RESUMO
The mitogen-activated protein kinase (MAPK) cascade is one of the most ubiquitous signal transduction systems and is rapidly activated by various stimuli, such as cellular stress and death. The Caco-2 cell line is an in vitro model for colon cancer studies. We investigated the activation status of the ERK1/2, p38, JNK1/2, and ERK5 kinases and their respective upstream intracellular activators in Caco-2 cells induced to proliferate by 10% fetal bovine serum (FBS). The states of phosphorylation of the above MAPKs and their upstream kinases, MEK1/2, MKK3/6, MKK4, and MKK7, respectively, were studied by Western blot analysis. Phosphorylation was barely detectable before serum stimulation, and the stimulation of cell proliferation by the addition of FBS increased MEK1/2 and ERK1/2 phosphorylation 2 to 3 fold after 3 min. FBS stimulated p38 and MKK3/6 to the same extent within 2 min of treatment and JNK1/2 and its upstream kinases MKK4 and MKK7 5-fold (3 min). Addition of FBS also rapidly phosphorylated ERK5 (2 to 3.5-fold between 2 and 5 min) and the transcription factor CREB. Incubation of Caco-2 cells with FBS was followed by a rapid induction of c-Fos and c-Myc expression. Studies with ERK1/2 specific inhibitor PD98059, p38 MAPK inhibitor SB203580, or JNK inhibitor SP600125 showed that FBS regulates Caco-2 cell proliferation via the three MAPK pathways.
Assuntos
Proliferação de Células , Neoplasias do Colo/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células CACO-2 , Neoplasias do Colo/patologia , Humanos , Sistema de Sinalização das MAP Quinases , Quinases de Proteína Quinase Ativadas por Mitógeno , FosforilaçãoRESUMO
The mitogen activated protein kinases (MAPKs) have been classified into at least six subfamilies, among which ERK1/2, JNK1/2 and p38 MAPK are the most extensively studied. The steroid hormones 1alpha,25-dihydroxy-Vitamin D(3) and 17beta-estradiol promote biological responses through activation of MAPK cascades in various cell types. We previously reported that 1alpha,25(OH)(2)D(3) rapidly (within 1 min) activates p38 MAPK in C2C12 skeletal muscle cells. In this work, using the same muscle cell line, we demonstrate that 1alpha,25(OH)(2)D(3) or 17beta-estradiol phosphorylate and activate ERK1/2 and p38 MAPK after longer treatment intervals, maximal effects seen at 90 and 30 min (ERK1/2) and at 60 and 15 min (p38 MAPK) for these hormones, respectively. Hormone-dependent ERK and p38 activation was abolished by MAPK specific inhibitors U0126 and SB203580. 1alpha,25(OH)(2)D(3) and 17beta-estradiol also induced the phosphorylation of CREB and Elk-1 transcription factors in an ERK1/2-dependent manner. Simultaneous addition of both hormones potentiated CREB phosphorylation. 1alpha,25(OH)(2)D(3)- and 17beta-estradiol-induced c-fos expression, which was mediated by p38 phosphorylation. The action of 17beta-estradiol on c-fos levels was also dependent on ERK1/2. These results suggest that MAPK signalling pathways play an important role in regulating early gene expression through CREB and Elk-1 activation in skeletal muscle cells.