Increase in epidermal growth factor receptor protein induced in osteoblastic cells after exposure to flow of culture media

Toshiko Ogata

Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 101-0062, Japan

Submitted 4 November 2002 ; accepted in final form 16 April 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To investigate how bone cells respond to mechanical stimuli, we subjected osteoblastic cells to fluid flow. We and others already reported that in a culture system of osteoblast-like cells, ERK1/2, Shc, and other proteins were tyrosine-phosphorylated by medium flow and the early response gene, egr-1 or c-fos mRNA, increased. These are the same as events found after stimulation by various growth factors. Moreover, because there were also reports suggesting that growth factor signaling is involved in the responses to mechanical stimuli, we examined the change in epidermal growth factor (EGF) receptor in the cells exposed to medium flow. The results demonstrated that EGF receptor protein increased after exposure to medium flow. This increase did not occur without serum in media, and the addition of EGF restored it. Furthermore, leupeptin blocked this increase. These results suggest that degradation of EGF-occupied EGF receptor by leupeptin-sensitive protease(s) in endosomes decreased with exposure to medium flow. This was presumed to participate, at least in part, in signaling of fluid flow.

mechanical stimuli; epidermal growth factor receptor; leupeptin; proteolysis


IT IS WELL DOCUMENTED that mechanical stimuli are essential to maintain homeostasis of bone and that osteoblasts can sense the mechanical stimuli (10, 19, 22, 27, 30, 33). However, the mechanism to transduce the mechanical stimuli to the intracellular signals has not yet been elucidated. Several studies have suggested that growth factor signaling is involved in the signal transduction of mechanical stimuli. A few growth factor receptors are activated by mechanical stress (9, 17, 18, 29). Shc and ERK1/2 are tyrosine-phosphorylated by flow of culture media and egr-1 or c-fos mRNA increases (10, 23, 24, 33). Furthermore, we had found that the upregulation of egr-1 mRNA and tyrosine-phosphorylation of Shc and ERK1/2 by fluid flow were not induced without serum and that the responses were recovered by the addition of epidermal growth factor (EGF) (23, 24). These findings raised the question of how activation of EGF receptor by EGF participates in this response to fluid flow. Although we first examined tyrosine phosphorylation of the EGF receptor by fluid flow, we could not observe the upregulation of tyrosine phosphorylation of the EGF receptor, perhaps due to experimental errors too large to estimate small changes induced by fluid flow (unpublished observation). However, during the experiments, we noticed that the amount of EGF receptor protein increased after exposure to fluid flow.

The EGF signaling begins with EGF binding to the EGF receptor, followed by dimerization and tyrosine phosphorylation of the EGF receptor. The activated EGF receptor recruits other signal transduction-related molecules and tyrosine-phosphorylates them. The activated EGF receptor is internalized via clathrin-coated pits and degraded by proteasomes or in lysosomes. The internalization and the degradation of the EGF receptor have generally been considered to be a downregulation of the EGF signal. However, evidence is now accumulating to show that EGF receptor trafficking and EGF receptor degradation regulate the EGF signal transduction (8) and that the regulation is performed in the intracellular compartments, especially in early endosomes, and cross talk with other signals also occurs there. For example, the EGF receptor incorporated in early endosomes exists in an activated form and binds Shc (3, 14, 25) and continues participating in the signaling of EGF (16). Cathepsin B degrades the EGF receptor in the endosomes and negatively regulates EGF signaling (3). PKC regulation on EGF signaling or differences between EGF and TGF-{alpha} signaling are due to diversity in sorting of the EGF receptor in early endosomes (4, 34). This evidence suggests a possibility that the increase in EGF receptor found in this study might regulate signal transduction and that elucidation of the mechanism might lead to solving the mechanism of signaling of mechanical stimuli. Therefore, we investigated the increase in the EGF receptor induced by fluid flow.

