Selective internaiization of the apical plasma membrane and rapid

redistribution of lysosomal enzymes and mannose 6-phosphate receptors

during osteoclast inactivation by calcitonin

ROLAND BARON*, LYNN NEFF

Yale University School of Medicine, Departments of Cell Biology and Orthopaedics, New Haven, CT, USA

WILLIAM BROWN

Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY, USA

DANIEL LOUVARD

Department of Molecular Biology, Pasteur Institute, Paris, France

and PIERRE J. COURTOY

International Institute for Cellular Pathology, Brussels, Belgium

* Author for correspondence at: Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT06510, USA

Summary

The effects of inhibition of bone resorption by thepeptide hormone calcitonin have been studied atthe level of the osteoclast. Although not epithelial,the osteoclast is polarized with the secretion of newlysynthesized lysosomal enzymes and of acid occurringspecifically at the apical pole, facing the bonecompartment. The membranes composing the apical(ruffled-border) and basolateral domains containtopologically restricted antigens, a 100xl03Mr lyso-somal membrane protein and the Na+,K+-ATPase,respectively. It was found that calcitonin induces arapid (15-60 min) redistribution of the apical markeras well as of markers of the secretory compartment ofthe osteoclast (arylsulfatase and cation-independentmannose 6-phosphate (Man6-P) receptors). The apicalplasma membrane, in contrast to the basolateralmembrane, is selectively internalized. This internai-ization leads to the disappearance of the ruffledborder. The vesicular translocation of apical mem-branes is reminiscent of the events occurring in

gastric oxyntic cells and in kidney tubule interca-lated cells during the regulation of acid secretion. Inparallel, the synthesis of both the lysosomal enzymearylsulfatase and Man6P receptors is arrested. Theproducts that were already present in the secretorypathway seem to be rerouted to intracellularvacuoles instead of being targeted to the plasmamembrane, leading to marked accumulation of en-zymes in the inhibited cells. These results suggestthat the rapid inhibition of bone resorption bycalcitonin involves the vesicular translocation of theapical membranes and the rapid arrest in thesynthesis and secretion of lysosomal enzymes inosteoclasts.

Key words: osteoclast, calcitonin, bone resorption, lysosomalenzyme secretion, cation-independent mannose 6-phosphatereceptor, sodium pumps, lysosomal membrane proteins,vesicular translocation.

Introduction

Polarized epithelial cells that are involved in acidsecretion, such as the gastric oxyntic cell and the kidneyintercalated cell, can regulate their acid secretion by thevesicular insertion and removal of proton pumps fromtheir apical domain (Stetson and Steinmetz, 1983; Forte etal. 1977; Forte et al. 1981; Schwartz and Al-Awqati, 1986;Gluck et al. 1982; van Adelsberg and Al-Awqati, 1986). Inthe kidney tubule intercalated cell, this process ofvesicular translocation may even lead to a completeJournal of Cell Science 97, 439-447 (1990)Printed in Great Britain © The Company of Biologists Limited 1990

reversal in the polarity of acid secretion (Schwartz et al.1985; Brown et al. 1988). Vesicular translocation alsoregulates the insertion of glucose transporters in responseto insulin in adipocytes (Lienhard, 1983; Karnicli et al.1981; Blok et al. 1988) and has been implicated in theformation of the apical pole in cultured kidney epithelialcells (Vega-Salas et al. 1988).

Although not an epithelial cell, the osteoclast ispolarized and secretes acid into an apical compartmentduring the resorption of bone (Baron et al. 1985; Blair et al.1989). This cell is polarized both in terms of plasma

