TRPV1 antagonists that cause hypothermia, instead of hyperthermia, in rodents: Compounds' pharmacological profiles, in vivo targets, thermoeffectors recruited and implications for drug development.

AIM: Thermoregulatory side effects hinder the development of transient receptor potential vanilloid-1 (TRPV1) antagonists as new painkillers. While many antagonists cause hyperthermia, a well-studied effect, some cause hypothermia. The mechanisms of this hypothermia are unknown and were studied herein. METHODS: Two hypothermia-inducing TRPV1 antagonists, the newly synthesized A-1165901 and the known AMG7905, were used in physiological experiments in rats and mice. Their pharmacological profiles against rat TRPV1 were studied in vitro. RESULTS: Administered peripherally, A-1165901 caused hypothermia in rats by either triggering tail-skin vasodilation (at thermoneutrality) or inhibiting thermogenesis (in the cold). A-1165901-induced hypothermia did not occur in rats with desensitized (by an intraperitoneal dose of the TRPV1 agonist resiniferatoxin) sensory abdominal nerves. The hypothermic responses to A-1165901 and AMG7905 (administered intragastrically or intraperitoneally) were absent in Trpv1-/- mice, even though both compounds evoked pronounced hypothermia in Trpv1+/+ mice. In vitro, both A-1165901 and AMG7905 potently potentiated TRPV1 activation by protons, while potently blocking channel activation by capsaicin. CONCLUSION: TRPV1 antagonists cause hypothermia by an on-target action: on TRPV1 channels on abdominal sensory nerves. These channels are tonically activated by protons and drive the reflectory inhibition of thermogenesis and tail-skin vasoconstriction. Those TRPV1 antagonists that cause hypothermia further inhibit these cold defences, thus decreasing body temperature. SIGNIFICANCE: TRPV1 antagonists (of capsaicin activation) are highly unusual in that they can cause both hyper- and hypothermia by modulating the same mechanism. For drug development, this means that both side effects can be dealt with simultaneously, by minimizing these compounds' interference with TRPV1 activation by protons.

Human ITCH is a coregulator of the hematopoietic transcription factor NF-E2.

We have cloned a new protein that interacts with the hematopoietic DNA-binding transcription factor, p45/NF-E2, by screening a human erythroleukemia cell cDNA library with the yeast two-hybrid approach. Predicted peptide sequence and chromosomal mapping identified the cloned molecule to be the product of the human ortholog of the mouse Itch gene, which has been implicated previously in the regulation of growth and differentiation of erythroid and lymphoid cells. Transfection experiments indicate that this human ITCH protein can act as a transcriptional corepressor of p45/NF-E2. Our data provide novel insights into the functional roles of the mammalian ITCH proteins in the development of hematopoietic cell lineages.

Loading of DNA-binding factors to an erythroid enhancer.

