No abstract

SummaryTo cope with manganese (Mn) deficiency, plants have evolved an efficient transport system to uptake and redistribute Mn. However, the underlying molecular mechanisms remain to be demonstrated.We carried out a forward genetic screen in a root high-affinity Mn transporter nramp1 mutant background in Arabidopsis thaliana and identified an uncharacterized Mn transport NRAMP2. We investigated the effect of nramp2 mutation on root growth and reactive oxygen species (ROS) accumulation and we also examined the NRAMP2 expression pattern, and the subcellular localization and transport activity of NRAMP2.Mutation of NRAMP2 impaired plant growth, while overexpression of NRAMP2 improved plant growth under low Mn conditions. In the nramp2-1nramp1 double mutant, Mn deficiency inhibited root cell elongation and root hair development, which was associated with increased hydrogen peroxide (H 2 O 2 ) accumulation. NRAMP2 is preferentially localized to the trans-Golgi network. NRAMP2 has Mn influx transport activity in yeast, and mutation of NRAMP2 led to greater Mn retention in roots.Our results suggest that under Mn-deficient conditions, increased accumulation of H 2 O 2 is partially responsible for the root growth inhibition and NRAMP2 is involved in remobilization of Mn in Golgi for root growth.

Androgens provide survival signals to prostate epithelial cells, and androgen ablation induces apoptosis in the prostate gland. However, the molecular mechanisms of actions of the androgen-signaling pathway in these processes are not fully understood. Here, we report that androgens induced expression of the cellular Fas/FasL-associated death domain protein-like inhibitory protein (c-FLIP) gene, which is a potent inhibitor of Fas/FasL-mediated apoptosis. The androgen receptor was recruited to the promoter of the c-FLIP gene in the presence of androgens. We found that c-FLIP promoter contained multiple functional androgen response elements. In addition, we show that c-FLIP overexpression accelerated progression to androgen independence by inhibiting apoptosis in LNCaP prostate tumors implanted in nude mice. Our results suggest that the androgen receptor affects survival and apoptosis of prostate cells through regulation of the c-FLIP gene in response to androgens.

Sindbis virus (SINV) is a mosquito-borne virus in the genus Alphavirus, family Togaviridae. Like most alphaviruses, SINVs exhibit lytic infection (apoptosis) in many mammalian cell types, but are generally thought to cause persistent infection with only moderate cytopathic effects in mosquito cells. However, there have been several reports of apoptotic-like cell death in mosquitoes infected with alphaviruses or flaviviruses. Given that apoptosis has been shown to be an antiviral response in other systems, we have constructed recombinant SINVs that express either proapoptotic or anti-apoptotic genes in order to test the effects of inducing or inhibiting apoptosis on SINV replication in mosquito cells. Recombinant SINVs expressing the pro-apoptotic genes reaper (rpr) from Drosophila or michelob_x (mx) from Aedes aegypti caused extensive apoptosis in cells from the mosquito cell line C6/36, thus changing the normal persistent infection observed with SINV to a lytic infection. Although the infected cells underwent apoptosis, high levels of virus replication were still observed during the initial infection. However, virus production subsequently decreased compared with persistently infected cells, which continued to produce high levels of virus over the next several days. Infection of C6/36 cells with SINV expressing the baculovirus caspase inhibitor P35 inhibited actinomycin D-induced caspase activity and protected infected cells from actinomycin D-induced apoptosis, but had no observable effect on virus replication. This study is the first to test directly whether inducing or inhibiting apoptosis affects arbovirus replication in mosquito cells.

