11 research outputs found
Skin-targeted inhibition of PPAR β/δ by selective antagonists to treat PPAR β/δ-mediated psoriasis-like skin disease in vivo
We have previously shown that peroxisome proliferator activating receptor ß/δ (PPAR β/δ is overexpressed in psoriasis. PPAR β/δ is not present in adult epidermis of mice. Targeted expression of PPAR β/δ and activation by a selective synthetic agonist is sufficient to induce an inflammatory skin disease resembling psoriasis. Several signalling pathways dysregulated in psoriasis are replicated in this model, suggesting that PPAR β/δ activation contributes to psoriasis pathogenesis. Thus, inhibition of PPAR β/δ might harbour therapeutical potential. Since PPAR β/δ has pleiotropic functions in metabolism, skin-targeted inhibition offer the potential of reducing systemic adverse effects. Here, we report that three selective PPAR β/δ antagonists, GSK0660, compound 3 h, and GSK3787 can be formulated for topical application to the skin and that their skin concentration can be accurately quantified using ultra-high performance liquid chromatography (UPLC)/mass spectrometry. These antagonists show efficacy in our transgenic mouse model in reducing psoriasis-like changes triggered by activation of PPAR β/δ. PPAR β/δ antagonists GSK0660 and compound 3 do not exhibit systemic drug accumulation after prolonged application to the skin, nor do they induce inflammatory or irritant changes. Significantly, the irreversible PPAR β/δ antagonist (GSK3787) retains efficacy when applied topically only three times per week which could be of practical clinical usefulness. Our data suggest that topical inhibition of PPAR β/δ to treat psoriasis may warrant further exploration
Adjacent single-stranded regions mediate processing of tRNA precursors by RNase E direct entry
The RNase E family is renowned for being central to
the processing and decay of all types of RNA in
many species of bacteria, as well as providing the
first examples of endonucleases that can recognize
50
-monophosphorylated ends thereby increasing
the efficiency of cleavage. However, there is
increasing evidence that some transcripts can be
cleaved efficiently by Escherichia coli RNase E via
direct entry, i.e. in the absence of the recognition of
a 50
-monophosphorylated end. Here, we provide
biochemical evidence that direct entry is central to
the processing of transfer RNA (tRNA) in E. coli, one
of the core functions of RNase E, and show that it is
mediated by specific unpaired regions that are
adjacent, but not contiguous to segments cleaved
by RNase E. In addition, we find that direct entry at a
site on the 50 side of a tRNA precursor triggers a
series of 50
-monophosphate-dependent cleavages.
Consistent with a major role for direct entry
in tRNA processing, we provide additional evidence
that a 50
-monophosphate is not required to
activate the catalysis step in cleavage. Other
examples of tRNA precursors processed via direct
entry are also provided. Thus, it appears increasingly
that direct entry by RNase E has a major role
in bacterial RNA metabolism
Mad4 is regulated by a transcriptional repressor complex that contains Miz-1 and c-Myc.
Myc and Mad family proteins are central regulators of cellular proliferation and differentiation. We show that various Mad family genes have distinct patterns of expression during the chemically induced differentiation of mouse erythroleukaemia (MEL) cells, suggesting that they each serve a different function. Mad4 RNA is highly induced and persists in terminally differentiated cells, in agreement with observations in other systems. Using reporter gene assays in stably transfected MEL cells, we show that induction of Mad4 is mediated by a 49 nt core promoter region. We demonstrate that the initiator element is required for Mad4 activation, and show that induction is associated with the loss from the initiator of a complex that contains Miz-1 and c-Myc. Miz-1 activates the Mad4 promoter in transient transfection assays, and this effect is antagonized by c-Myc. We therefore identify Mad4 as a novel target of transcriptional repression by c-Myc. These data suggest that the expression of Mad4 in proliferating undifferentiated cells is suppressed by the binding of a c-Myc-Miz-1 repressor complex at the initiator, and that the activation of Mad4 during differentiation results, at least in part, from a decrease in c-Myc-mediated repression
Absence of inflammatory changes induced by PPAR β/δ antagonists in skin after topical application.
