RESUMO
CASE: A 25-year-old woman with chronic anorexia nervosa and depression presented with sudden weakness and fatigue. Psychosocial history was notable for binge-starve cycles over the past year and a decline in overall well-being. Vitals on presentation were notable for hypothermia, hypotension, and bradycardia. Initial exam was significant for emaciation, lethargy, and lower extremity edema. Laboratory work-up revealed markedly elevated LFTs, hypoglycemia, thrombocytopenia and elevated INR and lipase. ECG showed sinus bradycardia with prolonged QTc. Ultrasound revealed normal liver and biliary tree. Serum acetaminophen, alcohol level, and urinary toxicology were unremarkable. Work up for infectious, autoimmune, and genetic causes of hepatitis was negative. Echocardiogram revealed left ventricular hypokinesis and EF 10-15%. Nutritional support was begun slowly, however electrolyte derangements began to manifest on hospital day 2, with hypophosphatemia, hypokalemia, hypocalcemia, and hypomagnesemia. Multiple medical and psychiatric disciplines were consulted, and aggressive electrolyte monitoring and repletion were done. The patient's overall clinical status improved slowly during her hospital course. Her liver enzymes trended down, and her QTc interval eventually returned toward the normal range. Repeat echocardiogram following treatment revealed improvement of her EF to 40%. DISCUSSION: Anorexia nervosa is an eating disorder characterized by extremely low body weight, fear of gaining weight or distorted perception of body image, and amenorrhea. Anorexia can lead to life threatening medical complications, and thus constitutes a major challenge to manage. Central to the pathogenesis of the refeeding syndrome is a weakened cardiopulmonary system, electrolytes abnormalities, hepatic dysfunction, liver hypoperfusion and failure. CONCLUSION: Given the clinical presentation, this patient likely presented on the brink of developing frank refeeding syndrome, with cardiac dysfunction and hypovolemia, leading to hepatic hypoperfusion and ischemic hepatitis. Subsequently, she developed electrolyte disturbances characteristic of refeeding syndrome, which were managed without major complication. Her hospital course is encouraging not only for her recovery, but for the collaboration of the different teams involved in her care, and it highlights the importance of a multidisciplinary approach to caring for patients with the potential dire complications of a complex psychiatric illness.
Assuntos
Anorexia Nervosa/sangue , Anorexia Nervosa/psicologia , Eletrólitos/sangue , Adulto , Alanina Transaminase/sangue , Anorexia Nervosa/complicações , Anorexia Nervosa/terapia , Aspartato Aminotransferases/sangue , Feminino , Coração/fisiologia , Humanos , Apoio Nutricional/métodos , Síndrome da Realimentação/etiologia , Síndrome da Realimentação/terapia , Resultado do Tratamento , Desequilíbrio Hidroeletrolítico/sangue , Desequilíbrio Hidroeletrolítico/etiologia , Desequilíbrio Hidroeletrolítico/terapiaRESUMO
Epidemiological studies indicate that patients suffering from atherosclerosis are predisposed to develop osteoporosis. Accordingly, atherogenic determinants such as oxidized low density lipoprotein (OxLDL) particles have been shown to alter bone cell functions. In this work, we investigated the cytotoxicity of lysophosphatidylcholine (lysoPC), a major phospholipid component generated upon LDL oxidation, on bone-forming MG-63 osteoblast-like cells. Cell viability was reduced by lysoPC in a concentration-dependent manner with a LC50 of 18.7±0.7 µM. LysoPC-induced cell death was attributed to induction of both apoptosis and necrosis. Since impairment of intracellular calcium homeostasis is often involved in mechanism of cell death, we determined the involvement of calcium in lysoPC-induced cytotoxicity. LysoPC promoted a rapid and transient increase in intracellular calcium attributed to mobilization from calcium stores, followed by a sustained influx. Intracellular calcium mobilization was associated to phospholipase C (PLC)-dependent mobilization of calcium from the endoplasmic reticulum since inhibition of PLC or calcium depletion of reticulum endoplasmic with thapsigargin prevented the calcium mobilization. The calcium influx induced by lysoPC was abolished by inhibition of transient receptor potential vanilloid (TRPV) channels with ruthenium red whereas gadolinium, which inhibits canonical TRP (TRPC) channels, was without effect. Accordingly, expression of TRPV2 and TRPV4 were shown in MG-63 cells. The addition of TRPV2 inhibitor Tranilast in the incubation medium prevent the calcium influx triggered by lysoPC and reduced lysoPC-induced cytotoxicity whereas TRPV4 inhibitor RN 1734 was without effect, which confirms the involvement of TRPV2 activation in lysoPC-induced cell death.
