Byung-Heon Lee

Identification of motifs for cell adhesion within the repeated domains of transforming growth factor-beta-induced gene, betaig-h3.

βig-h3 is a transforming growth factor-β-inducible cell adhesion molecule that has four characteristic homologous repeated domains. We made recombinant βig-h3 proteins, which were highly active in mediating human corneal epithelial (HCE) cell adhesion and spreading. The 2nd and the 4th repeated domains were sufficient to mediate HCE cell adhesion. A sequence analysis showed that aspartic acid (Asp) and isoleucine (Ile) of the 2nd and the 4th domains are highly conserved in many fasciclin 1 homologous (fas-1) domains. Substitution mutational study identified these two amino acids are essential for cell adhesion. Synthetic peptides containing Asp and Ile, NKDIL and EPDIM derived from the 2nd and the 4th domains, respectively, almost completely blocked cell adhesion mediated by not only wild type βig-h3 but also each of the 2nd and the 4th domains. These peptides alone were fully active in mediating cell adhesion. In addition, we demonstrated the functional receptor for βig-h3 is α3β1integrin. These results, therefore, establish the essential motifs within the 2nd and the 4th domains of βig-h3, which interact with α3β1 integrin to mediate HCE cell adhesion to βig-h3 and suggest that other proteins containing Asp-Ile in their fas-1 domains could possibly function as cell adhesion molecules.

Expression patterns of bone-related proteins during osteoblastic differentiation in MC3T3-E1 cells.

Bone formation involves several tightly regulated gene expression patterns of bone‐related proteins. To determine the expression patterns of bone‐related proteins during the MC3T3‐E1 osteoblast‐like cell differentiation, we used Northern blotting, enzymatic assay, and histochemistry. We found that the expression patterns of bone‐related proteins were regulated in a temporal manner during the successive developmental stages including proliferation (days 4–10), bone matrix formation/maturation (days 10–16), and mineralization stages (days 16 –30). During the proliferation period (days 4–10), the expression of cell‐cycle related genes such as histone H3 and H4, and ribosomal protein S6 was high. During the bone matrix formation/maturation period (days 10–16), type I collagen expression and biosynthesis, fibronectin, TGF‐β1 and osteonectin expressions were high and maximal around day 16. During this maturation period, we found that the expression patterns of bone matrix proteins were two types: one is the expression pattern of type I collagen and TGF‐β1, which was higher in the maturation period than that in both the proliferation and mineralization periods. The other is the expression pattern of fibronectin and osteonectin, which was higher in the maturation and mineralization periods than in the proliferation period. Alkaline phosphatase activity was high during the early matrix formation/maturation period (day 10) and was followed by a decrease to a level still significantly above the baseline level seen at day 4. During the mineralization period (days 16–30), the number of nodules and the expression of osteocalcin were high. Osteocalcin gene expression was increased up to 28 days. Our results show that the expression patterns of bone‐related proteins are temporally regulated during the MC3T3‐E1 cell differentiation and their regulations are unique compared with other systems. Thus, this cell line provides a useful in vitro system to study the developmental regulation of bone‐related proteins in relation to the different stages during the osteoblast differentiation.

Tumor-Targeting Peptide Conjugated pH-Responsive Micelles as a Potential Drug Carrier for Cancer Therapy

