We demonstrated the presence in ECs of a novel PECAM-1–dependent signaling pathway activated by FSS and HOS, both of which are thought to exert mechanical perturbation to the plasma membrane. ERK phosphorylation, a known early flow response of cultured ECs (Davies, 1995
; Tseng et al., 1995
) was found to be a downstream event of this signaling. The initial step of this signaling appeared to be tyrosine phosphorylation of PECAM-1, and we presented evidence suggesting that PECAM-1 might be a mechanoresponsive molecule. The single most important experimental evidence was to elicit this signaling response by applying tugging force directly to PECAM-1 on the cell surface. In such experiments, only the fraction of PECAM-1 that was bound to anti–PECAM-1ext–coated beads and pulled by magnetic force was tyrosine phosphorylated, and ERK was also phosphorylated under these conditions. When PECAM-1 expression was downregulated, the level of ERK phosphorylation by HOS was substantially reduced, suggesting that the PECAM-1–dependent signaling cascade played a significant role in the activation of ERK. Other results reported in this paper are consistent with the idea that PECAM-1 is a mechanotransducer and activates a signaling cascade in ECs, leading to ERK activation. Besides ERK activation, there are many other forms of EC responses to FSS. It is generally thought that more than one mechanosignaling pathway is involved in initiating various flow responses, and accordingly, several mechanotransduction schemes have been suggested. We propose that PECAM-1 is a new member of such mechanotransduction molecules in ECs. At present, it is not known how many of the known flow responses are regulated by PECAM-1 tyrosine phosphorylation. Currently, we are investigating what other FSS-induced events might depend on PECAM-1.
Using live BAECs expressing GFP-tagged SHP-2, we showed rapid, mechanical stimulus–dependent translocation of this signaling molecule to the intercellular junction. The similar translocation of endogenous SHP-2 as well as Gab1 was demonstrated by immunofluorescence microscopy. In ECs with reduced PECAM-1 expression, neither SHP-2 nor Gab1 translocated to the cell junction even though the cells were exposed to FSS or HOS. Thus, the translocation of these molecules to the cell junction depends on the expression of PECAM-1 and its tyrosine phosphorylation, which is induced by mechanical stresses. Several questions remain unanswered regarding the nature of the interaction among PECAM-1, SHP-2, and Gab1 at the cell junction. We have shown direct binding between pY- PECAM-1 and SHP-2 (Masuda et al., 1997
), and this is achieved presumably by a BTAM present in PECAM-1. Direct binding between Gab1 and SHP-2 is also reported and is mediated by a BTAM of Gab1 (Cunnick et al., 2000
). These investigators have shown that in cells stimulated by EGF, Gab1 binding activates the phosphatase activity of SHP-2, which is required for ERK activation. Similar molecular events could occur in ECs stimulated by FSS or HOS. However, in either case in which cells are treated by chemical (EGF) or mechanical (FSS and HOS) stimuli, the precise way in which the membrane protein (EGF receptor or PECAM-1), SHP-2, and Gab1 interact is not known. Because immunoprecipitation experiments did not show direct binding between PECAM-1 and Gab1 (unpublished data), the observed Gab1 accumulation at the cell junction may be due to its interaction with SHP-2. This may create competition between PECAM-1 and Gab1 for binding to SHP-2, because both use BTAM to bind to SHP-2, and such competition might be unfavorable for the observed accumulation of SHP-2 and Gab1 at the cell junction. It is possible that increased concentrations of SHP-2 in this region may work as a sink for Gab1, but it is also possible that some unknown molecules are involved in the observed Gab1 accumulation because it has several protein binding motifs. Attempts to identify other PECAM-1 and Gab1 binding proteins at the cell junction are ongoing. Another question is that for Gab1 to bind to SHP-2, two of Gab1's tyrosine residues (Tyr627 and Tyr659) must be phosphorylated (Cunnick et al., 2001
). If Gab1 does bind to SHP-2 in ECs exposed to FSS, how is Gab1 tyrosine phosphorylated? It is quite possible that the answer to this question holds a key to understanding FSS mechanosignaling.
