The hypoxia-inducible transcription factor-1 (HIF-1) is central to a number of pathological processes through the induction of specific genes such as vascular endothelial growth factor (VEGF). Even though HIF-1 is highly regulated by cellular oxygen levels, other elements of the inflammatory and tumor microenvironment were shown to influence its activity under normal oxygen concentration. Among others, recent studies indicated that transforming growth factor (TGF) ␤ increases the expression of the regulatory HIF-1␣ subunit, and induces HIF-1 DNA binding activity. Here, we demonstrate that TGF␤ acts on HIF-1␣ accumulation and activity by increasing HIF-1␣ protein stability. In particular, we demonstrate that TGF␤ markedly and specifically decreases both mRNA and protein levels of a HIF-1␣-associated prolyl hydroxylase (PHD), PHD2, through the Smad signaling pathway. As a consequence, the degradation of HIF-1␣ was inhibited as determined by impaired degradation of a reporter protein containing the HIF-1␣ oxygen-dependent degradation domain encompassing the PHD-targeted prolines. Moreover, inhibition of the TGF␤1 converting enzyme, furin, resulted in increased PHD2 expression, and decreased basal HIF-1␣ and VEGF levels, suggesting that endogenous production of bioactive TGF␤1 efficiently regulates HIF-1-targeted genes. This was reinforced by results from HIF-1␣ knock-out or HIF-1␣-inhibited cells that show impairment in VEGF production in response to TGF␤. This study reveals a novel mechanism by which a growth factor controls HIF-1 stability, and thereby drives the expression of specific genes, through the regulation of PHD2 levels.

To prepare for the DNA synthesis (S) phase of the cell cycle, transcription of many genes required for nucleotide biosynthesis increases. The promoters of several of these genes contain binding sites for the E2F family of transcription factors, and, in many cases, mutation of these sites abolishes growth-regulated transcription. The RNA levels of one family member, E2F1, increase about 15-fold at the G1/S-phase boundary and expression of E2F1 in quiescent cells activates transcription from some G1/S-phase-specific promoters, suggesting that E2F1 plays a critical role in preparing cells to enter S phase. To elucidate the signal transduction pathway leading to the activation of genes required for DNA synthesis, we are investigating the mechanism by which expression of E2F1 is regulated. To determine whether levels of E2F1 mRNA are controlled by changes in promoter activity, we have cloned and characterized the mouse E2F1 promoter. Sequence analysis revealed two sets of overlapping E2F-binding sites located between -12 and -40 relative to the transcription initiation site. We show that these sites bind cellular E2F and that an E2F1 promoter fragment can be activated up to 100-fold by coexpression of E2F proteins. We also show that the activity of this E2F1 promoter fragment increases -80-fold at the Gl/S-phase boundary and that this activation is, in part, regulated by Go-specific repression via the E2F sites. However, the E2F sites are not sufficient to mediate growth-regulated transcriptional activity; our results indicate that multiple DNA elements are required for transcription regulation of the E2F1 promoter at the G1/S-phase boundary.

The transcription rate of the dihydrofolate reductase (DHFR) gene increases at the G1/S boundary of the proliferative cell cycle. Through analysis of transiently and stably transfected NIH 3T3 cells, we have now demonstrated that DHFR promoter sequences extending from -270 to +20 are sufficient to confer similar regulation on a reporter gene. Mutation of a protein binding site that spans sequences from -16 to + 11 in the DHFR promoter resulted in loss of the transcriptional increase at the G1/S boundary. Purification of an activity from HeLa nuclear extract that binds to this region enriched for a 180-kDa polypeptide (HIP1). Using this HIP1 preparation, we have identified specific positions within the binding site that are critical for efficient protein-DNA interactions. An analysis of association and dissociation rates suggests that bound HIP1 protein can exchange rapidly with free protein. This rapid exchange may facilitate the burst of transcriptional activity from the DHFR promoter at the GI/S boundary. Cellular proliferation is controlled by a programmed series of events termed the cell cycle. Neoplasia can result when quiescent or differentiated cells escape cell cycle control and begin inappropriate proliferation. Therefore, understanding the checks and balances used by the cell to control entrance into S phase of the cell cycle is important in understanding the loss of proliferation control that leads to neoplasia. One widely used model system to study cell cycle progression is the reentry of quiescent cells into the proliferative cell cycle elicited by an increase in the level of serum growth factors. Murine 3T3 fibroblasts withdraw from the proliferative cell cycle into a quiescent Go state when the concentration of serum in the culture medium is low. The addition of high concentrations of serum to these quiescent cells initiates a series of events that results in the transduction of extracellular signals to the cell nucleus, with a concomitant change in the expression of growth-responsive genes. The first genes that are activated by the serum growth factors are termed immediate-early-response genes, many of which are transcription factors. Several hours later, at the transition from G1 to S phase, late-response genes are activated, followed by the onset of DNA synthesis and the activation of histone gene expression. Among the late-response genes are the genes for DNA polymerase alpha, thymidine kinase, thymidylate synthase, carbamoyl phosphate synthase-aspartate carbamoyltransferase-dihydroorotase, and dihydrofolate reductase (DHFR). Many of these genes are involved in nucleotide biosynthesis; for example, the DHFR gene encodes an enzyme involved in the production of glycine, purines, and thymidylate.Although much is known about the cis elements and trans-acting factors that are responsible for the activation of the early-response genes (see reference 32 for a review), few studies have focused on the activation of the late-response genes. Recently, several groups have analyzed the ability of the promo...

Hypoxia is a common tumorigenesis enhancer, mostly owing to its impact on gene expression of many angiogenic and invasion-related mediators, some of which are natural substrates for the proprotein convertase furin. Analysis of furin promoters revealed the presence of putative binding sites for hypoxia-inducible factor-1 (HIF-1), a transcription complex that plays a pivotal role in cellular adaptation to hypoxia. In fact, we demonstrate herein that the levels of fur mRNA, encoding furin, are remarkably increased upon hypoxic challenge. Cotransfection of a HIF-1␣ dominant negative form in wild-type (WT) cells or transfection of a furin promoter-reporter gene in HIF-1-deficient cells indicated the requirement of HIF-1 for furin promoter activation by hypoxia. Direct HIF-1 action on the furin promoter was identified as a canonical hypoxia-responsive element site with enhancer capability. The hypoxic/ HIF-1 regulation of furin correlated with an increased proteolytic activation of the substrates membrane-type 1 matrix metalloproteinase and transforming growth factor-␤1. Our findings unveil a new facet of the physiological consequences of hypoxia/HIF-1, through enhanced furin-induced proteolytic processing/activation of proproteins known to be involved in tumorigenesis.

Proprotein convertases (PCs) have been proposed to play a role in tumor necrosis factor-K K converting enzyme (TACE) processing/activation. Using the furin-de¢cient LoVo cells, as well as the furin-pro¢cient synoviocytes and HT1080 cells expressing the furin inhibitor K K 1 -PDX, we demonstrate that furin activity alone is not su⁄cient for e¡ective maturation and activation of the TACE enzyme. Data from in vitro and in vivo cleavage assays indicate that PACE-4, PC5/PC6, PC1 and PC2 can directly cleave the TACE protein and/or peptide. PC inhibition in macrophages reduced the release of soluble TNF-K K from transmembrane pro-TNF-K K. We therefore conclude that furin, in addition to other candidate PCs, is involved in TACE maturation and activation. ß