Development of secondary palate requires strict regulation of ECM remodeling: sequential distribution of RECK, MMP-2, MMP-3, and MMP-9.

We have evaluated RECK (reversion-inducing-cysteine-rich protein with Kazal motifs), MMP-2 (matrix metalloproteinase-2), MMP-3, and MMP-9 involvement during palate development in mice by using various techniques. Immunohistochemical features revealed the distribution of RECK, MMP-2, and MMP-3 in the mesenchymal tissue and in the midline epithelial seam at embryonic day 13 (E13), MMPs-2, -3, and -9 being particularly expressed at E14 and E14.5. In contrast, RECK was weakly immunostained at these times. Involvement of MMPs was validated by measuring not only their protein expression, but also their activity (zymograms). In situ hybridization signal (ISH) for RECK transcript was distributed in mesenchymal and epithelial regions within palatal shelves at all periods evaluated. Importantly, the results from ISH analysis were in accord with those obtained by real-time polymerase chain reaction. The expression of RECK was found to be temporally regulated, which suggested possible roles in palatal ontogeny. Taken together, our results clearly show that remodeling of the extracellular matrix is finely modulated during secondary palate development and occurs in a sequential manner.

Downregulation of the RECK-tumor and metastasis suppressor gene in glioma invasiveness.

Invasive behavior is the pathological hallmark of malignant gliomas, being responsible for the failure of surgery, radiation, and chemotherapy. Matrix metalloproteinases (MMPs) are essential for proper ECM remodeling and invasion. The tumor and metastasis suppressor RECK protein regulates at least three members of the MMPs family: MMP-2, MMP-9, and MT1-MMP. In order to mimic the in vivo invasion process, A172 and T98G, respectively, non-invasive and invasive human glioblastoma cell lines, were cultured onto uncoated (control) or type I collagen gel-coated surface, and maintained for up to 7 days to allow establishment of the invasive process. We show that the collagen substrate causes decreased growth rates and morphological alterations correlated with the invasive phenotype. Electronic transmission microscopy of T98G cells revealed membrane invaginations resembling podosomes, which are typically found in cells in the process of crossing tissue boundaries, since they constitute sites of ECM degradation. Real time PCR revealed higher RECK mRNA expression in A172 cells, when compared to T98G cells and, also, in samples obtained from cultures where the invasive process was fully established. Interestingly, the collagen substrate increases RECK expression in A172 cells and the same tendency is displayed by T98G cells. MMPs-2 and -9 displayed higher levels of expression and activity in T98G cells, and their activities are also upregulated by collagen. Therefore, we suggest that: (1) RECK downregulation is critical for the invasiveness process displayed by T98G cells; (2) type 1 collagen could be employed to modulate RECK expression in glioblastoma cell lines. Since a positive correlation between RECK expression and patients survival has been noted in several types of tumors, our results may contribute to elucidate the complex mechanisms of malignant gliomas invasiveness.

Transcriptional control of the RECK metastasis/angiogenesis suppressor gene.

The RECK gene is widely expressed in normal human tissues but is downregulated in tumor cell lines and oncogenically transformed fibroblasts. RECK encodes a membrane-anchored glycoprotein that suppresses tumor invasion and angiogenesis by regulating matrix-metalloproteinases (MMP-2, MMP-9 and MT1-MMP). Understanding of the transcriptional regulation of tumor/metastasis suppressor genes constitutes a potent approach to the molecular basis of malignant transformation. In order to uncover the mechanisms of control of RECK gene expression, the RECK promoter has been cloned and characterized. One of the elements responsible for the Ras-mediated downregulation of mouse RECK gene is the Sp1 site, to which Sp1 and Sp3 factors bind. Other regulatory events, such as DNA methylation of the RECK promoter and histone acetylation/deacetylation have been studied to understand the underlying mechanisms of RECK expression. Understanding of the mechanisms which control RECK gene transcription may lead to the development of new strategies for cancer prevention and treatment.

Gene supressor de tumor RECK: clonagem e caracterização do promotor e regulação de sua expressão / The RECK gene: promoter coning and characterization and its expression regulation

A expressão do gene RECK é ubíqua em tecidos normais e não detectável nas linhagens celulares tumorais testadas e em fibroblastos transformados por diversos oncogenes. Inicialmente isolado como um gene indutor da reversão fenotípica tumoral`setaïnormal em fibroblastos transformados por v-Ki-ras, o gene RECK codifica uma glicoproteína de membrana que suprime a invasão tumoral e metástase através da regulação da metaloprotease de matriz-9. Para entender os mecanismos de inibição da expressão do gene RECK, mediada por oncogenes, isolou-se e caracterizou-se a região 5'- flanqueadora do gene RECK de camundongo (mRECK). Ensaios de atividade promotora utilizando mutantes de deleção da região 5'- flanqueadora e o gene repórter luciferase, revelaram que a sequência de 52 pb, imediatamente ®upstream¼ ao gene, possui uma atividade promotora que é suprimida pelo produto do oncogene Ha-ras (V12)...