Jae Youn Yi

Type I Transforming Growth Factor β Receptor Binds to and Activates Phosphatidylinositol 3-Kinase

We have examined the interaction of transforming growth factor (TGF)β receptors with phosphatidylinositol 3-(PI3) kinase in epithelial cells. In COS7 cells, treatment with TGFβ increased PI3 kinase activity as measured by the ability of p85-associated immune complexes to phosphorylate inositides in vitro. Both type I and type II TGFβ receptors (TβR) associated with p85, but the association of TβRII appeared to be constitutive. The interaction of TβRI with p85 was induced by treatment with TGFβ. The receptor association with PI3 kinase was not direct as 35S-labeled rabbit reticulocyte p85 did not couple with fusion proteins containing type I and type II receptors. A kinase-dead, dominant-negative mutant of TβRII blocked ligand-induced p85-TβRI association and PI3 kinase activity. In TβRI-null R1B cells, TGFβ did not stimulate PI3 kinase activity. This stimulation was restored upon reconstitution of TβRI by transfection. In R1B and NMuMG epithelial cells, overexpression of a dominant active mutant form of TβRI markedly enhanced ligand-independent PI3 kinase activity, which was blocked by the addition of the TβRI kinase inhibitor LY580276, suggesting a causal link between TβRI function and PI3 kinase. Overexpressed Smad7 also prevented ligand-induced PI3 kinase activity. Taken together, these data suggest that 1) TGFβ receptors can indirectly associate with p85, 2) both receptors are required for ligand-induced PI3 kinase activation, and 3) the activated TβRI serine-threonine kinase can potently induce PI3 kinase activity.

Activated type I TGFβ receptor kinase enhances the survival of mammary epithelial cells and accelerates tumor progression

We have examined the effects of transforming growth factor-beta (TGFβ) signaling on mammary epithelial cell survival. Transgenic mice expressing an active mutant of Alk5 in the mammary gland (MMTV-Alk5T204D) exhibited reduced apoptosis in terminal endbuds and during postlactational involution. Transgene-expressing mammary cells contained lower Smad2/3 and higher c-myc levels than controls, high ligand-independent phosphatidylinositol-3 kinase (PI3K) and Akt activities, and were insensitive to TGFβ-mediated growth arrest. Treatment with a proteasome inhibitor increased Smad2/3 levels and ligand-independent Smad transcriptional reporter activity, as well as reduced both c-myc protein and basal cell proliferation. Treatment with an Alk5 kinase small-molecule inhibitor upregulated Smad2/3 levels, reduced PI3K activity, P-Akt, and c-myc, and inhibited cell survival. Although Alk5T204D-expressing mice did not develop mammary tumors, bigenic MMTV-AlkT204D × Neu mice developed cancers that were more metastatic than those occurring in MMTV-Neu transgenics. These data suggest that (1) TGFβ can signal to PI3K/Akt and enhance mammary epithelial cell survival in vivo before cytological or histological evidence of transformation, and (2) TGFβ signaling can provide epithelial cells with a ‘gain-of-function’ effect that synergizes with oncogene-induced transformation.

TXNIP potentiates Redd1-induced mTOR suppression through stabilization of Redd1.

The mammalian target of rapamycin (mTOR) is a highly conserved serine–threonine kinase activated in response to growth factors and nutrients. Because of frequent dysregulation of the mTOR signaling pathway in diverse human cancers, this kinase is a key therapeutic target. Redd1 is a negative regulator of mTOR, mediating dissociation of 14-3-3 from tuberous sclerosis complex (TSC)2, which allows formation of a TSC–TSC2 complex. In the present study, we identify TXNIP that inhibits mTOR activity by binding to and stabilizing Redd1 protein. Redd1 and TXNIP expression was induced by a synthetic glucose analog, 2-deoxyglucose (2-DG). Moreover, Redd1 expression in response to 2-DG was regulated by activating transcription factor 4 (ATF4). Overexpression of TXNIP was associated with reduced mTOR activity mediated by an increase in Redd1 level, whereas knockdown of TXNIP using small interfering RNA resulted in recovery of mTOR activity via downregulation of Redd1 during treatment with 2-DG. Interestingly, Redd1 was additionally stabilized via interactions with N-terminal-truncated TXNIP, leading to suppression of mTOR activity. Our results collectively demonstrate that TXNIP stabilizes Redd1 protein induced by ATF4 in response to 2-DG, resulting in potentiation of mTOR suppression. To the best of our knowledge, this is the first study to identify TXNIP as a novel member of the mTOR upstream that acts as a negative regulator in response to stress signals.

