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Activation of G Proteins Mediates Flow-Induced Prostaglandin E₂ Production in Osteoblasts

Department of Bioengineering, University of California, San Diego, La Jolla, California 92093-0412

Address all correspondence and requests for reprints to: Dr. John A. Frangos, Department of Bioengineering, University of California, San Diego, La Jolla, California 92093-0412.

	Abstract

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Interstitial fluid flow may play a role in load-induced bone remodeling. Previously, we have shown that fluid flow stimulates osteoblast production of cAMP, inositol trisphosphate (IP₃), and PGE₂. Flow-induced increases in cAMP and IP₃ were shown to be a result of PG production. Thus, PGE₂ production appears to be an important component in fluid flow induced signal transduction. In the present study, we investigated the mechanism of flow-induced PGE₂ synthesis. Flow induced a 20-fold increase in PGE₂ production in osteoblasts. Increases were also observed with AlF₄^- (10 mM) (98-fold), an activator of guanidine nucleotide-binding proteins (G proteins), and calcium ionophore A23187 (2 µM) (100-fold) in stationary cells. We then investigated whether flow stimulation is mediated by G proteins and increases in intracellular calcium. Flow-induced PGE₂ production was inhibited by the G protein inhibitors GDPßS (100 µM) and pertussis toxin (1 µg/ml) by 83% and 72%, respectively. Chelation of extracellular calcium by EGTA (2 mM) and intracellular calcium by quin-2/AM (30 µM) blocked flow stimulation by 87% and 67%, respectively. These results suggest that G proteins and calcium play an important role in mediating mechanochemical signal transduction in osteoblasts.

	Introduction

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BONE TISSUE actively remodels to maintain an optimal configuration for mineral homeostasis and mechanical integrity. This process is influenced by the local mechanical environment (1). The cellular responses and signaling pathways that dictate load-induced bone maintenance and remodeling, however, are poorly understood.

It has become increasingly evident that a significant and rapid flow of fluid occurs through the interstitial spaces of bone (2). Using markers such as colloidal thorium dioxide (3), horseradish peroxidase (4), and ferritin (5, 6, 7), it has been demonstrated that interstitial fluid (ISF) flow is driven from the endosteum toward the periosteal surface in cortical bone. ISF flow is driven by both the hydrostatic pressure gradient across the cortex, as well as skeletal loading (8, 9). Theoretical calculations based on these flow measurements suggest shear stresses on the order of 8–30 dynes/cm² within the cortical bone (10).

Changes in flow may be responsible for the induction of bone formation in regions of elevated fluid pressure due to fluid shifts and hypertension (6, 11). These observations are notable in that the localized remodeling is independent of mechanical loading. We have hypothesized that ISF flow mediates both local and systemic bone homeostasis by stimulating the release of autocrine/paracrine factors that alter the dynamic balance between osteoblast and osteoclast activity. In cultured osteoblasts, we have demonstrated that fluid flow increases levels of intracellular cAMP and IP₃ and increases production of NO and PGE₂ (12, 13, 14, 15).

In vivo, PG production mediates load-induced bone remodeling by both increasing the number of progenitor and osteoblast cells and by inhibiting osteoclast activity (9, 16, 17, 18). Furthermore, PGE₂ synthesis itself may mediate mechanical loading-induced remodeling (16). It is likely, therefore, that PG production represents an early event in the flow-induced signal transduction pathway for osteoblasts.

In the present study, we examined the initial events by which fluid flow induces PG production in osteoblasts. Using a series of inhibitors, activators and calcium chelators, we evaluated the cellular signaling pathways necessary for PGE₂ production in flow-stimulated osteoblasts.

The results presented here suggest that the flow-induced production of PGE₂ is mediated by G proteins. The majority of the G proteins involved in PGE₂ production were pertussis toxin-sensitive. In addition, chelation of extracellular or intracellular calcium also decreased PGE₂ production in flow-stimulated osteoblasts, suggesting a role for calcium in this signal transduction cascade.

