Bcl-X-L translocation in renal tubular epithelial cells in vitro protects distal cells from oxidative stress

Background. The molecular pathogenesis of different sensitivities of the renal proximal and distal tubular cell populations to ischemic injury, including ischemia-reperfusion (IR)-induced oxidative stress, is not well-defined. An in vitro model of oxidative stress was used to compare the survival of distal [Madin-Darby canine kidney (MDCK)] and proximal [human kidney-2 (HK-2)] renal tubular epithelial cells, and to analyze for links between induced cell death and expression and localization of selected members of the Bcl-2 gene family (anti-apoptotic Bcl-2 and Bcl-X-L, pro-apoptotic Bax and Bad), Methods. Cells were treated with 1 mmol/L hydrogen peroxide (H2O2) Or were grown in control medium for 24 hours. Cell death (apoptosis) was quantitated using defined morphological criteria. DNA gel electrophoresis was used for biochemical identification. Protein expression levels and cellular localization of the selected Bcl-2 family proteins were analyzed (West ern immunoblots, densitometry, immunoelectron microscopy). Results. Apoptosis was minimal in control cultures and was greatest in treated proximal cell cultures (16.93 +/- 4.18% apoptosis) compared with treated distal cell cultures (2.28 +/- 0.85% apoptosis, P < 0.001). Endogenous expression of Bcl-X-L and Bax, but not Bcl-2 or Bad, was identified in control distal cells, Bcl-X-L and Bax had nonsignificant increases (P > 0.05) in these cells. Bcl-2, Bax, and Bcl-X-L, but not Bad, were endogenously expressed in control proximal cells. Bcl-X-L was significantly decreased in treated proximal cultures (P < 0.05), with Bas and Bcl-2 having nonsignificant increases (P > 0.05). Immunoelectron microscopy localization indicated that control and treated hut surviving proximal cells had similar cytosolic and membrane localization of the Bcl-2 proteins. In comparison, surviving cells in the treated distal cultures showed translocation of Bcl-X-L from cytosol to the mitochondria after treatment with H2O2, a result that was confirmed using cell fractionation and analysis of Bcl-XL expression levels of the membrane and cytosol proteins. Bax remained distributed evenly throughout the surviving distal cells, without particular attachment to any cellular organelle. Conclusion. The results indicate that in this in vitro model, the increased survival of distal compared with proximal tubular cells after oxidative stress is best explained by the decreased expression of anti-apoptotic Bcl-X-L in proximal cells, as well as translocation of Bcl-X-L protein to mitochondria within the surviving distal cells.

Alterations in ciliary neurotrophic factor signaling in rapsyn deficient mice

Rapsyn is a key molecule involved in the formation of postsynaptic specializations at the neuromuscular junction, in its absence there are both pre- and post-synaptic deficits including failure to cluster acety]choline receptors. Recently we have documented increases in both nerve-muscle branching and numbers of motoneurons, suggesting alterations in skeletal muscle derived trophic support for motoneurons. The aim of the present study was to evaluate the contribution of target derived trophic factors to increases in motoneuron branching and number, in rapsyn deficient mice that had their postsynaptic specializations disrupted, We have used reverse transcription-polymerase chain reaction and Western blot to document the expression of known trophic factors and their receptors in muscle, during the period of synapse formation in rapsyn deficient mouse embryos. We found that the mRNA levels for ciliary neurotrophic factor (CNTF) was decreased in the rapsyn deficient muscles compared with litter mate controls although those for NGF, BDNF, NT-3 and TGF-beta2 did not differ. We found that both the mRNA and the protein expression for suppressor of cytokine signaling 3 (SOCS3) decreased although janus kinase 2 (JAK2) did not change in the rapsyn deficient muscles compared with litter mate controls. These results suggest that failure to form postsynaptic specializations in rapsyn deficient mice has altered the CNTF cytokine signaling pathway within skeletal muscle, the target for motoneurons. This alteration may in part, account for the increased muscle nerve branching and motoneuron survival seen in rapsyn deficient mice. (C) 2001 Wiley-Liss, Inc.

