Characterization of an arachidonic acid-deficient (Fads1 knockout) mouse model

Arachidonic acid (20:4Δ5,8,11,14, AA)-derived eicosanoids regulate inflammation and promote cancer development. Previous studies have targeted prostaglandin enzymes in an attempt to modulate AA metabolism. However, due to safety concerns surrounding the use of pharmaceutical agents designed to target Ptgs2 (cyclooxygenase 2) and its downstream targets, it is important to identify new targets upstream of Ptgs2. Therefore, we determined the utility of antagonizing tissue AA levels as a novel approach to suppressing AA-derived eicosanoids. Systemic disruption of the Fads1 (Δ5 desaturase) gene reciprocally altered the levels of dihomo-γ-linolenic acid (20:3Δ8,11,14, DGLA) and AA in mouse tissues, resulting in a profound increase in 1-series-derived and a concurrent decrease in 2-series-derived prostaglandins. The lack of AA-derived eicosanoids, e.g., PGE2, was associated with perturbed intestinal crypt proliferation, immune cell homeostasis, and a heightened sensitivity to acute inflammatory challenge. In addition, null mice failed to thrive, dying off by 12 weeks of age. Dietary supplementation with AA extended the longevity of null mice to levels comparable to wild-type mice. We propose that this new mouse model will expand our understanding of how AA and its metabolites mediate inflammation and promote malignant transformation, with the eventual goal of identifying new drug targets upstream of Ptgs2. Arachidonic acid (20:4Δ5,8,11,14, AA)-derived eicosanoids regulate inflammation and promote cancer development. Previous studies have targeted prostaglandin enzymes in an attempt to modulate AA metabolism. However, due to safety concerns surrounding the use of pharmaceutical agents designed to target Ptgs2 (cyclooxygenase 2) and its downstream targets, it is important to identify new targets upstream of Ptgs2. Therefore, we determined the utility of antagonizing tissue AA levels as a novel approach to suppressing AA-derived eicosanoids. Systemic disruption of the Fads1 (Δ5 desaturase) gene reciprocally altered the levels of dihomo-γ-linolenic acid (20:3Δ8,11,14, DGLA) and AA in mouse tissues, resulting in a profound increase in 1-series-derived and a concurrent decrease in 2-series-derived prostaglandins. The lack of AA-derived eicosanoids, e.g., PGE2, was associated with perturbed intestinal crypt proliferation, immune cell homeostasis, and a heightened sensitivity to acute inflammatory challenge. In addition, null mice failed to thrive, dying off by 12 weeks of age. Dietary supplementation with AA extended the longevity of null mice to levels comparable to wild-type mice. We propose that this new mouse model will expand our understanding of how AA and its metabolites mediate inflammation and promote malignant transformation, with the eventual goal of identifying new drug targets upstream of Ptgs2. Prostaglandins of the 2-series (e.g., PGE2) found in high abundance in colorectal tissue, are downstream products of cyclooxygenase-2 (Ptgs2). A significant increase in Ptgs2 gene expression has been shown to promote PGE2-dependent colon cancer development, in part by enhancing cell proliferation and repressing protective proapoptotic signaling pathways (1.Castellone M.D. Teramoto H. Williams B.O. Druey K.M. Gutkind J.S. Prostaglandin E2 promotes colon cancer cell growth through a GS-axin-β-catenin signaling axis.Science. 2005; 310: 1504-1510Crossref PubMed Scopus (764) Google Scholar, 2.Greenhough A. Wallam C.A. Hicks D.J. Moorghen M. Willams A.C. Paraskeva C. The proapoptotic BH2-only protein Bim is downregulated in a subset of colorectal cancers and is repressed by antiapoptotic COX-2/PGE2 signalling in colorectal adenoma cells.Oncogene. 2010; 29: 3398-3410Crossref PubMed Scopus (38) Google Scholar). Recent controversies associated with the role of aspirin and other Ptgs2 inhibitors indicate that more work is needed to elucidate the effects of PGE2 in cancer initiation, progression, and metastasis (3.Berg M. Soreide K. Will an aspirin a day keep the colorectal cancer away?.Nat. Rev. Clin. Oncol. 