Marija Schwirtlich

GABAA and GABAB receptors of distinct properties affect oppositely the proliferation of mouse embryonic stem cells through synergistic elevation of intracellular Ca2

Gamma‐amminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system of vertebrates, serves as an autocrine/ paracrine signaling molecule during development, modulating a number of calcium (Ca2+)‐dependent processes, including proliferation, migration, and differentiation, acting via 2 types of GABA receptors (GABARs): ionotropic GABAARs and metabotropic GABABRs. Here, we demonstrate that mouse embryonic stem cells (mESCs), which possess the capacity for virtually unlimited self‐renewal and pluripotency’ synthesize GABA and express functional GABAARs and GABABRs, as well as voltage‐gated calcium channels (VGCCs), ryanodine receptors (RyRs), and inwardly rectifying potassium (GIRK) channels. On activation, both GABAR types triggered synergistically intracellular calcium rise. Muscimol (a GABAAR agonist) induced single Ca2+ transients involving both VGCC‐mediated Ca2+ influx and intracellular stores, while baclofen (a GABABR agonist) evoked Ca2+ transients followed by intercellular Ca2+ waves and oscillations that were resistant to antagonists and entirely dependent on Ca2+ release from intracellular stores. Prolonged treatment with muscimol slightly inhibited, while baclofen or SR95531 (a GABAAR antagonist) significantly facilitated, mESC proliferation. GABAAR‐specific ligands also induced morphological and gene expression changes indicating a differentiation shift. Our data suggest that the interplay between GABARs and downstream (coupled) effectors differentially modulates mESC proliferation/differentiation through selective activation of second messenger signaling cascades.— Schwirtlich, M., Emri, Z., Antal, K., Maté, Z., Katarova, Z., Szabo, G. GABAA and GABAB receptors of distinct properties affect oppositely the proliferation of mouse embryonic stem cells through synergistic elevation of intracellular Ca2+. FASEB J. 24, 1218–1228 (2010). www.fasebj.org

GAD isoforms exhibit distinct spatiotemporal expression patterns in the developing mouse lens: Correlation with Dlx2 and Dlx5

Gamma‐aminobutyric acid (GABA), the major inhibitory neurotransmitter of the adult nervous system and its biosynthetic enzyme glutamic acid decarboxylase (GAD) are abundantly expressed in the embryonic nervous system and are involved in the modulation of cell proliferation, migration, and differentiation. Here we describe for the first time the expression of GABA and embryonic and adult GAD isoforms in the developing mouse lens. We show that the GAD isoforms are sequentially induced with specific spatiotemporal profiles: GAD65 and embryonic GAD isoforms prevail in primary fibers, while GAD67 is the predominant GAD expressed in the postnatal secondary fibers. This pattern correlates well with the expression of Dlx2 and Dlx5, known as upstream regulators of GAD. GABA and GAD are most abundant at the tips of elongating fibers and are absent from organelle‐free cells, suggesting their involvement is primarily in shaping of the cytoskeleton during fiber elongation stages. Developmental Dynamics 236:3532–3544, 2007.

Expression analysis of SOX14 during retinoic acid induced neural differentiation of embryonal carcinoma cells and assessment of the effect of its ectopic expression on SOXB members in HeLa cells.

SOX14 is a member of the SOXB2 subgroup of transcription factors implicated in neural development. Although the first SOX14 gene in vertebrates was cloned and characterized more than a decade ago and its expression profile during development was revealed in various animal model systems, the role of this gene during neural development is largely unknown. In the present study we analyzed the expression of SOX14 in human NT2/D1 and mouse P19 pluripotent embryonal carcinoma cells. We demonstrated that it is expressed in both cell lines and upregulated during retinoic acid induced neural differentiation. We showed that SOX14 was expressed in both neuronal and non-neuronal differentiated derivatives, as revealed by immunocytochemistry. Since it was previously proposed that increased SOXB2 proteins level interfere with the activity of SOXB1 counteracting partners, we compared expression patterns of SOXB members during retinoic acid induction of embryonal carcinoma cells. We revealed that upregulation of SOX14 expression is accompanied by alterations in the expression patterns of SOXB1 members. In order to analyze the potential cross-talk between them, we generated SOX14 expression construct. The ectopic expression of SOX14 was demonstrated at the mRNA level in NT2/D1, P19 and HeLa cells, while an increased level of SOX14 protein was detected in HeLa cells only. By transient transfection experiments in HeLa cells we showed for the first time that ectopic expression of SOX14 repressed SOX1 expression, whereas no significant effect on SOX2, SOX3 and SOX21 was observed. Data presented here provide an insight into SOX14 expression during in vitro neural differentiation of embryonal carcinoma cells and demonstrate the effect of its ectopic expression on protein levels of SOXB members in HeLa cells. Obtained results contribute to better understanding the role of one of the most conserved SOX proteins.

