Oxygen-dependent Regulation of HIF-1alpha Expression and Function

Hypoxia is a state of reduced oxygen levels below normal values. It occurs in the pathophysiology of many diseases and plays important roles in mammalian physiology and development. The hypoxia-inducible factors (HIFs) are transcription factors that mediate adaptive cellular responses to hypoxia by activating the transcription of target genes. HIF is a heterodimer consisting of an oxygen-regulated α subunit and a constitutively expressed HIF-1β (ARNT) subunit. Both HIF subunits belong to the bHLH/PAS domain-containing protein family. The bHLH and PAS domains mediate DNA binding and dimerization. HIF-α contains a unique oxygen-dependent degradation (ODD) domain and two transactivation domains (TADs: N-TAD and CTAD). The oxygen-dependent degradation of HIF-α is mediated by PHD-dependent hydroxylation, and subsequent pVHL-dependent ubiquitylation and proteasomal degradation. Under hypoxia, HIF-α is stabilized and accumulates in the nucleus where it dimerizes with ARNT and activates target gene expression by recruiting coactivators. FIH-1 inhibits the transcriptional activity of HIF-α by inhibiting the recruitments of coactivators such as CBP/p300 through the hydroxylation of Asparagine residue in CTAD. The aim of this study is to investigate in detail oxygen-dependent regulation of HIF-α degradation and function. In paper I, we identified residues that are critical for the degradation and transactivation functions of HIF-1α N-TAD. In paper II, we demonstrated that the expression of pVHL-interacting peptides activates endogenous HIF-1α function, providing a potential pro-angiogenic stimulus. The degradation of HIF-1α is regulated by subcellular compartmentalization in a cell-type-dependent manner. The compartment in which degradation of HIF-1α occurs dictates where the pVHL-interacting peptide must be located in order to efficiently increase HIF-1α function at normoxia. In paper III, we identified filamin A (FLNa) as a novel modulator of the HIF-1α pathway and presented a novel nuclear function of this actin-binding protein. Hypoxia enhances calpain-mediated cleavage of FLNa and the nuclear localization of the cleaved C-terminal fragment of FLNa. The C-terminal fragment of FLNa mediates the function of FLNa by facilitating the nuclear localization and enhancing the transcriptional activity of HIF-1α. FLNa promotes VEGF-A-mediated angiogenesis through activating HIF-1α-mediated hypoxic responses. In paper IV, we demonstrated how hypoxia controls the differentiation status of a cell and demonstrates a crosstalk between the HIF-1α and Notch signaling pathways. Hypoxia blocks differentiation in myoblasts, satellite cells and primary neural stem cells in a Notch-dependent manner. Hypoxia stabilizes the Notch intracellular domain, activates Notch-responsive promoters and increases expression of Notch-regulated downstream genes. The modulation of the Notch pathway by hypoxia is mediated by HIF-1α. HIF-1α interacts with Notch ICD, binds to Notch-responsive promoter, and a transcriptionally active form of HIF-α increases Notch signaling. The results from these studies contribute to a better understanding of the mechanism, which controls oxygen-dependent regulation and function of HIF-1α, and provide therapeutic implications by novel means to modulate the HIF-1α signaling pathway. LIST OF PUBLICATIONS This thesis is based on the following papers: I. Teresa Pereira, Xiaowei Zheng, Jorge L. Ruas, Keiji Tanimoto, and Lorenz Poellinger. Identification of residues critical for regulation of protein stability and the transactivation function of the hypoxia-inducible factor-1alpha by the von Hippel-Lindau tumor suppressor gene product. J Biol Chem. 2003 Feb 28;278(9):6816-23. II. Xiaowei Zheng, Jorge L. Ruas, Renhai Cao, Florian A. Salomons, Yihai Cao, Lorenz Poellinger, and Teresa Pereira. Cell-Type-Specific Regulation of Degradation of Hypoxia-Inducible Factor 1α: Role of Subcellular Compartmentalization. Mol Cell Biol. 2006 June; 26(12): 4628–4641. III. Xiaowei Zheng, Xianghua Zhou, Meit Bjorndahl, Hidetaka uramoto, Lakshmanan Ganesh, Elizabeth G. Nabel, Yihai Cao, Jan Boren, Teresa Pereira, Lorenz Poellinger, and Levent M. Akyurek. Filamin-A promotes VEGF-A activity through the HIF-1a-mediated hypoxic response. Manuscript. IV. Maria V. Gustafsson, Xiaowei Zheng, Teresa Pereira, Katarina Gradin, Shaobo Jin, Johan Lundkvist, Jorge L. Ruas, Lorenz Poellinger, Urban Lendahl and