Reggies/flotillins interact with Rab11a and SNX4 at the tubulovesicular recycling compartment and function in transferrin receptor and E-cadherin trafficking.

The lipid raft proteins reggie-1 and -2 (flotillins) are implicated in membrane protein trafficking but exactly how has been elusive. We find that reggie-1 and -2 associate with the Rab11a, SNX4, and EHD1-decorated tubulovesicular recycling compartment in HeLa cells and that reggie-1 directly interacts with Rab11a and SNX4. Short hairpin RNA-mediated down-regulation of reggie-1 (and -2) in HeLa cells reduces association of Rab11a with tubular structures and impairs recycling of the transferrin-transferrin receptor (TfR) complex to the plasma membrane. Overexpression of constitutively active Rab11a rescues TfR recycling in reggie-deficient HeLa cells. Similarly, in a Ca(2+) switch assay in reggie-depleted A431 cells, internalized E-cadherin is not efficiently recycled to the plasma membrane upon Ca(2+) repletion. E-cadherin recycling is rescued, however, by overexpression of constitutively active Rab11a or SNX4 in reggie-deficient A431 cells. This suggests that the function of reggie-1 in sorting and recycling occurs in association with Rab11a and SNX4. Of interest, impaired recycling in reggie-deficient cells leads to de novo E-cadherin biosynthesis and cell contact reformation, showing that cells have ways to compensate the loss of reggies. Together our results identify reggie-1 as a regulator of the Rab11a/SNX4-controlled sorting and recycling pathway, which is, like reggies, evolutionarily conserved.

Stage-specific integration of maternal and embryonic peroxisome proliferator-activated receptor delta signaling is critical to pregnancy success.

Successful pregnancy depends on well coordinated developmental events involving both maternal and embryonic components. Although a host of signaling pathways participate in implantation, decidualization, and placentation, whether there is a common molecular link that coordinates these processes remains unknown. By exploiting genetic, molecular, pharmacological, and physiological approaches, we show here that the nuclear transcription factor peroxisome proliferator-activated receptor (PPAR) delta plays a central role at various stages of pregnancy, whereas maternal PPARdelta is critical to implantation and decidualization, and embryonic PPARdelta is vital for placentation. Using trophoblast stem cells, we further elucidate that a reciprocal relationship between PPARdelta-AKT and leukemia inhibitory factor-STAT3 signaling pathways serves as a cell lineage sensor to direct trophoblast cell fates during placentation. This novel finding of stage-specific integration of maternal and embryonic PPARdelta signaling provides evidence that PPARdelta is a molecular link that coordinates implantation, decidualization, and placentation crucial to pregnancy success. This study is clinically relevant because deferral of on time implantation leads to spontaneous pregnancy loss, and defective trophoblast invasion is one cause of preeclampsia in humans.

Protracted treatment with MDMA induces heteromeric nicotinic receptor up-regulation in rat brain: an autoradiography study.

Previous studies indicate that 3,4-methylenedioxy-methamphetamine (MDMA, ecstasy) can induce heteromeric nicotinic acetylcholine receptor (nAChR, mainly of α4β2 subtype) up-regulation. In this study we treated Sprague-Dawley rats twice-daily for 10 days with either saline or MDMA (7 mg/kg) and killed them on day 11 to perform [125I]epibatidine binding autoradiograms on serial coronal slices. Results showed significant increases in nAChR density in the substantia nigra, ventral tegmental area, nucleus accumbens, olfactory tubercle, anterior caudate-putamen, somatosensory cortex, motor cortex, auditory cortex, retrosplenial cortex, laterodorsal thalamus nuclei, amygdala, postsubiculum and pontine nuclei. These increases ranged from 3% (retrosplenial cortex) to 30 and 33% (amygdala and substantia nigra). No increased α4 subunit immunoreactivity was found in up-regulated areas compared with saline-treated rats, suggesting a post-translational mechanism as occurs with nicotine. The percentage of up-regulation correlated positively with the density of serotonin transporters, according to the serotonergic profile of MDMA. The heteromeric nAChR increase in concrete areas could account, at least in part, for the reinforcing, sensitizing and psychiatric disorders observed after long-term treatment with MDMA.

Protracted treatment with MDMA induces heteromeric nicotinic receptor up-regulation in rat brain: an autoradiography study.