2011; 8: 130-131Crossref PubMed Scopus (5) Google Scholar). The preferred substrate for cyclooxygenase catalysis of either Ptgs1 or Ptgs2 is arachidonic acid (20:4Δ5,8,11,14, AA) (4.Smith W.L. Cyclooxygenases, peroxide tone and allure of fish oil.Curr. Opin. Cell Biol. 2005; 17: 174-182Crossref PubMed Scopus (133) Google Scholar). AA is derived metabolically from linoleic acid (18:2Δ9,12, LA), the major polyunsaturated fatty acid (PUFA) in the diet. Dietary LA is the major source of tissue dihomo-γ-linolenic acid (20:3Δ8,11,14, DGLA) and AA, and its metabolism is regulated by the complementary action of fatty acid desaturases (Fads1 and Fads2) (5.Mathias R.A. Vergara C. Gao L. Rafaels N. Hand T. Campbell M. Bickel C. Ivester P. Sergeant S. Barnes K.C. et al.FADS genetic variants in ω-6 polyunsaturated fatty acid metabolism in a homogeneous island population.J. Lipid Res. 2010; 51: 2766-2774Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) (see supplementary Fig. I). In addition to LA, dietary AA can contribute to tissue AA levels. However, compared with the dietary intake of LA (10–20 g/day), AA intake (100–500 mg/day) is a very minor contributor (6.Taber L. Chiu C.H. Whelan J. Assessment of the arachidonic acid content of foods commonly consumed in the American diet.Lipids. 1998; 33: 1151-1157Crossref PubMed Scopus (77) Google Scholar). AA-derived eicosanoids, including PGE2, function as mediators of immune inflammation. Despite the traditional belief that PGE2 acts as an immunosuppressant based on its inhibition of T-cell activation and cytokine [tumor necrosis factor (TNF)α, interleukin (IL)-12] production in vitro (7.van der Pouw Kraan T.C. Boeije L.C. Smeenk R.J. Wildenes J. Aarden L.A. Prostaglandin-E2 is a potent inhibitor of human interleukin 12 production.J. Exp. Med. 1995; 181: 775-779Crossref PubMed Scopus (633) Google Scholar), PGE2 has now emerged as an immunoactivator, which facilitates Th1 differentiation and Th17 cell expansion, two T-cell subsets involved in adaptively mediated inflammation (8.Sakata D. Yao C. Narumiya S. Prostaglandin E2, an immunoactivator.J. Pharmacol. Sci. 2010; 112: 1-5Crossref PubMed Scopus (97) Google Scholar). Therefore, PGE2 has the ability to influence the cytokine microenvironment, thereby skewing naïve T-cell differentiation, and ultimately function, toward inflammatory T-cell subsets. For example, it has been reported that PGE2 propagates inflammatory bowel disease (IBD) by enhancing the development and function of IL-17-producing Th17 cells (9.Barrie A. Khare A. Henkel M. Zhang Y. Barmada M.M. Duerr R. Ray A. Prostaglandin E2 and IL-23 plus IL-1β differentially regulate the Th1/Th17 immune response of human CD161(+) CD4(+) memory T cells.Clin. Transl. Sci. 2011; 4: 268-273Crossref PubMed Scopus (25) Google Scholar). In addition to its role in cytokine regulation, AA is a major constituent of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), and via the action of phospholipase C, results in the accumulation of inositol trisphosphate (IP3) and 1-stearoyl-2-AA-diacylglycerol to elicit intracellular Ca2+ mobilization (10.Poccia D. Larijani B. Phosphatidylinositol metabolism and membrane fusion.Biochem. J. 2009; 418: 233-246Crossref PubMed Scopus (53) Google Scholar). The hydrolysis of PI(4,5)P2 plays an important role in membrane rapid cytoskeletal remodeling (11.Hao J.J. Liu Y. Kruhlak M. Debell K.E. Rellahan B.L. Shaw S. Phospholipase C-mediated hydrolysis of PIP2 releases ERM proteins from lymphocyte membrane.J. Cell Biol. 2009; 184: 451-462Crossref PubMed Scopus (109) Google Scholar) and Ras/Erk, as well as Ca2+ signaling in lymphocytes (12.Huang Y.H. Sauer K. Lipid signaling in T cell development and function.Cold Spring Harb. Perspect. Biol. 2010; 2: a002428Crossref PubMed Scopus (50) Google Scholar). Most studies have targeted prostaglandin biosynthetic and degradation enzymes in an attempt to suppress AA-derived eicosanoid-mediated inflammation and tumor-promoting action (13.Wu W.K. Sung J.J. Lee C.W. Yu J. Cho C.H. Cyclooxygenase-2 in tumorigenesis of gastrointestinal cancers: an update on the molecular mechanisms.Cancer Lett. 2010; 295: 7-16Crossref PubMed Scopus (135) Google Scholar). Surprisingly, no investigators to date have attempted to target AA (substrate levels) as a way of modulating prostaglandin biosynthesis and tumor development. The controversies associated with the role of aspirin and Ptgs inhibitors indicate that more work is needed to elucidate the effects of eicosanoids in colon cancer and inflammatory diseases (3.Berg M. Soreide K. Will an aspirin a day keep the colorectal cancer away?.Nat. Rev. Clin. Oncol. 