Small Molecular Weight G-protein Research Articles

Regulation of Rac1 transcription by histone and DNA methylation in diabetic retinopathy

Cytosolic ROS, generated by NADPH oxidase 2 (Nox2) in diabetes, damage retinal mitochondria, which leads to the development of retinopathy. A small molecular weight G-protein essential for Nox2 activation, Rac1, is also transcriptionally activated viaactive DNA methylation-hydroxymethylation. DNA methylation is a dynamic process, and can also be regulated by histone modifications; diabetes alters retinal histone methylation machinery. Our aim is to investigate the role of histone methylation (H3K9me3) of Rac1 promoter in dynamic DNA methylation- transcriptional activation. Using human retinal endothelial cells in 20 mM D-glucose, H3K9me3 at Rac1 promoter was quantified by chromatin-Immunoprecipitation technique. Crosstalk between H3K9me3 and DNA methylation was examined in cells transfected with siRNA of histone trimethyl-transferase, Suv39H1, or Dnmt1, exposed to high glucose. Key parameters were confirmed in retinal microvessels from streptozotocin-induced diabetic mice, with intravitreally administered Suv39H1-siRNA or Dnmt1-siRNA. Compared to cells in normal glucose, high glucose increased H3K9me3 and Suv39H1 binding at Rac1 promoter, and Suv39H1-siRNA prevented glucose-induced increase 5 hydroxy methyl cytosine (5hmC) and Rac1 mRNA. Similarly, in diabetic mice, Suv39H1-siRNA attenuated increase in 5hmC and Rac1 mRNA. Thus, H3K9me3 at Rac1 promoter assists in active DNA methylation-hydroxymethylation, activating Rac1 transcription. Regulation of Suv39H1-H3K9 trimethylation could prevent further epigenetic modifications, and prevent diabetic retinopathy.

Adaptor Protein p66Shc: A Link Between Cytosolic and Mitochondrial Dysfunction in the Development of Diabetic Retinopathy.

Diabetes increases oxidative stress in the retina and dysfunctions their mitochondria, accelerating capillary cell apoptosis. A 66 kDa adaptor protein, p66Shc, is considered as a sensor of oxidative stress-induced apoptosis. In the pathogenesis of diabetic retinopathy, a progressive disease, reactive oxygen species (ROS) production by activation of a small molecular weight G-protein (Ras-related C3 botulinum toxin substrate 1 [Rac1])-Nox2 signaling precedes mitochondrial damage. Rac1 activation is facilitated by guanine exchange factors (GEFs), and p66Shc increases Rac1-specific GEF activity of Son of Sevenless 1 (Sos1). p66Shc also possesses oxidoreductase activity and can directly stimulate mitochondrial ROS generation. Our aim was to investigate the role of p66Shc in the development of diabetic retinopathy and mechanism of its transcription. High glucose increased p66Shc expression in human retinal endothelial cells, and elevated acetylated histone 3 lysine 9 (H3K9) levels and transcriptional factor p53 binding at its promoter. Glucose also augmented interactions between Rac1 and Sos1 and activated Rac1-Nox2. Phosphorylation of p66Shc was increased, allowing it to interact with peptidyl prolyl isomerase to facilitate its localization inside the mitochondria, culminating in mitochondrial damage. P66shc-small interfering RNA (siRNA) inhibited glucose-induced Rac1 activation and mitochondrial damage. Similar results are observed in retinal microvessels from diabetic rats. This is the first report identifying the role of p66Shc in the development of diabetic retinopathy and implicating increased histone acetylation in its transcriptional regulation. Thus, p66Shc has dual role in the development of diabetic retinopathy; its regulation in the early stages of the disease should impede Rac1-ROS production and, in the later stages, prevent mitochondrial damage and initiation of a futile cycle of free radicals.

