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Vol. 16, Issue 7, 3100-3106, July 2005
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* Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX 78229-3900;
Department of Radiology, University of Texas Health Science Center, San Antonio, TX 78229-3900; and
Department of Oral Biology, School of Dentistry, University of Missouri, Kansas City, MO 64108
Submitted October 19, 2004;
Revised March 18, 2005;
Accepted April 8, 2005
Monitoring Editor: Asma Nusrat
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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-glycyrrhetinic acid and carbenoxolone, even in cells without physical contact, suggesting the involvement of Cx43-hemichannels. Inhibitors of other channels, such as the purinergic receptor P2X7 and the prostaglandin transporter PGT, had no effect on PGE2 release. Cell surface biotinylation analysis showed that surface expression of Cx43 was increased by shear stress. Together, these results suggest fluid flow shear stress induces the translocation of Cx43 to the membrane surface and that unapposed hemichannels formed by Cx43 serve as a novel portal for the release of PGE2 in response to mechanical strain. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Keller et al., 1993; Baylink et al., 1995, 1996; Harada et al., 1995), and long-term release of PGE2 is significantly reduced in cultured osteoporotic bone cells compared with age-matched control cultures (Sterck et al., 1998). Prostaglandin seems to be essential for new bone formation in response to mechanical strain (Forwood, 1996); however, mice lacking the genes for enzymes required for prostaglandin synthesis, cyclooxygenase (COX)-1 and COX-2, do not seem to have any major bone developmental defects (Dinchuk et al., 1995; Langenbach et al., 1995). Therefore, prostaglandins seem to be more important in the mature skeleton that is exposed to greater mechanical strain than the developing skeleton.
Osteocytes are the major mechanosensory cells in bone (Aarden et al., 1994). These cells are ideally located in the bone to sense shear stress induced by fluid flowing in the canaliculi surrounding their dendritic processes. They have been shown to be more sensitive than other types of bone cells with respect to release of prostaglandins in response to fluid flow shear stress (Klein-Nulend et al., 1995). In our previous studies, we have reported that fluid flow increases the release of PGE2 and that the released PGE2 functions in an autocrine manner to stimulate gap junction function and connexin (Cx)43 expression (Cheng et al., 2001a).
A most intriguing question is how prostaglandin is rapidly released by the cell, especially by osteocytes in response to mechanical strain. As a charged organic anion at physiological pH, the diffusion rate of prostaglandin across plasma membranes is at least a magnitude lower than the response of PGE2 release observed in most cells (Schuster, 2002). A prostaglandin transporter, PGT, has been identified from rat and bovine cells (Kanai et al., 1995; Banu et al., 2003); however, this transporter mainly mediates the uptake of PGE2 into the cell, not its release (Chan et al., 1998). Therefore, the means by which PGE2 is rapidly released in not known.
In addition to being the major component of gap junction channels, connexins have recently been shown to exist and function in the form of unapposed halves of gap junction channels called hemichannels. These channels are localized at the cell surface, independent of physical contact with adjacent cells (Goodenough and Paul, 2003). These hemichannels, like gap junction channels, display low substrate selectivity and permit molecules with molecular masses <1 kDa to pass through. Hemichannels are regulated by extracellular Ca2+, and these Ca2+ regulated hemichannels control the osmotic volume of the cell (Quist et al., 2000; Gomez-Hernandez et al., 2003). The existence of functional hemichannels formed by Cx43 has been reported in several cell types, including neural progenitors, neurons, astrocytes, cardiomyocytes, osteoblasts, and osteocytes (Goodenough and Paul, 2003). Mechanical stimulation has been shown to open hemichannels in astrocytes (Stout et al., 2002) and osteoblasts (Romanello et al., 2003), although none of these studies used specific, physiologically relevant, defined forms of mechanical strain, such as fluid flow shear stress. In general, the regulation of the opening of hemichannels under normal physiological conditions remains uncharacterized.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Cell Culture and PGE2 Measurement
MLO-Y4 cells were cultured at various densities on collagen-coated (rat tail collagen type I; 0.15 mg/ml) surfaces, including plastic plates, polyester sheets, and glass slides. Cells were grown in 
-modified essential medium (MEM) supplemented with 2.5% FBS and 2.5% CS and incubated in 5% CO2 incubator at 37°C as described previously (Cheng et al., 2001a).
After the fluid flow treatment for 30 min or 2 h at 16 dynes/cm2, the conditioned medium was collected, and the extracellular PGE2 released into the medium was measured using a PGE2 EIA kit according to the manufacturer's instructions, whereas intracellular PGE2 was measured after the cells were thoroughly washed three times with phosphate-buffered saline (PBS) and lysed.