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Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Lisofosfatidilcolinas/farmacologia , Canais de Cátion TRPV/metabolismo , Apoptose/efeitos dos fármacos , Humanos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/genética , Fosfolipases Tipo C/metabolismo , ortoaminobenzoatos/farmacologiaRESUMO
OBJECTIVE: Clinical and in vitro studies suggest that altered osteogenesis or bone remodeling is involved in the progression and/or onset of osteoarthritis (OA). Wnt signaling plays a key role in osteogenesis via the canonical Wnt/ß-catenin signaling pathway. Two of the R-spondins, Rspo-1 and Rspo-2, a family of 4 proteins unrelated to other Wnt ligands that act as Wnt agonists, are present in bone tissues. The purpose of this study was to investigate the potential role of Rspo-1 and Rspo-2 in OA osteoblasts. METHODS: Primary human normal and OA osteoblasts were prepared from tibial plateaus. The expression of Rspo-1 and Rspo-2 was evaluated by quantitative reverse transcription-polymerase chain reaction analysis. Western blot analysis was used to determine Rspo-2, ß-catenin, and phospho-ß-catenin levels. Wnt/ß-catenin signaling was evaluated using the TOPflash T cell factor (TCF)/lymphoid enhancer factor (LEF) luciferase reporter assay. Mineralization was evaluated by alizarin red staining. RESULTS: The expression of Rspo-1 was similar in normal and OA osteoblasts, whereas the expression and production of Rspo-2 were reduced in OA osteoblasts due to elevated levels of transforming growth factor ß1 in these cells. The reduced Wnt-3a-dependent TOPflash TCF/LEF luciferase reporter activity in OA osteoblasts as compared to normal osteoblasts was corrected by the addition of recombinant human Rspo-2. Wnt-3a-dependent ß-catenin levels were also corrected in OA osteoblasts by Rspo-2 addition. Wnt-3a alone increased the mineralization of OA osteoblasts, which was further increased by Rspo-2. CONCLUSION: Reduced Rspo-2 levels in OA osteoblasts are responsible, at least in part, for their reduced Wnt/ß-catenin signaling and abnormal mineralization. As Rspo-2 is a secreted soluble protein, this could lead to potential new avenues of treatment of OA.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Osteoartrite do Joelho/metabolismo , Osteoblastos/metabolismo , Transdução de Sinais/fisiologia , Trombospondinas/metabolismo , Proteínas Wnt/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Feminino , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/patologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/patologia , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Via de Sinalização Wnt/fisiologia , Proteína Wnt3/farmacologia , beta Catenina/metabolismoRESUMO
Adequate bone formation is assured by the coordinated proliferation, migration, differentiation, and secretory functions of osteoblasts. Epidemiological studies have linked insufficient dietary magnesium (Mg) intake to osteoporosis. Here, we investigated the role of melastatin-like transient receptor potential 7 (TRPM7), a calcium (Ca) and Mg channel, in osteoblastic differentiation of the murine MC3T3 cell line. Osteoblastic differentiation was monitored by alkaline phosphatase activity, osteocalcin gene expression, and extracellular matrix mineralization. Gene expression of TRPM7 increased with osteoblastic differentiation, suggesting the importance of intracellular Ca/Mg homeostasis to cell differentiation. Alteration of intracellular Ca/Mg homeostasis by culture conditions with low extracellular Ca or Mg significantly reduced the osteoblastic differentiation markers alkaline phosphatase activity and osteocalcin gene expression. In accordance, matrix mineralization was reduced under low extracellular Ca or Mg levels. Nevertheless, expression of collagen type I, the predominant matrix protein, was increased in low-Mg culture conditions, indicating that dysfunction of matrix protein production cannot account for the reduced mineralization. Silencing TRPM7 expression during the differentiation period also reduced osteoblastic differentiation and the extent of matrix mineralization. Gene expression of osteoblastic transcription factor Runx2 was reduced by conditions of culture under low extracellular Ca or Mg levels, as well as by TRPM7 silencing. Our results indicate that intracellular Ca and Mg homeostasis ensured by TRPM7 expression is important for the osteoblastic differentiation of MC3T3 cells. Thus, Mg deficiency, a common condition among the population, may be associated with altered osteoblastic differentiation leading to inadequate bone formation and the development of osteoporosis.
