[Frontiers in Bioscience 2, d482-500, October 1, 1997]

	[Frontiers in Bioscience 2, d482-500, October 1, 1997] Reprints PubMed CAVEAT LECTOR
	PROSTAGLANDINS AND CANCER Susan M. Fischer The University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville, TX 78957 Received 9/3/97 Accepted 9/23/97 3. THE ARACHIDONATE CASCADE 3.1. Metabolic Pathways Arachidonic acid (5,8,11,14 eicosatetraenoic acid; 20:4n-6) is found esterified in membrane phospholipids and triglycerides in all mammalian tissues. In this bound form, arachidonic acid is not usually a substrate for metabolizing enzymes. The esterification of arachidonic acid in phospholipids has been postulated to serve both the function of sustaining membrane fluidity and of substrate storage (6). Oxidative metabolism of arachidonic acid to PGs, hydroxy-fatty acids and leukotrienes, collectively referred to as eicosanoids, depends on the availability of free, nonesterified fatty acid. Arachidonic acid is readily released as the free fatty acid by one or more of the phospholipase A₂s (PLA₂), which by definition hydrolyze the ester linkage at the sn-2 position of a phospholipid. In many respects, PLA₂s are among the more important enzymes involved in eicosanoid synthesis because the availability of free arachidonic acid is believed to be one of the rate-limiting steps for the formation of all the eicosanoids. Because eicosanoids are involved in a number of patho-physiological conditions in addition to carcinogenesis, an understanding of the mechanisms by which PLA₂ activity is regulated is of great interest, and is discussed briefly below. A second pathway for arachidonic acid release is via phospholipase C, which hydrolyzes the head group function of phospholipids and thus yields diacylglycerol (DAG). A DAG lipase can then release arachidonic acid in a subsequent reaction. In many tissues including murine keratinocytes both phospholipases A₂ and C appear to be operative following treatment with irritating agents such as tumor promoters (7). The levels of free arachidonic acid are normally very low since the liberated fatty acid is rapidly metabolized. Free arachidonic acid is a substrate for two distinctively different enzymatic pathways, one leading to the synthesis of PGs, the other to the hydroperoxy- and hydroxy-eicosatetraenoic acids referred to as HPETEs and HETEs, respectively. The two pathways have in common the insertion of molecular oxygen into the fatty acid, although PGs have, in addition, a cyclopentane ring. Details of the enzymatic reactions involved in arachidonic acid metabolism have recently been reviewed (8). Briefly, the enzymes responsible for the production of PGs are referred to as PG synthetases, which contain two active sites. The cyclooxygenase (COX) moiety introduces two molecules of oxygen into arachidonic acid to form the hydroperoxy endoperoxide, PGG₂, which is then reduced by the endoperoxidase moiety of the enzyme to the hydroxy endoperoxide, PGH₂. Prostaglandin synthetases or cyclooxygenases, as they are commonly called, are the target of many PG synthesis inhibitors, particularly the nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin and indomethacin (8). These inhibitors have been particularly useful in demonstrating an active role for eicosanoids in many physiological and pathological conditions. Their use in studies on the involvement of eicosanoids in tumor development will be described below. The intermediate endoperoxide generated by PG synthetase, PGH₂, is the substrate for several enzymes. The PG endoperoxide E and D isomerases produce PGE₂ and PGD_2, respectively (9, 10). Additional dehydration and isomerization of PGE₂ produce PGA₂, PGB₂ and PGC₂. PGF_2alpha arises either via nonenzymatic reduction of PGH₂ or from PGE₂ by way of a 9-keto-reductase enzyme (8). The endoperoxides can also give rise to the thromboxanes by way of thromboxane synthetase and to prostacyclin by way of an oxy-cyclase (11). The second major arachidonic acid metabolic pathway involves the lipoxygenases. These enzymes give rise to hydroperoxy products which can be reduced by glutathione peroxidase to hydroxy forms. A half dozen or so different lipoxygenases have been described as reviewed recently by Funk (12), each responsible for the insertion of molecular oxygen at a particular carbon. Hence, the 5-lipoxygenase generates the 5-HPETE, the 12-lipoxygenase, 12-HPETE, and so forth. The 5-HPETE is noteworthy in that it is the only HPETE that can be further metabolized to the leukotrienes, which historically have been referred to as the slow-reacting substances of anaphylaxis (13). Mammalian lipoxygenases are of the (S) type, i.e., they generate (S) hydroperoxy fatty acids (14). This feature has been used in many cases to distinguish enzymatic from nonenzymatic metabolism in which the (R) form is also produced. Arachidonic acid may also be metabolized by cytochrome P-450 type enzyme systems to produce a number of hydroxy and carboxy products, including the 19-hydroxy, 19-oxo, 20-hydroxy and 20-carboxy metabolites (15). With the possible exception of the formation of the 12 (R)-HETE in psoriatic scales, cytochrome P-450 derived products are probably of minimal importance in such tissues as skin(8), in part, because of the extremely low levels of cytochrome P-450 in normal skin and the suppression of carcinogen-induced cytochrome, P-450, cyp1a-1, by tumor promoting phorbol esters (16).However, in tissues such as the kidney and cornea, cytochrome P450 metabolism results in biologically active products that play a role in the physiology of these tissues. Arachidonic acid is not the only fatty acid that can be metabolized by PG synthetase and/or the lipoxygenases. Since fish oils that contain the n-3 fatty acids, eicosapentaenoic and docosahexaenoic acids, have been shown to reduce tumor development at several organ sites (18, 19), there has been considerable interest in the nature and biological activity of their metabolites. In the skin, eicosapentaenoic acid has been shown to compete with arachidonic acid for incorporation into cell membrane phospholipids (20). This results in both competitive inhibition of arachidonic acid metabolism as well as the production of such metabolites as PGE₃, PGD₃, and 12- and 15-hydroxyeicosapentaenoic acids (HEPES) (20).These metabolites are biologically less active than the corresponding arachidonic acid metabolites and presumably compete with them for receptor occupancy. The 15-lipoxygenase, distributed widely among various types of tissues, converts arachidonic acid to 15-HPETE which can then be reduced by glutathione peroxidase to 15-HETE (8). However, the preferential endogenous substrate is believed to be linoleic acid (18:2n-6) leading to the production of 13-hydroxyoctadecadienoic acid (13-HODE). For some organs this is significant because, for example, the upper layers of the epidermis contain high levels of linoleate-rich sphingolipids that contribute to the barrier function of the skin (21). Murine keratinocytes are also capable of synthesizing 9-HODE from linoleic acid (unpublished data); the function or activity of this eicosanoid is unknown at this time. Unlike eicosapentaenoic acid, linoleic acid cannot be metabolized by COX to PG-like products (22). The significance of the production of non-arachidonate eicosanoids to the function of skin or other organs is not well understood; however, the ability of some of them to modulate tumorigenesis indicates that they may, be at least as important as the arachidonate metabolites. This is an area in need of additional investigation. In addition to enzymatically generated products, arachidonic acid can also be converted to PG-like compounds referred to as isoprostanes (see reference 23 for review). Isoprostanes are generated by free radical catalyzed peroxidation of lipids and are found in vivo in both esterified and unesterified forms. Because isoprostanes are isomeric to COX-derived PGs it was thought that they should exert biological effects. Recently it has been shown that 8-iso-PGE₂ and 8-iso-PGF_2alpha are in fact potent vasoconstrictors in the renal vascular bed and are associated with a number of pathophysiological processes in this and other tissues (23). 3.2. Regulation of Metabolism 3.2.1. Phospholipase A₂ The regulatory mechanisms governing PLA₂ activation are not well understood; this is largely because of the number of different mechanisms that can elicit activity, and because of the recent identification of several different types of PLA₂s. An understanding of the mechanisms of activation and regulation of PLA₂ activity is crucial because this determines the types and amounts of fatty acid released, which in turn is rate-limiting for the production of biologically active eicosanoids. Several families of PLA₂ have been identified in mammalian tissues: the low-molecular weight (14kD) types I and II PLA₂, referred to as secretory or sPLA₂, and the high-molecular weight (85kD) cytosolic PLA₂ (cPLA₂) (24, 25). The sPLA₂s differ from the cPLA₂ in several significant ways. sPLA₂s are found either in the membrane or extracellularly and, with regards to both phospholipid and sn-2 fatty acid, have a broad range of substrate preferences (24).In tissues where they have been studied, activation of sPLA₂ has been observed