The Endogenous Fibroblast Growth Factor-2 Antisense Gene Product Regulates Pituitary Cell Growth and Hormone Production

Sylvia L. Asa, Lily Ramyar, Paul R. Murphy, Audrey W. Li and Shereen Ezzat

Department of Pathology and Laboratory Medicine (S.L.A., L.R.) Mount Sinai Hospital and Department of Laboratory Medicine and Pathobiology University of Toronto Toronto, Ontario, Canada M5G 2M9
Department of Physiology and Biophysics (P.R.M., A.W.L.) Dalhousie University Halifax, Nova Scotia, Canada B3H 4H7
Department of Medicine (S.E.) Mount Sinai Hospital and University of Toronto Toronto, Ontario, Canada M5G 2M9


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Basic fibroblast growth factor (bFGF; FGF-2) is one of 19 related members of a growth factor family with mitogenic and hormone-regulatory functions. In Xenopus laevis oocytes, a 1.5-kb FGF-2 antisense (GFG) RNA complementary to the third exon and 3'-untranslated region (UTR) of FGF-2 mRNA has been implicated in FGF-2 mRNA editing and stability. The human homolog has been cloned, and we localized this gene by yeast artificial chromosome (YAC), somatic cell, and radiation hybrid panels to the same chromosomal site as FGF-2 (chromosome 4, JO4513 adjacent to D4S430), confirming this as a human endogenous antisense gene. The full-length GFG antisense RNA encodes a 35-kDa protein, which is highly homologous with the MutT family of antimutator nucleosidetriphosphatases (NTPases). We show that human pituitary tumors express FGF-2 and its endogenous antisense partner GFG. While normal pituitary expresses GFG but not FGF-2, pituitary adenomas express FGF-2 and have reduced levels of GFG; aggressive and recurrent adenomas expressed more FGF than GFG mRNA. To examine the effects of this antisense gene in the pituitary, we transfected the pituitary-derived GH4 mammosomatotroph cell line with constructs encoding the full-length human GFG cDNA. Transiently and stably transfected cells expressed the 35-kDa GFG protein that was localized to the cytoplasm. These cells exhibited enhanced PRL expression as documented by transiently transfected PRL-luciferase reporter assay and by endogenous PRL protein. GFG expression in these cells did not alter endogenous FGF-2 expression but increased the proportion of the higher molecular mass 22-kDa form of GH. Moreover, GFG expression inhibited cell proliferation as shown by [3H]thymidine incorporation, proliferating cell nuclear antigen (PCNA) nuclear staining, and cell cycle analysis. We conclude that the GFG-encoded protein has divergent hormone-regulatory and antiproliferative actions in the pituitary that are independent of FGF-2 expression. GFG represents a novel mechanism involved in restraining pituitary tumor cell growth while promoting hormonal activity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Pituitary tumors are common neoplasms that occur in approximately 20% of the population on autopsy examination and imaging (1). They exhibit a wide range of biological behavior: some remain small and clinically insignificant, some are hormonally active, and a subpopulation grow and invade into the brain. Both invasiveness and hormone excess result in significant morbidity and mortality; however, these lesions almost never attain malignant potential as defined by the ability to metastasize.

The biology of these tumors provides an opportunity to identify novel mechanisms of dysregulated cell proliferation. Although they are monoclonal in origin, the molecular events responsible for pituitary cell transformation are not known (2). Their relatively indolent behavior is consistent with lack of somatic mutations that are characteristic of other human malignancies. Because these tumors form an integral part of the multiple endocrine neoplasia type 1 (MEN-1) syndrome, the MEN-1 tumor suppressor gene was a candidate gene responsible for these tumors. However, loss of heterozygosity with mutations and down- regulation of the menin gene are rare in the more common sporadic tumors. Activating mutations of the Gs{alpha} protein have been implicated in a minority of GH-producing adenomas. Hypothalamic stimulation has been implicated and appears to be important in promotion of tumor growth but not cell transformation. Mice that overexpress GH-releasing hormone (GHRH) and mice that lack dopaminergic D2 receptors that mediate inhibition of PRL secretion develop target adenohypophysial cell hyperplasia that then predisposes to subsequent neoplastic transformation of pituitary tumors. However, these models differ from the human disease, since patients with pituitary tumors do not exhibit underlying hyperplasia (2).

Growth factors have been implicated in pituitary tumorigenesis. The pituitary is the site of synthesis and the target of several growth factors that modulate hormone production and are believed to regulate, in part, pituitary cell growth (3, 4). At least 19 members of the fibroblast growth factor (FGF) family have been described with variable mitogenic, angiogenic, and hormone regulatory functions (5). Fibroblast growth factor-2 [FGF-2 or basic FGF (bFGF)] was, in fact, originally isolated from bovine pituitary (6) and is differentially expressed by pituitary adenoma cells with higher levels noted in the more aggressive tumors (7). FGF-2 is known to stimulate vascular endothelial growth factor (VEGF) expression as a mechanism of inducing angiogenesis, suggesting a possible indirect mechanism of tumorigenesis (8).

