Hypoxia Induced Factor in Chronic Kidney Disease: Friend or Foe?
Li Weiying,Zhao Yuliang,Fu Ping
Frontiers in medicine
Many studies have shown evidence that erythropoiesis-stimulating agents (ESAs), as a classic treatment for chronic kidney disease (CKD)-related anemia, have several disadvantages and may trigger various adverse events with long-term use. The hypoxia-induced factor (HIF) pathway has been intensively investigated in kidney disease, especially in CKD, as research has shown that HIF-mediated erythropoiesis might work as a potential therapeutic strategy for managing CKD-related anemia. Development of prolyl hydroxylase domain inhibitors (PHIs), as an effective HIF activator, is a valuable step toward finding a replacement for ESAs, which showed an effective erythropoiesis through a comprehensive and physiological approach by promoting erythropoietin production, increasing iron bioavailability and improving chronic inflammatory status. Heretofore no adverse events or obvious off-target effects have been reported in clinical trials of PHIs. Nevertheless, a cautious inspection with extended follow-up period is warranted to validate the safety of prolonged HIF elevation, especially considering its ambiguous role in fibrogenesis and inflammation responses and possible risks in accelerating vascular calcification and tumorigenesis. A weighed dosing strategy might be the key to circumvent the unexpected side-effect brought by pleotropic effects of HIF elevation and achieve a selective augmentation of HIF-mediated signaling pathway. New studies with longer follow-up period and adequate analysis about the risks for proinflammation, vascular calcification and tumorigenesis are needed to ensure the drugs are safe for long-term use before being widely accepted in daily clinical practice.
Roxadustat in the treatment of anaemia in chronic kidney disease.
Del Vecchio Lucia,Locatelli Francesco
Expert opinion on investigational drugs
INTRODUCTION:Anaemia is one of the hallmarks of advanced chronic kidney disease (CKD); it correlates with a lower quality of life and increased cardiovascular risk. Currently its management is based on iron and erythropoiesis-stimulating agents (ESAs) therapy. Given safety issues on ESA therapy and excessive iron use, anaemia management is still suboptimal. Areas covered: The inhibitors of the prolyl-hydroxylases domain (PHD) are oral drugs which activate the hypoxia-inducible factors (HIF) and stimulate the production of endogenous erythropoietin. Roxadustat (FG-4592) is a second-generation PHD inhibitor; it is undergoing now phase-III clinical development. Expert opinion: Phase-II clinical trials have shown that roxadustat is effective and save in the short term in either non-dialysis or dialysis CKD patients. Roxadustat is a chemical drug and thus has the potential of being cheaper than traditional ESAs. Given that the peaks of endogenous EPO are much lower than those observed with traditional ESA, it is possible to speculate the roxadustat (and more in general PHD inhibitors) will be safer than ESA on cardiovascular safety end-points. Considering that HIFs are involved in different pathways, with possible promotion of relevant side effects, their safety must be proven in long-term studies.
miR-210 expression in PBMCs from patients with systemic lupus erythematosus and rheumatoid arthritis.
Huang Q,Chen S-S,Li J,Tao S-S,Wang M,Leng R-X,Pan H-F,Ye D-Q
Irish journal of medical science
BACKGROUND:In hypoxic conditions, miRNA-210 plays an important role in regulating the expression of hypoxia-inducing factor-1α (HIF-1α) and the differentiation of T helper 17 (Th17) cells, and this may be involved in the development and function of the immune system. AIMS:This study was to investigate the miR-210 expression levels in peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) and its association with the clinical and laboratory features of both diseases. METHODS:Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to detect miR-210 expression levels in PBMCs from 35 patients with SLE, 38 patients with RA, and 35 healthy controls. RESULTS:Compared with the healthy controls, the miR-210 expression levels were significantly increased in patients with SLE (P = 0.001) and there was increased significantly expression of miR-210 in SLE with pleuritis (Z = -2.345, P = 0.019) and anti-SSB/La-positive group (Z = -2.076, P = 0.038). However, we have not found the significant correlation between the miR-210 levels and the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score (r = 0.091, P = 0.602). Although, no significant difference between miR-210 levels in RA patients and those in healthy controls was found (Z = -1.226, P = 0. 220). There was a significant decreased expression of miR-210 in active RA patients than inactive RA patients (Z = -4.011, P < 0.001). CONCLUSIONS:The dysregulation of miR-210 levels in SLE and RA patients suggests that miR-210 might play an important role in the pathogenesis of these diseases.
Effects of loading on chondrocyte hypoxia, HIF-1α and VEGF in the mandibular condylar cartilage of young rats.
Yu J,Liang F,Huang H,Pirttiniemi P,Yu D
Orthodontics & craniofacial research
OBJECTIVES:To investigate hypoxia-inducible factor 1-alpha (HIF-1α) and vascular endothelial growth factor (VEGF) expression under altered loading, and to explore the relationship between loading and hypoxia in the mandibular condylar cartilage of young rats. SETTING AND SAMPLE POPULATION:Eighty Sprague-Dawley rats. MATERIAL AND METHODS:The reduced loading group was fed soft food, and their incisors were cut to avoid occlusal contact. The increased loading group was fed hard food and had forced jaw-opening. Ten rats from each group (n = 10) were sacrificed at 12, 24, 48, and 96 hours after initiation of the experiment. Pimonidazole hydrochloride (Hypoxyprobe-1, HP-1) was used as a hypoxia marker to confirm the hypoxic state. Hypoxic chondrocytes as indicated by HP-1, HIF-1α and VEGF protein expressions were recognized by immunohistochemical detection. HIF-1α and VEGF mRNA expressions were detected by semi-quantitative RT-PCR. RESULTS:Hypoxyprobe-1 was confined in the upper layers of cartilage, and was most strongly expressed in the weight-bearing area of TMJ at 12 and 96 hours. Staining of HIF-1α and VEGF was most strongly expressed in the chondrocytes of the fibrous and proliferative layer at all time points. Furthermore, expressions were also displayed in the hypertrophic and calcified layers at 48 and 96 hours. The expressions of HIF-1α and VEGF mRNA were higher in the increased loading group than in the reduced loading group at 48 and 96 hours (P < . 05). CONCLUSION:Mechanical loading seems to directly induce weight-bearing area hypoxia followed by new vessel formation, which indicates that these factors are related and important for the development of cartilage.
Enhancement of bone regeneration with the accordion technique via HIF-1α/VEGF activation in a rat distraction osteogenesis model.
Xu Jia,Sun Yuxin,Wu Tianyi,Liu Yang,Shi Liu,Zhang Jinfang,Kang Qinglin,Chai Yimin,Li Gang
Journal of tissue engineering and regenerative medicine
Axial micromotion of bone fragments promotes callus formation and bone healing during the process of distraction osteogenesis (DO). This study investigated the effects of the combined axial compression and distraction (accordion) technique on bone regeneration in rat DO model. Male Sprague-Dawley rats (n = 62) underwent right tibial transverse osteotomy and were randomly divided into four groups after lengthening: control (no manipulation) and three experimental groups assigned on the basis of the period of accordion manoeuvres in the consolidation phase (Groups 1, 2, and 3 with accordion technique applied at Weeks 1, 3, and 5, respectively). Animals were terminated at 1 week after each accordion phase (i.e., Weeks 2, 4, and 6). Callus formation was monitored by X-ray radiography; new bone quality was evaluated by microcomputed tomography, histological analysis, and mechanical testing. Serum levels of hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) were measured. Callus formation after accordion manoeuvre at Week 3 (Group 2) increased significantly over time of consolidation. The microcomputed tomography and mechanical analysis revealed Group 2 had more newly formed bone and superior mechanical properties in contrast to the other groups at termination. Histomorphological and immunohistochemical analyses confirmed a greater degree of osteogenesis and angiogenesis corresponding to increased serum levels of HIF-1α and VEGF in Group 2. The accordion technique was effective in promoting bone consolidation via activation of HIF-1α/VEGF during DO. The accordion technique may be used in the middle phase of bone consolidation to promote bone formation in patients undergoing DO treatment.