	Materials and Methods

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Cell culture and flow experiments
Newborn rat calvarial osteoblasts were isolated using culture techniques adapted from Ecarot-Charrier et al. (19). This method takes advantage of selective migration of osteoblast onto glass chips and has been detailed elsewhere (13). Briefly, 4- to 6-day-old Sprague-Dawley rat pup calvaria were sprinkled with glass chips (300–500 µm). Osteoblasts explanted onto the glass chips, harvested after 8 days and plated onto fibronectin-coated (20 µg; Biomedical Technologies Inc., Stoughton, MA) 35 x 76 mm glass slides.

Osteoblasts grown to confluence on slides were subjected to fluid flow in a flow chamber as described earlier (20). Parallel-plate flow chambers with 250-µm gap widths were perfused by constant pressure head, steady flow loops. Total cell perfusate volume was approximately 15 ml. Cells were exposed to a defined shear stress of 24 dynes/cm² for 6 h. This level of shear has been shown to elicit a large increase in PGE₂ production (12), and is in the predicted physiological range (10).

Slides were rinsed with HBSS and incubated in complete media with or without: GDPßS (Sigma Chemical Co., St. Louis, MO), 100 µM, 24 h; pertussis toxin (Sigma), 1 µg/ml, 1 h; EGTA (Sigma), 2 mM, 30 min.; and quin 2/AM (Calbiochem, San Diego, CA), 30 µM, 30 min. Osteoblasts were then subjected to flow. The perfusion media consisted of M199 (Sigma) supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, Utah), 1% penicillin/streptomycin (Sigma), and 1% glutamine (Sigma) plus appropriate concentration of inhibitor or vehicle, except for quin 2/AM which was not present in the perfusion media. ATP free M199 was obtained from GIBCO. Cell viability was verified by trypan blue staining for each chemical treatment. In all cases, viability was greater than 98%.

Stationary controls were maintained in petri dishes and given fresh media in a manner identical to the positive treatments. We have shown previously that sham loading of the flow loop does not significantly stimulate the cells as measured by NO or cGMP production (21).

PGE₂ determination
One milliliter cell perfusate samples were taken from the flow system in intervals ranging from 5 min to 1 h for up to 6 h. An equal volume of fresh media were returned to the system, so that the total volume was constant. The media samples were assayed for PGE₂ by RIA. The assay buffer consisted of 100 mM NaCl, 10 mM disodium phosphate, 0.5 mM EDTA, and 0.15% BSA (all from Sigma). Rabbit anti-PGE₂ polyclonal antiserum was obtained from Chemicon (Temecula, CA). Tritiated PGE₂ was from New England Nuclear (Boston, MA). At the end of each experiment, the cell layer was scraped into 3 ml of 4 mM EDTA solution, and the total cellular protein was measured using a modified Lowry method (Sigma kit no. 5656). By assaying the levels of PGE₂ in the perfusate over a period of 6 h and taking into account the PGE₂ removed during sampling, cumulative PGE₂ production as a function of time was determined (20). Production rates were normalized by the total protein on each slide.

Statistics
The slope of the cumulative production curve represents the PGE₂ production rate. Slopes for long term production (0–6 h) were calculated by linear regression of all time points for each trial (14 (n = 2) or 21 (n = 3) points in regression). Short term (0–2 h) production rates were calculated by linear regression of 3 data points for each trial (6 or 9 points in regression). Note that n = 2 for all conditions except pertussis toxin, where n = 3. Error represents standard error of regression coefficients (22).

