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1.
Int J Mol Sci ; 24(18)2023 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-37762698

RESUMO

Low back pain (LBP) is a common musculoskeletal complaint that can impede physical function and mobility. Current management often involves pain medication, but there is a need for non-pharmacological and non-invasive interventions. Soft tissue manipulation (STM), such as massage, has been shown to be effective in human subjects, but the molecular mechanisms underlying these findings are not well understood. In this paper, we evaluated potential changes in the soft tissue levels of more than thirty pro- or anti-inflammatory cytokines following instrument-assisted STM (IASTM) in rats with chronic, induced LBP using Complete Freund's Adjuvant. Our results indicate that IASTM is associated with reduced soft tissue levels of Regulated on Activation, Normal T cell Expressed and Secreted (RANTES)/Chemokine (C-C motif) ligand 5 (CCL5) and increased soft tissue levels of Interleukin (IL)-4, which are pro-inflammatory and anti-inflammatory factors, respectively, by 120 min post-treatment. IASTM was not associated with tissue-level changes in C-X-C Motif Chemokine Ligand (CXCL)-5/Lipopolysaccharide-Induced CXC Chemokine (LIX)-which is the murine homologue of IL-8, CXCL-7, Granulocyte-Macrophage-Colony Simulating Factor (GM-CSF), Intercellular Adhesion Molecule (ICAM)-1, IL1-Receptor Antagonist (IL-1ra), IL-6, Interferon-Inducible Protein (IP)-10/CXCL-10, L-selectin, Tumor Necrosis Factor (TNF)-α, or Vascular Endothelial Growth Factor (VEGF) at either 30 or 120 min post-treatment. Combined, our findings raise the possibility that IASTM may exert tissue-level effects associated with improved clinical outcomes and potentially beneficial changes in pro-/anti-inflammatory cytokines in circulation and at the tissue level.

2.
Biochem Biophys Res Commun ; 524(4): 890-894, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32057362

RESUMO

Osteoporosis is a disease of low bone mass that places individuals at enhanced risk for fracture, disability, and death. Osteoporosis rates are expected to rise significantly in the coming decades yet there are limited pharmacological treatment options, particularly for long-term management of this chronic condition. The drug development pipeline is relatively bereft of new strategies, causing an urgent and unmet need for developing new strategies and targets for treating osteoporosis. Here, we examine a lesser-studied bone remodeling pathway, Neuromedin U (NMU), which is expressed in the bone microenvironment along with its cognate receptors NMU receptor 1 (NMUR1) and 2 (NMUR2). We independently corroborate a prior report that global loss of NMU expression leads to high bone mass and test the hypothesis that NMU negatively regulates osteoblast differentiation. Consistent with this, in vitro studies reveal NMU represses osteoblastic differentiation of osteogenic precursors but, in contrast, promotes osteoblastic marker expression, proliferation and activity of osteoblast-like cells. Phospho-profiling arrays were used to detail differential signaling outcomes that may underlie the opposite responses of these cell types. Collectively, our findings indicate that NMU exerts cell-type-specific responses to regulate osteoblast differentiation and activity.


Assuntos
Neuropeptídeos/genética , Osteoblastos/metabolismo , Osteoporose/genética , Fosfoproteínas/genética , Receptores Acoplados a Proteínas G/genética , Receptores de Neuropeptídeos/genética , Receptores de Neurotransmissores/genética , Animais , Densidade Óssea , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Diferenciação Celular , Linhagem Celular , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Neuropeptídeos/metabolismo , Osteoblastos/patologia , Osteogênese/genética , Osteoporose/metabolismo , Osteoporose/patologia , Fosfoproteínas/classificação , Fosfoproteínas/metabolismo , Fosforilação , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/metabolismo , Receptores de Neurotransmissores/metabolismo , Transdução de Sinais
3.
J Am Soc Nephrol ; 28(4): 1162-1174, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27837149