Cell Shear Stress Induced by Fluid Flow
Fluid flow experiments were performed as described previously (Cheng et al., 2001b). MLO-Y4 cells were cultured on collagen-coated surfaces for protein assay, PGE2 measurement and microscopic assay. Flow rate was continually monitored by an in-line flowmeter (Cole-Parmer Instrument, Vernon Hills, IL). Using this flow system, wall shear stress levels caused by steady laminar flow of 16 dynes/cm2 were generated by adjusting the channel height (using spacers) and medium flow rate (1–1.8 ml/s). All experiments were repeated at least three times.
Antisense Cx43 Oligodeoxynucleotide (ODN) Approach
Based on previous studies (Becker et al., 1999), Cx43 sense and antisense ODNs derived from nt 954 to 983 were synthesized at the DNA Core Laboratory of the University of Texas Health Science Center at San Antonio. MLO-Y4 cells were treated every 8 h in the absence or presence of fresh 50 µM sense or antisense ODN for a total of 24 h, and the expression of Cx43 was analyzed using immunofluorescence and Western blots.
Dye Uptake Assay
MLO-Y4 cells were grown at low initial plating density to ensure that the majority of the cells were not physically in contact. LY (Mr of 547 Da) was used as a tracer for hemichannel activity, and RD (Mr of 10 kDa) was used as a negative control. Cells maintained in Ca2+-free MEM were exposed to vehicle, 5 mM EGTA, 100 mM -GA, or glycyrrhetinic acid (GA) for 10 min. For fluid flow treatment, cells were treated in the absence or presence of 100 mM -glycyrrhetinic acid (-GA) or glycyrrhetinic acid (GA) under fluid flow shear stress at 16 dynes/cm2 for 10 min. After treatment, dye uptake experiments were conducted in the presence of 0.4% LY and 0.4% RD for 5 min, and cells were washed with medium containing 1.8 mM Ca2+ to close the hemichannels and then fixed with 1% paraformaldehyde. Similar fields were observed under the fluorescence microscope. Dye uptake was presented as a percentage of fluorescent cells.
Isolation of Chick Primary Osteocytes
Preparation of primary osteocytes from chick calvaria was based on previously published procedures (Tanaka et al., 1995) with some modifications. Briefly, calvarial bone was dissected from 16-d-old embryonic chicks and minced. The soft tissues and osteoid were removed by collagenase treatment followed by decalcification by using EDTA. The final particles were treated with collagenase and vigorously agitated to release osteocytes, followed by filtration through an 8-µm membrane filter to eliminate most of the larger sized osteoblast/fibroblast cells.
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). MLO-Y4 cells were labeled with or without 1 mg/ml EZ-link Sulfo-NHS-LC-Biotin in PBS at 4°C for 20 min. The cells were washed three times with PBS containing Ca2+, Mg2+, and glycine and lysed in lysis buffer (133 mM NaCl, 5 mM KCl, 1% dextrose, and 20 mM HEPES) plus 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, and proteases inhibitors. Cell lysates were mixed with equal volumes of monomeric avidin beads and incubated for 60 min at room temperature. The beads were then washed five times with PBS until no proteins could be detected by measurement of spectrometric absorbance at 280 nm. The biotinylated proteins were eluted by boiling for 5 min in sample loading buffer containing 1% SDS and 2% 2-mercaptoethanol, and equal volumes of each sample were loaded on SDS-PAGE and analyzed by Western blotting by using affinity-purified anti-Cx43 or anti--actin antibody.
Immunofluorescence Labeling and Fluorescence Microscopy
The cells cultured on the microscopic slides were washed three times with PBS, each wash of 5-min duration. This was followed by fixation in 2% paraformaldehyde in PBS for 30 min at room temperature after which the cells were washed three times with PBS of 5 min each, followed by incubation for 30 min in blocking solution containing 2% normal goat serum, 2% fish skin gelatin, and 1% bovine serum albumin in PBS, and then incubated overnight at 4°C with an affinity-purified anti-Cx43 antibody diluted 1:250 in blocking solution. Cells were washed three times; 5 min for each wash with PBS and then incubated for 1 h with fluorescein-conjugated goat anti-rabbit IgG diluted 1:250 in blocking solution. Fluorescence microscopy was performed using an Olympus B-MAX microscope (Olympus, Tokyo, Japan) and recorded on a "Spot II" digital camera (Diagnostic Instruments, Tokyo, Japan). For dye uptake and dye transfer results, LY was detected using the filter set for fluorescein and RD by using the filter set for rhodamine.
