Aberrant expression of LMO4 induces centrosome amplification and mitotic spindle abnormalities in breast cancer cells.

The LIM-only protein, LMO4, is a transcriptional modulator overexpressed in breast cancer. It is oncogenic in murine mammary epithelium and is required for G2/M progression of ErbB2-dependent cells as well as growth and invasion of other breast cancer cell types. However, the mechanisms underlying the oncogenic activity of LMO4 remain unclear. Herein, we show that LMO4 is expressed in all breast cancer subtypes examined and its expression level correlates with the degree of proliferation of such tumours. In addition, we have determined that LMO4 silencing induces G2/M arrest in cells from various breast cancer subtypes, suggesting that LMO4 action in the cell cycle is not restricted to a single breast cancer subtype. This arrest was accompanied by increased cell death, amplification of centrosomes, and formation of abnormal mitotic spindles. Consistent with its ability to positively and negatively regulate the formation of active transcription complexes, overexpression of LMO4 also resulted in an increase in centrosome number. Centrosome amplification has been shown to prolong the G2/M phase of the cell cycle and induce apoptosis; thus, we conclude that supernumerary centrosomes mediate the G2/M arrest and cell death in LMO4-deficient cells. Furthermore, the correlation of centrosome amplification with genomic instability suggests that the impact of dysregulated LMO4 on the centrosome cycle may promote LMO4-induced tumour formation.

Glutaredoxin regulates apoptosis in cardiomyocytes via NFkappaB targets Bcl-2 and Bcl-xL: implications for cardiac aging.

Cardiomyocyte apoptosis is a well-established contributor to irreversible injury following myocardial infarction (MI). Increased cardiomyocyte apoptosis is associated also with aging in animal models, exacerbated by MI; however, mechanisms for this increased sensitivity to oxidative stress are unknown. Protein mixed-disulfide formation with glutathione (protein glutathionylation) is known to change the function of intermediates that regulate apoptosis. Since glutaredoxin (Grx) specifically catalyzes protein deglutathionylation, we examined its status with aging and its influence on regulation of apoptosis. Grx1 content and activity are decreased by approximately 40% in elderly (24-mo) Fischer 344 rat hearts compared to adult (6-mo) controls. A similar extent of Grx1 knockdown in H9c2 cardiomyocytes led to increased apoptosis, decreased NFkappaB-dependent transcriptional activity, and decreased production (mRNA and protein) of anti-apoptotic NFkappaB target genes, Bcl-2 and Bcl-xL. Knockdown of Bcl-2 and/or Bcl-xL in wild-type H9c2 cells to the same extent ( approximately 50%) as observed in Grx1-knockdown cells increased baseline apoptosis; and knockdown of Bcl-xL, but not Bcl-2, also increased oxidant-induced apoptosis analogous to Grx1-knockdown cells. Natural Grx1-deficient cardiomyocytes isolated from elderly rats also displayed diminished NFkappaB activity and Bcl-xL content. Taken together, these data indicate diminution of Grx1 in elderly animals contributes to increased apoptotic susceptibility via regulation of NFkappaB function.

Glutaredoxin regulates autocrine and paracrine proinflammatory responses in retinal glial (muller) cells.

Protein S-glutathionylation is a reversible redox-dependent post-translational modification. Many cellular functions and signal transduction pathways involve proteins whose cysteine-dependent activities are modulated by glutathionylation. Glutaredoxin (Grx1) plays a key role in such regulation because it is a specific and efficient catalyst of deglutathionylation. We recently reported an increase in Grx1 in retinae of diabetic rats and in rat retinal Müller glial cells (rMC-1) cultured in high glucose. This up-regulation of Grx1 was concomitant with NFkappaB activation and induction of intercellular adhesion molecule-1 (ICAM-1). This proinflammatory response was replicated by adenoviral-directed up-regulation of Grx1 in cells in normal glucose. The site of regulation of NFkappaB was localized to the cytoplasm, where IkappaB kinase (IKK) is a master regulator of NFkappaB activation. In the current study, inhibition of IKK activity abrogated the increase in ICAM-1 induced by high glucose or by adenoviral-directed up-regulation of Grx1. Conditioned medium from the Müller cells overexpressing Grx1 was added to fresh cultures of Müller or endothelial cells and elicited increases in the Grx1 and ICAM-1 proteins in these cells. These effects correlate with a novel finding that secretion of interleukin-6 was elevated in the cultures of Grx overexpressing cells. Also, pure interleukin-6 increased Grx1 and ICAM-1 in the rMC-1 cells. Thus, Grx1 appears to play an important role in both autocrine and paracrine proinflammatory responses. Furthermore, IKKbeta isolated from Müller cells in normal glucose medium was found to be glutathionylated on Cys-179. Hence Grx-mediated activation of IKK via deglutathionylation may play a central role in diabetic complications in vivo where Grx1 is increased.

Molecular mechanisms and clinical implications of reversible protein S-glutathionylation.

