Mol Cell Biochem (2014) 388:51–59 DOI 10.1007/s11010-013-1898-x
Fibroblast growth factor acts upon the transcription of phospholipase C genes in human umbilical vein endothelial cells Vincenza Rita Lo Vasco • Martina Leopizzi Chiara Puggioni • Carlo Della Rocca • Rita Businaro
•
Received: 28 September 2013 / Accepted: 5 November 2013 / Published online: 16 November 2013 Ó Springer Science+Business Media New York 2013
Abstract Besides the control of calcium levels, the phosphoinositide-specific phospholipases C (PI-PLCs), the main players in the phosphoinositide signalling pathway, contribute to a number of cell activities. The expression of PI-PLCs is strictly tissue specific and evidence suggests that it varies under different conditions, such as tumour progression or cell activation. In previous studies, we obtained a complete panel of expression of PI-PLC isoforms in human umbilical vein endothelial cells (HUVEC), a widely used experimental model for endothelial cells (EC), and demonstrated that the expression of the PLC genes varies under inflammatory stimulation. The fibroblast growth factor (FGF) activates the PI-PLC c1 isoform. In the present study, PI-PLC expression in FGF-treated HUVEC was performed using RT-PCR, observed 24 h after stimulation. The expression of selected genes after stimulation was perturbed, suggesting that FGF affects gene transcription in PI signalling as a possible mechanism of regulation of its activity upon the AkT-PLC pathway. The most efficient effects of FGF were recorded in the 3–6h interval. To understand the complex events progressing in EC might provide useful insights for potential therapeutic strategies. The opportunity to manipulate the EC might offer a powerful tool of considerable practical and clinical importance.
V. R. Lo Vasco (&) Dipartimento Organi di Senso, Policlinico Umberto I, Facolta` di Medicina e Odontoiatria, Universita` di Roma ‘‘Sapienza’’, viale del Policlinico 155, 00185 Rome, Italy e-mail: [email protected] M. Leopizzi C. Puggioni C. Della Rocca R. Businaro Dipartimento di Scienze e Biotecnologie Medico Chirurgiche, Facolta` di Farmacia e Medicina-Polo Pontino, Universita` di Roma ‘‘Sapienza’’, Rome, Italy
Keywords Fibroblast growth factor FGF Phospholipase C Gene expression Angiogenesis Signal transduction
Introduction The morphology, the molecular expression and the activities of endothelial cells (EC) vary under the influence of the surrounding environment [1–3]. ECs react to different stimuli with finely tuned responses mediated by different signal transduction pathways, thus leading the endothelium to adapt [1]. The fibroblast growth factor (FGF) proteins constitute a large family of growth factors playing important roles in the regulation of a number of events, including angiogenesis [4]. In EC, basic FGF (also known as FGF-2 or FGF-b) acts as an autocrine effector, which induces cell growth, migration, DNA synthesis, plasminogen activation and metalloproteinase production [5]. Binding to fibroblast growth factor receptors [6] initiates several signal transduction cascades, including the phosphoinositide (PI) pathway, which comprises the Phosphoinositide-specific Phospholipase C (PI-PLC) family of enzymes [7, 8]. PI-PLC enzymes regulate the spatio-temporal balance of the PI metabolism [9, 10]. Once activated, PI-PLC cleaves the membrane phosphatidyl inositol 4,5 bisphosphate (PIP2) into inositol trisphosphate (IP3) and diacylglycerol, crucial molecules in the transduction of signals [11–13]. Thirteen mammalian PI-PLC enzymes were identified, divided into six sub-families on the basis of size, amino acid sequence, domain structure and mechanism of recruitment: b(1–4), c(1–2), d(1, 3–4), e(1), f(1) and
123
52
g(1–2) [14]. PI-PLC f is exclusively expressed in spermatid cells [14]. Isoforms within the sub-families share sequence similarity, a common domain organisation and a general regulatory mechanism. Different genes codify PIPLC enzymes. Gene sequence analysis studies demonstrated that each isoform has more than one alternative splicing variant [14]. The distribution of PI-PLCs is tissue specific, emphasising the hypothesis that each isoform bears a unique function in the modulation of physiological responses. These potentialities are too far to be highlighted. The existence of a complex interplay between FGF and PI-PLC signalling was suggested. In breast cancer, FGF induces the cell cycle to enter the M-phase. The event is controlled by ordered interaction of activated Akt with PIPLC c and checkpoint with forkhead and zinc finger domains (Chfr) [15]. Akt phosphorylates PI-PLC c before Chfr, allowing the cell cycle checkpoint release. The disruption of Akt interaction with PI-PLC c forces Akt to phosphorylate Chfr, promoting earlier G2/M transition. Anti-proliferative therapeutic strategies targeting Akt aim to maintain the Akt-PLC c interaction in order to avoid accelerated M-phase entry [15]. In our previous studies, we delineated the panel of expression of PI-PLC enzymes in Human Umbilical Vein EC (HUVEC) [16] and suggested that inflammation modifies the expression [17]. Due to the role of FGF upon gene expression [18, 19] and the described relationship between FGF and PI-PLC, in the present study, we analysed the effect of FGF upon the transcription of PLC genes, which codify for PI-PLC isoforms.
Materials and methods Cell culture HUVECs (Cambrex Corporation, Walkersville, MD–USA) were cultured as previously described [16]. Endothelial Cell Growth Supplement was added to the Endogro medium (Endogro, Millipore, MA-USA). Confluent monolayer ([60 %) was obtained after 6–9 days. Cells were then stimulated by adding to the medium of culture three different concentrations of basic FGF (Sigma, St. Louis, MO, USA): 5 ng/ml (indicated as the lowest dosage), 20 ng/ml (intermediate dosage) and 80 ng/ml (highest dosage). Contemporarily, untreated HUVECs were cultured for 3, 6 and 24 h. MTT test The cell viability was evaluated using the colorimetric microassay [20]. Briefly, tetrazolium salt (MTT: 3-[4,5-
123
Mol Cell Biochem (2014) 388:51–59
dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide, 5 mg/ml) (Sigma) was added to cell cultures and then incubated for 4 h. The plates were read with a multiwell scanning Elisa reader (wavelength 540 nm, reference length 630 nm) (Biorad Laboratories, Hercules, CA, USA). The relative absorbance was shown as a percent of the corresponding control. The relative absorbance was calibrated against control cell cultures plated at different densities, which were then counted in a Coulter ZM cell counter equipped with a Channelyser to control for dead cells. The accuracy of the MTT method was shown to be ±200 cells as previously described [20]. RT-PCR HUVECs, both treated and untreated negative controls, plated on six-well plates, were suspended in TRIzol reagent (Invitrogen Corporation, Carlsbad, CA, USA) 3, 6 and 24 h after FGF addiction to the medium of culture. Total RNA was isolated from samples following the manufacturer’s instructions. The obtained RNA purity was assessed using a UV/Vis spectrophotometer (SmartSpec 3000, Biorad Laboratories). 1 mg of total RNA was reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (Applied Byosystems, Carlsbad, CA, USA) according to the manufacturer’s instructions. Briefly, RT Buffer, dNTP Mix, RT Random Primers, Multiscribe Reverse Transcriptase, RNase Inhibitor and DEPC-treated distilled water were added in RNase-free tubes on ice. The RNA sample was added. The thermal cycler programme was as follows: 25 °C for 10 m; 37 °C for 120 m; and the reaction was stopped at 85 °C for 5 m. The final volume was 20 ml. For PCR reactions, the primer pairs (Bio Basic Inc., Amherst, NY, USA) used for PI-PLC isoforms are listed in Table 1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; OMIM *138400) housekeeping gene was used as a positive control. The following primer pairs for GAPDH were used (Bio Basic Inc): forward 50 -CGAGATCCCTCCAAAATC AA-30 and reverse 50 -GTCTTCTGGGTGGCAGTGAT-30 . The specificity of the primers was verified by searching in the NCBI database for possible homology to cDNAs of unrelated proteins. Each PCR tube contained the following reagents: 0.2 lM of both sense and antisense primers, 1–3 microl (about 1 mg) template cDNA, 0.2 mM dNTP mix, 2.5 U REDTaq Genomic DNA polymerase (Sigma, Germany) and 19 reaction buffer. MgCl2 was added at variable (empirical determination by setting the experiment) final concentration. The final volume was 50 ml. The amplification was started with an initial denaturation step at 94 °C for 2 min and was followed by 35 cycles consisting of denaturation (30 s) at 94 °C, annealing (30 s) at the appropriate temperature for each primer pairs and extension (1 min) at 72 °C. The PCR products were analysed by 1.5 % TAE
