Background
Vascular endothelial growth factor (VEGF) is essential for many angiogenic processes in both normal and pathological conditions [
1,
2]. The biological activities of VEGF are mediated mainly through two tyrosine kinase receptors, fms-like tyrosine kinase-1(Flt-1) and fetal liver kinase-1/kinase-insert domain receptor (Flk-1/KDR), whose expressions are mainly restricted to endothelial cells [
1,
2]. These receptors are membrane-spanning receptor tyrosine kinases that bind VEGF with high affinity. Flk-1 is now considered to be the main receptor involved in endothelial cell proliferation, migration, survival, and the dominant form in pulmonary vascular system [
2,
3]. In contrast, Flt-1 has a decoy effect on VEGF signaling, possibly with variations related to the vascular bed type [
2]. Both Flt-1- and Flk-1-deficient mice die
in utero between embryonic days (E) 8.5 and E 9.5 but have different phenotypes. Flt-1-deficient embryos showed an overgrowth of endothelial cells, disorganization of blood vessels [
4], and normal vascular development [
5], suggesting that the Flt-1 tyrosine kinase is not necessary for vasculogenesis during development. On the other hand, Flk-1-deficient mice lack both mature endothelial and hematopoietic cells, indicating that Flk-1 is crucial for vascular development of both endothelial and hematopoietic precursors [
6]. During later stages of embryonic development, Flk-1 is highly expressed on endothelial cells, but is down-regulated in most hematopoietic cells [
7]. In the adult, the expression level of Flk-1 is low, restricted to endothelial cells and transiently upregulated during angiogenesis [
8].
In vitro studies have shown that Flk-1 expression is temporally regulated by several growth factors [
2] and by shear stress [
9]. For example, both basic fibroblast growth factor (bFGF) and tumor necrosis factor-α(TNF-α) have been shown to induce expression of the endogenous Flk-1 gene and increase Flk-1 upstream promoter activity in cultured endothelial cells [
10,
11]. It has been known that shear stress induces Flk-1 expression through the CT-rich Sp1 binding site within Flk-1 promoter [
9]. Incubation of cells with the multifunctional angiogenic cytokine transforming growth factor β1 (TGF-β1) results in a rapid and marked decrease in Flk-1 expression levels and cell surface
125I-VEGF binding capacity [
12]. Because expression of Flk-1 is highly restricted to endothelial cells and tightly controlled during angiogenesis, further understanding of the potential factors that regulate the expression of Flk-1 in the lung and endothelium would provide general insights into the mechanisms of vascular development in health and diseases in the pulmonary circulation.
Hypoxia-induced mitogenic factor (HIMF) is a secreted protein from airway epithelial cells and alveolar type II cells and it is originally discovered in a mouse model of hypoxia-induced pulmonary hypertension [
13]. Subsequent studies showed that HIMF is a lung-specific growth factor participating in lung cell proliferation and modulation of compensatory lung growth [
13,
14]. HIMF possesses an angiogenic function that promotes vascular tubule formation in a matrigel plug model [
13], and is developmentally regulated and exhibits antiapoptotic functions [
15]. Moreover, our recent studies have indicated that HIMF modulates surfactant protein B and C expression in lung epithelial cells [
16]. We have also established that HIMF promotes VEGF production in alveolar type II cells, indicating HIMF may play critical roles in angiogenesis in the pulmonary system [
17]. In this study, we further investigated the molecular mechanisms of HIMF on Flk-1 expression in mouse lungs, and in cultured endothelial cells. The results showed that HIMF promotes expression of Flk-1 via activation of PI-3 kinase/Akt and NF-κB signaling pathways.
Materials and methods
Animal experiments
Adult male C57Bl/6 mice (10–12 weeks old) were obtained from Jackson Laboratories (Bar Harbor, ME). Recombinant HIMF protein was produced in TREx 293 cells and purified as previously described [
13]. Intratracheal instillation of HIMF protein or bovine serum albumin (BSA, Sigma, St. Louis, MO) were performed as previously reported [
14,
16]. All experiments followed the protocols approved by the Animal Care and Use Committee of Saint Louis University.