Assuntos
Diferenciação Celular/genética , Osteoblastos/fisiologia , Canais de Cátion TRPM/fisiologia , Células 3T3 , Animais , Cálcio/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Homeostase/efeitos dos fármacos , Homeostase/genética , Magnésio/farmacologia , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Interferência de RNA/fisiologia , RNA Interferente Pequeno/farmacologia , Homologia de Sequência , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismoRESUMO
The preservation of bone mass relies on adequate proliferation, differentiation, secretion of matrix proteins and rate of apoptosis of the bone-forming osteoblasts. Although growing body of evidence indicates that the transient receptor potential (TRP) channels play important roles in numerous cellular functions, limited information is available about the TRP channels in osteoblasts. Here, we inventoried the gene expression and addressed some roles of the TRP channels in various osteoblast-like cells. The transcripts of canonical TRP (TRPC) channels were revealed for TRPC1, TRPC3, TRPC4 and TRPC6 in human MG-63, SaOS and U2 OS osteoblasts while transcripts for TRPC2, TRPC4, TRPC6 and TRPC7 were observed in the murine MC3T3 osteoblasts. PCR products were shown for the melastatin-related TRP (TRPM) channels TRPM4, TRPM6, TRPM7 and TRPM8 in all cell lines. The TRPM1 was specifically expressed by murine MC3T3 cells while the TRPM3 transcripts were revealed solely in human osteoblast-like cells. Transcripts for TRPV2 and TRPV4 were shown in osteoblastic cells. By interfering RNA approaches, the TRPC1 channels in osteoblasts were shown to be responsible for the capacitative calcium entry (CCE) and for the stimulation of cell proliferation by platelet-derived growth factor. On the other hand, interfering RNA-mediated abrogation of the expression of TRPM7, known as calcium and magnesium channels, resulted in the reduction of both basal and growth factor-stimulated osteoblastic cell proliferation. Our results provide the first complete reference for the gene expression of TRP channels in osteoblasts and point to their importance in cell proliferation.
Assuntos
Osteoblastos/citologia , Canais de Potencial de Receptor Transitório/análise , Animais , Proliferação de Células , Expressão Gênica , Humanos , Camundongos , RNA Mensageiro/análise , Canais de Cátion TRPM/análise , Canais de Cátion TRPM/genética , Canais de Cátion TRPV/análise , Canais de Cátion TRPV/genética , Canais de Potencial de Receptor Transitório/genéticaRESUMO
Bone is a dynamic tissue that is continuously being remodeled throughout life. Specialized cells called osteoclasts transiently break down old bone (resorption process) at multiple sites as other cells known as osteoblasts are replacing it with new tissue (bone formation). Usually, both resorption and formation processes are in balance and thereby maintain skeletal strength and integrity. This equilibrium is assured by the coordination of proliferation, migration, differentiation, and secretory functions of the osteoblasts, which are essential for adequate formation and resorption processes. Disturbances of this equilibrium may lead to decreased bone mass (osteoporosis), increased bone fragility, and susceptibility to fractures. Epidemiological studies have linked insufficient dietary magnesium (Mg(2+)) intake in humans with low bone mass and osteoporosis. Here, we investigated the roles of Mg(2+) and melastatin-like transient receptor potential 7 (TRPM7), known as Mg(2+) channels, in human osteoblast cell proliferation and migration induced by platelet-derived growth factor (PDGF), which has been involved in the bone remodeling process. PDGF promoted an influx of Mg(2+), enhanced cell migration, and stimulated the gene expression of TRPM7 channels in human osteoblast MG-63 cells. The stimulation of osteoblast proliferation and migration by PDGF was significantly reduced under culture conditions of low extracellular Mg(2+) concentrations. Silencing TRPM7 expression in osteoblasts by specific small interfering RNA prevented the induction by PDGF of Mg(2+) influx, proliferation, and migration. Our results indicate that extracellular Mg(2+) and TRPM7 are important for PDGF-induced proliferation and migration of human osteoblasts. Thus Mg(2+) deficiency, a common condition among the general population, may be associated with altered osteoblast functions leading to inadequate bone formation and the development of osteoporosis.
