**4. Ceramide 1-phosphate and the control of cell growth and death**

The first report showing that C1P was biologically active was published in 1995 (Gomez-Munoz et al., 1995a). C1P was found to have mitogenic properties as it stimulated DNA synthesis and cell division in rat or mouse fibroblasts (Gomez-Munoz et al., 1995a; Gomez-Munoz et al., 1997). Subsequent studies using primary macrophages, demonstrated that like for most growth factors, the mechanisms whereby C1P exerted its mitogenic effects implicated stimulation of the mitogen-activated protein kinase kinase (MEK)/Extracellularly regulated kinases 1-2 (ERK1-2), phosphatidylinositol 3-kinase (PI3- K)/protein kinase B (PKB, also known as Akt), and c-Jun terminal kinase (JNK) pathways (Gangoiti et al., 2008b). In addition, C1P caused stimulation of the DNA binding activity of the transcription factor NF-κB, and the selective inhibitors of MEK, PI3-K, and JNK (PD98059, LY290042, and SP600125), respectively) completely blocked NF-κB activation. Another major target of PKB is glycogen synthase kinase-3β (GSK-3 β), which expression was increased in the presence of C1P. This led to up-regulation of cyclin D1, and c-Myc, two important markers of cell proliferation that are targets of GSK-3β.

In addition, we found that C1P-stimulated macrophage proliferation, involved activation of sphingomyelin synthase (SMS), an enzyme that catalyzes the transfer of phosphocholine from phosphatidylcholine (PC) to ceramide to synthesize sphingomyelin (SM). The other by-product of this reaction is diacylglycerol (DAG), which is a well-established activator of protein kinase C (PKC). Conventional and novel PKC isoforms respond to DAG by translocating to the plasma membrane so that these enzymes can then express their activity and act on signaling events. In this connection, C1P stimulated the translocation and activation of the alpha isoform of PKC (PKC-α) in macrophages, and this resulted to be essential for stimulation of cell growth by C1P (Gangoiti et al., 2010c).

In a more recent report, it has been demonstrated that another essential kinase involved in the regulation of cell proliferation by C1P is the mammalian target of rapamycin (mTOR) (Gangoiti et al., 2010a). Activation of this kinase was tested my measuring the phosphorylation state of its downstream target p70S6K after treatment with C1P. Activation of mTOR/ p70S6K was dependent upon prior activation of PI3-K, as selective inhibition of this kinase blocked mTOR phosphorylation and activation. In addition, C1P caused phosphorylation of PRAS40, a component of the mTOR complex 1 (mTORC1) that is absent in mTORC2, and inhibition of the small G protein Ras homolog enriched in brain (Rheb), which is also a specific component of mTORC1, completely blocked C1P-stimulated mTOR phosphorylation, DNA synthesis and macrophage growth. C1P also caused phosphorylation of another Ras homolog gene family member, RhoA, and inhibition of its downstream effector RhoA-associated kinase (ROCK) also blocked C1P-stimulated mTOR and cell proliferation. It was concluded that mTORC1, and RhoA/ROCK are essential components of the mechanism whereby C1P stimulates macrophage proliferation. However, phospholipase D (PLD), and cAMP are not involved in the mitogenic effect of C1P (Gomez-Munoz et al., 1995a; Gomez-Munoz et al., 1997). Concerning intracellular calcium levels, which have also been implicated in the regulation of cell proliferation, the situation is controversial. Although short-chain C1Ps failed to induce Ca2+ mobilization in fibroblasts (Gomez-Munoz et al., 1995a; Gomez-Munoz et al., 1997) or neutrophils (Rile et al., 2003), and natural C16-C1P did not alter intracellular Ca2+ concentrations in A549 cells (Pettus et al., 2004), C2-C1P- or C8-C1P, caused intracellular Ca2+ mobilization in calf pulmonary artery endothelial (CAPE) cells (Gijsbers et al., 1999), thyroid FRTL-5 (Hogback et al., 2003), or Jurkat T-cells (Colina et al., 2005), suggesting that regulation of Ca2+ homeostasis may be cell type specific.

