2:26 Nicotine inhibits bone morphogenic protein–2–induced proteoglycan and collagen 2 synthesis of disc nucleus cells

Abstract

Purpose of study: We test the hypothesis that nicotine can reverse bone morphogenic protein (BMP)-2 induced upregulation of proteoglycan synthesis and collagen 2 expression but not collagen 1.Methods used: Cells were isolated from nucleus pulposus of rat lumbar discs and were grown in monolayer culture. The cells were cultured in DMEM/F12 with 1% FBS and treated without added BMP-2 or nicotine (no treatment), 100 ng/ml of BMP-2 (BMP-2 only) or with 100 ng/ml of BMP-2 and increasing doses of nicotine (1, 10, 100 mcg/ml). The culture media was collected between day 4 and 7 after BMP-2/nicotine treatment and sulfated-glycosaminoglycan (s-GAG) content in the media was quantified using the DMMB to estimate proteoglycan production. The results were normalized by cell number at day 7. On day 7, mRNA was extracted, and reverse transcriptase polymerase chain reaction (RT-PCR) and real-time PCR were used to assess collagen 1 and 2 mRNA expression.of findings: By day 7, s-GAG production in the BMP-2–only group was increased 2.5-fold as compared with no treatment group. The s-GAG production monotonically decreased with increased concentration of nicotine down to 46% of BMP-2–only group (Fig. 1) . No change in collagen 1 expression with the addition of BMP-2 or nicotine was noted. However, significant increase in collagen 2 mRNA in the BMP-2–only group compared with no treatment group was noted. Collagen 2 mRNA downregulation was noted with nicotine concentrations of 100 mcg/ml with RT-PCR. In order to better quantitate changes in collagen 2 mRNA abundance, real-time PCR was used. This showed collagen 2 mRNA that decreased to 69% and 27% of BMP-2–only group with the addition of nicotine at 10 and 100 mcg/ml, respectively. The effect of nicotine on sGAG production and the relative abundance of mRNA as percentage of BMP-2 only is noted in Fig. 1.Relationship between findings and existing knowledge: BMP-2 and its receptor are normally found in the intervertebral disc, and BMP-2 has recently been shown to enhance the chondrocytic phenotype of disc cells (increase proteoglycan and collagen 2 synthesis). Epidemiologic human studies and in vivo rat experiments indicate that smoking may lead to disc degeneration. We show here that nicotine, an important component of cigarette smoke, can inhibit proteoglycan and collagen 2 synthesis induced by BMP-2. Of note, collagen 1 is not regulated by BMP-2 or nicotine at the tested concentrations, indicating a certain specificity in nicotine activity.Overall significance of findings: This study establishes the negative influence of nicotine on intervertebral disc cell response to BMP-2 at a cellular and molecular level.Disclosures: No disclosures.Conflict of interest: No conflicts.