Assuntos
Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Magnésio/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Canais de Cátion TRPM/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , Forma Celular , Humanos , Deficiência de Magnésio , Osteoblastos/citologia , Proteínas Serina-Treonina Quinases , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Canais de Cátion TRPM/genéticaRESUMO
Background There is limited information about cardiovascular complications among young adults (YA) with type 1 diabetes mellitus (T1DM) who are transitioning from pediatric to adult care. We aimed to study the prevalence and associated factors of dyslipidemia (DLD) and statin treatment in these patients. Methods We recruited 129 YA with T1DM aged 15-25 years. In a cross-sectional analysis, the prevalence of DLD (low-density lipoprotein cholesterol [LDL-C] ≥ 100 mg/dL, high-density lipoprotein cholesterol [HDL-C] <40 mg/dL [males] or <50 mg/dL [females], total cholesterol [TC] ≥200 mg/dL or triglycerides [TG] ≥150 mg/dL) was reported. Socioeconomic and clinical characteristics were compared between YA with and without DLD. We also assessed statin use among YA with DLD. Results DLD was found in 64% of YA, predominantly increased LDL-C (34.9%). Higher mean glycated hemoglobin (HbA1c) was associated with DLD (p < 0.043). Of all YA who met the criteria for statin therapy, only 42% had one prescribed. Conclusions The prevalence of DLD is high in YA with T1DM and is associated with poor glycemic control, and use of statin therapy in this high-risk population is low.
Assuntos
Diabetes Mellitus Tipo 1/complicações , Dislipidemias/tratamento farmacológico , Dislipidemias/epidemiologia , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Transição para Assistência do Adulto , Adolescente , Adulto , Biomarcadores/análise , Criança , Estudos de Coortes , Estudos Transversais , Dislipidemias/etiologia , Feminino , Seguimentos , Humanos , Masculino , Oklahoma/epidemiologia , Prognóstico , Fatores de Risco , Adulto JovemRESUMO
BACKGROUND: Osteoarthritis (OA) is a complex disease, which affects multiple tissues, namely the subchondral bone, articular cartilage and synovial membrane. Alterations of the subchondral bone include an increased, yet under mineralized osteoid matrix, abnormal osteoblast cell phenotype including elevated alkaline phosphatase (ALP) activity, increased release of osteocalcin (OC) and transforming growth factor ß-1 (TGF-ß1). Previous studies have demonstrated an inhibition of the canonical Wnt signaling (cWnt) pathway in OA osteoblasts (Ob). As resveratrol (RSV) has been shown to upregulate the Wnt signaling pathway in different cell systems, we hypothesized that RSV could be beneficial for OA Ob. METHOD: We prepared primary human Ob using the subchondral bone plate of tibial plateaus of OA patients undergoing total knee arthroplasty, or tibial plateaus of normal individuals at autopsy. Sirtuin 1 (Sirt1) expression in normal and OA subchondral bone tissue was evaluated by immunohistochemical analysis. Expression of genes was evaluated by qRT-PCR and protein production by western blot analysis. ALP activity and osteocalcin secretion were evaluated respectively with substrate hydrolysis and enzyme immunoassay. Mineralization levels were evaluated with alizarin red staining. Wnt/ß-catenin signaling was evaluated by target gene expression using the TOPflash TCF/lef luciferase reporter assay and intracellular signaling using ß-catenin levels in western blot analysis. Extracellular signal-regulated kinase (Erk)1/2 and the Smad1/5/8 pathways were evaluated by western blot analysis. RESULTS: Sirt1 expression and production were reduced in OA subchondral bone tissue compared to normal tissue. RSV upregulated Sirt1 and its activity, and reduced the expression of leptin. RSV increased Erk1/2 phosphorylation in OA Ob; however, it had no effect on Smad 1/5/8 phosphorylation. RSV had little effect on cell proliferation and only slightly affected the Bax/Bcl2 ratio. The expression of Runx2/Cbfa1 and peroxisome proliferator-activated receptor (PPAR)γ were not affected by increasing doses of RSV. The endogenous increased ALP activity and OC release observed in OA Ob compared to normal Ob were partly corrected only for ALP at high RSV levels but not for OC release. In contrast, RSV increased the mineralization of OA Ob. Moreover, whereas Wnt3a stimulates the Wnt/ß-catenin pathway in these cells, RSV further increased the response to Wnt3a. CONCLUSION: These data indicate that RSV promotes Sirt1 levels, inhibits the endogenous expression of leptin by OA osteoblasts and can promote the Wnt/ß-catenin and Erk1/2 signaling pathways, which are altered in these cells.