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Publications and source records attributed to J E Kudlow.
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We have previously shown that rat pancreatic islets contain a predominant 135 kDa O-glycosylated protein (p135) that is recognized by immunoprecipitation and Western blotting with anti-O-GlcNAc antibody. In this paper, we show that p135 is also detectable in other rat tissues including brain, heart, liver, spleen, and lung, but not kidney. To identify p135, the protein was purified from rat brain using a multistep procedure including selective absorption with anti-O-GlcNAc antibody. After electrophoresis, and Coomassie staining, the protein was excised from the gel for tryptic digestion. Next, O-methylisourea was used to convert lysine residues to homoarginine to increase the sequence coverage, and MALDI-TOF mass spectrometry detection was performed. MALDI-TOF identified p135 as rat O-GlcNAc transferase (OGT), an identity confirmed by LC/MS of individual peptides. The identification of p135 as OGT is consistent with previous reports of the tissue distribution of OGT, as well as reports that OGT is itself O-glycosylated.
The posttranslational modification of eukaryotic intracellular proteins by O-linked N-acetylglucosamine (O-GlcNAc) monosaccharides is essential for cell viability, yet its precise functional roles are largely unknown. O-GlcNAc transferase utilizes UDP-GlcNAc, the end product of hexosamine biosynthesis, to catalyze this modification. The availability of UDP-GlcNAc correlates with glycosylation levels of intracellular proteins as well as with transcriptional levels of some genes. Meanwhile, transcription factors and RNA polymerase II can be modified by O-GlcNAc. A linkage between transcription factor O-GlcNAcylation and transcriptional regulation therefore has been postulated. Here, we show that O-GlcNAcylation of a chimeric transcriptional activator containing the second activation domain of Sp1 decreases its transcriptional activity both in an in vitro transcription system and in living cells, which is in concert with our observation that O-GlcNAcylation of Sp1 activation domain blocks its in vitro and in vivo interactions with other Sp1 molecules and TATA-binding protein-associated factor II 110. Furthermore, overexpression of O-GlcNAc transferase specifically inhibits transcriptional activation by native Sp1 in cells. Thus, our studies provide direct evidence that O-GlcNAcylation of transcription factors is involved in transcriptional regulation.
Streptozotocin (STZ), an analogue of GlcNAc, inhibits purified rat spleen O-GlcNAc-selective N-acetyl-beta-D-glucosaminidase (O-GlcNAcase), the enzyme that removes O-GlcNAc from protein. We have shown previously that STZ increases pancreatic islet O-linked protein glycosylation. In light of these data, we investigated the possibility further that STZ causes beta-cell death by inhibiting O-GlcNAcase. In isolated islets, the time course and dose curve of STZ-induced O-glycosylation correlated with beta-cell toxicity. STZ inhibition of rat islet O-GlcNAcase activity also paralleled that of its beta-cell toxicity, with significant inhibition occurring at a concentration of 1 mM. In contrast, STZ inhibition of rat brain O-GlcNAcase and beta-TC3 insulinoma cell O-GlcNAcase was significantly right-shifted compared with islets, with STZ only significantly inhibiting activity at a concentration of 5 mM, the same concentration required for beta-TC3 cell toxicity. In comparison, N-methyl-N-nitrosourea, the nitric oxide-donating portion of STZ, did not cause increased islet O-glycosylation, beta-cell toxicity or inhibition of beta-cell O-GlcNAcase. Enhanced STZ sensitivity of islet O-GlcNAcase compared with O-GlcNAcase from other tissues or an insulinoma cell line suggests why actual islet beta-cells are particularly sensitive to STZ. Confirming this idea, STZ-induced islet beta-cell toxicity was completely blocked by GlcNAc, which also prevented STZ-induced O-GlcNAcase inhibition, but was not even partially blocked by glucose, glucosamine or GalNAc. Together, these data demonstrate that STZ's inhibition of beta-cell O-GlcNAcase is the mechanism that accounts for its diabetogenic toxicity.