In this article, we report that the amount of EGF receptor increased in osteoblastic cells exposed to medium flow, that the protease inhibitor leupeptin blocked the increase, and that the increase was not induced in the cells cultured in serum-free media. Furthermore, from the examination of the possible mechanisms of the increase, we have demonstrated that the increase in EGF receptor induced by fluid flow might be due to a decrease in the degradation by leupeptin-sensitive protease. Leupeptin-sensitive protease(s), of which cathepsin B is a candidate, might act only on the EGF-occupied EGF receptor, which exists in endosomes, probably early endosomes, as well as cathepsin B (3, 14, 15, 25). This colocalization of the EGF-occupied EGF receptor and the protease might be the reason that the increase in EGF receptor by fluid flow did not occur without EGF in culture media. These results suggest that prolongation of the life span of EGF receptor in the active form by exposure to fluid flow participates, at least in part, in signaling of fluid flow.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials. EGF (recombinant human) was obtained from R &D System (Minneapolis, MN). Filipin complex, chloroquine diphosphate salt, monodansylcadaverine (MDC), and phenylmethylsulfonyl fluoride (PMSF) were obtained from Sigma Chemical (St. Louis, MO). Z-Leu-Leu-Leu-H (aldehyde), also called MG-132, leupeptin, and CA074-methyl ester (ME) were obtained from Peptide Institute (Osaka, Japan). Herbimycin A was obtained from Kyowa Medex (Tokyo, Japan). Anti-EGF receptor polyclonal antibodies, EGFR(1005), and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). HRP-conjugated anti-phosphotyrosine antibodies (PY20) were obtained from BD Transduction Laboratories (Lexington, KY). A colloidal gold solution was obtained from Bio-Rad (Hercules, CA).

Cell culture. SV-HFO cells, a human osteoblastic cell line immortalized by simian virus 40, were kindly provided by Dr. H. Chiba (11) and were cultured in DMEM containing 10% fetal bovine serum, in 60-mm-diameter polystyrene tissue culture dishes without any additional treatment, in a culture chamber at 37°C under 5% CO2 in air. Media were replaced 2 or 5 h before the start of experiments, as indicated.

Fluid flow experiment. The flow of the culture media was generated by shaking the culture dishes on a shaker placed in a culture chamber. The shaker moved horizontally and in parallel (23). The amplitude was 3 cm and the rate was 60 shakes/min. Shaking was carried out for 1 or 5 min. Cells were lysed immediately before shaking and 0.1, 2, or 10 min after shaking. Media were replaced 2 or 5 h before shaking, and after that time the chamber was not disturbed until shaking. Nonshaken dishes as control were placed on the upper shelf in the same chamber and treated in the same manner as the shaken dishes.

