IGF1R signaling drives antiestrogen resistance through PAK2/PIX activation in luminal breast cancer.

Antiestrogen resistance in estrogen receptor positive (ER+) breast cancer is associated with increased expression and activity of insulin-like growth factor 1 receptor (IGF1R). Here, a kinome siRNA screen has identified 10 regulators of IGF1R-mediated antiestrogen with clinical significance. These include the tamoxifen resistance suppressors BMPR1B, CDK10, CDK5, EIF2AK1, and MAP2K5, and the tamoxifen resistance inducers CHEK1, PAK2, RPS6KC1, TTK, and TXK. The p21-activated kinase 2, PAK2, is the strongest resistance inducer. Silencing of the tamoxifen resistance inducing genes, particularly PAK2, attenuates IGF1R-mediated resistance to tamoxifen and fulvestrant. High expression of PAK2 in ER+ metastatic breast cancer patients is correlated with unfavorable outcome after first-line tamoxifen monotherapy. Phospho-proteomics has defined PAK2 and the PAK-interacting exchange factors PIXα/ß as downstream targets of IGF1R signaling, which are independent from PI3K/ATK and MAPK/ERK pathways. PAK2 and PIXα/ß modulate IGF1R signaling-driven cell scattering. Targeting PIXα/ß entirely mimics the effect of PAK2 silencing on antiestrogen re-sensitization. These data indicate PAK2/PIX as an effector pathway in IGF1R-mediated antiestrogen resistance.

Elucidation of Signaling Pathways from Large-Scale Phosphoproteomic Data Using Protein Interaction Networks.

Phosphoproteomic experiments typically identify sites within a protein that are differentially phosphorylated between two or more cell states. However, the interpretation of these data is hampered by the lack of methods that can translate site-specific information into global maps of active proteins and signaling networks, especially as the phosphoproteome is often undersampled. Here, we describe PHOTON, a method for interpreting phosphorylation data within their signaling context, as captured by protein-protein interaction networks, to identify active proteins and pathways and pinpoint functional phosphosites. We apply PHOTON to interpret existing and novel phosphoproteomic datasets related to epidermal growth factor and insulin responses. PHOTON substantially outperforms the widely used cutoff approach, providing highly reproducible predictions that are more in line with current biological knowledge. Altogether, PHOTON overcomes the fundamental challenge of delineating signaling pathways from large-scale phosphoproteomic data, thereby enabling translation of environmental cues to downstream cellular responses.

Annexin A1 sustains tumor metabolism and cellular proliferation upon stable loss of HIF1A.

Despite the approval of numerous molecular targeted drugs, long-term antiproliferative efficacy is rarely achieved and therapy resistance remains a central obstacle of cancer care. Combined inhibition of multiple cancer-driving pathways promises to improve antiproliferative efficacy. HIF-1 is a driver of gastric cancer and considered to be an attractive target for therapy. We noted that gastric cancer cells are able to functionally compensate the stable loss of HIF-1α. Via transcriptomics we identified a group of upregulated genes in HIF-1α-deficient cells and hypothesized that these genes confer survival upon HIF-1α loss. Strikingly, simultaneous knock-down of HIF-1α and Annexin A1 (ANXA1), one of the identified genes, resulted in complete cessation of proliferation. Using stable isotope-resolved metabolomics, oxidative and reductive glutamine metabolism was found to be significantly impaired in HIF-1α/ANXA1-deficient cells, potentially explaining the proliferation defect. In summary, we present a conceptually novel application of stable gene inactivation enabling in-depth deconstruction of resistance mechanisms. In theory, this experimental approach is applicable to any cancer-driving gene or pathway and promises to identify various new targets for combination therapies.

Annexin A1 expression in a pooled breast cancer series: association with tumor subtypes and prognosis.