Mechanical regulation of bone formation involves a complex biophysical process, yet the underlying mechanisms remain poorly understood. Polycystin-1 (PC1) is postulated to function as a mechanosensory molecule mediating mechanical signal transduction in renal epithelial cells. To investigate the involvement of PC1 in mechanical strain-induced signaling cascades controlling osteogenesis, PKD1 gene was stably silenced in osteoblastic cell line MC3T3-E1 by using lentivirus-mediated shRNA technology. Here, our findings showed that mechanical tensile strain sufficiently enhanced osteogenic gene expressions and osteoblastic proliferation. However, PC1 deficiency resulted in the loss of the ability to sense external mechanical stimuli thereby promoting osteoblastic osteogenesis and proliferation. The signal pathways implicated in this process were intracellular calcium and Akt/β-catenin pathway. The basal levels of intracellular calcium, phospho-Akt, phospho-GSK-3β and nuclear accumulation of active β-catenin were significantly attenuated in PC1 deficient osteoblasts. In addition, PC1 deficiency impaired mechanical strain-induced potentiation of intracellular calcium, and activation of Akt-dependent and Wnt/β-catenin pathways, which was able to be partially reversed by calcium ionophore A23187 treatment. Furthermore, applications of LiCl or A23187 in PC1 deficient osteoblasts could promote osteoblastic differentiation and proliferation under mechanical strain conditions. Therefore, our results demonstrated that osteoblasts require mechanosensory molecule PC1 to adapt to external mechanical tensile strain thereby inducing osteoblastic mechanoresponse, partially through the potentiation of intracellular calcium and downstream Akt/β-catenin signaling pathway.

The androgen receptor (AR) is a ligand-regulated and sequence-specific transcription factor that activates or represses expression of target genes. Here, we show that the N terminus of AR contains an inhibitory domain located in an 81-amino acid segment lying upstream of the DNA-binding domain (DBD). The inhibitory domain interacted directly with DBD and repressed DBD binding to the androgen response element. Mutations of the conserved amino acid residues (K520E and R538E) within the inhibitory domain decreased its inhibiting ability in vitro and increased AR trans-activation in vivo. These data demonstrate the existence of a novel inhibitory domain in the N-terminal part of AR, which might play important roles in the regulation of AR trans-activation.

Atopic dermatitis (AD) is characterized by a defective skin barrier, which increases the penetration of allergens and pathogens through the skin. The role of interleukin (IL)-17, a pro-inflammatory cytokine, in the pathogenesis of AD remains to be elucidated. The present study aimed to examine the effects of IL-17 on skin barrier proteins in the HaCaT cell line. The expression levels of filaggrin (FLG) and involucrin (IVL) were evaluated by reverse transcription-quantitative polymerase chain reaction and western blot analyses of the HaCaT cells following IL-17 simulation. The role of IL-17 was further examined by using small molecule inhibitors of extracellular signal-regulated kinase (ERK) and P38. Treatment of the HaCaT cells with IL-17 resulted in reduced expression levels of FLG and IVL at the mRNA and protein levels. In addition, the gene expression levels of FLG and IVL were significantly reduced in the HaCaT cells by IL-4. Treatment with the mitogen-activated protein kinase (MAPK) inhibitors, SB203580 and PD98059, significantly inhibited the effects of IL-17 on the gene and protein expression levels of FLG and IVL. Finally, the protein levels of phosphorylated ERK and P38 were significantly increased following IL-17 stimulation. Taken together, the results revealed that IL-17 reduced the expression of FLG and IVL in HaCaT cells, and this effect involved the P38/ERK MAPK signaling pathways.

While apoptosis regulation has been studied extensively in Drosophila melanogaster, similar studies in other insects, including disease vectors, lag far behind. In D. melanogaster, the inhibitor of apoptosis (IAP) protein DIAP1 is the major negative regulator of caspases, while IAP antagonists induce apoptosis, in part, by binding to DIAP1 and inhibiting its ability to regulate caspases. In this study, we characterized the roles of two IAP antagonists, Michelob_x (Mx) and IMP, in apoptosis in the yellow fever mosquito Aedes aegypti. Overexpression of Mx or IMP caused apoptosis in A. aegypti Aag2 cells, while silencing expression of mx or imp attenuated apoptosis. Addition of recombinant Mx or IMP, but not cytochrome c, to Aag2 cytosolic extract caused caspase activation. Consistent with this finding, AeIAP1 bound and inhibited both initiator and effector caspases from A. aegypti, and Mx and IMP competed with caspases for binding to AeIAP1. However, a difference was observed in the BIR domains responsible for Dronc binding by AeIAP1 versus DIAP1. These findings demonstrate that the mechanisms by which IAP antagonists regulate apoptosis are largely conserved between A. aegypti and D. melanogaster, although subtle differences exist.