Jorge Martínez

Cloning and expression of the panallergen profilin and the major allergen (Ole e 1) from olive tree pollen

BACKGROUND Olive tree (Olea europaea) pollen allergy is one of the main causes of allergy in Mediterranean countries and some areas of North America. OBJECTIVE To clone olive allergens and to characterize immunologically the purified recombinant allergens. METHODS Full-length complementary deoxyribonucleic acid (cDNA) strands encoding olive allergens (Ole e 1) were cloned by polymerase chain reaction amplification and sequenced. Recombinant proteins were produced in Escherichia coli by the use of two different expression systems. Immunoreactivity of the recombinant proteins was tested by ELISA and Western blot with serum from patients with allergy to olive. RESULTS Significant sequence polymorphism was found in both allergens. The panallergen profilin was expressed as a nonfusion protein and was purified to homogeneity after a single step of affinity chromatography with a poly-L-proline Sepharose column. One cDNA encoding an Ole e 1 isoform was expressed as a fusion protein consisting of the glutathione S-transferase of Schistosoma japonicum and Ole e 1. The fusion protein was purified to homogeneity by gel filtration chromatography and affinity chromatography with a glutathione-Sepharose column, and digested with thrombin. Both recombinant allergens shared B cell epitopes with the corresponding natural allergens. CONCLUSION IgE-reactive Ole e 1 and olive profilin expressed in bacteria were purified after simple chromatographic procedures and may be useful for diagnostic purposes.

Transforming growth factor-β1 modulates matrix metalloproteinase-9 production through the Ras/MAPK signaling pathway in transformed keratinocytes

Mouse transformed keratinocytes cultured in the presence of transforming growth factor-beta1 (TGF-beta1) acquire a set of morphological and functional properties giving rise to a more motile phenotype that expresses mesenchymal markers. In this work, we present evidence showing that TGF-beta1 stimulates cellular production of MMP-9 (Gelatinase B), a metalloproteinase that plays an important role in tumoral invasion. Our results demonstrate that TGF-beta1stimulates MMP-9 production and MMP-9 promoter activity in a process that depends of the activation of the Ras-ERK1,2 MAP kinase pathway. The latter was demonstrated by cellular transfection of TGF-beta1-sensitive cells with a RasN17 mutant gene, using PD 098059, a MEK 1,2 inhibitor, and treating cells with anti-sense oligodeoxinucleotides. The enhanced MMP-9 production proved to be an important factor in the acquisition of migratory and invasive properties as shown by the use of a specific inhibitor of MMP-9 (GM6001) that inhibits the TGF-beta1-stimulated invasive and migratory properties of these transformed keratinocytes.

Sequencing and high level expression in Escherichia coli of the tropomyosin allergen (Der p 10) from Dermatophagoides pteronyssinus

The cDNA encoding an allergen from the dust mite Dermatophagoides pteronyssinus has been cloned and sequenced. The allergen (Der p 10) is a tropomyosin that shared more than 65% identical residues with other invertebrate tropomyosins. The final recovery of recombinant Der p 10 from the culture media after a single purification step was as much as 26 mg/l. The recombinant allergen is reactive to shrimp antitropomyosin IgG antibodies and has a 5.6% frequency of IgE reactivity in sera from mite-allergic patients.

ERK 1,2 and p38 pathways are involved in the proliferative stimuli mediated by urokinase in osteoblastic SaOS-2 cell line.

Bone metastases from prostate origin generate an osteoblastic reaction that is expressed in vitro by increased osteoblast proliferation. The urokinase‐like plasminogen activator (u‐PA) present in the media conditioned by tumoral prostatic cells acting as a ligand of the cellular membrane receptor (u‐PAR), has been identified as the specific factor that modulates this proliferative reaction. The present study represents an effort to unravel the intracellular pathway by which u‐PA activates osteoblastic proliferation and to evaluate the role of cellular receptor u‐PAR in this proliferative phenomenon. Our results show that in vitro u‐PA stimulates proliferation of SaOS‐2 osteoblastic cells by activating the MAP kinase route of ERK 1 and 2 and the p38 pathway. These results are in accordance with the inhibition of intermediate activation and cell proliferation by PD 098059 and SB 203580, specific inhibitors of MEK and p38, respectively. We also show that SaOS‐2 cells increase their proliferative response when cells are plated onto vitronectin, the second natural ligand of u‐PAR, and that culturing SaOS‐2 cells in the presence of u‐PA represents a stimuli for u‐PAR expression. On the basis of these results we propose that osteoblastic cells respond to the prostate‐derived u‐PA stimuli in a very efficient manner that includes the utilization of two different signaling routes and the stimulation of the expression of the u‐PA receptor.

