Prostaglandins & Signal Transduction

-first a little history- from Colin Frunk's article in science
-1930's an experiment in which rats were not given fat in their diets and a lot of unpleasant things occurred -discovery of essential fatty acids
-another experiment identified a molecule with fatty acid properties and "vasodepressor" properties that stimulated smooth muscle activity- this activity was given the name "prostaglandin"
-30 yrs. Later the structures classical prostaglandins was revealed
-many studies since and some nobel prizes awarded


Ruey-Bing et al., Toll-like receptor-2 mediates lipopolysaccharide-induced cellular signalling, Nature, 395, 17sept. 98
-one of the ways cells were tested was inducing an immune response using LPS a gram negative bacteria.
So here is an article that talks about its receptor.
-has leucine rich extracellular domain and cytoplasmic domain with sequence homology to interleukin-1 receptor.
-activation of receptor leads to induction of genes via NF-kB pathway
-5 homolougues in humans
-pic ­p;(-)1 shows expression of toll2 receptors

Jesus Balsinde et al., Functional coupling between secretory phospholipase A2 and cox-2 and its regualtion by cytosolic group 4 phospholipaseA2, Journal of Biochemistry, 95 7951-7956 July 7, 1998.

This article illustrates how arachidonic acid(AA) is liberated from the membrane.
-Phospholipase A2(PLA2) is a family of lipolytic enzymes that hydrolyze fatty acid
Three PLA2's are known
1) secretory PLA2-sPLA2
2) cytosolic group4 PLA2-cPLA2
3) Ca2+ independent group 4 PLA2- iPLA2

- sPLA2 is the key player in releasing AA in activated cells, however it is thought to be activated by increased levels of AA. This initial increase is caused by cPLA2. This is thought to make room for sPLA2 attack (picture-1). sPLA2 also requires Ca2+ to be activated.

-Once AA is released from the membrane it is acted on by one of the COX isoforms to begin prostaglandin synthesis.

Hinz and Brune, Cyclooxygenase-2 10 years later, Journal of Pharmacology and Experimental Therapeutics,300 367-375, Feb. 2002
Bhattacharya et al., Proc. Natl. Acad. Sci. U.S.A., 95 15792 (1998)

The two isoforms of COX are
1) COX-1, this is constitutively expressed in most cells
2) COX-2, which is up-regulated by inflammatory responses

The mechanism by which COX turns AA into prostaglandinH-2(PGH2)-(picture2& picture3)
-these pictures illustrate that there is two parts to this mechanism
-the first being Cyclooxygenase and the second is peroxidase ultimately producing PGH2 which goes on to make?

Structure of COX's
(Picture4)
1)primary struc.of
-COX1, 576 amino acids long
-COX2, 587 amino acids long
60-65% sequence identity between COX's in a species, 85-90% identity among each isoform when compared to a different species.

3) 3-dimensional struc.
-Homodimers
-each monomer has three domainis
1) epidermal growth factor domain (EGF)-near n terminus
2) membrane binding domain(MBD)
3) C-terminal globular catalytic domain(large 460aa)

EGF- essential for folding, has disulfide bonds
MBD-membrane association has 4 alpha helices
Catalytic domain is where AA and NSAIDs bind.
So it has the cyclooxygenase and peroxidase activity here(pictures 5-6)
- a hydrophobic channel that originates in the MBD and extends to the core of the catalytic domain allows for the movement of AA from the lipid bilayer to the catalytic domain picture 5 allows us to see where aspirin does its job(acetylating Ser530) I should say that the hydrophobic pore in COX-2 is about 20% larger
- Generally hydrophobic residues in COX func. To position AA in the active site. Arg120 is the supposed to be the most crucial amino acid for this
- Picture7, shows why COX-2 has a larger pore. Ile434(COX-1)->V434(COX-2) and Ile523(COX-1)->V523(COX-2)

Schuster VL, Molecular Mechanisms of Prostagalndin Transport Annu. Rev. Physiol. 1998. 60.221-242
-Prostaglandin transporter(PGT) is 12 membrane spanning integral membrane protein
-humanPGT and ratPGT show 82% similarity
-extracellular glycosylation sites asparagine
-intracellular phosphorlation sites at serines
-transport is Cl-, and H+ independent
-possibly Na+ dependant

