Transglycosidase activity of glycosynthase-type mutants of a fungal GH20 ß-N-acetylhexosaminidase.

ß-N-Acetylhexosaminidases (CAZy GH20, EC 3.2.1.52) are exo-glycosidases specific for cleaving N-acetylglucosamine and N-acetylgalactosamine moieties of various substrates. The ß-N-acetylhexosaminidase from the filamentous fungus Talaromyces flavus (TfHex), a model enzyme in this study, has a broad substrate flexibility and outstanding synthetic ability. We have designed and characterized seven glycosynthase-type variants of TfHex mutated at the catalytic aspartate residue that stabilizes the oxazoline reaction intermediate. Most of the obtained enzyme variants lost the majority of their original hydrolytic activity towards the standard substrate p-nitrophenyl 2-acetamido-2-deoxy-ß-D-glucopyranoside (pNP-ß-GlcNAc); moreover, the mutants were not active with the proposed glycosynthase donor 2-acetamido-2-deoxy-d-glucopyranosyl-α-fluoride (GlcNAc-α-F) either as would be expected in a glycosynthase. Importantly, the mutant enzymes instead retained a strong transglycosylation activity towards the standard substrate pNP-ß-GlcNAc. In summary, five out of seven prepared TfHex variants bearing mutation at the catalytic Asp370 residue acted as efficient transglycosidases, which makes them excellent tools for the synthesis of chitooligosaccharides, with the advantage of processing an inexpensive, stable and commercially available pNP-ß-GlcNAc.

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 ß-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.

Tumor stiffness is unrelated to myosin light chain phosphorylation in cancer cells.

Many tumors are stiffer than their surrounding tissue. This increase in stiffness has been attributed, in part, to a Rho-dependent elevation of myosin II light chain phosphorylation. To characterize this mechanism further, we studied myosin light chain kinase (MLCK), the main enzyme that phosphorylates myosin II light chains. We anticipated that increases in MLCK expression and activity would contribute to the increased stiffness of cancer cells. However, we find that MLCK mRNA and protein levels are substantially less in cancer cells and tissues than in normal cells. Consistent with this observation, cancer cells contract 3D collagen matrices much more slowly than normal cells. Interestingly, inhibiting MLCK or Rho kinase did not affect the 3D gel contractions while blebbistatin partially and cytochalasin D maximally inhibited contractions. Live cell imaging of cells in collagen gels showed that cytochalasin D inhibited filopodia-like projections that formed between cells while a MLCK inhibitor had no effect on these projections. These data suggest that myosin II phosphorylation is dispensable in regulating the mechanical properties of tumors.

Matrix-specified differentiation of human decidua parietalis placental stem cells.

To create suitable biological scaffolds for tissue engineering and cell therapeutics, it is essential to understand the matrix-mediated specification of stem cell differentiation. To this end, we studied the effect of collagen type I on stem cell lineage specification. We altered the properties of collagen type I by incorporating carbon nanotubes (CNT). The collagen-CNT composite material was stiffer with thicker fibers and longer D-period. Human decidua parietalis stem cells (hdpPSC) were found to differentiate exclusively and rapidly towards neural cells on the collagen-CNT matrix. We attribute this accelerated neural differentiation to the enhanced structural and mechanical properties of collagen-CNT material. Strikingly, the collagen-CNT matrix, unlike collagen, imposes the neural fate by an alternate mechanism that may be independent of beta-1 integrin and beta-catenin. The study demonstrates the sensitivity of stem cells to subtle changes in the matrix and the utilization of a novel biocomposite material for efficient and directed differentiation of stem cells.

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

INTRODUCTION: Soluble 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. METHODS: sAPPα 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. RESULTS: Here 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. CONCLUSIONS: These 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.

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.

Human transcriptional coactivator with PDZ-binding motif (TAZ) is downregulated during decidualization.

Transcriptional coactivator with PDZ-binding motif (TAZ) is known to bind to a variety of transcription factors to control cell differentiation and organ development. However, its role in uterine physiology has not yet been described. To study its regulation during the unique process of differentiation of fibroblasts into decidual cells (decidualization), we utilized the human uterine fibroblast (HuF) in vitro cell model. Immunocytochemistry data demonstrated that the majority of the TAZ protein is localized in the nucleus. Treatment of HuF cells with the embryonic stimulus cytokine interleukin 1 beta in the presence of steroid hormones (estradiol-17 beta and medroxyprogesterone acetate) for 13 days did not cause any apparent TAZ mRNA changes but resulted in a significant TAZ protein decline (approximately 62%) in total cell lysates. Analysis of cytosolic and nuclear extracts revealed that the decline of total TAZ was caused primarily by a drop of TAZ protein levels in the nucleus. TAZ was localized on the peroxisome proliferator-activated receptor response element site (located at position -1200 bp relative to the transcription start site) of the genomic region of decidualization marker insulin-like growth factor-binding protein 1 (IGFBP1) in HuF cells as detected by chromatin immunoprecipitation. TAZ is also present in human endometrium tissue as confirmed by immunohistochemistry. During the secretory phase of the menstrual cycle, specific TAZ staining particularly diminishes in the stroma, suggesting its participation during the decidualization process, as well as implantation. During early baboon pregnancy, TAZ protein expression remains minimal in the endometrium close to the implantation site. In summary, the presented evidence shows for the first time to date TAZ protein in the human uterine tract, its downregulation during in vitro decidualization, and its localization on the IGFBP1 promoter region, all of which indicate its presence in the uterine differentiation program during pregnancy.

