[Show abstract][Hide abstract] ABSTRACT: Overwhelming oxidative stress and compromised tubular cell antioxidant response have been incriminated for cisplatin (Cis) -induced acute kidney injury (AKI). We hypothesized that Cis-induced KI was the outcome of the deactivated redox-sensitive stress response program (RSSRP). Wild (WT) and heterozygous p66ShcA(+/-) mice in groups of six were administered either normal saline (WT) or Cis (12.5mg/kg, intraperitoneal, Cis/WT). Renal biomarkers were collected and kidneys were harvested for renal histology. Cis/WT showed elevated blood urea nitrogen levels and enhanced tubular cell apoptosis, necrosis, and dilated tubules filled with casts when compared to Cis/p66(+/-). Cis/p66(+/-) developed only a clinically occult ARF (normal blood urea levels and only microscopic alterations). Immunoblots from the lysates of renal tissues of Cis/WT displayed enhanced expression of phospho-p66ShcA, and phospho-Foxo3A but attenuated expression of MnSOD and catalase; conversely, p66 deficit prevented these alterations in Cis milieu. In in vitro studies, Cis treated mouse proximal tubular cells (MPTCs) displayed enhanced phosphorylation of p66ShcA and no increase in tubular cell expression of MnSOD. In addition, renal tissues of Cis/WT and Cis-treated MPTCs displayed enhanced phosphorylation of p53 and Bax expression. However, MPTC partially silenced for p66ShcA displayed partial inhibition of Cis-induced tubular cell apoptosis as well as necrosis. These findings indicated that Cis-induced AKI was the outcome of the deactivated RSSRP (attenuated anti-oxidant response) and activation of pro-apoptotic (p53-induced Bax expression) pathway.
Experimental and Molecular Pathology 03/2013; · 2.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A fundamental problem in the identification of new molecular targets for therapeutic intervention in diabetic nephropathy has been the lack of an experimental mouse model that faithfully recapitulates human diabetic nephropathy.
Our laboratory, in collaboration with Drs Kakoki and Smithies at the University of North Carolina-Chapel Hill, has developed novel strains of Akita diabetic mice in which the p66 longevity gene has been deleted by homologous recombination. We chose to delete p66 because p66 controls mitochondrial metabolism and cellular responses to oxidative stress, aging, and apoptosis. The redox function of p66 is indispensable for the exponential increase in reactive oxygen species (ROS) associated with diabetes.
p66 null Akita mice express a protection phenotype in kidneys that includes marked attenuation of oxidative stress and glomerular/tubular injury and a striking reduction in urine albumin excretion. Furthermore, the p66 null mutation not only confers a survival advantage to podocytes but also prevents foot process effacement and retains the stationary phenotype. Sirtuin 1 (SIRT1) deacetylase and p66 share overlapping biological functions but induce divergent phenotypes, including opposite effects on longevity, ROS metabolism, cell senescence, and apoptosis. Exciting new data from our laboratory show that SIRT1 is upregulated in the kidneys of p66 null Akita mice and decreases acetylation of p53, which destabilizes the p53 protein and prevents the transcription of p53 proapoptosis genes. Conversely, SIRT1 activates the transcription of FOXO3a-dependent stress gene programs that detoxify ROS and promote the survival phenotype.
We will focus future research on translating these experimental findings in the mouse to clinical diabetic nephropathy.
