Genesis and reversal of the ischemic phenotype in epithelial cells.
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Digestive System↗
Biomedical subjects
S K Nigam
Publications and source records attributed to S K Nigam.
A study of electroencephalogram in meditators.
Electroencephalographic patterns were studied in 30 normal healthy individuals practicing meditation and compared with 10 normal healthy controls not practicing meditation. In this study, we found prominent alpha wave activity and increase it its voltage in meditators as compared to controls. Meditators had significantly more alpha rhythm as compared to control group. Percentage of alpha waves were higher in persons performing meditation with good coherence which suggested good homogenicity, uniformity and increased orderliness of brain.
Adolescent↗
Cell-cell dissociation upon epithelial cell scattering requires a step mediated by the proteasome.
During development, tissue repair, and tumor metastasis, both cell-cell dissociation and cell migration occur and appear to be intimately linked, such as during epithelial "scattering." Here we show that cell-cell dissociation during scattering induced by hepatocyte growth factor (HGF) or activation of the temperature-sensitive v-Src tyrosine kinase in MDCK cells can be blocked by inhibiting the proteasome with lactacystin and MG132. Although both proteins of the tight junction and the adherens junction redistributed during cell scattering, proteasome inhibitors largely prevented this process, resulting in the stabilization of Triton X-100-insoluble tight junction proteins as well as adherens junction proteins at sites of cell-cell contact. Proteasome inhibition also led to a decrease of E-cadherin turnover in (35)S-labeled cells. In addition, proteasome inhibition partly preserved cell polarity, as determined by the subcellular distribution of Na(+),K(+)-ATPase (basolateral marker) and gp135 (apical marker), and the structure of the subcortical actin ring, both of which are normally disrupted during scattering. However, cells were able to establish focal contacts, and single cell migration toward HGF was unaffected by proteasome inhibition in quantitative assays, indicating that cell-cell dissociation during scattering occurs independently of anchorage-dependent cell migration. Thus, a proteasome-dependent step during scattering induced by HGF and pp60(v-Src) appears to be essential for cell-cell dissociation, disassembly of junctional components, and (at least indirectly) it also plays a role in the loss of protein polarity.
Acetylcysteine↗
Branching morphogenesis independent of mesenchymal-epithelial contact in the developing kidney.
Whether mesenchymal-epithelial interactions leading to branching morphogenesis in developing epithelial tissues such as the kidney require direct cell-cell contact or are due to soluble mediators elaborated by the inducing tissue has been the subject of much debate. Here we demonstrate that ureteric bud (UB) epithelium, from which the kidney collecting system and upper urinary tract are derived, can undergo impressive three-dimensional branching morphogenesis when cultured in the appropriate extracellular matrix context in the absence of direct contact with mesenchymal tissue, indicating that the program for branching morphogenesis is inherent to the UB. Both a soluble factor in BSN cell-conditioned medium (BSN-CM) derived from an immortalized cell line thought to originate in the early metanephric mesenchyme and glial cell line-derived neurotrophic factor (GDNF) were required for early and later events in branching morphogenesis. In the absence of BSN-CM, the isolated UB did not survive; a similar result was obtained in the presence of neutralizing antibodies against glial cell line-derived neurotrophic factor. Preliminary analysis of key activity present in BSN-CM indicates that it is a heat-sensitive, heparin-binding factor with a probable molecular mass greater than 100 kDa. When the in vitro cultured UB was recombined with freshly isolated metanephric mesenchyme, nephric units were induced in the mesenchyme, and the UB branches underwent elongation. Our data suggest that, although UB branching morphogenesis per se does not require direct mesenchymal contact, such contact may play a key role in regulating branch elongation and establishing the pattern of branching. The results also suggest an approach to in vitro engineering of nephron.
Animals↗
Less degradation, more shock, please.
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Animals↗
Role of tyrosine phosphorylation in the reassembly of occludin and other tight junction proteins.