to occur through at least two distinct mechanisms: one mediated by elevated cAMP and the other by the inflammatory cytokines, interleukin-1 and tumor necrosis factor-alpha (26). It has been suggested that cell-associated sPLA₂ functions primarily in maintaining cell membrane homeostasis, extracellular sPLA₂ plays a role in inflammatory diseases, while cPLA₂ may be involved in the initiation of the inflammatory response (24). Due to its preference for arachidonic acid over other fatty acids, the activation of cPLA_2, in particular, may be critical for subsequent eicosanoid biosynthesis. Among the PLA₂s, the mechanisms by which cPLA₂ is activated is the best understood. Several kinds of regulatory mechanisms have been reported, including phopsphorylation, interaction with calcium, and receptor-induced interaction with GTP-binding proteins (27). Growth factor stimulation of cells has been reported to result in increased phosphorylation of cPLA₂ on serine residues by either p42 MAP kinase or protein kinase C (28-30). Specific regulators of PLA₂ have also been investigated; lipocortins (members of the annexin family of molecules) have been touted as negative modulators, while PLA₂-activating-protein (PLAP) has been suggested to be a positive regulator. Glucocorticoids have been linked to an increase in the amount of lipocortin, a protein with anti-PLA₂ activity (31). Glucocorticoids are known to suppress both arachidonic acid release and inflammation; this is believed to, at least, partially explain their mechanism of action as anti-tumor promoters in the mouse skin carcinogenesis model (32). However, the anti-inflammatory activity of lipocortin has not always been conclusive and appears to occur only in in vitro assays (31). Based on sequence, six members of this protein super-family have now been identified. A physiological role, however, has not been established for any of them and it was concluded that there is not sufficient evidence for a negative regulatory role for lipocortins (31). PLAP, which has homology to melittin, has recently been isolated and identified (33). PLAP increases PLA₂activity by increasing the V_max of a phosphatidylcholine-specific PLA₂(33). The importance of PLAP to eicosanoid synthesis was recently demonstrated by showing that stimulation by leukotriene D₄ of smooth muscle and endothelial cells resulted in increased PLAP mRNA and that the antisense cDNA for PLAP effectively blocked the activation of PLA₂(34). 3.2.2. Prostaglandin Synthases/Cyclooxygenases Although originally thought to be one enzyme, it has recently been shown that there are two PG synthetases, referred to as PG synthetase-1 and -2, or COX-1 and COX-2, respectively (24, 35-37).These two distinct gene products exhibit similar COX and peroxidase activities although they are differentially regulated (24, 35-37). While a variety of factors, including serum, growth factors and phorbol esters, can upregulate the mRNA levels for both COX-1 and COX-2, generally COX-2 responds in a much more dramatic fashion and thus has been referred to as a phorbol ester-inducible immediate early gene product.The molecular biology, including DNA and amino acid sequences of the PG synthetases, has recently been well reviewed and described (24, 35-37). While it has long been a tenet that the availability of free arachidonic acid controls PG synthesis, recent evidence suggests that significant regulation also occurs at the level of COX gene expression (36). For example, treatment of mouse skin or cultured keratinocytes with phorbol ester tumor promoters results in high PG production which could be due to either elevated substrate levels (phorbol esters activate cPLA₂) or increased expression of COX-1 or COX-2. Using cultured keratinocytes, phorbol esters were shown to significantly increase mRNA and protein levels for COX-2 but not for COX-1 (38). When COX-2 activity was inhibited with the selective inhibitor, NS-398, PG synthesis was reduced to control levels. Thus, induction of COX-2 appears to be the primary determinant of the increased PGE₂ in stimulated keratinocytes. These observations are similar to those reported for endothelial cells where phorbol esters were shown also to induce de novo synthesis of COX. This induction appears to be required for continued high levels of PG synthesis in the face of high levels of autoinactivation (39). 