RESUMO
The Activin A Receptor type I (ALK2) is a critical component of BMP-SMAD signaling that, in the presence of ligands, phosphorylates cytosolic SMAD1/5/8 and modulates important biological processes, including bone formation and iron metabolism. In hepatocytes, the BMP-SMAD pathway controls the expression of hepcidin, the liver peptide hormone that regulates body iron homeostasis via the BMP receptors ALK2 and ALK3, and the hemochromatosis proteins. The main negative regulator of the pathway in the liver is transmembrane serine protease 6 (TMPRSS6), which downregulates hepcidin by cleaving the BMP coreceptor hemojuvelin. ALK2 function is inhibited also by the immunophilin FKBP12, which maintains the receptor in an inactive conformation. FKBP12 sequestration by tacrolimus or its silencing upregulates hepcidin in primary hepatocytes and in vivo in acute but not chronic settings. Interestingly, gain-of-function mutations in ALK2 that impair FKBP12 binding to the receptor and activate the pathway cause a bone phenotype in patients affected by Fibrodysplasia Ossificans Progressiva but not hepcidin and iron metabolism dysfunction. This observation suggests that additional mechanisms are active in the liver to compensate for the increased BMP-SMAD signaling. Here we demonstrate that Fkbp12 downregulation in hepatocytes by antisense oligonucleotide treatment upregulates the expression of the main hepcidin inhibitor Tmprss6, thus counteracting the ALK2-mediated activation of the pathway. Combined downregulation of both Fkbp12 and Tmprss6 blocks this compensatory mechanism. Our findings reveal a previously unrecognized functional cross talk between FKBP12 and TMPRSS6, the main BMP-SMAD pathway inhibitors, in the control of hepcidin transcription.NEW & NOTEWORTHY This study uncovers a previously unrecognized mechanism of hepcidin and BMP-SMAD pathway regulation in hepatocytes mediated by the immunophilin FKBP12 and the transmembrane serine protease TMPRSS6.
Assuntos
Hepcidinas , Proteína 1A de Ligação a Tacrolimo , Humanos , Hepcidinas/genética , Hepcidinas/metabolismo , Ferro/metabolismo , Proteínas de Membrana/genética , Serina , Serina Endopeptidases/genética , Serina Proteases , Proteína 1A de Ligação a Tacrolimo/genéticaRESUMO
A recently approved drug that induces erythroid cell maturation (luspatercept) has been shown to improve anemia and reduce the need for blood transfusion in non-transfusion-dependent as well as transfusion-dependent ß-thalassemia (BT) patients. Although these results were predominantly positive, not all the patients showed the expected increase in hemoglobin (Hb) levels or transfusion burden reduction. Additional studies indicated that administration of luspatercept in transfusion-dependent BT was associated with increased erythropoietic markers, decreased hepcidin levels, and increased liver iron content. Altogether, these studies suggest that luspatercept may necessitate additional drugs for improved erythroid and iron management. As luspatercept does not appear to directly affect iron metabolism, we hypothesized that TMPRSS6-ASO could improve iron parameters and iron overload when co-administered with luspatercept. We used an agent analogous to murine luspatercept (RAP-GRL) and another novel therapeutic, IONIS TMPRSS6-LRx (TMPRSS6-ASO), a hepcidin inducer, to treat non-transfusion-dependent BT-intermedia mice. Our study shows that RAP-GRL alone improved red blood cell (RBC) production, with no or limited effect on splenomegaly and iron parameters. In contrast, TMPRSS6-ASO improved RBC measurements, ameliorated splenomegaly, and improved iron overload most effectively. Our results provide pre-clinical support for combining TMPRSS6-ASO and luspatercept in treating BT, as these drugs together show potential for simultaneously improving both erythroid and iron parameters in BT patients.