RESUMO

αKlotho (αKL) regulates mineral metabolism, and diseases associated with αKL deficiency are characterized by hyperphosphatemia and vascular calcification (VC). αKL is expressed as a membrane-bound protein (mKL) and recognized as the coreceptor for fibroblast growth factor-23 (FGF23) and a circulating soluble form (cKL) created by endoproteolytic cleavage of mKL. The functions of cKL with regard to phosphate metabolism are unclear. We tested the ability of cKL to regulate pathways and phenotypes associated with hyperphosphatemia in a mouse model of CKD-mineral bone disorder and αKL-null mice. Stable delivery of adeno-associated virus (AAV) expressing cKL to diabetic endothelial nitric oxide synthase-deficient mice or αKL-null mice reduced serum phosphate levels. Acute injection of recombinant cKL downregulated the renal sodium-phosphate cotransporter Npt2a in αKL-null mice supporting direct actions of cKL in the absence of mKL. αKL-null mice with sustained AAV-cKL expression had a 74%-78% reduction in aorta mineral content and a 72%-77% reduction in mineral volume compared with control-treated counterparts (P<0.01). Treatment of UMR-106 osteoblastic cells with cKL + FGF23 increased the phosphorylation of extracellular signal-regulated kinase 1/2 and induced Fgf23 expression. CRISPR/Cas9-mediated deletion of fibroblast growth factor receptor 1 (FGFR1) or pretreatment with inhibitors of mitogen-activated kinase kinase 1 or FGFR ablated these responses. In summary, sustained cKL treatment reduced hyperphosphatemia in a mouse model of CKD-mineral bone disorder, and it reduced hyperphosphatemia and prevented VC in mice without endogenous αKL. Furthermore, cKL stimulated Fgf23 in an FGFR1-dependent manner in bone cells. Collectively, these findings indicate that cKL has mKL-independent activity and suggest the potential for enhancing cKL activity in diseases of hyperphosphatemia with associated VC.


Assuntos
Glucuronidase/uso terapêutico , Hiperfosfatemia/tratamento farmacológico , Calcificação Vascular/tratamento farmacológico , Animais , Osso e Ossos/metabolismo , Doença Crônica , Nefropatias Diabéticas/complicações , Modelos Animais de Doenças , Feminino , Fator de Crescimento de Fibroblastos 23 , Glucuronidase/administração & dosagem , Glucuronidase/fisiologia , Hiperfosfatemia/etiologia , Proteínas Klotho , Masculino , Camundongos , Camundongos Knockout
4.
Curr Osteoporos Rep ; 12(3): 252-62, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24980542

RESUMO

Rapid and somewhat surprising advances have recently been made toward understanding the molecular mechanisms causing heritable disorders of hypophosphatemia. The results of clinical, genetic, and translational studies have interwoven novel concepts underlying the endocrine control of phosphate metabolism, with far-reaching implications for treatment of both rare Mendelian diseases as well as common disorders of blood phosphate excess such as chronic kidney disease (CKD). In particular, diseases caused by changes in the expression and proteolytic control of the phosphaturic hormone fibroblast growth factor-23 (FGF23) have come to the forefront in terms of directing new models explaining mineral metabolism. These hypophosphatemic disorders as well as others resulting from independent defects in phosphate transport or metabolism will be reviewed herein, and implications for emerging therapeutic strategies based upon these new findings will be discussed.


Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Hiperfosfatemia/metabolismo , Fosfatos/metabolismo , Raquitismo Hipofosfatêmico/metabolismo , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/genética , Homeostase , Humanos , Hiperfosfatemia/genética , Raquitismo Hipofosfatêmico/genética
5.
Biomedicines ; 12(4)2024 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-38672266

RESUMO

Casimersen (AMONDYS 45TM) is an antisense oligonucleotide of the phosphorodiamidate morpholino oligomer subclass developed by Sarepta therapeutics. It was approved by the Food and Drug Administration (FDA) in February 2021 to treat Duchenne muscular dystrophy (DMD) in patients whose DMD gene mutation is amenable to exon 45 skipping. Administered intravenously, casimersen binds to the pre-mRNA of the DMD gene to skip a mutated region of an exon, thereby producing an internally truncated yet functional dystrophin protein in DMD patients. This is essential in maintaining the structure of a myocyte membrane. While casimersen is currently continuing in phase III of clinical trials in various countries, it was granted approval by the FDA under the accelerated approval program due to its observed increase in dystrophin production. This article discusses the pathophysiology of DMD, summarizes available treatments thus far, and provides a full drug review of casimersen (AMONDYS 45TM).

