Biomedical subjects

J G Haggerty

Publications and source records attributed to J G Haggerty.

18 recordsLinked to original sources

PubMed1999

Growth and differentiation regulate CD44 expression on human keratinocytes.

Several members of the CD44 family of hyaluronan receptors are expressed on keratinocytes. To identify factors that might be important in regulating CD44 expression, we studied CD44 expression on keratinocytes growing in vitro under a variety of conditions and on cells isolated directly from epidermis. Using Western immunoblots and metabolic labeling, we showed that the pattern of CD44 proteins expressed by keratinocytes was strongly influenced by growth and differentiation. Many protein forms of CD44 are expressed on proliferating keratinocytes in preconfluent cultures, whereas only a few forms are expressed on differentiated cells and in confluent cultures. In preconfluent monolayers, at least four splice variants were identified, including epican, CD44H, CD44E, and a 180-kDa variant. In differentiated cells or in confluent cultures, by contrast, only epican and the 180-kDa protein variant were found. Synthesis of all variants is strongly downregulated when keratinocytes become confluent or when they differentiate. Epican is the predominant form of CD44 on keratinocytes under all conditions and is expressed as a heparan, chondroitin, or keratan sulfate proteoglycan. Preconfluent basal keratinocytes, but not confluent or differentiated keratinocytes, also express chondroitin sulfate proteoglycan forms of CD44E and of the 180-kDa core protein. The modal size of the epican expressed on differentiated keratinocytes is smaller than the size of the epican expressed on basal keratinocytes. Thus, cell confluence and differentiation regulate several aspects of CD44 expression on keratinocytes, suggesting nuances in function for the different protein forms.

Cell Differentiation↗

PubMed1999

Expression and localization of thymidine phosphorylase/platelet-derived endothelial cell growth factor in skin and cutaneous tumors.

Thymidine phosphorylase/platelet-derived endothelial cell growth factor (TPase/PD-ECGF) is a catabolic enzyme that has been shown to be chemotactic for endothelial cells in vitro and angiogenic in vivo. TPase/PD-ECGF expression is increased in a variety of tumors. In the skin, TPase is active in normal keratinocytes in vitro and in vivo. Our objective was to study the expression and localization of TPase/PD-ECGF by immunohistochemical analysis in normal skin and cutaneous tumors and to correlate this information with enzymatic activity of TPase. TPase/PD-ECGF expression was observed in keratinocytes with intense staining of the infundibulum of hair follicles but no staining of hair bulbs. Expression localized primarily to the nucleus of keratinocytes in the basal layer but was more intense and cytoplasrmic in suprabasal keratinocytes. Increased expression of TPase/PD-ECGF in differentiated cells was confirmed by in vitro studies of TPase activity. In cutaneous tumors, there was positive staining for TPase/ PD-ECGF in squamous cell carcinomas (10/10), eccrine poromas (3/4), eccrine syringomas (4/4), trichoepitheliomas (1/3), and tumors of the follicular infundibulum (2/3) and melanomas (5/8). There was no staining of any intradermal nevi (0/2), basal cell carcinomas (0/10) or Merkel cell carcinoma (0/1). We conclude TPase/PD-ECGF is found throughout the epidermis and its expression increases with differentiation of keratinocytes. In cutaneous tumors, expression of TPase/PD-ECGF may be linked to the cell of origin of the tumor as well as the tumor's degree of differentiation.

Antibody Specificity↗

PubMed1998

Beta-L-1,3-dioxolane-cytidine: a novel nucleoside that inhibits proliferation and induces differentiation of keratinocytes in vitro.

beta-L-1,3-Dioxolane-cytidine (L-(-)-OddC) is a novel L-nucleoside, and its antitumor activity is under investigation in clinical trials. To evaluate the potential of L-(-)-OddC for treating hyperproliferative diseases of the skin, we examined its activity in human keratinocytes in vitro. The dose of L-(-)-OddC that inhibited the rate of proliferation of keratinocytes by 50% was 50 nM. L-(-)-OddC was about as cytotoxic as 9-beta-D-arabinofuranosylcytosine but was about 1,000 time more potent than 3'-azidothymidine. L-(-)-OddC caused irreversible growth arrest and induced differentiation of keratinocytes. L-(-)-OddC altered morphology, increased the cell size of keratinocytes and increased the expression of involucrin. These data suggest that L-(-)-OddC may have potential as a therapeutic agent against hyperproliferative skin diseases.