Finally, it should be pointed out that C1P has been recently shown to be a key mediator in the development and survival of retina photoreceptors, and to also play a critical role in photoreceptor differentiation (Miranda et al., 2011)

Apart from its mitogenic effect, another mechanism by which C1P controls cell homeostasis is by prevention of apoptosis (reviewed in (Gangoiti et al., 2010b)). We previously demonstrated that natural C1P blocked apoptosis in bone marrow-derived macrophages (Gomez-Munoz et al., 2004; Gomez-Munoz et al., 2005), and this was confirmed by Mitra and co-workers (Mitra et al., 2007) who found that down-regulation of CerK in mammalian cells reduced growth, and promoted apoptosis. Also, downregulation of CerK blocked epithelial growth factor-induced cell proliferation. However, in contrast to these observations, it was reported that addition of the cell-permeable C2-ceramide to cells overexpressing CerK led to C2-C1P formation and stimulation of apoptosis (Graf et al., 2007). This controversy can be explained by the fact that overexpression of CerK would substantially increase the intracellular levels of C1P, especially when cells are supplied with high concentrations of exogenous cell permeable C2-ceramide; this action would cause overproduction of C2-C1P inside the cells, which is toxic at high concentrations (Gomez-Munoz et al., 1995a; Gomez-Munoz et al., 2004).

When cells become apoptotic, their metabolism undergoes important changes from early stages. For example, apoptotic bone marrow-derived macrophages express high acid sphingomyelinase (A-SMase) activity and show high levels of ceramides compared to nonapoptotic cells (Gomez-Munoz et al., 2003; Hundal et al., 2003). Of interest, inhibition of A-SMase activation resulted to be one of the mechanisms by which C1P blocks apoptosis (Gomez-Munoz et al., 2004). C1P also blocked the activity of A-SMase in cell-free systems (in vitro), suggesting that inhibition of this enzyme takes place by direct physical interaction of C1P with the enzyme.

Recent work by our group (Granado et al., 2009a) showed that ceramide levels are also increased in alveolar NR8383 macrophages when they become apoptotic. However, A-SMase activity was only slightly enhanced in these cells under apoptotic conditions. This suggested the intervention of a different pathway for ceramide generation in these cells. In subsequent work we demonstrated that the mechanism whereby ceramide levels increased in apoptotic alveolar macrophages involved activation of serine palmitoyltransferase (SPT), the key regulatory enzyme of the de novo pathway of ceramide synthesis. Like for A-SMase, inhibition of SPT activation by treatment with C1P prevented the alveolar macrophages from entering apoptosis. These findings led to conclude that C1P promotes macrophage survival by blocking ceramide accumulation, and action that can be brought about through inhibition of either A-SMase activity, or SPT, depending on cell type.

The prosurvival effect of C1P was highlighted by the demonstration that intracellular levels of C1P were substantially decreased when the cells became apoptotic. It was hypothesized

Although short-chain C1Ps failed to induce Ca2+ mobilization in fibroblasts (Gomez-Munoz et al., 1995a; Gomez-Munoz et al., 1997) or neutrophils (Rile et al., 2003), and natural C16-C1P did not alter intracellular Ca2+ concentrations in A549 cells (Pettus et al., 2004), C2-C1P- or C8-C1P, caused intracellular Ca2+ mobilization in calf pulmonary artery endothelial (CAPE) cells (Gijsbers et al., 1999), thyroid FRTL-5 (Hogback et al., 2003), or Jurkat T-cells (Colina et al.,

Finally, it should be pointed out that C1P has been recently shown to be a key mediator in the development and survival of retina photoreceptors, and to also play a critical role in

Apart from its mitogenic effect, another mechanism by which C1P controls cell homeostasis is by prevention of apoptosis (reviewed in (Gangoiti et al., 2010b)). We previously demonstrated that natural C1P blocked apoptosis in bone marrow-derived macrophages (Gomez-Munoz et al., 2004; Gomez-Munoz et al., 2005), and this was confirmed by Mitra and co-workers (Mitra et al., 2007) who found that down-regulation of CerK in mammalian cells reduced growth, and promoted apoptosis. Also, downregulation of CerK blocked epithelial growth factor-induced cell proliferation. However, in contrast to these observations, it was reported that addition of the cell-permeable C2-ceramide to cells overexpressing CerK led to C2-C1P formation and stimulation of apoptosis (Graf et al., 2007). This controversy can be explained by the fact that overexpression of CerK would substantially increase the intracellular levels of C1P, especially when cells are supplied with high concentrations of exogenous cell permeable C2-ceramide; this action would cause overproduction of C2-C1P inside the cells, which is toxic at high concentrations (Gomez-

When cells become apoptotic, their metabolism undergoes important changes from early stages. For example, apoptotic bone marrow-derived macrophages express high acid sphingomyelinase (A-SMase) activity and show high levels of ceramides compared to nonapoptotic cells (Gomez-Munoz et al., 2003; Hundal et al., 2003). Of interest, inhibition of A-SMase activation resulted to be one of the mechanisms by which C1P blocks apoptosis (Gomez-Munoz et al., 2004). C1P also blocked the activity of A-SMase in cell-free systems (in vitro), suggesting that inhibition of this enzyme takes place by direct physical interaction of

