In vitro study of glyphosate effects on thyroid cells.

Glyphosate is a pesticide, which contaminates the environment and exposes workers and general population to its residues present in foods and waters. In soil, Glyphosate is degraded in metabolites, amino-methyl-phosphonic acid (AMPA) being the main one. Glyphosate is considered a potential cancerogenic and endocrine-disruptor agent, however its adverse effects on the thyroid were evaluated only in animal models and in vitro data are still lacking. Aim of this study was to investigate whether exposure to Glyphosate could exert adverse effects on thyroid cells in vitro. Two models (adherent-2D and spheroid-3D) derived from the same cell strain Fisher-rat-thyroid-cell line-5 (FRTL-5) were employed. After exposure to Glyphosate at increasing concentrations (0.0, 0.1-0.25- 0.5-1.0-2.0-10.0 mM) we evaluated cell viability by WST-1 (adherent and spheroids), results being confirmed by propidium-iodide staining (only for spheroids). Proliferation of adherent cells was assessed by crystal violet and trypan-blue assays, the increasing volume of spheroids was taken as a measure of proliferation. We also evaluated the ability of cells to form spheroids after Glyphosate exposure. We assessed changes of reactive-oxygen-species (ROS) by the cell-permeant H2DCFDA. Glyphosate-induced changes of mRNAs encoding for thyroid-related genes (TSHR, TPO, TG, NIS, TTF-1 and PAX8) were evaluated by RT-PCR. Glyphosate reduced cell viability and proliferation in both models, even if at different concentrations. Glyphosate at the highest concentration reduced the ability of FRTL-5 to form spheroids. An increased ROS production was found in both models after exposure to Glyphosate. Finally, Glyphosate increased the mRNA levels of some thyroid related genes (TSHR, TPO, TG and TTF-1) in both models, while it increased the mRNAs of PAX8 and NIS only in the adherent model. The present study supports an adverse effect of Glyphosate on cultured thyroid cells. Glyphosate reduced cell viability and proliferation and increased ROS production in thyroid cells.

The transcription factor Pax6 contributes to the induction of GLT-1 expression in astrocytes through an interaction with a distal enhancer element.

The Na(+) -dependent glutamate transporter GLT-1 (EAAT2) shows selective expression in astrocytes, and neurons induce the expression of GLT-1 in astrocytes. In an unpublished analysis of GLT-1 promoter reporter mice, we identified an evolutionarily conserved domain of 467 nucleotides ~ 8 kb upstream of the GLT-1 translation start site that is required for astrocytic expression. Using in silico approaches, we identified Pax6 as a transcription factor that could contribute to the control of GLT-1 expression by binding within this region. We demonstrated the expression of Pax6 protein in astrocytes in vivo. Lentiviral transduction of astrocytes with exogenous Pax6 increased the expression of enhanced green fluorescent protein (eGFP) in astrocytes prepared from transgenic mice that use a bacterial artificial chromosome containing a large genomic region surrounding the GLT-1 gene to control expression of eGFP. It also increased GLT-1 protein and GLT-1-mediated uptake, whereas there was no effect on the levels of the other astroglial glutamate transporter, glutamate aspartate transporter (GLAST). Transduction of astrocytes with an shRNA directed against Pax6 reduced neuron-dependent induction of GLT-1 or eGFP. Finally, we confirmed Pax6 interaction with the predicted DNA-binding site in electrophoretic mobility assays and chromatin immunoprecipitation (ChIP). Together, these studies show that Pax6 contributes to the regulation of GLT-1 through an interaction with these distal elements and identify a novel role of Pax6 in astrocyte biology. The astroglial glutamate transporter GLT-1 shows selective expression in astrocytes and its expression can be induced by neurons. In this study, we demonstrate that Pax6 is expressed in astrocytes and binds to the GLT-1 promoter in vitro and in vivo. Exogenous expression of Pax6 increases GLT-1 and enhanced green fluorescent protein (eGFP) expression in astrocytes from a transgenic mouse line that uses the GLT-1 gene to drive eGFP expression, and an shRNA directed against Pax6 attenuates neuron-dependent induction of GLT-1/eGFP. We therefore conclude that Pax6 contributes to the neuron-dependent induction of GLT-1.

Paracrine Pax6 activity regulates oligodendrocyte precursor cell migration in the chick embryonic neural tube.

Homeoprotein transcription factors play fundamental roles in development, ranging from embryonic polarity to cell differentiation and migration. Research in recent years has underscored the physiological importance of homeoprotein intercellular transfer in eye field development, axon guidance and retino-tectal patterning, and visual cortex plasticity. Here, we have used the embryonic chick neural tube to investigate a possible role for homeoprotein Pax6 transfer in oligodendrocyte precursor cell (OPC) migration. We report the extracellular expression of Pax6 and the effects of gain and loss of extracellular Pax6 activity on OPCs. Open book cultures with recombinant Pax6 protein or Pax6 blocking antibodies, as well as in ovo gene transfer experiments involving expression of secreted Pax6 protein or secreted Pax6 antibodies, provide converging evidences that OPC migration is promoted by extracellular Pax6. The paracrine effect of Pax6 on OPC migration is thus a new example of direct non-cell autonomous homeoprotein activity.