Antimetabolites, Antineoplastic↗

PubMed1996

CD44 expression on epidermal melanocytes.

We examined CD44 expression on melanocytes to begin to understand what role CD44 might have in the normal behavior of melanocytes and to provide a basis for comparing CD44 expression in melanoma cells. CD44 was expressed on the entire surface of melanocytes and accentuated at the tips of dendritic processes. Two predominant forms of CD44 are expressed on cultured human foreskin melanocytes. One form has the covalent addition of chondroitin sulfate, whereas the other form has no chondroitin sulfate. Both use the hematopoietic, or CD44H, core protein. Using polymerase chain reaction primers that span the site where alternative splicing of CD44 occurs, we found only the cDNA coding CD44H. 12-O-Tetradecanoylphorbol 13-acetate increases the size of the chondroitin sulfate chain(s) attached to CD44 but not the proportion of CD44 molecules that carry chondroitin sulfate. Ninety percent of proteoglycans on melanocytes are chondroitin sulfate proteoglycans, and the CD44 chondroitin sulfate proteoglycan represented 10% of that total. These data show that CD44H is expressed as a "part-time" chondroitin sulfate proteoglycan on normal cultured melanocytes.

Base Sequence↗

PubMed1994

Response of stratified cultures of human keratinocytes to disruption of proteoglycan synthesis by p-nitrophenyl-beta-D-xylopyranoside.

Proteoglycans play a role in regulating proliferation and adhesion of cells to each other and to the basal lamina. Synthesis of proteoglycans is disrupted by beta-xylosides, which serve as alternate substrate sites for glycosaminoglycan chain attachment and therefore prevent glycosylation of the core protein. We have investigated the effects of p-nitrophenyl-beta-D-xylopyranoside (PNP-xyloside) on cultured human keratinocytes. Stratified cultures were incubated for 7 days with PNP-xyloside (0.05-2.0 mM). Concentrations as low as 0.05 mM increased the secretion of free chondroitin sulfate by 10-15-fold over untreated cultures. Cell-associated proteoglycan decreased as PNP-xyloside concentration increased. At 2 mM PNP-xyloside, heparan sulfate as well as chondroitin sulfate addition to core proteins was disrupted: the core protein of epican, a heparan sulfate form of CD44 found on keratinocytes, was detected immunologically but lacked heparan sulfate. 2.0 mM PNP-xyloside reduced the number of attached cells by 20-25% after 7 days, but had little effect on morphology or protein synthesis. These results indicate that intact proteoglycans are not critical for maintaining epidermal keratinocyte stratification, cell-cell adhesion, or growth.

Cell Division↗

PubMed1994

Epican, a heparan/chondroitin sulfate proteoglycan form of CD44, mediates cell-cell adhesion.

Epican is a heparan/chondroitin sulfate proteoglycan form of CD44 and is expressed on the surface of keratinocytes from the basal layer to the granular layer of the epidermis. To analyze the adhesive properties of epican apart from the influence of other adhesive molecules found on keratinocytes, mouse L cell fibroblasts were transfected with CD44Epican cDNA. The epican expressed on the surface of transfected L cells was predominantly a heparan or chondroitin sulfate proteoglycan. The CD44Epican-transfected L cells acquired: (a) a self-aggregating phenotype that required hyaluronan but was calcium-independent; and (b) a new capacity to adhere to keratinocytes, a property that was blocked by an anti-epican antibody. Both aggregation and adhesion of CD44Epican-transfected cells were completely prevented by pretreatment with hyaluronidase, but were totally restored by the addition of exogenous hyaluronan. Aggregation of transfected L cells was minimally influenced by other glycosaminoglycans, but adhesion of transfected L cells to keratinocytes was substantially inhibited by heparin.

