Effects of selective somatostatin analogs and cortistatin on cell viability in cultured human non-functioning pituitary adenomas.

Clinically "non-functioning" human pituitary adenomas (NFPA) constitute about 35% of pituitary adenomas. Somatostatin receptors (SSTR) expression in these adenomas has previously been described both in vitro and in vivo, without evidence for a correlation with tumor volume or the therapeutic efficacy of somatostatin analogs. This study was performed on 13 surgically removed pituitary macroadenomas, diagnosed before surgery as "non-functioning". In addition, 3 growth hormone (GH)-secreting adenomas served as controls. A specimen from each tumor was dispersed and digested to isolate and culture the tumor cells, and the in vitro effects of SSTR2 and SSTR5 selective analogs and Cortistatin (CST) (100nM) on cell viability were studied. The quantity of viable cells was estimated using the XTT method. RNA purification of tumor samples and subsequent RT-PCR studies for SSTR2 and SSTR5 expression were performed. Somatostatin analog with high affinity for SSTR2 reduced cell viability by 20-80% in 8 of 13 NFPAs studied, all expressing the SSTR2. The inhibitory effect on cell viability of SSTR5-selective analog was 15-80% in 10 of 13 NFPAs studied, all but three expressing the SSTR5. CST, however, effectively reduced cell viability in only 6 NFPAs. Cell viability was inhibited by all peptides studied in 2 out of 3 GH-secreting adenomas, expressing both receptors. The third adenoma responded to SSTR2 analog and expressed only SSTR2. These results suggest the involvement of SSTR2 and SSTR5 in the anti-proliferative effects of somatostatin; however, CST is less potent in reducing cell viability in these tumors.

Novel ghrelin analogs with improved affinity for the GH secretagogue receptor stimulate GH and prolactin release from human pituitary cells.

OBJECTIVE: Ghrelin, a recently identified 28-amino acid peptide is a potent GH secretagogue (GHS) produced predominantly by the stomach. Ghrelin stimulates GH secretion through binding to the GHS receptor in the hypothalamus and pituitary. In addition to the GH-releasing action, ghrelin has been found to be a powerful orexigenic factor. To assess the direct in vitro effects of ghrelin on human pituitary hormone secretion we have produced a panel of novel ghrelin analogs (molecular weight, 3323-3384; human native ghrelin, 3371) with enhanced affinity for the human GHS receptor (IC(50) 0.38-1.09 nM; human ghrelin, 1.2-2.2 nM). METHODS: The peptidic analogs were tested for their effect on GH secretion using dispersed human fetal pituitaries (21 to 23 weeks of gestation) and cultured GH- and prolactin (PRL)-secreting adenomas. The expression of the GHS receptor in normal (fetal and adult) human pituitary tissues, GH- and PRL-cell adenomas was established using RT-PCR. RESULTS: The effects of ghrelin, its analogs and GH-releasing hormone (GHRH) alone or in combination on GH and PRL secretion were compared at various concentrations. The ghrelin analogs stimulated GH release by 35-60% from human fetal pituitary cells (1-10 nM; P<0.05) and by 50-75% from cultured pituitary adenomas (10 nM; P<0.05). This releasing effect was dose-dependent, achieving maximal stimulation with analog concentrations at 100 nM. Human ghrelin was less potent as compared with its analogs in stimulating human GH, in keeping with the improved binding affinity of the analogs for the GHS-1a receptor. The ghrelin analogs and GHRH had comparable effects on GH secretion from both normal and adenomatous cells, and in combination produced an additive stimulatory effect on GH (150%; P<0.0001). In contrast, ghrelin and its analogs induced a comparable increase in PRL release ranging between 25 and 40% (P<0.05) from fetal cells and 30 and 70% (P<0.001) from cultured PRL-cell and mixed GH-PRL adenomas. CONCLUSIONS: Our results have demonstrated for the first time that ghrelin analogs with enhanced affinity for the GHS receptor are potent stimulators of GH secretion from human pituitary cells, and thus may possess potential clinical therapeutic benefits.

Involvement of the activation loop of ERK in the detachment from cytosolic anchoring.

