Nm23-H1 suppresses hepatocarcinoma cell adhesion and migration on fibronectin by modulating glycosylation of integrin beta1.
BACKGROUND
Nm23 gene was isolated as a metastatic suppressor gene. The antimetastatic effect of Nm23 has been an enigma for more than 10 years. Little is known about its molecular mechanisms. In this study we overexpressed Nm23-H1 in H7721 cells and observed reduction of cell adhesion, migration and extension of actin stress fibers in cells stimulated by fibronectin (Fn).


METHODS
pcDNA3/Nm23-H1 was introduced into H7721 cells, and expression of Nm23-H1 was monitored by RT-PCR and western blot. Cell adhesion, actin extension and wound-induced migration assays were done on dishes coated with fibronectin. Phosphorylation of focal adhesion kinase (FAK) and total amount of integrin alpha 5 and beta1 in Nm23-H1 transfected cells and control cells were measured by western blot. Flow cytometry was used to detect expression of surface alpha 5 and beta1 integrin. N-glycosylation inhibitor tunicamycin was used to deglycosylate the integrin beta1 subunit.


RESULTS
Overexpression of nm23-H1 in H7721 cells reduced cell adhesion, migration and extension of actin stress fibers on dishes coated with Fn. Phosphorylation of FAK in Nm23-H1 transfected cells was also attenuated. Integrin alpha 5 and beta1 gene messages were unaltered in nm23-H1 overexpressed cells as detected by RT-PCR. However, while cell surface integrin alpha 5 was unchanged, surface expression of beta1 integrin was downregulated. Western blot also showed that the total amounts of integrin alpha 5 and beta1 were unaltered, but the level of mature integrin beta1 isoform was decreased significantly. Furthermore, partially glycosylated precursor beta1 was increased, which indicated that the impaired glycosylation of integrin beta1 precursor might contribute to the loss of cell surface integrin beta1 in nm23-H1 overexpressed cells.


CONCLUSIONS
These results suggest that by modulating glycosylation of integrin beta1, nm23-H1 down-regulates integrin beta1 subunit on cell surface and mediates intracellular signaling and subsequent suppression of the invasive process, including cell adhesion and migration.
She
Shangyang
S
Clinical Laboratory, Guangxi Maternal and Child Health Hospital, Nanning 530003, China.
Xu
Boying
B
He
Min
M
Lan
Xiuwan
X
Wang
Qiuyan
Q
D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000517:prevention & control*; D002448:Cell Adhesion; D002465:Cell Movement; D003599:Cytoskeleton / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D016023:Integrins / Q000235:genetics / Q000378:metabolism; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000517:prevention & control*; D054778:NM23 Nucleoside Diphosphate Kinases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
D016023:Integrins; D054778:NM23 Nucleoside Diphosphate Kinases; D012333:RNA, Messenger; D051416:Focal Adhesion Protein-Tyrosine Kinases; C517938:NME1 protein, human