12-Hydroxyheptadecatrienoic acid

12-Hydroxyheptadecatrienoic acid
Names
IUPAC name
(5Z,8E,10E,12S)-12-Hydroxy-5,8,10-heptadecatrienoic acid
Identifiers
54397-84-1
ChemSpider 4446265
Jmol-3D images Image
PubChem 5283141
Properties
Molecular formula
 C17H28O3
Molar mass 280.40 g·mol−1
Except where noted otherwise, data is given for materials in their standard state (at 25 °C (77 °F), 100 kPa)
Infobox references

12-Hydroxyheptadecatrenoic acid (12-HHT, 12-hydroxy-heptadecatrienoic acid) is a 17 carbon metabolite of the 20 carbon polyunsaturated fatty acid, arachidonic acid. It was first detected and structurally defined by P. Wlodawer, B. Samuelsson, and M. Hamberg as a product of arachidonic acid metabolism made by from the microsomeal (i.e. endoplasmic reticulum) fraction of sheep seminal vesicle glands and from intact human platelets.[1][2] 12-HHT is less ambiguously termed 12-(S)-hydroxy-5Z,8E,10E-heptadecatrienoic acid to indicate the S stereoisomerism of its 12-hydroxyl residue and the Z, E, and E cis-trans isomerism of its three double bonds. The metabolite was for many years thought to be merely a biologically inactive byproduct of prostaglandin synthesis. More recent studies, however, have attached potentially important activity to it.

Production

Primary source

Cyclooxygenase-1 and cyclooxygenase-2 metabolize arachidonic acid to the 15-hydroperoxy, 20 carbon prostaglandin (PG) intermediate, PGG2, and then to the 15-hydroxy, 20 carbon intermediate, prostaglandin H2 (PGH2). Thromboxane synthase further metabolizes PGH2 to the 20 carbon product, Thromboxane A2, the 17 carbon product, 12-HHT, and the 3 carbon product Malonyldialdehyde. Platelets express cycloxygenase and thromoxane synthase enzymes, producing PGG2, PGH2, and TXA2 in response to platelet aggregating agents such as thrombin; these metabolites act as autocrines by feeding back to promote further aggregation of their cells of origin and as paracrine by recruiting other platlets into the response as well as exerting other effects such as contracting nearby blood vessels.[3] 12-HHT is a particularly abundant product of these responses, accounting for about one third of the total amount of arachidonic acid metabolites formed by physiologically stimulated human platelets.[4] The cyclooxygenases and to a lesser extent thromboxane synthase enzymes are expressed by a wide range of cell types and tissues.

Other sources

Various cytochrome P450 enzymes (e.g. CYP1A1, CYP1A2, CYP1B1, CYP2E1, CYP2S1, and CYP3A4) metabolize PGG2 and PGH2 to 12-HHT and MDA.[5][6][7][8] While the latter studies were conducted using recombinant cytochrome enzymes or sub-fractions of disrupted cells, the human monocyte, a form of blood circulating leukocyte, increases its expression of CYP2S1 when forced to differentiate into a macrophage phenotype by interferon gamma or lipopolysaccharide (i.e. endotoxin) cytokines; associated with these changes, the differentiated macrophage metabolized arachidonic acid to 12-HHT by a CYP2S1-dependent mechanism.[9] Future studies, therefore may show that cytochromes are responsible for 12-HHT and MDA production in vivo.

PGH2, particularly in the presence of ferrous iron (FeII), ferric iron (FeIII), or hemin, rearranges non-enzymatically to a mixture of 12-HHT and 12-HHT's 8-cis isomer, i,e, 12-(S)-hydroxy-5Z,8Z,10E-heptadecatrienoic acid.[10][11][12][13] This non-enzymatic pathway may explain findings that cells can make 12-HHT in excess of TXA2 and also in the absence of active cycloxygenase and/or thromboxane synthase enzymes.[14]

Further metabolism

12-HHT is further metabolized by 15-hydroxyprostaglandin dehydrogenase (NAD+) in a wide variety of human and other vertebrate cells to its 12-oxo (also termed 12-keto) derivative, 12-oxo-5Z,8E,10E-heptadecatrienoic acid (12-oxo-HHT or 12-keto-HHT).[15][16][17][18] Pig kidney tissue also converted 12-HHT to 12-keto-5Z,8E-heptadecadienoic acid (12-oxo-5Z,8E-heptadecadienoic acid) and 12-hydroxy-heptadecadienoic acid.[19]