6.
J Bone Miner Res ; 39(3): 298-314, 2024 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-38477790

RESUMO

Osteocytes sense and respond to mechanical force by controlling the activity of other bone cells. However, the mechanisms by which osteocytes sense mechanical input and transmit biological signals remain unclear. Voltage-sensitive calcium channels (VSCCs) regulate calcium (Ca2+) influx in response to external stimuli. Inhibition or deletion of VSCCs impairs osteogenesis and skeletal responses to mechanical loading. VSCC activity is influenced by its auxiliary subunits, which bind the channel's α1 pore-forming subunit to alter intracellular Ca2+ concentrations. The α2δ1 auxiliary subunit associates with the pore-forming subunit via a glycosylphosphatidylinositol anchor and regulates the channel's calcium-gating kinetics. Knockdown of α2δ1 in osteocytes impairs responses to membrane stretch, and global deletion of α2δ1 in mice results in osteopenia and impaired skeletal responses to loading in vivo. Therefore, we hypothesized that the α2δ1 subunit functions as a mechanotransducer, and its deletion in osteocytes would impair skeletal development and load-induced bone formation. Mice (C57BL/6) with LoxP sequences flanking Cacna2d1, the gene encoding α2δ1, were crossed with mice expressing Cre under the control of the Dmp1 promoter (10 kb). Deletion of α2δ1 in osteocytes and late-stage osteoblasts decreased femoral bone quantity (P < .05) by DXA, reduced relative osteoid surface (P < .05), and altered osteoblast and osteocyte regulatory gene expression (P < .01). Cacna2d1f/f, Cre + male mice displayed decreased femoral strength and lower 10-wk cancellous bone in vivo micro-computed tomography measurements at the proximal tibia (P < .01) compared to controls, whereas Cacna2d1f/f, Cre + female mice showed impaired 20-wk cancellous and cortical bone ex vivo micro-computed tomography measurements (P < .05) vs controls. Deletion of α2δ1 in osteocytes and late-stage osteoblasts suppressed load-induced calcium signaling in vivo and decreased anabolic responses to mechanical loading in male mice, demonstrating decreased mechanosensitivity. Collectively, the α2δ1 auxiliary subunit is essential for the regulation of osteoid-formation, femur strength, and load-induced bone formation in male mice.


The ability of bone to sense and respond to forces generated during daily physical activities is essential to skeletal health. Although several bone cell types contribute to the maintenance of bone health, osteocytes are thought to be the primary mechanosensitive cells; however, the mechanisms through which these cells perceive mechanical stimuli remains unclear. Previous work has shown that voltage sensitive calcium channels are necessary for bone to sense mechanical force; yet the means by which those channels translate the physical signal into a biochemical signal is unclear. Data within this manuscript demonstrate that the extracellular α2δ1 subunit of voltage sensitive calcium channels is necessary for load-induced bone formation as well as to enable calcium influx within osteocytes. As this subunit enables physical interactions of the channel pore with the extracellular matrix, our data demonstrate the need for the α2δ1 subunit for mechanically induced bone adaptation, thus serving as a physical conduit through which mechanical signals from the bone matrix are transduced into biochemical signals by enabling calcium influx into osteocytes.


Assuntos
Osteócitos , Osteogênese , Camundongos , Masculino , Feminino , Animais , Osteócitos/metabolismo , Osteogênese/genética , Cálcio/metabolismo , Microtomografia por Raio-X , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Fêmur/diagnóstico por imagem , Fêmur/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo
7.
JBMR Plus ; 8(2): ziad008, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38505532

RESUMO

Voltage-sensitive calcium channels (VSCCs) influence bone structure and function, including anabolic responses to mechanical loading. While the pore-forming (α1) subunit of VSCCs allows Ca2+ influx, auxiliary subunits regulate the biophysical properties of the pore. The α2δ1 subunit influences gating kinetics of the α1 pore and enables mechanically induced signaling in osteocytes; however, the skeletal function of α2δ1 in vivo remains unknown. In this work, we examined the skeletal consequences of deleting Cacna2d1, the gene encoding α2δ1. Dual-energy X-ray absorptiometry and microcomputed tomography imaging demonstrated that deletion of α2δ1 diminished bone mineral content and density in both male and female C57BL/6 mice. Structural differences manifested in both trabecular and cortical bone for males, while the absence of α2δ1 affected only cortical bone in female mice. Deletion of α2δ1 impaired skeletal mechanical properties in both sexes, as measured by three-point bending to failure. While no changes in osteoblast number or activity were found for either sex, male mice displayed a significant increase in osteoclast number, accompanied by increased eroded bone surface and upregulation of genes that regulate osteoclast differentiation. Deletion of α2δ1 also rendered the skeleton insensitive to exogenous mechanical loading in males. While previous work demonstrates that VSCCs are essential for anabolic responses to mechanical loading, the mechanism by which these channels sense and respond to force remained unclear. Our data demonstrate that the α2δ1 auxiliary VSCC subunit functions to maintain baseline bone mass and strength through regulation of osteoclast activity and also provides skeletal mechanotransduction in male mice. These data reveal a molecular player in our understanding of the mechanisms by which VSCCs influence skeletal adaptation.

