Characterization of the sodium/hydrogen exchanger NHA2

Cation/proton exchange has been recognized for decades in mammalian mitochondria, but the exchanger proteins have eluded identification. In this study, a cDNA from a human brain library, previously designated NHA2 in the genome, was cloned and characterized. The NHA2 transcript bears more similarity to prokaryotic than known eukaryotic sodium/proton exchangers, but it was found to be expressed in multiple mammalian organs and cultured cells. A mAb to NHA2 was generated and found to label an approximately 55-kD native protein in multiple tissues and cell lines. The specificity of this antibody was confirmed by demonstrating the loss of the native NHA2 band on immunoblots when cultured cells were treated with NHA2-specific small interfering RNA. Although NHA2 protein was detected in multiple organs, within each, its expression was restricted to specific cell types. In the kidney, co-localization with calbindin 28k and reverse transcription-PCR of microdissected tubules revealed that NHA2 is limited to the distal convoluted tubule. In cell lines, native NHA2 was localized both to the plasma membrane and to the intracellular compartment; immunogold electron microscopy of rat distal convoluted tubule demonstrated NHA2 predominantly but not exclusively on the inner mitochondrial membrane. Furthermore, co-sedimentation of NHA2 antigen and mitochondrial membranes was observed with differential centrifugation, and two mitochondrial markers co-localized with NHA2 in cultured cells. Regarding function, human NHA2 reversed the sodium/hydrogen exchanger-null phenotype when expressed in sodium/hydrogen exchanger-deficient yeast and restored the ability to defend high salinity in the presence of acidic extracellular pH. In summary, NHA2 is a ubiquitous mammalian sodium proton/exchanger that is restricted to the distal convoluted tubule in the kidney.

Hint2 is expressed in the mitochondria of H295R cells and is involved in steroidogenesis

Hint2 belongs to the superfamily of histidine triad hydrolase enzymes. Recently, it has been shown to influence the mitochondria-dependent apoptosis occurring in hepatocytes, but its mechanism of action is still obscure. Here, we demonstrate that Hint2 is expressed in the mitochondria of H295R cells and in normal adrenals, and that this protein is involved in steroidogenesis. The presence of Hint2 in H295R cells was revealed by RT-PCR and by immunoblot analysis of subcellular fractions. The protein appeared associated with mitochondrial membranes, probably facing the interior of the organelle. Hint2 overexpression in H295R cells had no effect on pregnenolone secretion elicited by angiotensin II or K+, whereas protein silencing with specific small interfering RNA resulted in a marked reduction of the steroidogenic response. The duration of the mitochondrial calcium signal induced by angiotensin II was also reduced upon Hint2 down-regulation with small interfering RNA, but not affected after its overexpression, suggesting that under basal conditions, Hint2 is optimally expressed, and not rate limiting in steroidogenesis. Moreover, Hint2 also appeared involved in Ca2+-independent pathways leading to steroid formation. Indeed, pregnenolone formation in response to either forskolin or a hydroxyl analog of cholesterol was markedly reduced after Hint2 silencing. Calcium-dependent and calcium-independent actions of Hint2 on steroidogenesis could be related to its ability to maintain a favorable mitochondrial potential. In conclusion, these data suggest that, in H295R cells, Hint2 is required for an optimal steroidogenic response, possibly because of a particular signalling function exerted within the mitochondria and that still remains to determine at the molecular level.

Transforming growth factor-beta2 upregulates sphingosine kinase-1 activity, which in turn attenuates the fibrotic response to TGF-beta2 by impeding CTGF expression

Transforming growth factor-beta2 (TGF-beta2) stimulates the expression of pro-fibrotic connective tissue growth factor (CTGF) during the course of renal disease. Because sphingosine kinase-1 (SK-1) activity is also upregulated by TGF-beta, we studied its effect on CTGF expression and on the development of renal fibrosis. When TGF-beta2 was added to an immortalized human podocyte cell line we found that it activated the promoter of SK-1, resulting in upregulation of its mRNA and protein expression. Further, depletion of SK-1 by small interfering RNA or its pharmacological inhibition led to accelerated CTGF expression in the podocytes. Over-expression of SK-1 reduced CTGF induction, an effect mediated by intracellular sphingosine-1-phosphate. In vivo, SK-1 expression was also increased in the podocytes of kidney sections of patients with diabetic nephropathy when compared to normal sections of kidney obtained from patients with renal cancer. Similarly, in a mouse model of streptozotocin-induced diabetic nephropathy, SK-1 and CTGF were upregulated in podocytes. In SK-1 deficient mice, exacerbation of disease was detected by increased albuminuria and CTGF expression when compared to wild-type mice. Thus, SK-1 activity has a protective role in the fibrotic process and its deletion or inhibition aggravates fibrotic disease.