BACKGROUND: Annexin A1 (ANXA1) is a protein related with the carcinogenesis process and metastasis formation in many tumors. However, little is known about the prognostic value of ANXA1 in breast cancer. The purpose of this study is to evaluate the association between ANXA1 expression, BRCA1/2 germline carriership, specific tumor subtypes and survival in breast cancer patients. METHODS: Clinical-pathological information and follow-up data were collected from nine breast cancer studies from the Breast Cancer Association Consortium (BCAC) (n = 5,752) and from one study of familial breast cancer patients with BRCA1/2 mutations (n = 107). ANXA1 expression was scored based on the percentage of immunohistochemical staining in tumor cells. Survival analyses were performed using a multivariable Cox model. RESULTS: The frequency of ANXA1 positive tumors was higher in familial breast cancer patients with BRCA1/2 mutations than in BCAC patients, with 48.6 % versus 12.4 %, respectively; P <0.0001. ANXA1 was also highly expressed in BCAC tumors that were poorly differentiated, triple negative, EGFR-CK5/6 positive or had developed in patients at a young age. In the first 5 years of follow-up, patients with ANXA1 positive tumors had a worse breast cancer-specific survival (BCSS) than ANXA1 negative (HRadj = 1.35; 95 % CI = 1.05-1.73), but the association weakened after 10 years (HRadj = 1.13; 95 % CI = 0.91-1.40). ANXA1 was a significant independent predictor of survival in HER2+ patients (10-years BCSS: HRadj = 1.70; 95 % CI = 1.17-2.45). CONCLUSIONS: ANXA1 is overexpressed in familial breast cancer patients with BRCA1/2 mutations and correlated with poor prognosis features: triple negative and poorly differentiated tumors. ANXA1 might be a biomarker candidate for breast cancer survival prediction in high risk groups such as HER2+ cases.

Tumor cell migration screen identifies SRPK1 as breast cancer metastasis determinant.

Tumor cell migration is a key process for cancer cell dissemination and metastasis that is controlled by signal-mediated cytoskeletal and cell matrix adhesion remodeling. Using a phagokinetic track assay with migratory H1299 cells, we performed an siRNA screen of almost 1,500 genes encoding kinases/phosphatases and adhesome- and migration-related proteins to identify genes that affect tumor cell migration speed and persistence. Thirty candidate genes that altered cell migration were validated in live tumor cell migration assays. Eight were associated with metastasis-free survival in breast cancer patients, with integrin ß3-binding protein (ITGB3BP), MAP3K8, NIMA-related kinase (NEK2), and SHC-transforming protein 1 (SHC1) being the most predictive. Examination of genes that modulate migration indicated that SRPK1, encoding the splicing factor kinase SRSF protein kinase 1, is relevant to breast cancer outcomes, as it was highly expressed in basal breast cancer. Furthermore, high SRPK1 expression correlated with poor breast cancer disease outcome and preferential metastasis to the lungs and brain. In 2 independent murine models of breast tumor metastasis, stable shRNA-based SRPK1 knockdown suppressed metastasis to distant organs, including lung, liver, and spleen, and inhibited focal adhesion reorganization. Our study provides comprehensive information on the molecular determinants of tumor cell migration and suggests that SRPK1 has potential as a drug target for limiting breast cancer metastasis.

Drug-induced endoplasmic reticulum and oxidative stress responses independently sensitize toward TNFα-mediated hepatotoxicity.

Drug-induced liver injury (DILI) is an important clinical problem. Here, we used a genomics approach to in detail investigate the hypothesis that critical drug-induced toxicity pathways act in synergy with the pro-inflammatory cytokine tumor necrosis factor α (TNFα) to cause cell death of liver HepG2 cells. Transcriptomics of the cell injury stress response pathways initiated by two hepatoxicants, diclofenac and carbamazepine, revealed the endoplasmic reticulum (ER) stress/translational initiation signaling and nuclear factor-erythroid 2 (NF-E2)-related factor 2 (Nrf2) antioxidant signaling as two major affected pathways, which was similar to that observed for the majority of ∼80 DILI compounds in primary human hepatocytes. Compounds displaying weak or no TNFα synergism, namely ketoconazole, nefazodone, and methotrexate, failed to synchronously induce both pathways. The ER stress induced was primarily related to protein kinase R-like ER kinase (PERK) and activating transcription factor 4 (ATF4) activation and subsequent expression of C/EBP homologous protein (CHOP), which was all independent of TNFα signaling. Identical ATF4 dependent transcriptional programs were observed in primary human hepatocytes as well as primary precision-cut human liver slices. Targeted RNA interference studies revealed that whereas ER stress signaling through inositol-requiring enzyme 1α (IRE1α) and activating transcription factor 6 (ATF6) acted cytoprotective, activation of the ER stress protein kinase PERK and subsequent expression of CHOP was pivotal for the onset of drug/TNFα-induced apoptosis. Whereas inhibition of the Nrf2-dependent adaptive oxidative stress response enhanced the drug/TNFα cytotoxicity, Nrf2 signaling did not affect CHOP expression. Both hepatotoxic drugs enhanced expression of the translational initiation factor EIF4A1, which was essential for CHOP expression and drug/TNFα-mediated cell killing. Our data support a model in which enhanced drug-induced translation initiates PERK-mediated CHOP signaling in an EIF4A1 dependent manner, thereby sensitizing toward caspase-8-dependent TNFα-induced apoptosis.

