Mechanical culture conditions effect gene expression: gravity-induced changes on the space shuttle.

Three-dimensional suspension culture is a gravity-limited phenomenon. The balancing forces necessary to keep the aggregates in suspension increase directly with aggregate size. This leads to a self-propagating cycle of cell damage by balancing forces. Cell culture in microgravity avoids this trade-off. We determined which genes mediate three-dimensional culture of cell and tissue aggregates in the low-shear stress, low-turbulent environment of actual microgravity. Primary cultures of human renal cortical cells were flown on the space shuttle. Cells grown in microgravity and ground-based controls were grown for 6 days and fixed. RNA was extracted, and automated gene array analysis of the expression of 10, 000 genes was performed. A select group of genes were regulated in microgravity. These 1,632 genes were independent of known shear stress response element-dependent genes and heat shock proteins. Specific transcription factors underwent large changes in microgravity including the Wilms' tumor zinc finger protein, and the vitamin D receptor. A specific group of genes, under the control of defined transcription factors, mediate three-dimensional suspension culture under microgravity conditions.

Substance P dependence of endosomal fusion during bladder inflammation.

Urinary bladder instillation of ovalbumin into presensitized guinea pigs stimulates rapid development of local bladder inflammation. Substance P is an important mediator of this inflammatory response, as substance P antagonists largely reverse the process. Vacuolization of the subapical endosomal compartment of the transitional epithelial cells lining the bladder suggests that changes in endosomal trafficking and fusion are also part of the inflammatory response. To test directly for substance P mediation of changes in endosomal fusion, we reconstituted fusion of transitional cell endosomes in vitro using both cuvette-based and flow cytometry energy transfer assays. Bladders were loaded with fluorescent dyes by a hypotonic withdrawal protocol before endosomal isolation by gradient centrifugation. Endosomal fusion assayed by energy transfer during in vitro reconstitution was both cytosol and ATP dependent. Fusion was confirmed by the increase in vesicle size on electron micrographs of fused endosomal preparations compared with controls. In inflamed bladders, dye uptake was inhibited 20% and endosomal fusion was inhibited 50%. These changes are partly mediated by the neurokinin-1 (NK1) receptor (NK1R), as 4 mg/kg of CP-96,345, a highly selective NK1 antagonist, increased fusion in inflamed bladders but had no effect on control bladders. The receptor-mediated nature of this effect was demonstrated by the expression of substance P receptor mRNA in rat bladder lumen scrapings and by the detection of the NK1R message in guinea pig subapical endosomes by Western blot analysis. The NK1Rs were significantly upregulated following induction of an inflammatory response in the bladder. These results demonstrate that 1) in ovalbumin-induced inflammation in the guinea pig bladder, in vitro fusion of apical endosomes is inhibited, showing endocytotic processes are altered in inflammation; 2) pretreatment in vivo with an NK1R antagonist blocks this inhibition of in vitro fusion, demonstrating a role for NK1R in this process; and 3) the NK1R is present in higher amounts in apical endosomes of inflamed bladder, suggesting changes in translation or trafficking of the NK1R during the inflammatory process. This suggests that NK1R can change the fusion properties of membranes in which it resides.

Nuclear factor kappa B mediates lipopolysaccharide-induced inflammation in the urinary bladder.

PURPOSE: The proteins which constitute the final common pathway linking receptors on cell surfaces to the inflammatory cascade have recently been identified and cloned. Central to activation of this inflammatory cascade is translocation from cytosol to nucleus of the nuclear transcription factor known as nuclear factor-kappa B (NF-kappaB). The purpose of this study was to determine whether NF-kappaB cascade plays a role in lipopolysaccharide (LPS)-induced inflammation of the mouse urinary bladder. MATERIALS AND METHODS: Bladder inflammation was induced in anesthetized mice by intravesical instillation of lipopolysaccharide (LPS) and quantified by morphometric analysis. The NK-1 receptors for substance P were quantified by flow cytometry. LPS-induced degradation of inhibitory IkappaB subunit was quantified by Western blotting analysis and translocation of NF-kappaB protein from cytosol to the nucleus was determined by confocal microscopy and Western blotting analysis. In addition, we determine the effect of lactacystin, a proteosome inhibitor, on LPS-induced IkappaB degradation and NF-kappaB translocation, NK-1 receptor fluorescence intensity, and bladder inflammation. RESULTS: LPS instillation into the mouse bladder resulted in time dependent loss of the inhibitory IkappaB subunit of the NF-kappaB protein complex. IkappaB cleavage was followed by translocation of NF-kappaB from the cytosol to the nucleus. This was associated with increased expression of an NF-kappaB dependent inflammatory component, the NK-1 receptor. Pretreatment of mouse bladders with the NF-kappaB inhibitor, lactacystin, prevented cleavage of IkappaB in a dose-dependent manner. Lactacystin prevented increases in the NF-kappaB dependent inflammatory cascade components such as the NK-1 receptor. At the whole tissue level, the marked inflammatory infiltrate and mucosal breakdown associated with LPS administration was completely abolished by lactacystin. CONCLUSION: NF-kappaB mediates many features of urinary bladder inflammation induced by LPS. The NF-kappaB cascade is an important target for anti-inflammatory management of cystitis.

