Enhancement of Connexin 43 Expression Increases Proliferation and Differentiation of an Osteoblast-like Cell Line

Experimental Cell Research 264, 397– 407 (2001) doi:10.1006/excr.2000.5145, available online at http://www.idealibrary.com on

Enhancement of Connexin 43 Expression Increases Proliferation and Differentiation of an Osteoblast-like Cell Line B. Gramsch,* H.-D. Gabriel,† M. Wiemann,* R. Gru¨mmer,† E. Winterhager,† D. Bingmann,* ,1 and K. Schirrmacher* *Department of Physiology and †Department of Anatomy, Medical School, University of Essen, D-45122 Essen, Germany

Bone cells form a functional syncytium as they are coupled by gap junctions composed mainly of connexin 43 (Cx43). To further understand the role of Cx43 in bone cell growth and differentiation, we stably transfected Cx45-expressing UMR 106-01 cells with Cx43 using an expression vector containing rat Cx43 cDNA. Three stably transfected clones were analyzed, all of which showed altered expression of Cx43 and/or Cx45 as was obvious from immunocytochemistry and Northern blotting. Double whole-cell patch clamping revealed single-channel conductances of 20 (Cx45) and 60 pS (Cx43). The overexpression of Cx43 led to an increase in dye coupling concomitant with elevated gap-junctional conductance. The phenotype of the transfected clones was characterized by an increased proliferation (4- to 7-fold) compared to controls. Moreover, a transfectant clone with 10- to 12-fold enhanced Cx43 expression showed a significantly increased calcium content of the extracellular matrix and enlarged mineralization nodules, while alkaline phosphatase was moderately increased. We conclude that enhanced gap-junctional coupling via Cx43 significantly promotes proliferation and differentiation of UMR cells. © 2001 Academic Press

Key Words: osteoblast-like cells; transfection; gap junctions; electric coupling; proliferation; differentiation.

INTRODUCTION

Recent studies have provided evidence that direct cell– cell communication via gap junctions is an important element promoting growth and differentiation in various tissues [1]. Also physiological functions of tissues, such as the proper functioning of pancreatic islet cells, obviously depend on gap junction coupling [2]. Gap junctions are composed of connexins, which belong to a multigene family with numerous members and are 1

To whom reprint requests should be addressed at the Institut fu¨r Physiologie, Universita¨t Essen, D-45122 Essen, Germany. Fax: ⫹49 201 723-4648. E-mail: [email protected].

differentially expressed in a tissue-specific manner [3]. Two hemichannels of neighboring cells, each composed of six specific protein subunits (21– 47 kDa) termed connexins [1], form a pore which allows ions and small molecules (⬍1 kDa) to pass. Consequently, gap junctions are involved in the transmission of bioelectrical signals such as ion currents as well as second messengers [4 – 6]. They may furthermore play a role in nutrition of cells remote from energy sources, thus enabling a metabolic cooperation. Gap-junctional communication is important also in bone cells [7], where the channels are involved in mechanical transmission [8 –10], induction of cytokines in osteoblasts by T cells [11], and coordination of hormonal responses [12, 13]. In osteoblast-like cells in vitro connexin 43 (Cx43) is the dominant connexin subtype and probably important for normal skeletal development [14 –16]. Furthermore, Cx45 was found to be expressed at a low level in osteoblast-like cells derived from calvaria explants [14, 17, 18]. As the biological significance and the interplay of these two connexin subtypes is currently not understood, cell culture models are increasingly employed to define this role. It was recently shown that Cx43-expressing ROS cells reduced the expression of osteocalcin and alkaline phosphatase, when cells were transfected with Cx45 as an additional connexin [19]. Another transformed osteoblastic cell line, UMR 106-01, expresses Cx45, which has a lower channel permeability than Cx43 [20 –22]. In a previous study on UMR 106-1 cells [23] the additional expression of the Cx43 gene increased production of osteocalcin and bone sialoproteins, both of which are involved in the formation of the extracellular matrix and calcification. These studies [19, 23] suggest that the type(s) of connexin to be expressed at least partly determines whether cells start to proliferate or undergo tissue differentiation. The present study was designed to further address this problem. We investigated whether transfection of Cx45-expressing UMR 106-01 cells with Cx43 alters not only dye and electric coupling but also proliferation, activity of alkaline phosphatase, and mineraliza-

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0014-4827/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

