Wei-Qing Han

Diversity of cultivated and uncultivated actinobacterial endophytes in the stems and roots of rice

A dual approach consisting of cultivation and molecular retrieval of actinobacterial 16S rRNA genes was used to characterize the diversity of actinobacterial community inhabiting interior of rice stems and roots. Streptomyces is the most frequently isolated genus from rice stems and roots. Forty-five clones chosen randomly among 250 clones in the 16S rRNA gene clone library from roots were affiliated with nine genera of actinobacteria and uncultured actinobacteria (Mycobacterium, Streptomyces, Micromonospora, Actinoplanes, Frankia, Dactylosporangium, Amycolatopsis, Corynebacterium, Rhodococcus, and uncultured actinobacterium). However, 33 clones from stems were affiliated with four genera and uncultured actinobacteria (Streptomyces, Mycobacterium, Nocardiodies, Janibacter, uncultured earthworm cast bacterium, uncultured earthworm intestine bacterium, and uncultured actinobacterium). Species similar to S. cyaneus were isolated from surface-sterilized roots and stems of rice and detected inside rice roots by culture-independent methods. Species similar to S. caviscabies, S. scabies, and S. turgidiscabies were simultaneously detected from the interior of rice stems by the culture-dependent and culture-independent methods. S. galilaeus was detected from the interior of rice stems and roots. These results indicated that some actinobacterial populations in rice stems were correlated with those in roots.

Hypoxia-inducible factor prolyl-hydroxylase-2 mediates transforming growth factor beta 1-induced epithelial-mesenchymal transition in renal tubular cells

Transforming growth factor beta 1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) in kidney epithelial cells plays a key role in renal tubulointerstitial fibrosis in chronic kidney diseases. As hypoxia-inducible factor (HIF)-1α is found to mediate TGF-β1-induced signaling pathway, we tested the hypothesis that HIF-1α and its upstream regulator prolyl hydroxylase domain-containing proteins (PHDs) are involved in TGF-β1-induced EMT using cultured renal tubular cells. Our results showed that TGF-β1 stimulated EMT in renal tubular cells as indicated by the significant decrease in epithelial marker P-cadherin, and the increase in mesenchymal markers α-smooth muscle actin (α-SMA) and fibroblast-specific protein 1 (FSP-1). Meanwhile, we found that TGF-β1 time-dependently increased HIF-1α and that HIF-1α siRNA significantly inhibited TGF-β1-induced EMT, suggesting that HIF-1α mediated TGF-β1 induced-EMT. Real-time PCR showed that PHD1 and PHD2, rather than PHD3, could be detected, with PHD2 as the predominant form of PHDs (PHD1:PHD2=0.21:1.0). Importantly, PHD2 mRNA and protein, but not PHD1, were decreased by TGF-β1. Furthermore, over-expression of PHD2 transgene almost fully prevented TGF-β1-induced HIF-1α accumulation and EMT marker changes, indicating that PHD2 is involved in TGF-β1-induced EMT. Finally, Smad2/3 inhibitor SB431542 prevented TGF-β1-induced PHD2 decrease, suggesting that Smad2/3 may mediate TGF-β1-induced EMT through PHD2/HIF-1α pathway. It is concluded that TGF-β1 decreased PHD2 expression via an Smad-dependent signaling pathway, thereby leading to HIF-1α accumulation and then EMT in renal tubular cells. The present study suggests that PHD2/HIF-1α is a novel signaling pathway mediating the fibrogenic effect of TGF-β1, and may be a new therapeutic target in chronic kidney diseases.

Visfatin-induced lipid raft redox signaling platforms and dysfunction in glomerular endothelial cells.

