RESUMO
Here, we explored the role of Prolyl 4-Hydroxylase Subunit Alpha 3 (P4HA3), the most recently identified member of the prolyl-4-hydroxylase (P4H) family, in head and neck squamous cell carcinoma (HNSCC) progression. P4HA3 is upregulated during cancer progression; however, its specific role in HNSCC progression remains elusive. Thus, this study aimed to elucidate the regulatory function of P4HA3 in HNSCC development and progression and to describe the underlying mechanisms. Initially, we analyzed the correlation between the expression of P4HA3 and the WNT pathway genes and clinicopathologic features in HNSCC based on microarray data from The Cancer Genome Atlas (TCGA). Next, we used Gene Oncology (GO) functional data to describe several potentially associated pathways in HNSCC. Then, we knocked down P4HA3 in SCC15 and SCC25 cells, two classic HNSCC cell lines, and assessed the resulting changes using RT-qPCR. Furthermore, we used Western blot to evaluate the regulatory role of P4HA3 in the epithelial-to-mesenchymal transition (EMT) and the WNT/ß-catenin signaling pathway. To explore the effect of P4HA3 knockdown on tumor progression, in vivo experiments were conducted using a murine model. Immunohistochemistry assays were then employed to identify proteins associated with EMT and the WNT/ß-catenin signaling pathway in tumor tissues. Upregulated P4HA3 in HNSCC patient tumor tissues was positively correlated with poor prognosis. Notably, P4HA3 knockdown significantly inhibited the proliferative and invasive abilities of HNSCC. The levels of genes and proteins associated with EMT and the WNT/ß-catenin signaling pathway were also markedly reduced by P4HA3 knockdown. Importantly, the in vivo experiments demonstrated that P4HA3 can promote subcutaneous tumorigenesis in nude mice and knockdown of P4HA3 induce a significant ihibitation of EMT and WNT/ß-catenin pathway detected by immunohistochemistry assay in tumor tissues. In summary, we demonstrated that P4HA3 is a promising diagnostic and therapeutic biomarker for HNSCC. As an oncogene, P4HA3 increases HNSCC proliferation by inducing the EMT and activating the WNT/ß-catenin signaling pathway.
Assuntos
Progressão da Doença , Transição Epitelial-Mesenquimal , Neoplasias de Cabeça e Pescoço , Carcinoma de Células Escamosas de Cabeça e Pescoço , Via de Sinalização Wnt , Humanos , Via de Sinalização Wnt/fisiologia , Via de Sinalização Wnt/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Animais , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/genética , Camundongos , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Pró-Colágeno-Prolina Dioxigenase/genética , Linhagem Celular Tumoral , Masculino , Camundongos Nus , Feminino , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Pessoa de Meia-IdadeRESUMO
Proline 4-hydroxylase 2 (P4HA2) is known for its hydroxylase activity, primarily involved in hydroxylating collagen precursors and promoting collagen cross-linking under physiological conditions. Although its overexpression influences a wide variety of malignant tumors' occurrence and development, its specific effects and mechanisms in oral squamous cell carcinoma (OSCC) remain unclear. This study focused on investigating the expression patterns, carcinogenic functions, and underlying mechanisms of P4HA2 in OSCC cells. Various databases, including TCGA, TIMER, UALCAN, GEPIA, and K-M plotter, along with paraffin-embedded samples, were used to ascertain P4HA2 expression in cancer and its correlation with clinicopathological features. P4HA2 knockdown and overexpression cell models were developed to assess its oncogenic roles and mechanisms. The results indicated that P4HA2 was overexpressed in OSCC and inversely correlated with patient survival. Knockdown of P4HA2 suppressed invasion, migration, and proliferation of OSCC cells both in vitro and in vivo, whereas overexpression of P4HA2 had the opposite effects. Mechanistically, the phosphorylation levels of the PI3K/AKT pathway were reduced following P4HA2 silencing. The study reveals that P4HA2 acts as a promising biomarker for predicting prognosis in OSCC and significantly affects metastasis, invasion, and proliferation of OSCC cells through the regulation of the PI3K/AKT signaling pathway.
