engleski

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 cell lines MC3T3- E1 and Py1a.However, treatment with LPS (10ug/ml), TGF-beta (1ng/ml), TNF-alpha (100ng/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 cell 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 1h and maximal at 6h.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-2 and U-2 OS cells.IN adition, TNF (100 ng/ml) stimulated the release of LIF protein from both these cellc and PMA (2.5 ng/ml) stimulated LIF protein in Saos-2 cells.These results show that several different human malignant and rodent immortalized clonal bone cell lines can express and regulate steady-state LIF mRNA levels and produce LIF protein but that primary cultures of fetal rat osteoblastic cells do not express this cytokine.Hence, LIF may regulate malignant osteogenic cell growth and function in bone but may not be an important regulator of normal bone metabolism.

LIF ; Bone metabolism ; Human osteosarcoma cell lines ; Osteoblast-enriched cell cultures ; Steady-state LIF mRNA levels

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