Current Molecular Medicine (v.13, #6)

An EJC Factor RBM8a Regulates Anxiety Behaviors by A. Alachkar, D. Jiang, M. Harrison, Y. Zhou, G. Chen, Y. Mao (887-899).
Neuroplasticity depends on the precise timing of gene expression, which requires accurate control ofmRNA stability and rapid elimination of abnormal mRNA. Nonsense-mediated mRNA decay (NMD) is an RNAsurveillance mechanism that ensures the speedy degradation of mRNAs carrying premature terminationcodons (PTCs). This mechanism relies on several key Exon Junction Complex (EJC) factors to distinguishPTCs from normal stop codons. NMD degrades not only aberrant transcripts carrying PTCs, but also normaltranscripts harboring a normal stop codon [1]. Intriguingly, mutations in an NMD factor, Upf3b, have beenfound in patients with autism [2, 3]. A binding partner of Upf3b, RBM8a, is located in the 1q21.1 copy-numbervariation (CNV) associated with mental retardation, autism [4], schizophrenia [5], and microcephaly [6].However, the functions of EJC factors and their roles in behavioral regulation are still elusive. RBM8a protein isa core component of the EJC that plays an important role in NMD. Recent genetic study indicated that RBM8again-of-function significantly associated with intellectual disability [7]. In this study we investigated the effect ofRBM8a overexpression on affective behaviors in mice. Lentivirus expressing RBM8a was infused into thehippocampus of adult mice to conduct behavioral studies including social interaction, open field, elevated plusmaze, and forced swimming tests. Our results showed that overexpression of RBM8a in the mouse dentategyrus (DG) leads to increased anxiety-like behavior, abnormal social interaction and decreased immobile timein forced swimming test (FST). To examine the underlying mechanism, we found that overexpressing RBM8ain cultured primary neurons lead to significant higher frequency of miniature excitatory postsynaptic currents(mEPSCs). To explore the underlying mechanism of RBM8a mediated behavioral changes, RNAimmunoprecipitation(RNA-IP) detected that RBM8a binds to CaMK2, GluR1 and Egr1 mRNA, suggesting thatRBM8a may target neuronal genes to regulate behaviors. This is the first study that demonstrates the key roleof RBM8a on the emotional behaviors in mice. These results reveal new neural mechanisms by which NMDmodulates behaviors and potentially provide a better understanding of pathophysiology underlying psychiatricdisorders.

Antimicrobial Potential of Lycosin-I, a Cationic and Amphiphilic Peptide from the Venom of the Spider Lycosa singorensis by H. Tan, X. Ding, S. Meng, C. Liu, H. Wang, L. Xia, Z. Liu, S. Liang (900-910).
Antimicrobial peptides (AMPs) are significant components of the innate immune system and playindispensable roles in the resistance to bacterial infection. Here, we investigated the antimicrobial activity oflycosin-I, a 24-residue cationic anticancer peptide derived from Lycosa singorensis with high structuralsimilarity to several cationic and amphiphilic antimicrobial peptides. The antimicrobial activity of lycosin-Iagainst 27 strains of microbes including bacteria and fungi was examined and compared with that of theXenopus-derived AMP magainin 2 using a microdilution assay. Lycosin-I inhibited the growth of mostmicroorganisms at low micromolar concentrations, and was a more potent inhibitor than magainin 2. Lycosin-Ishowed rapid, selective and broad-spectrum bactericidal activity and a synergistic effect with traditionalantibiotics. In vivo, it showed potent bactericidal activity in a mouse thigh infection model. High Mg2+concentrations reduced the antibacterial effect of lycosin-I, implying that the peptide might directly interact withthe bacterial cell membrane. Uptake of the fluorogenic dye SYTOX and changes in the surface of lycosin-Itreatedbacterial cells observed by scanning electron microscopy confirmed that lycosin-I permeabilized thecell membrane, resulting in the rapid bactericidal effect. Taken together, our findings indicate that lycosin-I is apromising peptide with the potential for the development of novel antibacterial agents.

