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
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ABSTRACT |
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mechanical stimuli; epidermal growth factor receptor; leupeptin; proteolysis
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- 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.
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MATERIALS AND METHODS |
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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.
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RESULTS |
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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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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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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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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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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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DISCUSSION |
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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.
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DISCLOSURES |
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FOOTNOTES |
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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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