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.

Manipulating actin dynamics affects human in vitro decidualization.

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 microM), 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.

Multipotent properties of myofibroblast cells derived from human placenta.

Human uterine fibroblasts (HuF) isolated from the maternal part (decidua parietalis) of a term placenta provide a useful model of in vitro cell differentiation into decidual cells (decidualization, a critical process for successful pregnancy). After isolation, the cells adhere to plastic and have either a small round or spindle-shaped morphology that later changes into a flattened pattern in culture. HuF robustly proliferate in culture until passage 20 and form colonies when plated at low densities. The cells express the mesenchymal cell markers fibronectin, integrin-beta1, ICAM-1 (CD54), and collagen I. Flow cytometry of HuF has detected the presence of CD34, a marker of the hematopoietic stem cell lineage, and an absence of CD10, CD11b/Mac, CD14, CD45, and HLA type II. Furthermore, they also express the pluripotency markers SSEA-1, SSEA-4, Oct-4, Stro-1, and TRA-1-81 as detected by confocal microscopy. Treatment for 14-21 days with differentiation-inducing media leads to the differentiation of HuF into osteoblasts, adipocytes, and chondrocytes. The presence of alpha-smooth muscle actin, calponin, and myosin light-chain kinase in cultured HuF implies their similarity to myofibroblasts. Treatment of the HuF with dimethyl sufoxide causes reversion to the spindle-shaped morphology and a loss of myofibroblast characteristics, suggesting a switch into a less differentiated phenotype. The unique abilities of HuF to exhibit multipotency, even with myofibroblast characteristics, and their ready availability and low maintenance requirements make them an interesting cell model for further exploration as a possible tool for regenerative medicine.

Increased phosphorylation of myosin light chain prevents in vitro decidualization.

Differentiation of stromal cells into decidual cells, which is critical to successful pregnancy, represents a complex transformation requiring changes in cytoskeletal architecture. We demonstrate that in vitro differentiation of human uterine fibroblasts into decidual cells includes down-regulation of alpha-smooth muscle actin and beta-tubulin, phosphorylation of focal adhesion kinase, and redistribution of vinculin. This is accompanied by varied adhesion to fibronectin and a modified ability to migrate. Cytoskeletal organization is determined primarily by actin-myosin II interactions governed by the phosphorylation of myosin light chain (MLC20). Decidualization induced by cAMP [with estradiol-17beta (E) and medroxyprogesterone acetate (P)] results in a 40% decrease in MLC20 phosphorylation and a 55% decline in the long (214 kDa) form of myosin light-chain kinase (MLCK). Destabilization of the cytoskeleton by inhibitors of MLCK (ML-7) or myosin II ATPase (blebbistatin) accelerates decidualization induced by cAMP (with E and P) but inhibits decidualization induced by IL-1beta (with E and P). Adenoviral infection of human uterine fibroblast cells with a constitutively active form of MLCK followed by decidualization stimuli leads to a 30% increase in MLC20 phosphorylation and prevents decidualization. These data provide evidence that the regulation of cytoskeletal dynamics by MLC20 phosphorylation is critical for decidualization.

Decidualization regulates the expression of the endometrial chorionic gonadotropin receptor in the primate.

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.

Human chorionic gonadotropin and decidualization in vitro inhibits cytochalasin-D-induced apoptosis in cultured endometrial stromal fibroblasts.

Endometrial apoptosis increases from the proliferative phase through the secretory phase and peaks at menses. However, with the onset of pregnancy, the corpus luteum is rescued and stromal cells, instead of undergoing apoptosis, reorganize the cytoskeleton and then begin to differentiate. We hypothesized that in the presence of hormones (estradiol-17beta and medroxyprogesterone acetate), chorionic gonadotropin (hCG) as an early embryonic signal, and induction of decidualization by dibutyryl-cAMP (dbcAMP), endometrial stromal cells are rescued by the regulation of proteins that inhibit apoptosis. The percentage of cells stained with annexin V, an early apoptotic marker, increased dramatically after cytoskeletal disruption with cytochalasin D compared with non-cytochalasin-D-treated controls (P < 0.05). However, treatment of cells with hCG or dbcAMP in the presence of hormones significantly (P < 0.05) decreased the percentage of annexin-V-stained cells compared with cells treated with cytochalasin D alone. This inhibition was further confirmed by immunodetection of cleaved caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. The inhibition of apoptosis by hCG and dbcAMP was via the intrinsic pathway because the cytochalasin-D-treated cells stained intensely for Bax, whereas the cells treated with hormones, hCG, or dbcAMP stained predominantly for Bcl-2. Treatment of cytochalasin-D-treated cells with hormones and dbcAMP resulted in an increase in the secretion of IGF-binding protein-1 (IGFBP-1) and prolactin. Treatment of cytochalasin-D-treated cells with recombinant IGFBP-1 and prolactin also inhibited apoptosis. These data suggest that under in vitro conditions, both hCG and the induction of decidualization play a direct role in preventing uterine stromal cells from undergoing apoptosis. Furthermore, this inhibition of apoptosis may be mediated in part by IGFBP-1 and prolactin and the alteration in the expression of Bcl-2 and Bax.

