44 MAPK and p38 MAPK regulate hepatocyte growth factor secretion from human astrocytoma cells

Molecular Brain Research 102 (2002) 73–82 www.elsevier.com / locate / bres

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PKC, p42 / 44 MAPK and p38 MAPK regulate hepatocyte growth factor secretion from human astrocytoma cells Naibedya Chattopadhyay*, Jacob Tfelt-Hansen, Edward M. Brown Endocrine-Hypertension Division and Membrane Biology Program, Department of Medicine, Brigham and Women’ s Hospital, Harvard Medical School, 221 Longwood Ave., Boston, MA 02115, USA Accepted 14 January 2002

Abstract Hepatocyte growth factor (HGF) and its receptor c-Met are expressed in inappropriately high abundance in gliomas and are further upregulated during the transition from low- to high-grade malignancy. In these cells HGF induces expression of c-Met via PKC, Ras and mitogen activated protein kinase (MAPK) pathway. Here we report that secretion and expression of HGF in U87 astrocytoma is increased by a PKC activator, PMA, an effect which is abolished by a PKC inhibitor, Go6976, specific for PKCa and PKCb1. Activating PKA by forskolin, on the other hand, had no effect. Furthermore, messenger molecule downstream of PKC, i.e. MEK mediates such effect of PKC as specific MEK inhibitors (PD98059 and U0126) abolished PMA induced HGF secretion by U87 cells. Accordingly, PMA induced rapid phosphorylation of MEK substrate, i.e. Erk1 / 2 (p42 / 44 MAPK). In addition, such effect of PKC is Ras-dependent as specific Ras inhibitor L-744,832 attenuated both PMA mediated induction of Erk 1 / 2 phosphorylation as well as HGF secretion. Moreover, a specific p38 MAPK inhibitor (SB203580) almost completely inhibited basal HGF secretion to an undetectable level. Increased secretion of HGF is most likely exerted at the transcriptional level since inhibitor of transcription, actinomycin D abolished such increase. Furthermore, when assessed by Northern blot analysis, PMA increased HGF transcripts while U0127 and SB203580 inhibited. Therefore, our data reveal that HGF secretion in U87 cells is regulated by Ras-dependent PKC, MEK cascade and in parallel by p38 MAPK pathway. Since the Raf–PKC–MEK cascade is used for HGF’s signaling via its receptor in astrocytoma cells, our data revealing similar regulatory mechanism for HGF secretion in these cells would help to explain the feed forward nature of HGF action in glioma cells that would further accentuate its basal secretion, exacerbating its effects on the progression of gliomas in an autocrine fashion.  2002 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Neuro-oncology Keywords: Glioma; Autocrine; Signaling; Mitogenesis; Therapeutics

1. Introduction Hepatocyte growth factor / scatter factor (HGF / SF) is a mesenchymal cell-derived, multifunctional cytokine having important roles in tissue development and regeneration as well as in tumor progression [6, for review 31]. HGF is produced predominantly by mesenchymal cells, while its receptor, c-Met, is expressed on neighboring epithelial cells, thereby enabling canonical paracrine interactions *Corresponding author. Tel.: 11-617-732-4093; fax: 11-617-7325764. E-mail address: [email protected] (N. Chattopadhyay).

[34]. The actions of HGF / SF (hereafter referred to simply as HGF) are transduced through the c-Met proto-oncogene product, a transmembrane tyrosine kinase. In the CNS, HGF has neurotrophic role by enhancing NGF dependent survival and growth of neurons [14]. Recent findings have suggested that HGF significantly contributes to the malignant progression of human gliomas by acting as a mitogen and a motogen. It also enhances their invasiveness as well as tumor-associated angiogenesis and therefore, represent a target for attenuating the growth, invasiveness and angiogenesis of gliomas [15,19,23,32,33]. In human brain tumors HGF and of c-Met are prevalent both in infiltrating tumor cells and in associated hyperplastic endothelial cells, implying, therefore, the existence of

