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Effects of cAMP on Intercellular Coupling and Osteoblast Differentiation
Biochemical and Biophysical Research Communications 282, 1138 –1144 (2001) doi:10.1006/bbrc.2001.4710, available online at http://www.idealibrary.com on
Effects of cAMP on Intercellular Coupling and Osteoblast Differentiation Milena Romanello,* Luigi Moro,* Doroti Pirulli,† Sergio Crovella,† and Paola D’Andrea* ,1 *Dipartimento di Biochimica, Biofisica e Chimica delle Macromolecole, Universita` di Trieste, via Licio Giorgieri 1, I-34127 Trieste, Italy; and †Dipartimento di Scienze della Riproduzione e dello Sviluyppo, Universita` di Trieste, via dell’Istria, 65/1-34137 Trieste, Italy
Received March 20, 2001
Bone-forming cells are organized in a multicellular network interconnected by gap junctions. Direct intercellular communication via gap junctions is an important component of bone homeostasis, coordinating cellular responses to external signals and promoting osteoblast differentiation. The cAMP pathway, a major intercellular signal transduction mechanism, regulates osteoblastic function and metabolism. We investigated the effects of this second messenger on junctional communication and on the expression of differentiation markers in human HOBIT osteoblastic cells. Increased levels of cAMP induce posttranslational modifications (i.e., phosphorylations) of connexin43 and enhancement of gap junction assembly, resulting in an increased junctional permeance to Lucifer yellow and to a positive modulation of intercellular Ca 2ⴙ waves. Increased intercellular communication, however, was accompanied by a parallel decrease of alkaline phosphatase activity and by an increase of osteocalcin expression. cAMP-dependent stimulation of cell-to-cell coupling induces a complex modulation of bone differentiation markers. © 2001 Academic Press Key Words: osteoblasts; gap junctions; cAMP; alkaline phosphatase; osteocalcin.
Cells are joined with intercellular channels that allow them to send messages and exchange molecules and signals (⬍1 kDa) that improve their homeostatic control (1). These channels are present as aggregates in gap junctions, specialized membrane structures that are found between most cell types. Intercellular channels are more complex than ion channels because they span two plasma membranes and each cell contributes with a half channel, or connexon, which interacts with another connexon from the adjacent cell. Each connexon, in turn, is an oligomer of subunit proteins, 1
To whom correspondence should be addressed. Fax: (39) (40) 676 3691. E-mail: [email protected]. 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
called connexins (Cx), which form a multigene family (1). A single cell type may express one or several connexins, which form channels with different molecular size, permeability and ionic selectivity (2, 3). Dynamic regulation of cell-cell communication takes place at different levels (e.g., transcription, translation, intracellular trafficking and gating of connexins) and is controlled by hormones and growth factors, through signaling pathways that have only begun to be understood. Up-regulation of gap junctional coupling by stimulation of the adenylyl cyclase/cAMP pathway has been demonstrated in a number of cell systems. Depending on the cell type, activation of protein kinase A either increases the rate of connexin targeting to the plasmamembrane (4 – 6), or stimulates protein transcription and translation (7–9). In human osteoblasts, the expression of connexins increases with cell maturation and differentiation (10); gap junctional communication sustains bone cells responsiveness to hormonal stimulation (11) and enhances their in vitro mineralization capacity (12). Finally, increased connexin expression stimulates the transcriptional activity of osteoblast-specific promoters (13), suggesting that the degree of cell-to-cell coupling is directly linked to the fully differentiated osteoblastic phenotype. In this study we show that, in human HOBIT osteoblastic cells, elevated cAMP levels stimulate cell-to-cell coupling due to an increased targeting of connexin43 (Cx43) to the plasmamembrane. cAMP stimulates phosphorylations of Cx43 and protein accumulation at appositional membranes. These modifications are accompanied by an increased intercellular diffusion of Lucifer yellow and by a positive modulation of intercellular Ca 2⫹ waves. cAMP-dependent increase of cellto-cell communication is accompanied by a parallel decrease in alkaline phosphatase expression and by a marked increase of osteocalcin gene (BGLAP) transcription.
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The relationship between intercellular coupling and bone differentiation appears, therefore, more complex than previously envisaged (13). MATERIALS AND METHODS Reagents. Fura2-AM, 18␣-glycyrrhetinic acid, Lucifer yellow CH, forskolin, IBMX, PMSF, aprotinin, chymostatin and leupeptin were obtained from Sigma (St. Louis, MO). The primary antibody employed was the polyclonal anti-rat Cx43, from Zymed Laboratories (San Francisco, CA). For immunoblot detection, the secondary antibody was a goat anti-rabbit IgG conjugated with alkaline phosphatase (Calbiochem, San Diego, CA). The chemiluminescence substrate and enhancer for alkaline phosphatase were CSPD and Nitro-Block, respectively (Tropix, Bedford, MA). For immunofluorescence, the second antibody was a fluoresceine (FITC)-conjugated affinity purified goat anti-rabbit IgG from Jackson ImmunoResearch Laboratories (West Grove, PA). Cell culture. HOBIT cells were kindly provided by Professor B. Lawrence Riggs (Mayo Foundation, Rochester, MN). Cells were grown in Dulbecco’s Minimal Essential Medium supplemented with 10% fetal calf serum, 2 mM L-glutamine and penicillin–streptomycin sulfate and cultured at 37°C. Immunofluorescence. Site-specific localization of Cx43 immunoreactivity was performed as previously described (14) on cultures grown on glass coverslips. Intercellular dye transfer. Glass capillaries prepared with a dualstep puller (Narishige, Tokyo, Japan) were filled with a 5% solution of Lucifer yellow dissolved in 0.33 M lithium chloride. Individual cells in confluent monolayers grown onto coverslips were pressureinjected with a pneumatic PLI-100 pico-injector (Medical Systems Corp., New York). 