Investigating the effect of arachidonate supplementation on the phosphoinositide content of MCF10a breast epithelial cells

AbstractPhosphoinositides in primary mammalian tissue are highly enriched in a stearoyl/arachidonyl (C38:4) diacylgycerol backbone. However, mammalian cells grown in culture typically contain more diverse molecular species of phosphoinositides, characterised by a reduction in arachidonyl content in the sn-2 position. We have analysed the phosphoinositide species in MCF10a cells grown in culture by mass spectrometry. Under either serum or serum starved conditions the most abundant species of PI, PIP, PIP2 and PIP3 had masses which corresponded to C36:2, C38:4, C38:3, C38:2 and C36:1 diacylglycerol backbones and the relative proportions of each molecular species were broadly similar between each phosphoinositide class (approx. 50%, 25%, 10%, 10% and 10% respectively, for the species listed above). Supplementing the culture medium with BSA-loaded arachidonic acid promoted a rapid increase in the proportion of the C38:4 species in all phosphoinositide classes (from approx. 25%–60% of total species within 24 h), but the total amount of all combined species for each class remained remarkably constant. Stimulation of cells, cultured in either normal or arachidonate-enriched conditions, with 2 ng/ml EGF for 90 s caused substantial activation of Class I PI3K and accumulation of PIP3. Despite the increased proportion of C38:4 PIP3 under the arachidonate-supplemented conditions, the total amount of all combined PIP3 species accumulating in response to EGF was the same, with or without arachidonate supplementation; there were however small but significant preferences for the conversion of some PIP2 species to PIP3, with the polyunsaturated C38:4 and C38:3 species being more favoured over other species. These results suggest the enzymes which interconvert phosphoinositides are able to act on several different molecular species and homoeostatic mechanisms are in place to deliver similar phosphoinositide pool sizes under quite different conditions of arachidonate availability. They also suggest enzymes regulating PIP3 levels downstream of growth factor stimulation (i.e. PI3Ks and PIP3-phosphatases) show some acyl selectivity and further work should be directed at assessing whether different acyl species of PIP3 exhibit differing signalling potential

BMX Acts Downstream of PI3K to Promote Colorectal Cancer Cell Survival and Pathway Inhibition Sensitizes to the BH3 Mimetic ABT-737

Evasion of apoptosis is a hallmark of cancer, and reversing this process by inhibition of survival signaling pathways is a potential therapeutic strategy. Phosphoinositide 3-kinase (PI3K) signaling can promote cell survival and is upregulated in solid tumor types, including colorectal cancer (CRC), although these effects are context dependent. The role of PI3K in tumorigenesis combined with their amenability to specific inhibition makes them attractive drug targets. However, we observed that inhibition of PI3K in HCT116, DLD-1, and SW620 CRC cells did not induce apoptotic cell death. Moreover, these cells were relatively resistant to the Bcl-2 homology domain 3 (BH3) mimetic ABT-737, which directly targets the Bcl-2 family of apoptosis regulators. To test the hypothesis that PI3K inhibition lowers the apoptotic threshold without causing apoptosis per se, PI3K inhibitors were combined with ABT-737. PI3K inhibition enhanced ABT-737-induced apoptosis by 2.3- to 4.5-fold and reduced expression levels of MCL-1, the resistance biomarker for ABT-737. PI3K inhibition enhanced ABT-737-induced apoptosis a further 1.4- to 2.4-fold in CRC cells with small interfering RNA-depleted MCL-1, indicative of additional sensitizing mechanisms. The observation that ABT-737-induced apoptosis was unaffected by inhibition of PI3K downstream effectors AKT and mTOR, implicated a novel PI3K-dependant pathway. To elucidate this, an RNA interference (RNAi) screen of potential downstream effectors of PI3K signaling was conducted, which demonstrated that knockdown of the TEC kinase BMX sensitized to ABT-737. This suggests that BMX is an antiapoptotic downstream effector of PI3K, independent of AKT

Lysophosphatidylinositol-acyltransferase-1 (LPIAT1) is required to maintain physiological levels of PtdIns and PtdInsP(2) in the mouse.

We disrupted the gene encoding lysophosphatidylinositol-acyltransferase-1 (LPIAT1) in the mouse with the aim of understanding its role in determining cellular phosphoinositide content. LPIAT1(-/-) mice were born at lower than Mendelian ratios and exhibited a severe developmental brain defect. We compared the phospholipid content of livers and brains from LPIAT1(-/-) and LPIAT1(+/+) littermates by LC-ESI/MS. In accord with previous studies, the most abundant molecular species of each phosphoinositide class (PtdIns, PtdInsP, PtdInsP2 and PtdInsP3) possessed a C38∶4 complement of fatty-acyl esters (C18∶0 and C20∶4 are usually assigned to the sn-1 and sn-2 positions, respectively). LPIAT1(-/-) liver and brain contained relatively less of the C38∶4 species of PtdIns, PtdInsP and PtdInsP2 (dropping from 95-97% to 75-85% of the total species measured for each lipid class) and relatively more of the less abundant species (PtdInsP3 less abundant species were below our quantification levels). The increases in the less abundant PtdIns and PtdInsP2 species did not compensate for the loss in C38∶4 species, resulting in a 26-44% reduction in total PtdIns and PtdInsP2 levels in both brain and liver. LPIAT1(-/-) brain and liver also contained increased levels of C18∶0 lyso-PtdIns (300% and 525% respectively) indicating a defect in the reacylation of this molecule. LPIAT1(-/-) brain additionally contained significantly reduced C38∶4 PC and PE levels (by 47% and 55% respectively), possibly contributing to the phenotype in this organ. The levels of all other molecular species of PC, PE, PS and PA measured in the brain and liver were very similar between LPIAT1(-/-) and LPIAT1(+/+) samples. These results suggest LPIAT1 activity plays a non-redundant role in maintaining physiological levels of PtdIns within an active deacylation/reacylation cycle in mouse tissues. They also suggest that this pathway must act in concert with other, as yet unidentified, mechanisms to achieve the enrichment observed in C38∶4 molecular species of phosphoinositides