Cell lysis and Western blotting. The dishes were taken out of the chamber and quickly washed three times with ice-cold PBS, and then 400 µl of ice-cold lysis buffer (1% Triton X-100, 10 mM Tris·HCl, pH 7.4, 10% glycerol, 150 mM NaCl, 1 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 50 mM NaF, 0.1 mM Na2MoO4, 1 mM Na3VO4, and 1 mM PMSF) was added to each dish. The cell lysates were passed through a 26G needle several times and then centrifuged for 20 min at 15,000 g. The supernatants containing 10 µg of total protein were used for 8% SDS-PAGE. The fractionated proteins were electrophoretically transferred onto polyvinylidene difluoride membranes (Immobilon; Millipore, Bedford, MA). In the immunoblotting with anti-EGF receptor antibodies, the membranes were incubated in a blocking buffer [washing buffer (see below) containing 5% nonfat dry milk] for 16 h and then in anti-EGF receptor antibody [EGFR(1005)] solution (66 ng/ml) diluted with blocking buffer for 2 h. The membranes were washed five times with a washing buffer (10 mM Tris·HCl, pH 7.6, 100 mM NaCl, 0.1% Tween 20) and then incubated with an HRP-conjugated anti-rabbit IgG antibody solution (133 ng/ml) diluted with blocking buffer. In the immunoblotting with anti-phosphotyrosine antibodies, the membranes were incubated in blocking buffer (washing buffer containing 1% BSA) for 16 h and then in HRP-conjugated anti-phosphotyrosine antibody (PY20) solution (100 ng/ml) diluted with blocking buffer for 4 h. After the membranes were washed five times with washing buffer, the immunoreactive proteins were detected using the ECL+ Western blotting detection system (Amersham Biosciences, Piscataway, NJ) on X-ray films.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
EGF receptor protein increases in osteoblastic cells exposed to medium flow. Human osteoblastic cells were exposed to medium flow generated by shaking culture dishes for 1 min as described in MATERIALS AND METHODS. At first, although we tried to examine the tyrosine phosphorylation of the EGF receptor after shaking by immunoprecipitation with anti-EGF receptor or anti-phosphotyrosine antibodies, variations in the results were too high to permit us to draw any conclusions. However, during the experiments, a change in the amount of EGF receptor protein after shaking was noticed. Western blotting of the cell lysates before and after the shaking with anti-EGF receptor antibodies showed that immunoreactive EGF receptor protein began to increase immediately after the shaking, and the increase continued for 10 min (compare the 6th to 8th lanes from the left with the 5th lane in Fig. 1A), whereas in the nonshaken cells, no such change in the EGF receptor protein was observed (see the 1st to 4th lanes in Fig. 1A). Relative intensity of the EGF receptor in the shaken cells increased significantly compared with that in the nonshaken cells 10 min after shaking as shown by the data in Fig. 1B, which were obtained from three independent experiments. Figure 2 shows that the EGF receptor protein increased at 10 min and the increase fell by 30 min. These two experiments were performed in EGF-containing but serum-free media.



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Fig. 1. Increase in epidermal growth factor (EGF) receptor protein induced in cells exposed to media flow. Culture media were replaced with serum-deprived DMEM containing EGF at a concentration of 10 ng/ml 5 h before shaking of culture dishes. The dishes in the shaken and nonshaken groups were placed in a chamber as described in MATERIALS AND METHODS. Shaking was performed for 1 min. The cells in both groups were lysed immediately (time 0) before shaking and 0.1, 2, and 10 min after shaking. A, top: Western blotting of the whole cell lysate with anti-EGF receptor antibodies; bottom: staining of total proteins on the filters with colloidal gold. EGFR/gold values are ratios of immunoreactive bands to anti-EGF receptor antibodies to total proteins stained with colloidal gold, normalized with the values at time 0 (immediately before shaking). B: relative intensity of EGF receptor bands normalized with the values at time 0 was obtained from 3 independent experiments. Values are presented as means ± SD. A significant difference was recognized between shaken and nonshaken cells 10 min after shaking of culture dishes for 1 min (*P < 0.01).

 


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Fig. 2. Time course of EGF receptor protein after shaking of culture dishes. The experiment was performed and the results presented as described in Fig. 1A, except that cell lysis was performed immediately before shaking and 10, 30, and 60 min after shaking. The peak was at about 10 min.

 

Increase in the EGF receptor after shaking does not occur in cells cultured in serum-free media without EGF. Because we had previously found that upregulation by medium flow of egr-1 mRNA and tyrosine phosphorylation of Shc and ERK1/2 was not induced in the cells cultured in serum-free media without EGF (23, 24), we examined whether the increase in EGF receptor after shaking was also not induced without EGF in the serum-free media. As shown in Fig. 3, no increase in the EGF receptor after shaking was observed.



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Fig. 3. No increase in EGF receptor protein after shaking was observed in cells cultured in serum-free media without EGF. The experiment was performed and the results presented as described in Fig. 1, except that cells were cultured in serum-free media without EGF for 5 h before shaking of culture dishes. A: Western blotting with anti-EGF receptor antibodies and staining with colloidal gold. B: relative intensity of EGF receptor bands normalized with the values at time 0 was obtained from 3 independent experiments. Values are presented as means ± SD. There was no difference between nonshaken cells and shaken cells.

