Growth suppression of MCF-7 cancer cell-derived xenografts in nude mice by caveolin-1

Caveolin-1 is an essential structural constituent of caveolae membrane domains that has been implicated in mitogenic signaling and oncogenesis. However, the exact functional role of caveolin-1 still remains controversial. In this report, utilizing MCF-7 human breast adenocarcinoma cells stably transfected with caveolin-1 (MCF-7/cav-1 cells), we demonstrate that caveolin-1 expression dramatically inhibits invasion and migration of these cells. Importantly, in vivo experiments employing xenograft tumor models demonstrated that expression of caveolin-1 results in significant growth inhibition of breast tumors. Moreover, a dramatic delay in tumor progression was observed in MCF-7/cav-1 cells as compared with MCF-7 cells. Histological analysis of tumor sections demonstrated a marked decrease in the percentage of proliferating tumor cells (Ki-67 assay) along with an increase in apoptotic tumor cells (TUNEL assay) in MCF-7/cav-1-treated animals. Our current findings provide for the first time in vivo evidence that caveolin-1 can indeed function as a tumor suppressor in human breast adenocarcinoma derived from MCF-7 cells rather than as a tumor promoter.     

Caveolin enhances hepatocellular carcinoma cell metabolism,migration, and invasion in vitro via a hexokinase 2-dependent mechanism

The development and progression of hepatocellular carcinoma (HCC) have been associated with abnormal cellular metabolism. Gene Expression Profiling Interactive Analysis RNA sequencing data revealed caveolin-1 (CAV-1) and hexokinase 2 (HK2) messenger RNA (mRNA) were significantly upregulated in human HCC compared with normal tissues, and high HK2 expression was associated with significantly poorer overall survival in HCC ( p < 0.05). CAV-1 and HK2 mRNA and protein expression were upregulated and positively correlated in 42 fresh human HCC tissues compared with tumor-adjacent normal tissues. Overexpression of CAV-1 or HK2 in SMMC-7721 and HepG2 HCC cells enhanced glucose and lactate metabolism and increased cell migration and invasion in transwell assays; knocking down CAV-1 or HK2 had the opposite effects. Overexpression of CAV-1 increased HK2 expression; overexpression of HK2 did not affect CAV-1 expression. Knocking down HK2 partially reversed the ability of CAV-1 to promote cellular metabolism, invasion, and migration in HCC, indicating CAV-1 enhances glycolysis, invasion, and metastasis in HCC cells via HK2-dependent mechanism. Further studies of the function and relationship between CAV-1 or HK2 expression are warranted to explore the potential of these proteins as metabolic targets for the treatment of HCC.     

Clathrin- and caveolin-1-independent endocytosis: entry of simian virus 40 into cells devoid of caveolae

Simian Virus 40 (SV40) has been shown to enter host cells by caveolar endocytosis followed by transport via caveosomes to the endoplasmic reticulum (ER). Using a caveolin-1 (cav-1)-deficient cell line (human hepatoma 7) and embryonic fibroblasts from a cav-1 knockout mouse, we found that in the absence of caveolae, but also in wild-type embryonic fibroblasts, the virus exploits an alternative, cav-1-independent pathway. Internalization was rapid (t1/2 = 20 min) and cholesterol and tyrosine kinase dependent but independent of clathrin, dynamin II, and ARF6. The viruses were internalized in small, tight-fitting vesicles and transported to membrane-bounded, pH-neutral organelles similar to caveosomes but devoid of cav-1 and -2. The viruses were next transferred by microtubule-dependent vesicular transport to the ER, a step that was required for infectivity. Our results revealed the existence of a virus-activated endocytic pathway from the plasma membrane to the ER that involves neither clathrin nor caveolae and that can be activated also in the presence of cav-1.     

