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Biomedical subjects

Joaquín Arribas

Publications and source records attributed to Joaquín Arribas.

17 recordsLinked to original sources
PubMed2007

Post-transcriptional up-regulation of ADAM17 upon epidermal growth factor receptor activation and in breast tumors.

ADAM17 is a transmembrane metalloprotease involved in the proteolytic release of the extracellular domain of many cell surface molecules, a process known as ectodomain shedding. Despite its likely participation in tumor progression and its current consideration as a therapeutic target, very little is known about the regulation of the expression of ADAM17. Here we show that long term treatment with epidermal growth factor (EGF) leads to a marked increase in the levels of ADAM17. EGF receptor activation does not affect the levels of the mRNA that encodes for, or the rate of synthesis of, ADAM17 but increases its half-life. The effect of EGF is biologically relevant because it increases the shedding of several substrates of ADAM17, including the desmosomal cadherin Dsg-2. Analysis of protein and mRNA levels in mammary tumor samples shows that in vivo the levels of ADAM17 can also be controlled post-transcriptionally. Finally, we show that both the shed extracellular domains of Dsg-2 and ADAM17 are frequently expressed in tumors, further supporting the participation of the metalloprotease in malignant progression.

ADAM Proteins↗
PubMed2006

Biosynthesis of tumorigenic HER2 C-terminal fragments by alternative initiation of translation.

The overactivation of the HERs, a family of tyrosine kinase receptors, leads to the development of cancer. Although the canonical view contemplates HER receptors restricted to the secretory and endocytic pathways, full-length HER1, HER2 and HER3 have been detected in the nucleoplasm. Furthermore, limited proteolysis of HER4 generates nuclear C-terminal fragments (CTFs). Using cells expressing a panel of deletion and point mutants, here we show that HER2 CTFs are generated by alternative initiation of translation from methionines located near the transmembrane domain of the full-length molecule. In vitro and in vivo, HER2 CTFs are found in the cytoplasm and nucleus. Expression of HER2 CTFs to levels similar to those found in human tumors induces the growth of breast cancer xenografts in nude mice. Tumors dependent on CTFs are sensitive to inhibitors of the kinase activity but do not respond to therapeutic antibodies against HER2. Thus, the kinase domain seems necessary for the activity of HER2 CTFs and the presence of these HER2 fragments could account for the resistance to treatment with antibodies.

Amino Acid Sequence↗
PubMed2006

Proteomic identification of desmoglein 2 and activated leukocyte cell adhesion molecule as substrates of ADAM17 and ADAM10 by difference gel electrophoresis.

In contrast with the early view of metalloproteases as simple extracellular matrix-degrading entities, recent findings show that they are highly specific modulators of different signaling pathways involved, positively or negatively, in tumor development. Thus, before considering a given metalloprotease a therapeutic target, it seems advisable to characterize its function by identifying its repertoire of substrates. Here, we present a proteomic approach to identify ADAM17 substrates by difference gel electrophoresis. We found that the shedding of the extracellular domain of the transferrin receptor and those of two cell-cell adhesion molecules, activated leukocyte cell adhesion molecule (ALCAM) and desmoglein 2 (Dsg-2), is increased in cells overexpressing ADAM17. Genetic evidence shows that while ADAM17 is responsible for the shedding of ALCAM, both ADAM17 and ADAM10 can act on Dsg-2. Activation of the epidermal growth factor receptor leads to the upregulation of the shedding of Dsg-2 and to the concomitant upregulation of ADAM17, but not ADAM10, supporting the ability of overexpressed ADAM17 to shed Dsg-2. These results unveil a role of ADAM10 and ADAM17 in the shedding of cell-cell adhesion molecules. Since loss of cell adhesion is an early event in tumor development, these results suggest that ADAM17 is a useful target in anticancer therapy.

ADAM Proteins↗
PubMed2005

Recycling of cell surface pro-transforming growth factor-{alpha} regulates epidermal growth factor receptor activation.