 

Increase in EGF receptor induced by shaking does not involve ligand-mediated internalization, chloroquine-sensitive endosomal proteolysis, proteasomal degradation, or herbimycin A-sensitive tyrosine kinase activity. As shown in Fig. 1, the increase in EGF receptor began immediately after shaking. This rapid response led us to speculate that a decrease in proteolysis, rather than an increase in synthesis, of the EGF receptor occurs after shaking. It is well known that EGF stimulation internalizes the EGF receptor within 1 min through clathrin-coated pits and that the receptor is delivered to endosomes within several minutes (12, 20). We also observed that the addition of EGF (50 ng/ml) decreased the amount of EGF receptor to 80% of the amount before the addition within 10 min (data not shown). If this pathway were disturbed by shaking, the increase in the protein might occur within a few minutes. Therefore, we examined the participation of this pathway by interfering with it. The cells were pretreated with MDC, an inhibitor of clathrin-dependent endocytosis (13), chloroquine, an inhibitor of endosomal proteolysis (5, 20, 34), MG-132, a proteasome inhibitor (20, 32), or herbimycin A, a tyrosine kinase inhibitor, in media containing EGF (10 ng/ml) but no serum. In untreated cells, which were not treated with any of the inhibitors, the relative intensity of the EGF receptor increased 1.2-fold on average at 2 min after shaking for 5 min. In all the pretreatments performed, the relative intensity also increased to a greater extent than in untreated cells (Fig. 4, A and B).



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Fig. 4. Effect of pretreatment with monodansylcadaverine (MDC), chloroquine, MG-132, or herbimycin A on the increase in EGF receptor after shaking. MDC (300 µM), chloroquine (200 µM), MG-132 (20 µM), or herbimycin A (3 µM) was added to serum-free media containing EGF (10 ng/ml). The cells were cultured in these media for 5 h before shaking of culture dishes, except for MDC, which was cultured for 2 h before shaking because of the toxicity. The untreated cells were cultured for 5 h in serum-free media containing EGF (10 ng/ml) but no inhibitors. Shaking was performed for 5 min. The cells were lysed immediately before shaking and 2 and 10 min after shaking. A: Western blotting was carried out with anti-EGF receptor antibodies. Afterward, total proteins on the filters were stained with colloidal gold (not shown). B: relative intensity of EGF receptor bands corrected with total proteins and normalized with the values at time 0 (immediate time before shaking). The experiments were performed in a pair of treated cells and in untreated cells, twice independently for each treatment. Consequently, the values for untreated cells are means ± SE of 8 independent experiments, whereas other values are means of 2 independent experiments.

 

Leupeptin-sensitive protease(s) is involved in the increase in the EGF receptor after shaking. The ligand-mediated degradation pathway did not seem to participate in this response, as shown in Fig. 4. Another presumption derived from the rapid response was that the EGF receptor translocated from the insoluble to the soluble fraction after shaking. However, because the mature EGF receptor of 170 kDa was almost completely extracted in the Triton X-100 lysis buffer and Western blotting of the insoluble fraction did not demonstrate any specific change in the EGF receptor after shaking (data not shown), it was still presumed that proteolysis is involved in this response. Therefore, we examined the shaking effect on the cells pretreated with a protease inhibitor, leupeptin, which is often used to protect the EGF receptor from degradation. In the absence of shaking, pretreatment with leupeptin increased the amount of EGF receptor to about 1.4-fold that in untreated cells (compare EGFR bands of the 4th lane from the left with the 1st lane in Fig. 5A; the numerical values are shown on the 1st and 2nd lanes from the left in Fig. 6A). A further increase in EGF receptor, upregulated by leupeptin, was not observed after shaking (compare the 5th and 6th lanes from the left with the 4th lane in Fig. 5A), whereas in untreated cells, the EGF receptor increased after shaking (compare the 2nd lane from the left with the 1st lane in Fig. 5A). As shown in Fig. 5B, there was a significant difference between untreated and leupeptin-treated cells at 2 min after shaking for 5 min.