After the simulation of anoxia by ATP depletion of MDCK cell monolayers with metabolic inhibitors, the tight junction (TJ) is known to become structurally perturbed, leading to loss of the permeability barrier. Peripheral TJ proteins such as zonula occludens 1 (ZO-1), ZO-2, and cingulin become extremely insoluble and associate into large macromolecular complexes (T. Tsukamoto and S. K. Nigam. J. Biol. Chem. 272: 16133-16139, 1997). For up to 3 h, this process is reversible by ATP repletion. We now show that the reassembly process depends on tyrosine phosphorylation. Recovery of transepithelial electrical resistance in ATP-replete monolayers was markedly inhibited by the tyrosine kinase inhibitor, genistein. Indirect immunofluorescence revealed a decrease in staining of occludin, a membrane component of the TJ, in the region of the TJ after ATP depletion, which reversed after ATP repletion; this reversal process was inhibited by genistein. Examination of the Triton X-100 solubilities of occludin and several nonmembrane TJ proteins revealed a shift of occludin and nonmembrane TJ proteins into an insoluble pool following ATP depletion. These changes reversed after ATP repletion, and the movement of insoluble occludin, ZO-1, and ZO-2 back into the soluble pool was again via a genistein-sensitive mechanism. Rate-zonal centrifugation analyses of detergent-soluble TJ proteins showed a reversible increase in higher density fractions following ATP depletion-repletion, although this change was not affected by genistein. In 32P-labeled cells, dephosphorylation of all studied TJ proteins was observed during ATP depletion, followed by rephosphorylation during ATP repletion; rephosphorylation of occludin was inhibited by genistein. Furthermore, during the ATP repletion phase, tyrosine phosphorylation of Triton X-100-insoluble occludin, which is localized at the junction, as well as ZO-2, p130/ZO-3 (though not ZO-1), and other proteins was evident; this tyrosine phosphorylation was completely inhibited by genistein. This indicates that tyrosine kinase activity is necessary for TJ reassembly during ATP repletion and suggests an important role for the tyrosine phosphorylation of occludin, ZO-2, p130/ZO-3, and possibly other proteins in the processes involved in TJ (re)formation.
Adenosine Triphosphate↗
Pretreatment with inducers of ER molecular chaperones protects epithelial cells subjected to ATP depletion.
We have investigated the potential cytoprotective role of endoplasmic reticulum (ER) molecular chaperones in a cultured cell model of renal ischemia. Madin-Darby canine kidney (MDCK) cells were pretreated with tunicamycin (an inducer of ER but not cytosolic molecular chaperones) for 12-16 h, followed by 6 h of ATP depletion. A rapid and severe depletion of cellular ATP was noted in both control and tunicamycin-treated cells. Trypan blue exclusion assays indicated that pretreatment of MDCK cells with tunicamycin reduced ATP depletion-induced cell damage by approximately 80% compared with nonpretreated controls. This apparent cytoprotective effect was also found following pretreatment with another inducer of ER molecular chaperones (i.e., A23187). For example, A23187 was found to reduce lactate dehydrogenase release by approximately 50% compared with untreated controls, whereas E-64, a cysteine protease inhibitor which may affect degradation of some proteins in the ER, had little or no effect on cell injury. Moreover, a fluorescent assay confirmed the marked reduction in cell damage following ATP depletion (up to 80% reduction in tunicamycin-pretreated cells). Together, these findings are consistent with the notion that induction of ER molecular chaperones leads to the acquisition of cytoprotection in the face of ATP depletion. However, inhibition of protein translation by cycloheximide was found to only partially attenuate the observed cytoprotective effect, raising the possibility that other, as yet to be identified, nonprotein synthesis-dependent mechanisms may also play a role in the observed cytoprotection.
Adenosine Triphosphate↗
A role for intracellular calcium in tight junction reassembly after ATP depletion-repletion.
