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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

To address the relation between osteoblast growth and cell-to-cell communication, we examined the effects of basic fibroblast growth factor (bFGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA), both potent stimulators of osteoblastic proliferation, on gap junctional intercellular communication between osteoblastic MC3T3-E1 cells. The level of intercellular communication was estimated by a photobleaching method. TPA inhibited the degree of intercellular communication in two different time-dependent manners. The early (<1 h) inhibition by TPA was consistent with an increase in the phosphorylation of connexin 43 (Cx43). The later inhibition was caused by reduction in the total amount of Cx43 on the plasma membrane, due to the decrease in the level of Cx43 transcripts. These qualitative and quantitative modulations by TPA were inhibited by a selective inhibitor of protein kinase C, GF109203X. bFGF also attenuated the gap junctional intercellular communication. However, short exposure (<5 h) to bFGF did not affect the communication. The fact that the growth factor immediately stimulated the phosphorylation of Cx43 indicates that the phosphorylation site(s) affected by bFGF was not involved in the inhibition of communication. The decrease in the intercellular communication level was detected by the longer exposure (>8 h) to bFGF and paralleled the decline in the Cx-mRNA level. This inhibitory effect of bFGF was abolished by the addition of a tyrosine kinase inhibitor, herbimycin A. Thus, gap junctional intercellular communication between osteoblasts was down-regulated by osteoblastic mitogens through different mechanisms of the modulation of Cx43.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Intercellular communication is mediated through cell-to-cell channels that provide a direct pathway for the exchange of various signaling molecules such as Ca2+, cyclic adenosine monophosphate (cAMP), inositol phosphates, and some morphogens, as well as low molecular weight components including inorganic ions and nutrients up to 1000 kD, between neighboring cells.1 The channels are well known to form a large cluster, the so-called gap junction, on plasma membranes. Gap junctional intercellular communication is considered to be an important mechanism for regulating events between cells during embryogenesis and during the normal functioning of organs and for modulating cell differentiation and growth.2–5 A functional gap junction channel consists of two membrane hemichannels (connexons), one in each cell, composed of hexamers of transmembrane proteins known as connexins (Cxs). More than a dozen different Cxs have been identified with molecular masses in the range of 26–50 kD and reveal similar topology and high homology in amino acid sequence.1 The expression of each Cx depends upon the type of organs and cells. Among these Cxs, Cx43 has been extensively studied and is widely expressed in many tissues, especially in heart, as well as in a number of established cell lines.6–8

Numerous morphological studies have shown that bone-forming cells such as osteoblasts and osteocytes contact each other, by which cell-to-cell contact extensive networks of bone-forming cells are formed.9,10 Large numbers of gap junctions exist at these contact sites. Therefore, signals of bone metabolism-regulating hormones and cytokines as well as mechanical loading can be synchronously transmitted into each osteoblast and osteocyte through these gap junctions.11 With respect to the Cx species, Cx43 has been reported to be predominantly expressed in osteoblastic cells.12–14 Recent studies showed that the expression of Cx43 by osteoblasts and the gap junctional intercellular communication between osteoblasts were up-regulated by many osteotropic hormones and factors.15–18 Furthermore, most recently, gap junctional intercellular communication has been shown to modulate responsiveness of osteoblasts to parathyroid hormone (PTH) in cellular networks.19 Therefore, cell-to-cell communication via gap junctions is considered to be critical for the coordinated osteoblast behavior necessary for bone metabolism in response to extracellular signals. However, down-regulation of gap junctional intercellular communication has been little investigated in osteoblasts. Aberrations in Cx expression have been implicated in cancer.20,21 In many cases, a negative relation between cell proliferation and gap junctional communication has been reported.22,23 Basic fibroblast growth factor (bFGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) are potent mitogens of osteoblasts,24,25 and these factors have been shown from in vivo and in vitro studies to suppress osteoblastic phenotypes such as high-level synthesis of type I collagen and alkaline phosphatase (ALP) activity.25,26

This study was undertaken to examine the probable negative effect of osteoblastic mitogens on gap junctional intercellular communication between these cells. We found that both TPA and bFGF down-regulated the gap junctional intercellular communication by qualitative and quantitative modulation of Cx43 via different signal transduction systems.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Materials

