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Background and purpose Therapeutic area guidelines (TAGs) published by the EMA and the FDA offer guidance in planning the launch of a trial in a certain indication. We assessed and compared the guidance on preclinical efficacy of all available TAGs from EMA and FDA. Experimental approach EMA and FDA websites and databases were searched for all TAGs. A mixed deductive and inductive approach was applied to analyse and cluster content for preclinical efficacy. Key results A total of 114 EMA and 120 FDA TAGs were identified, covering 126 indications. Our core finding is that 75% of EMA TAGs and 58% from the FDA TAGs do not offer any guidance on preclinical efficacy. TAGs varied widely on the extent, nature and detail of guidance. Conclusions and implications Guidance on preclinical efficacy in a consistent, comprehensive and explicit way that still allows for justified deviations is an important but neglected aspect of transparency for drug development. This transparency would help sponsors in designing preclinical studies and in negotiating more efficiently with regulators.

The Assay Guidance Manual (AGM) is an eBook of best practices for the design, development, and implementation of robust assays for early drug discovery. Initiated by pharmaceutical company scientists, the manual provides guidance for designing a "testing funnel" of assays to identify genuine hits using high-throughput screening (HTS) and advancing them through preclinical development. Combined with a workshop/tutorial component, the overall goal of the AGM is to provide a valuable resource for training translational scientists.

The Human Touch Responses to the Covid-19 pandemic by stakeholders throughout the health care ecosystem are contributing to rapid innovation, including the development of new equipment and drugs. L...

Human protection policies require favorable risk–benefit judgments prior to launch of clinical trials. For phase I and II trials, evidence for such judgment often stems from preclinical efficacy studies (PCESs). We undertook a systematic investigation of application materials (investigator brochures [IBs]) presented for ethics review for phase I and II trials to assess the content and properties of PCESs contained in them. Using a sample of 109 IBs most recently approved at 3 institutional review boards based at German Medical Faculties between the years 2010–2016, we identified 708 unique PCESs. We then rated all identified PCESs for their reporting on study elements that help to address validity threats, whether they referenced published reports, and the direction of their results. Altogether, the 109 IBs reported on 708 PCESs. Less than 5% of all PCESs described elements essential for reducing validity threats such as randomization, sample size calculation, and blinded outcome assessment. For most PCESs (89%), no reference to a published report was provided. Only 6% of all PCESs reported an outcome demonstrating no effect. For the majority of IBs (82%), all PCESs were described as reporting positive findings. Our results show that most IBs for phase I/II studies did not allow evaluators to systematically appraise the strength of the supporting preclinical findings. The very rare reporting of PCESs that demonstrated no effect raises concerns about potential design or reporting biases. Poor PCES design and reporting thwart risk–benefit evaluation during ethical review of phase I/II studies., Author summary To make a clinical trial ethical, regulatory agencies and institutional review boards have to judge whether the trial-related benefits (the knowledge gain) outweigh the trial-inherent risks. For early-phase human research, these risk–benefit assessments are often based on evidence from preclinical animal studies reported in so-called “investigator brochures.” However, our analysis shows that the vast majority of such investigator brochures lack sufficient information to systematically appraise the strength of the supporting preclinical findings. Furthermore, the very rare reporting of preclinical efficacy studies that demonstrated no effect raises concerns about potential design and/or reporting biases. The poor preclinical study design and reporting thwarts risk–benefit evaluation during ethical review of early human research. Regulators should develop standards for the design and reporting of preclinical efficacy studies in order to support the conduct of ethical clinical trials.

To study the role of the thyroid hormone (TH) in cerebellar development, we generated transgenic mice expressing a dominant-negative TH receptor (TR) in cerebellar Purkinje cells. A mutant human TRβ1 (G345R), which binds to the TH-response element but cannot bind to T3, was subcloned into exon 4 of the full-length L7/Pcp-2 gene, which is specifically expressed in Purkinje and retinal rod bipolar cells. The transgene was specifically expressed in Purkinje cells in the postnatal cerebellum. Purkinje cell dendrite arborization was significantly delayed in the transgenic mice. Surprisingly, granule cell migration was also significantly delayed. In the primary cerebellar culture, TH-induced Purkinje cell dendrite arborization was also suppressed. In quantitative real-time RT-PCR analysis, the expression levels of several TH-responsive genes were altered. The expression levels of inositol trisphosphate receptor type 1 and retinoic acid receptor-related orphan receptorα mRNAs, which are mainly expressed in Purkinje cells, and brain-derived neurotrophic factor mRNA, which is expressed in both Purkinje and granule cells, were significantly decreased. The expression levels of neurotrophin-3 and hairless mRNAs, which are mainly expressed in granule cells, and myelin basic protein mRNA, which is mainly expressed in oligodendrocytes, were also decreased. The motor coordination of transgenic mice was significantly disrupted. These results indicate that TH action through its binding to TR in Purkinje cells is required for the normal cerebellar development. TH action through TR in Purkinje cells is also important for the development of other subsets of cerebellar cells such as granule cells and oligodendrocytes.

Even with many new therapeutic options, the proportion of health care spending devoted to retail prescription medications remains about the same as it was in 1960. Relying on competitive markets to set drug prices and encourage innovation seems to be working.

Biomarkers can facilitate all aspects of the drug development process. However, biomarker qualification-the use of a biomarker that is accepted by the U.S. Food and Drug Administration-needs a clear, predictable process. We describe a multistakeholder effort including government, industry, and academia that proposes a framework for defining the amount of evidence needed for biomarker qualification. This framework is intended for broad applications across multiple biomarker categories and uses.

Modern approaches for research with human biospecimens employ a variety of substantially different types of ethics approval and informed consent. In most cases, standard ethics reporting such as “consent and approval was obtained” is no longer meaningful. A structured analysis of 120 biospecimen studies recently published in top journals revealed that more than 85% reported on consent and approval, but in more than 90% of cases, this reporting was insufficient and thus potentially misleading. Editorial policies, reporting guidelines, and material transfer agreements should include recommendations for meaningful ethics reporting in biospecimen research. Meaningful ethics reporting is possible without higher word counts and could support public trust as well as networked research., This Perspective shows that while ethics reporting on consent and approval for biospecimen research is widespread, for the majority of cases it is not meaningful; the authors make pragmatic suggestions of substantial changes needed to remedy this.