The epidermal growth factor receptor (EGFR) and its ligands EGF and transforming growth factor-alpha (TGF alpha) are expressed in the anterior pituitary, and overexpression of TGF alpha in the lactotrope cells of the pituitary gland in transgenic mice results in lactotrope hyperplasia and adenomata, suggesting a role for EGFR signaling in pituitary cell proliferation. To address the role of EGFR signaling in pituitary development in vivo, we blocked EGFR signaling in transgenic mice using the dominant negative properties of a mutant EGFR lacking an intracellular protein kinase domain (EGFR-tr). We directed EGFR-tr expression to GH- and PRL- producing cells using GH and PRL promoters, and a tetracycline-inducible gene expression system, to allow temporal control of gene expression. EGFR-tr overexpression in GH-producing cells during embryogenesis resulted in dwarf mice with pituitary hypoplasia. Both somatotrope and lactotrope development were blocked. However, when EGFR-tr overexpression was delayed to the postnatal period either by directing its expression with the PRL promoter or by delaying the onset of induction with tetracycline in the GH cells, no specific phenotype was observed. Lactotrope hyperplasia during pregnancy also occurred normally in the PRL-EGFR-tr mice. These data suggest that EGFR signaling is required for the differentiation and/or maintenance of somatomammotropes early in pituitary organogenesis but not later in life. (Molecular Endocrinology 15: 600-613, 2001)
An important link has recently been shown in vivo between beta-cell O-linked protein glycosylation and beta-cell apoptosis, with hyperglycemia having been demonstrated to reversibly increase beta-cell O-linked protein glycosylation by providing substrate for the glucosamine pathway. In contrast, the same study showed that the administration of streptozotocin to rats prior to the induction of hyperglycemia results in irreversible increases in O-glycosylation and subsequent beta-cell apoptosis. In light of these data, we investigated beta-cell O-glycosylation in vitro by exposing isolated rat islets to high glucose, glucosamine, or streptozotocin and analyzing the pattern of O-glycosylated proteins present. All three compounds acutely increased O-glycosylation of a predominate 135-kDa protein (p135); however, their ability to stimulate p135 O-glycosylation was only consistently observed when islets were isolated in the presence of high glucose and 1 mM L-glutamine. Islets isolated in low glucose and no added L-glutamine demonstrated no consistent increase in p135 O-glycosylation in response to glucose, glucosamine, or streptozotocin. These data suggest that during islet isolation, beta-cell enzymes responsible for regulating p135 O-glycosylation may be adversely affected by the absence of high glucose and glutamine, which together are necessary for O-linked N-acetylglucosamine synthesis. We propose that the combination of high glucose and glutamine during islet isolation generates UDP-N-acetylglucosamine and O-linked N-acetylglucosamine, thus providing substrate protection for these enzymes and preserving the ability of isolated islets to O-glycosylate p135.
O-linked N-acetylglucosamine (O-GlcNAc) modification has been described in many proteins, including nuclear pore glycoproteins. In the present study we investigated the effect of extracellular glucose on the level of modification of nuclear pore protein p62 by O-GlcNAc. We found that exposure of cells to a high concentration of glucose caused an increased level of modification of p62 with O-GlcNAc, whereas the modification of other proteins did not change detectably. The increased O-GlcNAc modification of p62 in response to glucose required the metabolism of glucose to glucosamine. The exposure of the cells to glucosamine itself also caused increased O-GlcNAc modification, whereas mannosamine and galactosamine did not. Furthermore, changes in glucose concentrations within the physiological range induced the O-GlcNAc modification in p62 in rat aortic smooth-muscle cells, indicating that this modification of p62 might occur in an intact animal. These results imply that the ambient glucose concentration has an impact on the level of O-GlcNAc modification of proteins such as p62, and that functional changes in some of these proteins might ensue.
