Question

1.

a. describe the HIF1A pathway

b. describe how the pathway functions in normoxia,

c. describe how the pathway functions in hypoxia,

d. define the sequence of the HIF response element (HRE)

e. describe medical significance of HIF1A pathway

Answer 1

Question a. describe the HIF1A pathway.

Answer: Hypoxia-inducible factor 1-alpha, also known as HIF-1-alpha, is a subunit of a heterodimeric transcription factor hypoxia-inducible factor 1 (HIF-1) that is encoded by the HIF1A gene. It is a basic helix-loop-helix PAS domain containing protein, and is considered as the master transcriptional regulator of cellular and developmental response to hypoxia. The dysregulation and overexpression of HIF1A by either hypoxia or genetic alternations have been heavily implicated in cancer biology, as well as a number of other pathophysiologies, specifically in areas of vascularization and angiogenesis, energy metabolism, cell survival, and tumor invasion.

Question: b. describe how the pathway functions in normoxia.

Answer: In normoxia, oxygen dependent hydroxylation of HIF-1α asparagine (N803) residue by factor inhibiting HIF-1 (FIH-1), also known as asparginyl hydroxylase, blocks the interaction between the two domains, abrogating the subsequent HIF-1α mediated gene transcription. Another oxygen-dependent major mechanism for negative regulation of HIF-1α pathway under normoxia is through controlling HIF-1α transactivation. This pathway represents another level of post-translational modifications of HIF-1α transactivation domain but does not involve the pVHL protein. The transcriptional activation of HIF-1α target genes is initiated through the cooperative binding of C-TAD in the HIF-1α and the co-activator CBP/p300.

Question: c. describe how the pathway functions in hypoxia.

Answer: The HIF pathway in hypoxia can help treat diverse disorders. HIF(s) are also upregulated under inflammatory conditions, suggesting their role in maintaining homeostatic conditions and protecting against cellular inflammation. The role of the HIF pathway varies under diverse conditions. For example, HIF inhibitors have been developed to treat cancer and ischemia, whereas HIF activators could be utilized for stroke and spinal cord injuries. Significant developments have been made towards understanding the roles of the HIFs under both physiological and pathophysiological conditions. The roles of HIF(s) are becoming clearer during physiological adaptation.

Question d: define the sequence of the HIF response element (HRE)

Answer: HREs are enhancers elements containing the consensus core sequence 5'-(A/G)CGT(G/C)(G/C)-3', localized at varying positions and orientations of the coding region of several hypoxia-regulated genes.

During hypoxia, the hypoxia inducible factor (HIF) binds to hypoxiaresponse elements (HRE) and induces the expression of hypoxia-inducible genes (HIG). A functionally active HRE typically contains an adjacent hypoxia ancillary sequence (HAS) in its downstream within the space of 7- 15 nt. Here, a computational tool 'HRE Finder' is discussed using an example data set. The tool provides the ability for mining canonical HREs and predicting functional HREs in the upstream region of genes, which can be an indispensable tool for genome-wide analysis and identification of hypoxia inducible genes in vertebrates. The hypoxia responsive element (HRE) sequences isolated from oxygen-responsive genes have been shown to selectively induce gene expression in response to hypoxia when placed upstream of a promoter. The levels of induced gene expression were dependent on the number of HRE copies and the oxygen tension. Hypoxiamediated cancer gene therapy strategies may represent a promising mean to significantly improve the efficacy of standard radiation therapy and chemotherapy approaches.

Question: e. describe medical significance of HIF1A pathway

Answer: HIF1A is known to regulate elements of the adenosine pathway. Specifically, HIF1A has been shown to regulate expression of the ADORA2B receptor, by binding to the HRE of its promoter, CD73, and equilibrative nucleotide transporters (ENTs) which allow adenosine transport across the cell membrane. This regulation ultimately leads to elevation of extracellular adenosine levels and enhancement of ADORA2B signaling.

The promoter of CD73 has also been found to have a HIF1 binding site and in vivo studies of a mouse model of hypoxia-induced intestinal injury showed CD73 inhibition increased barrier disruption, supporting a protective role for CD73 in hypoxia-mediated acute injury. Cyclic mechanical stretch of pulmonary epithelia and in vivo model of VILI has been shown to promote HIF1A stabilization and significant increases in ADORA2B which were reduced with HIF1A inhibition in vitro and epithelial-cell specific HIF1A deletion. These studies suggest a role for HIF1A in modulating players in the adenosine signaling pathway that contribute to tissue protection in acute lung injury.

Most recently, up-regulation of HIF1A along with ADORA2B, CD73, and ENT-1 has been demonstrated in tissue samples from patients with Group 3 pulmonary hypertension (pH), or PH associated with chronic lung disease, in association with increased succinate levels. As HIF1A has been shown to regulate ADORA2B expression and ADORA2B has been found to be elevated and localized to alternatively activated macrophages in IPF patients, recent investigations have focused on the role HIF1A may play in modulating ADORA2B expression on myeloid cells and macrophage differentiation.