Assuntos
Carcinoma de Células Escamosas , Movimento Celular , Proliferação de Células , Neoplasias Bucais , Invasividade Neoplásica , Fosfatidilinositol 3-Quinases , Pró-Colágeno-Prolina Dioxigenase , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Animais , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Camundongos Nus , Neoplasias Bucais/patologia , Neoplasias Bucais/metabolismo , Neoplasias Bucais/genética , Metástase Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Pró-Colágeno-Prolina Dioxigenase/genética , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Sensing the lowering of endoplasmic reticulum (ER) calcium (Ca2+), STIM1 mediates a ubiquitous Ca2+ influx process called the store-operated Ca2+ entry (SOCE). Dysregulated STIM1 function or abnormal SOCE is strongly associated with autoimmune disorders, atherosclerosis, and various forms of cancers. Therefore, uncovering the molecular intricacies of post-translational modifications, such as oxidation, on STIM1 function is of paramount importance. In a recent proteomic screening, we identified three protein disulfide isomerases (PDIs)-Prolyl 4-hydroxylase subunit beta (P4HB), protein disulfide-isomerase A3 (PDIA3), and thioredoxin domain-containing protein 5 (TXNDC5)-as the ER-luminal interactors of STIM1. Here, we demonstrated that these PDIs dynamically associate with STIM1 and STIM2. The mutation of the two conserved cysteine residues of STIM1 (STIM1-2CA) decreased its Ca2+ affinity both in cellulo and in situ. Knockdown of PDIA3 or P4HB increased the Ca2+ affinity of wild-type STIM1 while showing no impact on the STIM1-2CA mutant, indicating that PDIA3 and P4HB regulate STIM1's Ca2+ affinity by acting on ER-luminal cysteine residues. This modulation of STIM1's Ca2+ sensitivity was further confirmed by Ca2+ imaging experiments, which showed that knockdown of these two PDIs does not affect STIM1-mediated SOCE upon full store depletion but leads to enhanced SOCE amplitudes upon partial store depletion. Thus, P4HB and PDIA3 dynamically modulate STIM1 activation by fine-tuning its Ca2+ binding affinity, adjusting the level of activated STIM1 in response to physiological cues. The coordination between STIM1-mediated Ca2+ signaling and redox responses reported herein may have implications for cell physiology and pathology.
Assuntos
Cálcio , Proteínas de Neoplasias , Oxirredução , Pró-Colágeno-Prolina Dioxigenase , Isomerases de Dissulfetos de Proteínas , Molécula 1 de Interação Estromal , Molécula 1 de Interação Estromal/metabolismo , Molécula 1 de Interação Estromal/genética , Humanos , Isomerases de Dissulfetos de Proteínas/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Cálcio/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Pró-Colágeno-Prolina Dioxigenase/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/genética , Retículo Endoplasmático/metabolismo , Células HEK293 , Ligação Proteica , Sinalização do Cálcio , Molécula 2 de Interação Estromal/metabolismo , Molécula 2 de Interação Estromal/genéticaRESUMO
Aberrations in the Hedgehog (Hh) signaling pathway are significantly prevailed in various cancers, including B-cell lymphoma. A critical facet of Hh signal transduction involves the dynamic regulation of the suppressor of fused homolog (SUFU)-glioma-associated oncogene homolog (GLI) complex within the kinesin family member 7 (KIF7)-supported ciliary tip compartment. However, the specific post-translational modifications of SUFU-GLI complex within this context have remained largely unexplored. Our study reveals a novel regulatory mechanism involving prolyl 4-hydroxylase 2 (P4HA2), which forms a complex with KIF7 and is essential for signal transduction of Hh pathway. We demonstrate that, upon Hh pathway activation, P4HA2 relocates alongside KIF7 to the ciliary tip. Here, it hydroxylates SUFU to inhibit its function, thus amplifying the Hh signaling. Moreover, the absence of P4HA2 significantly impedes B lymphoma progression. This effect can be attributed to the suppression of Hh signaling in stromal fibroblasts, resulting in decreased growth factors essential for malignant proliferation of B lymphoma cells. Our findings highlight the role of P4HA2-mediated hydroxylation in modulating Hh signaling and propose a novel stromal-targeted therapeutic strategy for B-cell lymphoma.
Assuntos
Progressão da Doença , Proteínas Hedgehog , Linfoma de Células B , Pró-Colágeno-Prolina Dioxigenase , Proteínas Repressoras , Transdução de Sinais , Proteínas Hedgehog/metabolismo , Humanos , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Camundongos , Animais , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Hidroxilação , Comunicação Parácrina , Proliferação de Células , Cinesinas/metabolismo , Cinesinas/genética , Linhagem Celular Tumoral , Prolil HidroxilasesRESUMO
Heat stress (HS) poses a substantial threat to animal growth and development, resulting in declining performance and economic losses. The intestinal system is susceptible to HS and undergoes intestinal hyperthermia and pathological hypoxia. Hypoxia-inducible factor-1α (HIF-1α), a key player in cellular hypoxic adaptation, is influenced by prolyl-4-hydroxylase 2 (PHD2) and heat shock protein 90 (HSP90). However, the comprehensive regulation of HIF-1α in the HS intestine remains unclear. This study aims to explore the impact of HS on pig intestinal mucosa and the regulatory mechanism of HIF-1α. Twenty-four Congjiang Xiang pigs were divided into the control and five HS-treated groups (6, 12, 24, 48, and 72 h). Ambient temperature and humidity were maintained in a thermally-neutral state (temperature-humidity index (THI) < 74) in the control group, whereas the HS group experienced moderate HS (78 < THI <84). Histological examination revealed villus exfoliation after 12 h of HS in the duodenum, jejunum, and ileum, with increasing damage as HS duration extended. The villus height to crypt depth ratio (V/C) decreased and goblet cell number increased with prolonged HS. Quantitative real-time PCR, Western blot, and immunohistochemistry analysis indicated increased expression of HIF-1α and HSP90 in the small intestine with prolonged HS, whereas PHD2 expression decreased. Further investigation in IPEC-J2 cells subjected to HS revealed that overexpressing PHD2 increased PHD2 mRNA and protein expression, while it decreases HIF-1α. Conversely, interfering with HSP90 expression substantially decreased both HSP90 and HIF-1α mRNA and protein levels. These results suggest that HS induces intestinal hypoxia with concomitant small intestinal mucosal damage. The expression of HIF-1α in HS-treated intestinal epithelial cells may be co-regulated by HSP90 and PHD2 and is possibly linked to intestinal hyperthermia and hypoxia.
