Multiple Roles of Integrin-Linked Kinase in Epidermal Development, Maturation and Pigmentation Revealed by Molecular Profiling

Integrin-linked kinase (ILK) is an important scaffold protein that mediates a variety of cellular responses to integrin stimulation by extracellular matrix proteins. Mice with epidermis-restricted inactivation of the Ilk gene exhibit pleiotropic phenotypic defects, including impaired hair follicle morphogenesis, reduced epidermal adhesion to the basement membrane, compromised epidermal integrity, as well as wasting and failure to thrive leading to perinatal death. To better understand the underlying molecular mechanisms that cause such a broad range of alterations, we investigated the impact of Ilk gene inactivation on the epidermis transcriptome. Microarray analysis showed over 700 differentially regulated mRNAs encoding proteins involved in multiple aspects of epidermal function, including keratinocyte differentiation and barrier formation, inflammation, regeneration after injury, and fundamental epidermal developmental pathways. These studies also revealed potential effects on genes not previously implicated in ILK functions, including those important for melanocyte and melanoblast development and function, regulation of cytoskeletal dynamics, and homeobox genes. This study shows that ILK is a critical regulator of multiple aspects of epidermal function and homeostasis, and reveals the previously unreported involvement of ILK not only in epidermal differentiation and barrier formation, but also in melanocyte genesis and function

Calcium-Activated Potassium Channels BK and IK1 Are Functionally Expressed in Human Gliomas but Do Not Regulate Cell Proliferation

Gliomas are morbid brain tumors that are extremely resistant to available chemotherapy and radiology treatments. Some studies have suggested that calcium-activated potassium channels contribute to the high proliferative potential of tumor cells, including gliomas. However, other publications demonstrated no role for these channels or even assigned them antitumorogenic properties. In this work we characterized the expression and functional contribution to proliferation of Ca2+-activated K+ channels in human glioblastoma cells. Quantitative RT-PCR detected transcripts for the big conductance (BK), intermediate conductance (IK1), and small conductance (SK2) K+ channels in two glioblastoma-derived cell lines and a surgical sample of glioblastoma multiforme. Functional expression of BK and IK1 in U251 and U87 glioma cell lines and primary glioma cultures was verified using whole-cell electrophysiological recordings. Inhibitors of BK (paxilline and penitrem A) and IK1 channels (clotrimazole and TRAM-34) reduced U251 and U87 proliferation in an additive fashion, while the selective blocker of SK channels UCL1848 had no effect. However, the antiproliferative properties of BK and IK1 inhibitors were seen at concentrations that were higher than those necessary to inhibit channel activity. To verify specificity of pharmacological agents, we downregulated BK and IK1 channels in U251 cells using gene-specific siRNAs. Although siRNA knockdowns caused strong reductions in the BK and IK1 current densities, neither single nor double gene silencing significantly affected rates of proliferation. Taken together, these results suggest that Ca2+-activated K+ channels do not play a critical role in proliferation of glioma cells and that the effects of pharmacological inhibitors occur through their off-target actions

Myelin-mediated inhibition of oligodendrocyte precursor differentiation can be overcome by pharmacological modulation of Fyn-RhoA and protein kinase C signalling

Failure of oligodendrocyte precursor cell (OPC) differentiation contributes significantly to failed myelin sheath regeneration (remyelination) in chronic demyelinating diseases. Although the reasons for this failure are not completely understood, several lines of evidence point to factors present following demyelination that specifically inhibit differentiation of cells capable of generating remyelinating oligodendrocytes. We have previously demonstrated that myelin debris generated by demyelination inhibits remyelination by inhibiting OPC differentiation and that the inhibitory effects are associated with myelin proteins. In the present study, we narrow down the spectrum of potential protein candidates by proteomic analysis of inhibitory protein fractions prepared by CM and HighQ column chromatography followed by BN/SDS/SDS–PAGE gel separation using Nano-HPLC-ESI-Q-TOF mass spectrometry. We show that the inhibitory effects on OPC differentiation mediated by myelin are regulated by Fyn-RhoA-ROCK signalling as well as by modulation of protein kinase C (PKC) signalling. We demonstrate that pharmacological or siRNA-mediated inhibition of RhoA-ROCK-II and/or PKC signalling can induce OPC differentiation in the presence of myelin. Our results, which provide a mechanistic link between myelin, a mediator of OPC differentiation inhibition associated with demyelinating pathologies and specific signalling pathways amenable to pharmacological manipulation, are therefore of significant potential value for future strategies aimed at enhancing CNS remyelination

Two Distinct Modes of Hypoosmotic Medium-Induced Release of Excitatory Amino Acids and Taurine in the Rat Brain In Vivo