Mol Cell Biochem (2014) 388:51–59 Table 1 Primers for PI-PLC isoforms; in brackets, the corresponding gene and OMIM catalogue number
53
PI-PLC b1 (PLCB1; OMIM *607120)
forward 50 -AGCTCTCAGAACAAGCCTCCAACA-30 reverse 50 -ATCATCGTCGTCGTCACTTTCCGT-30
PI-PLC b2 (PLCB2; OMIM *604114)
forward 50 -AAGGTGAAGGCCTATCTGAGCCAA-30
PI-PLC b3 (PLCB3; OMIM *600230)
reverse 50 -CTTGGCAAACTTCCCAAAGCGAGT-30 forward 50 -TATCTTCTTGGACCTGCTGACCGT-30 reverse 50 -TGTGCCCTCATCTGTAGTTGGCTT-30
PI-PLC b4 (PLCB4; OMIM *600810)
forward 50 -GCACAGCACACAAAGGAATGGTCA-30 reverse 50 -CGCATTTCCTTGCTTTCCCTGTCA-30
PI-PLC c1 (PLCG1; OMIM *172420)
forward 50 -TCTACCTGGAGGACCCTGTGAA-30 reverse 50 -CCAGAAAGAGAG CGTGTAGTCG-30
PI-PLC c2 (PLCG2; OMIM *600220)
forward 50 -AGTACATGCAGATGAATCACGC-30 reverse 50 -ACCTGAATCCTGATTTGACTGC-30
PI-PLC d1 (PLCD1; OMIM *602142)
forward 50 -CTGAGCGTGTGGTTCCAGC-30 reverse 50 -CAGGCCCTCGGACTGGT-30
PI-PLC d3 (PLCD3; OMIM *608795)
forward 50 -CCAGAACCACTCTCAGCATCCA-30 reverse 50 -GCCA TTGTTGAGCACGTAGTCAG-30
PI-PLC d4 (PLCD4; OMIM *605939)
forward 50 -AGACACGTCCCAGTCTGGAACC-30 reverse 50 -CTGCTTCCTCTTCCTCATATTC-30
PI-PLC e (PLCE; OMIM *608414)
forward 50 -GGGGCCACGGTCATCCAC-30 reverse 50 -GGGCCTTCATACCGTCCATCCTC-30
PI-PLC g1 (PLCH1; OMIM *612835
forward 50 -CTTTGGTTCGGTTCCTTGTGTGG-30 reverse 50 -GGATGCTTCTGTCAGTCCTTCC-30
PI-PLC g2 (PLCH2; OMIM *612836)
forward 50 -GAAACTGGCCTCCAAACACTGCCCGCCG-30 reverse 50 -GTCTTGTTGGAGATGCACGTGCCCCTTGC-30
ethidium bromide-stained agarose gel electrophoresis (Agarose Gel Unit, BIO-rad Laboratories Inc, UK). A PCassisted CCD camera UVB lamp (Vilber Lourmaret, France) was used for gel documentation. Gel electrophoresis of the amplification products revealed single DNA bands with nucleotide lengths as expected for each primer pair. Optical densities were normalised to the mRNA content of GAPDH. To exclude possible DNA contamination during the RTPCR, RNA samples were amplified by PCR without reverse transcription. No band was observed, suggesting that there was no DNA contamination in the RNA preparation procedure (data not shown). We measured mRNA concentrations as follows: in untreated HUVECs, we measured the basal (t = 0) concentration, and after 3, 6 and 24 h to evaluate the effects of the progression of the cell cycle upon transcription. In HUVECs treated with different concentrations of FGF, we measured mRNA concentration after 3, 6 and 24 h. The PCR products were analysed and quantified with the Agilent 2100 bioanalyzer using the DNA 1000 LabChip kit (Agilent Technologies, Germany). All the experiment sets were repeated at least three times. Western blot Western blot analysis was performed following proteins’ extraction from 24 h cultured untreated HUVECs and from
HUVECs treated for 24 h with 5 ng/ml FGF, 20 ng/ml FGF and 80 ng/ml FGF. Briefly, cells were lysed in RIPA buffer (50 mM Tris pH = 7.5, NP-40, 0.1 % SDS, 100 mM NaCl, 50 mM NaF, 1 mM EDTA) supplemented with a set of protease inhibitors: 10 lg of leupeptin per ml, 10 lg of aprotinin per ml, 1 mM sodium benzamidine and 1 mM phenylmethylsulfonyl fluoride (all the reagents were obtained from Sigma). Proteins (50 lg) were separated on 12 % polyacrylamide, 0.1 % SDS gel (Biorad Laboratories, CA, USA). Then, incubation with polyclonal antibodies specific for each PIPLC isoform (Santa Cruz, CA, USA) followed. Antibody against GAPDH was used as protein loading control (Pierce Biotechnology, Rockford, IL, USA). Immunoreactive bands were visualised using the enhanced chemiluminescence method. All the experiment sets were repeated three times. Statistical analyses The mean concentrations of mRNA transcripts of each PIPLC isoform were compared both in untreated HUVECs and in FGF-treated HUVECs, separately taking into account FGF dosage and time of exposure. For each PLC gene, the increase/reduction of mRNA concentration in FGF-treated HUVECs was expressed as a percentage with respect to the basal mean mRNA concentration.
123
54
In order to evaluate the overall effect of different dosages of FGF during the 24-h observation period, the means of mRNA concentrations were compared using the t-test. Comparisons between means of different groups were performed using a two-tailed t-test. The difference between two subsets of data was considered statistically significant if the two-tailed t-test gave a significance level P (p value) less than 0.05.
Results MTT test The survival rate of FGF-treated HUVECs was comparable to the corresponding untreated controls. Optic microscopy observations identified the presence of 3–4 % multinucleated cells in HUVECs and 20 % multinucleated cells in FGF-treated HUVECs. No differences were identified taking into account the dosages of FGF. RT-PCR and multiliquid bioanalysis quantification The results were comparable in all the sets of experiments. Concentrations were comparable (non-significant standard deviations were calculated). GAPDH was correctly amplified. The mRNA concentrations for GAPDH transcript ranged from 2.9 to 4.0 ng/ml in the experiments and no significant standard deviation was identified. For PI-PLC isoforms, the following mRNA concentrations were measured (the indicated concentrations are intended as the mean obtained in the experiment sets). The mean values recorded at each time of observation are available in Table 2. Statistical analyses of transcript concentrations The differences in gene expression were referred as percentage of increase/decrease with respect to the corresponding basal mRNA concentration measured at t = 0 in untreated HUVECs (Table 2). The t-test indicated that FGF treatment plays a statistically significant (p values \0.05) effect upon the expression of most of the analysed PLC genes (Table 2), with the/ exception of PLCH1. Western blot The results were comparable in the sets of experiments. The GAPDH loading control was visualised as expected. FGF treatment induced reduction in the optic intensity of gel bands for the expressed PI-PLC isoforms 24 h after administration. PI-PLC d4 and PI-PLC e were not detected
123
Mol Cell Biochem (2014) 388:51–59
in HUVECs and in FGF-treated HUVECs. PI-PLC b4 and PI-PLC g2 were slightly detected in untreated HUVECs and undetected in FGF-treated HUVECs.
Discussion FGF induces cell proliferation in 24 h [21, 22], efficiently enhances angiogenesis [23–25] and affects gene regulation [26, 27], i.e. reducing the expression of genes during bone differentiation [28]. In the present experiments, in HUVECs, the mean mRNA concentrations of each PI-PLC enzyme differed after FGF administration, as indicated by statistically significant differences (Table 2). Therefore, the variation in the PLCs’ transcription was not exclusively due to the cell cycle progression and might be related to FGF. During the 24-h interval, FGF did not affect the cell survival. Multinucleated cells increased from 3–4 to 5–6 % in untreated HUVECs to about 20 % after FGF treatment. This observation will require further studies in order to verify whether the phenomenon might be due to increased mitosis rate induced by FGF. FGF overall reduced the expression of PLCB1 (Table 2; Fig. 1) and reduced the transcript of PLCB2 in a timedependent manner (Table 2; Fig. 1). The FGF-induced reduction of PLCB3 transcript did not fit with the dosage (Table 2; Fig. 1). FGF treatment blocked the transcription of PLCB4, although the basal expression was low (Table 2; Fig. 1), accordingly to the literature data [14]. FGF reduced the transcription of PLCG1 independent of the dose (Table 2; Fig. 1), whilst it affected the transcription of PLCG2 in a time- and dose-dependent manner (Table 2; Fig. 1). The expression of PLCD1 decreased independent of the dose of FGF, whereas PLCD3 decreased in a doseand time-dependent manner (Table 2; Fig. 1). Accordingly to previous data [16, 17], PI-PLC d4 and PI-PLC e enzymes were not detected (Table 2). The basal expression of PLCH1 in HUVECs was low, accordingly to the literature data, indicating mainly the presence of PI-PLC g members in the nervous system [14]. 3 and 6 h after the administration of FGF, no PLCH1 transcript was detected. The basal levels were restored 24 h after administration of the lowest and intermediate FGF dosages (Table 2; Fig. 1). After 24 h, the 80 ng/ml FGF administration induced, instead of the expected 191-bp peak, one 639-bp peak. The corresponding mRNA concentration was high. BLAST analysis and sequencing of the transcript revealed no homology with any other protein. However, the peak detected in the present investigation does not seem to be compatible with any of the four described splicing variants [14]. Actually, we do not have an explanation for this finding. The basal concentration of
