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The histamine H(1) receptor (H1R) gene is up-regulated in patients with allergic rhinitis. However, the mechanism and reason underlying this up-regulation are still unknown. Recently, we reported that the H1R expression level is strongly correlated with the severity of allergic symptoms. Therefore, understanding the mechanism of this up-regulation will help to develop new anti-allergic drugs targeted for H1R gene expression. Here we studied the molecular mechanism of H1R up-regulation in HeLa cells that express H1R endogenously in response to histamine and phorbol 12-myristate 13-acetate (PMA). In HeLa cells, histamine stimulation caused up-regulation of H1R gene expression. Rottlerin, a PKCδ-selective inhibitor, inhibited up-regulation of H1R gene expression, but Go6976, an inhibitor of Ca(2+)-dependent PKCs, did not. Histamine or PMA stimulation resulted in PKCδ phosphorylation at Tyr(311) and Thr(505). Activation of PKCδ by H(2)O(2) resulted in H1R mRNA up-regulation. Overexpression of PKCδ enhanced up-regulation of H1R gene expression, and knockdown of the PKCδ gene suppressed this up-regulation. Histamine or PMA caused translocation PKCδ from the cytosol to the Golgi. U0126, an MEK inhibitor, and DPQ, a poly(ADP-ribose) polymerase-1 inhibitor, suppressed PMA-induced up-regulation of H1R gene expression. These results were confirmed by a luciferase assay using the H1R promoter. Phosphorylation of ERK and Raf-1 in response to PMA was also observed. However, real-time PCR analysis showed no inhibition of H1R mRNA up-regulation by a Raf-1 inhibitor. These results suggest the involvement of the PKCδ/ERK/poly(ADP-ribose) polymerase-1 signaling pathway in histamine- or PMA-induced up-regulation of H1R gene expression in HeLa cells.

A soybean Kunitz trypsin inhibitor (KTI) interacts with cells as a negative modulator of the invasive cells. Using complementary pharmacological and genetic approaches, we provide novel findings regarding mechanisms by which KTI inhibits signaling pathways in ovarian cancer cells leading to invasion. Transforming growth factor-beta1 (TGF-beta1) directly activates Src kinase, which in turn activates ERK-phosphatidylinositol 3-kinase/Akt, the downstream targets of Src, for urokinase-type plasminogen activator (uPA) up-regulation in human ovarian cancer HRA cells. Preincubation of the HRA cells with KTI reduced the ability of TGF-beta1 to trigger the uPA expression at the gene level and at the protein level. To further elucidate the mechanism of the KTI-dependent suppressive effect of TGF-beta1-induced uPA expression and invasion, we investigated which signaling pathway transduced by KTI is responsible for this inhibitory effect. Here, we show that 1) KTI suppressed TGF-beta1-induced phosphorylation of Src, ERK1/2, and Akt by 40-60%; 2) KTI was insensitive to suppress the phosphorylation of ERK1/2 and Akt in the constitutively active (CA)-c-Src (Y529F) cells; 3) uPA expression was up-regulated in TGF-beta1-stimulated HRA cells and in unstimulated Y529F cells; 4) the addition of KTI reduced the TGF-beta1-induced increase of uPA gene and protein expression in the wild-type c-Src-transfected cells (in contrast, KTI could not inhibit uPA expression in the Y529F cells); and 5) CA-c-Src transfection resulted in a 2-fold increase in invasiveness, whereas KTI did not reduce invasion of the Y529F cells. Using additional complementary genetic approaches (CA-MEK1, CA-Akt, or kinase-dead-Akt), we conclude that KTI may suppress uPA expression and promotion of invasion possibly through one or more upstream targets of Src.

Urokinase-type plasminogen activator (uPA) degrades the extracellular matrix and plays critical roles in tumor invasion and metastasis. Matriptase, a membrane-bound serine protease, was shown to activate uPA in a uPA receptor-free, solution-based study. We now investigate whether matriptase affects activation of receptor-bound uPA and contributes to the invasiveness of HRA human ovarian cancer cells in vitro and tumor behavior in nude mice. Here we show the following. 1) uPA expression was effectively stimulated by TGF-beta1 in HRA cells. 2) Antisense (AS)-matriptase transfection achieved a marked inhibition of receptor-bound pro-uPA activation without altering expression of uPA and uPA receptor mRNA and proteins, irrespective of whether cells were stimulated with TGF-beta1. 3) Tumor cell receptor-bound pro-uPA could be efficiently cleaved by matriptase to generate enzymatically active two-chain uPA. Thus, matriptase can substitute for plasmin in the proteolytic activation of pro-uPA to enzymatically active uPA. 4) The AS-matriptase-treated cells had a decreased ability to invade an extracellular matrix layer, as compared with control cells. 5) When the AS-matriptase-treated cells were injected intraperitoneally into nude mice, the mice developed smaller tumors. Our data identify a novel role for matriptase for activation of receptor-bound uPA.

