While nicotine is not a carcinogen by itself, it has been shown to induce tumor cell proliferation and differentiation (31
). The mitogenic effects of nicotine in NSCLC are analogous to those of growth factors, and involve activation of multiple signaling pathways (7
). NAChRs appear to play an important role in mediating the effects of nicotine on cell proliferation and survival. Consistent with reports in lung fibroblasts and human bronchial epithelial cells (28
), nicotine upregulates α7 nAChR expression in NSCLC cells, which could amplify the effects of nicotine. We have reported that nicotine also stimulates NSCLC proliferation through the induction of fibronectin, a matrix glycoprotein highly expressed in acute and chronic forms of lung disease that has been implicated in the biology of lung cancer (19
Herein, we show that nicotine induces NSCLC cell proliferation by stimulating the expression of PPARβ/δ. As a member of the nuclear hormone receptor superfamily of transcription factors, PPARβ/δ has been implicated in several processes including insulin sensitivity, terminal differentiation, and tumor growth (15
). We report that silencing of PPARβ/δ inhibited, while overexpression of PPARβ/δ enhanced the mitogenic effect of nicotine, demonstrating a tumor promoting role for PPARβ/δ in mediating the effect of nicotine on cell growth. In line with this finding, one recent study showed that PPARβ/δ is strongly expressed in the majority of lung cancers, and activation of PPARβ/δ induces NSCLC cell proliferation and survival (34
). It should be highlighted that results implicating PPARβ/δ activation in the upregulation of lung carcinoma cell growth (20
) contradict those reported elsewhere in which a decrease in lung cancer cell proliferation was observed (35
). That particular work was performed in another lung carcinoma cell line (A549) and with the use of L-165041, a PPARβ/δ agonist. Note that L-165041 has also been shown to act as an agonist to PPARγ. which isknown to reduce tumor cell proliferation (36
Our observations that PPARβ/δ and α7 nAChR interact and that this is enhanced by nicotine are intriguing. This suggests the possibility that some PPARβ/δ recycles to cytoplasm and interacts with α7nAChR, which may be a potential mechanism for enhancing the stimulatory effect of nicotine on cell growth. This mechanism needs to be explored further.
The intracellular pathways mediating the effect of nicotine on PPARβ/δ expression in NSCLC have not been elucidated. The PI3-K/Akt pathway is a critical pathway in cancer because it contributes to tumor growth, invasion, metastasis and tumor angiogenesis (37
). Therefore, targeting this pathway may represent an attractive strategy for novel anticancer therapies. Akt serves at a key point in the PI3-K pathway and is likely important for the development and maintenance of lung cancer (6
). mTOR also plays a central role in modulating cellular proliferation and angiogenesis in normal tissues and neoplastic processes (38
). Nicotine activation of Akt increased phosphorylation of multiple downstream signals including mTOR. Moreover, nicotine was found to stimulate Akt-dependent proliferation in lung cancer cells (6
). The current report suggests that targeting these signaling pathways inhibits nicotine-induced PPARβ/δ expression. Together, our results highlight the involvement of α7 nAChR and PI3-K/mTOR signaling in mediating the stimulatory effect of nicotine on PPARβ/δ expression.
Several transcription factor binding sites within regions of the PPARβ/δ promoter have been characterized including regulatory elements for AP-2, C/EBP, and Sp1, among others (22
). Our findings demonstrate a critical role for AP-2α in mediating the effect of nicotine on the expression of PPARβ/δ. AP-2α proteins are known to be essential biological factors during development, cell growth, differentiation, and apoptosis (39
). Loss of AP-2α expression has been associated with several invasion and metastasis promoting events (39
). Conversely, overexpression of AP-2α has been associated with survival in colon cancer cells (41
). Our results demonstrated that a reduction in AP-2α gene expression is needed for nicotine to stimulate PPARβ/δ. Supershift and ChIP assays highlight the key role of AP-2α transactivation in the regulation of PPARβ/δ promoter activity by nicotine. Additional studies using sitedirected mutagenesis of key AP-2 sites are required to confirm their role in nicotine-induced PPARβ/δ expression. However, this is commitment with the work of others suggesting that AP-2 acts as a tumor suppressor.
Interestingly, our results also suggested a role for Sp1 in regulation of PPARβ/δ by nicotine. Sp1 regulates activation of many genes involved in tumor growth, apoptosis, and angiogenesis. Down-regulation of Sp1 activity inhibited urokinase receptor expression and reduced the migration of breast cancer cells (42
). Here, Mithramycin A, a Sp1 inhibitor (43
), appeared to block the inhibitory effect of nicotine on AP-2α protein expression via inhibition of Sp1 activity. Of note, while the PPARβ/δ -227 promoter construct showed induction by nicotine, the PPARβ/δ -587 and -445 promoter constructs did not, suggesting the presence of co-repressors. Moreover, since nicotine induced the interaction between Sp1 and the AP-2 cis-acting element, additional mechanisms that enhance the effects of nicotine may exist as demonstrated in previous studies where both Sp1 and AP-2 interaction was required for gene expression (44
). Competitive binding between Sp1 and other transcription factors has also been shown to be important in the control of several other genes (46
). Together, these studies suggest that the existence of functional Sp1 and its interaction with AP-2α influence the stimulatory effect of nicotine on expression of PPARβ/δ.
In summary, we have demonstrated that nicotine increases PPARβ/δ expression through α7 nAChR-mediated activation of PI3-K and mTOR pathways and inhibition of AP-2α expression and DNA binding activity in the PPARβ/δ gene promoter. Sp1 appears to modulate these processes. Nicotine also enhances the formation of the α7 nAChR- PPARβ/δ protein complex (). To our knowledge, this represents the first link between nicotine and the PPARβ/δ gene thereby unveiling a novel mechanism by which nicotine stimulates NSCLC cell growth.