Prolactin granulogenesis is associated with increased secretogranin expression and aggregation in the Golgi apparatus of GH4C1 cells.
The GH4C1 pituitary tumor cell line (GH cells) serves as a model system to study the role of the granins in the packaging of PRL into secretory granules. The number of secretory granules containing PRL and two members of the granin family, chromogranin-B (CgB) and secretogranin-II (SgII), can be hormonally manipulated. In the present study we have investigated whether 1) granulogenesis in GH cells is preceded by condensation of the granins and PRL in the Golgi; 2) granulogenesis is preceded by an increase in granin expression in GH cells; and 3) PRL and the granins aggregate in vitro under high calcium, low pH conditions. GH cells were treated for up to 3 days with 17 beta-estradiol (1 nM), insulin (300 nM), and epidermal growth factor (10 nM) and were fixed in 4% paraformaldehyde for immunocytochemistry or harvested for RNA isolation and Northern blot analysis. After 1 day of hormone treatment, there was a significant increase in staining for PRL and the granins in the Golgi apparatus, which was identified using an antibody to MG-160. After 3 days of hormone treatment, PRL and granin staining was also found in a perinuclear region that was not stained with anti-MG-160 antibody, most likely representing secretory granules. An increase in PRL and granin expression contributed to increased Golgi staining, as the steady state levels of CgB, SgII, and PRL mRNA increased 186 +/- 14%, 203 +/- 7%, and 337 +/- 5% above control levels, respectively, within 6 h after hormone treatment. An in vitro aggregation system was used to determine whether PRL and the granins coprecipitate under high calcium, low pH conditions, which are thought to be characteristic of the trans-Golgi and secretory granules. Aggregation of the granins CgB and SgII was negligible during overnight dialysis against a buffer containing 150 mM NaCl and 10 mM 2[N-morpholino]ethanesulfonic acid-NaOH (pH 5.5) in the absence of calcium. There was significant aggregation of PRL under these conditions. When dialysis was performed in the presence of 10 mM CaCl2, PRL, CgB, and SgII coaggregated. This study indicates that increased expression and aggregation of the granins is associated with PRL granulogenesis in hormone-treated GH cells. However, the role of the granins may not be obligatory, as some cells can store PRL in the absence of detectable levels of CgB and SgII, and PRL has the capacity to self-aggregate.
Thompson
M E
ME
Department of Pharmacology, University of South Alabama, Mobile 36688.
Zimmer
W E
WE
Haynes
A L
AL
Valentine
D L
DL
Forss-Petter
S
S
Scammell
J G
JG
D000818:Animals; D015152:Blotting, Northern; D002122:Calcium Chloride / Q000494:pharmacology; D002864:Chromogranins / Q000235:genetics / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004958:Estradiol / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D006056:Golgi Apparatus / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000494:pharmacology; D046911:Macromolecular Substances; D010911:Pituitary Neoplasms / Q000378:metabolism* / Q000648:ultrastructure; D011388:Prolactin / Q000378:metabolism*; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D014407:Tumor Cells, Cultured
D002864:Chromogranins; D007328:Insulin; D046911:Macromolecular Substances; D011506:Proteins; D012333:RNA, Messenger; D004958:Estradiol; D004815:Epidermal Growth Factor; D011388:Prolactin; D002122:Calcium Chloride