Data for each experiment was regressed according to the following formula (23):

If the slopes are not equal then significance can be determined by:

For example, to determine if GDPßS affected the flow-induced production of PGE₂, the cumulative production of flow, flow and GDPßS, no flow, and no flow with GDPßS were fitted to the following equations:

where Y_i is the cumulative production at times i = 0,1,2... 6 h

If ß(flow) = 0, flow did not stimulate PGE₂ production. If ß(flow, GDPßS) = 0, GDPßS did not affect flow-induced PGE₂ production. If ß(flow, GDPßS) was not equal to zero, then the significance is determined by Student’s t test where:

	Results

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G protein activation
Shear stress of 24 dynes/cm² stimulated PGE₂ production 20-fold (Fig. 1) in osteoblasts. The production was consistently biphasic, for both flow and flow/inhibitor experiments. In each case at approximately 2 h, the slope increased 3-fold (Figs. 2 and 3, and Table 1). Increases in PGE₂ were also seen with the G protein activator AlF₄^- (10 mM) and calcium ionophore A23187 (2 µM) in stationary culture (Fig. 1).

View larger version (56K): [in this window] [in a new window]   Figure 1. Effect of flow, G protein activation, and calcium on PGE2 production in osteoblasts. Osteoblasts were subjected to flow (24 dynes/cm2), 10 mM Al F4- for 1 h and 2 µM A23187 (calcium ionophore) for 1 h in comparison with stationary cultures. Error bars represent the standard deviation.

View larger version (16K): [in this window] [in a new window]   Figure 2. Effect of GDPßS on the cumulative production of PGE2 during 24 dynes/cm2 of shear stress. Osteoblasts were incubated in 100 µM GDPßS for 24 h and then exposed to flow with media containing 100 µM GDPßS. (-•-) flow, (--) flow + GDPßS, (--) stationary control, (--) stationary + GDPßS. GDPßS significantly inhibited the flow- induced increase in PGE2 production (P < 0.001).

View larger version (16K): [in this window] [in a new window]   Figure 3. Effect of pertussis toxin (PTX) on the cumulative production of PGE2 during 24 dynes/cm2 of shear stress. Osteoblasts were incubated in PTX (1 µg/ml) for 1 h and then exposed to flow. (-•-) flow, (--) flow +PTX, (--) stationary control, (--) stationary + PTX. PTX significantly inhibited the flow-induced increase in PGE2 production (P < 0.001).

Preincubation in 1 µg/ml PTX, a G_i/G_o inhibitor, for 1 h partially inhibited the long term (0–6 h) flow-induced PGE₂ production 72 ± 6% (Fig. 3 and Table 1). Longer preincubations with PTX (24 h) gave similar results (data not shown).

Calcium chelation
The shear stress signal transduction mechanism of osteoblasts was further examined to determine the role of calcium in flow-induced PGE₂ production. Chelating extracellular calcium with EGTA (2 mM) significantly attenuated the increase in PGE₂ production (83 ± 7%). Likewise, chelating intracellular calcium with quin 2/AM (30 µM, 30 min preincubation only) decreased the production of flow-induced PGE₂ by 65 ± 8% (Table 1).

Mass transfer
Osteoblasts were exposed to flow in the absence of ATP to determine what role, if any, the mass transfer of ATP plays in the mechanism of flow stimulation. Osteoblasts exposed to flow in the presence of ATP produced 5585 ± 350 pg PGE₂/mg protein·h which was statistically equivalent to the 5750 ± 620 pg PGE₂/mg protein·h produced in the absence of ATP. Likewise, PGE₂ production in stationary cultures was unaffected by the presence or absence of ATP.

	Discussion

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PGE₂ is an integral messenger in mechanochemical signal transduction and has been shown to regulate bone formation both in vivo and in vitro (15, 16, 18, 24, 25, 26). Previously, we demonstrated that PG production mediates flow stimulated increases in cAMP and IP₃ (12, 13). This study examined the cellular signaling pathways in the flow-induced production of PGE₂ in osteoblasts and suggested that G proteins mediate this response.

