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Biomedical subjects

Hong Sun

Publications and source records attributed to Hong Sun.

At least 55 records · Page 3Linked to original sources

Topographic organization in the auditory brainstem of juvenile mice is disrupted in congenital deafness.

There is an orderly topographic arrangement of neurones within auditory brainstem nuclei based on sound frequency. Previous immunolabelling studies in the medial nucleus of the trapezoid body (MNTB) have suggested that there may be gradients of voltage-gated currents underlying this tonotopic arrangement. Here, our electrophysiological and immunolabelling results demonstrate that underlying the tonotopic organization of the MNTB is a combination of medio-lateral gradients of low-and high-threshold potassium currents and hyperpolarization-activated cation currents. Our results also show that the intrinsic membrane properties of MNTB neurones produce a topographic gradient of time delays, which may be relevant to sound localization, following previous demonstrations of the importance of the timing of inhibitory input from the MNTB to the medial superior olive (MSO). Most importantly, we demonstrate that, in the MNTB of congenitally deaf mice, which exhibit no spontaneous auditory nerve activity, the normal tonotopic gradients of neuronal properties are absent. Our results suggest an underlying mechanism for the observed topographic gradient of neuronal firing properties in the MNTB, show that an intrinsic neuronal mechanism is responsible for generating a topographic gradient of time-delays, and provide direct evidence that these gradients rely on spontaneous auditory nerve activity during development.

Action Potentials↗

Role of NAD(P)H oxidase in alcohol-induced impairment of endothelial nitric oxide synthase-dependent dilation of cerebral arterioles.

BACKGROUND AND PURPOSE: Our goal was to determine whether NAD(P)H oxidase is involved in impaired endothelial nitric oxide synthase (eNOS)-dependent reactivity of cerebral arterioles during chronic alcohol consumption. METHODS: Sprague-Dawley rats were fed with an alcohol diet for 2 to 3 months. We determined the effects of acute and chronic treatment with an NAD(P)H oxidase inhibitor, apocynin, on responses of pial arterioles to eNOS-dependent agonists (acetylcholine and ADP) and an eNOS-independent agonist (nitroglycerin). Expression of NAD(P)H oxidase in pial arterioles was measured with the use of real-time polymerase chain reaction and Western blot analysis, and superoxide production was measured with the use of lucigenin-enhanced chemiluminescence. RESULTS: Vasodilation in response to acetylcholine and ADP, but not nitroglycerin, was significantly less in alcohol-fed rats. Treatment with apocynin did not alter vasodilation in non-alcohol-fed rats but significantly improved impaired vasodilation in alcohol-fed rats. In addition, an upregulation of p47phox in pial arterioles was found in alcohol-fed rats. Furthermore, alcohol consumption increased superoxide production under basal conditions and in the presence of ADP and NAD(P)H. CONCLUSIONS: Our findings suggest that NAD(P)H oxidase plays a role in chronic alcohol consumption-induced impairment of eNOS-dependent dilation of cerebral arterioles.

Acetophenones↗

Organization of the Caenorhabditis elegans small non-coding transcriptome: genomic features, biogenesis, and expression.

Recent evidence points to considerable transcription occurring in non-protein-coding regions of eukaryote genomes. However, their lack of conservation and demonstrated function have created controversy over whether these transcripts are functional. Applying a novel cloning strategy, we have cloned 100 novel and 61 known or predicted Caenorhabditis elegans full-length ncRNAs. Studying the genomic environment and transcriptional characteristics have shown that two-thirds of all ncRNAs, including many intronic snoRNAs, are independently transcribed under the control of ncRNA-specific upstream promoter elements. Furthermore, the transcription levels of at least 60% of the ncRNAs vary with developmental stages. We identified two new classes of ncRNAs, stem-bulge RNAs (sbRNAs) and snRNA-like RNAs (snlRNAs), both featuring distinct internal motifs, secondary structures, upstream elements, and high and developmentally variable expression. Most of the novel ncRNAs are conserved in Caenorhabditis briggsae, but only one homolog was found outside the nematodes. Preliminary estimates indicate that the C. elegans transcriptome contains approximately 2700 small non-coding RNAs, potentially acting as regulatory elements in nematode development.

Animals↗

Identification and characterization of survival-related gene, a novel cell survival gene controlling apoptosis and tumorigenesis.