Recombinant human bFGF was purchased from Upstate Biotechnology Inc. (Lake Placid, NY, U.S.A.), its purity being greater than 90% as determined by high performance liquid chromatography (HPLC) and sodium dodecyl sulfide polyacrylamide gel electrophoresis (SDS-PAGE). TPA and 5− (and-6-)carboxyfluorescein diacetate (CFDA) were obtained from Sigma (St. Louis, MO, U.S.A.) and Molecular Probes, Inc. (Eugene, OR, U.S.A.), respectively. Immunostaining reagents were purchased from Vector Laboratories Inc. (Burlingame, CA, U.S.A.). Monoclonal anti-Cx43 antibody raised against a synthetic peptide corresponding to positions 252–270 of rat native Cx43 was obtained from Chemicon International Inc. (Temecula, CA, U.S.A.). The amino acid sequence of the synthetic peptide is identical to that at the same positions of mouse Cx43. The 1.5-kb cDNA encoding rat Cx43 was kindly provided by Drs. Hiroshi Yamasaki (Centre International de Recherche sur le Cancer, Lyon, France) and Yozaburo Shibata (Kyushu University, Fukuoka, Japan). Herbimycin A and bisindolylmaleimide (GF 109203X), which are inhibitors of tyrosine kinases and protein kinase C (PKC),27,28 respectively, were obtained from Wako Pure Chemical Industries (Osaka, Japan).

Cell culture

MC3T3-E1 cells29,30 (2 × 104 and 2 × 105) were seeded in 35− and 100-mm dishes, respectively, and cultured for 3 days in alpha modified minimal essential medium (α-MEM) containing 10% fetal bovine serum (Filtron, Australia) until confluent. The cells were washed with α-MEM and then incubated in the serum-free α-MEM supplemented with 0.1% bovine serum albumin (Fraction V, Sigma) for one more day before treatment with TPA or bFGF and/or other agents.

Determination of intercellular communication

The cell-to-cell communication of MC3T3-E1 cells was fluorometrically analyzed by the method of Wade et al.31 with an anchored cell analysis and sorting (ACAS) 570 scanning laser microscope (Meridian Instrument, Okemos, MI, U.S.A.). After the cells had been treated or not with various concentrations of TPA or bFGF for the desired periods, they were labeled for 15 minutes with CFDA (10 μg/ml) in α-MEM. The dishes containing labeled cells were placed on the ACAS stage. The automated stage moved the sample in 1−μm steps in a two-dimensional grid pattern past the microscope objective that focused the excitation beam (488 nm) from an argon ion laser onto the sample. A photomultiplier tube captured the emission intensities at 530 nm at each addressed excitation point. Several cells were selected from each field for monitoring fluorescence transfer, and CFDA in their selected cells was bleached. Sufficient bleaching of fluorescence to measure recovery without causing cell damage observable at the light microscope level was the criterion used to determine laser strength, number of bleaches per cell, and bleach intensities. Unbleached cells were used as positive controls. Series of four postbleaching image scans were generated at 2-minute intervals to measure subsequent redistribution of intracellular fluorescence. Fluorescence levels were quantified by computer-assisted evaluation of the data. We considered the recovery percentage of fluorescence intensity as a magnitude of gap junctional intercellular communication.

