Burn-induced intestinal barrier failure has been proposed to be a potential cause of subsequent multiple organ failure after burn. Studies have shown that the increased iNOS activity is closely related to intestinal and pulmonary damage in rats after burn. Expression of iNOS and MMP-9 is regulated by nuclear factor NF-£eB activation, which is frequently a result of MAPKs pathway activation. This study was to investigate the role of ERKs in intestinal and pulmonary damage induced by burn in rats. In experiments, SD rats underwent 30 ~ 35 % TBSA burn. At various times after burn, intestinal mucosa and pulmonary proteins were assayed for ERKs and p38 phosphorylation by immunoblotting, nuclear extracts were assayed for NF-£eB activation by EMSA, intestinal and pulmonary iNOS, MMP-9 expressions were evaluated by RT-PCR, the FITC-dextran permeability was determined to assess the intestinal barrier function and the pulmonary microvascular dysfunction was quantitated by measuring the extravasation of Evans blue dye. The results show that burn induced ERKs and p38 phosphorylation, the expression of iNOS, and NF-£eB activation in intestinal mucosa and lung, but the expression of MMP-9 was attenuated. Treatment with MEK1/2 inhibitors, PD98059 (10 mg/kg i.p.) or U0126 (5 mg/kg i.p.) immediately after burn, attenuated the phosphorylation of intestinal mucosa and pulmonary ERKs, the activation of NF-£eB, the increase in intestinal permeability, and pulmonary microvascular dysfunction. Interestingly, the expression of iNOS in intestinal mucosa and pulmonary tissues was induced by PD98059 administration, but the expression of MMP-9 in intestinal mucosa was attenuated by PD98059 administration. These results suggest that the tissue damage is regulated by NF-£eB activation and the activation of NF-£eB is primarily mediated by signal pathway of ERKs in burn-injured rats, so the signal transduction pathway may involve ERKs and p38, NF-£eB, iNOS or MMP-9, then causes tissue damage. Further, burn-induced intestinal mucosa and pulmonary ERKs have different degree of activation. The p38 and ERKs phosphorylation showed a two-step activation in intestinal mucosa and pulmonary tissues after burn. Inhibition of intestinal and pulmonary ERKs in vivo afforded significant protection against burn-induced barrier failure. However, the data showed that iNOS may not play a major role in the burn-induced intestinal and pulmonary damage, and MMP-9 may have more affect on tissues damage.

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A osseointegração de implantes de titânio (Ti) é dependente da interação entre a superfície de Ti e células, a qual é modulada por diversas vias de sinalização intracelular. Sabe-se que as quinases reguladas por sinais extracelulares (ERKs), membros da família das proteínas quinases ativadas por mitógenos (MAPKs), atuam tanto na osteogênese, quanto na adipogênese e, portanto, podem estar envolvidas no processo de osseointegração de Ti. Nesse contexto, o objetivo do presente estudo foi avaliar se a interação entre células-tronco mesenquimais (CTMs) e superfícies de Ti usinada e com nanotopografia é modulada, ao menos em parte, por ERK1/2 e o consequente efeito da inibição dessas ERKs na diferenciação osteoblástica e adipocítica. Para isso, CTMs derivadas de medula óssea de ratos foram cultivadas sobre discos de Ti usinados e com nanotopografia em condições osteogênicas e adipogênicas, na presença ou não do inibidor de ERK1/2, PD98059, em concentração previamente determinada (25 μM) e foram avaliados parâmetros relacionados à diferenciação osteoblástica e adipocítica. Os resultados mostraram que a expressão gênica dos marcadores osteoblásticos RUNX2, osterix (OSX), fosfatase alcalina (ALP) e osteocalcina (OC) foi aumentada pela inibição da via de sinalização de ERK1/2 nas células crescidas sobre Ti usinado e apenas ALP e OC, naquelas crescidas sobre Ti com nanotopografia. A expressão proteica de RUNX2 foi discretamente maior nas células crescidas sobre Ti usinado, mas não sobre Ti com nanotopografia, quando ERK1/2 foram inibidas e essa inibição não afetou a formação de matriz extracelular mineralizada, independentemente da superfície de Ti avaliada. Com relação à diferenciação adipocítica, a inibição da via de sinalização de ERK1/2 aumentou a expressão gênica dos marcadores adipocíticos PPARγ, adiponectina (ADIPOQ) e proteína ligadora de ácido graxo do adipócito ( AP2) nas células crescidas sobre ambas as superfícies de Ti, com efeito mais acentuado na superfície