Diabetic retinopathy and transcriptional regulation of a small molecular weight G-Protein, Rac1

In diabetic retinopathy, increased cytosolic reactive oxygen species, produced by NADPH oxidase (Nox), damage mitochondria, and this accelerates apoptosis of retinal capillary cells, resulting in the histopathology. Activation of Nox2 is mediated by a small molecular weight GTPase, Rac1, and retinal Rac1 is activated in diabetes. Our goal is to investigate the molecular mechanism responsible for transcriptional activation of Rac1 in the development of diabetic retinopathy. Using retinal microvessels, the site of histopathology associated with diabetic retinopathy, from streptozotocin-induced diabetic rats, we investigated the binding of the nuclear transcriptional factor-kB (NF-kB) at Rac1 promoter. Since activation of NF-kB is regulated by its acetylation-deacetylation, the role of acetylation in Rac1 transcription was confirmed in the retina from diabetic mice overexpressing a deacetylase, Sirtuin 1. Diabetes increased the binding of p65 subunit of NF-kB at the Rac1 promoter. Overexpression of Sirtuin 1 prevented hyper-acetylation of p65, decreased its binding at the Rac1 promoter and ameliorated Rac1-Nox2 mediated mitochondrial damage. Thus, in diabetes Rac1 transcriptional activation in the retina is mediated by acetylation of NF-kB, and modulation of acetylation during the early stages of diabetic retinopathy has potential to inhibit/retard its development.

Calcium-Sensing Receptor: Trafficking, Endocytosis, Recycling, and Importance of Interacting Proteins.

The cloning of the extracellular calcium-sensing receptor (CaSR) provided a new paradigm in G-protein-coupled receptor (GPCR) signaling in which principal physiological ligand is a cation, namely, extracellular calcium (Ca(o)(2+)). A wealth of information has accumulated in the past two decades about the CaSR's structure and function, its contribution to pathology in disorders of calcium in humans, and CaSR-based therapeutics. The CaSR unlike many other GPCRs must function in the presence of its ligand, thus understanding the mechanisms such as anterograde trafficking and endocytic pathways of this receptor are complex and fallen behind other classical GPCRs. Factors controlling CaSR signaling include various proteins affecting the expression of the CaSR as well as modulation of its trafficking to and from the cell surface. The dimeric cell-surface CaSR links to various heterotrimeric G-proteins (G(q/11), G(i/o), G(12/13), and G(s)) to regulate intracellular second messengers, lipid kinases, various protein kinases, and transcription factors that are part of the machinery enabling the receptor to modulate the functions of the wide variety of cells in which it is expressed. This chapter describes key features of CaSR structure and function and discusses novel mechanisms by which the level of cell-surface receptor expression can be regulated including forward trafficking during biosynthesis, desensitization, internalization and recycling from the cell surface, and degradation. These processes are impacted by its interactions with several proteins in addition to signaling molecules per se (i.e., G-proteins, protein kinases, inositol phosphates, etc.) and include small molecular weight G-proteins (Sar1, Rabs, ARF, P24A, RAMPs, filamin A, 14-3-3 proteins, calmodulin, and caveolin-1). Moreover, CaSR signaling seems compartmentalized in cell-type-specific manner, and caveolin and filamin A likely act as scaffolds that bind signaling components and other key cellular elements (e.g., the cytoskeleton) to facilitate the interaction of the receptor with its signaling pathways. Regulatory mechanisms are still evolving to understand how defects in trafficking of CaSR contribute to pathology in disorders of calcium homeostasis.

The GAP Portion of Pseudomonas Aeruginosa Type III Secreted Toxin ExoS Upregulates Total and Surface Levels of Wild Type CFTR

