EXPERIMENTAL CELL RESEARCH 196, 72-81 (1991)

Enhanced Gap Junction Formation with LDL and Apolipoprotein B RITA A. MEYER, PAUL D. LAMPE, BARBARAMALEWICZ,* WOLFGANGJ. BAUMANN,* AND Ross G. JOHNSON’ Department of Genetics and Cell Biology, University of Minnesota, St. Paul, Minnesota 55108; and *The Hormel Institute, Austin, Minnesota 55912

nication [lo]. We are interested in identifying agents that control gap junction formation and elucidating the mechanisms involved in assembly. The regulation of gap junction channel permeability has been extensively studied (for reviews see [ll, 121) while the processes and components that modulate gap junction formation have only recently been investigated (see below). The mechanisms that govern junction assembly may be distinct from those involved in channel gating. Gap junction formation and the establishment of cell-cell communication are thought to occur in stages although the temporal sequence of events is unclear. The steps include: (1) transcriptional generation of the gap junction mRNA, (2) synthesis and post-translational modification of the gap junction protein (connexin), (3) formation of the connexons (hemichannels) consisting of six connexin monomers, (4) localization of the connexons to the appositional region in response to cell-cell contact, and (5) interactions between the connexons of adjacent cells to form functional intercellular channels. One example of the complexity of gap junction assembly is demonstrated when cell-cell contact occurs. Anticonnexin immunofluorescence studies of oocytes transfected with connexin cDNAs revealed a granular cytoplasmic staining which, upon pairing of two oocytes, shifted to the region of cell-cell apposition [13, 141. In other studies, increases in gap junction communication were correlated with increased expression of cell adhesion molecules (CAMS) when cells were transfected with CAM cDNA [15, 161. Our data illustrate another example of the complexity of gap junction assembly. Immunoblots of Novikoff cells probed with anti-connexin43 antibody revealed no detectable increase in gap junction protein levels. However, we observed increased intercellular dye permeability and increased numbers of aggregated gap junction particles. We investigated the effects of low density lipoprotein (LDL) and apolipoprotein B (apo-B) on gap junction assembly. LDL, a multicomponent particle composed of cholesterol, apoproteins, and phospholipids, is taken up by cells via receptor-mediated endocytosis which involves the binding of LDL to the LDL receptor followed by the internalization of the receptor-ligand complex

Gap junctions are plasma membrane specializations involved in direct cell-cell communication. Intercellular communication is dependent upon the assembly of gap junction structures and would be influenced by agents which alter the assembly process. We investigated the effects of low density lipoprotein (LDL) on gap junction assembly between cultured Novikoff cells using quantitative dye transfer and freeze-fracture electron microscopic methods. We observed a concentration-dependent increase in dye transfer (maximum effect at 2.6 Mg protein/ml) and a sixfold increase in the number of aggregated gap junction particles per cell. Immunoblots of Novikoff cells probed with anti-connexin43 antibody revealed no detectable increase in gap junction protein (connexin) levels. The influence of the different components of LDL on junction formation was also examined. First, we treated cells with cholesterol (O-160 a) in serum-free BSA media and observed a decrease in junction assembly. Second, we added apolipoprotein-B (apo-B) in phosphatidyl choline vesicles to the cells and observed a concentration-dependent increase in dye transfer (maximum effect at 2.6 bg protein/ml) and a fivefold increase in the number of aggregated gap junction particles per cell. The addition of phosphatidyl choline vesicles without apo-B had no effect on gap junction formation. Thus, we demonstrated that gap junction assembly can be modulated by LDL and apo-B treatments. o 1991 Academic PWES,IUC.

INTRODUCTION Cell-cell communication through gap junctions has been implicated in the control of cell proliferation, embryonic development, cell differentiation, and the regulation of differentiated function in postmitotic cells [l51. Gap junctions are dynamic structures with reported half-lives of 1.5-10 h [6-91. Therefore, factors that alter junction assembly are able to modulate cell-cell commu1 To whom correspondence and reprint requests should be addressed at Department of Genetics and Cell Biology, University of Minnesota, 250 Biological Sciences Center, 1445 Gortner Avenue, St. Paul, MN 55108. 0014~4827191 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