Renal endosomes contain angiotensin peptides, converting enzyme, and AT(1A) receptors.

Kidney cortex and proximal tubular angiotensin II (ANG II) levels are greater than can be explained on the basis of circulating ANG II, suggesting intrarenal compartmentalization of these peptides. One possible site of intracellular accumulation is the endosomes. In the present study, we tested for endosomal ANG I, ANG II, angiotensin type 1A receptor (AT(1A)), and angiotensin converting enzyme (ACE) activity and determined whether these levels are regulated by salt intake. Male Sprague-Dawley rats were fed chow containing either high or low dietary sodium for 10-14 days. Blood and kidneys were harvested and processed for measurement of plasma, kidney, and renal intermicrovillar cleft and endosomal angiotensin levels. Kidney ANG I averaged 179 +/- 20 fmol/g and ANG II averaged 258 +/- 36 fmol/g in rats fed a high-sodium diet and were significantly higher, averaging 347 +/- 58 fmol/g and 386 +/- 55 fmol/g, respectively, in rats fed a low-salt diet. Renal intermicrovillar clefts and endosomes contained ANG I and ANG II. Intermicrovillar cleft ANG I and ANG II levels averaged 8.4 +/- 2.6 and 74 +/- 26 fmol/mg, respectively, in rats fed a high-salt diet and 7.6 +/- 1.7 and 70 +/- 25 fmol/mg in rats fed a low-salt diet. Endosomal ANG I and ANG II levels averaged 12.3 +/- 4.4 and 43 +/- 19 fmol/mg, respectively, in rats fed a high-salt diet, and these levels were similar to those observed in rats fed a low-salt diet. Renal endosomes from rats fed a low-salt diet demonstrated significantly more AT(1A) receptor binding compared with rats fed a high-salt diet. ACE activity was detectable in renal intermicrovillar clefts and was 2.5-fold higher than the levels observed in renal endosomes. Acute enalaprilat treatment decreased ACE activity in renal intermicrovillar clefts by 90% and in renal endosomes by 84%. Likewise, intermicrovillar cleft and endosomal ANG II levels decreased by 61% and 52%, respectively, in enalaprilat-treated animals. These data demonstrate the presence of intact angiotensin peptides and ACE activity in renal intermicrovillar clefts and endosomes, indicating that intact angiotensin peptides are formed and/or trafficked through intracellular endosomal compartments and are dependent on ACE activity.

Select de novo gene and protein expression during renal epithelial cell culture in rotating wall vessels is shear stress dependent.

The rotating wall vessel has gained popularity as a clinical cell culture tool to produce hormonal implants. It is desirable to understand the mechanisms by which the rotating wall vessel induces genetic changes, if we are to prolong the useful life of implants. During rotating wall vessel culture gravity is balanced by equal and opposite hydrodynamic forces including shear stress. The current study provides the first evidence that shear stress response elements, which modulate gene expression in endothelial cells, are also active in epithelial cells. Rotating wall culture of renal cells changes expression of select gene products including the giant glycoprotein scavenger receptors cubulin and megalin, the structural microvillar protein villin, and classic shear stress response genes ICAM, VCAM and MnSOD. Using a putative endothelial cell shear stress response element binding site as a decoy, we demonstrate the role of this sequence in the regulation of selected genes in epithelial cells. However, many of the changes observed in the rotating wall vessel are independent of this response element. It remains to define other genetic response elements modulated during rotating wall vessel culture, including the role of hemodynamics characterized by 3-dimensionality, low shear and turbulence, and cospatial relation of dissimilar cell types.

Myeloma light chains are ligands for cubilin (gp280).

