A critical role for muscle ring finger-1 in acute lung injury-associated skeletal muscle wasting

Rationale: Acute lung injury (ALI) is a debilitating condition associated with severe skeletal muscle weakness thatpersists in humans long after lung injury has resolved. The molecular mechanisms underlying this condition are unknown. Objectives: To identify the muscle-specific molecular mechanisms responsible for muscle wasting in a mouse model of ALI. Methods:Changes in skeletal muscle weight, fiber size, in vivo contractile performance, and expression of mRNAs and proteins encoding muscle atrophy-associated genes for muscle ring finger-1 (MuRF1) and atrogin1 were measured. Genetic inactivation of MuRF1 or electroporation-mediated transduction of miRNA-based short hairpin RNAs targeting either MuRF1 or atrogin1 were used to identify their role in ALI-associated skeletal muscle wasting. Measurements and Main Results: Mice with ALI developed profound muscle atrophy and preferential loss of muscle contractile proteins associatedwith reducedmuscle function in vivo. Although mRNA expression of the muscle-specific ubiquitin ligases, MuRF1 and atrogin1, was increased in ALI mice, only MuRF1 protein levels were up-regulated. Consistent with these changes, suppression of MuRF1 by genetic or biochemical approaches prevented muscle fiber atrophy, whereas suppression of atrogin1 expression was without effect. Despite resolution of lung injury and down-regulation of MuRF1 and atrogin1, force generation in ALI mice remained suppressed. Conclusions: These data show that MuRF1 is responsible for mediating muscle atrophy that occurs during the period of active lung injury inALI mice and that, as in humans, skeletal muscle dysfunction persists despite resolution of lung injury

Disruption of thyroid hormone-mediated Xenopus laevis tadpole tail tip regression by hexabromocyclododecane (HBCD) and 2,2',3,3',4,4',5,5',6-nona brominated diphenyl ether (BDE206)

Thyroid hormone regulates amphibian metamorphosis, including the transformation of a tadpole into a froglet and regression of the tail. Xenopus laevis tadpole tail tips in organ culture (ex vivo) undergo regression when exposed to 3,3',5-triiodo-l-thyronine (T(3)) and interference by chemicals with this process was utilized as a bioassay to detect thyroid hormone disruption. In the present study the bioassay was further validated by investigating its response to compound induced T(3)-antagonism and - potentiation. Tadpole tail tips were exposed to two brominated flame retardants (BFRs) in presence or absence of T(3) at its EC(50) (20nM). T(3)-induced tail tip regression was antagonized by 2,2',3,3',4,4',5,5',6-nona brominated diphenyl ether (BDE206) and potentiated by hexabromocyclododecane (HBCD) in a concentration dependent manner, which was consistent with results obtained with a in vitro T(3)-dependent proliferation bioassay termed the T-screen. Neither compound induced any effect in the absence of T(3). The results indicate that studying possible hormone disrupting effects of agonistic, antagonistic or potentiating compounds should include combined exposure with the natural hormone at around its EC(50) concentration. The results obtained with the tail tip exposures were in accordance with the T-screen predictions, and occurred at BFR-concentrations that were only 5-50 times those of T(3). The bioassay proved to be suitable not only for detecting T(3)-agonism, but also for antagonism and potentiation

Thyroid hormone receptor isoform selectivity of thyroid hormone disrupting compounds quantified with an in vitro reporter gene assay

Some compounds, including brominated diphenyl ethers (BDEs), can interfere with thyroid hormone (TH) receptor (TR)-mediated TH-signalling. In this study, the TR isoform selectivity of some TH disrupting compounds was investigated with TR alpha/beta specific reporter gene assays. For this purpose, the effects of compounds on 3,3',5-triiodothyronine (T-3)-induced TR alpha- or TR beta-activation were tested in green monkey kidney fibroblast (CV-1) cells transiently transfected with Xenopus TRs and a luciferase reporter gene. The T-3-like BDE-OH and diiodobiphenyl (DIB) increased T-3-induced TR alpha-activation, but not T-3-induced TRP-activation. BDE28 (100 nM) did not act via TR alpha, but almost tripled T-3-induced TRP-activation relative to T-3 at its EC50. BDE206 (100 nM) was antagonistic on both TRs with a maximum repression -54% relative to T-3 at its EC50. Contrary to previous results obtained with the T-screen, HBCD was inactive. The present study illustrates the importance of testing potential TH disrupting compounds in model systems that enable independent characterization of effects on both T3-induced TRs. (c) 2006 Elsevier B.V. All rights reserved

Detection of thyroid hormone receptor disruptors by a novel stable in vitro reporter gene assay