3.2.3. Lipoxygenases The lipoxygenase products are considered to be biologically at least as important as the PGs because of their mediation of many aspects of inflammation. There are only limited reports on the regulation of specific lipoxygenases and in the context of cancer development this has been restricted primarily to the mouse skin model. Here, phorbol ester treatment of mouse skin was shown to have little effect on the 5-, 12- or 15-lipoxygenase pathways; however, a cytosolic 8-lipoxygenase activity was strongly induced (40). The 8(S)-lipoxygenase was recently cloned, allowing the demonstration that expression at the mRNA and protein level was greatly enhanced by phorbol esters (Alan Brash, Vanderbilt University; personal communication). While the exact function of 8-HPETE or 8-HETE is unknown, its importance in tumor promoter-elicited events is suggested by the finding that (i) an induction of 8-lipoxygenase or hyperplasia is not observed in the phorbol ester-resistant C57BL/6J mouse, but is in sensitive mice (40, 41)and (ii) application of the lipoxygenase inhibitor eicosatetraynoic acid maximally inhibits tumor promotion when applied at the time (18 h) of maximum induction of 8-lipoxygenase (42). Although papillomas and carcinomas show dramatically elevated levels, compared to normal skin, of both 8-HETE and 12-HETE, it is not yet known whether this is due to increased expression of the respective enzymes or increased substrate availability (43). 3.3. Receptors Elucidation of the function of the individual eicosanoids has been hampered by the fact that the same PGs often elicit opposing effects in different cells. This observation led to the search for receptors for the individual PGs, as well as other arachidonate metabolites. As described in detail in several recent reviews (44-46), specific receptors have been shown to exist for the PGs; the action of the PGs depends on the cell type, metabolic activity, location, and state of stimulation by other agonists (47). Three major subtypes of the PGE₂ receptor have so far been identified: EP₁, EP₂ and EP₃(48). The EP₃ receptor, which belongs to the seven-transmembrane-domain family (49), is currently the most well characterized; the mouse EP₃ receptor has been cloned and shown to inhibit adenylate cyclase via G protein coupling (50). Namba et al. (49) have recently shown that there are at least four isoforms of the EP₃ receptor, all produced by alternative splicing, and differing only at their carboxy-terminal tails. This allows for activation of different second messenger systems because of coupling to different G proteins (49). For example, PGE₂ causes down-regulation of EP_3alpha, but not EP_3beta, by sequestering it away form the cell surface (48). The EP₂ receptor for PGE₂ has also seven-transmembrane domains; unlike the EP₃ receptor family, it activates adenylate cyclase activity (51). Very recently, at least four isoforms of EP₂ have been identified, which are the result of alternative splicing (52). Activation of the third PGE receptor subtype, EP₁, can cause a rise in intracellular calcium concentration (44). Receptors have also been identified for thromboxane A₂, prostacyclin, PGF_2alpha , and PGD₂; the signaling pathwaysthey activate are slowly being elucidated (44-46). With regards to the lipoxygenase products, several receptors have been identified, eg., specific leukotriene C₄ receptors have been identified that mediate its mitogenic activity in cutaneous psoriatic lesions (53). Binding sites for 12-HETE have also been found in a human squamous cell carcinoma cell line, although the binding protein(s) has not been isolated or characterized (54, 55). Currently, little is known about whether or how the expression of specific receptors is regulated. There are several reports that suggest that, at least some receptors, can be regulated by exogenous agents. For example, the binding of 12-HETE was found to be inhibited by exposure to ultraviolet-B light exposure (54, 55). Recently Cameron et al. (56) reported that application of phorbol ester to mouse keratinocytes dramatically reduced the number of apparent PGE₂ binding sites, with little change in affinity (56). This dearth of information on modulation of receptor binding points to a clear need to characterize the eicosanoid receptors in normal and pathological states, with regards to distribution, function and regulation. While the classical PG receptors are cell surface receptors, a class of nuclear proteins may also function as receptors for at least some of the eicosanoids. The peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily that includes receptors for the steroid and retinoid hormones. PPARs, which affect the expression of many genes, and particularly those involved in the catabolism and storage of fatty acids, appear to represent another pathway or mechanism through which some eicosanoids exert their biological effects (57-59).

