INTRODUCTION

The endoplasmic reticulum (ER) consists of an extensivenetwork of interconnected membrane tubules spreadthroughout the cytoplasm and sheet-like cisternae, moreoften seen in the perinuclear region (Palade, 1956; Porteret al., 1945; Palade and Porter, 1954). The rough endo-plasmic reticulum (RER), a morphologically distinct sub-domain of the ER studded with ribosomes, is a componentof the endomembrane system involved in the biosynthesisof membrane and secretory proteins (Palade, 1975). Mor-phometric analysis has shown that an increase in secretoryactivity leads in many cell types to an increase in the sizeof the RER. For example, frog hepatocytes stimulated by17β-estradiol to secrete vitellogenin increase their RERcontent 4- to 5-fold (Bergink et al., 1977; Herbener et al.,1983; Rajasekaran et al., unpublished). Similarly, rat sem-inal vesicle epithelial cells induced by testosterone tosecrete plasma protein S and F (Falwell and Higgins, 1984),

aleurone cells stimulated by gibberellic acid to secrete α-amylase (Belanger et al., 1986) and resting B-lymphocytesstimulated to secrete immunoglobulins by either mitogensor specific antigen (de Vries et al., 1983; Shohat et al.,1973) enlarge their RER, but in all cases the increase wasnot quantified. Two studies with stable cell lines, the murineB cell line CH12 stimulated by lipopolysaccharide (LPS)to secrete IgM (Wiest et al., 1990) and the rat pancreaticacinar carcinoma AR42J cell line induced by dexametha-sone to secrete amylase (Logsdon et al., 1985; Swarovskyet al., 1988), have also reported an increase in the size ofthe RER.

An intriguing, but unexplained, finding is that in mostvery actively secreting cells the large stacked cisternal RER(SC-RER) predominates over the tubular and vesicular RER(TV-RER; Weiss, 1988). This is the case, for instance, inall the examples of induced secretion described above.Additional examples of large increases in this characteris-tic form of RER are found in the cells of the posterior silk

333Journal of Cell Science 105, 333-345 (1993)Printed in Great Britain © The Company of Biologists Limited 1993

A striking reorganization of the rough endoplasmicreticulum (RER) from a tubulo-vesicular (TV-RER) toa stacked cisternal (SC-RER) configuration wasobserved when the secretory activity of AR42J cells, acell line derived from a rat pancreatic acinar carcinoma,was induced by dexamethasone. Treatment with 10 nMdexamethasone resulted in a 6.6-fold increase in theintracellular and a 4.6-fold increase in the secreted amy-lase activity, respectively. On the basis of the morpho-metric analysis of thin-section electron micrographs ithas been previously reported that this increase in secre-tory activity is accompanied by a 2.4-fold or 30-foldincrease in the size of the RER. We have developed anew biochemical method to determine the size of theRER by quantifying the membrane-bound ribosomes.Using this procedure we did not detect any change inthe size of the RER after induction of an active secre-

tory state in AR42J cells. Electron microscopic obser-vation showed the predominance of SC-RER in dexam-ethasone-treated cells compared to the abundance ofTV-RER in control cells. Laser scanning confocalmicroscopy showed a patchy distribution of ER stain-ing in dexamethasone-treated cells compared to morebasal localization in control cells. On the basis of ourobservations we conclude that in AR42J cells theincrease in secretory activity induced by dexamethasoneis accompanied by a reorganization of the RER ratherthan by an increase in ER surface area, as reported byothers. Our results suggest that SC-RER is a biosyn-thetically more efficient form of the RER, which is foundpredominantly in actively secreting cells.

Key words: endoplasmic reticulum, ribophorin, boundribosomes, reorganization, dexamethasone, secretion

SUMMARY

Structural reorganization of the rough endoplasmic reticulum without size

expansion accounts for dexamethasone-induced secretory activity in

AR42J cells

Ayyappan K. Rajasekaran1,*, Takashi Morimoto2, David K. Hanzel3, Enrique Rodriguez-Boulan1 andGert Kreibich2

1Department of Cell Biology and Anatomy, Cornell University Medical College, New York, NY 10021, USA2Department of Cell Biology, New York University Medical Center, New York, NY 10016, USA3Molecular Dynamics, Sunnyvale, CA 94086, USA

*Author for correspondence

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gland, which is actively secreting large amounts of fibroinduring larval stages of the silkworm (Morimoto et al.,1968), and in chicken primary chondrocytes induced byascorbic acid to secrete collagen (Pacifici and Iozzo, 1988).It is unclear at present what mechanisms regulate the ampli-fication and structural changes of the RER during enhancedsecretory activity and what are the advantage(s) of the cis-ternal over the tubular form of the organelle. These strikingchanges in the morphology of the ER must be regulated byfactors yet to be identified. These factors may be either con-stitutively expressed in cells with a high secretory activityor induced upon stimulation of an active secretory state.

The mechanisms that control these remarkable structuralchanges of the RER can best be studied in cells that makelarge amounts of cisternal RER in response to a specificstimulus. The rat pancreatic acinar carcinoma cell line,AR42J, provides a useful model for this type of study, sincethese cells respond with a dramatic increase in secretoryactivity (5- to 20-fold) upon exposure to dexamethasone.Two previous reports of morphometric analyses indicatedthat the increase in the secretory activity of this cell line isaccompanied by a 2.4- (Logsdon et al., 1985) or 30-fold(Swarovsky et al., 1988) increase in the size of the RER.This discrepancy led us to reinvestigate the dexamethasone-induced changes in RER structure and size at the ultra-structural level and by a new biochemical assay thatallowed us to determine the size of the RER by quantitat-ing the amount of bound ribosomes (Rajasekaran et al.,unpublished). Our results indicate that the RER does notincrease in size in spite of a dramatic increase in the secre-tory activity after dexamethasone treatment. Rather, theincreased secretory activity of dexamethasone-induced cellsappears to depend on a change in the structure of the RERfrom a tubulo-vesicular to a cisternal configuration that isbiosynthetically more efficient. To our knowledge, this isthe first clear demonstration that reorganization of the RER,rather than an increase in surface area, is accompanied byan increase in secretory activity.

