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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 4, 2002 as doi:10.1096/fj.02-0049fje. |
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INSERM U343 et Laboratoire d’Immunologie, Université de Nice-Sophia Antipolis, France
2Correspondence: INSERM U343, Hôpital de l’Archet, 06202 Nice cedex 3, France. E-mail: u343{at}unice.fr
SPECIFIC AIMS
CD99, an integral transmembrane protein present on all human T cells, can powerfully regulate peripheral T cell adhesion or, alternatively, lead double-positive thymocytes to apoptosis. Our purpose was to investigate the molecular basis of these different functional outcomes.
PRINCIPAL FINDINGS
1. Normal T cells express two different CD99 isoforms according to their differentiation status
Cellular functions triggered via CD99 appear to be critically linked to the differentiation status of T cells. CD99 displays two surface isoforms generated by alternative splicing a long (L) 32 kDa and a short (S) 28 kDa form. To distinguish these forms, we compared surface biotinylated proteins to whole T cell lysates. Biotinylated surface proteins from T cells were subjected to immunoprecipitation with a CD99 antibody and revealed with streptavidin. On the other hand, CD99 molecules from unbiotinylated cells were subjected to immunoprecipitation and revealed by Western blot analysis. Double-positive thymocytes exhibited both the surface classical L isoform and the S isoform. In a marked contrast, single-positive thymocytes and peripheral T cells exhibited solely the classical L variant on their surface.
To ensure that the 28 kDa material present on the surface of T cell resulted from alternative splicing, we performed RT-PCR analysis using appropriate primers. RNA from mature thymocytes (SP) as well as peripheral T cells yielded only the RT-PCR product corresponding to the L isoform transcript; using RNA from immature thymocytes (DP), we detected PCR fragments corresponding to S and L splice variants.
2. T cell fate triggered via CD99, T cell adhesion, and apoptosis depends on CD99 isoform expression
To gather evidence that CD99 isoforms dictate cellular functional outcomes, we performed transfection experiments using a Jurkat CD99 fully deficient T cell line. These CD99-deficient cells were stably transfected with the cDNA encoding for either the 28 kDa short form solely, the 32 kDa L form solely, or both cDNAs. Immunoprecipitation experiments after cell biotinylation or not revealed the expected surface expression pattern on the CD99 transfected cells. RT-PCR experiments confirmed the respective mRNA expression.
We have previously shown that under flow conditions at physiological shear stress, CD99 up-regulates 
4ß1 integrin-dependent adhesion events. Moreover, CD99 induces, with no delay, mature and Jurkat T cells arrest on tumor necrosis factor-treated endothelial cells and on human recombinant VCAM-1 monolayers. We therefore investigated the adhesion properties of the different CD99 transfectants in an in vitro assay under physiological shear stress. We showed that transfection of the L form is sufficient to restore adhesion functions.
Moreover, we have shown that CD99 induces apoptosis of double-positive thymocytes and Jurkat cells in a Fas-independent manner. We therefore investigated apoptosis-inducing properties of the different CD99 transfectants. Strikingly, only the cells transfected with both the L and S isoforms could be induced to apoptosis via CD99 (Fig. 1
).
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3. CD99-induced apoptosis requires CD99 heterodimers and their localization in glycosphingolipidic rafts
Western blot experiments performed under nonreducing conditions have suggested that CD99 heterodimers could be formed when the L and S isoforms are coexpressed. The primary sequence of CD99 revealed an intracytoplasmic cysteine in position 155. Therefore, we transfected CD99-deficient Jurkat cells with a mutated cDNA encoding for CD99 long and short isoforms in which cysteine 155 was substituted by alanine (C155A). These C155A transfectants could not be induced to apoptosis via CD99. Thus, formation of covalent heterodimers is required for CD99 to be able to transduce a proapoptotic signal.