Glutamine:fructose-6-phosphate amidotransferase (GFAT) is the rate-limiting enzyme in glucosamine synthesis. Prior studies from our laboratory indicated that activation of adenylate cyclase was associated with depletion of O-GlcNAc modification. This finding and evidence that human GFAT (hGFAT) might be regulated by cAMP-dependent protein kinase (PKA) led us to investigate the role of PKA in hGFAT function. We confirmed that adenylate cyclase activation by forskolin results in diminished O-GlcNAc modification of several cellular proteins which can be overcome by exposure of the cells to glucosamine but not glucose, suggesting the PKA activation results in depletion of UDP-GlcNAc for O-glycosylation. To determine if GFAT is indeed regulated by PKA, we expressed the active form of the enzyme using a vaccinia virus expression system and showed that the activity of the enzyme was to decrease to undetectable levels by PKA phosphorylation. We mapped the PKA phosphorylation sites with the aid of matrix-assisted laser desorption ionization mass spectroscopy and showed that the protein was stoichiometrically phosphorylated at serine 205 and also phosphorylated, to a lesser extent at serine 235. Mutagenesis studies indicated that the phosphorylation of serine 205 by PKA was necessary for the observed inhibition of enzyme activity while serine 235 phosphorylation played no observable role. The activity of GFAT is down-regulated by cAMP, thus placing regulation on the hexosamine pathway that is in concert with the energy requirements of the organism. During starvation, hormones acting through adenylate cyclase could direct the flux of glucose metabolism into energy production rather than into synthetic pathways that require hexosamines.
The transcription factor Sp1 was previously shown to undergo proteasome-dependent degradation when cells were glucose-starved and stimulated with the adenylate cyclase inducer, forskolin. However, the control of the Sp1 degradation process is largely unknown. Using in vitro and in vivo interaction studies, we show in the present study that Sp1 interacts with human Sug1 [hSug1, also known as p45 or thyroid-hormone-receptor interacting protein ('TRIP1')], an ATPase subunit of the 26 S proteasome and a putative transcriptional modulator. This interaction with Sp1 occurs through the C-terminus of hSug1, the region that contains the conserved ATPase domain in this protein. Both in vitro studies, in reconstituted degradation assays, and in vivo experiments, in which hSug1 is overexpressed in normal rat kidney cells, show that full-length hSug1 is able to stimulate the proteasome-dependent degradation of Sp1. However, hSug1 truncations that lack either the N- or C-terminal domain of hSug1 act as dominant negatives, inhibiting Sp1 degradation in vitro. Also, an ATPase mutant of hSug1, while still able to bind Sp1, acts as a dominant negative, blocking Sp1 degradation both in vitro and in vivo. These results demonstrate that hSug1 is involved in the degradation of Sp1 and that ATP hydrolysis by hSug1 is necessary for this process. Our findings indicate that hSug1 is an exchangeable proteasomal component that plays a critical regulatory role in the proteasome-dependent degradation of Sp1. However, hSug1 is not the factor limiting Sp1 degradation in the cells treated with glucosamine. This and other considerations suggest that hSug1 co-operation with other molecules is necessary to target Sp1 for proteasome degradation.
The pancreatic beta cell can respond in the long term to hyperglycemia both with an increased capacity for insulin production and, in susceptible individuals, with apoptosis. When glucose-induced apoptosis offsets the increasing beta cell capacity, type 2 diabetes results. Here, we tested the idea that the pathway of glucose metabolism that leads to the modification of intracellular proteins with the O-linked monosaccharide N-acetylglucosamine (O-GlcNAc) is involved in the glucose-induced apoptosis. This idea is based on two recent observations. First, the beta cell expresses much more O-GlcNAc transferase than any other known cell, and second, that the beta cell-specific toxin, streptozotocin (STZ), itself a GlcNAc analog, specifically blocks the enzyme that cleaves O-GlcNAc from intracellular proteins. As a consequence, we now show that hyperglycemia leads to the rapid and reversible accumulation of O-GlcNAc specifically in beta cells in vivo. Animals pretreated with STZ also accumulate O-GlcNAc in their beta cells when hyperglycemic, but this change is sustained upon re-establishment of euglycemia. In concert with the idea that STZ toxicity results from the sustained accumulation of O-GlcNAc after a hyperglycemic episode, we established a low-dose STZ protocol in which the beta cells' toxicity of STZ was manifest only after glucose or glucosamine administration. Transgenic mice with impaired beta cell glucosamine synthesis treated with this protocol are resistant to the diabetogenic effect of STZ plus glucose yet succumb to STZ plus glucosamine. This study provides a causal link between apoptosis in beta cells and glucose metabolism through glucosamine to O-GlcNAc, implicating this pathway of glucose metabolism with beta cell glucose toxicity.
