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Peroxisome proliferator-activated receptor (PPAR)-[gamma] is highly expressed in the collecting duct (CD), yet little is known about the effects of PPAR-[gamma] ligands, thiazolidinediones (TZDs), on CD cell structure and function. M1 mouse cortical CD cells were treated with 5 [micro]M troglitazone (TRO) and rosiglitazone (ROSI). First, growth was measured by [[sup.3]H]thymidine and [[sup.3]H]leucine incorporation, as well as analysis of cyclin D I and the CDK inhibitor p27 by Western blot. [[sup.3]H]thymidine incorporation was reduced by 56 and 24% by TRO and ROSI at 6 h, and [[sup.3]H]leucine by 21 and 10%. A similar growth inhibition was also observed after 24 h for thymidine, but leucine was reduced by 48 and 24%, respectively. Likewise, cyclin D1 was diminished 60% by TRO, and p27 was elevated 1.6-and 1.7-fold in response to TRO and ROSI. Next, epithelial cell integrity was assessed by measuring different markers by Western blot analysis. While fibronectin and [alpha]-smooth muscle actin levels were unchanged, by 24 h E-cadherin was decreased by 50%, and [beta]-catenin levels were reduced 2-and 1.5-fold in response to TRO and ROSI, respectively. GW9662, a PPAR-[gamma] antagonist, did not reverse any of the TZD responses in M 1 cells. Of interest, phosho-p38 levels were also elevated 2-fold in response to TRO and 2.3-fold to ROSI, but MAPK inhibition by PD98059 or SB203580 caused an additive inhibition of cell growth and did not alter E-cadherin or [beta]-catenin in response to TZDs. Finally, apoptotic death was assessed by Western blot, but cleaved caspase-3 levels were unchanged from 15 min to 24 h in response to TZDs, and TRO did not affect cell viability or reactive oxygen species generation. Our data suggest that TZDs cause a disruption of M1 cell integrity that is preceded by an inhibition of cell growth. This response is independent of p38 or PPAR-[gamma] activation. M1 collecting duct cells; troglitazone; rosiglitazone doi: 10.1152/ajprenal.00735.2009.

In wild-type mice, a single injection of streptozotocin (STZ, 200 mg/kg body wt) caused within 4 days severe hyperglycemia, hypoinsulinemia, significant glucose intolerance, loss of body weight, and the disappearance of pancreatic [beta]-cells. However, in ATP-sensitive [K.sup.+] channel ([K.sub.ATP] channel)-deficient mice ([Kir6.2.sup.-/-] mice), STZ had none of these effects. Exposing isolated pancreatic islets to STZ caused severe damage in wild-type but not in [Kir6.2.sup.-/-] islets. Following a single injection, plasma STZ levels were slightly less in [Kir6.2.sup.-/-] mice than in wild-type mice. Despite the difference in plasma STZ, wild-type and [Kir6.2.sup.-/-] liver accumulated the same amount of STZ, whereas [Kir6.2.sup.-/-] pancreas accumulated 4.1-fold less STZ than wild-type pancreas. [Kir6.2.sup.-/-] isolated pancreatic islets also transported less glucose than wild-type ones. Quantification of glucose transporter 2 (GLUT2) protein content by Western blot using an antibody with an epitope in the extracellular loop showed no significant difference in GLUT2 content between wild-type and [Kir6.2.sup.-/-] pancreatic islets. However, visualization by immunofluorescence with the same antibody gave rise to 32% less fluorescence in [Kir6.2.sup.-/-] pancreatic islets. The fluorescence intensity using another antibody, with an epitope in the COOH terminus, was 5.6 times less in [Kir6.2.sup.-/-] than in wild-type pancreatic islets. We conclude that 1) [Kir6.2.sup.-/-] mice are STZ resistant because of a decrease in STZ transport by GLUT2 in pancreatic [beta]-cells and 2) the decreased transport is due to a downregulation of GLUT2 activity involving an effect at the COOH terminus. knockout; insulin; liver; glucose transporter; glucose tolerance; diabetic model

An updated view of member of Hu family of Au rich elements-binding proteins known as HuD and its role in multiple aspects of a neurons life including the initial phenotypic commitment and differentiation, as well as synaptic remodeling during learning paradigms and in response to injury and regeneration are presented. The current state of knowledge concerning the molecular and cellular events regulating the expression, activity and function of HuD in neurons is discussed.