Apoptosis plays a critical role in cellular homeostasis during development, immune responses, and tumorigenesis. Recent studies have identified a number of genes that control this process. We report here our identification of a novel cell survival-related gene (SRG) from a human expression cDNA library by functional cloning. SRG shows no significant nucleotide sequence homology to any known genes in the Genbank. Our fluorescence in situ hybridization analysis has estimated that SRG is located at 1p36, agreeing with the location at 1p36.22 in the human genome sequence. SRG encodes a putative protein of 172 amino acids, which is mainly located in the perinuclear region. Northern blotting analysis indicates that SRG is highly expressed in many human cancer cell lines although it is low in most tissues except liver and placenta. To investigate the function of SRG in apoptosis, we transfected SRG cDNA into BAF/BO3 and B16/F0 cells and induced apoptosis by cytokine/serum deprivation. We found that SRG-transfected cells are resistant to apoptosis induced by cytokine/serum deprivation. In addition, mice bearing SRG-transfected melanoma had more tumor formation and larger tumor growth. Melanoma transfected with antisense SRG showed significantly less tumor formation and smaller tumor growth. Interestingly, mouse SRG gene was also identified on chromosome 4 and blocking SRG expression with small interfering RNA promoted serum deprivation-induced apoptosis of NIH3T3 cells. Our results show that SRG is a novel cell survival gene that critically controls apoptosis and tumor formation.

Amino Acid Sequence↗

Genome-wide analysis of mammalian DNA segment fusion/fission.

As a powerful tool for gene function prediction, gene fusion has been widely studied in prokaryotes and certain groups of eukaryotes, but it has been little applied in studies of mammalian genomes. With the first fully sequenced mammalian genomes (human, mouse, rat) now available, we defined and collected a set of fusion/fission event-linked segments (FFLS) based on structured organized genomic alignment. The statistics of the sequence features highlighted the FFLSs against their random context. We found that there are three groups of FFLSs with different component pairs (i.e. gene-gene, gene-noncoding and noncoding-noncoding) in all three mammalian genomes. The proteins encoded by the components of FFLSs in the first group shown a strong tendency to interact with each other. The segmental components in the last two groups which did not contain any protein-coding genes, were found not only to be transcribed to some level, but also more conserved than the random background. Thus, these segments are possibly carrying certain biologically functional elements. We propose that FFLS may be a potential tool for prediction and analysis of function and functional interaction of genetic elements, including both genes and noncoding elements, in mammalian genomes. The full list of the FFLSs in the genomes of the three mammals is available as supporting information at doi:10.1016/j.jtbi.2005.09.016.

Animals↗

Inhibition of NADPH oxidase improves impaired reactivity of pial arterioles during chronic exposure to nicotine.

Our goals were to determine whether chronic exposure to nicotine alters nitric oxide synthase (NOS)-dependent reactivity of cerebral (pial) arterioles and to identify a potential role for NADPH oxidase in impaired NOS-dependent responses during chronic exposure to nicotine. We measured in vivo diameter of pial arterioles to NOS-dependent (acetylcholine and ADP) and -independent (nitroglycerin) agonists in saline-treated rats and rats chronically treated with nicotine (2 mg.kg(-1).day(-1) for 2 wk via an osmotic minipump). We found that NOS-dependent, but not -independent, vasodilatation was impaired in nicotine-treated compared with saline-treated rats. In addition, the production of superoxide anion (lucigenin chemiluminescence) was increased in rats treated with nicotine compared with saline-treated rats. Furthermore, using Western blot analysis, we found that chronic exposure to nicotine increased p47phox protein in the parietal cortex. Finally, we found that apocynin (40 mg.kg(-1).day(-1)) in the drinking water to inhibit NADPH oxidase alleviated impaired NOS-dependent cerebral vasodilatation in nicotine treated rats but did not alter NOS-dependent responses in saline treated rats and did not alter NOS-independent reactivity in saline- or nicotine-treated rats. These findings suggest that chronic exposure to nicotine impairs NOS-dependent dilatation of pial arterioles by a mechanism that appears to be related to the formation of superoxide anion via activation of NADPH oxidase.

Acetophenones↗

Evaluation of androgen receptor transcriptional activities of bisphenol A, octylphenol and nonylphenol in vitro.