TGFβ1-mediated epithelial to mesenchymal transition is accompanied by invasion in the SiHa cell line

It has recently been suggested by several investigators that the epithelial-mesenchymal transition-inducing capacity of TGFbetas contributes to invasive transition of tumors at later stages of carcinogenesis. In the present study, we examined the possibility of TGFbeta1-stimulated epithelial-mesenchymal transition in SiHa cell line, detailed molecular events in the process, and its possible contribution to the invasive transition of tumors. TGFbeta1-induced epithelial-mesenchymal transition of SiHa cells was based on morphological and biochemical criteria; actin stress fiber formation, focal translocalization of integrin alphav, talin, and vinculin, fibronectin-based matrix assembly at the cell periphery, and translocalization and down-regulation of E-cadherin. TGFbeta1 also stimulated surface expression of integrin alphavbeta3 and FAK activation. Focal translocalization of integrin alphav preceded actin reorganization and fibronectin matrix assembly, and functional blocking of the integrin suppressed actin stress fiber formation. Furthermore, induction of actin reorganization and fibronectin matrix assembly by TGFbeta1 were shown to be mutually independent events. These changes were irreversible because 5 minutes pulse exposure to TGFbeta1 was sufficient to stimulate progress of actin reorganization and fibronectin matrix assembly. In further studies with raft culture, TGFbeta1 was found to stimulate invasion of SiHa cells into a type I collagen gel matrix. In conclusion, TGFbeta1 stimulated epithelial-mesenchymal transition of SiHa cells, indicating a positive role in the invasive transition of tumors.

Substance P stimulates the recovery of bone marrow after the irradiation.

The therapeutic use of ionizing radiation (e.g., X‐rays and γ‐rays) needs to inflict minimal damage on non‐target tissue. Recent studies have shown that substance P (SP) mediates multiple activities in various cell types, including cell proliferation, anti‐apoptotic responses, and inflammatory processes. The present study investigated the effects of SP on γ‐irradiated bone marrow stem cells (BMSCs). In mouse bone marrow extracts, SP prolonged activation of Erk1/2 and enhanced Bcl‐2 expression, but attenuated the activation of apoptotic molecules (e.g., p38 and cleaved caspase‐3) and down‐regulated Bax. We also observed that SP‐decreased apoptotic cell death and stimulated cell proliferation in γ‐irradiated mouse bone marrow tissues through TUNEL assay and PCNA analysis. To determine how SP affects bone marrow stem cell populations, mouse bone marrow cells were isolated and colony‐forming unit (CFU) of mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) was estimated. SP‐pretreated ones showed higher CFUs of MSC and HSC than untreated ones. Furthermore, when SP was pretreated in cultured human MSC, it significantly decreased apoptotic cells at 48 and 72 h after γ‐irradiation. Compared with untreated cells, SP‐treated human MSCs showed reduced cleavage of apoptotic molecules such as caspase‐8, ‐9, ‐3, and poly ADP‐ribose polymerase (PARP). Thus, our results suggest that SP alleviates γ‐radiation‐induced damage to mouse BMSCs and human MSCs via regulation of the apoptotic pathway. J. Cell. Physiol. 226: 1204–1213, 2011.