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Osteoartrite/metabolismo , Osteoblastos/efeitos dos fármacos , Fenótipo , Estilbenos/farmacologia , Idoso , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Linhagem Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Osteoartrite/tratamento farmacológico , Osteoblastos/fisiologia , Resveratrol , Sirtuína 1/biossíntese , Estilbenos/uso terapêutico , Resultado do TratamentoRESUMO
OBJECTIVE: Clinical and in vitro studies suggest that subchondral bone sclerosis due to abnormal osteoblasts (Ob) is involved in the progression and/or onset of osteoarthritis (OA). Human Ob isolated from sclerotic subchondral OA bone tissue show an altered phenotype, a decreased canonical Wnt/ß-catenin signaling pathway (cWnt), and a reduced mineralization in vitro. In addition to the cWnt pathway, at least two non-canonical signaling pathways, the Wnt/PKC and Wnt/PCP pathway have been described. However, there are no reports of either pathway in OA Ob. Here, we studied the two non-canonical pathways in OA Ob and if they influence their phenotype. METHODS: Human primary subchondral Ob were isolated from the subchondral bone plate of tibial plateaus of OA patients undergoing total knee arthroplasty, or of normal individuals at autopsy. The expression of genes involved in non-canonical Wnt signaling was evaluated by qRT-PCR and their protein production by Western blot analysis. Alkaline phosphatase activity and osteocalcin secretion (OC) were determined with substrate hydrolysis and EIA, respectively. Mineralization levels were evaluated with Alizarin Red Staining, Wnt/PKC and Wnt/PCP pathways by target gene expression and their respective activity using the NFAT and AP-1 luciferase reporter assays. RESULTS: OA Ob showed an altered phenotype as illustrated by an increased alkaline phosphatase activity and osteocalcin release compared to normal Ob. The expression of the non-canonical Wnt5a ligand was increased in OA Ob compared to normal. Whereas, the expression of LGR5 was significantly increased in OA Ob compared to normal Ob, the expression of LGR4 was similar. Wnt5a directly stimulated the expression and production of LGR5, contrasting, Wnt5a did not stimulate the expression of LGR4. Wnt5a also stimulated the phosphorylation of both JNK and PKC, as well as the activity of both NFAT and AP-1 transcription factors. The inhibition of Wnt5a expression partially corrects the abnormal mineralization, OC secretion and ALPase activity of OA Ob. CONCLUSION: These data indicate that the alteration of Wnt5a, a non-canonical Wnt signaling activator, is implicated in the modified signalisation and phenotype observed in OA Ob.
Assuntos
Cálcio/metabolismo , Polaridade Celular , Osteoartrite/metabolismo , Osteoblastos/metabolismo , Proteína Quinase C/metabolismo , Proteínas Wnt/metabolismo , Proteína Wnt-5a/metabolismo , Idoso , Apoptose , Osso e Ossos , Proliferação de Células , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite/patologia , Osteoblastos/patologia , Receptores Acoplados a Proteínas G/metabolismo , Transdução de SinaisRESUMO
Eosinophilic gastroenteritis is an uncommon condition characterized by focal or diffuse infiltration of eosinophils in the gastrointestinal tract in the absence of secondary causes. The pathogenesis of this condition is not well understood and its clinical presentation depends on the segment and layer of the gastrointestinal tract affected. The definition of eosinophilic gastroenteritis may be difficult, as the normal ranges of eosinophil numbers in normal and abnormal gastric and intestinal mucosa are not standardized. We present the case of a 59-year-old male who came to the hospital with hypovolemic shock and lethargy secondary to severe diarrhea. Laboratory analysis was significant for peripheral eosinophilia, and pathology from both the duodenum and colon showed marked eosinophilic infiltration.
