somatostatin-28 and Pituitary-Neoplasms

Targeted overexpression of biologically active peptides represents a powerful approach to the functional dissection of neuroendocrine systems. However, the requirement to generate separate, biologically active and reporter molecules necessitates the use of internal ribosome entry site (IRES) technology, which often results in preferential translation of the second cistron. We report here a novel approach in which the proteolytic processing machinery of the regulated secretory pathway (RSP) has been exploited to generate multiple mature proteins from a monocistronic construct that encodes a single precursor. This was achieved by duplication of the pre-pro cleavage sites in pre-prosomatostatin cDNA. The duplicated site included 10 flanking amino acids on either side of the Gly-Ala cleavage position. This enabled the incorporation of a foreign protein-coding sequence (in this case, enhanced green fluorescent protein (eGFP)) between these sites. The pre-eGFP-prosomatostatin (PEPS) construct generated co-localized expression of fully processed eGFP and somatostatin to the RSP of transiently transfected AtT20 cells. This approach represents an advance upon bicistronic and other extant approaches to the targeting of multiple, biologically active proteins to neuroendocrine systems, and, importantly, permits the co-expression of fluorescent markers with biologically active neuropeptides. In this study, our demonstration of the fusion of the first 10 amino acids of the prosomatostatin sequence to the N-terminus of eGFP shows that this putative sorting sequence is sufficient to direct expression to the RSP.

Topics: Animals; Cell Line, Tumor; Genes; Green Fluorescent Proteins; Mice; Pituitary Neoplasms; Protein Engineering; Protein Precursors; Protein Processing, Post-Translational; Protein Transport; Somatostatin

We have previously shown that the somatostatin (SRIH) precursor (pro-SRIH) and SRIH are present in the normal human pituitary, whereas mature SRIH is never detected in GH-secreting adenomas associated with high plasma GH levels, and pro-SRIH is absent in 50% of them. Considering the fact that GHRH is present and released in vitro in higher amounts from GH adenomas than from normal human pituitaries, the possibility of a negative control exerted by GHRH on pituitary SRIH was investigated. When GH-secreting adenomas were incubated for 4 h in the presence of GHRH (10(-8) mol/L), the amount of pro-SRIH, quantified after Sephadex G-50 filtration of the acetic acid extracts, was significantly decreased. The percent inhibition was negatively correlated to the amount of endogenously released GHRH measured in the control incubation medium, suggesting a local negative feedback control exerted by pituitary GHRH on pituitary SRIH. This was further demonstrated when adenomas were incubated with a GHRH antibody. Indeed, the presence of the GHRH antibody resulted in a significant increase in the content of pro-SRIH in the adenoma. Similar results were obtained for in vitro SRIH release; exogenous GHRH induced an inhibition of SRIH release from GH-secreting adenomas, and the GHRH antibody had the opposite effect. Using a perifusion system, we showed the concomitance between the increase in GH release and the decrease in SRIH release after GHRH stimulation. Together, these results show in vitro a negative control exerted by GHRH (both exogenous and locally released) on adenomatous pituitary SRIH. This further amplifies the importance of autocrine or paracrine controls in these tumors.

Topics: Adenoma; Antibodies; Culture Techniques; Growth Hormone; Humans; Pituitary Neoplasms; Protein Precursors; Somatostatin

To investigate the relationship between prohormone processing and sorting of mature polypeptides into nascent secretory vesicles, we recently developed a permeabilized cell system that supports both these reactions (Xu, H., and Shields, D. (1993) J. Cell Biol. 122, 1169-1184). Rat anterior pituitary GH3 cells expressing high levels of prosomatostatin (proSRIF) were incubated at 20 degrees C; this temperature prevented exit from the trans-Golgi network and inhibited proSRIF processing. Following the 20 degrees C block, the cells were mechanically permeabilized and incubated at 37 degrees C, and proSRIF processing was determined. Cleavage of proSRIF to the mature hormone required ATP hydrolysis and was inhibited by chloroquine, a weak base, or carbonyl cyanide m-chlorophenylhydrazone, a protonophore. This suggested that a proton gradient and/or an acidic pH facilitated by a vacuolar H(+)-ATPase was required for prohormone processing. We have now utilized the permeabilized cell system in conjunction with the antibiotic bafilomycin A1, a specific inhibitor of vacuolar H(+)-ATPases, to elucidate the role of acidic pH in prohormone processing. Here we report that (i) proSRIF processing was inhibited in vivo and in vitro by low concentrations of bafilomycin A1, confirming the involvement of a vacuolar type ATPase in prohormone processing; (ii) the ATP requirement for processing could be circumvented in vitro by incubating permeabilized cells at acidic pH in the presence of protonophores, indicating that an acidic pH rather than a H+ gradient is necessary for processing; and (iii) a pH of between 6 and 6.2 in the trans-Golgi network was optimal for proSRIF cleavage. We also demonstrate that prohormone convertase 2 exhibited temperature-dependent activity in which proSRIF processing was inhibited at 20 degrees C in vitro. This result explains our previous observation that prohormone processing is inhibited when intact cells are incubated at 20 degrees C.