Animals↗

PubMed1992

The core protein of epican, a heparan sulfate proteoglycan on keratinocytes, is an alternative form of CD44.

Epican, a heparan sulfate proteoglycan, was recently identified on the surface of keratinocytes with the aid of a monoclonal antibody to its core protein. Using that antibody to screen a human keratinocyte cDNA library, a clone encoding the entire epican core protein was selected and sequenced. The core protein of epican is a form of CD44. The deduced protein sequence of 699 amino acids has a novel 339 amino acid domain inserted into the proximal extracellular domain of the standard, leukocyte form of CD44. The additional domain adds a number of potential N- and O-linked glycosylation sites and two proteolysis sites to this form of CD44.

Amino Acid Sequence↗

PubMed1992

Identification and characterization of a cell surface proteoglycan on keratinocytes.

Proteoglycans fill the intercellular space between keratinocytes but their structure and function are not well understood. We have identified and partially characterized one intercellular proteoglycan on human keratinocytes, for which we propose the name epican (epidermal intercellular proteoglycan). Monoclonal antibodies (MoAb) were generated from a mixture of keratinocyte proteoglycans. One, designated MoAb17, identified the core protein of an intercellular proteoglycan that had an apparent mobility of greater than 250 kDa on Western blots. The core protein itself had an apparent mobility of 180 kDa following deglycosylation with trifluoromethanesulfonic acid. Enzymatic deglycosylation revealed that most core protein molecules were substituted with heparan sulfate but that some carried chondroitin sulfate instead. Smaller forms of the core protein were more abundant in tissue-culture medium than in cell extracts. This proteoglycan was localized by immunofluorescence to the intercellular space of the epidermis and the surface of keratinocytes in vitro, particularly at cell-cell contacts. MoAb17 did not react with protoglycans extracted from other skin cells, nor did it bind to basement membranes or connective tissue. Comparison of Western immunoblots using MoAb17 and antibodies to core proteins of other proteoglycans suggested that epican is not related to syndecan but is a member of the CD44 family.

Antibodies, Monoclonal↗

PubMed1991

Increased Na(+)-H+ antiporter activity in apical membrane vesicles from mutant LLC-PK1 cells.

In whole cell experiments, the PKE20 mutant of the renal epithelial cell line LLC-PK1 displays a severalfold elevation of Na(+)-H+ antiporter activity at the apical surface (J.G. Haggerty, N. Agarwal, R.F. Reilly, E. A. Adelberg, and C.W. Slayman. Proc. Natl. Acad. Sci. USA 85: 6797-6801, 1988). The present study was undertaken to explore the properties of the mutant at the membrane level. Apical membrane vesicles were prepared by the magnesium-aggregation technique, with a similar enrichment (ca. 10-fold) of the marker enzyme gamma-glutamyltranspeptidase in vesicles from parent and mutant cell lines. In both cases, 22Na influx was stimulated by an inside-acid pH gradient, inhibited by ethylisopropylamiloride (EIPA), and unaffected by valinomycin, indicating that it was mediated by Na(+)-H+ antiport. Quantitatively, PKE20 vesicles showed a 4.2-fold increase in the maximal velocity of Na(+)-H+ antiporter activity compared with the parent, with only minor increases in the activity of two other Na(+)-dependent transporters (14-56% for alpha-methylglucoside and L-glutamate). Dose-response curves for EIPA indicated that the increased Na(+)-H+ antiport activity in PKE20 vesicles was due to an increased activity of the relatively amiloride-resistant form of the Na(+)-H+ antiporter with little or no change in the amiloride-sensitive form. No differences in polypeptide composition of the two vesicle preparations could be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Taken together, the results indicate that the mutation in PKE20 is expressed at the membrane level and is specific for the relatively amiloride-resistant Na(+)-H+ antiporter.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗

PubMed1988

Pharmacologically different Na/H antiporters on the apical and basolateral surfaces of cultured porcine kidney cells (LLC-PK1).