Over the past two decades, there has been growing concern over the lack of proper medication for children. This review attempts to evaluate the current progress of EU Pediatric Regulation made since 2007. The lack of properly evaluated pediatric medication has for long been a source of concern in the European Union. The drugs that were used in the past were often not properly evaluated, and dosage was arbitrarily calculated. Therefore, it was necessary to establish the Pediatric Regulation (EC no. 1901/2006) in the EU which would mandate research for pediatric drugs. Current legislations in place not only require mandatory research by pharma industry but also have guidelines to direct the quality of pediatric research performed. The main aim of this regulation was to advance high-quality research and development of pediatric drugs, thereby increasing the availability of safe and effective drugs for children. It also aimed to improve the information available on existing pediatric drugs. It has been 9 years since the pediatric regulation was framed. The pharma industry now sees pediatric research as an integral process of development. Drug companies which develop plans for a new drug, new form of drug, new indication, or new route of administration for adults are obliged to integrate in their development plan similar research for pediatric populations as well.It is hoped that the implementation of the current legislation will be reflected better in the future by the marketing of better and safer drugs for the pediatric population. The upcoming assessment to the European Commission in 2017 will further inform us on the impact after 10 years implementation of the legislation. What is Known: • The lack of properly evaluated pediatric medication has for long been a source of concern in the European Union. • Therefore, it was necessary to establish the EU Pediatric Regulation which would mandate research for pediatric drugs. What is New: • It has been 9 years since the pediatric regulation was framed, and the teething problems are slowly being overcome and the regulation is being used with increasing confidence. • As the Regulation is due for revision in 2017, this paper gives a current perspective on the impact of the regulation on availability and access to medicine for children.

Background: Diagnosis of oral cancer is conventionally carried out using white light endoscopy and histopathology of biopsy samples. However, oral tumours are mostly superficial and the lesion and its margins can be difficult to visualise under white light. We present clinical data on fluorescence diagnostic imaging of oral lesions using hypericin, a plant-based photosensitiser. Methods: Fluorescence images of lesions and normal tissue were captured using an endoscope after hypericin administration. The images were analysed to extract their colour parameters, which, along with the red-to-blue intensity ratios, were analysed and used to discriminate between tissue types. The results were correlated with those from histopathology. Results: The red-to-blue intensity ratio increased from normal to hyperplastic to cancerous tissue and was a good parameter to discriminate between these tissue types, with sensitivity and specificity levels of 90% and above. Conclusion: Our results show that hypericin fluorescence imaging has the potential to be used for the clinical diagnosis of oral cancer. Further study to enhance the clinical potential of this technique includes the development of a real-time image processing and analysis system interfaced to the endoscope to enable same-day cancer diagnosis and demarcation of lesion margins in a clinical setting.

We have determined the influences of polyvinylpyrrolidone (PVP) on the topical delivery of chlorin e6 (Ce6) in malignant bladder cells. The chick chorioallantoic membrane (CAM) was used to model the tumor spheroids that resemble small residual bladder tumors prior to vascularization. Macroscopic fluorescence imaging showed that Ce6-PVP-induced fluorescence had a higher sensitivity and specificity for delineating tumor from the adjacent normal CAM compared to Ce6 alone. Nonlinear regression analyses have shown that Ce6-PVP has a longer half-life in the tumor compared to Ce6. The uptake ratio of Ce6-PVP was found to have a 2-fold increase across the CAM when compared to that of Ce6, indicating that PVP was able to facilitate diffusion of Ce6 across the membrane. Confocal laser scanning microscopy further confirmed that Ce6-PVP has better penetration in the CAM as well as in the tumor cells compared to Ce6. The present work contributes to our understanding of the Ce6-PVP drug–polymer system by demonstrating for the first time that the presence of PVP facilitates the transport of Ce6 across the chorioallantoic membrane. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Tumor response to photodynamic therapy (PDT) is dependent on treatment parameters used. In particular, the light fluence rate may be an important determinant of the treatment outcome. In this clinical case report, we describe the response of angiosarcoma to PDT carried out using different fluence rates and drug and light doses. A patient with recurrent multifocal angiosarcoma of the head and neck was recruited for PDT. A new generation chlorin-based photosensitizer, Fotolon, was administered at a dose of 2.0 to 5.7 mg/kg. The lesions were irradiated with 665 nm laser light for a light dose of 65 to 200 J/cm2 delivered at a fluence rate of 80 or 150 mW/cm2. High dose PDT carried out at a high fluence rate resulted in local control of the disease for up to a year; however, the disease recurred and PDT had to be repeated. PDT of new lesions carried out at a lower fluence rate resulted in tumor eradication. More significantly, it also resulted in spontaneous remission of neighboring and distant untreated lesions. Repeat PDT carried out on a recurrent lesion at a lower fluence rate resulted in eradication of both treated and untreated lesions despite the lower total light dose delivered. Immunohistochemical examination of biopsy samples implies that PDT could have activated a cell-mediated immune response against untreated lesions. Subsequent histopathological examination of the lesion sites showed negative for disease. Our clinical observations show that lower fluence rate PDT results in better outcome and also indicate that the fluence rate, rather than the total light dose, is a more crucial determinant of the treatment outcome. Specifically, lower fluence rate PDT appears to activate the body's immune response against untreated lesions.

Formulations of hypericin (HY) with plasma protein have been conventionally used, but to date, no alternative pharmaceutical formulation has been developed for clinical use. Previously, it was reported that formulation of HY containing a biocompatible solvent and penetration enhancer, N-methyl pyrrolidone (NMP) was found to be effective for the delivery of HY across in vivo chick chorioallantoic membrane (CAM). This present study reports further investigations on the HY-NMP formulations in CAM implanted with human bladder cancer cells as a potential fluorescence diagnostic agent of cancer. The conventional formulation of HY (HY-HSA 0.5%) was included as control. The red-to-blue (I(R)/I(B)) intensity ratio of fluorescence images was used as a diagnostic algorithm, to differentiate the uptake of HY between tumor and adjacent regions on CAM. Results indicated that HY-NMP 0.05% was significantly better than HY-HSA 0.5%. The findings of the I(R)/I(B) ratios between tumor and adjacent tissues, indicated the potential of using NMP as an alternative to plasma protein in clinical fluorescence diagnosis with HY. The NMP formulations investigated were able to produce significantly higher contrast for tumor tissues and at earlier time points than was possible with HY-HSA 0.5%.

