Quantitative impedimetric NPY-receptor activation monitoring and signal pathway profiling in living cells.

Label-free and non-invasive monitoring of receptor activation and identification of the involved signal pathways in living cells is an ongoing analytic challenge and a great opportunity for biosensoric systems. In this context, we developed an impedance spectroscopy-based system for the activation monitoring of NPY-receptors in living cells. Using an optimized interdigital electrode array for sensitive detection of cellular alterations, we were able for the first time to quantitatively detect the NPY-receptor activation directly without a secondary or enhancer reaction like cAMP-stimulation by forskolin. More strikingly, we could show that the impedimetric based NPY-receptor activation monitoring is not restricted to the Y1-receptor but also possible for the Y2- and Y5-receptor. Furthermore, we could monitor the NPY-receptor activation in different cell lines that natively express NPY-receptors and proof the specificity of the observed impedimetric effect by agonist/antagonist studies in recombinant NPY-receptor expressing cell lines. To clarify the nature of the observed impedimetric effect we performed an equivalent circuit analysis as well as analyzed the role of cell morphology and receptor internalization. Finally, an antagonist based extensive molecular signal pathway analysis revealed small alterations of the actin cytoskeleton as well as the inhibition of at least L-type calcium channels as major reasons for the observed NPY-induced impedance increase. Taken together, our novel impedance spectroscopy based NPY-receptor activation monitoring system offers the opportunity to identify signal pathways as well as for novel versatile agonist/antagonist screening systems for identification of novel therapeutics in the field of obesity and cancer.

PCAF-dependent epigenetic changes promote axonal regeneration in the central nervous system.

Axonal regenerative failure is a major cause of neurological impairment following central nervous system (CNS) but not peripheral nervous system (PNS) injury. Notably, PNS injury triggers a coordinated regenerative gene expression programme. However, the molecular link between retrograde signalling and the regulation of this gene expression programme that leads to the differential regenerative capacity remains elusive. Here we show through systematic epigenetic studies that the histone acetyltransferase p300/CBP-associated factor (PCAF) promotes acetylation of histone 3 Lys 9 at the promoters of established key regeneration-associated genes following a peripheral but not a central axonal injury. Furthermore, we find that extracellular signal-regulated kinase (ERK)-mediated retrograde signalling is required for PCAF-dependent regenerative gene reprogramming. Finally, PCAF is necessary for conditioning-dependent axonal regeneration and also singularly promotes regeneration after spinal cord injury. Thus, we find a specific epigenetic mechanism that regulates axonal regeneration of CNS axons, suggesting novel targets for clinical application.

DNA methylation temporal profiling following peripheral versus central nervous system axotomy.

The regulatory mechanisms responsible for the gene expression pattern associated with axotomy-dependent signaling affecting the neuronal phenotype, including the axonal regenerative program, remain unclear. To further this understanding, we recently performed DNA methylation temporal profiling in lumbar dorsal root ganglia (DRG) after axotomy of the central spinal (non-regenerating) and of the peripheral sciatic nerve (regenerating) axonal branches. DNA methylation microarrays for mouse gene promoters and CpG islands (Roche/NimbleGen) were employed after immunoprecipitation of 5-methylcytosine-DNA. Here we provide a detailed data descriptor of this DNA methylation dataset, which allows in depth evaluation of the experimental design, assessment of data reproducibility and a full interactive operator-based systematic data analysis. In fact, we offer a methylation 'hit' scoring map of the whole microarray data in a workable spreadsheet that allows data sorting by genes, conditions or hits of interests that is ready for functional gene annotation and classification. This dataset allows investigators bioinformatic comparison to other epigenetic and gene expression datasets and further experimental characterization of the role of DNA methylation in axotomy-dependent pathways.

Epigenetic regulation of axon outgrowth and regeneration in CNS injury: the first steps forward.

