Overexpression of Hdac4 Down-regulates Vascular Endothelial Growth Factor Expression in Chondrosarcoma Cells by Modulating Runx2 Expression

Introduction Tumor progression and growth require an appropriate rate of blood vessel formation for the rate of neoplastic cellular proliferation. Angiogenesis is regulated by the balance of proand anti-angiogenic factors. The most potent pro-angiogenic factor is vascular endothelial growth factor (VEGF). Its expression is regulated by pathways related to the normal physiologic response to hypoxia (1) and genetic alterations related to the malignant phenotype. Reactivation of pathways normally operative during growth is a common theme in cancer biology. These pathways can give a selective growth advantage to the tumor cells. After growth is completed, some genes are turned off by epigenetic events such as DNA methylation and histone modification (2). Recent studies show that class II histone deacetylase genes (HDACs) are involved in cellular differentiation and developmental processes and their dysregulation may be involved in carcinogenesis. In the growth plate, HDAC4 inhibits the activity of the transcription factor runt-related gene 2 (Runx 2/Cbfa1) in the proliferative zone. HDAC4 decreases and Runx2 increases during growth plate maturation, which ultimately culminates in endochondral ossification and angiogenesis. In transgenic mice Runx2 increases lymphomagenesis, is expressed in normal murine endothelial cells stimulated by angiogenic cytokines, and in fibroblasts increases VEGF mRNA and protein levels. In tumors, reduced expression of class II HDACs is associated with poor prognosis in lung cancer patients (3). These reports suggest that Runx2 may play a role in carcinogenesis and angiogenesis. In this study, we test the hypotheses that HDAC4 expression is decreased while Runx2 and VEGF are increased in chondrosarcoma cells when compared with normal chondrocytes, and that transfection with an HDAC4 expression vector can reverse these abnormalities. Materials and Methods Cell lines and cell culture: Human chondrocytes isolated from adult articular cartilage and human chondrosarcoma (CS) cell lines CS-1 and JJ cells were cultured in DMEM, 1640 medium and complete medium (40% of DMEM, 40% of MEM, 20% of F12) with 10% FBS, respectively (1). Transfections: Cells were transiently transfected with expression constructs for HDAC4, Runx2, and/or Runx2 promoter/pGL3 luciferase reporter plasmid or empty vector using lipofectamin 2000 in 6 or 12 well plates 24 hr after seeding. Cells were then incubated for 48 hours and harvested for the following experiments. Real-time RT-PCR: RNA was isolated from the transfected and nontransfected cells, and treated by DNase. The quantification of mRNA for HDAC4, Runx2, and VEGF was performed by one-step real-time quantitative RT-PCR (Qiagen). Luciferase Assay: Co-transfections with Runx2 promoter/pGL3 luciferase reporter plasmid were performed with expression constructs for HDAC4-full length, the N-terminal and the C-terminal fragments of HDAC4, or empty vector. The Promega Dual LUC system was used to assess reporter gene expression. In this system cells are co-transfected with a pRL plasmid (Renilla LUC driven by a full SV40 promoter), which is used as an internal control to which the activity of the experimental reporter is normalized. Western Blot Analysis of VEGF expression: Cell lysates containing 20μg protein from transfections with HDAC4 plasmid or empty vector in CS-1 and JJ cell lines were separated via SDS-PAGE and anti-VEGF polyclonal antibody (Santa Cruz) was used to detect VEGF protein. Statistics: The differences were evaluated with one-way ANOVA, followed by the Student’s t-test. The null hypothesis of no difference was rejected at a significance level of 5%. Results HDAC4 mRNA level was reduced in CS cells when compared with normal chondrocytes as shown by real-time RT-PCR (Fig. 1), while Runx2 mRNA was upregulated in the CS cells. CS cell lines, JJ and CS-1, transfected with HDAC4-pcDNA3 expressed the Runx2 gene at 1/2 the level of those transfected with empty vector, and the VEGF gene at 1/3 (Fig 2). Overexpression of HDAC4 also decreased VEGF protein production as shown by Western blot, whereas transfection with Runx2 upregulated VEGF gene expression 1.5-2 fold. Luciferase assay showed that in CS-1, HDAC4 and its N-terminal fragment inhibit the activity of the Runx2 promoter, while the C-terminal fragment of HDAC4 has no effect (Fig 3). The same results were found in chondrocytes. All reported results are statistically significant.