The use of fluorescence diagnosis as a modern cancer diagnostic modality is rapidly gaining importance in the field of urology. It is based on the detection of distinctive light emission of tissues sensitized by fluorescent dyes, commonly referred to as photosensitizers, after irradiation with a specific light source. Therefore, the search for specific fluorescent dyes with high sensitivity and specificity for bladder cancer is constantly being sought after. The aim of this study is to investigate the use of a new formulation consisting a mixture of chlorin e6 and polyvinylpyrrolidone (Ce6-PVP) for the detection of human bladder cancer cells (MGH) implanted on the chick chorioallantoic membrane (CAM) model. Uptake kinetics studies were quantitatively determined for both systemic and topical administrations of Ce6-PVP to the normal CAM as well as the MGH human bladder tumor implanted on CAM using fluorescence imaging technique. Rapid elimination of Ce6-PVP was displayed following topical application compared to systemic administration in the normal CAM system. Ce6-PVP was found to localize selectively in the xenografted bladder tumor in contrast to the CAM tissue. Neither dark toxicity nor irritancy was observed on the CAM tissue at the dose of 2 mg/kg Ce6-PVP. In conclusion, the Ce6-PVP formulation appeared to have the potential as a fluorescent marker for fluorescence diagnosis of human bladder cancer.

Evaluations of the efficiency of a new formulation of chlorin consisting of a complex of trisodium salt chlorin e6 (Ce6) and polyvinylpyrrolidone (PVP) in photodynamic therapy (PDT) and fluorescence diagnosis was performed on poorly differentiated human bladder carcinoma murine model with the following specific aims: (i) to qualitatively evaluate the fluorescence accumulation in human bladder tumor, (ii) to determine fluorescence distribution of Ce6-PVP using the tissue extraction technique and fluorescence imaging technique, (iii) to compare the fluorescence distribution of Ce6, Ce6-PVP and Photofrin in skin of nude mice, and (iv) to investigate phototoxicity caused by different parameters (drug-light interval, drug dose, irradiation fluence rate and total light fluence) in PDT. The fluorescence of the Ce6-PVP formulation was determined either by fluorescence imaging measurements or by chemical extraction from the tissues displaying similar trends of distribution. Our results demonstrated that the Ce6-PVP formulation possesses less in vivo phototoxic effect compared to Ce6 alone. The phototoxicity revealed a strong dependence on the drug and light dosimetry as well as on the drug-light interval. In PDT, the Ce6-PVP compound was most toxic at the 1h drug-light interval at 200J/cm(2), while Ce6 alone was most toxic at a light dose of more that 50J/cm(2) at the 1 and 3h drug-light interval. We also confirmed that Ce6-PVP has a faster clearance compared to Ce6 alone or Photofrin. This eliminates the need for long-term photosensitivity precautions. In conclusion, the Ce6-PVP formulation seems to be a promising photosensitizer for fluorescence imaging as well as for photodynamic treatment.

Hypericin (HY) was examined for photodynamic therapy (PDT)-induced vascular damage using the chick chorioallantoic membrane (CAM) model. Clinically, plasma protein was used to solubilize HY. Upon binding to albumin, free HY available to be transported through the membrane may be limited. Hence, formulations containing a biocompatible solvent, N-Methyl pyrrolidone (NMP), have the potential to enhance HY delivery into solid tumors. At suitable concentrations, NMP and/or light irradiation did not produce antivascular damage. Hypericin-PDT effects showed to be HY and NMP concentrations-dependent. These findings indicate that NMP is a promising solvent and penetration enhancer for HY-PDT clinical applications.

Vitamin D receptor (VDR) ligands are therapeutic agents for the treatment of psoriasis, osteoporosis, and secondary hyperparathyroidism. VDR ligands also show immense potential as therapeutic agents for autoimmune diseases and cancers of skin, prostate, colon, and breast as well as leukemia. However, the major side effect of VDR ligands that limits their expanded use and clinical development is hypercalcemia that develops as a result of the action of these compounds mainly on intestine. In order to discover VDR ligands with less hypercalcemia liability, we sought to identify tissue-selective VDR modulators (VDRMs) that act as agonists in some cell types and lack activity in others. Here, we describe LY2108491 and LY2109866 as nonsecosteroidal VDRMs that function as potent agonists in keratinocytes, osteoblasts, and peripheral blood mononuclear cells but show poor activity in intestinal cells. Finally, these nonsecosteroidal VDRMs were less calcemic in vivo, and LY2108491 exhibited more than 270-fold improved therapeutic index over the naturally occurring VDR ligand 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in an in vivo preclinical surrogate model of psoriasis.

Steroid receptor coactivator-1 (SRC-1) plays a crucial role in nuclear receptor-mediated transcription including thyroid hormone receptor (TR)-dependent gene expression. Interaction of the TR-ligand binding domain and SRC-1 through LXXLL motifs is required for this action. However, potential interactions between the TRbeta1-N terminus (N) and SRC-1 have not been explored and thus are examined in this manuscript. Far-Western studies showed that protein construct containing TRbeta1-N + DNA binding domain (DBD) bound to nuclear receptor binding domain (NBD)-1 (amino acid residue, aa 595-780) of SRC-1 without ligand. Mammalian two-hybrid studies showed that NBD-1, as well as SRC-1 (aa 595-1440), bound to TRbeta1-N+DBD in the absence of ligand in CV-1 cells. However, NBD-2 (aa 1237-1440) did not bind to this protein. Glutathione-S-transferase pull-down studies showed that TRbeta1-N (aa 1-105) bound to the broad region of SRC-1-C terminus. Expression vectors encoding a series of truncations and/or point mutations of TRbeta1 were used in transient transfection-based reporter assays in CV-1 cells. N-terminal truncated TRbeta1 (DeltaN-TRbeta1) showed lower activity than that of wild-type in both artificial F2-thyroid hormone response element and native malic enzyme response element. These results suggest that there is the interaction between N terminus of TRbeta1 and SRC-1, which may serve a full activation of SRC-1, together with activation function-2 on TRbeta1-mediated transcription.

To effectively direct targeted repression, the class I histone deacetylases (HDACs) associate with many important regulatory proteins. In this paper we describe the molecular characterization of a member of the Jumonji domain 2 (JMJD2) family of proteins, and demonstrate its binding to both class I HDACs and the retinoblastoma protein (pRb). JMJD2 proteins are characterized by the presence of two leukemia-associated protein/plant homeodomain (LAP/PHD) zinc fingers, one JmjN, one JmjC (containing an internal retinoblastoma-binding protein 2 (RBBP2)-like sequence), and two Tudor domains. The first member of this group, JMJD2A, is widely expressed in human tissues and cell lines, and high endogenous expression of JMJD2A mRNA was found in several cell types, including human T-cell lymphotropic virus 1 (HTLV-1)-infected cell lines. JMJD2A and JMJD2B exhibit cell type-specific responses to the HDAC inhibitor trichostatin A. We show that the JMJD2A protein associates in vivo with pRb and class I HDACs, and mediates repression of E2F-regulated promoters. In HTLV-1 virus-infected cells, we find that JMJD2A binds to the viral Tax protein. Antibodies to JMJD2A recognize the native protein but also a half-sized protein fragment, the latter up-regulated in THP-1 cells during the G(2)/M phase of the cell cycle. The ability of JMJD2A to associate with pRb and HDACs and potentiate pRb-mediated repression of E2F-regulated promoters implies an important role for this protein in cell proliferation and oncogenesis.