Sulfhydryl chemistry plays a vital role in normal biology and in defense of cells against oxidants, free radicals, and electrophiles. Modification of critical cysteine residues is an important mechanism of signal transduction, and perturbation of thiol-disulfide homeostasis is an important consequence of many diseases. A prevalent form of cysteine modification is reversible formation of protein mixed disulfides (protein-SSG) with glutathione (GSH). The abundance of GSH in cells and the ready conversion of sulfenic acids and S-nitroso derivatives to S-glutathione mixed disulfides suggests that reversible S-glutathionylation may be a common feature of redox signal transduction and regulation of the activities of redox sensitive thiol-proteins. The glutaredoxin enzyme has served as a focal point and important tool for evolution of this regulatory mechanism, because it is a specific and efficient catalyst of protein-SSG deglutathionylation. However, mechanisms of control of intracellular Grx activity in response to various stimuli are not well understood, and delineation of specific mechanisms and enzyme(s) involved in formation of protein-SSG intermediates requires further attention. A large number of proteins have been identified as potentially regulated by reversible S-glutathionylation, but only a few studies have documented glutathionylation-dependent changes in activity of specific proteins in a physiological context. Oxidative stress is a hallmark of many diseases which may interrupt or divert normal redox signaling and perturb protein-thiol homeostasis. Examples involving changes in S-glutathionylation of specific proteins are discussed in the context of diabetes, cardiovascular and lung diseases, cancer, and neurodegenerative diseases.

Regulation by reversible S-glutathionylation: molecular targets implicated in inflammatory diseases.

S-glutathionylation is a reversible post-translational modification that continues to gain eminence as a redox regulatory mechanism of protein activity and associated cellular functions. Many diverse cellular proteins such as transcription factors, adhesion molecules, enzymes, and cytokines are reported to undergo glutathionylation, although the functional impact has been less well characterized. De-glutathionylation is catalyzed specifically and efficiently by glutaredoxin (GRx, aka thioltransferase), and facile reversibility is critical in determining the physiological relevance of glutathionylation as a means of protein regulation. Thus, studies with cohesive themes addressing both the glutathionylation of proteins and the corresponding impact of GRx are especially useful in advancing understanding. Reactive oxygen species (ROS) and redox regulation are well accepted as playing a role in inflammatory processes, such as leukostasis and the destruction of foreign particles by macrophages. We discuss in this review the current implications of GRx and/or glutathionylation in the inflammatory response and in diseases associated with chronic inflammation, namely diabetes, atherosclerosis, inflammatory lung disease, cancer, and Alzheimer's disease, and in viral infections.

Glutaredoxin regulates nuclear factor kappa-B and intercellular adhesion molecule in Müller cells: model of diabetic retinopathy.

Reversible S-glutathionylation of proteins is a focal point of redox signaling and cellular defense against oxidative stress. This post-translational modification alters protein function, and its reversal (deglutathionylation) is catalyzed specifically and efficiently by glutaredoxin (GRx, thioltransferase), a thioldisulfide oxidoreductase. We hypothesized that changes in glutaredoxin might be important in the development of diabetic retinopathy, a condition characterized by oxidative stress. Indeed, GRx protein and activity were increased in retinal homogenates from streptozotocin-diabetic rats. Also, incubation of rat retinal Müller cells (rMC-1) in normal glucose (5 mm) or diabetic-like glucose (25 mm) medium led to selective upregulation of GRx in contrast to thioredoxin, the other thioldisulfide oxidoreductase system. Under analogous conditions, NF-kappaB (p50-p65) translocated to the nucleus, and expression of ICAM-1 (intercellular adhesion molecule-1), a transcriptional product of NF-kappaB, increased. Proinflammatory ICAM-1 is increased in diabetic retinae, and it is implicated in pathogenesis of retinopathy. To evaluate the role of GRx in mediating these changes, intracellular GRx content and activity in rMC-1 cells were increased independently under normal glucose via infection with an adenoviral GRx1 construct (Ad-GRx). rMC-1 cells exhibited adenovirus concentration-dependent increases in GRx and corresponding increases in NF-kappaB nuclear translocation, NF-kappaB luciferase reporter activity, and ICAM-1 expression. Blocking the increase in GRx1 via small interfering RNA in rMC-1 cells in high glucose prevented the increased ICAM-1 expression. These data suggest that redox regulation by glutaredoxin in retinal glial cells is perturbed by hyperglycemia, leading to NF-kappaB activation and a pro-inflammatory response. Thus, GRx may represent a novel therapeutic target to inhibit diabetic retinopathy.

Glutaredoxin: role in reversible protein s-glutathionylation and regulation of redox signal transduction and protein translocation.

Reversible posttranslational modifications on specific amino acid residues can efficiently regulate protein functions. O-Phosphorylation is the prototype and analogue to the rapidly emerging mechanism of regulation known as S-glutathionylation. The latter is being recognized as a potentially widespread form of modulation of the activities of redox-sensitive thiol proteins, especially those involved in signal transduction pathways and translocation. The abundance of reduced glutathione in cells and the ready conversion of sulfenic acids and S-nitroso derivatives to S-glutathione mixed disulfides support the notion that reversible S-glutathionylation is likely to be the preponderant mode of redox signal transduction. The glutaredoxin enzyme has served as a focal point and important tool for evolution of this regulatory mechanism because of its characterization as a specific and efficient catalyst of protein-SSG de-glutathionylation (akin to phosphatases). Identification of specific mechanisms and enzyme(s) that catalyze formation of protein-SSG intermediates, however, is largely unknown and represents a prime objective for furthering understanding of this evolving mechanism of cellular regulation. Several proteomic approaches, including the use of cysteine-reactive fluorescent and radiolabel probes, have been developed to detect arrays of proteins whose cysteine residues are modified in response to oxidants, thus identifying them as potential interconvertible proteins to be regulated by redox signaling (glutathionylation). Specific criteria were used to evaluate current data on cellular regulation via S-glutathionylation. Among many proteins under consideration, actin, protein tyrosine phosphatase-1B, and Ras stand out as the best current examples for establishing this regulatory mechanism.