JOURNAL OF HONE AND MINERAL RESEARCH Volume 10, Nurnher 6, I995 Blackwell Science, Inc.

Cell-to-Cell Communication in Osteoblastic Networks: Cell Line-Dependent Hormonal Regulation of Gap

Junction Function

H..!. DONAHUE,' K.J. MCLEOD,' C.T. RUBIN,' J. ANDERSEN,' E.A. GRINI: E.L. HERTZBERG,5 and P.R. BRINK'

ABSTRACT

We have characterized the distribution, expression, and hormonal regulation of gap junctions in primary cultures of rat osteoblast-like cells (ROBS), and three osteosarcoma cell lines, ROS 17/23, UMR-106, and SAOS-2, and a continuous osteoblastic cell line, MC3T3-El. All cell lines we examined were functionally coupled. ROS 17/23 were the more strongly coupled, while ROB and MC3T3-El were moderately coupled and UMR-106 and SAOS-2 were weakly coupled. Exposure to parathyroid hormone (PTH) for I h increased functional coupling in ROB cells in a concentration-dependent manner. Furthermore, €TH(3-34), an analog of FTH with binds to the PTH receptor and thus attenuates PTH-stimulated CAMP accumulation, also attenuated PTH-stimulated functional coupling in ROB, This suggests that PTH increases functional coupling partly through a CAMP-dependent mechanism. A 1 h exposure to PTH did not affect coupling in ROS 17/23, UMR-106, MC3T3-E1, or SAOS-2. To examine whether connexin43 (Cx43), a specific gap junction protein, is present in functionally coupled osteoblastic cells. we characterized Cx43 distribution and expression. Indirect immunofluorescence with antibodies to Cx43 revealed that ROS 17/23, ROB, and to a lesser extent MC3T3-El and UMR-106, expressed Cx43 immunoreactivity. SAOS-2 showed little if any Cx43 immunoreactivity. Cx43 mRNA and Cx43 protein were detected by Northern blot analysis and immunoblot analysis, respectively, in all cell lines examined, including SAOS-2. Our findings suggest that acute exposure to PTH regulates gap junction coupling, in a cell-line dependent manner, in osteoblastic cells. Furthermore, Cx43 is expressed in osteoblastic cells regardless of the degree to which the cells are functionally coupled or the degree to which coupling is regulated by PTH.

INTRODUCTION

I I I -lo-( I I I ( ' O M M I I N I ( . A I I O N is critical to the coordinated C cell behavior necessary for bone development and re- modeling. Morphological studies have shown that gap junc- tions, channels that span the transmembranc space, exist between bone cells"' and thus are ideally situated to func- tion in intercellular communication. Gap junctions function i n intercellular communication by allowing small molecules

(molecular mass < I kD) to pass from cell to cell. C'onncxins (Cx) are the protein subunits that form gap junctions, and in mammalian species at least 12 conncxins have 1)een identified. The most widely expressed of these include ('x2h and Cx32, originally isolated from rat and ('x43 from rat (for rcview see Ref. 6).

While recent data suggest that osteoblastic cclls can com- municate with each other via gap junctions composcd o f Cx43,('-' I ) the role this intercellular communication plays

'Musculoskeletal Research Laboratory, Departmcnt o f Orthopedics, The Pennsylvania State University Collcge o l Medicine. llcr\hcy,

'Department o l Orthopedics, State University of New York at Stony Brook, Stony Brook, Ncw York. 'Department of Ohstctrics and Gynecology, State University of New York at Stony Brook. Stony Brook, New York. 'Depnrtmcnt of Biophysics and Physiology, State University o f New York at Stony Brook, Stony Brook, New York. 'Departments o f Neuroscience and of Anatomy and Structural Biology, Albert Einstein Collcge of Medicine. Bronx, New Yorh.

Pennsylvania.