72

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[ 17-201. The LDL receptor recycles to the cell surface from the endosome after receptor-ligand dissociation while LDL is delivered to a lysosome. The classic function of LDL is the delivery of cholesterol to cells. The cholesterol liberated from LDL in the lysosome affects at least three processes: (1) it suppresses 3 hydroxy-3methylglutaryl CoA reductase (HMG CoA reductase), the rate controlling enzyme in endogenous cholesterol biosynthesis, (2) it activates acyl-CoA cholesterol acyltransferase (ACAT), which reesterifies excess cholesterol, and (3) it suppresses the synthesis of new LDL receptors and results in decreased amounts of the receptor on the cell surface [17-201. In the present study, we treated Novikoff cells with LDL and observed a concentration-dependent increase in intercellular dye permeability and increased numbers of aggregated gap junction particles. APO-B (in phosphatidyl choline vesicles) added to the cells increased junction assembly while cholesterol added in serum-free BSA media reduced assembly. The rapid onset (1 h) and extent of enhanced assembly with LDL and apo-B were indistinguishable and occurred at the same protein concentration (2.5 pg protein/ml). Our results suggest that the protein component of LDL, apo-B, can effectively replace LDL in eliciting enhanced gap junction assembly. Therefore, the trigger for altered junction assembly is the protein component rather than the cholesterol component of LDL. MATERIALS

AND

METHODS:

Cell line origin. The Novikoff hepatoma cell line (NlSl-67) was derived from a chemically induced rat liver tumor [21] and adapted to culture. The cells are probably not a hepatocyte cell line. Pitot and Potter [22] suggested the line may be of bile duct origin. We observed a reaction of Novikoff cell membranes with an antibody to A-CAM that reacts with cells of mesodermal origin [23, 241. No reaction was observed with anti-E cadherin antibodies that react with epithelial cells such as hepatocytes or bile duct cells [25] (unpublished data). A nonhepatocyte cell origin for Novikoff cells is also supported by immunoblots of Novikoff plasma membranes reacted with anti-connexin antibodies. Anti-connexin43 antibodies which react with heart and cells of mesodermal origin label Novikoff membrane proteins at 29, 43, and 45 kDa. Connexin32 C-terminal antibodies which react with hepatocytes do not cross-react with the Novikoff membrane proteins. Northern blots demonstrated that Novikoff cells contain RNA which hybridizes with a probe for connexin43 but not probes for connexin32 or 26 (another hepatocyte connexin) using high stringency washes (Finis and Johnson, unpublished). Our result suggests an origin from Kupffer or endothelial cells. Cell dissociation, recovery, and reaggregation. Cells in logarithmic growth were centrifuged from standard growth medium (S210 f 10% newborn calf serum, GIBCOIBRL), resuspended in S210 with 10 mA4 EDTA at 6 X lo6 cells/ml, and placed in a 37°C gyratory shaker incubator (200 rpm) for 15 min. The EDTA treatment was repeated resulting in greater than 95% single cells. The cells were “recovered” in S210 with 5% fatty acid-free bovine serum albumin (BSA; Sigma) for 90 min, which eliminated remnants of previously existing junctions [26, 271. Some cells were reaggregated by centrifugation at 30g for 5 min to yield loose pellets in 50 ml round bottom tubes. Cell pellets used for electron microscopy were incubated for 60 min at 37°C in a

GAP JUNCTION

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73

5% CO, atmosphere. Other recovered cells (2.5 ml at 8 X 10’ per ml) were settled out in 60 mm petri dishes for 60 min and used for microinjection experiments. Experiments were performed in triplicate except where noted. The electron microscopy required a multilayered cell population to minimize microscope time, while cell pairs were ideal for unambiguous dye permeability data on a single cell-cell interface. The two sets of data were qualitatively consistent. However, the values cannot be compared in a quantitative sense because the cells for electron microscopy and dye injection were not reaggregated in the same manner. Freeze-fracture and electron microscopy. Cell pellets were fixed in 2.5% glutaraldehyde in S210 medium for freeze-fracture and electron microscopy. Further processing was performed as previously reported [26, 271. We examined two replicas per treatment in each experiment. Methods of quantitation have previously been described [26, 271. Briefly, an “interface” was defined as a fractured membrane area comprising at least 57 pm2 (i.e., filling the screen at 10,000X on our microscope) and containing an indication of cell apposition. Interfaces were scored according to the presence or absence of one or more formation plaques or gap junctions. “Formation plaques” [28] were defined as specialized membrane areas with clusters or arrays of 9-11 nm intramembranous particles. Formation plaque area and particle number measurements were performed as reported in detail elsewhere [26,27]. Measurements were made with the aid of an Apple II computer equipped with a Houston Instruments Hipad digitizing tablet and morphometry software developed for use with the Novikoff cell system.