Finally, it was of interest to check whether the membrane localization of CD99 heterodimer would be distinct from CD99 single chain molecules. Lipid rafts isolation on saccharose gradient (Fig. 2
A) revealed that the CD99 chains are located in the light fractions only on double transfected cells, namely, with cells displaying CD99 heterodimers. Accordingly, these results are confirmed by CD99 labeling with "O662" mAb and glycosphingolipidic rafts stained by cholera toxin B. Only cells expressing both CD99 isoforms (L+S) showed colocalization of CD99 and GM1 ganglioside (Fig. 2B
).
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Cholesterol depletion experiments show that this localization is required for the induction of apoptosis.
The rapid exposition of phosphatidylserine to the outer leaflet of the plasma membrane observed under CD99 engagement is associated to a rapid and specific degradation of sphingomyelin, restricted to the glycosphingolipidic raft-enriched fractions.
CONCLUSION
Remarkably, we had previously described that CD99 D44 mAb reacts differentially with different T cell subsets. This mAb defined an extracellular CD99 epitope that displays no correlation with expression of the two isoforms described in this study.
Analysis of the intracytoplasmic segment of the two CD99 isoforms reveals remarkable residues. In the common intracytoplasmic fragment of the L and S isoforms, we noticed a lysine triplet (K151K152K153) that has been shown to bind the ezrin/radixin/moesin molecules known to functionally link some surface proteins to the actin cytoskeleton. Such an interaction could explain why expression of one CD99 isoform alone is sufficient to trigger adhesion events. It is striking that 4ß1 activation and induction of actin polymerization occur very quickly, allowing immediate T cell arrest on inflamed endothelium under physiological shear stress. We had demonstrated that this arrest is mediated by the interaction between 4ß1 integrin and VCAM-1 and observed no effect of CD99 on the Lß2 integrin/ICAM-1 pathway within T cells in contrast with CD99 effects described in a B cell line. CD99 was recently shown to play a major role in monocyte extravasation.
In the CD99 L form, a serine (S168) belonging to a consensus site for protein kinases C (PKC) can be observed. PKC have been implicated in both signaling pathways leading to cell adhesion and apoptosis events. PKC induce focal adhesion kinase phosphorylation upon CD3/CD28 engagement, leading to actin polymerization and T cell spreading. As for apoptosis, in fibroblasts, tumor necrosis factor induces PKC translocation and dephosphorylation by a specific phosphatase: ceramide-activated protein phosphatase. This pathway could be triggered upon CD99 engagement only when the heterodimeric forms are expressed in the glycosphingolipic rafts, where they induce sphingomyelin degradation leading to ceramide production.
Indeed, another clue to the mechanism by which the CD99 isoform expression dictates distinct functional events is localization of the CD99 heterodimeric forms restricted to glycosphingolipidic rafts; no CD99 could be detected within rafts on cells expressing only a single chain. After disrupting rafts with a cholesterol depleting agent, we could no longer observe apoptosis upon CD99 stimulation. Sphingomyelin degradation occurs solely in the cells carrying CD99 heterodimers in contrast to cells carrying only a single chain. Thus, a sphingomyelinase preferentially harbored in the glycosphingolipidic rafts is activated upon CD99 heterodimer engagement. CD99-induced apoptosis is restricted to double-positive thymocytes. Since this population undergoes a selection process, it is likely that the CD99 proapoptotic form plays an important role and that T cell selection might depend on the peculiar architectural organization of CD99. It must nevertheless be emphasized that in double-positive thymocytes displaying heterodimers, CD99 monomeric forms are also present outside glycosphingolipid-rich domains. This may explain why adhesion events can also be triggered in this T cell population.
The last question raised by these observations is to understand why these functional effects that appear to be independent of each other are mediated by the same molecular species as a surface receptor. In fact, many receptors that can induce apoptotic signals can also trigger cell activation, depending on the circumstances, e.g., CD3 and CD40. Fewer receptors have the capacity to induce either apoptosis or to modulate adhesion (e.g., CD2 and CD47). Although the conjunction of the distinct functional outcomes via a single receptor might be coincidental, it is tempting to speculate that this represents a coordinated mechanism.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0049fje; to cite this article, use FASEB J. (October 4, 2002) 10.1096/fj.02-0049fje 
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