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tion [24 –26]. Part of this study has been published as an abstract (B. Gramsch et al., 2000, Pflu¨gers Arch. Eur. J. Physiol. 439(Suppl.), R371). MATERIALS AND METHODS Cell cultures. In order to compare gap-junctional properties of endogenous Cx43 with exogenous Cx43 and endogenous Cx45 of UMR cells, calvarial fragments of 2- to 4-day-old neonatal rats were explanted onto collagen-coated coverslips and placed in plastic tubes (Nunclon, Germany) which contained 2 ml of high growth enhancement medium (high GEM; ICN, Germany), 24 mM bicarbonate, 10% heat-inactivated fetal calf serum (FCS), 1% glutamine (200 mM), and 1% penicillin (5000 IU/ml)/streptomycin (5000 ␮g/ml). After 2 weeks 5% instead of 10% FCS was used. Nutrient medium was exchanged twice a week. Rat osteoblast-like (ROB) cells were investigated 2 to 6 weeks after explantation. The osteosarcoma cell line UMR-106/01 (UMR) was a generous gift from T. H. Steinberg (Washington University School of Medicine, St. Louis, MO). UMR cells were cultured in high GEM (see above) supplemented with nonessential amino acids (Gibco) and Na ⫹ pyruvate (Sigma). UMR cells were grown in tissue culture flasks (Falcon) and passaged once or twice a week using 0.05% trypsin/EDTA (Sigma) in phosphate-buffered saline (PBS; pH 7.2). For alkaline phosphatase and mineralization assays UMR cells were plated in 96-well plates and 12-well plates, respectively. Different initial densities (cells/cm 2) were seeded such that subconfluency or confluency was reached simultaneously in all cultures when cell properties were analyzed. Plasmid construction and transfection of UMR cells with Cx43. To create the expression vector pPH4 (rat cx43) the open reading frame of the Cx43 gene was amplified by polymerase chain reaction and cloned into the pBK-RSV vector (Stratagene, La Jolla, CA), which also comprises a Geneticin selection marker (for details see [27]). To transfect UMR cells, 10 ␮g of the expression vector pPH4 and 50 ␮l LipofectAmine in OptiMEM (serum-free medium; Gibco) were added to 4 ⫻ 10 6 cells, which were kept in OptiMEM. After 6 –12 h the medium was replaced by fresh OptiMEM without additives and cells were cultured for 2 days. Thereafter, selection of resistant clones was carried out with 500 ␮g/ml Geneticin for 2–3 weeks with the medium being changed every 2–3 days. Stably Cx43transfected UMR cells were selected by standard methods, expanded in 24-well plates, and characterized by indirect immunocytochemistry. Immunofluorescence labeling. Connexins were localized using indirect immunofluorescence. Cells were fixed in acetone/methanol (1/1) for 5 min, rinsed in PBS containing 0.5% (w/v) bovine serum albumin (PBS–BSA). Fixed cells were incubated for 90 min with an anti-Cx43 antibody (Chemicon, U.S.A.) raised against a 19-aminoacid C-terminal peptide sequence of the cytoplasmic domain of mouse Cx43 or with an anti-Cx45 antibody whose immunogen was a 6His fusion protein containing the carboxyl terminus of Cx45 (a gift from T. H. Steinberg). Antisera were diluted 1:40 and 1:500 in PBS, respectively. Controls were performed without the primary antibody or with the preimmune serum instead. Subsequently, cells were washed in PBS–BSA and incubated with FITC- or rhodamine-conjugated goat anti-rabbit IgG (1:40; Sigma) for 45 min. After cells were rinsed three times in PBS–BSA and, thereafter, in distilled water, cultures were coverslipped with glycerol containing 0.1% phenylenediamine to avoid bleaching. Cultures were examined with an Axiophot microscope (Zeiss) equipped with the appropriate epifluorescence filter sets. Northern blot analysis. Total RNA was isolated from subconfluent cell monolayers using a Qiagen RNeasy Midi kit. RNA samples were quantified spectrophotometrically at 260 nm; 5–10 ␮g RNA was separated on denaturing agarose gels which were blotted onto nylon

