Justine S. Fitzgerald

Trophoblast invasion: the role of intracellular cytokine signalling via signal transducer and activator of transcription 3 (STAT3)

Trophoblast cells display a very unique capability: they physiologically invade into the surrounding tissue. This capability is widely associated with tumours, and, indeed, the invasive behaviour of both is rather similar. The imposing difference is that trophoblast cell invasion is temporally and locally controlled in contrast to unlimited tumour invasion. It initiates immediately after embryo implantation into the endometrium. Parallel to tumours, trophoblasts secrete proteases, such as matrix metalloproteinases, which dissolve the extracellular matrix and the surrounding tissue. Thereby, these proteases prepare and allow true invasion of trophoblasts. The invasive capacities of trophoblasts are positively and negatively regulated by numerous cytokines including leukaemia inhibitory factor (LIF), interleukin-6, hepatocyte growth factor, granulocyte macrophage-colony stimulating factor and others. They interact via specific receptors with the trophoblast cells, in which they activate intracellular signalling cascades. These will then induce expression of invasion relevant genes. One of these signalling pathways is the Janus kinase/signal transducers and activators of transcription (STAT) pathway. Especially phosphorylated STAT3 enhances invasiveness of tumours and trophoblast cells, where it is mainly activated by LIF. One of its most efficient physiological antagonists is suppressor of cytokine signalling 3. The balance of these two intracellular molecules seems to be a key regulator of tumour and trophoblast invasion.

Signal Transduction in Trophoblast Invasion

During the first trimester of pregnancy, well-differentiated primary cells of the placenta known as trophoblast cells grow in an invasive and destructive fashion similar to malignancies, but limited in space and time. The comparison of trophoblast cells with their malignant counterpart, human choriocarcinoma cells, offers an attractive model to understand the origin or development of malignant growth. Several cytokines and growth factors are known to influence trophoblast migration (e.g. EGF, IGF-2, HGF), proliferation (e.g. leptin, HGF, GM-CSF) and/or invasion (e.g. leukemia inhibitory factor, LIF), each factor utilizing at least one pathway for intracellular signaling in the trophoblast. Two pathways that are crossed especially often mediate the signals of these factors and are simultaneously well established in terms of tumor invasion: the Janus kinase-signal transducers and activators of transcription (Jak-Stat) and receptor-associated tyrosine kinase-mitogen-activated protein kinase (RTK-MAPK) pathways. These two pathways are detrimental for reproduction in general, and in part for placenta development, as a series of knockout experiments demonstrate. Aspects of each pathway are also implicated to be involved in trophoblast invasion, e.g. STAT3 is constitutively activated in invasive first trimester trophoblast cells, and activated ERK is detectable in intermediate trophoblast cells, an invasive phenotype. Interaction at several intersection points between the pathways has been described in several cell systems so that the same would seem to be possible in trophoblast cells. In this review, some of the possible areas of interaction are alluded to.

Evidence for a Correlation between Trophoblast Invasiveness and STAT3 Activity

Problem: Extravillous trophoblast cells are capable of invading decidual tissue during early pregnancy. This property is reminiscent of cancer cells. The invasiveness of trophoblasts, however, extends only to a well‐regulated limit. Signal transduction processes underlying this phenomenon are as yet poorly characterized. Many factors involved in trophoblast invasiveness are known to trigger intracellular signaling cascades in other cell types that ultimately lead to the activation of signal transducers and activators of transcription (STATs). STAT3 activity was recently found related to the malignant phenotype of different tumor cells and potentially contributes to their invasive properties.

Governing the invasive trophoblast: current aspects on intra- and extracellular regulation.

Citation Fitzgerald JS, Germeyer A, Huppertz B, Jeschke U, Knöfler M, Moser G, Scholz C, Sonderegger S, Toth B, Markert UR. Governing the invasive trophoblast: current aspects on intra‐ and extracellular regulation. Am J Reprod Immunol 2010

REVIEW ARTICLE: Governing the Invasive Trophoblast: Current Aspects on Intra- and Extracellular Regulation

Citation Fitzgerald JS, Germeyer A, Huppertz B, Jeschke U, Knöfler M, Moser G, Scholz C, Sonderegger S, Toth B, Markert UR. Governing the invasive trophoblast: current aspects on intra‐ and extracellular regulation. Am J Reprod Immunol 2010

Inhibition of term decidual NK cell cytotoxicity by soluble HLA-G1

Objectives  Soluble (s)HLA‐G1 is produced by trophoblast cells. Aim was to analyze the capacities and mechanisms of sHLA‐G1 to regulate interleukin (IL)‐2‐induced cytotoxicity of natural killer (NK) cells from human deciduas.