The gonadotropin-releasing hormone receptor (GnRHR) is expressed primarily in the gonadotropes of the anterior pituitary. Pituitary homeobox 1 (Pitx-1) has been shown to activate pituitary-specific gene expression by direct DNA binding and/or protein-protein interaction with other transcription factors. We hypothesized that Pitx-1 might also dictate tissue-specific expression of the mouse GnRHR (mGnRHR) gene in a similar manner. Pitx-1 activated the mGnRHR gene promoter, and transactivation was localized to sequences between −308 and −264. Pitx-1 bound to this region only with low affinity. This region includes an activating protein 1 (AP-1) site, which was previously shown to be important for mGnRHR gene expression. Further characterization indicated that an intact AP-1 site was required for full Pitx-1 responsiveness. Furthermore, Pitx-1 and AP-1 were synergistic in the activation of the mGnRHR gene promoter. A Pitx-1 homeodomain (HD) point mutation, which eliminated DNA binding ability, caused only a partial reduction of transactivation, whereas deletion of the HD completely prevented transactivation. Pitx-1 interacted directly with c-Jun, and the HD was sufficient for this interaction. While the point mutation in the Pitx-1 HD did not affect interaction with c-Jun, deletion of the HD eliminated the interaction. Taken together, our studies indicate that Pitx-1 can direct transactivation of the mGnRHR gene, in part by DNA binding and in part by an action of Pitx-1 as a cofactor for AP-1, augmenting AP-1 activity through a novel protein-protein interaction between c-Jun and the HD of Pitx-1.

We have recently shown that in colon cancer cells, Vitamin D receptor (VDR) interacts with the catalytic subunit of Ser/Thr protein phosphatases, PP1c and PP2Ac, and induces their enzymatic activity in a ligand-dependent manner. The VDR-PP1c and VDR-PP2Ac interactions were ligand independent in vivo, and 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3))-mediated increase in VDR-associated phosphatase activity resulted in dephosphorylation and inactivation of p70S6 kinase in colon cancer cells. Here, we demonstrate that in myeloid leukemia cells, 1,25(OH)(2)D(3) treatment increased the Thr389 phosphorylation of p70S6 kinase. Accordingly, 1,25(OH)(2)D(3) decreased VDR-associated Ser/Thr protein phosphatase activity by dissociating VDR-PP1c and VDR-PP2Ac interactions. Further, 1,25(OH)(2)D(3) increased the association between VDR and Thr389 phosphorylated p70S6 kinase. Finally, by using non-secosteroidal VDR ligands, we demonstrate a separation between transactivation and p70S6 kinase phosphorylation activities of VDR and show pharmacologically that p70S6 kinase phosphorylation correlates with HL-60 cell differentiation.

The vitamin D receptor (VDR) belongs to the thyroid hormone/retinoid receptor subfamily of nuclear receptors and functions as a heterodimer with retinoid X receptor (RXR). The RXR-VDR heterodimer, in contrast to other members of the class II nuclear receptor subfamily, is nonpermissive where RXR does not bind its cognate ligand, and therefore its role in VDR-mediated transactivation by liganded RXR-VDR has not been fully characterized. Here, we show a unique facet of the intermolecular RXR-VDR interaction, in which RXR actively participates in vitamin D3-dependent gene transcription. Using helix 3 and helix 12 mutants of VDR and RXR, we provide functional evidence that liganded VDR allosterically modifies RXR from an apo (unliganded)- to a holo (liganded)-receptor conformation, in the absence of RXR ligand. As a result of the proposed allosteric modification of RXR by liganded VDR, the heterodimerized RXR shows the "phantom ligand effect" and thus acquires the capability to recruit coactivators steroid receptor coactivator 1, transcriptional intermediary factor 2, and amplified in breast cancer-1. Finally, using a biochemical approach with purified proteins, we show that RXR augments the 1,25-dihydroxyvitamin D3-dependent recruitment of transcriptional intermediary factor 2 in the context of RXR-VDR heterodimer. These results confirm and extend the previous observations suggesting that RXR is a significant contributor to VDR-mediated gene expression and provide a mechanism by which RXR acts as a major contributor to vitamin D3-dependent transcription.

Nuclear receptors mediate gene activation through ligand-dependent interaction with coactivators. We previously cloned and characterized thyroid hormone receptor-binding protein, TRBP (NcoA6: AIB3/ASC-2/RAP250/PRIP/TRBP/NRC), as an LXXLL-containing coactivator that associates with coactivator complexes through its C terminus. To search for protein factors involved in TRBP action, we identified a distinct set of proteins from HeLa nuclear extract that interacts with the C terminus of TRBP. Analysis by mass spectrometric protein sequencing revealed a DNA-dependent protein kinase (DNA-PK) complex including its catalytic subunit and regulatory subunits, Ku70 and Ku86. DNA-PK is a heterotrimeric nuclear phosphatidylinositol 3-kinase that functions in DNA repair, recombination, and transcriptional regulation. DNA-PK phosphorylates TRBP at its C-terminal region, which directly interacts with Ku70 but not Ku86 in vitro. In addition, in the absence of DNA, TRBP itself activates DNA-PK, and the TRBP-stimulated DNA-PK activity has an altered phosphorylation pattern from DNA-stimulated activity. An anti-TRBP antibody inhibits TRBP-induced kinase activity, suggesting that protein content of TRBP is responsible for the stimulation of DNA-independent kinase activity. Furthermore, in DNA-PK-deficient scid cells, TRBP-mediated transactivation is significantly impaired, and nuclear localization of TRBP is altered. The activation of DNA-PK in the absence of DNA ends by the coactivator TRBP suggests a novel mechanism of coactivator-stimulated DNA-PK phosphorylation in transcriptional regulation.

