Hypoxia induces peroxisome proliferator-activated receptor γ expression via HIF-1-dependent mechanisms in HepG2 cell line.

Hypoxia-inducible factor-1 (HIF-1) can activate expression of a broad range of genes in response to hypoxia. It has been shown that the levels of peroxisome proliferator-activated receptor γ (PPARγ) are influenced by changes in oxygen tension, and PPARγ plays a critical role in metabolism regulation and cancers. In this research, we observed an increased PPARγ mRNA and protein levels in company with increased HIF-1 protein levels in HepG2 cells in hypoxia as compared with in normoxia. Enforced expression of HIF-1α induced PPARγ1 and PPARγ2 expression, while knockdown of HIF-1α by small interference RNA deduced PPARγ1 and PPARγ2 expression in HepG2 cells under hypoxic conditions. By dual-luciferase reporter assay and chromatin immunoprecipitation assay we confirmed a functional hypoxic response element (HRE) localized at 684bp upstream of the transcriptional start site (TSS) of PPARγ1 and a functional HRE localized at 204bp downstream of the TSS of PPARγ2 in HepG2 cells. Additionally we observed an increase and co-presence of PPARγ and HIF-1α, and a highly positive correlation between PPARγ expression and HIF-1α expression (r=0.553, p<0.0001), in the same tumor tissue areas of hepatocellular carcinoma patients. Our data suggested a new mechanism of hepatocellular carcinoma cells response to hypoxia.

Multimodal mechanisms of pathogenic variants in the signal peptide of FIX leading to hemophilia B.

Signal peptide (SP) is essential for protein secretion, and pathogenic variants in the SP of FIX have been identified in hemophilia B (HB). However, the underlying mechanism for the genotype-phenotype correlation of these variants has not been well studied. Here we systematically examined the effects of 13 pathogenic point variants in the SP of FIX using different approaches. Our results showed that these point variants lead to HB by missense variants and/or aberrant pre-mRNA splicing. The missense variants in h-region mainly affected the co-translational translocation function of the SP, and those in c-region caused FIX deficiency mainly by disturbing the co-translational translocation and/or cleavage of the SP. Almost absolute aberrant pre-mRNA splicing was only observed in variants of c.82T>G, but a slight change of splicing patterns was found in variants of c.53G>T, c.77C>A, c.82T>C, and c.83G>A, indicating that these variants might have different degree to affect pre-mRNA splicing. Although two 6-nt deletion aberrant pre-mRNA splicing products caused FIX deficiency by disturbing the SP cleavage, but they could produce some functional mature FIX and vitamin K could increase the secretion of functional FIX. Taken together, our data indicated that pathogenic variants in the SP of FIX caused HB through diverse molecular mechanisms or even a mixture of several mechanisms, and vitamin K availability could be partially attributed to varying bleeding tendencies in patients carrying the same variant in the SP.

Hypoxia-inducible factor-1 (HIF-1) promotes LDL and VLDL uptake through inducing VLDLR under hypoxia.

Metabolism under hypoxia is significantly different from that under normoxia. It has been well elucidated that HIF-1 (hypoxia-inducible factor-1) plays a central role in regulating glucose metabolism under hypoxia; however, the role of HIF-1 in lipid metabolism has not yet been well addressed. In the present study we demonstrate that HIF-1 promotes LDL (low-density lipoprotein) and VLDL (very-LDL) uptake through regulation of VLDLR (VLDL receptor) gene expression under hypoxia. Increased VLDLR mRNA and protein levels were observed under hypoxic or DFO (deferoxamine mesylate salt) treatment in MCF7, HepG2 and HeLa cells. Using dual-luciferase reporter and ChIP (chromatin immunoprecipitation) assays we confirmed a functional HRE (hypoxia-response element) which is localized at +405 in exon 1 of the VLDLR gene. Knockdown of HIF1A (the α subunit of HIF-1) and VLDLR, but not HIF2A (the α subunit of HIF-2), attenuated hypoxia-induced lipid accumulation through affecting LDL and VLDL uptake. Additionally we also observed a correlation between HIF-1 activity and VLDLR expression in hepatocellular carcinoma specimens. The results of the present study suggest that HIF-1-mediated VLDLR induction influences intracellular lipid accumulation through regulating LDL and VLDL uptake under hypoxia.

Hypoxia-inducible factor 1-mediated human GATA1 induction promotes erythroid differentiation under hypoxic conditions.

