Aquaporin-1 expression in proximal tubule epithelial cells of human kidney is regulated by hyperosmolarity and contrast agents.

Primary cells of renal proximal tubule epithelium (S1 segment) of human kidney (HRPTE cells) up-regulate aquaporin-1 (AQP-1) expression in response to hyperosmolarity. NaCl and D(+)-raffinose increased (2-2.5 fold) AQP-1 expression when medium osmolarity was 400 and 500 mOsm/kg.H2O. Urea did not have this effect. Unlike our previous findings with mIMCD-3 cells, vasopressin (10(-8)M) did not affect AQP-1 expression in HRPTE cells in isosmolar or NaCl-enriched hyperosmolar conditions. Furthermore, HRPTE cells increased (3-4 fold) AQP-1 expression when exposed to hyperosmolar Reno-60 and Hypaque-76 (diatrizoates, ionic) contrast agents at 400 and 500 mOsm/kg.H2O. Isosmolar (290 mOsm/kg H2O) Visipaque (iodixanol, non-ionic) at 10% (v/v) concentrations also increased AQP-1 expression, and 25% v/v of Visipaque rendered morphological alterations of HRPTE cells and a 3-fold increase in AQP-1 expression after 24h exposure. Finally, semi-quantitative RT-PCR of HRPTE cells subjected to various isosmolar or hyperosmolar conditions demonstrated up-regulation of AQP-1 mRNA and protein levels. Our results suggest AQP-1 up-regulation in HRPTE cells exposed to environmental stresses such as hyperosmolarity and high doses of isosmolar contrast agents.

Integrin alpha2beta1 recognizes laminin-2 and induces C-erb B2 tyrosine phosphorylation in metastatic human melanoma cells.

Previous studies have shown that tumor cells with metastatic propensity secrete more of the laminin alpha2 chain than non-metastatic tumor cells do, and that laminin-2, which contains the alpha2 chain, promotes cell adhesion better than laminin-1 (Jenq et al. (1994). Differentiation, 58, 29-36). The current studies were designed to determine whether a correlation exists between the expression of the laminin-2 isoform and the metastatic phenotype in melanoma cells. We found that expression of the laminin-2 isoform was upregulated in the metastatic melanoma cell lines tested. Cell attachment studies showed that metastatic melanoma cells attached more efficiently to laminin-2 substrates. Studies on integrin expression revealed that the presence of alpha2beta1 integrin correlated with expression of the laminin-2 isoform in metastatic melanoma cells; anti-integrin alpha2 antibody prevented cell attachment to laminin-2 substrates. The data suggest that the alpha2beta1 integrin is the receptor mediating cell attachment to the laminin-2 isoform. This interaction, mediated by the alpha2beta1 integrin, stimulates secretion of the 72 kD type IV collagenase and induces a specific 185 kD protein tyrosine phosphorylation. The 185 kD tyrosine-phosphorylated protein was identified as the p185/C-erb B2 oncoprotein by immunoprecipitation. These studies suggest that upregulation of expression of the laminin-2 chain correlates with the metastatic phenotype of melanoma cells and provides evidence that the specific p185/C-erb B2 tyrosine phosphorylation may be involved in integrin-mediated signaling during tumor cell invasion and metastasis.

Aquaporin-1: an osmoinducible water channel in cultured mIMCD-3 cells.

The expressions of aquaporin-1 (AQP-1) in cultured mIMCD-3 cells were studied. There was no detectable AQP-1 in cells grown in serum-containing growth medium (SM, 297 +/- 2 mOsm/kg. H2O). When SM was supplemented with NaCl (406 +/- 2 mOsm/kg. H2O), cellular AQP-1 was induced. A further increase in medium osmolarity with NaCl (493 +/- 3 mOsm/kg. H2O) had conferred cells an 2.5 to 3-fold increase of AQP-1 expression over those grown in the 406 +/- 2 mOsm/kg. H2O medium. Moreover, AQP-1 was found to be translocated from cytosol to membrane. In addition, exposing the mIMCD-3 cells to vasopressin (AVP, 10(-8) M) and/or NaCl-supplemented serum-free media (496 +/- 3 mOsm/kg. H2O) for 6h did not render them to produce AQP-1. However, AQP-1 was induced after 24h of incubation, with an 1.5-fold additive effect by AVP. Our RT-PCR data had confirmed the NaCl inducibility and AVP synergism in AQP-1 expression at both mRNA and protein levels. This suggests a new role for cellular AQP-1 and AVP in overcoming osmotic stress in an in vitro system.

Hypoxic effects on the expression of mineralocorticoid and glucocorticoid receptors in human renal cortex epithelial cells.