The regulation of FGF-2 gene expression is poorly understood. However, one possible mechanism involves posttranscriptional regulation of the FGF-2 mRNA by interaction with an FGF antisense RNA. A 1.5-kb FGF-2 antisense (GFG) RNA complementary to the third exon and 3'-untranslated region (UTR) of FGF-2 mRNA has been indirectly implicated in FGF-2 mRNA editing and stability (reviewed in Ref. 9). FGF antisense gene expression has been identified in a number of species including avian, rodent, and human (10, 11, 12). Indirect evidence supports the hypothesis that the antisense RNA may regulate FGF-2 in these species. Steady-state levels of the antisense RNA are inversely related to the level of FGF-2 mRNA during embryonic development and, in a variety of tumor cell lines (11, 13, 14, 15, 16). We recently demonstrated that expression of the FGF antisense RNA in rat C6 glioma cells suppressed FGF-2 expression and inhibited cell proliferation (17).

In addition to its putative role as a regulatory RNA, the antisense transcript also encodes a translated 35 kDa protein (GFG) with homology to the MutT family of antimutator nucleotide hydrolases (NTPases) (14). Although GFG can partially complement function in MutT-deficient Escherichia coli (18), the physiological function of GFG in mammalian cells is unknown.

We hypothesized that human GFG may play a role in modulating the proliferative and hormone-regulatory actions of FGF in the pituitary and may be implicated in the unique biological behavior of pituitary adenomas.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GFG Is Localized to Chromosome 4.60 FGF as an FGF Antisense Gene
The complementary nature of the FGF-2 and GFG mRNAs (Fig. 1AGo) has led to the assumption that they are convergently transcribed from the same gene locus. However, chromosomal localization of the GFG gene has not previously been reported. To determine the chromosomal localization of the human GFG gene, we used a PCR-based approach with gene-specific primers. A screen of a somatic cell hybrid panel identified the predicted product on chromosome 4, and a radiation hybrid panel confirmed this localization with product located on chromosome 4 at 4.60 cR from WI-6416. Positive yeast artificial chromosomes (YACs) included 846e11, 945b5, 940b4, 820g2, and 802g6; the common regions indicate localization between WI-7035 and WI-4402. These sites encompass D4S430 (Fig. 1BGo), the site of the FGF-2 gene (19).



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Figure 1. GFG Gene Structure and Chromosomal Localization

A, GFG gene structure, protein structure, and approach to RT-PCR analysis. Shown is the relationship between FGF-2 and GFG sequences. The FGF-2 gene cartoon indicates the open reading frame (ORF) and 3'-UTRs. The shaded boxes represent the GFG exons that are 100% complementary to the hatched areas of the FGF-2 3'UTR. The primers (P) used for RT-PCR are indicated by the arrows. A splice variant that lacks exons 2 and 3 (spl 2/3) retains the regions of mRNA homology with FGF-2 but yields a truncated protein due to frame shift and in the ORF that results in a premature stop codon; this variant was used as a negative control in all experiments. B, GFG chromosomal localization. Using a PCR-based approach to screen somatic cell hybrid and radiation hybrid panels, human GFG was localized to chromosome 4. Further detailed localization utilizing a panel of YAC clones restricted this gene between WI-7035 and WI-4402. These sites encompass D4S430, the site of the FGF-2 gene (Ref. 19; arrow).

 
Expression of FGF and GFG in the Pituitary and Pituitary Adenomas
Using RT-PCR, GFG mRNA was examined in 10 normal pituitaries and in 24 primary human pituitary tumors. These included seven somatotroph, seven lactotroph, one mammosomatotroph, three corticotroph, and six gonadotroph adenomas. Whereas normal pituitary expressed GFG but did not express FGF-2, most of the adenomas expressed both FGF-2 and GFG (Fig. 2AGo). GFG transcripts of the predicted size were identified in all but one adenoma. In comparing the expression of GFG to FGF-2, GFG mRNA levels were less than those of FGF-2 in 15 of the 24 tumors, all of which were macroadenomas with suprasellar extension. GFG and FGF-2 mRNA levels were nearly the same in four tumors. GFG mRNA levels were greater than those of FGF-2 in five tumors that included four microadenomas (three PRL and one ACTH-producing) and only one macroadenoma (gonadotroph) with suprasellar extension; none of these have recurred during 7 yr of follow-up. In contrast, 10 of the 15 tumors with higher FGF-2 than GFG mRNA levels have exhibited recurrence. The expression pattern of GFG was not related to specific cell types of the pituitary tumors but did correlate with less aggressive behavior.