2011; 8: 130-131Crossref PubMed Scopus (5) Google Scholar). Therefore, we generated a novel genetic model, i.e., the Fads1 (Δ5 desaturase) knockout mouse, to determine the role of AA-derived 2-series eicosanoids in mucosal physiology and inflammation. This model allows for the specific investigation of AA deficiency without the underlying complications of essential fatty acid (LA and DGLA) deficiency. Mutant Fads1 mice were generated using a gene-trapping technique (14.Durick K. Mendlein J. Xanthopoulos K.G. Hunting with traps: genome-wide strategies for gene discovery and functional analysis.Genome Res. 1999; 9: 1019-1025Crossref PubMed Scopus (31) Google Scholar). Mice (strain C57BL/6) were cloned from an ES cell line (IST11525H2; Texas Institute for Genomic Medicine, TIGM). The ES cell clone contained a retroviral insertion in the Fads1 gene identified from the TIGM gene trap database, and was microinjected into C57BL/6 host blastocysts to generate germline chimeras using standard procedures. The retroviral OmniBank Vector 76 (Fig. 1) contained a splice acceptor sequence (SA) followed by a 5′ selectable marker β-geo, a functional fusion between the β-galactosidase and neomycin resistance genes, for identification of successful gene trap events followed by a polyadenylation signal (pA). Insertion of the retroviral vector into the Fads1 gene led to the splicing of the endogenous upstream exons into this cassette to produce a fusion transcript that was used to generate a sequence tag (OST) of the trapped gene by 3′ RACE (15.Zambrowicz B.P. Friedrich G.A. Buxton E.C. Lilleberg S.L. Person C. Sands A.T. Disruption and sequence identification of 2,000 genes in mouse embryonic stem cells.Nature. 1998; 392: 608-611Crossref PubMed Scopus (400) Google Scholar). Chimeric males were bred to C57BL/6 females for germline transmission of the mutant Fads1 allele. Three genotypes [wild-type (Wt), heterozygous (Het) and null (Null)] of Fads1 mice were derived from heterozygous males and females. All procedures followed the guidelines approved by Public Health Service and the Institutional Animal Care and Use Committee at Texas A&M All were of In a the was with AA, to determine the of dietary AA on the of mice. Mice were at the of For was using a and tissue was to determine the of the gene trap using Y. L.A. L. Y. S. et effects of dietary fatty are in of knockout 2009; PubMed Scopus Google Scholar). (Fads1 Vector For were from mouse using the of et J. M. A for the and of from Biol. Full Text PDF PubMed Google Scholar) and in acid were by as of to linoleic acid into arachidonic of Google Scholar). Fads1 and expression levels in colon and were determined by on an was from using and with was using levels were to expression using from for for and for in colon and were by the of J. M. A for the and of from Biol. 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PubMed Scopus Google Scholar). were using of were Fads1 knockout mice were generated by a gene-trapping technique in and was by shown in Fig. Fads1 gene was in and the gene trap was in and that targeted in the in Fads1 and expression levels in mouse colon and were by shown in Fig. Fads1 expression levels were altered as in and mice compared with In expression levels were the the source of AA for the Fads1 were an of was no significant of on Fig. to mice to at weeks of with no 12 weeks of (Fig. The at of was weeks and of a no between and mice were mice at weeks of this in mice of the were no of or as as 12 to of the mice. mice lack of or of no between and mice (e.g., and Fig. were Mice were In an attempt to the levels of AA or were to the diet. The of supplementation extended the longevity of