Western blotting analysis

Crude gap junctional preparations were obtained by a NaOH fractionation according to the method of Brissette et al.32 After having been treated with TPA or bFGF, the cells were washed twice with phosphate buffered saline (PBS) containing 5 mM EDTA and 0.1 mM sodium orthovanadate and scrapped into 0.1 M sodium bicarbonate (pH 7.0) buffer containing 2 mM EDTA, 10 mM NaF, 1 mM sodium orthovanadate, and protease inhibitors (1 mM aminoethyl-benzene sulfonylfluoride, 10 μg/ml aprotinin, 1 μg/ml pepstatin, and 1 μg/ml leupeptin). An equal volume of 40 mM NaOH was added to the cell suspension, after which the sample was homogenized with a glass-Teflon homogenizer, incubated on ice for 30 minutes, and centrifuged at 100,000 g for 30 minutes. The NaOH-insoluble pellets were washed with the bicarbonate buffer and then dissolved in Laemmli's sample buffer. Protein concentration in the sample was determined with a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, U.S.A.). Samples with an equal amount of proteins were electrophoresed through SDS-PAGE (12.5%) and transferred onto a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% skim milk, the membrane was incubated with 2.5 μg/ml of anti-Cx43 antibody. Immunoreactive Cx43 bands were visualized by avidin-biotin-peroxidase complex method using immunostaining reagents (Vector) following the manufacturer's instructions. Dephosphorylation of Cx43 was carried out by treatment of cell extracts with 2.5 units of ALP (Boehringer Mannheim, Indianapolis, IN, U.S.A.) in Laemmli's sample buffer for 2 h at 37°C. The sample was boiled for 10 minutes at 95°C and subjected to Western blot analysis with anti-Cx43.

Northern blotting analysis for Cx43

The cells were homogenized in 4 M guanidinium thiocyanate. The total RNA was extracted with phenol/chloroform, precipitated with isopropyl alcohol, and washed with 80% ethanol. After quantitation at 260 nm, 10 μg of the total RNA was run on a 1% agarose-2.2 M formaldehyde gel and transferred to a nylon membrane. cDNA probes encoding rat Cx43 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were labeled with [α-32P]dCTP by use of a random-primed DNA labeling kit (Takara, Kyoto, Japan). The membrane was hybridized overnight at 55°C with the labeled cDNA probes. After having been washed, the membrane was exposed to a X-ray film at −80°C. Signals for Cx43 transcripts were quantified by an image-analyzing densitometer (B.I. Systems Corp., Ann Arbor, MI, U.S.A.).

Assay for DNA synthesis

DNA synthesis in MC3T3-E1 cells was measured as reported previously.25 After serum depletion for 1 day, the cells were treated with various concentrations of TPA and bFGF for 24 h and labeled for the last 1 h with 14.8 kBq of [3H]thymidine. At the end of the labeling period, the cells were treated with trichloroacetic acid (final concentration 10%). The amount of [3H]thymidine incorporated into the trichloroacetic acid-insoluble materials was counted and regarded as the level of DNA synthesis.

Statistical methods

Data were presented as the means ± SE, and differences were assessed for statistical significance by Student's t-test.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Intercellular communication between MC3T3-E1 cells at different stages of culture

Intercellular communication through functional gap junctions was assessed in MC3T3-E1 cells at the confluent stage of culture by use of the ACAS system. After the fluorescent dye had been uniformly incorporated into the cells, CFDA in the selected cells was bleached by the laser beam (Fig. 1). As shown in Figs. 1A and 1B, after bleaching, the fluorescence intensity was time-dependently recovered, indicating the influx of the dye from adjacent cells. Thus, the amount of recovery of the dye intensity from photobleaching in 1 minute can be considered as the level of intercellular communication through gap junctions. The level of intercellular communication was low at the early growing stage, increased with increasing duration of the culture time, and reached a maximum at the confluent stage (data not shown).

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Figure FIG. 1. Restoration of dye fluorescence in photobleached osteoblastic MC3T3-E1 cells as a result of intercellular communication. The cells at confluence were uniformly labeled with CFDA. (A) The fluorescence intensity in the cells was monitored by an ACAS 570 work station before, and 0, 2, and 4 minutes after the selected cells (#1–7) had been photobleached. C in (A) indicates the control. (B) Time course of fluorescence recovery after the photobleaching. The percentage of the fluorescence (% Prebleach) was calculated in comparison to the fluorescence intensity in the cells before photobleaching taken as 100%.