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Fig. 5. Pretreatment with leupeptin blocked the increase in the EGF receptor after shaking of culture dishes. The cells were cultured in serum-free media containing EGF (10 ng/ml) and leupeptin (2 mM) for 2 h. The untreated cells were cultured in serum-free media containing only EGF for 2 h. Shaking was performed for 5 min. The cells were lysed immediately before shaking and 2 and 10 min after shaking. The experiment was performed and the results presented as described in Fig. 1. A: Western blotting with anti-EGF receptor antibodies and staining with colloidal gold. B: relative intensity of EGF receptor bands normalized with the values at time 0 was obtained from 4 independent experiments. Values are presented as means ± SD. A significant difference was recognized between leupeptin-treated and untreated cells 2 min after shaking of culture dishes for 5 min (*P < 0.05).

 


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Fig. 6. Effect of leupeptin and other protease inhibitors on EGF receptor proteolysis. After cells were cultured in EGF-containing (10 ng/ml; A) or EGF-free serum-free media (B) for 5 h, leupeptin (2 mM), PMSF (2 mM), CA074-methyl ester (ME; 1 µM), MG-132 (20 µM), or vehicle (C1, H2O; C2, methanol; C3, DMSO) was added to the media. The cells were lysed 2 h after the addition. Western blotting with anti-EGF receptor antibodies and staining with colloidal gold are shown. These experiments were performed and the results presented as described in Fig. 1A. The experiment was performed twice independently, and similar results were observed between the 2 experiments.

 

Effect of leupeptin and other protease inhibitors on EGF proteolysis. To examine which leupeptin-sensitive protease(s) participates in the increase in EGF receptor after shaking, we tested the effect of various inhibitors on EGF receptor proteolysis. Leupeptin is an inhibitor of serine and cysteine proteases and is reported to inhibit the activity of trypsin, plasmin, papain, and cathepsin B (2, 21). Therefore, the proteolytic effects on the EGF receptor of PMSF, an inhibitor of serine proteases including trypsin and plasmin, CA074-ME, a membrane-permeable inhibitor of cathepsin B (3, 6), and MG-132, an inhibitor of calpain as well as proteasomes (32), were compared with the effect of leupeptin. As shown in Fig. 6A, in the cells cultured in EGF-containing serum-free media, all of these protease inhibitors increased the amount of EGF receptor, but the electrophoretic mobility of the EGF receptor was lower in the cells treated with PMSF or MG-132 than in the control, whereas the electrophoretic mobility of the EGF receptor in the cells treated with leupeptin or CA074-ME was no different from that in the control. This result suggests that the effects of leupeptin and CA074-ME on EGF receptor proteolysis are different from the effects of PMSF and MG-132. Furthermore, we examined the possibility that leupeptin-sensitive protease(s) relates to the necessity of EGF in EGF receptor increase after shaking. As shown in Fig. 6B, in the cells cultured in serum-free media without EGF, leupeptin and CA074-ME did not increase the amount of EGF receptor protein, whereas MG-132 induced both an increase in the amount of EGF receptor and a decrease in the electrophoretic mobility, similar to that in cells cultured in EGF-containing serum-free media. This result suggests that leupeptin-sensitive protease and CA074-ME-sensitive protease, which is presumed to be cathepsin B, act on only the EGF-occupied EGF receptor, unlike MG-132-sensitive protease, and that this might be the reason for the necessity of EGF in EGF receptor increase after shaking.

Enhancement of tyrosine phosphorylation by leupeptin and the similarity to that induced by shaking and EGF addition. Because it was presumed that this increase in the EGF receptor is due to a decrease in the proteolysis of the EGF receptor by leupeptin-sensitive protease, we examined whether such a decrease in the proteolysis takes part in EGF signaling. Western blotting with anti-phosphotyrosine antibodies showed that tyrosine phosphorylation of various proteins was enhanced in cells treated with leupeptin (Fig. 7A). The enhanced proteins were similar to those induced by shaking or EGF addition (Fig. 7, compare A with B and C). This suggests that the increase in the EGF receptor induced by shaking found in this study participates, at least in part, in the signaling of medium flow.



