Gianpaolo Rando

Amino Acid-Dependent Activation of Liver Estrogen Receptor Alpha Integrates Metabolic and Reproductive Functions via IGF-1

Throughout evolution, organisms have devised strategies to limit fertility in case of prolonged starvation. In mammals, the liver plays a central role in the orchestration of mechanisms allowing for the maintenance of energy homeostasis. We here demonstrate that dietary amino acids regulate the transcriptional activity of hepatic estrogen receptor alpha (ERα) through an mTOR-dependent mechanism. As a result of ERα activation, hepatic IGF-1 mRNA and blood IGF-1 are increased. Conversely, calorie restriction or selective ablation of ERα in the liver decrease blood IGF-1 to levels inadequate for the correct proliferation of the lumen epithelium in the uterus and the progression of the estrous cycle. We propose that the liver acts as critical mediator of energetic and reproductive functions responsible for the blockade of the estrous cycle in case of protein scarcity. Our findings may provide novel insights to understand the cause of selected forms of infertility and metabolic alterations in women after menopause.

The conundrum of estrogen receptor oscillatory activity in the search for an appropriate hormone replacement therapy.

By the use of in vivo imaging, we investigated the dynamics of estrogen receptor (ER) activity in intact, ovariectomized, and hormone-replaced estrogen response element-luciferase reporter mice. The study revealed the existence of a long-paced, noncircadian oscillation of ER transcriptional activity. Among the ER-expressing organs, this oscillation was asynchronous and its amplitude and period were tissue dependent. Ovariectomy affected the amplitude but did not suppress ER oscillations, suggesting the presence of tissue endogenous oscillators. Long-term administration of raloxifene, bazedoxifene, combined estrogens alone or with basedoxifene to ovariectomized estrogen response element-luciferase mice showed that each treatment induced a distinct spatiotemporal profile of ER activity, demonstrating that the phasing of ER activity among tissues may be regulated by the chemical nature and the concentration of circulating estrogen. This points to the possibility of a hierarchical organization of the tissue-specific pacemakers. Conceivably, the rhythm of ER transcriptional activity translates locally into the activation of specific gene networks enabling ER to significantly change its physiological activity according to circulating estrogens. In reproductive and nonreproductive organs this hierarchical regulation may provide ER with the signaling plasticity necessary to drive the complex metabolic changes occurring at each female reproductive status. We propose that the tissue-specific oscillatory activity here described is an important component of ER signaling necessary for the full hormone action including the beneficial effects reported for nonreproductive organs. Thus, this mechanism needs to be taken in due consideration to develop novel, more efficacious, and safer hormone replacement therapies.

Differential effect of pure isoflavones and soymilk on estrogen receptor activity in mice.

BACKGROUND Because of the complexity of estrogen receptor (ER) physiological activity, the interaction of pure isoflavones or soy-based diets on ER needs to be clearly demonstrated. OBJECTIVES To investigate the effects of the administration of isoflavones as a pure compound or as a component of diet on the ER transcriptional activity in adult mice. METHODS Effects of acute (6 h) and chronic (21 days) oral administration of soy milk, pure genistein and a mix of genistein and daidzein was studied in living ERE-Luc mice. In this animal model, the synthesis of luciferase is under the state of ER transcriptional activity. Luciferase activity was measured in living mice by daily bioluminescence imaging sessions and in tissue extracts by enzymatic assay. RESULTS Acute, oral administration of genistein or soymilk caused a significant increase of ER activity in liver. In a 20 day long treatment, soymilk was more potent than genistein in liver and appeared to extend its influence on ER transcriptional activity in other tissues, such as the digestive tract. A mixture of pure genistein and daidzein at the same concentration as in soymilk failed to induce significant changes during acute and chronic studies suggesting an important, uncharacterized role of the soymilk matrix. Consistent with this observation, synergistic effects of the matrix plus isoflavones were observed in MCF-7 cells stably transfected with the ERE-luc construct. CONCLUSIONS This study underlines the limitations of the analysis of single food components in the evaluation of their effects on estrogen receptor activity and advocates the necessity to use complex organisms for the full comprehension of the effects of compounds altering the endocrine balance.

An innovative method to classify SERMs based on the dynamics of estrogen receptor transcriptional activity in living animals.

Using a mouse model engineered to measure estrogen receptor (ER) transcriptional activity in living organisms, we investigated the effect of long-term (21 d) hormone replacement on ER signaling by whole-body in vivo imaging. Estrogens and selective ER modulators were administered daily at doses equivalent to those used in humans as calculated by the allometric approach. As controls, ER activity was measured also in cycling and ovariectomized mice. The study demonstrated that ER-dependent transcriptional activity oscillated in time, and the frequency and amplitude of the transcription pulses was strictly associated with the target tissue and the estrogenic compound administered. Our results indicate that the spatiotemporal activity of selective ER modulators is predictive of their structure, demonstrating that the analysis of the effect of estrogenic compounds on a single surrogate marker of ER transcriptional activity is sufficient to classify families of compounds structurally and functionally related. For more than one century, the measure of drug structure-activity relationships has been based on mathematical equations describing the interaction of the drug with its biological receptor. The understanding of the multiplicity of biological responses induced by the drug-receptor interaction demonstrated the limits of current approach and the necessity to develop novel concepts for the quantitative analysis of drug action. Here, a systematic study of spatiotemporal effects is proposed as a measure of drug efficacy for the classification of pharmacologically active compounds. The application of this methodology is expected to simplify the identification of families of molecules functionally correlated and to speed up the process of drug discovery.