membrane (Hybond-N; Amersham, Brunswick, Germany) and fixed for 2 h at 80°C. Hybridization with DNA was carried out as described [28]. The plasmid F9-7 containing 2.1 kb mouse Cx45 cDNA, Cx43m-3c with a 1.3-kb mouse Cx43 cDNA fragment [29], and an actin cDNA fragment [30] were used as probes. Filters were washed in 1% sodium citrate buffer (SCB)/0.1% sodium dodecyl sulfate (SDS) and afterward in 0.5% SCB/0.1% SDS for 30 min at 55°C. To quantify signal intensity of RNA bands, the autoradiography films were photographed with a digital color camera (Nikon, Lucia System) and converted into 8-bit black and white images. After background correction, size and intensity of each band were measured and expressed together as integral intensity according to the Lucia software. Values were normalized taking the most intense signal as 100%. Proliferation assay. The tetrazolium dye (MTT) assay was used to measure cell proliferation [31]. Cells were seeded into 96-well plates using 200 ␮l/well of cell suspension containing 10 5 cells/ml medium plus 10% FCS. On day 4, 20 ␮l of a solution made of 5 mg MTT/ml PBS was added to each well and the plates were returned to the incubator for a period of 5 h. Medium was removed from each well, 200 ␮l of dimethyl sulfoxide was added to dissolve the crystals, and the plates were rocked for 10 min. Absorbance of each well was determined at 540 and 690 nm using a dual-wavelength plate reader (Dynatech MR 7000, Germany). Measurements were run in quadruplicates. Five independent experiments were run for each clone. Activity of alkaline phosphatase. Alkaline phosphatase (ALP) activity was measured using p-nitrophenyl phosphate as a substrate (Sigma; 104-S). Cells grown to confluence in 96-well plates were rinsed in PBS and incubated in substrate solution (4 mg p-nitrophenyl phosphate per milliliter of 100 mM Tris–HCl, pH 10.3) for 5 min. Reaction was stopped by 0.1 N NaOH. Thereafter, absorbance was measured at a wavelength of 405 nm in a plate reader (Dynatech MR 7000). Measurements were run in quadruplicate in four independent experiments for each clone. Mineralization assay. This test was performed in 12-well plates. In each well 1 ⫻ 10 5 (parental cells and vector control), 2 ⫻ 10 4 (clone 37), or 5 ⫻ 10 4 (clones 20 and 5) cells were seeded to compensate for differences in proliferation and to obtain confluent cell layers at the day of the assay. Mineralization was induced by adding 50 ␮g/ml ascorbic acid and 5 mM ␤-glycerophosphate to the medium at day 5. Cultures were then maintained for another 48 h. For microscopic analysis cells were rinsed with PBS, fixed in 10% paraformaldehyde for 20 min, and rinsed again three times with double-distilled water. Cells were covered with 40 mM Alizarin Red S (AR-S; Sigma) at pH 9.0 (1 h), pH 7.0 (10 min), and pH 4.2 (10 min), washed in between with PBS, and finally fixed with 5% sodium thiosulfate. Distribution of the red dye was qualitatively evaluated with bright-field and phase-contrast optics. Quantification of the calcium mineral content with Alizarin Red S. Cultures were briefly rinsed with PBS and fixed in ice-cold 70% (v/v) ethanol for 1 h. Cultures were washed several times with doubledistilled water and stained with 40 mM AR-S, pH 4.2, for 10 min at room temperature. Cultures were then rinsed five times with water followed by a 15-min wash with PBS to reduce nonspecific AR-S stain. Stained cultures were then subjected to a quantitative destaining procedure using 10% (w/v) cetylpyridinium chloride (CPC) in 10 mM sodium phosphate (pH 7.0), for 1 h at room temperature. Aliquots of these AR-S extracts were diluted 10-fold in 10% CPC solution, and the AR-S concentration was determined by absorbance measurement at 570 nm on a multiplate reader (Dynatech MR 7000). An AR-S calibration curve was created with CaCl 2 (0 –200 mM) dissolved in the same diluent. Three independent experiments were performed. Dye injection into single cells. Micropipettes were backfilled at their tips with 4% (w/v) aqueous Lucifer Yellow CH (Sigma) and topped with 1 M LiCl. Dye was applied iontophoretically using a