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Fig. 7. Effect of leupeptin, shaking of culture dishes, and EGF on the tyrosine phosphorylation. Western blotting of whole cell lysates with anti-phosphotyrosine antibodies is shown. A: after the cells were cultured in serum-free media containing EGF (10 ng/ml) without (c) or with leupeptin (2 mM) for 2 h, the cells were lysed. B: after the cells were cultured in serum-free media containing EGF (10 ng/ml) for 5 h, the cells were subjected to shaking for 1 min. The cells were lysed immediately before shaking and 2 min after shaking. C: after the cells were cultured in serum-free media without EGF for 5 h, EGF was added to the media at a concentration of 50 ng/ml. The cells were lysed immediately before and 5 min after the addition of EGF.

 

Pretreatment with filipin blocked both the increase in EGF receptor protein after shaking and the leupeptin effect on EGF receptor proteolysis. The results described above suggest the possibility that the degradation of the EGF receptor by leupeptin-sensitive protease decreases after shaking. However, the mechanism is unclear. Recently, some authors have reported that a cholesterol-enriched domain, caveolae or raft, on the membrane senses the fluid flow (26, 28). Therefore, we interfered with the domain with a cholesterol binding reagent, filipin (31). As shown in Fig. 8, A and B, this treatment blocked the increase in the EGF receptor protein induced in untreated cells after shaking. If the blocking were due to destruction of a sensor of fluid flow, we thought that the increase in the EGF receptor by leupeptin treatment would also probably occur in the cells treated with filipin. However, as shown in Fig. 9, the increase by leupeptin treatment was not observed in the presence of filipin, whereas the leupeptin effect was observed in the presence of MDC or MG-132. This result suggests that the effect of filipin, which blocks the increase in the EGF receptor induced by shaking, as shown in Fig. 8, might not be due to blocking of the sensing of the fluid flow but, rather, to disturbance of the EGF receptor proteolysis system.



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Fig. 8. Effect of pretreatment with filipin on the increase in the EGF receptor after shaking of culture dishes. The cells were cultured in serum-free media containing EGF (10 ng/ml) and filipin complex (5 mg/ml) for 5 h. The untreated cells were cultured in serum-free media containing only EGF. Shaking was performed for 5 min. The cells were lysed immediately before shaking and 2 and 10 min after shaking. The experiments were performed and the results presented as described in Fig. 1. A: Western blotting with anti-EGF receptor antibodies and staining with colloidal gold. B: relative intensity of EGF receptor bands normalized with the values at time 0 was obtained from 4 independent experiments. Values are presented as means ± SD. A significant difference was recognized between cells treated with and without filipin at 2 min (*1P < 0.05) and 10 min (*2P < 0.05) after shaking of culture dishes for 5 min.

 


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Fig. 9. Effect of filipin on the leupeptin effect on EGF receptor proteolysis. Cells were cultured in media containing EGF (10 ng/ml) and MDC (300 µM), MG-132 (20 µM), filipin complex (5 mg/ml), or vehicle (DMSO) for 5 h (2 h in the case of MDC). Afterward, leupeptin (2 mM) or H2O was added to the media. The cells were lysed 2 h after the addition. The experiment was performed twice independently; similar results were observed between the 2 experiments.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Many researchers have reported that various responses are induced in cells subjected to mechanical stimuli. They involve an increase in intracellular Ca2+ concentration or inositol 1,4,5-trisphosphate level, release of prostaglandin or nitric oxide, or upregulation of tyrosine phosphorylation of Shc, ERK1/2, or focal adhesion kinase (FAK) (7, 10, 19, 22, 27, 30, 33), even if referring only to responses in osteoblasts and within several minutes from the stimulation. However, the trigger that induces these responses is unclear, although it must be on the cell surface. We found that medium flow generated by shaking culture dishes induced an increase in the amount of EGF receptor protein in osteoblastic cells (Fig. 1). This response occurred within a few minutes from the stimulation, leading us to presume that proteolysis of the EGF receptor is involved. Furthermore, this response did not occur in cells cultured in EGF-free media (Fig. 3) or in media containing EGF but treated with leupeptin (Fig. 5).

