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

 

Abstract

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

 

Supplement

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.

 

ACKNOWLEDGEMENTS

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

 

REFERENCES

  1. Farquhar MG 1957 “Corticotrophs” of the rat adenohypophysis as revealed by electron microscopy. Anat Rec 127:291
  2. Allaerts W, Vankelecom H 2005 History and perspectives of pituitary folliculo-stellate cell research. Eur J Endocrinol 153:1-12
  3. Morris J, Christian H 2011 Folliculo-stellate cells: paracrine communicators in the anterior pituitary. Open Neuroendocrinol J 4:77-89
  4. Cardin J, Carbajal ME, Vitale ML 2000 Biochemical and morphological diversity amongst folliculo-stellate cells of the mink (Mustela vison) anterior pituitary. Gen Comp Endocrinol 120:75-87
  5. Zheng GF, Solinet S, Pelletier R-M, Vitale ML 2005 Actin cytoskeleton remodelling in the anterior pituitary folliculo-stellate cell line TtT/GF: Implication of the actin-binding protein cortactin. J Mol Histol 36:461-474
  6. Vitale ML, Barry A 2015 Biphasic effect of bFGF on anterior pituitary folliculo-stellate TtT/GF cell coupling,and connexin 43 expression and phosphorylation. J Neuroendocrinol 27:787-801
  7. Soji T, Mabuchi Y, Kurono C, Herbert DC 1997 Folliculo-stellate cells and intercellular communication within the rat anterior pituitary gland. Micr Res Tech 39:138-149
  8. Vitale ML, Cardin J, Gilula NB, Carbajal ME, Pelletier R-M 2001 Dynamics of connexin 43 levels and distribution in the mink (Mustela vison) anterior pituitary is associated with seasonal changes in anterior pituitary prolactin content. Biol Reprod 64:625-633
  9. Fletcher WA, Anderson NC, Everett JW 1975 Intercellular communication in the rat anterior pituitary gland. An in vivo and in vitro study. J Cell Biol 67:469-476
  10. Wilfinger WW, Larsen WJ, Downs TR, Wilbur DL 1984 An in vitro model for studies of intercellular communication in cultured rat anterior pituitary cells. Tissue Cell 16:483-497
  11. Yamamoto T, Hossain MZ, Hertzberg EL, Uemura H, Murphy LJ, Nagy JI 1993 Connexin 43 in rat pituitary: localization at pituicyte and stellate cell gap junction and with gonadotrophs. Histochemistry 100:53-64
  12. Morand I, Fonlupt P, Guerrier A, Trouillas J, Calle A, Remy C, Rousset B, Munari-Silem Y 1996 Cell-to-cell communication in the anterior pituitary: evidence for gap junction-mediated exchanges between endocrine cells and folliculostellate cells. Endocrinology 137:3356-3367
  13. Lauf U, Giepmans BNG, López P, Braconnot S, Chen S-C, Falk MM 2002 Dynamic trafficking and delivery of connexons to the plasma membrane and accretion to gap junctions in living cells. Proc Natl Acad Sci (USA) 99:10446-10451
  14. Dbouk HA, Mroue RM, El-Sabban ME, Talhouk RS 2009 Connexins: a myriad of functions extending beyond assembly of gap junctions. Cell Commun Signal 7:1-17
  15. Inoue K, Matsumoto H, Koyama C, Shibata K 1992 Establishment of a folliculo-stellate like cell line from a murine thyrotropic pituitary tumor. Endocrinology 131:3110-3116
  16. Fortin M-E, Pelletier R-M, Meilleur M-A, Vitale ML 2006 Modulation of GJA1 turnover and intercellular communication by pro-inflammatory cytokines in the anterior pituitary folliculo-stellate cell line TtT/GF. Biol Reprod 74:2-12