Previous studies indicate that 3,4-methylenedioxy-methamphetamine (MDMA, ecstasy) can induce heteromeric nicotinic acetylcholine receptor (nAChR, mainly of α4β2 subtype) up-regulation. In this study we treated Sprague-Dawley rats twice-daily for 10 days with either saline or MDMA (7 mg/kg) and killed them on day 11 to perform [125I]epibatidine binding autoradiograms on serial coronal slices. Results showed significant increases in nAChR density in the substantia nigra, ventral tegmental area, nucleus accumbens, olfactory tubercle, anterior caudate-putamen, somatosensory cortex, motor cortex, auditory cortex, retrosplenial cortex, laterodorsal thalamus nuclei, amygdala, postsubiculum and pontine nuclei. These increases ranged from 3% (retrosplenial cortex) to 30 and 33% (amygdala and substantia nigra). No increased α4 subunit immunoreactivity was found in up-regulated areas compared with saline-treated rats, suggesting a post-translational mechanism as occurs with nicotine. The percentage of up-regulation correlated positively with the density of serotonin transporters, according to the serotonergic profile of MDMA. The heteromeric nAChR increase in concrete areas could account, at least in part, for the reinforcing, sensitizing and psychiatric disorders observed after long-term treatment with MDMA.

Protracted treatment with MDMA induces heteromeric nicotinic receptor up-regulation in rat brain: an autoradiography study.

Previous studies indicate that 3,4-methylenedioxy-methamphetamine (MDMA, ecstasy) can induce heteromeric nicotinic acetylcholine receptor (nAChR, mainly of α4β2 subtype) up-regulation. In this study we treated Sprague-Dawley rats twice-daily for 10 days with either saline or MDMA (7 mg/kg) and killed them on day 11 to perform [125I]epibatidine binding autoradiograms on serial coronal slices. Results showed significant increases in nAChR density in the substantia nigra, ventral tegmental area, nucleus accumbens, olfactory tubercle, anterior caudate-putamen, somatosensory cortex, motor cortex, auditory cortex, retrosplenial cortex, laterodorsal thalamus nuclei, amygdala, postsubiculum and pontine nuclei. These increases ranged from 3% (retrosplenial cortex) to 30 and 33% (amygdala and substantia nigra). No increased α4 subunit immunoreactivity was found in up-regulated areas compared with saline-treated rats, suggesting a post-translational mechanism as occurs with nicotine. The percentage of up-regulation correlated positively with the density of serotonin transporters, according to the serotonergic profile of MDMA. The heteromeric nAChR increase in concrete areas could account, at least in part, for the reinforcing, sensitizing and psychiatric disorders observed after long-term treatment with MDMA.

Coregulator Control of Androgen Receptor Action by a Novel Nuclear Receptor-Binding Motif

The androgen receptor (AR) is a ligand-activated transcription factor that is essential for prostate cancer development. It is activated by androgens through its ligand-binding domain (LBD), which consists predominantly of 11 α-helices. Upon ligand binding, the last helix is reorganized to an agonist conformation termed activator function-2 (AF-2) for coactivator binding. Several coactivators bind to the AF-2 pocket through conserved LXXLL or FXXLF sequences to enhance the activity of the receptor. Recently, a small compound-binding surface adjacent to AF-2 has been identified as an allosteric modulator of the AF-2 activity and is termed binding function-3 (BF-3). However, the role of BF-3 in vivo is currently unknown, and little is understood about what proteins can bind to it. Here we demonstrate that a duplicated GARRPR motif at the N terminus of the cochaperone Bag-1L functions through the BF-3 pocket. These findings are supported by the fact that a selective BF-3 inhibitor or mutations within the BF-3 pocket abolish the interaction between the GARRPR motif(s) and the BF-3. Conversely, amino acid exchanges in the two GARRPR motifs of Bag-1L can impair the interaction between Bag-1L and AR without altering the ability of Bag-1L to bind to chromatin. Furthermore, the mutant Bag-1L increases androgen-dependent activation of a subset of AR targets in a genome-wide transcriptome analysis, demonstrating a repressive function of the GARRPR/BF-3 interaction. We have therefore identified GARRPR as a novel BF-3 regulatory sequence important for fine-tuning the activity of the AR.

Peroxisome proliferator-activated receptor beta/delta activation inhibits hypertrophy in neonatal rat cardiomyocytes.

