Therapeutic nuclear shuttling of YB-1 reduces renal damage and fibrosis.

Virtually all chronic kidney diseases progress towards tubulointerstitial fibrosis. In vitro, Y-box protein-1 (YB-1) acts as a central regulator of gene transcription and translation of several fibrosis-related genes. However, it remains to be determined whether its pro- or antifibrotic propensities prevail in disease. Therefore, we investigated the outcome of mice with half-maximal YB-1 expression in a model of renal fibrosis induced by unilateral ureteral obstruction. Yb1+/- animals displayed markedly reduced tubular injury, immune cell infiltration and renal fibrosis following ureteral obstruction. The increase in renal YB-1 was limited to a YB-1 variant nonphosphorylated at serine 102 but phosphorylated at tyrosine 99. During ureteral obstruction, YB-1 localized to the cytoplasm, directly stabilizing Col1a1 mRNA, thus promoting fibrosis. Conversely, the therapeutic forced nuclear compartmentalization of phosphorylated YB-1 by the small molecule HSc025 mediated repression of the Col1a1 promoter and attenuated fibrosis following ureteral obstruction. Blunting of these effects in Yb1+/- mice confirmed involvement of YB-1. HSc025 even reduced tubulointerstitial damage when applied at later time points during maximum renal damage. Thus, phosphorylation and subcellular localization of YB-1 determines its effect on renal fibrosis in vivo. Hence, induced nuclear YB-1 shuttling may be a novel antifibrotic treatment strategy in renal diseases with the potential of damage reversal.

The glutamate transporter subtypes EAAT4 and EAATs 1-3 transport glutamate with dramatically different kinetics and voltage dependence but share a common uptake mechanism.

Here, we report the application of glutamate concentration jumps and voltage jumps to determine the kinetics of rapid reaction steps of excitatory amino acid transporter subtype 4 (EAAT4) with a 100-micros time resolution. EAAT4 was expressed in HEK293 cells, and the electrogenic transport and anion currents were measured using the patch-clamp method. At steady state, EAAT4 was activated by glutamate and Na+ with high affinities of 0.6 microM and 8.4 mM, respectively, and showed kinetics consistent with sequential binding of Na(+)-glutamate-Na+. The steady-state cycle time of EAAT4 was estimated to be >300 ms (at -90 mV). Applying step changes to the transmembrane potential, V(m), of EAAT4-expressing cells resulted in the generation of transient anion currents (decaying with a tau of approximately 15 ms), indicating inhibition of steady-state EAAT4 activity at negative voltages (<-40 mV) and activation at positive V(m) (>0 mV). A similar inhibitory effect at V(m) < 0 mV was seen when the electrogenic glutamate transport current was monitored, resulting in a bell-shaped I-V(m) curve. Jumping the glutamate concentration to 100 muM generated biphasic, saturable transient transport and anion currents (K(m) approximately 5 microM) that decayed within 100 ms, indicating the existence of two separate electrogenic reaction steps. The fast electrogenic reaction was assigned to Na+ binding to EAAT4, whereas the second reaction is most likely associated with glutamate translocation. Together, these results suggest that glutamate uptake of EAAT4 is based on the same molecular mechanism as transport by the subtypes EAATs 1-3, but that its kinetics and voltage dependence are dramatically different from the other subtypes. EAAT4 kinetics appear to be optimized for high affinity binding of glutamate, but not rapid turnover. Therefore, we propose that EAAT4 is a high-affinity/low-capacity transport system, supplementing low-affinity/high-capacity synaptic glutamate uptake by the other subtypes.

Differential modulation of the glutamate transporters GLT1, GLAST and EAAC1 by docosahexaenoic acid.

At present, the ability of polyunsaturated fatty acids (PUFAs) to regulate individual glutamate transporter subtypes is poorly understood and very little information exists on the mechanism(s) by which PUFAs achieve their effects on the transport process. Here we investigate the effect of cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) on the activity of the mammalian glutamate transporter subtypes, GLT1, GLAST and EAAC1 individually expressed in human embryonic kidney (HEK) cells. Exposure of cells to 100 muM DHA increased the rate of d-[(3)H]aspartate uptake by over 72% of control in HEK(GLT1) cells, and by 45% of control in HEK(EAAC1) cells. In contrast, exposure of HEK(GLAST) cells to 200 muM DHA resulted in almost 40% inhibition of d-[(3)H]aspartate transport. Removal of extracellular calcium increased the inhibitory potential of DHA in HEK(GLAST) cells. In contrast, in the absence of extracellular calcium, the stimulatory effect of DHA on d-[(3)H]aspartate uptake in HEK(GLT1) and HEK(EAAC1) cells was abolished, and significant inhibition of the transport process by DHA was observed. Inhibition of CaM kinase II or PKC had no effect on the ability of DHA to inhibit transport into HEK(GLAST) cells but abolished the stimulatory effect of DHA on d-[(3)H]aspartate transport into HEK(GLT1) and HEK(EAAC1) cells. Inhibition of PKA had no effect on the modulation of d-[(3)H]aspartate transport by DHA in any of the cell lines. We conclude that DHA differentially modulates the GLT1, GLAST and EAAC1 glutamate transporter subtypes via different mechanisms. In the case of GLT1 and EAAC1, DHA appears to stimulate d-[(3)H]aspartate uptake via a mechanism requiring extracellular calcium and involving CaM kinase II and PKC, but not PKA. In contrast, the inhibitory effect of DHA on GLAST does not require extracellular calcium and does not involve CaM kinase II, PKC or PKA.

Cloning, transport properties, and differential localization of two splice variants of GLT-1 in the rat CNS: implications for CNS glutamate homeostasis.

At least two splice variants of GLT-1 are expressed by rat brain astrocytes, albeit in different membrane domains. There is at present only limited data available as to the spatial relationship of such variants relative to the location of synapses and their functional properties. We have characterized the transport properties of GLT-1v in a heterologous expression system and conclude that its transport properties are similar to those of the originally described form of GLT-1, namely GLT-1alpha. We demonstrate that GLT-1alpha is localized to glial processes, some of which are interposed between multiple synapse types, including GABAergic synapses, whereas GLT-1v is expressed by astrocytic processes, at sites not interposed between synapses. Both splice variants can be expressed by a single astrocyte, but such expression is not uniform over the surface of the astrocytes. Neither splice variant of GLT-1 is evident in brain neurons, but both are abundantly expressed in some retinal neurons. We conclude that GLT-1v may not be involved in shaping the kinetics of synaptic signaling in the brain, but may be critical in preventing spillover of glutamate between adjacent synapses, thereby regulating intersynaptic glutamatergic and GABAergic transmission. Furthermore, GLT-1v may be crucial in ensuring that low levels of glutamate are maintained at extrasynaptic locations, especially in pathological conditions such as ischemia, motor neurone disease, and epilepsy.