Role of EBAF/Lefty in implantation and uterine bleeding.
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Collagen↗
Biomedical subjects
S Tabibzadeh
Publications and source records attributed to S Tabibzadeh.
Lefty proteins exhibit unique processing and activate the MAPK pathway.
Lefty polypeptides, novel members of the transforming growth factor-beta (TGF-beta) superfamily, are involved in the formation of embryonic lateral patterning. Members of the TGF-beta superfamily require processing for their activation, suggesting cleavage to be an essential step for lefty activation. Transfection of different cell lines with lefty resulted in expression of a 42-kDa protein, which was proteolytically processed to release two polypeptides of 34 and 28 kDa. Since members of the proprotein convertase (PC) family cleave different TGF-beta factors and are involved in the establishment of embryonic laterality, we studied their role in lefty processing. Cotransfection analysis showed that PC5A processed the lefty precursor to the 34-kDa form in vivo, whereas furin, PACE4, PC5B, and PC7 had a limited activity. None of these PCs showed activity in the processing of the lefty polypeptide to the 28-kDa lefty form. The mutation of the consensus sequences for PC cleavage in the lefty protein allowed the lefty cleavage sites to be identified. Mutations of the sequence RGKR to GGKG (amino acids 74-77) and of RHGR to GHGR (amino acids 132-135) prevented the proteolytic processing of the lefty precursor to the 34- and 28-kDa forms, respectively. To identify the biologically active form of lefty, we studied the effect of lefty treatment on pluripotent P19 cells. Lefty did not induce Smad2 or Smad5 phosphorylation, Smad2/Smad4 heterodimerization, or nuclear translocation of Smad2 or Smad4, but activated the MAPK pathway in a time- and dose-dependent fashion. Further analysis showed the 28-kDa (but not the 34-kDa) polypeptide to induce MAPK activity. Surprisingly, the 42-kDa lefty protein was also capable of inducing MAPK activity, indicating that the lefty precursor is biologically active. The data support a molecular model of processing as a mechanism for regulation of lefty signaling.
3T3 Cells↗
Lefty inhibits receptor-regulated Smad phosphorylation induced by the activated transforming growth factor-beta receptor.
Transforming growth factor-beta (TGF-beta) is a pleiotropic cytokine that regulates growth and differentiation of diverse types of cells. TGF-beta actions are directed by ligand-induced activation of TGF-beta receptors with intrinsic serine/threonine kinase activity that trigger phosphorylation of receptor-regulated Smad (R-Smad) protein. Phosphorylated R-Smad proteins bind to Smad4, and the complexes formed move into the nucleus, where they act as components of a transcriptional complex. Here, we show that TGF-beta signaling is inhibited by lefty, a novel member of the TGF-beta superfamily. Lefty perturbed TGF-beta signaling by inhibiting the phosphorylation of Smad2 following activation of the TGF-beta receptor. Moreover, lefty inhibited the events that lie downstream from R-Smad phosphorylation, including heterodimerization of R-Smad proteins with Smad4 and nuclear translocation of the R-Smad.Smad4 complex. Lefty repressed TGF-beta-induced expression of reporter genes for the p21, cdc25, and connective tissue growth factor promoters and of a reporter gene driven by the Smad-binding element. Similarly, lefty inhibited both BMP-mediated Smad5 phosphorylation and gene transcription. The action of lefty does not appear to depend on protein synthesis, including synthesis of inhibitory Smad proteins. Thus, lefty provides a repressed state of TGF-beta- or BMP-responsive genes and participates in negative modulation of TGF-beta and BMP signaling by inhibition of phosphorylation of R-Smad proteins.
Animals↗
Endometrial osteopontin, a ligand of beta3-integrin, is maximally expressed around the time of the "implantation window".
