Interaction of the TNFR-receptor associated factor TRAF1 with I-kappa B kinase 2 (IKK2, IKK-beta, IKBKB) and TRAF2 indicating a dose dependent regulatory function of TRAF1 for NF-kappa B signaling

IKK2 is one of the most crucial signaling kinases for activation of the transcription factor NF-kappa B. Since many NF-kappa B activating pathways converge at the level of IKK2, we searched for interaction partners of this kinase using the C-terminal part (aa 466-756) as bait in a yeast two-hybrid system. We identified the N-terminal part (aa 1-228) of the TNF-receptor associated factor TRAF1 as putative interaction partner, which was subsequently confirmed in mammalian cells by coimmunoprecipitation experiments. However, this interaction seemed weaker than the interaction between TRAF1 and TRAF2, an important activating adapter molecule of NF-kappa B signaling indicating that relative levels of IKK2, TRAF1 and TRAF2 might be important for the final biological readout. Reporter gene and kinase assays using ectopic expression of TRAF1 indicated that it can have both activating and inhibiting functions for IKK2 and NF-kappa B. Co-expression of fluorescently tagged TRAF1 and TRAF2 at different ratios implied that TRAF1 can affect clustering and presumably the activating function of TRAF2 in a dose dependent manner

The actin-binding protein profilin II in neuronal plasticity

Activity-dependent plasticity in neurons involves changes in synaptic transmission and connectivity. These changes lead to altered neuronal circuit properties and are thought to underlie learning and memory. Transcription and protein synthesis are indispensable in order to maintain changes in neural circuitry over periods of several hours or longer. Therefore signaling from the synapse to the nucleus is required to control activity-dependent expression of RNA and proteins which have to be transported back to the activated synaptic sites. The small actin-binding protein profilin has been shown to accumulate in postsynaptic dendritic spines of pyramidal neurons as a necessary element in activity-dependent stabilization of synaptic morphology, a putative anatomical correlate of changes in transmission strength. In this work I show that profilin also enters the nucleus in an NMDA receptor and Ca2+ dependent manner. However, in contrast to spine targeting, nuclear enrichment is reversible within minutes after removal of the stimulus. Nuclear accumulation of profilin is likely coupled to activity-dependent actin polymerization at the cell cortex which also takes place in response to NMDA receptor stimulation. Nuclear profilin has been implicated in different steps of gene expression including transcription and pre-mRNA splicing. Activity-dependent nuclear and synaptic accumulation suggests profilin to be involved in different aspects of neuronal plasticity. To this end, I introduce approaches to elucidate profilin function in experience-dependent plasticity and gene expression

Functional Remodeling of Benign Human Prostatic Tissues In Vivo by Spontaneously Immortalized Progenitor and Intermediate Cells

Tissue remodeling or regeneration is believed to initiate from multipotent stem and progenitor cells. We report here the establishment of two spontaneously immortalized adult non-tumorigenic human prostate epithelial cell lines, NHPrE1 and BHPrE1. NHPrE1 (CD133high/CD44high/OCT4high/PTENhigh) was characterized as a putative progenitor cell, and BHPrE1 (p63high/p53high/p21(WAF1)high/RBhigh) was characterized as a putative epithelial intermediate cell. Genomic analysis demonstrated an abnormal karyotype with genomic rearrangements including PTEN amplification in NHPrE1 and CTNNB1 (β-catenin) amplification in BHPrE1 cells. Embedded three-dimensional culture of NHPrE1 showed greater branching than BHPrE1. A tissue recombination-xenografting model was utilized to compare remodeling of human prostatic tissues in vivo. A series of tissue recombinants, made by mixing different ratios of human prostatic epithelial cells and inductive rat urogenital sinus mesenchyme, were grafted to the renal capsule of severe combined immunodeficient mice. Both cell lines were able to regenerate benign secretory ductal-acinar architecture in vivo, containing intact basal and luminal epithelial layers confirmed by the expression of appropriate CK profiles. Prostate-specific antigen, 15-lipoxygenase-2, androgen receptor, and NKX3.1 proteins were appropriately expressed in the regenerated epithelia. Regeneration of benign prostatic glandular structures could be achieved using as few as 10 NHPrE1 cells, whereas 200,000 BHPrE1 cells were required to achieve prostatic architecture. This suggests a greater proportion of progenitor/stem cells in NHPrE1 than in BHPrE1. These cell lines provide important data on progenitor and intermediate cell phenotypes and represent significant new tools for the elucidation of molecular mechanisms of human prostatic regeneration, pathogenesis, and carcinogenesis

Preserved Morphology and Physiology of Excitatory Synapses in Profilin1-Deficient Mice

Profilins are important regulators of actin dynamics and have been implicated in activity-dependent morphological changes of dendritic spines and synaptic plasticity. Recently, defective presynaptic excitability and neurotransmitter release of glutamatergic synapses were described for profilin2-deficient mice. Both dendritic spine morphology and synaptic plasticity were fully preserved in these mutants, bringing forward the hypothesis that profilin1 is mainly involved in postsynaptic mechanisms, complementary to the presynaptic role of profilin2. To test the hypothesis and to elucidate the synaptic function of profilin1, we here specifically deleted profilin1 in neurons of the adult forebrain by using conditional knockout mice on a CaMKII-cre-expressing background. Analysis of Golgi-stained hippocampal pyramidal cells and electron micrographs from the CA1 stratum radiatum revealed normal synapse density, spine morphology, and synapse ultrastructure in the absence of profilin1. Moreover, electrophysiological recordings showed that basal synaptic transmission, presynaptic physiology, as well as postsynaptic plasticity were unchanged in profilin1 mutants. Hence, loss of profilin1 had no adverse effects on the morphology and function of excitatory synapses. Our data are in agreement with two different scenarios: i) profilins are not relevant for actin regulation in postsynaptic structures, activity-dependent morphological changes of dendritic spines, and synaptic plasticity or ii) profilin1 and profilin2 have overlapping functions particularly in the postsynaptic compartment. Future analysis of double mutant mice will ultimately unravel whether profilins are relevant for dendritic spine morphology and synaptic plasticity

