c-Jun Amino-Terminal Kinase and Mitogen Activated Protein Kinase 1/2 Mediate Hepatocyte Growth Factor-Induced Migration of Brain Endothelial Cells

Abstract

Hepatocyte growth factor (HGF) influences several components of the angiogenic response, including endothelial cell migration. While recent studies indicate a crucial role of HGF in brain angiogenesis, the signaling pathways that regulate brain endothelial cell migration by HGF remain largely uncharacterized. Herein, we report that HGF stimulates human brain microvascular endothelial cell (HBMEC) migration in a dose- and time-dependent manner. Challenge of HBMECs with HGF activates the c-jun amino-terminal kinase (JNK), increases phosphorylation of the proline-rich tyrosine kinase 2 (Pyk-2) at Tyr402, and activates c-Src. Inhibition of JNK by SP600125 or expression of a dominant negative JNK1 construct abrogates the migratory response of HBMECs to HGF. Treatment of HBMECs with the Src inhibitor PP2 markedly decreases HGF-stimulated JNK activation and migration. Moreover, expression of a mutant Pyk-2 construct prevents HGF-induced Pyk-2 phosphorylation at Tyr402 and stimulation of HBMEC migration. Activation of the extracellular signal regulated kinase (ERK) pathway was also examined. Stimulation of HBMECs by HGF leads to rapid activation of ERK1/2, phosphorylation of Raf-1 at Ser338 and Tyr340/341, and MEK1/2 at Ser222. Inhibition of ERK activation by UO126 and PD98059 markedly decreases HGF-stimulated HBMEC migration. HGF also activates AKT, while inhibition of AKT by LY294002 induces a modest decrease of HGF-induced HBMEC migration. These results highlight a model whereby JNK and ERK play a critical role in regulation of brain endothelial cell migration by HGF.

Keywords: HGF, Brain, Endothelial cells, Migration, JNK, ERK, Pyk-2, Src, Raf, MEK

Introduction

Endothelial cell migration is a complex process critical during angiogenesis. This process is regulated by highly orchestrated molecular events allowing endothelial cells to leave the vascular bed, infiltrate the basement membrane, and reach the interstitial space where they differentiate into mature blood vessels. The ability of endothelial cells to migrate in response to external stimuli is essential for physiological and pathological angiogenesis. Conversely, inappropriate migration can compromise new blood vessel formation and vascular stability, increasing vascular permeability and tissue damage. Understanding intracellular events orchestrating endothelial cell migration in response to extracellular signals is vital for therapeutic modulation of angiogenesis.

Hepatocyte growth factor (HGF), also known as scatter factor, is a multifunctional growth factor that potently stimulates migration of various cell types, including vascular endothelial cells. HGF also regulates other angiogenic responses such as proliferation and differentiation. Evidence indicates HGF stimulates angiogenesis during pathological conditions like inflammation, ischemia, and tumor growth. HGF acts directly on endothelial cells by binding to c-Met, a tyrosine kinase receptor composed of a 50-kDa extracellular α subunit and a 145-kDa transmembrane β subunit containing multiple tyrosine phosphorylation sites linking c-Met to downstream signaling pathways. Activation of phosphatidylinositol-3-kinase regulates migratory response of human umbilical vein endothelial cells to HGF. HGF induces cytoskeletal-localized events, including phosphorylation of focal adhesion kinase and its association with paxillin, reducing permeability of pulmonary vascular endothelial cells. Recent studies suggest a critical role for HGF in brain angiogenesis; however, mechanisms by which HGF influences brain endothelial cell angiogenic responses remain largely uncharacterized.

Members of the mitogen-activated kinase (MAPK) family, including c-jun amino-terminal kinases (JNKs) and extracellular signal regulated kinases (ERK), regulate diverse cellular functions including motility. Activation of JNK is involved in neuronal cell migration and tumor-associated angiogenesis. The role of JNK in brain endothelial cell migration is not established. This study dissects signaling pathways regulating brain endothelial cell migration by HGF, demonstrating that HGF activates JNK, ERK, and AKT, and that inhibition of JNK and ERK markedly decreases HGF-stimulated brain endothelial cell migration, indicating a critical role for these kinases.

Materials and Methods
Reagents

Endothelial cell medium, attachment factor, and growth factor supplements were obtained from Cell Systems (Kirkland, WA). HGF was purchased from R&D Systems (Minneapolis, MN). Fetal bovine serum from Hyclone (Logan, UT). M199 medium from Gibco (Grand Island, NY). Antibodies for myelin basic protein, enolase, fibronectin, β-actin from Sigma (St. Louis, MO). Phospho-ERK (Thr202/Tyr204), Flag antibodies, and chemiluminescence systems from Santa Cruz Biotechnology. Phospho-Raf-1 (Ser338, Tyr340/341) antibodies from Upstate Biotechnology. Phospho-MEK1/2 (Ser222), phospho-Pyk-2 (Tyr402) antibodies from Biosource. c-Met, phospho-c-Met (Tyr1234/1235), phospho-AKT (Ser473) antibodies from Cell Signaling. Radioactive ATP from Amersham Life Science. Inhibitors SP600125, U0126, PP2, PD98059, LY294002 from Calbiochem. Protein G/A agarose beads and pp60src antibodies from Oncogene Science. Transwell chambers from Costar/Corning. Lipofectamine/Plus reagent, gels, and markers from Invitrogen. X-ray films from Phenix Research Products.

