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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 13, 2005 as doi:10.1096/fj.05-4574fje. |
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,¶
,||,¶,#,1
,1
Departments of
* Chemical and Biomolecular Engineering and
Biomedical Engineering, The Johns Hopkins University, Baltimore, Maryland, USA;
Department of Pharmacology, The Johns Hopkins School of Medicine, Baltimore, Maryland, USA; Departments of
Dermatology and
|| Medicine, Brigham and Women’s Hospital;
¶ Harvard Skin Disease Research Center, Harvard Medical School, Boston, Massachusetts, USA; and
# Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Masachusetts, USA
1 Correspondence: K. K.: E-mail: kkonsta1{at}jhu.edu; R. S.: E-mail: rsackstein{at}rics.bwh.harvard.edu
SPECIFIC AIMS
Accumulating evidence suggests a link between selectin binding activity and metastatic spread in vivo. Therefore, from a molecular perspective, elucidating the specific selectin counter-receptor(s) on tumor cells and characterizing their biochemical nature are critical steps toward understanding and preventing metastasis.
PRINCIPAL FINDINGS
1. CD44v isoforms on LS174T colon carcinoma cells possess P-, L-, and E-selectin ligand activity as evidenced by blot rolling assays
Previous studies reveal that LS174T colon carcinoma cells possess novel glycoprotein ligands for E-, P-, and L-selectin, distinct from PSGL-1. Recently, we identified the presence of sialofucosylated CD44v isoforms on LS174T colon carcinoma cells and documented their participation in E-selectin-mediated adhesion under flow. Accordingly, we sought to examine the ability of these isoforms to support P- and L-selectin-mediated adhesion. We first employed the blot rolling assay, a technique that functionally identifies adhesion molecules under physiological shear conditions, to evaluate the selectin binding capabilities of SDS-PAGE resolved LS174T membrane lysates. P-selectin transfected CHO cells and human peripheral blood lymphocytes were perfused over HECA-452 immunostained blots to evaluate the respective P- and L-selectin binding properties over separated membrane protein bands. Both CHO-P and human lymphocytes interacted and adhered to the Western blot, indicating the retention of P- and L-selectin ligand activity in the membrane lysate. While the HECA-452 stained blot indicated the presence of prominent sialofucosylated antigens distributed at 
150 and 225 kDa, CHO-P cells and lymphocytes reproducibly rolled predominantly over the 150 kDa region. Controls, consisting of CHO-P cells or lymphocytes suspended in flow medium containing 5 mM EDTA, or mock transfectants at physiological Ca2+/Mg2+ levels, had negligible adhesion to any regions of the blot confirming specificity for P- and L-selectin ligand activity.
To test whether CD44v isoforms from LS174T cells function as P- and L-selectin ligands, we immunoprecipitated CD44 from LS174T membrane lysates using an anti-CD44 mAb (2C5), and separated the variant from the standard isoforms by SDS-PAGE. Western blot analysis with the anti-CD44 mAb 2C5 revealed the presence of a relatively small level of standard CD44 (CD44 s) at 100 kDa, while a more intense CD44 signal observed at 150 kDa corresponded to variant isoforms of CD44. A second lane, stained with HECA-452, identified the presence of sialofucosylated epitopes solely on the variant isoforms of CD44. Blot rolling analysis confirmed that human lymphocytes adhered only to the higher molecular weight regions on the blot at levels similar to those found to support adhesion using whole membrane lysate. CHO-P cells also adhered appreciably to this same region, indicating that specific glycoforms of CD44v function as ligands for P-selectin.
Clearance of CD44 from membrane lysates of LS174T cells by repeated immunoprecipitation dramatically decreased P-selectin ligand activity and abrogated L-selectin-dependent binding as revealed by blot rolling analysis. The residual CHO-P interactions were also more transient in nature and consisted of mostly fast rolling cells as opposed to stationary cells that were observed for the whole lysate. These experiments suggest that CD44v isoforms serve as the major P- and L-selectin ligands on LS174T colon carcinoma cells.
2. LS174T CD44 compared with HL-60 CD44s exhibits a markedly higher affinity for selectins
We next employed a cell-free flow-based adhesion assay to evaluate the adhesion capabilities of microspheres coated with CD44 immunoprecipitated from LS174T cells vs. the CD44s immunopurified from HL-60 cells to selectin substrates. This assay allows direct comparison of adhesion properties using equivalent levels of bound adhesion molecules, whose expression levels on live cells may vary dramatically with time or cell type. Control experiments included BSA-coated microspheres, or the addition of 5 mM EDTA to the perfusion buffer (Fig. 1
A). Specificity was assessed through the use of nonspecific IgG-coated polystyrene dishes, and by using selectin-functionalized dishes preincubated with the respective function-blocking anti-selectin antibodies (anti-L-selectin DREG-56, anit-P-selectin AK-4, anti-E-selectin 68-5H11) (Fig. 1A
). In accord with experiments using live LS174T cells, the efficiency of LS174T CD44-coated microsphere recruitment to selectins varied, with E-selectin supporting the greatest adhesion and L-selectin the least (Fig. 1A
). Along these lines, LS174T CD44-bearing beads exhibited slow, intermediate and fast rolling on E-, P-, and L-selectin substrates, respectively (Fig. 1B
).