The HS-40 enhancer is the major cis-acting regulatory element responsible for the developmental stage- and erythroid lineage-specific expression of the human alpha-like globin genes, the embryonic zeta and the adult alpha2/alpha/1. A model has been proposed in which competitive factor binding at one of the HS-40 motifs, 3'-NA, modulates the capability of HS-40 to activate the embryonic zeta-globin promoter. Furthermore, this modulation was thought to be mediated through configurational changes of the HS-40 enhanceosome during development. In this study, we have further investigated the molecular basis of this model. First, human erythroid K562 cells stably integrated with various HS-40 mutants cis linked to a human alpha-globin promoter-growth hormone hybrid gene were analyzed by genomic footprinting and expression analysis. By the assay, we demonstrate that factors bound at different motifs of HS-40 indeed act in concert to build a fully functional enhanceosome. Thus, modification of factor binding at a single motif could drastically change the configuration and function of the HS-40 enhanceosome. Second, a specific 1-bp, GC-->TA mutation in the 3'-NA motif of HS-40, 3'-NA(II), has been shown previously to cause significant derepression of the embryonic zeta-globin promoter activity in erythroid cells. This derepression was hypothesized to be regulated through competitive binding of different nuclear factors, in particular AP1 and NF-E2, to the 3'-NA motif. By gel mobility shift and transient cotransfection assays, we now show that 3'-NA(II) mutation completely abolishes the binding of small MafK homodimer. Surprisingly, NF-E2 as well as AP1 can still bind to the 3'-NA(II) sequence. The association constants of both NF-E2 and AP1 are similar to their interactions with the wild-type 3'-NA motif. However, the 3'-NA(II) mutation causes an approximately twofold reduction of the binding affinity of NF-E2 factor to the 3'-NA motif. This reduction of affinity could be accounted for by a twofold-higher rate of dissociation of the NF-E2-3'-NA(II) complex. Finally, we show by chromatin immunoprecipitation experiments that only binding of NF-E2, not AP1, could be detected in vivo in K562 cells around the HS-40 region. These data exclude a role for AP1 in the developmental regulation of the human alpha-globin locus via the 3'-NA motif of HS-40 in embryonic/fetal erythroid cells. Furthermore, extrapolation of the in vitro binding studies suggests that factors other than NF-E2, such as the small Maf homodimers, are likely involved in the regulation of the HS-40 function in vivo.

Distinction between AP1 and NF-E2 factor-binding at specific chromatin regions in mammalian cells.

Specific nuclear factor-DNA complexes formed within the promoters and enhancers are essential for transcriptional regulation. For eukaryotic systems, however, some DNA motif(s) are capable of binding to a family of related factors, thus making it difficult to identify the factor actually binding on the chromatic DNA in vivo and modulating the local transcription processes. To resolve this matter, we have refined a chromatin immunoprecipitation assay. Using the assay, we could directly link the regulatory functions of two members of the AP1/NF-E2 transcription factor family and their stable binding in vivo within distinct chromatin regions. The study demonstrated the feasibility of a general scheme in the determination of the identity of specific factor(s), among a group of family members, bound at unique sequence(s) in living mammalian cells.

Derepression of human embryonic zeta-globin promoter by a locus-control region sequence.

A multiple protein-DNA complex formed at a human alpha-globin locus-specific regulatory element, HS-40, confers appropriate developmental expression pattern on human embryonic zeta-globin promoter activity in humans and transgenic mice. We show here that introduction of a 1-bp mutation in an NF-E2/AP1 sequence motif converts HS-40 into an erythroid-specific locus-control region. Cis-linkage with this locus-control region, in contrast to the wild-type HS-40, allows erythroid lineage-specific derepression of the silenced human zeta-globin promoter in fetal and adult transgenic mice. Furthermore, zeta-globin promoter activities in adult mice increase in proportion to the number of integrated DNA fragments even at 19 copies/genome. The mutant HS-40 in conjunction with human zeta-globin promoter thus can be used to direct position-independent and copy number-dependent expression of transgenes in adult erythroid cells. The data also supports a model in which competitive DNA binding of different members of the NF-E2/AP1 transcription factor family modulates the developmental stage specificity of an erythroid enhancer. Feasibility to reswitch on embryonic/fetal globin genes through the manipulation of nuclear factor binding at a single regulatory DNA motif is discussed.

Interaction of WW domains with hematopoietic transcription factor p45/NF-E2 and RNA polymerase II.

NF-E2 is an erythroid-specific transcription factor required for expression of several erythroid-specific genes. By Far-Western blotting and yeast two-hybrid assay, we demonstrate that p45, the large subunit of NF-E2, is capable of binding to a specific set of WW domain-containing proteins, including the ubiquitin ligase hRPF1. This binding is mediated through the interaction between the WW domains and a PY motif located within the amino-terminal region of p45. Interestingly, the carboxyl-terminal domain of mammalian RNA polymerase II binds a similar set of WW domains to which p45 interacts with. We discuss the data in terms of possible new pathways through which the processes of transcriptional regulation by NF-E2 could be regulated in erythroid and megakaryote cells.