Hypoxia Signaling Modulation

Can you induce hypoxia in vitro?

Conventionally, the study of hypoxic responses has relied on the use of monolayer cell cultures exposed to low oxygen conditions (e.g., 0.02-5% O2) by incubation in gas-controlled chambers or incubators with a specific mixture of gases (e.g., 95% N2, 5% CO2- anoxia). However, studying hypoxia signaling using hypoxia-mimetic agents can forgo gas exchange requirements.

Prolyl Hydroxylase (PHD) Inhibitors Promote HIF Signaling

Under normoxic conditions, the first step of HIF degradation is proline hydroxylation in the oxygen dependent degradation domain (ODDD) by the prolyl hydroxylase (PHD) family of enzymes. Hydroxylated HIF-1 alpha will then bind to the von Hippel-Lindau protein (VHL), where HIF-1 alpha is ubiquitinated and targeted to the proteasome. Inhibiting the initial hydroxylation step by PHDs promotes HIF-1 alpha expression, independent of oxygen levels.

	Mechanism	Bio-Techne Products
Cobalt Chloride (CoCl2)
	Binding to the iron-binding domain of PHD, preventing co-factor binding.	CoCl2 treated lysates
Deferoxamine/Desferrioxamine (DFO/DFX)	Iron chelator. PHD require iron as co-factor for activity. Inhibit the activity of PHDs	Bio-Techne Catalog # 5764
Dimethyloxalylglycine (DMOG)	Competitive inhibitor of PHD enzymes	Bio-Techne Catalog # 4408

Western Blot: HeLa cells were either left untreated (-) or treated with CoCl2 and probed for HIF-1 alpha expression with Rabbit Anti-HIF-1 alpha (Novus Catalog # NB100-134) and Tubulin expression as loading control. HIF-1 alpha expression is readily detectable in CoCl2 treated cells. Cell Lysate used is HeLa CoCl2 Treated/Untreated Cell Lysate (Novus Catalog # NBP2-36450). The specificity of NB100-134 has been validated by both Biological and Orthogonal Strategies.

Molecules to Directly Target HIF Signaling

Other biochemical based approaches rely on the use of small molecules to target different steps in HIF signaling. Using inhibitors of key steps or enzymes involved in the regulation of HIF expression helps to uncover how hypoxia-induced signaling operates in specific systems or models under study.

Small Molecules: HIF Modulators

	Small Molecule	Mechanism	Bio-Techne Catalog #
Inhibitors	TAT-cyclo-CLLFVY	Selective HIF-1 dimerization inhibitor	5582
	TC-S 7009	High affinity and selective HIF-2α inhibitor	5243
	Echinomycin	Highly potent and selective HIF-1α inhibitor	5520
	Evofosfamide	Hypoxia-activated DNA alkylating agent prodrug	7507
Inducers	VH 298	Inhibitor of E3 ubiquitin ligase VHL, Inhibits interaction between VHL and HIF-1α	6156
	ML 228	HIF pathway activator, Iron chelator, PHD independent	4565

Find Out More About HIF Signaling

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Molecules to Indirectly Modulate Hypoxia Signaling

Modulating proteins that play a role in HIF expression is another way to promote or inhibit the cellular response to hypoxia and modulate HIF expression. For example, STAT3 upregulates HIF1 mRNA expression, so inhibiting STAT3 would prevent HIF1 upregulation and inhibit the hypoxic response. Similarly, activating STAT3 would promote HIF1 expression and downstream targets of hypoxia signaling.

	Small Molecule	Mechanism	Bio-Techne Catalog #
Inhibitors	CGP 37157	Antagonize mitochondrial Na+/Ca2+ exchange	1114
	Stattic	Selective STAT3 inhibitor; STAT 3 upregulates HIF1 expression at mRNA level	2798
	Rapamycin	mTOR inhibitor; mTOR upregulates HIF1 expression at protein level	1292
	Torin 1		4247
	LC2	KRAS Protac®	7420
Inhibitors	Prostratin	Activates NF-kB and PKC	5739
	Colivelin	STAT3 activator	3945

Learn More About PROTAC^® and Targeted Protein Degradation from Tocris

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