MINIREVIEW

Structure, biochemistry and biology of hepoxilins

An update

Santosh Nigam, Maria-Patapia Zafiriou, Rupal Deva, Roberto Ciccoli andRenate Roux-Van der Merwe*

Eicosanoid & Lipid Research Division and Centre for Experimental Gynecology & Breast Research, Charite – University Medical Centre

Benjamin Franklin, Berlin, Germany

Keywords

glutathione peroxidases; hepoxilin A3;

hepoxilin A3 synthase; insulin secretion;

IRE1a; lung fibrosis, RINm5F cells

Correspondence

S. Nigam, Eicosanoid & Lipid Research

Division and Centre for Experimental

Gynecology & Breast Research, Charite –

University Medical Centre Benjamin

Franklin, D-12200 Berlin, Germany

Fax ⁄ Tel: +49 30 8445 2467

E-mail: santosh.nigam@charite.de

*Present address

Department of Food Technology, Tschwane

University of Technology, Pretoria, South

Africa

(Received 12 Janurary 2007, revised 30 April

2007, accepted 29 May 2007)

doi:10.1111/j.1742-4658.2007.05910.x

Hepoxilins are biologically relevant epoxy-hydroxy eicosanoids synthesized

through the 12S-lipoxygenase (12S-LOX) pathway of the arachidonic acid

(AA) metabolism. The pathway is bifurcated at the level of 12S-hydro-

peroxy-eicosatetraenoic acid (12S-HpETE), which can either be reduced to

12S-hydro-eicosatetraenoic acid (12S-HETE) or converted to hepoxilins.

The present review gives an update on the biochemistry, biology and clin-

ical aspects of hepoxilin-based drug development. The isolation, cloning

and characterization of a rat leukocyte-type 12S-LOX from rat insulinoma

RINm5F cells revealed a 12S-LOX possessing an intrinsic 8S ⁄R-hydroxy-11,12-epoxyeicosa-5Z,9E,14Z-trienoic acid (HXA3) synthase activity. Site-

directed mutagenesis studies on rat 12S-LOX showed that the HXA3

synthase activity was impaired when the positional specificity of AA was

altered. Interestingly, amino acid Leu353, and not conventional sequence

determinants Met419 and Ile418, was found to be a crucial sequence deter-

minant for AA oxygenation. The regulation of HXA3 formation is depend-

ent on the cellular overall peroxide tone. Cellular glutathione peroxidases

(cGPxs) compete with HXA3 synthase for 12S-HpETE as substrate either

to reduce to 12S-HETE or to convert to HXA3, respectively. Therefore,

RINm5F cells, which are devoid of GPxs, are capable of converting AA or

12S-HpETE to HXA3 under basal conditions, whereas cells overexpressing

cGPx are unable to do so. HXA3 exhibits a myriad of biological effects,

most of which are associated with the stimulation of intracellular calcium

or the transport of calcium across the membrane. The activation of

HXA3–G-protein-coupled receptors explains many of the extracellular

effects of HXA3, including AA- and diacylglycerol (DAG) release in

human neutrophils, insulin secretion in rat pancreatic b-cells or islets, and

synaptic actions in the brain. The availability of stable analogs of HXA3,

termed 10-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid deriv-

atives (PBTs), recently made several animal studies possible and explored

the role of HXA3 as a therapeutic in treatment of diseases. Thus, PBT-3

Abbreviations

AA, arachidonic acid; cGPx, cellular glutathione peroxidase; COX, cyclooxygenase; ER, endoplasmic reticulum; GPCR, G-protein coupled

receptor; GPx, glutathione peroxidase; 12S-HETE, 12S-hydroxy-eicosatetraenoic acid; 12S-HpETE, 12S-hydroperoxy-eicosatetraenoic acid;

HSP, heat shock protein; HXA3, 8S ⁄ R-hydroxy-11,12-epoxyeicosa-5Z,9E,14Z-trienoic acid (hepoxilin A3); HXB3, 10S ⁄ R-hydroxy-11,12-

epoxyeicosa-5Z,8Z,14Z-trienoic acid; IRE1a, endoplasmic reticulum-resident transmembrane protein kinase; 12S-LOX, 12S-lipoxygenase;

LT, leukotriene; PBT, 10-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid derivative; PGE2, prostaglandin E2; PHGPx, phospholipid

hydroperoxide glutathione peroxidase; UPR, unfolded protein response.

FEBS Journal 274 (2007) 3503–3512 ª 2007 The Authors Journal compilation ª 2007 FEBS 3503

Introduction

Hepoxilins are bioactive epoxy-hydroxy products of the

arachidonic acid (AA) metabolism via the 12S-lipoxyg-

enase (12S-LOX) pathway. Following the dioxygena-

tion of AA to 12S-hydroperoxy-eicosatetraenoic acid

(12S-HpETE), the recently uncovered enzyme, hepoxi-

lin A3 (HXA3) synthase, readily converts 12S-HpETE

to the bioactive compound, 8S ⁄R-hydroxy-11,12-epoxy-eicosa-5Z,9E,14Z-trienoic acid (hepoxilin A3), trivially

called HXA3, and inactives 10S ⁄R-hydroxy-11,12-epoxyeicosa-5Z,8Z,14Z-trienoic acid (HXB3) (Fig. 1)

[1,2]. Hepoxilins are formed by various types of cells,

tissues and organs, including platelets [3–5], neutroph-

ils [6], rat insulinoma cells [7], lung [8,9], pancreatic

islets [10,11], brain [12], aorta [13] and pineal glands

[14]. Hepoxilins exert a myriad of biological actions

and regulate processes [15 and references therein], but

clues for the biosynthesis of hepoxilins and their pleio-

tropic actions were, until recently, unknown.

induced apoptosis in K562 tumour cells and inhibited growth of K562

CML solid tumours in nude mice. HXA3 inhibited bleomycin-evoked lung

fibrosis and inflammation in mice and the raised insulin level in the circula-

tion of rats. At low glucose concentrations (0–3 mM), HXA3 also stimula-

ted insulin secretion in RINm5F cells through the activation of IRE1a, anendoplasmic reticulum-resident kinase. The latter regulates the protein fold-

ing for insulin biosynthesis. In conclusion, HXA3-mediated signaling may

be involved in normal physiological functions, and hepoxilin-based drugs

may serve as therapeutics in diseases such as type II diabetes and idio-

pathic lung fibrosis.

Arachidonic Acid (AA)

GSSG

2GSH

GPx- 1 GPx-4 (PHGPx )

Hx A 3 synthase

Hx B 3

Epoxide hydrolase

8(S) TrX A 3 8(R) TrXA 3

PL

12(S/R)-HpETE

12(S/R)-HETE 11(S), 12(S) -HXA 3

COOH

COOH

OOH

COO H

OH

COOH

O

O H

COOH

OH

O

COOH

OH

OH O H

COOH

OH

OH O H

Fig. 1. Schematic pathways depicting the cellular biosynthesis and catabolism of hepoxilins.