Immunohistochemical and immunofluorescent staining for Flk-1
Lung samples were processed and immunostained as previously described [
13,
15,
16]. Briefly, the sections were incubated for 1 hour with anti-Flk-1 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 1:200 dilution) followed by a 2-hour incubation with goat anti-rabbit antibodies conjugated with HRP or FITC (1: 400 dilution, Bio-Rad, Hercules, CA). For immunofluorescent staining, the cells were examined directly under a fluorescent microscope after secondary antibody incubation and washing. For immunohistochemical staining, DAB substrate (Dako, Carpinteria, CA) was used to generate dark brown precipitate in the cells of the tissues. The images were taken with a Sony color digital DXC-S500 camera (Sony Electronics, Oradell, NJ), using Image Pro-Express software (Media Cybernetics, Silver Spring, MD).
Western blot for HIMF, Flk-1, VEGF, and GAPDH
Tissue collection, homogenization, and protein electrophoresis were performed as previously described [
14‐
16]. Protein (50 μg) or 40 μl of medium supernatant (for HIMF expression assay in cultured cells) from each sample was subjected to 4–20% pre-cast polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA). HIMF, Flk-1, VEGF, and GAPDH were detected with 1:1000, 1:500, 1:500 and 1:1000 dilutions of antibodies, respectively, followed by 1:3000 dilution of goat anti-rabbit HRP-labeled antibody (Bio-Rad). ECL substrate kit (Amersham, Piscataway, NJ) was used for the chemiluminscent detection of the signals with autoradiography film (Amersham).
Real-time RT-PCR for HIMF, Flk-1, and VEGF
Total RNA was isolated with RNeasy Mini Kit (Qiagen Inc., Valencia, CA). The reverse transcription reactions were conducted with Transcriptor First Strand cDNA Synthesis Kit (Roche, Indianapolis, IN). Real-time PCR with SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) was performed using ABI Prism 7700 Sequence Detector (Applied Biosystems). The PCR primers were the following: for mouse HIMF 5'-ATGAA GACTACAACTTGTTCCC-3' (positions 104 to 125 of second exon) and 5'-TTAGGACAGT TGGCAGCAGCG-3' (positions 419 to 439 of fourth exon) amplifying a 336-bp fragment; for mouse Flk-1 5'-GCATCACCAGCAGCCAGAG-3' and 5'-GGGCCATCCACTTCAAAGG-3' amplifying a 327-bp fragment between positions 3095 and 3421; for mouse VEGF 5'-TGGAT GTCTACCAGCGAAGC-3' and 5'-ACAAGGCTCACAGTGATTTT-3' amplifying a 308-bp fragment between positions 522 and 829; for mouse GAPDH, 5'-GCCAAGGTCATCCATGA CAACTTTGG-3' and 5'-GCCTGCTTCACCACCTTCTTGATGTC-3' amplifying a 314-bp fragment between positions 532 and 845.
Cell culture and stimulation with HIMF
SVEC 4–10, an SV40-transformed murine endothelial cell line [
18], was obtained from the ATCC (CRL-2181) and grown in Dulbecco's Minimal Eagles Medium (DMEM, Gibco Laboratories, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, Gibco), penicillin (100 U/ml) and streptomycin (100 μg/ml). Cells were maintained at 37°C in a humidified atmosphere of 5% CO
2. After the cells reached 80–90% confluency, the cells were fed with a medium supplemented with 0.1% FBS and 2 mmol/L L-glutamine. Thirty-three hours later, cells were incubated in serum-free DMEM for 4 h, and pretreated with LY294002, SB203580, PD98059 or U0126 (Calbiochem, La Jolla, CA) as indicated, then stimulated with different concentrations of HIMF protein for specified periods, with or without Actinomycin D (5 μg/ml, Sigma).