The integrity of the tight junction (TJ), which is responsible for the permeability barrier of the polarized epithelium, is disrupted during ischemic injury and must be reestablished for recovery. Recently, with the use of an ATP depletion-repletion model for ischemia and reperfusion injury in Madin-Darby canine kidney cells, TJ proteins such as zonula occludens-1 (ZO-1) were shown to reversibly form large complexes and associate with cytoskeletal proteins (T. Tsukamoto and S. K. Nigam, J. Biol. Chem. 272: 16133-16139, 1997). In this study, we examined the role of intracellular calcium in TJ reassembly after ATP depletion-repletion by employing the cell-permeant calcium chelator 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM). Lowering intracellular calcium during ATP depletion is associated with significant inhibition of the reestablishment of the permeability barrier following ATP repletion as measured by transepithelial electrical resistance and mannitol flux, marked alterations in the subcellular localization of occludin by immunofluorescent analysis, and decreased solubility of ZO-1 and other TJ proteins by Triton X-100 extraction assay, suggesting that lowering intracellular calcium potentiates the interaction of TJ proteins with the cytoskeleton. Coimmunoprecipitation studies indicated that decreased solubility may partly result from the stabilization of large TJ protein-containing complexes with fodrin. Although ionic detergents (SDS and deoxycholate) appeared to cause a dissociation of ZO-1-containing complexes from the cytoskeleton, sucrose gradient analyses of the solubilized proteins suggested that calcium chelation leads to self-association of these complexes. Together, these results raise the possibility that intracellular calcium plays an important facilitatory role in the reassembly of the TJ damaged by ischemic insults. Calcium appears to be necessary for the dissociation of TJ-cytoskeletal complexes, thus permitting functional TJ reassembly and paracellular permeability barrier recovery.
Adenosine Triphosphate↗
Evolution of gene expression patterns in a model of branching morphogenesis.
Branching morphogenesis of the ureteric bud in response to unknown signals from the metanephric mesenchyme gives rise to the urinary collecting system and, via inductive signals from the ureteric bud, to recruitment of nephrons from undifferentiated mesenchyme. An established cell culture model for this process employs cells of ureteric bud origin (UB) cultured in extracellular matrix and stimulated with conditioned media (BSN-CM) from a metanephric mesenchymal cell line (H. Sakurai, E. J. Barros, T. Tsukamoto, J. Barasch, and S. K. Nigam. Proc. Natl. Acad. Sci. USA 94: 6279-6284, 1997.). In the presence of BSN-CM, the UB cells form branching tubular structures reminiscent of the branching ureteric bud. The pattern of gene regulation in this model of branching morphogenesis of the kidney collecting system was investigated using high-density cDNA arrays. Software and analytical methods were developed for the quantification and clustering of genes. With the use of a computational method termed "vector analysis," genes were clustered according to the direction and magnitude of differential expression in n-dimensional log-space. Changes in gene expression in response to the BSN-CM consisted primarily of differential expression of transcription factors with previously described roles in morphogenesis, downregulation of pro-apoptotic genes accompanied by upregulation of anti-apoptotic genes, and upregulation of a small group of secreted products including growth factors, cytokines, and extracellular proteinases. Changes in expression are discussed in the context of a general model for epithelial branching morphogenesis. In addition, the cDNA arrays were used to survey expression of epithelial markers and secreted factors in UB and BSN cells, confirming the largely epithelial character of the former and largely mesenchymal character of the later. Specific morphologies (cellular processes, branching multicellular cords, etc.) were shown to correlate with the expression of different, but overlapping, genomic subsets, suggesting differences in morphogenetic mechanisms at these various steps in the evolution of branching tubules.
Animals↗
Folding of secretory and membrane proteins.
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Cystic Fibrosis↗
Involvement of Galphai2 in the maintenance and biogenesis of epithelial cell tight junctions.