Failure after glaucoma filtration surgery is attributed to fibrosis at the operated site. To understandthe wound healing process after glaucoma filtration surgery, we have developed a mouse model for glaucomafiltration surgery which closely mimics the clinical response. In this study, we describe a systematic analysis ofthe wound healing response in vivo. Our data revealed that the post-surgical tissue response was separableinto two distinguishable phases. The early ?acute inflammatory? phase was characterized by significantlyincreased transcript expression of Vegfa, Cxcl1, Cxcl5, Ccl2, Ccl3, Ccl4, Gmcsf and specific Mmps as well asgreater infiltration of monocytes/macrophages and T cells. The late ?fibrotic? phase was characterized by anincreased expression of Tgfb2 and extracellular matrix genes as well as a notable reduction of infiltratinginflammatory cells. Significantly, more mitotic cells were observed at both time points post-surgery.Subconjunctival fibroblasts may be involved in both phases since they have the capacity to reiterate the in vivogene expression profiles upon either pro-inflammatory or pro-fibrotic cytokine stimulation. Given that thecellular and molecular targets that govern the early and late phases of wound healing are distinct and timesensitive, a multi-targeted therapeutic approach to sequentially inhibit inflammation and fibrogenesis at thecritical time point may lead to improved surgical outcomes in glaucoma filtration surgery.

Common Variant in VEGFA and Response to Anti-VEGF Therapy for Neovascular Age-Related Macular Degeneration by L. Zhao, S. Grob, R. Avery, A. Kimura, D. Pieramici, J. Lee, M. Rabena, S. Ortiz, J. Quach, G. Cao, H. Luo, M. Zhang, M. Pei, Y. Song, P. Tornambe, M. Goldbaum, H. Ferreyra, I. Kozak, K. Zhang (929-934).
Age-related macular degeneration (AMD) is a leading cause of visual impairment in agingpopulations in industrialized countries. Here we investigated whether the genotype of vascular endothelialgrowth factor A (VEGFA) gene is associated with response to anti-VEGF therapy. 223 eyes with neovascularAMD were treated with intravitreal anti-VEGF therapy. Responders were defined as patients who had animprovement in best corrected visual acuity (BCVA) of at least 5 letters or one line on the EDTRS visual acuitychart along with resolution of intraretinal or subretinal fluid over 12 months. Patients who did not meet thedefinition of responders were classified as poor-responders. The vision of responders (n = 148) improved whilethe vision of poor-responders (n = 75) worsened (P <0.001). Responders on average had a decrease in centralfoveal thickness (CFT), while poor-responders had an increase in CFT (P <0.001). Compared with theresponder group, the poor-responder group had a higher frequency of the risk (T) allele (Allelic P = 0.019) andTT genotype (P = 0.002 under a recessive model) for the VEGFA-rs943080 polymorphism. VEGFA expressionwas 1.8-fold higher in cells with the VEGFA rs943080 TT genotype than in cells with the VEGFA rs943080 CCgenotype (P = 0.012). Age, gender, smoking, diabetes mellitus, and hypertension did not play a significant rolein treatment response, but BMI was found to be significantly different between responders and poorresponders(P = 0.033). In conclusion, we demonstrated a potential pharmacogenetic relationship between theVEGFA gene and treatment response to anti-VEGF therapy.</p><p>The studies are registered at under the identifiers NCT00474695 ( and NCT01464723 (