Hypoxia inducible factor-1 (HIF-1α) reduced inflammation in spinal cord injury via miR-380-3p/ NLRP3 by Circ 0001723.
Li Xigong,Lou Xianfeng,Xu Sanzhong,Du Junhua,Wu Junsong
BACKGROUND:Spinal cord injury (SCI) is a severe central nervous system trauma. The present study aimed to evaluate the effect of HIF-1α on inflammation in spinal cord injury (SCI) to uncover the molecular mechanisms of anti-inflammation. RESULTS:HIF-1α was reduced in SCI model rats and HIF-1α activation reduced TNF-α, IL-1β, IL-6 and IL-18 levels in SCI model rats. Meanwhile, Circ 0001723 expression was down-regulated and miR-380-3p expression was up-regulated in SCI model rats. In vitro model, down-regulation of Circ 0001723 promoted TNF-α, IL-1β, IL-6 and IL-18 levels, compared with control negative group. However, over-expression of Circ 0001723 reduced TNF-α, IL-1β, IL-6 and IL-18 levels in vitro model. Down-regulation of Circ 0001723 suppressed HIF-1α protein expressions and induced NLRP3 and Caspase-1 protein expressions in vitro model by up-regulation of miR-380-3p. Next, inactivation of HIF-1α reduced the pro-inflammation effects of Circ 0001723 in vitro model. Then, si-NLRP3 also inhibited the pro-inflammation effects of Circ 0001723 in vitro model via promotion of autophagy. CONCLUSIONS:We concluded that HIF-1α reduced inflammation in spinal cord injury via miR-380-3p/ NLRP3 by Circ 0001723.
Salidroside improves angiogenesis-osteogenesis coupling by regulating the HIF-1α/VEGF signalling pathway in the bone environment.
Guo Qiaoyun,Yang Jing,Chen Yumeng,Jin Xin,Li Zongmin,Wen Xiaochang,Xia Qun,Wang Yue
European journal of pharmacology
Angiogenesis is essential for bone formation during skeletal development. HIF-1α and the HIF-responsive gene VEGF (vascular endothelial growth factor) are reported to be a key mechanism for coupling osteogenesis and angiogenesis. Salidroside (SAL), a major biologically active compound of Rhodiola rosea L., possesses diverse pharmacological effects. However, whether SAL can protect against bone loss via the HIF-1α/VEGF pathway, specifically by inducing angiogenesis-osteogenesis coupling in vivo, remains unknown. Therefore, in the present study, we employed primary human umbilical vein endothelial cells (HUVECs) and the permanent EA.hy926 human endothelial cell line to determine the cellular and molecular effects of SAL on vascular endothelial cells and the HIF-1α-VEGF signalling pathway in the coupling of angiogenesis-osteogenesis. The in vitro study revealed that the HUVECs and EA.hy926 cells treated with conditioned medium from osteoblast cells (MG-63 cells) treated with SAL or treated directly with SAL showed enhanced proliferation, migration and capillary structure formation. However, supplementation with an anti-VEGF antibody during the treatment of endothelial cells (ECs) significantly reversed the pro-angiogenic effect of SAL. Moreover, SAL upregulated HIF-1α expression and increased its transcriptional activity, consequently upregulating VEGF expression at the mRNA and protein levels. In addition, our in vivo analysis demonstrated that SAL can stimulate endothelial sprouting from metatarsal bones. Thus, our mechanistic study demonstrated that the pro-angiogenic effects of SAL involve HIF-1α-VEGF signalling by coordinating the coupling of angiogenesis-osteogenesis in the bone environment. Therefore, we have discovered an ideal molecule that simultaneously enhances angiogenesis and osteogenesis and thereby accelerates bone healing.
The peptidyl prolyl isomerase, PIN1 induces angiogenesis through direct interaction with HIF-2α.
Choi Min-A,Saeidi Soma,Han Hyeong-Jun,Kim Su-Jung,Kwon Nayoung,Kim Do-Hee,Min Sang-Hyun,Choi Bu Young,Surh Young-Joon
Biochemical and biophysical research communications
PIN1, the peptidyl-prolyl isomerase (PPIase), is an enzyme that changes the conformation of phosphoproteins. The conformational change induced by PIN1 alters the function and stability of the target proteins. PIN1 is overexpressed in many different types of malignancies, including breast, lung, cervical, brain and colorectal tumors. PIN1 overexpression has been associated with activation of multiple oncogenic signaling pathways during tumor development. Hypoxia-inducible factor 2α (HIF-2α), a transcription factor activated in hypoxia, plays a role in erythropoiesis, glycolysis, tissue invasion, metastasis and angiogenesis. In this study, we found the direct interaction between HIF-2α and PIN1 in colorectal cancer HCT116 cells. Notably, serine 16 and lysine 63 residues of PIN1 were critical for its interaction with HIF-2α. When PIN1 protein was silenced by transient transfection of PIN1 short interfering RNA, the expression of HIF-2α was attenuated under a hypoxic condition. Moreover, genetic and pharmacologic inhibition of PIN1 abrogated the expression of vascular endothelial growth factor and angiogenesis. The cycloheximide chase experiment revealed the stabilization of HIF-2α by PIN1. Both WW and PPIase domains of PIN1 appear to be critical for its interaction with HIF-2α.
Up-regulated HIF-2α contributes to the Osteoarthritis development through mediating the primary cilia loss.
Yang Qining,Zhou Yongwei,Cai Pengfei,Fu Weicong,Wang Jinhua,Wei Qiang,Li Xiaofei
BACKGROUNDS:Up-regulated HIF-2α (hypoxia induced factor 2) had been demonstrated to contribute to Osteoarthritis (OA) development via inducing the expression of matrix-degrading enzymes. However, the HIF-2α also could promote primary cilia loss through HIF-2α/AURKA (Aurora kinase A)/NEDD9 pathway. And the primary cilia dysfunction is another characteristic of the OA. Thus, we investigated here whether the HIF-2α also contributes the OA development through mediating the primary cilia loss. METHODS:The primary chondrocytes were isolated from the experimental OA mice induced by destabilization of the medial meniscus (DMM). Chondrocytes were cultured under normoxia (21% O) or hypoxia (2% O) conditions. The HIF-1α and HIF-2α expressions were assessed by western blot. The cilia formation was counted by immuno-staining the acetylated tubulin. The contribution of HIF-1α or HIF-2α to the primary cilia loss was assessed by knocking-down the HIF-1α or HIF-2α individually. The HIF-2α/AURKA/NEDD9 pathway was validated through over-expressing or knocking-down specific components of the pathway and then counting the primary cilia number. Finally, the pathway was further confirmed in the OA mice. RESULTS:Hypoxia could induce the expression of both HIF-1α and HIF-2α, and also reduce the number of primary cilia on the chondrocytes isolated from the experimental OA mice. Knocking-down or over-expressing HIF-1α or HIF-2α individually showed that the HIF-2α could induce the primary cilia reduction rather than the HIF-1α. Manipulating the HIF-2α expression could positively affect the AURKA and NEDD9 expression. Manipulating the AURKA and NEDD9 expressions could reverse the function of HIF-2α on primary cilia. In the mice, knocking-down both AURKA and NEDD9 could alleviate the OA development significantly. CONCLUSION:Up-regulated HIF-2α contributes to the Osteoarthritis development through mediating the primary cilia loss, which might be developed as therapeutic targets for OA treatment.