Sphingosine kinase 1 is pivotal for Fc epsilon RI-mediated mast cell signaling and functional responses in vitro and in vivo

Mast cell degranulation is pivotal to allergic diseases; investigating novel pathways triggering mast cell degranulation would undoubtedly have important therapeutic potential. FcepsilonRI-mediated degranulation has contradictorily been shown to require SphK1 or SphK2, depending on the reports. We investigated the in vitro and in vivo specific role(s) of SphK1 and SphK2 in FcepsilonRI-mediated responses, using specific small interfering RNA-gene silencing. The small interfering RNA-knockdown of SphK1 in mast cells inhibited several signaling mechanisms and effector functions, triggered by FcepsilonRI stimulation including: Ca(2+) signals, NFkappaB activation, degranulation, cytokine/chemokine, and eicosanoid production, whereas silencing SphK2 had no effect at all. Moreover, silencing SPHK1 in vivo, in different strains of mice, strongly inhibited mast cell-mediated anaphylaxis, including inhibition of vascular permeability, tissue mast cell degranulation, changes in temperature, and serum histamine and cytokine levels, whereas silencing SPHK2 had no effect and the mice developed anaphylaxis. Our data differ from a recent report using SPHK1(-/-) and SPHK2(-/-) mice, which showed that SphK2 was required for FcepsilonRI-mediated mast cell responses. We performed experiments in mast cells derived from SPHK1(-/-) and SPHK2(-/-) mice and show that the calcium response and degranulation, triggered by FcepsilonRI-cross-linking, is not different from that triggered in wild-type cells. Moreover, IgE-mediated anaphylaxis in the knockout mice showed similar levels in temperature changes and serum histamine to that from wild-type mice, indicating that there was no protection from anaphylaxis for either knockout mice. Thus, our data strongly suggest a previously unrecognized compensatory mechanism in the knockout mice, and establishes a role for SphK1 in IgE-mediated mast cell responses.

EpCAM-targeted delivery of nanocomplexed siRNA to tumor cells with designed ankyrin repeat proteins

Specific delivery to tumors and efficient cellular uptake of nucleic acids remain major challenges for gene-targeted cancer therapies. Here we report the use of a designed ankyrin repeat protein (DARPin) specific for the epithelial cell adhesion molecule (EpCAM) as a carrier for small interfering RNA (siRNA) complementary to the bcl-2 mRNA. For charge complexation of the siRNA, the DARPin was fused to a truncated human protamine-1 sequence. To increase the cell binding affinity and the amount of siRNA delivered into cells, DARPin dimers were generated and used as fusion proteins with protamine. All proteins expressed well in Escherichia coli in soluble form, yet, to remove tightly bound bacterial nucleic acids, they were purified under denaturing conditions by immobilized metal ion affinity chromatography, followed by refolding. The fusion proteins were capable of complexing four to five siRNA molecules per protamine, and fully retained the binding specificity for EpCAM as shown on MCF-7 breast carcinoma cells. In contrast to unspecific LipofectAMINE transfection, down-regulation of antiapoptotic bcl-2 using fusion protein complexed siRNA was strictly dependent on EpCAM binding and internalization. Inhibition of bcl-2 expression facilitated tumor cell apoptosis as shown by increased sensitivity to the anticancer agent doxorubicin.

Cathepsin W expressed exclusively in CD8+ T cells and NK cells, is secreted during target cell killing but is not essential for cytotoxicity in human CTLs

OBJECTIVE: Cathepsin W (CatW, lymphopain) is a putative cysteine protease with restricted expression to natural killer (NK) cells and CD8(+) T cells and so far unknown function and properties. Here, we characterize in detail, the regulation of human CatW during T-cell development in response to different stimuli and its functional involvement in cytotoxic lymphocyte effector function. MATERIALS AND METHODS: Western blots and real time polymerase chain reaction of sorted, unstimulated, and stimulated cell subsets (thymocytes, T cells, NK cells) and their culture supernatants were used to study regulation and expression of CatW. Primary CD8(+) T cells and short-term T-cell lines were transfected with small interfering RNA to study the involvement of CatW in effector function such as target cell killing and interferon-gamma production. RESULTS: Levels of CatW expression correlate closely with cytotoxic capacity both during development and in response to factors influencing cytotoxicity. Furthermore, CatW is secreted during specific target cell killing. However, knockdown of CatW expression by small interfering RNA neither influences target cell killing nor interferon-gamma production. CONCLUSION: Despite being expressed in the effector subset of CD8(+) and NK cells and of being released during target cell killing, our functional inhibition studies exclude an essential role of CatW in the process of cytotoxicity.

