Alteration of p66shc is associated with endothelial dysfunction in the abdominal aortic coarctation of rats

To examine the role of p66shc in endothelial dysfunction, we investigated the endothelium-dependent relaxation, protein expression and superoxide production in abdominal aortic coarctation rats. Endothelium-dependent relaxation to acetylcholine was impaired only in the aortic segments above the aortic coarctation (35.0+/-7.1% vs. 86.6+/-6.0% for sham control at 1 microM Ach). The aortic segments exposed to increased blood pressure showed a decreased phosphorylation of endothelial nitric oxide synthase, an increased phosphorylation of p66shc, and an increased superoxide production. Angiotensin II elicited a significantly increased phosphorylation of p66shc in the endothelial cells. Taken together, these findings suggest that the increased phosphorylation of p66shc is one of the important mediators in the impaired endothelium-dependent relaxation of aortic coarctation rats.     

Opposite effects of the p52shc/p46shc and p66shc splicing isoforms on the EGF receptor-MAP kinase-fos signalling pathway.

Shc proteins are targets of activated tyrosine kinases and are implicated in the transmission of activation signals to Ras. The p46shc and p52shc isoforms share a C-terminal SH2 domain, a proline- and glycine-rich region (collagen homologous region 1; CH1) and a N-terminal PTB domain. We have isolated cDNAs encoding for a third Shc isoform, p66shc. The predicted amino acid sequence of p66shc overlaps that of p52shc and contains a unique N-terminal region which is also rich in glycines and prolines (CH2). p52shc/p46shc is found in every cell type with invariant reciprocal relationship, whereas p66shc expression varies from cell type to cell type. p66shc differs from p52shc/p46shc in its inability to transform mouse fibroblasts in vitro. Like p52shc/p46shc, p66shc is tyrosine-phosphorylated upon epidermal growth factor (EGF) stimulation, binds to activated EGF receptors (EGFRs) and forms stable complexes with Grb2. However, unlike p52shc/p46shc it does not increase EGF activation of MAP kinases, but inhibits fos promoter activation. The isolated CH2 domain retains the inhibitory effect of p66shc on the fos promoter. p52shc/p46shc and p66shc, therefore, appear to exert different effects on the EGFR-MAP kinase and other signalling pathways that control fos promoter activity. Regulation of p66shc expression might, therefore, influence the cellular response to growth factors.     

Tumor interactions with soluble factors and the nervous system

Members of Shc (src homology and collagen homology) family, p46shc, p52shc, p66shc have known to be related to cell proliferation and carcinogenesis. Whereas p46shc and p52shc drive the reaction forward, the role of p66shc in cancers remains to be understood clearly. Hence, their expression in cancers needs to be evaluated carefully so that Shc analysis may provide prognostic information in the development of carcinogenesis. In the present study, the expression of p66shc and its associate targets namely Eps8 (epidermal pathway substrate 8), Rac1 (ras-related C3 botulinum toxin substrate1) and Grb2 (growth factor receptor bound protein 2) were examined in fresh tissue specimens from patients with esophageal squamous cell carcinoma and esophageal adenocarcinoma using western blot analysis. A thorough analysis of both esophageal squamous cell carcinoma and adenocarcinoma showed p66shc expression to be significantly higher in both types of carcinomas as compared to the controls. The controls of adenocarcinoma show a higher basal expression level of p66shc as compared to the controls of squamous cell carcinoma. The expression level of downstream targets of p66shc i.e., eps8 and rac1 was also found to be consistently higher in human esophageal carcinomas, and hence correlated positively with p66shc expression. However the expression of grb2 was found to be equal in both esophageal squamous cell carcinoma and adenocarcinoma. The above results suggest that the pathway operated by p66shc in cancers does not involve the participation of Ras and Grb2 as downstream targets instead it operates the pathway involving Eps8 and Rac1 proteins. From the results it is also suggestive that p66shc may have a role in the regulation of esophageal carcinomas and represents a possible mechanism of signaling for the development of squamous cell carcinoma and adenocarcinoma of esophagus.     