A screen for apoptotic synergism between clinical relevant nephrotoxicant and the cytokine TNF-α.

Nephrotoxicity remains one of the main reasons for post-market drug withdrawal. Tumour necrosis factor α (TNF-α) secretion has been shown to underlie the nephrotoxicity induced by some of these drugs. Yet, there is currently no reliable and sensitive in vitro assay available to screen for nephrotoxicants of which toxicity largely depends on TNF-α secretion. Therefore, we developed and applied a sensitive fluorescence-based in vitro assay for TNF-α-mediated nephrotoxicity screening using mouse immortalized proximal tubular epithelial cells (IM-PTECs). Our assay allows rapid evaluation of TNF-α-mediated toxicant-induced apoptosis and necrosis using fixed endpoint and live cell measurements. To evaluate our assay, sixteen nephrotoxicants and two control non-nephrotoxicants were used. Out of the sixteen nephrotoxicants, eight induced cell death, of which five induced apoptosis as well as necrosis. Moreover, TNF-α significantly enhanced apoptotic cell death induced by cisplatin, cyclosporine A, tacrolimus and azidothymidine. These nephrotoxicants are known to induce inflammation in vivo which has been linked to an enhancement of nephrotoxicity for cisplatin, cyclosporine A and tacrolimus, confirming the functionality of our assay. Overall, our assay allows rapid and sensitive measurement of apoptosis and necrosis induced by a combination of nephrotoxicants and inflammatory components such as TNF-α and can be used as an alternative assay for nephrotoxicity prediction in vitro.

The nuclear factor κB family member RelB facilitates apoptosis of renal epithelial cells caused by cisplatin/tumor necrosis factor α synergy by suppressing an epithelial to mesenchymal transition-like phenotypic switch.

Cis-diamminedichloroplatinum(II) (cisplatin)-induced renal proximal tubular apoptosis is known to be preceded by actin cytoskeleton reorganization, in conjunction with disruption of cell-matrix and cell-cell adhesion. In the present study, we show that the proinflammatory cytokine tumor necrosis factor α (TNF-α) aggravated these cisplatin-induced F-actin and cell adhesion changes, which was associated with enhanced cisplatin-induced apoptosis of immortalized proximal tubular epithelial cells. TNF-α-induced RelB expression and lentiviral small hairpin RNA (shRNA)-mediated knockdown of RelB, but not other nuclear factor κB members, abrogated the synergistic apoptosis observed with cisplatin/TNF-α treatment to the level of cisplatin-induced apoptosis. This protective effect was associated with increased stress fiber formation, cell-matrix, and cell-cell adhesion in the shRNARelB (shRelB) cells during cisplatin/TNF-α treatment, mimicking an epithelial-to-mesenchymal phenotypic switch. Indeed, gene array analysis revealed that knockdown of RelB was associated with upregulation of several actin regulatory genes, including Snai2 and the Rho GTPase proteins Rhophilin and Rho guanine nucleotide exchange factor 3 (ARHGEF3). Pharmacological inhibition of Rho kinase signaling re-established the synergistic apoptosis induced by combined cisplatin/TNF-α treatment of shRelB cells. In conclusion, our study shows for the first time that RelB is required for the cisplatin/TNF-α-induced cytoskeletal reorganization and apoptosis in renal cells by controlling a Rho kinase-dependent signaling network.