8.
Life (Basel) ; 13(4)2023 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-37109447

RESUMO

Global loss of the neuropeptide Neuromedin-U (NMU) is associated with increased bone formation and high bone mass in male and female mice by twelve weeks of age, suggesting that NMU suppresses osteoblast differentiation and/or activity in vivo. NMU is highly expressed in numerous anatomical locations including the skeleton and the hypothalamus. This raises the possibility that NMU exerts indirect effects on bone remodeling from an extra-skeletal location such as the brain. Thus, in the present study we used microinjection to deliver viruses carrying short-hairpin RNA designed to knockdown Nmu expression in the hypothalamus of 8-week-old male rats and evaluated the effects on bone mass in the peripheral skeleton. Quantitative RT-PCR confirmed approximately 92% knockdown of Nmu in the hypothalamus. However, after six weeks, micro computed tomography on tibiae from Nmu-knockdown rats demonstrated no significant change in trabecular or cortical bone mass as compared to controls. These findings are corroborated by histomorphometric analyses which indicate no differences in osteoblast or osteoclast parameters between controls and Nmu-knockdown samples. Collectively, these data suggest that hypothalamus-derived NMU does not regulate bone remodeling in the postnatal skeleton. Future studies are necessary to delineate the direct versus indirect effects of NMU on bone remodeling.

10.
Int J Mol Sci ; 13(11): 14385-400, 2012 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-23203070

RESUMO

Live-cell microscopy is now routinely used to monitor the activities of the genetically encoded biosensor proteins that are designed to directly measure specific cell signaling events inside cells, tissues, or organisms. Most fluorescent biosensor proteins rely on Förster resonance energy transfer (FRET) to report conformational changes in the protein that occur in response to signaling events, and this is commonly measured with intensity-based ratiometric imaging methods. An alternative method for monitoring the activities of the FRET-based biosensor proteins is fluorescence lifetime imaging microscopy (FLIM). FLIM measurements are made in the time domain, and are not affected by factors that commonly limit intensity measurements. In this review, we describe the use of the digital frequency domain (FD) FLIM method for the analysis of FRET signals. We illustrate the methods necessary for the calibration of the FD FLIM system, and demonstrate the analysis of data obtained from cells expressing "FRET standard" fusion proteins. We then use the FLIM-FRET approach to monitor the changes in activities of two different biosensor proteins in specific regions of single living cells. Importantly, the factors required for the accurate determination and reproducibility of lifetime measurements are described in detail.


Assuntos
Técnicas Biossensoriais , Rastreamento de Células/métodos , Microscopia de Fluorescência/métodos , Animais , Humanos
11.
Endocrinology ; 164(1)2022 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-36412119

RESUMO

Diabetes results from insufficient insulin production by pancreatic islet ß-cells or a loss of ß-cells themselves. Restoration of regulated insulin production is a predominant goal of translational diabetes research. Here, we provide a brief overview of recent advances in the fields of ß-cell proliferation, regeneration, and replacement. The discovery of therapeutic targets and associated small molecules has been enabled by improved understanding of ß-cell development and cell cycle regulation, as well as advanced high-throughput screening methodologies. Important findings in ß-cell transdifferentiation, neogenesis, and stem cell differentiation have nucleated multiple promising therapeutic strategies. In particular, clinical trials are underway using in vitro-generated ß-like cells from human pluripotent stem cells. Significant challenges remain for each of these strategies, but continued support for efforts in these research areas will be critical for the generation of distinct diabetes therapies.


