Functional Analysis of a Breast Cancer-Associated Mutation in the Intracellular Domain of the Metalloprotease ADAM12

A recently identified breast cancer-associated mutation in the metalloprotease ADAM12 alters a potential dileucine trafficking signal, which could affect protein processing and cellular localization. ADAM12 belongs to the group of A Disintegrin And Metalloproteases (ADAMs), which are typically membrane-associated proteins involved in ectodomain shedding, cell-adhesion, and signaling. ADAM12 as well as several members of the ADAM family are over-expressed in various cancers, correlating with disease stage. Three breast cancer-associated somatic mutations were previously identified in ADAM12, and two of these, one in the metalloprotease domain and another in the disintegrin domain, were investigated and found to result in protein misfolding, retention in the secretory pathway, and failure of zymogen maturation. The third mutation, p.L792F in the ADAM12 cytoplasmic tail, was not investigated, but is potentially significant given its location within a di-leucine motif, which is recognized as a potential cellular trafficking signal. The present study was motivated both by the potential relevance of this documented mutation to cancer, as well as for determining the role of the di-leucine motif in ADAM12 trafficking. Expression of ADAM12 p.L792F in mammalian cells demonstrated quantitatively similar expression levels and zymogen maturation as wild-type (WT) ADAM12, as well as comparable cellular localizations. A cell surface biotinylation assay demonstrated that cell surface levels of ADAM12 WT and ADAM12 p.L792F were similar and that internalization of the mutant occurred at the same rate and extent as for ADAM12 WT. Moreover, functional analysis revealed no differences in cell proliferation or ectodomain shedding of epidermal growth factor (EGF), a known ADAM12 substrate between WT and mutant ADAM12. These data suggest that the ADAM12 p.L792F mutation is unlikely to be a driver (cancer causing)-mutation in breast cancer

PKCα and PKCδ Regulate ADAM17-Mediated Ectodomain Shedding of Heparin Binding-EGF through Separate Pathways

Epidermal growth factor receptor (EGFR) signalling is initiated by the release of EGFR-ligands from membrane-anchored precursors, a process termed ectodomain shedding. This proteolytic event, mainly executed by A Disintegrin And Metalloproteases (ADAMs), is regulated by a number of signal transduction pathways, most notably those involving protein kinase C (PKC). However, the molecular mechanisms of PKC-dependent ectodomain shedding of EGFR-ligands, including the involvement of specific PKC isoforms and possible functional redundancy, are poorly understood. To address this issue, we employed a cell-based system of PMA-induced PKC activation coupled with shedding of heparin binding (HB)-EGF. In agreement with previous studies, we demonstrated that PMA triggers a rapid ADAM17-mediated release of HB-EGF. However, PMA-treatment also results in a protease-independent loss of cell surface HB-EGF. We identified PKCα as the key participant in the activation of ADAM17 and suggest that it acts in parallel with a pathway linking PKCδ and ERK activity. While PKCα specifically regulated PMA-induced shedding, PKCδ and ERK influenced both constitutive and inducible shedding by apparently affecting the level of HB-EGF on the cell surface. Together, these findings indicate the existence of multiple modes of regulation controlling EGFR-ligand availability and subsequent EGFR signal transduction

Reversal of type 2 diabetes: normalisation of beta cell function in association with decreased pancreas and liver triacylglycerol

The presence of Fc-receptor-blocking factors in the sera of normal and insulin-dependent diabetic pregnant women was investigated by means of an antibody-dependent cell-mediated cytotoxicity assay. Sera from normal pregnant women induced a significant depression of antibody dependent cell-mediated cytotoxicity when compared with sera from normal and diabetic non-pregnant women (p less than 0.0001; p less than 0.002, respectively). The effect of sera from diabetic pregnant women, however, was not different from that observed with sera from normal and diabetic non-pregnant women. Thus, we confirm the presence of Fc-receptor-blocking factors in the sera of normal pregnant women. The higher cytotoxicity levels measured in the presence of sera from pregnant women with insulin-dependent diabetes suggests that the titres of such factors are reduced in this conditio

Cirurgia gastrointestinal no tratamento da diabete tipo 2 Gastrointestinal surgery for the treatment of type 2 diabetes