Transfection and stable cell lines
HIMF cDNA vector, dominant-negative mutants of IKKα [IKKα (K44A)], IKKβ [IKKβ (K44A)], IκBα super-repressor [IκBα (S32A/S36A)] and phosphatidylinositol 3-kinase (PI-3K) dominant negative mutant (Δp85) were previously described [
16,
19,
20]. HIMF cDNA or dominant-negative mutants were transfected into SVEC 4–10 cells with Lipofectamine 2000 (Life Technologies, Inc., Gaithersburg, MD). Stable cell lines, SVEC-HIMF, and their transfection control (vector only) cells SVEC-Zeo, were selected with Zeocin (400 μg/ml). HIMF expression was validated by Western blot and real-time RT-PCR analyses.
Dual-luciferase reporter assay for Flk-1 and NF-κB
Mouse Flk-1 5'-flanking regions (-258/+299, -96/+299, -71/+299, and -36/+299 bp; GenBank accession No. AF153057) were amplified by PCR from genomic DNA obtained from SVEC 4–10 and subcloned into the KpnI-HindIII site of pGL3-Basic (Invitrogen, Carlsbad, CA), a firefly luciferase reporter vector. Mutagenesis and deletion of NF-κB binding site within Flk-1 promoter were performed using the GeneTailor Site-Directed Mutagenesis System (Invitrogen). Mutation and deletion oligonucleotides for NF-κB binding site were designed as follows: forward mutation 5'-TATCGATAGGTACCGGACGCACCGAGTCCCCACCCCT, forward deletion 5'-TATCGATAGGTACCGGACGCACCCCACCCCT, reverse 5'-TGCGTC CGGTACCTATCGATAGAG AAATGTT. The DNA constructs were verified by sequence analysis. The NF-κB firefly luciferase reporter vector, pNFκB-Luc (Stratagene, La Jolla, CA), is designed to measure the binding of transcription factors to the κ enhancer. It contains five tandem repeats of NF-κB binding sites (TGGGGACTTTCCGC) as promoters upstream of the luciferase transcription start site in the vector. The expression of luciferase gene in the reporter plasmid is controlled by these NF-κB binding sequences. Only when there is activated NF-κB in the nucleus (translocated NF-κB), the luciferase transcription and translation start. By measuring the luciferase activity in the transfected cell lysats, it provides an indirect evidence of NF-κB activation in the nucleus. Cells were co-transfected with each reporter construct and the renilla luciferase vector pRL-TK (Promega, Madison, WI), with or without HIMF protein stimulation, and then treated with passive lysis buffer according to the dual-luciferase assay manual (Promega). The luciferase activity was measured with a luminometer (Lumat LB9507, Berthold Tech., Bad Wildbad, Germany). The firefly luciferase signal was normalized to the renilla luciferase signal for each individual analysis to eliminate the variations of transfection efficiencies.
Phosphorylation assay for IKK, IκBα, Akt, and MAPK
SVEC 4–10 cells were treated with HIMF as described above. Protein (50 μg) from each sample was subjected to 4–20% pre-cast polyacrylamide gel (Bio-Rad) electrophoresis and transferred to nitrocellulose membranes (Bio-Rad), and then probed with rabbit anti-mouse antibodies against phospho-specific and non-phosphorylated IKK, IκBα, Akt, ERK1/2, p38 kinase, and JNK mitogen-activated protein kinase (MAPK) (1:500 dilutions, Santa Cruz Biotechnology), followed by 1:3000 dilution of goat anti-rabbit HRP-labeled antibody (Bio-Rad). ECL substrate kit (Amersham) was used for the chemiluminscent detection of the signals with autoradiography film (Amersham).
Statistical analysis
Unless otherwise stated, all data were shown as mean ± standard error of the mean (SEM). Statistical significance (P < 0.05) was determined by t test or analysis of variance (ANOVA) followed by assessment of differences using SigmaStat 2.03 software (Jandel, Erkrath, Germany).
Discussion
Endothelial cell tyrosine kinase receptors are of fundamental importance in transmission of both differentiation and angiogenic signals from the extracellular environment to the endothelium. Five endothelial cell-specific tyrosine kinase receptors, each of which has a specific role in blood vessel formation, have been identified. These include Tie-1, Tie-2 (also known as Tek), Flt-1, Flt-4, and Flk-1/KDR [
24]. While the ligands for Tie-1 and Tie-2 have not yet been identified, Flk-1 and Flt-1 are receptors for VEGF [
1,
2], an endothelial cell-specific mitogen whose importance in both physiological and pathological angiogenesis is well established [
1,
2]. One of the important functions of Flk-1 is the stimulation of vascular endothelial cell survival, growth, and promotion of angiogenesis. In the lung, Flk-1 also plays central roles in alveolar formation. It is worthy to note that coordinated alveolar development and angiogenesis are critical for lung maturation as a gas exchange organ [
25‐
27]. Inhibition of Flk-1 by specific inhibitor SU5416 resulted in decreased alveolarization in developing lung [
25,
27], emphysema [
26], and severe hypoxic pulmonary hypertension in adult [
28], indicating the fundamental roles of Flk-1 in lung development and maintenance of homeostasis in the pulmonary circulation. Although VEGF receptors have been characterized extensively at the level of expression, high affinity VEGF binding, phosphorylation, and other signal transduction properties, very little is known about factors which regulate its expression in endothelial cells [
2,
24]. An understanding of the mechanisms that underlie the transcriptional regulation of the Flk-1/KDR gene might provide important information about the molecular basis of endothelial cell differentiation, vascular development, and further assist our understanding in pulmonary angiogenesis. In the present study, we found that HIMF enhances Flk-1 expression in mouse lung tissues and endothelial cell line by activation of the PI-3K/Akt-NF-κB signaling pathway. In addition, our recent studies indicated that VEGF expression in lung epithelial cells can be induced by HIMF via the same signaling pathway [
17], suggesting that additional transcription factors are involved in HIMF-mediated cell type-specific modulation of VEGF and its receptor Flk-1. Furthermore, HIMF, as it has dual function in upregulation of VEGF in epithelial cells and its receptor in endothelial cells, may serve as a coordinator in the control of pulmonary development and maturation, which certainly warrants further investigation.
Both mouse (Flk-1) and human (KDR) genes reveal a class II promoter structure, characterized by the absence of a TATA box and by the presence of several conserved
cis-regulatory elements, including Sp1-, AP-2-, NF-κB-, and GATA-binding sites [
22,
29]. The upstream NF-κB site has been demonstrated to be the important one in mediating basal expression of the Flk-1/KDR promoter [
30]. In addition, an overlapping palindromic GATA sequence plays a role in mediating constitutive promoter activity [
30]. It has been previously shown that TNF-α activates NF-κB function to enhance human KDR expression [
11], while TGF-β inhibits Flk-1/KDR expression through a mechanism that involves reduced binding of GATA-2 to a palindromic GATA site in the 5'-UTR [
30]. These findings indicate that the binding of specific sets of transcription factors to the promoter region is necessary to modulate the expression of Flk-1 in response to different stimuli. In the current study, we found that HIMF protein upregulated Flk-1 expression by enhancing the Flk-1 promoter activity, rather than stabilizing Flk-1 mRNA posttranscriptionally. Moreover, the NF-κB activity was induced by HIMF administration or HIMF overexpression. Impairing NF-κB binding to the Flk-1 promoter via site-directed mutation or deletion abolishes HIMF-induced Flk-1 transcription, demonstrating a critical role of NF-κB in HIMF-mediated Flk-1 upregulation. In addition, we also found that deletion of binding sites for transcription factors E-box, Sp-1, and AP-2 partially attenuated HIMF-induced Flk-1 transcription, indicating that these transcription factors in the Flk-1 promoter also participate in HIMF-induced Flk-1 upregulation. The activation and interaction of these transcription factors and their correlation with NF-κB activity warrant our further study in the future.