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Figure 2. GFG Expression in the Pituitary

A, GFG mRNA. RNA extracted from human pituitary tumors (T) and normal pituitary (N) was reverse transcribed (RT). The integrity of RNA and the efficiency of the RT reaction were first confirmed by PCR for the housekeeping gene PGK-1 (lower panel). Negative controls omitted reverse transcriptase (-RT) or replaced template with water. The size of PCR products was determined by comparison with concurrently electrophoresed standards (Kb), and the identity of all PCR products was confirmed by sequencing. GFG mRNA was identified in normal pituitary and in pituitary tumors. Normal pituitary (N) did not express FGF-2; some tumors (T) expressed FGF-2 as well as GFG. High levels of GFG are seen in some adenomas that have lower levels of FGF-2 (e.g. T3; Ref. 7) and vice versa (T1; Refs. 2, 5, 9, 13–15). B, GFG protein examined by Western blotting analysis. Normal pituitaries (N) and one of two pituitary tumors (T7) contain a strong band of immunoreactive protein at 35 kDa. A pituitary tumor that did not express GFG mRNA (T20) contains no GFG protein, nor does the rat-derived GH4 cell line.

 
Translation and Cytoplasmic Localization of Pituitary GFG
We have previously reported that the FGF antisense RNA is translated into a 35-kDa nuclear protein in rat tissues (11). To investigate the translation of GFG and to identify its cellular source in the pituitary, we examined multiple adenomas and nontumorous pituitary by Western blotting and immunohistochemistry. Nontumorous pituitary and pituitary tumors that expressed GFG mRNA contained a protein of the predicted size detected with anti-GFG on Western blotting (Fig. 2BGo). The one tumor that did not express GFG mRNA was devoid of GFG immunoreactive protein, and the rat GH4C1 pituitary tumor cell line did not express endogenous GFG protein (Fig. 2BGo).

Using immunohistochemistry, nontumorous pituitary exhibited cytoplasmic GFG protein throughout the gland with variable levels detectable in individual cells (Fig. 3aGo). Localization did not correlate with hormone content; double staining for pituitary hormones showed colocalization of GFG with each of the pituitary hormones (data not shown). Pituitary tumors exhibited variable intensity of diffuse cytoplasmic staining for GFG (Fig. 3aGo) that correlated with mRNA expression but not with tumor type. More aggressive and recurrent adenomas lacked detectable GFG immunoreactivity.



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Figure 3. Immunolocalization of GFG

A, By immunohistochemistry, nontumorous pituitary (N) exhibits cytoplasmic GFG protein (brown); a pituitary tumor (T, lower right) exhibits reduced cytoplasmic staining for GFG (immunohistochemistry with fast green nuclear counterstain). B, Using confocal microscopy, GH4 cells transfected with full-length GFG cDNA have diffuse cytoplasmic distribution of this protein; cells in which nuclei are visualized show a negative nuclear image, confirming predominant cytoplasmic localization. C, Control cells transfected with empty vector are negative. D, Since nuclear staining was not identified using either method of localization, Western blots were performed on subcellular fractions of pituitary cells. This confirms predominant reactivity in cytoplasmic fractions and little or none in nuclear fractions.

 
The subcellular localization of GFG was determined by confocal microscopy. GH4 cells stably transfected with full-length GFG cDNA exhibited a pattern of positive staining that is characteristic of a diffuse cytoplasmic distribution of this protein (Fig. 3bGo). No staining was identified in untransfected cells (Fig. 3cGo) or in cells transfected with an untranslated splice variant that lacks exons 2 and 3 (see Fig. 1aGo), resulting in a truncated protein due to a frame shift with an in-frame premature stop codon (17). Nuclear staining was not identified in any of these cells using either method of localization. To corroborate these findings, we examined Western blots of subcellular fractionated protein and confirmed cytoplasmic residence of GFG in both primary pituitary tumors and in transfected GH4 cells (Fig. 3dGo).

GFG Inhibits Cell Proliferation in Vitro
To determine whether GFG is of oncogenic significance, we produced stably transfected lines of GH4 cells expressing GFG and measured cellular proliferation by [3H]thymidine incorporation. Compared with control cells transfected with empty vector or the untranslated splice variant, cells expressing GFG exhibited reduced proliferative activity, ranging from 65 to 80% of control. Among 12 clones that expressed GFG, the degree of inhibition of thymidine incorporation was proportional to levels of GFG protein expression. The highest GFG stably expressing clone demonstrated the greatest reduction of proliferative activity (Fig. 4AGo). Inhibition of proliferation was further confirmed by proliferating cell nuclear antigen (PCNA) labeling indices that were 30% in control cells and were reduced to 8–12% in 12 stably transfected cell lines (Fig. 4AGo).