The involvement of proteolysis of the EGF receptor and the necessity of EGF in culture media indicated a possible involvement of the ligand-dependent internalization and degradation pathway. However, in this pathway, the increase in EGF receptor and the enhancement in EGF signaling are contradictory because EGF occupation on the EGF receptor promotes degradation of the EGF receptor. Next, it has been presumed that the delay in internalization via clathrin-coated pits of the EGF-occupied EGF receptor occurred after shaking. However, we also excluded this possibility because the increase in EGF receptor after shaking also occurred in the cells that suffered a disturbance of clathrin-dependent internalization by MDC (13) (Fig. 4). Moreover, if the regulation of the EGF receptor occurred on the plasma membrane, it cannot be explained why leupeptin blocked the increase in the EGF receptor after shaking but chloroquine and MG-132 did not (Fig. 4), because all the three inhibitors are thought to work inside the cells. Chloroquine is thought to inhibit proteolysis at the late stage of endosomes (5, 20, 34), probably due to inhibition of acidification of endosomes. MG-132 is an inhibitor of proteasomes (20, 32) that is thought to degrade the ubiquitinated EGF receptor downstream of the endosomes. If leupeptin works in early endosomes, the difference among the effects of leupeptin, chloroquine, and MG-132 on the increase in the EGF receptor after shaking can be explained. In Fig. 6, we show that the leupeptin-sensitive protease might be cathepsin B. Cathepsin B is expressed in osteoblast-like cells, and most of the activity is detected in the cells (1). Although this protease has been considered a lysosomal enzyme, recent studies have provided evidence that cathepsin B activity is present in endosomes, probably in early endosomes (3, 15). If this protease works in endosomes, the EGF receptor is not degraded by this protease in EGF-free media because the EGF nonoccupied EGF receptor is on the plasma membrane and does not exist in endosomes (14, 25). This colocalization of leupeptin-sensitive protease and EGF-occupied EGF receptor might be necessary for the increase in EGF receptor after shaking. For these reasons, it is probable that the upregulation of EGF signaling by medium flow occurred as a result of the increase in the amount of activated EGF receptor in the endosomes caused by a decrease in proteolysis.

If so, how is the decrease in proteolysis induced? One possibility is that the signal to decrease the protease activity is transmitted from cell surface to early endosomes. Another is that the EGF-occupied EGF receptor undergoes a change to avoid proteolysis in early endosomes, such as threonine phosphorylation of the EGF receptor by PKC, which promotes sorting to recycling endosomes from early endosomes (4). To solve this question, we tested whether the cholesterol-enriched domain, caveolae or raft, takes part in the decrease in proteolysis of the EGF receptor by fluid flow, because some reports have suggested that the domain is a sensor of fluid flow (26, 28). Filipin, a reagent that disrupts the domain (31), blocked the increase in the EGF receptor after shaking (Fig. 8). However, this finding does not necessarily mean that this domain is a sensor for the decrease in the proteolysis of the EGF receptor after shaking, because no increase in the EGF receptor by leupeptin treatment was observed in the presence of filipin (Fig. 9). The filipin effect on the increase in EGF receptor after exposure to fluid flow might mean that the EGF-occupied EGF receptor does not exist in endosomes in the presence of filipin or that the proteolysis in the endosomal compartment is disturbed by filipin.

In summary, fluid flow induced an increase in the EGF receptor in osteoblastic cells. This induction seemed to involve a decrease in the degradation of the EGF-occupied EGF receptor by leupeptin-sensitive protease and seemed to occur in endosomes, probably early endosomes. The mechanism still remains to be solved.


    DISCLOSURES
 
This study was supported by Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science (13671493).


    FOOTNOTES
 

Address for reprint requests and other correspondence: T. Ogata, Dept. of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental Univ., 3-10 Kandasurugadai 2-chome, Chiyodaku, Tokyo 101-0062, Japan (E-mail: oga.mph{at}mri.tmd.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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