of dye. Dye injection studies were performed to Microinjection evaluate intercellular dye permeability between reaggregated cells that had been settled out in petri dishes. One cell of a cell pair was microinjected with a 4-ms pulse of 1% aqueous Lucifer yellow CH (Sigma) under 25 psi using a General Valve picospritzer. We recorded dye injection and subsequent cell-to-cell transfer on a Zeiss IM microscope equipped with a Dage SIT camera, Panasonic video recorder and TV monitor. The degree of dye transfer was estimated by measuring the time elapsed from the beginning of dye injection into the impaled cell to detection of fluorescence in the adjacent cell. These “transfer time intervals” [lo, 291 were determined by analysis of the videotapes. LDL (Sigma) was added Low density lipoprotein (LDL) treatment. to the cells in S210 media (GIBCOIBRL) containing 5% fatty acidfree BSA. We added a range of LDL concentrations (0.5, 1.25,2.5,5, 10, and 20 pg protein/ml) to the cells. LDL was either present in the recovery and reaggregation periods (2.5 h) or in the reaggregation period alone (1 h). We evaluated junction formation by measuring dye transfer times to examine intercellular dye permeability and quantitative freeze-fracture and electron microscopy to document junction size and number. We performed experiments to evaluate the Cholesterol treatment. effect on gap junction formation of added cholesterol in serum-free BSA media. Cholesterol dissolved in alcohol was forcefully injected into 10 ml of media containing 5% fatty acid-free BSA at 37°C and vortexed for 30 min. The final culture medium always contained less than 0.05% ethanol. We added cholesterol (20-150 PM) to the cells during both the recovery and reaggregation periods (2.5 h). Apolipoprotein-B (ape-B) in phsphutidyl choline vesicles treatment. Lyophilized apo-B (Sigma, >97% pure) was rehydrated in 10 mM deoxycholate and the pH was adjusted to 7.4. Phosphatidyl choline (PC, Sigma type IIIe, 396 rg) and N-(lissamine rhodamine B sulfonyl)phosphatidyl ethanolamine (R-PE, Avanti Polar lipids, 4 pg) were dried in a stream of nitrogen and dissolved in 900 pl PBS (137 m&f NaCl, 3 mM KCl, 10 m&f phosphate, pH 7.4) containing 10 mM deoxycholate. APO-B (100 pg/lOO ~1) was added to the phospholipids. A control mixture of 396 rg PC and 4 pg R-PE was also dried and

74

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dissolved in 1 ml of the deoxycholate, PBS buffer. Both samples were dialyzed against 1 liter of PBS for 48 h with two changes to form vesicles. The amount of reconstituted vesicles added to the cells is reported as the final apo-B in phosphatidyl choline vesicles concentration (0.5, 1.25, 2.5, or 5 pg protein/ml) or the equivalent amount (equal volumes) for the control vesicles without apo-B. The vesicle preparations were examined by freeze-fracture and showed mostly single vesicles of 20-24 nm size. Zmmunoblot analysis. SDS-PAGE was performed on 10% polyacrylamide gels [30] and the proteins were transferred to nitrocellulose (Biotrace NT, Gelman). The blots were blocked with 3% BSA in Tris-buffered saline (25 miV Tris-HCl, 150 m&f NaCl, pH 7.4) and incubated with either a rabbit pofyclonal anti-peptide antibody made to the N-terminus of heart connexin43 at 2 pg protein/ml (kindly provided by B. Yancey) [31] or a rabbit polyclonal anti-LDL receptor antibody at 8 rg protein/ml (kindly provided by R. Anderson [20]). The blots were next incubated with a goat anti-rabbit IgG conjugated to alkaline phosphatase and developed with 5bromo-4-chloro-3-indolyl-phosphate and nitroblue tetrazolium (KPL). ZDL receptor detecrion. Novikoff plasma membranes were isolated from untreated cells by a two-phase polymer method [32]. The plasma membranes were washed and used for immunoblots. Connerin detection. Whole cell samples were sonicated in 10 mM EDTA (Sigma), 2 mM phenylmethylsulfonyl fluoride (PMSF, Sigma), 1 mg/ml pepstatin (Boehringer-Mannheim), and 1 mg/ml leupeptin (Boehringer-Mannheim). Proteins from 5 X 10s cells were solubilized in Laemmli sample buffer [30], loaded in each lane, and used for immunoblots.

