RESUMEN
Regulation of gene transcription in vascular smooth muscle cells (VSMCs) by serum response factor (SRF) plays a crucial role in vascular development and in the pathophysiology of vascular diseases. Nevertheless, the regulation of specific genes by SRF in vascular diseases is poorly understood. Therefore, we investigated the regulation of smooth muscle myosin light chain kinase (smMLCK) by using spontaneously hypertensive rats (SHR) as an experimental model. We found that smMLCK expression in blood vessels increases during the development of hypertension and is always greater in blood vessels from SHR compared with normotensive rats. Analysis of the DNA sequences of the promoters isolated from SHR and normotensive rats revealed that SHR contain a 12-base pair insertion adjacent to the CArG box. This insertion increases SRF binding to the CArG box and positively regulates SRF-dependent promoter activity. The increase in smMLCK expression was blocked by dominant-negative SRF, dominant-negative Ras, or antisense oligonucleotides to ERK. In vivo, inhibiting MEK decreased smMLCK expression and blood pressure in SHR partly by decreasing SRF binding to the smMLCK promoter. These data provide novel insight into the regulation of smMLCK expression at the molecular level and demonstrate the importance of SRF in regulating smMLCK promoter activity in SHR.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Hipertensión/enzimología , Mutagénesis Insercional , Quinasa de Cadena Ligera de Miosina/genética , Quinasa de Cadena Ligera de Miosina/metabolismo , Regiones Promotoras Genéticas/genética , Factor de Respuesta Sérica/metabolismo , Animales , Secuencia de Bases , Presión Sanguínea/fisiología , Células COS , Células Cultivadas , Chlorocebus aethiops , Hipertensión/fisiopatología , Intrones/genética , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Datos de Secuencia Molecular , Músculo Liso Vascular/citología , Músculo Liso Vascular/enzimología , Cadenas Ligeras de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/antagonistas & inhibidores , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Ratas , Ratas Endogámicas SHR , Transducción de SeñalRESUMEN
Previous short-term studies have correlated an increase in the phosphorylation of the 20-kDa light chain of myosin II (MLC20) with blebbing in apoptotic cells. We have found that this increase in MLC20 phosphorylation is rapidly followed by MLC20 dephosphorylation when cells are stimulated with various apoptotic agents. MLC20 dephosphorylation is not a consequence of apoptosis because MLC20 dephosphorylation precedes caspase activation when cells are stimulated with a proapoptotic agent or when myosin light chain kinase (MLCK) is inhibited pharmacologically or by microinjecting an inhibitory antibody to MLCK. Moreover, blocking caspase activation increased cell survival when MLCK is inhibited or when cells are treated with tumor necrosis factor alpha. Depolymerizing actin filaments or detaching cells, processes that destabilize the cytoskeleton, or inhibiting myosin ATPase activity also resulted in MLC20 dephosphorylation and cell death. In vivo experiments showed that inhibiting MLCK increased the number of apoptotic cells and retarded the growth of mammary cancer cells in mice. Thus, MLC20 dephosphorylation occurs during physiological cell death and prolonged MLC20 dephosphorylation can trigger apoptosis.
Asunto(s)
Apoptosis , Cadenas Ligeras de Miosina/metabolismo , Miosina Tipo II/metabolismo , Quinasa de Cadena Ligera de Miosina/antagonistas & inhibidores , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Caspasa 3 , Inhibidores de Caspasas , Caspasas/metabolismo , Células Cultivadas , Citoesqueleto/metabolismo , Humanos , Ratones , Quinasa de Cadena Ligera de Miosina/inmunología , FosforilaciónRESUMEN
We have previously shown that ML-7, which inhibits myosin light chain kinase (MLCK), induces apoptosis in transformed and non-transformed cells. We have extended these studies and found that ML-7 stimulates the ability of etoposide to induce apoptosis in Mm5MT mouse mammary adenocarcinoma cells and Mat-Ly-Lu rat prostate cancer cells in vitro. ML-7 was also found to have a chemopreventive effect using an in vitro mouse mammary organ culture model. In vivo experiments demonstrated that ML-7 retards the growth of mammary tumours in mice and prostate tumours in rats. Moreover, ML-7 significantly stimulates the ability of etoposide to prevent the growth of established mammary tumours in mice and prostate tumours in rats. These results provide evidence for the efficacy of ML-7 as an adjuvant to etoposide in these models and warrants further development.