Stressor categorization: acute physical and psychological stressors elicit distinctive recruitment patterns in the amygdala and in medullary noradrenergic cell groups

It has been hypothesized that the brain categorizes stressors and utilizes neural response pathways that vary in accordance with the assigned category. If this is true, stressors should elicit patterns of neuronal activation within the brain that are category-specific. Data from previous Immediate-early gene expression mapping studies have hinted that this is the case, but interstudy differences in methodology render conclusions tenuous. In the present study, immunolabelling for the expression of c-fos was used as a marker of neuronal activity elicited in the rat brain by haemorrhage, immune challenge, noise, restraint and forced swim. All stressors elicited c-fos expression in 25-30% of hypothalamic paraventricular nucleus corticotrophin-releasing-factor cells, suggesting that these stimuli were of comparable strength, at least with regard to their ability to activate the hypothalamic-pituitary-ad renal axis. In the amygdala, haemorrhage and immune challenge both elicited c-fos expression in a large number of neurons in the central nucleus of the amygdala, whereas noise, restraint and forced swim primarily elicited recruitment of cells within the medial nucleus of the amygdala. In the medulla, all stressors recruited similar numbers of noradrenergic (A1 and A2) and adrenergic (C1 and C2) cells. However, haemorrhage and immune challenge elicited c-fos expression In subpopulations of A1 and A2 noradrenergic cells that were significantly more rostral than those recruited by noise, restraint or forced swim. The present data support the suggestion that the brain recognizes at least two major categories of stressor, which we have referred to as 'physical' and 'psychological'. Moreover, the present data suggest that the neural activation footprint that is left in the brain by stressors can be used to determine the category to which they have been assigned by the brain.

Basic fibroblast growth factor and fibroblast growth factor receptors in adult olfactory epithelium

Basic fibroblast growth factor (FGF2) stimulates proliferation of the globose basal cells, the neuron:ll precursor in the olfactory epithelium. The present study investigates the expression of basic fibroblast growth factor and fibroblast growth factor receptors in the adult olfactory epithelium. FGF2 immunoreactivity was expressed widely in the olfactory epithelium, with the highest density of immunoreactivity in the supporting cells. In contrast, most cells in the epithelium expressed FGF2 mRNA. Fibroblast growth factor receptor-1 (FGFr1) immunoreactivity was densest in the basal cell and neuronal layers of the olfactory epithelium and on the apical surface of supporting cells. In the lamina propria FGF2 immunoreactivity and mRNA were densest in cells close to the olfactory nerve bundles. FGFr1 immunoreactivity was heaviest on the olfactory ensheathing cells. Using reverse transcriptase-polymerase chain reaction analysis, the olfactory epithelium was shown to express only three receptor splice variants, including one (FGFr1c) with which basic fibroblast growth factor has high affinity. Other receptor splice variants were present in the lamina propria. Taken together, these observations indicate endogenous sources of FGF? within the olfactory epithelium and lamina propria and suggest autocrine and paracrine pathways via which FGF2 might regulate olfactory neurogenesis. The observation of only three receptor splice variants in the olfactory epithelium limits the members of the fibroblast growth factor family which could act in the olfactory epithelium. The widespread distribution of receptors suggests that fibroblast growth factors may have roles other than proliferation of globose basal cells. (C) 2001 Published by Elsevier Science B.V.

Angiotensin-converting enzyme inhibition attenuates the progression of rat hepatic fibrosis

Background & Aims: There is a significant relationship between inheritance of high transforming growth factor (TGF)-beta1 and angiotensinogen-producing genotypes and the development of progressive hepatic fibrosis in patients with chronic hepatitis C. In cardiac and renal fibrosis, TGF-beta1 production may be enhanced by angiotensin II, the principal effector molecule of the renin-angiotensin system. The aim of the present study was to determine the effects of the angiotensin converting enzyme inhibitor, captopril, on the progression of hepatic fibrosis in the rat bile duct ligation model. Methods: Rats were treated with captopril (100 mg kg(-1) day(-1)) commencing 1 or 2 weeks after bile duct ligation. Animals with bile duct ligation only and sham-operated animals sewed as controls. Four weeks after bile duct ligation, indices of fibrosis were assessed. Results: Cap topril treatment significantly reduced hepatic hydroxyproline levels, mean fibrosis score, steady state messenger RNA levels of TGF-beta1 and procollagen alpha1(I), and matrix metalloproteinase 2 and 9 activity. Conclusions: Captopril significantly attenuates the progression of hepatic fibrosis in the vat bile duct ligation model, and its effectiveness should be studied in human chronic liver diseases associated with progressive fibrosis.