Inadequate axonal sprouting and lack of regeneration limit functional recovery following neurologic injury, such as stroke, brain, and traumatic spinal cord injury. Recently, the enhancement of the neuronal regenerative program has led to promising improvements in axonal sprouting and regeneration in animal models of axonal injury. However, precise knowledge of the essential molecular determinants of this regenerative program remains elusive, thus limiting the choice of fully effective therapeutic strategies. Given that molecular regulation of axonal outgrowth and regeneration requires carefully orchestrated waves of gene expression, both temporally and spatially, epigenetic changes may be an ideal regulatory mechanism to address this unique need. While recent evidence suggests that epigenetic modifications could contribute to the regulation of axonal outgrowth and regeneration following axonal injury in models of stroke, and spinal cord and optic nerve injury, a number of unanswered questions remain. Such questions require systematic investigation of the epigenetic landscape between regenerative and non-regenerative conditions for the potential translation of this knowledge into regenerative strategies in human spinal and brain injury, as well as stroke.

A novel cross-talk between endothelin and ErbB receptors controlling glutamate transporter expression in astrocytes.

The endothelin and epidermal growth factor (EGF) systems are central to the control of reactive brain processes and are thought to partly exert these tasks by endothelin-induced transactivation of the epidermal growth factor receptor (EGFR) Here we show that beyond EGFR transactivation, endothelins prevent the ligand-induced internalization of the EGFR. We unravel that endothelins abrogate internalization of the EGFR by either promoting the formation of "internalization-deficient" EGFR/ErB2-heterodimers or by activating c-Abl kinase, a negative regulator of EGFR internalization. We further provide evidence that this cross-talk is operational in the control of astrocytic glutamate transport. Specifically, we establish that the inhibitory effects exerted by endothelins on basal as well as EGF-induced expression of the major astroglial glutamate transporter subtype, glutamate transporter 1, are a direct consequence of the endothelin-dependent retention of the EGFR at the cell surface. Together our findings unravel a previously unknown cross-talk between endothelin and epidermal growth factor receptors, which may have implications for a variety of pathological conditions.

The serum and glucocorticoid inducible kinases SGK1-3 stimulate the neutral amino acid transporter SLC6A19.

The neutral amino acid transporter SLC6A19 (B(0)AT1) plays a decisive role in transport of neutral amino acids in the kidney and intestine. Recently, mutations in SLC6A19 were identified that result in severe neutral aminoaciduria known as Hartnup disorder. SLC6A19 expression and function is controlled by the brush-border angiotensin-converting enzyme 2 (ACE2). Beyond that the mechanisms regulating SLC6A19 function are unknown. The SLC6A19 sequence contains a conserved putative phosphorylation site for the serum and glucocorticoid inducible kinase isoforms SGK1-3, kinases known to regulate a variety of channels and transporters. The present study explored the role of SGK1-3 in the regulation of SLC6A19. As shown by two-electrode voltage clamp in the Xenopus oocyte expression system, leucine-induced currents in SLC6A19 expressing oocytes were activated by the protein kinases SGK1-3. The putative phosphorylation site on the transporter is not essential for SLC6A19 regulation by the kinases. As determined by quantitative immunoassay and electrophysiology, the kinases increase SLC6A19 currents by increasing the cell surface expression of the protein without altering the affinity of the carrier. Following inhibition of carrier insertion into the cell membrane by treatment with brefeldin A (BFA), the leucine-induced current declined significantly slower in Xenopus oocytes expressing SLC6A19 together with SGK1 than in oocytes expressing SLC6A19 alone, a finding pointing to SGK-mediated transporter stabilization in the plasma membrane. Coexpression of ACE2 markedly increased leucine-induced currents in SLC6A19 expressing oocytes that were further enhanced by SGK1-3 kinases. In conclusion, SGK isoforms are novel potent stimulators of SLC6A19 and may thus participate in the regulation of neutral amino acid transport in vivo.

Regulation of the glutamate transporter EAAT4 by PIKfyve.

