Enhanced Proteolytic Processing of Recombinant Human Coagulation Factor VIII B-Domain Variants by Recombinant Furins.

Recombinant human factor VIII (rFVIII) is used in replacement therapy for hemophilia A. Current research efforts are focused on bioengineering rFVIII molecules to improve its secretion efficiency and stability, limiting factors for its efficient production. However, high expression yield in mammalian cells of these rFVIII variants is generally associated with limited proteolytic processing. Non-processed single-chain polypeptides constitute non-natural FVIII molecule configurations with unpredictable toxicity and/or antigenicity. Our main objective was to demonstrate the feasibility of promoting full-proteolytic processing of an rFVIII variant retaining a portion of the B-domain, converting it into the smallest natural activatable form of rFVIII, while keeping its main advantage, i.e., improved secretion efficiency. We generated and employed a CHO-DG44 cell clone producing an rFVIII variant retaining a portion of the B-domain and the FVIII native cleavage site between Arg(1648) and Glu(1649). By bioengineering CHO-DG44 cells to express stably the recombinant human endoproteases PACE, PACE-SOL, PCSK5, PCSK6, or PCKS7, we were able to achieve complete intra- or extracellular proteolytic processing of this rFVIII variant. Additionally, our quantitative data indicated that removal of the B-domain segment by intracellular proteolytic processing does not interfere with this rFVIII variant secretion efficiency. This work also provides the first direct evidence of (1) intracellular cleavage at the Arg(1648) FVIII processing site promoted by wild-type PACE and PCSK7 and (2) proteolytic processing at the Arg(1648) FVIII processing site by PCSK6.

ADAM23 negatively modulates alpha(v)beta(3) integrin activation during metastasis.

The ADAM23 gene is frequently silenced in different types of tumors, and, in breast tumors, silencing is correlated with tumor progression, suggesting that it might be associated with the acquisition of a metastatic phenotype. ADAM23 exerts its function mainly through the disintegrin domain, because its metalloprotease domain is inactive. Analysis of ADAM23 binding to integrins has revealed a specific interaction with alpha(v)beta(3) integrin mediated by the disintegrin domain. Altered expression of alpha(v)beta(3) integrin has been observed in different types of tumors, and expression of this integrin in the activated form has been shown to promote metastasis formation. Here, we investigated the possibility that interaction between ADAM23 and alpha(v)beta(3) integrin might negatively modulate alpha(v)beta(3) activation during metastatic progression. ADAM23 expression was knocked down using short hairpin RNA in the MDA-MB-435 cell line, which has been extensively used as a model for alpha(v)beta(3) integrin activation. Ablation of ADAM23 enhanced alpha(v)beta(3) integrin activation by at least 2- to 4-fold and ADAM23 knockdown cells showed enhanced migration and adhesion to classic alpha(v)beta(3) integrin ligands. Ablation of ADAM23 expression also enhanced pulmonary tumor cell arrest in immunodeficient mice. To complement our findings with clinical evidence, we showed that silencing of ADAM23 gene by DNA promoter hypermethylation in a collection of 94 primary breast tumors was significantly associated with lower distant metastases-free and disease-specific survivals and was an independent prognostic factor for poor disease outcome. Our results strongly support a functional role of ADAM23 during metastatic progression by negatively modulating alpha(v)beta(3) integrin activation.

Prion protein ablation increases cellular aggregation and embolization contributing to mechanisms of metastasis.

Cellular Prion Protein (PrP(C)) is a cell surface protein highly expressed in the nervous system, and to a lesser extent in other tissues. PrP(C) binds to the extracellular matrix laminin and vitronectin, to mediate cell adhesion and differentiation. Herein, we investigate how PrP(C) expression modulates the aggressiveness of transformed cells. Mesenchymal embryonic cells (MEC) from wild-type (Prnp(+/+)) and PrP(C)-null (Prnp(0/0)) mice were immortalized and transformed by co-expression of ras and myc. These cells presented similar growth rates and tumor formation in vivo. When injected in the tail vein, Prnp(0/0)ras/myc cells exhibited increased lung colonization compared with Prnp(+/+)ras/myc cells. Additionally, Prnp(0/0)ras/myc cells form more aggregates with blood components than Prnp(+/+)ras/myc cells, facilitating the arrest of Prnp(0/0)ras/myc cells in the lung vasculature. Integrin alpha(v)beta(3) is more expressed and activated in MEC and in transformed Prnp(0/0) cells than in the respective Prnp(+/+) cells. The blocking of integrin alpha(v)beta(3) by RGD peptide reduces lung colonization in transformed Prnp(0/0) cells to similar levels of those presented by transformed Prnp(+/+) cells. Our data indicate that PrP(C) negatively modulates the expression and activation of integrin alpha(v)beta(3) resulting in a more aggressive phenotype. These results indicate that PrP(C) may have main implications in modulating metastasis formation.