OBJECTIVE: To analyze endometrial mRNA and protein expression of osteopontin and its receptor, beta3-integrin, throughout the menstrual cycle. DESIGN: Study by immunohistochemistry, RNase protection assay, and ELISA. SETTING: Academic research unit. PATIENT(S): Forty-five regularly cycling women without endometrial pathology. INTERVENTION(S): Expression of endometrial osteopontin and beta3-integrin mRNA was analyzed by RNase protection assay in endometrium, endometrial epithelial cells, stromal cells, and endometrial leukocytes (CD45) and by immunohistochemistry in frozen sections of endometrium throughout the menstrual cycle. Concentration of osteopontin was studied in uterine secretions by ELISA. MAIN OUTCOME MEASURE(S): mRNA and protein expression of osteopontin and beta3-integrin. RESULT(S): Osteopontin mRNA and protein was weakly expressed in the proliferative phase and maximally expressed in the mid to late secretory phases in endometrium, endometrial epithelial cells, and endometrial leukocytes and in uterine secretions. Beta3-integrin mRNA and protein were expressed in stromal cells throughout the menstrual cycle and were maximally expressed in epithelial cells in the mid to late secretory phases. CONCLUSION(S): Expression of osteopontin and its receptor, beta3-integrin, in human endometrial glands and osteopontin secretion into the uterine cavity around the time of the "implantation window" suggest a role for these proteins in endometrial function and implantation.
Adult↗
"Literature update 1999, part 3".
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Animals↗
Progestin regulation of human endometrial function.
A well-orchestrated sequence of events enables the human endometrium to become receptive to embryo implantation during a defined period in the menstrual cycle. Ovarian steroids, oestradiol and progesterone, regulate many of these functions in a highly co-ordinated manner. There are no experimental systems for critically examining the regulation of endometrial functions by steroid hormones, especially those responses restricted to the epithelium. Using an experimental xenograft model where the steroid responses of normal endometrium could be predictably recapitulated, the role of progesterone in the regulation of alpha crystallin B in the glandular epithelial cells of human endometrium was established.
Animals↗
Regulated expression of cytokines in human endometrium throughout the menstrual cycle: dysregulation in habitual abortion.
It is widely assumed that, after ovulation, the human endometrium undergoes specific changes and becomes receptive to the implantation of embryo during the mid-secretory phase. When implantation does not take place, further changes occur which eventually result in the shedding of human endometrium. The present study was carried out to examine whether there are changes in the cytokine gene expression in human endometrium which are correlated with endometrial function in various phases of the menstrual cycle. The RNase protection assay was performed on carefully dated endometria from normal subjects to characterize the expression of cytokines which potentially contribute to endometrial function. These included: tumour necrosis factor (TNF), interleukin (IL)-1beta, IL-6, IL-8, leukaemia inhibitory factor (LIF), transforming growth factor beta1 (TGF-beta1), macrophage colony stimulating factor (MCSF or colony stimulating factor-1), and vascular endothelial growth factor (VEGF) mRNAs. A low level of expression of these cytokine mRNAs was found during the proliferative and early secretory phase. Expression of cytokine mRNA increased during the mid-secretory phase and rose to a peak in the late secretory phase. The level of cytokine mRNA expression during gestation was most akin to that observed during the mid-secretory phase. Individuals with habitual abortion presented with an abnormal expression of IL-1beta and IL-6 mRNA in endometrium, during the mid-secretory phase. Taken together, these findings are consistent with a progressive rise in the expression of cytokines in human endometrium during the secretory phase in natural cycles. Furthermore, the findings show that habitual abortion is associated with the abnormal expression of IL-1beta and IL-6 in the mid-secretory phase.
Abortion, Habitual↗
Identification of a prodromal period in Crohn's disease but not ulcerative colitis.
OBJECTIVES: Irritable bowel syndrome, a common gastrointestinal diagnosis, has not been clearly studied in inflammatory bowel disease. Some of the residual symptoms in subjects treated with Crohn's disease and ulcerative colitis are thought to be related to irritable bowel syndrome. The aims of this study were 1) to describe the duration and nature of complaints before the diagnosis of Crohn's disease and ulcerative colitis (prodromal period), and 2) to determine the role of IBS in this prodromal period. METHODS: A total of 66 patients with confirmed inflammatory bowel disease were enrolled in the study. The subjects received a questionnaire to ascertain the nature and duration of symptoms preceding the diagnosis of Crohn's disease or ulcerative colitis, including features described under the Rome criteria for irritable bowel syndrome. RESULTS: Of the 66 subjects analyzed, 45 had Crohn's disease and 21 had ulcerative colitis. The prodromal period was 7.7 +/- 10.7 yr for Crohn's disease and 1.2 +/- 1.8 yr for ulcerative colitis (p < 0.05). Once patients meeting the Rome criteria for irritable bowel syndrome during the prodrome were excluded, the duration of the prodromal period (non-IBS) for ulcerative colitis dropped to 0.8 +/- 1.3 yr compared to 6.9 +/- 9.8 yr in the Crohn's disease group (p < 0.05). The symptoms of the non-IBS prodrome in subjects with Crohn's disease were bloating, diarrhea, stomach pain, heartburn, fever, weight loss, and fatigue. Further analysis demonstrated that subjects whose Crohn's disease initially began as colonic disease had a longer prodrome than with small bowel. In the non-IBS Crohn's group, there was also a correlation between the age at the time of diagnosis and the duration of prodrome (r = 0.67, p < 0.0001). CONCLUSIONS: There is a significant prodromal period before the time of diagnosis of Crohn's disease that is not found in ulcerative colitis even after exclusion of subjects with IBS.