SHP-2(C/S) has no phosphatase activity, but other sites, such as BTAM, are intact (Noguchi et al., 1994
). When this mutant was expressed in ECs, it was associated with the cell junction regardless of FSS or HOS. This result provides certain insights into the PECAM-1–dependent signaling. Although its physiological function is not understood, a small fraction of PECAM-1 is always tyrosine phosphorylated in ECs (Masuda et al., 1997
; Osawa et al., 1997
). Thus, via their BTAM, both SHP-2 and SHP-2(C/S) should bind to pY-PECAM-1 present in unstimulated ECs. By its phosphatase activity, however, SHP-2 would dephosphorylate PECAM-1, causing it to detach from PECAM-1, and no appreciable build-up of SHP-2 would occur at the EC junction. On the other hand, SHP-2(C/S) with no phosphatase activity should bind to PECAM-1 but would not dephosphorylate it. Consequently, the mutant SHP-2 would remain bound, causing its gradual accumulation at the cell junction. In another signaling system in which SHP-2 is involved, similar effects have been observed (Oh et al., 1999
There is an interesting corollary to the proposed on–off interaction between PECAM-1 and SHP-2. When PECAM-1 is tyrosine phosphorylated by FSS, SHP-2 binds to it. The bound SHP-2 would then dephosphorylate PECAM-1. We suggest that signaling is initiated at some point during these processes. If ECs are still under FSS, PECAM-1 would be rephosphorylated, followed by binding of SHP-2 and dephosphorylation by the bound SHP-2, resulting in another wave of signaling. This cycling allows the system to continuously respond to FSS. We showed earlier that the expression of proteins containing phosphotyrosine is augmented in the area of aorta where FSS is experimentally increased (Kano et al., 2000
). One interpretation of this result is that ECs, although they have been under increased FSS for 1 wk, maintain increased levels of signaling. The protein(s) responsible for the observed increase in phosphotyrosine is not fully identified, but a part of it could be due to pY-PECAM-1. These data support the idea that ECs continuously monitor FSS levels, and the proposed cyclic interaction between PECAM-1 and SHP-2 appears to be a good feature for a system that works continuously.
Extracellularly, PECAM-1 of an EC binds to PECAM-1 of neighboring ECs primarily in a homophilic manner (Albelda et al., 1990
). Although a large fraction of PECAM-1 is extractable by Triton, ~30% is associated with the cytoskeleton (Ayalon et al., 1994
). Indeed, PECAM-1 binds to β- and γ-catenins (Kusano et al., 1998
; Ilan et al., 2000
), suggesting its interaction with actin filaments. Being anchored both extracellularly and intracellularly, PECAM-1 may not be able to move freely as FSS or HOS deforms the cell surface. Thus, when the cell surface is mechanically stressed, the force of stress may act on PECAM-1, which then is tyrosine phosphorylated. This hypothesis is supported by our study using antibody-coated magnetic beads. Our hypothesis predicts that PECAM-1 signaling would be inhibited if PECAM-1's extracellular or intracellular anchoring was disrupted. PECAM-1 of sparsely cultured BAECs would not be engaged, and indeed, PECAM-1 phosphorylation was severely inhibited in such cells. However, sparse ECs responded to HOS as if they were in a confluent monolayer if cells were cultured on the anti–PECAM-1–coated surface. Under these conditions, PECAM-1 would be externally anchored, enabling it to signal. Internal anchoring of PECAM-1 may be modulated by treating cells with drugs that affect actin filaments. Our preliminary results indicate that ECs treated with cytochalasin D or phalloidin caused a significant decrease or increase in pY-PECAM-1 levels, respectively (Masuda et al., 1998
). These results are consistent with our hypothesis.
Our pharmacological studies indicate that PECAM-1 tyrosine phosphorylation does not depend on activities of various ion channels, PKC, or the Gq-coupled receptors and is not achieved by stimulating ECs with Ca2+
ionophores, NO analogs, or growth factors (Harada et al., 1995
; Masuda et al., 1998
). To date, no known signaling event has been found to precede PECAM-1 tyrosine phosphorylation in ECs, suggesting that PECAM-1 may initiate a mechanosignaling process. PECAM-1 is localized at interendothelial adhesion, a site proposed for mechanosignal transduction in ECs (Davis, 1993
; Kano et al., 2000
, Fujiwara et al., 2001
is our working model for PECAM-1–initiated mechanosignal transduction. We propose that mechanical force acts on PECAM-1 at the cell–cell adhesion and causes phosphorylation of PECAM-1 at two tyrosine residues (Y663 and Y686). Because PECAM-1 is not an autophosphorylating protein, involvement of some kinase(s) is postulated. However, the precise mechanism of PECAM-1 tyrosine phosphorylation is not known at this time. One possible scenario might be that mechanical force causes conformational change in PECAM-1. Without mechanical force acting on it, PECAM-1 might be in a “closed” conformation in which the tyrosine residues would not be available to kinase(s). However, when mechanical force acts on the molecule, PECAM-1's conformation might become “open” so that Tyr663 and Tyr686 would now be available to the kinase. Whether or not such a mechanical stress–induced conformational change occurs in PECAM-1 needs to be investigated. When PECAM-1 is tyrosine phosphorylated, SHP-2 binds to it. This activates SHP-2's enzymatic activity, which then activates, together with Gab1, an ERK signaling pathway.