Impairments in signal transduction, leading to the regulation of cell proliferation, differentiation, or migration are frequently the cause of cancer. Since the accurate spatial and temporal location of their components is crucial to ensure the correct regulation of these signaling pathways, it could be anticipated that defects in intracellular trafficking are at the base of certain neoplasias. However, the trafficking of many components of pathways frequently up-regulated in cancers, such as the epidermal growth factor receptor (EGFR) pathway, are largely unknown. Here, we show that the pro-transforming growth factor-alpha (pro-TGF-alpha), a prototypical EGFR ligand, is endocytosed from the cell surface via a clathrin-dependent pathway. Internalized pro-TGF-alpha does not progress to the lysosome; instead, it is delivered to the cell surface via recycling endosomes. To analyze the functional meaning of the internalization of pro-TGF-alpha, we used a deletion construct that is normally transported to the cell surface but is deficiently endocytosed. Due to this impairment, the levels of this construct at the cell surface are dramatically augmented. Consequently, the deletion construct displays a higher EGFR-activating ability, revealing a link between the trafficking of pro-TGF-alpha and the signaling by the EGFR and opening the possibility that defects in the trafficking of the growth factor may contribute to the development of tumors.

Animals↗
PubMed2005

N-terminal cleavage of proTGFalpha occurs at the cell surface by a TACE-independent activity.

ProTGFalpha (transforming growth factor alpha precursor) maturation and conversion into soluble TGFalpha is a complex process that involves three proteolytic steps. One, that occurs co-translationally, eliminates the signal sequence. Another, occurring at the juxta-membrane domain, solubilizes TGFalpha. A third cleavage removes the N-terminal extension of proTGFalpha. This latter step has been poorly studied, mainly because of the rapid kinetics of this cleavage. In the present study, we have designed a strategy to analyse several aspects regarding this N-terminal cleavage. In vivo treatment with the hydroxamate-based metalloprotease inhibitors BB3103 or TAPI-2 (tumour necrosis factor-alpha protease inhibitor 2) reversibly induced accumulation of forms of proTGFalpha that included the N-terminal extension. N-terminal shedding was rapid, and occurred at the cell surface. However, the machinery responsible for the N-terminal cleavage was inactive in other cellular sites, such as the endoplasmic reticulum. Experiments of proTGFalpha expression and maturation in cells deficient in TACE (tumour-necrosis-factor-alpha-converting enzyme) activity indicated that this protease was dispensable for N-terminal processing of proTGFalpha in vivo, but was required for regulated cleavage at the C-terminus. These findings indicate that TACE is not involved in N-terminal processing of proTGFalpha, and suggest differences in the machineries that control the cleavage at both ends of TGFalpha within its precursor.

ADAM Proteins↗
PubMed2004

Combined epidermal growth factor receptor targeting with the tyrosine kinase inhibitor gefitinib (ZD1839) and the monoclonal antibody cetuximab (IMC-C225): superiority over single-agent receptor targeting.

PURPOSE: The epidermal growth factor receptor (EGFR) is abnormally activated in cancer and two classes of anti-EGFR agents, monoclonal antibodies and low-molecular-weight tyrosine kinase inhibitors, have shown antitumor activity in patients. Because these two classes of antireceptor agents target the EGFR at different sites, we decided to explore whether the combined administration of gefitinib, a tyrosine kinase inhibitor, and cetuximab, a monoclonal antibody, had superior antitumor activity than either agent given alone. EXPERIMENTAL DESIGN: We studied the effects of the combination of gefitinib and cetuximab in a panel of human cancer cell lines and in an EGFR-dependent human tumor xenograft model (A431). The effects of these two agents on EGFR signaling, proliferation, apoptosis, and vascularization were evaluated. In addition, we analyzed, with cDNA arrays, changes in gene expression profiles induced by both agents. RESULTS: The combined treatment with gefitinib and cetuximab resulted in a synergistic effect on cell proliferation and in superior inhibition of EGFR-dependent signaling and induction of apoptosis. In a series of in vivo experiments, single-agent gefitinib or cetuximab resulted in transient complete tumor remission only at the highest doses. In contrast, suboptimal doses of gefitinib and cetuximab given together resulted in a complete and permanent regression of large tumors. In the combination-treated tumors, there was a superior inhibition of EGFR, mitogen-activated protein kinase, and Akt phosphorylation, as well as greater inhibition of cell proliferation and vascularization and enhanced apoptosis. Using cDNA arrays, we found 59 genes that were coregulated and 45 genes differentially regulated, including genes related to cell proliferation and differentiation, transcription, DNA synthesis and repair, angiogenesis, signaling molecules, cytoskeleton organization, and tumor invasion and metastasis. CONCLUSIONS: Our findings suggest both shared and complementary mechanisms of action with gefitinib and cetuximab and support combined EGFR targeting as a clinically exploitable strategy.