Western Blotting
The protein concentration of crude membrane samples of MLO-Y4 cells was determined using the MicroBCA assay according to the manufacturer's instructions (Pierce Chemical). Western blotting was performed as described previously (Cheng et al., 2001b), and membranes were incubated with a 1:250 dilution of affinity-purified Cx43 antibody or a 1:5000 dilution of monoclonal anti--actin antibody. The primary antibody was detected using peroxidase-conjugated secondary anti-rabbit or anti-mouse antiserum followed by use of a chemiluminescence reagent kit (ECL) according to the manufacturer's instructions. The intensity of Cx43 bands was quantified by densitometry (NIH Image).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). To determine whether gap junction channels and hemichannels play a role in this process, we applied -GA, an inhibitor known to block gap junction channels as well as unapposed hemichannels, to cells exposed to fluid flow shear stress (Figure 1). The release of PGE2 stimulated by fluid flow was significantly suppressed by this inhibitor. We found that -GA or GA at a concentration of 100 µM does not interfere with PGE2 quantitation (our unpublished data). These result suggests that the release of PGE2 is likely to occur through the function of either gap junctions, hemichannels, or both.
Almost all chemical inhibitors for gap junctions are known to block gap junction channels as well as unapposed hemichannels. To distinguish the relative contributions of these two different types of channels, cells were plated at decreasing densities to the point where few cells were in contact, 2.0 x 103 cells/cm2 or lower (Figure 2A). Interestingly, cultures at lower cell densities (0.9 x 103 and 2 x 103 cells/cm2) released significantly more PGE2 per cell than cells cultured at normal and higher densities (7.5 x 103 cells/cm2 and higher) (Figure 2B), suggesting that at high cell density, i.e., more gap junctions, the release of PGE2 is suppressed, although the increase in response to fluid flow shear stress is significant. The inhibitor -GA significantly blocked PGE2 release at both lower (2 x 103 cells/cm2) and normal (7.5 x 103 cells/cm2) cell densities in response to fluid flow shear stress (Figure 2C). The observation that the blockage of the release of PGE2 at a density where few gap junction channels could physically exist suggests that hemichannels are the likely mechanism responsible for the release of PGE2 in response to fluid flow shear stress.
Because the precise blocking mechanism for all of the chemical inhibitors of gap junctions and hemichannels is not well understood (Goodenough and Paul, 2003), we used a Cx43 antisense approach to further verify that release of PGE2 is a connexin-mediated event. An antisense ODN was derived from nt 954 to 983 of the Cx43 coding sequence (Becker et al., 1999). Immunofluorescent staining and Western blots showed that expression of Cx43 was greatly reduced after antisense ODN treatment compared with nontreated and sense ODN control (Figure 2, D and E). The increased release of PGE2 induced by fluid flow shear stress was also significantly attenuated upon treatment of cultures with antisense Cx43 ODN at both lower and normal cell densities (Figure 2F). The observation of a blockade of PGE2 release by antisense Cx43 ODN in cells without gap junctions strongly suggests that hemichannels formed by Cx43 are responsible for the release of PGE2 from osteocytes.
Another effective channel blocker, carbenoxolone (Plotkin et al., 2002), was used to further confirm the role of hemichannels in the release of PGE2. Similar to the effects by -GA and Cx43 antisense, the fluid flow-induced release of PGE2 from cells without physical contact was significantly inhibited by carbenoxolone (Figure 3A). The amount of intracellular PGE2 was also increased in response to fluid flow; however, there was no discernible increase in the intracellular PGE2 levels in carbenoxolone-treated cells (Figure 3B), which implies a potential feedback inhibition of biosynthesis by the substrate. Alternatively, metabolic instability of intracellular PGE2 due to oxidation (Schuster, 1998) may contribute to the lack of increase in intracellular PGE2 in the presence of a hemichannel inhibitor. Together, these results suggest that fluid flow induced the opening of hemichannels, which leads to a release of intracellular stores of PGE2.