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bFGF and TPA down-regulate intercellular communication between MC3T3-E1 cells

A negative relationship between gap junctional intercellular communication and cell growth has been demonstrated in a variety of cells, especially with respect to cell transformation.32,33 Thus, we next examined the effects of bFGF and TPA, potent mitogens of osteoblastic cells, on DNA synthesis and intercellular communication of quiescent MC3T3-E1 cells. Both agents dose-dependently stimulated the DNA synthesis in the cells (Fig. 2) as reported previously.24,25 In contrast, by 24 h treatment with bFGF or TPA, the level of communication was attenuated in a dose-dependent manner (Fig. 2). Significant decreases in the level by bFGF and TPA were observed at 10 pM and 10 nM, respectively. This dose dependency was consistent with that of each agent to stimulate the DNA synthesis of these same cells. However, the profile of the time-dependent decrease in intercellular communication elicited by bFGF differed from that by TPA (Fig. 3). TPA decreased the communication level to less than 10% of the initial level as early as 1 h after TPA addition. Thereafter, the decreased level recovered up to about 50% of the initial level within 4 h, and the low level maintained until 24 h (Fig. 3A). However, bFGF did not affect the intercellular communication level within 1 h after its addition, but a decline in the intercellular communication level was detected from 8 h after the addition of bFGF and continued until 24 h (Fig. 3B). Thus, the inhibitory effects of TPA and bFGF on the intercellular communication between MC3T3-E1 cells seem to result from different mechanisms.

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Figure FIG. 2. Effects of TPA and bFGF on the level of intercellular communication between MC3T3-E1 cells and the DNA synthesis. The cells were treated with various concentrations of (A) TPA or (B) bFGF for 24 h. After the treatments, the level of intercellular communication (○) was measured. The values are means ± SE for 14–40 cells. *p < 0.01 versus untreated cells. For determination of the DNA synthesis (•), the cells were labeled with [3H]thymidine, and the incorporated amount was measured. The values were means ± SD for four cultures. *p < 0.01 versus untreated cells.

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Figure FIG. 3. The time course of inhibitory effects of TPA and bFGF on the level of intercellular communication between MC3T3-E1 cells. The cells were treated with (•) or without (○) TPA (10 nM, A) or bFGF (0.3 nM, B) for the indicated times. Then the level of intercellular communication was measured. Values are means ± SE for 20–38 cells. *p < 0.01 versus cells before treatment.

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Inhibitory effect of TPA and bFGF on the levels of Cx43 and its transcript in MC3T3-E1 cells

Cx43 has been reported to be predominantly expressed and to be a functional Cx in osteoblastic cells.12–14 Thus, we next examined whether the modulation of Cx43 by TPA and bFGF reflects the down-regulation of intercellular communication among the MC3T3-E1 cells. Western blotting analysis using an anti-Cx43 antibody showed that at least four immunoreactive Cx43 molecules with different molecular masses were present on the plasma membrane of quiescent MC3T3-E1 cells (Fig. 4). Among these Cx43s, the 43-kD one was the most abundant, with the 41−, 45−, and 46-kD Cx43s in lesser amounts. ALP treatment increased the density of the 41-kD form band at the expense of the other three forms, whose band densities were diminished (Fig. 4C), indicating the 43−, 45−, and 46-kD Cxs to be phosphorylated forms of 41-kD Cx43.

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Figure FIG. 4. Western blotting analysis for Cx43 on the plasma membrane of MC3T3-E1 cells. The cells were treated (lanes 3, 5, and 7) or not (lanes 2, 4, and 6) with TPA (10 nM, [A]) or bFGF (0.5 nM [B]). Then the plasma membrane of the cells was prepared, and Western blotting analysis for Cx43 was performed. (C) Sample (lane 1) from untreated cells was dephosphorylated by ALP-treatment (lane 2) prior to the Cx43-Western blotting analysis as described in Materials and Methods.

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One-hour treatment with TPA at 10 nM, at which concentration and at which time the phorbol ester decreased the intercellular communication level in MC3T3-E1 cells (Fig. 3A), caused the mobility shift of immunoreactive Cx43 to high molecular weight forms (Fig. 4A). In addition, longer exposure (6 and 24 h) to TPA resulted in a decrease in the level of all forms of Cx43. The addition of bFGF also induced the mobility shift of Cx43 on SDS-PAGE gels in the early period of the treatment (<1 h) (Fig. 4B). The pattern of Cx43 modulation by bFGF was apparently similar to that by TPA, whereas bFGF, unlike TPA, did not affect the level of intercellular communication (Fig. 3B). Longer treatment (24 h) of bFGF caused the decline in the level of Cx43, consistent with the decrease of intercellular communication by the longer exposure. However, the level of Cx43 transcripts was not changed within 1 h after the addition of TPA or bFGF, but the amount was decreased by the longer exposure (Fig. 5).

