Systemic long-term inactivation of hypoxia-inducible factor prolyl 4-hydroxylase 2 ameliorates aging-induced changes in mice without affecting their life span.

Hypoxia inactivates hypoxia-inducible factor (HIF) prolyl 4-hydroxylases (HIF-P4Hs), which stabilize HIF and upregulate genes to restore tissue oxygenation. HIF-P4Hs can also be inhibited by small molecules studied in clinical trials for renal anemia. Knowledge of systemic long-term inactivation of HIF-P4Hs is limited but crucial, since HIF overexpression is associated with cancers. We aimed to determine the effects of systemic genetic inhibition of the most abundant isoenzyme HIF prolyl 4-hydroxylase-2 (HIF-P4H-2)/PHD2/EglN1 on life span and tissue homeostasis in aged mice. Our data showed no difference between wild-type and HIF-P4H-2-deficient mice in the average age reached. There were several differences, however, in the primary causes of death and comorbidities, the HIF-P4H-2-deficient mice having less inflammation, liver diseases, including cancer, and myocardial infarctions, and not developing anemia. No increased cancer incidence was observed due to HIF-P4H-2-deficiency. These data suggest that chronic inactivation of HIF-P4H-2 is not harmful but rather improves the quality of life in senescence.

A long hypoxia-inducible factor 3 isoform 2 is a transcription activator that regulates erythropoietin.

Hypoxia-inducible factor (HIF), an αß dimer, is the master regulator of oxygen homeostasis with hundreds of hypoxia-inducible target genes. Three HIF isoforms differing in the oxygen-sensitive α subunit exist in vertebrates. While HIF-1 and HIF-2 are known transcription activators, HIF-3 has been considered a negative regulator of the hypoxia response pathway. However, the human HIF3A mRNA is subject to complex alternative splicing. It was recently shown that the long HIF-3α variants can form αß dimers that possess transactivation capacity. Here, we show that overexpression of the long HIF-3α2 variant induces the expression of a subset of genes, including the erythropoietin (EPO) gene, while simultaneous downregulation of all HIF-3α variants by siRNA targeting a shared HIF3A region leads to downregulation of EPO and additional genes. EPO mRNA and protein levels correlated with HIF3A silencing and HIF-3α2 overexpression. Chromatin immunoprecipitation analyses showed that HIF-3α2 binding associated with canonical hypoxia response elements in the promoter regions of EPO. Luciferase reporter assays showed that the identified HIF-3α2 chromatin-binding regions were sufficient to promote transcription by all three HIF-α isoforms. Based on these data, HIF-3α2 is a transcription activator that directly regulates EPO expression.

HIF-P4H-2 deficiency protects against skeletal muscle ischemia-reperfusion injury.

We show here that mice hypomorphic for hypoxia-inducible factor prolyl 4-hydroxylase-2 (HIF-P4H-2) (Hif-p4h-2 (gt/gt)), the main regulator of the stability of the HIFα subunits, have normoxic stabilization of HIF-1α and HIF-2α in their skeletal muscles. The size of the capillaries, but not their number, was increased in the skeletal muscles of the Hif-p4h-2 (gt/gt) mice, whereas the amount of glycogen was reduced. The expression levels of genes for glycolytic enzymes, glycogen branching enzyme 1 and monocarboxylate transporter 4, were increased in the Hif-p4h-2 (gt/gt) skeletal muscles, whereas no significant increases were detected in the levels of any vasculature-influencing factor studied. Serum lactate levels of the Hif-p4h-2 (gt/gt) mice recovered faster than those of the wild type following exercise. The Hif-p4h-2 (gt/gt) mice had elevated hepatic phosphoenolpyruvate carboxykinase activity, which may have contributed to the faster clearance of lactate. The Hif-p4h-2 (gt/gt) mice had smaller infarct size following limb ischemia-reperfusion injury. The increased capillary size correlated with the reduced infarct size. Following ischemia-reperfusion, glycogen content and ATP/ADP and CrP/Cr levels of the skeletal muscle of the Hif-p4h-2 (gt/gt) mice were higher than in the wild type. The higher glycogen content correlated with increased expression of phosphofructokinase messenger RNA (mRNA) and the increased ATP/ADP and CrP/Cr levels with reduced apoptosis, suggesting that HIF-P4H-2 deficiency supported energy metabolism during ischemia-reperfusion and protection against injury. Key messages: HIF-P4H-2 deficiency protects skeletal muscle from ischemia-reperfusion injury. The mechanisms involved are mediated via normoxic HIF-1α and HIF-2α stabilization. HIF-P4H-2 deficiency increases capillary size but not number. HIF-P4H-2 deficiency maintains energy metabolism during ischemia-reperfusion.

