The human prolactin gene upstream promoter is regulated in lymphoid cells by activators of T-cells and by cAMP

Prolactin (PRL) is produced in human thymocytes, T-cells and endometrium. I n these extrapituitary tissues, PRL gene transcription is directed by an al ternative upstream promoter, and it is thought to act as a locally produced cytokine, with relevance for immune regulation and modulation of T-cell fu nction. We have studied PRL transcriptional regulation in the human T-lymph oblastoid Jurkat cell line transfected with a fragment of the upstream prom oter linked to luciferase. A cAMP analogue (cptAMP) increased promoter acti vity rapidly and dose dependently. This increase was resistant to inhibitio n by cyclosporin A and thus independent of calcineurin phosphatase (CN). T- cell activation by phorbol myristate acetate (PMA) failed to enhance promot er activity but phytohaemagglutinin (PHA) alone or PHA+PMA increased it, an d cptAMP acted in synergy with PMA or PHA to increase it further. H-89, a c AMP-dependent protein kinase A (PKA) inhibitor, inhibited the effect of cpt AMP as did transfection with protein kinase inhibitor PKI, an expression ve ctor of the specific inhibitor of PKA. A single point mutation in the CRE ( cAMP response element) located at -25 bp in the PRL upstream promoter (TGAC GT to TGCCGT) failed to reduce the response to cptAMP, while mutations or d eletion of four nucleotides in the CRE to TACTCT diminished the response to cAMP by more than half. We conclude that activity of the human PRL upstream extrapituitary promoter can be induced by activators of T-cells, as well as by a cAMP analogue. Th e signal is transmitted by PKA and the effect of cAMP is independent of CN. It is partly dependent on an intact proximal CRE motif but a more upstream enhancer may contribute to promoter regulation.