LPS-Induced G-CSF Expression in Macrophages Is Mediated by ERK2, but Not ERK1

Granulocyte colony-stimulating factor (G-CSF) selectively stimulates proliferation and differentiation of neutrophil progenitors which play important roles in host defense against infectious agents. However, persistent G-CSF production often leads to neutrophilia and excessive inflammatory reactions. There is therefore a need to understand the mechanism regulating G-CSF expression. In this study, we showed that U0126, a MEK1/2 inhibitor, decreases lipopolysaccharide (LPS)-stimulated G-CSF promoter activity, mRNA expression and protein secretion. Using short hairpin RNA knockdown, we demonstrated that ERK2, and not ERK1, involves in LPS-induced G-CSF expression, but not LPS-regulated expression of TNF-α. Reporter assays showed that ERK2 and C/EBPβ synergistically activate G-CSF promoter activity. Further chromatin immunoprecipitation (ChIP) assays revealed that U0126 inhibits LPS-induced binding of NF-κB (p50/p65) and C/EBPβ to the G-CSF promoter, but not their nuclear protein levels. Knockdown of ERK2 inhibits LPS-induced accessibility of the G-CSF promoter region to DNase I, suggesting that chromatin remodeling may occur. These findings clarify that ERK2, rather than ERK1, mediates LPS-induced G-CSF expression in macrophages by remodeling chromatin, and stimulates C/EBPβ-dependent activation of the G-CSF promoter. This study provides a potential target for regulating G-CSF expression.     

Both Loved and Feared: Third Party Punishers Are Viewed as Formidable and Likeable,but These Reputational Benefits May Only Be Open to Dominant Individuals

Third party punishment can be evolutionarily stable if there is heterogeneity in the cost of punishment or if punishers receive a reputational benefit from their actions. A dominant position might allow some individuals to punish at a lower cost than others and by doing so access these reputational benefits. Three vignette-based studies measured participants'' judgements of a third party punisher in comparison to those exhibiting other aggressive/dominant behaviours (Study 1), when there was variation in the success of punishment (Study 2), and variation in the status of the punisher and the type of punishment used (Study 3). Third party punishers were judged to be more likeable than (but equally dominant as) those who engaged in other types of dominant behaviour (Study 1), were judged to be equally likeable and dominant whether their intervention succeeded or failed (Study 2), and participants believed that only a dominant punisher could intervene successfully (regardless of whether punishment was violent or non-violent) and that subordinate punishers would face a higher risk of retaliation (Study 3). The results suggest that dominance can dramatically reduce the cost of punishment, and that while individuals can gain a great deal of reputational benefit from engaging in third party punishment, these benefits are only open to dominant individuals. Taking the status of punishers into account may therefore help explain the evolution of third party punishment.     

VEGFR2 Trafficking,Signaling and Proteolysis is Regulated by the Ubiquitin Isopeptidase USP8

Vascular endothelial growth factor A (VEGF‐A) regulates many aspects of vascular function. VEGF‐A binding to vascular endothelial growth factor receptor 2 (VEGFR2) stimulates endothelial signal transduction and regulates multiple cellular responses. Activated VEGFR2 undergoes ubiquitination but the enzymes that regulate this post‐translational modification are unclear. In this study, the de‐ubiquitinating enzyme, USP8, is shown to regulate VEGFR2 trafficking, de‐ubiquitination, proteolysis and signal transduction. USP8‐depleted endothelial cells displayed altered VEGFR2 ubiquitination and production of a unique VEGFR2 extracellular domain proteolytic fragment caused by VEGFR2 accumulation in the endosome–lysosome system. In addition, perturbed VEGFR2 trafficking impaired VEGF‐A‐stimulated signal transduction in USP8‐depleted cells. Thus, regulation of VEGFR2 ubiquitination and de‐ubiquitination has important consequences for the endothelial cell response and vascular physiology.      

