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Synthesis of acetylcholine (ACh) by non-neuronal cells is now well established and plays diverse physiologic roles. In neurons, the Na+-dependent, high affinity choline transporter (CHT1) is absolutely required for ACh synthesis. In contrast, some non-neuronal cells synthesize ACh in the absence of CHT1 indicating a fundamental difference in ACh synthesis compared to neurons. The aim of this study was to identify choline transporters, other than CHT1, that play a role in non-neuronal ACh synthesis. ACh synthesis was studied in lung and colon cancer cell lines focusing on the choline transporter-like proteins, a five gene family choline-transporter like protein (CTL)1–5. Supporting a role for CTLs in choline transport in lung cancer cells, choline transport was Na+-independent and CTL1–5 were expressed in all cells examined. CTL1, 2, and 5 were expressed at highest levels and knockdown of CTL1, 2, and 5 decreased choline transport in H82 lung cancer cells. Knockdowns of CTL1, 2, 3, and 5 had no effect on ACh synthesis in H82 cells. In contrast, knockdown of CTL4 significantly decreased ACh secretion by both lung and colon cancer cells. Conversely, increasing expression of CTL4 increased ACh secretion. These results indicate that CTL4 mediates ACh synthesis in non-neuronal cell lines and presents a mechanism to target non-neuronal ACh synthesis without affecting neuronal ACh synthesis.
Synthesis and secretion of acetylcholine (ACh) to act as an autocrine or paracrine hormone by non-neuronal tissues is now well established (Wessler and Kirkpatrick 2008). Choline acetyltransferase (ChAT) and ACh synthesis has been demonstrated in airway epithelial cells (Klapproth et al. 1997; Proskocil et al. 2004; Wessler and Kirkpatrick 2008), colon epithelial cells (Porter et al. 1996; Cheng et al. 2008; Yajima et al. 2011), keratinocytes (Kurzen et al. 2007), glia (Wessler et al. 1997), lymphocytes (Kawashima and Fujii 2000), and ovarian follicular cells (Mayerhofer and Kunz 2005) among other cell types. Non-neuronal ACh plays multiple physiologic and pathological roles, affecting growth, development, secretion, and ciliary movement among many actions (Lips et al. 2007; Wessler and Kirkpatrick 2008; En-Nosse et al. 2009; Novotny et al. 2011; Hollenhorst et al. 2012). In both lung cancer and colon cancer, ACh acts as an autocrine growth factor for cancer growth and development (Song et al. 2003, 2007, 2008). The wide-spread expression of ACh in non-neuronal tissues makes it important to understand how non-neuronal ACh synthesis differs from neuronal ACh synthesis.
There are clear similarities and differences between neuronal and non-neuronal cholinergic signaling. Both neuronal and non-neuronal cell types express choline acetyltransferase, the vesicular ACh transporter, cholinesterases, nicotinic, and muscarinic receptors (Proskocil et al. 2004; Wessler and Kirkpatrick 2008). Regulation of ACh secretion is obviously different as non-neuronal cells generally are not excitable and secretion is not triggered by action potentials. Another clear difference is transport of choline that is used for ACh synthesis.
In neurons, choline used for ACh synthesis is transported from extracellular spaces via a Na+-dependent, high-affinity choline transporter (CHT1) (Apparsundaram et al. 2000; Okuda et al. 2000) and in the absence of CHT1, neurons cannot synthesize ACh (Ferguson et al. 2004). In contrast, colon epithelial cells (Yajima et al. 2011) and lung cancer cells can synthesize ACh in the absence of CHT1 (Song et al. 2003; Song and Spindel 2008). Thus, choline transport in non-neuronal cells cannot be solely dependent on CHT1 and must by necessity utilize other choline transporters such as the recently described family of five choline transporters designated as the choline transporter-like proteins 1–5 (CTL1–5) (O'Regan et al. 2000; Traiffort et al. 2005). The CTLs are Na+-independent and have an intermediate-affinity for choline and hemicholinium-3 (HC-3) as compared to CHT1 (O'Regan et al. 2000; Inazu et al. 2005; Traiffort et al. 2005). CTL1 in particular has been shown to transport choline in renal tubule epithelia (Yabuki et al. 2009), keratinocytes (Uchida et al. 2009), neuroblastoma (Machova et al. 2009; Yamada et al. 2011), and lung adenocarcinoma cells (Nakamura et al. 2010). CTL2 has also been shown to transport choline in lung adenocarcinoma cells (Nakamura et al. 2010). CTL3 has recently been demonstrated to be expressed in neutrophils and has been identified as the human neutrophil alloantigen-3a (Greinacher et al. 2010). Although it has been suggested that CTL1 may be linked to non-neuronal ACh synthesis, this has only been proposed on the frequent co-expression of CTL1 with non-neuronal ACh (Yamada et al. 2011).