  17. Lampe PD, Lau AF 2004 The effects of connexin phosphorylation on gap junctional communication. Int J Biochem Cell Biol 36:1171-1186
  18. Hervé J-C, Sarrouilhe D 2002 Modulation of junctional communication by phosphorylation: protein phosphatases, the missing link in the chain. Biol Cell 94:423-432
  19. Hervé J-C, Derangeon M, Bahbouhi B, Mesnil M, Sarrouilhe D 2007 The connexin turnover, an important modulating factor of the level of cell-to-cell junctional communication: comparison with other integral membrane proteins. J Membrane Biol 217:21-33
  20. Klotz L-O 2012 Posttranscriptional regulation of connexin-43 expression. Arch Biochem Biophys 524:23-29
  21. Meilleur M-A, Akpovi CD, Pelletier R-M, Vitale ML 2007 Tumor Necrosis Factor-α-induced anterior pituitary folliculostellate TtT/GF cell uncoupling is mediated by connexin 43 dephosphorylation. Endocrinology 148:5913-5924
  22. Ferrara N, Schweigerer L, Neufeld G, Mitchell R, Gospodarowicz D 1987 Pituitary follicular cells produce basic fibroblast growth factor. Proc Natl Acad Sci (USA) 84:5773-5777
  23. Baird A, Mormède P, Ying S-Y, Wehrenberg WB, Ueno N, Ling N, Guillemin R 1985 A nonmitogenic pituitary function of fibroblast growth factor: regulation of thyrotropin and prolactin secretion. Proc Natl Acad Sci (USA) 82:5545-5549
  24. Schweppe RE, Frazer-Abel AA, Gutierrez-Hartmann A, Bradford AP 1997 Functional components of fibroblast growth factor (FGF) signal transduction in pituitary cells. J Biol Chem 272:30852-30859
  25. Hentges S, Boyadjieva N, Sarkar DK 2000 Transforming growth factor-3β stimulates lactotrope cell growth by increasing basic fibroblast growth factor from folliculo-stellate cells. Endocrinology 141:859-867
  26. Jackson TA, Koterwas DM, Morgan MA, Bradford AP 2003 Fibroblasts growth factors regulate prolactin transcription via an atypical Rac-dependent signaling pathway. Mol Endocrinol 17:1921-1930
  27. Vlotides G, Chen Y-H, Ren S-G, Melmed S 2009 Fibroblast growth factor-2 autofeedback regulation in pituitary folliculostellate TtT/GF cells. Endocrinology 150:3252-3258
  28. Koike K, Zhang ZX, Sakamoto Y, Kand Y, Murakami K, Miyake A, Inoue M 1997 The pituitary folliculo-stellate cell line TtT/GF augments basal and TRH-induced prolactin secretion by GH3 cells. Life Sci 61:2491-2497
  29. Lewis BM, Pexa A, Francis K, Verma V, McNicol AM, Scanlon M, Deussen A, Evans WH, Rees DA, Ham J 2006 Adenosine stimulates connexin 43 expression and gap junctional communication in pituitary folliculostellate cells. FASEB J 20:E2036-E2045
  30. Pelletier R-M, Akpovi CD, Chen L, Day R, Vitale ML 2011 Cx43 expression, phosphorylation and distribution in the normal and autoimmune orchitic testis with a look at gap junctions joining germ cell-to-germ cell. Am J Physiol Regul Integr Comp Physiol 300:R121-R139
  31. Pelletier R-M, Akpovi CD, Chen L, Kumar NM, Vitale ML 2015 Complementary regulation of Cx46 and Cx50 expression and phosphorylation in development and  following gene deletion in mouse and in normal and orchitic mink testes. Am J Physiol Regul Integr Comp Physiol 309 :R255-R276
  32. Mitašiková M, Lin H, Soukup T, Imanaga I, Tribulová N 2009 Diabetes and thyroid hormones affect connexin-43 and PKC-ε expression in rat heart atria. Physiol Res 58:211-217

 

 

Multiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier SchönmannMultiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier Schönmann