ATP citrate lyase knockdown impacts cancer stem cells in vitro.

ATP citrate lyase (ACL) knockdown (KD) causes tumor suppression and induces differentiation. We have previously reported that ACL KD reverses epithelial-mesenchymal transition (EMT) in lung cancer cells. Because EMT is often associated with processes that induce stemness, we hypothesized that ACL KD impacts cancer stem cells. By assessing tumorsphere formation and expression of stem cell markers, we showed this to be the case in A549 cells, which harbor a Ras mutation, and in two other non-small-cell lung cancer cell lines, H1975 and H1650, driven by activating EGFR mutations. Inducible ACL KD had the same effect as stable ACL KD. Similar effects were noted in another well-characterized Ras-induced mammary model system (HMLER). Moreover, treatment with hydroxycitrate phenocopied the effects of ACL KD, suggesting that the enzymatic activity of ACL was critical. Indeed, acetate treatment reversed the ACL KD phenotype. Having previously established that ACL KD impacts signaling through the phosphatidylinositol 3-kinase (PI3K) pathway, not the Ras-mitogen-activated protein kinase (MAPK) pathway, and that EMT can be reversed by PI3K inhibitors, we were surprised to find that stemness in these systems was maintained through Ras-MAPK signaling, and not via PI3K signaling. Snail is a downstream transcription factor impacted by Ras-MAPK signaling and known to promote EMT and stemness. We found that snail expression was reduced by ACL KD. In tumorigenic HMLER cells, ACL overexpression increased snail expression and stemness, both of which were reduced by ACL KD. Furthermore, ACL could not initiate either tumorigenesis or stemness by itself. ACL and snail proteins interacted and ACL expression regulated the transcriptional activity of snail. Finally, ACL KD counteracted stem cell characteristics induced in diverse cell systems driven by activation of pathways outside of Ras-MAPK signaling. Our findings unveil a novel aspect of ACL function, namely its impact on cancer stemness in a broad range of genetically diverse cell types.

Cell surface glypicans are low-affinity endostatin receptors.

Endostatin, a collagen XVIII fragment, is a potent anti-angiogenic protein. We sought to identify its endothelial cell surface receptor(s). Alkaline phosphatase- tagged endostatin bound endothelial cells revealing two binding affinities. Expression cloning identified glypican, a cell surface proteoglycan as the lower-affinity receptor. Biochemical and genetic studies indicated that glypicans' heparan sulfate glycosaminoglycans were critical for endostatin binding. Furthermore, endostatin selected a specific octasulfated hexasaccharide from a sequence in heparin. We have also demonstrated a role for endostatin in renal tubular cell branching morphogenesis and show that glypicans serve as low-affinity receptors for endostatin in these cells, as in endothelial cells. Finally, antisense experiments suggest the critical importance of glypicans in mediating endostatin activities.

Hgs (Hrs), a FYVE domain protein, is involved in Smad signaling through cooperation with SARA.

Smad proteins are effector molecules that transmit signals from the receptors for the transforming growth factor beta (TGF-beta) superfamily to the nucleus; of the Smad proteins, Smad2 and Smad4 are essential components for mouse early embryogenesis. We demonstrated that Hgs, a FYVE domain protein, binds to Smad2 in its C-terminal half and cooperates with another FYVE domain protein, the Smad anchor for receptor activation (SARA), to stimulate activin receptor-mediated signaling through efficient recruitment of Smad2 to the receptor. Furthermore, a LacZ knock-in allele of the C-terminal half-deletion mutant of mouse Hgs was created by gene targeting. The introduced mutation causes an embryonic lethality between embryonic days 8.5 and 10.5. Mutant cells showed significantly decreased responses to stimulation with activin and TGF-beta. These findings suggest that the two FYVE domain proteins, Hgs and SARA, are prerequisites for receptor-mediated activation of Smad2.

Expression of transfected human Na+/H+ exchanger (NHE-1) in the the basolateral membrane of opossum kidney cells.

