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The Journal of Immunology, 2002, 168: 2091-2095.
Copyright © 2002 by The American Association of Immunologists

Cutting Edge

Cutting Edge: Internalization of Transduced E-Selectin by Cultured Human Endothelial Cells: Comparison of Dermal Microvascular and Umbilical Vein Cells and Identification of a Phosphoserine-Type Di-leucine Motif 1

Martin S. Kluger2,*,{dagger}, Stephen L. Shiao*, Alfred L. M. Bothwell*,§ and Jordan S. Pober*,{dagger},{ddagger},§

* Interdepartmental Program in Vascular Biology and Transplantation, Departments of {dagger} Dermatology and {ddagger} Pathology, and § Section of Immunology, Yale University School of Medicine, New Haven, CT 06536


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Persistent E-selectin expression on human dermal microvascular endothelial cells (HDMEC), believed to mediate skin-specific T cell homing, results from a slow rate of surface protein internalization after cytokine induction. Following transduction of unactivated HDMEC with E-selectin cDNA, the rate of internalization was largely independent of increasing levels of surface protein expression, leading to prolonged t1/2 values of over 4 h, comparable to that observed following cytokine induction. In HUVEC, the rate of internalization increased with surface expression level, leading to an essentially constant t1/2 of under 2 h. Thus, the internalization process rather than cytokine responsiveness or E-selectin structure underlies the difference in endothelial cell behavior. Mutational analysis of the cytoplasmic region demonstrated a role for a di-leucine-type motif involving I588 and L589 but not for a putative tyrosine-type motif. Control of E-selectin surface expression appears to be phosphoserine dependent, since alanine but not aspartic acid substitution for S581 slows E-selectin internalization.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
E-selectin (CD62E) is an endothelial cell (EC)3-specific type I transmembrane surface protein (1) that mediates rolling of blood leukocytes in microvessels at sites of inflammation (2). Following injection of TNF, onset of E-selectin expression is usually transient, peaking at 4–6 h, coincident with neutrophil recruitment (3). When expression persists, as may occur on dermal venular EC (4), E-selectin is displayed concomitantly with other molecules (e.g., chemokines) that mediate T cell recruitment, thereby favoring extravasation of those T cells that express E-selectin ligands. In support of this idea, memory T cells expressing an E-selectin ligand, designated cutaneous lymphocyte Ag-1 (CLA-1), are enriched within inflamed dermis compared with peripheral blood. It has been proposed that CLA-1+ memory T cells use persistent E-selectin on dermal EC as an address signal for skin homing (4).

Most of our knowledge about E-selectin regulation has come from experiments using cultured human EC, which behave similarly to EC in situ. For example, in HUVEC cultures, E-selectin expression is basally absent, but is rapidly (2 h) induced by TNF (5). Surface expression is transient, falling to 10% of peak levels by 24 h, due to both a cessation in synthesis and to rapid internalization of E-selectin protein (6, 7). Shedding does not significantly contribute to the loss of E-selectin from the cell surface (8). Human dermal microvascular EC (HDMEC) show sustained E-selectin expression compared with HUVEC, but this property seems to be a general feature of cultured EC derived from microvascular sources rather than a skin-specific characteristic (9). Our previous work also established that slower E- selectin endocytosis, rather than more persistent synthesis, underlies sustained E-selectin surface expression in cultured HDMEC (9). Slower E-selectin clearance by HDMEC could not be explained by nonspecific lethargy of plasma membrane internalization, since receptor-mediated endocytosis of low-density lipoprotein complexed with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine percholate (DiI-LDL) is more rapid in HDMEC than HUVEC (9).

Two different, peptide-length signal motifs within the cytoplasmic domain of transmembrane proteins have been found to facilitate endocytosis of most cell surface receptors through associations with adaptor proteins on coated pits (10). The first of these consists of two adjacent leucine residues (or a leucine plus a second hydrophobic residue) plus a nearby, typically upstream negative residue, either a constitutively charged aspartic acid or else a conditionally phosphorylated serine (10, 11). In E-selectin, the two final residues I588L589 are conserved across species (i.e., in human, bovine, pig, rabbit, and rat but not mouse (12)) and are just downstream of a potentially phosphorylated serine residue, S581 (13). The second signal motif is YXX{phi} (where Y is a critical tyrosine residue, X is any amino acid, and {phi} is an amino acid with a bulky side chain). The E-selectin cytoplasmic region from Y582 through P585 fulfills this criteria and is also conserved across multiple species (12, 14).