We thus undertook to determine which of the CTL's is positively linked to ACh synthesis in two different non-neuronal systems, examining both small cell lung carcinoma (SCLC) cells using the H82 cell line that synthesizes ACh but does not express CHT1 and the H508 colon cancer cell line that similarly synthesizes ACh but does not express detectable levels of CHT1. This was done by a combination of pharmacologic characterization of choline transport and characterizing the effect of knockdown of the different CTL's and expression of CTL4 on ACh synthesis. In this article, we show that of the CTLs, only CTL4 appears positively linked to ACh synthesis in two different non-neuronal cell types.
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Acetylcholine is synthesized from choline and acetyl-CoA by the action of ChAT. For ACh synthesis to proceed, choline must be transported into the cell by a choline transporter that is linked in some manner to ChAT. Choline is an organic cation and does not freely cross cell membranes. Its uptake from extracellular spaces requires specific choline transporters present on cell membranes. In neurons, the high affinity choline transporter CHT1 is absolutely required for ACh synthesis (Apparsundaram et al. 2000; Okuda et al. 2000; Ferguson et al. 2004), but as shown in Table 1, H82 cells and the majority of SCLC cell lines examined do not express CHT1 yet synthesize ACh (Song et al. 2003); therefore, lung cancers must utilize a different choline transporter than CHT1 for ACh synthesis. This is not unique to lung cells as colon crypt cells also lack CHT1 but synthesize ACh (Yajima et al. 2011). Thus, two quite different types of epithelial cells do not utilize CHT1 for choline transport for ACh synthesis and therefore rely on other choline transporters for this purpose. Multiple choline transporters have been identified in humans and other species (Kleinzeller et al. 1994; Pfeil et al. 2003), therefore the purpose of this study was to begin to identify the choline transporters that are linked to non-neuronal ACh synthesis.
As shown in Fig. 1, ACh secretion by H82 SCLC cells is directly related to media choline concentration (Fig. 1a–c) and there is a clear correlation between choline concentration and ACh secretion (Fig. 1d). The increased ACh secretion caused by increased choline levels was not caused by increased ChAT activity, nor was the increase caused by changes in cholinesterase levels since the changes were seen in the presence of neostigmine, a cholinesterase inhibitor. In this regard, H82 cells are similar to neurons where choline also increases ACh synthesis (Ulus et al. 1989) although obviously other significant differences such as the mechanisms underlying ACh secretion are present (Song and Spindel 2008). This is reflected by the relatively low ACh levels in H82 cell pellets in which ACh in cell pellets is only 1/10 as much as is in the media, suggesting a degree of constitutive secretion.
Recently, a new family of intermediate affinity choline transporters, the CTLs has been shown to be widely expressed in mammalian tissues. Five CTL genes have been described (CTL1–5) and complex alternative spicing also occurs (O'Regan et al. 2000; Traiffort et al. 2005). In human and rat, CTL1-4 mRNA are mainly detected in peripheral tissues, while CTL1 is also widely expressed throughout the nervous system (Traiffort et al. 2005). The CTLs are Na-independent and inhibited by HC-3 at millimolar concentrations as opposed to the nanomolar inhibition of CHT1 by HC-3. This pharmacology of the CTLs is similar to what we observed in H82 cells in which choline transport was also primarily Na+-independent with a Km of 8.2 μM and was inhibited by μM concentrations of HC-3 (Fig. 2). Consistent with a role for the CTL's in choline transport in lung cells, RT-PCR showed that CTL1-5 are expressed in essentially all lung cancer cell lines tested (Table 1). Quantitation by real-time PCR showed abundant levels of CTL1, 2 and 5 in all cell lines and tumors while levels of CTL3 and 4 were significantly lower. This distribution is similar to that reported by Tomi et al. who found levels of CTL3 and CTL4 were 100-fold lower than levels of CTL1 in rat retinal capillary endothelial cells (Tomi et al. 2007).
The roles of CTL 1, 2, and 5 in transporting choline in H82 cells was confirmed by sRNA knockdown, in which knockdown of each of those transporter subtypes decreased choline transport by ~ 30% (Fig. 3b). This confirms that the multiple CTLs expressed by SCLC can indeed transport choline. Knockdown of CTL4 did not affect choline uptake, suggesting CTL4 does not significantly contribute to total choline uptake consistent with its lower levels of expression. Our findings are consistent with those of Nakamura al (Nakamura et al. 2010) who also saw a decrease in choline transport by knockdown of CTL1 and CTL2 in A549 lung adenocarcinoma cells and Machova et al. who showed CTL1 knockdown decreased choline transport in neuroblastoma cells (Machova et al. 2009). Thus, our and others' findings clearly demonstrate a role for the CTL's in choline transport.