439

membrane composition and in terms of acid and lysosomalenzyme secretion (Baron et al. 1985; Baron et al. 1988).First, a 100xl03Mr membrane protein, otherwise ex-pressed in the limiting membrane of the lysosomes andendosomes of all cell types (Reggio et al. 1984), is presentat, and restricted to, the apical plasma membrane domainof the active osteoclast or ruffled border (Baron et al. 1985).Second, newly synthesized lysosomal enzymes, packagedinto transport vesicles and bound to mannose 6-phosphate(Man6P) receptors, are vectorially transported and se-creted into an apical extracellular compartment formed bythe attachment of the osteoclast to the bone matrix that itresorbs (Baron et al. 1988). This compartment is activelyacidified by the osteoclast (Baron et al. 1985) via the actionof apical proton-pumps (Baron et al. 1985; Blair et al.1989), themselves coupled to other ion-transport systemspresent at the basolateral surface (Baron et al. 19866; Tetiet al. 1989). These observations suggest that the functionof the osteoclast depends upon the polarized distribution ofa number of membrane proteins, which are essential inmaintaining the ion gradients required for bone resorption(Baron, 1989), and upon the synthesis, vectorial transportand polarized secretion of lysosomal enzymes into the boneresorbing compartment.

Like acid secretion in epithelial cells, bone resorption istightly regulated (Nijweide et al. 1986). Osteoclast activityis rapidly inhibited by calcitonin, a 32 amino acid hormonesecreted by the parafollicular cells of the thyroid. Thishypocalcemic peptide hormone has dramatic effects on theactivity and morphology of the osteoclast (Holtrop et al.1974; Lucht, 1973; Kallio et al. 1972; Baron and Vignery,1981) as well as profoundly altering its cytoskeleton(Chambers and Moore, 1983; Chambers etal. 1984; Hunteret al. 1989). The present study was designed to investigatewhether the rapid effects of calcitonin on bone resorptioninvolved alterations in the functional polarity of theosteoclast. This question was addressed using markers ofthe apical and basolateral membranes (100xl03Mr lyso-somal membrane protein and the Na+,K+-ATPase, re-spectively; Baron et al. 1985, 19866), as well as markers ofthe secretory pathway of the osteoclast (the lysosomalenzyme arylsulfatase and the cation-independent (CI)Man6P receptor; Baron et al. 1988). The results suggestthat calcitonin induces a rapid internalization of theapical ruffled-border membrane without affecting thedistribution of basolateral sodium pumps. The synthesisand secretion of lysosomal enzymes are arrested and theenzymes already present in the exocytic pathway at thetime of hormone action accumulate into intracellularvacuolar structures limited by membranes lacking lyso-somal or endosomal markers. The combined effect of thesemembrane and secretory alterations leads to an arrest ofbone resorption.

Materials and methods

Ultrastructural localizationPurified synthetic salmon calcitonin was obtained from Dr JamesTretter (Rorer Central Research, Ft Washington, PA). Thepreparation had a specific activity of 4500 MRC units mg"1. 5-day-old Wistar rat pups were injected subcutaneously with 0.001MRC unitg"1 of body weight (0.2ngg-1) in a total volume of0.1ml saline. Four animals were injected with saline beforeperfusion (control group), and 16 animals were treated withcalcitonin (4 at each time point). All animals were anesthetizedand killed 15, 30, 60 and 90min after injection of the hormone byperfusion of the fixative (formaldehyde 2 %-0.75 M lysine-0.01 M

sodium periodate (PLP) for immunocytochemistry or 1.5%glutaraldehyde in 0 . 1 M cacodylate buffer for enzyme cyto-chemistry) via the femoral arteries. After dissection of theperfused posterior limbs, the growth plate area was cut andprocessed for enzyme cytochemistry or for immunocytochemistryas previously described (Baron et al. 1985; Baron et al. 1988).

ImmunocytochemistryThe femoral distal and the tibial proximal growth plates werequickly dissected out and fixation was pursued by immersion at4°C for 15min in formaldehyde-glutaraldehyde followed by a 15-min quenching in 0.1 M phosphate-buffered saline (PBS) or 4 h inPLP, respectively. The preparations were next incubated for 1 h inPBS containing 1 % dimethyl sulfoxide, and frozen in liquidnitrogen. Frozen sections (40 /im) were prepared on a BrightCryostat (Huntingdon, England) equipped with Jung K tungstencarbide knives and a special holder. The sections were furtherfixed in PLP for an additional 2h and washed (PBS) beforeincubation. All sections were incubated and processed asdescribed by Brown and Farquhar (1984): briefly, cryostatsections were incubated in primary antibodies diluted in PBSwith 0.1 % BSA and 0.05 % Saponin overnight at 4°C, washed andincubated in secondary antibodies for 2h at 20°C; after washing,the sections were further fixed in 2.5% glutaraldehyde in 0 . 1 Mcacodylate+7% sucrose for 30-45 min on ice. After furtherwashing, they were reacted with di-amino benzidine (DAB) on icein the presence of H2O2 (0.1%).