Structure, biochemistry and biology of hepoxilins S. Nigam et al.

3504 FEBS Journal 274 (2007) 3503–3512 ª 2007 The Authors Journal compilation ª 2007 FEBS

Isolation and characterization ofhepoxilin A3 synthase

The biosynthetic mechanism for hepoxilin formation

has always been highly controversial. It was generally

accepted that the 12S-LOX-mediated dioxygenated

product of AA, 12S-HpETE, undergoes isomerization

to form biologically active HXA3 and inactive HXB3

[15]. However, arguments against the isomerization

reaction were (a) heat sensitivity of the HXA3 forma-

tion in mammalian cells [16], (b) specificity of 12S-

HpETE, but not of 12R-HpETE, as a substrate for

HXA3 formation in rat insulinoma cells and rat pineal

glands [7,17], (c) the formation of HXA3 in mamma-

lian cells only in association with a high expression

level of 12S-LOX activity [7,17] and (d) the absence of

HXA3 formation in cells containing abundantly gluta-

thione peroxidase (GPx), including cellular GPx

(cGPx) and membrane-bound phospholipid hydroper-

oxide GPx (PHGPx) [18]. The latter observations were

reported earlier in selenium-deficient platelets, which

converted AA to the novel hydroxy-epoxy compounds

trioxilins A3 and B3 [19].

GPxs are selenoenzymes that are responsible for

reducing the cellular peroxide tone. In cells,

AA-derived 12S-HpETE is thus preferably reduced by

cGPx and PHGPx to 12S-hydro-eicosatetraenoic acid

(12S-HETE). In fact, this reaction hindered the detec-

tion of HXA3 in cells abundantly equipped with GPxs.

Thus, human platelets, which exhibit high 12S-LOX

activity, are incapable of converting AA or 12S-

HpETE to HXA3 as a result of the presence of GPxs.

However, inhibition of GPxs by iodoacetate easily

revealed the formation of HXA3 from the above sub-

strates in human platelets [18]. PHGPx is more potent

than cGPx to reduce the cellular peroxide tone.

Although PHGPx constitutes barely 2% of the total

12S-HpETE reductase activity, it is capable of com-

pletely reducing 12S-HpETE to 12S-HETE, as shown

in platelets of cGPx gene knockout mice [20]. This also

shows that the contribution of cGPx to the cellular

antioxidant mechanism and to the defense against

hyperoxic insult is very limited.

Rat insulinoma cells, RINm5F, which contain leu-

kocyte-type 12S-LOX and are depleted of cGPx and

PHGPx, were found to produce selectively significant

amounts of HXA3, but not HXB3, from AA or 12S-

HpETE [7]. Conversely, cells stably transfected with

cGPx or PHGPx failed to produce any HXA3, sug-

gesting the presence of a specific enzyme for HXA3

formation regulated by GPxs [7]. Other lines of evi-

dence for the presence of a putative HXA3 synthase

in RINm5F were (a) abrogation of HXA3 formation

when cells were heat inactivated or depleted of 12S-

LOX activity [21], (b) colocalization of 12S-LOX and

HXA3 synthase activities in the 100 000 g cytosolic

fraction, which were abolished by heat-denaturation

[21], (c) co-immunoprecipitation of 12S-LOX and

HXA3 activities by a specific anti-12S-LOX immuno-

globulin [21] and (d) highly regio- and enantioselec-

tive synthesis of the 8(S)- but not of the 8(R)-HXA3

epimer upon incubation of RINm5F cells with AA

or 12S-HpETE [21]. These observations pinpointed

the presence of an HXA3 synthase activity exhibited

by rat 12S-LOX and led us to isolate, clone and

sequence HXA3 synthase from RINm5F cells [21].

Comparison of the sequence of HXA3 synthase with

the known rat 12S ⁄ 15S-LOX sequence (accession no.

NM_031010) using the Blast server identified the

HXA3 synthase as an intrinsic activity of the authen-

tic rat 12S-LOX [21]. Investigation of other 12S ⁄15S-LOX isoforms with respect to intrinsic HXA3

synthase failed to exhibit any activity in human 5S-

LOX or rabbit reticulocyte 15S-LOX. However,

human platelet-type 12S-LOX showed ample intrinsic

HXA3 synthase activity [15,18], indicating that the

positional specifity of amino acid residues in 12S-

LOX isoforms may be the determinant for such an

activity [21].

Positional specificity of AA oxygenationdetermines the extent of HXA3 synthaseactivity in rat 12S-lipoxygenase

Of all LOXs studied to date, only rabbit reticulocyte-

type 15-LOX has been extensively investigated. Its

crystal structure is known and shows a two-domain

protein comprising (a) an N-terminal domain, which

resembles the b-barrel domain of mammalian lipases

and is required to adjust the enzyme in the vicinity of

the substrate, and (b) a large C-terminal catalytic

domain, which contains the nonheme iron liganded by

three histidine residues and a carboxy-terminal isoleu-

cine [22]. The oxygenation rate of various AA isomers

by 12S- or 15S-LOX depends upon the distance of the

double allylic methylene from the x-end of the AA iso-

mer [23]. Detailed structural studies on 15-LOX, which

allowed easy transformation of 15-LOX into 12-LOX

and vice versa, implicate the 12S ⁄ 15S-LOX enzyme

complex as a single enzyme, but with either dominant

12S-LOX- or 15S-LOX activity in different organs

[23].

Based on the structural studies with rabbit reticu-

locyte-type 15-LOX, it was proposed that the shape

and size of the AA-binding pocket is crucial for

positional specificity of the amino acid residues [24].

S. Nigam et al. Structure, biochemistry and biology of hepoxilins

FEBS Journal 274 (2007) 3503–3512 ª 2007 The Authors Journal compilation ª 2007 FEBS 3505

Met419 and Ile418 were identified as critical sequence

determinants in rabbit reticulocyte-type 15-LOX for

the AA oxygenation. Their mutation to residues with

a smaller side chain, such as Val, gave rise to a

mutant possessing the porcine leukocyte-type 12S-

LOX activity. Conversely, reverse mutation of these

amino acids in porcine leukocyte-type 12S-LOX ren-

dered functional properties of rabbit leukocyte-type

15S-LOX to 12S-LOX mutants [24–26]. However,

upon application of these mutations in rat 12S-LOX,

no alteration of positional specificity was observed

for the conventional sequence determinants Met419

and Ile418 [27]. Instead, Leu353 was identified as a

crucial sequence determinant for positional specificity

of the AA oxygenation in rat 12S-LOX [24]. Fur-

thermore, all mutants of rabbit 15S-LOX in which

amino acid residues were exchanged with space-filling

site chains, showed comparable HXA3 synthase and

12S-LOX activities (Table 1) [21]. Strikingly, the rab-

bit 15S-LOX mutant I418A exhibited 12S-LOX and

HXA3 synthase activities, whereas the reverse mutant

of rat 12S-LOX A418I failed to show any 15S-LOX

activity. However, it retained almost completely 12S-

LOX and HXA3 synthase activities. As expected, the

rat 12S-LOX mutants, L353F and V593I, exhibited

15S-LOX activity, but also retained residual 12S-

LOX and HXA3 synthase activities. These findings

revealed that 12S-LOX activity is inevitably required

for HXA3 synthase expression.