Efforts examining E-selectin endocytosis utilizing transfected Chinese hamster ovary (CHO) (12) or COS (15) cells may be misleading because endocytosis of the same surface protein may vary among different cell types (16) and, in the case of E-selectin, internalization differs significantly even among human EC types (9). Adenoviral transduction typically leads to very high levels of expression likely to saturate normal cell trafficking pathways (17). We express E-selectin protein on human EC within the range of physiological expression by retroviral transduction to demonstrate differences in the density dependence of E-selectin internalization in HDMEC vs HUVEC and to identify a cytoplasmic phosphoserine-type di-leucine motif regulating E-selectin internalization.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Abs and reagents

Mouse mAbs used in FACS analysis were H4/18, anti-E-selectin (5), K16/16, nonbinding control (5) W6/32 anti-MHC class I (1), and E1/6, anti-VCAM-1 (gift from M. P. Bevilacqua, Pharmaceut, Boulder, CO); mAbs used for immunoblotting were M2 anti-Flag (Kodak, New Haven, CT) and anti-human {beta}-actin (Sigma-Aldrich, St. Louis, MO). Fibronectin was isolated from human plasma and TNF obtained from R&D Systems (Minneapolis, MN). Unless otherwise specified, all other reagents were from Sigma-Aldrich.

DNA constructs

The translated region of human E-selectin cDNA (6) was modified by a Flag epitope tag after E-selectin extracellular residue M10 (amino acid numbering as done previously (12) omitting the 21-aa signal peptide). Cytoplasmic tail mutations were introduced by PCR, confirmed for each cloned E-selectin insert by nucleotide sequencing, and recombined into the retroviral vector pLZRS-BMNZ (18). Retroviral vectors were transfected (Lipofectamine-Plus; Invitrogen, San Diego, CA) into the Phoenix-Ampho packaging cell line (gift from Dr. G. P. Nolan, Stanford University, Palo Alto, CA) and puromycin-resistant cells developed as a source of retrovirus.

EC cultures and retroviral transduction

HDMEC isolated from reduction mammoplasties obtained with Institutional Review Board approval (9) were purified by anti-CD-31-biotin mini-MACS (Miltenyi Biotec, Auburn, CA). HDMEC cultured in EGM2-MV growth medium (Clonetics, San Diego, CA) were >99% positive for von Willebrand factor and >95% positive for E-selectin expression following TNF treatment. HUVEC isolated from umbilical cords (19) with Institutional Review Board approval were pooled and cultured as previously described (9). In early experiments, HUVEC were maintained in HDMEC medium, but later in their own medium since medium choice did not affect their E-selectin internalization rate. After drug-free transduction of EC was performed by three to six serial incubations with retrovirus (20), EC expression of transduced genes was stable over multiple passages. The intensity of transgene expression correlated with the number of viral transductions, permitting development of homogenous EC lines expressing surface E-selectin over a 10-fold range.

Immunoassays

Immunoblot analysis was performed as previously described (21). E-selectin was not significantly shed from the surface of HUVEC transfectants as measured by a sandwich ELISA (R&D Systems; data not shown). For FACS analysis of E-selectin surface clearance, replicate cultures of EC transductants (passages four to six) were incubated in the presence of 10 µg/ml cycloheximide (CHX). Cells treated in this manner were viable by trypan blue exclusion assay and were able to proliferate following removal of CHX (data not shown). Immunostaining was as previously described (9) using the F(ab')2 of goat anti-mouse AlexaFluor 488 secondary reagent (Molecular Probes, Eugene, OR). In FACS analysis, K16/16 nonbinding control Ab was used to correct mean fluorescence intensity (MFI) values. Half-life data are presented ±SEM, and significance was determined with a two-tailed Student’s t test assuming equal variances using Microsoft Excel 98 (Redmond, WA).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
E-selectin internalization by transduced HDMEC and HUVEC

Slower internalization rates of E-selectin protein expressed on TNF-treated HDMEC vs HUVEC we had observed previously (9) could arise from differences in the structure of endogenous E-selectin protein expressed by these two cell types, from differences in the TNF responsiveness, or from differences in the process of internalization. To examine these possibilities, we generated stable HDMEC and HUVEC transductants from the same E-selectin cDNA. Without drug selection, our retroviral transduction protocol produced stable, fairly homogenous levels of E-selectin expression on both EC types. Our E-selectin cDNA constructs bore an extracellular Flag tag, situated far from the intracellular region mediating internalization (Table IGo). Wild-type (WT) E-selectin surface expression was detected on transduced but not on mock-transduced HDMEC and HUVEC by indirect immunofluorescence with anti-E-selectin mAb (Fig. 1Go). (We also detected transduced E-selectin surface protein by specific Ab recognition of the Flag epitope, but this recognition is trypsin sensitive.) Transduced EC types were negative for VCAM-1 and positive for basal MHC class I expression (data not shown). Cumulatively these observations suggest that transduction resulted in surface expression of Flag-tagged E-selectin without causing EC activation.