Protein phosphatase 2A and phosphoprotein SET regulate androgen production by P450c17

Cytochrome P450c17 catalyzes 17 alpha-hydroxylation needed for cortisol synthesis and 17,20 lyase activity needed to produce sex steroids. Serine phosphorylation of P450c17 specifically increases 17,20 lyase activity, but the physiological factors regulating this effect remain unknown. Treating human adrenal NCI-H295A cells with the phosphatase inhibitors okadaic acid, fostriecin, and cantharidin increased 17,20 lyase activity, suggesting involvement of protein phosphatase 2A (PP2A) or 4 (PP4). PP2A but not PP4 inhibited 17,20 lyase activity in microsomes from cultured cells, but neither affected 17 alpha-hydroxylation. Inhibition of 17,20 lyase activity by PP2A was concentration-dependent, could be inhibited by okadaic acid, and was restored by endogenous protein kinases. PP2A but not PP4 coimmunoprecipitated with P450c17, and suppression of PP2A by small interfering RNA increased 17,20 lyase activity. Phosphoprotein SET found in adrenals inhibited PP2A, but not PP4, and fostered 17,20 lyase activity. The identification of PP2A and SET as post-translational regulators of androgen biosynthesis suggests potential additional mechanisms contributing to adrenarche and hyperandrogenic disorders such as polycystic ovary syndrome.

Hypoxia up-regulates expression of Eph receptors and ephrins in mouse skin.

Eph receptor tyrosine kinases and their ligands (ephrins) are key players during the development of the embryonic vasculature; however, their role and regulation in adult angiogenesis remain to be defined. Both receptors and ligands have been shown to be up-regulated in a variety of tumors. To address the hypothesis that hypoxia is an important regulator of Ephs/ephrins expression, we developed a mouse skin flap model of hypoxia. We demonstrate that our model truly represents segmental skin hypoxia by applying four independent methods: continuous measurement of partial cutaneous oxygen tension, monitoring of tissue lactate/pyruvate ratio, time course of hypoxia-inducible factor-1alpha (HIF-1alpha) induction, and localization of stabilized HIF-1alpha by immunofluorescence in the hypoxic skin flap. Our experiments indicate that hypoxia up-regulates not only HIF-1alpha and vascular endothelial growth factor (VEGF) expression, but also Ephs and ephrins of both A and B subclasses in the skin. In addition, we show that in Hep3B and PC-3 cells, the hypoxia-induced up-regulation of Ephs and ephrins is abrogated by small interfering RNA-mediated down-regulation of HIF-1alpha. These novel findings shed light on the role of this versatile receptor/ligand family in adult angiogenesis. Furthermore, our model offers considerable potential for analyzing distinct mechanisms of neovascularization in gene-targeted mice.

Double-target antisense U7 snRNAs promote efficient skipping of an aberrant exon in three human beta-thalassemic mutations.

We have used three beta-thalassemic mutations, IVS2-654, -705 and -745, that create aberrant 5' splice sites (5' ss) and activate a common cryptic 3' ss further upstream in intron 2 of the human beta-globin gene to optimize a generally applicable exon-skipping strategy using antisense derivatives of U7 small nuclear RNA (snRNA). Introducing a modified U7 snRNA gene carrying an antisense sequence against the cryptic 3' ss into cultured cells expressing the mutant beta-globin genes, restored correct beta-globin mRNA splicing for all three mutations, but the efficiency was much weaker for IVS2-654 than for the other mutations. The length of antisense sequence influenced the efficiency with an optimum of approximately 24 nucleotides. Combining two antisense sequences directed against different target sites in intron 2, either on separate antisense RNAs or, even better, on a single U7 snRNA, significantly enhanced the efficiency of splicing correction. One double-target U7 RNA was expressed on stable transformation resulting in permanent and efficient suppression of the IVS2-654 mutation and production of beta-globin. These results suggest that forcing the aberrant exon into a looped secondary structure may strongly promote its exclusion from the mRNA and that this approach may be used generally to induce exon skipping.