Fenofibrate is clinically successful in treating hypertriglyceridemia and mixed hyperlipidemia presumably through peroxisome proliferator-activated receptor alpha (PPARalpha)-dependent induction of genes that control fatty acid beta-oxidation. Lipid homeostasis and cholesterol metabolism also are regulated by the nuclear oxysterol receptors, liver X receptors alpha and beta (LXRalpha and LXRbeta). Here we show that fenofibrate ester, but not fenofibric acid, functions as an LXR antagonist by directly binding to LXRs. Likewise, ester forms, but not carboxylic acid forms, of other members of the fibrate class of molecules antagonize the LXRs. The fibrate esters display greater affinity for LXRs than the corresponding fibric acids have for PPARalpha. Thus, these two nuclear receptors display a degree of conservation in their recognition of ligands; yet, the acid/ester moiety acts as a chemical switch that determines PPARalpha versus LXR specificity. Consistent with its LXR antagonistic activity, fenofibrate potently represses LXR agonist-induced transcription of hepatic lipogenic genes. Surprisingly, fenofibrate does not repress LXR-induced transcription of various ATP-binding cassette transporters either in liver or in macrophages, suggesting that fenofibrate manifests variable biocharacter in the context of differing gene promoters. These findings provide not only an unexpected mechanism by which fenofibrate inhibits lipogenesis but also the basis for examination of the pharmacology of an LXR ligand in humans.

The nuclear receptor superfamily comprises a large number of ligand-activated transcription factors that are involved in numerous biological processes such as cell proliferation, differentiation, and homeostasis. ROR(alpha) (NR1F1) and Reverb(alpha) (NR1D1) are two members of this family whose biological functions are largely unknown. In addition, no ligand has been yet identified for these two receptors; therefore, they are referred as orphan receptors. Here, we show that ROR(alpha) and Reverb(alpha) are expressed with a similar tissue distribution and are both induced during the differentiation of rat L6 myoblastic cells. Ectopic expression of ROR(alpha)1 in L6 cells significantly induces Reverb(alpha) expression as demonstrated by Northern blot analysis. Using reverse transcription-PCR to analyze Reverb(alpha) gene expression from staggerer mice, we found that there was a significant reduction of Reverb(alpha) mRNA in the skeletal muscle comparing it with the wild-type mice, which suggests that ROR(alpha) is involved in the regulation of Reverb(alpha) gene expression. Transient transfection assays using the Reverb(alpha) promoter demonstrate that ROR(alpha) regulates the Reverb(alpha) gene at the transcriptional level. Furthermore, mutagenesis experiments indicate that ROR(alpha) regulates Reverb(alpha) transcription via a monomeric ROR response element located in the Reverb(alpha) gene promoter. Electrophoretic mobility shift assays show that ROR(alpha) binds strongly to this site in a specific-manner. Finally, overexpression of GRIP-1/TIF-2, but not SRC-1, potentiates ROR(alpha)-stimulated Reverb(alpha) promoter activity in transient transfection experiments. Together, our results identify Reverb(alpha) as a novel target gene for ROR(alpha).

We provide evidence of a cross-talk between nuclear receptor and Ser/Thr protein phosphatases and show that vitamin D receptor (VDR) interacts with the catalytic subunit of protein phosphatases, PP1c and PP2Ac, and induces their enzymatic activity in a ligand-dependent manner. PP1c specifically interacts with VDR but not retinoic acid receptor alpha and retinoid X receptor alpha in yeast. Although VDR-PP1c and VDR-PP2Ac interaction is ligand-independent in vivo, 1alpha,25-dihydroxy-vitamin D(3) induces VDR-associated phosphatase activity. Further, VDR modulation of PP1c/PP2Ac activity results in a rapid and specific dephosphorylation and inactivation of their substrate, p70 S6 kinase (p70(S6k)). Finally, we demonstrate that the endogenous VDR, PP1c or PP2Ac, and p70(S6k) are present in a ternary complex in vivo, and the interaction of p70(S6k) with the VDR-PP complex is modulated by the phosphorylation state of the kinase. Since p70(S6k) is essential for G(1)-S transition, our results provide a molecular basis of 1alpha,25-dihydroxyvitamin D(3)-induced G(1) block in colon cancer cells.

The response of pituitary gonadotropes to GnRH correlates directly with the concentration of GnRH receptors (GnRHRs) on the cell surface, which is mediated in part at the level of GnRHR gene expression. We have previously localized GnRH responsiveness in the mouse GnRHR (mGnRHR) gene promoter to two elements: activating protein-1 and sequence underlying responsiveness to GnRH-1. This study was designed to investigate potential synergy between GnRH and activin A in transcriptional activation of the mGnRHR gene. In functional transfection studies using alphaT3-1 cells, GnRH agonist stimulation of the mGnRHR gene promoter (-765/+62) resulted in a 10.9-fold increase in activity, which was further increased 2-fold (to 21.3-fold) following activin pretreatment. Activin pretreatment alone had no effect. Deletion of region -387/-308 or mutation of a putative SMAD-binding element at -331/-324 (5'-GTCTAG[T]C-3') abrogated the augmented response to GnRH in the presence of activin but not the response to GnRH alone. Overexpression of SMAD2 and SMAD3 along with SMAD4 increased transcriptional activity of the mGnRHR gene, which was further increased by GnRH agonist stimulation. These data demonstrate that activin augments GnRH-mediated transcriptional activation of the mGnRHR gene and suggest that this effect may be mediated through SMAD transcription factors.

Ligand-dependent gene transcription mediated by the nuclear receptors involves the recruitment of transcriptional coactivators to the ligand-binding domain (LBD), which leads to interaction with the basal transcription machinery, and ultimately with RNA polymerase II. Although most of these coactivators are ubiquitously expressed, a tissue-selective coactivator, PGC-1, has recently been characterized. Because PGC-1 and the retinoid X receptors (RXRs) possess an overlapping tissue distribution, we investigated whether PGC-1 is a coactivator for the retinoid X receptors. In a transient transfection assay, PGC-1 augments ligand-stimulated RXR transcription. Furthermore, PGC-1 efficiently enhances the RXR element-driven reporter gene transcription by all three RXR isoforms. An immunoprecipitation assay reveals that PGC-1 and RXRalpha interact in vivo. In addition, a glutathione S-transferase pull-down assay showed that this interaction requires the presence of the LXXLL motif of PGC-1. We demonstrate further, in a mammalian two-hybrid assay, that this physical interaction also requires the presence of the AF-2 region of RXR to interact with the LXXLL motif of PGC-1, which is consistent with our protein-protein interaction results. A time-resolved fluorescence assay shows that a peptide within the NR box of PGC-1 is efficiently recruited by a ligand-bound RXRalpha in vitro. Finally, PGC-1 and TIF2 synergistically enhance ligand-activated RXRalpha transcriptional activity. Taken together, these results indicate that PGC-1 is a bona fide coactivator for RXRalpha.