Regulation of hepoxilin A3 synthaseactivity by cellular glutathioneperoxidases

As multifunctional enzymes, LOXs exhibit, besides

inherent oxygenase activity, hydroperoxidase [28,29],

leukotriene synthase [30,31] and lipoxin synthase [32–

34] activities. Unlike the dioxygenase reaction, HXA3

synthesis from 12S-HpETE follows the hydroperoxi-

dase reaction [28,29] and does not require the insertion

of molecular oxygen. This involves two steps (a)

homolytic cleavage of the peroxy group forming rad-

ical intermediates and (b) radical stabilization via the

formation of secondary products, such as epoxy-

hydroxy compounds, ketodienes, short-chain aldehydes

and alkanes. For the conversion of exogenous 12S-

HpETE by recombinant rat 12S-LOX to HXA3, both

steps appear to be enzyme-controlled. Thus, the

exhaustion of 12S-HpETE to produce to HXA3 could

be observed by monitoring the UV absorption at

235 nm. This reaction was completely blocked by the

12S-LOX inhibitor, 4-(2-oxapentadeca-4-yne) phenyl-

propanoic acid [35].

Various reports in the literature have pinpointed the

role of selenium-dependent glutathione peroxidases

cGPx and PHGPx for the regulation of AA metabolism

[18,36–39]. PHGPx is not only capable of reducing free

hydroperoxy fatty acids, but also the esterified ones in

membrane phospholipids [40]. In this way it differs

completely from cGPx, which is unable to reduce the

esterified hydroperoxy fatty acids. The GPxs reduce

cellular peroxides, including 12S-HpETE, thereby alle-

viating the formation of HXA3 [7]. Differential expres-

sion levels of GPxs may therefore be responsible for

the absence of hepoxilins in various cell types despite

the presence of leukocyte-type or platelet-type 12S-

LOX [7]. The conversion by rat insulinoma cells

RINm5F, which are depleted of cGPx and PHGPx, of

AA or 12S-HpETE to HXA3, and its abrogation in

cells overexpressing cGPx, clearly indicate the regula-

tory role of GPxs for HXA3 biosynthesis (Fig. 1).

Biological actions and therapeutic roleof hepoxilin A3 in human diseases

Hepoxilins exhibit numerous biological actions, which

have been intensively investigated previously [15 and

references therein]. Most of the HXA3 actions reported

to date have, as their basis, the stimulation of intracel-

lular calcium [6,41,42] or increased calcium transport

across the membrane [43]. Thus, HXA3-mediated

AA and diacylglycerol release in human polyform

neutrophils [44], targeting of human neutrophils to

Table 1. 8S ⁄ R-Hydroxy-11,12-epoxyeicosa-5Z,9E,14Z-trienoic acid

(hepoxilin A3) (HXA3) synthase and 12S ⁄ 15S-lipoxygenase (LOX)

activities of rat 12S-LOX (wild-type) and its mutants. Rat 12S-LOX

from pancreatic RINm5F cells and its mutant isoforms were

expressed in Escherichia coli and the supernatants obtained follow-

ing cell lysis were used for determination of the enzyme activity, as

described previously [20]. The LOX and HXA3 synthase activities

(lgÆmL)1 in culture medium per 30 min)1) are expressed as the

ratio of 12-hydro-eicosatetraenoic (HETE) to 15-HETE and HXA3

(determined as trioxilin A3s), respectively. Values given represent

the mean of two separate experiments.

12S-LOX isoform

12-HETE ⁄15-HETE

HXA3 synthase

activity

12S-LOX (wild-type)a 88 ⁄ 12 0.85 ± 0.15

12S-LOX (A418I)a 87 ⁄ 13 0.58 ± 0.10

12S-LOX (K417Q)a 94 ⁄ 6 0.80 ± 0.20

12S-LOX (L353F)a 25 ⁄ 75 0.20 ± 0.15

12S-LOX (V593I) 52 ⁄ 48 0.32 ± 0.10

aThe activities of HXA3 isoforms were taken from a previous publi-

cation [21].

Structure, biochemistry and biology of hepoxilins S. Nigam et al.

3506 FEBS Journal 274 (2007) 3503–3512 ª 2007 The Authors Journal compilation ª 2007 FEBS

migrate across intestinal epithelia at the site of inflam-

mation [45], stimulation of insulin secretion from

pancreatic islets [10], presynaptic release of 5-hydroxy-

tryptamine and modification of K+ channel activity in

Aplysia [46], mimicking of synaptic actions in brain

[47,48] and inhibition of norepinephrine release from

mammalian brain [49], have been reported to be cal-

cium-dependent actions. The formation of HXA3 in

pineal glands to regulate melatonin biosynthesis also

requires the stimulation of intracellular Ca2+ [14,17].

The role of HXA3 in the modulation of apoptosis

is controversial. Whereas both isomers 8S- and

8R-HXA3 induce the expression of the cell-protective

heat shock protein 72 (HSP72) in human neutrophils

[50] and of the cell-protective heat shock protein 90

(HSP90) in rat pancreatic b-cells (M. P. Zafiriou,

R. Deva, Y. Ishijo, A. Baran, A. Siafaka-Kapadai,

M. P. Roux-Van der Merwe, R. Ciccoli & S. Nigam,

unpublished), a stable synthetic analogue of HXA3,

called 10-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-

trienoic acid (PBT-3), was found to induce apoptosis in

the CML cell line K562 in vitro [51] and to inhibit

growth of K562 CML solid tumours in vivo in nude

mice [52,53]. The release of cytochrome c from mito-

chondria and, in turn, the activation of caspase-3

through caspase-9, was described as a pathway for the

induction of apoptosis. This divergence in action of

HXA3 as an anti- or a pro-apoptotic agent may be

attributed to the specific cell type and organ experiment-

ally used and ⁄or to the chemical properties of PBT-3.