In vivo infusion of interleukin-1beta and chorionic gonadotropin induces endometrial changes that mimic early pregnancy events in the baboon.

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.

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.

Inhibition of matrix metalloproteinases prevents the synthesis of insulin-like growth factor binding protein-1 during decidualization in the baboon.

During pregnancy in the primate, uterine stromal fibroblasts are transformed into decidual cells. Decidualization is associated with extensive remodeling of the extracellular matrix (ECM). Matrix metalloproteinases (MMPs) play a pivotal role in ECM degradation. We hypothesized that MMPs also contribute to regulation of IGF binding protein-1 (IGFBP-1), a biochemical marker of primate decidual cells. We reported that IL-1beta (10 ng/ml) with steroid hormones [36 nm estradiol-17beta, 1 microm medroxyprogesterone acetate (P), and 100 ng/ml relaxin] induces in vitro IGFBP-1 synthesis. This study demonstrates that IL-1beta also induces stromelysin-1 (MMP-3) mRNA and synthesis of the latent form of MMP-3 (pro-MMP-3) protein in baboon stromal fibroblasts. In contrast, hormones (particularly P) negatively regulate MMP-3 because their addition decreases IL-1beta-induced pro-MMP-3 protein. The ERK and p38 MAPK pathways induced by IL-1beta regulate pro-MMP-3 because inhibitors PD98059 (20 microm) and SB203580 (1 microm) prevent its synthesis. The nuclear factor-kappaB inhibitory peptide, SN50 (50 microg/ml), or proteasome inhibitor, MG-132 (1 microm), did not inhibit pro-MMP-3 synthesis but appeared to enhance it. The role of MMPs in IGFBP-1 induction was investigated using a broad-spectrum MMP inhibitor, doxycycline, and specific MMP-3 inhibitor, N-Isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid (NNGH). Both inhibitors caused the dose-dependent decrease of IGFBP-1. alpha-Smooth muscle actin, which is down-regulated during decidualization, was partially up-regulated by doxycycline or N-Isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid. This suggests that alpha-smooth muscle actin is modulated by changes in ECM caused by the action of MMPs/MMP-3. Disruption of actin filaments enhances IGFBP-1 induction. Thus, our data imply that IL-1beta-induced MMPs and particularly MMP-3 may up-regulate IGFBP-1 by disrupting the actin cytoskeleton as a result of ECM degradation.

Signal transduction pathways activated by chorionic gonadotropin in the primate endometrial epithelial cells.

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.

Endometrial function: cell specific changes in the uterine environment.

The uterus undergoes dynamic changes during the cycle and these events are largely driven by ovarian steroids. However, in the presence of an embryo, an additional series of changes that are not otherwise observed predominate. The ability of the embryo to modulate the uterine environment is restricted to a specific time of the cycle which is termed the 'window of receptivity'. Changes that occur within this window of receptivity and immediately following implantation can be divided into three distinct phases. The first phase, regulated by estrogen and progesterone, is characterized primarily by changes in both the luminal and glandular epithelial cells in preparation for blastocyst apposition and attachment. If the action of progesterone is antagonized, these changes are inhibited and the uterus is maintained in a pre-receptive state. The second phase is in the further modulation of these steroids induced changes by embryonic signals. In the primate, infusion of chorionic gonadotropin in a manner that mimics blastocyst transit, results in the endoreplication and plaque formation in the luminal epithelium. The glandular epithelium responds by increasing transcriptional and post-translational modifications of secretory proteins and the stromal fibroblasts initiate their differentiation process into a decidual phenotype. The final phase is associated with trophoblast invasion and remodeling of the endometrial stromal compartment. The most dramatic effect is on the stromal fibroblasts, which in response to embryonic stimuli, differentiate into decidual cells, the major cell type of the gestational endometrium. Thus, during the window of receptivity, signals from the embryo can dramatically alter the morphological and functional characteristics of the uterine endometrium. We suggest that these changes are critical to ensure prolonged maintenance of endometrial function during gestation and facilitate trophoblast invasion.