Enzyme cytochemistryGrowth plates were dissected out and further fixed by immersionin glutaraldehyde containing 7% sucrose and 10% dimethylsulfoxide for 1 h, frozen in liquid nitrogen, and 40-jum thick frozensections were prepared. The sections were decalcified in 4%EDTA, 5% polyvinylpyrrolidone and 7% sucrose, pH7.4, at 4°Cfor 15-20 h, washed in 0 . 1 M cacodylate for 24-48 h, andincubated in the appropriate medium. Arylsulfatase was demon-strated using p-nitrocathecol sulfate as a substrate. Controlpreparations were incubated without substrate. Sections werepostfixed in 1 % OSO4 at 4°C for 1 h, dehydrated, and embedded inPolybed. All grids were stained with uranyl acetate and leadcitrate.

AntibodiesAntibodies against the cation-independent Man6P receptor (R2)were as previously described (Brown and Farquhar, 1984). Theaffinity-purified antibodies specifically recognize only the CIMan6P receptor by immunoprecipitation and immunoblotting(Brown and Farquhar, 1984; Brown and Farquhar, 1987). Theantibody was used at a concentration of 50^/gml""1 for theimmunocytochemical procedures.

Antibodies against the 100xl03Mr lysosomal membraneprotein were as described by Reggio et al. (1984). The purifiedantiserum recognized a 100xl03Mr integral membrane proteinpresent in lysosomes and in other compartments along theendocytic pathway (endosomes, coated vesicles) as well as theruffled-border membrane in osteoclasts (Baron et al. 1985). Theantiserum was used at a dilution of 1:100.

Antibodies against the rat Na+,K+-ATPase were as describedby Sweadner and Gilkeson (1985). Briefly, the rabbit polyclonalantibody K2 was raised against purified Na+,K+-ATPase from ratrenal medulla and was determined by Western blotting torecognize both the alpha and the beta subunits of the sodiumpump, but more specifically the kidney form of the alpha subunit(Sweadner and Gilkeson, 1985). The antiserum was used at adilution of 1:50.

Controls were incubated with rabbit non-immune serum or inthe absence of the primary antibodies and processed in parallel.

Results

Distribution of the membrane and secretory markers inuntreated osteoclastsThe distribution of arylsulfatase, the CI Man6P receptor

440 R. Baron et al.

and the 100xl03Mr lysosomal membrane protein inosteoclasts from control animals was as described pre-viously (Baron et al. 1985; Baron et al. 1988). The100xl03Mr protein was restricted to the apical ruffled-border membrane (Fig. 1A). This antigen could not bedetected at the sealing-zone or at the basolateral mem-brane. In contrast, using an antibody that recognizes thekidney isoform (alpha 1+beta) of the Na+,K+-ATPase, wefound this molecule mostly restricted to the basolateraldomain of the osteoclast membrane (Fig. 2A). The stainingof osteoclast membranes was abolished when this anti-

serum was preabsorbed with purified enzyme, therebyconfirming its specificity.

Arylsulfatase and the CI Man6P receptor were found toco-distribute in all elements of the exocytic pathway(Figs 3A and 5A), i.e. the endoplasmic reticulum (ER), theGolgi cisternae and the transport vesicles found in thetrans-Go\gi area and towards the ruffled-border mem-brane. A few secondary lysosomes, present in the basalportion of the cell, were stained for the enzyme and the100 x 103 Mr lysosomal membrane protein but, as expected,not for the Man6P receptor. No accumulation of the

m.