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autocrine / paracrine HGF-c-Met signaling in glioma tumorogenesis [24]. Transfecting HGF into glioma cells expressing c-Met results in considerably larger and more vascularized intracranial tumors in vivo than is observed with HGF-negative, control clones, owing to enhanced vascular endothelial growth factor (VEGF) expression [7]. Conversely, downregulating the HGF-c-Met signaling pathway by transfecting glioblastoma cells with chimeric transgenes consisting of U1 small nuclear RNA, a hammerhead ribozyme, antisense sequences and neutralizing antibodies to HGF significantly suppresses tumor progression [2,9]. Therefore, the autocrine / paracrine actions of HGF, acting via c-Met, play an important roles in the tumor progression. Although much understanding has taken place regarding the mechanism of actions of HGF, current knowledge on the mechanism of its secretion is relatively poor. Among the various factors regulating HGF secretion in other cell types include for example angiotensin II and transforming growth factor b (TGFb) downregulating HGF expression in vascular smooth muscle cells (VSMCs) [26], dexamethasone in human fibroblasts [12] and agonists of retinoic acid- and retinoid X receptors in U87 human astrocytoma cells [10]. In addition, HGF secretion by human skin fibroblast is regulated by both PKA and PKC mediated pathways [12,22] while in endometrial stromal cells it has been reported to be only through PKC [27]. In this study we sought to determine the regulation of secretion and expression of HGF by various second messenger agents so as to gain insights into the signaling pathways that are involved in its constitutive secretion and help explain the mechanisms underlying the autocrine mode of its action. Our study revealed involvement of Ras-dependent activation of PKC and MEK pathway in addition to a parallel pathway mediated via p38 MAPK in the secretion of HGF by human malignant astrocytoma cells (U87). Its regulation of secretion by PKC, MEK pathway sets a stage for a ‘vicious’ feed forward mechanism by which c-Met mediated activation of MAPK pathway results in increased turn over and synthesis of HGF in these cells.

Go6976 and SB-203580 were obtained from Calbiochem (La Jolla, CA, USA). HGF ELISA kit was purchased from R&D Systems (Minneapolis, MN, USA).

2.2. Determination of HGF secretion For studying HGF secretion, U87 cells were grown to 70–75% confluency in complete growth medium in 24well plates. They were then incubated with DMEM containing 0.2% BSA overnight with various concentrations of inhibitors and activators as described in the Results section, following which HGF was measured in the conditioned media using an ELISA. The latter employs a quantitative sandwich enzyme-linked immunoassay technique, utilizing a monoclonal antibody specific for HGF that is bound to microtiter wells. Assay sensitivity was 125 pg / ml. The HGF content of each well was normalized by determining protein concentration.

2.3. Northern blot analysis To study whether various inhibitors and activators exert inhibitory effects on the expression of HGF mRNA, we performed Northern blot analysis. For HGF mRNA determination, aliquots of 2.5 mg poly (A1) enriched RNA were utilized, which had been extracted [10] following exposure of the cells to various modulators at the doses and times shown in the results section. RNA samples were denatured and electrophoresed in 2.2 M formaldehyde in 1% agarose gels along with an 0.24 Kb–9.5 Kb RNA ladder (Gibco / BRL) and transferred overnight to nylon membranes (Duralon, Stratagene, La Jolla, CA, USA). cDNA probe for HGF was obtained from Dr. T. Nakamura (Osaka University School of Medicine, Osaka, Japan). A 2.3-kilo-base-pair (kbp) fragment of human HGF was subcloned into pBlueScript SK(2) and was digested with BamH I and Kpn I in order to excise a 2.2-kbp fragment, which was then used for probing the Northern blots using standard technique [10]. Specific radioactive signals were analyzed on a Molecular Dynamics Phosphorimager (Sunnyvale, CA, USA) with the IMAGEQUANT program.