8 min after injection, cells were fixed with 4% formaldehyde in PBS for 15–20 min. Protein immunoblot analysis. HOBIT cells grown to confluence were washed with ice-cold PBS added with 10 mM NaF and 0.1 mM NaVO 4. Proteins were solubilized in lysis buffer (in mM: 50 Tris, pH 6.8, 2% SDS, 0.5 NaVO 4, 10 NaF, 2 PMSF, 10 g/ml aprotinin, 10 g/ml chymostatin and 10 g/ml leupeptin). Prior to electrophoresis, protein concentration was quantitated by the bicinchoninic acid method (Pierce, Rockford, IL) with bovine serum albumin as standard. For each sample 20 g of total proteins were separated on 13% SDS gel and then transferred to nitro-cellulose. Immunoblot was performed as previously described (14). Quantitation of Cx43 bands was performed after densitometric scanning of developed films (Gel DOC 2000, Bio-Rad) using the image-analysis software MultiAnalyst/PC (Bio-Rad). Levels are expressed as fold change of individual band intensities. Ca 2⫹ imaging. HOBIT cells grown onto coverslips were loaded at 37°C with fura-2 AM (5 M), dissolved in Pluronic (Molecular Probes, Eugene, OR) 20% (1:2) and added to the culture medium. After 60 min the loading solution was removed and the cells bathed in a solution containing (mM): NaCl 125, KCl 5, MgSO 4 1, KH 2PO 4 0.7, CaCl 2 2, glucose 6, Hepes-NaOH buffer 25, pH 7.4. Video microscopy and Ca 2⫹measurements were carried out as previously described (15). Mean values in discrete areas of interest were calculated from sequence of images. Quantitative temporal analyses in spatially restricted areas were thus obtained throughout the experiment. Mechanical stimulation. Mechanical stimulation of single cells in confluent monolayers was performed as previously described (15). Experiments were rejected when the cell membrane was damaged, as revealed by a leak of the fluorescent probe from the cell. Stimulations were performed under continuous fluid flow of 3 ml/min. Alkaline phosphatase assay. Cells were seeded at the density of 1 ⫻ 10 5/well into 24-well plates. After reaching confluence, the cells were treated, in serum free medium, with different drugs for 24 or
48 h. At the end of incubation, the cells, washed with PBS, were added with 0.1 M Tris buffer, pH 9.8, containing 0.2% (v/v) Triton X-100 and stored at ⫺80°C for 30 min, after which samples were thawed and scraped. Alkaline phosphatase was measured on total cell lysates resuspended in 0.1 M Tris buffer, pH 9.8, 1 mM MgCl 2 and 6 mM para-nitrophenylphosphate; the reaction was allowed to proceed at 37°C for 60 min and blocked by addition of 40 mM NaOH. Optical densities were measured at 410 nm. An aliquot of the remaining lysate was employed for determination of protein concentration with bicinchoninic acid method. Statistical analysis. Data were analyzed by analysis of variance (ANOVA) and the significance of the difference of the average values between groups was estimated by Scheff e` test. Quantitative PCR. Total RNA was extracted from Hobit cells using the SV total RNA isolation System (Promega, Madison, WI), following manufacturer’s instructions. Two-step RT quantitative PCR was performed using the TaqMan Gold RT-PCR kit protocol (Applied Biosystems, Foster City, CA) with SYBR Green PCR Master Mix. Primers for cDNA amplification were: forward 5⬘-AGCAAAGGTGCAGCCTTTGT-3⬘; reverse 5⬘-CACAGTCCGGATTGAGCTCA3⬘. Real-time PCR was performed using the following temperatures: initial hold at 95°C for 10 min then 95°C for 15 s, 60°C for 60 s for 45 cycles. Four replicates of each RT-PCR were run on ABI PRISM 7700. TaqMan GAPDH Control Reagents (Applied Biosystems) were used as endogenous reference in order to normalize the quantitative data. Two types of PCR controls were performed: (a) no template controls (NTC) with no target DNA and (b) no amplification control (NAC) with the addition of 1 l SDS (0.5% w/v) in order to ensure the denaturation of the DNA polymerase. Quantitative results were analyzed following the comparative Ct method (Applied Biosystems).
RESULTS In this study we employed HOBIT cells, a line derived from normal adult human osteoblastic cells, which retain most of the osteoblastic differentiation markers, including the expression of osteocalcin, alkaline phosphatase, type I collagen, osteopontin 1␣ and the sensitivity to steroid hormones (16). HOBIT cells express Cx43 (17) and are functionally coupled through gap junctions, as demonstrated by the intercellular diffusion of Lucifer yellow microinjected into a single cell (11.4 ⫾ 1.8 fluorescent cells, mean ⫾ SE, n ⫽ 35, Fig. 1A). 18␣-glycyrrhetinic acid (18GA, 40 M, preincubations of 60 min), a reversible, nontoxic inhibitor of gap junctions, totally prevented intercellular dye transfer (1 ⫾ 0.2 fluorescent cells, n ⫽ 17, P ⬍ 0.00001, Fig. 1A). Differentiated osteoblasts exhibit elevated alkaline phosphatase activity correlating with high levels of enzyme expression (18, 19). To evaluate whether the inhibition of cell-to-cell coupling could interfere with the expression of alkaline phosphatase, we treated cells with 18GA (40 M) for 24 h; the treatment did not affect cell viability (not shown), but induced a significant decrease of alkaline phosphatase activity with respect to controls (23.5%, P ⬍ 0.05, Fig. 1B), confirming that gap junctional-dependent communication is required for the expression of bone differentiation markers (10, 19). To verify the relationship between the degree of intercellular coupling and the expression of osteoblastic
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FIG. 1. Effect of 18␣-glycyrrhetinic acid on intercellular coupling and alkaline phosphatase activity. (A) Intercellular dye transfer. Cells were grown onto glass coverslips until confluent. Fluorescence micrographs were taken 8 –10 min after microinjection of Lucifer yellow into one cell. Injections were performed in untreated (control) cells, and in cultures pretreated (60 min) with 18␣-glycyrrhetinic acid (18GA). Bar ⫽ 30 m. (B) Alkaline phosphatase activity. Cells were grown onto plastic dishes until confluent. Alkaline phosphatase activity was measured from whole cell lysates obtained from untreated (control) cells and from cultures incubated 24 h with 18␣-glycyrrhetinic acid (18GA, 40 M). Values represent the mean ⫾ SE of three independent experiments.