bmB bm D

Fig. 1. Effects of calcitonin onthe apical ruffled-bordermembrane of the osteoclast,traced with the antibodies to the100xl03Mr lysosomal membraneprotein. In control cells (A), theapical membrane of theosteoclast, facing the bonematrix (bm), exhibits extensiveruffling and contains the100xl03Mr antigen (filledarrows); numerous vesicles andlarger vacuoles are seen in theunderlying cytoplasm of the cell,most of which are limited byunstained membranes. Onelarger vacuole is labeled alongits limiting membrane (openarrow). In sharp contrast, theapical membrane of cells treatedwith calcitonin (B-D) show amarked decrease in apicalruffling. Fifteen minutes aftertreatment (B), the cell stillcontains numerous unstainedvacuoles but the ruffled border(filled arrows) exhibits much lessruffling; small vacuolescontaining the 100xl03Mrprotein are seen in thecytoplasm under the apicalmembrane (open arrows); inaddition, the basolateralmembrane of the treatedosteoclasts shows extensivefolding (top, curved arrow).(C) The apical portion of anosteoclast 30 min after calcitonintreatment and at the samemagnification as A; note thedecreased ruffling (filled arrows,bottom), the decreased number ofvacuoles and the presence of alarge vacuole heavily stained forthe 100xl03Mr antigen (openarrow, top; compare with similarstructure in A). By 60 min aftercalcitonin treatment (D), theruffled border is almost absentfrom the apical portion of thecell (bottom) and the 100xl03Mrantigen is observed only withinsmall cytoplasmic vacuoles, afeature rarely observed inuntreated cells, bm, bone matrix.Bars: A-C, 1/im; D, 0.25//m.

Redistribution of secretory and plasma membrane proteins 441

enzyme and/or the Man6P receptor was usually observedin cytoplasmic vacuoles other than the small transportvesicles and tubular elements of the trans Golgi network.

These observations therefore further established thepolarized distribution of plasma membrane proteins, theco-distribution of lysosomal enzymes and the CI Man6Preceptor along the exocytic pathway and the vectorialtransport and secretion of enzymes into the apicalsubosteoclastic bone compartment in osteoclasts.

Effects of calcitonin on the morphology of the osteoclastRapid changes occurred in the morphology of the osteo-clasts after treatment with calcitonin. The most dramaticchange was a marked decrease in apical ruffling, whichwas associated with an increase in membrane folding ofthe basolateral surface of the cells (Figs IB and 2C). By 30and 60min, the membrane interface between osteoclastsand the bone matrix was essentially smooth and was linedon its cytoplasmic surface by a layer of filamentousmaterial resembling the sealing zone, normally restrictedto the periphery of the contact zone between the osteoclastand the bone surface (Fig. 4). The total surface area ofcontact between the cells and the matrix most oftenseemed to be reduced (see Fig. 2C), the osteoclast goingfrom a relatively flattened to a more rounded-up configur-ation.

Effects of calcitonin on the distribution of the apical andbasolateral markersCalcitonin also had striking effects on the distribution ofapical plasma membrane proteins. Immunolocalization ofthe 100xl03Mr apical membrane protein after 15 and

30min indicated that it was found in vacuoles withstaining reaction along their luminal side (Fig. IB). By60min, the 100xlfJ3Mr antigen was not detectable on theapical plasma membrane (Fig. ID), which exhibited amarkedly decreased degree of ruffling (Fig. 1B-D). Thisantigen was instead found in intracellular vacuolesmorphologically resembling endosomes or lysosomes(Fig. 1C and D). In contrast, and despite the increasedfolding of this membrane domain, the distribution of theNa+,K+-ATPase at the plasma membrane was not affectedby treatment with calcitonin (Fig. 2). We interpreted theseresults as suggesting that the membrane composing theruffled border is rapidly internalized after calcitonintreatment while the basolateral membrane is not.

