In cerebellar granule neurons, a BH3-only Bcl-2 family member, death protein 5/harakiri, is up-regulated in a JNK-dependent manner during apoptosis induced by potassium deprivation. However, it is not clear whether c-Jun is directly involved in the induction of dp5. Here, we showed that the up-regulation of dp5, but not fas ligand and bim, after potassium deprivation was suppressed by the expression of a dominant negative form of c-Jun. Deletion analysis of the 5-flanking sequence of the dp5 gene revealed that a major responsive element responsible for the induction by potassium deprivation is an ATF binding site located at ؊116 to ؊109 relative to the transcriptional start site. Mutation of this site completely abolished promoter activation. Furthermore, a gel shift assay showed that a specific complex containing c-Jun and ATF2 recognized this site and increased in potassium-deprived cerebellar granule neurons. Chromatin immunoprecipitation demonstrated that c-Jun was able to bind to this site in vivo. Finally, we demonstrated that knockdown of Dp5 by small interfering RNA rescued neurons from potassium deprivation-induced apoptosis. Taken together, these results suggest that dp5 is a target gene of c-Jun and plays a critical role in potassium deprivation-induced apoptosis in cerebellar granule neurons.The Bcl-2 family proteins can be divided into three major subgroups (1). Antiapoptotic proteins, such as Bcl-2, Bcl-X L , and Mcl-1, typically share four conserved motifs termed Bcl-2 homology (BH) 3 domains and inhibit mitochondrial cytochrome c release and apoptosis. Multidomain proapoptotic proteins, the second subgroup, such as Bax, Bak, and Bok, typically have three BH domains but promote cytochrome c release and apoptosis. The third, and the most structurally diverse subgroup, is the BH3-only proteins, including Dp5/HRK (death protein 5/harakiri), Bim (Bcl2-interacting mediator of cell death), Bid, Bad, Puma, and Noxa, which share the BH3 domain. The BH3-only proteins are critical initiators of apoptosis. Upon challenge, BH3-only proteins translocate to mitochondria and promote the chromec release by neutralizing the antiapoptotic action of Bcl-2 family members. BH3-only proteins are stringently regulated at the transcriptional and post-translational levels during apoptosis, such as Dp5, Bim, and Puma, depending on the cell type and apoptotic stimulus (2-6). Among the BH3-only proteins, Dp5 is of particular interest to studies of apoptosis in the nervous system. In rodents, the expression of Dp5 is largely restricted to and is developmentally regulated in the nervous system (2, 7). Dp5 is the first found BH3-only protein to be induced by NGF deprivation in sympathetic neurons (2). dp5 is highly homologous to the human gene harakiri (HRK) cloned by a two-hybrid screen with Bcl-2 and Bcl-X L (3). As well as being induced in NGF-deprived sympathetic neurons, the induction of dp5 is also observed in cerebellar granule neurons (CGNs) deprived of potassium, cortical neurons exposed to toxic concentrations of amyl...

The activator protein 1 (AP-1) transcription factor c-Jun is crucial for neuronal apoptosis. However, c-Jun dimerization partners and the regulation of these proteins in neuronal apoptosis remain unknown. Here we report that c-Jun-mediated neuronal apoptosis requires the concomitant activation of activating transcription factor-2 (ATF2) and downregulation of c-Fos. Furthermore, we have observed that c-Jun predominantly heterodimerizes with ATF2 and that the c-Jun/ATF2 complex promotes apoptosis by triggering ATF activity. Inhibition of c-Jun/ATF2 heterodimerization using dominant negative mutants, small hairpin RNAs, or decoy oligonucleotides was able to rescue neurons from apoptosis, whereas constitutively active ATF2 and c-Jun mutants were found to synergistically stimulate apoptosis.