TNF-α-mediated NF-κB survival signaling impairment by cisplatin enhances JNK activation allowing synergistic apoptosis of renal proximal tubular cells.

Cisplatin-induced nephrotoxicity is an important limiting factor for cisplatin use. Tumor necrosis factor-α (TNF-α) is known to contribute to cisplatin-induced nephrotoxicity by inducing an inflammatory process aggravating the primary injury, thereby resulting in acute kidney injury (AKI). The present study investigates the pathways synergistically activated by cisplatin and TNF-α responsible for TNF-α-enhanced cisplatin-induced renal cell injury. To do so, immortalized renal proximal tubular epithelial cells (IM-PTECs) were co-treated with TNF-α and cisplatin. Under these conditions, cisplatin induced dose-dependent apoptosis in IM-PTECs, which was significantly enhanced by TNF-α. Transcriptomic analysis revealed that cisplatin inhibited the typical TNF-α response and cisplatin/TNF-α treatment up-regulated cell death pathways while it down-regulated survival pathways compared to cisplatin alone. In concordance, the gene expression levels of kidney injury markers combined with activation of specific inflammatory mediators were enhanced by cisplatin/TNF-α treatment, resembling the in vivo cisplatin-induced nephrotoxicity response. Furthermore, combined cisplatin/TNF-α treatment inhibited NF-κB nuclear translocation and NF-κB-mediated gene transcription leading to enhanced and prolonged JNK and c-Jun phosphorylation. JNK sustained activation further inhibited NF-κB signaling via a feedback loop mechanism. This led to an alteration in the transcription of the NF-κB-induced anti-apoptotic genes c-IAP2, Bcl-XL, Bruce and Bcl2 and pro-apoptotic genes Bfk and Xaf1 and consequently to sensitization of the IM-PTECs toward cisplatin/TNF-α-induced toxicity. In conclusion, our findings support a model whereby renal cells exposed to both cisplatin and TNF-α switch into a more pro-apoptotic and inflammatory program by altering their NF-κB/JNK/c-Jun balance.

Macrophage ABCA2 deletion modulates intracellular cholesterol deposition, affects macrophage apoptosis, and decreases early atherosclerosis in LDL receptor knockout mice.

OBJECTIVE: The ABCA2 transporter shares high structural homology to ABCA1, which is crucial for the removal of excess cholesterol from macrophages and, by extension, in atherosclerosis. It has been suggested that ABCA2 sequesters cholesterol inside the lysosomes, however, little is known of the macrophage-specific role of ABCA2 in regulating lipid homeostasis in vivo and in modulating susceptibility to atherosclerosis. METHODS: Chimeras with dysfunctional macrophage ABCA2 were generated by transplantation of bone marrow from ABCA2 knockout (KO) mice into irradiated LDL receptor (LDLr) KO mice. RESULTS: Interestingly, lack of ABCA2 in macrophages resulted in a diminished lesion size in the aortic root (-24.5%) and descending thoracic aorta (-36.6%) associated with a 3-fold increase in apoptotic cells, as measured by both caspase 3 and TUNEL. Upon oxidized LDL exposure, macrophages from wildtype (WT) transplanted animals developed filipin-positive droplets in lysosomal-like compartments, corresponding to free cholesterol (FC) accumulation. In contrast, ABCA2-deficient macrophages displayed an abnormal diffuse distribution of FC over peripheral regions. The accumulation of neutral sterols in lipid droplets was increased in ABCA2-deficient macrophages, but primarily in cytoplasmic clusters and not in lysosomes. Importantly, apoptosis of oxLDL loaded macrophages lacking ABCA2 was increased 2.7-fold, probably as a consequence of the broad cellular distribution of FC. CONCLUSIONS: Lack of functional ABCA2 generates abnormalities in intracellular lipid distribution/trafficking in macrophages consistent with its lysosomal sequestering role, leading to an increased susceptibility to apoptosis in response to oxidized lipids and reduced atherosclerotic lesion development.

cAMP signalling protects proximal tubular epithelial cells from cisplatin-induced apoptosis via activation of Epac.

