High hopes for RANKL: will the mouse model live up to its promise?

The steroid hormones, estrogens and progesterone are key drivers of postnatal breast development and are linked to breast carcinogenesis. Experiments in the mouse mammary gland have revealed that they rely on paracrine factors to relegate their signal locally and to amplify it. In particular, RANKL is a key mediator of progesterone action. Systemic inhibition of RANKL blocked proliferation in the mammary epithelium with potential clinical implications: a RANKL-inhibiting antibody, Denosumab (Amgen), has been approved by the US Food and Drug Administration for osteoporosis treatment. Two publications now provide evidence that progestin-driven mouse mammary tumorigenesis can be blocked by ablating RANK signaling. Can the osteoporosis drug help breast cancer patients? The burning question now is whether the role of this pathway is conserved in the human breast and whether RANKL signaling has a role in the pathogenesis of one or more subtypes of breast cancer

What signals operate in the mammary niche?

Adult stem cells reside in a specialized microenvironment, the niche, which controls their behavior. As mammary stem cells, and consequently their niches, are still poorly defined, we look at better-characterized adult mammalian stem cell niches in the hematopoietic system and the skin. We attempt to define the mammary stem cell niche functionally, based on the widely used mammary fat pad reconstitution assay. We note that the concept of the niche needs to be extended from the specialized microenvironment described in the hematopoietic system, to a model that takes into account the macroenviroment, as recently shown in the skin, and systemic clues as we will illustrate for the mammary gland where the reproductive hormones are major determinants of stem cell activation. In fact, in the mammary gland a special type of stem cells is determined only during pregnancy. Reproductive hormones act on hormone receptor positive cells, sensor cells, in the mammary epithelium to induce paracrine signaling that leads to activation of stem cells. Some of the downstream mediators are in common with other niches such as Wnt and possibly Notch signaling. Other signals are specific to the mammary gland such as amphiregulin and RANKL

The small GTP-binding protein RhoA regulates c-Jun by a ROCK-JNK signaling axis

RhoA regulates the actin cytoskeleton and the expres- sion of genes associated with cell proliferation. This includes c-fos and c-jun, which are members of the AP1 family of transcription factors that play a key role in normal and aberrant cell growth. Whereas RhoA stimulates the c-fos SRE by a recently elucidated mechanism that is dependent on actin treadmilling, how RhoA regulates c-jun is still poorly understood. We found that RhoA stimulates c-jun expression through ROCK, but independently from the ability of ROCK to promote actin polymerization. Instead, we found that ROCK activates JNK, which then phosphor- ylates c-Jun and ATF2 when bound to the c-jun pro- moter. Thus, ROCK represents a point of signal diver- gence downstream from RhoA, as it promotes actin reorganization and the consequent expression from the c-fos SRE, while a parallel pathway connects ROCK to JNK, thereby stimulating c-jun expression. Ultimately, these pathways converge in the nucleus to regulate AP1 activity

Investigating the complex interplay between fibroblast activation protein α-positive cancer associated fibroblasts and the tumor microenvironment in the context of cancer immunotherapy

