Hormonal induction of secretory granules in a pituitary tumor cell line

GH4C1 cells are a rat pituitary tumor cell strain that secretes PRL and GH but contains almost no secretory granules. Treatment of GH4C1 cells with a combination of estradiol (1 nM), insulin (300 nM), and epidermal growth factor (10 nM) increased the cellular content of PRL by more than 30-fold above control levels but only increased PRL accumulation in the medium 6-fold. To determine whether the increase in intracellular PRL was accompanied by an increase in secretory granules, we compared the numbers of granules in ultrathin sections from untreated GH4C1 cells and from cells treated with the combined hormone regimen and found a nearly 50-fold increase in granule number. Only 75% of the granules stained for PRL by the protein-A gold technique; the other 25% stained for neither PRL nor GH. The occasional granules found in untreated GH4C1 cells stained for PRL. The data demonstrate that the number of granules in GH4C1 cells can be regulated by hormone treatment and that the increase in intracellular PRL is found in storage granules.     

Growth hormone synthesis and secretion by a somatomammotroph cell line derived from normal adult pituitary of the rat

A somatomammotroph cell line derived from male rat anterior pituitary (rPCO) has been established without the use of transforming agents and has been maintained in culture for more than 1 yr (45 passages) using Minimum Essential Medium supplemented with 10% horse serum, 5 nM T3, 2 nM corticosterone, and 0.1 nM GH-releasing hormone (GRH). Peroxidase and immunofluorescent staining revealed immunoreactive GH in 99% of rPCO cells and immunoreactive PRL in 72% of cells. Within individual cells, GH and PRL appear to be colocalized. The storage capacity for GH in rPCO cells represented 40% of the daily hormone production. In serum-free medium containing 5 nM cortisol, GH secretion was stimulated 10- and 25-fold by 50 pM and 50 nM T3, respectively. GRH (1 nM) stimulated GH secretion 8-fold in the absence of T3, although no effect was observed in the presence of 50 nM T3. Qualitatively similar changes occurred in GH mRNA responses to T3 and GRH. Other secretory proteins were detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of culture medium, several of which were present in concentrations similar to that of GH. Nine separate cell lines were cloned from rPCO cells by limiting dilution, all of which secreted GH and PRL. GH secretion varied 6-fold between clones, and the GH to PRL ratio varied more than 200-fold. These rPC cell lines provide a new model for studying the control of GH and PRL gene expression, including hormone synthesis, processing, and secretion. They may also be useful for identifying other pituitary secretory products and as a source for the production of protein hormones.     

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.     

Thyroid hormone and glucocorticoid regulation of receptor and target gene mRNAs in pituitary GH3 cells

We have examined the effects of triiodothyronine (T₃), in dose-response and time-course studies, on T₃ receptor (T₃R) and β and glucocorticoid receptor (GR) mRNAs in rate pituitary GH₃ cells, in parallel with T₃ actions on expression of the growth hormone (GH) target gene. Modulatory influences of dexamethasone (dex) on T₃ action were studied by treatment with dex before and during T₃ treatment. T₃ treatment (1–100 nM) for 24 h reduced T₃R mRNA, while the presence of dex (1 μM) enhanced the T₃ effect on T₃R mRNA and induced T₃ inhibition of T₃R β mRNA. Stimulatory effects of T₃ treatment on GH mRNA and release were seen in the face of inhibition of T₃R mRNAs; these effects on GH were also enhanced by the presence of dex. T₃ treatment for 24 h increased GR mRNA; this effect was inhibited by the presence of dex. We next examined the influence of dex on GR and T₃R and β mRNAs, in parallel with effects of dex on the prolactin (PRL) target gene. Modulatory influences of T₃ on dex action were studied by treatment of cells with T₃ before and during dex treatment. Treatment with dex (0.1–10 μM) for 24 h reduced GR mRNA, an action enhanced by the presence of T₃ (100 nM). Dex treatment resulted in inhibition of PRL mRNA and release despite parallel inhibition of GR mRNA by dex; these effects were enhanced by the presence of T₃. In contrast to actions on GR, dex has no effect on T₃R mRNAs. These effects of T₃ and dex on receptor mRNAs suggest that glucocorticoid modulation of T₃ action is not related to direct actions on T₃R synthesis. In contrast, the mechanism of T₃ modulation of glucocorticoid action may be due in part to alteration of GR mRNA expression. Effects of T₃ and dex on target gene expression were observed in the presence of parallel reduction of their respective receptor mRNAs. This provides new evidence that interactions between these hormones are likely to be mediated by mechanisms other than regulation of receptor gene expression.     