phenotype, in a next series of experiments we stimulated cell-to-cell coupling by increasing the intracellular levels of cAMP. Stimulation of cAMP pathway, obtained by the contemporary administration of the adenylyl cyclase activator forskolin (FK, 20 M) and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 1 mM) for increasing times up to 24 h, did not increase Cx43 expression levels, as assayed in Western blot experiments (Fig. 2A), but induced a time-dependent accumulation of slower-migrating
Cx43 bands (Figs. 2A and 2B), presumably reflecting protein posttranslational modifications. It has been well documented, in fact, that Cx43 appears on Western blots as multiple bands, as a result of different degree of protein phosphorylation (20). From the functional viewpoint, high levels of Cx43 phosphorylation are associated to an increased rate of protein translocation from non-junctional stores to junctional plaques (4, 21). In HOBIT cells, the cAMP-dependent accumulation of slower-migrating Cx43 bands was
FIG. 2. Effect of increased cAMP on Cx43 expression and localization. (A) Western blot analysis of Cx43 protein from HOBIT cells. Cells grown to confluence onto plastic dishes were treated with forskolin (FK, 20 M) and 3-isobutyl-1-methylxanthine (IBMX, 1 mM) for increasing times (0, 2, 6, 24 h). All samples are from 20 g of whole lysates (see Materials and Methods). Cx43 bands are labeled according to Musil et al. (20): NP (not phosphorylated) represents the fastest migrating form, P1 and P2 the slower migrating (phosphorylated) forms. Experiments were repeated four times. (B) Densitometric analysis of the three Cx43 bands from Western blot. Levels are expressed as fold change of individual band intensities. (C) Immunofluorescence staining of Cx43 on confluent HOBIT cells cultures. Confluent cells grown onto glass coverslips were fixed, permeabilized, and incubated with a polyclonal anti-Cx43 antibody. A fluoresceine-conjugated secondary antibody was used for visualization. The two micrographs represent the results obtained with untreated (control) cells and cultures treated for 24 h with FK (20 M) ⫹ IBMX (1 mM). Bar ⫽ 30 m. Experiments were repeated six times. 1140
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FIG. 3. Effect of increased cAMP on intercellular coupling. (A) Intercellular dye transfer. Cells were grown onto glass coverslips until confluent. Fluorescence micrographs were taken 8 –10 min after microinjection of Lucifer yellow into one cell. Injections were performed in untreated (control) cells, and in cultures treated for 24 h with FK (20 M) ⫹ IBMX (1 mM). Bar ⫽ 20 m. (B) Ca 2⫹ imaging recording of mechanically induced intercellular Ca 2⫹ waves. Confluent cells grown onto glass coverslips were loaded with fura2-AM. Intercellular Ca 2⫹waves were induced by mechanical stimulation of a single cell (see the spatial map on the left). The kinetics of calcium wave propagation in control and FK ⫹ IBMX-treated cells is reported (right). Five cells in a row from the stimulated cell were analysed from imaging sequences. Data are presented as relative fluorescence ratio.
maximal at 24 h and was accompanied by an increased Cx43 immunostaining at cell-to cell interfaces, as revealed by immunofluorescence (Fig. 2C). The increased immunostaining involved both the extent and the size of highly fluorescent zones, possibly corresponding to an increase in the number and size of gap junctional plaques. Cell treatment for increasing times (up to 48 h) did not further modify Cx43 electrophoretic pattern, nor protein localization (not shown). The possibility that such modifications in Cx43 intracellular distribution could result in altered intercellular coupling was next evaluated. Functional analysis of gap junction permeability was carried out with the dye transfer technique. Compared to controls (11.4 ⫾ 1.8 fluorescent cells, mean ⫾ SE, n ⫽ 35, Fig. 3A), cells treated for 24 h with FK and IBMX show an increased intercellular diffusion of Lucifer yellow (28.4 ⫾ 2.5 fluorescent cells, n ⫽ 19, P ⬍ 0.0001, Fig. 3A), indicating that the high level of gap junctional immunostaining is accompanied by an up-regulation of intercellular coupling. Propagation of intercellular Ca 2⫹ waves following focal cell stimulation is often observed in gap junctions coupled (15, 22, 23). In calcium imaging experiments we induced intercellular Ca 2⫹ waves by mechanically stimulating a single HOBIT cell. In control cells the waves involved 20.7 ⫾ 1.4 cells, (n ⫽ 98, Fig.3B), while in cultures stimulated for 24 h with FK and IBMX, Ca 2⫹ waves involved 39.9 ⫾ 3.9 cells (n ⫽ 27, P ⬍ 0.0001, Fig. 3B), indicating a cAMP-dependent increase in metabolic intercellular coupling. Taken together,
these results demonstrated that, in HOBIT cells, elevated cAMP levels trigger an enhancement of gap junctional assembly associated to an increased rate of intercellular communication. The consequences of this increase on the expression of the osteoblastic phenotype were next evaluated. In HOBIT cells treatments increasing the intracellular cAMP concentration induced a significant inhibition of alkaline phosphatase activity (Fig. 4). The inhibition was evident after 24 h (33.6% inhibition with respect to controls, P ⬍ 0.05, Fig. 4A) and became more pronounced at 48 h (45% inhibition, P ⬍ 0.05, Fig. 4B), while the inhibition due to 18GA (40 M) did not increased after 48 h treatment (22.5% inhibition with respect to controls, P ⬍ 0.05, Fig. 4B). At this time point, the effect of cAMP-increasing agents was additive with that of 18GA (72% inhibition, Fig. 4B) suggesting that cAMP elevation and gap junctions inhibition lead to alkaline phosphatase down regulation by two independent mechanisms. Finally, the effect of elevated cAMP levels on expression of osteocalcin was measured by quantitative comparative RT-PCR. We evaluated the expression of BGLAP (osteocalcin) gene in control cells and in cells treated 48 h with FK ⫹ IBMX. RT quantitative PCR showed that osteocalcin mRNA was more expressed in cells treated with FK ⫹ IBMX (⌬⌬Ct value of ⫺3.84) than in non treated cells. ⌬⌬Ct value (⫺3.84) indicates that BGLAP expression increased 14.32-fold following exposure to cAMP increasing agents.