88 1

882 DONAHUE ET AL.

in bone function is unknown. Gap junctions may modulate bonc development and rcmodeling by coordinating the re- sponse of bone cell networks to physical or hormonal sig- nals.‘l’- IJ) This would occur if hormones affect the degree to which bone cells communicate via gap junctions ( i t . , are functionally coupled), as has been reported for several other cell Indeed, it has recently been reported that parathyroid hormone (PTH), a potent regulator of bone metabolism, stimulates functional coupling in a trans- formed osteoblastic cell line and primary cultures of osteo- blast-like cells.(’)

In this study we have characterized the distribution, ex- pression, and hormonal regulation of gap junctions in pri- mary cultures of rat osteoblast-like cells and four cell lines that express phenotypic characteristics of osteoblasts: ROS I7/2.8 and UMR-106, two transformed cell lines derived from rat osteosarcomas; SAOS-2, a transformed cell line from a human osteosarcoma; and MC3T3-E1, an immor- talized preosteoblastic cell line derived from mouse cal- varia. We examined the degree of functional coupling in these cell lines, relative to each other, because significant differences in functional coupling have been reported be- tween primary cultures of osteoblasts and transformed os- teoblastic cell lines.(”))

We chose to examine the effect of PTH on functional coupling for several reasons. First, PTH regulates bone remodeling through initiation of osteoblastic bone forma- tion as well as activation of osteoclastic bone resorption. Second, the effect of PTH o n osteoblasts is direct via bind- ing to PTH receptors, whereas the effect on osteoclasts is indirect, requiring prior interaction of PTH with osteo- blasts. This indirect effect of PTH on osteoclasts may be supported by direct physical contact between osteoblasts and osteoclasts, suggesting a role for gap junctions.‘”’ Fi- nally, the mechanism by which PTH regulates osteoblasts, and ultimately osteoclasts, includes the mobilization of CAMP and cytosolic calcium ion (Ca’+)(’’-’’) both of which are rcgulators of gap junctional coupling in other cell types.“”’ Utilizing Cd~bOXyflUOreSCein dye transfer tech- nique. we quantified functional coupling in osteoblastic cell networks in the presence or absence of rat PTH fragment (1-34).

METHODS

Reugents

Rat parathyroid fragment 1-34 (rPTH[1-34]) and an amino-truncated analog, PTH(3-34), were purchased from Bachem, Inc. (Torrance, CA). Dulbecco’s modified Eagle’s media (DMEM), modified Eagle’s media (MEM), McCoy’s media, Ham’s F12, sodium pyruvate, L-glutamine, and pen- icillin/streptomycin were purchased from Gibco (Grand Is- land, NY). Fetal bovine serum (FBS) was purchased from Hyclone Laboratories (Logan, UT).

Polyclonal antibodies prepared to a synthetic peptide corresponding to amino acids 346-363 of rat Cx43 de- scribed earlier(”2772x’ were affinity purified using a Beck- man Ultraffinity EP column to which peptide was coupled according to the suppliers protocol. Bound antibody was

eluted with 0.1 M glycine-HCI, pH 2.5, neutralized imme- diately with I M Tris base, dialyzed overnight against Tris-buffered saline, aliquoted and stored at -80°C prior to use. Anti-actin antibody was purchased from Sigma (St. Louis, MO; Cat. #A2668) and fluorescein isothiocyanate (FITC) linked goat-anti-rabbit IgG was purchased from CappeVOrganon Teknika (Malvern, PA; Cat. #55646). Carboxyfluorescein and FITC were purchased from Molec- ular Probes (Eugene, OR). All other reagents were pur- chased from Fisher Scientific (Pittsburgh, PA) and were of tissue culture grade.

Cell cu fture

Cell lines: ROS 1712.8 cells, supplied by Dr. R. Majeska, were cultured in Ham’s F-12 with 10% FBS, 2 mM L- glutamine, 1 mM sodium pyruvate, 100 IU/ml of penicillin and 100 p d m l of streptomycin. UMR-106 cells (American Type Culture Collection) were cultured in MEM with 10% FBS, 1% nonessential amino acids, 20 mM HEPES, 100 IU/ml of penicillin, and 100 p d m l of streptomycin. SAOS-2 cells (American Type Culture Collection) were cultured in McCoy’s 5A media, 10% FBS, 100 lU/ml of penicillin, and 100 p d m l of streptomycin. MC3T3-El cells were provided by Dr. D. Yamaguchi and were cultured in MEM with 10% FBS, 2.2 g/l of NaHCO,, and penicillinlstreptomycin.