Assuntos
Células Epiteliais , Proteínas de Choque Térmico HSP90 , Resposta ao Choque Térmico , Subunidade alfa do Fator 1 Induzível por Hipóxia , Intestino Delgado , Animais , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Choque Térmico HSP90/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Suínos , Intestino Delgado/metabolismo , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Pró-Colágeno-Prolina Dioxigenase/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Linhagem CelularRESUMO
The overexpression of PDIA1 in cancer has spurred the quest for effective inhibitors. However, existing inhibitors often bind to only one active site, limiting their efficacy. In our study, we developed a PROTAC-mimetic probe dPA by combining PACMA31 (PA) analogs with cereblon-directed pomalidomide. Through protein profiling and analysis, we confirmed dPA's specific interaction with PDIA1's active site cysteines. We further synthesized PROTAC variants with a thiophene ring and various linkers to enhance degradation efficiency. Notably, H4, featuring a PEG linker, induced significant PDIA1 degradation and inhibited cancer cell proliferation similarly to PA. The biosafety profile of H4 is comparable to that of PA, highlighting its potential for further development in cancer therapy. Our findings highlight a novel strategy for PDIA1 inhibition via targeted degradation, offering promising prospects in cancer therapeutics. This approach may overcome limitations of conventional inhibitors, presenting new avenues for advancing anti-cancer interventions.
Assuntos
Antineoplásicos , Proliferação de Células , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Sondas Moleculares/química , Sondas Moleculares/farmacologia , Sondas Moleculares/síntese química , Estrutura Molecular , Pró-Colágeno-Prolina Dioxigenase , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Isomerases de Dissulfetos de Proteínas/metabolismo , Relação Estrutura-Atividade , Peptídeos/química , Peptídeos/farmacologiaRESUMO
Prolyl 4-hydroxylase beta subunit (P4HB) plays a vital role in bone formation. This study intends to clarify the role of P4HB in the therapeutic effect of Icariin (ICA) on osteoporosis. Herein, in vivo and in vitro models were constructed by performing ovariectomy (OVX) in rats and inducing osteogenic differentiation in bone marrow stem cells (BMSCs), respectively. Hematoxylin and eosin staining and micro-computed tomography analysis were performed to evaluate osteoporosis in OVX rats. Alizarin Red staining, alkaline phosphatase staining, and the ALP activity test were employed to assess osteogenesis. m6A dot blotting and methylated RNA immunoprecipitation were used to determine m6A modification. We found that P4HB was downregulated in bone tissues of patients with osteoporosis and OVX rats. P4HB facilitated osteogenic differentiation of BMSCs. What's more, ICA upregulated P4HB expression, promoted osteogenic differentiation of BMSCs, and alleviated osteoporosis in OVX rats, which were reversed by knocking down P4HB. ICA enhanced the stability and m6A modification of P4HB. METTL14 mediated m6A modification of P4HB mRNA. In addition, METTL14 knockdown overturned the promotive effects of ICA on P4HB m6A level and BMSC osteogenic differentiation. To sum up, ICA elevated the METTL14-mediated m6A modification of P4HB to facilitate BMSC osteogenic differentiation.
Assuntos
Diferenciação Celular , Flavonoides , Metiltransferases , Osteogênese , Ratos Sprague-Dawley , Animais , Osteogênese/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Ratos , Feminino , Flavonoides/farmacologia , Metiltransferases/metabolismo , Metiltransferases/genética , Humanos , Osteoporose/patologia , Osteoporose/metabolismo , Osteoporose/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Ovariectomia , Regulação para Cima/efeitos dos fármacos , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Pró-Colágeno-Prolina Dioxigenase/genética , Células da Medula Óssea/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células Cultivadas , Adenosina/análogos & derivados , Adenosina/metabolismoRESUMO
Prolyl 4-hydroxylases (P4Hs) are a family of key modifying enzymes in collagen synthesis. P4Hs have been confirmed to be closely associated with tumor occurrence and development. However, the expression of P4Hs in head and neck cancer (HNSC) as well as its relationship with prognosis and tumor immunity infiltration has not yet been analyzed. We investigated the transcriptional expression, survival data, and immune infiltration of P4Hs in patients with HNSC from multiple databases. P4H1-3 expression was significantly higher in HNSC tumor tissues than in normal tissues. Moreover, P4HA1 and P4HA2 were associated with tumor stage, patient prognosis, and immune cell infiltration. P4HA3 was related to patient prognosis and immune cell infiltration. Correlation experiments confirmed that P4HA1 may serve as a prognosis biomarker and plays a role in the progression of nasopharyngeal carcinoma. These findings suggest that P4HA1-3 may be a novel biomarker for the prognosis and treatment of HNSC, which is expected to support the development of new therapies for patients with head and neck tumors and improve patient outcomes.