Adult↗
Dysregulated expression of ebaf, a novel molecular defect in the endometria of patients with infertility.
We recently described the expression of ebaf, a novel member of the transforming growth factor-beta superfamily in human endometrium. ebaf messenger ribonucleic acid was expressed in late secretory and menstrual endometria. Here, we show that ebaf is secreted as 42-, 34-, 28-, and 14-kDa proteins into the conditioned medium of transfected cells, endometrial fluid, and serum. The amount of secreted proteins was markedly reduced during the implantation window in the endometria and sera of normal fertile subjects. The expression of ebaf was dysregulated in the endometria of a subset of women with infertility during the receptive phase of the menstrual cycle. Abundant secreted protein was present in the endometria of these women during the implantation window. During the critical period of endometrial receptivity, ebaf protein was more abundant in patients with endometriosis who did not conceive than in patients who became pregnant. These findings show that ebaf is a secreted product and is released into body fluids. Some types of infertility are associated with dysregulated expression of ebaf in human endometrium, suggesting that a molecular defect in uterine receptivity may be identified using such a marker protein.
Adult↗
An experimental model for the endometriosis in athymic mice.
Endometriosis is an adhesion disorder characterized by the presence of endometrial tissue in ectopic sites outside the uterus. The disease is associated with dysmenorrhea, pelvic pain and infertility. Although endometriosis is the most common gynecologic disorder, relatively little is known regarding its etiology, pathogenesis and the course of the disease. This situation is primarily due to the absence of experimental systems to examine the mechanism of endometrial cell adhesion, role of inflammatory cells and the interactions of epithelial, and stromal cells with the peritoneum and ovarian tissue leading to the development of this disorder. Dissociated human endometrial cells were suspended in peritoneal fluids of individuals with and without endometriosis and were injected into the peritoneal cavity of athymic mice. This led to development of ectopic adhesions of endometrial cells at the peritoneal and ovarian surfaces. Endometrial cells which were marked with fluorescent lipophylic dyes, prior to intraperitoneal injection, could be visualized without surgery at such sites. The studies demonstrate a model for endometriosis in athymic mice.
Adult↗
Multiprobe RNase protection assay with internally labeled radioactive probes, generated by RT-PCR and nested PCR.
RNase Protection Assay (RPAs) is a highly sensitive and reproducible method of quantitating the levels of specific mRNA transcripts. The introduction of the commercially available Multiprobe RPAs allow comparing and quantifying the expression of up to different mRNA species in a single sample of 1-20 micrograms of total RNA. To generate probes which are not commercially available, we prepared highly specific probes by RT-PCR and nested PCR. Then, after ligation of a T7 promoter, another PCR was performed with a primer set consisting of a specific sense primer and antisense T7 primer. Only the antisense strand of the double stranded PCR-product contained the T7-promoter sequence on its 5' end, allowing in vitro transcription and internal labeling with [alpha-32]UTP. Probe concentration was determined in a scintillation counter and equal counts were introduced in the assay. In vitro transcription of the PCR generated probes resulted in radioactive probes with a very high specific activity, allowing simultaneous analysis of 70 different RNA samples. RPA could be performed under the same conditions as recommended for the commercially available probe sets, avoiding time consuming optimization of reaction conditions. Negative controls consisted of yeast RNA and sense RNA probes. Positive controls were single stranded templates, generated by asymmetric PCR. Dilution series revealed a high reproducibility and the potential of this technique to semi-quantitate mRNA in different RNA samples. In conclusion, probes may be generated by RT-PCR and nested PCR that will work with the commercially available Multiprobe RPAs. The high probe yield allows analysis of a great number of samples using the same set of probes with a high reproducibility.
Antigens, CD↗
From endometrial receptivity to infertility.