Characterization of recombinant Mercurialis annua major allergen Mer a 1 (profilin)

BACKGROUND Two major allergens (Mer a 1A and Mer a 1B), tentatively identified as profilin, have been described in the euphorbiacea, Mercurialis annua. OBJECTIVES We sought to clone and characterize these major allergens from M. annua pollen and to obtain the immunologically active and soluble recombinant allergen, which could then be used for diagnostic procedures and therapy. METHODS Isolation of cDNA clones was performed by polymerase chain reaction amplification with degenerate primers. Expression in Escherichia coli BL21 (DE3) was carried out with a vector based in the T7 expression system, and the recombinant allergen was isolated by affinity chromatography on poly-(L-proline)-Sepharose. Electrophoretic (sodium dodecylsulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and 2-dimensional polyacrylamide gel electrophoresis) and immunochemical methods (Western blot and ELISA) were used for the characterization of the recombinant allergen. RESULTS Two cDNA inserts coding for M. annua pollen profilin (Mer a 1) were cloned and sequenced. Full-length Mer a 1 cDNA was expressed in E. coli as nonfusion protein. The final yield of recombinant Mer a 1 from the culture media after a single purification step on poly-(L-proline)-Sepharose was as much as 5 mg per liter. The reactivity of recombinant Mer a 1 with IgE antibodies present in sera from patients allergic to M. annua, Olea europaea, and Ricinus communis pollens was comparable to that of the natural counterparts, but latex profilin had no cross-reactivity with M. annua profilin. Recombinant Mer a 1 was shown to share B-epitopes with sunflower profilin. CONCLUSION This approach is suitable for the production of defined and purified recombinant allergens, which could allow more detailed immunologic characterization of these proteins and the development of much more accurate diagnostic measures and specific anti-allergic treatments.

Genistein and Curcumin Block TGF-β1-Induced u-PA Expression and Migratory and Invasive Phenotype in Mouse Epidermal Keratinocytes

Transforming growth factor-β1 (TGF-β1) stimulates migration/invasion of mouse transformed keratinocytes and increases urokinase (u-PA) expression/secretion. In this report, we analyzed the biological behavior of two naturally occurring inhibitors of protein tyrosine kinases, genistein and curcumin, that could abrogate the enhancement of u-PA levels induced by TGF-β1 in transformed keratinocytes. Our results showed that genistein and curcumin blocked this response in a dose-dependent manner and also inhibited the TGF-β1-induced synthesis of fibronectin, an early responsive gene to the growth factor. Both compounds also reduced TGF-β1-stimulated cell migration and invasiveness. These results suggest that a tyrosine kinase-signaling pathway should be involved in TGF-β1-mediated increased malignancy of transformed keratinocytes and that genistein and curcumin could play an important role in inhibiting tumor progression.

NOX4-dependent ROS production by stromal mammary cells modulates epithelial MCF-7 cell migration

Background:The influence of the stromal microenvironment on the progression of epithelial cancers has been demonstrated. Unravelling the mechanisms by which stromal cells affect epithelial behaviour will contribute in understanding cellular malignancy. It has been proposed that redox environment has a role in the acquisition of malignancy. In this work, we studied the influence of epithelial cells on the stromal redox status and the consequence of this phenomenon on MCF-7 cell motility.Methods:We analysed in a co-culture system, the effect of RMF-EG mammary stromal cells on the migratory capacity of MCF-7 cell line. To test whether the NOX-dependent stromal redox environment influences the epithelial migratory behaviour, we knocked down the expression of NOX4 using siRNA strategy. The effect of TGF-β1 on NOX4 expression and activity was analysed by qPCR, and intracellular ROS production was measured by a fluorescent method.Results:Migration of MCF-7 breast epithelial cells was stimulated when co-cultured with RMF-EG cells. This effect depends on stromal NOX4 expression that, in turn, is enhanced by epithelial soluble factors. Pre-treatment of stromal cells with TGF-β1 enhanced this migratory stimulus by elevating NOX4 expression and intracellular ROS production. TGF-β1 seems to be a major component of the epithelial soluble factors that stimulate NOX4 expression.Conclusions:Our results have identified that an increased stromal oxidative status, mainly provided by an elevated NOX4 expression, is a permissive element in the acquisition of epithelial migratory properties. The capacity of stromal cells to modify their intracellular ROS production, and accordingly, to increase epithelial motility, seems to depend on epithelial soluble factors among which TGF-β1 have a decisive role.

