Intracellular Pathways Involved in the Apoptotic and Antiproliferative Actions of Prolactin on Lactotropes

NATALY DE DIOS; MARTIN IRIZARRI; SANTIAGO ORRILLO; MARIA FLORENCIA GOTTARDO; JULIA HALPERIN; FLORENCE BOUTILLON; ADRIANA SEILICOVICH; VINCENT GOFFIN; DANIEL PISERA; JIMENA FERRARIS

Boston

Congreso; Endocrine Society's 98th Annual Meeting and Expo; 2016

Endocrine Society

In contrast to what is observed inmany other target tissues, prolactin (PRL) induces apoptosis and inhibitsproliferation of lactotropes, which is assumed to maintain anterior pituitaryhomeostasis (1,2). Our aim was to identify the signaling pathways involved inthese unusual effects. Since somatolactotrope GH3 cells and lactotropes secretePRL constitutively, we used a pure PRL receptor antagonist (Δ1?9-G129R-hPRL) toinhibit all the PRL receptor-mediated effects. GH3 cells and rat anteriorpituitary primary cell cultures were incubated with Δ1?9-G129R-hPRL in thepresence or in the absence of various kinase inhibitors targeting canonicalsignaling pathways of the PRL receptor (AG490, a JAK2 inhibitor, and PD98950, aMEK inhibitor). We determined the phosphorylation status of STAT5, ERK1/2 andAkt and the expression of Bax and Bcl-2 by western blot. Phospho-STAT5 was alsoanalyzed using immunofluorescence. We used GH3 cells to evaluate the effects ofkinase inhibitors and/or Δ1?9-G129R-hPRL on cell apoptosis (hypodiploidy-FACSand TUNEL assay) and proliferation (BrdU incorporation). Together, our resultssuggest that PRL induces apoptosis through the activation of MEK-relatedpathways and the regulation of the balance of Bcl-2 family proteins. On theother hand, JAK2/STAT5 may mediate the antiproliferative effect of PRL onanterior pituitary cells. In contrast to what is observed in many other target tissues, prolactin (PRL) induces apoptosis and inhibits proliferation of lactotropes, which is assumed to maintain anterior pituitary homeostasis (1,2). Our aim was to identify the signaling pathways involved in these unusual effects. Since somatolactotrope GH3 cells and lactotropes secrete PRL constitutively, we used a pure PRL receptor antagonist (Δ1?9-G129R-hPRL) to inhibit all the PRL receptor-mediated effects. GH3 cells and rat anterior pituitary primary cell cultures were incubated with Δ1?9-G129R-hPRL in the presence or in the absence of various kinase inhibitors targeting canonical signaling pathways of the PRL receptor (AG490, a JAK2 inhibitor, and PD98950, a MEK inhibitor). We determined the phosphorylation status of STAT5, ERK1/2 and Akt and the expression of Bax and Bcl-2 by western blot. Phospho-STAT5 was also analyzed using immunofluorescence. We used GH3 cells to evaluate the effects of kinase inhibitors and/or Δ1?9-G129R-hPRL on cell apoptosis (hypodiploidy-FACS and TUNEL assay) and proliferation (BrdU incorporation). Together, our results suggest that PRL induces apoptosis through the activation of MEK-related pathways and the regulation of the balance of Bcl-2 family proteins. On the other hand, JAK2/STAT5 may mediate the antiproliferative effect of PRL on anterior pituitary cells. - See more at: http://press.endocrine.org/doi/abs/10.1210/endo-meetings.2016.NP.17.FRI-495#sthash.nWCKRVCL.dpufIn contrast to what is observed in many other target tissues, prolactin (PRL) induces apoptosis and inhibits proliferation of lactotropes, which is assumed to maintain anterior pituitary homeostasis (1,2). Our aim was to identify the signaling pathways involved in these unusual effects. Since somatolactotrope GH3 cells and lactotropes secrete PRL constitutively, we used a pure PRL receptor antagonist (Δ1?9-G129R-hPRL) to inhibit all the PRL receptor-mediated effects. GH3 cells and rat anterior pituitary primary cell cultures were incubated with Δ1?9-G129R-hPRL in the presence or in the absence of various kinase inhibitors targeting canonical signaling pathways of the PRL receptor (AG490, a JAK2 inhibitor, and PD98950, a MEK inhibitor). We determined the phosphorylation status of STAT5, ERK1/2 and Akt and the expression of Bax and Bcl-2 by western blot. Phospho-STAT5 was also analyzed using immunofluorescence. We used GH3 cells to evaluate the effects of kinase inhibitors and/or Δ1?9-G129R-hPRL on cell apoptosis (hypodiploidy-FACS and TUNEL assay) and proliferation (BrdU incorporation). Together, our results suggest that PRL induces apoptosis through the activation of MEK-related pathways and the regulation of the balance of Bcl-2 family proteins. On the other hand, JAK2/STAT5 may mediate the antiproliferative effect of PRL on anterior pituitary cells. - See more at: http://press.endocrine.org/doi/abs/10.1210/endo-meetings.2016.NP.17.FRI-495#sthash.VqjirhyT.dpuf