PBT-3 is chemically an HXA3 cyclopropane analogue

and has been used as a thromboxane A2 receptor antag-

onist. It does not inhibit 12S-LOX, phospholipase A2

and cyclooxygenases (COXs) [54]. COXs and 12S-LOX,

however, have been documented as survival enzymes in

the literature and their inhibition has been shown to

cause carcinoma cell apoptosis [55–59]. Therefore, it can

be speculated that PBT-3-mediated apoptosis is inde-

pendent of the modulation of eicosanoid-converting

enzymes. By contrast, the up-regulation of cell protec-

tion by HSP72 (an inducible form of HSP70) in human

neutrophils [50] and HSP90 and protein phosphatase 5

in pancreatic RINm5F cells by HXA3 (M. P. Zafiriou

et al., unpublished) are fully in agreement with previous

reports, in which the expression of HSP72 and HSP90

has been shown to regulate negatively the heat stress-

induced cell damage in breast cancer cells [60,61]. More-

over, transfection of a 12-LOX promoter construct with

the HSP90-binding site into RINm5F cells exhibited sig-

nificant 12S-LOX up-regulation and cell proliferation

upon challenge with HXA3. Indeed, both effects were

abrogated by the HSP90 inhibitor, geldanamycin (M.-P.

Zafiriou & S. Nigam unpublished), suggesting that

HXA3 is an anti-apoptotic agent. It must be stressed

here that HSP72 and HSP90 serve as markers of HXA3

challenge and are probably not protective per se,

because cell transfectants that overexpressed HSP72 and

HSP90 demonstrated enhanced apoptosis in response to

12S-HpETE (M.-P. Zafiriou & S. Nigam, unpublished).

The actions of HXA3, at least in the human neutro-

phil, appear to occur via the activation of intracellular

receptors [41,42,44], which translocate Ca2+ within the

cell from the endoplasmic reticulum to mitochondria

[62]. HXA3 binding to the unidentified receptor in

human neutrophils was found to be specific [63],

although no additional evidence of the existence of

such a receptor in any cell type has been provided.

The only report on the involvement of a G-protein-

coupled receptor (GPCR) dates back to 1990, in which

fMet-Leu-Phe-triggered AA and diacylglycerol release

in human neutrophils was blocked by pertussis toxin

[42,44,64].

Among the plethora of biological effects of hepoxilin

A3, the regulation of insulin secretion and inhibition of

lung fibrosis are of vital clinical importance. The avail-

ability of stable analogues of HXA3, known as PBTs,

recently made it possible to conduct in vivo studies

in animals. The stability of the molecule HXA3 was

achieved by stabilizing the unstable epoxide ring with

a methylene group to form a cyclopropane ring. The

compounds thus obtained were resistant to catabolic

reactions and were utilized for in vivo studies [65].

Induction of insulin secretion by HXA3

in vitro and in vivo

Early studies in the rat revealed that HXA3 is capable

of releasing insulin from isolated [10] or perifused

Langerhans’ islets in vitro [11]. Following bolus injec-

tion of HXA3 isomers (100 lg of HXA3 per rat), an

enhanced circulating level of insulin was observed

within 20 min. The insulin release by both 8S- and 8R-

HXA3 epimers was dependent on intracellular Ca2+-

release as well as on the glucose status of the rat. The

latter was inevitably required, because fasted rats failed

to show any changes in insulin blood level [66].

Whereas HXA3 isomers augmented insulin levels in

20 min, injection of glucose alone required barely 30 s

to enhance the blood insulin level. The delayed

response by HXA3 isomers compared with the glucose

injection is probably a result of the slow uptake of

HXA3 by the organ [66].

Current investigations on HXA3-mediated regulation

of insulin biosynthesis and release in rat insulinoma

cells RINm5F in our laboratory focus primarily on

the specific role of the endoplasmic reticulum

S. Nigam et al. Structure, biochemistry and biology of hepoxilins

FEBS Journal 274 (2007) 3503–3512 ª 2007 The Authors Journal compilation ª 2007 FEBS 3507

(ER)-resident transmembrane protein kinase, IRE1a[67–70]. The proinsulin produced in the ER by clea-

vage of the signal peptide from preproinsulin under-

goes a protein folding in the lumen of the ER and

forms three –S–S– bonds. The so-formed proinsulin

translocates to the Golgi apparatus and appears there

as packaged secretory granules. In the Golgi apparatus

it is converted to insulin and released through exocyto-

sis [71,72]. Any fluctuation in blood glucose levels

results in an alteration of proinsulin folding in the ER

of b-cells [73,74], which impairs the homeostasis of

b-cells and leads to ER stress. To counteract the ER

stress, b-cells respond with the up-regulation of unfol-

ded protein response (UPR)-specific genes. IRE1a in

the ER serves as the upstream regulator of UPR-

specific gene expressions [75,76]. Lipson et al. [77]

recently showed that postprandial hyperglycemia

(around 10 mm glucose concentration) activates IRE1aand enhances the biosynthesis of proinsulin in pancre-

atic b-cells. Nevertheless, under chronic exposure to

high glucose (around 25 mm) cells showed ER stress

and hyperactivation of IRE1a, but the insulin secretion

was suppressed [77]. Normally, IRE1a is stabilized by

HXA3-mediated up-regulation of HSP90, a critical

component of the transcriptional arm of the UPR [78].

Preliminary data from our laboratory revealed that at

low glucose concentrations (0–3 mm), but not above

10 mm glucose, endogenously produced 8S-HXA3 [7]

activates significantly IRE1a and insulin secretion

(M.-P. Zafiriou, R. Deva, R. Ciccoli, K. C. Chang,

A. Siafaka-Kapadai, R. Roux-Van der Merwe &

S. Nigam, unpublished results). In Fig. 2, the effects of

high or low glucose on the insulin secretion in

RINm5F cells are schematically summarized. Support

for the role of HSP90 was achieved by using geldana-

mycin, an inhibitor of HSP90, as well as an HSP90-

mutant construct. Both agents promoted dissociation

of HSP90 from IRE1a, thereby reducing markedly

the half-life of IRE1a (M.-P. Zafiriou & S. Nigam,

unpublished).

HXA3-mediated protection frompulmonary fibrosis

Idiopathic fibrotic lung disease is a progressive pul-

monary disorder hallmarked by inflammation, mesen-

chymal cell proliferation and accumulation of

extracellular matrix proteins (e.g. collagen), which

finally leads to respiratory failure and death [79]. Con-

sidering the poor outcome and limited therapeutic

options available, a dire need for an alternative patho-

biological aspect is required, which may then be trans-

lated into a promising therapeutic approach. Screening

of the results of earlier studies showed that idiopathic

lung fibrosis involves the interplay of several lipid

mediator cascades, including those triggered by plate-

let-derived growth factor [80], transforming growth

factor-b [81] and interleukin-8 [82]. AA-converting

enzymes have been reported to affect the lung function

through eicosanoids. One of these eicosanoids – pros-

taglandin E2 (PGE2) – has been found to exhibit bron-

chodilatory, anti-inflammatory and antifibrotic actions

on the lung through PGE2 receptors (EP). Indeed,

mice in which the COX-1 gene was knocked out

Blood glucose

O2- ions/ROS

12HpETE HXA3

ß-cell growthApoptosis

Insulinsecretion

InsulinIRE1aCOX-2PGE2

High GlucoseHigh Glucose Low GlucoseLow Glucose

Blood glucose

O2- ions/ROS

12HpETE HXA3

ß-cell growthApoptosis

Insulinsecretion

InsulinIRE1aCOX-2PGE2

Postulated role of HXAPostulated role of HXA33 in the regulation of glucosein the regulation of glucose--sensitive insulin secretionsensitive insulin secretion

Fig. 2. Postulated role of 8S ⁄ R-hydroxy-

11,12-epoxyeicosa-5Z,9E,14Z-trienoic acid

(hepoxilin A3) (HXA3) in the regulation of

glucose-sensitive insulin secretion.