Mutations in palmitoyl-protein thioesterase 1 alter exocytosis and endocytosis at synapses in Drosophila larvae

Infantile-onset neuronal ceroid lipofuscinosis (INCL) is a severe pediatric neurodegenerative disorder produced by mutations in the gene encoding palmitoyl-protein thioesterase 1 (Ppt1). This enzyme is responsible for the removal of a palmitate group from its substrate proteins, which may include presynaptic proteins like SNAP-25, cysteine string protein (CSP), dynamin, and synaptotagmin. The fruit fly, Drosophila melanogaster, has been a powerful model system for studying the functions of these proteins and the molecular basis of neurological disorders like the NCLs. Genetic modifier screens and tracer uptake studies in Ppt1 mutant larval garland cells have suggested that Ppt1 plays a role in endocytic trafficking. We have extended this analysis to examine the involvement of Ppt1 in synaptic function at the Drosophila larval neuromuscular junction (NMJ). Mutations in Ppt1 genetically interact with temperature sensitive mutations in the Drosophila dynamin gene shibire, accelerating the paralytic behavior of shibire mutants at 27 °C. Electrophysiological work in NMJs of Ppt1-deficient larvae has revealed an increase in miniature excitatory junctional potentials (EJPs) and a significant depression of evoked EJPs in response to repetitive (10 hz) stimulation. Endocytosis was further examined in Ppt1-mutant larvae using FM1–43 uptake assays, demonstrating a significant decrease in FM1–43 uptake at the mutant NMJs. Finally, Ppt1-deficient and Ppt1 point mutant larvae display defects in locomotion that are consistent with alterations in synaptic function. Taken together, our genetic, cellular, and electrophysiological analyses suggest a direct role for Ppt1 in synaptic vesicle exo- and endocytosis at motor nerve terminals of the Drosophila NMJ.     

Invasion of host cells by JC virus identifies a novel role for caveolae in endosomal sorting of noncaveolar ligands

Invasion of glial cells by the human polyomavirus, JC virus (JCV), leads to a rapidly progressing and uniformly fatal demyelinating disease known as progressive multifocal leukoencephalopathy. The endocytic trafficking steps used by JCV to invade cells and initiate infection are not known. We demonstrated that JCV infection was inhibited by dominant defective and constitutively active Rab5-GTPase mutants that acted at distinct steps in endosomal sorting. We also found that labeled JCV colocalized with labeled cholera toxin B and with caveolin-1 (cav-1) on early endosomes following internalization by clathrin-dependent endocytosis. JCV entry and infection were both inhibited by dominant defective mutants of eps15 and Rab5-GTPase. Expression of a dominant-negative scaffolding mutant of cav-1 did not inhibit entry or infection by JCV. A single-cell knockdown experiment using cav-1 shRNA did not inhibit JCV entry but interfered with a downstream trafficking event important for infection. These data show that JCV enters cells by clathrin-dependent endocytosis, is transported immediately to early endosomes, and is then sorted to a caveolin-1-positive endosomal compartment. This latter step is dependent on Rab5-GTPase, cholesterol, caveolin-1, and pH. This is the first example of a ligand that enters cells by clathrin-dependent endocytosis and is then sorted from early endosomes to caveosomes, indicating that caveolae-derived vesicles play a more important role than previously realized in sorting cargo from early endosomes.     

SorLA in Glia: Shared Subcellular Distribution Patterns with Caveolin-1

SorLA is an established sorting and trafficking protein in neurons with demonstrated relevance to Alzheimer’s disease (AD). It shares these roles with the caveolins, markers of membrane rafts microdomains. To further our knowledge on sorLA’s expression and traffic, we studied sorLA expression in various cultured glia and its relation to caveolin-1 (cav-1), a caveolar microdomain marker. RT-PCR and immunoblots demonstrated sorLA expression in rat C6 glioma, primary cultures of rat astrocytes (PCRA), and human astrocytoma 1321N1 cells. PCRA were determined to express the highest levels of sorLA’s message. Induction of differentiation of C6 cells into an astrocyte-like phenotype led to a significant decrease in sorLA’s mRNA and protein expression. A set of complementary experimental approaches establish that sorLA and cav-1 directly or indirectly interact in glia: (1) co-fractionation in light-density membrane raft fractions of rat C6 glioma, PCRA, and human 1321N1 astrocytoma cells; (2) a subcellular co-localization distribution pattern in vesicular perinuclear compartments seen via confocal imaging in C6 and PCRA; (3) additional confocal analysis in C6 cells suggesting that the perinuclear compartments correspond to their co-localization in early endosomes and the trans-Golgi; and; (4) co-immunoprecipitation data strongly supporting their direct or indirect physical interaction. These findings further establish that sorLA is expressed in glia and that it shares its subcellular distribution pattern with cav-1. A direct or indirect cav-1/sorLA interaction could modify the trafficking and sorting functions of sorLA in glia and its proposed neuroprotective role in AD.     