We examined whether calcineurin-NFAT (nuclear factors of activated T cells) signaling plays a role in specifically directing the expression of utrophin in the synaptic compartment of muscle fibers. Immunofluorescence experiments revealed the accumulation of components of the calcineurin-NFAT signaling cascade within the postsynaptic membrane domain of the neuromuscular junction. RT-PCR analysis using synaptic vs. extrasynaptic regions of muscle fibers confirmed these findings by showing an accumulation of calcineurin transcripts within the synaptic compartment. We also examined the effect of calcineurin on utrophin gene expression. Pharmacological inhibition of calcineurin in mice with either cyclosporin A or FK506 resulted in a marked decrease in utrophin A expression at synaptic sites, whereas constitutive activation of calcineurin had the opposite effect. Mutation of the previously identified NFAT binding site in the utrophin A promoter region, followed by direct gene transfer studies in mouse muscle, led to an inhibition in the synaptic expression of a lacZ reporter gene construct. Transfection assays performed with cultured myogenic cells indicated that calcineurin acted additively with GA binding protein (GABP) to transactivate utrophin A gene expression. Because both GABP- and calcineurin-mediated pathways are targeted by peroxisome proliferator-activated receptor-[gamma]-coactivator-1[alpha] (PGC-1[alpha]), we examined whether this coactivator contributes to utrophin gene expression. In vitro and in vivo transfection experiments showed that PGC-1[alpha] alone induces transcription from the utrophin A promoter. Interestingly, this induction is largely potentiated by coexpression of PGC-1[alpha] with GABP. Together, these studies indicate that the synaptic expression of utrophin is also driven by calcineurin-NFAT signaling and occurs in conjunction with signaling events that involve GABP and PGC-1[alpha]. synaptic gene expression; Duchenne muscular dystrophy; nuclear respiratory factor 2

Neonatal sciatic nerve injury is known to result in an extensive loss of lumbar motor neurons as well as the disappearance of their respective muscle fibers in the hindlimb musculature. The loss of motor neurons and muscle fibers can be prevented by immediate administration of target-derived neurotrophic factors to the site of injury. In the present study, we investigated the role of ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) in the survival and maturation of a subset of motor neurons innervating the extensor digitorum longus (EDL) and tibialis anterior (TA) muscles. We have shown that combined administration of CNTF and BDNF prevented the loss of motor units after neonatal nerve injury and contributed to the maintenance of muscle mass. Importantly, this combined neurotrophin regimen also prevented the disappearance of muscle fibers that express myosin heavy chain IIB (MyHC IIB) in both EDL and TA muscles 3 mo after neonatal sciatic nerve crush. In parallel studies, we observed a higher level of BDNF in EDL muscle during the critical period of development when motor neurons are highly susceptible to target removal. Given our previous findings that combined administration of CNTF with neurotrophin-3 (NT-3) or neurotrophin-4/5 (NT-4/5) did not result in the rescue of MyHC IIB fibers in EDL, the present results show the importance of muscle-derived BDNF in the survival and maturation of a subpopulation of motor neurons and of MyHC IIB muscle fibers during neonatal development of the neuromuscular system. motor neurons; neuromuscular development; neurotrophins

Martineau, Louis C., Lyne I. McVeigh, Bernard J. Jasmin, and Chris R. J. Kennedy. p38 MAP kinase mediates mechanically induced COX-2 and PG [EP.sub.4] receptor expression in podocytes: implications for the actin cytoskeleton. Am J Physiol Renal Physiol 286: F693-F701, 2004. First published December 9, 2003; 10.1152/ajprenal.00331.2003.--A dynamic cytoskeleton allows podocytes to withstand significant mechanical stress on elevation of intraglomerular capillary pressure ([P.sub.gc]). However, vasoactive hormones, such as prostaglandin [E.sub.2] (PG[E.sub.2]), may challenge the integrity of the actin cytoskeleton, alter podocyte morphology, and compromise glomerular permeability. PG[E.sub.2] synthesis correlates with the onset of proteinuria and increased [P.sub.gc] following reduced nephron mass. We investigated the interplay among mechanical stress, cyclooxygenase (COX), E-prostanoid (EP) receptor expression, and the actin cytoskeleton, using an in vitro model of cell stretch. Immortalized mouse podocytes grown on flexible silicone membranes were cyclically stretched (5% elongation, 0.5 Hz) for 2 h. [EP.sub.4] and COX-2 mRNA increased three-and sevenfold above nonstretched controls, whereas [EP.sub.1] and COX-1 levels were unchanged. Six hours of stretch resulted in a threefold increase in PG[E.sub.2]-stimulated cAMP accumulation, a measure of [EP.sub.4] receptor function, and an increase in COX-2 protein. The stretch-induced effects on COX-2/[EP.sub.4] expression and [EP.sub.4]-induced cAMP production were attributable to p38 MAP kinase, as blockade of this pathway, but not of ERK or JNK, abrogated the response. These stretch-induced changes in expression were transcriptionally dependent as they were actinomycin D sensitive. Finally, we investigated the influence of enhanced [EP.sub.4] signaling on the actin cytoskeleton. Addition of PG[E.sub.2] resulted in actin filament depolymerization observable only in stretched cells. Our results indicate that key components of the eicosanoid pathway are upregulated by mechanically stimulated p38 MAP kinase in podocytes. Enhanced [EP.sub.4] receptor signaling may undermine podocyte cytoskeletal dynamics and thereby compromise filtration barrier function under conditions of increased [P.sub.gc]. eicosanoids; adenosine 3',5'-cyclic monophosphate; mechanical stimulation