Acidic conditions (pH~1.1-1.5) cause 12-HHT to rearrange in a time- and temperature process to its to its 5-cis isomer, 12-hydroxy-5E,8E,10E-heptadecatrienoic acid.[20]

Activities and clinical significance

Early studies

Fourteen years after the first publication on its detection in 1973, 12-HHT was reported to stimulate fetal bovine aortic and human umbilical vein endotheleal cells to metabolize arachidonic acid to Prostacyclin I2 (PGI2), a powerful inhibitor of platelet activation and stimulator of Vasodilation (see Prostacyclin synthase); it did not, however, alter arachidonic acid metabolism in human platelets.[21] Shortly thereafter, 12-HHT was reported to inhibit the chemotaxis-blocking effect of a human monocyte-derived factor on human moncytes[22] while the immediate metabolite of 12-HHT, 12-oxo-HT was reported to stimulate the chemotasis of human neutrophils.[23] and to inhibit platelet aggregation responses to various agents by stimulating platelets to raise their levels of Cyclic adenosine monophosphate (cAMP), an intracellular signal that serves broadly to inhibit platelet activation.[24] These studies were largely overlooked; in 1998 and 2007 publications, for example, 12-HHT was regarded as either inactive or without significant biological activity.[25][26] Nonetheless, this early work suggested that 12-HHT may serve as a contributor to monocyte- and neutrophil-based inflammatory responses and 12-oxo-HT may serve as a counterpoise to platelet aggregation responses elicited or promoted by TXA2. Relevant to the latter activity, a later study showed that this inhibitory effect was due to the ability of 12-oxo-HT to act as a partial antagonist of the Thromboxane receptor: it blocks TXA2 binding to its receptor and thereby the responses of platelets and possibly other tissues to TXA2 as well as agents that depend on stimulating TXA2 production for their activity.[27] Thus, 12-HHT forms simultaneously with, and by stimulating PGI2 production, inhibits TXA2-mediated platelet activation responses while 12-oxo-HT blochs TXA2 receptor binding to block not only TXA2-induced thrombosis and blood clotting but possibly also vasospasm and other actions of TXA2. In this view, thromboxane synthase leads to the production of a broadly active arachidonic acid metabolite, TXA2, plus two other arachidonic acid metabolites, 12-HHT and 12-oxo-HT, that can serve indirectly to stimulate PGI2 production or directly as a receptor antagonist to moderate TXA2's action, respectively. This strategy may be essential to limit the potentially very deleterious thrombotic and vasospastic activities of TXA2.

12-HHT is a BLT2 receptor agonist

Leukotriene B4 (i.e. LTB4) is an arachidonic acid metabolite made by the 5-lipoxygenase enzyme pathway. It activates cells through both its high affinity (Dissociation constant [Kd] of 0.5-1.5 nM) Leukotriene B4 receptor 1 ('BLT1 receptor) and its low affinity BLT2 receptor (Kd=23 nM); both receptors are G protein coupled receptors that, when ligand-bound, activate cells by releasing the Gq alpha subunit and pertussis toxin-sensitive Gi alpha subunit from Heterotrimeric G proteins.[28][29] BLT1 receptor has a high degree of ligand-binding specificity: among a series of hydroxylated eicosanoid metabolites of arachidonic acid, it binds only LTB4, 20-hydroxy-LTB4, and 12-epi-LTB4; among this same series, BLT2 receptor has far less specificity in that it binds not only LTB4, 20-hydroxy-LTB4, and 12-epi-LTB4 but also 12(R)-HETE and 12(S)-HETE (i.e. the two stereoisomers of 12-Hydroxyeicosatetraenoic acid) and 15(S)-HpETE and 15(S)-HETE (i.e. the two stereoisomers of 15-Hydroxyicosatetraenoic acid).[30] The BLT2 receptor's relative affinities for finding LTB4, 12(S)-HETE, 12(S)-HpETE, 12(R)-HETE, 15(S)-HETE, and 20-hydroxy-LTB4 are ~100, 10, 10, 3, 3, and 1, respectively. All of these binding affinities are considered to be low and possibly indicating that some unknown ligand(s) might bind BLT2 with high affinity. In 2009, 12-HHT was found to bind with the BLT2 receptor with ~10-fold higher affinity than LTB4; 12-HHT did not bind to the BLT1 receptor.[31] Thus, the BLT1 receptor exhibits exquisite specificity, binding 5(S),12(R)-dihydroxy-6Z,8E,10E,14Z-eicosatetraenoic acid (i.e. LTB4) but not LTB4's 12(S) or 6Z isomers while the BLT2 receptor exhibits a promiscuous finding pattern.[32] Formyl peptide receptor 2 is a relevant and well-studied example of promiscuous receptors. Initially identified as a second and low affinity receptor for the neutrophil tripeptide chemotactic factor, N-formyl-met-leu-phe, subsequent studies showed that it was a high affinity receptor for the arachidonic acid metabolite, lipoxin A4, but also bound and was activated by a wide range of peptides, proteins, and other agents.[33] BLT2 may ultimately prove to have binding specificity for a similarly broad range of agents.