Urokinase-type plasminogen activator (uPA) has been implicated in tumor cell invasion and metastasis. We reported previously that transforming growth factor (TGF)-beta1 induces a dose- and time-dependent up-regulation of uPA mRNA and protein in highly invasive human ovarian cancer cell line HRA, leading to invasion. To further elucidate the mechanism of the invasive effect of TGF-beta1, we investigated which signaling pathway transduced by TGF-beta1 is responsible for this effect. Here, we show that 1) nontoxic concentrations of TGF-beta1 activated Src kinase; 2) TGF-beta1 rapidly phosphorylates ERK1/2 and Akt, but not p38; 3) pharmacological Src inhibitor PP2 or antisense (AS) c-Src oligodeoxynucleotide (ODN) treatment reduced TGF-beta1-induced phosphorylation of ERK1/2 and Akt by 85-90% compared with controls; 4) pharmacological inhibition of MAPK by PD98059 abrogated TGF-beta1-mediated Akt stimulation, whereas TGF-beta1-induced ERK1/2 stimulation was not inhibited by PI3K inhibitor LY294002 or AS-PI3K ODN transfection; 5) up-regulation of uPA mRNA in response to TGF-beta1 was almost totally blocked by PP2 and PD98059 and partially ( approximately 55%) by LY294002; 6) TGF-beta1-induced uPA mRNA up-regulation was inhibited by treatment with AS ODNs to c-Src or PI3K by 90 or 60%, respectively, compared with control ODN treatment; and 7) blockade of the release of the transcription factor NF-kappaB by pyrrolidinedithiocarbamate reduced the TGF-beta1-induced activation of the uPA gene by approximately 65%. In addition, curcumin, a blocker of the transcriptional factor AP-1, partially (35%) canceled this effect. Taken together, these data support a role for TGF-beta1 activation of two distinct pathways (Src-MAPK-PI3K-NF-kappaB-dependent and Src-MAPK-AP-1-dependent) for TGF-beta1-dependent uPA up-regulation and promotion of invasion.

Using a cDNA microarray analysis, we previously found that exposure of a highly invasive ovarian cancer cell line HRA with bikunin, a Kunitz-type protease inhibitor, or bikunin gene overexpression markedly reduced phosphoinositide kinase (PI3K) p85 gene expression, demonstrating that PI3K may be a candidate bikunin target gene. To clarify how reduced levels of PI3K may confer repressed invasiveness, we transfected HRA cells with PI3K p85 antisense-oligodeoxynucleotide (AS-ODN) and compared the properties of the transfected cells with those of parental cells and sense (S)-ODN cells. We have also demonstrated previously that transforming growth factor-beta1 (TGF-beta1) stimulates urokinase-type plasminogen activator (uPA)-dependent invasion and metastasis of HRA cells. Here, we show that 1) TGF-beta1 induced a rapid increase of the PI3K activity that was accompanied by increased expression (5-fold) of the uPA mRNA; 2) pharmacological inhibition of PI3K or AS-PI3K ODN transfection inhibited TGF-beta1-stimulated Akt phosphorylation; 3) both PI3K pharmacological inhibitors and forced expression of AS-PI3K ODN reduced TGF-beta1-stimulated uPA mRNA and protein expression by approximately 70% compared with controls; 4) concentrations of PI3K inhibitors, sufficient to inhibit uPA up-regulation, inhibited TGF-beta1-dependent HRA cell invasion; 5) the AS-PI3K ODN cells had a decreased ability to invade the extracellular matrix layer as compared with controls; and 6) when the AS-PI3K ODN cells were injected intraperitoneally into nude mice, the mice developed smaller intraperitoneal tumors and showed longer survival. We conclude that PI3K plays an essential role in promoting uPA-mediated invasive phenotype in HRA cells. Our data identify a novel role for PI3K as a bikunin target gene on uPA up-regulation and invasion.