Some epithelial cells have Na+/H+ exchanger (NHE) activity in both apical and basolateral membranes. Amiloride-sensitive NHE-1 is generally identified in the basolateral membrane. The renal cell line, OK7a, targets amiloride-resistant NHE predominantly to the apical membrane. It is controversial whether the transfected NHE-1 is targeted preferentially to the basolateral membrane in OK7a cells, when human NHE-1 is chronically expressed under control of constitutively active promoters. We tried to identify the membranes in which the transfected human NHE-1 could be detected following acute expression in OK7a cells. We have always observed small Na(+)-dependent pH recovery in the basolateral membrane in OK7a cells. It is, however, controversial whether or not OK7a cells express NHE activity in the basolateral membrane. We also characterized Na(+)-dependent pH recovery in the basolateral membrane. It was not inhibited by [4,4'diisothiocyanatostilbene-2,2'-disulfonic acid] (DIDS), [4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid] (SITS), or contralateral amiloride. Li+ but not K+, chol+, or NMG+ could replace Na+. These results are consistent with the presence of the NHE in the basolateral membrane. NHE activities were predominant in the apical membrane and those in both membranes were resistant to amiloride analogs. After stable transfection with human NHE-1 in a vector utilizing the metallothionein promoter, overnight induction with Zn(2+)increased the NHE activity and its sensitivity to amiloride only in the basolateral membrane in OK7a cells. We conclude that the transfected human NHE-1 is exclusively targeted to the basolateral membrane of OK7a cells during acute induction.

Role of p300, a transcriptional coactivator, in signalling of TGF-beta.

BACKGROUND: Smad proteins are novel transcriptional regulators mediating the signalling of the transforming growth factor-beta (TGF-beta) superfamily. Coactivators such as p300/CBP promote transactivation by various transcription factors through a direct interaction with them. Adenoviral oncoprotein E1A, which binds p300, was shown to inhibit the signalling of TGF-beta. These findings raise the possibility that p300 may be involved in TGF-beta signalling. RESULTS: We investigated whether p300 is involved in transactivation by Smads. p300 enhanced the Smad-induced transactivation of p3TP-Lux, a TGF-beta responsive reporter. E1A inhibited this enhancement, and the inhibition required its ability to bind p300/CBP. p300 and Smad3, as well as Smad2, interacted in vivo in a ligand-dependent manner. The binding region in Smad3 was its C-terminal half that was previously shown to possess an intrinsic transactivation activity. The binding region in p300 was mapped to its C-terminal 678 amino acids. The minimal Smad2/3-interacting region, as well as the rest of the p300, inhibited the transactivation of p3TP-Lux in a dominant-negative fashion. CONCLUSION: p300 interacted with Smad2 and Smad3 in a ligand-dependent manner, and enhanced the transactivation by Smads. Our results present the molecular basis of the transactivation by Smad proteins.

Na+/H+ exchanger (NHE) in the basolateral membrane is encoded by NHE-1 mRNA in LLC-PK1 clone 4 cells.

Several isoforms of Na+/H+ exchanger (NHE-1-5) have been identified. LLC-PK1 clone 4 (CL4) expresses the amiloride-sensitive type of NHE predominantly in the basolateral membrane, which is believed to be NHE-1. It is not clear whether CL4 expresses NHE in the apical membrane and which side of NHE is encoded by the NHE-1 mRNA. Using acidified CL4 cells on the filter membrane, we examined Na(+)-dependent pH recovery of the apical and basolateral membranes separately. Na+ applied to the apical membrane recovered cell pH. Na(+)-dependent pH recovery in the apical membrane was not inhibited by SITS, DIDS, or contralateral amiloride. Li+ but not K+, chol+, or NMG+ could replace Na+. These data are consistent with the presence of NHE in the apical membrane. Transfection with an antisense oligonucleotide corresponding to the 5' terminal site of NHE-1 cDNA of CL4 decreased NHE activity in the basolateral membrane but not in the apical membrane. We conclude that CL4 expresses NHE activities in both apical and basolateral membranes and that NHE-1 mRNA encodes NHE only in the basolateral membrane.