MR-1 Modulates Proliferation and Migration of Human Hepatoma HepG2 Cells through Myosin Light Chains-2 (MLC2)/Focal Adhesion Kinase (FAK)/Akt Signaling Pathway

Abstract

The key of cell migration process on solid substrates is phosphorylation of myosin light chain-2 (MLC2), which is implicated in a variety of intracellular functions. The previous data show that MLC2 interacts with a novel human gene, myofibrillogenesis regulator 1 (MR-1). Here, we reported that MR-1 was specially overexpressed in human hepatoma HepG2 cells. Transient treatment of cells with small interfering RNA (siRNA) against MR-1 or stable transfection of cells with plasmid expressing MR-1-siRNA led to inhibitions of cell proliferation, migration, and adhesion. Following down-regulation of MR-1, the phosphorylations of MLC2, focal adhesion kinase (FAK), and Akt were dramatically decreased, and the formation of stress fiber was destroyed by MR-1-siRNAs in hepatoma HepG2 cells. In addition, exogenous MR-1-induced as well as inherent phosphorylations of FAK and Akt were decreased by MLC kinase (MLCK) inhibitor, and F-actin polymerization inhibitor also decreased phosphorylations of FAK and Akt. Correspondingly, MR-1-enhanced migration of cells was also inhibited by these two inhibitors. These indicated that MLC2 activation and intact actin cytoskeleton were pivotal for MR-1 function. In vivo data showed that MR-1-siRNA markedly inhibited growth of human HepG2. This study suggested that overexpression of MR-1 was associated with cancer cell proliferation and migration through MLC2 and that MR-1 might be a potential cancer therapeutic target.Cancer progression from a primary tumor to secondary metastasis is a highly complex process involving alteration of gene expression, acquisition of cell motility, interaction with extracellular matrix (ECM), 3 change in cell adhesion, and expression of ECM-degrading protease (1). Cell migration is a critical step in tumor metastasis. Cancer cells move within tissues during invasion and metastasis by their own motility, and cell migration involves multiple processes that are regulated by various signaling molecules (2). It results from a dynamic interplay between the substrate and cytoskeleton protein located at the focal adhesion complex (3). It is known that cells exert force propelling the cell forward by contraction of the actin cytoskeleton through activation of myosin II (4). The actin-myosin II interaction in non-muscle cells is regulated by the phosphorylation of MLC2 at serine-19 (5). MLC2 dephosphorylation can induce apoptosis (6), and inhibitor of MLCK can abrogate MLC2 phosphorylation, cell polarization, and migration (7). MLC2 is also involved in the activation of mid-G 1 phase cyclin D1 expression (8, 9). It has been reported that hyperphosphorylated MLC2 induces stress fiber formation and integrin clustering that link cell surface cytoskeletal proteins such as FAK to actin (10, 11). FAK is a member of the focal adhesions that mediates integrin-mediated signal transduction associated with a variety of cellular functions including cellular proliferation, migration, and adhesion (12). Downstream signaling pathways implicated in FAK signaling include the Jun N-termin...

A Pathogenic Relationship Between a Regulator of the Actin Cytoskeleton and Serum Response Factor

“…It is also a possibility that SRF affects cell proliferation through changes in signaling that occur due to F-actin accumulation. The filamentous actin cytoskeleton shows stress fiber formation in cancer cells (Barkan et al 2008;Guan et al 2008;Ren et al 2008), which can serve as a signal to promote proliferation (Barkan et al 2008;Gray et al 2008). Further investigation is necessary to determine contributing roles for upregulated SRF target genes or the F-actin accumulation in the hyperproliferative phenotype in Dstn corn1 cornea.…”

CCN1 (CYR61) and CCN3 (NOV) signaling drives human trophoblast cells into senescence and stimulates migration properties

“…FAK is involved in integrinmediated signal transduction pathways of the extracellular matrix and plays an important role in the regulation of cell proliferation, migration, and invasion. 53 It is known that the activation of Akt and ERK1/2 is related to cell migration and the activation of FAK [54][55][56][57] and that these kinases are involved in trophoblast migration and invasion (reviewed by Chakraborty et al 8 ). In SGHPL-5 cells, phosphorylation and thereby activation of FAK occurs only after treatment with glycosylated CCN3.…”

Knockdown of Chk1 sensitizes human colon carcinoma HCT116 cells in a p53-dependent manner to lidamycin through abrogation of a G2/M checkpoint and induction of apoptosis

“…Equal amounts of mRNA were subjected to RT-PCR analysis using a SuperScript TM One-step RT-PCR kit (Invitrogen, Carlsbad, CA, USA) as recently described. 48 Following oligonucleotide primers were used: Chk1, 5'-AGA TAT GAA GCG TGC CGT AG-3' and 5'-CCA GCG AGC ATT GCA GTA AG-3'; Chk2, 5'-ATG TCT CGG GAG TCG GAT GT-3' and 5'-TCT TGC TGT ATG TTC GGT AT-3'. As an internal control, GAPDH was amplified using the primers 5'-CGG AGT CAA CGG ATT TGG TCG TAT-3' and 5'-GTC TTC ACC ACC ATG GAG AAG GCT-3'.…”

“…Whole cell lysates was used for immunoblotting as previously described. 48 Briefly, Cells grown in wells were washed with cold PBS, and then lysed with Laemmli sample buffer containing protease and phosphatase inhibitor. Floating cells or cell fragments were collected and lysed with the above lysates.…”