Most of the genes induced by hypoxia are regulated by a family of transcription factors termed hypoxia-inducible factors (HIF). Under normoxic conditions, HIF␣proteins are very unstable due to hydroxylation by a recently described family of proline hydroxylases termed EGL-Nine homologs (EGLN). Upon hydroxylation, HIF␣ is recognized by the product of the tumor suppressor vhl and targeted for proteosomal degradation. Since EGLNs require oxygen to catalize HIF hydroxylation, this reaction does not efficiently occur under low oxygen tension. Thus, under hypoxia, HIF␣ escapes from degradation and transcribes target genes. The mRNA levels of two of the three EGLNs described to date are induced by hypoxia, suggesting that they might be novel HIF target genes; however, no proof for this hypothesis has been reported. Here we show that the induction of EGLN1 and -3 by hypoxia is found in a wide range of cell types. The basal levels of EGLN3 are always well below those of EGLN1 and EGLN2, and its induction by hypoxia is larger than that found for EGLN1. The inhibitor of transcription, actinomycin D, prevents the increase of EGLN3 mRNA induced by hypoxia, indicating that it is due to enhanced gene expression. Interestingly, EGLN1 and EGLN3 mRNAs were also triggered by EGLN inhibitors, suggesting the involvement of HIF␣ in the control of its transcription. In agreement with this possibility, pVHL-deficient cell lines, which present high HIF activity under normoxia, also showed dramatically increased normoxic levels of EGLN3. Moreover, the overexpression of an oxygen-insensitive mutant form of HIF␣ resulted in increased normoxic levels of EGLN3 mRNA. Finally, hypoxic induction of EGLNs was not observed in cells lacking functional HIF␣.Mammalian cells are critically dependent on oxygen for survival; thus, it is not surprising that an evolutionarily conserved machinery able to sense oxygen levels is present in almost every cell type studied. The activity of a variety of signaling molecules has been found to be altered in response to changes in oxygen tension, including ion channels (1), kinases (2-7), and transcription factors (2, 8 -13). Among them, the most common and best understood response is the activation of a group of basic helix-loop-helix-Per/Aryl hydrocarbon receptor translocator/Sim domain transcription factors, called hypoxiainducible factors (HIF) 1 (14). In fact, HIF is responsible for the induction of the vast majority of genes up-regulated in response to decreased oxygen tension. HIF transcription factors are heterodimers of a constitutively expressed ␤ subunit (HIF-␤; also known as the aryl hydrocarbon receptor translocator) and an oxygen-regulated ␣ subunit (HIF-␣). Although there are three types of ␣ subunits, HIF-1␣, HIF-2␣ (also known as the endothelial Per/Aryl hydrocarbon receptor translocator/Sim domain), and HIF-3␣, encoded by different genes, it is currently accepted that they are regulated in a similar fashion by oxygen availability. The expression of HIF␤ and ␣ subunits genes, their mRNA stability, ...

Hypoxia-inducible factors (HIF) are heterodimeric (␣/␤) transcription factors that play a fundamental role in cellular adaptation to low oxygen tension. In the presence of oxygen, the HIF-␣ subunit becomes hydroxylated at specific prolyl residues by prolyl hydroxylases. This post-translational modification is recognized by the von Hippel-Lindau (VHL) protein, which targets HIF-␣ for degradation. In the absence of oxygen, HIF-␣ hydroxylation is compromised and this subunit is stabilized. We have previously shown that the hypoxia-induced accumulation of HIF-␣ protein is strongly impaired by the inhibitor of diacylglycerol kinase, R59949. Here, we have investigated the mechanisms through which this inhibitor exerts its effect. We found that R59949 inhibits the accumulation of HIF-1/2␣ protein without affecting the expression of their mRNAs. We also determined that R59949 could only block the accumulation of HIF-␣ in the presence of VHL protein. In agreement with this, the binding of VHL to endogenous HIF-␣ was significantly enhanced after R59949 treatment, even under hypoxic conditions. In addition, we found that R59949 could stimulate prolyl hydroxylase both at 21% O 2 as well as at 1% O 2 . Taken together, these results reveal that R59949 is an activator of HIF prolyl hydroxylases. This is of particular interest when we consider that, to date, mainly inhibitors of these enzymes have been described.

In mammalians, advancing age is associated with sarcopenia, the progressive and involuntary loss of muscle mass and strength. Hyperphosphatemia is an aging-related condition involved in several pathologies. The aim of this work was to assess whether hyperphosphatemia plays a role in the age-related loss of mass muscle and strength by inducing cellular senescence in murine myoblasts and to explore the intracellular mechanism involved in this effect. Cultured mouse C2C12 cells were treated with 10 mM beta-glycerophosphate (BGP] at different periods of time to induce hyperphosphatemia. BGP promoted cellular senescence after 24 h of treatment, assessed by the increased expression of p53, acetylated-p53 and p21 and senescence associated β-galactosidase activity. In parallel, BGP increased ILK expression and activity, followed by mTOR activation and autophagy reduction. Knocking-down ILK expression increased autophagy and protected cells from senescence induced by hyperphosphatemia. BGP also reduced the proliferative capacity of cultured myoblasts. Old mice (24-months-old] presented higher serum phosphate concentration, lower forelimb strength, higher expression of p53 and ILK and less autophagy in vastus muscle than young mice (5-months-old]. In conclusion, we propose that hyperphosphatemia induces senescence in cultured myoblasts through ILK overexpression, reducing their proliferative capacity, which could be a mechanism involved in the development of sarcopenia, since old mice showed loss of muscular strength correlated with high serum phosphate concentration and increased levels of ILK and p53.