In this study, we have developed a human androgen receptor (hAR) reporter gene assay using African monkey kidney cell line CV-1 transiently transfected with the constructed reporter gene plasmid pMMTV-CAT and the hAR expression plasmid AR/pcDNA3.1. The assay displayed appropriate response to the known AR agonist 5alpha-dihydrotestosterone (DHT) and AR antagonist flutamide. DHT induced AR-mediated transcriptional activity in a concentration-dependent manner with median effective concentration (EC50) value of (3.90+/-1.43)x10(-10)M. Flutamide exhibited potent antiandrogenic activity with median inhibitory concentration (IC50) value of (1.02+/-0.35)x10(-7)M. Bisphenol A (BPA) and alkylphenols (APs) belong to the industrial chemicals that have received considerable attention due to high production and widespread usage. We investigated BPA, 4-octylphenol (OP) and 4-nonylphenol (NP) for their agonistic and antagonistic activities by the AR reporter gene assay. BPA showed significant inhibitory effects on the transcriptional activity induced by DHT with IC50 value of (7.46+/-1.23)x10(-7)M. OP and NP exhibited lower antiandrogenic activities than BPA with IC(50) of (9.71+/-3.82)x10(-5)M and (2.02+/-0.90)x10(-5)M, respectively. On the other hand, we failed to find AR-mediated androgenic activities of the three tested chemicals. In conclusion, BPA, OP and NP all act as AR antagonists, and BPA possesses the highest activity.

Androgen Antagonists↗

Androgen receptor activities of p,p'-DDE, fenvalerate and phoxim detected by androgen receptor reporter gene assay.

In this study, we have developed a transient human androgen receptor (hAR) reporter gene assay using African monkey kidney cell line CV-1. The assay displayed appropriate response to the known androgen receptor (AR) agonist 5alpha-dihydrotestosterone (DHT) and AR antagonist nilutamide. DHT induced AR-mediated transcriptional activity in a concentration-dependent manner with median effective concentration (EC(50)) value of 3.90 x 10(-10) M. Nilutamide exhibited potent antiandrogenic activity with median inhibitory concentration (IC(50)) value of 8.90 x 10(-7) M. The transcription could not be activated by glucocorticoid receptor (GR) agonist dexamethasone, which suggested that the assay system be highly specific to androgenic compounds without cross-talk to GR agonist. The assay shows acceptable repeatability to DHT with intra coefficient of variability (CV) of 9.3% and inter CV of 10.9%. We applied this assay to evaluate the androgenic and antiandrogenic activities of some pesticides including organochlorine p,p'-dichlorodiphenyldichloroethylene (DDE), pyrethroid fenvalerate and organophosphorus phoxim. The results showed that p,p'-DDE exhibited potent antiandrogenic activity. Fenvalerate acted as weak AR antagonist and phoxim did not show AR antagonistic activity. We failed to find AR agonistic effects of the three pesticides. The present study provided a promising tool to screen for AR agonists and antagonists.

Androgen Antagonists↗

Effects of renal failure on drug transport and metabolism.

Renal failure not only alters the renal elimination, but also the non-renal disposition of drugs that are extensively metabolized by the liver. Although reduced metabolic enzyme activity in some cases can be responsible for the reduced drug clearance, alterations in the transporter systems may also be involved in the process. With the development of renal failure, the renal secretion of organic ions mediated by organic anion transporters (OATs) and organic cation transporters (OCTs) is decreased. 3-Carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) and other organic anionic uremic toxins may directly inhibit the renal excretion of various drugs and endogenous organic acids by competitively inhibiting OATs. In addition, the expression of OAT1 and OCT2 was reduced in chronic renal failure (CRF) rats. Renal failure also impairs the liver uptake of drugs and organic anions, such as bromosulphophthalein (BSP), indocyanine green (ICG), and thyroxine, where organic anion transport polypeptides (OATPs) are the major transporters. Most previous studies have been done in animals or cell culture, very often in rat models, but these are presumed to reflect the presentation of advanced renal disease in humans as well. Recent studies demonstrate that the uremic toxins CMPF and indoxyl sulfate (IS) can directly inhibit rOatp2 and hOATP-C in hepatocytes. The protein content of the liver uptake transporters Oatp1, 2, and 4 were significantly decreased in CRF rats. Decreased activity of the intestinal efflux transporter, P-glycoprotein (P-gp), was also observed in CRF rats, with no significant change of protein content, suggesting that uremic toxins may suppress P-gp function. However, increased protein levels of multidrug resistance-associated protein (MRP) 2 in the kidney and MRP3 in the liver were found in CRF rats, suggesting an adaptive response that may serve as a protective mechanism. Increases in drug areas under the curve (AUCs) in subjects with advanced renal disease for drugs that are not renally excreted are consistent with uremic toxin effects on either intestinal or hepatic cell transporters, metabolizing enzymes, or both. In conclusion, alterations of drug transporters, as well as metabolic enzymes, in patients with renal failure can be responsible for reduced drug clearance.

