Zuzana Strakova

Signal Transduction Pathways Activated by Chorionic Gonadotropin in the Primate Endometrial Epithelial Cells

Abstract 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 E2 (PGE2) was released into the media and increased significantly 2 h following CG stimulation. CG-stimulated PGE2 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 PGE2 release as the possible result of cyclooxygenase-2 activation.

The Role of Chorionic Gonadotropin (CG) in Blastocyst Implantation

Implantation is a complex spatio-temporal interaction between the genotypically different embryo and the mother. Success of this event requires the synchronization of development and effective biochemical communications from both sides. Chorionic gonadotropin (CG), which is a major embryonic signal in the primate, is a glycoprotein hormone synthesized and secreted by the trophoblast. Various isoforms exist in plasma, urine, and blastocyst culture medium, a result of posttranslational modifications. The exponential secretion of CG and its long circulatory half-life extends the life span of corpus luteum to maintain the supply of progesterone during the first 6-8 weeks of pregnancy. To study the direct effects of CG in the uterus, we used the baboon (Papio anubis) as a non-human primate model. In vivo stimulation with CG during the window of uterine receptivity results in further morphologic and biochemical modifications of the receptive endometrium. These are characterized by the plaque reaction in the luminal epithelium, an increase in glycodelin expression and secretion by the glandular epithelium, and the differentiation of subepithelial stromal fibroblasts characterized by expression of the alpha smooth muscle actin (alpha SMA). Pretreatment with progesterone receptor antagonist (PRa) completely or partially inhibits these effects. The signal transduction pathway activated by CG in primate endometrial epithelial cells involves the protein kinase A (PKA)-independent phosphorylation of extracellular signal regulated kinase (ERK 1/2). This alternate signal transduction pathway may prevent CG Receptor (R) downregulation at the implantation site and enhance epithelial cell proliferation and differentiation. Thus, our results suggest that CG plays an important role in implantation in addition to its luteotrophic role.

Chorionic gonadotropin and uterine dialogue in the primate

Implantation is a complex spatio-temporal interaction between the growing embryo and the mother, where both players need to be highly synchronized to be able to establish an effective communication to ensure a successful pregnancy. Using our in vivo baboon model we have shown that Chorionic Gonadotropin (CG), as the major trophoblast derived signal, not only rescues the corpus luteum but also modulates the uterine environment in preparation for implantation. This response is characterized by an alteration in both the morphological and biochemical activity in the three major cell types: luminal and glandular epithelium and stromal fibroblasts. Furthermore, CG and factors from the ovary have a synergistic effect on the receptive endometrium. Novel local effects of CG which influence the immune system to permit the survival of the fetal allograft and prevent endometrial cell death are also discussed in this review. An alternate extracellular signal-regulated kinase (ERK) activation pathway observed in epithelial endometrial cells and the possibility of differential expression of the CG/LH-R isoforms during gestation, open many questions regarding the mechanism of action of CG and its signal transduction pathway within the primate endometrium.

Soluble amyloid precursor protein: a novel proliferation factor of adult progenitor cells of ectodermal and mesodermal origin

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.

Chorionic Gonadotropin Regulates Prostaglandin E Synthase via a Phosphatidylinositol 3-Kinase-Extracellular Regulatory Kinase Pathway in a Human Endometrial Epithelial Cell Line: Implications for Endometrial Responses for Embryo Implantation

Successful implantation necessitates modulation of the uterine environment by the embryo for a specific period of time during the menstrual cycle. Infusion of chorionic gonadotropin (CG) into the oviducts of baboons to mimic embryo transit induces a myriad of morphological, biochemical, and molecular changes in the endometrium. Endometrial epithelial cells from both baboons and humans when stimulated by CG in vitro, activates a cAMP-independent MAPK pathway leading to prostaglandin E(2) (PGE(2)) synthesis. This study shows that in the human endometrial cell line, HES, CG, acting via its G-protein coupled receptor, phosphorylates protein kinase B, c-Raf, and ERK1/2 in a phosphatidylinositol 3-kinase (PI3K)-dependent manner. Furthermore, ERK1/2 phosphorylation is independent of the signaling paradigms of Galpha(s), Galpha(I), and epidermal growth factor receptor (EGFR) transactivation, typical of gonadal cells, indicating an alternative signaling pattern in the endometrium. After phosphorylation by CG, ERK1/2 translocates to the nucleus in a time-dependent manner. Downstream of ERK1/2, CG activates the nuclear transcription factor, Elk1, also in a PI3K-MAPK-dependent manner. Lastly, we show that in HES cells, this pathway regulates the expression of the microsomal enzyme PGE(2) synthase (mPTGES), a terminal prostanoid synthase responsible for PGE(2) synthesis. CG regulates the mPTGES promoter and also induces mPTGES synthesis in HES cells via the PI3K-ERK1/2 pathway. We suggest that this alternative PI3K-ERK-Elk pathway activated by CG regulates prostaglandin production by the endometrial epithelium and serves as an early trigger to prepare the endometrium for implantation.