RACIONAL: Evidências científicas demonstram o controle metabólico da diabete tipo 2 obtido com diversas intervenções sobre o sistema gastrointestinal, principalmente as operações bariátricas. OBJETIVOS: Revisar os dados da literatura referentes aos efeitos da cirurgia gastrointestinal na diabete tipo 2, especialmente os relacionados ao controle metabólico e sua fisiopatologia. MÉTODOS: Foi realizada pesquisa no Medline em páginas da internet procurando referências de artigos de maior relevância e estudos apresentados e publicados nos anais da conferência de Roma sobre os efeitos da cirurgia gastrointestinal no tratamento da diabete tipo 2 em 2007. Revisão da literatura - Entre as operações bariátricas, as derivações biliopancreáticas apresentam as mais altas taxas de controle da diabete tipo 2, seguidas pelo bypass gástrico e pela banda gástrica. Esse controle está relacionado à perda de peso e redução da ingestão de alimentos. As derivações biliopancreáticas e o bypass gástrico apresentam ainda efeito importante promovido por modificações hormonais. Os hormônios mais significativos são: o GLP-1, o GIP, o PYY, a grelina, a leptina, o IGF-1 e a adiponectina. Além dos efeitos sobre o controle do apetite no hipotálamo, os hormônios apresentam ação sobre as células &#946;, promovem a secreção de insulina e diminuem sua resistência periférica. Duas hipóteses foram formuladas para explicar as modificações desses hormônios: a do intestino anterior, em que a exclusão do duodeno e do jejuno proximal previne a secreção de algum agente ainda não identificado que promove a resistência periférica à insulina; e a do intestino posterior, em que a derivação intestinal promove a passagem rápida do quimo até o intestino distal e induz à secreção precoce dos hormônios que promovem o controle da diabete. Além das operações bariátricas tradicionais, novos procedimentos promissores foram desenvolvidos nos últimos anos. Entre eles estão o bypass duodeno-jejunal, a interposição ileal e as ressecções intestinais associadas à gastrectomia vertical. Esses novos procedimentos ainda são considerados experimentais. CONCLUSÃO: Os procedimentos cirúrgicos sobre o trato gastrointestinal, principalmente as cirurgias bariátricas, proporcionam controle metabólico da diabete tipo 2. Entre as cirurgias bariátricas os melhores resultados são obtidos com as derivações biliopancreáticas seguidas pelo bypass gástrico e pela banda gástrica. Novos procedimentos cirúrgicos, ainda considerados experimentais, estão demonstrando resultados preliminares favoráveis ao controle metabólico da diabete tipo 2.<br>BACKGROUND: The ability of gastrointestinal surgical interventions, mainly bariatric surgery, to promote the control of type 2 diabetes, has already been well documented. AIM: To review the literature related to the effects of gastrointestinal surgery regarding type 2 diabetes, especially in relation to metabolic control and its physiopathology. METHODS: Literature was reviewed on Medline, pages on the internet, references from relevant articles and studies presented and published on the annals of the International Conference on Gastrointestinal Surgery to Treat Type 2 Diabetes, which occurred in Rome in 2007. CONCLUSIONS: Among all bariatric surgeries, biliopancreatic diversion, presented the best control rates for type 2 diabetes followed by gastric bypass and gastric banding. This control is related to weight loss and reduction on food intake. Biliopancreatic diversion and gastric bypass also presented important modifications in gut hormones. The most significant ones being: GLP-1, GIP, PYY, ghrelin, leptin, IGF-1, adiponectin. These hormones promote loss of appetite, promote actions over ß cells, increase the secretion of insulin, and increase insulin sensitivity. Two theories have been formulated to explain the changes observed on these hormones: the foregut theory, where the bypass of the duodenum and proximal jejunum avoids the secretion of an unknown factor that induces insulin resistance; and the hindgut theory, where the early presentation of food to the ileum anticipates the production of hormones that control diabetes. Recently, new promising procedures have been developed. Among them are the duodenal-jejunal bypass, ileal interposition, and intestinal resection associated to vertical gastrectomy. These new procedures are still considered experimental