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FIG. 4. Effect of increased cAMP on alkaline phosphatase activity. Cells were grown onto plastic dishes until confluent. (A) Alkaline phosphatase activity was measured from whole cell lysates obtained from untreated (control) cells and from cultures incubated with FK (20 M) ⫹ IBMX (1 mM) for 24 h. Values are represented as the mean ⫾ SE of three independent experiments. (B) Alkaline phosphatase activity from cell lysates obtained from untreated (control) cells and from cultures incubated for 48 h with FK ⫹ IBMX, with 18GA (40 M), or with the combination of the three drugs (FK ⫹ IBMX ⫹ 18GA). Data, presented as percentage of control activity, represent the mean ⫾ SE of three independent experiments.
DISCUSSION Intercellular communication is a fundamental property of skeletal tissues and is thought to control several physiological functions of bone cells, such as the coordination of tissue responses to mechanical stimulations (24), sensitivity to hormones and growth factors (11) and the expression of differentiation markers (13). Gap junctions are abundant in bone cells, and at least two different gap junction proteins, Cx43 and Cx45, have been identified in human osteoblast (2). The expression of Cx43 is associated to elevated levels of metabolic coupling (25) and to a high degree of cell differentiation (13, 19), while Cx43-null mice display delayed ossification and osteoblast dysfunction (26) thus pointing to a fundamental physiological role of this protein for the function of bone tissue. HOBIT cells represent a useful model for studying osteoblast physiology, due to the elevated expression of bone differentiation markers such as alkaline phosphatase, osteopontin 1␣ and osteocalcin (16). HOBIT cells express Cx43 and functional gap junctions capable of sustaining the intercellular transfer of Lucifer yellow and the spreading of intercellular Ca 2⫹ waves. Similarly to other osteoblastic cells (10), inhibition of gap junctional intercellular communication by 18GA decreases the expression of differentiation markers (alkaline phosphatase). Given the crucial role of cell-tocell coupling in sustaining the differentiated phenotype, we evaluated whether treatments known to positively modulate gap junctions could enhance HOBIT cells differentiation. Elevated cAMP levels, induced by simultaneous activation of adenylyl cyclase and inhibition of cAMP phosphodiesterase, led to increased gap junction per-
meability and metabolic cell coupling, secondary to changes in the cellular distribution of Cx43. These effects of cAMP on intercellular communication have been demonstrated in other cell types (4 – 6), but are different from those described in preosteoblastic cells, where cAMP is known to stimulate Cx43 mRNA transcription and translation (8, 9). The reasons for this discrepancy are likely to depend on the milieu of transcription factors present at a particular developmental state of the cells along the osteoblastic differentiation pathway. Thus, cAMP may increase Cx43 gene expression in proliferating and maturing preosteoblastic cells (9), but may fail to up-regulate protein expression in differentiated cells (8). In HOBIT cells cAMP-dependent upregulation of intercellular coupling is accompanied by a decrease in the expression of alkaline phosphatase and by a marked increase of osteocalcin mRNA level. This appears in partial contrast with previous findings in other osteoblastic cells, in which the overexpression of Cx43 induced a marked increase of both alkaline phosphatase and osteocalcin gene transcription (10, 13). A crucial difference appears to be the mechanism responsible for gap junctions upregulation. In a recent study, Lecanda et al. (13) showed that overexpression of Cx43, obtained by transfecting Cx43 cDNA into the poorly differentiated MC3T3-E1 osteoblasts was sufficient for activating the transcription of bone specific genes. Conversely, overexpression of Cx45 in the differentiated ROS17/2.8 cell line significantly decreased the basal transcription of alkaline phosphatase and osteocalcin genes. In this study, we show that increased cAMP levels upregulate intercellular coupling due to an enhanced
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rate of Cx43 delivery at gap junctional plaques. cAMP is a second messenger involved in several pathways of bone cells signal transduction and exerts pleiotropic effects on osteoblasts metabolism. Among the molecular targets of cAMP-dependent pathway, the osteoblast specific transcription factor Cbfa1 has been recently described (18). Cbfa1 is a master regulator of osteoblastic differentiation, directly controlling the activation of osteoblast specific genes such as osteopontin, osteocalcin and alkaline phosphatase (27). In cells treated with cAMP elevating agents, the half-life of Cbfa1 is shortened due to cAMP-dependent stimulation of ubiquitin/ proteasome degradation (18). This decreases Cbfa1 steady-state levels and ultimately leads to the downregulation of Cbfa1-controlled genes (18). This mechanism could account for the decreased alkaline phosphatase activity that we recorded in HOBIT cells following stimulation with cAMP increasing agents. However, the increase in the osteocalcin mRNA level suggests a more complex interplay between the signals deriving from the cAMP-dependent Cbfa1 degradation and those arising from the increased cell-to-cell coupling. Although further work is needed to establish the correct relationship between the degree of osteoblast differentiation and the level of intercellular communication, our data suggest that functional coupling, although necessary, might not be sufficient for sustaining the differentiated osteoblastic phenotype. Finally, a speculation can be attempted on the relationship among the increased cAMP concentration, the decreased alkaline phosphatase activity and the stimulated transcription of osteocalcin. The high level of osteocalcin mRNA would suggest that the cells are well differentiated, while the decrease of alkaline phosphatase could be related to a decreased mineralization capacity. As alkaline phosphatase is involved in the formation of mineralized nodules, a modulation of this process via cAMP, leading to a decrease of alkaline phosphatase, could be a way to control the mineralization process. This conclusion is supported by the data published by Tintut and co-workers (18) who found that the inhibition of alkaline phosphatase in MC3T3-E1 cells treated with forskolin is associated to a parallel decrease of matrix mineralization.
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ACKNOWLEDGMENTS We thank Bianca Pani, Valentina Veronesi, and Massimiliano Bicego for helpful discussions. The work was supported by University of Trieste, by MURST-PRIN2000, and by Regione Friuli Venezia Giulia.