Engraftment of embryonic stem cell-derived myogenic progenitors in a dominant model of muscular dystrophy.

Muscular dystrophies (MDs) consist of a genetically heterogeneous group of disorders, recessive or dominant, characterized by progressive skeletal muscle weakening. To date, no effective treatment is available. Experimental strategies pursuing muscle regeneration through the transplantation of stem cell preparations have brought hope to patients affected by this disorder. Efficacy has been demonstrated in recessive MD models through contribution of wild-type nuclei to the muscle fiber heterokaryon; however, to date, there has been no study investigating the efficacy of a cell therapy in a dominant model of MD. We have recently demonstrated that Pax3-induced embryonic stem (ES) cell-derived myogenic progenitors are able to engraft and improve muscle function in mdx mice, a recessive mouse model for Duchenne MD. To assess whether this therapeutic effect can be extended to a dominant type of muscle disorder, here we transplanted these cells into FRG1 transgenic mice, a dominant model that has been associated with facioscapulohumeral muscular dystrophy. Our results show that Pax3-induced ES-derived myogenic progenitors are capable of significant engraftment after intramuscular or systemic transplantation into Frg1 mice. Analyses of contractile parameters revealed functional improvement in treated muscles of male mice, but not females, which are less severely affected. This study is the first to use Frg1 transgenic mice to assess muscle regeneration as well as to support the use of a cell-based therapy for autosomal dominant types of MD.

Association between the development of the body axis and the craniofacial skeleton studied by immunohistochemical analyses using collagen II, Pax9, Pax1, and Noggin antibodies.

STUDY DESIGN: Immunohistochemical analyses on the axial skeleton from wild type mice. OBJECTIVE: In the clinic, we have previously observed cervical spine defects associated with deviations in the posterior part of the occipital bone and with morphologic and functional variations in the craniofacial skeleton. As examples, cervical spine fusions occurred frequently in patients with mandibular overjet and even more frequently and more caudally in the cervical spine in patients with sleep apnoea. The aims of the present study were to elucidate this association between the spine and the cranium by comparing gene expression domains of important developmental genes known to be involved in vertebral column formation with gene expression in the craniofacial region. SUMMARY OF BACKGROUND DATA: This is the first study looking specifically on gene expression in the basilar part of the occipital bone that is formed around the cranial part of the notochord, thus connecting the spine and the craniofacial skeleton. METHODS: The material consisted of 4 mouse embryos p.c. day 13.5, NMRI wild-type mice, from the same litter. The body axis, the cranial base, and the craniofacial area were studied by immunohistochemical analyses using Collagen II, Pax9, Pax1, and Noggin antibodies. RESULTS: Pax1 expression was highly similar in the posterior part of the occipital bone and in the vertebral column, indicating that the basilar part of the occipital bone from a developmental standpoint can be considered the uppermost vertebra. Pax9 and Noggin expression domains were in accordance with those described previously. CONCLUSION: The present study supports that the basilar part of the occipital bone may be regulated by similar developmental mechanisms as the vertebral column and may thus be regarded the uppermost vertebra. Thus, the clinically observed association between the cervical column and the craniofacial area has been proved by immunohistochemical methods.

Pax4 paired domain mediates direct protein transduction into mammalian cells.

Pax4, a paired-box transcription factor, is a key regulator of pancreatic islet cell growth and differentiation. Here, we report for the first time that the Pax4 protein can permeate into various cell types including pancreatic islets. The paired domain of Pax4 serves as a novel protein transduction domain (PTD). The Pax4 protein can transduce in a dose- and time-dependent manner. The cellular uptake of Pax4 PTD can be completely blocked by heparin, whereas cytochalasin D and amiloride were partially effective in blocking the Pax4 protein entry. Transduced intact Pax4 protein functions similarly to the endogenous Pax4. It inhibits the Pax6 mediated transactivation and protects Min6 cells against TNFalpha-induced apoptosis. These data suggest that Pax4 protein transduction could be a safe and valuable strategy for protecting islet cell growth in culture from apoptosis and promoting islet cell differentiation.

Agonistic and antagonistic action of AP2, Msx2, Pax6, Prox1 AND Six3 in the regulation of Sox2 expression.

Sox2 transcription factor is expressed in neural tissues and sensory epithelia from the early stages of development. Particularly, it is known to activate crystallin gene expression and to be involved in differentiation of lens and neural tissues. However, its place in the signaling cascade is not well understood. Here, we report about the response of its promoter to the presence of other transcription factors, AP2alpha, Msx2, Pax6, Prox1 and Six3, in a transient reporter gene assay using HEK293 cells as recipient cells. Taking our data together, AP2, Pax6 and PROX1 can activate the Sox2 promoter. Msx2 has an inhibitory effect, whereas Six3 does not affect the Sox2 promoter. These data indicate a common activating cascade at least for AP2, Pax6, Prox1 and Sox2.