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PECAM-1 (CD31) engagement activates a phosphoinositide-independent, nonspecific cation channel in endothelial cells ¹

Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA; and
^* Department of Biomedical Science, Cornell University School of Veterinary Medicine, Ithaca, New York, USA

³Correspondence: University of Pennsylvania School of Medicine, 838 BRBII/III, 421 Curie Blvd., Philadelphia, PA 19104, USA. E-mail: christoo{at}mail.med.upenn.edu

SPECIFIC AIMS

Previous studies have demonstrated that antibody engagement of the highly expressed adhesion molecule PECAM-1 in endothelial cells activates prolonged calcium transients mediated through plasmalemmal calcium-conducting channels. Using whole-cell patch clamp techniques in human umbilical vein endothelial cells (HUVEC) and in an endothelia-like mesothelioma-derived cell line (REN) stably transfected with wild-type (RHP) and mutant PECAM-1, three specific aims were addressed: 1) to establish the ionic and gating characteristics of the plasmalemmal calcium conducting channels that underlie PECAM-1-dependent calcium signals, 2) to further define the mode of PECAM-1 engagement necessary to elicit endothelial cell PECAM-1-dependent calcium signals, and 3) to establish the signal transduction pathways that regulate PECAM-1-dependent ionic signaling.

PRINCIPAL FINDINGS

1. PECAM-1 engagement, not cross-linking, activates ionic signaling in HUVEC and PECAM-1 transfected REN cells
Puffer pipette application of mAb 4G6 (20 µg/ml) to voltage-clamped HUVEC and RHP cells elicited a large inward current with a delay in onset of 1–3 min and prolonged time course (Fig. 1 ). The kinetics were similar to previously described PECAM-1-activated calcium transients in these cells. The mean peak current amplitude was 636 ± 33 pA in HUVEC and 666 ± 32 pA in RHP cells at a holding potential of -60 mV. Currents were evoked in 67% of HUVEC (n=12) and 76% of RHP (n=76), whereas addition of mAb 4G6 to untransfected REN cells never activated current (n=10). No currents were observed after exposure of HUVEC (n=8) or RHP (n=10) to control antibody directed against ICAM-1 or vehicle alone. Exposure of RHP cells to 4G6 Fab fragments (20 µg/ml) elicited an identical current in six of eight cells tested (75%), demonstrating that PECAM-1 engagement, rather than cross-linking or dimerization, was sufficient for current activation. Experiments showing that the current could not be reconstituted in transfected murine fibroblasts (n=7) or Xenopus oocytes (n=2) expressing PECAM-1 suggest PECAM-1 alone does not form the channel.

View larger version (45K): [in this window] [in a new window]   Figure 1. A) PECAM-1 activates a voltage-independent, nonrectifying nonselective cation channel. HUVEC and RHP cells voltage clamped at a holding potential of -60 mV in HBSS solution were puffed with 20 µg/ml mAb 4G6 for 3–5 min. Reversal potential, voltage gating, and rectification were assessed for both cell types after an instantaneous voltage ramp protocol from -60 mV to +50 mV and a 20 mV/20 mS incremental step gradient protocol. RHP cells demonstrate a Vrev = 0 with an ohmic I/V relationship derived from instantaneous voltage ramp (below current tracing) and step gradient trials (not shown) consistent with a nonselective, nonrectifying channel. Step gradient trials manifested no voltage dependence (not shown). B) HUVEC similarly demonstrated a Vrev = 0 with a linear I/V relationship during step gradient trials and instantaneous voltage ramp protocols. Step gradient trials manifested no voltage dependence. RHP cells puffed with mAb 4G6 in TRIS-HCL bath solution (symmetric anion and asymmetric impermeant cation conditions) manifested a characteristic inward current with a left shift in Vrev and decreased inward slope conductance (below current tracing). RHP cells puffed with mAb 4G6 in CsCl bath with CsAcetate pipette solutions (symmetric cation and asymmetric impermeant anion conditions) manifested an inward current with characteristic kinetics and no shift in Vrev. These results suggest that the PECAM-1-dependent current is mediated solely by a nonspecific cation channel without voltage-dependent gating or rectification.