Background: Pseudomonas aeruginosa (PA) infections account for a large percentage of fatal hospital acquired pneumonias. One of the PA Type III secreted toxin (TTST) ExoS, a bifunctional protein with N-terminal GTPase activating protein (GAP) and C-terminal ADP rybosyl transferase (ADPRT) activities, significantly contributes to PA virulence by targeting small molecular weight G-proteins (SMWGP). In this study, we have looked at one of the mechanisms by which the GAP portion of ExoS (ExoS-GAP) mediates cellular toxicity. Methods: The effects of ExoS-GAP on CFTR trafficking were studied in CFBE41o- Kir 2.2 and MDCK cell lines stably expressing CFTR using a transient transfection system. Results: Transient transfection of ExoS-GAP increased the total and surface protein levels of mature wild type CFTR in epithelial cells stably expressing wild type (WT) CFTR. The effect of ExoS-GAP was specific to CFTR in bronchial epithelial cells since it did not affect the total protein levels of Na⁺/K⁺ATPase, another membrane protein. A point mutation in the ExoS GAP domain (R146K), known to disrupt its catalytic GAP activity, abolished the effect of ExoS-GAP on WT CFTR. Lysosomal inhibition studies with Bafilomycin A1 indicate that ExoS-GAP decreased lysosomal degradation of the mature WT CFTR with concomitant increase in the total levels of mature WT CFTR. However, ExoS-GAP did not increase the total protein levels of ∆F508CFTR. Conclusion: The GAP portion of the PA TTST ExoS increases the total and surface levels of wild type CFTR in vitro mammalian cell system. The effect of ExoS-GAP on WT CFTR total protein levels provides new insight into understanding the virulent pathophysiology of PA infections.

Rab-geranylgeranyl transferase regulates glucose-stimulated insulin secretion from pancreatic β cells

A growing body of evidence implicates essential roles for small molecular weight G-proteins (e.g., Cdc42, Rac1, Arf6 and Rab3A and Rab27A) in islet β-cell function including glucose-stimulated insulin secretion (GSIS). One of the known mechanisms for optimal activation of small G-proteins involves post-translational prenylation, which is mediated by farnesyltransferase (FTase) and geranylgeranyl transferases (GGTases I and II). The FTase catalyzes incorporation of a 15-carbon farnesyl group while the GGTase mediates incorporation of a 20-carbon geranylgeranyl group into the C-terminal cysteines of G-proteins. The FTase, GGTase I and GGTase II prenylate Ras, Cdc42/Rac1, and Rab G-proteins, respectively. While considerable evidence exists on FTase/GGTase I-mediated regulation of GSIS, very little is known about GGTase II (also referred to as Rab GGTase; RGGT) and its regulatory proteins in the cascade of events leading to GSIS. Herein, we provide the first immunological evidence to suggest expression of α- and β-subunits of RGGT in clonal INS 832/13 β-cells, normal rat islets and human islets. Furthermore, Rab escort protein1 (REP1), which has been shown to be critical for prenylation of Rab G-proteins, is also expressed in these cells. Furthermore, evidence is presented to suggest that siRNA-mediated knockdown of α- or β-subunits of RGGT and REP1 markedly attenuates GSIS in INS 832/13 cells. These findings provide the first evidence in support of key roles for RGGT and its regulatory proteins in GSIS.

Diabetic retinopathy and signaling mechanism for activation of matrix metalloproteinase‐9

In the pathogenesis of diabetic retinopathy, H-Ras (a small molecular weight G-protein) and matrix metalloproteinase-9 (MMP9) act as pro-apoptotic, accelerating the apoptosis of retinal capillary cells, a phenomenon that predicts its development and the activation of MMP9 is under the control of H-Ras. The goal of this study is to elucidate the cellular mechanism by which H-Ras activates MMP9 culminating in the development of diabetic retinopathy. Using isolated retinal endothelial cells, the effect of regulation of H-Ras downstream signaling cascade, Raf-1, MEK, and ERK, was investigated on glucose-induced activation of MMP9. In vitro results were confirmed in the retina obtained from diabetic mice manipulated for MMP9 gene, and also in the retinal microvasculature obtained from human donors with diabetic retinopathy. Regulation of Raf-1/MEK/ERK by their specific siRNAs and pharmacologic inhibitors prevented glucose-induced activation of MMP9 in retinal endothelial cells. In MMP9-KO mice, diabetes had no effect on retinal MMP9 activation, and H-Ras/Raf-1/MEK signaling cascade remained normal. Similarly, donors with diabetic retinopathy had increased MMP9 activity in their retinal microvessels, the site of histopathology associated with diabetic retinopathy, and this was accompanied by activated H-Ras signaling pathway (Raf-1/ERK). Collectively, these results suggest that Ras/Raf-1/MEK/ERK cascade has an important role in the activation of retinal MMP9 resulting in the apoptosis of its capillary cells. Understanding the upstream mechanism responsible for the activation of MMP9 should help identify novel molecular targets for future pharmacological interventions to inhibit the development/progression of diabetic retinopathy.