[Show abstract][Hide abstract] ABSTRACT: Candidate genes have been identified that confer increased risk for diabetic glomerulosclerosis (DG). Mice heterozygous for the Akita (Ins2(+/C96Y)) diabetogenic mutation with a second mutation introduced at the bradykinin 2 receptor (B2R(-/-)) locus, express a disease phenotype that approximates human DG. Src homology 2 domain transforming protein 1 (p66) controls mitochondrial metabolism and cellular responses to oxidative stress, aging and apoptosis. We generated p66-null Akita mice to test whether inactivating mutations at the p66 locus will rescue kidneys of Akita mice from disease-causing mutations at the Ins2 and B2R loci. Here we show null mutations at the p66 and B2R loci interact with the Akita (Ins2(+/C96Y)) mutation, independently and in combination, inducing divergent phenotypes in the kidney. The B2R(-/-) mutation induces detrimental phenotypes, as judged by increased systemic and renal levels of oxidative stress, histology and urine albumin excretion, whereas the p66-null mutation confers a powerful protection phenotype. To elucidate the mechanism(s) of the protection phenotype, we turned to our in vitro system. Experiments with cultured podocytes revealed previously unrecognized crosstalk between p66 and the redox sensitive transcription factor p53 that controls hyperglycemia-induced ROS metabolism, transcription of p53 target genes (angiotensinogen, angiotensin II type-1 receptor; bax) angiotensin II generation and apoptosis. RNA-interference targeting p66 inhibits all of the above. Finally, protein levels of p53 target genes were upregulated in kidneys of Akita mice, but unchanged in p66-null Akita mice. Taken together, p66 is a potential molecular target for therapeutic intervention in DG.
[Show abstract][Hide abstract] ABSTRACT: Human immunodeficiency virus (HIV)-1 has been reported to cause tubular cell injury both in in vivo and in vitro studies. In the present study, we evaluated the role of oxidative stress in the induction of apoptosis in HIV gene expressing mouse tubular cells in in vivo (Tg26, a transgenic mouse model of HIV-associated nephropathy) and in vitro (tubular cells were transduced with pNL4-3: ΔG/P-GFP, VSV.G psueudo typed virus) studies. Although Tg26 mice showed enhanced tubular cell reactive oxygen species (ROS) generation and apoptosis, renal tissue did not display a robust antioxidant response in the form of enhanced free radical scavenger (MnSOD/catalase) expression. Tg26 mice not only showed enhanced tubular cell expression of phospho-p66ShcA but also displayed nuclear Foxo3a translocation to the cytoplasm. These findings indicated deactivation of tubular cell Foxo3A-dependent redox-sensitive stress response program (RSSRP) in Tg26 mice. In in vitro studies, NL4-3 (pNL4-3: ΔG/P-GFP, VSV.G pseudotyped virus)-transduced mouse proximal tubular cells (NL4-3/MPTEC) displayed enhanced phosphorylation of p66ShcA. NL4-3/MPTECs also displayed greater (P < 0.01) ROS generation when compared with empty vector-transduced tubular cells; however, both diminution of p66ShcA and N-acetyl cysteine attenuated NL4-3-induced tubular cell ROS generation as well as apoptosis. In addition, both antioxidants and free radical scavengers partially inhibited HIV-induced tubular cell apoptosis. NL4-3/MPTEC displayed deactivation of RSSRP in the form of enhanced phosphorylation of Foxo3A and attenuated expression of superoxide dismutase (SOD) and catalase. Since both SOD and catalase were able to provide protection against HIV-1-induced tubular cell apoptosis, it suggests that HIV-1-induced proapoptotic effect may be a consequence of the deactivated RSSRP.