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Figure 4. Effect of GFG on Pituitary Cell Proliferation

A, [3H]Thymidine incorporation by GH4C1 cells and PCNA labeling indices reflect cell proliferation. The results illustrated represent the mean of triplicate wells for each treatment group from three independent experiments. The rate of thymidine incorporation and of PCNA labeling by cells transfected with full-length GFG is significantly (P < 0.01) diminished from those transfected with empty vector or the splice variant spl 2/3, combined as control. Cell proliferation in the various clones (GFG-1, GFG-7, and GFG-12) was reduced in proportion to levels of GFG expression as shown on Western blot. B, Cell cycle distribution using FACS analysis in cells stably transfected with GFG is compared with that of cells transfected with empty vector. In this representative experiment, S-phase fractions were 5.1% in control cells and reduced to 1.1%, 1.4%, and 2.0% in proportion to GFG expression of the clones as shown in panel a. Similar results were obtained in transient transfections and cells transfected with the splice variant spl 2/3 did not differ from control cells transfected with empty vector.

 
To determine whether the reduction in cell proliferation was attributed to or associated with enhanced apoptosis, we analyzed transfected and control cells using the Apoptag nick end labeling method. Apoptosis was identified in less than 1% of cells in both groups (not shown).

Flow cytometric evaluation of cell cycle in GH4C1 cells showed at least a 50% reduction in S phase fractions in the cell lines stably transfected with GFG (Fig. 4BGo). Again, the degree of reduction of the proportion of cells in S phase was proportional to levels of GFG protein expression in the various clones.

To exclude the possibility of clonal selection as an independent cause of reduced cell proliferation, we performed the experiments on cells transiently transfected with the GFG expression vector, the empty vector, or the untranslated splice variant. Again there was persistent reduction of thymidine incorporation (<75% of control) as well as a marked reduction (>50%) in S phase fraction in cells transiently transfected with GFG compared with control cells expressing empty vector or the untranslated splice variant. ß-Galactosidase staining revealed a transfection efficiency of more than 50%, and trypan blue exclusion by more than 90% of cells exclude the possibility that the reduction in cell proliferation was attributed to or associated with nonspecific toxicity. Western blotting confirmed expression of the transfected protein (Fig. 5AGo).



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Figure 5. GFG Expression Does Not Inhibit FGF-2 Expression in Pituitary Cells

Western blot analysis of GFG and FGF-2 in cells transiently transfected (panel A) or stably transfected (panel B) with GFG (clones 1, 2, 7, and 12), the splice variant (spl 2/3), or empty vector (pcDNA) show no difference in levels of FGF-2 protein in cell lysates. There was also no change in FGF-2 levels in culture media (not shown).

 
GFG Does Not Inhibit FGF-2 Expression in the Pituitary
To determine whether the mechanism of inhibition of cell proliferation is by inhibition of FGF-2 expression, we examined FGF-2 protein and mRNA levels in control and transfected cells. There was no difference in the levels of FGF-2 mRNA or of FGF-2 protein in cell lysates (Fig. 5Go) and culture media (not shown). Moreover, there was no change in FGF-2 mRNA or of FGF-2 protein in the cells transfected with the splice variant that retains mRNA homology with FGF-2 mRNA, excluding RNA interactions as the mechanism of inhibition of cell proliferation.

Conversely, to determine whether FGF regulates GFG translation, we exposed transfected cells expressing GFG to FGF-2. There was no change in GFG protein in the cells exposed to FGF stimulation, excluding FGF modulation of GFG directly.

GFG Regulates Pituitary Hormones
Since FGF is known to stimulate expression of the PRL gene in GH4C1 cells, we used this model to determine whether GFG plays a role in hormone production. Compared with cells stably or transiently transfected with empty vector or the untranslated splice variant, those expressing GFG showed significantly enhanced PRL-luciferase activity (Fig. 6Go, A and C) (P < 0.05) and had markedly increased PRL immunoreactivity in cell lysates and media as determined by Western blotting (Fig. 6BGo). GFG transfection resulted in no increase in GH-luciferase activity, which was even slightly reduced (Fig. 6Go, A and C). Total GH immunoreactivity as determined by Western blotting and densitometry was unchanged; however, GFG expression was associated with an increase of the 22-kDa form over the 20 kDa form of GH (Fig. 6BGo).