Mol Cell Biochem (2014) 388:51–59
55
Table 2 (Basal): mRNA concentrations of PLC genes in untreated HUVECs intended in nanograms per millilitre mRNA (ng/ml) Untreated
Basal (t=0) 6.27 FGF 5 ng
FGF 20 ng
FGF 80 ng
% 5 ng treated versus untreated
% 20 ng treated versus untreated
% 80 ng treated versus untreated
PLCB1 3h
6.2
1.59
1.14
5.88
\74.64
\81.81
\6.22
6h
6.26
2.22
1.48
3.97
\64.59
\76.39
\37.8
24 h
6.29
\39.07
\78.3
\58.7
Two-tailed t-test
3.82
1.36
2.59
p = 0.00114
p = 0.04593
p = 0.04593
mRNA (ng/ml) Untreated
Basal (t=0) 4.84 FGF 5 ng
FGF 20 ng
FGF 80 ng
% 5 ng treated versus untreated
% 20 ng treated versus untreated
% 80 ng treated versus untreated
\80.37
PLCB2 3h
4.84
0
0
0.95
\\\
\\\
6h
5
0
0
0.4
\\\
\\\
\91.73
24 h
4.9
2.74
2.4
2.93
\43.39
\50.4
\39.46
p = 0.00346
p = 0.00171
p = 0.00149
% 80 ng treated versus untreated
Two-tailed t-test mRNA (ng/ml)
Basal (t=0) 5.51
Untreated
FGF 5 ng
FGF 20 ng
FGF 80 ng
% 5 ng treated versus untreated
% 20 ng treated versus untreated
3h
5.4
0.78
0
1.79
\85.84
\\\
\67.51
6h
5.5
0
1.04
2.7
\\\
\81.12
\51
24 h
5.4
\35.57
\53.54
\10.89
PLCB3
Two-tailed t-test
3.55
2.56
4.91
p = 0.00673
p = 0.00103
p = 0.03048
mRNA (ng/ml)
Basal (t=0) 1.83
Untreated
FGF 5 ng
FGF 20 ng
FGF 80 ng
3h
1.8
0
0
0
6h
1.9
0
0
0
24 h
1.85
0
0
0
PLCB4
mRNA (ng/ml) Untreated
Basal (t=0) 20.15 FGF 5 ng
FGF 20 ng
FGF 80 ng
% 5 ng treated versus untreated
% 20 ng treated versus untreated
% 80 ng treated versus untreated
PLCG1 3h
20.15
0.72
1.12
2
\96.43
\94.44
\90.07
6h
20.1
8.54
3.19
5.6
\57.62
\84.17
\72.21
24 h
20.1
\48.73
\34.69
\58.86
% 20 ng treated versus untreated
% 80 ng treated versus untreated
\\\
Two-tailed t-test
10.33
13.16
8.29
p = 0.00234
p = 0.0051
p = 0.0002
FGF 5 ng
FGF 20 ng
FGF 80 ng
mRNA (ng/ml) Untreated
Basal (t=0) 4.39 % 5 ng treated versus untreated
PLCG2 3h
4.39
0
0
0
\\\
\\\
6h
4.4
0
0
0
\\\
\\\
\\\
24 h
4.2
3.54
1.85
0.73
\19.36
\57.86
\83.37
p = 0.02398
p = 0.00081
p = 0.0135
Two-tailed t-test
123
56
Mol Cell Biochem (2014) 388:51–59
Table 2 continued mRNA (ng/ml) Untreated
Basal (t=0) 5.92 FGF 5 ng
FGF 20 ng
FGF 80 ng
% 5 ng treated versus untreated
% 20 ng treated versus untreated
% 80 ng treated versus untreated
PLCD1 3h
5.8
0
0
0
\\\
\\\
\\\
6h
6.2
0
0
0
\\\
\\\
\\\
24 h
5.92
\67.9
\80.57
\69.59
% 20 ng treated versus untreated
% 80 ng treated versus untreated
Two-tailed t-test
1.9
1.15
1.8
p = 0.05289
p = 0.01864
p = 0.0473
mRNA (ng/ml) Untreated
Basal (t=0) 17.47 FGF 5 ng
FGF 20 ng
FGF 80 ng
% 5 ng treated versus untreated
PLCD3 3h
18
0
0
1.01
\\\
\\\
\\\
6h
18.1
1.37
0
0.83
\92.16
\\\
\\\
24 h
17.1
\49.17
\74.53
\74.53
Two-tailed t-test
8.88
4.45
0.54
p = 0.12583
p = 0.03275
p = 0.00027
mRNA (ng/ml)
Basal (t=0) 0
Untreated
FGF 5 ng
FGF 20 ng
FGF 80 ng
3h
0
0
0
0
6h
0
0
0
0
24 h
0
0
0
0
PLCD4
mRNA (ng/ml)
Basal (t=0) 0
Untreated
FGF 5 ng
FGF 20 ng
FGF 80 ng
3h
0
0
0
0
6h
0
0
0
0
24 h
0
0
0
0
PLCE
mRNA (ng/ml) Untreated
Basal (t=0) 2.43 FGF 5 ng
FGF 20 ng
FGF 80 ng
% 5 ng treated versus untreated
% 20 ng treated versus untreated
% 80 ng treated versus untreated
PLCH1 3h
2.4
0
0
0
\\\
\\\
\\\
6h
2.4
0
0
0
\\\
\\\
\\\
24 h
2.41
[2.06
\9.05
[701.23
Two-tailed t-test
2.48
2.21
*0–19.47
p = 0.43447
p = 0.36932
p = 0.78404
mRNA (ng/ml)
Basal (t=0) 1.82
Untreated
FGF 5 ng
FGF 20 ng
FGF 80 ng
3h
1.88
0
0
0
6h
1.8
0
0
0
24 h
1.82
0
0
0
PLCH2
FGF dose of b-FGF used to treat HUVECs. FGF dosages are intended in nanograms per millilitre. T = time of exposure to FGF intended in hours. mRNA mean transcript concentrations intended in nanograms per millilitre. \ = reduced with respect to basal mRNA concentration in untreated cells, intended in percentage. [ = increased with respect to basal mRNA concentration in untreated cells, intended in percentage. \\\ = abolished transcription; 100 % reduction of the transcript concentration. (t-test). One-tailed t-test results. Ratio basal/FGF treatment. p value considered significant when \0.05
123
Mol Cell Biochem (2014) 388:51–59
57
Fig. 1 Histograms of PLC genes’ expression in quiescent HUVEC (UT) and after HUVEC treatment with different concentrations of FGF
PI-PLC g2 in untreated HUVECs was low, accordingly to the literature data [14], and no PLCH2 transcript was detected after FGF treatment (Table 2; Fig. 1).
The present results corroborate the hypothesis that FGF acts upon the regulation of gene expression [28–30]. Beside the immediate effect upon the activity of PI-PLC
123
58
enzymes, widely demonstrated by a number of functional studies [9], our data suggest that FGF also acts upon the expression of PLC genes. FGF might contribute to address a complex, coordinated crosstalk amongst the isoforms. In fact, FGF acts contemporarily upon the expression of a number of PLC genes, reducing the transcription in most cases. As PI-PLC isoforms contribute to regulate the apoptosis [14], FGF probably affects this phenomenon by modifying the expression of the PLC genes. The greatest efficacy of FGF in reducing the PLC transcription was evident for PLCG1, PLCD1 and PLCD3 (Table 2; Fig. 1). Interestingly, PI-PLC c1, ubiquitously expressed, regulates a multitude of cell functions in a number of tissues, although its role in the regulation of cell proliferation remains controversial [15, 31–35]. Literature data suggest that FGF induces the G2/M transition acting upon the Akt/ PLC c1 axis [15]. Our results suggest that FGF might act, just affecting the expression of PLCG1. The PI-PLC d family members are the most basic due to their very simple structure and high sensitivity to calcium [14]. They regulate the activity of the other PI-PLC subfamilies’ members [14]. PI-PLC d1 probably bears antioncogene functions [36, 37] that strongly suggest a role in the cell cycle control. Plcd1 was expressed in quiescent rat astrocytes and unexpressed in the tumour counterpart [38]. Both PI-PLC d1 and d3 decreased in different cell types after inflammatory stimulation [17, 39, 40]. Our results also indicate that FGF dosage did not act linearly upon PLC genes. We observed linear correspondence between mRNA reduction and the increasing FGF dose exclusively for PLCG2. Interestingly, PI-PLC c2, mainly expressed in haematopoietic elements, plays a key role in the regulation of the immune response [41–43]. Regarding the exposure time, the most efficient effects of FGF were in the 3–6-h interval. Therefore, FGF mainly affects the transcription of PLC genes shortly after administration. With notable exceptions (PLCD3 and PLCH1), after 24 h, the effect of FGF upon genes’ transcription was less intense. Interestingly, activation of PIPLC g1 enhances the GPCR-mediated calcium pathway [44] and probably plays as a sensor activated by very small calcium increases. PI-PLC g1 is probably involved in the finest regulation of signalling [14, 45]. Increasing evidence indicates that PI-PLC g isoforms might be of critical importance and their expression after FGF treatment will deserve further studies. In fact, PI-PLC g isoforms were recently analysed in selected human osteosarcoma cell lines [46] and in human skin fibroblasts [47]. In HUVECs, PI-PLC g isoforms are slightly, although inconstantly, expressed [16] and the expression varied under inflammatory stimulation [17].
123
Mol Cell Biochem (2014) 388:51–59
Studies addressed to investigate the relationship of PIPLC enzymes with FGF and to highlight the mechanisms, which regulate PLC gene expression, might provide promising perspectives. Investigations are also required in order to delineate the relationship between the FGFinduced variation in PLC expression and the cell cycle control. The growing interest directed to understand the complex events progressing in EC might provide useful insights for potential therapeutic strategies. The opportunity to understand the complex network that regulates angiogenesis and the ability to manipulate the EC might offer a powerful tool of considerable practical and clinical importance. Acknowledgements This investigation was funded by the Iniziative Universitarie 2011 Funding of ‘Sapienza’ University of Rome. The authors thank the ‘Serena Talarico Association’ for precious support to this research.
