Ki-Sung Lee

Dual Transcriptional Regulation of the Escherichia coli Phosphate-Starvation-Inducible psiE Gene of the Phosphate Regulon by PhoB and the Cyclic AMP (cAMP)-cAMP Receptor Protein Complex

We have shown that the Escherichia coli phosphate-starvation-inducible psiE gene is regulated by both phosphate and the carbon source by using both lacZ and chloramphenicol acetyltransferase gene (cat) fusions. Yet, under all conditions tested, a single transcriptional start site lying 7 bp downstream of a predicted -10 region was revealed by primer extension analysis. DNase I footprinting showed that the PhoB transcriptional-activator protein protects two predicted pho boxes lying upstream of and near the -35 promoter region. Similar analysis showed that the cyclic AMP (cAMP)-cAMP receptor protein (cAMP-CRP) complex binds a region that overlaps with the downstream pho box. These results, together with measurements of the in vivo psiE promoter activity under various conditions, show that expression of the psiE gene is under direct positive and negative control by PhoB and cAMP-CRP, respectively.

Activation of phospholipase D by 8-Br-cAMP occurs through novel pathway involving Src, Ras, and ERK in human endometrial stromal cells

We investigated the mechanism of 8‐Br‐cAMP‐mediated phospholipase D (PLD) activation using a primary cell culture system of human endometrial stromal cells (ES cells). PLD activity was increased by the treatment of ES cells with 8‐Br‐cAMP, maximally at 5 min. To determine whether the effects of 8‐Br‐cAMP on PLD occurred as a consequence of PKC activation, ES cells were preincubated for 15 min with RO320432 (1 μM) and GF109203X (1 μM), the PKC inhibitors, or they were pretreated for 24 h with phorbol myristate acetate (100 nM) to downregulate PKC. However, these treatments had no effects on PLD activation induced by 8‐Br‐cAMP. Furthermore, 8‐Br‐cAMP had no effects on the subcellular distribution of PKC α and PKC βI, confirming no involvement of PKC. 8‐Br‐cAMP activated ERK1/2, maximally at 5 min, and PD98059 (MEK inhibitor: 50 μM) and transfection of ES cells with dominant negative (DN)‐MEK completely inhibited 8‐Br‐cAMP‐induced PLD activation, suggesting that ERK1/2 mediates the PLD activation. To investigate the involvement of protein kinase A (PKA), Src, and Ras in 8‐Br‐cAMP‐induced PLD activation, we used PKA inhibitor, H89 and Rp‐cAMPs, and transfections of DN‐Src and DN‐Ras. H‐89 and Rp‐cAMPs completely blocked 8‐Br‐cAMP‐mediated PLD and ERK activation, implying the involvement of PKA in this PLD activation. In addition, transfection of ES cells with DN‐Src, or DN‐Ras partially inhibited 8‐Br‐cAMP‐induced ERK1/2 and consequently PLD activation, whereas cotransfection of DN‐Src and DN‐Ras completely inhibited ERK1/2 and PLD activation, suggesting that Src and Ras independently regulate ERK/PLD activation. Taken together, these results demonstrate a novel pathway in ES cells that 8‐Br‐cAMP activate PLD through PKA and ERK1/2 and this ERK/PLD activation by 8‐Br‐cAMP is mediated by Src and Ras, separately.

A Novel Role of Hippocalcin in bFGF-Induced Neurite Outgrowth of H19-7 Cells

Hippocalcin is a Ca2+‐binding protein that is expressed mainly in pyramidal nerve cells of the hippocampus. However, its functions and mechanism in the brain remain unclear. To elucidate the role of hippocalcin, we used a conditionally immortalized hippocampal cell line (H19‐7) and showed that bFGF treatment increased the expression of hippocalcin during bFGF‐induced neurite outgrowth of H19‐7 cells. Overexpression of hippocalcin dramatically elongated neurites and increased the expression of basic helix–loop–helix transcription factor, that is, NeuroD without bFGF stimulation. Treatment of the cells with hippocalcin siRNA completely blocked bFGF‐induced neurite outgrowth and NeuroD expression. bFGF stimulation resulted in activation of phospholipase C–γ (PLC‐γ) and an increased level of intracellular Ca2+. Hippocalcin expression by bFGF stimulation was fully blocked by both the PLC‐γ inhibitor U73122 and BAPTA‐AM, a chelator of intracellular Ca2+, suggesting that hippocalcin expression by bFGF is dependent on PLC‐γ and Ca2+. Moreover, both U73122 and BAPTA‐AM completely blocked bFGF‐induced neurite outgrowth and NeuroD expression. Taken together, these results suggest for the first time that bFGF induces hippocalcin expression in H19‐7 cells through PLC‐γ activation, which leads to neurite outgrowth.

