Clinical Science (2005) 109, (177–182) (Printed in Great Britain)

Specific effect of arachidonic acid on inducible nitric oxide synthase mRNA expression in human osteoblastic cells

Giovanna PRIANTE*, Estella MUSACCHIO*, Elisa PAGNIN†, Lorenzo A. CALÒ† and Bruno BAGGIO*

*Department of Medical and Surgical Sciences, University of Padua, via Giustiniani 2, 35128 Padova, Italy, and †Clinical and Experimental Medicine, University of Padua, via Giustiniani 2, 35128 Padova, Italy

Key words: arachidonic acid, bone metabolism, fatty acid, nitric oxide synthase, osteoblast.

Abbreviations: AA, arachidonic acid; ASA, acetyl salicylic acid; COX, cyclo-oxygenase; EPA, eicosapentaenoic acid; FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDL, high-density lipoprotein; HI-FCS, heat-inactivated FCS; IL, interleukin; NO, nitric oxide; iNOS, inducible NO synthase; OA, oleic acid; PG, prostaglandin; PKC, protein kinase C; PUFA, polyunsaturated fatty acid; RT, reverse transcription; TK, tyrosine kinase; TNFa, tumour necrosis factor a.

Correspondence: Professor Bruno Baggio (email bruno.baggio@unipd.it).

A specific modulatory effect of PUFAs (polyunsaturated fatty acids) on gene expression of some cytokines involved in bone remodelling has been reported previously. In particular, although a direct action of AA (arachidonic acid) on bone cytokine gene expression has been shown in human osteoblastic cells, OA (oleic acid) and EPA (eicosapentaenoic acid) were ineffective. Since the NO (nitric oxide) system has also been shown to have an important modulatory activity on osteoblasts, osteoclasts and bone metabolism, in the present study we have investigated the effects of PUFAs on iNOS (inducible NO synthase) gene expression in a human osteoblast-like cell line. AA induced a significant increase in iNOS mRNA expression, whereas EPA and OA had no stimulatory effects but instead caused a significant inhibition of AA-induced iNOS gene expression. Blocking of the COX (cyclo-oxygenase) pathway did not inhibit AA-induced iNOS expression. AA action was inhibited instead by the addition of calphostin C and genistein, inhibitors of PKC (protein kinase C) and tyrosine kinases respectively. Experiments performed with specific anti-cytokine antibodies showed a significant decrease in iNOS expression in AA-treated osteoblastic cells, suggesting that both cytokine-dependent and -independent mechanisms account for the effects of AA on iNOS gene expression. In conclusion, our investigation clearly shows specific effects of PUFAs on iNOS expression in human osteoblast-like cells with a cytokine-dependent and -independent mechanism. These results might have clinical relevance and are of interest for understanding the reported beneficial effects of dietary PUFA manipulation on the prevention and/or treatment of primary and secondary bone disease.

Received 20 December 2004/14 February 2005; accepted 1 April 2005

Published as Immediate Publication 1 April 2005, doi: 10.1042/CS20040369

©2005 The Biochemical Society