Particle size reduction can be used for enhancing the dissolution of poorly water-soluble drugs in order to enhance bioavailability. In nanosuspensions, the particle size of the drug is reduced to nanometer size. Nanosuspensions after downstream processing into drug products have successfully shown its impact on formulation design, the augmentation of product life cycle, patent life, and therapeutic efficacy. Formulation considerations for the nanosuspension formulation, its processing into a solid form, and aspects of material characterization are discussed. Technology assessments and feasibility of upstream processes for nanoparticle creation, and subsequently transformation into a drug product via the downstream processes have been reviewed. This paper aims to bridge formulation and process considerations along with patent reviews and may provide further insight into understanding the science and the white space. An analysis of current patent outlook and future trends is described to fully understand the limitations and opportunities in intellectual property generation.

Nuclear receptors are ligand-activated transcription factors, which have the potential to integrate internal metabolic events in an organism, with consequences for control of behaviour. Previous studies from this laboratory have shown that thyroid hormone receptor (TR) isoforms can inhibit oestrogen receptor (ER)alpha-mediated induction of preproenkephalin (PPE) gene expression in the hypothalamus. Also, thyroid hormone administration inhibits lordosis, a behaviour facilitated by PPE expression. We have examined the effect of multiple ligand-binding TR isoforms on the ER-mediated induction of the PPE gene in transient transfection assays in CV-1 cells. On a natural PPE gene promoter fragment containing two putative oestrogen response elements (EREs), both ER alpha and beta isoforms mediate a four to five-fold induction by oestrogen. Cotransfection of TR alpha 1 along with ER alpha inhibited the ER alpha transactivation of PPE by approximately 50%. However, cotransfection with either TR beta 1 or TR beta 2 expression plasmids produced no effect on the ER alpha or ER beta mediated induction of PPE. Therefore, under these experimental conditions, interactions with a single ER isoform are specific to an individual TR isoform. Transfection with a TR alpha 1 DNA-binding mutant could also inhibit ER alpha transactivation, suggesting that competition for binding on the ERE may not be the exclusive mechanism for inhibition. Data with the coactivator, SRC-1, suggested that coactivator squelching may participate in the inhibition. In dramatic contrast, when ER beta is cotransfected, TR alpha 1 stimulated ER beta-mediated transactivation of PPE by approximately eight-fold over control levels. This is the first study revealing specific interactions among nuclear receptor isoforms on a neuroendocrine promoter. These data also suggest that the combinatorics of ER and TR isoforms allow multiple forms of flexible gene regulations in the service of neuroendocrine integration.

Thyroid hormone (TH) plays an important role in brain development. The rodent cerebellum has been an excellent model for the study of the molecular mechanisms of TH action in brain. However, most studies have utilized the rat rather than the mouse. Considering the usefulness of mice with regard to diverse genetic models, the study of TH effect on mouse cerebellar development is needed. Thus, we examined the effect of perinatal hypothyroidism on the expression of neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) genes, which play critical roles in cerebellar development. Newborn mice were rendered hypothyroid by administering methimazole and perchlorate in drinking water to their mothers. The growth of hypothyroid mice was retarded, which was reversed by daily thyroxine administration. NT-3 and BDNF gene expression was depressed in the perinatal hypothyroid cerebellum. Furthermore, the expression of retinoid-receptor-related orphan nuclear hormone receptor-alpha (RORalpha), an orphan nuclear receptor that plays critical roles in Purkinje cell development, was also decreased. Morphologically, disappearance of the external granule cell layer was retarded and arborization of Purkinje cell dendrite was decreased, events that were also observed in hypothyroid rats. These results indicate that the mouse cerebellum is comparable to the rat cerebellum as a model for the examination of the molecular mechanisms of TH action in brain development.

OBJECTIVE: The acute decrease in iodide organification in the thyroid in response to excess iodide is termed the acute Wolff-Chaikoff effect and normal organification resumes in spite of continued high plasma iodide concentrations (escape from the acute Wolff-Chaikoff effect). We have recently reported that large doses of iodide given to rats chronically decrease the sodium/iodide symporter (NIS) mRNA and protein, suggesting that escape is due to a decrease in NIS and subsequent iodide transport. We have now studied the effect of excess iodide on NIS in FRTL-5 cells to further explore the mechanisms whereby excess iodide decreases NIS. DESIGN: FRTL-5 cells were employed and were incubated in the presence or absence of various concentrations of iodide. NIS mRNA and protein and the turnover of NIS were assessed. METHODS: NIS mRNA was measured by Northern analysis, NIS protein by Western analysis and NIS turnover by pulse-chase labeling experiments. RESULTS: Iodide (10(-) mol/l) had no effect on NIS mRNA in FRTL-5 cells at 24 and 48 h compared with cells cultured in the absence of iodide. However, excess iodide decreased NIS protein by 50% of control values at 24 h and by 70% at 48 h. This effect of iodide was dose dependent. Pulse-chase experiments demonstrated that there was no effect of iodide on new NIS protein synthesis and that the turnover of NIS protein in the presence of iodide was 27% faster than in the absence of added iodide. CONCLUSIONS: Excess iodide does not decrease NIS mRNA in FRTL-5 cells but does decrease NIS protein, suggesting that in this in vitro thyroid cell model iodide modulates NIS, at least in part, at a post-transcriptional level. This iodide-induced decrease in NIS protein appears to be due, at least partially, to an increase in NIS protein turnover.

