Production of leukemia inhibitory factor mRNA and protein by malignant and immortalized bone cells

Leukemia inhibitory factor (LIF) is a recently characterized glycoprotein with complex biologic activities on bone cells. We tested various rodent and human immortalized and malignant bone cell lines and primary osteoblast-enriched cell cultures from fetal rat calvarial digests for expression of LIF mRNA and LIF protein. Both human and rodent immortalized 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 MC3T3-E1 and Py1a. However, treatment with LPS (10 micrograms/ml), TGF-beta (1 ng/ml), TNF-alpha (100 ng/ml) or inhibitors of protein synthesis (cycloheximide, emetine, puromycin, and anisomycin) induced the expression of LIF message in these cells. In contrast, primary osteoblast-enriched cells did not express LIF mRNA in Northern blot assays either constitutively or after treatment with TNF-alpha or cycloheximide. The human osteosarcoma cells lines U-2 OS and Saos-2 constitutively expressed LIF mRNA and did not respond to LPS treatment. However, phorbol myristate acetate (PMA), an activator of protein kinase C, was a potent stimulator of LIF message in Saos-2 but not U-2 OS cells. The effects of PMA (0.5 ng/ml) on LIF mRNA in Saos-2 cells were detectable at 1 h and maximal at 6 h. TNF-alpha (100 ng/ml) and inhibitors of protein synthesis also increased LIF mRNA in both Saos-2 and U-2 OS cells. LIF protein was also detected constitutively in the conditioned medium from both Saos and U-2 OS cells.(ABSTRACT TRUNCATED AT 250 WORDS)