IL-1β during in vitro decidualization in primate

Abstract The sequence of biochemical and molecular events associated with decidualization in the primate remain unclear. In the baboon, the sequential changes during this period in vivo are characterized by the downregulation of α-smooth actin followed by induction of cyclooxygenase-2 (COX-2) at the implantation site and the expression of insulin growth factor binding protein-1 (IGFBP-1). IGFBP-1 is the predominant protein in decidualized cells and is considered to be biochemical marker of decidualization. In the baboon the expression of IGFBP-1 requires the presence of a conceptus in vivo or N 6 , 2′- O -dibutyryladenosine 3:5′-cyclic monophosphate (dbcAMP) in the presence of hormones in vitro. In addition IL-1β, as a possible conceptus-mediated factor, can induce IGFBP-1 expression in the presence of hormones following 3 days of incubation. However, if IL-1β and dbcAMP are added together, IGFBP-1 expression is inhibited which resulted in IL-1β being considered to be ‘inhibitory’ to decidualization. Current data suggest that IL-1β can activate multiple signaling pathways that either positively (no exogenous cAMP) or negatively (in presence of exogenous cAMP) regulate IGFBP-1 gene expression and decidualization in vitro. Signaling pathways activated by IL-1β following 10 min of stimulation result in the phosphorylation of mitogen-activated protein kinase (MAPK, specifically p38 MAPK) and also lead to NF-κB activation. The expression of COX-2 and matrix metalloproteinase-3 (MMP-3) genes follows after 4–6 h. The steroid hormones, particularly progesterone, which are critical for IGFBP-1 expression, modulate the activity of IL-1β by down-regulating MMP-3 activity. Disruption of actin filaments enhances IGFBP-1 induction during decidualization. IL-1β induced MMP-3 may upregulate IGFBP-1 by initiation of cytoskeletal reorganization through degradation of extracellular matrix (ECM). Inhibition of IL-1β induced pathways leads to reduction of IGFBP-1 expression, suggesting that IL-1β may be involved in the events leading to decidualization in baboons.

Manipulating actin dynamics affects human in vitro decidualization.

Abstract The differentiation of uterine stromal fibroblasts into decidual cells is critical for establishing pregnancy. This process, called decidualization, requires the reorganization of the actin cytoskeleton, which mainly depends on actin dynamics and the phosphorylation status of the myosin light chain. We manipulated actin dynamics with jasplakinolide (100 nM) and latrunculin B (1 μM), both of which significantly inhibited the synthesis of decidualization markers induced by 6 days of treatment with embryo-mimicking stimulus interleukin 1beta (IL1B) and steroid hormones (SHs; 17beta-estradiol and medroxyprogesterone acetate) in the human uterine fibroblast (HuF) in vitro model. However, only jasplakinolide had long-lasting effects on the G-actin:F-actin ratio and prevented decidualization induced by the artificial stimulus cAMP (and SHs). Actin-binding protein cofilin mainly colocalized with G-actin in the nucleus as well as the cytoplasm. Only some spots of colocalization between cofilin and F-actin were detected in the cytoplasm. Brief extraction of cytosolic proteins from living cells revealed that in cells treated with IL1B or cAMP (and SHs) for 6 days, cofilin was mainly detected in the nucleus. The translocation of cofilin from cytosol to nucleus was also detected in HuFs treated for 12 days with SHs, IL1B and SHs, and cAMP and SHs. The same significant translocation was confirmed in primary baboon stromal uterine fibroblasts. We conclude that changes in actin dynamics, particularly the stabilization of F-actin, have a significant negative impact on decidualization, and the translocation of cofilin to the nucleus is a key feature of this process in the primate.