Adipokines have been reported to contribute to glomerular injury during obesity or diabetes mellitus. However, the mechanisms mediating the actions of various adipokines on the kidney remained elusive. The present study was performed to determine whether acid sphingomyelinase (ASM)-ceramide associated lipid raft (LR) clustering is involved in local oxidative stress in glomerular endothelial cells (GECs) induced by adipokines such as visfatin and adiponectin. Using confocal microscopy, visfatin but not adiponectin was found to increase LRs clustering in the membrane of GECs in a dose and time dependent manner. Upon visfatin stimulation ASMase activity was increased, and an aggregation of ASMase product, ceramide and NADPH oxidase subunits, gp91(phox) and p47(phox) was observed in the LR clusters, forming a LR redox signaling platform. The formation of this signaling platform was blocked by prior treatment with LR disruptor filipin, ASMase inhibitor amitriptyline, ASMase siRNA, gp91(phox) siRNA and adiponectin. Corresponding to LR clustering and aggregation of NADPH subunits, superoxide (O(2)(-)) production was significantly increased (2.7 folds) upon visfatin stimulation, as measured by electron spin resonance (ESR) spectrometry. Functionally, visfatin significantly increased the permeability of GEC layer in culture and disrupted microtubular networks, which were blocked by inhibition of LR redox signaling platform formation. In conclusion, the injurious effect of visfatin, but not adiponectin on the glomerular endothelium is associated with the formation of LR redox signaling platforms via LR clustering, which produces local oxidative stress resulting in the disruption of microtubular networks in GECs and increases the glomerular permeability.

Reconstitution of lysosomal NAADP-TRP-ML1 signaling pathway and its function in TRP-ML1−/− cells

It is well known that the mutation of TRP-ML1 (transient receptor potential-mucolipin-1) causes mucolipidosis IV, a lysosomal storage disease. Given that lysosomal nicotinic acid adenine dinucleotide phosphate (NAADP)-Ca(2+) release channel activity is associated with TRP-ML1, the present study was designed to test the hypothesis that NAADP regulates lysosome function via activation of TRP-ML1 channel activity. Using lysosomal preparations from wild-type (TRP-ML1(+/+)) human fibroblasts, channel reconstitution experiments demonstrated that NAADP (0.01-1.0 μM) produced a concentration-dependent increase in TRP-ML1 channel activity. This NAADP-induced activation of TRP-ML1 channels could not be observed in lysosomes from TRP-ML1(-/-) cells, but was restored by introducing a TRP-ML1 transgene into these cells. Microscopic Ca(2+) fluorescence imaging showed that NAADP significantly increased intracellular Ca(2+) concentration to 302.4 ± 74.28 nM (vs. 180 ± 44.13 nM of the basal) in TRP-ML1(+/+) cells, but it had no effect in TRP-ML1(-/-) cells. If a TRP-ML1 gene was transfected into TRP-ML1(-/-) cells, the Ca(2+) response to NAADP was restored to the level comparable to TRP-ML1(+/+) cells. Functionally, confocal microscopy revealed that NAADP significantly enhanced the dynamic interaction of endosomes and lysosomes and the lipid delivery to lysosomes in TRP-ML1(+/+) cells. This functional action of NAADP was abolished in TRP-ML1(-/-) cells, but restored after TRP-ML1 gene was rescued in these cells. Our results suggest that NAADP increases lysosomal TRP-ML1 channel activity to release Ca(2+), which promotes the interaction of endosomes and lysosomes and thereby regulates lipid transport to lysosomes. Failure of NAADP-TRP-ML1 signaling may be one of the important mechanisms resulting in intracellular lipid trafficking disorder and consequent mucolipidosis.

TRAIL-Death Receptor 4 Signaling via Lysosome Fusion and Membrane Raft Clustering In Coronary Arterial Endothelial Cells: Evidence from ASM Knockout Mice