Use of PB-Cre4 Mice for Mosaic Gene Deletion

<div><p>Transgene expression from short promoters in transgenic animals can lead to unwanted transgene expression patterns, often as a byproduct of random integration of the expression cassette into the host genome. Here I demonstrate that the often used PB-Cre4 line (also referred to as “Probasin-Cre”), although expressing exclusively in the male prostate epithelium when transmitted through male mice, can lead to recombination of loxP-flanked alleles in a large variety of tissues when transmitted through female mice. This aberrant Cre activity due to Cre expression in the oocytes leads to different outcomes for maternally or paternally transmitted loxP-flanked alleles: Maternally inherited loxP-flanked alleles undergo recombination very efficiently, making female PB-Cre4 mice an efficient monoallelic “Cre deleter line”. However, paternally inherited loxP-flanked alleles are inefficiently recombined by maternal PB-Cre4, giving rise to mosaic expression patterns in the offspring. This mosaic recombination is difficult to detect with standard genotyping approaches of many mouse lines and should therefore caution researchers using PB-Cre4 to use additional approaches to exclude the presence of recombined alleles. However, mosaic recombination should also be useful in transgenic “knockout” approaches for mosaic gene deletion experiments.</p> </div

Different degrees of mosaicism in transgenic livers.

<p>The numbers above the images indicate the percentage of EGFP positive cells (top row) and the numbers of animals (out of n = 15 animals with any degree of mosaicisim, i.e. EGFP fluorescence) within the respective group (row below). Examples for tissues of the respective groups are shown below the numbers. Scale bars, 100 µm.</p

Maternal PB-Cre4 leads to mosaic deletion of paternally transmitted loxP-flanked alleles.

<p>A, breeding scheme. Male mice carrying a <i>Pten</i> flox and a <i>Pten</i>+allele were bred to female mice carrying the Cre transgene. In the absence of aberrant Cre activity, an equal distribution of <i>Pten</i> +/+ and <i>Pten</i> flox/+ offspring is expected. B, results from the breeding: Maternal PB-Cre4 and paternal <i>Pten</i> flox/+ lead to offspring in which the floxed allele is still present in roughly the expected frequency in tail biopsies, but a delta allele can be detected as well, indicating a mosaic deletion.</p

Maternal PB-Cre4 leads to efficient recombination of maternally transmitted loxP-flanked alleles.

<p>A, breeding scheme: male wildtype mice with two <i>Pten</i> wildtype alleles (marked “+”) were bred to female mice carrying the ARR2PB-Cre transgene (PB-Cre4 line) and a loxP-flanked <i>Pten</i> allele (“flox”) as well as a wildtype allele. The expected outcome (depicted here) concerning the <i>Pten</i> status of the offspring would be equal numbers of <i>Pten</i> +/+ (one wildtype allele each from the father and the mother animal) and <i>Pten</i> flox/+ (one wildtype allele from the father, one floxed allele from the mother) animals. B, Scheme of the <i>Pten</i> allele and primers (red arrows) used for their detection. A PCR using primers 2 and 3 yields a band for the+allele and a larger band for the floxed allele. In case of recombination, the sequence between the two loxP sites (blue triangles) is deleted, yielding a delta allele which can be detected by a PCR of primers 1 and 3. C, examples of PCRs with primers 2/3 (left) distinguishing between +/+ and flox/+ genotypes, and PCRs with primers 1/3, detecting delta alleles (right). For the left image, tail biopsies from offspring mice where one parental animal carried a <i>Pten</i> flox allele and none carried the PB-Cre4 transgene were used, while for the right image, tail biopsies from offspring mice of the breeding indicated in A were used. D, In the offspring of the breeding in A, no flox/+ combination was detected, but delta/+ was seen in nearly equal numbers as +/+.</p

Cloning of the PB-Cre4 integration site.

<p>A, scheme of the genome walking procedure used. Genomic DNA was cut using blunt-end cutters, followed by ligation of adaptor oligonucleotides. Nested PCR was done using primers within the adaptors as well as gene-specific primers. The gene-specific primers in the ARR2PB sequence primed twice because of the two identical androgen-responsive regions. The corresponding results for different blunt-end cutters are shown in B. Bands obtained for SmaI and PvuII were sequenced. The sequence for the SmaI band (arrow) is shown in C. D, PCR primers were designed within the putative flanking (upstream) sequence and within the ARR2PB (again priming twice), yielding a double band as expected. Bands were sequenced again, confirming the integration site.</p

Maternal PB-Cre4 leads to mosaicism of paternally transmitted alleles which can be visualized in reporter animals (part II).

<p>Organs from the same animal as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053501#pone-0053501-g003" target="_blank">Figure 3</a> are shown to demonstrate mosaicism in a variety of tissues. Controls were used as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053501#pone-0053501-g003" target="_blank">Figure 3</a>. Scale bars, 100 µm.</p