Matrix Metalloproteinases MMP2 and MMP9 Are Produced in Early Stages of Kidney Morphogenesis but Only MMP9 Is Required for Renal Organogenesis In Vitro

We analyzed matrix metalloproteinase (MMP) production by 11-d embryonic mouse kidneys and the effects of these enzymes on subsequent renal organogenesis. In vivo, immunolocalization of metalloproteinases by laser scanning confocal microscopy and zymograms of kidney lysates showed that the mesenchyme of embryonic kidneys synthesized both MMP9 and MMP2 enzymes. In vitro, embryonic kidneys also secreted both enzymes when cultured in a medium devoid of hormone, growth factor, and serum for 24 h during which T-shaped branching of the ureter bud appeared. We then evaluated the role of MMP2 and MMP9 in kidney morphogenesis by adding anti-MMP2 or anti-MMP9 IgGs to the culture medium of 11-d kidneys for 24 or 72 h. Although it inhibited activity of the mouse enzyme, anti-MMP2 IgGs had no effect on kidney morphogenesis. In contrast, anti-MMP9 IgGs with enzyme-blocking activity impaired renal morphogenesis, in a concentration-dependent manner, by inhibiting T-shaped branching and further divisions of the ureter bud. This effect was irreversible, still observed after inductive events and reproduced by exogenous tissue inhibitor of metalloproteinase 1 (TIMP1), the natural inhibitor of MMP9. These data provide the first demonstration of MMP9 and MMP2 production in vivo by 11-d embryonic kidneys and further show that MMP9 is required in vitro for branching morphogenesis of the ureter bud.     

Akt Phosphorylates Both Tsc1 and Tsc2 in Drosophila,but Neither Phosphorylation Is Required for Normal Animal Growth

Akt, an essential component of the insulin pathway, is a potent inducer of tissue growth. One of Akt''s phosphorylation targets is Tsc2, an inhibitor of the anabolic kinase TOR. This could account for part of Akt''s growth promoting activity. Although phosphorylation of Tsc2 by Akt does occur in vivo, and under certain circumstances can lead to reduced Tsc2 activity, the functional significance of this event is unclear since flies lacking Akt phosphorylation sites on Tsc2 are viable and normal in size and growth rate. Since Drosophila Tsc1, the obligate partner of Tsc2, has an Akt phosphorylation motif that is not conserved in mammals, we investigate here whether Akt redundantly phosphorylates the Tsc complex on Tsc1 and Tsc2. We provide evidence that Akt phosphorylates Tsc1 at Ser533. We show that flies lacking Akt phosphorylation sites on Tsc1 alone, or on both Tsc1 and Tsc2 concurrently, are viable and normal in size. This shows that phosphorylation of the Tsc1/2 complex by Akt is not required for Akt to activate TORC1 and to promote tissue growth in Drosophila.     

All Akt Isoforms (Akt1, Akt2, Akt3) Are Involved in Normal Hearing,but Only Akt2 and Akt3 Are Involved in Auditory Hair Cell Survival in the Mammalian Inner Ear

The kinase Akt is a key downstream mediator of the phosphoinositide-3-kinase signaling pathway and participates in a variety of cellular processes. Akt comprises three isoforms each encoded by a separate gene. There is evidence to indicate that Akt is involved in the survival and protection of auditory hair cells in vitro. However, little is known about the physiological role of Akt in the inner ear—especially in the intact animal. To elucidate this issue, we first analyzed the mRNA expression of the three Akt isoforms in the inner ear of C57/BL6 mice by real-time PCR. Next, we tested the susceptibility to gentamicin-induced auditory hair cell loss in isoform-specific Akt knockout mice compared to wild-types (C57/BL6) in vitro. To analyze the effect of gene deletion in vivo, hearing and cochlear microanatomy were evaluated in Akt isoform knockout animals. In this study, we found that all three Akt isoforms are expressed in the cochlea. Our results further indicate that Akt2 and Akt3 enhance hair cell resistance to ototoxicity, while Akt1 does not. Finally, we determined that untreated Akt1 and Akt2/Akt3 double knockout mice display significant hearing loss, indicating a role for these isoforms in normal hearing. Taken together, our results indicate that each of the Akt isoforms plays a distinct role in the mammalian inner ear.     