Airnary cultures of rat osteohlust-like cells: Rat osteoblast- like cells (ROBS) were isolated from tibiae and femora of 4-month-old male F344 rats. The bones were removed from anesthetized rats, stripped of soft tissue and periosteum, and placed in cold DMEM. The epiphysis remained intact ensuring that only cells from the subperiosteal surface were isolated. Once all bones had been isolated from a given rat, the whole nonminced bones were placed in 5 ml of 2 mgiml of collagenase (Type 1, Worthington) in phosphate buff- ered saline (PBS). Bones in collagenase were incubated in a shaking water bath for 1.5 h at 37°C and then removed from the collagenase solution. The cells from this first digestion were collected by centrifugation and not used in these experiments. The bones were then returned to the collagenase solution and the incubation continued for an additional 1.5 h. The cells from the second digestion were collected by centrifugation and placed in T-25 tissue culture flasks (25 cm2 surface area) and fed with DMEM, 10% FBS, and 100 lU/ml of penicillin and 100 pgiml of strepto- mycin. The cells were allowed to reach confluency (1-2 weeks) and then subcultured into culture dishes appropri- ate for experiments. Experiments were completed on cells in the first or second passage. Table 1 summarizes the phenotypic characteristics of ROB isolated in this manner.

Cell coupling

Cells were grown to confluency in 3.0 cm diameter, round tissue culture dishes. A single cell was selected for dye loading based on morphology and the number of neighbor- ing cells with which it was in immediate physical contact. Only plump cuboidal cells in physical contact with at least five cells were loaded. The selection of cells based on morphology was particularly important for ROB cells that

CELL-TO-CELL COMMUNICATION IN OSTEOULASTIC CELLS 883

ROS ROB RSFB -

Dcx-stim AP 30 00 0

PTH-stim CAMP 5000 550 40 (% over bas:il)

(%I over basal)

(% over basal) PTH-stim [Ca' ' Ii 500 400 20

Type I collagen mRNA + + n.1. PTH stim I L - 0 m R N A + + AI' stain + -1

-

-

- Phenotypic chaiactcristics were determincd in ROS 17iZ.X

(ROS). primary culturcs of rat ostcohlast-like cells (ROB) and rat skin fihrohlasts (RSFl3). KSFB were isolated a n d cultured as prc- viously descrihcd.""' For clexamcthasonc-stimulated alkaline phos- phatasc activity (dcx-stim AP) cells were exposed for 24 h to I0 M dcxanicthasonc and alkaline phosphatax activity determined hy quantifying conversion of p-nitrophcnyl phosphate t o p-nitrophenoi+ph(,sphate. ('ytocheniical cvidcncc for alkaline phosphatasc activity ( A P stain) was obtained using a commercially av;iil;ihlc kit (Signxi # M - R ) . PTH-stimulated CAMP accumulation (PI'H-stim CAMP) wits dctci-mined following ii 15 min exposure lo 10 M rPI'H( 1-34), PTli-stimulated cytosolic calcium ion con- centr;ition (PTH-stim (Ca' ' ],) was measured in acquorin loaded cells during cxpoaurc t o 10 M rPTH(1-34). as previously de- scribed.""' PTH-stim IL-h mRNA expression (PTki-stim I L A mRNA) was quantified hy reverse transcriptax polymerase chain reaction as previously dcscrihcd.' '"' Type I collagen was dcter- mined by Northern hlot analysis.

displaycd two distinct morphologies: plump cuboidal and stellate. Plump cuboidal cells were considered more osteo- blastic because they consistently stained strongly for alka- line phosphatase while stellate cells did not. Furthermore, stellate cells were often not coupled and did not display Cx43 immunorcactivity.

Coupling was examined in cclls bathed i n PBS (1.8 mM CaCI, and 0.8 mM Mg,SO,) as previously (,-carboxyfluorcscein (0-CF, M W 376) wxs dissolved in I20 mM potassium citrate (pH 9.0) to yield a final stock con- centration of 20 mM 6-CF, arid was titrated to pl-l 7.0. Stock 6-CF solution was stored in the dark at -20°C. Microelec- trodc tips (0.1 pni O.D.) were backfilled with stock dye solution and cells imp:iled. The fluorescent probe filled the cell by simple diffusion. Typical filling times ranged from 30 s t o 2 min. Imp;ilcnients were monitored o n the stage o f a n inverted microscope (Nikon Diaphot. Melville, NY) by low light phase contrast and epifluoresccnt imaging. Epi- fluorcsccncc illumination was provided by a 100 W mercury lamp power source and a 400 nm excitation filter. A small percentage o f impaled cells were damaged such that dye diffused out o f the cell i n t o the surrounding mcdia; these cclls were not included in the analysis. Five minutes after a n individual cell was loaded. the number o f neighboring cells containing dye wcrc counted. Histograms depicting the fre- qucncy with which individual dye loaded cclls were coupled to a given number of neighboring cells were developed for each cell line. The coupling experiments were done with

cells in PBS to which had been added vehicle, I0 I" t o 10 "

M rat PTH(1-34), or I0 " M rPTH(1-34) and 10 " M rPTH(3-34), 1 h prior t o the start of the cxpcrimcnts. We chose to incubate cells in PTH 1 h prior t o experiments to ensure sufficient time for PTH t o aff'cct coupling through trmscriptional, translational, or post-translatioii~il mechanisms.