References 1. Christ M, Wehling M (1998) Cardiovascular steroid actions: swift swallows or sluggish snails? Cardiovasc Res 40:34–44 2. Grienddling KK, Alexander WR (1996) Endothelial control of the cardiovascular system: recent advances. FASEB J 10:283–292 3. Gloe T, Pohl U (2002) Laminin binding conveys mechanosensing in endothelial cells. News Physiol Sci 17:166–169 4. Folkman J, Klagsbrun M (1987) Angiogenic factors. Science 235(4787):442–447 5. Sato Y, Rifkin DB (1988) Autocrine activities of basic fibroblast growth factor: regulation of endothelial cell movement, plasminogen activator synthesis, and DNA synthesis. J Cell Biol 107(3):1199–1205 6. Maric D, Fiorio PA, Chang YH et al (2007) Self-renewing and differentiating properties of cortical neural stem cells are selectively regulated by basic fibroblast growth factor (FGF) signaling via specific FGF receptors. J Neurosci 27(8):1836–1852 7. McFarlane S, McNeill L, Holt CE (1995) FGF signaling and target recognition in the developing Xenopus visual system. Neuron 15(5):1017–1028 8. Maffucci T, Falasca M (2007) Phosphoinositide 3-kinasedependent regulation of phospholipase Cgamma. Biochem Soc Trans 35:229–230 9. Berridge MJ, Dupont G (1994) Spatial and temporal signalling by calcium. Curr Opin Cell Biol 6(2):267–274 10. Divecha N, Irvine RF (1995) Phospholipid signaling. Cell 80(2):269–278 11. Hisatsune C, Nakamura K, Kuroda Y et al (2005) Amplification of Ca2? signaling by diacylglycerolmediatedinositol 1,4,5-trisphosphate production. J Biol Chem 280(12):11723–11730 12. Rhee SG (2001) Regulation of phosphoinositide-specific phospholipase C. Annu Rev Biochem 70:281–312 13. Bunney TD, Katan M (2011) PLC regulation: emerging pictures for molecular mechanisms. Trends Biochem Sci 36(2):88–96 14. Suh PG, Park J, Manzoli L et al (2008) Multiple roles of phosphoinositide-specific phospholipase C isozymes. BMB Rep 41: 415–434 15. Browaeys-Poly E, Perdereau D, Lescuyer A et al (2009) Akt interaction with PLC(gamma) regulates the G(2)/M transition triggered by FGF receptors from MDA-MB-231 breast cancer cells. Anticancer Res 29(12):4965–4969
Mol Cell Biochem (2014) 388:51–59 16. Lo Vasco VR, Pacini L, Di Raimo T et al (2011) Expression of phosphoinositide-specific phospholipase C isoforms in HUVEC. J Clin Pathol 64(10):911–915 17. Lo Vasco VR, Leopizzi M, Chiappetta C et al (2013) Lypopolysaccharide downregulates the expression of selected phospholipase C genes in cultured endothelial cells. Inflammation 36(4):862–868. doi:10.1007/s10753-013-9613-3 18. Shiba H, Nakamura S, Shirakawa M et al (1995) Effects of basic fibroblast growth factor on proliferation, the expression of osteonectin (SPARC) and alkaline phosphatase, and calcification in cultures of human pulp cells. Dev Biol 170(2):457–466 19. Shiba T, Nishimura D, Kawazoe Y et al (2003) Modulation of mitogenic activity of fibroblast growth factors by inorganic polyphosphate. J Biol Chem 278(29):26788–26792 20. Mosmann T (1993) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63 21. Raballo R, Rhee J, Lyn-Cook R et al (2000) Basic fibroblast growth factor (Fgf2) is necessary for cell proliferation and neurogenesis in the developing cerebral cortex. J Neurosci 20(13):5012–5023 22. Yang J, Meyer M, Muller AK et al (2010) Fibroblast growth factor receptors 1 and 2 in keratinocytes control the epidermal barrier and cutaneous homeostasis. J Cell Biol 188(6):935–952 23. Falasca M, Logan SK, Lehto VP et al (1998) Activation of phospholipase C gamma by PI 3-kinase induced PH domainmediated membrane targeting. EMBO J 17:414–422 24. Cao R, Jensen LD, Soll I et al (2008) Hypoxia-induced retinal angiogenesis in zebrafish as a model to study retinopathy. PLoS ONE 3(7):e2748 25. Zhao G, Li WY, Chen D et al (2011) A novel, selective inhibitor of fibroblast growth factor receptors that shows a potent broad spectrum of antitumor activity in several tumor xenograft models. Mol Cancer Ther 10(11):2200–2210 26. Webber CA, Hyakutake MT, McFarlane S (2003) Fibroblast growth factors redirect retinal axons in vitro and in vivo. Dev Biol 263(1):24–34 27. Chang CH, Huang YL, Shyu MK et al (2013) Sphingosine-1phosphate induces VEGF-C expression through a MMP-2/FGF-1/ FGFR-1-dependent pathway in endothelial cells in vitro. Acta Pharmacol Sin 34(3):360–366 28. Hughes-Fulford M, Li CF (2011) The role of FGF-2 and BMP-2 in regulation of gene induction, cell proliferation and mineralization. J Orthop Surg Res 9(6):8 29. Murakami M, Nguyen LT, Hatanaka K et al (2011) FGFdependent regulation of VEGF receptor 2 expression in mice. J Clin Invest 121(7):2668–2678 30. Ji QS, Ermini S, Baulida J et al (1998) Epidermal growth factor signaling and mitogenesisin Plcg1 null mouse embryonic fibroblasts. Mol Biol Cell 9:749–757 31. Smith MR, Liu YL, Kim H et al (1990) Inhibition of serum- and ras-stimulated DNA synthesis by antibodies to phospholipase C. Science 247:1074–1077 32. Wang Z, Gluck S, Zhang L et al (1998) Requirement for phospholipase C-gamma1 enzymatic activity in growth factor-induced mitogenesis. Mol Cell Biol 18:590–597
59 33. Oh JE, Kook JK, Park KH et al (2003) Phospholipase C-gamma1 is required for subculture-induced terminal differentiation of normal human oral keratinocytes. Int J Mol Med 11:491–498 34. Kolsch V, Charest PG, Firtel RA (2008) The regulation of cell motility and chemotaxis by phospholipid signaling. J Cell Sci 121:551–559 35. Stallings JD, Zeng YX, Narvaez F et al (2008) Phospholipase C-delta 1is linked to pro liferation, DNA synthesis and cyclin E levels. J Biol Chem 283:13992–14001 36. Stallings JD, Tall EG, Pentyala S et al (2005) Nuclear translocation of phospholipase C-delta1 is linked to the cell cycle and nuclear phosphatidylinositol 4,5-bisphosphate. J Biol Chem 280:22060–22069 37. Lo Vasco VR, Fabrizi C, Artico M et al (2007) Expression of phosphoinositide-specific phospholipase C isoenzymes in cultured astrocytes. J Cell Biochem 100(4):952–959 38. Lo Vasco VR, Fabrizi C, Fumagalli L et al (2010) Expression of phosphoinositide specific phospholipase C isoenzymes in cultured astrocytes activated after stimulation with Lipopolysaccharide. J Cell Biochem 109(5):1006–1012 39. Lo Vasco VR, Leopizzi M, Chiappetta C et al (2012) Expression of phosphoinositide-specific phospholipase C enzymes in normal endometrium and in endometriosis. Fertil Steril 98(2):410–414 40. Marshall AJ, Niiro H, Yun TJ et al (2000) Regulation of B-cell activation and differentiation by the phosphatidylinositol 3-kinase and phospholipase Cgamma pathway. Immunol Rev 176:30–46 41. Homma Y, Takenawa T, Emori Y et al (1989) Tissue- and cell type-specific expression of mRNAs for four types of inositol phospholipid-specific phospholipase C. Biochem Biophys Res Commun 164:406–412 42. Yu P, Constien R, Dear N et al (2005) Autoimmunity and inflammation due to a gain-of-function mutation in phospholipase C gamma 2 that specifically increases external Ca2? entry. Immunity 22:451–465 43. Kim JK, Choi JW, Lim S et al (2011) Phospholipase C-g1 is activated by intracellular Ca(2?) mobilization and enhances GPCRs/PLC/Ca(2?) signaling. Cell Signal 23(6):1022–1029 44. Stewart AJ, Morgan K, Farquharson C et al (2007) Phospholipase C-eta enzymes as putative protein kinase C and Ca2? signalling components in neuronal and neuroendocrine tissues. Neuroendocrinology 86(4):243–248 45. Lo Vasco VR, Leopizzi M, Chiappetta C et al (2013) Expression of phosphoinositide-specific phospholipase C enzymes in human osteosarcoma cell lines. J Cell Commun Signal 7(2):141–150. doi:10.1007/s12079-013-0194-6 46. Lo Vasco VR, Leopizzi M, Chiappetta C et al (2013) Expression of phosphoinositide-specific phospholipase C enzymes in human skin fibroblasts. Connect Tissue Res 54(1):1–4 47. Lo Vasco VR, Fabrizi C, Panetta B et al (2010) Expression pattern and sub cellular distribution of phosphoinositide specific phospholipase C enzymes after treatment with U-73122 in rat astrocytoma cells. J Cell Biochem 110(4):1005–1012
123
Fibroblast growth factor acts upon the transcription of phospholipase C genes in human umbilical vein endothelial cells Vincenza Rita Lo Vasco • Martina Leopizzi Chiara Puggioni • Carlo Della Rocca • Rita Businaro
•
Received: 28 September 2013 / Accepted: 5 November 2013 / Published online: 16 November 2013 Ó Springer Science+Business Media New York 2013
Abstract Besides the control of calcium levels, the phosphoinositide-specific phospholipases C (PI-PLCs), the main players in the phosphoinositide signalling pathway, contribute to a number of cell activities. The expression of PI-PLCs is strictly tissue specific and evidence suggests that it varies under different conditions, such as tumour progression or cell activation. In previous studies, we obtained a complete panel of expression of PI-PLC isoforms in human umbilical vein endothelial cells (HUVEC), a widely used experimental model for endothelial cells (EC), and demonstrated that the expression of the PLC genes varies under inflammatory stimulation. The fibroblast growth factor (FGF) activates the PI-PLC c1 isoform. In the present study, PI-PLC expression in FGF-treated HUVEC was performed using RT-PCR, observed 24 h after stimulation. The expression of selected genes after stimulation was perturbed, suggesting that FGF affects gene transcription in PI signalling as a possible mechanism of regulation of its activity upon the AkT-PLC pathway. The most efficient effects of FGF were recorded in the 3–6h interval. To understand the complex events progressing in EC might provide useful insights for potential therapeutic strategies. The opportunity to manipulate the EC might offer a powerful tool of considerable practical and clinical importance.