Dephosphorylation of Synaptosomal Proteins P96 and P139 Is Regulated by Both Depolarization and Calcium, but Not by a Rise in Cytosolic Calcium Alone

Depolarization of intact synaptosomes activates calcium channels, leads to an influx of calcium, and increases the phosphorylation of several neuronal proteins. In contrast, there are two synaptosomal phosphoproteins labeled in intact synaptosomes with 32Pi, termed P96 and P139, which appear to be dephosphorylated following depolarization. Within intact synaptosomes P96 was found in the cytosol whereas P139 was present largely in membrane fractions. Depolarization-stimulated dephosphorylation was fully reversible and continued for up to five cycles of depolarization/repolarization, suggesting a physiological role for the phenomenon. The basal phosphorylation of these proteins was at least partly regulated by cyclic AMP, since dibutyryl cyclic AMP produced small but significant increases in P96 and P139 labeling, even in the presence of fluphenazine at concentrations that inhibited calcium-stimulated protein kinases. Depolarization-dependent dephosphorylation was independent of a rise in intracellular calcium, since agents such as guanidine and low concentrations of A23187, which increase intracellular calcium without activating the calcium channel, did not initiate P96 or P139 dephosphorylation. These agents did sustain increases in the phosphorylation of a number of other proteins including synapsin I and protein III. The results suggest that the phosphorylation of these two synaptosomal proteins is intimately linked to the membrane potential and that their dephosphorylation is dependent on both the mechanism of calcium entry and calcium itself, rather than simply on a rise in intracellular free calcium.     

Activity, but not Expression, of Soluble and Cell Wall-Bound Acid Invertases Is Induced by Abscisic Acid in Developing Apple Fruit

The present experiment, involving both the in vivo injection of abscislc acid （ABA） Into apple （Malus domestica Brohk.） fruits and the in vivo Incubation of fruit tissues in ABA-contalnlng medium, revealed that ABA activates both soluble and cell wall-bound acid invertases. Immunoblottlng and enzyme-linked Immunosorbent assays showed that this ABA-induced acid invertase activation is Independent of the amount of enzyme present. The acid Invertase activation induced by ABA is dependent on medium pH, time course, ABA dose, living tissue and developmental stage. Two isomers of cls-（＋）-ABA, （-）-ABA and trans- ABA, had no effect on acid invertases, showing that ABA-induced acid invertase activation is specific to physiologically active cis-（＋）ABA. Protein kinase inhlbltors K252a and H7 as well as acid phosphatase Increased the ABA-Induced effects. These data indicate that ABA specifically activates both soluble and cell wall-bound acid Invertases by a posttranslational mechanism probably Involving reversible protein phosphorylatlon, and this may be one of the mechanisms by which ABA Is Involved In regulating fruit development.     

Lipocalin 2 Expression and Secretion Is Highly Regulated by Metabolic Stress,Cytokines, and Nutrients in Adipocytes

Lipocalin 2 (Lcn2) has been recently characterized as a new adipokine having a role in innate immunity and energy metabolism. Nonetheless, the metabolic regulation of Lcn2 production in adipocytes has not been comprehensively studied. To better understand the Lcn2 biology, we investigated the regulation of Lcn2 expression in adipose tissue in response to metabolic stress in mice as well as the control of Lcn2 expression and secretion by cytokines and nutrients in 3T3-L1 adipocytes. Our results showed that the mRNA expression of Lcn2 was upregulated in white and brown adipose tissues as well as liver during fasting and cold stress in mice. Among pro-inflammatory cytokines TNFα, IL-1β, and IL-6, IL-1β showed most profound effect on Lcn2 expression and secretion in 3T3-L1 adipocytes. Insulin stimulated Lcn2 expression and secretion in a dose-dependent manner; this insulin effect was significantly abolished in the presence of low concentration of glucose. Moreover, insulin-stimulated Lcn2 expression and secretion was also attenuated when glucose was replaced by 3-O-methyl-d-glucose or by blocking NFκB pathway activation. Additionally, we showed that palmitate and oleate induced Lcn2 expression and secretion more significantly than EPA, while phytanic acid reduced Lcn2 production. Our results demonstrated that Lcn2 production in adipocytes is highly responsive to metabolic stress, cytokines, and nutrient signals, suggesting an important role of Lcn2 in adipocyte metabolism and inflammation.     