Assuntos
Biomarcadores Tumorais , Neoplasias de Cabeça e Pescoço , Imunoterapia , Pró-Colágeno-Prolina Dioxigenase , Humanos , Biomarcadores Tumorais/metabolismo , Prognóstico , Neoplasias de Cabeça e Pescoço/terapia , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/imunologia , Neoplasias de Cabeça e Pescoço/diagnóstico , Imunoterapia/métodos , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Pró-Colágeno-Prolina Dioxigenase/genética , Regulação Neoplásica da Expressão Gênica , Feminino , Masculino , Carcinoma Nasofaríngeo/terapia , Carcinoma Nasofaríngeo/imunologia , Carcinoma Nasofaríngeo/diagnóstico , Carcinoma Nasofaríngeo/patologia , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/metabolismo , Carcinoma Nasofaríngeo/mortalidadeRESUMO
Cell metabolism reprogramming to sustain energy production, while reducing oxygen and energy consuming processes is crucially important for the adaptation to hypoxia/ischemia. Adaptive metabolic rewiring is controlled by hypoxia-inducible factors (HIFs). Accumulating experimental evidence indicates that timely activation of HIF in brain-resident cells improves the outcome from acute ischemic stroke. However, the underlying molecular mechanisms are still incompletely understood. Thus, we investigated whether HIF-dependent metabolic reprogramming affects the vulnerability of brain-resident cells towards ischemic stress. Methods: We used genetic and pharmacological approaches to activate HIF in the murine brain in vivo and in primary neurons and astrocytes in vitro. Numerous metabolomic approaches and molecular biological techniques were applied to elucidate potential HIF-dependent effects on the central carbon metabolism of brain cells. In animal and cell models of ischemic stroke, we analysed whether HIF-dependent metabolic reprogramming influences the susceptibility to ischemic injury. Results: Neuron-specific gene ablation of prolyl-4-hydroxylase domain 2 (PHD2) protein, negatively regulating the protein stability of HIF-α in an oxygen dependent manner, reduced brain injury and functional impairment of mice after acute stroke in a HIF-dependent manner. Accordingly, PHD2 deficient neurons showed an improved tolerance towards ischemic stress in vitro, which was accompanied by enhanced HIF-1-mediated glycolytic lactate production through pyruvate dehydrogenase kinase-mediated inhibition of the pyruvate dehydrogenase. Systemic treatment of mice with roxadustat, a low-molecular weight pan-PHD inhibitor, not only increased the abundance of numerous metabolites of the central carbon and amino acid metabolism in murine brain, but also ameliorated cerebral tissue damage and sensorimotor dysfunction after acute ischemic stroke. In neurons and astrocytes roxadustat provoked a HIF-1-dependent glucose metabolism reprogramming including elevation of glucose uptake, glycogen synthesis, glycolytic capacity, lactate production and lactate release, which enhanced the ischemic tolerance of astrocytes, but not neurons. We found that strong activation of HIF-1 in neurons by non-selective inhibition of all PHD isoenzymes caused a HIF-1-dependent upregulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 redirecting glucose-6-phosphate from pentose phosphate pathway (PPP) to the glycolysis pathway. This was accompanied by a reduction of NADPH production in the PPP, which further decreased the low intrinsic antioxidant reserve of neurons, making them more susceptible to ischemic stress. Nonetheless, in organotypic hippocampal cultures with preserved neuronal-glial interactions roxadustat decreased the neuronal susceptibility to ischemic stress, which was largely prevented by restricting glycolytic energy production through lactate transport blockade. Conclusion: Collectively, our results indicate that HIF-1-mediated metabolic reprogramming alleviates the intrinsic vulnerability of brain-resident cells to ischemic stress.
Assuntos
Astrócitos , Carbono , Subunidade alfa do Fator 1 Induzível por Hipóxia , Prolina Dioxigenases do Fator Induzível por Hipóxia , AVC Isquêmico , Neurônios , Animais , Feminino , Masculino , Camundongos , Astrócitos/metabolismo , Astrócitos/efeitos dos fármacos , Encéfalo/metabolismo , Isquemia Encefálica/metabolismo , Carbono/metabolismo , Reprogramação Celular/efeitos dos fármacos , Modelos Animais de Doenças , Glicólise/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , AVC Isquêmico/metabolismo , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Pró-Colágeno-Prolina Dioxigenase/genéticaRESUMO
Mammalian target of rapamycin (mTOR) kinase functions as a central regulator of cell growth and metabolism, and its complexes mTORC1 and mTORC2 phosphorylate distinct substrates. Dysregulation of mTOR signaling is commonly implicated in human diseases, including cancer. Despite three decades of active research in mTOR, much remains to be determined. Here, we demonstrate that prolyl 4-hydroxylase alpha-2 (P4HA2) binds directly to mTOR and hydroxylates one highly conserved proline 2341 (P2341) within a kinase domain of mTOR, thereby activating mTOR kinase and downstream effector proteins (e.g. S6K and AKT). Moreover, the hydroxylation of P2341 strengthens mTOR stability and allows mTOR to accurately recognize its substrates such as S6K and AKT. The growth of lung adenocarcinoma cells overexpressing mTORP2341A is significantly reduced when compared with that of cells overexpressing mTORWT. Interestingly, in vivo cell growth assays show that targeting P4HA2-mTOR significantly suppresses lung adenocarcinoma cell growth. In summary, our study reveals an undiscovered hydroxylation-regulatory mechanism by which P4HA2 directly activates mTOR kinase, providing insights for therapeutically targeting mTOR kinase-driven cancers.