PTH-stimulutcd CAMP uccumulution in ROB

ROB cells were grown to confluency in (,-well multiwcll plates. PTH stimulation was carried out in culture inedi;i t o which had been added I mM isobutylmethylxanthcnc (IBMX). The cells were stimulated at 37°C' for IS min with I W " to 10 ") M rPTH( 1-34). The reaction was stopped by the addition of boiling sodium acetate buffer (0.05 M t 1 mM IBMX, pH 5. I ) . Cells were scraped from the wells and transferred with the buffer to glass tubes. Each well was washed with another 0.5 ml of buffer, which was pooled with the scraped cclls. The glass tubes were placed on ;I

boiling water bath for 5 min and subsequently transferred to ice f(>r I 0 min. The cold tubes were centrifuged at 3OO(!q lor 10 min and the supernatant frozcn at --20°C f o r later analysis of total CAMP (cells and media) by RIA kit (Incstar, Stillwater, MN).

lmmunostuining with untihodies to gup junction proteins

Cells were plated at ;I density of 2.5 x IO' o n t o 25 inin

glass covcrslips and grown to conflucncy. The coverslips were then rinsed in cold PBS and fixcd for IS min with 4% paraformaldehydc in 0. I M PBS at 4°C. The covcrslipc were washed briefly in PBS and left to soak in PBS for at least 00 min at 4°C. They were then drained o f excess buffer and placed cell side up in a covcrcd box lined with dampened filter paper to prevent drying. Antibody to Cx43 was diluted 1:250 in 0.1 M PBS containing 0.3% 'Triton-X 100. Fifty microliters of the antibody solution was then pipcttcd o n t o each coverslip. For a negative control, 50 p1 o f the huller with Triton-X 100 was used instead of the antibody. Anti- actin antibody at a dilution of 1 : l O O was used a s ii positive control. The coverslips were incubated overnight a t 4°C'. After incubation, coverslips were washcd twice for ii total time o f 30 min in 0.1 M PBS. Excess buffer was draincd off the covcrslips and they were then incubated with 100-fold dilution o f FITC-linked goat-anti-rabbit IgG for 45 min at room temperature in the dark to inhibit fading. To test for autofluorcsccnce, one o f the coverslips was incubated with antibodies to Cx43 without FITC. Instead, the coverslip was incubatcd in 0.1 M PBS. Aftcr the incubation period, the coverslips were washed twice for IS min each with buffer and then mounted o n t o glass slides using ;I S0:SO mixture o f buffer and glycerin. The slides were examined under ;I hluc filter (excitation = 495 nm; emission = 510 nm) and pho- tographed with black and white film.

884 DONAHUE ET AL.

Detection of Cx43 mRNA

Cells were grown to confluency in 10 cm diameter round tissue culture dishes. Total RNA was isolated by dissolving the cells in 4 M guanidinium thiocydnate, 2.5 mM sodium nitrate, pH 7.0,0.5% sarcosyl and 0.1 M P-mercaptoethanol at room Chromosomal DNA was sheared by passing the extract through a 20-G needle at least four times and then l/lOth volume of 2 M sodium acetate, pH 4.0, was added to decrease the pH of the extract. Total RNA was purified by phenolichloroform extraction, precip- itated with ethanol, dried and resuspended in sterile filtered double deionized water. The concentration of RNA in each sample was determined by absorbance at 260 nm. Samples (20 pg) were normalized by reprecipitating the appropriate volume for each sample, and precipitates were subjected to Northern blot analysis as previously described.'")