Animals↗
PubMed2004

Inactivating mutations block the tumor necrosis factor-alpha-converting enzyme in the early secretory pathway.

The ectodomain of different transmembrane molecules is released by a proteolytic event known as shedding. The metalloprotease disintegrin proTNF-alpha converting enzyme (TACE) is responsible for the shedding of various proteins, including protransforming growth factor-alpha (proTGF-alpha) and amyloid-beta precursor protein (APP). Inactive TACE accumulates in the early secretory pathway of cell mutants (M1 and M2) defective in proTGF-alpha and APP shedding. Although previous evidences indicated that the component mutated in M1 and M2 cells is different from TACE, recent results show the existence of two heterozygous point mutations in TACE from M2 cells. Here, we show that wild-type TACE stably transfected in M2 cells is processed, transported to the cell surface, and rescues the proTGF-alpha and APP shedding-defective phenotype. Furthermore, M1 cells also express mutant TACE and transfection with wild-type TACE restores the wild-type phenotype. Therefore, different inactivating mutations result in the accumulation of TACE in the early secretory pathway, emphasizing the importance of the initial steps in the biosynthesis of TACE.

ADAM Proteins↗
PubMed2004

Melanoma metastasis is associated with enhanced expression of the syntenin gene.

Primary cutaneous melanomas and melanoma metastases were examined for differential gene expression using subtractive suppression hybridization in a search for any genes associated with metastasis. Generating a subtracted library of candidate genes up-regulated in melanoma metastases, this library contained 8 different cDNAs, among them a cDNA fragment of the syntenin gene which was overexpressed in further independent melanoma resection specimens on cDNA Southern blots when compared to acquired melanocytic nevi from which melanomas are known to arise. Upon immunohistochemistry, the syntenin protein expression was detected in the cytoplasm of primary cutaneous melanomas and melanoma metastases. Melanoma metastases exhibited higher proportions of tumour cells positive for syntenin immunostaining in comparison to acquired melanocytic nevi or non-metastasizing primary melanomas which was statistically significant. In addition to melanoma, gastric cancer tissues exhibited a higher syntenin mRNA expression on a matched tumour/normal cDNA array than their normal counterparts which was statistically significant. Altogether, we here first describe the detection of the syntenin protein in resection specimens of melanocytic lesions. We conclude that melanoma metastasis is associated with increased expression of the syntenin gene which may participate in signal transduction and cell adhesion via the multifunctional protein-binding properties of its tandem PDZ domains.

Blotting, Northern↗
PubMed2003

The shedding of betaglycan is regulated by pervanadate and mediated by membrane type matrix metalloprotease-1.