Proximal tubule cells of the kidney contain, on their apical surface, an amiloride-sensitive Na/H antiporter that functions in Na reabsorption and proton secretion. We have investigated the localization of the antiporter in a cloned cell line of porcine renal origin, LLC-PK1/Cl4, which is often considered to be a useful model of the proximal tubule. Transport measurements were performed with differentiated monolayers grown on Nuclepore filters, permitting independent access to the apical and basolateral cell surfaces. In control experiments with LLC-PK1/Cl4 monolayers, three marker transport systems showed the expected polarity: 87% of ouabain-sensitive Rb uptake was at the basolateral surface, and 99% of Na-dependent alpha-methylglucoside transport and 93% of Na-dependent D-aspartate (L-glutamate) transport were at the apical surface. By contrast, the monolayers displayed significant Na/H antiporter activity (assayed as ethylisopropylamiloride-sensitive 22Na uptake) at both cell surfaces, with an apical uptake rate amounting to 44% and a basolateral rate amounting to 56% of the total. Significantly, the apical and basolateral antiporters could readily be distinguished from one another on the basis of ethylispropylamiloride sensitivity. The apical system had an IC50 of 13 microM, close to that reported for kidney brush border vesicle preparations, whereas the basolateral system had an IC50 of 44 nM, similar to values seen in undifferentiated LLC-PK1 cells and other cultured cell lines. The PKE20 mutant, previously selected from LLC-PK1/Cl4 on the basis of resistance to ethylisopropylamiloride, was found to overexpress the more resistant antiporter both during rapid growth and on its apical cell surface at confluence; normal amounts of the more sensitive antiporter were seen on the basolateral surface of confluent PKE20 cells. Taken together, these results suggest that there are two distinct forms of the Na/H antiporter, which are under separate genetic control.

Animals↗

PubMed1988

LLC-PK1 mutant with increased Na+-H+ exchange and decreased sensitivity to amiloride.

LLC-PK1 cells contain a well-characterized Na+-H+ antiporter that is sensitive to ethylisopropylamiloride (EIPA) in the submicromolar range. Using a modification of the method of Franchi et al. (J. Biol. Chem. 261: 14614-14620, 1986), we have selected mutants that can recover from an acid load in the presence of 100 microM EIPA. One such mutant, designated PKE20, has been studied in detail. The maximal velocity (Vmax) for the Na+-H+ antiporter, assayed as EIPA-sensitive 22Na+ uptake, has increased from 44 nmol.min-1.10(6) cells-1 in the parent cells to 106 nmol.min-1.10(6) cells-1 in PKE20. No detectable change has occurred in the Km for Na+ (118 mM in the parent, 111 mM in the mutant) or in the dependence of Na+ uptake on intracellular pH. However, the PKE20 antiporter exhibits a greatly decreased sensitivity to amiloride and its derivatives, with drops in inhibitory potency ranging from 25-fold (amiloride) to 100-fold (EIPA). The mutation is specific for the antiporter; measurements of Na+-K+ pump and Na+-dependent amino acid uptake show only small changes, which appear to result from minor antiporter-induced alterations in internal Na+ concentration. PKE20 cells should prove useful in experiments to identify and isolate the antiporter protein.

Amiloride↗

PubMed1987

Stimulation by serum of the Na+/H+ antiporter in quiescent pig kidney epithelial (LLC-PK1) cells and role of the antiporter in the reinitiation of DNA synthesis.

LLC-PK1 cells can be brought into a classical quiescent state by depriving them of serum for 6 days. At this time, pulse-labeling with [3H]-thymidine shows that only 3% of the cells are synthesizing DNA, but the quiescent cells can be stimulated with serum to re-enter the cell cycle at a point early in G1. The rate of amiloride-sensitive 22Na+ uptake (as a measure of the Na+/H+ antiporter) is relatively low during quiescence; it rises 2- to 3-fold within 4 h after serum addition. This increase in antiporter activity appears to be required for the resumption of DNA synthesis in the absence of bicarbonate, because ethylisopropylamiloride (EIPA) blocks [3H]-thymidine incorporation when serum is added to cells in bicarbonate-free medium. In the presence of bicarbonate, however, EIPA has no effect on [3H]-thymidine incorporation, indicating that another (bicarbonate-dependent) transport system can substitute for the antiporter under these conditions.