Although myeloma light chains are known to undergo receptor-mediated endocytosis in the kidney, the molecular identity of the receptor has not been characterized. We examined the interaction between cubilin (gp280) and four species of light chains isolated from the urine of patients with multiple myeloma. Four lines of evidence identify cubilin, a giant glycoprotein receptor, which is restricted in distribution to endocytic scavenger pathways and which has potent effects on endosomal trafficking, as a potentially physiologically relevant binding site for light chains: 1) light chains coeluted during immunoaffinity purification of cubilin; 2) polyclonal antisera to cubilin but not control sera, displaced human light chain binding from rat renal brush-border membranes; 3) cubilin bound to multiple species of light chains during surface plasmon resonance; 4) anti-cubilin antiserum interfered with light chain endocytosis by visceral yolk sac epithelial cells. However, both binding of light chains to brush-border membranes and endocytosis of light chains by yolk sac epithelial cells were only partially inhibited by anticubilin antibodies, suggesting presence of additional or alternate binding sites for light chains. Excess light chain had a potent inhibitory effect on endosomal fusion in vitro. Binding showed dose and time-dependent saturability with low-affinity, high-capacity equilibrium binding parameters. These data demonstrate that cubilin plays a role in the endocytosis and trafficking of light chains in renal proximal tubule cells.

Membrane potential mediates H(+)-ATPase dependence of "degradative pathway" endosomal fusion.

In some epithelial cell lines, the uptake and degradation of proteins is so pronounced as to be regarded as a specialized function known as "degradative endocytosis." The endosomal pathways of the renal proximal tubule and the visceral yolk sac share highly specialized structures for "degradative endocytosis." These endosomal pathways also have a unique distribution of their H(+)-ATPase, predominantly in the subapical endosomal pathway. Previous studies provide only indirect evidence that H(+)-ATPases participate in endosomal fusion events: formation of vesicular intermediates between early and late endosomes is H(+)-ATPase dependent in baby hamster kidney cells, and H(+)-ATPase subunits bind fusion complex proteins in detergent extracts of fresh rat brain. To determine directly whether homotypic endosomal fusion is H(+)-ATPase dependent, we inhibited v-type H(+)-ATPase during flow cytometry and cuvette-based fusion assays reconstituting endosomal fusion in vitro. We report that homotypic fusion in subapical endosomes derived from rat renal cortex, and immortalized visceral yolk sac cells in culture, is inhibited by the v-type H(+)-ATPase specific inhibitor bafilomycin A1. Inhibition of fusion by H(+)-ATPase is mediated by the membrane potential as collapsing the pH gradient with nigericin had no effect on homotypic endosomal fusion, while collapsing the membrane potential with valinomycin inhibited endosomal fusion. Utilizing an in vitro reconstitution assay this data provides the first direct evidence for a role of v-type H(+)-ATPase in mammalian homotypic endosomal fusion.

The intrinsic factor-vitamin B12 receptor and target of teratogenic antibodies is a megalin-binding peripheral membrane protein with homology to developmental proteins.

The present report shows the molecular characterization of the rat 460-kDa epithelial glycoprotein that functions as the receptor facilitating uptake of intrinsic factor-vitamin B12 complexes in the intestine and kidney. The same receptor represents also the yolk sac target for teratogenic antibodies causing fetal malformations in rats. Determination of its primary structure by cDNA cloning identified a novel type of peripheral membrane receptor characterized by a cluster of eight epidermal growth factor type domains followed by a cluster of 27 CUB domains. In accordance with the absence of a hydrophobic segment, the receptor could be released from renal cortex membranes by nonenzymatic and nonsolubilizing procedures. The primary structure has no similarity to known endocytic receptors but displays homology to epidermal growth factor and CUB domain proteins involved in fetal development, e.g. the bone morphogenic proteins. Electron microscopic immunogold double labeling of rat yolk sac and renal proximal tubules demonstrated subcellular colocalization with the endocytic receptor megalin, which is expressed in the same epithelia as the 460-kDa receptor. Furthermore, megalin affinity chromatography and surface plasmon resonance analysis revealed a calcium-dependent high affinity binding of the 460-kDa receptor to megalin, which thereby may mediate its vesicular trafficking. Due to the high number of CUB domains, accounting for 88% of the protein mass, we propose the name cubilin for the novel receptor.

Gentamicin inhibits rat renal cortical homotypic endosomal fusion: role of megalin.