Cx43 EXPRESSION ALTERS OSTEOBLAST PHENOTYPE 20-nA hyperpolarizing current for 10 s supplied by a current source (List Electronic, Germany). Spreading of the dye from the injected into the adjacent cells was examined under epifluorescence illumination. Three minutes after application of Lucifer yellow the number of dye-filled coupled cells was counted in reference to all cells directly neighboring the injected cell. The Student t test was used to determine statistical differences between parental and transfected UMR cells. Electrophysiological recordings. Pairs of cells were prepared from confluent cultures by trypsin treatment: ROB cells were rinsed with PBS three times. One milliliter of trypsin solution (0.05% trypsin/0.02% EDTA) was added to the cells for 30 – 40 s. Thereafter, solution was aspirated and enzyme treatment was continued at 37°C for 2–3 min. Trypsinization was stopped with medium containing 10% FCS (v/v) when about 90% of the cells had detached from the substrate. Cells were sedimented at 1800 rpm for 5 min, resuspended in Hepes-buffered minimum essential medium (ICN), plated into 35-mm dishes (Falcon), and allowed to settle 20 –30 min before patching. UMR and transfected UMR cells were trypsinized the same way but were then seeded directly for patch-clamp experiments without further centrifugation steps. Total junctional conductance (g j) of selected single ROB and UMR cell pairs was determined using the double whole-cell voltage-clamp technique [32]. Patch pipettes were made from soft-glass capillaries (Hilgenberg, Germany) in a two-step pulling procedure (P-87; Flaming/Brown, Sutter Instruments) and filled with a solution containing 135 mM CsCl, 0.5 mM CaCl 2, 1 mM MgCl 2, 5 mM Na 2-ATP, 10 mM EGTA, 10 mM Hepes (pH 7.2 with CsOH). Recordings were performed on cell pairs at room temperature in a saline composed of 160 mM NaCl, 7 mM CsCl, 0.1 mM CaCl 2, 1 mM MgCl 2, 0.6 mM MgSO 4, 10 mM Hepes (pH 7.2 with NaOH). Pipette resistances were 2–3 M⍀ as measured in the bath saline. After whole-cell conditions were achieved, transjunctional potentials (V j) were evoked by stepping the holding potential of one cell (V 2 ) from a common holding potential (V 2 ⫽ V 1 ⫽ 0 mV, when V 1 is the holding potential of the nonstepped cell) to a new value (V 2⬘ ). Appropriate compensation for series resistance was always made. Input resistances (R i) of the cells ranged from 50 to 500 M⍀, while cells with R i ⬍ 50 M⍀ were not evaluated. An EPC9 and an EPC8 amplifier (HEKA Electronics, Germany) were used to set the voltage V 2⬘ in cell 1 to defined values and to record resulting junctional currents from cell 2 and vice versa. Current signals of cell 1 and cell 2 were filtered at 2.3 kHz. Data were acquired and digitized with an IBM-compatible PC-AT using PULSE software (HEKA Electronics). To measure g j, PULSE software delivered rectangular voltage pulses (⫺10 mV, 100 ms) in one of the two cells and vice versa. Single-channel conductance (g j) was measured during exposure of cells to 2–3 mM heptanol [33], which reduces g j to near-zero values. Under these conditions unitary current changes during continuous voltage stimulation of ⫾40, ⫾50, or ⫾60 mV were measured. Events were analyzed offline using the TAC program (Bruxton Corp., U.S.A.). Statistics. We performed the unpaired Student t test for the data obtained from studies on proliferation and alkaline phosphatase and with the mineralization assay. Differences were considered to be significant for P ⬍ 0.05.

RESULTS

To examine whether changes of gap-junctional intercellular communication influence proliferation and differentiation properties of osteoblast-like cells, we stably transfected UMR 106-01 cells with an additional Cx43 gene(s) to increase Cx43 gap junction protein expression. This strategy was chosen since UMR cells predominantly express Cx45, but only low amounts of

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endogenous Cx43. We obtained about a hundred transfectant colonies, 3 of which were selected and cloned for further experiments as they showed stable expression of Cx43 and Cx45 (see below). The coupling strength of transfectants (mediated by Cx43 and Cx45) was measured by double whole-cell patch clamping and compared with that of ROB cells and UMR cells. Results of these electrophysiological studies were correlated with the transfectants’ proliferation, activity of bone-specific alkaline phosphatase, and mineralization. For all studies, parental UMR cells and those transfected with the pPH4 vector lacking the Cx43 gene served as controls. Characterization of UMR Cells Stably Transfected with Cx43 Immunocytochemistry. Expression of Cx45 and Cx43 protein was examined in parental and transfected UMR cells by indirect immunocytochemistry. Both types of connexins were visible as a punctate reaction pattern at the cell boundaries (Fig. 1). As expected, parental UMR cells expressed endogenous Cx45 (Fig. 1A) but very little amounts of Cx43 (Fig. 1C). After transfection we found an increased expression of Cx43 and a widely unchanged expression of Cx45. The three clones selected for this study were characterized as follows: clone 37 showed a 5- to 10-fold larger number of labeled Cx43 dots (compare Figs. 1C and 1E), while its Cx45 was widely unchanged (compare Figs. 1A and 1G). Thus, this clone showed the strongest amplification of Cx43 expression. Clone 20 was characterized by high amounts of Cx45 and low amounts of Cx43 (Figs. 1M and 1O). Finally, clone 5 expressed low amounts of Cx45 and also moderate amounts of Cx43 (Figs. 1I and 1K). Northern blotting. Analysis of connexin expression by Northern blotting showed that transfection with pPH4 resulted in increased amounts of Cx43 mRNA. The mRNA derived from the inserted Cx43 fragment banded at 1.8 kb, whereas the endogenous Cx43 mRNA gave rise to a 3.0-kb band, such that a Cx43-selective cDNA probe detected two bands only in pPH4-transfected cells (Fig. 2). In general, findings corresponded to what was seen by immunocytochemistry: clone 37 showed strongly increased Cx43 expression (11.7-fold vs control) and almost unchanged Cx45 expression (1.6-fold vs control). Clone 20 had an increased Cx45 signal (3.2-fold vs control) and a weak Cx43 signal (1.3-fold vs control), composed of the endogenous and exogenous band. Clone 5 had both a weak Cx45 (2-fold vs control) and a weak Cx43 signal (1.2-fold vs control), the latter also being composed of a double band. Interestingly, transfection led to amplification of endogenous Cx43 mRNA in clone 37, which is a common phenomenon possibly linked to the random insertion of the transfected sequence into the genome [34].