MATERIALS AND METHODS

Cell cultureAR42J cells (ATCC, CRL 1492) were obtained from the Ameri-can type culture collection (Rockville, MD). AR42J cells werealso provided by Dr Craig Logsdon (University of Michigan,USA) and Dr Horst Kern (University of Marburg, Germany). Cellswere maintained at 37°C as subconfluent monolayer cultures inDMEM containing 10% fetal calf serum supplemented with glu-tamine (2 mM), penicillin (100 i.u./ml), streptomycin (100 µg/ml),Fungizone (2 µg/ml) and polymyxin B (50 µg/ml). Cells obtainedfrom ATCC required insulin (4 µg/ml; Irvine Scientific, SantaAnna, CA), EGF (2 ng/ml; Boehringer Mannheim, Indianapolis,IN) and HEPES (20 mM, pH 7.4) for normal growth. Cells fromDr Logsdon’s and Dr Kern’s laboratories were maintained at 5%CO2 while the cells from the ATCC were maintained at 10% CO2.The doubling times of these cultures was about 24 h. Cells weregrown in 75 cm2 flasks (Corning, NY) and fed twice a week. Cellswere detached with trypsin (0.25%) in Hanks’ buffer containingEDTA (2 mM, pH 8; trypsin-EDTA) and plated at a density of5×106 to 6×106, 2×106 to 3×106 or 0.5×106 to 106 cells per 100mm, 60 mm or 35 mm dish (Corning, NY), respectively. The

medium was changed 2-3 h before the addition of dexamethasone(Sigma Chemical Co., St. Louis, MO) to a final concentration 10nM. Dexamethasone was disolved in 50% ethanol (5 µM stocksolution) and added 24-36 h after splitting the cells. An equiva-lent volume (2 µl/ml) of 50% ethanol was added to control cul-tures. The medium was changed once at 36 h and dexamethasoneor 50% ethanol was added immediately to experimental or con-trol cultures, respectively. Cells were harvested at 72 h after theinitial addition of dexamethasone. RU 38486 (kindly provided byDr D. Philibert, Roussel Uclaf, France) was dissolved in 50%ethanol and added (1 µM final concentration) 1 h prior to theaddition of dexamethasone.

Measurement of intracellular and secretedamylase contentIntracellular and secreted amylase was measured according to themethod of Logsdon et al. (1985), with the following changes. Cellsgrown in 60 mm dishes for 69 h in the presence of dexametha-sone were washed twice with MEM containing no phenol red.Cells were then incubated for 3 h in the same medium (1.2 ml)containing BSA (5 mg/ml) and soybean trypsin inhibitor (0.1mg/ml; Sigma Chemical Co., St. Louis, MO). The media wereremoved, PMSF (Sigma Chemical Co., St. Louis, MO) was addedto a final concentration of 1 mM and the cells were stored at−20°C. The cells were washed twice with PBS and scraped in 1ml of 50 mM sodium phosphate (pH 6.9) and 50 mM NaCl con-taining 1 mM PMSF. Cells kept on ice were then sonicated threetimes for 15 s each with cooling intervals of 10 s, using a W185cell disrupter (Heat System Ultrasonics Inc., Plainview, NY)equipped with a microtip. A sample of 500 µl was used for DNAanalysis. The remaining 500 µl was used to measure amylaseactivity as described in the Worthington enzyme manual (Wor-thington, 1988).

Biochemical analysisTo determine cell numbers the cells were washed twice in PBSand trypsinized for 3-4 min with trypsin-EDTA in Hanks’ buffer(see above). After the cells were detached, normal growth medium5 times the volume of the trypsin-EDTA-containing buffer wasadded. Cells were recovered by centrifugation, resuspended in thenormal growth medium and counted using a hemocytometer. Theprotein content of the cell lysates was quantified according to themethod of Lowry et al. (1951). RNA was analyzed according tothe procedure described by Munro and Fleck (1966). The precip-itate of the alkaline digest with cold PCA (perchloroacetic acid)was suspended in 0.6 M PCA, incubated in boiling water for 10min, chilled to about 4°C in ice water, and centrifuged at 3000r.p.m. for 15 min in the cold. The supernatant was transferred intoa clean tube, the pellet was resuspended in 0.6 M PCA and thesuspension was centrifuged. The supernatant was combined withthe previous one and used for DNA quantification by either UVabsorbance or the diphenylamine colorimetric method of Burton(1968).

Metabolic labelling, cell lysis andimmunoprecipitationAR42J cells grown in 60 mm dishes in the absence or presenceof dexamethasone for 72 h were washed twice in methionine-freeDMEM and starved for 30 min. The medium was replaced by thesame medium (1.2 ml/dish) containing 125 µCi/ml of [35S]methio-nine (specific activity 1209 Ci/mmol). After 5 min of labelling themedium was removed and the dishes were cooled to 0°C. Lysisof the cells and immunoprecipitation of amylase was done asreported earlier (Tsao et al., 1992). A rabbit anti-human amylaseantibody (Sigma Chemical Co., St. Louis, MO) diluted 200-foldwas used for immunoprecipitation.