[Show abstract][Hide abstract] ABSTRACT: In HIV patients, antiretroviral medications trigger metabolic abnormalities, including insulin resistance. In addition, the inflammatory cytokine tumor necrosis factor-α (TNFα), which is elevated in human immunodeficiency virus encephalitis (HIVE), also induces insulin resistance and inflicts neuronal damage in vitro. In differentiated PC12 cells and rat cortical neurons, high glucose (HG; 25 mM) triggers reactive oxygen species (ROS) accumulation, contributing to the retraction of neuronal processes, with only a minimal involvement of neuronal apoptosis. In the presence of TNFα, HG-treated neurons undergo massive apoptosis. Because mammalian homolog of the Forkhead family of transcription factors, Forkhead box O transcription factor 3a (FOXO3a), controls ROS metabolism, we asked whether FOXO3a could affect the fate of differentiated neurons in the paradigm of HIVE. We observed FOXO3a nuclear translocation in HG-treated neuronal cultures, accompanied by partial loss of mitochondrial potential and gradual retraction of neuronal processes. Addition of TNFα to HG-treated neurons increased expression of the FOXO-dependent proapoptotic gene Bim, which resulted in extensive apoptotic death. Insulin-like growth factor-I (IGF-I) significantly lowered intracellular ROS, which was accompanied by IGF-I-mediated FOXO3a nuclear export and decrease in its transcriptional activity. The clinical relevance of these findings is supported by detection of nuclear FOXO3a in TUNEL-positive cortical neurons from HIVE, especially in brain areas characterized by elevated TNFα.
Journal of Neuroscience Research 02/2011; 89(2):183-98. · 2.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Glomerular visceral epithelial cells (podocytes) play a critical role in the pathogenesis of human immunodeficiency virus (HIV)-associated nephropathy. A key question concerns the mechanism(s) by which the HIV-1 genome alters the phenotype of the highly specialized, terminally differentiated podocytes. Here, using an in vitro system of conditionally immortalized differentiated human podocytes (CIDHPs), we document a pivotal role for the p66ShcA protein in HIV-1-induced reactive oxygen species generation and CIDHP apoptosis. CIDHP transfected with truncated HIV-1 construct (NL4-3) exhibit increased reactive oxygen species metabolism, DNA strand breaks, and a 5-fold increase in apoptosis, whereas the opposite was true for NL4-3/CIDHP co-transfected with mu-36p66ShcA (micro-36) dominant negative expression vector or isoform-specific p66-small interfering RNA. Phosphorylation at Ser-36 of the wild type p66ShcA protein, required for p66ShcA redox function and inhibition of the potent stress response regulator Foxo3a, was unchanged in micro-36/NL4-3/CIDHP but increased in NL4-3/CIDHP. Acute knockdown of Foxo3a by small interfering RNA induced a 50% increase in micro-36/NL4-3/CIDHP apoptosis, indicating that Foxo3a-dependent responses promote the survival phenotype in micro-36 cells. We conclude that inhibition of p66ShcA redox activity prevents generation of HIV-1 stress signals and activation of the CIDHP apoptosis program.
Journal of Biological Chemistry 05/2009; 284(24):16648-58. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Apoptotic myocyte cell death, diastolic dysfunction, and progressive deterioration in left ventricular pump function characterize the clinical course of diabetic cardiomyopathy. A key question concerns the mechanism(s) by which hyperglycemia (HG) transmits danger signals in cardiac muscle cells. The growth factor adapter protein p66ShcA is a genetic determinant of longevity, which controls mitochondrial metabolism and cellular responses to oxidative stress. Here we demonstrate that interventions which attenuate or prevent HG-induced phosphorylation at critical position 36 Ser residue (phospho-Ser36) inhibit the redox function of p66ShcA and promote the survival phenotype. Adult rat ventricular myocytes obtained by enzymatic dissociation were transduced with mutant-36 p66ShcA (mu-36) dominant-negative expression vector and plated in serum-free media containing 5 or 25 mM glucose. At HG, adult rat ventricular myocytes exhibit a marked increase in reactive oxygen species production, upregulation of phospho-Ser36, collapse of mitochondrial transmembrane potential, and increased formation of p66ShcA/cytochrome-c complexes. These indexes of oxidative stress were accompanied by a 40% increase in apoptosis and the upregulation of cleaved caspase-3 and the apoptosis-related proteins p53 and Bax. To test whether p66ShcA functions as a redox-sensitive molecular switch in vivo, we examined the hearts of male Akita diabetic nonobese (C57BL/6J) mice. Western blot analysis detected the upregulation of phospho-Ser36, the translocation of p66ShcA to mitochondria, and the formation of p66ShcA/cytochrome-c complexes. Conversely, the correction of HG by recombinant adeno-associated viral delivery of leptin reversed these alterations. We conclude that p66ShcA is a molecular switch whose redox function is turned on by phospho-Ser36 and turned off by interventions that prevent this modification.