Rac1 leads to phosphorylation-dependent increase in stability of the p66shc adaptor protein: role in Rac1-induced oxidative stress

The rac1 GTPase and the p66shc adaptor protein regulate intracellular levels of reactive oxygen species (ROS). We examined the relationship between rac1 and p66shc. Expression of constitutively active rac1 (rac1V12) increased phosphorylation, reduced ubiquitination, and increased stability of p66shc protein. Rac1V12-induced phosphorylation and up-regulation of p66shc was suppressed by inhibiting p38MAPK and was dependent on serine 54 and threonine 386 in p66shc. Phosphorylation of recombinant p66shc by p38MAPK in vitro was also partly dependent on serine 54 and threonine 386. Reconstitution of p66shc in p66shc-null fibroblasts increased intracellular ROS generated by rac1V12, which was significantly dependent on the integrity of residues 54 and 386. Overexpression of p66shc increased rac1V12-induced apoptosis, an effect that was also partly dependent on serine 54 and threonine 386. Finally, RNA interference-mediated down-regulation of endogenous p66shc suppressed rac1V12-induced cell death. These findings identify p66shc as a mediator of rac1-induced oxidative stress. In addition, they suggest that serine 54 and threonine 386 are novel phosphorylatable residues in p66shc that govern rac1-induced increase in its expression, through a decrease in its ubiquitination and degradation, and thereby mediate rac1-stimulated cellular oxidative stress and death.     

p66shc negatively regulates insulin-like growth factor I signal transduction via inhibition of p52shc binding to Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 leading to impaired growth factor receptor-bound protein-2 membrane recruitment

Our previous studies have indicated an essential role of p52shc in mediating IGF-I activation of MAPK in smooth muscle cells (SMC). However, the role of the p66 isoform of shc in IGF-I signal transduction is unclear. In the current study, two approaches were employed to investigate the role of p66shc in mediating IGF-I signaling. Knockdown p66shc by small interfering RNA enhanced IGF-I-stimulated p52shc tyrosine phosphorylation and growth factor receptor-bound protein-2 (Grb2) association, resulting in increased IGF-I-dependent MAPK activation. This was associated with enhanced IGF-I-stimulated cell proliferation. In contrast, knockdown of p66shc did not affect IGF-I-stimulated IGF-I receptor tyrosine phosphorylation. Overexpression of p66shc impaired IGF-I-stimulated p52shc tyrosine phosphorylation and p52shc-Grb2 association. In addition, IGF-I-dependent MAPK activation was also impaired, and SMC proliferation in response to IGF-I was inhibited. IGF-I-dependent cell migration was enhanced by p66shc knockdown and attenuated by p66shc overexpression. Mechanistic studies indicated that p66shc inhibited IGF-I signal transduction via competitively inhibiting the binding of Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) to SHP substrate-1 (SHPS-1), leading to the disruption of SHPS-1/SHP-2/Src/p52shc complex formation, an event that has been shown previously to be essential for p52shc phosphorylation and Grb2 recruitment. These findings indicate that p66shc functions to negatively regulate the formation of a signaling complex that is required for p52shc activation in response to IGF-I, thus leading to attenuation of IGF-I-stimulated cell proliferation and migration.     

Antagonism of p66shc by melanoma inhibitory activity

The p66shc protein governs oxidant stress and mammalian lifespan. Here, we identify melanoma inhibitory activity (MIA), a protein secreted by melanoma cells, as a novel binding partner and antagonist of p66shc. The N-terminal collagen homology-2 (CH2) domain of p66shc binds to the Src Homology-3 (SH3)-like domain of MIA in vitro. In cells, ectopically expressed MIA and p66shc colocalize and co-precipitate. MIA also co-precipitates with the CH2 domain of p66shc in vivo. MIA expression in vivo suppresses p66shc-stimulated increase in endogenous hydrogen peroxide (H(2)O(2)), and inhibits basal and H(2)O(2)-induced phosphorylation of p66shc on serine 36 and H(2)O(2)-induced death. In human melanoma cells expressing MIA, endogenous MIA and p66shc co-precipitate. Downregulation of MIA in melanoma cells increases basal and ultraviolet radiation (UVR)-induced phosphorylation of p66shc on serine 36, augments endogenous H(2)O(2) levels, and increases their susceptibility to UVR-induced death. These findings show that MIA binds to p66shc, and suggest that this interaction antagonizes phosphorylation and function of p66shc.     