SOX18 Is a Novel Target Gene of Hedgehog Signaling in Cervical Carcinoma Cell Lines

Although there is much evidence showing functional relationship between Hedgehog pathway, in particular Sonic hedgehog, and SOX transcription factors during embryonic development, scarce data are available regarding their crosstalk in cancer cells. SOX18 protein plays an important role in promoting tumor angiogenesis and therefore emerged as a promising potential target in antiangiogenic tumor therapy. Recently it became evident that expression of SOX18 gene in tumors is not restricted to endothelium of accompanying blood and lymphatic vessels, but in tumor cells as well.In this paper we have identified human SOX18 gene as a novel target gene of Hedgehog signaling in cervical carcinoma cell lines. We have presented data showing that expression of SOX18 gene is regulated by GLI1 and GLI2 transcription factors, final effectors of Hedgehog signaling, and that modulation of Hedgehog signaling activity in considerably influence SOX18 expression. We consider important that Hedgehog pathway inhibitors reduced SOX18 expression, thus showing, for the first time, possibility for manipulationwith SOX18 gene expression. In addition, we analyzed the role of SOX18 in malignant potential of cervical carcinoma cell line, and showed that its overexpression has no influence on cells proliferation and viability, but substantially promotes migration and invasion of cells in vitro. Pro-migratory effect of SOX18 suggests its role in promoting malignant spreading, possibly in response to Hedgehog activation.

GABAergic signaling in primary lens epithelial and lentoid cells and its involvement in intracellular Ca2+ modulation

Primary lens epithelial cell (LEC) cultures derived from newborn (P0) and one-month-old (P30) mouse lenses were used to study GABA (gamma-aminobutyric acid) signaling expression and its effect on the intracellular Ca2+ ([Ca2+]i) level. We have found that these cultures express specific cellular markers for lens epithelial and fiber cells, all components of the functional GABA signaling pathway and GABA, thus recapitulating the developmental program of the ocular lens. Activation of both GABA-A and GABA-B receptors (GABAAR and GABABR) with the specific agonists muscimol and baclofen, respectively induces [Ca2+]i transients that could be blocked by the specific antagonists bicuculline and CGP55845 and were dependent on extracellular Ca2+. Bicuculline did not change the GABA-evoked Ca2+ responses in Ca2-containing buffers, but suppressed them significantly in Ca2+-free buffers suggesting the two receptors couple to convergent Ca2+ mobilization mechanisms with different extracellular Ca2+ sensitivity. Prolonged activation of GABABR induced wave propagation of the Ca2+ signal and persistent oscillations. The number of cells reacting to GABA or GABA+bicuculline in P30 mouse LEC cultures expressing predominantly the synaptic type GABAAR did not differ significantly from the number of reacting cells in P0 mouse LEC cultures. The GABA-induced Ca2+ transients in P30 (but not P0) mouse LEC could be entirely suppressed by co-application of bicuculline and CGP55845. The GABA-mediated Ca2+ signaling may be involved in a variety of Ca2+-dependent cellular processes during lens growth and epithelial cell differentiation.

Crosstalk between SOXB1 proteins and WNT/β-catenin signaling in NT2/D1 cells

During early vertebrate embryogenesis, the expression of SOXB1 proteins is precisely regulated by a number of different mechanisms, including Wnt/β-catenin signaling. This is essential for controlling the balance between stemness and differentiation in embryonic stem cells. In the present study, we analyzed the molecular mechanism of LiCl action in NT2/D1 cells and examined the crosstalk between SOXB1 proteins and Wnt signaling in this model system. We have shown that LiCl increases β-catenin level, induces its translocation to the nucleus and consequently up-regulates β-catenin/Tcf-dependent transcription in NT2/D1 cells. Our results also suggest that LiCl treatment leads to increased expression of SOX2 and SOX3 proteins in NT2/D1 cells through activation of canonical Wnt signaling. Finally, we have detected a negative feedback loop between β-catenin and SOX2 expression in NT2/D1 cells. Since β-catenin and SOX2 have been linked to processes of self-renewal and pluripotency, our results have implications for future research on the maintenance of stemness and lineage commitment of embryonic stem cells.

