Decreased tumorigenicity of c-Myc-transformed fibroblasts expressing active USF2.

USF is a small family of basic helix-loop-helix leucine zipper (bHLH-zip) transcription factors with DNA binding specificities similar to that of the c-Myc oncoprotein. Evidence for a role of USF in growth control includes inhibition of c-Myc-dependent cellular transformation in vitro and loss of USF transcriptional activity in many cancer cell lines. However, a direct effect of USF on the tumorigenicity of an established cell line has never been demonstrated. Here, cell lines derived from rat embryo fibroblasts transformed by c-Ha-Ras and either c-Myc or E1A were used as model system to investigate the tumor suppression ability of USF. Overexpression of USF2 stimulated transcription and inhibited colony formation in c-Myc-transformed, but not E1A-transformed, fibroblasts. Stable clones expressing high USF2 levels were constructed from c-Myc-transformed fibroblasts. In two of these clones, overexpressed USF2 did not activate transcription, and there was no significant change in the transformed phenotype. In contrast, a clone that expressed transcriptionally active USF2 exhibited altered morphology and a strongly decreased ability to proliferate in semisolid medium. The ability of these cells to form tumors in nude mice was also decreased by a factor of more than 30 as compared to the parental cell line or cells overexpressing transcriptionally inactive USF2. Cotransfection assays with USF- or Myc-specific dominant-negative mutants indicated that active USF2 inhibited cellular transformation by preventing transcriptional repression by c-Myc.

Evidence for a cancer-specific switch at the CDK4 promoter with loss of control by both USF and c-Myc.

USF and c-Myc are basic helix-loop-helix transcription factors with similar DNA-binding specificities, but antagonistic effects on cellular transformation. In order to determine how these opposite functions correlate with the transcriptional activities of the two factors on particular downstream targets, we investigated the roles of USF and c-Myc in expression of CDK4, a known direct target of c-Myc. Overexpression of either c-Myc or USF2, but not USF1, stimulated the expression of CDK4 promoter-driven reporter genes in the non-tumorigenic mammary epithelial MCF-10A cells. Dominant-negative mutants specific to either Myc or USF family proteins inhibited reporter gene activity as well as endogenous CDK4 expression, demonstrating involvement of both USF and Myc in CDK4 transcriptional control. In contrast, in two different breast cancer cell lines where USF is transcriptionally inactive and c-Myc is overexpressed, CDK4 promoter activity was no longer responsive to either transcription factor. Accordingly, chromatin immunoprecipitation revealed significantly lower levels of both USF and c-Myc bound to the endogenous CDK4 promoter in breast cancer cells than in MCF-10A cells, with a concomitant decrease in associated histone H3 acetylation. These results suggest that a major switch in the transcriptional control of CDK4 occurs during breast carcinogenesis, with likely alteration of cell cycle regulation.

Diminished milk synthesis in upstream stimulatory factor 2 null mice is associated with decreased circulating oxytocin and decreased mammary gland expression of eukaryotic initiation factors 4E and 4G.

Previous studies have suggested that upstream stimulatory factors (USFs) regulate genes involved with cell cycle progression. Because of the relationship of USFs to an important oncogene in breast cancer, c-myc, we chose to determine the importance of USF to normal mammary gland development in the mouse. Expression of USF in the mammary gland throughout development demonstrated only modest changes. Mutation of the Usf2 gene was associated with reduced fertility in females, but had no effect on prepartum mammary gland development. However, lactation performance in Usf2-/- females was only half of that observed in Usf2+/+ females, and both lactose and nitrogen were decreased in milk from Usf2-/- dams. This decrease was associated with diminished mammary tissue wet weight and luminal area by d 9 of lactation and with a decreased protein-DNA ratio. This decrease was associated with reduced abundance of the eukaryotic initiation factors eIF4E and eIF4G. Blood oxytocin concentrations on d 9 postpartum were also lower in Usf2-/- mice than Usf2+/+ mice. In contrast, the mutation had no effect on blood prolactin concentrations, mammary cell proliferation or apoptosis, mammary tissue oxytocin receptors, or milk protein gene expression. The mutation had only modest effects on maternal behavior. These data support the idea that USF is important to physiological processes necessary for the establishment and maintenance of normal lactation and suggest that USF-2 may impact lactation through both systemic and mammary cell-specific mechanisms.

The IGF2 receptor is a USF2-specific target in nontumorigenic mammary epithelial cells but not in breast cancer cells.