TNFα Signals via p66Shc to Induce E-Selectin,Promote Leukocyte Transmigration and Enhance Permeability in Human Endothelial Cells

Endothelial cells participate in inflammatory events leading to atherogenesis by regulating endothelial cell permeability via the expression of VE-Cadherin and β-catenin and leukocyte recruitment via the expression of E-Selectins and other adhesion molecules. The protein p66^Shc acts as a sensor/inducer of oxidative stress and may promote vascular dysfunction. The objective of this study was to investigate the role of p66^Shc in tumor necrosis factor TNFα-induced E-Selectin expression and function in human umbilical vein endothelial cells (HUVEC). Exposure of HUVEC to 50 ng/ml TNFα resulted in increased leukocyte transmigration through the endothelial monolayer and E-Selectin expression, in association with augmented phosphorylation of both p66^Shc on Ser³⁶ and the stress kinase c-Jun NH₂-terminal protein kinase (JNK)-1/2, and higher intracellular reactive oxygen species (ROS) levels. Overexpression of p66^Shc in HUVEC resulted in enhanced p66^Shc phosphorylation on Ser³⁶, increased ROS and E-Selectin levels, and amplified endothelial cell permeability and leukocyte transmigration through the HUVEC monolayer. Conversely, overexpression of a phosphorylation-defective p66^Shc protein, in which Ser³⁶ was replaced by Ala, did not augment ROS and E-Selectin levels, nor modify cell permeability or leukocyte transmigration beyond those found in wild-type cells. Moreover, siRNA-mediated silencing of p66^Shc resulted in marked reduction of E-Selectin expression and leukocyte transmigration. In conclusion, p66^Shc acts as a novel intermediate in the TNFα pathway mediating endothelial dysfunction, and its action requires JNK-dependent phosphorylation of p66^Shc on Ser³⁶.     

Sos-mediated activation of rac1 by p66shc

The Son of Sevenless 1 protein (sos1) is a guanine nucleotide exchange factor (GEF) for either the ras or rac1 GTPase. We show that p66shc, an adaptor protein that promotes oxidative stress, increases the rac1-specific GEF activity of sos1, resulting in rac1 activation. P66shc decreases sos1 bound to the growth factor receptor bound protein (grb2) and increases the formation of the sos1-eps8-e3b1 tricomplex. The NH(2)-terminal proline-rich collagen homology 2 (CH2) domain of p66shc associates with full-length grb2 in vitro via the COOH-terminal src homology 3 (C-SH3) domain of grb2. A proline-rich motif (PPLP) in the CH2 domain mediates this association. The CH2 domain competes with the proline-rich COOH-terminal region of sos1 for the C-SH3 domain of grb2. P66shc-induced dissociation of sos1 from grb2, formation of the sos1-eps8-e3b1 complex, rac1-specific GEF activity of sos1, rac1 activation, and oxidative stress are also mediated by the PPLP motif in the CH2 domain. This relationship between p66shc, grb2, and sos1 provides a novel mechanism for the activation of rac1.     

Distinct mechanisms of taxol-induced serine phosphorylation of the 66-kDa Shc isoform in A549 and RAW 264.7 cells

