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We hypothesized that nucleophosmin (NPM), a nucleolar phosphoprotein, is critical for Bax-mediated cell death. To test this hypothesis, Bax activation was induced by metabolic stress. During stress, nucleolar NPM translocated into the cytosol, NPM-Bax complexes formed, and both NPM and Bax accumulated in mitochondria. Expression of a cytosol-restricted NPM mutant (NPM-ΔNLS), but not a nucleus-restricted NPM mutant, increased NPM-Bax complex formation, mitochondrial NPM and Bax accumulation, mitochondrial membrane injury, caspase 3 activation, and ischemia-induced cell death. Coexpression of NPM-ΔNLS with constitutively active Bax mutants caused nearly universal cell death in the absence of metabolic stress, whereas expression of active Bax or NPM-ΔNLS alone did not. A Bax peptide that disrupts NPM-Bax interaction significantly reduced cell death caused by exposure to metabolic inhibitors in vitro and preserved kidney function after ischemia in vivo. Thus, NPM-Bax interaction enhances mitochondrial Bax accumulation, organelle injury, and cell death. NPM-Bax complex formation is a novel target for preventing ischemic tissue injury. [ABSTRACT FROM AUTHOR]

Although heat shock protein 72 kDa (HSP72) protects tubular epithelium from a variety of acute insults, its role in chronic renal injury and fibrosis is poorly characterized. In this study, we tested the hypothesis that HSP72 reduces apoptosis and epithelial-to-mesenchymal transition (EMT), important contributors to tubular cell injury in vitro and in vivo. In rats, orally administered geranylgeranylacetone (GGA), an agent that selectively induces HSP72, markedly reduced both apoptosis and cell proliferation in tubular epithelium and decreased both interstitial fibroblast accumulation and collagen I deposition after unilateral ureteric obstruction, a model of chronic renal tubulointerstitial fibrosis and dysfunction. In cultured renal NRK52E cells, exposure to TGF-β1 induced EMT and apoptosis, major causes of renal fibrosis and tubular atrophy, respectively. Exposure to a pan-caspase inhibitor (ZVAD-FMK) prevented TGF-β 1-induced apoptosis but did not reduce EMT. In contrast, selective HSP72 expression in vitro inhibited EMT caused by TGF-β1 as indicated by preserving the E-cadherin expression level and α-smooth muscle actin induction. Small interfering RNA directed against HSP72 blocked the cytoprotective effects of HSP72 overexpression on EMT in TGF-β1-exposed cells. Taken together, our data indicate that HSP72 ameliorates renal tubulointerstitial fibrosis in obstructive nephropathy by inhibiting both renal tubular epithelial cell apoptosis and EMT. [ABSTRACT FROM AUTHOR]

Heat shock protein (Hsp) 90 is a ubiquitously expressed chaperone that stabilizes expression of multiple signaling kinases involved in growth regulation, including ErbB2, Raf-1, and Akt. The chaperone activity of Hsp90 requires ATP, which binds with ∼10-fold lower affinity than ADP. This suggests that Hsp90 may be a physiological ATP sensor, regulating the stability of growth signaling cascades in relation to cellular energy charge. Here we show that lowering ATP concentration by inhibiting glycolysis or mitochondrial respiration in isolated myocytes triggers rapid dissociation of Hsp90 from ErbB2 and degradation of ErbB2 along with other client proteins. The effect of disrupting Hsp90 chaperone activity by ATP depletion was similar to the effect of the pharmacological Hsp90 inhibitor geldanamycin. ATP depletion-induced disruption of Hsp90 chaperone activity was associated with cellular resistance to growth factor activation of intracellular signaling. ErbB2 degradation was also induced by the physiological stress of β-adrenergic receptor stimulation in electrically stimulated cells. These results support a role for Hsp90 as an ATP sensor that modulates tissue growth factor responsiveness under metabolically stressed conditions and provide a novel mechanism by which cellular responsiveness to growth factor stimulation is modulated by cellular energy charge. [ABSTRACT FROM AUTHOR]