To determine if one of the CTL's was specifically linked to ACh synthesis in lung cells, CTL1-5 were individually knocked-down with siRNAs. As seen in Fig. 3c, knockdown of CTL 1, 2, 3, 5 and CHT1 did not decrease ACh synthesis. Only the knockdown of CTL4 significantly reduced ACh synthesis and secretion. This suggests that in SCLC, of the five CTL's, only CTL4 is specifically coupled to ACh synthesis; similar to CHT1 which in neurons is specifically coupled to ACh synthesis. Confirming the linkage of CTL4-mediated choline transport to ACh synthesis in lung cell lines, expression of CTL4 in H82 cells increased both choline transport and ACh synthesis (Fig. 4a and b). An unexpected finding was that ACh synthesis after knockdown of CTL1 and CTL2 actually increased. One possible explanation is that in the absence of CTL1 and CTL2, more choline is transported by CTL4 thus leading to increased ACh synthesis. Another possibility is that CTL1 and 2 play a role in ACh reuptake as has been proposed for the organic cation transporter N1 (OCTN1) (Pochini et al. 2011) and therefore the CTL1 and 2 knockdowns increased media ACh levels by inhibiting reuptake.
It is interesting to note that stimulation of ACh secretion and cell growth occurs at choline levels higher than the saturating levels for choline transport shown in Fig. 2. This suggests that that CTL4 may have lower affinity for choline than some of the other CTL's or reflect the mechanism by which choline for ACh synthesis is segregated from the general pool of intracellular choline.
To rule out the possibility that lung cancer cells are unique among non-neuronal cell types in their use of CTL4 for ACh synthesis, we examined a second epithelial cell type known to synthesize ACh, namely colonic epithelial cells as exemplified by colon cancer cells which have been clearly shown to synthesize ACh (Porter et al. 1996; Yajima et al. 2011). As shown in Fig. 5b, 7 of 7 colon cancer cell lines expressed CTL4. CHT1 was generally undetectable in these lines (data not shown). Colon cancer cells consistently expressed higher levels of CTL4 than did lung cancer cells (Fig. 5b). Confirming the role of CTL4 in ACh synthesis in colon cancer cells knockdown of CTL4 significantly decreased levels of ACh synthesis and secretion in H508 colon cancer cells which express relatively high levels of CTL-4 and CTL4 transduction increased levels of intracellular ACh in Caco-2 colon cancer cells which express relatively low levels of CTL4. Increased levels of secretion of ACh into the media in Caco-2 cells transduced with CTL4 was not seen which likely represents the relatively low levels of ChAT that are expressed by those cells and thereby likely limiting ACh synthesis. This finding confirms the role of CTL4 in two diverse types of epithelial cells in non-neuronal ACh synthesis and suggests that CTL4 is likely involved in ACh synthesis by cell types that do not depend on CHT1.
It is important to note that CTL-4 may not be the only choline transporter linked to non-neuronal ACh synthesis. For example, the organic cation transporter 1 (OCT1) has also been suggested to play such a role as well as being involved in ACh secretory processes (Lips et al. 2007; Yajima et al. 2011). Other choline or cation transporters may also turn out to be linked to non-neuronal ACh synthesis role as well and there may be tissue specificity in the choline transporters used for ACh synthesis in different tissues. The finding of the linkage of CTL-4 to non-neuronal ACh synthesis suggests apparent compartmentalization of choline transport and ACh synthesis. In neurons, compartmentalization appears to derive from localization of CHT1 to synaptic vesicles, endosomes, or the cell membrane (Ribeiro et al. 2006; Cuddy et al. 2012). Whether similar localization of CTL4 in analogous compartments (e.g., secretory vesicles, endosomes, or cell membrane) plays a role in non-neuronal ACh synthesis remains to be determined. Regardless of the mechanism of linkage, it is clear that not just any choline transporter will support non-neuronal ACh synthesis as knockdowns of CTL1, 2, 5, and CHT1 did not affect ACh secretion in the cell lines tested.
Identification of the choline transporters linked to non-neuronal ACh synthesis has clear importance both for a fundamental understanding of the mechanisms of non-neuronal ACh synthesis but also to provide a way to target non-neuronal ACh synthesis without targeting neuronal ACh synthesis. An example is shown in Fig. 6 in which the growth of a lung cancer cell line can be inhibited by knocking down levels of CTL4 while knockdown of CTL1 or 5 had no effect on cell growth. This is consistent with the multiple reports that, choline uptake is increased in lung cancers compared to normal cells (Ackerstaff et al. 2003; Glunde et al. 2006) which may partially underlie the increased levels of ACh in lung cancer compared to normal lung (Song and Spindel 2008; Song et al. 2008). The data shown in Fig. 6 suggest that increased choline can stimulate cell proliferation through both nicotinic and muscarinic mechanisms, and it is likely that increased choline also stimulates cell growth secondary to increased phospholipid synthesis. These data also suggest that other diseases found to be characterized by increased non-neuronal ACh synthesis could also potentially be treated by CTL4 knockdown.
In summary, our data show that the choline transporter CTL4 appears to be specifically linked to non-neuronal ACh synthesis and secretion as exemplified by lung and colon cancer cells. In contrast the choline transporters CHT1, CTL1, CTL2, and CTL5 do not appear necessary for non-neuronal ACh synthesis and/or secretion. These findings suggest some degree of functional linkage of CTL4 to ACh synthesis and secretion in non-neuronal cells and that therefore, CTL4 can be targeted as a way to change non-neuronal ACh secretion without affecting the choline transported in neurons by CHT1 that is needed for neuronal ACh synthesis.