J Neuroendocrinol. 2015 Oct;27(10):787-801.

Biphasic Effect of Basic Fibroblast Growth Factor on Anterior Pituitary Folliculostellate TtT/GF Cell Coupling, and Connexin 43 Expression and Phosphorylation.

María Leiza Vitale and Ahmed Barry

Département de pathologie et biologie cellulaire, Faculté de Médecine, Université de Montréal, Montreal, Québec, H3T 1J4, Canada


Corresponding author:

María L. Vitale

Department of Pathology and Cell Biology

Faculty of Medicine

Université de Montréal

Montréal, Québec, Canada

Tel: 1 (514) 343-7543

Fax: 1 (514) 343-5755

Email: maria.leiza.vitale@umontreal.ca



Basic fibroblast growth factor (bFGF) is a mitogenic and differentiating cytokine. In the anterior pituitary, folliculostellate (FS) cells constitute the major source of bFGF. bFGF affects endocrine cell proliferation and secretion in the anterior pituitary. In addition, bFGF increases its own expression by acting directly on FS cells. FS cell Cx43-mediated gap junction intercellular communication allows the establishment of an intrapituitary network for the transmission of information. In the present study, we assessed how bFGF regulates FS cell coupling. Time course studies were carried out on the FS cell line TtT/GF. Short-term bFGF treatment induced a transient cell uncoupling and the phosphorylation in Ser368 of membrane-bound Cx43 without modifying Cx43 levels. We demonstrated the involvement of the protein kinase C (PKC) isoform α in the phosphorylation of Cx43 in S368. Moreover, we showed that bFGF induced PKCα activation by stimulating its expression, phosphorylation and association with the plasma membrane. The long-term incubation with bFGF increased TtT/GF cell coupling, total Cx43 levels and Cx43 accumulation at the cell membrane of cytoplasmic projections. The Cx43 level increase was a result of the stimulation of Cx43 gene transcription as mediated by the extracellular-regulated kinase 1/2 signalling pathway. Taken together, the data show that bFGF modulates TtT/GF cell coupling by activating different pathways that lead to opposite effects on Cx43 phosphorylation and expression depending on the duration of the exposure of the cells to bFGF. A short-term bFGF exposure reduces cell-to-cell communication as a mean of desynchronising FS cells. By contrast, long-term exposure to bFGF enhances cell-to-cell communication and facilitates coordination among FS cells.

KEYWORDS: bFGF; connexion 43; folliculostellate cells; gap junction; hypophysis

PMID: 26265106



The endocrine cells of the anterior pituitary gland secrete hormones that control and coordinate growth, reproduction and basal metabolism.  The deregulation of anterior pituitary hormone secretion is associated with several diseases and metabolic disorders.  Typically, the anterior pituitary endocrine cells contain secretory granules where the hormones are stored.  The endocrine cells are organized into follicles surrounded by the non-endocrine so-called folliculo-stellate cells first identified by Farquhar in 1957 and described as chromophobe, agranular cells (1).  Aside from being Anon-endocrine and devoid of secretory granules, the folliculo-stellate cells secrete cytokines and growth factors (2;3).  Our aim is to uncover the contribution of the folliculo-stellate cells in the control of hormone secretion in the pituitary.

Each folliculo-stellate cell is characterized by cytoplasmic projections that give it a stellate shape.  By favouring folliculo-stellate cell-folliculo-stellate cell and folliculo-stellate cell-endocrine cell contacts the cytoplasmic projections provide the anatomical basis for the establishment of a intercellular network within the anterior pituitary parenchyma.  Our research has shown that cytoplasmic projection development responds to cytokines and growth factors (4-6).  In addition to the projections, the folliculo-stellate cells are connected among themselves and to endocrine cells by gap junctions (7-12).  Gap junctions are intercellular channels that permit exchanges of small molecules between communicating cells.  Thus, gap junctions help coordinate metabolic and electrical activities of the cells involved in the junction.  The connexins are structural proteins of gap junctions.  Six connexin molecules assemble to form a hemichannel or connexon (13).  The docking of two connexons from neighboring cells results in the formation of a gap junction channel (13).  To date, 20 connexin isoforms have been identified in human and mouse tissues (14).  The folliculo-stellate cells and the cells of the folliculo-stellate cell line, TtT/GF (15), express the gap junction protein connexin 43 (Cx43) (11;16).  We reported that Cx43-positive gap junctions are associated with the cytoplasmic projections of the folliculo-stellate cells in vivo and in vitro {Vitale, Cardin, et al. 2001 833 /id}.



Figure 1Figure 1. Distribution of folliculostellate cells and Cx43 in the anterior pituitary. Left panel: Folliculostellate cells in the anterior pituitary are characterized by the expression of the protein S-100.  S-100 labelling of mink anterior pituitary depicts the stellate-shape of the cells (arrows) whose nuclei are situated near the centre of the follicles. The cells send thin cytoplasmic projections surrounding adjacent cells of the follicles (open arrowheads).  Right panel: Cx43 labelling in mink anterior pituitary.  A wide distribution of a spotty Cx43 labelling can be observed throughout the tissue (arrowheads).