Animals↗

Microsurgical resection of craniopharyngioma of the third ventricle via an improved transventricular approach.

BACKGROUND: Craniopharyngioma of the third ventricle is difficult to treat and its therapeutic regimens and operative approaches have been controversial. This study was undertaken to probe indications for microsurgical resection of craniopharyngioma of the third ventricle via an improved transventricular approach, its surgical procedures and therapeutic effects, and prevention of postoperative complications. METHODS: Fifty-one patients with craniopharyngioma of the third ventricle were treated from January 2000 to October 2004 by an improved transventricular approach for removing the tumor via the interventricular foramen, the intermedius of the septum pellucidum or choroid fissure. Symptoms and signs of the patients, and results of imaging, operation, and follow-up were analyzed. RESULTS: Of the 51 patients who had received the improved transventricular resection, 4 underwent a combined approach with an entrance of the pterion. Forty patients (78.43%) underwent total resection and others subtotal resection, without an operative death. Epileptic seizures were found in 3 patients (5.88%) and subdural effusion in the operative field in 4 (7.84%). All patients showed good general conditions after operation, and follow-up for an average of 27.52 months showed relapse of the tumour in 8 patients (15.69%). CONCLUSIONS: Microsurgical resection of craniopharyngioma of the third ventricle by an improved transventricular approach has advantages of operative safety and efficacy, lower mortality and disability, and less complications.

Adolescent↗

Activation of NHE3 by dexamethasone requires phosphorylation of NHE3 at Ser663 by SGK1.

Glucocorticoids stimulate Na+ absorption by activation of the epithelial Na+/H+ exchanger NHE3 in the kidney and intestine. It has been thought that glucocorticoid-induced activation of NHE3 is solely dependent on transcriptional induction of the NHE3 gene. While the transcriptional regulation remains an essential part of the chronic effect of glucocorticoids, a previous study by us identified the serum- and glucocorticoid-inducible kinase 1 (SGK1) as an important component of the activation of NHE3 by glucocorticoids. In this work, we have demonstrated phosphorylation of NHE3 by SGK1 as the key mechanism for the stimulation of the transport activity by glucocorticoids. By using in vitro SGK1 kinase assay and site-directed mutagenesis, we have identified Ser663 of NHE3 to be the major site of phosphorylation by SGK1. Ser663 is invariantly conserved in all NHE3 proteins from several species, and the mutation of Ser663 to Ala blocks the effect of dexamethasone, demonstrating the importance of phosphorylation at Ser663. We also show that phosphorylation of NHE3 precedes the changes in NHE3 activity, and the increased activity is associated with an increased amount of NHE3 proteins in the surface membrane. These data reveal that dexamethasone activates NHE3 activity by phosphorylating the NHE3 protein, which initiates trafficking of the protein into the plasma membrane.

Amino Acid Sequence↗

Atomistic deformation modes in strong covalent solids.

We report on a first-principles study of the structural deformation modes in diamond, cubic boron nitride (c-BN), and cubic BC2N. We show that (i) the diamond C-C bonds remain strong up to the breaking point, leading to the large and nearly identical shear and tensile strength, (ii) c-BN exhibits a shear failure mode different from that in diamond and a significant softening in the B-N bonds at large tensile strains long before the bond breaking, and (iii) cubic BC2N displays a large disparity between the shear and tensile strength, contrary to the expectation for the hybrid of diamond and c-BN. We examine the microscopic bond-breaking processes to elucidate the atomistic mechanisms for the deformation modes and the implications for material strength.

Journal Article↗

LPA2 receptor mediates mitogenic signals in human colon cancer cells.

Lysophosphatidic acid (LPA) is a mediator of multiple cellular responses. LPA mediates its effects predominantly through the G protein-coupled receptors LPA1, LPA2, and LPA3. In the present work, we studied LPA2-mediated signaling using human colon cancer cell lines, which predominantly express LPA2. LPA2 activated Akt and Erk1/2 in response to LPA. LPA mediated Akt activation was inhibited by pertussis toxin (PTX), whereas Erk1/2 activation was completely inhibited by a blocker of phospholipase Cbeta, U-73122. LPA also induced interleukin-8 (IL-8) synthesis in the colon cancer cells by primarily activating LPA2 receptor. We also found that LPA2 interacts with Na+/H+ exchanger regulatory factor 2 (NHERF2). Activation of Akt and Erk1/2 was significantly attenuated by silencing of NHERF2 expression by RNA interference, suggesting a pivotal role of NHERF2 in LPA2-mediated signaling. We found that expression of LPA2 was elevated, whereas expression of LPA1 downregulated in several types of cancers, including ovarian and colon cancer. We conclude that LPA2 is the major LPA receptor in colon cancer cells and cellular signals by LPA2 are largely mediated through its ability to interact with NHERF2.