Positive Surgical Margin,HPV Persistence,and Expression of Both TPX2 and PD-L1 Are Associated with Persistence/Recurrence of Cervical Intraepithelial Neoplasia after Cervical Conization

Objective

To determine the clinicopathologic and immunohistochemical predictors of the persistence/recurrence of cervical intraepithelial neoplasia (CIN) after cervical conization.

Methods

Medical records of 502 patients who received cervical conization treatment of CIN between 2005 and 2012 were reviewed. The clinicopathologic parameters were analyzed using Cox hazard regression. Fifty patients with CIN persistence/recurrence were matched to 50 cases without CIN persistence/recurrence. These 100 cervical specimens were assessed for expression of insulin-like growth factor II messenger RNA (mRNA)-binding protein 3 (IMP3), targeting protein for xenopus kinesin-like protein 2 (TPX2), and programmed cell death-1 ligand-1 (PD-L1) using immunohistochemical staining.

Results

Multivariate analysis found that the independent predictors of CIN persistence/recurrence were positive surgical margin (hazard ratio 5.777, 95% confidence interval 2.334–14.301, p < 0.001) and human papilloma virus persistence for 6 months (hazard ratio 20.685, 95% confidence interval 7.350–57.657, p < 0.001). Co-expression of TPX2 and PD-L1 was significantly higher in CIN persistence/recurrence group than the group without CIN persistence/recurrence (p = 0.013). The depth of glandular involvement (GI) was less than 3mm in about 86.8% (59/68) CIN2-3 lesions, However, No statistically significant associations between GI and persistence/recurrence were observed (P = 0.58).

Conclusion

Positive surgical margin, HPV persistence, and expression of both TPX2 and PD-L1 are associated with persistence/recurrence of cervical intraepithelial neoplasia after cervical conization.     

TIA-1 Self-Multimerization,Phase Separation,and Recruitment into Stress Granules Are Dynamically Regulated by Zn2+

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Hypoxia Induced Aggressiveness of Prostate Cancer Cells Is Linked with Deregulated Expression of VEGF, IL-6 and miRNAs That Are Attenuated by CDF

Tumor hypoxia with deregulated expression of hypoxia inducing factor (HIF) and its biological consequence leads to poor prognosis of patients diagnosed with solid tumors, resulting in higher mortality, suggesting that understanding of the molecular relationship of hypoxia with other cellular features of tumor aggressiveness would be invaluable for developing newer targeted therapy for solid tumors. Emerging evidence also suggest that hypoxia and HIF signaling pathways contributes to the acquisition of epithelial-to-mesenchymal transition (EMT), maintenance of cancer stem cell (CSC) functions, and also maintains the vicious cycle of inflammation, all of which contribute to radiation therapy and chemotherapy resistance. However, the detailed mechanisms by which hypoxia/HIF drive these events are not fully understood. Here, we have shown that hypoxia leads to increased expression of VEGF, IL-6, and CSC marker genes such as Nanog, Oct4 and EZH2, and also increased the expression of miR-21, an oncogenic miRNA, in prostate cancer (PCa) cells (PC-3 and LNCaP). The treatment of PCa cells with CDF, a novel Curcumin-derived synthetic analogue previously showed anti-tumor activity in vivo, inhibited the productions of VEGF and IL-6, and down-regulated the expression of Nanog, Oct4, EZH2 mRNAs, as well as miR-21 under hypoxic condition. Moreover, CDF treatment of PCa cells led to decreased cell migration under hypoxic condition. Taken together, these results suggest that the anti-tumor effect of CDF is in part mediated through deregulation of tumor hypoxic pathways, and thus CDF could become useful for cancer therapy.     