Assuntos
Diabetes Mellitus , Células Secretoras de Insulina , Humanos , Regeneração/fisiologia , Células Secretoras de Insulina/metabolismo , Proliferação de Células , Diabetes Mellitus/terapia , Diabetes Mellitus/metabolismo , Insulina/metabolismo , Diferenciação Celular
12.
Physiol Rep ; 10(11): e15307, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35656701

RESUMO

Ferric citrate (FC) is an approved therapy for chronic kidney disease (CKD) patients as a phosphate (Pi) binder for dialysis-dependent CKD, and for iron deficiency anemia (IDA) in non-dialysis CKD. Elevated Pi and IDA both lead to increased FGF23, however, the roles of iron and FGF23 during CKD remain unclear. To this end, iron and Pi metabolism were tested in a mouse model of CKD (0.2% adenine) ± 0.5% FC for 6 weeks, with and without osteocyte deletion of Fgf23 (flox-Fgf23/Dmp1-Cre). Intact FGF23 (iFGF23) increased in all CKD mice but was lower in Cre+ mice with or without FC, thus the Dmp1-Cre effectively reduced FGF23. Cre+ mice fed AD-only had higher serum Pi than Cre- pre- and post-diet, and the Cre+ mice had higher BUN regardless of FC treatment. Total serum iron was higher in all mice receiving FC, and liver Tfrc, Bmp6, and hepcidin mRNAs were increased regardless of genotype; liver IL-6 showed decreased mRNA in FC-fed mice. The renal 1,25-dihydroxyvitamin D (1,25D) anabolic enzyme Cyp27b1 had higher mRNA and the catabolic Cyp24a1 showed lower mRNA in FC-fed mice. Finally, mice with loss of FGF23 had higher bone cortical porosity, whereas Raman spectroscopy showed no changes in matrix mineral parameters. Thus, FC- and FGF23-dependent and -independent actions were identified in CKD; loss of FGF23 was associated with higher serum Pi and BUN, demonstrating that FGF23 was protective of mineral metabolism. In contrast, FC maintained serum iron and corrected inflammation mediators, potentially providing ancillary benefit.


Assuntos
Fatores de Crescimento de Fibroblastos , Ferro , Insuficiência Renal Crônica , Animais , Ácido Cítrico , Modelos Animais de Doenças , Eletrólitos , Compostos Férricos , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/metabolismo , Humanos , Ferro/metabolismo , Camundongos , Minerais , RNA Mensageiro/metabolismo , Insuficiência Renal Crônica/metabolismo
13.
Biomolecules ; 12(12)2022 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-36551284

RESUMO

Our understanding of how osteocytes, the principal mechanosensors within bone, sense and perceive force remains unclear. Previous work identified "tethering elements" (TEs) spanning the pericellular space of osteocytes and transmitting mechanical information into biochemical signals. While we identified the heparan sulfate proteoglycan perlecan (PLN) as a component of these TEs, PLN must attach to the cell surface to induce biochemical responses. As voltage-sensitive calcium channels (VSCCs) are critical for bone mechanotransduction, we hypothesized that PLN binds the extracellular α2δ1 subunit of VSCCs to couple the bone matrix to the osteocyte membrane. Here, we showed co-localization of PLN and α2δ1 along osteocyte dendritic processes. Additionally, we quantified the molecular interactions between α2δ1 and PLN domains and demonstrated for the first time that α2δ1 strongly associates with PLN via its domain III. Furthermore, α2δ1 is the binding site for the commonly used pain drug, gabapentin (GBP), which is associated with adverse skeletal effects when used chronically. We found that GBP disrupts PLN::α2δ1 binding in vitro, and GBP treatment in vivo results in impaired bone mechanosensation. Our work identified a novel mechanosensory complex within osteocytes composed of PLN and α2δ1, necessary for bone force transmission and sensitive to the drug GBP.


Assuntos
Proteoglicanas de Heparan Sulfato , Mecanotransdução Celular , Proteoglicanas de Heparan Sulfato/metabolismo , Gabapentina/farmacologia , Proteínas da Matriz Extracelular/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo
14.
J Cell Physiol ; 226(4): 1044-51, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20857415