The effects of both mild and severe hypoxic conditions on the expression of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) in the primary human renal cortex epithelial cells (RCEC) were investigated. Slot blot analyses were performed with human beta-actin cDNA and the cDNA probes for unique regions of MR and GR. The steady-state mRNA level of the house-keeping beta-actin gene remains the same in RCEC under either normoxic or hypoxic conditions. However, there was a 45% reduction in MR expression and a 30% increase in GR expression, under mild hypoxic condition (15% O2) over 3 days. Furthermore, cells subjected to a severe hypoxic condition (0.5% O2) for 24 hours exerted a 22% reduction in MR expression and a 64% increase in GR expression. Changes in MR and GR expression may, in part, explain the physiological changes in adrenal cortical function observed in hypoxic conditions.

Integrin expression on cell adhesion function and up-regulation of P125FAK and paxillin in metastatic renal carcinoma cells.

Integrins from normal human renal cortex epithelial cells (RCEC) and from four renal carcinoma lines (metastatic Caki-1, non-metastatic Caki-2, metastatic ACHN, and non-metastatic 769-P) were compared by immunoprecipitation with specific anti-integrin antibodies. Integrin alpha 2 was present in normal RCEC, but absent in all four tumor lines. There was a 2.0-3.0 fold decrease of alpha 3 and beta 1 in localized tumor lines, and a further 5.0-7.0 fold decrease in metastatic lines over their expression in normal renal cells. No alpha V was detected in Caki-1 cells. The greatest adhesion of all cells occurred in the presence of a stimulatory anti-alpha 3 antibody, mediated by specific matrix proteins employed as substrates, while anti-beta 1 treatment dramatically inhibited cell attachment on collagen IV, plasma fibronectin, laminin and merosin substrates. In addition, the mRNA expression of focal adhesion kinase (p125FAK) and paxillin were up-regulated (2.0-2.5 fold increase) in the metastatic Caki-1 cells over normal RCEC. The alteration of integrin subunits alpha 2, alpha 3, alpha V, beta 1, as well as p125FAK and paxillin may contribute to the pathogenicity and/or metastatic propensity of renal epithelial tumors. The up-regulation of paxillin independently or in concert with p125FAK as shown in this study indicates its significant role as a potential marker of metastasis in renal carcinoma cells.

Expression of the alpha 2-subunit of laminin correlates with increased cell adhesion and metastatic propensity.

Previous studies have indicated that laminin from neoplastic cells of high tumorigenicity is less active in promoting cell adhesion than aminin from normal cells or tissues. In the present study, we tested the hypothesis that laminin of metastatic tumor cells differs from that of nonmetastatic cells. Accordingly, we determined the subunit composition of laminin in highly metastatic, ras-transformed cells (4R) and compared it with laminin produced by nonmetastatic cells transformed with ras plus E1a (RE4). Metastatic 4R cells produced three to four times more of the alpha 2-subunit of laminin than RE4 cells did. Furthermore, the highly metastatic human melanoma cells (1205 and A2058) made and secreted into the medium, laminin containing significantly more of the alpha 2-subunit than laminin from the highly tumorigenic but nonmetastatic melanoma WM793 or HT1080 fibrosarcoma cells. Using HT1080 cells, laminin (250 ng/well) from 4R cells showed more adhesion promoting activity (68%) than laminin from RE4 cells (39%). Similarly, laminin isolated from human placenta, which expresses both the alpha 1 beta 1 gamma 1 and alpha 2 beta 1 gamma 1 isoforms, promoted cell adhesion better (63%) than EHS laminin (26%), which contains only the former isoform, at 250 ng/well. In addition, both 4R and RE4 cells attached more efficiently to 4R laminin-coated substratum than to RE4 laminin at 0.3 and 0.6 microgram/well.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a major cell wall antigen and potential adhesin in three strains of Candida albicans.

Selected strains of Candida albicans were examined to reveal the surface antigenicity and biochemical nature of major cell wall proteins that also were shown to serve as cellular adhesins on human buccal epithelial cells. Confirmation of the adhesive properties of these cells was made by scanning electron microscopy and immunofluorescence microscopy. Particular attention was directed at the clinical isolate KM-302. By means of indirect immunofluorescence staining, the KM-302 blastoconidia absorbed rabbit anti-C. albicans ATCC-32354 serum, revealing specific localization of surface antigens on germ tubes and pseudohyphae. Extracellular polymeric material and the cell wall extract of C. albicans KM-302 blastoconidia were found to contain a major surface antigen of 49 kDa that exhibited 42% adhesion inhibition in vitro. Of considerable significance is that immunogold localization by electron microscopy showed the antigen to be almost exclusively cell wall bound. This major antigen, identified in affinity and gel filtration chromatography fractions, was composed of 4% carbohydrate and 95.7% protein and had an isoelectric point of 6.1. The major antigen also showed a high level of similarity with that of C. albicans strain SC-5314 inasmuch as the major antigen of that strain had carbohydrate and protein compositions of 4 and 95.5%, respectively. Both of these strains also possessed the same percent of adhesion inhibition on human buccal epithelial cells.