[Frontiers in Bioscience 2, d482-500, October 1, 1997]
Reprints
PubMed
CAVEAT LECTOR

PROSTAGLANDINS AND CANCER

Susan M. Fischer

The University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville, TX 78957

Received 9/3/97 Accepted 9/23/97

3. THE ARACHIDONATE CASCADE

3.1. Metabolic Pathways

Arachidonic acid (5,8,11,14 eicosatetraenoic acid; 20:4n-6) is found esterified in membrane phospholipids and triglycerides in all mammalian tissues. In this bound form, arachidonic acid is not usually a substrate for metabolizing enzymes. The esterification of arachidonic acid in phospholipids has been postulated to serve both the function of sustaining membrane fluidity and of substrate storage (6). Oxidative metabolism of arachidonic acid to PGs, hydroxy-fatty acids and leukotrienes, collectively referred to as eicosanoids, depends on the availability of free, nonesterified fatty acid. Arachidonic acid is readily released as the free fatty acid by one or more of the phospholipase A₂s (PLA₂), which by definition hydrolyze the ester linkage at the sn-2 position of a phospholipid. In many respects, PLA₂s are among the more important enzymes involved in eicosanoid synthesis because the availability of free arachidonic acid is believed to be one of the rate-limiting steps for the formation of all the eicosanoids. Because eicosanoids are involved in a number of patho-physiological conditions in addition to carcinogenesis, an understanding of the mechanisms by which PLA₂ activity is regulated is of great interest, and is discussed briefly below.

A second pathway for arachidonic acid release is via phospholipase C, which hydrolyzes the head group function of phospholipids and thus yields diacylglycerol (DAG). A DAG lipase can then release arachidonic acid in a subsequent reaction. In many tissues including murine keratinocytes both phospholipases A₂ and C appear to be operative following treatment with irritating agents such as tumor promoters (7).

The levels of free arachidonic acid are normally very low since the liberated fatty acid is rapidly metabolized. Free arachidonic acid is a substrate for two distinctively different enzymatic pathways, one leading to the synthesis of PGs, the other to the hydroperoxy- and hydroxy-eicosatetraenoic acids referred to as HPETEs and HETEs, respectively. The two pathways have in common the insertion of molecular oxygen into the fatty acid, although PGs have, in addition, a cyclopentane ring. Details of the enzymatic reactions involved in arachidonic acid metabolism have recently been reviewed (8). Briefly, the enzymes responsible for the production of PGs are referred to as PG synthetases, which contain two active sites. The cyclooxygenase (COX) moiety introduces two molecules of oxygen into arachidonic acid to form the hydroperoxy endoperoxide, PGG₂, which is then reduced by the endoperoxidase moiety of the enzyme to the hydroxy endoperoxide, PGH₂. Prostaglandin synthetases or cyclooxygenases, as they are commonly called, are the target of many PG synthesis inhibitors, particularly the nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin and indomethacin (8). These inhibitors have been particularly useful in demonstrating an active role for eicosanoids in many physiological and pathological conditions. Their use in studies on the involvement of eicosanoids in tumor development will be described below.

The intermediate endoperoxide generated by PG synthetase, PGH₂, is the substrate for several enzymes. The PG endoperoxide E and D isomerases produce PGE₂ and PGD_2, respectively (9, 10). Additional dehydration and isomerization of PGE₂ produce PGA₂, PGB₂ and PGC₂. PGF_2alpha arises either via nonenzymatic reduction of PGH₂ or from PGE₂ by way of a 9-keto-reductase enzyme (8). The endoperoxides can also give rise to the thromboxanes by way of thromboxane synthetase and to prostacyclin by way of an oxy-cyclase (11).

The second major arachidonic acid metabolic pathway involves the lipoxygenases. These enzymes give rise to hydroperoxy products which can be reduced by glutathione peroxidase to hydroxy forms. A half dozen or so different lipoxygenases have been described as reviewed recently by Funk (12), each responsible for the insertion of molecular oxygen at a particular carbon. Hence, the 5-lipoxygenase generates the 5-HPETE, the 12-lipoxygenase, 12-HPETE, and so forth. The 5-HPETE is noteworthy in that it is the only HPETE that can be further metabolized to the leukotrienes, which historically have been referred to as the slow-reacting substances of anaphylaxis (13). Mammalian lipoxygenases are of the (S) type, i.e., they generate (S) hydroperoxy fatty acids (14). This feature has been used in many cases to distinguish enzymatic from nonenzymatic metabolism in which the (R) form is also produced.