Structure, biochemistry and biology of hepoxilins S. Nigam et al.

3508 FEBS Journal 274 (2007) 3503–3512 ª 2007 The Authors Journal compilation ª 2007 FEBS

showed a diminished PGE2 level in bronchoalveolar

lavage and hyper-reagibility towards inhaled allergens

[83]. Moreover, COX-2 null mice rapidly produced

lung fibrosis after administration of bleomycin or

vanadium pentoxide [84,85]. Recently, Peters-Golden

and coworkers [86] showed another group of lipid

mediators – leukotrienes (LTs) – as potent activators

of lung fibrosis. Because LTs have been shown to exert

strong pro-inflammatory and bronchoconstrictive

effects on the lung through their specific receptors,

these authors showed by assessing collagenase levels in

lung digests that mice ablated of the 5-LOX gene had

reduced lung inflammation. These mice also showed

reduction of bleomycin-induced lung fibrosis, as

assessed by Masson’s trichrome staining and total

hydroxyproline levels in the lung [86]. Recently, eleva-

ted levels of interleukin-13 and LTB4, but not of

cysteinyl LTs, have been reported to co-exist at the

sites of T helper 2 cell inflammation and fibrosis in the

lung [85]. Whether the 5-LOX pathway influences the

fibrotic response directly by producing LTs, or indi-

rectly by producing protective mediators, such as

PGE2, is far from clear.

To study the oxidant-mediated epithelial cell injury

as a cause of idiopathic pulmonary fibrosis [87], Pace-

Asciak and coworkers [88] used a stable analogue of

HXA3 PBT-1 together with bleomycin intratracheally

in a lung injury model and assessed the reduction in

pulmonary fibrosis by a staining method. PBT-1 was

also intradermally applied to assess vascular permeabil-

ity in the skin. Both bleomycin-provoked lung fibrosis

and permeability changes in the skin were abolished by

PBT-1 at a low dosis of 10 lg per mouse [65]. In con-

clusion, the in vivo data on stable hepoxilin analogues

show their use in suppression of chronic lung inflam-

mation and lung fibrosis in a mouse model. More

investigations on other animal models are required to

initiate preliminary clinical studies in humans.

Future directions

Most of the past work on hepoxilins was carried out

in Pace-Asciak’s laboratory in Toronto and at our

laboratory in Berlin. These studies provided strong

support for the notion that hepoxilin A3 functions as a

second messenger in the regulation of a variety of bio-

logical effects. It is of interest to know whether these

effects are directly or indirectly triggered by the

up-regulation of cytokines. The pro- and anti-apoptot-

ic role of HXA3 in various tissues and cell types has to

be judged correctly to improve the understanding of its

specific role in tumour cell proliferation and apoptosis.

A number of HXA3 actions in human neutrophils have

been shown to be mediated by the GPCR, a receptor

for which HXA3 is believed to be a ligand; however,

the GPCR has not yet been characterized. Hence,

future challenges should focus on the isolation and

characterization of the GPCR and intracellular signa-

ling pathways triggered by the HXA3–GPCR complex

to define the intracellular targets of HXA3. By addres-

sing all these issues we may be able to boost the devel-

opment of specific tools and novel therapeutics based

on hepoxilins.

Acknowledgements

The authors wish to thank the ‘Deutsche Forschungsg-

emeinschaft, Bonn’ for supporting the work on hepoxi-

lins by a grant (Ni-242 ⁄ 27-1). The authors are also

grateful to Dr P. Shankaranarayanan and Dr M. Suth-

erland for their valuable contributions.

References

1 Pace-Asciak CR (1994) Hepoxilins: a review on their

cellular actions. Biochim Biophys Acta 1215, 1–8.

2 Pace-Asciak CR, Granstrom E & Samuelsson B (1983)

Arachidonic acid epoxides. Isolation and structure of

two hydroxy epoxide intermediates in the formation of

8,11,12- and 10,11,12-trihydroxyeicosatrienoic acids.

J Biol Chem 258, 6835–6840.

3 Bryant RW & Bailey JM (1978) Isolation of a new lip-

oxygenase metabolite of arachidonic acid, 8. 11, 12-tri-

hydroxy-5,9,14-eicosatrienoic acid from human platelets.

Fed Proc 37, 31–37.

4 Bryant RW & Bailey JM (1980) Altered lipoxygenase

metabolism and decreased glutathione peroxidase activ-

ity in platelets from selenium-deficient rats. Biochem

Biophys Res Commun 92, 268–276.

5 Pace-Asciak CR (1993) Hepoxilins. Gen Pharmacol 24,

805–810.

6 Dho S, Grinstein S, Corey EJ, Su WG & Pace-Asciak

CR (1990) Hepoxilin A3 induces changes in cytosolic

calcium, intracellular pH and membrane potential in

human neutrophils. Biochem J 266, 63–68.

7 Shankaranarayanan P, Ciccoli R & Nigam S (2003) Bio-

synthesis of hepoxilins: evidence for the presence of a

hepoxilin synthase activity in rat insulinoma cells. FEBS

Lett 538, 107–112.

8 Pace-Asciak CR, Mizuno K & Yamamoto S (1983) A

comparison of leukotriene and prostaglandin binding to

human myometrium. Prostaglandins 25, 79–84.

9 Pace-Asciak CR, Mizuno K, Yamamoto S, Granstrom

E & Samuelsson B (1983) Oxygenation of arachidonic

acid into 8,11,12- and 10,11,12-trihydroxyeicosatrienoic

acid by rat lung. Adv Prostagl Thrombox Leukotr Res

11, 133–139.

S. Nigam et al. Structure, biochemistry and biology of hepoxilins

FEBS Journal 274 (2007) 3503–3512 ª 2007 The Authors Journal compilation ª 2007 FEBS 3509

10 Pace-Asciak CR & Martin JM (1984) Hepoxilin, a new

family of insulin secretagogues formed by intact rat

pancreatic islets. Prostagl Leukotr Med 16, 173–180.