Cellular prion protein expression in astrocytes modulates neuronal survival and differentiation.

The functions of cellular prion protein (PrP(C)) are under intense debate and PrP(C) loss of function has been implicated in the pathology of prion diseases. Neuronal PrP(C) engagement with stress-inducible protein-1 and laminin (LN) plays a key role in cell survival and differentiation. The present study evaluated whether PrP(C) expression in astrocytes modulates neuron-glia cross-talk that underlies neuronal survival and differentiation. Astrocytes from wild-type mice promoted a higher level neuritogenesis than astrocytes obtained from PrP(C)-null animals. Remarkably, neuritogenesis was greatly diminished in co-cultures combining PrP(C)-null astrocytes and neurons. LN secreted and deposited at the extracellular matrix by wild-type astrocytes presented a fibrillary pattern and was permissive for neuritogenesis. Conversely, LN coming from PrP(C)-null astrocytes displayed a punctate distribution, and did not support neuronal differentiation. Additionally, secreted soluble factors from PrP(C)-null astrocytes promoted lower levels of neuronal survival than those secreted by wild-type astrocytes. PrP(C) and stress-inducible protein-1 were characterized as soluble molecules secreted by astrocytes which participate in neuronal survival. Taken together, these data indicate that PrP(C) expression in astrocytes is critical for sustaining cell-to-cell interactions, the organization of the extracellular matrix, and the secretion of soluble factors, all of which are essential events for neuronal differentiation and survival.

STI1 promotes glioma proliferation through MAPK and PI3K pathways.

Gliomas are tumors derived from glia or their precursors within the central nervous system. Clinically, gliomas are divided into four grades and the glioblastoma multiforme (GBM), also referred as grade IV astrocytoma, is the most aggressive and the most common glioma in humans. The prognosis for patients with GBM remains dismal, with a median survival of 9-12 months. Despite their striking heterogeneity, common alterations in specific cellular signal transduction pathways occur within most GBMs. Previous work from our group identified the co-chaperone stress-inducible protein 1 (STI1) as a cell surface ligand for cellular prion (PrP(C)), which leads to the activation of several signal transduction pathways, some of which modulate cell survival. In the present work, we used thymidine incorporation assays to investigate the effect of STI1 upon proliferation of the human glioblastoma-derived cell line A172. Here we report that STI1 is secreted by and induces proliferation in tumor cells, an effect that is modulated by the Erk and PI3K pathways, and that, in contrast to glioma cells, STI1 does not induce proliferation of normal glia. In addition, our data suggest the involvement of PrP(C) in STI1-induced proliferation of A172 cells. These results provide initial evidence of a new functional role for STI1 on the physiology of human gliomas, and may lead to the identification of new therapeutic targets in these tumors.

Interaction of cellular prion and stress-inducible protein 1 promotes neuritogenesis and neuroprotection by distinct signaling pathways.

Understanding the physiological function of the cellular prion (PrPc) depends on the investigation of PrPc-interacting proteins. Stress-inducible protein 1 (STI1) is a specific PrPc ligand that promotes neuroprotection of retinal neurons through cAMP-dependent protein kinase A (PKA). Here, we examined the signaling pathways and functional consequences of the PrPc interaction with STI1 in hippocampal neurons. Both PrPc and STI1 are abundantly expressed and highly colocalized in the hippocampus in situ, indicating that they can interact in vivo. Recombinant STI1 (His6-STI1) added to hippocampal cultures interacts with PrPc at the neuronal surface and elicits neuritogenesis in wild-type neurons but not in PrPc-null cells. This effect was abolished by antibodies against either PrPc or STI1 and was dependent on the STI1 domain that binds PrPc. Binding of these proteins induced the phosphorylation/activation of the mitogen-activated protein kinase, which was essential for STI1-promoted neuritogenesis. His6-STI1, but not its counterpart lacking the PrPc binding site, prevented cell death via PKA activation. These results demonstrate that two parallel effects of the PrPc-STI1 interaction, neuritogenesis and neuroprotection, are mediated by distinct signaling pathways.