V. R. Lo Vasco (&) Dipartimento Organi di Senso, Policlinico Umberto I, Facolta` di Medicina e Odontoiatria, Universita` di Roma ‘‘Sapienza’’, viale del Policlinico 155, 00185 Rome, Italy e-mail: [email protected] M. Leopizzi C. Puggioni C. Della Rocca R. Businaro Dipartimento di Scienze e Biotecnologie Medico Chirurgiche, Facolta` di Farmacia e Medicina-Polo Pontino, Universita` di Roma ‘‘Sapienza’’, Rome, Italy
Keywords Fibroblast growth factor FGF Phospholipase C Gene expression Angiogenesis Signal transduction
Introduction The morphology, the molecular expression and the activities of endothelial cells (EC) vary under the influence of the surrounding environment [1–3]. ECs react to different stimuli with finely tuned responses mediated by different signal transduction pathways, thus leading the endothelium to adapt [1]. The fibroblast growth factor (FGF) proteins constitute a large family of growth factors playing important roles in the regulation of a number of events, including angiogenesis [4]. In EC, basic FGF (also known as FGF-2 or FGF-b) acts as an autocrine effector, which induces cell growth, migration, DNA synthesis, plasminogen activation and metalloproteinase production [5]. Binding to fibroblast growth factor receptors [6] initiates several signal transduction cascades, including the phosphoinositide (PI) pathway, which comprises the Phosphoinositide-specific Phospholipase C (PI-PLC) family of enzymes [7, 8]. PI-PLC enzymes regulate the spatio-temporal balance of the PI metabolism [9, 10]. Once activated, PI-PLC cleaves the membrane phosphatidyl inositol 4,5 bisphosphate (PIP2) into inositol trisphosphate (IP3) and diacylglycerol, crucial molecules in the transduction of signals [11–13]. Thirteen mammalian PI-PLC enzymes were identified, divided into six sub-families on the basis of size, amino acid sequence, domain structure and mechanism of recruitment: b(1–4), c(1–2), d(1, 3–4), e(1), f(1) and
123
52
g(1–2) [14]. PI-PLC f is exclusively expressed in spermatid cells [14]. Isoforms within the sub-families share sequence similarity, a common domain organisation and a general regulatory mechanism. Different genes codify PIPLC enzymes. Gene sequence analysis studies demonstrated that each isoform has more than one alternative splicing variant [14]. The distribution of PI-PLCs is tissue specific, emphasising the hypothesis that each isoform bears a unique function in the modulation of physiological responses. These potentialities are too far to be highlighted. The existence of a complex interplay between FGF and PI-PLC signalling was suggested. In breast cancer, FGF induces the cell cycle to enter the M-phase. The event is controlled by ordered interaction of activated Akt with PIPLC c and checkpoint with forkhead and zinc finger domains (Chfr) [15]. Akt phosphorylates PI-PLC c before Chfr, allowing the cell cycle checkpoint release. The disruption of Akt interaction with PI-PLC c forces Akt to phosphorylate Chfr, promoting earlier G2/M transition. Anti-proliferative therapeutic strategies targeting Akt aim to maintain the Akt-PLC c interaction in order to avoid accelerated M-phase entry [15]. In our previous studies, we delineated the panel of expression of PI-PLC enzymes in Human Umbilical Vein EC (HUVEC) [16] and suggested that inflammation modifies the expression [17]. Due to the role of FGF upon gene expression [18, 19] and the described relationship between FGF and PI-PLC, in the present study, we analysed the effect of FGF upon the transcription of PLC genes, which codify for PI-PLC isoforms.
Materials and methods Cell culture HUVECs (Cambrex Corporation, Walkersville, MD–USA) were cultured as previously described [16]. Endothelial Cell Growth Supplement was added to the Endogro medium (Endogro, Millipore, MA-USA). Confluent monolayer ([60 %) was obtained after 6–9 days. Cells were then stimulated by adding to the medium of culture three different concentrations of basic FGF (Sigma, St. Louis, MO, USA): 5 ng/ml (indicated as the lowest dosage), 20 ng/ml (intermediate dosage) and 80 ng/ml (highest dosage). Contemporarily, untreated HUVECs were cultured for 3, 6 and 24 h. MTT test The cell viability was evaluated using the colorimetric microassay [20]. Briefly, tetrazolium salt (MTT: 3-[4,5-
123
Mol Cell Biochem (2014) 388:51–59
dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide, 5 mg/ml) (Sigma) was added to cell cultures and then incubated for 4 h. The plates were read with a multiwell scanning Elisa reader (wavelength 540 nm, reference length 630 nm) (Biorad Laboratories, Hercules, CA, USA). The relative absorbance was shown as a percent of the corresponding control. The relative absorbance was calibrated against control cell cultures plated at different densities, which were then counted in a Coulter ZM cell counter equipped with a Channelyser to control for dead cells. The accuracy of the MTT method was shown to be ±200 cells as previously described [20]. RT-PCR HUVECs, both treated and untreated negative controls, plated on six-well plates, were suspended in TRIzol reagent (Invitrogen Corporation, Carlsbad, CA, USA) 3, 6 and 24 h after FGF addiction to the medium of culture. Total RNA was isolated from samples following the manufacturer’s instructions. The obtained RNA purity was assessed using a UV/Vis spectrophotometer (SmartSpec 3000, Biorad Laboratories). 1 mg of total RNA was reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (Applied Byosystems, Carlsbad, CA, USA) according to the manufacturer’s instructions. Briefly, RT Buffer, dNTP Mix, RT Random Primers, Multiscribe Reverse Transcriptase, RNase Inhibitor and DEPC-treated distilled water were added in RNase-free tubes on ice. The RNA sample was added. The thermal cycler programme was as follows: 25 °C for 10 m; 37 °C for 120 m; and the reaction was stopped at 85 °C for 5 m. The final volume was 20 ml. For PCR reactions, the primer pairs (Bio Basic Inc., Amherst, NY, USA) used for PI-PLC isoforms are listed in Table 1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; OMIM *138400) housekeeping gene was used as a positive control. The following primer pairs for GAPDH were used (Bio Basic Inc): forward 50 -CGAGATCCCTCCAAAATC AA-30 and reverse 50 -GTCTTCTGGGTGGCAGTGAT-30 . The specificity of the primers was verified by searching in the NCBI database for possible homology to cDNAs of unrelated proteins. Each PCR tube contained the following reagents: 0.2 lM of both sense and antisense primers, 1–3 microl (about 1 mg) template cDNA, 0.2 mM dNTP mix, 2.5 U REDTaq Genomic DNA polymerase (Sigma, Germany) and 19 reaction buffer. MgCl2 was added at variable (empirical determination by setting the experiment) final concentration. The final volume was 50 ml. The amplification was started with an initial denaturation step at 94 °C for 2 min and was followed by 35 cycles consisting of denaturation (30 s) at 94 °C, annealing (30 s) at the appropriate temperature for each primer pairs and extension (1 min) at 72 °C. The PCR products were analysed by 1.5 % TAE
Mol Cell Biochem (2014) 388:51–59 Table 1 Primers for PI-PLC isoforms; in brackets, the corresponding gene and OMIM catalogue number
53
PI-PLC b1 (PLCB1; OMIM *607120)
forward 50 -AGCTCTCAGAACAAGCCTCCAACA-30 reverse 50 -ATCATCGTCGTCGTCACTTTCCGT-30
PI-PLC b2 (PLCB2; OMIM *604114)
forward 50 -AAGGTGAAGGCCTATCTGAGCCAA-30
PI-PLC b3 (PLCB3; OMIM *600230)
reverse 50 -CTTGGCAAACTTCCCAAAGCGAGT-30 forward 50 -TATCTTCTTGGACCTGCTGACCGT-30 reverse 50 -TGTGCCCTCATCTGTAGTTGGCTT-30
PI-PLC b4 (PLCB4; OMIM *600810)
forward 50 -GCACAGCACACAAAGGAATGGTCA-30 reverse 50 -CGCATTTCCTTGCTTTCCCTGTCA-30
PI-PLC c1 (PLCG1; OMIM *172420)
forward 50 -TCTACCTGGAGGACCCTGTGAA-30 reverse 50 -CCAGAAAGAGAG CGTGTAGTCG-30
PI-PLC c2 (PLCG2; OMIM *600220)
forward 50 -AGTACATGCAGATGAATCACGC-30 reverse 50 -ACCTGAATCCTGATTTGACTGC-30
PI-PLC d1 (PLCD1; OMIM *602142)
forward 50 -CTGAGCGTGTGGTTCCAGC-30 reverse 50 -CAGGCCCTCGGACTGGT-30
PI-PLC d3 (PLCD3; OMIM *608795)