RESUMO

Fluid shear stress protects cells from TNF-α-induced apoptosis. Oscillatory fluid shear stress (OFSS) is generally perceived as physiologically relevant biophysical signal for bone cells. Here we identify several cellular mechanisms responsible for mediating the protective effects of OFSS against TNF-α-induced apoptosis in vitro. We found that exposure of MC3T3-E1 osteoblast-like cells to as little as 5 min of OFSS suppressed TNF-α-induced activation of caspase-3, cleavage of PARP and phosphorylation of histone. In contrast, H(2)O(2)-induced apoptosis was not inhibited by OFSS suggesting that OFSS might not be protecting cells from TNF-α-induced apoptosis via stimulation of global pro-survival signaling pathways. In support of this speculation, OFSS inhibition of TNF-α-induced apoptosis was unaffected by inhibitors of several pro-survival signaling pathways including pI3-kinase (LY294002), MAPK/ERK kinase (PD98059 or U0126), intracellular Ca2+ release (U73122), NO production (L-NAME), or protein synthesis (cycloheximide) that were applied to cells during exposure to OFSS and during TNF-α treatment. However, TNF-α-induced phosphorylation and degradation of IκBα was blocked by pre-exposure of cells to OFSS suggesting a more specific effect of OFSS on TNF-α signaling. We therefore focused on the mechanism of OFSS regulation of TNF-receptor 1 (TNFR1) signaling and found that OFSS (1) reduced the amount of receptor on the cell surface, (2) prevented the association of ubiquitinated RIP in TNFR1 complexes with TRADD and TRAF2, and (3) reduced TNF-α-induced IL-8 promoter activity in the nucleus. We conclude that the anti-apoptotic effect of OFSS is not mediated by activation of universal pro-survival signaling pathways. Rather, OFSS inhibits TNF-α-induced pro-apoptotic signaling which can be explained by the down-regulation of TNFR1 on the cell surface and blockade of TNFR1 downstream signaling by OFSS.


Assuntos
Osteoblastos/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Reologia , Transdução de Sinais , Estresse Mecânico , Animais , Apoptose/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Endocitose/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Peróxido de Hidrogênio/farmacologia , Proteínas I-kappa B/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Inibidor de NF-kappaB alfa , Óxido Nítrico/biossíntese , Osteoblastos/efeitos dos fármacos , Osteoblastos/enzimologia , Regiões Promotoras Genéticas/genética , Biossíntese de Proteínas/efeitos dos fármacos , Reologia/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitinação/efeitos dos fármacos
15.
Life (Basel) ; 11(6)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203792

RESUMO

The authors present a stereotypical case presentation of X-linked hypophosphatemia (XLH) and provide a review of the pathophysiology and related pharmacology of this condition, primarily focusing on the FDA-approved medication burosumab. XLH is a renal phosphate wasting disorder caused by loss of function mutations in the PHEX gene (phosphate-regulating gene with homologies to endopeptidases on the X chromosome). Typical biochemical findings include elevated serum levels of bioactive/intact fibroblast growth factor 23 (FGF23) which lead to (i) low serum phosphate levels, (ii) increased fractional excretion of phosphate, and (iii) inappropriately low or normal 1,25-dihydroxyvitamin D (1,25-vitD). XLH is the most common form of heritable rickets and short stature in patients with XLH is due to chronic hypophosphatemia. Additionally, patients with XLH experience joint pain and osteoarthritis from skeletal deformities, fractures, enthesopathy, spinal stenosis, and hearing loss. Historically, treatment for XLH was limited to oral phosphate supplementation, active vitamin D supplementation, and surgical intervention for cases of severe bowed legs. In 2018, the United States Food and Drug Administration (FDA) approved burosumab for the treatment of XLH and this medication has demonstrated substantial benefit compared with conventional therapy. Burosumab binds circulating intact FGF23 and blocks its biological effects in target tissues, resulting in increased serum inorganic phosphate (Pi) concentrations and increased conversion of inactive vitamin D to active 1,25-vitD.

16.
Life (Basel) ; 11(10)2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34685399

RESUMO

Neuromedin-U (NMU) is an evolutionarily conserved peptide that regulates varying physiologic effects including blood pressure, stress and allergic responses, metabolic and feeding behavior, pain perception, and neuroendocrine functions. Recently, several lines of investigation implicate NMU in regulating bone remodeling. For instance, global loss of NMU expression in male and female mice leads to high bone mass due to elevated bone formation rate with no alteration in bone resorption rate or observable defect in skeletal patterning. Additionally, NMU treatment regulates the activity of osteoblasts in vitro. The downstream pathway utilized by NMU to carry out these effects is unknown as NMU signals via two G-protein-coupled receptors (GPCRs), NMU receptor 1 (NMUR1), and NMU receptor 2 (NMUR2), and both are expressed in the postnatal skeleton. Here, we sought to address this open question and build a better understanding of the downstream pathway utilized by NMU. Our approach involved the knockdown of Nmur1 in MC3T3-E1 cells in vitro and a global knockout of Nmur1 in vivo. We detail specific cell signaling events (e.g., mTOR phosphorylation) that are deficient in the absence of NMUR1 expression yet trabecular bone volume in femora and tibiae of 12-week-old male Nmur1 knockout mice are unchanged, compared to controls. These results suggest that NMUR1 is required for NMU-dependent signaling in MC3T3-E1 cells, but it is not required for the NMU-mediated effects on bone remodeling in vivo. Future studies examining the role of NMUR2 are required to determine the downstream pathway utilized by NMU to regulate bone remodeling in vivo.