CD24 enhances DNA damage-induced apoptosis by modulating NF-κB signaling in CD44-expressing breast cancer cells

Cluster of differentiation 24 (CD24) is a small glycosylphosphatidylinositol-linked cell surface molecule that is expressed in a variety of human carcinomas, including breast cancer. To determine the role of CD24 in breast cancer cells, we expressed CD24 in CD24-negative/low and cluster of differentiation 44 (CD44)-positive MDA-MB-231 metastatic breast cancer cells. Forced expression of CD24 resulted in a decrease in c-Raf/mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)/mitogen-activated protein kinase signaling and reduced cell proliferation. Apoptosis induced by DNA damage was greatly enhanced in MDA-MB-231 CD24 cells as compared with MDA-MB-231 vec cells. CD24 expression efficiently attenuated DNA damage-induced nuclear factor-kappaB (NF-κB) signaling in MDA-MB-231 cells. However, in CD24-positive and CD44-negative/low MCF-7 cells, knockdown of CD24 did not significantly affect DNA damage-induced apoptosis nor NF-κB signaling. Silencing of CD24 in CD24/CD44-double-positive MDA-MB-468 cells partially rescued DNA damage-induced apoptosis. Transient transfection studies with 293T cells also revealed that CD24 attenuated cell viability and NF-κB signaling only when CD44 was cotransfected. These data indicate that CD24 expression potentiated DNA-induced apoptosis by suppressing antiapoptotic NF-κB signaling in CD44-expressing cells.

Reduced activity of topoisomerase II in an Adriamycin-resistant human stomach-adenocarcinoma cell line

Abstract A human stomach-adenocarcinoma cell line (MKN-45) was selected for resistance to Adriamycin by stepwise exposure to increasing concentrations of this agent. The resulting cell line (MKN/ADR) exhibited a high level of cross-resistance to topoisomerase II (topo II)-targeted drugs such as Adriamycin, mitoxantrone, and etoposide but showed no cross-resistance to other chemotherapeutic agents such as cisplatin, carboplatin, 5-fluorouracil, or mitomycin-C. P-glycoprotein encoded by the mdr-1 gene was not overexpressed in the MKN/ADR cell line. The doubling time of the MKN/ADR cell line (2.1 days) increased only slightly as compared with that of the MKN cell line (1.7 days). The patterns of cross-resistance to various chemotherapeutic agents led us to examine the cellular contents of topo II in both the drug-sensitive and the drug-resistant cells. Extractable topo II enzyme activity was 3-fold lower in MKN/ADR cells as compared with the parental MKN cells. Levels of topoisomerase I (topo I) catalytic activity were similar in both wild-type MKN and drug-resistant MKN/ADR cells. Southern-blot analysis of genomic DNA probed with topo IIα or IIβ showed no sign of either gene rearrangement or hypermethylation. Northern-blot analysis revealed that both topo IIα and topo IIβ mRNA transcripts were essentially identical in the MKN and MKN/ADR cells. In contrast, Western-blot analysis revealed an approximately 20-fold lower level of topo IIα in drug-resistant cells as compared with drug-sensitive cells, whereas topo IIβ levels were similar in both lines. Moreover, the amount of in vivo topo IIα-DNA covalent complexes formed in the presence of etoposide was also approximately 20-fold lower in drug-resistant cells. No mutation was detected in the promoter region of the topo IIα gene in resistant cells as compared with sensitive cells. Thus, low levels of topo IIα polypeptide cannot be ascribed to changes in the mRNA levels. Collectively, the data suggest that a quantitative reduction in topo IIα may contribute to the resistance of MKN cells to Adriamycin and other topo II-targeted drugs.