Nanomolar concentrations of Taxol, and other antimitotic agents that interact with microtubules, mediate serine phosphorylation of the 66-kDa Shc isoform (p66shc) in A549 human lung carcinoma cells, 9-18 h after drug treatment. This event coincides with the release of PARP cleavage fragments that are early indicators of apoptosis. Taxol-induced serine phosphorylation of p66shc results from a MEK-independent signaling pathway that is activated in A549 cells that have a prolonged or abnormal mitotic phase of the cell cycle [Cancer Res. 60 (2000) 5171]. In contrast, in murine macrophage RAW 264.7 cells, micromolar concentrations of Taxol but not other microtubule-interacting agents induced serine phosphorylation of p66shc that correlated with the phosphorylation of Raf-1 and extracellular signal-regulated kinase (ERK1/2), within 15-30 min after Taxol treatment. This event also was induced by lipopolysaccharide (LPS). The MEK-inhibitor, U0126, that specifically inhibits the activation of ERK also blocked the phosphorylation of p66shc and Raf-1, suggesting that these processes were MEK-dependent, quite different from that which was observed in A549 cells. Taxol also induced phosphorylation of p38 and JNK MAP kinases within 8-15 min after drug treatment. It is known that Taxol, but not other microtubule-interacting agents, induces the production of cytokines, such as tumor necrosis factor alpha (TNF-alpha) in mouse macrophages. The time course of Taxol-induced TNF-alpha expression coincides with that of Taxol-induced p66shc phosphorylation, and U0126 inhibits significantly Taxol-induced TNF-alpha expression in RAW 264.7 cells. Our data indicate that the Taxol-induced serine phosphorylation of p66shc in RAW 264.7 cells is microtubule-independent and may be related to increased TNF-alpha expression after Taxol and LPS treatment. It is concluded that the mechanisms involved in Taxol-induced p66shc phosphorylation are distinct in A549 and RAW 264.7 cells.     

野生型p53基因对内皮细胞细胞间粘附分子-1表达的影响

细胞间粘附分子－１（ＩＣＡＭ－１）是介导白细胞与内皮细胞粘附的重要粘附分子．为研究野生型ｐ５３基因对内皮细胞ＩＣＡＭ－１表达的影响,分别采用流式细胞术和ＲＴ－ＰＣＲ／ＨＰＬＣ方法测定ＩＣＡＭ－１蛋白及ｍＲＮＡ水平．静息状态的内皮细胞表面结构性地表达有少量的ＩＣＡＭ－１,在肿瘤坏死因子α（ＴＮＦα,１０～１０００Ｕ／ｍｌ）诱导下,其表达呈剂量依赖性增加．将ｐ５３基因导入内皮细胞,则显著抑制ＴＮＦα诱导的内皮细胞表面ＩＣＡＭ－１的表达．ｐ５３基因的导入对静息状态内皮细胞表面结构性表达的ＩＣＡＭ－１影响较小．ｐ５３基因主要通过降低ＩＣＡＭ－１的ｍＲＮＡ水平而抑制内皮细胞表面ＩＣＡＭ－１的表达,但对蛋白的抑制程度小于对ｍＲＮＡ的抑制程度．提示：ｐ５３基因对内皮细胞ＩＣＡＭ－１表达的影响除转录水平控制外,还存在转录后水平的调控     

Alpha-tocopherol protects against monocyte Mac-1 (CD11b/CD18) expression and Mac-1-dependent adhesion to endothelial cells induced by oxidized low-density lipoprotein

Alpha-tocopherol supplementation is reported to protect against cardiovascular disease and to influence cells involved in atherogenesis, such as monocytes. Interactions between monocytes and vascular endothelial cells occur early in atherogenesis, and adhesion is mediated by integrins. We evaluated the effects of alpha-tocopherol on expression of Mac-1 (CD11b/CD18) by monocytes after stimulation with oxidized low-density lipoprotein (LDL), which is implicated as a potent chemotactic agent in atherogenesis. Incubation of whole blood with oxidized LDL (100 microg/ml) increased Mac-1 expression on monocytes, and preincubation with alpha-tocopherol reduced this upregulation in a concentration dependent manner. In another experiment, whole blood was obtained from healthy adult volunteers after 10 days of alpha-tocopherol administration (600 mg/day) and was incubated with oxidized LDL (100 microg/ml). There was a decrease in the upregulation of Mac-1 compared with that measured before administration. Adherence of oxidized LDL-stimulated monocytes to human umbilical vein endothelial cells was reduced by pretreatment with alpha-tocopherol, and was also inhibited by an anti-CD18 monoclonal antibody. Experiments with protein kinase C inhibitors suggested that reduction of Mac-1 upregulation by alpha-tocopherol was secondary to a decrease of protein kinase C activity. In conclusion, alpha-tocopherol suppressed the upregulation of Mac-1 expression on monocytes by oxidized LDL.