HIF-1α contributes to Ang II-induced inflammatory cytokine production in podocytes.
Huang Hao,Fan Yanqin,Gao Zhao,Wang Wei,Shao Ning,Zhang Lu,Yang Yingjie,Zhu Weifang,Chen Zhaowei,Hu Jijia,Ding Guohua
BMC pharmacology & toxicology
BACKGROUND:Studies have indicated that changed expression of hypoxia-inducible factor-1α (HIF-1α) in epithelial cells from the kidney could affect the renal function in chronic kidney disease (CKD). As Angiotensin II (Ang II) is a critical active effector in the renin-angiotensin system (RAS) and was proved to be closely related to the inflammatory injury. Meanwhile, researchers found that Ang II could alter the expression of HIF-1α in the kidney. However, whether HIF-1α is involved in mediating Ang II-induced inflammatory injury in podocytes is not clear. METHODS:Ang II perfusion animal model were established to assess the potential role of HIF-1α in renal injury in vivo. Ang II stimulated podocytes to observe the corresponding between HIF-1α and inflammatory factors in vitro. RESULTS:The expression of inflammatory cytokines such as MCP-1 and TNF-α was increased in the glomeruli from rats treated with Ang II infusion compared with control rats. Increased HIF-1α expression in the glomeruli was also observed in Ang II-infused rats. In vitro, Ang II upregulated the expression of HIF-1α in podocytes. Furthermore, knockdown of HIF-1α by siRNA decreased the expression of MCP-1 and TNF-α. Moreover, HIF-1α siRNA significantly diminished the Ang II-induced overexpression of HIF-1α. CONCLUSION:Collectively, our results suggest that HIF-1α participates in the inflammatory response process caused by Ang II and that downregulation of HIF-1α may be able to partially protect or reverse inflammatory injury in podocytes.
HIF-1α-TWIST pathway restrains cyclic mechanical stretch-induced osteogenic differentiation of bone marrow mesenchymal stem cells.
Liu Ying,Huang Xia,Yu Haibo,Yang Jing,Li Yazhen,Yuan Xiao,Guo Qingyuan
Connective tissue research
: Mechanical strain plays a crucial role in bone formation and remodeling. Hypoxia-inducible factor (HIF)-1α and TWIST are upstream of master regulators of osteogenesis, including runt-related transcription factor 2 (RUNX2) and bone morphogenetic proteins (BMPs). This study investigated the effect of the HIF-1α-TWIST pathway on cyclic mechanical stretch-induced osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) and the underlying mechanism. : BMSCs were isolated from bone marrow derived from the femurs and humeri of Sprague-Dawley rats. Osteogenic differentiation of BMSCs was induced by applying cyclic mechanical stretch using the Flexcell Tension System. HIF-1α and TWIST were knocked down using recombinant lentiviral vectors. Osteogenic differentiation was evaluated by real-time qPCR, western blotting, and the alkaline phosphatase (ALP) activity assay. : Cyclic mechanical stretch increased ALP activity and expression of HIF-1α and TWIST in BMSCs. Knockdown of HIF-1α decreased TWIST expression in stretched BMSCs. Moreover, knockdown of HIF-1α or TWIST enhanced cyclic mechanical stretch-induced osteogenic differentiation of BMSCs. In addition, knockdown of TWIST increased expression of RUNX2 and BMP2 in stretched BMSCs. : The HIF-1α-TWIST signaling pathway inhibits cyclic mechanical stretch-induced osteogenic differentiation of BMSCs. This finding may facilitate cell and tissue engineering for clinical applications.
ITRAQ-based quantitative proteomic analysis of MG63 in response to HIF-1α inducers.
Chen Chunxia,Hao Xuehui,Geng Zhirong,Wang Zhilin
Journal of proteomics
Non-healing fractures constitute a serious clinical problem. HIF-1α is a crucial regulator in response to hypoxia and is proven to be pivotal in bone growth; however, the mechanism still needs further research. In this study, iTRAQ was used to study the effects of two HIF-1α inducers on the expression of proteins in MG63 cells. A total of 841 proteins were significantly changed after treatment with HIF-1α inducers. Among these, 12 proteins were functionally involved in the HIF-1 and VEGF signaling pathways. We then studied the protein and gene expression of the twelve proteins by western blot and RT-PCR, respectively. The results confirmed that VEGF, TFRC, ERK1/2, iNOS, GLUT1, ALDOA, ENO1 and IP3R1 were markedly upregulated, while NF-κB, RCN1, PLCγ1 and CaMKII were significantly downregulated upon treatment with HIF-1α inducers. Meanwhile, the intracellular levels of Ca, NO and ROS were closely related and significantly changed. Up-regulation of HIF can maintain high levels of Ca and NO while reducing ROS and protect cells from apoptosis induced by low serum. This study presents a new way to study the regulation of HIF on bone growth by investigating the Ca, NO and ROS levels. SIGNIFCANCE: We found that the regulation of Ca and NO proteins are tightly associated with HIF pathway using iTRAQ method. Furthermore, the concentration of Ca, NO and ROS are closely related in low serum cultured cells. Up-regulation of HIF pathway can maintain high levels of Ca and NO while reducing ROS damage.
HIF-1α Regulates Glucocorticoid-Induced Osteoporosis Through PDK1/AKT/mTOR Signaling Pathway.
Xu Wen-Ning,Zheng Huo-Liang,Yang Run-Ze,Jiang Lei-Sheng,Jiang Sheng-Dan
Frontiers in endocrinology
Long-term and high dose glucocorticoid treatment can cause decreased viability and function of osteoblasts, which leads to osteoporosis and osteonecrosis. In this study, we investigated the role and mechanism of action of HIF-1α in glucocorticoid-induced osteogenic inhibition in MC3T3-E1 cells. Our results showed that HIF-1α protein expression was reduced when MC3T3-E1 cells were exposed to dexamethasone (Dex) at varying concentrations ranging from 10 to 10 M. PDK1 expression was also decreased in MC3T3-E1 cells after dexamethasone treatment. MC3T3-E1 cells when treated with the glucocorticoid receptor antagonist RU486 along with dexamethasone showed enhanced HIF-1α expression. In addition, upregulated expression of HIF-1α was capable of promoting the osteogenic ability of MC3T3-E1 cells and PDK1 expression. However, the HIF-1α antagonist 2-methoxyestradiol (2-ME) had a reverse effect in MC3T3-E1 cells exposed to dexamethasone. Furthermore, the PDK1 antagonist dichloroacetate could repress the osteogenic ability of MC3T3-E1 cells, although HIF-1α was upregulated when transduced with adenovirus-HIF-1α construct. The PDK1 agonist PS48 was able to promote the osteogenic ability of MC3T3-E1 cells treated with dexamethasone. Importantly, the protein levels of p-AKT and p-mTOR were increased in MC3T3-E1 cells treated with dexamethasone after PS48 treatment. , the PDK1 agonist PS48 could maintain the bone mass of mice treated with dexamethasone. This study provides a new understanding of the mechanism of glucocorticoid-induced osteoporosis.