Vascular smooth muscle cell phenotypic modulation in culture is associated with reorganisation of contractile and cytoskeletal proteins

Smooth muscle cells (SMC) exhibit a functional plasticity, modulating from the mature phenotype in which the primary function is contraction, to a less differentiated state with increased capacities for motility, protein synthesis, and proliferation. The present study determined, using Western analysis, double-label immunofluorescence and confocal microscopy, whether changes in phenotypic expression of rabbit aortic SMC in culture could be correlated with alterations in expression and distribution of structural proteins. Contractile state SMC (days 1 and 3 of primary culture) showed distinct sorting of proteins into subcellular domains, consistent with the theory that the SMC structural machinery is compartmentalised within the cell. Proteins specialised for contraction (alpha -SM actin, SM-MHC, and calponin) were highly expressed in these cells and concentrated in the upper central region of the cell. Vimentin was confined to the body of the cell, providing support for the contractile apparatus but not co-localising with it. In line with its role in cell attachment and motility, beta -NM actin was localised to the cell periphery and basal cortex. The dense body protein alpha -actinin was concentrated at the cell periphery, possibly stabilising both contractile and motile apparatus. Vinculin-containing focal adhesions were well developed, indicating the cells' strong adhesion to substrate. In synthetic state SMC (passages 2-3 of culture), there was decreased expression of contractile and adhesion (vinculin) proteins with a concomitant increase in cytoskeletal proteins (beta -non-muscle [NM] actin and vimentin). These quantitative changes in structural proteins were associated with dramatic chan-es in their distribution. The distinct compartmentalisation of structural proteins observed in contractile state SMC was no longer obvious, with proteins more evenly distributed throughout die cytoplasm to accommodate altered cell function. Thus, SMC phenotypic modulation involves not only quantitative changes in contractile and cytoskeletal proteins, but also reorganisation of these proteins. Since the cytoskeleton acts as a spatial regulator of intracellular signalling, reorganisation of the cytoskeleton may lead to realignment of signalling molecules, which, in turn, may mediate the changes in function associated with SMC phenotypic modulation. (C) 2001 Wiley-Liss, Inc.

A soluble form of CD83 is released from activated dendritic cells and B lymphocytes, and is detectable in normal human sera

CD83 is an inducible glycoprotein expressed predominantly by dendritic cells (DC) and B lymphocytes. Expression of membrane CD83 (mCD83) is widely used as a marker of differentiated/ activated DC but its function and ligand(s) are presently unknown. We report the existence of a soluble form of CD83 (sCD83). Using both a sCD83-specific ELISA and Western blotting, we could demonstrate the release of sCD83 by mCD83(+) B cell and Hodgkin's disease-derived cell lines, but not mCD83(-) cells. Inhibition of de novo protein synthesis did not affect the release of sCD83 during short-term (2 h) culture of cell lines although mCD83 expression was significantly reduced, suggesting sCD83 is generated by the release of mCD83. Isolated tonsillar B lymphocytes and monocyte-derived DC, which are mCD83(low), released only low levels of sCD83 during culture. However, the differentiation/activation of these populations both up-regulated mCD83 and increased sCD83 release significantly. Analysis of sera from normal donors demonstrated the presence of low levels (121 +/- 3.6 pg/ml) of circulating sCD83. Further studies utilizing purified sCD83 and the analysis of sCD83 levels in disease may provide clues to the function and ligand(s) of CD83.

Transcriptional modulation of mouse mu-opioid receptor distal promoter activity by Sox18

Previously, we reported the presence of dual promoters, referred to as distal (DP) and proximal, with a negative regulatory element between them in the mouse mu -opioid receptor (mor) gene. Here we have identified a positive regulatory element influencing mor DP transcription, which contains multiple consensus binding motifs for Sox factors (sex-determining Sry-like high mobility group box-containing genes). In gel supershift assays, the Sox family member Sox18 bound directly to the multiple Sox consensus binding motifs of the mor DP enhancer. Overexpression of Sox18 cDNA increased luciferase activity regulated by the mor DP, and did so in a Sox18 concentration-dependent manner. In contrast, overexpression of another Sox member, Sox5, triggered no such trans-activation of mor DP-driven luciferase activity or DNA-protein binding activity. These results suggest that Sox18 directly and specifically stimulates mor gene expression, by trans-activating the mor DP enhancer.

