MondoA-Mlx heterodimers are candidate sensors of cellular energy status: mitochondrial localization and direct regulation of glycolysis.

Transcription factors can be sequestered at specific organelles and translocate to the nucleus in response to changes in organellar homeostasis. MondoA is a basic helix-loop-helix leucine zipper transcriptional activator similar to Myc in function. However, unlike Myc, MondoA and its binding partner Mlx localize to the cytoplasm, suggesting tight regulation of their nuclear function. We show here that endogenous MondoA and Mlx associate with mitochondria in primary skeletal muscle cells and erythroblast K562 cells. Interaction between MondoA and the mitochondria is salt and protease sensitive, demonstrating that it associates with the outer mitochondrial membrane by binding a protein partner. Further, endogenous MondoA shuttles between the mitochondria and the nucleus, suggesting that it communicates between these two organelles. When nuclear, MondoA activates transcription of a broad spectrum of metabolic genes, including those for the glycolytic enzymes lactate dehydrogenase A, hexokinase II, and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3. Regulation of these three targets is mediated by direct interaction with CACGTG sites in their promoters. Consistent with its regulation of glycolytic targets, MondoA is both necessary and sufficient for glycolysis. We propose that MondoA communicates information about the intracellular energy state between the mitochondria and the nucleus, resulting in transcriptional activation of glycolytic target genes.

TGF-beta induces novel Lef-1 splice variants through a Smad-independent signaling pathway.

The lymphoid enhancer-binding factor (Lef-1) transcription factor is best known for the ability to transduce Wnt signals during development and to mediate excessive Wnt signaling in certain types of cancer. We recently identified and characterized a novel Wnt-like effect of transforming growth factor-beta (TGF-beta) on beta-catenin, the binding partner of Lef-1. Therefore, we sought to determine the effect of TGF-beta on expression of the Lef/T-cell-specific transcription factor (TCF) components of the Wnt pathway. We found that TGF-beta markedly induced Lef-1 mRNA expression in cell lines originating from fetal lung (Mv1Lu) and newborn skin (Balb/MK), tissues that normally express Lef-1 during development. Lef-1 induction was temporally related to but independent of TGF-beta-induced G1 cell cycle arrest. Furthermore, the induction of Lef-1 was independent of both new protein synthesis and Smad-mediated signaling. Using TGF-beta-treated Mv1Lu cells, we identified multiple splice forms of Lef-1, including novel variants that lack both exons 2 and 3. We conclude that the induction of Lef-1 has permissive effects on the well-characterized TGF-beta signal that inhibits c-myc expression and induces a G1 arrest.

TGF-beta targets the Wnt pathway components, APC and beta-catenin, as Mv1Lu cells undergo cell cycle arrest.

The highly coordinated interaction of TGF-beta and Wnt signaling pathways is critical for normal development. However, the effects of TGF-beta on APC and beta-catenin, two key mediators of Wnt signaling in epithelial cells, have been largely unknown. We determined the effect of TGF-beta on APC and beta-catenin expression in Mv1Lu, a nontransformed epithelial cell line, in which TGF-beta signaling causes a G(1) cell cycle arrest. We found that TGF-beta rapidly reduced APC protein levels through a post-transcriptional mechanism. Further, TGF-beta increased beta-catenin mRNA and protein levels, and increased beta-catenin nuclear accumulation. Finally, retrovirus-mediated overexpression of beta-catenin discernibly enhanced the ability of TGF-beta to induce a G(1) cell cycle arrest. This is the first report demonstrating that TGF-beta mimics the effect of Wnt signaling on beta-catenin in Mv1Lu cells, and that reduction of APC and nuclear accumulation of beta-catenin have cooperative effects on mechanisms that mediate TGF-beta-induced cell cycle arrest.

The human papilloma virus E7 oncoprotein inhibits transforming growth factor-beta signaling by blocking binding of the Smad complex to its target sequence.

The human papillomavirus (HPV) oncoprotein E7 is implicated in the etiology of cervical cancer associated with infection by HPV. HPV-positive cells develop resistance to TGF-beta growth inhibitory activity through the inhibition of hypophosphorylation of pRb by papillomavirus type 16 E7 oncoprotein. In this study, we examined whether E7, in addition to its well known effects on pRb, might directly target the Smad proteins that mediate TGF-beta signaling. Here, we show that E7 significantly blocks both Smad transcriptional activity and the ability of TGF-beta to inhibit DNA synthesis. We found that E7 interacts constitutively with Smad2, Smad3, and Smad4. Confocal microscopic studies confirm that E7 and Smads co-localize in vivo. Using a canonical Smad DNA binding sequence, we found that E7 blocks Smad3 binding to its target sequence on DNA. These results suggest that suppression of Smad-mediated signaling by E7 may contribute to HPV-associated carcinogenesis.