A. K. Rajasekaran and others

335Reorganization of RER in AR42J cells

Northern blot analysisCells treated with or without dexamethasone for different timeperiods were washed once with PBS and total RNA was isolatedusing the acid/phenol/guanidinium thiocyanate procedure (Chom-czynski and Sacchi, 1987). RNA was dissolved in autoclavedwater and the concentration was determined by measuring theabsorbance at 260 nm. After electrophoretic separation on a 1.5%formaldehyde gel the RNA was transferred to a GeneScreenhybridization transfer membrane (NEN, Boston, MA), accordingto the procedure specified by the manufacturer. The filters werebaked at 80°C for 2 h and hybridized with full-length ribophorinI (RI), or ribophorin II (RII) cDNAs labelled with [32P]dCTP(specific activity 3000 Ci/mmole) using the BRL nick-translationkit (BRL, Gaithersburg, MD). The filters were washed four timesfor 10 min each at room temperature with 2× SSC containing 0.1%SDS, once for 15 min at 65°C with 0.1× SSC containing 0.1%SDS and then exposed at −70°C using Kodak X-Omat AR film.

Western blot analysisCell lysates were prepared from control or dexamethasone-treatedcultures according to the method of Tsao et al. (1992). Proteinsamples were separated by SDS-PAGE (10%) according to themethod of Laemmli (1970) and transferred to nitrocellulose paperat 200 mA for 12-15 h. The blots were blocked for 1 h in bufferA (10% Carnation milk in PBS) and incubated with rabbit anti-rat RI and RII polyclonal antibodies in buffer B (buffer A con-taining 0.3% Tween 20) for 2 h. The blots were washed in bufferC (PBS containing 0.3% Tween 20) 8 times for 5 min each andincubated in buffer B containing 50,000 c.p.m./ml of 125I-ProteinA (specific activity 2.59-3.70 MBq/µg) for 60-90 min. After wash-ing (8 times, 5 min each) in buffer C the blots were exposed toKodak X-Omat AR film.

Quantification of free and membrane-boundpolysomes in AR42J cells All steps were carried out at 4°C unless otherwise specified. Threeplates were selected from control and treated with dexamethasonefor cell counting to obtain the average cell number per plate. Thesame number of cells were used per sample (about 300 millioncells). Prior to harvesting, the cells were treated with cyclohex-imide (final concentration 1 mM) to stop protein synthesis andwashed twice in PBS containing the same concentration of cyclo-heximide. Washed control and dexamethasone-treated cells werehomogenized in a homogenizing solution (10 mM Tris-HCl, pH7.5, 10 mM KCl, 1 mM MgCl2) using a Dounce homogenizer.The homogenate was quickly mixed with 2.5 M sucrose to bringthe homogenate sucrose concentration to 0.25 M. Post-nuclearsupernatant (PNS) was obtained by centrifuging the homogenateon a 1 ml of 1 M sucrose-LSB (50 mM Tris-HCl, pH 7.5, 50 mMKCl, 5 mM MgCl2) cushion in a 15 ml Corex tube at 2500 r.p.m.for 5 min at 4°C in a Sorvall HB-4 rotor. The nuclear pellet wasstored on ice for further analysis. The PNS was adjusted to a finalconcentration of 2.1-2.2 M and 1 mM of sucrose and MgCl2,respectively. The mixture (2.1 M.S-PNS) was used to makesucrose step gradients as follows: 2.5 M sucrose-LSB (1.5 ml),2.1 M.S-PNS (8.0 ml), 1.99 M sucrose-LSB (1.5 ml) and 0.7 Msucrose-LSB (1.5 ml). The gradients were centrifuged in a SW-41 rotor (Beckman, USA) at 4°C for 20 h or longer at 36,000r.p.m.

The band at the interface between the 1.99 M and 0.7 M sucroselayers (membrane fraction) and the rest (free ribosome fraction)were diluted 3-fold with LSB and centrifuged at 4°C for 3 and 5h, respectively, at 40,000 r.p.m. in a 60Ti rotor. The membraneand free ribosome pellets were resuspended in 1 ml of LSB andused for further analysis.

Immunofluorescence and laser scanningconfocal microscopyCells grown on polylysine-coated glass coverslips were treatedwith dexamethasone for 72 h. The control cells were treated withthe same volume of 50% ethanol. Cells were washed once in PBS,fixed with 4% paraformaldehyde for 30 min and permeabilizedwith 0.2% Triton X-100 for 5 min. Then cells were washed withPBS and blocked with 2% BSA in PBS for 15 min. The endo-plasmic reticulum was visualized using an ER antibody kindlyprovided by Dr Daniel Louvard. Cells were incubated at 37°Cwith 1:50 diluted ER antibody for 30 min, washed with PBS 3times (10 min each) and further incubated for 30 min at 37°C withan anti-rabbit biotin-conjugated goat antibody (Vectar Labs,Burlingame, CA). After washing as described above, the cellswere incubated with streptavidin-conjugated Texas red (Tx-R) for30 min at 37°C, washed 6 times (10 min each) with PBS andmounted on glass slides using FITC-guard (Testog Inc., IL) as themounting medium.

Cells, fixed and stained as described above, were examined ina PHOIBOS 1000 laser scanning confocal microscope (Sarastro,Stockholm, Sweden). Tx-R was excited with an argon laser. Theemitted signals were collected and used to create three-dimen-sional reconstructions of serial confocal sections using the pro-gram Vanis (Sarastro, Stockholm, Sweden).

Electron microscopyCells grown on 60 mm dishes were washed twice with 0.1 Mcacodylate buffer (pH 7.4) and fixed with 2% glutaraldehyde in0.1 M cacodylate buffer for 2-4 h. At 30 min after addition of thefixative the cells were scraped and spun for 4-5 min in amicrofuge. The pellet was washed, fixed with 2% osmium tetrox-ide, processed by conventional procedures for electron microscopyand viewed with a Philips 300 electron microscope at 80 kV.

RESULTS

Effect of dexamethasone on the growthcharacteristics and content of intracellularand secreted amylase Treatment of AR42J cells with dexamethasone led to aninhibition of cell growth as measured by cell counting andDNA determination (Fig. 1A). Pretreatment of the cellswith the antiglucocorticoid agent RU 38486 (Moguilewskyand Philbert, 1984; Baulieu, 1989) abolished these bio-chemical changes (Fig. 1A). Furthermore, the RNA andprotein content, when normalized to DNA, was practicallyunchanged (1.2-fold higher) in dexamethasone-treated andin control cells (Fig. 1B).