The near total inhibition of shear-induced PGE₂ stimulation by GDPßS shows that a G protein-dependent pathway mediated flow responses. More specifically, PTX-sensitive G proteins appear to be responsible for the majority of the flow-induced prostaglandin production in osteoblasts. Similar findings were reported for flow-induced PGI₂ production in endothelial cells (28).

In osteoblasts, changes in intracellular calcium have been associated with PTH, vitamin D metabolites, PGE₂, and endothelin-1 stimulation (28, 29, 30, 31, 32). Calcium can either be released from intracellular stores or obtained from the extracellular media. This study suggests that calcium from both pools is necessary for flow-induced PGE₂ production. It is likely, however, that quin2 also chelates calcium of extracellular origin which has diffused into the osteoblasts. This calcium dependence is similar to thrombin-induced PGE₂ synthesis (33). Chelating either intracellular or extracellular calcium stores significantly blocked PGE₂ stimulation (65 ± 8% and 83 ± 7% respectively). The large degree of inhibition by extracellular calcium chelation and the G protein dependence of the flow responses suggests that G proteins may directly activate calcium ion channels. Alternatively, the observed effect can be due to decreases in intracellular calcium associated with a net efflux upon extracellular chelation. Intracellular calcium is likely to act as a cofactor for the activation of phospholipase A₂ and diacylglycerol lipase.

Flow may influence osteoblast function through shear stress, streaming potentials, or enhanced mass transfer of a regulatory substance(s). In endothelial cells, some responses to flow appear to be dependent on the presence of ATP in the flow media (34, 35). This implies that flow is not directly stimulating the cells but is merely enhancing the mass transfer of ATP to the cell surface. It has been hypothesized that in the presence of exogenous ATP, flow may stimulate cells by increasing the ATP levels near the cell membrane thus overcoming local degradation by ectonucleases (34, 35). We show, however, that flow-induced production of PGE₂ was not related to the presence or absence of ATP in the flow media. This suggests that flow-induced PGE₂ production is stimulated by mechanical forces rather than increased mass transfer of ATP. Moreover, we have previously shown that the flow-induced production of cAMP in osteoblasts was mediated by shear stress and not mass transfer (13).

Flow-stimulated PGE₂ production was consistently marked by a biphasic response. At approximately 2 h after the initiation of fluid flow, production rates increased 2- to 3-fold above the short term (0–2 h) production rate. we have shown previously that the biosynthetic machinery necessary to produce PGE₂ is upregulated by flow within 2 h (36). This biphasic response is consistent with cyclooxygenase induction (37). Taken together, this suggests that flow-stimulated AA release and not cyclooxygenase induction is the rate limiting step in flow-induced PGE₂ production.

It is increasingly evident that fluid flow plays an important physiological and pathological role in bone maintenance and remodeling. We have proposed that fluid flow, induced by either mechanical loading or vascular pressure gradients across the cortex, stimulates second messengers that can selectively alter the balance between osteoblast and osteoclast activity.

From the pharmacological data presented here, it is possible to delineate the probable events leading to flow-induced PG production. We propose that fluid flow activates (directly or indirectly) G proteins and causes changes in intracellular calcium levels either by release from intracellular stores or by calcium influx. Arachidonate, which is liberated via phospholipases, is then metabolized to PGE₂ (Fig. 4).

View larger version (30K): [in this window] [in a new window]   Figure 4. Proposed scheme for mechanochemical signal transduction in osteoblasts.

	Acknowledgments

 
This work was supported by the National Heart, Lung, and Blood Institute Grant HL-40696 and NASA Grant NAGW-4558. K. M. Reich was supported by a NASA National Space Grant College Fellowship.

	Footnotes

 
This work was supported by National Heart, Lung, and Blood Institute Grant HL-40696 and NASA Grant NAGW-4558.

Received August 19, 1996.

	References

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Reich, K. M. Articles by Frangos, J. A. Search for Related Content PubMed PubMed Citation Articles by Reich, K. M. Articles by Frangos, J. A.