Siamak Tabibzadeh

Life emerged on Earth in an anaerobic environment, bathed in noxious gases. Among these gases, the role of hydrogen sulfide is significant since this gas, was required as a building block of life, contributed to abiogenic generation of organic compounds that gave rise to life and drove adaptations of life throughout its entire evolutionary path. During evolution, hydrogen sulfide contributed to sustaining life in face of harsh environmental conditions. Modern cells still utilize hydrogen sulfide as a signaling molecule, in pro and anti-inflammatory responses, for acquisition of tolerance against damage, in directing repair responses, as a source of energy and in modifying their genetic makeup and function to acquire a phenotype reminiscent of early life forms.

LEFTY is expressed in normal endometrium in cells that decidualize. To understand the importance of this expression, we have studied the effect of LEFTY on decidualization in vitro and in vivo. Exposure of human uterine fibroblast (HuF) cells to recombinant LEFTY blocked the induction of the decidual differentiation-specific marker genes, IGFBP1 (IGF-binding protein 1) and PRL (prolactin) in response to medroxyprogesterone acetate, estradiol, and prostaglandin E2. The inhibitory effect was associated with decreased induction of the transcription factors ETS1 and FOXO1, both of which are essential for decidualization. Overexpression of LEFTY in decidualized HuF cells with an adenovirus that transduced LEFTY caused a marked decrease in IGFBP1 secretion, and withdrawal of medroxyprogesterone acetate from decidualized cells resulted in a decrease in IGFBP1 secretion and an increase in LEFTY expression. Moreover, overexpression of LEFTY in decidualized cells reprogrammed the cells to a less differentiated state and attenuated expression of decidual markers. Uterine decidualization was markedly attenuated and litter size was significantly reduced by retroviral transduction of LEFTY in the uterine horns of pregnant mice or by induction of LEFTY expression by doxycycline treatment in Tet-On conditional LEFTY transgenic pregnant mice. In addition, administration of the contraceptive agent drospirenone to ovariectomized mice induced a marked increase in LEFTY expression and inhibited decidualization. Taken together, these finding indicate that LEFTY acts as a molecular switch that modulates both the induction of decidual differentiation and the maintenance of a decidualized state. Because decidual cells express abundant amounts of LEFTY, the action of LEFTY on decidualization occurs by an autocrine mechanism.

Stem cells are functionally defined by their ability to self-renew and generate a progeny capable of creation or reconstitution of various tissues. Microarray analysis has shown a member of the transforming growth factor (TGF)-beta superfamily, Lefty, to be the single most abundant inhibitor in stem cells and in maternal decidua that supports embryo implantation. Lefty is regulated by pathways such as Smad (Sma and Mad [mothers against decapentaplegic]) and WNT (wingless-type) and by the transcriptional factor Oct3/4 (octamer-binding transcription factor 3/4), which support "stemness." Lefty is also induced upon exit from the state of stemness, including forced in vitro differentiation, and leukemia inhibitory factor withdrawal. Lefty is a candidate in cell-fate decisions because of its unique ability to modulate the expression of TGF-beta family proteins such as Nodal and by blanket inhibition of the activity of members of this family which require EGF-CFC (epidermal growth factor-Cripto, Frl-1, and Cryptic) as a coreceptor.

BACKGROUND: Reproductive tissues are unique structures that exhibit cyclic stromal remodelling during menstrual cycles in humans. Ebaf/lefty participates in tissue remodelling of human endometrium by induction of matrix metalloproteases (MMP).METHODS: We describe the temporal expression and spatial distribution of lefty and tissue remodelling events in mouse endometrium. RT–PCR and real-time PCR were used to identify mRNA expression and western blots to analyse Lefty protein. Immunolocalization was performed with specific antibodies and horseradish peroxidase staining. RESULTS: Lefty was expressed in endometrium throughout the estrous cycle. Expression of MMP (MMP-2, -3, -7 and -14) was higher at estrus, metestrus and/or diestrus while collagen content of endometrium decreased in these phases. During pregnancy, lefty levels were higher on days 3–5 and were minimal by day 9. Similarly, expression of endometrial MMP was higher on days 3 and 5 of pregnancy and was low on day 9. During pregnancy, loss of collagen was initiated on day 3, persisted to day 5, and led to a significantly reduced collagen on day 9. Immunoreactive lefty decorated basal laminae, and was associated with extracellular matrix in stroma. CONCLUSIONS: Regulated expression and spatial distribution of lefty in mouse endometrium confines its biological impact on tissues that undergo remodelling during estrous cycle and pregnancy.

BACKGROUND: Endometrium is a unique tissue that is prepared for implantation of blastocyst during each menstrual cycle. In humans, if implantation does not occur or fails, endometrium is shed. METHODS and RESULTS: We identified ebaf/lefty, as a key cytokine, highly expressed in human endometrium during the non-receptive phase of tissue remodelling. Lefty was increased in the endometria of a number of patients with ‘unexplained infertility’ during the receptive phase, suggesting dysregulation of lefty as a potential factor contributing to infertility. Here, we showed that induction of a similar state of lefty overexpression in endometrium, by in vivo gene delivery, decreased implantation in pregnant mice. This state of overexpression could be induced by a retroviral vector transducing lefty or by liposome-mediated introduction of a lefty expression vector. Analysis of endometrium showed increased lefty after in vivo gene transfer. CONCLUSION: These findings suggest that induction of a state of lefty overexpression in endometrium leads to reduced implantation.