Streptozotocin has been widely used to create animal models of diabetes. Structurally, streptozotocin resembles N-acetylglucosamine, with a nitrosourea group corresponding to the acetate present in N-acetylglucosamine. Streptozotocin has recently been shown to inhibit O-GlcNAc-selective N-acetyl-beta-d-glucosaminidase, which removes O-linked N-acetylglucosamine from proteins. Compared to other cells, beta-cells express much more of the enzyme O-GlcNAc transferase, which catalyzes addition of O-linked N-acetylglucosamine to proteins. This suggests why beta-cells might be particularly sensitive to streptozotocin. In this report, we demonstrate that both streptozotocin and glucose stimulate O-glycosylation of a 135 kD beta-cell protein. Only the effect of glucose, however, was blocked by inhibition of fructose-6-phosphate amidotransferase, suggesting that glucose acts through the glucosamine pathway to provide UDP-N-acetylglucosamine for p135 O-glycosylation. The fact that both glucose and streptozotocin stimulate p135 O-glycosylation provides a possible mechanism by which hyperglycemia may cause streptozotocin-like effects in beta-cells and thus contribute to the development of type 2 diabetes.
This report examines a major barrier to suicide gene therapy in cancer and other diseases: namely, bystander cell killing. Existing vectors for in vivo gene delivery are inefficient and often transduce or transfect less than 1% of target cells. The E. coli PNP gene brings about cellular necrosis under conditions when 1 in 100 to 1 in 1000 cells express the gene product in vitro. In vivo bystander killing at or near this magnitude has not been reported previously. In the present experiments, transfection of cells with the E. coli PNP gene controlled by a SV40 promoter resulted in 30 nmol 6-methyl purine deoxyriboside (MeP-dR) converted per milligram tumor cell extract per hour (or conversion units (CU)). This level of expression led to elimination of entire populations of tumor cells in vitro after treatment with MeP-dR. Much earlier killing was observed using a tat transactivated E. coli PNP vector (approximately seven-fold higher activity, 230 CU). In vivo effects on tumor growth were next examined. Human ovarian tumors transfected with E. coli PNP were excised 5 days after i.p. implantation from the peritoneal cavities of mice in order to determine both E. coli PNP enzymatic activity and the fraction of cells expressing the gene. PNP activity at 5 days after gene transfer was approximately 170 CU and was expressed in approximately 0.1% of the tumor cells as judged by in situ hybridization. The expression of E. coli PNP at this level produced a 30% increase in life span (P < 0.001) and 49% reduction in tumor size (P < 0.005) after MeP-dR treatment, as compared with control tumors. Our observations lead to the conclusion that pronounced bystander killing by E. coli PNP is conferred in vivo, and that vectors capable of transgene expression in as few as one in 1000 cells can produce substantial antitumor effects if expression on a per cell basis is very high.