An oligonucleotide (22 mer; 5'-TCTCCAGGTCAT CAGGCCGAGG-3') was made that hybridizes to a highly conserved coding region of a human Cx43 mRNA.('4) The oligonucleotide was purified using an Applied Biosystems Oligonucleotide Purification Cartridge following the man- ufacturers directions. Forty-five picomoles of the oligonu- cleotide was labeled as previously described,(33) and greater than 1 x 10" cpm/ml were incubated with a nylon mem- brane containing the RNA samples using the conditions described by Fishman et al.(34)

Detection of Cx43 protein

Cells were grown to confluency in 100 mm tissue culture dishes and then scraped off the dish with a rubber police- man. The cell suspension was washed with PBS, the super- natant removed, and the pellet frozen at -20°C until anal- ysis. Fifty micrograms of cell protein, as determined by the Biorad method, was resolved by SDS-PAGE, and the pres- ence of Cx43 was evaluated by Western blots using rabbit antibody to Cx43, as previously described.(*") In brief, pro- teins were electrophoretically transferred to nitrocellulose membranes that were then blocked in PBS containing 5% (wiv) non-fat dry milk and 10 mM sodium azide. Blots were screened using a 1:1000 dilution of rabbit antibody to amino acids 346-360 of the Cx43 sequence. The sheets were then incubated with diluted antibody, washed, incubated with '251-Protein A, rewashed, and dried. Visualization of anti- body binding was carried out by exposure to X-ray film at -80°C in the presence of an intensifying screen. Preim- mune serum has previously been shown to show no binding in numerous cell system^,('^.^^.-^^) including systems where cells of the osteoblastic lineage were shown to be posi- t i ~ e . ' ~ ~ ) Furthermore, affinity purified antibodies, prepared using a column to which peptide antigen had been conju- gated. have not differed in patterns of immunoreactivity when compared with the antiserum used in these studies.

Statistics

The effect of PTH on coupling in cell lines was deter- mined on frequency data using X2 analysis (contingency tables) as described by Zar."') Dose-dependent effect of PTH and PTH analog o n coupling in ROB cells was deter-

E H " f UMR MC-3T3 SAOS

Number of coupled cells per cell

FIG. 1. Quantitative analysis of intercellular communica- tion in five types of osteoblast-like cells. Individual cells were loaded with carboxyfluorescein and the number of cells containing dye was quantified 5 min later. The histo- grams depict the frequency with which individual cells were coupled to a given number of neighboring cells. Data are normalized to 100% to allow comparisons among groups. Zero neighboring cells coupled means the loaded cell was not coupled. The numbers within each panel are mean +- SD number of cells coupled per loaded cell. The total number of cells loaded for each cell line were: ROS 1712.8 = 21, ROB = 53, UMR-106 = 26, MC3T3-EI = 14, and SAOS-2 = 11.

mined on frequency data using ANOVA. p values < 0.05 were considered significant.

RESULTS

Cell coupling studies revealed that all of the cell lines examined were functionally coupled to some degree; how- ever, not all individual cells loaded were coupled. Typically, following dye loading, if the cell was coupled the dye would rapidly (within 15 s) diffuse into adjoining cells. Diffusion would continue, depending on the degree to which the particular cell was coupled, for up to 5 min. After 10 min, if the cell was highly coupled, the dye had diffused or faded to such a degree that quantification of the number of cells containing dye was difficult, therefore we routinely counted the number of cells containing dye at 5 min. Figure 1 shows a quantitative analysis of cell coupling within each cell line. The degree to which cells were coupled varied among the cell lines examined. ROS 17/22 cells were clearly the more highly coupled cells with 95% of the loaded cells couplcd (mean 5 SD number of cells coupled per injection = 10.5

5.7; significantly greater than other cell types, p < 0.05). ROB (55%; 4.9 ? 5.9 cells coupled), MC3T3-El (43%; 2.6 ? 3 3 , and UMR-106 (50%; 1.2 ? 1.6) were more moder-

CELL-TO-CELL COMMUNICATION IN OSTEOBLASTIC CELLS 885

2 1 I

U

cont. -lOM -8M -6M analog

FIG. 2. Concentration-dependent effect of PTH on inter- cellular communication in ROB cclls. Cells were exposed to I 0 I" t o I0 " M r P r H ( 1-34) or l o - " M rPTH( 1-34) and I 0 " M PTH(3-34) f o r 1 h prior to being loaded with carboxyfluorcscein and the percentage of cells coupled cal- culated. Values ;ire the ratios of proportion of cells coupled in the presence of PTH t o the proportion coupled in the presence of vehicle control (:+- SE of proportion). Control = vehicle control, values = log [PTH]; analog = PTH(1- 34) plus PTH(3-34). *Significantly greater than vehicle con- trol, p < 0.05. The total number o f cells loaded for each group was: control ==: 53; - 10 M = 28; -8 M = IS; -6 M = 22; analog = 39.

ately coupled, whilc SAOS-2 (45%; 0.9 5 1.0) were weakly coupled. All cell lines were at similar confluency when examined.