Decidualization Regulates the Expression of the Endometrial Chorionic Gonadotropin Receptor in the Primate

Abstract Chorionic gonadotropin (CG) plays an important role in establishing a receptive endometrium by directly modulating the function of both endometrial stromal and epithelial cells in the baboon. The focus of this study was to characterize changes in CG receptor (LHCGR, also known as CG-R) expression during the menstrual cycle and early pregnancy, particularly during decidualization. LHCGR was localized by using a peptide-specific antibody generated against the extracellular domain. Immunostaining was absent in any of the cell types during the proliferative phase of the cycle. In contrast, during the secretory phase, both luminal and glandular epithelial cells stained positively. Stromal staining was confined to the cells around spiral arteries (SAs) and in the basalis layer. This stromal staining pattern persisted at the implantation site between Days 18 and 25 of pregnancy and after CG infusion. However, as pregnancy progressed (Days 40 to 60), staining for LHCGR was dramatically decreased in the stromal cells. These data were confirmed by nonisotopic in situ hybridization. To confirm whether the loss of LHCGR was associated with a decidual response, stromal fibroblasts were decidualized in vitro, and cell lysates obtained after 3, 6, and 12 days of culture were analyzed by Western blotting. LHCGR protein decreased with the onset of decidualization in vitro, confirming the in vivo results. Addition of CG to decidualized cells resulted in the reinduction of LHCGR in the absence of dbcAMP. We propose that CG acting via its R on stromal cells modulates SA in preparation for pregnancy and trophoblast invasion. As pregnancy progresses, further modification of SA by migrating endovascular trophoblasts and subsequent decidualization results in the downregulation of LHCGR. This inhibition of LHCGR expression also coincides with the decrease of measurable CG in peripheral circulation.

Cofilin and Slingshot Localization in the Epithelium of Uterine Endometrium Changes During the Menstrual Cycle and in Endometriosis

Regulation of the actin cytoskeleton is essential for epithelial cell polarity and protein trafficking within human uterine epithelium. The actin-binding protein cofilin is involved in regulation of actin dynamics by promoting actin branching and cytoskeleton reorganization. Dual immunohistochemical staining of cofilin and G-actin (represented by DNAse I staining) revealed cofilin-G-actin colocalization in the apical side of luminal epithelial cells of human uterine endometrium during the proliferative phase of the menstrual cycle. Interestingly, during the secretory phase of the menstrual cycle, cofilin was only present on the basolateral side. To determine whether the disease endometriosis causes a different pattern of actin remodeling, we investigated an established baboon model of induced endometriosis. The cofilin pattern in the secretory phase of baboons with endometriosis was similar to the proliferative phase in normal animals; cofilin was observed in the apical parts of luminal and glandular epithelium. A phosphatase regulating the activity of cofilin, slingshot (SSH1), revealed a similar staining pattern within these tissues. These patterns were confirmed through quantitative image analysis. Quantification of messenger RNA (mRNA) detected upregulated SSH1 and suggested a progesterone resistance-related pattern of nuclear steroid hormone receptors, but no change in membrane progesterone receptors (mPR alpha or mPR beta) was observed in endometriosis. Our data indicate that the severe dyssynchrony during menstrual cycle phases in endometriosis is connected with improper cytoskeleton rearrangements. We suggest that cofilin-mediated actin reorganization in uterine epithelial cells might be important in preparation for blastocyst implantation; dysregulation of this reorganization may lead to decreased fertility in endometriosis.

Two-way regulation between cells and aligned collagen fibrils: local 3D matrix formation and accelerated neural differentiation of human decidua parietalis placental stem cells.

It has been well established that an aligned matrix provides structural and signaling cues to guide cell polarization and cell fate decision. However, the modulation role of cells in matrix remodeling and the feedforward effect on stem cell differentiation have not been studied extensively. In this study, we report on the concerted changes of human decidua parietalis placental stem cells (hdpPSCs) and the highly ordered collagen fibril matrix in response to cell-matrix interaction. With high-resolution imaging, we found the hdpPSCs interacted with the matrix by deforming the cell shape, harvesting the nearby collagen fibrils, and reorganizing the fibrils around the cell body to transform a 2D matrix to a localized 3D matrix. Such a unique 3D matrix prompted high expression of β-1 integrin around the cell body that mediates and facilitates the stem cell differentiation toward neural cells. The study offers insights into the coordinated, dynamic changes at the cell-matrix interface and elucidates cell modulation of its matrix to establish structural and biochemical cues for effective cell growth and differentiation.