Dense-core granules: a specific hallmark of the neuronal/neurosecretory cell phenotype

Abstract

Expression of dense-core granules, a typical exocytic organelle, is widely believed to be controlled by coordinate gene expression mechanisms specific to neurones and neurosecretory cells. Recent studies in PC12 cells, however, have suggested the number of granules/cells depends on the levels of only one of their cargo proteins, chromogranin A, regulating the metabolism of the other proteins, and thus the composition of the organelles, by an on/off switch mechanism. In addition, transfection of chromogranin A was reported to induce appearance of dense-core granules in the non-neurosecretory fibroblasts of the CV-1 line. Here the role of chromogranin A has been reinvestigated using not the heterogeneous PC12 line but several clones isolated therefrom. In these clones, investigated as such or after transfection with chromogranin A antisense sequences, the ratio between chromogranin A and its secretory protein mate, chromogranin B, was not constant but highly and apparently randomly variable. Variability of the chromogranin A/chromogranin B ratio was seen by confocal immunofluorescence also among the cells of single clones and subclones and among the granules of single cells. Moreover, stable and transient transfections of chromogranin A in a PC12 clone characterised by a low number of dense-core granules (one fifth of the reference clone) failed to modify significantly the number of the organelles, despite the several-fold increase of the granin. Finally, in three types of non-neurosecretory cells (CV-1, adenocarcinoma TS/A and a clone of PC12 incompetent for secretion) the transfected chromogranin A accumulated mostly in the Golgi/transGolgi area and was released rapidly from resting cells (constitutive secretion) as revealed by both immunofluorescence during cycloheximide treatment and pulse-chase experiments. Only a minor fraction was sorted to discrete organelles that were not dense-core granules, but primarily lysosomes because they contained no chromogranin B, and were largely positive for the late endosomal-lysosomal markers, lamp1 and lamp3. Dense-core granules are therefore true hallmarks of neurones and neurosecretory cells. Their number/cell appears independent of chromogranin A and their composition does not appear to be constant; in particular, they exhibit considerable, and so far unexplained variability in the chromogranin A/chromogranin B ratio.

ANXA7 expression represents hormone‐relevant tumor suppression in different cancers

“…36,37 Our current study identified abundant ANXA7 expression in neuroendocrine tissues which contain dense-core granules as true exocytotic hallmarks. 38 A routine marker of neuroendocrine differentiation in cancer, Chromogranin A can improve diagnostic accuracy of prostate-specific antigen 39 by identifying the neuroendocrine differentiation in prostate cancer leading to the androgeninsensitive cells arrested in G0-phase and resistant to apoptosis. 40 Remarkably, androgen-resistant prostate cancer was characteristic of more drastic ANXA7 loss that was clearly demonstrated in our present and previous studies.…”

Beta cell chromogranin B is partially segregated in distinct granules and can be released separately from insulin in response to stimulation

“…Immunocytochemistry, image analysis and immunoelectron microscopy Immunofluorescence was performed as described [31]. Image deconvolutions and 3D cell reconstructions were performed on 150 nm optical Z-stack sections acquired by Deltavision wide-field microscopy system and processed with softWoRx3.4.5 software (Applied Precision, Issaquah, WA, USA).…”

“…sodium deoxycholate, 50 mmol/l Tris pH 7.4 and 150 mmol/l NaCl added with protease inhibitors, were loaded on to a 0.22 µm pore size nitrocellulose membrane (Biorad, Milan, Italy) in a dot-blot apparatus. Proteinloaded membranes were air-dried and then processed as for western blotting [31] using a mixture of two anti-CgB antibodies: the pAb PE-11 [27] (1:400) and the mAb CIRO (1:1,000). Chemiluminescence of bound antibodies, revealed by the Western Femto Signal (Pierce Biotech, Rockford, IL, USA), was acquired every 30 s in a UVP Bioimaging System (UVP, Upland, CA, USA).…”

Gene expression profiling of HUH7-ins: Lack of a granulogenic function for Chromogranin A.

“…Two members of the granin family, chromogranin A (CgA) and B (CgB), have been described as regulating SGB in neuroendocrine cells, 1,2 although the level of influence these proteins exert is disputed. [3][4][5] Overexpression of these granins in fibroblasts initiated the formation of putative dense-core granules, and a reduction in expression both lowered the number of granules present in the cytoplasm of the neuroendocrine cell line PC12…”