Mitochondria-Targeted Antioxidant Peptide SS31 Protects the Retinas of Diabetic Rats by J. Huang, X. Li, M. Li, J. Li, W. Xiao, W. Ma, X. Chen, X. Liang, S. Tang, Y. Luo (935-945).
Oxidative stress is one of the main contributors in the pathogenesis of diabetic retinopathy. The aimof this study is to investigate the effects of SS31 which is a mitochondria-targeted antioxidant peptide on theretinas of streptozotocin (STZ)-induced diabetic rats. Two weeks after induction of diabetes, SS31 (3 mg/kg) orthe same volume of normal saline (N.S) was injected subcutaneously into the back of diabetic rats every day.Four months later, the integrity of inner blood retinal barrier (iBRB) was measured by Evans blue perfusion.The expression and distribution of claudin-5, occludin, acrolein, 8-OHdG and nitrotyrosine in the rat retinaswere detected by immunofluorescent staining. Retinal ultrastructures were observed by transmission electronmicroscopy. The protein level of VEGFR2, Trx-2, Bcl-2, Bax, caspase-3, p53, and NF-κB in the rat retinas wereassayed by western blot. Four months after subcutaneous injection, the diabetic rats treated with SS31 hadbetter structures of retinal ganglion cells, thinner capillary basement membrane, less iBRB leakage, moreuniform staining of claudin-5 and occludin in the retinal vessels, lower levels of acrolein, 8-OHdG, nitrotyrosine,Bax, caspase-3, p53, and NF-κB, and higher levels of Trx-2 and Bcl-2 in the retinas than those treated withN.S. In conclusion, SS31 could protect the retinal structures and inhibit the breakdown of iBRB by reducingoxidative damage, increasing Trx-2 and Bcl-2 expression, and decreasing p53, NF-κB, Bax, caspase-3, andVEGFR2 expression in the retinas of diabetic rats. SS31 could be a potential new treatment for diabeticretinopathy and other oxidative stress-related diseases.

Objective: Intravitreal glucocorticoids and anti-vascular endothelial growth factor (VEGF) therapiesare novel strategies for the treatment of advanced diabetic retinopathy, a condition with inflammatory andneuropathic elements. In contrast with anti-VEGF therapy, glucocorticoids may also exert neuroprotectiveeffects. How glucocorticoids protect retinal neurons is unknown. The aims of the study are to investigate theanti-apoptotic actions of glucocorticoids on diabetic retinal neurons, and characterize the signalling pathwaysinvolved.</p><p>Research Design and Methods: The regulation of gene expression of the four p38 mitogen-activated proteinkinase (MAPK) isoforms (α, β, δ and γ) and the glucocorticoid receptor (GR) in the retinas was evaluated usingquantitative RT-PCR, Western blot and immunohistochemistry. Phosphorylation of all isoforms p38MAPK(Thr180/Tyr182) and GR (S-211) was further evaluated. Apoptosis was confirmed by immunolocalization ofactive CASPASE-3 and the subsequent cleavage of poly (ADP-ribose) polymerase (PARP) followingintravitreal injection of triamcinolone acetonide (IVTA), in an early diabetic rat model (26 days after induction ofdiabetes).</p><p>Results: IVTA significantly down-regulated mRNA expression of Caspase 3. Activation of CASPASE-3, thesubsequent cleavage of PARP-1 and phosphorylation of p38MAPK induced by diabetes were attenuated byIVTA treatment, concomitantly with activation by phosphorylation of the glucocorticoid receptor (GR S-211).</p><p>Conclusions: IVTA activates the GR and exerts neural protective effects on retinal neurons. Inhibition of thep38MAPK pathway and activation of GR play a critical anti-apoptotic role in retinal neurons of diabetesfollowing IVTA treatment. Both the anti-inflammatory and anti-apoptotic effects of glucocorticoids may bemediated through inhibition of the p38MAPK pathway in diabetic retinopathy.