11 Pace-Asciak CR, Martin JM, Corey EJ & Su WG

(1985) Endogenous release of hepoxilin A3 from isolated

perifused pancreatic islets of Langerhans. Biochem Bio-

phys Res Commun 128, 942–946.

12 Pace-Asciak CR (1990) Hepoxilin A3 blocks the release

of norepinephrine from rat hippocampal slices. Biochem

Biophys Res Commun 173, 949–953.

13 Laneuville O, Corey EJ, Couture R & Pace-Asciak CR

(1991) Hepoxilin A3 (HxA3) is formed by the rat aorta

and is metabolized into HxA3-C, a glutathione conju-

gate. Biochim Biophys Acta 1094, 60–68.

14 Reynaud D, Delton I, Gharib A, Sarda N, Lagarde M

& Pace-Asciak CR (1994) Formation, metabolism, and

action of hepoxilin A3 in the rat pineal gland. J Neuro-

chem 62, 126–133.

15 Nigam S & Zafiriou MP (2005) Hepoxilin A3 syn-

thase, A review. Biochem Biophys Res Commun 338,

161–168.

16 Pace-Asciak CR, Reynaud D & Demin P (1993) Enzy-

matic formation of hepoxilins A3 and B3. Biochem Bio-

phys Res Commun 197, 869–873.

17 Reynaud D, Demin P & Pace-Asciak CR (1994) Hep-

oxilin A3 formation in the rat pineal gland selectively

utilizes (12S)-hydroperoxyeicosatetraenoic acid

(HPETE), but not (12R)-HPETE. J Biol Chem 274,

23976–23980.

18 Sutherland M, Shankaranarayanan P, Schewe T &

Nigam S (2001) Evidence for the presence of phospho-

lipid hydroperoxide glutathione peroxidase in human

platelets: implications for its involvement in the regula-

tory network of the 12-lipoxygenase pathway of arachi-

donic acid metabolism. Biochem J 353, 91–100.

19 Bryant RW, Simon TC & Bailey JM (1983) Hydro-

peroxy fatty acid formation in selenium deficient rat

platelets: coupling of glutathione peroxidase to the lip-

oxygenase pathway. Biochem Biophys Res Commun 117,

183–189.

20 Ho YS, Magnenat JL, Bronson RT, Cao J, Gargano

M, Sugawara M & Funk CD (1997) Mice deficient in

cellular glutathione peroxidase develop normally and

show no increased sensitivity to hyperoxia. J Biol Chem

272, 16644–16651.

21 Nigam S, Shankaranarayanan P, Ciccoli R, Ishdorj G,

Schwarz K, Petrucev B, Kuhn H & Haeggstrom JZ

(2004) The rat leukocyte-type 12-lipoxygenase exhibits

an intrinsic hepoxilin A3 synthase activity. J Biol Chem

279, 29023–29030.

22 Gilmore SA, Villasenor A, Fletterick R, Sigel E &

Browner MF (1997) The structure of mammalian

15-lipoxygenase reveals similarity to the lipases and the

determinants of substrate specificity. Nat Struct Biol 4,

1003–1009.

23 Kuhn H, Sprecher H & Brash AR (1990) On singular

or dual positional specificity of lipoxygenases. The num-

ber of chiral products varies with alignment of methy-

lene groups at the active site of the enzyme. J Biol

Chem 265, 16300–16305.

24 Borngraber S, Browner M, Gilmore S, Gerth C, Anton

M, Fletterick R & Kuhn H (1999) Shape and specificity

in mammalian 15-lipoxygenase active site. The func-

tional interplay of sequence determinants for the reac-

tion specificity. J Biol Chem 274, 37345–37350.

25 Sloane DL, Leung R, Craik CS & Sigal E (1991) A pri-

mary determinant for lipoxygenase positional specificity.

Nature 354, 149–152.

26 Suzuki H, Kishimoto K, Yoshimoto T, Yamamoto S,

Kanai F, Ebina Y, Miyatake A & Tanabe T (1994)

Site-directed mutagenesis studies on the iron-binding

domain and the determinant for the substrate oxygen-

ation site of porcine leukocyte arachidonate 12-lipoxyge-

nase. Biochim Biophys Acta 1210, 308–316.

27 Watanabe T & Haeggstrom JZ (1993) Rat 12-lipoxyge-

nase: mutations of amino acids implicated in the posi-

tional specificity of 15- and 12-lipoxygenases. Biochem

Biophys Res Commun 192, 1023–1029.

28 Garssen GJ, Veldink GA, Vliegenthart JF & Boldingh J

(1976) The formation of threo-11-hydroxy-trans-12: 13-

epoxy-9-cis-octadecenoic acid by enzymic isomerisation

of 13-L-hydroperoxy-9-cis, 11-transoctadecadienoic acid

by soybean lipoxygenase-1. Eur J Biochem 62, 33–36.

29 Bryant RW & Bailey JM (1979) Isolation of a new lip-

oxygenase metabolite of arachidonic acid, 8. 11, 12-tri-

hydroxy-5,9,14-eicosatrienoic acid from human platelets.

Prostaglandins 17, 9–18.

30 Shimizu T, Radmark O & Samuelsson B (1984) Enzyme

with dual lipoxygenase activities catalyzes leukotriene

A4 synthesis from arachidonic acid. Proc Natl Acad Sci

USA 81, 689–693.

31 Schwarz K, Gerth C, Anton M & Kuhn H (2000)

Alterations in leukotriene synthase activity of the

human 5-lipoxygenase by site-directed mutagenesis

affecting its positional specificity. Biochemistry 39,

14515–14521.

32 Kuhn H, Wiesner R, Alder L, Schewe T & Stender H

(1986) Formation of lipoxin B by the pure reticulocyte

lipoxygenase. FEBS Lett 208, 248–252.

33 Serhan CN, Takano T, Gronert C, Chiang N & Clish

CB (1999) Lipoxin and aspirin-triggered 15-epi-lipoxin

cellular interactions anti-inflammatory lipid mediators.

Clin Chem Lab Med 37, 299–309.

34 Romano M, Chen XS, Takahashi Y, Yamamoto S &

Funk CD (1993) Lipoxin synthase activity of human

platelet 12-lipoxygenase. Biochem J 296, 127–133.

35 Moody JS & Marnett LJ (2002) Kinetics of inhibition

of leukocyte 12-lipoxygenase by the isoform-specific

inhibitor 4-(2-oxapentadeca-4-yne) phenylpropanoic

acid. Biochemistry 41, 10297–10303.

Structure, biochemistry and biology of hepoxilins S. Nigam et al.

3510 FEBS Journal 274 (2007) 3503–3512 ª 2007 The Authors Journal compilation ª 2007 FEBS

36 Hatzelman A, Schatz M & Ullrich V (1989) Involve-

ment of glutathione peroxidase activity in the stimula-

tion of 5-lipoxygenase activity by glutathione-depleting

agents in human polymorphonuclear leukocytes. Eur J

Biochem 180, 527–533.