ET AL.

20

10

0 0

1.25

2.5

3.75

5

10

20

Added LDL (pg protein/ml) in BSA Cell-cell dye transfer times (R + SEM) observed between cells treated for 2.5 h with a range of LDL concentrations. One cell of a pair of cells was injected with 1% Lucifer yellow. The level of dye transfer (dye transfer time) was estimated by measuring the time elapsed from the beginning of dye injection into the impaled cell to detection of fluorescence in the adjacent cell. We observed increased intercellular dye permeability, reflected by the shortest mean dye transfer times, between cells treated with 2.5 pg protein/ml LDL compared to cells treated with BSA. Treatments with higher and lower concentrations of LDL resulted in longer dye transfer times indicating less intercellular dye permeability.

LDL receptor and junction protein measurements with LDL treatment. Cells were dissociated, recovered, and then reaggregated for 1 and 2.5 h at 37°C with LDL (2.5 pg protein/ml) or BSA as a control. Samples of the cells at each time point were processed to measure LDL receptor levels on the surfaces of intact cells. The treated cells (5 X lo6 per sample) were resuspended in 2.5 ml cold PBS (4°C) with anti-LDL receptor antibody (16 rg protein/ml) and kept on ice for 1 h. The samples were washed in cold PBS three times and incubated with I261 goat anti-rabbit IgG for 1 h on ice. The samples were again washed three times in cold buffer and counted on a gamma counter. Two independent experiments were performed. Parallel samples of the cells at each time point and treatment were processed for immunoblots to measure connexin amounts in whole cell samples. Cell adhesion evaluation. The numbers of single cells, cell pairs, triplets, and groups of 4 or more cells were counted on samples that were used for dye injection. The samples were fixed in 2.5% glutaraldehyde, stored overnight at 4°C and countedusing an ocular eyepiece with a grid on a Zeiss microscope. Five fields were counted per sample and at least two samples were averaged per treatment.

RESULTS

We investigated gap junction assembly between Novikoff cells which were dissociated, recovered, and reaggregated. We used freeze-fracture electron microscopy to assay for junction size. We observed small junctions (less than 60 particles) on 2% of the cell interfaces between cells that were dissociated and recovered, but not reaggregated. In contrast, we observed junctions with 250-1300 particles on 60-70% of the cell interfaces between cells that were dissociated, recovered, and reaggregated for 60 min. This procedure yielded little carry over of preexisting junctions into the formation experiments.

Novikoff cells were treated with a range of LDL concentrations for 2.5 h during the recovery and reaggregation periods. We observed a concentration dependent increase in intercellular dye permeability (Fig. 1) with microinjection of 1% Lucifer yellow. LDL at 2.5 pg protein/ml elicited the greatest increase in dye permeability, reflected by the shortest mean dye transfer time (Fig. 1). Higher and lower concentrations of LDL resulted in longer dye transfer times indicating less intercellular dye permeability. Treatment of the cells with LDL (2.5 pg protein/ml) for 1 h also resulted in increased junction permeability (dye transfer times; LDL, 13.6 + 1.4 s; BSA control, 32.4 f 2.1 s), demonstrating that the onset of the response elicited by LDL was rapid. Freeze-fracture and electron microscopy were performed to determine whether the increased intercellular dye permeability we observed was associated with an increase in the number of aggregated gap junction particles, in contrast to an effect on channel gating. A sixfold increase in the number of aggregated gap junction particles per interface was observed in freeze-fracture samples of cells treated for 1 and 2.5 h with LDL compared to BSA control cells (Fig. 2). We also observed an increase in formation plaque area per interface with LDL treatment (Table 1). Thus, we demonstrated enhanced gap junction formation with LDL in both the l- and the 2.5-h treatments. A correlation between the number of reaggregated junction particles and intercellular dye permeability was observed. The morphometrically calculated num-

LDL

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TABLE Quantitative

Freeze-Fracture

Formation plaque area (pm*) per interface (ii + SEM)

1 h BSA (control) 1 h LDL (2.5 pg/ml)

27 36

0.23 k 0.20 0.88 * 0.14

2.5 h BSA (control) 2.5 h LDL (1.25 *g/ml) 2.5 h LDL (2.5 pg/ml)

