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IntroductionSoluble amyloid precursor protein α (sAPPα) is a proteolyte of APP cleavage by α-secretase. The significance of the cleavage and the physiological role of sAPPα are unknown. A crystal structure of a region of the amino terminal of sAPPα reveals a domain that is similar to cysteine-rich growth factors. While a previous study implicates sAPPα in the regulation of neural progenitor cell proliferation in the subventricular zone of adult mice, the ubiquitous expression of APP suggests that its role as a growth factor might be broader.MethodssAPPα and α-secretase activities were determined in neural progenitor cells (NPCs), mesenchymal stem cells (MSC) and human decidua parietalis placenta stem cells (hdPSC). Inhibition of α-secretase was achieved by treatment with the matrixmetalloproteinase inhibitor GM6001, and proliferation was determined using clonogenic and immunocytochemical analysis of cell-lineage markers. Recovery of proliferation was achieved by supplementing GM6001-treated cells with recombinant soluble APPα. Expression of APP and its cellular localization in the subventricular zone was determined by Western blot and immunohistochemical analyses of APP wild type and knockout tissue. Alterations in pERK and pAKT expression as a function of soluble APPα production and activity in NPCs were determined by Western blot analysis.ResultsHere we show that sAPPα is a proliferation factor of adult NPCs, MSCs and hdpPSC. Inhibition of α-secretase activity reduces proliferation of these stem cell populations in a dose-dependent manner. Stem cell proliferation can be recovered by the addition of sAPPα in a dose-dependent manner, but not of media depleted of sAPPα. Importantly, sAPPα operates independently of the prominent proliferation factors epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), but in association with ERK signaling and MAP-kinase signaling pathways. Levels of sAPPα and putative α-secretase, ADAM10, are particularly high in the subventricular zone of adult mice, suggesting a role for sAPPα in regulation of NPCs in this microenvironment.ConclusionsThese results determine a physiological function for sAPPα and identify a new proliferation factor of progenitor cells of ectodermal and mesodermal origin. Further, our studies elucidate a potential pathway for sAPPα signaling through MAP kinase activation.

Successful implantation requires synergism between the developing embryo and the receptive endometrium. In the baboon, infusion of chorionic gonadotropin (CG) modulates both morphology and physiology of the epithelial and stromal cells of the receptive endometrium. This study explored the signal transduction pathways activated by CG in endometrial epithelial cells from baboon (BE) and human (HES). Incubations of BE and HES cells with CG did not significantly alter adenylyl cyclase activity or increase intracellular cAMP when compared with Chinese hamster ovarian cells stably transfected with the full-length human CG/luteinizing hormone (LH) receptor (CHO-LH cells). However, in BE and HES cells, CG induced the phosphorylation of several proteins, among them, extracellular signal-regulated protein kinases 1 and 2 (ERK 1/2). Phosphorylation of ERK 1/2 in uterine epithelial cells was protein kinase A (PKA) independent. This novel signaling pathway is functional because, in response to CG stimulation, prostaglandin E(2) (PGE(2)) was released into the media and increased significantly 2 h following CG stimulation. CG-stimulated PGE(2) synthesis in epithelial cells was inhibited by a specific mitogen-activated protein kinase (MEK 1/2) inhibitor, PD 98059. In conclusion, immediate signal transduction pathways induced by CG in endometrial epithelial cells are cAMP independent and stimulate phosphorylation of ERK 1/2 via a MEK 1/2 pathway, leading to an increase in PGE(2) release as the possible result of cyclooxygenase-2 activation.

Both human chorionic gonadotropin (hCG) and IL-1beta induce changes in the endometrium that are associated with the establishment of pregnancy. We investigated the synergistic effect of these two embryonic signals on endometrial function using a baboon model of simulated pregnancy. Recombinant hCG (30 IU/d) was infused between d 6 and 10 post ovulation (PO) to mimic blastocyst transit. On the expected day of implantation (d 10 PO), IL-1beta (12 ng/d) or IL-1 receptor antagonist (IL-1Ra; 12 ng/d) was infused for an additional 5 d. Endometria were harvested on d 15 PO. Both hCG and hCG plus IL-1beta induced marked differences in the distribution of alpha-smooth muscle actin, proliferation marker Ki67, decidualization marker IGF-binding protein-1, and cyclooxygenase-1. The most marked effect of IL-1beta was the induction of IGF-binding protein-1 protein in stromal cells close to the apical surface, whereas cyclooxygenase-1 was down-regulated in the glandular epithelium. Protein arrays of uterine flushings showed significant suppression of death receptors, Fas and TNF receptor 1, in the hCG- with or without IL-1beta-treated groups, suggesting an inhibition of apoptosis. Additionally, cytotoxic T lymphocyte antigen-4, matrix metalloproteinase-3, and IL-4 were suppressed in treated animals compared with controls. However, no differences were observed in cytokine profile between hCG-treated and hCG- plus IL-1beta-treated baboons. This study confirms that in preparation for pregnancy, the primate endometrium undergoes both morphological and functional changes, which are modulated by hCG and IL-1beta, that lead to the inhibition of apoptosis and the development of an immunotolerant environment. These changes suggest that infusion of IL-1beta at the time of implantation into the nonpregnant baboon treated with hCG synergizes with hCG and mimics the early endometrial events associated with the presence of an embryo.

Oxytocin (OT) induces PG synthesis by both uterine endometrial and amnion cells. We showed previously that CHO cells stably transfected with the rat oxytocin receptor (CHO-OTR cells) also synthesize PGE2 in response to OT. In the present work we have demonstrated that OTRs are coupled to both Gi and Gq/11, using immunoprecipitation of solubilized OTR complexes and ADP ribosylation. OT treatment caused the rapid phosphorylation of extracellular signal-regulated protein kinase 2 (ERK2 or p42MAPK), which was partially inhibited by pertussis toxin (PTX), consistent with OTR-Gi coupling. The PTX-insensitive portion of ERK2 phosphorylation was linked to Gq, as inhibitors of both phospholipase C (U-73122) and protein kinase C (GF-109203X) blocked OT-induced ERK2 phosphorylation. OT-stimulated c-fos expression was also mediated by ERK2 phosphorylation. The ERK- c-fospathway has been shown to be associated with cell proliferation, but OT had no effect on [3H]thymidine uptake by CHO-OTR cells. However, inhibition of OT-induced ERK2 phosphorylation with an ERK kinase inhibitor (PD-98059) markedly reduced OT-stimulated PGE2synthesis, pointing to the importance of ERK2 activation in OT action.