37 Schnurr K, Belkner J, Ursini F, Schewe T & Kuhn H

(1996) The selenoenzyme phospholipid hydroperoxide

glutathione peroxidase controls the activity of the

15-lipoxygenase with complex substrates and preserves

the specificity of the oxygenation products. J Biol Chem

271, 4653–4658.

38 Weitzel F & Wendel A (1993) Selenoenzymes regulate

the activity of leukocyte 5-lipoxygenase via the peroxide

tone. J Biol Chem 268, 62888–62892.

39 Imai H, Narashima K, Arai M, Sakamoto H, Chiba N

& Nakagawa Y (1998) Suppression of leukotriene for-

mation in RBL-2H3 cells that overexpressed phospholi-

pid hydroperoxide glutathione peroxidase. J Biol Chem

273, 1990–1997.

40 Chambers SJ, Lambert N & Williamson G (1994) Puri-

fication of a cytosolic enzyme from human liver with

phospholipid hydroperoxide glutathione peroxidase

activity. Int J Biochem 26, 1279–1286.

41 Reynaud D, Demin PM, Sutherland M, Nigam S & Pace-

Asciak CR (1999) Hepoxilin signaling in intact human

neutrophils: biphasic elevation of intracellular calcium by

unesterified hepoxilin A3. FEBS Lett 446, 236–238.

42 Sutherland M, Schewe T & Nigam S (2000) Biological

actions of the free acid of hepoxilin A3 on human neu-

trophils. Biochem Pharmacol 59, 435–440.

43 Derewlany LO, Pace-Asciak CR & Radde IC (1984)

Hepoxilin A, hydroxyepoxide metabolite of arachidonic

acid, stimulates transport of 45Ca2+ across the guinea

pig visceral yolk sac. Can J Physiol Pharmacol 62,

1466–1469.

44 Nigam S, Nodes J, Cichon G, Corey EJ & Pace-Asciak

CR (1990) Receptor-mediated action of hepoxilin A3

releases diacylglycerol and arachidonic acid from human

neutrophils. Biochem Biophys Res Commun 171, 944–948.

45 Mrsny RJ, Gewirtz AT, Siccardi D, Savidge T, Hurley

BP, Madara JL & McCormick BA (2004) Identification

of Hepoxilin A3 in inflammatory events: a required role

in neutrophil migration across intestinal epithelia. Proc

Natl Acad Sci USA 101, 7421–7426.

46 Belardetti F, Campbell WB, Falck JR, Demontis G &

Rosolowski M (1989) Products of heme-catalyzed trans-

formation of the arachidonate derivative 12-HpETE open

S-type K+ channels in Aplysia. Neuron 3, 497–505.

47 Pace-Asciak CR, Laneuville O, Su WG, Corey EJ,

Gurevich N, Wu PH & Carlen PL (1990) A glutathione

conjugate of hepoxilin A3: formation and action in the

rat central nervous system. Proc Natl Acad Sci USA 87,

3037–3041.

48 Carlen PL, Gurevich N, Wu PH, Su WG, Corey EG &

Pace-Asciak CR (1989) Actions of arachidonic acid and

hepoxilin A3 on mammalian hippocampal CA1 neurons.

Brain Res 497, 171–176.

49 Pace-Asciak CR, Wong L & Corey EJ (1990) Hepoxilin

A3 blocks the release of norepinephrine from rat hippo-

campal slices. Biochem Biophys Res Commun 173, 949–

953.

50 Lin Z, Laneuville O & Pace-Asciak CR (1991) Hepoxi-

lin A3 induces heat shock protein (HSP72) expression in

human neutrophils. Biochem Biophys Res Commun 179,

52–56.

51 Qiao N, Lam J, Reynaud D, Abdelhaleem M & Pace-

Asciak CR (2003) The hepoxilin analog PBT-3 induces

apoptosis in BCR-ABL-positive K562 leukemia cells.

Anticancer Res 23, 3617–3622.

52 Li X, Qiao N, Reynaud D, Abdelhaleem M & Pace-

Asciak CR (2005) The hepoxilin analog, PBT-3, inhibits

growth of K-562 CML solid tumours in vivo in nude

mice. In Vivo 19, 185–189.

53 Li X, Qiao N, Renaud D, Abdelhaleem M & Pace-

Asciak CR (2005) PBT-3, a hepoxilin stable analog,

causes long term inhibition of growth of K562 solid

tumours in vivo. Biochem Biophys Res Commun 338,

156–160.

54 Pace-Asciak CR, Reynaud D, Demin P, Aslam R &

Sun A (2002) A new family of thromboxane receptor

antagonists with secondary thromboxane synthase inhi-

bition. J Pharm Exp Ther 301, 618–624.

55 Leung HW, Yang WH, Lai MY, Lin CJ & Lee HZ

(2007) Inhibition of 12-lipoxygenase during baicalein-

induced human lung nonsmall carcinoma H460 cell

apoptosis. Food Chem Toxicol 45, 403–411.

56 Ikemoto S, Sugimura K, Kuratukuri K & Nakatani T

(2004) Antitumour effects of lipoxygenase inhibitors on

murine bladder cancer cell line (MBT-2). Anticancer Res

24, 733–736.

57 Brown JR & DuBois RN (2005) COX-2: a molecular

target for colorectal cancer prevention. J Clin Oncol 23,

2840–2855.

58 Gasparini G, Longo R, Sarmiento R & Morabito A

(2003) Inhibitors of cyclooxygenase 2: a new class of

anticancer agents? Lancet Oncol 4, 605–615.

59 Rao CV & Reddy BS (2004) NSAIDS and chemopre-

vention. Curr Cancer Drug Targets 4, 29–42.

60 Kiang JG, Gist ID & Tsokos GC (2000) Regulation of

heat shock protein 72 kDa and 90 kDa in human breast

cancer MDA-MB-231 cells. Mol Cell Biochem 204, 169–

178;.

61 Trieb K, Kohlbeck R, Lang S, Klinger H, Blahovec H

& Klotz R (2000) Heat shock protein 72 expression

in chondrosarcoma correlates with differentiation.

J Cancer Res Clin Oncol 126, 667–670.

62 Mills L, Reynaud D & Pace-Asciak CR (1997) Hepoxi-

lin-evoked intracellular reorganization of calcium in

human neutrophils: a confocal microscopy study. Exp

Cell Res 230, 237–241.

S. Nigam et al. Structure, biochemistry and biology of hepoxilins

FEBS Journal 274 (2007) 3503–3512 ª 2007 The Authors Journal compilation ª 2007 FEBS 3511

63 Reynaud D, Demin P & Pace-Asciak CR (1996) Hep-

oxilin A3-specific binding in human neutrophils. Bio-

chem J 313, 537–541.