Amiloride↗

PubMed1986

Isolation and characterization of a Na-H antiporter-deficient mutant of LLC-PK1 cells.

LLC-PK1 is an established cell line derived from pig kidney epithelium; it differentiates in vitro to form a polarized epithelial sheet, capable of the vectorial transport of solutes and water. We have used a modification of the "proton-suicide" method of Pouyssegur et al. (Proc. Natl. Acad. Sci. USA. 81:4833-4837, 1984) to isolate a mutant of LLC-PK1 cells that is deficient in Na-H antiporter activity. The mutant grows normally at pH 7.0 and above in the presence and absence of bicarbonate; at pH 6.5, however, it requires bicarbonate for growth. Alkalinization of the cytoplasm by the Na-H antiporter thus appears to be essential for growth at acidic pH in the absence of bicarbonate. The antiporter is also essential for the formation of domes (a consequence of vectorial water transport) in the absence of bicarbonate.

Amiloride↗

PubMed1985

Na+/H+ exchanger activity in the pig kidney epithelial cell line, LLC-PK1: inhibition by amiloride and its derivatives.

Rapidly growing pig-kidney-derived epithelial cells, LLC-PK1, lack detectable amiloride-sensitive Na+/H+ exchange activity when assayed directly. A large 22Na uptake is induced when the cells are acid-loaded prior to assay by incubation with buffer containing ammonium chloride or nigericin. The acid-stimulated sodium uptake is sensitive to amiloride, with half-maximal inhibition at 3.5-4.5 microM in buffer containing 15 mM sodium ion. There is simple competitive interaction between amiloride and sodium ion when the amiloride concentration is below 25 microM and the sodium ion concentration is above 20 mM. Derivatives of amiloride which carry substituents on the 5-amino group are 35- to 175-fold more inhibitory than amiloride itself.

Amiloride↗

PubMed1985

Mild proteolytic digestion restores exocytotic activity to N-ethylmaleimide-inactivated cell surface complex from sea urchin eggs.

The Ca2+-stimulated release of cortical vesicle (cortical granule) contents from the cell surface complex (CSC) of the sea urchin egg is an in vitro model for exocytosis. To gain insight into the molecular mechanism of exocytosis we investigated the sensitivity of this model to sulfhydryl modification and proteolytic digestion. Our findings include the following: (a) Proteolytic treatment with trypsin or pronase of CSC prepared from the eggs of Strongylocentrotus purpuratus increased the free Ca2+ concentration required to elicit exocytosis. Although a small increase in the Ca2+ threshold was detected after mild proteolysis, high concentrations of trypsin (0.5 mg/ml) and prolonged incubation (3 h) were required to render the CSC unresponsive to high concentrations of Ca2+ (0.5 mM). Despite the severity of the proteolytic digestions required to inactivate the CSC, the individual cortical vesicles remained intact, as gauged by the latency of ovoperoxidase, a cortical vesicle enzyme. (b) As previously shown (Haggerty, J. C., and R. C. Jackson, 1983, J. Biol. Chem. 258:1819-1825), cortical exocytosis can be blocked by sulfhydryl-modifying reagents such as N-ethylmaleimide (NEM). In this report we demonstrate that NEM inhibits by increasing the Ca2+ threshold required for exocytosis. When CSC that had been completely inactivated by NEM modification was briefly digested, on ice, with a low concentration of trypsin (or several other proteases), exocytotic activity was restored. Although the Ca2+ threshold of the reactivated CSC was slightly higher than that of untreated CSC, it was nearly identical to that of control CSC, which was trypsinized but not treated with NEM. We discuss the significance of these results with regard to the molecular mechanism of exocytosis.

Animals↗

PubMed1983

Release of granule contents from sea urchin egg cortices. New assay procedures and inhibition by sulfhydryl-modifying reagents.