Dexamethasone had a profound effect on the productionand release of amylase, a major secretory product ofinduced AR42J cells; 24 h after this treatment an approx.6.6-fold increase in intracellular amylase activity and anapprox. 4.6-fold increase in the secreted amylase activitywas observed. These values did not change significantly atlater times (Fig. 2; Fig. 3B). This correlated with a 6-foldincrease in the rate of incorporation of [35S]methionine intoamylase (Fig. 3A). On the other hand the rate of incorpo-ration of [35S]methionine into total TCA-insoluble mater-ial increased 1.8-fold after 24 h (Fig. 3A) and 2.4-fold after72 h of dexamethasone treatment (Table 1). On the basisof our results and on published data (Swarovsky et al.,1988) we estimate that, after dexamethasone treatment,

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amylase and total secretory proteins (including chy-motrypsinogen, trypsinogen, procarboxypeptidase andlipase; Swarovsky et al., 1988) constitute 23% and approx.50%, respectively, of the total newly synthesized protein.This indicates that the 1.8- to 2.4-fold increase in the incor-

poration into total protein may be primarily accounted forby the increase in the synthesis of secretory proteins. Theeffect of dexamethasone on the increase in amylase secre-tion was completely reversed by the antiglucocorticoid RU38486 (Fig. 2).

A. K. Rajasekaran and others

Fig. 1. Effect of dexamethasone on the growth of AR42J cells.(A) Cells grown in 60 mm dishes in the absence (CO) or presence(DX) of dexamethasone (10 nM) for 72 h were trypsinized andcounted using a hemocytometer. Cells were treated with RU38486 (RU) or with RU 38486 prior (1 h) to dexamethasoneaddition (RU+DX). DNA was quantified both by UV absorptionat 260 nm and by the diphenylamine reaction. (B). RNA wasdetermined by measuring the absorbance at 260 nm and proteinconcentration was determined according to the method of Lowryet al. (1951) after 72 h of growth in the absence (CO) or presence(DX) of dexamethasone. The values of RNA and protein werenormalized to DNA. Bar indicates average of 3 differentdeterminations.

Fig. 2. Effect of dexamethasone on the amylase activity in AR42Jcells. Duplicate cultures, grown in the absence (CO) or in thepresence of dexamethasone (DX), RU 38486 (RU), or RU 38486plus dexamethasone (RU+DX), were harvested and amylaseactivity in cell lysates and media was determined using starch assubstrate. The amylase activity in cell lysates or media weredetermined from the same dish and normalized to the DNAcontent of the respective culture. Bars correspond to the averagevalues obtained from duplicate dishes in two differentexperiments.

A

B

Fig. 3. Induction of amylase and total protein synthesis afterdexamethasone treatment. (A) Rate of synthesis of total proteinand amylase: duplicate cultures grown in 60 mm dishes were keptas controls or incubated with dexamethasone (10 nM). Cells werelabelled for 5 min with 125 µCi/ml of [35S]methionine and thenlysed with 500 µl of lysis buffer; 250 µl was used for DNAanalysis and the rest was used to determine total protein synthesisby TCA precipitation, and amylase synthesis byimmunoprecipitation. The radioactivity values recovered in eitherTCA precipitates or immunoprecipitates were normalized withrespect to the DNA content. Values are expressed as fold increaserelative to those of controls and are the means of two experimentsdone in duplicate. (B) Intracellular and secreted amylase activity:cells grown in 60 mm dishes were treated with dexamethasone (10nM). The cell lysate and medium were collected at different timepoints to assay for amylase activity. The DNA content fromrespective culture dishes was also obtained and used for thenormalization of the enzyme activities. Values are the average oftwo experiments done in duplicate.

337Reorganization of RER in AR42J cells

Effect of dexamethasone on the level of RER-resident proteins, ribophorin I and II in AR42JcellsThe increase in secretory activity in AR42J cells wasreported to be accompanied by an increase in the size ofthe RER (Logsdon et al., 1985; Swarovsky et al., 1988).

To determine whether dexamethasone induced an increasein the amount of RER, levels of the RER-specific proteinsribophorin I and II were measured, which were shown toincrease proportionally with respect to the size of the RER(Wiest et al., 1990). Ribophorins I and II are RER-specificintegral membrane proteins (Kreibich et al., 1978a) thatare found in the RER in a 1:1 stochiometric ratio withrespect to bound ribosomes (Marcantonio et al., 1984) andare in close proximity to these bound ribosomes (Kreibichet al., 1978b; Yu et al., 1990). Recently it has been shownthat the ribophorins are part of a heterotrimeric complexthat includes a 48 kDa polypeptide that has oligosaccha-ryl transferase activity (Kelleher et al., 1992). The contentof ribophorin mRNA and protein were quantified bynorthern and western blot analyses, respectively, atdifferent times after addition of the drug. Neither themRNA (Fig. 4) nor the protein (Fig. 5) levels of ribo-phorin I or ribophorin II changed after 24 h or 72 h (Fig.6), which are the times when the morphological changesof the RER were observed. Thus, the induction of secre-tory activity by dexamethasone in AR42J cells is notaccompanied by an increase in the levels of a characteris-tic RER marker.

