In the mammary gland, during the different phases of the reproductive cycle, moments of tissue growth with increased cellular proliferation are followed by moments of tissue involution, with augmented cellular apoptosis. These phases are regulated both by endocrine hormones (estrogens, progesterone, etc) and by local acting growth factors (EGFs, IGFs, etc). The downstream cellular signaling of these factors induces the expression of immediate early genes (IEGs) and late genes that monitor different steps within the cell cycle. An altered cellular response to these signals may cause deregulated proliferation within this tissue, with increased cancer susceptibility. The aim of our work is to understand, in mammary epithelial cells, the expression kinetics of three important IEGs (EGR1, FOS, JUN) in response to different stimuli. We are also focusing our research on which intracellular signaling pathway, involved in mammary growth or differentiation, is able to modulate the expression of EGR1, FOS and JUN. Mammary epithelial cells obtained from different species of domestic animals are grown in a starving medium in order to obtain a synchronization in the G0/G1 phase of the cell cycle. Subsequently cells are stimulated with various stimuli (growth medium, estrogen, progesterone, EGF, IGFs, etc.) at different time points and IEGs expression is analyzed by real-time PCR and/or western-blot. Treatment with UO126 (ERK 1/2 inhibitor) and/or Wortmannin (PI3K-akt inhibitor) in combination with different stimuli are used to investigate the relative importance of these two important signaling pathways on IEGs RNA and protein expression. We have observed that expression of EGR1 and FOS is strongly upregulated following stimulation with growth medium, EGF and phorbol 12-myristate 13-acetate. JUN is also upregulated, but to a lower levels. Following addition of growth medium we have observed an increase in EGR1 protein expression associated with ERK1/2 and PI3K-akt signaling pathway activation. When ERK1/2 inhibitor is added together to stimuli that upregulate IEGs we observe that UO126 abolishes EGR1 and FOS upregulation leaving JUN expression mostly unaltered. Western blot analysis suggests a similar trend in EGR1 protein expression. This result indicates that IEGs upregulation following phorbol 12-myristate 13-acetate (an activator of protein kinase C which is downstream G-protein coupled receptors) stimulus is driven exclusively by ERK1/2 phosphorilation. PI3K-Akt pathway inhibition with Wortmannin does not influence IEGs RNA expression and EGR1 protein levels. We conclude that the ERK1/2, but not the PI3K-akt pathway, is the most important signaling event that drives the modulation of both EGR1 and FOS in mammary epithelial cells of domestic animals. On the other hand, JUN Expression is probably controlled through different signaling pathways.

MODULATION OF IMMEDIATE EARLY GENES IN MAMMARY EPITHELIAL CELLS / Santino, Paolo; Martignani, Eugenio; Miretti, Silvia; Baratta, Mario; Accornero, Paolo. - (2015), pp. 1-457. (Intervento presentato al convegno LXIX convegno SISVet tenutosi a Perugia nel 15-17 Giugno 2015).

MODULATION OF IMMEDIATE EARLY GENES IN MAMMARY EPITHELIAL CELLS

Baratta Mario;
2015-01-01

Abstract

In the mammary gland, during the different phases of the reproductive cycle, moments of tissue growth with increased cellular proliferation are followed by moments of tissue involution, with augmented cellular apoptosis. These phases are regulated both by endocrine hormones (estrogens, progesterone, etc) and by local acting growth factors (EGFs, IGFs, etc). The downstream cellular signaling of these factors induces the expression of immediate early genes (IEGs) and late genes that monitor different steps within the cell cycle. An altered cellular response to these signals may cause deregulated proliferation within this tissue, with increased cancer susceptibility. The aim of our work is to understand, in mammary epithelial cells, the expression kinetics of three important IEGs (EGR1, FOS, JUN) in response to different stimuli. We are also focusing our research on which intracellular signaling pathway, involved in mammary growth or differentiation, is able to modulate the expression of EGR1, FOS and JUN. Mammary epithelial cells obtained from different species of domestic animals are grown in a starving medium in order to obtain a synchronization in the G0/G1 phase of the cell cycle. Subsequently cells are stimulated with various stimuli (growth medium, estrogen, progesterone, EGF, IGFs, etc.) at different time points and IEGs expression is analyzed by real-time PCR and/or western-blot. Treatment with UO126 (ERK 1/2 inhibitor) and/or Wortmannin (PI3K-akt inhibitor) in combination with different stimuli are used to investigate the relative importance of these two important signaling pathways on IEGs RNA and protein expression. We have observed that expression of EGR1 and FOS is strongly upregulated following stimulation with growth medium, EGF and phorbol 12-myristate 13-acetate. JUN is also upregulated, but to a lower levels. Following addition of growth medium we have observed an increase in EGR1 protein expression associated with ERK1/2 and PI3K-akt signaling pathway activation. When ERK1/2 inhibitor is added together to stimuli that upregulate IEGs we observe that UO126 abolishes EGR1 and FOS upregulation leaving JUN expression mostly unaltered. Western blot analysis suggests a similar trend in EGR1 protein expression. This result indicates that IEGs upregulation following phorbol 12-myristate 13-acetate (an activator of protein kinase C which is downstream G-protein coupled receptors) stimulus is driven exclusively by ERK1/2 phosphorilation. PI3K-Akt pathway inhibition with Wortmannin does not influence IEGs RNA expression and EGR1 protein levels. We conclude that the ERK1/2, but not the PI3K-akt pathway, is the most important signaling event that drives the modulation of both EGR1 and FOS in mammary epithelial cells of domestic animals. On the other hand, JUN Expression is probably controlled through different signaling pathways.
2015
MODULATION OF IMMEDIATE EARLY GENES IN MAMMARY EPITHELIAL CELLS / Santino, Paolo; Martignani, Eugenio; Miretti, Silvia; Baratta, Mario; Accornero, Paolo. - (2015), pp. 1-457. (Intervento presentato al convegno LXIX convegno SISVet tenutosi a Perugia nel 15-17 Giugno 2015).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11381/2899480
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