Caco-2 Cells↗

Factors predictive of tumor growth, tumor decalcification, choroidal neovascularization, and visual outcome in 74 eyes with choroidal osteoma.

OBJECTIVE: To evaluate choroidal osteoma for tumor growth, tumor decalcification, related choroidal neovascularization, visual acuity loss, and poor visual acuity. DESIGN: Retrospective nonrandomized single-center case series. SETTING: Ocular Oncology Service at Wills Eye Hospital, Thomas Jefferson University, Philadelphia, Pa. PARTICIPANTS: There were 74 eyes of 61 patients with choroidal osteoma evaluated between January 1, 1977, and January 1, 2003. MAIN OUTCOME MEASURES: The 5 outcome measures included tumor growth, tumor decalcification, related choroidal neovascularization, visual acuity loss of 3 or more Snellen lines, and poor visual acuity of 20/200 or worse. RESULTS: At 5 and 10 years, Kaplan-Meier analysis revealed tumor growth in 22% and 51% of eyes, tumor decalcification in 28% and 46% of eyes, choroidal neovascularization in 31% and 31% of eyes, visual acuity loss in 26% and 45% of eyes, and poor visual acuity in 45% and 56% of eyes, respectively. The clinical factor predictive of tumor growth was absent overlying retinal pigment epithelial alterations. The factor predictive of decalcification was irregular tumor surface. Of the 15 tumors that showed partial decalcification at the first visit, there was no further tumor growth in any case. Of the remaining 12 tumors that later developed decalcification, tumor growth, if it occurred, was along the margin opposite the decalcification. No tumor showed growth in the region of decalcification. Factors predictive of choroidal neovascularization included irregular tumor surface and subretinal hemorrhage. In 6 eyes that had both choroidal neovascularization and tumor decalcification, the neovascularization was detected prior to or at the same time as the decalcification. The factor predictive of visual acuity loss was presence of subretinal fluid whereas the factors predictive of poor visual acuity included symptoms and tumor decalcification. A comparison of eyes with subfoveal vs extrafoveal choroidal osteoma showed poor visual acuity in 15 (34%) of 44 eyes and 3 (10%) of 30 eyes, respectively. Eyes with decalcified choroidal osteomas manifested poor visual acuity in 13 (48%) of 27 eyes whereas those with nondecalcified tumors showed poor visual acuity in 5 (11%) of 47 eyes. CONCLUSIONS: Choroidal osteoma showed evidence of growth in 51% of eyes and decalcification in nearly 50% of eyes by 10 years. Tumors with any degree of decalcification at the initial visit showed no further growth. Overall, poor visual acuity of 20/200 or worse was found in 56% of eyes by 10 years, and decalcified subfoveal choroidal osteomas displayed a particularly poor visual prognosis.

Adolescent↗

Sclerochoroidal calcification in a patient with classic Bartter's syndrome.

PURPOSE: To report sclerochoroidal calcification in a patient with classic Bartter's syndrome. DESIGN: Observational case report. METHODS: A 42-year-old woman with a 26-year history of classic Bartter's syndrome was found to have bilateral fundus tumors. The patient presented initially with quivering lips and hand stiffness at age 6 years but was not diagnosed until age 16 years. Treatment included magnesium and potassium supplementation and Amiloride therapy. RESULTS: On ocular examination, there were multifocal, yellow-white, geographic, solid choroidal lesions along the superior and inferior retinal vascular arcades in both eyes. Ultrasonography showed echogenic, placoid calcified lesions at the level of the sclera and choroid, consistent with bilateral sclerochoroidal calcification. CONCLUSIONS: Sclerochoroidal calcification can be associated with classic Bartter's syndrome.

Adult↗

Human neutrophil collagenase expression is C/EBP-dependent during myeloid development.