Betaglycan is a membrane-anchored proteoglycan that binds transforming growth factor-beta (TGF-beta) via its core protein. A soluble form of betaglycan can be released by proteolytic cleavage (also known as shedding) of the membrane-bound form, yielding soluble betaglycan. The mechanism leading to the generation of soluble betaglycan is poorly understood. Because the membrane and soluble forms of betaglycan have opposite effects regulating the availability of TGF-beta, it is important to characterize the shedding of betaglycan further. Here we present evidence showing that in certain cell types, pervanadate, a general tyrosine phosphatase inhibitor, induces the release of the previously described fragment that encompasses almost the entire extracellular domain of betaglycan (sBG-120). In addition, treatment with pervanadate unveils the existence of a novel 90-kDa fragment (sBG-90). Noticeably, the cleavage that generates sBG-90 is mediated by a tissue inhibitor of metalloprotease-2-sensitive protease. Overexpression of all membrane type matrix metalloproteases (MT-MMPs) described to date indicates that MT1-MMP and MT3-MMP are endowed with ability to generate sBG-90. Furthermore, the patterns of expression of different MT-MMPs in the cell lines used in this study suggest that MT1-MMP is the protease involved in the shedding of sBG-90. Overexpression of MT1-MMP in COS-1 cells, which do not express detectable levels of this metalloprotease, confirms the feasibility of this hypothesis. Unexpectedly, during the course of these experiments, we observed that MT2-MMP decreases the levels of MT1-MMP and betaglycan. Finally, binding competition experiments indicate that, similar to the wild type membrane betaglycan, sBG-90 binds the TGF-beta2 isoform with greater affinity than TGF-beta1, suggesting that once released, it could affect the cellular availability of TGF-beta.

Animals↗
PubMed2003

TACE is required for the activation of the EGFR by TGF-alpha in tumors.

The factors and mechanisms that transduce the intracellular signals sent upon activation of the receptor for the epidermal growth factor (EGFR) and related receptors are reasonably well understood and, in fact, are the targets of anti-tumor drugs. In contrast, less is known about the mechanisms implicated in sending the signals that activate these receptors. Here we show that when its proteolytic shedding is prevented, the transmembrane form of the transforming growth factor-alpha (proTGF-alpha) interacts with, but does not activate, the EGFR. Thus, shedding seems to control not only the availability of the soluble form of the growth factor (TGF-alpha) but also the activity of the transmembrane form. The activity of the protease responsible for the shedding of proTGF-alpha, tumor necrosis factor-alpha converting enzyme (TACE), is required for the activation of the EGFR in vivo and for the development of tumors in nude mice, indicating a crucial role of TACE in tumorigenesis. In agreement with this view, TACE is dramatically overexpressed in the majority of mammary tumors analyzed. Collectively, this evidence points to TACE as a promising target of anti-tumor therapy.

ADAM Proteins↗
PubMed2002

The proteasome is a major autoantigen in multiple sclerosis.

Multiple sclerosis seems to be an autoimmune disease of unknown aetiology affecting the white matter of the CNS. It is generally accepted that the autoimmune response is directed against specific components of myelin. We show here that proteasome, a ubiquitous protease complex composed of 14 different subunits, is a target for autoantibodies (IgG and IgM classes) present in the serum (66%, 73 out of 110) and in the CSF (61%, 16 out of 26) of patients with multiple sclerosis. Using recombinant proteasomal subunits we demonstrate the presence of specific autoantibodies against subunits C2, C8, C9 and C5 in multiple sclerosis patients. Recombinant C2 constructs allow us to localize an immunodominant autoepitope recognized by the sera of multiple sclerosis patients within the C-terminal of C2 proteasomal subunit (251-DEPAEKADEPMEH-263). In addition, two constructs of the recombinant proteasomal subunits C2 and C8 were also used to study the proliferation of peripheral blood mononuclear cells from multiple sclerosis patients; 12 out of 30 (40%) multiple sclerosis patients show positive proliferation with one or both of these recombinant subunits. The high prevalence of anti-proteasome autoantibodies in multiple sclerosis sera compared with sera from patients with other chronic inflammatory conditions: systemic lupus erythematosus (35%, 35 out of 100), primary Sjogren's syndrome (16%, 5 out of 31), vasculitis (0 out of 20), sarcoidosis (7%, 1 out of 13) and Behcet's disease (19%, 4 out of 21) suggest that humoral autoreactivity to proteasome could be a useful test in multiple sclerosis patients that may be of help in the diagnosis and/or progression of this chronic inflammatory disease. Finally, these results suggest that some global abnormality in B and/or T cell function is also involved in the chronic inflammatory response observed in multiple sclerosis patients, as it is frequently observed in other human organ-specific autoimmune diseases.

Adult↗