85 22 48

0.31 2 0.04 1.05 t 0.10 0.99 + 0.14

46 14 16 16 38 12

0.28 0.21 0.15 0.24 0.12 0.04

55 41 43

0.23 f 0.04 0.93 k 0.12 0.89 * 0.10

2.5 2.5 2.5 2.5 2.5 2.5

h h h h h h

20 pM cholesterol 40 PM cholesterol 60 pM cholesterol 80 pM cholesterol 100 pM cholesterol 150 pM cholesterol

1 h PC vesicles 1 h apo-B (1.25 gg/ml) 1 h apo-B (2.5 pg/ml)

ber of gap junction channels seen by electron microscopy has been proposed to correlate with junction permeability if the channels were in the open state [33,34]. Previous studies have also demonstrated that increases in the number and permeability of gap junctions correlated with increased time of cell reaggregation [26, 27, 331. Therefore, the Novikoff cell system provides a good model for the investigation of gap junction assembly. LDL is composed of several components including cholesterol and apolipoprotein B. We examined the effect of cholesterol added to the cells in serum-free BSA media. A range of cholesterol concentrations (O-150 PM) was added to the cells in fatty acid-free BSA for 2.5 h. We previously reported an increase in gap junction assembly with 20 pM added cholesterol in serum containing media [lo]. In the present study, we observed no significant change in the number of aggregated gap junction particles at 20 pM added cholesterol in serumfree BSA media and up to a 75% decrease with the higher cholesterol levels (40-150 pM; Fig. 3). A reduction was also seen in the area of the formation plaques (Table 1) and the percentage of cells displaying junctions (BSA control, 65%; 100 pM cholesterol, 49%; 150 pM, 43%). Therefore, cholesterol added to the cells in serum-free BSA media reduced gap junction formation. We next examined apolipoprotein-B, the major protein component of LDL and the component recognized by the LDL receptor. APO-B in phosphatidyl choline (PC) vesicles was added to Novikoff cells during the

Analysis

Number of interfaces*

Treatment”

FIG. 2. The number of aggregated gap junction particles observed in cells treated with LDL and assayed by quantitative freezefracture and electron microscopy. We observed a sixfold increase in the number of aggregated gap junction particles per cell interface (x -t SEM) with the addition of 2.5 pg protein/ml LDL for either the lor 2.5-h treatment times compared with BSA control cell samples.

1

+ t f f f f

0.04 0.07 0.03 0.06 0.02 0.01

’ All treatments were performed in S210 media containing 5% fatty acid-free BSA without serum. Treatments were for 1 h during the reaggregation period or for 2.5 h during the recovery and reaggregation periods. * An interface is defined as a fractured membrane comprising at least 57 pm* with an indication of cell-cell apposition.

reaggregation period (1 h). We quantitated a concentration-dependent increase in cell-cell communication with added apo-B (maximum at 2.5 pg protein/ml) (Fig. 4). We observed greater dye transfer between apo-Btreated cells compared to control cells (Fig. 5). We observed no change in dye transfer times with the addition of control PC vesicles lacking apo-B (Fig. 4).

z

BSA

20

40

60

Added Cholesterol

80

100

150

(PM) in BSA

FIG. 3. The number of aggregated gap junction particles observed in cells treated with a range of cholesterol concentrations in serum-free BSA media and assayed by quantitative freeze-fracture and electron microscopy. A concentration-dependent reduction of gap junction formation was observed with a 2.5-h treatment of cholesterol in serum-free BSA media.

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FIG. 4. Cell-cell dye transfer times (ii f SEM) observed between cells treated for 1 h with PC vesicles or apo-B/PC vesicles. One cell of a pair of cells was injected with 1% Lucifer yellow. The level of dye transfer (dye transfer time) was estimated by measuring the time elapsed from the beginning of dye injection into the impaled cell to detection of fluorescence in the adjacent cell. We observed increased intercellular permeability, reflected by the shortest mean dye transfer times, between cells treated with 2.5 pg protein/ml apo-B compared with control BSA-treated cells. Treatments with higher and lower concentrations of apo-B resulted in longer dye transfer times indicating less intercellular dye permeability. Control samples included a range of PC vesicle concentrations which corresponded to equivalent lipid concentration of vesicles containing apo-B. No differences were observed among the PC samples; therefore, the data were pooled and labeled as PC in the graph.