Dystrophic cardiomyopathy: role of TRPV2 channels in stretch-induced cell damage

Aims Duchenne muscular dystrophy (DMD), a degenerative pathology of skeletal muscle, also induces cardiac failure and arrhythmias due to a mutation leading to the lack of the protein dystrophin. In cardiac cells, the subsarcolemmal localization of dystrophin is thought to protect the membrane from mechanical stress. The absence of dystrophin results in an elevated stress-induced Ca2+ influx due to the inadequate functioning of several proteins, such as stretch-activated channels (SACs). Our aim was to investigate whether transient receptor potential vanilloid channels type 2 (TRPV2) form subunits of the dysregulated SACs in cardiac dystrophy. Methods and results We defined the role of TRPV2 channels in the abnormal Ca2+ influx of cardiomyocytes isolated from dystrophic mdx mice, an established animal model for DMD. In dystrophic cells, western blotting showed that TRPV2 was two-fold overexpressed. While normally localized intracellularly, in myocytes from mdx mice TRPV2 channels were translocated to the sarcolemma and were prominent along the T-tubules, as indicated by immunocytochemistry. Membrane localization was confirmed by biotinylation assays. Furthermore, in mdx myocytes pharmacological modulators suggested an abnormal activity of TRPV2, which has a unique pharmacological profile among TRP channels. Confocal imaging showed that these compounds protected the cells from stress-induced abnormal Ca2+ signals. The involvement of TRPV2 in these signals was confirmed by specific pore-blocking antibodies and by small-interfering RNA ablation of TRPV2. Conclusion Together, these results establish the involvement of TRPV2 in a stretch-activated calcium influx pathway in dystrophic cardiomyopathy, contributing to the defective cellular Ca2+ handling in this disease.

Nucleolar proteins regulate stage-specific gene expression and ribosomal RNA maturation in Trypanosoma brucei

Different life-cycle stages of Trypanosoma brucei are characterized by stage-specific glycoprotein coats. GPEET procyclin, the major surface protein of early procyclic (insect midgut) forms, is transcribed in the nucleolus by RNA polymerase I as part of a polycistronic precursor that is processed to monocistronic mRNAs. In culture, when differentiation to late procyclic forms is triggered by removal of glycerol, the precursor is still transcribed, but accumulation of GPEET mRNA is prevented by a glycerol-responsive element in the 3' UTR. A genome-wide RNAi screen for persistent expression of GPEET in glycerol-free medium identified a novel protein, NRG1 (Nucleolar Regulator of GPEET 1), as a negative regulator. NRG1 associates with GPEET mRNA and with several nucleolar proteins. These include two PUF proteins, TbPUF7 and TbPUF10, and BOP1, a protein required for rRNA processing in other organisms. RNAi against each of these components prolonged or even increased GPEET expression in the absence of glycerol as well as causing a significant reduction in 5.8S rRNA and its immediate precursor. These results indicate that components of a complex used for rRNA maturation can have an additional role in regulating mRNAs that originate in the nucleolus.

RNA interference in mammalian cell systems

In the last decade, few areas of biology have been transformed as thoroughly as RNA molecular biology. Without any doubt, one of the most significant advances has been the discovery of small (20-30 nucleotide) noncoding RNAs that regulate genes and genomes. The effects of small RNAs on gene expression and control are generally inhibitory, and the corresponding regulatory mechanisms are therefore collectively subsumed under the heading of RNA silencing and/or RNA interference. Two primary categories of these small RNAs - short interfering RNAs (siRNAs) and microRNAs (miRNAs) - act in both somatic and germline lineages of eukaryotic species to regulate endogenous genes and to defend the genome from invasive nucleic acids. Recent advances have revealed unexpected diversity in their biogenesis pathways and the regulatory mechanisms that they access. Our understanding of siRNA and miRNA-based regulation has direct implications for fundamental biology as well as disease aetiology and treatment as it is discussed in this review on 'new techniques in molecular biology'.

RNA editing in kinetoplastids

RNA editing in kinetoplastid protozoa is a post-transcriptional process of uridine insertion or deletion in mitochondrial mRNAs. The process involves two RNA species, the pre-edited mRNA and in most cases a trans-acting guide RNA (gRNA). Sequences within gRNAs define the position and extend of mRNA editing. Both mRNAs and gRNAs are encoded by mitochondrial genes in the kinetoplast DNA (kDNA), which consists of thousands of small circular DNA molecules, called minicircles, encoding thousands of gRNAs, catenated together and with a few mRNA encoding larger circles, the maxicircles, to form a huge DNA network. Editing has been shown to result in translatable mRNAs of bona fide mitochondrial genes as well as novel alternatively edited transcripts that are involved in the maintenance of the kDNA itself. RNA editing occurs within large protein-RNA complexes, editosomes, containing gRNA, preedited and partially edited mRNAs and also structural and catalytically active proteins. Editosomes are diverse in both RNA and protein composition and undergoe structural remodeling during the maturation. The compositional and structural diversity of editosomes further underscores the complexity of the RNA editing process.