Hypoxia-inducible factor promotes erythropoiesis through coordinated cell type-specific hypoxia responses. GATA1 is essential to normal erythropoiesis and plays a crucial role in erythroid differentiation. In this study, we show that hypoxia-induced GATA1 expression is mediated by HIF1 in erythroid cells. Under hypoxic conditions, significantly increased GATA1 mRNA and protein levels were detected in K562 cells and erythroid induction cultures of CD34(+) haematopoietic stem/progenitor cells. Enforced HIF1α expression increased GATA1 expression, while HIF1α knockdown by RNA interference decreased GATA1 expression. In silico analysis revealed one potential hypoxia response element (HRE). The results from reporter gene and mutation analysis suggested that this element is necessary for hypoxic response. Chromatin immunoprecipitation (ChIP)-PCR showed that the putative HRE was recognized and bound by HIF1 in vivo. These results demonstrate that the up-regulation of GATA1 during hypoxia is directly mediated by HIF1.The mRNA expression of some erythroid differentiation markers was increased under hypoxic conditions, but decreased with RNA interference of HIF1α or GATA1. Flow cytometry analysis also indicated that hypoxia, desferrioxamine or CoCl(2) induced expression of erythroid surface markers CD71 and CD235a, while expression repression of HIF1α or GATA1 by RNA interference led to a decreased expression of CD235a. These results suggested that HIF1-mediated GATA1 up-regulation promotes erythropoiesis in order to satisfy the needs of an organism under hypoxic conditions.

Hypoxic induction of human erythroid-specific δ-aminolevulinate synthase mediated by hypoxia-inducible factor 1.

Hypoxia-inducible factor 1 (HIF1) is a heterodimeric basic helix-loop-helix transcription factor that regulates many key genes. δ-Aminolevulinate synthase (ALAS) catalyzes the first and rate-limiting reaction in the heme biosynthetic pathway. In this study, we show that hypoxia-induced expression of erythroid-specific ALAS2 is mediated by HIF1 in erythroid cells. Under hypoxic conditions, significantly increased ALAS2 mRNA and protein levels were detected in K562 cells and erythroid induction cultures of CD34+ hematopoietic stem/progenitor cells. Enforced HIF1α expression increased the level of ALAS2 expression, while HIF1α knockdown by RNA interference decreased the level of ALAS2 expression. In silico analysis revealed three potential hypoxia-response elements (HREs) that are located 611, 621, and 741 bp downstream of the ALAS2 gene. The results from reporter gene and mutation analysis suggested that these elements are necessary for a maximal hypoxic response. Chromatin immunoprecipitation and polymerase chain reaction showed that the HREs could be recognized and bound by HIF1α in vivo. These results demonstrate that the upregulation of ALAS2 during hypoxia is directly mediated by HIF1. We hypothesize that HIF1-mediated ALAS2 upregulation promotes erythropoiesis to satisfy the needs of an organism under hypoxic conditions. This may be accomplished via increased heme levels and an interaction between ALAS2 and erythropoietin.

Analysis of two sequence variants in peroxisome proliferator activated receptor gamma gene in Tajik population at high altitudes and Han population at low altitudes in China.

The Tajik people in China have resided at high altitude for thousands of years. We analyzed the Pro12Ala (C > G) polymorphism in exon B and the 161C > T polymorphism in exon 6 of peroxisome proliferator activated receptor gamma gene (PPARG) in Chinese Tajik population living at high altitude and Chinese Han population living at low attitude. Significant higher frequencies of the CG and GG genotypes and G allele of the Pro12Ala (C > G) polymorphism were observed in the Tajik population than that in the Han population (P < 0.0001), which suggested the G allele was associated with high-altitude adaptation in the dominate model. The significant differences were remained in both of the male and female groups after stratified by gender, and the differences were more pronounced in men (G versus C, OR = 7.700) than in women (OR = 5.056). No significant difference was observed for the 161C > T polymorphism in the two populations. The frequencies of haplotypes GT (P < 0.0001) and GC (P < 0.05) were significantly higher, while the frequency of CT (P < 0.0001) was significantly lower in the Tajik population than that in the Han population. Our results suggest that PPARG is a candidate gene for high-altitude adaptation in the Chinese Tajik population.

Analysis of two sequence variants in peroxisome proliferator-activated receptor gamma gene in a Tibetan population at high altitude and a Han population at low altitude in China.