Substance P accelerates intestinal tissue regeneration after γ-irradiation–induced damage

Radiation therapy causes varying degrees of damage to biological systems. Many groups are investigating the mechanism underlying radiation‐induced cellular damage but there are limited therapeutic solutions for affected patients. Recent studies show that substance P (SP) participates in cell proliferation. In the present study, we characterized the mechanism underlying SP‐induced cellular signaling in radiation‐induced damage of the intestine. Exposure of Caco‐2 cells to SP increases cell proliferation and Erk phosphorylation in a time‐ and dose‐dependent manner. The proliferation of cells exposed to γ‐irradiation is also stimulated by exposure to SP, a phenomenon that may result from inhibition of apoptosis because SP activates Akt and inhibits the cleavage of caspase‐3. The effect of SP on cell proliferation and protection was confirmed by investigations in mice. Proliferating cell nuclear antigen staining shows that cell proliferation in radiation‐damaged mouse intestine increases significantly upon exposure to SP. Furthermore, terminal deoxynucleotidyl transferase‐mediated dUTP‐fluorescein nick end labeling assay reveals fewer cells stained in SP‐treated mice compared with untreated controls. These findings show the potential for SP‐induced acceleration of intestinal wound healing and reveal that the mechanism underlying this process involves activation of Erk and Akt and inhibition of caspase‐3 cleavage.

Induction of galectin-1 by TGF-β1 accelerates fibrosis through enhancing nuclear retention of Smad2.

Fibrosis is one of the most serious side effects in cancer patients undergoing radio-/ chemo-therapy, especially of the lung, pancreas or kidney. Based on our previous finding that galectin-1 (Gal-1) was significantly increased during radiation-induced lung fibrosis in areas of pulmonary fibrosis, we herein clarified the roles and action mechanisms of Gal-1 during fibrosis. Our results revealed that treatment with TGF-β1 induced the differentiation of fibroblast cell lines (NIH3T3 and IMR-90) to myofibroblasts, as evidenced by increased expression of the fibrotic markers smooth muscle actin-alpha (α-SMA), fibronectin, and collagen (Col-1). We also observed marked and time-dependent increases in the expression level and nuclear accumulation of Gal-1. The TGF-β1-induced increases in Gal-1, α-SMA and Col-1 were decreased by inhibitors of PI3-kinase and p38 MAPK, but not ERK. Gal-1 knockdown using shRNA decreased the phosphorylation and nuclear retention of Smad2, preventing the differentiation of fibroblasts. Gal-1 interacted with Smad2 and phosphorylated Smad2, which may accelerate fibrotic processes. In addition, up-regulation of Gal-1 expression was demonstrated in a bleomycin (BLM)-induced mouse model of lung fibrosis in vivo. Together, our results indicate that Gal-1 may promote the TGF-β1-induced differentiation of fibroblasts by sustaining nuclear localization of Smad2, and could be a potential target for the treatment of pulmonary fibrotic diseases.

Reconstruction of basement membrane in skin equivalent; role of laminin-1.

Abstract To reconstruct the basement membrane in a skin equivalent, the epidermodermal interface was coated with porcine type IV collagen and mouse laminin-1 at various ratios before keratinocyte seeding. Laminin-1, a component of the basement membrane, induced massive infiltration of keratinocytes into the dermal equivalent, while type IV collagen induced discrete demarcation between dermal and epidermal compartments without any infiltrating cells. Immunohistochemical staining indicated that the laminin-induced infiltrating cells expressed endogenous type IV collagens at the cell periphery, which were not incorporated into the basement membrane structure. The infiltrating cells did not express fibronectin receptor α 5 β 1 integrin but showed MMP-9 secretion and cell surface associated MMP-2. However, when laminin-1 was preincubated with type IV collagen, laminin-1-induced keratinocyte infiltration as well as MMP-9 induction were almost completely suppressed to basal levels. Therefore, replenishment of the type IV collagen lattice seemed to cause laminin-stimulated cells to anchor to the lattice, in a similar manner to the basal cells on the basement membrane of normal skin. Our study suggests that the molar ratio of basement membrane components may determine the behavior of basal cells within the wound healing microenvironment, which is probably regulated either by extracellular matrix deposition or degradation.