Glycogen synthase kinase-3 (GSK-3) plays a critical role in neuronal apoptosis. The two mammalian isoforms of the kinase, GSK-3␣ and GSK-3␤, are inhibited by phosphorylation at Ser-21 and Ser-9, respectively. Depolarization, which is vital for neuronal survival, causes both an increase in Ser-21/9 phosphorylation and an inhibition of GSK-3␣/␤. However, the role of GSK-3 phosphorylation in depolarization-dependent neuron survival and the signaling pathway contributing to GSK-3 phosphorylation during depolarization remain largely unknown. Using several approaches, we showed that both isoforms of GSK-3 are important for mediating neuronal apoptosis. Nonphosphorylatable GSK-3␣/␤ mutants (S21A/S9A) promoted apoptosis, whereas a peptide encompassing Ser-9 of GSK-3␤ protected neurons in a phosphorylation-dependent manner; these results indicate a critical role for Ser-21/9 phosphorylation on depolarization-dependent neuron survival. We found that Ser-21/9 phosphorylation of GSK-3 was mediated by Ca 2؉ / calmodulin-dependent protein kinase II (CaMKII) but not by Akt/PKB, PKA, or p90 RSK . CaMKII associated with and phosphorylated GSK-3␣/␤. Furthermore, the pro-survival effect of CaMKII was mediated by GSK-3 phosphorylation and inactivation. These findings identify a novel Ca 2؉ /calmodulin/CaMKII/ GSK-3 pathway that couples depolarization to neuronal survival.The survival or death of neurons is critical for the establishment of appropriate neural circuitry during brain development (1, 2). Considerable evidence supports that electrical activity plays a crucial role in neuronal survival (3, 4). For example, pharmacological blockade of electrical activity in rat brain induces extensive apoptotic neurodegeneration (5, 6). Deafferentiation of the cerebellar granule layer in adult rats resulted in massive and typical apoptosis of cerebellar granule neurons (CGNs), 3 suggesting the importance of afferent input-related factors for survival of CGNs in vivo (7). In culture, survival of rat CGNs can be maintained by electrical activity, which is effected by depolarizing concentrations of extracellular potassium [KCl] o ϭ 25 mM KCl ((25 K) or potassium depolarization) (8, 9). Lowering [KCl] o to 5 mM KCl ((5 K) or potassium deprivation) triggers typical apoptosis (10). Presumably, this recapitulates the naturally occurring neuronal death that takes place in the newborn rat cerebellum (11). These characteristics, along with an abundant neuronal population and up to 98% homogeneity, make cultured CGNs an excellent and extensively studied model for deciphering the signaling mechanisms that underlie depolarization-dependent neuron survival (4).It has been well documented that depolarizing conditions (such as elevated [KCl] o ) sustain neuronal survival by causing the influx of Ca 2ϩ through L-type Ca 2ϩ channels (8, 12, 13), implicating Ca 2ϩ as a necessary second messenger for survival signaling. When activated by elevated Ca 2ϩ , Ca 2ϩ /calmodulindependent protein kinase II (CaMKII) has been reported to mediate the depolarization-...

IntroductionThis technical note describes a new arthroscopic technique to repair the peripheral attachment lesion of the posterior horn of the medial meniscus. The operation was performed under arthroscopy using a standard anterior portal.Surgical TechniqueA FasT-Fix needle was inserted obliquely close to the tibial plateau and the first implant was inserted into the joint capsule depending on its bending angle underneath the meniscus. The second implant was inserted through 1/3 periphery of the meniscus into the meniscocapsular area. The pre-tied self-sliding knot was tensioned to achieve secure fixation of the posterior meniscal peripheral attachment at the original attachment point.Materials and MethodsFrom August 2011 to February 2014, 23 knees were diagnosed as ramp lesion, underwent meniscal repair using FasT-Fix technique.ResultsAll patients were followed up for average 14 months. The Lysholm score improved from preoperative 64.4±4.52 to postoperative 91.2±4.60.ConclusionsWe believe that the FasT-Fix technique via the standard anterior portal can be a more convenient and less traumatic alternative for repair of the peripheral attachment lesion of the posterior horn of the medial meniscus in the anterior cruciate ligament deficient knee.