Implantation of the blastocyst in endometrium requires establishment of a coordinated molecular dialogue between the embryo and the endometrium. Factors instrumental in the preparation of a receptive endometrium are derived from the hypothalamic-pituitary-gonadal axis. These factors modulate the expression of genes that drive the endometrium throughout the characteristic menstrual cycles. During each menstrual cycle, a series of coordinated, architectural, morphological, cytochemical, and molecular changes ultimately lead to the preparation of a receptive endometrium during the putative "receptive period" or "implantation window." It is during this critical period that a proper dialogue can be established between an intrusive blastocyst and a receptive endometrium. If, for any reason, this dialogue is not established or is perturbed, the embryo is aborted. The natural fate of the receptive endometrium, in the absence of implantation, is development of a second set of changes that ultimately lead to menstruation. The identity of the molecular repertoire that makes endometrium receptive to implantation and/or lead to menstruation is being revealed and broadly includes cytokines, heat shock factors, adhesion molecules and matrix metalloproteases. We identified a novel gene of the transforming growth factor-beta, superfamily of molecules, the so-called endometrial bleeding--associated factor or ebaf, whose expression is confined to the late secretory and menstrual phases. Various forms of female infertility were associated with dysregulated expression of ebaf during the implantation window. The findings show an occult molecular defect of endometrial receptivity that seems to be due to dysregulated and premature expression of a member of the premenstrual molecular repertoire. The dysregulated expression of ebaf may assist in the identification, prognostication, and monitoring of treatment of infertile women.
Embryo Implantation↗
Regulation of TNF-alpha mRNA expression in endometrial cells by TNF-alpha and by oestrogen withdrawal.
During each menstrual cycle, the human endometrium undergoes a series of orchestrated and well controlled changes in anticipation of the arrival of the blastocyst. In the absence of implantation, the endometrium is shed. The underlying basis of the menstrual bleeding is not clear, however, it seems to be related to steroid hormone withdrawal. We showed that tumour necrosis factor-alpha (TNF-alpha) is released by human endometrium and that endometrial epithelial cells are a major source of TNF-alpha mRNA and protein. We show here that TNF-alpha mRNA shows a specific menstrual cycle-dependent expression. The expression of TNF-alpha is mostly minimal throughout the proliferative, early and mid-secretory phases. Expression of TNF-alpha mRNA, however, is increased in the human endometrium in the late secretory phase and during endometrial bleeding. Such a menstrual cycle-dependent expression suggests that specific signals regulate the expression of TNF-alpha mRNA in the human endometrium. In vitro, the expression of TNF-alpha mRNA in endometrial epithelial cells could be regulated by exogenous TNF-alpha. This induced expression was both time- and dose-dependent. In vitro, the TNF-alpha mRNA expression was not altered by oestrogen, progesterone, or both, in the endometrial epithelial cells under conditions that maintain the steroid hormone receptors. However, in vivo, oestrogen withdrawal led to an enhanced expression of TNF-alpha in endometrial epithelial cells. These findings suggest that the up-regulation of TNF-alpha in human endometrium in the late secretory phase may be related to the falling serum oestrogen concentration at the end of the menstrual cycle as well as the potentiating effect of released TNF-alpha on its own mRNA expression.
Adult↗
Heat shock proteins in human endometrium throughout the menstrual cycle.
Human endometrium, in response to steroid hormones, undergoes characteristic cycles of proliferation, secretory changes, and tissue shedding. Human endometrium expresses a molecular repertoire which includes the heat shock proteins (Hsps) Hsp27, Hsp60, Hsp70, Hsp90, and alpha crystallin B chain. The expression of Hsp27, Hsp60, and the constitutive form of Hsp70 (Hsc70) shows a sharp increase in human endometrium after ovulation. The maximal expression of the molecular chaperone, alpha crystallin B chain, occurs during the secretory phase. In view of known functions of the Hsps, it is likely that these proteins are involved in protection of the endometrial proteins against factors with the potential to lead to protein denaturation. Tumor necrosis factor-alpha (TNF-alpha) is a cytotoxic cytokine that is produced in progressive amounts during the secretory phase. The function of the Hsps may be to protect cells against the cytotoxic damage of TNF-alpha, particularly during the critical period of "implantation window."
Endometrium↗
Temporal and site-specific expression of transforming growth factor-beta4 in human endometrium.
