Yong-Bae Kim

Tetraspanin TM4SF5 mediates loss of contact inhibition through epithelial-mesenchymal transition in human hepatocarcinoma

The growth of normal cells is arrested when they come in contact with each other, a process known as contact inhibition. Contact inhibition is lost during tumorigenesis, resulting in uncontrolled cell growth. Here, we investigated the role of the tetraspanin transmembrane 4 superfamily member 5 (TM4SF5) in contact inhibition and tumorigenesis. We found that TM4SF5 was overexpressed in human hepatocarcinoma tissue. TM4SF5 expression in clinical samples and in human hepatocellular carcinoma cell lines correlated with enhanced p27Kip1 expression and cytosolic stabilization as well as morphological elongation mediated by RhoA inactivation. These TM4SF5-mediated effects resulted in epithelial-mesenchymal transition (EMT) via loss of E-cadherin expression. The consequence of this was aberrant cell growth, as assessed by S-phase transition in confluent conditions, anchorage-independent growth, and tumor formation in nude mice. The TM4SF5-mediated effects were abolished by suppressing the expression of either TM4SF5 or cytosolic p27Kip1, as well as by reconstituting the expression of E-cadherin. Our observations have revealed a role for TM4SF5 in causing uncontrolled growth of human hepatocarcinoma cells through EMT.

Cell Adhesion-dependent Cofilin Serine 3 Phosphorylation by the Integrin-linked Kinase·c-Src Complex

Integrin-linked kinase (ILK) is involved in signal transduction by integrin-mediated cell adhesion that leads to dynamic actin reorganization. Actin (de)polymerization is regulated by cofilin, the Ser3 phosphorylation (pS3cofilin) of which inhibits its actin-severing activity. To determine how ILK regulates pS3cofilin, we examined the effects of ILK on pS3cofilin using normal RIE1 cells. Compared with suspended cells, fibronectin-adherent cells showed enhanced pS3cofilin, depending on ILK expression and c-Src activity. The ILK-mediated pS3cofilin in RIE1 cells did not involve Rho-associated kinase, LIM kinase, or testicular protein kinases, which are known to be upstream of cofilin. The kinase domain of ILK, including proline-rich regions, appeared to interact physically with the Src homology 3 domain of c-Src. In vitro kinase assay revealed that ILK immunoprecipitates phosphorylated the recombinant glutathione S-transferase-cofilin, which was abolished by c-Src inhibition. Interestingly, epidermal growth factor treatment abolished the ILK effects, indicating that the linkage from ILK to cofilin is biologically responsive to extracellular cues. Altogether, this study provides evidence for a new signaling connection from ILK to cofilin for dynamic actin polymerization during cell adhesion, depending on the activity of ILK-associated c-Src.

Syndecan-2 overexpression regulates adhesion and migration through cooperation with integrin α2

Syndecan-2, a transmembrane heparan sulfate proteoglycan, is known to serve as an adhesion receptor, but details of the regulatory mechanism governing syndecan-2 cell adhesion and migration remain unclear. Here, we examined this regulatory mechanism, showing that overexpression of syndecan-2 enhanced collagen adhesion, cell migration and invasion of normal rat intestinal epithelial cells (RIE1), and increased integrin alpha2 expression levels. Interestingly, RIE1 cells transfected with either syndecan-2 or integrin alpha2 showed similar adhesion and migration patterns, and a function-blocking anti-integrin alpha2 antibody abolished syndecan-2-mediated adhesion and migration. Consistent with these findings, transfection of integrin alpha2 siRNA diminished syndecan-2-induced cell migration in HCT116 human colon cancer cells. Taken together, these results demonstrate a novel cooperation between syndecan-2 and integrin alpha2beta1 in adhesion-mediated cell migration and invasion. This interactive dynamic might be a possible mechanism underlying the tumorigenic activities of colon cancer cells.