IntroductionThis study investigates the role of Fibroblast Activation Protein (FAP)-positive cancer-associated fibroblasts (FAP+CAF) in shaping the tumor immune microenvironment, focusing on its association with immune cell functionality and cytokine expression patterns.MethodsUtilizing immunohistochemistry, we observed elevated FAP+CAF density in metastatic versus primary renal cell carcinoma (RCC) tumors, with higher FAP+CAF correlating with increased T cell infiltration in RCC, a unique phenomenon illustrating the complex interplay between tumor progression, FAP+CAF density, and immune response.ResultsAnalysis of immune cell subsets in FAP+CAF-rich stromal areas further revealed significant correlations between FAP+ stroma and various T cell types, particularly in RCC and non-small cell lung cancer (NSCLC). This was complemented by transcriptomic analyses, expanding the range of stromal and immune cell subsets interrogated, as well as to additional tumor types. This enabled evaluating the association of these subsets with tumor infiltration, tumor vascularization and other components of the tumor microenvironment. Our comprehensive study also encompassed cytokine, angiogenesis, and inflammation gene signatures across different cancer types, revealing heterogeneous cellular composition, cytokine expressions and angiogenic profiles. Through cytokine pathway profiling, we explored the relationship between FAP+CAF density and immune cell states, uncovering potential immunosuppressive circuits that limit anti-tumor activity in tumor-resident immune cells.ConclusionsThese findings underscore the complexity of tumor biology and the necessity for personalized therapeutic and patient enrichment approaches. The insights gathered from FAP+CAF prevalence, immune infiltration, and gene signatures provide valuable perspectives on tumor microenvironments, aiding in future research and clinical strategy development

Resting cells rely on the DNA helicase component MCM2 to build cilia

Minichromosome maintenance (MCM) proteins facilitate replication by licensing origins and unwinding the DNA double strand. Interestingly, the number of MCM hexamers greatly exceeds the number of firing origins suggesting additional roles of MCMs. Here we show a hitherto unanticipated function of MCM2 in cilia formation in human cells and zebrafish that is uncoupled from replication. Zebrafish depleted of MCM2 develop ciliopathy-phenotypes including microcephaly and aberrant heart looping due to malformed cilia. In non-cycling human fibroblasts, loss of MCM2 promotes transcription of a subset of genes, which cause cilia shortening and centriole overduplication. Chromatin immunoprecipitation experiments show that MCM2 binds to transcription start sites of cilia inhibiting genes. We propose that such binding may block RNA polymerase II-mediated transcription. Depletion of a second MCM (MCM7), which functions in complex with MCM2 during its canonical functions, reveals an overlapping cilia-deficiency phenotype likely unconnected to replication, although MCM7 appears to regulate a distinct subset of genes and pathways. Our data suggests that MCM2 and 7 exert a role in ciliogenesis in post-mitotic tissues

