Nicotinic regulation of c-fos and osteopontin expression in human-derived osteoblast-like cells and human trabecular bone organ culture.
Long-term in vivo studies have highlighted smoking as a risk factor in postmenopausal osteoporosis, bone fracture incidence, and increased nonunion rates. In contrast, there are few data postulating the effects of smoking at the cellular level in human skeletal tissue. In this study, we present novel evidence demonstrating that the nicotinic receptor alpha4 subunit is present in human primary bone cells by using reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, we demonstrate direct cellular effects of nicotine on primary human bone cells and blockage of these effects with a nicotinic receptor antagonist, D-tubocurarine. Nicotine effects on cell proliferation were biphasic with toxic, antiproliferative effects at high levels of nicotine (>1 mmol/L) and stimulatory effects at very low levels (0.01-10 micromol/L) after 72 h. This nicotine-induced increase in cell proliferation was inhibited in a dose-dependent manner by the addition of D-tubocurarine. In addition, proliferation effects from low-level treatment correlated with an upregulation of expression of the AP-1 transcription factor, c-fos, within 1 h, which was blocked by incubation with D-tubocurarine. To determine in situ bone cell responses within their trabecular matrix, cores of human bone isolated from biopsies were perfused with 0.1 micromol/L nicotine for 24 h. Western analysis of proteins isolated from the cores highlighted an increase in osteopontin, a bone matrix protein implicated in regulating resorption, which was partially inhibited by the addition of D-tubocurarine. To conclude, our results suggest that nicotine has a direct effect on human bone cells in modulating proliferation, upregulation of the c-fos transcription factor, and the synthesis of the bone matrix protein, osteopontin.
Walker
L M
LM
Centre for Science and Technology in Medicine, School of Postgraduate Medicine, Keele University, North Staffordshire Hospital, Stoke on Trent, UK.
Preston
M R
MR
Magnay
J L
JL
Thomas
P B
PB
El Haj
A J
AJ
D001483:Base Sequence; D017931:DNA Primers; D005786:Gene Expression Regulation; D016762:Genes, fos; D006801:Humans; D009538:Nicotine / Q000494:pharmacology*; D009924:Organ Culture Techniques; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D053495:Osteopontin; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D011978:Receptors, Nicotinic / Q000235:genetics; D012795:Sialoglycoproteins / Q000235:genetics*; D014403:Tubocurarine / Q000494:pharmacology
D017931:DNA Primers; D012333:RNA, Messenger; D011978:Receptors, Nicotinic; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; D053495:Osteopontin; D009538:Nicotine; D014403:Tubocurarine