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  		Table I. E-selectin construct cytoplasmic domains and surface protein t1/2 in HUVEC1

 


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  		FIGURE 1. FACS analysis of E-selectin surface expression. EC transduced with either Flag-tagged WT E-selectin (top) or mock-transduced (bottom) were immunostained with H4/18 anti-E-selectin (filled) or negative control K16/16 (open). Corrected MFI values measured for H4/18 following WT transduction were 211 (HDMEC) and 271 (HUVEC). Expression patterns are typical of the multiple transductants generated.

 
Since internalization accounts for virtually all clearance of E-selectin from the cultured EC surface (Ref. 8 and data not shown), we monitored the internalization rate of E-selectin by FACS analysis of surface expression using timed 37°C incubations in the presence of the protein synthesis inhibitor CHX. Transduced HDMEC were more than twice as slow as HUVEC to remove E-selectin protein from their surface (Fig. 2GoA). In two comparisons, using different preparations of transduced EC, the measured t1/2 of E-selectin for HDMEC and HUVEC were, respectively, 4.0 vs 1.8 h and 4.0 vs 1.6 h. These values are strikingly similar to the average half-lives of 4.3 and 1.6 h observed previously for TNF-treated HDMEC and HUVEC, respectively (9). As expected, there was no significant clearance of MHC class I on either EC type assayed (Fig. 2GoB). These new data show that E-selectin internalization is slower on HDMEC than on HUVEC in the absence of TNF treatment, ruling out the possible explanation that this difference arises from a differential response to TNF. Since both EC types were transduced with the same cDNA, the difference in internalization cannot arise from structural differences due to alternative splicing. By immunoblot, the size of the transduced E-selectin proteins is indistinguishable between HDMEC and HUVEC (Fig. 2GoC), suggesting that differences in E-selectin structure from posttranslational processing are also unlikely to account for differences in E-selectin clearance. Thus, intrinsic differences in the E-selectin internalization pathway between HDMEC and HUVEC is the likeliest explanation for differences in duration of surface expression.



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  		FIGURE 2. Slower clearance of surface E-selectin by HDMEC than HUVEC. A and B, EC lines stably expressing E-selectin were subjected to 2.5 h of timed 37°C incubations in the presence of CHX. The level of protein expression on the EC surface (corrected MFI logarithmic y-axis) was determined by FACS analysis for each incubation point (x-axis, hours) following immunofluorescence staining with either mAb H4/18 (A) or W6/32 (B). The t1/2 of surface E-selectin protein on HDMEC (4.1 h, y = 383.42e-0.1748x, r2 = 0.9156) was more than twice that of HUVEC (1.6 h, y = 272.03e-0.4467x, r2 = 0.9858). Representative of two independent experiments. C, Immunoblot analysis of Flag-E-selectin protein. Anti-Flag Ab M2 immunostaining (arrow) showed bands of equal mobility for HDMEC or HUVEC WT E-selectin, yet a shift in mobility was observed in the {Delta}25C truncation mutant. Anti-{beta}-actin staining (arrowhead) produced bands of equal mobility and intensity.

 
The creation of stable transductants allowed us to examine whether the rate of internalization depends on the density of cell surface expression. Specifically, we developed a series of HUVEC and HDMEC transductants with stable levels of WT E-selectin surface expression varying across an ~10-fold range. In HUVEC, the rate of internalization of E-selectin molecules was highly dependent on the level of E-selectin expression and rose proportionally with expression level (fitting a first-order equation y = 0.2822x - 1.2729, r2 = 0.9746; three experiments pooled). However, the t1/2 of E-selectin surface protein on HUVEC was constant (1.9 ± 0.5 h), i.e., independent of surface density (Fig. 3GoA). The behavior of HDMEC was remarkably different. Specifically, the rate of E-selectin clearance was constant, i.e., independent of expression level, except at the lowest measured levels of E-selectin expression (from two experiments). Consequently, the t1/2 of E-selectin increased as the level of expression increased, reaching 4.6 h at the highest levels of expression (Fig. 3GoB).