MAPKs mediated regulatory events acting upon the c-Fos early response gene

La inducción de la expresión génica involucra procesos bioquímicos en los que participan múltiples caminos de transducción de señales. Dependiendo del tipo de estímulo distintas proteínas quinasas se activan provocando en última instancia la fosforilación de factores de transcripción y por lo tanto la regulación de la expresión génica. Las MAPKs (Mitogen Activated Protein Kinases) son importantes propagadoras de las señales que van desde la membrana celular al núcleo. Son un grupo amplio de enzimas quinasas entre las cuales se destacan, por ser las más estudiadas hasta el momento, ERK1/2, JNK y p38. Las quinasas JNK y p38 forman una subfamilia dentro de las MAPKs y se las denomina SAPKs (Stress Activated Protein Kinases) ya que son generalmente activadas por estrés. Por otro lado, las quinasas ERK1/2 responden principalmente a señales mitogénicas. Los miembros de las familias de factores de transcripción que forman AP-1 son codificados por genes de respuesta temprana. El nivel de expresión de estos genes aumenta rápida y transitoriamente en respuesta tanto a estímulos mitogénicos como a estímulos que producen estrés celular. Ejemplos típicos de miembros de AP-1 son los productos de los proto-oncogenes c-Jun y c-Fos, cuya actividad prolongada o descontrolada desencadena proliferación desmedida que puede dar lugar a una neoplasia. Las MAPK cumplen un papel importante tanto en la activación de promotores de genes tempranos como en la modificación post-traduccional de las proteínas codificadas por estos genes al agregarle grupos fosfato, tal como en el caso ampliamente estudiado de la fosforilación de c-Jun por JNK. El factor de transcripción c-Fos heterodimeriza con proteínas de la familia Jun para formar el factor de transcripción AP-1. Su actividad está finamente regulada a nivel transcripcional, a nivel de la vida media del mensajero transcripto, a nivel de la vida media de la proteína y a nivel de modificaciones post-traduccionales que se adicionan sobre la proteína. A lo largo de este trabajo de tesis nos hemos propuesto investigar la regulación de c-Fos por MAPKs en respuesta a dos tipos de estímulos diferentes. Estudiamos la interacción de c-Fos con las p38 SAPKs, su efecto sobre la fosforilación del producto de este proto-oncogén ante un estímulo de estrés, radiación UV y la regulación de la actividad transcripcional de c-Fos por SAPKs. Por otra parte, estudiamos la regulación de c-Fos desencadenada por un estímulo mitogénico, haciendo hincapié tanto en la regulación de la transcripción de esta proteína como en modificaciones post traduccionales en respuesta al agonista del receptor muscarínico 1, Carbacol. Observamos que la exposición de células HEK 293 a radiación UV produce la activación de las cuatro isoformas de las p38 SAPKs las cuales, como consecuencia de dicha activación, fosforilan a c-Fos en su dominio de transactivación, de modo análogo a lo ampliamente descripto para JNK y c-Jun, produciendo un aumento en su capacidad de transactivar genes con sitios de unión AP-1. Además, observamos que el agonista del receptor muscarínico1, Carbacol, induce la activación del promotor de c-fos actuando a través de la activación de la MAPK ERK2. Esto produce un aumento en la cantidad de proteína c-Fos presente en las células que sería fosforilada por múltiples MAPKs en su dominio de transactivación activando su función como factor de transcripción. Estos resultados proveen nueva información acerca de la complejidad de las respuestas de los productos de genes de respuesta temprana, a estímulos externos actuando a través de la activación de MAPKs.Regulation of gene expression involves biochemical processes exerted through signal transduction pathways. Depending on the sources of stimuli, a variety of protein kinase cascades activate and finally phosphorylate distinct transcription factors that ultimately regulate the transcription of several genes. The mitogen-activated protein kinases (MAPKs) are a family of serine/threonine kinases that play an essential role in signal transduction by modulating gene transcription in the nucleus in response to changes in the cellular environment. They include the extracellular signal-regulated protein kinases ERK1/2, JNKs and p38s. While the former responds mostly to mitogenic signals, JNK and p38 are generally ignited by sources of stress and have been named SAPKs (Stress Activated Protein Kinases). Transcription factors belonging to the AP-1 family are coded by early responsive genes (ERGs). Its expression levels rise quickly and transiently in response to both mitogenic stimuli and stress. Typical examples of AP-1 family members are the products of the proto-oncogenes c-jun and c-fos. Its prolonged expression or activation has been linked to cellular transformation. MAPKs play an important role both as activators of ERG promoters or in the post-translational modification of its protein products, adding phosphate groups to key residues, as is the case for c-Jun and JNK. The product of the proto-oncogene c-fos forms heterodimers with members of the Jun family to form the transcription factor AP-1. Its activity is tightly regulated at different levels, promoter activity, stability of the transcript, and posttranslational modification or half-life of the protein product. During this thesis work, we have characterized the regulation of the transcription factor c-Fos at different levels by MAPKs in response to stress and mitogenic stimuli. Our findings indicate that as a consequence of the activation of stress pathways induced by UV light, endogenous c-Fos becomes a substrate of p38 MAPKs and, for the first time, provide evidence that support a critical role for p38 MAPKs in mediating stress-induced c-Fos phosphorylation and gene transcription activation, acting concomitantly with the activation of c-Jun by JNK/ MAPKs and thereby contributing to the complexity of AP1-driven gene transcription regulation. We also demonstrate that the G-Protein Coupled Receptor M1 regulates c-fos at multiple levels, activating the c-fos promoter through ERK2 MAPK and modulating the expression and activation of the c-Fos protein through several MAPKs.Fil: Tanos, Tamara. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Isolation of putative stem cells present in human adult olfactory mucosa

