Phorbol ester enhancement of IL-3-dependent proliferation of primitive hematopoietic progenitors of mice in culture.

Protein kinase C (PKC) is a Ca++- and phospholipid-dependent protein kinase activated by diacylglycerol that is either released from cell membranes in response to certain growth factors or mimicked by 12-O-tetradecanoyl phorbol-13-acetate (TPA). We studied the effects of TPA on interleukin-3 (IL-3)-dependent colony formation of mouse bone marrow cells from mice injected with 5-fluorouracil 2 days before examination in order to clarify the significance of PKC in the proliferation of primitive hematopoietic progenitors. Although TPA alone did not support colony formation, TPA in combination with IL-3 increased colony numbers from 1.5 to 2 times that formed with IL-3 and vehicle. TPA increased not only the granulocyte/macrophage colonies, but also the multilineage colonies. A sequential colony count showed that TPA, unlike IL-6, did not hasten the appearance of colonies. Because TPA enhanced IL-3-dependent colony formation derived from lineage-negative marrow cells obtained from mice that received 5-FU 2 days before, it is possible that it might act directly on primitive progenitors. Prolonged pretreatment of marrow cells with TPA prevented TPA-augmented colony growth. Calphostin C, a specific PKC inhibitor, and certain specific tyrosine kinase inhibitors, such as genistein and herbimycin A, abrogated the enhancing effects of TPA on IL-3-dependent colony formation. These data suggest that TPA had a direct effect on the primitive progenitors and enhanced IL-3-dependent colony formation via activation of PKC and certain tyrosine kinases.