Effects of calcitonin on the distribution of proteinsassociated with the exocytic pathwayAs early as 15min after injection of the hormone, amarked change in the distribution of enzymes and CIMan6P receptors was observed. Arylsulfatase accumu-lated in numerous vacuoles, most of which were largerthan the Golgi transport vesicles observed in controlanimals (Figs 3 and 4). No clathrin-like coating could beobserved along the membranes of these vacuoles, incontrast to the transport vesicles seen in untreated cells.By 30-60 min, the accumulation of enzyme-loadedvacuoles was even more dramatic, particularly in trans-Golgi regions and in areas adjacent to the bone surface(Figs 3D and 4). The endoplasmic reticulum and the Golgicisternae were progressively depleted in enzyme (Fig. 3B).By 90 min new biosynthetic activity could again bedetected in these organelles. Interestingly, the increase in

I

Fig. 2. Effects of calcitonin on the basolateral membrane of the osteoclast, traced with antibodies to the Na+,K+-ATPase. Inuntreated cells (A) the Na+,K+-ATPase is restricted to the basolateral domain (filled arrows) and is not found at the attachmentzone (open arrow) or the apical ruffled-border membrane (not shown). (B and C) The distribution of the Na+,K+-ATPase 30 and60 min, respectively, after treatment with calcitonin; in (B) the contrast between the labeling of the basolateral surface (top arrows)and the ruffled-border membrane (rb, bottom) is well demonstrated. In (C) in a cell identified as an osteoclast by its highbasolateral concentration of Na+,K+-ATPase (filled arrows), the decrease in the apical membrane ruffling is apparent (open arrow,bottom) compared with untreated cells (Fig. 1A) or earlier time points (Fig. 2B), but no redistribution of the basolateral membraneis observed, bm, bone matrix. Bars, 1 ,um.

442 R. Baron et al.

the number of intracellular vacuoles stained for arylsulfa-tase, a marker of both primary and secondary lysosomes,was much larger than that observed for the 100xl03Mrmembrane protein, a marker of secondary lysosomes andendosomes (compare Figs 1 and 3-4). These resultssuggest that the majority of the arylsulfatase-positivevacuoles observed after treatment with calcitonin werenot endocytic in nature but, rather, part of the deliverysystem between the Golgi and the plasma membrane, i.e.secretory in nature.

The expression of the Man6P receptor followed the sametime course as the lysosomal enzymes but its distributiondiffered in two ways. First, only the enzyme could beobserved in the intracellular vacuoles that accumulatedimmediately after calcitonin treatment. Second,30-60 min after treatment Man6P receptors were detectedin tubuloreticular structures highly reminiscent of theER, most often accumulating towards the basolateralmargins of the cell (Fig. 5B and C). The more centrallylocated cisternae of the rough ER were usually depleted inimmunoreactive Man6P receptor (Fig. 5B), in contrast tothe untreated cells (Fig. 5A). By 90 min, Man6P receptors

could again be detected in all cisternae of the ER, therebyindicating new biosynthetic activity.

Discussion

This study demonstrates that inhibition of bone resorptionby calcitonin is associated with a rapid internalization ofthe apical plasma membrane and a redistribution of newlysynthesized lysosomal enzymes and mannose 6-phosphatereceptors in the osteoclast. These effects are observedwithin 15—30 min and are maximal after around l h . Theinternalization of the apical plasma membrane is selec-tive, since the basolateral membrane domain, althoughincreasing in folding, is not affected by calcitonin.

Osteoclasts are known to be the prime target forcalcitonin and to express a large number of calcitoninreceptors (Warshawsky et al. 1980; Nicholson et al. 1986).Administration of the hormone in vivo is accompanied by arapid (within minutes) decrease in serum calcium (Baronand Vignery, 1981). This rapid effect is attributed, for themost part, to the inhibition of osteoclastic bone resorption,

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Fig. 3. Effects of calcitonin onthe distribution of arylsulfatasein the exocytic pathway of theosteoclast. (A and C) Controlcells; in A, the lysosomal enzymeis found in the endoplasmicreticulum (ER), including theperinuclear cisterna (openarrow), in the perinuclear Golgistacks (filled arrows) and innumerous small transportvesicles on the trans side of theGolgi; in C, which shows aregion of the cell closer to thebone matrix (bm), only smalltransport vesicles are seen. Incontrast, calcitonin-treated cellsshow an accumulation ofarylsulfatase in numerous andlarger vacuoles (B and D); Bshows an area equivalent to A at30 min after calcitonintreatment; the Golgi cisternaeare depleted (filled arrow), ERcisternae including theperinuclear envelope (large openarrow), are rarely stained and,in addition to the smalltransport vesicles, largercytoplasmic vacuoles are stained(small open arrows). (D) An areacomparable to C but with astriking accumulation of largervacuoles containing arylsulfatasein the cytoplasm of the osteoclast60 min after calcitonintreatment. Bars, 1 ;un.