Heat shock protein 70 (Hsp70) is a potent antiapoptotic agent. Here, we tested whether it directly regulates renal cell survival and organ function in a model of transient renal ischemia using Hsp70 knockout, heterozygous, and wild-type mice. The kidney cortical Hsp70 content inversely correlated with tubular injury, apoptosis, and organ dysfunction after injury. In knockout mice, ischemia caused changes in the activity of Akt and glycogen synthase kinase 3-β (kinases that regulate the proapoptotic protein Bax), increased active Bax, and activated the proapoptotic protease caspase 3. As these changes were significantly reduced in the wild-type mice, we tested whether Hsp70 influences ischemia-induced apoptosis. An Hsp70 inducer, geranylgeranylacetone, increased Hsp70 expression in heterozygous and wild-type mice, and reduced both ischemic tubular injury and organ dysfunction. When administered after ischemia, this inducer also decreased tubular injury and organ failure in wild-type mice but did not protect the knockout mice. ATP depletion in vitro caused greater mitochondrial Bax accumulation and death in primary proximal tubule cells harvested from knockout compared with wild-type mice and altered serine phosphorylation of a Bax peptide at the Akt-specific target site. In contrast, lentiviral-mediated Hsp70 repletion decreased mitochondrial Bax accumulation and rescued Hsp70 knockout cells from death. Thus, increasing Hsp70 either before or after ischemic injury preserves renal function by attenuating acute kidney injury. [ABSTRACT FROM AUTHOR]

The cytoprotective effect of heat stress proteins on epithelial cell detachment, an important cause of acute, ischemic renal failure, was examined after ATP depletion by evaluating focal adhesion complex (FAC) integrity. The intracellular distribution of FAC proteins (paxillin, talin, and vinculin) was assessed by immunohistochemistry before, during, and after exposure of renal epithelial cells to metabolic inhibitors. The resulting ATP depletion caused reversible re-distribution of all three proteins from focal adhesions to the cytosol. Paxillin, a key adaptor protein, was selected as a surrogate marker for FAC integrity in subsequent studies. Prior heat stress increased hsp72, a molecular chaperone, in beth the Triton X-100-soluble and -insoluble protein fractions. Compared with ATP depleted control, heat stress significantly decreased paxillin and hsp72 shift from the Triton X-100 soluble to the insoluble protein fraction (an established marker of denaturation and aggregation); increased paxillin-hsp72 interaction detected by co-immunoprecipitation; enhanced paxillin extractability from Triton X-100-insoluble precipitates, increased the reformation of focal adhesions, and improved cell attachment (p < 0.05). To determine whether hsp72 mediates protection afforded by heat stress, cells were infected with adenovirus containing human hsp72 or empty vector. Hsp72 overexpression increased its interaction with paxillin and improved focal adhesion reformation during recovery, mimicking the protective effects of heat stress. These data suggest that hsp72 facilitates the reassembly of focal adhesions and improves cell attachment by reducing paxillin denaturation and increasing its re-solubilization after ATP depletion. [ABSTRACT FROM AUTHOR]

Tests the hypothesis that hsp72 interacts with focal adhesion kinase (FAK), preventing caspase-3-mediated degradation during ATP depletion. Background on prior heat stress of the selective overexpression of hsp72 presents apoptosis caused by exposure to metabolic inhibitors by protecting the mitochondrial membrane; Increased interaction between hsp72 and FAK.

Evaluates the localization of alpha[sub 2micromole]-globulin (A[sub 2])/A[sub 2]-fragment in the proximal tubule cell. Immunoblot analysis of renal cortical homogenates; Accumulation of A[sub 2]fragment in the cytosol of the proximal tubule epithelial cell; Use of ligand binding, uptake and degradation assays.

The function of site-specific phosphorylation of nucleophosmin (NPM), an essential Bax chaperone, in stress-induced cell death is unknown. We hypothesized that NPM threonine 95 (T95) phosphorylation both signals and promotes cell death. In resting cells, NPM exclusively resides in the nucleus and T95 is nonphosphorylated. In contrast, phosphorylated T95 NPM (pNPM T95) accumulates in the cytosol after metabolic stress, in multiple human cancer cell lines following γ-radiation, and in postischemic human kidney tissue. Based on the T95 phosphorylation consensus sequence, we hypothesized that glycogen synthase kinase-3β (GSK-3β) regulates cytosolic NPM translocation by phosphorylating T95 NPM. In a cell-free system, GSK-3β phosphorylated a synthetic NPM peptide containing T95. In vitro, bidirectional manipulation of GSK-3β activity substantially altered T95 phosphorylation, cytosolic NPM translocation, and cell survival during stress, mechanistically linking these lethal events. Furthermore, GSK-3β inhibition in vivo decreased cytosolic pNPM T95 accumulation in kidney tissue after experimental ischemia. In patients with acute kidney injury, both cytosolic NPM accumulation in proximal tubule cells and NPM-rich intratubular casts were detected in frozen renal biopsy tissue. These observations show, for the first time, that GSK-3β promotes cell death partly by phosphorylating NPM at T95, to promote cytosolic NPM accumulation. T95 NPM is also a rational therapeutic target to ameliorate ischemic renal cell injury and may be a universal injury marker in mammalian cells.