The permeability and gating activity of gap junctions are modulated not only by the type of connexins making the junctions but by turnover of the connexin molecules as well.  Acute effects on the function of gap junctions are usually caused by post-translational modifications of the connexin proteins.  One of the most common of these modifications is the alteration of the phosphorylation status of specific amino-acid residues catalyzed by various kinases and phosphatases (17;18).  The long-lasting effects on gap junction-mediated intercellular communication implicate changes in the expression, subcellular distribution and degradation of connexins (19;20).

Cytokines, hormones and growth factors modulate intercellular communication within tissues by acting on the connexins that make the junctions.  We showed that this is also the case in the anterior pituitary folliculo-stellate cells. We demonstrated that intercellular communication and the Cx43 protein are targets of cytokines and growth factors in TtT/GF cells.  We showed that treating the cells with either pro-inflammatory cytokines such as tumor necrosis α (TNF-α) and interleukin 1β (IL-1β) (16;21) or with growth factors such as the basic fibroblast growth factor (bFGF) (6) alters the expression, localization and phosphorylation status of Cx43, and TtT/GF cell coupling.  Of particularly significant is the impact of bFGF on gap junctions.  In the pituitary gland, the folliculo-stellate cells are the principal source of this mitogenic and differentiating cytokine (22).  The bFGF stimulates both lactotrope cell proliferation and prolactin secretion (23-26).  In addition to these paracrine effects, bFGF acts in an autocrine fashion on folliculo-stellate cells by stimulating its own synthesis and the proliferation of the cells (27).  We showed that bFGF also regulates folliculo-stellate-folliculo-stellate cell coupling.  A short-term incubation of TtT/GF cells with bFGF transiently diminishes cell coupling.  As stated above, acute effects on gap junction function are generally the consequence of changes in the phosphorylation status of specific amino acids in connexin proteins.  For instance, TNFα rapidly decreases TtT/GF cell coupling by stimulating the phosphatase PP2A-dependent dephosphorylation of the serine 368 residue (S368) of Cx43 and the concomitant the tyrosine phosphorylation of the protein (21).  The bFGF-induced cell uncoupling is due to the protein kinase C-isoform α (PKCα)-mediated phosphorylation of S368 in Cx43 molecules assembled in connexons at the cell membrane (6).  By contrast to the acute effect of bFGF, the sustained treatment of TtT/GF cells with the growth factor enhances cell coupling.  This effect is accompanied by the up-regulation of Cx43 gene transcription, Cx43 mRNA translation, and connexon assembly and localization to the cell membrane.  The bFGF-evoked Cx43 level increase is mediated by the extracellular regulated kinases 1/2 (Erk1/2).  Therefore, the early and late effects of bFGF on Cx43 and TtT/GF cells coupling are not only opposite but most importantly they are depend on different signalling pathways.



Figure 2Figure 2. Expression of non-phosphorylated Cx43 in control and cytokine bFGF -stimulated TtT/GF cells. Control TtT/GF cell cultures were incubated in serum containing media (A) or serum starved for 24h (B).  Serum starved cells were incubated for 2 h with 10ng/ml bFGF (C).  Cell cultures were next processed for immunofluorescence microscopy with antibodies against Cx43 non-phosphorylated in S368 (NP(S368)-Cx43).  In serum culture cells, NP(S369)-Cx43labelling occurs at the perinuclear region and cytoplasmic projections.  Serum starved cells exhibit a polygonal shape. NP(S368)-Cx43 labelling is mainly associated with the cell membrane.  Incubation of serum-starved cells with bFGF alters the cell=s shape and increases NP(S368)-Cx43 labelling in the perinuclear area and cell membrane projections.


Modulation of gap junction-mediated communication may be a key factor in the regulation of anterior pituitary hormone secretion.  We have reported that Cx43 expression and the number of Cx43-bearing gap junctions increase in the anterior pituitary during periods of enhanced prolactin secretion (8).  bFGF enhances prolactin synthesis (24;26), the basal and TRH-stimulated prolactin release (23) and the proliferation of lactotroph (23;25).  Co-culturing TtT/GF cells and the prolactin secreting GH3 cells increases both basal and TRH-induced prolactin secretion (28).  Co-culturing TtT/GF cells with the prolactin secreting MMQ cells results in increased Cx43 expression in TtT/GF cells (29).  Collectively these experimental data highlights a functional relation linking bFGF, Cx43 mediated-folliculo-stellate cell coupling and prolactin secretion.

The deregulation of gap junction proteins has been associated with cell/tissue dysfunction (30-32).  Coordination and synchronization among folliculo-stellate cell is necessary for satisfactory and timely hormone secretion (2;3).  The biphasic effect of cytokines and growth factors on the function of gap junctions in folliculo-stellate cells illustrates a fine control in communication among folliculo-stellate cells. The correct adjustment of cell communication to various durations of cytokine or growth factor challenge may restrict or improve the transfer of information within the cellular network as a mechanism to regulate the coordination among participating cells in the anterior pituitary.



Figure 3

Figure 3. Diagram showing cytokine/growth factor effects on Cx43 in FS cells. A short-term challenge of TtT/GF-FS cells with cytokines or growth factors induces uncoupling of cells by modifying the phosphorylation state of Cx43.  By contrast, a long-term exposition of the cells to the same factors increases Cx43 levels and cell coupling.



This work was funded by the Natural Sciences and Engineering Research Council of Canada (RGPIN 194652).



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