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) and a prostaglandin transporter, PGT (Chan et al., 1998), could potentially provide alternative or additional pathways for the exit of PGE2 from the cell. To determine whether the release of PGE2 by fluid flow could be mediated by either of these two mechanisms, two commonly used antagonists, oxidized ATP (oATP), specific for P2X7 (North, 2002), and 4,4'-diisothiocyanatostilbene 2,2'-disulfonate (DIDS), specific for PGT (Chan et al., 1998), were applied, and the intracellular as well as extracellular levels of released PGE2 were determined (Figure 4). Only the hemichannel inhibitor, not the inhibitors for either P2X7 or PGT, blocked the release of PGE2 (Figure 4A). Similar to the observation in Figure 3B, the elevated intracellular PGE2 level induced by fluid flow was not altered by -GA or by the other inhibitors for P2X7 receptor and PGT transporter (Figure 4B).
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). Compared with unstressed (Figure 5, A and C) and EGTA-treated (Figure 5A, EGTA) cells, fluid flow shear-stressed cells (Figure 5A, FF) increased the uptake of LY. The number of cells taking up the dye upon fluid flow treatment is even higher than those treated by EGTA. The induction of the opening of hemichannels by fluid flow was blocked by -GA (Figure 5A, FF + -GA). Quantification of dye uptake showed a significant increase in the number of cells that took up the dye when subjected to fluid flow compared with unstressed control cells, and this increase was significantly inhibited by -GA (Figure 5B). Hemichannel activity in primary osteocytes also was observed using the dye uptake assay (Figure 5C). Similar to MLO-Y4 cells, fluid flow induced the uptake of LY in primary osteocytes. These results suggest that fluid flow shear stress significantly stimulates the opening of functional hemichannels in osteocytes.
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Because fluid flow shear stress increased the activity of hemichannels to allow the release of PGE2, it would be expected that the expression level of the hemichannel-forming Cx43 protein would be increased on the cell surface as well. Therefore, cell surface expression of Cx43 was assessed by determining the amount of Cx43 that can be biotinylated due to exposure on the cell surface (Figure 6A) (n = 3). More biotinylated Cx43 protein was observed from lysates of cells subjected to fluid flow shear stress compared with the control sample (Figure 6A, lanes 3 and 4), even though the total amount of Cx43 being subjected to precipitation by avidin beads in the fluid flow sample was less than that of its control (Figure 6A, lanes 1 and 2). In addition, the highly biotinylated form of Cx43 (top band) seems to be more dominant in samples subjected to fluid flow, implying greater extracellular exposure of Cx43 in response to shear stress. The relative ratio of biotinylated to total Cx43 was determined and showed an increase in the amount of surface-biotinylated Cx43 in cells subjected to fluid flow shear stress compared with controls (Figure 6A, right). Lysates of the cells without biotin treatment were applied to avidin beads to control for nonspecific binding (Figure 6B). Nonbiotinylated Cx43 was not precipitated by the beads (Figure 6B, lane 2). To eliminate the possibility that intracellular domains of Cx43, due to incomplete quenching, became accessible to the biotinylating reagent after the cells were lysed, a parallel control biotinylation assay was conducted for -actin, a protein only expressed intracellularly (Figure 6C). No biotinylation of -actin was detected (Figure 6C, lane 2). Together, the data show an approximate doubling in surface Cx43 expression in response to fluid flow shear stress.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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-GA and carbenoxolone, can block the release of PGE2 even at low cell densities with minimal cell contact, which prevents any gap junction formation. The release of PGE2 was only inhibited by -GA and carbenoxolone, but not by inhibitors of other channels such as the purinergic receptor P2X7 or the prostaglandin transporter PGT. A Cx43 antisense ODN also inhibited the release of PGE2 at low cell density. Dye uptake analysis, indeed, showed that hemichannels were induced to open by fluid flow shear stress (Jiang and Cherian, 2003). Moreover, we found that fluid flow stimulated the increased expression of Cx43 on the cell surface. Together, these results suggest that fluid flow shear stress stimulates the movement of Cx43 to the cell membrane and induces the opening of Cx43-hemichannels likely to be involved in the exit of intracellular PGE2 from the cell.
Osteocytes and osteocyte-like MLO-Y4 cells express large amounts of Cx43, and this protein is located on the plasma membrane and predominantly, in the cytoplasm. Our previous study showed that in the presence of fluid flow shear stress, Cx43 seems to migrate toward the plasma membrane (Cheng et al., 2001b). In the present study, biotinylation analysis revealed that Cx43 expression on the cell surface is doubled, most likely to accommodate an increased demand for hemichannel function in response to fluid flow shear stress. Because gap junction-forming Cx43 is not accessible to the cross-linking reagent biotin (Lampe, 1994), and thus cannot be surface-biotinylated, the biotinylated Cx43 is likely the component of hemichannels on the cell surface.