[Show abstract][Hide abstract] ABSTRACT: The stimulatory G protein Gsalpha transmits signals from activated beta-adrenergic receptors via the cyclic AMP-PKA pathway, targeting the key regulatory protein phospholamban. We hypothesized that mice with intrinsic activation of cardiac Gsalpha are resistant to the development of the diabetic cardiomyopathy phenotype. Accordingly, streptozotocin (STZ)-diabetes mellitus was induced in genetically engineered mice with cardiac-specific Gsalpha overexpression and in nontransgenic (NTG) littermates. At 8 weeks, Gsalpha diabetic mice showed no impairment of LV contractility nor increase in myocyte apoptosis, whereas NTG diabetic mice showed a 30% decrease in +dP/dt and -dP/dt with sustained (3-fold) myocyte loss by apoptosis. To assess the level of myocardial reactive oxygen species, we measured malondialdehyde, a surrogate marker of oxidative stress, which was increased in the hearts of NTG and Gsalpha diabetic mice. In addition, chronic hyperglycemia also increased the activity of catalase and superoxide dismutase in the hearts of NTG and Gsalpha diabetic mice. Hearts of NTG diabetic mice, but not Gsalpha mice, showed increased expression of proapoptosis Bax, downregulation in Bcl2, and an increase in the Bax/Bcl2 ratio. Hearts of NTG diabetic mice showed 60% reduction in phosphorylation at the critical Ser16 residue of phospholamban, whereas phosphorylation at Ser16 was restored in hearts of Gsalpha-diabetic mice. We conclude that cardiac-specific overexpression of Gsalpha compensates for the loss of cardiac function in diabetes mellitus.
Canadian Journal of Physiology and Pharmacology 04/2008; 86(3):122-30. · 1.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hyperglycemia triggers an exponential increase in reactive oxygen species (ROS) at the cellular level. Here, we demonstrate induction of the oxidant-resistant phenotype in mesangial cells by silencing the wild-type (WT) p66ShcA gene. Two approaches were employed to inhibit WTp66ShcA in SV40 murine mesangial cells and normal human mesangial cells: transient transfection with isoform-specific p66ShcA short-intervening RNA and stable transfection with mutant 36 p66ShcA expression vector. At high ambient glucose (HG), p66ShcA-deficient cells exhibit resistance to HG-induced ROS generation and attenuation in the amplitude of the kinetic curves for intracellular ROS metabolism, indicative of the pivotal role of WTp66ShcA in the generation of HG oxidant stress. We next examined phosphorylation and subcellular distribution of FKHRL1 (FOXO3a), a potent stress response regulator and downstream target of WTp66ShcA redox function. At HG, cell extracts of p66ShcA-deficient cells analyzed by immunoblotting show attenuation of FOXO3a phosphorylation at Thr-32, and indirect immunofluorescence of p66ShcA-deficient cells, cotransfected with HA-FOXO3a, show predominant HA-FOXO3a nuclear localization. Conversely, parental cells at HG show upregulation of phos-Thr-32 and nuclear export of HA-FOXO3a. To determine whether inhibition of cross talk between WTp66ShcA and FOXO3a confers protection against oxidant-induced DNA damage, DNA strand breaks (DSB) and apoptosis were examined. At HG, p66ShcA-deficient cells exhibit increased resistance to DSB and apoptosis, while parental cells show a striking increase in both parameters. We conclude that knockdown of WTp66ShcA redox function prevents HG-dependent FOXO3a regulation and promotes the survival phenotype.