We have developed a rapid turbidimetric assay for the release of cortical granule contents from cortices prepared from eggs of the sea urchin, Strongylocentrotus purpuratus. The decrease in turbidity of cortex suspensions which occurs when the free calcium ion concentration is increased to 0.38-0.62 microM can be followed spectrophotometrically. Kinetic experiments demonstrate that this calcium-triggered turbidity change occurs rapidly and with no detectable lag period. Evidence indicating that the observed decrease in turbidity results from the release of cortical granule contents was obtained by correlating the free calcium ion concentration required to initiate the turbidity change with the free calcium ion concentration required in microscopic and enzymatic assays. All three assays exhibited similar calcium dependence. In the microscopic assay, morphological changes are used to assess the extent of cortical granule exocytosis. The enzymatic assay is based upon the latency of ovoperoxidase, a cortical granule enzyme. Ovoperoxidase catalyzed a 30-125-fold increase in the incorporation of [125I]iodine into trichloroacetic acid-precipitable cortex protein at and above threshold calcium ion concentrations. We have utilized the turbidimetric assay to screen several potential inhibitors of the cortical reaction. In confirmation of previous reports, we find that the phenothiazine drugs, chlorpromazine and trifluoperazine, are inhibitory. Sulfhydryl-modifying reagents, N-ethylmaleimide and sodium tetrathionate, are also inhibitory. Inhibition of cortical granule enzyme release by N-ethylmaleimide was confirmed with the ovoperoxidase latency assay.

Animals↗

PubMed1981

Restoration of 125I-alpha-bungarotoxin binding activity to the alpha subunit of Torpedo acetylcholine receptor isolated by gel electrophoresis in sodium dodecyl sulfate.

The four subunits (alpha, beta, gamma, delta) of the acetylcholine receptor from Torpedo californica have been isolated by preparative gel electrophoresis in sodium dodecyl sulfate. After removal of the sodium dodecyl sulfate by dialysis of the polypeptides against a cholate-containing buffer, the alpha subunit, but not the other chains, binds 125I-alpha-bungarotoxin in a saturable manner. The binding affinity, 0.1-0.2 microM, is approximately 10(4)-fold lower than that observed for native acetylcholine receptor. For three preparations of alpha subunit, 1 mol of subunit bound 0.87, 0.38, and 0.33 mol of 125I-alpha-bungarotoxin at saturation. The binding was inhibited by cholinergic ligands, although the apparent affinities of these ligands for alpha were 50-100-fold lower than that found for the native receptor. These results indicate that at least part of the alpha-bungarotoxin binding site resides on the alpha subunit.

Animals↗

PubMed1980

Inhibition of electron transfer in the cytochrome b-c, segment of the mitochondrial respiratory chain by a synthetic analogue of ubiquinone.

A synthetic analogue of ubiquinone, 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole, inhibits oxidation of succinate and NADH-linked substrates by rat liver mitochondria. Inhibition occurs both in the presence (state 3) and absence (state 4) of ADP. With isolated succinate-cytochrome c reductase complex from bovine heart mitochondria the quinone analogue inhibits succinate-cytochrome c reductase and ubiquinol-cytochrome c reductase activities but does not inhibit succinate-ubiquinone reductase activity. Inhibition of cytochrome c reductase activities is markedly dependent on pH in the range pH 7-8. At pH 7.0 inhibition occurs with an apparent Ki less than or equal to 1 x 10(-8) M, while at pH 8.0 the apparent Ki is more than an order of magnitude greater than this. Spectrophotometric titrations of 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole show a visibly detectable pKa at pH 6.5 attributable to ionization of the 6-hydroxy group. These results indicate that this quinone derivative is a highly specific and potent inhibitor of electron transfer in the b-c1 segment of the respiratory chain. Because of the structural analogy, it is likely that the mechanism of inhibition involves disruption of normal ubiquinone function. In addition, this inhibition depends on protonation of the ionizable hydroxy group of the inhibitory analogue or on protonation of a function group in the b-c1 segment.

Animals↗