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 TGF1 converting enzyme, furin, resulted in increased PHD2 expression, and decreased basal HIF-1␣ and VEGF levels, suggesting that endogenous production of bioactive TGF1 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.
Chronic hypoxia and inflammatory cytokines are hallmarks of inflammatory joint diseases like rheumatoid arthritis (RA), suggesting a link between this microenvironment and central pathological events. Because TACE/ADAM17 is the predominant protease catalyzing the release of tumor necrosis factor ␣ (TNF␣), a cytokine that triggers a cascade of events leading to RA, we examined the regulation of this metalloprotease in response to hypoxia and TNF␣ itself. We report that low oxygen concentrations and TNF␣ enhance TACE mRNA levels in synovial cells through direct binding of hypoxia-inducible factor-1 (HIF-1) to the 5 promoter region. This is associated with elevated TACE activity as shown by the increase in TNF␣ shedding rate. By the use of HIF-1-deficient cells and by obliterating NF-B activation, it was determined that the hypoxic TACE response is mediated by HIF-1 signaling, whereas the regulation by TNF␣ also requires NF-B activation. As a support for the in vivo relevance of the HIF-1 axis for TACE regulation, immunohistological analysis of TACE and HIF-1 expression in RA synovium indicates that TACE is up-regulated in both fibroblast-and macrophage-like synovial cells where it localizes with elevated expression of both HIF-1 and TNF␣. These findings suggest a mechanism by which TACE is increased in RA-affected joints. They also provide novel mechanistic clues on the influence of the hypoxic and inflammatory microenvironment on joint diseases.Tumor necrosis factor-␣ converting enzyme (TACE) 2 or ADAM17 was initially described as the predominant enzyme responsible for the physiological cleavage of membrane-anchored tumor necrosis factor-␣ (TNF␣), releasing it in soluble form (1, 2). This enzyme belongs to the ADAM (a disintegrin and metalloprotease domain) family of transmembrane, multidomain zinc metalloproteinases (3) and is expressed in a wide variety of cell types, including TNF␣ non-producing cells (1). Beside TNF␣, TACE was also shown to solubilize a wide variety of proteins including the receptors TNFR-I and TNFR-II (4), interleukin-1RII (5), interleukin-6R (6), and macrophage/colony-stimulating factor-R (7), the cytokine transforming growth factor-␣ (4), members of the membrane-bound epidermal growth factor family (4), the Notch receptor (8), the chemokine fractalkine (9), L-selectin (4), and the -amyloid precursor protein (10). The importance of TACE substrates in a variety of physiological functions, including development, is underscored by the fact that in vivo inhibition of TACE or disruption of the TACE gene results in the death of mice between embryonic day 17.5 and the first day after birth, due to a number of developmental defects. In addition, the implication of TACE substrates in immunoregulation has made this enzyme an efficient therapeutic target in the treatment of a number of pathological conditions including airway inflammation, cancer, and arthritis.Because of the pathophysiological importance of TACE-mediated shedding, several studies have addressed the mechanism of TACE regulation. Sur...
Huntingtin-interacting protein 1 (HIP1) and HIP12 are orthologues of Sla2p, a yeast protein with essential functions in endocytosis and regulation of the actin cytoskeleton. We now report that HIP1 and HIP12 are major components of the clathrin coat that interact but differ in their ability to bind clathrin and the clathrin adaptor AP2. HIP1 contains a clathrin-box and AP2 consensus-binding sites that display high affinity binding to the terminal domain of the clathrin heavy chain and the ear domain of the AP2 ␣ subunit, respectively. These consensus sites are poorly conserved in HIP12 and correspondingly, HIP12 does not bind to AP2 nor does it demonstrate high affinity clathrin binding. Moreover, HIP12 co-sediments with F-actin in contrast to HIP1, which exhibits no interaction with actin in vitro. Despite these differences, both proteins efficiently stimulate clathrin assembly through their central helical domain. Interestingly, in both HIP1 and HIP12, this domain binds directly to the clathrin light chain. Our data suggest that HIP1 and HIP12 play related yet distinct functional roles in clathrin-mediated endocytosis.Huntington disease, a neurological disorder characterized by selective loss of striatal and cortical neurons and manifest clinically by chorea and intellectual decline, results from polyglutamine expansion of huntingtin into the pathologic range of beyond 35 repeats. In an effort to understand the disease mechanism, several groups have identified proteins that associate with huntingtin. One such protein, HIP1, was identified in a yeast two-hybrid screen (1, 2). Binding of HIP1 to huntingtin is dramatically reduced following polyglutamine expansion, strongly implicating this interaction in the disease process. Subsequent to the identification of HIP1, a highly related protein (HIP12/HIP1R) was identified based on its homology to HIP1 (3-5). In contrast to HIP1, HIP12 does not bind directly to huntingtin (5).HIP1 and HIP12 are orthologues of yeast Sla2p, a protein that functions in both endocytosis and regulation of the actin cytoskeleton (6 -9). Each of these proteins contains an ENTH (epsin N-terminal homology) domain that is thought to be involved in clathrin-mediated endocytosis through binding to phosphatidylinositol 4,5-bisphosphate-containing membranes (10 -13). The central portion of these proteins consists of a helical domain with a high probability to form coiled-coil interactions, followed by a C-terminal talin-homology domain. The talin-homology domain has been shown to bind F-actin in Sla2p and HIP12 suggesting a function in linking membrane attachment and clathrin-coated vesicle (CCVs) 1 formation with actin dynamics (4,14).Recent studies have shown that both HIP1 and HIP12 are enriched on clathrin-coated pits and CCVs and co-localize with markers of endocytosis including clathrin, the clathrin adaptor AP2 and Rab5 (4, 15-17). HIP1 binds directly to the terminal domain of the clathrin heavy chain through a type I clathrin box with the sequence LMDMD and to the ear domain of the ␣ subu...
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