OBJECTIVE: Peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) is the predominant PPAR subtype in cardiac cells and plays a prominent role in the regulation of cardiac lipid metabolism. However, the role of PPARbeta/delta activators in cardiac hypertrophy is not yet known. METHODS AND RESULTS: In cultured neonatal rat cardiomyocytes, the selective PPARbeta/delta activator L-165041 (10 micromol/L) inhibited phenylephrine (PE)-induced protein synthesis ([(3)H]leucine uptake), induction of the fetal-type gene atrial natriuretic factor (ANF) and cardiac myocyte size. Induction of cardiac hypertrophy by PE stimulation also led to a reduction in the transcript levels of both muscle-type carnitine palmitoyltransferase (50%, P<0.05) and pyruvatedehydrogenase kinase 4 (30%, P<0.05), and these changes were reversed in the presence of the PPARbeta/delta agonist L-165041. Stimulation of neonatal rat cardiomyocytes with PE and embryonic rat heart-derived H9c2 cells with lipopolysaccharide (LPS) enhanced the expression of the nuclear factor (NF)-kappaB-target gene monocyte chemoattractant protein 1 (MCP-1). The induction of MCP-1 was reduced in the presence of L-165041, suggesting that this compound prevented NF-kappaB activation. Electrophoretic mobility shift assay (EMSA) revealed that L-165041 significantly decreased LPS-stimulated NF-kappaB binding activity in H9c2 myotubes. Finally, coimmunoprecipitation studies showed that L-165041 strongly enhanced the physical interaction between PPARbeta/delta and the p65 subunit of NF-kappaB, suggesting that increased association between these two proteins is the mechanism responsible for antagonizing NF-kappaB activation by PPARbeta/delta activators. CONCLUSION: These results suggest that PPARbeta/delta activation inhibits PE-induced cardiac hypertrophy and LPS-induced NF-kappaB activation.

Phorbol 12-myristate 13-acetate induces surface expression of T3 on human immature T cell lines with and without concomitant expression of the T cell antigen receptor complex.

The T3 complex is known to be expressed on the cell surface of mature T cells together with either the alpha-beta heterodimeric T cell receptor (TCR) or the TCR gamma protein. In a number of immature T cell malignancies, however, T3 has been described exclusively in the cytoplasm. We have investigated five such T cell lines with cytoplasmic T3 and could demonstrate by biosynthetic labeling the presence of the alpha and beta chains of the TCR in the cytoplasm of two of them, CEM and Ichikawa. No surface TCR alpha-beta protein could be detected by staining with the WT31 antibody. These observations, therefore, argue against the concept that expression of the TCR alpha chain controls the surface expression of the T3/TCR complex. Interestingly, phorbol 12-myristate 13-acetate (PMA) induced cell surface expression of T3 protein in these two cell lines only. Moreover, on surface-iodinated CEM cells no association of T3 and TCR molecules could be demonstrated after treatment with PMA, and expression of TCR alpha and beta chains was limited to the cytoplasm. In Ichikawa cells, however, PMA induced surface expression of a mature T3/TCR complex. Our findings indicate that separate regulatory mechanisms may exist for the surface expression of the T3 proteins and for the assembly of the T3/TCR complex.

The Wnt receptor FZD1 mediates chemoresistance in neuroblastoma through activation of the Wnt/beta-catenin pathway.

The development of chemoresistance represents a major obstacle in the successful treatment of cancers such as neuroblastoma (NB), a particularly aggressive childhood solid tumour. The mechanisms underlying the chemoresistant phenotype in NB were addressed by gene expression profiling of two doxorubicin (DoxR)-resistant vs sensitive parental cell lines. Not surprisingly, the MDR1 gene was included in the identified upregulated genes, although the highest overexpressed transcript in both cell lines was the frizzled-1 Wnt receptor (FZD1) gene, an essential component of the Wnt/beta-catenin pathway. FZD1 upregulation in resistant variants was shown to mediate sustained activation of the Wnt/beta-catenin pathway as revealed by nuclear beta-catenin translocation and target genes transactivation. Interestingly, specific micro-adapted short hairpin RNA (shRNAmir)-mediated FZD1 silencing induced parallel strong decrease in the expression of MDR1, another beta-catenin target gene, revealing a complex, Wnt/beta-catenin-mediated implication of FZD1 in chemoresistance. The significant restoration of drug sensitivity in FZD1-silenced cells confirmed the FZD1-associated chemoresistance. RNA samples from 21 patient tumours (diagnosis and postchemotherapy), showed a highly significant FZD1 and/or MDR1 overexpression after treatment, underlining a role for FZD1-mediated Wnt/beta-catenin pathway in clinical chemoresistance. Our data represent the first implication of the Wnt/beta-catenin pathway in NB chemoresistance and identify potential new targets to treat aggressive and resistant NB.