American journal of physiology. Renal physiology 03/2007; 292(2):F523-30. · 3.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The IGF-1R is a genetic determinant of oxidative stress and longevity. Hyperglycemia induces an exponential increase in the production of a key danger signal, reactive oxygen intermediates, which target genomic DNA. Here, we report for the first time that ligand activation of the IGF-1R prevents hyperglycemia-induced genotoxic stress and enhances DNA repair, maintaining genomic integrity and cell viability. We performed single gel electrophoresis (comet assay) to evaluate DNA damage in serum-starved SV40 murine mesangial cells (MMC) and normal human mesangial cells (NHMC), maintained at high ambient glucose concentration. Hyperglycemia inflicted an impressive array of DNA damage in the form of single-strand breaks (SSBs) and double-strand breaks (DSBs). The inclusion of IGF-1 to culture media of MMC and NHMC prevented hyperglycemia-induced DNA damage. To determine whether DNA damage was mediated by reactive oxygen species (ROS), ROS generation was evaluated, in the presence of IGF-1, or the free radical scavenger n-acetyl-cysteine (NAC). IGF-1 and NAC inhibited hyperglycemic-induced ROS production and hyperglycemia-induced DNA damage. We next asked whether IGF-1 promotes the repair of DSB under hyperglycemic conditions, by homologous recombination (HRR) or nonhomologous end joining (NHEJ). Repair of DSB by NHEJ and HRR was operative in MMC maintained under hyperglycemic conditions. IGF-1 increased HRR by nearly twofold, whereas IGF-1 did not affect DNA repair by NHEJ. IGF-1R enhancement of HRR correlated with the translocation of Rad51 to foci of DNA damage. Inhibition of Rad51 expression by short interfering RNA experiments markedly decreased percentage of MMC positive for Rad51 nuclear foci and increased hyperglycemic DNA damage. We conclude that the activated IGF-1R rescues mesangial cells from hyperglycemia-induced danger signals that target genomic DNA by suppressing ROS and enhancing DNA repair by HRR.
American journal of physiology. Renal physiology 12/2005; 289(5):F1144-52. · 3.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Diabetes mellitus is complicated by the development of a primary cardiomyopathy, which contributes to the excess morbidity and mortality of this disorder. The protein kinase C (PKC) family of isozymes plays a key role in the cardiac phenotype expressed during postnatal development and in response to pathological stimuli. Hyperglycemia is an activating signal for cardiac PKC isozymes that modulate a myriad of cell events including cell death and survival. The epsilon-isozyme of the PKC family transmits a powerful survival signal in cardiac muscle cells. Accordingly, to test the hypothesis that endogenous activation of cardiac PKC-epsilon will protect against hyperglycemic cell injury and left ventricular dysfunction, diabetes mellitus was induced using streptozotocin in genetically engineered mice with cardiac-specific expression of the PKC-epsilon translocation activator [psiepsilon-receptors for activated C kinase (psiepsilon-RACK)]. The results demonstrate a striking PKC-epsilon cardioprotective phenotype in diabetic psiepsilon-RACK (epsilon-agonist) mice that is characterized by inhibition of the hyperglycemia apoptosis signal, attenuation of hyperglycemia-mediated oxidative stress, and preservation of parameters of left ventricular pump function. Hearts of diabetic epsilon-agonist mice exhibited selective trafficking of PKC-epsilon to membrane and mitochondrial compartments, phosphorylation/inactivation of the mitochondrial Bad protein, and inhibition of cytochrome c release. We conclude that activation of endogenous PKC-epsilon in hearts of diabetic epsilon-agonist mice promotes the survival phenotype, attenuates markers of oxidative stress, and inhibits the negative inotropic properties of chronic hyperglycemia.