Exposurc of ROB cells t o PTH significantly altercd the degree o f coupling in ROB cclls. Figure 2 shows the con- centration-dependent effect of PTH o n functional coupling in ROB cells exposcd t o I 0 " I t o 10 '' M rPTH( 1-34), At I 0 "', I 0 ', and 10 " M PTH, the percentage of coupled cells relative t o control (coupling ratio) increased signifi- cantly. The EC,,, for PTH-stimulated functional coupling was between 10 ' and 10 I" M. Exposure to 10 " M rPTH( 1-34) alone f o r I h had no effect o n the degree o f coupling in ROS 17/23, UMR-106, MC3T3-E1, or SAOS-2 cclls (data not shown).

To explore the mechanism by which PTH stimulates coupling, we examined the effects of PTH(3-34), an analog o f PTH that blocks PTH-stimulated CAMP accumulation o n coupling. ROB cells werc preincubated for 1 h in ICYf' PTH and 10 " M analog, and coupling quantified. As shown in Fig. 2, the analog significantly attenuated PTH( 1- 34) stimulated gap junctional coupling. We also examined PTH-stimulated CAMP accumulation for the purpose of comparing EC,,, f o r PIH-stimulated CAMP accumulation and functional coupling. PTH stimulated CAMP accumula- tion in ROB in a concentration-dependant manner (Fig. 3). The EC,,, for PTH-stimulated CAMP accumulation was approximately 10 '' M, similar to the EC,,, for PTH-stim- ulatcd functional coupling.

KOS 1712.8, ROB and, to a lcsscr extent MC3T3-El and UMR-106, displayed Cx43 immunoreactivity at the inter- face of adjoining cells (Fig. 4). Additionally, considerable Cx43 was detected in the perinuclear region o f ROB and ROS cells. In somc preparations, fluorescence appearcd between thc pcrinuclcar region and the nearest cell-cell interface, as would occur if Cx43 proteins werc bcing trans-

8

i 6

c2

s $ 4

a z * 9

Q v

0

FIG.3. C

*

cent. -lOM -9M -8M -7M -6M

Nncentration-deDendent effect o f PTH on CP P accumulation in ROB cells. Cells were exposcd to rPTH( I- 34) for 15 min and total CAMP quantified by RIA. Values are peak CAMP levels over basal t SE. Cont. vehicle control; values = log[PTH]. "Significantly greater Ihan ve- hicle control, p < 0.05, n = 10.

ported from the perinuclear region to the membrane. This is best visualized in Fig. 3C. SAOS-2 cells displaycd littlc. if any, Cx43 immunoreactivity at the interface o f adjoining cells or at the perinuclear region.

Each cell line was subjected to Northern blot malysis to detect Cx43 mRNA, and Western blot analysis to dctcct Cx43 protein. Northern blot analysis revealed that a11 cclls examined expressed Cx43 mRNA (Fig. 5). Western blot analysis revealed the presence of a 41 and ii 43 k l l hand, which is consistent with the presencc of both the phosphor- ylated and unphosphorylated Cx43 protein in ROB, ROS, UMR, and SAOS-2 cells (Fig. 6) . '3 ' ) I n MC3T3, wc ilc- tected only the band corresponding to thc un-phosphoiy- lated Cx43. In ROB, ROS, and to some degree UMR, higher weight bands were evident. Thcsc bands may corre- spond t o protcin aggregates or more highly phosphorylated forms of Cx43.

DISCUSSION

Previous studies have shown morphologic cvidcncc of gap junctions within bone tissue.") Additionally, Hoh et al.(4") reported the extraction of Cx43 mRNA from whole bone, but it was not clear which cells were producing Cx43 mRNA. More recently, Schirrmacher et al."' havc identi- fied Cx43 in osteoblastic cells using indircct immunofltio- rescence confirmed with Western blot analysis and Schiller et al.'" have detected Cx43 mRNA in osteoblastic cclls. Our results confirm these reports and localize Cx43 to the perinuclear region and appositional membrancs of adjoin- ing osteoblastic cells. Furthermore, close examination o f individual micrographs such as that shown in Fig. 4((7, rcvcal Cx43 immunoreactivity between the perinuclear re- gion and appositional membranes. However, there is no evidence of Cx43 immunoreactivity between the pcrinu- clear region and nonappositional membranes. Onc cxpl:i- nation for this observation is that Cx43 i h transported prcl- erentially from the perinuclear area to thc appositioiial