Effect of LPIAT1 knockout on brain and liver lyso-phospholipid molecular species.

<p>Brains (<b>A</b>) or livers (<b>B</b>) from 13 day old littermates expressing (LPIAT1^+/+ (WT)) or lacking (LPIAT1^−/− (KO)) LPIAT1 were ground, homogenized and lipids extracted from 0.5 mg wet weight as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058425#s2" target="_blank">Materials and Methods</a>. Lyso-phospholipids were targeted and were detected by MRM mass spectrometric anaylsis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058425#s2" target="_blank">Materials and Methods</a>. Data are expressed as moles/mg protein, normalized to relevant internal standards. Shown are mean ± SD, n = 4 for both WT and KO. Data were analyzed by T-test. *p≤0.05, **p≤0.005.</p

Generation of LPIAT1 knockout first mice.

<p>(<b>A</b>) Schematic representation of LPIAT^{tm1a(KOMP)Wtsi} gene targeting vector, constructed by the High throughput gene targeting group at the Sanger Center as a ‘knock-out first’ allele that abrogates expression of the targeted allele. Shown are expected enzymatic digest fragments for EcoRI, HindIII and AseI digests of the correctly targeted allele, which were confirmed by Southern analysis of three ES cell clones and WT BL6 genomic DNA control using a [³²P]-oligonucleotide probe to a 600bp region of the Neomycin gene within the targeting cassette (<b>B</b>). (<b>C</b>) Genotyping of mice was performed by PCR amplification to identify the presence of LPIAT1 WT allele (+) and/or targeted KO allele (−) from LPIAT1^+/−, LPIAT1^+/+ and LPIAT1^−/− mice. (<b>D</b>) Western blot analysis was performed on 50 µg wet weight brain tissue from three pairs of LPIAT1^+/+ (WT) and LPIAT1^−/− (KO) 13 day old littermates as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058425#s2" target="_blank">Materials and Methods</a>. Shown is a representative immunoblot, with the arrow indicating the position of LPIAT1 protein.</p

Effect of LPIAT1 knockout on brain phosphoinositide lipid molecular species.

<p>Brains from 13 day old littermates expressing (LPIAT1^+/+ (WT)) or lacking (LPIAT1^−/− (KO)) LPIAT1 were homogenised and lipid extracted from 0.5 mg wet weight as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058425#s2" target="_blank">Materials and Methods</a>. Targeted molecular species of PtdIns (<b>A</b>), PtdInsP (<b>B</b>), PtdInsP₂ (<b>C</b>) and PtdInsP₃ (<b>D</b>) were detected by MRM mass spectrometric analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058425#s2" target="_blank">Materials and Methods</a>. Data are expressed as moles/mg protein, normalized to relevant internal standards. Shown are mean ± SD, n = 4 for both WT and KO. Data were analyzed by T-test. *p≤0.05, **p≤0.005.</p

Numbers of genotypes in litters from LPIAT1^+/− crosses.

<p>Numbers of genotypes in litters from LPIAT1^+/− crosses.</p

Effect of LPIAT1 knockout on liver phospholipid molecular species.

<p>Livers from 13 day old littermates expressing (LPIAT1^+/+ (WT)) or lacking (LPIAT1^−/− (KO)) LPIAT1 were homogenized and lipids extracted from 0.5 mg wet weight as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058425#s2" target="_blank">Materials and Methods</a>. Targeted molecular species of PC (<b>A</b>), PE (<b>B</b>), PS (<b>C</b>) and PA (<b>D</b>) were detected by MRM mass spectrometric analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058425#s2" target="_blank">Materials and Methods</a>. Data are expressed as moles/mg protein, normalized to relevant internal standards. Shown are mean ± SD, n = 4 for both WT and KO. Data were analyzed by T-test.</p

Effect of LPIAT1 knockout on brain phospholipid molecular species.

<p>Brains from 13 day old littermates expressing (LPIAT1^+/+ (WT)) or lacking (LPIAT1^−/− (KO)) LPIAT1 were homogenized and lipids extracted from 0.5 mg wet weight as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058425#s2" target="_blank">Materials and Methods</a>. Targeted molecular species of PC (<b>A</b>), PE (<b>B</b>), PS (<b>C</b>) and PA (<b>D</b>) were detected by MRM mass spectrometric analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058425#s2" target="_blank">Materials and Methods</a>. Data are expressed as moles/mg protein, normalized to relevant internal standards. Shown are mean ± SD, n = 4 for both WT and KO. Data were analyzed by T-test. *p≤0.05, **p≤0.005.</p