Synthesis and evaluation of the HIF-1α inhibitory activity of 3(5)-substituted-4-(quinolin-4-yl)- and 4-(2-phenylpyridin-4-yl)pyrazoles as inhibitors of ALK5.
Li Yan-Wei,Li Xiang-Yu,Li Shanji,Zhao Li-Min,Ma Juan,Piao Hu-Ri,Jiang Zhe,Jin Cheng Hua,Jin Xuejun
Bioorganic & medicinal chemistry letters
The transcription factor hypoxia-inducible factor-1α (HIF-1α) plays an important role in apoptosis, metastasis, and proliferation and is recognized as an important potential therapeutic target for cancer. Six series of 3(5)-(6-methylpyridin-2-yl)-4-(quinolin-4-yl)pyrazoles (11a-d, 12a-d, and 18a-d) and 3(5)-(6-methylpyridin-2-yl)-4-(2-phenyl-pyridin-4-yl)pyrazoles (19a-d, 20a-d, and 21a-d) were synthesized and evaluated for activin receptor-like kinase 5 (ALK5) and HIF-1α inhibitory activity at the enzyme and cell levels. The effect of the lead compound 20d (J-1012) on HIF-1α activation in HCT116 cells was investigated. J-1012 markedly decreased the hypoxia-induced or TNF-induced accumulation of HIF-1α protein dose-dependently. Analysis revealed that J-1012 inhibited HIF-1α protein synthesis, without affecting the degradation of HIF-1α protein. Furthermore, by inhibiting the activation of HIF-1α, J-1012 suppressed the metastasis and proliferation and promoted apoptosis of HCT116 cells. These results suggest that J-1012 may be a potential therapeutic agent against human colon cancer.
Substance P participates in periodontitis by upregulating HIF-1α and RANKL/OPG ratio.
Yan Kaixian,Lin Qin,Tang Kailiang,Liu Shuang,Du Yi,Yu Xijiao,Li Shu
BMC oral health
BACKGROUND:Both substance P and hypoxia-inducible factor 1 alpha (HIF-1α) are involved in inflammation and angiogenesis. However, the relationship between substance P and HIF-1α in rat periodontitis is still unknown. METHODS:Ligation-induced rat periodontitis was established to observe the distribution and expression of substance P and HIF-1α by immunohistochemistry. Rat gingival fibroblasts were cultured and stimulated with Porphyromonas gingivalis lipopolysaccharide (LPS). Recombinant substance P was applied to elaborate the relationship between substance P and HIF-1α in gingival fibroblasts in vitro. Primary mouse bone marrow-derived macrophages (BMMs) were isolated and cultured to observe the effect of substance P on receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis by TRAP staining. Western blotting was used to investigate the expression of HIF-1α, osteoprotegerin (OPG) and RANKL. RESULTS:Rat experimental periodontitis was successfully established 6 weeks after ligation. Gingival inflammatory infiltration and alveolar bone loss were observed. Positive expression of substance P was found in the infiltrating cells. Higher HIF-1α levels were observed in periodontitis compared to that of normal tissues. Substance P upregulated the level of HIF-1α in gingival fibroblasts with or without 1 μg/ml LPS in vitro (*P < 0.05). Substance P upregulated the expression of HIF-1α in RANKL-stimulated BMMs in vitro. Substance P also increased the RANKL/OPG ratio in gingival fibroblasts (*P < 0.05). Both 10 nM and 50 nM substance P promoted RANKL-induced osteoclast differentiation (*P < 0.05). CONCLUSION:Substance P participates in periodontitis by upregulating HIF-1α and the RANKL/OPG ratio.
Expression and distribution of HIF-1α, HIF-2α, VEGF, VEGFR-2 and HIMF in the kidneys of Tibetan sheep, Plain sheep and goat.
Yang K,Zhang Z,Li Y,Chen S,Chen W,Ding H,Tan Z,Ma Z,Qiao Z
BACKGROUND:The objective of this study was to detect the expression and distribution characteristics of five proteins [the hypoxia-inducible factor 1α (HIF-1α), HIF-2α, vascular endothelial growth factor (VEGF), VEGF-2 receptor (VEGFR-2) and hypoxia-induced mitogenic factor (HIMF)] in kidney of Tibetan sheep, plain sheep and goat. The results will provide the basic information for the comparative study of sheep breeds living at different altitudes. MATERIALS AND METHODS:The kidney tissues were collected from healthy adult Tibetan sheep, plain sheep and goats and made into paraffin sections. Histological characteristics were assessed by H&E staining. Expressions of HIF-1α, HIF-2α, VEGF, VEGFR-2 and HIMF proteins were measured by immunohistochemical staining. RESULTS:Immunohistochemistry results showed that the positive expression signals of HIF-1α, HIF-2α, VEGF and VEGFR-2 were detected in epithelial cells of renal tubules and collecting tubules, renal corpuscles in the kidneys of the three sheep breeds. Positive expression signals of HIMF were detected in epithelial cells of proximal tubules and distal tubules in Tibetan sheep and epithelial cells of distal tubules in goat. Immunostaining intensity of HIF-1α, HIF-2α, VEGF and VEGFR-2 proteins in Tibetan sheep was signiﬁcantly higher than that in both plain sheep and goat (p < 0.05). Immunostaining intensity of HIMF in Tibetan sheep was higher than goat (p < 0.05). Positive expression signals of HIMF were not detected in plain sheep. CONCLUSIONS:The expression and distribution characteristics of HIF-1α, HIF-2α, VEGF, VEGFR-2 and HIMF in the studied kidney tissues suggested that these proteins may be related to the physiological regulation of Tibetan sheep kidney in hypoxia environment, and therefore might be important regulating proteins for Tibetan sheep to adapt to high altitude hypoxia environment.
SENP1/HIF-1α axis works in angiogenesis of human dental pulp stem cells.
Zhou Jie,Sun Cong
Journal of biochemical and molecular toxicology
Dental diseases seriously affect the quality of life. Studying the mechanism of dental pulp stem cells (DPSCs) had far-reaching significance for the therapy of dental diseases. In this paper, we studied the small ubiquitin-like modifier-specific protease 1 (SENP1) and hypoxia-inducible factor (HIF)-1α functions in angiogenesis under anoxic conditions. Flow cytometry was used to identify and sort DPSCs. Reverse transcription-quantitative polymerase chain reaction and Western blot were carried out to analyze the expression of von Willebrand factor, vascular endothelial growth factor receptor 2, vascular endothelial cadherin, and CD31. Besides, si-SENP1 and si-HIF-1- levels were changed by cell transfection. Tube formation ability was carried out by tubulogenesis assay. Furthermore, the levels of HIF-1α and SENP1 were also tested by Western blot. We obtained DPSCs and induced them to differentiate into vessels and form tubules in vitro. On the basis of this, we demonstrated hypoxia enhanced HIF-1α and SENP1 expression. si-HIF-1α downregulated SENP1 expression and angiogenesis ability under hypoxia, and si-SENP1 downregulated HIF-1α expression and angiogenesis ability under hypoxia. Under hypoxia, SENP1 and HIF-1α formed a positive feedback loop and played a momentous part in angiogenesis.