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FIG. 1. Immunocytochemical localization of Cx43 and Cx45 gap junction proteins in parental and transfected UMR cells. Gap junction plaques appear as an intensely stained punctate pattern mostly at the cell boundaries (arrows). Parental UMR cells (A–D) express high amounts of Cx45 (A) and low amounts of Cx43 (C). After transfection with Cx43 clone 37 (E–H) they exhibited a drastically increased expression of Cx43 (E), while Cx45 was widely unchanged (G). Clone 5 (I–L) expressed low amounts of both Cx43 (I) and Cx45 (K). Clone 20 (M–P) expressed low amounts of Cx43 (M) but high amounts of Cx45 (O). At the right side of each fluorescent picture the corresponding phase-contrast image (B, D, F, H, J, L, N, P) is shown. Bars represent 25 ␮m.

Dye coupling. Transfected UMR cells grown to near confluence exhibited a strong dye coupling upon Lucifer yellow injection. Figure 3 illustrates a typical dyetransfer experiment carried out on cells transfected with clone 37. Within 1–3 min after dye injection into a single cell, dye spread out into all six directly neighboring cells (Figs. 3A and 3B). In contrast, parental cells showed poor dye coupling and in most cases Lucifer yellow was restricted to the injected cell (Figs. 3C and 3D). In parental cells, 6.4 ⫾ 3.3% (8 injections) of

the directly neighboring cells were stained compared to 69.5 ⫾ 5.2% in transfected cells (12 injections). Electric coupling. Intercellular communication via gap junction channels between transfectants or controls was determined using the double whole-cell patch-clamp technique [32]. We measured total junctional conductance in single cell pairs (Fig. 4A) as well as single-channel openings (Fig. 4B). Results were compared with gj values measured between ROB cells

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FIG. 1—Continued

known to express mainly Cx43. Single ROB cell pairs exhibited gj values of 42.3 ⫾ 5.3 nS (mean ⫾ SE; n ⫽ 15), whereas parental UMR cells showed lower conductances of 20.3 ⫾ 4.7 nS (n ⫽ 23). The clone 37 showed gj values even larger than that of ROB cells (50.4 ⫾ 5.8 nS; n ⫽ 11). Clones 20 and 5 exhibited mean gj values of 31.3 ⫾ 4.7 (n ⫽ 6) and 10.2 ⫾ 2.9 nS (n ⫽ 7), respectively. To investigate whether Cx43 gap junction channels between transfected UMR cell pairs were operative, their single-channel conductance was measured (Fig. 4B). In well-coupled cell pairs singlechannel recordings were obtained by washing with saline containing 2 mM heptanol. Single-channel recordings from UMR cells derived from clone 37

demonstrated two step-like transitions of 20 and 60 pS in response to a transjunctional potential of ⫺50 mV typical for Cx45 and Cx43 channels, respectively (Fig. 4B). Adhesion and Proliferation Cx43-transfected UMR cells exhibited altered cell adhesion properties compared to parental cells (Fig. 5). Parental cells initially formed clusters of round cells which had no or only weak contact to the culture dish for more than 1 day after seeding. These clusters remained small (diameters ⬍100 ␮m) although slow proliferation occurred 1 day after seeding (Fig. 5B). Even-