[Show abstract][Hide abstract] ABSTRACT: Recruitment of the protein kinase C (PKC) family of isozymes is an integral component of the signaling events that direct cardiac phenotype expressed during postnatal development and in response to pathologic stimuli. Hyperglycemia is a potent activating signal for cardiac PKC isozymes and induces the apoptosis program in cardiac muscle cells. To determine whether cardiac PKC isozymes modulate transmission of the hyperglycemia apoptosis signal, we have employed isozyme-specific peptide modulators to selectively inhibit (PKC betaI/betaII, zeta and epsilon) or activate (PKCepsilon). PKC peptides were delivered to primary cultures of serum starved adult rat ventricular myocytes (ARVM), by conjugation to the homeodomain of drosophila antennapedia. As expected, hyperglycemia induced a 35% increase in ARVM apoptosis. Peptide inhibitors of PKC betaI/betaII and zeta blocked transmission of the hyperglycemia apoptosis signal, whereas the isozyme specific inhibitor of PKCepsilon (epsilonV1-2) did not alter the magnitude of glucose-induced ARVM apoptosis. Alternatively, the PKCepsilon translocation activator (psi epsilonRACK) abolished hyperglycemia-induced apoptosis, strongly suggesting a cardioprotective role for PKCepsilon in this system. Therefore, we conclude that cardiac PKC isozymes modulate hyperglycemia-induced apoptosis and activation of cardiac PKCepsilon protects ARVM from the hyperglycemia-induced death signal.
Molecular and Cellular Biochemistry 02/2005; 268(1-2):169-73. · 2.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recruitment of the protein kinase C (PKC) family of isozymes is an integral component of the signaling events that direct cardiac phenotype expressed during postnatal development and in response to pathologic stimuli. Hyperglycemia is a potent activating signal for cardiac PKC isozymes and induces the apoptosis program in cardiac muscle cells. To determine whether cardiac PKC isozymes modulate transmission of the hyperglycemia apoptosis signal, we have employed isozyme-specific peptide modulators to selectively inhibit (PKC I/II, and ) or activate (PKC). PKC peptides were delivered to primary cultures of serum starved adult rat ventricular myocytes (ARVM), by conjugation to the homeodomain of drosophila antennapedia. As expected, hyperglycemia induced a 35% increase in ARVM apoptosis. Peptide inhibitors of PKC I/II and blocked transmission of the hyperglycemia apoptosis signal, whereas the isozyme specific inhibitor of PKC (V1-2) did not alter the magnitude of glucose-induced ARVM apoptosis. Alternatively, the PKC translocation activator (RACK) abolished hyperglycemia-induced apoptosis, strongly suggesting a cardioprotective role for PKC in this system. Therefore, we conclude that cardiac PKC isozymes modulate hyperglycemia-induced apoptosis and activation of cardiac PKC protects ARVM from the hyperglycemia-induced death signal. (Mol Cell Biochem 268: 169–173, 2005)
Molecular and Cellular Biochemistry 12/2004; 268(1):169-173. · 2.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aging and diabetes mellitus (DM) both affect the structure and function of the myocardium, resulting in increased collagen in the heart and reduced cardiac function. As part of this process, hyperglycemia is a stimulus for the production of advanced glycation end products (AGEs), which covalently modify proteins and impair cell function. The goals of this study were first to examine the combined effects of aging and DM on hemodynamics and collagen types in the myocardium in 12 dogs, 9-12 yr old, and second to examine the effects of the AGE cross-link breaker phenyl-4,5-dimethylthazolium chloride (ALT-711) on myocardial collagen protein content, aortic stiffness, and left ventricular (LV) function in the aged diabetic heart. The alloxan model of DM was utilized to study the effects of DM on the aging heart. DM induced in the aging heart decreased LV systolic function (LV ejection fraction fell by 25%), increased aortic stiffness, and increased collagen type I and type III protein content. ALT-711 restored LV ejection fraction, reduced aortic stiffness and LV mass with no reduction in blood glucose level (199 +/- 17 mg/dl), and reversed the upregulation of collagen type I and type III. Myocardial LV collagen solubility (%) increased significantly after treatment with ALT-711. These data suggest that an AGE cross-link breaker may have a therapeutic role in aged patients with DM.
