Serum-free DMEM Medium Research Articles

Transforming Growth Factor β Suppresses Human Telomerase Reverse Transcriptase (hTERT) by Smad3 Interactions with c-Myc and the hTERT Gene

Telomerase underpins stem cell renewal and proliferation and is required for most neoplasia. Recent studies suggest that hormones and growth factors play physiological roles in regulating telomerase activity. In this report we show a rapid repression of the telomerase reverse transcriptase (TERT) gene by transforming growth factor beta (TGF-beta) in normal and neoplastic cells by a mechanism depending on the intracellular signaling protein Smad3. In human breast cancer cells TGF-beta induces rapid entry of Smad3 into the nucleus where it binds to the TERT gene promoter and represses TERT gene transcription. Silencing Smad3 gene expression or genetically deleting the Smad3 gene disrupts TGF-beta repression of TERT gene expression. Expression of the Smad3 antagonist, Smad7, also interrupts TGF-beta-mediated Smad3-induced repression of the TERT gene. Mutational analysis identified the Smad3 site on the TERT gene promoter, mediating TERT repression. In response to TGF-beta, Smad3 binds to c-Myc; knocking down c-Myc, Smad3 does not bind to the TERT gene, suggesting that c-Myc recruits Smad3 to the TERT promoter. Thus, TGF-beta negatively regulates telomerase activity via Smad3 interactions with c-Myc and the TERT gene promoter. Modifying the interaction between Smad3 and TERT gene may, thus, lead to novel strategies to regulate telomerase.

A Formylated Hexapeptide Ligand Mimics the Ability of Wnt-5a to Impair Migration of Human Breast Epithelial Cells

Loss of Wnt-5a protein expression is associated with shorter recurrence-free survival in breast carcinoma patients and increased motility in mammary cell lines. Based on sequence analysis of Wnt-5a, we identified 14 peptide fragments and investigated their ability to mimic the effects of Wnt-5a on mammary cell adhesion and migration. Two of these peptides significantly increased adhesion and impaired migration in the non-tumorigenic HB2 breast epithelial cell line and in the MDA-MB-468 breast cancer cell line, both of which show little endogenous expression of the Wnt-5a protein. We removed two amino acids at a time from the N terminus of the shorter of these two peptides to identify the shortest peptide that still inhibited migration. The influence on tumor cell adhesion was gradually lost and was no longer detectable when only six amino acids remained. However, formylation of the N-terminal methionine of this hexapeptide restored its effect on adhesion and reduced tumor cell motility via a Frizzled-5 receptor-dependent mechanism, even at a low pH such as encountered in breast tumor tissue. This formylated hexapeptide ligand induced a rapid cytosolic calcium signal, whereas it did not affect the cellular levels of unphosphorylated beta-catenin or active JNK. The novel formyl-Met-Asp-Gly-Cys-Glu-Leu peptide ligand is not only a valuable experimental tool but has also a potential role in antimetastatic treatment of the 50% of human breast cancer patients that have reduced endogenous Wnt-5a protein expression.

A Conserved Sequence within the Propeptide Domain of Membrane Type 1 Matrix Metalloproteinase Is Critical for Function as an Intramolecular Chaperone

The propeptide domain of secreted matrix metalloproteinases (MMPs) is responsible for maintaining the latency of these proteinases. Recently, the propeptide domain of the prototype membrane type matrix metalloproteinase (MT1-MMP) was demonstrated to act as an intramolecular chaperone (Cao, J., Hymowitz, M., Conner, C., Bahou, W. F., and Zucker, S. (2000) J. Biol. Chem. 275, 29648-29653). In the current study, the role of an unique four-amino acid sequence in the propeptide domain of MT1-MMP was examined. The sequence (42)YGYL(45) is conserved in the propeptide domain of all six members of the MT-MMP subfamily, but not in secreted MMPs. Mutant MT1-MMP cDNAs coding for alanine substitutions (single and double amino acid sequences) in this conserved propeptide region were transfected into COS-1 cells deficient in endogenous MT1-MMP. As demonstrated by immunofluorescence, mutant MT1-MMP protein was synthesized and displayed on the plasma membrane of transfected cells. Alanine substitutions within the (42)YGYL(45) sequence proved to be detrimental for enzyme function in terms of activation of proMMP-2 and binding TIMP-2 to the cell surface (MT1-MMP serves as a cell surface receptor for TIMP-2). In contrast to wild-type MT1-MMP-transfected cells, mutant MT1-MMP-transfected cells were incapable of degrading and migrating on a fibronectin substrate. These data indicate that the conserved (42)YGYL(45) sequence within the propeptide domain of MT-MMPs is required for intramolecular chaperone function of these intrinsic membrane proteinases.