Dynamic regulation of caveolin-1 trafficking in the germ line and embryo of Caenorhabditis elegans

Caveolin is the major protein component required for the formation of caveolae on the plasma membrane. Here we show that trafficking of Caenorhabditis elegans caveolin-1 (CAV-1) is dynamically regulated during development of the germ line and embryo. In oocytes a CAV-1-green fluorescent protein (GFP) fusion protein is found on the plasma membrane and in large vesicles (CAV-1 bodies). After ovulation and fertilization the CAV-1 bodies fuse with the plasma membrane in a manner reminiscent of cortical granule exocytosis as described in other species. Fusion of CAV-1 bodies with the plasma membrane appears to be regulated by the advancing cell cycle, and not fertilization per se, because fusion can proceed in spe-9 fertilization mutants but is blocked by RNA interference-mediated knockdown of an anaphase-promoting complex component (EMB-27). After exocytosis, most CAV-1-GFP is rapidly endocytosed and degraded within one cell cycle. CAV-1 bodies in oocytes appear to be produced by the Golgi apparatus in an ARF-1-dependent, clathrin-independent, mechanism. Conversely endocytosis and degradation of CAV-1-GFP in embryos requires clathrin, dynamin, and RAB-5. Our results demonstrate that the distribution of CAV-1 is highly dynamic during development and provides new insights into the sorting mechanisms that regulate CAV-1 localization.     

Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities.

Caveolin-1 is the principal structural protein of caveolae membranes in fibroblasts and endothelia. Recently, we have shown that the human CAV-1 gene is localized to a suspected tumor suppressor locus, and mutations in Cav-1 have been implicated in human cancer. Here, we created a caveolin-1 null (CAV-1 -/-) mouse model, using standard homologous recombination techniques, to assess the role of caveolin-1 in caveolae biogenesis, endocytosis, cell proliferation, and endothelial nitric-oxide synthase (eNOS) signaling. Surprisingly, Cav-1 null mice are viable. We show that these mice lack caveolin-1 protein expression and plasmalemmal caveolae. In addition, analysis of cultured fibroblasts from Cav-1 null embryos reveals the following: (i) a loss of caveolin-2 protein expression; (ii) defects in the endocytosis of a known caveolar ligand, i.e. fluorescein isothiocyanate-albumin; and (iii) a hyperproliferative phenotype. Importantly, these phenotypic changes are reversed by recombinant expression of the caveolin-1 cDNA. Furthermore, examination of the lung parenchyma (an endothelial-rich tissue) shows hypercellularity with thickened alveolar septa and an increase in the number of vascular endothelial growth factor receptor (Flk-1)-positive endothelial cells. As predicted, endothelial cells from Cav-1 null mice lack caveolae membranes. Finally, we examined eNOS signaling by measuring the physiological response of aortic rings to various stimuli. Our results indicate that eNOS activity is up-regulated in Cav-1 null animals, and this activity can be blunted by using a specific NOS inhibitor, nitro-l-arginine methyl ester. These findings are in accordance with previous in vitro studies showing that caveolin-1 is an endogenous inhibitor of eNOS. Thus, caveolin-1 expression is required to stabilize the caveolin-2 protein product, to mediate the caveolar endocytosis of specific ligands, to negatively regulate the proliferation of certain cell types, and to provide tonic inhibition of eNOS activity in endothelial cells.     

Loss of caveolin-1 gene expression accelerates the development of dysplastic mammary lesions in tumor-prone transgenic mice

Caveolin-1 is the principal structural component of caveolae microdomains, which represent a subcompartment of the plasma membrane. Several independent lines of evidence support the notion that caveolin-1 functions as a suppressor of cell transformation. For example, the human CAV-1 gene maps to a suspected tumor suppressor locus (D7S522/7q31.1) that is frequently deleted in a number of carcinomas, including breast cancers. In addition, up to 16% of human breast cancers harbor a dominant-negative mutation, P132L, in the CAV-1 gene. Despite these genetic associations, the tumor suppressor role of caveolin-1 still remains controversial. To directly assess the in vivo transformation suppressor activity of the caveolin-1 gene, we interbred Cav-1 (-/-) null mice with tumor-prone transgenic mice (MMTV-PyMT) that normally develop multifocal dysplastic lesions throughout the entire mammary tree. Herein, we show that loss of caveolin-1 gene expression dramatically accelerates the development of these multifocal dysplastic mammary lesions. At 3 wk of age, loss of caveolin-1 resulted in an approximately twofold increase in the number of lesions (foci per gland; 3.3 +/- 1.0 vs. 7.0 +/- 1.2) and an approximately five- to sixfold increase in the total area occupied by these lesions. Similar results were obtained at 4 wk of age. However, complete loss of caveolin-1 was required to accelerate the appearance of these dysplastic mammary lesions, because Cav-1 (+/-) heterozygous mice did not show any increases in foci development. We also show that loss of caveolin-1 increases the extent and the histological grade of these mammary lesions and facilitates the development of papillary projections in the mammary ducts. Finally, we demonstrate that cyclin D1 expression levels are dramatically elevated in Cav-1 (-/-) null mammary lesions, consistent with the accelerated appearance and growth of these dysplastic foci. This is the first in vivo demonstration that caveolin-1 can function as a transformation suppressor gene.     

Loss of parkin promotes lipid rafts-dependent endocytosis through accumulating caveolin-1: implications for Parkinson’s disease

Background

Parkinson’s disease (PD) is characterized by progressive loss of midbrain dopaminergic neurons, resulting in motor dysfunctions. While most PD is sporadic in nature, a significant subset can be linked to either autosomal dominant or recessive mutations. PARK2, encoding the E3 ubiquitin ligase, parkin, is the most frequently mutated gene in autosomal recessive early onset PD. It has recently been reported that PD-associated gene products such as PINK1, α-synuclein, LRRK2, and DJ-1, as well as parkin associate with lipid rafts, suggesting that the dysfunction of these proteins in lipid rafts may be a causal factor of PD. Therefore here, we examined the relationship between lipid rafts-related proteins and parkin.

Results

We identified caveolin-1 (cav-1), which is one of the major constituents of lipid rafts at the plasma membrane, as a substrate of parkin. Loss of parkin function was found to disrupt the ubiquitination and degradation of cav-1, resulting in elevated cav-1 protein level in cells. Moreover, the total cholesterol level and membrane fluidity was altered by parkin deficiency, causing dysregulation of lipid rafts-dependent endocytosis. Further, cell-to-cell transmission of α-synuclein was facilitated by parkin deficiency.

Conclusions

Our results demonstrate that alterations in lipid rafts by the loss of parkin via cav-1 may be a causal factor of PD, and cav-1 may be a novel therapeutic target for PD.

     

Characterization of rotavirus cell entry

While recently we have learned much about the viral and cellular proteins involved in the initial attachment of rotaviruses to MA104 cells, the mechanism by which these viruses reach the interior of the cell is poorly understood. For this study, we observed the effects of drugs and of dominant-negative mutants, known to impair clathrin-mediated endocytosis and endocytosis mediated by caveolae, on rotavirus cell infection. Rotaviruses were able to enter cells in the presence of compounds that inhibit clathrin-mediated endocytosis as well as cells overexpressing a dominant-negative form of Eps15, a protein crucial for the assembly of clathrin coats. We also found that rotaviruses infected cells in which caveolar uptake was blocked; treatment with the cholesterol binding agents nystatin and filipin, as well as transfection of cells with dominant-negative caveolin-1 and caveolin-3 mutants, had no effect on rotavirus infection. Interestingly, cells treated with methyl-beta-cyclodextrin, a drug that sequesters cholesterol from membranes, and cells expressing a dominant-negative mutant of the large GTPase dynamin, which is known to function in several membrane scission events, were not infected by rotaviruses, indicating that cholesterol and dynamin play a role in the entry of rotaviruses.     

Caveolin-1 maintains activated Akt in prostate cancer cells through scaffolding domain binding site interactions with and inhibition of serine/threonine protein phosphatases PP1 and PP2A

Previously it has been reported that caveolin-1 (cav-1) has antiapoptotic activities in prostate cancer cells and functions downstream of androgenic stimulation. In this study, we demonstrate that cav-1 overexpression significantly reduced thapsigargin (Tg)-stimulated apoptosis. Examination of the phosphatidylinositol 3-kinase (PI3-K)/Akt signaling cascade revealed higher activities of PDK1 and Akt but not PI3-K in cav-1-stimulated cells compared to control cells. We subsequently found that cav-1 interacts with and inhibits serine/threonine protein phosphatases PP1 and PP2A through scaffolding domain binding site interactions. Deletion of the cav-1 scaffolding domain significantly reduces phosphorylated Akt and cell viability compared with wild-type cav-1. Analysis of potential substrates for PP1 and PP2A revealed that cav-1-mediated inhibition of PP1 and PP2A leads to increased PDK1, Akt, and ERK1/2 activities. We demonstrate that increased Akt activities are largely responsible for cav-1-mediated cell survival using dominant-negative Akt mutants and specific inhibitors to MEK1/MEK and show that cav-1 increases the half-life of phosphorylated PDK1 and Akt after inhibition of PI3-K by LY294002. We further demonstrate that cav-1-stimulated Akt activities lead to increased phosphorylation of multiple Akt substrates, including GSK3, FKHR, and MDM2. In addition, overexpression of cav-1 significantly increases translocation of phosphorylated androgen receptor to nucleus. Our studies therefore reveal a novel mechanism of Akt activation in prostate cancer and potentially other malignancies.     

Astragaloside IV enhances taxol chemosensitivity of breast cancer via caveolin-1-targeting oxidant damage

Accumulating evidence suggests that caveolin-1 (CAV-1) is a stress-related oncotarget and closely correlated to chemoresistance. Targeting CAV-1 might be a promising strategy to improve chemosensitivity for breast cancer treatment. Astragaloside IV (AS-IV), a bioactive compound purified from Astragalus membranaceus, has been shown to exhibit multiple bioactivities, including anticancer. However, the involved molecular targets are still ambiguous. In this study, we investigated the critical role of CAV-1 in mediating the chemosensitizing effects of AS-IV to Taxol on breast cancer. We found that AS-IV could enhance the chemosensitivity of Taxol with minimal direct cytotoxicity on breast cancer cell lines MCF-7 and MDA-MB-231, as well as the nontumor mammary epithelial cell line MCF-10A. AS-IV was further demonstrated to aggravate Taxol-induced apoptosis and G2/M checkpoint arrest. The phosphorylation of mitogen-activated protein kinase (MAPK) signaling extracellular signal-regulated kinase (ERK) and c-Jun N-terminal Kinase (JNK), except p38, was also abrogated by a synergistic interaction between AS-IV and Taxol. Moreover, AS-IV inhibited CAV-1 expression in a dose-dependent manner and reversed CAV-1 upregulation induced by Taxol administration. Mechanism study further demonstrated that AS-IV treatment triggered the eNOS/NO/ONOO⁻ pathway via inhibiting CAV-1, which led to intense oxidant damage. CAV-1 overexpression abolished the chemosensitizing effects of AS-IV to Taxol by inhibiting oxidative stress. In vivo experiments further validated that AS-IV increased Taxol chemosensitivity on breast cancer via inhibiting CAV-1 expression, followed by activation of the eNOS/NO/ONOO⁻ pathway. Taken together, our findings not only suggested the potential of AS-IV as a promising candidate to enhance chemosensitivity, but also highlighted the significance of CAV-1 as the target to reverse cancer drug resistance.     

Caveolae-Dependent and -Independent Uptake of Albumin in Cultured Rodent Pulmonary Endothelial Cells

Although a critical role for caveolae-mediated albumin transcytosis in pulmonary endothelium is well established, considerably less is known about caveolae-independent pathways. In this current study, we confirmed that cultured rat pulmonary microvascular (RPMEC) and pulmonary artery (RPAEC) endothelium endocytosed Alexa488-labeled albumin in a saturable, temperature-sensitive mode and internalization resulted in co-localization by fluorescence microscopy with cholera B toxin and caveolin-1. Although siRNA to caveolin-1 (cav-1) in RPAEC significantly inhibited albumin uptake, a remnant portion of albumin uptake was cav-1-independent, suggesting alternative pathways for albumin uptake. Thus, we isolated and cultured mouse lung endothelial cells (MLEC) from wild type and cav-1^-/- mice and noted that ~ 65% of albumin uptake, as determined by confocal imaging or live cell total internal reflectance fluorescence microscopy (TIRF), persisted in total absence of cav-1. Uptake of colloidal gold labeled albumin was evaluated by electron microscopy and demonstrated that albumin uptake in MLEC from cav-1^-/- mice was through caveolae-independent pathway(s) including clathrin-coated pits that resulted in endosomal accumulation of albumin. Finally, we noted that albumin uptake in RPMEC was in part sensitive to pharmacological agents (amiloride [sodium transport inhibitor], Gö6976 [protein kinase C inhibitor], and cytochalasin D [inhibitor of actin polymerization]) consistent with a macropinocytosis-like process. The amiloride sensitivity accounting for macropinocytosis also exists in albumin uptake by both wild type and cav-1^-/- MLEC. We conclude from these studies that in addition to the well described caveolar-dependent pulmonary endothelial cell endocytosis of albumin, a portion of overall uptake in pulmonary endothelial cells is cav-1 insensitive and appears to involve clathrin-mediated endocytosis and macropinocytosis-like process.     

Transmitochondrial embryonic stem cells containing pathogenic mtDNA mutations are compromised in neuronal differentiation

Objectives:  Defects of the mitochondrial genome (mtDNA) cause a series of rare, mainly neurological disorders. In addition, they have been implicated in more common forms of movement disorders, dementia and the ageing process. In order to try to model neuronal dysfunction associated with mitochondrial disease, we have attempted to establish a series of trans mitochondrial mouse embryonic stem cells harbouring pathogenic mtDNA mutations.
Materials and methods:  Trans mitochondrial embryonic stem cell cybrids were generated by fusion of cytoplasts carrying a variety of mtDNA mutations, into embryonic stem cells that had been pretreated with rhodamine 6G, to prevent transmission of endogenous mtDNA. Cybrids were differentiated into neurons and assessed for efficiency of differentiation and electrophysiological function.
Results:  Neuronal differentiation could occur, as indicated by expression of neuronal markers. Differentiation was impaired in embryonic stem cells carrying mtDNA mutations that caused severe biochemical deficiency. Electrophysiological tests showed evidence of synaptic activity in differentiated neurons carrying non-pathogenic mtDNA mutations or in those that caused a mild defect of respiratory activity. Again, however, neurons carrying mtDNA mutations that resulted in severe biochemical deficiency had marked reduction in post-synaptic events.
Conclusions:  Differentiated neurons carrying severely pathogenic mtDNA defects can provide a useful model for understanding how such mutations can cause neuronal dysfunction.     

Dynamin 2 and c-Abl Are Novel Regulators of Hyperoxia-mediated NADPH Oxidase Activation and Reactive Oxygen Species Production in Caveolin-enriched Microdomains of the Endothelium

Reactive oxygen species (ROS) generation, particularly by the endothelial NADPH oxidase family of proteins, plays a major role in the pathophysiology associated with lung inflammation, ischemia/reperfusion injury, sepsis, hyperoxia, and ventilator-associated lung injury. We examined potential regulators of ROS production and discovered that hyperoxia treatment of human pulmonary artery endothelial cells induced recruitment of the vesicular regulator, dynamin 2, the non-receptor tyrosine kinase, c-Abl, and the NADPH oxidase subunit, p47^phox, to caveolin-enriched microdomains (CEMs). Silencing caveolin-1 (which blocks CEM formation) and/or c-Abl expression with small interference RNA inhibited hyperoxia-mediated tyrosine phosphorylation and association of dynamin 2 with p47^phox and ROS production. In addition, treatment of human pulmonary artery endothelial cells with dynamin 2 small interfering RNA or the dynamin GTPase inhibitor, Dynasore, attenuated hyperoxia-mediated ROS production and p47^phox recruitment to CEMs. Using purified recombinant proteins, we observed that c-Abl tyrosine-phosphorylated dynamin 2, and this phosphorylation increased p47^phox/dynamin 2 association (change in the dissociation constant (K_d) from 85.8 to 6.9 nm). Furthermore, exposure of mice to hyperoxia increased ROS production, c-Abl activation, dynamin 2 association with p47^phox, and pulmonary leak, events that were attenuated in the caveolin-1 knock-out mouse confirming a role for CEMs in ROS generation. These results suggest that hyperoxia induces c-Abl-mediated dynamin 2 phosphorylation required for recruitment of p47^phox to CEMs and subsequent ROS production in lung endothelium.     