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Figure FIG. 5. Effects of TPA and bFGF on the level of Cx43 transcripts in MC3T3-E1 cells. The cells were incubated without (lanes 2, 4, and 6) or with (lanes 3, 5, and 7) TPA (10 nM, A) or bFGF (0.5 nM, B). Total RNA was extracted from the cells, and Northern blotting analyses were carried out. The lower panel in (A) and (B) shows the relative intensity of Cx43 transcripts per GAPDH transcripts densitometrically measured.

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Differential mechanism of down-regulation of Cx43 by TPA and bFGF

The above results suggested that TPA and bFGF down-regulated intercellular communication through the modulation of Cx43 in MC3T3-E1 cells by different mechanisms. On the one hand, TPA is a potent activator of PKC, and on the other hand, signals of bFGF are transmitted through the receptor tyrosine kinase cascade. Thus, we examined the relationship between the down-regulation of Cx43 by TPA and bFGF and their signal transduction systems. Both early and prolonged inhibitions of intercellular communication by TPA were completely negated by the addition of GF 109203X, a potent and specific PKC inhibitor (Fig. 6A). In contrast, herbimycin A, a tyrosine kinase inhibitor, little blocked the inhibitory effect of TPA on the communication level (Fig. 6B). Consistently, the simultaneous exposure of the cells to TPA and GF 109203X for 1 h inhibited the mobility shift of Cx43 by TPA to high molecular weight forms (Fig. 7A). In addition, by longer exposure to TPA and GF 109203X, amounts of both Cx43 transcripts and protein were returned to control levels (Figs. 7A and 7B). In contrast to the case for TPA, the addition of herbimycin A completely blocked inhibition of intercellular communication induced by bFGF. In addition, GF 109203X also partially, but not completely, reduced the inhibitory effect of the factor (Fig. 8). Herbimycin A inhibited the mobility shift of Cx43 by a short exposure of bFGF and the quantitative decrease in Cx43 by the longer exposure (Fig. 9A). Herbimycin A also recovered the level of Cx43 transcripts down-regulated by longer exposure to bFGF (Fig. 9B).

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Figure FIG. 6. Effects of protein kinase inhibitors on the inhibition of intercellular communication by TPA in MC3T3-E1 cells. The cells were pretreated or not for 2 h with GF 109203X (0.25 μM [A]) or herbimycin A (1.74 μM [B]) and then incubated for 1 h (open bar) or 24 h (closed bar) with or without TPA (50 nM) in the absence or presence of (A) GF 109203X or (B) herbimycin A. Values are means ± SE for 20–40 cells. *p < 0.01 versus untreated cells.

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Figure FIG. 7. Effect of GF 109203X on the modulation of Cx43 protein and its transcript levels by TPA in MC3T3-E1 cells. For Western blotting analysis for Cx43 (A), the cells were pretreated (lanes 3 and 6) or not (lanes 1, 2, 4, and 5) with GF 109203X (1 μM) for 2 h and then incubated for 1 h or 24 h with or without TPA (10 nM) in the absence or presence of GF 109203X (A). In the case of Northern blotting analysis for Cx43 transcripts (B), the cells were pretreated (lane 3) or not (lanes 1 and 2) with GF 109203X (1 μM) for 2 h prior to incubation for 12 h with (lanes 2 and 3) or without (lane 1) TPA (10 nM) in the absence (lanes 1 and 2) or presence (lane 3) of GF 109203X. The lower panel in (B) shows the relative intensity of Cx43 transcripts per GAPDH transcripts densitometrically measured.