Urokinase expression and binding activity associated with the transforming growth factor β1-induced migratory and invasive phenotype of mouse epidermal keratinocytes

Transforming growth factor β1(TGF‐β1) is a stimulator of malignant progression in mouse skin carcinogenesis. TGF‐β1 exerts a differential effect on cultured nontumorigenic (MCA3D cell line) and transformed (PDV cell line) keratinocytes. Whereas MCA3D cells are growth arrested and committed to die in the presence of the factor, it induces a reversible epithelial‐fibroblastic conversion in PDV cells. This conversion is associated in vivo with a squamous‐spindle cell carcinoma transition. Here we have investigated the role of urokinase (uPA) during malignant progression of transformed epidermal keratinocytes. We show that the levels of uPA expression/secretion, and the uPA binding activity to the cell surface, correlate with the invasive and malignant potentials of mouse epidermal cell lines. TGF‐β1 enhanced uPA production, the number of uPA cell surface binding sites, and the expression of the plasminogen activator inhibitor PAI‐1, in transformed PDV cells, but had no major effect on nontumorigenic MCA3D keratinocytes. Increased uPA production depended on the presence of the factor in the culture medium and occurred concomitantly to the stimulation of the migratory and invasive abilities of PDV cells. Synthetic peptides containing the amino terminal sequence of the mature mouse uPA inhibited the binding of uPA to the cell surface and decreased TGF‐β1‐induced cell motility and invasiveness. These results demonstrate that the uPA system mediates at least part of the migratory and invasive phenotype induced by TGF‐β1 in transformed keratinocytes, and suggest a role for uPA on the changes that lead to the appearance of spindle carcinomas. J. Cell. Biochem. 74:61–73, 1999.

RAC1 activity and intracellular ROS modulate the migratory potential of MCF-7 cells through a NADPH oxidase and NFκB-dependent mechanism

In the present study, we demonstrated that changes in Rac1 activity associated with the production of intracellular ROS modulate the migratory properties in MCF-7 and T47D human mammary cell lines. We also described that the NFkappaB pathway exerts a downstream control on the expression of the ROS-dependent cellular migratory potential. These results emphasize the importance of redox balance in the acquisition of malignancy and support previous data sustaining that an oxidative environment predisposes cells to activate signal-transduction pathways actively involved in cellular oncogenesis. Our data also provides evidence that NADPH oxidase could constitute the main source of intracellular ROS in response to changes in Rac1 activity. We suggest that Rac1 plays a role in cellular migration not only limited to its known function in reorganization of the actin cytoskeleton, but also as part of the intracellular machinery that controls the redox balance.

PROSTATE-DERIVED SOLUBLE FACTORS BLOCK OSTEOBLAST DIFFERENTIATION IN CULTURE

Bone metastasis is a common event and a major cause of morbidity in prostate cancer patients. After colonization of bone, prostate cells induce an osteoblastic reaction which is not associated with marrow fibrosis (i.e., osteoblast but not fibroblast proliferation). In the present study we test the hypothesis that the tumoral prostatic cell line (PC‐3) secretes factors that block the osteoblast differentiation process, resulting in an increase of the relative size of the proliferative cell pool. Our results, using fetal rat calvaria cells in culture, show that conditioned medium from PC‐3 cells (PC‐3 CM) stimulates osteoblast proliferation and inhibits both alkaline phosphatase (AP) activity (an early differentiation marker) and the mineralization process, measured as calcium accumulation (late differentiation marker). The inhibition of the expression of AP and mineralization depends on the presence of PC‐3 CM during the proliferative phase of culture and suggests that both processes occur in a nonsimultaneous fashion. The inhibitory effect of PC‐3 CM was not reverted by dexamethasone, which would indicate that prostatic‐derived factors and the glucocorticoid do not share a common site of action. Measurement of the proliferative capacity of subcultures from control and treated cells demonstrates that PC‐3 CM treatment induces the maintenance of the proliferative potential that characterizes undifferentiated precursor cells.