Background Ischemic AKI lacks a urinary marker for early diagnosis and an effective therapy. Differential nucleophosmin (NPM) phosphorylation is a potential early marker of ischemic renal cell injury and a therapeutic target. Methods Differential NPM phosphorylation was assessed by mass spectrometry in NPM harvested from murine and human primary renal epithelial cells, fresh kidney tissue, and urine before and after ischemic injury. The biologic behavior and toxicity of NPM was assessed using phospho-NPM mutant proteins that either mimic stress-induced or normal NPM phosphorylation. Peptides designed to interfere with NPM function were used to explore NPM as a therapeutic target. Results Within hours of stress, virtually identical phosphorylation changes were detected at distinct serine/threonine sites in NPM harvested from primary renal cells, tissue, and urine. A phosphomimic NPM protein that replicated phosphorylation under stress localized to the cytosol, formed monomers that interacted with Bax, a cell death protein, coaccumulated with Bax in isolated mitochondria, and significantly increased cell death after stress; wild-type NPM or a phosphomimic NPM with a normal phosphorylation configuration did not. Three renal targeted peptides designed to interfere with NPM at distinct functional sites significantly protected against cell death, and a single dose of one peptide administered several hours after ischemia that would be lethal in untreated mice significantly reduced AKI severity and improved survival. Conclusions These findings establish phosphorylated NPM as a potential early marker of ischemic AKI that links early diagnosis with effective therapeutic interventions., (Copyright © 2019 by the American Society of Nephrology.)

Ischemia activates Bax, a proapoptotic BCL2 protein, as well as the prosurvival beta-catenin/Wnt signaling pathway. To test the hypothesis that beta-catenin/Wnt signaling regulates Bax-mediated apoptosis after induction of metabolic stress, which occurs during renal ischemia, we infected immortalized and primary proximal tubular epithelial cells with adenovirus to express either constitutively active or dominant negative beta-catenin constructs. Constitutively active beta-catenin significantly decreased apoptosis and improved cell survival after metabolic stress. Furthermore, active beta-catenin decreased Bax activation, oligomerization, and translocation to mitochondria, and reduced both organelle membrane injury and apoptosis. Dominant negative beta-catenin had the opposite effects. Because Akt regulates Bax, we examined the effects of the beta-catenin mutants on Akt expression and activation. Constitutively active beta-catenin increased Akt-1 expression and activation before and after stress, and treatment with a phosphatidylinositol-3 kinase inhibitor antagonized the protective effects of beta-catenin on Akt activation, Bax inhibition, and cell survival. In addition, beta-catenin significantly increased the rate of phosphorylation at Bax serine(184), an Akt-specific target. Taken together, these results suggest that beta-catenin/Wnt signaling promotes survival of renal epithelial cells after metabolic stress, in part by inhibiting Bax in a phosphatidylinositol-3 kinase/Akt-dependent manner.

Activation of the nuclear transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) inhibits growth and survival of hepatocellular carcinoma (HCC) cell lines. To further investigate the function of PPARgamma in HCC, PPARgamma expression patterns in primary tumors were examined, and the responses of two HCC cell lines to PPARgamma activation and inhibition were compared. PPARgamma expression was increased in HCC and benign-appearing peritumoral hepatocytes compared with remote benign hepatocytes. Both compound PPARgamma inhibitors and PPARgamma small interfering RNAs prevented HCC cell lines from adhering to the extracellular matrix. Loss of adhesion was followed by caspase-dependent apoptosis (anoikis). PPARgamma inhibitors had no effect on initial beta1 integrin-mediated adhesion, or on total focal adhesion kinase levels but did reduce focal adhesion kinase phosphorylation. The PPARgamma inhibitor T0070907 was significantly more efficient at causing cancer cell death than the activators troglitazone and rosiglitazone. T0070907 caused cell death by reducing adhesion and inducing anoikis, whereas the activators had no direct effect on adhesion and caused cell death at much higher concentrations. In conclusion, PPARgamma overexpression is present in HCC. Inhibition of PPARgamma function causes HCC cell death by preventing adhesion and inducing anoikis-mediated apoptosis. PPARgamma inhibitors represent a potential novel treatment approach to HCC.