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Figure 6. Effect of GFG on Pituitary Hormone Genes

A, Cells were transiently cotransfected with GFG cDNA, the GFG splice variant spl 2/3, or empty vector and with a -422 PRL-luciferase or a -320 GH-Luciferase reporter plasmid. The effect of GFG expression is expressed as fold induction of light emission directed by the promoter activity integrated over 15 sec compared with empty vector transfected cells. Each value represents the mean fold change in three wells compared with an equal number of wells of cells transfected with empty vector, and all experiments were performed in triplicate. PRL-luciferase activity shows significant increases (P < 0.05) whereas GH-luciferase activity is slightly reduced. B, Cell lysates from clones stably expressing GFG were compared with those transfected with empty vector. Western blotting shows increases in endogenous PRL protein expression that parallel levels of GFG expression in three separate clones. Total GH immunoreactivity is not altered; however, GFG expression was associated with an increase of the 22 kDa form of GH relative to the 20 kDa form. C, Cells stably transfected with GFG cDNA or empty vector were transiently transfected with a -422 PRL-luciferase or a -320 GH-Luciferase reporter plasmid. The effect of GFG expression is expressed as fold induction of light emission directed by the promoter activity integrated over 15 sec compared with empty vector transfected cells. Changes in endogenous protein were determined by densitometric evaluation of Western blots as shown in panel B. Each value represents the mean fold change in three wells compared with an equal number of wells of cells transfected with empty vector, and all experiments were performed in triplicate. PRL-luciferase activity and protein show significant increases (P < 0.05) whereas GH-luciferase activity is slightly reduced and total GH protein is not significantly altered.

 
After stimulation with FGF, PRL-luciferase activity and PRL content of cells transfected with empty- vector increased on average 4-fold. Cells transfected with GFG had a similar response to FGF treatment (data not shown). Addition of FGF did not alter GH-luciferase or GH protein.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Basic fibroblast growth factor (bFGF, also known as FGF-2) is 1 of 19 members of the FGF family that has potent mitogenic, angiogenic, and hormone-regulatory functions. Expression of FGF-2 is considered to be tightly regulated, with low levels of mRNA detected in most normal tissues outside the central nervous system (CNS) (20). FGF-2 immunoreactivity was described originally in the non-hormone-producing bovine pituitary folliculo-stellate cells (6); since FGF-2 has been shown to regulate PRL secretion (21, 22), it was implicated in paracrine regulation within the pituitary. Inappropriate expression of FGF-2 is noted in many types of human solid tumors (23). In the human pituitary, FGF-2 is produced by adenohypophysial cells that comprise pituitary adenomas (7, 24, 25). The transcriptional and posttranscriptional control mechanisms governing FGF-2 expression, however, remain to be determined.

In Xenopus laevis oocytes, a 1.5-kb FGF-2 antisense RNA complementary to the third exon and 3'-UTR of the FGF-2 mRNA has been suggested to play a role in regulating FGF-2 mRNA editing and stability (26). In addition, however, the antisense RNA encodes a distinct protein of poorly defined function. We have previously shown that this predicted protein, with homology to the MutT family of nucleotide hydrolases, is expressed in non-CNS tissue in the postnatal period (11). Here we show, for the first time, that the human pituitary expresses the FGF-AS mRNA and that the predicted protein GFG product is efficiently translated and can be detected by Western blotting and immunohistochemistry. Moreover, we demonstrate that this protein is localized in the cytoplasm in pituitary cells. This is in contrast to the predominantly nuclear localization of GFG detected in rat C6 glioma cells. Subcellular distribution of GFG may be determined by alternative splicing, as has been reported for BRCA1 (27) and the human MutY homolog (28). C6 cells predominantly express a 28-kDa isoform of GFG that is derived by alternative splicing to remove exon 2 of the FGF antisense pre-mRNA (17). Alternatively, nuclear vs. cytoplasmic distribution may be a result of lineage-dependent cell-specific factors, as has been reported for p53 (29) and for the homeodomain protein OTX2 (30).

Pituitary-derived FGF-2 has been shown to stimulate replication of PRL-secreting cells but also may inhibit DNA synthesis in pituitary adenoma cells (31), suggesting that some forms of the growth factor or its receptor may act as growth inhibitors. Elevated blood concentrations of bFGF-like immunoreactivity have been documented in patients with MEN-1 (32) and in patients with sporadic pituitary adenomas (7). The FGF-related hst has been found in transforming DNA of human PRL-secreting tumors (33), which also facilitates lactotroph proliferation (34). Transgenic mice expressing bFGF under the control of the GH and the glycoprotein {alpha}-subunit promoters developed hyperplasia of several adenohypophysial cell types but not frank adenomatous changes (35). FGF-2 or homolog family members have therefore been suggested to play an important role in pituitary tumor cell replication.