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor, death receptor 4 (DR4), have been implicated in the development of endothelial dysfunction and atherosclerosis. However, the signaling mechanism mediating DR4 activation leading to endothelial injury remains unclear. We recently demonstrated that ceramide production via hydrolysis of membrane sphingomyelin by acid sphingomyelinase (ASM) results in membrane raft (MR) clustering and the formation of important redox signaling platforms, which play a crucial role in amplifying redox signaling in endothelial cells leading to endothelial dysfunction. The present study aims to investigate whether TRAIL triggers MR clustering via lysosome fusion and ASM activation, thereby conducting transmembrane redox signaling and changing endothelial function. Using confocal microscopy, we found that TRAIL induced MR clustering and co-localized with DR4 in coronary arterial endothelial cells (CAECs) isolated from wild-type (Smpd1+/+) mice. Furthermore, TRAIL triggered ASM translocation, ceramide production, and NADPH oxidase aggregation in MR clusters in Smpd1+/+ CAECs, whereas these observations were not found in Smpd1−/− CAECs. Moreover, ASM deficiency reduced TRAIL-induced O2−⋅ production in CAECs and abolished TRAIL-induced impairment on endothelium-dependent vasodilation in small resistance arteries. By measuring fluorescence resonance energy transfer, we found that Lamp-1 (lysosome membrane marker protein) and ganglioside GM1 (MR marker) were trafficking together in Smpd1+/+ CAECs, which was absent in Smpd1−/− CAECs. Consistently, fluorescence imaging of living cells with specific lysosome probes demonstrated that TRAIL-induced lysosome fusion with membrane was also absent in Smpd1−/− CAECs. Taken together, these results suggest that ASM is essential for TRAIL-induced lysosomal trafficking, membrane fusion and formation of MR redox signaling platforms, which may play an important role in DR4-mediated redox signaling in CAECs and consequently endothelial dysfunction.

Lysosome fusion to the cell membrane is mediated by the dysferlin C2A domain in coronary arterial endothelial cells

Dysferlin has recently been reported to participate in cell membrane repair in muscle and other cells through lysosome fusion. Given that lysosome fusion is a crucial mechanism that leads to membrane raft clustering, the present study attempted to determine whether dysferlin is involved in this process and its related signalling, and explores the mechanism underlying dysferlin-mediated lysosome fusion in bovine coronary arterial endothelial cells (CAECs). We found that dysferlin is clustered in membrane raft macrodomains after Fas Ligand (FasL) stimulation as detected by confocal microscopy and membrane fraction flotation. Small-interfering RNA targeted to dysferlin prevented membrane raft clustering. Furthermore, the translocation of acid sphingomyelinase (ASMase) to membrane raft clusters, whereby local ASMase activation and ceramide production – an important step that mediates membrane raft clustering – was attenuated. Functionally, silencing of the dysferlin gene reversed FasL-induced impairment of endothelium-dependent vasodilation in isolated small coronary arteries. By monitoring fluorescence quenching or dequenching, silencing of the dysferlin gene was found to almost completely block lysosome fusion to plasma membrane upon FasL stimulation. Further studies to block C2A binding and silencing of AHNAK (a dysferlin C2A domain binding partner), showed that the dysferlin C2A domain is required for FasL-induced lysosome fusion to the cell membrane, ASMase translocation and membrane raft clustering. We conclude that dysferlin determines lysosome fusion to the plasma membrane through its C2A domain and it is therefore implicated in membrane-raft-mediated signaling and regulation of endothelial function in coronary circulation.

Requirement of translocated lysosomal V1 H+-ATPase for activation of membrane acid sphingomyelinase and raft clustering in coronary endothelial cells

The activation of translocated lysosomal H+-ATPase is attributed to FasL-induced formation and maintenance of an acid microenvironment around the endothelial cell membrane, which facilitates the activation of ASM and production of ceramide, thereby leading to MR clustering and redox signaling platform formation.

Triggering role of acid sphingomyelinase in endothelial lysosome-membrane fusion and dysfunction in coronary arteries

The present study determined whether activation of acid sphingomyelinase (ASM) drives membrane proximal lysosomes to fuse to the cell surface, facilitating membrane lipid rafts (LRs) clustering in coronary arterial endothelial cells (CAECs) and leading to endothelial dysfunction. By confocal microscopy, the activators of ASM, phosphatidylinositol (PI), and bis (monoacylglyceryl) phosphate (Bis), and an inducer of ASM, butyrate, were found to increase LRs clustering in bovine CAECs, which was blocked by lysosome fusion inhibitor vacuolin-1. However, arsenic trioxide (Ars), an inducer of de novo synthesis of ceramide, had no such effect. Similarly, vacuolin-1-blockable effects were observed using fluorescence resonance energy transfer detection. Liquid chromatography-electrospray ionization-tandem mass spectrometry analysis demonstrated that all of these treatments, even Ars, increased ceramide production in CAECs. When ASM gene was silenced, all treatments except Ars no longer increased ceramide levels. Furthermore, dynamic fluorescence monitoring in live cells showed that PI and Bis stimulated lysosome-membrane fusion in CAECs. Functionally, PI and Bis impaired endothelium-dependent vasodilation in perfused coronary arteries, which was blocked by vacuolin-1 and a lysosome function inhibitor, bafilomycine. FasL (Fas ligand), a previously confirmed lysosome fusion stimulator as a comparison, also produced a similar effect. It is concluded that ASM activation serves as a triggering mechanism and driving force, leading to fusion of membrane proximal lysosomes into LR clusters on the cell membrane of CAECs, which represents a novel mechanism mediating endothelial dysfunction during death receptor activation or other pathological situation.