Assuntos
Adenocarcinoma de Pulmão , Proliferação de Células , Neoplasias Pulmonares , Serina-Treonina Quinases TOR , Humanos , Hidroxilação , Serina-Treonina Quinases TOR/metabolismo , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/tratamento farmacológico , Animais , Camundongos , Linhagem Celular Tumoral , Transdução de Sinais , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Pró-Colágeno-Prolina Dioxigenase/genéticaRESUMO
BACKGROUND: Hypoxia is a pathological hallmark in most cancers, including glioblastoma (GBM). Hypoxic signaling activation and post-translational modification (PTM) of oncogenic proteins are well-studied in cancers. Accumulating studies indicate glycolytic enzyme PGK1 plays a crucial role in tumorigenesis, yet the underlying mechanisms remain unknown. METHODS: We first used ChIP assays to uncover the crosstalk between HIF1α and ATF3 and their roles in P4HA1 regulation. Protein degradation analysis, LC-MS/MS, and in vitro succinate production assays were performed to examine the effect of protein succinylation on GBM pathology. Seahorse assay measured the effects of PGK1 succinylation at K191/K192 or its mutants on glucose metabolism. We utilized an in vivo intracranial mouse model for biochemical studies to elucidate the impact of ATF3 and P4HA1 on aerobic glycolysis and the tumor immune microenvironment. RESULTS: We demonstrated that HIF1α and ATF3 positively and negatively regulate the transcription of P4HA1, respectively, leading to an increased succinate production and increased activation of HIF1α signaling. P4HA1 expression elevated the succinate concentration, resulting in the enhanced succinylation of PGK1 at the K191 and K192 sites. Inhibition of proteasomal degradation of PGK1 by succinylation significantly increased aerobic glycolysis to generate lactate. Furthermore, ATF3 overexpression and P4HA1 knockdown reduced succinate and lactate levels in GBM cells, inhibiting immune responses and tumor growth. CONCLUSIONS: Together, our study demonstrates that HIF1α/ATF3 participated in P4HA1/succinate signaling, which is the major regulator of succinate biosynthesis and PGK1 succinylation at K191 and K192 sites in GBM. The P4HA1/succinate pathway might be a novel and promising target for aerobic glycolysis in GBM.
Assuntos
Fator 3 Ativador da Transcrição , Neoplasias Encefálicas , Glioblastoma , Subunidade alfa do Fator 1 Induzível por Hipóxia , Fosfoglicerato Quinase , Transdução de Sinais , Ácido Succínico , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioblastoma/genética , Fosfoglicerato Quinase/metabolismo , Fosfoglicerato Quinase/genética , Animais , Fator 3 Ativador da Transcrição/metabolismo , Fator 3 Ativador da Transcrição/genética , Camundongos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/genética , Ácido Succínico/metabolismo , Regulação Neoplásica da Expressão Gênica , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Pró-Colágeno-Prolina Dioxigenase/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Proliferação de CélulasRESUMO
Oxygen sensors enable cells to adapt to limited oxygen availability (hypoxia), affecting various cellular and tissue responses. Prolyl-4-hydroxylase domain 1-3 (PHD1-3; also called Egln1-3, HIF-P4H 1-3, HIF-PH 1-3) proteins belong to the Fe2+- and 2-oxoglutarate-dependent dioxygenase superfamily and utilise molecular oxygen (O2) alongside 2-oxoglutarate as co-substrate to hydroxylate two proline residues of α subunits of the dimeric hypoxia inducible factor (HIF) transcription factor. PHD1-3-mediated hydroxylation of HIF-α leads to its degradation and inactivation. Recently, various PHD inhibitors (PHI) have entered the clinics for treatment of renal anaemia. Pre-clinical analyses indicate that PHI treatment may also be beneficial in numerous other hypoxia-associated diseases. Nonetheless, the underlying molecular mechanisms of the observed protective effects of PHIs are only partly understood, currently hindering their translation into the clinics. Moreover, the PHI-mediated increase of Epo levels is not beneficial in all hypoxia-associated diseases and PHD-selective inhibition may be advantageous. Here, we summarise the current knowledge about the relevance and function of each of the three PHD isoforms in vivo, based on the deletion or RNA interference-mediated knockdown of each single corresponding gene in rodents. This information is crucial for our understanding of the physiological relevance and function of the PHDs as well as for elucidating their individual impact on hypoxia-associated diseases. Furthermore, this knowledge highlights which diseases may best be targeted by PHD isoform-selective inhibitors in case such pharmacologic substances become available.
