2 and the signal transducers and activators of transcription (STATs) in HEK293 cells stably expressing the GH receptor (293GHR), HuH7 (hepatoma) and T-47D (breast cancer) cells. 293GHR cells were transiently transfected with an estrogen receptor-α expression plasmid and luciferase reporters with binding elements for STAT3 and STAT5 or the β-casein promoter. GH stimulated the reporter activities by four- to sixfold. Cotreatment with 17β-estradiol (E2) resulted in a dose-dependent reduction in the response of all three reporters to GH to a maximum of 49–66% of control at 100 nM (P < 0.05). No reduction was seen when E2 was added 1–2 h after GH treatment. Similar inhibitory effects were observed in HuH7 and T-47D cells. E2 suppressed GH-induced JAK2 phosphorylation, an effect attenuated by actinomycin D, suggesting a requirement for gene expression. Next, we investigated the role of the suppressors of cytokine signaling (SOCS) in E2 inhibition. E2 increased the mRNA abundance of SOCS-2 but not SOCS-1 and SOCS-3 in HEK293 cells. The inhibitory effect of E2 was absent in cells lacking SOCS-2 but not in those lacking SOCS-1 and SOCS-3. In conclusion, estrogen inhibits GH signaling, an action mediated by SOCS-2. This paper provides evidence for regulatory interaction between a sex steroid and the GH/JAK/STAT pathway, in which SOCS-2 plays a central mechanistic role.

Molecular mechanism of vitamin D receptor action.

Abstract. The vitamin D system is unique in that distinct calcium homeostatic functions and cell growth regulatory activities are mediated through a single ligand, calcitriol, acting through a specific receptor exhibiting ubiquitous tissue expression, the vitamin D receptor (VDR). The VDR is a member of a superfamily of nuclear steroid hormone receptors which regulate gene transcription by interacting with response elements in gene promoters. Structure-function analysis of the VDR protein has defined distinct domains involved in DNA binding, ligand binding, receptor dimerisation and gene transactivation, including a C-terminal activation function domain (AF-2) that is important for cofactor interaction. A model for regulation of gene transcription by the VDR is evolving and proposes VDR interaction with various components of the basal transcriptional machinery, including newly defined coactivators and corepressors, which may act to regulate gene transcription by altering histone acetylation and chromatin structure. This review describes the vitamin D endocrine system and the role of the VDR in regulating this system, including the molecular basis for the diverse actions of synthetic calcitriol analogues in the treatment of autoimmune disease and cancer.

Ski-interacting protein interacts with Smad proteins to augment transforming growth factor-beta-dependent transcription.

Transforming growth factor-β (TGF-β) signaling requires the action of Smad proteins in association with other DNA-binding factors and coactivator and corepressor proteins to modulate target gene transcription. Smad2 and Smad3 both associate with the c-Ski and Sno oncoproteins to repress transcription of Smad target genes via recruitment of a nuclear corepressor complex. Ski-interacting protein (SKIP), a nuclear hormone receptor coactivator, was examined as a possible modulator of transcriptional regulation of the TGF-β-responsive promoter from the plasminogen activator inhibitor gene-1. SKIP augmented TGF-β-dependent transactivation in contrast to Ski/Sno-dependent repression of this reporter. SKIP interacted with Smad2 and Smad3 proteins in vivo in yeast and in mammalian cells through a region of SKIP between amino acids 201–333. In vitro, deletion of the Mad homology domain 2 (MH2) domain of Smad3 abrogated SKIP binding, like Ski/Sno, but the MH2 domain of Smad3 alone was not sufficient for protein-protein interaction. Overexpression of SKIP partially overcame Ski/Sno-dependent repression, whereas Ski/Sno overexpression attenuated SKIP augmentation of TGF-β-dependent transcription. Our results suggest a potential mechanism for transcriptional control of TGF-β signaling that involves the opposing and competitive actions of SKIP and Smad MH2-interacting factors, such as Ski and/or Sno. Thus, SKIP appears to modulate both TGF-β and nuclear hormone receptor signaling pathways.

Growth Hormone Deficiency: Strategies and Indications to Continue Growth Hormone Therapy in Transition from Adolescence to Adult Life

The most common practice in children with growth hormone (GH) deficiency is to discontinue GH treatment in adolescence after attainment of final height. Childhood-onset GH deficiency (GHD) that continues into adulthood and is not treated may be associated with more severe consequences than GHD acquired as an adult. This raises the question of the importance of GH for continuing tissue maturation after longitudinal growth has stopped. Data from recent studies suggest that muscle and bone maturation is arrested when GH treatment is discontinued at final height in adolescents in whom severe GHD continues into adulthood. These patients also develop, even in the short term, well-known cardiovascular risk factors associated with GHD in adults. Retesting for GHD is crucial in adolescence because a considerable number of patients will not have severe GHD according to the criteria set for adults. Continuing replacement therapy in these patients is warranted, but cost-benefit comparisons of treatment are still under debate and a lack of acceptance, and hence reimbursement, for such treatment is still common. In this review, the management and organization of transition, with and without continuing GH replacement therapy, are also discussed.