The production of LTB4 and expression of BLT1 by human tissues are largely limited to bone marrow-derived cells such as the neutrophil, eosinophil, mast cell, and various types of lymphocytes[34][35] and accordingly are regarded primarily as contributing to the many human defensive and pathological (ulcerative colitis, arthritis, asthma, etc.) Inflammatory responses which are mediated by these cell types. Drugs that inhibit LTB4 production or binding to BLT1 are in use or development for the latter diseases.[36][37][38] In contrast, production of 12-HH2 by the cyclooxygenase/thromboxane synthase pathway and the expression of BLT2 receptors by human tissues appears be far wider and more robust than that of the LTB4/BLT2 receptor axis.[39][40][41] Recent studies indicate that the role(s) of the 12-HHT/BLT2 receptor axis in human physiology and pathology may be very different than those of the LTB4/BLT1 receptor axis.

Recent studies on 12-HHT/BLT2 receptor activities

Inflammation

12-HHT stimulates chemotactic responses in mouse bone marrow mast cells, which naturally express BLT2 receptors, as well as in Chinese hamster ovary cell made to express these receptors by transfection but not in sham-transfected Chinese hamster ovary cells which normally do not express these receptors.[42] This finding suggest However, the 12-HHT/BLT2 receptor pathway may oppose rather than mimic or support the pro-inflammatory actions of the LTB4/BLT1 receptor pathway. The human skin immortalized keratinocyte cell line HaCaT, which expresses BLT2 receptors, responds to ultraviolet B (UVB) radiation by generating toxic reactive oxygen species which in turn cause these cells to die by activating apoptotic pathways in a BLT2 receptor-dependent reaction. Topical treatment of mouse epidermal skin with a BLT2 receptor antagonist, LY255283, protects against UVB radiation-induced apoptosis and BLT2-overexpressing transgenic mice exhibited significantly more extensive skin apoptosis in response to UVB irradiation.[43] Furthermore, 12-HHT inhibits HaCaT cells from synthesizing interleukin-6 (IL-6), a pro-inflammatory cytokine associated with cutaneous inflammation, in response to UVB radiation.[44] Opposition between the LTB4/BLT1 receptor and 12-HHT/BLT2 receptor pathways occurs in another setting. In a mice model of ovalbumin-induced allergic airway disease, 12-HHT and its companion cyclooxygenase metabolites, Prostaglandin E2 and Prostaglandin D2, but not 12 other lipoxygenase or cycloxygenase mebolites showed a statistically significantly increase in bronchoalveolar lavage fluid levels after intratracheal ovalbumin challenge; after this challenge, only 12-HHT, among the monitored BLT2 receptor-activating ligands (LTB4, the 12(S) stereoisomer of 12-HETE, and 15(S)-HETE) attained levels capable of activating BLT2 receptors. Also, BLT2 knockout mice exhibited a greatly enhanced response to ovalabumin challenge. Finally, BLT2 receptor expression was significantly reduced in allergy-regulating CD4+ T cells form patients with asthma compared to healthy control subjects.[45] Unlike LTB4 and its BLT1 receptors, which are implicated in contributing to allergen-based airway disease in mice and humans,[46] 12-HHT and BLT2 receptors appear to suppress this disease in mice and may do so in humans.[47][48] These findings recomment further studies to probe the role of 12-HHT and its BLT2 receptors in human neutrophil-based inflammation and eosinophil/mast cell-based allergic inflammatory diseases.