RESUMO
INTRODUCTION: Wnt/ß-catenin (cWnt) signaling plays a key role in osteogenesis by promoting the differentiation and mineralization of osteoblasts, activities altered in human osteoarthritic subchondral osteoblast (OA Ob). Sclerostin (SOST) has been shown to alter cWnt signaling. Sirtuin 1 (SIRT1) acts as a novel bone regulator and represses SOST levels in Ob. However the role of SIRT1 and SOST in OA Ob remains unknown. Herein, we explored the role played by SIRT1 and SOST on the abnormal mineralization and cWnt signaling in OA Ob. METHODS: Primary human normal and OA Ob were prepared from tibial plateaus. SOST levels were evaluated by immunohistochemistry, the expression and production of genes by qRT-PCR and WB analysis. Their inhibitions were performed using siRNA. cWnt signaling was measured by the TOPflash TCF/lef luciferase reporter assay. Mineralization was determined by alizarin red staining. RESULTS: SOST levels were significantly increased in OA Ob compared to normal and were linked with elevated TGF-ß1 levels in these cells. SIRT1 expression was significantly reduced in OA Ob compared to normal yet not modified by TGF-ß1. Specific inhibition of SIRT1 increased TGF-ß1 and SOST expressions in OA Ob, while stimulating SIRT1 activity with ß-Nicotinamide mononucleotide reduced the expression of TGF-ß1 and SOST, and increased mineralization in OA Ob. Resveratrol also reduced SOST expression in OA Ob. Reduced cWnt signaling, ß-catenin levels, and mineralization in OA Ob were all corrected via reducing SOST expression. CONCLUSION: These data indicate that high level of SOST is responsible, in part, for the reduced cWnt and mineralization of human OA Ob, which in turn is linked with abnormal SIRT1 levels in these pathological cells.
Assuntos
Proteínas Morfogenéticas Ósseas/genética , Marcadores Genéticos/genética , Osteoartrite/genética , Osteoartrite/patologia , Osteoblastos/metabolismo , Osteoblastos/patologia , Sirtuína 1/metabolismo , Via de Sinalização Wnt , Proteínas Adaptadoras de Transdução de Sinal , Idoso , Animais , Proteínas Morfogenéticas Ósseas/biossíntese , Proteínas Morfogenéticas Ósseas/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Calcificação Fisiológica/genética , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Fenótipo , Fator de Crescimento Transformador beta1/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genéticaRESUMO
INTRODUCTION: Bone remodelling and increased subchondral densification are important in osteoarthritis (OA). Modifications of bone vascularisation parameters, which lead to ischemic episodes associated with hypoxic conditions, have been suspected in OA. Among several factors potentially involved, leptin and dickkopf-related protein 2 (DKK2) are good candidates since they are up-regulated in OA osteoblasts (Obs). Therefore, in the present study, we investigated the hypothesis that hypoxia may drive the expression of leptin and DKK2 in OA Obs. METHODS: Obs from the sclerotic portion of OA tibial plateaus were cultured either under 20% or 2% oxygen tension in the presence or not of 50 nM of 1,25-dihydroxyvitamin D3 (VitD3). The expression of leptin, osteocalcin, DKK2, hypoxia-inducible factors (Hif)-1α and -2α was measured by real-time polymerase chain reaction and leptin production by enzyme-linked immunosorbent assay (ELISA). The expression of Hif-1α, Hif-2α, leptin and DKK2 was reduced using silencing (si) RNA technique. Signalling pathway of hypoxia-induced leptin was investigated by western blotting and mitogen-activated protein kinase (MAPK) inhibitors. RESULTS: As expected, hypoxia stimulated the expression of Hif-1 and Hif-2. The expression of leptin and DKK2 in Obs was