Redistribution of secretory and plasma membrane proteins 443

Fig. 4. Accumulation of vacuoles containing arylsulfatase inosteoclasts treated with calcitonin for 30-60 min. Thelysosomal enzyme has accumulated in a large number ofcytoplasmic vacuoles of irregular size and shape. (A) A lowmagnification of an osteoclast 30 min after calcitonintreatment. (B) A higher magnification of the region facing thebone matrix, where the accumulation of arylsulfatase-positivestructures is extreme. (C) A similar area in another cell withless pronounced accumulation of smaller vacuoles. Note that inboth B and C the osteoclasts do not have a ruffled border;instead, the interface between the cell and the bone matrix issmooth, with an extended zone of filamentous material liningthe cytoplasmic face of the membrane (arrows, C). Suchfeatures are normally restricted to the periphery of theattachment zone (sealing zone) in untreated cells (see Fig. 2A);bm, bone matrix. Bars: A, l,um; B and C, 0.5 /an.

Fig. 5. Effects of calcitonin on the distribution of the cation-independent mannose 6-phosphate (Man6P) receptor inosteoclasts. In untreated osteoclasts, immunoreactive Man6Preceptors are mostly colocalized with lysosomal enzymes in thebiosynthetic and exocytic pathway; (A) shows the presence ofthe Man6P receptors in cisternae of the endoplasmic reticulumin a control cell. Sixty minutes after calcitonin treatment (Band C), the endoplasmic reticulum is largely depleted inimmunoreactive Man6P receptors (B, left); instead, Man6Preceptors are found in an ER-like network of irregularcisternae towards the basolateral margins of the cell (arrows atright) and are still visible in the Golgi (open arrows). Similarfeatures are observed in C with, in addition, some largerendocytic vacuoles (arrows), also at the basolateral margins ofthe cell. Bar, 1 /an.

444 R. Baron et al.

although this hormone also affects mineral transport bythe kidney. It has long been established that treatmentwith calcitonin induces rapid changes in osteoclastmorphology with a marked decrease in the extent of theruffled border (Kallio et al. 1972; Holtrop et al. 1974), aswell as an apparent decrease in the extent of the contactwith the bone surface (Baron and Vignery, 1981). This isfollowed also, within hours, by a decrease in the number ofthese cells (Holtrop et al. 1974; Baron and Vignery, 1981).In vitro studies have shown that the most immediate effectof calcitonin on isolated osteoclasts is on the cytoskeletonwith a disruption of the tubulin network (Hunter et al.1989), an arrest in the cell motility and an overallretraction of the cell (Chambers and Magnus, 1982;Chambers et al. 1984; Kanehisa, 1989). These effects areassociated with calcium entry (Malgaroli et al. 1989) and aprolonged inhibition of the bone resorbing activity of theosteoclast (Kanehisa, 1989).

Our results confirm and extend these observations byshowing that, in addition to the cytoskeletal effects,calcitonin inhibits bone resorption by: (1) the selectivetranslocation of membrane from the apical domain,retrieving the transport systems that normally allow theacidification of the subosteoclastic bone-resorbing com-partment (Baron et al. 1985; Blair et al. 1989); and (2) anarrest in the secretion of lysosomal enzymes that arererouted to intracellular vacuoles.