2. Materials and methods

2.4. Western blot analysis 2.1. Materials All routine culture media were obtained from GibcoBRL (Grand Island, NY, USA). U87 cells were purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA) and were maintained in monolayer culture in Dulbecco’s minimum essential medium (DMEM) supplemented with 10% fetal bovine serum. Forskolin, phorbol 12-myristate 13-acetate (PMA), PD98059, U0126 and L-744,832 were purchased from Biomol (Plymouth Meeting, PA, USA); actinomycin D,

For the determination of ERK1 / 2 phosphorylation, monolayers of serum starved U87 cells were incubated at 37 8C in serum-free medium containing 0.2% BSA, with varying concentrations of PMA for different times. At the end of the incubation, whole cell lysates were used for western analysis by following standard method [18] using a polyclonal antiserum against phosphorylated ERK1 / 2 [phospho-p44 / 42 MAPK (Thr 202 / Tyr 204 ) antibody] (Cell Signaling Technology, Beverly, MA, USA). The bands were visualized by chemiluminescence (NEN Life Science,

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Boston, MA, USA) and quantitation was performed using densiometer (Molecular Dynamics).

indicate a statistically significant difference between groups.

2.5. Statistics

3. Results

Results are expressed as the mean6S.E.M. Statistical evaluation for differences between group means was done using one-way analysis of variance (ANOVA) followed by Fisher’s protected least significant difference (PLSD). For all statistical tests, values of P,0.05 were considered to

3.1. HGF secretion is PKC regulated Fig. 1A shows that a specific PKC activator, PMA dose dependently increases HGF secretion with maximal effect at 100 nM which is completely obliterated by pretreatment

Fig. 1. (A) Concentration dependent increase in HGF secretion by PKC activator, PMA in U87 cells which is significantly reduced by preincubating the cells with Go6976, a PKCa and b1 inhibitor. Results are representative of three independent experiments; P,0.05. (B) Specific PKA activator forskolin had no effect on HGF secretion.

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of the cells for 4 h with 1 mM Go6976, a cell permeable and specific inhibitor for PKCa and PKCb1 [13,25]. Specific PKA activator, forskolin had no effect even at 50 mM concentration (Fig. 1B).

(MEK-2 inhibitor), we observed concentration dependent inhibition of HGF secretion with maximal inhibition at 5 and 10 mM, respectively (Fig. 2A). In addition, specific p38 MAPK inhibitor, SB203580 also inhibited HGF secretion with maximal inhibition at 5 mM (Fig. 2B).

3.2. Different MAP kinases regulate basal secretion of HGF

3.3. PKC and MEK are functionally coupled

As MEK is downstream of PKC, we studied whether or not this pathway is involved in the secretion of HGF. Using U0126 (MEK-1 and 2 inhibitor) and PD98059

A 100-nM concentration of PMA, which results in maximum stimulation of HGF, results in rapid phosphorylation of Erk1 / 2 (Fig. 3A). Phosphorylation occurred as

Fig. 2. (A) Concentration dependent inhibition of basal HGF secretion by specific MEK inhibitors in U87 cells. (B) Specific p38 MAPK inhibitor, SB203580 inhibits basal HGF secretion in U87 cells. Results are representative of three independent experiments; P,0.05.

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Fig. 3. (A) PMA (100 nM) results in rapid and sustained phosphorylation of p44 / 42 MAPK in U87 cells. (B) PMA induced secretion of HGF is attenuated by preincubating U87 cells with MEK 1 and 2 inhibitor, U0126 for 3 h. Results are representative of three independent experiments; P,0.05.

early as 5 min and remained until 15 min following PMA incubation of U87 cells. Furthermore, preincubating the cells with 2.5 mM U0126 (MEK1 and 2 inhibitor) for 3 h completely abolished PMA stimulated HGF secretion by HGF cells (Fig. 3B) thereby suggesting that these two pathways are functionally coupled in regulating HGF secretion.

3.4. Ras dependent activation of PKC and HGF secretion In order to study whether or not PKC and MEK-1 mediated secretion of HGF is Ras dependent, we preincubated U87 cells with 20 mM Ras inhibitor L-744,832 for 30 min following which the cells were exposed to 100 nM PMA. Fig. 4A shows that while PMA alone results in robust phosphorylation of Erk1 / 2, preincubation with L744,832 abolishes such activation. Accordingly, preincubation of U87 cells for 3 h with 20 mM L-744,832 abolished PMA induced stimulation of HGF (Fig. 4B).

