Mouse mammary tumors display Stat3 activation dependent on leukemia inhibitory factor signaling

Introduction: It has been demonstrated that leukemia inhibitory factor (LIF) induces epithelium apoptosis through Stat3 activation during mouse mammary gland involution. In contrast, it has been shown that this transcription factor is commonly activated in breast cancer cells, although what causes this effect remains unknown. Here we have tested the hypothesis that locally produced LIF can be responsible for Stat3 activation in mouse mammary tumors. Methods: The studies were performed in different tumorigenic and non-tumorigenic mammary cells. The expression of LIF and LIF receptor was tested by RT-PCR analysis. In tumors, LIF and Stat3 proteins were analyzed by immunohistochemistry, whereas Stat3 and extracellular signal-regulated kinase (ERK)1/2 expression and phosphorylation were studied by Western blot analysis. A LIF-specific blocking antibody was used to determine whether this cytokine was responsible for Stat3 phosphorylation induced by conditioned medium. Specific pharmacological inhibitors (PD98059 and Stat3ip) that affect ERK1/2 and Stat3 activation were used to study their involvement in LIF-induced effects. To analyze cell survival, assays with crystal violet were performed. Results: High levels of LIF expression and activated Stat3 were found in mammary tumors growing in vivo and in their primary cultures. We found a single mouse mammary tumor cell line, LM3, that showed low levels of activated Stat3. Incidentally, these cells also showed very little expression of LIF receptor. This suggested that autocrine/paracrine LIF would be responsible for Stat3 activation in mouse mammary tumors. This hypothesis was confirmed by the ability of conditioned medium of mammary tumor primary cultures to induce Stat3 phosphorylation, activity that was prevented by pretreatment with LIF-blocking antibody. Besides, we found that LIF increased tumor cell viability. Interestingly, blocking Stat3 activation enhanced this effect in mammary tumor cells. Conclusion: LIF is overexpressed in mouse mammary tumors, where it acts as the main Stat3 activator. Interestingly, the positive LIF effect on tumor cell viability is not dependent on Stat3 activation, which inhibits tumor cell survival as it does in normal mammary epithelium. © 2007 Quaglino et al.; licensee BioMed Central Ltd.Fil:Quaglino, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Schere-Levy, C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Romorini, L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Kordon, E.C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Assembling and function of scaffolding proteins of the post-synaptic density

Romorini Stefano, PhD in Pharmacology, Chemotherapy and medical Toxicology Summary of the PhD thesis "Assembling and function of scaffolding proteins at the post-synaptic density" Number, location and type of synapses are tightly regulated by several cellular and molecular mechanisms. At excitatory synapses a central role is occupied by scaffolding proteins that organize the structure of the dendritic spines (dendritic protrusions, on top of which glutamatergic synapses are allocated), in particular we have analyzed the formation of a complex constituted by three NMDA receptor linked scaffold (PSD95, GKAP and Shank) as a key regulatory event in the assembling and function of glutamatergic post-synaptic density. We demonstrated as the targeting of Shank (known to be involved in spines development and maturation) to the synapse is driven by the formation of this complex and how the interaction of this complex with the adhesion molecule NLG1 is able to induce the formation of new synapses. We also found a role for GKAP in maintaining the number of dendritic spines and the structure of the post-synaptci density

Hypothyroid phenotype is contributed by mitochondrial complex I inactivation due to translocated neuronal nitric-oxide synthase

Although transcriptional effects of thyroid hormones have substantial influence on oxidative metabolism, how thyroid sets basal metabolic rate remains obscure. Compartmental localization of nitric-oxide synthases is important for nitric oxide signaling. We therefore examined liver neuronal nitric-oxide synthase-α (nNOS) subcellular distribution as a putative mechanism for thyroid effects on rat metabolic rate. At low 3,3′,5-triiodo-L-thyronine levels, nNOS mRNA increased by 3-fold, protein expression by one-fold, and nNOS was selectively translocated to mitochondria without changes in other isoforms. In contrast, under thyroid hormone administration, mRNA level did not change and nNOS remained predominantly localized in cytosol. In hypothyroidism, nNOS translocation resulted in enhanced mitochondrial nitric-oxide synthase activity with low O2 uptake. In this context, NO utilization increased active O2 species and peroxynitrite yields and tyrosine nitration of complex I proteins that reduced complex activity. Hypothyroidism was also associated to high phospho-p38 mitogen-activated protein kinase and decreased phospho-extracellular signal-regulated kinase 1/2 and cyclin D1 levels. Similarly to thyroid hormones, but without changing thyroid status, nitric-oxide synthase inhibitor Nω-nitro-L-arginine methyl ester increased basal metabolic rate, prevented mitochondrial nitration and complex I derangement, and turned mitogen-activated protein kinase signaling and cyclin D1 expression back to control pattern. We surmise that nNOS spatial confinement in mitochondria is a significant downstream effector of thyroid hormone and hypothyroid phenotype. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.Fil:Franco, M.C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Antico Arciuch, V.G. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Galli, S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Romorini, L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina