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Cam Patterson

Publications and source records attributed to Cam Patterson.

At least 55 records · Page 3Linked to original sources
PubMed2004

Retrograde transport of the glucocorticoid receptor in neurites requires dynamic assembly of complexes with the protein chaperone hsp90 and is linked to the CHIP component of the machinery for proteasomal degradation.

Here, we have used a chimera of green fluorescent protein (GFP) and the glucocorticoid receptor (GR) to study retrograde movement of a model soluble (i.e., non-vesicle-associated) protein in axons and dendrites of cultured NT2-N neurons. It is known that in non-neuronal cells, the GFP-GR moves from cytoplasm to the nucleus in a steroid-dependent manner by a rapid, hsp90-dependent mechanism. When rapid movement is inhibited by geldanamycin (GA), a specific inhibitor of the protein chaperone hsp90, the GFP-GR translocates slowly to the nucleus by diffusion. Here we show that GFP-GR expressed in hormone-free neurons is localized in both cytoplasm and neurites, and upon treatment with dexamethasone (DEX), it moves to the nucleus. In neurites, movement by diffusion is not possible, and we show that movement of the GFP-GR from neurites is blocked by geldanamycin, suggesting that the hsp90-dependent movement machinery is required for retrograde movement. In cells treated with both dexamethasone and geldanamycin, the GFP-GR becomes concentrated in fluorescent globules located periodically along the neurites. Carboxyl terminus of Hsc70-interacting protein (CHIP), the E3 ubiquitin ligase for the GR, also concentrates in the same loci in a steroid-dependent and geldanamycin-dependent manner. If geldanamycin is removed, the GFP-GR exits the globules and continues its retrograde movement. However, in the continued presence of geldanamycin, the GFP-GR in the globules undergoes proteasomal degradation, suggesting that the globules function as degradasomes. This is the first evidence for a linkage between receptor trafficking along neurites and receptor degradation by the proteasome.

Axons↗
PubMed2004

Expansion of protein interaction maps by phage peptide display using MDM2 as a prototypical conformationally flexible target protein.

Expanding on the possible protein interaction partners in a biochemical pathway is one key molecular goal in the post-genomic era. Phage peptide display is a versatile in vitro tool for mapping novel protein-protein interfaces and the advantage of this technique in expanding protein interaction maps is that in vitro manipulation of the bait protein conformational integrity can be controlled carefully. Phage peptide display was used to expand on the possible types of binding proteins for the conformationally responsive protein MDM2. Peptides enriched differ depending upon whether MDM2 is ligand-free, zinc-bound, or RNA-bound, suggesting that MDM2 conformational changes alter the type of peptide ligands enriched. Classes of putative/established MDM2-binding proteins identified by this technique included ubiquitin-modifying enzymes (F-box proteins, UB-ligases, UBC-E1) and apoptotic modifiers (HSP90, GAS1, APAF1, p53). Of the many putative MDM2 proteins that could be examined, the impact of HSP90 on MDM2 activity was studied, since HSP90 has been linked with p53 protein unfolding in human cancers. Zinc ions were required to reconstitute a stable MDM2-HSP90 protein complex. Zinc binding converted MDM2 from a monomer to an oligomer, and activated MDM2 binding to its internal RING finger domain, providing evidence for a conformational change in MDM2 protein when it binds zinc. Reconstitution of an HSP90-MDM2 protein complex in vitro stimulated the unfolding of the p53 tetramer. A p53 DNA-binding inhibitor purified from human cells that is capable of unfolding p53 at ambient temperature in vitro contains co-purifying pools of HSP90 and MDM2. These data highlight the utility of phage peptide display as a powerful in vitro method to identify regulatory proteins that bind to a conformationally flexible protein like MDM2.

Amino Acid Sequence↗
PubMed2004

p68RacGAP is a novel GTPase-activating protein that interacts with vascular endothelial zinc finger-1 and modulates endothelial cell capillary formation.

The endothelium is required for maintenance of vascular integrity and homeostasis during vascular development and in adulthood. However, little is known about the coordinated interplay between transcription factors and signaling molecules that regulate endothelial cell-dependent transcriptional events. Vascular endothelial zinc finger-1 (Vezf1) is a zinc finger-containing transcription factor that is specifically expressed within the endothelium during vascular development. We have previously shown that Vezf1 potently activates transcription of the endothelin-1 promoter. We now report the identification of p68RacGAP, a novel Vezf1-interacting 68-kDa RhoGAP domain-containing protein. p68RacGAP mRNA is highly expressed in vascular endothelial cells by Northern blot analysis, and immunohistochemical staining of adult mouse tissues identified p68RacGAP in endothelial cells, vascular smooth muscle cells, and epithelial cells in vivo. Rac1 and Vezf1 both bind avidly to p68RacGAP, suggesting that p68RacGAP is not only a GTPase-activating protein for Rac1 but that p68RacGAP may also be part of the protein complex that binds to and modulates Vezf1 transcriptional activity. Functionally p68RacGAP specifically activates the GTPase activity of Rac1 in vivo but not Cdc42 or RhoA. In addition, p68RacGAP potently inhibits Vezf1/DB1-mediated transcriptional activation of the human endothelin-1 promoter and modulates endothelial cell capillary tube formation. Taken together, these data suggest that p68RacGAP is a multifunctional regulatory protein that has a Rac1-specific GTPase-activating activity, regulates transcriptional activity of the endothelin-1 promoter, and is involved in the signal transduction pathway that regulates endothelial cell capillary tube formation during angiogenesis.

Amino Acid Sequence↗
PubMed2004

PDTC, metal chelating compound, induces G1 phase cell cycle arrest in vascular smooth muscle cells through inducing p21Cip1 expression: involvement of p38 mitogen activated protein kinase.

Pyrrolidine dithiocarbamate (PDTC), a metal chelating compound, is known to induce cell death in vascular smooth muscle cells (VSMC). However, the molecular mechanism for PDTC-induced VSMC death is not well understood. Addition of PDTC reduced cell growth and DNA synthesis on VSMC in low density conditions. However, in serum depleted medium, PDTC did not affect the cell viability, suggesting that certain factors in serum may mediate the cytotoxic effect of PDTC. Several metal chelators prevented the cell death induced by PDTC. In a serum-deprived condition, addition of exogenous metals, copper, iron, and zinc, restored the cytotoxic effect of PDTC. These data indicate that metals such as copper, iron, and zinc in serum may mediate the cytotoxic effect of PDTC. At low VSMC density in 10% FBS, treatment of PDTC, which induced a cell-cycle block in G1-phase, induced down-regulation of cyclins and CDKs and up-regulation of the CDK inhibitor p21 expression, whereas up-regulation of p27 or p53 by PDTC was not observed. Finally, we determined PDTC-mediated signaling pathway involved in VSMC death. Among relevant pathways, PDTC induced marked activation of p38MAPK and JNK. Expression of dominant negative p38MAPK and SB203580, a p38MAPK specific inhibitor, blocked PDTC-dependent p38MAPK, growth inhibition, and p21 expression. These data demonstrate that the p38MAPK pathway participates in p21 induction, which consequently leads to decrease of cyclin D1/cdk4 and cyclin E/cdk2 complexes and PDTC-dependent VSMC growth inhibition. In conclusion, an understanding of the molecular mechanisms of PDTC in VSMC provides a theoretical basis for clinical approaches using antioxidant therapies in atherosclerosis.

Animals↗
PubMed2004

Age-related changes in matrix metalloproteinase-9 regulation in cultured mouse aortic smooth muscle cells.

We previously reported that aortic smooth muscle cells (SMC) from aged mice have an age-related decline in proliferative capacity compared with those derived from young mice. Here we investigated matrix metalloproteinase-9 (MMP-9) regulation in both young and aged SMC. Zymography, immunoblot, and northern blot analysis showed that MMP-9 expression is significantly reduced in response to tumor necrosis factor-alpha stimulation with increasing in vitro age. Mutational analysis, gel shift assays and supershift assays demonstrated that the lower MMP-9 expression in aged SMC is associated with lower activities of NF-kappaB and AP-1. Since mitogen-activated protein kinase ERK1/2 induce MMP-9 expression, we examined whether U0126, an ERK1/2 inhibitor, influenced MMP-9 expression in aged SMC. Treatment with U0126 successfully inhibited MMP-9 expression in both young and aged SMC. Finally, to analyze the causal relationship between replicative senescence and MMP-9 expression, we stably overexpressed the MMP-9 gene in aged SMC and we showed no alteration of the proliferative capacity of the transduced cells. Taken together, these results suggest that down-regulation of MMP-9 expression in SMC may play a role in vascular remodeling during in vitro aging.

Aging↗
PubMed2004

A vascular gene trap screen defines RasGRP3 as an angiogenesis-regulated gene required for the endothelial response to phorbol esters.

We identified Ras guanine-releasing protein 3 (RasGRP3) as a guanine exchange factor expressed in blood vessels via an embryonic stem (ES) cell-based gene trap screen to identify novel vascular genes. RasGRP3 is expressed in embryonic blood vessels, down-regulated in mature adult vessels, and reexpressed in newly formed vessels during pregnancy and tumorigenesis. This expression pattern is consistent with an angiogenic function for RasGRP3. Although a loss-of-function mutation in RasGRP3 did not affect viability, RasGRP3 was up-regulated in response to vascular endothelial growth factor (VEGF) stimulation of human umbilical vein endothelial cells, placing RasGRP3 regulation downstream of VEGF signaling. Phorbol esters mimic the second messenger diacylglycerol (DAG) in activating both protein kinase C (PKC) and non-PKC phorbol ester receptors such as RasGRP3. ES cell-derived wild-type blood vessels exposed to phorbol myristate acetate (PMA) underwent extensive aberrant morphogenesis that resulted in the formation of large endothelial sheets rather than properly branched vessels. This response to PMA was completely dependent on the presence of RasGRP3, as mutant vessels were refractory to the treatment. Taken together, these findings show that endothelial RasGRP3 is up-regulated in response to VEGF stimulation and that RasGRP3 functions as an endothelial cell phorbol ester receptor in a pathway whose stimulation perturbs normal angiogenesis. This suggests that RasGRP3 activity may exacerbate vascular complications in diseases characterized by excess DAG, such as diabetes.

Amino Acid Sequence↗
PubMed2004

Atrogin-1/muscle atrophy F-box inhibits calcineurin-dependent cardiac hypertrophy by participating in an SCF ubiquitin ligase complex.

Calcineurin, which binds to the Z-disc in cardiomyocytes via alpha-actinin, promotes cardiac hypertrophy in response to numerous pathologic stimuli. However, the endogenous mechanisms regulating calcineurin activity in cardiac muscle are not well understood. We demonstrate that a muscle-specific F-box protein called atrogin-1, or muscle atrophy F-box, directly interacts with calcineurin A and alpha-actinin-2 at the Z-disc of cardiomyocytes. Atrogin-1 associates with Skp1, Cul1, and Roc1 to assemble an SCF(atrogin-1) complex with ubiquitin ligase activity. Expression of atrogin-1 decreases levels of calcineurin A and promotes its ubiquitination. Moreover, atrogin-1 attenuates agonist-induced calcineurin activity and represses calcineurin-dependent transactivation and NFATc4 translocation. Conversely, downregulation of atrogin-1 using adenoviral small interfering RNA (siRNA) expression enhances agonist-induced calcineurin activity and cardiomyocyte hypertrophy. Consistent with these cellular observations, overexpression of atrogin-1 in hearts of transgenic mice reduces calcineurin protein levels and blunts cardiac hypertrophy after banding of the thoracic aorta. These studies indicate that the SCF(atrogin-1) ubiquitin ligase complex interacts with and represses calcineurin by targeting calcineurin for ubiquitin-mediated proteolysis, leading to inhibition of cardiac hypertrophy in response to pathologic stimuli.

Actinin↗
PubMed2004

Universal mouse reference RNA derived from neonatal mice.

A reproducible, transcriptionally diverse common reference RNA is required for accurate comparisons of data generated from most spotted microarray experiments in different experiments. Several methods have been proposed to make such a reference RNA, such as pooling RNAs isolated from multiple cell lines or tissues, amplifying pooled RNAs, or synthesizing RNAs or DNAs complementary to microarray features. We report an approach to prepare a large amount of mouse reference RNA from whole neonatal mice. This approach is simple, quick, reliable, reproducible, and inexpensive. The whole mouse reference RNA is highly representative when compared to two commercially available universal mouse reference RNAs isolated and pooled from multiple cell lines or organs.

Animals↗
PubMed2003

Catecholamine-induced vascular wall growth is dependent on generation of reactive oxygen species.

Alpha1-adrenoceptor-dependent proliferation of vascular smooth muscle cells (VSMCs) is strongly augmented by vascular injury, and may contribute to intimal growth and lumen loss. Because reactive oxygen species (ROS) are increased by injury and have been implicated as second messengers in proliferation of VSMCs, we investigated the role of ROS in catecholamine-induced VSMC growth. Rat aortae were isolated 4 days after balloon injury, maintained in organ culture under circumferential wall tension, and exposed to agents for 48 hours. The antioxidants N-acetylcysteine (NAC, 10 mmol/L) and Tiron (5 mmol/L) and the flavin-inhibitor diphenylene iodonium (DPI, 20 micromol/L) abolished norepinephrine-induced increases in protein synthesis and DNA content in media. In aortic sections, norepinephrine augmented ROS production (dihydroethidium confocal microscopy), which was dose-dependently inhibited by NAC, Tiron, and DPI. In cultured VSMCs, phenylephrine caused time- and dose-dependent ROS generation (aconitase activity), had similar efficacy to thrombin (1 U/mL), and was eliminated by the superoxide dismutase (SOD) mimetic Mn-(III)-tetrakis-(4-benzoic-acid)-porphyrin-chloride (200 micromol/L) and Tiron. Phenylephrine-induced ROS production and increases in DNA and protein content were blocked by prazosin (0.3 micromol/L) and abolished in p47phox-/- cells. PEG-SOD (25 U/mL) had little effect, whereas PEG-catalase (50 U/mL) eliminated phenylephrine-induced proliferation in VSMCs. DPI (10 micromol/L) and apocynin (30 micromol/L) abolished phenylephrine-stimulated mitogenesis, whereas inhibitors of other intracellular ROS sources had not effect. Furthermore, PE increased p47phox expression (RT-PCR). These data demonstrate that the trophic effect of catecholamines on vascular wall cells is dependent on a ROS-sensitive step that we hypothesize consists of activation of the NAD(P)H-dependent vascular oxidase.

Adrenergic alpha-1 Receptor Agonists↗
PubMed2003

CHIP activates HSF1 and confers protection against apoptosis and cellular stress.

Induction of molecular chaperones is the characteristic protective response to environmental stress, and is regulated by a transcriptional program that depends on heat shock factor 1 (HSF1), which is normally under negative regulatory control by molecular chaperones Hsp70 and Hsp90. In metazoan species, the chaperone system also provides protection against apoptosis. We demonstrate that the dual function co-chaperone/ubiquitin ligase CHIP (C-terminus of Hsp70-interacting protein) regulates activation of the stress-chaperone response through induced trimerization and transcriptional activation of HSF1, and is required for protection against stress-induced apoptosis in murine fibroblasts. The consequences of this function are demonstrated by the phenotype of mice lacking CHIP, which develop normally but are temperature-sensitive and develop apoptosis in multiple organs after environmental challenge. CHIP exerts a central and unique role in tuning the response to stress at multiple levels by regulation of protein quality control and transcriptional activation of stress response signaling.

Amino Acid Substitution↗
PubMed2003

Chaperone-dependent regulation of endothelial nitric-oxide synthase intracellular trafficking by the co-chaperone/ubiquitin ligase CHIP.

Endothelial nitric-oxide synthase (eNOS), the enzyme responsible for production of endothelial NO, is under tight and complex regulation. Proper cellular localization of eNOS is critical for optimal coupling of extracellular stimulation with NO production. In addition, the molecular chaperone Hsp90 interacts with eNOS and positively regulates eNOS activity. Hsp90 is modulated by physical interaction with its co-chaperones. CHIP (carboxyl terminus of Hsp70-interacting protein) is such a co-chaperone that remodels the Hsp90 heterocomplex and causes protein degradation of some Hsp90 substrates through the ubiquitin-protein isopeptide ligase activity of CHIP. Here we show that CHIP incorporated into the eNOS.Hsp90 complex and specifically decreased soluble eNOS levels in transiently transfected COS cells. Surprisingly, in contrast to the effects of the Hsp90 inhibitor geldanamycin, which induces eNOS ubiquitylation and its subsequent protein degradation, CHIP did not target eNOS for ubiquitylation and proteasome-dependent degradation. Instead, CHIP partitioned soluble eNOS into an insoluble and inactive cellular compartment, presumably through its co-chaperone activity. This effect seems to be due to displacement of eNOS from the Golgi apparatus, which is otherwise required for trafficking of eNOS to the plasmalemma and subsequent activation. Consistent with observations from overexpression studies, eNOS localization to the membrane and activity were increased in mouse lung endothelial cells lacking CHIP. Taken together, these results demonstrate a novel co-chaperone-dependent mechanism through which eNOS trafficking is regulated and suggest a potentially generalized role for CHIP in protein trafficking through the Golgi compartment.

Animals↗
PubMed2003

Defective valvulogenesis in HB-EGF and TACE-null mice is associated with aberrant BMP signaling.

Heparin-binding epidermal growth factor (HB-EGF) and betacellulin (BTC) are activating ligands for EGF receptor (EGFR/ErbB1) and ErbB4. To identify their physiological functions, we disrupted mouse HB-EGF and BTC alleles by homologous recombination. Most HB-EGF(-/-) mice died before weaning, and survivors had enlarged, dysfunctional hearts and reduced lifespans. Although BTC(-/-) mice were viable and fertile and displayed no overt defects, the lifespan of double null HB-EGF(-/-)/BTC(-/-) mice was further reduced, apparently due to accelerated heart failure. HB-EGF(-/-) newborns had enlarged and malformed semilunar and atrioventricular heart valves, and hypoplastic, poorly differentiated lungs. Defective cardiac valvulogenesis was the result of abnormal mesenchymal cell proliferation during remodeling, and was associated with dramatic increases in activated Smad1/5/8. Consistent with the phenotype, HB-EGF transcripts were localized to endocardial cells lining the margins of wild-type valves. Similarly defective valvulogenesis was observed in newborn mice lacking EGFR and tumor necrosis factor-alpha converting enzyme (TACE). These results suggest that cardiac valvulogenesis is dependent on EGFR activation by TACE-derived soluble HB-EGF, and that EGFR signaling is required to regulate bone morphogenetic protein signaling in this context.

ADAM Proteins↗
PubMed2003

AtCHIP, a U-box-containing E3 ubiquitin ligase, plays a critical role in temperature stress tolerance in Arabidopsis.

The Arabidopsis gene AtCHIP encodes a protein with three tetratricopeptide repeats and a U-box domain, which is structurally similar to the animal CHIP proteins, a new class of E3 ubiquitin ligases. Like animal CHIP proteins, AtCHIP has E3 ubiquitin ligase activity in vitro. AtCHIP is a single-copy gene, and its transcript is up-regulated by several stress conditions such as low and high temperatures. However, increased AtCHIP expression alone was not correlated with increased stress tolerance; in fact, overexpression of AtCHIP in Arabidopsis rendered plants more sensitive to both low- and high-temperature treatments. Higher electrolyte leakage was observed in leaves of AtCHIP overexpression plants after chilling temperature treatment, suggesting that membrane function is likely impaired in these plants under such a condition. These results indicate that AtCHIP plays an important role in plant cellular metabolism under temperature stress conditions.

Acclimatization↗
PubMed2003

Thrombin and vascular development: a sticky subject.

Formation of the vasculature is an essential step in embryogenesis. It was observed decades ago that the vasculature and the intravascular blood compartment, which uses the former as a means of transportation, develop in a close spatial and temporal relationship. In this review, we discuss the role of the blood coagulation system as a tool to coordinate angiogenesis. Several mouse models lacking coagulation factors result in impaired thrombin generation and display a phenotype of disturbed cardiovascular development. Similar phenotypes are observed in mouse models of impaired thrombin binding to its cellular receptor, protease-activated receptor-1, or of disrupted signaling via G proteins. Most interestingly, the available data provide evidence that thrombin signaling in vascular development cannot be explained by a model based only on the classic extrinsic and intrinsic coagulation pathways. Because angiogenesis in adults follows the same signaling patterns as angiogenesis in embryos, it is important to learn about these pathways, hoping that they may serve as therapeutic targets in cardiovascular disease.

Animals↗
PubMed2003

C-terminal Hsp-interacting protein slows androgen receptor synthesis and reduces its rate of degradation.

The androgen receptor (AR) is a member of the nuclear receptor superfamily that requires the action of molecular chaperones for folding and hormone binding. C-terminal Hsp-interacting protein (Chip) is a cochaperone that interacts with Hsp70 and Hsp90 molecular chaperones via a tetratricopeptide domain and inhibits chaperone-dependent protein folding in vitro. Chip also stimulates protein degradation by acting as an E3 ubiquitin ligase via a modified ring finger domain called a U box. We analyzed whether Chip affected AR levels using a transient transfection strategy. Chip overexpression led to a large decrease in AR steady state levels and increased levels of AR ubiquitinylation. However, Chip effects were not fully reversed by proteasome inhibitors, suggesting that mechanisms alternative to or in addition to proteasome-mediated degradation were involved. This hypothesis was supported by the finding that Chip overexpression reduced the rate of AR degradation, consistent with an effect on AR folding, perhaps leading to aggregation. The possibility that Chip affected AR folding was further supported by the finding that the effects of exogenous Chip were reproduced by a mutant lacking the U box. These results are discussed in terms of the role played by molecular chaperones in AR biogenesis.

Acetylcysteine↗
PubMed2003

Evidence for association of coronary sinus levels of hepatocyte growth factor and collateralization in human coronary disease.

The therapeutic use of angiogenic factors to protect ischemic myocardium is limited by our incomplete understanding of their endogenous production. We determined the association between angiogenic factors and collateral formation in patients with coronary artery disease (CAD). A total of 71 patients underwent catheterization with sampling of the pulmonary artery, aorta, and coronary sinus (CS) to determine the levels of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). VEGF and HGF levels were not different in the three vascular sites, suggesting that the heart is not a major source of these cytokines in the circulation. CS VEGF and HGF levels were similar in patients with and without CAD. Elevated CS HGF levels were associated with collateral formation, whereas VEGF levels were not. Additionally, CS HGF was significantly elevated in patients with left ventricular dysfunction. These data map for the first time the concentration of endogenous angiogenic factors in the coronary circulation and support further studies to determine whether HGF may be an endogenous cardioprotective angiogenic factor.

Aged↗