Avaliação da participação de prion celular em fenômenos associados a tumorigênese e metástase experimentais / Evaluation of prion protein participation in experimental tumorigenesis and metastasis related processes

O prion celular (PrPc) exerce papel fundamental nas encefalopatias espongiformes. No entanto, suas funções fisiológicas ainda não foram totalmente compreendidas. Nosso grupo mostrou, previamente, que PrPc se liga a laminina (LN}, uma proteína da matriz extracelular, e que esta interação medeia adesão neuronal, formação e manutenção de neuritos (GRANER et ai. 2000a). Sabendo-se que a matriz extracelular e em particular, LN, pode influenciar os processos tumorais e metastáticos, decidimos investigar a possível participação de PrPc nestes eventos. Para tanto, células embrionárias mesenquimais (CEM) de camundongos tipo selvagem (Prnp+1 +) ou deficientes do gene de PrPc (Prnp010) foram transfectadas com um vetor que codifica a expressão dos oncogenes rase myc. Foram selecionados três clones transformados a partir de cada grupo de células: CEM Prnp+J+ raslmyc e Prnp010 ras/myc, que possuíam expressão similar de ambas as oncoproteínas. Os clones Prnp+l+ ras/myc e Prnp010 ras/myc apresentaram tempos de duplicação semelhantes in vitro, assim como exibiram doses tumorais mínimas semelhantes quando injetados por via subcutânea no dorso de camundongos tipo selvagem. Os clones Prnp+l+ ras/myc e Prnp010 ras/myc foram testados em modelo de metástase experimental (colonização pulmonar), injetando-se células na veia lateral da cauda de camundongos tipo selvagem. Após 21 dias, o número de colônias pulmonares presentes na superfície pleural foram quantificados. Os clones Prnp010 raslmyc produziram um número de colônias pulmonares estatisticamente superior ao dos clones Prnp +I+ raslmyc. Ensaios de esteróides multicelulares mostraram que os clones Prnp010 ras/myc apresentam uma maior adesão célula-célula do que aqueles Prnp+l+ raslmyc. Entretanto, nenhuma alteração na expressão de ICAM-1 (CD54) ou caderinas foi encontrada entre eles. Por outro lado, experimentos de imunofluorescência, mostraram que células dos clones Prnp010 raslmyc assim como sua parenta! CEM Prnp010 apresentam a integrina avf33 funcionalmente mais ativada do que seus equivalentes Prnp+1 +. Estes dados sugerem que a ausência de PrPc leva a uma regulação positiva na atividade de avf33 integrina. Por sua vez, sabe-se que o aumento da atividade desta integrina pode ser responsável pela maior adesão das células tumorais a vitronectina e componentes sanguíneos como as plaquetas, com conseqüente aumento na formação de êmbolos. Desta forma, variações na expressão de PrPc podem contribuir na sinalização celular, em particular naquela envolvida com a ativação da integrina avf33, que por sua vez participa na formação de êmbolos e conseqüente aumento na colonização de células tumorais (AU)

The cellular prion (PrPc) has a criticai role in spongiform encephalopathies. However, its physiological functions are not completely understood. We have reviously shown that PrPc binds the extracellular matrix protein, laminin (LN), and this interaction affects neuronal cell adhesion, neurite formation and maintenance (GRANER et ai. 2000a). The extracellular matrix and in particular, LN, can affect the tumoral and metastatic processes, therefore we decided to investigate a possible participation of PrPc in such processes. Thus, mesenchimal embryonic cells from wild-type (Prnp+1 +) or PrPc gene ablated mice (Prnp010) were transfected with a vector containing the expression sequences of ras and myc oncogenes. We isolated three transformed clones from each genotype (Prnp+l+ and Prnp010) with similar expression of both oncoproteins. Prnp+r+raslmyc or Prnp010ras/myc clones presented the same doubling time in vitro as well as similar minimum tumoral doses when injected subcutaneously in the dorsal region of wild type mice. In lung colonization assays, an experimental model of metastasis, Prnp+1 +ras/myc or Prnp010raslmyc clones were injected in the lateral tail vein of wild-type mice and the number of pulmonary colonies were counted after 21 days. Prnp010ras/myc clones exhibited a significant higher number of lung colonies than those presented by Prnp+1 +raslmyc ones. Multicellular spheroids from Prnp010ras/myc clones showed a higher cell-cell interaction and a lower cellular scattering when compared with the Prnp +I+ raslmyc ones.However, alterations in the expression of ICAM-1 (CD54) and cadherins were not found between the Prnp010ras/myc and Prnp+1 +raslmyc clones. On the other hand, immunofluoresce assays revealed that Prnp010 ras/myc clones and the mesenchimal embryonic Prnp010 parenta! cells presented a higher avf33 activity when compared to the Prnp +I+ ones. These data suggest that the PrPc depletion leads to a positive regulation of avJ33 integrin activity. lt is well known that tumoral cell adhesion mediated by avJ33, can mediate tumoral cell adhesion to vitronectin and other blood components, such platelets, increasing embolization capacity. Thus, alterations on PrPc expression can regulate cellular signaling, particularly, that involved in av~3 integrin activation which participates on cellular embolization and consequently elevation on pulmonar colonization (AU)