In this study we have found that the normal pituitary and the less aggressive pituitary adenomas expressed relatively more GFG than FGF-2. This may be of functional significance inasmuch as we have demonstrated that GFG expression is associated with diminished cell replication. We also show here that expression of GFG results in restrained pituitary cell growth, an effect supported by diminished thymidine incorporation, PCNA cell labeling, as well as reduced entry into the S-phase of the cell cycle. While we noted that the more aggressive and recurrent adenomas had reduced GFG expression, we did not observe a strict discordant pattern between FGF-2 and GFG expression in primary pituitary adenomas. Moreover, forced expression of GFG in GH4 pituitary cells did not result in parallel reduction in FGF-2 mRNA or protein expression. The inhibition of proliferation was not seen with transfection of a splice variant of GFG that retains the mRNA homology with FGF-2 mRNA but fails to express functional GFG protein. Taken together, these data indicate that GFG plays a direct antiproliferative role in pituitary cell replication that is independent of FGF-2 expression.

FGF-2 and FGF-4 have been shown to induce PRL gene expression (36). In contrast to other systems, however, this FGF effect is independent of Ras or Raf kinase activation. FGF induction of the PRL gene is dependent on mitogen-activated protein (MAP) kinase activity with defined Ets binding sites (36). Antagonism of FGF-2 expression would, therefore, have been expected to result in inhibition of PRL. Instead, we demonstrate that GFG expression results in PRL stimulation, as shown by endogenous PRL levels and transfected PRL-promoter activity. These data further support the notion that GFG expression plays a hormone-regulatory role that is independent of FGF-2 expression. Further studies are underway to determine whether FGF and GFG mediate their effects on the PRL gene through similar signaling cascades.

In contrast to PRL, FGFs have not been shown to regulate the GH gene. In the current study we did not identify stimulation of GH-promoter activity by GFG. Instead, GFG expression was associated with an increase of the 22-kDa isoform relative to the 20-kDa form of GH. Pituitary GH mRNA undergoes alternative splicing into a 20-kDa and 22-kDa isoforms. GFG-induced alteration in the ratio of 22/20 kDa GH isoforms suggests a possible role for GFG in regulating GH mRNA splicing. This finding would suggest that in some systems, GFG may play a role in regulating pituitary gene splicing. It will be particularly interesting to determine whether GFG regulates other non-FGF-related gene splicing. The 20-kDa form of GH has been shown to be a weaker agonist for hPRLR than 22-kDa hGH (37). In the case of GH, the relative increase in the 22-kDa form may serve to enhance responsiveness to PRL action in addition to the direct stimulatory role of GFG on PRL expression.

Our current findings of the expression of the endogenous FGF antisense gene extend our understanding of the role of FGF in modulating the balance of FGF function in the pituitary. The observation that GFG leads to restrained pituitary cell growth coupled with the induction of PRL gene expression are consistent with the common occurrence of small pituitary tumors in patients with hyperprolactinemic disorders.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Isolation of Human Pituitary Tissues and Pituitary Cell Lines
Fresh human pituitary tumors were obtained at the time of surgery, and normal pituitary tissue was obtained at autopsy within 6 h postmortem. Tumors were functionally classified based on circulating hormone levels. Tumor size, extrasellar extension, and postoperative recurrence were determined by magnetic resonance imaging. One piece was fixed in formalin and embedded in paraffin for histological and immunohistochemical analysis and classification using accepted criteria (1); a second piece was snap frozen in liquid nitrogen and stored at -70 C. The rat pituitary tumor-derived GH4C1 mammosomatotroph cell line was obtained from ATCC (Manassas, VA) and cultured in Ham’s F10 medium supplemented with 12.5% horse serum and 2.5% FCS with antibiotics.

mRNA Analysis by RT-PCR
Total RNA was extracted by the guanidinium isothiocyanate method. One microgram of DNase-treated RNA was used for reverse transcription. This was performed using 2.5 U/ml of murine leukemia virus reverse transcriptase, 2.5 mM MgCl2, 1 mM deoxynucleoside triphosphate (dNTP), 2.5 mM random hexamers, and 1 U/ml of RNase inhibitor. The integrity of RNA from each sample was assessed by amplification of the PGK-1 housekeeping gene as previously described (38). PCR analyses were performed with primers within the coding human sequences to amplify a 301-bp fragment corresponding to GFG exons 4 and 5, which is common to all three GFG splice variants (17), and a 375-bp fragment of the coding region of FGF-2 (7). All primers were designed to span at least one intron to permit the exclusion of genomic DNA contamination. PCR conditions were optimized to ensure product linearity. The identity of all PCR products was confirmed by Southern blotting hybridization and by sequencing.

Transfection and Plasmid Constructs
Plasmids containing full-length human GFG cDNA or an untranslated GFG splice variant that lacks exons 2 and 3 but retains the regions of homology to FGF-2 (17) were prepared by subcloning into the pcDNA3.1 (Invitrogen, San Diego, CA) eukaryotic expression vector. The resulting products were subjected to sequencing for confirmation of sequence fidelity before transfection with lipofectamine (Life Technologies, Inc., Gaithersburg, MD) into wild-type GH4C1 cells. Stably transfected cells were isolated by G418 selection. As inhibition of FGF-2 expression might bias the selection of transfectant lines in favor of FGF-2 independence, cells were grown and passaged in the presence and absence of added FGF-2. GFG expression was confirmed by Western blotting and immunohistochemistry. To establish transfection efficiency and to allow comparison within and between experiments, 20 ng/well of pSV-ß-galactosidase control vector (Promega Corp., Madison WI) was included with each transient transfection and measured on cell lysates by colorimetric analysis or in culture by light microscopy.