Herein, we prepared tumor-targeting peptide (AP peptide; CRKRLDRN) conjugated pH-responsive polymeric micelles (pH-PMs) in cancer therapy by active and pH-responsive tumor targeting delivery systems, simultaneously. The active tumor targeting and tumoral pH-responsive polymeric micelles were prepared by mixing AP peptide conjugated PEG-poly(d,l-lactic acid) block copolymer (AP-PEG-PLA) into the pH-responsive micelles of methyl ether poly(ethylene glycol) (MPEG)-poly(beta-amino ester) (PAE) block copolymer (MPEG-PAE). These mixed amphiphilic block copolymers were self-assembled to form stable AP peptide-conjugated and pH-responsive AP-PEG-PLA/MPEG-PAE micelles (AP-pH-PMs) with an average size of 150 nm. The AP-pH-PMs containing 10 wt % of AP-PEG-PLA showed a sharp pH-dependent micellization/demicellization transition at the tumoral acid pH. Also, they presented the pH-dependent drug release profile at the acidic pH of 6.4. The fluorescence dye, TRITC, encapsulated AP-pH-PMs (TRITC-AP-pH-PMs) presented the higher tumor-specific targeting ability in vitro cancer cell culture system and in vivo tumor-bearing mice, compared to control pH-responsive micelles of MPEG-PAE. For the cancer therapy, the anticancer drug, doxorubicin (DOX), was efficiently encapsulated into the AP-pH-PMs (DOX-AP-pH-PMs) with a higher loading efficiency. DOX-AP-pH-PMs efficiently deliver anticancer drugs in MDA-MB231 human breast tumor-bearing mice, resulted in excellent anticancer therapeutic efficacy, compared to free DOX and DOX encapsulated MEG-PAE micelles, indicating the excellent tumor targeting ability of AP-pH-PMs. Therefore, these tumor-targeting peptide-conjugated and pH-responsive polymeric micelles have great potential application in cancer therapy.

RGD peptides released from beta ig-h3, a TGF-beta-induced cell-adhesive molecule, mediate apoptosis.

βig-h3 is a transforming growth factor-β (TGF-β)-induced cell-adhesive molecule and has an RGD sequence at its C-terminus. A previous report suggested that βig-h3 normally undergoes carboxy-terminal processing that results in the loss of the RGD sequence. RGD peptides appear to play various roles in cell function. Here we show that the RGD peptides released from βig-h3 may facilitate TGF-β-induced apoptosis. We found that carboxy-terminal cleavage of βig-h3 occurred after its secretion, and that overexpression of the wild-type βig-h3 induced apoptosis, unlike the C-terminal deleted but RGD-containing mutant βig-h3, which is resistant to C-terminal processing. The βig-h3-induced apoptosis was abolished by either deletion of the RGD sequence or mutation of RGD to RAE. Synthetic peptides of ERGDEL and GRGDSP derived from βig-h3 and fibronectin, respectively, also induced apoptosis, unlike ERGEEL and GRGESP. Culture supernatants of cells overexpressing βig-h3 filtered to isolate molecules smaller than 3 kDa also induced apoptosis. A fusion protein composed of the N-terminal 100 amino acids of fibronectin and the RGD-containing C-terminal part of βig-h3 was also subjected to C-terminal cleavage and overexpression resulted in apoptosis. The anti-βig-h3 antibody blocks TGF-β-induced apoptosis. Thus, βig-h3 may be important in regulating cell apoptosis by providing soluble RGD peptides.

Poly-cyclodextrin and poly-paclitaxel nano-assembly for anticancer therapy

Effective anticancer therapy can be achieved by designing a targeted drug-delivery system with high stability during circulation and efficient uptake by the target tumour cancer cells. We report here a novel nano-assembled drug-delivery system, formed by multivalent host-guest interactions between a polymer-cyclodextrin conjugate and a polymer-paclitaxel conjugate. The multivalent inclusion complexes confer high stability to the nano-assembly, which efficiently delivers paclitaxel into the targeted cancer cells via both passive and active targeting mechanisms. The ester linkages between paclitaxel and the polymer backbone permit efficient release of paclitaxel within the cell by degradation. This novel targeted nano-assembly exhibits significant antitumour activity in a mouse tumour model. The strategy established in this study also provides knowledge for the development of advanced anticancer drug delivery.