Role of Matrix Metalloproteinase-9 in the Development of Diabetic Retinopathy and Its Regulation by H-Ras

Diabetes activates a small molecular weight G-protein, H-Ras, in the retina and its capillary cells, and H-Ras activation is implicated in the apoptosis of retinal capillary cells. Matrix metalloproteinase (MMP)-9 is regulated by H-Ras, and in diabetes its activation is associated with increased vascular permeability. The goal of this study was to investigate the role of sustained activation of MMP-9 in the pathogenesis of diabetic retinopathy and to illustrate the mechanism through which it is upregulated in diabetes. Retinal MMP-9 activation and its tissue inhibitor, TIMP-1, were quantified in streptozotocin-induced diabetic rats. Inhibition of H-Ras by simvastatin on diabetes-induced activation of H-Ras was evaluated. The mechanism by which diabetes regulates retinal MMP-9 was confirmed by determining the effect of genetic or pharmacologic regulation of H-Ras on its activation in retinal endothelial cells. In rats, MMP-9 was activated and expression of TIMP-1 was decreased in the retina and its microvasculature at both 2 months and 12 months of diabetes. In retinal endothelial cells, high glucose activated MMP-9, and inhibition of its activation (by pharmacologic inhibitor or siRNA) ameliorated accelerated apoptosis. Inhibition of H-Ras, both in diabetic rats (simvastatin) and in isolated endothelial cells (H-Ras siRNA), abrogated the activation of MMP-9 and prevented the reduction of TIMP-1. Hyperglycemia-induced activation of MMP-9 accelerates apoptosis of retinal capillary cells, a phenomenon that predicts the development of diabetic retinopathy, and the activation of MMP-9 is downstream of H-Ras. Characterizing the role of MMP-9 in the development of diabetic retinopathy will help explore novel molecular targets for future pharmacological interventions.

Translocation of H-Ras and its implications in the development of diabetic retinopathy

H-Ras, a small molecular weight G-protein, undergoes post-translational modifications enabling its translocation from cytosol to the membrane. Hyperglycemia increases apoptosis of retinal capillary cells via activation of H-Ras, which can be ameliorated by farnesylation inhibitors. Our aim is to investigate the mechanism of retinal H-Ras activation in diabetes. H-Ras and Raf-1 were quantified in the retinal membrane and cytosol fractions obtained from streptozotocin-induced diabetes rats, and the role of post-translation modification was determined by investigating the effect of simvastatin on diabetes-induced alterations. The effect of H-Ras-siRNA on membrane translocation and apoptosis was also determined in bovine retinal endothelial cells (BRECs). Diabetes increased expressions of H-Ras and Raf-1 in the retinal membranes, and simvastatin prevented such translocation. Glucose-exposure of BRECs increased membrane H-Ras expression and H-Ras-siRNA prevented this translocation, and also decreased their apoptosis. Thus, membrane translocation of H-Ras is a plausible mechanism responsible for accelerated apoptosis of retinal capillary cells in diabetes.

Rab7 Regulates Late Endocytic Trafficking Downstream of Multivesicular Body Biogenesis and Cargo Sequestration