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FIG. 2. Northern blot analysis of connexin gene expression in Cx43-transfected UMR 106-01 cells compared to UMR parental cells, vector control cells, and heart tissue. 5 ␮g of total RNA was used for each lane and probed with a Cx43-, a Cx45-, or a ␤-actin-specific probe. Banding of ribosomal RNA is indicated on the right. UMR parental cells and vector control cells express high amounts of Cx45 and low amounts of Cx43. Transfected cells show a 1.8-kb fragment characteristic of the RNA transcribed from the transfected gene (termed “exogenous”) and the original 3.0-kb fragment (termed “endogenous”). Note that the endogenous Cx43 message is also increased in clone 37.

tually, cultures grew to subconfluence after 4 days (Fig. 5C). In contrast, the Cx43-transfected UMR cells were firmly attached within 8 h after seeding and exhibited

an elongated or polygonal shape. Furthermore, Cx43 transfectants proliferated without delay and formed subconfluent areas after 2– 4 days in culture (Figs. 5A and 5D). Eight to 10 weeks after transfection we studied whether modification in gap-junctional intercellular coupling affects growth of Cx43 transfectants (clones 5, 20, 37) compared to controls. Using the MTT cell proliferation assay we found that all transfected cells grew at a significantly higher rate than parental UMR cells or vector controls (Fig. 6A; P ⬍ 0.05). The proliferation rate of clone 37 was enhanced fourfold while clones 20 and 5 grew about sevenfold faster than parental UMR cells or vector controls. In parallel experiments it was confirmed that the plating efficiencies (number of live cells 2 h after plating) of parental cells and all clones were in the same range and that the optical densities delivered by the MTT procedure correlated linearly with the cell numbers (tested for growth periods of 4 days, data not shown). Therefore, results from the MTT assay of different clones were directly comparable. Activity of Alkaline Phosphatase As the expression of ALP is an important marker for the process of early differentiation of bone cells, we tested ALP activity of Cx43 transfectants. Comparison was based on equal cell numbers found under confluent conditions of all clones and parental cells. Figure 6B

FIG. 3. Dye coupling of Cx43-transfected UMR cells vs controls. Fluorescence images of Lucifer yellow injected into transfected UMR cells from clone 37 (A) and parental cells (C). (B and D) The corresponding phase-contrast pictures. Arrows point to the injected cells. Note that extensive dye transfer to all primary neighbors occurred in cells of clone 37 (A), whereas dye coupling was absent in a confluent layer of parental UMR cells (C). Bars represent 20 ␮m.

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the strongly Cx43-expressing clone 37 compared to parental UMR or vector controls (P ⬍ 0.05, Fig. 6C). However, no increased calcium content was found for clones 20 and 5 (P ⬍ 0.05), both of which had comparatively low amounts of Cx43, but differed with respect to Cx45 (Fig. 2). However, the enhanced expression and protein level of Cx45 found in clone 20 had no influence on the cell physiological properties of UMR cells so far tested. Staining with Alizarin Red S was also used to evaluate the quality of mineralization nodules. It was obvious and in line with the quantitative data that Cx43 transfectants of clone 37 formed larger nodules covering a larger area than those of parental UMR cells (Fig. 7). DISCUSSION

FIG. 4. Electric coupling and single-channel events in parental and transfected UMR cells. (A) Total junctional conductance (g j) measured in ROB cell pairs as well as in parental and transfected UMR cells. Each data point (filled circles) represents the g j of one single cell pair. Open circles with error bars represent means ⫾ SE, numbers in parentheses give the numbers of cell pairs investigated. In parental UMR cell pairs g j was low compared to the majority of ROB cells. Transfected UMR clones showed g j values in the rank order clone 37 ⬎ clone 20 ⬎ clone 5. (B) Double whole-cell patchclamp recording from a Cx43-transfected UMR cell pair (clone 37) after exposure to 2 mM heptanol revealing single-channel conductances. Cell 1 was held at H ⫽ 0 mV, while cell 2 was stepped from 0 to ⫺50 mV (V j ⫽ ⫺50 mV). Simultaneous deflections of equal amplitude and opposite polarity show current flow through open gap junction channels. Numerous transitions between closed (c) and open (o) channel states are visible. Single-channel conductances of 20 and 60 pS were found, indicative of gap junctions composed of Cx45 and Cx43, respectively.

shows that the activity of alkaline phosphatase of Cx43 transfectants was not significantly increased vs parental cells except for clone 37, which possessed the highest expression of Cx43 and showed a slight (1.5-fold) increase in ALP activity. Mineralization To examine the ability of UMR cells and of transfectants to form mineralization nodules, cells were grown in the presence of 5 mM ␤-glycerophosphate and 50 mg/ml ascorbic acid [11]. To quantify the degree of mineralization we photometrically measured the calcium content using the Alizarin Red S assay (see Materials and Methods) under conditions equal to those chosen for the ALP assay. With this method we found that the calcium content was significantly higher for