The signaling network of transforming growth factor beta1, protein kinase Cdelta, and integrin underlies the spreading and invasiveness of gastric carcinoma cells

ABSTRACT Integrin-mediated cell adhesion and spreading enables cells to respond to extracellular stimuli for cellular functions. Using a gastric carcinoma cell line that is usually round in adhesion, we explored the mechanisms underlying the cell spreading process, separate from adhesion, and the biological consequences of the process. The cells exhibited spreading behavior through the collaboration of integrin-extracellular matrix interaction with a Smad-mediated transforming growth factor β1 (TGFβ1) pathway that is mediated by protein kinase Cδ (PKCδ). TGFβ1 treatment of the cells replated on extracellular matrix caused the expression and phosphorylation of PKCδ, which is required for expression and activation of integrins. Increased expression of integrins α2 and α3 correlated with the spreading, functioning in activation of focal adhesion molecules. Smad3, but not Smad2, overexpression enhanced the TGFβ1 effects. Furthermore, TGFβ1 treatment and PKCδ activity were required for increased motility on fibronectin and invasion through matrigel, indicating their correlation with the spreading behavior. Altogether, this study clearly evidenced that the signaling network, involving the Smad-dependent TGFβ pathway, PKCδ expression and phosphorylation, and integrin expression and activation, regulates cell spreading, motility, and invasion of the SNU16mAd gastric carcinoma cell variant.

Integrin signaling and cell spreading mediated by phorbol 12‐myristate 13‐acetate treatment

Spreading of SNU16mAd gastric carcinoma cells was previously shown to be regulated via a signaling network from transforming growth factor β1 (TGFβ1) to integrins signaling, through a mediation of protein kinase C δ (PKCδ). However, in the previous study, the roles of PKCδ appeared complicated. In this study to clarify the roles of PKCδ in the spreading of the gastric carcinoma cells, we questioned if PKC activation via phorbol 12‐myristate 13‐acetate (PMA) treatment could mimic the TGFβ1 effects. An acute PMA treatment increased phosphorylations of focal adhesion (FA) kinase, paxillin, c‐Src, and cofilin, just as TGFβ1 did. Furthermore, cell spreading mediated by TGFβ1‐ or acute PMA treatment correlated with activation of RhoA, which regulates actin reorganization and FA formation. However, stress fiber formation was prominent in TGFβ1‐treated cells, compared to cortical actin organization in PMA‐treated cells. Altogether, these observations indicate that acute PMA treatment could mimic the TGFβ1 mechanisms for cell spreading through subtly different effects on actin reorganization. J. Cell. Biochem.

PKCδ and cofilin activation affects peripheral actin reorganization and cell-cell contact in cells expressing integrin α5 but not its tailless mutant

Integrin-mediated cell adhesion transduces signaling activities for actin reorganization, which is crucially involved in cellular function and architectural integrity. In this study, we explored the possibility of whether cell-cell contacts might be regulated via integrin-α5β1-mediated actin reorganization. Ectopic expression of integrin α5 in integrin-α5-null intestinal epithelial cells resulted in facilitated retraction, cell-cell contact loss, and wound healing depending on Src and PI3K (phosphoinositide 3-kinase) activities by a reagent that affects actin organization. However, cytoplasmic tailless integrin α5 (hereafter referred to as α5/1) expression caused no such effects but rather sustained peripheral actin fibers, regardless of Src and PI3K signaling activities. Furthermore, integrin α5 engagement with fibronectin phosphorylated Ser643 of PKCδ, upstream of FAK and Src and at a transmodulatory loop with PI3K/Akt. Pharmacological PKCδ inactivation, dominant-negative PKCδ adenovirus or inactive cofilin phosphatase (SSH1L mutant) retrovirus infection of α5-expressing cells sustained peripheral actin organization and blocked the actin reorganizing-mediated loss of cell-cell contacts. Meanwhile, wild-type PKCδ expression sensitized α5/1-expressing cells to the actin disruptor to induce cell scattering. Altogether, these observations indicate that integrin α5, but not α5/1, mediates PKCδ phosphorylation and cofilin dephosphorylation, which in turn modulate peripheral actin organization presumably leading to an efficient regulation of cell-cell contact and migration.