Determination of the free and membrane-bound polysomes in AR42J cellsWe have recently developed a biochemical procedure tomeasure the RER, which is based on the quantitation ofbound polysomes (Rajasekaran et al., unpublished). Thelevels of bound polysomes can be expected to reflect theamount of RER and provide an independent estimate of thesize of this organelle. We applied this procedure to AR42Jcells at three time points after dexamethasone addition (48,72 and 96 h) when the change in the structure of the RERwas very prominent (see below). The same numbers of con-trol and treated cells from three different sources (see Ma-terials and Methods) were processed for cell fractionation,so that their sedimentation profiles can be directly com-

Table 1. Effect of dexamethasone on the total proteinand amylase synthesis

Control Dexamethasone

Parameter 24 h 72 h 24 h 72 h

Total incorporation (c.p.m.) 63,909 56,371 112,902 134,135Fold increase 1.8 2.38

Amylase (c.p.m.) 3,407 4,420 19,467 30,723Fold increase 5.7 6.95% of total 5.33 7.85 17.2 22.9

Cells were grown in 60 mm dishes for 24 h or 72 h in the absence orpresence of dexamethasone. At 24 h or 72 h the cells were labelled with[35S]methionine (125 µCi/ml) for 5 min and then lysed in 500 µl of thelysisbuffer. 250 µl was used for the quantification of DNA and the rest forthe determination of total incorporation of [35S]methionine by TCAprecipitation and incorporation into amylase by immunoprecipitation. Thevalues were normalized to DNA and are the average of two experimentsdone in duplicate.

Fig. 4. Northern blot analysis of ribophorins in AR42J cells. TotalRNA was extracted from cultures kept as control (DEX−) ortreated, for the time periods indicated, with dexamethasone(Dex+). The RNA loaded onto agarose gels was normalized withrespect to the DNA content of the cultures and 7-9 µg per lanewere separated electrophoretically. RI (A) and RII (C) mRNAswere detected by hybridizing GeneScreen membrane blots withthe respective full-length nick-translated cDNA probes. Blotswere exposed to X-ray film overnight at −70°C.(B) and (D) showethidium bromide staining of 18 S and 28 S rRNA of therespective gels after transfer to a GeneScreen membrane.

Fig. 5. Western blot analysis of ribophorins in AR42J cells.AR42J cells kept as controls (Dex−) or treated with 10 nMdexamethasone (Dex+) were grown for up to 72 h. Aliquots ofcell lysates corresponding to 100-130 µg protein afternormalization with respect to DNA were electrophoreticallyseparated and transferred to nitrocellulose filters and probed withrabbit antisera (1:20 dilution) directed against rat RI or RII. Blotswere then incubated with 125I-labelled Protein A and the bandscorresponding to RI and RII were visualized by autoradiography.

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pared without any further normalization. To this effect,postnuclear supernatants were subfractionated into totalmembranes and free ribosomes. These two fractions werecollected on sucrose density gradients, treated with high saltand puromycin and the absorbance profiles of the gradientswere obtained to quantify the ribosome content. As shownin Fig. 7, there was no detectable difference in the amountsof free and membrane-bound polysomes between controland treated AR42J cells. In particular, the approximate 1:1ratio of free to bound ribosomes is not affected by the dex-amethasone treatment. The nuclear fraction did not containany detectable amount of bound ribosomes, suggesting thatcontamination by unbroken cells was negligible and that allthe bound ribosomes were contained in the membrane frac-tion. Western blot analysis of membranes collected fromthe peak fraction did not show any difference in theribophorin I content, when comparing control and dexam-ethasone-treated cells (data not shown).

We also carried out studies in order to determinewhether the surface area of the RER increased without acorresponding increase in bound ribosomes. AR42J cellskept as control or treated for 72 h with dexamethasonewere labelled with [1 4C]uridine for 24 h and the total mem-brane fractions were prepared from post-nuclear super-natants as described in Materials and Methods. The mem-brane fractions were then subjected to isopycnic densitygradient centrifugation. The gradients were then fraction-ated (500 µl aliquots) and for each fraction the density ofsucrose and the radioactivity incorporated into ribosomeswas determined. As shown in Fig. 8 (A,B) the density dis-tributions of rough microsomes derived from control ortreated cells were similar, indicating that the surface areaof the RER did not change after dexamathasone treatment.The finding that the radioactivity contained in the mem-

brane fraction (Fig. 8A,B) is indeed incorporated intorRNA was confirmed by experiments showing that almostall radioactivity was distributed in proportion to the UVabsorbance of large and small ribosomal subunits, whenthe total membranes were treated with puromycin in highsalt buffer (Fig. 8C,D). These data provide independent

A. K. Rajasekaran and others

Fig. 6. Effect of dexamethasone on the levels of ribophorins I andII after dexamethasone treatment. Autoradiograms obtained afterdifferent exposure times of either northern or western blots of RIand RII werescanned with a transmission scanning densitometer(model GS300 connected to a computer with a GS370 integrator;Hoefer Instruments, San Francisco, CA) to determine the areaunder each peak. The values obtained in dexamethasone-treatedsamples were divided by the control values to obtain values forthe relative levels of RI and RII expression for each time point.

ml from the top

Fig. 7. Sucrose density gradient analysis of membrane and freepolysome fractions from control and dexamethasone-treatedAR42J cells.3×108 cells from control or dexamethasone-treatedcultures were used in this analysis. Total cellular membranes andfree polysomes were resuspended in 1 ml of LSB (50 mM Tris-HCl, pH 7.4, 50 mM KCl, 5 mM MgCl2). Aliquots (100 µl) ofeach fraction were adjusted to the buffer condition of HSB (500mM KCl,50 mM Tris-HCl, pH 7.4, 5 mM MgCl2) containing 1mM puromycin; final volume 300 µl. After incubation at 37°C for5 min the samples were loaded onto a continous (7.5% to 22.5%)sucrose density gradient in HSB, made on top of 1 ml of a2 Msucrose cushion, and centrifuged in a SW41 rotor at 36,000 r.p.m.for 3 h at 20°C. The nuclear pellet was resuspended in 1 ml LSBcontaining Triton X-100 (0.5% final concentration) and RNase (1µg/ml) was added. After a brief centrifugation in amicrofuge, 300µl of the supernatant was loaded onto a continous sucrose (10% to30%) LSB density gradient made on top of 1 ml of a 2 M sucrosecushion and centrifuged using a SW41 rotor at 36,000 r.p.m. for 3h at 4°C. After centrifugation the gradientswere withdrawn fromthe top using an Autodensiflow apparatus (Buchler instruments,NJ), and the absorbance was continuously recorded at 254 nmwith an Isco UA-5 absorbance and fluorescence detector (IscoInc., Lincoln NB). 40 S, 60 S are small and large ribosomalsubunits, respectively; mb, membrane.