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Effects of cAMP on Intercellular Coupling and Osteoblast Differentiation Milena Romanello,* Luigi Moro,* Doroti Pirulli,† Sergio Crovella,† and Paola D’Andrea* ,1 *Dipartimento di Biochimica, Biofisica e Chimica delle Macromolecole, Universita` di Trieste, via Licio Giorgieri 1, I-34127 Trieste, Italy; and †Dipartimento di Scienze della Riproduzione e dello Sviluyppo, Universita` di Trieste, via dell’Istria, 65/1-34137 Trieste, Italy
Received March 20, 2001
Bone-forming cells are organized in a multicellular network interconnected by gap junctions. Direct intercellular communication via gap junctions is an important component of bone homeostasis, coordinating cellular responses to external signals and promoting osteoblast differentiation. The cAMP pathway, a major intercellular signal transduction mechanism, regulates osteoblastic function and metabolism. We investigated the effects of this second messenger on junctional communication and on the expression of differentiation markers in human HOBIT osteoblastic cells. Increased levels of cAMP induce posttranslational modifications (i.e., phosphorylations) of connexin43 and enhancement of gap junction assembly, resulting in an increased junctional permeance to Lucifer yellow and to a positive modulation of intercellular Ca 2ⴙ waves. Increased intercellular communication, however, was accompanied by a parallel decrease of alkaline phosphatase activity and by an increase of osteocalcin expression. cAMP-dependent stimulation of cell-to-cell coupling induces a complex modulation of bone differentiation markers. © 2001 Academic Press Key Words: osteoblasts; gap junctions; cAMP; alkaline phosphatase; osteocalcin.
Cells are joined with intercellular channels that allow them to send messages and exchange molecules and signals (⬍1 kDa) that improve their homeostatic control (1). These channels are present as aggregates in gap junctions, specialized membrane structures that are found between most cell types. Intercellular channels are more complex than ion channels because they span two plasma membranes and each cell contributes with a half channel, or connexon, which interacts with another connexon from the adjacent cell. Each connexon, in turn, is an oligomer of subunit proteins, 1
To whom correspondence should be addressed. Fax: (39) (40) 676 3691. E-mail: [email protected]. 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
called connexins (Cx), which form a multigene family (1). A single cell type may express one or several connexins, which form channels with different molecular size, permeability and ionic selectivity (2, 3). Dynamic regulation of cell-cell communication takes place at different levels (e.g., transcription, translation, intracellular trafficking and gating of connexins) and is controlled by hormones and growth factors, through signaling pathways that have only begun to be understood. Up-regulation of gap junctional coupling by stimulation of the adenylyl cyclase/cAMP pathway has been demonstrated in a number of cell systems. Depending on the cell type, activation of protein kinase A either increases the rate of connexin targeting to the plasmamembrane (4 – 6), or stimulates protein transcription and translation (7–9). In human osteoblasts, the expression of connexins increases with cell maturation and differentiation (10); gap junctional communication sustains bone cells responsiveness to hormonal stimulation (11) and enhances their in vitro mineralization capacity (12). Finally, increased connexin expression stimulates the transcriptional activity of osteoblast-specific promoters (13), suggesting that the degree of cell-to-cell coupling is directly linked to the fully differentiated osteoblastic phenotype. In this study we show that, in human HOBIT osteoblastic cells, elevated cAMP levels stimulate cell-to-cell coupling due to an increased targeting of connexin43 (Cx43) to the plasmamembrane. cAMP stimulates phosphorylations of Cx43 and protein accumulation at appositional membranes. These modifications are accompanied by an increased intercellular diffusion of Lucifer yellow and by a positive modulation of intercellular Ca 2⫹ waves. cAMP-dependent increase of cellto-cell communication is accompanied by a parallel decrease in alkaline phosphatase expression and by a marked increase of osteocalcin gene (BGLAP) transcription.
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The relationship between intercellular coupling and bone differentiation appears, therefore, more complex than previously envisaged (13). MATERIALS AND METHODS Reagents. Fura2-AM, 18␣-glycyrrhetinic acid, Lucifer yellow CH, forskolin, IBMX, PMSF, aprotinin, chymostatin and leupeptin were obtained from Sigma (St. Louis, MO). The primary antibody employed was the polyclonal anti-rat Cx43, from Zymed Laboratories (San Francisco, CA). For immunoblot detection, the secondary antibody was a goat anti-rabbit IgG conjugated with alkaline phosphatase (Calbiochem, San Diego, CA). The chemiluminescence substrate and enhancer for alkaline phosphatase were CSPD and Nitro-Block, respectively (Tropix, Bedford, MA). For immunofluorescence, the second antibody was a fluoresceine (FITC)-conjugated affinity purified goat anti-rabbit IgG from Jackson ImmunoResearch Laboratories (West Grove, PA). Cell culture. HOBIT cells were kindly provided by Professor B. Lawrence Riggs (Mayo Foundation, Rochester, MN). Cells were grown in Dulbecco’s Minimal Essential Medium supplemented with 10% fetal calf serum, 2 mM L-glutamine and penicillin–streptomycin sulfate and cultured at 37°C. Immunofluorescence. Site-specific localization of Cx43 immunoreactivity was performed as previously described (14) on cultures grown on glass coverslips. Intercellular dye transfer. Glass capillaries prepared with a dualstep puller (Narishige, Tokyo, Japan) were filled with a 5% solution of Lucifer yellow dissolved in 0.33 M lithium chloride. Individual cells in confluent monolayers grown onto coverslips were pressureinjected with a pneumatic PLI-100 pico-injector (Medical Systems Corp., New York). 