also stimulated 7-fold and 1.8-fold respectively (p<0.05) under hypoxia. Interestingly, whereas VitD3 stimulated leptin and DKK2 expression 2- and 4.2-fold under normoxia, it further stimulated it to 28- and 6.2-fold under hypoxia (p<0.05). The hypoxia-induced leptin production was confirmed by ELISA, particularly in presence of VitD3 (p<0.02). Compared to Obs incubated in the presence of siScramble RNAs, siHif-2α inhibited VitD3-stimulated leptin mRNA and protein levels by 70% (p=0.004) and 60% (p<0.02), respectively while it failed to significantly alter the expression of DKK2. SiHif-1α has no effect on these genes. Immunoblotting showed that VitD3 greatly stabilized Hif-2α under hypoxic condition. The increase in leptin expression under hypoxia was also regulated, in addition to the role of Hif-2α, by p38 MAPK (p<0.03) and PI 3-kinase (p<0.05). Finally, we demonstrated that the expression of leptin and DKK2 were not related to each other under hypoxia. CONCLUSIONS: Hypoxic conditions via Hif-2 regulation trigger Obs to produce leptin particularly under VitD3 stimulation whereas DKK2 is mainly regulated by VitD3 rather than hypoxia.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/genética , Leptina/genética , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Vitamina D/farmacologia , Idoso , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Osso e Ossos/patologia , Hipóxia Celular , Células Cultivadas , Feminino , Expressão Gênica/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Immunoblotting , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Leptina/metabolismo , Masculino , Pessoa de Meia-Idade , Osteoartrite/patologia , Osteocalcina/genética , Osteocalcina/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Vitaminas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Exposition to cadmium (Cd) has been linked to bone metabolism alterations and occurrence of osteoporosis. Despite its known renal toxicity which indirectly disrupts bone metabolism through impairment of vitamin D synthesis, increasing evidence argues for the direct action of Cd on bone-forming osteoblasts. Indeed, accumulation of Cd in osteoblasts and metal-induced cell death has been documented but little is known about the intracellular mechanisms of protection against this stress. In this work, we investigated the protection afforded by thiol-containing proteins against Cd cytotoxicity in MC3T3 osteoblastic cells. Viability of MC3T3 cells was reduced by Cd in a concentration-dependent manner with a LC(50) of 7.6±1.1µM. Depletion of glutathione by l-buthionine sulphoximine (BSO) increased cell sensitivity to Cd cytotoxicity, suggesting the involvement of thiol-containing peptides as a mechanism of protection. Accordingly, Cd was shown to promote progressive depletion of reduced thiol content and to stimulate the production of reactive oxygen species (ROS). Interestingly, low non cytotoxic concentrations of Cd increased the gene expression of macrophage migration inhibitory factor (MIF), also a thiol-containing protein. Inhibition of the transcription factor NFκB prevented Cd-dependent upregulation of MIF expression and consequently, increased Cd cytotoxicity in osteoblasts. Moreover, MIF deficient mouse osteoblasts were more sensitive to Cd cytotoxicity than the corresponding control cells. By gel-filtration chromatography, we demonstrated that MIF acts as a thiol-containing protein and thereby promotes Cd complexation. In accordance with its binding ability, addition of recombinant MIF to the culture medium reduced Cd cytotoxicity. Overall, upregulation of MIF expression by Cd may protect against the cytotoxicity of this metal in the osteoblasts.