Arachidonic acid may also be metabolized by cytochrome P-450 type enzyme systems to produce a number of hydroxy and carboxy products, including the 19-hydroxy, 19-oxo, 20-hydroxy and 20-carboxy metabolites (15). With the possible exception of the formation of the 12 (R)-HETE in psoriatic scales, cytochrome P-450 derived products are probably of minimal importance in such tissues as skin(8), in part, because of the extremely low levels of cytochrome P-450 in normal skin and the suppression of carcinogen-induced cytochrome, P-450, cyp1a-1, by tumor promoting phorbol esters (16).However, in tissues such as the kidney and cornea, cytochrome P450 metabolism results in biologically active products that play a role in the physiology of these tissues.

Arachidonic acid is not the only fatty acid that can be metabolized by PG synthetase and/or the lipoxygenases. Since fish oils that contain the n-3 fatty acids, eicosapentaenoic and docosahexaenoic acids, have been shown to reduce tumor development at several organ sites (18, 19), there has been considerable interest in the nature and biological activity of their metabolites. In the skin, eicosapentaenoic acid has been shown to compete with arachidonic acid for incorporation into cell membrane phospholipids (20). This results in both competitive inhibition of arachidonic acid metabolism as well as the production of such metabolites as PGE₃, PGD₃, and 12- and 15-hydroxyeicosapentaenoic acids (HEPES) (20).These metabolites are biologically less active than the corresponding arachidonic acid metabolites and presumably compete with them for receptor occupancy.

The 15-lipoxygenase, distributed widely among various types of tissues, converts arachidonic acid to 15-HPETE which can then be reduced by glutathione peroxidase to 15-HETE (8). However, the preferential endogenous substrate is believed to be linoleic acid (18:2n-6) leading to the production of 13-hydroxyoctadecadienoic acid (13-HODE). For some organs this is significant because, for example, the upper layers of the epidermis contain high levels of linoleate-rich sphingolipids that contribute to the barrier function of the skin (21). Murine keratinocytes are also capable of synthesizing 9-HODE from linoleic acid (unpublished data); the function or activity of this eicosanoid is unknown at this time. Unlike eicosapentaenoic acid, linoleic acid cannot be metabolized by COX to PG-like products (22). The significance of the production of non-arachidonate eicosanoids to the function of skin or other organs is not well understood; however, the ability of some of them to modulate tumorigenesis indicates that they may, be at least as important as the arachidonate metabolites. This is an area in need of additional investigation.

In addition to enzymatically generated products, arachidonic acid can also be converted to PG-like compounds referred to as isoprostanes (see reference 23 for review). Isoprostanes are generated by free radical catalyzed peroxidation of lipids and are found in vivo in both esterified and unesterified forms. Because isoprostanes are isomeric to COX-derived PGs it was thought that they should exert biological effects. Recently it has been shown that 8-iso-PGE₂ and 8-iso-PGF_2alpha are in fact potent vasoconstrictors in the renal vascular bed and are associated with a number of pathophysiological processes in this and other tissues (23).

3.2. Regulation of Metabolism

3.2.1. Phospholipase A₂

The regulatory mechanisms governing PLA₂ activation are not well understood; this is largely because of the number of different mechanisms that can elicit activity, and because of the recent identification of several different types of PLA₂s. An understanding of the mechanisms of activation and regulation of PLA₂ activity is crucial because this determines the types and amounts of fatty acid released, which in turn is rate-limiting for the production of biologically active eicosanoids. Several families of PLA₂ have been identified in mammalian tissues: the low-molecular weight (14kD) types I and II PLA₂, referred to as secretory or sPLA₂, and the high-molecular weight (85kD) cytosolic PLA₂ (cPLA₂) (24, 25). The sPLA₂s differ from the cPLA₂ in several significant ways. sPLA₂s are found either in the membrane or extracellularly and, with regards to both phospholipid and sn-2 fatty acid, have a broad range of substrate preferences (24).In tissues where they have been studied, activation of sPLA₂ has been observed to occur through at least two distinct mechanisms: one mediated by elevated cAMP and the other by the inflammatory cytokines, interleukin-1 and tumor necrosis factor-alpha (26). It has been suggested that cell-associated sPLA₂ functions primarily in maintaining cell membrane homeostasis, extracellular sPLA₂ plays a role in inflammatory diseases, while cPLA₂ may be involved in the initiation of the inflammatory response (24).