HIF-1β Positively Regulates NF-κB Activity via Direct Control of TRAF6.
D'Ignazio Laura,Shakir Dilem,Batie Michael,Muller H Arno,Rocha Sonia
International journal of molecular sciences
NF-κB signalling is crucial for cellular responses to inflammation but is also associated with the hypoxia response. NF-κB and hypoxia inducible factor (HIF) transcription factors possess an intense molecular crosstalk. Although it is known that HIF-1α modulates NF-κB transcriptional response, very little is understood regarding how HIF-1β contributes to NF-κB signalling. Here, we demonstrate that HIF-1β is required for full NF-κB activation in cells following canonical and non-canonical stimuli. We found that HIF-1β specifically controls TRAF6 expression in human cells but also in . HIF-1β binds to the gene and controls its expression independently of HIF-1α. Furthermore, exogenous TRAF6 expression is able to rescue all of the cellular phenotypes observed in the absence of HIF-1β. These results indicate that HIF-1β is an important regulator of NF-κB with consequences for homeostasis and human disease.
Vav1 is Essential for HIF-1α Activation via a Lysosomal VEGFR1-Mediated Degradation Mechanism in Endothelial Cells.
Hong Jaewoo,Min Yongfen,Wuest Todd,Lin P Charles
The vascular response to hypoxia and ischemia is essential for maintaining homeostasis during stressful conditions and is particularly critical for vital organs such as the heart. Hypoxia-inducible factor-1 (HIF-1) is a central regulator of the response to hypoxia by activating transcription of numerous target genes, including vascular endothelial growth factor (VEGF). Here we identify the guanine nucleotide exchange factor (GEF) Vav1, a regulator of the small Rho-GTPase and cell signaling in endothelial cells, as a key vascular regulator of hypoxia. We show that Vav1 is present in the vascular endothelium and is essential for HIF-1 activation under hypoxia. So, we hypothesized that Vav1 could be a key regulator of HIF-1 signaling. In our findings, Vav1 regulates HIF-1α stabilization through the p38/Siah2/PHD3 pathway. In normoxia, Vav1 binds to vascular endothelial growth factor receptor 1 (VEGFR1), which directs Vav1 to lysosomes for degradation. In contrast, hypoxia upregulates Vav1 protein levels by inhibiting lysosomal degradation, which is analogous to HIF-1α regulation by hypoxia: both proteins are constitutively produced and degraded in normoxia allowing for a rapid response when stress occurs. Consequently, hypoxia rapidly stabilizes Vav1, which is required for HIF-1α accumulation. This shows that Vav1 is the key mediator controlling the stabilization of HIF1α in hypoxic conditions. With this finding, we report a novel pathway to stabilize HIF-1, which shows a possible reason why clinical trials targeting HIF-1 has not been effective. Targeting Vav1 can be the new approach to overcome hypoxic tumors.
Overexpression of bHLH domain of HIF-1 failed to inhibit the HIF-1 transcriptional activity in hypoxia.
Sadeghi Fatemeh,Kardar Gholam Ali,Bolouri Mohammad Reza,Nasri Farzad,Sadri Maryam,Falak Reza
BACKGROUND:Hypoxia inducible factor-1 (HIF-1) is considered as the most activated transcriptional factor in response to low oxygen level or hypoxia. HIF-1 binds the hypoxia response element (HRE) sequence in the promoter of different genes, mainly through the bHLH domain and activates the transcription of genes, especially those involved in angiogenesis and EMT. Considering the critical role of bHLH in binding HIF-1 to the HRE sequence, we hypothesized that bHLH could be a promising candidate to be targeted in hypoxia condition. METHODS:We inserted an inhibitory bHLH (ibHLH) domain in a pIRES2-EGFP vector and transfected HEK293T cells with either the control vector or the designed construct. The ibHLH domain consisted of bHLH domains of both HIF-1a and Arnt, capable of competing with HIF-1 in binding to HRE sequences. The transfected cells were then treated with 200 µM of cobalt chloride (CoCl) for 48 h to induce hypoxia. Real-time PCR and western blot were performed to evaluate the effect of ibHLH on the genes and proteins involved in angiogenesis and EMT. RESULTS:Hypoxia was successfully induced in the HEK293T cell line as the gene expression of VEGF, vimentin, and β-catenin were significantly increased after treatment of untransfected HEK293T cells with 200 µM CoCl. The gene expression of VEGF, vimentin, and β-catenin and protein level of β-catenin were significantly decreased in the cells transfected with either control or ibHLH vectors in hypoxia. However, ibHLH failed to be effective on these genes and the protein level of β-catenin, when compared to the control vector. We also observed that overexpression of ibHLH had more inhibitory effect on gene and protein expression of N-cadherin compared to the control vector. However, it was not statistically significant. CONCLUSION:bHLH has been reported to be an important domain involved in the DNA binding activity of HIF. However, we found that targeting this domain is not sufficient to inhibit the endogenous HIF-1 transcriptional activity. Further studies about the function of critical domains of HIF-1 are necessary for developing a specific HIF-1 inhibitor.
Borneol promotes apoptosis of Human Glioma Cells through regulating HIF-1a expression via mTORC1/eIF4E pathway.
Wang Zeng,Li Qinglin,Xia Liang,Li Xia,Sun Caixing,Wang Qiong,Cai Xinjun,Yang Guonong
Journal of Cancer
The main reason for the failure of malignant glioma treatment is local tumor recurrence. Tumor cells in hypoxic microenvironment activate HIF-1 α transcription, and thus promoting tumor invasion and metastasis is one of the important reasons. In our previous study, we clearly established that borneol opens the blood-brain tumor barrier and its related mechanism. However, the effects of borneol itself on glioma proliferation have not yet been elucidated. Therefore, in this study, we evaluated the effect of borneol on glioma by constructing SD rat brain glioma model and human primary cultured glioma cell model. We found that borneol could suppress the proliferation of primary glioma cells and the tumor volume of SD rat brain glioma. Further, we measured the apoptosis effect induced by borneol in human primary cultured glioma cells. The results showed that the higher the concentration of borneol, the higher the apoptosis rate of human primary cultured glioma cells, but the effect was reversed after transfection of HIF-1 overexpression plasmid; In addition, borneol could downregulate the expression of Bcl-2 and upregulation the expression of Bax and caspase-3, similarly, the effect was also reversed after transfection of HIF-1 overexpression plasmid, suggesting that the apoptosis effect induced by borneol in human primary cultured glioma cells is mediated via HIF-1α. Moreover, the bioinformatics analysis of correlation between HIF-1α and apoptosis-related factors based on CGGA database showed that there was a positive correlation between the expression of eIF4E and HIF-1 α (P < 0.05), and in patients with high expression of eIF4E and HIF-1α had poor survival and prognosis (P<0.001). It was further discovered that in the human primary cultured glioma cells borneol regulated HIF-1a expression via mTORC1/eIF4E pathway. In conclusion, the findings of the present study suggest that HIF-1α may be a key factor in borneol induced apoptosis of glioma cells, and mTORC1 / eIF4E pathway is involved in the HIF-1α regulation by borneol in malignant glioma. Our results not only reveal the target and molecular mechanism and action of borneol leading to promote apoptosis in glioma cells, but also provide experimental basis and theoretical support for the clinical application of borneol.