886 DONAHUE ET AL.

FIG. 4. Photomicrograph of Cx43 localization in osteoblastic cells. Anti-Cx43 immunoreactivity in (A) subconfluent ROS 17/23 cells, (B) confluent ROS 17/22 cells, (C) ROB cells, note localization of Cx43 in the perinuclear region as well as in appositional membranes (arrows), (D) MC3T3-EI cells, (E) UMR cells, and (F) SAOS-2. No SAOS-2 cells examined stained positively for Cx43. n = 8 to 20 for each cell type. Scale bar = SO p n .

membrane. This is consistent with the finding that, in oo- cytes, contact between cells causes movement of connexin to the appositional membrane.(") Detection of Cx43 mRNA and Cx43 protein in all of the cell lines examined confirmed the existence of gap junctions composed of Cx43 in osteoblastic cells. Interestingly, Cx43 mRNA and Cx43 protein, as determined by Northern and Western blot anal- ysis, respectively, were detected in SAOS-2 cells despite the lack of Cx43 immunoreactivity at appositional membranes in these cells. Thus, SAOS-2 cells appear to have levels of Cx43 below that of the other cell lines examined and below the detection limits of our indirect immunofluorescent pro- cedure. Furthermore, since SAOS-2 did express Cx43 mRNA, it would suggest that (343 gene expression does not assure formation of functional gap junctions in all cell lines.

The results of our cell coupling experiments arc consis- tent with and extend previous studies showing dye coupling and electrical coupling between bone cells both in sitd4" and in vitro.('-''' A percentage of dye-loaded cells were coupled in each cell type we examined. ROS 17/22 was the more strongly coupled cell line, while ROB and MC3T3-El

were moderately coupled and UMR-106 was weakly cou- pled. Interestingly, SAOS-2 cells that expressed Cx43 mRNA and had detectable Cx43 protein by Western blot analysis, but had no detectable Cx43 immunoreactivity at appositional membranes, were poorly coupled. These find- ings are similar to those of Civitelli et al.(") and suggest that SAOS-2 may be coupled by gap junctions containing con- nexins other than Cx43. Indeed, Civitelli et al.(") have re- ported that SAOS-2 express Cx4S mRNA. Another possi- bility is that insufficient Cx43 was present within SAOS-2 cells to detect by indirect immunofluorescence. Lack of Cx43 immunostaining in SAOS-2 is most likely not due to species-specific differences because the polyclonal antibody was made to a peptide with an amino acid sequence highly conserved between rat and human.('") However, it is pos- sible that there may be a species-specific difference in the tertiary structure of Cx43 that could account for the lack of Cx43 immunoreactivity in SAOS-2. This could result in stearic hindrances that may have rendered the epitope for the Cx43 antibody inaccessible for immunostaining but not for Western blot analysis.

PTH increased functional coupling in ROB cells in a

CELL-TO-CELL COMMUNICATION IN OSTEOBLASTIC CELLS 887

- Cx43mRNA

FIG. 5. Northern blot analysis o f the steady-state levels o f Cx43 mRNA in ostcoblastic cell lines. Twenty micrograms o f total RNA from each cell line was subjected to Northern blot analysis ;is described in text. The size of the Cx43 mRNA (arrow) wiis judged to be the expected 3.1-3.2 kb based o n its mobility rclative to the position of marker KNAs on t h e blot. MC = MC3T3: ROS = ROS 17/23; LJMR = UMR-106; SAOS = culture rat osteoblasts.

a m cn 0 U

SAOS-2; ROB = primary

Y C u m m 0 0 U K

FIG. 6. Western blot analysis o f Cx43 protein in osteo- blastic cell lines. Fifty micrograms of protein from each cell type was subjected t o Western blot analysis as described in text. The location o f the 43 kD band is consistent with the location of phosphorylated Cx43 protein while the 41 kD band is consistent with the unphosphorylated Cx43 protein. The protein from ROB (two rats) was analyzed in a sepa- rate gel.

concentration-dependent manner. The effect o f PTH was specific for ROB since neither ROS 1712.8. UMR-106, MC3T3-EI. n o r SAOS-2 responded in 21 similar manner. I t is perhaps n o t surprising that SAOS-2 did not respond t o PTH since we find n o cvidencc o f Cx43 imniunorcactivity at appositional membranes in these cells. However, it is less clear why ROS 17/28 cells, which express abundant Cx43 at appositional meml,ranes and have abundant PTH rccep- tors. did not respond. Perhaps since coupling in ROS 17/23 cells was miiximal in the absence of PTH. we were unable to detect further PTH-stimulated coupling.

