PRODUCTION OF LEUKEMIA INHIBITORY FACTOR MESSENGER-RNA AND PROTEIN BYMALIGNANT AND IMMORTALIZED BONE-CELLS

Leukemia inhibitory factor (LIF) is a recently characterized glycoprot ein with complex biologic activities on bone cells. We tested various rodent and human immortalized and malignant bone cell lines and primar y osteoblast-enriched cell cultures from fetal rat calvarial digests f or expression of LIF mRNA and LIF protein. Both human and rodent immor talized and malignant cells expressed a single 4.4 kb mRNA transcript that hybridized to a human LIF cDNA probe in Northern blots. LIF mRNA was undetectable in unstimulated rodent osteoblast-like cells lines MC 3T3-E1 and Py1a. However, treatment with LPS (10 mug/ml), TGF-beta (1 ng/ml), TNF-alpha (100 ng/ml) or inhibitors of protein synthesis (cycl oheximide, emetine, puromycin, and anisomycin) induced the expression of LIF message in these cells. In contrast, primary osteoblast-enriche d cells did not express LIF mRNA in Northern blot assays either consti tutively or after treatment with TNF-alpha or cycloheximide. The human osteosarcoma cells lines U-2 OS and Saos-2 constitutively expressed L IF mRNA and did not respond to LPS treatment. However, phorbol myrista te acetate (PMA), an activator of protein kinase C, was a potent stimu lator of LIF message in Saos-2 but not U-2 OS cells. The effects of PM A (0.5 ng/ml) on LIF mRNA in Saos-2 cells were detectable at 1 h and m aximal at 6 h. TNF-alpha (100 ng/ml) and inhibitors of protein synthes is also increased LIF mRNA in both Saos-2 and U-2 OS cells. LIF protei n was also detected constitutively in the conditioned medium from both Saos and U-2 OS cells. In addition, TNF (100 ng/ml) stimulated the re lease of LIF protein from both these cells and PMA (2.5 ng/ml) stimula ted LIF protein in Saos-2 cells. These results show that several diffe rent human malignant and rodent immortalized clonal bone cell lines ca n express and regulate steady-state LIF mRNA levels and produce LIF pr otein but that primary cultures of fetal rat osteoblastic cells do not express this cytokine. Hence, LIF may regulate malignant osteogenic c ell growth and function in bone but may not be an important regulator of normal bone metabolism.