The processes of ovarian cancer dissemination are characterized by altered local proteolysis, cellular proliferation, cell attachment, and invasion, suggesting that the urokinase-type plasminogen activator (uPA) and its specific inhibitor (plasminogen activator inhibitor type-1 (PAI-1)) could be involved in the pathogenesis of peritoneal dissemination. We showed previously that expression of uPA and PAI-1 in the human ovarian cancer cell line HRA can be down-regulated by exogenous bikunin (bik), a Kunitz-type protease inhibitor, via suppression of transforming growth factor-beta1 (TGF-beta1) up-regulation and that overexpression of the bik gene can specifically suppress the in vivo growth and peritoneal dissemination of HRA cells in an animal model. We hypothesize that the plasminogen activator system in mesothelial cells can be modulated by HRA cells. To test this hypothesis, we used complementary techniques in mesothelial cells to determine whether uPA and PAI-1 expression are altered by exposure to culture media conditioned by HRA cells. Here we show the following: 1) that expression of PAI-1, but not uPA, was markedly induced by culture media conditioned by wild-type HRA cells but not by bik transfected clones; 2) that by antibody neutralization the effect appeared to be mediated by HRA cell-derived TGF-beta1; 3) that exogenous TGF-beta1 specifically enhanced PAI-1 up-regulation at the mRNA and protein level in mesothelial cells in a time- and concentration-dependent manner, mainly through MAPK-dependent activation mechanism; and 4) that mesothelial cell-derived PAI-1 may promote tumor invasion possibly by enhancing cell-cell interaction. This represents a novel pathway by which tumor cells can regulate the plasminogen activator system-dependent cellular responses in mesothelial cells that may contribute to formation of peritoneal dissemination of ovarian cancer.

Bikunin, a Kunitz-type protease inhibitor, could potentially suppress tumor cell invasion and metastasis. Our previous study revealed that overexpression of bikunin in a human ovarian cancer cell line, HRA, resulted in a down-regulation in uPA and uPAR gene expression. For identifying the full repertoire of bikunin-regulated genes, a cDNA microarray hybridization screening was conducted using mRNA from bikunin-treated or bikunin-transfected HRA cells. A number of bikunin-regulated genes were identified, and their regulation was confirmed by Northern blot analysis. Our screen identified 11 bikunin-stimulated genes and 29 bikunin-repressed genes. The identified genes can indeed be classified into distinct subsets. These include transcriptional regulators, oncogenes/tumor suppressor genes, signaling molecules, growth/cell cycle, invasion/metastasis, cytokines, apoptosis, ion channels, extracellular matrix proteins, as well as some proteases. This screen identified suppression of several genes such as CDC-like kinase, LIM domain binding, Ets domain transcription factor, Rho GTPase-activating protein, tyrosine phosphorylation-regulated kinase, hyaluronan-binding protein, matriptase, and pregnancy-associated plasma protein-A (PAPP-A), which have previously been implicated in enhancing tumor promotion. Northern blot analysis confirmed that several genes including matriptase and PAPP-A were down-regulated by bikunin by approximately 9-fold. Further, genetic inhibition of matriptase or PAPP-A could lead to diminished invasion. These results show that bikunin alters the pattern of gene expression in HRA cells leading to a block in cell invasion.

Bikunin is a Kunitz-type protease inhibitor, acting at the level of tumor invasion and metastasis. The goal of this study was to investigate the effect of bikunin-dependent signal transduction involved in the expression of a plasminogen activator (PA) system and invasion. We report here the following. 1) The human ovarian cancer cell line HRA produced secreted and cell-associated urokinase-type PA (uPA) and PA inhibitor type 1 (PAI-1). The plasma membrane of the cells showed enzymatically active uPA even in the presence of high level of PAI-1, as measured by zymography, Western blot, chromogenic assay, enzyme-linked immunosorbent assay, and Northern blot. 2) HRA cells leading to invasion are induced through up-regulation of uPA expression. 3) HRA cells specifically released transforming growth factor-beta type 1 (TGF-beta1) participating in an autocrine/paracrine regulation of cell invasion. 4) Elimination of endogenous TGF-beta1 could induce change in uPA/PAI-1 expression, which could in turn modify the invasive behavior of the cells. 5) The constitutive expression of TGF-beta1 as well as up-regulation of the PA system observed in HRA cells was inhibited by preinoculation of the cells with bikunin or calcium channel blocker SK&F 96365 but not with nifedipine or verapamil, with an IC(50) of approximately 100 nm for bikunin or approximately 30 microm for SK&F 96365, respectively, as measured by enzyme-linked immunosorbent assay. Bikunin showed no additive effect on SK&F 96365-mediated suppression of TGF-beta1 expression. 6) The ability of TGF-beta1 to elevate free intracellular Ca(2+), followed by activation of Src and ERK, was reduced by preincubation of the cells with bikunin. In conclusion, bikunin could inhibit the constitutive expression of TGF-beta1 and TGF-beta1-mediated, Src- and ERK-dependent, PA system signaling cascade, at least in part, through inhibition of a non-voltage-sensitive calcium channel.