2. Engagement of PECAM-1 in endothelial cells activates a plasmalemmal, nonspecific, voltage-independent cation channel that conducts calcium
The ion selectivity and voltage dependence of the PECAM-1 current were examined in HUVEC and RHP. Using standard solutions, the reversal potential of the evoked current, determined by voltage ramp and voltage step protocols, was 0 mV, close to the theoretical monovalent cation equilibrium potential of -1.2 mV. The current-voltage relationship of the PECAM-1 current was ohmic, with no evidence of voltage-dependent gating or rectification. Replacement of extracellular Na⁺ ions with impermeant TRIS resulted in a left shift of the current reversal potential and a decrease in the slope conductance of the inward current, consistent with a nonselective cation current (Fig. 1) . Conversely, asymmetric impermeant anion and symmetric cation solutions (CsAcetate pipette and CsCl bath) did not shift the reversal potential nor alter the slope conductance (Fig. 1) . At a holding potential of -60 mV, PECAM-1 ligations in HUVEC and RHP cells, bathed in extracellular solution containing 100 mM Ca²⁺ as the only permeant cation, activated a slowly developing Ca²⁺ current with kinetics similar to those observed in monovalent cation solutions. The average peak amplitude of the current in 100 mM Ca²⁺ was 737 ± 48 pA, and the current reversal potential was shifted to positive potentials, as predicted for Ca²⁺-permeant channels. These data indicate that PECAM-1 engagement results in the gating of voltage-independent, calcium-permeant nonselective cation (NSC) channels in HUVEC and RHP cells and suggests that these plasmalemmal NSC channels mediate the PECAM-1-dependent endothelial cell calcium transients previously observed in whole-cell fluorometry studies.

3. Homophilic engagement of PECAM-1 activates ionic signaling in endothelial cells
Homophilic (PECAM-1–PECAM-1) engagement of PECAM-1 is known to play an important role in neutrophil–endothelial interaction and neutrophil transmigration. A soluble bivalent PECAM-1/immunoglobulin (Ig) chimeric protein consisting of two PECAM-1 extracytoplasmic domains fused to the IgG hinge region, known to bind homophilically to cellular PECAM-1, was added to HUVEC, RHP, and REN cells. In voltage-clamped cells, PECAM-1/Ig chimeric protein (150 µg/ml) activated currents indistinguishable from mAb 4G6-induced currents in 54% HUVEC (n=13) and 62% RHP (n=18). Exposure of untransfected REN cells to PECAM-1/Ig chimera failed to elicit a detectable current (n=8). Thus, homophilic engagement of PECAM-1 on HUVEC and RHP triggers ionic signaling.

4. Activation of the PECAM-1-dependent cation channel requires src kinase activity and intact cytoplasmic and transmembrane domains
Voltage-clamped RHP cells pretreated with genistein and then treated with mAb 4G6 failed to activate a current, confirming a regulatory role for tyrosine kinase activity in the PECAM-1-dependent current. Since PECAM-1 is known to act as a substrate for src family tyrosine kinases, we sought to determine whether the src kinase might play a physiologic role in the PECAM-1-dependent response. In voltage-clamped RHP cells dialyzed with Sc-18 (an src neutralizing rabbit polyclonal antibody) and treated with mAb 4G6, no current was elicited (n=6). However, in RHP cells dialyzed with an irrelevant (anti-thymidine kinase) rabbit polyclonal antibody, current identical to nondialyzed cells was elicited in five of six cells tested. To examine the role of the PECAM-1 intracellular domain in this response, REN cells stably transfected with a mutant PECAM-1 construct consisting of the PECAM-1 extracellular domain fused to the ICAM-1 transmembrane and cytoplasmic domains (PITC) were studied. In all trials, PITC cells treated with mAb 4G6 failed to manifest current (n=13). These findings suggest that src kinase activity, possibly directed toward PECAM-1 intracellular tyrosine-containing motifs as well as other potential intermediates, is necessary for current activation.