We have followed the redistribution of the apical plasmamembrane with antibodies to an integral protein of100xl03Mr normally restricted to this domain of the cellsurface in the osteoclast (Baron et al. 1985) and, in all celltypes, to the limiting membranes of intracellular vacuolesthat belong to the endocytic pathway (Reggio et al. 1984).As previously reported (Kallio et al. 1972; Lucht, 1973;Holtrop et al. 1974), we observed that the ruffling of theapical membrane decreases rapidly after calcitonin treat-ment. Our results however suggest that the disappearanceof the ruffled border is not merely due to the detachment ofthe cells from their substratum, permitting the lateraldiffusion of proteins in the plane of the membrane, butrather (or also) to a specific internalization process of theapical membrane by vesicular translocation, comparableto the observations made in the kidney tubule and thegastric oxynitic cells (Gluck et al. 1982; Stetson andSteinmetz, 1983; Schwartz et al. 1985; Schwartz and Al-Awqati, 1986). The specificity of this internalizationprocess is further demonstrated by the fact that, incontrast to the apical domain, the basolateral plasmamembrane, traced here with antibodies to the sodiumpump, is not internalized. This membrane domain showsinstead an increase in folding, as was observed during theinactive phases of the egg-laying cycle in birds, duringwhich osteoclasts stop resorbing the bone matrix (Miller,1977). These changes may reflect the transition from themore rigid attached cell to the more fluid configuration of arounded-up cell on its substratum. It is noteworthy thatthe sodium pumps remained restricted to the basolateraldomain and were not redistributed to the apical mem-brane (Fig. 2C). We attribute this maintenance of restric-ted domains to the peristence of the zone of adherence ofthe osteoclast to its bone substratum, the presence of theadhesion apparatus (podosomes; Zambonin-Zallone et al.1989) probably preventing the lateral diffusion of proteinsin the plane of the membrane (Gumbiner and Louvard,1985).

The internalization of the apical plasma membrane,traced here with antibodies to the 100xl03Mr protein,

could also reflect a normal flow of membrane traffic. If thiswere the case, the changes in distribution observed aftertreatment with calcitonin would represent the lastinternalization events before the hormone effect isestablished. In both cases this would effectively removethe apical membrane transporters from the cell surface.

In parallel with this selective internalization of apicalplasma membrane proteins we observed a strikingredistribution of lysosomal enzymes. Arylsulfatase de-creased in the endoplasmic reticulum and the Golgi andaccumulated in a large number of vacuoles throughout thecell cytoplasm instead of being released in the bone-resorbing compartment by fusion with the apical mem-brane. Studies performed in organ and cell culture havepreviously suggested that calcitonin inhibits the secretionof lysosomal enzymes in culture medium (Vaes, 1972;Eilon and Raisz, 1978; Chambers et al. 1987). It was nothowever possible to determine in these experimentalconditions whether these changes resulted from effects onthe biosynthesis of the enzymes or on their secretion. Thefact that we observe a progressive decrease in enzymecontent and in immunoreactive Man6P receptors, despitethe longer half-life of this latter, in the endoplasmicreticulum and the Golgi cisternae of the treated cellssuggests that the hormone induces an inhibition of theirbiosynthesis. The enzymes that were already synthesizedand present along the exocytic pathway at the time ofhormone binding seem to be rerouted to accumulate inintracellular structures instead of being secreted. Theseresults suggest that calcitonin induces not only an arrestin the synthesis but also an arrest in the secretion oflysosomal enzymes.

The vacuoles in which the residual lysosomal enzymesaccumulated were found to be uncoated and to lackimmunoreactive Man6P receptors and lysosomal100xl03Mr membrane protein. This suggests that, de-spite their enzymatic content, these vacuoles do not belongto the endocytic pathway (Reggio et al. 1984; Brown et al.1986) but rather to the secretory pathway, formingstorage-granule-like structures. The effects of calcitoninon the secretory component of the osteoclast functiontherefore involves: (1) a rapid change in targeting of thetransport vesicles loaded with lysosomal enzymes from anapical surface domain to a vacuolar intracellular location,causing an arrest in secretion; and (2) a concomitant arrestin the synthesis of lysosomal enzymes.