3.5. PKC activator and various MAPK inhibitors act on HGF mRNA In order to investigate whether or not the PKC activator, PMA exerts its stimulatory effect on HGF transcription, we used transcriptional inhibitor, actinomycin D (10 mg / ml). U87 cells were coincubated with actinomycin D and PMA (100 nM) for 16 h and supernatants were used for HGF assay. Fig. 5A shows that while PMA stimulates HGF secretion by more than 2-fold, such an effect is completely abolished in the presence of actinomycin D thereby suggesting a transcriptional effect. On the other hand, since MEK and p38 MAPK inhibitors inhibit HGF secretion, we performed Northern blot analysis in order to test whether or not these agents have an effect on the mRNA expression of HGF in U87 cells. Because overnight incubation of PMA or these inhibitors resulted in either stimulation or inhibition of HGF secretion, respectively, we used a similar time for studying their effects on HGF mRNAs. Previously, we have shown that U87 cells express 6.0-, 3.0- and 1.5-kb HGF tran-

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Fig. 4. (A) Specific Ras inhibitor, L-744,832 inhibits PMA induced phosphorylation of p44 / 42 MAPK in U87 cells. Lanes: 15vehicle, 25100 nM PMA for 5 min, 35100 nM PMA for 5 min following preincubation with 20 mM L-744,832 for 30 min. (B) PMA induced secretion of HGF is attenuated by preincubating U87 cells with L-744,832 for 3 h. Results are representative of three independent experiments; P,0.05.

scripts [10]. Fig. 5B shows that 100 nM PMA increases all three transcripts and the total increase of HGF mRNAs by PMA was 2.5-fold compared to vehicle treated control cells. Interestingly, with MEK inhibitors, a differential effect on the HGF transcripts were observed; being 3.4fold less in the presence of U0126 while no change was observed with PD98059. The p38 MAPK inhibitor, SB203580 had a modest yet significant inhibitory effect on HGF transcripts (inhibited by 1.3-fold) (Fig. 5C).

4. Discussion Our data for the first time revealed the regulation of secretion of HGF in a glioma derived cell which might underlie its autocrine mode of action in these cells. Several recent reports have shown that HGF, and its receptor, c-Met, are expressed at abnormally high levels in malignant astrocytes compared to their normal counterparts and

exert a strong proliferative and motogenic actions in an autocrine / paracrine fashion [33]. Hence, inhibition of HGF production and / or neutralization of its protein product represent major chemotherapeutic strategies to control glioma growth. Moreover, it has recently been shown that HGF in glioma cells via Raf–PKC–MEK pathway induces expression of its own receptor, c-Met [1] which is contrary to a previously reported ligand-induced downregulation in liver cells [20]. A similar signaling profile for c-Met has been reported in other cells [4,17,30]. Therefore, although the literature is replete with the signaling pathways used by the HGF via c-met, there is a scarcity of data on the mechanisms by which HGF secretion and expression is regulated in general, and particularly in glioma cells where its overexpression results in mitogenesis, motogenesis and angiogenesis finally resulting in malignant progression of these tumors. It has previously been demonstrated that the proliferation of various established glioma cell lines correlated

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Fig. 5. (A) Effect of actinomycin D (10 mg / ml) on PMA induced HGF secretion. (B) Effects of various pharmacological modulators on HGF mRNA expression after 24 h. U87 cells were incubated with various modulators as described in Materials and methods: Lanes: 15vehicle treated control; 25100 nM PMA, 355 mM U0126, 4510 mM PD98059 and 555 mM SB203580. (C) Densitometric analysis of total of three HGF transcripts expressed in arbitrary units. Results show the average of two independent experiments.