5. Activation of the PECAM-1-dependent channel is independent of IP3-mediated store release, PI turnover, and intracellular calcium elevation and manifests trivalent block
The PECAM-1-dependent nonspecific cation channel and the current it mediates are kinetically similar to the nonspecific depletion-activated (DAC) or store-operated (SOC) plasmalemmal cation channels that mediate capacitative calcium entry in endothelial cells after phosphoinositide-mediated intracellular calcium store release. To determine the relationship of the PECAM-1-dependent cation current to such IP₃-mediated intracellular calcium store release events, RHP cells were dialyzed with 5 mg/ml heparin, an IP₃ receptor antagonist, and currents were measured after engagement with mAb 4G6 (n=4) or thrombin (1.5U/ml) (n=5). Ligation with mAB 4G6 evoked typical NSC currents in heparin-dialyzed cells, suggesting that the PECAM-1 activated NSC is neither IP₃ gated nor dependent on IP₃-mediated intracellular calcium store release. Conversely, in control experiments, thrombin (1.5 U/ml) activated a large inward store-operated (calcium release-activated) current (n=9) very similar to the PECAM-1-activated current in time course, reversal potential (Vrev=0), mean peak current amplitude (730±47pA), and current-voltage relationships, but was completely blocked by dialysis with heparin. The role of phosphoinositide (PI) metabolism leading to other potential intermediates such as IP₄ in the PECAM-1 response was further evaluated with PI turnover assays using tritiated myoinositol in HUVEC and RHP cells. Engagement with mAb 4G6 failed to elicit significant stimulation of phosphoinositide formation in either cell type, whereas addition of thrombin yielded a robust increase in phosphoinositide turnover, further excluding a role for phosphoinositide mediated signaling in the PECAM-1-mediated current.

The role of Ca²⁺-activated nonselective ion channels in the PECAM-1-dependent current was further evaluated in voltage-clamped RHP cells that were also calcium clamped by dialysis with 10 mM EGTA and 0 mM Ca²⁺ through the patch pipette (resulting in a calculated [Ca²⁺]_i of less than 1 nM). Currents similar to those in nondialyzed cells were observed in Ca²⁺-clamped RHP cells when puffed with 20 µg/ml mAb 4G6 for 3–5 min. No spontaneous current activation was observed. Thus, despite the similarity in current amplitude and kinetics between PECAM-1 and store depletion-induced ionic signaling (as seen with thrombin stimulation), PECAM-1 currents are independent of IP₃ receptor-mediated store release, calcium gating, or phosphoinositide metabolism.

We sought to characterize the response of the PECAM-1 NSC channel to the trivalent cation La⁺³, an inhibitor of DAC/SOC plasmalemmal NSC channels. RHP cells activated with mAb 4G6 (n=4) or thrombin (1.5 U/ml) (n=6) were puffed with solution containing 50 µM LaCl₃ and currents were recorded. Both currents were rapidly inhibited by La⁺³, though inhibition of the PECAM-1 response was not complete at La⁺³ concentrations sufficient to completely block thrombin-mediated inward current. This suggests a more complex current response or different subtype of La⁺³-sensitive channel may underlie the PECAM-1 response.

CONCLUSIONS AND SIGNIFICANCE

The purpose of this study was to define the characteristics of endothelial cell (EC) Ca²⁺-permeant channels and signal transduction events that underlie Ca²⁺ transients associated with ligation of PECAM-1. We have established the presence of a large, prolonged, nonspecific cation current that is activated in a cell type-specific manner by PECAM-1 engagement with anti-PECAM-1 mAbs and by homophilic engagement with soluble PECAM-1. This response requires the cytoplasmic and transmembrane domains and is blocked by inhibitors of src kinase function. Despite the remarkable similarity of the PECAM-1-activated current to endothelial DAC/SOC currents, the PECAM-1-dependent response is substantially different as it bypasses the initial intracellular calcium release phase and activates through an IP₃/Ca⁺²-independent signaling pathway. The direct activation by an Ig superfamily protein of a plasmalemmal NSC channel similar in kinetic characteristics to the DAC/SOC family of NSC channels has not been previously reported and represents a potentially important alternate calcium-signaling pathway in endothelial cells.