The antiproliferative activities of the USF proteins and the frequent loss of USF function in cancer cells suggest a role for these ubiquitous transcription factors in tumor suppression. However, the cellular targets that mediate the effects of USF on cellular proliferation and transformation remain uncharacterized. IGF2R, with multiple functions in both normal growth and cancer, was investigated here as a possible USF target in both nontumorigenic and tumorigenic breast cell lines. The 5'-flanking sequences of the human IGF2R gene contain multiple, highly conserved E boxes almost identical to the consensus USF DNA-binding sequence. These E boxes were found to be essential for IGF2R promoter activity in the nontumorigenic mammary epithelial cell line MCF-10A. USF1 and USF2 bound the IGF2R promoter in vitro, and both USF1 and USF2, but not c-Myc, were present within the IGF2R promoter-associated chromatin in vivo. Overexpressed USF2, but not USF1, transactivated the IGF2R promoter, and IGF2R mRNA was markedly decreased by expression of a USF-specific dominant negative mutant, identifying IGF2R as a USF2 target. IGF2R promoter-driven expression was USF-independent in both MCF-7 and MDA-MB-231 breast cancer cell lines, suggesting that a defect in USF function may contribute to down-regulation of IGF2R expression in cancer cells.

Involvement of upstream stimulatory factors 1 and 2 in RANKL-induced transcription of tartrate-resistant acid phosphatase gene during osteoclast differentiation.

Tartrate-resistant acid phosphatase (TRAP) plays an important role in bone resorption. TRAP expression in osteoclasts is regulated by receptor activator of NF-kappaB (RANKL), a potent activator of osteoclast differentiation. However, the molecular mechanism underlying the RANKL-induced TRAP expression remains unknown. Here we show that two regions in the mouse TRAP promoter (one at -1858 to -1239 and the other at -1239 to -1039, relative to the translation start site) are implicated in RANKL-induced TRAP transcription in RAW264.7 cells. A detailed characterization of the region at -1239 to -1039 identifies a 12-bp sequence, AGCCACGTGGTG, that specifically binds nuclear proteins from RAW264.7 cells and primary bone marrow macrophages (BMMs) in an electrophoretic mobility shift assay (EMSA). Moreover, the binding is significantly enhanced in EMSA with nuclear extracts from RANKL-treated RAW264.7 cells and BMMs, suggesting that the 12-bp sequence may be involved in RANKL-induced TRAP transcription. Various assays reveal that nuclear proteins binding to the 12-bp sequence are upstream stimulatory factors (USF) 1 and 2. Importantly, mutation of the USF-binding site partially blocks RANKL-induced TRAP transcription in RAW264.7 cells, confirming that USF1 and USF2 are functionally involved in RANKL-induced TRAP transcription. In summary, our data show that USF1 and USF2 play a functional role in RANKL-dependent TRAP expression during osteoclast differentiation.

Severe iron deficiency anemia in transgenic mice expressing liver hepcidin.

We recently reported the hemochromatosis-like phenotype observed in our Usf2 knockout mice. In these mice, as in murine models of hemochromatosis and patients with hereditary hemochromatosis, iron accumulates in parenchymal cells (in particular, liver and pancreas), whereas the reticuloendothelial system is spared from this iron loading. We suggested that this phenotypic trait could be attributed to the absence, in the Usf2 knockout mice, of a secreted liver-specific peptide, hepcidin. We conjectured that the reverse situation, namely overexpression of hepcidin, might result in phenotypic traits of iron deficiency. This question was addressed by generating transgenic mice expressing hepcidin under the control of the liver-specific transthyretin promoter. We found that the majority of the transgenic mice were born with a pale skin and died within a few hours after birth. These transgenic animals had decreased body iron levels and presented severe microcytic hypochromic anemia. So far, three mosaic transgenic animals have survived. They were unequivocally identified by physical features, including reduced body size, pallor, hairless and crumpled skin. These pleiotropic effects were found to be associated with erythrocyte abnormalities, with marked anisocytosis, poikylocytosis and hypochromia, which are features characteristic of iron-deficiency anemia. These results strongly support the proposed role of hepcidin as a putative iron-regulatory hormone. The animal models devoid of hepcidin (the Usf2 knockout mice) or overexpressing the peptide (the transgenic mice presented in this paper) represent valuable tools for investigating iron homeostasis in vivo and for deciphering the molecular mechanisms of hepcidin action.