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with the PKC activity [5,8]. More recently, it has been shown that malignant gliomas including U87 cells express high levels of ‘conventional’ PKC isozymes (specially PKCa) and the ‘novel’ form (PKCd) [21]. Furthermore, upregulating PKCa makes these cells more proliferative while PKCd renders them apoptotic to etoposide [21]. Since our data revealed that PKC activation was closely associated with a robust increase in HGF secretion, we sought to determine the relative involvement of PKCa in this process by using a cell permeable agent, Go6976, that is specific for inhibiting PKCa- and -b1 activities [25]. Our data show that in U87 cells that are expressing high levels of PKCa, Go6976 completely obliterated PMA induced HGF secretion. Therefore, given the high expression of PKCa in U87 cells our data strongly suggest its relatively greater regulatory role in HGF secretion than other isozymes, which in turn results in increased cellular proliferation. Regulation of HGF secretion by PKC has been shown in human endometrial stromal cells [27] as well as by PKA in fibroblasts [12]. However, our study shows that forskolin has no effect on HGF secretion from U87 cells. In an effort to identify downstream effectors of PKC activation that result in increased secretion of HGF, we investigated the classic MAPK cascade (Raf→MEK→ERK). This pathway has been shown to mediate a number of downstream effects of PKC activation in a variety of cells. We first show that two specific inhibitors of MEK, PD98059 and U0126 completely obliterated basal HGF secretion. Not surprisingly, U0126, a dual inhibitor of MEK 1 and 2 was more effective than PD98059 which inhibits MEK 1 alone. That such effect is mediated by PKC was revealed by PMA induced phosphorylation of Erk1 / 2 (p42 / 44 MAPK), an MEK substrate. Furthermore, MEK inhibitor, U0126 prevented PMA stimulated HGF secretion thereby suggesting involvement of PKC mediated MAP kinase pathway regulating HGF secretion in these cells. Since any PKC dependent cellular event could be either Ras-dependent or -independent, we used a specific Ras inhibitor, L-744,832. Our data revealed that HGF secretion in these cells is Ras dependent as PMA induced phosphorylation of Erk and HGF secretion were abolished by L-744,832. HGF has been shown to activate the MEK pathway among other MAPKs via PKC activation in gliomas and in other tumor cells. In gliomas, such an event also results in the induction of c-met expression [1]. Our study reveals that the messenger molecules produced as a result of HGF’s action on its receptor in turn increase its own secretion which then would exacerbate HGF actions of mitogenesis, motogenesis and angiogenesis in these tumor cells. Inhibition of basal level of HGF secretion in the presence of p38 MAPK inhibitor in addition to PKC suggests that the activation of p38 MAPK and PKC occurs

in parallel with the MEK / Erk cascade. Gliomas express high levels of epidermal growth factor receptor (EGFR) which are implicated in the mitogenic and anti-apoptotic functions. Activation of p38 MAPK by EGFR, which has been shown in other systems [16], might also be the mechanism for p38 MAPK regulated HGF secretion in these cells however, this is beyond the scope of the present study. Scarcely available reports show that these two mechanistically distinct pathways (i.e. MEK / Erk cascade and p38 MAPK) together regulate secretion and production of prostaglandin E2 release from human bronchial epithelial cells in response to proinflammatory cytokines [28], tumor necrosis factor in murine macrophage cell lines, and PGE-2 synthesis by J774 monocyte / macrophage cell lines [3]. In addition, a recent report showing HGFinduced proliferation of H441 cells from lung adenocarcinoma is regulated both by p42 / p44 and p38 MAPKs [4]. It is possible that as in H441 cells the p38 MAPK pathway might also be activated by HGF in glioma cells simultaneously in parallel with p42 / 44 MAPK which would imply a further aggravation of the feed forward mechanism of HGF action. Given such regulation of HGF secretion by glioma cells, a recently available systemically applicable p38 MAPK inhibitor [29] along with strategy to down regulate PKC activity might be a potential adjuvant therapy for gliomas. Activating PKC by PMA stimulates HGF secretion by acting at the transcriptional level since inhibitor of transcription, actinomycin D completely abolished such stimulation. Multiple mRNA species transcribed from a single HGF gene encode at least three distinct proteins: the full-length HGF protein (encoded by 6.0-kb transcript) and two truncated HGF isoforms that encompass the N-terminal (N) domain through kringle 1 (NK1, encoded by the 3.0 Kb transcript) and kringle 2 (NK2, encoded by the 1.5 Kb transcript), respectively [11]. NK1 and NK2 are natural splice variants of HGF; all interact with the receptor, c-Met. These variants have been reported to have agonistic or antagonistic activity compared to HGF depending on the target cell studied and the presence of full length HGF in assays of cell proliferation and motility [31]. We previously observed expression of all three HGF transcripts in U87 cells however, of the two splice variants observed, 1.5-kb transcript was about 8-fold more abundant than the 3.0-kb transcript [10]. Although the functions of these splice variants have not yet been determined in astrocyte-derived cells, our results show that PKC activation by PMA upregulates all three HGF transcripts. In contrast, U0126 downregulated all three transcripts while PD98059 had no significant effect and SB203580 downregulated 3.0- and 1.5-kb transcripts more than the 6.0-kb mRNA. Since all three HGF mRNAs are derived from single gene therefore, MEK and p38 MAPK likely regulate its expression both at transcriptional and post-transcriptional levels, an issue which is not within the purview of this study. Taken together, our data show that HGF secretion by a

