Maimon E. Hubbi

The hypoxia-inducible transcription factors HIF-1 and HIF-2 regulate the response to hypoxia. Both proteins are dimers of an alpha subunit and a shared beta subunit. Under hypoxic conditions, the alpha subunits are stabilized, and the transactivation ability of the HIF-1 transcription factor is induced. Accordingly, assessment of HIF-1α protein levels and HIF transcriptional activity serve as an indirect indicator of hypoxia. In this series of protocols, I describe three methods to probe the HIF pathway. Key words Hypoxia Hypoxia-inducible factor Western blot Reporter assay qRT-PCR

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PDF file - 98K, Figure S1. HIF-1α is required for PLOD induction under hypoxia in mouse embryo fibroblasts.

PDF file - 135K, Figure S2. Efficiency of PLOD2 knockdown in MDA-MB-435 cells.

PDF file - 135K, Figure S2. Efficiency of PLOD2 knockdown in MDA-MB-435 cells.

PDF file - 98K, Figure S1. HIF-1α is required for PLOD induction under hypoxia in mouse embryo fibroblasts.

A major factor in the progression to heart failure in humans is the inability of the adult heart to repair itself after injury. We recently demonstrated that the early postnatal mammalian heart is capable of regeneration following injury through proliferation of preexisting cardiomyocytes^1,2 and that Meis1, a three amino acid loop extension (TALE) family homeodomain transcription factor, translocates to cardiomyocyte nuclei shortly after birth and mediates postnatal cell cycle arrest³. Here we report that Hoxb13 acts as a cofactor of Meis1 in postnatal cardiomyocytes. Cardiomyocyte-specific deletion of Hoxb13 can extend the postnatal window of cardiomyocyte proliferation and reactivate the cardiomyocyte cell cycle in the adult heart. Moreover, adult Meis1–Hoxb13 double-knockout hearts display widespread cardiomyocyte mitosis, sarcomere disassembly and improved left ventricular systolic function following myocardial infarction, as demonstrated by echocardiography and magnetic resonance imaging. Chromatin immunoprecipitation with sequencing demonstrates that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and cell cycle. Finally, we show that the calcium-activated protein phosphatase calcineurin dephosphorylates Hoxb13 at serine-204, resulting in its nuclear localization and cell cycle arrest. These results demonstrate that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and proliferation and provide mechanistic insights into the link between hyperplastic and hypertrophic growth of cardiomyocytes.

Hypoxia is a physiological cue that impacts diverse physiological processes, including energy metabolism, autophagy, cell motility, angiogenesis, and erythropoiesis. One of the key cell-autonomous effects of hypoxia is as a modulator of cell proliferation. For most cell types, hypoxia induces decreased cell proliferation, since an increased number of cells, with a consequent increase in O₂ demand, would only exacerbate hypoxic stress. However, certain cell populations maintain cell proliferation in the face of hypoxia. This is a common pathological hallmark of cancers, but can also serve a physiological function, as in the maintenance of stem cell populations that reside in a hypoxic niche. This review will discuss major molecular mechanisms by which hypoxia regulates cell proliferation in different cell populations, with a particular focus on the role of hypoxia-inducible factors.