Estrogen receptors (ER) and thyroid hormone receptors (TR) are members of the nuclear receptor family of transcription factors that induce or repress the expression of target genes. Previous behavioral studies in female rodents have demonstrated that thyroid hormones can antagonize the effects of estrogen in the central nervous system (CNS), particularly by attenuating estrogen's ability to facilitate reproductive behaviors. Additional molecular studies have suggested a mechanism for this antagonism by showing that ligand-activated ER alpha and TRs have the potential to interact in their transcriptional controls. Although the expression patterns of ER alpha and TRs in the rodent brain appear to overlap in behaviorally relevant areas, it remained to be determined whether these two classes of proteins coexist in vivo at the level of single neurons. To address this possibility, we employed a highly sensitive double-label in situ hybridization technique using digoxigenin and (35)S-labeled cRNA probes to analyze, in detail, the expression of ER alpha mRNA with TR alpha 1 and TR alpha 2 mRNAs in the same neurons of the ovariectomized (OVX) adult mouse brain. Our results demonstrate that a large majority of the ER alpha-positive neurons also expresses TR alpha 1 and TR alpha 2 mRNAs. Quantitative examination of the cellular expression in the ventromedial and arcuate nuclei of the hypothalamus (VMH and Arc) showed that 81.5% and 80.5% of the neurons endowed with ER alpha mRNA also contain TR alpha 1 and TR alpha 2 mRNAs, respectively. In the amygdala, more than 60.5% and 67% of ER alpha-positive cells also contain TR alpha 1 and TR alpha 2 mRNAs, respectively. These findings provide the first anatomical evidence that ER and TR can be found in the same neurons, including hypothalamic neurons. This coexpression of ER alpha and TR provides the cellular basis for a new level of neuronal integration in a brain region where estrogens control female reproductive behaviors.

Thyroid hormone receptor (TR), a ligand-mediated transcription factor, binds to a DNA sequence known as a thyroid-hormone response element (TRE) to activate or repress transcription of target genes. Recently, studies have shown that Ca2+/calmodulin-dependent protein kinases (CaMKs) may be involved in regulating gene transcription via phosphorylation of specific transcription factors, including RORalpha, a retinoic acid-related orphan nuclear hormone receptor. In this light, we examined the effect of CaMK type IV (CaMKIV) and RORalpha, which also shown to influence thyroid hormone action, on TR-mediated transcription using a transient transfection assay. Expression vectors containing TR, vitamin D receptor (VDR), and estrogen receptor (ER) were cotransfected in CV-1 cells with RORalpha and/or constitutively active CaMKIV and thymidine kinase promotor-luciferase reporter vector containing their cognate response elements. When CaMKIV or RORalpha was co-transfected with TR, the T3-induced transcription was significantly augmented compared to that induced by TR alone. When both were co-transfected with TR, T3-induced transcription was augmented additively. In contrast, the augmentation by CaMKIV or ROR on ligand-induced transcription was not detected with VDR and ER. Hence, these results indicate that the augmentation mediated by CaMKIV and RORalpha is specific for TR-mediated transcription on TRE. Our results suggest that CaMKIV, as well as RORalpha, play important roles in TR-mediated transcription on TREs.

Nuclear hormone receptors activate gene transcription through ligand-dependent association with coactivators. Specific LXXLL sequence motifs present in these cofactors are sufficient to mediate these ligand-induced interactions. A thyroid hormone receptor (TR)-binding protein (TRBP) was cloned by a Sos-Ras yeast two-hybrid system using TRβ1-ligand binding domain as bait. TRBP contains 2063 amino acid residues, associates with TR through a LXXLL motif, and is ubiquitously expressed in a variety of tissues and cells. TRBP strongly transactivates through TRβ1 and estrogen receptor in a dose-related and ligand-dependent manner, and also exhibits coactivation through AP-1, CRE, and NFκB-response elements, similar to the general coactivator CBP/p300. The C terminus of TRBP binds to CBP/p300 and DRIP130, a component of the DRIP/TRAP/ARC complex, which suggests that TRBP may activate transcription by means of such interactions. Further, the association of TRBP with the DNA-dependent protein kinase (DNA-PK) complex and DNA-independent phosphorylation of TRBP C terminus by DNA-PK point to a potential connection between transcriptional control and chromatin architecture regulation.

Thyroid hormone (TH) plays an important role in the postnatal development of the rodent cerebellum, particularly within the first 2 weeks of postnatal life. This action is exerted through the regulation of specific genes during development and is mediated by coactivator and corepressor proteins that determine transcriptional repression or activation, respectively. Thus, we hypothesized that the effect of TH on rodent cerebellar development could be influenced by the relative amounts of coactivator and corepressor proteins in vivo. These ratios might be modulated in an age-specific manner and/or by hormones to generate the "critical period" of TH action. To examine this hypothesis, we cloned rat complementary DNA fragments corresponding to coactivators (SRC1, TIF2 and TRAM1) and corepressors (N-CoR and SMRT), and studied the ontogenic changes in their corresponding messenger RNAs in rat cerebellum of normal and hypothyroid rats during postnatal development, using a RNase protection assay. We found an increased expression of SRC1 and TIF2, as well as of N-CoR, during rat cerebellar development but no change in the expression of SMRT and TRAM1 genes. However, thyroid hormone status did not affect the expression of coactivator and corepressor genes in the cerebellum. These results indicate that coactivator and corepressor messenger RNAs exhibit differential expression through cerebellar development but are not regulated by TH during this period.

Thyroid hormones influence both bone formation and bone resorption. Clinical data and animal studies provide evidence of skeletal site heterogeneity (hip vs. spine) of bone responses to thyroid hormones. In vitro studies also demonstrate direct effects of thyroid hormones on cells of the osteoblast lineage. Transcriptional regulation by thyroid hormone is mediated by ligand-dependent transcription factors called thyroid hormone receptors (TRs). Two genes, c-ErbAalpha and c-ErbAbeta, generate at least four TR isoforms in the rat: TRalpha(1), c-erbAalpha(2), TRbeta(1), and TRbeta(2). Although functional TRs have been identified in cells of the osteoblast lineage, it is still not known if TR isoform expression in bone differs depending upon which skeletal site is examined. We have used ribonuclease protection assay and Northern blot analysis to simultaneously examine the expression of TR isoform mRNAs in adult rat femoral and vertebral bone. TRalpha(1), c-erbAalpha(2), and TRbeta(1) are expressed in both femur and vertebra whole bone. Bone marrow cells from both skeletal sites were also cultured under conditions whereby the osteoprogenitors differentiated into osteoblasts and formed a mineralized extracellular matrix. TRalpha(1), c-erbAalpha(2), and TRbeta(1) mRNAs are each expressed in both femoral and vertebral osteoblast cultures. The presence of TRalpha(1), c-erbAalpha(2), and beta(1) proteins was confirmed by Western analysis of nuclear protein extracts from femoral and vertebral cell cultures. These results indicate that the three predominant TR isoforms are highly expressed in bone and osteoblasts from femurs and vertebrae. Whether there are distinct mechanisms of thyroid hormone action mediated by TRalpha(1), c-erbAalpha(2), and TRbeta(1) at these separate skeletal sites remain to be shown.

