This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hoggard, N. Articles by Martial, J. A. Search for Related Content PubMed PubMed Citation Articles by Hoggard, N. Articles by Martial, J. A.

Pit-1 Binding Sequences Permit Calcium Regulation of Human Prolactin Gene Expression

Department of Medicine, University of Manchester Manchester M13 9PT, United Kingdom
Laboratoire de Biologie Moleculaire et de Génie Génétique, Institut de Chimie, Université de Liege B-4000 Sart Tilman, Belgium

Address requests for reprints to: Dr. N. Hoggard, Department of Medicine, Stopford Building, University of Manchester, Manchester, M13 9PT, United Kingdom.

Abstract

This study examines the regulation of the human PRL (hPRL) gene promoter by intracellular calcium. Deletants of the 5'-flanking region of the hPRL gene and constructs consisting of the thymidine kinase promoter linked to the first or second proximal Pit-1 binding site were fused to the bacterial chloramphenicol acetyl transferase (CAT) reporter gene.

With the complete 5-kilobase pair (kbp) hPRL promoter sequence the calcium channel agonist Bay K8644 induced a significant 2-fold increase in CAT reporter gene expression and the antagonist verapamil a 4.5-fold reduction, using GH₃ cells cultured in physiological levels of calcium. The transcriptional response to calcium influx was similar with a series of 5'-deleted hPRL-CAT constructs including those that comprised the proximal (up to 740 bp) or distal (–1300- to –1700-bp) sequences alone. When treating cells cultured in low calcium conditions the induction with the hPRL promoter increased to 5-fold on the addition of exogenous calcium and Bay K8644. The pituitary-specific expression of the hPRL gene is conferred by the interaction of the pituitaryspecific factor Pit-1 with several binding sites located in the 5'-flanking DNA, of which three are located in the proximal region. This suggested that Pit-1 binding sites may be involved in the calcium response. The presence of either the first or the second proximal Pit-1 binding site alone linked to a thymidine kinase promoter was sufficient to confer a significant 2-fold increase in reporter gene response to exogenous calcium and Bay K8644, while the constructs containing multiple Pit-1 sites linked to a thymidine kinase promoter conferred a further increase. This transcriptional response was lost when the constructs were transfected into the (calcium-responsive) nonpituitary HIT-T15 cell line. The calcium response was also lost when point mutations were introduced into a Pit-1 binding site.

Our data indicate that the hPRL gene promoter is responsive to raised intracellular calcium. This response involves Pit-1 binding sites and appears to require the presence of Pit-1 protein.

FOOTNOTES

These studies were supported by European Community Grant SCI-0245CTT, SPPS Belgium Sciences de la Vie Grant BIO/15, and by grants from the Wellcome Trust and the Rotary Club, Liege, Belgium (to M.B.)

Received for publication June 5, 1991. Revision received August 29, 1991. Accepted for publication September 5, 1991.

This article has been cited by other articles:

				S. L. Shorte, G. M. Leclerc, R. Vazquez-Martinez, D. C. Leaumont, W. J. Faught, L. S. Frawley, and F. R. Boockfor PRL Gene Expression in Individual Living Mammotropes Displays Distinct Functional Pulses That Oscillate in a Noncircadian Temporal Pattern Endocrinology, March 1, 2002; 143(3): 1126 - 1133. [Abstract] [Full Text] [PDF]

				D. W. McFerran, J. A. Stirland, A. J. Norris, R. A. Khan, N. Takasuka, Z. C. Seymour, M. S. Gill, W. R. Robertson, A. S. I. Loudon, J. R. E. Davis, et al. Persistent Synchronized Oscillations in Prolactin Gene Promoter Activity in Living Pituitary Cells Endocrinology, July 1, 2001; 142(7): 3255 - 3260. [Abstract] [Full Text] [PDF]

				P. Kievit, J. D. Lauten, and R. A. Maurer Analysis of the Role of the Mitogen-Activated Protein Kinase in Mediating Cyclic-Adenosine 3',5'-Monophosphate Effects on Prolactin Promoter Activity Mol. Endocrinol., April 1, 2001; 15(4): 614 - 624. [Abstract] [Full Text]

				I. Manfroid, J. A. Martial, and M. Muller Inhibition of Protein Phosphatase PP1 in GH3B6, but Not in GH3 Cells, Activates the MEK/ERK/c-fos Pathway and the Human Prolactin Promoter, Involving the Coactivator CBP/p300 Mol. Endocrinol., April 1, 2001; 15(4): 625 - 637. [Abstract] [Full Text]

				J. R. E. Davis, J. McVerry, G. A. Lincoln, S. Windeatt, P. R. Lowenstein, M. G. Castro, and A. S. McNeilly Cell Type-Specific Adenoviral Transgene Expression in the Intact Ovine Pituitary Gland after Stereotaxic Delivery: An in VivoSystem for Long-Term Multiple Parameter Evaluation of Human Pituitary Gene Therapy Endocrinology, February 1, 2001; 142(2): 795 - 801. [Abstract] [Full Text] [PDF]

				N. Takasuka, M. R. H. White, C. D. Wood, W. R. Robertson, and J. R. E. Davis Dynamic Changes in Prolactin Promoter Activation in Individual Living Lactotrophic Cells Endocrinology, March 1, 1998; 139(3): 1361 - 1368. [Abstract] [Full Text] [PDF]

				P. W. Howard and R. A. Maurer A Composite Ets/Pit-1 Binding Site in the Prolactin Gene Can Mediate Transcriptional Responses to Multiple Signal Transduction Pathways J. Biol. Chem., September 8, 1995; 270(36): 20930 - 20936. [Abstract] [Full Text] [PDF]