RSK2 protein suppresses integrin activation and fibronectin matrix assembly and promotes cell migration.

Modulation of integrin activation is important in many cellular functions including adhesion, migration, and assembly of the extracellular matrix. RSK2 functions downstream of Ras/Raf and promotes tumor cell motility and metastasis. We therefore investigated whether RSK2 affects integrin function. We report that RSK2 mediates Ras/Raf inactivation of integrins. As a result, we find that RSK2 impairs cell adhesion and integrin-mediated matrix assembly and promotes cell motility. Active RSK2 appears to affect integrins by reducing actin stress fibers and disrupting focal adhesions. Moreover, RSK2 co-localizes with the integrin activator talin and is present at integrin cytoplasmic tails. It is thereby in a position to modulate integrin activation and integrin-mediated migration. Activation of RSK2 promotes filamin phosphorylation and binding to integrins. We also find that RSK2 is activated in response to integrin ligation to fibronectin. Thus, RSK2 could participate in a feedback loop controlling integrin function. These results reveal RSK2 as a key regulator of integrin activity and provide a novel mechanism by which it may promote cell migration and cancer metastasis.

Transmembrane prolyl 4-hydroxylase is a fourth prolyl 4-hydroxylase regulating EPO production and erythropoiesis.

An endoplasmic reticulum transmembrane prolyl 4-hydroxylase (P4H-TM) is able to hydroxylate the α subunit of the hypoxia-inducible factor (HIF) in vitro and in cultured cells, but nothing is known about its roles in mammalian erythropoiesis. We studied such roles here by administering a HIF-P4H inhibitor, FG-4497, to P4h-tm(-/-) mice. This caused larger increases in serum Epo concentration and kidney but not liver Hif-1α and Hif-2α protein and Epo mRNA levels than in wild-type mice, while the liver Hepcidin mRNA level was lower in the P4h-tm(-/-) mice than in the wild-type. Similar, but not identical, differences were also seen between FG-4497-treated Hif-p4h-2 hypomorphic (Hif-p4h-2(gt/gt)) and Hif-p4h-3(-/-) mice versus wild-type mice. FG-4497 administration increased hemoglobin and hematocrit values similarly in the P4h-tm(-/-) and wild-type mice, but caused higher increases in both values in the Hif-p4h-2(gt/gt) mice and in hematocrit value in the Hif-p4h-3(-/-) mice than in the wild-type. Hif-p4h-2(gt/gt)/P4h-tm(-/-) double gene-modified mice nevertheless had increased hemoglobin and hematocrit values without any FG-4497 administration, although no such abnormalities were seen in the Hif-p4h-2(gt/gt) or P4h-tm(-/-) mice. Our data thus indicate that P4H-TM plays a role in the regulation of EPO production, hepcidin expression, and erythropoiesis.

Roles of the human hypoxia-inducible factor (HIF)-3α variants in the hypoxia response.

The hypoxia-inducible transcription factor (HIF) controls (in an oxygen-dependent manner) the expression of a large number of genes whose products are involved in the response of cells to hypoxia. HIF is an αß dimer that binds to hypoxia response elements (HREs) in its target genes. Human HIF-α has three isoforms, HIF-1α, HIF-2α and HIF-3α, of which the roles of HIF-3α are largely unknown, although it is usually regarded as a negative regulator of HIF-1α and HIF-2α. The human HIF-3α locus is subject to extensive alternative splicing, leading to at least seven variants. We analyzed here the effects of the long variants and the short variant HIF-3α4 on the hypoxia response. All these variants were found to interact with HIF-ß, HIF-1α and HIF-2α. The long HIF-3α variants were localized in the nucleus in hypoxia, while HIF-3α4 was cytoplasmic. Interaction of the HIF-3α variants with HIF-1α inhibited the nuclear translocation of both. None of the long HIF-3α variants was capable of efficient induction of an HRE reporter in overexpression experiments, but instead inhibited the transcriptional activation of the reporter by HIF-1 and HIF-2. Unexpectedly, siRNA knock-down of the endogenous HIF-3α variants led to downregulation of certain HIF target genes, while overexpression of individual long HIF-3α variants upregulated certain HIF target genes in a variant and target gene-specific manner under conditions in which HIF-ß was not a limiting factor. These data indicate that the HIF-3α variants may have more versatile and specific roles in the regulation of the hypoxia response than previously anticipated.