Reversal by Growth Hormone of Homocysteine- induced Epithelial-to-Mesenchymal Transition through Membrane Raft-Redox Signaling in Podocytes

Epithelial-to-Mesenchymal Transition (EMT) is an important pathogenic mechanism mediating glomerular injury or sclerosis in a variety of renal and systemic diseases such as hyperhomocysteinemia (hHcys). The present study was designed to test whether Hcys-induced EMT in podocytes is reversed by growth hormone (GH), a hormone regulating cell differentiation and growth and to explore the cellular and molecular mechanism mediating its action. It was found that Hcys induced significant EMT in podocytes, as shown by marked decreases in slit diaphragm-associated protein P-cadherin and zonula occludens-1 as epithelial markers and by dramatic increases in the expression of mesenchymal markers, fibroblast specific protein-1 and α-smooth muscle actin, which were detected by all examinations via immunocytochemistry, real time RT-PCR and Western blot analysis. When podocytes were treated with GH at 25 ng/mL, however, Hcys failed to induce podocyte EMT. Using electromagnetic spin resonance spectrometry, Hcys-induced superoxide (O2.-) production via NADPH oxidase was found to be significantly inhibited by GH (66%). Functionally, GH was shown to substantially inhibit Hcys-induced increases in the permeability of podocyte monolayers and to block the decrease in podocin expression in these cells. In addition, NADPH oxidase subunit, gp91phox and GH receptors aggregated in membrane raft clusters, which produced O2.- in response to Hcys and could be blocked by GH, membrane raft disruptors filipin and MCD or NADPH oxidase inhibitor, apocynin. It is concluded that Hcys-induced podocyte EMT is associated with transmembrane membrane raft-redox signaling and that GH reverses this Hcys-induced EMT protecting podocytes from functional disturbance.

The PDE1A-PKCα Signaling Pathway Is Involved in the Upregulation of α-Smooth Muscle Actin by TGF-β1 in Adventitial Fibroblasts

Background: Increasing evidence has suggested that differentiation of adventitial fibroblasts (AFs) to myofibroblasts plays an important role in arterial remodeling. The molecular mechanisms by which myofibroblast formation is regulated still remain largely unknown. This study aimed to evaluate the role of cyclic nucleotide phosphodiesterase 1A (PDE1A) in the formation of adventitial myofibroblasts induced by transforming growth factor (TGF)-β1. Methods and Results: AFs were cultured by the explant method. Western blot and immunocytochemistry were applied for α-smooth muscle actin (SMA) or protein kinase C (PKC) α protein analysis. Results showed that TGF-β1 upregulated PDE1A protein expression in rat aortic AFs and pharmacological inhibition of PDE1A blocked TGF-β1-induced α-SMA expression, a marker of myofibroblast formation, suggesting that the upregulation of PDE1A may mediate TGF-β1-induced AF transformation. Moreover, calphostin C (a PKC inhibitor) inhibited TGF-β1-induced α-SMA expression, whereas phorbol-12-myristate-13-acetate (a PKC activator) induced it. Finally, the upregulation of PKCα expression by TGF-β1 was also inhibited by PDE1A inhibition. Conclusions: Taken together, our data suggest that TGFβ1 induces α-SMA expression and myofibroblast formation via a PDE1A-PKCα-dependent mechanism. Our study thus unveils a novel signaling mechanism underlying TGF-β1-induced adventitial myofibroblast formation.