64 Pace-Asciak CR & Nigam S (1991) Hepoxilins modulate

second messenger systems in the human neutrophil. Adv

Exp Med Biol 314, 133–139.

65 Pace-Asciak CR, Li X, Qiao N, Renaud D, Demin P &

Abdelhaleem M (2006) Hepoxilin analogs, potential new

therapeutics in disease. Curr Pharm Des 12, 963–969.

66 Pace-Asciak CR, Demin PM, Estrada M & Liu GY

(1999) Hepoxilins raise circulating insulin levels in vivo.

FEBS Lett 461, 165–168.

67 Cox JS, Shamu CE & Walter P (1993) Transcriptional

induction of genes encoding endoplasmic reticulum resi-

dent proteins requires a transmembrane protein kinase.

Cell 73, 1197–1206.

68 Brewer JW, Cleveland JL & Hendershot LM (1997) A

pathway distinct from the mammalian unfolded protein

response regulates expression of endoplasmic reticulum

chaperones in non-stressed cells. EMBO J 16, 7207–

7216.

69 Wang XZ, Harding HP, Zhang Y, Jolicoeur EM,

Kuroda M & Ron D (1998) Cloning of mammalian

Ire1 reveals diversity in the ER stress responses. EMBO

J 17, 5708–5717.

70 Harding HP, Zhang Y & Ron D (1999) Protein transla-

tion and folding are coupled by an endoplasmic-reticu-

lum-resident kinase. Nature 397, 271–274.

71 Rhodes CJ (2004) Processing of the insulin molecule. In

Diabetes Mellitus (LeRoith D, Taylor SI & Olefsky JM,

eds), pp. 27–50. Lippincott Williams & Wilkins, Phil-

adelphia, PA.

72 Rhodes CJ, Schoelson S & Halban PA (2005) Insulin

biosynthesis, processing, and chemistry. In Joslin’s Dia-

betes Mellitus (Kahn CR, Weir GC, King GL, Jacobson

AM, Moses AC & Smith RJ, eds), pp. 65–82. Joslin

Diabetes Center, Boston, MA.

73 Harding HP, Novoa I, Zhang Y, Zeng H, Wek R, Sha-

pira M & Ron D (2000) Regulated translation initiation

controls stress-induced gene expression in mammalian

cells. Mol Cell 6, 1099–1108.

74 Kaufmann RJ (2002) Orchestrating the unfolded protein

response in health and disease. J Clin Invest 110, 1389–

1398.

75 Harding HP, Calfon M, Urano F, Novoa I & Ron D

(2002) Transcriptional and translational control in the

mammalian unfolded protein response. Annu Rev Cell

Dev Biol 18, 575–599.

76 Mori K (2000) Tripartite management of unfolded pro-

teins in the endoplasmic reticulum. Cell 101, 451–454.

77 Lipson KL, Fonseca SG, Ishigaki S, Nguyen LX, Foss

E, Bortell R, Rossini AA & Urano F (2006) Regulation

of insulin biosynthesis in pancreatic beta cells by an

endoplasmic reticulum-resident protein kinase IRE1.

Cell Metab 4, 245–254.

78 Marcu MG, Doyle M, Bertolotti A, Ron D, Hendershot

L & Neckers L (2002) Heat Shock Protein 90 modulates

the unfolded protein response by stabilizing IRE1a. Mol

Cell Biol 22, 8506–8513.

79 Panos R & King T (1991) Idiopathic pulmonary fibro-

sis. In Immunologically Mediated Pulmonary Diseases

(Lynch JP, DeRemee RA III, eds), pp. 1–39. JB Lippin-

cott, Philadelphia, PA.

80 Martinet Y, Rom W, Grotendorst G, Martin G & Crys-

tal R (1987) Exaggerated spontaneous release of plate-

let-derived growth factor by alveolar macrophages from

patients with idiopathic pulmonary fibrosis. N Engl J

Med 317, 202–209.

81 Kalil N, O’Connor R, Unruh H, Warren P, Kemp A,

Bereznay O & Flanders & Greenberg A (1990) Increased

production immunohistochemical localization of trans-

forming growth factor beta (TGF-b) in idiopathic pul-

monary fibrosis. Am J Respir Cell Mol Biol 5, 155–162.

82 Carre P, Mortenson R, King T Jr, Noble P, Sable C &

Riches D (1991) Increased expression of interleukin-8

gene by alveolar macrophages in idiopathic pulmonary

fibrosis: a potential mechanism for the recruitment and

activation of neutrophils in lung fibrosis. J Clin Invest

88, 1802–1810.

83 Gavett SH, Madison Madison SL, Chulada PC, Scar-

borough PE, Qu W, Boyle JE, Tiano HF, Lee CA,

Lanngenbach RVL & Roggli & Zeldin DC (1999) Aller-

gic lung responses are increased in prostaglandin H syn-

thase-deficient mice. J Clin Invest 104, 721–732.

84 Keerthisingam CB, Jenkins RG, Harrison NK, Hernan-

dez-Rodriguez NA, Booth H, Laurent GJ, Hart SL,

Foster ML & McAnulty RJ (2001) Cyclooxygenase-2

deficiency results in a loss of the anti-proliferative

response to transforming growth factor-beta in human

fibrotic lung fibroblasts and promotes bleomycin-

induced pulmonary fibrosis in mice. Am J Pathol 158,

1411–1422.

85 Rice A, Zhang P, Moomaw C, Morgan D, Langenbach

R, Bradbury A, Chulada PC, Zeldin DC & Bonner JC

(2000) Differential pulmonary fibrotic responses in pros-

taglandin H synthase-deficient mice following metal-

induced lung injury. Am J Respir Crit Care Med 161,

A752.

86 Peters-Golden M, Bailie M, Marshall T, Wilke C, Phan

SM, Gaelen GB & Moore BB (2002) Protection from

Pulmonary Fibrosis in Leukotriene-Deficient Mice. Am

J Respir Crit Care Med 165, 229–235.

87 Shim YM, Zhu Z, Zheng T, Lee CG, Homer RJ, Ma B

& Elias JA (2006) Role of 5-lipoxygenase in IL-13-

induced pulmonary inflammation and remodeling.

J Immunol 177, 1918–1924.

88 Jankov RP, Luo X, Demin P, Aslam R, Hannam V,

Tanswell AK & Pace-Asciak CR (2002) Hepoxilin ana-

logs inhibit bleomycin-induced pulmonary fibrosis in the

mouse. J Pharm Exper Ther 301, 435–440.

Structure, biochemistry and biology of hepoxilins S. Nigam et al.

3512 FEBS Journal 274 (2007) 3503–3512 ª 2007 The Authors Journal compilation ª 2007 FEBS