Wound healing

High dose aspirin treatemet (which inhibits cyclooxygenases to block 12-HHT production), thromboxane synthase knockout, and BLT2 receptor knockout but not TXA2 receptor knockout impair the closure by keratinocyte-based re-epithelialization of experimentally induced wounds in mice while a synthetic BLT2 receptor agonist accelerats wound closure not only in this mouse model but also in the db/db mouse model of obesity, diabetes, and dyslipidemia due to leptin receptor deficiency. 12-HHT accumulated in the wounds of the former mouse model. Companion studies using an in vitro scratch test assay indicated that 12-HHT stimulated human and mouse keratinocyte migration by a BLT2 receptor-dependent mechanism that involved the production of tumor necrosis factor α and metalloproteinases.[49] These results indicate that the 12-HHT/BLT2 receptor axis is a critical contributor to wound healing in mice and possibly humans. The axis operates by recruiting the movement of keratinocytes to close the wound. This mechanism may underlie the suppression of wound healing that may accompany the high dose intake of asperin or, based on mouse studies, other non-steroidal anti-inflammatory agents (NSAID) in humans.[50][51] Syntheic BLT2 agonists may be useful for speeding the healing of chronic ulcerative wounds, particularly in patients with, for example diabetics, that have impaired wound healing.[52][53][54]

Cancer

12-HHT and, based on studies using the TXA2 synthetic analog, I-BOP ([1S-(1 alpha,2 beta (5Z),3 alpha(1E,3S*),4 alpha)]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptan-2-yl]-5-heptenoic acid),[55] TXA2 stimulate the PC3 human prostate cancer cell line to activate several pro-growth and pro-survival signaling pathways: Protein kinase B (also termed AKT), Phosphoinositide 3-kinase, Protein kinase C, Proto-oncogene tyrosine-protein kinase Src, and, by causing proteolytic cleavage and release of the EGFR receptor ligand from HB-EGF), EGFR.[56] Additionally, cultured non-malignant PWR-1E prostate and PC3 cancer cells die by engaging suicidal apoptosis pathways when detached from surfaces, a process termed anoikis. This suicidal reaction is accompanied by an increase in the expression of BLT2 receptors, activation of NADPH oxidase (NOX), NOX-mediated increase in the production of Reactive oxygen species (ROS), and ROS-induced activation of the pro-survival transcription factor, NF-κB. The forced ectopic expression of the BLT2 receptor as well as the stimulation of this receptor by either 12(S)-HETE') or the synthetic BLT2 receptor agonist, CAY-10583, inhibited while BLT2 receptor mRNA Gene knockdown by mRNA interference as well as pharmacological inhibition of this receptor by LY255283 enhanced these cells' anoikis response to surface detachment.[57] Finally, LNCaP and CWR22rv-1 human prostate cancer cell lines, unlike PC-3 cells, require exogenous androgen for their survival and thereby mimic the androgen dependency shown by human prostate cancers in their early, untreated stage. Both cell lines overexpress BLT2 receptors compared to the PWR-1E non-malignant human prostate cell line. Treatment with BLT2 receptor antagonist Ly255283 caused both cell lines to become apoptotic and BLT2 receptor mRNA knockdown caused LNCaP but not PWR-1E cells to become apoptotic. These responses were due to the loss of BLT2 constitutional activity in activating NOX4 which in turn generate reactive oxygen species that activate NF-κB to up-regulate the expression of androgen receptors.[58] 12-HETE has also been shown to increase the survival of PC-3 cells by maintaining high levels of phosphorylated Rb Retinoblastoma protein, an effect which reduces the ability of retinoblastoma protein to inhibit the synthesis of DNA and thereby cell division.[59] These results suggest that 12-HHT can act through BLT4 receptors to promote the the survival and growth of human prostate cancer cells in vitro and may act similarly in the human disease.

Many human cancers overexpess BLT2 receptors when compared to their normal tissue counterparts. These include thyroid gland follicular carcinoma, kidney renal cell carcinoma, bladder transitional cell carcinoma, esophagus squamous cell carcinoma, colon adenocarcinoma, ovary serous carcinoma, uterus cervix carcinoma, and pancreatic ductal carcinoma.[60] The human mammary epethelial MCF-10A cell line expresses BLT2 protein.[61][62] Similar to prostate cancers, these latter as well as other cancers in which BLT2 receptors have been suggested to drive survival, growth, and metastasis (see Leukotriene B4 receptor 2), the offending ligand requires identification. As the strongest known activator of BLT2 receptors, 12-HHT presents the possibility of being this ligand for many of these cancers.

See also

References

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