Protective effect of rhodioloside and bone marrow mesenchymal stem cells infected with HIF-1-expressing adenovirus on acute spinal cord injury.
Ha Xiao-Qin,Yang Bo,Hou Huai-Jing,Cai Xiao-Ling,Xiong Wan-Yuan,Wei Xu-Pan
Neural regeneration research
Rhodioloside has been shown to protect cells from hypoxia injury, and bone marrow mesenchymal stem cells have a good effect on tissue repair. To study the effects of rhodioloside and bone marrow mesenchymal stem cells on spinal cord injury, a rat model of spinal cord injury was established using the Infinite Horizons method. After establishing the model, the rats were randomly divided into five groups. Rats in the control group were intragastrically injected with phosphate buffered saline (PBS) (5 μL). PBS was injected at 6 equidistant points around 5 mm from the injury site and at a depth of 5 mm. Rats in the rhodioloside group were intragastrically injected with rhodioloside (5 g/kg) and intramuscularly injected with PBS. Rats in the mesenchymal stem cell (MSC) group were intramuscularly injected with PBS and intramuscularly with MSCs (8 × 10/mL in a 50-μL cell suspension). Rats in the Ad-HIF-MSC group were intragastrically injected with PBS and intramuscularly injected with HIF-1 adenovirus-infected MSCs. Rats in the rhodioloside + Ad-HIF-MSC group were intramuscularly injected with MSCs infected with the HIF-1 adenovirus and intragastrically injected with rhodioloside. One week after treatment, exercise recovery was evaluated with a modified combined behavioral score scale. Hematoxylin-eosin staining and Pischingert's methylene blue staining were used to detect any histological or pathological changes in spinal cord tissue. Levels of adenovirus IX and Sry mRNA were detected by real-time quantitative polymerase chain reaction and used to determine the number of adenovirus and mesenchymal stem cells that were transfected into the spinal cord. Immunohistochemical staining was applied to detect HIF-1 protein levels in the spinal cord. The results showed that: (1) compared with the other groups, the rhodioloside + Ad-HIF-MSC group exhibited the highest combined behavioral score (P < 0.05), the most recovered tissue, and the greatest number of neurons, as indicated by Pischingert's methylene blue staining. (2) Compared with the PBS group, HIF-1 protein expression was greater in the rhodioloside group (P < 0.05). (3) Compared with the Ad-HIF-MSC group, Sry mRNA levels were higher in the rhodioloside + Ad-HIF-MSC group (P < 0.05). These results confirm that rhodioloside combined with bone marrow mesenchymal stem cells can promote the recovery of spinal cord injury and activate the HIF-1 pathway to promote the survival of bone marrow mesenchymal stem cells and repair damaged neurons within spinal cord tissue. This experiment was approved by the Animal Ethics Committee of Gansu University of Traditional Chinese Medicine, China (approval No. 2015KYLL029) in June 2015.
High levels of HIF-1ɑ in hypoxic dental pulps associated with teeth with severe periodontitis.
Yu Xijiao,Jiang Huan,Cheng Guannan,Shang Wenzhi,Zhang Shanyong
Journal of molecular histology
In this study we investigated the expression of HIF-1ɑ in dental pulps of the teeth with severe periodontitis. The expression of HIF-1ɑ in dental pulps of the teeth with severe periodontitis was detected by immunohistochemistry, immunofluorescence and real-time PCR. Bone marrow macrophages (BMMs) were cultured under hypoxia in vitro. HIF-1ɑ, osteoclast-specific factors (NFATc1, CTSK and c-fos) and RANKL-induced osteoclastogenesis were evaluated by immunofluorescence, TRAP staining and western blotting. High levels of HIF-1ɑ protein were detected in dental pulps of teeth with severe periodontitis, whereas few positive HIF-1ɑ expressions were detected in healthy dental pulps. Hypoxia occurred in the dental pulps in response to heavy periodontitis. Many HIF-1ɑ-positive infiltratory inflammatory cells were observed around blood vessels. Tooth internal resorption was found in some teeth with severe periodontitis. The HIF-1ɑ levels were upregulated in BMMs under hypoxia, which also promoted osteoclast formation and resorption by NFATc1, CTSK and c-fos. Teeth with severe periodontitis show hypoxic dental pulps and increased potential of osteoclastic differentiation.
Hif-1alpha stabilisation is protective against infection in zebrafish comorbid models.
Schild Yves,Mohamed Abdirizak,Wootton Edward J,Lewis Amy,Elks Philip M
The FEBS journal
Multi-drug-resistant tuberculosis is a worldwide problem, and there is an urgent need for host-derived therapeutic targets, circumventing emerging drug resistance. We have previously shown that hypoxia-inducible factor-1α (Hif-1α) stabilisation helps the host to clear mycobacterial infection via neutrophil activation. However, Hif-1α stabilisation has also been implicated in chronic inflammatory diseases caused by prolonged neutrophilic inflammation. Comorbid infection and inflammation can be found together in disease settings, and it remains unclear whether Hif-1α stabilisation would be beneficial in a holistic disease setting. Here, we set out to understand the effects of Hif-1α on neutrophil behaviour in a comorbid setting by combining two well-characterised in vivo zebrafish models - TB infection (Mycobacterium marinum infection) and sterile injury (tailfin transection). Using a local Mm infection near to the tailfin wound site caused neutrophil migration between the two sites that was reduced during Hif-1α stabilisation. During systemic Mm infection, wounding leads to increased infection burden, but the protective effect of Hif-1α stabilisation remains. Our data indicate that Hif-1α stabilisation alters neutrophil migration dynamics between comorbid sites and that the protective effect of Hif-1α against Mm is maintained in the presence of inflammation, highlighting its potential as a host-derived target against TB infection.
HIF-1α induces hypoxic apoptosis of MLO-Y4 osteocytes via JNK/caspase-3 pathway and the apoptotic-osteocyte-mediated osteoclastogenesis in vitro.