Growth hormone (GH)-releasing factor does not regulate GH release or GH mRNA levels in GH3 cells

Previous studies have shown that GH-releasing factor (GRF) regulates both GH production and GH mRNA levels in primary cultures of rat pituitary cells. Investigations were carried out to ascertain the ability of GRF to regulate GH production or mRNA levels in a clonal strain of rat pituitary tumor (GH3) cells. Incubation of the cells with GRF at 1-1000 nM for 4 h to 10 days did not result in a stimulation of GH or PRL production, nor did it affect the cytoplasmic levels of the corresponding mRNAs. The lack of response to GRF was not affected by dexamethasone, T3, or serum. We conclude that GH3 cells do not provide a useful model system for studies of the mechanism(s) of action of GRF on either GH release or GH gene expression.     

Age-related decrease of growth hormone and prolactin gene expression in the mouse pituitary

The effects of aging on pituitary GH, PRL, and alpha-tubulin messenger RNA (mRNA) levels were measured in 3-, 12-, and 27-month-old male C57BL/6J mice by dot-blot hybridization. The amount of GH and PRL mRNA in the pituitary deceased dramatically with age. However, total poly(A+) RNA (mRNA), as measured by hybridization with radioactively labeled oligo-(dT), was not altered during aging. In addition, there were no age-related changes in the level of alpha-tubulin mRNA. Thus, the effects of aging on GH and PRL mRNA levels are specific; the levels of the majority of cellular mRNAs are not altered with age. GH and mRNA levels decreased 35% between 3 and 12 months (P less than 0.05) and a total of 75% after 27 months (P less than 0.01). PRL mRNA levels decreased 65% between 12 and 27 months (P less than 0.01), although there was no significant decrease before 12 months. Whereas T3 is the most potent regulator of GH gene expression, we did not detect any significant age-related change in serum T3 levels. These results suggest that factors other than T3 play a role in the age-related decline in GH and PRL gene expression.     

Dopaminergic control of prolactin mRNA accumulation in the pituitary of the male rat

Dopaminergic control of the expression of the prolactin gene was investigated by administration of biomoergocryptine (CB154) to male rats. The effects of the drug on the following parameters were measured: (i) circulating levels of GH and PRL; (ii) synthesis of GH and PRL measured by pulse labeling pituitary fragments in vitro; (iii) GH and PRL mRNA activities; and (iv) content of PRL mRNA. After 1 day of CB154 administration, serum PRL fell to undetectable levels whereas it took 3 days to observe a 50% reduction in PRL synthesis. This effect was accounted for by a parallel decrease in PRL mRNA activity and content. GH synthesis and GH mRNA were not affected by the treatment. Our results show that the dopaminergic inhibition of PRL production involves regulation at a pre-translational level.     

Inhibitory effects of serum and stimulatory effects of estrogen on prolactin mRNA levels in GH3 rat pituitary tumor cells.

We investigated the effects of serum and estrogen on prolactin (PRL) mRNA accumulation in GH3 cells under different cell culture conditions. Hybridization analysis of GH3 cellular RNA indicated that PRL mRNA levels decreased more than 20-fold in cells cultured for 1 week in medium containing dextran-charcoal-treated serum (stripped serum). No effects on actin mRNA levels were observed under these conditions. Furthermore, this inhibition of PRL mRNA accumulation depended on the concentration of stripped serum in the medium. Although incubation in stripped-serum medium inhibited cell growth, these culture conditions did not appear to irreversibly affect the GH3 cell population. These data indicate that a potent inhibitor of PRL mRNA accumulation is present in stripped serum. GH3 cells grown in stripped-serum medium were shown to be responsive to estrogen. Treatment of these cells with 10(-9) M estradiol resulted in a 6.6-fold stimulation of PRL gene expression. However, estrogen had no effect on cell growth under these conditions, suggesting that estrogen stimulates PRL gene expression and cell proliferation by independent mechanisms.     

Effects of endocrine disrupters on the expression of growth hormone and prolactin mRNA in the rainbow trout pituitary

It is now widely accepted that chemical pollutants in the environment can interfere with the endocrine system of animals, thus affecting development and reproduction. Some of these endocrine disrupters (EDs) can have estrogenic or antiestrogenic effects. Most studies to date have focused on the effects of EDs on the reproductive system and sex hormones and only limited information exists on how EDs may affect pituitary gland function. A rainbow trout (Oncorhynchus mykiss) pituitary gland culture system was used for studying the effects of EDs on growth hormone (GH) and prolactin (PRL) mRNA expression. We determined that the pituitary glands actively synthesized and secreted GH and PRL over the experimental time-course. In addition, we found that treatment with 17beta-estradiol (positive control) increased levels of GH and PRL mRNA, in a concentration-dependent manner. Treatment of pituitary glands with 500 and 1000 nM of a xenoestrogen, o,p'-DDT (o,p'-dichlorodiphenyltrichloroethane), resulted in a significant induction of GH and PRL mRNA, with a 20-fold increase for PRL and 3-fold increase for GH following treatment with 1000 nM o,p'-DDT. Co-incubation of pituitary glands with ICI 182 780 (a selective estrogen receptor antagonist) and o,p'-DDT resulted in inhibition of PRL mRNA levels; however, the stimulatory effect of DDT on GH mRNA was not seen in this experiment, nor was the inhibitory effect of ICI 182 780 observed with GH mRNA. To the contrary, ICI 182 780 (2.5 nM) had a stimulatory effect on GH mRNA levels. TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), which is known to exert antiestrogenic effects, had an estrogenic-like effect that resulted in a concentration-dependent increase in the levels of GH and PRL mRNA. Co-incubation of pituitaries with TCDD and alpha-napthoflavone (ANF), which is an inhibitor of the aryl hydrocarbon receptor (AhR), caused an inhibition of TCDD-induced PRL mRNA at the higher and lower concentrations, but these effects were less consistent on GH mRNA levels. However, the responses of PRL and GH mRNA to co-incubation with TCDD and ANF, at the various concentrations, were bi-phasic wherein stimulation was seen at the low concentrations and inhibition at the high concentrations. Combined, these results suggest that o,p'-DDT and TCDD are xenoestrogens and that their effects on the expression of GH and PRL genes in the rainbow trout pituitary are modulated, in part, through the ER and AhR, respectively.     