Although signal transduction and calcium transients elicited by Ig superfamily members such as ICAM-1 and ICAM-3 appear to require cross-linking, some integrin-mediated calcium signaling events apparently do not. Similarly, although PECAM-1 is predominantly localized to the cell–cell border, it is also present on the luminal EC surface and has been shown to exist in equilibrium between monomeric and dimeric states. In agreement with prior whole-cell fluorometry data showing activation of PECAM-1-mediated calcium transients in confluent HUVEC and RHP with Fab fragments (with no augmentation by cross-linking), we found efficient activation of the NSC current with Fab fragments in isolated HUVEC and RHP cells. This suggests that cell confluence, homophilic cell–cell border interactions, and PECAM-1 dimerization do not play a role in this signal.

PECAM-1 itself does not appear to form a functional channel because expression of PECAM-1 in murine fibroblasts and Xenopus oocytes is not sufficient to elicit PECAM-1 regulated current. Rather, it is likely that that PECAM-1 acts as a regulatory trigger in (at least) a 2-component system in which tyrosine phosphorylation regulates either the channel or as yet unidentified intermediate(s) (Fig 2 ). This paradigm is similar to that mediating ß₁ integrin-regulated calcium signals involving voltage-independent, calcium permeable channels that interact with membrane-associated proteins such as integrin-associated protein and calreticulin. Similarly, endothelial cell homologues of the Drosophila transient receptor potential (Trp) retinal proteins, as well as Trp-like (Trpl) protein, appear to function as components in multimeric nonspecific cation channels mediating capacitative current responses after agonist-activated IP₃-dependent and thapsigargin-induced store release. The actual relationship of any of these systems to the PECAM-1-activated NSC current response remains unknown and is an active area of investigation.

View larger version (27K): [in this window] [in a new window]   Figure 2. PECAM-1 activates a nonselective cation channel through an alternate signaling pathway that is independent of [Ca+2]i release and PI turnover, but is tyrosine kinase-dependent. After PECAM-1 engagement, at least one central downstream component (possibly the PECAM-1 cytoplasmic domain) undergoes tyrosine phosphorylation by src kinase. Interaction through the PECAM-1 intracellular domain with the channel (model 1) or with one or more intermediaries (model 2) either directly activates the NSC or inhibits a channel suppressor, thus indirectly activating the channel. PECAM-1 activation of the NSC requires a minimal two component system or three component system if SHP-2 participates, either as an adaptor or phosphatase, in this process.

The recent description of a unique prolonged calcium response activated specifically by engagement of PECAM-1 adds a new dimension to the role of this cell adhesion protein as a regulator of endothelial cell ion channel activity. We have expanded on these initial findings and provided the first electrophysiologic evidence of an endothelial calcium-permeant NSC channel directly activated by homophilic engagement of an Ig superfamily cell adhesion molecule. Whether PECAM-1 directly activates the NSC channel (Fig. 2 , model 1), functions in a multicomponent system (Fig. 2 , model 2), or instead acts to sequester a constitutively active channel inhibitor (thus indirectly activating the PECAM-1-dependent current) is currently unknown. Although beyond the scope of this paper, defining the critical regulatory PECAM-1 cytoplasmic motifs, the molecular identity of the PECAM-1 activated channel, and establishing the role of potential adaptor and phosphotyrosine regulating proteins, such as SHP-2, will be the next steps in better understanding this new signaling pathway.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0467fje ; to cite this article, use FASEB J. (March 20, 2001) 10.1096/fj.00-0467fje

² Both authors contributed equally to this manuscript.

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