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Figure FIG. 8. Effects of protein kinase inhibitors on the inhibition of intercellular communication by bFGF in MC3T3-E1 cells. The cells were incubated for 2 h with or without GF 109203X (0.25 μM [A]) or herbimycin A (1.74 μM [B]), and then incubated for 24 h with or without bFGF (0.5 nM) in the absence or presence of (A) GF 109203X or (B) herbimycin A. Values are means ± SE for 20–40 cells. *p < 0.01 versus untreated cells.

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Figure FIG. 9. Effect of herbimycin A on the modulation of Cx43 protein and transcript levels by bFGF in MC3T3-E1 cells. For Western blotting analysis for Cx43 (A), the cells were pretreated (lanes 3 and 6) or not (lanes 1, 2, 4, and 5) with herbimycin A (1.74 μM) for 2 h and then incubated for 1 h or 24 h with or without bFGF (0.5 nM) in the absence (lanes 1, 2, 4, and 5) or presence (lanes 3 and 6) of (A) herbimycin A. In the case of Northern blotting analysis for (B) Cx43 transcripts, the cells were pretreated (lane 3) or not (lanes 1 and 2) with herbimycin A (1.74 μM) for 2 h prior to incubation for 12 h with (lanes 2 and 3) or without (lane 1) bFGF (0.5 nM) in the absence (lanes 1 and 2) or presence (lane 3) of herbimycin A. The lower panel in (B) shows the relative intensity of Cx43 transcripts per GAPDH transcripts densitometrically measured.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

A variety of Cxs contribute to the formation of gap junctions in most tissues and cells, the structures of which are involved in embryogenesis, proliferation, and differentiation.2–5 So far, more than 12 species of Cxs have been identified.1 Cx43 has been reported to be the most predominantly expressed in several cell lines with osteoblastic phenotypes,12–14 though the existence of Cx45 was also demonstrated in osteoblastic UMR106–01 cells.34 MC3T3-E1 cells specifically produce a large amount of Cx43.14 Taken together, available data suggest that Cx43 is the principal Cx constructing osteoblastic gap junctions. Furthermore, hormonal regulation of gap junctional intercellular communication between osteoblastic cells has been investigated in relation to osteoblastic differentiation. PTH and prostaglandin E2 (PGE2) have been demonstrated to stimulate the Cx43 expression and/or gap junctional intercellular communication in cultures of human and rat osteoblastic cell lines.15–17 These effects are mediated by elevation of the intracellular cyclic adenosine monophosphate (cAMP) content. cAMP-elevating agents such as PTH, PGE2, and forskolin have been demonstrated to have anabolic effects on osteoblastic differentiation.35–37 In addition, transforming growth factor-β and retinoic acid, which stimulate matrix synthesis,38,39 also increased the Cx43 expression in osteoblastic cells.18 Those results suggest a positive correlation between osteoblast functions and gap junctional intercellular communication. However, data on down-regulation of osteoblastic intercellular communication are totally lacking. Many studies have indicated that TPA and bFGF are potent stimulators of osteoblastic proliferation and suppressers of osteoblastic differentiation.24–26 We showed by the data presented here that both TPA and bFGF inhibited intercellular communication in MC3T3-E1 cells, indicating a negative relationship between the osteoblastic proliferation and gap junctional intercellular communication. This down-regulation resulted from the qualitative and quantitative modulations of Cx43 by these mitogens.

At least three different phosphorylated forms of Cx43 have been widely demonstrated in a variety of cells7,8,40 on the basis of differences in migration on SDS-PAGE gels, with the magnitude of the difference being dependent on the degree of phosphorylation. The physiological significance of Cx43 phosphorylation is controversial to date. Conversion of 42-kD unphosphorylated Cx43 into 44− and 46-kD phosphorylated forms has been indicated to contribute to the Cx43 trafficking into the plasma membrane and the formation of gap junctional plaques on the membrane.41 cAMP-elevating agents such as forskolin have been reported to stimulate gap junctional intercellular communication coupled to the Cx43 phosphorylation.42,43 Indeed, as the differentiation of MC3T3-E1 cells progressed with increasing culture time, the phosphorylated forms of Cx43 accumulated on the plasma membrane in parallel with the increase in intercellular communication level (unpublished data). However, as demonstrated in this study, down-regulating agents of intercellular communication such as TPA and bFGF also induced the Cx phosphorylation in an apparently similar manner on SDS-PAGE patterns. Although phosphorylation sites in response to various factors remain to be clarified, several different sites seem to exist in the intracellular C-terminal domain of Cx43, judged from the high content of serine, threonine, and tyrosine in this region44,45; and thus differential regulation of intercellular communication is likely to be in part attributable to the heterogeneity of Cx phosphorylation sites.