The small molecular weight G-protein RAB7 is localized to both early and late endosomes and has been shown to be critical for trafficking through the endocytic pathway. The role of RAB7 in the endocytic pathway has been controversial, with some groups reporting that it regulates trafficking from early to late endosomes and others ascribing its role to trafficking between late endosomes and lysosomes. In this study, we use RNA interference to identify the exact step RAB7 regulates in the movement of the epidermal growth factor receptor (EGFR) from the cell surface to the lysosome. In the absence of RAB7, trafficking of the EGF.EGFR complex through the early endosome to the late endosome/multivesicular body (LE/MVB) does not change, but exiting from the LE/MVB is blocked. Ultrastructural analysis reveals that RAB7 is not required for formation of intraluminal vesicles of the LE/MVB, since RAB7-deficient cells have an increased number of enlarged LE/MVBs densely packed with intraluminal vesicles. Biochemical data indicate that the EGFR complex is sequestered in these intraluminal vesicles. Together, these data provide evidence that RAB7 is required for the transfer of cargo from the LE/MVB to the lysosome and for endocytic organelle maintenance.

Diabetes regulates small molecular weight G-protein, H-Ras, in the microvasculature of the retina: Implication in the development of retinopathy

Retinopathy, a largely microvascular complication, affects over 80% of patients with diabetes for 20 years. The purpose of this study is to investigate the effect of diabetes on the activation of H-Ras, a small molecular weight G-protein that regulates cell fate, in the retinal microvessels. Microvessels were prepared from freshly isolated retina from streptozotocin diabetic rats or 30% galactose-fed rats by hypotonic lysis method. Ras activation was quantified by Raf-1 binding assay, and the activation of the signaling proteins, Raf-1 and mitogen activated protein (MAP) kinase, by quantifying their gene transcripts (RTPCR) and/or by protein expression (western blot). Two months of diabetes or experimental galactosemia activated H-Ras (Raf-binding assay) in the retinal microvessels by over 40% and 70% respectively compared to the values obtained from normal rat retinal microvessels. In the same diabetic rats the gene transcripts of H-Ras and its effector protein Raf-1 were elevated by 30% and 135% respectively with their protein expressions elevated by about 25% each, and this was paralleled by similar increases in the protein expressions of H-Ras and Raf-1 in experimentally galactosemic rats. Diabetes increased the gene expression of Ras-Raf-1 downstream signaling protein MAP kinase by over 50%, and that of nuclear transcriptional factor by 25–30%. This activation of H-Ras in retinal microvessels implies that its signaling pathway, in part, could be contributing to the microvascular pathology characteristic of diabetic retinopathy. Comparable activation of H-Ras and its signaling cascade in the retinal microvessels from experimentally galactosemic rats suggests that H-Ras activation is not due to insulin deficiency. Regulation of Ras function could provide important target in the complex approach to inhibit the pathogenesis of diabetic retinopathy.

Role of Raf-1 kinase in Diabetes-induced Accelerated Apoptosis of Retinal Capillary Cells

Small molecular weight G-proteins serve as fundamental signaling switches that regulate cell fates by coupling receptor activation to downstream effector pathways. H-Ras, a small molecular weight G-protein, in its active form, recruits Raf. Activated Raf via a signaling transduction pathway regulates apoptosis. Our previous studies have shown that H-Ras has an important role in the loss of retinal capillary cells in diabetes. The purpose of this study is to investigate the role of Raf-1 in the development of diabetic retinopathy. Bovine retinal endothelial cells were incubated in 5 mM or 20 mM glucose in the presence of Raf-1 kinase inhibitor (10μM of GW5074), activator (200μM of ZM336374) or mitogen activated protein kinase inhibitor (30μM of PD098059) for five days. Apoptosis of endothelial cells was analyzed by ELISA and activation of Raf-1 and its downstream signaling proteins by determining genes and protein expressions. Inhibition of Raf-1 kinase repressed glucose-induced apoptosis of the cells by 75%, and this was accompanied by attenuation of activation of MAP kinase, ERK-1, nuclear transcriptional factor and caspase-3. In contrast, ZM336374 further increased glucose-induced apoptosis by 50%, and activated the signaling molecules and caspase 3 by over 30%. Further, PD098059 alone also attenuated glucose-induced apoptosis of retinal endothelial cells. These findings demonstrate that accelerated loss of retinal capillary cells in diabetes is mediated via Raf-1 kinase activation. Modulation of Raf-1 kinase activity could, in part, regulate apoptosis of retinal endothelial cells, which may ultimately contribute to the development of diabetic retinopathy.