Knocking off the suppressors of cytokine signaling (SOCS): their roles in mammalian pregnancy

This review discusses the possible role of the suppressor of cytokine signaling (SOCS) proteins in mammalian reproduction. SOCS are regulatory proteins that are rapidly transcribed in response to intracellular Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling, a cascade governing biological functions including cytokine-induced immunological responses and reproductive processes. For instance STAT3 appears to mediate trophoblast invasion induced by LIF. The SOCS family includes 8 members (cytokine-inducible SH2 protein [CIS] and SOCS1-7) that orchestrate distinct reactions by antagonizing STAT activation. Emerging evidence points to a role of some family members in synchronizing Th1/Th2 cell profiles, the balance in which is considered vital to pregnancy maintenance. The reproductive phenotypes of mutant mice harboring targeted disruption of SOCS gene isoforms offer insights for reproductive immunology, trophoblast function and human pregnancy. CIS transgenic mice display impaired responses to IL-2 and resemble STAT5 deficient mice, except they are fertile. SOCS1 deficiency leads to an overabundance of IFNgamma signaling, yet SOCS1 null mutant mice are able to reproduce. Lack of SOCS3 is embryonically lethal due to placental insufficiency, while SOCS3 over-expression leads to elevated Th2 responses. SOCS3 seems to be vital for reproduction by regulating LIF-driven trophoblast differentiation. SOCS5 inhibits IL-4 signaling, yet the SOCS5 transgenic mouse has no conspicuous reproductive phenotype. SOCS-6 and SOCS-7 null mutant mice display growth retardation. In summary, SOCS proteins are avidly involved in fine regulation of immunological and other vital cellular responses. Many of the above phenotypes present contradictions to accepted reproductive immunological paradigms.

HTR8/SVneo Cells Display Trophoblast Progenitor Cell-Like Characteristics Indicative of Self-Renewal, Repopulation Activity, and Expression of “Stemness-” Associated Transcription Factors

Introduction. JEG3 is a choriocarcinoma—and HTR8/SVneo a transformed extravillous trophoblast—cell line often used to model the physiologically invasive extravillous trophoblast. Past studies suggest that these cell lines possess some stem or progenitor cell characteristics. Aim was to study whether these cells fulfill minimum criteria used to identify stem-like (progenitor) cells. In summary, we found that the expression profile of HTR8/SVneo (CDX2+, NOTCH1+, SOX2+, NANOG+, and OCT-) is distinct from JEG3 (CDX2+ and NOTCH1+) as seen only in human-serum blocked immunocytochemistry. This correlates with HTR8/SVneos self-renewal capacities, as made visible via spheroid formation and multi-passagability in hanging drops protocols paralleling those used to maintain embryoid bodies. JEG3 displayed only low propensity to form and reform spheroids. HTR8/SVneo spheroids migrated to cover and seemingly repopulate human chorionic villi during confrontation cultures with placental explants in hanging drops. We conclude that HTR8/SVneo spheroid cells possess progenitor cell traits that are probably attained through corruption of “stemness-” associated transcription factor networks. Furthermore, trophoblastic cells are highly prone to unspecific binding, which is resistant to conventional blocking methods, but which can be alleviated through blockage with human serum.

Understanding the link between the IL-6 cytokine family and pregnancy: implications for future therapeutics

Cytokines are involved in almost all processes during the menstrual cycle, the fertilization period and pregnancy. They are expressed in numerous reproduction-related body fluids and tissues. Disorders of cytokine expression patterns may cause pregnancy pathologies. Therefore, cytokines have the potential as new biomarkers in different body compartments for a variety of such pathologies. Furthermore, cytokines may also serve to treat fertility and pregnancy disorders. The IL-6-like family of cytokines is an intensively investigated group of cytokines with well-accepted functions in fertility and pregnancy. This article summarizes current knowledge on IL-6-like cytokines in regard of their role in reproduction and their potential for new strategies in the treatment of reproductive pathologies.

Intranuclear crosstalk between extracellular regulated kinase1/2 and signal transducer and activator of transcription 3 regulates JEG-3 choriocarcinoma cell invasion and proliferation.

Invasiveness of trophoblast and choriocarcinoma cells is in part mediated via leukemia inhibitory factor- (LIF-) induced activation of signal transducer and activator of transcription 3 (STAT3). The regulation of STAT3 phosphorylation at its ser727 binding site, possible crosstalk with intracellular MAPK signaling, and their functional implications are the object of the present investigation. JEG-3 choriocarcinoma cells were cultured in presence/absence of LIF and the specific ERK1/2 inhibitor (U0126). Phosphorylation of signaling molecules (p-STAT3 (ser727 and tyr705) and p-ERK1/2 (thr 202/tyr 204)) was assessed per Western blot. Immunocytochemistry confirmed results, but also pinpointed the location of phosphorylated signaling molecules. STAT3 DNA-binding capacity was studied with a colorimetric ELISA-based assay. Cell viability and invasion capability were assessed by MTS and Matrigel assays. Our results demonstrate that LIF-induced phosphorylation of STAT3 (tyr705 and ser727) is significantly increased after blocking ERK1/2. STAT3 DNA-binding capacity and cell invasiveness are enhanced after LIF stimulation and ERK1/2 blockage. In contrast, proliferation is enhanced by LIF but reduced after ERK1/2 inhibition. The findings herein show that blocking ERK1/2 increases LIF-induced STAT3 phosphorylation and STAT3 DNA-binding capacity by an intranuclear crosstalk, which leads to enhanced invasiveness and reduced proliferation.