Hippocalcin increases phospholipase D2 expression through extracellular signal‐regulated kinase activation and lysophosphatidic acid potentiates the hippocalcin‐induced phospholipase D2 expression

We have previously isolated a 22 kDa protein from a rat brain which was found to be involved in activating phospholipsae D (PLD), and identified the protein as hippocalcin through sequence analysis. Nevertheless, the function of hippocalcin for PLD activation still remains to be resolved. Here, we proposed that hippocalcin was involved in extracellular signal‐regulated kinase (ERK)‐mediated PLD2 expression. To elucidate a role of hippocalcin, we made hippocalcin transfected NIH3T3 cells and showed that the expression of PLD2 and basal PLD activity were increased in hippocalcin transfected cells. We performed PLD assay with dominant negative PLD2 (DN‐PLD2) and hippocalcin co‐transfected cells. DN‐PLD2 suppressed increase of basal PLD activity in hippocalcin transfected cells, suggesting that increased basal PLD activity is due to PLD2 over‐expression. Hippocalcin is a Ca2+‐binding protein, which is expressed mainly in the hippocampus. Since it is known that lysophosphatidic acid (LPA) increases intracellular Ca2+, we investigated the possible role of hippocalcin in the LPA‐induced elevation of intracellular Ca2+. When the intracellular Ca2+ level was increased by LPA, hippocalcin was translocated to the membrane after LPA treatment in hippocalcin transfected cells. In addition, treatment with LPA in hippocalcin transfected cells markedly potentiated PLD2 expression and showed morphological changes of cell shape suggesting that increased PLD2 expression acts as one of the major factors to cause change of cell shape by making altered membrane lipid composition. Hippocalcin‐induced PLD2 expression potentiated by LPA in hippocalcin transfected cells was inhibited by a PI‐PLC inhibitor, U73122 and a chelator of intracellular Ca2+, BAPTA–AM suggesting that activation of hippocalcin caused by increased intracellular Ca2+ is important to induce over‐expression of PLD2. However, downregulation of PKC and treatment of a chelator of extracellular Ca2+, EGTA had little or no effect on the inhibition of hippocalcin‐induced PLD2 expression potentiated by LPA in the hippocalcin transfected cells. Interestingly, when we over‐express hippocalcin, ERK was activated, and treatment with LPA in hippocalcin transfected cells significantly potentiated ERK activation. Specific inhibition of ERK dramatically abolished hippocalcin‐induced PLD2 expression. Taken together, these results suggest for the first time that hippocalcin can induce PLD2 expression and LPA potentiates hippocalcin‐induced PLD2 expression, which is mediated by ERK activation. J. Cell. Biochem. 97: 1052–1065, 2006.

Der f 2 activates phospholipase D in human T lymphocytes from Dermatophagoides farinae specific allergic individuals: involvement of protein kinase C-alpha.

The major house-dust mite allergen, Der f 2, stimulates the phospholipase D (PLD) in T lymphocytes from Dermatophagoides farinae specific allergic individuals. PLD activity increased more than two-fold in T cells from allergic patients compared with those cells from normal controls with maximal responses within 30 min after exposure of Der f 2. A well-known PLD activator PKC-α was found to be translocated to membrane from cytosol in Der f 2-treated T cells from Dermatophagoides farinae specific allergic individuals. Down-regulation of PKC-α with phorbol myristate acetate pretreatment for 24 h abolished Der f 2-induced PLD activation. Ro 320432, PKC inhibitor also reduced the effects of Der f 2-induced PLD activation suggesting that PKC-α acts as upstream activator of PLD in Der f 2-treated T cells. Taken together, the present data suggest that Der f 2 can stimulate PLD activity through the PKC-α activation in T cells from Dermatophagoides farinae allergic individuals

Olfactomedin 4 suppresses tumor growth and metastasis of mouse melanoma cells through downregulation of integrin and MMP genes

Olfactomedin 4 (OLFM4) is highly expressed in gastrointestinal cancers and has an anti-apoptotic function. The roles of OLFM4 in tumor growth and metastasis and how it functions in these processes remain elusive. We investigated the function of OLFM4 in tumor growth and metastasis using B16F10 mouse melanoma cells as an experimental system. Our results showed that OLFM4 had no positive effect on cell viability or cell cycle progression in B16F10 cells. However, it significantly suppressed the tumorigenicity of B16F10 cells, i.e., intradermal primary tumor growth and lung metastasis. OLFM4 also suppressed the migration and invasion of B16F10 cells in vitro. For further insight into the mechanisms underlying OLFM4-mediated suppression of tumor progression, we examined the effect of OLFM4 on the expression of integrin and matrix metalloproteinase (MMP), both of which are involved in tumor progression. Overexpression of OLFM4 clearly reduced the expression levels of integrin α1, integrin α4, integrin α5, integrin α6, and MMP9. Moreover, forced expression of MMP9 attenuated the inhibitory activity of OLFM4 on migration and invasiveness. Our findings provide the experimental evidence that OLFM4 may function as a tumor suppressor and an anti-metastatic gene during tumor progression.