Due to its preference for arachidonic acid over other fatty acids, the activation of cPLA_2, in particular, may be critical for subsequent eicosanoid biosynthesis. Among the PLA₂s, the mechanisms by which cPLA₂ is activated is the best understood. Several kinds of regulatory mechanisms have been reported, including phopsphorylation, interaction with calcium, and receptor-induced interaction with GTP-binding proteins (27). Growth factor stimulation of cells has been reported to result in increased phosphorylation of cPLA₂ on serine residues by either p42 MAP kinase or protein kinase C (28-30).

Specific regulators of PLA₂ have also been investigated; lipocortins (members of the annexin family of molecules) have been touted as negative modulators, while PLA₂-activating-protein (PLAP) has been suggested to be a positive regulator. Glucocorticoids have been linked to an increase in the amount of lipocortin, a protein with anti-PLA₂ activity (31). Glucocorticoids are known to suppress both arachidonic acid release and inflammation; this is believed to, at least, partially explain their mechanism of action as anti-tumor promoters in the mouse skin carcinogenesis model (32). However, the anti-inflammatory activity of lipocortin has not always been conclusive and appears to occur only in in vitro assays (31). Based on sequence, six members of this protein super-family have now been identified. A physiological role, however, has not been established for any of them and it was concluded that there is not sufficient evidence for a negative regulatory role for lipocortins (31).

PLAP, which has homology to melittin, has recently been isolated and identified (33). PLAP increases PLA₂activity by increasing the V_max of a phosphatidylcholine-specific PLA₂(33). The importance of PLAP to eicosanoid synthesis was recently demonstrated by showing that stimulation by leukotriene D₄ of smooth muscle and endothelial cells resulted in increased PLAP mRNA and that the antisense cDNA for PLAP effectively blocked the activation of PLA₂(34).

3.2.2. Prostaglandin Synthases/Cyclooxygenases

Although originally thought to be one enzyme, it has recently been shown that there are two PG synthetases, referred to as PG synthetase-1 and -2, or COX-1 and COX-2, respectively (24, 35-37).These two distinct gene products exhibit similar COX and peroxidase activities although they are differentially regulated (24, 35-37). While a variety of factors, including serum, growth factors and phorbol esters, can upregulate the mRNA levels for both COX-1 and COX-2, generally COX-2 responds in a much more dramatic fashion and thus has been referred to as a phorbol ester-inducible immediate early gene product.The molecular biology, including DNA and amino acid sequences of the PG synthetases, has recently been well reviewed and described (24, 35-37).

While it has long been a tenet that the availability of free arachidonic acid controls PG synthesis, recent evidence suggests that significant regulation also occurs at the level of COX gene expression (36). For example, treatment of mouse skin or cultured keratinocytes with phorbol ester tumor promoters results in high PG production which could be due to either elevated substrate levels (phorbol esters activate cPLA₂) or increased expression of COX-1 or COX-2. Using cultured keratinocytes, phorbol esters were shown to significantly increase mRNA and protein levels for COX-2 but not for COX-1 (38). When COX-2 activity was inhibited with the selective inhibitor, NS-398, PG synthesis was reduced to control levels. Thus, induction of COX-2 appears to be the primary determinant of the increased PGE₂ in stimulated keratinocytes. These observations are similar to those reported for endothelial cells where phorbol esters were shown also to induce de novo synthesis of COX. This induction appears to be required for continued high levels of PG synthesis in the face of high levels of autoinactivation (39).