ERK Signaling Pathway Regulates Embryonic Survival and Eye Development in Goldfish, Carassius auratus by L. Li, L. Wang, T.-T. Li, X. Li, X.-Q. Huang, X.-W. Chen, Z.-L. Li, X.-M. Lv, F.-Y. Liu, Z.-W. Luo, M. Liu, X.-H. Hu, W.-F. Hu, Z.-X. Huang, M. Yi, S.-J. Liu, Y.-Z. Liu, D.W.-C. Li (959-967).
The extracellular signal-regulated kinase (ERK) is one of the three major types of mitogen-activatedprotein kinases. Previous studies showed that ERKs mediate various signaling pathways for cell proliferation,differentiation, survival and transformation in mammals. In the present study, we use goldfish as a modelsystem and demonstrate that ERK kinases play important roles in promoting embryonic survival and regulatedevelopment of eye and trunk in vertebrates. ERKs are highly expressed in multiple tissues including lensepithelial cells, lens fiber cells, retina, brain, muscle and heart of adult goldfish. Injection of the dominantnegative ERK mutant (DNM-ERK) into the fertilized eggs of goldfish significantly inhibited ERK activity atblastula stage, and completely blocked ERK activity at gastrula and later stages. As a result, the blastula cellswere induced into apoptosis, and majority of the injected embryos were lethal at embryonic stages. At themolecular level, inhibition of ERK activity by DNM-ERKs suppressed phosphorylation of Bad at Ser-112 topromote apoptosis. Similar results were observed when MEK activity was inhibited by U0126 treatment. Thesurvived embryos display significant abnormality in the phenotypes of both eye and trunk. Associated with theabnormality in the eye development, phosphorylation in Pax-6 and expression of HSF4 were significantlydecreased and expression of the β-crystallin gene was also downregulated. These results provide novelinformation regarding the roles of ERKs in regulating vertebrate development.

p53 Directly Regulates αA- and βA3/A1-Crystallin Genes to Modulate Lens Differentiation by W.-K. Ji, X.-C. Tang, M. Yi, P.-Q. Chen, F.-Y. Liu, X.-H. Hu, W.-F. Hu, S.-J. Fu, J.-F. Liu, K.-L. Wu, M.-X. Wu, X.-L. Liu, L.-X. Luo, S. Huang, Z.-Z. Liu, M.-B. Yu, Y.-Z. Liu, D.W.-C. Li (968-978).
It is well established that the tumor suppressor p53 plays major roles in regulating apoptosis and cellcycle progression. In addition, recent studies have demonstrated that p53 is actively involved in regulating celldifferentiation in muscle, the circulatory system and various carcinoma tissues. We have recently shown thatp53 also controls lens differentiation. Regarding the mechanism, we reveal that p53 directly regulates c-Mafand Prox1, two important transcription factors to control cell differentiation in the ocular lens. In the presentstudy, we present further evidence to show that p53 can regulate lens differentiation by controlling expressionof the differentiation genes coding for the lens crystallins. First, the αA and βA3/A1 gene promoters or intronsall contain putative p53 binding sites. Second, gel mobility shifting assays revealed that the p53 protein innuclear extracts from lens epithelial cells directly binds to the p53 binding sites found in these crystallin genepromoters or introns. Third, exogenous wild type p53 induces dose-dependent expression of the luciferasereporter gene driven by different crystallin gene promoters and the exogenous dominant negative mutant p53causes dose-dependent inhibition of the same crystallin genes. Fourth, ChIP assays revealed that p53 binds tocrystallin gene promoters in vivo. Finally, in the p53 knockout mouse lenses, expression levels of variouscrystallins were found down-regulated in comparison with those from the wild type mouse lenses. Together,our results reveal that p53 directly regulates expression of different sets of genes to control lens differentiation.

The LIM Protein fhlA is Essential for Heart Chamber Development in Zebrafish Embryos by H. Xie, X. Fan, X. Tang, Y. Wan, F. Chen, X. Wang, Y. Wang, Y. Li, M. Tang, D. Liu, Z. Jiang, X. Liu, W. Yuan, G. Li, X. Ye, J. Zhou, X. Mo, Y. Deng, X. Wu (979-992).
Four-and-a-half LIM proteins FHL1-3 play important roles in cardiovascular pathophysiology.However, their roles in heart development remain unclear. Here, we report that fhlA, the zebrafish homolog ofhuman FHL1, was found to be expressed around the 22-somite stage. After 24 hpf, expression was restrictedto the heart. fhlA knockdown caused an enlarged cardiac chamber phenotype with up-regulated expression ofthe cardiac markers, but fhlA overexpression reduced the sizes of the cardiac chambers and down-regulatedexpression of the markers. The morphology associated with the cmlc2, amhc, and vmhc expression patterns atthe 22 somite and 24 hpf stages included a broadened domain in embryos lacking fhlA and a smaller domainin embryos overexpressing fhlA. The changes in the sizes of the chambers were attributed to the changes inthe number of ventricular and atrial cells. Loss of fhlA caused a longer heart period and pause betweenheartbeats in M-modes than in controls, but fhlA overexpression caused shorter systolic and diastolic intervals.Abnormal cardiac chambers and physiological function were found to be largely rescued. We also showed theexpression of fhlA in the heart to be increased by retinoic acid (RA) and decreased by the RA synthaseinhibitor DEAB. Both fhlA and RA signaling caused a phenotype characterized by the morphological alterationsin the chamber sizes, suggesting that the role of fhlA in heart development is probably regulated by RAsignaling. Taken together, these results showed that fhlA regulates the size of the heart chamber by reducingthe number of cardiac cells.