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Cádmio/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Células 3T3 , Animais , Poluentes Ambientais/toxicidade , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Oxirredutases Intramoleculares/genética , Fatores Inibidores da Migração de Macrófagos/genética , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Nitrilas/farmacologia , Transdução de Sinais , Sulfonas/farmacologiaRESUMO
The Wnt signaling pathway is crucial for osteogenesis and regulates terminal osteoblast differentiation. Although osteoarthritic (OA) osteoblasts show an abnormal phenotype and poor in vitro mineralization, the mechanism leading to this situation still remains unknow. Recent evidence indicates that Wnt signaling may be altered in OA osteoblasts. In this study we determined whether an alteration of the Wnt/ß-catenin signaling pathway is responsible for the abnormal phenotype of OA osteoblasts. Expression of the Wnt signaling antagonist Dickkopf-1 (DKK1) was similar in normal and OA osteoblasts, whereas DKK2 expression was higher in OA osteoblasts than in normal osteoblasts. OA osteoblasts showed a decrease of Wnt3a-dependent Wnt/ß-catenin signaling, measured by the TOPflash reporter assay and by Western blot analysis, compared with normal osteoblasts. Correcting DKK2 levels in OA osteoblasts by siRNA techniques enhanced Wnt/ß-catenin signaling. Elevated DKK2 levels could be explained by elevated transforming growth factor ß1 (TGF-ß1) in OA osteoblasts, and exogenous TGF-ß1 increased DKK2 expression in normal osteoblasts, whereas ablating TGF-ß1 expression in OA osteoblasts reduced DKK2 expression. Inhibiting TGF-ß1 or DKK2 expression corrected the abnormal phenotype of OA osteoblasts. In vitro mineralization of OA osteoblasts also was increased by DKK2 siRNA. We conclude that elevated TGF-ß1 levels in OA osteoblasts can stimulate DKK2 expression, which, in turn, is responsible, at least in part, for their abnormal phenotype.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Osteoartrite/metabolismo , Osteoartrite/patologia , Osteoblastos/metabolismo , Osteoblastos/patologia , Idoso , Fosfatase Alcalina/metabolismo , Animais , Proteína Morfogenética Óssea 2/farmacologia , Calcificação Fisiológica/efeitos dos fármacos , Feminino , Humanos , Ligantes , Masculino , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoblastos/enzimologia , Osteocalcina/metabolismo , Fenótipo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Exposure to cadmium (Cd) disrupts bone metabolism, causing osteoporosis. Impaired vitamin D metabolism was initially proposed as the underlying mechanism, yet recent studies argue for the direct effect of Cd on bone cells. This study aimed at characterizing (109)Cd uptake and cytotoxicity in MC3T3-E1 osteoblasts. Time-dependent accumulation of (109)Cd was observed with a 50% lethal concentration (LC(50)) of 9.6 ± 1.2 µM at 24-h. Reducing extracellular calcium (Ca) or magnesium (Mg) increased Cd cytotoxicity. The presence of Ca, Mg, zinc or gadolinium decreased (109)Cd uptake suggesting the involvement of non-selective cationic channels. The Mg-sensitive part of (109)Cd uptake increased at acidic pH, a condition known to stimulate TRPM7 channel activity. Stimulating TRPM7 channel activity by cellular Mg starvation enhanced (109)Cd uptake. Silencing TRPM7 channel expression abolished the Mg-sensitive and the Mg starvation-induced uptake indicating that TRPM7 is involved in Cd transport in osteoblasts.
Assuntos
Cádmio/toxicidade , Osteoblastos/efeitos dos fármacos , Canais de Cátion TRPM/fisiologia , Animais , Cádmio/farmacocinética , Cálcio/farmacologia , Células Cultivadas , Inativação Gênica , Magnésio/farmacologia , Camundongos , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/genéticaRESUMO
The outcome of malarial anemia is determined by a complex interplay between pro-inflammatory and anti-inflammatory cytokines, its severity associated with accumulation of hemozoin (Hz) in macrophages, elevated IL-10 responses and impaired IL-12 production. Although free heme contributes to malarial anemia by inducing oxidative damage of red blood cells (RBCs) and enhancing their clearance by phagocytes, its impact on IL-12/IL-10 interactions has not been fully characterized. Herein, the effect of hemin (HE) on IL-12 and IL-10 responses was studied in murine bone marrow-derived macrophages (BMDM) and compared with synthetic Hz. Our data reveal that HE induces modest inhibition of IL-12p70 responses to lipopolysaccharide (LPS) whereas Hz significantly impairs IL-12p70 responses to IFNgamma/LPS through down-regulation of IL-12p35 and p40 gene expression. Although reactive oxygen species (ROS) are generated after short-term exposure to HE and Hz, prolonged exposure to these iron protoporphyrins has opposite effects on the cellular redox status, HE being the only compound able to promote persistent ROS production. Accordingly, the inhibitory effect of HE on IL-12p70 seems sustained by redox-dependent induction of IL-10 and is partially controlled by the p38 mitogen-activated protein kinase (MAPK) signalling pathway. Indeed, treatment with n-acetylcysteine (NAC) or with the p38 MAPK inhibitor SB203580 inhibits IL-10 responses and significantly restores IL-12p70 responses to IFNgamma/LPS in HE-conditioned BMDM. Our results suggest that oxidant stress induced by free heme may potentially contribute to sustained production of IL-10 and down-regulation of IL-12 responses in malaria.