Connective Tissue Growth Factor (CTGF/CCN2) Is Negatively Regulated during Neuron-Glioblastoma Interaction

<div><p>Connective-tissue growth factor (CTGF/CCN2) is a matricellular-secreted protein involved in complex processes such as wound healing, angiogenesis, fibrosis and metastasis, in the regulation of cell proliferation, migration and extracellular matrix remodeling. Glioblastoma (GBM) is the major malignant primary brain tumor and its adaptation to the central nervous system microenvironment requires the production and remodeling of the extracellular matrix. Previously, we published an <em>in vitro</em> approach to test if neurons can influence the expression of the GBM extracellular matrix. We demonstrated that neurons remodeled glioma cell laminin. The present study shows that neurons are also able to modulate CTGF expression in GBM. CTGF immnoreactivity and mRNA levels in GBM cells are dramatically decreased when these cells are co-cultured with neonatal neurons. As proof of particular neuron effects, neonatal neurons co-cultured onto GBM cells also inhibit the reporter luciferase activity under control of the CTGF promoter, suggesting inhibition at the transcription level. This inhibition seems to be contact-mediated, since conditioned media from embryonic or neonatal neurons do not affect CTGF expression in GBM cells. Furthermore, the inhibition of CTGF expression in GBM/neuronal co-cultures seems to affect the two main signaling pathways related to CTGF. We observed inhibition of TGFβ luciferase reporter assay; however phopho-SMAD2 levels did not change in these co-cultures. In addition levels of phospho-p44/42 MAPK were decreased in co-cultured GBM cells. Finally, in transwell migration assay, CTGF siRNA transfected GBM cells or GBM cells co-cultured with neurons showed a decrease in the migration rate compared to controls. Previous data regarding laminin and these results demonstrating that CTGF is down-regulated in GBM cells co-cultured with neonatal neurons points out an interesting view in the understanding of the tumor and cerebral microenvironment interactions and could open up new strategies as well as suggest a new target in GBM control.</p> </div

Downregulation of CTGF inhibits GBM95 cell migration.

<p>Representative images of HE stained migrated cells. GBM95 cells (A) GBM95 platted with P0 neurons (B) Graph showing the percentage of cells that migrate in each condition (C). GBM95 transfected with control siRNA (D) or siRNA to downregulate CTGF expression (E). Statistical analysis of the effects of downregulation of CTGF on the GBM95 migration (F). Transwell migration of co-cultures of GBM95 with P0 neurons and GBM95 siRNA to downregulate CTGF shows a significant reduction in motility when CTGF levels are reduced. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055605#s3" target="_blank">Results</a> are mean±SD from three experiments each performed in duplicate. The numbers of invasive cells were significant different from the control group (*p<0.05 and **p<0.01).</p

Inhibition of CTGF in GBM cells by P0 neurons do not require TGFβ/SMAD2/3 signaling.

<p>(A) Western blot analysis of the cell extracts from GBM95, GBM02 or U87 and GBM95, GBM02 or U87 co-cultured with P0 neurons. From top to bottom, the pictures show membranes reacted with anti-phospho SMAD2/3 and anti-α tubulin antibodies. Molecular weight in kDa is shown on the left side of the picture. (B–D) Graphs show Firefly/Renilla luciferase activity of GBM95 (B), GBM02 (C) or U87 (D) cells transfected with 3TP-lux and Renilla plasmids were cultured alone or P0 neurons, in presence or absence of TGFβ-1 (1 nM). Each point represents the average of three independent experiments done in triplicate.*p<0.05 (in comparison to the control).</p

CTGF promoter activity is inhibited when neurons are co-cultured with GBM cells.

<p>Graph shows Firefly/Renilla luciferase activity of GBM95 or U87 cells transfected with CTGF-lux and Renilla plasmids co-cultured with different amounts of freshly dissociated neurons. Note that 5×10⁴ neurons were able to inhibit more than 40% of luciferase activity (comparing first to third bar, p<0.05) and 2×10⁵ neurons were able to inhibit more than 50% of luciferase activity controlled by the CTGF promoter (A) (comparing first to fourth bar, p<0.01). As a positive control 0.5 nM of TGFβ-1 was used and we could observed an increase of almost 40% of luciferase activity (comparing first to fifth bar, p<0.05). 1×10⁵ P0 neurons were able to inhibit almost 40% of luciferase activity when co-cultured with U87 cells (B) (p<0.05).</p

Curculio sp.

<p>Representative images of HE stained migrated cells. U87 cells (A) U87 platted with P0 neurons (B) Graph showing the percentage of cells that migrate in each condition (C). U87 were transfected with control siRNA (D) or siRNA to down regulate CTGF expression (E). Statistical analysis of the effects with P0 neurons and downregulation of CTGF on the GBM02 migration (F). Downregulation of CTGF dramatically reduced U87 cell migration in vitro. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055605#s3" target="_blank">Results</a> are mean±SD from three experiments each performed in duplicate. *p<0.05 and **p<0.01, statistically significant difference.</p

CTGF immunoreactivity is decreased and CTGF mRNA is down-regulated in co-cultures of GBM cells with neurons.

