Altered expression of genes encoding growth factors and developmental regulators in ILK-deficient epidermis.

<p>(<b>A, B</b>) Expression levels of selected transcripts encoding proteins associated with growth factors or pigmentation (panel A), or key factors in developmental epidermal pathways (panel B) in ILK-deficient <i>K14Cre-Ilk^f/f</i> epidermis, determined by qPCR. The results are expressed as the mean+SEM (n = 5). The differences in expression in ILK-deficient epidermis for each of transcripts shown reached statistical significance (<i>P</i><0.001, Student's t test) relative to the corresponding transcript levels in ILK-expressing <i>K14Cre-Ilk^f/+</i> epidermis (set to 1). (<b>C</b>) Protein lysates prepared from the skin of 3 day-old <i>K14Cre-Ilk^f/f</i> or <i>K14Cre-Ilk^f/+</i> mice (two animals of each genotype) were resolved by denaturing gel electrophoresis, followed by immunoblot analysis using antibodies against LGR5, or γ-tubulin, used as loading control. Lysates prepared from wild type mouse brain were used as a positive control for LGR5 expression.</p

Validation of the differential expression of selected genes in ILK-deficient epidermis.

<p>Validation of the differential expression of selected genes in ILK-deficient epidermis.</p

Targeted inactivation of <i>Ilk</i> and loss of ILK transcripts and protein in mouse epidermis.

<p>(<b>A</b>) The skin of 3 day-old <i>K14Cre-Ilk^f/+</i> and <i>K14Cre-Ilk^f/f</i> mice (4 each, from two different litters) was isolated and treated with dispase to separate the epidermis from the dermis. Epidermal lysates were prepared and analyzed by immunoblot with antibodies against ILK or γ-tubulin, used as loading control. (<b>B</b>) Total RNA from <i>K14Cre-Ilk^f/+</i> and <i>K14Cre-Ilk^f/f</i> mice (5 each) was used to interrogate GeneChip Mouse Gene 1.0 ST Arrays, and the signal intensity corresponding to individual <i>Ilk</i> exons was analyzed. The average intensity values obtained for each exon in RNA from <i>K14Cre-Ilk^f/f</i> epidermis are shown relative to those in <i>K14Cre-Ilk^f/+</i> tissues, which have been set to 1.0.</p

Analysis of hair follicle- and keratinocyte differentiation-associated transcripts in ILK-deficient epidermis.

<p>(<b>A</b>) Paraffin sections of skin isolated from 3 day-old mice of the indicated genotype were processed for histological analysis, and stained with hematoxylin and eosin. Bar, 40 µm (<b>B, C</b>) Expression levels of selected transcripts encoding proteins present in the hair follicle (panel B) or in differentiated keratinocytes (panel C) in ILK-deficient <i>K14Cre-Ilk^f/f</i> epidermis, determined by qPCR. The results are expressed as the mean+SEM (n = 5), and asterisks indicate <i>P</i><0.001 (Student's t test) relative to the corresponding transcript levels in ILK-expressing <i>K14Cre-Ilk^f/+</i> epidermis (set to 1). (<b>D</b>) Protein lysates prepared from the skin of 3 day-old mice of the indicated genotypes were resolved by denaturing gel electrophoresis, followed by immunoblot analysis using antibodies against anti-S100A3. The blots were also probed with antibodies against GAPDH, used as loading control.</p

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 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

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

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

Effect of hypoosmotic or isoosmotic low [NaCl] medium on amino acid levels measured in the rat cortex <i>in vivo</i>.

<p>(a–c) Microdialysis probes, implanted in the rat frontoparietal cortex, were perfused with hypoosmotic medium (−95 mM NaCl, −65% osmolarity) or isoosmotic low NaCl medium (−95 mM NaCl +167 mM mannitol) for one hour. In these experiments, the rat brain was perfused with both the hypoosmotic and isoosmotic medium on opposite sides of the cortex. The data represent average dialysate levels of glutamate (a), aspartate (b), and taurine (c) ±SEM from 5 rats. ** p<0.01, hypoosmotic vs. isoosmotic low [NaCl], repeated measures ANOVA. (d–e) In several experiments dialysate levels of glutamine (d, N = 5), and asparagine (e, n = 3) were additionally measured on the “hypoosmotic” side of the brain.</p

Effect of DNDS on swelling-activated D-[³H]aspartate release from cultured astrocytes and swelling-activated [³H]taurine uptake in cortical synaptosomes.

<p>(a) Cultured astrocytes preloaded with D-[³H]aspartate were superfused with hypoosmotic medium in the presence or absence of 2 mM DNDS. The data are the mean values of five experiments for each group ±SEM. *** p<0.001 hypo, vs. DNDS. (b) Release of preloaded [³H]taurine from cortical synaptosomes was measured under isoosmotic (BASAL) and hypoosmotic (HYPO) conditions in the presence or absence of 2 mM DNDS. The data are the mean values of integral 10-min [³H]taurine release ±SEM of three experiments performed in quadruplicate. ***p<0.001 vs. isoosmotic control (BASAL), ^###p<0.001 vs. hypoosmotic control (HYPO).</p