Biochimica et Biophysica Acta, 1134 (1992)297-302 297 © 1992 Elsevier Science PublishersB.V. All rightsreserved0167-4889/92/$05.00 BBAMCR 13136 Stimulatory and inhibitory effects of 1,25-dihydroxyvitamin D 3 on thymocyte mitogenesis induced by phorbol ester and calcium ionophore Yael Zilberman a, Amiram Ravid b, Uri A. Liberman ¢, Abraham Novogrodsky b and Ruth Koren c a Department of Pharmacology. The Hebrew University-Hadassah Medical School, lerusa~m (Israel), b Rogof/Medical Research Institute and c Metabolic Disease Unit, Beilinson Medical Center, Petah.Tdcva (Israel) (Received 17 August1991) Keywords: T lymphocyte;1,25-Dihydroxyvitamin D-3;Calciumionophore; Phorboiester Mouse medullary thymocytes have specific receptors for 1,25-dihydroxyvitamin D 3 ~,I,25(OH)2D3).The mitogenic stimulation of these cells by phytohemagglutinin in the presence or absence of the phorbol ester TPA is inhibited by 1,25(OH)2D 3. The calcium ionophore A23187 did not reverse the inhibition by 1,25(OH)2D 3 of phytohemagglutinin. Stimulation of thymocyteswith either TPA or A23187 alone did not result in proliferation. Co-stimulation of the thymocytes with TPA and A23187 induces cell proliferation. 1,25(OH)2D 3 markedly enhanced the TPA and A23187-induced cell proliferation even when added 4 h after the initiation of the culture. In contrast, DNA synthesis by thymocytes incubated for 4 h in the presence of TPA and A23187 and then cultured in medium containing 1,25(OH)2D 3 but in the absence of both TPA and A23187, was inh~ited by 1,25(OH)zD 3. The extem of inhibition was comparable to the inhibition of lectin-induced stimulation by the hormone. Using monoclonal antibodies to neutralize IL-2 and block IL-2 receptors we showed that 1,25(OH)2D 3 enhanced the IL-2-independent component of the A23187- and TPA-induced mitogenesis. In conclusion: (1) The nature and presence of the mitogenic signal determines whether 1,25(OH)2D 3 enhances or inhibits thymocyte stimulation. (2) Both stimulatoff and inhibitor/actions of 1,25(OH)2D 3 seem to take place at points distal to the initial increase in intracellular calcium or activation of protein kinase C. Intreduetiou 1,25-Dihydroxyvitamin D 3 (1,25(OH)2D3), the most active metabolite of vitamin D, is a hormone responsi- ble for calcium and phosphorous homeostasis [1]. In addition to its 'classical' target organs, the intestine and kidney, it also affects cells of the immune system, such as macrophages and lymphocytes as well as hematopoietic cells [2]. The hormone interacts with an intracellular receptor which in turn activates transcrip- tion of specific genes [I]. 1,25(OH)2D3 inhibits lym- phocyte functions such as T-cell mitogenesis, lym- phokine production and antibody production by acti- vated B cells [2]. Receptors for 1,25(OH)2D 3 are found in activated but not in resting peripheral blood lym- phocytes [3,4], as well as in freshly isolated medullary mouse thymocytes [5]. It has been shown that Correspondence: Y. Zilbennan, Deparlmeotof Pharmacology,The Hebrew University-Hadassah Medical School, P.O. Box 1172, Jerusalem 91-010, Israel. 1,25(OH)2D 3 inhibits the mitogenic response of thy- mocytes to phytohemagglutinin (PHA), alone or in combination with interleukin 1, TPA or intefleukin 2 (IL-2) [5]. The signal transduction step in thymoojtes stimu- lated with mitogenic lectins is thought to involve activa- tion of a phosphatidylinositol bisphosphate (PlP2)- specific phospholipase C [6]. Breakdown of PIP2 re- sults in intracellular accumulation of inositol phos- phates and diacylglycerol which are thought to be two crucially important intracellular second messengers in the mitogenic pathway. Inositt; trisphosphate releases calcium from intraceilular stores and diacylglycerol ac- tivates protein kinase C. The calcium iouophore, A23187, and the phothol ester, TPA, bypass the signal transductio,x step and directly increase the intracellular calcium concentration and activate protein kinase C, respectively. The notion that activation of plmspho- lipase C is of importance for the mitogenic cascade of events is supported by the fact that stimulation of thymocytes with A23187 and TPA results in DNA synthesis.