ATP6V0C Competes with Von Hippel-Lindau Protein in Hypoxia-Inducible Factor 1α (HIF-1α) Binding and Mediates HIF-1α Expression by Bafilomycin A1

Abstract

HIF-1␣ not only enables cells to survive under hypoxic conditions but also promotes cell cycle arrest and apoptosis. Therefore, its expression should be controlled at optimal levels in growing tumors. We recently reported that bafilomycin A1 exorbitantly expressed HIF-1␣ and induced the p21 WAF1/Cip1 -mediated growth arrest of tumors (Mol Pharmacol 70: 1856 -1865, 2006). In the present study, we addressed the mechanism underlying bafilomycin-induced HIF-1␣ expression. Bafilomycin stabilized HIF-1␣ under normoxic conditions without changes in intracellular pH. However, when ATP6V0C, the target protein of bafilomycin, was knocked down, this bafilomycin effect was significantly attenuated. Inversely, ATP6V0C expression increased HIF-1␣ levels in a gene dosedependent manner. ATP6V0C competed with Von HippelLindau protein in HIF-1␣ binding by directly interacting with HIF-1␣, which was stimulated by bafilomycin. In confocal images, ATP6V0C was normally present in the cytoplasm but was translocated in company with HIF-1␣ to the nucleus by bafilomycin. The N-terminal end (amino acids 1-16) of HIF-1␣ was identified as the ATP6V0C-interacting motif. These results suggest that ATP6V0C, a novel regulator of HIF-1␣, mediates HIF-1␣ expression by bafilomycin.HIF-1␣ contains the basic helix-loop-helix/periodicity-aryl hydrocarbon receptor nuclear translocator (ARNT)-simpleminded domains at the N terminus (Jiang et al., 1996) and two transactivation domains at the C terminus (Jiang et al., 1997). It also has the Pro-Ser-Thr-rich oxygen-dependent degradation domain (ODDD, aa. 401-603) in the middle part, which is responsible for destabilization of HIF-1␣ protein (Huang et al., 1998). Under normoxic conditions, HIF-1-prolyl hydroxylases (PHD1-3) (Bruick and McKnight, 2001;Epstein et al., 2001) hydroxylate two proline residues (Pro402 and Pro564) within the ODDD (Ivan et al., 2001;Jaakkola et al., 2001;Masson et al., 2001) and, in turn, the von Hippel-Lindau tumor suppressor protein (pVHL) binds to the modified HIF-1␣, resulting in ubiquitination and proteasomal degradation of HIF-1␣ (Maxwell et al., 1999;Kamura et al., 2000). Because PHDs require molecular oxygen as a substrate, the proline hydroxylation is limited under hypoxic conditions, and thereby HIF-1␣ is stabilized. In the nucleus, HIF-1␣ binds ARNT to form a HIF-1 complex, which up-regulates many genes essential for cellular adaptation to hypoxia (Semenza, 2000).HIF-1␣ is frequently found to be overexpressed in human tumors (Zhong et al., 1999), and its levels in tumor specimens are correlated with tumor hypervascularity, aggressiveness, and poor prognosis (Birner et al., 2000, Zagzag et al., 2000. The essential roles of HIF-1␣ in tumor growth and angiogenesis have also been demonstrated in J.-H.L. and J.-W.P. contributed equally to this work. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.doi:10.1124/mol.106.030296. ABBREVIATIONS:HIF-1␣, hypoxia-inducible factor 1␣; ARNT, aryl hydrocarbon receptor nuclear ...

Combined effects of EGFR tyrosine kinase inhibitors and vATPase inhibitors in NSCLC cells

“…The induction of the Hif-1α protein was observed in the cells treated with bafilomycin A1 (Fig. 4A), consistent with previous studies (Lim et al, 2006(Lim et al, , 2007. Lim et al have reported that ATP6V0C, a component of vATPase, binds to the N-terminal end of Hif-lα, perturbing its structure so that it is unfavorable for pVHL binding, resulting in its stabilization (Lim et al, 2007).…”

“…4A), consistent with previous studies (Lim et al, 2006(Lim et al, , 2007. Lim et al have reported that ATP6V0C, a component of vATPase, binds to the N-terminal end of Hif-lα, perturbing its structure so that it is unfavorable for pVHL binding, resulting in its stabilization (Lim et al, 2007). Bafilomycin stimulates ATP6V0C binding to Hif-lα (Lim et al, 2007).…”

“…Lim et al have reported that ATP6V0C, a component of vATPase, binds to the N-terminal end of Hif-lα, perturbing its structure so that it is unfavorable for pVHL binding, resulting in its stabilization (Lim et al, 2007). Bafilomycin stimulates ATP6V0C binding to Hif-lα (Lim et al, 2007). Specially, we found that Hif-1α expression in the H460 cells treated with vATPase inhibitors was greater than that in the A549 cells treated with these inhibitors (Fig.…”

V-ATPase is a candidate therapeutic target for Ewing sarcoma

“…4A). In particular, we choose the V 0 c as it has been successfully targeted by siRNA strategies also in other cancers [13,29,30], and since it is the target of BF-1 [31]. V-ATPase is formed by two different domains, the V 1 domain and the intramembrane V 0 domain, that can be reversibly dissociated [15], and the expression pattern of a subunit of the V c domain should be similar to the expression pattern of a subunit of the V 1 domain.…”

COMMD1: A Novel Protein Involved in the Proteolysis of Proteins

“…The main HIF-1 inhibitors, such as VHL, PHDs, amplified in osteosarcoma-9 (OS-9) and factor inhibiting HIF-1 (FIH-1) regulate HIF-1a stability or HIF-1 activity in an oxygen-dependent manner, [70][71][72][73][74][75] whereas the proteins heat shock protein 90 (HSP90), histone deacetylase inhibitors, receptor of activated protein C kinase (RACK1), ATP6V0C (ATPase, H+ transporting, lysosomal, V0 subunit C) and JAB1/CSN5 (COP9, subunit 5) control HIF-1 activity in an oxygen-independent fashion. [76][77][78][79][80][81][82][83] The molecular mechanism of HSP90 and RACK1 in regulating HIF-1a stability has recently been discussed by Liu and Semenza. 79 Increasing Increasing evidence suggests that HIF-1 HIF-1a protein stability/expression can also protein stability/expression can also be increased by tumor-specific genetic alterations in oncogene or tumor suppressor genes independent of O 2 concentration.…”