Mutation p.S335X in GATA4 Reduces its DNA Binding Affinity and Enhances Cell Apoptosis Associated with Ventricular Septal Defect by F. Yang, M. Wu, Y. Li, G.-Y. Zheng, H.-Q. Cao, W. Sun, R. Yang, H. Zhang, Y.-H. Sheng, X.-Q. Kong, X.-L. Tian, L. Zhou (993-999).
Genetic mutations in GATA4, a transcriptional factor, have been found to cause congenital heartdiseases. The underlying mechanism, however, remains largely unknown. We previously reported 7heterozygous variants in patients with ventricular septal defects (VSD). Here we functionally characterized a denovo mutation p.S335X and demonstrated that this mutation led to the pre-termination of its translation,producing a truncated GATA4 lacking a conservative region at C-terminus. Truncated GATA4 did not disturbits subcellular localization; however, it delayed the cardiomyocyte differentiation in P19cl6 model andprohibited Bcl2 expression that led to apoptosis proved by fragmented genomic DNA and positive TUNELstaining in H9C2 cells. By ChIP assay, we showed that GATA4 without C-terminus reduced its DNA bindingaffinity and suppressed the expressions of its target genes. These findings suggest that C-terminus of GATA4is critical to maintain DNA binding, and genetic mutations in this region may affect genes important for myocyteapoptosis and differentiation associated with congenital heart defects.

N-acetyl-L-cysteine (NAC) improves antioxidant potentials of RBCs to provide protection againstoxidative stress induced hemolysis. The antioxidant mechanism of NAC to reduce oxidative stress in RBC,studied through inactivation of pro-oxidant MetHb. NAC causes irreversible inactivation of the MetHb in anH2O2 dependent manner, and the inactivation follows the pseudo- first- order kinetics. The kinetic constantsare ki = 8.5μM, kinact = 0.706 min-1 and t1/2 = 0.9 min. Spectroscopic studies indicate that MetHb accepts NACas a substrate and oxidizes through a single electron transfer mechanism to the NACox. The single e- oxidationproduct of NAC has been identified as the 5, 5?- dimethyl-1- pyrroline N- oxide (DMPO) adduct of the sulfurcentered radical (aN = 15.2 G and aH=16. 78 G). Binding studies indicate that NACox interacts at the hememoiety and NAC oxidation through MetHb is essential for NAC binding. Heme-NAC adduct dissociated fromMetHb and identified (m/z 1011.19) as 2:1 ratio of NAC/heme in the adduct. TEMPO and PBN treatmentreduces NAC binding to MetHb and protects against inactivation confirms the role of thiyl radical in theinactivation process. Furthermore, scavenging thiyl radicals by TEMPO abolish the protective effect of NAC inhemolysis. Current work highlights antioxidant mechanism of NAC through NAC thiyl radical generation, andMetHb inactivation to exhibit protection in RBC against oxidative stress induced hemolysis.