The stimulating effects of HIMF on Flk-1 upregulation in SVEC 4–10 cells can only maintain for 24 hours. The dramatic decrease of NF-κB activity at 48 hour time point might be a result of HIMF degradation because we only administered the HIMF protein at the beginning of the experiment. These effects parallel with the activation of IKK and increased PI-3K activities as we showed that blocking IKK or PI-3K abolished HIMF-induced NF-κB activity and decreased Flk-1 mRNA production. The quick degradation or lost activity of HIMF further indicates that HIMF is a cytokine-like molecule and an early response gene to hypoxia, inflammation or other stress related stimuli [
13,
14].
NF-κB is composed of heterodimers of DNA-binding subunits (p50 and p52) and subunits with transcriptional activity (RelA, RelB, or c-Rel) [
31]. In unstimulated cells, binary complexes of these subunits are restricted to the cytoplasm by interaction with members of a family of inhibitory proteins, inhibitors of κB (IκBs) [
32]. In response to extracellular stimuli, phosphorylation of IκBα on serines 32 and 36 and of IκBβ on serines 19 and 23 facilitate their ubiquitination on neighboring lysine residues, thereby targeting these proteins for rapid degradation by the proteosome [
32]. Dissociation from IκBs unmasks the nuclear localization sequence of NF-κB, permitting it to move into the nucleus, bind the promoters of target genes, and subsequently alter gene expression [
33]. Although NF-κB can be activated by different stimuli, a high molecular weight IκB kinase (IKK) complex, termed IKK signalsome, serves as the key point that converges diverse upstream signals [
23]. Activated IKK complexes phosphorylate IκB proteins, promoting their dissociation from NF-κB [
23]. In the present study, we found that HIMF administration induced phosphorylation of IKK and IκBα. Moreover, transfection of the dominant-negative mutants of IKKα and IKKβ, and an IκBα super-repressor abolished HIMF-induced NF-κB activation. These data support the notion that HIMF activates NF-κB through phosphorylation of IKK and IκBα.
Phosphatidylinositol 3-kinase (PI-3K) is a heterodimer of an 85-kDa (p85) adaptor subunit and a 100-kDa (p110) catalytic subunit [
34]. PI-3K activation has been linked to a number of biological processes such as cell survival, membrane trafficking, and insulin-stimulated glucose transport [
35]. The serine-threonine protein kinase Akt is a downstream target of PI-3K-generated signals. A number of different growth factors have been shown to rapidly activate Akt via PI-3K signaling, such as platelet derived growth factor, epidermal growth factor, bFGF, insulin, and insulin-like growth factor 1 [
36]. Akt may affect NF-κB through multiple mechanisms. It has been demonstrated previously that TNF-α activates Akt, which phosphorylates and activates IKKα, thus promoting NF-κB function [
37]. Interleukin-1 can also increase the transactivation potential of the RelA subunit of NF-κB through a mechanism in which Akt has been implicated [
38]. Our results demonstrated that HIMF induced Akt phosphorylation in SVEC 4–10 cells. The time-course of Akt phosphorylation is compatible with that of NF-κB activation in HIMF stimulated cells. Pretreatment of cells with LY294002, a PI-3K specific inhibitor, attenuated HIMF-induced Akt phosphorylation. Further, transfection of Δp85 blocked HIMF-induced phosphorylation of the IKK and IκBα, NF-κB activation, and thus prevented upregulation of Flk-1. These results provided strong evidence of HIMF induced cell signaling in endothelial cells via PI-3K/Akt, which cross talks with NF-κB, in the mediation of Flk-1 upregulation.
In summary, the current studies indicated that HIMF enhances Flk-1 expression in mouse lung tissues and endothelial cells in a PI-3K/Akt-NF-κB signaling pathway-dependent manner, which at least in part, elucidated the molecular mechanisms of transcriptional regulation of the Flk-1/KDR gene and contributed to our better understanding of the functions of HIMF in pulmonary angiogenesis and maintenance of pulmonary vascular homeostasis.