forward 50 -CCAGAACCACTCTCAGCATCCA-30 reverse 50 -GCCA TTGTTGAGCACGTAGTCAG-30
PI-PLC d4 (PLCD4; OMIM *605939)
forward 50 -AGACACGTCCCAGTCTGGAACC-30 reverse 50 -CTGCTTCCTCTTCCTCATATTC-30
PI-PLC e (PLCE; OMIM *608414)
forward 50 -GGGGCCACGGTCATCCAC-30 reverse 50 -GGGCCTTCATACCGTCCATCCTC-30
PI-PLC g1 (PLCH1; OMIM *612835
forward 50 -CTTTGGTTCGGTTCCTTGTGTGG-30 reverse 50 -GGATGCTTCTGTCAGTCCTTCC-30
PI-PLC g2 (PLCH2; OMIM *612836)
forward 50 -GAAACTGGCCTCCAAACACTGCCCGCCG-30 reverse 50 -GTCTTGTTGGAGATGCACGTGCCCCTTGC-30
ethidium bromide-stained agarose gel electrophoresis (Agarose Gel Unit, BIO-rad Laboratories Inc, UK). A PCassisted CCD camera UVB lamp (Vilber Lourmaret, France) was used for gel documentation. Gel electrophoresis of the amplification products revealed single DNA bands with nucleotide lengths as expected for each primer pair. Optical densities were normalised to the mRNA content of GAPDH. To exclude possible DNA contamination during the RTPCR, RNA samples were amplified by PCR without reverse transcription. No band was observed, suggesting that there was no DNA contamination in the RNA preparation procedure (data not shown). We measured mRNA concentrations as follows: in untreated HUVECs, we measured the basal (t = 0) concentration, and after 3, 6 and 24 h to evaluate the effects of the progression of the cell cycle upon transcription. In HUVECs treated with different concentrations of FGF, we measured mRNA concentration after 3, 6 and 24 h. The PCR products were analysed and quantified with the Agilent 2100 bioanalyzer using the DNA 1000 LabChip kit (Agilent Technologies, Germany). All the experiment sets were repeated at least three times. Western blot Western blot analysis was performed following proteins’ extraction from 24 h cultured untreated HUVECs and from
HUVECs treated for 24 h with 5 ng/ml FGF, 20 ng/ml FGF and 80 ng/ml FGF. Briefly, cells were lysed in RIPA buffer (50 mM Tris pH = 7.5, NP-40, 0.1 % SDS, 100 mM NaCl, 50 mM NaF, 1 mM EDTA) supplemented with a set of protease inhibitors: 10 lg of leupeptin per ml, 10 lg of aprotinin per ml, 1 mM sodium benzamidine and 1 mM phenylmethylsulfonyl fluoride (all the reagents were obtained from Sigma). Proteins (50 lg) were separated on 12 % polyacrylamide, 0.1 % SDS gel (Biorad Laboratories, CA, USA). Then, incubation with polyclonal antibodies specific for each PIPLC isoform (Santa Cruz, CA, USA) followed. Antibody against GAPDH was used as protein loading control (Pierce Biotechnology, Rockford, IL, USA). Immunoreactive bands were visualised using the enhanced chemiluminescence method. All the experiment sets were repeated three times. Statistical analyses The mean concentrations of mRNA transcripts of each PIPLC isoform were compared both in untreated HUVECs and in FGF-treated HUVECs, separately taking into account FGF dosage and time of exposure. For each PLC gene, the increase/reduction of mRNA concentration in FGF-treated HUVECs was expressed as a percentage with respect to the basal mean mRNA concentration.
123
54
In order to evaluate the overall effect of different dosages of FGF during the 24-h observation period, the means of mRNA concentrations were compared using the t-test. Comparisons between means of different groups were performed using a two-tailed t-test. The difference between two subsets of data was considered statistically significant if the two-tailed t-test gave a significance level P (p value) less than 0.05.
Results MTT test The survival rate of FGF-treated HUVECs was comparable to the corresponding untreated controls. Optic microscopy observations identified the presence of 3–4 % multinucleated cells in HUVECs and 20 % multinucleated cells in FGF-treated HUVECs. No differences were identified taking into account the dosages of FGF. RT-PCR and multiliquid bioanalysis quantification The results were comparable in all the sets of experiments. Concentrations were comparable (non-significant standard deviations were calculated). GAPDH was correctly amplified. The mRNA concentrations for GAPDH transcript ranged from 2.9 to 4.0 ng/ml in the experiments and no significant standard deviation was identified. For PI-PLC isoforms, the following mRNA concentrations were measured (the indicated concentrations are intended as the mean obtained in the experiment sets). The mean values recorded at each time of observation are available in Table 2. Statistical analyses of transcript concentrations The differences in gene expression were referred as percentage of increase/decrease with respect to the corresponding basal mRNA concentration measured at t = 0 in untreated HUVECs (Table 2). The t-test indicated that FGF treatment plays a statistically significant (p values \0.05) effect upon the expression of most of the analysed PLC genes (Table 2), with the/ exception of PLCH1. Western blot The results were comparable in the sets of experiments. The GAPDH loading control was visualised as expected. FGF treatment induced reduction in the optic intensity of gel bands for the expressed PI-PLC isoforms 24 h after administration. PI-PLC d4 and PI-PLC e were not detected
123
Mol Cell Biochem (2014) 388:51–59
in HUVECs and in FGF-treated HUVECs. PI-PLC b4 and PI-PLC g2 were slightly detected in untreated HUVECs and undetected in FGF-treated HUVECs.
Discussion FGF induces cell proliferation in 24 h [21, 22], efficiently enhances angiogenesis [23–25] and affects gene regulation [26, 27], i.e. reducing the expression of genes during bone differentiation [28]. In the present experiments, in HUVECs, the mean mRNA concentrations of each PI-PLC enzyme differed after FGF administration, as indicated by statistically significant differences (Table 2). Therefore, the variation in the PLCs’ transcription was not exclusively due to the cell cycle progression and might be related to FGF. During the 24-h interval, FGF did not affect the cell survival. Multinucleated cells increased from 3–4 to 5–6 % in untreated HUVECs to about 20 % after FGF treatment. This observation will require further studies in order to verify whether the phenomenon might be due to increased mitosis rate induced by FGF. FGF overall reduced the expression of PLCB1 (Table 2; Fig. 1) and reduced the transcript of PLCB2 in a timedependent manner (Table 2; Fig. 1). The FGF-induced reduction of PLCB3 transcript did not fit with the dosage (Table 2; Fig. 1). FGF treatment blocked the transcription of PLCB4, although the basal expression was low (Table 2; Fig. 1), accordingly to the literature data [14]. FGF reduced the transcription of PLCG1 independent of the dose (Table 2; Fig. 1), whilst it affected the transcription of PLCG2 in a time- and dose-dependent manner (Table 2; Fig. 1). The expression of PLCD1 decreased independent of the dose of FGF, whereas PLCD3 decreased in a doseand time-dependent manner (Table 2; Fig. 1). Accordingly to previous data [16, 17], PI-PLC d4 and PI-PLC e enzymes were not detected (Table 2). The basal expression of PLCH1 in HUVECs was low, accordingly to the literature data, indicating mainly the presence of PI-PLC g members in the nervous system [14]. 3 and 6 h after the administration of FGF, no PLCH1 transcript was detected. The basal levels were restored 24 h after administration of the lowest and intermediate FGF dosages (Table 2; Fig. 1). After 24 h, the 80 ng/ml FGF administration induced, instead of the expected 191-bp peak, one 639-bp peak. The corresponding mRNA concentration was high. BLAST analysis and sequencing of the transcript revealed no homology with any other protein. However, the peak detected in the present investigation does not seem to be compatible with any of the four described splicing variants [14]. Actually, we do not have an explanation for this finding. The basal concentration of
Mol Cell Biochem (2014) 388:51–59
55
Table 2 (Basal): mRNA concentrations of PLC genes in untreated HUVECs intended in nanograms per millilitre mRNA (ng/ml) Untreated
