Pyeung-Hyeun Kim

Mechanisms underlying TGF-β1-induced expression of VEGF and Flk-1 in mouse macrophages and their implications for angiogenesis

TGF‐β induces vascular endothelial growth factor (VEGF), a potent angiogenic factor, at the transcriptional and protein levels in mouse macrophages. VEGF secretion in response to TGF‐β1 is enhanced by hypoxia and by overexpression of Smad3/4 and hypoxia‐inducible factor‐1α/β (HIF‐1α/β). To examine the transcriptional regulation of VEGF by TGF‐β1, we constructed mouse reporters driven by the VEGF promoter. Overexpression of HIF‐1α/β or Smad3/4 caused a slight increase of VEGF promoter activity in the presence of TGF‐β1, whereas cotransfection of HIF‐1α/β and Smad3/4 had a marked effect. Smad2 was without effect on this promoter activity, whereas Smad7 markedly reduced it. Analysis of mutant promoters revealed that the one putative HIF‐1 and two Smad‐binding elements were critical for TGF‐β1‐induced VEGF promoter activity. The relevance of these elements was confirmed by chromatin immunoprecipitation assay. p300, which has histone acetyltransferase activity, augmented transcriptional activity in response to HIF‐1α/β and Smad3/4, and E1A, an inhibitor of p300, inhibited it. TGF‐β1 also increased the expression of fetal liver kinase‐1 (Flk‐1), a major VEGF receptor, and TGF‐β1 and VEGF stimulated pro‐matrix metalloproteinase 9 (MMP‐9) and active‐MMP‐9 expression, respectively. The results from the present study indicate that TGF‐β1 can activate mouse macrophages to express angiogenic mediators such as VEGF, MMP‐9, and Flk‐1.

Encapsulated Bifidobacterium bifidum potentiates intestinal IgA production.

We asked whether Bifidobacterium bifidum regulates the synthesis of IgA by mucosal lymphoid cells. B. bifidum alone, but not Clostridium perfringens, significantly induced total IgA and IgM synthesis by both mesenteric lymph nodes (MLN) and Peyers patch (PP) cells. We, further, investigated the mucosal antibody production following peroral administration of B. bifidum to mice. Ingested B. bifidum significantly increased the number of Ig (IgM, IgG, and IgA) secreting cells in the culture of both MLN and spleen cells. Nonetheless, B. bifidum itself does not induce the own specific antibody responses, implying that B. bifidum does not provoke unnecessary immune reaction. Subsequently, it was found that encapsulation of B. bifidum further augments the total IgA production in the culture of both MLN and spleen cells. Finally, we found that the immuno-stimulating activity of B. bifidum is due to its cellular components but not due to any actively secreting component(s) from bacteria.

TGF-β1 and IFN-γ stimulate mouse macrophages to express BAFF via different signaling pathways

B cell‐activating factor belonging to the TNF family (BAFF) is primarily expressed by macrophages and dendritic cells and stimulates the proliferation, differentiation, and survival of B cells and their Ig production. In the present study, we examined the pathways by which TGF‐β1 and IFN‐γ induce BAFF expression to see if TGF‐β1 and IFN‐γ regulate B cell differentiation via macrophages. We found that TGF‐β1 stimulated mouse macrophages to express BAFF and that a typical TGF‐β signaling pathway was involved. Thus, Smad3 and Smad4 promoted BAFF promoter activity, and Smad7 inhibited it, and the BAFF promoter was shown to contain three Smad‐binding elements. Importantly, TGF‐β1 enhanced the expression of membrane‐bound and soluble forms of BAFF. IFN‐γ further augmented TGF‐β1‐induced BAFF expression. IFN‐γ caused phosphorylation of CREB, and overexpression of CREB increased IFN‐γ‐induced BAFF promoter activity. Furthermore, H89, a protein kinase A (PKA) inhibitor, abrogated the promoter activity. Neither Stat1α (a well‐known transducing molecule of IFN‐γ) nor AG490 (a JAK inhibitor) affected BAFF expression in response to IFN‐γ. Taken together, these results demonstrate that TGF‐β1 and IFN‐γ up‐regulate BAFF expression through independent mechanisms, i.e., mainly Smad3/4 and PKA/CREB, respectively.