<p>Immunocytochemistry showing CTGF expression (red) in GBM 95 cultures (A, C) and co-cultures of GBM95 with E18 (D, F) or P0 neurons (G, I). Stained neuron β tubulin III (green) are shown in (E, H). The arrowhead shows a CTGF stained E18 neuron (D). Notice that CTGF staining decreased in co-cultures (comparing A with D and G). Bar 50 µm. Densitometry of the immunocytochemistry images show a decrease in the intensity of CTGF staining of GBM95, GBM02 or U87 co-cultured with P0 neurons (J–L) (p<0.05). RT-PCR analysis of CTGF expression in GBM95 co-cultured with E18 or P0 neurons (M). Base pairs numbers appears on the right side of each gel. GAPDH was used as loading control. Histogram expressing arbitrary units (A.U) products obtained from CTGF over GAPDH of the PCR bands (N). CTGF expression of GBM95 co-cultured with P0 neurons was decreased when compared to pure GBM95 cultures.</p

Exogenous CTGF protein increases the number of Sox2-positive cells of a neural progenitor culture.

<p>Immunostaining showing Sox2 (A and D) expression of untreated and CTGF-treated cells. C and F show merged pictures of Sox2 immunostaining together with nuclei-DAPI staining. Scale bars 10 μm. G shows the percentage of cells that were positive for Sox2.</p

Recombinant CTGF increases expression and deposition of fibronectin.

<p>Immunostaining and immunoblotting for fibronectin and laminin. (A-D) Untreated or CTGF-treated progenitor neural cells immunostained for fibronectin (A, B) and laminin (C, D). (E) Immunoblotting for fibronectin and laminin of untreated (lane 1) or CTGF-treated neural progenitor cells (lane 2). CTGF incubation of neural progenitor cells for 120 h increased the expression of fibronectin threefold (compare untreated versus CTGF bars of graph in E). Scale bar 50 μm.</p

Exogenous CTGF protein increases GFAP and reduces Sox2 expression in human cancer stem cells.

<p>Immunoblotting showing GFAP, β-tubulin III and Sox2 expression in untreated and 1 nM and 5 nM CTGF-treated cells. CTGF incubation of human glioma stem cells for 120 h increased GFAP and reduced Sox2 expression in a dose-dependent manner. Alpha-tubulin was used as a loading control. **<i>P</i> < 0.001 and *<i>P</i> < 0.005.</p

Connective-Tissue Growth Factor (CTGF/CCN2) Induces Astrogenesis and Fibronectin Expression of Embryonic Neural Cells <i>In Vitro</i>

<div><p>Connective-tissue growth factor (CTGF) is a modular secreted protein implicated in multiple cellular events such as chondrogenesis, skeletogenesis, angiogenesis and wound healing. CTGF contains four different structural modules. This modular organization is characteristic of members of the CCN family. The acronym was derived from the first three members discovered, cysteine-rich 61 (CYR61), CTGF and nephroblastoma overexpressed (NOV). CTGF is implicated as a mediator of important cell processes such as adhesion, migration, proliferation and differentiation. Extensive data have shown that CTGF interacts particularly with the TGFβ, WNT and MAPK signaling pathways. The capacity of CTGF to interact with different growth factors lends it an important role during early and late development, especially in the anterior region of the embryo. ctgf knockout mice have several cranio-facial defects, and the skeletal system is also greatly affected due to an impairment of the vascular-system development during chondrogenesis. This study, for the first time, indicated that CTGF is a potent inductor of gliogenesis during development. Our results showed that <i>in vitro</i> addition of recombinant CTGF protein to an embryonic mouse neural precursor cell culture increased the number of GFAP- and GFAP/Nestin-positive cells. Surprisingly, CTGF also increased the number of Sox2-positive cells. Moreover, this induction seemed not to involve cell proliferation. In addition, exogenous CTGF activated p44/42 but not p38 or JNK MAPK signaling, and increased the expression and deposition of the fibronectin extracellular matrix protein. Finally, CTGF was also able to induce GFAP as well as Nestin expression in a human malignant glioma stem cell line, suggesting a possible role in the differentiation process of gliomas. These results implicate ctgf as a key gene for astrogenesis during development, and suggest that its mechanism may involve activation of p44/42 MAPK signaling. Additionally, CTGF-induced differentiation of glioblastoma stem cells into a less-tumorigenic state could increase the chances of successful intervention, since differentiated cells are more vulnerable to cancer treatments.</p></div