[Show abstract][Hide abstract] ABSTRACT: The activated insulin-like growth factor-1 receptor (IGF-1R) protects cells from a wide range of apoptotic stimuli. Hyperglycemia promotes the intracellular generation of superoxide anion and hydrogen peroxide, both of which have been linked to the activation of the mitochondrial apoptosis program. Here, we report for the first time that ligand activation of the IGF-1R protects normal human mesangial cells and SV40 murine mesangial cells from the glycol-oxidant-induced apoptosis program. The IGF-1R antiapoptosis program was dependent on the recruitment of both Akt/PKB and the ERK subfamily of mitogen-activated protein kinases. IGF-1 treatment also protected the redox potential of mesangial cells maintained at high ambient glucose concentration, by inhibiting the generation of reactive oxygen intermediates and preserving mitochondrial transmembrane potential. IGF-1R survival signals targeted the Bcl-2 family of proteins to protect against glucose-induced apoptosis and oxidative stress. IGF-1-treated cells exhibited a decrease in the Bax/Bcl-2 ratio; increased phosphorylation/inactivation of Bad at Ser112 and Ser136; inhibition of cytochrome c release; perturbations directionally opposed to the initiation of the apoptosis program. In addition, we demonstrate IGF-1R-activated ERK signaling modules phosphorylate Ser112 of the mitochondrial Bad protein, establishing a direct link between surface IGF-1R and the survival program in mitochondria. Our findings indicate that in mesangial cells maintained at high ambient glucose concentration, IGF-1 activates a survival program that maintains the integrity of mitochondria and prevents the expression of the genetic program for apoptosis.
American journal of physiology. Renal physiology 12/2003; 285(5):F1013-24. · 3.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Reactive oxygen species are recognized as important mediators of biological responses. Hyperglycemia promotes the intracellular generation of superoxide anion and hydrogen peroxide. In several cell lines, oxidant stress has been linked to the activation of death programs. Here, we report for the first time that high ambient glucose concentration induces apoptosis in murine and human mesangial cells by an oxidant-dependent mechanism. The signaling cascade activated by glucose-induced oxidant stress included the heterodimeric redox-sensitive transcription factor NF-kappaB, which exhibited an upregulation in p65/c-Rel binding activity and suppressed binding activity of the p50 dimer. Recruitment of NF-kappaB and mesangial cell apoptosis were both inhibited by antioxidants, implicating oxidant-induced activation of NF-kappaB in the transmission of the death signal. The genetic program for glucose-induced mesangial cell apoptosis was characterized by an upregulation of the Bax/Bcl-2 ratio. In addition, phosphorylation of the proapoptotic protein Bad was attenuated in mesangial cells maintained at high-glucose concentration, favoring progression of the apoptotic process. These perturbations in the expression and phosphorylation of the Bcl-2 family were coupled with the release of cytochrome c from mitochondria and caspase activation. Our findings indicate that in mesangial cells exposed to high ambient glucose concentration, oxidant stress is a proximate event in the activation of the death program, which culminates in mitochondrial dysfunction and caspase-3 activation, as the terminal event.
American journal of physiology. Renal physiology 04/2003; 284(3):F455-66. · 3.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The focus of this review will be recent advances in the molecular biology of protein kinase C (PKC) signaling in cardiac myocytes
and the application of these novel techniques to study the pathobiology of diabetic cardiac myopathy. The PKC family of serine/threonine
kinases have been implicated in a diverse array of biologic responses in health and disease. Compelling evidence has linked
PKC signaling to hyperglycemia mediated cell injury. Although the cardiac myocyte has not been traditionally considered a
major target cell for insulin, high ambient concentration of glucose promotes the activation of cardiac PKC isozymes, that
target physiologically relevant intracellular substrates. The availability of genetically engineered mice, with targeted activation
of distinct PKC isozyme (PKCe) in cardiac muscle cells and the development of selective peptide PKC modulators, provides an
approach to examine PKC signaling events in the diabetic heart, and to explore the in vivo and in vitro consequences of this
signaling cascade. The rapid growth of knowledge in this area is critical to the development of therapeutic strategies with
the potential to arrest or reverse the progression of diabetic cardiomyopathy.