A stable luciferase reporter gene assay was developed based on the thyroid hormone responsive rat pituitary tumor GH3 cell line that constitutively expresses both thyroid hormone receptor isoforms. Stable transfection of the pGL4CP-SV40-2xtaDR4 construct into the GH3 cells resulted in a highly sensitive cell line (GH3.TRE-Luc), which was further optimized into an assay that allowed the detection of Triiodothyronine (T3) and Thyroxine (T4) concentrations in the picomolar range after only 24 h of exposure. The greater than 20-fold induction of T3 relative to the solvent control is illustrative of the high responsiveness of the system. The assay was validated by the quantification of the agonistic effect of the natural hormones (T3 and T4), the acetic acid derivatives of T3 (triiodothyroaceticacid, or Triac) and T4 (tetraiodothyroacetic acid, or Tetrac), hydroxy polybrominated diphenylethers (OH-PBDEs), hydroxy polychlorinated biphenyls (OH-PCBs) and the antagonistic action of sodium arsenite (NaAsO2). The putative antagonist Amiodarone, Bisphenol A (BPA) and its halogenated derivatives (TCBPA and TBBPA) for which effects reported in the literature are not consistent, showed comparable dose–response curves with a slight agonistic effect (5% of T3-max) followed by a slight antagonistic effect. The magnitude and reproducibility of the responses to various compounds confirms this assay as a promising tool for the identification and quantification of specific thyroid hormone receptor disrupting potency of compounds

Identification of thyroid hormone receptor active compounds using a quantitative high-throughput screening platform

To adapt the use of GH3.TRE-Luc reporter gene cell line for a quantitative high-throughput screening (qHTS) platform, we miniaturized the reporter gene assay to a 1536-well plate format. 1280 chemicals from the Library of Pharmacologically Active Compounds (LOPAC) and the National Toxicology Program (NTP) 1408 compound collection were analyzed to identify potential thyroid hormone receptor (TR) agonists and antagonists. Of the 2688 compounds tested, eight scored as potential TR agonists when the positive hit cut-off was defined at =10% efficacy, relative to maximal triiodothyronine (T3) induction, and with only one of those compounds reaching =20% efficacy. One common class of compounds positive in the agonist assays were retinoids such as all-trans retinoic acid, which are likely acting via the retinoid-X receptor, the heterodimer partner with the TR. Five potential TR antagonists were identified, including the antiallergy drug tranilast and the anxiolytic drug SB 205384 but also some cytotoxic compounds like 5-fluorouracil. None of the inactive compounds were structurally related to T3, nor had been reported elsewhere to be thyroid hormone disruptors, so false negatives were not detected. None of the low potency (>100µM) TR agonists resembled T3 or T4, thus these may not bind directly in the ligand-binding pocket of the receptor. For TR agonists, in the qHTS, a hit cut-off of =20% efficacy at 100 µM may avoid identification of positives with low or no physiological relevance. The miniaturized GH3.TRE-Luc assay offers a promising addition to the in vitro test battery for endocrine disruption, and given the low percentage of compounds testing positive, its high-throughput nature is an important advantage for future toxicological screening

A multi-tiered, in vivo, quantitative assay suite for environmental disruptors of thyroid hormone signaling

The essential role of thyroid hormone (TH) signaling in mammalian development warrants the examination of man-made chemicals for its disruption. Among vertebrate species, the molecular components of TH signaling are highly conserved, including the thyroid hormone receptors (TRs), their heterodimer binding partners the retinoid-X receptors (RXRs), and their DNA recognition sequences (TREs). This molecular conservation allows examination of potential TH disruption in the tractable, in vivo model system of amphibian metamorphosis. Metamorphosis requires TH signaling for both instigation and progression, and it provides dramatic and well-characterized phenotypes involving different cell fates. Here we describe a quantitative, precocious-metamorphosis assay suite we developed using one-week post-fertilization (PF) Xenopus laevis tadpoles in order to assess disruption of TH signaling. Tadpoles at this developmental stage (Nieuwkoop-Faber (NF)-48) are competent to respond to TH hormone, although not yet producing TH, along many metamorphic pathways, and they are uniform in size. This allowed us to quantify changes in morphology associated with natural metamorphosis (e.g. gill and tail resorption, brain expansion, and craniofacial remodeling) after five days of treatment. Using the same tadpoles from morphological measurements, we quantified a 20-fold increase in TH-induced cellular proliferation in the rostral head region by whole-mount immunocytochemistry. At the molecular level, we used F3-generation tadpoles from a transgenic X. laevis line, which expresses luciferase under the control of a native TRE, to assess the ability of compounds to disrupt TR function. The luciferase reporter showed over 10-fold activation by physiologic concentrations of TH. We used the synthetic TR antagonist NH-3 to demonstrate the feasibility of our assay suite to measure inhibition of TH activity at the level of the receptor. Finally, we assessed the capabilities of suspected TH-disrupting chemicals tetrabrominated diphenyl ether 47 (BDE-47) and tetrabromobisphenol A (TBBPA). We found that BDE-47 displays general toxicity rather than TH disruption, as it did not increase brain width nor affect the TRE-luciferase reporter. However, TBBPA, a suspected TR antagonist, although not effective in antagonizing cell proliferation, significantly inhibited the TRE-luciferase reporter, suggesting that it bears closer scrutiny as a TH disruptor. Overall the assay suite has important advantages over the classical tadpole metamorphosis assays with respect to the uniformity of animal size, small test volume, reproducibility, and short test period. The assays are performed before endogenous TH production and free feeding start, which further reduces complexity and variability