SummaryHyperphosphatemia is related to some pathologies, affecting vascular cell behavior. This work analyzes whether high concentration of extracellular phosphate induces endothelial senescence through up‐regulation of endothelin‐1 (ET‐1), exploring the mechanisms involved. The phosphate donor β‐glycerophosphate (BGP) in human endothelial cells increased ET‐1 production, endothelin‐converting enzyme‐1 (ECE‐1) protein, and mRNA expression, which depend on the AP‐1 activation through ROS production. In parallel, BGP also induced endothelial senescence by increasing p16 expression and the senescence‐associated β‐galactosidase (SA‐ß‐GAL) activity. ET‐1 itself was able to induce endothelial senescence, increasing p16 expression and SA‐ß‐GAL activity. In addition, senescence induced by BGP was blocked when different ET‐1 system antagonists were used. BGP increased ROS production at short times, and the presence of antioxidants prevented the effect of BGP on AP1 activation, ECE‐1 expression, and endothelial senescence. These findings were confirmed in vivo with two animal models in which phosphate serum levels were increased: seven/eight nephrectomized rats as chronic kidney disease models fed on a high phosphate diet and aged mice. Both models showed hyperphosphatemia, higher levels of ET‐1, and up‐regulation in aortic ECE‐1, suggesting a direct relationship between hyperphosphatemia and ET‐1. Present results point to a new and relevant role of hyperphosphatemia on the regulation of ET‐1 system and senescence induction at endothelial level, both in endothelial cells and aorta from two animal models. The mechanism involved showed a higher ROS production, which probably activates AP‐1 transcription factor and, as a result, ECE‐1 expression, increasing ET‐1 synthesis, which in consequence induces endothelial senescence.

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Background Hyperphosphatemia has been related to the development of sarcopenia in aging mice. We describe the intracellular mechanisms involved in the impairment of the myogenic differentiation promoted by hyperphosphatemia and analyse these mechanisms in the muscle from older mice. Methods C 2 C 12 cells were grown in 2% horse serum in order to promote myogenic differentiation, in the presence or absence of 10 mM beta-glycerophosphate (BGP) for 7 days. Troponin T, paired box 7 (Pax-7), myogenic factor 5 (Myf5), myogenic differentiation 1 (MyoD), myogenin (MyoG), myocyte enhancer factor 2 (MEF2C), P300/CBP-associated factor (PCAF), histone deacetylase 1 (HDAC1), fibronectin, vimentin, and collagen I were analysed at 48, 72, and 168 h, by western blotting or by immunofluorescence staining visualized by confocal microscopy. Studies in mice were performed in 5-and 24-month-old C57BL6 mice. Three months before sacrifice, 21-month-old mice were fed with a standard diet or a low phosphate diet, containing 0.6% or 0.2% phosphate, respectively. Serum phosphate concentration was assessed by a colorimetric method and forelimb strength by a grip test. Fibrosis was observed in the tibialis anterior muscle by Sirius Red staining. In gastrocnemius muscle, MyoG, MEF2C, and fibronectin expressions were analysed by western blotting. Results Cells differentiated in the presence of BGP showed near five times less expression of troponin T and kept higher levels of Pax-7 than control cells indicating a reduced myogenic differentiation. BGP reduced Myf5 about 50% and diminished MyoD transcriptional activity by increasing the expression of HDAC1 and reducing the expression of PCAF. Consequently, BGP reduced to 50% the expression of MyoG and MEF2C. A significant increase in the expression of fibrosis markers as collagen I, vimentin, and fibronectin was found in cells treated with BGP. In mice, serum phosphate (17.24 ± 0.77 mg/dL young; 23.23 ± 0.81 mg/dL old; 19.09 ± 0.75 mg/dL old with low phosphate diet) correlates negatively (r = À0.515, P = 0.001) with the muscular strength (3.13 ± 0.07 gf/g young; 1.70 ± 0.12 gf/ g old; 2.10 ± 0.09 gf/g old with low phosphate diet) and with the expression of MyoG (r = À0.535, P = 0.007) and positively with the expression of fibronectin (r = 0.503, P = 0.001) in gastrocnemius muscle. The tibialis anterior muscle from old mice showed muscular fibrosis. Older mice fed with a low phosphate diet showed improved muscular parameters relative to control mice of similar age. Conclusions Hyperphosphatemia impairs myogenic differentiation, by inhibiting the transcriptional activity of MyoD, and enhances the expression of fibrotic genes in cultured myoblasts. Experiments carried out in older mice demonstrate a close relationship between age-related hyperphosphatemia and the decrease in the expression of myogenic factors and the increase in factors related to muscle fibrosis.

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