The TPA-induced gating mechanism of the gap junctional intercellular communication between osteoblastic MC3T3-E1 cells seems to be composed of two steps: the early and late responses. By short-term (<1 h) exposure to TPA, the level of intercellular communication was reduced, consistent with the induction of the phosphorylation of Cx43. With respect to this early TPA inhibition, Lampe46 demonstrated that the decrease in intercellular communication was due mainly to the blockage of Cx43 assembly in the plasma membrane by TPA, and not to stimulated dissociation or turnover of Cx43. Thereby, the formation of the gap junctional plaque responsible for intercellular communication was negatively affected. In MC3T3-E1 cells, the total amount of Cx43 in the plasma membrane was not affected, but rather increased by TPA, excluding the possible stimulation of Cx43 dissociation or turnover on the plasma membrane. However, the effect of TPA on channel closing remains to be examined. In some cells, the inhibition of communication by TPA has been reported to be transient and be reversed to the basal level during the long-term treatment.47 However, long-term exposure to TPA also induced the reduction of intercellular communication between MC3T3-E1 cells. Since the levels of Cx43 proteins and transcripts were decreased from 6 h after TPA addition, the late inhibition by TPA is a result of the decrease in biosynthesis of Cx43. Determining whether the quantitative reduction is caused by transcriptional or post-transcriptional regulation will require further studies. Since both the early phosphorylation and the late quantitative reduction of Cx43 by TPA were abolished anyway by a specific inhibitor of PKC, the down-regulation of gap junctional intercellular communication by TPA is PKC dependent in MC3T3-E1 cells.

Accumulating evidence indicates a possible negative relationship between the tyrosine kinase cascade and regulation of gap junctional intercellular communication. Among the tyrosine kinases, the src-gene product has been extensively investigated in this respect. Several laboratories have demonstrated a decrease in gap junctional communication in cells transformed by the src oncogene of the Rous sarcoma virus.48 The decrease in the communication was associated with tyrosine phosphorylation of Cx43.48,49 In the studies presented here, we demonstrated that bFGF, the receptor of which belongs to the family of receptor tyrosine kinases, quantitatively down-regulated the expression of Cx43 at both protein and mRNA levels, resulting in the decrease in gap junctional intercellular communication between MC3T3-E1 cells. Although the growth factor also transiently stimulated the phosphorylation of Cx43, this phosphorylation, unlike that induced by TPA, was not involved in the decrease in the intercellular communication. Since the bFGF-induced Cx43 phosphorylation was reduced by the addition of herbimycin A, a potent tyrosine kinase inhibitor, tyrosine residues in Cx43 are likely to be phosphorylated in response to bFGF. Our results are not consistent with the observations obtained from the src studies. The inhibition of Cx43 expression by bFGF seems to be dependent on some tyrosine kinase cascade, though the components downstream from the bFGF receptor have not yet been identified. Since GF 109203X partially inhibited the down-regulation of intercellular communication by bFGF, the activation of PKC through a cross-talk with tyrosine kinase cascade may in part contribute to the bFGF action. To our knowledge, this study is the first to indicate the inhibitory effect of bFGF on gap junctional intercellular communication. In contrast, bFGF has been demonstrated to stimulate Cx43 expression and intercellular communication of cardiac fibroblasts and of microvascular endothelial cells.50,51 Although we do not know the reason for this apparent discrepancy, this difference may be due to different types of cells. In any event, the negative regulation of gap junctional intercellular communication by TPA and bFGF, using each different mechanism, seems to play a role in controlling osteoblastic proliferation.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

We thank Drs. Hiroshi Yamasaki and Yozaburo Shibata for kindly providing the cDNA of Cx43.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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