8 min after injection, cells were fixed with 4% formaldehyde in PBS for 15–20 min. Protein immunoblot analysis. HOBIT cells grown to confluence were washed with ice-cold PBS added with 10 mM NaF and 0.1 mM NaVO 4. Proteins were solubilized in lysis buffer (in mM: 50 Tris, pH 6.8, 2% SDS, 0.5 NaVO 4, 10 NaF, 2 PMSF, 10 g/ml aprotinin, 10 g/ml chymostatin and 10 g/ml leupeptin). Prior to electrophoresis, protein concentration was quantitated by the bicinchoninic acid method (Pierce, Rockford, IL) with bovine serum albumin as standard. For each sample 20 g of total proteins were separated on 13% SDS gel and then transferred to nitro-cellulose. Immunoblot was performed as previously described (14). Quantitation of Cx43 bands was performed after densitometric scanning of developed films (Gel DOC 2000, Bio-Rad) using the image-analysis software MultiAnalyst/PC (Bio-Rad). Levels are expressed as fold change of individual band intensities. Ca 2⫹ imaging. HOBIT cells grown onto coverslips were loaded at 37°C with fura-2 AM (5 M), dissolved in Pluronic (Molecular Probes, Eugene, OR) 20% (1:2) and added to the culture medium. After 60 min the loading solution was removed and the cells bathed in a solution containing (mM): NaCl 125, KCl 5, MgSO 4 1, KH 2PO 4 0.7, CaCl 2 2, glucose 6, Hepes-NaOH buffer 25, pH 7.4. Video microscopy and Ca 2⫹measurements were carried out as previously described (15). Mean values in discrete areas of interest were calculated from sequence of images. Quantitative temporal analyses in spatially restricted areas were thus obtained throughout the experiment. Mechanical stimulation. Mechanical stimulation of single cells in confluent monolayers was performed as previously described (15). Experiments were rejected when the cell membrane was damaged, as revealed by a leak of the fluorescent probe from the cell. Stimulations were performed under continuous fluid flow of 3 ml/min. Alkaline phosphatase assay. Cells were seeded at the density of 1 ⫻ 10 5/well into 24-well plates. After reaching confluence, the cells were treated, in serum free medium, with different drugs for 24 or
48 h. At the end of incubation, the cells, washed with PBS, were added with 0.1 M Tris buffer, pH 9.8, containing 0.2% (v/v) Triton X-100 and stored at ⫺80°C for 30 min, after which samples were thawed and scraped. Alkaline phosphatase was measured on total cell lysates resuspended in 0.1 M Tris buffer, pH 9.8, 1 mM MgCl 2 and 6 mM para-nitrophenylphosphate; the reaction was allowed to proceed at 37°C for 60 min and blocked by addition of 40 mM NaOH. Optical densities were measured at 410 nm. An aliquot of the remaining lysate was employed for determination of protein concentration with bicinchoninic acid method. Statistical analysis. Data were analyzed by analysis of variance (ANOVA) and the significance of the difference of the average values between groups was estimated by Scheff e` test. Quantitative PCR. Total RNA was extracted from Hobit cells using the SV total RNA isolation System (Promega, Madison, WI), following manufacturer’s instructions. Two-step RT quantitative PCR was performed using the TaqMan Gold RT-PCR kit protocol (Applied Biosystems, Foster City, CA) with SYBR Green PCR Master Mix. Primers for cDNA amplification were: forward 5⬘-AGCAAAGGTGCAGCCTTTGT-3⬘; reverse 5⬘-CACAGTCCGGATTGAGCTCA3⬘. Real-time PCR was performed using the following temperatures: initial hold at 95°C for 10 min then 95°C for 15 s, 60°C for 60 s for 45 cycles. Four replicates of each RT-PCR were run on ABI PRISM 7700. TaqMan GAPDH Control Reagents (Applied Biosystems) were used as endogenous reference in order to normalize the quantitative data. Two types of PCR controls were performed: (a) no template controls (NTC) with no target DNA and (b) no amplification control (NAC) with the addition of 1 l SDS (0.5% w/v) in order to ensure the denaturation of the DNA polymerase. Quantitative results were analyzed following the comparative Ct method (Applied Biosystems).
RESULTS In this study we employed HOBIT cells, a line derived from normal adult human osteoblastic cells, which retain most of the osteoblastic differentiation markers, including the expression of osteocalcin, alkaline phosphatase, type I collagen, osteopontin 1␣ and the sensitivity to steroid hormones (16). HOBIT cells express Cx43 (17) and are functionally coupled through gap junctions, as demonstrated by the intercellular diffusion of Lucifer yellow microinjected into a single cell (11.4 ⫾ 1.8 fluorescent cells, mean ⫾ SE, n ⫽ 35, Fig. 1A). 18␣-glycyrrhetinic acid (18GA, 40 M, preincubations of 60 min), a reversible, nontoxic inhibitor of gap junctions, totally prevented intercellular dye transfer (1 ⫾ 0.2 fluorescent cells, n ⫽ 17, P ⬍ 0.00001, Fig. 1A). Differentiated osteoblasts exhibit elevated alkaline phosphatase activity correlating with high levels of enzyme expression (18, 19). To evaluate whether the inhibition of cell-to-cell coupling could interfere with the expression of alkaline phosphatase, we treated cells with 18GA (40 M) for 24 h; the treatment did not affect cell viability (not shown), but induced a significant decrease of alkaline phosphatase activity with respect to controls (23.5%, P ⬍ 0.05, Fig. 1B), confirming that gap junctional-dependent communication is required for the expression of bone differentiation markers (10, 19). To verify the relationship between the degree of intercellular coupling and the expression of osteoblastic
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FIG. 1. Effect of 18␣-glycyrrhetinic acid on intercellular coupling and alkaline phosphatase activity. (A) Intercellular dye transfer. Cells were grown onto glass coverslips until confluent. Fluorescence micrographs were taken 8 –10 min after microinjection of Lucifer yellow into one cell. Injections were performed in untreated (control) cells, and in cultures pretreated (60 min) with 18␣-glycyrrhetinic acid (18GA). Bar ⫽ 30 m. (B) Alkaline phosphatase activity. Cells were grown onto plastic dishes until confluent. Alkaline phosphatase activity was measured from whole cell lysates obtained from untreated (control) cells and from cultures incubated 24 h with 18␣-glycyrrhetinic acid (18GA, 40 M). Values represent the mean ⫾ SE of three independent experiments.
