Regulatory mechanisms controlling human E-cadherin gene expression.

In cancer cells, loss of E-cadherin gene expression caused dysfunction of the cell-cell junction system, triggering cancer invasion and metastasis. Therefore, E-cadherin is an important tumor-suppressor gene. To understand how E-cadherin gene expression is regulated in cancer cells, we have used E-cadherin-positive and -negative expressing cells to find out the possible up- or down regulating transcription factors in human E-cadherin regulatory sequences. Functional analysis of human E-cadherin regulatory sequences constructs indicated that AML1, Sp1, and p300 may play important roles in promoting E-cadherin expression. In addition, we found there are four HNF3-binding sites in human E-cadherin regulatory sequences. The exogenous HNF3 can enhance the E-cadherin promoter activity in metastatic breast cancer cells and the metastatic breast cancer cells stably transfected with HNF3 showed re-expression of E-cadherin. The HNF3 stable transfectants changed from mesenchymal-like into epithelial morphology. The transwell assays showed the re-expressed E-cadherin reduced cell motility of metastatic breast cancer cells. These results suggested HNF3 may play important roles in the upregulation of the E-cadherin promoter, with the consequent re-expression of E-cadherin, thus reducing the metastatic potential of breast cancer cells. These findings suggested HNF3 plays important roles in the upregulation of the E-cadherin gene and may be able to reduce the motility of metastatic breast cancer cells.

Translational event mediates differential production of tumor necrosis factor-alpha in hyaluronan-stimulated microglia and macrophages.

Recent evidence has demonstrated that hyaluronan synthase 2 mRNA is up-regulated after brain ischemia. After a cerebral ischemic event, microglia and macrophages are the major inflammatory cells and are activated by hyaluronan (HA). However, it is unclear how these cells compare with regard to HA responsiveness. We show here that peritoneal macrophages and RAW 264.7 macrophages produced more than five- and 10-fold more tumor necrosis factor-alpha (TNF-alpha) than primary microglia and BV-2 microglia, respectively. Antibody blockade study showed that CD44, Toll-like receptor-4 receptor and the receptor for HA-mediated motility were responsible for HA-induced TNF-alpha release. Furthermore, HA induced higher levels of phosphorylated MAPK in RAW 264.7 cells when compared with BV-2 cells. HA-mediated TNF-alpha production required p38 MAPK, extracellular-regulated kinase and c-Jun N-terminal kinase phosphorylation in both cell types. The levels of HA-induced TNF-alpha mRNA expression in BV-2 cells were only twofold lower compared with RAW 264.7 cells, suggesting that a translational event is involved in the differential production of TNF-alpha. Western blot analysis revealed that HA treatment resulted in more rapid phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and more effective dissociation of 4E-BP1 from eukaryotic initiation factor 4E in RAW 264.7 cells than in BV-2 cells. Additionally, HA-induced phosphorylation of 4E-BP1 was dependent on MAPK signaling, indicating that RAW 264.7 cells exhibited higher levels of hyperphosphorylated 4E-BP1 possibly due to the overactivation of MAPK. The results suggest that resident microglia and blood-derived monocytes/macrophages exhibit differential sensitivities in response to extracellular mediators after brain ischemia.