We previously found that bikunin (bik), a Kunitz-type protease inhibitor, suppresses phorbol ester (PMA)-stimulated expression of urokinase-type plasminogen activator (uPA). In the present study, we tried to answer this mechanism using human chondrosarcoma HCS-2/8 cells. Our results showed the following novel findings: (a) the standard form of CD44 (CD44s; 85 kDa) is expressed in both unstimulated and PMA-stimulated cells, while CD44v isoforms containing epitope v9 (110 kDa) are strongly up-regulated in response to treatment with PMA; (b) CD44v isoforms containing epitope v9 present on the same cell exclusively form aggregates in stimulated cells; (c) induction of uPA mRNA expression could be achieved by using a second cross-linker antibody to cross-link Fab monomers of anti-CD44; (d) co-treatment of stimulated cells with anti-CD44 mAb alone or anti-CD44v9 mAb alone suppresses PMA-induced clustering of CD44, which results in inhibition of uPA overexpression; (e) bikunin efficiently disrupts PMA-induced clustering of CD44, but does not prevent PMA-induced up-regulation of CD44v isoforms containing epitope v9; and (f) after exposure to bik, approximately 150-kDa band is mainly detected with immunoprecipitation and this band is shown to be a heterodimer composed of the 110-kDa v9-containing CD44v isoforms and a 45-kDa bik receptor (bik-R). In conclusion, we provide, for the first time, evidence that the bik-R can physically interact with the CD44v isoforms containing epitope v9 and function as a repressor to down-regulate PMA-stimulated uPA expression, at least in part, by preventing clustering of CD44v isoforms containing epitope v9.

Urinary trypsin inhibitor (UTI), a Kunitz-type protease inhibitor, interacts with cells as a negative modulator of the invasive cells. Human ovarian cancer cell line, HRA, was treated with phorbol ester (PMA) to evaluate the effect on expression of urokinase-type plasminogen activator (uPA), since the action of uPA has been implicated in matrix degradation and cell motility. Preincubation of the cells with UTI reduced the ability of PMA to trigger the uPA expression at the gene level and at the protein level. UTI-induced down-regulation of PMA-stimulated uPA expression is irreversible and is independent of a cytotoxic effect. Down-regulation of uPA by UTI is mediated by its binding to the cells. We next asked whether the mechanism of inhibition of uPA expression by UTI was due to interference with the protein kinase C second messenger system. An assay for PKC activity demonstrated that UTI does not directly inhibit the catalytic activity of PKC and that PMA translocation of PKC from cytosol to membrane was inhibited by UTI, indicating that UTI inhibits the activation cascade of PKC. PMA could also activate a signaling pathway involving MEK1/ERK2/c-Jun-dependent uPA expression. When cells were preincubated with UTI, we could detect suppression of phosphorylation of these proteins. Like several types of PKC inhibitor, UTI inhibited PMA-stimulated invasiveness. We conclude that UTI markedly suppresses the cell motility possibly through negative regulation of PKC- and MEK/ERK/c-Jun-dependent mechanisms, and that these changes in behavior are correlated with a coordinated down-regulation of uPA which is likely to contribute to the cell invasion processes.

Urinary trypsin inhibitor (UTI), a Kunitz-type protease inhibitor, directly binds to some types of cells via cell-associated UTI-binding proteins (UTI-BPs). Here we report that the 40-kDa protein (UTI-BP(40)) was purified from the cultured human chondrosarcoma cell line HCS-2/8 by UTI affinity chromatography. Purified UTI-BP(40) was digested with trypsin, and the amino acid sequences of the peptide fragments were determined. The sequences of six tryptic fragments of UTI-BP(40) were identical to subsequences present in human link protein (LP). Authentic bovine LP and UTI-BP(40) displayed identical electrophoretic and chromatographic behavior. The UTI-binding properties of UTI-BP(40) and LP were indistinguishable. Direct binding and competition studies strongly demonstrated that the NH(2)-terminal fragment is the UTI-binding part of the LP molecule, that the COOH-terminal UTI fragment (HI-8) failed to bind the NH(2)-terminal subdomain of the LP molecule, and that LP and UTI-BP(40) exhibited significant hyaluronic acid binding. These results demonstrate that UTI-BP(40) is identical to LP and that the NH(2)-terminal domain of UTI is involved in the interaction with the NH(2)-terminal fragment of LP, which is bound to hyaluronic acid in the extracellular matrix.