Convergence of Ca2+-desensitizing mechanisms activated by forskolin and phenylephrine pretreatment, but not 8-bromo-cGMP

Contractile stimuli can sensitize myosin to Ca²⁺ by activating RhoA kinase (ROK) and PKC that inhibit myosin light chain phosphatase (MLCP) activity. Relaxant stimuli, acting through PKA and PKG (cyclic nucleotide-dependent protein kinases), and pretreatment with contractile agents such as phenylephrine (PE), can desensitize myosin to Ca²⁺. It is unknown precisely how these stimuli cause Ca²⁺ desensitization. To test the hypothesis that PKA, PKG, and PE pretreatment signaling systems converge to cause relaxation by inhibition of ROK in intact, isolated tissues, we examined the effects of forskolin (FSK; PKA activation), 8-bromo-cGMP (8br-cGMP; PKG activation), and PE pretreatment on KCl-induced force maintenance in rabbit arteries, a response nearly completely dependent on ROK activation. PE pretreatment and agents activating PKA and PKG caused Ca²⁺ desensitization by inhibiting KCl-induced tonic force and MLC phosphorylation without inhibiting intracellular [Ca²⁺]. At pCa 5 in -escin-permeabilized muscle, FSK and 8b-cGMP accelerated the relaxation rate when tissues were returned to pCa 9, suggesting that both agents can elevate MLCP activity. However, a component of the Ca²⁺ desensitization attributed to PKG activation in intact tissues appeared to involve a MLC phosphorylation-independent component. Inhibition of KCl-induced tonic force by the ROK inhibitor, Y-27632, and by PE pretreatment, were synergistically potentiated by 8b-cGMP, but not FSK. FSK and PE pretreatment, but not 8b-cGMP, inhibited the KCl-induced increase in site-specific myosin phosphatase target protein-1 phosphorylation at Thr⁸⁵³. These data support the hypothesis that PKA and PE pretreatment converge on a common Ca²⁺-desensitization pathway, but that PKG can act by a mechanism different from that activated by PKA and PE pretreatment. vascular smooth muscle; Ca²⁺ sensitization; RhoA kinase; signal transduction     

Albumin Stimulates the Activity of the Human UDP-Glucuronosyltransferases 1A7, 1A8, 1A10, 2A1 and 2B15, but the Effects Are Enzyme and Substrate Dependent

Human UDP-glucuronosyltransferases (UGTs) are important enzymes in metabolic elimination of endo- and xenobiotics. It was recently shown that addition of fatty acid free bovine serum albumin (BSA) significantly enhances in vitro activities of UGTs, a limiting factor in in vitro–in vivo extrapolation. Nevertheless, since only few human UGT enzymes were tested for this phenomenon, we have now performed detailed enzyme kinetic analysis on the BSA effects in six previously untested UGTs, using 2–4 suitable substrates for each enzyme. We also examined some of the previously tested UGTs, but using additional substrates and a lower BSA concentration, only 0.1%. The latter concentration allows the use of important but more lipophilic substrates, such as estradiol and 17-epiestradiol. In five newly tested UGTs, 1A7, 1A8, 1A10, 2A1, and 2B15, the addition of BSA enhanced, to a different degree, the in vitro activity by either decreasing reaction’s K _m, increasing its V _max, or both. In contrast, the activities of UGT2B17, another previously untested enzyme, were almost unaffected. The results of the assays with the previously tested UGTs, 1A1, 1A6, 2B4, and 2B7, were similar to the published BSA only as far as the BSA effects on the reactions’ K _m are concerned. In the cases of V _max values, however, our results differ significantly from the previously published ones, at least with some of the substrates. Hence, the magnitude of the BSA effects appears to be substrate dependent, especially with respect to V _max increases. Additionally, the BSA effects may be UGT subfamily dependent since K _m decreases were observed in members of subfamilies 1A, 2A and 2B, whereas large V _max increases were only found in several UGT1A members. The results shed new light on the complexity of the BSA effects on the activity and enzyme kinetics of the human UGTs.