Megalin, a giant glycoprotein receptor heavily concentrated in the early endosomal pathway of renal proximal tubular cells, binds gentamicin with high affinity and delivers the drug to lysosomes. Utilizing an in vitro reconstitution assay we tested whether gentamicin-induced vacuolation is associated with inhibition of early endosomal fusion, as well as whether megalin plays a role in mediating these effects. Pretreatment of rats with gentamicin inhibited rat renal proximal tubular homotypic endosomal fusion. Administered simultaneously, gentamicin and polymers of polyaspartic acid, which protect against the hemodynamic effects of gentamicin nephrotoxicity, had no net effect on fusion. Polyaspartic acid alone had no effect on fusion. Antisera to the tail of the megalin/gentamicin receptor inhibited fusion, whereas non-specific controls had no effect. Peptides matching homologous NPXY repeat sequence motifs in the cytosolic tail stimulated endosomal fusion, whereas reverse sequence control peptides had no effect. These data suggest that gentamicin inhibition of endosomal fusion in the renal proximal tubule is a damage mechanism mediated by specific peptide sequences in the cytosolic tail of the giant gentamicin-binding receptor megalin and that receptors can effect the fusion properties of membranes in which they reside.

Lack of glyconeogenesis in pancreatic islets: expression of gluconeogenic enzyme genes in islets.

Studies were performed to obtain evidence for glyconeogenesis from pyruvate to the triose phosphates in pancreatic islets. Inability to show this evidence would be consistent with the fact that glyceraldehyde, but not pyruvate, is a potent insulin secretagogue. Synthesis of 14C-labelled glucose from 14C-labelled pyruvate could not be detected. Since this might have been due to lack of sensitivity required to measure 14C-glucose production in such a scarce tissue as islets, cDNA probes were used to estimate the relative expression of genes coding for gluconeogenic enzymes. Islets expressed pyruvate carboxylase mRNA, but even islets from rats which had been starved (a condition which induces phosphoenolpyruvate carboxykinase (PEPCK) in liver, kidney and adipose tissue) showed no PEPCK mRNA. This is consistent with our previous work showing the absence of PEPCK enzyme activity in islets. Therefore, islets can convert pyruvate to oxalacetate, but since they lack PEPCK, neither the beta nor alpha cell can convert oxalacetate to phosphoenolpyruvate and carry out glyconeogenesis. Pyruvate carboxylase mRNA was increased in islets that possessed the capacity for glucose-induced insulin release versus islets that lacked the capacity to respond to glucose, such as islets from fed rats (versus starved rats) and in islets cultured at a high concentration of glucose (versus at low glucose). Pyruvate carboxylase, therefore, must be involved in pyruvate metabolism and not glyconeogenesis in the pancreatic islet.

Pyruvate dehydrogenase and pyruvate carboxylase. Sites of pretranslational regulation by glucose of glucose-induced insulin release in pancreatic islets.

It has been shown previously that glucose-induced insulin release is completely absent in rat pancreatic islets that had been cultured for 1 day at low glucose (1 mM) and that it is restored by culturing islets for a 2nd day at high (20 mM) glucose (MacDonald, M. J., Fahien, L. A., McKenzie, D. I., and Moran, S. M. (1991) Am. J. Physiol. 259, E548-E554). It has been suggested that the incapacitation of glucose's insulinotropism is due to down-regulation of the synthesis of enzymes that process glucose's metabolic signal for insulin release. In the current study, results of metabolic, enzymic, and molecular biologic experiments were each consistent with (an) intramitochondrial site(s) of down-regulation in islets cultured at low glucose. Glucose metabolism was inhibited 80% in islets cultured at 1 mM glucose. The suppression of release of 14CO2 from [6-14C]glucose greater than from [U-14C]glucose greater than [3,4-14C]glucose greater than from [1-14C]glucose in islets cultured at low glucose indicated a mitochondrial site of down-regulation because C-6 of glucose can only be converted to CO2 in the citric acid cycle, whereas C-1 can be released as CO2 in the 6-phosphogluconate dehydrogenase [corrected] reaction, and C-6 of glucose dwells in the citric acid cycle longer than carbons 2-5 of glucose. Since carbons 3 and 4 of glucose can be decarboxylated in the pyruvate dehydrogenase reaction, incomplete suppression of CO2 formation from these carbons is consistent with suppression of pyruvate carboxylation as well as decarboxylation. Formation of 3HOH from [5-3H]glucose was equal in the two groups of islets, indicating that glycolysis as far as phosphoenolpyruvate was intact. This idea was supported by assays which showed that activities of enzymes of the glycolytic pathway between glucokinase/hexokinase and pyruvate kinase were equal in both types of islets. Additional studies indicated that regulation by glucose was at transcription of genes coding for some mitochondrial enzymes. Glucokinase, malic enzyme, and fumarase mRNAs were not affected by glucose, whereas the pyruvate dehydrogenase E1 alpha subunit and pyruvate carboxylase mRNAs were decreased 85-90% in islets cultured at 1 mM glucose. Pyruvate dehydrogenase enzyme activity was decreased to a similar extent in these islets. About 24 h was required for maximal (de)induction of pyruvate dehydrogenase E1 alpha and pyruvate carboxylase mRNAs, and the amounts of transcripts were proportional to the concentrations of glucose between 1 and 20 mM.(ABSTRACT TRUNCATED AT 400 WORDS)

Glucose regulates leucine-induced insulin release and the expression of the branched chain ketoacid dehydrogenase E1 alpha subunit gene in pancreatic islets.