The excitatory amino-acid transporter EAAT4 (SLC1A6), a Na(+),glutamate cotransporter expressed mainly in Purkinje cells, serves to clear glutamate from the synaptic cleft. EAAT4 activity is stimulated by the serum and glucocorticoid inducible kinase SGK1. SGK1-dependent regulation of the Na(+),glucose transporter SGLT1 (SLC5A1) and the creatine transporter CreaT (SLC6A8) has recently been shown to involve the mammalian phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3). The present experiments thus explored whether SGK1-dependent EAAT4-regulation similarly involves PIKfyve. In Xenopus oocytes expressing EAAT4, but not in water injected oocytes, glutamate induced a current which was significantly enhanced by coexpression of PIKfyve and SGK1. The glutamate induced current in Xenopus oocytes coexpressing EAAT4 and both, PIKfyve and SGK1, was significantly larger than the current in Xenopus oocytes expressing EAAT4 together with either kinase alone. Coexpression of the inactive SGK1 mutant (K127N)SGK1 did not significantly alter glutamate induced current in EAAT4-expressing Xenopus oocytes and abolished the stimulation of glutamate induced current by coexpression of PIKfyve. The stimulating effect of PIKfyve was abrogated by replacement of the serine with alanine in the SGK consensus sequence ((S318A)PIKfyve). Furthermore, coexpression of (S318A)PIKfyve significantly blunted the stimulating effect of SGK1 on EAAT4 activity. The observations disclose that PIKfyve indeed participates in the regulation of EAAT4.

Regulation of the glutamate transporter EAAT2 by PIKfyve.

The Na(+),glutamate cotransporter EAAT2 is expressed in astrocytes and clears glutamate from the synaptic cleft. EAAT2 dependent currrent is stimulated by the serum and glucocorticoid inducible kinase SGK1. Phosphorylation targets of SGK1 include the human phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3). Nothing is known, however, on the role of PIKfyve in the regulation of EAAT2. The present experiments thus explored, whether PIKfyve expression modifies EAAT2 dependent currrent and protein abundance in the cell membrane. In Xenopus oocytes expressing EAAT2 but not in water injected oocytes application of glutamate (2 mM) induced an inward current (I(glu)). Coexpression of either, SGK1 or PIKfyve, significantly enhanced I(glu) in EAAT2 expressing oocytes. I(glu) was significantly higher in Xenopus oocytes coexpressing EAAT2, SGK1 and PIKfyve than in Xenopus oocytes expressing EAAT2 and either, SGK1 or PIKfyve, alone. Additional coexpression of the inactive mutant of the serum and glucocorticoid inducible kinase (K127N)SGK1 did not significantly alter I(glu) in EAAT2 expressing oocytes and significantly decreased I(glu) in oocytes coexpressing EAAT2 together with PIKfyve. The stimulating effect of PIKfyve on I(glu) was abrogated by replacement of the serine in the SGK consensus sequence by alanine ((S318A)PIKfyve). Furthermore, additional coexpression of (S318A)PIKfyve virtually abolished I(glu) in Xenopus oocytes coexpressing SGK1 and EAAT2. Confocal microscopy reveals that PIKfyve enhances the EAAT2 protein abundance in the cell membrane. The observations disclose that PIKfyve indeed participates in the regulation of EAAT2.

Regulation of the Na(+)-coupled glutamate transporter EAAT3 by PIKfyve.