The novel somatostatin analog SOM230 is a potent inhibitor of hormone release by growth hormone- and prolactin-secreting pituitary adenomas in vitro

To determine the inhibitory profile of the novel somatostatin (SRIF) analog SOM230 with broad SRIF receptor binding, we compared the in vitro effects of SOM230, octreotide (OCT), and SRIF-14 on hormone release by cultures of different types of secreting pituitary adenomas. OCT (10 nM) significantly inhibited GH release in seven of nine GH-secreting pituitary adenoma cultures (range, -26 to -73%), SOM230 (10 nM) in eight of nine cultures (range, -22 to -68%), and SRIF-14 (10 nM) in six of six cultures (range, -30 to -75%). The sst analysis showed predominant but variable levels of somatostatin receptor (sst)(2) and sst(5) mRNA expression. In one culture completely resistant to OCT, SOM230 and SRIF-14 significantly inhibited GH release in a dose-dependent manner with an IC(50) value in the low nanomolar range. In the other cultures, SOM230 showed a lower potency of GH release inhibition (IC(50), 0.5 nM), compared with OCT (IC(50), 0.02 nM) and SRIF-14 (IC(50), 0.02 nM). A positive correlation was found between sst(2) but not sst(5) mRNA levels in the adenoma cells and the inhibitory potency of OCT on GH release in vivo and in vitro, and the effects of SOM230 and SRIF-14 in vitro. In three prolactinoma cultures, 10 nM OCT weakly inhibited prolactin (PRL) release in only one (-28%), whereas 10 nM SOM230 significantly inhibited PRL release in three of three cultures (-23, -51, and -64.0%). The inhibition of PRL release by SOM230 was related to the expression level of sst(5) but not sst(2) mRNA. Several conclusions were reached. First, SOM230 has a broad profile of inhibition of tumoral pituitary hormone release in the low nanomolar range, probably mediated via both sst(2) and sst(5) receptors. The higher number of responders of GH-secreting pituitary adenoma cultures to SOM230, compared with OCT, suggest that SOM230 has the potency to increase the number of acromegalic patients which can be biochemically controlled. Second, compared with OCT, SOM230 is more potent in inhibiting PRL release by mixed GH/PRL-secreting adenoma and prolactinoma cells.     

Sequence and expression of hamster prolactin and growth hormone messenger RNAs

Complementary DNAs encompassing the complete protein-encoding regions for PRL and GH of the Syrian Golden hamster were sequenced and used as probes to examine the expression of hamster PRL and GH messenger RNA (mRNA)s. The complementary DNA (cDNA) for hamster PRL encodes a 226 amino acid preprotein which, by analogy to rat and mouse PRLs, is predicted to be processed to yield a 197 amino acid secreted protein. The hamster GH cDNA codes for a 216 amino acid preprotein predicted to yield a 190 amino acid secreted protein. Both hamster proteins are highly homologous to the corresponding rat and mouse hormones. For the secreted proteins, hamster PRL has 82% amino acid identity with rat PRL and 72% identity with mouse PRL. The rodent GH sequences are more strongly conserved, with 97-98% sequence identity between hamster, rat, and mouse GHs. The hamster hormones contain the highly conserved cysteine residues (six in hamster PRL and four in hamster GH) present in other mammalian PRLs and GHs. Neither hamster PRL nor hamster GH contains cysteine residues corresponding to the unique pair of cysteines present in hamster placental lactogen-II. The hamster PRL and GH cDNAs each hybridized to pituitary mRNAs of approximately 1 kilobase. Expression of hamster PRL and GH mRNAs was compared between 2 days of the estrous cycle (proestrus and estrus) and early, mid, and late pregnancy (days 5, 10, and 15). PRL mRNA levels in cycling hamsters were approximately 50% of those in pregnant hamsters. No other significant differences in PRL or GH mRNA levels were observed, suggesting that differences in circulating PRL and GH protein levels during the estrous cycle and pregnancy in the hamster are the result largely of factors other than changes in mRNA levels.