usinada, sem afetar a formação de acúmulo lípidico. Em conclusão, os resultados mostraram que a inibição de ERK1/2 favoreceu a diferenciação osteoblástica de CTMs crescidas sobre a superfície de Ti usinada, mas não sobre Ti com nanotopografia. Além disso, a inibição de ERK1/2 favoreceu a diferenciação adipocítica de CTMs crescidas sobre as superfícies de Ti com nanotopografia e usinada, sendo o efeito mais acentuado na usinada. Considerando aplicações terapêuticas, esses resultados são relevantes para direcionar o desenvolvimento de superfícies de biomateriais que atuem em vias de sinalização que sabidamente modulam o processo de osteogênese. / Osseointegration of titanium (Ti) implants depends on interaction between Ti surface and cells, which is modulated by several intracellular signaling pathways. Extracellular signal-regulated kinases (ERKs) are members of mitogen-activated protein kinases (MAPKs) family and act on both osteogenesis and adipogenesis and, therefore, may be involved in the process of Ti osseointegration. In this context, the aim of this study was to evaluate if the interaction between mesenchymal stem cells (MSCs) and Ti surfaces, either machined or with nanotopography, is modulated, at least in part, by ERK1/2 and the effect of ERK1/2 inhibition on osteoblast and adipocyte differentiation. Rat bone marrow MSCs were cultured on Ti discs either machined or with nanotopography under osteogenic and adipogenic conditions, in presence or not of the ERK1/2 inhibitor, PD98059, at a concentration previously determined (25 μM) and it was evaluated parameters related to osteoblast and adipocyte differentiation. The results showed that gene expression of the bone markers RUNX2, osterix (OSX), alkaline phosphatase (ALP) and osteocalcin (OC) was increased by ERK1/2 signaling inhibition in cells grown on machined Ti and only ALP and OC in cells grown on Ti with nanotopography. RUNX2 protein expression was slightly higher in cells grown on machined Ti, but not on Ti with nanotopography, when ERK1/2 signaling was inhibited and such inhibition did not affect extracellular matrix mineralization, irrespective of the evaluated Ti surface. Regarding adipocyte differentiation, ERK1/2 signaling inhibition increased gene expression of the adipose tissue markers PPARγ, adiponectin (ADIPOQ) and adipocyte fatty acid-binding protein (AP2) in cells grown on both Ti surfaces, with more prominet effect on machined one, without affecting lipid accumulation. In conclusion, our results showed that ERK1/2 signaling inhibition favored osteoblast differentiation of MSCs grown on machined Ti, but not on Ti with nanotopography. In addition, ERK1/2 signaling inhibition favored adipocyte differentiation of MSCs grown on both Ti surfaces, being more noticeable on machined one. Considering therapeutical applications, these results are relevant to drive the development of biomaterial surfaces to act on signaling pathways that regulate the process of osteogenesis.

A osseointegração de implantes de titânio (Ti) é dependente da interação entre a superfície de Ti e células, a qual é modulada por diversas vias de sinalização intracelular. Sabe-se que as quinases reguladas por sinais extracelulares (ERKs), membros da família das proteínas quinases ativadas por mitógenos (MAPKs), atuam tanto na osteogênese, quanto na adipogênese e, portanto, podem estar envolvidas no processo de osseointegração de Ti. Nesse contexto, o objetivo do presente estudo foi avaliar se a interação entre células-tronco mesenquimais (CTMs) e superfícies de Ti usinada e com nanotopografia é modulada, ao menos em parte, por ERK1/2 e o consequente efeito da inibição dessas ERKs na diferenciação osteoblástica e adipocítica. Para isso, CTMs derivadas de medula óssea de ratos foram cultivadas sobre discos de Ti usinados e com nanotopografia em condições osteogênicas e adipogênicas, na presença ou não do inibidor de ERK1/2, PD98059, em concentração previamente determinada (25 μM) e foram avaliados parâmetros relacionados à diferenciação osteoblástica e adipocítica. Os resultados mostraram que a expressão gênica dos marcadores osteoblásticos RUNX2, osterix (OSX), fosfatase alcalina (ALP) e osteocalcina (OC) foi aumentada pela inibição da via de sinalização de ERK1/2 nas células crescidas sobre Ti usinado e apenas ALP e OC, naquelas crescidas sobre Ti com nanotopografia. A expressão proteica de RUNX2 foi discretamente maior nas células crescidas sobre Ti usinado, mas não sobre Ti com nanotopografia, quando ERK1/2 foram inibidas e essa