Human T-cell leukemia virus type I (HTLV-I) is the etiologic agent of adult T-cell leukemia (ATL), and hypercalcemia frequently associated with ATL is mediated by parathyroid hormone-related peptide (PTHRP). The present study was undertaken to clarify the role of cAMP second messenger system in the regulation of human PTHRP gene expression in ATL cells, using an HTLV-I-infected T-cell line, MT-2. Forskolin and dibutyryl cAMP (Bt2cAMP) caused a marked and transient increase in the steady-state level of PTHRP mRNA. The effects of these agents were dose-dependent, and the maximal effects were observed at 3 h. Nuclear runoff transcription assay showed that forskolin and Bt2cAMP caused an increase in the transcription rate of the human PTHRP gene. In contrast, the stability of PTHRP mRNA was only modestly increased by these agents. Forskolin and Bt2cAMP also increased the secretion of PTHRP by MT-2 cells, as determined by both a newly established immunoradiometric assay using two antibodies against human PTHRP-(1-34) and PTHRP-(50-83) and a radioimmunoassay using an antibody against human PTHRP-(109-141). Prostaglandin E1 (PGE1) caused a marked stimulation of intracellular cAMP production in MT-2 cells, whereas PGE2 and PGF2 alpha had only modest effects. The ability of these PGs to stimulate cAMP production correlated well with their ability to increase PTHRP mRNA level and the secretion of PTHRP. Indomethacin did not affect the basal level of cAMP production or PTHRP mRNA, suggesting that endogenous PG was not involved in the basal production of cAMP or PTHRP. When PGE1 was given to MT-2 cells together with interleukin 2, which is another stimulator of PTHRP gene expression, PTHRP secretion was synergistically stimulated. These results suggest that the transcription of the human PTHRP gene is enhanced through a cAMP-dependent pathway by PGE1 and that PGE1, as well as interleukin 2, plays an important role in the overexpression of the human PTHRP gene in HTLV-I-infected T cells leading to the development of hypercalcemia in ATL patients.