Osteoblast-like cells secrete a vast number of proteins for the formation of an extracellular matrix and have the capabilities to express bone-specific alkaline phosphatase and to mineralize the extracellular osteoid [24 –26]. Recently, Lecanda and co-workers [23] reported that expression of osteocalcin and bone sialoprotein of osteoblastic osteosarcoma cells UMR 106-01 was modulated by gap-junctional communication such that the additional expression of Cx43 was accompanied by an increased expression of these two bonespecific proteins [23]. Beyond this, altered connexin expression may likewise influence the degree of contraction in rat osteoblast-populated collagen lattices [8]. In these and similar reports on the physiological functions of gap junction channels additional nonendogenous gap junction proteins were experimentally expressed [27, 28, 35]. In our study we employed this strategy to investigate the role of an additional Cx43 for growth and differentiation of UMR cells, which are weakly coupled, as they predominantly express Cx45 and only very little endogenous Cx43. After inserting Cx43, which is the main connexin in bone and is expressed also in ROB cells [16], we obtained UMR subclones, three of which were selected for this study. Based on the analyses of these clones our main findings were (1) that functional gap junctions composed of Cx43 were abundant after transfection, (2) that transfection with Cx43 led to accelerated growth and adhesion of all clones, and (3) that expression of ALP and mineralization was enhanced only in clone 37, which strongly expressed Cx43. Characterizing the subclones’ coupling properties with the double whole-cell patch clamp technique [32] confirmed the increased Cx43 coupling in transfected UMR cells. However, despite the high overall expression at least in clone 37, we found a considerable variation of the total junctional conductance (gj) among selected cell pairs. This is in line with an often inhomogeneous connexin expression pattern [27], which

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FIG. 5. Growth and adhesion of Cx43-transfected UMR cells and controls. Cx43 transfectants after 2 (A) and 4 days (D) in culture. Cells appear flattened and firmly attached to the culture dish. Parental cells 2 (B) and 4 days (C) after plating. Note that parental UMR cells form clusters of round cells and have not reached confluence after 4 days. Bar represents 50 ␮m.

was also seen in our immunocytochemical study. Inhomogeneous gj values may also reflect different states of connexin phosphorylation [36]. A functional interference between Cx45 and Cx43 was unlikely because single-channel conductances characteristic of Cx45 and Cx43 [22, 27, 37] coexisted within transfected cells. Nevertheless, there was a clear tendency showing that gj values were large in clone 37, which also showed a strong dye coupling due to a more than 10-fold improved Cx43 expression (see Figs. 1 and 2). This clone, therefore, resembled the previously characterized ROB cells [16] with respect to intercellular communication properties. Expression of Cx43 was less effective in UMR clones 20 and 5, which contained low levels of exogenous Cx43 mRNA. Interestingly, clone 20 expressed more Cx45 than control cells. Although we do not know whether this was indirectly related to transfection per se, the clone was incorporated into the analyses as it provided an additional control and permitted some insight into effects of additional Cx45. In all clones transfection with Cx43 led to increased proliferation. As proliferation was measured by the MTT assay, which provides a measure of live cells at the end of an experiment, and no signs of cell death or cell detachment were obvious during the period of exponential growth (not shown), we conclude that the results from the MTT assay indeed reflect the increased proliferation rates of clones 5, 20, and 37. It was a striking finding that the strongly Cx43-coupled

clone 37 exhibited the highest rate of mineralization and ALP activity and that it grew faster than (vector) control cells. However, clones 5 and 20, the latter overexpressing Cx45, grew even faster than clone 37, while ALP and mineralization remained unchanged. From this comparison we conclude that both growth and differentiation are favored by Cx43. There might by a gene dose effect which underlies the differentiation process exemplified in our study by enhanced mineralization and/or ALP expression. However, the enhanced proliferation obvious in clones 5 and 20 seems to depend on the presence of Cx43. At present, this phenomenon cannot be interpreted and further studies are needed to clarify whether this effect of Cx43 is cell type specific or may be generalized. Increased proliferation of clone 37 was surprising as many studies suggest an important role for gap junctions in growth control, in which strong coupling could down-regulate [27, 38] or even arrest [35] cell proliferation. Furthermore, it should be taken into account that reduced proliferation by restoration of cell– cell communication has been proven in malignant tumor cell lines, but not in benign ones [39]. Augmented proliferation of Cx43-transfected UMR cells could be due to insertion of multiple connexin channels with larger conductance than the endogenous Cx45 channel, allowing for an improved exchange of signaling molecules (e.g., Ca 2⫹ or inositol 3-phosphate) between cells [22, 40] even if only few Cx43 channels are expressed as is the case with clones