The reason we did not detect a response to PTH in UMR-I00 cells is less clear. Schiller et al.'" found that a 1 h pre-exposure t o PTll-stimulated Cx43 mRNA expression and a 2.5-6 h pre-exposure stimulated functional coupling in UMR-I06 cells. However, we found that ;i 1 h pre- exposure t o PTH did not affect coupling i n UMR-106 cells.

This may be partly explained by our finding that UMK-I00 cells express relatively little Cx43 at appositional mein- brancs and by previous reports that, relative t o other osteo- Mastic cells, UMR-I00 cells are weakly ~ o u p l c d . ' ~ " ' 'l'hus, UMR-106 cells may be relatively insensitive t o PTH rcgu- lation of gap junction coupling. Therefore, a longer expo- sure to PTH may be required t o stimulatc functionid cou- pling in UMR-106 cells. However, we found thal ;in exposure t o PTH for 1 h was sufficient to stiniulntc tunc- tional coupling in ROB cells, suggesting that ROB cell\ are more sensitive to PTH, in that they respond more acutely, than do UMR-106 cells. Indeed, we found n o effect of I ~ I I I , following a 1 h exposure, o n functional coupling in m y o f the transformed or immortalized cell lines we examined. This suggests that ROB cells may respond more acutely to PTH, with respect to the stimulation of gap junction coii- pling, than either transformed or immortalized ecll lines.

The precise mechanism by which PTII stimulates lunc- tional coupling in ROB cells is unclear. Previous studies suggest that PTH does not affect gap junction distribution as determined by electron microscopy."" Thus. the effect of PTH o n functional coupling may be partly due [ ( I an interaction with pre-existing gap junctions. This effect could he mediated by one of several second messengers that I'TIl activates, and which arc known t o affect gap junctionid coupling. These include cytosolic Ca' ' , ( . '2 .J7.4J) hydrogen ion,("5.4") and CAMP,''^' the cytosolic levels of which arc all increased by PTH. However, thc effect of cytosolic (':I' ' and hydrogen ion on gap junctions is to decrease lunction;il coupling."") Thus, we do not believe these ions arc medi- ating the effect of PTH o n functional coupling in ROB cells. On the other hand, in several cell types CAMP mediates hormonal-stimulated functional coupling."5 I"' Addition- ally, we found that the PTH analog, Prll(3-34), which blocks PTH-stimulated CAMP accumulation,'2J) also blocks PTH-stimulated gap junctional coupling. Finally, Schiller ct al.") have recently shown that forskolin mimics the ability of PTH to stimulate functional coupling in osteoblastic UMR-106 cells. Therefore we believe that the effect o f PTH on functional coupling in ROB cells is due in part to an increase in CAMP. In support of this concept, we lotind that the lZC,,,'s for PTH-stimulated CAMP iiccumulatictn and functional coupling were similar.

In conclusion, we have found that Cx43, 21 specific gap junction protein, is expressed in osteoblastic cell networks. Additionally, gap junctional coupling takes place t o variouh degrees in osteoblastic cells. Finally, an acute I h exposure t o PTH is capable of increasing functional coupling in primary cultures of rat ostcoblast-like cells hut not in trans- formed or immortalized cell lines.

ACKNOWLEDGMENTS

We thank Lourdcs Porres and Denise Coiichman for excellent technical assistance. This work was supported by NIH grants HL3I299 (PRB), AGIO190 (HJD); GM300h7 (ELH), a grant from the American Federation for Aging Research (HJD) and Electric Power Research Institute grant RFP 2065-21 (KJM). ELH is ;I recipient o f a <';irecr

888 DONAHUE ET AL.

Scientist Award f r o m t h e I rma T. Hirschl Trust . Port ions of this work w e r e presented a t t h e Nat ional Meet ing of the Endocr ine Society, S a n Antonio , TX, 1992 a n d t h e A m e r - ican Society of B o n e a n d Mineral Research, Minneapolis, MN, 1992. This manuscr ipt is dedica ted t o t h e m e m o r y of Harry 0. D o n a h u e .

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Received in original form May 31, 1994; in revised form Ilcccnihci- 27, 1994; accepted January 7, 1995.