Beta ig-h3 promotes renal proximal tubular epithelial cell adhesion, migration and proliferation through the interaction with α3β1 integrin

Betaig-h3 (βig-h3) is a secretory protein composed of fasciclin I-like repeats containing sequences that allows binding of integrins and glycosaminoglycans in vivo. Expression of βig-h3 is responsive to TGF-β and the protein is found to be associated with extracellular matrix (ECM) molecules, implicating βig-h3 as an ECM adhesive protein of developmental processes. We previously observed predominant expression of βig-h3 expression in the basement membrane of proximal tubules of kidney. In this study, the physiological relevance of such localized expression of βig-h3 was examined in the renal proximal tubular epithelial cells (RPTEC). RPTEC constitutively expressed βig-h3 and the expression was dramatically induced by exogenous TGF-β1 treatment. βig-h3 and its second and fourth FAS1 domain were able to mediate RPTEC adhesion, spreading and migration. Two known α3β1 integrin-interaction motifs including aspartatic acid and isoleucine residues, NKDIL and EPDIM in βig-h3 were responsible to mediate RPTEC adhesion, spreading, and migration. By using specific antibodies against integrins, we confirmed that α3β1 integrin mediates the adhesion and migration of RPTECs on βig-h3. In addition, it also enhanced proliferation of RPTECs through NKDIL and EPDIM. These results indicate that βig-h3 mediates adhesion, spreading, migration and proliferation of RPTECs through the interaction with α3β1 integrin and is intimately involved in the maintenance and the regeneration of renal proximal tubular epithelium.

A new atherosclerotic lesion probe based on hydrophobically modified chitosan nanoparticles functionalized by the atherosclerotic plaque targeted peptides

We developed a new imaging probe for atherosclerotic lesion imaging by chemically conjugating an atherosclerotic plaque-homing peptide (termed the AP peptide) to hydrophobically modified glycol chitosan (HGC) nanoparticles. The AP peptide was previously discovered by using an in vivo phage display screening method. HGC nanoparticles were labeled with the near-infrared (NIR) fluorophore Cy5.5, yielding nanoparticles 314 nm in diameter. The binding characteristics of nanoparticles to cytokine (TNF-alpha)-activated bovine aortic endothelial cells (BAECs) were studied in vitro under static conditions and in a dynamic flow environment. AP-tagged HGC-Cy5.5 nanoparticles (100 microg/ml, 2 h incubation) bound more avidly to TNF-alpha-activated BAECs than to unactivated BAECs. Nanoparticles were mostly located in the membranes of BAECs, although some were taken up by the cells and were visible in the cytoplasm, suggesting that the AP peptides in HGC nanoparticles retained target selectivity for activated BAECs. Binding selectivity of AP-tagged HGC-Cy5.5 nanoparticles was also studied in vivo. NIR fluorescence imaging demonstrated that AP-tagged HGC-Cy5.5 nanoparticles bound better to atherosclerotic lesions in a low-density lipoprotein receptor-deficient (Ldlr(-/-)) atherosclerotic mouse than to such lesions in a normal mouse. These results suggest that the newly designed AP-tagged HGC-Cy5.5 nanoparticles may be useful for atherosclerotic lesion imaging, and may also be employed to elucidate pathophysiological changes, at the molecular level, on atherosclerotic endothelium.

Targeting Bladder Tumor Cells In vivo and in the Urine with a Peptide Identified by Phage Display

Bladder cancer is one of the most common tumors of the genitourinary tract. Here, we use phage display to identify a peptide that targets bladder tumor cells. A phage library containing random peptides was screened for binding to cells from human bladder tumor xenografts. Phage clones were further selected for binding to a bladder tumor cell line in culture. Six clones displaying the consensus sequence CXNXDXRX/RC showed selective binding to cells from primary human bladder cancer tissue. Of these, the CSNRDARRC sequence was selected for further study as a synthetic peptide. Fluorescein-conjugated CSNRDARRC peptide selectively bound to frozen sections of human bladder tumor tissue, whereas only negligible binding to normal bladder tissue was observed. When the fluorescent peptide was introduced into the bladder lumen, in a carcinogen-induced rat tumor model, it selectively bound to tumor epithelium. Moreover, when the peptide was intravenously injected into the tail vein, it homed to the bladder tumor but was not detectable in normal bladder and control organs. Next, we examined whether the peptide can detect tumor cells in urine. The fluorescent peptide bound to cultured bladder tumor cells but not to other types of tumor cell lines. Moreover, it bound to urinary cells of patients with bladder cancer, while showing little binding to urinary cells of patients with inflammation or healthy individuals. The CSNRDARRC peptide may be useful as a targeting moiety for selective delivery of therapeutics and as a diagnostic probe for the detection of bladder cancer. (Mol Cancer Res 2007;5(1):11–19)