inibição não afetou a formação de matriz extracelular mineralizada, independentemente da superfície de Ti avaliada. Com relação à diferenciação adipocítica, a inibição da via de sinalização de ERK1/2 aumentou a expressão gênica dos marcadores adipocíticos PPARγ, adiponectina (ADIPOQ) e proteína ligadora de ácido graxo do adipócito ( AP2) nas células crescidas sobre ambas as superfícies de Ti, com efeito mais acentuado na superfície usinada, sem afetar a formação de acúmulo lípidico. Em conclusão, os resultados mostraram que a inibição de ERK1/2 favoreceu a diferenciação osteoblástica de CTMs crescidas sobre a superfície de Ti usinada, mas não sobre Ti com nanotopografia. Além disso, a inibição de ERK1/2 favoreceu a diferenciação adipocítica de CTMs crescidas sobre as superfícies de Ti com nanotopografia e usinada, sendo o efeito mais acentuado na usinada. Considerando aplicações terapêuticas, esses resultados são relevantes para direcionar o desenvolvimento de superfícies de biomateriais que atuem em vias de sinalização que sabidamente modulam o processo de osteogênese. / Osseointegration of titanium (Ti) implants depends on interaction between Ti surface and cells, which is modulated by several intracellular signaling pathways. Extracellular signal-regulated kinases (ERKs) are members of mitogen-activated protein kinases (MAPKs) family and act on both osteogenesis and adipogenesis and, therefore, may be involved in the process of Ti osseointegration. In this context, the aim of this study was to evaluate if the interaction between mesenchymal stem cells (MSCs) and Ti surfaces, either machined or with nanotopography, is modulated, at least in part, by ERK1/2 and the effect of ERK1/2 inhibition on osteoblast and adipocyte differentiation. Rat bone marrow MSCs were cultured on Ti discs either machined or with nanotopography under osteogenic and adipogenic conditions, in presence or not of the ERK1/2 inhibitor, PD98059, at a concentration previously determined (25 μM) and it was evaluated parameters related to osteoblast and adipocyte differentiation. The results showed that gene expression of the bone markers RUNX2, osterix (OSX), alkaline phosphatase (ALP) and osteocalcin (OC) was increased by ERK1/2 signaling inhibition in cells grown on machined Ti and only ALP and OC in cells grown on Ti with nanotopography. RUNX2 protein expression was slightly higher in cells grown on machined Ti, but not on Ti with nanotopography, when ERK1/2 signaling was inhibited and such inhibition did not affect extracellular matrix mineralization, irrespective of the evaluated Ti surface. Regarding adipocyte differentiation, ERK1/2 signaling inhibition increased gene expression of the adipose tissue markers PPARγ, adiponectin (ADIPOQ) and adipocyte fatty acid-binding protein (AP2) in cells grown on both Ti surfaces, with more prominet effect on machined one, without affecting lipid accumulation. In conclusion, our results showed that ERK1/2 signaling inhibition favored osteoblast differentiation of MSCs grown on machined Ti, but not on Ti with nanotopography. In addition, ERK1/2 signaling inhibition favored adipocyte differentiation of MSCs grown on both Ti surfaces, being more noticeable on machined one. Considering therapeutical applications, these results are relevant to drive the development of biomaterial surfaces to act on signaling pathways that regulate the process of osteogenesis.

Lithium, a drug used for the treatment of bipolar disorder, has been shown to affect different aspects of neuronal development such as neuritogenesis, neurogenesis and survival. The underlying mechanism responsible for lithium's influence on neuronal development, however, still remains to be elucidated. In the present study, we demonstrate that lithium increases the phosphorylation of extracellular-signal regulated kinases (ERKs) and protein kinase B (Akt) and promotes neurite outgrowth in mouse N2a neuroblastoma cells (N2a). The inactivation of mitogen-activated protein kinase kinase (MEK)/ERKs signaling with a MEK inhibitor inhibits neurite outgrowth, but it enhances Akt activation in lithium-treated N2a cells. Furthermore, the inactivation of phosphoinositide-3-kinase (PI3K)/Akt signaling with a PI3K inhibitor increases both lithium-induced ERKs activation and lithium-induced neurite outgrowth. Taken together, our study suggests that lithium-induced neurite outgrowth in N2a cells is regulated by cross-talk between the MEK/ERKs and PI3K/Akt pathways and requires the activation of the MEK/ERKs signaling.