Acute renal failure due to ischemia/reperfusion involves disruption of integrin-mediated cellular adhesion and activation of the extracellular signal-regulated kinase (ERK) pathway. The dynamics of focal adhesion organization and phosphorylation during ischemia/ reperfusion in relation to ERK activation are unknown. In control kidneys, protein tyrosine-rich focal adhesions, containing focal adhesion kinase, paxillin, and talin, were present at the basolateral membrane of tubular cells and colocalized with short F-actin stress fibers. Unilateral renal ischemia/reperfusion caused a reversible protein dephosphorylation and loss of focal adhesions. The focal adhesion protein phosphorylation rebounded in a biphasic manner, in association with increased focal adhesion kinase, Src, and paxillin tyrosine phosphorylation.

The ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) induces apoptosis in S49 mouse lymphoma cells. A variant cell line, S49AR, made resistant to ALP, was found previously to be impaired in ALP uptake via lipid-raft-mediated endocytosis. In the present paper, we report that these cells display cross-resistance to Fas/CD95 ligation [FasL (Fas ligand)], and can be gradually resensitized by prolonged culturing in the absence of ALP. Fas and ALP activate distinct apoptotic pathways, since ALP-induced apoptosis was not abrogated by dominant-negative FADD (Fas-associated protein with death domain), cFLIP(L) [cellular FLICE (FADD-like interleukin 1beta-converting enzyme)-inhibitory protein long form] or the caspase 8 inhibitor Z-IETD-FMK (benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone). ALP-resistant cells showed decreased Fas expression, at both the mRNA and protein levels, in a proteasome-dependent fashion. The proteasome inhibitor MG132 partially restored Fas expression and resensitized the cells to FasL, but not to ALP. Resistant cells completely lacked SM (sphingomyelin) synthesis, which seems to be a unique feature of the S49 cell system, having very low SM levels in parental cells. Lack of SM synthesis did not affect cell growth in serum-containing medium, but retarded growth under serum-free (SM-free) conditions. SM deficiency determined in part the resistance to ALP and FasL. Exogenous short-chain (C12-) SM partially restored cell-surface expression of Fas in lipid rafts and FasL sensitivity, but did not affect Fas mRNA levels or ALP sensitivity. We conclude that the acquired resistance of S49 cells to ALP is associated with down-regulated SM synthesis and Fas gene transcription and that SM in lipid rafts stabilizes Fas expression at the cell surface.

Renal ischemia/reperfusion (I/R) injury is associated with cell matrix and focal adhesion remodeling. Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase that localizes at focal adhesions and regulates their turnover. Here, we investigated the role of FAK in renal I/R injury, using a novel conditional proximal tubule-specific fak-deletion mouse model. Tamoxifen treatment of FAK loxP/loxP //␥GT-Cre-ER T2 mice caused renal-specific fak recombination (FAK ⌬loxP/⌬loxP ) and reduction of FAK expression in proximal tubules. In FAK ⌬loxP/⌬loxP mice compared with FAK loxP/loxP controls, unilateral renal ischemia followed by reperfusion resulted in less tubular damage with reduced tubular cell proliferation and lower expression of kidney injury molecule-1, which was independent from the postischemic inflammatory response. Oxidative stress is involved in the pathophysiology of I/R injury. Primary cultured mouse renal cells were used to study the role of FAK deficiency for oxidative stress in vitro. The conditional fak deletion did not affect cell survival after hydrogen peroxide-induced cellular stress, whereas it impaired the recovery of focal adhesions that were disrupted by hydrogen peroxide. This was associated with reduced c-Jun N-terminal kinase-dependent phosphorylation of paxillin at serine 178 in FAK-deficient cells, which is required for focal adhesion turnover. Our findings support a role for FAK as a novel factor in the initiation of c-Jun N-terminal kinase-mediated cellular stress response during renal I/R injury and suggest FAK as a target in renal injury protection.

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S49 mouse lymphoma cells undergo apoptosis in response to the ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine), FasL (Fas ligand) and DNA damage. S49 cells made resistant to ALP (S49(AR)) are defective in sphingomyelin synthesis and ALP uptake, and also have acquired resistance to FasL and DNA damage. However, these cells can be re-sensitized following prolonged culturing in the absence of ALP. The resistant cells show sustained ERK (extracellular-signal-regulated kinase)/Akt activity, consistent with enhanced survival signalling. In search of a common mediator of the observed cross-resistance, we found that S49(AR) cells lacked the PtdIns(3,4,5)P(3) phosphatase SHIP-1 [SH2 (Src homology 2)-domain-containing inositol phosphatase 1], a known regulator of the Akt survival pathway. Re-sensitization of the S49(AR) cells restored SHIP-1 expression as well as phosphoinositide and sphingomyelin levels. Knockdown of SHIP-1 mimicked the S49(AR) phenotype in terms of apoptosis cross-resistance, sphingomyelin deficiency and altered phosphoinositide levels. Collectively, the results of the present study suggest that SHIP-1 collaborates with sphingomyelin synthase to regulate lymphoma cell death irrespective of the nature of the apoptotic stimulus.

Edelfosine (ALP, Et-18-OCH3) is a prototype alkyl-lysophospholipid that has effective antitumoral activity. It inserts in the membrane and accumulates in lipid rafts leading to apoptosis in S49 mouse T cell lymphoma cells. A variant cell line, made resistant to ALP (S49AR) shows in impaired uptake of ALP. S49AR cells are not only resistant to a variety of ALP analogues, but also to DNA damage and Fas/CD95 death receptor induced apoptosis, suggesting another cause for resistance in addition to impaired ALP uptake. Here we studied the ALP resistance of S49AR. We observe an upregulation of phosho-PKB/Akt and phospho-ERK1/2 which are key proteins in the canonical survival signaling pathways. However inhibition of ERK and/or PKB/Akt using pharmacological inhibitors has no effect on the resistance of the S49AR cells to apoptotic stimuli. PKB/Akt and ERK1/2 activation is regulated by PI(3,4,5)P3 formation. The levels of phosphoinositides in the various cell lines were measured with HPLC. The cell lines resistant to apoptotic inducing agents show decreased levels of the diverse phosphopoinositides. SHIP-1 is a SH2 domain containing inositol phosphatase that removes the 5′ phosphate of PI(3,4,5)P3 and is mainly expressed in hemotopoietic cells. It decreases the pool of PI(3,4,5)P3 thereby negatively regulating the activation of PKB/Akt. We see down regulation of SHIP-1 both on gene expression and protein level in the S49AR compared to S49 cells. Other components of the phosphoinositide metabolism, like PI3Kinase and SHIP2, did not show any changes. Resensitization of the S49AR cells to ALP leads to restoration of SHIP-1 expression. Knocking down SHIP-1 using siRNA causes resistance to various ALPs, DNA damage and Fas/CD95 death receptor induced apoptosis. Microarray analysis show that the S49AR and S49siSHIP cells show remarkably similar expression profile which are distinct from the expression profiles shown by S49 and S49mock cells. Concluding, SHIP-1 is downregulated in ALP resistant T cell lymphoma cells concurrent with a general downregulation of phosphoinositide levels in the cells which is associated with the resistance to ALP induced apoptosis. How ALP resistance is caused is yet unknown and is the scope of current research. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4025.

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