Myosin Phosphatase Regulatory Pathways

In many ways, the smooth muscle cell (SMC) has been at the forefront in defining cellular signaling pathways. For example, the initial description of agonist-dependent intracellular calcium ([Ca2+]i) transients was made on vertebrate vascular SMC using aqueorin as the indicator.1 Delineation of nitric oxide (NO) and cGMP signaling was first accomplished in smooth muscle.2,3 And more recently, insights into the general mechanisms of gene transcription that dictate phenotypic traits of cells are being defined by smooth muscle specific gene expression.4,5 It should come as no surprise then that the interactions among many signaling pathways in cells should be explained first in the SMC. Perhaps the reason for the central position of the SMC in defining paradigms in cellular biology is that there are well-defined physiologic end points amenable to measurement (ie, contractile force) and even better described and easily quantified biochemical pathways that underlie said physiologic end points (ie, myosin regulatory light chain phosphorylation). From a historical perspective, the role of protein phosphorylation in smooth muscle contraction as a paradigm for cell signaling follows only that for the control of glycogen metabolism (see 6,7 for reviews). Increases in the levels of cytosolic [Ca2+]i initiate smooth muscle contraction by binding to the universal intracellular Ca2+ receptor protein, calmodulin (CaM), which in turn binds to and activates smooth muscle myosin light chain kinase (MLCK). Activated MLCK catalyzes the phosphorylation serine-19 of the regulatory myosin light chain (MLC) thereby increasing cross-bridge cycling and the rate of tension development. Dephosphorylation of MLC is catalyzed by smooth muscle specific myosin light chain phosphatase (MLCP). Decreases in cytosolic [Ca2+]i and MLC dephosphorylation are considered salient events in smooth muscle relaxation. What has become apparent only within the last decade is the fact that …

Hyperglycemia Inhibits Retinoic Acid–Induced Activation of Rac1, Prevents Differentiation of Cortical Neurons, and Causes Oxidative Stress in a Rat Model of Diabetic Pregnancy

Diabetes is a risk factor for neuronal dysfunction. Impairment in signaling mechanisms that regulate differentiation of neurons is hypothesized to be one of the main causes of neuronal dysfunction. Retinoic acid, a physiologically active retinoid synthesized from vitamin A, regulates neuronal differentiation during embryonic development and is required for maintenance of plasticity in differentiated neurons. To date, little is known about the molecular events underlying hyperglycemia-induced complications in the central nervous system (CNS). Here, we provide evidence, in a diabetes rat model, of hyperglycemia-induced oxidative stress along with apoptotic stress in developing cortical neurons isolated from 16-day-old rat embryos. We also demonstrate impaired retinoic acid signaling that is involved in neuronal differentiation. Retinoic acid-induced neurite outgrowth and expression of neuronal markers were reduced in this model. The activation of small-molecular weight G-protein, Rac1, that mediates these effects was also reduced. Retinoic acid applied at a physiological concentration significantly decreased hyperglycemia-induced oxidative stress and thus supported the antioxidant defense system. These results suggest that diabetes-induced neuronal complications during pregnancy might be due to impaired retinoic acid signaling, and exogenously administered retinoic acid may be useful against CNS complications associated with diabetes.

Small molecular weight G-protein, H-Ras, and retinal endothelial cell apoptosis in diabetes

