Here, we sought to determine whether the Δ15 PECAM-1 isoform is expressed at the protein level in human and murine tissues and whether human Δ15 PECAM-1 produces different signals compared with WT PECAM-1. Because the Δ15 form of PECAM-1 ends in a unique C-terminus, we were able to develop a novel antibody specific for forms of PECAM-1 that lack exon 15 ().
Using the Δ15 PECAM-1-specific antibody, we demonstrate here that a variety of human and murine tissues do indeed express Δ15 PECAM-1 protein (). Initially, tissue lysates were examined for reactivity with the Δ15 PECAM-1 antibody. The results from these studies merely indicated that this protein was synthesized by cells, but they did not demonstrate that the protein traffics to the cell surface. Therefore, we also immunoprecipitated PECAM-1 from whole cells and subsequently immunoblotted for Δ15 PECAM-1 to determine whether Δ15 PECAM-1 is present at the surface of primary cells and cell lines. Notably, human brain tissue expresses a substantial amount of Δ15 PECAM-1. The cells on which Δ15 PECAM-1 is expressed in the brain could include not only endothelium but also microglial or other central nervous system cells. Human ovary and testes also express Δ15 PECAM-1. When compared with the total amount of PECAM-1 present in human testes tissue, the amount of the Δ15 isoform expressed appears to be proportionally large. The role that PECAM-1 plays in human reproductive tissues is largely unknown, but prior work has demonstrated that PECAM-1 is expressed in human spermatozoa and that PECAM-1 might in part be responsible for capacitation-associated signaling cascades (Nixon et al., 2005
). In addition, PECAM-1 expression has been described in subpopulations of cytotrophoblasts (Zhou et al., 1997a
; Zhou et al., 1997b
). HUVECs also have a relatively small, but easily detectable, amount of Δ15 PECAM-1 protein, and human cancer cell lines can express substantial amounts of Δ15 PECAM-1 on the cell surface. Taken together, these data suggest that the proportion of Δ15 PECAM-1 expressed varies in different vascular beds.
Most strikingly, murine, but not human, platelets and lung tissue from C57BL/6 mice express a considerable proportion of PECAM-1 that lacks exon 15 (). This might have wide-ranging implications for the way in which prior studies of PECAM-1 function in murine tissues have been interpreted, most of which have been performed in the C57BL/6 strain. Interpretation of these studies was based on the assumption that the majority of PECAM-1 in WT mice is the full-length form. It is not known whether other murine strains also express such large proportions of exon-15-deficient PECAM-1 protein, although, in platelets from FVB/N and C129 strains, the majority of PECAM1
mRNA lacks exon 15 (Wang and Sheibani, 2002
). Interestingly, studies examining the effect of PECAM-1 on leukocyte transmigration found that C57BL/6, but not FVB/N, mice are uniquely able to compensate for the loss of PECAM-1 function (Schenkel et al., 2004
). It is possible that WT mouse strains express a different array of isoforms of PECAM-1 in leukocytes and/or in endothelial cells, and this in part accounts for the differences in leukocyte transmigration seen between strains. PECAM-1 has also been implicated in modulating platelet signaling (Falati et al., 2006
; Jones et al., 2001
; Patil et al., 2001
), as well as murine megakaryocytopoiesis (Dhanjal et al., 2007
; Wu et al., 2007
). Once again, it is not known whether certain alternatively spliced isoforms of PECAM-1 are more efficient than others at eliciting these responses. Because the data in demonstrate that isoforms lacking exon 15 are differentially expressed at the protein level in a variety of human and murine tissues, re-examination of the ability of Δ15 PECAM-1 to signal might be warranted.
Many of the functions of PECAM-1 stem from its ability to bind homophilically to other PECAM-1 molecules and to signal by means of its cytoplasmic ITIMs. Thus, we sought to determine whether the Δ15 isoform of PECAM-1 can similarly perform these functions. Prior studies of murine Δ15 PECAM-1 in Madin-Darby canine kidney (MDCK) cells indicated that it is unable to localize to adherens junctions when compared with murine Δ14,15 PECAM-1 (Sheibani et al., 2000
). Unfortunately, these studies did not compare Δ15 PECAM-1 with WT PECAM-1, and the human counterparts of these isoforms were not examined in human cells. In the present investigation, human Δ15 PECAM-1 became concentrated normally at cell-cell borders in human cells, and cells expressing Δ15 PECAM-1 largely exhibited a similar morphology to those cells expressing WT PECAM-1 (). In addition, we determined that human Δ15 PECAM-1 can signal through its ITIM domains (). This suggests that the functions of human PECAM-1 that are mediated merely by extracellular region engagement and/or ITIM-mediated signaling might be largely intact in cells expressing the Δ15 PECAM-1 isoform.
Both ITIM-mediated signals and homophilic binding have previously been shown to be required for PECAM-1 to protect efficiently against programmed cell death (Gao et al., 2003
). However, even though Δ15 PECAM-1 retains the ability to both signal through its ITIM domains () and localize to cell-cell borders (), it surprisingly lacks the full cytoprotective function when compared with WT PECAM-1, when apoptosis is induced both by overexpression of Bax and by treatment with a chemotherapy agent (). This suggests a novel role for the C-terminal region of the PECAM-1 cytoplasmic domain in PECAM-1-mediated cytoprotection. While it is unclear what this role might be, it is tempting to speculate that failure to recruit a cytosolic binding partner that normally associates with an amino acid sequence found in WT, but not Δ15, PECAM-1 accounts for this difference. Identification of such proteins is the subject of current investigations.
A diminished ability to exhibit cytoprotection is one of several potential functional variations between WT and Δ15 PECAM-1. Further studies will be necessary to determine whether expression of Δ15 PECAM-1 has additional functional consequences. Even if further differences are uncovered between these PECAM-1 isoforms, the phenotypic effect might be minimal unless the Δ15 PECAM-1 is expressed at high levels or exhibits dominant-negative or positive effects. However, our results demonstrate that large relative fractions of exon-15-deficient PECAM-1 are expressed in at least a few cell types. It is in these tissues where functional differences are more likely to result in a change of cellular phenotype.
Taken together, the data in the present investigation demonstrate, first, that Δ15 PECAM-1 is expressed in both murine and human tissues and cell lines and, second, that Δ15 PECAM-1 retains homophilic binding characteristics and is able to signal through its ITIM domains, yet, despite this, it fails to offer appreciable cytoprotection against apoptosis when compared with WT PECAM-1. These studies suggest a novel role for the C-terminal region of the PECAM-1 cytoplasmic domain in cytoprotective signaling and highlight a need for further studies on the implications of the expression of this, sometimes abundant, alternatively spliced isoform of PECAM-1. The Δ15-specific antibody against PECAM-1 described here is one tool that can aid in the characterization of the expression of PECAM-1 isoforms in cancers and other tissues.