phenotype, in a next series of experiments we stimulated cell-to-cell coupling by increasing the intracellular levels of cAMP. Stimulation of cAMP pathway, obtained by the contemporary administration of the adenylyl cyclase activator forskolin (FK, 20 M) and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 1 mM) for increasing times up to 24 h, did not increase Cx43 expression levels, as assayed in Western blot experiments (Fig. 2A), but induced a time-dependent accumulation of slower-migrating
Cx43 bands (Figs. 2A and 2B), presumably reflecting protein posttranslational modifications. It has been well documented, in fact, that Cx43 appears on Western blots as multiple bands, as a result of different degree of protein phosphorylation (20). From the functional viewpoint, high levels of Cx43 phosphorylation are associated to an increased rate of protein translocation from non-junctional stores to junctional plaques (4, 21). In HOBIT cells, the cAMP-dependent accumulation of slower-migrating Cx43 bands was
FIG. 2. Effect of increased cAMP on Cx43 expression and localization. (A) Western blot analysis of Cx43 protein from HOBIT cells. Cells grown to confluence onto plastic dishes were treated with forskolin (FK, 20 M) and 3-isobutyl-1-methylxanthine (IBMX, 1 mM) for increasing times (0, 2, 6, 24 h). All samples are from 20 g of whole lysates (see Materials and Methods). Cx43 bands are labeled according to Musil et al. (20): NP (not phosphorylated) represents the fastest migrating form, P1 and P2 the slower migrating (phosphorylated) forms. Experiments were repeated four times. (B) Densitometric analysis of the three Cx43 bands from Western blot. Levels are expressed as fold change of individual band intensities. (C) Immunofluorescence staining of Cx43 on confluent HOBIT cells cultures. Confluent cells grown onto glass coverslips were fixed, permeabilized, and incubated with a polyclonal anti-Cx43 antibody. A fluoresceine-conjugated secondary antibody was used for visualization. The two micrographs represent the results obtained with untreated (control) cells and cultures treated for 24 h with FK (20 M) ⫹ IBMX (1 mM). Bar ⫽ 30 m. Experiments were repeated six times. 1140
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FIG. 3. Effect of increased cAMP on intercellular coupling. (A) Intercellular dye transfer. Cells were grown onto glass coverslips until confluent. Fluorescence micrographs were taken 8 –10 min after microinjection of Lucifer yellow into one cell. Injections were performed in untreated (control) cells, and in cultures treated for 24 h with FK (20 M) ⫹ IBMX (1 mM). Bar ⫽ 20 m. (B) Ca 2⫹ imaging recording of mechanically induced intercellular Ca 2⫹ waves. Confluent cells grown onto glass coverslips were loaded with fura2-AM. Intercellular Ca 2⫹waves were induced by mechanical stimulation of a single cell (see the spatial map on the left). The kinetics of calcium wave propagation in control and FK ⫹ IBMX-treated cells is reported (right). Five cells in a row from the stimulated cell were analysed from imaging sequences. Data are presented as relative fluorescence ratio.
maximal at 24 h and was accompanied by an increased Cx43 immunostaining at cell-to cell interfaces, as revealed by immunofluorescence (Fig. 2C). The increased immunostaining involved both the extent and the size of highly fluorescent zones, possibly corresponding to an increase in the number and size of gap junctional plaques. Cell treatment for increasing times (up to 48 h) did not further modify Cx43 electrophoretic pattern, nor protein localization (not shown). The possibility that such modifications in Cx43 intracellular distribution could result in altered intercellular coupling was next evaluated. Functional analysis of gap junction permeability was carried out with the dye transfer technique. Compared to controls (11.4 ⫾ 1.8 fluorescent cells, mean ⫾ SE, n ⫽ 35, Fig. 3A), cells treated for 24 h with FK and IBMX show an increased intercellular diffusion of Lucifer yellow (28.4 ⫾ 2.5 fluorescent cells, n ⫽ 19, P ⬍ 0.0001, Fig. 3A), indicating that the high level of gap junctional immunostaining is accompanied by an up-regulation of intercellular coupling. Propagation of intercellular Ca 2⫹ waves following focal cell stimulation is often observed in gap junctions coupled (15, 22, 23). In calcium imaging experiments we induced intercellular Ca 2⫹ waves by mechanically stimulating a single HOBIT cell. In control cells the waves involved 20.7 ⫾ 1.4 cells, (n ⫽ 98, Fig.3B), while in cultures stimulated for 24 h with FK and IBMX, Ca 2⫹ waves involved 39.9 ⫾ 3.9 cells (n ⫽ 27, P ⬍ 0.0001, Fig. 3B), indicating a cAMP-dependent increase in metabolic intercellular coupling. Taken together,
these results demonstrated that, in HOBIT cells, elevated cAMP levels trigger an enhancement of gap junctional assembly associated to an increased rate of intercellular communication. The consequences of this increase on the expression of the osteoblastic phenotype were next evaluated. In HOBIT cells treatments increasing the intracellular cAMP concentration induced a significant inhibition of alkaline phosphatase activity (Fig. 4). The inhibition was evident after 24 h (33.6% inhibition with respect to controls, P ⬍ 0.05, Fig. 4A) and became more pronounced at 48 h (45% inhibition, P ⬍ 0.05, Fig. 4B), while the inhibition due to 18GA (40 M) did not increased after 48 h treatment (22.5% inhibition with respect to controls, P ⬍ 0.05, Fig. 4B). At this time point, the effect of cAMP-increasing agents was additive with that of 18GA (72% inhibition, Fig. 4B) suggesting that cAMP elevation and gap junctions inhibition lead to alkaline phosphatase down regulation by two independent mechanisms. Finally, the effect of elevated cAMP levels on expression of osteocalcin was measured by quantitative comparative RT-PCR. We evaluated the expression of BGLAP (osteocalcin) gene in control cells and in cells treated 48 h with FK ⫹ IBMX. RT quantitative PCR showed that osteocalcin mRNA was more expressed in cells treated with FK ⫹ IBMX (⌬⌬Ct value of ⫺3.84) than in non treated cells. ⌬⌬Ct value (⫺3.84) indicates that BGLAP expression increased 14.32-fold following exposure to cAMP increasing agents.