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FIG. 6. Proliferation, alkaline phosphatase (ALP) activity, and mineralization of Cx43-transfected UMR clones (5, 20, 37) vs parental cells (UMR) and vector controls (vector). (A) Proliferation measured as optical density of reduced MTT 4 days after seeding identical cell numbers per well. (B) Comparison of ALP activity in confluent cultures; ALP activity was optically measured at 405 nm using p-nitrophenyl phosphate as a substrate. (C) Calcium mineral content was optically measured after dissolving bound Alizarin Red S at 570 nm. Optical values were converted into millimolar by means of a standard curve made up with CaCl 2. Error bars represent SEM; values which are significantly different (P ⬍ 0.05) from parental UMR cells and vector controls are labeled with “*”.

5 and 20. It is furthermore tempting to speculate that larger, hitherto unknown, molecules unable to pass Cx45 channels can be exchanged in transfected cells weakly coupled by Cx43 and that these molecules induce proliferation. In the case of extensive Cx43 coupling, differentiation processes may be turned on by

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strongly increased intracellular signals or by increased exchange of substrates needed, e.g., for augmented protein synthesis. Increased proliferation of Cx43 transfectants may be linked as well to improved cell adhesion, which was observed in all three clones (see Fig. 5). Also Bowman and co-workers [8] have described changes in cell morphology of osteoblastic cell lines after Cx43 transfection. They reported that the majority of elongated cells was highly coupled and responsible for a maximal collagen lattice contraction. Thus, Cx43 gap-junctional coupling might help to keep osteoblastic cells elongated while a lack of functional gap junctions may favor a rounded phenotype, typical of parental UMR cells (see Fig. 5). Cell shape and adhesion properties may also be linked to cell adhesion molecules, as during bone formation, e.g., N-cadherin seems to be associated with the establishment of cell– cell contacts which may precede the formation of Cx43-mediated gap-junctional coupling [41]. As formation of hard bone tissue is the main task of bone cells, we have selected the capacity of transfected UMR cells to mineralize the extracellular matrix [24 – 26] as a parameter for differentiation. Mineralization of the extracellular matrix started after cells had grown to confluence [42], when ␤-glycerophosphate and ascorbic acid were added to the medium [24, 43]. Light-microscopic investigations of the degree of mineralization revealed pronounced nodule formation in Cx43 transfectants of clone 37— but not of clones 5 and 20. Again, a causal relationship between enhanced Cx43 coupling and augmented mineralization, which is an important marker of differentiation, appears obvious. In that particular, clone 37 bone-specific ALP was slightly (1.5-fold, not significant) increased and this is in line with assumptions that increased ALP can be linked to increased mineralization [42, 44 – 46]. Many studies using various types of osteoblasts have demonstrated a reciprocal relationship between decay in cell growth and the subsequent increase in the expression of tissue-specific genes (e.g., ALP, fibronectin,

FIG. 7. Mineralization nodules in parental UMR cells (A) and Cx43 cells transfected with clone 37 (B) after 7 days in culture. Note that the sizes of nodules (arrows) and areas covered by them are much larger in Cx43-transfected UMR cells. Bar represents 50 ␮m.

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type I collagen, osteocalcin, and osteopontin) during mineralization [46, 47]. In contrast to these studies our results show for clone 37 that an increased expression of Cx43 apparently promoted cell proliferation and differentiation, while in clones 5 and 20 growth was widely unchanged. The latter findings are in line with those investigations [8, 23] which show that improved intercellular communication favors cell differentiation in bone. In summary, the present study shows that additional Cx43 expression can enhance bone cell growth and differentiation and suggests a role for Cx43 as a channel used by bone cells for the passage of differentiation signals. We thank Dr. T. H. Steinberg (Washington University School of Medicine, St. Louis, MO) for providing the UMR 106-01 cells and the anti Cx45 antibodies. We are also indebted to O. Traub and H. Hennemann (Institut fu¨r Genetik, Bonn, Germany) for providing the Cx constructs, and P. Hellmann (Institut fu¨r Anatomie, Essen, Germany) for the vector pPH4. This study was supported by an interdisciplinary research grant (IFORES 107446-0) from the Medical Faculty of the University of Essen to K.S. and E.W.

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