Osteocyte dendritic processes are surrounded by canaliculi, which form an extensive three-dimensional network or syncytium in the bone matrix. The release of PGE2 from mechanically stimulated osteocytes has been reported in previous publications by us and others (Klein-Nulend et al., 1995; Ajubi et al., 1996; Cheng et al., 2001a). We also have shown that increases in release of PGE2 are correlated with increases in magnitude of fluid flow shear stress (Cheng et al., 2001a). PGE2 is an anabolic factor that can increase bone mass in animals (Jee et al., 1985; Keller et al., 1993; Baylink et al., 1995; Harada et al., 1995; Baylink et al., 1996). The use of indomethacin, the inhibitor of prostaglandin synthase (COX-1 and COX-2), blocks bone formation in response to mechanical strain (Forwood, 1996). Conversely, prostaglandin also stimulates osteoclast formation and activation (Collins and Chambers, 1991, 1992; Kaji et al., 1996). It is possible that extracellular release of PGE2 into the bone fluid could reach the bone surface and have effects not only on osteoclasts but also on osteoblasts or any other cell on the bone surface. Osteoclastic bone resorption is coupled to bone formation and is responsible for bone remodeling. Bone remodeling in response to mechanical strain has been shown to be due to prostaglandin synthesis (Reich et al., 1990; Reich and Frangos, 1991). In our previous studies (Cheng et al., 2001a), we showed that the PGE2 released in response to fluid flow shear stress also acted in an autocrine/paracrine manner to stimulate gap junction-mediated communication. Therefore, not only could prostaglandin be having an effect on cells on the surface of bone but also on osteocytes downstream of the released prostaglandin.
Prostaglandin signaling mechanisms through their EP receptors is well known (Breyer et al., 2001), and a new function and signaling pathway has recently been identified—that of spinal PGE2 acting as an inflammatory pain-sensitizing molecule through the glycine receptor to boost neuronal activity (Harvey et al., 2004). However, the pathway(s) for the exiting of prostaglandins from the cell remains largely unknown. Prostaglandins are charged anions that diffuse poorly across membrane bilayers (Schuster, 2002). Even though the human prostaglandin transporter gene hPGT is up-regulated by fluid flow mechanical stimuli in endothelial cells (Topper et al., 1998), studies exclude the possible involvement of PGT in the release of PGE2 (Chan et al., 1998; Schuster, 2002). PGT, instead, is suggested to play a role in metabolic oxidation clearance through the reuptake of the released PGE2 from the extracellular milieu and matrix. It has been suggested that PGE2 exits the cell by a channel-like diffusion process, not through a specific transporter-facilitated process (Chan et al., 1998). A potential channel that could be responsible for the efflux of PGE2 is the purinergic receptor P2X7, the only type of purinergic receptor that acts as a channel and permits passage of molecules up to Mr of 800 Da (North, 2002). However, specific inhibitors for P2X7 failed to block the release of intracellular PGE2 in the present study, which excludes this possibility. Moreover, opening of P2X7 channels requires a high concentration of ATP (close to 1 mM) (North, 2002). We found that conditioned medium collected from fluid flow-stimulated cultured cells failed to induce dye uptake (our unpublished data), indicating that the amount of ATP released is not sufficient to activate P2X7 channels. Additionally, P2X7 channels are less permeable for anionic molecules such as PGE2 because these channels selectively allow passage of cationic molecules over anionic molecules. Hemichannels, like gap junction channels, seem to be passive, energy independent, and less selective with regard to the charge of the substrate (Goodenough and Paul, 2003), thus probably permitting rapid release of anionic PGE2 in response to mechanical stimulation.
The present study provides evidence that hemichannels play an important role in osteocyte function and response to mechanical strain under physiological conditions. It provides a solution to a long-standing, important issue of how the highly charged, anionic prostaglandins traverse the plasma membrane and exit the cell under physiological conditions. The function of hemichannels is likely to offer a general mechanism for the rapid exit of not just prostaglandins, but presumably other extracellular regulatory factors, when cells are activated and challenged by physiological stimuli, such as mechanical stimulation.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Abbreviations used: -GA, 18 -glycyrrhetinic acid; Cx43, connexin 43; DIDS, 4,4'-diisothiocyanatostilbene 2,2'-disulfonate; LY, Lucifer yellow; oATP, oxidized ATP; ODN, oligodeoxynucleotide; PGE2, prostaglandin E2; RD, rhodamine dextran.
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: Jean X. Jiang (jiangj{at}uthscsa.edu).
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