Basal (t=0) 6.27 FGF 5 ng
FGF 20 ng
FGF 80 ng
% 5 ng treated versus untreated
% 20 ng treated versus untreated
% 80 ng treated versus untreated
PLCB1 3h
6.2
1.59
1.14
5.88
\74.64
\81.81
\6.22
6h
6.26
2.22
1.48
3.97
\64.59
\76.39
\37.8
24 h
6.29
\39.07
\78.3
\58.7
Two-tailed t-test
3.82
1.36
2.59
p = 0.00114
p = 0.04593
p = 0.04593
mRNA (ng/ml) Untreated
Basal (t=0) 4.84 FGF 5 ng
FGF 20 ng
FGF 80 ng
% 5 ng treated versus untreated
% 20 ng treated versus untreated
% 80 ng treated versus untreated
\80.37
PLCB2 3h
4.84
0
0
0.95
\\\
\\\
6h
5
0
0
0.4
\\\
\\\
\91.73
24 h
4.9
2.74
2.4
2.93
\43.39
\50.4
\39.46
p = 0.00346
p = 0.00171
p = 0.00149
% 80 ng treated versus untreated
Two-tailed t-test mRNA (ng/ml)
Basal (t=0) 5.51
Untreated
FGF 5 ng
FGF 20 ng
FGF 80 ng
% 5 ng treated versus untreated
% 20 ng treated versus untreated
3h
5.4
0.78
0
1.79
\85.84
\\\
\67.51
6h
5.5
0
1.04
2.7
\\\
\81.12
\51
24 h
5.4
\35.57
\53.54
\10.89
PLCB3
Two-tailed t-test
3.55
2.56
4.91
p = 0.00673
p = 0.00103
p = 0.03048
mRNA (ng/ml)
Basal (t=0) 1.83
Untreated
FGF 5 ng
FGF 20 ng
FGF 80 ng
3h
1.8
0
0
0
6h
1.9
0
0
0
24 h
1.85
0
0
0
PLCB4
mRNA (ng/ml) Untreated
Basal (t=0) 20.15 FGF 5 ng
FGF 20 ng
FGF 80 ng
% 5 ng treated versus untreated
% 20 ng treated versus untreated
% 80 ng treated versus untreated
PLCG1 3h
20.15
0.72
1.12
2
\96.43
\94.44
\90.07
6h
20.1
8.54
3.19
5.6
\57.62
\84.17
\72.21
24 h
20.1
\48.73
\34.69
\58.86
% 20 ng treated versus untreated
% 80 ng treated versus untreated
\\\
Two-tailed t-test
10.33
13.16
8.29
p = 0.00234
p = 0.0051
p = 0.0002
FGF 5 ng
FGF 20 ng
FGF 80 ng
mRNA (ng/ml) Untreated
Basal (t=0) 4.39 % 5 ng treated versus untreated
PLCG2 3h
4.39
0
0
0
\\\
\\\
6h
4.4
0
0
0
\\\
\\\
\\\
24 h
4.2
3.54
1.85
0.73
\19.36
\57.86
\83.37
p = 0.02398
p = 0.00081
p = 0.0135
Two-tailed t-test
123
56
Mol Cell Biochem (2014) 388:51–59
Table 2 continued mRNA (ng/ml) Untreated
Basal (t=0) 5.92 FGF 5 ng
FGF 20 ng
FGF 80 ng
% 5 ng treated versus untreated
% 20 ng treated versus untreated
% 80 ng treated versus untreated
PLCD1 3h
5.8
0
0
0
\\\
\\\
\\\
6h
6.2
0
0
0
\\\
\\\
\\\
24 h
5.92
\67.9
\80.57
\69.59
% 20 ng treated versus untreated
% 80 ng treated versus untreated
Two-tailed t-test
1.9
1.15
1.8
p = 0.05289
p = 0.01864
p = 0.0473
mRNA (ng/ml) Untreated
Basal (t=0) 17.47 FGF 5 ng
FGF 20 ng
FGF 80 ng
% 5 ng treated versus untreated
PLCD3 3h
18
0
0
1.01
\\\
\\\
\\\
6h
18.1
1.37
0
0.83
\92.16
\\\
\\\
24 h
17.1
\49.17
\74.53
\74.53
Two-tailed t-test
8.88
4.45
0.54
p = 0.12583
p = 0.03275
p = 0.00027
mRNA (ng/ml)
Basal (t=0) 0
Untreated
FGF 5 ng
FGF 20 ng
FGF 80 ng
3h
0
0
0
0
6h
0
0
0
0
24 h
0
0
0
0
PLCD4
mRNA (ng/ml)
Basal (t=0) 0
Untreated
FGF 5 ng
FGF 20 ng
FGF 80 ng
3h
0
0
0
0
6h
0
0
0
0
24 h
0
0
0
0
PLCE
mRNA (ng/ml) Untreated
Basal (t=0) 2.43 FGF 5 ng
FGF 20 ng
FGF 80 ng
% 5 ng treated versus untreated
% 20 ng treated versus untreated
% 80 ng treated versus untreated
PLCH1 3h
2.4
0
0
0
\\\
\\\
\\\
6h
2.4
0
0
0
\\\
\\\
\\\
24 h
2.41
[2.06
\9.05
[701.23
Two-tailed t-test
2.48
2.21
*0–19.47
p = 0.43447
p = 0.36932
p = 0.78404
mRNA (ng/ml)
Basal (t=0) 1.82
Untreated
FGF 5 ng
FGF 20 ng
FGF 80 ng
3h
1.88
0
0
0
6h
1.8
0
0
0
24 h
1.82
0
0
0
PLCH2
FGF dose of b-FGF used to treat HUVECs. FGF dosages are intended in nanograms per millilitre. T = time of exposure to FGF intended in hours. mRNA mean transcript concentrations intended in nanograms per millilitre. \ = reduced with respect to basal mRNA concentration in untreated cells, intended in percentage. [ = increased with respect to basal mRNA concentration in untreated cells, intended in percentage. \\\ = abolished transcription; 100 % reduction of the transcript concentration. (t-test). One-tailed t-test results. Ratio basal/FGF treatment. p value considered significant when \0.05
123
Mol Cell Biochem (2014) 388:51–59
57
Fig. 1 Histograms of PLC genes’ expression in quiescent HUVEC (UT) and after HUVEC treatment with different concentrations of FGF
PI-PLC g2 in untreated HUVECs was low, accordingly to the literature data [14], and no PLCH2 transcript was detected after FGF treatment (Table 2; Fig. 1).
The present results corroborate the hypothesis that FGF acts upon the regulation of gene expression [28–30]. Beside the immediate effect upon the activity of PI-PLC
123
58
enzymes, widely demonstrated by a number of functional studies [9], our data suggest that FGF also acts upon the expression of PLC genes. FGF might contribute to address a complex, coordinated crosstalk amongst the isoforms. In fact, FGF acts contemporarily upon the expression of a number of PLC genes, reducing the transcription in most cases. As PI-PLC isoforms contribute to regulate the apoptosis [14], FGF probably affects this phenomenon by modifying the expression of the PLC genes. The greatest efficacy of FGF in reducing the PLC transcription was evident for PLCG1, PLCD1 and PLCD3 (Table 2; Fig. 1). Interestingly, PI-PLC c1, ubiquitously expressed, regulates a multitude of cell functions in a number of tissues, although its role in the regulation of cell proliferation remains controversial [15, 31–35]. Literature data suggest that FGF induces the G2/M transition acting upon the Akt/ PLC c1 axis [15]. Our results suggest that FGF might act, just affecting the expression of PLCG1. The PI-PLC d family members are the most basic due to their very simple structure and high sensitivity to calcium [14]. They regulate the activity of the other PI-PLC subfamilies’ members [14]. PI-PLC d1 probably bears antioncogene functions [36, 37] that strongly suggest a role in the cell cycle control. Plcd1 was expressed in quiescent rat astrocytes and unexpressed in the tumour counterpart [38]. Both PI-PLC d1 and d3 decreased in different cell types after inflammatory stimulation [17, 39, 40]. Our results also indicate that FGF dosage did not act linearly upon PLC genes. We observed linear correspondence between mRNA reduction and the increasing FGF dose exclusively for PLCG2. Interestingly, PI-PLC c2, mainly expressed in haematopoietic elements, plays a key role in the regulation of the immune response [41–43]. Regarding the exposure time, the most efficient effects of FGF were in the 3–6-h interval. Therefore, FGF mainly affects the transcription of PLC genes shortly after administration. With notable exceptions (PLCD3 and PLCH1), after 24 h, the effect of FGF upon genes’ transcription was less intense. Interestingly, activation of PIPLC g1 enhances the GPCR-mediated calcium pathway [44] and probably plays as a sensor activated by very small calcium increases. PI-PLC g1 is probably involved in the finest regulation of signalling [14, 45]. Increasing evidence indicates that PI-PLC g isoforms might be of critical importance and their expression after FGF treatment will deserve further studies. In fact, PI-PLC g isoforms were recently analysed in selected human osteosarcoma cell lines [46] and in human skin fibroblasts [47]. In HUVECs, PI-PLC g isoforms are slightly, although inconstantly, expressed [16] and the expression varied under inflammatory stimulation [17].
123
Mol Cell Biochem (2014) 388:51–59
Studies addressed to investigate the relationship of PIPLC enzymes with FGF and to highlight the mechanisms, which regulate PLC gene expression, might provide promising perspectives. Investigations are also required in order to delineate the relationship between the FGFinduced variation in PLC expression and the cell cycle control. The growing interest directed to understand the complex events progressing in EC might provide useful insights for potential therapeutic strategies. The opportunity to understand the complex network that regulates angiogenesis and the ability to manipulate the EC might offer a powerful tool of considerable practical and clinical importance. Acknowledgements This investigation was funded by the Iniziative Universitarie 2011 Funding of ‘Sapienza’ University of Rome. The authors thank the ‘Serena Talarico Association’ for precious support to this research.