Relationship of XIST expression and responses of ovarian cancer to chemotherapy

Expression profiling to characterize cancer pharmacology has become a new approach to discover novel molecular targets for prognostic markers and cancer therapy. In a study to compare the global RNA expression profiles between primary and recurrent ovarian tumors from the same patient, we have identified XIST (inactive X chromosome-specific transcripts) as the most differentially expressed gene that was down-regulated in the recurrent tumor. XIST encodes a spliced noncoding polyadenylated transcript that is unique in being expressed exclusively from the inactive X chromosome and is involved in the X-inactivation process. Subsequent characterization of XIST expression in a panel of female cancer cell lines showed that the expression level of XIST correlates significantly with Taxol sensitivity. The clinical relevance of this observation is demonstrated by the strong association between XIST RNA levels and disease-free periods of ovarian cancer patients in a group of 21 ovarian cancer cases with Taxol in the therapeutic regiments. Cytogenetic studies on ovarian cancer cell lines indicated that loss of inactive X chromosome is one mechanism for the loss of XIST transcripts in the cell lines. Our data suggest that XIST expression may be a potential marker for chemotherapeutic responses in ovarian cancer.

RNA trafficking and stabilization elements associate with multiple brain proteins

Two of the best understood somatic cell mRNA cytoplasmic trafficking elements are those governing localization of beta-actin and myelin basic protein mRNAs. These cis-acting elements bind the trans-acting factors fibroblast ZBP-1 and hnRNP A2, respectively. It is not known whether these elements fulfil other roles in mRNA metabolism. To address this question we have used Edman sequencing and western blotting to identify six rat brain proteins that bind the beta-actin element (zipcode). All are known RNA-binding proteins and differ from ZBP-1. Comparison with proteins that bind the hnRNP A2 and AU-rich response elements, A2RE/A2RE11 and AURE, showed that AURE and zipcode bind a similar set of proteins that does not overlap with those that bind A2RE11. The zipcode-binding protein, KSRP, and hnRNP A2 were selected for further study and were shown by confocal immunolluorescence microscopy to have similar distributions in the central nervous system, but they were found in largely separate locations in cell nuclei. In the cytoplasm of cultured oligodendrocytes they were segregated into separate populations of cytoplasmic granules. We conclude that not only may there be families of trans-acting factors for the same cis-acting element, which are presumably required at different stages of mRNA processing and metabolism, but independent factors may also target different and multiple RNAs in the same cell.

Heterogeneous nuclear ribonucleoprotein A3, a novel RNA trafficking response element-binding protein

The cis-acting response element, A2RE, which is sufficient for cytoplasmic mRNA trafficking in oligodendrocytes, binds a small group of rat brain proteins. Predominant among these is heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a trans-acting factor for cytoplasmic trafficking of RNAs bearing A2RE-like sequences. We have now identified the other A2RE-binding proteins as hnRNP A1/A1(B), hnRNP B1, and four isoforms of hnRNP A3. The rat and human hnRNP A3 cDNAs have been sequenced, revealing the existence of alternatively spliced mRNAs. In Western blotting, 38-, 39-, 41 -, and 41.5-kDa components were all recognized by antibodies against a peptide in the glycine-rich region of hnRNP A3, but only the 41- and 41.5-kDa bands bound antibodies to a 15-residue N-terminal peptide encoded by an alternatively spliced part of exon 1. The identities of these four proteins were verified by Edman sequencing and mass spectral analysis of tryptic fragments generated from electrophoretically separated bands. Sequence-specific binding of bacterially expressed hnRNP A3 to A2RE has been demonstrated by biosensor and UV cross-linking electrophoretic mobility shift assays. Mutational analysis and confocal microscopy data support the hypothesis that the hnRNP A3 isoforms have a role in cytoplasmic trafficking of RNA.