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well established human astrocytoma cell line, U87, is regulated by the Raf–PKC–MEK pathway with the likely involvement of PKCa in the process as well as by p38 MAPK cascade. Such an effect is exerted both at the secretion and transcript levels. Since such signaling cascade is used by c-Met mediated signaling and induction of c-Met expression in glioma cells, our results reveal that this in turn results in increased secretion of HGF as well resulting in ‘vicious’ feed forward effects of HGF actions. However, p38 MAPK, exerting its action in the secretion and expression of HGF requires detailed studies.

Acknowledgements Generous grant supports by Pfizer /American Federation for Aging Research (N.C.) for this work was provided by the National Institutes of Health (DK41415, DK48330 and DK52005 to E.M.B.) and by the St. Giles Foundation (to E.M.B.).

References [1] R. Abounader, S. Ranganathan, B.Y. Kim, C. Nichols, J. Laterra, Signaling pathways in the induction of c-met receptor expression by its ligand scatter factor / hepatocyte growth factor in human glioblastoma, J. Neurochem. 76 (2001) 1497–1508. [2] R. Abounader, S. Ranganathan, B. Lal, K. Fielding, A. Book, H. Dietz, P. Burger, J. Laterra, Reversion of human glioblastoma malignancy by U1 small nuclear RNA / ribozyme targeting of scatter factor / hepatocyte growth factor and c-met expression, J. Natl. Cancer Inst. 91 (1999) 1548–1556. [3] S.J. Ajizian, B.K. English, E.A. Meals, Specific inhibitors of p38 and extracellular signal-regulated kinase mitogen-activated protein kinase pathways block inducible nitric oxide synthase and tumor necrosis factor accumulation in murine macrophages stimulated with lipopolysaccharide and interferon-g, J. Infect. Dis. 179 (1999) 939– 944. [4] V. Awasthi, R.J. King, PKC, p42 / p44 MAPK, and p38 MAPK are required for HGF-induced proliferation of H441 cells, Am. J. Physiol. Lung Cell Mol. Physiol. 279 (2000) L942–949. [5] G.H. Baltuch, W.T. Couldwell, J.G. Villemure, V.W. Yong, Protein kinase C inhibitors suppress cell growth in established and lowpassage glioma cell lines. A comparison between stuarosporine and tamoxifen, Neurosurgery 33 (1993) 495–501. [6] L. Beviglia, K. Matsumoto, C.S. Lin, B.L. Ziober, R.H. Kramer, Expression of the c-Met / HGF receptor in human breast carcinoma: correlation with tumor progression, Int. J. Cancer 74 (1997) 301– 309. [7] A.A. Book, S. Ranganathan, R. Abounader, E. Rosen, J. Laterra, Scatter factor / hepatocyte growth factor gene transfer increases rat blood–glioma barrier permeability, Brain Res. 833 (1999) 173–180. [8] M. Bredel, I.F. Pollack, The role of protein kinase C (PKC) in the evolution and proliferation of malignant gliomas, and the application of PKC inhibition as a novel approach to anti-glioma therapy, Acta Neurochir. (Wien) 139 (1997) 1000–1013. [9] B. Cao, Y. Su, M. Oskarsson, P. Zhao, E.J. Kort, R.J. Fisher, L.M. Wang, G.F. Vande Woude, Neutralizing monoclonal antibodies to hepatocyte growth factor / scatter factor (HGF / SF) display antitumor activity in animal models, Proc. Natl. Acad. Sci. USA 98 (2001) 7443–7448.