Differential phosphorylation of mitogen-activated protein kinase families by epidermal growth factor and ultraviolet B irradiation in SV40-transformed human keratinocytes

SV-40 transformed human keratinocytes (SVHK cells) were stimulated with epidermal growth factor (EGF) and ultraviolet B (UVB) irradiation. Following the stimulation, cell growth, apoptosis, and the activities of mitogen-activated protein (MAP) kinase families were analyzed. EGF (100 ng/ml) increased SVHK cell number compared with control cells cultured in serum-free DMEM medium. The EGF-stimulated cells did not show DNA fragmentation. In contrast, UVB irradiation (40 mJ/cm 2) markedly decreased viable cell number that was accompanied with DNA fragmentation. EGF stimulated extracellular signal-regulated kinase (ERK) and stress-activated protein kinase/c-Jun N-terminal kinase (JNK). Following the EGF stimulation, phosphorylated ERK and JNK were detected by phospho-p42/44 MAP kinase antibody and phospho-SAPK/JNK antibody, respectively. On the other hand, UVB irradiation stimulated the phosphorylation of p38 and JNK but not of ERK. The stimulation of ERK and JNK induced by EGF was observed earlier than the stimulation of p38 and JNK induced by UVB. PD98059, a specific MAP kinase kinase (MAPKK) 1 (also referred to as MEK1) inhibitor, inhibited EGF-dependent cell proliferation, that was associated with the inhibition of ERK and JNK phosphorylation. In contrast, UVB-induced overall cell death was not significantly affected by PD98059, that inhibited phosphorylation of JNK but not of p38. PD98059, however, significantly augmented UVB-induced cell death earlier time points (30 min–2 h). These results indicate that ERK and JNK are activated following EGF stimulation that might be associated with cell proliferation. On the other hand, UVB-induced apoptosis seems to be mostly associated with the activation of p38. JNK stimulation might provide an anti-apoptotic tonus during the UVB-induced, p38-associated SVHK cell death.

Urokinase Plasminogen Activator/Urokinase-specific Surface Receptor Expression and Matrix Invasion by Breast Cancer Cells Requires Constitutive p38α Mitogen-activated Protein Kinase Activity

Overexpression of urokinase plasminogen activator (uPA) and its receptor (uPAR) has been well documented in a wide variety of tumor cells. In breast cancer, expression of uPA/uPAR is essential for tumor cell invasion and metastasis. However, the mechanism responsible for uPA/uPAR expression in cancer cells remains unclear. In the studies reported here, we show that endogenous p38 MAPK activity correlates well with breast carcinoma cell invasiveness. Treatment of highly invasive BT549 cells with a specific p38 MAPK inhibitor SB203580 diminished both uPA/uPAR mRNA and protein expression and abrogated the ability of these cells to invade matrigel, suggesting that p38 MAPK signaling pathway is involved in the regulation of uPA/uPAR expression and breast cancer cell invasion. We also demonstrated that SB203580-induced reduction in uPA/uPAR mRNA expression resulted from the de- stabilization of uPA and uPAR mRNA. Finally, by selectively inhibiting p38alpha or p38beta MAPK isoforms, we demonstrate that p38alpha, rather than p38beta, MAPK activity is essential for uPA/uPAR expression. These studies suggest that p38alpha MAPK signaling pathway is important for the maintenance of breast cancer invasive phenotype by promoting the stabilities of uPA and uPAR mRNA.

Molecular Cloning of a Novel Potassium-dependent Sodium-Calcium Exchanger from Rat Brain

We have isolated a novel cDNA clone from rat cerebral cortex encoding a protein of 670 amino acids (NCKX2) that has significant similarity to the 1199-amino acid-long Na/Ca-K exchanger of bovine rod outer segment (NCKX1). NCKX2 transcripts are 10.5 kilobase pairs in length and are expressed abundantly in neurons throughout the brain and with much lower abundance in selected other tissues. The predicted topology of the rat NCKX2 protein is very similar to that of bovine NCKX1, beginning with a solitary transmembrane segment (M0), which is removed as a "signal peptide" in bovine NCKX1, an extracellular loop, a cluster of five transmembrane spanning segments (M1 to M5), a long cytoplasmic loop, and a final hydrophobic cluster (M6 to M11). Within the hydrophobic clusters, rat NCKX2 shares 80% identity and 91% similarity with bovine NCKX1. The two larger hydrophilic loops are much shorter in NCKX2 than in NCKX1, accounting largely for the difference in length between the two proteins, and are dissimilar in sequence except for a 32-amino acid stretch with 69% identity in the cytosolic loop. NCKX2 was epitope-tagged in the extracellular domain and was shown to be expressed at the surface of transfected HEK cells. Analysis of NCKX2 function by fluorescent imaging of fura-2-loaded transfected cells demonstrated that NCKX2 is a potassium-dependent sodium/calcium exchanger.