Preparation and Physical Characterization of Alginate Microparticles Using Air Atomization Method

Alginate microparticles were prepared using an air atomization method and varying formulation and processing conditions. Thereafter, the size and surface morphology of alginate microparticles were characterized. The trapping efficiencies of the ketoconazole, acetaminophen, vitamin C, and Bifidobacteria bifidum as model core materials were then determined. The air atomization process produced free-flowing and small-size microparticles after the freeze-drying process. The size distribution and surface morphology varied depending on the concentration of wall-forming materials and processing conditions. Generally, the geometric mean size increased as the concentration of alginate and poly-l-lysine and the delivery rate increased, but the air pressure decreased. Most of all, the ratio of delivery rate of alginate solution and air pressure could affect the size and surface morphology of alginate microparticles. However, the geometric mean size of alginate poly-l-lysine microparticles reproducibly ranged from about 80 to 130 μm. The microparticles were irregularly spherical or elliptical. The trapping efficiencies of ketoconazole, acetaminophen, vitamin C, and bifidobacteria were determined to be 71.5%, 60.1%, 1.6%, and 31%, respectively, when alginate concentration (1.5%), poly-l-lysine concentration (0.02%), air pressure (0.75 bar), delivery rate (8 ml/min), and spraying distance (45 cm) were applied. The current microencapsulation process using the air atomization method provides an alternative to entrapping small molecules and macromolecules without using harmful organic solvents. In addition, the small-size and free-flowing alginate microparticles containing active substances can be used as an intermediate in pharmaceutical applications.

High aldehyde dehydrogenase activity enhances stem cell features in breast cancer cells by activating hypoxia-inducible factor-2α

High aldehyde dehydrogenase (ALDH) activity has been recognized as a marker of cancer stem cells (CSCs) in breast cancer. In this study, we examined whether inhibition of ALDH activity suppresses stem-like cell properties in a 4T1 syngeneic mouse model of breast cancer. We found that ALDH-positive 4T1 cells showed stem cell-like properties in vitro and in vivo. Blockade of ALDH activity reduced the growth of CSCs in breast cancer cell lines. Treatment of mice with the ALDH inhibitor diethylaminobenzaldehyde (DEAB) significantly suppressed 4T1 cell metastasis to the lung. Recent evidence suggests that ALDH affects the response of stem cells to hypoxia; therefore, we examined a possible link between ALDH and hypoxia signaling in breast cancer. Hypoxia-inducible factor-2α (HIF-2α) was highly dysregulated in ALDH-positive 4T1 cells. We observed that ALDH was highly correlated with the HIF-2α expression in breast cancer cell lines and tissues. DEAB treatment of breast cancer cells reduced the expression of HIF-2α in vitro. In addition, reduction of HIF-2α expression suppressed in vitro self-renewal ability and in vivo tumor initiation in ALDH-positive 4T1 cells. Therefore, our findings may provide the evidence necessary for exploring a new strategy in the treatment of breast cancer.

Regulation of Neutrophil Adhesion by Pituitary Growth Hormone Accompanies Tyrosine Phosphorylation of Jak2, p125FAK, and Paxillin

Neutrophil adhesion is fundamentally important during the onset of inflammatory responses. The adhesion signaling pathways control neutrophil arrest and extravasation and influence neutrophil shape and function at sites of inflammation. In the present study the intracellular signaling pathways for the adhesion of human neutrophils by pituitary growth hormone (GH) were examined. Pituitary GH triggered the tyrosine phosphorylation of Janus kinase 2 (Jak2) and STAT3 in neutrophils. In addition, pituitary GH treatment resulted in the morphological changes and the tyrosine phosphorylation of focal adhesion kinase (p125FAK) and paxillin. Preincubation with genistein, a tyrosine kinase inhibitor, blocked the GH-stimulated adhesion and Jak2, STAT3, p125FAK, and paxillin phosphorylation. Confocal microscopy revealed that pituitary GH stimulates the focal localization of p125FAK, paxillin, phosphotyrosine, and filamentous actin filament into the membrane rufflings and uropods of human neutrophils. Immunoprecipitation experiments revealed a physical association of Jak2 with p125FAK via STAT3 in vivo. Also an in vitro kinase assay showed an augmentation of p125FAK autophosphorylation as a result of pituitary GH treatment. These results suggest that pituitary GH modulates neutrophil adhesion through tyrosine phosphorylation of Jak2, p125FAK, and paxillin and actin polymerization.

TIMP-2 disrupts FGF-2-induced downstream signaling pathways

We have previously reported that tissue inhibitor of metalloproteinases-2 (TIMP-2), an endogenous inhibitor of matrix metalloproteinase, modulates angiogenic responses through the MMP inhibition-independent activity. In this study, we investigate the molecular mechanisms of TIMP-2-mediated growth inhibition in response to fibroblast growth factor-2 (FGF-2). Pre-treatment with a protein tyrosine phosphatase inhibitor orthovanadate or expression of a dominant negative Shp-1 mutant fails to induce TIMP-2 inactivation of FGF-2 signaling pathways in human microvascular endothelial cells. We also show that TIMP-2 inhibition of FGF-2-induced p42/44(MAPK) activation and cell proliferation is associated with TIMP-2 binding to integrin alpha3beta1 on endothelial cell surfaces, as demonstrated by use of anti-integrin alpha3 or beta1 blocking antibodies, or disruption of integrin alpha3 expression by siRNA. Collectively, our results indicate that TIMP-2 inhibits FGF-2 signaling pathways through association with integrin alpha3beta1 and Shp-1-dependent inhibition of p42/44(MAPK) signaling, which in turn, results in suppression of FGF-2-stimulated endothelial cell mitogenesis.