339Reorganization of RER in AR42J cells

evidence for the conclusion that dexamethasone treatmentdoes not cause an increase in the size of the RER in AR42Jc e l l s .

Morphological characteristics of AR42J cellsafter dexamethasone treatmentThe results from our biochemical analysis led us to studythe effect of dexamethasone on the morphology of AR42Jcells. It was found that both control and dexamethasone-treated cells grew as small three-dimensional aggregates,rather than as monolayers. However, the size of the aggre-gates was smaller after drug treatment. At the ultrastruc-tural level the most striking difference between control anddexamethasone-treated cells was the appearance of secre-tory granules, and a change in the structure of the RER.Whereas in control cells secretory granules were rarely seenand the RER appeared as tubules, vesicles and isolatedsmall cisternae (Fig. 9A), in dexamethasone-treated cellsthe secretory granules were very prominent and the RERappeared predominantly as large stacked cisternae. Laserscanning confocal microscopy showed that the cells werepartially polarized and that amylase-containing granuleswere mainly localized at the apical pole (Rajasekaran et al.,unpublished results). Although the cisternal stacks werealready seen after 24 h of dexamethasone treatment (Fig.9B), they became very prominent at 72 and 96 h (Fig. 9C,D)after addition of the drug. Pretreatment of the cells with theantiglucocorticoid agent RU 38486 resulted in a controlphenotype (Fig. 9E), indicating that the effect of dexam-ethasone was specific and was mediated by the glucocorti-coid receptor. No significant changes in the morphology ofother organelles were detected.

Confocal microscopy of control anddexamethasone-treated cellsIn order to determine whether dexamethasone treatment ofAR42J cells causes changes in the cellular distribution ofthe ER, laser scanning confocal microscopy of control andtreated cells was performed on cells labelled with an anti-body specific for the ER. In en face views of control cells,the endoplasmic reticulum had a rather uniform distributionwith an apparent concentration in the perinuclear region thatis caused, in part, by the increased cell thickness at thislevel (Fig. 10a). After dexamethasone treatment, however,the ER staining was not uniform, but appeared to be con-centrated in discrete regions of the cell (Fig. 10c). In sideviews of control cells, obtained by a 90° rotation, the ERappeared to be preferentially localized at the base (Fig.10b), while in dexamethasone-treated cells, ER patcheswere observed at various levels (Fig. 10d). These patchesmay represent the clumps of SC-RER observed at the ultra-structural level (Fig. 9C,D).

DISCUSSION

This report demonstrates that a cell can drastically increaseits protein-synthetic and secretory activity without anincrease in the size of its RER or of its bound polysomepopulation. The increased rate of secretory protein produc-tion is accompanied by the reorganization of the RER froma tubulo-vesicular/small cisternal configuration (TV-RER)to that of large stacked cisternae (SC-RER) characteristicof ‘professional’ secretory cells. These results suggest thatthe organization of the RER into large cisternae allows

Fig. 8. Comparison of the isopycnic density gradient profile oftotal membranes from control and treated AR42J cells. Controlcells (A) and cells treated with dexamethasone (B) for 72 hgrown in two 10 cmplates were labelled with [14C]uridine (3µCi/10 cm dish, specific activity 532.9 mCi/mmole) for 24 h.Labelled cells were mixed with the corresponding unlabelledcells and total membranes were prepared as described inMaterials and Methods. The total membranes wereresuspended in LSB and a 120 µl aliquot from each sample waslayered on top of a 0.7 M to 2.0 M sucrose linear densitygradient made in SW41 tubes and centrifuged for 28 h at 4°C.The gradients were fractionated into 0.5 ml aliquots. 50 and100 µl from each fraction was used for the determination of therefractive index and the radioactivity, respectively. To verifythat the [14C]uridine-labelled material in total membranes wasincorporated into ribosomes, the total membranes of controlcells were treated with either LSB (C) or HSB and puromycin(D) and subjected to sucrose density gradient centrifugationusing LSB and HSB gradients as described in the legend toFig. 7.

340

secretory proteins made on bound polysomes to be synthe-sized at a higher rate.

Dexamethasone treatment of rat pancreatic acinar carci-noma AR42J cells resulted in a 6.6-fold increase in intra-cellular amylase and a 4.4-fold increase in secreted amylaseactivity. Since amylase constitutes about 23% of the newlysynthesized proteins and dexamethasone increases the syn-thesis and secretion of several other secretory proteins

(Swarovsky et al., 1988), the observed 2.4-fold increase intotal protein synthesis may be primarily due to an increasedsynthesis of secretory proteins. The greater capacity of thesecells to synthesize secretory proteins appears to be in partdue to increased levels of the corresponding mRNAs(Swarovsky et al., 1988); in the case of amylase it is clearthat the effect of dexamethasone on the mRNA content isat the transcriptional level (Logsdon et al., 1987).