The ErbB2 receptor tyrosine kinase (RTK) is expressed in basal cells of squamous epithelia and the outer root sheath of hair follicles. We previously showed that constitutive expression of activated ErbB2 directed to these sites in the skin by the keratin 14 (K14) promoter produces prominent hair follicle abnormalities and striking skin hyperplasia in transgenic mice. However, perinatal lethality precluded the establishment of a transgenic line for analysis of ErbB2 function in adult animals. To investigate the significance of ErbB2 signaling in epithelial tissues during and post development, we developed a K14-rtTA/TetRE-ErbB2 'Tet-On' bitransgenic mouse system. These mice were normal until the ErbB2 transgene was induced by exposure to doxycycline (Dox). Prenatal induction resulted in perinatal death. Postnatally, ErbB2 transgene expression was observed at 4 h after the initiation of Dox, and reached a plateau at 24 h. Skin hyperplasia followed after 2 days and these changes reverted to normal upon Dox withdrawal. In adults, as in the neonates, prolonged ErbB2 induction caused prominent skin and hair follicle hyperplasias. Severe hyperplasias in the cornea, eye lids, tongue and esophagus were also observed. ErbB2 transgene induction was accompanied by increased expression of TGFalpha, a ligand of epidermal growth factor receptor (EGFR), and to a lesser extent, EGFR, further enhancing RTK signal transduction. We conclude that ErbB2 plays important roles in both development and maintenance of hair follicles and diverse squamous epithelia and that this ligand-inducible and tissue-specific 'Tet-On' transgenic mouse system provides a means to study transgenes with perinatal toxicity.
The transcription factor Sp1 is important for the expression of many cellular genes. Previously, it was shown that reduced O-glycosylation of Sp1 is associated with increased proteasome susceptibility. Sp1 undergoes proteasome-dependent degradation in cells stressed with glucose deprivation and adenylate cyclase activation, and this process is blocked in cells treated with glucosamine. In this study, using a reconstituted in vitro system, we identified the principal structural determinant in Sp1 that targets Sp1 for proteasome-dependent degradation. We found by using deletion analysis that the N-terminal 54 amino acids of Sp1 is required for Sp1 degradation. This element can act as an independent processing signal by directing degradation of an unrelated protein. Recognition of this Sp1 element by the proteasome-dependent system is saturable, and ubiquitination of this element is not required for recognition. Time course experiments revealed that Sp1 degradation is a two-step process. First, a discrete endoproteolytic cleavage occurs downstream of the target region immediately C-terminal to Leu56. The Sp1 sequence C-terminal to the cleavage site is subsequently degraded, whereas the N-terminal peptide remains intact. The identification of this Sp1 degradation-targeting signal will facilitate the identification of the critical proteins involved in the control of Sp1 proteasome-dependent degradation and the role of OGlcNAc in this process.
Transforming growth factor-alpha (TGF-alpha) is a member of the epidermal growth factor family. It activates signal transduction pathways leading to cell proliferation through the interaction with cell surface epidermal growth factor receptor. The overexpression of TGF-alpha has been found in many types of cancers and is thought to be involved in the genesis and maintenance of these tumors. Recent results also implicate this growth factor in the development of certain diabetic complications, such as atherosclerosis. The function of TGF-alpha can be tightly controlled at the level of transcription of its gene. We have previously characterized the proximal TGF-alpha promoter and identified two neighboring regulatory elements that appeared to cooperate with each other in the regulation of TGF-alpha transcription. The transcription factor that functions through the distal element was identified as AP-2, a protein that was found to be induced by the oncoprotein, Ras. However, what factor binds and controls the proximal regulatory element (PRE) is still unclear. Here, we report the purification and preliminary characterization of the PRE-binding transcription factor TEF1 by sequence-specific DNA-affinity chromatography from rat kidney nuclear extracts. The purified TEF1 migrates on the SDS-PAGE at a molecular mass of about 36 kDa. It specifically interacts with the PRE and was able to strongly activate transcription from the TGF-alpha promoter in HeLa cell nuclear extracts in an in vitro transcription assay. The UV cross-linking experiment confirmed that this 36 kDa protein is indeed the protein that specifically binds the PRE. We also show that the spacing between the AP-2 and the TEF1 sites in the TGF-alpha promoter has little effect on the transcription from the TGF-alpha promoter. The purification of TEF1 furthers our understanding of how TGF-alpha expression is regulated and may help us understand the upstream signaling events that lead to the elevated expression of this growth factor.