BACKGROUND AND PURPOSE: Nephrotoxicity is the principal dose-limiting factor for cisplatin chemotherapy and is primarily associated with proximal tubular epithelial cells, including disruption of cell adhesions and induction of apoptosis. Cell adhesion and survival is regulated by, amongst other factors, the small GTPase Rap and its activator, the exchange protein directly activated by cAMP (Epac). Epac is particularly enriched in renal tubule epithelium. This study investigates the cytoprotective effects of cAMP-Epac-Rap signalling in a model of cisplatin-induced renal cell injury. EXPERIMENTAL APPROACH: The Epac-selective cAMP analogue 8-pCPT-2'-O-Me-cAMP was used to activate the Epac-Rap signalling pathway in proximal tubular epithelial cells. Cells were exposed to cisplatin, in the presence or absence of 8-pCPT-2'-O-Me-cAMP, and nephrotoxicity was determined by monitoring cell-cell junctions and cell apoptosis. KEY RESULTS: Activation of Epac-Rap signalling preserves cell-cell junctions and protects against cell apoptosis of mouse proximal tubular cells during cisplatin treatment. Activation with the Epac-selective cAMP analogue 8-pCPT-2'-O-Me-cAMP or receptor-mediated induction of cAMP both induced cytoprotection against cisplatin, whereas a PKA-selective cAMP analogue was not cytoprotective. 8-pCPT-2'-O-Me-cAMP mediated cytoprotection was blocked by RNAi-mediated silencing of Epac-Rap signalling in these cells. In contrast, 8-pCPT-2'-O-Me-cAMP did not protect against cisplatin-induced cell death of cancer cells that lacked Epac1 expression. CONCLUSIONS AND IMPLICATIONS: Our study identifies activation of Epac-Rap signalling as a potential strategy for reducing the nephrotoxicity associated with cisplatin treatments and, as a result, broadens the therapeutic window of this chemotherapeutic agent.

Focal adhesion kinase signaling mediates acute renal injury induced by ischemia/reperfusion.

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)//γGT-Cre-ER(T2) mice caused renal-specific fak recombination (FAK(ΔloxP/ΔloxP)) and reduction of FAK expression in proximal tubules. In FAK(ΔloxP/ΔloxP) 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.

Diclofenac inhibits tumor necrosis factor-α-induced nuclear factor-κB activation causing synergistic hepatocyte apoptosis.

UNLABELLED: Drug-induced liver injury (DILI) is an important clinical problem. It involves crosstalk between drug toxicity and the immune system, but the exact mechanism at the cellular hepatocyte level is not well understood. Here we studied the mechanism of crosstalk in hepatocyte apoptosis caused by diclofenac and the proinflammatory cytokine tumor necrosis factor α (TNF-α). HepG2 cells were treated with diclofenac followed by TNF-α challenge and subsequent evaluation of necrosis and apoptosis. Diclofenac caused a mild apoptosis of HepG2 cells, which was strongly potentiated by TNF-α. A focused apoptosis machinery short interference RNA (siRNA) library screen identified that this TNF-α-mediated enhancement involved activation of caspase-3 through a caspase-8/Bid/APAF1 pathway. Diclofenac itself induced sustained activation of c-Jun N-terminal kinase (JNK) and inhibition of JNK decreased both diclofenac and diclofenac/TNF-α-induced apoptosis. Live cell imaging of GFPp65/RelA showed that diclofenac dampened the TNF-α-mediated nuclear factor kappaB (NF-κB) translocation oscillation in association with reduced NF-κB transcriptional activity. This was associated with inhibition by diclofenac of the TNF-α-induced phosphorylation of the inhibitor of NF-κB alpha (IκBα). Finally, inhibition of IκB kinase ß (IKKß) with BMS-345541 as well as stable lentiviral short hairpin RNA (shRNA)-based knockdown of p65/RelA sensitized hepatocytes towards diclofenac/TNF-α-induced cytotoxicity. CONCLUSION: Together, our data suggest a model whereby diclofenac-mediated stress signaling suppresses TNF-α-induced survival signaling routes and sensitizes cells to apoptosis.