A. K. Rajasekaran and others

Fig. 9. Changes in the structure ofthe RER in dexamethasone-treatedAR42J cells. Cells were grown for72 h in the absence (A) or in thepresence of dexamethasone (10nM) for 24 h (B) or 72 h (C,D).Cells were also treated with theantiglucocorticoid agent RU38486 (1 µM for 1 h prior todexamethasone treatment andgrown for 72 h (E). Note thepresence of smaller RER cisternaeand tubules, and the absence ofsecretory granules in control (A)and RU 38486-treated cells (E)and the alignment of smallcisternae and tubules to formlarger stacked cisternae after 24 hof dexamethasone treatment (B).Large stacked cisternae andsecretory granules are prominentin cells treated withdexamethasone for 72 h (C,D). N,nucleus; M, mitochondria; G,Golgi complex; SG, secretorygranules; arrowheads, RER. Bars,1 µm.

341Reorganization of RER in AR42J cells

Fig. 9C-E

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Do the increased levels of secretory protein synthesisreflect an increased amount of RER and bound polysomesor an increased efficiency of the existing ribosomes with-out an increase in their number?

To address this problem it was necessary to utilize a reli-able method to quantify the RER after induction of thesecretory activity by dexamethasone. In previous work onAR42J cells the size of the RER was determined using mor-phometric procedures (Logsdon et al., 1985; Swarovsky etal., 1988). These methods, while reliable, are tedious andlaborious and are subject to considerable sampling errors.We utilized, instead, two biochemical procedures to deter-mine the size of the RER. The first involved the measure-ment of RNA and protein levels of ribophorins by north-ern and western blot analyses, respectively. Ribophorins Iand II are present in the RER in a 1:1 ratio with respect tobound ribosomes (Marcantonio et al., 1984), to which theycan be cross-linked (Kreibich et al., 1978b). Recent evi-dence indicates that they are components of the oligosac-charyl transferase (Kelleher et al., 1992), which is part ofthe protein-translocating apparatus in the RER. Further-

more, a good correlation has been shown betweenribophorin levels and the amount of RER in CH12 cells(Wiest et al., 1990) and in B lymphocytes at different stagesof development (Zhou et al., unpublished results). Ourresults showed no change in the levels of ribophorins I andII after induction with dexamethasone, and thereforesuggest that the number of ribosomes bound to the RERdid not change in spite of an induction of secretory activityby dexamethasone.

To determine directly the effect of dexamethasone on theamount of bound polysomes, we utilized sucrose densitygradient analysis of a total cell membrane fraction, fromwhich ribosomes are released with puromycin in a bufferof high ionic strength. This is a very sensitive method thatcan detect changes of only 0.25-fold in the size of the RER,as measured by the amount of bound polysomes recoveredform microsomes (Rajasekaran et al., unpublished). Usingthis procedure the 4.2-fold increase in the population ofmembrane-bound ribosomes in Xenopus laevis hepatocytes,caused by steroid hormone treatment, is in good agreementwith a 4-fold increase obtained by morphometric analysis

A. K. Rajasekaran and others

Fig. 10. Visualization of the ER by laser scanning confocal microscopy in AR42J cells. Cells were grown on polylysine-coated coverslipsin the absence (a,b) or presence (c,d) of dexamethasone for 72 h and fluorescently labelled as described in Materials and Methods.Brightly fluorescent cells were selected for optical sectioning. 3-D reconstruction of the optical sections was performed from 35-40sections taken throughout the cell at 0.2 µm intervals. (a) and (c) show the en face view of the control and dexamethasone-treated cells,respectively. (b) and (d) give the view of the same cell from the side, rotated 90° latitudinally. Arrows indicate the the bottom of the cellattached to the coverslip. Note that in control cells ER is found predominantly in the perinuclear region (a) and at the basal pole of the cell(b).After dexamethasone treatment (c,d) a redistribution of the ER staining is observed. The large poorly stained blue areas represent thenucleus. Bar, 5 µm.

343Reorganization of RER in AR42J cells

performed on hepatocytes from a different species, Ranapipiens (Herbener et al., 1983). A similar correlationbetween an increase in the size of the RER and the amountsof bound polysomes was obtained by stereological analy-sis of thin-section electron micrographs of hepatocytesderived from rats treated with phenobarbital (Staubli et al.,1969). Our results, both on the quantification of ribophorinlevels and the direct determination of the amount of mem-brane-bound ribosomes support the conclusion that thelarge steroid-induced increase in secretory activity occurswithout an enlargement in the population of bound ribo-somes.

We then considered the possibility that dexamethasonetreatment causes an increase in the size of the RER surfacearea without a change in the amount of membrane-boundribosomes. However, this would result in a decrease in thebuoyant density of the rough microsomal fraction. Usingsucrose density gradient analysis Wibo et al. (1971) haveshown a correlation between the density of rat liver micro-somes and the number of bound ribosomes associated withthe microsomal vesicles (see also Amar-Costesec et al.,1984). Since the total membrane fractions from control andtreated AR42J cells had a very similar isopycnic densitydistribution in sucrose density gradients (Fig. 8), this pos-sibility was ruled out. In addition, a significant increase inthe amount of the bound ribosomes would have been easilydetected by a biochemical determination of total RNA. Ouranalysis of the RNA content shows no significant differ-ence between control and dexamethasone-treated cells (Fig.1b). Finally, since the ratio of free to membrane-bound ribo-somes in dexamethasone-treated cells is 1:1 (Fig. 7), whichis the same as in control cells, the possibility that free ribo-somes are converted into bound ribosomes can be excluded.On the basis of all of these criteria we conclude that dex-amethasone treatment of AR42J cells causes a largeincrease in the synthesis and secretion without an increasein the size of the RER.

Our results thus contradict those reported by Logsdon etal. (1985) and Swarovsky et al. (1988), who, respectively,reported 2.4-fold and 30-fold increases in RER, using mor-phometric analysis. The reason for the different resultsobtained by these two groups, or between these groups andus is not clear. Biased sampling or too small a number ofsamples can lead to a considerable error; these factors werenot extensively discussed by these authors. The fact thattwo independent biochemical procedures failed to detect anincrease in the amount of bound polysomes and that no shiftin the density distribution of the bound polysomes observedare very strong arguments against an increase in the size ofthe RER.