Figure 8. A model for mechanosignal transduction by PECAM-1. The model depicts the following scenario. When an EC is under no mechanical stress, PECAM-1 is in a closed state in which Tyr663 and Tyr686 are unavailable to kinase. When mechanical force acts on PECAM-1, (more ...)
It is still possible that this signaling is not specific for PECAM-1. Force applied to one of many membrane proteins tethered to the cytoskeleton may activate this or a similar signaling cascade. We attempted to test this possibility by using antibodies against other cell surface proteins, but were unsuccessful for technical reasons. Experiments were possible with poly-l–coated beads, but they provide only a partial answer to the issue. Poly-l beads bind to any negatively charged surface molecules. Although they bind avidly, they are likely adherent to a variety of molecules including phospholipids and random surface molecules, none of which may be clustered under the beads in high enough concentrations to generate a specific signal. Although we have provided reasonable evidence implicating PECAM-1 as a mechanoresponsive molecule, we have not proven that it is the actual molecule that converts the mechanical force of shear stress into the chemical signal of protein tyrosine phosphorylation.
Several important questions remain to be investigated. First, what is the mechanism of PECAM-1 tyrosine phosphorylation? More specifically, what is the PECAM-1 kinase? Although Src family kinases are possible candidates, as they phosphorylate PECAM-1 in vitro (Osawa et al., 1997
) and in cells overexpressing it (Cao et al., 1998
; Lu et al., 1997
), Src family kinase inhibitors (herbimycin A, erbstatin, and PP1) had no effect on PECAM-1 tyrosine phosphorylation by HOS (unpublished data). The expression of PECAM-1 kinase may be limited to ECs because PECAM-1 transfected into non-ECs, including ECV304, COS7, HEK293, and L cells, failed to be phosphorylated when these cells were treated with HOS, although they formed tight monolayers just like ECs, expressing exogenous PECAM-1 at their cell junction (unpublished data). Work is now in progress to identify PECAM-1 tyrosine kinase.
Another set of questions is concerned with the molecular link between PECAM-1 and the actin cytoskeleton. β-Catenin may play a role in this as it can bind to PECAM-1. However, our preliminary results indicated that the affinity between the two proteins was not strong (Kusano et al., 1998
). The actin cytoskeleton that could anchor PECAM-1 includes the cortical actin filament network, the actin bundle associated with the adherens junction, and the apical stress fiber system. One of these actin structures could play the primary role, but it is also possible that all or a combination of them may be necessary. Based on their three-dimensional distribution, we have proposed that the apical stress fibers that run between the apical (where FSS exerts its force) and lateral (where the PECAM-1–dependent signaling is likely to take place) regions of cells may play a role in this signaling (Kano et al., 2000
). Morphological as well as biochemical characterization of the link between PECAM-1 and actin filaments is needed.
A third question is the physiological significance of PECAM-1 mechanosignal transduction. Preliminary results indicate that PECAM-1 in ECs is tyrosine phosphorylated in vivo (unpublished data). When a short coarctation was made in guinea pig aorta for 1 wk and the blood vessel was stained with antiphosphotyrosine, increased staining was noted at the cell–cell border (Kano et al., 2000
). At present, it is not known how these observations relate to the physiology and pathology of blood vessels. PECAM-1–null mice exhibit no apparent developmental abnormalities, although some minor defects are noted in adult mice (Duncan et al., 1999
; Mahooti et al., 2000
; Thompson et al., 2001
). However, this does not necessarily mean that PECAM-1 plays only a minor role in vivo. In fact, it plays a critical role in leukocyte extravasation, and more recent studies indicate its involvement in signaling (Ji et al., 2002
). The primary sequence of its cytoplasmic domain is highly conserved among mammalian species, suggesting that PECAM-1 function is of advantage for survival. ECs might have a backup system for PECAM-1's function, and this is done by expressing other proteins with the same function. The presence of functionally redundant molecules would make knockout mice appear near normal. EC responses to FSS are not usually life or death types of reactions, even though FSS affects a wide variety of EC functions. Many are transient and their long-term effects are difficult to assess. It appears that the FSS effects on ECs that have significant implications for an organism are not acute types and thus must be determined using a long time span. The life span of a mouse may not be long enough for chronic effects of PECAM-1 deletion to become obvious. Therefore, whether or not there are detrimental effects of the PECAM-1–null condition in man is still an open question.