Song Xiwen,Tang Yi,Zhu Jie,Tian Yuanye,Song Zhaohui,Hu Xiu,Hong Chaoyue,Cai Yun,Kang Feiwu
Tissue & cell
Apoptotic osteocytes were found in the hypoxic bone microenvironment in osteoporosis, osteotomy, orthodontic tooth movement and periodontitis, and played a key role in bone remolding and the differentiation of osteoclasts. Hypoxia inducible factor-1α(HIF-1α), as a transcription factor under hypoxic conditions, has been confirmed to participate in cell apoptosis. However, the effect of HIF-1α on osteocytes apoptosis and the osteocyte-mediated osteoclast formation remains elusive. Here, we hypothesized that HIF-1α was involved in osteocytes apoptosis. Our results showed that CoCl increased the MLO-Y4 cells apoptosis by upregulating the proapoptotic gene expression of caspase-3. Moreover, siRNA-mediated knockdown of HIF-1α decreased the phosphorylation by JNK and the activation of caspase-3 to inhibit the cell apoptosis in MLO-Y4. Furthermore, SP600125, an inhibitor of JNK, reversed CoCl-induced the increased apoptosis of MLO-Y4 cells in term of reducing the expression of caspase-3. These findings revealed that HIF-1α served as a pro-apoptotic factor in the apoptosis of MLO-Y4 cells cultured with CoCl, by activating the JNK/caspase-3 signaling pathway. Besides, the osteocyte-mediated osteoclastogenesis was reduced with the decline of the expression of HIF-1α and caspase-3 in MLO-Y4 cells. Our study provided an idea for a more comprehensive understanding of HIF-1α and the process of bone remodeling.
Aristolochic acid induces renal fibrosis by arresting proximal tubular cells in G2/M phase mediated by HIF-1α.
Zhao Hao,Jiang Na,Han Yachun,Yang Ming,Gao Peng,Xiong Xiaofen,Xiong Shan,Zeng Lingfeng,Xiao Ying,Wei Ling,Li Li,Li Chenrui,Yang Jinfei,Tang Chengyuan,Xiao Li,Liu Fuyou,Liu Yu,Sun Lin
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Renal tubulointerstitial fibrosis (TIF) is a common pathological feature of aristolochic acid (AA) nephropathy (AAN). G2/M arrest of proximal tubular cells (PTCs) is implicated in renal fibrosis of AAN, but the upstream regulatory molecule remains unknown. Hypoxia inducible factor-1α (HIF-1α) promotes renal fibrosis in kidney disease, but the role of HIF-1α in AAN is unclear. Evidence shows that HIF-1α and p21, a known inducer of cellular G2/M arrest, are closely related to each other. To investigate the role of HIF-1α in renal fibrosis of AAN and its effects on p21 expression and PTCs G2/M arrest, mice with HIF-1α gene knockout PTCs (PT-HIF-1α-KO) were generated, and AAN was induced by AA. In vitro tests were conducted on the human PTCs line HK-2 and primary mouse PTCs. HIF-1α and p21 expression, fibrogenesis, and G2/M arrest of PTCs were determined. Results showed that HIF-1α was upregulated in the kidneys of wild-type (WT) AAN mice, accompanied by p21 upregulation, PTCs G2/M arrest and renal fibrosis, and these alterations were reversed in PT-HIF-1α-KO AAN mice. Similar results were observed in HK-2 cells and were further confirmed in primary PTCs from PT-HIF-1α-KO and WT mice. Inhibiting p21 in HK-2 cells and primary PTCs did not change the expression of HIF-1α, but G2/M arrest and fibrogenesis were reduced. These data indicate that HIF-1α plays a key role in renal fibrosis in AAN by inducing PTCs G2/M arrest modulated through p21. HIF-1α may serve as a potential therapeutic target for AAN.
Correlation between the HIF-1α/Notch signaling pathway and Modic changes in nucleus pulposus cells isolated from patients with low back pain.
Xiong Zekang,Ding Jun,Zhou Jinge,Yao Sheng,Zheng Jin,Guo Xiaodong
BMC musculoskeletal disorders
BACKGROUND:The HIF-1α/Notch signaling pathway regulates cell proliferation, apoptosis, and metabolism in the intervertebral discs (IVDs) and is implicated in disc degeneration. The nucleus pulposus (NP) is an important structure adjacent to the IVDs. However, the role of the HIF-1α/Notch signaling pathway in NP cells obtained from patients with different Modic changes (MCs) remains unclear. The purpose of the present study was to investigate the role of HIF-1α and components of the Notch pathway in the NP obtained from patients with various MCs. METHODS:A total of 85 NP tissue samples were collected from patients undergoing diskectomy for the treatment of low back pain. The NP tissues were divided into four groups based on the adjacent endplate degeneration, namely, MC I, II, III, and negative MC groups. The expression of HIF-1α and Notch-related components was measured and compared. RESULTS:The expression of HIF-1α, Notch1, and Notch2 was gradually increased in the MC I and MC II groups compared with that in the negative MC group. HIF-1α and Notch-related components were rarely detected in the MC III group. CONCLUSIONS:The expression of HIF-1α/Notch increased in the NP cells of patients with MC I and MC II. HIF-1α and Notch-related components are potential biomarkers and the HIF-1α/Notch signaling pathway may serve as a promising therapeutic target for disc degeneration in patients with MCs.
GSK343, an inhibitor of EZH2, mitigates fibrosis and inflammation mediated by HIF-1α in human peritoneal mesothelial cells treated with high glucose.
Wang Qinglian,Xu Liang,Zhang Xianzheng,Liu Dan,Wang Rong
European journal of pharmacology
Inflammation and fibrosis in peritoneal mesothelial cells caused by long-term peritoneal dialysis (PD) are the main reasons why patients withdraw from peritoneal dialysis treatment. However, the related mechanism is still unclear. In the current study, we revealed that the expression of EZH2 was positively related to EMT and fibrosis in an in vitro model using human peritoneal mesothelial cells (HPMCs) stimulated with high glucose. Moreover, EZH2 also exhibited a positive correlation with HIF-1α expression. Using an sh-RNA lentivirus specific to EZH2, the EZH2 inhibitor GSK343 and rescue experiments of HIF-1α, we showed that EZH2 was an inducer of inflammation and fibrosis mediated by HIF-1α. Mechanistically, we revealed that on the one hand, EZH2 could increase the trimethylation of H3K4 at the HIF-1α gene promoter and directly activate HIF-1α transcription, as demonstrated by co-IP and ChIP-RT-PCR experiments. On the other hand, we verified that EZH2 could increase the trimethylation of H3K27 at the miR-142 gene promoter, which repressed the expression of miR-142. Combining bioanalysis and dual-luciferase assays, we found that miR-142 could regulate HIF-1α expression by directly binding to its mRNA 3'-UTR. Inhibition of miR-142 could rescue the protective effect of GSK343 on inflammation and fibrosis. In conclusion, our current study revealed that EZH2 plays a vital role in peritoneal fibrosis mediated by HIF-1α and related mechanisms. To our knowledge, this is the first study to demonstrate the effect of the EZH2-HIF-1α interaction and miR-142 on peritoneal fibrosis and inflammation and to suggest EZH2 and miR-142 as potential targets for the treatment of peritoneal fibrosis in patients with PD.
Hypoxic Regulation of Mitochondrial Metabolism and Mitophagy in Nucleus Pulposus Cells Is Dependent on HIF-1α-BNIP3 Axis.