OBJECTIVE: Human neutrophil collagenase (HNC) is one of several secondary granule proteins (SGP) expressed late in the myeloid maturation pathway. SGPs are encoded by unlinked and functionally diverse genes that are hypothesized to be coordinately regulated at the transcriptional level and demonstrate uniform dysregulation in leukemic cells. In support of the hypothesis that tissue and stage-specific expression of SGP genes is regulated by shared factor(s), we sought to identify factors responsible for positive regulation of the SGP genes. METHODS: Using 5' deletion analysis, we identified a minimal HNC promoter located within the first 193 bp upstream of the transcription start site. Three CCAAT enhancer binding protein (C/EBP) sites were identified within this region and their functional importance was confirmed by mutational analysis, gel retardation, and oligonucleotide pulldown assays. Using chromatin immunoprecipitation (ChIP), we demonstrated that C/EBPalpha binds to the SGP gene promoters lactoferrin and HNC in nonexpressing cells. Upon induction of maturation, C/EBPalpha binds to these promoters and this binding correlates with the expression of both SGP genes. CONCLUSION: We conclude that in the later stages of myeloid development, SGP genes are coordinately upregulated, and that members of the C/EBP family of transcription factors, in particular C/EBPalpha and C/EBPepsilon, play specific and unique roles in upregulating their expression.

Animals↗

Sex difference in nitric oxide synthase-dependent dilatation of cerebral arterioles during long-term alcohol consumption.

BACKGROUND: Although long-term alcohol consumption impairs both endothelial nitric oxide synthase (NOS) (eNOS)-dependent and neuronal NOS (nNOS)-dependent reactivity of cerebral arterioles in male rats, the influence of sex on alcohol consumption-induced impairment of NOS-dependent cerebral vasodilation has not been examined. Therefore, the authors' first goal was to determine whether long-term alcohol consumption alters eNOS-dependent and nNOS-dependent reactivity of pial arterioles in female rats. Their second goal was to examine potential mechanisms for impaired responses of pial arterioles during long-term alcohol consumption. METHODS: Male and female rats were fed liquid diets with or without alcohol for 2-3 months. The authors measured the in vivo diameter of pial arterioles in response to eNOS-dependent agonists (acetylcholine and ADP), nNOS-dependent agonists (kainate and NMDA), and NOS-independent agonists (nitroglycerin and diethylaminodiazen (NONOate)) in the absence and presence of tetrahydrobiopterin (BH4). RESULTS: Topical application of acetylcholine (1.0 and 10 microM), ADP (10 and 100 microM), kainate (100 and 300 microM), and NMDA (100 and 300 microM) produced dose-related dilatation of pial arterioles. However, the magnitude of vasodilation in response to acetylcholine and ADP was significantly less in alcohol-fed male and female rats, and the magnitude of vasodilation in response to kainate and NMDA was significantly less in alcohol-fed male rats. Nitroglycerin and NONOate produced similar vasodilation in non-alcohol-fed and alcohol-fed male and female rats. Application of BH4 did not alter vasodilation in non-alcohol-fed rats but improved impaired eNOS-dependent vasodilation in alcohol-fed male and female rats. CONCLUSIONS: Our findings suggest that long-term alcohol consumption impairs eNOS-dependent but not nNOS-dependent dilatation of cerebral arterioles in female rats and that impaired eNOS-dependent vasodilation may be related to a deficiency or alteration in the utilization of BH4.

Alcohol Drinking↗

Host adaptor proteins Gab1 and CrkII promote InlB-dependent entry of Listeria monocytogenes.

The bacterial surface protein InlB mediates internalization of Listeria monocytogenes into mammalian cells through interaction with the host receptor tyrosine kinase, Met. InlB/Met interaction results in activation of the host phosphoinositide (PI) 3-kinase p85-p110, an event required for bacterial entry. p85-p110 activation coincides with tyrosine phosphorylation of the host adaptor Gab1, and formation of complexes between Gab1 and the p85 regulatory subunit of PI 3-kinase. When phosphorylated in response to agonists, Gab1 is known to recruit several Src-homology 2 (SH2) domain-containing proteins including p85, the tyrosine phosphatase Shp2 and the adaptor CrkII. Here, we demonstrate that Gab1.p85 and Gab1.CrkII complexes promote entry of Listeria. Overexpression of wild-type Gab1 stimulated entry, whereas Gab1 alleles unable to recruit all SH2 proteins known to bind wild-type Gab1 inhibited internalization. Further analysis with Gab1 alleles defective in binding individual effectors suggested that recruitment of p85 and CrkII are critical for entry. Consistent with this data, overexpression of wild-type CrkII stimulated bacterial uptake. Experiments with mutant CrkII alleles indicated that both the first and second SH3 domains of this adaptor participate in entry, with the second domain playing the most critical role. Taken together, these findings demonstrate novel roles for Gab1 and CrkII in Listeria internalization.

Animals↗