Assuntos
Proteínas de Membrana , Serina Endopeptidases , Talassemia beta , Talassemia beta/tratamento farmacológico , Talassemia beta/terapia , Camundongos , Animais , Proteínas de Membrana/genética , Proteínas Recombinantes de Fusão/uso terapêutico , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Fragmentos Fc das Imunoglobulinas/farmacologia , Humanos , Fator de Crescimento Transformador beta/metabolismo , Sobrecarga de Ferro/tratamento farmacológico , Sobrecarga de Ferro/etiologia , Hepcidinas , Ferro/metabolismo , Feminino , Masculino , Quimioterapia Combinada , Receptores de Activinas Tipo IIRESUMO
The mol-ecule of the title NCNHCS pincer N-heterocyclic carbene palladium(II) complex, [PdBr(C21H25N3S)]Br, exhibits a slightly distorted square-planar coordination at the palladium(II) atom, with the five-membered chelate ring nearly planar. The six-membered chelate ring adopts an envelope conformation. Upon chelation, the sulfur atom becomes a stereogenic centre with an RS configuration induced by the chiral carbon of the precursor imidazolium salt. There are intra-molecular C-Hâ¯Br-Pd hydrogen bonds in the structure. The two inter-stitial Br atoms, as the counter-anion of the structure, are both located on crystallographic twofold axes and are connected to the complex cations via C-H⯷Br hydrogen bonds.
RESUMO
Synaptojanin proteins are evolutionarily conserved regulators of vesicle transport and membrane homeostasis. Disruption of synaptojanin function has been implicated in a wide range of neurological disorders. Synaptojanins act as dual-functional lipid phosphatases capable of hydrolyzing a variety of phosphoinositides (PIPs) through autonomous SAC1-like PIP 4-phosphatase and PIP2 5-phosphatase domains. The rarity of an evolutionary configuration of tethering two distinct enzyme activities in a single protein prompted us to investigate their individual and combined roles in budding yeast. Both PIP and PIP2 phosphatase activities are encoded by multiple gene products and are independently essential for cell viability. In contrast, we observed that the synaptojanin proteins utilized both lipid-phosphatase activities to properly coordinate polarized distribution of actin during the cell cycle. Expression of each activity untethered (in trans) failed to properly reconstitute the basal actin regulatory activity; whereas tethering (in cis), even through synthetic linkers, was sufficient to complement these defects. Studies of chimeric proteins harboring PIP and PIP2 phosphatase domains from a variety of gene products indicate synaptojanin proteins have highly specialized activities and that the length of the linker between the lipid-phosphatase domains is critical for actin regulatory activity. Our data are consistent with synaptojanin possessing a strict requirement for both two-domain configuration for some but not all functions and provide mechanistic insights into a coordinated role of tethering distinct lipid-phosphatase activities.
Assuntos
Actinas , Proteínas do Tecido Nervoso , Monoéster Fosfórico Hidrolases , Humanos , Actinas/genética , Actinas/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , LipídeosRESUMO
Periodontal disease is the pathological outcome of the overwhelming inflammation in periodontal tissue. Cellular senescence has been associated with chronic inflammation in several diseases. However, the role of cellular senescence in the pathogenesis of periodontal disease remained unclear. This study aimed to investigate the role and the mechanism of cellular senescence in periodontal disease. Using single-cell RNA sequencing, we first found the upregulated level of cellular senescence in fibroblasts and endothelial cells from inflamed gingival tissue. Subsequently, human gingival fibroblasts isolated from healthy and inflamed gingival tissues were labeled as H-GFs and I-GFs, respectively. Compared to H-GFs, I-GFs exhibited a distinct cellular senescence phenotype, including an increased proportion of senescence-associated ß-galactosidase (SA-ß-gal) positive cells, enlarged cell morphology, and significant upregulation of p16INK4A expression. We further observed increased cellular reactive oxygen species (ROS) activity, mitochondrial ROS, and DNA damage of I-GFs. These phenotypes could be reversed by ROS scavenger NAC, which suggested the cause of cellular senescence in I-GFs. The migration and proliferation assay showed the decreased activity of I-GFs while the gene expression of senescence-associated secretory phenotype (SASP) factors such as IL-1ß, IL-6, TGF-ß, and IL-8 was all significantly increased. Finally, we found that supernatants of I-GF culture induced more neutrophil extracellular trap (NET) formation and drove macrophage polarization toward the CD86-positive M1 pro-inflammatory phenotype. Altogether, our findings implicate that, in the inflamed gingiva, human gingival fibroblasts acquire a senescent phenotype due to oxidative stress-induced DNA and mitochondrial damage, which in turn activate neutrophils and macrophages through the secretion of SASP factors.
