Post-translational processing of proopiomelanocortin (POMC) in mouse pituitary melanotroph tumors induced by a POMC-simian virus 40 large T antigen transgene.

M J Low,B Liu,G D Hammer,M Rubinstein,R G Allen

doi:10.1016/s0021-9258(19)74558-x

M J Low, B Liu + Show 3 more

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https://doi.org/10.1016/s0021-9258(19)74558-x

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Abstract

Mice harboring a transgene composed of proopiomelanocortin (POMC) gene promoter sequences (nucleotides -706 to +64) ligated to the simian virus (SV) 40 early gene encoding large T antigen developed large POMC-expressing pituitary tumors. Histologically the tumors arose from the intermediate lobe, contained nuclear SV40 T antigen and POMC peptides, but did not express other pituitary hormones. POMC processing in the pituitary tumors was indistinguishable from normal mouse intermediate lobe melanotrophs and was characterized by high proportions of acetylated and carboxyl-terminal shortened beta-endorphins, and amino-terminal acetylated alpha-melanocyte-stimulating hormone, and virtually no adrenocorticotropic hormone (ACTH)(1-39), beta-lipotropin, or POMC. The tumors contained abundant levels of mRNA for the prohormone convertase PC2 and undetectable levels of PC1. Normal mouse neurointermediate lobe also has a high ratio of PC2/PC1 expression that is distinct from the relative abundance of PC1 in anterior lobe and AtT-20 corticotroph cells. In contrast, extracts from tumors transplanted subcutaneously in nude mice contained predominantly nonacetylated forms of beta-endorphin(1-31) and -(1-27), very little ACTH(1-39), almost no corticotropin-like intermediate peptide or alpha-melanocyte-stimulating hormone, and higher proportions of intact POMC. Surprisingly, despite the less efficient proteolytic cleavage, a transplanted tumor expressed both PC1 and PC2. These studies are the first biochemical documentation of a melanotroph pituitary tumor in a rodent species and provide a new model for the investigation of pituitary oncogenesis and the molecular basis of tissue-specific prohormone post-translational processing.

Highlights

From the $ Vollum Institute for Advanced BiomedicalResearch, **Centerfor Research on Occupationaland Environmental Toxicology, and the §Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, Oregon 97201
POMC process- terior and intermediate lobes ing in the pituitary tumors was indistinguishable froof mthe pituitary gland, as well as POMC secreting neurons normal mouse intermediate lobe melanotropbs and was have characteristic processing phenotypes that appear to be characterized by high proportions of acetylated and regulated during development and possibly in response to carboxyl-terminal shortened &endorphins, aanmdinoterminaalcetylateda-melanocyte-stimulatinghormone, and virtually no adrenocorticotropic hormone (ACTH)(1-39), 8-lipotropin, or POMC
Mature corticotrophs and the AtT-20 cell line derived from a mouse corticotroph tumor secrete predominantly ACTH[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39], @-lipotropin,and small amounts of nonacetylated @-endorphin[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31].Melanotrophs contained abundant levels ofmRNA for the prohor- have more complete proteolytic processing of POMC and mone convertase PC2 and undetectablelevels of PC1. secrete acetylated a-MSH, corticotropin-like intermediate

Summary

RESULTS

DNA for SV40 large T antigen sequences developed obvious pituitary tumors. Founder mouse No 427 was bred successfully prior to succumbing to its pituitary tumor and established a pedigree that has maintained the pituitary tumor phenotype unchanged for 15 generations through successive crosses to outbred Swiss Webster females. Microscopic abnormalities of the pituitary intermediate lobes were evident as early as 3 weeks of age and consisted of multifocal nodules of SV40 Tag immunoreactive melanotrophs. At 3 weeks of age ( l e f t )multifocal nodulesof abnorsitive cells were demonstrated occasionally in the anterior malcells are presentin a normalsizedintermediatelobe lobes (Fig. 1 and 2). At 6 weeks of age many of the mice had adiffuse hyperplasia of the intermediate lobe visible to the unaided eye, the general architecture of the pituitary remained intact. Larger tumors sometimes contained tiny residual islands of immunohistochemically normal anterior lobe cells buried within them but therweas no evidence of hormone expression other than POMC peptides in the neoplastic cells (data not shown). Transformed cells from the large tumors varied widely in theircontent of Tag, ACTH-like and pendorphin-like peptides by immunohistochemical staining (Fig. 2). The tumor bearing mice invariably rapidly lost weight in their final week

POMC Processing in Mouse MelanToutrmooprhs

ACTE RIA

SUBCUTANEOUSLY TRANSPLANTED PITUITARYTUMORS