Polarized epithelial cells have highly developed tight junctions (TJ) to maintain an impermeant barrier and segregate plasma membrane functions, but the mechanisms that promote TJ formation and maintain its integrity are only partially defined. Treatment of confluent monolayers of Madin-Darby canine kidney (MDCK) cells with AlF4- (activator of heterotrimeric G protein alpha subunits) results in a 3-4-fold increase in transepithelial resistances (TER), a reliable indicator of TJ integrity. MOCK cells transfected with activated Galpha0 (Q205L) have acclerated TJ formation (Denker, B. M., Saha, C. , Khawaja, S., and Nigam, S. J. (1996) J. Biol. Chem. 271, 25750-25753). Galphai2 has been localized within the tight junction, and a role for Galphai2 in the formation and/or maintenance of the tight junction was studied by transfection of MDCK cells with vector without insert (PC), wild type Galphai2, or a GTPase-deficient mutant (constitutively activated), Q205Lalphai2. Tryptic conformational analysis confirmed expression of a constitutively active Galphai2 in Q205Lalphai2-MDCK cells, and confocal microscopy showed a similar pattern of Galphai2 localization in the three cell lines. Q205Lalphai2-MDCK cells had significantly higher base-line TER values than wild type Galphai2- or PC-MDCK cells (1187 +/- 150 versus 576 +/- 89 (Galphai2); 377 +/- 52 Omega.cm2 (PC)), and both Galphai2- and Q205Lalphai2-transfected cell lines more rapidly develop TER in the Ca2+ switch, a model widely used to study the mechanisms of junctional assembly. Treatment of cells with AlF4- during the Ca2+ switch had little effect on the kinetics of TER development in Galphai2- or Q205Lalphai2-MDCK cells, but PC cells reached half-maximal TER significantly sooner in the presence of AlF4- (similar times to Galphai2-transfected cells). Base-line TER values obtained after the switch were significantly higher for all three cell lines in the presence of AlF4-. These findings indicate that Galphai2 is important for both the maintenance and development of the TJ, although additional Galpha subunits are likely to play a role.
Aluminum Compounds↗
Four acylated triterpenoid saponins from Albizia procera.
Four new oleanane-type triterpene glycosides, proceraosides A-D (1-4), were isolated from the seeds of Albizia procera. Their structures were established by extensive NMR experiments and chemical methods. Compounds 1-3 comprised acacic acid as the aglycon and a monoterpenic carboxylic acid linked to a monoterpene quinovoside as the acyl moiety at C-21. The common oligosaccharide moiety linked to C-28 in 1-3 was determined as alpha-l-arabino- furanosyl-(1-->4)-[beta-D-glucopyranosyl-(1-->3)]-alpha-L-rhamnopyranosy l- (1-->2)-beta-D-glucopyranosyl ester. These compounds differed in the C-3-linked sugar unit or in the configuration of C-6' of the inner monoterpene moiety in the C-21-linked acyl unit. Compound 4 was established as the 16-deoxy analogue of 1.
Carbohydrate Sequence↗
In vitro branching tubulogenesis: implications for developmental and cystic disorders, nephron number, renal repair, and nephron engineering.
Branching tubulogenesis of the ureteric bud is critically important for kidney development. Recent findings using three-dimensional cell culture systems for in vitro branching tubulogenesis are likely to shed light on the mechanisms of ureteric bud morphogenesis. Here, we try to unify these findings with those obtained using genetic approaches and organ culture of the embryonic kidney into a working model of ureteric bud branching tubulogenesis. It appears that the balance between branching tubulogenesis facilitating growth factors such as epidermal growth factor receptor ligands, hepatocyte growth factor, insulin-like growth factors, and inhibitory growth factors such as transforming growth factor beta family members may regulate branching morphogenesis. Growth factors induce epithelial cell proliferation, migration, and modulate the expression of a variety of proteins. Downstream in the growth factor-mediated tubulogenesis pathway, extracellular proteases, protease inhibitors, extracellular matrix proteins, and integrins are likely to act as effectors and regulators of branching tubulogenesis. Discussed in some detail are the relevance of insights gleaned from in vitro models of branching tubulogenesis to congenital urogenital abnormalities, cystic kidney diseases, oligonephropathies and hypertension, tubular cell regeneration after injury, and tubular engineering.
Animals↗
Molecular structure and assembly of the tight junction.