The ultimate fate of both the enzymes and the Man6Preceptors remains uncertain. The lysosomal enzymes arenormally constitutively secreted by the osteoclast (Baronet al. 1985; Baron et al. 1988). Their rapid redistributioncould therefore represent a switch from a constitutivesecretion, with transport vesicles but no storage granules,to a regulated secretion where the secretory productsaccumulate in vacuoles before a new stimulation ofdischarge. It is noteworthy that such accumulation ofsecretory lysosomal enzymes is also observed in mono-nuclear precursors of the osteoclast during their latestages of differentiation, prior to their attachment to thebone surface (Baron et al. 1986a), and in differentiatingcells of the granulocyte/macrophage series in the bonemarrow (Bainton and Farquhar, 1966), to which theosteoclast is closely related (Nijweide et al. 1986). Finally,the fate of the Man6P receptors that were alreadysynthesized prior to hormone action might differ from thatof their ligands. Although they closely co-distribute inuntreated cells (Baron et al. 1988) we have not observedimmunoreactive Man6P receptors in the arylsulfatase-

Redistribution of secretory and plasma membrane proteins 445

positive vacuoles that accumulate after calcitonin treat-ment. Man6P receptors might therefore be rapidlydegraded or recycled after reaching this new delivery site.Conversely, we did not observe lysosomal enzymes in thereticular structures that contained Man6P receptors. Theexact nature of these structures is unclear but similarcisternae have been found in cells treated with Brefeldin A(Ulmer and Palade, 1990) and might represent themorphological counterpart of a degradative pathwaybypassing the Golgi (Chen et al. 1988; Lippincott-Schwartzet al. 1989; Nuchtern et al. 1989).

These results therefore clarify the mechanisms by whichcalcitonin inhibits bone resorption. The specific netinternalization of the apical membrane domain removesfrom the resorbing pole of the osteoclast a number offunctional proteins, among which are those associatedwith acidification (Baron et al. 1985; Blair et al. 1989). Thiswould dissipate the proton gradient at the apical pole ofthe cell, preventing the dissolution of the mineral phaseand the proteolytic action of the extracellular lysosomalenzymes on the organic phase of the bone matrix. Inparallel, the proteolytic degradation of the matrix isfurther stopped by the rapid multistep arrest in thesynthesis and secretion of the lysosomal enzymes.

Finally, these observations illustrate the fact that thedistribution and targeting of membrane and secretoryproteins are closely related to the functional state of thecells. They may rapidly be altered by regulatory agents.Although this concept is well established for agents suchas nocodazole, colchicine or Brefeldin A, our data suggestthat the effects of physiological agents such as hormonesmight involve similar redistributions. The rapid changesreported here in the osteoclast after calcitonin treatmentconstitute a new example of regulated changes infunctional cell polarity and targeting of secretory proteins.Other examples include reversibility of polarity inepithelial cells (Schwartz et al. 1985; Brown et al. 1988),insertion and removal of apical proton pumps in kidneyintercalated cells (Schwartz and Al-Awqati, 1986; Gluck etal. 1982; van Adelsberg and Al-Awqati, 1986) and gastricoxyntic cells (Stetson and Steinmetz, 1983; Forte et al.1977; Forte et al. 1981; Mercier et al. 1989), and insulin-regulated insertion of glucose transporters in adipocytes(Lienhard, 1983; Karnicli et al. 1981; Blok et al. 1988).

This work has been supported by a grant from the NIH(DE04724) and by a gift from the RORER PharmaceuticalCompany to Roland Baron. The authors are very grateful toKathleen Sweadner for the antibodies to the sodium pump and toMarilyn G. Farquhar for her advice and comments on themanuscript.

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BARON, R., NEFF, L., LOUVARD, D. AND COURTOY, P. J. (1985). Cell-mediated extracellular acidification and bone resorption: Evidence fora low pH in resorbing lacunae and localization of a 100 kD lysosomalmembrane protein at the osteoclast ruffled border. J. Cell Biol. 101,2210-2222.

BARON, R., NEFF, L., ROY, C, BOISVERT, A. AND CAPLAN, M. (19866).Evidence for a high and specific concentration of (Na+,K+)ATPase inthe plasma membrane of the osteoclast. Cell 46, 311-320.

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(Received 8 June 1990 - Accepted 18 July 1990)

Redistribution of secretory and plasma membrane proteins 447