Erythropoietin Decreases Renal Fibrosis in Mice with Ureteral Obstruction: Role of Inhibiting TGF-β–Induced Epithelial-to-Mesenchymal Transition

The inhibitory effects of recombinant human erythropoietin (rhEPO) were examined against (1) the progression of renal fibrosis in mice with complete unilateral ureteral obstruction and (2) the TGF-beta1-induced epithelial-to-mesenchymal transition (EMT) in MDCK cells. Unilateral ureteral obstruction was induced in BALB/c mice and rhEPO (100 or 1000 U/kg, intraperitoneally, every other day) or vehicle was administered from day 3 to day 14. Immunoblotting and immunohistochemistry revealed increased expressions of TGF-beta1, alpha-smooth muscle actin (alpha-SMA), and fibronectin and decreased expression of E-cadherin in the obstructed kidneys. In contrast, rhEPO treatment significantly attenuated the upregulation of TGF-beta1 and alpha-SMA and the downregulation of E-cadherin. MDCK cells were treated with TGF-beta1 (5 ng/ml) for 48 h to induce EMT, and the cells were then co-treated with TGF-beta1 and rhEPO for another 48 h. Increased expressions of alpha-SMA and vimentin and decreased expressions of zona occludens-1 and E-cadherin were observed after TGF-beta1 treatment, and these changes were markedly attenuated by rhEPO co-treatment. TGF-beta1 increased phosphorylated Smad-2 expression in MDCK cells, which was decreased by rhEPO co-treatment. In conclusion, rhEPO treatment inhibits the progression of renal fibrosis in obstructed kidney and attenuates the TGF-beta1-induced EMT. It is suggested that the renoprotective effects of rhEPO could be mediated, at least partly, by inhibition of TGF-beta1-induced EMT.

α5β1 integrin stimulates Bcl‐2 expression and cell survival through Akt, focal adhesion kinase, and Ca2+/calmodulin‐dependent protein kinase IV

CHO cells expressing α5β1 integrin are more resistant to apoptosis and express more Bcl‐2 than the same cells engineered to express αvβ1 or cytoplasmically truncated α5Δcβ1 integrin as their main fibronectin receptor. The Bcl‐2 up‐regulation by α5β1 is mediated, at least in part, by the focal adhesion kinase (FAK) and phosphatidylinositol‐3 kinase (PI3K)/Akt pathways. Here, we show that integrin‐mediated activation of Ca2+/calmodulin‐dependent protein kinase (CaMK) IV, and the NF‐κB and CREB transcription factors also enhance the integrin‐dependent regulation of Bcl‐2 expression in the α5β1cells. A forkhead transcription factor, which is inactivated by Akt, blocked Bcl‐2 expression. The FAK pathway was found to be defective in both the αvβ1 and α5Δcβ1 cells. These cell lines differed from one another in two Bcl‐2‐regulating pathways: adhesion through αvβ1 failed to activate Akt, allowing forkhead to suppress Bcl‐2 transcription, whereas α5Δcβ1 did not activate NF‐κB and CREB, presumably because CaMK IV was not activated. Our results indicate that three pathways, the FAK, PI3K/Akt, and CaMK IV mediate the survival‐supporting activity of α5β1 integrin.