A fivefold increase in the number of aggregated gap junction particles per interface was observed upon treatment of the cells for 1 h with apo-B (1.25 and 2.5 pg

FIG. 6. Fluorescent and phase contrast photographs of Novikoff cells taken 1 min after microinjection of dye, showing transfer between cells treated for 1 h with PC vesicles (A) or 2.5 pg protein/ml apo-B/PC vesicles (B). Dye transfer was greater in cells treated with apo-B compared to control BSA-treated cells reflecting higher levels of intercellular dye permeability. Bar = 28 pm.

ET AL.

2. zA

;: t-4

FIG. 6. The number of aggregated gap junction particles observed in cells treated for 1 h with PC vesicles or apo-B/PC vesicles assayed by quantitative freeze-fracture and electron microscopy. A fivefold increase in the number of aggregated gap junction particles was observed with the addition of apo-B-treated cells compared with the BSA controls.

protein/ml; Figs. 6 and 7). We observed a fourfold increase in the area of the formation plaques per interface comparing the apo-B-treated samples to the control PC vesicles (Table 1). APO-B treatment resulted in increased intercellular dye permeability., Thus, apo-B can effectively replace LDL in eliciting the enhancement of gap junction assembly. The addition of LDL or apo-B to the cells increased gap junction formation in all our experiments, but the magnitude of the increase varied among experimental series. The average increase in the number of aggregated gap junction particles per interface was &&fold in treated samples compared to controls. The values ranged from 4- to 8-fold. A similar range of variability was also observed in the dye transfer rates from one series of experiments to another yet consistent trends were observed. Samples were compared within a series so that treated samples were compared to concurrent controls. We performed a simple cell-cell adhesion measurement to determine whether apo-B was merely enhancing cell-cell adhesion and thereby affecting gap junction formation. The numbers of singles, pairs, triplets, and clusters of cells were counted after the cells had been settled onto petri dishes. We observed no significant changes with the various treatments (Fig. 8). Thus, the effect of apo-B in phosphatidyl choline vesicles on enhanced gap junction formation is not likely due to major changes in cell-cell adhesion. We examined LDL receptor labeling on Novikoff cells to determine whether there was a change in LDL recep-

LDL

AND

APO-B

ENHANCE

GAP JUNCTION

ASSEMBLY

FIG. 7. Freeze-fracture micrographs of a control cell (PC vesicle) formation plaque (A) compared to a formation plaque between apo-Bt PC vesicle-treated cells (B). The apo-B-treated cells showed a larger number of aggregated gap junction particles per plaque compared to control cell interfaces. The arrowhead (A) points to an area of aggregated pits on the E-face membrane. Bar = 0.36 pm.

tor number that correlated with the enhanced gap junction assembly. The anti-LDL receptor antibody recognized a single protein band with an approximate molecular weight of 140 kDa on immunoblots of Novikoff cell membranes (data not shown) [20,35]. The rabbit polyclonal anti-LDL receptor antibody was then used to label the receptors found on the surfaces of Novikoff cells that had been treated for 1 and 2.5 h with BSA or LDL. The amount of LDL receptors was estimated by using lz51-labeled goat anti-rabbit antibody. We observed no significant differences in the amount of ‘251-labeled goat anti-rabbit antibody that reacted with the anti-LDL re-

ceptor antibody (ranging from 29,000 to 31,000 dpmlmg cell protein). Thus, no change in LDL receptors on the cell surface was detected during the period in which an increase in junction assembly was observed. The increase in gap junction formation we observed could have been due to elevated levels of gap junction protein. As a first attempt to investigate this issue, immunoblots of whole reaggregated Novikoff cells were reacted with an antibody directed against an N-terminal peptide of connexin43 (Fig. 9). Proteins from whole cell preparations were probed following a l- or 2.5-h treatment with BSA or LDL. The antibody labeled bands

78

MEYER

q q E

q

0

g

4r 8

90 2

$

$2 ?CI

z 3’

‘96 3 -E m

mb 2

singles pairs

triplets 4&greater

g

5 2

FIG. 8. Percentage of single cells, pairs, triplets, and groups of four or more cells observed with PC vesicles, apo-B/PC vesicles, and LDL treatments for 1 h. We observed no significant differences in cell-cell adhesion among these treatments. The apo-B concentrations are given in micrograms protein per milliliter.