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  		FIGURE 3. Effect of E-selectin surface expression level on the rate of E-selectin clearance. Two series of EC transductants, HUVEC (A) and HDMEC (B), were developed with graduated levels of constitutive WT E-selectin surface expression (horizontal x-axis, units of corrected MFI). Half-lives (vertical y-axis, hours) were determined for transduced ({diamondsuit} and {blacksquare}) and TNF-treated ({diamond} and {square}) EC types. Half-lives for TNF-induced E-selectin approximated that of the highest expressing transductants.

 
In light of the observation that the E-selectin t1/2 in HDMEC depends upon expression level, we re-examined the behavior of TNF-induced E-selectin. EC treated with saturating amounts of TNF (100 U/ml) expressed higher levels of (endogenous) E-selectin than transduced EC. After maximal TNF treatment, the half-lives of E-selectin, 1.8 h for HUVEC and 4.7 h for HDMEC, were strikingly similar to that found for each of the maximally transduced cultures (Fig. 3Go).

Structural features of E-selectin contributing to internalization

We used our retroviral transduction approach to identify structural features important for internalization of the E-selectin protein. We performed this analysis exclusively in HUVEC for two reasons. First, the independence of t1/2 from surface concentrations in HUVEC allowed simple comparisons among multiple constructs, not all of which were expressed at identical levels. Second, the more rapid internalization rate in HUVEC allowed us to keep the span of CHX exposure to a duration that was not overtly harmful to these cells, even with mutants that are internalized more slowly than WT. Immunoblotting confirmed that all of the E-selectin constructs are expressed in HUVEC (Fig. 4GoA). Specifically, anti-Flag mAb M2 produced a single intense band in each lane at the size expected for E-selectin (110–116 kDa), but not in the lane corresponding to mock transduction with the pLZRS-negative control.



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  		FIGURE 4. A, Immunoblot of HUVEC transductants. After transduction, E-selectin protein expression was detected by anti-Flag Ab M2(arrow, 110–116 kDa), but not after mock transduction with the empty pLZRS vector (negative control). Loading control, anti-human {beta}-actin (arrowhead). Immunoblot analysis on constructs not shown yielded similar results. B, Control of E-selectin internalization by cytoplasmic residues S581 and I588L589. Over a 2.5-h time course of CHX incubation, the surface t1/2 of construct S/A+IL/AA ({blacktriangleup}) was slow (6.6 h, y = 145e-0.1046x, r2 = 0.74), like construct {Delta}25C ({blacksquare}). In contrast, WT E-selectin ({diamondsuit}) cleared rapidly (t1/2 = 1.9 h, y = 128.36e-0.3733x, r2 = 0.97).

 
We first examined the effect of deleting 25 cytoplasmic aa residues (construct {Delta}25C). This modification was sufficient to prevent E-selectin clearance over a 2.5-h CHX treatment time course in three independent experiments (Table IGo), showing that one or more amino acid motifs permitting efficient clearance of E-selectin from the EC surface likely reside within the cytoplasmic tail domain. We next examined the possible contributions of two distinct motifs within the cytoplasmic domain, initially considering whether the Y582 to P585 region of the E-selectin cytoplasmic domain may mediate surface protein internalization as it is homologous to functionally active tyrosine internalization motifs (14). However, the t1/2 of a Y582A/P585A mutant (1.5 and 1.6 h) was no slower than that of WT E-selectin (1.5 and 1.8 h) in two independent experiments. This result suggests that these residues do not constitute a functional internalization motif for E-selectin, confirming a report using transfected CHO cells (12). Although recognition of tyrosine-based endocytotic signals by adapter proteins requires a nonphosphorylated tyrosine (22), tyrosine phosphorylation is unlikely to explain our results since it does not occur on E-selectin (13). Recently, Owen and Evans (22) showed by x-ray crystallography that formation of a functional tyrosine internalization motif requires an extended {beta} strand conformation for interaction with the µ2 unit of the AP-2 adaptor protein. Our data would suggest that E-selectin fails to adapt the required {beta} strand conformation.