Chromosomal Localization
The human GFG gene chromosomal localization was determined by PCR of somatic cell hybrid and radiation hybrid panels by PCR and hybridization with a human full-length GFG cDNA. Further detailed localization used a panel of YAC clones for PCR.

Protein Extraction and Cell Fractionation
Total protein was extracted from total cell lysates and media and quantified. Cell fractionation was performed by the hypotonic/NP-40 lysis method. Briefly, cells were washed in Tris-buffered saline (TBS), swollen in homogenization buffer [10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM ethylene glycolbis, 1 mM dithiothreitol (DTT), and 0.5 mM phenylmethylsulfonyl fluoride (PMSF)], and vortexed in homogenization buffer containing 0.6% NP-40. Supernatant containing cytoplasm and plasma membranes were removed, and the pellet containing the nuclear fraction was suspended in a resuspension buffer (250 mM Tris, pH 7.8, 60 mM KCl, 1 mM DTT, and 1 mM PMSF).

Western Blot Analysis
Protein concentrations were determined by the Bio-Rad Laboratories, Inc. (Hercules, CA) protein assay. Equal amounts of protein (50 µg) from cell lysates or media were solubilized in 2.5x SDS-sample buffer and separated on SDS-8% polyacrylamide gel and transferred to nitrocellulose. Apparent molecular weights were determined by comparison with concurrently electrophoresed standards. GFG, FGF-2, PRL, and GH protein levels were determined using the following antibodies: polyclonal antisera raised against synthetic peptides corresponding to the C-terminal epitope of deduced GFG (14) applied at a dilution of 1:1500; a polyclonal antiserum that recognizes FGF-2 (Transduction Laboratories, Inc., Lexington, KY) applied at a dilution of 1:300 (of 100 µg/ml affinity-purified IgG); and polyclonal antisera to rat PRL or GH (donated by the National Hormone and Pituitary Program (NHPP), NIDDK, NICHHD, Bethesda, MD) applied at dilutions of 1:8,000 and 1:50,000, respectively. An actin control was performed using a monoclonal antibody (Sigma, St. Louis, MO) at 1:500.

Immunocytochemical Localization of GFG
For immunolocalization, a polyclonal antiserum that recognizes the MuT domain and another directed against the C-terminal tail of human GFG (14) was applied at a dilution of 1:300. Primary human tissues and tumors were fixed in formalin and embedded in paraffin; the immunolocalization was detected with the streptavidin-biotin-peroxidase complex technique and 3,3'-diaminobenzidine (DAB) and visualized with a light microscope. For colocalization with pituitary hormones, double staining was performed with the following primary antibodies and antisera: ACTH and GH [polyclonal antisera from DAKO Corp. (Carpinteria, CA) prediluted 1:15 and 1:1,500 respectively); PRL [prediluted monoclonal antibody from Biomeda Corp. (Foster City, CA)], {alpha}-subunit of glycoprotein hormones [monoclonal antibodies from Amac Inc. (Westbrook, ME) and Zymed Laboratories, Inc. (South San Francisco, CA) 1:200 and 1:4]; ß-TSH, ß-FSH, and ß-LH (monoclonal antibodies from Amac Inc., diluted 1:500, 1:400, and 1:400, respectively). For the double stain to colocalize GFG and hormone, the streptavidin-biotin-peroxidase method was used to detect one antigen, and a peroxidase-conjugated secondary antibody method was used for the other antigen to avoid cross-reaction; the second chromogen was cobalt DAB. The order and technique of primary antibody detection were reversed to accurately evaluate cross-reaction by the primary antibodies. For subcellular localization, transfected cells were grown on glass coverslips and fixed in 1% formalin in PBS, and the primary GFG antiserum was localized with fluorescein-tagged secondary antibody and visualized with a MRC 600 confocal microscope (Bio-Rad Laboratories, Inc.). The specificity of all reactions was verified by replacing the primary antibody with normal rabbit serum and by examining negative controls.

Mitogenic Assay
GH4C1 cells were transfected with GFG, the splice variant, or empty vector, and cell proliferation was measured by [3H]thymidine incorporation. Cells were grown in six-multiwell microtiter plates (5 x 104 cells per well), labeled with 1 Ci/ml [3H]thymidine for 6 h and collected, and the amount of trichloroacetic acid (TCA)-precipitable radioactivity associated with the cells was measured and normalized for total cell number.