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FIG. 4. Effect of increased cAMP on alkaline phosphatase activity. Cells were grown onto plastic dishes until confluent. (A) Alkaline phosphatase activity was measured from whole cell lysates obtained from untreated (control) cells and from cultures incubated with FK (20 M) ⫹ IBMX (1 mM) for 24 h. Values are represented as the mean ⫾ SE of three independent experiments. (B) Alkaline phosphatase activity from cell lysates obtained from untreated (control) cells and from cultures incubated for 48 h with FK ⫹ IBMX, with 18GA (40 M), or with the combination of the three drugs (FK ⫹ IBMX ⫹ 18GA). Data, presented as percentage of control activity, represent the mean ⫾ SE of three independent experiments.
DISCUSSION Intercellular communication is a fundamental property of skeletal tissues and is thought to control several physiological functions of bone cells, such as the coordination of tissue responses to mechanical stimulations (24), sensitivity to hormones and growth factors (11) and the expression of differentiation markers (13). Gap junctions are abundant in bone cells, and at least two different gap junction proteins, Cx43 and Cx45, have been identified in human osteoblast (2). The expression of Cx43 is associated to elevated levels of metabolic coupling (25) and to a high degree of cell differentiation (13, 19), while Cx43-null mice display delayed ossification and osteoblast dysfunction (26) thus pointing to a fundamental physiological role of this protein for the function of bone tissue. HOBIT cells represent a useful model for studying osteoblast physiology, due to the elevated expression of bone differentiation markers such as alkaline phosphatase, osteopontin 1␣ and osteocalcin (16). HOBIT cells express Cx43 and functional gap junctions capable of sustaining the intercellular transfer of Lucifer yellow and the spreading of intercellular Ca 2⫹ waves. Similarly to other osteoblastic cells (10), inhibition of gap junctional intercellular communication by 18GA decreases the expression of differentiation markers (alkaline phosphatase). Given the crucial role of cell-tocell coupling in sustaining the differentiated phenotype, we evaluated whether treatments known to positively modulate gap junctions could enhance HOBIT cells differentiation. Elevated cAMP levels, induced by simultaneous activation of adenylyl cyclase and inhibition of cAMP phosphodiesterase, led to increased gap junction per-
meability and metabolic cell coupling, secondary to changes in the cellular distribution of Cx43. These effects of cAMP on intercellular communication have been demonstrated in other cell types (4 – 6), but are different from those described in preosteoblastic cells, where cAMP is known to stimulate Cx43 mRNA transcription and translation (8, 9). The reasons for this discrepancy are likely to depend on the milieu of transcription factors present at a particular developmental state of the cells along the osteoblastic differentiation pathway. Thus, cAMP may increase Cx43 gene expression in proliferating and maturing preosteoblastic cells (9), but may fail to up-regulate protein expression in differentiated cells (8). In HOBIT cells cAMP-dependent upregulation of intercellular coupling is accompanied by a decrease in the expression of alkaline phosphatase and by a marked increase of osteocalcin mRNA level. This appears in partial contrast with previous findings in other osteoblastic cells, in which the overexpression of Cx43 induced a marked increase of both alkaline phosphatase and osteocalcin gene transcription (10, 13). A crucial difference appears to be the mechanism responsible for gap junctions upregulation. In a recent study, Lecanda et al. (13) showed that overexpression of Cx43, obtained by transfecting Cx43 cDNA into the poorly differentiated MC3T3-E1 osteoblasts was sufficient for activating the transcription of bone specific genes. Conversely, overexpression of Cx45 in the differentiated ROS17/2.8 cell line significantly decreased the basal transcription of alkaline phosphatase and osteocalcin genes. In this study, we show that increased cAMP levels upregulate intercellular coupling due to an enhanced
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rate of Cx43 delivery at gap junctional plaques. cAMP is a second messenger involved in several pathways of bone cells signal transduction and exerts pleiotropic effects on osteoblasts metabolism. Among the molecular targets of cAMP-dependent pathway, the osteoblast specific transcription factor Cbfa1 has been recently described (18). Cbfa1 is a master regulator of osteoblastic differentiation, directly controlling the activation of osteoblast specific genes such as osteopontin, osteocalcin and alkaline phosphatase (27). In cells treated with cAMP elevating agents, the half-life of Cbfa1 is shortened due to cAMP-dependent stimulation of ubiquitin/ proteasome degradation (18). This decreases Cbfa1 steady-state levels and ultimately leads to the downregulation of Cbfa1-controlled genes (18). This mechanism could account for the decreased alkaline phosphatase activity that we recorded in HOBIT cells following stimulation with cAMP increasing agents. However, the increase in the osteocalcin mRNA level suggests a more complex interplay between the signals deriving from the cAMP-dependent Cbfa1 degradation and those arising from the increased cell-to-cell coupling. Although further work is needed to establish the correct relationship between the degree of osteoblast differentiation and the level of intercellular communication, our data suggest that functional coupling, although necessary, might not be sufficient for sustaining the differentiated osteoblastic phenotype. Finally, a speculation can be attempted on the relationship among the increased cAMP concentration, the decreased alkaline phosphatase activity and the stimulated transcription of osteocalcin. The high level of osteocalcin mRNA would suggest that the cells are well differentiated, while the decrease of alkaline phosphatase could be related to a decreased mineralization capacity. As alkaline phosphatase is involved in the formation of mineralized nodules, a modulation of this process via cAMP, leading to a decrease of alkaline phosphatase, could be a way to control the mineralization process. This conclusion is supported by the data published by Tintut and co-workers (18) who found that the inhibition of alkaline phosphatase in MC3T3-E1 cells treated with forskolin is associated to a parallel decrease of matrix mineralization.
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ACKNOWLEDGMENTS We thank Bianca Pani, Valentina Veronesi, and Massimiliano Bicego for helpful discussions. The work was supported by University of Trieste, by MURST-PRIN2000, and by Regione Friuli Venezia Giulia.
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