We finally examined the putative di-leucine motif (I588L589) and the nearby serine (S581) for a role in internalization (Table IGo). The t1/2 of IL588,589AA (3.0 ± 0.4 h) was significantly (p < 0.05) greater than that that of WT E-selectin (1.9 ± 0.1 h) in four independent experiments. The t1/2 of S581A (3.8 ± 0.3 h) was also significantly (p < 0.01) greater than that of WT E-selectin (2.4 ± 0.2 h) in three independent experiments. Consequently, we next made an E-selectin mutant construct (S/A+IL/AA, Table IGo) with alanine mutations at both of these sites. Clearance of the S/A+IL/AA surface protein (t1/2 = 5.8 ± 0.5 h) was roughly twice as slow as either of the single site mutation E-selectin constructs (p < 0.05 and p < 0.01 for comparisons of S/A+IL/AA with IL588,589AA and with S581A, respectively; data from the same two series of experiments described above) and approached that of {Delta}25C (Fig. 4GoB). We conclude that rapid internalization of E-selectin surface protein involves a di-leucine motif comprised of three critical cytoplasmic residues, S581, I588, and L589. Our identification of a serine-type di-leucine motif is discrepant with an earlier report in which clearance of E-selectin on transiently transfected CHO cells did not appear to involve I588and L589 (12). This discrepancy may reflect a difference in E-selectin trafficking that is cell-type specific.

Since in other cell systems phosphorylation of a serine upstream of a di-leucine motif controls internalization (23), the finding of a serine-type di-leucine motif raised the possibility of endothelial regulation of the internalization rate. Because leukocyte binding to E-selectin can cause dephosphorylation of cytosolic serines (13), this would provide a means for leukocytes to sustain E-selectin expression on EC. To test the hypothesis that phosphorylation of E-selectin regulates internalization, we mutated S581 to a constitutively negative aspartic acid (construct S581D, Table IGo). By immunoblot, S581D mutant E-selectin protein was expressed comparably to WT E-selectin, yet FACS measurements showed only weak (but clearly shifted above negative control) S581D surface expression on the same transduced population (data not shown). The level of S581D surface expression we have been able to achieve in HUVEC, although too low to permit direct measurement of internalization, indirectly suggests a rapid rate of internalization, because (after similar degrees of transduction) among all other constructs their levels of steady-state surface expression were well correlated with the t1/2 value for clearance of E-selectin surface protein. Furthermore, combining the aspartic acid mutation at S581 with alanine mutation at I588 and L589 (S/D+IL/AA, Table IGo) sufficed to easily obtain high levels of constitutive surface expression after the usual number of retroviral incubations and demonstrated significantly faster internalization (t1/2 = 1.8 h) than did the triple alanine substitution (S/A+IL/AA; t1/2 = 4.6 h).

In summary, our studies provide two new insights into the regulation of E-selectin persistence on the cell surface. First, we demonstrate that following transduction, the internalization pathway for E-selectin in HDMEC but not HUVEC appears to be saturated at low levels of E-selectin expression. This difference occurs independently of E-selectin structural variations or differential responses to TNF activation and accounts for the slower rate of internalization seen following cytokine induction. Second, we have identified a role for a phosphoserine type of di-leucine motif in the internalization of E-selectin by HUVEC. Our observations support the hypothesis that leukocytes regulate E-selectin internalization by modulating serine phosphorylation at S581, providing a mechanism for sustaining the level of functional E-selectin on the endothelial surface.

Note added in proof. After submission of this manuscript, Hu et al. (24) showed that tyrosine phosphorylation may occur on E-selectin following cross-linking of H4/18. However, this observation does not affect our interpretation since we first immunostained with H4/18 after internalization had already occurred.


   Acknowledgments
 
We sincerely thank Louise Camera-Benson, Lisa Gras, Gwendolyn Davis, and Courtney Doyle for their assistance and gratefully acknowledge Dr. Mary Lou Gaeta and Dr. David Golan for their helpful discussions.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Grant RO3-AR45766 (to M.S.K.) and R37-HL36003 (to J.S.P.). Back

2 Address correspondence and reprint requests to Dr. Martin S. Kluger, Yale University School of Medicine, 295 Congress Avenue, Room 454, New Haven, CT 06536-0812. E-mail address: martin.kluger{at}yale.edu Back

3 Abbreviations used in this paper: EC, endothelial cell; CLA-1, cutaneous lymphocyte Ag-1; HDMEC, human dermal microvascular endothelial cell; CHX, cycloheximide; MFI, mean fluorescence intensity; WT, wild type; CHO, Chinese hamster ovary. Back

Received for publication November 27, 2001. Accepted for publication January 10, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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