A growing body of evidence suggests that endometriosis modulates the microenvironment of peritoneal cavity. Therefore, in this study, we compared the protein profile of peritoneal fluids from normal fertile women with those from patients with infertility, and patients with mild to severe endometriosis. Two-dimensional gel electrophoresis of peritoneal fluids from normal subjects exhibited a distinct and reproducible pattern of proteins in the size ranges of approximately 35 to 80 kD and pI close to 4.5 to 6.6. Infertility without evidence of endometriosis was not associated with changes in the relative abundance of proteins present in the peritoneal fluid. However, mild endometriosis was associated with a mild reduction in the amount of several peritoneal protein spots with the approximate molecular weights of 35-40 kD and pI close to 5.7-6.0. These changes became markedly apparent in the peritoneal fluid of patients who suffered from the severe form of this disease. Severe endometriosis was also associated with appearance of protein spots in the gels that were not detectable in the peritoneal fluids of normal subjects. Consistent with these data, enzyme-linked immunosorbent assay showed that moderate to severe endometriosis was associated with markedly elevated levels of IL-10 in the peritoneal fluid. Reverse transcription followed by polymerase chain reaction amplification using primers specific to IL-10 confirmed presence of IL-10 mRNA in cells derived from peritoneal fluids. These findings show that endometriosis is associated with disturbed secretion of proteins into the peritoneal cavity and with an elevated level of IL-10 in the peritoneal fluid. The studies also show cells resident in peritoneum as a major source of IL-10.

Human endometrium is a unique tissue that undergoes sequential phases of proliferation, and secretory changes followed by tissue shedding and bleeding during menstruation. Tissue remodeling is a distinct feature of human endometrium in the secretory phase which prepares endometrium for implantation during the "receptive phase" of the cycle. A discrete dissolution of extracellular matrix (ECM) by a host of enzymes called matrix metalloproteases (MMP) is required for a successful implantation. In the absence of implantation, as a result of progesterone withdrawal, human endometrium loses its receptive state in the premenstrual period and subsequently undergoes a generalized breakdown of ECM by MMPs during menstruation. The homeostasis of ECM of endometrium and the delicate balance between its synthesis and degradation appear to be mediated by reciprocal interaction between TGF-beta and ebaf (lefty) signaling. While TGF-beta acts as a pro-fibrogenic cytokine and maintains the integrity of ECM in endometrium, expression of lefty is associated with events that lead to destruction of ECM facilitating tissue shedding.

Both normal and neoplastic tissues have a stroma comprised of fibroblasts which deposit an extracellular matrix (ECM) enriched in collagen. In most normal tissues, the synthesis and breakdown of the ECM is maintained at a low level. However, in normal adult tissues such as endometrium, and in rapidly growing embryonic and neoplastic tissues, there is a significant increase in the synthesis and/or breakdown of ECM. The homeostasis of the ECM is maintained by a molecular repertoire which appears to consist of TGF-beta and lefty. TGF-beta acts as a pro-fibrogenic cytokine by increasing the synthesis of collagen and decreasing the degradation of ECM. Physiologic levels of TGF-beta maintains tissue homeostasis and aberrant over-expression of TGF-beta leads to tissue fibrosis. TGF-beta acts through a core Smad signaling pathway which is initiated by the binding of homo-dimeric TGF-beta protein to two type I and II receptors. The constitutively active receptor type II leads to phosphorylation of receptor type I which, in turn, causes the R-Smads to get phosphorylated. The downstream gene transcriptional activity of this event includes significant increase in connective tissue growth factor (CTGF) and collagen mRNA synthesis which leads to deposition of collagen in tissues. Lefty inhibits Smad2 phosphorylation initiated by TGF-beta or its receptor and prevents CTGF promoter activity driven by TGF-beta. Moreover, lefty inhibits CTGF and collagen mRNA synthesis and increases collagenolysis and elastolysis and as a result of these actions, lefty significantly reduces the amount of collagen deposited in tissues. Thus, TGF-beta and lefty might coordinately participate in the homeostasis of ECM in tissues.

Human endometrium is a unique tissue that undergoes sequential phases of proliferation, and secretory changes followed by tissue shedding and bleeding during menstruation. Tissue remodeling is a distinct feature of human endometrium in the secretory phase which prepares endometrium for implantation during the "receptive phase" of the cycle. A discrete dissolution of extracellular matrix (ECM) by a host of enzymes called matrix metalloproteases (MMP) is required for a successful implantation. In the absence of implantation, as a result of progesterone withdrawal, human endometrium loses its receptive state in the premenstrual period and subsequently undergoes a generalized breakdown of ECM by MMPs during menstruation. The homeostasis of ECM of endometrium and the delicate balance between its synthesis and degradation appear to be mediated by reciprocal interaction between TGF-beta and ebaf (lefty) signaling. While TGF-beta acts as a pro-fibrogenic cytokine and maintains the integrity of ECM in endometrium, expression of lefty is associated with events that lead to destruction of ECM facilitating tissue shedding.