Once transported out of membrane goes to receptor and causes response

Narumiya, Shuh, et. al., Genetic and pharmacological analysis of prostanoid receptor function, J. Clin. Invest, 108, july2001.25-30

-prostanoids(PGD2,PGE2,etc) work locally because they are unstable
-eight receptors
-four subtypes for PGE. Receptors(EP1-4)
-PGF receptor FP
-PGI receptor IP
-PGD receptor DP
-TxA receptor TP
-each has own gene and is G-protein coupled with X membrane spans.
-IP, DP, EP2 ,EP4 mediate cAMP rise, relaxant receptors
-TP, FP, EP1 induce Ca2+ mobilization therefore called contractile
-EP3 induces cAMP decline called inhibitory
picture 9 shows some phenotypes of mice with a gene knockout

Tilley SL. Et al., Mixed messages: modulation of inflammation and immune responses by prostaglandin and thromboxanes, J Clin. Invest., 108, july 2001

-TP receptor is highly expressed in thymus
-PGE2 binding to EP2 or EP4 inhibits wide range of B and T cell func.
-TXA2 may promote T cell activation
-PGE2 and PGI2 both serve is vasodilation. Used agonist for PGE2 & PGI2 called cicaprost and this produced systematic hypOtension
-it is possible that prostanoids might be ligands for nuclear receptors which directly effects transcription.
-picture 10 gives an idea of what the prostanoid does in the immune response

Now I will go through some papers that deal with 1-IP recptor, 2-EP2&4 , and others and the responses.


Murata T. et al., Altered pain perception and inflammatory respoanse in mice lacking Prostacyclin receptor, Nature,388 Aug97.
-the experiments they did showed the importance of IP receptor when activated by ligand.
-PGI2 inhibits platelet aggregation and causes vasodilation found mostly in endothelium
-used cicaprost in their one of their experiments
-in another experiment they used FeCl3, which is known to cause endothelium
picture 11&12 nicely show the results

Lawler, OA et al., Protein Kinase A-mediated Phosphorylation of serine357 of the mouse prostacyclin recptor regulates it s coupling to Gs-to Gi and to Gq-coupled effector signalling, Journal of Bio. Chem., 276,33596-33607,sep7 01

-actions of PGI2 counteract those of TXA2. So the levels of these prostanoids are the central maintenance to vessel tone and hemostasis!
-this is interesting in that Gs can trigger its phosphorylation and, in turn, favor its signalling to Gi and Gq
-PGI2 binds to IP receptor which is a GPCR. It couples directly to Gs.
GalphaS stimulates the formation of AC which we all know causes cAMP formation then PKA levels increase. Since PKA is a Kinase it phosphorylates.
In this instance PKA may Phosphorylate serine357 within the c-tail which may regulate the activity of Gi and Gq.
This phosphorylation favors the activity of Gi which in turn causes a decrease in cAMP
The Phosphorylation also favors activation of Gq pathway
-H-89 PKA inhibitor, U73122 PLC inhibitor
-if H-89 is used then Ca2+ is inhibited shows how Gs is coupled to Gq
-it is also worth noting that the IP GPCR is isoprenylated which is required for Gs and Gq mediated activation of AC and PLC
-picture 13 illustratres this pathway


Zou et al., High glucose via peroxynitrite causes tyrosine nitration and inactivation of prostacyclin synthase that is associated with thromboxane/prostaglandinH2 receptor-mediated apoptosis and adhesion molecule expression in cultured human aortic endothelial cells, Diabetes, 51,198-203,jan2002.