Assuntos
Prolina Dioxigenases do Fator Induzível por Hipóxia , Animais , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Oxigênio/metabolismo , Deleção de Genes , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Pró-Colágeno-Prolina Dioxigenase/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Hipóxia/metabolismoRESUMO
AIMS: Prolyl hydroxylase domain 2 (PHD2), encoded by the Egln1 gene, serves as a pivotal regulator of the hypoxia-inducible factor (HIF) pathway and acts as a cellular oxygen sensor. Somatic inactivation of Phd2 in mice results in polycythemia and congestive heart failure. However, due to the embryonic lethality of Phd2 deficiency, its role in development remains elusive. Here, we investigated the function of two egln1 paralogous genes, egln1a and egln1b, in zebrafish. MAIN METHODS: The egln1 null zebrafish were generated using the CRISPR/Cas9 system. Quantitative real-time PCR assays and Western blot analysis were employed to detect the effect of egln1 deficiency on the hypoxia signaling pathway. The hypoxia response of egln1 mutant zebrafish were assessed by analyzing heart rate, gill agitation frequency, and blood flow velocity. Subsequently, o-dianisidine staining and in situ hybridization were used to investigate the role of egln1 in zebrafish hematopoietic function. KEY FINDINGS: Our data show that the loss of egln1a or egln1b individually has no visible effects on growth rate. However, the egln1a; egln1b double mutant displayed significant growth retardation and elevated mortality at around 2.5 months old. Both egln1a-null and egln1b-null zebrafish embryo exhibited enhanced tolerance to hypoxia, systemic hypoxic response that include hif pathway activation, increased cardiac activity, and polycythemia. SIGNIFICANCE: Our research introduces zebrafish egln1 mutants as the first congenital embryonic viable systemic vertebrate animal model for PHD2, providing novel insights into hypoxic signaling and the progression of PHD2- associated disease.
Assuntos
Prolina Dioxigenases do Fator Induzível por Hipóxia , Hipóxia , Policitemia , Peixe-Zebra , Animais , Camundongos , Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Policitemia/genética , Pró-Colágeno-Prolina Dioxigenase/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
The interplay between metal ion binding and the activity of thiol proteins, particularly within the protein disulfide isomerase family, remains an area of active investigation due to the critical role that these proteins play in many vital processes. This research investigates the interaction between recombinant human PDIA1 and zinc ions, focusing on the subsequent implications for PDIA1's conformational stability and enzymatic activity. Employing isothermal titration calorimetry and differential scanning calorimetry, we systematically compared the zinc binding capabilities of both oxidized and reduced forms of PDIA1 and assessed the structural consequences of this interaction. Our results demonstrate that PDIA1 can bind zinc both in reduced and oxidized states, but with significantly different stoichiometry and more pronounced conformational effects in the reduced form of PDIA1. Furthermore, zinc binding was observed to inhibit the catalytic activity of reduced-PDIA1, likely due to induced alterations in its conformation. These findings unveil a potential regulatory mechanism in PDIA1, wherein metal ion binding under reductive conditions modulates its activity. Our study highlights the potential role of zinc in regulating the catalytic function of PDIA1 through conformational modulation, suggesting a nuanced interplay between metal binding and protein stability in the broader context of cellular redox regulation.
Assuntos
Pró-Colágeno-Prolina Dioxigenase , Isomerases de Dissulfetos de Proteínas , Humanos , Oxirredução , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Ligação Proteica , Isomerases de Dissulfetos de Proteínas/metabolismo , Zinco/química , Zinco/metabolismoRESUMO
Pacak-Zhuang syndrome is caused by mutations in the EPAS1 gene, which encodes for one of the three hypoxia-inducible factor alpha (HIFα) paralogs HIF2α and is associated with defined but varied phenotypic presentations including neuroendocrine tumors and polycythemia. However, the mechanisms underlying the complex genotype-phenotype correlations remain incompletely understood. Here, we devised a quantitative method for determining the dissociation constant (Kd) of the HIF2α peptides containing disease-associated mutations and the catalytic domain of prolyl-hydroxylase (PHD2) using microscale thermophoresis (MST) and showed that neuroendocrine-associated Class 1 HIF2α mutants have distinctly higher Kd than the exclusively polycythemia-associated Class 2 HIF2α mutants. Based on the co-crystal structure of PHD2/HIF2α peptide complex at 1.8 Å resolution, we showed that the Class 1 mutated residues are localized to the critical interface between HIF2α and PHD2, adjacent to the PHD2 active catalytic site, while Class 2 mutated residues are localized to the more flexible region of HIF2α that makes less contact with PHD2. Concordantly, Class 1 mutations were found to significantly increase HIF2α-mediated transcriptional activation in cellulo compared to Class 2 counterparts. These results reveal a structural mechanism in which the strength of the interaction between HIF2α and PHD2 is at the root of the general genotype-phenotype correlations observed in Pacak-Zhuang syndrome.