Thyroid hormone (TH) plays a critical role in normal cerebellar development. However, the molecular mechanisms of TH action in the developing cerebellum are not fully understood. This action could be exerted in part through brain-derived neurotropic factor (BDNF), as cerebellar BDNF messenger RNA (mRNA) expression is lower, and replacement of BDNF partially reverses the abnormal neurogenesis in the hypothyroid rat. The rat BDNF gene consists of four noncoding exons (exons I–IV), each of which is linked to a different promoter, and a protein-coding exon (exon V). To study promoter-specific regulation of the BDNF gene by TH, ribonuclease protection assay of each exon mRNA was performed using total developing rat cerebellar RNA. During cerebellar development, all exon mRNAs were detected, but with different expression patterns; among noncoding exon mRNAs, exon II mRNA was the most abundant. Daily TH replacement induced a 3-fold increase in exon II mRNA on postnatal day (P) 15. On P30, exon II mRNA was still much greater in the TH-replaced animal. Exon I mRNA was detected on P2 and P7. However, in contrast to exon II mRNA, TH treatment suppressed the expression of exon I mRNA on P2. Exon III and IV mRNAs were not detected on P2 and P7, but small amounts were observed starting on P15 in TH-replaced animals. They were not detected by P30 in hypothyroid animals. In contrast, in the cerebral cortex, although all exons are differentially regulated during development, the expression of each mRNA was not significantly altered by TH. These results indicate that TH regulates BDNF gene expression in a promoter-, developmental stage-, and brain region-specific manner, which may play an important role in region- and stage-specific regulation of brain development by TH.

GnRH plays a critical role in regulating mammalian reproductive development and function. At the level of the anterior pituitary, GnRH binds to the GnRH receptor (GnRHR) on the cell surface of pituitary gonadotropes. Here, it activates intracellular signal transduction pathways to effect both the synthesis and intermittent release of the gonadotropins LH and FSH. These hormones then enter the systemic circulation to regulate gonadal function, including steroid hormone synthesis and gametogenesis. The response of pituitary gonadotropes to GnRH correlates directly with the concentration of GnRHR on the cell surface, which is mediated, at least in part, at the level of gene expression. A number of endocrine, paracrine, and autocrine factors are known to regulate GnRHR gene expression. This article reviews in detail the role of the GnRHR in the hypothalamic-pituitary-gonadal axis and the factors mediating expression of this gene. A better understanding of the molecular mechanisms that regulate transcription of the GnRHR gene will further our knowledge about the role of this receptor in mammalian reproductive physiology in health and disease.

Ligand-dependent nuclear hormone receptors (NRs), such as retinoic acid and thyroid hormone receptors, play critical roles in diverse aspects of development. They enhance or repress transcription by recruiting an array of coactivator and corepressor proteins, which function as signaling intermediates between the NRs and the basal transcriptional machinery. To study the possible involvement of these cofactors on tissue-specific regulation of gene expression by NRs, we examined the expression of the coactivator SRC-1 mRNA during mouse embryogenesis by in situ hybridization (ISH). 35S-labeled riboprobe specific for SRC-1 mRNA was used for analysis. The distribution of this transcript was studied from 8.5 to 18.5 embryonic days (E8.5-E18.5) and in postnatal day 15 (P15). The SRC-1 transcript was largely ubiquitously expressed, even on E8.5. At E14.5 and E18.5, highest levels of SRC-1 transcript was found in the olfactory epithelium. Significant SRC-1 hybridization signal was also detected in the neocortex, anterior pituitary and heart. We conclude that (1) SRC-1 mRNA is widely expressed in the developing embryo, and (2) SRC-1 mRNA is expressed at the highest level in the olfactory epithelium, suggesting that this coactivator may be involved in the development and/or function of the olfactory system.

The peroxisome proliferator-activated receptors (PPARs) are a subgroup of nuclear receptors activated by fatty acids and eicosanoids. In addition, they are subject to phosphorylation by insulin, resulting in the activation of PPARalpha, while inhibiting PPARgamma under certain conditions. However, it was hitherto unclear whether the stimulatory effect of insulin on PPARalpha was direct and by which mechanism it occurs. We now demonstrate that amino acids 1-92 of hPPARalpha contain an activation function (AF)-1-like domain, which is further activated by insulin through a pathway involving the mitogen-activated protein kinases p42 and p44. Further analysis of the amino-terminal region of PPARalpha revealed that the insulin-induced trans-activation occurs through the phosphorylation of two mitogen-activated protein kinase sites at positions 12 and 21, both of which are conserved across evolution. The characterization of a strong AF-1 region in PPARalpha, stimulating transcription one-fourth as strongly as the viral protein VP16, is compatible with the marked basal transcriptional activity of this isoform in transfection experiments. However, it is intriguing that the activity of this AF-1 region is modulated by the phosphorylation of two serine residues, both of which must be phosphorylated in order to activate transcription. This is in contrast to PPARgamma2, which was previously shown to be phosphorylated at a single site in a motif that is not homologous to the sites now described in PPARalpha. Although the molecular details involved in the phosphorylation-dependent enhancement of the transcriptional activity of PPARalpha remain to be elucidated, we demonstrate that the effect of insulin on the AF-1 region of PPARalpha can be mimicked by the addition of triiodothyronine receptor beta1, a strong binder of corepressor proteins. In addition, a triiodothyronine receptor beta1 mutant deficient in interacting with corepressors is unable to activate PPARalpha. These observations suggest that the AF-1 region of PPARalpha is partially silenced by corepressor proteins, which might interact in a phosphorylation-dependent manner.

Inactivation of the melanocortin-4 receptor (MC4-R) by gene-targeting results in mice that develop maturity-onset obesity, hyperinsulinemia, and hyperglycemia. These phenotypes resemble common forms of human obesity, which are late-onset and frequently accompanied by NIDDM. It is not clear whether sequence variation of the MC4-R gene contributes to obesity in humans. Therefore, we examined the human MC4-R gene polymorphism in 190 individuals ascertained on obesity status. Three allelic variants were identified, including two novel ones, Thr112Met and Ile137Thr. To analyze possible functional alterations, the variants were cloned and expressed in vitro and compared with the wild-type receptor. One of the novel variants, Ile137Thr, identified in an extremely obese proband (BMI 57), was found to be severely impaired in ligand binding and signaling, raising the possibility that it may contribute to development of obesity. Furthermore, our results also suggest that sequence polymorphism in the MC4-R coding region is unlikely to be a common cause of obesity in the population studied, given the low frequency of functionally significant mutations.