with approximate molecular weights of 29, 43, and 45 kDa (Fig. 9). The 29-kDa protein is commonly observed and may be a degradation product of connexin43 [31]. The 43/45-kDa bands have been suggested to be nonphosphorylated and phosphorylated forms respectively of connexin43 [7, 36-381. We observed no obvious differences in immunolabeling intensities between the BSA control and LDL-treated cells. Immunoblots from four separate experiments were evaluated by a 1-D Analyst densitometry program on a Model 620 Video Densitometer (Bio-Rad Laboratories Inc). The areas under the peaks for each band were similar in the LDL-treated samples and the BSA control samples. LDL-treated sample values were 91-108% of the BSA control sample values. Concurrent samples of the whole Novikoff cell proteins (treated and controls) were electrophoresed in 10% polyacrylamide gels which were silver stained. The gels were analyzed by densitometry and the major protein bands (including a BSA band) were measured. No significant differences were observed in the protein profiles among the various samples indicating equal protein loading per lane. Our results suggest that LDL treatment can trigger modification of gap junction communication via the modulation of assembly in contrast to an alteration of connexin synthesis or turnover. DISCUSSION

The sizes of individual gap junctions as well as the total junction areas per cell vary substantially in different tissues [39], in different organisms [12, 401, and at different stages in embryonic development [39,41-441. The processes that produce gap junction variations

ET AL.

could act at different levels within cells, including message transcription [41,44,45], protein translation [8,44, 461, protein modification [7,36-38,471, protein translocation [13, 141, and turnover [48]. We have used Novikoff cells as a model system to quantify gap junction formation and inquire whether assembly is regulated. Novikoff cells are readily dissociated without the use of proteases and reaggregated to form functional junctions over a time course of minutes [28,26]. LDL treatment of the cells resulted in enhanced cell-cell communication and a sixfold increase in the number of aggregated gap junction particles without a detectable increase in levels of connexin43-related proteins. We previously reported that gap junction formation increased between Novikoff cells that were treated with 20 pM cholesterol in serum containing media [lo]. Cholesterol added to the cells in serum-free fattv acid-free BSA media resulted in reduced gap junction formation. Our results suggested that some serum component in conjunction with added cholesterol led to the increased gap junction assembly. We have also shown that colchicine, but not lumicol-

1234

FIG. 9. Immunoblot of whole reaggregated Novikoff cells treated for 1 or 2.5 h with BSA control or LDL. The primary antibody was a rabbit polyclonal antibody directed against an N-terminal peptide of connexin43. No annarent differences were observed in the intensity of the 29-, 43-, and 45-kDa protein bands (arrowheads) with the addition of LDL or BSA to the cells. Lane 1 = 1 h treatment with BSA, lane 2 = 1 h treatment with 2.5 pg protein/ml LDL, lane 3 = 2.5 h treatment with BSA, and lane 4 = 2.5 h treatment with 2.5 rgprotein/ ml LDL. Molecular weight markers are indicated on the left in kiloDaltons.

LDL

AND

APO-B

ENHANCE

chicine (an inactive analog), blocked the increased gap junction assembly observed with 20 PM cholesterol in serum (unpublished data). The results of the colchicine experiments suggested the involvement of microtubules either in the endocytosis of a component from the serum or in the exocytosis of connexin43 to the plasma membrane [7, 13, 141. The lack of enhanced junction assembly with cholesterol in serum-free BSA media along with the need for microtubules suggested that lipoproteins may be the factors in cholesterol-enriched serum that resulted in enhanced gap junction formation. LDL is a major lipoprotein in serum and is taken up by cells via receptor mediated endocytosis. We added LDL to the cells and observed a concentration-dependent increase in gap junction assembly. APO-B, the major protein component of LDL, also caused a five- to sixfold increase ,in gap junction formation and effectively replaced LDL in enhancing junction assembly. Our results suggest that the apoprotein component rather than the cholesterol component of LDL triggered the enhanced gap junction formation we observed with LDL treatment. This study was performed in serum-free fatty acidfree BSA containing media to eliminate the variability found in serum components. Gap junction formation in the Novikoff cell system has been shown to be serum concentration dependent. Optimal formation was observed in 5% serum with lower levels of formation at 10, 2, 1, and 0.5% serum. Five percent purified bovine serum albumin (BSA) protein can completely replace the whole newborn calf serum in supporting gap junction formation [49]. Therefore, we have replaced the 5% newborn calf serum with 5% BSA in this study. We observed no significant differences in the numbers of aggregated gap junction particles in samples reaggregated in serum containing media (580 + 99) and concurrent samples in serum-free BSA media (550 -t 87, Fig. 3). Other investigators have reported a loss of gap junctions [50], increased junction permeability [33,51, 521, or lack of response to agents [53] when experiments were performed in serum-free conditions. The results of our study may not be directly comparable with these other studies since we replaced serum with BSA. We do not know how 20 PM cholesterol added to serum resulted in enhanced gap junction formation. The addition of cholesterol to the serum may have reconstituted LDL into a form which is recognized by the LDL receptor [54-581. Serum complicates the interpretation of cellular responses to added agents. Serum has been reported to amplify the reduced junction permeability observed with the growth factor, EGF, which was likely due to the presence of EGF already in the serum [50]. Interactions of serum components can also occur. Insulin can modulate LDL receptor levels and insulin concentration varies among serum batches, thereby al-