Molecular imaging of nuclear factor-Y transcriptional activity maps proliferation sites in live animals

The activity of the nuclear factor-Y (NF-Y) transcription factor is restricted to proliferating cells in vitro. We engineered transgenic mice that enabled bioluminescence imaging of NF-Y activity in every area of the body. We visualized areas of proliferation, and we highlight for the first time a role of NF-Y activity in hepatocyte proliferation during liver regeneration.

Molecular Imaging, an Innovative Methodology for Whole-Body Profiling of Endocrine Disrupter Action

Endocrine disrupters (EDs) are environment and food contaminants known to alter metabolic functions of mammals by interfering with specific endocrine pathways. Many EDs act on steroid hormone target cells by interacting with intracellular receptors (IRs) like estrogen receptors, androgen receptors, and thyroid hormone receptors; other receptors may be engaged. IRs are ligand-operated transcription factors acting in concert with general or cell-specific coregulators. The newly acquired awareness on the panoply of IR functions has increased the concern on potential, unsought, harmful effects of EDs on human health and has questioned the capability of currently available methodologies to identify and study EDs in the environment and in the food chain. Indeed, current in vivo and in vitro methodologies restrict the analysis to very specific organs or cell systems, with obvious limitations in predicting the systemic metabolic consequences of ED exposure. The emphasis recently laid by Regulatory Authorities, including European Center for the Validation of Alternative Methods, on the generation of in vitro model systems for toxicological analyses discouraged the development of models suitable to envision the whole spectrum of ED body actions required when studying compounds acting through IRs. Molecular imaging now provides the opportunity to quantify ED effects in living organisms enabling, for the first time, to acquire a full comprehension of the systemic effects of acute and prolonged exposure to EDs, solving the issue of the potential harm due to repeated low-dose exposure. The systems here reviewed are of unquestionable toxicological relevance and need to be taken into consideration to improve the methodology currently available and in use.

Molecular imaging of NF-Y transcriptional activity maps proliferation sites in live animals

The activity of the nuclear factor-Y (NF-Y) transcription factor is restricted to proliferating cells in vitro. We engineered transgenic mice that enabled bioluminescence imaging of NF-Y activity in every area of the body. We visualized areas of proliferation, and we highlight for the first time a role of NF-Y activity in hepatocyte proliferation during liver regeneration.

Novel locally active estrogens accelerate cutaneous wound healing. A preliminary study.

New 17beta-estradiol (E2) derivatives 1-11 were synthesized from an estrone derivative by addition of organometallic reagents prepared from protected alpha,omega-alkynols and further elaboration of the addition products. The estrogenic activity of these novel compounds was determined using in vitro binding competition assay and transactivation analysis. Among the E2 derivatives synthesized, compound 2 showed the highest transactivation potency and was therefore tested for its ability to modulate cutaneous wound healing in vivo. Compound 2s ability to accelerate wound healing in ovariectomized mice and decrease the production of inflammatory molecules was comparable to that of E2. However, the activity of compound 2 was not superimposable to E2 with regard to the cells involved in the wound repairing process. When locally administered, compound 2 did not show any systemic activity on ER. This class of compounds with clear beneficial effects on wound healing and suitable for topical administration may lead to the generation of innovative drugs for an area of unmet clinical need.

Profiling of Drug Action Using Reporter Mice and Molecular Imaging

Reporter mice associated to molecular imaging represent a major asset for the study of the spatio-temporal effects of drugs in living animals. The field is still relatively young and so far the number of animals genetically modified to express a given reporter gene ubiquitously and under the control of specific drugs is still limited. For a reporter animal the indispensable elements for the application to drug research and development are (i) the short life of the reporter enabling to have a clear view of the onset as well as the termination of drug effects, (ii) the generalized, drug-dependent activation of the reporter, and (iii) imaging modality suitable for high-throughput analysis. Because of its relative cheapness and ease to perform, in addition to all the above considerations, bioluminescence-based imaging is now regarded as the best imaging technology to be applied to the field of drug research. We show here the application of reporter mouse systems for drug screening in living animals in order to compare drug potency on target and specificity of action.

Reporter mice for the study of intracellular receptor activity.

During the past decade the remarkable progress in molecular genetics and the possibility to engineer cells to express genes reporting on the activity of specific promoters has produced major changes in biological research. The description and validation of reporter mice for non-invasive assessment of biological and biochemical processes in living subjects and the results obtained with the models reporting on the activity of estrogen and peroxisome proliferator receptors clearly showed that such technologies have the potential to enhance our understanding of disease and drug activity. Although reporter-gene technology is in its infancy, reporter animals already represent a valuable tool for biomedical investigation. The present chapter aims at critically illustrating the methodology to be applied when dealing with reporter systems and in vivo imaging.