81

[10] N. Chattopadhyay, R.R. Butters, E.M. Brown, Agonists of the retinoic acid- and retinoid X-receptors inhibit hepatocyte growth factor secretion and expression in U87 human astrocytoma cells, Brain Res. Mol. Brain Res. 87 (2001) 100–108. [11] D.Y. Chigradze, J. Hepple, R.A. Byrd, R. Sowdhamini, T.L. Blundell, E. Gherardi, Insights into the structure of hepatocyte growth factor / scatter factor (HGF / SF) and implications for receptor activation, FEBS Lett. 430 (1998) 126–129. [12] E. Gohda, H. Kataoka, H. Tsubouchi, Y. Daikilara, I. Yamamoto, Phorbol ester-induced secretion of human hepatocyte growth factor by human skin fibroblasts and its inhibition by dexamethasone, FEBS Lett. 301 (1992) 107–110. [13] M. Gschwendt, S. Dieterich, J. Rennecke, W. Kittstein, H.J. Mueller, F.J. Johannes, Inhibition of protein kinase C m by various inhibitors. Differentiation from various protein kinase c isozymes, FEBS Lett. 392 (1996) 77–80. [14] M. Hamanoue, N. Takemoto, K. Matsumoto, T. Nakamura, K. Nakajima, S. Kohsaka, Neurotrophic effect of hepatocyte growth factor on central nervous system neurons in vitro, J. Neurosci. Res. 43 (1996) 554–564. [15] M. Jeffers, S. Rong, G.F. Vande Woude, Enhanced tumorigenicity and invasionmetastasis by hepatocyte growth factor / scatter factormet signalling in human cells concomitant with induction of the urokinase proteolysis network, Mol. Cell Biol. 16 (1996) 1115– 1125. [16] Y. Kanda, K. Mizuno, Y. Kuroki, Y. Watanabe, Thrombin-induced p38 mitogenactivated protein kinase activation is mediated by epidermal growth factor receptor transactivation pathway, Br. J. Pharmacol. 132 (2001) 1657–1664. [17] A. Karihaloo, D.A. O’Rourke, C. Nickel, K. Spokes, L.G. Cantley, Differential MAPK pathways utilized for HGF- and EGF-dependent renal epithelial morphogenesis, J. Biol. Chem. 276 (2001) 9166– 9173. [18] O. Kifor, R.J. MacLeod, R. Diaz, M. Bai, T. Yamaguchi, T. Yao, I. Kifor, E.M. Brown, Regulation of MAP kinase by calcium-sensing receptor in bovine parathyroid and CaR-transfected HEK293 cells, Am. J. Physiol. Renal Physiol. 280 (2001) F291–F302. [19] K. Lamszus, J. Laterra, M. Westphal, E.M. Rosen, Scatter factor / hepatocyte growth factor (SF / HGF) content and function in human gliomas, Int. J. Dev. Neurosci. 17 (1999) 517–530. [20] K.X. Liu, Y. Kato, I. Kino, T. Nakamura, Y. Sugiyama, Ligandinduced downregulation of receptor-mediated clearance of hepatocyte growth factor in rats, Am. J. Physiol. 275 (1998) E835–842. [21] R. Mandil, E. Ashkenazi, M. Blass, I. Kronfeld, G. Kazimirsky, G. Rosenthal, F. Umansky, P.S. Lorenzo, P.M. Blumberg, C. Brodie, Protein kinase Ca and protein kinase Cd play opposite roles in the proliferation and apoptosis of glioma cells, Cancer Res. 61 (2001) 4612–4619. [22] T. Matsunaga, E. Gohda, T. Takebe, Y.L. Wu, M. Iwao, H. Kataok, I. Yamamoto, Expression of hepatocyte growth factor is upregulated through activation of a cAMP-mediated pathway, Exp. Cell Res. 10 (1994) 326–335. [23] T. Moriyama, H. Kataoka, M. Koono, S. Wakisaka, Expression of hepatocyte growth factor / scatter factor and its receptor c-Met in brain tumors: evidence for a role in progression of astrocytic tumors, Int. J. Mol. Med. 3 (1999) 531–536. [24] K. Nabeshima, Y. Shimao, S. Sato, H. Kataoka, T. Moriyama, H. Kawano, S. Wakisaka, M. Koono, Expression of c-Met correlates with grade of malignancy in human astrocytic tumours: an immunohistochemical study, Histopathology 31 (1997) 436–443. [25] K. Nakajima, S. Honda, Y. Tohyama, Y. Imai, S. Kohsaka, T. Kurihara, Neurotrophin secretion from cultured microglia, J. Neurosci. Res. 65 (2001) 322–331. [26] N. Nakano, R. Morishita, A. Moriguchi, Y. Nakamura, S.I. Hayashi, M. Aoki, I. Kida, K. Matsumoto, T. Nakamura, J. Higaki, T. Ogihara, Negative regulation of local hepatocyte growth factor expression by angiotensin II and transforming growth factor-b in