Peroxisome proliferator-activated receptor alpha in the human breast cancer cell lines MCF-7 and MDA-MB-231

Peroxisome proliferator-activated receptor (PPAR) alpha is a ligand-activated transcription factor that has been linked with rodent hepatocarcinogenesis. It has been suggested that PPARalpha mRNA expression levels are an important determinant of rodent hepatic tumorigenicity. Previous work in rat mammary gland epithelial cells showed significantly increased PPARalpha mRNA expression in carcinomas, suggesting the possible role of this isoform in rodent mammary gland carcinogenesis. In this study we sought to determine whether PPARalpha is expressed and dynamically regulated in human breast cancer MCF-7 and MDA-MB-231 cells. Having established the presence of PPARalpha in both cell types, we then examined the consequence of PPARa activation, by its ligands Wy-14,643 and clofibrate, on proliferation. With real-time reverse transcriptase-polymerase chain reaction, we showed that PPARalpha mRNA was dynamically regulated in MDA-MB-231 cells and that PPARalpha activation significantly increased proliferation of the cell line. In contrast, PPARalpha expression in MCF-7 cells did not change with proliferation during culture and was present at significantly lower levels than in MDA-MB-231 cells. However, PPARalpha ligand activation still significantly increased the proliferation of MCF-7 cells. The promotion of proliferation in breast cancer cell lines following PPARalpha activation was in stark contrast to the effects of PPARgamma-activating ligands that decrease proliferation in human breast cancer cells. our results established the presence of PPARalpha in human breast cancer cell lines and showed for the first time that activation of PPARalpha in human breast cancer cells promoted proliferation. Hence, this pathway may be significant in mammary gland tumorigenesis. (C) 2002 Wiley-Liss, Inc.

Osteonectin/SPARC induction by ectopic beta(3) integrin in human radial growth phase primary melanoma cells

Expression of the beta(3) integrin subunit in melanoma in situ has been found to correlate with tumor thickness, the ability to invade and metastasize, and poor prognosis. Transition from the radial growth phase (RGP) to the vertical growth phase (VGP) is a critical step in melanoma progression and survival and is distinguished by the expression of beta(3), integrin. The molecular pathways that operate in melanoma cells associated with invasion and metastasis were examined by ectopic induction of the beta(3), integrin subunit in RGP SBcl2 and WM1552C melanoma cells, which converts these cells to a VGP phenotype. We used cDNA representational difference analysis subtractive hybridization between beta(3)-Positive and -negative melanoma cells to assess gene expression profile changes accompanying RGP to VGP transition. Fourteen fragments from known genes including osteonectin (also known as SPARC and BM-40) were identified after three rounds of representational difference analysis. Induction of osteonectin was confirmed by Northern and Western blot analysis and immunohistochemistry and correlated in organotypic cultures with the beta(3)-induced progression from RGP to VGP melanoma. Expression of osteonectin was also associated with reduced adhesion to vitronectin, but not to fibronectin. Osteonectin expression was not blocked when melanoma cells were cultured with anti-alpha(v)beta(3) LM609 mAb, mitogen-activated protein kinase, or protein kinase C inhibitors, indicating that other signaling pathway(s) operate through a(v)beta(3) integrin during conversion from RGP to VGP.

Gene complementation of airway epithelium in the cystic fibrosis mouse is necessary and sufficient to correct the pathogen clearance and inflammatory abnormalities

Increasingly, cystic fibrosis (CF) is regarded as an inflammatory disorder where the response of the lung to Pseudomonas aeruginosa is exaggerated as a consequence of processes mediated by the product of the CF gene, CFTR. Of importance to any gene-replacement strategy for treatment of CF is the identification of the cell type(s) within the lung milieu that need to be corrected and an indication whether this is sufficient to restore a normal inflammatory response and bacterial clearance. We generated G551D CF mice transgenically expressing the human CFTR gene in two tissue compartments previously demonstrated to mediate a CFTR-dependent inflammatory response: lung epithelium and alveolar macrophages. Following chronic pulmonary infection with P. aeruginosa, CF mice with epithelial-expressed but not macrophage-specific CFTR showed an improvement in pathogen clearance and inflammatory markers compared with control CF animals. Additionally, these data indicate the general role for epithelial cell-mediated events in the response of the lung to bacterial pathogens and the importance of CFTR in mediating these processes.