Polarized epithelial cells separate two extremely different cellular milieus. The tight junction (TJ) is the most apical component of the junctional complex and serves as the permeability barrier between these environments. The tight junctional complex appears to be a dynamic and regulated structure. Some of its protein components have been identified and include the transmembrane protein occludin. Nontransmembrane proteins on the cytosolic leaflet including ZO-1, ZO-2, cingulin, 7H6, and several unidentified phosphoproteins are also believed to be part of the TJ. Interactions of some of these proteins with the actin cytoskeleton are a major determinant of TJ structure and may also play a role in the regulation of TJ assembly. Recent progress using the "calcium switch" and the "ATP depletion-repletion" model of TJ formation offers new insight regarding how these structures form. TJ biogenesis appears to be regulated, in part, by classic signal transduction pathways involving heterotrimeric G proteins, release of intracellular Ca2+, and activation of protein kinase C. Although many of the details of the signaling pathways have yet to be defined, these observations may provide insight into how TJs form during tubular development. Furthermore, it may be possible to suggest potential therapeutic targets for intervention in a variety of diseases (e.g., ischemia, toxic injury to the kidney and other epithelial tissue) where TJ integrity has been compromised and reassembly is required.
Animals↗
Acute renal failure. I. Relative importance of proximal vs. distal tubular injury.
For more than 15 years, there has been an ongoing debate regarding the nephron segment(s) most severely injured in acute renal failure (ARF) induced by an ischemic or toxic insult. Although some investigators have argued that the proximal tubule (and particularly the S3 segment) is the major target of injury in ARF, others have held the view that damage to the distal nephron [particularly the medullary thick ascending limb (MTAL) segment] plays a more important role in this disease. In this discussion, the first of three on different aspects of ARF that have been hotly debated, we have invited several experts to discuss their opinions on this issue. The goals of this first discussion (and the subsequent two articles in this forum) are to establish areas of consensus in each area of controversy and also to identify unanswered questions that represent important areas for future research.
Animals↗
Tight junction proteins form large complexes and associate with the cytoskeleton in an ATP depletion model for reversible junction assembly.
A key feature of the ischemic epithelial cell phenotype is the disruption of tight junctions (TJ). In a Manin-Darby canine kidney cell model for ischemia-reperfusion/hypoxia-reoxygenation injury which employs inhibitors of glycolysis (2-deoxy-D-glucose) and oxidative phosphorylation (antimycin A), transepithelial electrical resistance, a measure of TJ integrity, dropped rapidly, correlating well with declining ATP levels. Although immunocytochemical studies revealed only subtle changes in the distribution of the TJ proteins, zonula occludens (ZO)-1, ZO-2, and cingulin, examination of the Triton X-100 solubilities of these proteins, an indicator of cytoskeletal association, revealed a striking shift of all three TJ proteins into the insoluble pool, consistent with increased cytoskeletal interaction during ATP depletion. In addition, rate-zonal centrifugation analysis of a detergent-soluble fraction showed an increase in the amount of ZO-1 and ZO-2 in high density fractions following ATP depletion, providing further evidence for association of TJ proteins into a large complex possibly involving the cytoskeleton. Analysis of immunoprecipitation data from [35S]methionine-labeled cells revealed that ATP depletion led to the association of a 240-kDa protein with the ZO-1-containing complex. Western blots of this protein immunoprecipitated with anti-ZO-1 antibodies confirmed its identity as fodrin, a protein believed to link membrane and other proteins to the actin-based cytoskeleton. Together, our data suggest that in the absence of major immunocytochemical changes, ATP depletion leads TJ proteins to form large insoluble complexes and associate with the cytoskeleton. We propose a model in which a key, potentially regulated, step in the generation of the ischemic epithelial cell phenotype is the interaction between TJ proteins and fodrin and/or other cytoskeletal proteins.
Adenosine Triphosphate↗
An in vitro tubulogenesis system using cell lines derived from the embryonic kidney shows dependence on multiple soluble growth factors.