Effects of Child- and Adolescent-Onset Endogenous Cushing Syndrome on Bone Mass, Body Composition, and Growth: A 7-Year Prospective Study Into Young Adulthood†

The long‐term effects on bone and fat mass in children with endogenous CS are unknown. In 14 children followed for 3–7 years into young adulthood after cure of CS, whereas bone mass largely recovered, persisting increases in total body and visceral fat suggests an increase risk of the metabolic syndrome.

Vitamin D Analogue‐Specific Recruitment of Vitamin D Receptor Coactivators

Synthetic ligands for the vitamin D receptor (VDR) are potential therapeutic agents for metabolic, neoplastic, and autoimmune disorders. Some of these ligands have similar or more potent antiproliferative, yet reduced hypercalcemic actions, than calcitriol. However, the mechanisms for these differential actions have not been clearly defined. We hypothesized that these gene‐ and tissue‐specific effects may relate to ligand‐directed selective recruitment of transcriptional coactivators. To identify key elements in ligand structure that facilitate VDR‐coactivator interactions, the current studies assessed the ability of the VDR to recruit the coactivators GRIP1 and RAC3 following activation by a series of 20‐R‐ and 20‐S (20‐epi)‐modified analogues. The strength of VDR‐coactivator interactions was ligand‐specific and did not always correlate with ligand‐receptor binding affinity. In general, the 20‐epi analogues enhanced these interactions, whereas the 20‐R‐modified analogues were less effective than calcitriol. The 16‐ene,23‐yne modification and fluorinated substituents to the side‐chain attenuated interaction with coactivators. The enhanced ability of the VDR to recruit GRIP1 following activation by the 20‐epi analogues was consistent with potentiation of 20‐epi analogue‐induced transactivation of the osteocalcin gene promoter by GRIP1. Overall, the structure of the ligand side‐chain as well as its orientation seemed to affect the avidity of coactivator binding. These results suggest that selective recruitment of coactivators may contribute to gene‐ and tissue‐specific effects of vitamin D analogues.

Glucocorticoid excess during adolescence leads to a major persistent deficit in bone mass and an increase in central body fat.

Endogenous Cushings syndrome (CS) in children causes growth retardation, decreased bone mass, and increased total body fat. No prospective controlled studies have been performed in children to determine the long‐term sequelae of CS on peak bone mass and body composition. A 15‐year‐old girl with Cushing disease (CD), and her healthy identical co‐twin, were followed for 6 years after the CD was cured. At the 6‐year follow‐up both twins had areal bone mineral density (BMD) and body composition determined by dual‐energy X‐ray absorptiometry (DXA) and three‐dimensional quantitative computed tomography (3DQCT). Z scores for height, weight, and body mass index (BMI) were −2.3, −0.8 and 0.2, and 1.2, 0.2, and −0.6, in the twin with CD and her co‐twin, respectively. In the twin with CD, areal BMD and bone mineral apparent density (BMAD) at different sites varied from 0.7 to 3 SD below her co‐twin. Volumetric lumbar spine bone density Z score was −0.75 and 1.0, and total body, abdominal visceral, and subcutaneous fat (%) was 42, 10, and 41 versus 26, 4, and 17 in the twin with CD and her co‐twin, respectively. The relationship between total body fat and L2‐L4 BMAD was inverse in the twin with CD (p < 0.05), which by contrast in her co‐twin was opposite and direct (p < 0.001). In the twin with CD, despite cure, there was a persistent deficit in bone mass and increase in total and visceral body fat. These observations suggest that hypercortisolism (exogenous or endogenous) during adolescence may have persistent adverse effects on bone and fat mass.

Disorders of sex development: Insights from targeted gene sequencing of a large international patient cohort

BackgroundDisorders of sex development (DSD) are congenital conditions in which chromosomal, gonadal, or phenotypic sex is atypical. Clinical management of DSD is often difficult and currently only 13% of patients receive an accurate clinical genetic diagnosis. To address this we have developed a massively parallel sequencing targeted DSD gene panel which allows us to sequence all 64 known diagnostic DSD genes and candidate genes simultaneously.ResultsWe analyzed DNA from the largest reported international cohort of patients with DSD (278 patients with 46,XY DSD and 48 with 46,XX DSD). Our targeted gene panel compares favorably with other sequencing platforms. We found a total of 28 diagnostic genes that are implicated in DSD, highlighting the genetic spectrum of this disorder. Sequencing revealed 93 previously unreported DSD gene variants. Overall, we identified a likely genetic diagnosis in 43% of patients with 46,XY DSD. In patients with 46,XY disorders of androgen synthesis and action the genetic diagnosis rate reached 60%. Surprisingly, little difference in diagnostic rate was observed between singletons and trios. In many cases our findings are informative as to the likely cause of the DSD, which will facilitate clinical management.ConclusionsOur massively parallel sequencing targeted DSD gene panel represents an economical means of improving the genetic diagnostic capability for patients affected by DSD. Implementation of this panel in a large cohort of patients has expanded our understanding of the underlying genetic etiology of DSD. The inclusion of research candidate genes also provides an invaluable resource for future identification of novel genes.