mice to levels comparable with and mice (Fig. the mice were to to acute intestinal inflammation J.S. L.A. L. et fish and to modulate and gene expression in J. 2011; PubMed Scopus (53) Google Scholar, S. C. B. mouse of intestinal 2: PubMed Scopus Google Scholar). All and mice the challenge. In mice a significant in the Fig. indicate that Fads1 mice are to an acute intestinal inflammatory challenge. fatty acid were in Fads1 mouse and (Fig. were in the levels of and AA in mice compared with and with AA to levels in in the levels of AA and in mice were comparable with the of in mice was from that in and mice (Fig. and AA are to and 2-series and Therefore, we the of gene in and AA levels with to the biosynthesis of and 2-series prostaglandins. Prostaglandins were from and and by (Fig. PGE2 levels were levels were in mice mice an has been reported that AA-derived cell proliferation in colon cancer cells Sung The effects of the cyclooxygenase-2 prostaglandin E2 on the proliferation of colon cancer Pharmacol. Exp. 2011; PubMed Scopus Google Scholar). intestinal PGE2 was in Fads1 null mice we the of cell proliferation in mouse colon shown in Fig. of the Fads1 gene the of cells in the of cells was in compared with and mice. mice an between and with the levels of AA and PGE2 (Fig. Fig. membrane PI(4,5)P2 is a of metabolism in T cells (12.Huang Y.H. Sauer K. Lipid signaling in T cell development and function.Cold Spring Harb. Perspect. Biol. 2010; 2: a002428Crossref PubMed Scopus (50) Google Scholar) and its is of we the of AA associated with the of the Fads1 gene altered the PI(4,5)P2 in T The of PI(4,5)P2 was by in Fads1 T cells compared with and (Fig. AA-derived eicosanoids an important role in immune Zhang P. R.J. The of T cell immune toward cell development by prostaglandin 2011; PubMed Scopus Google Scholar, D. and for disease J. PubMed Scopus Google Scholar), we the effects associated with a deficiency in AA-derived 2-series prostaglandins. For this we compared immune cell in Fads1 and mice. cells were and T-cell and were by shown in Fig. the of cells and of T cells were in mice. In was no in the of cell the In complementary were from cells and from and and were with agents designed to or T cells cell of T cells and from and mice contained levels of and T-cell activation (Fig. and supplementary of inflammatory T cells were from and and and with of plus of for were and cytokine levels were using a are the indicate significant In this we determined the utility of antagonizing tissue AA levels as a novel approach to suppressing AA-derived eicosanoids (e.g., PGE2) For this we have generated a genetic model, the Fads1 (Δ5 desaturase) knockout In tissues, LA is to AA by an sequence of gene and (Fads1 gene in which are to new and two are to the fatty acid P. of desaturases by and other 2011; PubMed Scopus Google Scholar). Dietary AA the and can by cyclooxygenase enzymes and to generate tumor-promoting 2-series (e.g., PGE2) Fig. of dietary acid in human and 1998; PubMed Scopus Google Scholar). that we the Fads1 fatty acid that Fads1 null mice have levels of AA compared with heterozygous and wild-type mice (Fig. and supplementary effects were in the and a of In Fads1 in the of dihomo-γ-linolenic acid (20:3Δ8,11,14, the prostaglandin substrate in (Fig. the in prostaglandin levels was associated with a profound in and AA-derived biosynthesis (Fig. derived e.g., with compared with PGE2 of dietary acid in human and 1998; PubMed Scopus Google Scholar, D. M. J. A. A. S. S. and PGE2 function through Lipid 2011; PubMed Scopus (50) Google Scholar). This is is of colon cancer cell proliferation J. S. P. of acid and prostaglandin on human colon cell Full Text PDF PubMed Scopus Google Scholar, H. S. A. C. of prostaglandin on growth factor production by human and colon cancer Exp. Clin. Res. Google Scholar) as to PGE2, which promotes growth (1.Castellone M.D. Teramoto H. Williams B.O. Druey K.M. Gutkind J.S. Prostaglandin E2 promotes colon cancer cell growth through a GS-axin-β-catenin signaling axis.Science. 