We recently identified a novel member of the transforming growth factor (TGF)-beta superfamily and showed that this gene, designated as endometrial bleeding associated factor (ebaf), or TGFbeta4, has a unique expression pattern in human endometrium. By Northern blot analysis, we showed that this gene was expressed in human endometrium during the late secretory and menstrual phases and was absent in proliferative, early and mid-secretory endometria. In this report, we show by in-situ hybridization that the mRNA of the TGF-beta4 is not expressed in the proliferative endometria. On the other hand, focal expression of the TGFbeta4 mRNA first appears in some endometrial glands in the mid-secretory phase. The TGFbeta4 mRNA is strongly expressed in the endometrial stroma during the late secretory and menstrual phases of the cycle. We raised a polyclonal rabbit antiserum against a peptide at the C terminal of the protein. Western blot analysis using affinity purified antiserum shows that the TGFbeta4 precursor detected in the endometrium as well as placenta is 41 kDa. Bands in the range of 45-51 kDa are also present in human endometrium, more predominantly during the late secretory phase. Immunohistochemical staining shows a low level of immunoreactivity for TGFbeta4 in the early, mid- and late proliferative and early and mid-secretory endometria. A strong immunoreactivity for TGFbeta4 is present in the stroma and to lesser extent in the endometrial glands in late secretory and menstrual endometria. The specificity of staining was shown by neutralizing the activity of the antibody with the synthetic peptide used for raising the antibody and by omitting the antibody. The findings show that TGFbeta4, both at the mRNA and protein levels, exhibits temporal and site specific expression in human endometrium.
Amino Acid Sequence↗
Implantation: from basics to the clinic.
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Adolescent↗
Distinct tumor specific expression of TGFB4 (ebaf)*, a novel human gene of the TGF-beta superfamily.
We recently identified a novel gene of the TGF-beta superfamily, endometrial bleeding associated factor, TGFB4 (ebaf), that, throughout the menstrual cycle, exhibited a defined expression in human endometrium. Here, we report on the expression of TGFB4 (ebaf) in normal and neoplastic human tissues. The expression of this gene was absent in a host of normal tissues including lung, stomach, small bowel, liver, kidney, breast, lymph node, spleen, ovary and fallopian tube. However, a weak expression of the 2.1 kb variant of the TGFB4 (ebaf) mRNA was observed in rectal, ovarian, and testicular tissues and the 2.1 and 2.5 kb TGFB4 (ebaf) mRNAs were observed in the pancreatic tissue. The expression of the mRNA of this gene was absent in sarcomas, Hodgkin's and non-Hodgkin's lymphomas, melanomas, squamous cell carcinomas, hepatocellular carcinomas, renal cell carcinomas, and adenocarcinomas of the breast, endometrium and lung. The expression of the TGFB4 (ebaf) mRNA was observed primarily in adenocarcinomas that exhibited a mucinous differentiation. This included colonic, duodenal, and ovarian adenocarcinomas. The expression of TGFB4 (ebaf) mRNA was absent in non- mucinous colonic, gastric and ovarian adenocarcinomas and adenocarcinomas of colon metastatic to the liver. However, some serous adenocarcinomas of the ovary also exhibited TGFB4 (ebaf) mRNA. The testicular tumors, seminomas and embryonal carcinomas, also expressed TGFB4 (ebaf) mRNA. These findings show that the TGFB4 (ebaf) mRNA has distinct tumor specific expression.
Adenocarcinoma↗
Detection of ebaf, a novel human gene of the transforming growth factor beta superfamily association of gene expression with endometrial bleeding.
Human endometrium is unique since it is the only tissue in the body that bleeds at regular intervals. In addition, abnormal endometrial bleeding is one of the most common manifestations of gynecological diseases, and is a prime indication for hysterectomy. Here, we report on a novel human gene, endometrial bleeding associated factor (ebaf), whose strong expression in endometrium was associated with abnormal endometrial bleeding. In normal human endometrium, this gene was transiently expressed before and during menstrual bleeding. In situ hybridization showed that the mRNA of ebaf was expressed in the stroma without any significant mRNA expression in the endometrial glands or endothelial cells. The predicted protein sequence of ebaf showed homology with and structural features of the members of TGF-beta superfamily. Fluorescence in situ hybridization showed that the ebaf gene is located on human chromosome 1 at band q42.1. Thus, ebaf is a novel member of the TGF-beta superfamily and an endometrial tissue factor whose expression is associated with normal menstrual and abnormal endometrial bleeding.
Adult↗