17.
BMC Res Notes ; 13(1): 400, 2020 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-32854782

RESUMO

OBJECTIVE: Soft tissue manual therapies are commonly utilized by osteopathic physicians, chiropractors, physical therapists and massage therapists. These techniques are predicated on subjecting tissues to biophysical mechanical stimulation but the cellular and molecular mechanism(s) mediating these effects are poorly understood. Previous studies established an in vitro model system for examining mechanical stimulation of dermal fibroblasts and established that cyclical strain, intended to mimic overuse injury, induces secretion of numerous pro-inflammatory cytokines. Moreover, mechanical strain intended to mimic soft tissue manual therapy reduces strain-induced secretion of pro-inflammatory cytokines. Here, we sought to partially confirm and extend these reports and provide independent corroboration of prior results. RESULTS: Using cultures of primary human dermal fibroblasts, we confirm cyclical mechanical strain increases levels of IL-6 and adding long-duration stretch, intended to mimic therapeutic soft tissue stimulation, after cyclical strain results in lower IL-6 levels. We also extend the prior work, reporting that long-duration stretch results in lower levels of IL-8. Although there are important limitations to this experimental model, these findings provide supportive evidence that therapeutic soft tissue stimulation may reduce levels of pro-inflammatory cytokines. Future work is required to address these open questions and advance the mechanistic understanding of therapeutic soft tissue stimulation.


Assuntos
Citocinas , Manipulações Musculoesqueléticas , Células Cultivadas , Fibroblastos , Humanos , Pele
18.
JCI Insight ; 4(4)2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30830862

RESUMO

The phosphaturic hormone FGF23 is elevated in chronic kidney disease (CKD). The risk of premature death is substantially higher in the CKD patient population, with cardiovascular disease (CVD) as the leading mortality cause at all stages of CKD. Elevated FGF23 in CKD has been associated with increased odds for all-cause mortality; however, whether FGF23 is associated with positive adaptation in CKD is unknown. To test the role of FGF23 in CKD phenotypes, a late osteoblast/osteocyte conditional flox-Fgf23 mouse (Fgf23fl/fl/Dmp1-Cre+/-) was placed on an adenine-containing diet to induce CKD. Serum analysis showed casein-fed Cre+ mice had significantly higher serum phosphate and blood urea nitrogen (BUN) versus casein diet and Cre- genotype controls. Adenine significantly induced serum intact FGF23 in the Cre- mice over casein-fed mice, whereas Cre+ mice on adenine had 90% reduction in serum intact FGF23 and C-terminal FGF23 as well as bone Fgf23 mRNA. Parathyroid hormone was significantly elevated in mice fed adenine diet regardless of genotype, which significantly enhanced midshaft cortical porosity. Echocardiographs of the adenine-fed Cre+ hearts revealed profound aortic calcification and cardiac hypertrophy versus diet and genotype controls. Thus, these studies demonstrate that increased bone FGF23, although associated with poor outcomes in CKD, is necessary to protect against the cardio-renal consequences of elevated tissue phosphate.


Assuntos
Osso e Ossos/metabolismo , Síndrome Cardiorrenal/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Fosfatos/sangue , Insuficiência Renal Crônica/metabolismo , Adenina/administração & dosagem , Adenina/toxicidade , Animais , Osso e Ossos/citologia , Síndrome Cardiorrenal/sangue , Síndrome Cardiorrenal/diagnóstico , Síndrome Cardiorrenal/etiologia , Modelos Animais de Doenças , Ecocardiografia , Feminino , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/sangue , Fatores de Crescimento de Fibroblastos/genética , Humanos , Camundongos , Camundongos Transgênicos , Osteócitos/metabolismo , Hormônio Paratireóideo/sangue , Hormônio Paratireóideo/metabolismo , Fosfatos/metabolismo , Insuficiência Renal Crônica/sangue , Insuficiência Renal Crônica/induzido quimicamente , Insuficiência Renal Crônica/complicações , Calcificação Vascular/sangue , Calcificação Vascular/etiologia , Calcificação Vascular/metabolismo
19.
BMC Res Notes ; 12(1): 331, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31186065