The increase in secretory activity of dexamethasone-treated AR42J cells must, therefore, reflect a biosyntheti-cally more efficient RER. On the other hand our morpho-logical results clearly show that this increased secretoryactivity is accompanied by a dramatic structural reorgani-zation and redistribution of the RER. Whereas the RER inuntreated cells was organized mainly as tubules, vesiclesand small cisternae (TV-RER) (Fig. 9A), 24 h after additionof dexamethasone, when the induction of secretion wasalmost maximal (Fig. 3A,B), both tubules and smaller cis-ternae had aligned to form the SC-RER (Fig. 9B), which

became the predominant RER type at later times (Fig.9C,D). At the whole-cell level, laser scanning confocalmicroscopy with antibodies against ER membrane proteinsshowed a redistribution of the ER in dexamethasone-treatedcells (Fig. 10c,d). Whereas, in control cells the ER stain-ing was concentrated mostly in basal regions of the cell,after drug treatment the ER staining appeared as discrete‘patches’ at various cell levels, presumably correspondingto the stacks of SC-RER observed at the ultrastructurallevel. A similar ‘patchy’ distribution of the SC-RER hasbeen reported in estrogen-treated Xenopus male livers(Bergink et al., 1977; Herbener et al., 1983; Rajasekaran etal., unpublished) and in the posterior silk gland of the silkworm during the fourth and fifth instars of the larval stage(Morimoto et al., 1968).

The dexamethasone-induced increase in amylase activityand reorganization of the RER were abolished by pretreat-ment of the cells with RU 38486, an antiglucocorticoidagent (Figs 1A,2 and 9E), suggesting that these processesare mediated by a glucocorticoid receptor. At the momentit is impossible to decide whether the increase in the syn-thesis of secretory proteins acts as a signal for the reorga-nization of the RER, or whether a common factor regulatesboth processes via the glucocorticoid receptor. The align-ment of RER elements (Fig. 9B) suggests the participationof cytoskeletal elements that is regulated by dexamethasoneand may be responsible in the generation and maintainanceof the SC-RER (Rajasekaran et al., unpublished results).

In the vast majority of actively secreting cells, or whenthe secretory state is induced, the RER appears as SC-RER(Weiss, 1988; Bergink et al., 1977; Herbener et al., 1983;Pacifici and Iozzo, 1988; Wiest et al., 1990 Morimoto etal., 1968; Rajasekaran et al., unpublished results). On thebasis of these and our results, we postulate that the SC-RER of secretory cells represents a more efficient arrange-ment of the components of the translation and translocationapparatus, thus allowing for an increase in the RER outputof secretory products. This hypothesis is based on the obser-vation that the amount of bound polysomes remains thesame in spite of a dramatic increase in the synthesis ofsecretory proteins. Two possible explanations may accountfor this observation. It is possible that some proteins thatare synthesized at significant levels prior to dexamethasonetreatment are either not synthesized or synthesized at amuch lower level after dexamethasone treatment; theseribosomes could then be utilized for the dexamethasone-induced synthesis of secretory proteins. Another possibilityis that a sufficient synthetic capacity is present within theexisting bound polysome population. Our preliminaryresults support the latter view. When the total membranesfrom control and dexamethasone-treated cells were brieflytreated with RNase about 2-fold more ribosomes werereleased from the control membranes, suggesting that theseribosomes are bound to the membrane via their mRNA butnot through the nacent chain and, hence, not active in pro-tein synthesis (Rajasekaran et al., unpublished results). Astructural reorganization of the RER to become SC-RERmay result in a more ordered distribution of these ribosomesand thus increase the efficiency of protein synthesis.

Furthermore, reorganization of TV-RER into SC-RERmay also represent a faster and energetically more efficient

344

mechanism of responding to increased demands for secre-tory products. Sea urchin eggs reorganize their non-corti-cal RER from a cisternal type to a more finely divided RERand back to a cisternal RER within 5-8 min of fertilization(Terasaki and Jaffe, 1991). In AR42J cells the reorganiza-tion of the ER was evident already within 24 h of dexam-ethasone treatment (Fig. 9B). In cases where the synthesisof new RER is well documented, this process usually takesmuch longer, e.g. more than 2 days in B cells induced byLPS (Wiest et al., 1990) and 5-15 days in frog hepatocytesinduced with estrogen (Bergink et al., 1977; Herbener etal., 1983; Rajasekaran et al., unpublished).

We have presented strong evidence that a dramatic reor-ganization of the RER from a tubulo-vesicular into a largestacked cisternal form, rather than an increase in the sizeof the RER, accounts for the increased protein synthetic andsecretory activity of AR42J cells stimulated by a gluco-corticoid. These results suggest an explanation for the pre-dominance of this form of RER in actively secreting cells.Additional work is needed to identify cytosolic factors thatcontrol this striking transformation of the RER.

We thank Dr David D. Sabatini for encouragement and sup-port. We also thank Dr Craig Logsdon and Dr Horst Kern for pro-viding us with AR42J cells, Dr Daniel Louvard for ER antibodyand Dr Philbert for RU 38486. Special thanks to Iwona Gumper,who carried out the thin-section electron microscopy. The help ofJ. Culkin, F. Forcino and H. Plesken with the preparation of theillustration is gratefully acknowledged. The help of the CornellUniversity Medical Art and Photography facility in the prepara-tion of photographs is acknowledged. We are grateful to Dr DiegoGravotta and members of the G.K. Lab. for helpful discussions.

This work was supported by National Institute of Health grantsGM21971 (G.K.), GM20277 (Dr David D.Sabatini) and GM34107 (E.R.B.) and by a grant from the American Cancer Soci-ety (CD-514; G.K.).

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(Received 14 December 1992 - Accepted 10 February 1993)