Annexin A1 regulates TGF-beta signaling and promotes metastasis formation of basal-like breast cancer cells.

Annexin A1 (AnxA1) is a candidate regulator of the epithelial- to mesenchymal (EMT)-like phenotypic switch, a pivotal event in breast cancer progression. We show here that AnxA1 expression is associated with a highly invasive basal-like breast cancer subtype both in a panel of human breast cancer cell lines as in breast cancer patients and that AnxA1 is functionally related to breast cancer progression. AnxA1 knockdown in invasive basal-like breast cancer cells reduced the number of spontaneous lung metastasis, whereas additional expression of AnxA1 enhanced metastatic spread. AnxA1 promotes metastasis formation by enhancing TGFbeta/Smad signaling and actin reorganization, which facilitates an EMT-like switch, thereby allowing efficient cell migration and invasion of metastatic breast cancer cells.

Annexin A2 phosphorylation mediates cell scattering and branching morphogenesis via cofilin Activation.

Dynamic remodeling of the actin cytoskeleton is required for cell spreading, motility, and migration and can be regulated by tyrosine kinase activity. Phosphotyrosine proteomic screening revealed phosphorylation of the lipid-, calcium-, and actin-binding protein annexin A2 (AnxA2) at Tyr23 as a major event preceding ts-v-Src kinase-induced cell scattering. Expression of the phospho-mimicking mutant Y23E-AnxA2 itself was sufficient to induce actin reorganization and cell scattering in MDCK cells. While Y23E-AnxA2, but not Y23A-AnxA2, enhanced Src- or hepatocyte growth factor (HGF)-induced cell scattering, short hairpin RNA-mediated knockdown of AnxA2 inhibited both v-Src- and HGF-induced cell scattering. Three-dimensional branching morphogenesis was induced in wild-type-AnxA2-expressing cells only in the presence of HGF, while Y23E-AnxA2 induced HGF-independent branching morphogenesis. Knockdown of AnxA2 prevented lumen formation during cystogenesis. The Y23E-AnxA2-induced scattering was associated with dephosphorylation/activation of the actin-severing protein cofilin. Likewise, inactive S3E-cofilin and constitutively active LIM kinase, a direct upstream kinase of cofilin, inhibited Y23E-AnxA2-induced scattering. Together, our studies indicate an essential role for AnxA2 phosphorylation in regulating cofilin-dependent actin cytoskeletal dynamics in the context of cell scattering and branching morphogenesis.

Extracellular signal-regulated kinase activation during renal ischemia/reperfusion mediates focal adhesion dissolution and renal injury.

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. Preceding phosphorylation of these focal adhesion proteins, reperfusion caused increased phosphorylation of ERK. The specific mitogen-activated protein kinase kinase 1/2 inhibitor U0126 prevented ERK activation and attenuated focal adhesion kinase, paxillin, and Src phosphorylation, focal adhesion restructuring, and ischemia/reperfusion-induced renal injury. We propose a model whereby ERK activation enhanced protein tyrosine phosphorylation during ischemia/reperfusion, thereby driving the dynamic dissolution and restructuring of focal adhesions and F-actin cytoskeleton during reperfusion and renal injury.

Proteomic analysis of alternative protein tyrosine phosphorylation in 1,2-dichlorovinyl-cysteine-induced cytotoxicity in primary cultured rat renal proximal tubular cells.