Recent work by our group (Granado et al., 2009a) showed that ceramide levels are also increased in alveolar NR8383 macrophages when they become apoptotic. However, A-SMase activity was only slightly enhanced in these cells under apoptotic conditions. This suggested the intervention of a different pathway for ceramide generation in these cells. In subsequent work we demonstrated that the mechanism whereby ceramide levels increased in apoptotic alveolar macrophages involved activation of serine palmitoyltransferase (SPT), the key regulatory enzyme of the de novo pathway of ceramide synthesis. Like for A-SMase, inhibition of SPT activation by treatment with C1P prevented the alveolar macrophages from entering apoptosis. These findings led to conclude that C1P promotes macrophage survival by blocking ceramide accumulation, and action that can be brought about through

The prosurvival effect of C1P was highlighted by the demonstration that intracellular levels of C1P were substantially decreased when the cells became apoptotic. It was hypothesized

inhibition of either A-SMase activity, or SPT, depending on cell type.

2005), suggesting that regulation of Ca2+ homeostasis may be cell type specific.

photoreceptor differentiation (Miranda et al., 2011)

Munoz et al., 1995a; Gomez-Munoz et al., 2004).

C1P with the enzyme.

that depletion of intracellular C1P could result in the release of A-SMase from inhibition, thereby triggering ceramide generation an apoptotic cell death (Gomez-Munoz et al., 2004). Once generated, ceramides act on different intracellular targets to induce apoptosis. One of these targets is protein kinase B (or Akt), a kinase that lies downstream of PI3-K, a major signaling pathway through which growth factors promote cell survival. Using two different experimental approaches, it was demonstrated that PI3-K was also a target of C1P (Gomez-Munoz et al., 2005). On one hand, PI3-K activation was demonstrated by immunoprecipitation of the enzyme from whole cell lysates and assayed in vitro using 32Pphosphatidylinositol. On the other hand, an in vivo approach provided evidence of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) formation in intact cells that were prelabeled with 32P-orthophosphate (Gomez-Munoz et al., 2005). PIP3 is a major product of PI3-K, and was shown to directly inhibit A-SMase (Testai et al., 2004). Therefore, it could be speculated that PI3-K activation might potentiate the inhibitory effect of C1P on A-SMase through generation of PIP3. C1P stimulated the phosphorylation of PKB, which was sensitive to inhibition by wortmannin or LY294002, thereby confirming that PI3-K was the enzyme responsible for its phosphorylation. These two PI3-K inhibitors also blocked the prosurvival effect of C1P, as expected (Gomez-Munoz et al., 2005). Another relevant finding was that C1P caused IkB phosphorylation and stimulation of the DNA binding activity of NF-kB in primary cultures of mouse macrophages (Gomez-Munoz et al., 2005). Of note, C1P up-regulated the expression of anti-apoptotic Bcl-XL, which is a downstream target of NFkB. The latter results provided the first evidence for a novel biological role of natural C1P in the regulation of cell survival by the PI3-K/PKB/NF-kB pathway in mammalian cells (Gomez-Munoz et al., 2005).

As mentioned above, C1P can be metabolized to ceramide by different phosphatases, and then further converted to sphingosine and S1P by the coordinated actions of ceramidases and sphingosine kinases. Therefore, it could be speculated that the effects of C1P might be mediated through C1P-derived metabolites. However, usually ceramides and C1P exert opposing effects, (i.e. on PLD activation, adenylyl cyclase inhibition, or Ca2+ mobilization), and C1P is not able to reproduce the effects of S1P (Gomez-Munoz et al., 1995a; Gomez-Munoz et al., 1995b; Gomez-Munoz et al., 1997; Gomez-Munoz, 1998). Also, ceramides can decrease the expression of Bcl-XL (Chalfant & Spiegel, 2005), whereas C1P causes its upregulation (Gomez-Munoz et al., 2005). Finally, no ceramidases capable of converting C1P into S1P have so far been reported to exist in mammalian cells, and S1P and C1P inhibit A-SMase through different mechanisms (Gomez-Munoz et al., 2003; Gomez-Munoz et al., 2004). Therefore, it can be concluded that C1P acts on its own right to regulate cell homeostasis. The above observations suggest that regulation of the enzyme activities involved in ceramide and C1P metabolism is essential for cell fate. Elucidation of the mechanisms controlling ceramide and C1P levels may help develop new molecular strategies for preventing metabolic disorders, or designing novel therapeutic agents for treatment of disease.