Assuntos
Policitemia , Prolil Hidroxilases , Humanos , Prolil Hidroxilases/genética , Hidroxilação , Policitemia/genética , Mutação , Pró-Colágeno-Prolina DioxigenaseRESUMO
The Von Hippel-Lindau (VHL) protein, which is frequently mutated in clear-cell renal cell carcinoma (ccRCC), is a master regulator of hypoxia-inducible factor (HIF) that is involved in oxidative stresses. However, whether VHL possesses HIF-independent tumor-suppressing activity remains largely unclear. Here, we demonstrate that VHL suppresses nutrient stress-induced autophagy, and its deficiency in sporadic ccRCC specimens is linked to substantially elevated levels of autophagy and correlates with poorer patient prognosis. Mechanistically, VHL directly binds to the autophagy regulator Beclin1, after its PHD1-mediated hydroxylation on Pro54. This binding inhibits the association of Beclin1-VPS34 complexes with ATG14L, thereby inhibiting autophagy initiation in response to nutrient deficiency. Expression of non-hydroxylatable Beclin1 P54A abrogates VHL-mediated autophagy inhibition and significantly reduces the tumor-suppressing effect of VHL. In addition, Beclin1 P54-OH levels are inversely correlated with autophagy levels in wild-type VHL-expressing human ccRCC specimens, and with poor patient prognosis. Furthermore, combined treatment of VHL-deficient mouse tumors with autophagy inhibitors and HIF2α inhibitors suppresses tumor growth. These findings reveal an unexpected mechanism by which VHL suppresses tumor growth, and suggest a potential treatment for ccRCC through combined inhibition of both autophagy and HIF2α.
Assuntos
Proteína Beclina-1 , Carcinoma de Células Renais , Neoplasias Renais , Proteína Supressora de Tumor Von Hippel-Lindau , Animais , Humanos , Camundongos , Autofagia , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Hidroxilação , Neoplasias Renais/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
BACKGROUND: Angiogenesis is essential for tissue repair in ischemic diseases, relying on glycolysis as its primary energy source. Prolyl 4-hydroxylase subunit alpha 1 (P4HA1), the catalytic subunit of collagen prolyl 4-hydroxylase, is a glycolysis-related gene in cancers. However, its role in glycolysis-induced angiogenesis remains unclear. METHODS: P4HA1 expression was modulated using adenoviruses. Endothelial angiogenesis was evaluated through 5-ethynyl-2'-deoxyuridine incorporation, transwell migration, and tube formation assays in vitro. In vivo experiments measured blood flow and capillary density in the hindlimb ischemia (HLI) model. Glycolytic stress assays, glucose uptake, lactate production, and quantitative reverse transcription-polymerase chain reaction (RT-PCR) were employed to assess glycolytic capacity. Transcriptome sequencing, validated by western blotting and RT-PCR, was utilized to determine underlying mechanisms. RESULTS: P4HA1 was upregulated in endothelial cells under hypoxia and in the HLI model. P4HA1 overexpression promoted angiogenesis in vitro and in vivo, while its knockdown had the opposite effect. P4HA1 overexpression reduced cellular α-ketoglutarate (α-KG) levels by consuming α-KG during collagen hydroxylation. Downregulation of α-KG reduced the protein level of a DNA dioxygenase, ten-eleven translocation 2 (TET2), and its recruitment to the fructose-1,6-biphosphatase (FBP1) promoter, resulting in decreased FBP1 expression. The decrease in FBP1 enhanced glycolytic metabolism, thereby promoting endothelial angiogenesis. CONCLUSIONS: Hypoxia-induced endothelial P4HA1 overexpression enhanced angiogenesis by promoting glycolytic metabolism reprogramming through the P4HA1/α-KG/TET2/FBP1 pathway. The study's findings underscore the significance of P4HA1 in post-ischemic angiogenesis, suggesting its therapeutic potential for post-ischemic tissue repair.
Assuntos
Angiogênese , Células Endoteliais , Animais , Humanos , Células Endoteliais/metabolismo , Colágeno/metabolismo , Hipóxia , Glicólise , Prolil Hidroxilases/metabolismo , Isquemia , Pró-Colágeno-Prolina Dioxigenase/genética , Pró-Colágeno-Prolina Dioxigenase/metabolismoRESUMO
It has been reported that prolyl 4-hydroxylase subunit alpha 1 (P4HA1) promoted tumor growth and metastasis of glioma; thus, targeting P4HA1 may be a promising therapeutic strategy against glioma. In consideration of the instability of siRNA in vivo, the chitosan-gelatin microspheres loaded with P4HA1 siRNA (P4HA1 siRNA@CGM) were employed. Firstly, the gel electrophoresis and hemolytic test were performed to assess the stability and blood compatibility of P4HA1 siRNA@CGM. Then, methyl thiazolyl tetrazolium (MTT), cell colony formation, Transwell assay, wound healing assay, gliosphere formation, tube formation, and Western blot were performed to assess the effects of P4HA1 siRNA@CGM on the biological functions of glioma. Finally, 125I-labeled P4HA1 siRNA@CGM was injected into the xenograft mice, radionuclide imaging was recorded, Ki67 and terminal deoxynucleoitidyl transferase-mediated nick end labeling (TUNEL) staining was performed to assess the effects of P4HA1 siRNA@CGM on tumor growth and apoptosis of glioma in vivo. The results showed that P4HA1 siRNA and P4HA1 siRNA@CGM not only markedly inhibited the proliferation, metastasis, gliosphere formation, and the protein levels of interstitial markers (N-cadherin and vimentin) and the transcription factors of epithelial-mesenchymal transition (EMT) (Snail, Slug, and Twist1) in glioma cells, but also inhibited the tube formation in human brain microvascular endothelial cells (HBMECs), and P4HA1 siRNA@CGM exhibited the better inhibitory effects than P4HA1 siRNA. Above results suggested the feasibility of P4HA1 siRNA@CGM in the clinical treatment of glioma.