2005; 310: 1504-1510Crossref PubMed Scopus (764) Google Scholar, D. The role of in intestinal inflammation and colorectal 2010; 29: PubMed Scopus Google Scholar). Therefore, we are in a to the of on mice that have no 2-series prostaglandin substrate (20:4Δ5,8,11,14, with the of AA in membrane it is that PIP2 levels were in T cells from mice (Fig. that PIP2 is of molecular C.H. of and and the in the molecular of in PubMed Scopus Google Scholar), we propose that the lack of AA substrate for the remodeling to a in its R. polyunsaturated fatty suppress remodeling T cell J. PubMed Scopus Google Scholar, H. S. A. C. of prostaglandin on growth factor production by human and colon cancer Exp. Clin. Res. Google Scholar, M. S. H. in between and fatty PubMed Scopus Google Scholar). PIP2 the of proteins and as a source of K. how of PI(4,5)P2 are in the Sci. 2010; PubMed Scopus Google Scholar), work is needed to how AA deficiency and signaling at the will how in response to of Disruption of the Fads1 genes in mice For example, of in and intestinal M. E.C. P. Zhang Y. Cho M. R.A. et of gene in mice and and intestinal Lipid Res. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. R.A. acid supplementation and in Lipid Res. 2010; 51: Full Text Full Text PDF PubMed Scopus Google B. B. C. M. S. et deficiency the role of and fatty J. PubMed Scopus Google Scholar). In we that null mice failed to thrive, dying off at weeks of with no 12 weeks of (Fig. In the in null mice were weeks of for females and weeks for males M. E.C. P. Zhang Y. Cho M. R.A. et of gene in mice and and intestinal Lipid Res. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. R.A. acid supplementation and in Lipid Res. 2010; 51: Full Text Full Text PDF PubMed Scopus Google B. B. C. M. S. et deficiency the role of and fatty J. PubMed Scopus Google Scholar). Fads1 mice to events were A factor for the of the null mice was the of AA in the M. M. R.A. acid supplementation and in Lipid Res. 2010; 51: Full Text Full Text PDF PubMed Scopus Google Scholar, B. B. C. M. S. et deficiency the role of and fatty J. PubMed Scopus Google Scholar). This have the mice to and and In addition, in the genetic of the mice have the All null mice were on a Fads1 mice were on a C57BL/6 In addition, between metabolically to the that eicosanoids are to for of the AA-derived of dietary acid in human and 1998; PubMed Scopus Google Scholar). In to Fads1 mice are to due to the of M. E.C. P. Zhang Y. Cho M. R.A. et of gene in mice and and intestinal Lipid Res. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). the of the of in Fads1 mice is this was supplementation of AA to the (Fig. to the underlying the of immune cell in Fads1 knockout mice (Fig. Fig. the of PI(4,5)P2 and PGE2 in immune cell function Zhang of with to regulate 2005; PubMed Scopus Google Scholar, in membrane Sci. 2009; PubMed Scopus Google M. N. Prostaglandin E2 and T or Cell Biol. 2011; of Google Scholar), it is that the of mediators to a disruption in immune cell The lack of AA-derived production is with the in intestinal crypt proliferation (Fig. and the of Fads1 mice to an acute intestinal inflammatory Fig. effects have been reported in mice and cyclooxygenase-2 deficiency M. A. T. S. A.T. PGE2 colon and mucosal Res. 2011; 4: PubMed Scopus Google Scholar, M.M. J. T. M. Liu C. et deficiency to intestinal and 2011; PubMed Scopus Google Scholar). PGE2 in the has a protective on the of the intestinal and its promotes a disease M.M. J. T. M. Liu C. et deficiency to intestinal and 2011; PubMed Scopus Google Scholar). the effects of Fads1 on the of AA-derived eicosanoids is it is to the of tissue AA to the specific role of In we have generated and a novel (Fads1 mouse mice this can by AA to the diet. in it will to membrane levels of indicate that and AA-derived mediators are reciprocally in this the role of AA-derived eicosanoids in and immune cell we are in a to our to determine at a the utility of antagonizing tissue AA levels as a novel approach to suppressing inflammation and The for the with arachidonic acid cyclooxygenase dihomo-γ-linolenic acid fatty acid (Δ5 desaturase) heterozygous interleukin linoleic acid major prostaglandin tumor necrosis factor wild-type

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