Toxicant exposure affects the activity of various protein tyrosine kinases. Using phosphotyrosine proteomics, we identified proteins that were differentially phosphorylated before renal cell detachment and apoptosis. Treatment of primary cultured rat proximal tubular epithelial cells with the model nephrotoxicant S-(1,2-dichlorovinyl)-L-cysteine (DCVC) resulted in early reorganization of F-actin stress fibers and formation of lamellipodia, which was followed by cell detachment from the matrix and apoptosis. This was prevented by genistein-mediated inhibition of protein tyrosine kinases and enhanced by inhibition of protein tyrosine phosphatases using vanadate. Phosphotyrosine proteomics revealed that DCVC-induced renal cell apoptosis was preceded by changes in the tyrosine phosphorylation status of a subset of proteins, as identified by matrix-assisted laser desorption ionization/time of flight-mass spectrometry (MS)/MS including actin-related protein 2 (Arp2), cytokeratin 8, t-complex protein 1 (TCP-1), chaperone containing TCP-1, and gelsolin precursor. The major differentially tyrosine-phosphorylated protein was Arp2, whereas phosphorylation of Arp3 was not affected. Arp2 was located in the lamellipodia that were formed before the onset of apoptosis. Because DCVC-induced cell detachment and apoptosis is regulated by tyrosine kinases, we propose that alterations in tyrosine phosphorylation of a subset of proteins, including Arp2, play a role in the regulation of the F-actin reorganization and lamellipodia formation that precede renal cell apoptosis caused by nephrotoxicants.

Phospho-proteomic analysis of cellular signaling.

Reversible protein phosphorylation plays an important role in the regulation of many different processes, such as cell growth, differentiation, migration, metabolism, and apoptosis. Identification of differentially phosphorylated proteins by means of phospho-proteomic analysis provides insight into signal transduction pathways that are activated in response to, for example, growth factor stimulation or toxicant-induced apoptosis. This review summarizes recent advances made in the field of phospho-proteomics and provides examples of how phospho-proteomic techniques can be combined to quantitatively investigate the dynamic changes in protein phosphorylation in time. By linking experimental data to clinical data (e.g., disease progression or response to therapy) new disease markers could be identified, which could then be validated for applications in disease diagnosis and progression or prediction of a response to drugs.

Cellular stress responses and molecular mechanisms of nephrotoxicity.

Increasing our knowledge on the molecular and cellular mechanisms of acute renal tubular pathologies will lead to potential novel therapeutic strategies either to prevent the initiation of renal failure or to promote the renal regeneration after injury. Currently many genomic- and proteomic-based techniques are available to identify genes, proteins or protein modifications in relation to renal toxicity. Although we are able to identify many genes and proteins at once, the actual role of the genes and proteins with respect to cellular toxicity needs to be defined in order to better understand the molecular basis of renal cell injury and repair. This review will focus on the relationship between changes in gene and protein expression, cellular perturbations, signal transduction, and mechanisms of toxicity. A focus is on the role of stress response proteins in repair of injured renal cells.

Heat shock protein 27 is the major differentially phosphorylated protein involved in renal epithelial cellular stress response and controls focal adhesion organization and apoptosis.

We used two-dimensional difference gel electrophoresis to determine early changes in the stress-response pathways that precede focal adhesion disorganization linked to the onset of apoptosis of renal epithelial cells. Treatment of LLC-PK1 cells with the model nephrotoxicant 1,2-(dichlorovinyl)-L-cysteine (DCVC) resulted in a >1.5-fold up- and down-regulation of 14 and 9 proteins, respectively, preceding the onset of apoptosis. Proteins included those involved in metabolism, i.e. aconitase and pyruvate dehydrogenase, and those related to stress responses and cytoskeletal reorganization, i.e. cofilin, Hsp27, and alpha-b-crystallin. The most prominent changes were found for Hsp27, which was related to a pI shift in association with an altered phosphorylation status of serine residue 82. Although both p38 and JNK were activated by DCVC, only inhibition of p38 with SB203580 reduced Hsp27 phosphorylation, which was associated with accelerated reorganization of focal adhesions, cell detachment, and apoptosis. In contrast, inhibition of JNK with SP600125 maintained cell adhesion as well as protection against apoptosis. Active JNK co-localized at focal adhesions after DCVC treatment in a FAK-dependent manner. Inhibition of active JNK localization at focal adhesions did not prevent DCVC-induced phosphorylation of Hsp27. Overexpression of a phosphorylation-defective mutant Hsp27 acted as a dominant negative and accelerated the DCVC-induced changes in the focal adhesions as well as the onset of apoptosis. Our data fit a model whereby early p38 activation results in a rapid phosphorylation of Hsp27, a requirement for proper maintenance of cell adhesion, thus suppressing renal epithelial cell apoptosis.