Analysis of transforming growth factor-beta1-induced Ig germ-line gamma2b transcription and its implication for IgA isotype switching.

Transforming growth factor (TGF)‐β1 directs class switch recombination (CSR) to IgG2b as well as to IgA. Smad3/4, Runx3 and p300 mediate TGF‐β1‐induced germ‐line (GL) α transcription leading to IgA expression. However, the molecular mechanisms by which TGF‐β1 induces IgG2b CSR are unknown. We used luciferase reporter plasmids to investigate how TGF‐β1 regulates the activity of the promoter for GL transcripts of IgG2b constant gene (GLγ2b promoter). Similarly to the GLα promoter, overexpression of Smad3/4 and Runx3 enhances TGF‐β1‐induced GLγ2b promoter activity. Mutation analysis of the promoter identified likely Smad‐ and Runx3‐binding sites. Also similar to the GLα promoter, overexpression of p300 enhances Smad3/4‐mediated promoter activity, whereas E1A represses promoter activity. Since these regulation mechanisms underlying both GLα and GLγ2b transcription are similar, we explored the possibility that TGF‐β1 induces IgA CSR via transitional IgG2b CSR. TGF‐β1 enhances the expression of both Iα‐Cμ and Iα‐Cγ2b circle transcripts, indicative of direct (Sμ→Sα) and sequential CSR (Sμ→Sγ2b→Sα).

Retinoic acid, acting as a highly specific IgA isotype switch factor, cooperates with TGF‐β1 to enhance the overall IgA response

The present study demonstrates that RA has activity of an IgA switch factor and is more specific than TGF‐β1. RA independently caused only IgA switching, whereas TGF‐β1 caused IgA and IgG2b switching. We found that RA increased IgA production and that this was a result of its ability to increase the frequency of IgA‐secreting B cell clones. Increased IgA production was accompanied by an increase of GLTα. RA activity was abrogated by an antagonist of the RAR. Additionally, RA affected intestinal IgA production in mice. Surprisingly, RA, in combination with TGF‐β1, notably enhanced not only IgA production and GLTα expression but also CCR9 and α4β7 expression on B cells. These results suggest that RA selectively induces IgA isotype switching through RAR and that RA and TGF‐β have important effects on the overall gut IgA antibody response.

IL-21 ensures TGF-β1-induced IgA isotype expression in mouse Peyer’s patches

It is well established that TGF‐β1 induces IgA and IgG2b class‐switching recombination in murine B cells. In the present study, we assessed the activity of IL‐21 along with TGF‐β1 in Ig synthesis by murine spleen B cells. IL‐21 showed antiproliferative activity on LPS‐activated splenic B cells, comparable with that of TGF‐β1. IL‐21 alone had little effect on IgA secretion and decreased other isotypes. Likewise, IL‐21 also did not alter the TGF‐β1‐induced IgA synthesis and concurrently diminished the syntheses of IgM and IgG2a, which were repressed by TGF‐β1. Unexpectedly, IL‐21 inhibited the TGF‐β1‐induced IgG2b production. This IL‐21 effect was examined using B cells from IL‐21R knockout mice, where the IgA production profile was paralleled by that seen in wild‐type B cells. However, the inhibitory effect of IL‐21 on TGF‐β1‐induced IgG2b synthesis was not seen in the IL‐21R−/− mouse, suggesting that IL‐21 causes TGF‐β1‐stimulated B cells to decrease IgG2b synthesis. Expression patterns of Ig germ‐line α(GLα)/GLγ2b transcripts under the influence of TGF‐β1 and IL‐21 were paralleled by IgA/IgG2b secretion. This was also observed in the activities of GLα and GLγ2b promoters. These results indicate that IL‐21 decreases IgG2b secretion mainly through inhibition of GLγ2b transcription and is ultimately associated with selective IgA secretion induced by TGF‐β1. Our results showed that IL‐21 was expressed in greater magnitude in Peyers patches (PP) than in spleen. These results suggest that IL‐21 has an important effect on selective IgA+ B cell commitment in PP.