References 1. Christ M, Wehling M (1998) Cardiovascular steroid actions: swift swallows or sluggish snails? Cardiovasc Res 40:34–44 2. Grienddling KK, Alexander WR (1996) Endothelial control of the cardiovascular system: recent advances. FASEB J 10:283–292 3. Gloe T, Pohl U (2002) Laminin binding conveys mechanosensing in endothelial cells. News Physiol Sci 17:166–169 4. Folkman J, Klagsbrun M (1987) Angiogenic factors. Science 235(4787):442–447 5. Sato Y, Rifkin DB (1988) Autocrine activities of basic fibroblast growth factor: regulation of endothelial cell movement, plasminogen activator synthesis, and DNA synthesis. J Cell Biol 107(3):1199–1205 6. Maric D, Fiorio PA, Chang YH et al (2007) Self-renewing and differentiating properties of cortical neural stem cells are selectively regulated by basic fibroblast growth factor (FGF) signaling via specific FGF receptors. J Neurosci 27(8):1836–1852 7. McFarlane S, McNeill L, Holt CE (1995) FGF signaling and target recognition in the developing Xenopus visual system. Neuron 15(5):1017–1028 8. Maffucci T, Falasca M (2007) Phosphoinositide 3-kinasedependent regulation of phospholipase Cgamma. Biochem Soc Trans 35:229–230 9. Berridge MJ, Dupont G (1994) Spatial and temporal signalling by calcium. Curr Opin Cell Biol 6(2):267–274 10. Divecha N, Irvine RF (1995) Phospholipid signaling. Cell 80(2):269–278 11. Hisatsune C, Nakamura K, Kuroda Y et al (2005) Amplification of Ca2? signaling by diacylglycerolmediatedinositol 1,4,5-trisphosphate production. J Biol Chem 280(12):11723–11730 12. Rhee SG (2001) Regulation of phosphoinositide-specific phospholipase C. Annu Rev Biochem 70:281–312 13. Bunney TD, Katan M (2011) PLC regulation: emerging pictures for molecular mechanisms. Trends Biochem Sci 36(2):88–96 14. Suh PG, Park J, Manzoli L et al (2008) Multiple roles of phosphoinositide-specific phospholipase C isozymes. BMB Rep 41: 415–434 15. Browaeys-Poly E, Perdereau D, Lescuyer A et al (2009) Akt interaction with PLC(gamma) regulates the G(2)/M transition triggered by FGF receptors from MDA-MB-231 breast cancer cells. Anticancer Res 29(12):4965–4969
Mol Cell Biochem (2014) 388:51–59 16. Lo Vasco VR, Pacini L, Di Raimo T et al (2011) Expression of phosphoinositide-specific phospholipase C isoforms in HUVEC. J Clin Pathol 64(10):911–915 17. Lo Vasco VR, Leopizzi M, Chiappetta C et al (2013) Lypopolysaccharide downregulates the expression of selected phospholipase C genes in cultured endothelial cells. Inflammation 36(4):862–868. doi:10.1007/s10753-013-9613-3 18. Shiba H, Nakamura S, Shirakawa M et al (1995) Effects of basic fibroblast growth factor on proliferation, the expression of osteonectin (SPARC) and alkaline phosphatase, and calcification in cultures of human pulp cells. Dev Biol 170(2):457–466 19. Shiba T, Nishimura D, Kawazoe Y et al (2003) Modulation of mitogenic activity of fibroblast growth factors by inorganic polyphosphate. J Biol Chem 278(29):26788–26792 20. Mosmann T (1993) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63 21. Raballo R, Rhee J, Lyn-Cook R et al (2000) Basic fibroblast growth factor (Fgf2) is necessary for cell proliferation and neurogenesis in the developing cerebral cortex. J Neurosci 20(13):5012–5023 22. Yang J, Meyer M, Muller AK et al (2010) Fibroblast growth factor receptors 1 and 2 in keratinocytes control the epidermal barrier and cutaneous homeostasis. J Cell Biol 188(6):935–952 23. Falasca M, Logan SK, Lehto VP et al (1998) Activation of phospholipase C gamma by PI 3-kinase induced PH domainmediated membrane targeting. EMBO J 17:414–422 24. Cao R, Jensen LD, Soll I et al (2008) Hypoxia-induced retinal angiogenesis in zebrafish as a model to study retinopathy. PLoS ONE 3(7):e2748 25. Zhao G, Li WY, Chen D et al (2011) A novel, selective inhibitor of fibroblast growth factor receptors that shows a potent broad spectrum of antitumor activity in several tumor xenograft models. Mol Cancer Ther 10(11):2200–2210 26. Webber CA, Hyakutake MT, McFarlane S (2003) Fibroblast growth factors redirect retinal axons in vitro and in vivo. Dev Biol 263(1):24–34 27. Chang CH, Huang YL, Shyu MK et al (2013) Sphingosine-1phosphate induces VEGF-C expression through a MMP-2/FGF-1/ FGFR-1-dependent pathway in endothelial cells in vitro. Acta Pharmacol Sin 34(3):360–366 28. Hughes-Fulford M, Li CF (2011) The role of FGF-2 and BMP-2 in regulation of gene induction, cell proliferation and mineralization. J Orthop Surg Res 9(6):8 29. Murakami M, Nguyen LT, Hatanaka K et al (2011) FGFdependent regulation of VEGF receptor 2 expression in mice. J Clin Invest 121(7):2668–2678 30. Ji QS, Ermini S, Baulida J et al (1998) Epidermal growth factor signaling and mitogenesisin Plcg1 null mouse embryonic fibroblasts. Mol Biol Cell 9:749–757 31. Smith MR, Liu YL, Kim H et al (1990) Inhibition of serum- and ras-stimulated DNA synthesis by antibodies to phospholipase C. Science 247:1074–1077 32. Wang Z, Gluck S, Zhang L et al (1998) Requirement for phospholipase C-gamma1 enzymatic activity in growth factor-induced mitogenesis. Mol Cell Biol 18:590–597
59 33. Oh JE, Kook JK, Park KH et al (2003) Phospholipase C-gamma1 is required for subculture-induced terminal differentiation of normal human oral keratinocytes. Int J Mol Med 11:491–498 34. Kolsch V, Charest PG, Firtel RA (2008) The regulation of cell motility and chemotaxis by phospholipid signaling. J Cell Sci 121:551–559 35. Stallings JD, Zeng YX, Narvaez F et al (2008) Phospholipase C-delta 1is linked to pro liferation, DNA synthesis and cyclin E levels. J Biol Chem 283:13992–14001 36. Stallings JD, Tall EG, Pentyala S et al (2005) Nuclear translocation of phospholipase C-delta1 is linked to the cell cycle and nuclear phosphatidylinositol 4,5-bisphosphate. J Biol Chem 280:22060–22069 37. Lo Vasco VR, Fabrizi C, Artico M et al (2007) Expression of phosphoinositide-specific phospholipase C isoenzymes in cultured astrocytes. J Cell Biochem 100(4):952–959 38. Lo Vasco VR, Fabrizi C, Fumagalli L et al (2010) Expression of phosphoinositide specific phospholipase C isoenzymes in cultured astrocytes activated after stimulation with Lipopolysaccharide. J Cell Biochem 109(5):1006–1012 39. Lo Vasco VR, Leopizzi M, Chiappetta C et al (2012) Expression of phosphoinositide-specific phospholipase C enzymes in normal endometrium and in endometriosis. Fertil Steril 98(2):410–414 40. Marshall AJ, Niiro H, Yun TJ et al (2000) Regulation of B-cell activation and differentiation by the phosphatidylinositol 3-kinase and phospholipase Cgamma pathway. Immunol Rev 176:30–46 41. Homma Y, Takenawa T, Emori Y et al (1989) Tissue- and cell type-specific expression of mRNAs for four types of inositol phospholipid-specific phospholipase C. Biochem Biophys Res Commun 164:406–412 42. Yu P, Constien R, Dear N et al (2005) Autoimmunity and inflammation due to a gain-of-function mutation in phospholipase C gamma 2 that specifically increases external Ca2? entry. Immunity 22:451–465 43. Kim JK, Choi JW, Lim S et al (2011) Phospholipase C-g1 is activated by intracellular Ca(2?) mobilization and enhances GPCRs/PLC/Ca(2?) signaling. Cell Signal 23(6):1022–1029 44. Stewart AJ, Morgan K, Farquharson C et al (2007) Phospholipase C-eta enzymes as putative protein kinase C and Ca2? signalling components in neuronal and neuroendocrine tissues. Neuroendocrinology 86(4):243–248 45. Lo Vasco VR, Leopizzi M, Chiappetta C et al (2013) Expression of phosphoinositide-specific phospholipase C enzymes in human osteosarcoma cell lines. J Cell Commun Signal 7(2):141–150. doi:10.1007/s12079-013-0194-6 46. Lo Vasco VR, Leopizzi M, Chiappetta C et al (2013) Expression of phosphoinositide-specific phospholipase C enzymes in human skin fibroblasts. Connect Tissue Res 54(1):1–4 47. Lo Vasco VR, Fabrizi C, Panetta B et al (2010) Expression pattern and sub cellular distribution of phosphoinositide specific phospholipase C enzymes after treatment with U-73122 in rat astrocytoma cells. J Cell Biochem 110(4):1005–1012
123