82

[27]

[28]

[29]

[30]

N. Chattopadhyay et al. / Molecular Brain Research 102 (2002) 73 – 82 blood vessels: potential role of HGF in cardiovascular disease, Hypertension 32 (1998) 444–451. K. Nasu, T. Sugano, N. Matsui, H. Narahara, Y. Kawano, I. Miyakawa, Expression of hepatocyte growth factor in cultured human endometrial stromal cells is induced through a protein kinase C-dependent pathway, Biol. Reprod. 60 (1999) 1183–1187. R. Newton, L. Cambridge, L.A. Hart, D.A. Stevens, M.A. Lindsay, P.J. Barnes, The MAP kinase inhibitors, PD098059, UO126 and SB203580, inhibit IL-1bdependent PGE(2) release via mechanistically distinct processes, Br. J. Pharmacol. 130 (2000) 1353–1361. J.A. Nick, S.K. Young, K.K. Brown, N.J. Avdi, P.G. Arndt, B.T. Suratt, M.S. Janes, P.M. Henson, G.S. Worthen, Role of p38 mitogen-activated protein kinase in a murine model of pulmonary inflammation, J. Immunol. 164 (2000) 2151–2159. R. Paumelle, D. Tulasne, C. Leroy, J. Coll, B. Vandenbunder, V. Fafeur, Sequential activation of ERK and repression of JNK by

[31]

[32]

[33]

[34]

scatter factor / hepatocyte growth factor in madin-darby canine kidney epithelial cells, Mol. Biol. Cell 11 (2000) 3751–3763. M.C. Stella, P.M. Comoglio, HGF: a multifunctional growth factor controlling cell scattering, Int. J. Biochem. Cell Biol. 31 (1999) 1357–1362. J.H. Uhm, N.P. Dooley, J.G. Villemure, V.W. Yong, Glioma invasion in vitro: regulation by metalloprotease-2 and protein kinase C, Clin. Exp. Metastasis 14 (1996) 421–433. W.C. Welch, P.L. Kornblith, G.K. Michalopoulos, B.E. Petersen, A. Beedle, S.M. Gollin, R.H. Goldfarb, Hepatocyte growth factor (HGF) and receptor (c-met) in normal and malignant astrocytic cells, Anticancer Res. 19 (1999) 1635–1640. S. Yi, J.R. Chen, J. Viallet, R.H. Schwall, T. Nakamura, M.S. Tsao, Paracrine effects of hepatocyte growth factor / scatter factor on nonsmall-cell lung carcinoma cell lines, Br. J. Cancer 77 (1998) 2162–2170.