The Na(+), glutamate cotransporter EAAT3 is expressed in a wide variety of tissues. It accomplishes transepithelial transport and the cellular uptake of acidic amino acids. Regulation of EAAT3 activity involves a signaling cascade including the phosphatidylinositol-3 (PI3)-kinase, the phosphoinositide dependent kinase PDK1, and the serum and glucocorticoid inducible kinase SGK1. Targets of SGK1include the mammalian phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3). The present experiments explored whether PIKfyve participates in the regulation of EAAT3 activity. To this end,EAAT3 was expressed in Xenopus oocytes with or without SGK1 and/or PIKfyve and glutamate-induced current (I(glu)) determined by dual electrode voltage clamp. In Xenopus oocytes expressing EAAT3 but not in water injected oocytes glutamate induced an inwardly directed I(glu). Coexpression of either, SGK1 orPIKfyve, significantly enhanced I(glu) in EAAT3 expressing oocytes. The increased I(glu) was paralleled by increased EAAT3 protein abundance in the oocyte cell membrane. I(glu) and EAAT3 protein abundance were significantly larger in oocytes coexpressing EAAT3, SGK1 and PIKfyve than in oocytes expressingEAAT3 and either, SGK1 or PIKfyve, alone. Coexpression of the inactive SGK1 mutant (K127N)SGK1 did not significantly alter I(glu) in EAAT3 expressing oocytes and completely reversed the stimulating effect ofPIKfyve coexpression on I(glu). The stimulating effect of PIKfyve on I(glu) was abolished by replacement of the serine by alanine in the SGK consensus sequence ((S318A)PIKfyve). Moreover, additional coexpression of(S318A)PIKfyve significantly blunted I(glu) in Xenopus oocytes coexpressing SGK1 and EAAT3. The observations demonstrate that PIKfyve participates in EAAT3 regulation likely downstream of SGK1.

NFAT-3 is a transcriptional repressor of the growth-associated protein 43 during neuronal maturation.

Transcription is essential for neurite and axon outgrowth during development. Recent work points to the involvement of nuclear factor of activated T cells (NFAT) in the regulation of genes important for axon growth and guidance. However, NFAT has not been reported to directly control the transcription of axon outgrowth-related genes. To identify transcriptional targets, we performed an in silico promoter analysis and found a putative NFAT site within the GAP-43 promoter. Using in vitro and in vivo experiments, we demonstrated that NFAT-3 regulates GAP-43, but unexpectedly, does not promote but represses the expression of GAP-43 in neurons and in the developing brain. Specifically, in neuron-like PC-12 cells and in cultured cortical neurons, the overexpression of NFAT-3 represses GAP-43 activation mediated by neurotrophin signaling. Using chromatin immunoprecipitation assays, we also show that prior to neurotrophin activation, endogenous NFAT-3 occupies the GAP-43 promoter in PC-12 cells, in cultured neurons, and in the mouse brain. Finally, we observe that NFAT-3 is required to repress the physiological expression of GAP-43 and other pro-axon outgrowth genes in specific developmental windows in the mouse brain. Taken together, our data reveal an unexpected role for NFAT-3 as a direct transcriptional repressor of GAP-43 expression and suggest a more general role for NFAT-3 in the control of the neuronal outgrowth program.

The C-terminal PDZ-binding motif in the Kv1.5 potassium channel governs its modulation by the Na+/H+ exchanger regulatory factor 2.

Kv1.5 belongs to the family of voltage-gated potassium (Kv) channels and contains a N- and a C-terminal PDZ-binding motif that might be recognized by PDZ domains on the scaffold proteins NHERF1 and NHERF2. Expression studies in Xenopus oocytes demonstrated that NHERF1 and NHERF2 activate Kv1.5, an effect requiring the C-terminal PDZ-binding motif on Kv1.5. NHERF2 enhances Kv1.5 activity and cell surface expression as determined by electrophysiology and immunoassays. NHERF2 elevates Kv1.5 abundance at the plasma membrane by decreasing channel internalization as proven by Brefeldin A experiments. Kv1.5 is stimulated by the serum and glucocorticoid inducible kinase SGK1, a kinase known to interact with the second PDZ domain of NHERF2. This study aims to identify if SGK1 and NHERF2 synergize to increase Kv1.5 currents. Expression of NHERF2 potentiated SGK1-mediated Kv1.5 activation, which was significantly attenuated by deletion of the second PDZ domain in NHERF2. Specificity of observed effects was verified by evaluating the influence of NHERFs on Kv1.3, a known SGK1 target that contains an internal PDZ binding motif. In summary, our results suggest that NHERFs might participate in the regulation of electrical excitability in part by controlling Kv1.5 surface abundance and by clustering signal transduction molecules to the channel.