Assuntos
Quitosana , Glioma , Humanos , Animais , Camundongos , RNA Interferente Pequeno , Gelatina , Radioisótopos do Iodo/metabolismo , Células Endoteliais , Linhagem Celular Tumoral , Glioma/metabolismo , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Pró-Colágeno-Prolina Dioxigenase/genética , Pró-Colágeno-Prolina Dioxigenase/metabolismoRESUMO
Collagen biosynthesis requires several co- and post-translational modifications of lysine and proline residues to form structurally and functionally competent collagen molecules. Formation of 4-hydroxyproline (4Hyp) in Y-position prolines of the repetitive -X-Y-Gly- sequences provides thermal stability for the triple-helical collagen molecules. 4Hyp formation is catalyzed by a collagen prolyl 4-hydroxylase (C-P4H) family consisting of three isoenzymes. Here we identify specific roles for the two main C-P4H isoenzymes in collagen hydroxylation by a detailed 4Hyp analysis of type I and IV collagens derived from cell and tissue samples. Loss of C-P4H-I results in underhydroxylation of collagen where the affected prolines are not uniformly distributed, but mainly present in sites where the adjacent X-position amino acid has a positively charged or a polar uncharged side chain. In contrast, loss of C-P4H-II results in underhydroxylation of triplets where the X-position is occupied by a negatively charged amino acid glutamate or aspartate. Hydroxylation of these triplets was found to be important as loss of C-P4H-II alone resulted in reduced collagen melting temperature and altered assembly of collagen fibrils and basement membrane. The observed C-P4H isoenzyme differences in substrate specificity were explained by selective binding of the substrate to the active site resulting in distinct differences in Km and Vmax values. Furthermore, our results clearly show that the substrate proline selection is not dependent on the collagen type, but the main determinant is the X-position amino acid of the -X-Pro-Gly- triplet. Although our data clearly shows the necessity of both C-P4H-I and II for normal prolyl 4-hydroxylation and function of collagens, the mRNA expression of the isoenzymes with various procollagens was, surprisingly, not tightly coordinated, suggesting additional levels of control. In conclusion, this study provides a molecular level explanation for the need of multiple C-P4H isoenzymes to generate collagen molecules capable to assemble into intact extracellular matrix structures.
Assuntos
Dipeptídeos , Isoenzimas , Prolil Hidroxilases , Prolil Hidroxilases/genética , Isoenzimas/genética , Colágeno Tipo I/genética , Pró-Colágeno-Prolina Dioxigenase/genética , Pró-Colágeno-Prolina Dioxigenase/química , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Colágeno/genética , Colágeno/metabolismo , Prolina/metabolismoRESUMO
OBJECTIVE: Although hypoxia-inducible factor-prolyl hydroxylase (HIF-PH) inhibitors have been developed for the treatment of renal anemia, their effects on cardiac and renal dysfunction remain unknown. We previously reported on Dahl salt-sensitive rats, in a rat model of salt-sensitive hypertension, that exhibited anemia and impaired expression of duodenal iron transporters after the development of hypertensive cardiac and renal dysfunction. Therefore, we investigated the effects of Roxadustat (FG-4592), an HIF-PH inhibitor, on anemia, iron regulation, and cardiac and renal dysfunction in Dahl salt-sensitive rats. METHODS: Six-week-old male Dahl salt-sensitive rats were fed a normal or high-salt diet for 8âweeks. A further subset of Dahl salt-sensitive rats, that were fed a high-salt diet, was administered Roxadustat for 8âweeks. RESULTS: Dahl salt-sensitive rats fed a high-salt diet developed hypertension, cardiac and renal dysfunction, and anemia after 8âweeks of feeding. Roxadustat increased hemoglobin and serum erythropoietin levels in Dahl salt-sensitive rats fed a high-salt diet. With regard to the iron-regulating system, Roxadustat lowered hepatic hepcidin gene expression and increased the gene expression of duodenal iron transporters, such as cytochrome b and divalent metal transporter 1 , in Dahl salt-sensitive rats fed a high-salt diet. Roxadustat did not affect the development of hypertension and cardiac hypertrophy in Dahl salt-sensitive rats with a high-salt diet; however, Roxadustat treatment attenuated renal fibrosis in these rats. CONCLUSIONS: Roxadustat ameliorated anemia with affecting the gene expression of the iron-regulating system, and did not affect cardiac hypertrophy but attenuated renal fibrosis in Dahl salt-sensitive rats fed a high-salt diet.