D609-sensitive tyrosine phosphorylation is involved in Fas-mediated phospholipase D activation

Both Fas and PMA can activate phospholipase D via activation of protein kinase Cβ in A20 cells. Phospholipase D activity was increased 4 fold in the presence of Fas and 2.5 fold in the presence of PMA. The possible involvement of tyrosine phosphorylation in Fas-induced activation of phospholipase D was investigated. In five minute after Fas cross-linking, there was a prominent increase in tyrosine phosphorylated proteins, and it was completely inhibited by D609, a specific inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC). A tyrosine kinase inhibitor, genistein, can partially inhibit Fas-induced phospholipase D activation. There were no effects of genistein on Fas-induced activation of PC-PLC and protein kinase C. These results strongly indicate that tyrosine phosphorylation may in part account for the increase in phospholipase D activity by Fas cross-linking and D609 can block not only PC-PLC activity but also tyrosine phosphorylation involved in Fas-induced phospholipase D activation.

The progesterone receptor as a transcription factor regulates phospholipase D1 expression through independent activation of protein kinase A and Ras during 8-Br-cAMP-induced decidualization in human endometrial stromal cells.

Decidualization is a biological and morphological process occurring in hES (human endometrial stromal) cells. Previously, we reported that PLD1 (phospholipase D1) plays an important role in cAMP-induced decidualization of hES cells. In the present study, we focused on how PLD1 expression is up-regulated during decidualization. Treatment with PKA (protein kinase A) inhibitors (Rp-cAMP or H89) or a Ras inhibitor (manumycin) partially inhibited PLD1 expression and decidua formation in response to cAMP treatment. Interestingly, dual inhibition of PKA and Ras completely inhibited PLD1 expression and cAMP-induced decidualization. These results suggest that PLD1 expression during decidualization is controlled additively by PKA and Ras. The use of inhibitors showed that extracellular-signal-regulated kinase, a downstream effector of Ras, was required for PLD activation and the morphological changes during decidualization, but not for the increase in PLD1 protein. Next, to investigate the regulator of the PLD1 gene at the transcriptional level, a promoter assay using deletion mutants of the PLD1 promoter was performed; the result indicated that PR (progesterone receptor) was a possible regulator of the PLD1 gene. In addition, chromatin immunoprecipitation assays on the PLD1 promoter identified PR as a transcription factor for PLD1 expression during 8-Br-cAMP-induced decidualization. Taken together, our findings demonstrate that PKA and Ras are novel regulators of PLD1 expression and also identify PR as a transcription factor for PLD1 expression during the decidualization of hES cells.

Major house dust mite allergen, Der p I, activates phospholipase D in human peripheral blood mononuclear cells from allergic patients: involvement of protein kinase C.

The major house-dust-mite allergen, Der p I, stimulates the phospholipase D (PLD) in peripheral blood mononuclear cells (PBMC) from allergic patients with maximal responses after 30 min exposure. At 30 min, Der p I stimulated PLD activity by 1.4-fold in mild, 1.6-fold in moderate and 2-fold in severe allergic patients over control values (p < 0.05). When the cells were pretreated for 24 h with phorbol myristate acetate to down-regulate protein kinase C (PKC), PLD stimulation by Der p I was largely abolished. These results indicate that in PBMC from allergic patients, Der p I can stimulate PLD activity, and that PKC activation is involved in this stimulation.

Advanced lipid extraction method for the determination of the phospholipase D activity.

Phospholipase D is a ubiquitous enzyme that plays an important role in various lipid mediatedcellular signaling pathways and produces rare phospholipids, phosphatidylethanol or phos-phatidylbutanol, instead of phosphatidic acid with unique catalytic activity transphosphatidyla-tion in the presence of primary alcohols. The reaction products, phosphatidylethanol or phosphatidylbutanol are used as markers ofin vitro phospholipase D activity in many studies. For the sensitive detection of the phospholipase D products, we developed an advanced lipid extraction method that facilitates recovery of the compounds. With the new method, the activity change of phospholipase D by agonists could be detected more easily and the recovery rate was also increased. The increase of detected enzyme activity change was about double fold compared to the conventional lipid extraction method. This method provides selective force for the phospholipase D products in the extraction procedure.