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While CD44s from HL-60 cells is capable of efficiently tethering to E-selectin under shear, its adhesion to P- and L-selectin is drastically lower than that of LS174T CD44 (Fig. 1A
). In addition, the interactions between HL-60 CD44s and P- or L-selectin consisted of mostly transient tethers rather than the smooth-rolling behavior exhibited by LS174T CD44-coated microspheres. It is noteworthy that despite having equal levels of adsorbed CD44, the site density of HECA-452-reactive epitopes was nearly 8-fold higher for LS174T CD44- vs. HL-60 CD44s-coated microspheres. Cumulatively, these data suggest that the lower glycosylation levels of HL-60 CD44s may be in part responsible for the low-affinity interactions between CD44s and P- or L-selectin. The incorporation of CD44 variant regions may provide new sites for glycosylation, resulting in unique LS174T CD44 adhesion characteristics.
3. The selectin-binding determinants on LS174T CD44 isoforms are sialofucosylated glycans presented on O-linked carbohydrates structures
Using highly selective enzymes which cleave specific carbohydrate moieties, we modified the LS174T CD44 adsorbed onto microspheres to characterize the structural linkage bearing P- and L-selectin binding determinants. In accord with previous findings using live cells, neuraminidase treatment of LS174T CD44-coated microspheres, which completely removed the sialic acid residue from sLex, nearly abrogated microsphere adhesion to P- and L-selectin, although adhesion to E-selectin was 60% of control (Fig. 2
A). Treatment with OSGE enzymatically cleaved CD44 from the microspheres, suggesting the presence of a high level of O-linked glycans on the LS174T CD44. This enzymatic intervention reduced HECA-452 reactivity down to basal levels, and abrogated microsphere binding to all selectin substrates under shear (Fig. 2A
). On the other hand, microspheres prepared with LS174T CD44 immunoprecipitated from N-glycosidase F-treated membrane lysate had only a minimal difference with respect to HECA-452 recognition, and interacted with selectin substrates at levels similar to the control (Fig. 2A
). The extent of enzymatic cleavage by N-glycosidase F in the cell lysate was monitored using SDS-PAGE and noting a mobility shift of treated CD44 on Western blots compared with untreated CD44.
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Alternatively, microspheres were generated using CD44 immunoprecipitated from LS174T cells that were cultured for 48 h in medium containing 1 mM DMJ, to disrupt N-linked processing, or 2 mM benzyl-GalNAc, to inhibit O-linked glycosylation. The impact of the DMJ and benzyl-GalNAc treatments was apparent by the reduction in molecular weight of CD44, as evidenced by its faster migration in the gel compared with the control. The efficacy of DMJ treatment was confirmed by assessing the sensitivity of CD44 from DMJ-treated cells to the enzyme endoglycosidase H, which only cleaves high mannose or hybrid N-linked glycans that lack further processing. The mobility of endoglycosidase H-treated CD44 matched that of CD44 treated with N-glycosidase F, an enzyme that cleaves high mannose, hybrid and complex oligosaccharides from glycoproteins. CD44 from DMJ-treated LS174T cells had levels of HECA-452-reactive epitopes similar to the control, while benzyl-GalNAc eliminated HECA-452-reactive sites. In concert with the enzymatic assays, and previous selectin adhesion studies using live LS174T cells treated with metabolic inhibitors, CD44 from DMJ-treated LS174T cells bound to E-, P-, or L-selectin at levels equivalent to the control, while CD44 from benzyl-GalNAc-treated cells were nearly incapable of adhesion (Fig. 2B
). Taken altogether, these data suggest that the selectin binding determinants on CD44 from LS174T cells are sialofucosylated structures displayed on O-linked glycans, akin to those on PSGL-1, but distinct from those on CD44s HCELL expressed on KG1a cells. This difference may allow LS174T CD44 to function as a ligand for E-, P-, and L-selectin under physiological flow conditions. The neuraminidase sensitivity of CD44-mediated tethering to P- and L-selectin (Fig. 2A
) coupled with Western blot analysis showing that only CD44v is HECA-452-reactive strongly suggests that CD44v rather than CD44s isoforms expressed on LS174T cells are primarily responsible for P- and L-selectin binding.
CONCLUSIONS AND SIGNIFICANCE
CD44-mediated adhesion has been implicated in numerous diverse pathophysiological phenomena, including cancer metastasis. Mounting evidence suggests that the up-regulation of CD44v confers metastatic potential in vivo and results in poor prognosis. While others have hypothesized that tumor cell adhesion to hyaluronic acid is a dominant factor regulating metastasis, CD44-mediated adhesion to the selectins has been largely overlooked. There have been several reports that link selectin binding activity with metastatic spread in vivo. However, the identity of the vast majority of the ligands responsible for selectin-mediated tumor-host cell interactions has remained a mystery. Our data suggest that incorporation of CD44v on tumor cells may provide new sites for glycosylation, resulting in unique LS174T CD44 adhesion characteristics. Cumulatively, our findings offer a unifying perspective on the apparent enhanced metastatic potential associated with CD44v overexpression on many types of tumor cells and the critical role of selectins in metastatic spread.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4574fje;
This article has been cited by other articles:
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S. L. Napier, Z. R. Healy, R. L. Schnaar, and K. Konstantopoulos Selectin Ligand Expression Regulates the Initial Vascular Interactions of Colon Carcinoma Cells: THE ROLES OF CD44V AND ALTERNATIVE SIALOFUCOSYLATED SELECTIN LIGANDS J. Biol. Chem., February 9, 2007; 282(6): 3433 - 3441. [Abstract] [Full Text] [PDF]
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M. M. Burdick, J. T. Chu, S. Godar, and R. Sackstein HCELL Is the Major E- and L-selectin Ligand Expressed on LS174T Colon Carcinoma Cells J. Biol. Chem., May 19, 2006; 281(20): 13899 - 13905. [Abstract] [Full Text] [PDF]
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30 rolling microspheres ± SD.
