Maintained PC1 and PC2 Expression in the AtT-20 Variant Cell Line 6T3 Lacking Regulated Secretion and POMC: Restored POMC Expression and Regulated Secretion after cAMP Treatment

Abstract

Two variant cell lines were recently established from parent AtT-20 cells. Whereas HYA.15.10.T.2 have a reduced level of secretory granules, HYA.15.6.T.3 are completely devoid of both the regulated pathway of secretion and of dense-core secretory granules. AtT-20 cells normally express the processing enzymes PC1, PC2, furin, carboxypeptidase E, and peptidylglycine alpha-amidating monooxygenase, as well as proopiomelanocortin, chromogranin B, and 7B2. We measured the expression of these mRNAs in both variant cell lines. Although some differences in mRNA level were noted, HYA.15.10.T.2 and HYA.15.6.T.3 cell lines maintained their expression of the processing enzymes and of 7B2. Furthermore, PC1 and PC2 were shown to be functionally active in the HYA.15.6.T.3 cells. In contrast, proopiomelanocortin and chromogranin B mRNA levels were no longer detectable in HYA.15.6.T.3 cells. Interestingly, stimulation of the HYA.15.6.T.3 cells with cAMP restored proopiomelanocortin mRNA, beta-endorphin immunoreactivity, and dense-core granules. Furthermore, at the ultrastructural level, beta-lipotropin immunoreactivity was detected in granules of cAMP-induced HYA.15.6.T.3 cells. Finally, depolarization of cAMP-induced HYA.15.6.T.3 cells with 56 mM potassium chloride resulted in a marked increase in the release of beta-endorphin immunoreactivity. These observations demonstrate that cAMP restores the regulated pathway of secretion in HYA.15.6.T.3 cells, which under untreated conditions do not demonstrate regulated release. These variant cell lines are unique models to understand better the relationship of the regulated pathway and the expression of the processing enzymes.

Neurosecretion Competence

“…The defective phenotype of NI clones is stable. In contrast to another reported cell model (12), it cannot be reverted by stimulatory treatments or by changes in growth conditions. However, the NI phenotype defect can be rescued by fusion of the NI cells with WT cells (regardless of the species or phenotype) and following transfection with a WT PC12 cDNA library (11,13).…”

Chromogranin B-induced Secretory Granule Biogenesis

“…In this PC12 variant, the genes for CGA and CGB were found to be down-regulated more than 10-fold (43). Further, in a mouse pituitary cell line AtT-20 variant (HYA.15.6.T.3), which was devoid of both regulated secretion and secretory granules, no CGB expression was detected (44). Using whole rats, the level of CGA expression in pituitary gonadotropes was shown to be closely related to the number of large secretory granules, whereas the number of small secretory granules was related to the level of secretogranin II expression (45).…”

Chromogranin A processing and secretion: specific role of endogenous and exogenous prohormone convertases in the regulated secretory pathway.

“…While Jung et al (43,44) studied cleavage-dependent trafficking of exogenous (transfected ELH) neuropeptides, our results document cleavage-dependent trafficking of endogenous (chromogranin A, ␤-endorphin) neuropeptides. By contrast, expression of PCs may not be entirely sufficient to ensure regulated secretion, since the AtT-20/myeloma fusion cell line HYA.15.6.T.3 (45), which expresses both PC1 and PC2, lacks dense core vesicles and a regulated pathway of secretion; in the HYA.15.6.T.3 line, regulated secretion of proopiomelanocortin can be restored by cyclic AMP treatment (45). Likewise, in at least some cell types, cleavage of chromogranin A may not be entirely necessary for its entry into the regulated secretory pathway; for example, chromogranin A is only partially processed in chromaffin vesicles, and intact chromogranin A is released from chromaffin cells in response to physiologic (nicotinic cholinergic) secretory stimulation (2,4,41).…”