Madhu Vedavathi,Boneski Paige K,Silagi Elizabeth,Qiu Yunping,Kurland Irwin,Guntur Anyonya R,Shapiro Irving M,Risbud Makarand V
Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research
Nucleus pulposus (NP) cells reside in an avascular and hypoxic microenvironment of the intervertebral disc and are predominantly glycolytic due to robust HIF-1 activity. It is generally thought that NP cells contain few functional mitochondria compared with cells that rely on oxidative metabolism. Consequently, the contribution of mitochondria to NP cell metabolism and the role of hypoxia and HIF-1 in mitochondrial homeostasis is poorly understood. Using mitoQC reporter mice, we show for the first time to our knowledge that NP cell mitochondria undergo age-dependent mitophagy in vivo. Mechanistically, in vitro studies suggest that, under hypoxic conditions, mitochondria in primary NP cells undergo HIF-1α-dependent fragmentation, controlled by modulating the levels of key proteins DRP1 and OPA1 that are involved in mitochondrial fission and fusion, respectively. Seahorse assays and steady state metabolic profiling coupled with [1-2- C]-glucose flux analysis revealed that in hypoxia, HIF-1α regulated metabolic flux through coordinating glycolysis and the mitochondrial TCA cycle interactions, thereby controlling the overall biosynthetic capacity of NP cells. We further show that hypoxia and HIF-1α trigger mitophagy in NP cells through the mitochondrial translocation of BNIP3, an inducer of receptor-mediated mitophagy. Surprisingly, however, loss of HIF-1α in vitro and analysis of NP-specific HIF-1α null mice do not show a decrease in mitophagic flux in NP cells but a compensatory increase in NIX and PINK1-Parkin pathways with higher mitochondrial number. Taken together, our studies provide novel mechanistic insights into the complex interplay between hypoxia and HIF-1α signaling on the mitochondrial metabolism and quality control in NP cells. © 2020 American Society for Bone and Mineral Research.
Orchestration of lincRNA-p21 and miR-155 in Modulating the Adaptive Dynamics of HIF-1α.
Sun Cheng-Yuan,Zhang Xiao-Peng,Liu Feng,Wang Wei
Frontiers in genetics
Hypoxia-inducible factor-1 (HIF-1) is the key regulator of cellular adaptive response to hypoxia. Accumulating evidence shows that HIF-1 induces some non-coding RNAs (ncRNAs) including lncRNAs and miRNAs to modulate its own activity, enclosing several feedback loops. How the two classes of ncRNAs are orchestrated in the HIF-1-dependent adaptive response to hypoxia is poorly understood. By selecting lincRNA-p21 and miR-155 as the representatives, we develop an integrated model of the HIF-1 network comprising interlinked positive and negative feedback loops to clarify the interplay between the two ncRNAs in the hypoxic response. By numerical simulations, we find that coordination of lincRNA-p21 and miR-155 shapes the adaptive dynamics of HIF-1α: lincRNA-p21 induction in the early phase stimulates the upregulation of HIF-1α via stabilizing it, while miR-155 induction in the late phase promotes the recovery of HIF-1α via enhancing the degradation of its mRNA. Moreover, HIF-1α-induced PHD2 plays an auxiliary role in the decline of HIF-1α. In addition, lincRNA-p21 and miR-155 modulate each other via regulating HIF-1α activity. Together, lincRNA-p21 and miR-155 coordinate in modulating HIF-1α dynamics, and our work may shed light on the role for ncRNAs in the cellular adaptation to hypoxia.
Short-wave enhances mesenchymal stem cell recruitment in fracture healing by increasing HIF-1 in callus.
Ye Dongmei,Chen Chen,Wang Qiwen,Zhang Qi,Li Sha,Liu Hongwei
Stem cell research & therapy
BACKGROUND:As a type of high-frequency electrotherapy, a short-wave can promote the fracture healing process; yet, its underlying therapeutic mechanisms remain unclear. PURPOSE:To observe the effect of Short-Wave therapy on mesenchymal stem cell (MSC) homing and relative mechanisms associated with fracture healing. MATERIALS AND METHODS:For in vivo study, the effect of Short-Wave therapy to fracture healing was examined in a stabilized femur fracture model of 40 SD rats. Radiography was used to analyze the morphology and microarchitecture of the callus. Additionally, fluorescence assays were used to analyze the GFP-labeled MSC homing after treatment in 20 nude mice with a femoral fracture. For in vitro study, osteoblast from newborn rats simulated fracture site was first irradiated by the Short-Wave; siRNA targeting HIF-1 was used to investigate the role of HIF-1. Osteoblast culture medium was then collected as chemotaxis content of MSC, and the migration of MSC from rats was evaluated using wound healing assay and trans-well chamber test. The expression of HIF-1 and its related factors were quantified by q RT-PCR, ELISA, and Western blot. RESULTS:Our in vivo experiment indicated that Short-Wave therapy could promote MSC migration, increase local and serum HIF-1 and SDF-1 levels, induce changes in callus formation, and improve callus microarchitecture and mechanical properties, thus speeding up the healing process of the fracture site. Moreover, the in vitro results further indicated that Short-Wave therapy upregulated HIF-1 and SDF-1 expression in osteoblast and its cultured medium, as well as the expression of CXCR-4, β-catenin, F-actin, and phosphorylation levels of FAK in MSC. On the other hand, the inhibition of HIF-1α was significantly restrained by the inhibition of HIF-1α in osteoblast, and it partially inhibited the migration of MSC. CONCLUSIONS:These results suggested that Short-Wave therapy could increase HIF-1 in callus, which is one of the crucial mechanisms of chemotaxis MSC homing in fracture healing.
Mechanical loading induces HIF-1α expression in chondrocytes via YAP.
Jing Xingzhi,Yang Xiaoxia,Zhang Weimin,Wang Shengjie,Cui Xingang,Du Ting,Li Tao
OBJECTIVES:To investigate the role of YAP in cyclic mechanical stress induced up-regulation of HIF-1α in rat cartilage chondrocytes. RESULTS:Our in vitro and in vivo experiments demonstrated that cyclic mechanical stress promoted HIF-1α and YAP proteins expression in a magnitude dependent manner. Cyclic mechanical stress at 4000μ strain exhibited most significant effect in promoting HIF-1α and YAP up-regulation. Activation of YAP using LPA significantly promoted HIF-1α stabilization and expression, while YAP siRNA treatment suppressed the up-regulation of HIF-1α induced by cyclic mechanical stress. CONCLUSION:Our results indicated that cyclic mechanical stress promoted HIF-1α stabilization and YAP is involved in mechanical stress induced HIF-1α up-regulation.
Zebrafish Hif3α modulates erythropoiesis via regulation of to facilitate hypoxia tolerance.
Cai Xiaolian,Zhou Ziwen,Zhu Junji,Liao Qian,Zhang Dawei,Liu Xing,Wang Jing,Ouyang Gang,Xiao Wuhan
Development (Cambridge, England)
The hypoxia-inducible factors 1α and 2α (HIF1α and HIF2α) are master regulators of the cellular response to O In addition to HIF1α and HIF2α, HIF3α is another identified member of the HIFα family. Even though the question of whether some HIF3α isoforms have transcriptional activity or repressive activity is still under debate, it is evident that the full length of HIF3α acts as a transcription factor. However, its function in hypoxia signaling is largely unknown. Here, we show that loss of in zebrafish reduced hypoxia tolerance. Further assays indicated that erythrocyte number was decreased because red blood cell maturation was impeded by disruption. We found that expression was downregulated in null zebrafish, as were several hematopoietic marker genes, including , , , and Hif3α recognized the hypoxia response element located in the promoter of and directly bound to the promoter to transactivate expression. Our results suggested that facilities hypoxia tolerance by modulating erythropoiesis via regulation.