Interactions between the ureteric bud (UB) and metanephric mesenchyme are crucial for tubulogenesis during kidney development. Two immortalized cell lines derived from the day 11.5 embryonic kidney, UB cells, which appear to be epithelial (cytokeratin-positive, E-cadherin-positive, and ZO-1-positive by immunostaining) and BSN cells, which are largely mesenchymal (vimentin-positive, but negative for cytokeratin, cell surface E-cadherin, and cell surface ZO-1), were used to establish an in vitro tubulogenesis system. BSN cells expressed hepatocyte growth factor (HGF) and transforming growth factor-beta1 mRNAs, and its conditioned medium (BSN-CM) contained factors capable of activating the epidermal growth factor (EGF) receptor (EGFR). When UB cells were cultured in an extracellular matrix gel in the presence of the embryonic kidney or BSN-CM, the UB cells underwent morphogenetic changes characteristic of early in vitro branching tubulogenesis. These changes were largely inhibited by a combination of neutralizing anti-HGF antibodies and the EGFR inhibitor tyrphostin AG1478, suggesting that EGFR ligands, together with HGF, account for much of this early morphogenetic activity. Nevertheless, there was a significant fraction of tubulogenic activity that could not be inhibited, suggesting the existence of other soluble factors. Whereas HGF, EGF, transforming growth factor alpha, basic fibroblast growth factor (bFGF), and insulin-like growth factor 1 (IGF-1), or a mixture of these growth factors, induced epithelial processes for up to 3 days, only IGF-1, possibly bFGF, and the mixture were able to sustain morphogenesis for longer periods, though not nearly to the same degree as BSN-CM. Moreover, only BSN-CM induced branching tubular structures with clear lumens, consistent with the existence of other soluble factors crucial for the formation and/or maintenance of branching tubular structures with lumens in vitro.
Animals↗
Proteasome inhibition leads to a heat-shock response, induction of endoplasmic reticulum chaperones, and thermotolerance.
The accumulation of misfolded proteins in the cytosol leads to increased expression of heat-shock proteins, while accumulation of such proteins in the endoplasmic reticulum (ER) stimulates the expression of many ER resident proteins, most of which function as molecular chaperones. Recently, inhibitors of the proteasome have been identified that can block the rapid degradation of abnormal cytosolic and ER-associated proteins. We therefore tested whether these agents, by causing the accumulation of abnormal proteins, might stimulate the expression of cytosolic heat-shock proteins and/or ER molecular chaperones and thereby induce thermotolerance. Exposure of Madin-Darby canine kidney cells to various proteasome inhibitors, including the peptide aldehydes (MG132, MG115, N-acetyl-leucyl-leucyl-norleucinal) and lactacystin, inhibited the degradation of short-lived proteins and increased markedly the levels of mRNAs encoding cytosolic heat-shock proteins (Hsp70, polyubiquitin) and ER chaperones (BiP, Grp94, ERp72), as shown by Northern blot analysis. However, inhibitors of cysteine proteases (E64), serine proteases (leupeptin), or metalloproteases (1, 10-phenanthroline) had no effect on the levels of these mRNAs. The relative efficacies of the peptide aldehyde inhibitors in inducing these mRNAs correlated with their potencies against the proteasome. Furthermore, reduction of the aldehyde group of MG132 decreased its inhibitory effect on proteolysis and largely prevented the induction of these mRNAs. Although treatment with the proteasome inhibitors caused rapid increases in mRNA levels (as early as 2 h after treatment with MG132), the inhibitors did not detectably affect total protein synthesis, total protein secretion, ER morphology, or the retention of ER-lumenal proteins, even after 18 h of treatment. Together, the findings suggest that inhibition of proteasome function induces heat-shock proteins and ER chaperones due to the accumulation of sufficient amounts of abnormal proteins and/or the inhibition of degradation of a key regulatory factor (e.g. heat-shock factor). Since expression of heat-shock proteins can protect cells from thermal injury, we tested whether the proteasome inhibitors might also confer thermotolerance. Treatment of cells with MG132 for as little as 2 h, markedly increased the survival of cells subjected to high temperatures (up to 46 degrees C). Thus, these agents may have applications in protecting against cell injury.
Acetylcysteine↗