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79

tering the potential response of the cells to added LDL [59]. We observed a biphasic concentration-dependent change in gap junction permeability with LDL or apo-B treatment. A similar biphasic response on gap junction permeability was reported when 3T3 cells were treated with a range of retinoic acid concentrations [60]. The mechanisms involved appear to be complex and may include two distinct and opposing forces [60]. Potential enhancement and inhibitory mechanisms have not yet been clarified for the effects of retinoids or for LDL and apo-B treatments in this study. We observed enhanced gap junction assembly without a detectable increase in levels of connexin43 related gap junction proteins. Our immunoblot results suggest that the increase in the number of aggregated gap junction particles may not result from a simple change in the synthesis or turnover of connexin43-related proteins. We have previously demonstrated that the enhanced gap junction assembly associated with 20 pM added cholesterol in serum containing media was abolished by protein synthesis inhibitors, but not RNA synthesis inhibitors [lo]. It was not clear whether the cycloheximide results demonstrated the need for gap junction protein synthesis or the synthesis of other proteins involved in assembly. Our present results suggest that the synthesis of the connexin43-related protein may not be required. The sensitivity to cycloheximide was likely due to the required synthesis of other proteins involved in junction formation (e.g., adhesion proteins). Immunoblot and Northern data suggested that connexin43 but not connexin32 or 26 are expressed in the Novikoff cells (Finis and Johnson, unpublished). However, we cannot exclude the presence of other undetermined connexins in Novikoff cells whose levels may change and thus be involved in the enhanced gap junction assembly triggered by LDL and apo-B treatments. More than one kind of connexin has been demonstrated to be present within a single cell or tissue [47, 61, 621. Other cellular events which may be involved in the enhanced gap junction assembly observed with LDL or apo-B treatments include the relocation of gap junction proteins from precursor pools. The existence of cytoplasmic precursor pools has been suggested by subcellular fractionation methods [63] and by immunofluorescent staining of cells with anti-connexin antibodies. Investigators observed punctate junctional staining at sites of cell-to-cell contact and prominent perinuclear cytoplasmic staining with immunofluorescence [7, 331 (unpublished in the Novikoff cell system). Gap junction precursors might also exist in the plasma membrane and redistribute upon cell-cell contact in a manner analogous to the lateral redistribution of acetylcholine receptors within the plane of the plasma membrane [64, 651. LDL added to the cells produced a rapid enhance-

80

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ment of gap junction assembly yet no change in the number of LDL receptors. APO-B increased junction assembly, while cholesterol in serum-free BSA media decreased gap junction assembly. Therefore, our data suggest that the intracellular events resulting from the liberation of cholesterol from LDL (e.g., a decrease in HMG CoA reductase or a decrease in LDL receptor synthesis [17, 191) are not likely involved. The enhanced gap junction assembly may instead be related to the initial events that occur in the receptor-mediated endocytosis of LDL and apo-B (e.g., receptor-ligand binding or receptor internalization [66, 671). Our study is one of a few that have described LDL activation of cellular components through a mechanism other than cholesterol metabolism [68-721. LDL has been reported to activate phosphatidylinositol turnover [70,71] which may result in a cascade of protein phosphorylation. Several studies have suggested that protein phosphorylation plays a key role in the regulation of junctions and possibly junction assembly [ 7, 36-38, 48, 73-751. Thanks are due to S. B. Yancey and R. Anderson for providing antibodies. This work was supported by NSF Grant DCB 8517726 and NIH Grant GM 37230.

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