We have demonstrated that the expressions of small molecular weight G-protein, H-Ras, and its effector protein, Raf-1, are increased in the retina in diabetes, and the specific inhibitors of Ras function inhibit glucose-induced apoptosis of retinal capillary cells. This study is to examine the contributory roles for H-Ras in glucose-induced apoptosis of retinal endothelial cells by genetic manipulation of functionally active H-Ras levels. Bovine retinal endothelial cells were transfected with the plasmids of either wild type (WT), constitutively active (V12) or dominant-negative (N17) H-Ras. Glucose-induced increase in apoptosis, nitric oxide (NO) levels and activation of NF-kappaB and caspase-3 were determined in these genetically manipulated cells. Exposure of bovine retinal endothelial cells to 20 mM glucose significantly increased H-Ras activation as determined by Raf-1 binding assay. Overexpression of V12 in the endothelial cells further increased their glucose-induced apoptosis by 40%, NO levels by about 50%, and activated NF-kappaB and caspase-3 by about 30-40% compared to the untransfected cells incubated in 20 mM glucose. In contrast, overexpression of the inactive mutant, N17, inhibited glucose-mediated increases in apoptotic cell death, NO levels and NF-kappaB and caspase-3 activation; the values were significantly different (p < 0.02) compared to those obtained from the untransfected cells incubated under similar conditions. Our findings demonstrate that H-Ras activation is important in the activation of the specific signaling events leading to the accelerated retinal capillary cell apoptosis in hyperglycemic conditions, suggesting the possible use of H-Ras inhibitors to inhibit the pathogenesis of diabetic retinopathy.

Interferon-γ enhances superoxide production in human mesangial cells via the JAK–STAT pathway

Immune reactive cytokines, such as interferon (IFN)-gamma, have multiple effects in glomerulonephritis. Superoxide anions (O(2)(-)), which are associated with the progression of glomerulonephritis, are mainly generated by nicotinamide adenine dinucleotide phosphate (reduced form) NAD(P)H oxidases. We determined the effects of IFN-gamma on O(2)(-) production, phosphorylation of signal transducer and activator of transcription (STAT)-1alpha, and the mRNA and protein expressions of p22phox and Nox1, components of NAD(P)H oxidases, in human mesangial cells (HMCs). Significant increases in O(2)(-) production with IFN-gamma were completely abolished by the flavin-containing enzyme inhibitor, diphenyleneiodonium (10 micromol/l), and the Janus-activated kinase (JAK)2 inhibitor, AG490 (100 micromol/l). Phosphorylated STAT-1alpha was detected after 5 min of IFN-gamma stimulation using Western blot analysis, and binding to the gamma-activating site was observed from 30 min to 4 h, thereafter by electrophoretic mobility shift assay (EMSA). Super-shift analysis in EMSA revealed that the main transcription factor was STAT-1alpha. IFN-gamma significantly increased the expression of p22phox mRNA and protein, although expression was inhibited by AG490. These data suggest that IFN-gamma stimulates O(2)(-) production in HMCs via the JAK-STAT pathway and NAD(P)H oxidase.

Rab7 Activity Affects Epidermal Growth Factor:Epidermal Growth Factor Receptor Degradation by Regulating Endocytic Trafficking from the Late Endosome

The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family. Ligand (epidermal growth factor or EGF) binding to the EGFR results in the coordinated activation and integration of biochemical signaling events to mediate cell growth, migration, and differentiation. One mechanism the cell utilizes to orchestrate these events is ligand-mediated endocytosis through the canonical clathrin-mediated endocytic pathway. Identification of proteins that regulate the intracellular movement of the EGF.EGFR complex is an important first step in dissecting how specificity of EGFR signaling is conferred. We examined the role of the small molecular weight guanine nucleotide-binding protein (G-protein) rab7 as a regulator of the distal stages of the endocytic pathway. Through the transient expression of activating and inactivating mutants of rab7 in HeLa cells, we have determined that rab7 activity directly correlates with the rate of radiolabeled EGF and EGFR degradation. Furthermore, when inhibitory mutants of rab7 are expressed, the internalized EGF.EGFR complex accumulates in high-density endosomes that are characteristic of the late endocytic pathway. Thus, we conclude that rab7 regulates the endocytic trafficking of the EGF.EGFR complex by regulating its lysosomal degradation.

Differential Activation of Stress Kinases Pathways and Elaboration of Inflammatory Cytokines MCP-1 and RANTES by Major and Minor Group Rhinovirus.