Oxidative Stress Protection by Novel Telomerase Activators in Mesenchymal Stem Cells Derived from Healthy and Diseased Individuals by A. Tichon, E. Eitan, B.G. Kurkalli, A. Braiman, A. Gazit, S. Slavin, E. Beith-Yannai, E. Priel (1010-1022).
Human Bone Marrow Mesenchymal Stem cells (hMSC) are a promising candidate for cytotherapy.However, the therapeutic potential is limited since the therapy requires ex-vivo cell culturing in whichdeterioration in cellular viability and aging is observed with time.</p><p>Telomerase ribonucleoprotein complex re-elongates telomeres and therefore promotes genomic integrity,proliferation and lifespan. Recently we showed that increasing telomerase reverse transcriptase (TERT)expression by novel compound confers resistance from apoptosis induced by oxidative stress. Here weinvestigated the possibility that a controlled induction of human TERT (hTERT) levels by chemical compounds(AGS-499 and AGS-500) might improve the functionality of hMSC derived from healthy and neurodegenerativediseased individuals. We demonstrate that AGS treatments of hMSC increased telomerase activity and hTERTlevels in a time and dose dependent manner. Prolonged treatments with the compounds increased theaverage telomeres length, without altering population doublings (PD) or inducing chromosomal aberrations.AGS treatments of hMSC protected the cells from apoptosis and DNA damages induced by H2O2, and from thetoxicity induced by long term exposure to DMSO. These AGS effects were shown to be mediated bytelomerase since they were not observed when TERT was depleted from hMSC or in mouse embryonic stemcells derived from TERT knockout mice. Furthermore, AGS compounds did not alter the functionality of hMSCas examined by their ability to differentiate into various lineages in the presence of the compounds. Theseresults suggest that pharmaceutical increase of telomerase may confer a beneficial therapeutic advantage inregenerative medicine when hMSC therapy is applied.

β2-AR-HIF-1α: A Novel Regulatory Axis for Stress-Induced Pancreatic Tumor Growth and Angiogenesis by T. Shan, J. Ma, Q. Ma, K. Guo, J. Guo, X. Li, W. Li, J. Liu, C. Huang, F. Wang, E. Wu (1023-1034).
The purpose of this study was to test the hypothesis that chronic stress in a negative social andpsychological state plays a critical role in pancreatic cancer development and progression. In this study, wecreated a new stress model system to determine the effects of chronic stress on pancreatic cancer progression.Here, we show that chronic stress not only causes depression in mice, most likely attributed to an elevated levelof epinephrine, but also induces pancreatic cancer progression. We provide evidence that the pancreatic cancerprogression induced by chronic stress could be blocked to a significant degree by β2-AR inhibitor ICI118 551 orHIF-1α inhibitor 2-methoxyestradiol. Moreover, establishment of pancreatic cancer in mice exposed to chronicstress was accompanied by up-regulation of the expression of MMP-2, MMP-9, and VEGF, mediated by a HIF-1α-dependent β-AR signaling pathway. Our data suggest that the β2-AR-HIF-1α axis regulates stress-inducedpancreatic tumor growth and angiogenesis. This study may have a therapeutic or preventive potential for thepatients with pancreatic cancer who are especially prone to psychosocial stress challenges.

Immune System Alterations by Aldosterone During Hypertension: From Clinical Observations to Genomic and Non-Genomic Mechanisms Leading to Vascular Damage by N. Munoz-Durango, M.F. Barake, N.A. Letelier, C. Campino, C.E. Fardella, A.M. Kalergis (1035-1046).
Hypertension is traditionally considered a disease in which elevated blood pressure contributes toinflammation and activation of the immune system, leading to cardiovascular injury and end-organ damage.Here, we discuss the effects of aldosterone on the immune system and aldosterone?s contribution to vascularpathogenesis. Studies in human have suggested a broader role for aldosterone, beyond elevating bloodpressure. Recent clinical data support the notion that aldosterone can directly alter the function of the immunesystem and cause vascular-damaging inflammation. Clinical observations have been reproduced inexperimental models of hypertension, further supporting the idea that an aberrant immune responsecontributes to the onset of hypertension. Such studies have shown that myeloid cells are required to induce thedisease and IL-17-producing CD4+ T cells may contribute to maintaining aldosterone-mediated hypertension.In addition, regulatory T cells diminish the inflammatory damage caused by aldosterone during hypertension.This is a very active area of research that could lead to new therapeutic targets for treating hypertension.