Key wordsProtein Kinase C (PKC)–Hyperglycemia–Diabetes Mellitus–Cardiac Myocytes–PKC Isozymes
[Show abstract][Hide abstract] ABSTRACT: The PKC family of serine/threonine kinases have been implicated in a diverse array of cellular responses. Adult cardiac myocytes express multiple PKC isozymes, which participate in the response of muscle cells to extracellular stimuli, modulate contractile properties, and promote cell growth and survival. Recently, the classification of this ubiquitous family of signaling molecules has been expanded from three to four subfamilies. This review will focus on the application of pharmacologic and molecular approaches to explore the biology of cardiac PKC isozymes. The availability of transgenic mice and peptide PKC modulators have been instrumental in identifying target substrates for activated cardiac PKC isozymes, as well as the identification of specific isozymes linked to distinct growth characteristics and cell phenotype. The rapid growth of knowledge in the area of PKC signaling and PKC substrate interactions, may result in the development of therapeutic modalities with the potential to arrest or reverse the progression of cardiovascular diseases.
Molecular and Cellular Biochemistry 10/2001; 225(1-):97-107. · 2.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Activation of the protein kinase C (PKC) family is a potential signaling mechanism by which high ambient glucose concentration modulates the phenotype and physiological function of cells. Recently, the cardiac renin angiotensin system (RAS) has been reported to promote PKC translocation in the diabetic heart via the angiotensin (ANG) II type 1 receptor (AT-1R). To evaluate the molecular events coupled with high glucose-induced PKC translocation and to examine the role of endogenously released ANG II in myocyte PKC signaling, primary cultures of adult rat ventricular myocytes were exposed to normal (5 mmol/l) or high (25 mmol/l) glucose for 12-24 h. Western blot analysis indicated that adult rat ventricular myocytes coexpress six PKC isozymes (alpha, beta(1,) beta(2,) delta, epsilon, and zeta). Translocation of five PKC isozymes (beta(1), beta(2), delta, epsilon, and zeta) was detected in response to 25 mmol/l glucose. Inhibition of phospholipase C with tricyclodecan-9-yl-xanthogenate blocked glucose-induced translocation of PKC-beta(2), -delta, and -zeta. Inhibition of tyrosine kinase with genistein blocked glucose-induced translocation of PKC-beta(1) and -delta, whereas chelation of intracellular Ca(2+) with 1,2-bis(2-aminophenoxy)ethane N,N,N,'N'-tetraacetic acid blocked translocation of PKC-beta(1) and -beta(2). Enzyme-linked immunosorbent assay performed on culture media from myocytes maintained in 25 mmol/l glucose detected a twofold increase in ANG II. Addition of an AT-1R antagonist (losartan; 100 nmol/l) to myocyte cultures blocked translocation of PKC-beta(1), -beta(2), -delta, and -epsilon. Phosphorylation of troponin (Tn) I was increased in myocytes exposed to 25 mmol/l glucose. Losartan selectively inhibited Tn I serine phosphorylation but did not affect phosphorylation at threonine residues. We concluded that 1) 25 mmol/l glucose triggers the release of ANG II by myocytes, resulting in activation of the ANG II autocrine pathway; 2) differential translocation of myocyte PKC isozymes occurs in response to 25 mmol/l glucose and ANG II; and 3) AT-1R-dependent PKC isozymes (beta(1), beta(2), delta, and epsilon) target Tn I serine residues.