Psoriasis vulgaris is characterized by hyperproliferation and incomplete terminal differentiation of epidermal keratinocytes. Despite the established role of Wnt pathways in the regulation of stem cell proliferation and differentiation, they have not yet been associated with the pathophysiology of psoriasis. Here, we took biopsies from uninvolved and from lesional skin of 20 patients with plaque-type psoriasis. The biopsies were used for microarray RNA expression profiling. Based on paired samples from 13 patients, we defined 179 genes that were more than 2-fold differentially expressed in lesional skin. This list included 16 genes with known or possible association to the canonical Wnt/beta-catenin or the non-canonical Wnt/Ca2+ pathway. The expression of Wnt5a was 4-fold higher in lesional skin. Other Wnt molecules were largely unchanged (Wnt4 and Wnt16), or tended to be expressed at lower levels (Wnt7b). The mRNA expression levels of two inhibitory factors related to Wnt signaling, frizzled-related protein, and dickkopf homolog 2, were reduced in lesional skin, as was mRNA expression of cyclin D1. These findings were confirmed by quantitative reverse transcription-PCR experiments. We conclude that Wnt5a and other Wnt pathway genes are differentially expressed in psoriatic plaques. Their functional contribution to the pathophysiology of psoriasis needs to be elaborated.

The estrogen receptor (ER) is a strong hormone-inducible transcription factor that regulates the expression of many genes. It was shown for the human progesterone receptor that the binding of hormone causes distinct conformational changes in the ligand binding domain (LBD) and that these changes in LBD conformation are crucial for events after DNA binding. We now show that conformational changes in the LBD of the human ER are a prerequisite for trans-activation. Under the appropriate conditions ER binds to its response element and activates transcription only in the presence of ligand. Binding of the ligand causes changes in the conformation of the LBD. Antihormones induce distinct conformational changes, the differences between the conformations lying in the carboxy-terminal end of the receptor. Changing the experimental conditions results in a receptor that can bind to DNA and activate transcription in a ligand-independent manner. Under these conditions the LBD has a transcriptionally active conformation in the absence of ligand. Taken together, our data indicate that the conformational change induced by ligand is required for converting a receptor to the transcriptionally active form.

Oligonucleotides provide novel reagents for inhibition of gene expression because of their high affinity binding to specific nucleotide sequences. We describe a 38 base, single-stranded DNA that forms a triple helix or 'triplex' on progesterone response elements of a target gene. This triplex-forming oligonucleotide binds with a Kd = 100 nM at 37 degrees C and physiological pH, and blocks binding of progesterone receptors to the target. Furthermore, it completely inhibited progesterone receptor-dependent transcription in vitro. To approach in vivo conditions, triplex-forming oligonucleotides were tested in cell transfection studies. The derivation of the oligonucleotides with cholesterol enhanced their cellular uptake and nuclear concentration by at least four-fold. The cholesterol-derivatized triplex-forming oligonucleotide specifically inhibited transcription of the PRE-containing reporter gene in cells when applied to the medium at micromolar concentrations. This is the first demonstration of steroid-responsive gene inhibition by triplex formation and joins the growing body of evidence indicating that oligonucleotides have therapeutic potential.

Several studies using microarrays have shown that changes in gene expression provide information about the mechanism of toxicity induced by xenobiotic agents. Nevertheless, the issue of whether gene expression profiles are reproducible across different laboratories remains to be determined. To address this question, several members of the Hepatotoxicity Working Group of the International Life Sciences Institute Health and Environmental Sciences Institute evaluated the liver gene expression profiles of rats treated with methapyrilene (MP). Animals were treated at one facility, and RNA was distributed to five different sites for gene expression analysis. A preliminary evaluation of the number of modulated genes uncovered striking differences between the five different sites. However, additional data analysis demonstrated that these differences had an effect on the absolute gene expression results but not on the outcome of the study. For all users, unsupervised algorithms showed that gene expression allows the distinction of the high dose of MP from controls and low dose. In addition, the use of a supervised analysis method (support vector machines) made it possible to correctly classify samples. In conclusion, the results show that, despite some variability, robust gene expression changes were consistent between sites. In addition, key expression changes related to the mechanism of MP-induced hepatotoxicity were identified. These results provide critical information regarding the consistency of microarray results across different laboratories and shed light on the strengths and limitations of expression profiling in drug safety analysis. minimal expression of single-cell necrosis with minimal mononuclear infiltrate without associated changes in clinical chemistry parameters (Waring et al. 2001). Thus, in the present study we chose 100 mg/kg/day as the high dose expected to elicit hepatotoxicity. A dose of 10 mg/kg/day was selected as the low dose with the expectation that no hepatotoxic effect would be observed.Male Sprague-Dawley rats were obtained from Charles River Laboratories, Inc. (Wilmington, MA). Rats were 57 days old and weighed 233.4-274.0 g at the start of the treatment. Upon arrival to Abbott Laboratories (Abbott Park, IL), all rats were acclimated for 6 days before treatment began. The two treatment groups comprising four rats each received the test compound at a concentration of 10 or 100 mg/kg, respectively. Animals in the equally sized control group received vehicle only.Rats were dosed once daily by gavage for 7 days. The dose volume was 10 mL/kg. Doses were milligram salt per kilogram per day and were calculated for each rat on the basis of the most recent body weight data available. Rats were fasted overnight after their last treatment, euthanized under halothane anesthesia and submitted for necropsy. Each rat received its last treatment approximately 24 hr before scheduled necropsy.In vivo observations, pathology, and sampling. All rats were observed twice each day during the pretreatment and t...

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS). Interferon-beta (IFN-beta) therapy for MS is hypothesized to cause short-term and long-term changes in gene expression that shift the inflammation from Th1 to Th2. In vivo gene induction to define kinetics of response to IFN-beta therapy in a large cohort of MS patients is described. Differential gene expression in peripheral blood mononuclear cells (PBMCs) obtained from relapsing-remitting MS patients (RRMS) was assessed using high content microarrays. Rapid onset of gene expression appeared within 4 h of subcutaneous IFN-beta administration, returning to baseline levels at 42 h in clinically stable RRMS. IFN-beta therapy in vivo rapidly but transiently induced strong upregulation of genes mediating immune modulation, IFN signaling, and antiviral responses. RT-PCR showed significant patient-to-patient variation in the magnitude of expression of multiple genes, especially for IFN-beta-inducible genes, such as MxA, IRF7, and CCL8, a Th1 product. Variation among patients in IFN-beta-induced RNA transcription was not explained by neutralizing antibodies or IFN receptor expression. Surprisingly, genes regulated in vivo by IFN-beta therapy do not support a simple Th1 to Th2 shift. A complex interplay between both proinflammatory and anti-inflammatory immune regulatory genes is likely to act in concert in the treatment of RRMS.