The olfactory mucosa (OM) has the unique characteristic of performing an almost continuous and lifelong neurogenesis in response to external injuries, due to the presence of olfactory stem cells that guarantee the maintenance of the olfactory function. The easy accessibility of the OM in humans makes these stem cells feasible candidates for the development of regenerative therapies. In this report we present a detailed characterization of a patient-derived OM, together with a description of cell cultures obtained from the OM. In addition, we present a method for the enrichment and isolation of OM stem cells that might be used for future translational studies dealing with neuronal plasticity, neuro-regeneration or disease modeling

Immunodiagnosis of fasciolosis using recombinant procathepsin L cystein proteinase

Cathepsin L1, a cysteine protease secreted by the gastrodermis of juvenile and adult Fasciola hepatica, was expressed in Escherichia coli as a fusion protein containing the proregion, supplied with six histidyl residues at the N-terminal end (rproCL1). In this study we tested its potential as antigen for the serologic diagnosis of F. hepatica infections by enzyme-linked immunosorbent assay (ELISA). The analyzed human sera included 16 positive samples, 99 negative controls and 111 from individuals affected by other parasitic and non parasitic diseases. The sensitivity and specificity of the rproCL1-ELISA were 100%. We also assessed the ability to detect antibodies in sera from 10 experimentally infected sheep, obtaining preliminary results that shown a response since the third week post infection in all the studied animals. Therefore, the recombinant rproCL1-based ELISA could be a standardized test for the accurate diagnosis of fasciolosis

Isolation of putative stem cells present in human adult olfactory mucosa

<div><p>The olfactory mucosa (OM) has the unique characteristic of performing an almost continuous and lifelong neurogenesis in response to external injuries, due to the presence of olfactory stem cells that guarantee the maintenance of the olfactory function. The easy accessibility of the OM in humans makes these stem cells feasible candidates for the development of regenerative therapies. In this report we present a detailed characterization of a patient-derived OM, together with a description of cell cultures obtained from the OM. In addition, we present a method for the enrichment and isolation of OM stem cells that might be used for future translational studies dealing with neuronal plasticity, neuro-regeneration or disease modeling.</p></div

Recombinant GRA4 or ROP2 protein combined with alum or the gra4 gene provides partial protection in chronic murine models of toxoplasmosis

Fil: Martin, Valentina. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina.Fil: Supanitsky, Alicia. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina.Fil: Echeverria, Pablo C. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina.Fil: Litwin, Silvana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Producción de Biológicos; Argentina.Fil: Tanos, Tamara. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina.Fil: de Roodt, Adolfo R. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Producción de Biológicos; Argentina.Fil: Guarnera, Eduardo. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina.Fil: Angel, Sergio O. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina.The efficacy of vaccination with Toxoplasma gondii recombinant GRA4 (rGRA4) and ROP2 (rRPO2) proteins and a mix of both combined with alum were evaluated in C57BL/6 and C3H mice. In C57BL/6 mice, rGRA4 and rGRA4-rROP2 immunizations generated similar levels of immunoglobulin G1 (IgG1) and IgG2a isotypes against GRA4, whereas immunizations with rROP2 and the mix induced a predominant IgG1 production against ROP2. All groups of C3H vaccinated mice exhibited higher levels of IgG1 than IgG2a. rGRA4-stimulated splenocytes from vaccinated mice produced primarily gamma interferon while those stimulated with rROP2 produced interleukin-4. Challenge of rGRA4- or rGRA4-rROP2-vaccinated mice from both strains with ME49 cysts resulted in fewer brain cysts than the controls, whereas vaccination with rROP2 alone only conferred protection to C3H mice. Immunization with a plasmid carrying the entire open reading frame of GRA4 showed a protective level similar to that of rGRA4 combined with alum. These results suggest that GRA4 can be a good candidate for a multiantigen anti-T. gondii vaccine based on the use of alum as an adjuvant