RESUMO
OBJECTIVE: With the growing interest in the role of fibroblasts in osteogenesis, this study presents a comparative evaluation of the osteogenic potential of fibroblasts derived from three distinct sources: human gingival fibroblasts (HGFs), mouse embryonic fibroblasts (NIH3T3 cells), and mouse subcutaneous fibroblasts (L929 cells). MC3T3-E1 pre-osteoblast cells were employed as a positive control for osteogenic behavior. DESIGN: Our assessment involved multiple approaches, including vimentin staining for cell origin verification, as well as ALP and ARS staining in conjunction with RT-PCR for osteogenic characterization. RESULTS: Our findings revealed the superior osteogenic differentiation capacity of HGFs compared to MC3T3-E1 and NIH3T3 cells. Analysis of ALP staining confirmed that early osteogenic differentiation was most prominent in MC3T3-E1 cells at 7 days, followed by NIH3T3 and HGFs. However, ARS staining at 21 days demonstrated that HGFs produced the highest number of calcified nodules, indicating their robust potential for late-stage mineralization. This late-stage osteogenic potential of HGFs was further validated through RT-PCR analysis. In contrast, L929 cells displayed no significant osteogenic differentiation potential. CONCLUSIONS: In light of these findings, HGFs emerge as the preferred choice for seed cells in bone tissue engineering applications. This study provides valuable insights into the potential utility of HGFs in the fields of bone tissue engineering and regenerative medicine.
Assuntos
Diferenciação Celular , Fibroblastos , Gengiva , Osteogênese , Animais , Camundongos , Fibroblastos/citologia , Fibroblastos/metabolismo , Células NIH 3T3 , Humanos , Gengiva/citologia , Engenharia Tecidual/métodos , Osteoblastos/citologia , Osteoblastos/metabolismoRESUMO
Manganese is an essential yet potentially toxic metal. Initially reported in 2012, mutations in SLC30A10 are the first known inherited cause of manganese excess. SLC30A10 is an apical membrane protein that exports manganese from hepatocytes into bile and from enterocytes into the lumen of the gastrointestinal tract. SLC30A10 deficiency results in impaired gastrointestinal manganese excretion, leading to manganese excess, neurologic deficits, liver cirrhosis, polycythemia, and erythropoietin excess. Neurologic and liver disease are attributed to manganese toxicity. Polycythemia is attributed to erythropoietin excess. The goal of this study was to determine the basis of erythropoietin excess in SLC30A10 deficiency. Here, we demonstrate that transcription factors hypoxia-inducible factor 1a (Hif1a) and 2a (Hif2a), key mediators of the cellular response to hypoxia, are both upregulated in livers of Slc30a10-deficient mice. Hepatic Hif2a deficiency corrected erythropoietin expression and polycythemia and attenuated aberrant hepatic gene expression in Slc30a10-deficient mice, while hepatic Hif1a deficiency had no discernible impact. Hepatic Hif2a deficiency also attenuated manganese excess, though the underlying cause of this is not clear at this time. Overall, our results indicate that hepatic HIF2 is a key determinant of pathophysiology in SLC30A10 deficiency and expand our understanding of the contribution of HIFs to human disease.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Fígado , Manganês , Policitemia , Animais , Policitemia/metabolismo , Policitemia/genética , Camundongos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fígado/metabolismo , Manganês/metabolismo , Manganês/toxicidade , Manganês/deficiência , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Humanos , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Eritropoetina/metabolismo , Eritropoetina/genética , Camundongos Knockout , Masculino , Hepatócitos/metabolismoRESUMO
SLC30A10 deficiency is a disease of severe manganese excess attributed to loss of SLC30A10-dependent manganese excretion via the gastrointestinal tract. Patients develop dystonia, cirrhosis, and polycythemia. They are treated with chelators but also respond to oral iron, suggesting that iron can outcompete manganese for absorption in this disease. Here we explore the latter observation. Intriguingly, manganese absorption is increased in Slc30a10-deficient mice despite manganese excess. Studies of multiple mouse models indicate that increased dietary manganese absorption reflects two processes: loss of manganese export from enterocytes into the gastrointestinal tract lumen by SLC30A10, and increased absorption of dietary manganese by iron transporters SLC11A2 (DMT1) and SLC40A1 (ferroportin). Our work demonstrates that aberrant absorption contributes prominently to SLC30A10 deficiency and expands our understanding of biological interactions between iron and manganese. Based on these results, we propose a reconsideration of the role of iron transporters in manganese homeostasis is warranted.