Topics: Animals; Anti-Bacterial Agents; Cell Line; Cell Membrane Permeability; Cysteine; Golgi Apparatus; Hydrogen-Ion Concentration; Intracellular Membranes; Kinetics; Macrolides; Pituitary Gland, Anterior; Pituitary Neoplasms; Protein Precursors; Protein Processing, Post-Translational; Proton-Translocating ATPases; Rats; Somatostatin; Sulfur Radioisotopes; Temperature; Vacuoles

Preprosomatostatin (preproSRIF) is a peptide hormone precursor that undergoes tissue-specific processing at either a single set of paired basic residues to yield SRIF-14 or, alternatively, at a monobasic site to produce SRIF-28, an NH2 terminally extended form of SRIF-14. Mammalian preproSRIFs are a family of precursors that are remarkably conserved from rat to humans. In five species, the signal peptide and propeptides are approximately 96% identical; this high degree of sequence identity may be indicative of functional conservation. Since the propeptide is approximately five times larger than SRIF-14, we hypothesized that it would be secreted as a separate polypeptide following proSRIF proteolytic processing. To test this idea, we raised polyclonal antibodies to the entire propeptide to follow its biosynthesis and secretion. Here we demonstrate that in transfected rat anterior pituitary GH3 cells both SRIF-14 and the intact 9.5-kDa propeptide were processed coordinately from proSRIF with identical kinetics. Treatment of the cells with chloroquine, a weak base which inhibits processing to mature SRIF-14, also inhibited the appearance of the 9.5-kDa propeptide. Approximately 40% of the propeptide was targeted to the regulated secretory pathway as determined by its quantitative secretion in response to secretagogues. We also examined the secretion of the SRIF propeptide independently of SRIF-14 by expressing a truncated "propeptide" in which SRIF-14 was deleted. Significantly, the SRIF propeptide was itself efficiently transported through the secretory pathway and secreted into the culture medium. This suggests that the propeptide possesses all the topogenic information necessary for intracellular transport. The coordinate secretion of the intact propeptide with mature SRIF-14 suggests that it might function as a novel bioactive peptide.

Topics: Amino Acid Sequence; Animals; Antibodies; Cell Line; Chromatography, High Pressure Liquid; Fishes; Humans; Kinetics; Methionine; Molecular Sequence Data; Pituitary Gland, Anterior; Pituitary Neoplasms; Protein Precursors; Protein Processing, Post-Translational; Rats; Somatostatin; Tumor Cells, Cultured

In contrast to normal human pituitaries, GH-secreting adenomas cannot process in vivo ProSRIH whereas they do it in vitro. The existence of an endogenous factor able to inhibit ProSRIH processing in vivo was postulated and such a role was analyzed for GHRH. Results showed that when GH adenomas are incubated in vitro with GHRH 10(-8) M, their ProSRIH contents are decreased, percent inhibition being negatively correlated to the amount of endogenously released GHRH. When incubation is performed in the presence of GHRH antibody in order to block the effect of endogenous GHRH, Pro-SRIH content is increased. The same effects are observed on SRIH release: inhibition by GHRH, stimulation by GHRH antibody. Normal rabbit serum had no effect. It may therefore be concluded that the absence of ProSRIH maturation observed in adenomas in vivo may be the consequence of the GHRH release that is known to be higher from GH adenomas than from normal pituitaries.

Topics: Adenoma; Adult; Aged; Antibodies; Growth Hormone; Growth Hormone-Releasing Hormone; Humans; Middle Aged; Pituitary Gland; Pituitary Neoplasms; Protein Precursors; Somatostatin