3.2.3. Lipoxygenases

The lipoxygenase products are considered to be biologically at least as important as the PGs because of their mediation of many aspects of inflammation. There are only limited reports on the regulation of specific lipoxygenases and in the context of cancer development this has been restricted primarily to the mouse skin model. Here, phorbol ester treatment of mouse skin was shown to have little effect on the 5-, 12- or 15-lipoxygenase pathways; however, a cytosolic 8-lipoxygenase activity was strongly induced (40). The 8(S)-lipoxygenase was recently cloned, allowing the demonstration that expression at the mRNA and protein level was greatly enhanced by phorbol esters (Alan Brash, Vanderbilt University; personal communication). While the exact function of 8-HPETE or 8-HETE is unknown, its importance in tumor promoter-elicited events is suggested by the finding that (i) an induction of 8-lipoxygenase or hyperplasia is not observed in the phorbol ester-resistant C57BL/6J mouse, but is in sensitive mice (40, 41)and (ii) application of the lipoxygenase inhibitor eicosatetraynoic acid maximally inhibits tumor promotion when applied at the time (18 h) of maximum induction of 8-lipoxygenase (42). Although papillomas and carcinomas show dramatically elevated levels, compared to normal skin, of both 8-HETE and 12-HETE, it is not yet known whether this is due to increased expression of the respective enzymes or increased substrate availability (43).

3.3. Receptors

Elucidation of the function of the individual eicosanoids has been hampered by the fact that the same PGs often elicit opposing effects in different cells. This observation led to the search for receptors for the individual PGs, as well as other arachidonate metabolites. As described in detail in several recent reviews (44-46), specific receptors have been shown to exist for the PGs; the action of the PGs depends on the cell type, metabolic activity, location, and state of stimulation by other agonists (47). Three major subtypes of the PGE₂ receptor have so far been identified: EP₁, EP₂ and EP₃(48). The EP₃ receptor, which belongs to the seven-transmembrane-domain family (49), is currently the most well characterized; the mouse EP₃ receptor has been cloned and shown to inhibit adenylate cyclase via G protein coupling (50). Namba et al. (49) have recently shown that there are at least four isoforms of the EP₃ receptor, all produced by alternative splicing, and differing only at their carboxy-terminal tails. This allows for activation of different second messenger systems because of coupling to different G proteins (49). For example, PGE₂ causes down-regulation of EP_3alpha, but not EP_3beta, by sequestering it away form the cell surface (48).

The EP₂ receptor for PGE₂ has also seven-transmembrane domains; unlike the EP₃ receptor family, it activates adenylate cyclase activity (51). Very recently, at least four isoforms of EP₂ have been identified, which are the result of alternative splicing (52). Activation of the third PGE receptor subtype, EP₁, can cause a rise in intracellular calcium concentration (44).

Receptors have also been identified for thromboxane A₂, prostacyclin, PGF_2alpha , and PGD₂; the signaling pathwaysthey activate are slowly being elucidated (44-46). With regards to the lipoxygenase products, several receptors have been identified, eg., specific leukotriene C₄ receptors have been identified that mediate its mitogenic activity in cutaneous psoriatic lesions (53). Binding sites for 12-HETE have also been found in a human squamous cell carcinoma cell line, although the binding protein(s) has not been isolated or characterized (54, 55).

Currently, little is known about whether or how the expression of specific receptors is regulated. There are several reports that suggest that, at least some receptors, can be regulated by exogenous agents. For example, the binding of 12-HETE was found to be inhibited by exposure to ultraviolet-B light exposure (54, 55). Recently Cameron et al. (56) reported that application of phorbol ester to mouse keratinocytes dramatically reduced the number of apparent PGE₂ binding sites, with little change in affinity (56). This dearth of information on modulation of receptor binding points to a clear need to characterize the eicosanoid receptors in normal and pathological states, with regards to distribution, function and regulation.

While the classical PG receptors are cell surface receptors, a class of nuclear proteins may also function as receptors for at least some of the eicosanoids. The peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily that includes receptors for the steroid and retinoid hormones. PPARs, which affect the expression of many genes, and particularly those involved in the catabolism and storage of fatty acids, appear to represent another pathway or mechanism through which some eicosanoids exert their biological effects (57-59).