This article shows a pathway that affects PGIS (PGI sythase) and because of this PGI2 synthesis is effected. I made a pedagogical diagram picture 14


Fiebich BL., Mechanisms pf prostaglandin E2- induced interleukin-6 release in astrocytes: possible involvement of EP4-like receptors, p38 mitogen-activation protein kinase and protein kinase C, J of Nuerochemistry,2001,79,950-958

-the differences in the expression of recptors plays a role in what the response will be
-microglial cells have EP1&2 but not EP3&4, astroglial cells have
EP1,2&3, but EP3 to lesser extent, primary astrocytes have all four receptors for PGE2.
-COX-2 is induced by interleukin-1beta in astrocytes
-like I said above PGE2 via EP2 receptor induces cAMP rise
-pic 15 shows a pathway in human astrocytoma cells and the involvement of the EP2 and EP4 receptors in the pathway leading to production of interleukin synthesis

Faouir WH et al., Prostaglandin E2 regulates the level and stability of cyclooxygenase2 mRNA through activation of p38 mitogen-activated protein kinase in interleukin-1beta-treated human synovial fibroblasts, Journal of Bio. Chem., 2001,276,31720-31731.

-interleukin-1(IL-1) is a cytokine associated with proinflammatory, immune, and arthritic diseases.
-COX-2 is one of the genes under IL-1 control.
-I made a diagram showing what they were saying in this paper(picture 16)
-picture 17 shows cox gene
and finally another paper

Fujino H et al., Phosphorylation of glcogen synthase kinase-3 and stimulation of T-cell factor signalling following activation of EP2 and EP4 prostanoid receptors by prostaglandin E2, Journal of Bio. Chem., 2002,277, 2614-2619.

-ways in which GSK3 can be inhibited so Tcf pathway can be carried out
-two forms of GSK3. Aplha(51Kda) and beta(47Kda) these have 85% homology
alpha is phosphorylated by AKT at serine21, beta at serine9
-PKA has been show to phorylate at these positions as well, a crosswalk between wnt and cAMP/PKA signalling.
-EP4 receptor(488 amino acids) larger than EP2(358 amino acids) receptor
- I made a diagram to show the what happens- picture 18

Southall MD et al., Prostaglandin receptor subtypes, EP3C and EP4, mediate the prostaglandin E2 induced cAMP production and sensitization of sensory nuerons, Journal of Bio. Chem., 2001,276,16083-19091.

-exposing sensory neurons to PGE2 increases ; cAMP, # of action potentials, and augments the relaese of neuropeptides.
-this paper shows EP3C and EP4 mediate PGE2 sensitization of sensory neurons
-sensory neurons can express EP1-4 and IP receptors
-PGE2 sensitization decreases the threshold for action potential
-This is thought to be done by cAMP

DISEASES
-Hansen-Petrik MB et al., ProstaglandinE2 protests intestinal tumors from non steroidal anti inflammatory drug induced regression in APC min/+ mice, Cancer research,2002 jan, 62,403-408
-Colorectal cancer is bad. SECOND leading cause of cancer mortality in U.S about sixty thousand people died in 1998 from this form of cancer.
-APC ^(min/+) is used to study colorectal cancer
-germ line vs. somatic mutantions
-NSAIDS block synthetic pathway of prostaglandins, treatment with NSAIDS shows some antitumomorigenic effects
-they found PGE2 is important in maintaining tumor integrity and NSAIDS may reduce tumor by modulating calcium levels
-two diagrams were made to show all this picture 19 and 20

Bibliography
-Ushikubi F. et al., impaired febrile response in mice lacking the prostaglaindin E receptor subtype EP3, nature,1998,395,281-
-Johnson PM et al., COX-2 inhibition attenuates anorexia during systemic inflammation without impairing cytokine production, AJP- endocrinology and metabolism,march 2002,282
-Yamada et al., Prostacyclin synthase gene transfer modulates cyclooxygenase-2 derived prostanoid synthesis and inhibits neointinal formation in rat balloon-injured ateries, Ateriosclerosis, Thrombosis, and vascular biology, feb 2002,22(2)
-Iverson et al., Arachidonic acid metabolism in skin health and disease, Prostaglandins and other lipid mediators,2000,63,25-42
-Frunk CD, Prostaglandins and leukotriens: Advances in Eicosanoid Biology,science,nov 2001,294

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This page was last updated on April 19, 2002

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