A cross-sectional analysis of patterns of obesity in a cohort of working nurses and midwives in Australia, New Zealand, and the United Kingdom

OBJECTIVE The aim of this study was to examine the prevalence of overweight and obesity and the association with demographic, reproductive work variables in a representative cohort of working nurses and midwives. DESIGN A cross sectional study of self reported survey data. SETTINGS Australia, New Zealand and the United Kingdom. METHODS Measurement outcomes included BMI categories, demographic (age, gender, marital status, ethnicity), reproductive (parity, number of births, mothers age at first birth, birth type and menopausal status) and workforce (registration council, employment type and principal specialty) variables. PARTICIPANTS 4996 respondents to the Nurses and Midwives e-Cohort study who were currently registered and working in nursing or midwifery in Australia (n=3144), New Zealand (n=778) or the United Kingdom (n=1074). RESULTS Amongst the sample 61.87% were outside the healthy weight range and across all three jurisdictions the prevalence of obesity in nurses and midwives exceeded rates in the source populations by 1.73% up to 3.74%. Being overweight or obese was significantly associated with increasing age (35-44 yrs aOR 1.71, 95% CI 1.41-2.08; 45-55 yrs aOR 1.90, 95%CI 1.56-2.31; 55-64 aOR 2.22, 95% CI 1.71-2.88), and male gender (aOR 1.46, 95% CI 1.15-1.87). Primiparous nurses and midwives were more likely to be overweight or obese (aOR 1.37, 95% CI 1.06-1.76) as were those who had reached menopause (aOR 1.37, 95% CI 1.11-1.69). Nurses and midwives in part-time or casual employment had significantly reduced risk of being overweight or obese, (aOR 0.81, 95% CI 0.70-0.94 and aOR 0.75, 95% CI 0.59-0.96 respectively), whilst working in aged carried increased risk (aOR 1.37, 95% CI 1.04-1.80). CONCLUSION Nurses and midwives in this study have higher prevalence of obesity and overweight than the general population and those who are older, male, or female primiparous and menopausal have significantly higher risk of overweight or obesity as do those working fulltime, or in aged care. The consequences of overweight and obesity in this occupational group may impact on their workforce participation, their management of overweight and obese patients in their care as well as influencing their individual health behaviours and risks of occupational injury and chronic disease.

Caveolin‐1 orchestrates the balance between glucose and lipid‐dependent energy metabolism: Implications for liver regeneration

Caveolin‐1 (CAV1) is a structural protein of caveolae involved in lipid homeostasis and endocytosis. Using newly generated pure Balb/C CAV1 null (Balb/CCAV1−/−) mice, CAV1−/− mice from Jackson Laboratories (JAXCAV1−/−), and CAV1−/− mice developed in the Kurzchalia Laboratory (KCAV1−/−), we show that under physiological conditions CAV1 expression in mouse tissues is necessary to guarantee an efficient progression of liver regeneration and mouse survival after partial hepatectomy. Absence of CAV1 in mouse tissues is compensated by the development of a carbohydrate‐dependent anabolic adaptation. These results were supported by extracellular flux analysis of cellular glycolytic metabolism in CAV1‐knockdown AML12 hepatocytes, suggesting cell autonomous effects of CAV1 loss in hepatic glycolysis. Unlike in KCAV1−/− livers, in JAXCAV1−/− livers CAV1 deficiency is compensated by activation of anabolic metabolism (pentose phosphate pathway and lipogenesis) allowing liver regeneration. Administration of 2‐deoxy‐glucose in JAXCAV1−/− mice indicated that liver regeneration in JAXCAV1−/− mice is strictly dependent on hepatic carbohydrate metabolism. Moreover, with the exception of regenerating JAXCAV1−/− livers, expression of CAV1 in mice is required for efficient hepatic lipid storage during fasting, liver regeneration, and diet‐induced steatosis in the three CAV1−/− mouse strains. Furthermore, under these conditions CAV1 accumulates in the lipid droplet fraction in wildtype mouse hepatocytes. Conclusion: Our data demonstrate that lack of CAV1 alters hepatocyte energy metabolism homeostasis under physiological and pathological conditions. (HEPATOLOGY 2011)