Much evidence has accumulated to support the idea that leucine can stimulate insulin release by allosterically activating glutamate dehydrogenase thus enhancing glutamate metabolism. It is less clear how the metabolism of leucine itself contributes to the signal for insulin release. We recently found that culturing pancreatic islets for 1 day at low glucose (1 mM) suppressed glucose-induced insulin release, but preserved leucine-induced insulin release. When islets were cultured at high glucose (20 mM), glucose-induced insulin release was preserved, but leucine-induced insulin release was suppressed (MacDonald, M. J., Fahien, L. A., McKenzie, D. I., and Moran, S. M. (1990) Am. J. Physiol., 259, E548-E554). The suppression of leucine-induced insulin release can be explained by glucose's suppression of the synthesis of the enzyme that catalyzes the first committed step of leucine metabolism, branched chain ketoacid dehydrogenase complex (BCKDH). High glucose suppressed the enzyme activity of the E1 component of the BCKDH complex, as well as the total activity of the BCKDH complex, to usually negligible levels in islets and decreased by an average of 90% the mRNA which encodes E1 alpha, the catalytic subunit of the E1 component of BCKDH, in islets and rat insulinoma cells. Time course studies showed that about 24 h in culture was required to maximally induce or suppress the expression of BCKDH E1 alpha. Culture at high glutamine with or without leucine mimicked to a lesser and more variable degree the effects of high glucose on leucine-induced insulin release and BCKDH E1 alpha mRNA. Leucine-plus-glutamine-induced insulin release was present after culture of islets with glucose and with or without any other secretagogue. Also, glutamate dehydrogenase transcripts and enzyme activity were not significantly altered by varying the concentration of glucose in the culture medium. Thus, leucine's insulinotropism via activation of glutamate dehydrogenase is constitutive. Preproinsulin mRNA levels were markedly increased at high glucose and glyceraldehyde phosphate dehydrogenase transcripts were either unaffected or slightly increased by glucose. Glutamine did not significantly effect the expression of genes other than BCKDH E1 alpha, and leucine had little or no effect on the expression of any of the four genes.(ABSTRACT TRUNCATED AT 400 WORDS)

Positioning of nucleosomes reconstituted with xeroderma pigmentosum and normal histones.

Positioning of nucleosomes was examined in a reconstituted system using a plasmid DNA and histones from normal human and xeroderma pigmentosum, complementation group A (XPA), lymphoblastoid cells. The present studies indicate that the arrangement of nucleosomes, composed of normal human histones, in a region near the SV40 origin of replication on the plasmid DNA, is nonrandom. The alignment of nucleosomes in this region was not affected by the presence of histone H1. No difference in nucleosome positioning was observed when the nucleosomes were composed of histones from XPA cells.

Enhancement of two apurinic/apyrimidinic endonuclease activities from normal but not xeroderma pigmentosum lymphoblastoid cells by nucleosome structure.

The influence of nucleosomes on the activity of two chromatin-associated apurinic/apyrimidinic (AP) DNA endonuclease activities, pIs 9.2 and 9.8, from normal and xeroderma pigmentosum, complementation group A (XPA), lymphoblastoid cells was examined. These AP endonuclease activities were studied on non-nucleosomal and nucleosomal plasmid pWT830/pBR322 DNA which had been reconstituted with core (H2A, H2B, H3, H4) or total (core plus H1) histones from normal or XPA cells. Both nucleosomal and non-nucleosomal DNA was rendered partially AP by alkylation with 12.5 mM methyl methanesulfonate, followed by heating it at 70 degrees C, to produce approximately three AP sites per DNA molecule. The activities of both normal lymphoblastoid AP endonuclease activities on nucleosomal AP DNA, reconstituted with core histones, was approximately 2.5 times greater than that on non-nucleosomal AP DNA. When histone H1 was added to the system, this increase was reduced. XPA AP endonuclease activities, on the other hand, did not show any increase in activity on nucleosomal AP DNA reconstituted with core histones. These differences between normal and XPA endonuclease activities on AP nucleosomal DNA were the same regardless of whether histones from normal or XPA cells were used in the reconstituted system.