A variety of physiological and pathological factors induce cellular swelling in the brain. Changes in cell volume activate several types of ion channels, which mediate the release of inorganic and organic osmolytes and allow for compensatory cell volume decrease. Volume-regulated anion channels (VRAC) are thought to be responsible for the release of some of organic osmolytes, including the excitatory neurotransmitters glutamate and aspartate. In the present study, we compared the in vivo properties of the swelling-activated release of glutamate, aspartate, and another major brain osmolyte taurine. Cell swelling was induced by perfusion of hypoosmotic (low [NaCl]) medium via a microdialysis probe placed in the rat cortex. The hypoosmotic medium produced several-fold increases in the extracellular levels of glutamate, aspartate and taurine. However, the release of the excitatory amino acids differed from the release of taurine in several respects including: (i) kinetic properties, (ii) sensitivity to isoosmotic changes in [NaCl], and (iii) sensitivity to hydrogen peroxide, which is known to modulate VRAC. Consistent with the involvement of VRAC, hypoosmotic medium-induced release of the excitatory amino acids was inhibited by the anion channel blocker DNDS, but not by the glutamate transporter inhibitor TBOA or Cd2+, which inhibits exocytosis. In order to elucidate the mechanisms contributing to taurine release, we studied its release properties in cultured astrocytes and cortical synaptosomes. Similarities between the results obtained in vivo and in synaptosomes suggest that the swelling-activated release of taurine in vivo may be of neuronal origin. Taken together, our findings indicate that different transport mechanisms and/or distinct cellular sources mediate hypoosmotic medium-induced release of the excitatory amino acids and taurine in vivo

Dependence of taurine and glutamate uptake on extracellular [Na⁺] in cultured astrocytes.

<p>Taurine and glutamate transport rates were measured in primary astrocyte cultures using [³H]taurine and d-[³H]aspartate. Extracellular concentrations of amino acids were adjusted to 10 µM using unlabeled taurine or l-glutamate. To compare glutamate versus taurine uptake, the values were normalized to uptake levels under basal conditions ([Na⁺]_o = 135 mM). Note that under basal conditions absolute d-[³H]aspartate uptake rate (nmols/mg protein) was ∼5-fold higher compared to taurine. Data are the mean values ±SEM of three experiments from each group.</p

Effect of the glutamate transporter inhibitor dl-TBOA on hypoosmotic medium induced amino acid release in the cortex and glutamate transporter reversal in cultured astrocytes.

<p>(a–b) Microdialysis probes implanted on opposite sides of the cortex were perfused with hypoosmotic medium in the presence or absence of 500 µM dl-TBOA, given 20 minutes prior to and during one hour hypoosmotic medium perfusion. The data represent average dialysate levels of glutamate (a), aspartate (b) ±SEM from 4 rats. ** p<0.01 HYPO vs. HYPO+TBOA. (c) DL-TBOA effectively prevented reversal of glutamate transporter in cultured astrocytes. Cultured astrocytes were superfused for one hour with 1 mM ouabain and additionally for 20 min high [KCl] (100 mM) plus ouabain to induce glutamate transporter reversal. 300 µM dl-TBOA was given 10 minutes prior to and during the high [KCl] perfusion in the presence of ouabain. The data are the average values ±SEM for three experiments in each group. ** p<0.01 KCl vs. KCl+TBOA.</p

Effect of H₂O₂ on hypoosmotic medium induced amino acid release in the cortex.

<p>(a–c) Two microdialysis probes implanted on opposite sides of the cortex were perfused with hypoosmotic medium in the presence or absence of 1 mM H₂O₂ given 20 minutes prior to and during one-hour hypoosmotic medium perfusion. The data represent the average dialysate levels ±SEM of glutamate (a), aspartate (b) and taurine (c) from 9 rats. ** p<0.01 HYPO vs. HYPO+H₂O₂. In separate experiments, rats were perfused with 1 mM H₂O₂ alone (N = 5).</p

Hypothetical explanation of the experimental data showing differences in taurine and glutamate release <i>in vivo</i>.

<p>A reduction in medium osmolarity (↓[osm]_e) in the rat cortex causes an increase in the extracellular levels of the excitatory amino acids glutamate and aspartate and the sulfonic acid taurine via a mechanism sensitive to the anion channel blocker DNDS. Despite these similarities, the excitatory amino acid and taurine release demonstrate different kinetics and are likely mediated by different transport pathways (1 and 2) and/or originate from different cellular sources. The taurine pathway (1) but not the excitatory amino acid pathway (2) is activated by isoosmotic lowering of [NaCl]_e. Conversely, the swelling-activated excitatory amino acid release pathway (2) but not the taurine pathway (1) is potentiated by H₂O₂. Alternative transport pathways that were considered in this study include: [Na⁺]_e-dependent taurine transporters (3), [Na⁺]_e/[K⁺]_i-dependent glutamate transporters in neurons (4), and in astrocytes (5), which are sensitive to TBOA; and vesicular glutamate release (6), which is sensitive to the voltage-gated Ca²⁺ channel blocker Cd²⁺. Based on the similarities of excitatory amino acid release <i>in vivo</i> and in cultured astrocytes, we speculate that glutamate release <i>in vivo</i> largely originates from glial cells. Similarities between taurine release <i>in vivo</i> and in synaptosomes suggest that taurine release may be of a neuronal origin.</p