The cytokine-inducible SH2 domain-containing protein CIS inhibits signaling from the growth hormone (GH) receptor (GHR) to STAT5b by a proteasome-dependent mechanism. Here, we used the GH-responsive rat liver cell line CWSV-1 to investigate the role of CIS and the proteasome in GH-induced GHR internalization. Cell-surface GHR localization and internalization were monitored in GH-stimulated cells by confocal immunofluorescence microscopy using an antibody directed against the GHR extracellular domain. In GH naïve cells, GHR was detected in small, randomly distributed granules on the cell surface and in the cytoplasm, with accumulation in the perinuclear area. GH treatment induced a rapid (within 5 min) internalization of GH⅐GHR complexes, which coincided with the onset of GHR tyrosine phosphorylation and the appearance in the cytosol of distinct granular structures containing internalized GH. GHR signaling to STAT5b continued for ϳ30 -40 min, however, indicating that GHR signaling and deactivation of the GH⅐GHR complex both proceed from an intracellular compartment. The internalization of GH and GHR was inhibited by CIS-R107K, a dominant-negative SH2 domain mutant of CIS, and by the proteasome inhibitors MG132 and epoxomicin, which prolong GHR signaling to STAT5b. GH pulse-chase studies established that the internalized GH⅐GHR complexes did not recycle back to the cell surface in significant amounts under these conditions. Given the established specificity of CIS-R107K for blocking the GHR signaling inhibitory actions of CIS, but not those of other SOCS/CIS family members, these findings implicate CIS and the proteasome in the control of GHR internalization following receptor activation and suggest that CIS-dependent receptor internalization is a prerequisite for efficient termination of GHR signaling. Growth hormone (GH)2 is an important regulator of somatic growth and cellular metabolism. GH exerts its action via the GH receptor (GHR), a transmembrane protein of the cytokine receptor superfamily that activates multiple intracellular signaling pathways, including the STAT, mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and protein kinase C pathways (1, 2). Upon binding of ligand, the dimeric GHR undergoes a conformational change (3, 4) and induces tyrosine phosphorylation, resulting in activation of the tyrosine kinase JAK2, which catalyzes tyrosine phosphorylation of GHR and of GHR-or JAK2-associated STAT1, STAT3, STAT5a, and STAT5b (5, 6). The tyrosine-phosphorylated STAT proteins dimerize and translocate to the nucleus, where they bind to specific DNA response elements upstream of GH target genes and activate gene transcription (7,8).Many physiological responses to GH are dependent on the temporal pattern of plasma GH stimulation, which is sex-dependent and subject to neuroendocrine control. In the rat model, the adult male plasma GH pattern is characterized by regular pulses of hormone every ϳ3.5-4 h, separated by well defined GH-free intervals, whereas a more continuous plasma GH profile i...

The effects of gonadal hormones, testosterone (Te) and estrogen (E2) as factors in the development of the immune system in two lines, high response (HC) and low response (LC), of broiler chickens divergently selected for early or late immune maturation were studied. For this purpose, plasma Te and E2 levels were tested and correlated with immune response. Also, the effects of exogenous administration of gonadal steroids testosterone propionate (TP), dihydrotestosterone (DHT), and estradiol 3-benzoate (EB), and the nonsteroidal androgen antagonist flutomide (Flu) and anti-estrogen tamoxifen (Tam) on the immune system were studied. Male chicks of the LC line had a higher level of endogenous Te during first 30 d posthatch. The administration of TP or DHT had no noticeable effect on the humoral immune response, whereas DHT suppressed growth of the bursa of Fabricius of both sexes of HC line. No differences in the endogenous E2 level were observed between sexes in either line. Administration of EB inhibited comb and testicle growth and enhanced significantly the humoral immune response to Escherichia coli and sheep erythrocytes (SRBC). The anti-androgen Flu and anti-estrogen Tam strongly inhibited humoral immune response to E. coli and SRBC antigen, whereas no effects on comb and testicle growth were observed. The experimental results suggest that gonadal hormones have similar principal posthatch effects in avian as in mammals; however, the gonadal steroids prehatch effects and the genetic-physiological-environmental effects require further study.

Background: Pituitary neuroendocrine tumors (PitNETs) represent approximately 15% of all intracranial tumors and usually are associated with severe comorbidities. Unfortunately, a relevant number of patients do not respond to currently available pharmacological treatments, that is, somatostatin analogs (SSAs) or dopamine-agonists (DA). Thus, novel, chimeric somatostatin/dopamine compounds (dopastatins) that could improve medical treatment of PitNETs have been designed. Objective: This study aims to determine the direct therapeutic effects of a new-generation dopastatin, BIM-065, on primary cell cultures from different PitNETs subtypes. Methods: Thirty-one PitNET-derived cell cultures (9 corticotropinomas, 9 somatotropinomas, 11 nonfunctioning pituitary adenomas [NFPAs], and 2 prolactinomas), were treated with BIM-065, and key functional endpoints were assessed (cell viability, apoptosis, hormone secretion, expression levels of key genes, free cytosolic [Ca2+]i dynamics, etc.). AtT-20 cell line was used to evaluate signaling pathways in response to BIM-065. Results: This chimeric compound decreased cell viability in all corticotropinomas and somatotropinomas tested, but not in NFPAs. BIM-065 reduced ACTH, GH, chromogranin-A and PRL secretion, and increased apoptosis in corticotropinomas, somatotropinomas, and NFPAs. These effects were possibly mediated through modulation of pivotal signaling cascades like [Ca2+]i kinetic and Akt- or ERK1/2-phosphorylation. Conclusions: Our results unveil a robust antitumoral effect in vitro of the novel chimeric compound BIM-065 on the main PitNET subtypes, inform on the mechanisms involved, and suggest that BIM-065 could be an efficacious therapeutic option to be considered in the treatment of PitNETs.

The effect of gonadal steroids (GS) on proliferation of lymphocytes and distribution of lymphocyte subpopulations in cell culture was examined. The involvement of protein kinase C (PKC) and calcium ionophore in the proliferative response was tested. Estradiol benzoate (EB) or testosterone propionate (TP) had no significant influence on proliferation of peripheral blood lymphocytes (PBL) when cells were not stimulated by mitogen. At high concentration (10-6 M), EB and dihydrotestosterone (DHT) decreased lymphocyte proliferative response to concanavalin A (ConA) and lipopolysaccharide (LPS) at 24 and 72 h of incubation. However, at physiological doses (10(-12) to 10(-16) M), EB significantly enhanced the proliferative response at 24 h of incubation, whereas DHT had no effect. The inhibitory effect of the high dose of EB or DHT on proliferation of T and B lymphocytes was independent of time of hormone presentation to the cells or age and gender of cell donor. In all cultures, pre-incubation of lymphocytes with 10(-6) M of EB or DHT significantly reduced their proliferative responses to ConA, phytohemagglutinin (PHA), and LPS. The percentage of CD3+ cells was significantly reduced by EB, whereas DHT had no such effect. In contrast to inhibition of proliferation in response to mitogens, 10(-6) M EB dramatically enhanced the proliferation of lymphocytes in response to the PKC activator, phorbol 12-myristate 13-acetate, and calcium ionophore, A23187. Results suggest that high doses of EB do not damage the viability or proliferation capability of lymphocytes and, therefore, suppress the proliferative response to mitogens in a different manner, perhaps by reducing gene transcription for receptors that recognize the mitogens, or suppressing some postreceptor events. The enhancement of proliferation in response to mitogens by low doses of EB may support this assumption, because the biphasic effects of steroids on gene transcription are well documented.

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