Drosophila adducin regulates Dlg phosphorylation and targeting of Dlg to the synapse and epithelial membrane

Adducin is a cytoskeletal protein having regulatory roles that involve actin filaments, functions that are inhibited by phosphorylation of adducin by protein kinase C. Adducin is hyperphosphorylated in nervous system tissue in patients with the neurodegenerative disease amyotrophic lateral sclerosis, and mice lacking β-adducin have impaired synaptic plasticity and learning. We have found that Drosophila adducin, encoded by hu-li tai shao (hts), is localized to the post-synaptic larval neuromuscular junction (NMJ) in a complex with the scaffolding protein Discs large (Dlg), a regulator of synaptic plasticity during growth of the NMJ. hts mutant NMJs are underdeveloped, whereas over-expression of Hts promotes Dlg phosphorylation, delocalizes Dlg away from the NMJ, and causes NMJ overgrowth. Dlg is a component of septate junctions at the lateral membrane of epithelial cells, and we show that Hts regulates Dlg localization in the amnioserosa, an embryonic epithelium, and that embryos doubly mutant for hts and dlg exhibit defects in epithelial morphogenesis. The phosphorylation of Dlg by the kinases PAR-1 and CaMKII has been shown to disrupt Dlg targeting to the NMJ and we present evidence that Hts regulates Dlg targeting to the NMJ in muscle and the lateral membrane of epithelial cells by controlling the protein levels of PAR-1 and CaMKII, and consequently the extent of Dlg phosphorylation.     

Disruption of caveolin-1 leads to enhanced nitrosative stress and severe systolic and diastolic heart failure

Although caveolin-1 is not expressed in cardiomyocytes, this protein is assumed to act as a key regulator in the development of cardiomyopathy. In view of recent discordant findings we aimed to elucidate the cardiac phenotype of independently generated caveolin-1 knockout mice (cav-1(-/-)) and to unveil causative mechanisms. Invasive hemodynamic measurements of cav-1(-/-) show a severely reduced systolic and diastolic heart function. Additionally, genetic ablation of caveolin-1 leads to a striking biventricular hypertrophy and to a sustained eNOS-hyperactivation yielding increased systemic NO levels. Furthermore, a diminished ATP content and reduced levels of cyclic AMP in hearts of knockout animals were measured. Taken together, these results indicate that genetic disruption of caveolin-1 is sufficient to induce a severe biventricular hypertrophy with signs of systolic and diastolic heart failure. Collectively, our findings suggest a causative role of a sustained nitrosative stress in the development of the pronounced cardiac impairment.     

Caveolin-1 in oncogenic transformation, cancer, and metastasis

Caveolae are 50- to 100-nm omega-shaped invaginations of the plasma membrane that function as regulators of signal transduction. Caveolins are a class of oligomeric structural proteins that are both necessary and sufficient for caveolae formation. Interestingly, caveolin-1 has been implicated in the pathogenesis of oncogenic cell transformation, tumorigenesis, and metastasis. Here, we review the available experimental evidence (gleaned from cultured cells, animal models, and human tumor samples) that caveolin-1 (Cav-1) functions as a "tumor and/or metastasis modifier gene." Genetic evidence from the study of Cav-1(-/-) null mice and human breast cancer mutations [CAV-1 (P132L)] supports the idea that caveolin-1 normally functions as a negative regulator of cell transformation and mammary tumorigenesis. In contrast, caveolin-1 may function as a tumor promoter in prostate cancers. We discuss possible molecular mechanisms to explain these intriguing, seemingly opposing, findings. More specifically, caveolin-1 phosphorylation (at Tyr14 and Ser80) and mutations (P132L) may override or inactivate the growth inhibitory activity of the caveolin-scaffolding domain (residues 82-101).