RESUMO

OBJECTIVE: The bone morphogenetic protein (BMP) signaling pathway comprises the largest subdivision of the transforming growth factor (TGFß) superfamily. BMP signaling plays essential roles in both embryonic development and postnatal tissue homeostasis. Dysregulated BMP signaling underlies human pathologies ranging from pulmonary arterial hypertension to heterotopic ossification. Thus, understanding the basic mechanisms and regulation of BMP signaling may yield translational opportunities. Unfortunately, limited tools are available to evaluate this pathway, and genetic approaches are frequently confounded by developmental requirements or ability of pathway components to compensate for one another. Specific inhibitors for type 2 receptors are poorly represented. Thus, we sought to identify and validate an antibody that neutralizes the ligand-binding function of BMP receptor type 2 (BMPR2) extracellular domain (ECD). RESULTS: Using a modified, cell-free immunoprecipitation assay, we examined the neutralizing ability of the mouse monoclonal antibody 3F6 and found a dose-dependent inhibition of BMPR2-ECD ligand-binding. Consistent with this, 3F6 blocks endogenous BMPR2 function in the BMP-responsive cell line HEK293T. The specificity of 3F6 action was confirmed by demonstrating that this antibody has no effect on BMP-responsiveness in HEK293T cells in which BMPR2 expression is knocked-down. Our results provide important proof-of-concept data for future studies interrogating BMPR2 function.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Especificidade de Anticorpos/imunologia , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/metabolismo , Anticorpos Neutralizantes/farmacologia , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/antagonistas & inibidores , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Células HEK293 , Humanos , Camundongos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia
20.
J Appl Physiol (1985) ; 126(4): 854-862, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30605400

RESUMO

Diabetic nephropathy (DN) is a primary cause of end-stage renal disease and is becoming more prevalent because of the global rise in type 2 diabetes. A model of DN, the db/db uninephrectomized ( db/db-uni) mouse, is characterized by obesity, as well as compromised renal function. This model also manifests defects in mineral metabolism common in DN, including hyperphosphatemia, which leads to severe endocrine disease. The FGF23 coreceptor, α-Klotho, circulates as a soluble, cleaved form (cKL) and may directly influence phosphate handling. Our study sought to test the effects of cKL on mineral metabolism in db/db-uni mice. Mice were placed into either mild or moderate disease groups on the basis of the albumin-to-creatinine ratio (ACR). Body weights of db/db-uni mice were significantly greater across the study compared with lean controls regardless of disease severity. Adeno-associated cKL administration was associated with increased serum Klotho, intact, bioactive FGF23 (iFGF23), and COOH-terminal fragments of FGF23 ( P < 0.05). Blood urea nitrogen was improved after cKL administration, and cKL corrected hyperphosphatemia in the high- and low-ACR db/db-uni groups. Interestingly, 2 wk after cKL delivery, blood glucose levels were significantly reduced in db/db-uni mice with high ACR ( P < 0.05). Interestingly, several genes associated with stabilizing active iFGF23 were also increased in the osteoblastic UMR-106 cell line with cKL treatment. In summary, delivery of cKL to a model of DN normalized blood phosphate levels regardless of disease severity, supporting the concept that targeting cKL-affected pathways could provide future therapeutic avenues in DN. NEW & NOTEWORTHY In this work, systemic and continuous delivery of the "soluble" or "cleaved" form of the FGF23 coreceptor α-Klotho (cKL) via adeno-associated virus to a rodent model of diabetic nephropathy (DN), the db/db uninephrectomized mouse, normalized blood phosphate levels regardless of disease severity. This work supports the concept that targeting cKL-affected pathways could provide future therapeutic avenues for the severe mineral metabolism defects associated with DN.


Assuntos
Nefropatias Diabéticas/sangue , Glucuronidase/metabolismo , Fosfatos/sangue , Animais , Glicemia/metabolismo , Linhagem Celular Tumoral , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/metabolismo , Nefropatias Diabéticas/metabolismo , Modelos Animais de Doenças , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/metabolismo , Hiperfosfatemia/sangue , Hiperfosfatemia/metabolismo , Proteínas Klotho , Camundongos , Osteoblastos/metabolismo , Ratos
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