The transcription factor Sp1 plays an important role in the expression of many cellular genes. In studies of proteins that associate with Sp1, a 62-kDa glycoprotein was found in immunoprecipitates of Sp1. This protein was detected in these immunoprecipitates by the monoclonal antibody, RL2, which was originally raised against nuclear pore proteins but was subsequently found to recognize an epitope that contains O-linked N-acetylglucosamine (O-GlcNAc). The association of this protein with Sp1 could be blocked by SDS denaturation of the protein complex. Western blot analysis of the Sp1 immunoprecipitate using antibodies to p62 nucleoporin indicated that this nuclear pore protein associates with Sp1. Furthermore, immunoprecipitation of p62 nucleoporin resulted in the coprecipitation of Sp1. Recombinant p62, expressed as a GST-fusion protein using a vaccinia virus system, also interacted with both recombinant and native Sp1. This interaction between p62 and Sp1 required the C-terminus of p62 and the C-terminus was able to bind Sp1, albeit less efficiently than native p62. A mammalian two-hybrid interaction assay was devised in which p62 was fused to the Gal4 DNA-binding domain. This system also indicated that p62, through its C-terminus, interacts with Sp1 in the living cell. We propose that this interaction of a nuclear pore protein with Sp1 may reflect the nuclear organization required to bring transcribable DNA in contact with the transcription factors.
Integrin-linked kinase (ILK) is a newly identified serine/threonine protein kinase implicated in integrin signaling. To investigate the functions of ILK in vivo, we have analyzed the expression and regulation of ILK in the skin, in which proper control of cell-extracellular matrix interactions and cell proliferation is essential for its normal development and homeostasis. We report here that ILK is abundantly expressed throughout the extracellular matrix-rich dermis. ILK mRNA was also detected in the hair follicles and the basal cells of the interfollicular epidermis. However, ILK expression is lost in the suprabasal layers of keratinocytes that are undergoing terminal differentiation. PINCH, an ILK-binding protein, exhibited a similar expression pattern in the skin. Recent studies have indicated that erbB-2, a member of the epidermal growth factor receptor family, plays a pivotal role in epidermal growth, differentiation, and hair follicle morphogenesis. Using a transgenic mouse system in which an activated erbB-2 is overexpressed in the epidermis, we show that ILK expression is regulated by erbB-2. The in vivo expression and regulation patterns of ILK, together with its biochemical activities, suggest an important role of ILK in coordinating the integrin signaling pathways and the growth factor signaling pathways in the development of the skin and the pathogenesis of skin diseases.
The erbB-2 proto-oncogene belongs to a receptor tyrosine kinase family that includes the epidermal growth factor receptor, erbB-2, erbB-3, and erbB-4. erbB-2 is expressed in basal cells of the squamous epithelia and the outer root sheath of the hair follicles, but its function in epidermal development has not been well studied. To investigate its role in the skin, we created transgenic mice harboring an activated erbB-2 oncogene under the control of the human keratin 14 promoter. The keratin 14 promoter directed its expression to cells in which erbB-2 is normally expressed, whereas the activated receptor gene ensured increased signaling. All transgenic founder mice exhibited extensive and striking skin phenotype, including epidermal hyperplasia, preneoplasia, papilloma, hyperkeratosis, and dyskeratosis. The majority of the hair follicles were replaced by bizarre hyperproliferative intradermal squamous invaginations, whereas the rest of the follicles exhibited severe hyperplasia and disorganization. All but one of the transgenic mice died before or shortly after birth, probably as a consequence of defects in the skin and esophagus. These observations demonstrate that the skin is sensitive to erbB-2 signaling, suggesting an important role for this receptor tyrosine kinase in epidermal growth, differentiation, and hair follicle morphogenesis.