BACKGROUND: The Tibetan people in China have lived at high altitude for thousands of years, raising the possibility that the Tibetans are genetically adapted to high altitude. In this study we analyzed the Pro12Ala (C>G) polymorphism in exon 2 and the 161C>T polymorphism in exon 6 of peroxisome proliferator-activated receptor gamma gene (PPARγ) in a Tibetan population and a Han population. MATERIAL/METHODS: We recruited 142 Tibetan volunteers who are permanent inhabitants in Qingzang plateau (higher elevation) and 266 Han volunteers who are permanent inhabitants in the plain (lower elevation). PCR/RFLP method was applied to examine the 2 polymorphisms in the 2 populations. RESULTS: Significantly higher Pro12Ala (C>G) CC genotype frequency and 161C>T CC genotype frequency were observed in the Tibetan population compared to the Han population (p<0.001). When the samples were stratified by sex, significant differences were only observed in females. The haplotypes constructed by Pro12Ala (C>G) and 161C>T were also analyzed. The frequency of the haplotype CC (p<0.0001) was significantly higher, while the frequency of the haplotype CT (p<0.0001) and GT (p<0.01) was significantly lower in the Tibetan population than in the Han population. CONCLUSIONS: Our results suggested that PPARγ might be a candidate gene for high-altitude adaptation; the Pro12Ala (C>G) CC genotype and/or the 161C>T CC genotype are possibly advantageous factors in the female Tibetan population. Alternatively, the difference of the Pro12Ala (C>G) genotype distribution and /or the difference of the 161C>T genotype distribution in the 2 populations may be due to the racial difference.

[Association of the polymorphisms of sodium transport related genes with essential hypertension].

OBJECTIVE: To investigate the association of the polymorphisms of rs4961 in alpha-adducin (ADD1) and rs28933400 in Na+/K+ -ATPase a2 (ATP1A2) genes, the products of which are important for sodium transport, with essential hypertension. METHODS: Mutagenically separated PCR (MS-PCR) was used to detect the genotypes of the two loci. The subjects were recruited randomly including 196 patients of essential hypertension and 192 healthy controls. RESULTS: The frequencies of genotypes and alleles of in the ADD1 gene were significantly different between the patients and controls respectively (P=0.03, P=0.04). There was significant relationship between the genotypes of rs4961 and systolic blood pressure and blood sodium concentration. However, there was no significant relationship between the rs4961 genotypes and diastolic blood pressure, body mass index, blood kalium and chlorine concentrations. There was no polymorphism at the rs28933400 locus in the subjects analyzed. CONCLUSION: The rs4961 polymorphism of the ADD1 gene is associated with essential hypertension, but the rs28933400 locus in the ATP1A2 gene may have no association with essential hypertension in the studied population.

[Cytogenetic studies of 131 patients with primary amenorrhea (including three novel abnormal karyotypes)].

The reason for primary amenorrhea is very complicated. One of the main causes is chromosome anomalies. Karyotype analysis of 131 patients with primary amenorrhea using the G-banding technique revealed that 83 patients (63.36%) have a normal female karyotype, and 48 patients (36.64%) have abnormal karyotypes including three novel abnormal karyotypes [46,X,t(X;1)(q22;p34); 46,X,t(X;5;6)(p11.2;q35;q16); 46,XX,t(4;9)(q21;p22),(6;10)(p25;q25), t(11; 14)(q23; q32)]. In addition, when the clinical features and karyotypes of 33 Turner's(TS) patients were compared with M.Elsheikh's data, significant differences were found in the proportion of short stature, webbed neck, low posterior hairline and cubitus valgus, which suggests that there are differences in clinical features of TS patients between westerner and easterner. Karyotype analysis of two patients with X-autosome translocations indicated that Xp11.2 and Xq22 may be associated with primary amenorrhea.

Hypoxia-inducible factor 1-mediated regulation of PPP1R3C promotes glycogen accumulation in human MCF-7 cells under hypoxia.

Hundreds of genes can be regulated by hypoxia-inducible factor 1 (HIF1) under hypoxia. Here we demonstrated a HIF1-mediated induction of protein phosphatase 1, regulatory subunit 3C gene (PPP1R3C) in human MCF7 cells under hypoxia. By mutation analysis we confirmed the presence of a functional hypoxia response element that is located 229bp upstream from the PPP1R3C gene. PPP1R3C induction correlates with a significant glycogen accumulation in MCF7 cells under hypoxia. Knockdown of either HIF1α or PPP1R3C attenuated hypoxia-induced glycogen accumulation significantly. Knockdown of HIF2α reduced hypoxia-induced glycogen accumulation slightly (but not significantly). Our results demonstrated that HIF1 promotes glycogen accumulation through regulating PPP1R3C expression under hypoxia, which revealed a novel metabolic adaptation of cells to hypoxia.