The Extracellular signal-regulated kinases (ERKs) are translocated into the nucleus in response to mitogenic stimulation. The mechanism of translocation and the residues in ERKs that govern this process are not clear as yet. Here we studied the involvement of residues in the activation loop of ERK2 in determining its subcellular localization. Substitution of residues in the activation loop to alanines indicated that residues 173-181 do not play a significant role in the phosphorylation and activation of ERK2. However, residues 176-181 are responsible for the detachment of ERK2 from MEK1 upon mitogenic stimulation. This dissociation can be mimicked by substitution of residues 176-178 to alanines and is prevented by deletion of these residues or by substitution of residues 179-181 to alanines. On the other hand, residues 176-181, as well as residues essential for ERK2 dimerization, do not play a role in the shuttle of ERK2 through nuclear pores. Thus, phosphorylation-induced conformational rearrangement of residues in the activation loop of ERK2 plays a major role in the control of subcellular localization of this protein.

Identification of a cytoplasmic-retention sequence in ERK2.

A key step in the signaling mechanism of the mitogen-activated protein kinase/extracellular signal-responsive kinase (ERK) cascade is its translocation into the nucleus where it regulates transcription and other nuclear processes. In an attempt to characterize the subcellular localization of ERK2, we fused it to the 3'-end of the gene expressing green fluorescent protein (GFP), resulting in a GFP-ERK2 protein. The expression of this construct in CHO cells resulted in a nuclear localization of the GFP-ERK2 protein. However, coexpression of the GFP-ERK2 with its upstream activator, MEK1, resulted in a cytosolic retention of the GFP-ERK2, which was the result of its association with MEK1, and was reversed upon stimulation. We then examined the role of the C-terminal region of ERK2 in its subcellular localization. Substitution of residues 312-319 of GFP-ERK2 to alanine residues prevented the cytosolic retention of ERK2 as well as its association with MEK1, without affecting its activity. Most important for the cytosolic retention are three acidic amino acids at positions 316, 319, and 320 of ERK2. Substitution of residues 321-327 to alanines impaired the nuclear translocation of ERK2 upon mitogenic stimulation. Thus, we conclude that residues 312-320 of ERK2 are responsible for its cytosolic retention, and residues 321-327 play a role in the mechanism of ERK2 nuclear translocation.

Detection of ERK activation by a novel monoclonal antibody.

The mitogen-activated protein kinase, ERK is activated by a dual phosphorylation on threonine and tyrosine residues. Using a synthetic diphospho peptide, we have generated a monoclonal antibody directed to the active ERK. The antibody specifically identified the active doubly phosphorylated, but not the inactive mono- or non- phosphorylated forms of ERKs. A direct correlation was observed between ERK activity and the intensity in Western blot of mitogen-activated protein kinases from several species. The antibody was proven suitable for immunofluorescence staining, revealing a transient reactivity with ERKs that were translocated to the nucleus upon stimulation. In conclusion, the antibody can serve as a useful tool in the study of ERK signaling in a wide variety of organisms.

Nuclear translocation of mitogen-activated protein kinase kinase (MEK1) in response to mitogenic stimulation.

Mitogen-activated protein kinase kinase (MEK) is a dual-specificity protein kinase that is located primarily in the cellular cytosol, both prior to and upon mitogenic stimulation. The existence of a nuclear export signal in the N-terminal domain of MEK [Fukuda, M., Gotoh, I., Gotoh, Y. & Nishida, E. (1996) J. Biol. Chem. 271, 20024-20028] suggests that there are circumstances under which MEK enters the nucleus and must be exported. Using mutants of MEK, we show that the deletion of the nuclear export signal sequence from constitutively active MEK caused constitutive localization of MEK in the nucleus of COS7 and HEK-293T cells. However, when the same region was deleted from a catalytically inactive MEK, cytoplasmic localization was observed in resting cells, which turned nuclear upon stimulation. Confocal microscopy of COS7 cells expressing the above mutants showed localization of the active MEK in the nuclear envelope and also in the cell periphery. The differences in cellular localization between the wild-type and mutant MEKs are not due to severe changes in specificity because the recombinant, constitutively active MEK that lacked its N-terminal region exhibited the same substrate specificity as the wild-type MEK, both in vitro and in intact cells. Taken together, our results indicate that upon mitogenic stimulation, MEK, like extracellular signal responsive kinase and p90(RSK), is massively translocated to the nucleus. Rapid export from the nucleus, which is mediated by the nuclear export signal, is probably the cause for the cytoplasmic distribution observed with wild-type MEK.