Hypoxia-inducible factor (HIF)-3alpha is subject to extensive alternative splicing in human tissues and cancer cells and is regulated by HIF-1 but not HIF-2.

The hypoxia-inducible transcription factors (HIFs) play a central role in the response of cells to hypoxia. HIFs are alphabeta dimers, the human alpha subunit having three isoforms. HIF-3alpha is unique among the HIF-alpha isoforms in that its gene is subject to extensive alternative splicing. Database analyses have predicted the generation of six HIF-3alpha splice variants that utilize three alternative transcription initiation sites. None of these variants is likely to act as an efficient transcription factor, but some of them have been reported to inhibit HIF-1 and HIF-2 functions. We analyzed here for the first time in detail whether these six variants are indeed generated in various human tissues and cell lines. We identified four novel variants, named here HIF-3alpha7 to HIF-3alpha10, whereas we obtained no evidence for the predicted HIF-3alpha3 and HIF-3alpha5. Distinct differences in the expression patterns of the variants were found between human tissues, the levels being particularly low in many cancer cell lines. Hypoxia upregulated transcription from all three alternative HIF-3alpha promoters. siRNA experiments showed that this induction is mediated specifically by HIF-1 and not by HIF-2. The tissue-specific differences in the expression patterns and levels of the HIF-3alpha variants can be expected to modulate the hypoxia response of various tissues and cell types to different extents during development and in pathological situations. A further level of regulation is brought about by the fact that the levels of the HIF-3alpha transcripts themselves are regulated by hypoxia and by changes in HIF-1 levels.

The partial female to male sex reversal in Wnt-4-deficient females involves induced expression of testosterone biosynthetic genes and testosterone production, and depends on androgen action.

Wnt-4 signaling has been implicated in female development, because its absence leads to partial female to male sex reversal in the mouse. Instead of Mullerian ducts, Wnt-4-deficient females have Wolffian ducts, suggesting a role for androgens in maintaining this single-sex duct type in females. We demonstrate here that testosterone is produced by the ovary of Wnt-4-deficient female embryos and is also detected in the embryonic plasma. Consistent with this, the expression of several genes encoding enzymes in the pathway leading to the synthesis of testosterone in the mouse is induced in the Wnt-4-deficient ovary, including Cyp11a, Cyp17, Hsd3b1, Hsd17b1, and Hsd17b3. Inhibition of androgen action with an antiandrogen, flutamide, during gestation leads to complete degeneration of the Wolffian ducts in 80% of the mutant females and degeneration of the cortical layer that resembles the tunica albuginea in the masculinized ovary. However, androgen action is not involved in the sexually dimorphic organization of endothelial cells in the Wnt-4 deficient ovary, because flutamide did not change the organization of the coelomic vessel. These data imply that Wnt-4 signaling normally acts to suppress testosterone biosynthesis in the female, and that testosterone is the putative mediator of the masculinization phenotype in Wnt-4-deficient females.

Wnt-4 deficiency alters mouse adrenal cortex function, reducing aldosterone production.

Wnt-4 is a signaling factor with multiple roles in organogenesis, a deficiency that leads to abnormal development of the kidney, pituitary gland, female reproductive system, and mammary gland. Wnt-4 is expressed in the cortical region of the developing adrenal gland from embryonic d 11.5 onward, especially in the outermost part. Expression of Cyp11B2 and preadipocyte factor 1 is lowered in the glands of Wnt-4 mutant animals, resulting in significantly reduced aldosterone production in the newborn mutants, suggesting that Wnt-4 may be needed for proper formation of the zona glomerulosa. On the other hand, both proopiomelanocortin-derived peptide beta-endorphin and corticosterone concentration levels are elevated in Wnt-deficient mice, and the expression of Cyp17 is altered in Wnt-4 mutant females, so that it mimics the pattern specific for males. Finally, some cells that are positive for Cyp21, which is normally expressed only in the adrenal gland, are found in the gonads of Wnt-4-deficient embryos, indicating that Wnt-4 may play a role in cell migration or in the sorting of adrenal and gonadal cells during early development. In summary, these results point to a role for Wnt-4 in adrenal gland development and function.