Given that virus-induced asthma has been resistant to therapy, knowledge concerning the cellular and physiological processes that are critical in the development of virus-induced asthma is likely to lead to the generation of new forms of intervention. In this regard, previous studies have implicated the macrophage as playing an important role in the elaboration of the inflammatory environment observed in rhinovirus-induced exacerbation of asthma. However, little is known about the cell signaling events that are initiated upon macrophage infection with rhinovirus. Major and minor group rhinovirus are genetically similar although they bind to two different cellular receptors. To determine if the attachment of major (HRV16) and minor group (HRV1a) rhinovirus to the human monocyte-derived macrophages elicits different kinetics of activation for ERK5 and p38, macrophages cells were exposed to HRV16 or HRV1a at an MOI of 10 for 15,30,60, 90 and 120 min. Cell lysates were generated, separated by SDS-PAGE and immunoblotted with anti-phospho-ERK5 or p38. For MAP kinase p38, major and minor group rhinovirus stimulated phosphorylation occurred after 15 min and sustained phosphorylation up to 60 min. Interestingly at the 90 and 120 min time points, there was no p38 phosphorylation for the major group rhinovirus and sustained phosphorylation stimulated by the minor group rhinovirus. A similar kinetics profile holds true for the MAP kinase ERK5. Furthermore, activation of these two MAP kinases and the small molecular weight G-protein RAC are important in the elaboration of the inflammatory mediators MCP-1 and RANTES.

GTP-binding protein-independent potentiation by mastoparan of IL-1beta-induced nitric oxide release from insulin-secreting HIT-T15 cells.

Our recent data implicated small molecular weight G-proteins (e.g., H-Ras) in interleukin 1beta (IL 1beta)-induced metabolic dysfunction and apoptotic demise of the islet beta cell (Tannous et al., Biochem Pharmacol 2001; 62:1459-1468, Kowluru and Morgan, Biochem Pharmacol, 2002; 63:1027-1035, Chen et al. Biochem Pharmacol, 2003; 66:1681-1694). Recently, we have shown that mastoparan, a tetradecapeptide from wasp venom, has been shown to directly activate islet endogenous G-proteins and regulate islet function (Amin et al., Endocrinology 2003; 144: 4508-4518). Herein, we investigated potential contributory roles, if any, of mastoparan (Mas)-sensitive G-proteins in IL-induced nitric oxide (NO) release from insulin-secreting HIT-T15 cells. While, ineffective by itself, Mas significantly potentiated IL-induced NO release from HIT-T15 cells. Interestingly, Mas-17, an inactive analog of Mas, also potentiated IL-induced NO release, suggesting that the potentiating effect of Mas may not involve activation of specific G-proteins. Such potentiating effects on IL-induced NO release were also demonstrable in the presence of another polycationic compound, melittin. Together, these findings suggest that Mas-induced potentiation of IL-induced NO release may in part be due to its amphiphilic and polycationic nature. These data also warrant caution in the use of Mas to study its regulation of cellular function without the use of an appropriate negative control, such as Mas-17.

Rab32 is an A-kinase anchoring protein and participates in mitochondrial dynamics

A-kinase anchoring proteins (AKAPs) tether the cAMP-dependent protein kinase (PKA) and other signaling enzymes to distinct subcellular organelles. Using the yeast two-hybrid approach, we demonstrate that Rab32, a member of the Ras superfamily of small molecular weight G-proteins, interacts directly with the type II regulatory subunit of PKA. Cellular and biochemical studies confirm that Rab32 functions as an AKAP inside cells. Anchoring determinants for PKA have been mapped to sites within the conserved α5 helix that is common to all Rab family members. Subcellular fractionation and immunofluorescent approaches indicate that Rab32 and a proportion of the cellular PKA pool are associated with mitochondria. Transient transfection of a GTP binding–deficient mutant of Rab32 promotes aberrant accumulation of mitochondria at the microtubule organizing center. Further analysis of this mutant indicates that disruption of the microtubule cytoskeleton results in aberrantly elongated mitochondria. This implicates Rab32 as a participant in synchronization of mitochondrial fission. Thus, Rab32 is a dual function protein that participates in both mitochondrial anchoring of PKA and mitochondrial dynamics.