Brain Endothelial Cells

Primary human brain microvascular endothelial cells (HBMECs) were purchased from Cell Systems. Cells maintained on attachment factor-coated flasks at 37°C, 5% CO₂, in medium with 10% fetal bovine serum and endothelial growth factor supplement. Used between passages 2 and 12.

Plasmids

pcDNA3-FLAG-JNK1 (APF) vector containing catalytically inactive dominant negative JNK1 mutant provided by Dr. R.J. David. pME18s-FLAG-Pyk-2 (Y402F) vector with mutated autophosphorylation site Tyr402 to alanine provided by Dr. T. Katagiri.

Migration Assays

HBMEC migration assessed using Boyden chamber (Transwell) assay. Cells suspended in M199 with 0.1% BSA, plated on fibronectin-coated filters, placed in upper chamber. Lower chamber contained serum-free medium with HGF or vehicle. Cells allowed to migrate at 37°C, 5% CO₂ for indicated times. Non-migrated cells removed; migrated cells fixed, stained, and counted in 5 random fields at 20x magnification. Pretreatment with kinase inhibitors SP600125, PP2, PD98059, U0126, LY294002 done where indicated.

Western Blot Analyses

HBMECs lysed in appropriate buffers for detection of ERK, Pyk-2, Raf-1, MEK1/2 phosphorylation. Proteins separated by gel electrophoresis, transferred to nitrocellulose membranes, immunoblotted with specific antibodies. Visualization by enhanced chemiluminescence.

JNK Assay

JNK activity measured by solid-phase kinase assay using GST-c-jun fusion protein substrate. Phosphorylated GST-c-jun detected by autoradiography and quantified by densitometry.

ERK Immunocomplex Kinase Assay

ERK immunoprecipitated from cell lysates and kinase activity measured using myelin basic protein (MBP) as substrate with radioactive ATP incorporation.

Src Immunocomplex Kinase Assay

Src kinase activity measured by immunoprecipitation and kinase assay using enolase as substrate.

Transient Transfection Assays

HBMECs transfected with Flag-Pyk-2Y402F or dominant negative JNK1 constructs using Lipofectamine/Plus reagent. Transfection efficiency monitored by EGFP expression. Migration assays performed 48 hours post-transfection.

Protein Determination

Protein concentration determined by Bradford assay.

Statistical Analyses

Data expressed as mean ± SE. Statistical significance assessed by Student’s t-test; P ≤ 0.05 considered significant.

Results
HGF Stimulates Migration of HBMECs

HGF induced a significant, dose-dependent increase in migration of HBMECs. At 10 ng/ml HGF, migration increased 1.8-fold compared to vehicle; at 25 and 50 ng/ml, migration increased 2.5- and 4.3-fold, respectively, reaching a plateau at 100 ng/ml. Migration was time-dependent, peaking at 8 hours of exposure. Migration required fibronectin-coated filters; no migration occurred on uncoated filters. Human aortic endothelial cells also migrated in response to HGF.

c-Met Phosphorylation in HBMECs

Western blot showed time-dependent phosphorylation of c-Met at Tyr1234/1235 upon HGF stimulation (50 ng/ml), detectable at 5, 15, and 30 minutes, with total c-Met protein levels unchanged.

Activation of JNK by HGF

JNK activity, measured by GST-c-jun phosphorylation, increased in a time-dependent manner after HGF stimulation, peaking at 30 minutes and sustained up to 120 minutes. No activation was observed with mutated GST-c-jun substrate. JNK inhibitor SP600125 completely blocked HGF-induced JNK activation.

Involvement of Src and Pyk-2 in HGF-Induced JNK Activation

HGF induced time-dependent Src activation, detected by kinase assay using enolase substrate, with phosphorylation evident at 5 minutes. Src activation preceded JNK activation. Src inhibitor PP2 markedly reduced HGF-induced JNK activation and Src activity. HGF also induced Pyk-2 phosphorylation at Tyr402, peaking at 15-30 minutes and sustained up to 60 minutes.

Inhibition of JNK, Src, and Pyk-2 Impairs HGF-Induced Migration

Pretreatment with JNK inhibitor SP600125 significantly reduced HGF-stimulated migration of HBMECs. Expression of dominant negative JNK1 similarly abrogated migration. Src inhibitor PP2 also significantly inhibited migration. Expression of mutant Pyk-2 (Y402F) construct prevented HGF-induced Pyk-2 phosphorylation and markedly reduced migration.

Discussion

This study demonstrates that HGF stimulates migration of human brain microvascular endothelial cells via activation of c-Met receptor and downstream signaling pathways involving JNK, ERK, Src, and Pyk-2 kinases. The activation of JNK and ERK is critical for the migratory response, as their inhibition significantly impairs migration. Src kinase acts upstream of JNK, and Pyk-2 phosphorylation is essential for HGF-induced migration. These findings elucidate key intracellular signaling events regulating brain endothelial cell migration during angiogenesis and suggest potential therapeutic targets for modulating UNC2250 angiogenic responses in brain pathologies.