Modulation mechanism of c-mpl promoter activity in megakaryoblastic cells

Abstract

Summary: Thrombopoietin receptor (c-Mpl) is considered to regulate megakaryocytopoiesis. In this study, we investigated an effect of activation of a protein kinase C (PKC) on c-mpl promoter activity to elucidate the underlying mechanisms of c-mpl gene expression in megakaryoblastic cells. PKC is a member of a family of serine/threonine protein kinases in the cytosol involved in cell growth and differentiation. Phorbol 12-myristate 13-acetate (PMA) is known as PKC activator, significantly enhanced the c-mpl promoter activity and PKC inhibitors (H7, GF109203) suppressed the up-regulation of PMAinduced promoter activity and reduced the steady level of its activity. These results strongly suggest that PKC plays the essential role in the modulation of c-mpl promoter activity of megakaryoblastic cells.

GATA-dependent regulation of TPO-induced c-mpl Gene Expression during Megakaryopoiesis

“…It was recently reported that the promoter activity of c-mpl was upregulated by TPO pretreatment and TPO-induced c-mpl promoter activity was modulated at transcription level through a PKC-dependent pathway in the CMK cells (Sunohara et al, 2003 11) , 2004 12) ). In this research, we investigated the role of GATA promoter element in TPO-induced c-mpl gene expression through a PKC-independent pathway by using a transient transfection assay system.…”

“…As described previously (Sunohara et al, 2003 11) , 2013 13) , 2009 14) , 2010 15) , 2008 16) ), the cells were cultured in RPMI 1640 medium (GIBCO, Grand Island, NY) containing 10% FCS, 2mM L-glutamine, 100 units/ml penicillin, 100µg/ ml streptomycin, and 0.25 µg/ml amphotericin B at 37 °C in humid air containing 5% CO 2 atmosphere. CMK cells were cultured in RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented with 10% fetal calf serum (FCS), 2mM L-glutamine, 100 units/ml penicillin, 100 µg/ ml streptomycin, and 0.25 µg/ml amphotericin B at 37 °C in humidified air containing 5% CO 2 atmosphere.…”

“…The construct [pGL3-c-mpl (-310)] has been reported previously (Sunohara et al, 2003 11) , 2013 13) , 2009 14) , 2010 15) ), the 310 bp upstream promoter region of c-mpl was amplified from the chromosomal DNA of the CMK cells by polymerase chain reaction using the following primer set, (forward 5'-CTA TTC TGA TGC TAT TAT ACC ATT C-3', reverse 5'-CTT GGC TGC TGA CTT GGG C-3'). The PCR product was cloned into pGEMTeasy vector (Promega, Madison, WI) and designated as pGEM-c-mpl-prom.…”

“…As described previously (Sunohara et al, 2003 11) , 2013 13) , 2009 14) , 2010 15) ), three reporter plasmids, pGL3-basic vector (as a negative control), pGL3-control vector in which the luciferase gene is driven under SV40 promoter (as a positive control, Promega) and the pGL3-c-mpl (-310) were used for transfection assay. A plasmid, Fig.…”

“…(Hong et al, 1998 10) ). Recently we suggested that TPO-induced c-mpl promoter activity was modulated at transcription level through a PKC-dependent pathway (Sunohara et al, 2003 11) , 2004 12) ) and that GATA(-77) is involved as a positive regulatory element in TPOinduced c-mpl gene expression in the megakaryoblastic CMK cells (Sunohara et al, 2013 13) ). In this research, we investigated role of GATA promoter element in TPOinduced c-mpl gene expression through a PKC-independent pathway by using a transient transfection assay system.…”