PSGs are among the most abundant proteins in the maternal serum, yet their function during pregnancy is still being explored [3
]. Previously, the role of PSGs centered on their functions as immunoregulators during pregnancy [6
]. Treatment of monocytes/macrophages with recombinant human and murine PSGs leads to induction of anti-inflammatory cytokines, including IL10 and TGFB1, while there is no induction of pro-inflammatory cytokines [7
]. In addition, human PSG1 was shown to induce alternative activation of monocytes and to enhance Th2-type immune responses [6
]. Other CEA family members that, unlike PSGs, are membrane-bound have been shown to be involved in angiogenesis and immunoregulation [34
]. For example, CEACAM1 has angiogenic properties [35
] and inhibits activated decidual lymphocytes [37
], and CEACAM6, expressed on placental trophoblast, has been shown to activate a distinct subset of regulatory CD8+ T cells [34
Recent studies have identified PSG expression at a unique site in nonpregnant mice. Kawano et al. [38
] reported expression of murine PSG18 in the follicle-associated epithelium of the Peyer's patches and suggested a role for this murine PSG in the interplay between epithelial cells and immune cells in mucosa-associated lymphoid tissue.
The important players in placentation include macrophages [11
], DCs [26
], uNK cells [30
], endothelial cells, and trophoblasts [39
]. Previous work with murine and human PSGs identified monocytes/macrophages as the target cells for protein binding and activity [5
]. As previously observed for other human and murine PSGs [5
], we found that macrophages up-regulate TGFB1 secretion in response to PSG23 in a dose-dependent manner. In addition, treatment of BMDCs with PSG23 resulted in up-regulation of TGFB1. TGFB1 has multiple roles during pregnancy, including regulation of extravillous trophoblast migration and proliferation [40
] and regulation of NK cell function [41
]. TGFB1 and VEGFA have additive effects on vasodilation [1
] and have been implicated in immunosuppression [43
]. Based on the observation that TGFB1 activated macrophages to express VEGF [29
], we explored the possible induction of angiogenic factors in response to PSG23 treatment. VEGFA, but not VEGFC, PlGF, Ang-1, or Ang-2, was strongly induced in macrophages and BMDCs upon PSG23 treatment. The induction of VEGFA was found to be TGFB1 dependent.
Our results indicated that PSG23 does not induce TGFB1 at the transcriptional level, which is in agreement with our previous observations [7
]. At the posttranscriptional level TGFB is synthesized as an inactive precursor complex comprised of the active TGFB protein bound to a propeptide latency-associated protein [45
]. It is possible that PSGs activate effector molecules, such as enzymes, which cleave the inactive precursor and release active TGFB in target cell types [46
]. The mechanisms involved in PSG up-regulation of TGFB1 warrant further investigation.
To extend our hypothesis that PSG23 is involved in angiogenesis, we investigated the effect of PSG23 on additional cell types involved in this process. PSG23 treatment of a murine endothelial cell line led to induction of both TGFB1 and VEGFA, and the induction of VEGFA was TGFB-dependent. In a murine trophoblast cell line, we saw that PSG23 treatment induced both TGFB1 and VEGFA. Similar to our results in murine macrophages, treatment with PSG23 did not result in up-regulation of other proangiogenic factors commonly associated with pregnancy. Further studies could elucidate whether PSGs have an effect on uNK cells, which are the most abundant leukocytes in pregnancy [30
] and are involved in angiogenesis and trophoblast chemoattraction [47
We previously reported cross-reactivity between human cells and murine PSGs [5
]; hence, we treated human monocytes and a human trophoblast cell line with PSG23. In the two cell types, PSG23 induced TGFB1 and VEGFA. Induction of proangiogenic factors in human cells with the mouse protein suggests that human PSGs may also play a role in placentation, although this has yet to be explored. Interestingly, a significant reduction in PSGs (SP1) was reported in women with subsequent preeclampsia or growth-retarded fetuses in the early second trimester of pregnancy [48
Previously, we demonstrated that PSG17, another member of the murine PSG family, induces TGFB1 in RAW 264.7 cells [5
]. Upon our observation of PSG23 induction of VEGFA in murine macrophages, we treated RAW cells with PSG17 and found that PSG17 also up-regulates VEGFA secretion (data not shown). Future experiments will be necessary to determine if PSG17 has an effect on endothelial cells and trophoblasts.
VEGFA is the most abundantly expressed VEGF family member during pregnancy and one of the key growth factors in angiogenesis [49
]. VEGFA binds to VEGF receptor-1 (VEGFR-1) and VEGFR-2 to induce the growth of new vessels [49
]. Macrophages, endothelial cells, and trophoblasts secrete and express receptors for VEGFA, thereby allowing for autocrine and paracrine activity of VEGFA during angiogenesis [2
]. Future studies will be necessary to determine whether PSG23 affects the expression of VEGF receptors.
We examined whether PSG23 binds to CD9, the PSG17 receptor. We could not demonstrate binding of PSG23 to CD9 by ELISA and FACS analysis. While we cannot conclusively rule out the possibility that PSG23 binds to CD9, this interaction, if it happens, may be of low affinity. Importantly, expression of CD9 in peritoneal macrophages was not required for induction of TGFB1 and VEGF. Therefore, we conclude that CD9 is not essential for PSG23 function. Tetraspanins are often organized together in a tetraspanin web on the cell surface, and many tetraspanin family members are expressed in the cell types shown to respond to PSG23 in this report [18
]. Therefore, the possibility that PSG23 may use another tetraspanin family member as its receptor should be explored.
Our findings indicate that PSGs, besides their immunoregulatory role, may be important in the processes of vasculogenesis and angiogenesis required for the establishment and maintenance of the fetoplacental blood supply. We are currently investigating whether other murine PSGs or human PSGs share the ability with PSG23 to induce proangiogenic factors.