Modulation of the voltage-gated potassium channel Kv1.5 by the SGK1 protein kinase involves inhibition of channel ubiquitination.

The serum and glucocorticoid inducible kinase SGK1 is involved in dexamethasone-induced inhibition of insulin secretion by increasing voltage-gated potassium channel (Kv) activity. SGK1 upregulates the Kv1.5 channel but the precise mechanism underlying the SGK1 dependent regulation of Kv1.5 has not been defined yet. The present study explored the signal transduction processes involved. Expression studies in Xenopus oocytes revealed that SGK1 promotes channel activity by interfering with the Nedd4-2 ubiquitination pathway, irrespective of the presence of putative SGK1 phosphorylation sites on Kv1.5. Expression of the ubiquitin ligase Nedd4-2 declined Kv1.5 currents by ubiquitinating and thereby reducing Kv1.5 plasma membrane expression. Increasing concentrations of SGK1 gradually compensated the inhibiting effect of Nedd4-2 on Kv1.5. Enhanced Kv1.5 surface abundance by SGK1 reflects decreased channel internalization as indicated by Brefeldin A experiments. In conclusion, Kv1.5 upregulation by SGK1 involves inhibition of channel ubiquitination by Nedd4-2 that leads to Kv1.5 stabilization in the plasma membrane. Our results suggest that the kinase might participate in the regulation of insulin secretion in part by controlling Kv1.5 surface abundance.

The peptide transporter PEPT2 is targeted by the protein kinase SGK1 and the scaffold protein NHERF2.

PEPT1 and PEPT2 are members of the family of proton-dependent oligopeptide transporters that mediate electrogenic uphill transport of small peptides and peptidomimetics into a variety of cells. Kinetic properties and substrate recognition sites of those transporters have been well defined previously. Little is known, however, about regulation of those transporters. Both PEPT isoforms contain putative phosphorylation sites for the serum and glucocorticoid inducible kinase SGK1 and a C-terminal PDZ binding motif that might be recognized by PDZ domains of the Na(+)/H(+) exchanger regulatory factors NHERF1 and NHERF2. Thus, the present study attempted to clarify the role of SGK1 and NHERFs in the modulation of PEPT isoforms. Expression studies in Xenopus oocytes with subsequent electrophysiology and immunoassays revealed that SGK1 and NHERF2, but not the NHERF1 isoform specifically enhance PEPT2 function and surface abundance. The kinase is effective through phosphorylation of (185)Ser within the SGK1 consensus site, since disruption of this site prevented transporter modulation by the kinase. NHERF2 failed to regulate the C-terminal deletion mutant (PEPT2DeltaC) indicating that the C-terminal PDZ-binding motif in PEPT2 governs transport modulation by NHERF2. Coexpression of NHE3 stimulates PEPT2 activity to a similar extent as coexpression of NHERF2. Dynasore experiments demonstrated that SGK1 and NHERF2 activate PEPT2 by stabilizing the transporter at the cell surface. In conclusion, the present results reveal two novel PEPT2 posttranslational modulators, SGK1 and NHERF2, which might regulate transport of oligopeptides and peptidomimetic drugs.

Oval cell proliferation in p16INK4a expressing mouse liver is triggered by chronic growth stimuli.

Terminal differentiation requires molecules also involved in aging such as the cell cycle inhibitor p16(INK4a). Like other organs, the adult liver represents a quiescent organ with terminal differentiated cells, hepatocytes and cholangiocytes. These cells retain the ability to proliferate in response to liver injury or reduction of liver mass. However, under conditions which prevent mitotic activation of hepatocytes, regeneration can occur instead from facultative hepatic stem cells.For therapeutic application a non-toxic activation of this stem cell compartment is required. We have established transgenic mice with conditional overexpression of the cell cycle inhibitor p16(INK4a) in hepatocytes and have provoked and examined oval cell activation in adult liver in response to a range of proliferative stimuli. We could show that the liver specific expression of p16(INK4a) leads to a faster differentiation of hepatocytes and an activation of oval cells already in postnatal mice without negative consequences on liver function.