Cell proliferation in transfected cells was also analyzed by PCNA labeling. Cultured cells were collected in pellets, fixed in formalin, and embedded in paraffin. Sections (4 µm-thick) were stained using the PCNA monoclonal antibody (Novocastra, Newcastle-Upon-Tyne, UK) applied at 1:4,000, and detected with the streptavidin-biotin-peroxidase complex technique and DAB. Labeling indices were determined by counting 1,000 cells for each sample, and the labeled cells were expressed as a percentage of total cells. All analyses were performed in triplicate.

Apoptosis and Toxicity Assays
The rate of apoptosis was measured in GH4C1 cells transfected with GFG, the splice variant or empty vector using DNA nick-end labeling. Cultured cells were collected in pellets, fixed in formalin, and embedded in paraffin. Nuclei of tissue sections were stripped of proteins by incubation with 20 µg/ml proteinase K (Sigma) for 15 min at room temperature. Endogenous peroxidase was inactivated with 2% H2O2 for 5 min. The sections were immersed in TdT buffer (Oncor, Intergen, Purchase, NY) (30 mM Trizma base, pH 7.2, 140 mM sodium cacodylate, 1 mM cobalt chloride). Sections were then incubated in TdT (30% solution) and biotinylated dUTP in TdT buffer (Oncor) in a humid atmosphere at 37 C for 60 min. The reaction was terminated by transferring the slides to TB buffer (300 mM sodium chloride, 30 mM sodium citrate) for 15 min. The sections were blocked with a 2% aqueous solution of human serum albumin (HSA) for 10 min, rinsed and detected with streptavidin peroxidase, diluted 1:20 in water, and incubated for 30 min at 37 C and with DAB for another 30 min at 37 C. Labeling indices were determined by counting 1,000 cells for each sample and expressed as the percentage of labeled cells. All analyses were performed in triplicate.

As this assay does not identify nonspecific toxicity, we performed trypan blue exclusion analysis of transfected cells.

Cell Cycle
To identify the cell cycle effects of GFG, cells were analyzed by fluorescence activated cell sorting (FACS). For cell cycle analysis, 1–3 x 106 cells were trypsinized, washed with PBS, and fixed with 80% ethanol for 1 h on ice. The fixed cells were washed with staining buffer (0.2% Triton X-100 and 1 mM EDTA, pH 8.0, in PBS) and resuspended in the staining buffer containing 100 µl (10 mg/ml) RNAse A (Sigma) and 50 µl (1 mg/ml) propidium iodide for 1 h. Cell cycle analysis was done by a FACScan (Becton Dickinson and Co., San Jose, CA) using the Cellquest Analysis program, and specific S-phase was analyzed using Modfit DNA Analysis program (Verity Software, Inc., Topsham, ME).

Hormonal Regulation
Expression of endogenous PRL and GH was determined by Western blotting of cell extracts and media from transiently and stably transfected and control cells as described above and normalized to actin. Relative concentrations were determined by densitometric analysis of autoradiographs.

PRL promoter activity was analyzed with reporter constructs pSV2A-rPRL-luc containing the 422-bp fragment of the rPRL promoter or 320 bp of the rGH promoter (kindly provided by Dr. H. Elsholtz, Toronto). To normalize for transfection efficiency variation within and between experiments, 20 ng/well of pSV-ß-galactoside control vector (Promega Corp.) was included with each transfection. The results were normalized to ß-galactosidase activity.

Stimulation of PRL by FGF was analyzed in GH4C1 cells transfected with GFG, the splice variant of GFG, or empty vector. Transfected cells were grown in six-multiwell microtiter plates (5 x 104 cells per well), preincubated for 48 h in serum-free defined media [insulin (5 g/ml), transferrin (5 g/ml)], and then treated with and without FGF-1 (Sigma, 50 ng/ml) and 10 U/ml of heparin in serum free medium for 24 h at 37 C.

Statistical Analyses
Data are expressed as mean ± SEM. Differences were examined by one-way ANOVA or Student’s t-test both with significance level of <0.05.


    ACKNOWLEDGMENTS
 
The authors gratefully acknowledge Jennifer Skaug and Kelvin So for invaluable technical assistance.


    FOOTNOTES
 
Address requests for reprints to: Sylvia L. Asa, M.D., Ph.D., Department of Pathology, University Health Network, Suite 4–302, 610 University Avenue, Toronto, Ontario, Canada M5G-2M9. E-mail: sylvia.asa{at}uhn.on.ca

This work was supported by the Medical Research Council of Canada Grants MT-14404 (to S.E.) and MT-14464 (to S.L.A.).

Received for publication November 17, 2000. Revision received December 18, 2000. Accepted for publication January 9, 2001.


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