Histone modifications on the promoters of human OCT4 and NANOG genes at the onset of neural differentiation of NT2/D1 cells

Transcription factors OCT4 and NANOG are main constituents of a functional network that controls proliferation and pluripotency maintenance of stem cells as well as early lineage decisions. We investigated expression profiles of OCT4 and NANOG during the early phases of neural differentiation using NT2/D1 cells induced by retinoic acid as an in vitro model system of human neurogenesis. We demonstrated decrease in OCT4 and NANOG mRNA and protein levels following exposure to retinoic acid. Next, by employing chromatin immunoprecipitation, we investigated profiles of selected H3 and H2B histone marks deposited on the promoters of the OCT4 and NANOG genes. We found decline in H3K4me3, H2BK5ac, and H2BK120ac on both promoters, which paralleled the decrease in OCT4 and NANOG expression. Moreover, we found that the H2BK16ac mark is differentially enriched on these two promoters, pointing to differences in epigenetic regulation of OCT4 and NANOG gene expression. Finally, based on our data, we suggest that the early response of pluripotency genes OCT4 and NANOG to the differentiation-inducing stimuli is mediated by dynamic changes in chromatin marks, while DNA methylation is acquired in the later stages of neurogenesis.

The overexpression of SOX2 affects the migration of human teratocarcinoma cell line NT2/D1

The altered expression of the SOX2 transcription factor is associated with oncogenic or tumor suppressor functions in human cancers. This factor regulates the migration and invasion of different cancer cells. In this study we investigated the effect of constitutive SOX2 overexpression on the migration and adhesion capacity of embryonal teratocarcinoma NT2/D1 cells derived from a metastasis of a human testicular germ cell tumor. We detected that increased SOX2 expression changed the speed, mode and path of cell migration, but not the adhesion ability of NT2/D1 cells. Additionally, we demonstrated that SOX2 overexpression increased the expression of the tumor suppressor protein p53 and the HDM2 oncogene. Our results contribute to the better understanding of the effect of SOX2 on the behavior of tumor cells originating from a human testicular germ cell tumor. Considering that NT2/D1 cells resemble cancer stem cells in many features, our results could contribute to the elucidation of the role of SOX2 in cancer stem cells behavior and the process of metastasis.

SOX2 Overexpression Affects Neural Differentiation of Human Pluripotent NT2/D1 Cells

SOX2 is one of the key transcription factors involved in maintenance of neural progenitor identity. However, its function during the process of neural differentiation, including phases of lineage-specification and terminal differentiation, is still poorly understood. Considering growing evidence indicating that SOX2 expression level must be tightly controlled for proper neural development, the aim of this research was to analyze the effects of constitutive SOX2 overexpression on outcome of retinoic acid-induced neural differentiation of pluripotent NT2/D1 cells. We demonstrated that in spite of constitutive SOX2 overexpression, NT2/D1 cells were able to reach final phases of neural differentiation yielding both neuronal and glial cells. However, SOX2 overexpression reduced the number of mature MAP2-positive neurons while no difference in the number of GFAP-positive astrocytes was detected. In-depth analysis at single-cell level showed that SOX2 downregulation was in correlation with both neuronal and glial phenotype acquisitions. Interestingly, while in mature neurons SOX2 was completely downregulated, astrocytes with low level of SOX2 expression were detected. Nevertheless, cells with high level of SOX2 expression were incapable of entering in either of two differentiation pathways, neurogenesis or gliogenesis. Accordingly, our results indicate that fine balance between undifferentiated state and neural differentiation depends on SOX2 expression level. Unlike neurons, astrocytes could maintain low level of SOX2 expression after they acquired glial fate. Further studies are needed to determine whether differences in the level of SOX2 expression in GFAP-positive astrocytes are in correlation with their self-renewal capacity, differentiation status, and/or their phenotypic characteristics.

SOX14 activates the p53 signaling pathway and induces apoptosis in a cervical carcinoma cell line

SOX14 is a member of the SOX family of transcription factors mainly involved in the regulation of neural development. Recently, it became evident that SOX14 is one of four hypermethylated genes in cervical carcinoma, considered as a tumor suppressor candidate in this type of malignancy. In this paper we elucidated the role of SOX14 in the regulation of malignant properties of cervical carcinoma cells in vitro. Functional analysis performed in HeLa cells revealed that SOX14 overexpression decreased viability and promoted apoptosis through altering the expression of apoptosis related genes. Our results demonstrated that overexpression of SOX14 initiated accumulation of p53, demonstrating potential cross-talk between SOX14 and the p53 signaling pathway. Further analysis unambiguously showed that SOX14 triggered posttranslational modification of p53 protein, as detected by the significantly increased level of phospho-p53 (Ser-15) in SOX14-overexpressing HeLa cells. Moreover, the obtained results revealed that SOX14 activated p53 protein, which was confirmed by elevated p21Waf1/Cip1, a well known target gene of p53. This study advances our understanding about the role of SOX14 and might explain the molecular mechanism by which this transcription factor could exert tumor suppressor properties in cervical carcinoma.