<p>(a) C57Bl/6j wild type mice were treated with ointments containing GSK0660 or compound 3 h applied twice daily to shaved dorsal skin for one week. Mice were sacrificed 1 h after the last ointment application and skin tissue processed for H&E based histology and mass spectrometry, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037097#s4" target="_blank">Methods</a>. Data shown represent average ± s.d. of n = 3 mice per data point (left) treated with GSK (blue columns) or compound 3 h (red). Representative histology sections of all treated mice are shown on right. The inset in the middle panel shows a section of GSK0660-treated epidermis showing apoptotic looking cells (marked by red arrow head). Horizontal bar represents 5 µm. (b) Representative H&E sections of C57Bl/6j wild type mice treated for one week with either GSK0660 (top) or GSK3787 (bottom). Red arrow-heads denoting apoptotic looking cells.</p
Half – life of GSK0660 after topical application to skin.
<p>42 mg of GSK0660 ointment was applied to dorsal skin of C57Bl/6j wild type mice. Mice were sacrificed at the time after drug application indicated in the figure and drug concentration determined by mass spectrometry, as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037097#s4" target="_blank">Methods</a>. Data shown represent average ± s.d. of n = 3 mice per data point.</p
Prevention of epidermal hyperplasia by transdermal application of selective PPAR β/δ antagonists.
<p>Both the PPAR β/δ agonist GW501516 (GW) and the antagonists GSK0660 (GSK) or compound 3 h were applied topically to the skin, as described in the text. Left: representative H&E-stained paraffin-sections of dorsal skin from PPAR β/δ transgenic mice after treatment with ointments containing the indicated drugs for twenty days, as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037097#s4" target="_blank">Methods</a>. Horizontal bar represents 5 µm. Right: quantification of H&E-based epidermal thickness observed in n = 4 mice per group, performed as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037097#s4" target="_blank">Methods</a>. * p<0.05 in a two-sided independent t-test.</p
Low systemic absorption of topically applied PPAR β/δ antagonists.
<p><b>A</b>. Peak blood concentrations of PPAR β/δ agonist GW501516, and antagonists GSK0660 and compound 3 h, respectively, at 1 h after topical application to skin. Left: Amount of drugs detected in systemic circulation, expressed as fraction of total amount applied, was calculated as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037097#s4" target="_blank">methods</a>. Right: Drug concentration expressed as molar concentration. <b>B</b>. GSK0660 concentration in blood (left) and total amount of circulating drug as fraction of amount applied (right, calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037097#s4" target="_blank">Methods</a>) at the indicated time points after drug application. The horizontal dashed line represents the reported IC50 for GSK0660 acting on PPAR β/δ reported previously (300 nmol/L). Data shown represent average ± s.d. of n = 3 mice per data point.</p
PPAR β/δ selective antagonists used in this study.
<p>Chemical structures and in vitro pharmacodynamic data shown are taken from the references listed. The structure of the PPAR β/δ selective agonist GW501516 used in this study is given for comparison.</p
Control of PPAR β/δ – mediated skin disease using reduced-frequency application of ointment containing an irreversible PPAR β/δ antagonist.
<p>Skin disease in PPAR β/δ transgenic mice was induced by i.p. injection of the agonist GW501516. Additionally, mice were shaved on their abdomen and were treated with vehicle-ointment or ointment containing either GSK0660 (twice daily) or GSK3787 at the indicated frequencies. Red arrow denotes apoptotic cells noted in the GW-only treatment group. <b>A</b>. Top: Representative H&E stains from 3 different mice in each treatment group. Horizontal bar represents 5 µm. Bottom: Quantification of epidermal thickness (p<0.01 in all treatment groups vs. GW-only). <b>B</b>. Quantification of dermal infiltrate. Data shown represent average ± s.d. of five mice per group.</p