Adhesion promoting property of laminin from normal tissue and from a tumorigenic cell line.

The cell adhesion promoting activity of laminin isolated from normal human placenta was compared with that isolated from mouse EHS tumor and from the cultures of a mouse epithelial cell line B82 and its tumorigenic derivative, B82HT. The adhesion promoting properties of commercial merosin isolated from placenta was also compared with the above preparations using the human fibrosarcoma HT1080 cells. Percent attachment was defined as (radioactivity extracted from attached cells)/(radioactivity in cells added to assay) x 100. HT1080 cells adhered more efficiently on laminin (0.5 micrograms/well), isolated from the B82 rather than B82HT cell conditioned medium, (82% vs 64%). Percent attachment of HT1080 cells on isolated native placental laminin or commercial merosin was significantly higher compared to laminin from the EHS tumor (at 0.75 micrograms/well, 69%, 73% and 20% respectively). In parallel experiments the steady-state levels of mRNAs for subunits A, M, B1 and B2 in cultures of B82 and B82HT cells were determined. The ratio of mRNA for the laminin subunits in B82 and B82HT cells was 1:0.9 for the A chain, 1:0.6 for the M chain, 1:0.4 for the B1 chain, and 1:0.3 for the B2 chain. Protein studies indicated that the M subunit is absent in laminin preparations from the EHS tumor whereas it is abundant in the laminin from placenta and in commercial merosin. Laminin isolated from B82 cells contains a higher proportion of the M subunit compared to that from B82HT cells. The data suggest that there are functional differences between the laminin found in normal tissue and that present in a solid tumor. Functional differences were noted between the laminins synthesized by the B82 cell line and its tumorigenic counterpart, B82HT. These differences may result from the lack of gene expression for the laminin subunit M by the EHS tumor and by the lower degree of gene expression for this subunit by B82HT cells. The possibility that the laminin synthesized by the tumorigenic cell line may be structurally different from that synthesized by the B82 cells should also be considered.

Enhanced Conversion of Lactose to Glycerol by Kluyveromyces fragilis Utilizing Whey Permeate as a Substrate.

Kluyveromyces fragilis (CBS 397) is a nonhalophilic yeast which is capable of lactose utilization from whey permeate and high glycerol production under anaerobic growth conditions. However, the optimum yields of glycerol (11.6 mg/ml of whey permeate medium) obtained in this study occurred only in the presence of 1% Na(2)SO(3) as a steering agent. The use of other concentrations of Na(2)SO(3), as well as 5% NaCl and 1% ascorbic acid, had no or detrimental effects on cell growth, lactose utilization, and glycerol production. Glycerol yields were greater in cultures grown from a light inoculum of K. fragilis than in cultures in which a resuspended mass of cells was introduced into the medium. The results of this study suggest that this strain of K. fragilis may be useful commercially in the utilization of cheese whey lactose and the concomitant production of glycerol.

Mitochondrial DNA in Candida pintolopesii, a yeast indigenous to the surface of the secreting epithelium of the murine stomach.

Candida pintolopesii 108-1 is an indigenous yeast which colonizes the surface of the secreting gastric mucosa of mice. We have been exploring the aerobic respiratory capacities of this organism in reference to its capacity to colonize the stomach surface, an environment that could contain little oxygen for microbial growth. In this paper, we report mitochondrial DNA and membranes in cells of a strain of this yeast isolated from the gastric epithelium of a mouse and compare the findings with those made by other investigators in studies of Saccharomyces cerevisiae. Putative mitochondrial DNA was isolated from crude lysates of C. pintolopesii and S. cerevisiae as fluorescing bands in CsCl gradients containing 4',6-diamidino-2-phenylindole. The DNA from C. pintolopesii hybridized with a 32P-labeled DNA probe for the 21S rRNA gene encoded by mitochondrial DNA in S. cerevisiae. Postvital cell staining with 4',6-diamidino-2-phenylindole and rhodamine 123 revealed mitochondrial DNA and membranes, respectively, in the cytoplasm of intact C. pintolopesii cells. The staining patterns were generally similar to those reported for S. cerevisiae. Finally, structures similar to those reported to be mitochondria in electron micrographs of S. cerevisiae were seen in preparations of C. pintolopesii cells examined by transmission electron microscopy. These data confirm findings from studies of its respiratory capacity published earlier that a strain of C. pintolopesii isolated directly from its native habitat has functional mitochondria.