The proapoptotic BH3-only protein Bim is a crucial regulator of neuronal apoptosis. Previous studies have indicated the involvement of the c-Jun, FOXO1/3a, and B/C-Myb transcription factors in the regulation of Bim during neuronal apoptosis. However, the mechanism underlying the transcriptional regulation of Bim in activity deprivation-induced neuronal apoptosis has remained unclear. The present study demonstrates that early growth response 1 (Egr-1), rather than c-Jun, FOXO1/3a, or B/C-Myb, directly transactivates Bim gene expression to mediate apoptosis of rat cerebellar granule neurons. We showed that Egr-1 was sufficient and necessary for neuronal apoptosis. Suppression of Egr-1 activity using dominant-negative mutant or knockdown of Egr-1 using small interfering RNAs led to a decrease in Bim expression, whereas overexpression of Egr-1 resulted in induction of Bim. Deletion and site-directed mutagenesis of the Bim promoter revealed that Bim transcriptional activation depends primarily on a putative Egr-binding sequence between nucleotides Ϫ56 and Ϫ47 upstream of the start site. We also showed that Egr-1 binding to this sequence increased in response to activity deprivation in vitro and in vivo. Moreover, inhibition of Egr-1 binding to the Bim promoter, by mithramycin A and chromomycin A3, reduced the activity deprivation-induced increases in Bim promoter activity and mRNA and protein levels and protected neurons from apoptosis, further supporting the Egr-1-mediated transactivation of Bim. Additionally, Bim overcame the Egr-1 knockdown-mediated inhibition of apoptosis, whereas Bim knockdown impaired the increase in apoptosis induced by Egr-1. These findings establish Bim as an Egr-1 target gene in neurons, uncovering a novel Egr-1/Bim pathway by which activity deprivation induces neuronal apoptosis.

Background/AimsStem cell therapy has been applied to treat a variety of autoimmune diseases, including Crohn’s disease (CD), but few studies have examined the use of umbilical cord mesenchymal stem cells (UC-MSCs). This trial sought to investigate the efficacy and safety of UC-MSCs for the treatment of CD.MethodsEighty-two patients who had been diagnosed with CD and had received steroid maintenance therapy for more than 6 months were included in this study. Forty-one patients were randomly selected to receive a total of four peripheral intravenous infusions of 1×106 UC-MSCs/kg, with one infusion per week. Patients were followed up for 12 months. The Crohn’s disease activity index (CDAI), Harvey-Bradshaw index (HBI), and corticosteroid dosage were assessed.ResultsTwelve months after treatment, the CDAI, HBI, and corticosteroid dosage had decreased by 62.5±23.2, 3.4±1.2, and 4.2±0.84 mg/day, respectively, in the UC-MSC group and by 23.6±12.4, 1.2±0.58, and 1.2±0.35 mg/day, respectively, in the control group (p<0.01, p<0.05, and p<0.05 for UC-MSC vs control, respectively). Four patients developed a fever after cell infusion. No serious adverse events were observed.ConclusionsUC-MSCs were effective in the treatment of CD and produced mild side effects.

Background: Pancreatic ductal adenocarcinoma (PDAC) is an almost universally lethal disease. Deregulation or dysfunction of miRNAs contribute to cancer development. The role of miR-940 in PDAC remains unclear. Methods: The level of miR-940 in PDAC tissues and cell lines was measured by qRT-PCR. MiR-940 was over-expressed by miRNAs mimics transfection and reduced by miRNAs antisense oligonucleotides (ASO) transfection. Cell proliferation was analyzed by MTT assay and cell apoptosis was evaluated by FACS analysis. Targeted genes were predicted by a bioinformatics algorithm and confirmed by a dual luciferase reporter assay. Myeloid differentiation primary response gene (88) (MyD88) protein level was assayed by immunohistochemistry and Western blot analysis. Results: Low miR-940 level and high MyD88 protein level in PDAC tissues were both correlated with low survival rate. Up-regulation of miR-940 inhibited PDAC cell lines growth while down-regulation induced cell growth. The 3' UTR of MyD88 was targeted by miR-940. Conclusions: Low level of miR-940 and high level of MyD88 in PDAC promoted PDAC cells growth which might be related to the low survival rate of PDAC patients. MiR-940 exerted its effect by targeting MyD88.

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